Detection of tumors by fluorescence tomography
using multifunctional gold (Au) nanoparticles
Sonu Bhaskar
Zaragoza, 2009
1
Instituto Universitario de
Nanociencia de Aragón
UNIVERSIDAD DE ZARAGOZA
Detection of tumors by fluorescence tomography using
multifunctional gold (Au) nanoparticles
Sonu Bhaskar
Presented to the Faculty of Graduate School of
Instituto de Investigación en Ingeniería de Aragón, I3A,
Universidad de Zaragoza, Zaragoza, España
In partial fulfillment of requirements for degree of the
Masters in Biomedical Engineering
Director of the thesis
Codirector
Dr. Jesús Martínez de la Fuente
Dra. Maria Valeria Grazu Bonavia
2
CERTIFICADO DE TRABAJO
D . Jesús Martínez
Martí nez de la Fuente,
Fuente , Investigador Principal, Agencia Aragonesa para
la Investigación y el Desarrollo (ARAID), Grupo de Biofuncionalización de
Nanopartículas y Superficies, Instituto Universitario de Nanociencia de Aragón
(INA), Universidad de Zaragoza y D ª . Valeria Grazu Bonavia,
Bonavia ,
Investigador , Instituto de Nanociencia de Aragon (INA),
CERTIFY:
That the present thesis entitled "Detection of tumors by fluorescence tomography
using multifunctional gold (Au) nanoparticles" has been made under our guidance
and supervision in the laboratory of Grupo de Biofuncionalización de
Nanopartículas y Superficies, Instituto Universitario de Nanociencia de Aragón
(INA), Universidad de Zaragoza during the academic years 2008-2009, towards
the partial fulfillment of degree in Master in Biomedical Engineering.
Engineering
Zaragoza. September, 2009
Fdo.: Jesús M. de la Fuente
Valeria Grazu Bonavia
3
CERTIFICADO DE TRABAJO
D. Jesús Martínez
Martí nez de la Fuente, Investigador Principal, Agencia Aragonesa para
la Investigación y el Desarrollo (ARAID), Grupo de Biofuncionalización de
Nanopartículas y Superficies, Instituto Universitario de Nanociencia de Aragón
(INA), Universidad de Zaragoza y Dª. Valeria Grazu Bonavia, Investigador ,
Instituto de Nanociencia de Aragon (INA),
CERTIFICAN: Que la memoria de tesis de Master en Ingeniería Biomédica
realizada por Don Sonu Bhaskar (NIE: X9290989R) con el titulo “Detección
de tumores por tomografía fluorescente mediante el uso de nanoparticulas de oro
multifuncionales’’ fue supervisada bajo nuestra dirección en el Laboratorio
de Biofuncionalización de Nanopartículas y Superficies de la Instituto
Universitario de Nanociencia de Aragón (INA) de la Universidad de Zaragoza
durante los años académicos 2008-2009, y se cumple las condiciones
exigidas para que su autor pueda optar al Master en Ingeniería Biomédica
Zaragoza, Septiembre de 2009
Fdo.: Jesús M. de la Fuente
Valeria Grazu Bonavia
4
To my parents...
5
Este Proyecto Fin de Máster
Má ster en Ingeni
Ingen i ería Biomédica se ha llevado a
cabo con ayuda de una beca predoctoral de formación del personal
investigador
concedida
por
la
Dirección General de Investigación,
Innovación y Desarrollo del Departamento de Ciencia, Tecnología y
Universidad del Gobierno de Aragon,
Aragon según resolución de 7 de agosto de
2008.
6
Abstract
Detection of tumors by fluorescence
fluorescence tomography using
multifunctional gold nanoparticles
The most commonly used techniques that are currently used for the detection of
tumors are Magnetic Resonance Imaging (MRI) and Positron Emission
Tomography (PET). However, the radioactive isotopes used for PET can produce
potentially harmful side effects associated with radiation. MRI allows for 3D
images with a resolution of about 50 cubic micrometers. This low resolution
implies the need for long term image acquisition muq which implies a slow data
acquisition. To prevent damage by the use of isotopes or long time image
acquisition, the study of light transmission through highly dispersive media such
as tissue is a target of recent research to use in medical diagnostic imaging.
The overall objective of this project is to design functionalised Gold (Au)
nanoparticles with a fluorescent marker that allows tomographic detection by
using fluorescent light in the near infrared spectral region (NIR). This would allow
an ability to penetrate deep inside through several inches due to the low
absorption capacity of the tissues in the spectral region between 700-850 nm.
During implementation of this project we have optimized the synthesis of
functionalised Gold (Au) nanoparticles with different fluorescent markers, namely
Fluorescin Isothiocynate and Rhodamine. The necessary criterion for a colloidal
system for to be used in biological systems: It must have a uniform size and
should not undergo aggregation in biological fluids. Furthermore, invitro
cytotoxicity and morphological studies have been performed for the analysis of
Cell-Nanoparticle interactions. The in vitro cytotoxicity as well as studies in vivo
fluorescent tomography has been carried out in collaboration with Institute of
Inhalation Biology (IHB), and at Institute of Biological and Medical Imaging (IBMI),
Helmholtz Zentrum Muenchen, and at Technical University of Munich (TUM),
Munich, Germany and the University Hospital-Lozano Blesa, Zaragoza.
7
Resumen
Detección de tumores por tomografía
tomografía fluorescente mediante el
uso de nanoparticulas de oro multifuncionales
Tanto la técnica de imagen por resonancia magnética (MRI) como la tomografía
por emisión de positrones son utilizadas actualmente para la detección de
tumores. Sin embargo, los isótopos radioactivos usados para PET pueden producir
efectos secundarios potencialmente dañinos asociados con el uso la radiación. La
MRI permite obtener imágenes 3D con una resolución cercana a los 50
micrómetros cúbicos. Esta baja resolución conlleva la necesidad de largos tiempos
de adquisición de imágenes lo que implica una muy lenta adquisición de datos.
Para evitar daños por el uso de isótopos o largos tiempos de adquisición de
imágenes, el estudio del transporte de luz a través de medios muy dispersivos
como son los tejidos, es un objetivo reciente de investigación hacia su uso en el
diagnóstico médico por imagen.
El objetivo general que se pretende alcanzar en este proyecto es el diseño de
nanoparticulas de oro funcionalizadas con un marcador fluorescente que permita
su detección por tomografía fluorescente utilizando luz en la región espectral del
infrarrojo cercano (NIR). Esto permitiría una capacidad de penetración de varios
centímetros debido a la baja capacidad de adsorción de los tejidos en la región
espectral entre 700-850 nm.
Durante la realización de este proyecto hemos optimizado la síntesis de
nanoparticulas de oro funcionalizadas con el marcador fluorescente. Las mismas
deben tener un tamaño uniforme y no pueden sufrir agregación en fluidos
biológicos, requisitos estrictamente necesarios para que un sistema coloidal pueda
ser usado en sistemas biológicos. Se ha realizado a su vez la evaluación in vitro
de la citotoxicidad y respuesta celular frente a las nanoparticulas. Tanto los
estudios in vitro de citotoxicidad como los estudios in vivo de tomografía
fluorescente se han realizado en colaboración con el Instituto de Inhalación
Biología (IHB), y en el Instituto de Imagen Biológica e Imagen Medica (IBMI),
Helmholtz Zentrum Muenchen, la Universidad de Munich (TUM), Munich, Alemania
y en el Hospital Clínico Universitario Lozano Blesa, Zaragoza.
8
ACKNOWLEDGEMENTS
I am indebted to many people who helped me through this journey of Master
degree in Biomedical Engineering. Foremost, I am grateful to my research
advisors Dr. Jesús Martínez
Martínez de la Fuente and Dra. Valeria Grazu for their constant
encouragement and unending enthusiasm to help me to learn many fascinating
things in nanotechnology world. I still remember myself attending one of their
courses during our Master Program, which stimulated my curiosity and interest to
delve into the enigmatic world of nanomedicine. They have been not only an
excellent teacher but also a mentor and a guide. They presented me the
opportunity to work in an interdisciplinary program that allowed me to interact
with researchers from diverse fields such as physics, chemistry, biology and
medicine. Moreover, coming from an engineering background, I was very naïve to
the field of biochemistry. So I am indeed very indebted to Dr. Jesus and Dr.
Valeria for their patience in bearing with me. I have learned many techniques and
above all, also loved the scientific environment at INA. Valeria has been an
excellent mentor, and a friend. She has been very humble and forgiving.
I had stimulating discussions with Jesús
Jesús and Valeria on research and experiments.
I found their tremendous enthusiasm and perseverance in solving a problem so
inspirational that I was willing to continue the project despite of getting negative
results during initial months. I would like to thank Pablo especially for helping me
with synthesis of nanoparticles.
This project could not have been completed without the support of our
collaborators: Dr. Furong Tian, Dr. Tobias Stoeger and Dr. Wolfgang Kreyling at
Institute of Inhalation Biology, Helmholtz Zentrum, Muenchen, Munich, Germany
and Dr. Daniel Razansky and Prof. Vasilis Ntziachristamos, at Institute of Biological
and Medical Imaging (IBMI),
(IBMI), Helmholtz Zentrum Muenchen, and Technical
University of Munich,
Munich, Munich, Germany, who assisted with imaging and biological
aspects of the project. I am also thankful to Dr. Furong Tian for funding my stay at
Munich.
I am greatful to all our lab members,, Pablo, Carlos, Beatriz, Iñaki, Mozos, Puertas
and everyone out there. They have been very kind and cooperative.
I am thankful to the friendly staff at INA for their assistance.
I would like to thank Dr Pedro C. Marijuán
Marijuán at Instituto Aragonés Ciencias de la
Salud, for his support in carrying out my research work at INA and also for his
valuable guidance and patience at various stages of the project. His insights into
the project helped me view the forest instead of individual trees. I am grateful to
9
Raquel del Moral, Amaia Calderón, Beatriz Poblador and Jorge Navarro for their
loving friendship and cheerfulness. I am also thankful to all my colleagues at
Instituto Aragonés Ciencias de la Salud (I+CS).
I appreciate the generous support from the funding agencies,, Diputación General
de Aragón, Gobierno de Aragón and Instituto
Instituto Aragonés
Aragonés Ciencias de la Salud to
accomplish this project.
At personal level, my family and friends inspired and supported me through out my
endeavors to pursue higher studies. I am indebted to my parents and brothers for
considering my dreams as theirs and providing me the strength and enthusiasm to
reach my goals. My father’s unrelenting support and my mother’s confidence in my
abilities gave me confidence to look beyond the horizon. I am grateful to them for
always being there for me.
This endeavor would not have started for my friends Hector Manuel Bhecerra,
Abebe, Fernando Naranjo Mayorga, Andres Galavis and others who were
responsible for my making my stay in Zaragoza a memorable experience. I extend
my thanks to Dr. Pablo Laguna, Director of the Biomedical Engineering program
for his support and patience in considering an English speaking alumnus for this
course. And I am also thankful to all the professors and fellow batchmates through
whom I learned a lot of new things in sciences and biomedical engineering in
particular. I would also like to thank Cristina Traid,
Traid at the International Students
office of Universidad de Zaragoza for her support and guidance during various
legal formalities in Spain.
I would like to thank my teachers, friends, colleagues
colleagues and labmates here in
Zaragoza, Bombay, Madras, Munich and Bangalore who inspired me to dream and
work towards making my dreams come true. Finally, I am greatful to the almighty.
Sonu Bhaskar
September, 2009
Zaragoza, Spain
10
Index
1. INTRODUCTION
INTRODUCTION....................................................
.................................................... ..............................
.......................................
..............................................
....................13
...........13
1.1. The innovation of nanotechnology.....................................
nanotechnology............................................
........
...........13
1.2. Simple multiple functionality of polymeric nanoparticles............
nanoparticles............17
1.3. Targeted constructs......................................................................
constructs......................................................................18
1.4. CoCo-encapsulation of multiple therapeutics..................................
therapeutics..................................18
2.
EXPERIMENTAL PROCEDURE.....................................
........................................
..................................
.........................
.......................
....................22
2.1. Nanoparticle synthesis and Invitro Studies.................................
Studies..................................
..................................22
2.1.1. Synthesis: Au@tiopronin.............................................................
Au@tiopronin......................................................................
.........................................................................22
............22
2.1.2. Materials and Methods......
Methods............................................................................
............................................................................23
......................................................................23
2.1.3. Synthesis of Gold Nanoparticles.............................................................
Nanoparticles.............................................................24
.............................................................24
2.1.4. Derivatization of Gold Nanoparticles. Au@tioproninAu@tiopronin-EDA.............
EDA...................
....................
.......24
2.1.5. Au@tioproninAu@tiopronin-PEG..........................................................................
PEG................................................................................
..................................................................................2
........24
..24
2.1.6.
6. Functionalisation Au2.1.
Au-PEG/AuPEG/Au-EDA nanoparticles
nanoparticles with fluorochromes
........................................................................................
................................................................................................................
..................................................
...................................27
...........27
2.1.7. Cell Culture
Culture..............................................................................................
..............................................................................................28
..............................................................................................28
2.1.8. Cell Viability..................................................................
Viability............................................................................................
............................................................................................2
..........................28
2.1.9. Clathrin Immunofluorescence and Cytoskeletal Observation................
Observation................29
................29
2.2. Mesoscopic Fluorescence Tomography based invivo imaging of
fluorescent nanoprobes...........................................................
nanoprobes......................................................................
........................................................................
.............29
3.
RESULTS AND DISCUSSION.....................................................................
......................................................................
......................32
3.1. UVUV-Vis spectra of Rhodamine Gold Nanoparticles at different
dilutions............................................................................................................
.............................................................................................................
...........................................3
...32
3.2. FTIR Spectra of AuAu-EDA and AuAu-PEG......................................
PEG......................................33
3.3. Cell Culture and Cytotoxicity assays................................................34
................................................34
3.4. Cell Morphology and Cytoskeleton Studies...................................
....................................
............................35
3.5. Invivo Imaging of fluorescent nanoparticles in mouse models
using fluorescent tomography ..................................
.............................................
.......38
..............................................38
11
4. CONCLUSION AND FUTURE PROSPECTS........................
.........................
...................
................4
.....42
Annexure
Annexure
Annexure
Annexure
Annexure
I ...................................................................................
.........................................................................................
.............................................
..............
.........
...........
....44
II ..................................................................................
.........................................................................................
....................................................
................
..........
....45
III ......................................
..........................................................................
...................................................................
..........................................
............
.......
.......
..........
....52
IV....
............................................................................
...........................................................................
........................................
............
..........
........
.......
........54
12
1.
INTRODUCTION
1.1. The inno
innovation of nanotechnology
Nanotechnology innovation has brought a variety of new possibilities into
biological discovery and clinical practice. In particular, nano-scaled carriers have
revolutionalized drug delivery, allowing for therapeutic agents to be selectively
targeted on an organ, tissue and cell specific level, also minimizing exposure of
healthy tissue to the drug. At the core of nano-scaled drug delivery systems,
three issues are very important, namely functionalization of nanocarriers, delivery
to target organs and in vivo imaging. To begin with, the latest developments on
highly specific conjugation strategies that are used to attach biomolecules to the
surface of nanoparticles are reviewed here and extensively in Annexure IV.
Besides drug carrying capabilities, the functionalisation of nanocarriers also
facilitate their transport to primary target organs. Potential treatments for Cancer,
such as Brain tumors sought-after application of nanomedicine. Likewise any
other drug delivery system, a number of parameters need to be registered once
functionalised nanoparticles are administered, for instance their efficiency in
organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo
imaging of nanomaterials is an exciting recent field that can provide real-time
tracking of those nanocarriers. We have recently reported in an extensive review
(see annexure IV) a range of systems suitable for in vivo imaging and monitoring
of drug delivery, with an emphasis on recent advances in optical and hybrid
imaging modalities, such as fluorescent and mesoscopic molecular tomography,
multispectral optoacoustic tomography and fluorescent protein tomography.
Overall, great potential is foreseen for nanocarriers in medical diagnostics,
therapeutics and molecular targeting. A proposed roadmap for ongoing and future
research directions is therefore discussed in detail with emphasis on the
development of novel approaches for functionalisation, targeting and imaging of
nano-based drug delivery systems, a cutting-edge technology poised to change
the ways medicine is administered.
The overall objective of this project is to design functionalised Gold (Au)
nanoparticles with a fluorescent marker that allows tomographic detection by
using fluorescent light in the near infrared spectral region (NIR). This would allow
an ability to penetrate deep inside through several inches due to the low
absorption capacity of the tissues in the spectral region between 700-850 nm.
13
Tumor targetting by
nanoparticles
Tumor cell uptake of
nanoparticles
Tumor detection for
diagnostics
Fluorescence
microscopy
Tumor therapy
Tumor free
mouse
Optical imaging
High-resolution Xray computed
tomography (micro-CT)
Fluorescence Molecular
Tomography (FMT)
Multimodality
imaging
Figure 1.1. Schematic representation of nanoparticle based tumor detection and therapy (Bhaskar, S,
et.al. 2009 [2.21])
[2.21]).
Figure 1.2. A schematic representation of nanoparticle formulations. A) Simple multi-functional
nanoparticles are formulated from a solid polymer core in which chemotherapeutic drugs and/or
alternate anticancer therapeutics (such as antiangiogenic drugs or multi-drug resistance
modulating drugs) are encapsulated. The core is surrounded by PEG chains, which promote
prolonged circulation and to which tumour-targeting ligands can be covalently attached. B)
Complex multi-functional nanoparticles include (from left to right) iron oxide nanoparticles, gold
nanoshells, Gd nanoparticles and quantum dots. Surface modification allows for covalent
14
attachment of tumourtargeting ligands and encapsulation of anticancer drugs (Figure from Vlerken
et al. 2006 [2.20]). Gd: Gadolinium; PEG: Poly(ethylene glycol); TOP: Tri-n-octyl phosphine;
TOPO: Tri-n-octyl phosphine oxide
Undoubtedly, the use of nanoparticles as drug delivery vehicles for anticancer
therapeutics has great potential to revolutionise the future of cancer therapy. As
tumour architecture causes nanoparticles to preferentially accumulate at the
tumour site, their use as drug delivery vectors results in the localisation of a
greater amount of the drug load at the tumour site; thus improving cancer therapy
and reducing the harmful nonspecific side effects of chemotherapeutics. In
addition, formulation of these nanoparticles with imaging contrast agents provides
a very efficient system for cancer diagnostics. Given the exhaustive possibilities
available to polymeric nanoparticle chemistry, research has quickly been directed
at multi functional nanoparticles, combining tumour targeting, tumour therapy and
tumour imaging in an all-in-one system, providing a useful multi-modal approach
in the battle against cancer. Here we discuss the properties of nanoparticles that
allow for such multiple functionality, as well as recent scientific advances in the
area of multi-functional nanoparticles for cancer therapeutics.
With the advancement in the field of oncology, the search for successful cancer
treatment is the quest for the ultimate cancer therapeutic. Although conventional
treatment options such as chemotherapy and radiation have experienced many
advances over the past decades, cancer therapy is still far from optimal.
Effectiveness of cancer therapy depends on a fine ratio that is determined by the
ability of the therapeutic to eradicate the tumour while affecting as few healthy
cells as possible. To this end, systemically administering bolus doses of powerful
chemotherapeutics often results in intense side effects due to the action of the
drugs on sites other than the intended target. With such nonspecific drug action,
the concentration of drug rendered available at the tumour site itself is potentially
beneath the minimal effective concentration, entering the patient into a vicious
predicament between choosing a near-toxic effective dose and a comfortable
ineffective dose. In order to overcome this challenge, decades of research have
focused on developing cancer-specific drugs or delivery systems that can
preferentially localize existing agents to the tumour site. Recent advances in
nanotechnology promises further developments in target-specific drug delivery
systems.
The boom in the nanotechnology research and industry has emerged into a
revolution, thereby making nanoparticles as a type of drug delivery vector with
great efficacy. Nanoparticles are colloidal systems of submicron (< 1 μM) size that
can be constructed from a large variety of materials in a large variety of
compositions. Commonly defined nanoparticle vectors include: liposomes,
micelles, dendrimers, solid lipid nanoparticles, metallic nanoparticles,
semiconductor nanoparticles and polymeric nanoparticles, although the scope of
15
nanoparticle formulations that have been applied to cancer therapy is far more
elaborate. Despite the large variety of formulations available, this review will
focus primarily on natural and synthetic polymer-based solid core nanoparticles,
including metal and nanocrystal formulations, due to their role in the multiple
functionality of the vector. Depending on the chemical composition of the
nanoparticles, these can carry a wide variety of compounds, making them efficient
drug delivery vehicles. In addition, there also exists the ability to introduce to the
particle a metallic core or shell, giving the particle optical, magnetic, or
hyperthermic properties; or to covalently bind antibodies or lectins, whereby
enhancing targeting efficiency of the particle. Such variant properties allow for
the trend towards multiple functionality of nanoparticles that will be discussed in
this review. Examples of biocompatible and biodegradable polymers that have
been used to prepare nanoparticles for tumour-targeted delivery include
poly(D,L-lactide-co-glycolide), poly(ε-caprolactone), and poly(β-amino esters)
[1-4].
Nanoparticles are excellent tumour-targeting vehicles because of a unique
inherent property of solid tumours. Due to the rapid growth of solid tumours,
many tumours present with fenestrated vasculature and poor lymphatic drainage,
resulting in an enhanced permeability and retention (EPR) effect [5], which allows
nanoparticles to accumulate specifically at the tumour site (Figure
Figure 1.1.
1.1.).
.1. Although
nanoparticles protect the drug from rapid metabolism and clearance, as well as
nonspecific recognition and distribution, stealth-shielding nanoparticles (using
PEG surface modification [6]), in addition, will help to avoid uptake by the
reticuloendothelial system [7] and mononuclear phagocytes [8]. Altogether, this
results in the property of nanoparticles to circulate for prolonged periods of time,
allowing them to eventually reach the tumour vasculature where, guided by the
EPR effect, they specifically extravasate through the fenestrated capillaries to
accumulate drugs at the tumour mass. It has been shown that nanoparticle and
polymer conjugate delivery can allow concentrations of the drug near the vicinity
of the tumour to reach 10- to 100-fold higher than when administering free drug
[6]. Beyond the passive tumour-targeting properties by the EPR effect,
intratumoural localisation of nanoparticles can be further improved by active
targeting through conjugation of the particle with tumour-specific recognition of
small molecules, such as folic acid [9], thiamine [10] and even antibodies or
lectins [11]. In addition, at the tumour site, nanoparticles offer one further
advantage: they can be endocytosed/ phagocytosed, enhancing cell internalisation
of the drug, and leading to delivery of the drug closer to the intracellular site of
action [8].
It was soon discovered that tumour-specific accumulation of nanoparticles
provided not the means for drug delivery to the tumour, but also an opportunity to
further conjugate a metallic core or shell to exploit for optical imaging or MRI in
tumour diagnostics, guided hyperthermia therapy and guided radiation therapy.
16
After the use of first-generation nanoparticles as either drug delivery carriers,
imaging agents or guided therapy agents alone, the possibility to combine these
differing properties soon emerged. Subsequently, by exploiting the varied
chemistry of the polymeric nanoparticle, one could encapsulate multiple drugs and
tag on tumour-specific targeting moieties; therefore truly multifunctionalising the
vector. One could also envision sequential delivery of drugs based on their
location in the nanoparticles (e.g., surface bound versus encapsulation in the
matrix). By confining drug molecules to a specific location, the delivery is further
optimised because a particular agent is available where it has the highest effect.
In continuation of the introduction, the paper as described in Annexure IV will
focus on such recent progress surrounding the multiple functionality of
nanoparticles for improved cancer therapy, advancing from simple multiple
functionality of the nanoparticle by inclusion of targeting moieties and
coencapsulation of variant therapeutics, to complex multiple functionality of the
nanoparticle by combining targeting, imaging and therapy together into one
system.
1.2.
1.2. Simple multiple functionality of polymeric nanoparticles
One of the greatest step forward in the field of Polymer chemistry are the
scientific developments that allow for many variations, whereby polymeric
nanoparticles can be easily manipulated without the loss of their desired physical,
chemical, and biological properties. In one manner, this principle can be used to
greatly improve the function of the nanoparticle in cancer therapy through the
attachment of tumour-specific targeting moieties (e.g., antibodies or receptor
ligands), directed at cell surface markers unique to the cancer cell. Alternatively,
this principle can be used to improve the function of the nanoparticle for
simultaneous delivery of a combination of drugs to the cancer cell, creating a
multivalent therapeutic strategy. Such manipulations of the nanoparticle
formulation allow for simple multiple functionality directed at enhancing cancer
therapy (Figure
Figure 1.2.).
1.2.
1.3. Targeted constructs
One of the major challenges in nanomedicine is targeted detection and therapy. To
this end, despite preferential accumulation of nanoparticles in the tumour mass by
the EPR effect, the functionality of these nanoparticles by an inclusion of tumourtargeting moieties enhances tumour-specific localisation of the nanoparticle and
its payload. In addition, it allows for targeting of the nanoparticles to much smaller
and earlier stage tumours, as well as to the cancerous cells that do not belong to a
17
solid tumour mass, such as metastatic cells and cancerous leukocytes. Using the
expression of specific recognition markers by the tumour, bioconjugation of the
nanoparticles with antibodies directed against such tumour markers improves
localisation of the particles specifically at the cancer cells. Two tumour markers
most commonly used as targets for directed therapy are the folic acid receptor
and the EGFR-2 (erbB2/HER2), as their implication in tumorigenesis results in
their over-expression on the cancer cell surface of a wide variety of tumour
types [12-16].
Hence, the choice of antibodies and tumour cell surface markers becomes an
important aspect in nanomedicine approaches towards cancer. For example, folic
acid-coated polymeric nanoparticles showed enhanced localisation and
internalisation of nanoparticles intended for drug delivery to the breast cancer
cells [12], whereas on the other spectrum, folic acid coating also improved
localisation and internalisation of magnetite nanoparticles intended for tumour
imaging of breast cancer cells [17]. Similarly, tagging the anti-HER2 to the
nanoparticle surface greatly improved cell internalisation of gelatin/albumin [15]
and gold nanoparticles [18,19], regardless of the fact that the nanoparticles differ
in structure and intended function. Along these lines, delivery of the therapeutic
can be enhanced by the functionality of nanoparticles with targeting moieties
directed against any number of tumour-specific markers.
1.4. CoCo-encapsulation of multiple therapeutics
As cancer research has progressed, it has became evident that therapy with
cytotoxic drugs was not the only effective option for cancer treatment [2.20]. On
one hand, an alternate strategy arose that has opened up a different direction of
anticancer drug development, mainly therapy directed at inhibiting angiogenesis at
the tumour mass. However, on the other hand, the necessity to design alternate
drugs directed at mechanisms of multi-drug resistance (MDR), has emerged as
multi-drug resistant cancers are unresponsive to conventional chemotherapeutics.
Given such multivalent cancer therapy, nanoparticles provide a good platform to
coadminister anticancer therapeutics directed at different targets, which can all
converge for maximum cell-toxic effect.
- Complex multiple functionality of nanoparticles
The interdisciplinary nature of nanomedicine has brought in scientists from
different domains such as biochemistry, medicine, engineering e.t.c... Beyond the
ordinary use of nanoparticles as a mere vector for delivery of either therapeutic
drugs or imaging contrast agents, it seemed obvious to combine these roles and
18
create all-inclusive nanoparticle formulations that can carry imaging and drug
delivery capabilities; specifically targeted to the tumour site by passive and/or
active targeting.
Furthermore, inherent properties of the core imaging agents, such as iron oxide,
gold, gadolinium and quantum dots, allowed for these nanoparticles to also
function in alternate anticancer therapies, such as hyperthermia, radiation and
photodynamic therapy. Hereby, the possibilities emerged to develop nanoparticles
that simultaneously image and treat cancer; a more complex approach to multiple
functionality.
- Gold nanoparticles
Due to its biocompatibity, Gold nanoparticles have gained tremendous attention in
nanotechnology. Gold nanoparticles are another class of metal nanoparticles that
have found a niche in the tumour-imaging and guided hyperthermia market.
Figure 1.3. Schema showing the multifunctional nanoparticle for tumor detection and therapy.
For more information of the state of the art and future perpectives of
multifunctional nanoparticles, please refer here [1.21, 1.22].
19
In this project, our goal is to design functionalised Gold (Au) nanoparticles with a
fluorescent marker such as Rhodamine e.t.c., and that allows tomographic
detection by using fluorescent light in the near infrared spectral region (NIR).
During implementation of this project we have optimized the synthesis of
functionalised Gold (Au) nanoparticles with different fluorescent markers, namely
Fluorescin Isothiocynate and Rhodamine. The necessary criterion for a colloidal
system for to be used in biological systems: It must have a uniform size and
should not undergo aggregation in biological fluids. Furthermore, invitro
cytotoxicity and morphological studies have been performed for the analysis of
Cell-Nanoparticle interactions. The in vitro cytotoxicity as well as studies in vivo
fluorescent tomography has been carried out in the Clinical Hospital-Lozano
Blesa, Instituto Aragones Ciencias de la Salud, Zaragoza and at Helmholtz Zentrum
Muenchen, Munich, Germany.
References
1. 1. Fonseca C, Simoes S, Gaspar R: Paclitaxel-loaded PLGA nanoparticles:
preparation, physicochemical characterization and in vitro anti-tumoral activity. J.
Control. Release (2002) 83(2):273-286.
83
1.2. Chawla JS, Amiji MM: Biodegradable poly(epsilon-caprolactone) nanoparticles
for tumor-targeted delivery of tamoxifen. Int. J. Pharm. (2002) 249(1-2):127-138.
249
1.3. Potineni A, Lynn DM, Langer R, Amiji MM: Poly(ethylene oxide)-modified
poly(beta-amino ester) nanoparticles as a pH-sensitive biodegradable system for
paclitaxel delivery. J. Control. Release (2003) 86(2-3):223-234.
86
1.4. Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM: Polymeric systems for
controlled drug release. Chem. Rev. (1999) 99:3181-3198.
99
1.5. Maeda H, Wu J, Sawa T Matsumura Y, Hori K: Tumor vascular permeability
and the EPR effect on macromolecular therapeutics: a review. J. Control. Release
(2000) 65:271-284.
65
1.6. Kaul G, Amiji M: Long-circulating poly(ethylene glycol)-modified gelatin
nanoparticles for intracellular delivery. Pharm. Res. (2002) 19(7):1061-1067.
19
1.7. Brannon-Peppas L, Blanchette JO: Nanoparticle and targeted systems for
cancer therapy. Adv. Drug Del. Rev. (2004) 56:1649-1659.
56
1.8. Brigger I, Dubernet C, Couvreur P: Nanoparticles in cancer therapy and
diagnosis. Adv. Drug Del. Rev. (2002) 54:631-651.
54
1.9. Reddy JA, Allagadda VM, Leamon CP: Targeting therapeutic and imaging
agents to folate receptor positive tumors. Curr. Pharm. Biotechnol. (2005)
6(2):131-150.
20
1.10. Cascante M, Centelles JJ, Veech RL, Lee WN, Boros LG: Role of thiamin
(vitamin B-1) and transketolase in tumor cell proliferation. Nutr. Cancer. (2000)
36(2):150-154.
36
1.11. Park JW, Benz CC, Martin FJ: Future directions of liposome- and
immunoliposome-based cancer therapeutics. Semin. Oncol. (2004)
31(6
31 Suppl. 13):196-205.
1.12. Reddy JA, Low PS: Folate-mediated targeting of therapeutic and imaging
agents to cancers. Crit. Rev. Ther. Drug Carrier Syst. (1998) 15(6):587-627.
15
1.13. Stella B, Arpicco S, Peracchia MT et al.: Design of folic acid-conjugated
nanoparticles for drug targeting. J. Pharm. Sci. (2000) 89(11):1452-1464.
89
1.14. Hilgenbrink AR, Low PS: Folate receptor-mediated drug targeting: from
therapeutics to diagnostics. J. Pharm. Sci. (2005) 94(10):2135-2146.
94
1.15. Wartlick H, Michaelis K, Balthasar S, Strebhardt K, Kreuter J, Langer K:
Highly specific HER2-mediated cellular uptake of antibody-modified nanoparticles
in tumor cells. J. Drug Target. (2004) 12(7):461-471.
12
1.16. Bianco AR: Targeting c-erbB2 and other receptors of the c-erbB family:
rationale and clinical applications. J. Chemother. (2004) 16(Suppl.
4):52-54.
16
1.17. Zhang Y, Zhang J: Surface modification of monodisperse magnetite
nanoparticles for improved intracellular uptake to breast cancer cells. J. Coll.
Inter. Sci. (2005) 283:352-357.
283
1.18. El-Sayed IH, Huang X, El-Sayed MA: Surface Plasmon resonance scattering
and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer
diagnostics: applications in oral cancer. Nano Lett. (2005) 5(5):829-834.
1.19. Loo C, LOwery A, Halas N, West J, Drezek R: Immunotargeted nanoshells for
integrated cancer imaging and therapy. Nano Lett. (2005) 5(4):709-711.
1.20. Van Vlerken LE, Amiji MM. Multi-functional polymeric nanoparticles for
tumour-targeted drug delivery. Expert Opin Drug Deliv. 3(2):205-16; Mar, 2006.
1.21. Bhaskar, S., Tian, F., Stoeger, T., V., Kreyling, W., de la Fuente, J M., Grazu,
Estrada, G., Ntziachristos, V., Razansky, D. Multifunctional nanocarriers for drug
delivery across the blood-brain barrier. 2009. Journal of Fiber Toxicology.
Submitted. Invited Publication.
1.22. Bhaskar, S., Tian, F., Stoeger, T., V., Kreyling, W., de la Fuente, J M., Grazu,
Estrada, G., Ntziachristos, V., Razansky, D.Multifunctionalization of nanocarriers
for in vivo neuroimaging : a survey. In Proceedings of 2nd European Conference
for Clinical Nanomedicine. 27-29 April 2009. Basel, Switzerland. (Poster).
21
2. EXPERIMENTAL PROCEDURE
Nanoparticles preparation and functionalisation during this study was carried out
at the Lab of Biomaterial Surfaces and Biofunctionalisation, at Aragon Institute of
Nanosciences (INA).
2.1. Nanoparticle synthesis and Invitro Studies
2.1.1. Synthesis: Au@tiopronin
Nanoparticles are prepared using the reaction by the Codissolution of HauCl4 and
tiopronin in the presence of methanolic/acetic acid mixture, which gives a stable
ruby-red solution. Then the reducing agent NaBH4 is added, which produces a
dark brown solution. The suspension is shaken for about 30 min, and then the
solvent is removed. The nanoparticles are purified by dialysis and characterized
by 1H-NMR, FTIR, UV-visible and transmission electron microscopy (TEM).
Usually, gold nanoparticles with core diameter of 2.8 nm are produced. The
reaction utilizes the water-soluble carbodiimide N-[3-(dimethylamino) propyl]N’-ethylcarbodiimide hydrochloride (EDC) to catalyze reactions between the
nanoparticle acid groups and any of the amine groups of the peptide. This results
in Nanoparticles with core diameter around 5 nm. Moreover, the same
Au@tiopronin nanoparticles may be functionalized with Poly ethylene glycol (PEG)
using NH2-(R)-NH2, where R=PEG, in the presence of EDC and NHS. Then, CONHRNH2 gets attached to the Gold core as pioneered by the Dr. Jesus lab
[2.1].
Figure 2.1. Preparation of multifunctional biodegradable gold nanoparticle for tumor targeting
22
The stability of gold nanoparticles in physiological conditions is required for
applications in cell biology. Although several groups have prepared water-soluble
gold nanoparticles, [2.2] it appears that the resultant particles are not particularly
amenable to a broad range of applications; their synthetic scope is limited, or they
aggregate in physiological conditions.
Although inorganic and metal nanoparticles can be prepared from various
materials by several methods, the coupling and functionalisation with biological
components has only been carried out with a limited number of chemical methods
[2.3-2.6].To apply gold colloids in newly developed biological assay systems,
simple and easy means of anchoring different ligand biomolecules onto particle
surfaces are required.
A key issue in evaluating the utility of these materials is assessing their potential
toxicity, either due to their inherent chemical composition or as a consequence of
their nanoscale properties [2.7, 2.8].One can modify these nanoparticles to better
suit biological systems via modification of their surface layer for enhanced
aqueous solubility, biocompatibility, and biorecognition.
In this study, gold nanoparticles were synthesized using different alkanethiolate
capping agents. These NPs have been derivatized with two different diamine
functionalized linkers, ethylenediamine (EDA) and poly(ethylene glycol) bis(3ami- nopropyl) terminated (PEG). The influence on HeLa tumoral cells in vitro
was assessed in terms of stability, cytotoxicity, and fluorescent observation of
cytoskeleton elements F-actin as well as clathrin. The results show that only
nanoparticles protected with the non-natural amino acid tiopronin are stable.
2.1.2. Materials and Methods
Materials. All of the chemicals were of reagent grade and were used without
further purification. Hydrogen tetrachloroaureate(III) trihydrate (99.9+%) (product
no.: 484385), (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride,
2-[N-morpholino] ethanesulfonic acid (99.5%) (product no.: M3671), L-cysteine
(>97%) (product no.: W326305), poly(ethylene glycol) bis(3- aminopropyl)
terminated (product no.: 452572), and 11-mercap- toundecanoic acid (95%)
(product
no.:
450561)
were
purchased
from
Sigma-Aldrich;
N-(2
mercaptopropionyl)glycine (>98%) (product no.: 63794), N hydroxysuccinimide
(>97%) (product no.: 56480), and ethylendiamine (75-80%) (product no.: 03783)
were from Fluka; NaBH4 (98%) (product no.: 5859) was from Lancaster. Buffers
were prepared according to standard laboratory procedure. Other chemicals were
reagent grade and used as received.
23
General Procedures. UV spectra were carried out with a UV/vis VARIAN (Cary
100 BIO) spectrometer in milliQ water. Infrared spectra of solid NPs samples
pressed into a KBr plate were recorded from 4000 to 750 cm-1 with a JASCO
FT/IR 410 model spectrometer.
2.1.3. Synthesis of Gold Nanoparticles
Au@tiopronin. Hydrogen tetrachloroaureate(III) trihydrate (0.15 g; 0.4 mmol; 1
equiv) and N-(2-mercaptopropionyl)glycine (tiopronin) (0.19 g; 1.2 mmol; 3 equiv)
were codissolved in 20 mL of 6:1 methanol/acetic acid, resulting in a ruby red
solution. Sodium borohydrate (0.30 g; 8.0 mmol; 20 equiv) in 7.5 mL of H2O was
subsequently added via rapid stirring. The resultant black suspension was stirred
for an additional 30 min after cooling, with the solvent removed under vacuum at
40° c. The crude sample was completely insoluble in methanol but reasonably
soluble in water. It was purified by dialysis, in which the pH of 130 mg of crude
product dissolved in 20 mL of water (NANOpure) was adjusted to 1 by dropwise
addition of concentrated hydrochloric acid. This solution was loaded into 15 cm
segments of seamless cellulose ester dialysis membrane (Sigma, MWCO ) 10.000),
placed in 4 L beakers of water, and stirred slowly, recharging with freshwater ca.
every 10 h over the course of 72 h. The dark blue Au@tiopronin solutions were
collected from the dialysis tubes and were lyophilized. The product materials
were found to be spectroscopically clean and produced a yield of 96 mg.
2.1.4. Derivatization of Gold Nanoparticles. Au@tioproninAu@tiopronin-EDA
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (10 mg;
0.05 mmol) and N-hydroxysuccinimide (NHS) (15 mg; 0.125 mmol) were added to
4 mL of the above Au@tiopronin solution (10 mg) in 2-[Nmorpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The reaction was
permitted 30 min. Subsequently, ethylenediamine (0.012 mL; 0.17 mmol) was
added and the mixture was stirred a further 24 h. This solution was loaded into 6
cm segments of dialysis membrane, placed in 4 L beakers of water, and stirred
slowly, recharging with freshwater ca. every 10 h over the course of 24 h. The
dark blue Au@tiopronin-EDA solutions were collected from the dialysis tubes and
were lyophilized producing a yield of 5.5 mg.
2.1.5. Au@tioproninAu@tiopronin-PEG
(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (20 mg;
0.1 mmol) and N- hydroxysuccinimide (NHS) (30 mg; 0.25 mmol) were added to 4
mL
of
the
above
Au@tiopronin
solution
(20
mg)
in
2-[Nreaction
was
morpholino]ethanesulfonic acid (MES) (50 mM, pH 6.5). The
permitted 30 min. Subsequently, poly(ethylene glycol) bis(3-aminopropyl)
24
terminated (554 mg) was added and the mixture was stirred a further 24 h. This
solution was loaded into 6 cm segments of dialysis membrane, placed in 4 L
beakers of water, and stirred slowly, recharging with freshwater ca. every 10 h
over the course of 24 h. The dark blue Au@tiopronin-PEG solutions were
collected from the dialysis tubes and were lyophilized producing a yield of 99 mg.
See Figure 2.2.
25
H
COOH
N
SH
HAuCl4
+
O
MeOH.AcOH 6:1
NaBH4
COOH
COOH
COOH
S
S
COOH
S
S
COOH
S
S
S
S
COOH
COOH
COOH
Au@Tiopronin
2
CONHRNH2
2
CO
NH
RN
H
CONHRNH2
CON
CONHRNH2
H2
HRN
Au@Tiopronin-EDA
(R= -CH2-CH2- )
H2
CONHRNH2
N
HR'
2
NH
CON
R
NH
CO
CONHR'NH2
S S S
S
S
S
S
S
NH2R'NH2
CONHR'NH2
NH2RNH2
CO
NH
R'
NH
CONHRNH2
EDC
NHS
S S S
S
S
S
S
S
H2
R 'N
H
N
CO
CONHR'NH2
CONHR'NH2
CONHR'NH2
Au@Tiopronin-PEG
(R= -(CH2)3(OCH2-CH2)32-34 )(CH2)3-)
Figure 2.2.
2.2. The chemical method of producing Au nanoparticles of the size with average mean
diameter as 2.8 nm. In this case. EDC acts as a catalyst and facilitates the reaction between the
carboxylic acid groups present on Au @Tiopronin and the amine group containing molecules like
EDA and PEG. The resultant nanoparticle is watersoluble and highly stable in phsyiological
conditions.
26
2.1.6.
2.1.6. Functionalisation of AuAu-PEG/AuPEG/Au-EDA nanoparticles
nanoparticles with fluorochromes
The next step in functionalisation of nanoparticles is to decorate it with a
fluorochrome such as Rhodamine and fluorescin.
Rhodamine functionalisation
Au-PEG-Rhodamine:
Au nanoparticle was added to bicarbonate solution at 50mM, followed by the
addition of Rhodamine stock solution. The quantity and chemical specifications of
the solutions are shown as below,
1 mg Au-PEG+0.5 ml Bicarbonate Solution (50 mM, pH 10) + 20 µl Rhodamine
stock solution.
Rhodamine Stock: 2mg Rhodamine + 75 µl Ethanol + 75 µl bicarbonate
Au-PEG: 1 mg + 480 µl bicarbonate + 20 µl Rhodamine Stock
Similarly for Au-EDA-Rhodamine, similar protocol was followed and detail is
listed below,
[0.2 mg Au-EDA+ 0.5 ml bicarbonate+20µl Rd Stock] × 5 times = [1 mg AuEDA + 2.4 ml bicarbonate + 100 µl Rhodamine Stock]
The samples were left for two hours in a stirring position, so that they get
properly mixed. To optimize the quantity of sufficient Rhodamine for the
activation of the nanoparticles, we took varying amounts of Rhodamine
concentrations to functionalize Au NPs.
Fluorescin Isothiocynate Functionalisation
First of all, we prepared PEG NP and EDA NP solutions by adding bicarbonate.
Also its important to ensure that the pH of bicarbonate is 10. Then, we prepared
Fluorescin stock solution and stirred it well. The composition of Fluorescin stock
solution is, 2mg Fluorescin + 75 µl Ethanol + 75 µl Bicarbonate (10 pH, 50 mM).
Then, Fluorescin stock solution was added to the EDA and PEG nanoparticle
solutions.
Protocols
PEG-NP Fluorescin: 1mg PEG Np + 480 µl Bicarbonate + 20 µl Fluorescin Stock
EDA-Np Fluorescin: 1mg EDA NP + 2.4 ml Bicarbonate + 100 µl Fluorescin Stock
The solutions were properly mixed with a pipette. Then, the resultant solution
was wrapped in an aluminum foil and stirred for two hours.
The above fluorochrome functionalized nanoparticle solutions were then made to
pass through ultra filtration to remove access of fluorochrome from the
27
nanoparticle solution. After ultra filtration, the samples were loaded into mini
dialysis tubes (Cut off 8 kDa, Max= 2ml) and left for 24 hours for dialysis. It is
important to keep the membrane of the dialysis tube wet until its used, by dipping
the cap in a small volume of water kept in a plate. Finally, the solutions were
lyophilized to procure Au-Np-Alexafluor/Fluorescin. UV Vis spectra for the AuPEG Rhodamine Nps were obtained.
2.1.7.. Cell Culture
2.1.7
Refer to Annexure II for more information and detailed methodology.
HeLa cells were seeded onto glass coverslips (13 mm diameter) at a density of 1
× 104 cells per well in 1 mL of complete medium. The medium used was 71%
Dulbeccos Modified Eagles Medium (DMEM) (Sigma, UK), 17.5% Medium 199
(Sigma, UK), 9% foetal calf serum (FCS) (Life Technologies, UK), 1.6% 200 mM
L-glutamine (Life Technologies, UK), and 0.9% 100 mM sodium pyruvate (Life
Technologies, UK). The cells were incubated at 37 ˚ C with a 5% CO2 atmosphere
for 24 h. At this time point the cells were incubated in complete medium
supplemented with 0.05 mg mL-1 of gold nanoparticles for a further 24 h. All
control cells were cultured in the absence of any particles.
For more detailed description, kindly see the annexure II and III.
2.1.8. Cell Viability
To determine cell cytotoxicity/viability, the cells were plated at a density of 1 ×
104 cells/well in a 96-well plate at 37˚ C in 5% CO2 atmosphere. After 24 h of
culture, the medium in the wells was replaced with fresh medium containing
nanoparticles in varying concentrations. After 24 h, 20 µL of MTT dye solution (5
mg/mL in phosphate buffer pH 7.4, MTT Sigma-Aldrich, UK) was added to each
well. After 4 h of incubation at 37˚ C and 5% CO2 for exponentially growing cells
and 15 min for steady-state confluent cells, the medium was removed and
formazan crystals were solubilized with 200 µL of DMSO, and the solution was
vigorously mixed to dissolve the reacted dye. The absorbance of each well was
read on a microplate reader (Dynatech MR7000 instruments) at 550 nm. The
spectrophotometer was calibrated to zero absorbance, using culture medium
without cells. The relative cell viability (%) related to control wells containing cell
culture medium without nanoparticles was calculated by [A]test/[A]control ×
100.
28
2.1.9.
2.1.9. Clathrin Immunofluorescence and Cytoskeletal Observation
After 24 h culture with the nanoparticles, cells were fixed in 4%
formaldehyde/PBS, with 1% sucrose at 37 ˚ C for 15 min. The samples were then
washed with PBS, and permeabilizing buffer was added at 4˚ C for 5 min. The
samples were then incubated at 37˚ C for 5 min in 1% BSA/PBS. This was
followed by the addition of anti-vinculin, anti-tubulin, or anti-clathrin primary
antibody (1:100 in 1% BSA/PBS; monoclonal anti-human raised in mouse (IgG1),
Sigma, UK) for 1 h (37˚ C Simultaneously, rhodamine conjugated phalloidin was
added for the duration of this incubation (1:100 in 1% BSA/PBS. Molecular Probes,
OR). The samples were then washed in 0.5% Tween 20/PBS, and a secondary,
biotin conjugated antibody (1:50 in 1% BSA/PBS, monoclonal horse anti-mouse
(IgG), Vector Laboratories, UK) was added for 1 h (37˚ C followed by more
washing. A FITC conjugated streptavidin third layer was added (1:50 in 1%
BSA/PBS, Vector Laboratories, UK) at 4˚ C for 30 min, and given a final wash.
Samples were then viewed by fluorescence microscopy (Nikon, Eclipse TE 20002000S). Detailed protocol is given in annexure II.
2.2. Mesoscopic Fluorescence
Fluorescence Tomography based invivo imaging of
fluorescent nanoprobes
The experiments were conducted at the Institute of Inhalation Biology (IHB),
Helmholtz Zentrum Muenchen, Munich and the imaging experiments were
conducted at the Institute of Biological and Medical Imaging (IBMI), HZM, Munich,
Germany.
For this experiment, we aimed at, first of all organising a functional set up of
Mesocopic Fluorescence Tomography (MFT) and then to use it to detect
fluorescent nanoparticles invivo. Fortunately, the technique on fluorescent
nanoparticle diagnostic in living animal has recently been developed by Dr. Vasilis
Ntziachristos at the Centre of Molecular Imaging and Research, Massachussets
General Hospital & Harvard Medical School and lately relocated to the Institute
for Biological and Medical Imaging (IBMI) at the Helmholtz Centre Munich
(HMGU). Using MFT whole-body three-dimensional visualization of the
morphogenesis of GFP-expressing salivary glands and wing imaginal discs in
living Drosophila melanogaster pupae was reported [2.10] in vivo and over time.
The method extends whole-body optical imaging to the mesoscopic depth scale
therefore can potentially be used for monitoring of drug delivery in isolated
organs or embryos [2.11] as well as in other important model organisms smaller
than mice, e.g. fishes, worms or flies.
To assess the efficacy of this tomographic technique on mouse models, we
conducted an experiment to visualise
29
The animal experiments were conducted complying to the German federal
guidelines for the use and care of laboratory animals and under the due approval
from the HMGU Institutional Animal Care and Use Committee.
The mouse was sacrificed after anaesthesia and the different organs were
dissected. Brain of mice was first fixed in 4% formaldehyde and embedded in
agarose gel (2% agar in barbitone buffer). The sample was prepared for
visualization in a MFT set up. Nanoparticles labeled with red fluorescent protein
(DsRed).
Figure 2.3. Spectra of DsDs-Red fluorescent protein [2.9]
Mesoscopic Fluorescence Tomography (MFT) was recently developed to operate
in the 0.5mm-1cm regime with focus on enabling in-vivo observation of common
biological model organisms [2.10]. In a typical MFT set up (shown in figure), the
microscope is horizontally mounted, and the sample lies vertically on a highspeed rotation stage. A laser beam is focused by way of a low-numericalaperture objective in close proximity to the center area of the sample’s surface.
The sample is then rotated and images are captured with a CCD mounted on the
microscope. The technique utilizes a modified laboratory microscope and multiprojection illumination to collect data at 360-degree projections. It employs the
Fermi simplification to the Fokker-Plank solution of the photon transport
equation, combined with geometrical optic principles in order to allow in vivo
whole-body visualization of non-transparent three-dimensional structures in
samples up to several millimeters in size.
Images were recorded and processed with Winview and ImajeJ software for
posterior analysis.
30
References
2.1. de la Fuente, M., Berry C.C., Riehle, M.O., Curtis A.S.G. Nanoparticle
targeting at cells. Langmuir, Vol.22, No.7. 3286-3293. 2006
2.2. Ackerson, C. J.; Jadzinsky, P. D.; Kornberg, R. D. J. Am. Chem. Soc.
2005
2005,127, 6550-6551.
2.3. Ghosh, S. S.; Kao, P. M.; McCue, A. W.; Chapelle, H. L. Bioconjugate Chem.
1990,
1990 1, 71-76.
2000,
2.4. Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000
1025-1026.
2.5. Shenton, W.; Davies, S. A.; Mann, S. AdV. Mater. 1999,
1999 11, 449-452.
2.6. Connolly, S.; Fitzmaurice, D. AdV. Mater. 1999,
1999 11, 1202-1205.
2.7. Rojo, J.; Díaz V.; de la Fuente J.M.; Segura I.; Barrientos A.G.; Riese H.H.;
Bernad A.; Penadés S., ChemBioChem, 2004, 5, 291-297.
2.8. Bernad, A.; Penade´s, S. ChemBioChem 2004,
2004 5, 291-297.
2.9. Patterson et al. Fluorescent protein spectra. Cell Science at a Glance, 837838,114 (5).
). 2001
2.10. Vinegoni C, Pitsouli C, Razansky D, Perrimon N, Ntziachristos V: In vivo
imaging of Drosophila melanogaster pupae with mesoscopic fluorescence
tomography. Nat Meth 2008, 5(1):45-47.
2.11. Tian F, Razansky D, Estrada G,Semmler-Behnke M, Beyerle A,Kreyling W,
Ntziachristos V, Stoeger T. Surface modification and size dependence in particle
translocation during early embryonic development. Inhalation Toxicology 2009 21,
92-96.
31
2. RESULTS AND DISCUSSIONS
The overall objective of this project is to design functionalised Gold (Au)
nanoparticles with a fluorescent marker that allows tomographic detection by
using fluorescent light in the near infrared spectral region (NIR). This would allow
an ability to penetrate deep inside through several inches due to the low
absorption capacity of the tissues in the spectral region between 700-850 nm.
The results of our experimental studies are enlisted here:
I.
We succefully prepared water soluble gold nanoparticles. Au@Tiopronin
is highly stable in physiological conditions. Parallel studies in our lab
have shown that the mean diameter of these Au@Tiopronin
nanoparticles, prepared using the Murray et. al procedure comes around
2.8 nm. Purification of Au@Tiorpronin, Au-PEG, and Au-EDA
nanoparticles were done using dialysis followed by liophilisation.
II.
The carbodiimide N-[3-(dimethylamino)propyl]-N -ethylcarbodiimide
hydro-chloride (EDC) plays the role of a catalyst by catlaysing the
reactions between the amine groups of EDA/PEG and the carboxylic
acid groups present on the tiopronin Au@nanoparticles.
III.
Ultraviolet-visible spectrophotometry (UV/VIS) Results : Au@Tiopronin
UV-Vis spectra showed negligible surface plamsa resonance. The main
reason behind this observation may be attributed to the relatively
smaller size of the clusters. The plasmon resonance is non-existent for
tiopronin-capped NPs.
3.1. UVUV-Vis spectra of Rhodamine Gold Nanoparticles
Nanoparticles at different
dilutions
32
Fig. 3.1. The shift in the absorbance value with decrease in the dilution suggests a bathscomic
pattern which may be attributed to conjugation of double and triple bonds, thereby shifting the
absorption maximum to longer wavelengths.
wavelengths.
3.2. FTIR Spectra of AuAu-EDA and
and AuAu-PEG
Fourier transform infrared (FTIR) spectrometry is a useful tool for identifying
both organic and inorganic chemicals. It can be utilized to quantify some
components of an unknown mixture and can be used to analyze liquids, solids, or
gases. Data are collected and converted from an interference spectrum. FTIR
spectrum shows a plot of Fourier Transform of the intensity of light as a function
of wavenumber. The peaks of this spectrum may help us to identify the chemical
composition of a substance. We can see that the Au@Tiopronin and Au@PEG
have remarkably different features. The presence of 2881 and 1114 cm-1 bands,
refers to the location of PEG coating on the nanoparticle surface, because these
bands correspond to the OCH2 and CH2OCH2 stretching modes. On the contrary,
FTIR spectra of AU@Tipronin and Au@Tiopronin-EDA nanoparticles have a lot of
similarity, as they both have similar coatings. But, we can observe some irregular
bands in the spectral range of 1000-1400 wavelenght. Moreover, there is also a
representation of the spectra of the same type of nanoparticle during different
time frame, using same method of preparation. The explicability of the graphs
show that the method is quite robust and reproducible.
33
Figure 3.2.
3.2. FTIR spectra of AuAu-EDA nanoparticles produced at two different time lines.
Interestingly, the band pass lines in between 1000 to 1500 are consistent with both of them.
Figure 3.3.
3.3. Au@tipronin UV Vis Spectra [De
[De la Fuente, Jesús,
Jes s, et. Al, Langmuir, 2001]
34
Figure 3.4. FTIR spectra of AuAu-PEG nanoparticles produced at two different time lines.
lines The
frequent band pass lines depicting high intensity wavelengths in the initial wavenumber range of
<1000 cm-1.
3.3
Cell Culture and Cytotoxicity assays
HeLa cells were cultured in low-glucose DMEM. Media contained fetal calf serum
(10 %), l-glutamine (2.9 mgmL-1), streptomycin (1 mgmL-1), and penicillin (1000
unitsml-1). L929 cells were cultured in RPMI 1640 medium with newborn calf
serum (5 %), lglutamine (2.9 mgmL-1), streptomycin (1 mgmL-1), and penicillin
(1000 unitsml). All cells were cultured at 37C in water-saturated air
supplemented with 5% CO2. Culture media were changed every 3 days. Cells
were passaged once a week. Cell numbers were estimated using a cell counter
(Schaerfe cellcounting system, Germany).
The metabolic activity and proliferation of HeLa cells was measured after 24 h
culture. The bar graph in Figure 3.5. shows that the cell proliferation was more
favorable in case of EDA and PEG coated particles than with Au@Tiopronin.
The figure below shows the cytotoxicity profiles of the three Au nanoparticles
when incubated with Hela Cells as determined by MTT assay. Relative cell
viability (%) related to control wells containing cells without nanoparticles was
calculated by [Abs]test/[Abs]control × 100 (n=3). Results are represented as
mean ± standard deviation.
35
Cytotoxicity profiles
Au@Tio
Au@Tio-EDA
Au@Tio-PEG
Percentage Viability
120
100
80
Au@Tio
60
Au@Tio-EDA
40
Au@Tio-PEG
20
0
Percentage Viability
A very little or negligible cytotoxicity was observed. Most importantly, PEG
coated Au@Tiopronin seems to be least toxic to the cells.
Cytotoxicity profiles
150
100
50
0
1
2
3
4
5
6
100
95
90
92
90
80
Au@Tio-EDA 100 100 92
Au@Tio-PEG 100 97 95
90
95
85
95
97
90
Au@Tio
Conce ntra tion (µM)
Concentration (µM)
Figure 3.5. Cytotoxicity profiles of Au@Tiopronin, Au@TioproninAu@Tiopronin-EDA and Au@TioproninAu@Tiopronin-PEG
when incubated with HeLa cells as determined by MTT assay.
3.4. Cell Morphology and Cytoskeleton Studies
The use of Phalloidin for cytoskeleton colouring gives very nice view of the
cytoskeleton (F-Actin and Tubulin), especially in cytoskeleton of control cells.
10 µm
36
10 µm
10 µm
Figure 3.6.
3.6. Cytoskeleton (F-Actin, β-Tubulin and Vinculin)
Vinculin) fluorescent staining for control cells.
Phalloidin colors the cytoskeleton of the cells. Well defined focal adhesion found in all the
samples.
37
10 µm
Figure 3.7.
3.7. Cytoskeleton
Cytoskeleton labeled cells incubated with PEGPEG-AuAu-Rhodamine nanoparticles.
nanoparticles. Because of
lot of quenching, it’s difficult to figure out the location of nanoparticles. [Scale: 10 µ m]
10 µm
Figure 3.8.
3.8. As we can observe that cytoskeleton fluorescent staining is quite obvious.
Unfortunately, nothing much can be said about the location of nanoparticles.
38
3.5. Invivo Imaging of fluorescent nanoparticles in mouse models using
fluorescent tomography
Figure. 3.9. MFT image of the mouse brain fixed in agar.
As described before, the mouse brain was dissected and was fixed in agar. This
image was taken by a Mesoscopic fluorescence tomography instrument at Institute
of Biological and Medical imaging, Helmholtz Zentrum Munich, Germany under the
guidance of Dr. Daniel Razansky and Dr. Furong Tian. General schema of a
fluorescent tomography technique instrument is shown below.
Figure 3.10.
3.10. Experimental Set up of Fluorescence
Fluorescence tomography instrument.
The imager consists of a laser diode, a beam splitter that divided light to a
reference channel and an optical switch. Optical source fibers and fiber bundles
39
are employed to illuminate and collect light respectively from the optical imaging
bore. The fiber bundles and reference fiber are arranged on a grid and imaged
with a CCD camera using appropriate filters. The optical imaging chamber in the
FMT imager is used for planar, transillumination, and tomographic acquisitions of
small animals.
In a typical MFT set up, the microscope is horizontally mounted, and the mouse
sample lies vertically on a high-speed rotation stage. A laser beam is focused by
way of a low-numerical-aperture objective in close proximity to the center area
of the brain’s surface. The sample is then rotated and images are captured with a
CCD mounted on the microscope.
Figure.3.11.
Figure.3.11. Overview of the experimental configuration
configuration of MFT.
Figure 3.12.
3.12. The image taken with MFT set up after the administration of fluorescent nanoparticles
functionalized with DsRed fluorescent protein in the spinal cord of the sacrificed mouse.
40
Figure 3.13.
3.13. MFT image taken by the illumination
illumination of Agar fixed sample of mouse brain in the laser
light. We can visually see the presence of fluorescent DsRed nanoparticles which were directly
administered in the healthy brain after dissection.
In this experiment the laser beam was focused by way of a low-numericalaperture objective in close proximity to the centre area of the mouse brain
surface. The mouse brain was then rotated and images were captured with a CCD
mounted on the microscope. The technique utilizes a modified laboratory
microscope and multi-projection illumination to collect data at 360-degree
projections. It employs the Fermi simplification to the Fokker-Plank solution of
the photon transport equation, combined with geometrical optic principles in order
to allow in vivo whole-body visualization of non-transparent three-dimensional
structures in samples up to several millimetres in size. With this technique we
could operate in the 0.5mm-1cm regime, thereby enabling in-vivo observation of
common biological model organisms.
We also explored the bioluminescence imaging set up and the brief report on the
bioluminescence imaging experiment with cancer model of mouse using luciferase
gene reporter has also been explained in Annexure I. In context of this
experiment using MFT, as we can see optical imaging has unique advantages
compared to other imaging modalities, including simplicity, low-cost and small
size. The light radiation is non-ionizing, and therefore reasonable doses can be
repeatedly employed without harm to the animal or patient. Optical contrast
methods offer the potential to differentiate between soft tissues, due to their
different absorption that are indistinguishable using other modalities. Also,
specific absorption by natural chromophores (such as oxy-haemoglobin) allows
functional information to be obtained. The use of extrinsically-administered
“switchable” and “tumor-selective” fluorescent optical agents further advances
the application possibilities by allowing visualization of otherwise invisible cellular
and sub-cellular processes. Since many of the probes are developed to fluoresce
41
in the near-infrared (NIR) optical window, where optical absorption is very low so
that light can penetrate deeply, fluorescence imaging has been successfully
translated from a microscopy level to whole body small animal imaging and
clinics. To this end, optical tomographic approaches in diffusive objects have been
applied in tissues with dimensions that are normally larger than 1 cm while
offering spatial resolution on the order of 1 mm. Therefore, optical imaging
methods were so far inadequate for non-invasive in vivo imaging of intact
developing insects, animal embryos or small animal extremities, i.e. when working
at mesoscopic dimensions between the penetration limits of modern optical
microscopy (0.5-1mm) and the diffusion-imposed limits in optical macroscopy
(>1cm). Mesoscopic Fluorescence Tomography (MFT) was recently developed to
operate in the 0.5mm-1cm regime with focus on enabling in-vivo observation of
common biological model organisms.
During implementation of this project we have optimized the synthesis of
functionalised Gold (Au) nanoparticles with different fluorescent markers, namely
Fluorescin Isothiocynate and Rhodamine. The necessary criterion for a colloidal
system for to be used in biological systems: It must have a uniform size and
should not undergo aggregation in biological fluids. Furthermore, invitro
cytotoxicity and morphological studies have been performed for the analysis of
Cell-Nanoparticle interactions. The in vitro cytotoxicity (Clinical Hospital-Lozano
Blesa) as well as studies in vivo fluorescent tomography (in collaboration with
Institute of Inhalation Biology (IHB), and at Institute of Biological and Medical
Imaging (IBMI), Helmholtz Zentrum Muenchen, and at Technical University of
Munich (TUM), Munich, Germany) has been carried out.
Our results show promise for fluorescent Au nanoparticles, with little or negligible
cytotoxicity, as a candidate for diagnostic and therapeutic agent in invitro and
invivo experiments. Moreover, optical imaging using fluorescence tomography is
not only less prone to irradiation effects and cell damage but also can be used
effectively to wards diagnostics and therapy of cancer.
We are further exploring the application of these multifunctional nanoparticles in
detection and therapy of brain tumours (especially Glioblastomas) by effective
conjugation strategies in order to facilitate effective crossing of Blood Brain
Barrier (BBB), which acts as a major impediment by protecting the brain from
entry of substances. To this end, due to its biocompatibility, small size, less
agglutination, and other properties, multifunctional Gold nanoparticles comes out
as a promising candidate in the field of nanomedicine for diagnostics and therapy
of cancer in general, and brain cancer in particular.
42
4. CONCLUSION AND FUTURE PERSPECTIVES
Gold nanoparticles play an important role in invitro and invivo detection of tumors.
Most importantly, the chemistry of biofunctionality of these gold nanoparticles
hence becomes of paramount importance. In this thesis work, we have
demonstrated that the nanoparticle uptake can be avoided using adequate
functionalisation, thereby increasing their adhesion to their cell surface.
Moreover, we have also explored the technique of Mesoscopic fluorescence
tomography and how far can it be helpful in tracing nanoparticles.
We have also reported methodology to optimise functionalisation and increased
biocompatibity of nanoparticles.
In this study we reported synthesis of gold Nanoparticles using different
alkanethiolate capping agents, These NPs have been derivitazed with two
different diamine functional linkers, EDA and PEG. We also functionalised the NPs
with fluorochrome and UV-Vis and FTIR studies were done to study their
characteristics.
The delivery of drugs to tumors especially those of the Central nervous system
has been a challenging area of research for many years. The two major obstacles,
overcoming the physiological barriers of the brain and achieving high drug
concentrations within the tumor bed, have prompted an intensive search for
alternative routes of drug delivery. Within the confines of these limitations,
multifunctional nanoparticles have allowed a new approach for delivering
pharmaceutical agents to the brain.
The overall objective of this work is towards the synthesis of a biodegradable and
multifunctional nanoparticle that can pursue advances within the field of in vivo
imaging of multifunctional nanoparticle based diagnostics and therapeutic
approaches and to delineate the challenges associated with the utilization of
immunotherapies that are unique to tumors, and more especially to brain tumors.
Immunotherapy of CNS tumors is complicated by multiple factors, such as, tumor
heterogeneity, immunosuppression, and immunologic privilege. The study of
immunotherapies requires a thorough knowledge of immunological response
within the CNS and its potential consequences, including the induction of
autoimmune disorders, is mandatory.
Imaging of function and molecular activity is at the frontier of current research
efforts to detect and study cancer noninvasively. Optical imaging offers
complementary features to those of established radiological imaging techniques,
primarily the quantitative imaging of haemoglobin saturation and concentration,
43
and the selective imaging of specific gene expression with high sensitivity,
because background signals can be suppressed using enzyme-activated
fluorescence probes. Introduction of multispectral optoacoustic tomography has
removed major limitations of optical imaging methods related to light scattering,
enabling high resolution visualization of optical biomarkers deep in diffuse living
tissues.
The advancement of effective conjugation strategies, complemented by discovery
of new target receptors and ligands in context of brain tumor therapy, is needed
for the advancement of nanotherapeutics. Alternatives route of drug
administrations will also be an important factor in order to make these nanocarrier
systems carrying drug reach specific areas in the brain and thereby account for
greater drug accumulation, and hence enhanced therapeutic benefits.
Moreover, the proper combination of optical contrast techniques with conventional
techniques like CT and MRI can definitely enhance the ways we can image
structure and function quantitatively in neurological disease models. It is also
important in terms of drug development and in-vivo imaging applications as the
proper combination can enable an excellent spatial and temporal resolution,
thereby facilitating a unique way to keep a track on disease progression as well
as on the histological changes in the target tissues following systematic
chemotherapy.
To summarize, the future prospects of the multifunctional nanocarrier systems
are; developing of a multimodal nanosystem based imaging platform for
diagnostics in neurodegenerative pathologies; application of nanoprobes carrying
multiple diagnostic, therapeutic or targeting molecules against brain specific
diseases/disorders/social concerns such as brain tumor, Neuro AIDS, obesity,
drug addiction, etc., thereby fostering the translation of the above techniques into
clinical trials; enhancing the understanding of the BBB, elucidation of mechanisms
governing its structure, composition, and structural changes in response to
various natural BBB transporters, undesirable toxins, infective viruses like HIV-1,
and potential BBB disrupting molecules; study of the efficiency and kinetics of
BBB crossing of various BBB transporting molecules such as growth factors,
insulin, transferrin, etc during the crossing of nanoprobes. This may contribute
towards a state of the art cutting edge nanoimaging based workbench that would
competitively evaluate the efficiency of a number of such molecules in a
multiplexed, 'high throughput manner'; and, finally, testing and validating the
nanoimaging system in varying tumoral animal models particularly brain tumor. We
believe that imaging methods using optical wavelengths, such as fluorescence
molecular tomography, Mesoscopic fluorescence tomography and multispectral
optoacoustic tomography, will play a vital role in our further understanding of
tumoroogenesis, in early detection of tumors, and in the design of effective
treatments.
44
ANNEXURE I
Bioluminescence
Bioluminescence Imaging in Mouse Tumoral Models
These experiments were carried out in collaboration with Dr. Eduardo Romanos,
Unidad Mixta de Investigación, Hospital Universitario-Lozano Blesa, Instituto
Aragones Ciencias de la Salud. It included orientation and practice session on
anaesthesia, animal handling protocols followed by Bioluminescence Imaging
experiment.
Bioluminescence is light produced by a chemical reaction within an organism.
organism.
Bioluminescent imagers, because they don’t require an exogenous light source,
and because mammals exhibit little or no endogenous bioluminescence, have
extremely little background noise to contend with. They thus have the potential
for very high sensitivity. Yet they are limited by the researcher’s ability to
engineer or inject an appropriate reporter, and so fluorescence may be the
modality of choice.
Observations with in vivo carcinoma cancer Mouse model
The mouse was anaesthetized before doing the imaging. The mouse cancer model
of Carcinoma was then injected with a luciferase gene reporter.
Figure I.1.
I.1. Bioluminescence imaging in invivo set up posterior to Luciferase reporter gene injection
in anaesthetised mouse tumour model.
model The luciferase catalyzes the oxidation of luciferin, thereby
resulting in light and an inactive "oxyluciferin". Into the system, either through the diet or
by internal synthesis
45
ANNEXURE
ANNEXURE II
Cell Culture methodology
First.1. First of all, the FAN and the UV light was switched on for 15 minutes to
sterilise the UV-Laminar flow cabin.
Step.2. In two bottles, we took 80 ml of media (DMEM+SBF+antibodies) and PBS.
They were tightly covered with paraffin, and then kept in a bath for warming for
next 15 minutes (To ensure that it is in sterile conditions, precautions should be
taken every time it is taken in and out of the laminar flow).
Step.3. Sterile test tubes were prepared and the pipettes were washed with
ethanol.
Step.4. Trypsin (enzyme) and medium was taken out. 10 ml of PBS was added. It
was then washed properly.
Step.5. PBS was taken out and Trypsin was added (1.5 mL).
Detachment
Detachment of cells (with Trypsin)
Step.6. 15 mL of Trypsin was added into the bottle.
Step.7. The bottle was kept in the incubator for 3 minutes with its top closed.
Step.8. We ensured that the bottle is checked for the white suspension every 1
minute. This is done to see if the enzyme Trypsin detached cells in the wells at 37
°C.
Step.9. The UV Laminar flow was washed with ethanol and 10 ml of media was
put. The solution was resuspended well and the 10 mL of the media containing
cells was kept in another tube.
Step.10. The tube was then kept for ultra centrifugation. Parameters: 1500 RPM;
and for 5 minutes.
Step.11. Then, we took bigger tubes and resuspended it well using an electrical
vibrator.
Step.12. CELL COUNTING: We prepared 5 times dilution of the cell sample (100
µL of cell solution + 400 µL of media), The dilution can be made in lesser
magnitude depending upon the experimental design and protocol.
Step.13. We put 400 µL of media and then 100 v of cell solution in an eppendorf
and it was resuspended well.
Step.14. In an another eppendorf, we took another 50 µL of the prepared dilution.
Step.15. 50 µL of Trypsin blue was added and then it was resuspended properly.
Step.16. We took 6 µL of the eppendorf on to a Neubaimer sample (Counting
Chamber).
Step.17. The microscope was used to count the number of cells in each chamber.
46
The figure II.1 is a schematic description of the cell chamber seen through the
microscope for counting.
Figure II.1.
II.1. Cell counting with the microscope.
Calculations
Number of Cells in each counting chamber unit=
[(53+65)/2]/ 4 = 14.75 * 10000 * No. of times of dilution
=14.75 * 10000 * (5*2)
=14, 75, 000 [1 mL of media + Cells]
5000 cells---- 100 µL
14, 75, 000 cells----------1mL(=1000 µL)
So, we have the stock of 14, 75, 000 cells/mL.
We need 5000 cells/well--- 100 µL dilutions.
5000 cells------100 µL
x-------------1000 µL
Concentration of the diluted solution is 50000 cells/mL.
C1 * V1 = C2 * V2
1475000 cells/mL * V1 = 50000 cells/ml * 15 mL
V1 = [50000 * 15 ] / [1475000]
So, we have, 0.5 mL stock + 14.5 mL of media = 50, 000 cells/mL solution.
47
[100 µL = 5000 cells]
Cell Labelling:
We needed 1 mL of labelling. So, in total we needed to prepare= 12 mL (for MTT)
+ 1 mL (for cell labelling) = 13 mL of solution.
But to make it sufficient, we prepared 15 mL of solution.
14, 75, 000 cells--------------- 1 mL of solution
No. of cells in 15 mL= 1475000*15= 22125000
Therefore, number of cells= 1475000/5000= 295
The cell solution was prepared and transferred to an Elisa plate using a multicanal
pipette. We used Elisa plate with 24 wells. 1 mL of media was kept in each of
these wells. Round microscope cover glasses of diameter=12 mm was kept in
each well. And then, it was later sterilised. After putting the sterilised glass round
plates in each well, we put 100 µL of cell solution in each well from the tray.
Then, it was resuspended well.
Sterilisation of cover microscope
microscope glass plates
We used Piraña solution [H2SO4: H2O2][3:1]. It was washed well in round plates.
First of all, it was washed with ethanol 2 times in a UV chamber. The ethanol was
put and then left under UV light in UV laminar flow chamber for 15 minutes. Then,
it was washed with water on serial bases.
Immunolabelling
First of all, the HeLa cells were kept with a fixing solution for 15 minutes (4%
formaldehyde /PBS, 15 minutes, 37°C. In between, Phalloidin solution was
prepared in the following way. As we needed 200 µl of Phalloidin for each well, so
for 8 wells, we needed 200 × 8 = 1600 µl ~2000 µl to make 100 times dilution of
Phalloidin, so for 20 µl, we need 2000 µl of the sample ~ 2 ml of the Phalloidin
sample.
So, PBS/BSA (1980 µl) + Phalloidin (20 µl) ---> 2000 µl.
Note: If one’s sample has fluorescent tag, we should use the contrast. For
example, in our case we have Rhodamine (Rd), so we will need Phalloidin (Green).
48
In case of Alexafluor, we will need Phalloidin red. Phalloidin is used to colour the
cytoskeleton. And the NP with fluorescent, if it has quenching, the green colour of
the cytoskeleton will disappear.
Solutions: Fixative: 10 ml (38%) Formaldehyde+ 90 ml PBS with 2 % sucrose
PBS/1%BSA: IgBSA into 100 ml PBS.
PBS/0.5% Tween: 0.5 ml Tween 20 into 100 ml of PBS.
1. NP Solution: We took stock of 1mg/ml of each NP, namely PEG Rd, EDA Rd
and PEG. 3 ml of each was taken with media and then filtered and sterilised.
For Peg-Rd, EDA-Rd, MTT table was performed.
After 5 minutes, we took all the sample out of the wells. And then put 200 µl of
Phalloidin in each well (not 1ml). It was left for incubation for almost an hour.
After 1 hour of incubation, the stain as taken from each one of the well. It was
then washed with PBS 0.5 % Tween for 5 times (by replacing it one after
another). Finally, all the solutions were removed from all the wells. The cells
were then transferred to a microscope slide. Beforehand, we washed the slide
with ethanol and then with optical paper (pelco lens paper) or optical lens paper.
Step 1, preliminary we put a drop to stain the media for each well. Step 2, we put
a chop of mounting medium for fluorescence (with DAPI). Step 3, we covered it
with a square plate glass. Finally, in the step 4, we painted the boundary with nail
polish. The plates were wrapped in aluminium foil till the nail polish for some
time. Eventually, all the prepared slide plates were kept in a refrigerator.
Details of the equipment used during the experiments:
1. UV/Vis SPECTROPHOTOMER
MANUFACTURER: VARIAN
MODEL: Cary 100 BIO
APPLICATION:
•
Cell and bacteria growing control.
•
Proteins purification.
•
Biomolecules concentration measurement.
•
Proteic characterization.
•
Ptotein-protein, protein-binding (kd) interaction.
49
•
Kinetic studies: enzimatics, red-ox and denaturation-renaturation
processes.
TECHNICAL SPECIFICATIONS:
•
Photometric range > de 3.7 Abs (+/- 0.002 Abs).
•
Operating range: 190-900nm ( +/- 0.2nm, 3000nm/min).
•
A phase locked wavelength drive prevents peak shifts and peak suppression
at high scan speeds.
•
Variable slits provide optimum control over spectral resolution.
•
The sealed optics prevents exposure to corrosive environments.
•
Quartz overcoating protects the optics from the environment and allows
cleaning without damage to their reflective surface.
Figure II.2. Photo of the UvUv-Vis Spectrophotometer
•
Although the Cary 100 has a double beam design you can select to operate
it in single, double or dual-single beam modes.
•
The Accessory Controller offers centralized accessory control, making it
easier to service the instrument because of the single set of accessory
electronics. In addition, the controller allows you to communicate with nonVarian accessories.
50
•
•
Double choppers ensure that the sample and reference beam strike the
detector at the same point, eliminating any errors due to non-uniformity of
the detector.
The large sample compartment gives you more flexibility in sample size (6
reference and 6 sample cells capacity).
•
•
•
Temperature range: -10-100ºC (PELTIER thermostatted cell holder with
cell shake).
Temperature variation: The temperature control of the Peliter
thermostatted cell holders (either the multicell or single cell versions) is
extremely stable over time, with a typical variation being ±0.05 °C. The cell
to cell variation is also minimal with a maximum difference of 0.2 °C at 37
°C. The temperature inside the cuvette can be monitored with the
Temperature Probe accessory. Temperature range: -10 to 100ºC.
Cary WinUV Bio Software Package:
o
o
o
o
o
o
o
o
o
o
o
o
Scanning software with Maths module
Simple Reads module
Advanced Reads module
Concentration module (with built-in protein
concentration methods)
Kinetics module
Enzyme Kinetics module
Scanning Kinetics module
RNA/DNA module
Thermal denaturation and renaturation module
Instrument Validate module
Applications Development Language (ADL)
2. LAMINAR FLOW CABIN
MANUFACTURER: TELSTAR
MODEL: PVPV-30/70
APPLICATION: Vertical laminar flow cabin for the secure handling of molecular
biology and microbiology samples.
51
Figure II.3. Photo of the Laminar flow cabin along with the schematic design.
TECHNICAL SPECIFICATIONS:
•
•
•
•
•
•
•
•
Sterile area, class 10.
Air ultrafiltration by vertical laminar flow (caudal 900 m3/h).
70% of air recycling by descendent one-direction ultra filtered air flow.
Hepa filter (H14).
Electrical plug.
Double air flow.
Microprocessor control.
Digital display for parameters selection:
o Working time.
o UV lamp ON/OFF/TIME PROGRAM.
o Alarm.
2. INVERTED MICROSCOPE
MANUFACTURER:
MANUFACTURER: NIKON
MODEL: Eclipse TE 20002000-S
APPLICATIONS:
•
Images capture and visualization for all advanced life cell applications.
52
Figure
Figure II.3. Photo of the Inverted Microscope.
TECHNICAL SPECIFICATIONS:
•
•
•
•
•
Two-ports design microscope that permits integrating various pieces of
imaging equipment in your desired combination and placement.
Extendible optical configuration: Taking advantage of infinity optics, the
TE2000’s extendible design allows the distance between the objective and
tube lenses to be extended up to 80mm (max.). This feature enables the
researcher to add optional attachments without modifying the microscope.
For example, by using optional stage risers you can add a laser light source,
without affecting the performance or stability for standard epi-fluorescence
or DIC techniques.
Unprecedented signal to noise ratio by eliminating stray Light: The new
Noise Terminator technology directs deviated stray light out of the
objective light collection path. This results in images of high contrast and
unparalleled S/N ratio during fluorescence observation using advanced
techniques such as evanescence wave microscopy (TIRF) increasing the
contrast and extending the detection level limit.
CFI60 optical system:: The TE2000 adopts CFI60 infinity optics, known for
crisp, clear images at any magnification, while providing higher NA’s and
longer working distances..
Greater Z-axis precision: The model TE2000-E features a motorized focus
that is precisely controlled by high-precision Z-axis readout. This feature
53
•
•
•
•
•
•
•
is perfect for research that requires comprehensive 3D information about
the specimen.
Auto Descending Nosepiece.
User-friendly, ergonomic design.
Hg source C-LHG1. 100W
CCD Camera. Colour, 2MP, DS-2.
UV-2E/C (DAPI), B-2E/C (FITO), G-2E/C (TRITC).
Accessories for light polarization.
Objectives: LWD 10x, 20x, 40x.
54
ANNEXURE III
MTT Essay
The MTT assay laboratory tests and standard colorimetric assays (an assay which
measures changes in color) for measuring the activity of enzymes that reduce
MTT or MTS + PMS to formazan,
formazan giving a purple color. It can also be used to
determine cytotoxicity of potential medicinal agents and other toxic materials,
since those agents would result in cell toxicity and therefore metabolic
dysfunction and therefore decreased performance in the assay.
Procedure
First of all, the UV Laminar Flow was switched ON 15 minutes before the
experiment. The cells ere inspected as to to observe whether there was
confluence or not. The tray was then kept back in the incubator. PBS and media
was taken in separate bottles. The media to be heated was kept in bath at 37 ° for
15 minutes. Meanwhile, MTT solution was prepared (5mg/ml). But to use it
extensively, we prepared more [3 ml of PbS= 5*3= 15 mg]. 15 mg of MTT was
measured in an eppendorf. Before mixing it with 3 ml of PbS, at first only 1 ml of
PbS was added and the solution was resuspended well, and then it was
transferred to a bigger tube and 2 ml of rest of the PbS was added The tube was
warpped in an aluminium foil.
Then, the media was taken out from the wells of the tray, using a multicanal
pipette. 100 µL of media was added in each well and then 20 µL of PbS was added.
After 2-3 hours, the media+MTT was pipetted out from each well slowly.
Moreover, 100 µL of DMSO (DImethyl Sulfoxide) was added in the bigger
chamber. The solution was resuspended well. It was then read using UV
spectrophotometer.
55
1
2
3
4
5
6
7
8
9
10 11
12
A
B
Cntrl
C
1
D
0.5
E
0.25
F
0.1
G
0.05
H
Au-PEG-Rd
Au-EDA-Rd
Figure III.1. Set up showing wells in an ELISA plate during an MTT assay with AuAuPEGPEG-Rd and Au.Au.-EDAEDA-Rd nanoparticles.
56
ANNEXURE IV
Bhaskar, S.,
S., Tian, F., Stoeger, T., V., Kreyling, W., de la Fuente, J M., Grazu,
Estrada, G., Ntziachristos, V.,
V., Razansky, D. Multifunctional nanocarriers for drug
delivery across the bloodblood-brain barrier. 2009.
2009. Journal of Fiber Toxicology.
Submitted. Invited Publication.
Multifunctional nanocarriers for drug delivery across the blood-brain barrier
*
Sonu Bhaskar1,2, Furong Tian3*, Tobias Stoeger3, Wolfgang Kreyling3, Jesús Mª de la Fuente1,
Valeria Grazú1, Giovani Estrada4, Vasilis Ntziachristos5, and Daniel Razansky5
Submitted to the Journal of Fiber Toxicology, 2009. [Invited]
1
Instituto Universitario de Nanociencia de Aragón (INA), Universidad de Zaragoza, Zaragoza,
Spain
2
Zaragoza University Hospital, and Instituto Aragones de Ciencias de la Salud (IACS), Zaragoza,
Spain
3
Institute of Lung Biology and Disease, Helmholtz Zentrum München, 85764, Neuherberg,
Germany
4
Institute of Bioinformatics, Helmholtz Zentrum München, Neuherberg, Germany
5
Institute for Biological and Medical Imaging, Helmholtz Zentrum München, and Technische
Universität München, Germany
Email
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
57
Corresponding author:
* Dr Furong Tian
Institute of Lung Biology and Disease
Helmholtz Zentrum München
Ingolstädter Landstraße 1, 85764 Neuherberg, Germany
[email protected]
Abstract
Nanotechnology innovation has brought a variety of new possibilities into biological discovery and
clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing
for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also
minimizing exposure of healthy tissue to the drug. In this review we discuss and analyze three
issues, which are considered to be at the core of nano-scaled drug delivery systems, namely
functionalization of nanocarriers, delivery to target organs and in vivo imaging. To begin with, the
latest developments on highly specific conjugation strategies that are used to attach biomolecules
to the surface of nanoparticles are reviewed. Besides drug carrying capabilities, the
functionalisation of nanocarriers also facilitate their transport to primary target organs. We
highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier
(BBB), a tightly packed layer of endothelial cells surrounding the brain preventing high-molecular
weight molecules from entering the brain. The BBB has several transport molecules such as
growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of
brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as
stroke, tumours and Alzheimer’s, are therefore a much sought-after application of nanomedicine.
Likewise any other drug delivery system, a number of parameters need to be registered once
functionalised nanoparticles are administered, for instance their efficiency in organ-selective
targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an
exciting recent field that can provide real-time tracking of those nanocarriers. We review a range
of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on
recent advances in optical and hybrid imaging modalities, such as fluorescent and mesoscopic
molecular tomography, multispectral optoacoustic tomography and fluorescent protein
tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics,
therapeutics and molecular targeting. A proposed roadmap for ongoing and future research
58
directions is therefore discussed in detail with emphasis on the development of novel approaches
for functionalisation, targeting and imaging of nano-based drug delivery systems, a cutting-edge
technology poised to change the ways medicine is administered.
Keywords: functionalisation, nanoparticles, drug delivery, in vivo imaging, BBB.
1. Introduction
Nanotechnology has brought a new generation of lightweight materials with superior mechanical
and electrical properties. Engineered nanoparticles (NP) are normally embedded in the matrix of
other composites to enhance certain characteristics. Biology and medicine, however, usually
employ dispersed nanoparticles, for instance as fluorescent biological labels [1-3], drug and gene
delivery agents [4, 5], bio-detection of pathogens [6], detection of proteins [7], probing of DNA
structure [8], tissue engineering [9, 10], tumor destruction via heating (hyperthermia) [11],
separation and purification of biological molecules and cells [12], MRI contrast enhancement [13]
and phagokinetic studies [14]. The ability of the engineered nanoparticles to interact with cells and
tissues at a molecular level gives them a distinct advantage over other polymeric or
macromolecular substances.
The advent of nanotechnology made its first mark on consumer products, and little or nothing was
known about potential medical applications. Nanoparticles have long been noticed to pass across
the blood–brain barrier (BBB) [15], a tightly packed layer of endothelial cells surrounding the
brain preventing high-molecular weight molecules from passing through. This in itself is a leading
advantage for drug delivery systems across the BBB, that can pave the way for effective treatments
of many central nervous system disorders. This advantage, however, was not fully exploited till
two decades later.
Despite the advances and breakthroughs in nanotechnology based approaches, their efficacy
towards the treatment of neurological disorders, like brain tumor, stroke, Alzheimer’s disease,
have been largely constrained. As such, keeping in mind the paucity of therapies for such
debilitating disorders, advances in the targeting of drugs to the central nervous system (CNS) will
be the main stay for the future success and development of nanotechnology based theradiagnostics
(application of nanoparticles in therapy and diagnostics) in neurology. To this end, efficient
59
delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain, is
hindered by the blood–brain barrier, the blood–CSF barrier, or other specialized CNS barriers [16].
As a result, the global market for drugs for the CNS is greatly under-penetrated and would have to
grow by over 500% just to be comparable to the global market for cardiovascular drugs [17]. Only
a small class of drugs or small molecules with high lipid solubility and low molecular mass of <
400–500 Daltons actually goes across the BBB [18]. For instance, in a recent study of the
comprehensive medicinal chemistry (CMC) database [19], over 7,000 drugs were analyzed and
only 5% of these drugs affected the CNS, treating primarily depression, schizophrenia, and
insomnia. The average molecular mass of the CNS active drug was 357 Daltons. Another similar
study found 12% of drugs active upon the CNS, but only 1% of the total numbers of drugs were
active in the CNS for diseases other than affective disorders [20]. Modern ageing societies require
therefore a broader spectrum of treatments for neurological disorders.
Functionalisation of nanoparticles is indeed the first and perhaps foremost step towards nano-scale
drug delivery systems. Nanoparticles should inherit a number of desirable characteristics from
their functionalisation. Drug-carrying capabilities are as important as transport, organ targeting and
eventual excretion. Affinity of functional groups to tissue specific transport methods is clearly a
challenging problem. It is known that some transport molecules such as growth factors, insulin and
transferrin can potentially increase the efficiency and kinetics of drugs across a range of tissues.
Once nanomaterials are enhanced with drug-carrying and transport capabilities, in-vivo imaging
markers, such as fluorescent dyes for optical imaging, is the next landmark to achieve. No review
on functionalisation of nanocarriers is complete without mentioning imaging technologies capable
of their effective visualization. Beyond improvements in overall image quality and spatial
resolution, imaging modalities have been entrusted with the challenge of capturing dynamic
processes involving various biological system components as well as their respective interactions.
For example, the ability to resolve and monitor transmigration ability of various types of
biomolecules across the BBB in vivo is a daunting challenge. Fluorescence-based imaging
techniques have become an integral part of modern biological discovery process, especially in the
pre-clinical small-animal-based research. Initially, fluorescence imaging was limited to ex vivo and
in vitro applications [22, 23] with an exception of several intravital microscopy and photographic
imaging approaches [21]. Although helpful in some cases, these methods fall short to the potential
of more recent trans-illumination and tomographic techniques that allow non-invasive fluorescence
60
images in vivo [201] Powerful opportunities are found when those techniques are co-registered
with precise in-vivo anatomical views of the brain provided by magnetic resonance imaging or Xray CT. An additional enormous potential lies ahead with the recent advances of high resolution
optoacoustic molecular imaging approaches, such as multispectral optoacoustic tomography
(MSOT) [211]. All these are expected to facilitate the development of novel imaging-based
diagnostic and therapeutic nanoprobes for early diagnosis and therapy of various disorders of the
brain following systematic administration. In this review, we highlight some of the ongoing trends
in molecular and fluorescence tomographic imaging of live animals and present insights into
exploiting targeting of brain tumors for therapeutic and diagnostics purpose.
As a field on its own, nano-drug delivery requires proper functionalisation, profound knowledge of
the range of possible routes to and from the central nervous system, as well as ways to verify
whether drugs and nanocarriers reach their final destination. We proceed to review some of the
most exciting trends in functionalisation, delivery and imaging of nanomaterials.
2. Nanoparticle mediated brain delivery systems
Before starting with the functionalisation of nanoparticles, it is important to keep in mind a range
of useful properties we wish to have in any drug delivery across the BBB. In this context, owing to
their small size, customizable surface, improved solubility, targeted drug delivery and
multifunctionality, nanoparticles have emerged as potential drug delivery carriers to tissues
throughout the body [24]. Yet passing the BBB is particularly difficult. The proper design of such
engineered ‘nanocarriers’ becomes very important in transversing the impermeable membranes to
facilitate drug delivery. At the same time, it is also required to retain the drug stability and ensure
that early degradation of drugs from the nanocarriers does not take place.
Therefore, for drugs to be successfully delivered to their target, many factors such as its size,
biocompatibility, target specific affinity, avoidance of reticuloendothelial systems, stability in
blood, or ability to facilitate controlled drug release need to be considered during manufacture of
the NPs. Ideal conditions, or wish-list, of any drug are difficult to meet simultaneously. As for
nanocarriers to serve as good candidates for drug delivery across the blood brain barrier can be
summarized as follows[25, 26].
•
particle diameter less than 100 nanometers;
61
•
non-toxic, biodegradable and biocompatible;
•
stable in blood (i.e., no opsonisation by proteins);
•
BBB-targeted (i.e., use of cell surface, ligands, and receptor mediated endocytosis);
•
no activation of neutrophils, non-inflammatory;
•
no platelet aggregation;
•
avoidance of the reticuloendothelial systems;
•
prolonged circulation time;
•
scalable and cost effective with regard to manufacturing process;
•
scalable and cost effective with regard to manufacturing process;
•
amenable to small molecules, peptides, proteins or nucleic acids;
•
controlled drug release or should exhibit modulation of drug release profiles.
From materials science perspective, the design of such nanocarriers become more complicated
when it comes to drug delivery to the brain because of its immunologically privileged
characteristics which restricts the entry of most pharmaceutical compounds across the BBB. As
such, the applicability of nanotechnology in CNS drug delivery has been grossly limited and this
may be attributed to the scarcity of strategies that can allow localized and controlled delivery of
drugs across the BBB to the desired site of injury or impairment.
2.1. Functionalisation of NPs
One of the most important challenges in nano-based diagnostics and drug delivery is the
functionalisation of nanoparticles. Firstly, we need to develop effective conjugation strategies to
combine, in a highly controlled way, specific biomolecules to the surface of nanoparticles. Figure
1 shows an example of a PEGylated, multilayer nanoparticle (polyethylene glycol, PEG, a popular
choice for biocompatible nanocarriers.
Some of the most prominent candidate biomolecules are cell penetrating peptides (CPP) such as
SynB vectors, penetratin [27] and Tat [28-30] that facilitate enhanced intracellular delivery [31],
fluorescent dyes (rhodamine, alexa, Cy5.5), tumoral markers for brain and gene therapeutic agents
for genetic therapy such as siRNA [32-37]. Figure 2 show two kinds of mouse tumour models,
namely Xenograft and genetically engineered mouse model (GEMM) [38].
2.2. Multifunctional NPs for brain specificity
62
Functionalisation itself requires a profound knowledge of the target organ and its transport
mechanisms. The BBB has several transport molecules that can potentially increase the efficiency
and kinetics of nanocarriers towards brains [39], such as, growth factors (e.g. epidermal growth
factor [40], vascular endothelial growth factor [41], basic fibroblast growth factor [42], insulin-like
growth factors (IGF-I and -II) [43]), biotin-binding proteins (avidin, streptavidin, or neutravidin)
[44], insulin [45, 46], albumin [47-49], leptin [50, 51], iron binding protein p97
(melanotransferrin) [52], lactoferrin [39, 53], transferrin [54, 55] and Angiopep-2 [56].
Agent / condition
Effect on BBB
Reference
Bradykinin, RMP-7
transient increase of permeability, activates
[57]
B2 receptors
VEGF, HIF-1,
increase of permeability and leakage
[58, 59]
TNF-alpha, IL-1beta
moderate increase of permeability
[60]
Tat, Nef, gp120 + IFN-
HIV-1-associated dysfunction
[28-30, 61,
Deferoxamine,
gamma
Low magnetic field (0.15
62 ]
moderated increase of permeability
[63, 64]
metalloproteinases
increase of permeability
[65]
LTC4
leukotriene-induced permeability
[66, 67 ]
Lipopolysaccharide
Enhance the passage of regulatory proteins
[68, 69 ]
P85
increase permeability by inhibiting the drug [70]
T)
efflux transporter Pgp
endothelin-1
dramatic increase of permeability after
[71 ]
intracisternal administration
tPA
Increase permeability via Akt
[72 ]
phosphorylation
PTX
increased permeability by altering
[73 ]
endothelial plasticity and angiogenesis
63
Moreover, biocompatible coating of non-viral gene delivery systems e.g. by poly (ethylene
glycol) (PEG) attachment for siRNA delivery [34] show significant advantage in brain targeting.
By altering the surface of polymeric nanoparticles on coating them with different hydrophilic
surfactants such as polysorbate 80 (Tween® 80) or other polysorbates with 20 polyoxyethylene
units, Kreuter et al. [74]
and Schroeder et al. [75, 76] have demonstrated significant brain
targeting of nanoparticles.
2.3. Need of surfactants for BBB transport
NP-mediated drug transport to the brain strongly depends on the type of surfactant.. In one study
[77], 12 different surfactants were coated onto the surface of poly(butylcyanoacrylate) (PBCA)
NPs were injected intravenously into mice to evaluate the influence of surfactant on the analgesic
effects. The authors reported that only the NPs with polysorbate 20, 40, 60 and 80 coatings
produced significant effect and the maximum effect was observed for the PBCA NP bearing
polysorbate 80 coating. PBCA NPs coated with surfactants have been successfully used in the
delivery of number of drugs across the BBB [78, 79], including the peptides (hexapeptide dalargin
[76, 77] and the dipeptide kytorphin), anti-tumor antibiotic doxorubicin (DOX) [80], loperamide,
the NMDA receptor antagonist MRZ 2/576 [81], and tubocurarine.
Calvo et al. [82] employed a novel strategy by using PEGylated polycyanoacrylate nanoparticles
(PEG PHDCA) as vector for drug delivery in experimental model of Prion disease. The work
showed that the PEG PHDCA particles produced a higher uptake by the spleen and the brain
which are both the target tissues of PrPres (an abnormal isoform which is characterized by the
accumulation of the host-encoded Prion protein (PrP) in the brain of experimental Prion diseases
mice) in comparison to the non-PEGylated nanoparticles.
Wilson et al. [83, 84] have used
polymeric nanoparticles for drug delivery of anti-Alzheimer's drugs such as tacrine [83] and
rivastigmine [84] in the brain of rats.
Toxicity of conjugated drug-nanocarriers has always been a concern. Gelperina et al. [80] studied
the toxicity of DOX bound to polysorbate 80-coated PBCA NPs in healthy rats and rats with
intracranial glioblastoma. No drug-induced mortality occurred with a dose of 3×1.5 mg/kg of the
64
DOX NP formulation on days 2, 5, 8 after tumor implantation. They concluded that the toxicity of
DOX bound to NP was similar or even lower than that of free DOX. Other studies [85, 86] aimed
at investigating the toxicological profile of doxorubicin bound to nanoparticles employing different
dose regimens correlates with the results of this study. Based on the above findings, Pereverzeva et
al. [85] hypothesized that the lower toxicity of the nanoparticulate formulation may be due to the
altered biodistribution of the drug mediated by the nanoparticles. Wang et al. [87] applied a unique
1% polysorbate-80 coated gemcitabine PBCA nanoparticles (GCTB-PBCA-NPs) to investigate its
inhibitory effects in C6 glioma cells in vitro and in vivo with Sprague Dawley (SD) rats. They
observed significant increase in the survival time of the rats injected with the formulation
compared with the saline control (P < 0.05).
In an interesting approach, You et al. [88] investigated feedback regulated paclitaxel delivery by
using pH-Sensitive poly (N,N-dimethylaminoethyl methacrylate (DMAEMA)/2-hydroxyethyl
methacrylate (HEMA)) nanoparticles for the triggered release of paclitaxel within a tumour
microenvironment. Driven by the fact that the tumours exhibit a lower extracellular pH than
normal tissues, the authors found that the paclitaxel release from DMAEMA/HEMA particles can
be actively triggered by small, physiological changes in pH (within 0.2-0.6 pH units). It seems to
be a promising way to facilitate drug delivery by regulating the tumour microenvironment. Further
studies are thus required to explore other factors within tumour microenvironment that can be
exploited to enable controlled release of drugs in brain tumours.
2.4. Lipid Nanoparticles
Liposomes and related lipid structures have long been employed for drug delivery. Lipid
nanoparticles, however, are alternative carrier system to traditional colloidal carriers, such as
emulsions, liposomes and polymeric particles. These novel carriers have been employed for brain
tumour targeting purposes, see [89, 90] for a detailed review. Nanoparticles based on solid lipids
comes in different types such as "solid lipid nanoparticles" (SLN), "nanostructured lipid carriers"
(NLC) and "lipid drug conjugate" (LDC) [91]. The breakthrough in advanced conjugation
strategies have further led to the emergence of the newer forms of SLN such as polymer-lipid
hybrid nanoparticles, nanostructured lipid carriers and long-circulating SLN [92]. Because of its
physiochemical characteristics, solid lipid NPs (SLN) have been very successful in comparison to
65
polymeric NPs due to the lower cytotoxicity, higher drug loading capacity, and best production
scalability [93].
Back in 2002, Olbrich et al. [94] reported, for the first time, the use of LDC-polysorbate 80
nanoparticles for brain delivery of diminazene to treat second stage human African
trypanosomiasis (HAT). They obtained nanoparticles with a very high drug load of 33% (w/w)
despite the highly water-soluble drug diminazenediaceturate. They concluded that by transforming
water-soluble hydrophilic drugs into LDC and formation of nanoparticles allows prolonged drug
release and targeting to specific sites by intravenous injection. In an another study published in the
same year, Wang et al. [95] synthesized 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine (DO-FUdR) and
incorporated it into solid lipid nanoparticles (DO-FUdR-SLN) by a thin-layer ultrasonication
technique in order to deliver the drug 5-fluoro-2'-deoxyuridine (FUdR) to the brain. With the
average particle size of 76 nm, drug loading of 29.02% and entrapment efficiency of 96.62%, DOFUdR-SLN proved to be very efficient in in vivo brain targeting.
More recently, Kuo et al. [96] evaluated the permeability of anti-human immunodeficiency virus
(HIV) agents, including stavudine (D4T), delavirdine (DLV), and saquinavir (SQV), across an in
vitro model of BBB and incorporating them with PBCA NPs, methylmethacrylatesulfopropylmethacrylate (MMA-SPM) NPs, and SLNs. Their experimental results revealed an
enhanced BBB permeability. Their work suggests that the PBCA, MMA-SPM, and SLNs hold a
lot of promise for the drug delivery and clinical applications in neuro-AIDS treatment.
2.5. Alternatives routes to drug delivery to the brain
No review of drug delivery across BBB is complete without looking at the broad picture of
administration routes. A direct drug administration to the brain region, painless and safe, will
definitively change the scenario. However, in the meantime intravenous administration is most
popular choice in clinical studies. Some approaches, however, that have been gaining considerable
attention, such as oral route [97], inhalation or intra-tracheal instillation (IT) [98], intranasal drug
delivery [100, 101], convection-enhanced diffusion [112] and intrathecal/intraventricular drug
delivery systems in addition to the conventional modes like intravenous administration. Therefore,
the administration route of NPs becomes an important criteria of consideration so as to overcome
66
the physiological barriers of the brain and to achieve high drug concentrations within the brain
[103].
Interestingly, Semmler-Behnke et al. [99] have recently reported the uptake of 1.4 and 18 nm gold
nanoparticles in secondary target organs like the brain following intra-tracheal or intravenous
application. Moreover, Wang et al. [104] used fluorescence-labeled bovine serum albumin (FBSA)
loaded in biodegradable poly(lactic acid-co-glycolic acid) (PLGA) for intraspinal administration of
Glial cell line derived neurotrophic factor (GDNF) following contusive spinal cord injury (SCI)
and for in vitro study. PLGA-FBSA nanoparticles were well absorbed by neurons and glia,
indicating that PLGA as a considerable nanovehicle for the delivery of neuroprotective
polypeptide into injured spinal cord. Also, local administration of PLGA-GDNF effectively
preserved neuronal fibers and led to the hind limb locomotor recovery in rats with SCI. The
research opened a new route nanocarrier administration by intraspinal administration.
Two different modes of nanoparticle administration in brain tumor mouse models are shown in
figure 2. Once administered the nanoparticles, reach the site of tumor, e.g. brain, thereby locating
them. Once they cross the BBB, the specific ligands or peptides get attached to the specific surface
markers expressed on the tumors. Hence, by functionalising nanoparticles with fluorescent dyes
could naturally provide in vivo imaging of the ongoing biological events during the drug
administration as well may act as potential diagnostics labels for early detection and location of
brain tumors.
3. Blood Brain Barrier: A gateway to neurological diseases
Treatment of neurological diseases such as brain tumors, inborn metabolic errors (e.g., lysosomal
storage diseases), infectious diseases and aging, is a daunting challenge due to the unique
environment of CNS [105, 106]. The advancement of pharmacological drug delivery to the brain
has been constrained due the existence of protective barriers which restricts the passage of foreign
particles into the brain. Therefore, the efficient design of non-invasive nanocarrier systems that can
facilitate controlled and targeted drug delivery to the specific regions of the brain is a major
challenge in drug development and delivery for the neurological diseases [26, 107]. It becomes
crucial to understand the structural composition as well as the functions of the factors that regulate
67
permeability of the substances across the BBB. For that reason, we will briefly discuss the main
transporters that mediate the transport of substances across the brain.
3.1. Physiology of the Blood Brain Barrier
Figure 3 gives an overview of the two main immunological barriers, namely blood-brain barrier
and blood cerebrospinal fluid barrier (BCSF) and their different components. We can see how
BBB acts as a neuroprotective shield by protecting the brain from most substances in the blood,
supplying brain tissues with nutrients, and filtering harmful compounds from the brain back to the
bloodstream [108]. BBB is constituted by the brain endothelial cells which form the anatomical
substrate called, cerebral microvascular endothelium which regulates the transport of solutes and
other substances including drugs in and out of the brain, leukocyte migration, and maintains the
homeostasis of the brain microenvironment, which is crucial for neuronal activity and proper
functioning of CNS. The cerebral microvascular endothelium, together with astrocytes, pericytes,
neurons, and the extracellular matrix, constitute a "neurovascular unit" that is essential for the
health and function of the CNS [109]. The transport of solutes and other substances across BBB is
strictly constrained through both physical tight junctions (TJs) and adherents junctions (AJs) and
metabolic barriers (enzymes, diverse transport systems) and hence excluding very small,
electrically neutral and lipid soluble molecules. Thus, conventional pharmacological drugs or
chemotherapeutic agents are unable to pass through the barrier.
TJs between endothelial cells of the BBB possess also an intricate complex of transmembrane
proteins (junctional adhesion molecule-1, occludin, and claudins) with cytoplasmic accessory
proteins (zonula occludens-1 and -2, cingulin, AF-6, and 7H6)
[109] and hence acts as
physiological and pharmacological barrier, thereby preventing influx of molecules from the
bloodstream into the brain. As shown in Figures 3 and 4, BBB is characterized by two membranes,
namely luminal and abluminal, facing blood capillary and brain interstitial fluids (ISF),
respectively. Another especial feature of BBB is the structural differences that exist between the
endothelia of the brain capillaries and endothelia in other capillaries, such as tight junctions
between adjacent endothelial cells [110], a lack of fenestrations (perforations) [111-114] and a lack
of pinocytotic vesicles [115, 116]. Furthermore, in addition to the BBB and BCSF, there exists
other CNS barrier shielding the delicate brain tissue from the outer world, but which may play a
role in drug transport, such as the blood tumor barrier [117] and the blood retina barrier [118],
formed of pigment epithelium enclosing the retina, and thereby acting as a barrier interface
68
between the systemic blood vessels of the neighboring choroid and the retina. Finally targeting of
tumor tissue is often constricted by the so called blood tumor barrier [117].
Moreover, blood–cerebrospinal fluid barrier is the second important feature of the CNS next to the
BBB, and is formed by the epithelial cells of the choroid plexus. BCSF controls the penetration of
molecules within the interstitial fluid of the brain parenchyma by closely regulating the exchange
of molecules between the blood and CSF. Previous reports have demonstrated the following
mechanisms of transport pertaining to the choroid plexus; facilitated diffusion (efflux) and active
transport into the CSF, as well as active transport (efflux) from CSF to the blood [119-121].
3.2. Role of efflux transporters
The treatment of intractable CNS disorders such as HIV dementia, epilepsy, CNS-based pain,
meningitis and brain cancers depend mainly on the ways to achieve higher drug concentration in
the targeted tissues of the brain. The ability of a substance to penetrate the BBB or be transported
across BBB is mainly dependent on its physiochemical properties. The total brain exposure, and
thus the pharmacological efficacy of a drug or drug candidate, depends on its drug uptake which
in turn depends on a combination of factors, including the physical barrier presented by the BBB
and the BCSF and the affinity of the substrate for specific transport systems located at both sides
of these interfaces [105, 106]. The efflux transporters present in the BBB and BCSF, limit brain
penetration as well as the intra- and extracellular distribution of a variety of endogenous and
exogenous compounds [107].
The efflux transporters role, both as a homeostatic agents against endogenous substances and
protective agents against the exogenous substances, have been extensively studied and three
classes of transporters have been implicated in the efflux of drugs from the brain: multidrug
resistance transporters, monocarboxylic acid transporters, and organic ion transporters [125].
Kabanov et al. [126] have reviewed the inhibition of efflux transporters by Pluronic® block
copolymers to enhance penetration of drugs for CNS delivery. Drug efflux transporters not only
cause elimination of the drugs from the brain but also affects its absorption and tissue distribution
[122]. Owing to the growing emphasis on identification and discovery of influx transport proteins
(from blood to brain) and efflux transport proteins (from brain to blood) in last years, BBB is now
considered to be a dynamic interface that controls the influx and efflux of a wide variety of
69
substances, including endogenous nutrients and exogenous compounds to maintain a favourable
environment for the CNS [127].
To this end, Deguchi and co-workers [128] demonstrated that the rat organic anion transporter 3
(rOat3) mediated brain-to-blood transport of uremic toxins, as well as that rat organic anion
transporting polypeptide (rOatp2) is involved in efflux of 3-carboxy-4-methyl-5-propyl-2furanpropionate. Sun et al. [129] investigated the transport of carbamazepine and drug interactions
with cultured rat brain microvascular endothelial cells (rBMEC) as an in vitro model of the bloodbrain barrier (BBB). They concluded that some specific ABC (ATP-binding cassette, ABC) efflux
transporters may be involved in the transport of carbamazepine across the BBB.
The fact that many of the lipophilic drugs show negligible brain uptake can be attributed to the
substrates of drug efflux transporters such as the organic anion transporting polypeptides [128,
130] and the BBB active drug efflux transporters of the ATP-binding cassette (ABC [100]) gene
family, e.g. P-glycoprotein (Pgp), multidrug resistance proteins (MRPs) and breast cancer
resistance protein (BCRP) [126, 131, 132], that are overexpressed by the endothelial or epithelial
cells of these barriers [131]. The combined action of these carrier systems results in rapid efflux of
xenobiotics from the CNS and they also account for the cellular localization, specificity,
regulation, and potential inhibition at the BBB and BCSF barriers.
Efflux transporters act as a major impediment factor to CNS access by restricting a number of
solutes. The future of CNS drug delivery is highly dependent on novel strategies towards
modulation of these efflux transporters by designing nanocarriers with tuned affinity for these
transporters [126, 131, 132]. The following section brings a more detailed account of transport
mechanisms.
4. Mechanisms of transport in and out from the brain
A schematic overview of transport mechanisms across the blood brain barrier is shown in Figure 4.
There are different mechanisms by which solutes move across membranes in and out of the brain;
but nevertheless, all these different mechanisms can be categorized into two basic forms. Firstly,
the transport may occur due to diffusion, either simply diffusion or facilitated across aqueous
channels. The primary bioenergy comes from a concentration gradient across the membranes,
between cells (i.e., paracellular) or across cells (i.e., transcellular). This passive diffusion accounts
70
for the passive transport of solutes through the cell membrane, depending upon size and
lipophilicity of the substances [134].Secondly, active transport is mediated by a carrier such as
proteins. The movement may be caused due to the molecular affinity, fluid streams or magnetic
fields.
Transports of solutes, drugs and other particles follow different mechanisms as shown in Figure 4
and discussed shortly. Cell migration, in particular that from blood leukocytes like
monocytes/macrophages, and T cells [133] circulating through the capillary bed may cross through
the BBB driven by chemotaxis, and thereby modifying the functionality of tight junctions. Passive
diffusion accounts for the passive transport of solutes through the cell membrane, depending upon
the lipophilicity of the substances [134].
Carrier mediated transport (CMT) or carrier-mediated influx are forms of diffusion which may be
passive or active, depending on the context, and involve the unidirectional transport of drugs from
the blood to the brain. It is mainly instrumental in the transport of many essential polar molecules,
with the help of carrier systems or transporters, such as glucose (GLUT1 glucose transporter),
amino acids (the LAT1 large neutral amino acid transporter, the CAT1 cationic amino acid
transporter), carboxylic acids (the MCT1 monocarboxylic acid transporter) and nucleosides (the
CNT2 nucleoside transporter) into the brain.
Active efflux transport or carrier mediated efflux involve efflux or extrusion of drugs from the
brain in the presence of efflux transporters such as P-glycoprotein, multidrug resistance protein
protein, breast cancer resistance protein and other transporters [135]. In contrast to the carrier
mediated transport, the active efflux transport causes the active efflux of drugs from brain back to
blood. It acts as a major obstacle in pharmacological drug delivery to the CNS. Interestingly,
Banks et al. [136] demonstrated that endogenous peptides like Tyr-Pro-Trp-Gly-NH2, transported
from the brain to the blood by peptide transport system-1 (PTS-1), are transported via active
efflux.
Receptor mediated transport is mainly employed in the transport of macromolecules like peptides
and proteins across the BBB by conjugating the substance with ligands such as lactoferrin,
transferrin and insulin [40, 45, 54]. It is an important transport mechanism of predominant interest
in drug delivery. Next, adsorptive mediated transport is a type of endocytosis induced by
71
conjugating the particle to cationised ligands or peptides such as albumin [137, 138]. Due to
electrostatic interaction with the anionic sites present on the membrane, the cationised ligand
conjugated nanoparticles takes the adsorptive mediated transport to enter the brain.
Finally, tight junction (TJ) modulation is caused by the relaxation of junctions, which facilitates
selective aqueous diffusion across paracellular junctions in the BBB. Mahajan et al. [139] reported
the modulation of tight junction using methamphetamine. Further, they also demonstrated
modulation of TJs using Morphine and HIV-1 Tat via the activation of pro-inflammatory
cytokines, intracellular Ca2+ release, and activation of myosin light chain kinase [140]. Their
studies revealed decreased transendothelial electric resistance and enhanced transendothelial
migration across the BBB. Similar observations are known about cocaine on BBB permeability,
which indeed worsen HIV dementia. Further studies are needed towards the development of novel
anti-HIV-1 therapeutics that target specific TJ proteins, such as ZO-1, JAM-2, Occludin, Claudin-3
and Claudin-5.
One important question in nano drug delivery, however often neglected, is about the fate of the
nanocarriers themselves. What happens when nanocarriers (hopefully still carrying the drugs)
succeeded in getting access to the central nervous system via BBB? What are the underlying
mechanisms that control how these nanocarriers release the therapeutic drugs upon reaching the
CNS or the target region? Many of these mechanisms are still not well understood. Dramatic
differences can be obtained depending on functionalisation, dosages, administration and so on. The
main mechanisms involving active targeting [141] are shown in Figure 5. BBB permeability of
drugs can be highly increased by active targeting, a non invasive way to transport drugs to target
organs using site-specific ligands. Nanocarriers conjugated to ligands capable of recognizing brain
capillary endothelial cells and cerebral tumoural cells have emerged as a major breakthrough in
CNS drug delivery and Neuro-oncology in particular [141]. The role of endocytosis in targeted
brain delivery has been recently reviewed by Bareford et al. [142] and they predicted that by
efficient targeting of conjugated nanocarrier systems to the endolysosomal pathway; significant
improvement of the drug delivery for the treatment of lysosomal storage diseases, cancer, and
Alzheimer's disease can be accomplished. Next, we will discuss about the two main mechanisms
of endocytosis mediated transport of nanocarrier systems.
4.1. Receptor mediated endocytosis
72
Receptor mediated endocytosis (RME) or clathrin-dependent endocytosis is an energy mediated
transport. It selectively uptake, by the receptors expressed on the brain endothelium cells,
nanocarriers conjugated to different types of ligands, including hormones, growth factors,
enzymes, viruses and plasma proteins. By direct and indirect conjugation of endogenous [143] and
chimeric peptides [144] to nanocarriers or receptors of BBB, significant improvement in drug
delivery has been reported [141]. Upon binding to the receptors, ligand conjugated nanocarrier gets
collected in specialized areas of the plasma membrane known as coated pits. The receptor
dissociates from the ligand conjugated nanocarrier and due to the acidification of the vesicle, the
nanocarrier complex degrades, hence releasing the drug to the organ. Furthermore, the coated pits
invaginate to form coated vesicles and then clathrin and associated proteins dissociate from the
vesicle membrane, and these proteins form new coated pits at the cell surface [54]. Receptorspecific ligands have been shown to be very effective to transport endogenous peptides like insulin
[46] and transferrin [54], albumin [145], and opioid peptides (e.g. deltorphins [146, 147], [Dpenicillamine2,5] enkephalin (DPDPE) and deltorphin II [130]).
Mechanisms underlying the ligand conjugated nanocarrier based transport of drugs such as
neuropeptides have been proposed such as the adsorption of apolipoprotein E, tight junction
modulation and P-glycoprotein inhibition [88]. Kreuter et al. [89, 148, 149] suggested that the
apolipoproteins B and E may be chiefly involved in the transport of nanoparticle-bound drugs into
the brain. They concluded that by coating the nanoparticles with polysorbate 80, apolipoproteins B
and E get adsorbed onto the nanoparticle surface from the blood after injection and thus seem to
mimic lipoprotein particles that could be taken up by the brain capillary endothelial cells via
receptor-mediated endocytosis.
After endocytosis, drugs may be released within the endothelium cells and undergo further
transportation into the brain by diffusion or through transcytosis [106]. For instance, Liu et al.
[150] used chelator-nanoparticle system and the chelator-nanoparticle system complexed with iron
to devise effective therapeutic strategy for Alzheimer’s disease which is characterized by
dyshomeostasis of metal ions with abnormally high levels of iron in affected areas of the brain.
They reported preferential adsorbtion of apolipoprotein E and apolipoprotein A-I in the in vitro
studies, thereby suggesting the RME transport of chelators and chelator-metal complexes by the
nanoparticles across the BBB. Further studies are needed to investigate whether these metal
73
chelators conjugated to nanoparticles can play a role in solubilizing amyloid-[beta] deposits in
Alzheimer disease. This can open new pathways to the treatment of neurodegerative diseases and
also to study the ways of neural repair using efficiently conjugated nanocarrier system.
Kim et al. [151] recently reported the blocking of low-density lipoprotein receptors (LDLR). Their
study is based on brain endothelial cells involving cellular internalization of Poly(methoxypolyethyleneglycol cyanoacrylate-co-hexa-decyl-cyanoacrylate) (PEG-PHDCA) nanoparticles
preincubated with apolipoprotein E. It strengthens the hypothesis of the preponderant role of the
LDLR-mediated transport in the endocytosis of PEG-PHDCA nanoparticles. Using protamineoligonucleotide nanoparticles (proticles) coated with Apolipoprotein A-I (apoA-I), Kratzer et al.
[152] observed increased particle uptake and transcytosis in an in vitro model of the BBB. These
findings were further supplemented by Petri et al. [153] who used Poly(butyl cyanoacrylate)
nanoparticles coated with poloxamer 188 (Pluronic® F68) bounded to doxorubicin and reported
enhanced anti-tumour effect of doxorubicin against an intracranial glioblastoma in rats. They
hypothesized that this may be facilitated by the interaction of apolipoprotein A-I, present on the
surface of the nanoparticles, with the scavenger receptor class B, type I (the prime receptor for
high density lipoprotein/apoA-I that is expressed on brain capillary endothelial cells (BCEC)
[152]) (SR-BI). Further research is required to reveal the mechanisms behind the interaction
between SR-B1 and apoA-1 and their possible role in enhancing the drug delivery via RME
pathway. Moreover, the possibility of more than one mechanism, implicated in the interaction of
nanocarrier based drug delivery systems with the brain endothelial cells, cannot be ruled out [154].
In a novel approach, Demeule et al. [56] reported the design of a family of Kunitz domain-derived
peptides called Angiopeps as a potential brain drug delivery system. Using a in vitro model of the
BBB and in situ brain perfusion, they demonstrated that these peptides, and in particular
Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than other
receptors such as transferrin, lactoferrin, and avidin. Furthermore, they suggested that the
Angioprep-2 endocytosis may be mediated by the low-density lipoprotein receptor-related protein1 (LRP1).
4.2. Absorptive-mediated endocytosis
74
Absorptive-mediated endocytosis (AME) [137, 155] is a transport mechanism that has gained
significant importance, and many new drug delivery technologies focuses on AME. The
underlying principle of AME based transport is the electrostatic interaction between a positively
charged substance (e.g. cationized peptide such as albumin [137]) and the negatively charged sites
on the brain endothelial cell (BEC) surface (e.g. glycoprotein [156]). Dos Santos et al. [157]
studied the nature and distribution of anions on the brain endothelial cell (BEC) surface in vitro
and in situ and found that the the predominant anion detected on BEC was heparan sulphate (HS)
in comparison to the anionic locations observed in endothelia from aorta and epididymal fat microvessels.
The hypothesis that the phagocytic cells of the innate immune system, mainly neutrophils and
monocytes, can be exploited as transporters of drugs to the brain has been studied by Afergan et al.
[158] in vitro and in rats and rabbits by utilizing negatively-charged nano-sized liposomes with
double-radiolabeled 3H (in the membrane) and 14C-serotonin (in the core), and fluorescent
markers (membrane and core). They observed a higher brain uptake of liposomal serotonin,
0.138% ± 0.034 and 0.097% ± 0.011, vs. 0.068% ± 0.02 and 0.057% ± 0.01, 4 h and 24 h after IV
administration in rats, serotonin liposomes and in solution, respectively. They concluded that
monocytes act as key players for the transport of serotonin liposomes.
Alkaloids like cocaine are well-known stimulants of the central nervous system, and its effect upon
the BBB has been studied extensively. Alas, little exploited for drug delivery, it actually relaxes
tight junctions and induces leukocyte migration. For instance, Liu et al. [143] reported enhanced
BBB permeability and pharmacological activity of the endogenous opioid receptor agonist,
endomorphin (EM)-1. A series of EM-1 analogs were tested, e.g. N-terminal cationization, Cterminal chloro-halogenation, and unnatural amino acid (D-Ala, Sar, and D-Pro-Gly) substitutions
in position 2. They found that in comparison with EM-1, the four D-Ala-containing tetrapeptides
and the chloro-halogenated D-Pro-Gly-containing pentapeptide elicited significant and prolonged
central-mediated analgesia upon subcutaneous administration. This fact might be interpreted as
more peptides reaching the CNS, thus bringing greater analgesic effect. They also reported that the
guanidino-[D-Ala2, p-Cl-Phe4]EM-1 showed 3 times more analgesia than the parent peptide
following intra cerebral-ventricular injection.
75
Absorptive-mediated transport (AME) based transport has been exploited to facilitate gene
delivery into brain tumours. Lu et al. [156], for instance, has incorporated plasmid pORF-hTRAIL
(pDNA) into cationic albumin-conjugated PEGylated nanoparticles (CBSA-NP) to evaluate the
efficacy of CBSA-NP-hTRAIL as a nonviral vector for gene therapy of gliomas. They observed
that 30 minutes after IV administration of CBSA-NP-hTRAIL to BALB/c mice bearing IC C6
gliomas.
These
nanoparticles
co-localized
with
glycoproteins
in
brain
and
tumour
microvasculature. And, more importantly, cells accumulated in tumour cells. In addition, they
reported apoptosis of brain tumour cells in vivo and significantly delayed tumour. The above
results suggest Absorptive-mediated transport is a very promising route of drug and gene delivery
across BBB for CNS disorders. More investigation is required to explore other anionic sites on the
BEC surface that can be used to design efficient strategies for delivery using nanocarrier systems
through absorptive-mediated transport. Despite of possessing a lower affinity than RME, AME
provides a higher capacity than receptor-mediated endocytosis.
4.3. In vivo pharmacokinetics and biodistribution NP mediated drug delivery system
Within the confines of size and charge dependent requirements to effectively deliver drugs via NP
carrier systems, there are other challenges that need further attention. Although, much of the work
has been focused towards drug delivery with NPs, relatively few studies have focused on the
interaction of NPs and their hosts in terms of biodistribution, organ accumulation, degradation
and/or toxicology like possible damage of cellular structures,. Nanomedicine may find itself at
crossroads. It might not be wise to ignore possible adverse effects and toxicity [4,8,159,165 and
167] of nanocarriers.
Till recently, no pan-European initiative was addressing these concerns. Noteworthy, the European
Commission has established the Registration, Evaluation, Authorisation and Restriction of
Chemical substances (REACH) which provide safety regulation on substances. Further, Borm et
al. [160] have extensively reviewed the potential risks of use of nanoparticles, in a review report
commissioned under the European Centre for Ecotoxicology and Toxicology of Chemicals
(ECETOC). We do expect similar commissions worldwide shortly. Nanodrug delivery is seen in
its infancy, and works are mostly focusing on particular aspects rather than holistic approaches,
e.g. ADME or DMPK. Well-established research protocols like absorption, distribution,
metabolism and elimination (ADME), and drug metabolism and pharmacokinetics (DMPK) will
surely be part of nanodrug delivery research in the near future [161].
76
On the distribution side, for instance, Kreyling et al. [162] have extensively studied translocation
kinetics [109, 162-165] and particle size dependency [148] of NPs. In general smaller NPs show
superior translocation kinetics but because of their small size might on the other hand cause
toxicological effects [reviewed by Oberdörster [223]]. It is all about a trade-off between drug
potency and immunologic surveillance; for example that NPs of size<100nm need to be used to
circumvent macrophage clearance in the lungs [166]. Furthermore, several authors have reported
that intrinsic characteristics of NPs, such as aspect ratio and surface area, can be pro-oxidant and
pro-inflammatory [109, 167-169]. Here the ultra high surface to mass ratio together with new and
often unexpected nanosize specific material properties related to extreme radii of curvature deserve
closer attention [224, 225]. Therefore, the use of biopersistent carbon-based, e.g. single or multiwall carbon nanotubes, or metallic nanocarriers is debatable. These important findings need not
discourage genuine efforts in nanodrug delivery, but strength the selection process of materials,
shapes and surface treatments [3,4,8,165]. Biodegradable, non-toxic multi-block co-polymers like
those based on poly(image-lysine), PEG copolyester and nanogels (e.g. polyethylenimine-PEG)
are thus advantageous.
Depending on their functionalisation, biodegradable nanocarriers can take a number of paths
within tissues. What are the possible trajectories nanocarriers take inside the brain?
Pharmacokinetics and excretion are key points that demand exhaustive research. Figure 6 shows
the main ways drugs and nanocarriers take within the extra cellular space of the brain. Following
their release, drugs can take different mechanisms by which they may be transported within (and
outside) the brain. One of the mechanisms is their transport by diffusion due to drug concentration
gradients as shown in Figure 6 (i); or they may be transported because of the convection due to
fluid pressure gradients (ii). Figure 6 (iii, a) shows drug migration into ventricular space via pial or
ependymal surface. The drug molecules may also undergo circulation in the sub-arachnoid mater
or ventricular spaces (iii, b). Subsequently, it is possible to diffuse back into the brain interstitium
(iii, c). The drug molecules may also undergo permeation through the endothelium (iv, a);
followed by the circulation in the cerebral blood vessels (iv, b); and eventually may re-enter the
brain interstitium by permeation (iv, c) [170].
The exact path, drugs and biodegradable nanocarriers take, depends on many factors and its in vivo
imaging is perhaps the next milestone for nanodrug delivery. In the next section, we will discuss
about the scope of nanoimaging and its convergence with other contemporary in vivo imaging
77
techniques, such as fluorescence tomography and PET. The field aims towards developing cutting
edge therapeutic neuroimaging.
5. Towards development of neurodiagnostic nanoimaging platform
With the advent of multifunctional nanoparticles, the field of brain imaging is encountering a
drastic change in the ways one can monitor events at molecular and cellular level as well as to
track the development of neurological diseases, cancerous formations etc. One important aspect is
development of suitable imaging platforms that can be used to trace these agents in-vivo. Many of
the well-established modalities like positron emission tomography (PET), single photon emission
computed tomography (SPECT), magnetic resonance imaging (MRI), computed tomography (CT),
as well as a variety of optical-contrast-based imaging approaches, such as bioluminescence
imaging, fluorescence molecular tomography, and optoacoustic tomography, have gained
considerable interest and applicability in neurological research. In the following section, we will
focus on some of the commonly used techniques with a special emphasis on the fast evolving
optical and optoacoustic in-vivo imaging techniques as well as some trends in multimodality
imaging approaches. To better introduce the reader into the modern light-based imaging
modalities, we further provide a brief overview of their basic principles of operation and main
performance characteristics for the mostly recent techniques.
5.1 Traditional whole-body imaging modalities
Over the last three decades, X-Ray CT, magnetic resonance imaging (MRI), and positron emission
tomography (PET) have been commonly utilized for visualization of distribution and therapeutic
effects of drugs.
X-Ray CT has emerged as a major imaging modality for imaging pharmacokinetics, drug delivery
and/or treatment monitoring, mainly based on indirect tracking of morphological changes. Most
common CT contrast agents are based on small iodinated molecules. They are effective in
absorbing X-rays, but non-specific distribution and rapid pharmacokinetics have rather limited
their microvascular and targeting performance. Rabin et.al. [171] reported enhanced in vivo
78
imaging of the vasculature, the liver and lymph nodes in mice using a polymer-coated Bi2S3
nanoparticle formulation as an injectable CT imaging agent. As such, nanoparticles and their
bioconjugates are expected to become an important adjunct to in vivo imaging of molecular targets
and pathological conditions. Maier-Hauff et. al. [172] used CT in order to noninvasively monitor
the local drug release in a rabbit radiofrequency (RF) ablation model. Overall, the application of
nanoparticle based imaging probes to X-ray CT imaging could have a significant impact on health
care, owing to the ubiquitous nature of CT in the clinical setting as well as the increasing use and
development of micro-CT and hybrid systems that combine positron-emission tomography (PET)
and single photon-emission CT (SPECT) with X-ray CT.
With the distinct advantage of functional-imaging capabilities as well as better contrast among soft
tissues in comparison to the computed tomography (CT), MRI has emerged as a potential tool in
oncological imaging and imaging of the diseased nervous system [173]. As such, the choice of
contrast agents is of paramount importance in the field of in vivo imaging. Manganese is gaining
importance as T1 contrast agent for MRI as a neural tracer, and Leergaard et al. [174] used it to
make an in vivo study of the three-dimensional (3-D) connectivity patterns in the rat
somatosensory system. MRI has played a major role in advancement of nanomedicine. To this end,
the magnetic nanoparticles (MNPs) have been gaining considerable interest as contrast agents for
MRI and as carriers for drug delivery [175]. Superparamagnetic iron oxide nanoparticles
(SPIONs), paramagnetic contrast agent (gadolinium) or perfluorocarbons have already been
established as major players in tracking single or clusters of labeled cells within target tissues
[176]. This may be attributed to their unique magnetic properties (higher magnetic moments, nonfouling surfaces) and the ability to function at the cellular and molecular level of biological
interactions. Recently, Feng et al. [177] reported significant magnetic resonance signal
enhancement in T2 weighted image of lymph nodes using monodisperse MNPs (synthesized via
chitosan-poly(acrylic acid) (CS-PAA) template) during MRI experiments with rabbits.
Multifunctional nanoplatforms, based on protein cage architectures loaded with imaging agents
(fluorophore and MRI contrast agent) onto cells, have also been developed for both diagnostics
and targeted treatment of recalcitrant bacterial infections [178]. Fiandaca et al. [179], by including
gadolinium-loaded liposomes (GDL) with adeno-associated viral vectors (AAV), reported real
time MRI imaging and tracking of convection-enhanced delivery (CED) of viral vectors to the
three different regions of non-human primate brain (corona radiata, putamen and thalamus).
79
Positron emission tomography (PET) is a noninvasive imaging technology that enables the
determination of biodistribution of positron emitter-labeled compounds. PET has many advantages
over other imaging modalities, because of its high sensitivity of the measurement and easy
applicability to humans. For instance, Ukrami et. al. [180] designed labelled lipid nanoparticles to
study in vivo distribution of liposome-encapsulated haemoglobin determined by Positron Emission
Tomography. Plotkin et. al. [181] employed PET for targeting the intra-tumourally injected
magnetic nanoparticles in patients with glioblastoma. Indeed, since its introduction in the late 70’s,
PET has became a powerful imaging modality with the ability for highly sensitive detection of
molecular tracers and is currently utilized in diagnosis, therapy monitoring, and imaging gene
expression using diverse reporter genes and probes. However, high costs and other complications
associated with PET and SPECT equipment limit their applicability. Moreover, the images
acquired by these techniques have poor spatial resolution and hence accurate identification of
regions of uptake is difficult to achieve.
In summary, low sensitivity, high costs and/or low spatial resolution associated with the existing
well-accepted imaging modalities, promoted the search for new approaches for in-vivo
visualization of brain-targeting nanocarriers.
5.2. Optical imaging
Optical imaging has unique advantages compared to other imaging modalities, including
simplicity, low-cost and small size. Optical wavelengths offer many probing mechanisms that can
be used for variety of interrogations, from intrinsic functional information on blood oxygenation to
molecular sensing [182]. The light radiation is non-ionizing, and therefore reasonable doses can be
repeatedly employed without harm to the animal or patient. Optical contrast methods offer the
potential to differentiate between soft tissues, due to their different absorption that are
indistinguishable using other modalities. Also, specific absorption by natural chromophores (such
as oxy-haemoglobin) allows functional information to be obtained. The use of extrinsicallyadministered “switchable” and “tumor-selective” fluorescent optical agents further advances the
application possibilities by allowing visualization of otherwise invisible cellular and sub-cellular
processes [183-185]. During the last decade, a large number of commercially available fluorescent
probes and markers are increasingly being offered, from non-specific fluorescent dyes and
fluorescent proteins to targeted or activatable photoproteins and fluorogenic-substrate-sensitive
80
fluorochromes [186] to enable a highly potent field for biological imaging. So far, these contrast
mechanisms were proven efficient in a number of clinical and small-animal applications, including
probing of tissue hemodynamics [187], gene expression profiling [188], detecting protease upregulation associated with cancer growth and inflammation [189, 190] continuous monitoring of
the efficacy of anti-cancer treatments and other therapeutic drugs [191]. Since many of the probes
are developed to fluoresce in the near-infrared (NIR) optical window, where optical absorption is
very low so that light can penetrate deeply, fluorescence imaging has been successfully translated
from a microscopy level to whole body small animal imaging and clinics [192-193]. The
combination of such probes with optical imaging may yield a unique, highly sensitive technology
for in vivo and real-time imaging of the expression patterns for various enzymes, which are
crucially involved in tumor formation and metastasis. A good example are various breast cancer
cell lines that have been identified to over-express specific enzymes such as matrix
metalloproteinases [194], which are not over expressed in normal cells.
Despite these advantages, early optical imaging systems were confronted with limitations
associated with single projection viewing, non-quantitative ability and low resolution, especially
for non-superficial optical contrast. An important physical difference between microscopy of cell
monolayers or thin, 5 – 20 micron histological slices and small animal or human imaging is
scattering: thick tissues diffuse light and significantly reduce the resolution and the overall image
fidelity. When light encounters thick tissues, photons interact with cellular interfaces and
organelles leading to multiple changes in direction (scattering events) within the specimen under
investigation [195]. The detected light therefore loses information on its origin and propagation
path, blurring the images and destroying spatial resolution. Even state-of-the-art multiphoton
microscopy [196] is usually limited to superficial imaging up to a depth of 0.5-1 mm in most living
tissues. Recent efforts to image entire embryos for example required naturally transparent
specimen [197] or special chemical treatment to clear them from scattering, which is only suitable
for post-mortem imaging. Some other macroscopic photographic approaches like epi-fluorescence
suffer from similar light diffusion limitations and therefore have low penetration depth, lack
quantification abilities, and overall cannot accurately provide depth and size information [194].
Whole-body fluorescence molecular tomography (FMT) techniques overcomes the said limitations
by providing high sensitivity, penetration depth, and good quantification performance [198, 189191]. FMT illuminates the sample under investigation at multiple projections and utilizes
mathematical models of photon propagation in tissues to reconstruct the underlying imaging
81
contrast. In contrast to microscopic three-dimensional “tissue-sectioning” imaging, tomography
and reconstruction here implies the formulation of a mathematical inverse problem, whose
algebraic solution (minimization) yields the reconstructed images, in analogy to methods used in
X-ray CT, Single Photon Emission Tomography (SPECT) or Positron Emission Tomography
(PET). Several different implementations, developed over the past years, have been successfully
used to three-dimensionally image bio-distribution of fluorochromes in entire animals, molecular
pathways of cancer and cardiovascular disease, offering quantitative imaging. Fluorescence
tomography of whole animals and human organs works optimally in the near-IR region where the
lower tissue attenuation allows the penetration of photons over several centimeters [199]. Figure
7(a) gives a general schematic of state-of-the-art free-space FMT imager system for in vivo
tomographic imaging of small animals [200]. The main components include laser diode source,
CCD camera and rotation stage, onto which the animal is being mounted. Filters in front of the
CCD are used to separate captured images into intrinsic (excitation) and fluorescence (emission)
wavelengths. The scanhead is responsible for scanning a focused laser beam upon the object while
the images are collected by the camera in a transillumination mode. The object is also rotated for
collection of the tomographic data.
FMT systems were so far successfully used in a variety of molecular imaging studies of brain
disease. In one of the studies [201], using near-infrared fluorescent molecular beacons and
inversion techniques that take into account the diffuse nature of photon propagation in tissue,
three-dimensional in vivo images of a protease activity in orthopic gliomas were obtained. In this
study, 2×105 cells (9L or HT1080) were stereotactically implanted into unilateral brain
hemispheres of nude mice. Animals were then intravenously injected with the cathepsin-B imaging
probe (2 nmol Cy 5.5 per animal). FMT images and its superimposition on MR images have been
shown in figure 7 b and c. The experiments presented the ability of FMT to resolve fluorochromes
in deep tissues and follow their response over time. An important aspect to longitudinal in-vivo
studies is the fact that, as opposed to planar two-dimensional imaging, FMT is inherently
quantitative. Additional advantages include the fact that no ionizing radiation is required, that
beacons and fluorochromes are usually stable and do not decay like isotopes and that the
technology is relatively inexpensive compared with other tomographic imaging systems. It is also
conceivable to perform multi-wavelength imaging to obtain information from multiple targets or to
validate measurements similar as in comparative hybridization experiments.
82
Fluorescence Protein Tomography (FPT) is a variation of FMT that is optimized for the in vivo 3D
tomographic imaging of fluorescence proteins in mice. The method expands the everyday
fluorescent protein tagging techniques to the 3D non-invasive in-vivo small animal imaging field.
In a recent work [202], FPT has been demonstrated to be highly successful in non-invasive
imaging of lung tumor progression (as shown in figure 7 d-i) of superficial and deep-seated FP
activity in small animals in vivo, as well as imaging of gene delivery using a herpes virus vector.
As evident from the good congruence observed between the location seen on FPT and
corresponding anatomical micro-CT images obtained with the animals under similar placement
conditions, FPT has been very effective in in vivo detection for deeper tissues in the live animals.
To this end, optical tomographic approaches in diffusive objects have been applied in tissues with
dimensions that are normally larger than 1 cm while offering spatial resolution on the order of 1
mm. Therefore, optical imaging methods were so far inadequate for non-invasive in vivo imaging
of intact developing insects, animal embryos or small animal extremities, i.e. when working at
mesoscopic dimensions between the penetration limits of modern optical microscopy (0.5-1mm)
and the diffusion-imposed limits in optical macroscopy (>1cm). Mesoscopic Fluorescence
Tomography (MFT) was recently developed to operate in the 0.5mm-1cm regime with focus on
enabling in-vivo observation of common biological model organisms [203]. In a typical MFT set
up (shown in figure 7 j), the microscope is horizontally mounted, and the sample lies vertically on
a high-speed rotation stage. A laser beam is focused by way of a low-numerical-aperture objective
in close proximity to the center area of the sample’s surface. The sample is then rotated and images
are captured with a CCD mounted on the microscope. The technique utilizes a modified laboratory
microscope and multi-projection illumination to collect data at 360-degree projections. It employs
the Fermi simplification to the Fokker-Plank solution of the photon transport equation, combined
with geometrical optic principles in order to allow in vivo whole-body visualization of nontransparent three-dimensional structures in samples up to several millimeters in size. Using MFT
whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary
glands and wing imaginal discs in living Drosophila melanogaster pupae was reported [203] in
vivo and over time. The method extends whole-body optical imaging to the mesoscopic depth scale
therefore can potentially be used for monitoring of drug delivery in isolated organs or embryos
[204] as well as in other important model organisms smaller than mice, e.g. fishes, worms or flies.
83
5.3. Multimodality optical imaging
As expected, every imaging or functionalisation technology comes with its own pros and cons. The
major imaging challenge of optical imaging is to overcome the effects of light scattering that
dramatically reduces spatial resolution. Since optical imaging does not automatically provide the
reference anatomical images, mapping the precise distribution of molecular markers requires using
an additional high resolution reference modality. Additional challenges arise from highly
heterogeneous optical properties of common biological tissues that might somewhat compromise
image reconstruction accuracy and quantification abilities.
Some of these complications can possibly be mitigated by a marriage between non-invasive optical
molecular imaging and other high resolution anatomical imaging modalities such as MRI
(magnetic resonance imaging), or X-Ray CT. The latter combination was recently employed to
study the progression of Alzheimer’s disease in vivo using a fluorescent oxazine dye to quantify
amyloid-[beta] plaques in a transgenic murine model [205]. The authors reported very accurate
signal localization and correlation of in vivo results to ex vivo studies, thereby emphasizing that
FMT is not only a potential tool to study in vivo molecular functions, but it can also provide
precise mapping of those functions onto high resolution animal anatomy, simultaneously provided
by X-Ray CT. Furthermore, the CT information was used to build a more precise forward model in
the FMT image reconstruction process, which also improved spatial resolution and quantification
performance of FMT.
Another example of multimodal imaging is McCann et al. [206]. They combined FMT and MRI to
study structure and function of small rodents. Three-dimensional multimodal images were fused to
provide a volumetric model of living mouse brains. Interestingly, this approach allows continuous
monitoring of tumour morphology, progression and protease activity. Again, high-resolution
information taken from another imaging modality can be implemented into the diffuse optical
tomography inversion scheme to improve the quantification accuracy of the optical method.
84
All in all, we do see a promising future for nanodrug delivery and monitoring with multimodality
optical imaging. A successful therapy usually requires a combination of elements rather than a
single magic solution. New developments like light guidance using optical fibre makes optical
imaging compatible with many other radiological methods, such as mammography, ultrasound,
MRI, and positron emission tomography [207]. The development of fully hybrid modalities offers
the potential of simultaneously scanning the brain, under identical physiologic and geometric
conditions. This can produce an increased number of features that may augment the diagnostic
value of any stand-alone technique.
5.4. Optoacoustic imaging
Indeed, recently developed fluorescence molecular tomography approaches in diffuse living
specimen can improve our ability to probe complex biological interactions dynamically and to
study disease and treatment response over time in the same animal, thus offering the potential to
accelerate the basic research and drug discovery using fewer animals.
The main challenge for optical imaging of diffuse tissues is degradation of the spatial resolution,
which is always exchanged for penetration. As the size of the imaged object grows, imaging
resolution quickly deteriorates [203]. It is therefore possible to perform optical tomography, e.g.
FMT, through entire mice with high sensitivity, but low resolution of about 1mm or worse [182,
208]. Optoacoustic (or photoacoustic) tomography is an alternative hybrid imaging modality that
has recently demonstrated unprecedented high-resolution imaging of chromophore distribution and
vasculature deep in tissues of small animals [209-211].
When optoacoustic interrogation is
applied, high optical absorption contrast can be simultaneously combined with good spatial
resolution, not limited by light scattering in tissue. This is due to the fact that optoacoustic imaging
relies on detection of ultrasonic signals induced by absorption of pulsed light. The amplitude of the
generated broadband ultrasound waves reflects local optical absorption properties of tissue. Unlike
classical optical imaging, the spatial resolution here is not determined nor limited by light
diffusion, therefore such performances cannot be achieved by any other optical imaging
technology developed so far. Originally, optoacoustic imaging of tissues targeted endogenous
tissue contrast, primarily resolving oxy- and deoxy-hemoglobin and different vascular structures.
Wang et al [209] demonstrated high resolution imaging of vascular anatomy in the mouse brain
with capability to visualize, with high spatial resolution, functional parameters, e.g. blood
85
oxygenation levels and hemodynamics due to stimuli, deep in an intact living mouse brain.
However, recently good contrast was also obtained from other biological tissues that do not
contain haemoglobin, like fat, bones, and other internal structures [212]. The method was so far
used for high-resolution whole-body visualization of several optically diffusive model organisms
whose sizes may vary from sub-millimeter up to a centimeter range, e.g. insects, fishes, and small
mammals [211, 212]. The method offers a platform for mesoscopic imaging with resolution that
can practically become on the order of 20 microns with improved detection technology.
Advantageously, spatial resolution in optoacoustics is relatively constant for the entire penetration
range of several millimeters to centimeter of tissue and is also not limited by light diffusion but
only by the useful bandwidth of ultrasonic detector, which in turn can be further improved over
time to attain better performance.
In addition to offering rich intrinsic tissue contrast, optoacoustic imaging can also be used to
visualize exogenous molecular and functional markers. Naturally, almost all materials in nature
absorb light therefore can become potential candidates for providing contrast in optoacoustic
imaging. For high contrast imaging, of special interest are compounds having high molar
extinction coefficient. Several dedicated agents were so far exploited for enhancing contrast in
optoacoustics. Gold nanoparticles and nanoshells [213], were shown to increase optoacoustic
signals in-vivo. LacZ gene encoding for the X-gal chromogenic substrate expression and other
possible chromogenic assays are of potential interest [214]. Furthermore, a recent longitudinal
study has demonstrated that single-walled carbon nanotubes (SWNT) conjugated with cyclic ArgGly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumour.
Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight
times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes
[215].
Clearly, many other dedicated contrast agents could potentially be developed for optoacoustic
imaging applications, however, additional studies are be required in order to address a variety of
efficiency, BBB penetration capabilities, dosing, safety and toxicity concerns associated with those
new contrast agents. Instead, many widely adopted optical contrast agents, such as fluorochromes,
can be readily used by applying multispectral optoacoustic tomography (MSOT) [211]. MSOT
uses pulsed illumination at multiple wavelengths so that distinct spectral signatures from certain
86
biomarkers can be resolved over background tissue absorption by applying spectral processing.
The MSOT principle of operation relies on the spectral identification of known reporter molecules,
such as common fluorochromes or other chromophores within the background tissue absorption,
typically due to hemoglobin, but also melanin and other natural tissue absorbers. Therefore
molecules with spectra that are different than the ones of background tissue are best suited for
MSOT imaging. In this way, various additional molecularly-relevant information contained in the
optical spectrum can potentially be resolved such as fluorogenic or chromogenic bio-markers
associated with gene expression, morphogenesis or decease progression. The method is capable of
high resolution 3D visualization of molecular probes, such as common optical molecular probes
[216] and fluorescent proteins [211], located deep in scattering living tissues. It can therefore
simultaneously deliver anatomical, functional and molecular information with both high resolution
and penetration capabilities.
In conclusion, multi-spectral optoacoustic tomography (MSOT) is a rapidly emerging field in the
imaging sciences that can overcome major limitations of optical imaging while retaining its
contrast and sensitivity advantages [217]. It is therefore expected to drastically expand the
capabilities of photonic imaging in the field of in-vivo imaging of drug delivery markers.
6. Conclusion and future perspectives
Drug delivery across the blood-brain barrier is already one of industry's most sought-after routes.
Many ageing disorders and tumours require drugs acting on the central nervous system, and the
number of patients looking for efficient treatments is constantly increasing. Longer life expectancy
should also match better old-age life [218], however, current therapies fall short of the
population’s expectations. Anatomic features prevent most drugs to be delivered to the CNS across
the BBB. By overcoming the physiological barriers of the brain, achieving higher drug
concentration will become indeed feasible, which prompts an intensive search for alternative drug
delivery routes.
Multifunctional nanoparticles allow for a new approach for delivering pharmaceutical agents into
the brain. We reviewed a range of endogenous molecular pathways represented by growth factors,
e.g. insulin and transferrin, which when taken advantage of, can increase the efficiency and
87
kinetics of nanocarriers across the BBB. Multifunctional nanocarriers or their combination with
other drugs will drive the search for targeting specific areas in the brain and hence enhance
therapies. Nanomedicine has yet to make its mark in clinical studies, and we believe therefore that
the accumulated experience in the field has reached its critical mass.
Here, we have reviewed part of this exciting progress and research advances within the context of
drug delivery and in vivo imaging of multifunctional nanoparticles. Those nanocarriers can indeed
be functionalized with drugs as well as fluorescent substances therefore their diagnostics and
therapeutic potential is enormous. Imaging of function and molecular activity is at the frontier of
current research efforts to detect and study a variety of diseases, such as cancer, in a less invasive
way. A range of imaging techniques was reviewed. We described the established radiological
imaging techniques and highlighted the recent developments in optical and optoacoustic molecular
imaging approaches that exploit intrinsic optical contrast and exogenous markers for in-vivo gene
expression profiling and visualization of different molecular pathways. Imaging of optical contrast
can provide high sensitivity because background signals can effectively be suppressed by using
smart bio-markers e.g. enzyme-activated fluorescence probes. Moreover, a proper combination of
optical techniques with conventional techniques like CT and MRI can definitely enhance the ways
one can quantitatively monitor structure, function and molecular pathways, key features of
neurological diseases.
For a successful nanomedicine approach, all three elements (functionalisation, targeting and
imaging) have to be further developed. The interest in BBB has steadily grown in recent years, as
can be seen from over 5000 papers now listed in PubMed. From the vast literature we concentrate
on the inter-relations between functionalization, targeting and imaging; each of these issues
deserving comprehensive reviews on their own. Their proper combination can enhance spatial and
temporal resolution, thereby facilitating a unique way to keep track on disease progression as well
as on the histological changes in the target tissues. Nanodrug delivery and multimodal imaging
could, in principle, treat and monitor tumour status, thus increasing the patient's likelihood of
survival.
Multifunctional nanocarriers for drug targeting and in vivo imaging are mature fields, with bright
prospects to bring much-needed treatments for neurodegenerative pathologies. However, from a
broader perspective, nanocarriers loaded with multiple diagnostic, therapeutic or targeting
88
molecules can pave the way for successfully dealing with a number of other diseases. Application
of multifunctional nanocarriers is one of the main driving forces behind our renewed interest in the
BBB. Moreover, it has helped to understand the mechanisms that govern structural and
composition changes in response to various natural BBB transporters, undesirable toxins, infective
viruses like HIV-1, and potential BBB disrupting molecules. Clinical translation of these findings
should be fully exploited as to introduce nano-based medicine, a cutting-edge technology poised to
change how medicine is administered.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SB conceptualized the manuscript and wrote the draft. FT, DR and TS contributed in the draft and
concept of the paper. All other co-authors contributed in the revision process. All the other authors
contributed with their experience in the field of nanoparticle functionalisation, toxicology, in vivo
imaging and BBB in the conception and critical review of the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
The first author would like to acknowledge the fellowship support from the Deputación General
de Aragón (DGA), Spain. Support from the German Research Foundation (DFG) Research Grant
RA 1848/1 (Daniel Razansky) is also acknowledged.
References
1.
Bruchez M, Jr., Moronne M, Gin P, Weiss S, Alivisatos AP: Semiconductor Nanocrystals
as Fluorescent Biological Labels. Science 1998, 281(5385):2013-2016.
2.
Chan WC, Nie S: Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic
Detection. Science 1998, 281(5385):2016-2018.
89
3.
Tian F, Prina-Mello A, Estrada G, Beyerle A, Moeller W, Schulz H, Kreyling W,
Stoeger T: A novel assay for the quantification of internalized nanoparticles in macrophages.
Nanotoxicology 2008, 2(4): 232-242.
4.
Cui D, Tian F, Coyer S, Wang J, Pan B, Gao F, He R, Zhang Y. Effects of Antisense-mycconjugated Single-Walled Carbon Nanotubes on HL-60 cells. Journal of Nanoscience and
Nanotechnology, 2007, 7:1639-1641.
5.
Pantarotto D, Partidos CD, Hoebeke J, Brown F, Kramer E, Briand J-P, Muller S, Prato M,
Bianco A: Immunization with Peptide-Functionalized Carbon Nanotubes Enhances VirusSpecific Neutralizing Antibody Responses. Chemistry & Biology 2003, 10(10):961-966.
6.
Edelstein RL, Tamanaha CR, Sheehan PE, Miller MM, Baselt DR, Whitman LJ, Colton RJ:
The BARC biosensor applied to the detection of biological warfare agents. Biosensors and
Bioelectronics 2000, 14(10-11):805-813.
7.
Nam J-M, Thaxton CS, Mirkin CA: Nanoparticle-Based Bio-Bar Codes for the
Ultrasensitive Detection of Proteins. Science 2003, 301(5641):1884-1886.
8.
Cui D, Tian F, Kong Y, Ozkan C, Titushikin I, Gao H. Effects of single-walled carbon
nanotubes on the polymerase chain reaction. Nanotechnology 2004, 15(1) 154-157.
9.
de la Isla A, Brostow W, Bujard B, Estevez M, Rodriguez JR, Vargas S, Castaño VM:
Nanohybrid scratch resistant coatings for teeth and bone viscoelasticity manifested in
tribology. Materials Research Innovations 2003, 7(2):110-114.
10.
Ma J, Huifen W, Kong LB, Peng KW: Biomimetic processing of nanocrystallite
bioactive apatite coating on titanium. Nanotechnology 2003, 14( ):619-623.
11.
Shinkai M, Yanase M, Suzuki M, Hiroyuki H, Wakabayashi T, Yoshida J, Kobayashi T:
Intracellular hyperthermia for cancer using magnetite cationic liposomes. Journal of
Magnetism and Magnetic Materials 1999, 194(1-3):176-184.
12.
Molday RS, Mackenzie D: Immunospecific ferromagnetic iron-dextran reagents for the
labeling and magnetic separation of cells. Journal of Immunological Methods 1982, 52(3):353367.
13.
Weissleder R, Elizondo G, Wittenberg J, Rabito CA, Bengele HH, Josephson L:
Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents
for MR imaging. Radiology 1990, 175(2):489-493.
14.
Parak W, Boudreau R, Gros ML, Gerion D, Zanchet D, Micheel C, Williams S, Alivisatos
AP, Larabell C: Cell Motility and Metastatic Potential Studies Based on Quantum Dot
Imaging of Phagokinetic Tracks. Advanced Materials 2002, 14(12):882-885.
15.
Czerniawska A: Experimental investigations on the penetration of 198Au from nasal
mucous membrane into cerebrospinal fluid. Acta Otolaryngol 1970, 70(1):58-61.
90
16.
Neuwelt E, Abbott NJ, Abrey L, Banks WA, Blakley B, Davis T, Engelhardt B, Grammas
P, Nedergaard M, Nutt J et al: Strategies to advance translational research into brain barriers.
The Lancet Neurology 2008, 7(1):84-96.
17.
Pardridge WM: Why is the global CNS pharmaceutical market so under-penetrated?
Drug Discovery Today 2002, 7(1):5-7.
18.
Pardridge WM: Brain Drug Targeting: The Future of Brain Drug Development.
Cambridge, U.K. : Cambridge University Press; 2001.
19.
Ghose AK, Viswanadhan VN, Wendoloski JJ: A Knowledge-Based Approach in
Designing Combinatorial or Medicinal Chemistry Libraries for Drug Discovery. 1. A
Qualitative and Quantitative Characterization of Known Drug Databases. Journal of
Combinatorial Chemistry 1999, 1(1):55-68.
20.
Lipinski CA: Drug-like properties and the causes of poor solubility and poor
permeability. Journal of Pharmacological and Toxicological Methods 2000, 44(1):235-249.
21.
Taggart DP, Choudhary B, Anastasiadis K, Abu-Omar Y, Balacumaraswami L, Pigott DW:
Preliminary experience with a novel intraoperative fluorescence imaging technique to
evaluate the patency of bypass grafts in total arterial revascularization. The Annals of
Thoracic Surgery 2003, 75(3):870-873.
22.
Chen J, Tung C-H, Allport JR, Chen S, Weissleder R, Huang PL: Near-Infrared
Fluorescent Imaging of Matrix Metalloproteinase Activity After Myocardial Infarction.
Circulation 2005, 111(14):1800-1805.
23.
Sosnovik DE, Schellenberger EA, Nahrendorf M, Novikov MS, Matsui T, Fred GD,
Grazette RL, Rosenzweig A, Weissleder R, Josephson L: Magnetic resonance imaging of
cardiomyocyte apoptosis with a novel magneto-optical nanoparticle. Magnetic Resonance in
Medicine 2005, 54(3):718-724.
24.
Singh R, Lillard Jr JW: Nanoparticle-based targeted drug delivery. Experimental and
Molecular Pathology, In Press.
25.
Lockman PR, Mumper RJ, Khan MA, Allen DD: Nanoparticle Technology for Drug
Delivery Across the Blood-Brain Barrier. Drug Development and Industrial Pharmacy 2002,
28(1):1 - 13.
26.
Olivier J-C: Drug Transport to Brain with Targeted Nanoparticles. NeuroRX 2005,
2(1):108-119.
27.
Mortensen LJ, Oberdörster G, Pentland AP, DeLouise LA: In Vivo Skin Penetration of
Quantum Dot Nanoparticles in the Murine Model: The Effect of UVR. Nano Letters 2008,
8(9):2779-2787.
28.
Berry CC: Intracellular delivery of nanoparticles via the HIV-1 tat peptide.
Nanomedicine 2008, 3:357-365.
91
29.
de la Fuente JM, Berry CC: Tat Peptide as an Efficient Molecule To Translocate Gold
Nanoparticles into the Cell Nucleus. Bioconjugate Chemistry 2005, 16(5):1176-1180.
30.
Rao KS, Reddy MK, Horning JL, Labhasetwar V: TAT-conjugated nanoparticles for the
CNS delivery of anti-HIV drugs. Biomaterials 2008, 29(33):4429-4438.
31.
Scherrmann J-M, Temsamani J: The use of Pep: Trans vectors for the delivery of drugs
into the central nervous system. International Congress Series 2005, 1277:199-211.
32.
Hu Q, Chen C, Yan J, Yang X, Shi X, Zhao J, Lei J, Yang L, Wang K, Chen L et al:
Therapeutic application of gene silencing MMP-9 in a middle cerebral artery occlusioninduced focal ischemia rat model. Experimental Neurology 2009, In Press.
33.
Gonzalez-Alegre P: Therapeutic RNA interference for neurodegenerative diseases:
From promise to progress. Pharmacology & Therapeutics 2007, 114(1):34-55.
34.
Pardridge WM: shRNA and siRNA delivery to the brain. Adv Drug Deliv Rev. 2007 Mar
30;59(2-3):141-52.
35.
Hoyer D, Dev KK: RNA interference as a therapeutic strategy for treating CNS
disorders. Drug Discovery Today: Therapeutic Strategies 2006, 3(4):451-456.
36.
Xie FY, Woodle MC, Lu PY: Harnessing in vivo siRNA delivery for drug discovery and
therapeutic development. Drug Discovery Today 2006, 11(1-2):67-73.
37.
Leung RKM, Whittaker PA: RNA interference: From gene silencing to gene-specific
therapeutics. Pharmacology & Therapeutics 2005, 107(2):222-239.
38.
Sharpless NE, DePinho RA: The mighty mouse: genetically engineered mouse models
in cancer drug development. Nat Rev Drug Discov 2006, 5(9):741-754.
39.
Hu K, Li J, Shen Y, Lu W, Gao X, Zhang Q, Jiang X: Lactoferrin-conjugated PEG-PLA
nanoparticles with improved brain delivery: In vitro and in vivo evaluations. Journal of
Controlled Release 2009, 134(1):55-61.
40.
Maratos-Flier E, Kao CY, Verdin EM, King GL: Receptor-mediated vectorial
transcytosis of epidermal growth factor by Madin-Darby canine kidney cells. J Cell Biol
1987, 105(4):1595-1601.
41.
Ikeda E, Takubo K, Kodama T, Okada Y: Brain-Specific Expression of Vascular
Endothelial Growth Factor 146 Correlates with the Blood-Brain Barrier Induction in Quail
Embryos. Developmental Neuroscience 2008, 30(5):331-339.
42.
Deguchi Y, Okutsu H, Okura T, Yamada S, Kimura R, Yuge T, Furukawa A, Morimoto K,
Tachikawa M, Ohtsuki S et al: Internalization of basic fibroblast growth factor at the mouse
blood-brain barrier involves perlecan, a heparan sulfate proteoglycan. Journal of
Neurochemistry 2002, 83(2):381-389.
92
43.
Reinhardt RR, Bondy CA: Insulin-like growth factors cross the blood-brain barrier.
Endocrinology 1994, 135(5):1753-1761.
44.
Townsend SA, Evrony GD, Gu FX, Schulz MP, Brown Jr RH, Langer R: Tetanus toxin C
fragment-conjugated nanoparticles for targeted drug delivery to neurons. Biomaterials 2007,
28(34):5176-5184.
45.
King GL, Johnson SM: Receptor-mediated transport of insulin across endothelial cells.
Science 1985, 227(4694):1583-1586.
46.
Witt KA, Huber JD, Egleton RD, Davis TP: Insulin Enhancement of Opioid Peptide
Transport across the Blood-Brain Barrier and Assessment of Analgesic Effect. J Pharmacol
Exp Ther 2000, 295(3):972-978.
47.
Pardridge WM, Triguero D, Buciak J, Yang J: Evaluation of cationized rat albumin as a
potential blood-brain barrier drug transport vector. J Pharmacol Exp Ther 1990, 255(2):893899.
48.
Lu W, Zhang Y, Tan Y-Z, Hu K-L, Jiang X-G, Fu S-K: Cationic albumin-conjugated
pegylated nanoparticles as novel drug carrier for brain delivery. Journal of Controlled Release
2005, 107(3):428-448.
49.
Lu W, Tan Y-Z, Hu K-L, Jiang X-G: Cationic albumin conjugated pegylated
nanoparticle with its transcytosis ability and little toxicity against blood-brain barrier.
International Journal of Pharmaceutics 2005, 295(1-2):247-260.
50.
Pan W, Barron M, Hsuchou H, Tu H, Kastin AJ: Increased Leptin Permeation across the
Blood-Brain Barrier after Chronic Alcohol Ingestion. Neuropsychopharmacology 2007,
33(4):859-866.
51.
Banks WA, DiPalma CR, Farrell CL: Impaired transport of leptin across the bloodbrain barrier in obesity* - relationship to plasma levels and adiposity in humans. Peptides
1999, 20:1341-1345.
52.
Karkan D, Pfeifer C, Vitalis TZ, Arthur G, Ujiie M, Chen Q, Tsai S, Koliatis G, Gabathuler
R, Jefferies WA: A Unique Carrier for Delivery of Therapeutic Compounds beyond the
Blood-Brain Barrier. PLoS ONE 2008, 3(6):e2469.
53.
Fillebeen C, Descamps L, Dehouck M-P, Fenart L, Benaissa M, Spik G, Cecchelli R,
Pierce A: Receptor-mediated Transcytosis of Lactoferrin through the Blood-Brain Barrier. J
Biol Chem 1999, 274(11):7011-7017.
54.
Roberts RL, Fine RE, Sandra A: Receptor-mediated endocytosis of transferrin at the
blood-brain barrier. J Cell Sci 1993, 104(2):521-532.
55.
Ulbrich K, Hekmatara T, Herbert E, Kreuter J: Transferrin- and transferrin-receptorantibody-modified nanoparticles enable drug delivery across the blood-brain barrier (BBB).
European Journal of Pharmaceutics and Biopharmaceutics 2009, 71(2):251-256.
93
56.
Demeule M, Currie J-C, Bertrand Y, Ch, Christian, Nguyen T, gina A, Gabathuler R,
Castaigne J-P, liveau R: Involvement of the low-density lipoprotein receptor-related protein in
the transcytosis of the brain delivery vector Angiopep-2. Journal of Neurochemistry 2008,
106:1534-1544.
57.
Lumenta DB, Plesnila N, Kläsner B, Baethmann A, Pruneau D, Schmid-Elsaesser R,
Zausinger S: Neuroprotective effects of a postischemic treatment with a bradykinin B2
receptor antagonist in a rat model of temporary focal cerebral ischemia. Brain Research 2006,
1069(1):227-234..
58. Chi OZ, Hunter C, Liu X, Weiss HR:Effects of deferoxamine on blood-brain barrier
disruption and VEGF in focal cerebral ischemia. Neurology Research 2008, 30(3):288-293.
59. Ay I, Francis JW, Brown RH Jr: VEGF increases blood-brain barrier permeability to
Evans blue dye and tetanus toxin fragment C but not adeno-associated virus in ALS mice.
Brain Research 2008, 1234:198-205.
60. Brabers NA, Nottet HS: Role of the pro-inflammatory cytokines TNF-alpha and IL-1beta
in HIV-associated dementia. European Journal of Clinical Investigation 2006, 36(7):447-58.
61. Khan NA, Di Cello F, Stins M, Kim KS: Gp120-mediated cytotoxicity of human brain
microvascular endothelial cells is dependent on p38 mitogen-activated protein kinase
activation. Jounral for Neurovirology. 2007 Jun;13(3):242-51.
62. Annunziata P: Blood-brain barrier changes during invasion of the central nervous system
by HIV-1. Old and new insights into the mechanism. Journal of Neurology 2003, 250(8):901906.
63. Prato FS, Wills JM, Roger J, Frappier H, Drost DJ, Lee TY, Shivers RR, Zabel P: Blood-brain
barrier permeability in rats is altered by exposure to magnetic fields associated with
magnetic resonance imaging at 1.5 T. Microscopical Research and Technology1994, 27(6):528534.
64. McKay JC, Prato FS, Thomas AW: A literature review: the effects of magnetic field
exposure on blood flow and blood vessels in the microvasculature. Bioelectromagnetics. 2007,
28(2):81-98.
65. Adibhatla RM, Hatcher JF. Tissue plasminogen activator (tPA) and matrix
metalloproteinases in the pathogenesis of stroke: therapeutic strategies. CNS Neurol Disord
Drug Targets. 2008 Jun;7(3):243-253.
66. Kusuhara H, Suzuki H, Naito M, Tsuruo T, Sugiyama Y: Characterization of efflux
transport of organic anions in a mouse brain capillary endothelial cell line. The Journal of
Pharmacology Experimental Therapeutic 1998, 285(3):1260-1265.
94
67. Baba T, Black KL, Ikezaki K, Chen KN, Becker DP. Intracarotid infusion of leukotriene C4
selectively increases blood-brain barrier permeability after focal ischemia in rats. Journal of
Cerebral Blood Flow & Metabolism1991, 11(4):638-643.
68. Ivey NS, Martin EN Jr, Scheld WM, Nathan BR:.A new method for measuring blood-brain
barrier permeability demonstrated with Europium-bound albumin during experimental
lipopolysaccharide (LPS) induced meningitis in the rat. J Neurosci Methods. 2005, 142(1):9195.
69. Nonaka N, Hileman SM, Shioda S, Vo TQ, Banks WA: Effects of lipopolysaccharide on
leptin transport across the blood-brain barrier. Brain Research. 2004, 30;1016(1):58-65.
70. Batrakova EV, Zhang Y, Li Y, Li S, Vinogradov SV, Persidsky Y, Alakhov VY, Miller DW,
Kabanov AV: Effects of pluronic P85 on GLUT1 and MCT1 transporters in the blood-brain
barrier. Pharmacology Research. 2004, 21(11):1993-2000.
71. Narushima I, Kita T, Kubo K, Yonetani Y, Momochi C, Yoshikawa I, Ohno N, Nakashima T:
Highly enhanced permeability of blood-brain barrier induced by repeated administration of
endothelin-1 in dogs and rats. Pharmacology Toxicology. 2003, 92(1):21-6.
72. An J, Zhang C, Polavarapu R, Zhang X, Zhang X, Yepes M: Tissue-type plasminogen
activator and the low-density lipoprotein receptor-related protein induce Akt
phosphorylation in the ischemic brain. Blood 2008, 112(7):2787-2794.
73. Lu C, Pelech S, Zhang H, Bond J, Spach K, Noubade R, Blankenhorn EP, Teuscher C:
Pertussis toxin induces angiogenesis in brain microvascular endothelial cells. Journal of
Neuroscience Research 2008, 86(12):2624-2640.
74. Kreuter J, Alyautdin RN, Kharkevich DA, Ivanov AA: Passage of peptides through the
blood-brain barrier with colloidal polymer particles (nanoparticles). Brain Research 1995,
674(1):171-174.
75.
Schröeder U, Sabel BA: Nanoparticles, a drug carrier system to pass the blood-brain
barrier, permit central analgesic effects of i.v. dalargin injections. Brain Research 1996,
710(1-2):121-124.
76.
Schröeder U, Sommerfeld P, Sabel BA: Efficacy of Oral Dalargin-loaded Nanoparticle
Delivery across the Blood-Brain Barrier. Peptides 1998, 19(4):777-780.
77.
Kreuter J, Petrov VE, Kharkevich DA, Alyautdin RN: Influence of the type of surfactant
on the analgesic effects induced by the peptide dalargin after its delivery across the bloodbrain barrier using surfactant-coated nanoparticles. Journal of Controlled Release 1997,
49(1):81-87.
78.
Kreuter J: Nanoparticulate systems for brain delivery of drugs. Advanced Drug Delivery
Reviews 2001, 47(1):65-81.
79.
Kreuter J: Application of nanoparticles for the delivery of drugs to the brain.
International Congress Series 2005, 1277:85-94.
95
80.
Gelperina SE, Khalansky AS, Skidan IN, Smirnova ZS, Bobruskin AI, Severin SE,
Turowski B, Zanella FE, Kreuter J: Toxicological studies of doxorubicin bound to polysorbate
80-coated poly(butyl cyanoacrylate) nanoparticles in healthy rats and rats with intracranial
glioblastoma. Toxicology Letters 2002, 126(2):131-141.
81.
Friese A, Seiller E, Quack G, Lorenz B, Kreuter J: Increase of the duration of the
anticonvulsive activity of a novel NMDA receptor antagonist using poly(butylcyanoacrylate)
nanoparticles as a parenteral controlled release system. European Journal of Pharmaceutics
and Biopharmaceutics 2000, 49(2):103-109.
82.
Calvo P, Gouritin B, Brigger I, Lasmezas C, Deslys J-P, Williams A, Andreux JP, Dormont
D, Couvreur P: PEGylated polycyanoacrylate nanoparticles as vector for drug delivery in
prion diseases. Journal of Neuroscience Methods 2001, 111(2):151-155.
83.
Wilson B, Samanta MK, Santhi K, Kumar KPS, Paramakrishnan N, Suresh B: Targeted
delivery of tacrine into the brain with polysorbate 80-coated poly(n-butylcyanoacrylate)
nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics 2008, 70(1):75-84.
84.
Wilson B, Samanta MK, Santhi K, Kumar KPS, Paramakrishnan N, Suresh B: Poly(nbutylcyanoacrylate) nanoparticles coated with polysorbate 80 for the targeted delivery of
rivastigmine into the brain to treat Alzheimer's disease. Brain Research 2008, 1200:159-168.
85.
Pereverzeva E, Treschalin I, Bodyagin D, Maksimenko O, Kreuter J, Gelperina S:
Intravenous tolerance of a nanoparticle-based formulation of doxorubicin in healthy rats.
Toxicology Letters 2008, 178(1):9-19.
86.
Couvreur P, Kante B, Grislain L, Roland M, Speiser P: Toxicity of
polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. Journal of
Pharmaceutical Sciences 1982, 71(7):790-792.
87.
Wang C-X, Huang L-S, Hou L-B, Jiang L, Yan Z-T, Wang Y-L, Chen Z-L: Antitumour
effects of polysorbate-80 coated gemcitabine polybutylcyanoacrylate nanoparticles in vitro
and its pharmacodynamics in vivo on C6 glioma cells of a brain tumour model. Brain
Research 2009, In Press.
88.
You J-O, Auguste DT: Feedback-regulated paclitaxel delivery based on poly(N,Ndimethylaminoethyl
methacrylate-co-2-hydroxyethyl
methacrylate)
nanoparticles.
Biomaterials 2008, 29(12):1950-1957.
89.
Blasi P, Giovagnoli S, Schoubben A, Ricci M, Rossi C: Solid lipid nanoparticles for
targeted brain drug delivery. Advanced Drug Delivery Reviews 2007, 59(6):454-477.
90.
Joshi MD, Müller RH: Lipid nanoparticles for parenteral delivery of actives. European
Journal of Pharmaceutics and Biopharmaceutics 2009, 71(2):161-172.
91.
Wissing SA, Kayser O, Müller RH: Solid lipid nanoparticles for parenteral drug
delivery. Advanced Drug Delivery Reviews 2004, 56(9):1257-1272.
96
92.
Wong HL, Bendayan R, Rauth AM, Li Y, Wu XY: Chemotherapy with anticancer drugs
encapsulated in solid lipid nanoparticles. Advanced Drug Delivery Reviews 2007, 59(6):491504.
93.
Date AA, Joshi MD, Patravale VB: Parasitic diseases: Liposomes and polymeric
nanoparticles versus lipid nanoparticles. Advanced Drug Delivery Reviews 2007, 59(6):505521.
94.
Olbrich C, Gessner A, Kayser O, Müller RH: Lipid-Drug-Conjugate (LDC)
Nanoparticles as Novel Carrier System for the Hydrophilic Antitrypanosomal Drug
Diminazenediaceturate. Journal of Drug Targeting 2002, 10:387-396.
95.
Wang J-X, Sun X, Zhang Z-R: Enhanced brain targeting by synthesis of 3',5'dioctanoyl-5-fluoro-2'-deoxyuridine and incorporation into solid lipid nanoparticles.
European Journal of Pharmaceutics and Biopharmaceutics 2002, 54(3):285-290.
96.
Kuo Y-C, Su F-L: Transport of stavudine, delavirdine, and saquinavir across the
blood-brain
barrier
by
polybutylcyanoacrylate,
methylmethacrylatesulfopropylmethacrylate, and solid lipid nanoparticles. International Journal of Pharmaceutics
2007, 340(1-2):143-152.
97.
Rae CS, Wei Khor I, Wang Q, Destito G, Gonzalez MJ, Singh P, Thomas DM, Estrada
MN, Powell E, Finn MG et al: Systemic trafficking of plant virus nanoparticles in mice via the
oral route. Virology 2005, 343(2):224-235.
98.
Elder A, Gelein R, Silva V, Feikert T, Opanashuk L, Carter J, Potter R, Maynard A, Ito Y,
Finkelstein J, Oberdörster G. Translocation of inhaled ultrafine manganese oxide particles to
the central nervous system. Environ Health Perspect. 2006 Aug;114(8):1172-8.
99.
Semmler-Behnke M, Kreyling WG, Lipka J, Fertsch S, Wenk A, Takenaka S, Schmid G,
Brandau W: Biodistribution of 1.4- and 18-nm Gold Particles in Rats. Small 2008, 4(12):21082111.
100. Wang X, Chi N, Tang X: Preparation of estradiol chitosan nanoparticles for improving
nasal absorption and brain targeting. European Journal of Pharmaceutics and
Biopharmaceutics 2008, 70(3):735-740.
101. Wang J, Liu Y, Jiao F, Lao F, Li W, Gu Y, Li Y, Ge C, Zhou G, Li B et al: Timedependent translocation and potential impairment on central nervous system by intranasally
instilled TiO2 nanoparticles. Toxicology 2008, 254(1-2):82-90.
102. MacKay JA, Deen DF, Szoka Jr FC: Distribution in brain of liposomes after convection
enhanced delivery; modulation by particle charge, particle diameter, and presence of steric
coating. Brain Research 2005, 1035(2):139-153.
103. Patel MM, Goyal BR, Bhadada SV, Bhatt JS, Amin AF: Getting into the Brain:
Approaches to Enhance Brain Drug Delivery. CNS Drugs 2009, 23:35-58.
97
104. Wang Y-C, Wu Y-T, Huang H-Y, Lin H-I, Lo L-W, Tzeng S-F, Yang C-S: Sustained
intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles
following contusive spinal cord injury. Biomaterials 2008, 29(34):4546-4553.
105. Begley DJ: Delivery of therapeutic agents to the central nervous system: the problems
and the possibilities. Pharmacology & Therapeutics 2004, 104(1):29-45.
106. Chen Y, Dalwadi G, Benson HAE: Drug Delivery Across the Blood-Brain Barrier.
Current Drug Delivery 2004, 1:361-376.
107. Juillerat-Jeanneret L: The targeted delivery of cancer drugs across the blood-brain
barrier: chemical modifications of drugs or drug-nanoparticles? Drug Discovery Today 2008,
13(23-24):1099-1106.
108. Persidsky Y, Ramirez S, Haorah J, Kanmogne G: Blood–brain Barrier: Structural
Components and Function Under Physiologic and Pathologic Conditions. Journal of
Neuroimmune Pharmacology 2006, 1(3):223-236.
109. Hawkins BT, Davis TP: The Blood-Brain Barrier/Neurovascular Unit in Health and
Disease. Pharmacol Rev 2005, 57(2):173-185.
110. Brightman MW, Reese TS: Junctions between intimately apposed cell membranes in
the vertebrate brain. J Cell Biol 1969, 40(3):648-677.
111. Saunders NR, Ek CJ, Habgood MD, Dziegielewska KM: Barriers in the brain: a
renaissance? Trends in Neurosciences 2008, 31(6):279-286.
112. Saunders N, Knott G, Dziegielewska K: Barriers in the Immature Brain. Cellular and
Molecular Neurobiology 2000, 20(1):29-40.
113. Saunders N, Habgood MD, Dziegielewska K: Barrier mechanisms in the brain, II.
Immature brain. Clinical and Experimental Pharmacology and Physiology 1999, 26(2):85-91.
114. Saunders N, Habgood MD, Dziegielewska K: Barrier mechanisms in the brain, I. Adult
brain. Clinical and Experimental Pharmacology and Physiology 1999, 26(1):11-19.
115. Reese TS, Karnovsky MJ: Fine structural localization of a blood-brain barrier to
exogenous peroxidase. J Cell Biol 1967, 34(1):207-217.
116. Stewart PA: Endothelial Vesicles in the Blood–Brain Barrier: Are They Related to
Permeability? Cellular and Molecular Neurobiology 2000, 20(2):149-163.
117. Decleves X, Amiel A, Delattre J-Y, Scherrmann J-M: Role of ABC Transporters in the
Chemoresistance of Human Gliomas. Current Cancer Drug Targets 2006, 6:433-445.
118. Burkhard S, Heiko S: The Blood-brain Barrier and the Outer Blood-retina Barrier.
Medicinal Chemistry Reviews - Online 2005, 2:11-26.
98
119. Spector R: Thymidine transport and metabolism in choroid plexus: effect of diazepam
and thiopental. J Pharmacol Exp Ther 1985, 235(1):16-19.
120. Spector R, Boose B: Accumulation of Pantothenic Acid by the Isolated Choroid Plexus
and Brain Slices In Vitro. Journal of Neurochemistry 1984, 43(2):472-478.
121. Zeuthen T: Secondary active transport of water across ventricular cell membrane of
choroid plexus epithelium of Necturus maculosus. J Physiol 1991, 444(1):153-173.
122. Graff CL, Pollack GM: Drug Transport at the Blood-Brain Barrier and the Choroid
Plexus. Current Drug Metabolism 2004, 5:95-108.
123.
569.
Pardridge W: Blood-brain barrier biology and methodology. J Neurovirol 1999, 5:556–
124. Potschka H, Löscher W: Efflux Transporters in the Brain. In: Handbook of
Neurochemistry and Molecular Neurobiology. 2007: 461-483.
125. Taylor EM: The Impact of Efflux Transporters in the Brain on the Development of
Drugs for CNS Disorders. Clinical Pharmacokinetics 2002, 41:81-92.
126. Kabanov AV, Batrakova EV, Miller DW: Pluronic® block copolymers as modulators of
drug efflux transporter activity in the blood-brain barrier. Advanced Drug Delivery Reviews
2003, 55(1):151-164.
127. Tsuji A: Small Molecular Drug Transfer across the Blood-Brain Barrier via CarrierMediated Transport Systems. NeuroRX 2005, 2(1):54-62.
128. Deguchi T, Isozaki K, Yousuke K, Terasaki T, Otagiri M: Involvement of organic anion
transporters in the efflux of uremic toxins across the bloodbrain barrier. Journal of
Neurochemistry 2006, 96:1051-1059.
129. Sun J-j, Xie L, Liu X-d: Transport of carbamazepine and drug interactions at bloodbrain barrier. Acta Pharmacologica Sinica 2006, 27:249-253.
130. Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A, Meier PJ: Organic AnionTransporting Polypeptides Mediate Transport of Opioid Peptides across Blood-Brain
Barrier. J Pharmacol Exp Ther 2000, 294(1):73-79.
131. Löscher W, Potschka H: Role of drug efflux transporters in the brain for drug
disposition and treatment of brain diseases. Progress in Neurobiology 2005, 76(1):22-76.
132. Löscher W, Potschka H: Blood-Brain Barrier Active Efflux Transporters: ATPBinding Cassette Gene Family. NeuroRX 2005, 2(1):86-98.
133. Engelhardt B: Molecular mechanisms involved in T cell migration across the blood–
brain barrier. Journal of Neural Transmission 2006, 113(4):477-485.
99
134. Fischer H, Gottschlich R, Seelig A: Blood-Brain Barrier Permeation: Molecular
Parameters Governing Passive Diffusion. Journal of Membrane Biology 1998, 165(3):201-211.
135. Ikumi Tamai AT: Transporter-mediated permeation of drugs across the blood-brain
barrier. Journal of Pharmaceutical Sciences 2000, 89(11):1371-1388.
136. Banks WA, Kastin AJ, Ehrensing CA: Endogenous peptide Tyr-Pro-Trp-Gly-NH2 (TyrW-MIF-1) is transported from the brain to the blood by peptide transport system-1. Journal
of Neuroscience Research 1993, 35(6):690-695.
137. Kumagai AK, Eisenberg JB, Pardridge WM: Absorptive-mediated endocytosis of
cationized albumin and a beta- endorphin-cationized albumin chimeric peptide by isolated
brain capillaries. Model system of blood-brain barrier transport. Journal of Biological
Chemistry 1987, 262(31):15214-15219.
138. Sai Y, Kajita M, Tamai I, Wakama J, Wakamiya T, Tsuji A: Adsorptive-mediated
endocytosis of a basic peptide in enterocyte-like Caco-2 cells. Am J Physiol Gastrointest Liver
Physiol 1998, 275(3):G514-520.
139. Mahajan SD, Aalinkeel R, Sykes DE, Reynolds JL, Bindukumar B, Adal A, Qi M, Toh J,
Xu G, Prasad PN et al: Methamphetamine alters blood brain barrier permeability via the
modulation of tight junction expression: Implication for HIV-1 neuropathogenesis in the
context of drug abuse. Brain Research 2008, 1203:133-148.
140. Mahajan S, Aalinkeel R, Sykes D, Reynolds J, Bindukumar B, Fernandez S, Chawda R,
Shanahan T, Schwartz S: Tight Junction Regulation by Morphine and HIV-1 Tat Modulates
Blood–Brain Barrier Permeability. Journal of Clinical Immunology 2008, 28(5):528-541.
141. Béduneau A, Saulnier P, Benoit J-P: Active targeting of brain tumours using
nanocarriers. Biomaterials 2007, 28(33):4947-4967.
142. Bareford LM, Swaan PW: Endocytic mechanisms for targeted drug delivery. Advanced
Drug Delivery Reviews 2007, 59(8):748-758.
143. Liu H-M, Liu X-F, Yao J-L, Wang C-L, Yu Y, Wang R: Utilization of Combined
Chemical Modifications to Enhance the Blood-Brain Barrier Permeability and
Pharmacological Activity of Endomorphin-1. J Pharmacol Exp Ther 2006, 319(1):308-316.
144. Dollinger S, Lober S, Klingenstein R, Korth C, Gmeiner P: A Chimeric Ligand Approach
Leading to Potent Antiprion Active Acridine Derivatives:&nbsp; Design, Synthesis, and
Biological Investigations. Journal of Medicinal Chemistry 2006, 49(22):6591-6595.
145. Tabernero A, Velasco A, Granda B, Lavado EM, Medina JM: Transcytosis of Albumin in
Astrocytes Activates the Sterol Regulatory Element-binding Protein-1, Which Promotes the
Synthesis of the Neurotrophic Factor Oleic Acid. J Biol Chem 2002, 277(6):4240-4246.
146. Fiori A, Cardelli P, Negri L, Savi MR, Strom R, Erspamer V: Deltorphin transport across
the blood brain barrier. Proceedings of the National Academy of Sciences of the United States of
America 1997, 94(17):9469-9474.
100
147. Thomas SA, Abbruscato TJ, Hau VS, Gillespie TJ, Zsigo J, Hruby VJ, Davis TP:
Structure-Activity Relationships of a Series of [D-Ala2]Deltorphin I and II Analogues; in
Vitro Blood-Brain Barrier Permeability and Stability. J Pharmacol Exp Ther 1997,
281(2):817-825.
148. Kreuter J, Shamenkov D, Petrov V, Ramge P, Cychutek K, Koch-Brandt C, Alyautdin R:
Apolipoprotein-mediated Transport of Nanoparticle-bound Drugs Across the Blood-Brain
Barrier. Journal of Drug Targeting 2002, 10:317-325.
149. Kreuter J: Influence of the Surface Properties on Nanoparticle-Mediated Transport of
Drugs to the Brain. Journal of Nanoscience and Nanotechnology 2004, 4:484-488.
140. Liu G, Men P, Harris PLR, Rolston RK, Perry G, Smith MA: Nanoparticle iron
chelators: A new therapeutic approach in Alzheimer disease and other neurologic disorders
associated with trace metal imbalance. Neuroscience Letters 2006, 406(3):189-193.
151. Kim H, Gil S, Andrieux K, Nicolas V, Appel M, Chacun H, Desma, le D, Taran F, Georgin
D et al: Low-density lipoprotein receptor-mediated endocytosis of PEGylated nanoparticles
in rat brain endothelial cells. Cellular and Molecular Life Sciences CMLS 2007, 64:356-364.
152. Kratzer I, Wernig K, Panzenboeck U, Bernhart E, Reicher H, Wronski R, Windisch M,
Hammer A, Malle E, Zimmer A et al: Apolipoprotein A-I coating of protamine-oligonucleotide
nanoparticles increases particle uptake and transcytosis in an in vitro model of the bloodbrain barrier. Journal of Controlled Release 2007, 117(3):301-311.
153. Petri B, Bootz A, Khalansky A, Hekmatara T, Müller R, Uhl R, Kreuter J, Gelperina S:
Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly(butyl
cyanoacrylate) nanoparticles: Revisiting the role of surfactants. Journal of Controlled Release
2007, 117(1):51-58.
154. Kreuter J, Hekmatara T, Dreis S, Vogel T, Gelperina S, Langer K: Covalent attachment of
apolipoprotein A-I and apolipoprotein B-100 to albumin nanoparticles enables drug
transport into the brain. Journal of Controlled Release 2007, 118(1):54-58.
155. Murata H, Futami J, Kitazoe M, Yonehara T, Nakanishi H, Kosaka M, Tada H, Sakaguchi
M, Yagi Y, Seno M et al: Intracellular Delivery of Glutathione S-transferase-fused Proteins
into Mammalian Cells by Polyethylenimine-Glutathione Conjugates. J Biochem 2008,
144(4):447-455.
156. Lu W, Sun Q, Wan J, She Z, Jiang X-G: Cationic Albumin-Conjugated Pegylated
Nanoparticles Allow Gene Delivery into Brain Tumors via Intravenous Administration.
Cancer Res 2006, 66(24):11878-11887.
157. dos Santos W, Rahman J, Klein N, Male D: Distribution and analysis of surface charge
on brain endothelium in vitro and in situ. Acta Neuropathologica 1995, 90(3):305-311.
158. Afergan E, Epstein H, Dahan R, Koroukhov N, Rohekar K, Danenberg HD, Golomb G:
Delivery of serotonin to the brain by monocytes following phagocytosis of liposomes. Journal
of Controlled Release 2008, 132(2):84-90.
101
159. Chen X, Schluesener HJ: Nanosilver: A nanoproduct in medical application. Toxicology
Letters 2008, 176(1):1-12.
160. Borm P, Robbins D, Haubold S, Kuhlbusch T, Fissan H, Donaldson K, Schins R, Stone V,
Kreyling W, Lademann J et al: The potential risks of nanomaterials: a review carried out for
ECETOC. Particle and Fibre Toxicology 2006, 3(1):11.
161. Hagens WI, Oomen AG, de Jong WH, Cassee FR, Sips AJAM: What do we (need to)
know about the kinetic properties of nanoparticles in the body? Regulatory Toxicology and
Pharmacology 2007, 49(3):217-229.
162. Kreyling WG: Translocation and accumulation of nanoparticles in secondary target
organs after uptake by various routes of intake. Toxicology Letters 2006, 164(Supplement
1):S34-S34.
163. Kreyling W: Parameters of nanoparticles determining distribution and accumulation
in secondary target organs. Toxicology Letters 2007, 172(Supplement 1):S35-S35.
164. Kreyling WG, Semmler-Behnke M, Moller W: Ultrafine Particle–Lung Interactions:
Does Size Matter? Journal of Aerosol Medicine 2006, 19(1):74-83.
165. Tian F, Cui D, Schwarz H, Estrada G, Kobayashic H: Cytotoxicity of single-wall carbon
nanotubes on human fibroblasts. Toxicology in vitro 2006, 20(7): 1202-1212
166. Wiebert P, Sanchez-Crespo A, Seitz J, Falk R, Philipson K, Kreyling WG, Moller W,
Sommerer K, Larsson S, Svartengren M: Negligible clearance of ultrafine particles retained in
healthy and affected human lungs. Eur Respir J 2006, 28(2):286-290.
167. Beyerle A, Höbel S, Czubayko F, Schulz H, Kissel T, Aigner A, Stoeger T: In vitro
cytotoxic and immunomodulatory profiling of low molecular weight polyethylenimines for
pulmonary application. Toxicology in Vitro 2009, 23(3):500-508.
168. Stoeger T, Reinhard C, Takenaka S, Schroeppel A, Karg E, Ritter B, Heyder J, Schulz H:
Instillation of six different ultrafine carbon particles indicates a surface area threshold dose
for acute lung inflammation in mice. Environ Health Perspect 2006 114(3):328-333.
169. Stoeger T, Takenaka S, Frankenberger B, Ritter B, Karg E, Maier K, Schulz H, Schmid O:
Deducing In Vivo Toxicity of Combustion-Derived Nanoparticles from a Cell-Free Oxidative
Potency Assay and Metabolic Activation of Organic Compounds. Environ Health Perspect
2009, 117:54-60.
170. Brodeur G: Molecular Basis For Heterogeneity in Human Neuroblastomas. Eur J
Cancer 1995, 31(A):505-510.
171. Ntziachristos V, Bremer C, Weissleder R: Fluorescence imaging with near-infrared
light: new technological advances that enable in vivo molecular imaging. Eur Radiol 2003,
13(1):195–208.
102
171. Rabin O, Manuel Perez J, Grimm J, Wojtkiewicz G, Weissleder R: An X-ray computed
tomography imaging agent based on long-circulating bismuth sulphide nanoparticles. Nat
Mater 2006, 5(2):118-122.
172. Maier-Hauff K, Rothe R, Scholz R, Gneveckow U, Wust P, Thiesen B, Feussner A, von
Deimling A, Waldoefner N, Felix R et al: Intracranial Thermotherapy using Magnetic
Nanoparticles Combined with External Beam Radiotherapy: Results of a Feasibility Study
on Patients with Glioblastoma Multiforme. Journal of Neuro-Oncology 2007, 81(1):53-60.
173. Alonzi R, Hoskin P: Functional Imaging in Clinical Oncology: Magnetic Resonance
Imaging- and Computerised Tomography-based Techniques. Clinical Oncology 2006,
18(7):555-570.
174. Leergaard TB, Bjaalie JG, Devor A, Wald LL, Dale AM: In vivo tracing of major rat
brain pathways using manganese-enhanced magnetic resonance imaging and threedimensional digital atlasing. NeuroImage 2003, 20(3):1591-1600.
175. Sun C, Lee JSH, Zhang M: Magnetic nanoparticles in MR imaging and drug delivery.
Advanced Drug Delivery Reviews 2008, 60(11):1252-1265.
176. Liu W, Frank JA: Detection and quantification of magnetically labeled cells by cellular
MRI. European Journal of Radiology 2009, In Press.
177. Feng B, Hong RY, Wu YJ, Liu GH, Zhong LH, Zheng Y, Ding JM, Wei DG: Synthesis of
monodisperse magnetite nanoparticles via chitosan-poly(acrylic acid) template and their
application in MRI. Journal of Alloys and Compounds 2009, 473(1-2):356-362.
178. Suci PA, Berglund DL, Liepold L, Brumfield S, Pitts B, Davison W, Oltrogge L, Hoyt KO,
Codd S, Stewart PS et al: High-Density Targeting of a Viral Multifunctional Nanoplatform to
a Pathogenic, Biofilm-Forming Bacterium. Chemistry & Biology 2007, 14(4):387-398.
179. Fiandaca MS, Varenika V, Eberling J, McKnight T, Bringas J, Pivirotto P, Beyer J,
Hadaczek P, Bowers W, Park J et al: Real-time MR imaging of adeno-associated viral vector
delivery to the primate brain. NeuroImage 2009, In Press.
180. Urakami T, Kawaguchi AT, Akai S, Hatanaka K, Koide H, Shimizu K, Asai T, Fukumoto
D, Harada N, Tsukada H, Oku N: In Vivo Distribution of Liposome-Encapsulated Hemoglobin
Determined by Positron Emission Tomography. Artificial Organs 2009, 33(2):164-168.
181. Plotkin M, Gneveckow U, Meier-Hauff K, Amthauer H, Feußner A, Denecke T,
Gutberlet M, Jordan A, Felix R, Wust P: 18F-FET PET for planning of thermotherapy using
103
magnetic nanoparticles in recurrent glioblastoma. International Journal of Hyperthermia 2006,
22(4):319 - 325.
182. Ntziachristos V, Ripoll J, Wang LV, Weissleder R: Looking and listening to light: the
evolution of whole-body photonic imaging. Nat Biotech 2005, 23(3):313-320.
183. Achilefu S, Dorshow RB, Bugaj JE, Rajagopalan R: Novel receptor-targeted fluorescent
contrast agents for in vivo tumor imaging. Invest Radiol 2000, 35(8):479-485.
184. Bugaj JE, Achilefu S, Dorshow RB, Rajagopalan R: Novel fluorescent contrast agents for
optical imaging of in vivo tumors based on a receptor-targeted dye-peptide conjugate
platform. Journal of Biomedical Optics 2001, 6(2):122-133.
185. Weissleder R, Tung C-H, Mahmood U, Bogdanov A: In vivo imaging of tumors with
protease-activated near-infrared fluorescent probes. Nat Biotech 1999, 17(4):375-378.
186. Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature 452 (7187):
580–9 (2008).
187. Ntziachristos V, Yodh AG, Schnall MD, Chance B. MRI-Guided Diffuse Optical
Spectroscopy of Malignant and Benign Breast Lesions. Neoplasia 4(4): 347–354 (2002).
188. Grimm J, Kirsch DG, Windsor SD, Kim CF, Santiago PM, Ntziachristos V, Jacks T,
Weissleder R. Use of gene expression profiling to direct in vivo molecular imaging of lung
cancer. Proc Natl Acad Sci. 102(40): 14404-14409 (2005).
189. Ntziachristos V., Tung C.-H., Bremer C., and Weissleder R. Fluorescence molecular
tomography resolves protease activity in vivo. Nature Med. 8, 757-760 (2002).
190. Sosnovik DE, Nahrendorf M., Deliolanis N., Novikov M., Aikawa E., Josephson L.,
Rosenzweig A., Weissleder R., Ntziachristos V. Fluorescence tomography and magnetic
resonance imaging of myocardial macrophage infiltration in infarcted myocardium in vivo.
Circulation 115, 1384-1391 (2007).
191. Ntziachristos V, Schellenberger EA, Ripoll J, Yessayan D, Graves E, Bogdanov A,
Josephson L, Weissleder R: Visualization of antitumor treatment by means of fluorescence
molecular tomography with an annexin V-Cy5.5 conjugate. Proceedings of the National
Academy of Sciences of the United States of America 2004, 101(33):12294-12299.
192. Corlu A, Choe R, Durduran T, Rosen MA, Schweiger M, Arridge SR, Schnall MD, Yodh
AG: Three-dimensional in vivo fluorescence diffuse optical tomography of breast cancer in
humans. Opt Express 2007, 15(11):6696-6716.
104
193. Graves E, Ripoll J, Weissleder R, Ntziachristos V: A submillimeter resolution
fluorescence molecular imaging system for small animal imaging. Med Phys 2003, 30(5):901911.
194. Weissleder R, Ntziachristos V: Shedding light onto live molecular targets. Nat Med
2003, 9(1):123-128.
195. Ntziachristos V., Leroy-Willig A., Tavitian B. (Editors). Textbook of in vivo Imaging in
Vertebrates, Wiley, NY (2007).
196. F Helmchen, W Denk, Deep tissue two-photon microscopy, Nature Methods, (2005).
197. Huisken, J. et al. Optical sectioning deep inside live embryos by selective plane
illumination microscopy. Science 305, 1007 (2004).
198. Ntziachristos V, Weissleder R: Experimental three-dimensional fluorescence
reconstruction of diffuse media by use of a normalized Born approximation. Opt Lett 2001,
26(12):893-895.
199. Ntziachristos V, Bremer C, Weissleder R: Fluorescence imaging with near-infrared
light: new technological advances that enable in vivo molecular imaging. Eur Radiol 2003,
13(1):195–208.
200. Deliolanis et al., Free-space fluorescence molecular tomography utilizing 360 geometry
projections, Optics Letters 32(4), pp. 382-384 (2007).
201. Ntziachristos V, Tung C-H, Bremer C, Weissleder R: Fluorescence molecular
tomography resolves protease activity in vivo. Nat Med 2002, 8(7):757-761.
202. Zacharakis G, Kambara H, Shih H, Ripoll J, Grimm J, Saeki Y, Weissleder R, Ntziachristos
V: Volumetric tomography of fluorescent proteins through small animals in vivo. Proc Natl
Acad Sci U S A 2005, 102(51):18252–18257.
203. Vinegoni C, Pitsouli C, Razansky D, Perrimon N, Ntziachristos V: In vivo imaging of
Drosophila melanogaster pupae with mesoscopic fluorescence tomography. Nat Meth 2008,
5(1):45-47.
204. Tian F, Razansky D, Estrada G,Semmler-Behnke M, Beyerle A,Kreyling W, Ntziachristos
V, Stoeger T. Surface modification and size dependence in particle translocation during early
embryonic development. Inhalation Toxicology 2009 In press.
205. Hyde D, de Kleine R, MacLaurin SA, Miller E, Brooks DH, Krucker T, Ntziachristos V:
Hybrid FMT-CT imaging of amyloid-[beta] plaques in a murine Alzheimer's disease model.
NeuroImage 2009, 44(4):1304-1311.
206. McCann CM, Waterman P, Figueiredo J-L, Aikawa E, Weissleder R, Chen JW: Combined
magnetic resonance and fluorescence imaging of the living mouse brain reveals glioma
response to chemotherapy. NeuroImage 2009, 45(2):360-369.
105
207. Ntziachristos V, Ma X, Chance B: Time-correlated single photon counting imager for
simultaneous magnetic resonance and near-infrared mammography. Review of Scientific
Instruments 1998, 69(12):4221-4233.
208. Razansky, D. & Ntziachristos, V. Hybrid photoacoustic fluorescence molecular
tomography using finite-element-based inversion. Med. Phys. 34, 4293-4301 (2007).
209.
Wang X, Pang Y, Ku G, Xie X, Stoica G, Wang LV: Noninvasive laser-induced
photoacoustic tomography for structural and functional imaging of the brain Nature
Biotechnology 2003, 21: 803-806.
210. Zhang HF, Maslov K, Stoica G, Wang V:Functional photoacoustic microscopy for highresolution and noninvasive imaging. Nature Biotechnology 2006, 24: 848-851.
211. Razansky D, Distel M, Vinegoni C, Ma R, Perrimon N, Koster RW, Ntziachristos V:
Multispectral opto-acoustic tomography of deep-seated fluorescent proteins in vivo. Nature
Photonics 2009.
212 RazanskyD, VinegoniC, Ntziachristos V: Imaging of mesoscopic-scale organisms using
selective-plane optoacoustic tomography. Physics in medicine and biology. 2009, 54(9): 27692777.
213. Rayavarapu RG, Petersen W, Ungureanu C, Post JN, van Leeuwen TG, Manohar S:
Synthesis and Bioconjugation of Gold Nanoparticles as Potential Molecular Probes for LightBased Imaging Techniques. International Journal of Biomedical Imaging 2007.
214.
Li L, Zemp RG, Lungu G, Stoica G, Wang L: Photoacoustic imaging of lacZ gene
expression in vivo. Journal of Biomedical Optics 2007, 12: 020504
215. de la Zerda A, Zavaleta C, Keren S, Vaithilingam S, Bodapati S, Liu Z, Levi J, Ma T-J,
Oralkan O, Cheng Z, Chen X, Dai H, Khuri-Yakub BT, Gambhir SS. Photoacoustic Molecular
Imaging in Living Mice Utilizing Targeted Carbon Nanotubes. Nature Nanotech. 3, 557-62
(2008).
216 Razansky D, Vinegoni C, and Ntziachristos V. Multispectral photoacoustic imaging of
fluorochromes in small animals Optics Letters 2007, 32: 289-293.
217 RazanskyD, Baeten J, Ntziachristos V: Sensitivity of molecular target detection by
multispectral optoacoustic tomography (MSOT). Medical physics. 2009, 36(3): 939-945.
218. Jagger C, Gillies C, Moscone F, Cambois E, Van Oyen H, Nusselder W, Robine JM:
Inequalities in healthy life years in the 25 countries of the European Union in 2005: a crossnational meta-regression analysis Lancet 2008, 372(9656):2124-2131.
219. Begley DJ: The blood-brain barrier: principles for targeting peptides and drugs to the
central nervous system. J Pharm Pharmacol 1996, 48(2):136-46.
220. Bradbury M, Begley DJ, Kreuter J (Eds): The Blood-Brain Barrier and Drug Delivery to
the CNS Informa Healthcare, USA 2000.
106
221. Begley D, Brightman M: Structural and functional aspects of the blood-brain barrier.
Prog Drug Res 2003, 61:39-78.
222. Ohtsuki S, Terasaki T: Contribution of Carrier-Mediated Transport Systems to the
Blood–Brain Barrier as a Supporting and Protecting Interface for the Brain; Importance for
CNS Drug Discovery and Development. Pharmaceutical Research 2007, 24(9):1745-1758.
223. Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline
evolving from studies of ultrafine particles. Environ Health Perspect. 2005 Jul;113(7):823-39.
224. Nel AE, Mädler L, Velegol D, Xia T, Hoek EM, Somasundaran P, Klaessig F, Castranova V,
Thompson M. Understanding biophysicochemical interactions at the nano-bio interface. Nat
Mater. 2009 Jul;8(7):543-57.
225. Nel A, Xia T, Mädler L, Li N. Toxic potential of materials at the nanolevel. Science. 2006
Feb 3;311(5761):622-7.
107
Figure legends
Figure 1: Schematic representation of a multifunctional NP for diagnostics and drug delivery.
Polyethylene glycol (PEG) copolymers are one of the most popular vehicles for drug delivery. The
NPs can be functionalized with suitable fluorescent markers, antibodies against tumoural marker,
gene delivery agents and drug molecules coated with a form of PEG. The antibody is using a long
linking molecule that allows the antibody to stick to PEG coatings. In contrast, cell penetrating
peptides (CPP), employed to trigger rapid cell uptake, are attached using a short linkers.
Figure 2: Mouse tumour models [38], the Xenograft and genetically engineered mouse model
(GEMM). In Xenograft mouse models, cancer cells are generally injected subcutaneously into
immunodeficient mice. Oncogenes in GEMM are activated and/or tumour-suppressor genes
(TSGs) are inactivated somatically.
Figure 3: Overview of the two main barriers in the CNS: blood-brain barrier and blood
cerebrospinal fluid barrier (BCSF). ISF: Interstitial Fluid. CSF: Cerebrospinal fluid. Adapted from
[219, 220].
Figure 4: Potential transport mechanisms across BBB (Adapted from [221, 222]).
Figure 5: Mechanisms of drug transport through the BBB using nanocarriers conjugated to
receptor-specific ligands and cationized ligands; (1) Receptor-mediated endocytosis of the
nanocarrier; (1a) Exocytosis of the nanocarrier; (1b) Dissociation of the receptor from the ligandconjugated nanocarrier and acidification of the vesicle leading to the degradation of the nanocarrier
and the release of the drug into the brain; (1c and 1d) Recycling of receptors at the luminal
cytoplasmic membrane; (2a) Adsorptive-mediated endocytosis of the nanocarrier conjugated to
Cationized ligands; (2b) Exocytosis of positively charged nanocarriers. (Adapted from [141]).
Figure 6 : Fate of drug released from the ‘Nanocarrier’ systems into the brain (Adapted from
[170]).
Figure 7: (a) Schematic of free-space 360 degree projection FMT imaging system (from [200]). (b
and c) FPT of lung cancer in imaging tumour growth and progression and the corresponding x-ray
CT images. (Adapted from [202]). (d, e, f, g, h and I ) In vivo FMT of cathepsin B expression
levels. (d and e), Axial and sagittal MR slices, shown in green after gadolinium enhancement. (f, g
108
and I) Consecutive FMT slices obtained from the volume of interest shown on e by thin white
horizontal lines. (h) Superposition of the MR axial slice passing through the tumour d onto the
corresponding FMT slice f after appropriate translation of the MR image to the actual dimensions
of the FMT image (Adapted from [201]). (j) Overview of the experimental configuration of MFT
(Adapted from [203]).
Figures
109
Figure 1.
Figure 2
110
Figure 3.
111
Figure 4.
Figure 5.
112
Figure 6.
113
Figure 7.
114