Cell-penetrating peptides
Uptake, stability and biological activity
Tina Holm
©Tina Holm, Stockholm 2011
ISBN 978-91-7447-269-1
Printed in Sweden by Universitetsservice, Stockholm 2011
Distributor: Department of Neurochemistry, Stockholm University
To my family
List of publications
I.
T. Holm, H. Johansson, P. Lundberg, M. Pooga, M. Lindgren,
and Ü. Langel
Studying the uptake of cell-penetrating peptides.
Nature Protocols. (2006) 1(2): 1001-1005
II.
T. Holm, S. Netzereab, M. Hansen, Ü. Langel, and M. Hällbrink
Uptake of cell-penetrating peptides in yeasts.
FEBS letters. (2005) 579: 5217-5222
III.
H. J. Johansson, S. EL Andaloussi, T. Holm, M. Mäe, J. Jänes,
T. Maimets, and Ü. Langel
Characterization of a Novel Cytotoxic Cell-penetrating Peptide Derived From p14ARF Protein.
Mol Ther. (2008) Jan:16(1):115-23
IV.
S. EL Andaloussi, H. J. Johansson, T. Holm, and Ü. Langel
A Novel Cell-Penetrating Peptide, M918, for Efficient Delivery of Proteins and Peptide Nucleic Acids.
Mol Ther. (2007) Oct:15(10):1820-6
V.
T. Holm*, H. Räägel*, S. EL Andaloussi, M. Hein, M. Mäe, M.
Pooga, Ü. Langel
Retro-inversion of certain cell-penetrating peptides causes
severe cellular toxicity.
BBA Biomembranes. (2010) Nov 9. In press
*These authors contributed equally to this work
Additional publications
S. EL Andaloussi, T. Holm, and Ü. Langel
Cell-penetrating peptides: mechanisms and applications.
Current Pharmaceutical Design. (2005) 11;28(3): 597-611
Bodor,N., Tóth-Sarudy,É., Holm,T., Pallagi,I., Vass,E., P.
Buchwald,P., and Langel,Ü (2007)
Novel, Cell–Penetrating molecular transporters with flexible
backbones and permanently charged side chains.
J.Pharm.Pharmacol. (2007) 59(8): 1065-1076
T. Holmström, A. Mialon, M. Kallio, Y. Nymalm, L. Mannermaa,
T. Holm, H. Johansson, E. Black, D. Gillespie, T. Salminen, Ü. Langel, B. Valdez, and J. Westermarck
c-Jun supports ribosomal RNA processing and nucleolar localization of RNA helicase DDX21.
J Biol Chem. (2008) Mar14:283(11):7046-53
P. Lundin, H. Johansson, P. Guterstam, T. Holm, M. Hansen, Ü.
Langel, S. EL Andaloussi
Distinct uptake routes of cell-penetrating peptide conjugates.
Bioconjug Chem. (2008) Dec:19(12):2535-42
S. Jones, T. Holm, I. Mäger, Ü. Langel, J. Howl
Characterisation of bioactive cell penetrating peptides from human Cytochrome c: Intracellular translocation, protein mimicry
and the development of a novel apoptogenic agent.
Chem Biol. (2010) Jul 30;17(7):735-44
T. Holm, S. EL Andaloussi, Ü. Langel
Comparison of cell-penetrating peptide uptake methods.
Chapter 15. Cell-Penetrating Peptides: Methods and Protocols. Methods Mol Biol. (2011) 683:207-17
Abstract
Cell-penetrating peptides (CPPs) have emerged as a group of remarkable
delivery vectors for various hydrophilic macromolecules, otherwise excluded from cells due to the protective plasma membrane.
Unbiased conclusions regarding e.g. uptake mechanism,
intracellular distribution and cargo delivery efficacy is complicated by the
use of different methodological parameters by different laboratories. The
first paper in this thesis introduced unifying protocols enabling comparison
of results from different research groups. One of these methods, HPLC, was
used in paper II to investigate CPP uptake and degradation in yeasts. Both
parameters varied depending on peptide and yeast species; however pVEC
emerged as a promising delivery vector in yeast since it internalized into
both species tested without concomitant degradation.
Protein mimicry was another investigated phenomenon and in
paper III a 22-mer peptide from the p14Arf protein (Arf (1-22)) was found to
be sufficient for retaining its function as a tumor suppressor. This peptide
comprised a combination of apoptogenic property and CPP in one unity, thus
providing opportunity to conjugate cytotoxic agents boosting the tumoricidal
activity. Surprisingly, a partially inverted control peptide to Arf (1-22),
called M918, was found to be an extraordinary CPP. In paper IV, it was
shown to be superior to well-established CPPs in delivery of both peptide
nucleic acids and proteins. Albeit the promising results these two peptides
displayed, their utility in vivo, as with all peptides, is hampered by rapid
degradation. With the aim of improving their stability, Arf (1-22) and M918
were synthesized with D-amino acids in the reverse order, a modification
called retro-inverso (RI) isomerization. Their cell-penetrating ability was
retained, but the treated cells displayed unexpected morphological alterations
indicative of apoptosis.
The presented results demonstrate the versatility of CPPs, functioning as
vectors in both yeast and mammalian cells and as protein mimicking peptides with biological activity. Their potential as drug delivery agents is obvious; however, peptide degradation is an issue that requires further improvements before clinical success is in reach.
Contents
List of publications ................................................................................... v
Additional publications............................................................................................. vi
Abstract ................................................................................................... vii
Abbreviations............................................................................................ x
Introduction ............................................................................................ 11
The cellular plasma membrane ............................................................................. 11
Yeast cell wall ..................................................................................................... 11
Endocytosis ............................................................................................................... 13
Clathrin mediated endocytosis......................................................................... 13
Caveolae-mediated endocytosis ...................................................................... 14
Macropinocytosis ................................................................................................ 14
Clathrin- and caveolae-independent uptake pathways ............................... 15
Cell-penetrating peptides ....................................................................................... 16
History .................................................................................................................. 17
Mechanisms of membrane translocation ....................................................... 17
Cargo delivery..................................................................................................... 20
Peptide degradation ................................................................................................ 21
Stability of CPPs ................................................................................................. 21
Strategies to increase peptide stability .......................................................... 22
Protein delivery and mimicry ................................................................................ 23
Protein delivery................................................................................................... 23
Protein mimicry .................................................................................................. 23
Peptides with dual functions: CPP and biological activity ........................... 26
Aims of the study................................................................................... 28
Methodological considerations ............................................................. 29
Solid phase peptide synthesis ............................................................................... 29
Purification and identification of synthesized peptides and PNA..................... 30
Peptide-PNA conjugation ........................................................................................ 30
Choice of cells .......................................................................................................... 30
Yeasts (paper II) ................................................................................................ 30
Breast cancer cells (paper III-V) ..................................................................... 31
HeLa cells (paper I, III-V) ................................................................................ 31
Astrocytoma cells (paper IV) ........................................................................... 31
Chinese hamster ovarian cells (paper IV) ..................................................... 31
HPLC in determination of peptide uptake and degradation ............................. 32
Quantitative uptake by spectrofluorometry ........................................................ 32
Microscopy ................................................................................................................ 33
Splice correction ...................................................................................................... 33
Cell viability .............................................................................................................. 34
LDH ....................................................................................................................... 34
Wst-1 .................................................................................................................... 34
Cell morphology ................................................................................................. 34
Apoptosis .................................................................................................................. 35
Results and discussion .......................................................................... 37
Studying the uptake of cell-penetrating peptides (Paper I) ............................ 37
Uptake of cell-penetrating peptides in yeasts (Paper II) ................................. 38
Characterization of a novel cytotoxic cell-penetrating peptide derived from
p14Arf protein (Paper III) ...................................................................................... 39
A novel cell-penetrating peptide, M918, for efficient delivery of proteins and
peptide nucleic acids (Paper IV) ........................................................................... 40
Retro-inversion of certain cell-penetrating peptides causes severe cellular
toxicity (Paper V)..................................................................................................... 41
Conclusions ............................................................................................. 43
Populärvetenskaplig sammanfattning på svenska ............................ 44
Acknowledgements ................................................................................ 46
References .............................................................................................. 48
Abbreviations
ASMase
AP-2
Arf
Boc
CHO
CLIC
CME
CPP
ECM
FACS
GAG
GFP
GAG
HA2
HDM2
LDH
MALDI-TOF
MβCD
Npys
PI
PI3K
PNA
RI
RP-HPLC
SCOs
SV40
TFA
Acid sphingomyelinase
Adaptor protein 2
Alternative reading frame
tert-Butyloxycarbonyl
Chinese hamster ovarian cells
Clathrin-independent carriers
Clathrin-mediated endocytosis
Cell-penetrating peptide
Extracellular matrix
Fluorescence-activated cell sorter
Glycosaminoglycan
Green fluorescent protein
Glycosaminoglycan
Hemagglutinin peptide 2
Human double minute
Lactate dehydrogenase
Matrix-assisted laser desorption/ionization time-of-flight
Methyl-β-cyclodextrin
3-nitro-2-pyridinesulphenyl
Propidium iodide
Phosphatidylinositol-3-phosphate
Peptide nucleic acid
Retro-inverso
Reversed phase high performance liquid chromatography
Splice correcting oligonucleotides
Simian virus 40
Trifluoro acetic acid
Introduction
The cellular plasma membrane
Modern-day organisms fall into three distinct evolutionary lineages - bacteria, archaea and eukaryotes. Bacteria and archaea are collectively classified
as prokaryotes, which lack a defined nucleus and have a simple sub cellular
organization. The cells of eukaryotes have a membrane-limited nucleus,
numerous specialized organelles, and a complex cytoskeleton.
Both single-cell eukaryotes, e.g. yeast, and cells of multicellular organisms
are surrounded by a membrane composed of lipids with a hydrophilic head
and a hydrophobic tail. The head groups face the cytoplasm and the extracellular space and the tails assemble into a hydrophobic interior, thus forming a
lipid bilayer. Different carbohydrates (in the form of glycoproteins and glycolipids) and proteins are inserted into this bilayer, functioning as e.g. receptors and anchoring points to the extracellular matrix (ECM) (in case of
mammalian cells) and cell wall (in the case of yeasts). The protein, carbohydrate and lipid composition varies between species and cell types and determine the function of the cell. For example, cholesterol is the most abundant
sterol in higher eukaryotes, while the yeast plasma membrane mainly contains ergosterol [1].
The plasma membrane functions as a protective barrier that regulates what is
transported in and out of the cell. To carry out these functions, the plasma
membrane contains specific transport proteins that permit the passage of
certain small molecules. Several of these proteins use the energy released by
ATP hydrolysis to pump ions and other molecules into or out of the cell
against their concentration gradients. For larger molecules, like proteins,
internalization occurs via other mechanisms, collectively known as endocytosis.
Yeast cell wall
The yeast cell wall maintains the structure and the rigidity of the cell but
is freely permeable for solutes smaller than 600 Da [1]. It can be schematically organized into two layers: a structural inner layer and an outer layer,
which composition varies with growth stage and species [2] (Fig. 1). Although the cell wall composition varies between species, a common central
core exists that is composed of a branched β-1,3-glucan cross-linked to chi11
tin. This glucan-chitin complex is covalently bound to other polysaccharides,
which specific composition varies between species [3]. The cell wall is actually a dynamic structure that continuously changes in response to the environment during cell cycle [2].
Figure 1. The yeast cell wall is composed of layers of different polysaccharides. A
mannose-protein layer is facing the plasma membrane and the extracellular space.
β-1-3-glucan cross linked to chitin makes up the central core and on top of this
structure is a layer often composed of β-1-3-glucan.
12
Endocytosis
Endocytosis is the process by which components situated in or in close
proximity to the plasma membrane are internalized into the cell via vesicles
of different character (Fig. 2). There are two distinct mechanisms of endocytosis: phagocytosis and pinocytosis. Phagocytosis involves ingestion of large
molecules like bacteria and cell debris through specialized cells, while pinocytosis involves ingestion of fluids and smaller particles and is carried out by
practically all cells [4]. The endocytosis literature is not consistent in terms
of classification of different endocytic pathways and authors have presented
different classification suggestions. One such classification is presented by
Conner et al and includes the categories; clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and clathrin- and caveolaeindependent endocytosis [4], all of which are examples of pinocytosis. The
last group encompasses more than one pathway and is divided with respect
to its dynamin dependence. The intracellular fate of an internalized molecule
is dependent on which of these pathways that is used. After internalization,
the most common fates of molecules are degradation or recycling back to the
cell surface, but many alternative pathways, such as trafficking to other organelles or translocation into the cytosol, also occur [5].
Clathrin mediated endocytosis (CME)
CME occurs constitutively in all mammalian cells and fulfills crucial
physiological roles, including nutrient uptake and intracellular communication. For most cell types, CME serves as the main mechanism of internalization for macromolecules and plasma membrane constituents.
Clathrin is composed of three-legged structures called triskelions that organize to form basket-like lattices on the cytoplasmic side of the plasma
membrane [5]. The assembly of clathrin triskelions induces deformation of
the plasma membrane into a coated pit of ~120 nm. Budding of the pit from
the membrane requires the binding of a GTPase called dynamin. Dynamin
binds at the neck of the pit to mediate membrane fission and formation of a
vesicle. This clathrin-coated vesicle is then uncoated and the clathrin triskelia recycled [4]. The vesicle fuses with acidic (pH ~6) early endosomes
where recycling receptors, such as transferrin and low density lipoprotein,
are localized. The presence of such receptors is a convenient and widely
used marker of early endosomes. The early endosome can mature into late
endosomes (pH <6), which then fuses with lysosomal vesicles (pH ~5). Lysosomes contain acid hydrolases and other enzymes that mediate degradation
of the endocytosed material [5].
13
Caveolae-mediated endocytosis
Caveolae has been demonstrated to be important in a variety of cellular functions including endocytic processes, signal transduction, and cholesterol and
lipid homeostasis [6]. Unlike clathrin-coated pits, caveolae are abundant in
some cell types (adipocytes, endothelia, muscle) but undetectable in others
(lymphocytes and many neuronal cells) [7].
Caveolae are bulb-shaped invaginations of the plasma membrane that are
formed by the cholesterol binding dimeric protein caveolin. Recently, information has gathered that also a protein family called cavins is involved in the
formation and function of caveolae [8]. These small vesicles (~50-60 nm in
diameter) contain only little fluid-phase liquid and internalize slowly [4].
Caveolin and cavin mediates the membrane curvature needed for invagination and vesicle formation. As in CME, dynamin is recruited to the neck of
the “bulb” where it catalyzes membrane fission enabling the vesicle to pinch
off from the plasma membrane [4]. The general belief has, up to last year,
been that these vesicles deliver its contents to pH-neutral “caveosomes”.
However, the group that first suggested the existence of the caveosome, revealed findings in the November issue of Journal of Cell Biology that questions the existence of this unique organelle [9]. They found that caveosomes
correspond to late endosomal compartments. Several toxins, e.g. cholera and
tetanus, are internalized into cells via caveolae vesicles and are therefore
commonly used as markers for this endocytic pathway [4,7].
Macropinocytosis
Macropinocytosis is another type of clathrin-independent endocytosis
pathway and it is the most effective way for cells to ingest large amounts of
extracellular fluid. It operates in macrophages and many tumor cells constitutively, and in other cells after stimulation with growth factors or phorbol
esters [10]. Membrane ruffling frequently leads to the formation of large and
heterogenous dynamic vesicular structures called macropinosomes, which
are sometimes as large as 5 µm in diameter [5]. These vesicles are formed by
an actin-driven process yielding membrane protrusions that collapse onto
and fuse with the plasma membrane, thereby trapping large quantities of
extracellular liquid [4]. Little is known about this fusion process and since
no specific cargo molecules for macropinocytosis have been identified, it has
been difficult to follow the intracellular fate of macropinosomes [11]. Also,
macropinosomes have been reported to reach different intracellular compartments depending on cell type. In macrophages, they move towards the
centre of the cell and change from an early-endosome-like organelle to a
late-endosome like organelle, and then merge completely with the lysosomal
compartment. They rarely fuse again with the plasma membrane [12]. In
contrast, macropinosomes in human epidermoid A431 cells does not fuse
14
with early or late endosomes, but eventually recycle most of its content to
the cell surface [13].
Although macropinocytosis accompanies seemingly chaotic membrane
ruffling, it is likely to be a highly controlled and regulated process [4]. Unlike clathrin coated pits, macropinosomes have no discernible coat and do
not concentrate receptors. Rather, they most likely contain membrane compositionally similar to plasma membrane [10].
Figure 2. Different endocytosis routes into the cell. Membrane protrusions fuse with
the membrane again to form large macropinosomes, which intracellular fate depends on cell type. In the other pathways smaller vesicles are formed from invaginations of the plasma membrane. Clathrin-and caveolae-independent endocytosis have
both dynamin-dependent and independent pathways.
Clathrin- and caveolae-independent uptake pathways
Some clathrin-and caveolae-independent uptake pathways are constitutive, whereas others are triggered by specific signals. They also differ in
their mechanisms and kinetics of formation, associated molecular machinery
and cargo destination [14]. Kirkham et al, and many others, denote clathrin15
independent vesicles CLICs (CLathrin-Independent Carriers) and presume
them to be highly dynamic structures [15]. Their shape and size varies depending on cargo. Simian virus 40 (SV40) has been shown to be internalized
by host cells via caveolae but in cells lacking caveolae, endocytosis of SV40
still occurs indicating that caveolae-independent pathways exist [16]. Damm
et al further showed that internalization occurred in small, tight-fitting vesicles and was independent of clathrin and dynamin [16]. However, other
CLICs exist that are dependent on dynamin. For example, Lamaze et al reported that the interleukin 2-receptor internalized into cells devoid of clathrin in a dynamin-dependent fashion [17] and Sauvonnet et al showed that
dynamin is required for the clathrin-independent uptake of γC cytokine receptor [18].
Table 1 Endocytosis pathways
CME
Caveolaemediated
Vesicle
Clathrin
Caveolincoating
triskelions
1[5], ca[4,5]
[4]
vins[8]
~60 nm[4]
Macropinocytosis
CLIC-D
CLICDI
No [5]
No [15]
No[15,16]
>1µm[4]
~90 nm[4]
~90 nm[4]
pHneutral,
intermediate
organelles[16]
No[16]
Vesicle
size
Intracellular destination
~120 nm
Late endosomes or
lysosomes[5]
Late endosomes or
lysosomes[9]
Lysosome[12]
or recycling to
cell surface[13]
depending on
cell type
Late endosomes
or lysosomes[18]
Dynamin
dependent
Actin
dependent
Examples
of markers
Yes[18]
Yes[19]
No[15]
Yes[18]
Yes[18,20]
Yes[20,21]
Yes[20]
Yes[17,18]
Possible[16]
Transferrin[4,5]
LDL[4,5]
Cholera
toxin B[19]
SV40[16,21]
No specific
ligand, dextran is used as
marker for
fluid-phase
endocytosis[22]
γc cytokine receptor[18]
IL-2[17]
SV40[16]
CME: clathrin-mediated endocytosis; CLIC-D: clathrin-independent carriers dynamin dependent; CLIC-DI: clathrin-independent carriers dynamin independent LDL:
low density lipoprotein; SV40: simian virus 40; IL-2: interleukin-2
16
Cell-penetrating peptides
History
Cell-penetrating peptides (CPPs) are short cationic and/or amphipathic peptides that can be conjugated or complexed with large macromolecules and
even microscopic particles to facilitate their cell entry. These peptides are
usually less than 30 amino acids in length and internalization is observed in
virtually all cells, albeit with different efficiencies that depend on the CPP,
the cargo and the cell-type.
Although the first report of a polycationic peptide capable of traversing the
cellular plasma membrane was published already in 1965 [23], it was not
until 1994 when the group of Prochiantz reported the cell-penetrating properties of penetratin that the potential of this new class of peptides was acknowledged [24]. Later a report from Lebleu’s laboratory revealed that a
short highly cationic peptide (Tat 48-60) from the Tat protein was sufficient
for cell penetration [25]. From that date an ever increasing number of new
CPPs have been found and characterized, some of which are presented in
Table 2. However, the mechanism of uptake is still not fully elucidated. A
summary of the knowledge gained this far is presented below.
Table 2 A selection of CPPs
Name
Sequence
Protein derived:
Penetratin
RQIKIWFQNRRMKWKK
Tat (48-60) GRKKRRQRRRPPQ
pVEC
LLIILRRRIRKQAHAHSK
Designed:
Polyarginine (R)n
Transportan GWTLNSAGYLLGKINLKALAALAKKIL
TP10
AGYLLGKINLKALAALAKKIL
Ref.
[24]
[25]
[26]
[27]
[28]
[29]
All peptides have an amidated C-terminus.
Mechanisms of membrane translocation
The uptake mechanism of CPPs has been a matter of great controversy, initially reported to be an energy-independent process of direct penetration
across the plasma membrane. The model that was proposed suggested that
polycationic CPPs interacted ionically with the negatively charged phospholipids of the cell membrane, causing an invagination that led to a local reorganization of the lipid bilayer, resulting in formation of inverted micelles.
These would contain the CPP-cargo conjugates, which were released on the
cytosolic face of the membrane [30]. However, the outer surfaces of verte17
brate plasma membranes are essentially neutral and electrostatic interactions
between cationic peptides and eukaryotic cells are much more likely to involve the abundant negatively charged components present on the membrane
surface and the ECM rather than the phospholipids of the bilayer [31].
Involvement of proteoglycans
Proteoglycans are the major components of the ECM and they contain a core
protein to which one or more glycosaminoglycan (GAG) chains are covalently coupled [32]. CPP binding induce clustering of GAGs, which triggers
intracellular signaling events and cytoskeletal remodeling preceding cellular
uptake. Letoha et al recently showed that syndecan-4, a transmembrane proteoglycan, initiates the uptake of Tat, penetratin, and Arg8 [33], and many
others have reported that GAGs are involved in the uptake of CPPs [34-37].
Interestingly, it was recently suggested that the GAGs do not initiate the
internalization process, instead these act merely as sites of CPP accumulation and other components are responsible for the subsequent uptake. This
conclusion originated from the finding that cellular delivery of Tat-Cre recombinase into GAG deficient or wild type cells did not differ significantly
[38].
Uptake via endocytosis
Since the first reports of the inverted micelle model described above, the
prevailing view has shifted and today most CPPs are reported to use the endocytic route to gain access to the intracellular milieu. Some examples of
endocytic uptake of CPPs are listed in Table 3. It must be noted that the cargo, which is not included in Table 3, also influences the uptake mechanism,
as discussed further in the next section.
Table 3 Endocytic uptake of a selection of CPPs
CPP
Endocytic pathway
Penetratin
Macropinocytosis
Tat (47-57) Macropinocytosis
Transportan Caveolae-mediated
CME
TP-10
Caveolae-mediated
CME
M918
Macropinocytosis and partly CME
Ref
[39],[40]
[22],[39]
[41]
[39]
[41]
[39]
paper IV,[39]
CME: clathrin-mediated endocytosis
To determine the involvement of different endocytic pathways in the uptake
of CPPs most researchers use pharmacological inhibitors or markers specific
for a certain pathway. A summary of commonly exploited inhibitors and
markers and their respective pathway is presented in Table 4.
18
Table 4 A selection of inhibitors for endocytic mechanisms
Inhibitor
Mechanism influenced
Macropinocytosis
Wortmannin
Inhibits PI3K dependent vesicle formation
Amiloride
Inhibits Na+/H+ exchange
Cytochalasin D Inhibits F-actin elongation
Clathrin-mediated endocytosis
Wortmannin
Inhibits PI3K dependent vesicle formation
Sucrose
Induces abnormal clathrin polymerization
Chlorpromazine Disrupts traffic of the clathrin-coated pit component
AP-2
Caveolae/lipid raft mediated endocytosis
MβCD
Depletes cellular cholesterol
Ref.
[42]
[43]
[44]
[42]
[45]
[46]
[47]
PI3K: phosphatidylinositol 3-kinase; AP-2: adaptor protein 2; MβCD: methyl-βcyclodextrin
Endocytosis-independent uptake
Several studies provide evidence that other, non-endocytic, mechanisms are
involved in the uptake of certain CPPs [48-50]. One example is the MPG
peptide that, both by itself and in complex with DNA, penetrates the membrane by a mechanism dependent on the GTPase Rac 1. Rac 1 activation
stimulates actin network remodeling, leading to increased membrane fluidity
and membrane fusion [50,51]. Furthermore, there is also the possibility that
one particular CPP might utilize different pathways depending on cargo [52]
and concentration [53]. The influence of cargo on the uptake mechanism of
Tat was investigated by Tünnerman et al and they found that Tat-protein
conjugates were internalized by endocytosis, while Tat-peptide conjugates
utilized a rapid mechanism dependent on membrane potential. They concluded that increasing the positive charge of the CPP itself and decreasing
the size of the cargo allows rapid internalization to occur in addition to the
slower process of endocytosis [52]. Duchardt et al showed that above a critical peptide concentration threshold, internalization occurs through a highly
efficient non-endocytic pathway originating from “nucleation zones”, spatially restricted membrane regions, leading to a rapid cytoplasmic distribution of the peptide. They further showed that, at concentrations below this
threshold, penetratin, Tat and Arg9 actually utilize a combination of macropinocytosis, CME and caveolae-mediated endocytosis depending on the
availability of the different pathways [53]. Recently the same group came a
step closer to resolving the mechanism of direct penetration when they
showed that cationic CPP interaction with the plasma membrane induced
translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the
19
membrane. It was shown that cationic CPP import depended on ASMaseinduced formation of ceramide, which changed the lipid composition of the
plasma membrane [54].
Cargo delivery
CPPs have been reported to deliver cargos ranging from small molecules and
imaging agents to oligonucleotides, peptides, proteins, and even nanoparticles and plasmids [55]. Peptide and protein delivery by CPPs is described
in more detail later and oligonucleotides as CPP cargo, both covalently and
non-covalently conjugated, was thoroughly reviewed recently by Ezzat et al
[56]. The assessment of cellular response following delivery of biologically
active cargos is imperative, since uptake mechanism and subcellular localization of free CPP and CPP-cargo is not necessarily the same. Merely analyzing uptake and not a concomitant biological response might lead to misinterpretations regarding efficiency of a certain CPP [57] or importance of
different mechanisms in CPP uptake [38]. Examples of cargos generating a
positive read out for evaluation of uptake are Cre-recombinase [22], plasmids [58], luciferin [59], and splice correcting oligonucleotides [60]. The
splice correction assay will be described in the methodological part of this
thesis.
Lysosomal entrapment
In most cases the cargo ends up in endolysosomal vesicles [57]. Escape from
these vesicles is necessary for the cargo to reach its intracellular compartment and exert its biological function. Chloroquine is a weak base that prohibits maturation of endosomes, thereby giving CPP-cargo opportunity to
escape to the cytosol. Improved cellular response to cargo activities upon
chloroquine treatment has been reported repeatedly [61-63]. Unfortunately
chloroquine is toxic at higher concentrations and is not suitable for in vivo
situations. Other successful approaches to increase endosomal escape are
chemical modifications of the CPP [64,65] or addition of viral fusogenic
HA2 peptide of the influenza virus hemagglutenin which destabilizes the
endosomal membrane [22].
Although this part of the CPP field has been full of conflicting
results, the “take home message” is that there is not one single pathway exclusively employed by any CPP, but rather a predisposition for some mechanisms and inhibition of one pathway may lead to up-regulation of another
[66]. For most CPPs, the uptake mechanism still needs to be confirmed and
remains controversial, partly due to the fact that different methods, which are
not comparable from one lab to another, have been employed to this aim.
20
Peptide degradation
A major hurdle to be solved before peptides will gain success in clinical
trials is their rapid degradation in vivo. Once the peptide enters the plasma of
a subject it becomes vulnerable to attack by proteases. Endoproteases, e.g.
trypsin and chymotrypsin, cleave the protein backbone adjacent to aromatic
and basic residues, while exopeptidases catalyze the removal of residues
from the N-terminus (aminopeptidases) or C-terminus (carboxypeptidases).
One approach to protect peptides from endopeptidases is to modify the peptide backbone, which can be done in a number of ways, e.g. β-peptides, peptoids, and retro-inverso peptides. Strategies for evading degradation by exopeptidases include C-terminal amidation and N-terminal acetylation, commonly known as “capping”.
Cells have several intracellular pathways for protein degradation, but the
major pathway is enzymatic degradation in lysosomes, as discussed above.
Cytosolic proteolytic mechanisms involve the proteasomes, which degrade
proteins flagged by ubiquitin molecules. All the backbone modifications
mentioned above will also rescue peptides from degradation in these compartments, since endogenous mechanisms only recognize natural peptide
backbones.
Stability of CPPs
When taking into account the enormous amount of articles published the
last 15 years in the field of CPPs, it is astonishing that so few have analyzed
the degradation of these peptides more thoroughly. There are, however, several reports regarding degradation of the CPP pVEC. Elmquist and Langel
analyzed the stability of pVEC, as well as its all-D-counterpart, intracellularly and in media with different composition. As expected, all-D-pVEC was
resistant to degradation in serum, intracellularly, extracellularly and in presence of trypsin or carboxypeptidase A and B, while the degradation pattern
of pVEC varied depending on exposure [67]. This variability was further
extended to apply also for different organisms, where it varied from no degradation in yeast to rapid degradation in Sf9 insect cells (paper II and [68]).
Also penetratin was included in these studies and, in contrast to pVEC, this
peptide was almost completely degraded in all investigated organisms (bacteria, insect Sf9 cells and yeasts). The degradation of penetratin was further
investigated in chinese hamster ovarian (CHO) cells where it was shown that
extracellular degradation is a critical determinant for uptake, since decreasing the extracellular degradation increased the uptake [69]. Trehin et al analyzed the metabolic degradation kinetics and cleavage patterns of penetratin,
Tat (47-57) and a novel human calcitonin (hCT) derived CPP in epithelial
models [70]. Although informative in many ways, the authors report of a
half life of Tat (47-57) of up to 24 h, which seems unlikely considering the
21
many cleavage sites for proteases within the Tat sequence. Grunewald et al
estimated the half-life of Tat (47-57) to 3 min. However, when conjugated to
a cargo the half life was increased to 10 min [71].
Strategies to increase peptide stability
As aforementioned, there are several methods to increase the stability of
peptides by synthesizing non-natural peptide mimics, e.g. β-peptides, peptoids, and retro-inverso peptides. The latter was used in paper V and will be
described below.
Retro-inverso peptides:
In the early 1980’s, Chorev and Goodman carried out extensive research
on the chemistry of peptide backbone modifications [72]. They hypothesized
that exchanging a natural peptide sequence with all-D amino acids (inverso)
and reversing the order (retro) would yield a peptide with the same stereochemistry as the parent peptide (figure 3). These peptidomimetics were
named retro-inverso (RI) peptides, where the amino acid side chains are
properly arranged in space, but the amine and carbonyl of the amide peptide
bond are reversed, thus conferring resistance towards proteases.
Figure 3. A dipeptide presented in All-L, All-D, All-L-retro and All-D-retro configuration.
In an extensive review from 2005, Chorev presents a number of successful implementations of the RI concept [73]. However, RI modification of
natural peptides has a history of mixed success and several groups have reported that their RI-peptides lost the biological activity of the parent Lpeptide ([74] and references therein).
Also CPPs have been subjected to this modification, with the aim of producing transporters with increased stability. Tat is by far the most commonly
exploited CPP for RI modification. Wender et al found that the L-, D- and RI
22
forms of Tat (49-57) showed similar cellular uptake in serum-free medium,
while in the presence of serum the D-form was modestly more active and the
RI form much more active than the L-form, indicating the likely role of proteolysis in limiting the activity of natural peptides [27]. The uptake mechanism of RI-Tat has been analyzed [75] and RI-Tat conjugates have been implicated in neuroprotection [76], as anti-HIV agent [77] and as tumor suppressant [78][79]. The RI-p53 restoring peptide reported by Snyder et al [80]
was later used by Takayama et al to determine the efficiency of their “penetration accelerating sequence” in conjugates with Arg8 or a CPP called FHV
[81]. Interestingly, the mutated p53 peptide used by Snyder et al as control
(i.e. not toxic against tumor cells), showed antiproliferative effects in parity
with wild type p53 peptide in the article by Takayama et al.
In a recent article it was shown that oligomerization of α-Synuclein, a
protein involved in Parkinson’s disease, was inhibited by cell-penetrating RI
β-Synuclein peptides. The classification of these peptides as CPPs is questionable since very high concentrations (50 and 250 µM) and long incubation times (4 h) were needed to detect uptake, but their biological activity is
certainly very promising considering the involvement of α-Synuclein fibrils
in Parkinson’s disease [82].
Protein delivery and mimicry
Protein delivery
There are now literally hundreds of reports of successful cellular delivery of
both functional peptides and proteins in vitro. Utilizing CPPs as transporting
vehicles is a more straight forward strategy than recombinant protein expression from plasmids, which is time consuming and laborious, and has proven
successful repeatedly, as recently reviewed in [56]. This can be achieved by
simply co-incubating the CPP and protein, yielding complexes which are
internalized by cells. Pep-1 was the first CPP reported to promote protein
delivery without covalent conjugation [83]. Streptavidin and avidin are two
proteins with high affinity to biotin and they have therefore been utilized as
model proteins in studies of protein delivery efficacy by CPP-biotin, since it
simplifies the chemical conjugation [84-86].
Protein mimicry
The large surface area of peptides gives them advantages over small molecules in their ability to disrupt specific signaling pathways by inhibiting
targeted protein-protein interactions. The most common application of pep-
23
tide inhibitors is in cancer research, but some also exert anti-inflammatory or
neuroprotective activity [87].
Protein-protein interactions are especially important in the cellular
processes that govern oncogene and tumor suppressor functions in general,
as well as regulation of the cell proliferation cycle and the cell death machinery. Regulators of these interactions therefore have tremendous potential as
a molecular strategy for the discovery of new cancer drugs [88]. As the bioavailability of these peptides is generally low because of poor cellular uptake, utilizing CPPs as transporting vehicles is an attractive alternative. The
apoptogenic 7 amino acid peptide Smac fused to Tat was shown to potentiate
the activity of cytotoxic drugs on a human glioma xenograft model in vivo
[89]. Another study showed that apoptosis was induced by a cell permeable
peptide called Shepherdin, which disrupts the interaction between survivin
and Hsp90, leading to massive death of tumor cells. Shepherdin by itself or
its scrambled peptide did not have any effect, but when fused to Tat or penetratin several hallmarks of apoptosis were detected [90].
Arf tumor suppressor
p53/HDM2
p53 is a stress-activated tumor suppressor that is involved in cellular
processes like DNA repair, cell-cycle arrest, and activation of the apoptotic
program [88]. The central role of p53 in protecting cells against malignant
transformation makes it an attractive target in the search for novel cancer
drugs. Human double minute-2 (HDM2) is a negative regulator of p53 and
the p53-HDM2 interaction is the most advanced protein-protein interaction
drug target in oncology [88]. HDM2 governs the activity of p53 by several
mechanisms; i) it is a p53-specific ubiquitin E3 ligase and thus promotes the
proteasomal degradation of p53, ii) it blocks transcriptional activity of p53
by binding to the N-terminal transactivation domain of p53, iii) it promotes
the nuclear export of p53 [91]. Over-expression of HDM2 is therefore an
efficient strategy exploited by tumor cells to prevent accumulation and activation of p53. Molecules which bind to HDM2 and block its interaction with
p53, including antibodies [92], or peptides based on p53 [93] elevate the
levels of p53 protein and its transcriptional activity. One well studied interacting partner of HDM2, acting as an antagonist of its ligase activity, is the
nucleolar protein Arf (known as p14Arf in humans and p19Arf in mouse).
The tumor suppressor Arf
The Ink4a/Arf locus encodes two tumor suppressor proteins, p16Ink4a and
Arf, and it is from the alternative reading frame generating Arf protein the
name derives [94]. The two most frequently inactivated tumor suppressor
genes in human cancer, irrespective of tumor type, site, and patient age, are
p53 and Ink4a [95]. Furthermore, Arf-null mice develop tumors early in life
24
[96]. Obviously, the potential of exploiting the antagonizing activity of
p14Arf towards HDM2 is of considerable interest in the development of
novel cancer therapies.
Peptide mimetics of p14Arf
Several studies have shown that the conserved N-terminus of p14Arf is
sufficient for binding HDM2, thereby blocking its E3 ligase activity and
ultimately elevating the levels and activity of p53 [97-99] See Table 5.
Table 5 N-terminal peptides of p14Arf with retained activity
p14Arf Peptide
Sequence
1-14
MVRRFLVTLRIRRA
1-14
MVRRFLVTLRIRRA
16-30
GPPRVRVFVVHIPRL
1-20
MVRRFLVTLRIRRACGPPRV
1-22
MVRRFLVTLRIRRACGPPRVRV
Ref.
[100]
[99]
[99]
[98]
[97]
The primary structure of Arf is unusual. The mouse protein is composed of
169 amino acids and the human protein of only 132 amino acids. They are
both composed of more than 20% arginine residues, making them highly
basic [101]. Clearly, this is a favorable feature in the search for bioactive
CPPs.
Indirect biological functions of p14Arf
The Arf response is a complex mechanism. Although it was first believed
that the main function of Arf was to suppress aberrant cell growth by inducing the p53 pathway and thereby mediating tumor suppression, there is now
ample evidence that Arf also displays p53-independent activities. These
functions are heterogenous and although several potential regulators have
been identified, the molecular basis of p53-independent Arf signaling remains largely unknown. These functions of Arf, such as p53-independent
cell cycle arrest or apoptosis, are reviewed in [101].
25
Figure 4. p14Arf increases the levels and the activity of the tumor suppressor 53 by
binding to its negative regulator HDM2.
Peptides with dual functions: CPP and biological activity
Biological activity of CPPs, other than its cargo delivery capacity, has up to
recently, been an unwanted phenomenon. However, when taking into account that several of the most widely used CPPs derive from proteins it is
not surprising that they also might have some intracellular targets, which
could be activated or inactivated upon binding. This was indeed reported to
be the case with penetratin, which decreased the transcriptional activity of
NFkB [102] and reduced the expression of p38 MAP kinase mRNA [103].
Tat (48-60) had the same effect on p38 MAP kinase [103] and polyargininecontaining peptides also inhibit the mammalian endoprotease furin [104].
However, once the cellular target and biological response is known this feature can be exploited to yield CPPs with dual functions. When synthesizing
peptides, short sequences are preferred to avoid aggregation before complete
synthesis etc. Also, shorter peptides allow addition of a second executor in
the same sequence, possibly yielding peptides with synergistic activity.
Thus, a CPP and cargo with the same biological effect might yield synergistic cellular response and exclude the need for extra transporting moieties.
This concept is particularly suitable for apoptosis inducing CPPs such as
p14Arf (1-22) (paper III) and Cyt c77-101 [105]. Indeed, the apoptogenic po26
tency of Cyt c77-101 was significantly enhanced when modified with a nucleoporin mimetic peptidyl motif [105]. Table 6 presents a summary of biologically active CPPs.
CPPs are nowadays an established group of delivery vectors, but the concept of biologically active CPPs was just recently born. Once the issue with
peptide instability has been solved, biologically active CPPs could have a
tremendous impact on this research field and hold a great potential for the
future.
Table 6 Biological effects of CPPs
CPP
Biological activity
Side effects
Penetratin
Decreased transcriptional activity of
NFκB
Penetratin
Decreased p38 MAP kinase mRNA
expression
Tat (48-60)
Decreased p38 MAP kinase mRNA
expression
Arg8
Proteasomal inhibitor
Arg9
Furin inhibitor
Arg9
Actin reorganization
Intended biological activity
Arf(1-22)
Apoptogenic
CytC77-101
Apoptogenic
mPrP(1-28)
Decreased formation of prions
bPrP(1-30)
Decreased formation of prions
Ref
[102]
[103]
[103]
[106]
[107]
[108]
Paper III
[105]
[109]
[109]
27
Aims of the study
28

Paper I: Present a unifying protocol for analysis of CPP uptake,
enabling comparison of data from different groups.

Paper II: Determine the uptake and intracellular stability of three
different CPPs in two commonly used yeast species.

Paper III: Characterize the novel CPP p14Arf possessing a combination of efficient drug delivery vector and apoptogenic peptide in one unity.

Paper IV: Evaluate the novel CPP M918 as a cellular transporter
of peptide nucleic acid (PNA) and proteins.

Paper V: Improve the stability of p14Arf, and thereby its efficacy as an apoptosis inducing agent.
Methodological considerations
Solid phase peptide synthesis
In 1963 Bruce Merrifield revolutionalized peptide synthesis by introducing
the concept of solid phase peptide synthesis (SPPS) [110]. The use of a solid
support, called resin, upon which the peptide chain is assembled, makes it
possible to drive every amino acid coupling step to completion. Excess reagents can easily be washed away and by anchoring the peptide to a support
the solubility in organic solvent is increased. The process of amino acid assembly is repetitive and easily automated.
Amino acid side chains are protected, thus ensuring that the activated carboxylic acid is only reacting with the α-amino group. However, if the αamino group was free the amino acids not yet coupled to the peptide could
react with each other. Therefore, the α-amino group is protected with either
tert-Butyloxycarbonyl (Boc) or 9-fluorenylmethyl-oxycarbonyl (Fmoc),
depending on what chemistry is applied. This protective group is removed
before every new amino acid coupling, while the side chain protective
groups are present until the final cleavage from the resin.
In this thesis only Boc chemistry was used, both for peptide and PNA synthesis. The Boc group was removed with trifluoro acetic acid (TFA) and the
peptide and side chain protective groups were cleaved with the very strong
acid hydrogen fluoride. To neutralize the reactive carbocations formed during the cleavage reaction, paracresol was added as a scavenger (together
with parathiocresol if the peptide contained methionine or cystein). 5,6Carboxyfluorescein was activated with N-hydroxybenzotriazole and diisopropylcarbodiimide and coupled to the N-terminus of the peptides prior to
final cleavage. PNA (paper III-V) was also assembled in a step wise manner,
but with PNA monomers instead of amino acids and with other activators, as
described in detail in the papers. After peptides and PNA were cleaved from
the resin, scavengers were removed by ether extraction in 10% acetic acid,
followed by filtration to separate the resin and lyophilization to remove the
solvent.
29
Purification and identification of synthesized peptides
and PNA
Peptides were purified with semi-preparative reversed phase high performance liquid chromatography (RP- HPLC) in a C18 column using a gradient
of acetonitrile and water containing 0.1 % TFA. The fraction containing the
right product was identified with matrix-assisted laser desorption ionization
time-of-flight (MALDI-TOF) mass spectrometry using α-cyanohydroxycinnamic acid as matrix.
Peptide-PNA conjugation
Peptide-PNA conjugates used in paper III-V were synthesized with disulfide
bond. There are other methods to link PNA to peptides, e.g. maleimide
coupling or continuous synthesis. However, to be able to analyze the biological effect of the PNA it is preferable if it is detached from the CPP once
inside the cell since the peptide might interfere with target binding. This can
be achieved by using disulfide bond for conjugation, since this linker is reduced in the cytoplasm, releasing the PNA. The reaction requires an activated thiol, which can be achieved with the 3-nitro-2-pyridinesulphenyl
(Npys) group on cystein side chain [111]. The peptides are synthesized with
N-terminal Cys(Npys) and PNAs are synthesized with cystein attached to the
N-terminal lysine. Two lysines are added to both the C- and N-terminus of
PNA to improve solubility, which is otherwise a major issue with PNA. To
form covalent conjugates, CPP and PNA were mixed in 20% acetonitrile/water containing 0.1% TFA and stirred over night. The conjugates were
purified with semi-preparative RP-HPLC and identified with MALDI-TOF
mass spectrometry.
Choice of cells
Yeasts (paper II)
Candida albicans and Saccharomyces cerevisiae are two widely used yeast
species that function as model systems for many genetic studies, due to their
rapid growth and ease of genetic manipulation. They are both commensal
yeasts, meaning that they are present in the human body without causing any
harm. However, in immunocompromised individuals C. albicans may become invasive and cause a condition called candidiasis.
30
Breast cancer cells (paper III-V)
In paper III-V the human breast cancer cell lines MCF-7 and MDA MB 231
were studied. Both are isolated from pleural effusions and are of adenocarcinoma origin. However, they differ in their expression of the tumor suppressor p53. MCF-7 expresses wild type p53, while MDA MB 231 has a dysfunctional p53 protein and this difference was exploited in paper III to investigate the p53 dependence of the p14Arf peptide. None of the cell lines express p14Arf protein, making these cell lines suitable models for studying
the effects of p14Arf peptide. Since paper V is a direct follow up study of
paper III, the same cell lines were used. MCF-7 cells were also used in paper
IV, since the initial discovery of the superior CPP properties of M918 originates from paper III utilizing this cell line.
HeLa cells (paper I, III-V)
The human cervical cancer cell line HeLa is probably the most widely used
cell line in laboratories all over the world. It is therefore convenient to include this cell line to enable comparison of results between different research
groups. HeLa cells are easy to work with since they proliferate rapidly and
are easy to transfect. The splice correction assay (paper III-V) depends upon
a special HeLa cell line called HeLa pLuc 705. This cell line was developed
by Kole and colleagues in 1998 and is described in more detail in the section
describing the splice correction assay.
Astrocytoma cells (paper IV)
Hifko and VEGF+ cells are genetically engineered transformed murine astrocytes, where the hypoxia inducible factor (Hif-1) is deleted and the VEGF
receptor over expressed, respectively [112]. These cells were used in a parallel project where we targeted astrocytomas, therefore they were included in
this study as well.
Chinese hamster ovarian cells (paper IV)
Chinese hamster ovarian cells (CHO)-K1 is a widely used cell-line. It is a
sub-clone from a parental cell-line initiated from a biopsy of an ovary of an
adult Chinese hamster [113]. One mutant GAG-deficient variant of CHOK1, pgsA-745, has been widely used in CPP internalization studies. In paper
IV this cell-line is referred to as CHO2242. The pgsA-745 clone harbors a
mutation that decreases the activity of xylosyl-transferase, the first enzyme
in GAG synthesis, and hence these cells are devoid of all GAGs [114].
31
HPLC in determination of peptide uptake and
degradation
One excellent method of determining peptide degradation, and simultaneously receiving quantitative information regarding intracellular uptake of CPPs,
was developed by Ohelke et al [115]. By chemically modifying peptides
bound to the extracellular face of the plasma membrane, making them more
hydrophobic, it is possible to distinguish these from truly internalized peptides by means of RP-HPLC, since modified peptides have a longer retention
time. From the chromatograms it is possible to determine not only the
amount of internalized peptide in total, but also the amount of intact and
degraded peptides. The protocol for this assay is described in paper I and it
was used in paper II and III to determine the uptake and degradation of CPPs
in yeasts and to analyze the intracellular stability of p14Arf (1-22), respectively. Although being an outstanding method for determining peptide uptake and degradation, it is claimed by Ohelke et al to be restricted to peptides
containing primary amines, i.e. lysine. However, the p14Arf (1-22) peptide
contains no lysines, and we were still able to track the intact and degraded
peptide. We hypothesized that also cystein is compatible with this method,
and recently this was confirmed by Aubry et al. They utilized the same method to assess uptake of thiol-containing peptides [116].
Quantitative uptake by spectrofluorometry
Quantitative uptake by spectrofluorometry is a convenient and easy method
to determine the amount of internalized, fluorophore-conjugated CPP. In this
assay fluoresceinyl peptides are incubated with cells followed by a mild
trypsin treatment to remove peptides bound to the outside of the plasma
membrane. Following centrifugation, lysis and measurement of fluorescence, the fluorescence is normalized to protein content in each well, to account for variances in cell density. Different endocytosis inhibitors can be
employed to evaluate the contribution of various uptake pathways [57]. A
selection of inhibitors is presented in Table 4.
A disadvantage with this method is that peptides trapped in endosomes cannot be discriminated from truly internalized peptides. Several methods are
therefore needed in conjunction to surely determine the cellular uptake.
However, it is an excellent initial screening tool; peptides that show no cellular uptake can be excluded from further studies, while the ones generating a
positive result are further investigated.
32
Microscopy
Before 2001 most studies on cellular internalization of CPPs were conducted
using fluorescence microscopy on fixed cells. However, after the discovery
by Lundberg et al that cell fixation could cause artifactual results, caution is
nowadays given to working with fixed cells [117]. It is becoming increasingly common to use confocal microscopy on live cells, since this technique
enables visualization of one focal plane of the cell.
Microscopy is the method providing most information regarding the intracellular localization of fluorophore labeled peptides. It is a perfect complement
to the method presented in the previous section, since it discriminates between membrane bound and intracellular peptide, as well as peptides trapped
in endosomes. In mechanistic studies of CPP internalization pathways, fluorescent markers for different endocytic routs enable measurement of colocalization between CPP and the marker. This is described in paper I where
we show that the fluid phase marker dextran co-localizes with the CPP TP10
in HeLa cells, indicating that macropinocytosis is involved in the uptake of
this peptide.
Peptide degradation complicates the picture since fluorescence inside cells
might arise from fluorescent degradation products rather than intact peptides.
Therefore, microscopy should always be complemented with other methods,
e.g. HPLC described above, to complete the picture of cellular internalization of CPPs
Splice correction
The use of splice correcting oligonucleotides (SCOs) as a research tool to
assess the cargo delivery capacity of CPPs has become common in recent
years. However, this method and the HeLa pLuc 705 cells upon which the
assay is dependent, was developed already in 1998 [60]. These cells are stably transfected with a luciferase reporter gene, which is interrupted by an
intron from β-globin pre-mRNA containing an aberrant splice site. The aberrant splice site activates a cryptic splice site and under normal conditions
non-functional luciferase is produced. Upon introduction of SCOs binding
complementary to the aberrant splice site, splicing is re-directed and functional luciferase is produced. By coupling different CPPs to SCOs complementary to the cryptic splice site, their capacity as drug delivery vectors can
be assessed. This assay gives information whether fractions of CPP-SCO
conjugates have escaped endosomes, making it superior to many other assays based on fluorescence techniques. Another major advantage with this
method is that it generates a positive biological read-out. A number of different SCOs exist [56], however, in this thesis only PNA was utilized. PNA
is an uncharged oligonucleotide analog with a peptide backbone which binds
strongly to RNA and DNA [118].
33
Cell viability
LDH
A widely used method to determine membrane damage is to measure the
amount of lactate dehydrogenase (LDH) leaking out from cells. This method
is based on the production of fluorescent product (resorufin) following administration of substrate (NAD+ and diaphorase) for the enzymatic reaction
and presence of the LDH enzyme catalyzing the reaction. Since LDH cannot
penetrate the plasma membrane, it is only present in the extracellular medium surrounding cells with damaged membrane. The amount of fluorescence produced is proportional to the number of leaky cells.
Wst-1
The LDH assay is utilized to determine acute toxicity like membrane damage. To investigate more long term effects on cells treated with peptides the
Wst-1 assay is suitable. Mitochondrial dehydrogenases convert tetrazolium
salts to formazan in viable cells. A decrease in the number of viable cells
leads to a reduction in overall enzyme activity and a decrease in the amount
of formazan dye produced, which is measured as reduced absorbance in the
sample. The same principle is involved in the historically more widely used
MTT assay, but Wst-1 is an improved variant with a single add-and-measure
approach.
Cell morphology
Actin cytoskeleton and focal adhesions
Cells treated with trypsin normally obtain a globular form and detach from
the underlying surface. However, in paper V we observed that cells treated
with RI-CPPs were unable to do so and remained unchanged. The actin cytoskeleton is the structure responsible for cell rigidity and movement and the
actin filaments terminate with focal adhesions at the cell membrane [119].
Therefore, to visualize the cytoskeleton we used a rabbit antibody that binds
to the focal adhesion protein zyxin and phalloidin that binds to polymerized
actin. Secondary antibody goat-anti-rabbit-Alexa-594 yields red fluorescence
and phalloidin-Alexa-488 fluoresces green.
34
Apoptosis
Apoptosis, or programmed cell death, is a highly regulated process of cell
suicide. This process follows certain patterns which can be tracked with different methods. In contrary to necrotic cell death, the apoptotic cells forms
membrane blebs and are initially not detaching from the cell culture plastics.
AnnexinV staining
Early in the apoptotic process phosphatidyl serine, normally present on the
inside of the plasma membrane, is displayed on the cell surface. Annexin V
is a molecule that specifically binds phosphatidyl serine and by utilizing
fluorescently labeled Annexin V, apoptotic cells can be detected with FACS
or microscopy. To enable distinction between apoptotic and necrotic cells,
propidium iodide (PI) is usually included. PI binds nucleic acids with high
affinity, but only cells with damaged membrane are stained. However, in the
later phases of the apoptotic process also these cells might be permeable to
PI making it hard to distinguish late apoptotic from necrotic cells. This method was utilized in paper III and would have been suitable to use also in
paper V. However, to be able to analyze the cells with FACS they have to be
trypsinized to detach from the cell plastics. Since the RI-peptides displayed
the unexpected characteristic of gluing the cells to the plastics, this method
could not be utilized. Attempts with scraping the cells only resulted in a
large population of PI positive cells, even in cells not treated with peptides.
Nuclear condensation
The nuclei of apoptotic cells display characteristic fragmentation and condensation of the chromatin. This can be visualized with fluorescence microscopy after staining the cells with a nuclear dye, e.g. Hoechst or DAPI. To
be able to quantify the number of condensed nuclei these have to be counted
manually, which is a difficult and time consuming task. In paper V a software (kindly provided by Tobias Holm ♥) was used to keep track of counted
cells and the proportion of normal and apoptotic cells. Therefore a large
number of cells could be counted.
Mitochondrial membrane potential
Mitochondria have a central role in apoptosis, and the mitochondrial membrane potential is a prerequisite for cellular viability. A fall in mitochondrial
membrane potential is one of the earliest events in apoptosis and is correlated with an uncoupling of electron transport from ATP production. MitoTracker® is a mitochondrion-selective probe that accumulates in active mito-
35
chondria, thus a decrease in fluorescence is detected upon lowered membrane potential.
Caspase-3
Caspases are crucial mediators of apoptosis. Caspase-3 is a central death
protease responsible for many of the characteristic morphological features of
apoptosis, such as chromatin condensation and membrane blebbing [120].
Detection of activated caspase-3 is a common assay to show induction of
apoptosis and was utilized in paper V. Following treatment with RI-peptides
at different concentrations for 4 h, expression of active caspase-3 was visualized with rabbit polyclonal Caspase-3 antibody and goat anti-rabbit-Alexa594 secondary antibody.
36
Results and discussion
Studying the uptake of cell-penetrating peptides (Paper
I)
The research field of CPPs is relatively new, but the fast increase in number
of published articles yearly show the great interest the community has in this
group of peptides. When more research groups become involved the need for
unifying methods is highly appreciated to enable comparison of data from
different laboratories.
Paper I provides detailed protocols for three different methods of analyzing
cellular uptake of CPPs. The first method, spectrofluorometry, enables quantification of CPP uptake. It is performed in 12- or 24 well plates, thus making it suitable for high throughput screening of new CPP candidates. The
importance of trypsination is highlighted by demonstrating that the uptake of
both pVEC and TP10 is 3-fold higher without trypsination, showing that
peptides bound to the outside of the cell are falsely interpreted as internalized if this step is omitted. Since this method precludes discrimination between biologically available peptides and those trapped in endosomes it is
recommended to also use confocal microscopy to get more detailed information regarding intracellular localization. In paper I the necessary preparations
of the samples for microscopy is described. It is also presented how to include markers for endocytosis to enable co-localization studies, in this case
dextran as a marker for macropinocytosis. A flaw with this method is that
only the fluorophore is detected, thus it cannot be ascertained that the fluorescence originates from intact CPP and not from degradation fragments.
Therefore, the third presented method is excellent in combination with the
earlier two. By use of RP-HPLC it is possible to discriminate between internalized and extracellularly bound peptides as well as intact and degraded
peptides. This method is unique in its ability to descriminate between intact
and degraded CPPs, as well as the amount of degradation products.
These three methods are recommended to use in conjunction, since there is
no single method available that gives a complete picture of the uptake pattern. Furthermore, the presented protocols are suitable for initial screening
and characterization of new CPPs, but in the end all promising candidates
need to be further investigated for capability of delivering cargos with retained biological activity.
37
Uptake of cell-penetrating peptides in yeasts (Paper II)
The uptake of CPPs in mammalian cells is a well studied phenomenon and it
has been shown that CPPs internalize all cell types. However, reports showing uptake into yeast cells are scarce. The cell wall of yeasts is thick and
yeast cells are very resistant towards treatment otherwise harmful to both
mammalian and bacterial cells [68].
In paper II we show that the CPPs penetratin, pVEC and (KFF)3K are taken up into two different yeast species, C. albicans and S. cerevisiae. Not
only internalization, but also degradation of the peptides was investigated
and shown to be different depending both on peptide and yeast species. The
most dramatic divergence was the uptake of penetratin in the two species. In
S. cerevisiae penetratin was the CPP with highest uptake, while in C. albicans the uptake was negligible. However, when subtracting the degraded
fragments from the total penetratin uptake the intracellular pool of intact
penetratin was negligible in both yeast species. (KFF)3K was a moderate
CPP in both species, even when looking at the total uptake. pVEC was the
most promising candidate, since it displayed both the highest uptake and
highest stability in both species. Surprisingly, L- and D-pVEC were equal in
S. cerevisiae, both in terms of stability and uptake. In contrary, the uptake of
D-pVEC in C.albicans was about 5-fold higher than L-pVEC at 10 µM,
although no degradation products of L-pVEC could be detected. However, in
this study the extracellular degradation of peptides was not determined,
which could be one explanation to the divergent and unexpected results. If
C. albicans expresses some species specific proteases, this might explain the
higher uptake of D-pVEC compared to pVEC, penetratin and (KFF)3K,
which are all composed of L-amino acids.
When analyzing time dependence of uptake for up to 60 min there was a
weak but visible pattern that the L-peptides reached a plateau. D-pVEC, on
the other hand, showed unsaturated uptake within the same time frame. One
explanation might be the higher stability of D-pVEC resulting in accumulation of peptide over time.
In summary, we show that CPPs, in addition to mammalian cells and bacteria, also internalizes into S. cerevisiae and C. albicans. Historically, studies
with yeasts have yielded basic insight into cellular processes as diverse as
mitochondrial genetics, membrane biogenesis and gene silencing, applicable
to all eukaryotes [121]. Yeast cells share most of the structural and functional features of higher eukaryotes, which has rendered yeast an ideal model for
eukaryotic cell biology. Utilizing CPPs as delivery agents might aid such
studies in the future. Furthermore, considering the pathogenicity of C. albicans, cytotoxic agents conjugated to CPPs could be used as antifungal
agents.
38
Characterization of a novel cytotoxic cell-penetrating
peptide derived from p14Arf protein (Paper III)
One feature common for many CPPs is their net positive charge, which also
is characteristic for the tumor suppressor protein p14Arf. This gave rise to
the idea of searching for cell-penetrating sequences within this protein, with
retained biological activity. Peptides from the N-terminus of p14Arf have
been reported by others to be sufficient for binding the HDM2 target and
increase p53 activity (Table 5). This prompted us to synthesize several Nterminal peptides and investigate if these sequences could have dual properties of being both cell-penetrating and having the biological effect of the
p14Arf protein.
Interestingly, Arf (1-22) was internalized to the same extent as the established CPP TP10 and was therefore further analyzed. The uptake displayed a
vesicular pattern and was partially co-localized with transferrin, indicating
that endocytosis, and more specifically CME, was the major mechanism of
internalization. Importantly, Arf (1-22) conjugated to splice correcting PNA
induced expression of functional luciferase, verifying its capability of delivering functional cargo to the nucleolus. Splicing was further increased
when cells were pre-treated with chloroquine, corroborating the involvement
of endocytosis in the uptake. Having established the cell-penetrating and
cargo delivery ability of Arf (1-22), the cellular effects were next analyzed.
Viability was decreased by approximately 50% in both MCF-7 and MDA
MB 231 cells following 25 µM Arf (1-22) treatment. This effect could be
reached with only 5 µM Arf (1-22) when chloroquine was added, stressing
the importance of endosomal escape.
MDA MB 231 cells were included as control since these cells are p53
deficient, thus expected to be non-responsive to the tumor suppressive functions of Arf (1-22). Intriguingly, MDA MB 231 cells were as affected as
MCF-7 cells, indicating that cellular pathways independent of p53 status
were activated. Indeed, p14Arf can trigger apoptosis by mechanisms that do
not require the expression of a wild-type p53 protein [122] [123]. Moreover,
Arf interacts with and antagonizes the transcriptional function of Myc and
E2F1 independently of p53, both proteins being potent oncogenes required
for cell cycle progression [124].
The altered viability was not an effect of membrane damage, since no
significant leakage of LDH was detected up to 50 µM. However, scrambled
Arf(1-22) did cause leakage at higher concentrations, which could be the
reason for the reduced viability observed at 25 µM. Chromatin condensation
was evident after Arf (1-22) treatment, indicative of apoptosis. FACS analysis of Annexin V binding showed approximately 30% early apoptotic MCF7 cells following 25 µM Arf (1-22) treatment. A partially inverted control
peptide (M918, see paper IV) was inactive in the above mentioned viability
39
and apoptosis assays, supporting specific interaction of Arf (1-22) with target proteins.
The results presented in this paper show that Arf (1-22) is a promising
candidate for drug delivery in tumor cells. Since it is an efficient CPP capable of delivering bioactive cargo, and at the same time possessing an apoptosis inducing effect per se, synergistic effects on tumor cell viability might be
achieved by conjugation with cytotoxic cargos.
A novel cell-penetrating peptide, M918, for efficient
delivery of proteins and peptide nucleic acids (Paper IV)
CPPs have emerged as alternatives for efficient delivery of bioactive cargos
such as proteins and PNA. Transfection agents composed of cationic lipids
are efficient but high delivery efficacy is often accompanied by toxicity.
Therefore, novel peptides with high transporting capacity without concomitant toxicity are highly desired. M918 is one such peptide. It was discovered
when it was synthesized as a control peptide to Arf (1-22) in paper III. Its
uptake in HeLa cells was significantly higher than the well characterized
CPPs penetratin and TP10, and it internalized into all investigated cell-lines.
No adverse effects on membrane integrity or cell proliferation were detected.
Two different approaches for transduction of the proteins avidin and streptavidin were employed; co-incubation and conjugation via biotinstreptavidin/avidin binding. With the co-incubation strategy CPPs and proteins were simply mixed before adding them to the cells. M918 induced the
highest increase in uptake of fluoresceinyl streptavidin. Avidin contains
more positive charges than streptavidin, thus counteracting the electrostatic
interactions needed for complex formation between CPP and protein. Therefore, CPPs does not increase uptake of avidin as much as streptavidin. On
the other hand, when mixing biotinyl CPPs with avidin or streptavidin, nearly irreversible bonds are formed and now avidin reached higher intracellular
levels with all three CPPs. M918 and TP10 were most efficient, however, at
25 µM TP10 caused 70% leakage of LDH, thus being very toxic at higher
concentrations.
When treating cells with M918 at 4 °C, the uptake was significantly reduced. Also sucrose and chloroquine had an inhibitory effect on uptake of
M918 and transferrin, indicating involvement of CME. Microscopy images
of cells treated with M918 together with transferrin showed partial colocalization, but this was also the case with dextran, which is a marker for
fluid phase endocytosis. Intriguingly, GAGs on the cell surface were not a
prerequisite, since uptake of M918 in CHO cells devoid of GAGs was not
significantly reduced. This observation is disparate from many reports claim-
40
ing GAGs to be the initial interacting partner of CPPs preceding cellular
uptake, as described in the introduction.
However, merely measuring the uptake and localization of M918 per se is
not giving true information regarding its capacity as delivery agent. The
splice correcting assay described earlier provides a positive read-out regarding bioavailability of the cargo. PNA, coupled to CPP with disulfide bond,
binds to the aberrant splice site and induces expression of luciferase upon
delivery to the nucleolus. M918 was more efficient than TP10 and penetratin
in PNA delivery at 5 µM concentration. This read-out system was employed
together with inhibitors of different endocytosis routs to further elucidate
which uptake mechanism M918 utilizes. Decreased splicing at 4 °C indicates
involvement of endocytosis in general, and the reduction after treatment with
cytochalasin D and Wortmannin indicates macropinocytosis in particular.
Together with the observed partial co-localization with dextran, macropinocytosis is suggested to be the major entry route. Splicing was increased after
addition of the endosome buffering agent chloroquine, thus a large fraction
of conjugates were trapped in endosomes and not biologically available before addition of chloroquine.
Put together, these results reveal M918 to be a highly efficient delivery vector capable of transporting diverse cargos, such as PNA and proteins, into
cells in a non-toxic fashion.
Retro-inversion of certain cell-penetrating peptides
causes severe cellular toxicity (Paper V)
Inspired by the promising results presented in paper III, the goal was to increase the stability of Arf (1-22) to enable decreased administration concentrations and incubation time. Several approaches exist to increase peptide
stability; however in paper V the RI isomerization was used. Since it has
been reported that p14Arf (1-14) retains the activity of the original p14Arf
(1-22) peptide [100], it was also used in paper V. Indeed, the cytotoxicity of
RI-Arf (1-14) was increased compared to its L-counterpart. However, a control peptide believed to be inactive was as cytotoxic as RI-Arf (1-14), indicating that the RI-isomerization could induce toxicity per se. This phenomenon was evident also when investigating the splice correcting capacity of the
recently discovered CPP M918 and its RI-counterpart, conjugated to splice
correcting PNA, as described in paper IV. There was no difference in delivery capacity between the two isomers at 2 and 5 µM, but at 10 µM the
splicing of RI-M918-PNA dropped to the same level as 2 µM while the
splicing of M918-PNA increased further.
Cells treated with RI-peptides also displayed trypsin insensitivity. To further
investigate this phenomenon different morphological examinations were
41
conducted, as well as measurements of apoptosis. Tat and penetratin, the two
most widely used CPPs, were synthesized in RI-mode and included. Also a
truncated version of penetratin was synthesized. RI-Arf (1-14), RI-M918,
and RI-penetratin decreased the metabolic activity in all three cell-lines
(MCF-7, MDA MB 231, and HeLa), as determined with the Wst-1 assay,
without rupturing the plasma membrane since no significant leakage of LDH
could be detected. RI-Tat and RI-short penetratin did not affect viability;
neither did the L-peptides. The same pattern was observed when investigating the morphological alterations; treatment with the longer RI-peptides was
detrimental to the focal adhesions and the actin cytoskeleton, while Lpeptides where harmless.
To investigate if RI-peptides induced apoptosis, firstly, the mitochondrial
membrane potential probe MitoTracker® was utilized. During early apoptotic
events the inner mitochondrial transmembrane potential is disrupted. The
results revealed that RI-Arf (1-14) and RI-penetratin exhibited a detrimental
effect on the mitochondrial network already after 3 h, suggesting this to be
the initiating event preceding the morphological alterations described above.
The longer RI-peptides also induced chromatin condensation visualized by
Hoechst staining and, as a more specific marker for apoptosis, increased
caspase-3 expression. None of these effects were evident upon RI-Tat, RIshort penetratin or L-peptide treatment, suggesting that longer RI-peptides
induced the observed cellular alterations. The importance of peptide length
on cellular toxicity has been demonstrated before [125]. The shortening of
MAP peptide by four amino acid residues either N- or C-terminally, corresponding to removal of one of the α-helix turns, eliminated its toxic effects
[126]. This raises the question whether there is a correlation between helicity
(a membrane interacting secondary structure) and cytotoxicity. Indeed, the
concept of “stapled” peptides, where an introduced molecule stabilizes the
helical structure of a peptide, shows that these peptides possess strong intracellular activities resulting in apoptosis of tumor cells [127]. The toxic peptides presented in paper V all contain amino acids commonly found in helical structures, whereas RI-Tat is highly cationic, counteracting formation of
helixes. Furthermore, both RI-Tat and RI-short penetratin are likely too short
to form a secondary structure. The observed differences in toxicity might
also stem from differences in intracellular levels of the peptides, since Tat
per se is not a very efficient CPP [128-130] and its uptake has been demonstrated to be about 50 times lower than penetratin at 5 µM [131].
In summary, we suggest that RI-isomerization of longer CPPs causes cytoxicity displayed as diminished metabolic activity and mitochondrial membrane potential, leading to gross morphological alterations resulting from
depolymerized actin cytoskeleton. These changes ultimately culminate in
apoptosis.
42
Conclusions
 Paper I: The presented protocols provide three different methods to
determine the cellular uptake of CPPs. If used in parallel these assays provide information regarding amount, degradation as well as
intracellular distribution of internalized fluorophore labeled CPPs.
 Paper II: The three investigated CPPs are internalized into both
S.cerevisiae and C. albicans with different efficacy. However, the
intracellular stability varies from complete stability, as with pVEC,
to almost complete degradation, as with penetratin.
 Paper III: Arf (1-22) is an efficient CPP with the ability to transport
bioactive cargo and, at the same time, reduces the viability of cancer cells. The dual cell-penetrating and apoptogenic ability presented by this peptide is attractive in the search for improved therapeutic agents, since cytotoxic drugs may be conjugated, possibly
yielding a synergistic effect on tumor cells.
 Paper IV: M918 has emerged as an outstanding CPP capable of
transporting both PNA and proteins into cells in a non-toxic fashion, with an efficiency superior to most established CPPs.
 Paper V: Retro-inversion of certain CPPs causes toxicity not attributed to the inherent function of the original natural peptide. This
modification also renders the treated cells resistant to trypsination.
These results advance the current knowledge about CPP uptake and stability in different organisms. The finding of a novel CPP with superior vector abilities is highly appreciated and a promising tool for improved cargo
delivery. Furthermore, the design of a CPP with inherent biological function
opens a new avenue in drug discovery. However, applying the commonly
exploited RI modification on this and other CPPs generates cytotoxic peptides, a phenomenon which has not been reported before. This provides a
warning to other researchers attempting to increase peptide stability with this
modification.
43
Populärvetenskaplig sammanfattning på
svenska
Kroppen är uppbyggd av celler som är omslutna av ett skyddande fetthölje
kallat plasmamembran. Läkemedel består ofta av vattenlösliga substanser
som inte kan tränga igenom plasmamembranet utan måste transporteras över
det för att kunna ha sin verkan inne i cellen. Denna transport kan utföras på
olika sätt, men de verktyg som presenteras och analyseras i den här
avhandlingen kallas för cell-penetrerande peptider (CPPer). 1994 var det en
forskargrupp i Frankrike som upptäckte den första CPPen och sedan dess har
flera hundra nya rapporterats. Det som är så anmärkningsvärt med dessa
peptider, som är små fragment av proteiner, är att de inte bara kan ta sig över
plasmamebranet, utan också föra med sig andra substanser som annars skulle
vara exkluderade från cellen. Till och med jästceller, som förutom
plasmamembran också omsluts av en tjock vägg av olika kolhydrater, kan
penetreras av CPPer. Detta kan utnyttjas inom grundforskning när man vill
undersöka funktionen av olika proteiner. Genom att koppla olika substanser
till CPPer kan dessa tas upp i cellen och inaktivera proteinet av intresse och
man kan då undersöka hur det påverkar cellen. Det finns även arter av jäst
som kan orsaka sjukdom och då kan man koppla på giftiga molekyler på
CPPer som dödar jästen.
CPPer används inom många olika forskningsområden,
framför allt inom olika sjukdomar som diabetes och cancer. Cancerceller har
förmågan att dela sig oändligt många gånger och mycket forskning inriktas
därför på att döda dessa, utan att skada normala celler. I den här
avhandlingen presenteras en peptid som har förmågan att både döda
cancerceller och vara en CPP, vilket är något helt nytt. Det visade sig även
att en peptid som användes som kontroll, det vill säga inte var skadlig för
celler, var en mycket effektiv CPP. Den var faktiskt mer effektiv än de
etablerade CPPer den jämförts med och den kunde transportera både
proteiner och modifierade nukleinsyror in i celler. Ett stort problem när
peptider ska användas som läkemedel är att de snabbt bryts ner i kroppen.
Med syfte att öka stabiliteten av de två peptiderna presenterade ovan
syntetiserades de på ett sätt som gör att nedbrytningsenzymer inte känner
igen dem. Det visade sig dock att dessa modifierade peptider orsakade
irreversibla skador på celler, vilket inte har rapporterats förut och kan
44
fungera som ett varnande exempel för andra forskare som tänkt utnyttja
samma metod.
CPPer som forskningsområde har ökat explosionsartat det
senaste decenniet och fler och fler forskningsgrupper använder CPPer inom
sina respektive områden. Data som kommer från olika laboratorier är svåra
att jämföra beroende på att olika parametrar använts, tex celltyp,
peptidkoncentration och inkuberingstider. Därför publicerade vi tre olika
protokoll som med fördel kan användas parallellt för att ge en mer komplett
bild av CPPers upptag i celler och som kan underlätta jämförelse av data.
45
Acknowledgements
Nu har jag då kommit till den sista och viktigaste delen av avhandlingen.
Efter en rad misslyckade projekt, några mer framgångsrika, mycket jobb och
två mammaledigheter börjar jag äntligen närma mig slutet! Jag hade inte
kommit så här långt om det inte var för en rad personer. Först och främst vill
jag tacka min handledare Ülo för att ha gett mig frihet att göra det jag tycker
är spännande, även om det inneburit sidospår. Tack för att du alltid tar dig
tid. Mattias, vilken tur jag hade att du behövde hjälp i ett projekt när jag var
precis ny! Tack för trevligt sällskap på många middagar. Jag vill även tacka
alla lärare för stimulerande föreläsningar. Ett stort tack till den
administrativa personalen Ulla, Marie-Louise, Birgitta och Siv för att ni får
institutionen att fungera. Ett särskilt tack går till Siv för att du bryr dig och
orkar se till att saker blir gjorda.
Alla tidigare och nuvarande doktorander; ni har verkligen gjort de här
åren roliga och minnesvärda! Det är några jag vill tacka särskilt:
Ulla, du har funnits där från dag 1! Tack för allt, både på jobbet och
utanför. Du är verkligen inspirerande! Helena, vem hade kunnat tro att vi
skulle bli släkt en dag? Du är så härligt annorlunda och jag blir glad bara av
att prata med dig! Maarja, du är en genomgod människa. Jag är så glad att
jag fått lära känna dig. Samir, var ska jag börja? Din närvaro har inspirerat
så många och du kommer gå långt. Tack för alla diskussioner, stunder på
gymmet, fester och lekstunder med barnen. Henke, tack för allt du lärde mig
när jag var ny och för alla gym-besök. Pontus och Linda, oj va jag har
saknat att ha er på rummet! Det blev verkligen tomt när ni slutade Peter J
och Jonas, det har varit mysigt att ständigt höra era diskussioner tvärs över
korridoren. Tack Peter G, för alla kluriga inlägg på seminarierna, Florén,
för sällskap och roliga samtal sena kvällar på labbet, Katri, för hjälp på
synteslabbet och Maria, för att du först fick mig intresserad av cellpenetrerande peptider. Emelia, Mats and Yang thank you for always helping out when the HPLC doesn’t want the same thing I do. Rania, lycka till
med din lilla flicka. Johan, du är en klippa! Tack för all hjälp med ditten och
datten. Andrés, Staffan, Kariem, Henrik och Kristin, ni har några tuffa
men roliga år framför er!
46
Johanna, du är verkligen en rolig kompis. Ibland skulle jag vilja vara lika
tuff som du! Caroline, tack för sällskap många tidiga morgonar. Jag saknar
ditt skratt. Veronica, jag är så glad att du är kvar. Karro, jag beundrar din
självständighet. Marie, du är en kämpe!
Tack Malin, Nina, Tom, Kristin, Linda T, Sofia, Jessica och Helena G
för alla roliga pratstunder i lunchrummet. Xin, thank you for always being so
cheerful.
Tack igen till Maarja, Kerstin och Samir för att ni tog er tid och
korrekturläste min avhandling. Helin and Margus, thank you for a fruitful
collaboration that resulted in paper V.
Tobias, tänk att du gjorde ett cell-räknar program till mig när jag hade typ
miljoner celler jag skulle sitta och räkna! Tack också för bilderna i
avhandlingen. Du är bäst!
Mina älsklingstjejer som har följts åt ända sedan vi började på
universitetet: Anne, Banafsheh, Karin, Christine och Emma! Jag längar
alltid till våra träffar och går därifrån med öronen trötta av allt prat.
Peter och Bettan, det känns verkligen som om jag haft en liten hejarklack
där borta i Asien.Ni är underbara! Min nya familj: Miriam och Torbjörn,
tack för att ni alltid bryr er! Det finns inga bättre svärföräldrar. Mattson,
Martin, Ullis och Micke, tack för allt roligt vi har gjort.
Tusen tack till Miriam, mamma och pappa för all barnvaktshjälp de
senaste veckorna. Utan er hade det inte gått.
Min älskade syster! Tack för att du alltid finns där och för att du faktiskt
har försökt sätta dig in i vad det är jag gör egentligen. En gång hörde jag dig
berätta för en kompis vad det är jag jobbar med och du hade ju verkligen
fattat! Mamma och pappa! Nu blir jag rörd när jag ska försöka
skriva....Tack för allt stöd, för att ni alltid finns där och för att ni alltid
uppmuntrat mig att göra det jag brinner för. Ni är dessutom underbara
morföräldrar!
Elvine och Einar, mina älsklingar! Ni har fått mig att förstå vad som är
viktigast i livet. Tobias, orden räcker inte till för att beskriva vad du betyder
för mig. I svåra stunder finns du där och delar tyngden, i alla lyckliga
stunder gör du glädjen dubbel. Du är mitt allt!
47
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