GHENT UNIVERSITY
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and Physical Pharmacy
Academic year 2009-2010
EFFECTS OF ENDOCYTOSIS INHIBITORS ON TRANSFECTION
MEDIATED BY POLYPLEXES AND LIPOPLEXES CARRYING mRNA
Laura WAYTECK
First Master of Drug Development
Promoter
Prof. dr. K. Braeckmans
Commissioners
Prof. dr. F. De Vos
Prof dr. N. Sanders
Instructor
Dr. Joanna Rejman
GHENT UNIVERSITY
FACULTY OF PHARMACEUTICAL SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and Physical Pharmacy
Academic year 2009-2010
EFFECTS OF ENDOCYTOSIS INHIBITORS ON TRANSFECTION
MEDIATED BY POLYPLEXES AND LIPOPLEXES CARRYING mRNA
Laura WAYTECK
First Master of Drug Development
Promoter
Prof. dr. K. Braeckmans
Commissioners
Prof. dr. F. De Vos
Prof dr. N. Sanders
Instructor
Dr. Joanna Rejman
COPYRIGHT
"The author and the promoters give the authorization to consult and to copy parts of
this thesis for personal use only. Any other use is limited by the laws of copyright, especially
concerning the obligation to refer to the source whenever results from this thesis are cited."
May , 2010
Promoter
Prof. dr. K. Braeckmans
Author
Laura Wayteck
First of all, I would like to thank my promoter, Prof .Dr. K. Braeckmans, for giving me the
opportunity to work and learn in his research group.
Especially, I would like to thank my instructor, Dr. Joanna Rejman, whose encouragement
and guidance enabled me to write this thesis. Joanna, thank you for your enthusiasm,
kindness and patience during this couple of months. Without your help and corrections, this
thesis would not be completed.
Dziękuję Ci bardzo. Cieszę się, że mogłam pracowad właśnie z Tobą.
Furthermore, I gratefully thank Prof. Dr. J. Demeester, Prof. Dr. S. De Smedt and all the
lab staff for always being ready to help and for their useful advice . I really want to say that it
was a real pleasure working with them.
Finally, thanks to my parents and friends for the support. Also a special thanks to my
fellow thesis students for the nice time we spend in the lab and during our lunch breaks.
In short: I had a very nice time in the lab. It was a most instructive experience.
Thanks to all of you!
CONTENTS
1.
INTRODUCTION ........................................................................................................... 1
1.1.
GENE DELIVERY..................................................................................................... 1
1.1.1.
General ......................................................................................................... 1
1.1.2.
Genetic material ........................................................................................... 1
1.1.3.
Gene delivery systems ................................................................................. 2
1.2.
TRANSFECTION ..................................................................................................... 6
1.2.1.
Binding of the carrier system to the cell surface and internalization ........ 6
1.2.2.
Escape of the nucleic acids from the endosomal compartment .............. 10
1.2.3.
Gene expression ......................................................................................... 13
1.3.
mMSCs ................................................................................................................ 13
1.4.
INHIBITORS ......................................................................................................... 14
1.4.1.
Methyl-β-cyclodextrin ................................................................................ 14
1.4.2.
Chlorpromazine .......................................................................................... 14
1.4.3.
Filipin........................................................................................................... 14
1.4.4.
Genistein ..................................................................................................... 15
2.
OBJECTIVES................................................................................................................ 16
3.
MATERIALS AND METHODS...................................................................................... 17
3.1.
PLASMID PURIFICATION ..................................................................................... 17
3.2.
PREPARATION OF IN VITRO TRANSCRIBED mRNA ............................................. 17
3.3.
CELL CULTURE..................................................................................................... 18
3.4.
MTT BASED COLORIMETRIC ASSAY .................................................................... 18
3.4.1.
Principle ...................................................................................................... 18
3.4.2.
Practical ...................................................................................................... 18
3.5.
LUCIFERASE ASSAY ............................................................................................. 19
3.5.1.
Principle ...................................................................................................... 19
3.5.2.
Practical ...................................................................................................... 20
3.6.
3.6.1.
Polyplexes ................................................................................................... 21
3.6.2.
Lipoplexes ................................................................................................... 21
3.7.
TOXICITY TEST..................................................................................................... 22
3.7.1.
Inhibitors..................................................................................................... 22
3.7.2.
Polyplexes and lipoplexes .......................................................................... 24
3.8.
4.
PREPARATION OF COMPLEXES........................................................................... 21
TRANSFECTION ................................................................................................... 24
RESULTS ..................................................................................................................... 27
4.1.
TOXICITY TEST..................................................................................................... 27
4.1.1.
Inhibitors..................................................................................................... 27
4.1.2.
Polyplexes: linPEI........................................................................................ 30
4.1.3.
Lipoplexes : DOTAP/DOPE ......................................................................... 33
4.2.
TRANSFECTION ................................................................................................... 37
4.2.1.
Polyplexes : linPEI ....................................................................................... 37
4.2.2.
Lipoplexes : DOTAP/DOPE ......................................................................... 40
5.
DISCUSSION ............................................................................................................... 45
6.
GENERAL CONCLUSIONS ........................................................................................... 47
7.
REFERENCES .............................................................................................................. 48
LIST OF ABBREVIATIONS
ATP: Adenosine triphosphate
CCV: Clathrin-coated vesicles
CDE: Clathrin-dependent endocytosis
CIE: Clathrin-independent endocytosis
DC-Chol: 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl])-cholesterol
DNA: Deoxyribonucleic acid
DOPE: 1,2 dioleoyl-sn-glycero-3-phospho-ethanolamine
DOTAP: N-(1-(2,3-dioleoyloxy)propyl,N,N,N-trimethylammonium chloride
FBS: Fetal bovine serum
GAGs: Glycosaminoglycans
GS1: Gemini surfactant 1
GTPases: Guanine triphosphatases
IMDM: Iscove’s Modified Dulbecco’s Medium
LB: Luria-Bertani
linPEI: Linear polyethyleneimine
MbCD: Methyl-β-cyclodextrin
mMSCs: Murine mesenchymal stem cells
mRNA: Messenger ribonucleic acid
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NADH: Nicotinamide adenine dinucleotide
NADPH: Nicotinamide adenine dinucleotide phosphate
PAMAM: Poly-amidoamine
PBS: Phosphate-buffered saline
pDNA: Plasmid Deoxyribonucleic acid
PEI: Polyethyleneimine
PLL: Poly(L-lysine)
RLU: Relative light units
RNasin : Ribonuclease inhibitor
SAINT-2: N-methyl-4(dioleyl)methyl-pyridinium-chloride
INTRODUCTION
1.
INTRODUCTION
1.1. GENE DELIVERY
1.1.1. General
Through the years, gene therapy has become a promising tool for the treatment of both
inheritable and acquired diseases. In some cases, genetic diseases can be treated
symptomatically but so far they have been basically incurable. The initial goal of gene
therapy was to replace a deficient gene with its correct copy. Later, this goal was extended
to genetic defects different from inherited disorders because of the possibility to modulate
the regulation of gene expression involved in numerous acquired diseases. Nowadays it is
known that gene therapy could not only be useful for the replacement of defect genes but it
could also allow the modulation of the expression of genes involved in the physiology of
malignant cells. Furthermore, it is also possible by means of gene therapy to introduce
functions into cells that are not originally present but could serve a therapeutic purpose
(Wasungu, 2006).
In current gene therapy strategies, there are two essential components: an effective
therapeutic gene (Vacik et al., 1999) and an efficient and safe delivery system (Shaffer et al.,
1998).
1.1.2. Genetic material
The genetic material to be delivered can be presented to the cells as part of the genome
of a virus (see 1.1.3.1.) or it can be formulated as an individual nucleic acid such as a plasmid
DNA (pDNA) or a messenger RNA (mRNA). Non-viral gene transfer thus far has been
performed largely with pDNA. mRNA has recently emerged as an attractive and promising
alternative for pDNA because it has several advantages over pDNA (Yamamoto et al.,2008).
1
INTRODUCTION
Unlike pDNA:

mRNA exerts its function in the cytoplasm, so it does not have to cross the nuclear
membrane.

mRNA does not bear the risk of insertional mutagenesis.

it does not require the use and determination of a promoter.

mRNA can also be used in non-dividing cells.

mRNA does not bear the risk of vector-induced immunogenicity, thus it can be
administered repeatedly.
1.1.3. Gene delivery systems
In gene therapy, the ‘drugs’ used are nucleic acids, which have their target sites mostly
present inside cells. As a consequence, it is essential for these molecules to cross the plasma
membrane (Bally et al., 1999). The plasma membrane of living cells restricts the entry of
large hydrophilic or charged molecules. By nature, genetically active molecules, i.e. nucleic
acids, are both large and charged, and thus experience the plasma membrane as a first
hurdle on their way to exert their action as a ‘drug’ (Khalil et al.,2006). It should not come as
a surprise therefore that the presentation of a naked DNA to cells will not result in efficient
gene expression (Li et al., 2007). Therefore, a carrier system is required for the efficient
delivery of genetic material into the cells. Such carrier systems are not only designed to
deliver the gene efficiently inside the cells, they also need to protect the nucleic acids from
extracellular as well as intracellular degradation and premature clearance from the
circulation in vivo (Han et al., 2000).
Gene delivery systems can be roughly divided into two main categories: viral and nonviral gene delivery systems.
2
INTRODUCTION
1.1.3.1. Viral vectors
Recombinant viruses (lentiviruses, adenoviruses) are commonly used for gene delivery.
Those viruses have some of their genes replaced by the relevant therapeutic gene
(Yamamoto et al., 2008).
Viruses have the ability to penetrate into cells and use the host-cell machinery for the
production of viral proteins. Viral delivery, however, may pose problems in terms of safety
because of the immunogenicity of the virus-derived particles and the possibility that the viral
genome integrates into the host genome, resulting in potential oncogenicity (Wasungu,
2006a). Furthermore, there are limitations to the large-scale production of viral vectors (Li
and Huang, 2000). Based on these disadvantages, non-viral gene delivery vectors have
emerged as alternative delivery systems.
1.1.3.2. Non-viral vectors
Non-viral gene delivery systems can be divided into two major categories: the polymerbased and the lipid-based systems. Cationic polymers and lipids spontaneously interact with
the negatively charged nucleic acids to form polyplexes and lipoplexes, respectively (Zuhorn,
2002). The most important shortcoming of these non-viral vectors is their relatively low
transfection efficiency compared to viral vectors. On the other hand, non-viral delivery
systems are characterized by low immunogenicity, low production cost and high
reproducibility. Finally, non-viral vectors are more readily amenable to chemical
modifications, which might be exploited to improve their therapeutic performance (Park et
al., 2006).

Cationic lipids
Cationic lipids have an amphiphilic character, implying that they consist of a hydrophilic
part and a hydrophobic part. The hydrophilic region consists of a (charged) cationic
3
INTRODUCTION
headgroup and a hydrophobic region usually consisting of two hydrocarbon chains (see
Fig.1.1). These two regions are attached to each other by a linker (for example glycerol).
FIGURE 1.1: THE GENERAL STRUCTURE OF A CATIONIC LIPID. THE STRUCTURE CONSISTS OF A CATIONIC
HEADGROUP AND A HYDROPHOBIC REGION ATTACHED TO EACH OTHER BY A LINKER. THE HYDROPHOBIC
REGION CONSISTS OF TWO HYDROCARBON CHAINS. X,Y AND Z REPRESENT A NUMBER OF POSSIBLE
CHEMICAL MOIETIES DEPENDING ON THE SPECIFIC LIPID.
(http://www.promega.com/paguide/chap12.htm(27-04-10))
Because of their amphiphilic character, they easily form vesicular structures in water,
called liposomes. When suspended in water, liposomes interact with the nucleic acids
because of the electrostatic interactions and thus form lipid-nucleic acid complexes. It is
important to know that the addition of nucleic acids to preformed cationic liposomes
triggers structural changes in both liposomes and the nucleic acids (Wasungu and Hoekstra,
2006b). In the past decades a host of different cationic lipids have been designed, e.g.
DOTAP, SAINT-2, DC-Chol and GS1.
In our experiments we used the cationic lipid DOTAP mixed with DOPE.
The
hydrophilic
region
of
DOTAP
(N-(1-(2,3-dioleoyloxy)propyl,N,N,N-
trimethylammonium chloride) consists of a quaternary ammonium polar headgroup linked
by a glycerol to the hydrophobic region, consisting of two oleoyl chains (see Fig.1.2). DOTAP
has a cylindrical shape and therefore adopts a lamellar structure in a bilayer (Wasungu and
Hoekstra, 2006b).
4
INTRODUCTION
FIGURE 1.2: STRUCTURE OF DOTAP. DOTAP CONSISTS OF A QUATERNARY AMMONIUM POLAR
HEADGROUP LINKED BY A GLYCEROL TO THE HYDROPHOBIC REGION, CONSISTING OF TWO OLEOYL
CHAINS (Wasungu and Hoekstra, 2006b).
The molecule DOPE (1,2-dioleoyl-sn-glycerol-3-phospho-ethanolamine) is zwitterionic at
neutral or acidic pH. DOPE forms an inverted hexagonal phase because of its conical shape
due to the relatively small polar headgroup (Wasungu and Hoekstra, 2006b; Zuhorn et al.,
2007). The structure of neutral DOPE is shown in Fig.1.3.
The function of DOPE in combination with DOTAP is discussed later (see 1.2.2.1.).
FIGURE 1.3: STRUCTURE OF NEUTRAL DOPE. THE MOLECULE DOPE BECOMES ZWITTERIONIC AT NEUTRAL
OR ACIDIC pH (http://www.promega.com/paguide/chap12.htm(27-04-10)).

Cationic polymeric vector
There is a large variety of cationic polymeric vectors like PLL, PEI, PAMAM etc., with very
different structural features. None of these has emerged as a generally preferable structure
(Gebhart et al., 2001). The one we use in our experiments is PEI, in particular linear PEI
(linPEI).
PEI (polyethylenimine) occurs as branched or linear molecules. linPEI is a linear polymer
made of repeating units of –CH2-CH2-NH- (see Fig.1.4). At physiological pH, 90 % of the imino
groups of the linPEI are protonated. These cationic polymers do not have hydrophobic
regions and they can condense the nucleic acids more efficiently than cationic lipids. This
results in the formation of smaller particles than with cationic lipids (Lungwitz et al., 2005).
5
INTRODUCTION
FIGURE 1.4: STRUCTURE OF LINEAR PEI. LinPEI IS A LINEAR POLYMER MADE OF REPEATING UNITS OF –
CH2-CH2-NH- (Park et al., 2006).
1.2. TRANSFECTION
Transfection can be defined as a procedure leading to expression of an exogenous
nucleic acid in cells. The process of cellular transfection can be divided into three main steps
(Zuhorn, 2002):
1. Binding of the carrier/nucleic acid complex to the cell surface followed by
internalization of the carrier system.
2. Escape of the nucleic acids from the endosomal compartment.
3. Protein production.
1.2.1. Binding of the carrier system to the cell surface and internalization
The first barrier encountered by a nucleic acid complex is the cell membrane. A surplus
positive charge (cationic lipid or polymer) over negative charge (nucleic acid) results in a net
cationic complex. This net positive charge of the complex may facilitate the binding of the
complex to the negatively charged cell membrane by electrostatic interactions (Zuhorn,
2002). In order to achieve a more specific interaction of the complexes with the cell surface,
a ligand can be incorporated in the complex allowing specific interactions with receptors on
the cell membrane.
Internalization of the complexes occurs through a process called endocytosis (Midoux et al.,
2008; Wattiaux et al., 2000).
6
INTRODUCTION
1.2.1.1. Endocytosis
Endocytosis is the internalization of extracellular material by cells through the
invagination of the plasma membrane leading to the formation of vesicular intracellular
structures (Khalil et al., 2006). Endocytosis can be classified in two main categories:
phagocytosis or cell eating and pinocytosis or cell drinking. Pinocytosis occurs in most animal
cells while phagocytosis is primarily conducted by specialized cells such as macrophages,
monocytes and neutrophils. The latter uptake mechanism can also occur in other cell types,
yet, under specific conditions. Research on the different pinocytosis pathways is still ongoing
and knowledge about it is still far from complete. As a consequence, the subdivision of
pinocytosis is not yet firmly established. Based on present-day insights, pinocytosis is
subdivided into: clathrin-dependent endocytosis (CDE), clathrin-independent endocytosis
(CIE) and macropinocytosis (Vercauteren et al., 2009). Fig.1.5 represents a classification
overview of the different endocytosis pathways.
These pathways differ in the composition of the coat, the size of the endocytic vesicles
pinched off from the plasma membrane and the fate of the internalized particles (Khalil et
al., 2006).

Phagocytosis
This endocytic pathway is active primarily in specialized animal cells. The interaction
between the ligand on the surface of the particle and the specific receptor on the phagocyte
initiates the internalization of the particle. This binding triggers actin assembly and the
formation of cell surface protrusions. These protrusions enclose the particle and finally
engulf it (Khalil et al.,2006). After internalization, the particle is present in a phagosome
which matures by a series of fusion and fission events (see Fig.1.7). This process results in
the formation of mature phagolysosomes where internalized particles are degraded (Allen
and Aderem, 1996).
7
INTRODUCTION
Endocytosis
phagocytosis
pinocytosis
macropinocytosis
clathrin
dependent
endocytosis
clathrinindependent
endocytosis
dynamin
dependent
caveolae
mediated
dynamin
independent
RhoA mediated
ARF6 or cdc42
mediated
Flotillin-1
mediated
FIGURE 1.5: OVERVIEW OF DIFFERENT ENDOCYTIC PATHWAYS (Vercauteren et al., 2009).

Clathrin-dependent endocytosis (CDE)
CDE is the best characterized endocytic pathway. After binding of a particle to a cell
surface receptor, particle-receptor complexes are clustered in clathrin-coated pits. These
pits then invaginate and pinch off from the cell membrane, forming clathrin-coated vesicles
(CCV). The clathrin on these vesicles depolymerizes turning the CCVs into early endosomes.
The early endosomes fuse with each other and become late endosomes which eventually
fuse with lysosomes (Khalil et al., 2006). This latter step should be avoided in the gene
delivery process as it will lead to the intralysosomal enzymatic degradation of the nucleic
acids (see Fig.1.6).
8
INTRODUCTION
FIGURE 1.6: CLATHRIN-DEPENDENT ENDOCYTOSIS. THE LIGAND FIRST BINDS TO A CELL SURFACE
RECEPTOR. PARTICLE-RECEPTOR COMPLEXES ARE CLUSTERED IN CLATHRIN-COATED PITS. THE PITS
INVAGINATE AND PINCH OFF FROM THE CELL MEMBRANE AND FORM CCV WHICH TURN INTO EARLY
ENDOSOMES. EARLY ENDOSOMES FUSE WITH EACH OTHER AND BECOME LATE ENDOSOMES WHICH
EVENTUALLY FUSE WITH LYSOSOMES (Khalil et al.,2006).

Clathrin-independent endocytosis (CIE)
The CIE mechanism has not been completely elucidated. The current subdivision is based
on the role of dynamin and several small GTPases. As a result, CIE is sub-classified into
dynamin-dependent and dynamin-independent CIE pathways. The best characterized CIE
route is the caveolae-mediated pathway. This is a dynamin-dependent CIE pathway.
Caveolae are membrane microdomains characterized by the presence of the protein
caveolin (Matveev et al., 2001; Harris et al., 2002). They form flask-shaped invaginations.
The invaginated vesicles are called caveosomes (see Fig.1.7). This pathway seems to be
advantageous for the purpose of nucleic acid gene delivery because the caveolar uptake
does not lead to the lysosomal compartment and thus avoids intralysosomal degradation
(Gabrielson and Pack, 2009). This pathway is a promising gene delivery mechanism especially
if the uptake efficiency can be increased, possibly through the use of specific receptors and
ligands for caveolae (Harris et al., 2002; Ferrari et al., 2003).
9
INTRODUCTION
FIGURE 1.7: PHAGOCYTOSIS, MACROPINOCYTOSIS AND CLATHRIN-INDEPENDENT ENDOCYTOSIS.
PHAGOCYTOSIS IS ACTIVE IN SPECIALIZED CELLS AND ENDS UP IN PHAGOLYSOSOMES.
MACROPINOCYTOSIS IS USUALLY ACCOMPANIED BY CELL SURFACE RUFFLING AND FORMS LARGE
ENDOCYTIC VESICLES, CALLED MACROPINOSOMES. CAVEOLAE ARE MEMBRANE MICRODOMAINS,
CHARACTERIZED BY THE PRESENCE OF THE PROTEIN CAVEOLIN. THE FLASK-SHAPED INVAGINATIONS
FORM CAVEOSOMES (Khalil et al, 2006).

Macropinocytosis
Macropinocytosis is a nonselective pathway for the uptake of suspended
macromolecules. This pathway is usually accompanied by cell surface ruffling (see Fig.1.7)
and forms large endocytic vesicles, called macropinosomes. These vesicles can have sizes up
to 5 µm (Vercauteren et al., 2009). This pathway has some benefits in case of gene delivery
such as the (possible) avoidance of intralysosomal degradation and the increased uptake of
macromolecules (Khalil et al., 2006; Swanson and Watts, 1995).
1.2.2. Escape of the nucleic acids from the endosomal compartment
After their endocytic uptake into the cells, the complexes need to escape from the
endosomal compartment before it fuses with lysosomes, where the nucleic acids would be
degraded.
10
INTRODUCTION
1.2.2.1. Endosomal release of nucleic acids from lipoplexes
For the release of the nucleic acids from lipoplexes located in endosomes, a close
contact between the complex and the inside aspect of the endosomal membrane is
required. In more detail, while in the endosomes of the cell, the complex initiates
destabilization of the endosomal membrane. As a result, the anionic lipids of the endosomal
membrane form charge-neutralized ion pairs with the cationic lipids of the lipoplex (see
Fig.1.8). Now, the nucleic acids can dissociate from the complex and are released into the
cytoplasm (Xu and Szoka, 1996). Conceivably, bigger lipoplexes (200-400 nm - 1 µm) expose
a larger surface area for interaction with the membrane of the endosomes than smaller
lipoplexes (200-250 nm). Moreover, small lipoplexes usually bear nucleic acid protrusions,
which may hamper the close contact between the endosomal membrane and the lipoplex.
These two conditions will cause nucleic acids to be released more slowly from small than
from large lipoplexes. Thus, nucleic acids carried by small lipoplexes will end up in and be
degraded by lysosomes to a larger extent than those carried by larger ones. As a
consequence, transfection efficiencies of larger lipoplexes will be higher than that of smaller
ones.
Apart from the size of the lipoplexes, it seems that the transfection is more efficient
when the lipids in the lipoplex are in a hexagonal phase. The inclusion of helper lipids such as
DOPE in the lipoplex (in addition to, for example, DOTAP) promotes the conversion of the
lamellar lipid phase in the lipoplex into a hexagonal phase. Such a hexagonal structure may
induce additional perturbations in the endosomal membrane, which will further facilitate
the release of the nucleic acids into the cytoplasm (Zuhorn, 2002; Wasungu and Hoekstra,
2006b).
1.2.2.2. Endosomal escape of nucleic acids from polyplexes
For the explanation of endosomal escape of nucleic acids from polyplexes, particularly
containing PEI, a ‘proton sponge hypothesis’ was proposed. PEI is unique due to its high
buffering capacity. This results in an intrinsic ability to destabilize the endosomal membrane
11
INTRODUCTION
(Huth et al., 2006). In addition, the hypothesis suggests that PEI becomes more protonated
at low pH. It is known that in the endosomes the pH is low, which would allow the imino
groups of PEI to become protonated. It was proposed that this protonation triggers an
endosomal influx of protons and chloride ions. The resulting osmotic influx of water would
cause the endosome to swell, ultimately leading to rupture of the endosomal membrane
and release of the polyplex in the cytosol (Behr., 1997; Sonawane et al., 2003).
FIGURE 1.8: MECHANISM OF UPTAKE AND ENDOSOMAL RELEASE OF NUCLEIC ACIDS FROM LIPOPLEXES.
LIPOPLEXES ARE INTERNALIZED BY ENDOCYTOSIS (STEP 1). IN THE EARLY ENDOSOME, MEMBRANE
DESTABILIZATION RESULTS IN ANIONIC PHOSPHOLIPID FLIP-FLOP (STEP 2). AS A RESULT, THE ANIONIC
LIPIDS OF THE ENDOSOMAL MEMBRANE FORM CHARGE-NEUTRALIZED ION PAIRS WITH THE CATIONIC
LIPIDS OF THE LIPOPLEX (STEP 3). THE NUCLEIC ACIDS CAN DISSOCIATE FROM THE LIPOPLEXES AND ARE
RELEASED INTO THE CYTOPLASM (STEP 4) (Xu and Szoka, 1996).
After escape of polyplexes from the endosome, the nucleic acids have to dissociate from
the polymer. Earlier studies (Itaka et al., 2004) demonstrate that pDNA dissociates from
polyamine gene vectors (such as PEI) in the cytoplasm after endosomal release. Cytoplasmic
12
INTRODUCTION
proteins have been suggested to dissociate pDNA from PEI (Okuda et al., 2004), but it was
demonstrated that this dissociation only occurs at low PEI/pDNA ratios which are not
optimal for transfection. Huth and colleagues investigated if RNA, which is present in the
cytoplasm in high concentration, could dissociate pDNA from the complexes. In addition,
RNA is structurally similar to pDNA and it is also negatively charged. The results
demonstrated that the replacement of pDNA by RNA is the critical factor in the dissociation
of pDNA from the complexes (Huth et al., 2006).
1.2.3. Gene expression
The nucleic acids, released from the endosomes and complexes, are now present in the
cytosol. In case of DNA, it still has to cross the nuclear envelop and be subsequently
transcribed into mRNA. The nuclear envelope thus presents an additional barrier that can
limit transfection efficiency. In case of mRNA, the transfer to the nucleus is not required as
its translation into proteins occurs in the cytoplasm. The mRNA, released from lipo- and
polyplexes is free to reach the ribosomes, where new proteins can be synthesized.
1.3. mMSCs
Murine mesenchymal stem cells (mMSCs) were used in all experiments described in this
thesis. These cells are ‘adult’ stem cells, also called bone marrow stromal cells. The three
most important characteristics of stem cells are: they are capable of dividing and renewing
themselves for long periods, they have no special function and they can give rise to
specialized cell types. The mesenchymal stem cells are present in the bone marrow and can
generate bone cells, cartilage cells, fat cells and stromal cells that support blood formation
(http://stemcells.nih.gov/info/basics/ (29-04-10)).
13
INTRODUCTION
1.4. INHIBITORS
The treatment of cells with endocytosis inhibitors that can inhibit a certain endocytic
pathway is useful in determining which uptake pathway(s) is/are employed by a specific
particle. The following four inhibitors were used in the experiments: methyl-β-cyclodextrin,
chlorpromazine, filipin and genistein.
1.4.1. Methyl-β-cyclodextrin
This inhibitor depletes the cell membrane of cholesterol and has been shown to perturb
internalization by both clathrin- and caveolae-mediated pathway (Rodal et al., 1999).
1.4.2. Chlorpromazine
This molecule dissociates clathrin and AP2 adaptor protein complexes from the cell
surface and thus inhibits clathrin-mediated endocytosis. In literature, there is no evidence
that chlorpromazine influences lipid raft or caveolae-mediated endocytosis (Ivanov, 2008).
1.4.3. Filipin
Filipin binds cholesterol and thus is able to change properties of cholesterol-rich
membrane domains such as in caveolae. As a consequence, it causes aberrations in the
caveolar shape and inhibits the internalization by lipid rafts. In contrast to methyl-βcyclodextrin, it does not interfere with internalization of transferrin, a classical ligand of
clathrin-mediated endocytosis, which demonstrates that it has no effect on clathrinmediated endocytosis. As a result, filipin appears to be a selective inhibitor of the lipid
raft/caveolae pathway (Orlandi and Fishman, 1998; Ros-Baro et al., 2001; Smart and
Anderson, 2002).
14
INTRODUCTION
1.4.4. Genistein
Genistein is a tyrosine kinase inhibitor. It causes local disruption of the actin network at
the site of endocytosis and inhibits the recruitment of dynamin II. These two events are
indispensable in the caveolae-mediated uptake and so genistein inhibits the caveolaemediated endocytosis (Parton et al., 1994; Nabi and Le, 2003).
15
OBJECTIVES
2.
OBJECTIVES
For a successful transfection of murine mesenchymal stem cells with mRNA, an efficient
carrier system is needed. In these experiments linear PEI and DOTAP/DOPE were tested as
possible non-viral carriers. Eukaryotic cells take up a variety of substances using different
endocytic pathways. Therefore it is possible that the way by which mRNA complexes are
internalized might have an effect on their intracellular processing and, subsequently, on
their transfection efficiency.
The main objective of this work was to establish if there is any difference in the
mechanism of uptake of DOTAP lipoplexes and linPEI polyplexes, carrying mRNA, that might
determine their transfection efficiency.
Therefore, it was important to know which endocytosis pathways are involved in the
uptake of the two different carrier systems. Thus, four different inhibitors (methyl-βcyclodextrin, chlorpromazine, filipin and genistein ), inhibiting different endocytic pathways,
were tested. In this way it is possible to determine to what extent a particular internalization
pathway is employed for transfection mediated by either of the two types of mRNA carriers.
There is a possibility that such inhibitors are toxic to the cells. Therefore, the first part of
the thesis is focused on determining at which concentration these inhibitors are still nontoxic to the cells. Subsequently, toxicity of these inhibitors in the presence of lipo- and
polyplexes was studied. An MTT assay was employed to study cell toxicity. Finally, the
transfection efficiency of mRNA, encoding luciferase, carried by the two different systems in
the presence of the four endocytosis inhibitors was tested using the luciferase assay.
16
MATERIALS AND METHODS
3.
MATERIALS AND METHODS
3.1. PLASMID PURIFICATION
Bacteria are grown in Luria Bertani-medium (LB-medium). To prepare the medium 5 g
NaCl (Lab M Limited, Bury, UK), 2.5 g Yeast extract (Lab M Limited, Bury, UK), and 5 g
Tryptone (Lab M Limited, Bury, UK) are dissolved in 500 ml of water. After the preparation,
the medium is autoclaved. Before the medium is used to grow bacteria, an antibiotic
(ampicillin, 0.1 mg/ml; Duchefa Biochem, Haarlem, The Netherlands) is added. Plasmid DNA
(Luc-A50) is isolated from the bacteria using the QIAfilter plasmid purification kit (Qiagen,
Venlo, The Netherlands) according to the manufacturer’s instructions. The isolated pDNA is
stored in TE buffer (10 mM Tris·Cl, pH 8.0; 1mM EDTA). pDNA is further purified using the
QIAquick PCR purification kit (Qiagen). The plasmid is linearized with the restriction enzyme
– DraI - (overnight, 37°C) and purified again using the Qiaquick PCR purification kit. After
adding an RNase inhibitor (1 U/µl, RNasin, Promega, Leiden, The Netherlands) the template
DNA is stored at – 20°C.
3.2. PREPARATION OF IN VITRO TRANSCRIBED mRNA
The in vitro transcription of mRNA is done using the T7 mMessage mMachine kit
(Ambion, Lennik, Belgium) producing a capped mRNA according to the manufacturer’s
instructions. Briefly, 6 µl buffer, 30 µl nucleotide mix, 10 µl water, 9 µl linearized plasmid
DNA and 5 µl T7 RNA polymerase are mixed together and incubated for 3h at 37°C.
Subsequently, 3 µl DNase is added. After 15 min, 30 µl water and 25 µl LiCl (lithium chloride)
are added to the samples, which are further incubated for 2h at -20°C. After centrifugation
(15 min, 14.000 RPM, 4°C), the pellet is washed with 1 ml 70% ethanol. After the subsequent
centrifugation (15 min, 14.000 RPM, 4°C), the supernatant is discarded and the pellet is resuspended in 20 µl water. The mRNA concentration is determined by measuring the
absorbance at 260 nm. The produced mRNA is stored at -80°C at a concentration of 1µg/µl.
17
MATERIALS AND METHODS
3.3. CELL CULTURE
The murine mesenchymal stem cells are cultured in 75-cm² flasks in a MSC medium. This
medium consists of Iscove’s Modified Dulbecco’s Medium (IMDM) (Lonza, Merelbeke,
Belgium) supplemented with 10 % horse serum, 10 % heat inactivated fetal bovine serum
(FBS) , 2 mM glutamine, 100 U/ml penicillin and streptomycin (Invitrogen, Merelbeke,
Belgium). The cells are cultivated at 37°C in a humidified 5 % CO2-containing atmosphere.
Cells are seeded in 24-well plates (30.000 cells/well) 24 h before the test.
3.4.
MTT BASED COLORIMETRIC ASSAY
3.4.1. Principle
The assay is based on the conversion of the yellow tetrazolium salt MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan crystals by
metabolically active cells. This is an intracellular reduction process that involves the pyridine
nucleotide cofactors NADH and NADPH. The reduction is catalyzed by reductases, which are
active only in viable cells. The amount of formazan produced corresponds to the number of
viable cells. The formazan crystals formed are solubilized with a buffer and the colored
solution can be quantified by measuring absorbance at 600 nm.
3.4.2. Practical
The MTT assay is performed using a ‘Cell Proliferation Kit’ according to the
manufacturer’s instructions (Roche Applied Science, Vilvoorde, Belgium). Briefly, after
removing the media, 500 µl regular mMSC medium is added to each well. Subsequently, 50
µl MTT labeling reagent is added (containing the yellow tetrazolium salt MTT) and, after a 4h-incubation, 500 µl solubilizing solution is added and incubation is continued overnight. The
absorbance at 600 nm is measured by UV-spectrophotometry (UV-1800 Shimadzu
18
MATERIALS AND METHODS
spectrophotometer, Shimadzu, Deurne Antwerpen, Belgium). The reference wavelength is
690 nm.
3.5. LUCIFERASE ASSAY
3.5.1. Principle
3.5.1.1. Luciferase assay
The efficiency of transfection of a vector containing the gene that encodes luciferase is
assayed by a luciferase assay. The expression of the protein is measured by assaying the
activity of the enzyme. Luciferin is oxidized to oxyluciferin, catalyzed by firefly luciferase with
ATP-Mg2+ as a cofactor (see Fig.3.1). The reaction produces a flash of light, which decays
quickly after enzyme and substrate are combined.
FIGURE 3.1: REDUCTION REACTION OF LUCIFERIN TO OXYLUCIFERIN CATALYZED BY FIREFLY LUCIFERASE
(http://www.promega.com/paguide/chap8.htm(29-04-10)).
The luciferase kit contains a cell lysis buffer and a luciferase substrate. The lysis buffer is
a detergent that disintegrates the cell membrane so that the enzyme is released.
3.5.1.2. Protein assay
Bio-Rad Protein Assay is based on the method of Bradford and is a dye-binding assay.
The dye which is used in this assay is Coomassie® brilliant Blue G-250. When the dye binds to
protein the absorbance maximum shifts from 465 nm to 595 nm. This dye binds primarily to
19
MATERIALS AND METHODS
basic and aromatic amino acid residues. The absorbance is measured with a
spectrophotometer at 595 nm against a standard curve.
3.5.2. Practical
3.5.2.1. Luciferase assay
After removing the culture medium, the cells are washed with PBS (Phosphate-buffered
saline ) (Invitrogen, Merelbeke, Belgium). The cells are incubated for 30 min with 100 µl of
Cell Culture Lysis Reagent (Promega, Leiden, The Netherlands). The lysates are transferred to
1.5-ml tubes and centrifuged (5 min, 14.000 RPM). 40 µl of each supernatant solution is used
to measure luciferase activity in a GloMax®-96 Microplate Luminometer (Promega, Leiden,
The Netherlands). 100 l of luciferase substrate (Promega, Leiden, The Netherlands) is
injected into each sample.
3.5.2.2. Protein assay
To prepare a standard curve bovine serum albumin (Thermo scientific – Pierce Protein
Research Products, Erembodegem Aalst, Belgium) is used. The standard solution is diluted
10 times resulting in a concentration of 0.2 mg/ml. From this solution the following dilutions
in nuclease-free water (Ambion, Lennik, Belgium) are prepared: 2.0; 3.0; 4.0; 5.0; 6.0; 7.0;
8.0; 9.0; 10.0 µg/ml and 200 µl of Bradford substrate (Biorad, Nazareth Eke, Belgium) is
added. The absorbance is measured with a spectrophotometer at 595 nm. The values are
used to prepare a standard curve. To estimate the protein content 10 µl of each supernatant
solution is transferred into a new Eppendorff tube and 790 µl nuclease-free water and 200 µl
Bradford reagent are added. The absorbance at 595 nm is measured in a
spectrophotometer. The results are expressed as relative light units (RLU) per milligram of
protein.
20
MATERIALS AND METHODS
3.6. PREPARATION OF COMPLEXES
3.6.1. Polyplexes
Linear Polyethyleneimine (jetPEITM) is purchased from PolyPlus Transfection (Illkirch,
France). The concentration of the stock solution is 7.5 mM.
6 µl of linPEI is dispersed in 44 µl of sodium chloride solution (150 mM)(PolyPlus
Transfection, Illkirch, France). This 50 µl solution is then added to 50 µl of mRNA solution,
containing 4 µl mRNA (1µg/l) in 46 µl sodium chloride solution. After 10 min. of incubation
at room temperature, 900 µl of OptiMem (Invitrogen, Merelbeke, Belgium) or inhibitor
solution in OptiMem is added. This 1 ml solution is divided among 2 wells (500µl / well) (see
Fig.3.2).
The size of linPEI polyplexes carrying mRNA measured at 37°C is 414±12 nm.
3.6.2. Lipoplexes
A mixture of DOTAP and DOPE (molar ratio 1:1; in chloroform) is purchased from Avanti
Polar Lipids (Alabaster, Alabama, USA). To make DOTAP/DOPE liposomes, 100µl of a
DOTAP/DOPE chloroform solution (10mg/ml) is transferred to a sterile glass flask. A lipid film
is formed on the glass surface by evaporating the solvent under nitrogen atmosphere. The
addition of 1 ml of nuclease-free water in the presence of sterile glass beads is followed by
sonication, yielding DOTAP/DOPE liposomes. The total lipid concentration in these liposome
dispersions is 1mg/ml.
20 µl of this liposome dispersion is dissolved in 30 µl OptiMem. This 50 µl solution is
then added to 50 µl of mRNA solution, containing 4 µl mRNA (1µg/µl) in 46 µl OptiMem.
After 10 min of incubation at room temperature, 900 µl of inhibitor solution in OptiMem is
added. This 1 ml solution is divided among 2 wells (500µl / well) (see Fig.3.3).
21
MATERIALS AND METHODS
The size of DOTAP/DOPE lipoplexes carrying mRNA measured at 37°C is 487±18 nm.
FIGURE 3.2: PREPARATION OF linPEI/mRNA POLYPLEXES
3.7. TOXICITY TEST
3.7.1. Inhibitors
This toxicity test is performed to test the toxicity of an inhibitor on the cells. First, an
inhibitor stock solution is prepared. To prepare inhibitor solutions with different
concentrations, different amounts of the stock solution are dissolved in OptiMem. Finally,
they are filter-sterilized through a 0.22 µm filter. The toxicity of four inhibitors is tested
22
MATERIALS AND METHODS
(methyl-β-cyclodextrin, chlorpromazine, filipin III and genistein; all from Sigma-Aldrich®,
Bornem, Belgium).
FIGURE 3.3: PREPARATION OF DOTAP/DOPE/mRNA LIPOPLEXES.
The experiments are performed in 24-well plates (30 000 cells/well). The cells are
incubated with the different inhibitor solutions for 3h. After incubation, the medium is
removed and fresh regular mMSC culture medium is added. The plates are further incubated
for 24h. Cell viability is evaluated then by the MTT assay. The MTT assay principle has been
described above (see 3.4.). Fig.3.4 demonstrates the scheme of the toxicity test with the
inhibitors.
23
MATERIALS AND METHODS
FIGURE 3.4: TOXICITY TEST WITH INHIBITORS. DIFFERENT INHIBITOR CONCENTRATIONS IN OPTIMEM ARE
ADDED TO mMSCs AND INCUBATED FOR 3h. SUNSEQUENTLY, THE MEDIUM IS REFRESHED. THE CELL
VIABILITY IS EVALUATED BY AN MTT ASSAY 24h LATER (SEE 3.4.).
3.7.2. Polyplexes and lipoplexes
This test starts in the same way as the test described above, that is by preparing the
different inhibitor solutions and adding them to the 24-well plates. The plates are preincubated with an inhibitor for 1h. In the mean time the complexes are made (see 3.6.).
After the incubation, the complex solutions are added to the cells (see also 3.6.) and
incubated for 2h. Finally, the medium is removed and fresh regular mMSC culture medium is
added. After 24h of incubation, the cell viability after transfection is evaluated by an MTT
assay. (see Fig.3.5 and Fig.3.6).
3.8. TRANSFECTION
A similar procedure as the toxicity test with polyplexes and lipoplexes is applied, except
that after 24h of incubation, the transfection efficiency is tested instead of the cell viability.
The transfection efficiency is evaluated by the luciferase assay. Fig.3.5 and Fig.3.6 represent
24
MATERIALS AND METHODS
schemes for the performance of the toxicity test after transfection and the transfection test
with linPEI polyplexes and DOTAP/DOPE lipoplexes, respectively.
FIGURE 3.5: TOXICITY TEST AFTER TRANSFECTION AND TRANSFECTION TEST WITH linPEI POLYPLEXES.
FIRST, mMSCs ARE INCUBATED FOR 1h WITH DIFFERENT INHIBITOR CONCENTRATIONS. IN THE MEAN
TIME, THE POLYPLEXES ARE MADE. AFTER THE INCUBATION, THE COMPLEXES ARE ADDED TO THE CELLS
AND INCUBATED FOR 2h. SUBSEQUENTLY, THE MEDIUM IS REFRESHED. AFTER 24h, AN MTT ASSAY IS
CARRIED OUT TO MEASURE THE TOXICITY AFTER TRANSFECTION. TO EVLUATE TRANSFECTION EFFICIENCY,
A LUCIFERASE ASSAY IS CARRIED OUT.
25
MATERIALS AND METHODS
FIGURE 3.6: TOXICITY TEST AFTER TRANSFECTION AND TRANSFECTION TEST WITH DOTAP/DOPE
LIPOPLEXES. FIRST, mMSCs ARE INCUBATED FOR 1h WITH DIFFERENT INHIBITOR CONCENTRATIONS. IN THE
MEAN TIME, THE LIPOPLEXES ARE MADE. AFTER THE INCUBATION, THE COMPLEX SOLUTIONS ARE ADDED
TO THE CELLS AND INCUBATED FOR 2h. SUBSEQUENTLY, THE MEDIUM IS REFRESHED. AFTER 24h, AN MTT
ASSAY IS CARRIED OUT TO MEASURE THE TOXICITY AFTER TRANSFECTION. TO EVLUATE TRANSFECTION
EFFIECINCY, A LUCIFERASE ASSAY IS CARRIED OUT.
26
RESULTS
4.
RESULTS
4.1. TOXICITY TEST
4.1.1. Inhibitors
To establish an optimal protocol for the use of endocytosis inhibitors for murine
mesenchymal stem cells, it is imperative to evaluate their cellular toxicity. The toxicity of
four inhibitors was tested: methyl-β-cyclodextrin, chlorpromazine, filipin and genistein.
4.1.1.1. Methyl-β-cyclodextrin
The toxicity of methyl-β-cyclodextrin was tested for three inhibitor concentrations: 0.5,
1.0 and 1.5 mg/ml. The results are shown in Fig.4.1.
cell viability (%)
120
100
80
60
40
20
0
0,5
1,0
1,5
Methyl-β-cyclodextrin concentration (mg/ml)
FIGURE 4.1: CELL VIABILITY OF mMSCs AFTER INCUBATION WITH DIFFERENT CONCENTRATIONS OF
METHYL-β-CYCLODEXTRIN. CELL VIABILITY WAS DETERMINED BY AN MTT ASSAY (SEE 3.4.). RESULTS ARE
EXPRESSED AS PERCENTAGES OF THE VIABILITY OF UNTREATED CELLS (100%). GRAPHS REPRESENT MEANS
± SD; n ≥6.
The results show no significant toxicity for any of the three inhibitor concentrations
tested.
27
RESULTS
4.1.1.2. Chlorpromazine
The cell toxicity of chlorpromazine was tested for six concentrations: 2.5, 5.0, 7.5, 10.0,
12.5 and 15.0 µg/ml. The results of the test are shown in Fig.4.2.
cell viability (%)
120
100
80
60
40
20
0
2,5
5,0
7,5
10,0
12,5
15,0
Chlorpromazine concentration (µg/ml)
FIGURE 4.2: CELL VIABILITY OF mMSCs AFTER INCUBATION WITH DIFFERENT CONCENTRATIONS OF
CHLORPROMAZINE. CELL VIABILITY WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS
PERCENTAGES OF THE VIABILITY OF UNTREATED CELLS (100%). GRAPHS REPRESENT MEANS ± SD; n ≥3.
The data demonstrate that at concentrations between 2.5 and 10.0 µg/ml the inhibitor
was not toxic to mMSCs. The cell viability of the cells treated with the inhibitor at a
concentration of 12.5 µg/ml was diminished by about 20%. A higher degree of toxicity
(about 60%) was observed for a concentration of 15.0 µg/ml.
4.1.1.3. Filipin
Fig.4.3 represents the viability of mMSCs treated with filipin at concentrations of 0.20,
0.30, 0.40, 0.50, 0.60 and 0.70 µg/ml.
No toxicity to mMSCs was observed for inhibitor concentrations between 0.20 and 0.50
µg/ml. Toxicity of more than 20% was observed for a concentration of 0.60 µg/ml. The
inhibitor concentration of 0.70 µg/ml decreased cell viability by 50%.
28
RESULTS
cell viability (%)
120
100
80
60
40
20
0
0,20
0,30
0,40
0,50
0,60
0,70
Filipin concentration (µg/ml)
FIGURE 4.3: CELL VIABILITY OF mMSCs AFTER INCUBATION WITH DIFFERENT CONCENTRATIONS OF
FILIPIN. CELL VIABILITY WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS PERCENTAGES
OF THE VIABILITY OF UNTREATED CELLS (100%). GRAPHS REPRESENT MEANS ± SD; n ≥ 3.
4.1.1.4. Genistein
The toxicity of genistein was tested for six inhibitor concentrations: 100, 200, 300, 400,
500 and 600 µM. The results are shown in Fig.4.4.
cell viability (%)
120
100
80
60
40
20
0
100
200
300
400
500
600
Genistein concentration (µM)
FIGURE 4.4: CELL VIABILITY OF mMSCs AFTER INCUBATION WITH DIFFERENT CONCENTRATIONS OF
GENISTEIN. CELL VIABILITY WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS
PERCENTAGES OF THE VIABILITY OF UNTREATED CELLS (100%). GRAPHS REPRESENT MEANS ± SD; n ≥ 6.
No toxicity was observed for any of the inhibitor concentrations tested.
29
RESULTS
Conclusions - toxicity of the inhibitors:

The tested concentrations of methyl-β-cyclodextrin were not toxic to mMSCs.

Chlorpromazine was toxic to mMSCs at concentrations ≥ 12.5 µg/ml.

Filipin was toxic to mMSCs at concentrations ≥ of 0.6 µg/ml.

The tested concentrations of genistein were not toxic to mMSCs.
4.1.2. Polyplexes: linPEI
The toxicity of the inhibitors in the presence of polyplexes, containing linPEI and mRNA,
was tested on mMSCs. The polyplexes were prepared as described in 3.6.1. The toxicity of
the complexes as such was determined first. Polyplexes alone were mildly toxic to mMSCs
(0-20%). The three or four highest concentrations which were not toxic to mMSCs (see
4.1.1.) were used in the toxicity tests with complexes and the transfection tests.
4.1.2.1. Methyl-β-cyclodextrin
The toxicity of the linPEI polyplexes in the presence of three concentrations of methyl-βcyclodextrin (0.5; 1.0; 1.5 mg/ml) was tested.
cell viability (%)
120
100
80
60
40
20
0
0,5
1,0
1,5
Methyl-β-cyclodextrin concentration (mg/ml)
FIGURE 4.5: TOXICITY OF METHYL-β-CYCLODEXTRIN IN THE PRESENCE OF linPEI/mRNA COMPLEXES.
VIABILTY OF mMSCs WAS DETERMINED BY AN MTT ASSAY (SEE 3.4.). RESULTS ARE EXPRESSED AS A
PERCENTAGE OF THE VIABILITY OF CELLS INCUBATED WITH THE POLYPLEXES ALONE (100%). GRAPHS
REPRESENT MEANS ± SD; n ≥ 8.
30
RESULTS
The data shown in Fig.4.5 demonstrate that there was no toxicity observed for mMSCs
treated with 0.5 and 1.0 mg/ml of inhibitor solutions in the presence of linPEI polyplexes.
Mild toxicity (25%) was determined for a concentration of 1.5 mg/ml.
4.1.2.2. Chlorpromazine
Toxicity of four chlorpromazine concentrations (2.5, 5.0, 7.5 and 10.0 µg/ml) in the
presence of linPEI polyplexes was tested on mMSCs.
120
% cell viability
100
80
60
40
20
0
2,5
5,0
7,5
10,0
Chlorpromazine concentration (µg/ml)
FIGURE 4.6: TOXICITY OF CHLORPROMAZINE IN THE PRESENCE OF linPEI/mRNA COMPLEXES. VIABILTY
OF mMSCs WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS A PERCENTAGE OF THE
VIABILITY OF CELLS INCUBATED WITH THE POLYPLEXES ALONE (100%). GRAPHS REPRESENT MEANS ± SD; n
≥ 4.
Chlorpromazine at a concentration of 2.5 µg/ml, incubated with mMSCs in the presence
of linPEI lipoplexes, proved to be non toxic. At concentrations 5.0 and 7.5 µg/ml, the cell
viability was decreased by about 20%. The toxicity increased to 70% for chlorpromazine at a
concentration of 10 µg/ml (Fig.4.6).
4.1.2.3. Filipin
The toxicity of the polyplexes in the presence of four concentrations of filipin (0.3; 0.4;
0.5 and 0.6 µg/ml) was tested.
31
RESULTS
cell viability (%)
120
100
80
60
40
20
0
0,30
0,40
0,50
0,60
Filipin concentration (µg/ml)
FIGURE 4.7: TOXICITY OF FILIPIN IN THE PRESENCE OF linPEI/mRNA COMPLEXES. VIABILTY OF mMSCs
WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS A PERCENTAGE OF THE VIABILITY OF
CELLS INCUBATED WITH THE POLYPLEXES ALONE (100%). GRAPHS REPRESENT MEANS ± SD; n ≥ 6.
None of the inhibitor concentrations tested showed any toxicity to mMSCs in the
presence of polyplexes (Fig.4.7).
4.1.2.4. Genistein
Toxicity of four genistein concentrations (300, 400, 500 and 600 µM) in the presence of
linPEI polyplexes was tested on mMSCs.
cell viability (%)
120
100
80
60
40
20
0
300
400
500
600
Genistein concentration (µM)
FIGURE 4.8: TOXICITY OF GENISTEIN IN THE PRESENCE OF linPEI/mRNA COMPLEXES. VIABILTY OF mMSCs
WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS A PERCENTAGE OF THE CELL VIABILITY
OF CELLS INCUBATED WITH THE POLYPLEXES ALONE (100%). GRAPHS REPRESENT MEANS ± SD; n ≥ 6.
32
RESULTS
As shown in Fig.4.8, genistein at a concentration ≤ 400 µM, incubated in the presence of
polyplexes, showed no toxic effects to mMSCs. Under the same conditions, the higher
concentrations of the inhibitor (500-600 µM) caused mild toxicity to the cells (about 10%).
Conclusions - toxicity of linPEI polyplexes:

Methyl-β-cyclodextrin, incubated with mMSCs in the presence of linPEI polyplexes,
was toxic to the cells at a concentration of 1.5 mg/ml.

Chlorpromazine, incubated with mMSCs in the presence of linPEI polyplexes, was
mildly toxic to the cells at concentrations 5.0 µg/ml and 7.5 µg/ml. Robust toxicity
was observed for the highest concentration of chlorpromazine (10.0 µg/ml).

Filipin, incubated with mMSCs in the presence of linPEI polyplexes, was not toxic to
the cells at any concentration tested.

Genistein, incubated with mMSCs in the presence of linPEI polyplexes, was mildly
toxic to the cells at concentrations ≥ of 500 µM.
4.1.3. Lipoplexes : DOTAP/DOPE
The same tests as described earlier for linPEI polyplexes were performed for lipoplexes
containing DOTAP/DOPE and mRNA. The lipoplexes were prepared as described in 3.6.2. The
toxicity of these complexes was determined first. The lipoplexes alone were mildly toxic to
mMSCs (10-30%).
4.1.3.1. Methyl-β-cyclodextrin
The toxicity of the lipoplexes in the presence of three methyl-β-cyclodextrin
concentrations was tested: 0.5, 1.0 and 1.5 mg/ml.
33
RESULTS
cell viability (%)
120
100
80
60
40
20
0
0,5
1,0
1,5
Methyl-β-cyclodextrin concentration (mg/ml)
FIGURE 4.9: TOXICITY OF METHYL-β-CYCLODEXTRIN IN THE PRESENCE OF DOTAP-DOPE/mRNA
COMPLEXES. VIABILTY OF mMSCs WAS DETERMINED BY AN MTT ASSAY (SEE 3.4.). RESULTS ARE
EXPRESSED AS A PERCENTAGE OF THE VIABILITY OF CELLS INCUBATED WITH THE LIPOPLEXES ALONE
(100%). GRAPHS REPRESENT MEANS ± SD; n ≥ 8.
As shown in Fig.4.9., methyl-β-cyclodextrin incubated with mMSCs in the presence of
DOTAP/DOPE lipoplexes showed no toxicity to the cells at any concentration tested.
4.1.3.2. Chlorpromazine
The toxicity of chlorpromazine in the presence of DOTAP/DOPE lipoplexes, was tested
for four inhibitor concentrations: 2.5, 5.0, 7.5 and 10.0 µg/ml.
120
cell viability (%)
100
80
60
40
20
0
2,5
5,0
7,5
10,0
Chlorpromazine concentration (µg/ml)
FIGURE 4.10: TOXICITY OF CHLORPROMZINE IN THE PRESENCE OF DOTAP-DOPE/mRNA COMPLEXES.
VIABILTY OF mMSCs WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS A PERCENTAGE OF
THE VIABILITY OF CELLS INCUBATED WITH THE LIPOPLEXES ALONE (100%). GRAPHS REPRESENT MEANS ±
SD; n ≥ 4.
34
RESULTS
Chlorpromazine at a concentration of 2.5 µg/ml, incubated with mMSCs in the presence
of DOTAP/DOPE lipoplexes, proved to be non toxic.
At concentrations of 5.0 and 7.5 µg/ml, the cell viability was decreased by about 20%.
The toxicity increased to 30% for chlorpromazine at a concentration of 10.0 µg/ml (Fig.4.10).
4.1.3.3. Filipin
The cell viability was also determined after treatment of mMSCs with filipin and
DOTAP/DOPE lipoplexes. Four concentrations of the inhibitor were tested (0.30, 0.40, 0.50
cell viability (%)
and 0.60 µg/ml).
140
120
100
80
60
40
20
0
0,30
0,40
0,50
0,60
Filipin concentration (µg/ml)
FIGURE 4.11: TOXICITY OF FILIPIN IN THE PRESENCE OF DOTAP-DOPE/mRNA COMPLEXES. VIABILTY OF
mMSCs WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS A PERCENTAGE OF THE
VIABILITY OF CELLS INCUBATED WITH THE LIPOPLEXES ALONE (100%). GRAPHS REPRESENT MEANS ± SD; n
≥ 6.
As shown in Fig.4.11 no toxicity was observed for mMSCs incubated with DOTAP/DOPE
lipoplexes and filipin at a concentration ≤ 0.40 µg/ml. Incubation of the cells with lipoplexes
and filipin at a concentration of 0.50 µg/ml reduced cell viability by 15%. The highest
concentration of filipin tested decreased cell viability by 25%.
35
RESULTS
4.1.3.4. Genistein
The cell viability determined after treatment of mMSCs with genistein and DOTAP/DOPE
lipoplexes is shown in Fig.4.12. Four concentrations of the inhibitor were tested (300, 400,
500 and 600 µM).
cell viability (%)
100
80
60
40
20
0
300
400
500
600
Genistein concentration (µM)
FIGURE 4.12: TOXICITY OF GENISTEIN IN THE PRESENCE OF DOTAP-DOPE/mRNA COMPLEXES. VIABILTY
OF mMSCs WAS DETERMINED BY AN MTT ASSAY. RESULTS ARE EXPRESSED AS A PERCENTAGE OF THE
VIABILITY OF CELLS INCUBATED WITH THE LIPOPLEXES ALONE (100%). GRAPHS REPRESENT MEANS ± SD; n
≥ 10.
As shown in Fig.4.12, at a concentration of 300 µM a decrease of 30% in cell viability was
observed. Toxicity further increased to approximately 50%, 60%, 70% for 400, 500, 600 µM
of genistein, respectively.
Conclusions - toxicity of DOTAP/DOPE:

Methyl-β-cyclodextrin, incubated with mMSCs in the presence DOTAP/DOPE
lipoplexes, was not toxic to the cells.

Chlorpromazine, incubated with mMSCs in the presence of DOTAP/DOPE lipoplexes,
was mildly toxic to the cells at concentrations ≥ 5.0 µg/ml.

Filipin, incubated with mMSCs in the presence of DOTAP/DOPE lipoplexes, was
slightly toxic to the cells at ≥ 0.5 µg/ml.

Genistein, incubated with mMSCs in the presence of DOTAP/DOPE lipoplexes, was
toxic to the cells at all concentrations tested.
36
RESULTS
4.2. TRANSFECTION
After having established the effects of different endocytosis inhibitors on the viability of
MSCs, the effects of these compounds on cellular transfection mediated by lipo- and
polyplexes were investigated. To this end, transfection efficiency of two different carriers in
the presence of inhibitors blocking different endocytic pathways was tested. The
experimental setup is described in section 3.8. Luciferase activity was normalized with
respect to amount of protein and expressed as relative light units per mg of protein (RLU/mg
protein). The final results are presented as percent of transfection efficiency relative to
control cells treated with complexes in absence of inhibitor (100% transfection efficiency).
4.2.1. Polyplexes : linPEI
The polyplexes were made as described in 3.6.1. The transfection tests were performed
in presence of one of the four inhibitors.
4.2.1.1. Methyl-β-cyclodextrin
The transfection efficiency of mRNA complexed with linPEI was tested in mMSCs in the
presence of three concentrations of methyl-β-cyclodextrin: 0.5, 1.0 and 1.5 mg/ml.
The transfection efficiency of linPEI polyplexes was strongly reduced by methyl-βcyclodextrin. A reduction of about 45 and 55% was observed for concentrations of 0.5 and
1.0 mg/ml, respectively. At a concentration of 1.5 mg/ml, the transfection efficiency was
reduced by 80% (Fig.4.13).
37
Transfection efficiency (%)
RESULTS
120
100
80
60
40
20
0
0,0
0,5
1,0
1,5
Methyl-β-cyclodextrin concentration (mg/ml)
FIGURE 4.13: TRANSFECTION MEDIATED BY linPEI POLYPLEXES IN THE PRESENCE OF METHYL-βCYCLODEXTRIN. mMSCs, PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE POLYPLEXES
AND SCREENED FOR LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE
ASSAY (SEE 3.5.) THE LEVEL OF LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N= 4; * = p<0.01 ; ** =
p<0.001).
4.2.1.2. Chlorpromazine
The transfection efficiency of mRNA complexed with linPEI was evaluated in the
Transfection efficiency (%)
presence of four chlorpromazine concentrations: 2.5, 5.0, 7.5 and 10.0 µg/ml.
140
120
100
80
60
40
20
0
0,0
2,5
5,0
7,5
10,0
Chlorpromazine concentration (µg/ml)
FIGURE 4.14: TRANSFECTION MEDIATED BY linPEI POLYPLEXES IN THE PRESENCE OF CHLORPROMAZINE.
mMSCs, PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE POLYPLEXES AND SCREENED
FOR LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE ASSAY. THE
LEVEL OF LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N ≥ 6; * = p<0.01 ; ** = p<0.001).
38
RESULTS
At a concentration of 2.5 µg/ml, no decrease in transfection efficiency was observed.
Upon further increase of the inhibitor concentration, transfection efficiency decreased from
85% at 5.0 µg/ml to about 15% at 10.0 µg/ml (Fig.4.14).
4.2.1.3. Filipin
The inhibitory effect of filipin on transfection efficiency of mRNA complexes with linPEI
was evaluated after treatment of mMSCs with three inhibitor concentrations: 0.3; 0.4 and
Transfection efficiency (%)
0.5 µg/ml.
140
120
100
80
60
40
20
0
0,0
0,3
0,4
0,5
Filipin concentration (µg/ml)
FIGURE 4.15: TRANSFECTION MEDIATED BY linPEI POLYPLEXES IN THE PRESENCE OF FILIPIN. mMSCs,
PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE POLYPLEXES AND SCREENED FOR
LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE ASSAY. THE LEVEL OF
LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N ≥ 4; * = p<0.01 ; ** = p<0.001).
Inhibition by filipin steadily increased from 30% to 50% with the inhibitor concentration
increasing from 0.3 to 0.5 µg/ml (Fig.4.15).
4.2.1.4. Genistein
The transfection efficiency of mRNA complexed with linPEI was tested in the presence of
genistein for four inhibitor concentrations: 300, 400, 500 and 600 µM.
39
RESULTS
Transfection efficiency (%)
140
120
100
80
60
40
20
0
0
300
400
500
600
Genistein concentration (µM)
FIGURE 4.16: TRANSFECTION MEDIATED BY linPEI POLYPLEXES IN THE PRESENCE OF GENISTEIN. mMSCs,
PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE POLYPLEXES AND SCREENED FOR
LUCIFERASE. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE ASSAY. THE LEVEL OF
LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N= 4; * = p<0.01 ; ** = p<0.001).
The transfection efficiency was strongly reduced by genistein. At inhibitor
concentrations between 300 and 600, transfection efficiency decreased from about 40 to 4%
(Fig.4.16).
Conclusions - transfection with linPEI polyplexes:

Methyl-β-cyclodextrin strongly reduced transfection at concentrations ≥ 0.5 mg/ml.

Chlorpromazine strongly reduced transfection at concentrations ≥ 7.5 µg/ml.

Filipin significantly reduced transfection at concentrations ≥ 0.5 µg/ml.

Transfection is strongly reduced by all genistein concentrations tested.
4.2.2. Lipoplexes : DOTAP/DOPE
The lipoplexes were made as described in 3.6.2. The transfection tests were performed
in presence of one of the four inhibitors.
40
RESULTS
4.2.2.1. Methyl-β-cyclodextrin
The transfection efficiency of mRNA complexed with DOTAP/DOPE lipoplexes was tested
in the presence of three concentrations of methyl-β-cyclodextrin (0.5, 1.0 and 1.5 mg/ml)
Transfection efficiency (%)
with mMSCs as target cells.
160
140
120
100
80
60
40
20
0
0,0
0,5
1,0
1,5
Methyl-β-cyclodextrin concentration (mg/ml)
FIGURE 4.17: TRANSFECTION MEDIATED BY DOTAP/DOPE LIPOPLEXES IN THE PRESENCE OF METHYL-βCYCLODEXTRIN. mMSCs, PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE LIPOPLEXES
AND SCREENED FOR LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE
ASSAY (SEE 3.5). THE LEVEL OF LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N = 8; * = p<0.01 ; ** =
p<0.001).
At a concentration of 0.5 mg/ml, no decrease in transfection efficiency was observed.
Concentrations 1.0 and 1.5 mg/ml reduced transfection efficiency by about 40 and 70 %,
respectively (Fig.4.17).
4.2.2.2. Chlorpromazine
The effect of chlorpromazine on transfection efficiency of mRNA complexed with
DOTAP/DOPE lipoplexes was studied with four inhibitor concentrations: 2.5, 5.0, 7.5 and
10.0 µg/ml.
41
Transfection efficiency (%)
RESULTS
140
120
100
80
60
40
20
0
0,0
2,5
5,0
7,5
10,0
Chlorpromazine concentration (µg/ml)
FIGURE 4.18: TRANSFECTION MEDIATED BY DOTAP/DOPE LIPOPLEXES IN THE PRESENCE OF
CHLORPROMAZINE. mMSCs, PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE
LIPOPLEXES AND SCREENED FOR LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A
LUCIFERASE ASSAY. THE LEVEL OF LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N = 8; * = p<0.01 ;
** = p<0.001).
At a concentration of 2.5 µg/ml, no decrease in transfection efficiency was observed.
Upon further increase of the inhibitor concentration, transfection efficiency decreased from
60% at 5.0 µg/ml to about 10% at 10.0 µg/ml (Fig.4.18).
4.2.2.3. Filipin
The inhibitory effect of filipin on transfection efficiency of mRNA complexes with
DOTAP/DOPE was evaluated after treatment of mMSCs with four inhibitor concentrations:
0.3; 0.4; 0.5 and 0.6 µg/ml.
At a concentration of 0.3 mg/ml, no decrease in transfection efficiency was observed. A
reduction of about 15% was observed for a concentration of 0.4 µg/ml. At inhibitor
concentrations 0.5 and 0.6 µg/ml, transfection efficiency was decreased by 80 and 95%,
respectively (Fig.4.19).
42
Transfection efficiency (%)
RESULTS
140
120
100
80
60
40
20
0
0,0
0,3
0,4
0,5
0,6
Filipin concentration (µg/ml)
FIGURE 4.19: TRANSFECTION MEDIATED BY DOTAP/DOPE LIPOPLEXES IN THE PRESENCE OF FILIPIN.
mMSCs, PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE LIPOPLEXES AND SCREENED
FOR LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE ASSAY. THE
LEVEL OF LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N = 4; * = p<0.01 ; ** = p<0.001).
4.2.2.4. Genistein
The effect of genistein on transfection efficiency of mRNA complexed with DOTAP/DOPE
lipoplexes was evaluated in the presence of four inhibitor concentrations: 300, 400, 500 and
Transfection efficiency (%)
600 µM.
140
120
100
80
60
40
20
0
0
300
400
500
600
Genistein concentration (µM)
FIGURE 4.20: . TRANSFECTION MEDIATED BY DOTAP/DOPE LIPOPLEXES IN THE PRESENCE OF GENISTEIN.
mMSCs, PRETREATED WITH THE INHIBITOR, WERE TRANSFECTED WITH THE LIPOPLEXES AND SCREENED
FOR LUCIFERASE ACTIVITY. TRANSFECTION EFFICIENCY WAS MEASURED BY A LUCIFERASE ASSAY. THE
LEVEL OF LUCIFERASE IN CONTROL CELLS WAS SET AS 100%, (N = 7; * = p<0.01 ; ** = p<0.001).
43
RESULTS
The transfection efficiency was reduced by 60 % at a concentration of 300 µM to as
much as 80 % at a concentration of 600 µM (Fig.4.20).
Conclusions - transfection with DOTAP/DOPE lipoplexes:

MbCD reduced transfection efficiency at concentrations ≥1.0 mg/ml.

Chlorpromazine strongly reduced transfection efficiency at concentrations ≥ 7.5
µg/ml.

Filipin strongly reduced transfection efficiency at concentrations ≥ 0.5 µg/ml.

Transfection efficiency was strongly reduced by genistein at all inhibitor
concentrations tested.
44
DISCUSSION
5.
DISCUSSION
The main objective of this project was to establish if there is any difference in the
mechanism of uptake of DOTAP lipoplexes and linPEI polyplexes, carrying mRNA, that might
determine their transfection efficiency.
The data demonstrate that transfection mediated by linPEI polyplexes carrying mRNA is
strongly reduced by inhibition of the caveolar pathway with genistein and to lower extent
with filipin. Also chlorpromazine, an inhibitor of clathrin-mediated endocytosis, strongly
affected transfection mediated by these polyplexes.
Interestingly, these data are different from those earlier published for pDNA. Earlier
studies on A549 pneumocytes and HeLa cells provided evidence that even though PEI/DNA
polyplexes are internalized by both clathrin-mediated and caveolae-mediated endocytosis,
only the latter leads to effective transfection (Rejman et al., 2005; Gabrielson, 2009).
More recent studies in HUH-7, COS-7 and HeLa cells confirmed that PEI/DNA polyplexes
are internalized by both clathrin- and caveolae-dependent pathways (von Gersdorff et al.,
2006). After studying transfection efficiency in HUH-7 cells, von Gersdorff and colleagues
suggested, in contrast to Rejman et al., that not only caveolar uptake but also clathrindependent uptake of PEI/DNA polyplexes contribute to transfection. Although the authors
did not take this into consideration, this observation was likely due to the fact that these
cells, like HepG2 cells, lack endogenous caveolins (Fujimoto et al., 2000). With HeLa cells,
which do possess caveolins, the findings of Rejman and colleagues were confirmed by von
Gersdorff et al.
The data for DOTAP/DOPE lipoplexes carrying mRNA demonstrate that transfection is
strongly reduced by inhibition of the caveolar pathway with genistein and to a lesser extent
with filipin. Also chlorpromazine, an inhibitor of the clathrin-mediated endocytosis pathway,
reduced transfection.
45
DISCUSSION
Earlier studies performed on different cell lines with pDNA lipoplexes demonstrated that
clathrin-mediated endocytosis rather than caveolae-mediated endocytosis is the major
uptake pathway leading to transfection (Zuhorn et al., 2007). More recent studies on A549
pneumocytes and HeLa cells provided strong evidence that DOTAP/DNA complexes
exclusively internalized by clathrin-mediated endocytosis are fully transfection effective
(Rejman et al., 2005).
46
CONCLUSION
6.
GENERAL CONCLUSIONS
Transfection of mMSCs by both linPEI and DOTAP/DOPE was affected by the caveolae
inhibitors as well as the clathrin inhibitors. Moreover, the results presented in this thesis
indicate that the mechanism of lipo- and polyplex internalization and their further
intracellular processing depend not only on the carrier but on the nucleic acid as well.
47
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