Cross-linking as a tool for enhancement of transfection efficiency of cationic vectors

European Polymer Journal 69 (2015) 110–120

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Cross-linking as a tool for enhancement of transfection efficiency of cationic vectors Ekaterina D. Maximova a,b, Evgeny B. Faizuloev b, Alexandra A. Nikonova b, Svetlana L. Kotova c, Anna B. Solov’eva c, Vladimir A. Izumrudov a,d, Ekaterina A. Litmanovich a, Elena V. Kudryashova a, Nickolay S. Melik-Nubarov a,⇑ a

Chemistry Department, M.V. Lomonosov Moscow State University, Vorobiovy Gory, 119991 Moscow, Russia I.I. Mechnikov Institute of Vaccines and Sera, RAS, M. Kazenniy per. 5A, 105064 Moscow, Russia N.N. Semenov Institute of Chemical Physics RAS, Kosygina St. 4, 119991 Moscow, Russia d A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St. 28, Moscow 119991, Russia b c

a r t i c l e

i n f o

Article history: Received 12 February 2015 Received in revised form 18 May 2015 Accepted 21 May 2015

Keywords: Nanogels Degree of cross-linking Transfection siRNA

a b s t r a c t A series of nanogel particles with the growing cross-linking degree was synthesized in reverse micelles by copolymerization of a cationic monomer N,N-dimethylaminoethylme thacrylate with increasing amounts of N,N0 -methylenebisacrylamide (MBA). The growth of MBA content in the studied range of 0.2–15% (mol) resulted in a regular decrease of the particles’ size with a narrowing in their size distribution which occurred at the first of the cross-linker addition. In the second region, where the MBA content was more than 5% (mol), the size distribution was very narrow. The cross-linked polymers prepared at 2–5% of the cross-linker amount demonstrated a minimum size of the polyplexes and enhancement in the transfection with plasmid DNA and small interfering RNA targeted to the luciferase gene. The revealed changes in the properties of nanogels occurring upon the changes in the cross-linking degree appear to be a platform for a controlled manufacturing of efficient cross-linked cationic vectors. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction In the last two decades, nano- and micro-sized cross-linked macromolecules, i.e. nanogels and microgels, have been extensively investigated as drug carriers [1–3] or molecular devices for temperature sensing [4], controlled release of cytotoxic agents [5] or insulin [6], enzyme immobilization for industrial biosynthesis [7], as interface carriers [8] and for fabrication of nano-arranged water-soluble catalytic systems [9,10]. The ways of nanogels synthesis discussed thus far in the literature include cross-linking of water-soluble polymers in solution with subsequent fractionation [1,11], cross-linking of aggregated polymer systems [12,13], precipitation polymerization of water-soluble monomers forming polymers with LCST (N-isopropylacrylamide [14–16], N-dimethylacrylamide [17], etc.) and template polymerization [18]. Among the above experimental procedures, the use of reverse micelles formed from certain surfactants in water-immiscible solvent continues to hold a firm place in the nanogels preparation. The implementation of reverse micelles was conducted by Speiser et al. [19] and further developed by Levashov et al. [20,21]. The water cavities of micelles are

⇑ Corresponding author. E-mail address: [email protected] (N.S. Melik-Nubarov). http://dx.doi.org/10.1016/j.eurpolymj.2015.05.024 0014-3057/Ó 2015 Elsevier Ltd. All rights reserved.

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unique nanoreactors for a large variety of polymerization, biocatalytic and other processes which occur in aqueous environment or at the interface [22,23]. This approach was further improved by insertion of cross-linkages susceptible to degradation under slightly acidic [24,25] or reductive [26,27] conditions. In spite of the fact that the use of cationic nanogels for delivery of plasmid DNA and siRNAs into eukaryotic cells is well documented [28–31], much less efforts were mounted to elucidate the influence of cross-linking degree on nanogels interaction with nucleic acids. Meanwhile, this factor could determine a balance between osmotic pressure inside the gel matrix and the polymer elasticity which control the size of the particles and their capacity to interact with oppositely charged biomacromolecules and linear polyelectrolytes. Thus, the optimal size of polyethyleneimine nanogels cross-linked via the photo-Fenton reaction was about 80–110 nm, while larger and smaller particles exhibited a lower transfection capacity [32]. The toxic effect of the PEGylated nanogels derived from poly(2-(N,N-diethylamino)ethyl methacrylate) [33] and uncovered with PEG nanogels composed of poly(2-(N,N-dimethylamino)ethyl methacrylate) [30] was significantly reduced when the cross-linking density was increased. It was also found that an increase in the degree of cross-linking decelerated diffusion of small molecules into nanogel core [34]. Encouraged by these findings, we have performed a comprehensive study on the plasmid DNA and siRNA interaction with cationic nanogels prepared in reverse micelles. Of special interest was to study the manner in which the cross-linking degree determines molecular and functional properties of the nanogels. The clearly observed non-monotonic changes in the nanogels properties as a function of the cross-linking degree appears to be a platform for controlled manufacturing of efficient cross-linked cationic vectors. 2. Experimental section 2.1. Materials N,N-dimethylaminoethylmethacrylate (DMAEMA) (Merck, Whitehouse Station, USA) was dried over calcium hydride at room temperature for 2 h and then distilled under reduced pressure. Cyclohexane (Merck), Brij-O10 (monooleyl ether of decapolyethylene glycol), branched polyethyleneimine (Mr  25 kDa), ammonium persulfate, 3-(4,5-dimethylthiazo l-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and N,N0 -methylenebisacrylamide (MBA) and SYBR Green I were purchased from Sigma–Aldrich (St. Louis, USA), tert-octylphenoxypolyoxyethylene Triton X-100 was purchased from Serva (Heidelberg, Germany), Lipofectamine 2000 was from Invitrogen (Carlsbad, USA), fetal calf serum was from HyClone Lab. Ltd., (Logan, USA); all of those were used as received. A plasmid DNA pGL3, containing the firefly luciferase gene, was prepared according to the standard protocols using a Promega (Fitchburg, USA) kit. The sequences of oligonucleotides used for the preparation of siRNA targeted to firefly luciferase and respiratory syncytial virus (RSV) phosphoprotein P [35] genes were (50 –30 ) UUUCCGUCAUCGUCUUUCCdTdT, GGAAGACGAUGACGGAAAdTdT, and UCUUGCAGUUAUAUUAUCGdTdT, CGAUAAU AUAACUGCAAGAdTdT, correspondingly. They were synthesized in Syntol Ltd. (Moscow, Russia). The solvents and buffer components were of the highest purity and were used without additional purification. 2.2. Synthesis of linear poly-N,N-dimethylaminoethylmethacrylate (PDMAEMA) and nanogels Linear PDMAEMA was synthesized by radical polymerization under argon atmosphere within 24 h, using a 1.6 M toluene solution of the monomer in the presence of 5 mM 2,20 -azobis(iso-butironitrile) as an initiator (the molar ratio of monomer:initiator was 320:1). The polymer was precipitated in hexane, dried under vacuum, dialyzed against distilled water and freeze-dried, obtaining the polyamine with 90% yield. According to GPC in DMF, Mn of the polymer was 17,500 g/mol, Mw – 34,200 g/mol, Mw/Mn = 1.95. The nanogel synthesis was performed by the method originally developed for acrylamide systems [20] and further applied for the synthesis of PDMAEMA nanogels as specified previously [30]. Briefly, 1 mL of 2 M water solutions of DMAEMA containing MBA at different monomer/cross-linker molar feed ratios ranging from 400 to 7 were neutralized by concentrated HCl to pH 7.0 and mixed with 30 mL of 0.3 M cyclohexane solution of a non-ionic detergent Brij-O10. Heating the mixture at 45 °C under intensive shaking resulted in a spontaneous formation of a transparent and stable microemulsion. The polymerization was initiated by addition of 0.2 mL of 1.7 M (NH4)2S2O8 solution, the system being immediately clarified by intensive shaking within no more than 30 s. The polymerization was allowed to proceed for 12–16 h at 45 °C and the polymer was precipitated in an excess of acetone, which was a good solvent for Brij-O10. The polymer pellet was washed by 6 subsequent cycles of thorough grinding with glass stick in fresh portions of acetone and centrifugation, and finally dried under vacuum. To remove traces of sulfate, sulfite and other impurities the resulting white powder was dissolved in water, dialyzed extensively against distilled water and freeze-dried. The yield of nanogels was about 0.3 g (90%). 2.3. Fourier-transform infrared (FTIR) spectroscopy IR spectra were recorded at 22 °C using a Bruker Tensor 27 spectrometer equipped with a liquid nitrogen cooled MCT detector and a thermostatic cell BioATR-II with ZnSe attenuated total reflection (ATR) element (Bruker, Germany). The

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FTIR spectrometer was purged with a constant flow of dry air. FTIR spectra were acquired from 1000 to 2000 cm1 with 1 cm1 spectral resolution and with a digital background subtraction. For each spectrum, 100 scans were accumulated at 20 kHz scanning speed and averaged. The spectral data were processed using the Bruker software system ‘‘Opus 7.0.’’ (Bruker, Germany). The concentrations of both Brij-O10 and nanogel were 1 mg/mL. 2.4. Fluorescence intercalator displacement assay The efficiency of the polycations binding with nucleic acids was estimated by fluorescence of intercalating dye SYBR Green I measured in black 96-well plates. The series of a polycation 2-fold dilutions (50 lL) were prepared in 96-well plate and equal volume of nucleic acid solution (100 lM of bases) containing SYBR Green I (final dilution 1:20,000) was added in 10 mM HEPES buffer, pH 7.2. The plates were incubated for 10 min at r.t. at continuous shaking and fluorescence intensity was measured at kex = 480 nm and kem = 520 nm. Fluorescence signal of in the presence of a polycation was normalized to that in its absence. Data were shown as a dependence of the normalized fluorescence on [N]/[P] ratio, where [N] is molar concentration of all amino groups of polycations and [P] – molar concentration of phosphate groups of nucleic acid. 2.5. Hydrodynamic radius measurements The nanogel particle size was measured by dynamic light scattering (DLS) using a scattered light goniometer PhotoCor (‘‘PhotoCor Corp.’’, USA) with a He–Ne-laser (k = 633 nm, 15 mV) light source [36]. The scattered light was collected at an angle of 90° in the photon counting mode. The dynamical viscosity of cyclohexane and water were taken as 0.894 and 0.692 cP respectively, and their refractive indices were 1.426 and 1.333, respectively. The autocorrelation functions were analyzed using DynaLS software version 2.7.1. (Alango Ltd.) using the cumulant analysis for unimodal distributions and Tikhonov regularization algorithms. Polydispersity index (PDI) was calculated as a ratio of the second cumulant to the squared average decay rate of the correlation function. 2.6. Z-potential measurements Z-potential of the polyplexes was measured using a ZetaSizer NanoZS instrument (Malvern) with a HeNe laser (633 nm), collecting the scattered light at an angle of 17 °C. The data were analyzed using a Dispersion Technology software, version 5.10, The polyplexes were analyzed in 5 mM Tris-acetate buffer at pH 7.0 at 37 °C. Measurement of Z-potential of each polyplex sample was carried out by three repeated cycles with 5 runs for each. 2.7. Atomic force microscopy For AFM imaging, 10 lL of 50 lM (by DMAEMA repeat units) aqueous solutions of the nanogels were dried on freshly cleaved mica in the dust protected chamber. Polyplexes were preformed by mixing of 12 lL of 53 lM (by moles of bases) solution of pGL3 with 5 or 10 lL of 900 lM (by repeat units) solutions of the polycations and adjusted volume to 50 lL with distilled water to obtain samples with N/P ratio 7.5 and 15. The samples were allowed to stay for 1 h at room temperature and then were incubated on freshly cleaved mica until dry (about 24 h). Atomic force microscopy imaging of the prepared samples was performed in the semicontact mode with a Solver P47 AFM instrument (NT-MDT, Russia). We used NSG10 probes (NT-MDT, Russia) with a nominal spring constant of 11.8 N/m, nominal resonant frequency of 240 kHz and a nominal tip radius of 10 nm, as well as TESP probes by Bruker with a nominal spring constant of 42 N/m, nominal resonant frequency of 320 kHz and a nominal tip radius of 8 nm for finer imaging. Typically, 14  14 lm images were obtained at a scan rate of 1 Hz, with a 512  512 pixels resolution. The images were flattened using the instrument built-in image processing software Nova (NT-MDT, version 1.0.26.1443). The heights of particles were measured and collated using a Grain Analysis tool in the software delivered with the instrument or using the ImageJ 1.48v software (Wayne Rasband, NIH). 2.8. Cell culturing Chinese hamster ovarian cells CHO were cultured in MEM supplemented with 4 mM of glutamine, 5% of FCS and 0.04 mg/mL gentamycin at 37 °C in atmosphere containing 5% CO2. CHO-luc cells were prepared according to the previously published protocol [37] by permanent transfection of CHO cells with a plasmid containing the firefly luciferase gene under the control of an eukaryotic promoter and neomycin resistance gene allowing selection of the transfected cells in the neomycin-containing medium. The cells were maintained in culture in MEM medium as described above. 2.9. MTT-assay The day before the experiment, the cells were seeded on a 96-well plate (Costar, USA) at a density of 3000–4000 cells per well. The next day, the culturing medium was removed and 0.2 mL of the polymer solutions of varying concentrations in the serum-free medium were placed in the wells for 1 h. In control wells (100% of surviving cells), the assayed compounds were

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replaced with an equal volume of the appropriate medium. Then, the polymer solutions were removed and the cells were cultured in 0.2 mL of fresh complete medium containing 10% of serum for 2 days. The amount of remaining living cells was assayed by addition of 0.05 mL of MTT solution (1 mg/mL) in the MEM culturing medium for 3–4 h. Then, the medium was removed, violet crystals of formazan were dissolved in 0.1 mL of DMSO, and the optical density at 550 nm was measured with a Multiscan photometer (Titertek, USA). The portion of survived cells was calculated as a ratio of the optical density in a well with a certain polymer concentration to that in the control well. All measurements were done in triplicates. 2.10. siRNA preparation Equimolar amounts of complementary linear oligoribonucleotides were mixed, incubated for 2 min at 50 °C, cooled to room temperature and diluted with ribonuclease-free water to the final concentration of 17 lM. 2.11. Preparation of polymer/nucleic acid complexes for transfection experiments An appropriate amount of a polycation (linear PDMAEMA or nanogels) was adjusted to 50–60 lL with Opti-MEM™ medium in 0.2 mL tube. The plasmid DNA and siRNA solutions were prepared in separate tubes (0.002 mg/50 lL). The solutions of the polycation and the nucleic acid were thoroughly mixed together at a fixed molar ratio between the amine groups of the polycation ([N]) and the phosphate groups of the nucleic acid ([P]) – [N]/[P], and incubated for 20 min at room temperature before addition to the cells (CHO cells in the case of pDNA and CHO-luc, cells, stable-transfected by the luciferase gene in the case of siRNA). As far as polyelectrolyte complexes obtained by mixing of oppositely charged components never be in equilibrium state, standardization of conditions of their preparation is of primary importance for obtaining reproducible results. Measurement of size distribution by DLS showed that 20 min incubation is sufficient for formation of polyplexes exhibiting reproducible size. 2.12. Transfection CHO and CHO-luc cells were seeded in a 12-well plate 24 h before the experiment (3  105 cells/well). The cells were rinsed two times by a serum-free medium and 1 mL of Opti-MEM™ containing 10 mM HEPES, and the solutions of the preformed complexes were added to the cells. The samples were incubated for 2–4 h, the complexes were removed, and the complete medium without complexes was placed into the wells. After the incubation within 24–48 h at 37 °C the cells were collected with the help of scrapers and were lyzed in a buffer containing 0.025 M Tris-phosphate, pH 7.4, 2 mM dithiothreitol, 2 mM EGTA, 1% TritoneX-100 and 10% of glycerol. In the case of siRNA, the results of the transfection efficiency estimation were standardized on the luciferase activity in wells, treated by a complex with the same composition but containing non-specific small interfering RNA targeted to RSV phosphoprotein P. The cell lyzate luciferase activity was measured according to the standard protocol [38] with the help of Luciferase Assay System (‘‘Promega’’, USA) reagent kit. 3. Results and discussion 3.1. Synthesis of nanogels and their characterization The synthesis of nanogels was performed in reverse micelles formed spontaneously by mixing of an aqueous solution and a solution of the surfactant in water-immiscible solvent. The inner cavity of a micelle is filled with water and the wall is

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covered by polar headgroups of the surfactant (Fig. 1a). About 30 years ago it became apparent that these thermodynamically stable and spontaneously formed systems can be exploited as nano-sized reactors for versatile chemical and biochemical processes [39,40]. In particular, it was reported that radical polymerization of water-soluble acrylamide/ methylene-bisacrylamide mixture in the micelles of sodium diisooctylsulfosuccinate (Aerosol OT) resulted in formation of nano-sized hydrogel particles [19,20]. So far as the negative charge of Aerosol OT micelles makes them inapplicable for synthesis of cationic nanogels, in the current work a non-charged Brij-O10 has been exploited. This surfactant forms small micelles which solubilize components of the cell-free translation system without a noticeable loss of ribosome activity [41]. Purification from the surfactant was accomplished by repeated polymer precipitation in the excess of acetone. The composition of the synthesized nanogels was evaluated by attenuated total reflection vibrational spectroscopy (FTIR-ATR) which revealed broad bands corresponding to carbonyl groups (1711 cm1), a-methyl and methylene groups (1486 cm1) of DMAEM repeat units and amide groups of MBA (1605 cm1) (Fig. 2, curve 1). This finding confirms that both the monomer and the cross-linker are introduced into the polymeric particles. The absence of any absorbance near 1100 cm1 which corresponds to C–O–C groups of polyethylene oxide of Brij-O10 [42] suggests that the sample does not contain even traces of the surfactant which exhibits clearly defined absorbance band in this region (Fig. 2, curve 2). The employment of a micellar microreactor allows one to restrict the lengthening of the growing chains by the size of the water cavity of reverse micelles thus preventing from the formation of a macroscopic gel. The Brij-O10 reversed micelles that were prepared in cyclohexane at a water/surfactant molar ratio of 7.4 possessed the mean hydrodynamic radii of about 10 nm and a relatively smoothed appearance of the MM distribution (Fig. S1 in Supporting Information). The sample of linear PDMAEMA manufactured in the reverse micelles was characterized by a broad peak positioned at 70 nm (Fig. 3a, curve 1). Noteworthy, the cross-linked polymer prepared at 5% of MBA consisted of a notably narrower size distribution with a peak located between the BrijO-10 micelles and the linear polyamine (cf. Figs. S1 and 3a). The finding that the hydrodynamic size of equilibrium coils of all polycations isolated from reverse micelles and dissolved in water exceeded the size of the micellar microreactor is readily explained by the polyelectrolyte swelling. A closer examination (Fig. 3b) evidenced the decisive influence of cross-linking on the size and specifically, on the size distribution (polydispersity index, PDI) of the polymer particles. The growth of the MBA content in the studied range of 0.2–15% (mol) resulted in a regular decrease of the particles size (curve 1) with a narrowing in their size distribution (curve 2) which occurred when the amount of the added cross-linker increased up to 5%. At a higher MBA content, the distribution remained very narrow. The evaluation of the size and the size distribution using the regularization procedure following the Tikhonov algorithm and cumulant analysis produced virtually the same results, thus indirectly confirming the very narrow size distribution of the nanogels. The reason for the revealed abrupt change in the PDI values at a low MBA content is not fully understood and calls for future investigation. Thus it is not inconceivable that the successive narrowing of the size distribution up to vicinity of the inflection point, at 4–5% (mol) MBA may result from the cross-linking of several propagating chains into one molecule within one reverse micelle that becomes more probable as the amount of the cross-linker increases. The nanogels obtained in the presence of relatively low amounts of the cross-linker contain large and small molecules, whereas the samples prepared at intermediate or high MBA amounts consisted of only one macromolecule which involved all the initial chains. It is clear that the narrowing of PDI occurs when the probability of cross-linking exceeds that of termination. In any case, these findings imply that the developed route for the nanogels synthesis allows a one-step preparation of polymer nanogels with molecular characteristics that meet the requirement imposed for practical implementation, e.g. for delivery of nucleic acids. As follows from Table 1 in Supporting Information S2, in contrast to similar systems, this approach provides preparation of nanogels particles with a controllable small size and narrow size distribution without fractionation of the products. Note that this route is applicable for all water-soluble monomers and polymers and can be used for cross-linking and polymerization strategies which can proceed in aqueous milieu.

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3.2. Binding of nanogels to nucleic acids The ability of nanogels to interact with nucleic acids was studied using intercalating dye exclusion assay. To this end, Sybr Green intercalating dye was used. This dye was shown to exhibit 500-fold higher sensitivity of detection of double-stranded DNA as compared to conventional ethidium bromide [43]. Intercalation of this dye into double-stranded nucleic acid results in a bright fluorescence at 525 nm, while its exclusion from double helix due to competitive binding of a polycations is accompanied by a sharp quenching. The fluorescence of SYBR Green complexes with plasmid DNA (Fig. 4a) and siRNA (Fig. 4b) was completely quenched upon addition of nearly equivalent quantities of linear PDMAEMA (curve 1) and branched PEI (curve 6). In contrast, complete displacement of SYBR green by nanogels required addition of 2–7-fold molar excess of repeat units with respect to plasmid DNA phosphate groups (Fig. 4a, curves 2–5). Complete binding of short siRNA by nanogels required even higher N/P ratios ranging between 7 to 40-fold (Fig. 4b, curves 2–5). In the both cases, N/P ratio corresponding to complete exclusion of the dye regularly increased with the growth of MBA content in nanogels, indicating that a considerable fraction of amino groups in nanogels remains inaccessible for ion-pairing with nucleic acids. So, the obtained results indicate that at high N/P ratios nanogels bind with both siRNA and plasmid DNA, although only superficial groups of nanogel particles are involved in ion pairing.

3.3. DNA condensation by the nanogels as assessed by DLS The important requirement to vectors for nucleic acids delivery is their ability to form complexes of a size which does not exceed 100–150 nm since such particles are captured most effectively by clathrin-coated endosomes [44]. Formation of cationic polyplexes soluble under physiological conditions (pH 7.4, 0.15 M NaCl) requires addition of excessive amounts of polycations [45,46] because in these systems the most efficient transfection occurs, when the polyplexes are in equilibrium with unbound chains. Accordingly, the size distributions of the plasmid DNA polyplexes with linear PDMAEMA and corresponding nanogels were estimated at a different and large excess of the linear and cross-linked polyamines.

Fig. 4. Evaluation of plasmid DNA (a) and siRNA (b) (50 lM of phosphate, both) interaction with linear PDMAEMA (1), nanogels prepared at 2%, (2), 5% (3), 8(4), 12% (5) and polyethyleneimine (6) by SYBR Green I exclusion from its complex with nucleic acids. Concentration of nucleic acid was 50 lM.

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The hydrodynamic radius of the polyplexes depended on the MBA amount non-monotonically (Fig. 5) with a clearly defined minimum at 3–5% (mol) of MBA, i.e. in the vicinity of the inflection point, that was discussed above in detail (Fig. 3b). In this region, the hydrodynamic radius of the nanogels polyplexes proved to be 2–3-fold lesser as compared to that of the linear PDMAEMA, but the further growth in the MBA amount up to 8%, 12% and 15% was accompanied by a considerable increase in the size of polyplexes (Fig. 5). So, the ability of cationic nanogels to induce DNA condensation depends not only on the N/P ratio as it was established previously [47–49], but is also determined by the degree of cross-linking of a nanogel macromolecule. The revealed enhanced DNA-condensing capability of the samples prepared at 3–5% of MBA may be rationalized by taking into account two factors, i.e. the restricted accessibility of the nanogels amino groups for the ion pairing and the decreased flexibility of highly cross-linked particles. The nanogels with the cross-linking degree ranging between 0.25% and 2% of MBA are characterized by a relatively broad molecular mass distribution and contain a considerable number of small particles that participate in the ion pairing but do not induce effective DNA condensation (Fig. 3b). This may be the reason why nanogels containing 2–5% of the MBA cross-linker induce more pronounced condensation of DNA as compared to those prepared at the lower MBA contents (0.25–1%). With the further elevation of the cross-linking degree, the number of positively charged groups inaccessible to the DNA phosphate groups increases and hence, the formation of the compact structures seems to be hampered due to the electrostatic repulsion between the DNA-bound nanogels, that could be optimized by increase in the size of the complex particles. The AFM analysis data visualize the studied systems and provide another evidence in support of the important role of the cross-linking.

3.4. AFM analysis of the polyplexes The AFM examination of DNA complexes with linear PDMAEMA (Fig. 6a and g) and nanogels prepared at 2% (Fig. 6b and h) and 12% of MBA (Fig. 6c and i) evidenced complete DNA condensation at N/P = 7.5 (Fig. 6a–c) and N/P = 15 (Fig. 6g–i). The particles in the samples were flattened on the surface of mica, i.e. their height (about 10–30 nm) was much smaller as compared with the diameter which ranged from 60 to 200 nm. Most likely, the flattening is caused by the sampling protocol which includes drying of the solutions and consequently, flattening of the spherical particles in the z-direction. Assuming that the x–y dimensions and the shape of a dried and a swollen flattened particle are proportional, we revealed that in all the studied systems there were two types of coexistent spherical particles, namely, bare nanogels and polyplexes, which were quite different in their sizes, depending on the cross-linking degrees. Thus, in polyplexes prepared from linear PDMAEMA at N/P = 7.5 and 15, the height of the majority of the particles ranged between 5 and 10 nm, and only a small fraction was 20–30 nm high (Fig. 6a and d). These fractions were attributed to the excessive polymer and to the polyplexes, correspondingly, both components being in equilibrium. In consistence with the results presented above, the sample of polyplexes formed by the nanogels prepared at 2% of the MBA cross-linker contained considerably smaller particles (with the height of about 7–10 nm) than those formed by linear PDMAEMA (Fig. 6b and e). In contrast, the polyplexes formed by tightly cross-linked nanogels prepared at 12% of MBA were strongly inclined to aggregation (see marked areas in Fig. 6c), and the formed particles had the height of about 20–30 nm (Fig. 6f). This propensity to aggregation for the polyplexes prepared from tightly cross-linked nanogels is in accordance with the DLS results (Fig. 5). The increase in the charge ratio N/P from 7.5 to 15 resulted in a growth of the fraction of unbound cationic particles, whereas the amount of the polyplex particles remained unchanged and hence, their content in the samples decreased. These regularities were inherent in all the studied systems (cf. Fig. 6a–c and g–i).

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Fig. 6. The AFM images (a–c, g–i) and size distributions (d–f, j–q) of polyplexes formed by DNA with linear PDMAEMA (a, d, g, j) and nanogels prepared at 2% MBA (b, e, h, k) and 12% MBA (c, f, i, q) at N/P = 7.5 (a–f) and 15 (g–q). Circles and arrows in panel (c) show aggregates composed of several spherical particles.

So, the AFM analysis of polyplexes formed by nanogels has revealed a spherical shape of the complexes that confirms estimates of the polyplex sizes made from the DLS data on the basis of the Stokes–Einstein equation. Moreover, evaluation of the size of polyplexes made from the AFM assay shows that nanogels prepared at 2% of the MBA cross-linker manifest

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the most pronounced ability for DNA condensation, in complete agreement with the data obtained from the DLS measurements. 3.5. Transfection with plasmid DNA Evaluation of the polycations transfection activity was conducted using a plasmid DNA containing the firefly luciferase gene. The activity of the nanogels (Fig. 7a) depended non-monotonically on the degree of cross-linking with a clearly defined maximum at 2–5% (mol) of MBA, i.e. in the vicinity of the above mentioned inflection point. This finding points to the important role of the cross-linking degree in the transfection which provides a versatile unconventional control of the cationic vectors functionality. Even though the maximal transfection efficacy of the nanogels was far from the efficacy of commercially available standard transfectants PEI 25 kDa and Lipofectamine 2000 (Fig. 7a), the revealed peculiar ability of slightly cross-linked nanogels to have a pronounced beneficial effect on transfection seems to be important from the fundamental point of view and can be feasible, e.g. in the preparation of functional nanogels. The toxicity of nanogels-based polyplexes is considerably lower than that of PEI-based polyplexes (Fig. 7b). Thus, the polyplexes of nanogels prepared with 2% of MBA at the optimal N/P ratio of 15, ensured the viability of 82% of cells in the culture, while in the presence of PEI-based polyplexes only 50% of the cells remained alive. As far as cytotoxicity of polyamines is mainly conditioned by their interaction with anionic centers on biological membranes [50], it is reasonable to suggest that the decrease in the nanogels cytotoxicity is determined by poor accessibility of the amino groups for ion pairing. The inability of a nanogel to spread over lipid bilayers to form a maximal number of ion pairs with negatively charged carbohydrates and proteins on the cell surface opposes formation of domains in cell membranes and therefore, to a lesser extent initiates apoptotic processes characteristic for polycations. Contrary to the poor delivery of the target plasmid DNA to the cell, the experimental results on transfection with siRNAs proved to be extremely encouraging. 3.6. Transfection with siRNAs To study the ability of the cationic nanogels to facilitate the transport of siRNAs into eukaryotic cells, we employed model cells originated from the CHO line persistently transfected with the firefly luciferase gene according to the reported technique [37]. Non-specific toxic effects of siRNA and the cationic carriers were inferred by the control polyplexes containing siRNA targeted to the P-gene of respiratory-syncytial virus (RSV). At N/P = 15, the nanogels prepared at 2% and 5% of MBA suppressed 90% and 80% of the luciferase activity, respectively, i.e. quite close to 92% value exhibited by polyplexes based on Lipofectamine 2000 (Fig. 8). It is seen that the further increase in the cross-linking degree considerably decreases activity of the nanogels in the siRNA delivery. This finding agrees well with the analogous trend revealed in the study of the plasmid DNA delivery (Fig. 6a) and is apparently conditioned by the enhanced rigidity of highly cross-linked nanogels. The polyplexes prepared at the lower N/P ratio of 7.5 (Fig. 8b) were much less effective similar to the case of the plasmid DNA, indicating that the 7.5-fold excess of the polycations is insufficient to ensure their stability in the presence of cells and culturing medium.

Fig. 7. Firefly luciferase activity (a) and cytotoxicity (b) of CHO cells transfected by pGL3-LUC complexes with linear PDMAEMA and nanogels prepared at various cross-linker contents (marked by percentage) and different charge feed ratio [N]/[P], i.e. 7.5 (1), 15 (2) and 30 (3) at a DNA concentration of 2 lg/mL. Lipofectamine 2000 and nascent DNA were used as the positive and negative controls, respectively.

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(a)

1

2

(b)

80 60 40 20

100

Remaining luciferase activity, %

Remaining luciferase activity, %

100

80 60 40 20

0

% % % 2% 5 % 000 l2 l5 l8 MA 1 l1 AE n o g e n o g e n o g e g e l g e ine 2 M o o a a a D N N N N a n N a n tam rP c ea ofe Lin Lip

0

Lin

ea

rP

A DM

A % % % % % 00 EM g e l 2 g e l 5 g e l 8 e l 1 2 e l 1 5 e 20 o o g g o n i n n n n o a n o tam Na Na Na c Na N ofe Lip

Fig. 8. Residual luciferase activity in CHO-LUC1 cells treated with polyplexes formed by siRNA targeted to firefly luciferase gene (1) and to P-protein of RSV (2) at N/P = 7.5 (a) and 15 (b).

Importantly, no considerable suppression of luciferase activity was caused by control polyplexes with siRNA targeted to the P-gene of RSV (bars 2 in Fig. 8), indicating that only small if any toxicity was induced by nanogels at N/P = 7.5 and 15 in the agreement with results obtained with plasmid DNA. So, the polyplexes formed by short siRNA molecules with slightly cross-linked nanogels corresponding to 2–5% (mol) MBA display a much higher transfection efficacy as compared with either linear PDMAEMA or tightly cross-linked nanogels. This regularity is in accordance with those revealed for the transfection of CHO cells with a plasmid DNA. However, in contrast to the plasmid DNA, the nanogels were very effective in the transfection of cells with siRNA. Most probably this difference is caused by the fact that the gene silencing induced by the RNA interference occurs in the cytoplasm and does not require penetration of the nucleic acid into the nucleus. It means that nanogels with intermediate cross-linking degrees are very efficient in the nucleic acid binding, condensation and overcoming of the lysosome/endosome barrier. The latter effect may be provoked by ‘‘proton sponge mechanism’’ [51], according to which weak polybases possessing groups titratable at pH 5–6 inherent for inner milieu of acidic compartments can induce endosomes disruption due to osmotic swelling. At the same time, these carriers are useless for the nucleic acids delivery into the nuclei. The disclosed regularities may be of primary importance for the construction of cationic vectors highly efficient in the delivery of nucleic acids into the cell cytoplasm.

4. Conclusions Herein we report the development of a synthesis of cationic nanogels in reverse micelles of the non-ionic detergent Brij-O10 yielding particles of a controlled size prepared at 0.2–15% (mol) of the MBA cross-linker. We revealed the following distinctive properties of the nanogels obtained at 2–5% (mol) of MBA: (i) a very narrow molecular-mass distribution of the nanogels, (ii) a pronounced minimum in the size of the corresponding polyplexes, (iii) maximum enhancement of the nanogels functionality in transfection with a plasmid DNA bearing the gene of firefly luciferase and, (iv) the most effective suppression of expression of the reporter enzyme by the nanogel binding with small interfering RNA targeted to the gene of luciferase. The revealed extreme character of the nanogels functionality appears to be a platform for the controlled development of efficient cross-linked cationic vectors.

Acknowledgement This work was supported by the Russian Foundation for Basis Research (Grant No. 12-03-00255a).

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.eurpolymj.2015.05.024.

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