Cell-penetrating peptides

Cell-penetrating peptides
Uptake mechanism and the role of receptors
Henrik Helmfors
Cover: Henrik Helmfors
© Henrik Helmfors, Stockholm 2015
All papers were reprinted with permission
ISBN: 978-91-7649-259-8
Printed in Sweden by Holmbergs, Malmö 2015
Distributor: Department of Neurochemistry
To Linda
enes are the major regulators of biological processes in every living
thing. Problems with gene regulation can cause serious problems for
the organism; for example, most cancers have some kind of genetic component. Regulation of biological processes using oligonucleotides can potentially be a therapy for any ailment, not just cancer. The problem so far has
been that the targets for oligonucleotide-based therapies all reside on the inside of cells, because the cellular plasma membrane is normally impermeable
to large and charged molecules (such as oligonucleotides) a delivery method
is needed. Cell-penetrating peptides are a class of carrier molecules that are
able to induce the cellular membrane into taking them and their cargo molecules into the cells. Understanding how and why cell-penetrating peptides
work is one of the first and most important steps towards improving them
to the point where they become useful as carriers for oligonucleotide-based
therapies. This thesis is comprised of four scientific papers that are steps toward finding an uptake mechanism for cell-penetrating peptides that have
been non-covalently complexed with oligonucleotides. In Paper I, we show
that the scavenger receptors are responsible for uptake of the cell-penetrating
peptide PepFect14 in complex with a short single-stranded oligonucleotide.
Paper II expands upon this first finding and shows that the same receptors
are key players in the uptake of several other cell-penetrating peptides that
have been complexed with either, long double-stranded plasmid DNA or
short double-stranded RNA. Paper III improves the luciferase-based assay
for short oligonucleotide delivery by increasing the throughput 4-fold and
reducing the cost by 95 %. The fourth manuscript uses the assay developed
in paper III to investigate the effects on cell-penetrating peptide-mediated
delivery by each of the constituents of a 264-member library of ligands for
G-protein coupled receptors. We identify three ligands that dose-dependently increase the luciferase expression compared to control cells. These three
ligands are one positive-, one negative allosteric modulator of metabotropic
glutamate receptor 5 and one antagonist of histamine receptor 3.
List of publications
This thesis is based on tree papers and one manuscript, in the text referred
to as Paper I, II, III and IV
Ezzat, K., Helmfors, H., Tudoran, O., Juks, C., Lindberg,
S., Padari, K., EL Andaloussi, S., Pooga, M., Langel, Ü.:
Scavenger receptor-mediated uptake of cell-penetrating peptide
nanocomplexes with oligonucleotides. FASEB J. 26, 1172–1180
Lindberg, S., Regberg, J., Eriksson, J., Helmfors, H., MuñozAlarcón, A., Srimanee, A., Figueroa, R.A., Hallberg, E., Ezzat,
K., Langel, Ü.: A convergent uptake route for peptide- and
polymer-based nucleotide delivery systems. J Control Release.
206, 58–66 (2015).
Helmfors, H., Eriksson, J., Langel, Ü.: Optimized luciferase
assay for cell-penetrating peptide-mediated delivery of short
oligonucleotides. Anal Biochem. 484, 136–142 (2015).
Helmfors, H., Langel, Ü.: GPCR-ligands influence the short
oligonucleotide transfection efficacy of the cell-penetrating
peptide; Pepfect14, Manuscript.
Additional publications
Publications not included in this thesis.
Eriksson, J., Helmfors, H., Langel, Ü.: A High-Throughput
Kinetic Assay for RNA-Cleaving Deoxyribozymes. PLoS ONE.
10, e0135984 (2015).
Helmfors, H., Lindberg, S., Langel, Ü.: SCAR A Involvement
in the Uptake of Nanoparticles Formed by Cell-Penetrating
Peptides. In: Langel, Ü. (ed.) Cell-Penetrating Peptides. pp.
163–174. Springer New York, New York, NY (2015).
VII. Kim, T.K., Sul, J.-Y., Helmfors, H., Langel, Ü., Kim, J., Eberwine,
J.: Dendritic glutamate receptor mRNAs show contingent local
hotspot-dependent translational dynamics. CellReports. 5,
114–125 (2013).
VIII. Lindberg, S., Muñoz-Alarcón, A., Helmfors, H., Mosqueira,
D., Gyllborg, D., Tudoran, O., Langel, Ü.: PepFect15, a novel
endosomolytic cell-penetrating peptide for oligonucleotide
delivery via scavenger receptors. Int J Pharm. 441, 242–247
Muñoz-Alarcón, A., Helmfors, H., Webling, K., Langel, Ü.:
Cell-Penetrating Peptide Fusion Proteins. In: Fusion Proteins:
Applications and Challenges. pp. 397–411. John Wiley & Sons,
Inc., Hoboken, NJ, USA (2013).
Helmfors, H., Langel, Ü.: Recent developments in applications of
cell-penetrating peptides Uptake mechanisms and oligonucleotide
delivery. Chim Oggi. 30, 10–12 (2012).
Andersson, O., Helmfors, H., Kanmert, D., Enander, K.: A
multiple-ligand approach to extending the dynamic range
of analyte quantification in protein microarrays. Biosens
Bioelectron. 24, 2458–2464 (2009).
* Paper I in this thesis has previously been included in my licentiate thesis. ISBN 978-91-7447-782-5
1 I ntro d u c t i o n. ..................................................................
1. 1 T h e ra p e u t i c O l i g o n u c l e o t i d es....................................
1. 1 .1 P l a s m i d s. ............... ......................................
1. 1 .2 R N A i. ...........................................................
1. 1 .3 A n t i s e n s e. .............. ......................................
1. 1 .4 G l y b e ra. .......................................................
1. 2 C e l l - P e n e t ra t i n g P e p t i d e s. .. ......................................
1. 3 O l i g o n u c l e o t i d e De l i v e ry. .... ......................................
1. 4 E n d o c y t o s i s ....................... ......................................
1. 4 .1 C l a t h ri n - M e d i a t e d E n d ocyt osi s.......................
1. 4 .2 C a v e o l a r E n d o c y t o s is....................................
1. 4 .3 O t h e r E n d o c y t o t i c P at hways . . .........................
1. 5 C P P e n t ry a n d e n d o c y t o s i s. ......................................
1. 6 U p t a k e M e c h a n i s m s . ...............................................
1. 7 S c a v e n g e r R e c e p t o rs. ..............................................
1. 8 H i g h - t h ro u g h p u t A s s a y De v el op ment.. ........................
1. 9 G - p ro t e i n c o u p l e d re c e p t o rs.....................................
2 Aim.. . . ...................................... ......................................
2. 1 P a p e r I .............................. ......................................
2. 2 P a p e r I I. ............................ ......................................
2. 3 P a p e r I I I. ........................... ......................................
2. 4 P a p e r I V. .................................................................
3 M e thod s.........................................................................
3. 1 S o l i d - P h a s e P e p t i d e S y n t h esi s..................................
3. 2 C e l l C u l t u re a n d Tre a t m e n t .. ......................................
3. 3 Dy n a m i c L i g h t S c a t t e ri n g. .........................................
3. 4 Lu c i f e ra s e. ........................ ......................................
3. 5 S C A R A I n h i b i t o rs. ....................................................
3. 6 S p l i c e - C o rre c t i o n A s s a y. ..........................................
3. 7 S C O De l i v e ry. .................... ......................................
3. 8 P l a s m i d De l i v e ry. .....................................................
3. 9 s i R N A De l i v e ry. .................. ......................................
3. 10 F l u o re s c e n c e M i c ro s c o p y.... ......................................
3. 11 To x i c i t y A s s a y s. ................. ......................................
4 Re s ult s a n d Di s c u s s i o n. .................................................
4. 1 P a p e r I & I I S C A R A s a n d C PP U p t ak e . . .......................
4. 2 P a p e r I I I : C P P De l i v e ry A s s ay . . ..................................
4. 3 P a p e r I V: G P C R L i g a n d s I n f l uence U p t ak e..................
4. 4 S u m m a ry a n d C o n c l u s i o n .... ......................................
5 Future Ou t l o o k. ........................ ......................................
6 P o p ulä r v e t e n s k a p l i g s a m m a n fa ttn in g.............................
7 Ac k now l e d g m e n t s. .........................................................
8 Re f e ren c e s............................... ......................................
adaptor protein 2
acid sphingomyelinase
C. elegans
Caenorhabditis elegans
caveolin 1
clathrin independent endocytosis
clathrin- and dynamin-independent carrier
clathrin-mediated endocytosis
cell-penetrating peptide
clustered regularly interspaced short palindromic repeats
D. melanogaster
Drosophila melanogaster
danger associated molecular patterns
dynamic light scattering
deoxyribonucleic acid
double-stranded RNA
E. coli
Escherichia coli
CLIC-GPI-anchored protein-enriched early endosomal compartments
high-performance liquid chromatography
heparan sulphate
low-density lipoprotein
lipoprotein lipase deficiency
matrix-assisted laser desorption/ionization - time of flight
Milli-Q ultrapure water
molar ratio
messenger RNA
membrane translocating sequence
neurophilin-1 receptor
pathogen associated molecular patterns
plasmid DNA
phosphatidylinositol 3-kinase
phosphaidyl inositol polyphosphate
peptide nucleic acid
Poly C
polycytidylic acid
Poly I
polyinosinic acid
primary miRNA
phosphorothioate 2’-O-methyl
protein transduction domains
Ras homolog gene family, member A
RNA induced silencing complex
ribonucleic acid
RNA interference
RNase H
ribonuclease H
scavenger receptor class A
splice-correcting oligonucleotide
short interfering RNA
solid-phase peptide synthesis
scavenger receptor
splice-switching oligonucleotide
transmission electron microscopy
trifluoroacetic acid
3′-untranslated region
I ntroduction
umans have always used selective breeding to exert control over the
genome of plants and animals. Then, ~70 years ago, science uncovered that DNA contains the genetic information [1, 2]. With those discoveries of the makeup and structure of the genome an entirely new scientific
field emerged, it came to be known as molecular biology, a term likely coined
by William Astbury in 1961. Ever since, our ability for exerting ever-finer
control over the genome has grown. Already in the 1970s genes were transferred between organisms [3] and eventually transgenic plants, bacteria and
even mammals became commonplace. At the turn of the millennium the
sequencing of the human genome [4] was completed. This was a monumental effort, and it probably is the largest breakthrough in the biomedical sciences of the last half-century. We are finally beginning to reap the benefits
of having cheaper and faster DNA sequencing. The control over the genome
can now potentially be so fine that a single error in the genetic code can be
corrected. In the coming decades genetic sequencing will be included in the
panel of common lab tests. This in turn will bring even more data about disease. The genetic contribution of each person’s illness will become especially
interesting. This will bring truly personalized medicine. Mutations (genetic
errors) within individuals suffering from hereditary disorders, mutations in
individual tumors in cancer patients and other spontaneous mutations are
all possible targets for gene-therapy.
Right now medicine is at the tipping point where gene therapy is just
becoming possible, the first human treatment with gene therapy has already
been approved in the European Union [5]. Alipogene tiparvovec treats adult
patients with an adeno-associated virus-based gene therapy against familial
lipoprotein lipase deficiency (LPLD). Now that the door has been opened,
more therapies will become available, for many more conditions. These new
therapies could work in four different ways. First, a gene could be restored
like in the LPLD case above, to replace a lost or non-functional gene, as is
common in hereditary diseases [6] via delivery of plasmid DNA (pDNA).
Second, altering gene function by silencing a disease-causing gene, where
a promising technique would be to use the RNA interference pathway [7],
which in a therapeutic case would likely be mediated by short interfering
RNAs (siRNA). Third, altering gene function by interfering with the splicing machinery either via anti-microRNAs or splice-switching oligonucleotides (SSOs). In recent years a fourth way has received a lot of attention;
the CRISPR/Cas system of editing genes [8, 9] has opened new possibilities
of longer lasting genetic treatments; CRISPR/Cas also requires delivery of
pDNA, the benefit of this technique is that it is simple and user friendly
compared to the gene-editing techniques that preceded it.
All these potential ON-based therapies have in common that they are
based on the same kind of charged, water-soluble, macromolecules that all
need to be on the inside of cells in order to exert their effect. However, the
plasma membrane of the cell is exceptionally good at keeping exactly these
kinds of molecules in place on either side of the membrane. By comparison
conventional therapeutic drugs are small, hydrophobic molecules, these two
properties alone are usually enough for them to be able to passively diffuse the plasma membrane. Because the cell membrane is not pearmable to
hydrophilic and/or large molecules, a method for safely delivering siRNA,
SSOs and pDNA across it is needed.
Therapeutic Oligonucleotides
Before the delivery problem can be addressed, the types of molecules that
need to be delivered have to be presented. Loss-of-function in a gene is not
only associated with hereditary diseases such as hemophilia, cystic fibrosis
and muscular dystrophy or the LPLD case mentioned above and described
more in detail below, but it is also implicated in several cancers. A treatment that could possibly restore the correct phenotype is delivery of pDNA
containing a functional gene. In the LPLD case, a successful method of
delivering that gene is based on using a virus where the genomic material has
been replaced with the gene of interest. The oligonucleotide-based therapy
that is easiest to understand is based on delivery of a plasmid, where successful delivery could restore a protein function that has been lost due to a
mutation or illness.
1.1.1 P l a s m i d s
A plasmid is a circular piece of DNA that is not part of the chromosomal
DNA; it has the ability to replicate independently and can carry genes. The
term plasmid was coined in 1952 by Joshua Lederberg [10], and defined as “a
generic term for any extra-chromosomal hereditary determinant”. The definition has since been narrowed, in order to exclude viruses, to genetic elements
that can replicate autonomously and that exist outside of the chromosome.
The first applications for plasmids in the treatment of human diseases were
indirect. Almost 40 years ago plasmids were used to completely transform
the production of insulin; from previously having to be harvested from animals into being manufactured in Escherichia coli (E. coli) [11]. The next use
of plasmids for the treatment of a human condition was to produce another
protein, the human growth hormone. The methodology for production of
both was the same, a plasmid was constructed and the protein was expressed
in E. coli [12]. The protein was then harvested, purified and later injected in
patients. Continuing with the insulin example, it would be much better to
give the patients lacking the ability to produce insulin, the ability back rather
than them having to inject insulin several times a day. Patients with type
1 diabetes have lost the insulin producing beta-cells, luckily it is possible
to produce insulin in other cells, an early example, from 1983, are AtT20
(mouse pituitary corticotroph) cells [13]. Of course insulin secretion has to
be tightly regulated [14], which is a different problem. The first attempts at
using plasmids in vivo did not utilize any transfection vector, plasmid DNA
was simply injected into the muscle [15, 16]. However, this method suffers
from low efficacy, only about 1 % of the cells are transfected [17]. The low
efficacy of plasmids when used alone indicates that there is large room for
improvement of the transfection efficacy, most likely by delivery vectors.
1.1. 2 R N A i
One of the most attractive potential therapies that has been introduced in
the last couple of decades is the ability to activate the RNA interference pathway by introducing synthetic siRNA. The first step towards the discovery of
this pathway was the realization in 1993 that the lin-4 gene, which is known
to control the timing of Caenorhabditis elegans (C. elegans) larval development, did not code for a protein, but produced two small RNAs of 22 and
61 nucleotides (nt) respectively. These two RNAs regulate the translation of
lin-14 gene [18]. In 1998, a previously hidden more general gene-regulation
mechanism was uncovered in that nematode (C. elegans), RNA interference
(RNAi). Andrew Fire and Craig Mello were awarded the 2006 Nobel Prize
in medicine for their discovery. Their realization was that double-stranded
RNA was significantly more potent in regulation of gene-expression than
single-stranded antisense RNA [7]. The RNAi process is guided by small
RNAs that include both siRNA and microRNA (miRNA). The two different RNAs have different effects and different origins, miRNA is expressed
from the endogenous hairpin-shaped transcripts [19] into longer primary
miRNA (pri-miRNA) [20]. The transcription of these genes occurs in a way
analogous to that of messenger RNA (mRNA). The pri-miRNA is cleaved
in the nucleus by an RNase called Drosha into a precursor miRNA of about
65-bp [21]. After cleavage the pre-miRNA is exported from the nucleus to
the cytoplasm by Exportin5 [22]. The final step, that produces the short ~22bp RNA duplex by cleaving the precursor, is carried out by another RNase
III enzyme named Dicer [23, 24]. Dicer is highly conserved across almost
all eukaryotes including yeast, plants and animals. Some organisms have
multiple Dicer homologues, in Drosophila melanogaster (D. melanogaster)
Dicer 1 is involved in miRNA biogenesis whereas Dicer 2 produces siRNA
[25]. miRNAs act as guide molecules in post-transcriptional gene regulation by base-pairing with the target mRNAs, usually in the 3’ untranslated
region (uTR). Binding of a miRNA to the target mRNA typically leads to
translational repression [26]. Other types of regulation by miRNA, such
as translational activation [27] and heterochromatin formation [28], have
also been described. Currently the online repository for miRNAs, miRBase
[29] contains 1881 human miRNAs, suggesting that the post-transcriptional
regulatory mechanism mediated by small RNAs is even more general than
previously appreciated. A simplified scheme of the process can be found in
Figure 1.
The siRNA pathway is different because usually it is not transcribed from
the chromosomal DNA but instead can begin when long double-stranded
RNA (dsRNA) is processed into short siRNA duplexes; these fragments are
21 nt long [30, 31]. The source of the dsRNA can either be endogenous
miRNA like above or typically exogenous dsRNA. They are both processed
into suitable ssRNA by a ribonuclease III enzyme (Dicer) [32, 33]. Next the
ssRNA duplexes are unwound and one strand, the so-called guide strand, is
loaded into the RNA Induced Silencing Complex (RISC). This complex then
searches the transcriptome for targets. Targets that are fully complimentary
to the guide strand are then cleaved approximately 10-11 nucleotides from
the 5’ end of the guide strand, by one of the components of the RISC complex, an argonaute protein. Humans have four argonaute proteins, Ago1-4.
Only Ago2 cleaves bound target mRNA [34, 35]. The enzymatic cleavage of
mRNA is what makes this process so efficient at repressing translation.
Using this method it is potentially possible to target any protein and
thereby any pathway, all that is necessary is to introduce a 21 bp dsRNA
(same length as the Dicer cleavage fragments) with a specific target and
that will suppress expression of any protein. It is therefore not surprising
that siRNA has received some attention from the biotech/pharmaceutical industry, a recent review lists over 50 clinical trials using 26 different
siRNAs [36]. The targets for siRNA therapies are very diverse, from dry eye
syndrome to Ebola infection.
Exportin 5
mRNA degradation
mRNA translational repression
Figure 1. Simplified illustration of the RNAi process.
One limitation of “naked” or “free” siRNA is that they are not taken into
cells to any large extent and they are rapidly filtered via the kidneys [37].
Another problem with siRNA, as with all small oligonucleotides, is degradation by serum nucleases. Delivery of RNAi therapeutics remains one of the
most significant challenges to overcome [38]. siRNA can be delivered either
as 21 bp oligonucleotides or as a plasmid expressing short hairpin RNA [39]
that is then processed by Dicer the same way as above to form 21 bp siRNAs.
1.1. 3 A n t i s e n s e
The idea behind using a complimentary oligonucleotide to suppress translation has been known since at least 1978, when a short single-strand 13 nt
oligonucleotide was used to inhibit viral production in chick embryo fibroblasts [40]. This mechanism is similar to the RNAi pathway described above
where the ON binds to the target mRNA and blocks translation or initiates
degradation, except that the ON is not double-stranded and that Dicer and
AGO enzymes are not recruited. The degradation of mRNA is performed by
ribonuclease H (RNase H). A major drawback of this technique is that the
process is not catalytic as the ON is also cleaved by RNase H. In order to
recruit RNase H the ON has to be designed to span a RNase H cleavage site
[41]. Antisense ONs can also block a cryptic splice site and restore proper
splicing to an mRNA transcript [42].
1.1. 4 G l y b e r a
The only approved gene-delivery vector (in EU/USA) is based on the nonintegrating and non-replicating adeno-associated virus (serotype 1), and it
compensates for the rare (affecting 1 in 1 million people) LPLD; a disease
where patients lack the ability to produce the enzyme necessary to break
down fat from digested food and suffer from severe and/or multiple pancreatitis attacks despite dietary restriction of fat. The therapy is administered
as a one-time series of intramuscular injections in the legs. The virus carries
the human gene LPL variant S447X, a mutation associated with several gainof-function effects, for example; reduced triglyceride levels, increased highdensity lipoprotein and a reduced risk of cardiovascular disease. The Worlds’
first approved gene therapy, in China over ten years ago [43] is also based on
viral delivery, recombinant human serotype 5 adenovirus that delivers p53
[44]. A virus is of course one way that nature has always delivered functional
ONs; unfortunately viral delivery has many drawbacks, not least being that
our immune system has been trained against them for eons and that they
can induce an immune response [45]. Another problem is the limited space
available to load in the viral capsid, for example in adenovirus the limit is
about 5kb [46].
Cell-Penetrating Peptides
The discovery of cell-penetrating peptides (CPPs) followed from the realization that the HIV-TAT protein was able to enter cells and translocate into
the nucleus [47, 48], and in 1991 the antennapedia homeodomain from D.
melanogaster was also shown to spontaneously cross the plasma membrane
[49]. The discovery of those two proteins capable of transducing the cell
membrane eventually lead to the identification of the minimal part required
for translocation. In the antennapedia homedomain, the minimal part was
found to be located in the third helix, as that part of the protein alone was
able to cross the cell membrane. This 16-mer peptide was named Penetratin
[50]. A few years later the minimal cell-penetrating portion of the HIV-TAT
peptide was also identified [51]. In the years since these initial discoveries
many more CPPs have been identified, they have some common properties;
they are poly-cationic like polyarginine [52] and/or amphipathic in nature
(although, a poly-anionic CPP also exists [53]). Another characteristic property of CPPs is that they usually have less than 30 amino acids.
In 1998, our research group introduced the concept of CPPs when we
demonstrated the ability of transportan [54], a chimeric peptide based on
the N-terminal part of the neuropeptide galanin and the wasp venom peptide mastoparan, to cross the plasma membrane and bring covalently linked
peptide nucleic acids (PNA) into the cells [55]. Ever since then the number
of CPP publications has been growing each year. Figure 2. shows a timeline
of important CPP discoveries.
Figure 2. Timeline of CPP discoveries.
There is some disagreement as to what these peptides should be called;
there are those who insist on calling these molecules protein transduction
domains (PTDs), presumably because they were first identified in protein
domains. Later, peptides not derived from protein domains were found to
also penetrate cells, hence the name, cell-penetrating peptides (CPPs). Just
to add to the confusion, the name membrane translocating sequence (MTS)
has also been used for these peptides [56]. The field has more or less settled
on calling these molecules CPPs, although the other names are still used
CPPs can be broadly divided into categories based on either their origin
or chemical properties. The origins are perhaps interesting for historical
purposes but classification by chemical properties like charge, hydrophobicity and amphipathicity is more informative. The archetype for cationic
CPPs would be poly-arginine, as it only contains cationic residues. Other
examples of cationic CPPs are TAT [51] and (RxR)4 [57, 58]. CPPs with only
hydrophobic residues are more rare but at least one (1) does exist [59]. Many
CPPs can be categorized as amphipathic, examples include penetratin [50]
and transportan [54].
In the end of the 1990s the group of Prof. Dowdy introduced the concept
of CPP-fusion proteins, where a TAT-peptide was expressed as a chimera together with another protein; the protein remained active and was transduced
into cells in culture [60] and into cells of live animals [61].
However, covalent linking of peptides and cargo has many drawbacks,
where the major one is laborious synthesis of the constructs. Since the peptides are cationic and ONs are anionic, the two should form electrostatic
complexes in solution. It was the group of Prof. Divita that showed this for
the first time. Oligoarginine was introduced in 2000 [62]. Just a year later
in 2001 Futaki et al. showed that stearylation at N-terminal of the oligoarginine peptide improved its delivery efficacy for ON cargo about 100-fold.
Since then more peptides that use this strategy have been developed [63-66].
We introduced the category of CPPs collectively known as PepFects, they
are all derived from the transportan peptide, and they are all modified by
a fatty acid at the N-terminal, but they incorporate different modifications
like endosomolytic moieties and unnatural amino acids and fusions with
targeting peptides, all of them are successful in delivering ON based cargoes
[64, 65, 67-70].
Much of the most recent research has been aimed at elucidating the
mechanism by which these peptides enter the cells, different groups have
shown different mechanisms for the peptides that they are working with,
there is currently nothing like a universal mechanism that applies to all
CPPs. The lack of a universal and well-understood mechanism is further
discussed below.
Delivery strategy
Nucleic acid
[92, 93]
Covalent conjugation
In vitro,
in vivo
In vivo
Covalent conjugation
In vivo
Covalent conjugation
In vitro
In vitro
In vitro
In vivo
Bee venom
Covalent conjugation
In vitro,
in vivo
Galanin +
In vivo
[101, 102]
2´-OMe ON
In vitro
In vivo
siRNA, 2’OMe ON,
In vitro
[65, 69,
LNA, antimiRNA, 2´OMe ON
In vitro
siRNA, 2’OMe ON,
In vitro
[63, 78]
Oligo Lys
Covalent conjugation
In vivo
Oligo Arg
In vitro
[52, 109]
In vitro
In vivo
Ex vivo
In vivo
Pep-3, PEGPep-3
In vivo
In vitro
In vitro
[115, 116]
In vitro
[120, 121]
HIV-1 gp41 +
NLS of SV40
large T antigen
(RX)8B, (RB)8B,
(RXR)4, R9F2
Covalent conjugation
In vitro,
in vivo
Covalent conjugation
Ex vivo
In vitro
In vitro,
in vivo
Table 1. List of peptides used for delivering nucleic acids.
CPPs have been an active area of research for over 20 years and have
been on the list of potentially exploitable vectors for gene-therapy almost
the entire time. CPPs possess the ability to cross the cellular membrane and
bring in cargo that would otherwise be unable to enter into the cytosol. The
type of cargo carried by CPPs now includes almost every kind of biomacromolecule from plasmids [69], siRNA [64] and other ONs [65, 68] to proteins
[71], peptides [72] and peptide nucleic acids [66], for more ON examples
see; Table 1. Many other therapeutic and potentially therapeutic substances
and molecules [73-76], have also been delivered using CPPs. None of the
types of cargo mentioned above, much like ONs, are normally taken up into
cells in any significant quantities. Versatile carriers like CPPs are rare and
interesting from a purely scientific point of view where understanding both
the mechanism and perhaps also the reason they work can be a worthwhile
pursuit in itself. Additionally, it is not difficult to imagine an applicationcentric view of CPPs where delivering a molecule in order to treat disease is
the ultimate goal. In the years since the CPPs were discovered most of the
research focus has been on the last part, developing new CPPs and developing new applications for them, so much so that currently (September 2015)
the online repository for CPPs lists over 800 members [77].
The use of cell-penetrating peptides as delivery vectors is attractive, not
least because of the absorption, distribution, metabolism, and excretion
(ADME) properties of peptides; they are cleared from the cells by the standard metabolism of all cells, also they are relatively non-toxic, they don’t
illicit an immune response in vivo and they successfully transfect entire
populations of cells in vitro [63, 78]. That is not to say that there are not
any toxic peptides, for example the conus venoms are a group of highly toxic
peptides that target ion channels [79].
Oligonucleotide Delivery
Since the molecules described above cannot efficiently enter cells by
themselves, a way to aid the entry has been the aim of many different research projects. The first chemical-based transfection method, using calcium
phosphate, was discovered in 1973 [80]. This method precipitates the plasmid together with the calcium phosphate onto the cells that are grown in a
monolayer. The cells then take up some of the precipitate through what is
believed to be an endocytotic mechanism [81]. The second method, based
on poly-L-lysine emerged in the end of the following decade [82, 83]. Today
lipid-based transfection systems are the most commonly used and could
be regarded as the standard methods for transfection in molecular biology
labs all over the world [84]. There are several commercial vendors of these
systems. As each new manufacturer of oligonucleotide products comes to the
market they seem to simultaneously introduce new “lipofection” reagents,
some of the trade names are Lipofectamine™, Dharmafect™, HiPerFect™,
ViaFect™, FuGENE™.
Non-chemical methods rely on using external forces to manipulate the
cells into taking up the ON. The most direct method is to use microinjection,
where a cell is injected with the foreign macromolecule by inserting a very
small needle through the plasma membrane [85]; needless to say, this is an
extremely time consuming way that requires a certain level of concentration
and a very steady hand. Electroporation is the most well-known method,
using a strong electrical field to increase the permeability of the cellular
membrane [86]. Sonoporation is a similar method where ultrasound is used
to generate pores in the membrane through the cavitation of gas bubbles
on the surface, which in theory could allow for directed transfection but
has side-effects like inducing cell-death and microvascular hemorrhage and
disruption of tissue structure [87], which can be useful in vivo if cell-death
is a desired outcome. An old technique that relies on generating transient
pores is the optical transfection method where a highly focused laser “burns”
a small transient hole in the plasma membrane [88], this could be useful for
single-cell experiments. One of the more imaginative techniques from the
1980s is the so-called gene-gun. Here a particle of a heavy element like gold
or tungsten is coated with DNA and then literarily shot from a 22. caliber
nail gun at a cell; it has been mostly used in plants, and was originally used
in an onion [89]. A recent application for this gene-gun technique was to
label neurons, not with genetic material but with a dye [90].
A more recent idea is to use microfluidics to “squeeze” the cells to induce
compression and shear forces that result in transient holes, which enable the
diffusion of macromolecules into the cytosol [91]. This method could enable
high-throughput transfection of all kinds of cells in combination with all
kinds of macromolecules, as it is independent of transfection reagent and
very efficient. However, because of the need to pass cells through a microfluidic device, an in vivo application is difficult to imagine.
There are only limited successes when it comes to delivery of oligonucleotides in humans, but they do exist and one of them is the above-mentioned
Endocytosis is the process by which all cells absorb molecules that are unable to passively diffuse the cellular membrane, for example water and large
polar molecules. The name encompasses a variety of processes that each have
different modes of action. Endocytosis has been divided into clathrin-mediated
endocytosis, caveolae-mediated endocytosis, macropinocytosis, phagocytosis,
and flotillin-dependent endocytosis, and other even less defined pathways. A
simpler endocytosis classification is either phagocytosis or pinocytosis [125].
The best understood and most researched method is the clathrin-mediated
endocytosis [126-128]; it is the process by which eukaryotic cells take up and
recycle receptors, internalize nutrients, antigens, growth factors and even
pathogens [129, 130]. The different pathways are further described below.
1. 4 .1 C l a t h r i n - M e d i a t e d E n d o c y t o s i s
The introduction of glutaraldehyde fixation in the 1960s allowed for visualization of uptake of material inside the cell. Using electron microscopy,
images of vesicles with protein coats in different tissues [131] were generated.
A decade later clathrin was identified as being the major protein component
of the coat around those vesicles, described as “vesicles in a basket” [132, 133].
The pathway was named clathrin-mediated endocytosis (CME), after the major
component. CME occurs constitutively in all mammalian cells, as it is involved
in the uptake of nutrients. CME modulates signaling receptor levels [134], it
can control the strength of synaptic transmission and CME is also involved
in protein recycling from vesicles. The first step in CME is that the cellular
membrane begins forming a pit, where cargo molecules cluster. The emerging
pit is coated with the protein clathrin, and because clathrin does not interact
directly with neither cargo nor cell membrane, adaptor proteins are necessary to
mediate the interaction. The clathrin-coated vesicle is three-layered where adaptor proteins attach to phosphatidyl-inositol-polyphosphate head groups on the
inner membrane and then to clathrin [135]. The main such adaptor protein is
thought to be adaptor protein 2 (AP2) [136], upon binding to a budding vesicle
AP2 changes conformation, which in turn recruits the clathrin to bind [137].
AP2 is one of at least 5 adaptor proteins [138], out of the five only AP1 and AP2
bind to clathrin, the other adaptor proteins are also involved in trans-Golgi
trafficking. When the invagination in the membrane is fully formed and the
coat is fully assembled, vesicle scission is catalyzed by assembly of the GTPase
dynamin at the neck of the pit. The size of the clathrin-coated vesicles differs
between species and tissues. CME has been implicated in other processes like
the uptake of micro-bubbles containing pDNA generated using sonoporation
[139]. Many receptors like glutamate receptor (NMDR) [140] and g-protein
coupled receptors (GPCRs) are known to be endocytosed via clathrin coated
pits where the internalization is regulated via adaptor proteins [141].
In order to study CME, many different methods of inhibiting CME have
been employed, the two most common are treatment with chlorpromazine
[142] and potassium depletion [143]. Chlorpromazine has been used to study
CPP-uptake [144-151]. However, chlorpromazine may have effects on the biogenesis of large intracellular vesicles such as phagosomes, macropinosomes,
and granules [152], because of this chlorpromazine should be complemented
with other, more specific, tools that do not affect other cellular processes.
Other points that are important when considering CME is that clathrin
function is not limited to only endocytosis, but it is crucial in many other
processes like mitosis and protein secretion from the trans-Golgi network. It
has been suggested that when looking to target CME with RNAi, it is more
specific towards endocytosis to knock down the α or µ2-subunit of AP2 than
to target clathrin itself [153].
1. 4 . 2 C a ve o l a r E n d o c y t o s i s
Caveolae were first identified by electron microscopy in the 1950s [154]
and are now defined as characteristically regular, flask-shaped or spherical
pits, without any obvious coat on the plasma membrane [155]. The size of the
pits is approximately 60–80 nm diameter [156]. The main protein component
of caveolae, Caveolin 1 (CAV1) is a more recent discovery, from 1992 [157],
than that of clathrin in CME, which is from 1976 [132]. The clathrin coat
can be seen from electron micrographs whereas caveolin cannot. Up until
the early 1990s there was no consensus as to whether clathrin-independent
endocytosis (CIE) existed at all [158]. The linking of caveolae to CIE finally
resolved this issue with the finding that dynamin is recruited to caveolae, as
well as clathrin-coated pits, which suggest that the caveolae can bud off the
membrane [159]. The role of the caveolin proteins for specific endocytosis
of cargo remains to some extent unresolved. SV40 virus and CAV1 clearly
co-localize when the virus is internalized in cultured cells indicating that
CAV1 is involved in the uptake of SV40 [160]. In CAV1 knockout mice the
rate of SV40 internalization is increased, suggesting that CAV1 inhibits the
uptake of SV40 [161]. Further confounding the role of CAV1 in endocytosis, there are seemingly conflicting reports involving the endocytosis and
transcytosis of albumin, where in one case CAV1 is required for albumin to
be transported [162], and in the other case for CAV1 knockout mice show
normal levels of albumin, indicating that transport occurs normally also in
the absence of CAV1 [163]. What is known is that Caveolin proteins play
crucial roles in many processes, CAV1 [163] and CAV3 [164] are involved
in the formation of caveolae and CAV2 seems to not have any effect on the
formation of caveolae [165]. Additionally CAV1 seems to be involved in lipid
regulation, expression of CAV1 facilitates the uptake of fatty acids into cells
and increases the levels of free cholesterol in cells and rate of cholesterol
export from cells [166-168]. More recently caveolin has been found in other
cellular organelles like mitochondria, nuclei and endoplasmic reticulum
[169]. Caveolin proteins interact with many signaling and transmembrane
proteins, like GPCRs, Src kinases, endothelial nitric oxide synthethase, adenylyl cyclases, protein kinase A, mitogen activated kinases and ion channels
[170]. Like CAV1 and CAV3 the cavin family of proteins (cavin 1-4) are also
involved in the formation of caveolae (cavin1), plays a role in the membrane
invagination (cavin2), is responsible for trafficking and budding of vesicles
(cavin3). Cavin4 is specific for muscle cells [170-173]. Caveolae in smooth
muscle cells do not take part in endocytosis, a reason could be that these
caveolae lack tyrosine resides that can be phosphorylated [174], but it does
not fully explain why muscle cells are not using caveolae for endocytosis.
Caveolin-1 and CDC42 (a small GTPase), play an important role in the
endocytosis of silica coated iron oxide nanoparticles and of superparamagnetic iron oxide nanoparticles with negative surface charge and a diameter
around 17 to 30 nm in HeLa cells in vitro [175].
1. 4 . 3 O t h e r E n d o c y t o t i c P a t hw a y s
Clathrin- and caveolar endocytosis recruit proteins that coat the vesicles,
but there are endocytotic pathways that are independent of these two mechanisms. Glycosylphosphatidylinositol (GPI) anchored proteins were one of the
first cargo proteins to be identified as markers for a pathway independent
of both clathrin and caveolin. That pathway is now termed clathrin- and
dynamin-independent carrier (CLIC)-GPI-anchored protein-enriched early
endosomal compartments (GEEC), CLIC-GEEC. The internalization via
this pathway is unaffected by inhibition of clathrin or dynamin but it is
sensitive to inhibition of the small GTP-binding protein CDC42 [176]. The
pathway is most active on the leading edge of migrating cells [177]. The carriers then fuse with early endosomes through a mechanism that is dependent
on phosphatidylinositol 3-kinase (PI3K) activity [178].
Other forms of clathrin independent endocytosis include a pathway associated with Arf6 [179-181], one that is dependent on dynamin and Rho
[182], and others that depend on flotillin [183].
Macropinocytosis originates in ruffled regions on the plasma membrane,
an invagination of the cell membrane forms a pocket that pinches off into
the cell to form a vesicle (0.5–5 µm in diameter). The vesicle is filled with
a comparatively large volume of extracellular fluid and molecules (roughly
about 100 clathrin-coated vesicles). The filling of the macropinosomes is
non-specific [184]. The pathway is likely to involve most of the molecular
players that are common to all the pathways described above, except caveolae
[185]. Simultaneously it is clear that the macropinosomes are substantially
larger than all the other pathways pointing towards a mechanistic difference.
From the discussion above it is obvious that the pathways to some extent
overlap, for example dynamin is involved in multiple pathways, endocytosis
of GPCRs seems to occur both through caveloae and clathrin-coated pits,
and there is significant crosstalk between the different pathways cavin1 and
cavin3 are potent inhibitors of the CLIC/GEEC pathway [186]. For an illustration of some of the overlapping proteins in the different endocytotic
pathways see Figure 3. Even the “well-defined” clathrin pathway may turn
out to be more than one pathway, because there is evidence of cargo sorting at all stages of the internalization process [187], it logically follows that
the protein constitution of the vesicles must be non-identical. The clathrinindependent pathways may in turn be revealed as red herrings as there is
some doubt as to if these pathways contribute to endocytosis in mammalian
cells at all [188].
Other implicated protein
Small G-protein depencence
Figure 3. Illustration of the overlap between the different endocytotic pathways; the ovals
represent a defined pathway, the diamonds a protein, the cylinders represent Small Gproteins, note especially that dynamin is implicated in 4 out of the 7 pathways, and actin in all
but 2 pathways.
Adapted from Table 1. in Doherty, G.J., McMahon, H.T.: Mechanisms of Endocytosis. Annu Rev Biochem. 78, 857–902
(2009). [254]
CPP entry and endocytosis
The endocytotic uptake route of CPPs is not yet fully understood, for
example; siRNA knockdown of Caveolin-1, and flotillin-1 showed no effect
on the uptake of the cationic peptides TAT and R8 [145]. There are reports
that TAT uses CME to enter cells [189] although this result relies mostly
on inhibition of CME using chlorpromazine which as discussed earlier may
have side effects. For TAT and larger cargo like TAT-fusion proteins the caveolar endocytotic route is implicated because of co-localization with CAV1
[190]. While TAT-fusion peptide uptake in cells expressing a dominant
negative mutant of dynamin, was not inhibited suggesting that neither the
caveolar nor the clathrin pathway is necessary for uptake [191]. Other CPPcargo complexes are taken up via all of the previously mentioned endocytic
pathways, with the exception of phagocytosis [192].
A method that could provide insight also for CPP-based delivery was
described recently in a paper that presents a way to visualize and detect
the release of siRNA from endosomes and correlating it with knockdown
of a GFP-reporter for lipid based delivery vectors. The escape occurs early
in the uptake process and only a small portion of siRNA escapes, it will be
interesting to se if this is similar for CPPs [193].
Uptake Mechanisms
For many CPP-based delivery systems, including PepFects, stearylpolyarginine, and TAT-DRBD, endocytosis has been suggested as the main
mechanism behind the uptake into cells [64, 65, 94, 194, 195]. For other
systems, like the CADY-peptide, the group of Prof. Divita has shown that
the translocation of CADY:siRNA complexes is independent of both endocytosis and/or surface proteoglycans and is probably energy-independent,
suggesting that the uptake mechanism must be direct translocation [119].
However, one of the papers in this thesis partly question direct translocation
by demonstrating that the CADY-peptide when in complex with siRNA is
also taken up through scavenger receptors. It is, of course, entirely possible
that CPPs take different routes into the cells when in complex with cargo
and when alone.
The arginine-rich peptide TAT has been shown to interact with the
membrane of giant unilamellar vesicles, it induces a specific conformational
change causing a membrane curvature that leads to the formation of pores
that allows the peptide to enter [196]. When the TAT-peptide is coupled
to cargo that is too large for these pores, the peptide was proposed to interact with actin inside of the vesicle and induce conformational changes,
reminiscent of membrane the blebbing associated with macropinocytosis
[196]. Verdurmen et al. showed that the endocytosis independent entry of
poly-arginines depends on a CPP-induced translocation of acid sphingomyelinase (ASMase) to the outer leaflet of the plasma membrane and ceramide
formation via hydrolysis of sphingomylin. This changes the composition of
the plasma membrane and allows for entry into the cytosol, the mechanism
of enzyme activation is not yet clear. The threshold for direct CPP translocation could be lowered through addition of ASMase and it enhances the
translocation of poly-arginine coupled to low-molecular weight cargos, but
not high-molecular weight cargos [197].
When studying the uptake mechanism of PepFects, the peptides and ONs
are complexed prior to treatment. The complexation is done with the peptide
in excess, in a molar-ratio (MR) ranging from just a few to tens of times
more peptide than ON [64, 65]. It could be expected that these complexes
would have an excess of positive charge and that this charge is important
for at least the initial interaction with the negatively charged cell membrane if not the entire translocation. However, the particles formed by the
CPP PepFect14, and splice-correcting oligonucleotides (SCOs) form complexes with a negative surface charge in bio-relevant media, as determined
by ζ-potential measurements [65, 198]. A particle with net negative charge
should not interact with the negative plasma membrane or glycosaminoglycans on the cell-surface. This contradiction is further explored later in the
In the early days of CPP research the uptake mechanisms of CPPs were
thought to be mostly independent of any endocytosis pathway and rely on
direct penetration of the cellular membrane. However, in 2003 a re-evaluation of the methodology used to determine the uptake mechanism dispelled
much of the previously held beliefs. The earlier results were likely caused
by artifacts related to the fixation of cells [199]. Because of this, imaging
and fluorescence activated cell sorting (FACS) is now done on live cells that
have been heparin washed and trypsinized. In the following decade, reports
indicating one or another kind of endocytotic mechanism as responsible for
the uptake of CPPs has steadily increased in number.
It had been known since before the artifacts due to cell fixation came to
light that internalization of the most famous and well-studied CPPs, the
cationic TAT-peptide requires cell-surface heparan sulphate (HS) proteoglycans [200], this is also true for other arginine-rich peptides [201]. This is the
first step of interaction with the cell membrane that then stimulates uptake.
The picture is, however, not as clear as that because there are reports that
state the opposite i.e. HS-interaction inhibits the uptake of cationic CPPs
[202]. On the other hand, when it comes to the other major class of CPPs,
the amphipathic ones, cell-surface clustering of HS-proteoglycans neither
contributes to, nor inhibits the uptake [203]. The endocytotic mechanism
by which the TAT-peptide is then taken up after the initial interaction with
cell-surface proteoglycans has been reported to be either; macropinocytotis
[201], lipid raft caveolar endocytosis [190], CME [189], or all three mechanisms simultaneously [144]. More recently a report detailing the ways in
which the TAT-peptide can interact with, and be internalized by, cells,
added some clarity to this apparent lack of agreement. The TAT-peptide
can bind to HS-proteoglycans and become internalized by endocytosis; it
can also interact with the membrane and generate saddle-splay curvature
and in this way induce pore-like structures in the membrane. If the cargo
is sufficiently small the peptide can be internalized directly, larger cargo is
anchored to the membrane via the peptide and a strong interaction between
TAT and actin can remodel the cytoskeleton and promote other pathways
like macropinocytosis [196].
Direct translocation for arginine-rich CPPs is likely related to the ability of
the guanidinium moieties on arginines to form hydrogen bonds with membrane lipids [204]. Each cationic guanidinium head group has a rigid planar
array of hydrogen bond donors that allows for highly effective bidentate
hydrogen bond formation with negatively charged carboxylates, phosphates,
and sulfates [205]. Direct penetration is also supported by the ability of the
arginine-rich TAT-peptide to induce pores in artificial membranes [206].
In 2012 Tanaka et al [207] reported that chemokine receptor type
4 (CXCR4) acts to stimulate the uptake of a dodecamer of arginine.
Stimulation of CXCR4 with the intrinsic ligand SDF-1α induces macropinocytosis. Additionally they showed that siRNA knockdown of the receptor
prevents the formation of lamellipodia, further indicating that for oligoarginine macropinocytosis is one uptake route.
Peptides that bind to prostate cancer cells may bind to the neurophilin-1
receptor (NRP-1) and then be quickly internalized. These peptides have a
C-terminal consensus sequence of R/KXXR/K which is similar to sequences
found in many CPPs [208]. This is the same consensus sequence that is
found in the C-terminal end of VEGF-A165 and some semaphorins that are
known to bind to NRP-1.
We have reported that our most recent generation of CPPs, peptides that
have been modified using both a N-terminal stearylation, phosphorylated
tyrosine or a side-chain extension of the peptide chain were also are taken
up through differing endocytotic routes. Inhibitors for the different pathways revealed that NickFect1 is endocytosed via both the CME pathway
and through macropinocytosis, whereas NickFect51 is internalized mainly
through macropinocytosis [209].
Scavenger Receptors
After their discovery of the low density lipoprotein receptors (LDL receptor) [210, 211], the Nobel laureates Brown and Goldstein went on to discover
the scavenger receptors (SR) in patients who lack LDL receptors [212]. SRs
are a family of cell-surface glycoproteins that where first recognized to bind
modified lipoproteins such as acetylated and oxidized LDLs. Initially these
receptors were thought to only scavenge acetylated LDL into macrophages
[213]. This first definition has since grown to encompass receptors that can
bind a diverse set of ligands, like modified endogenous proteins and lipoproteins; since these modified molecules are potential hazards to the cells,
they are called “danger-associated molecular patterns” (DAMPs). In addition
to DAMPs, the receptors promiscuously bind several different exogenous
molecules like bacterial lipopolysaccharides (LPS) [214], microorganisms
like Staphylococcus aureus bacteria [215], PrP106–126 prion protein [216],
and viral RNA [217], HCV virus [218], together these have been called
“pathogen associated molecular patterns” (PAMPs) [219]. Other foreign
particles that are not necessarily PAMPs like silver-nanoparticles [220] are
also recognized by scavenger receptors. Also, they recognize several sulfated
polysaccharides (dextran sulfate and fucoidan but not chondroitin sulfate)
and polyribonucleotides (polyinosinic (poly I) and polyguanilic (poly G)
acid but not polyadenilic (poly A) or polycytidylic acid (poly C)) [221]. SR
class A (SCARA) was found to recognize the exchangeable apolipo-proteins
A-I and E in both lipid-free and lipid-associated form, suggesting the shared
amphipathic alpha helix as a potential recognition motif [222].
Since SRs recognize so many different PAMPs, it is not surprising that
they are highly expressed on macrophages, where they were also initially
identified. Later, SRs were found in many different cell types, where subtypes were identified in cells such as smooth muscle cells, epithelial and
endothelial cells, splenic dendritic cells, and fibroblasts. The number of
cell-surface receptors classified as scavenger receptors is continually growing from the first suggestion of classes A-F in 1997 [223] to now include
19 receptors that are divided into classes A through I [219]. The receptors
share the ability to bind poly-anionic ligands with low specificity. The term
scavenger receptor used to be defined as: an extracellular glycoprotein (either
soluble or membrane-bound) involved in the recognition and/or endocytosis
of negatively charged molecules [224]. Today many more functions for the
receptors have been reported and they are generally considered a sub-class
of the membrane-bound pattern recognition receptors [219]. An emerging
role for SRs is cellular adhesion where SCAR As were found to contribute
to the majority of the macrophage adhesion to the extracellular matrix both
in the presence and absence of serum and almost all of the adhesion when
divalent ions were chelated [225]. The class A scavenger receptors are multimeric receptors with a cytoplasmic domain, a trans-membrane domain and
collagenous domain and some have an additional cysteine-rich domain or a
c-type lectin domain [226].
The SRs ability to take up viral DNA and other negatively charged molecules are the properties that made us suspect that SRs could be involved in
the uptake of CPP:ON complexes. We have shown, in several papers, that
SCAR As are involved in the uptake of CPP:ON complexes using both a
number of pharmacological inhibitors and by knocking down the receptor
using siRNA [68, 69, 198, 209, 227], two of those publications are included
in this thesis.
High-throughput Assay Development
The invention and standardization of the micro-plate has been a paradigm
shift in the life sciences. The subsequent technological developments have
brought various plate-readers, which now enable “everyone” to do some form
of higher throughput experiments. For example, even a simple experiment
like determination of protein concentration in all 96 wells of a standard
cell-culture plate using the much cited Lowry protein assay [228], increases
throughput almost 100-fold compared to using cuvettes. The increasing
number of wells in the standard format micro-plates enables ever-higher
throughput; a state-of-the-art RNAi HTS project that uses 1536-well plates
will be able to go through all the known human genes in less than 15 plates.
However, using plates with such small wells necessarily requires that no human be involved in any of the liquid handling steps, robots and high levels
of automation are necessary for repeatable experiments. The more difficult
problem is the evaluation of data, it is not trivial to determine what is a
“hit” when there are thousands of data points. The first question to answer
is whether the assay is robust; this is done by evaluation of the so-called
Z’-score [229], which compares the averages and standard deviations of the
positive and negative controls see Equation 1. A score > 0.5 is considered
an excellent assay, whereas 0 < Z’ < 0.5 is considered a marginal assay and
score below zero means that the variation between the negative and positive
controls overlap and that screening is essentially impossible. If the normally
accepted value of p < 0.05 was used, over a thousand hits would be identified
just by chance in a whole human genome siRNA scan, or over a dozen in
the 264-member library used here. To put it differently; almost five hits by
chance for each 96-well plate. From this it is obvious that a threshold set too
low will give many false positives, while a threshold set too high will exclude
many potential hits. In this work a hit was further investigated if it deviated
more than 3 standard deviations from the mean of the positive controls in at
least three consecutive experiments. This approach still leaves an acceptable
one in one-thousand chance of a false positive.
Z ' = 1−
3δ + + 3δ −
µ+ − µ−
Equation 1. Z’-score.
Mean (µ) and standard deviation (δ) of the
positive and negative controls.
G-protein coupled receptors
G-protein coupled receptors (GPCRs) are the largest family of cell-surface
receptors, they perform many diverse functions that include for example
vision in the eye, taste on the tongue, they regulate mood and alertness. The
ligands that bind to the receptors are diverse and include small molecules
and peptides. The receptors have in common that they span the membrane
seven times and on the inside of cells they associate with G-proteins. The
2012 Nobel Prize in chemistry was awarded to Brian Kobilka and Robert
Lefkowitz for their work “crucial for understanding how G-protein-coupled
receptors function”. The receptors can become internalized upon binding
a ligand and there is mounting evidence that the signaling function of the
receptors continues even after internalization [230].
A im
he primary aim of our research group is to use CPPs to improve the
delivery of therapeutic ONs; to this end this thesis includes a couple
of different projects aimed at identifying the uptake mechanism for peptidebased delivery vectors and methodologies for evaluation and comparison of
as many peptides as possible.
The over-arching aims are:
Development of new CPP vectors for gene delivery applications.
Application of CPPs in vitro and eventually in vivo.
The aims for each paper are detailed below.
Paper I
Our finding that surface charge was negative for CPP:ON complexes
in cell-media was perplexing and counter to the hypothesis that cellular
uptake of CPPs was initiated via the opposite charges of the CPPs and cell
membrane attracting each other. The aim here was to identify a receptor or
other mediator of an interaction between a negatively charged particle and
the negatively charged cell-membrane.
Paper II
The findings of paper I suggested that SCARAs were responsible for the
uptake of PepFect CPP:ON complexes, the main aim of the study was to
determine if this uptake mechanism is also responsible for the uptake of
other CPP:ON and cationic-polymer:ON complexes.
Paper III
The aim of this paper was to miniaturize our standard 24-well plate assay
for delivery of short ONs, i.e. the assay used in paper I and II, and simultaneously reduce cost by developing a homemade luciferase assay buffer. And
reduce complexity and decreased time required for experiments, in order
to make the assay amiable to high-throughput screening. Additionally this
development was necessary in order to perform the experiments required for
paper IV.
Paper IV
The aim of this study was to use a library of GPCR ligands to stimulate
cell-surface receptors and look for changes that affect the uptake of CPP:ON
complexes. The secondary aim was to perhaps identify a novel mechanism
either for uptake or endosomal escape.
M ethods
Solid-Phase Peptide Synthesis
olid-phase peptide synthesis (SPPS) is the most common method for
producing peptides for both research and therapeutic purposes, it is a
technique that earned its inventor, Robert Bruce Merrifield [231] the Nobel
Prize in chemistry in 1984. The peptide is attached via its C-terminus to a
solid phase, which allows for filtration to be used to remove spent reagents.
The 9-fluorenylmethyloxycarbonyl (Fmoc) scheme was used in this work.
The process is a stepwise, iterative cycle of addition followed by de-protection
of each amino acid. Addition occurs via the carboxylic acid to a free amine
on the anchored peptide chain, using the Fmoc method the peptide is attached to the solid support via an acid labile linker. In order to make the
reaction specific, a protection group is used on the α-amine of the amino
acid being attached; it is protected with the Fmoc group. This group is easily
removed (or de-protected) using a base like for example piperidine or piperazine. The amino acid side-chains are also protected with acid labile groups.
After addition of each amino acid the Fmoc group is removed and then the
next amino acid is coupled. When the peptide is ready the solid support and
side chains are removed using acid, yielding an unprotected peptide.
In the event that one of the side chains has to be functionalized further,
an orthogonal protecting group can be used. The peptide is first synthesized
to completion and the side chain is then de-protected in conditions that only
affect that one specific site. In this work all side-chain modifications have
been performed at a lysine. In this case the lysine is protected with the acid
labile 4-methyltrityl (MTT)-group that can be removed without cleaving the
resin, using weak acid.
All peptides were synthesized using one of two automated peptide synthesis machines (Syro II, Multisyntech GmBH or Alstra+, Biotage AB, Uppsala,
Sweden). Stearic acid modification was carried out either in a manual step
after automated synthesis or coupled under microwave heating conditions in
the Alstra+; 5,6 carboxy-fluorescein was coupled manually after removal of
the acid-labile side-chain protecting group from lysine 7 on PepFect 14. All
peptides were synthesized using a H-Rink-Amide-ChemMatrix resin (PCAS
Biomatrix, St-Jean-sur-Richelieu (province of Quebec), Canada), this kind of
resin produces peptides with amidated C-termini. The peptides were cleaved
from the resin, using 95 % tri-fluoroacetic acid (TFA), 2.5 % triisopropylsilane (TIS) and 2.5 % H2O, precipitated in ether and lyophilized. Crude peptides were purified using semi-preparative reversed-phase high performance
liquid chromatography (HPLC) and analyzed using matrix-assisted laser
desorption/ionization - time of flight (MALDI-TOF) mass spectrometry.
After purification the peptides where lyophilized again and reconstituted in
ultra-pure water (Milli Q, Merck Millipore) before use. All the peptides that
were used in this thesis
were synthesized in this
manner, except for the
CADY peptide, which
was a kind gift providdPF14
ed by Dr. Gilles Divita
and Dr. Sebastien
S-(RxR)4 Stearyl-RxRRxRRxRRxR-NH2
Deshayes. Table 2 lists
the peptides used in
Table 2. a Four chloroquine-analogs coupled via a
this thesis. Figure 4
lysine tree. b 5,6 Carboxy-Flourescein (only for labeled
shows the structure of
PF14) * small caps = d-amino acid, x= amino hexanoic
PF14, it was used in all
acid, O=Ornithine, Ac= acetylation, Cya = Cysteamine
papers included in the
Figure 4. The structure of PepFect14.
Cell Culture and Treatment
The first cell line, HeLa, that was propagated indefinitely in vitro came
from a cervical cancer patient named Henrietta Lacks. The man who first
generated it in the 1950s, George O. Gay, named the cell line HeLa after
the patient. Mrs. Lack’s cells have been an essential tool in the life sciences
ever since. More information and a popularized story of Mrs. Lacks and her
cells can be found in the book “The Immortal Life of Henrietta Lacks” by
Rebecca Skloot.
HeLa pLuc705 cells are a gift from Prof. Ryszard Kole (University of
North Carolina, Chapel Hill, NC, USA). The HeLa pLuc705 cell line is
stably transfected with a luciferase-encoding gene interrupted by a mutated
β-globin intron 2. These cells were used in all the papers included in this
work and are further described below.
The cells used for siRNA knockdown in paper II were U2OS-SAMP1-YFP
cells, these cells stably express a yellow fluorescent protein (YFP) fusion of
the inner nuclear membrane protein SAMP1 [232].
The cells used in the siRNA knockdown experiments in paper III (U87
MG Luc cells were provided to us from Dr. Kaido Kurrikoff (University of
Tartu, Tartu, Estonia); these cells stably express the luciferase gene in high
Cells were grown at 37°C, 5 % CO2 , in Dulbecco’s modified Eagle’s medium with glutamax supplemented with 0.1 mM non-essential amino acids,
10 % fetal bovine serum, 200 U/ml penicillin, and 200 µg/ml streptomycin
(Invitrogen, Stockholm, Sweden).
HeLa pLuc705 cells (7.5×10 4) were seeded 24 h prior to experiments into
24-well plates. PF14 was mixed with PS-2’-OMe SCOs at molar ratio 5
in MQ-water in 10 % of the final treatment volume (i.e., 10 µl). The final
concentrations were 200 nM SCO and 1 µM PF14/well. Complexes were
allowed to form for 30–45 min at room temperature.
Dynamic Light Scattering
In order to further characterize the physiochemical properties of the
particles that formed when mixing the peptides with ONs, dynamic light
scattering (DLS) was used. DLS is also known as photon correlation spectroscopy and as quasi-elastic light scattering [233]. This is a method that
allows for the determination of the size distribution of small particles in solution. It is built on measuring the intensity of light scattering of particles in
Brownian motion [234] where time-dependent fluctuations are correlated to
size. If particles are small compared to the wavelength of the laser used then
the light scatters in all directions (Rayleigh scattering [235]). In a way, it is
amazing that the initial explanation of scattering from 1871 was derived at a
time when everyone believed in the luminiferous ether. Lord Rayleigh went
on to use the preferential scattering of blue light (shorter wavelength) by the
atmosphere to explain why the sky is blue and the sunsets are red [236]. Due
to the Brownian motion the distance between the particles is constantly
changing, therefore scattered light intensity fluctuates. The time-scale of the
fluctuations is directly related to the translational diffusion coefficient of
the scattering particles, which in turn is related to their size [237]. From
this an autocorrelation function that relates the fluctuations in intensity to
the size can be used for size determination. One drawback of this method
is that intensity peaks have to be separated by a factor of at least two for the
method to be able to distinguish between them, if they are closer they will
yield a single broader peak [238]. Another problem is that the method is
more sensitive to larger species as they give higher intensity.
The ζ-potential is the difference in potential between the stationary layer
of fluid attached to a dispersed particle and that of the dispersion medium.
From an instrumental aspect it is fortunate that the electrophoretic velocity
is proportional to the measurable electrophoretic mobility. This allows for
the use of the above-described method of DLS to be adapted to electrophoretic light scattering. Here the Doppler effect on particles undergoing
electrophoresis shifts the frequency of the light scattered from the particles.
In this work an instrument that can do both types of measurements was
used; Zetasizer Nano ZS (Malvern Instruments, Malvert, UK).
PF14-SCO nano-complexes were prepared as described above and diluted
in MQ-water, 150 mM NaCl solution, Opti-MEM (Invitrogen), or OptiMEM with 10 % serum into a final volume of 1 ml. Samples were assessed
in disposable low-volume cuvettes. Data was converted to relative intensity
plots, from which the mean hydrodynamic diameter was derived.
Luciferase from firefly is an enzyme that catalyzes a reaction that emits
light; it is what gives the flies their characteristic glow. The gene that encodes
it can be used as a reporter gene in many different types of delivery assays.
Because the reaction produces light, is a convenient and easy way to measure
differences in gene expression. All that is needed is addition of the substrate
for the luciferase enzyme to the lysed sample, For example delivering a luciferase encoding plasmid to a cell that does not normally produce the enzyme
gives a measurable increase light output that is proportional to the amount
of luciferase present. Conversely, delivering siRNA to a cell that already
expresses luciferase will result in a decrease in the amount of enzyme and
therefore a reduction in light output. The assay described below (Section 3.6)
is another way to use luciferase as reporter gene in an ON assay.
SCARA Inhibitors
Scavenger receptor-specific “inhibitors” were used in paper I and II in order
to elucidate the effect of this family of receptors on the uptake of CPP:ON
complexes; the inhibitors used are fucoidan, dextran sulfate and Polyinosinic
acid (Poly I). Chemically similar molecules were used as controls. Fucoidan
is a sulfated polysaccaride found in brown algae, dextran sulfate is a sulfated
analog of dextran and Poly I is a polymer of the inosinic monophosphate.
The controls used where galactose for fucoidan and chondroitin sulfate for
dextran sulfate, and polycytidylic acid (Poly C) was used as control for the
Poly I. These molecules have been used previously in studies of SRs [221,
Splice-Correction Assay
The splice-correction assay that was developed by Kole et al. [42] provides
an elegant quantitative assessment of cellular delivery efficiency of SCOs.
The assay uses HeLa cells that have been stably transfected with pLuc705,
a plasmid containing a luciferase-encoding gene interrupted by a mutated
intron from a β-thalassemic globin gene. The intronic mutations activate
a cryptic splice site that produces non-functional luciferase. Masking the
mutated site with an antisense ON re-orients the splicing machinery to produce functional luciferase. Subsequent luminescence measurement allows
for quantification of uptake using an instrument as simple as a luminometer.
SCO Delivery
In papers I, III and IV SCOs were delivered, using non-covalent complexation with CPPs, to cells that stably express an incorrectly spliced, and nonfunctional, luciferase. The successful delivery results in functional luciferase.
In the first two papers a 24-well assay was used and in Papers III and IV a
96-well assay was used. In the 96-well assay the CPP:SCO complexes were
already present in the micro-plate wells when cells were seeded. In paper IV
a library of GPCR receptors was also present in the micro-plate wells prior
to seeding the cells. The cells and the assay are described above.
Plasmid Delivery
CPPs and two cationic polymers were used to deliver the pGL3 plasmid,
which expresses the luciferase gene. Plasmid delivery was performed again
by non-covalent complexation with the selected CPPs and polymers. In this
case the cells were pre-treated with SCARA inhibitors (and their controls)
to assess the effects of removal of the SCARAs from the uptake process of
CPP:pGL3 and polymer:pGL3 complexes. Additionally, plasmids expressing
the SCARA-3 and -5 genes were used to over-express the receptors and study
the effect on the uptake. In the over-expression experiments, a commercial
transfection reagent (LipoFectamie 2000) was used to transfect HeLa cells
with the SCARA genes before addition of the CPP/Polymer:pGL3 complexes.
siRNA Delivery
In Paper II, siRNA knockdown of SCAR A-3 and -5 was performed to
study the effects of the SCAR As on uptake. In the experiments, U2OSSAMP1-YFP cells were transfected with siRNA targeting SCAR A-3 and
-5, in this case by using a commercial transfection reagent, (Lipofectamine
RNAiMAX). After knockdown of the SCAR A receptors, the cells were
treated with CPP:ON complexes. In paper II, siRNA against green fluorescent protein (which also targets yellow fluorescent protein (YFP)) was delivered using both CADY and s-RxR4. Subsequent analysis of YFP knockdown
was performed using FACS.
3.10 Fluorescence Microscopy
One of the experiments in paper II uses fluorescence microscopy. A technique that is now an essential tool in life sciences because it allows for visualization and identification of cells, sub-microscopic cellular components and
fluorescently labeled molecules, today the method is no longer limited by
diffraction, as super-resolution microscopy [242] is becoming more common,
although that is not used in this thesis it will become a tool in CPP research.
Fluorescence microscopy enables the study of single molecules as well as
allowing for the identification of several target molecules simultaneously. In
this thesis, the technique has been applied to study the cellular uptake of
labeled CPPs and ON-cargo and to investigate the role played by SCARA in
the uptake of PF14-Fluorescein alone or PF14-Fluorescein in complex with
Alexa568-labelled siRNA.
3.11 Toxicity Assays
To exclude that the cell-penetrating and delivering activity of the peptides
in vitro is caused by toxicity, the WST-1 assay was used. The same assay
was also used for the fourth paper to exclude that the effects of the added
ligands (SCAR A inhibitors and GPCR library) on splice-correction were
due to toxicity. This assay measures cell viability as a function of mitochondrial metabolic activity. The activity of the mitochondrial dehydrogenases is
measured by their conversion of tetrazolium salts to formazan, cell-viability
and cell-proliferation is directly correlated to the amount of formazan dye
formed, more cells results in more formazan dye. The dye can be elegantly
quantified spectrophotometrically, using UV-absorption at 420 nm, more intense staining means more active mitochondrial dehydrogenase. The WST-1
reagent and the formazan product are both water-soluble, which means that
measurements can be performed 1-4 h after addition, making it a very rapid
and convenient assay compared to the commonly used MTT assay, which
requires solubilization of the formazan salt.
R esults and Discussion
Paper I & II SCARAs and CPP Uptake
n Paper I: We show involvement of the scavenger receptor class A for
the uptake of PepFect14:splice-correcting oligonucleotide complexes. In
Paper II: We show that the findings from paper I, namely the involvement
of the scavenger receptors in uptake, can be expanded to peptides and polymers and ON cargo that are different from PepFect14. When we first started
characterizing the physiochemical properties of the CPP:ON complexes we
found, much to our surprise, that even though the peptide was always in
molar excess (for example, at MR 5:1 for PF14, there would be 30 positive
charges from the five peptides to the 18 negative charges from the one SCO)
there was still a net negative surface charge (ζ-potential) in most dispersion
media. We also found that the surface charge was highly dependent on the
dispersion medium. In pure water the surface charge was entirely dependent
on the ratio of charges between the peptide and the oligonucleotide, being
negative at MR3, neutral at MR5 and positive at MR10. However, when
the dispersion medium was changed to better mimic the conditions that
the complexes would encounter in a cell (like isotonic NaCl or serum-free/
serum-containing medium) the ζ-potential became negative for all MRs. As
this change in surface charge happens even in the absence of serum, it would
be unlikely that the serum proteins are responsible for the change, but that
salt concentration and pH is. The net negative surface charge should cause
the CPP:ON complexes to be repelled by the negatively charged cell membrane and the negatively charged GAGs on the cell-surface.
We believed that something must have been mediating this interaction,
that there was something between the cell-surface and the complexes.
Literature searches gave us the SCAR As as potential candidates; because
they are known to bind with low specificity to polyanionic ligands (section
1 SCARAs). The simplest conceivable experiment at the time was to use the
splice-correction assay and pre-treat the cells with some ligands that competitively bind to the receptors. Even at low concentrations of the SCARA
ligands, fuciodan, dextran sulfate and poly i, the splice-switching effect of
PF14:SCOs was completely abolished in a dose-dependent manner. The next
question was of course which, if any, of the SCARA receptors are expressed
in the HeLa pLuc705 cells, we performed real-time PCR and found that
SCARA-5 and SCARA-3 (with its two splice variants) are expressed whereas
some of the others could not be detected.
Once the receptors had been identified the next step was to try to knock
them down with siRNA. Knockdown of SCARA-3 and SCARA-5 resulted
in an inhibition of the luminescence signal from luciferase by more than 50
%. In order to try to gather more evidence immunofluorescence and transmission electron microscopy (TEM) were performed in order to directly visualize changes in membrane association and localization at the cell-surface.
The TEM images clearly show the effects of the absence of SCARAs, when
the receptors are not available, because of pretreatment with dextran sulfate,
CPP:ON complexes are not associated with the cell membrane.
This is the first time that a specific receptor is demonstrated to be involved in the uptake of cell-penetrating peptides; in Paper I we demonstrated
SCARA involvement using both a number of pharmacological inhibitors and
by knocking down the receptor using siRNA and thus abolishing uptake
of PF14/SCO complexes. We later identified SCARAs as a receptor for the
Pepfect15 and the NickFect class of CPPs.
In Paper II, we went further with this investigation into the SCAR A
receptors and their impact on the uptake of CPP:ON complexes. The initial
results from the first paper cover only one of the several hundred CPPs that
have been reported. The whole ensemble of CPPs is, as can be seen from
(Section 1.2), a heterogeneous collection of peptides with the common ability to transport cargo into cells. We did not at the time know if the effects
we had seen were valid only for some peptides that all derive from the transportan peptide, or if what we had discovered was a more general mechanism.
We also included two cationic polymers to broaden the scope even further.
The discovery that SCAR As are involved in the uptake of CPP:ON
complexes offers new and interesting information that can be used both in
scavenger receptor research as well as in the development of CPP technology.
Original research on these receptors identified them in macrophages, later
they were identified in many different cell types, where different subtypes
were identified in different cells such as smooth muscle cells, epithelial and
endothelial cells, splenic dendritic cells, and fibroblasts.
The SCAR As are clearly very promiscuous and bind to all manner of
ligands, without any discernible preference for one thing over the other,
except maybe for dextran sulfate. At first it could seem as if linking one of
these ligands to the CPP, in a manner analogous to the endosomal-escape
moieties in PF6 [64] and PF15 [68], would be a possible way to enhance
uptake. However, such a modification could diminish the ability of the peptides to form complexes with ONs. Because of the opposite charge of CPPs
and dextran sulphate, the CPP would lose some of its ability to complex
ONs via charge interactions, as some of the charges on the CPP would be
Paper III: CPP Delivery Assay
In this paper we develop an assay that is cheaper, faster and higher
throughput than what we previously used, going from commercially sourced
reagents and 24-well plates to homemade reagents and 96-well plates while
reducing cost by 95 %. The assay that was developed in this work was born
out of necessity. The original intention was to use the library in Paper IV,
to determine if stimulation of other cell-surface receptors may have effects
other than the inhibitory effect on uptake that saturation of SCARAs has.
Discovering any effects would be interesting and discovering a ligand that
increased the uptake could eventually lead to an improvement of delivery
efficacy. The assumption was that simply scaling the assay based on the
treatment volume differences between 24- and 96-well plates would yield a
satisfactory experiment. That assumption turned out to be completely incorrect. The difference in volume is 5-fold and the difference in surface area is
approximately 6-fold, whereas there are 4 times more wells in a 96-well than
in a 24-well plate. The problem is then what, if any, scaling factor should be
used. In the initial attempts to run the library, consisting of 264 ligands, in
the 96-well format we used the difference in treatment volume as the scaling
factor. That did not yield any usable data. The major problem was that, all
of a sudden, there was much more variance within one experiment and experiments performed independently yielded sometimes opposite results. This
more or less forced the development and optimization of the assay. Initially,
the experiments were performed in way that was analogous to that for the
24-well plates. The cells were seeded on the first day, treated on the second
day and finally the luciferase measurement was performed on the third day.
Two things were quickly realized; first, the commercially sourced reagent is
not ideal when using a luminometer without reagent injectors, because the
luciferase signal changes by approximately 20 % during the time it takes to
read one plate. Second, this was not the only source of variance. The first
problem led to the development of a homemade luciferase reagent, which is
more stable during measurement. Fortunately this homemade reagent is significantly less costly; representing a discount of more than 95 % compared
to the commercial reagent used previously.
With a more stable luciferase reagent, it became clear what at least one
of the additional sources of variance was. Seeding ten-thousand cells on
the first day, treating the cells on the next day and measuring 24 hours
later caused large row and column effects. These effects are currently not
explained, even though attempts at discovering the source of the row effects
were made. However these problems are avoidable by seeding fewer cells
and treating the cells on the day of seeding. Overall, this assay is successful
at yielding experiments with low variation and a good separation between
positive and negative controls as evidenced by the excellent Z’ scores.
Paper IV: GPCR Ligands Influence Uptake
We use the above assay and a library of 264 GPCR ligands to identify
receptor ligands that increase the observed luminescence from luciferase
in the splice-correction assay. We also determined that the inhibition of
luciferase activity, initially observed in a library screen, was most likely due
to toxicity. Three ligands (or about 1 % of the library) have a stimulatory
effect on the luminescence from luciferase in the splice-correction assay,
two of them target the metabotropic glutamate receptor 5 (mGluR5) and
one targets histamine H3. We also investigate 5 ligands that seem to have
an inhibitory effect, all of them target estrogen receptors. The observed increase in luminescence from luciferase is fairly large and repeatable across
multiple experiments. The decrease we observed is disappointingly difficult
to separate from toxicity. It is not surprising that high toxicity results in a
decrease of luciferase activity, if not for any other reason than that there are
fewer viable cells in the treated wells. The over 3-fold increase we observed
in luminescence is interesting, not least for the potential to improve the
delivery efficacy of CPPs. However, that the increase of luminescence is due
to the ON being delivered more efficiently is yet to be confirmed. The result
can be an unspecific effect of something other than delivery. Stimulating the
receptor could trigger a signaling event, which in turn may cause anything
from increased transcription to reorganization at the cell-membrane, for
example it has been shown that GPCRs can signal from endosomes [243,
244]. The observed effect could be related to the stability of the endosomes.
The least exciting possibility is that the identified ligands may simply act to
disrupt the endosomes like in the case for other lysosomotropic amines [245].
The increase and decrease in transfection efficacy can also be related to
the endocytotic process of caveolae, there are indications that both SCARA
[246] and mGluR5 [247] are localized within caveolae on the cell membrane,
as are scavenger receptors class B [248], and many other GPCRs [249], as
well as the Na+/K+ ATPase [250]. Effects on caveolae that are involved in
cell-membrane lipid organization and stability, receptor signaling regulation,
and even mechanosensing of the cell membrane [251] could be a lead into
what is happening that causes the uptake to change. This study leaves many
unanswered questions, about the uptake of CPPs and about the complexities
of the workings of transport at the cellular membrane. An illustration of the
discussion above can be found in Figure 5.
Biological effect
Endosomal escape
Figure 5. GPCR internalization is mostly associated with clathrin-coated
pits, CPPs can be taken up via multiple endocytotic mechanisms and
SRs are also associated with clathrin and caveolae, what role if any the
receptors play in endosomal escape has not yet been explored.
Summary and Conclusion
This thesis presents three findings; first scavenger receptors are involved
in, if solely not responsible for, the uptake of many types of ONs in complex
with a number of different cationic molecules; we have shown this for the
peptides developed by us and by others as well as for polymers.
Second, this thesis contains an optimized assay for the delivery of short
ONs that will allow for the use of cell-penetrating peptides in high-throughput screening of for example siRNA libraries. Additionally the methods
used for evaluation of transfection efficacy and optimization of transfection
conditions for CPPs will be much higher throughput using this assay. This
method is faster, cheaper and more efficient that previously used methods.
Third, the assay above was used to screen a library of GPCR-ligands, looking for changes in uptake of CPPs. From the library, we identified ligands
that increase the uptake efficacy as measured by the luciferase-splicing assay.
The long-term goal of this scientific endeavor is to use CPPs to safely deliver ON-based molecules as therapies. The contribution of this work towards
that goal is to try to understand the uptake mechanism of cell-penetrating
peptides. Identifying first the SCARAs negative modulators of uptake and
then the mGlu5R and Histamine H3 receptors as positive modulators could
induce new directions to take. Understanding the uptake should reasonably
provide insight into how CPPs can be improved from where they stand today.
Future Outlook
he aim of this work was to clarify the uptake mechanism further,
and the first two papers seem to do that, as the involvement of the
SCAR As is quite clear and independent of which peptide or in fact what
delivery system is used. Recent studies suggest that it is the nano-particulate
form, not the composition of the particles that is important for the recognition and uptake by SCARA receptors [252]. We see similar results in Paper
II; the uptake of fluorescently labeled PF14 alone is also inhibited by the
SCARA ligands, even when no ONs are present. It is likely that PF14 forms
complexes with itself even in the absence of ONs.
The last manuscript suggests the possibility that the mechanism is more
convoluted and perhaps affected by other cellular processes. Currently we
don’t have a working hypothesis of the complete mechanism. Looking at
the normal course of events when a GPCR is activated can provide some
suggestions at what to investigate next; one possibility is to look for activation of protein kinases, which in turn activate other cellular responses.
Effects on the recruitment of caveolin and trafficking of caveolae should be
further investigated. Perhaps the effect we observe using the mGluR5 and
Histamine H3 ligands is due of the release of the CPP:ON complex from
the endosomes, this could perhaps be visualized using the endosome release
assay mentioned earlier [193].
Another aspect that is not yet fully investigated by us, is which endocytotic pathway PF:ON complexes is using. Studies using different pharmacological inhibitors and smaller cargo than ONs [149, 253] indicate that both
the clathrin and caveolin pathways are involved. The investigation using the
NickFect family of peptides revealed, using the pharmacological inhibitors
chlorpromazine and nystatin, that NF1 and NF51 were utilizing different
pathways [209]. Because of the overlap between the different pathways
(Section 1.4) and because of the overlap of the effects of chlorpromazine
[153] it will be difficult to answer definitely which pathway(s) are involved.
We knocked down Caveolin and Clathrin light- or heavy-chain using siRNA
without finding conditions from which useful information could be drawn.
The most recent suggestions are to use siRNA to target the adaptor proteins, the cavin proteins and Arf6. Super-resolution microscopy is another
possibility to further explore the content of the vesicles that contain the
endocytosed CPPs. This in combination with other new techniques like the
endosome escape assay, will eventually uncover the hidden machinery that
surrounds the uptake of CPPs and when all is revealed it will be easier to
design new peptides that take advantage of the new insights to become even
better transfection vectors.
Populärvetenskaplig sammanfattning
NA innehåller all biologisk information som behövs för att åstadkomma alla proteiner i en cell. Olika celler i kroppen utför olika
uppgifter; ögonen ser, lungorna tar upp syre från inandningsluften och hjärtat pumpar blodet ut till kroppens alla delar. Allt det här arbetet utförs av
olika specialiserade proteiner vars utseende, uppbyggnad och funktion bestäms av våra gener, av vårt DNA. Kostnaden för DNA-sekvensering sjunker
snabbare än det någonsin gjort för en tidigare teknologi, jämförelsen som
ligger närmast är beräkningskraft i datorer (Moores lag). En dag kommer
sekvensering bli lika vanligt som ett blodprov. Med all kunskap om hur
generna påverkar sjukdom som redan finns, och som ytterligare utökas när
fler sjukdomstillstånd kartläggs, är det naturligt att vilja påverka dem, till
exempel genom att stänga av en gen som inte borde vara aktiv, eller tvärtom.
För att kunna göra det behöver man föra in oligonukleotid-baserade molekyler i cellen, oligonukleotider är uppbyggda av DNA eller RNA. Det största
problemet är att cellen är mycket bra på att hålla sådana molekyler på en
sida av cellmembranet. För att göra det möjligt för dem att komma in i cellen behövs någon form av bärare eller transportör. Vi arbetar med en klass
av peptider som fungerar just som sådana transportörer. Cell-penetrerande
peptider (CPPer) har till skillnad från oligonukleotider positivt elektrisk
laddning och kan därmed skapa elektrostatiska komplex tillsammans med
dem. Ett sådant komplex kan sedan tas upp av cellen. Innan det här arbetet
var det känt att cellerna kunde ta upp komplexen och att det mest troligen
skedde genom en process som kallas endocytos. Den processen är det naturliga sättet för celler att ta upp näring och kommunicera med andra celler.
Cellmembranet har negativ laddning och man trodde att den första kontakten som inledde upptaget av CPPer var en elektrostatisk interaktion mellan
dem. Till vår förvåning upptäckte vi i Artikel I att komplexen mellan CPPer
och oligonuleotider inte hade positiv laddning. Det i sin tur ledde oss till att
tro att det borde finnas någonting i cell-membranet som fungerar som en
länk mellan en negativ partikel och det negativa cellmembranet. I Artikel I
identifierar vi Scavenger receptor class A som den länken, i alla fall när det
gäller en viss peptid och en bestämd typ av oligonukleotid. I Artikel II visar vi att mekanismen som medieras genom Scavenger receptorerna är mer
generell och gäller många olika typer av oligonuleotider och flera olika cellpenetrerande peptider samt ett par andra positivt laddade polymerer som
även de används för att få in oligonukleotider i celler.
För att göra det enklare, snabbare och billigare att utvärdera CPPers effektivitet som oligonukleotidbärare har vi i Artikel III utvecklat vår metod,
till att ha hälften så många steg, ta en dag mindre i anspråk och spara 95 %
av kostnaderna, samtidigt som den låter oss mäta 4 gånger så många prov
Denna metod användes sedan i Artikel IV till att undersöka om stimulans av den största familjen av receptorer på cellytan, G-proteinkopplade
receptorer, kunde påverka upptaget av CPP:oligo komplexen. Ur ett bibliotek
med 264 olika substanser identifierade vi tre stycken som var och en ökade
upptaget med upp till tre gånger.
Sammanfattningsvis innehåller den här avhandligen en metod som är
optimerad för att kunna göra experiment med CPPer snabbare och billigare
och samtidigt i större skala. En beskrivning av hur SCARA receptorer är inblandade i upptag av CPP:oligo komplex samt en indikation på att upptaget
ytterligare kan förbättras genom att ta andra receptorer i beaktande.
his is the section where I admit that none of the pages in this book
would have been possible without Linda my wife, who with her patience as I commuted back and forth the first four years and always with
her love and support made sure that I could finish this, these words are not
enough to express the gratitude and love that I feel.
Ülo, my supervisor who can motivate and inspire in ways I would have
thought were impossible. Thank you for letting me do this, and thank you
for you generosity.
My parents who must not have noticed that I stayed in school for what
must have seemed an indefinitely long time instead of getting a “real job”.
Thank you for everything.
Markus and Sara thank you for a place to stay on the days where the
Swedish railway system refused to cooperate, and thank you for your convincing interest in this work.
Staffan. It would have been a lot worse without you, thank you for the
company in the lab, the office and the conference bars.
Andrés who can talk more sense and more nonsense than anyone I know.
You have been great discussion partner irregardless of the subject. Scientific,
non-scientific or just entertaining subjects have all been memorable. Never
stop talking.
Jakob who knows all the detailed minutiae of the inner workings of the
academic bureaucracy. And without whom we all would have incapable of
distinguishing between an excellent meal and a merely good one. Thank
you for knowing the difference. And thank you for reading and correcting
my thesis.
Jonas who is the most ambitious and smartest scientist that I know, he
read this thesis and corrected the errors, saw the inconsistent parts found the
wrongly used words and made it better, thank you.
Tõnis really put an impressive amount of time and effort into reading this
thesis, the input I got made it a lot better and more readable, thank you.
Kristin we started together in the same group, I was lucky enough to get
the CPP project while you were lucky enough to get a real neurochemistry
project, thank you for always being so kind.
Carmine you are the nicest person I know, we have the same idea about
what a synthesis-lab fume hood should look like, but because you are so nice
and kind I worry that you won’t be as ruthless as may be necessary towards
the unlabeled tubes.
Ying you seem to always be in a hurry, but I am happy that you decided
to stay in our group your are good addition to it.
Daniel thank you for all the discussions, stay on the surfboard.
Kariem you started the SCAR A project, it is your idea, thank you for
letting me be a part of it.
Johan lured me into becoming the computer responsible person, thank
you for that.
Moataz thank you for all the discussions.
Marie-Louise whom I consider one of the few “adults” at the department,
thank you for knowing everything and making sure that all the inmates at
this asylum are well taken care of, we would not know what to do without
The master-thesis students that I have supervised, Aimee, Diogo, Chetan,
Sini, Maxime, Maria you all taught me what to expect, In most cases what
I thought beforehand was not enough, because all of you exceeded my expectations you were all better smarter and in less need of me then I though
you would be.
Thanks to all of my co-authors without whom the publications would not
Lastly it is more than likely that I have forgotten some friends, co-workers
students and colleagues. Doing a PhD is crazy, fun, interesting and full of
freedom in a way that won’t come again, but it is not the work itself but the
people that you work with that make it a worthwhile pursuit, so thank you
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