Chapter 4
Evaluation of digitally encoded
layer-by-layer coated
microparticles for reverse
transfection
Keywords: reverse transfection, microarrays, gene deliver, encoded microcarriers, LbL
assembly
0
Abstract
‘Reverse transfection’ of DNA or siRNA or the ‘reverse infection’ by viral vectors
has been proposed as a useful tool for simultaneous high-throughput analysis of the
function of many different genes on a solid surface. Recently, ‘reverse transfection’ has
been used to create transfected cell arrays. The aim of the present work is to introduce
encoded microcarriers, coated with polyelectrolyte multilayers as a delivery platform for
immobilized nucleic acids or (non-)viral complexes and to evaluate their possible use as
microarray (system).
Previously, we described that our encoded microcarriers are suitable to grow cells on
(ref). Additionally, we showed that the coating layers together with the cell layer do not
hamper the decoding of the beads. We observed that the code inside the beads remained
detectable even when the cells covering the microcarriers exhibited green or red
fluorescence due to the expression of GFP and RFP respectively.
Subsequently, these encoded microcarriers can be used as a cell-based microarray
loaded with cells keeping the digital code in the microcarriers readable. The surfaces of
the encoded microcarriers with polyelectrolytes are suitable for immobilizing DNA,
siRNA or adenovirus and subsequently growing cells on top of them. It has further been
shown that the cells growing on the polyelectrolyte layer can become transduced with
adenoviral particles hosted by the polyelectrolyte layer. In conclusion, these digitally
encoded microparticles can be used as transfection microarray for screening different
carriers for gene delivery.
1
1. Introduction
Gene therapy involves the insertion of corrective genetic material into cells and
tissues to treat diseases of genetic origin. These genetic based diseases can be treated by
adding the DNA that encodes the absent protein or by using the RNA interference
(RNAi) mechanism to inhibit a specific protein (ref 1"). To be effective, these nucleic
acids should be delivered to a well defined compartment of the target cells. The
development of non-toxic and highly efficient delivery system currently is the greatest
challenge in these therapies (ref 1`). In general, two types of delivery vehicles can be
distinguished, namely viral and non-viral vectors.
Viral vector such as retroviruses and adenoviruses are still the most efficient gene
delivery vehicles because of their capacity to carry and efficiently deliver foreign genes
(ref 2, 2'). However, there are serious concerns about the clinical application of these
vectors, because of risk of insertional mutagenesis, immunogenicity and cytotoxicity (ref
3). Hence, non-viral vectors have attracted a great deal of interest for safe delivery of
therapeutic nucleic acids into target cells (ref B, 11). Although non-viral vectors are
advantageous for their safety profile, the clinical applicability of them is still limited due
to their poor in vivo efficacy (73, 74).
Currently, non-viral gene delivery mostly involves the complexation of nucleic acids
with cationic polymers or lipids (ref B,C). To overcome the various encountered barriers
before the nucleic acids reach their intracellular target region, numerous cationic lipids
and polymers have been developed to package DNA. In vitro studies typically employ
bolus addition of nucleic acids, complexed or not, but this kind of delivery can be limited
2
by degradation or aggregation of nucleic acids (ref). Recently, immobilizing DNA onto
the surface of a culture substrate prior to cell seeding has been proposed. This method is
termed solid-phase delivery (7), reverse transfection (8) or substrate mediated delivery
(9.10) and is an alternative delivery system to improve transfection efficiency. Similarly,
also viral vectors are mostly used free in solution. Alternatively, immobilizing viral
vectors on a solid surface can result in transduction of target cells (ref).
In general, solid-phase delivery is based on two different approaches: immobilization
on the one hand and sustained release on the other. By immobilizing nucleic acids to the
cell culture substrate prior to cell seeding, DNA maintains in the cell microenvironment
for subsequent cellular internalization. This strategy is similar to the strategy used by
some viruses to infect target cells as they often first associate with cells to enhance their
transfection efficiency (ref virus 9,10).
Different strategies were developed to immobilize DNA on the substrate such as
gelatin entrapment (8), specific binding of DNA complexes to substrate through the
biotin-avidin interaction (9,10,10'), or non specific adsorption (10'', 10''').
Additionally to immobilization, creating a sustained release system offers the
potential to enhance gene transfer by increasing DNA concentrations in the cellular
microenvironment (Ref. Luo). The ‘Layer-by-Layer’ (LbL) coating which is composed of
polyelectrolytes (PEs) has opened up new opportunities for binding naked or precomplexed DNA to a solid surface. Multilayer films of naked pDNA and degradable
polyamine have shown to be efficient in the release of pDNA (ref 12,13). Jessel N. et al
proposed LbL films made from poly(L-glutamic acid) (PLGA) and poly(L-lysin) (PLL)
3
containing DNA and cationic cyclodextrins. This type of immobilized DNA offers the
possibility for delivery of different DNA molecules aimed at cell transfection (ref 14). In
the latter approach, poly electrolyte multilayer films are used as a carrier for polymerprecomplexed DNA (ref 15,16). Also viral vectors can be immobilized on solid surfaces
by embedding in multilayered polyelectrolyte films (viral ref 1 ), a specific binding of
viral vectors to substrate such as streptavidin and biotin (viral ref 2,3) or non specific
absorption (viral ref 4).
‘Reverse transfection’ of nucleic acids (in naked form or complexed with (non-)viral
carrier) provides a mode of transfection, useful in the development of research tools such
as cell-based arrays. Ziauddin and Sabatini have recently described a cell based
microarray system for identifying the cellular functions of gene products based on reverse
transfection (8). Such microarrays have features (spots) that are clusters of mammalian
cells expressing defined DNA constructs, which direct the increase or decrease in the
function of specific gene products. The results from array techniques show high
correlation with the transfection efficiency measured by a traditional assay. The
advantage of this method is its multiplex capacity which allows the screening of large
libraries on a broad range of cells.
Additionally, the immobilization of viruses on a solid surface could be used for other
in vitro applications, such as the virus arrays (13,14 virus). The recombinant viral vector
library (viral 17, 18) can be arrayed by spotting on a solid surface. The target cells are
plated on top of these arrays. This should result in an array of cells, expressing the
recombinant genes of interest. Therefore expression and functional analysis of many
4
different genes can be performed with a virus array. This delivery system of virus also
offers a highly site-specific delivery for viral particles to target tissues or cells.
Similarly, the reverse transfection approach also was used to knock down genes.
Mousses et al. arrayed 100 to 700 μm spots of synthetic small interfering RNAs
(siRNAs) on glass slides for parallel knockdown of different genes (ref Mosses).
In this chapter, we aimed to develop a non-positional transfection microarray to be
able to simultaneously screen different candidates for gene delivery on different cell
types. Here we propose to evaluate a) whether our LbL coated encoded beads are suitable
to be used as a platform for immobilizing viral vectors or naked or precomplexed nucleic
acids and b) whether adenoviral particles and nucleic acids immobilized on LbL coating
are able to selectively transfect the cells that grow on top of the coated microcarriers.
5
2. Materials and Methods
Materials
Non-magnetic fluorescent carboxylated microspheres (CFP-40052-100, ø = 39
µm) were purchased from Spherotech (Libertyville, Illinois, USA). Poly (allylamine
hydrochloride) (PAH; 70 kDa) and sodium poly (styrene sulfonate) (PSS; 70 kDa)
were
obtained
from
Sigma
Aldrich
(Steinheim,
Germany).
4,4’-
Trimethylenedipiperidine and anhydrous dichloromethane (CH2Cl2), were purchased
from Sigma-Aldrich (Bornem, Belgium). 1,4-Butanediol diacrylate was purchased
from
Alfa
Aesar
Organics
(Karlsruhe,
Germany).
1,2-Dioleolyl-3-
trimethylammoniumpropane (chloride salt) (DOTAP), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (AL, USA).
Linear poly (ethylenimine) (LPEI, MW 22 KDa) was a kind gift from Prof. E.
Wagner (Ludwig Maximilian University, Munich, Germany).
The recombinant adenoviral vectors Ad-RFP and Ad-GFP, which express the red
fluorescent protein (RFP) and green fluorescent protein (GFP) respectively, were
obtained from Vector Biolabs (Philadelphia, USA). CHO-320 cells were a kind gift
from Prof. Y.J. Schneider (Catholic University of Louvain). Vero-1 and CHO cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
Merelbeke, Belgium) containing 2 mM L-glutamine (L-Gln), 10% heat inactivated
fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). HuH-7_eGFPLuc
cells stably expressing eGFP-Luciferase were generated by transfecting HuH-7 cells
with the vector pEGFPLuc (Clontech, Palo Alto, USA) as previously described
41
.
DsRed2-C1 pDNA was obtained from Promega (Leiden, The Netherlands)\, amplified
in Escherichia coli and purified by the Qiagen Plasmid Mega Kit (Venlo, The
Netherlands). TOTO-3 was purchased from Invitrogen for labeling the pDNA.
6
siRNA against EGFP and Dsred, negative siRNA control and Cy5 labeled siRNA
were purchased from Dharmacon (Chicago, USA). All siRNAs were purchased in
their annealed form, dissolved in RNase free water at the concentration of 20μM,
aliquotized and stored at -80°C.
1. Layer-by-Layer (LbL) coating of the microcarriers
The polystyrene microspheres were coated with the polyectrolytes PSS and PAH
as reported previously [33]. Briefly, the microspheres were suspended in 1 ml PAH
solution (2 mg/ml in 0.5 M NaCl) and continuously vortexed (1000 rpm, 25°C) for 15
min. The non-adsorbed PAH was removed by repeated centrifugation and washing.
Subsequently, the microspheres were dispersed in deionised water containing CrO2
nanoparticles (NPs, <500nm). The CrO2 NPs are used later on to position the
microspheres in a magnetic field to allow the reading of the code, as described in
detail elsewhere [33]. The microsphere dispersion was continuously shaken for 15
min and the excess of CrO2 NPs was removed by repeated centrifugation/washing
steps. The next polyelectrolyte layers were applied similarly. As illustrated in Figure
2A, the microspheres were coated with 5 or 6 layers in the following order: PAH /
CrO2 NP / PAH / PSS / PAH / PSS. Finally, the resulting LbL coated microspheres
were resuspended in 1 ml of ultrapure demiwater at ± 400 000 microspheres/ml and
subsequently encoded.
2. Encoding of the microcarriers
The LbL coated microspheres were encoded by spatial selective photobleaching
as described previously [23]. Briefly, an in-house-developed encoding device was
used, being a microscopy platform equipped with an Aerotech ALS3600 scanning
stage, a SpectraPhysics 2060 Argon laser and an Acousto-Optic-Modulator
(AA.MQ/A0.5-VIS, A.A-Opto-Electronique, Orsay Cedex, France). The encoding
7
process consists of two steps, a writing step (i.e. the photobleaching process) and a
magnetizing step, during which the CrO2 loaded microspheres are exposed to an
external magnetic field sufficient to provide them with a magnetic memory. The
microspheres were fixed on a grid during the encoding process to prevent rotation
between the two steps.
3. Synthesis of PbAE1
PbAE1 was synthesized as described previously by Vandenbroucke et al. 2008
(ref R). Briefly, 37.8 mmol 1,4-butanediol diacrylate and 37.8 mmol 4,4’trimethylenedipiperidine were separately dissolved in 50 ml CH2Cl2. The 4,4’trimethylenedipiperidine solution was added dropwise to the 1,4-butanediol diacrylate
solution under vigorous stirring. The reaction mixture was placed in an oil bath at
50°C and the polymerisation was allowed to proceed during 48 hrs under a nitrogen
atmosphere. After cooling to room temperature, the reaction product was precipitated
in diethyl ether saturated with HCl. The precipitate was filtered and thoroughly
washed with diethyl ether. A white powder was obtained after overnight drying under
vacuum. Subsequently, the polymer was dissolved in acetate buffer (100 mM, pH 5.4)
at a stock solution concentration of 0.5 mg/ml and filtered through a 0.22 µm
membrane syringe filter prior to use.
4. Preparation of cationic liposomes
Cationic liposomes composed of DOTAP: DOPE in a 1:1 molar ratio were
prepared as described previously (12 N). Briefly, lipids were dissolved in chloroform
and mixed. The chloroform was subsequently removed by rotary evaporation at 37°C
followed by flushing the obtained lipid film with nitrogen during 30 min at room
temperature. The dried lipid film were then hydrated by adding Hepes buffer (20 mM,
pH 7.4) till a final lipid concentration of 10 mM. After mixing in the presence of glass
8
beads, liposome formation was allowed overnight at 4°C. Thereafter, the formed
liposomes were extruded 11 times through two stacked 100 nm polycarbonate
membrane filters (Whatman, Brentfort, UK) at room temperature using an Avanti
Mini-Extruder (Avanti Polar Lipids).
5. Preparation of polyplexes and lipoplexes
Polyplexes were prepared at an N/P ratio of 10 or 15 as optimized previously (ref
Far) and lipoplex at an +/- ratio of. ?. For polyplex formation, different volumes of the
polymer stock solution (0.5 mg/ml), dependent on the desired ratio, were added in one
step to 1 µg pDNA at 1 mg/ml in HEPES buffer and subsequently HEPES buffer was
added to reach a final volume of 40 µl. The mixture was vortexed for 10 sec and the
polyplexes were allowed to equilibrate for 30 min at room temperature prior to use.
For lipoplex formation, the liposomes were mixed with the appropriate amount of
pDNA, in a +/- charge ratio of ? and incubated at room temperature for 30 min. The
final concentration of pDNA in the LPX dispersion was 0.126 µg/µl.
6. Preparation of siRNA complex
PbAE:siRNA complexes at an N:P ratio of 30:1 were formed by adding an equal
volume of PbAE solution to 0.5 µM siRNA, followed by vigorously mixing. The
resulting PbAE:siRNA complexes were incubated at room temperature for at least
30 min before addition to the cells.
7. Size and zeta potential measurements
The average particle size and the zeta potential () of the lipo- and polyplexes and
siRNA complex were measured by photon correlation spectroscopy (PCS) (Autosizer
4700, Malvern, Worcestershire, UK) and by particle electrophoresis (Zetasizer 2000,
Malvern, Worcestershire, UK), respectively. The lipoplex dispersion was diluted
40-fold in 20 mM Hepes buffer pH 7.4 and PbAE:siRNA complex was diluted 2-fold
9
in 0.1 M acetate buffer pH 5.4 before the particle size and zeta potential were
measured.
8. Loading of the microcarriersurface with naked pDNA, polyplexes,
lipoplexes, naked siRNA, PbAE:siRNA complexes and adenoviral particles
For immobilizing adenoviral particles, naked DNA or siRNA, DNA and siRNA
complexes, an appropriate amount of sample was added to 10 µl of LbL coated
(encoded) microcarriers suspended in DMEM. The mixture was continuously
vortexed (1000 rpm, 25°C) for 3 hrs. Subsequently, the microcarriers were separated
from the free (i.e. unbound) Ad-RFP viral particles or nucleic acids (free or
complexed) by repeated centrifugation and washing 3 times with DMEM. Cells were
then grown on the loaded microcarriers as described above.
9. Evaluation of immobilized DNA and siRNA
The pDNA and siRNA binding to the microcarrier surface was monitored by
confocal laser scanning microscopy (CLSM; Bio-Rad MRC1024) using TOTO-3
labeled DNA or Cy5 labeled siRNA, excited with 647 nm.
10. Growing cells on the microcarriers and transfection
The LbL coated (encoded) beads were dispersed in cell culture medium (DMEM
containing about 50 % FBS). Approximately 100 µl volume of the microsphere
dispersion was applied in polycarbonate Erlenmeyer shake-flasks (Corning) that were
treated with a silicone solution (Sigmacoat®; Sigma) following the manufacturer’s
protocol. The silicone treatment should prevent the attachment of cells to the surface
of the flasks. Subsequently, an appropriate amount of cell suspension (in DMEM
supplemented with 2% P/S, 1% L-GLn and 10% FBS) was added to the flasks for 3
hrs at 37°C and 5% CO2 to allow the attachment of cells to the surface of the beads.
During this 3 hrs period the flasks were not shaken to allow the initial attachment of
10
the cells. Subsequently we began to agitate the flasks on an orbital shaker under
appropriate conditions to prevent the precipitation of the microcarriers. After 24 hours
the cells loaded microcarriers were studied under the microscope to evaluate
transfection efficiency.
11. Decoding of the cell loaded microcarriers
The cell loaded microspheres were decoded using a Bio-Rad MRC1024
microscope equipped with a 60x water immersion objective lens and a magnet. To
visualize the beads and to read their code upon magnetic orientation an excitation
light of 488 nm was used. To visualize the fluorescence in the cells at the surface of
the microcarriers, excitation wavelengths of 567 nm and 647 nm were used.
11
3. Results and Discussion
1. Synthesis of PbAE
To develop efficient biodegradable siRNA carriers, Vandenbroucke et al. showed
for the first time that biodegradable PbAE polymers were able to induce efficient
siRNA-mediated gene silencing of a viral (transduced) gene in primary rat
hepatocytes and a stably expressed gene in hepatoma cells without causing significant
cytotoxicity (ref R.). They showed that, both PbAEs studied (PbAE1 and PbAE2)
were able to form relatively small siRNA complexes with a positive surface charge.
The extent of gene silencing of the PbAE:siRNA complexes depended on the N:P
ratio. The gene silencing increased as a function of the N:P ratio and a gene silencing
of ~75 % was obtained at a N:P ratio 30:1. PbAE1: siRNA complexes with a higher
N:P ratios were not tested due to cytotoxicity concerns.
Incubating cell-covered encoded microcarriers for several days causes overgrowth
of cells around the microcarriers. Therefore it is necessary to use a carrier that can
release the siRNA fast enough.
We chose PbAE1 (ratio 30:1) for immobilizing on encoded microcarriers because
in contrast to PbAE2, it has faster degradation kinetics with higher transfection
efficiency.
2. Characterization of polyplexes and Lipoplex
see chapter…?
3. Evaluation immobilize DNA and siRNA
In literature, the plasmid DNA has been immobilized on glass (Ziauddin and
Sabatini, 2001) or tissue culture polystyrene (Segara et al., 2003; Bengali et al., 2004)
or silicon and quartz substrates (Lynn et al., 2004). We have loaded nucleic acids and
12
viral particles on polystyrene encoded beads, either with or without modifying the
surface with LbL.
We succeeded to load pDNA and siRNA, either naked or complexed (Linear
PEI/pDNA, PbAE1/siRNA, DOTAP:DOPE/pDNA) and Adenovirus on memobeads
and show the different loading capacity between naked beads and LBL coated beads.
The presence of the fluorescently labeled pDNA and siRNA on the beads was
demonstrated using a fluorescence microscope. We evaluated the influence of the LbL
coating, surface charge of micro- carriers and composition of the LbL coating on
immobilized pDNA and siRNA. In general we used naked and LbL coated
memobeads. Naked beads were carboxyl-functionalized and have a negative surface
charge. To introduce a positive charge on the naked beads, a single layer of PAH was
applied.To study the impact of surface charge on LbL coated beads, two kinds were
used: a) last layer was PAH and B) last layer was PSS.
The results show that the surface charge has an important role for loading naked
pDNA. Figures 2 and 3 show that beads (either naked or LbL coated) with a positive
layer of PAH at the surface have the best loading efficiency. On the other hand, figure
4 shows that the LbL coating causes a more homogeneous loading of naked pDNA
compared to naked PAH-coated beads. LbL coating and surface charge also influence
loading of pDNA in form of complexes, but in all cases inhomogeneous loading with
aggregation was observed (figure 5 and 6).
Figure 7 and 8 show that loading of siRNA in naked form is more depending on LbL
coating than surface charge: siRNA loading on LbL coated beads is twice as high
compared to loading on naked PAH-coated beads. Figure 9 and 10 show that also
siRNA in form of complexes shows aggregation and couldn’t homogeneously coat
microcariers.
13
4. Transfection efficiency of the cells on the microcarriers by pDNA and siRNA
immobilized in the LbL surface
Nucleic acids were immobilized on beads by incubation on LbL coated
microcarriers. To evaluate the transfection efficiency of these immobilized nucleic
acids, cells were grown on surface of these microcarrers. Although the presence of the
nucleic acids were demonstrated in the former paragraphe by the homogeneous
fluorescent coat on microcariers, but none of them showed significant transfection
activity (figure 11). Only polyplex(pDNA/LPei) results in low level of gene
expression (data not shown). It is hypothesized that strong binding between
biomaterials and LbL coated microcarriers doesn't allow the release of the genetic
material or the uptake by cells. This is an important requirement to have transfection
only at the contact site between the beads and cells.
We have studied the release of nucleic acids from by incubating microcarriers
with immobilized nucleic acids in culture medium up to 4 days .
5. Transduction of the cells on the microcarriers by adenoviral vectors
immobilized in the LbL surface
We investigated whether adenoviral vectors (bearing the genetic code for GFP or
RFP) immobilized in the LbL coating surrounding the encoded beads could transduce
the cells grown on the surface of the beads. As Figure 12 shows, after 24 hours a large
percentage of cells that had settled onto the virus-coated microcarriers expressed RFP
and thus became transduced. Importantly, only the cells attached to the surface of the
beads became transduced, as one can see when comparing the transmission and
fluorescence images in Figure 13. It proves that the adenoviral particles did not detach
14
from the microcarrier’s surface during the time of the experiment, which would have
resulted in the transduction of the free cells.
Figure 14 shows the outcome of transduction experiments on Vero-1 cells grown
in 96 well plates. The cells were tranduced with the ‘wash water’ obtained in the
preparation of the adenoviral coated microcarriers, thus containing the free adenoviral
particles which did not become incorporated in the LbL surface. Clearly, washing the
viral coated beads three times is sufficient to remove all free adenoviral particles as
this solution did not transfect cells anymore (see Fig. 14D). Note that the Ad-RFP
loaded beads used in Figure 12 were washed three times and that the transduction of
the cells on the beads could therefore only arrived from Ad-RFP particles
immobilized in the coating of the beads (as there were no viral particles remaining in
the surrounding solvent). Cells on the surface of encoded microcarriers allow
identification of which viral-construct transducted the cells, and thereby showing
which protein target is expressed in the cells on a specific microcarrier. This may
become an interesting tool; making cell-based expression arrays with promises for
proteomics and drug discovery.
Different strategies for immobilizing viral vectors in solid surfaces maintaining
their ability to infect cells have been reported. Recently, bioactive adenoviral vectors
embedded in multilayered polyelectrolyte films on a flat surface were shown to
efficiently transfect different cell lines (viral ref 1). Also Fischlechner et al suggested
virus coating of LbL coated colloids[43,44] and introduced these materials for use in
multiplex suspension arrays to detect virus specific antibodies.[45] Hobson D.A et al
proposed using the extremely tight interaction between streptavidin and biotin to
immobilize adenoviral vectors on the surface of wells and microparticles (viral ref
2,3).
15
3. Conclusions
As addressed in recent reports (11-12,article 5) the technology of living-cell
microarrays suffers from important constraints and limitations mainly due to low
transfection/transduction efficiency. Therefore, in this chapter, we examined if
encoded LbL coated microcarriers would be suitable to be used as a platform for
immobilizing viral vectors or naked or precomplexed nucleic acids.
We have shown that LbL coating could have a beneficial role in immobilizing
nucleic acids on microcarriers. Additionally, we also studied the delivery of
immobilized nucleic acids from the surface of an encoded 40 µm polystyrene bead.
No significant transfection efficiency could be observed with in case of naked pDNA
or siRNA, lipoplexes or PbAE:siRNA complexes. Linear PEI containing polyplexes
resulted in low level of gene expression. This rather limited transfection efficiency is
probably caused by the limited nucleic acids (complexes) release from the
microcarrier surface. In contrast, adenoviral particles immobilized in the LbL coating
were able to selectively tranduce the cells that were grow on top of the ‘adenovirus
coating’. We showed that adenoviral particles immobilized in the polyelectrolyte
layer retained their ability to infect cells. Importantly, only the cells at the surface of
the microcarriers, thereby in close contact with the adenoviral particles, became
transduced, while free cells (i.e. cells present in the dispersion but not attached to a
microcarrier) were not transduced.
In conclusion, we showed in pilot experiments that LbL coated microcarriers are
valuable tool to use as a cell based microarray.
16
ACKNOWLEDGMENTS
Ghent University (BOF) acknowledged for their support. We would like to thank
Dr. R. E. Vandenbroucke and Dr. B.G. De Geest for helpful discussions.
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24
Figures
Microsphere (λ exc488nm)
Merged image (λ exc488/567nm)
Figure 1 : Confocal image of a green-fluorescent encoded polystyrene bead (A) and 2
optical sections of a green-fluorescent encoded polystyrene bead covered with cells
expressing red fluorescent protein (B and C).
Naked beads
LbL coated
LbL coated
(last layer PSS)
(Last layer PAH
25
PAH coated beads
Figure 2.naked pDNA immobilized on microcarriers with different surface properties.
Green and red fluorescent image of green microcarriers loaded with red (yoyo3)
labeled naked pDNA, the insert is the transmission image
160
Fluorescence (A.U.)
140
120
100
80
60
40
20
0
pDNA loading on
Naked beads
pDNA loading on LBL() coated beads
pDNA loading on
LBL(+) coated bead
pDNA loading on PAH
coated beads
Figure 3. naked pDNA loading on microcarriers of different surface properties
Figure 4. fluorescent image of a green microsphere coated with red (yoyo3)
labeled naked pDNA
26
Naked beads
LbL coated
(last layer PAH)
LbL coated
(last layer PSS)
Figure 5. Fluorescent images of green microspheres coated with red (yoyo3) labeled
pDNA polyplexes (DNA/Lpei)
Naked beads
LbL coated beads
last layer ( PAH)
LbL coated beads
last layer (PSS)
Figure 6. fluorescent images of green microspheres loaded with red (yoyo3) pDNA
lipoplexes
27
Naked siRNA on naked beads
on PAH coated beads
on LbL coated beads
Figure 7. fluorescent images of green microcarriers loaded with red (cy5) labeled
siRNA
80
Fluorescence (A.U.)
70
60
50
40
30
20
10
0
siRNA loading on Naked bead
siRNA loading on PAH coated
beads
28
siRNA loading on LBL coated
beads
Figure 8. siRNA loading on microcarriers of different surface properties
Control beads
siRNA complex
LbL coated
LbL coated
load on naked beads
(last layer PSS)
(last layer PAH)
Figure 9. fluorescent images of green microcarriers loaded with red (cy5) labeled
siRNA complexed with PbAE1
siRNA complex on LbL coated beads
Non complex siRNA on LbL coated beads
Figure10. fluorescent images of green microcarriers loaded with red (cy5) labeled
siRNA, either complexed (A) or free (B)
29
Transfection
Figure 11. growing cells on beads after loading genetic materials (reverse
transfection)
A
B
C
D
Figure 12. (A-C) Transmission (top) and merged red/green fluorescence (bottom; λex
= 567 nm and λex
=
488 nm) images of Ad-RFP coated microcarriers loaded with
30
Vero-1 cells. In (D) microcarriers were used which did not contain viral particles in
their coating (negative control). Note that non-encoded microcarriers were used. The
scale bar represents 10 µm.
A
B
Figure 13. (A) Transmission and (B) merged green/red fluorescence images (λex 561
nm and λex 488 nm) of a dispersion containing respectively Ad-RFP coated
microcarriers loaded with Vero-1 cells and “free Vero-1 cells”. Non -encoded
microcarriers were used. The scale bar represents 50 µm.
Figure 14. Transmission (top) and red fluorescence (bottom) images of Vero-1 cells
seeded in 96 well plates transfected with respectively (A) the virus dispersion used to
coat the microcarriers and (B) the first, (C) the second and (D) the third wash water as
obtained during the preparation of the Ad-RFP coated microcarriers
31