Poly(vinyl benzyl trimethylammonium chloride) - ACS Publications

Article pubs.acs.org/JPCB

Poly(vinyl benzyl trimethylammonium chloride) Homo and Block Copolymers Complexation with DNA Emi Haladjova,*,§ Grigoris Mountrichas,‡ Stergios Pispas,‡ and Stanislav Rangelov*,§ §

Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St. bl.103A, Sofia 1113, Bulgaria Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vass. Constantinou Ave., 116 35 Athens, Greece

‡

S Supporting Information *

ABSTRACT: In this work we focus on the use of novel homo and block copolymers based on poly(vinyl benzyl trimethylammonium chloride) as gene delivery vectors. The homopolymers and block copolymers were synthesized by RAFT polymerization schemes and molecularly characterized. DNA/ polymer complexes (polyplexes) in a wide range of N/P (amino-to-phosphate groups) ratios were prepared. The ability of the novel polymers to form complexes with linear DNA was investigated by light scattering, zeta potential, and ethidium bromide fluorescence quenching measurements. The resulting polyplexes were in the size range of 80−300 nm and their surface potential changed from negative to positive depending on the N/P ratio. The stability of polyplexes was monitored by changes in their hydrodynamic parameters in the presence of salt. The novel vector systems were visualized by transmission electron microscopy. The influence of factors such as molar mass, content, and chemical structure of the polycationic moieties as well as presence of a hydrophilic poly[oligo(ethylene glycol) methacrylate] block on the structure and stability of the polyplexes, kinetics of their formation, and effectiveness of the (co)polymers to shrink and pack DNA was discussed.

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INTRODUCTION Gene therapy holds promise for treating a wide range of diseases.1−4 Of particular interest is the research on nonviral DNA delivery systems, which show important advantages vs viral systems that are usually associated with an immunological response and safety risks.5,6 The documented dangers of the viral systems have motivated the exploration for synthetic gene delivery systems, which are safer, less pathogenic and less immunogenic alternatives.7−9 The requirements for any effective synthetic vector system includes the ability to (i) condense the bulky structure of DNA to appropriate size for cellular internalization (e.g., about 100 nm), (ii) neutralize the negatively charged phosphate backbone of DNA to prevent charge repulsion against the anionic cell surface, and (iii) protect the DNA from both extracellular and intracellular nuclease degradation.5,10 Cationic polymers are a major class of nonviral DNA delivery systems.5,10−14 They are able to condense the large genes into smaller structures via electrostatic interactions and to mask the negative charges on DNA chain, which are necessary requirements for successful transfection into many types of cells. The resulting particulate structures are called polyplexes and typically are in the nanoscale range. These properties make polymer vectors promising systems for effective gene delivery. For the preparation of successful synthetic vector systems, however, the physicochemical parameters of the obtained © 2016 American Chemical Society

polyplexes are also essential. The amino to phosphate groups (N/P) ratio, size, stability, surface potential, salinity of the solution are only part of the parameters that can influence the behavior of the resulting delivery systems. A large number of cationic polymers such as polyethylenimine,15−17 poly(L-lysine),18−20 poly(dimethylaminoethyl methacrylate),21−23 chitosan,24,25 etc., have been used to form polyplexes. Although their transfection efficiencies have typically been found to be satisfactory, cytotoxicity issues often limit their clinical use.26,27 Nevertheless, the use of synthetic polymers offers a number of advantages and has no limitations in the possibility of polymer structure modification or attachment of ligands for cell-specific targeting. Poly(vinyl benzyl trimethylammonium chloride), PVBTMAC, is a water-soluble polymer with a strong positive charge at every repeating unit (Scheme 1a). A feature of this polymer is the relatively large benzyl group that offers a hydrophobic area making the polymer ideal for a number of demanding applications for which the specific hydrophilic/ lyophilic balance (HLB) is important. In particular, the HLB of the polymer is equal to 9.8 in the scale 0 to 20,28,29 that is, it is considered as more hydrophobic. However, the covalent Received: December 21, 2015 Revised: February 11, 2016 Published: February 16, 2016 2586

DOI: 10.1021/acs.jpcb.5b12477 J. Phys. Chem. B 2016, 120, 2586−2595

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The Journal of Physical Chemistry B Scheme 1. Chemical Structures of (a) Poly(vinyl benzyl trimethylammonium chloride) and (b) Poly[oligo(ethylene glycol) methacrylate]-b-poly(vinyl benzyl trimethylammonium chloride)

Table 1. Molecular Characteristics of the PVBTMAC Homopolymers and PVBTMAC-POEGMA Block Copolymers code PVBTMAC-20K PVBTMAC-40K PVBTMACPOEGMA-2 PVBTMACPOEGMA-4

Mn (co) polymer g mol‑1

Mn PVBTMAC g mol‑1

Mn POEGMA g mol‑1

PVBTMAC %

20 700 39 600 22 400

20 700 39 600 4200

18 200

100 100 19

33 600

15 400

18 200

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eluent showed indications of polymer adsorption on the columns, whereas the SEC analysis in aqueous media showed the presence of some aggregates (see the Supporting Information). Therefore, Mn of the polymers was calculated based on the stoichiometry of the polymerization reaction. The weight percent of PVBTMAC in the block copolymers was determined by UV−vis spectroscopy measurements in water (using a calibration curve made from PVBTMAC homopolymers, see the Supporting Information). Polyplex Formation. The polyplexes were formed by mixing (co)polymer (1 mg.mL−1) and DNA (100 μg.mL−1) aqueous solutions at ambient temperature under vortexing. The amounts of polymer and DNA solutions were selected to give N/P ratios in the 0.5−8 range. Methods. Ethidium Bromide Quenching Assay. In order to investigate the complexation ability of the cationic polymer with DNA, the fluorescence of ethidium bromide has been studied in solutions at various N/P ratios. In particular, an initial solution of DNA (1 × 10−4 mg.mL−1) was prepared followed by addition of ethidium bromide ([EB] = [P]/4). Subsequently, the DNA aqueous solution was titrated using a concentrated polymer solution, up to an N/P ratio equal to 8. The titration was followed by fluorescence spectroscopy. Solutions of polyplexes were measured at a double-grating excitation and a single-grating emission spectrofluorometer, Fluorolog-3 Jobin Yvon-Spex spectrofluorometer (model GL321), excitation at 535 nm, monitoring the emission at 600 nm. Dynamic Light Scattering. Light scattering measurements were conducted on an ALV/CGS-3 compact goniometer system (ALVGmbH), equipped with a ALV-5000/EPP multiτ digital correlator with 288 channels and an ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. A JDS Uniphase 22 mW He−Ne laser (λ = 632.8 nm) was used as the light source. Measurements were performed at an angle of 90°. Solutions were filtered through 0.45 μm hydrophilic PTFE filters (Millex-LCR from Millipore) before measurements. Zeta-Potential Measurements. Electrophoretic light scattering measurements were performed at 25 °C on a ZetaPlus Analyzer (Brookhaven Instruments Corporation) equipped with a 35 mW solid-state laser, operating at λ = 660 nm. ζpotential values were determined, using the Smolukowski equation relating the ionic mobilities with surface charge. Final measurements are the average of the ten repeated ones with an error smaller than ±2 mV. Transmission Electron Microscopy. The samples were examined using a HRTEM JEOL JEM-2100 transmission electron microscope operating at 200 kV. They were prepared by depositing a drop of the solution onto a carbon grid.

conjugation of poly(ethylene oxide) moieties to the PVBTMAC cationic polymer chain widely opens possibilities for stabilization of the polyplexes formed by DNA and the resulting copolymers. In this paper we investigate novel cationic PVBTMAC homopolymers and block copolymers with oligoethylene glycol methacrylate (Scheme 1b) as gene delivery vector systems. DNA/polymer complexes (polyplexes) at a wide range of N/P ratios are prepared and studied under various environmental conditions in terms of structure and stability.

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EXPERIMENTAL SECTION Materials. DNA sodium salt from salmon testes, molar mass ∼2000 bp, was received from Sigma-Aldrich. DNA sodium salt from salmon sperm, molar mass ∼113 bp, was purchased from Acros. The monomers vinyl benzyl trimethylammonium chloride (VBTMAC) and oligo(ethylene glycol) methacrylate (OEGMA) as well as ethidium bromide were purchased from Aldrich. Monomers were purified by passing through an inhibitor removing column. Synthesis of PVBTMAC Based Homo and Block Copolymers. The synthesis of homopolymers and block copolymers was performed by RAFT polymerization in aqueous media. In particular, for the synthesis of a homopolymer of Mn = 40K, VBTMAC monomer (4.0 g), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD) (28.0 mg), 4,4′azobis(4-cyanovaleric acid) (ACVA) (7.84 mg) and 25 mL distilled water were introduced in a 50 mL round-bottom flask. The solution was degassed for 20 min prior to heating using an oil bath. The polymerization reaction took place at 70 °C for 24 h. Afterward, the solution was cooled and opened in air and the polymer was isolated by precipitation in tetrahydrofuran. The polymerization yield was typically ca. 89%. In the case of block copolymers, a similar procedure was applied, using oligo(ethylene glycol) methyl ether methacrylate, OEGMA, (Mn ≈ 475) as the monomer, CPAD as the chain transfer agent and ACVA as the radical initiator for the polymerization of OEGMA. The polymerization reaction for the POEGMA block took place at 70 °C for 24 h. The polymerization of the second block (PVBTMAC) was performed by using ACVA as initiator and POEGMA as the chain transfer agent. All polymers were purified by dialysis against water using Spectra/Por 7 dialysis membranes (MWCO 3500). The molecular characteristics of the homo- and copolymers are given in Table 1. Attempts to analyze the polymers by size exclusion chromatography using DMF as the 2587


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The Journal of Physical Chemistry B Scheme 2. Synthetic Route for the Synthesis of PVBTMAC-POEGMA Block Copolymers

Figure 1. Variations of the hydrodynamic radii (Rh, open squares) and ζ-potential (closed circles) with the N/P ratio of polyplexes formed by PVBTMAC homopolymers and DNA: (a) PVBTMAC-20K and small DNA; (b) PVBTMAC-40K and small DNA; (c) PVBTMAC-20K and large DNA; (d) PVBTMAC-40K and large DNA. ζ-potential standard deviation ±2 mV.

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RESULTS

yield (about 90%). The synthesis of homopolymers was realized in a simple one pot reaction leading to PVBTMAC of different molecular weights. The molecular weight of the homopolymers was easily controlled by tuning the monomer/ chain transfer agent ratio. The molar mass characteristics of the polymers are given in Table 1. Block copolymers are composed of POEGMA and PVBTMAC (see Scheme 1 for the chemical structures).

Synthesis of PVBTMAC Based Homo and Block Copolymers. The synthesis of the homopolymers as well as of the block copolymers was conducted in aqueous media by RAFT polymerization. The absence of organic solvents and heavy metals (as catalyst) promotes the use of this kind of polymerization in applications of biological interest. In addition, the described polymerization procedure is characterized by high 2588


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The Journal of Physical Chemistry B

Figure 2. Variations of the hydrodynamic radii (Rh, open squares) and ζ-potential (closed circles) with the N/P ratio of polyplexes formed by PVBTMAC-POEGMA block copolymers and DNA: (a) PVBTMAC-POEGMA-2 and small DNA; (b) PVBTMAC-POEGMA-4 and small DNA; (c) PVBTMAC-POEGMA-2 and large DNA; (d) PVBTMAC-POEGMA-4 and large DNA. ζ-potential standard deviation ±2 mV.

polycations displace water molecules that are bound by hydrogen bonds with DNA.33 Therefore, the longer the DNA, the more water molecules are released and more compact particles are finally observed. The effect of polycation chain length, however, was marginal: the polyplex particles of PVBTMAC-40K were somewhat smaller particularly at higher N/P ratios implying more pronounced complexation ability. The situation with the effects of DNA molar mass and polycation chain length was just opposite if block copolymers are used (Figure 2): a fully missing or marginal effect for DNA molar mass and considerably more pronounced one for the PVBTMAC chain length. In particular, PVBTMAC-POEGMA4, that is the copolymer of the higher PVBTMAC content, invariably formed smaller, better defined, and stable polyplex particles than PVBTMAC-POEGMA-2. The polycation block of the latter is obviously too short to enable collapsing and shrinking of DNA into such small and well-defined structures. The variations of the ζ-potential of the polyplexes prepared with PVBTMAC-based homopolymers and block copolymers were also examined. Independently from the polymer chain architecture and DNA molar mass, the ζ-potential vs N/P ratio curves followed the typical sigmoidal pattern. The positive sections of these curves are better pronounced compared to the negative ones implying that the addition of even very small amounts of polymer, that is, at very low N/P ratios, drastically influence the surface potential of the resulting polyplex particles. An interesting observation was that in the negative sections of the curves, at equal N/P ratios, the ζ-potential of the polyplexes of PVBTMAC-40K and PVBTMAC-POEGMA-4

POEGMA is a methacrylic polymer exhibiting properties similar to poly(ethylene glycol) (PEG) due to the presence of short PEG chains at every repeating unit. PEG is a highly recommended for bioapplications because it is a biocompatible, water-soluble polymer, which can also add stealth properties to some colloidal nanosystems.30−32 The use of OEGMA facilitates the synthesis of block copolymers by RAFT polymerization thus avoiding the limitations of the expensive tailor-made linear PEGs, which are available as chain transfer agents at a limited number of molecular weights. POEGMA also provides a larger density of PEG chains, which may play an important role as a hydrophilic layer (corona) on the surface of the polyplex particles. The reaction route for the synthesis of PVBTMAC-POEGMA, followed in this work, is depicted in Scheme 2. The molar mass characteristics of the block copolymers synthesized are given in Table 1. Size, Shape, and ζ-Potential of Polyplexes. The ability of novel PVBTMAC-based homopolymers and block copolymers to condense DNA molecules was examined. Polyplexes at N/P ratios in the 0.5−8.0 range were prepared with DNAs of low (Mw ≈ 113 bp) and high (Mw ≈ 2000 bp) molar masses and investigated by dynamic and electrophoretic light scattering. The results for the polyplexes with homopolymers are displayed in Figure 1. As seen, the molar mass of DNA strongly influenced the size variations of the resulting polyplex particles (cf. a with c and b with d in Figure 1). Those of the large DNA were smaller at most of the N/P ratios studied. This could be related with the condensed phase of DNA double helices, which leads to the restructuring of water molecules giving rise to hydration forces. When complexation takes place, 2589


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Figure 3. TEM images of polyplexes formed from large DNA and PVBTMAC-40K homopolymer (a) and PVBTMAC-POEGMA-4 block copolymer (b) at N/P ratio of 4.

Figure 4. Ethidium bromide fluorescence quenching in polyplexes formed from PVBTMAC-based polymers with small (a) and large (b) DNA.

cations the fluorescence intensity decreases due to inhibition of its binding with DNA. Therefore, quenching of ethidium bromide fluorescence is frequently used to monitor the formation of polyplexes. Figure 4 represents typical curves of dependencies of the relative fluorescence intensity on the N/P ratio of polyplexes of the investigated homo- and block copolymers based on PVBTMAC with DNA. The curves exhibit a well-pronounced decrease of the fluorescent intensity being steeper for the polyplexes with small DNA. The significant quenching of the fluorescence is conditioned by displacement of intercalated ethidium bromide from the double helix and indicates strong complexation ability of the investigated (co)polymers. Whereas the curves of the polyplexes with small DNA are grouped together with a small but important exception of the polyplex small DNA:PVBTMAC-POEGMA-2 (Figure 4a), those of the polyplexes with large DNA are shifted (Figure 4b), which implies that the polymer molar mass and polycation chain length strongly influence the effectiveness of the polymer species to displace the fluorescent dye. It must be noted here and discussed later that the fluorescence measurements were performed immediately after the preparation of the polyplexes, which most probably are still far from the equilibrium. Behavior of Polyplexes in the Presence of Salt. The effective gene delivery vector systems must be designed with consideration of their interactions with body fluids, where nuclease degradation can occur. Therefore, the stability of the resulting polyplexes in the presence of salt was investigated. For this, the effect of ionic strength on the particle size was monitored by dynamic light scattering measurements (Figure 5). The experiments were performed on dispersions of

were less negative than those of their counterparts, which is in conformity with their higher effectiveness with regard to the complexation with DNA suggested above. On the other hand, the polyplexes prepared with block copolymers, particularly at higher N/P ratios, that is, in the positive sections of the curves, exhibited lower ζ-potential compared to the polyplexes with homopolymers. Obviously, the introduction of a hydrophilic nonionic block such as POEGMA in the gene delivery vectors not only stabilized the size of the particles but also reduced their positive charge. This could be attributed to the shielding effect34−36 of the POEGMA moieties. Although the POEGMA blocks in the two copolymers were of the same chain length, the polyplexes of PVBTMAC-POEGMA-2 were characterized with somewhat more positive ζ-potential than those of PVBTMAC-POEGMA-4. We can speculate here on the role of the polycationic block, which for PVBTMAC-POEGMA-2 is very short and presumably of lower effectiveness to compact DNA assuming that it is pulled to the surface of the particles by the hydrophilic POEGMA. The obtained vector systems were colloidaly stable for more than 1 week, preserving their size (see the Supporting Information). Their morphology was visualized by TEM. Figure 3 shows representative images of polyplex particles formed from PVBTMAC-40K homopolymer (Figure 3a) and PVBTMAC-POEGMA-4 block copolymer (Figure 3b). As seen the particles have spherical shape and their average diameters were close to those determined by DLS. Ethidium Bromide Quenching Assay. Ethidium bromide is a fluorescent compound interacting with DNA by intercalation between the base pairs.37 Due to this intercalation the dye exhibits strong fluorescence. Upon complexation with 2590


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Figure 5. Variations of the hydrodynamic radii (Rh) with NaCl concentration of PVBTMAC-based polyplexes formed with small (a and c) and large (b and d) DNA at N/P ratios of 0.5 (a and b) and 4.0 (c and d).

DNA packaging and delivery, which are relevant to gene transfection. The complexation of polyelectrolytes and, in particular, the formation of polyplexes are complicated processes in which a variety of parameters may influence the structure, stability, and effectiveness of the complex. Such parameters are polymer chain length and topology,38,39 charge density,40 N/P ratio,41 ionic strength,42 solvent polarity,43 order of mixing and preparation protocol.44−46 In the present contribution we systematically studied the complexation of two DNA samples−one small, 113 bp from salmon sperm and one large, 2000 bp from salmon testes−with polymer samples, representing two homoPVBTMAC polymers of different chain lengths and two block copolymers PVBTMAC-POEGMA of different PVBTMAC contents (Table 1). The total molar mass was in the 20−40 kDa range, whereas the PVBTMAC content varied from about 19 and 46% for the two copolymers to 100% for homopolymers. Precipitation was observed only in one case−the pair small DNA/homoPVBTMAC 20k, that is, the shortest partners, around the neutralization point, N/P ≈ 1 (Figure 1a). The precipitation was rather fast: within a few minutes after mixing the two solutions, a thin layer of solid material was deposited on the bottom of the vial. A fraction of the polyplex, however, presumably of different composition, remained in the solution, which made it possible to measure the ζ-potential. Within the time scale of the measurements at this ratio, no precipitate was formed for the pairs in which at least one of the partners was longer (Figure 1 b−d). Tiny fractions of solid material were

polyplexes containing increasing amounts of NaCl in the range of 0.01 to 0.3 M. As seen from Figure 5a,b at N/P ratio of 0.5, that is, in deficiency of polymer, the polyplexes were not sensitive to the presence of salt. This was not surprising because the effective diameter of neat DNA double helix decreases by increasing the ionic strength.33 At conditions of polymer in excess in the polyplex, that is, at higher N/P ratios, however, a critical salt concentration of about 0.2 M exists, at which the size of the polyplexes, prepared from the homopolymers started to increase (Figure 5c,d). This is probably due to the fact that the Na+ ions displace the polycations from their interactions with phosphate groups, causing destabilization of polymer binding to DNA. As a result the structure of complexes became looser and the polyplexes tend to disintegrate. Considering the moderate hydrophobicity of PVBTMAC (HLB = 9), one may expect a secondary aggregation of the partially dissociated complexes, which is seen as a sharp increase in particle size. In contrast, no change in particle size was observed for the polyplexes, prepared from the block copolymers, implying a stabilization effect of the POEGMA blocks. As seen from Figure 5c,d, the complexes formed with POEGMA modified cationic polymers retain their radii to around 45 and 85 nm for those prepared with small and large DNA, respectively.

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DISCUSSION Studies on electrostatic interactions between DNA and oppositely charged polyelectrolytes or copolymers containing polycationic moieties undoubtedly facilitate the elucidation of 2591


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homopolymers indicated the shielding effect of the POEGMA moieties. Only small variations in the size of polyplex particles with POEGMA-PVBTMAC-4 (Figure 2b,d) were observed in contrast to those with POEGMA-PVBTMAC-2 (Figure 2a,c). The common feature for the latter polyplexes was the initial formation of large particles which rapidly decreased in size (Figure 2a,c). Those of large DNA stabilized at about 75 nm at N/P ≥ 2, whereas the results obtained for the polyplexes with small DNA were less stable and scattered (large standard deviations and appearance of an additional mode in the correlation functions during the process of DLS measurements, which eventually was not separated by the software). The results clearly indicated that POEGMA-PVBTMAC-4 was able to effectively shrink and pack DNA independently from the molar mass and secondary structure of the latter presumably due to more efficient interactions of its longer cationic PVBTMAC block with the oppositely charged DNA. When the copolymer with shorter PVBTMAC block, POEGMAPVBTMAC-2, was used, DNA initially preserved its genuine structure. Larger quantities of this copolymer were needed in order to fully collapse and pack the DNA molecule in the polyplex. From the interpretation of the results so far, it was deduced that the copolymer POEGMA-PVBTMAC-4 was more effective in shrinking and packing DNA, whereas the effectiveness of the homopolymers was influenced by the molar mass, respectively, structure of DNA. At first glance, however, the data from the ethidium bromide quenching study (Figure 4) do not support these conclusions. The reason for these discrepancies is that the fluorescence measurements were done during the titration of DNA-ethidium bromide solution with polymer, i.e., the results represent the kinetics of the systems during the initial formation of the polyplexes. In that aspect, they reflect more the kinetics of polyplex formation rather than their equilibrium state. Indeed, in the cases with the small (Figure 4a) and large (Figure 4b) DNA, the copolymer POEGMA-PVBTMAC-2, that is, the copolymer with the shortest PVBTMAC moiety (molar mass of only 4200, Table 1), was the most effective in displacement of the intercalated dye. Its effectiveness can be rationalized in terms of fast kinetics of the processes of relaxation and redistribution of the polyelectrolyte chains in the polyplex that is associated with the short PVBTMAC moiety. Complete fluorescence quenching of ethidium bromide, however, was not achieved even at the highest N/P ratios. The concept of faster kinetics of displacement exhibited by the polymers with shorter polycationic sequences is clearly demonstrated by the polyplexes with large DNA (Figure 4b); the curves are nicely spaced according to the ability of the (co)polymer to displace the intercalated dye, which is associated with the length of the PVBTMAC moiety in the following order of decreasing displacement effectiveness: POEGMA-PVBTMAC-2 > POEGMA-PVBTMAC-4 > PVBTMAC-20K > PVBTMAC-40K. The situation slightly changed with time implying formation of kinetically frozen polyplex particles.41,50 A close correlation between the efficiency of cell transfection of polyplexes delivering DNA and the tolerance of the polyplexes to the addition of salt has been recently reported.51 The addition of salt to polyelectrolyte complexes typically leads to charge screening of the components and to a decrease of the number of interpolyelectrolyte salt bonds within the complex,52 which is followed by dissociation of the polyelectrolyte complex

detected after prolonged, e.g., more than a week, staying. The fast precipitation experienced by the small DNA/PVBTMAC 20k complex can be attributed to the faster processes of relaxation and redistribution of the polyelectrolyte chains inside the complex and chain exchange between complex nanoparticles exhibited for the shortest partners. The results obtained are in conformity with a recent study on long-term kinetics of DNA interacting with polycations.47 In contrast, no precipitation occurred at any N/P ratio for the polyplexes with the block copolymers. The samples remained optically clear displaying stable and reproducible values for both size and potential for many weeks (Figure 2). Here, the role of the POEGMA block providing colloidal stability was apparent. The closer inspection of the data presented in Figure 1 revealed that the homopolymer molar mass hardly had an effect on the size and surface potential: the variations of the latter two parameters with the N/P ratio followed the same curve pattern, whereas the values changed within the experimental error. For all combinations of partners, the ζ-potential tended to level off at a value of about +45 ± 5 mV at N/P ≈ 2. The curve patterns of size variation, however, were markedly different for the polyplexes with small (Figure 1a,b) and large (Figure 1c,d) DNA. As seen, at deficiency of polymer, that is, at the lowest N/P ratios, the sizes of polyplexes were very close to what was expected for the pristine DNAs−100−150 nm for circularized DNA (the large DNA, Figure 1c,d) and about 50 nm, which is consistent with the persistent length of a 140−150 bp DNA (Figure 1a,b). We can assume that at those ratios DNA is barely wrapped with polymer chains, which is in conformity with the negative values of ζ-potential. The size of the polyplexes with large DNA rapidly decreased with increasing N/P and leveled off at about 50 nm thus indicating a rapid transformation of the structure of DNA upon complexation. This observation is in conformity with earlier studies showing that long-chain DNA is in random coil conformation in the complex.48,49 It is also noteworthy that stabilization of both size and ζ-potential was observed at the same N/P ratio, N/P ≈ 2. In contrast, the complexes with short DNA initially increased their dimensions and then slowly and gradually tended to decrease in size (Figure 1 a and b). To explain this specificity, we can recall the stiff molecules (high persistent length) of the short oligonucleotides. At deficiency of polymer, such rigid, rod-like structures presumably align parallel and/or consecutively to each other thus giving rise to an initial sharp increase in size of the complex particles. Such alignment could also explain the precipitation observed for the complex with the shortest partners around the neutralization point. Beyond the neutralization region, at conditions of an increasing excess of polymer, the polyplex particles slowly disintegrated due to repulsion between the polymer chains. Very high polymer contents (N/P = 8) were needed to fully collapse and pack the DNA molecules. Similar to the homopolymers, the block copolymer polyplexes have analogous ζ-potential variations pattern: sharp transition from negative to positive values within a narrow N/P interval followed by leveling off at higher N/P ratios (Figure 2). The transitions were somewhat shifted to lower N/P values for the complexes based on POEGMAPVBTMAC-4, that is, the copolymer with the longer PVBTMAC block, implying more effective interactions with DNA. The lower (up to 20 mV) net values of the surface potential compared to those of the polyplexes with 2592


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conditions slightly beyond the physiological salt contents. The features of the PVBTMAC moieties, in particular, the large volume of the charged groups and the moderate hydrophobicity, played a decisive role in shrinking and compacting DNA. It was found that the longer polycationic block was more efficient in collapsing DNA independently from the molar mass and secondary structure of the latter. To achieve the same effect, larger quantities of the (co)polymers with shorter polycationic moieties were needed. The (co)polymers of longer PVBTMAC moieties, however, were less effective in displacement of EtBr, which was associated with the slow kinetics and formation of kinetically frozen polyplex particles that slowly changed with time.

to the initial polyions. The degree of polymerization, charge density, and substituents of the polycations as well as the type of added cations and anions have been shown important factors influencing stability of the polyplexes.53 It has been shown, however, that the polyplex formed by DNA and poly(L-lysine) of degree of polymerization of 700 monomer units remained quite stable even in 1.3 M NaCl solution.54 Furthermore, polyplexes formed by polycations with quaternary amine groups such as poly(N-alkyl-4-vinylpyridinium bromides) and poly(N,N-dimethyldiallyl ammonium chloride) are the least tolerant to dissociation by added salt. 54 Hydrophobic interactions provided by specific groups of the polycation may also noticeably contribute to the polyplex stability.53,55 Considering the chemical structure of PVBTMAC (Scheme 1), one would anticipate that the polyplexes based on (co)polymers containing such polycationic moieties are stable and tolerant to the addition of salts such as NaCl. Indeed, as Figure 5 shows, no indications for polyplex dissociation or quanitative precipitation were detected. The dimensions of the polyplex particles at N/P = 0.5 remained stable upon the addition of NaCl up to concentrations of 0.30 M (Figure 5a and b) independently from the type of the polyanion (small or large DNA) and polycation (homo- or block copolymers with different lengths of PVBTMAC). The same applied for the polyplexes with copolymers at N/P = 4 (Figure 5c and d). The markedly different situation for the polyplexes with homopolymers−the sharp increase of dimensions at NaCl concentration slightly beyond the physiological salt content (Figure 5c and d) − can be considered as the onset of precipitation. The addition of salt has been reported to favor the formation of insoluble polyelectrolyte complexes at conditions of slight excess of either nucleic acid or polycation.42 In the absence of hydrophilic moieties such as POEGMA, these polyplexes underwent aggregation, which is reflected by a sharp and sudden increase in dimensions at certain critical salt concentration. The latter is lower for the polyplexes with the homopolymer of higher molar mass which is in line with the expectations. Something similar was happening with the polyplexes based on the copolymers. However, the long POEGMA block exercised a stabilizing effect and prevented from aggregation, which ultimately lead to precipitation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b12477. SEC curves of PVBTMAC-POEGMA block copolymers in water and DMF; UV−vis calibration curve for determination of POEGMA-PVBTMAC block copolymer composition; ATR-FTIR characterization of PVBTMAC, POEGMA homopolymers and PVBTMAC-POEGMA block copolymer; Stability of polyplexes formed by PVBTMAC homopolymers and PVBTMAC-POEGMA block copolymers with small and large DNA. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Fund (Bulgaria) Project T-02/7. Partial financial support from the Greek General Secretariat for Research and Technology and the European Commission, through the European Fund for Regional Development, NSRF 2007-2013 action “Development of Research CentersKPHPIS”, Project 447963 “New Multifunctional Nanostructured Materials and DevicesPOLYNANO” is also acknowledged.

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CONCLUSION Novel PVBTMAC homopolymers as well as PVBTMACPOEGMA diblock copolymers were synthesized by RAFT polymerization. The polymerizations were conducted in aqueous media at conditions free of organic solvents and heavy metals, which is beneficial for applications of biological interest. The complexation of the (co)polymers with two DNA samples was systematically investigated focusing on parameters such as polycation chain length, polymer composition and architecture, N/P ratio, and ionic strength that might influence the structure, stability, and effectiveness of the resulting polyplexes. With the exception of the complex small DNA/ PVBTMAC-20k, that is, the shortest partners, precipitation around the neutralization point at N/P ≈ 1 was not observed. This was attributed to slow processes of relaxation, redistribution, and chain exchange of polyelectrolyte chains and, particularly for the complexes with block copolymers, formation of a hydrophilic POEGMA layer providing colloidal stability. The shielding effect of POEGMA was also manifested in lowering the net values of the surface potential and enhancement of the stability of the polyplex particles at

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REFERENCES

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