RANDOMLY BRANCHED POLY(2-DIMETHYLAMINOETHYL
METHACRYLATE) POLYELECTROLYTES AS
GENE TRANSFECTION AGENTS
John M. Laymana, Anjali A. Hiranib, Mathew G. McKeea, cPhillip F. Britt,
c
Joseph M. Pickel, Yong Woo Leeb, and Timothy E. Longa
CH3
H2C
CH2
Towards this end, linear and branched cationic polyelectrolytes based on
poly(2-N,N’-dimethylaminoethyl
methacrylate)
(PDMAEMA)
were
synthesized and comparatively analyzed for plasmid complexation efficiency.
Several investigators have examined the complexation and transfection of
branched versus linear cationic polyelectrolytes5,6 including star topologies.7,8
Although these references describe the importance of molecular topology,
most of these sources describe linear versus branched polymers categorically.
We attempt to describe the degree of branching using the well established,
semi-quantitative, g’ value, which is the ratio of intrinsic viscosities of
branched and linear polymers at similar molar mass. This work builds on
earlier efforts in our laboratories which suggested linear PDMAEMA was
more efficient.9 Investigators have already shown that the topology of the
DNA plasmid significantly influences linear gene transfection effienciey.10
Cl
+
CH2
CH2
CH2
n
CH2
N
H3C
O
CH2
O
n
CH3 O
C
C
C O
C CH2
O
n
CH3
CH2
o
APS, 60 C, N2
H2O, pH5
CH2
n
C O
O
C O
H3C C
CH3
CH2
CH3
O
O
CH2
O
C CH2
C O
O
CH2
C
O
H3C C
C
H3C CH
CH2
CH2
Cl
H3C
Figure 1. Electrostatic complexation between anionic DNA and cationic
polyelectrolyte to form a polyplex.
NH
Cl
CH2
a
Introduction
Gene therapy has received considerable attention as a potential avenue
to treat a number of acquired or inherited genetic disorders. In gene therapy,
extra-chromosomal circular DNA segments, or plasmids, are transferred
through the cellular membrane and expressed by the cell’s existing protein
synthesis machinery. For efficacious gene therapy, transfection agents are
needed to escort plasmids through the cell membrane since naked DNA is
negatively charged and expanded in aqueous solution. 1 The negative charge
from the phosphodiester bond in the DNA backbone interacts unfavorably
with the lipid bilayer of the cell membrane. Furthermore, mutual charge
repulsion along the polymeric nucleotide results in a chain-extended
configuration of the macromolecule. Viral vectors, which utilize an
inactivated virus (typically adeno and retro viruses), are extremely efficient
carrier molecules and can even target cells by their natural tendency to infect
certain cell types. However, viral vectors are limited to small plasmids and
although modified viruses are inactivated, host immune response is a frequent
complication.1,2 Non-viral agents, such as cationic polyelectrolytes, are an
attractive replacement to viruses due to absence of immunogenic risk and the
ability to tailor the macromolecular architecture. Non-viral vectors function
by electrostatically screening the anionic charges on the DNA plasmid to form
a plasmid-polymer complex, or polyplex (Figure 1). This action neutralizes
the net charge and alleviates mutual charge driven chain extension, thus
condensing the hydrodynamic radius of the polyplex. The elimination of net
ionic charges and smaller size of the polyplex considerably increase the
probability of gene transfection.3 Cationic polyelectrolytes have also been
shown to protect against enzymatic degradation from the host. Although nonviral vectors possess numerous advantages, several investigators have shown
that transfer efficiencies are considerably lower when compared to viral
vectors. 4
CH3
H3C
NH
C
Macromolecular Science and Engineering, Macromolecules and Interfaces Institute,
b
School of Biomedical Engineering and Sciences,
Virginia Tech, Blacksburg, VA 24061-0131
c
Oak Ridge National Laboratory, Center for Nanophase Material Sciences,
Oak Ridge, TN 37831-6197
CH3
H3C
NH
H3C
H2
C
H2
C O
CH2
C
O
C
CH3
n
Figure 2.
Synthesis of branched PDMAEMA through free radical
polymerization.
Experimental
Materials. 2-(N,N’-dimethylamino)ethyl methacrylate (DMAEMA,
Sigma-Aldrich) was passed through a neutral alumina column to remove
inhibitor. Ammonium persulfate (APS, 99.99%, Sigma-Aldrich) was used asreceived as the initiator in aqueous synthesis. 2,2’-Azobisisobutyronitrile
(AIBN,98%, Sigma-Aldrich) used as received in THF synthesis. The
branching agents ethylene glycol dimethacrylate (EG-DMA, Sigma-Aldrich)
and poly(ethylene glycol) dimethacrylate (PEG-DMA, MW=500 g/mol,
Sigma-Aldrich) were passed through a neutral alumina column to remove
inhibitor. 1-Dodecanthiol (Aldrich, 98%) was used as a chain transfer agent.
All other solvents and reagents were used as received from commercial
sources without further purification.
Instrumentation. 1H NMR spectra were obtained at room temperature
using a Varian Unity 400 spectrometer operating at 400 MHz. Samples were
dissolved in D2O for NMR experiments. Aqueous SEC experiments were run
in a buffer solution consisting of 0.7M sodium nitrate (Sigman-Aldrich), 0.1
M TRIS (Sigma-Aldrich) adjusted to pH 6.5 with acetic acid (Fisher).
Samples were analyzed at 0.8 mL/min through Shodex OHPAK 804 and
802.5 columns. Instrumentation consisted of an Agilent 1100 series
quaternary pump, Precision Detectors PD2020 light scattering detector,
Viscotek 270 viscosity detector, and a Wyatt Optilab REX referactive index
detector. Conventional calibration was performed using a series of 7 Pullulan
(Shodex) standards ranging from 5-400 kg/mol.
Solution rheology
experiments were conducted using a Bohlin VOR strain-controlled solution
rheometer at 25 ± 0.5 oC using a concentric cylinder geometry.
Synthesis of linear and branched Poly(2-N,N’-dimethylaminoethyl
methacrylate):
Linear and randomly branched PDMAEMA were
synthesized by conventional free radical polymerization methods as described
previously.9 Briefly, PDMAEMA was synthesized via solution radical
polymerization using either APS or AIBN (0.1-3wt% monomer) as the
initiator, depending upon solvent used. In aqueous synthesis, which was used
to produce higher molecular weight polymers, the pH of the monomer
solution was adjusted to 5.0 using 10 M HCl. In organic solvent based
synthesis, the polymer product was converted to an ionized form by dissolving
the neutral form in DI water that was adjusted to a pH of 5.0 using 10M HCl
under magnetic stirring. Branched PDMAEMA was synthesized by adding
the appropriate amount of PEG-DMA or EG-DMA (0.1-2wt% monomer)
(Figure 2) and synthesized by the same procedure used to produce linear
PDMEMA. A chain transfer agent (1-dodecanethiol) was used to prevent
gelation in reactions with higher concentrations of EG-DMA.
Cell culture. Human brain microvascular endothelial cells (HBMEC)
were isolated, cultivated, and purified as previously described.11 These cells
were positive for factor VIII-Rag, carbonic anhydrase IV, Ulex Europeus
Agglutinin I, and took up fluorescently labeled low-density lipoprotein and
expressed gamma glutamyl transpeptidase, demonstrating their brain
endothelial cell characteristics. Contamination of non-endothelial cells such
as pericytes and glial cells were less than 1%. HBMEC were cultured in
RPMI 1640-based medium with 10% fetal bovine serum (Mediatech), 10%
NuSerum (Becton Dickinson), 30 g/ml of endothelial cell growth
supplement (ECGS; Becton Dickinson), 15 U/ml of heparin (Sigma-Aldrich),
2 mM L-glutamine, 2 mM sodium pyruvate, nonessential amino acids,
vitamins, 100 U/ml of penicillin, and 100 g/ml of streptomycin (all reagents
Results and Discussion
1
H NMR spectra of linear and branched PDMAEMA, which were
prepared by free radical methods, verified the chemical structure of each
sample. Furthermore, FT IR was used to observe the conversion of the neutral
PDMEAMA to the ionized form by observing the nitrogen-hydrogen stretch
in the IR-spectrum. Aqueous SEC results show molar masses of linear and
branched polymers between 25,000-106 g/mol. Aqueous solution rheology
also confirmed the synthesis of a polyelectrolyte.
100
(wt% EGDMA)
O= 0
▲= 0.75
■=1.0
10
1.18
1.28
hsp
2.20
0.55
0.78
1
0.87
0.1
0.1
1
10
100
c (wt% )
Figure 3.
Aqueous
PDMAEMA·HCl.
solution
rheology
of
linear
and
branched
The scaling relationships of the specific viscosity as a function of
concentration, in both the unentangled and entangled regimes, match wellestablished values for polyelectrolytes.13 Interestingly, increasing the degree
of branching (lower g’ value) results in a suppression of the polyelectrolyte
effect. The scaling values in both regimes appear to approach the neutral limit
(Figure 3). This suppression may result from the inability of branched
polyelectrolytes to change conformation upon changes in the charge
environment, unlike the linear analog. The inability of branched PDMAEMA
to change conformation, and thus decrease in hydrodynamic volume, may
explain its capability as a gene transfection agent.
MTT conversion assay shows limited cytotoxicity of DNA/PDMAEMA
particles over the times, concentrations, N/P ratios, and cell type tested. The
only noticeable toxicity was observed in DNA/polymer particles with N/P
ratios of 8 (DNA 0.2μg/mL) or higher and exposure times of > 12 h.
Exposure to PDMAEMA polymer without the addition of DNA resulted in
significant toxicity with viabilities down to 50% at higher concentrations of
both linear and branched PDMAEMA. Since negligible toxicity was observed
for N/P ratios up to 8 (Figure 4), all cells were exposed to transfection
solutions for 12 h. Transfection using pRL-SV40 showed a tendency for
linear PDMAEMA to transfect better than branched PDMAEMA at similar
molar mass. Additionally, the degree of branching (g’) had a significant
impact on the transfection efficiency using PDMAEMA as a gene transfer
12 hour exposure
agent. Further transfection results will be disused during the presentation.
Cell Viability (% Control)
from Mediatech). Cultures were incubated at 37 C in a humid atmosphere of
5% CO2.
Cell Viability Assay. Cell viability was determined with the standard 3[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) conversion
assay as we previously described.12 Briefly, HBMEC cells were plated at a
concentration of approximately 5.0x104 cells/well on a 24-well plate 24 h
prior to each experiment. The pRL-SV40 Renilla luciferase expression
plasmid (Promega) was diluted in 2.5 mL basal RPMI media to a final
concentration of 0.4 μg/mL and incubated at room temperature for 30 min. At
the same time, the appropriate type and amount of polymer was diluted in 2.5
mL basal RPMI to the final concentrations corresponding to the various
nitrogen/phosphorus (N/P) ratios and allowed to incubate for 30 min at room
temperature. To complex the plasmid DNA with the PDMAEMA, the vector
and corresponding polymer solutions were mixed and incubated for 10 min at
room temperature. Prior to each assay, HBMEC cells were washed with
approximately 1 mL of basal RPMI. After washing, 1mL of the appropriate
plasmid/polymer solution was placed in each well. After 4, 12, 24, and 48
hour exposure times, the cells were rinsed with approximately 1mL of HBSS,
followed by the addition of 1 mL of RPMI 1640 medium containing 0.5
mg/ml of MTT (Sigma-Aldrich). After incubation for 4 h at 37oC, the medium
was aspirated and the formazan product was solubilized with DMSO.
Absorbance at 570 nm was measured for each well using a SPECTRAmax
190 microplate reader (Molecular Devices Corp.).
Renilla Luciferase Expression Vector Assay. HBMEC cells were
plated on 12 well plates 24 h prior to transfection. DNA/polymer solutions
were prepared using the same procedure used in cell viability assay
experiments. All solutions were allowed to transfect for 12 h at 37oC, 5%
CO2. After 12 h, the transfection solution was replaced with complete RPMI
growth media. The cells were then incubated for 24 h at 37oC, 5% CO2 to
allow for protein expression. After incubation, the cells were rinsed with
approximately 1 mL PBS and 100 μL of lysis buffer was added. Immediately
after adding lysis buffer, each well was scraped and incubated for 30 min at
room temperature with gentle mixing. The lysate mixture was then subjected
to two -80oC/37oC freeze/thaw cycles. Luciferase activity was measured
using the Renilla luciferase assay kit (Promega) and luminometer (Molecular
Devices Corp.).
100
80
60
40
20
0
C
L8
B8
LC
BC
Figure 4. Cell viability after 12 hour exposure to plasmid/polymer complexes
and polymer only controls. C represents an untreated control, L8 and B8
correspond to N/P ratios of 8 for the linear (L, Mw=396k g/mol) and branched
(B, Mw=352k g/mol) polymers. LC and BC are the linear and branched
polymer only controls at the concentration used for the highest N/P ratio.
Conclusions
Conventional free radical polymerization was used to synthesize linear
and randomly branched cationic polyelectrolytes based on PDMAEMA.
Increasing the degree of branching in PDMAEMA results in a suppression of
the polyelectrolyte effect.
The cytotoxicty of linear and branched
PDMAEMA was low at the concentrations and polymer architectures tested.
The topology of the PDMAEMA gene transfer agent was found to have an
affect on gene transfection in HBMEC cells.
Acknowledgements
This material is based upon work supported in part by the
Macromolecular Interfaces with Life Sciences (MILES) Integrative
Graduate Education and Research Traineeship (IGERT) of the National
Science Foundation under Agreement No. DGE-0333378.
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