International Conference on
Nanotheranostics
ICoN 2013
Conference Program and Summaries
26-28 September 2013
Golden Bay Beach Hotel
Larnaca, Cyprus
Organizers
European Union
http://europa.eu/index_en.htm
FP7
http://www.cordis.europa.eu/fp7
Marie Curie Actions - Industry Academia Partnerships and Pathways
http://ec.europa.eu/research/mariecurieactions/about-mca/actions/iapp/
European Federation of Biotechnology
http://www.efb-central.org
Cyprus Medical Association
http://www.cyma.org.cy
EPOS-Iasis Research & Development Ltd
http://www.epos-iasis.com
University of Cyprus
http://www.ucy.ac.cy
Cyprus University of Technology
http://www.cut.ac.cy
1 | 2013 International Conference on Nanotheranostics (ICoN 2013)
EUROPEAN FEDERATION
BIOT E CH NOLO G
Section on Medicines Developm
Contents
Welcome .................................................................................................................................. 3
Committees .............................................................................................................................. 5
Keynote Speakers .................................................................................................................... 6
Program at a glance ................................................................................................................. 7
Sessions ................................................................................................................................... 8
Thursday 26/9/2013............................................................................................................................. 8
Plenary Session ................................................................................................................................ 8
Session 1 – Imaging and Characterization of Nanotheranostic Agents .......................................... 8
Session 2 – Novel Nanotheranostic Approaches ............................................................................. 9
Poster Session and Reception ......................................................................................................... 9
Friday 27/9/2013 ................................................................................................................................ 10
Session 3 – Biocompatibility and Toxicity of Nanotheranostic Agents ......................................... 10
Session 4 – Drug Delivery to Solid Tumors and Through Barriers ................................................ 10
Session 5 – Delivery of Nanotheranosis ........................................................................................ 11
Cultural Activities and Conference Dinner .................................................................................... 11
Saturday 28/9/2013 ........................................................................................................................... 12
Session 6 – Cancer Theranostics ................................................................................................... 12
Short Course: Nanotheranostics: all-in-one personalized medicine ............................................. 12
Session 7 – Materials and Tissues in Nanotheranostics ................................................................ 13
Round Table Discussion and Student Awards ............................................................................... 13
Abstracts ................................................................................................................................ 14
Author Index ........................................................................................................................... 67
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2 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Welcome
Dear Delegates of the ICoN2013 Conference,
It is a great pleasure and an extreme honor to welcome all of you
to Cyprus on the occasion of the 2013 International Conference
on Nanotheranostics.
In the vast field of nanomedicine, “Theranostics” combine
therapeutics and diagnostics, aiming to provide a comprehensive
platform for diagnosis, therapy and monitoring of the patient,
leading to customized approaches and personalized treatment.
Emerging nanotechnology discoveries provide a unique
opportunity to design and develop such combination agents,
permitting the delivery of therapeutics and concurrently allowing the detection modality to be
used not only before or after but also throughout the entire treatment regimen, defining new
supra-disciplinary fields in major clinical specialties such as Radiology, Surgery,
Gynaecology and Oncology, to mention few. That is why a Nanotheranostics Conference is
an important initiative and it is expected to provide the forum for idea exchange and
discussion to create a potential high-impact nanomedicine paradigm. The ICoN 2013
Conference aims to provide the optimal venue to expand nanotheranostics research in a
multidisciplinary environment which will bring together all the key researchers in the
nanotheranostics field.
International scientific events at the interface between cutting edge biotechnology and clinical
translational research, through such an undertaking, are highly significant since they define
in the most appropriate way the contribution of medical, pharmaceutical, scientific and
academic entities towards the national and pan-European planning for knowledge- and
innovation-based economies. Within the framework of the EC-FP7 Marie-Curie, IndustryAcademia Pathways and Partnership Program, the technology transfer entity EPOS-Iasis,
R&D, the University of Cyprus, the Cyprus University of Technology, the European
Federation of Biotechnology and the Cyprus Medical Association, have joined forces towards
a trans-disciplinary event that is expected to pave the way towards effective collaborations in
translational nanomedicine.
ICoN2013 brings together high-caliber researchers, pioneers in the field and a promising
population of young scientists at the dawn of their career, from across Europe, USA, Asia
and Africa. It combines thoughtfully topic-oriented plenary and keynote lectures with
innovative research papers. Major thematic areas include (i) the roadmap of
nanotheranostics development in a patient-oriented approach, (ii) emerging challenges for
nanotheranostic applications, (iii) toxicology, regulatory aspects and ethics and, most
importantly (iv) an emphasis session on cancer nanotheranostics. A specifically targeted and
specially designed seminar for clinicians is featured, thus underlining the fact that
nanotheranostics are currently mature enough for strategic clinical applications. It is,
therefore, evident that the establishment of the International Conference on
Nanotheranostics promises exciting new pathways towards clinical and research excellence
and we expect that ICoN 2013 will set the grounds for the establishment of a lasting intersectoral and trans-disciplinary network on novel diagnostics and therapeutics both locally
and on an international dimension.
Cyprus, an island of nano-scale dimensions from a global prospective, but with tremendous
potentials when it comes to creativity and effectiveness, is certainly the place for a
Nanotheranostics Conference to be. I take the opportunity to wholeheartedly express the
gratitude of the Organizing Committee and myself to His Excellency the President of the
3 | 2013 International Conference on Nanotheranostics (ICoN 2013)
House of Representatives of the Republic of Cyprus, Mr Yiannakis Omirou who, having
acknowledged the significance and the anticipated impact of such an event, has put
ICoN2013 under his auspices. This event would not have been possible without the endless
efforts of all vice – chairs and members of the Organizing Committee nor without the input of
the renowned members of the Scientific Committee. I feel deeply honored by their
collaboration.
Cyprus, a sunbathed island, the birthplace of Aphrodite and the key passage to Europe, has
been an excellent example of hardships and endurance throughout its ten-thousand year
history. Being at the crossroads of world’s civilizations it has assimilated all passing traits of
culture onto its main Hellenic trunk. While at ICoN2013 you are strongly encouraged to take
the unique opportunity to experience the turbulent, but still exciting, history of Cyprus and
enjoy the warm hospitality of its people.
I wish you all a rewarding experience and ICoN2013 and I am looking forward to welcoming
you at ICoN2015.
Andreani D Odysseos
General Chair
Costas Pitris
Vice Chair
George Potamitis
Vice Chair
4 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Committees
General Chair:
Vice Chairs:
Andreani Odysseos, European Federation of Biotechnology, EPOSIasis Research and Development Ltd
Costas Pitris, University of Cyprus
George Potamitis, Cyprus Medical Association
Local Organizing Committee
Co-Chairs:
Treasurer:
Secretary:
Social Activities:
Andreas Anayiotos, Cyprus Technological University
Triantafyllos Stylianopoulos, University of Cyprus
Theodora Krasia, University of Cyprus
Costas Pitsillides, Cyprus Technological University
Maria Kokonou, EPOS-Iasis Research and Development Ltd
Scientific Committee
Bogos (Pavlos) Agianian, Democritus University of Thrace, Greece
Andreas Anayiotos, Cyprus Technological University
Rena Bizios, University of Texas, USA
Kenneth A. Dawson, University College Dublin, Ireland
Haris Doumanidis, University of Cyprus, Cyprus
Kostas Kostarelos, UCL School of Pharmacy, UK
Theodora Krasia, University of Cyprus
Jan Mollenhauer, University of Southern Denmark, Denmark
Costas Pitris, University of Cyprus
Claus Rebholz, University of Cyprus
Jean-Michel Siaugue, Université Pierre et Marie Curie, France
Alex Strongilos, Proactina SA, Greece
Triantafyllos Stylianopoulos, University of Cyprus
Chrysa Tziakouri-Shiakalli, Cyprus Medical Association
5 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Keynote Speakers
A number of keynote presentations will be delivered by distinguished speakers in field. Below
is an initial partial list (in alphabetical order):
Bogos (Pavlos) Agianian
Democritus University of Thrace
Greece
Michael Averkiou
University of Cyprus
Cyprus
Rena Bizios
University of Texas at San Antonio
USA
Kenneth A. Dawson
University College Dublin
Ireland
George Kordas
NCSR Democritos
Greece
Kostas Kostarelos
UCL School of Pharmacy
United Kingdom
Jan Mollenhauer
University of Southern Denmark
Denmark
Jean-Michel Siaugue
Université Pierre et Marie Curie
France
Triantafyllos Stylianopoulos
University of Cyprus
Cyprus
6 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Program at a glance
International Conference on Nanotheranostics (ICoN 2013)
Thursday
26/9/2013
09.00-11.00
Welcome and
Plenary Session
11.00-11.30
11.30-13.30
16.45-17.15
17.15-18.30
Saturday
28/9/2013
Session 3
Session 6
Biocompatibility and
Toxicity of
Nanotheranostic
Agents
Cancer Theranostics
Coffee Break
Session1
Session 4
Imaging and
Characterization of
Nanotheranostic
Agents
Drug Delivery to Solid Nanomedicine
Tumors and Through
Barriers
13.30-14.30
14.30-16.45
Friday
27/9/2013
Short Course
Lunch Break
Session 2
Session 5
Session 7
Novel
Nanotheranostic
Approaches
Delivery of
Nanotheranosis
Materials and Tissues
in Nanotheranostics
Cultural / Social
Events
Coffee Break
Coffee Break
Poster Session
Roundtable
Discussion
Student Awards
20.00-23.00
Conference Dinner
7 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Sessions
Thursday 26/9/2013
Plenary Session
09.00-09.15
Welcome
Andreani Odysseos, Conference Chair, EFB, EPOS-Iasis R&D Ltd
09.15-09.30
Welcome by the Cyprus Medical Association
Dr. Andreas Demetriou, CyMA President
09.30-09.50
European Commission’s Marie Curie Program in Horizon 2020
Dr. Audrey Arfi, European Commission Scientific Oficcer
09.50-10.00
Opening by the President of the House of Representatives
H.E. Mr. Yiannakis Omirou
10.00-10.45
Opportunities Offered by Nucleic Acid-Based Nano-Devices in Cancer
Therapy and Theranostics
Jan Mollenhauer, Ines Block, Angela Riedel, Steffen Schmidt, Helle Christiansen, Birgitte
Brinkmann Olsen, Helge Thisgaard, Poul Flemming Hoilund-Carlsen, Stefan Vogel and
Jesper Wengel, University of Southern Denmark, Denmark (p. 15)
10.45-11.30
Nanoscale Interface Between Engineered Matter and Living
Organisms: Understanding the Biological Identity of Nanosized
Materials
Kenneth Dawson, UC Doublin, Ireland (p. 16)
11.30 – 11.45 Coffee Break
Session 1 – Imaging and Characterization of Nanotheranostic Agents
Session Chair: Pavlos Agianian, Democritus University of Thrace, Greece
11.45-12.35
Assessing biological specificity of nanoparticles in vitro: test tubes,
chips or cells
Pavlos Agianian, Democritus University of Thrace, Greece (p. 17)
12.35-12.55
In-vitro release study and dynamic in-vivo imaging of pH- and
Magnetic field sensitive hybrid microspheres
Eleni Efthimiadou, NCSR Demokritos, Greece (p. 18)
12.55-13.15
Preparation and characterization of new elements for the conception
of nanohybrids for multicolored bioimaging
Morgane Rivoal and Andreani Odysseos, EPOS Iasis R&D Ltd, Cyprus (p. 19)
13.15-13.35
Size Dependent Biological Profiles of Pegylated Gold Nanorods for
Biomedical Applications
Federica Scaletti, University of Florence, Italy (p. 20)
13.35 – 14.30 Lunch
8 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Session 2 – Novel Nanotheranostic Approaches
Session Chair: Jean-Michel Siaugue, UPMC, France
14.30-15.20
Fluorescent and magnetic silica core shell nanoparticles for
biomedical applications
Jean-Michel Siaugue, UPMC, France (p. 21)
15.20-15.40
Two different approaches for the synthesis of functionalized silica
coated magnetic nanoparticles
Ana B. Davila Ibañez, Verónica Salgueiriño and Jean-Michel Siaugue, UPMC, France (p. 22)
15.40-16.00
Theragnosis combining ferrocenyl derivatives, carbohydrates and
nanovectors
Jeremy Malinge, Jean-Michel Siaugue, Christine Ménager, Matthieu Sollogoub, Yongmin
Zhang, Gérard Jaouen and Anne Vessières-Jaouen, UPMC, France (p. 23)
16.00-16.20
Chitosan – linear aldehyde nanoparticles obtained from reverse
micellar method
Krzysztof Gawlik, Iga Wasiak and Tomasz Ciach, Warsaw University of Technology, Poland
(p. 24)
16.20-17.00
New Routes Towards the Synthesis of Natural Products and Designed
Derivatives
Elias Couladouros, Agricultural University of Athens, Greece (p. 26)
Poster Session and Reception
17.30-19.00
Session Chair: Dr. Costas Pitris, University of Cyprus, Cyprus
1
Hemocompatibility of Albumin microspheres as drug delivery system, in vitro
study
Mohamed Elblbesy, University of Tabuk, Saudi Arabia (p.27)
2
Hemocompatibility of silver nanoparticles
Julie Laloy, Valentine Minet, Lutfiye Alpan, Bernard Chatelain, François Mullier and Jean-Michel Dogné,
University of Namur, Belgium (p. 28)
3
From docking to synthesis and grafting: Preliminary results for new
Anilinoquinazolines as potential EGFR inhibitors in multifunctional nanocarriers
Fotini Liepouri, Pavlos Agianian, Vassiliki Garefalaki, Elias Couladouros, Andreani Odysseos and
Alexandros Strongilos, Proactina SA, Greece (p. 29)
4
Theoretical investigation of a new metal nanoparticle for combined imaging and
therapy applications
Myria Angelidou and Costas Pitris, University of Cyprus, Cyprus (p. 30)
5
Surface Enhanced Raman Spectroscopy (SERS) for Point-Of-Care Diagnosis of
Urinary Tract Infections
Katerina Hadjigeorgiou, Evdokia Kastanos and Costas Pitris, University of Cyprus, Cyprus (p. 32)
6
Activity, anti-cancer effect and nanodelivery of new anilinoquinazoline EGFR
inhibitors
Eftychia Angelou, Fotini Liepouri, Maria Pavlaki, Andreani Odysseos, Jean-Michel Siaugue, Alexandros
Strongilos and Pavlos Agianian. Democritus University of Thrace, Greece (p. 34)
9 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Friday 27/9/2013
Session 3 – Biocompatibility and Toxicity of Nanotheranostic Agents
Session Chair: Cyrill Bussy, University of Manchester, UK
09.00-09.50
Toxicity and Safety of Carbon Nanomaterials for Biomedical
Applications
Cyrill Bussy and Kostas Kostarelos, University of Manchester, UK (p. 35)
09.50-10.30
Guidelines proposal for studying hemocompatibility of manufactured
nanoparticles and impact on fibrinolysis
Julie Laloy, Lutfiye Alpan, Valentine Minet and Jean-Michel Dogné, University of Namur,
Belgium (p. 34)
10.30-10.50
Rat whole-body exposure model to nanoaerosol: Development with
silicon carbide nanoparticles and study of their toxicity
Julie Laloy, Omar Lozano, Lutfiye Alpan, Valentine Minet, Olivier Toussaint, Bernard
Masereel, Jean-Michel Dogné and Stephane Lucas, University of Namur, Belgium (p. 37)
11.00-11.30 Coffee Break
Session 4 – Drug Delivery to Solid Tumors and Through Barriers
Session Chair: Triantafyllos Stylianopoulos, University of Cyprus, Cyprus
11.30-12.20
EPR-effect: a barrier to the effective delivery of large nanomedicines
to solid tumors
Triantafyllos Stylianopoulos, University of Cyprus, Cyprus (p. 39)
12.20-12.40
Strategies to improve nanomedicine delivery to solid tumors
Konstantinos Soteriou, Eva-Athena Economides and Triantafyllos Stylianopoulos, University
of Cyprus, Cyprus (p. 40)
12.40-13.00
Methods for delivery of living cells to the respiratory system via
aerosol route
Tomasz R. Sosnowski, Ewelina Tomecka, Warsaw University of Technology, Poland (p. 42)
13.00-13.20
Enhancement of Drug Absorption Across Intestinal Membrane Using
Magnetic Beads
Anjali Seth, David Lafargue and Christine Ménager, UPMC, France (p. 44)
13.30-14.30 Lunch
10 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Session 5 – Delivery of Nanotheranosis
Session Chair: Mike Averkiou, University of Cyprus, Cyprus
14.30-15.20
Ultrasound and microbubbles for monitoring therapies targeting
tumor vascularity
Mike Averkiou, University of Cyprus, Cyprus (p. 46)
15.20-15.40
Tunable magnetic/ICG small protocells as a platform for drug delivery
Jean-Sébastien Thomann, Gaëlle Corne, Didier Arl, Naoufal Bahlawane and Damien
Lenoble, CRP Gabriel lippmann, Luxemburg (p. 47)
15.40-16.00
Ultrasound-induced temperature elevation for in-vitro controlled
release of temperature-sensitive liposomes
Christophoros Mannaris, Jean-Michele Escoffre, Ayache Bouakaz, Marie-Edithe Meyre and
Michalakis Averkiou, University of Cyprus, Cyprus (p. 49)
16.00-16.20
Biodegradable Dextran Nanoparticles as Potential Drug and
Fluorescent Marker Carrier
Tomasz Ciach, Magdalena Janczewska and Iga Wasiak, Warsaw University of Technology,
Poland (p. 52)
16.20-16.40
Engineering carbon nanotubes based scaffolds for the efficient
delivery of new tyrosine kinase inhibitors
Davide Giust, Kostas Kostarelos, UCL School of Pharmacy, UK (p. 54)
16.45 – 17.15 Coffee Break
Cultural Activities and Conference Dinner
17.15-18.30
20.30-23.00
Cultural Activities
Conference Dinner
11 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Saturday 28/9/2013
Session 6 – Cancer Theranostics
Session Chair: Costas Pitsillides, Cyprus University of Technology, Cyprus
09.00-09.50
Folic Acid functionalized Quatro-NanoContainers as targeted agent: In
vitro and In vivo study
George Kordas, NCSR Demokritos (p. 55)
09.50-10.10
Doxorubicin-loaded and Antibody-Conjugated Liposome-QD Hybrid
Vesicles for Targeted Cancer Theranostics
Bowen Tian, Wafa Al-Jamal, Bowen Tian and Kostas Kostarelos, University of Manchester,
UK (p. 56)
10.10-10.30
Monitoring tumor burden by multicolor in vivo flow cytometry
Costas Pitsillides, Konstantinos Kapnisis and Andreas Anayiotos, Cyprus University of
Technology, Cyprus (p. 57)
10.30-10.50
Role of the Cell-Division Cycle on Nanoparticle Cellular Accumulation
and Implications for Cancer Targeting
Christoffer Åberg and Kenneth A. Dawson, UC Doublin, Ireland (p.59)
11.00-11.30 Coffee Break
Short Course: Nanotheranostics: all-in-one personalized medicine
Session Chairs: George Potamitis, Chrysa Tziakouri-Shiakalli, Cyprus Medical Association
This short course will provide an overview of the major concepts behind the newly created field of
nanotheranostics. Nanotheranostic agents have a number of significant advantages over current approaches: (i)
Nanotheranostic agents can be customized to the disease and personalized to the patient. (ii) Active targeting
and localization allows for better treatment with much less intense side effects compared to current regimens. (iii)
The integration of therapy and monitoring provides real-time information on whether or not the specific treatment
regimen is working for the specific patient. Given these attributes, it is not surprising that the field of
nanotheranostics is considered the future of treatment of highly inhomogeneous and variable diseases such as
cancer and chronic inflammatory disorders.
11.30-11.45
Introduction
Going to the lower limits: nanotechnology and nanomedicine
Theranostics: all-in-one personalized medicine
Andreani Odysseos, EFB, EPOS-Iasis R&D Ltd, Cyprus
11.45-12.05
Theranostic Nanoparticles
Rena Bizios, University of Texas at San Antonio, USA
12.05-12.25
Clinical applicability of Optical Imaging
Costas Pitris, University of Cyprus, Cyprus
12.25-12.45
Nanotheranostics at the clinical fore
Image-guided therapy: paving the way of nanotheranostic agents to
the clinic
Monitoring therapy by imaging
Andreani Odysseos, EFB, EPOS-Iasis R&D Ltd, Cyprus
12 | 2013 International Conference on Nanotheranostics (ICoN 2013)
12.45-13.00
Image-guided quantification of drug delivery: a revolution in
Radiology (Positron Emission Tomography (PET) and Magnetic
Resonance Imaging (MRI))
Radiation-based therapies
Costas Pitris, University of Cyprus, Cyprus
13.00-13.30
Discussion of Clinical Challenges and Prospects
13.30-14.30 Lunch
Session 7 – Materials and Tissues in Nanotheranostics
Session Chair: Theodora Krasia-Christoforou, University of Cyprus
14.30-15.20
Challenges in Nanotheranostics: A Materials Perspective
Rena Bizios, University of Texas at San Antonio, USA (p. 60)
15.20-15.40
Magnetoactive electrospun nanocomposite membranes in drug
delivery and hyperthermia applications
Ioanna Savva, Andreani Odysseos, Loucas Evaggelou, Oana Marinica, Eugeniu Vasile,
Ladislau Vekas, Yiannis Sarigiannis and Theodora Krasia-Christoforou, University of Cyprus,
Cyprus (p. 61)
15.40-16.00
Electrospun PEO/PLLA Fibrous Meshes for Sustained Tyrosine Kinase
Inhibitors Delivery in Situ
Maria Kokonou, Fotios Mpekris, Triantafyllos Stylianopoulos, Jean-Michel Siaugue and
Andreani Odysseos, University of Cyprus, Cyprus (p.62)
16.00-16.20
Drug delivery through reconstructed bronchial mucus modified by
functional carrier particles (FCPs)
Marcin Odziomek, Tomasz Sosnowski and Leon Gradoń, Warsaw University of Technology,
Poland (p. 64)
16.20-16.40
Agglomeration of Theophylline Nanoparticles: a New Protocol for
Pulmonary Drugs Administration
Heba Salem, Mohamed Abdelrahim, Kamal Abo Eid and Mohamed Sharaf, The University of
Beni Suef, Egypt (p. 66)
16.45 – 17.15 Coffee Break
Round Table Discussion and Student Awards
Session Chair: Andreani Odysseos, EFB, EPOS-Iasis R&D Ltd, Cyprus
17.15-18.15
Round Table Discussion: Challenges and Opportunities in
Nanotheranostics
Moderator: Andreani Odysseos
Panel Members: K. Dawson, J. Mollenhauer, H. Doumanides, K. Kostarellos
18.15-18.30
Best Student Paper Award
13 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Abstracts
14 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Opportunities Offered by Nucleic Acid-Based NanoDevices in Cancer Therapy and Theranostics
Jan Mollenhauer1, Ines Block1, Angela Riedel1, Steffen Schmidt1, Helle Christiansen1, Birgitte
Brinkmann Olsen2, Helge Thisgaard2, Poul Flemming2, Stefan Vogel1, Jesper Wengel1
1
2
NanoCAN - University of Southern Denmark, Denmark
Nuclear Medicine, Odense University Hospital, Denmark
[email protected]
Nucleic acids offer fascinating opportunities for the targeted design of nano-devices with therapeutical and
theranostics applications in cancer. Evolutionarily, RNAs are thought to have presented the first generation of
functional biomolecules and nowadays are considered for therapeutical applications as short interfering RNAs
(siRNAs), while, technically, immense progress has been achieved by DNA-based nano-engineering, commonly
known now as DNA-origami. In conjunction with the ability to create aptamers, nucleic acid-based targeting
molecules that function like nano-sized antibodies, the nucleic acid world essentially offers the building blocks
for the conception of nano-drugs and –theranostics.
Within our efforts, we concentrate on the identification of novel targets in breast cancer stem cells, based on
which we aim at designing nucleic acid-based nano-drugs, consisting of aptamers and siRNAs. These can also be
linked to imaging molecules in a simple fashion. Collectively, this may enable to develop personalized regimen,
in which a molecular trait of an individual patient`s tumor is targeted, causing selective (synthetic) lethality to
tumor cells but not to normal cells. On an individualized basis, tumor targeting could be assessed in advance and
therapy response can be monitored during treatment by implementation of imaging molecules. We reflect the
present state of the art of the individual research lines, i.e. of our efforts in identifying novel breast cancer genes,
systematic screening for active siRNAs, and experimental imaging devices.
[Back to Plenary Session]
15 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Nanoscale Interface Between Engineered Matter and
Living Organisms: Understanding the Biological Identity
of Nanosized Materials
Kenneth Dawson
Ireland Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Ireland.
[email protected]
Nanoscale materials can interact with living organisms in a qualitatively different manner than small molecules.
Crucially, biological phenomena such as immune clearance, cellular uptake and biological barrier crossing are
all determined by processes on the nanometer scale. Harnessing these endogeneous biological processes (for
example in creation of new nanomedicines or nanodiagnostics) will therefore require us to work on the
nanoscale. This ensures that nanoscience, biology and medicine will be intimately connected for generations to
come, and may well provide the best hope of tacking currently intractable diseases.
These same scientific observations lead to widespread concern about the potential safety of nanomaterials in
general. Early unfocussed concerns have diminished, leaving a more disciplined and balanced scientific
dialogue. In particular a growing interest in understanding the fundamental principles of bionanointeractions
may offer insight into potential hazard, as well as the basis for therapeutic use.
Whilst nanoparticle size is important, the detailed nature of the nanoparticle interface is key to understanding
interactions with living organisms. This interface may be quite complex, involving also adsorbed proteins from
the biological fluid (blood, or other), leading to a ‘protein corona’ on the nanoparticle surface that determines its
“biological identity”. We discuss how this corona is formed, how it is a determining feature in biological
interactions, and indeed how in many cases can undermine efforts at targeting nanoparticles using simple
grafting strategies. Thus, nanoparticle interactions with living organisms cannot be fully understood without
explicitly accounting for the interactions with its surroundings, i.e. the nature of the corona.
References
1.
2.
3.
4.
Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials.
Nature Nanotechnology 2012, 7, 779-786.
Kim, J. A.; Aberg, C.; Salvati, A.; Dawson, K. A. Role of Cell Cycle on the Cellular Uptake and Dilution of Nanoparticles in a Cell
Population. Nature Nanotechnology 2012, 7, 62-68.
Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Baldelli Bombelli, F.; Dawson, K. A. Physical-Chemical Aspects of
Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. Journal of the American Chemical Society
2011, 133, 2525-2534.
Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the NanoparticleProtein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proceedings of the National
Academy of Sciences 2007, 104, 2050-2055.
[Back to Plenary Session]
16 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Assessing biological specificity of nanoparticles in vitro:
test tubes, chips or cells?
Pavlos (Bogos) Agianian
Department of Molecular Biology and Genetics Democritus University of Thrace, Dragana, 68100 Alexandroupoli, Greece.
e-mail: [email protected]
The hope of applying nanotherapy to cure human diseases has sparked a new era of collaboration between
biologists, chemist and medical doctors. Irrespective of disease or nanoplatform used, biological targeting and
specificity of action, combined with low toxicity, is commonly desired. To achieve this goal, a big number of
functionalized nanoparticles (NPs) are produced for each biological target, creating a need for efficient and
cost effective in vitro methods to screen the functionality of NPs, before they can be applied to animal models
for further biological studies.
I will critically present in vitro methods of assessing the biological specificity of NPs, emphasizing on
solution methods, on chip biosensor analysis using Surface Plasmon Resonance (SPR) and cellular imaging. I
will elaborate on results from functionalized NPs for targeted drug delivery of the anticancer drugs taxol and
ixabepilone and for theranostic applications targeting the EGFR receptor.
[Back To Session 1]
17 | 2013 International Conference on Nanotheranostics (ICoN 2013)
In-vitro release study and dynamic in-vivo imaging of pHand Magnetic field sensitive hybrid microspheres
Eleni K. Efthimiadoua*, Christos Tapeinosa, Eirini Fragogeorgib,c, George Loudosc and George Kordasa*
a
Sol-Gel Laboratory, Institute for Advanced Materials, Physicochemical processes, Nanotechnology & Microsystems, NCSR
“Demokritos”, 153 10 Aghia Paraskevi Attikis, Greece
b
Department of Medical Instruments Technology, Technological Educational Institute, GR 122 10 Athens, Greece
c
Radiochemical/ Radiopharmacological Quality Control Laboratory, Institute of Nuclear and Radiological Sciences and Technology,
Energy & Safety, N.C.S.R. ‘Demokritos’, 15310 Aghia Paraskevi, Greece
[email protected]
This paper deals with the synthesis, characterization and property evaluation of drug-loaded magnetic
microspheres with pH-responsive cross-linked polymer shell. The synthetic procedure consists of 3 steps, of
which the first two comprises the synthesis of a Poly Methyl Methacrylate (PMMA) template and the
synthesis of a shell, using Acrylic Acid (AA) and Methyl Methacrylate (MMA) as monomers, and Divinyl
Benzene (DVB) as cross-linker. The third step of the procedure refers to the formation of magnetic
nanoparticles on the microsphere’s surface. AA that attaches pH-sensitivity in the microspheres and magnetic
nanoparticles in the inner and the outer surface of the microspheres, enhance the efficacy of this intelligent
Drug Delivery System (DDS), which constitutes a promising approach towards cancer therapy. A number of
experimental techniques were used to characterize the resulting microspheres. In order to investigate the invitro controlled release behaviour of the synthesized microspheres, we studied the DOX release percentage
under different pH conditions and under external magnetic field. Hyperthermia caused by an Alternating
Magnetic Field (AFM) is used in order to study the Doxorubicin (DOX) release behaviour from
microspheres with pH functionality. The in vivo fate of these hybrid-microspheres was tracked by labelling
them with the γ-emitting radioisotope 99mTc after being intravenously injected in normal mice. According
to our results, microcontainers present a pH depending and a magnetic heating, release behaviour. As
expected, labelled nanocarriers were mainly found in the mononuclear phagocyte system (MPS). The
highlights of the current research are: (i) to illustrate the advantages of controlled release by combining
hyperthermia and pH-sensitivity and (ii) to provide non invasive, in vivo information on the spatiotemporal
bio-distribution of these microcontainers by dynamic γ imaging.
Scheme 1. In- vitro and in-vivo study of fabricated MCs
1.
2.
Ganta, S.; Devalapally, H.; Shahiwala, A.; Amiji, M., A review of stimuli-responsive nanocarriers for drug and gene
delivery. J Control Release 2008, 126, (3), 187-204.
Motornov, M.; Roiter, Y.; Tokarev, I.; Minko, S., Stimuli-responsive nanoparticles, nanogels and capsules for integrated
multifunctional intelligent systems. Progress in Polymer Science 2010, 35, (1-2), 174-211.
[Back To Session 1]
18 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Preparation and characterization of new elements for the
conception of nanohybrids for multicolored bioimaging
a
M. Rivoala, A. D. Odysseosa
EPOS-Iasis, R&D, 5 Karyatidon street, Nicosia 2028, Cyprus
[email protected]
Currently, the development of organic/inorganic nanohybrid materials arouses the enthusiasm of many
researchers owing to their potential applications and in particular in bioimaging applications (Fig. 1).
Fig. 1: Principle of organic/inorganic nanohybrids for multicolored bioimaging
The aim of this work was to prepare and characterize new inorganic and organic components for the future
design of new multi-functional nanohybrids with properties responding to the current challenges. For this
purpose, we have prepared nanoparticles of zinc oxide (ZnO) as the inorganic component by laser ablation [1].
The surface of these nanoparticles can be modified by an organic component bearing the carboxylic group as an
anchor. First, we synthesized and characterized a number of viologen derivatives, well known as strong electron
acceptors, involving the anchoring groups. The nanohybrids of ZnO/viologens were prepared and characterized
by various spectroscopic techniques. In parallel, we recently have developed efficient synthetic routes toward a
series of new heterocycles (Fig. 2) possessing the electron donating properties: derivatives of
dibenzo[2,3:5,6]pyrrolizino[1,7-bc]indolo[1,2,3-lm]carbazole [2].
a
b
c
Fig. 2: Molecular and crystal structure of a a) new fluorescent heterocycle substituted with b) -MeO and c) -COOH groups.
These new molecules exhibit high thermal stability and strong fluorescence in the visible range. Those
derivatives can also serve as the efficient electron donating moieties in two-photon absorbing (TPA)
chromophores. Their one- and two-photon (Near-infrared) absorption properties and electron donor ability were
investigated experimentally and by means of quantum mechanical calculations.
Here we report on the preparation and properties of inorganic ZnO NPs and organic components: a series of
original viologens and hitherto unknown parent heterocyclic system together with a novel series of derivatives
and their potential to be functionalized to highly selective, quinazoline-based Tyrosine Kinase Inhibitors
towards a new generation of fluorescent nanotheranostic agents for ERBB-related malignancies. The
experimental details, the results and the perspectives will be presented.
This work was supported by ANR (French Agency for National Research), project NEM, ANR-09-BLAN-0107. We thank the French
Ministry of Education and Ecole Doctorale des Sciences Chimiques (ED 250, Marseille) for a fellowship to M.R. This work has been partially
supported by the Marie-Curie IAPP project NANORESISTANCE- Grant agreement no.: 286125
[1] Chelnokov E., Rivoal M., Colignon Y., Gachet D., Bekere L., Thibaudau F., Giorgio S., Khodorkovsky V, Marine W. “Band gap tuning
of ZnO nanoparticles via Mg doping by femtosecond laser ablation in liquid environment”. Applied Surface Science, 2012, 258, 23,
9408- 9411.
[2] M. Rivoal, L. Bekere, D. Gachet, V. Lokshin, W. Marine, V. Khodorkovsky. “Substituted dibenzo[2,3:5,6]-pyrrolizino[1,7bc]indolo[1,2,3-lm]carbazoles: a series of new electron donors”. Tetrahedron, 2013, 69, 3302-3307.
[Back To Session 1]
19 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Size Dependent Biological Profiles of Pegylated Gold
Nanorods for Biomedical Applications
F. Tatinia, I. Landinib, F. Scalettic, L. Massaic, S. Centid, F. Rattoa S. Nobilib, G. Romanod, F. Fusid, L.
Messoric, E. Minib, R. Pinia
a
Institute of Applied Physics "Nello Carrara" National Research Council, Via Madonna del Piano 10, Sesto Fiorentino, 50019, Italy.
Department of Health Sciences, Section of Clinical Pharmacology and Oncology University of Florence viale Pieraccini , 6, 50139
Florence , Italy.cDepartment of Chemistry, University of Florence , 500I9 Sesto Fiorentino, Italy.dDepartment of Clinical Physiopathology,
Viale G. Pieraccini 6, Firenze 50139, Italy
b
federica.scaletti@unift. it
Gold nanoparticles (GNP) have attracted a widespread attention due to their unique physicochemical
properties; they exhibit tremendous potential for biomedical applications from the detection of protein/DNA
interactions to drug delivery and cancer therapeutics and diagnostics. In particular gold nanorods (GNRs) are
attracting special attention due to their unique optical properties, which include a surface Plasmon absorption
band in the visible region and a second tunable absorption in the NIR, making them good agents for
photothermal treatment of cancers [1].
Crucial features of GNRs include their coating, shape and size. The kind of coating is critical to modulate the
interface between particles and biological systems, to enable the attachment of additional functional molecules
such as ligands for biochemical targets of interest and to determine the stability of the particles within culture
media and body fluids.
Most studies identified polyethylene glycol (PEG) as the coating of choice for GNRs because of lack of
toxicity [2], camouflage from immune systems, versatility for further functionalization and conferment of
stability in the blood.
An important requirement for the biomedical applications of GNRs is their solubility and stability in
physiological buffers. Previous studies [3,4] showed that gold nanoparticles exposed to biological fluids may
become coated with proteins. Inturn, adsorption of proteins on gold nanoparticles may modify their characteristic
conformation in solution, cause a loss of biological activity, elicit an altered immune response and modify
particle biodistribution and cellular uptake.
Here we present an extensive survey on the characterization, stability, proteins interaction, toxicity and
cellular uptake of PEG-coated GNRs of different size. In particular five distinct sizes of particles were
synthetized, characterized and studied.
Till now, the effect of size on the cytotoxicity and cellular uptake of PEG-coated GNRs has never been
reported. Therefore the understanding of such correlation provides useful hints for the selection of promising
products for biomedical applications.
Acknowledgements
We gratefully acknowledge Chiara Gabbiani for her work on the first stage of the project and Francesco Rugi for
the ICP-aes measurements, Beneficentia Stiftung (Vaduz, Liechetenstein) and Regione Toscana,
''NANOTREAT' project, for generous financial support.
References
[I] F. Ratto, P. Matteini, F. Rossi, L. Menabuoni, N. Tiwari, S. K. Kulkarni, R. Pini; "Photothermal effects in connective tissues mediated
by laser-activated gold nanorods "Nanomed.: Nanotec. Bioi. Med.S, 143-151 (2009).
[2] A M. Alkilany, A Shatanawi, T. Kurtz, R. B. Caldwell, R. W. Caldwell "Toxicity and cellular uptake of gold nanorods in vascular
endothelium and smooth muscles of isolated rat blood vessel: importance of surface modification" Small. 8, 1270-1278 (20 12).
(3] S. Chakraborty, P. Joshi, V. Shanker, Z. A Ansari, S. P. Singh, P. Chakrabarti "Contrasting effect of gold nanopartic/es and nanarods
with different surface modifications on the structure and activity of bovine serum albumin " Langmuir, 27, 7722-7731(2011).
[4] T. T. Moghadam, B. Ranjbar, K. Khajeh, S. M. Etezad, K. Khalifeh, M. R. Ganjalikhany "interaction of lysozyme with gold nanorods:
c01![ormation and activity investigations" Int. J. of Bioi. Macromol., 49, 629-636 (2011).
[Back To Session 1]
20 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Fluorescent and magnetic silica core shell nanoparticles
for biomedical applications
Ana B. Dávila-Ibañez1, Maria Kokonou1,2, Efstathia Mylouli1,3, Christine Ménager1, Jean-Michel Siaugue1
1
PECSA laboratory (Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques), UPMC, 75005, Paris, (France)
[email protected]
2
EPOS-Iasis Research and Development Ltd., 2028 Nicosia, Cyprus
3
pro-ACTINA S.A., GR-19400 Koropi Attikis (Industrial Zone) Athens, Greece
[email protected]
In the context of biomedical applications, we have developed a fluorescent and magnetic nanometric platform
made of a maghemite core embedded in a fluorescent and twice fonctionnalized silica shell. Synthesis,
functionalization and physico-chemical characterization of multifunctionalized magnetic core shell nanoparticles
(Fe2O3@SiO2(Fluorescent dyes)-NH2/PEG) were performed using either non size sorted or size sorted magnetic
nanoparticles and different fluorescent dyes like fluorescein, rhodamine and near-IR emitting dye.
This nanometric platform was used for the grafting of bleomycin, an antitumor antibiotic. The covalent
anchoring of bleomycin was performed thanks to reductive amination (A). Grafted bleomycin kept its capacity to
induce DNA cleavage (B). Moreover, selectivity and specificity of supported bleomycin to DNA were not
altered. Both bleomycin activated nanoparticles and nude nanoparticles internalization in human cancer cells
(HT1080) were studied at different times. Interestingly, confocal fluorescence microscopy and electronic
transmission microscopy indicated strong interactions between nanoparticles and cells nuclei, with major
accumulation near the nucleus (C,D). Some nanoparticles were observed too into the nucleus (E). Cells viability
assays were also carried out. Bleomycin activated nanoparticles were able to induce a cytotoxic effect whereas
nude nanoparticles did not thus demonstrating the role played by the grafted anticancer drug [1].
Bleomycin grafting (A). DNA cleavage (B). Nanoparticles translocation into human fibroblastom (TEM (C, E), fluorescent confocal
microscopy (D)).
Those core-shell nanoparticles were also used as a colloidal immunosupport in a homogeneous sandwich
immunoassay. PEG chains prevent non-specific adsorption and amino groups enable covalent grafting of αLactalbumin (α-Lac) antigen, thus allowing the capture of the target model analyte (goat anti α-lac
immunoglobulin G (IgG)). In comparison with classic ELISA test, the incubation time for target analyte capture
was accelerated 200 fold and the limit of detection was 20 times lower. This nanometric homogenous
immunoassay was successfully applied for IgG determination in real matrix like serum with good agreement
with ELISA test and further integrated in a microsystem to develop an immunoassay in a lab on chip [2].
Those nanoparticles are also good candidates for multimodal imaging (MRI/fluorescence) applications. We
used them to decorate model and biological membranes. We obtained notably human red blood cells with core
shell nanoparticles adsorbed onto their surface, thus magnetic and fluorescent human red blood cells, which
could be usefull for the detection of hemorrhage [3].
References
[1] T. Georgelin, S. Bombard, J.-M. Siaugue and V. Cabuil. Nanoparticle-Mediated Delivery of Bleomycin, Angew. Chem. Int. (2010), 49,
8897.
[2] B. Teste, F. Malloggi, J.-M. Siaugue, A. Varenne, F. Kanoufi, S. Descroix. Microchip integrating magnetic nanoparticles for allergy
diagnosis. Lab Chip (2011), 11, 4207.
[3] M. Laurencin , N. Cam , T. Georgelin , O. Clément , G.Autret , J.-M. Siaugue , C. Ménager, Human Erythrocytes Covered with
Magnetic Core–Shell Nanoparticles for Multimodal Imaging. Adv. Healthcare Mater ( 2013),, DOI: 10.1002/adhm.201200384
Acknowledgments
The authors gratefully acknowledge EU for funding through FP7 IAPP/NANORESISTANCE/Grant Agreement Number: 286125.
[Back to Session 2]
21 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Two different approaches for the synthesis of
functionalized silica coated magnetic nanoparticles
Ana B. Dávila-Ibañez1, Verónica Salgueiriño2, Jean-Michel Siaugue1
1
PECSA laboratory (Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques), UPMC, 75005, Paris, (France)
2
Departamento de Física Aplicada, Universidade de Vigo, 36310, Vigo (Spain)
[email protected]
Magnetic nanoparticles show great promise for a huge range of applications. The major advantages correspond
to their magnetic nature and ease of biofunctionalization. Herein will be presented the synthesis of
nanomaterials with magnetic properties that later will be functionalized in such a way to direct their possible
applications.
Magnetic materials were silica coated by two different approaches to provide them higher stabilities, as well
as to facilitate their functionalization. By the first approach we were able to synthesized silica coated
magnetic nanoparticles that later were functionalization with DNA[1, 2] to increase their biocompatibility,
follow by cytotoxicity studies that revealed the key role that surface functionalization plays in regulating the
mechanisms involved of the chemical degradation of the nanocoposites. By the second approach FIR
fluorescent dye-doped magnetic nanoparticles amino functionalized were synthesized providing magnetic
composites that could be used for therapeutic and diagnosis applications [3].
Different magnetic nanoparticles were synthesized, magnetite (Fe3O4), cobalt ferrite (CoFe2O4) and
maghemite (γ-Fe3O4) nanoparticles. They were characterized in terms of their magnetic behavior and
chemistry nature to later functionalize them. Magnetic nanoparticles were silica coated, firstly by reverse
microemulsion methods, providing silica coated magnetic nanoparticles with different shell thickness; and
secondly by a modified Stober method affording PEG and amino functionalized silica coated magnetic
nanoparticles. In terms of their functionalization two main approaches were carried on. In one hand the
synthesis of bio-functionalized magnetic composites by the deposition of DNA fragments on their surface. In
the other hand, the synthesis of dye-doped magnetic nanoparticles by the encapsulation of the dye into the
silica shell during the silica coating magnetic nanoparticles procedure[3]..
Fig 1. Image on the left shows the DNA functionalized silica coated magnetic nanoparticles once inside the cells where
morphological changes have started due cell environment. Images on the rigth shows dye-doped silica coated magnetic
nanoparticles (top) and the emission fluorescent spectrum (bottom) of these composites as function of the concentration of the
encapsulated dye.
References
[1] A. B. Davila-Ibáñez, V. Salgueiriño, V. Martínez-Zorzano, R. Mariño-Fernández, A. García-Lorenzo, M. Maceira-Campos, M. MuñozÚbeda, E. Junquera, E. Aicart, J. Rivas, F. J. Rodríguez-Berrocal, J. L. Legido, Magnetic Silica Nanoparticle Cellular Uptake and
Cytotoxicity regulated by Electrostatic Polyelectrolytes-DNA Loading at their Surface ACS Nano (2012), 6, 747.
[2] A. B. A. B. Davila-Ibáñez, Niklaas J. Buurma and V. Salgueiriño, Assesment of DNA complexation onto polyelectrolites- coated
magnetic silica nanoparticles, Nanoescale, (2013), 5, 4797.
[3] T. Georgelin, S. Bombard, J.-M. Siaugue and V. Cabuil. Nanoparticle-Mediated Delivery of Bleomycin, Angew. Chem. Int. (2010), 49,
8897.
Acknowledgments
The authors gratefully acknowledge EU for funding through FP7 IAPP/NANORESISTANCE/Grant Agreement
Number: 286125.
[Back to Session 2]
22 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Theragnosis combining ferrocenyl derivatives,
carbohydrates and nanovectors
Malinge Jérémy, Siaugue Jean-Michel(1), Ménager Christine(1), Sollogoub Matthieu(2), Zhang Yongmin(2),
Jaouen Gérard(3) & Vessières-Jaouen Anne(3).
(1)-PECSA, UPMC ; (2)-IPCM, Equipe GOBS, UPMC ; (3)-Laboratoire Friedel, ENSCP
[email protected]
Over the past decades, theragnosis platforms have emerged as a powerful tool for the simultaneous
diagnostic and therapy of cancer. Gathering, in a single vector, an imaging agent, a targeting unit and an efficient
drug is still a challenging task and a tremendous amount of work is currently being published in the scientific
community.
Ferrocifen derivatives are specifically cancer-cell-activated prodrugs and have proven useful for untreatable
cancer cells [1]. However, with these polyphenol species, formulation and drug delivery remain challenging.
The strategy employed in our work is based upon the use of magnetic liposomes as the central scaffold
(Figure). This vector offers structural versatility in terms of membrane composition. For instance, the project
aims to functionalize the outer membrane with bioavailable units to target infected tissues and a fluorescent
phospholipids to provide bimodal imagery tools (fluorescence and MRI). In addition, the presence of iron
nanoparticles inside the liposome allows active targeting under magnetic field and possible hyperthermia [2].
Formulation of the drug, liposome synthesis and first results (in vitro and in vivo) will be presented.
Figure 1. a) Schematic representation of the aimed theragnostic platform; b) TEM image of an isolated liposome
[1]
[2]
M. Görmen, P. Pigeon, S. Top, E. Hillard, M. Huché, C. Hartinger, F. de Montigny, M-A. Plamont, A. Vessières, G. Jaouen,
“Synthesis, Cytotoxicity, and COMPARE Analysis of Ferrocene and [3]Ferrocenophane Tetrasubstituted Olefin Derivatives against
Human Cancer Cells” ChemMedChem 5, 2039-2050 (2010).
G. Béalle, R. Di Corato, J. Kolosnjaj-Tabi, V. Dupuis, O. Clément, F. Gazeau, C. Wilhelm, C. Ménager, “Ultra magnetic liposomes
for MR imaging, targeting, and hyperthermia” Langmuir 28, 11834–11842 (2012)
[Back to Session 2]
23 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Chitosan – linear aldehyde nanoparticles obtained from
reverse micelle method
Gawlik K. Wasiak I. Ciach T.
Warsaw University of Technology, Faculty of Chemical and Process Engineering, Biomedical Engineering Laboratory,
Warynskiego Street 1, 00 -645 Warsaw, Poland
[email protected]
Introduction
Drug delivery systems can be defined as means to introduce therapeutic agents into the body. These systems
have been considered as the key to the clinical success of numerous drugs, they assure delivery of therapeutic
agent into the body. Ideal drug delivery system provides means to efficiently deliver active agent to the ill tissue
only, assuring efficacy and decreasing side effects. It is especially important in the case of cancer treatment,
when we sometimes administer very toxic drugs in nearly lethal doses. Recent researches suggest that
nanotechnology poses tolls that could overcome the disadvantages of current treatment. Nanoparticulate drug
delivery systems are purposely engineered and constructed objects that are measured in nanometer size. Their
size usually ranges from a few to several hundred nanometers. Nanoparticles can be made using a various raw
materials including polymer, lipids, viruses, noble metals, semiconductors, magnetic or organometalic
compounds. Biomedical application of nanoparticles include drug carriers, labeling and tracking agents, vectors
for gene therapy, hyperthermia treatments and magnetic resonance imaging contrast agents. This definition
includes monolithic nanoparticles in which the drug is adsorbed dissolved or dispersed throughout the matrix
and nanocapsules in which the active agent is confined to an aqueous or oily core surrounded by shell-like wall.
Alternatively the active agent can be covalently attached to the surface or into the matrix [1]. The unusual
property on nanoparticles in biological systems comes from the fact, that most of the interactions in cell and
between cells are performed in the same level of size. This can be a benefit but also a potential source of danger.
Nanoparticles can be biodegradable and nonbiodegradable. The first type will disappear from the body after
performing the desired action, due to hydrolysis or other biological process. The second type will probably stay
in our body for very long time or even forever, what pose a potential danger on such objects. That’s why
biodegradable – organic nanoparticles are frequently considered as safe, in comparison with inorganic. Perfect
materials for medical nanoparticle preparation are biodegradable polymers. They can be fabricated into various
shapes and sizes, with tailored, pore morphologies, mechanical properties and degradation kinetics to suit a
variety of application. By selecting the appropriate polymer type, molecular weight and copolymer blend ratio,
the degradation rate of nanoparticles can be controlled to achieve the desired type and rate of release of the
encapsulated drug. Biodegradable nanoparticles can be prepared from variety of materials such as non
biodegradable polymers, proteins, synthetic biodegradable polymers and polysaccharides. They protect
entrapped drug against degradation and control its site specific delivery. However, the main drawback of
conventional NPs is their non specific interaction with the cells and plasma proteins, leading to drug
accumulation in no target organs [2]. Therefore, polysaccharides coatings are attractive alternative to PEG once
their possess many recognition functions, allowing specific mucoadhesion or receptor recognition, as well as
providing neutral coatings with low surface energy, preventing non specific protein adsorption. The presence of
saccharide on the NP surface can also increase their uptake by cancer calls due to the Warburg effect – glucose
demand is about 200 times higher for cancer cells then for normal.
Chitosan is a biodegradable natural polymer with great potential for pharmaceutical applications due to its
biocompatibility, high charge density, non-toxicity and mucoadhesive propertiey [3]. Chitosan has been used as
a drug carrier for sustained release preparations and improvement of bioavailability for hydrophobic drugs, and
as a vehicle for directly compressed tablets, disintegrant, binder and granulating agent. Chitosan is a well-known
natural polysaccharide that is usually obtained from shells of crustaceans such as crab, shrimp, and crawfish. It
is a copolymer of 2-acetamido-2-deoxy-D-glucose (N-acetyl-glucosamine,GluNAc) and 2-amino-2-deoxy-Dglucose (N-glucosamine,GluN) units randomly or block distributed throughout the biopolymer chain depending
on the processing method used to derive the biopolymer. Chitosan is a partially N-deacetylated derivative of
chitin. The term chitosan is usually used when glucosamine units predominate or the polymers become soluble
in a dilute acid solution. The biological properties of chitosan include biocompatibility, biodegradability, nontoxicity, hemostaticity, antitumoral and antiviral activity. Chitosan is degraded by lysozyme present in the
various mammalian tissues and leads to production of N-acetyl-D-glucosamine and D-glucosamine, which also
plays an important physiological role in in vivo biochemical processes. In addition, chitosan has a special
feature of adhering to the mucosal surface and transiently opening the tight junction between epithelial cells [4].
Thus, chitosan nanoparticles are potential delivery system for hydrophilic drugs due to its outstanding
physicochemical and biological properties. Although chitosan should be useful for even more numerous
applications, its use suffers severe limitations because it is insoluble in neutral or alkaline media owing to its
rigid and compact crystalline structure and strong intra- and intermolecular hydrogen bonds.
24 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Chitosan has both reactive amino and hydroxyl groups that can be used to chemically alter its properties
under mild reaction conditions. The presence of amino groups leads to the possibility of several chemical
modifications, including the preparation of Schiff bases (–RC=N–) by reaction with aldehydes and ketones. The
reaction of chitosan with aromatic aldehydes to produce the corresponding Schiff bases has been described [5].
Schiff base compounds containing an imine group, are usually formed by the condensation of a primary amine
with an active carbonyl. Its attractiveness as analytical reagents raises from the fact that they enable simple and
inexpensive determinations of various organic and inorganic substances. The imine linkage formed by this
reaction is fairly stable in neutral and alkaline solution, but it is rapidly hydrolyzed under acidic conditions.
Schiffe base can be easily converted into N-alkyl derivative by reduction with sodium borohydride. The aim of
this paper was to evaluated reverse micellar method for chitosan- linear aldehyde nanoparticles preparation
Methods
The present work describes the preparation of chitosan nanoparticles obtained by reverse micellar method.
Nanoparticles were prepared from chitosan (MW = 200 kDa with degree of deacetylation = 95%) and different
linear aldehydes (oktanal, dekanal and dodekanal). Briefly the solution of 0.01 M surfactant in hexane were
prepared. 50 -150 µl of chitosan solution and 10 µl of aldehyde and liquor ammonium were added to the
surfactant solution. After 24 h reaction, the solvent was evaporated, and the dry mass resuspended in Tris–Cl
buffer (pH 7.4) by sonicaton. To this, 1 ml of 30% CaCl solution was added drop wise to precipitate the
surfactant. The precipitate was pelleted by centrifugation. The size and concentration of the nanoparticles were
investigated as a function of the preparation conditions. The surfactant and aldehyde type, ammonium,
chitosan, and aldehyde concentration was evaluated.
Reasults
Proposed method allows for preparation of stabile chitosan nanoparticles. The size range of the obtained
nanoparticles was between 100 and 200 nm. Obtained results shows that there is no significant influence of
chitosan concentration in the range between 50 -150 µl on nanoparticles size. The diameter of nanoparticles
increase with linear aldehyde C atoms number and liquor ammonium concentration, even at to 40%.
Nanoparticles concentration decrease with C atoms number in aldehyde and bee not change with chitosan
concentration. However increasing ammonium concentration results in increasing nanoparticles concentration
up to 20 %. Use of anionic surfactant (SDS) is promoting nanoparticles preparation.
References
[1] C. Duncan, “The dawning era of polymer therapeutic,” Nat Rev Drug Disc 2, 347-360 (2003).
[2] A. Mahapatro, D.K. ASingh “Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines”
J.Nanobio.9, 55 (2011)
[3] Xiao-Ying Yinga, Dan Cuia, Lian Yub, Yong-Zhong Dua, “Solid lipid nanoparticles modified with chitosan oligosaccharides for the
controlled release of doxorubicin” Carbohydr Polym 84, 1357-1364 (2011)
[4] F. Qian, F. Cui, J. Ding, C. Tang, and C. Yin, “Chitosan graft copolymer nanoparticles for oral protein drug delivery: preparation and
characterization,” Biomacromolecules, 7, 2722–2727 (2006).
[5] X. Jin, J. Wang, J. Bai “Snthesis and antimicrobial activity of Schiff base from chitsan and citral” Cabohydr Res 344, 825-829 (2009)
[Back to Session 2]
25 | 2013 International Conference on Nanotheranostics (ICoN 2013)
New Routes Towards the Synthesis of Natural Products
and Designed Derivatives
E. A. Couladouros
Agricultural University of Athens, Chemical Labs, Iera Odos 75, Athens, 118 55, Greece. and
Pro-Actina S.A., Archimidous 59, Koropi Attikis, 19400, Greece.
[email protected]
Chemical synthesis is one out of the most important steps both for the discovery of a new pharmaceutical and
for its exploitation to become a drug. Bioactive natural products of complicated structure are the outmost
challenge in this field. Apart from the purely academic interest, such endeavours may lead to the development of
general and convergent routes for the preparation of designed and diversified libraries, thus facilitating their
further exploitation. In this respect, short reaction sequences having a high degree of diversification and a
suitable handle for solid phase application are most desirable.
New synthetic methodologies applied on five families of natural products will be presented:
a.
b.
c.
d.
e.
An approach for the asymmetric synthesis of all naturally occurring tocotrienols.
Novel solid supported synthesis of “unnatural aminocyclitols”.
A short asymmetric synthesis of the highly potent antibiotic abyssomicin C.
Recent progress towards the development of a general method for the synthesis of polyprenylated
phloroglucinol derivatives.
The application of the “polymorphic scaffold” approach in the synthesis of sugars
[Back to Session 2]
26 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Hemocompatibility of Albumin Microspheres as Drug
Delivery System, In vitro Study
Mohamed A. Elblbesy
University of Tabuk, Saudi Arabia
E‐mail: [email protected]
Purpose
The main objective of the present work is to evaluate the Hemocompatibility of albumin microspheres. Albumin
microspheres were prepared by coacervation method. The characteristics of Albumin microspheres such as
particle size, zeta potential, particle morphologie, entrapment efficiency and drug loading were evaluated. In
vitro release study prolonged duration (50% total cumulative percentage at the end of 24 hours, 85% at 72 hrs).
Conclusion
That coacervation method is well suited to produce albumin microspheres and the preparative variables of the
procedure can be fine-tuned depending on the clinical application.
[Back to the Poster Session]
27 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Hemocompatibility of Silver Nanoparticles
Julie Laloy, Valentine Minet, Lutfiye Alpan, Bernard Chatelain, François Mullier and Jean-Michel Dogné
Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS),
University of Namur
[email protected]
Background
The presence of silver nanoparticles (Ag NPs) in consumer products like disinfectants, deodorants, antimicrobial
sprays, etc increased in the past few years. Ag NPs are also used for medical applications as antimicrobial agents
for wound dressings, catheters, and orthopedic and cardiovascular implants. As the clinical use of silver
nanoparticles increases, a better understanding of their safety when exposed to the bloodstream is needed. There
is a serious lack of information on the biological effects of Ag NPs on human blood cells.
Aim
The objectives of this study are to determine the impact of Ag NPs on erythrocyte integrity, platelet function and
blood coagulation.
Methods
Erythrocytes integrity was assessing based on measurement of haemoglobin release at 550 nm. Activation and
aggregation of the platelets was determined by optical aggregometry in combination with electron microscopy
observations. The calibrated thrombin generation test was used to study the impact of Ag NPs on coagulation
cascade. A particular attention was made on the potential interference of Ag NPs with the detection methods
used in these tests.
Results
With optical aggregometer, Ag NPs have no impact at on platelet function at low concentration. An impact on
platelet function could be observed at high concentration using electron microscopy. Ag NPs can also induce
hemolysis at concentration higher than 0.3 µg/ml. These NPs have also a procoagulant potential observed by an
increase in the thrombin generation.
Conclusions
Ag NPs have a high hemolytic potential, an impact on coagulation and on platelet aggregation. Maximal
precautions must be taken with the use of Ag NPs in medical applications.
[Back to the Poster Session]
28 | 2013 International Conference on Nanotheranostics (ICoN 2013)
From docking to synthesis and grafting: Preliminary
results for new Anilinoquinazolines as potential EGFR
inhibitors in multifunctional nanocarriers
Fotini Liepouri1, Pavlos Agianian2, Vasssiliki Gerafalaki2, Elias Couladouros1,4 Andreani Odysseos3,
Alexandros Strongilos1
1. Pro-Actina S.A., Archimidous 59, Koropi Attikis, 19400, Greece.
2. Democritus University of Thrace, Dragana 68100, Alexandroupoli Evros, Greece.
3. EPOS-Iasis, 5 Karyatidon Street, Suite 202, 2028 Nicosia, Cyprus.
4. Agricultural University of Athens,75 Iera Odos, 11855 Athens, Greece
[email protected]
Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor that constitutes a major biological target
for the development of anti-cancer drugs.
EGFR upon activation by its natural substrate EGF, mediates cell growth and proliferation pathways through
activation of its intracellular component which is a Tyrosine Kinase. EGFR’s overexpressions and mutant forms
found in cancer cells, make it a significant receptor whose inactivation would serve significantly to control cell
growth and proliferation.
Here in we present the roadmap towards optimized synthesis of EGFR kinase inhibitors as potential grafting
agents on nanoactuators, such as metal nanoparticles and carbon nanotubes. Bibliography search resulted in a
number of biologically active scaffolds that inhibit EGFR. Among the most potent and promising ones are
Anilinoquinazolines[1-3], of type I [Figure 1]. Furthermore, various Anilinoquinazolines carrying a Michael
acceptor inhibit the kinase irreversibly through formation of a chemical bond with a neighboring cysteine residue
in the active site of the kinase. Based in that rational, Anilinoquinazolines are selected as the most promising
scaffold for synthesis.
Figure 1: EGFR inhibitors carrying the Anilinoquinazoline moiety
In order to rationalize chemical synthesis of these derivatives in terms of their efficacy in EGFR binding and
inhibition, thus not compromising valuable resources, we initially aimed at in silico protein-ligand docking by
using Autodock 4.0. The advantage of this targeted approach is a higher chance to obtain functional EGFR
inhibitor derivatives in shorter times and most importantly, to be able to quickly reject molecules with significant
stereochemical issues that would prevent them from approaching the kinase’s binding cavity. The in silico
approach seems even more necessary if we consider that the constructs we finally aim at, contain large organic
parts.
After obtaining insight from our extended docking studies in a number of designed analogues, we moved on
to selected chemical synthesis of 12 new analogues. Determination of the EGFR- inhibitory activity and
assessment of their biological activity has been undertaken in order to identify their potential to serve as
promising grafting agents onto nanocarriers for drug delivery systems
The authors gratefully acknowledge EU for funding through FP7 IAPP/NANORESISTANCE/Grant Agreement
Number: 286125
References
[1] Gordon W. Rewcastle,†, Brian D. Palmer,†, Alexander J. Bridges,‡, H. D. Hollis Showalter,‡, Li Sun,‡, James Nelson,‡, Amy
McMichael,‡, Alan J. Kraker,‡, David W. Fry,‡ and, and William A. Denny*,† “Tyrosine Kinase Inhibitors. 9. Synthesis and Evaluation
of Fused Tricyclic Quinazoline Analogues as ATP Site Inhibitors of the Tyrosine Kinase Activity of the Epidermal Growth Factor
Receptor,”Journal of Medicinal Chemistry 39 (4), 918-928 (1996).
[2] Jeff B. Smaill,†, Gordon W. Rewcastle,†, Joseph A. Loo,‡, Kenneth D. Greis,§, O. Helen Chan,‡, Eric L. Reyner,‡, Elke Lipka,‡, H. D.
Hollis Showalter,‡, Patrick W. Vincent,‡, William L. Elliott,‡ and, and William A. Denny*,† “Tyrosine Kinase Inhibitors. 17. Irreversible
Inhibitors of the Epidermal Growth Factor Receptor: 4-(Phenylamino)quinazoline- and 4-(Phenylamino)pyrido[3,2-d]pyrimidine-6acrylamides Bearing Additional Solubilizing Functions” Journal of Medicinal Chemistry 43 (7), 1380-1397 (2000).
[3] Srivastava, Sanjay K.; Kumar, Vivek; Agarwal, Shiv K.; Mukherjee, Rama; Burman, Anand C., “Synthesis of Quinazolines as Tyrosine
Kinase Inhibitors” Anti-Cancer Agents in Medicinal Chemistry 9 (3), 246-275 (2009).
[Back to the Poster Session]
29 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Theoretical investigation of a new metal nanoparticle for
combined imaging and therapy applications
Myria Angelidou, Costas Pitris
KIOS Research Center for Intelligent Systems and Networks,
Dep. of Electrical and Computer Engineering, University of Cyprus
75 Kallipoleos St., 1678 Nicosia, Cyprus
[email protected], [email protected]
1. Introduction
Metal nanoparticles (NPs) have unique optical properties, due to the phenomenon of surface plasmon resonance
(SPR). By varying the size, shape, and material, the SPR wavelengths can range from visible to near-infrared
(NIR). [1] Most of the research effort focuses on noble metals, such as silver and gold, since they exhibit strong
absorption and scattering plasmon bands which can be exploited in both imaging and therapy applications [2].
The optical response, of metal nanoparticles, was extensively explored using computational methods. The noble
NPs were found to have absorption, or scattering or overlapping spectra [3, 4].
Tissue absorption in the NIR window (650-900nm) is minimal, and thus favorable to optimal light
penetration [5]. Imaging applications can significantly benefit from using NIR light, which minimizes absorption
from biomolecules and therefore allows deeper penetration of the incident light into living tissue. Photothermal
applications, on the other hand, could benefit from NPs having strong absorption with limited scattering, for
better efficiency. Here, we propose a new, complex metal nanoparticle which has the unique property of
distinctly separated absorption and scattering spectra. Its absorption peak is at the wavelength (λ) of 635nm, and
its scattering peak at λ = 785nm, both in the range of the optical window for biological applications. This could
be beneficial for combined imaging and therapy, since the laser wavelengths used for each application can be
decoupled for increased efficacy and safety.
2. Methodology
The Discrete Dipole Appoximation (DDA) method was used to calculate the optical response (absorption,
scattering, and extinction) of various metal nanoparticles. The DDA can address with any arbitrary shape,
composition and external medium as long as some criteria are satisfied [6]. The efficiency factors are used to
explore the various spectral characteristics, such as wavelength maximum and peak value. These factors are
defined as Qabs sca  Cabs sca  reff2 and Qext  Qabs  Qsca where Cabs/sca is the absorption and scattering cross
sections, and reff is the effective radius, which represents the radius of a sphere having a volume equal to that of
the simple or complex nanoparticle. The complex dielectric function for the various metals was specified and
obtained from the E.D. Palik [7] tabutaled data. For small sized nanoparticles, such as nanospheres, the dielectric
function was modified to include the surface damping effect, as described in ref. [8]. All the calculations
assumed water as the external medium.
3. Results
3.1 Simple nanoparticles
First, the efficiency factors were calculated for various simple metal NPs, such as silver (Ag), gold (Au),
aluminum (Al) and nickel (Ni). The NPs had nanospheres, nanocubes and tetrahedral shapes, and various sizes.
From the calculations it was found that simple nanostructures have mainly scattering or overlapping spectra.
(Data not shown.) Since none of the simple nanostructures provided with distinct spectra, combinations were
considered for further exploration. The Au nanocube, with reff = 74.4nm, was chosen as the imaging
nanoparticle, since it provides the stronger scattering with less absorption in the NIR range even though
absorption and scattering overlap in the visible.
3.2 Complex nanoparticles
To obtain the desired property, of distinctly separated absorption and scattering spectra, small nanospheres and
the Au nanocube, chosen from previous calculations, were combined to create a complex nanoparticle. The small
nanospheres were chosen as the therapy nanoparticle since they provide with adequate absorption in the visible
wavelength range [2]. The small nanospheres were considered to be arranged on the front face of the nanocube,
perpendicular to the propagation x-axis, as also shown in Fig. 1(a, b). Parameters such as the number, size
(diameter), and material (Ag or Au) of the small nanospheres were varied in order to obtain the optimum
nanostructure with distinctly separated absorption and scattering spectra.
30 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Figure 1. Example of a complex nanoparticle shape as viewed from (a) the side, and (b) front.
Figure 2(a) show the spectra of the Au nanocube chosen as the scattering nanoparticle. Figure 2(b) show the
optical response of the complex nanoparticle comprised of the Au nanocube combined with 16 (30nm) Ag
nanospheres. Other combinations, such as 4 (60nm), and 64(15nm) Ag nanospheres are not shown. It is clear,
from fig. 2 that the addition of a layer of small nanospheres, in front of the nanocube, affected the spectra. Table
1 provides with the ratios of absorption to scattering and vice versa for the various complex NPs, for better
comparison between them. We require high ratio for both wavelengths. From the two ratios, the combination
with 16 Ag provided with the highest values, and proved to exhibit the best separation between the spectra, and
at the desired wavelength range. The gold nanosphere combinations gave similar results, except in the case of 16
Au nanospheres, where the ratio at λ = 635nm was small compared to the counterpart.
Figure 2. Absorption and scattering spectra of (a) simple Au nanocube (reff = 74.4nm) and (b) the complex nanoparticle (nanocube plus 16
(30nm) Ag spheres).
Table 1. Ratio of absorption to scattering, and vice versa, for the two wavelengths of interest.
Cube plus 4 Ag spheres
Cube plus 4 Au spheres
Cube plus 16 Ag spheres
Cube plus 16 Au spheres
Cube plus 64 Ag spheres
Cube plus 64 Au spheres
Qabs/Qsca, at λ = 635nm
1.781
1.773
2.366
1.583
1.992
1.647
Qsca /Qabs, at λ = 785nm
1.354
1.317
3.233
3.242
1.947
1.729
When the complex nanoparticle was rotated relative to the incident propagation and polarization, the separation
between absorption and scattering was lost and scattering was significantly enhanced in the visible range. Also,
when the small nanospheres covered all nanocube surfaces again the separation was again lost and replaced by a
flat absorption spectrum and a broad scattering spectrum having two peaks at visible and NIR wavelengths.
(Data not shown.)
4. References
[1] P. K. Jain, X. Huang, I. H. El-Sayed and M. A. El-Sayed, “Review of some interesting surface plasmon resonance-enhanced properties
of noble metal nanoparticles and their applications to biosystems,” Plasmonics 2, 107-118 (2007).
[2] N. G. Khlebtsov and L. A. Dykman, “Optical properties and biomedical applications of plasmonic nanoparticles,” J. Quant. Spectrosc.
Radiat. Transfer 111, 1-35 (2010).
[3] I. O. Sosa, C. Noguez and R. G. Barrera, “Optical properties of metal nanoparticles with arbitrary shapes,” J. Phys. Chem. B 107, 62696275 (2003).
[4] P. K. Jain, K. S. Lee, I. H. El-Sayed and M. A. El-Sayed, “Calculated absorption and scattering properties of gold nanoparticles of
different size, shape, and composition: applications in biological imaging and biomedicine,” J. Phys. Chem. C 110, 7238-7248 (2006).
[5] R. Weissleder, “A clearer vision for in vivo imaging,” Nat. Biotechnol. 19, 316-317 (2001).
[6] B. T. Draine and P. J. Flatau, “Discrete-dipole approximation for scattering calculations,” J. Opt. Soc. Am. A 11, 1491-1499 (1994).
[7] E. D. Palik, Handbook of optical constants of solids, (Academic Press, Orlando, 1985).
[8] M. Angelidou and C. Pitris, “Investigation of nanostructure scattering and absorption for combined optical diagnostic and therapeutic
applications,” Proc. SPIE 8231, 8231081-8231087 (2012).
[Back to the Poster Session]
31 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Surface Enhanced Raman Spectroscopy (SERS) for PointOf-Care Diagnosis of Urinary Tract Infections
Katerina Hadjigeorgioua, Evdokia Kastanosb, Costas Pitrisa
a
KIOS Research Center for Intelligent System and Networks, Department of Electrical and Computer Engineering, University of Cyprus, 75
Kallipoleos St, Nicosia, Cyprus, 1678
b
University of Nicosia, Department of Life and Health Sciences, 46 Makedonitissas Avenue, Nicosia 1700, Cyprus.
[email protected]
1. Introduction
Urinary tract infections (UTIs) are very common, especially in women, resulting in billions of dollars in health
care costs every year in the US alone [1]. The current, standard, method of diagnosis for a UTI is by quantitative
urine culture which requires 24 hours to produce results. [2]. Antibiotic susceptibility testing requires an
additional 24 hour period. Due to the prolonged period of diagnosis, broad spectrum antibiotics are prescribed
before any definitive results are obtained. This practice leads to many undesirable consequences such as
unsuccessful treatments, chronic infections, rising health care costs, and, most importantly, increased antibiotic
resistance [2].
Surface Enhanced Raman Spectroscopy (SERS) has had a variety of bioanalytical applications in recent
years including the detection and identification of bacteria [3-7]. The aim of this work was the development of a
rapid identification and antibiogram method using SERS spectra obtained from bacteria incubated with various
antibiotics and colloidal silver nanoparticles. Bacteria were classified with an accuracy of ~ 94% and sensitivity
or resistance to antibiotics was determined with 81-100% accuracy using SERS spectra after just 4 hours of
exposure. These results may lead to the development of a fast and accurate diagnosis and antibiogram tool for
UTI based on SERS.
2. Methodology
Sample Preparation and Data Acquisition
Clinical bacterial isolates from patients with UTI were identified by biochemical tests. Liquid antibiograms were
used to specify the susceptibility of each bacterium to each of the 7 antibiotics used in this study. Bacterial
samples containing 2x105 cells/ml (determined optically) were used for the classification and antibiogram
studies. This concentration was chosen since it is equivalent to the minimum concentration found in urine
samples from UTIs.
For the purposes of this study, 16 bacterial strains were chosen, 7 E. coli, 4 K. pneumonia, and 5 Proteus
spp., and were treated separately with amoxicillin, amoxicillin/clavulanic acid (augmentin), cefaclor, cefazoline,
ceftriaxone, cefuroxime, and ciprofloxacin. 50 μl of each bacterial sample was mixed with an equal volume of
each antibiotic or PBS (as the control) and incubated for 0 and 4 hours at 37 oC. 20 μl of the incubated bacteriaantibiotic solutions (105 cells/ml) was mixed with 10μl of silver nanoparticles spotted on glass slides and allowed
to dry. SERS spectra were collected directly from the spots. SERS spectra were collected using the iRaman
system (BWTek, Inc) with a laser source at 532nm and 3.0cm-1 resolution. The power at the sample was
approximately 50 mW and the exposure time was 20s x 12 averages (4 min total.) The SERS spectra consisted of
data ranging from 300 cm-1 to 3000 cm-1 inclusive.
Analysis of SERS spectra
For classification of the bacterial species, the SERS spectra collected from the 16 bacterial strains at time 0
without any exposure to antibiotics were used. Spectra were pre-processed by removing cosmic spikes and
filtering to remove the background and the high frequency noise (Figure 1). The features used for the
classification were the spectrum itself and ratios of means of spectral segments (Figure 2). The classification
method used was Linear Discriminant Analysis. Principal component analysis (PCA) of the data preceded the
classification. A leave-one-out cross-validation (LOOC) procedure was performed and the results for each of the
classification exercises were recorded.
For the classification of bacteria as sensitive or resistant to any of the seven antibiotics, the SERS spectra
collected from the 16 bacterial strains after 4 hours of exposure either to each antibiotic or PBS (control) were
used. Pre-processing was performed as before. In addition, the corresponding control spectrum was subtracted
from the spectrum with antibiotic exposure. This procedure removes any background, bacterial, and/or antibiotic
contributions to the spectra leaving the data solely reflecting the changes due to the antibiotic activity. Each
sample was then classified as resistant, sensitive, or intermediate using the same feature creation, data reduction,
and classification techniques as before. A LOOC procedure was also performed.
32 | 2013 International Conference on Nanotheranostics (ICoN 2013)
E.Coli
Klebsiella
Proteus
0.16
Avg. E.Coli Ratios
Avg. Klebsiella Ratios
Avg. Proteus Ratios
6
Numerator Segment
arbitary stacked counts
0.18
0.14
0.12
0.1
0.08
0.06
0.04
5
4
3
2
0.02
1
500
1000
1500
2000
wavenumber (cm-1)
2500
3000
Figure 1. Average and filtered SERS spectra of the bacteria used in
this study. The background was removed.
Unknown: E.Coli (Correctly Classified)
Manova Score 2
94
E.Coli
Klebsiella
Proteus
Unknown
A
92
90
88
254
256
258
260
262
Manova Score 1
264
266
268
Manova Score 2
Unknown:
Klebsiella
(Misclassified)
Figure 3. Results
of bacterial
species
classification. An uknown
50
E. Coli is correctly classified.
E.Coli
49
B
48
1
2
3
4
5
61
2
3
4
5
Denominator Segment
61
2
3
4
5
6
Figure 2. Ratios of means of spectral segments used as features for
species classification. The average for each species is presented
here.
Table 1. Antibiotic sensitivity classification results.
%Correct
Antibiotic
Classification
Amoxil
87.50
Augmentin
93.75
Cefaclor
81.25
Cefazolin
81.25
Ceftriaxone
93.75
Cefuroxime
100.00
Ciprofloxacin
87.50
Mean
89,29
Klebsiella
Proteus
Unknown
47
3. Results
46
Following
the methodology described above, the classification of the bacterial species yielded 93.75 % correct
classification
rate (i.e.
only
1358of the
16362samples was misclassified.) Figure 3 shows two such examples of the LOOC
45
348
350
352
354
356
360
process. Following
theScoremethodology
described in section 2.2, each bacterium was classified as resistant,
Manova
1
intermediately sensitive, or sensitive to each of the 7 antibiotics used in the study. The spectra were correctly
classified between 81 and 100 % depending on the antibiotic (Table 1.) It is interesting to notice that the algorithm
is not only successful for antibiotics affecting the bacterial cell wall but also other types (e.g. ciprofloxacin which
acts on the bacterial DNA.)
4. Conclusions
The use of SERS for UTI diagnosis and antibiogram is presented in this study. The bacterial species can be
correctly classified with high accuracy (~94 % correct classification) even at low bacterial concentrations. In
addition, this method is able to distinguish the SERS spectra of bacteria that are treated and are sensitive to an
antibiotic from bacteria that resistant to an antibiotic, after a 4 hour incubation time with correct classification rate
of ~ 81-100%.
Experiments are currently under way to repeat this study using a larger number of samples belonging to a
greater number of species which will include both gram negative and gram positive bacteria. In addition, studies are
currently under way to determine what the shortest time of exposure. In order for this method to develop into a
point-of-care diagnostic the test time must be as short as possible. In addition, experiments have to be performed
directly on urine samples.
5. References
[1] Litwin M. S., Saigal C. S., "Introduction", [Urologic Diseases in America], DHHS, PHS, NIH, NIDDK, NIH publication, Washington, DC,
07-5512, 3-7 (2007).
[2] Morgan, M. G., McKenzie, H., "Controversies in the laboratory diagnosis of community-acquired urinary tract infection," Eur. J. Clin.
Microbiol. Infect. Dis. 12, 491-504 (1993).
[3] Zeiri, L., Bronk, B. V., Shabtai, Y., Eichler, J., Efrima, S., “Surface-enhanced Raman spectroscopy as a tool for probing specific
biochemical components in bacteria”, Appl. Spectrosc. 58, 33-40 (2004).
[4] Guzelian, A. A., Sylvia, J. M., Janni, J. A., Clauson, S. L., Spencer, K. M., “SERS of whole-cell bacteria and trace levels of biological
molecules”, Proc. SPIE 4577, 182 (2002).
[5] Premasiri, W. R., Moir, D. T., Klempner, M. S., Krieger, N., Jones, G. II, Ziegler, L. D., “Characterization of the surface enhanced raman
scattering (SERS) of bacteria”, J. Phys. Chem. B; 109, 312-320 (2005).
[6] Jarvis, R. M., Goodacre, R., “Characterisation and identification of bacteria using SERS”, Chem. Soc. Rev. 37, 931-936 (2008).
[7] Walter,A.,Marz,A.,Schumacher,W.,Rosch,P.,Popp,J., “Towards a fast, high specific and reliable discrimination of bacteria on strain level by
means of SERS in a microfluidic device”, Lab Chip 11, 1013-1021 (2011).
[Back to the Poster Session]
33 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Activity, anti-cancer effect and nanodelivery of new
anilinoquinazoline EGFR inhibitors
Eftychia Angelou1, Fotini Liepouri2, Maria Pavlaki1, Andreani Odysseos3, Jean-Michel Siaugue4 ,
Alexandros Strongilos2, Pavlos Agianian1
1. Department of Molecular Biology and Genetics, Democritus University of Thrace, Dragana, 68100 Alexandroupoli, Greece.
2. Pro-Actina S.A., Archimidous 59, Koropi Attikis, 19400, Greece.
3. EPOS-Iasis, 5 Karyatidon Street, Suite 202, 2028 Nicosia, Cyprus.
4. PECSA laboratory (Physico-chimie des Electrolytes, Colloïdes et Sciences Analytiques),
5. UPMC, 75005 Paris, France
[email protected]
Epidermal Growth Factor Receptor (EGFR) is a ubiquitous tyrosine kinase that modulates cell physiology (1).
Binding of physiological ligands, such as EGF, to EGFR alter cell differentiation, proliferation and migration and
control cell survival. In a number of malignancies, including those of the lung, breast, colon, and kidney, EGFR is
overexpressed or hyperactivated leading to aberrant cell signaling (2). Targeting EGFR with tyrosine kinase
inhibitors (TKIs) constitutes an important line of defense against EGFR-positive cancers, however, inevitably,
resistance develops. Recently discovered second generation TKIs, like the drugs gefitinib and lapatinib, are effective
against some resistant tumors (3). Chemically, they both comprise an anilinoquinazoline scaffold that specifically
recognize the ATP-binding pocket of the cytoplasmic, kinase domain of EGFR.
We aim to develop new anilinoquinazoline EGFR inhibitors and use them to direct nanodevices to cancer cells
and EGFR-resistant tumors for both targeted therapy and imaging (theranostics). A dozen of new type-I
anilinoquinazoline derivatives have been synthesized. We show their EGFR kinase inhibitory activity in vitro and
how they affect cell survival of HeLa and MCF-7 cells, untreated or after stimulation with EGF. Using confocal
microscopy, we present preliminary imaging data that demonstrate the interaction of promising fluorescently labeled
nanosystems, with living cells. We finally illustrate Surface Plasmon Resonance (SPR)- based approaches to quickly
screen the functional loading of the nanosystems in grafted anilinoquinazolines. The potential of using these results
for the construction of efficient, EGFR-specific, cancer theranostic applications is discussed.
Figure 1. Viability of EGF stimulated HeLa cells as a function of increasing concentrations (nM) of a new anilinoquinazoline
derivative
Acknowledgements
This work is supported from FP7-IAPP-NANORESISTANCE project (grant number 286125)
References
1.
2.
3.
Yarden Y., Sliwkowski M. X. (2001) Nat. Rev. Mol. Cell Biol. 2, 127–137.
Rowinsky E. K. (2004) Annu. Rev. Med. 55, 433–457.
Rosa D.D., Ismael G., Lago L.D., Awada A. (2008) Cancer Treat. Rev. 34, 61–80.
[Back to the Poster Session]
34 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Toxicity and Safety of Carbon Nanomaterials for
Biomedical Applications
Cyrill Bussy and Kostas Kostarelos
The University of Manchester, Manchester, United Kingdom.
[email protected]
The dramatic development of nanoscience and nanotechnology in recent years has offered numerous opportunities
and innovative solutions in various fields and applications. Among the different types of novel materials discovered
at the nanoscale, carbon-based nanomaterials are a superfamily that includes amongst many others, fullerenes,
carbon nanotubes (CNTs), and graphene. In terms of scope of applications, two of these carbon nanomaterials seem
to be developed more widely and maturing faster than the rest: carbon nanotubes and graphene. Both CNT and
graphene materials have outstanding electronic, mechanical, electrical, and optical properties and a chemically
tunable surface that have made them attractive candidates for a broad range of applications, spanning from
composites and electronics to nanomedicine. Biosensors, tissue engineering, as well as components for the design
of various types of drug delivery and release systems are among the potential applications of graphene and CNTs in
biomedicine. However, questions have been raised regarding a potential toxicity upon human exposure. In this
context, the importance of toxicity profiling and physicochemical characteristics in relation to safety considerations
for carbon nanomaterials based products candidate for biomedical applications cannot be overemphasized.
We will focus on how the two most popular forms of carbon nanomaterials (carbon nanotubes, graphene) used
in biotechnology and medicine would fair toxicologically in terms of their drug development potential. We will
highlight key physicochemical parameters for safety considerations and will also emphasize the importance of
toxicological profiling during the development phase of biomedical products based on carbon nanomaterials. A
comparison with clinically used nanomedicines and nanomedicine products in clinical trials will be given in order
to understand where carbon nanomaterials stand.
[Back to Session 3]
35 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Guidelines Proposal for Studying Hemocompatibility of
Manufactured Nanoparticles and Impact on Fibrinolysis
Julie Laloy, Lutfiye Alpan, Valentine Minet, Jean-Michel Dogné
Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS),
University of Namur
[email protected]
1. Background
Nanosciences and nanotechnologies are in constant evolution. Development of new therapeutic and diagnostic
agents using nanotechnologies to reach their pharmaceutical target require the knowledge of biocompatibility of
nanoparticles (NPs) with the blood compounds. Hemostasis is the ensemble of physiological phenomena which
prevent and lead to stop bleeding; it maintains the vascular integrity. A dysfunction of the hemostasis can lead to
slow down or even to completely stop the circulation of the blood. It is therefore primordial to study the
hemocompatibility of NPs.
2. Aims
The aim of this study is the evaluation of the biocompatibility of manufactured NPs on hemolysis, platelet function
and coagulation.
3. Methods
Erythrocytes integrity was studied by determination of haemoglobin release. For platelet function, six platelet
function tests were investigated: light transmission aggregometry, whole-blood impedance aggregometry, platelet
function analyser-100 (PFA-100) and Cone-and-Plate(let) analyser (Impact-R®), transmission- and field emission
gun scanning electron microscopy (FEG-SEM). Several existing methods of clotting times and thrombin generation
assays were evaluated in human normal pool plasma for the impact of NPs on coagulation. Five NPs (carbon
nanotubes, carbon black, silicon dioxide, copper oxide and silicon carbide) with different physicochemical
properties were used.
4. Results
The Impact-R® with scanning electronic microscopy support are the reference methods to investigate the potential
impact of NPs on platelet function. For coagulation, the calibration thrombin generation test is the reference method
to investigate the procoagulant activity of NPs.
5. Conclusion
We suggest guidelines for testing NP hemocompatibility to respond to a request of scientific community due to lack
of recommendations for the evaluation of nanomaterial hemocompatibility.
[Back to Session 3]
36 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Rat whole-body exposure model to nanoaerosol:
Development with silicon carbide nanoparticles and study of
their toxicity.
Julie Laloy, Omar Lozano, Lütfiye Alpan, Valentine Minet, Olivier Toussaint, Bernard Masereel, JeanMichel Dogné & Stéphane Lucas.
University of Namur, 61 rue de Bruxelles, Namur, 5000 Belgium
[email protected]
Background
Air represents the major route of human exposure to manufactured nanoparticles (NPs). Humans are exposed
every day to diesel exhaust particles containing NPs. In manufacturing industries, workers can also be
accidentally exposed to NPs due to the failure of ventilation system or individual protective equipment. The
assessment of the potential risks of NPs from air exposure on different tissues and organs, especially the
respiratory tract, is thus needed. The physicochemical property of NPs most considered for pulmonary tract
exposure is their size. The size and shape of particles determine their aerodynamic properties that govern entry,
depth of penetration, and deposition in the lung. Inhalation model is closer to the reality than instillation for
reproduction of working conditions, dose generated, and profile of deposition in the pulmonary system.
Inhalation is also more complex to implement and requires expansive equipments as aerosol generator and
analyzer.
Aims
The objectives of this work are: 1. The design, development, characterization and validation of a rat airborne
nanoaerosol whole-body exposure model. 2. To study the pulmonary impact of silicon carbide (SiC) NPs on rats
exposed to a dry aerosol in this whole- body exposure model in an acute toxicity protocol.
Methods
An airborne nanoaerosol whole-body exposure model was designed and developed to allow the simultaneous
exposure of six rats to a nanoaerosol and six rats to clean filtered air (Figure 1). The aerosol was generated using
®
®
a RBG-1000 and analyzed with a detection system ELPI (Electrical Low Pressure Impactor) for real-time
measurement of the NP aerosol concentration and particle size distribution. Complete physicochemical
®
®
characterization of the generated aerosol was performed by ELPI , another aerosol analyzer (Aerotrak ) and
scanning electron microscopy. SiC NPs are selected for the model development and the pulmonary toxicity
study. For the acute toxicity study, Sprague-Dawley rats were exposed during 6 hours to SiC NPs dry aerosol or
filtered air in the model.
Results
The aerosol generated in three individual experiments is stable and reproducible during 6 hours in the model.
A continuous monitoring of the NPs concentration in atmosphere during the experiment is however supported.
After rat exposure during 6 hours to SiC nanoaerosol, macrophages containing SiC NPs were observed in the
lungs with no histological toxicity observed.
37 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Figure 1. Scheme of the whole-body exposure model.
Conclusion
This is the first model developed and validated that allows the integrated assessment of safety NPs on biochemical,
histopathological parameters and biopersistance in different tissues in acute but also subacute and chronic exposure.
[Back to Session 3]
38 | 2013 International Conference on Nanotheranostics (ICoN 2013)
EPR-effect: a barrier to the effective delivery of large
nanomedicines to solid tumors
Triantafyllos Stylianopoulos
Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia 1678, Cyprus
[email protected]
The Enhanced Permeability and Retention (EPR) effect is based on the leakiness of tumor blood vessels that
enables nanoparticles to enter the tumor as well as on the absence of functional lymphatics in the tumor interior
that allows particles to stay in the tissue for a long time. The leakiness (i.e., hyper-permeability) of the tumor
vasculature is mainly due to large openings in the endothelial lining that comprises the neoplastic tumor vessel
wall [1]. These openings might be hundreds of nanometers in size, contrary to the openings of the normal vessel
wall, whose size is less than 10 nm [2, 3]. Therefore, the rationale for employing the EPR effect to treat cancer is
that particles with a size larger than 10 nm will not be able to extravasate to normal tissues - reducing adverse
effects - and would selectively pass through the openings of the tumor vessels. This reasoning, however, does not
guarantee that nanoparticles will reach tumors in amounts sufficient to cause cure.
The EPR was introduced by two independent studies more than two decades ago [4, 5]. The first study was
published by the research group of Hiroshi Maeda [4] and focused on the bright sight of the EPR, i.e., the selective
delivery of nanoparticle formulations to tumor and not to normal tissues. The second study was published by the
research group of Rakesh Jain [5] and apart from the great promise of EPR, they pointed out the potential barriers
that it might have to the effective delivery of the nanomedicines to the tumors. Since then the scientific community
was heavily based on the first study neglecting the barriers to the delivery of large size nanoparticles that are posed
by the abnormal tumor microenvironment.
Nowadays, despite the enormous scientific effort only three nanoparticle formulations have been widely
approved for treatment of solid tumors. These are: Doxil® a 100 nm pegylated liposomal doxorubicin that has
been approved for the treatment of HIV-related Kaposi's sarcomas, metastatic ovarian cancers and metastatic
breast cancers, DaunoXome® a 50 nm liposomal daunorubicin, approved for HIV-related Kaposi's sarcomas and
Abraxane® a 10 nm (following plasma disintegration) albumin-bound paclitaxel, which has been given approval
for metastatic breast cancers. These drugs are associated with significantly less adverse effects compared to
conventional chemotherapy, presumably due to the EPR effect. The increase in overall survival is, however,
modest in many cases [1, 6-8] largely because of limited delivery. Therefore, even though current nanomedicines
have succeeded in preventing delivery to normal tissues, they cannot ensure effective delivery in tumors.
In my talk, I will discuss how the EPR effect inhibits the effective delivery of large nanoparticles to solid
tumors and will provide strategies to overcome these barriers and improve intratumoral distribution. I will also
present design rules that optimize nanoparticle accumulation into tumor tissues.
References
[1] Jain RK, Stylianopoulos T. Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol 7, 653-64 (2010).
[2] Hobbs SK,Monsky WL, Yuan F et al. Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment. Proc
Natl Acad Sci U S A 95, 4607-12 (1998).
[3] Sarin H. Physiologic upper limits of pore size of different blood capillary types and another perspective on the dual pore theory of
microvascular permeability. J Angiogenes Res 2, 14 (2010).
[4] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of tumoritropic
accumulation of proteins and the antitumor agent smancs. Cancer Res 46, 6387-6392 (1986).
[5] Gerlowski LE, Jain RK. Microvascular permeability of normal and neoplastic tissues. Microvasc Res 31, 288-305 (1986).
[6] O'Brien MER, Wigler N, Inbar M et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal
doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol 15,
440-9 (2004).
[7] Gradishar WJ,Tjulandin S, Davidson N et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor
oil-based paclitaxel in women with breast cancer. J Clin Oncol 23, 7794-803 (2005).
[8] Gill PS, Wernz J, Scadden DT et al. Randomized phase III trial of liposomal daunorubicin versus doxorubicin, bleomycin, and vincristine
in AIDS-related kaposi's sarcoma. J Clin Oncol 14, 2353-64 (1996).
[Back to Session 4]
39 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Strategies to improve nanomedicine delivery to solid tumors
Konstantinos Soteriou, Eva-Athena Economides, Triantafyllos Stylianopoulos
Department of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus
[email protected]
Introduction
Failure of standard nanomedicine formulations to prolong the life of cancer patients is in large part due to the
inability of these drugs to penetrate deep into the tumor tissue and distribute homogeneously in the tumor
interstitial space. As a result cancer nanomedicines that have been approved for clinical use (e.g., Doxil®,
DaunoXome®) often provide modest survival benefits, despite the considerably less adverse effects compared to
conventional chemotherapy that they are associated with [1]. The leakiness of the tumor blood vessels has
served as the rationale for the use of nanoparticle formulations in cancer but at the same time it contributes to the
elevation of the interstitial fluid pressure (IFP), which hinders the transport of nanomedicines from the
vascular to the interstitial space (i.e., transvascular transport). Strategies to reduce IFP include anti-VEGF
treatment to improve pericyte coverage and thus, decrease the leakiness of the vessels, and anti-fibrotic treatment
to deplete extracellular fibers and decrease the resistance with which fluid can escape from the tumor interstitial
space [2]. Mathematical models were developed to investigate the potentials of both therapeutic strategies and
model predictions were validated with in vivo experiments in orthotopic murine mammary adenocarcinomas.
We found that anti-VEGF treatment improves the delivery of nanomedicines in a size- dependent manner,
favoring the transport of particles less than 20 nm in diameter. Anti-fibrotic treatment was able to improve the
transvascular transport even for large particles with sizes similar to that of currently used nanotherapeutics, such
as Doxil®.
Results
Fig. 1. Model predictions for the effect of anti-VEGF treatment on nanomedicine delivery. Anti-VEGF reduces the size of the
pores of the vessel wall, which improves the penetration of only small particles [2].
Equations
Vascular transport:
(1)
where v is the fluid velocity, cv is the intravascular concentration of the nanoparticle and Δcv is the
concentration difference that corresponds to a vascular length Δx.
40 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Transvascular transport:
(2)
where Pe is the Péclet number across the vessel wall, P is the vascular permeability of the nanoparticle through the
pores of the wall, Lp the hydraulic conductivity, P the vascular permeability, and σ the reflection coefficient.
Interstitial transport:
(3)
where ci is the concentration of the nanoparticle in the interstitial space, D is the diffusion coefficient, and vi is the
interstitial fluid velocity.
References
[1] Jain RK, Stylianopoulos T, “Delivering nanomedicine to solid tumors,” Nature Reviews Clinical Oncology 7, 653-664 (2010).
[2] Chauhan VP, Stylianopoulos T, “Normalization of tumor blood vessels imporves the delivery of nanomedicines in a size-dependent
manner,” Nature Nanotechnology 7, 383-388 (2012).
[Back to Session 4]
41 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Methods for delivery of living cells to the respiratory system
via aerosol route
Tomasz R. Sosnowski1, Ewelina Tomecka2
1
Faculty of Chemical and Process Engineering, Warsaw University of Technology, Waryńskiego 1, 00-645, Warsaw, Poland
2
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland
[email protected]
1.
Introduction
Aerosol route is a convenient and widely accepted method of delivering therapeutics to the lung surface where
they act at a local level (e.g. in curing pulmonary diseases) but can be also transferred to the circulation and
produce systemic effects. As the lungs have a very huge surface area (up to 100 m2), designing a delivery
system as an air dispersion (aerosol) is rational since such a method allows to distribute a drug in different parts of
the lungs without employing excessive doses.
Respiratory tract is exposed to environment and its cellular structure can be damaged by accidental
inhalation of hot gases or irritants [1]. In such cases, the vital breathing function can be seriously defected
leading to life-threatening conditions. A possible acceleration in the recovery can be brought by recellurization of
the lung surface i.e. by seeding the progenitor cells which might at least partially substitute functions of
damaged epithelium. Similar healing method was successfully implemented in recovery from burns and ulcers of
the skin [2,3], and it can be also considered as a future strategy in the lung therapy, also with the application of
stem cells [1]. Our recent studies done with very simple biological models (bacteria and yeast cells) indicated that
spraying of biocolloids with conventional inhalers (nebulizers) is ineffective as the average droplets
produced by these devices are too small to carry whole cells. In addition, all tested methods were too destructive for
biological material due to high hydrodynamic stresses associated with liquid atomization [4]. Based on these
studies only two devices could be selected as potentially applicable for cell spraying, and in preliminary tests
they were demonstrated to maintain 65-95% survival rate of sprayed murine fibroblasts. In a very recent paper by
Kardia et al. [5] it was shown that sprayed fibroblasts proliferate after atomization done by the same devices as the
one employed in our research. This result indicated no cell inactivation due to spraying.
In the current study we present more comprehensive tests of technical possibilities of cell atomization in
order to suggest suitable methods for the generation and delivery of aerosols containing living cells to the lungs.
2.
Materials and methods
The model cell line BALB/c3T3 (fibroblasts) was purchased from European Collection of Cell Cultures (UK).
The cells were suspended in DMEM (Dulbescco’s Modified Eagle’s Medium – Sigma Life Sciences, USA) at the
concentration of 4.35·105 ml-1. Biocolloid was sprayed from two mechanical devices: Microsprayer® Aerosolizer
model IA-1B (Penn-Century Inc., Wyndmor, PA, USA) and nasal atomizer (Coster, Italy) - Fig. 1. Collected
material was analyzed with three assays: (a) vital staining with trypan blue (0.4%) and subsequent cell counting
(Countess® Invitrogen, Korea), (b) fluorescent test in multi-well plate using calceine-AM (2 mM) with fluorescence
evaluation by SynergyTM Mx Multi-Mode Microplate Reader (BioTek, USA) at time = 0 and 24 hours after
spraying, and (c) direct fluorescence microscopic observations (IX7® inverted microscope, Olympus, Japan) after
cell staining with calceine-AM and propidium iodide.
Fibroblasts were also incubated in DMEM for 3 days at 37C at 5% CO2 (HERAcell® 150 – Thermo
Electron Corp. USA) for microscopic evaluation of cell proliferation.
sprayin
g tip
Fig.1. Atomizing devices: Microsprayer® Aerosolizer and nasal atomizer.
3.
Results
The size of droplets emitted from both devices were characterized previously using Spraytec aerosol
spectrometer (Malvern, UK) and it was shown that aerosols were log-normally distributed with median diameter of
47-49 m and 79-82 m, respectively [4]. This allows to expect that the fibroblasts (with an average size of 1020 m in suspension) can be safely surrounded by a liquid inside a droplet generated from a nozzle. The
cumulative results of cell survival tests are presented in Fig. 2, where relative viability means the current
viability as a percentage of cell viability in the initial sample. It follows that at least 75% of cells are viable after
spraying with both methods, and the viability is maintained at similar level 24 hrs after spraying. Cells have
ability to proliferate during next 72 hrs what is demonstrated in Fig.3. This result suggests that biological
42 | 2013 International Conference on Nanotheranostics (ICoN 2013)
activity of cells is not significantly altered by atomization which is in agreement both with results of other
authors [5] and with our data from MTT tests presented recently [4].
Fig.2. Cell survival (a) and proliferation (b) after spraying with both atomizing devices.
4.
Discussion and conclusion
Although two tested atomizing methods are suitable for generation of aerosols containing living cells, it should be
noted that they cannot be directly applied to deliver dispersed biocolloids to the respiratory system. The reason
is that the droplets are too big for their effective penetration to the bronchial tree. On the other hand, spraying
methods which allow to produce finer droplets (e.g. nebulization) cannot be used as such aerosols contain no
living cells [4]. Therefore it is proposed that cell delivery to the respiratory system should be accomplished
by a special techniques where the aerosol is generated either in situ (inside the trachea - e.g. by Microsprayer
device or similar atomizer) or outside the body but with specific inhalation maneuver to avoid droplets
deposition in the upper airways. Bioaerosol should be gently pushed into the bronchial tree e.g. via
endotracheal tube [6], allowing the droplets with cells at least partly penetrate the lower airways. In case of
intubated patients with massive lung damage, which might need this kind of treatment, both approaches of
aerosol delivery seem reasonable.
5.
Acknowledgment
Work supported by governmental funds for science in the years 2010-13 (Project No. NN209 023339).
6.
References
[1] D.J. Angelini et al., "Chemical warfare agent and biological toxin-induced pulmonary toxicity: could stem cells provide potential
therapies?", Inhalation Toxicol. 25, 37–62 (2013).
[2] G. Gravante et al., "A randomized trial comparing ReCell ® system of epidermal cells delivery versus classic skin grafts for the
treatment of deep partial thickness burns", Burns 33, 966-972 (2007).
[3] R.S. Kirsner et al., "Spray-applied cell therapy with human allogeneic fibroblasts and keratinocytes for the treatment of chronic venous leg
ulcers: a phase 2, multicentre, double-blind, randomised, placebo-controlled trial", The Lancet 380, 977-985 (2012).
[4] T.R. Sosnowski et al., "Spraying of cell colloids in medical atomizers", Chem. Eng. Transact. 32, 2257-2262 (2013).
[5] E. Kardia et al., "Aerosol-based delivery of fibroblast cells for treatment of lung diseases", J. Aerosol Med. Pulm, Drug Deliv.
doi:10.1089/jamp.2012.1020 (2013).
[6] J. Mazela et al., "Aerosolized albuterol sulfate delivery under neonatal ventilatory conditions: in vitro evaluation of a novel ventilator
circuit patient interface connector", J. Aerosol Med. Pulm, Drug Deliv. doi: 10.1089/jamp.2012.0992 (2013).
[Back to Session 4]
43 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Enhancement of Drug Absorption Across Intestinal
Membrane Using Magnetic Beads
Anjali Seth,1,2 David Lafargue,2 Cécile Poirier,2 Jean-Manuel Péan,2 Christine Ménager1
1
University Pierre et Marie Curie, Laboratoire PECSA, UMR 7195 4 Place Jussieu, 75005 Paris, France
TECHNOLOGIE SERVIER, Formulation Galénique 25-27 Rue Eugène Vignat, 45000 Orléans, France
2
[email protected]
Magnetic beads were prepared to study the impact of an external magnetic field on drug penetration across
intestinal membrane. Chitosan-alginate core-shell beads (Fig. 1.) were loaded with a low membrane
permeability drug and magnetic nanoparticles (MNPs). They were prepared by standard extrusion crosslinking
process.
Fig. 1. Magnetic core-shell chitosan-alginate beads.
Ex-vivo experiments were performed with Ussing chambers (Fig. 2.) using mice’s jejunum as model membrane.
A magnet was placed on the acceptor chamber wall to create a magnetic field. Apparent permeability was
measured on magnetic beads in comparison with different controls (free drug, magnetic beads without magnet,
drug loaded non-magnetic beads).
Fig. 2. Ussing chambers to measure drug apparent permeability.
Spherical beads with an average diameter of 1.5 mm were prepared with a drug experimental encapsulation ratio
of 4.5% w/w and a loading efficiency of 70%. The MNPs encapsulation ratio was 1.5% w/w. No MNPs release
from beads was detected during the experiments. Dissolution kinetics showed that 100% of the drug was
released within two hours without any influence of the magnetic field. When a magnetic field was applied,
magnetic beads were accumulated onto the intestinal membrane and the apparent drug permeability was
increased threefold in comparison with free drug or non-magnetic beads (Fig. 3.).
44 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Fig. 3. Permeation studies of the drug across non-stripped rat jejunal membrane. Effect of magnetic beads maintained near the
membrane with a magnet (■) in comparaison with the controls, magnetic beads free in the donor compartment (□), non-magnetic
beads free in the donor compartment (●) and free drug (○).
The absorption enhancement was due to the local over-concentration of the drug close to its absorption site and
not to a membrane’s permeability change. The use of magnetic carriers to localize a drug near its absorption
window enabled to enhance significantly its permeation. This approach opens new perspectives in the field of low
permeable drugs for oral administration.
[Back to Session 4]
45 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Ultrasound and microbubbles for monitoring therapies
targeting tumor vascularity
Mike Averkiou
Department of Mechanical and Manufacturing Enigineering, University of Cyprus, Nicosia 1678, Cyprus
[email protected]
Imaging is a key factor in the accurate monitoring of response to cancer therapies targeting tumor vascularity to
inhibit its growth and dissemination. Dynamic contrast enhanced ultrasound (DCE-US) is a relatively new
quantitative method with the advantage of being non-invasive, widely available, portable, cost effective, highly
sensitive and reproducible using microbubble contrast agents that are truly intravascular.
Advances in nonlinear imaging techniques have enabled ultrasound imaging to visualize the macro- and
microvasculature in real time. The image intensity of a region of interest (ROI) in the tumor is proportional to the
microbuble concentration. Metrics of blood flow and blood volume may be extracted from indicator dilution
models. The present talk will concentrate on the bolus injection method for contrast delivery and the analysis of the
wash-in and wash-out of the microbubbles in the ROI.
A review of current work in this area ill be presented and the issues described above will be discussed. Results
from clinical trials with liver cancer patients undergoing vascular targeted therapies will be presented.
[Back to Session 5]
46 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Tunable magnetic/ICG small protocells as a platform for
drug delivery
Thomann Jean-Sébastien, Corne Gaëlle, Arl Didier, Bahlawane Naoufal and Lenoble Damien
Département Science et Analyse des Matériaux (SAM), CRP Gabriel Lippmann, 41, rue du Brill L-4422 Belvaux, Luxembourg
[email protected]
1.
Introduction
Protocells have been reported as a promising drug delivery system due their high payload capability and their
versatility of usage [1]. Protocells are constituted by a mesoporous silica core (MSNPs) surrounded by a lipid
bilayer. The lipid bilayer plays the role of biocompatible gatekeeper which enables easy and efficient surface
functionalization. Brinker et al. [1] was the first to describe their morphological structure and to study their
physico-chemical stabilities. However, the size of reported protocells is typically in the range of 120 nm and
restricted to drug delivery function. The influence of MSNPs pores size on the protocell stability has not yet
been investigated. In our work, we propose to add imaging capability via building multi-phased small protocells
(MSNPs having diameter from 20nm up to 50 nm) incorporating a magnetic core and green indocyanine. The
pore size of the MSNPs will be carefully tuned and thoroughly characterized. This nanoparticle will offer
bimodal imaging capabilities combined with a drug delivery platform. The tailored inorganic carrier properties
that include size, pores geometry, magnetic and near-infrared imaging responses will be reported.
2.
Methods
The synthesis of magnetic MSNPs is achieved using the silatrane route originally published by Möller et al [2].
The properties of the magnetic core are controlled by adjusting the concentration of Fe3O4 NPs and the used
surfactant CTACl (cetyl trimethyl ammonium chloride). The ratio between reactants and solvents is a key
parameter for tuning the size of the MSNPs [3]. ICG is grafted on MSNPs by using Aminopropyl tetraethyl
silicate (APTES) functionalization [4]. The pores size is engineered using a selective chemical etching with
NaBH4 [5]. The lipid coating is applied using ultra-sonication or mixing lipid vesicles with MSNPs. Physical
and chemical characterizations are carried out with Nanoparticle Tracking Analysis, Transmission Electronic
Microscopy (TEM), Scanning Electronic Microscopy (SEM), Fourier transform infrared microscopy, X-Ray
Diffraction.
3.
Results
Figure 1: Small multi-phased protocells. A schematic view of the targeted protocells geometry is presented in the left-hand
side. Two sizes of NP (SEM pictures: A, B) were obtained by varying the dilution of reactant. The number of magnetic
nanoparticle loaded in MSNPs can be tuned by varying the ratio between CTAB, TEOS (triethylorthosilicate) and Fe3O4
nanoparticles (TEM picture C and D). After the synthesis of MSNPs, the pore size can be increased from 3 nm (TEM picture E)
up to 5 nm (TEM picture F) using NaBH4.
47 | 2013 International Conference on Nanotheranostics (ICoN 2013)
4.
Conclusion
We have designed multifunctional nano-objects for enabling the deployment of theragnostic approach in cancer
therapy. By combining dual in vivo imaging and drug loading (via encapsulation of paclitaxel or cis-platinum), the
proposed new generation of protocells is particularly appealing to tackle the current challenges of cancer
nanomedicine. In particular, the MSNPs small size and porosity demonstrated in this study should enable the
design of multi-layers coating while keeping the overall size below 100nm. In one hand, this should favor the
passive targeting of diseases cells based on the EPR. On the other hand, this design should enable a precise
control of the drug payload. In last, the lipid bilayer would enable various bio-conjugation scenarii.
5.
References
[1] Ashley, C. E.; Carnes, E. C.; Phillips, G. K.; Padilla, D.; Durfee, P. N.; Brown, P. A.; Hanna, T. N.; Liu, J.; Phillips, B.; Carter, M. B.;
Carroll, N. J.; Jiang, X.; Dunphy, D. R.; Willman, C. L.; Petsev, D. N.; Evans, D. G.; Parikh, A. N.; Chackerian, B.; Wharton, W.; Peabody,
D. S.; Brinker, C. J. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers. Nat
Mater 2011, 10 (5), 389-397.
[2] Moller, K.; Kobler, J.; Bein, T. Colloidal Suspensions of Nanometer-Sized Mesoporous Silica. Adv. Funct. Mater. 2007, 17 (4), 605612.
[3] Urata, C.; Aoyama, Y.; Tonegawa, A.; Yamauchi, Y.; Kuroda, K. Dialysis process for the removal of surfactants to form colloidal
mesoporous silica nanoparticles. Chem. Commun. 2009, 0 (34), 5094-5096.
[4] Quan, B.; Choi, K.; Kim, Y. H.; Kang, K. W.; Chung, D. S. Near infrared dye indocyanine green doped silica nanoparticles for
biological imaging. Talanta 2012, 99 (0), 387-393.
[5] Jia, L.; Shen, J.; Li, Z.; Zhang, D.; Zhang, Q.; Duan, C.; Liu, G.; Zheng, D.; Liu, Y.; Tian, X. Successfully tailoring the pore size of
mesoporous silica nanoparticles: Exploitation of delivery systems for poorly water-soluble drugs. International Journal of Pharmaceutics
2012, 439, 81-91.
[Back to Session 5]
48 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Ultrasound-induced temperature elevation for in-vitro
controlled release of temperature-sensitive liposomes
Christophoros Mannaris1, Jean-Michele Escoffre2, Ayache Bouakaz2, M. E Meyre3 and Michalakis
Averkiou1
1-Dept. of Mechanical and Manufacturing Engineering, University of Cyprus, Nicosia, Cyprus,
2-UMRS INSERM U930, CNRS ERL3106, Université François Rabelais-Tours, France,
3-Nanobiotix, Paris, France
1.
Introduction
Drug loaded thermosensitive liposomes (TSL) release their payload with mild hyperthermia near their phase
transition temperature (Tm = 43-45 °C) [1]. Focused ultrasound may be used to non-invasively induce local mild
hyperthermia in a region of interest with high accuracy [2]. In combination, ultrasound induced temperature
elevation for localized drug delivery using TSL shows potential in improving the efficacy of drug delivery in a
lesion while at the same time reducing undesired side effects.
In spite of several reports for in-vivo drug delivery using ultrasound and TSL [3-5], reports for in-vitro work
are scarce mainly due to the difficulty in finding an appropriate in-vitro experimental setup. One of the main
problems often encountered is that cell culture media have a very low absorption coefficient and are thus unable to
be heated with ultrasound. Finding a biocompatible medium with high absorption coefficient has not been possible
thus far.
In the present work, an in-vitro method that allows activation of TSL using ultrasound is presented. A single
element focused transducer is used to induce the required temperature elevation in Opti-MEM® cell culture
medium via thermal conduction and activate drug loaded TSL. A significant release of doxorubicin from TSL is
achieved.
2.
Materials and methods
Experimental setup
Figure 1 shows a schematic of the experimental setup. A small volume sample holder containing Opti-MEM® cell
culture medium and TSL solution was placed inside a plastic cuvette filled with 99% glycerol that has a high
absorption coefficient (α=5.7 Νp/m/MHz). Mylar acoustic windows on the cuvette and holder allowed ultrasound
to propagate through thus avoiding heating of the plastic walls. A single element focused transducer (center
frequency 1.1 MHz, 50 mm diameter and 50 mm focus) was used to heat up the glycerol in the cuvette. The OptiMEM®/TSL solution in the sample holder reached the required temperature for activation of the TSL via thermal
conduction. The experiments were carried out in a 37°C water bath.
Sample Holder
Cuvette
Mylar acoustic
windows
Figure 1: Schematic of experimental setup
Ultrasound Exposure
Detailed characterization of the ultrasound field was done in water using a 0.4 mm element membrane hydrophone
(Precision Acoustics Ltd, Dorchester, UK). The acoustic field experienced by the TSL in the sample holder was
also measured with the cuvette in place using a 0.5 mm needle hydrophone placed in the middle of the cuvette (the
rear Mylar window was removed). Any diffraction effects due to the presence of the cuvette were negligible. The
sample holder was placed at the focus of the transducer. Ultrasound was applied for 15 minutes at 1.1 MHz
frequency, 45% duty cycle and 1.4 MPa peak negative pressure.
Activation protocol
Thermosensitive lipososomes [DPPC:HSPC:Chol:DPPE-PEG (50:25:15:3)] from Nanobiotix were diluted in
OptiMEM® to a concentration of 3 μg/mL. The experiments were separated in the following categories:
1. OptiMEM + US: To check if US exposure alters the parameters of the medium.
2. Negative control: Samples kept at room temperature (0% release).
3. Positive control: Samples in 45°C water bath for 15 minutes (100% release).
49 | 2013 International Conference on Nanotheranostics (ICoN 2013)
4.
5.
6.
TSL + US (no heating): Same exposure conditions applied but without heating to check if US alone
induces any release from TSL. This was done by replacing the glycerol in the cuvette with water.
Since water has very low absorption, it does not heat up with US.
TSL + US + Heating: TSL activation experiment at p=1.4 MPa.
TSL + US + Heating*: TSL activation experiment at p=1.8 MPa.
Analysis of results
Analysis of the results was done using a 96-wells plate spectrofluorometer. The excitation wavelength, λex was 485
nm and the emission wavelength, λem was 580 nm. Each category was repeated at least three times and the results
are presented as mean ± standard deviation. The % release of doxorubicin was evaluated using equation 1 below
[1]:
(1)
where, Iexp, Ineg and Ipos are the fluorescence intensities of the experiment, negative control and positive control
respectively.
3.
Results
ΔΤ (°C)
A theoretical model based on the Pennes’ Bioheat equation was initially used to calculate the ultrasound
parameters required for temperature elevation in glycerol under conditions for drug activation (5-8°C). Fine-wire
(50μm) thermocouple readings were in close agreement with our theoretical predictions as shown in figure 2. A
temperature elevation of 6-7°C was obtained in the sample holder within 6 minutes before reaching a plateau.
Experiment
Theory
Input [MPa]
Figure 2: Comparison of experimental with predicted values for temperature elevation in glycerol as a function of input
pressure (f=1.1 MHz, 45% DC)
The results for TSL activation and release are shown in Figures 3 and 4. Figure 3 shows the fluorescence
measurements from each category whereas figure 4 shows the calculated % release [using equation (1)]. It is noted
that ultrasound induced almost as much release as the positive control while further increasing the pressure (and
temperature) does not show any added benefit. It is also noted that ultrasound does not affect the medium
properties (OptiMEM = OptiMEM + US) and that US alone does not influence the TSL or cause any release (NEG
Control = TSL + US).
Figure 3: Fluorescence measurements of the different categories.
50 | 2013 International Conference on Nanotheranostics (ICoN 2013)
4.
Discussion
% DOX Release
An ultrasound method for in-vitro drug release from thermosensitive liposomes has been developed. Water-based
cell culture media present difficulties in ultrasound heating but our method overcomes this issue. Temperature
elevation of 5-8 degrees (needed for the activation of the TSL) is reached with focused ultrasound at 1.1 MHz and
an 80% drug release was achieved with our method. Heating by thermal conduction approach mimics in-vivo
conditions where ultrasound is used to induce hyperthermia in tissue and the TSL (in blood) would be heated by
conduction.
Further improvements of the method will allow for faster and more uniform heating. A broader acoustic field
(very low focussing gain or even unfocused sources) will result in larger treatment areas. The current setup may be
used with cells in suspension; one possible modification is to allow testing of seeded cell cultrures (perhaps in
OptiCell).
Figure 4: Induced doxorubicin % release from thermosensitive liposomes.
5.
[1]
[2]
[3]
[4]
[5]
References
M. de Smet, et al., "Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance," Journal of Controlled
Release, vol. 143, pp. 120-127, 2010.
M. O. Köhler, et al., "Volumetric HIFU ablation under 3D guidance of rapid MRI thermometry," Medical Physics, vol. 36, pp.
3521-3535, 2009.
S. Dromi, et al., "Pulsed-high intensity focused ultrasound and low temperature - Sensitive liposomes for enhanced targeted
drug delivery and antitumor effect," Clinical Cancer Research, vol. 13, pp. 2722-2727, 2007.
R. Staruch, et al., "Localised drug release using MRI-controlled focused ultrasound hyperthermia," International Journal of
Hyperthermia, vol. 27, pp. 156-171, 2011.
A. Yudina, et al., "Ultrasound-mediated intracellular drug delivery using microbubbles and temperature-sensitive liposomes,"
Journal of Controlled Release, vol. 155, pp. 442-448, 2011.
[Back to Session 5]
51 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Biodegradable Dextran Nanoparticles as Potential Drug
and Fluorescent Marker Carrier
Janczewska M. Wasiak I. Pieczykolan J. Ciach T.
Warsaw University of Technology, Faculty of Chemical and Process Engineering, Biomedical Engineering Laboratory,
Warynskiego Street 1, 00 -645 Warsaw, Poland
1.
Introduction
Cancer is one of the biggest challenges of contemporary medicine. Despite the enormous scientific and
financial effort undertaken in recent years cancer remains one of the major death cause in western civilization.
Thanks to this effort various new drugs have been developed, but still many types of cancer in metastasis are
virtually untreatable. A lot of hope for new treatment methods are set on nanotechnology. This is because all the
cellular machinery and transport phenomena are happening at this size range. Nanoparticles seem to be a perfect
drug carrier due to possible targeted cancer drug delivery, what is a key to minimize main side effects of
chemotherapeutic agents and reduce tumor expansion. They are defined as submicron structures that can
covalently or physically bind therapeutic agent and administrate it directly to the tumor cells [1]. Nanoparticles
can be biodegradable and nonbiodegradable. The first type will disappear from the body after performing the
desired action, due to hydrolysis or other biological process. The second type will probably stay in our body for
very long time or even forever, what pose a potential danger on such objects. That’s why biodegradable –
organic nanoparticles are frequently considered as safe, in comparison with inorganic. Nanoparticles designed as
drug carriers should have diameter in a range of over twenty up to even hundreds of nanometers. The exact size
can differ according to the administrated drug structure and nanoparticle destination. Size is one of the crucial
nanoparticle features as it strongly influence their fate after administrating into the bloodstream. Another
important characteristic is NPs surface charge that is defined by zeta potential. These two agents decide whether the
nanoparticle will manage to avoid opsonization and removal from the bloodstream shortly after intravenous
application. Thus nanoparticles having size below a hundred nanometers and slightly charged surface seem to be a
perfect tool for targeted therapy. Depending on nanoparticles preparation method it is possible to obtain
nanocapsules or nanospheres. Nanocapsules characterize with core – shell structure, where polymeric shell
encloses often liquid core where drug is dispersed, whereas nanospheres consist of covalently modified polymeric
chain where the active agent is attached.
Biocompatibility and bioavailability of nanoparticles is necessary for them to be effective and safe as a drug
carrier. Nanoparticles made from natural polymers such as chitosan, dextran, cellulose or starch are regarded as
more biocompatibile due to their low toxicity and high biodegradability. [2] Dextran is already widely used in
medical and pharmaceutical field. It is common component of eye drops and is often used as hematopoietic
replacement agent. It is naturally synthesized by lactic - acid bacteria such as Leuconostoc mesenteroides and
Streptococcus mutan and is enzymatically degradated. Because of easy degradation there is no risk of
cumulating polymer inside organs what is particularly important when it comes to nanoparticles. [3]
Preparation nanoparticles from polysaccharide gives an enhanced targeting properties thanks to increased
requisition for glucose molecules. As Otto Warburg proven, cancerous cells are producing energy mainly by
glycolysis. Following this thought, composed from the saccharide nanoparticles have a better chance to be up
taken by malignant cells. [4] Nanoparticles are mostly integrated by endocytosis, phagocytosis and
macropinocytosis, however there is still a lack of exact information about the actual uptake mechanism.
Generally, it is claimed that the most probable path of intracellular integration is either clathrin or caveolae
mediated endocytosis. [5]
Due to its polysaccharide structure dextran can be easily oxidized to polyaldehydedextran in the reaction
with sodium periodate in aqueous solution. In this reaction glucose rings are open and oxidized without breaking the
polysaccharide backbone. The oxidation of dextran chain opens further possibilities for its modification.
Aldehyde groups afterwards, can form covalent links with amines, creating Schiff base. Further Amadori
rearrangement can stabilize obtained compound. If the covalently linked groups are lipophylic we can observe a
molecular selfassembly leading to nanoparticle formation, what is the main topic of this paper. The same
chemical reaction was employed to attach anticancer drug and fluorescent probes. Attaching anticancer drug by
Schiff base assures that the drug will be released inside a tumor cells after internalization, due to decreased pH in
lisozomes.
The aim of this paper is to obtain and characterize dextran nanoparticles designed for drug delivery and
diagnosis. Nanoparticles were synthesized, adjusting proportions of reagents in order to obtain particles about
100 nm. The cytotoxicity was tested in vitro on A549 human lung carcinoma, MCF10A human breast cells and
52 | 2013 International Conference on Nanotheranostics (ICoN 2013)
MES-SA/Dx5 human uterine sarcoma cell lines. Futhermore, in vivo studies were conducted on mouse model of
multidrug resistant human uterine sarcoma (MES-SA/Dx5).
2.
Methods
Nanoparticles were obtained from 40 and 70 kDa dextran (Nobilus Ent), which was oxidized in aqueous
solution with sodium periodate. The number of aldehyde groups in dextran chain was determined by the reaction
with hydroxylamine hydrochloride and titration with sodium hydroxide nominated solution. The product was
purified by dialysis against pure water for three days and dried overnight in 50ºC. Nanoparticles were prepared
by mixing solution of polyaldehydedextran (PAD), dodecylamine hydrochloride and common anticancer drug –
doxorubicin hydrochloride (Sequoia Research Products Ltd). In some cases fluorescent (9-aminoacridine) and
non fluorescent (trypan blue) dies were also covalently linked. Reaction was carried out in 30ºC in water bath
for 1,5 h. Product was purified by dialysis against pure water for an hour in order to remove non reacted
substances. The diameter of nanoparticles was measured using Nanoparticle Tracking Analysis (NTA) with
NanoSight. Subsequently, product was lyophilized and redissolved in order to examine if nanoparticles are
stable after dry storage. Cell lines were grown and nanoparticles were tested for cytotoxicity by test MTT. To
obtain fluorescent nanoparticles, fluorescent agent was covalently attached.
To improve fluorescent properties, fluorescent nanocristals were precipitated inside polysaccharide
nanoparticles. Fluorescent nanocrystals were precipitated by mixing 9-aminoacridine stearate tetrahydrofuran
solution with water containing polysaccharide nanoparticles.
3.
Results
Developed nanoparticles are bioavailable and biodegradable. Size range of synthesized particles was about 70
to 100 nm, depending on polysaccharides and coiling agents used in synthesis, and was suitable for a drug
carrier. Developed method is quick and safe as it does not concerns usage of any organic solvents. Nanocarrier
gave no therapeutic response in cytoxicity tests, whereas nanoparticles with drug attached gave therapeutic
effect comparable to toxic influence of doxorubicin. To confirm in vitro experiments the in vivo studies were
conducted on the same type of carcinoma cell line. Too large dosage and too frequent administration turned out
to be fatal for representative group of mice. However a medium dosage of 7,5 mg/kg injected intravenously
every 6 days was not toxic for mouse organism in comparison with free doxorubicin administrated in the same
way. The Tumor Growth Inhibiton (TGI) was defined and calculated and it oscillated at 29% for NPs with
Doxorubicin (7,5 mg/kg) in 35 day of experiment. Free doxorubicin in the same dosage showed a fatal influence
on studied group.
Results of cancer cell staining with a use of fluorescent nanoparticles showed that polysaccharide
nanoparticles are efficiently entrapped by the cancer cells, even dies that never enters the intact cell (trypan
blue) can be efficiently transported into the cell interior. Organic fluorescent nanocristals precipitated inside
polysaccharide nano-shells can serve as an efficient cancer detection and staining technique. Fluorescent organic
nanocrystals due to its high quantum efficiency can be a safe and biodegradable alternative to quantum dots.
4.
Acknowledgements
Presented research was partially supported by EU within the frame of uroNanoMed project FonDiag.
International patent pending by Nanovelos company (polysaccharide nanoparticles).
5.
References
[1] I. Brigger, C. Dubernet, P. Couvreur, “Nanoparticles in cancer therapy and diagnosis,” Adv. Drug. Del. Rev. 54, 631-651
(2002).
[2] A. Aumelas et al., “Nanoparticles of hydrophobically modified dextrans as potential drug carrier systems,” Coll. Surf. B: Bioint. 59
74- 80 (2007).
[3] S. Daoud-Mohammed et al., “Spontaneous association of hydrophobized dextran and poly-β-cyclodextrin into
nanoassemblies. Formation and interaction with a hydrophobic drug,” J. Coll. Int. Sci. 307, 83-93 (2007).
[4] E. Christ, “The Warburg effect and its role in cancer detection and therapy” in Program in Biotechnology Department of
Biological Sciences, (Columbia University, 2009).
[5] Ruthrotha B. Selvi et al., “ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors,” J.
Nanobio. 10:35, (2012).
[Back to Session 5]
53 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Engineering carbon nanotubes based scaffolds for the
efficient delivery of new tyrosine kinase inhibitors
Davide Giust, Kostas Kostarelos
UCL School of Pharmacy - 29-39 Brunswick Square - London - WC1N 1AX
[email protected]; [email protected]
Over-expression and/or co-expression of epidermal growth factor receptors (EGFRs) subtypes has been
found in numerous tumor types, including colon, breast, ovarian, non-small lung and other malignant
cancers [1]. Tyrosine Kinase Inhibitors (TKis) selective for the EGFR and inhibiting the related Tyrosine
Kinase activity have been approved for current therapy of different malignant forms. Nevertheless in
most cases, patients survival is strictly related to the decreased activity of these inhibitors due to receptors
mutation and/or decreased penetration inside cells [2]. In this sense, carbon nanotubes (CNTs) represent
good candidates to improve the delivery of Tkis. CNTs are allotropic form of carbon made of graphene
enrolled sheets with sp2 hybridized carbon, displaying unique chemical and physical properties useful for
the delivery of bio-active molecules [3]. The aim of this project is to use modified-CNTs to improve the
delivery of new synthesized TKis. This will achieve by the meaning of different functionalization
approaches. This involves the non-covalent attachment of Tkis to the side wall of MWNT upon π-π
stacking interactions, as well as the formation of covalent and covalent cleavable bonds [4-5] (Scheme 1).
All CNTs-Tkis will be further investigated in vitro as well as new synthesized Tkis. Human Glioblastoma
has been chosen as in vitro model among the new therapeutic challenges for these type therapeutic agents.
These malignant cells are in fact, one of the most challenging in cancer therapy due to the onset of
multi drug resistance and bad prognosis in all patients [6]. The project, still in progress, is at the halfway between the synthesis and the in vitro screening of the CNT-TKis complexes. The most active
among the new synthesized TKis have been identified. By the non-covalent interaction between the CNT
and TKis, no improvement of the activity was found compared to that one of TKi alone. Some synthetic
trials are actually on going to get covalently conjugates CNTs-TKis and evaluate their the activity in vitro.
Scheme 1. Synthetic approach used to functionalized CNTs with the Tkis.
References
[1] A. Arora and E. M. Scholar, “Role of Tyrosine Kinase Inhibitors in Cancer Therapy”, The Journal of Pharmacology and Experimental
Therapeutics, 315(3): 971-979 (2005).
[2] T. E. Taylor, F. B. Furnari and W. K. Cavenee, Curr Cancer Drug Targets, 12(3): 197–209 (2012).
[3] G. Pastorin, W. Wu, S. Wieckowski, J. P. Briand, K. Kostarelos, M. Prato, A. Bianco, “Double functionalisation of carbon nanotubes for
multimodal drug delivery”, Chem. Commun. 1182–1184 (2006).
[4] H. Ali-Boucetta, K. T. Al-Jamal, D. McCarthy, M. Prato, A. Bianco, K. Kostarelos, “Multiwalled carbon nanotube-doxorubicin
supramolecular complexes for cancer therapeutics”, Chem Commun (Camb), 4:459–461 (2008).
[5] C. Samori, H. Ali-Boucetta, R. Sainz, et al. “Enhanced anticancer activity of multi-walled carbon nanotube-methotrexate conjugates using
cleavable linkers”, Chem Commun (Camb), 46:1494–1496 (2010).
[6] A. D. Joshi, W. Loilome, I-Mei Siu, B. Tyler, G. L. Gallia, G. J. Riggins, “Evaluation of Tyrosine Kinase Inhibitor Combinations for
Glioblastoma Therapy”, PLOS ONE, 7 (10) (2012).
[Back to Session 5]
54 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Folic Acid functionalized Quatro-NanoContainers as
targeted agent: In vitro and In vivo study
Eleni K. Efthimiadoua*, Christos Tapeinos a , Eirini Fragogeorgib,c , George Loudosb, and George Kordas
*a
a
Sol-Gel Laboratory, Institute for Advanced Materials, Physicochemical processes, Nanotechnology & Microsystems, NCSR
“Demokritos”, 153 10 Aghia Paraskevi Attikis, Greece
b
Department of Medical Instruments Technology, Technological Educational Institute, GR 122 10 Athens, Greece
c
Radiochemical/ Radiopharmacological Quality Control Laboratory, Institute of Nuclear and Radiological Sciences and Technology,
Energy & Safety, N.C.S.R. ‘Demokritos’, 15310 Aghia Paraskevi, Greece
Nanocontainers (NCs) can serve as highly versatile platforms for the controlled functionalization and delivery
in the last decade as DDS. The responsiveness of the functionalized NCs could be attained by choosing
monomers with specific sensitivities, such as, sensitivity in temperature NIPAAm, HPMA, DMAEMA),
pH (e.g. AA, MAA) and reductive-oxidizing (redox) environments, which are desired for selective
release in tumor area. Nanospheres present many advantages such as stability, presence of many active
groups that can be easily functionalized with specific molecules for targeted therapy and coated with
polymers improving their circulation time in bloodstream and inducing therefore, stealthy properties. For the
targeted therapy through the NCs, the small molecule of folic acid has been employed. Because folate
receptors are known to be overexpressed and actively internalized through folate receptor mediated
endocytosis in various types of cancer cells, folic acid has been considered to be a suitable ligand for
improving the cellular uptake of drugs and macromolecules into cells. The NCs were modified by
carbodiimide chemistry with FITC and/or Folic Acid. Ovarian cells, HeLa, which overexpress the folic acid
receptor, were incubated for 72 h with the empty and drug loaded NCs aiming at studying the targeted
internalization mechanism with and without folic acid.
Without Folic Acid
With Folic Acid
Figure 1. Cellular trafficking of FITC-labeled and FA- FITC-labeled NCs in HeLa cells. The cells were incubated for
1h with the FITC-labeled NCs in the presence of LysoTracker Red (10 min) at 37 oC, followed by live cell imaging. In
all experiments cells were treated with either 3 μM folic acid functionalized NCs.
References
1.
2.
Brannon-Peppas, L.; Blanchette, J. O., Nanoparticle and targeted systems for cancer therapy. Advanced Drug Delivery Reviews
2012, 64, 206-212.
Brigger, I.; Dubernet, C.; Couvreur, P., Nanoparticles in cancer therapy and diagnosis. Advanced Drug Delivery Reviews
2012, 64, 24-36.
[Back to Session 6]
55 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Doxorubicin-loaded and Antibody-Conjugated LiposomeQD Hybrid Vesicles for Targeted Cancer Theranostics
Bowen Tian1, Wafa Al-Jamal2, KostasKostarelos2
1. University of Nottingham, United Kingdom
2. University College London, United Kingdom
[email protected]
Quantum dot (QD) have been extensively explored for in vitro and in vivo imaging due to their superior
fluorescence properties compared to organic fluorophores. The hydrophobic nature of QD hinders their
biomedical applications in biological milieu, therefore many efforts have been made to make-water soluble QD
by substituting the organic surface ligands with hydrophilic moieties. However such surface modifications
adversely affected the QD optical properties and colloidal stability. Previously, our group offered an alternative
approach to improve QD hydrophilicity by incorporating hydrophobic QD into the liposome lipid bilayer which
efficiently labelled cancer cells in vitro and in vivo[1]. In this study, we report the engineering of the multimodal
liposome-QD hybrids (L-QD) for targeted cancer theranostics. L-QD was loaded with doxorubicin (Dox) using
the osmotic gradient technique, achieving high loading efficiency compared to liposome alone[2]. Structural
elucidation using cryogenic electron microscopy (cryo-EM) clearly showed that QD were incorporated into the
lipid bilayer and Dox were encapsulated into the liposome aqueous core. Furthermore, the surface of Dox-loaded
hybrids were functionalized with anti-MUC-1 antibody for active targeting, using the post-insertion technique.
The specific binding of antibody-targeted hybrids was studied against MUC-1 epitope by surface plasmon
resonance (BIACORE) showing higher binding affinity compared to antibody alone due to multivalent effect. In
addition, cellular uptake studies of the antibody-targeted hybrids were conducted using confocal laser scanning
microscopy (CLSM). The antibody-targeted hybrids showed high binding and uptake by human breast cancer
cell lines (MCF-7) that overexpress MUC-1 receptors in contract to human pulmonary adenocarcinoma cells
(Calu-6) exhibiting low level of MUC-1 expression. Finally, cytotoxicity assays indicated higher toxicity of
antibody-targeted hybrids in MCF-7 compared to Calu-6 cells. In conclusion, MUC-1 antibody-targeted L-QD
hybrids encapsulating doxorubicin are thought to constitute a potential multimodal system for the simultaneous
delivery of therapeutic and diagnostic agents to cancer cells in vitro and in vivo.
References
1.
2.
Al-Jamal, W. T., Al-Jamal, K. T., Tian, B., Lacerda, L., Bornans, P. H., Frederik, P. M., Kostarelos, K., "Lipid-quantum dot bilayer
vesicles enhance tumor cell uptake and retention in vitro and in vivo", Acs Nano, 2008. 2(3): p. 408.
Tian, B., Al-Jamal, W. T., Al-Jamal, K. T., Kostarelos K., "Doxorubicin-loaded lipid-quantum dot hybrids: surface topography and
release properties", International Journal of Pharmaceutics, 2011. 416(2): p.443.
[Back to Session 6]
56 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Monitoring tumor burden by multicolor in vivo flow
cytometry
Costas Pitsillides, Konstantinos Kapnisis and Andreas Anayiotos
Department of Mechanical Engineering and Materials Science and Engineering, Cyprus University of Technology, Limassol, Cyprus
[email protected]; [email protected]; [email protected]
1. Introduction
In vivo measurement of tumor burden, both in cancer research models and in patients, is an important parameter
for the accurate assessment of disease progression and the response to therapeutic intervention [1]. Several in
vivo imaging modalities have been utilized in the assessment of tumor burden, including functional magnetic
resonance imaging, computer tomography and positron emission tomography [2, 3], fluorescence imaging [4, 5],
intravital microscopy [6] and bioluminescence imaging [7]. More recently, the detection/quantification of
circulating cancer cells has been explored as a method to evaluate tumor burden in the context of assessing
disease stage, prognosis as well as monitoring disease progression following therapeutic intervention in cancer
patients [8, 9]. Clinically, various ex vivo assays have been developed to detect cancer cells shed in circulation
by primary tumors, including breast cancer, prostate cancer and small-cell lung cancer [10, 11].
In vivo flow cytometry has been developed as a method for real-time detection of circulating cancer cells
injected into the circulation of experimental animals. The method does not require extraction of blood samples
and is therefore well suited for long-term monitoring of circulating tumor cells. In this report, we report on the
development of a multichannel in vivo flow cytometer to detect and quantify circulating cancer cells as a means
of assessing the tumor burden in animal models.
2. Materials and methods
Development of a multichannel in vivo flow cytometer Multichannel in vivo flow cytometry combines the
principles of confocal detection and flow cytometry in order to enable the real-time detection of fluorescently
labeled cells circulating in a live animal. The system can be applied for the dynamic and simultaneous
monitoring of multiple populations of circulating cells, which can be targeted and labeled with multiple
fluorescent markers and probes. To accomplish this, light from up to three separate excitation lasers was focused
by a cylindrical lens and then imaged across a blood vessel to form an excitation slit. Figure 1 illustrates the
concept of the multichannel in vivo flow cytometer. When fluorescently labeled cells were flown through the
excitation slit, the emitted fluorescence signal emitted was confocally detected by a photomultiplier tube in each
of the detection channels of the system. The collected PMT signal was sent to an analog-to-digital converter to
be digitized and then stored on a PC to be analyzed by Matlab software in order to identify cell peaks and extract
quantitative information on the number of cells passing.
Figure 1. Schematic of the multichannel in vivo flow cytometer
The excitation lasers chosen were the 633 nm Helium-Neon laser, preferred due to its good penetrating
capability through tissue and blood, as well as the 561 nm (Cobolt AB, Solna, Sweden) and 488 nm (Coherent
Inc, Santa Clara, CA, USA) diode-pumped, solid-state lasers, chosen for their ability to excite fluorescence in a
host of commonly used fluorochromes such as green fluorescent protein (GFP), fluorescein, and the yellow/red
fluorescent protein variants (YFP/RFP). Arteries (with average diameter of 40-50 µm), rather than veins, were
chosen for experiments because of faster blood flow and absence of cell-endothelium interactions.
57 | 2013 International Conference on Nanotheranostics (ICoN 2013)
In vivo detection of circulating cancer cells
Approx. 106 cells/ml from the MDA-MB-231 breast adenocarcinoma cell line were incubated with the Vybrant
series of lipophilic fluorescent probes (each at a concentration of 5 µg/ml) and then separately injected through
the tail vein of anesthetized male, 10-12 week old, CD1 mice in order to demonstrate the in vivo capabilities of
the system. The anesthetized animals were placed on an imaging platform within 5 minutes after injection of the
cells and an appropriate arteriole in the ear was chosen from which to obtain circulating cell count.
Figure 2. Breast adenocarcinoma cells labeled with the Vybrant DiD fluorescent probe and detected in vivo
3. Results and discussion
We have developed the multichannel in vivo flow cytometer for the dynamic monitoring of circulating cells and
have demonstrated its capabilities in detecting and quantifying fluorescently labeled breast adenocarcinoma cells
directly injected in the circulation of experimental animals. The system was designed and built with the ability to
simultaneously assess the circulation kinetics of several distinct cell populations in a single animal. This will
allow for a more efficient in vivo investigation of complex biological processes by enabling the simultaneous
monitoring of the multiple cell populations that might be participating and interacting in such processes.
To better approximate the tumor environment in vivo, a mouse tumor model will be developed through the
adoptive transfer of fluorescent protein expressing cancer cells in immune-compromised mice. Once the cells
migrate to tumor growth areas and the tumors are established, the animals will be monitored long term using the
multichannel in vivo flow cytometer in order to quantify the fluorescently labeled tumor-shed cells in
circulation. Tumor burden will also be assessed via whole body reflectance imaging and the results will be
compared to data on circulating cancer cells in order to validate the method for the in vivo assessment of tumor
burden in animals. By quantifying circulating tumor cells, in cancer disease models that include a circulating cell
component, the in vivo flow cytometer can be used to non- invasively track tumor burden and thus assess
important cancer treatment parameters such as the tumor growth and the response to therapeutic intervention.
4. References
[1] G. P. Schmidt, H. Kramer, M. F. Reiser, and C. Glaser, “Whole-body magnetic resonance imaging and positron emission tomographycomputed tomography in oncology,” Top. Magn. Reson. Imaging, vol. 18, no.3, pp. 193-202, Jun. 2007.
[2] T. Beyer, et al, “A combined PET/CT scanner for clinical oncology,” J. Nucl. Med., vol. 41, no. 8, pp. 1369-1379, Aug. 2000.
[3] H. U. Kauczor, C. Zechmann, B. Stieltjes, and M. A. Weber, “Functional magnetic resonance imaging for defining the biological target
volume,” Cancer Imaging, vol. 6, no. 1, pp. 51-55, Jun. 2006.
[4] C. S. Mitsiades, et al, “Fluorescence imaging of multiple myeloma cells in a clinically relevant SCID/NOD in vivo model: biologic and
clinical implications,” Cancer Res., vol. 63, no. 20, pp. 6689-6696, Oct. 2003.
5] H. Yamamoto, et al, “Quantitative assessment of small intraosseous prostate cancer burden in SCID mice using fluorescence imaging,”
Prostate, vol. 67, no. 1, pp. 107-114, Jan. 2007.
[6] J. Condeelis and J. E. Segall, “Intravital imaging of cell movement in tumours,” Nat. Rev. Cancer, vol. 3, no. 12, pp. 921-930, Dec.
2003.
[7] C. M. Deroose, et al, “Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, smallanimal CT, and bioluminescence imaging,” J. Nucl. Med., vol. 48, no. 2, pp. 295-303, Feb. 2007.
[8] M. Cristofanilli, “Circulating tumor cells, disease progression, and survival in metastatic breast cancer,” Semin. Oncol., vol. 33, no. 3,
pp. S9-14, Jun. 2006.
[9] J. B. Smerage and D. F. Hayes, “The measurement and therapeutic implications of circulating tumour cells in breast cancer,” Br. J.
Cancer, vol. 94, no. 1, pp. 8-12, Jan. 2006.
[10] Y. P. Sher, et al, “Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with
multiple marker genes,” Clin. Cancer Res., vol. 11, no. 1, pp. 173-179, Jan. 2005.
[11] K. Pachmann, et al, “Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows
detection of patients at risk of early relapse,” J. Clin. Oncol., vol. 26, no. 8, pp. 1208-1215, Mar. 2008.
[Back to Session 6]
58 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Role of the Cell-Division Cycle on Nanoparticle Cellular
Accumulation and Implications for Cancer Targeting
Christoffer Åberg, Kenneth A. Dawson
Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
[email protected]; [email protected]
The role of nanoparticle size and surface properties on nanoparticle uptake by cells has been extensively studied,
both to investigate potential hazards of nanoparticles, but also to develop new and improved medicines. Of
particular importance in such studies is the so-called 'biomolecular corona' which denotes the biomolecules that
associate (often strongly) with nanoparticles in realistic biological milieux [1]. For example, in a realistic
biological milieu, such as in the presence of serum, nanoparticle cellular uptake is vastly reduced compared to in
the absence of serum [2]. Furthermore, the impacts are also altered significantly, with cytotoxic responses
essentially completely mitigated in the more realistic milieu [3].
Going beyond physicochemical properties of the nanoparticles and their dispersion, less attention has been
given to biological variables, for instance the evolution of the cell population. In vitro cell-nanoparticle studies
are typically performed on cell lines, where the cells continuously divide. We have shown experimentally how
this evolution of the cell population leads to a characteristic ranking during continuous exposure, where cells in
the G2/M phase have a higher intracellular nanoparticle load, followed by cells in the S phase, and finally cells
in G0/G1 phase with the lowest load [4]. Several other phenomena can also be traced back to an evolving cell
population.
The evolution of the cell population can be described by a simple model and also simulated numerically
[4,5]. Experimental data provides all parameters of the model and subsequent parameter-free studies can be used
to validate the model, to excellent agreement. Upon this basis, we extend the model to include nanoparticle
uptake and accumulation. The whole range of experimental observables can be calculated, and compared with
experimental data. We demonstrate notable agreement with several careful experiments.
The model can also be used when the nanoparticles cause functional impacts on the cells. We demonstrate
the approach by elucidating limits in targeting nanoparticles towards cancer cells [5]. The model places rather
stringent demands on the targeting efficiency required to reach therapeutic nanoparticle loads in dividing
(cancerous) cells compared to slow-dividing (healthy) cells, and can be used as a framework for optimizations.
References
[1] M. P. Monopoli, C. Åberg, A. Salvati and K. A. Dawson, “Biomolecular coronas provide the biological identity of nanosized materials,”
Nature Nanotechnology 7, 779-786 (2012).
[2] A. Lesniak, A. Salvati, M. Santos-Martinez, M. Radomski, K. A. Dawson and C. Åberg, “Nanoparticle adhesion to the cell membrane
and its effect on nanoparticle uptake efficiency,” Journal of the American Chemical Society 135, 1438-1444 (2013).
[3] A. Lesniak, F. Fenaroli, M. P. Monopoli, C. Åberg, K. A. Dawson and A. Salvati, “Effects of the presence or absence of a protein corona
on silica nanoparticle uptake and impact on cells,” ACS Nano 6, 5845–5857 (2012).
[4] J. A. Kim, C. Åberg, A. Salvati and K. A. Dawson, “Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell
population,” Nature Nanotechnology 7, 62-68 (2012).
[5] C. Åberg, J. A. Kim, A. Salvati and K. A. Dawson, “Theoretical framework for nanoparticle uptake and accumulation kinetics in
dividing cell populations,” Europhysics Letters 101, 38007 (2013).
[Back to Session 6]
59 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Challenges in Nanotheranostics: A Materials Perspective
Rena Bizios
University of Texas at San Antonio, USA
[Back to Session 7]
60 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Magnetoactive electrospun nanocomposite membranes in
drug delivery and hyperthermia applications
Ioanna Savva1, Andreani D. Odysseos2, Loucas Evaggelou1, Oana Marinica3, Eugeniu Vasile4, Ladislau
Vekas5, Yiannis Sarigiannis6 and Theodora Krasia-Christoforou1
1University of Cyprus, Department of Mechanical and Manufacturing Engineering, Nicosia, Cyprus
2 EPOS-Iasis, R&D, Department of Biomedical Research, Nicosia, Cyprus
3National Center for Engineering of Systems with Complex Fluids, University ‘‘Politehnica’’ Timisoara, Timisoara, Romania
4 METAV Research & Development, Bucharest, Romania
5Center for Fundamental and Advanced Technical Research, Romanian Academy, Timisoara Branch, Timisoara, Romania
6 Department of Materials Science, University of Patras, 26504 Patras, Greece.
Magnetoactive, polymer-based fibrous nanocomposites belonging to the broad category of stimuli-responsive
materials consist of magnetic nanoparticles (MNP) embedded within a polymeric fibrous matrix. The presence of
MNP within these materials allows for the manipulation of their properties by an externally applied magnetic
field, rendering them useful in numerous technological and biomedical applications including sensing, magnetic
separation, catalysis and magnetic drug delivery.
Electrospinning is a simple, versatile and low cost technique employed for the production of continuous
(nano)fibers of various materials with diameters from a few nm up to a few micrometers [1]. The fiber
morphology and dimensional characteristics depend on the polymer and solution properties as well as on
processing parameters. The large surface-to-volume ratio, the existing flexibility on the selection of the surface
functionality and the freedom on materials’ design, comprise representative characteristics that render the
electrospun polymeric (nano)fibers and the resulting membranes ideal for numerous applications.
The present work focuses on the fabrication of electrospun magnetoactive fibrous nanocomposite membranes
based on the water soluble and biocompatible poly(ethylene oxide) (PEO), the biocompatible and biodegradable
poly(L-lactide) (PLLA) and pre-formed oleic acid coated magnetite nanoparticles (OA.Fe3O4). Scanning and
transmission electron microscopy techniques, reveal the presence of continuous fibers of approximately 2 μm in
diameter, with the magnetic nanoparticles being evenly distributed within the fibers, retaining at the same time
their nanosized diameters (~5 nm). Assessment of the magnetic properties of these materials by vibrating sample
magnetometry discloses superparamagnetic behaviour at ambient temperature. For the first time the
biocompatibility and biodegradability of PEO/PLLA and the tunable magnetic activity of the OA.Fe3O4 are
combined in the same drug delivery system, with N-acetyl-p-aminophenol (acetaminophen) as a proof-ofconcept pharmaceutical (Figure 1). Drug release kinetic profiles showed that the presence of OA.Fe3O4 as well
as the protein content of the release medium as critical parameters for the release rate. Furthermore, their heating
ability under alternating current (AC) magnetic field conditions is evaluated using frequency of 110 kHz and
corresponding magnetic field strength of 25mT (19.9 kA/m), which is very close to the typical values of 100 kHz
and 20mT used in medical treatments.
Fig. 1. Schematic presentation of the drug release process and photograph of the drug-loaded
PEO/PLLA/OA.Fe3O4/acetaminophen membrane immersed in an aqueous solution.
References
[1] Z. M. Huang, Y. Z. Zhang, M. Kotaki et al. Comp. Sci. Technol. 2003, 63, 2223.
[Back to Session 7]
61 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Electrospun PEO/PLLA Fibrous Meshes for Sustained
Tyrosine Kinase Inhibitors Delivery in Situ
Maria Kokonou1,2, Fotios Mpekris3, Triantafyllos Stylianopoulos3, Jean-Michel Siaugue2 and Andreani
Odysseos1
1EPOS-Iasis Research and Development Ltd., 2028 Nicosia, Cyprus
2Physicochimie des Electrolytes, Colloïdes et Sciences Analytiques (PESCA), Université Pierre et Marie Curie Univ Paris 06, 75252 Paris
Cedex 05, France
3Department of Mechanical and Manufacturing Engineering, University of Cyprus, 1678 Nicosia, Cyprus
[email protected], [email protected]
1. Background
Tyrosine Kinase Inhibitors (TKI) comprise a promising targeted molecular therapeutic solution for tumors
associated with aberrant expression of mutated forms of Tyrosine Kinase Receptors (TKR). Gefinitib (Iressa) is
an epidermal growth factor receptor (EGFR) inhibitor, in particular the first selective inhibitor of EGFR tyrosine
kinase domain. Electrospun fibrous, biocompatible and biodegradable membranes [1] are a very promising
nanosystem for localized drug delivery in situ, due to their very high surface to volume ratio, their wide range of
materials, easy fabrication and tailored physical, chemical and mechanical properties.
In this work, electrospun polyethylene oxide (PEO)/poly(L-lactid) acid (PLLA) nanofibers are introduced as
nanosystems for sustained Gefinitib delivery in solid tumors in situ. Gefinitib is blended to the polymeric
solution prior to electrospinning procedure, in two different ways: pristine or grafted on functionalized
fluorescent magnetic nanoparticles (NPs) intended for targeted drug delivery [2,3].
2. Experimental
PEO/PLLA polymeric solutions were formed with variable ratio (100/0, 90/10, 70/30, 50/50, 30/70, 10/90), in
order to control their biodegradation rate, which is the main parameter that determines the drug release duration
and rate. A wide parametric study of the electrospinning process was realized to find the optimum parameters
that result in solid, fibrous meshes with cylindrical fibers isotropically oriented and uniform in diameter. These
characteristics are essential in achieving a homogeneous and controlled drug release. Then the polymeric
solutions were loaded with Gefinitib in one group of experiments, and with maghemite NPs in a second group of
experiments, and fibrous meshes were fabricated again under the optimum electrospinning parameters. These
meshes were characterized in terms of geometrical characteristics, mechanical properties and drug release rate by
scanning electron microscopy (SEM), dynamic mechanical analysis (DMA) and UV-visible spectroscopy
respectively. Mechanical characterization of electrospun membranes intended to be used as implants for
localized drug delivery is important, since elasticity of the membranes determine their applicability and
functionality.
3. Results
For low concentrations of PLLA there is great uniformity on the geometry of the fibers, which are cylindrical,
with fiber diameter at 3.7 ± 0.35 μm. Above 50% beads start to form (Fig. 1). The introduction of Gefinitib
reduced diameters to 2.12 ± 0.35 μm, improved the homogeneity of the meshes and prevented the formation of
beads at high PLLA concentrations, while introduction of the magnetic nanoparticles did not affect significantly
the geometry of the fibers.
Fig. 1 PEO/PLLA (90/10, 50/50, 10/90) electrospun fibers
62 | 2013 International Conference on Nanotheranostics (ICoN 2013)
The elastic modulus of the meshes was found to be 115.8±38.7 kPa (100/0), 98.3±23.2 kPa (90/10), 131.7
±11.6 kPa (70/30), 171.2 ± 54.2 kPa (50/50), 42.8 kPa (10/90), i.e. it was in the range of 42.8 to 171 kPa with the
70/30 and 50/50 meshes being the stiffest with statistically significant difference (p<0.05) compared to the 90/10
meshes but not the 100/0. This range is comparable with electrospun polyurethane meshes and an order of
magnitude lower than cellulose acetate meshes [4]. while drug release duration increased with increasing PLLA
concentration as expected, since PLLA does not hydrolyze, from 2 hours (pure PLLA) up to > 6 months (PLLA>
50%).
4. Conclusions
TKI-loaded PEO/PLLA fibrous membranes were successfully fabricated by electrospinning, with tailored drug
release durations from a few hours up to several months. Introduction of the drug took place with blending of the
drug in the polymer solution or by grafting the drug on functionalized nanoparticles and then blending in the
polymer solution, enhancing this way functionality of the membranes. None of the two ways of drug
introduction affected the structure of the membranes. Especially in the case of pristine drug blending,
improvement of the homogeneity of the fibers was observed. These membranes are important for localized drug
delivery or functionalized nanoparticles delivery on solid tumors in situ, where targeting of the nanoparticles is
not possible, allowing this way the local administration of multifunctional, both therapeutic and tracking
schemes.
3. References
[1] A. J. Meinel, O. Germershaus, T. Luhmann, H. P. Merkle, L. Meinel, “Electrospun matrices for localized drug delivery: Current
technologies and selected biomedical applications”, Eur. J. Pharm. Biopharm. 81(1), 1-13 (2012).
[2] T. Georgelin, S. Bombard, J.-M. Siaugue and V. Cabuil, “Nanoparticle-Mediated Delivery of Bleomycin,” Angew. Chem. Int. 49,
8897¬8901 (2010).
[3] A. B. Davila-Ibanez, V. Salgueirino, V. Martinez-Zorzano, R. Mariño-Fernández, A. García-Lorenzo, M. Maceira-Campos, M. MuñozUbeda, E. Junquera, E. Aicart, J. Rivas, F. J. Rodriguez_Berrocal and J. L. Legido, “Magnetic Silica Nanoparticle Cellular Uptake and
Cytotoxicity Regulated by Electrostatic Polyelectrolytes – DNA Loading at Their Surface”, ACS Nano 6(1), 747-759 (2012).
[4] T. Stylianopoulos, M. Kokonou, S. Michael, A. Tryfonos, C. Rebholz, A. D. Odysseos and C. C. Doumanidis, “Tensile Mechanical
Properties and Hydraulic Permeabilities of Electrospun Cellulose Acetate Fiber Meshes”, J. Biomed. Mat. Res. PART B 100B(8),
2222¬2230 (2012).
4. Acknowledgements
The authors gratefully acknowledge EU for funding through FP7 IAPP/NANORESISTANCE/Grant Agreement
Number: 286125
[Back to Session 7]
63 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Drug delivery through reconstructed bronchial mucus
modified by functional carrier particles (FCPs)
Marcin Odziomek, Tomasz R. Sosnowski, Leon Gradoń
Faculty of Chemical and Process Engineering, Warsaw University of Technology, 1 Waryńskiego Street, 00-645 Warsaw, Poland
[email protected], [email protected], [email protected]
1. Introduction
Mucus covering surface of the tracheobronchial tree is the part of natural biological barrier that protects
organism against inhaled foreign particles. Strange matter deposited on the mucus surface is removed towards
upper respiratory tract by flow induced by ciliated epithelial cells. As the result of this highly efficient
mechanism drug particles are removed before they start to interact with receptors localized in the membranes of
the epithelial cells. The importance of this problem is especially pronounced in diseased conditions when the
mucus thickness and its viscoelasticity is significantly increased [1]. The effectiveness of treatment can be
improved by altering mucus structure by mucoactive substances, e.g. mucolytics [2].
The aim of the research was analysis of the rheology and mass transfer through the mucus modified by, so
called, functional carrier particles (FCPs) and their individual components. FCPs are microparticles prepared by
spray drying technique, and they are expected potentially useful in aerosol therapy as carriers of inhalation drugs
[3]. They are composed of a mucolytic (N-acetylcysteine, NAC) capable of reducing disulphur bonds between
mucin molecules, and a low molecular-weight dextran which acts as an osmotic agent and the stabilizer of FCPs.
2. Methods
The bronchial mucus was reconstructed according to the modified procedure of McGill and Smyth [4]. Raw
mucin – main component of natural mucus (Sigma Aldrich, Germany) was dissolved in the phosphate buffer (7.4
pH) to the final concentration of 20 % w/w. A small amount (0,05% w/w) of sodium azide was added to protect
the samples from microbial contamination.
Rhodamine B (POCH, Poland) was selected as a model substance for mass transfer studies. Its molar mass
(479 g/mol) is similar to the molar mass of many drugs used in the treatment of respiratory tract diseases (e.g.
disodium cromoglycate: 468.4 g/mol). Moreover, Rhodamine B is a fluoresecent dye which significantly
simplifies selectivity and sensitivity of quantitative determination of transferred material by spectrofluorimetry.
The effective diffusion coefficient De of Rhodamine B in the mucus was estimated using made in-house
plexiglass diffusion chambers (Fig. 1).
Fig. 1.Schame of diffusion chamber.
A layer of mucus sample (thickness – 2 µm) was placed between two drug-permeable membranes (PVDF,
Millipore, USA) with a pore size 0.1 µm. The donor compartment of the diffusion chamber was filled with a
Rhodamine B solution (0,05 mg/ml), whereas the receiver part with the pure phosphate buffer (pH 7,4). In order
to reduce of the local mass transfer resistance at the membranes, both solutions were continuously agitated.
Samples for analysis were taken from the receiver compartment at regular time intervals during 6 hours. The
content of Rhodamine B in these samples was determined quantitatively using fluorescence spectrometer
(Lumina - Thermo Scientific, USA). The effective diffusion coefficient De was calculated using the solution of
Fick’s law, with the justified assumptions [5].
The viscosity of samples at physiological value of share rate was measured using rotational rheometer (Smart
- Fungilab, Spain).
64 | 2013 International Conference on Nanotheranostics (ICoN 2013)
3. Results and discussion
Viscosity and diffusivity results (Fig. 2) indicate that mucus structure is destructed by the mucolytic (reduction
of mucus viscosity) which has an influence on the mass transfer rate through the mucus even for a relatively
small molecules like Rhodamine B (molecular mass, Mr = 479). The effective diffusion coefficient of
Rhodamine B calculated for unmodified mucus (20-25•10-10 m2/s) is approximately 5 times lower than in a
pure buffer solvent (119•10-10 m2/s). De value increases to 42•10-10 m2/s for mucus modified by FCPs (1%
w/w content), which is slightly less than after modification of mucus structure by pure NAC (2% w/w content:
De = 55•10-10 m2/s). Presence of dextran (needed as FCPs stabilizer), caused only slight increase of mucus
viscosity and, in consequence, a small decrease of mass transfer rate.
Fig. 2. A – dynamic viscosity of mucus modified by FCPs and their components, B – effective diffusion coefficient of
rhodamine B in the model mucus
4. Conclusions
Our results indicate that FCPs may be an useful concept for inhalation drug delivery to diseased lungs.
Simultaneous reduction of viscosity and the increment of mass transfer across the mucus layer may help to
achieve a therapeutic effect quicker. A more comprehensive description of mechanisms of the investigated
phenomena should be obtained by incorporating the results of theoretical predictions from molecular dynamics
simulations, which are currently underway.
5. Acknowledgments
This work was supported by a grant from National Science Centre based on decision DEC2011/03/N/ST8/04912.
6. References
[1] R. A. Cone, “Barrier properties of mucus”, Adv. Drug Deliv. Rev. 61, 75-85 (2009)
[2] M. Stern, N. J. Caplen, J. E. Browning, U. Griesenbach, F. Sorgi, L. Huang, D. C. Gruenert, C. Marriot, R. G. Crystal, M. G. Geddes,E.
W. Alton, “The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. Gene Therapy. 5, 91-98 (1998)
[3] M. Odziomek, T. R. Sosnowski, L. Gradoń, “Conception, preparation and properties of functional carrier particles for pulmonary drug
delivery”, Int. J. Pharm. 433, 51-59 (2012)
[4] S. L. McGill, H. D. Smyth, “Disruption of the Mucus Barrier by Topically Applied Exogenous Particles”, Mol. Pharm. 7(6), 2280-2288
(2010)
[5] M. A. Desai, P. Vadgama, “Estimation of Effective Diffusion Coefficients of Model Solutes Through Gastric Mucus: Assessment of a
Diffusion Chamber Technique Based on Spectrophotometric Analysis. Analyst. 116, 1113-1116 (1991).
[Back to Session 7]
65 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Agglomeration of Theophylline Nanoparticles: a New
Protocol for Pulmonary Drugs Administration
Salem HF1, Abdelrahim ME1, Abo Eid K2, Sharaf MA3
1Faculty of pharmacy, University of Beni Suef, Beni Suef, Egypt
2Faculty of Science, Helwan University, Cairo, Egypt
3Department of Chemistry, the American University in Cairo, New Cairo, Egypt
[email protected]
The aim of this study is to formulate theophylline as a dry powder aerosol for treatment of pulmonary
obstruction. Dry powder aerosols provide improved stability, ease of administration, and higher bioavailability
compared to traditional dosage forms. Particles in a size range of 1 to 5µm are facilitating the deposition of the
drug into lung; however they retard its dissolution in lung due to limited dissolution medium there. Using
nanoparticles, which are agglomerated by addition of electrolytes, may act as an excellent way to deliver
theophylline. Therefore, theophylline was precipitated with stearic acid to form a nanosuspension of theophyllin
stabilized with stearic acid. This was followed by a quantitative addition of electrolyte to destroy the electrostatic
repulsion between the particles. Achieving agglomerated nanoparticles of a controlled size was the final results
of this process. Characterization of both nanoparticles and the agglomerates was carried out using SEM, PCS
and zetasizer for studying the morphology, the particles size and the surface charge of the particles consequently.
SEM revealed formation of well defined self assembled structures of hollow agglomerates. It also revealed
formation of nanoparticles in size range of 190 to 230nm with relatively low polydispersity index. The zeta
potential was measured and found to be 34mV. However, for agglomerates were in the size of 2 to 5 µm with
zetapotential much lower than that of the nanoparticles. The nanoparticle agglomerates revealed enhanced
dissolution in relative original drug species suggesting the efficiency of such formulation approach for enhancing
dissolution of poorly water-soluble pulmonary medicines. The aerodynamic characterization of agglomerated
nanoparticles was determined using Anderson cascade impactor. All of this indicated the efficiency of using
controlled agglomeration of the nanoparticles as a way for treatment pulmonary obstruction diseases.
[Back to Session 7]
66 | 2013 International Conference on Nanotheranostics (ICoN 2013)
Author Index
Alexandros Strongilos, 10, 12
Ana B. Davila Ibañez, 10
Andreani Odysseos, 9, 10, 12, 13, 14
Andreas Anayiotos, 13
Angela Riedel, 9
Anjali Seth, 11
Anne Vessières-Jaouen, 10
Ayache Bouakaz, 12
Bernard Chatelain, 10, 11
Bernard Masereel, 11
Birgitte Brinkmann Olsen, 9
Bowen Tian, 13
Christine Ménager, 10, 11
Christoffer Åberg, 13
Christophoros Mannaris, 12
Costas Pitris, 10, 13
Costas Pitsillides, 13
Cyrill Bussy, 11
Damien Lenoble, 12
David Lafargue, 11
Didier Arl, 12
Eftychia Angelou, 12
Eleni Efthimiadou, 9
Elias Couladouros, 10
Eugeniu Vasile, 14
Eva-Athena Economides, 11
Evdokia Kastanos, 10
Ewelina Tomecka, 11
Federica Scaletti, 9
Fotini Liepouri, 10, 12
Fotios Mpekris, 14
François Mullier, 10, 11
Gaëlle Corne, 12
George Kordas, 13
Gérard Jaouen, 10
Heba Salem, 14
Helge Thisgaard, 9
Helle Christiansen, 9
Iga Wasiak, 10, 12
Ines Block, 9
Ioanna Savva, 14
Jan Mollenhauer, 9
Jean-Michel Dogné, 10, 11
Jean-Michel Siaugue, 10, 12, 14
Jean-Michele Escoffre, 12
Jean-Sébastien Thomann, 12
Jeremy Malinge, 10
Jesper Wengel, 9
Julie Laloy, 10, 11
Kamal Abo Eid, 14
Katerina Hadjigeorgiou, 10
Kenneth A. Dawson, 13
Kenneth Dawson, 9
Konstantinos Kapnisis, 13
Konstantinos Soteriou, 11
Kostas Kostarelos, 11, 13
Krzysztof Gawlik, 10
Ladislau Vekas, 14
Leon Gradoń, 14
Loucas Evaggelou, 14
Lutfiye Alpan, 10, 11
Magdalena Janczewska, 12
Marcin Odziomek, 14
Maria Kokonou, 14
Maria Pavlaki, 12
Marie-Edithe Meyre, 12
Matthieu Sollogoub, 10
Michalakis Averkiou, 12, 14
Mohamed Abdelrahim, 14
Mohamed Elblbesy, 10
Mohamed Sharaf, 14
Morgane Rivoal, 9
Myria Angelidou, 10
Naoufal Bahlawane, 12
Oana Marinica, 14
Olivier Toussaint, 11
Omar Lozano, 11
Pavlos Agianian, 9, 10, 12
Poul Flemming Hoilund-Carlsen, 9
Rena Bizios, 6, 7
Stefan Vogel, 9
Steffen Schmidt, 9
Stephane Lucas, 11
Theodora Krasia-Christoforou, 14
Tomasz Ciach, 10, 12
Tomasz R. Sosnowski, 11
Tomasz Sosnowski, 14
Triantafyllos Stylianopoulos, 11, 14
Valentine Minet, 10, 11
Vassiliki Garefalaki, 10
Verónica Salgueiriño, 10
Wafa Al-Jamal, 13
Yiannis Sarigiannis, 14
Yongmin Zhang, 10
67 | 2013 International Conference on Nanotheranostics (ICoN 2013)