European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Research paper

Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole)

linear oligomers for gene therapy applications
D. Velasco a,, G. Rthor b, B. Newland b, J. Parra c, C. Elvira a, A. Pandit b, L. Rojo d, J. San Romn a
a

Institute of Polymer Science & Technology, Madrid, Spain

Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Ireland
c
Unidad de Investigacin CHA-CSIC, vila, Spain
d
Department of Materials, Imperial College, London, United Kingdom
b

a r t i c l e

i n f o

Article history:
Received 19 March 2012
Accepted in revised form 8 August 2012
Available online xxxx
Keywords:
Oligomers
Low polydispersities
Cationic polymers
Non-viral vectors
Acrylics
pH sensitive

a b s t r a c t
Nonviral methods for gene delivery are becoming ever more prevalent along with the need to design new
vectors that are highly effective, stable in biological uids, inexpensive, and facile to produce. Here, we
synthesize our previously reported monomer N-ethyl pyrrolidine methacrylamide (EPA) and evaluate
its effectiveness in gene vector applications when copolymerized with 1-vinylimidazole (VI). A range
of these novel linear cationic copolymers were synthesized via free radical polymerization with low
molecular weights (oligomers) and low polydispersities showing two pKa values as the two co-monomers
are cationic. DNApolymer polyplexes had average sizes between 100 and 250 nm and zeta-potentials
between 10 and 25 mV, and a strong dependence of composition on the size on the zeta-potential was
observed. The cytotoxicity of the homopolymers, oligomers, and polyplexes toward human broblasts
and 3T3 mouse broblasts was evaluated using the MTT and AlamarBlue assays, proving that formulations could be made with toxicity as low as low molecular weight linear poly (dimethylaminoethyl methacrylate) (PDMAEMA). The transfection capability of the polyplexes measured using the G-luciferase
marker gene far superseded PDMAEMA when evaluated in biological conditions. Furthermore, blood
compatibility studies showed that these new oligomers exhibit no signicant hemolysis or platelet activation above PBS controls. These new EPA based oligomers with low toxicity and ease of scalability show
high transfection abilities in serum conditions, and blood compatibility showing its potential for systemic
gene delivery applications.
2012 Elsevier B.V. All rights reserved.

1. Introduction
The proton-sponge hypothesis for endosomal escape, whereby
cationic polymers buffer in the low pH of the endosome to facilitate its swelling and subsequent rupture is a well-known concept
and has become an integral part of polymeric vehicle design [1].
However, polymers designed in such a way often suffer from several drawbacks, most predominant of which is high cytotoxicity. It
is therefore imperative to design advanced biocompatible materials that mimic the proton-sponge mechanism, allowing efcient
endosomal escape without increasing toxicity [2]. Here, it is also
important to note the role of the imidazole heterocycle in gene
delivery. The incorporation of imidazole moieties represents a
promising option for the improvement of endolysosomal escape

Corresponding author. Institute of Polymer Science & Technology, CSIC and

CIBER-BBN Juan de la Cierva, 3, 28006, Madrid, Spain. Tel.: +34 915 622 900x332;
fax: +34 915 644 853.
E-mail address: diegovb@ictp.csic.es (D. Velasco).

and enhancement of the efciency of polymers without increasing

toxicity [38].
Systemic gene therapies suffer from several drawbacks such as a
lack of stability, degradation, and clearance of the carrier from the
blood stream, making the elucidation of the interactions of new
vector systems with blood components essential [9,10]. For these
reasons, a substantial part of current research into systemic biomaterials is focused on the design and preparation of polymers with
blood compatibility properties. The strategies developed involve:
the preparation of amphiphilic polymers [1113]; hyperbranched
polymers [14,15]; copolymers of lactic-co-glycolic [16]; polyesters
[17] and poly (ethylene glycol) [18,19]. Another strategy is based
upon the introduction of amidoamine groups in the polyamides
backbone, which selectively adsorb heparin from plasma or blood,
giving stable complexes without any adverse effect on plasma proteins and blood cells [20]. High-molecular weight polymers tend to
form extremely stable DNA-polyplex aggregates, whereas homologous shorter polycations show enhanced hemocompatibility and
reduced toxic effects [21]. However, these complexes formed between short multivalent polycations and DNA molecules tend to

0939-6411/$ - see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejpb.2012.08.002

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

lack in stability under physiological or serum conditions leading to

lower transfection efciencies. For these reasons, new materials as
non-viral gene transfer vectors for gene therapy applications are required, which combine all of the above desired properties.
A very promising approach to achieve this goal arises through
the use of novel linear cationic oligomers derived from N-ethyl
pyrrolidine methacrylamide (EPA) and 1-vinylimidazole (VI) produced via the highly scalable, conventional free radical polymerization technique. This paper presents the synthesis, characterization,
complexation ability, blood compatibility and the impressive
transfection capability of these (EPA-co-VI) oligomers, recently
developed in our laboratories, and their evaluation to be used as
potential non-viral gene carriers under serum conditions.

infrared (ATR-FTIR) spectroscopies. 1H and 13C NMR spectra were

recorded in an INOVA-300 spectrophotometer. The spectra were
recorded in deuterium oxide (10% w/v). Using trimethylsilane
(TMS) as internal standard, ATR-FTIR spectra were recorded on a
PerkinElmer-Spectrum One spectrophotometer, with an ATR
attachment.
2.4. Chromatographic techniques

2. Materials and methods

Number and weight average molecular weighs were calculated

by Size Exclusion Chromatography (SEC) with a Shimadzu SIL 20AHT SEC with an isocratic pump serial LC-20D connected to a differential refractometric detector (serial RID-10A). Calibration of SEC
was carried out with a monodisperse polyethylene glycol standard
in the range of 1.0  103500  103 obtained from Scharlab.

2.1. Materials

2.5. MALDITOF

1-vinylimidazole (VI; SigmaAldrich) was distilled under reduced

pressure. N-ethyl pyrrolidine methacrylamide (EPA) was synthesized
as reported previously [22]. 2,2 Azobisisobutyronitrile (AIBN, Merck)
was recrystallized from methanol (mp 194 C). Ethyl a-bromoisobutyrate (EBr), (1,1,4,7,7-Pentamethyl-diethylenetriamine) (PMDTA),
L-ascorbic acid (AA), 2-(dimethylamino) ethyl methacrylate (DMAEMA), and copper (II) chloride (CuCl2) were purchased from Sigma.
The Gaussian princeps luciferase (G-luc) plasmid and accompanying
analysis kit was purchased from New England Biolabs. All the
solvents used were puried by standard procedures.

Matrix-assisted laser desorption ionization/time-of-ight mass

spectrometry (MALDITOF/MS) was performed on Voyager-DE
PRO equipment (Applied Biosystems). Samples were dissolved in
Milli-Q water (3 mM) and mixed with trans-3-indoleacrylic acid
as the matrix at a ratio of 1:2 (v/v). Mass spectra were recorded
in the positive ion mode using a nitrogen laser (337 nm) and measured in the linear mode.

2.2. Synthesis of poly (EPA-co-VI) copolymers

Poly (EPA-co-VI) copolymers were obtained by free radical polymerization with molar ratio feeds of EPA/VI monomers of 80:20,
50:50, and 20:80, respectively. The appropriate co-monomer mixtures were dissolved in N,N-dimethylformamide ([M] = 1 mol/L),
and the mixture solution was deoxygenated with N2 for 15 min.
Then, AIBN (1 wt% with respect to the monomers) was added to
the solution and the reaction medium transferred to an oven at
50 C for 24 h. The copolymers were puried by dialysis using
membranes Spectra/Por (cut-off 5001000 Da). pEPA and pVI
homopolymers were obtained following the same procedure described above for the p(EPA-co-VI) copolymers.
Linear PDMAEMA (10 kDa, PDI: 1.097) was synthesized by activated atom transfer radical polymerization according to an already
published protocol [23]. Briey, the reaction was carried out within
a two necked round bottomed ask containing the monomer DMAEMA and initiator ethyl a-bromoisobutyrate (EBr) (50:1 wt%), by
the addition of CopperI/PMDTA (1,1,4,7,7-Pentamethyl-diethylenetriamine) and leaving the reaction to proceed for 6 h at 50 C under
argon atmosphere. Aliquots of reaction mixture were withdrawn at
the start and end of the reaction time, diluted in DMF and run
through a silica gel column to remove copper catalyst and analyzed
by gel permeation chromatography. The reaction was stopped by
exposing the solution to the air, and the product obtained was collected from the reaction mixture by precipitation in hexane followed by drying under laminar ow. The polymer obtained was
then re-dissolved using rstly acetone and then adding distilled
water. The polymer solution was then reduced to pH 5 by the drop
wise addition of 1 M hydrochloric acid under constant stirring. This
was then dialyzed against distilled water for several days, before
being freeze dried for subsequent studies.
2.3. Spectroscopic techniques
The polymers were characterized by nuclear magnetic resonance (NMR) and attenuated total reectance fourier transform

2.6. Thermal analysis

Glass transition temperatures (Tg) were determined by Differential Scanning Calorimetry (DSC) using a PerkinElmer DSC-7 calorimeter. The samples (68 mg) were run under nitrogen
atmosphere and heated from 0 to 200 C at 10 C min1 taking as
Tg the onset point of the corresponding thermal transition.
2.7. Determination of the dissociation constants (pKa)
The dissociation constant values of the polymers were determined by acidbase titration of the polymer solution in a 25 ml
of ionic strength saline buffer (0.1 M NaCl). Diluted aqueous
solutions of NaOH (0.1 M) were used to complete the titration.
13 ml of a 0.1 N HCl solution was added to ensure the ionization
of the amine groups of the copolymers. The changes of pH were
measured with a Schott GC841 pH meter.
2.8. In vitro evaluation of cytotoxicity of the homopolymers and
copolymers
The in vitro biological performance of the studied homopolymers and copolymers was assessed with a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and human
skin dermal broblasts (DPK-SKDF-HS Dominon Pharmakine),
and serial dilutions of each formulations were made. Cellular viability was determined by dissolving the corresponding polymer
(2.5 mg/ml) in free-serum supplemented DMEM (Dulbeccos Modied Eagles Medium). This stock solution was successively diluted
with serum-free medium, to obtain twelve dilutions of each formulation (from 2.5 mg/ml to 0.001 mg/ml). As initial incubation of the
transfection agent is usually carried out in serum free-media, the
same strategy was used performed for the cytotoxicity study herein. Fibroblasts (subculture 11) were seeded at a density of 1  105 cells/ml in complete medium in a sterile 96-well culture plate and
incubated at 37 C in humidied air with 5% CO2 for 24 h. Then, the
medium was replaced with the corresponding dilution and incubated for 48 h (n = 3). A solution of MTT was prepared in warm
PBS (5 mg/ml). 100 ll of the solution was added to each well,
and the plates were incubated at 37 C for 4 h. Excess medium

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

and MTT were removed, and DMSO (Scharlau) was added to all
wells in order to dissolve the formazan produced by viable cells.
This was mixed for 10 min and the absorbance was measured with
a Biotek ELX808IU detector using a test wavelength of 570 nm and
a reference wavelength of 630 nm. The relative cell viability was
calculated from the equation:

Relative viability 100 

ODS  ODB
ODC  ODB ec:x

where ODS, ODB, and ODC are the optical density of formazan production for the sample, blank (culture medium without cells), and
control (free-serum supplemented culture medium), respectively.
A doseresponse curve of cellular viability was plotted in each case
to delineate the concentration that depressed MTT-formazan production by 50%, called the half maximal inhibitory concentration
or IC50 value.
2.9. Blood compatibility analysis
Human blood was drawn from healthy volunteers into vacutainers containing either EDTA or sodium citrate. Oligomers were
tested to elucidate the effect of the composition on various components of blood and determine their effect on erythrocytes, coagulation, and the complement system according to an already
published protocol [24]. Ethical approval was granted by the Human Ethics Committee of the National University of Ireland,
Galway.
2.10. Platelet activation
Whole blood was centrifuged at 85g for 15 min to remove platelet-rich supernatant. The remaining blood was again centrifuged
for 10 min at 140g and mixed with the previous extracted plasma
to get platelet rich plasma (PRP). Platelet poor plasma (PPP) was
obtained by centrifuging the remaining blood for 5 min at 800g.
The PRP was then diluted 1:100 with 1% ammonium oxalate to
get a platelet concentration of 6  108/ml. 300 ll of PRP was incubated with 40 lg of oligomers from all different sizes and surface
modications for 1 h at 37 C. The supernatant was then centrifuged at 2000g for 10 min. Platelet activation was measured by
the concentration of sP-selectin levels in the plasma and was
determined using an ELISA kit (Human soluble P-selectin Immunoassay, R&D Systems, Minneapolis, MN, #BBE 6) according to the
manufacturers protocol. Both PPP and PRP were used as controls.

they fell on the standard curve; sample values above the top end
of the curve were retested following further dilution. The measurements were done in duplicates.
2.12. Plasma clotting time
Howells method was employed to investigate plasma recalcication time. Blood was collected in a sodium-citrate vacutainers. It
was then centrifuged at 3000 rpm at 8 C, for 20 min to obtain the
platelet-poor plasma (PPP). 0.1 ml of the PPP and 40 lg of samples
suspended in PBS were incubated at 37 C for 5 min in a 96 well
plate. 0.1 ml of 0.025 M CaCl2 solution was then added and the
plasma solution was monitored for clotting by measuring the
absorbance using at 405 nm.
2.13. Hemolysis
EDTA-anticoagulated blood was centrifuged for 5 min at a speed
of 900g. The serum fraction was removed, and the volume was
raised to its original using 150 mM NaCl. This step was repeated
twice and the nal suspension was diluted 1:10 with 100 mM
phosphate buffer. 2  108 red blood cells/ml were incubated with
the oligomers each at a nal concentration of 100 lg/ml. PBS was
used as a negative control, whereas Triton X-100 1% (w/v) was
used as a positive control. All samples were incubated under gentle
agitation for 2 h at 37 C and centrifuged at 900g for 5 min. The
absorbance of the supernatant was measured for release of hemoglobin at 545 nm. The percentage of hemolysis was calculated as
follows:

Haemolysis %

Aa  Ac  100
Apc

where Aa, Ac, and Apc are the absorbance of test sample, absorbance
of control, and highest absorbance for positive control, respectively.
2.14. Polyplex formation and characterization
Polymer/DNA complexes (polyplexes) were prepared at room
temperature in pre-ltered PBS 1 h prior to use. Various quantities
of polymer were added to the DNA (with gentle vortexing) to form
nitrogen/phosphate (N/P) ratios from 2 up to 30 with no post-ltration performed so as not to lose material.
2.15. Agarose gel electrophoresis

2.11. Complement system

To assess complement activation, the cleavage of complement
component C3 was monitored by measuring the formation of its
activation peptides, C3a and C3a des arg, using a commercial C3a
enzyme immunoassay kit (BD Bioscience). Activation studies were
performed using pooled citrated plasma isolated by centrifugation
from whole blood donations. Equal volumes of plasma and polymer solution in saline were incubated at 37 C for 1 h. Briey, the
samples were diluted with the dilution buffer provided in the kit
and added to a microtiter plate coated with a monoclonal antibody
specic for human C3a and C3a des arg. After 1 h incubation at
room temperature to allow any C3a in the sample to bind to the
monoclonal antibody, the plates were washed and incubated with
peroxidase-conjugated rabbit anti-C3a for 15 min. Following a nal
wash step, the chromogenic substrate was added to detect bound
C3a. Absorbance was measured at 450 nm. The sample C3a concentrations were calculated using a standard curve with net absorbance values plotted on the y-axis for each C3a concentration
indicated on the x-axis. Sample values were accepted as valid if

To assess the polyplex formation via migration properties, a

0.9% (w/v) agarose gel was used, to which 100 V was applied for
30 min. Naked DNA (used as a control) and polyplex samples were
mixed with a sucrose loading dye prior to loading to the agarose
wells. Afterward, UV light (G:BOX Chemi XL, UK) was used to visualize the bands.
2.16. Determination of average particle size and zeta potential
Polyplexes were sized using dynamic light scattering (DLS) (NanoZS Malvern Instruments), and the surface charge was analyzed
using a Zetamaster system (Malvern Instruments). Polyplexes were
made up in distilled water to a nal volume of 800 ll with 25 lg of
DNA and added to a clear disposable zeta cell as reported previously [25]. Three polyplex preparations were made with three
readings obtained for each measurement, all carried out at 25 C
allowing 1 min of equilibrium time before acquiring data. ALV-Correlator Control Software was used for data acquisition using a
counting time from 300 s to 600 s for each sample.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

2.17. Transfection studies

Transfection experiments were performed with 3T3 broblasts
by using a plasmid encoding for a cell secreted gaussian luciferase
as a reporter gene. Cells were routinely grown in cell culture medium, DMEM (Sigma), supplemented with 10% fetal bovine serum
(FBS, Gibco), 200 mM L-glutamine (Sigma), 100 units/ml penicillin,
and 100 lg/ml streptomycin (Sigma). 15,000 cells were seeded on
a 48 well-plate 24 h prior to the addition of complexes. Different
polymer/plasmid DNA molar ratios ranging from 2/1 to 20/1 (N/
P) were used to prepare the polyplexes. A poly (dimethylaminoethyl methacrylate)/DNA formulations prepared at different ratios
(18/1, 22/1, 27/1, 31/1, and 36/1) (N/P) were used as controls. The
incubation of the polyplexes with the cells was performed either in
the presence or absence of serum. In an absence of serum transfection experiment, the cells were incubated with desired amounts of
polyplexes (200 ll dispersion with 1 lg plasmid DNA per well) for
4 h at 37 C in a humidied 5% CO2-containing atmosphere. Then,
free-serum medium was removed and fresh culture medium was
added. In the presence of serum transfection experiment, cells
were incubated with desired amounts of polyplexes in a culture
serum medium. Cells were cultured for 2 days. Cell medium was
collected and analyzed for the production of luciferase protein.
The assay was performed using the Gaussia princeps luciferase assay kit (New England Biolabs) according to the supplier instructions. The luciferase expression was quantied by measuring
luminescence (RLU/15,000 cells) with a plate reader (VarioSkan).
2.18. Cell viability of the polyplexes
Cell viability of the polyplexes was measured using the AlamarBlue cell metabolic assay. The mouse 3T3 cell line (also referred
to as NIH/3T3) is commonly used to analyze the transfection capabilities of new polymers, so this cell line was chosen again here for
this study to allow ease of data comparison [26,27]. This assay was
performed after 2 days. Cells were washed with Hanks Balanced
Salt Solution (HBSS). AlamarBlue (BioSource International,
Invitrogen, Ireland), diluted by a factor of 10 in HBSS, was added
to each well. After 3 h of incubation, the absorbance of each sample
was measured in a 96-well plate, at wavelengths of 550 and
595 nm, using a microplate reader (VICTOR3 V Multilabel
Counter, PerkinElmer BioSignal Inc, USA). The percentage of
AlamarBlue reduction was calculated using a correlation factor
RO in accordance with the suppliers instructions.
2.19. Statistical analysis of the data
Unless otherwise stated, all of the in vitro polymer and polyplex
characterization experiments were performed in triplicate, with an
n number of three for each experiment and an average taken for
analysis. Analysis of variance (ANOVA) was performed by using
Statistica 6.0 software (Statsoft, Tulsa, USA). The statistical analysis
of the results was done by the application of one-way ANOVA and a
value of p < 0.05 was considered signicant where mentioned in
text.

O
N

N
H

N
1-vinylimidazole (VI)

N-ethyl pyrrolidine methacrylamide (EPA)

AIBN
DMF
T = 50 C

H
N

N
N
p(EPA-co-VI)
Scheme 1. Free radical copolymerization reaction of the VI and EPA monomers.

polymers, could buffer the endosome and potentially induce its

rupture [3]. Herein, three oligomers of EPA/VI were prepared in
order to increase the buffering capacity and to give more biocompatibility to the EPA monomer. The yield of the EPA and VI copolymerizations and their respective homopolymers after 24 h of
reaction was about 8590%. The free radical polymerization of
EPA and VI monomers was carried out following well known polymerization procedures (Scheme 1) [28].
The EPA and VI feed molar fractions were 0.8/0.2, 0.5/0.5, and
0.2/0.8, respectively. The composition of the systems was determined with the signals (Fig. 1) that appear between 6.7 and
7.3 ppm to the CHb2 ACHANACHACHAN and (NACHAN) protons
of VI that increase in intensity with the increase in the molar feed
composition of this monomer in the oligomers.
1
H NMR signals assignments (D2O) (ppm) and FTIR signals
assignments, stretching vibrations, m (cm1) (see also Fig. 1):
PolyEPA. 1H NMR spectrum (D2O): dH 1.8 (CHb2 ), 1.1 and 1.0
(CHa3 ), 4.2 (NHCH2), 3.3 (CH2N), 2.6 (CH2NCH2 cycle), 1.8 (CH2CH2
cycle). ATR-FTIR spectrum (cm1, the most characteristic bands):
3346 (NH), 29642814 (CH), 1630 (CO), 1141 (OCH2), 1203 (NCH2).

3. Results and discussion

3.1. Copolymerization and characterization of EPA and VI oligomers
Important research has been moving toward the development
of polycation-based gene-delivery systems designed to minimize
nuclease degradation through the design of vectors with the capacity to escape the endosome/lysosome. Polymers with buffering
capacities between 7.2 and 5.0, such as imidazole-containing

Fig. 1. 1H NMR spectra of the poly (EPA-co-VI) copolymers.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx
Table 1
Characterization of the homopolymers and copolymers: Molar feed and nal compositions, molecular weights, glass temperature, and dissociation constants.
SAMPLE

Molar feed composition

Molar copolymer composition

Mw (Da) [SEC]

Polydispersity index

DSC Tg (C)

Titration pKa

Poly-EPA
80 EPA 20 VI
50 EPA 50 VI
20 EPA 80 VI
Poly-VI

EPA: 0.80 VI: 0.20

EPA: 0.50 VI: 0.50
EPA: 0.20 VI: 0.80

EPA: 0.84 VI: 0.16

EPA: 0.57 VI: 0.43
EPA: 0.19 VI: 0.81

121,900
914
983
933
200,000

2.5
1.2
1.3
1.2
3.1

141
132
151
157
167

5.6
5.7,6.3
5.6,6.5
5.6,6
6.1

Poly VI. 1H NMR spectrum (D2O): dH 1.9 (CHb2 ), 3.0 (CHb2 ACH),
7.0 (CHb2 ACHANACHACHAN), 6.7 (CHb2 ACHANACHACHAN), 7.3
(NACHAN). ATR-FTIR spectrum (cm1, the most characteristic
bands): 3101 (N@CH), 2952 (CHsp3), 1497 (C@C).
Poly (EPA-co-VI). ATR-FTIR spectrum (cm1, the most characteristic bands): 3346 (NH), 29642814 (CH), 1630 (CO), 1141 (OCH2),
1203 (NCH2) 3101 (N@CH), 2952 (CHsp3), 1497 (C@C).
Table 1 shows a summary of the polymer system characterization. No differences were found between the molar feed and nal
compositions. Surprisingly, for a conventional radical polymerization, the copolymers showed much lower molecular weights
(9001000 Da) and polydispersities (1) in comparison with their
respective homopolymers EPA and VI (121,000 and 200,000 Da),
respectively. These results constitute one of the most relevant results exposed in this work as controlled low molecular weight
and near 1 polydispersity cationic copolymers are achieved by
standard free radical copolymerization avoiding the use of more
complicated reactions and conditions such as RAFT or ATRP
polymerization techniques [29,30]. In fact, the obtaining of such
near monodisperse copolymers has been conrmed by an independent technique, MALDITOF (Fig. 2), showing molecular weights

between 3000 and 5000 Da and polydispersities between 1.1 and

1.2. These low average molecular weight and polydispersity index
values are expected according to reported free radical copolymerizations of vinyl imidazole monomers and methacrylic related
comonomers [3133]. These show a deviation from classical mechanism due to a partial coordination between imidazole monomeric
units and propagating radicals, which stimulate competing reactions and the occurrence of degradative reactions. This leads to
the formation of a relatively unreactive radicals responsible of
the low molecular weight values reported for vinyl imidazole
based copolymers ranged between 300 and 4000 Da [3436]. Additionally, different spectroscopic studies, taking into consideration
end group analysis, have conrmed that controlled weight
distribution of near monodisperse imidazole containing macromolecular entities can be obtained when appropriate radical polymerization parameters are chosen [37,38].
Tg values of the corresponding polymers were found to be between 141 and 167 C. The Tg of the copolymers increased when
the VI monomer increased in the composition as the Tg of the VI
homopolymer was 167 C, higher in comparison with the EPA
homopolymer. The ionisable character of the polymers was studied

Fig. 2. MALDITOF spectra of the (A) 80 EPA/20 VI, (B) 50 EPA/50 VI, and (C) 20 EPA/80 VI copolymers and their Mn, Mw, and PDI values.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

Table 2
Half maximal inhibitory concentration (IC50) values obtained for the formulations
evaluated.
Formulation

IC50 (mean 95% condence interval) (mg/ml)

PVI
PDMAEMA
PEPA
80EPA20VI
50EPA50VI
20EPA80VI

0.570 0.638
0.548 0.359
0.008 0.002
0.057 0.029
0.347 0.267
0.526 0.262

by the determination of the dissociation constants pKa in ionic

strength buffered. It was observed that two pKa values (between
5 and 6) were obtained for the copolymers with an increase in
the buffering capacity in comparison with the homopolymer EPA
as can be seen in the Table 1 (also see Supporting material).

3.2. In vitro evaluation of cytotoxicity of homopolymers and oligomers

Biological activity of homopolymers and copolymers was evaluated on human broblasts cultures by using the MTT assay to obtain the doseresponse curves of relative cell viability and IC50
values for each compound against this cell line. The doseresponse
curve obtained from the relative cellular viability values of the culture medium corresponds to the PVI formulation, which is similar
to that of the poly (DMAEMA) formulations (Table 2). Indeed, the
IC50 concentration values for PVI do not differ signicantly neither
obtained values for poly (DMAEMA) (F1,4 = 0.018; p = 0.90).
In the case of the poly-EPA, the doseresponse curve indicated
that it is a system more toxic in comparison with the PVI and poly
(DMAEMA); this fact is supported with the inhibitory concentration values obtained for this system, which are signicantly lower
than the values for PVI (F1,4 = 14.375; p < 0.05) and poly (DMAEMA)
(F1,4 = 41.751; p < 0.01). In accordance with this observation,
copolymers richer in EPA are more toxic than the other two
copolymers, indeed the IC50 concentration values for the

80EPA20VI copolymer differs signicantly in comparison with

the other two copolymers (F1,4 = 21.588; p < 0.01) to 50EPA50VI
and F1,4 = 58.377; p < 0.01 to 20 EPA 80 VI). Besides, the IC50
obtained for the 80EPA20VI system is signicant higher than that
obtained for EPA (F1,4 = 53.849; p < 0.01) and signicantly lower
than that of the PVI (F1,4 = 11.949; p < 0.05), and as Fig. 3 shows,
its cytotoxicity is placed between the EPA and PVI polymers.
The 50EPA50VI and 20EPA80VI systems have a doseresponse
curve similar to that obtained for PVI; furthermore, the IC50 values
of these copolymers do not differ signicantly compared to the obtained value for PVI (F1,4 = 1.932 and p = 0.24 with respect to
50EPA50VI; F1,4 = 0.078, p = 0.279 with respect to 20EPA80VI).
3.3. Blood compatibility
One of the major drawbacks of synthetic carriers for gene delivery is their low circulation time due to their rapid clearance from
the blood stream [39]. Consequently, the understanding of their
interactions with blood components appears to be essential. Several parameters were investigated to determine the interactions
of our new polymers with blood components and their potential
application as gene delivery vehicle via systemic administration.
Red blood cell lysis is a well known method to determine interactions between particles and erythrocytes. During this experiment,
PBS (Phosphate Buffer Saline) and TritonX were used as negative
(0%) and positive (100%) controls, respectively. No signicant differences have been observed between conditions when compared
to each other and negative controls It is also worth noting that
all copolymers have a negligible effect on hemolysis (less than
0.1%) (Fig. 4A).
Another key parameter of blood incompatibility is represented
by platelet activation after interaction with particles. Indeed, cationic polymers are described to induce platelet aggregation and
activation, which could lead to thrombotic complications in vivo
[40]. The release of soluble P-Selectin was quantied to evaluate
the platelet activation after incubation with all copolymers. PBS
was used as negative control, and the results show that there is

Fig. 3. Comparison of doseresponse curves (MTT assay) of PVI, PDMAEMA, and EPA and the copolymers 80EPA20VI, 50EPA20VI, and 20EPA80VI against human broblasts.
Each point represents the mean and vertical lines represent the standard deviation (n = 3). From these diagrams, the IC50 values were determined.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

Fig. 6. Plasma recalcication time, quantied using calculation of the point at

which the recalcication prole reaches half of the maximum absorbance value.
Data are represented as the mean standard deviation.

Fig. 4. (A) Hemolysis after incubation with human erythrocytes. (B) Platelet
activation as indicated by sP-Selectin release. Data are represented as the
mean standard deviation (n = 3).

no signicant difference between our polymers and PBS (Fig. 4B).

Complement system activation is also another parameter to be
analyzed in the eld of synthetic carriers for delivery as they could
lead to the activation of the immune system [24]. After incubation
with all copolymers, the release of C3a has been investigated using
PBS as negative control and insulin (a potent complement activator) as positive control [14], and no signicant differences have
been observed between our samples and the control (Fig. 5).
For the clotting process studies, plasma re-calcication proles
are used to mimic the intrinsic coagulation system in vitro. To
quantify plasma re-calcication proles, T1/2max was calculated as
the time at which half the saturate absorbance was reached [24].
PBS was used as a negative control in this study. It was observed
that the clotting time is signicantly longer when VI copolymer
drops from 80% to 50% (Fig. 6). Moreover, it appears to be undeterminable when this ratio decreases to 20%.
EPA/VI copolymer ratios exerted no signicant inuence on
most of the blood compatibility parameters we have evaluated
through this investigation, except for plasma re-calcication time.
Indeed, while the EPA copolymer ratio increases, the kinetics of the

Fig. 5. Complement system activation as indicated by C3a release. Data are

represented as the mean standard deviation.

re-calcication slows down to reach a non-measurable time with

80 EPA 20 VI polymer, which indicates that the clotting time of
polyplexes made with a higher ratio of EPA is longer than the others. These results are consistent with investigation reported in the
literature, which show an increase in the blood coagulation time
when synthetic polymers are used (poly(ethyleneglycol), functionalized polyethylene terephthalate, sulfate polymers). It could be
explained by a suppression of the intrinsic blood coagulation system because of the steric repulsion to proteins that reach the surface [41,42].
3.4. Polyplex formation and characterization
Agarose gel electrophoresis (Fig. 7) showed that EPA and VI
homopolymers were not able to complex DNA at low N/P ratios.
This phenomenon is due to the disposition of the EPA homopolymer chains in solution and the lack of total ionization of the VI
homopolymer due to its pKa (6.5). Since it is known that 5% of
the imidazole groups are only protonated at pH 7.2 (3), this effect
of poor complexation ability is more pronounced than for poly(E-

Fig. 7. Electrophoretic mobility of plasmid DNA in polymer/DNA polyplexes with

2:1, 4:1, 6:1, 8:1, 10:1, 15:1, and 20:1 P/N ratios.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

Hydrodynamic Diameter (nm)

300

80 EPA 20 VI
50 EPA 50 VI
20 EPA 80 VI

250
200
150
100
50
0

2:1

4:1

6:1

8:1

10:1

15:1

pared at different polymer/plasmid DNA ratios ranging from 2:1

to 20:1 (N/P) were used. Polyplexes were incubated with the cells
in the presence and absence of 10% serum during 4 h. Non-transfected cells, cells treated with plasmid alone, and cells treated with
complexed linear poly (DMAEMA) were used as negative and positive controls, respectively. Different N/P ratios (18:1, 22:1, 27:1,
31:1, and 36:1) of poly (DMAEMA) (Fig. 9C) were selected due to
their highest transfection. Low transfection levels were observed
after treatment with the EPA and VI homopolymers (data not
shown) in comparison with the copolymers. This phenomenon
can be attributed to the lack of total complexation between the
homopolymers and DNA until 10/1 ratio (N/P). In the case of the
copolymers, the highest transfection efciency in 3T3 broblasts
was obtained with 80 EPA/20 VI copolymer from the ratio 6:1 to

20:1

N:P Ratio

Zeta Potential (mV)

30
25

80 EPA 20 VI
50 EPA 50 VI
20 EPA 80 VI

20
15
10
5
0

2:1

4:1

6:1

8:1

10:1

15:1

20:1

30:1

N:P Ratio
Fig. 8. Average size (A) and zeta potential (B) of copolymer/DNA polyplexes
measured at different P/N ratios.

PA) that contains both secondary and tertiary amines. On the other
hand, the copolymers bound DNA at all ratios and the complexation can be explained by a possible change in the disposition of
the chains in solution, which facilitates the ionic interaction between the tertiary amines of EPA and the phosphate groups of
DNA.
A degree of variation between the formulations was noticed for
all of the copolymers, a phenomenon previously experienced.
However, the polyplexes were in the size range of between 90
and 300 nm with a general trend toward larger diameter with
increasing N/P ratio (Fig. 8A). The size of the 80EPA/20VI (170
300 nm) polyplexes was higher in comparison with other compositions due to a possible aggregation between the complexes. This
can be attributed to a decrease in the charge in its structure
(Fig. 4B) as lower ionization degree in that copolymer composition
with lower content of imidazole monomer gives less repulsion between copolymers chains. The size of the 50EPA/50VI and 20EPA/
80VI polyplexes was between 90 and 240 nm, increasing the size
of the polyplexes with the increase in the N/P ratios, showing lower average size copolymers richer in VI except in the 6/1 N/P ratio.
Zeta-potential measurements (Fig. 8B) of the polyplexes were between 10 and 25 mV. The 80EPA/20VI zeta-potential was lower
compared to 50EPA/50VI and 20EPA/80VI compositions. This is related to the size data presented previously, which shows a lower
content of imidazole implies a decrease in the charge.
3.5. Transfection studies
The transfection efciency of the polymers complexed to DNA
(polyplexes) was studied using 3T3 broblasts. Polyplexes pre-

Fig. 9. Transfection efciency of polymer/DNA polyplexes in 3T3 in comparison

with (poly dimethylaminoethyl methacrylate)/DNA polyplexes without (A) and
with serum (B) at day 2. (C) Transfection efciency of (poly dimethylaminoethyl
methacrylate)/DNA polyplexes without and with serum at day 2. Asterisk denotes
signicant difference between the groups (One-way ANOVA, p < 0.05).

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

(EPA), which may inhibit serum-induced transfection reduction

[50].
3.6. Cell viability of the polyplexes
Cell metabolic activity in the presence and absence of serum
after treatment by the 80 EPA 20 VI and 50 EPA 50 VI polyplexes
was evaluated by the AlamarBlue (AB) assay in order to evaluate
whether the toxicity of the polyplexes might affect the transfection
efciency. Fig. 10 presents the metabolic activity for all the polyplexes at different polymer/plasmid DNA ratios ranging from 2:1
to 20:1 (N/P). The data were normalized to the non-treated cells
(indicating a 100% cell metabolic activity). Synthesized polyplexes
showed an absence or low toxicity 2 days after treatment in the
presence and absence of serum. No signicant difference inmetabolic activity was noted between poly (DMAEMA) treated cells,
cells alone, and 80 EPA 20 VI and 50 EPA 50 VI systems treated
cells, indicating these polyplexes cause little to no cytotoxicity at
the concentrations administered.
4. Conclusions

Fig. 10. Cell metabolic activity of the polyplexes after 2 days in the presence (A)
and absence (B) of serum. Different polymer ratios were tested 2:1, 4:1, 6:1, 8:1,
10:1, 15:1, and 20:1 polymer/DNA ratios. No statistically signicant difference was
observed between the groups (One-Way ANOVA p < 0.05).

15:1, being the highest at 10:1. For 50 EPA/50VI copolymer, higher

transfections at 10:1 N/P ratios were found in comparison with
poly (DMAEMA), whereas no transfection was found for 20 EPA/
80 VI (data not shown) indicating that there is a minimum concentration of EPA monomer at which the copolymers become ineffective for gene delivery (Fig. 9A). It can be noted that it was necessary
to use higher N/P ratios of PDMAEMA to obtain higher transfections in comparison with the 80 EPA/20 VI and 50 EPA/50 VI
copolymers. This phenomenon has been observed previously with
PDMAEMA in comparison with PEI, a Superfect dendrimer and LipoFectin [25], which could be very interesting in terms of obtaining
higher transfections and less toxic systems.
The transfection of the 80 EPA/20 VI and 50 EPA/50 VI copolymers in the presence of serum was also studied. Although polymeric transfection agents may exhibit reduced transfection
capability in the presence of serum, perhaps due to the copolymer
architecture or molecular weight [43], we found that the reverse is
true for both the 80 EPA 20 and the 50 EPA 50, where greater transfection was observed in the presence of serum as reported elsewhere [4449]. To check if the reduced cytotoxicity of the EPA
containing polyplexes could be the reason for improved transfection efciency in the presence of serum, cytotoxicity studies were
performed (Fig. 10) with polyplexes with and without addition of
serum and no differences were found.
While 80 EPA/20 VI and 50 EPA/50 VI copolymers were able to
mediate signicantly higher transfection with respect to serumfree transfection, the performance of PDMAEM decreased signicantly as can be seen in Fig. 9B. Moreover, a trend was observed
in the transfection of the 80 EPA/20 VI and 50 EPA/50 VI copolymers in presence of serum where an increase in transfection level
when the N/P ratio was increased being the transfection of 50 EPA/
50 VI higher due to a possible decrease in the active amine content

Three different compositions of new cationic copolymers of Nethyl pyrrolidine methacrylamide (EPA) and 1-vinylimidazole
(VI) (80/20, 50/50 and 20/80) have been prepared by radical polymerization and characterized for their application as gene vectors.
Low molecular weight copolymers with low polydisperisty were
obtained being one of the most relevant results exposed in this
work. Blood compatibility has also been checked and showed no
signicant difference compared to controls except for re-calcication time. The respective homopolymers, poly-EPA and poly-VI,
complexed DNA only at high N/P ratios of 10:1 and 20:1, respectively. In comparison, the three copolymers formed complexes
from a 2:1 ratio upwards. DNApolymer polyplexes average sizes
between 100 and 250 nm and f-potentials between 10 and
25 mV. Biological activity of homopolymers, copolymers, and polyplexes was evaluated on human broblasts and 3T3 mouse broblasts by using the MTT assay and AlamarBlue assays. 80/20
and 50/50 copolymers produced transfection capabilities in serum-free media from the N/P ratios 8:1 to 15:1, which were comparable to poly (DMAEMA) that needed higher N/P ratios to reach
similar transfection values. More impressively, 80/20 and 50/50
copolymers also showed the ability to retain and increase their
transfection properties in the presence of serum when the N/P ratios increased, while poly (DMAEMA) decreased signicantly.
Acknowledgments
The authors thank nancial support from the EU project SURFACET, the NoE, EXPERTISSUES, and the CICYT project MAT 201018155. Diego Velasco thanks the grant I3P from the CSIC and Rafaela Galera for her support. Carlos Elvira would like to acknowledge
to PIE-CSIC programme (200660I022) for nancial support. The
authors would like to thank Science Foundation of Ireland, Strategic Research Cluster (SRC), Grant number 07/SRC/B1163.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejpb.2012.08.002.
References
[1] A.K. Varkouhi, M. Scholte, G. Storm, J. Haisma, Endosomal escape pathways for
delivery of biological, J. Control. Release 151 (2011) 220228.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002

10

D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxxxxx

[2] D.W. Pack, A.S. Hoffman, S. Pun, P. Stayton, Design and development of
polymers for gene delivery, Nat. Rev. Drug Discovery 4 (2005) 581593.
[3] P. Midoux, C. Pichon, J. Yaouanc, P. Jaffrs, Chemical vectors for gene delivery: a
current review on polymers, peptides and lipids containing histidine or
imidazole as nucleic acids carriers, Br. J. Pharmacol. 157 (2009) 166178.
[4] V.B. Morris, C.P. Sharma, Folate mediated histidine derivative of quaternised
chitosan as a gene delivery vector, Int. J. Pharm. 389 (2010) 176185.
[5] R.K. June, K. Gogoi, A. Eguchi, X. Cui, S.F. Dowdy, Synthesis of a pH-sensitive
nitrilotriacetic linker to peptide transduction domains to enable intracellular
delivery of histidine imidazole ring-containing macromolecules, J. Am. Chem.
Soc. 132 (2010) 1068010682.
[6] C.K. Wang, S. Jiang, S.H. Pun, Localized cell uptake of His-tagged polyplexes
immobilized on NTA self-assembled monolayers, Langmuir 26 (2010) 15445
15452.
[7] V. Bennevault-Celton, A. Urbach, O. Martin, C. Pichon, P. Gugan, P. Midoux,
Supramolecular assemblies of histidinylated a-cyclodextrin in the presence of
DNA scaffold during CDplexes formation, Bioconjug. Chem. 22 (2011) 2404
2414.
[8] J. Gu, X. Wang, X. Jiang, Y. Chen, L. Chen, X. Fang, X. Sha, Self-assembled
carboxymethyl poly (L-histidine) coated poly (b-amino ester)/DNA complexes
for gene transfection, Biomaterials 33 (2012) 644658.
[9] P.P. Kundu, V. Sharma, Synthetic polymeric vectors in gene therapy, Curr. Opin.
Solid State Mater. Sci. 12 (2008) 89102.
[10] J.H. Park, S. Lee, J.H. Kim, K. Park, K. Kim, I.C. Kwon, Polymeric nanomedicine
for cancer therapy, Prog. Polym. Sci. 33 (2008) 113137.
[11] J.N. Kizhakkedathu, C.K. Nisha, S.V. Manorama, S. Maiti, Water-soluble
nanoparticles from random copolymer and oppositely charged surfactant. 3.
Nanoparticles of poly (ethylene glycol)-based cationic random copolymer and
fatty acid salts, Macromol. Biosci. 5 (2005) 549558.
[12] C.K. Nisha, S.V. Manorama, M. Ganguli, S. Maiti, J.N. Kizhakkedathu, Complexes
of poly (ethylene glycol)-based cationic random copolymer and calf thymus
DNA: a complete biophysical characterization, Langmuir 20 (2004) 2386
2396.
[13] M. Ganguli, K.N. Jayachandran, S. Maiti, Nanoparticles from cationic copolymer
and DNA that are soluble and stable in common organic solvents, J. Am. Chem.
Soc. 126 (2004) 2627.
[14] R.K. Kainthan, M. Gnanamani, M. Ganguli, T. Ghosh, D.E. Brooks, S. Maiti, J.N.
Kizhakkedathu, Blood compatibility of novel water soluble hyperbranched
polyglycerol-based multivalent cationic polymers and their interaction with
DNA, Biomaterials 27 (2006) 53775390.
[15] R.K. Kainthan, J. Janzen, E. Levin, D.V. Devine, D.E. Brooks, Biocompatibility
testing of branched and linear polyglycidol, Biomacromolecules 7 (2006) 703
709.
[16] L. He, L. Yang, Y. Duan, L. Deng, X. Sun, Z. Gu, Z. Zhang, Cytotoxicity and
hemocompatibility of a family of novel MeOPEGPoly (D, L-lactic-co-glycolic
acid)PEGOMe triblock copolymer nanoparticles, J. Appl. Polym. Sci. 113
(2009) 29332944.
[17] D. Motlagh, J. Allen, R. Hoshi, J. Yang, K. Lui, G. Ameer, Hemocompatibility
evaluation of poly(diol citrate) in vitro for vascular tissue engineering, J.
Biomed. Mater. Res. A 82 (2007) 907916.
[18] D. Wang, J. Ji, C. Gao, G. Yu, L. Feng, Surface coating of stearyl poly(ethylene
oxide) coupling-polymer on polyurethane guiding catheters with poly(ether
urethane) lm-building additive for biomedical applications, Biomaterials 22
(2001) 15491562.
[19] S. Zhu, F. Qian, Y. Zhang, C. Tang, Ch. Yin, Synthesis and characterization of PEG
modied N-trimethylaminoethylmethacrylate chitosan nanoparticles, Eur.
Polym. J. 43 (2007) 22442253.
[20] R.K. Dey, A.R. Ray, Synthesis, characterization, and blood compatibility of
polyamidoamines copolymers, Biomaterials 24 (2003) 29852993.
[21] L. Wan, Y. You, Y. Zou, D. Oupicky, G. Mao, DNA release dynamics from
bioreducible poly(amido amine) polyplexes, J. Phys. Chem. B 113 (2009)
1373513741.
[22] D. Velasco, C. Elvira, J. San Romn, New stimuli-responsive polymers derived
from morpholine and pyrrolidine, J. Mater. Sci.: Mater. Med. 19 (2008) 1453
1458.
[23] B. Newland, H. Tai, Y. Zheng, D. Velasco, A. Di Luca, S.M. Howdle, C. Alexander,
W. Wang, A. Pandit, A highly effective gene delivery vector-hyperbranched
poly (2-(dimethylamino) ethyl methacrylate) from in situ deactivation
enhanced ATRP, Chem. Commun. 46 (2010) 46984700.
[24] C.D. Biraja, G. Rethore, M. Monaghan, K. Fitzgerald, W. Gallagher, A. Pandit, The
inuence of size and charge of chitosan/polyglutamic acid hollow spheres on
cellular internalization, viability and blood compatibility, Biomaterials 31
(2010) 81888197.
[25] B. Newland, Y. Zheng, Y. Jin, M. Abu-Rub, H. Cao, W. Wang, A. Pandit, Single
cyclized molecule versus single branched molecule: a simple and efcient 3D
Knot polymer structure for nonviral gene delivery, J. Am. Chem. Soc. 134
(2012) 47824789.

[26] M. Breuing, U. Lungwitz, R. Liebl, A. Goepferich, Breaking up the correlation

between efcacy and toxicity for nonviral gene delivery, Proc. Natl. Acad. Sci.
USA 104 (2007) 1445414459.
[27] I. Kap Ko, A. Ziady, S. Lu, Y. Jik Kwon, Acid-degradable cationic methacrylamide
polymerized in the presence of plasmid DNA as tunable non-viral gene carrier,
Biomaterials 29 (2008) 38723881.
[28] H.G. Elias, Macromolecules, Chemical Structures and Synthesis, vol. 10, WileyVCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2005 (Chapter 10).
[29] Y. Zheng, H. Cao, B. Newland, Y. Dong, A. Pandit, W. Wang, 3D single cyclized
polymer chain structure from controlled polymerization of multi-vinyl
monomers: beyond Flory-Stockmayer theory, J. Am. Chem. Soc. 133 (2011)
1313013137.
[30] Y. Zheng, B. Newland, H. Tai, A. Pandit, W. Wang, Single cyclized molecule
structures from RAFT homopolymerization of multi-vinyl monomers, Chem.
Commun. 48 (2012) 30853087.
[31] N. Pekel, N. Sahiner, O. Gven, Z. Rzaev, Synthesis and characterization of Nvinylimidazoleethyl methacrylate copolymers and determination of
monomer reactivity ratios, Eur. Polym. J. 37 (2001) 24432451.
[32] N. Pekel, Z. Rzaev, O. Gven, Synthesis and characterization of Poly(Nvinylimidazole-co-acrylonitrile) and determination of monomer reactivity
ratios, Macromol. Chem. Phys. 205 (2004) 10881095.
[33] V.V. Annenko, E.N. Danilovtseva, H. Tenhu, V. Aseyev, S.P. Hirvonen, A.I.
Mikhaleva, Copolymers of 1-vinylimidazole and (meth)acrylic acid: synthesis
and polyelectrolyte properties, Eur. Polym. J. 40 (2004) 10271032.
[34] C.H. Bambord, E. Schoeld, Non-classical free-radical polymerization:
degradative addition to monomer in the polymerization of 1-vinylimidazole,
Polymer 22 (1981) 12271235.
[35] A. Chapiro, Peculiar aspects of the free-radical polymerization of 1vinylimidazole, Radiat. Phys. Chem. Int. J. Radiat. Appl. Instrum. C. 40 (1992)
8993.
[36] M.G. Joshi, F. Rodriguez, Photoinitiated polymerization of 1-vinylimidazole, J.
Appl. Polym. Sci. 29 (1984) 13451354.
[37] B.B. Dambatta, J.R. Ebdon, T.N. Huckerby, Unusual inuences of temperature
and medium on the tacticity of radically polymerized poly(n-vinyl imidazole),
Eur. Polym. J. 20 (1984) 645652.
[38] M.R. Simmons, C.S. Patrickios, Near-monodisperse, catalytically active,
imidazole-containing homooligomers: synthesis by group transfer
polymerization and solution characterization, J. Polym. Sci., Part A: Polym.
Chem. 10 (1999) 15011512.
[39] R.I. Mahato, Biomaterials for Delivery and Targeting of Proteins and Nucleic
Acids, CRC Press, Boca Raton, USA, 2004 (Chapter 6).
[40] G.N. Sushkevich, B.V. Dubovik, V.P. Baluda, V.S. tlis, F.N. Shomina, Synthetic
polycations as activators of the endogenous mechanism of platelet
aggregation, Bull. Exp. Biol. Med. 83 (1977) 627630.
[41] J. Wang, C.J. Pan, N. Huang, H. Sun, P. Yang, Y.X. Leng, J.Y. Chen, G.J. Wan, P.K.
Chu, Surface characterization and blood compatibility of poly(ethylene
terephthalate) modied by plasma surface grafting, Surf. Coat. Technol. 196
(2005) 307311.
[42] L. He, L. Yang, Y. Duan, L. Deng, X. Sun, Z. Gu, Z. Zhang, Cytotoxicity and
hemocompatibility of a family of novel MeOPEGpoly (D, L-lactic-co-glycolic
acid)PEGOMe triblock copolymer nanoparticles, J. Appl. Polym. Sci. 113
(2009) 29332944.
[43] M. Ahmed, R. Narain, The effect of polymer architecture, composition, and
molecular weight on the properties of glycopolymer-based non-viral gene
delivery systems, Biomaterials 32 (2011) 52795290.
[44] S. Carrabino, S. Di Gioia, E. Copreni, M. Conese, Serum albumin enhances
polyethylenimine-mediated gene delivery to human respiratory epithelial
cells, J. Gene Med. 7 (2005) 15551564.
[45] J. Morales-Sanfrutos, A. Megia-Fernandez, F. Hernandez-Mateo, M.D. GironGonzalez, R. Salto-Gonzalez, F. Santoyo-Gonzalez, Alkyl sulfonyl derivatized
PAMAM-G2 dendrimers as nonviral gene delivery vectors with improved
transfection efciencies, Org. Biomol. Chem. 9 (2011) 851864.
[46] D. Velasco, E. Collin, J. San Roman, A. Pandit, C. Elvira, End functionalized
polymeric system derived from pyrrolidine provide high transfection
efciency Eur, J. Pharm. Biopharm. 79 (2011) 485494.
[47] G. Nchinda, K. Uberla, O. Zschornig, Characterization of cationic lipid DNA
transfection complexes differing in susceptibility to serum inhibition, BMC
Biotechnol. 2 (2002) 110.
[48] Q.R. Chen, L. Zhang, S.A. Stass, A.J. Mixson, Copolymer of histidine and lysine
markedly enhances transfection efciency of liposomes, Gene. Ther. 7 (2000)
16981705.
[49] L. Garcia, M. Bunuales, N. Duzgunes, C. Tros de Ilaruya, Serum-resistant
lipopolyplexes for gene delivery to liver tumour cells, Eur. J. Pharm. Biopharm.
67 (2007) 5866.
[50] B. Yang, Y. Sun, W. Yi, J. Yang, C. Liu, H. Cheng, J. Feng, X. Zhang, R. Zhuo, A
linear-dendritic cationic vector for efcient DNA grasp and delivery, Acta
Biomater. 8 (2012) 21212132.

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene
therapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.1016/j.ejpb.2012.08.002