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Journal of Pharmaceutical Technology &
Drug Research
ISSN 2050-120X | Volume 3 | Article 2
Special Section | Pharmaceutics | Original
Open Access
Safety and efficacy of amine-containing methacrylate
polymers as nonviral gene delivery vectors
Noura H. Abd Ellah1,3, Sarah J. Potter1, Leeanne Taylor2, Neil Ayres2, Mona M. Elmahdy3, Gihan N. Fetih3, El-Sayed A. Ibrahim3 and
Giovanni M. Pauletti1*
*Correspondence: gm.pauletti@uc.edu
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James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH 45267, USA.
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
3
Faculty of Pharmacy, Assiut University, 71515 Assiut, Egypt.
1
2
Abstract
Background: Nonviral polymeric delivery systems are explored to enhance clinical development of nucleic
acids as therapeutic entities for effective management of debilitating conditions such as cancer. This study
was to compare safety and efficacy of quaternary amine-containing methacrylate polymer Eudragit® RL
PO (ERL) and poly[N-(2-hydroxypropyl)methacrylamide]-poly(N,N-dimethylaminoethyl methacrylate)
copolymer (pHPMA-b-pDMAEMA), which contains secondary and tertiary amines, as effective gene
carriers.
Methods: Polyplexes of pAcGFP1-C1 with ERL or pHPMA-b-pDMAEMA were fabricated at different
N/P ratios. Formation of DNA/catiomer nanostructures was monitored by ethidium bromide intercalation
and agarose gel retardation. Particle size, zeta potential and cytotoxicity of different polyplexes were
characterized. Transfection efficiency in presence and absence of serum was assessed using confocal
microscopy.
Results: pHPMA-b-pDMAEMA demonstrated at least a 10-fold greater DNA condensation capacity
per weight unit than ERL. However, DNA intercalation with pHPMA-b-pDMAEMA was reduced in
presence of serum-free cell culture media, whereas polyplex formation with ERL was equivalent in
phosphate-buffered saline, pH 7.4 and serum-free cell culture media. Cellular safety of HeLa cells was
not compromised by polyplexes fabricated with either polymer up to N/P=4. However, ERL alone was
more toxic. In absence of serum, pHPMA-b-pDMAEMA polyplexes at N/P=4 induced equivalent
transgene expression as control TurboFect™ polyplexes. In contrast, ERL-containing nanoassemblies
failed to produce measurable transgene expression. Inclusion of serum significantly decreased transfection
efficiency of pHPMA-b-pDMAEMA-containing polyplexes by ~30% at N/P=4 and ~50% at N/P=2.
Conclusion: Polyplexes fabricated with secondary and tertiary amine-containing pHPMA-b-pDMAEMA
copolymer represent more effective gene delivery systems than nanoassemblies composed of quaternary
amine-containing ERL and should be further explored for clinical applications.
Keywords: Transfection, cell survival, polymethacrylic acid, polyplex, Eudragit® RL PO
Introduction
Recent advances in molecular understanding of patho-physiological processes continue to fuel great interest in clinical
exploration of gene therapy. Increased safety concerns associated with highly effective viral vectors mandate development
of novel nonviral gene carriers in order to revolutionize clinical
management of debilitation genetic disorders such as cystic
fibrosis, diabetes, hemophilia, and cancers [1]. Due to the
high molecular weight and negative charge associated with
genetic material, transfer of these therapeutic moieties across
biological membranes is limited. Consequently, one of the major
challenges of gene therapy is to deliver therapeutically active
genetic material into desired target cells. To enhance stability
and efficiency of gene delivery systems, genetic material is
© 2014 Pauletti et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
http://www.hoajonline.com/journals/pdf/2050-120X-3-2.pdf
generally encapsulated into a vector that protects the payload
from degradation and, simultaneously, facilitates effective
intracellular delivery. Viral vectors were demonstrated to
achieve high transfection efficiency in vitro and in vivo. Unfortunately, clinical development of virus-based gene delivery
systems is restricted due to undesired immune responses
and cellular toxicity [1,2]. Nonviral vectors, in contrast, are
considered less immunogenic and generally exhibit favorable
safety profiles [2]. Fundamental to the design of a synthetic
gene delivery system is its ability to neutralize the negative
charge of genetic material in order to prevent charge repulsion at the anionic cell surface. Furthermore, synthetic carriers
must successfully condense the bulky DNA structure to the
nanoscales for effective cellular internalization and to protect
nucleic acids from enzymatic degradation mediated by both
extra- and intracellular nucleases [3]. Cationic polymers and
lipids spontaneously form electrostatically-driven association
complexes (i.e., polyplexes or lipoplexes) when combined
with DNA/RNA structures [4]. Previous research demonstrated
that gene delivery systems carrying excess positive charge
interact more efficiently with negatively charged cell membrane components such as proteoglycans and glycosaminoglycans, thus, enhancing cellular uptake and transfection
[5]. However, it was also reported that these highly positive
charged comp-lexes might be disrupted before reaching
their targets due to the effect of other charged molecules in
the serum or the extracellular matrix [6]. The stoichiometric
ratio of positively charged amine groups in a cationic polymer and negatively-charged phosphate groups in DNA/RNA
moieties (i.e., N/P ratio) is a key determinant of effective gene
delivery due to its impact on particle size and zeta potential
of nanoassemblies [7].
At the cellular level, safety and efficiency of these carriers
are strongly affected by the architecture of the polymer used
[8]. The amino groups in cationic polymers play a critical role in
defining physicochemical properties of association complexes
and transfection efficacy [9,10]. In particular, secondary and
CH3
H3C CH3
Cl
C
H2
ERL
Materials
The poly [N-(2-hydroxypropyl)methacrylamide]-poly (N,N-
S
S
C
O
O
Materials and methods
S
C
CH2
N
H3C
O
tertiary amine groups present in the polymer backbone are
predicted to enhance endosomal escape due to the “proton
sponge” effect, which is a prerequisite for transfer of genetic
material into the nucleus after cell internalization via endocytosis [11,12]. The objective of this study was to compare safety
and efficacy of nonviral gene delivery systems fabricated with
the commercially available, quaternary amine-containing
ERL and the secondary/tertiary amine-containing pHPMA-bpDMAEMA, which was synthesized by RAFT polymerization
(Figure 1). ERL is a FDA-approved excipient and widely used in
marketed pharmaceutical products to control pH-independent
drug release. Consequently, it is one of the logical first choices
of excipients when considering formulation approaches for
gene delivery systems as its safety profile in humans is already
established [13]. pHPMA-b-pDMAEMA is a representative
examples of the growing class of experimental, water-soluble
diblock copolymers that are synthesized by various polymer
chemists to overcome apparent safety and efficiency challenges associated with existing catiomers.
Chemically, pHPMA-b-pDMAEMA contains ~30% secondary and tertiary amino groups per gram that are predicted to
greatly enhance endosomal escape of internalized polyplexes
and, consequently, increase transfection efficiency [12]. In
contrast to pDMAEMA, which was demonstrated to induce
substantial cytotoxic effects [14], copolymerization with the
hydrophilic pHPMA moiety significantly increased cellular
safety [12]. For formulators, however, the lack of FDA-approval
for pHPMA-b-pDMAEMA or similar experimental copolymers
is a significant concern, as time- and cost-intensive safety
studies must be completed before initial clinical evaluation
with this novel excipient can begin. This strategy is only justified if the use of a novel excipient is predicted to dramatically
improve safety and/or efficacy of the formulation in patients.
O
CH3
C
doi: 10.7243/2050-120X-3-2
OH
n
O
O
O
NH
OH
O
O
O
CH3
C2H5
N
pHPMA-b-pDMAEMA
Figure 1. Schematic representation of the chemical structure of ERL and pHPMA-b-pDMAEMA, respectively.
2
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
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dimethylaminoethyl methacrylate) diblock copolymer
(pHPMA-b-pDMAEMA) was synthesized by reversible additionfragmentation chain transfer (RAFT) polymerization using
a procedure adapted from Duvall and colleagues [15]. The
average molecular weight of pHPMA-b-pDMAEMA determined
by gel permeation chromatography was=21.2 kDa. Eudragit® RL
PO (ERL) was a gift from Evonik Industries (Parsippany, NJ). The
pAcGFP1-C1 expression plasmid was obtained from Clontech
Laboratories (Mountain View, CA) and amplified according
to the manufacturer`s protocols. TurboFect™ Transfection
Reagent, DRAQ5™, 1kb DNA ladder, Dulbecco’s modified Eagle’s
medium (DMEM), phosphate-buffered saline pH 7.4 (PBS),
agarose, agarose gel loading dye, ethidium bromide (EtBr),
Tris/EDTA (TE) buffer, and cell supplements such as trypsin/
EDTA, penicillin-streptomycin, L-glutamine, and non-essential
amino acids were purchased from ThermoFisher Scientific
(Pittsburgh, PA). Tris-acetate-EDTA (TAE) running buffer and
CellTiter-Glo® cytotoxicity assay kit were purchased from
Promega (Madison, WI). Branched polyethylenimine 25 kDa
(PEI25k) was purchased from Sigma Aldrich (St. Louis, MO).
Heat-inactivated fetal bovine serum (FBS) was obtained from
Atlanta Biologicals (Atlanta, GA). All other chemicals were of
analytical grade and used as received.
doi: 10.7243/2050-120X-3-2
after 30 min destaining in water at λ=254 nm using the UVP
Bioimaging System (UVP, Upland, CA).
Polyplexes fabrication
Association complexes between pAcGFP1-C1 and pHPMA-bpDMAEMA, ERL were formed by combining 1 µg of pAcGFP1-C1
with various polymer amounts in PBS and SFM resulting in
polyplexes with N/P ratios up to 4. Electrostatic association was
allowed for 1hr at RT with occasional vortexing (20 seconds
every 15 minutes). Stock solutions of ERL were prepared in
95% (v/v) ethanol. For subsequent experiments, final ethanol
concentration was ≤1% (v/v). pHPMA-b-pDMAEMA solutions
were directly prepared in aqueous vehicles.
Physicochemical properties of polyplexes
Particle size distribution and zeta potential of fabricated polyplexes were estimated by dynamic laser light scattering using
the Zetasizer Nano-ZS (Malvern Instruments, Worcestershire,
U.K.) according to the manufacturer’s instructions. All particle size values reported in this study refer to the equivalent
hydrodynamic diameter.
Cellular safety
Viability of HeLa cells after exposure to the various polymers
in the presence and absence of pAcGFP1-C1 was quantified
HeLa cells were obtained from the American Type Culture Col- using the CellTiter-Glo® luminescent assay, which measures
lection (Manassas, VA) and maintained at 37°C in a humidified total cellular ATP. For these experiments, HeLa cells were
5% (v/v) CO2 atmosphere using DMEM supplemented with seeded in white 96-well plate at density of 1×104 cells/well.
10% (v/v) FBS, 1% (w/v) L-glutamine, 100 IU/ml penicillin, 100 Following an overnight attachment, cells were washed with
µg/ml streptomycin, and 1% (v/v) non-essential amino acids. prewarmed SFM and incubated for 4 hrs at 37°C in the presence
of various polyplex or polymer concentrations. Subsequently,
Ethidium bromide intercalation
cells were washed using FBS-containing DMEM and incubated
The ability of cationic polymers to condense pDNA was moni- for additional 44 hrs in maintenance media. Following addition
tored by fluorescence quenching of the pDNA-EtBr interaction of the CellTiter-Glo® reagent, luminescence was quantified
as described previously [16]. 1 µg of pDNA suspended in 50 µL using the POLARstar microplate reader (BMG Labtech, Cary,
of either PBS or serum-free DMEM (SFM) was combined with NC). Cells incubated with a 1% (v/v) Triton X-100 solution
50 µL of an aqueous EtBr solution (5 µg/mL) and incubated prepared in PBS were used as negative control. Cell viability
at room temperature (RT) for 15 min. Polymer aliquots were was normalized to vehicle-treated controls.
sequentially added and incubated for 30 min at RT before
fluorescence emission was quantified at λ=590 nm (EX=544 Acid-base titration
nm) using the POLARstar microplate reader (BMG Labtech, Endosomal buffering capacity of pHPMA-b-pDMAEMA and ERL
Cary, NC).
was estimated by acid-base titration as described by Cai and
colleagues [19]. Briefly, polymers were dissolved or suspended
Agarose gel retardation
at 20 mg/L in 50 mM NaCl, and pH value of this solution was
Electrophoretic mobility of polymer/pDNA complexes was adjusted to pH 10 using 0.1N NaOH. Acid-base titration was
determined by agarose gel electrophoresis [17,18]. Catiomer/ accomplished by incremental addition of 10 μL aliquots of
pDNA complexes containing 0.4 μg of pAcGFP1-C1 were com- 0.1N HCl (150 μL total). The pH value was determined at RT
bined with 2 µL of the gel loading dye, and 20 µL of this suspen- following each addition. PEI25k was used as a positive control.
sion were loaded onto a 0.5% (w/v) agarose gel. pAcGFP1-C1
plasmid without polymer was used as a control. Separation In vitro transfection efficiency
was carried out for 100 min at 90 V in TAE running buffer HeLa cells were seeded in 16-well chamber slides at density of
using the Power Pac® 200 (Bio-Rad, Hercules, CA). Following 1×104 cells/well. Following an overnight attachment, cells were
electrophoresis, gels were stained for 40 min using a 0.005% washed with prewarmed SFM and incubated for 4 hrs at 37°C
(v/v) aqueous EtBr solution. pDNA bands were visualized with polyplexes suspended in SFM (0.2 μg DNA/well). Naked
Cell culture
3
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
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Relative Fluorescence %
DNA and the transfection reagent TurboFect™ were used as
controls. Subsequently, cells were washed with FBS-containing
DMEM and incubated for additional 44 hrs in maintenance
media to allow transgene expression. Cells were washed 3x
with PBS and counterstained with the red DRAQ5 nuclear
stain. The percentage of positive, green fluorescent cells was
determined in three randomly selected sections (Zeiss LSM510
Confocal Microscope, Zeiss, Germany). To assess the impact of
serum on transfection efficiency, polyplexes were incubated
in FBS-containing DMEM.
doi: 10.7243/2050-120X-3-2
100
80
60
40
20
0
0.0
Statistical analysis
Experiments were performed at least in triplicate, and results
are reported as mean±standard deviation (SD). Statistical difference among various treatment groups was assessed using
one-way ANOVA or two-sided Student’s t-test for pairwise
comparison. A probability of p<0.05 was considered statistically
significant (GraphPad Prism 6.0, GraphPad, San Diego, CA).
Results and discussion
Polymer/pDNA interactions
Polyplex formation is thermodynamically driven by electrostatic
interactions between negatively charged phosphate groups of
nucleic acids and positively charged amino groups present on
the polymers [20]. To quantify the stoichiometric relationship
of this interaction for each polymer, fluorescence quenching
of EtBr/pDNA complexes in the presence of various polymer
amounts was measured. Decreasing fluorescence intensities
determined after addition of increasing amounts of pHPMAb-pDMAEMA experimentally underlined the ability of this
cationic polymer to electrostatically bind negatively charged
pDNA in PBS and SFM (Figure 2A). pHPMA-b-pDMAEMA is
estimated to contain on a molar basis ~30% cationic centers
in form of secondary and tertiary amine groups. In PBS, effective condensation of 1 µg of pDNA was achieved in the presence of 513 ng of this diblock polymer (N/P=1). These results
were consistent with agarose gel retardation data where the
fluorescence intensity of EtBr/pDNA bands associated with
relaxed and coiled nucleic acid strands dramatically decreased
following addition of polymer amounts >128 ng (Figure 2B).
In the presence of 513 ng of pHPMA-b-pDMAEMA (Figure 2B,
Lane 4), electrophoretic mobility of EtBr/pDNA was visually
absent suggesting effective pDNA condensation with this secondary and tertiary amine groups containing polymer [16,19].
In contrast, the stoichiometry of pDNA/pHPMA-b-pDMAEMA
interactions in SFM was significantly different. Based on EtBr
intercalation results (Figure 2A), it appears that the presence
of media components interferes with effective condensation
of pDNA with this cationic polymer. Addition of pHPMA-bpDMAEMA amounts >0.5 µg was only moderately successful
in increasing EtBr displacement greater than 40%. Even in
the presence of 1.5 µg of this polymer, electrostatic neutrality (i.e., N/P=1) was never reached. Agarose gel retardation
assay confirmed the inability of pHPMA-b-pDMAEMA to fully
1
2
3
0.5
1.0
pDMAEMA-b-pHPMA [μg]
1
4
2
1.5
1
4
bp
10,000
5,000
3,000
Figure 2. Vehicle effect on polyplex formation with
pHPMA-b-pDMAEMA. EtBr displacement from pDNA
in PBS ( ) and SFM ( ) was quantified in the presence
of 0-1.5 μg of pHPMA-b-pDMAEMA using fluorescence
spectrophotometry (Panel A). Results are shown as mean±SD
(n=3). Representative agarose gel pictures after electrophoretic
separation of pDNA/pHPMA-bpDMAEMA polyplexes
prepared at different mass ratios with 1 μg of pAcGFP1-C1
in PBS and SFM are shown in Panel B and C, respectively.
Lane 1: DNA control, Lane 2: 128 ng polymer, Lane 3: 256 ng
polymer, Lane 4: 513 ng polymer.
condensate pDNA in SFM by the presence of bright, fluorescent EtBr-stained pDNA bands (Figure 2C). It is hypothesized
that zwitterionic amino acids in SFM afforded electrostatic
shielding of positively charged amine groups in the DMAEMA
moieties, thereby reducing the pDNA condensation ability of
this polymer [21,22]. Similar binding experiments were performed with ERL that contains on a molar basis ~10% cationic
centers in form of quaternary ammonium groups [23]. Figure 3
summarizes the results of EtBr intercalation and agarose gel
retardation assays performed with ERL. Increasing polymer
amounts consistently reduced fluorescence signal of EtBr/
pDNA suggesting efficient electrostatic interactions between
the negatively charged pDNA and the positively charged ERL
(Figure 3A). In PBS, complete quenching equivalent to N/P=1
was achieved using 7 µg of ERL. Interestingly, the stoichiometry of pDNA displacement from EtBr in the presence of ERL
was not affected by buffer compositions, most likely due to
involvement of quaternary ammonium groups. The estimated
amount of ERL required for fabrication of ionically balanced
polyplexes with 1 µg of pDNA was not significantly different
between PBS and SFM (p=0.882). Agarose gel retardation
assays confirmed these results where no electrophoretic
mobility of pDNA was observed in the presence of 7 µg of
4
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
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Table 1. Physicochemical properties of pHPMA-b-pDMAEMA/
pAcGFP1-C1 polyplexes fabricated in PBS.
100
Relative Fluorescence %
doi: 10.7243/2050-120X-3-2
40
40
40
20
0
0
N/P
0.25
Size [nm] Zeta potential [mV]
192±66
-9.2±8.5
0.5
1
2
4
235±17
144±2.9
111±1.4
104±3.0
-3.9±0.6
-0.3±0.5
-2.7±2.1
-2.0±3.0
Data are shown as mean±SD (n=3)
0
2
2
3
4
ERL [μg]
4 5 6
6
1
2 3
8
4
5 6
bp
10,000
5,000
3,000
Figure 3. Vehicle effect on polyplex formation with ERL.
EtBr displacement from pDNA in PBS ( ) and SFM ( ) was
quantified in presence of 0-8 μg of ERL using fluorescence
spectrophotometry (Panel A). Results are shown as mean±SD
(n=3). Representative agarose gel pictures after electrophoretic
separation of pDNA/ERL polyplexes prepared at different mass
ratios with 1 μg of pAcGFP1-C1 in PBS and SFM are shown
in Panel B and C, respectively. Lane 1: DNA control, Lane 2:
1.75 μg polymer, Lane 3: 3.5 μg polymer, Lane 4: 7 μg polymer,
Lane 5: 14 μg polymer, Lane 6: 28 μg polymer.
ERL irrespective of the buffer system used to fabricate these
polyplexes (Figures 3B and 3C). The favorable condensation
ability of pHPMA-b-pDMAEMA when compared to ERL may
be the result of increased interactions between the increased
number of cationic centers engineered in the polymer and
negatively charged pDNA. In addition, the presence of amide
group in HPMA moieties may facilitate greater intrapolymeric
interactions [14].
Physicochemical properties of polyplexes
Cellular internalization of electrostatically stabilized polyplexes
is a necessary prerequisite for successful gene delivery. Previous studies identified the critical relationship between
particle size and cellular uptake rates [24]. Consequently,
physicochemical properties such as size and zeta potential
were quantified for polyplexes fabricated with pHPMA-bpDMAEMA and ERL at different stoichiometric ratios. Since
pDNA/pHPMA-b-pDMAEMA condensation was incomplete
(see Figure 2C), physiochemical properties of these polyplexes
were only determined in PBS. The results summarized in
Tables 1 and 2 indicate that the mean diameter of fabricated
polyplexes becomes generally smaller in the presence of
increasing polymer. At charge neutrality (i.e., N/P=1), polyplexes
fabricated in PBS with pHPMA-b-pDMAEMA were significantly
Table 2. Physicochemical properties of ERL/pAcGFP1-C1
polyplexes fabricated in PBS and SFM.
N/P ratio
0.25
0.5
1
2
4
10
Size [nm]
PBS
SFM
834±112
764±73
732±37
717±4.0
598±20
617±28
366±8.0
170±4.0
182±4.0
115±6.0
163±5.0
123±2.0
Zeta potential [mV]
PBS
SFM
-33±4.0
-28±3.0
-35±3.0
-24±2.0
-5.0±1.0 -1.3±0.7
+26±3.0 +32±2.0
+26±2.0 +30±0.0
+24±1.0 +29±1.0
Data are shown as mean±SD (n=3)
smaller in size than corresponding nanoparticles prepared
with ERL. It is predicted that greater density of positively
charged amine groups in pHPMA-b-pDMAEMA induces more
effective condensation of pDNA into electrostatically-stabilized
nanoassemblies [25].
In addition to particle size, surface charge of polymer/pDNA
nanoassemblies is recognized as an important determinant
of cellular uptake. In general, positively charged composites
are electrostatically attracted to negatively charged cell surfaces, which can augment cellular uptake [24]. However, the
excessive positively charged nanoparticles are reported to
induce greater cell membrane damage and increased platelet
aggregation suggesting a less favorable safety profile [13].
The zeta potential of polyplexes fabricated with increasing
polymer concentrations shifts towards the positive range
due to neutralization of the negative charge associated with
nucleic acids (Tables 1 and 2). The surface charge of ERL/pDNA
nanoassemblies at N/P<1 was significantly more negative
than that of pHPMA-b-pDMAEMA/pDNA polyplexes (-20 to
-30 mV vs. -9 to -4 mV, respectively). It is hypothesized that the
greater presence of methacrylate moieties in ERL contributes
to this difference in zeta potential. Following neutralization,
however, pHPMA-b-pDMAEMA complexes prepared with excess cationic polymers (N/P>1) failed to reach zeta potentials
significantly greater than 0 mV. This may result from shielding
effects induced by phosphate buffer ions that exhibit high
affinity for primary amines present in pHPMA-b-pDMAEMA
[26]. In addition, it is suggested that HPMA moieties may also
contribute to charge shielding which is required to decrease
the interaction with the negatively charged blood components,
5
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
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With DNA
Without DNA
100
4
2
NA
pD
1
0
pHPMA-b-pDMAEMA/DNA (N/P) ratio
With DNA
Without DNA
100
0
NA
pD
4
50
2
B
50
1
Clinical success of gene delivery systems critically depends
on an acceptable safety profile as well as therapeutic efficacy.
Undesired cellular effect induced by polyplexes may result
from chemical features of excipients use to fabricate these
colloidal drug delivery systems and/or from physicochemical
properties associated with these electrostatically stabilized
nanoassemblies. The impact of pHPMA-b-pDMAEMA and ERL
particles fabricated in the presence and absence of pDNA was
assessed on HeLa cell viability by quantifying total cellular
ATP levels using the CellTiter-Glo® assay.
Irrespective whether or not pDNA was included in the
experiment, pHPMA-b-pDMAEMA did not significantly
compromise cellular safety up to concentrations required for
N/P=4 polyplexes (Figure 4A). In contrast, exposure of cells
to increasing ERL concentrations significantly reduced cell
viability by ~20% when compared to media control using
polymer concentrations required to fabricate polyplexes at
N/P=4 (Figure 4B). However, inclusion of pDNA ameliorated
the observed negative effect of ERL on cell viability (Figure 4B).
Electrostatically stabilized pDNA/polymer complexes not
only reduce excess positive charge of ERL but also alter the
size distribution of these colloids that interact with the cells.
Inclusion of pDNA produced larger particles than ERL alone
(Table 2), which is hypothesized to decrease cellular uptake and,
consequently, cytotoxicity [28]. Previously, it was reported that
a high positive charge density in polymers used to fabricate
nonviral gene delivery systems compromises cell viability [29].
pHPMA-b-pDMAEMA carries on a molar basis approximately
3-times the number of cationic centers than ERL. As cellular
safety of HeLa cells was not compromised after incubation
with pHPMA-b-pDMAEMA-containing polyplexes, it seems
that particle size and zeta potential have a greater impact
on viability of this cancer cell line than the positive charge
density of the polymer. These findings are consistent with
results reported earlier by Cai and co-workers [19].
A
Cell Viability
[% Normalized to meda control]
Cellular safety
Cell Viability
[% Normalized to meda control]
thereby reducing zeta potential of fabricated polyplexes [27].
doi: 10.7243/2050-120X-3-2
ERL/DNA (N/P) ratio
Figure 4. Cellular safety of methacrylate-based polymers
in HeLa cells in the presence and absence of pDNA.
Nanoparticles were formed in PBS or SFM with and without
1 μg of pAcGFP1-C1 using polymer compositions equivalent
to N/P=1, 2 and 4, respectively. HeLa cells were incubated for
4 hrs in SFM with nanoassemblies fabricated with pHPMAb-pDMAEMA (Panel A) and ERL (Panel B). Cell viability
was quantified 48 hrs post-treatment using the 2 CellTiterGlo® assay. Results are normalized to media control and
shown as mean±SD (n=3). *Significantly different (p<0.05).
an unbuffered NaCl solution demonstrate rapid decrease in
solution pH upon addition of 0.1N HCl increments (Figure 5).
PEI25k, which exhibits an excellent buffering capacity in the
Polyplex buffering capacity
lysosomal pH range between pH 5.0–7.4, effectively delays
Transgene expression after incubation with a nonviral gene rapid acidification that correlates with high transfection efdelivery system critically depends on effective release of in- ficiency in vitro [29]. The pH profile of pHPMA-b-pDMAEMA
ternalized polyplexes from the endosome [30]. During the after incremental 0.1 N HCl additions demonstrated a similar
transition from early to late endosome, the luminal pH of buffer capacity as measured for the PEI25k solution, which
endocytic vesicles rapidly decreases to approximately pH 5 in implies an effective “proton sponge” effect under endosomesupport of physiological degradation. The presence of weak relevant acidic conditions. These results suggest that secondbases in the endosomal compartment neutralizes ATPase- ary and tertiary amines present in pHPMA-b-pDMAEMA are
generated protons and increases the osmotic pressure that freely accessible to protonation despite the methacrylamide
ultimately induces vesicle rupture [31]. This “proton sponge” polymer backbone. Buffer capacity of solutions prepared
effect is predicted to enhance endosomal escape of nonviral with ERL was significantly reduced due to the presence of
gene delivery systems, thereby augmenting transfection quaternary ammonium groups that are unable to change the
efficiency. Experimentally, the buffer capacity is considered degree of ionization as a function of environmental pH. The
a suitable parameter to predict “proton sponge” effects of rapid decrease in pH profile observed with ERL following HCl
cationic polymers. The results from acid-base titrations of addition implies minimal buffer capacity and, consequently,
6
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
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No Transfection
No Transfection
50
2
0
50
100
0.1N Hcl [μl]
150
200
Figure 5. Polymer buffer capacity. Titration of a basic 20 μg/
mL solution of pHPMA-bpDMAEMA (▲), ERL (☐), PEI25k
( ), and 5 mM NaCl ( ) was performed using 0.1 N HCl.
The pH value of the mixture was measured after each
addition of HCl increment. Results are shown as average±SD
(n=3).
In vitro transfection efficiency
To assess transfection efficiency of polyplexes prepared with
the different methacrylate-based polymers, HeLa cells were
incubated with these nonviral delivery systems of the pAcGFP1C1 reporter gene in the presence and absence of 10% FBS. The
commercially available TurboFect™ transfection reagent was
used as positive control. Visual quantitation of the percentage
of GFP-expressing cells under various conditions is summarized in Figure 6. Combination of the pAcGFP1-C1 expression
plasmid with TurboFect™ according to manufacturer’s recommendation resulted in a mean transfection efficiency of nearly
80%. No transgene expression was observed with pDNA only.
Polyplexes prepared with ERL up to N/P=4 failed to induce
effective GFP expression, which was primarily attributed to the
limited buffering capacity of this quaternary amine-containing
polymer under acidic conditions that may have restricted
effective endosomal escape. In general, pDNA association
complexes fabricated with the various methacrylate-based
polymers at N/P=1 were ineffective in inducing significant
transgene expression. It is hypothesized that weak interactions at charge neutrality facilitate rapid dissociation of the
electrostatically stabilized nanoassemblies in the presence
of polyanions at the cell surface [34]. Polyplexes comprised
of pHPMA-b-pDMAEMA resulted in a high GFP expression
(Figure 6). Comparison of different N/P ratios demonstrated
ER
L
Tu
pH
N r
PM ak bofe
A- ed p ct
bpD DN
A
M
AE
M
A
4
ER
L
0
6
0
10%FBS
No Transfection
8
N/P=1
N/P=2
N/P=4
Serum Free
T
pH
N u
PM ak rbo
A- ed p fect
bpD DNA
M
AE
M
A
Polymer solution pH
pHPMA-b-pDMAEMA
ERL
NaCL
100
No Transfection
PEI
10
Transfection efficiency %
inefficient endosomal escape mediated by “proton sponge”
effects. This endosomal escape inhibition due to the lack of
buffering tertiary amine moieties was reported previously
[32,33].
doi: 10.7243/2050-120X-3-2
Figure 6. Transfection efficiency of methacrylate-based
polyplexes in HeLa cells in the presence and absence of serum.
Nanoassemblies were formed in PBS or SFM with 1 μg of
pAcGFP1-C1 using polymer compositions equivalent to N/
P=1, 2 and 4, respectively. HeLa cells were incubated for 4
hrs in the presence and absence of 10% FBS with polyplexes
fabricated with pHPMA-b-pDMAEMA and ERL. TurboFect™/
pDNA polyplexes prepared according to manufacturer’s
instructions were used as positive control. GFP-expressing
cells were identified 48 hrs post-treatment by confocal
microscopy. Transfection efficiency for each polymer was
quantified by determining the percentage of GFP-positive cells
in three random sections. Data are shown as mean±SD (n=3).
maximal transfection efficiency of HeLa cells around 65% at
N/P=4, which was not significantly different from polyplexes
prepared with TurboFect™.
Transfection efficiency of gene delivery systems under in
vivo conditions is generally reduced due surface adsorption
of plasma proteins that can alter surface charge and hydrodynamic radius of polyplexes [35]. To assess the impact of plasma
proteins on pAcGFP-1/C1 polyplexes fabricated with different
methacrylate-based catiomers, transfection efficiency in HeLa
cells was determined in the presence of 10% FBS. Polyplexes
fabricated at N/P=1 and ERL polyplexes remained ineffective
in inducing significant transgene expression. This implies that
proposed surface adsorption of plasma proteins does not
enhance but rather impede effective internalization and/or
endosomal escape. In comparison to the results obtained with
the same pHPMA-b-pDMAEMA polyplexes in the absence of
serum, inclusion of plasma proteins reduced transfection efficiency by at least 30% (Figure 6). The transfection efficiency of
pHPMA-b-pDMAEMA polyplexes at N/P=4 was 36±8%, which
is not significantly different from the results using the positive control TurboFect™. Despite the negative effect of serum
on transfection efficiency, the predicted impact of pHPMAb-pDMAEMA polyplexes as nonviral gene delivery systems
7
Aba Ellah et al. Journal of Pharmaceutical Technology & Drug Research 2014,
http://www.hoajonline.com/journals/pdf/2050-120X-3-2.pdf
remains superior to those fabricated with the FDA-approved
ERL polymer.
Conclusion
Comparative assessment of methacrylate-containing cationic
polymers revealed that polyplex formation with pAcGFP1-C1
plasmid is significantly influenced by the composition of
the polymer and the fabrication vehicle. Physicochemical
properties such as size and zeta potential of electrostatically
stabilized nanoassemblies depend on charge density engineered into the polymer. In the absence of pDNA, cellular
safety profile of secondary and tertiary amine-containing
pHPMA-b-pDMAEMA polymer was superior to that of
quaternary ammonium-containing ERL. However, electrostatically stabilized association complexes with pDNA
ameliorate this material-dependent cytotoxicity effect.
Polyplexes fabricated with pHPMA-b-pDMAEMA at N/P≥2
successfully induced transgene expression in HeLa cells in the
presence and absence of serum, which may be facilitated by
an effective endosomal escape due to substantial buffering
capacity associated with a “proton sponge” effect of secondary
and tertiary amino groups. Quaternary amine-containing
ERL polyplexes, in contrast, failed to induce GFP expression
in HeLa cells even in the absence of 10% FBS. Future in vivo
studies will have to demonstrate whether secondary and
tertiary amine-containing methacrylate-based polymers such
as pHPMA-b-pDMAEMA provide a significant advantage with
respect to safety and/or efficacy of nonviral gene delivery
systems that would justify the additional time and expense
required to obtain FDA regulatory approval for this novel
excipient.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Authors’ contributions
NHA SJP LT NA MME GNF EAI GMP
Research concept and design
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Collection and/or assembly of data
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Data analysis and interpretation
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Writing the article
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Acknowledgement
The authors would like to thank Dr. Helen Jones and Charles
Klanke (Cincinnati Children`s Hospital Medical Center, Cincinnati,
USA) for providing the GFP-expression plasmid. This research was
supported in part by a predoctoral fellowship from the Egyptian
Ministry of Higher Education awarded to Noura H. Abd Ellah.
Publication history
EIC: James Radosevich, University of Illinois, USA.
Received: 10-Oct-2014 Final Revised: 24-Nov-2014
Accepted: 05-Dec-2014 Published: 11-Dec-2014
doi: 10.7243/2050-120X-3-2
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doi: 10.7243/2050-120X-3-2
Citation:
Abd Ellah NH, Potter SJ, Taylor L, Ayres N, Elmahdy
MM, Fetih GN, Ibrahim E-SA and Pauletti GM. Safety
and efficacy of amine-containing methacrylate
polymers as nonviral gene delivery vectors. J Pharm
Technol Drug Res. 2014; 3:2.
http://dx.doi.org/10.7243/2050-120X-3-2
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