european journal of pharmaceutics and biopharmaceutics volume 82 issue 3 2012 [doi...

European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxx–xxx

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European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier .com/locate /e jpb

Research paper

Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole)linear oligomers for gene therapy applications

D. Velasco a,⇑, G. Réthoré b, B. Newland b, J. Parra c, C. Elvira a, A. Pandit b, L. Rojo d, J. San Román a

a Institute of Polymer Science & Technology, Madrid, Spainb Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Irelandc Unidad de Investigación CHA-CSIC, Ávila, Spaind Department of Materials, Imperial College, London, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 March 2012Accepted in revised form 8 August 2012Available online xxxx

Keywords:OligomersLow polydispersitiesCationic polymersNon-viral vectorsAcrylicspH sensitive

0939-6411/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ejpb.2012.08.002

⇑ Corresponding author. Institute of Polymer ScieCIBER-BBN Juan de la Cierva, 3, 28006, Madrid, Spainfax: +34 915 644 853.

E-mail address: [email protected] (D. Velasco).

Please cite this article in press as: D. Velasco et atherapy applications, Eur. J. Pharm. Biopharm. (

Nonviral methods for gene delivery are becoming ever more prevalent along with the need to design newvectors that are highly effective, stable in biological fluids, inexpensive, and facile to produce. Here, wesynthesize our previously reported monomer N-ethyl pyrrolidine methacrylamide (EPA) and evaluateits effectiveness in gene vector applications when copolymerized with 1-vinylimidazole (VI). A rangeof these novel linear cationic copolymers were synthesized via free radical polymerization with lowmolecular weights (oligomers) and low polydispersities showing two pKa values as the two co-monomersare cationic. DNA–polymer polyplexes had average sizes between 100 and 250 nm and zeta-potentialsbetween 10 and 25 mV, and a strong dependence of composition on the size on the zeta-potential wasobserved. The cytotoxicity of the homopolymers, oligomers, and polyplexes toward human fibroblastsand 3T3 mouse fibroblasts was evaluated using the MTT and AlamarBlue™ assays, proving that formula-tions could be made with toxicity as low as low molecular weight linear poly (dimethylaminoethyl meth-acrylate) (PDMAEMA). The transfection capability of the polyplexes measured using the G-luciferasemarker gene far superseded PDMAEMA when evaluated in biological conditions. Furthermore, bloodcompatibility studies showed that these new oligomers exhibit no significant hemolysis or platelet acti-vation above PBS controls. These new EPA based oligomers with low toxicity and ease of scalability showhigh transfection abilities in serum conditions, and blood compatibility showing its potential for systemicgene delivery applications.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

The proton-sponge hypothesis for endosomal escape, wherebycationic polymers buffer in the low pH of the endosome to facili-tate its swelling and subsequent rupture is a well-known conceptand has become an integral part of polymeric vehicle design [1].However, polymers designed in such a way often suffer from sev-eral drawbacks, most predominant of which is high cytotoxicity. Itis therefore imperative to design advanced biocompatible materi-als that mimic the proton-sponge mechanism, allowing efficientendosomal escape without increasing toxicity [2]. Here, it is alsoimportant to note the role of the imidazole heterocycle in genedelivery. The incorporation of imidazole moieties represents apromising option for the improvement of endolysosomal escape

ll rights reserved.

nce & Technology, CSIC and. Tel.: +34 915 622 900x332;

l., Low polydispersity (N-ethyl p2012), http://dx.doi.org/10.101

and enhancement of the efficiency of polymers without increasingtoxicity [3–8].

Systemic gene therapies suffer from several drawbacks such as alack of stability, degradation, and clearance of the carrier from theblood stream, making the elucidation of the interactions of newvector systems with blood components essential [9,10]. For thesereasons, a substantial part of current research into systemic bioma-terials is focused on the design and preparation of polymers withblood compatibility properties. The strategies developed involve:the preparation of amphiphilic polymers [11–13]; hyperbranchedpolymers [14,15]; copolymers of lactic-co-glycolic [16]; polyesters[17] and poly (ethylene glycol) [18,19]. Another strategy is basedupon the introduction of amidoamine groups in the polyamidesbackbone, which selectively adsorb heparin from plasma or blood,giving stable complexes without any adverse effect on plasma pro-teins and blood cells [20]. High-molecular weight polymers tend toform extremely stable DNA-polyplex aggregates, whereas homolo-gous shorter polycations show enhanced hemocompatibility andreduced toxic effects [21]. However, these complexes formed be-tween short multivalent polycations and DNA molecules tend to

yrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene6/j.ejpb.2012.08.002

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

lack in stability under physiological or serum conditions leading tolower transfection efficiencies. For these reasons, new materials asnon-viral gene transfer vectors for gene therapy applications are re-quired, which combine all of the above desired properties.

A very promising approach to achieve this goal arises throughthe use of novel linear cationic oligomers derived from N-ethylpyrrolidine methacrylamide (EPA) and 1-vinylimidazole (VI) pro-duced via the highly scalable, conventional free radical polymeriza-tion technique. This paper presents the synthesis, characterization,complexation ability, blood compatibility and the impressivetransfection capability of these (EPA-co-VI) oligomers, recentlydeveloped in our laboratories, and their evaluation to be used aspotential non-viral gene carriers under serum conditions.

2. Materials and methods

2.1. Materials

1-vinylimidazole (VI; Sigma–Aldrich) was distilled under reducedpressure. N-ethyl pyrrolidine methacrylamide (EPA) was synthesizedas reported previously [22]. 2,2’ Azobisisobutyronitrile (AIBN, Merck)was recrystallized from methanol (mp 194 �C). Ethyl a-bromoisobu-tyrate (EBr), (1,1,4,7,7-Pentamethyl-diethylenetriamine) (PMDTA),L-ascorbic acid (AA), 2-(dimethylamino) ethyl methacrylate (DMA-EMA), and copper (II) chloride (CuCl2) were purchased from Sigma.The Gaussian princeps luciferase (G-luc) plasmid and accompanyinganalysis kit was purchased from New England Biolabs. All thesolvents used were purified by standard procedures.

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

Poly (EPA-co-VI) copolymers were obtained by free radical poly-merization with molar ratio feeds of EPA/VI monomers of 80:20,50:50, and 20:80, respectively. The appropriate co-monomer mix-tures 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 tothe solution and the reaction medium transferred to an oven at50 �C for 24 h. The copolymers were purified by dialysis usingmembranes Spectra/Por� (cut-off 500–1000 Da). pEPA and pVIhomopolymers were obtained following the same procedure de-scribed above for the p(EPA-co-VI) copolymers.

Linear PDMAEMA (10 kDa, PDI: 1.097) was synthesized by acti-vated atom transfer radical polymerization according to an alreadypublished protocol [23]. Briefly, the reaction was carried out withina two necked round bottomed flask containing the monomer DMA-EMA and initiator ethyl a-bromoisobutyrate (EBr) (50:1 wt%), bythe addition of CopperI/PMDTA (1,1,4,7,7-Pentamethyl-diethylene-triamine) and leaving the reaction to proceed for 6 h at 50 �C underargon atmosphere. Aliquots of reaction mixture were withdrawn atthe start and end of the reaction time, diluted in DMF and runthrough a silica gel column to remove copper catalyst and analyzedby gel permeation chromatography. The reaction was stopped byexposing the solution to the air, and the product obtained was col-lected from the reaction mixture by precipitation in hexane fol-lowed by drying under laminar flow. The polymer obtained wasthen re-dissolved using firstly acetone and then adding distilledwater. The polymer solution was then reduced to pH 5 by the dropwise addition of 1 M hydrochloric acid under constant stirring. Thiswas then dialyzed against distilled water for several days, beforebeing freeze dried for subsequent studies.

2.3. Spectroscopic techniques

The polymers were characterized by nuclear magnetic reso-nance (NMR) and attenuated total reflectance fourier transform

Please cite this article in press as: D. Velasco et al., Low polydispersity (N-ethyl ptherapy applications, Eur. J. Pharm. Biopharm. (2012), http://dx.doi.org/10.101

infrared (ATR-FTIR) spectroscopies. 1H and 13C NMR spectra wererecorded in an INOVA-300 spectrophotometer. The spectra wererecorded in deuterium oxide (10% w/v). Using trimethylsilane(TMS) as internal standard, ATR-FTIR spectra were recorded on aPerkin–Elmer-Spectrum One spectrophotometer, with an ATRattachment.

2.4. Chromatographic techniques

Number and weight average molecular weighs were calculatedby Size Exclusion Chromatography (SEC) with a Shimadzu SIL 20A-HT SEC with an isocratic pump serial LC-20D connected to a differ-ential refractometric detector (serial RID-10A). Calibration of SECwas carried out with a monodisperse polyethylene glycol standardin the range of 1.0 � 103–500 � 103 obtained from Scharlab.

2.5. MALDI–TOF

Matrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI–TOF/MS) was performed on Voyager-DEPRO equipment (Applied Biosystems). Samples were dissolved inMilli-Q water (3 mM) and mixed with trans-3-indoleacrylic acidas the matrix at a ratio of 1:2 (v/v). Mass spectra were recordedin the positive ion mode using a nitrogen laser (337 nm) and mea-sured in the linear mode.

2.6. Thermal analysis

Glass transition temperatures (Tg) were determined by Differ-ential Scanning Calorimetry (DSC) using a Perkin–Elmer DSC-7 cal-orimeter. The samples (6–8 mg) were run under nitrogenatmosphere and heated from 0 to 200 �C at 10 �C min�1 taking asTg the onset point of the corresponding thermal transition.

2.7. Determination of the dissociation constants (pKa)

The dissociation constant values of the polymers were deter-mined by acid–base titration of the polymer solution in a 25 mlof ionic strength saline buffer (0.1 M NaCl). Diluted aqueoussolutions of NaOH (0.1 M) were used to complete the titration.1–3 ml of a 0.1 N HCl solution was added to ensure the ionizationof the amine groups of the copolymers. The changes of pH weremeasured with a Schott GC841 pH meter.

2.8. In vitro evaluation of cytotoxicity of the homopolymers andcopolymers

The in vitro biological performance of the studied homopoly-mers and copolymers was assessed with a MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and humanskin dermal fibroblasts (DPK-SKDF-HS Dominon Pharmakine),and serial dilutions of each formulations were made. Cellular via-bility was determined by dissolving the corresponding polymer(2.5 mg/ml) in free-serum supplemented DMEM (Dulbecco’s Mod-ified Eagle’s Medium). This stock solution was successively dilutedwith serum-free medium, to obtain twelve dilutions of each formu-lation (from 2.5 mg/ml to 0.001 mg/ml). As initial incubation of thetransfection agent is usually carried out in serum free-media, thesame strategy was used performed for the cytotoxicity study here-in. Fibroblasts (subculture 11) were seeded at a density of 1 � 105 -cells/ml in complete medium in a sterile 96-well culture plate andincubated at 37 �C in humidified air with 5% CO2 for 24 h. Then, themedium was replaced with the corresponding dilution and incu-bated for 48 h (n = 3). A solution of MTT was prepared in warmPBS (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



D. Velasco et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2012) xxx–xxx 3

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

Relative viability ¼ 100� ðODS � ODBÞðODC � ODBÞðec:xÞ

where ODS, ODB, and ODC are the optical density of formazan pro-duction for the sample, blank (culture medium without cells), andcontrol (free-serum supplemented culture medium), respectively.A dose–response curve of cellular viability was plotted in each caseto delineate the concentration that depressed MTT-formazan pro-duction by 50%, called the half maximal inhibitory concentrationor IC50 value.

2.9. Blood compatibility analysis

Human blood was drawn from healthy volunteers into vacu-tainers containing either EDTA or sodium citrate. Oligomers weretested to elucidate the effect of the composition on various compo-nents of blood and determine their effect on erythrocytes, coagula-tion, and the complement system according to an alreadypublished protocol [24]. Ethical approval was granted by the Hu-man Ethics Committee of the National University of Ireland,Galway.

2.10. Platelet activation

Whole blood was centrifuged at 85g for 15 min to remove plate-let-rich supernatant. The remaining blood was again centrifugedfor 10 min at 140g and mixed with the previous extracted plasmato get platelet rich plasma (PRP). Platelet poor plasma (PPP) wasobtained by centrifuging the remaining blood for 5 min at 800g.The PRP was then diluted 1:100 with 1% ammonium oxalate toget a platelet concentration of 6 � 108/ml. 300 ll of PRP was incu-bated with 40 lg of oligomers from all different sizes and surfacemodifications for 1 h at 37 �C. The supernatant was then centri-fuged at 2000g for 10 min. Platelet activation was measured bythe concentration of sP-selectin levels in the plasma and wasdetermined using an ELISA kit (Human soluble P-selectin Immuno-assay, R&D Systems, Minneapolis, MN, #BBE 6) according to themanufacturer’s protocol. Both PPP and PRP were used as controls.

2.11. Complement system

To assess complement activation, the cleavage of complementcomponent C3 was monitored by measuring the formation of itsactivation peptides, C3a and C3a des arg, using a commercial C3aenzyme immunoassay kit (BD Bioscience). Activation studies wereperformed using pooled citrated plasma isolated by centrifugationfrom whole blood donations. Equal volumes of plasma and poly-mer solution in saline were incubated at 37 �C for 1 h. Briefly, thesamples were diluted with the dilution buffer provided in the kitand added to a microtiter plate coated with a monoclonal antibodyspecific for human C3a and C3a des arg. After 1 h incubation atroom temperature to allow any C3a in the sample to bind to themonoclonal antibody, the plates were washed and incubated withperoxidase-conjugated rabbit anti-C3a for 15 min. Following a finalwash step, the chromogenic substrate was added to detect boundC3a. Absorbance was measured at 450 nm. The sample C3a con-centrations were calculated using a standard curve with net absor-bance values plotted on the y-axis for each C3a concentrationindicated on the x-axis. Sample values were accepted as valid if


they fell on the standard curve; sample values above the top endof the curve were retested following further dilution. The measure-ments were done in duplicates.

2.12. Plasma clotting time

Howell’s method was employed to investigate plasma recalcifi-cation time. Blood was collected in a sodium-citrate vacutainers. Itwas then centrifuged at 3000 rpm at 8 �C, for 20 min to obtain theplatelet-poor plasma (PPP). 0.1 ml of the PPP and 40 lg of samplessuspended in PBS were incubated at 37 �C for 5 min in a 96 wellplate. 0.1 ml of 0.025 M CaCl2 solution was then added and theplasma solution was monitored for clotting by measuring theabsorbance using at 405 nm.

2.13. Hemolysis

EDTA-anticoagulated blood was centrifuged for 5 min at a speedof 900g. The serum fraction was removed, and the volume wasraised to its original using 150 mM NaCl. This step was repeatedtwice and the final suspension was diluted 1:10 with 100 mMphosphate buffer. 2 � 108 red blood cells/ml were incubated withthe oligomers each at a final concentration of 100 lg/ml. PBS wasused as a negative control, whereas Triton X-100 1% (w/v) wasused as a positive control. All samples were incubated under gentleagitation for 2 h at 37 �C and centrifuged at 900g for 5 min. Theabsorbance of the supernatant was measured for release of hemo-globin at 545 nm. The percentage of hemolysis was calculated asfollows:

Haemolysis ð%Þ ¼ ðAa� AcÞ � 100Apc

where Aa, Ac, and Apc are the absorbance of test sample, absorbanceof control, and highest absorbance for positive control, respectively.

2.14. Polyplex formation and characterization

Polymer/DNA complexes (polyplexes) were prepared at roomtemperature in pre-filtered PBS 1 h prior to use. Various quantitiesof polymer were added to the DNA (with gentle vortexing) to formnitrogen/phosphate (N/P) ratios from 2 up to 30 with no post-fil-tration performed so as not to lose material.

2.15. Agarose gel electrophoresis

To assess the polyplex formation via migration properties, a0.9% (w/v) agarose gel was used, to which 100 V was applied for30 min. Naked DNA (used as a control) and polyplex samples weremixed with a sucrose loading dye prior to loading to the agarosewells. Afterward, UV light (G:BOX Chemi XL, UK) was used to visu-alize the bands.

2.16. Determination of average particle size and zeta potential

Polyplexes were sized using dynamic light scattering (DLS) (Na-noZS Malvern Instruments), and the surface charge was analyzedusing a Zetamaster system (Malvern Instruments). Polyplexes weremade up in distilled water to a final volume of 800 ll with 25 lg ofDNA and added to a clear disposable zeta cell as reported previ-ously [25]. Three polyplex preparations were made with threereadings obtained for each measurement, all carried out at 25 �Callowing 1 min of equilibrium time before acquiring data. ALV-Cor-relator Control Software was used for data acquisition using acounting time from 300 s to 600 s for each sample.



N

N1-vinylimidazole (VI)

O

NH

N

N-ethyl pyrrolidine methacrylamide (EPA)

+

AIBNDMFT = 50 ºC

N

N

OHN

N

p(EPA-co-VI)

Scheme 1. Free radical copolymerization reaction of the VI and EPA monomers.


2.17. Transfection studies

Transfection experiments were performed with 3T3 fibroblastsby using a plasmid encoding for a cell secreted gaussian luciferaseas a reporter gene. Cells were routinely grown in cell culture med-ium, 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 ona 48 well-plate 24 h prior to the addition of complexes. Differentpolymer/plasmid DNA molar ratios ranging from 2/1 to 20/1 (N/P) were used to prepare the polyplexes. A poly (dimethylamino-ethyl methacrylate)/DNA formulations prepared at different ratios(18/1, 22/1, 27/1, 31/1, and 36/1) (N/P) were used as controls. Theincubation of the polyplexes with the cells was performed either inthe presence or absence of serum. In an absence of serum transfec-tion experiment, the cells were incubated with desired amounts ofpolyplexes (200 ll dispersion with 1 lg plasmid DNA per well) for4 h at 37 �C in a humidified 5% CO2-containing atmosphere. Then,free-serum medium was removed and fresh culture medium wasadded. In the presence of serum transfection experiment, cellswere incubated with desired amounts of polyplexes in a cultureserum medium. Cells were cultured for 2 days. Cell medium wascollected and analyzed for the production of luciferase protein.The assay was performed using the Gaussia princeps luciferase as-say kit (New England Biolabs) according to the supplier instruc-tions. The luciferase expression was quantified by measuringluminescence (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 Alamar-Blue™ cell metabolic assay. The mouse 3T3 cell line (also referredto as NIH/3T3) is commonly used to analyze the transfection capa-bilities of new polymers, so this cell line was chosen again here forthis study to allow ease of data comparison [26,27]. This assay wasperformed after 2 days. Cells were washed with Hank’s BalancedSalt Solution (HBSS). AlamarBlue™ (BioSource� International,Invitrogen, Ireland), diluted by a factor of 10 in HBSS, was addedto each well. After 3 h of incubation, the absorbance of each samplewas measured in a 96-well plate, at wavelengths of 550 and595 nm, using a microplate reader (VICTOR3 V™ MultilabelCounter, PerkinElmer BioSignal Inc, USA). The percentage ofAlamarBlue™ reduction was calculated using a correlation factorRO in accordance with the supplier’s instructions.

2.19. Statistical analysis of the data

Unless otherwise stated, all of the in vitro polymer and polyplexcharacterization experiments were performed in triplicate, with ann number of three for each experiment and an average taken foranalysis. Analysis of variance (ANOVA) was performed by usingStatistica 6.0 software (Statsoft, Tulsa, USA). The statistical analysisof the results was done by the application of one-way ANOVA and avalue of p < 0.05 was considered significant where mentioned intext.

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

3. Results and discussion

3.1. Copolymerization and characterization of EPA and VI oligomers

Important research has been moving toward the developmentof polycation-based gene-delivery systems designed to minimizenuclease degradation through the design of vectors with the capac-ity to escape the endosome/lysosome. Polymers with bufferingcapacities between 7.2 and 5.0, such as imidazole-containing


polymers, could buffer the endosome and potentially induce itsrupture [3]. Herein, three oligomers of EPA/VI were prepared inorder to increase the buffering capacity and to give more biocom-patibility to the EPA monomer. The yield of the EPA and VI copoly-merizations and their respective homopolymers after 24 h ofreaction was about 85–90%. The free radical polymerization ofEPA and VI monomers was carried out following well known poly-merization procedures (Scheme 1) [28].

The EPA and VI feed molar fractions were 0.8/0.2, 0.5/0.5, and0.2/0.8, respectively. The composition of the systems was deter-mined with the signals (Fig. 1) that appear between 6.7 and7.3 ppm to the ðCHb

2ACHANACHACHANÞ and (NACHAN) protonsof VI that increase in intensity with the increase in the molar feedcomposition of this monomer in the oligomers.

1H NMR signals assignments (D2O) (ppm) and FTIR signalsassignments, stretching vibrations, m (cm�1) (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 (cm�1, the most characteristic bands):3346 (NH), 2964–2814 (CH), 1630 (CO), 1141 (OCH2), 1203 (NCH2).



Table 1Characterization of the homopolymers and copolymers: Molar feed and final 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 – – 121,900 2.5 141 5.680 EPA 20 VI EPA: 0.80 VI: 0.20 EPA: 0.84 VI: 0.16 914 1.2 132 5.7,6.350 EPA 50 VI EPA: 0.50 VI: 0.50 EPA: 0.57 VI: 0.43 983 1.3 151 5.6,6.520 EPA 80 VI EPA: 0.20 VI: 0.80 EPA: 0.19 VI: 0.81 933 1.2 157 5.6,6Poly-VI – – 200,000 3.1 167 6.1


Poly VI. 1H NMR spectrum (D2O): dH 1.9 (CHb2), 3.0 (CHb

2ACH),7.0 (CHb

2ACHANACHACHAN), 6.7 (CHb2ACHANACHACHAN), 7.3

(NACHAN). ATR-FTIR spectrum (cm�1, the most characteristicbands): 3101 (N@CH), 2952 (CHsp3), 1497 (C@C).

Poly (EPA-co-VI). ATR-FTIR spectrum (cm�1, the most character-istic bands): 3346 (NH), 2964–2814 (CH), 1630 (CO), 1141 (OCH2),1203 (NCH2) 3101 (N@CH), 2952 (CHsp3), 1497 (C@C).

Table 1 shows a summary of the polymer system characteriza-tion. No differences were found between the molar feed and finalcompositions. Surprisingly, for a conventional radical polymeriza-tion, the copolymers showed much lower molecular weights(900–1000 Da) and polydispersities (�1) in comparison with theirrespective homopolymers EPA and VI (121,000 and 200,000 Da),respectively. These results constitute one of the most relevant re-sults exposed in this work as controlled low molecular weightand near 1 polydispersity cationic copolymers are achieved bystandard free radical copolymerization avoiding the use of morecomplicated reactions and conditions such as RAFT or ATRPpolymerization techniques [29,30]. In fact, the obtaining of suchnear monodisperse copolymers has been confirmed by an indepen-dent technique, MALDI–TOF (Fig. 2), showing molecular weights

Fig. 2. MALDI–TOF spectra of the (A) 80 EPA/20 VI, (B) 50 EPA/50 VI, an


between 3000 and 5000 Da and polydispersities between 1.1 and1.2. These low average molecular weight and polydispersity indexvalues are expected according to reported free radical copolymer-izations of vinyl imidazole monomers and methacrylic relatedcomonomers [31–33]. These show a deviation from classical mech-anism due to a partial coordination between imidazole monomericunits and propagating radicals, which stimulate competing reac-tions and the occurrence of degradative reactions. This leads tothe formation of a relatively unreactive radicals responsible ofthe low molecular weight values reported for vinyl imidazolebased copolymers ranged between 300 and 4000 Da [34–36]. Addi-tionally, different spectroscopic studies, taking into considerationend group analysis, have confirmed that controlled weightdistribution of near monodisperse imidazole containing macromo-lecular entities can be obtained when appropriate radical polymer-ization parameters are chosen [37,38].

Tg values of the corresponding polymers were found to be be-tween 141 and 167 �C. The Tg of the copolymers increased whenthe VI monomer increased in the composition as the Tg of the VIhomopolymer was 167 �C, higher in comparison with the EPAhomopolymer. The ionisable character of the polymers was studied

d (C) 20 EPA/80 VI copolymers and their Mn, Mw, and PDI values.



Table 2Half maximal inhibitory concentration (IC50) values obtained for the formulationsevaluated.

Formulation IC50 (mean ± 95% confidence interval) (mg/ml)

PVI 0.570 ± 0.638PDMAEMA 0.548 ± 0.359PEPA 0.008 ± 0.00280EPA20VI 0.057 ± 0.02950EPA50VI 0.347 ± 0.26720EPA80VI 0.526 ± 0.262


by the determination of the dissociation constants pKa in ionicstrength buffered. It was observed that two pKa values (between5 and 6) were obtained for the copolymers with an increase inthe buffering capacity in comparison with the homopolymer EPAas 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 evalu-ated on human fibroblasts cultures by using the MTT assay to ob-tain the dose–response curves of relative cell viability and IC50

values for each compound against this cell line. The dose–responsecurve obtained from the relative cellular viability values of the cul-ture medium corresponds to the PVI formulation, which is similarto that of the poly (DMAEMA) formulations (Table 2). Indeed, theIC50 concentration values for PVI do not differ significantly neitherobtained values for poly (DMAEMA) (F1,4 = 0.018; p = 0.90).

In the case of the poly-EPA, the dose–response curve indicatedthat it is a system more toxic in comparison with the PVI and poly(DMAEMA); this fact is supported with the inhibitory concentra-tion values obtained for this system, which are significantly lowerthan 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 twocopolymers, indeed the IC50 concentration values for the

Fig. 3. Comparison of dose–response curves (MTT assay) of PVI, PDMAEMA, and EPA andEach point represents the mean and vertical lines represent the standard deviation (n =


80EPA20VI copolymer differs significantly in comparison withthe other two copolymers (F1,4 = 21.588; p < 0.01) to 50EPA50VIand F1,4 = 58.377; p < 0.01 to 20 EPA 80 VI). Besides, the IC50

obtained for the 80EPA20VI system is significant higher than thatobtained for EPA (F1,4 = 53.849; p < 0.01) and significantly lowerthan 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 dose–responsecurve similar to that obtained for PVI; furthermore, the IC50 valuesof these copolymers do not differ significantly compared to the ob-tained value for PVI (F1,4 = 1.932 and p = 0.24 with respect to50EPA50VI; 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 deliv-ery is their low circulation time due to their rapid clearance fromthe blood stream [39]. Consequently, the understanding of theirinteractions with blood components appears to be essential. Sev-eral parameters were investigated to determine the interactionsof our new polymers with blood components and their potentialapplication as gene delivery vehicle via systemic administration.Red blood cell lysis is a well known method to determine interac-tions between particles and erythrocytes. During this experiment,PBS (Phosphate Buffer Saline) and TritonX were used as negative(0%) and positive (100%) controls, respectively. No significant dif-ferences have been observed between conditions when comparedto each other and negative controls It is also worth noting thatall copolymers have a negligible effect on hemolysis (less than0.1%) (Fig. 4A).

Another key parameter of blood incompatibility is representedby platelet activation after interaction with particles. Indeed, cat-ionic polymers are described to induce platelet aggregation andactivation, which could lead to thrombotic complications in vivo[40]. The release of soluble P-Selectin was quantified to evaluatethe platelet activation after incubation with all copolymers. PBSwas used as negative control, and the results show that there is

the copolymers 80EPA20VI, 50EPA20VI, and 20EPA80VI against human fibroblasts.3). From these diagrams, the IC50 values were determined.



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

Fig. 6. Plasma recalcification time, quantified using calculation of the point atwhich the recalcification profile reaches half of the maximum absorbance value.Data are represented as the mean ± standard deviation.


no significant difference between our polymers and PBS (Fig. 4B).Complement system activation is also another parameter to beanalyzed in the field of synthetic carriers for delivery as they couldlead to the activation of the immune system [24]. After incubationwith all copolymers, the release of C3a has been investigated usingPBS as negative control and insulin (a potent complement activa-tor) as positive control [14], and no significant differences havebeen observed between our samples and the control (Fig. 5).

For the clotting process studies, plasma re-calcification profilesare used to mimic the intrinsic coagulation system in vitro. Toquantify plasma re-calcification profiles, T1/2max was calculated asthe time at which half the saturate absorbance was reached [24].PBS was used as a negative control in this study. It was observedthat the clotting time is significantly longer when VI copolymerdrops from 80% to 50% (Fig. 6). Moreover, it appears to be undeter-minable when this ratio decreases to 20%.

EPA/VI copolymer ratios exerted no significant influence onmost of the blood compatibility parameters we have evaluatedthrough this investigation, except for plasma re-calcification time.Indeed, while the EPA copolymer ratio increases, the kinetics of the

Fig. 5. Complement system activation as indicated by C3a release. Data arerepresented as the mean ± standard deviation.


re-calcification slows down to reach a non-measurable time with80 EPA 20 VI polymer, which indicates that the clotting time ofpolyplexes made with a higher ratio of EPA is longer than the oth-ers. These results are consistent with investigation reported in theliterature, which show an increase in the blood coagulation timewhen synthetic polymers are used (poly(ethyleneglycol), function-alized polyethylene terephthalate, sulfate polymers). It could beexplained by a suppression of the intrinsic blood coagulation sys-tem because of the steric repulsion to proteins that reach the sur-face [41,42].

3.4. Polyplex formation and characterization

Agarose gel electrophoresis (Fig. 7) showed that EPA and VIhomopolymers were not able to complex DNA at low N/P ratios.This phenomenon is due to the disposition of the EPA homopoly-mer chains in solution and the lack of total ionization of the VIhomopolymer due to its pKa (6.5). Since it is known that 5% ofthe imidazole groups are only protonated at pH 7.2 (3), this effectof poor complexation ability is more pronounced than for poly(E-

Fig. 7. Electrophoretic mobility of plasmid DNA in polymer/DNA polyplexes with2:1, 4:1, 6:1, 8:1, 10:1, 15:1, and 20:1 P/N ratios.



2:1 4:1 6:1 8:1 10:1 15:1 20:10

50

100

150

200

250

300

Hyd

rody

nam

ic D

iam

eter

(nm

)

N:P Ratio

80 EPA 20 VI50 EPA 50 VI20 EPA 80 VI

30

25

20

15

10

5

02:1 4:1 6:1 8:1 10:1 15:1 20:1 30:1

N:P Ratio

Zeta

Pot

entia

l (m

V)

80 EPA 20 VI50 EPA 50 VI20 EPA 80 VI

A

B

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

Fig. 9. Transfection efficiency of polymer/DNA polyplexes in 3T3 in comparisonwith (poly dimethylaminoethyl methacrylate)/DNA polyplexes without (A) andwith serum (B) at day 2. (C) Transfection efficiency of (poly dimethylaminoethylmethacrylate)/DNA polyplexes without and with serum at day 2. Asterisk denotessignificant difference between the groups (One-way ANOVA, p < 0.05).


PA) that contains both secondary and tertiary amines. On the otherhand, the copolymers bound DNA at all ratios and the complexa-tion can be explained by a possible change in the disposition ofthe chains in solution, which facilitates the ionic interaction be-tween the tertiary amines of EPA and the phosphate groups ofDNA.

A degree of variation between the formulations was noticed forall of the copolymers, a phenomenon previously experienced.However, the polyplexes were in the size range of between 90and 300 nm with a general trend toward larger diameter withincreasing N/P ratio (Fig. 8A). The size of the 80EPA/20VI (170–300 nm) polyplexes was higher in comparison with other composi-tions due to a possible aggregation between the complexes. Thiscan be attributed to a decrease in the charge in its structure(Fig. 4B) as lower ionization degree in that copolymer compositionwith lower content of imidazole monomer gives less repulsion be-tween copolymers chains. The size of the 50EPA/50VI and 20EPA/80VI polyplexes was between 90 and 240 nm, increasing the sizeof the polyplexes with the increase in the N/P ratios, showing low-er average size copolymers richer in VI except in the 6/1 N/P ratio.Zeta-potential measurements (Fig. 8B) of the polyplexes were be-tween 10 and 25 mV. The 80EPA/20VI zeta-potential was lowercompared to 50EPA/50VI and 20EPA/80VI compositions. This is re-lated to the size data presented previously, which shows a lowercontent of imidazole implies a decrease in the charge.

3.5. Transfection studies

The transfection efficiency of the polymers complexed to DNA(polyplexes) was studied using 3T3 fibroblasts. Polyplexes pre-


pared at different polymer/plasmid DNA ratios ranging from 2:1to 20:1 (N/P) were used. Polyplexes were incubated with the cellsin the presence and absence of 10% serum during 4 h. Non-trans-fected cells, cells treated with plasmid alone, and cells treated withcomplexed linear poly (DMAEMA) were used as negative and posi-tive 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 totheir highest transfection. Low transfection levels were observedafter treatment with the EPA and VI homopolymers (data notshown) in comparison with the copolymers. This phenomenoncan be attributed to the lack of total complexation between thehomopolymers and DNA until 10/1 ratio (N/P). In the case of thecopolymers, the highest transfection efficiency in 3T3 fibroblastswas obtained with 80 EPA/20 VI copolymer from the ratio 6:1 to



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 significant difference wasobserved between the groups (One-Way ANOVA p < 0.05).


15:1, being the highest at 10:1. For 50 EPA/50VI copolymer, highertransfections at 10:1 N/P ratios were found in comparison withpoly (DMAEMA), whereas no transfection was found for 20 EPA/80 VI (data not shown) indicating that there is a minimum concen-tration of EPA monomer at which the copolymers become ineffec-tive for gene delivery (Fig. 9A). It can be noted that it was necessaryto use higher N/P ratios of PDMAEMA to obtain higher transfec-tions in comparison with the 80 EPA/20 VI and 50 EPA/50 VIcopolymers. This phenomenon has been observed previously withPDMAEMA in comparison with PEI, a Superfect dendrimer and Lip-oFectin [25], which could be very interesting in terms of obtaininghigher transfections and less toxic systems.

The transfection of the 80 EPA/20 VI and 50 EPA/50 VI copoly-mers in the presence of serum was also studied. Although poly-meric transfection agents may exhibit reduced transfectioncapability in the presence of serum, perhaps due to the copolymerarchitecture or molecular weight [43], we found that the reverse istrue for both the 80 EPA 20 and the 50 EPA 50, where greater trans-fection was observed in the presence of serum as reported else-where [44–49]. To check if the reduced cytotoxicity of the EPAcontaining polyplexes could be the reason for improved transfec-tion efficiency in the presence of serum, cytotoxicity studies wereperformed (Fig. 10) with polyplexes with and without addition ofserum and no differences were found.

While 80 EPA/20 VI and 50 EPA/50 VI copolymers were able tomediate significantly higher transfection with respect to serum-free transfection, the performance of PDMAEM decreased signifi-cantly as can be seen in Fig. 9B. Moreover, a trend was observedin the transfection of the 80 EPA/20 VI and 50 EPA/50 VI copoly-mers in presence of serum where an increase in transfection levelwhen 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


(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 serumafter treatment by the 80 EPA 20 VI and 50 EPA 50 VI polyplexeswas evaluated by the AlamarBlue™ (AB) assay in order to evaluatewhether the toxicity of the polyplexes might affect the transfectionefficiency. Fig. 10 presents the metabolic activity for all the poly-plexes at different polymer/plasmid DNA ratios ranging from 2:1to 20:1 (N/P). The data were normalized to the non-treated cells(indicating a 100% cell metabolic activity). Synthesized polyplexesshowed an absence or low toxicity 2 days after treatment in thepresence and absence of serum. No significant difference inmeta-bolic activity was noted between poly (DMAEMA) treated cells,cells alone, and 80 EPA 20 VI and 50 EPA 50 VI systems treatedcells, indicating these polyplexes cause little to no cytotoxicity atthe concentrations administered.

4. Conclusions

Three different compositions of new cationic copolymers of N-ethyl pyrrolidine methacrylamide (EPA) and 1-vinylimidazole(VI) (80/20, 50/50 and 20/80) have been prepared by radical poly-merization and characterized for their application as gene vectors.Low molecular weight copolymers with low polydisperisty wereobtained being one of the most relevant results exposed in thiswork. Blood compatibility has also been checked and showed nosignificant difference compared to controls except for re-calcifica-tion time. The respective homopolymers, poly-EPA and poly-VI,complexed DNA only at high N/P ratios of 10:1 and 20:1, respec-tively. In comparison, the three copolymers formed complexesfrom a 2:1 ratio upwards. DNA–polymer polyplexes average sizesbetween 100 and 250 nm and f-potentials between 10 and25 mV. Biological activity of homopolymers, copolymers, and poly-plexes was evaluated on human fibroblasts and 3T3 mouse fibro-blasts by using the MTT assay and AlamarBlue™ assays. 80/20and 50/50 copolymers produced transfection capabilities in ser-um-free media from the N/P ratios 8:1 to 15:1, which were compa-rable to poly (DMAEMA) that needed higher N/P ratios to reachsimilar transfection values. More impressively, 80/20 and 50/50copolymers also showed the ability to retain and increase theirtransfection properties in the presence of serum when the N/P ra-tios increased, while poly (DMAEMA) decreased significantly.

Acknowledgments

The authors thank financial support from the EU project SUR-FACET, the NoE, EXPERTISSUES, and the CICYT project MAT 2010-18155. Diego Velasco thanks the grant I3P from the CSIC and Rafa-ela Galera for her support. Carlos Elvira would like to acknowledgeto PIE-CSIC programme (200660I022) for financial support. Theauthors would like to thank Science Foundation of Ireland, Strate-gic Research Cluster (SRC), Grant number 07/SRC/B1163.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejpb.2012.08.002.

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