Nonviral Plasmid DNA Carriers Based on N,N ... - ACS Publications

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Nonviral Plasmid DNA Carriers Based on N,N′‑Dimethylaminoethyl Methacrylate and Di(ethylene glycol) Methyl Ether Methacrylate Star Copolymers Barbara Mendrek,† Łukasz Sieroń,‡ Iwona Ż ymełka-Miara,† Paulina Binkiewicz,§ Marcin Libera,† Mario Smet,∥ Barbara Trzebicka,† Aleksander L. Sieroń,‡ Agnieszka Kowalczuk,† and Andrzej Dworak*,† †

Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Sklodowskiej 34, 41-819 Zabrze, Poland Department of General, Molecular Biology and Genetics, Medical University of Silesia, Medykow 18, 40-752 Katowice, Poland § University of Occupational Safety Management in Katowice, ul. Bankowa 8, 40-007 Katowice, Poland ∥ Department of Chemistry, University of Leuven, Celestijnenlaan, 200F, B-3001 Leuven (Heverlee), Belgium ‡

S Supporting Information *

ABSTRACT: Star polymers with random and block copolymer arms made of cationic N,N′-dimethylaminoethyl methacrylate (DMAEMA) and nonionic di(ethylene glycol) methyl ether methacrylate (DEGMA) were synthesized via atom transfer radical polymerization (ATRP) and used for the delivery of plasmid DNA in gene therapy. All stars were able to form polyplexes with plasmid DNA. The structure and size of the polyplexes were precisely determined using light scattering and cryo-TEM microscopy. The hydrodynamic radius of a complex of DNA with star was dependent on the architecture of the star arms, the DEGMA content and the number of amino groups in the star compared to the number of phosphate groups of the nucleic acid (N/P ratio). The smallest polyplexes (Rh90° ∼ 50 nm) with positive zeta potentials (∼15 mV) were formed of stars with N/P = 6. The introduction of DEGMA into the star structure caused a decrease of polyplex cytotoxicity in comparison to DMAEMA homopolymer stars. The overall transfection efficiency using HT-1080 cells showed that the studied systems are prospective gene delivery agents. The most promising results were obtained for stars with random copolymer arms of high DEGMA content.

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INTRODUCTION

synthesized by controlled radical polymerizations were used as gene delivery systems. Linear (co)polymers,10,18,19 star polymers,7−9,11−13 comb-like structures,20 and other branched polymers6,21 have been successfully applied in gene therapy experiments. Several groups found that the polycationic stars exhibited enhanced transfection compared with linear analogues.8,11−13,22,23 Unfortunately, cationic polymers in high concentrations are toxic for most of the cell lines. While the transfection efficiency increased with the polymer concentration, the cell viability dropped.11,12,22 The solution to this problem for different nonviral vectors may be the introduction of a poly(ethylene glycol) (PEG) component into the polyplex structure, which frequently results in the decrease of cytotoxicity11,22,24 and the increase of the transfection efficiency.22 Moreover, the presence of PEG prevents serum-induced aggregation and accumulation of the star carriers at the cell surface. 11,22,24 This so-called “PEGylation” could be performed via the introduction of

Gene therapy is the treatment of genetic disorders by the introduction of genetic information into cells. The process of introducing DNA/RNA into eukaryotic cells is called transfection. Gene therapy is a promising approach for the treatment of many diseases such as genetic defects or cancer.1,2 Nonviral vectors based upon lipids,3 inorganic materials4 and polymers5 for gene delivery are currently some of the most investigated agents. Polymeric vectors based primarily on polycationic macromolecules are able to condense nucleic acids into nanosized structures called polyplexes. The polymer protects the genetic material against degradation while simultaneously improving the cellular uptake of the polyplex. Many cationic polymers have been investigated in transfection experiments; among other polymers are polyethylenimine,6−8 poly(N,Ndimethylaminoethyl methacrylate) (PDMAEMA),9−13 poly(Llysine) 14,15 and poly(N,N-dimethylaminopropylacrylamide).16,17 Currently, the major problem in nonviral gene delivery procedures is low transfection efficiency; therefore, it is important to find new solutions to this issue. Well-defined cationic polymers that have different topologies and are mainly © 2015 American Chemical Society

Received: July 15, 2015 Revised: September 10, 2015 Published: September 16, 2015 3275

DOI: 10.1021/acs.biomac.5b00948 Biomacromolecules 2015, 16, 3275−3285

Article

Biomacromolecules Table 1. Properties of the P(DMAEMA-ran-DEGMA) and PDMAEMA-b-PDEGMA Stars sample

Structure of arms

DEGMA contentd [mol %]

R1 R2 R3 R4 B1 B2

P(DMAEMA-ran-DEGMA) P(DMAEMA-ran-DEGMA) P(DMAEMA-ran-DEGMA) P(DMAEMA-ran-DEGMA) PDMAEMA-b-PDEGMA PDMAEMA-b-PDEGMA

11 20 30 40 8 21

Mn GPC‑MALLSa [g/mol] 230 254 350 226 183 372

000 000 000 000 000 000

Mw/Mn

dn/dcb

DParmc

2.70 2.65 2.39 2.55 2.48 2.76

0.083 0.083 0.072 0.076 0.077 0.073

46 51 71 43 36 76

a

Measured by GPC-MALLS in DMF. bCalculated from eq 1. cCalculated from Mn measured by GPC-MALLS assuming 28 initiating groups of PArOx and from NMR ratio DMAEMA:DEGMA. dCalculated from 1H NMR.

PEG to cationic polymers in order to obtain block,19,24−26 graft,20,27 or random copolymers.19,27 Currently oligo(ethylene glycol) methacrylates (OEGMA) are also used for PEGylation reactions of linear polymers serving as nucleic acid carriers.18,19,26,27 The structure of copolymers (e.g., random or block) and the PEG architecture influences the size, stability, and transfection efficiency of polymer/DNA complexes.19,27 Generally, linear copolymers with polycationic and PEG blocks form more compact complexes and demonstrate better longterm stability in the solution than random copolymers,26 they also exhibit a higher gene expression efficiency than random copolymers.27 To the best of our knowledge, there are only a few reports concerning the incorporation of methacrylates of oligo(ethylene glycols) into the structure of stars and subsequent transfection studies involving these macromolecules.11,22,28,29 Neoh et al.22 showed that the presence of oligo(ethylene glycol) ethyl ether methacrylate (Mn = 246 g/mol) as the second block in the arms of 4-arm PDMAEMA stars reduced toxicity when compared with homopolymer DMAEMA stars and branched polyethylenimine (PEI). The incorporation of this oligomer did not significantly influence the condensation capability of the DNA. Of more importance is that these stars improved the in vitro transfection of HEK293 cells in the presence and absence of serum. The influence of the star architecture on the ability to transfect HeLa cells was investigated for stars with 30 and 60 arms of PDMAEMA-poly[hexa(ethylene glycol) methyl ether methacrylate] (PHEGMA), possessing 10% mol of HEGMA.11 Independent of the architecture, all of the copolymer stars were less toxic to HeLa cells when compared with DMAEMA homopolymer stars. The highest transfection efficiency exhibited stars with HEGMA units in outer part of the arms, while the smallest efficiency was observed in the case of stars with random copolymer arms of HEGMA and DMAEMA.11 The use of branched polymers with low cytotoxicity for the construction of polyplexes, when compared with linear analogues, provides a high density of polymer chains, a large number of suitable functional groups and satisfactory intensity of gene expression. Research has often shown the positive impacts of branched architecture on transfection efficiency, but general conclusions based only upon the topology of cationic polymers are not possible. The introduction of components that improve biocompatibility appears to be an interesting solution that can significantly improve the transfection of star polyplexes. In this paper, we described the synthesis of the DMAEMA-di(ethylene glycol) methyl ether methacrylate star polymers using atom transfer radical polymerization (ATRP) via the “core first” method. The star polymer properties in solution and their ability to condense the plasmid DNA were studied for different star structures (random or block) and

different compositions of the arms (from 8 to 40 mol % of DEGMA comonomer). The size and structure of the obtained star−DNA polyplexes were thoroughly characterized. Subsequently, transfection and toxicity experiments were performed using HT-1080 cells and compared with the cell transfection “gold-standard” branched polyethylenimine.

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EXPERIMENTAL SECTION

Materials. Chemicals. Di(ethylene glycol) methyl ether methacrylate (DEGMA, 95%), branched PEI of Mn = 25 000 g/mol, 10 mg/mL ethidium bromide in H2O, 1,2-dichlorobenzene (99%), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 97%), copper(I) bromide (CuBr, 99.999%), copper(II) bromide (CuBr2, 99%), p-xylene (99%), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA, 99%), Dulbecco’s Modified Eagle’s Medium (DMEM) and kanamycin sulfate were purchased from Sigma-Aldrich and used as received. Lysogeny Broth (LB) medium for cultivation of the bacteria was purchased from QBIOgene (USA). Anisole (99%) was purchased from Sigma-Aldrich and purified by distillation prior to use. Phosphate buffered saline (PBS) purchased from Sigma-Aldrich was diluted 10fold to pH = 7.37. N,N-Dimethylaminoethyl methacrylate (DMAEMA, 99%) was purchased from Merck and purified by distillation prior to use. DOWEX MARATHON MSC ion-exchange resin was purchased from Sigma-Aldrich and transformed into the H+ type using 1.6 M HNO3. Methanol (99.8%) and tetrahydrofuran (THF, 99.8%) were purchased from POCh and used as received. Fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, amphotericin B, trypsin (0.25% in 1 mM EDTA) and phosphate buffered saline (PBS) were purchased from PAA Laboratories GmbH and used for the cell culture experiments. AlamarBlue Cell Viability Reagent was purchased from Invitrogen. Plasmid DNA pMetLuc2-control vector (pDNA) with 4784bp that was purchased from Clontech was prepared using the Plasmid DNA Maxi Kit (Omega Bio-Tek, USA). Synthesis of Poly(arylene oxindole) Core. The synthesis and characterization of the poly(arylene oxindole) (PArOx) core was described in our previous paper.30 PArOx with 28 bromoester groups was used as the macroinitiator of the ATRP of DMAEMA and DEGMA. The molar mass of PArOx was Mn = 21 000 g/mol and Mw/ Mn = 2.17. Synthesis of Star Polymers with Poly[N,N-dimethylaminoethyl methacrylate-ran-di(ethylene glycol) methyl ether methacrylate] Arms (P(DMAEMA-ran-DEGMA)). PArOx (50 × 10−3 g, 2.4 × 10−6 mol), CuBr (9.5 × 10−3 g, 6.6 × 10−5 mol), and CuBr2 (7 × 10−4 g, 3.1 × 10−6 mol) were dissolved in 0.5 mL of 1,2dichlorobenzene in a Schlenk flask under nitrogen with a magnetic stirrer and degassed using freeze−vacuum−thaw cycles. Then, HMTETA (15.2 × 10−3 g, 6.6 × 10−5 mol, and 18 × 10−3 mL; [CuBr]:[CuBr2]:[HMTETA] in the ratio of 1:0.05:1) was added to the solution, and degassed. Next, DMAEMA (0.16 g, 1 × 10−3 mol, 0.17 mL) and DEGMA (61 × 10−3 g, 3.33 × 10−4 mol, 0.06 mL; monomers to 1,2-dichlorobenzene ∼1:2 v/v) were added to the solution, and it was degassed twice. The flask was placed in an oil bath at 40 °C. Samples were taken during the polymerization process and analyzed without purification using gel permeation chromatography to determine the molar mass and dispersity. After the desired molar mass 3276


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Biomacromolecules was obtained, THF (5 mL) was added, and the solution was passed through a column with DOWEX-MSC-1 ion-exchange resin to remove the copper. The subsequent solution without copper was dialyzed, first against methanol and then against water (SpectraPor membrane with MWCO 1000 g/mol), and dried by lyophilization to prevent polymer aggregation (stars R1-R4, Table 1). Synthesis of Star Polymers with Poly[N,N-dimethylaminoethyl methacrylate-b-di(ethylene glycol) methyl ether methacrylate] Arms (PDMAEMA-b-PDEGMA). The conditions of DEGMA polymerization were previously established.31 Purified PDMAEMA star polymer (10 × 10−2 g), CuBr (1.33 × 10−3 g, 9.3 × 10−6 mol) and CuBr2 (1 × 10−4 g, 4.65 × 10−7 mol) were dissolved in 6 mL of anisole in a Schlenk flask under nitrogen with a magnetic stirrer and degassed using freeze−vacuum−thaw cycles. After the second degassing, DEGMA (0.18 g, 9.3 × 10−4 mol, 0.17 mL), which was degassed separately, was added to the solution (monomer to anisole ∼1:35 v/v). Next, PMDETA (3.22 × 10−3 g, 1.86 × 10−5 mol, 3.88 × 10−3 mL) was added to the stirred mixture [CuBr]:[CuBr2]: [PMDETA] in the ratio of 1:0.05:2). The solution was again degassed using freeze−vacuum−thaw cycles. The flask was placed in an oil bath and thermostated at 40 °C. The next steps were the same as for the synthesis of random copolymer stars (stars B1 and B2, Table 1). Plasmid DNA Preparation (pDNA). The plasmid pMetLuc2control vector (Clontech) was amplified in the Escherichia coli DH5 alpha strain and cultured in LB Broth-Miller medium (QBIOgene, USA) with 50 μg/mL of kanamycin. The plasmid DNA was purified using the Plasmid DNA Maxi Kit (Omega Bio-Tek, USA) according to manufacturer’s protocol. The DNA concentration was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc.). Formation of Complexes of Polymers with Plasmid DNA. A suitable amount of star or PEI solution in PBS (c = 0.5 mg/mL) was added to a fixed volume of plasmid DNA solution in PBS (400 μL, c = 0.02 mg/mL) to achieve the desired nitrogen/phosphate (N/P) ratios. The obtained solutions were mixed and incubated at room temperature for 30 min. Polyplexes prepared by this method in PBS solutions were used for dynamic light scattering (DLS) and zeta potential measurements. For agarose gel electrophoresis and all biological experiments, star or PEI polyplexes were prepared with 330 ng of plasmid DNA in a total volume of 20 μL of serum and supplements free DMEM medium. An appropriate volume of polymer in DMEM (c = 10 μg/ mL) was added to the DNA solution to reach different N/P ratios, which was subsequently vortexed and incubated for 30 min at RT to allow polyplex formation (polyplexes R1-pDNA−R4-pDNA, B1pDNA, B2-pDNA, and PEI-pDNA). Agarose Gel Electrophoresis. Two microliters of loading buffer was added to the star polyplex solution of the appropriate N/P in DMEM, and the entire reaction volume was loaded onto 1.5% agarose gel containing 1 μg/mL ethidium bromide. Electrophoresis was conducted at 150 V for 10 min in TAE (Tris-acetate EDTA) running buffer. The gel was analyzed on a UV illuminator (Syngene) to visualize the location of the polyplexes compared with naked plasmid DNA. Cell Culture. HT-1080 human fibrosarcoma cells (ATCC#CCL121) were cultured in DMEM medium supplemented with 10% FBS, 1% L-glutamine, penicillin, streptomycin and amphotericin B at 37 °C and 5% CO2. After reaching 90% confluency, the cells were passaged to three 175 cm2 bottles. Cytotoxicity of Polymers and Polyplexes. Seventeen hours prior to the cytotoxicity experiments, HT-1080 cells were seeded in DMEM growth medium in 24-well plates at a density of 8 × 104 cells per well. The next day, the medium was discarded and replaced with 500 μL of fresh complete culture medium supplemented with various concentrations of star polymer or PEI. The plates were incubated for an additional 24 h, then the medium was removed, and the plates were rinsed with warm PBS buffer. After adding 400 μL of 10% alamarBlue reagent in complete growth medium to each well, the plates were incubated for 1 h. Then, the fluorescence emission was monitored at 590 nm using VICTOR Multilabel Plate Reader (PerkinElmer, USA)

with a 560 nm excitation source. The cells viability (in %) was calculated by comparing treated cells with control cells that were not treated with the polymer and which represented to 100% of viability. The experiments were repeated four times. The stars or PEI polyplexes cytotoxicity assay was similarly performed to that described for the star polymers but for 12-well plates format. The polyplexes were prepared by combining 1 μg of DNA (in a total volume of 100 μL of serum and supplement-free DMEM) with an appropriate amount of the star polymer or PEI (corresponding to the different N/P ratios), and the solution was vortexed and then incubated for 30 min at room temperature. Transfection Efficiency Studies. Transfection experiments were performed on HT-1080 cells using the plasmid pMetLuc2-control vector. The HT-1080 cells were seeded in DMEM complete growth medium in 12-well plates at a density of 16 × 105 cells per well up to 17 h prior to the transfection experiments. Shortly before transfection, the medium was discarded and replaced with 1 mL of fresh and prewarmed supplemented DMEM. Prepared star and PEI polyplex solutions of the appropriate N/P were added to the cells in the culture medium. The plates were incubated for an additional 48 h at 37 °C. After this incubation time, the medium was aspirated, the cells were washed with warm PBS buffer, 0.5 mL of fresh medium was added, and the cells were incubated for 8 h. Then, 50 μL of culture medium was collected for secreted luciferase activity detection. Metrida secreted luciferase activity in the culture medium was measured using Ready-To-Glow Secreted Luciferase Reporter Assay kit (Clontech,) according to the manufacturer’s instruction. The transfection efficiency was evaluated based on the average of three luminescence intensity measurements performed in 96-well plates with a 1 s exposition time on VICTOR Multilabel Plate Reader (PerkinElmer, USA). Measurements. NMR spectra were recorded using a Bruker Ultrashield 600 spectrometer (600 MHz for 1H). The resonances were presented in ppm and referenced to the tetramethylsilane (TMS) peak. The molar masses and molar mass dispersities of the star polymers were determined using gel permeation chromatography (GPCMALLS) with a differential refractive index detector (Δn-2010 RI WGE Dr. Bures) and a multiangle laser light scattering detector (DAWN EOS from Wyatt Technologies). GPC was performed in DMF at 45 °C with a nominal flow rate of 1 mL/min using the following set of columns: GRAM gel guard, GRAM 100 Å, GRAM 1000 Å and GRAM 3000 Å (Polymer Standard Service). The results were evaluated using the ASTRA 5 software from Wyatt Technologies. The refractive index increments (dn/dc) of the stars were calculated from eq 1: ⎛ dn ⎞ ⎛ dn ⎞ dn = wPArOx ⎜ ⎟ + wDMAEMA ⎜ ⎟ ⎝ ⎠ ⎝ dc ⎠PDMAEMA dc dc PArOx ⎛ dn ⎞ + wDEGMA ⎜ ⎟ ⎝ dc ⎠PDEGMA

(1)

where wPArOx is the weight ratio of the core, wDMAEMA is the weight ratio of DMAEMA in the star polymer, wDEGMA is the weight ratio of DEGMA, (dn/dc)PArOx is the refractive index increment of the PArOx core, (dn/dc)PDMAEMA is the refractive index increment of the PDMAEMA, and (dn/dc)PDEGMA is the refractive index increment of the PDEGMA. The refractive index increments of PArOx (dn/dc = 0.149 mL/g), linear PDMAEMA (dn/dc = 0.056 mL/g) and linear PDEGMA (dn/ dc = 0.073 mL/g) were independently measured in DMF using a SEC3010 dn/dc WGE Dr. Bures differential refractive index detector. These values were used for calculating the refractive index increment of stars R1−R4 and B1−B2 (Table 1) according to eq 1. DLS measurements were performed for the star polymers and their polyplexes on a Brookhaven BI-200 goniometer with vertically polarized incident light with λ = 632.8 nm (supplied by a He−Ne laser operating at 35 mW) and a Brookhaven BI-9000 AT digital autocorrelator. The autocorrelation functions were analyzed using the constrained regularized algorithm CONTIN. The measurements were 3277


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Biomacromolecules Scheme 1. Synthesis of the P(DMAEMA-ran-DEGMA) (A) and PDMAEMA-b-PDEGMA Stars (B)

made at a 90° angle at 25 °C. The dispersity of particle sizes was given μ as 22 , where Γ̅ is the average value of relaxation rates Γ, and μ2 is its

close to unity. It may be assumed that DMAEMA and DEGMA (the monomer of a similar structure to OEGMA) will form arm chains with a random distribution of monomer units. Stars with block arms of DMAEMA and DEGMA were obtained in a two-pot method using the reactivity of an endcapped brominated star PDMAEMA macroinitiator (route B, Scheme 1). The second monomerDEGMAwas introduced into the ATRP polymerization system after the purification of PDMAEMA stars using conditions estimated in our previous study.31 Hyperbranched PArOx with a degree of branching equal to 100% and 2-bromoester groups, which initiated the ATRP of DMAEMA and DEGMA, was used as the core. The number of initiating groups was 28, as determined by the Frey equation,33 which relates the number of dendritic and terminal units to the degree of polymerization. The core was obtained via polycondensation of the 5-bromo-1-[4-(4-phenoxybenzoyl)benzyl]-isatin (AB2 monomer). Then, the functional groups were modified to 2-bromoester groups to introduce the initiating functions. The absolute molar mass of the core measured by GPC-MALLS was Mn = 21 000 g/mol and Mw/ Mn = 2.17.9 All polymerization processes were carried out to low monomer conversions in order to prevent radical star−star coupling reactions. The molar masses and molar mass dispersities of the stars were measured by gel permeation chromatography with multiangle laser light scattering detection (GPC-MALLS). The molar mass distributions of all stars were monomodal (Figure 1, for clarity only random star copolymers are shown). As expected, the molar mass of the stars increased with the increasing degree of polymerization (DP) of the arms. In the case of block copolymer stars syntheses, no measurable amount of star macroinitiator has been detected in the reaction product, which indicated the quantitative extension of star arms by the DEGMA comonomer. The 1H NMR spectra of the stars acquired in chloroform (Figure 2, sample R3) displayed peaks corresponding to αmethyl groups and methylene groups in the methacrylate backbone of DMAEMA and DEGMA at δ = 0.9−1.15 ppm (a) and 1.8−1.9 ppm (b), respectively. Proton signals from the methylene groups in pendant chains of DMAEMA were found at δ = 2.6−2.65 ppm (d) and δ = 4.0−4.1 ppm (e), and signals from the methyl protons of the DMAEMA amino group were

Γ̅

second moment. These values were obtained from cumulant analysis. Before DLS analysis, the star polymer solutions were passed through membrane filters with nominal pore sizes of 0.2 μm (ANATOP 25 PLUS, Whatman). The solvents used to prepare the solutions for DLS were filtered through a 0.1 μm membrane filter (ANATOP 25 PLUS, Whatman) in a laminar flow cabinet prior to use. The obtained solutions of polyplexes were not filtered. Zeta potential measurements were performed in triplicate on a Zetasizer Nano ZS 90 (Malvern Instruments) in disposable folded capillary cells. Cryogenic transmission electron microscopy images (cryo-TEM) were obtained using a Tecnai F20 TWIN microscope (FEI Company, USA) equipped with a field emission gun, which operates at an acceleration voltage of 200 kV. Images were recorded on an Eagle 4k HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA). Specimens were prepared by vitrification of aqueous polymer solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Germany). Prior to use, the grids were activated for 15 s in oxygen/argon plasma using a Fischione 1020 plasma cleaner (E.A. Fischione Instruments, Inc., USA). Samples were prepared by applying a droplet (2.1 μL) of solution to the grid, blotting with filter paper, and immediately freezing in liquid ethane using a fully automated Vitrobot Mark IV (FEI Company, USA) blotting device. After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a Gatan 626 cryoTEM-holder (Gatan Inc., USA) and analyzed in the TEM at −178 °C.

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RESULTS AND DISCUSSION Synthesis of the Star Polymers with P(DMAEMA-ranDEGMA) and PDMAEMA-b-PDEGMA Arms. Star polymers with a hyperbranched PArOx core and arms made of random (route A, Scheme 1) or block (route B, Scheme 1) copolymers of DMAEMA and DEGMA were obtained via ATRP. The polymerization process was carried out under similar conditions to those used in the synthesis of stars with homopolymer DMAEMA arms, previously described.9 Here, two types of stars were synthesized: stars with random copolymer arms (Table 1, samples R1-R4) and stars with block copolymer arms (Table 1, samples B1 and B2). A one-pot method was used to obtain stars with random copolymer arms (route A, Scheme 1). According to the studies of Lang et al.,32 the values of DMAEMA and OEGMA (Mn = 475 g/mol) reactivity ratios estimated in ATRP process are 3278


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Biomacromolecules

moieties are visible. In water, a solvent only for the hydrophilic arms of the star, signals from aromatic protons of the strongly hydrophobic hyperbranched core could not be observed. The molar ratios of comonomers in the arms of the star were calculated from NMR spectra. The ratio of DMAEMA to DEGMA were determined from the spectra recorded in chloroform by comparing peak integrals from the six protons of two methyl groups in DMAEMA at δ = 2.25−2.35 ppm (peak c, Figure 2) to the three protons of the methoxy group at the end of pendant chains in DEGMA at δ = 3.4 ppm (peak f, Figure 2). The content (mol %) of DEGMA in the copolymer arms of stars, as shown in Table 1, varied from 8 to 40%. Solution properties of the star polymers with P(DMAEMA-ran-DEGMA) and PDMAEMA-b-PDEGMA arms. The size of a star molecule in solution plays a key role in determining many of its properties, especially with respect to potential medical applications. The hydrodynamic radii of all studied stars were investigated in acetone, water, PBS buffer and culture medium DMEM (applied in transfection experiments) using DLS. The values of hydrodynamic radii Rh90 measured at 25 °C and averaged from a minimum of three measurements are summarized in Table 2. The same table shows the contour length calculated for a totally extended arm chain by multiplying the DP of the arm times 0.252 nm (the length of one monomer repeating unit containing two carbon atoms in the backbone34). The size distributions of all star polymers in acetone showed μ one broad peak with a dispersity of the particles sizes 22 from

Figure 1. Chromatograms (RI traces) of stars with random copolymer arms (DMF, 1 mL/min).

at δ = 2.25−2.35 ppm (c). Proton signals of methylene groups in pendant chains of DEGMA were found at δ = 3.5−3.7 ppm (g) and δ = 4.0−4.1 ppm (e′), and the signal from the methyl group at the end of pendant chains of DEGMA was found at δ = 3.4 ppm (f). The signals corresponding to the aromatic protons of the core were observed in the range of 7.0−7.8 ppm. The amphiphilic character of the stars could be observed when their NMR spectra were recorded in two solvents with different affinities for the components of the stars (Figure 2, sample R3). In chloroform, a good solvent for both the core and the arms, all peaks corresponding to particular chemical

Γ̅

0.2−0.25. Acetone is a good solvent for both the copolymer arms and the PArOx core. The differences between the measured values of the hydrodynamic radii of stars in acetone and the calculated contour length of the arms are small and do

Figure 2. 1H NMR spectra of the R3 star in D2O (A) and CDCl3 (B) (600 MHz). 3279


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Biomacromolecules Table 2. Hydrodynamic Radii of the Synthesized Stars in Different Solvents sample R1 R2 R3 R4 B1 B2 a

Mn GPC‑MALLS [g/mol] 230 254 350 226 183 372

000 000 000 000 000 000

DParm

contour length of arma [nm]

Rh90[nm] acetoneb

Rh90[nm] PBSb

Rh90[nm] DMEMb

46 51 71 43 36 76

11.6 12.9 17.9 10.8 9.1 19.2

13.6 14.1 14.4 14.2 13.0 14.8

20.5 31.0 19.0 17.2 18.0 22.0

24.5 26.3 20.0 18.0 18.0 28.0

Calculated for fully stretched arms. bc = 1 mg/mL.

Table 3. Hydrodynamic Radii of the Synthesized Stars in Aqueous Solutions of Different pH Rh90[nm] sample

pH = 2.5

pH = 3.8

pH = 4.1

pH = 6.2

pH ∼ 7.0

pH = 9.0

pH = 10.0

pH = 11.0

pH = 12.0

pH = 13.0

R1 R2 R3 R4 B1 B2

12.2, 20.4 9.8, 21.5 22.1 14.5 24.2 25.7

10.6, 20.5 9.4, 25.9 23.5 13.5 20.2 25.5

9.5, 22.4 9.4, 29.7 20.2 14.8 19.8 23.5

9.5, 27.1 6.8, 30.0 20.1 16.8 19.1 22.1

7.6, 33.5 6.8, 30.5 19.5 18.8 14.5 23.4

18.6 14.6 13.3 9.5 11.6 21.6

19.4 21.3 13.1 10.0 13.7 16.2

25.6 22.9 13.2 9.2 12.7 16.4

24.8 25.2 13.6 10.3 10.2 16.8

27.0 26.3 14.8 10.7 12.4 17.7

not permit one to conclude whether the stars exists in solution as isolated molecules or as aggregates of a few stars. The solution behavior was also investigated in culture medium DMEM (used in transfection experiments) and PBS buffer, which was used in the size and zeta potential measurements of polyplexes (Table 2). The results show that the sizes of all stars in these solutions were greater than those measured in acetone, suggesting that the macromolecules are aggregated; this behavior is similar to homopolymer DMAEMA stars with a PArOx core in DMEM.9 In an aqueous solution, the polyelectrolyte properties of PDMAEMAfull protonation at acidic pH and complete deprotonation of amine groups at alkaline pHcause this polymer to be sensitive to changes in pH. The change in the degree of protonation is manifested by an extension of polymeric chains in the protonated state (through electrostatic repulsion forces) or chain shrinkage caused by the reduction of charge.35 The star polymers were directly dissolved in deionized water, and the pH of the solutions was adjusted by adding appropriate amounts of 1 M KOH or 1 M HCl. For all obtained stars, either one or two populations (samples R1 and R2 at pH ≤ 7) of particles were observed in light scattering measurements, corresponding to star polymer molecules and/or star aggregates (Table 3). It was found that all star polymers are soluble in water throughout the whole pH range. However, a change in the star size occurs under the influence of pH. The variations are not sufficient to clearly indicate whether star macromolecules are present in solution as single stars or small aggregates of multiple stars. It should be noted that the sizes of all star aggregates are on the order of tens of nanometers, which does not limit the applications of stars in medicine and biology. The zeta potential is an indicator of the surface charge of nanoparticles; therefore, its value depends strongly on solution pH. The zeta potential was measured for aqueous star solutions at various pH values (Figure 3). For all star polymers, independent of DEGMA content and arm architecture, the values of zeta potential decreased with increasing pH. The zeta potential for star polymers exhibited positive values in acidic pH (Figure 3) due to the protonation

Figure 3. Dependence of the zeta potential with changes in pH for star polymers.

of amine groups of DMAEMA and displayed negative values in alkaline pH. Uchida et al.32 claimed that the negative values of zeta potential at high pH might be a result of the adsorption of OH¯ anions on the deprotonated star arms with increased OH¯ concentration in the solution. The isoelectric point of the stars was located between pH 9 and 11. The positive values of zeta potential (20−40 mV) for stars at physiological condition (pH ∼ 7.4) indicate that they could be used in polyplex formations with negatively charged DNA. Star/Plasmid DNA Complexes. A 4784 base-pair plasmid DNA encoding luciferase was condensed with star polymers into nanoparticles suitable for cellular uptake. It should be noted that in numerous previous studies, the thorough analysis of polyplex properties is not sufficiently elucidated or is even omitted. Often there is a lack of information on the structure and size of polyplexes present in a solution. Here, the ability to condense pDNA by the stars into polyplexes was confirmed following agarose gel electrophoresis, while DLS, zeta potential 3280


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Biomacromolecules measurements and cryo-TEM imaging were used to characterize the obtained star/plasmid DNA complexes. The polyplexes were formed at different N/P ratios in PBS buffer at pH = 7.4. The appropriate amount of polymer solution was added to a fixed amount of pDNA, then the solutions were incubated at room temperature for 30 min. The N/P ratio at which the pDNA is completely bound by a polycationic star was estimated using agarose gel electrophoresis. Naked pDNA was used as a control (the first lane at N/P = 0 in Figure 4). The complete condensation of the

Figure 5. Average hydrodynamic radii of polyplexes with random (R1pDNA to R4-pDNA) and block (B1-pDNA and B2-pDNA) star copolymers and PEI-pDNA at different N/P ratios in PBS buffer at pH = 7.4 (the lines are guides for the eye). The value of Rh90 of naked DNA is approximately 100 nm.

At low N/P values, the Rh90 of the polyplexes formed by random star copolymers with the highest DEGMA content (R3-pDNA and R4-pDNA) increased with N/P up to N/P = 2. Then, the sizes decreased with a further increase of this ratio. The R h 90 of R3-pDNA and R4-pDNA decreased to approximately 50 nm at N/P ≥ 8, significantly lower than Rh90 of naked pDNA. The maximum in the size of R3-pDNA and R4-pDNA polyplexes at N/P = 2 may be assigned to the aggregation of polyplexes, facilitated by the fact that at this N/P there is practically no overall surface charge (zeta potential close to zero, Figure 6). The Tam group24 also observed a jump in size for polyplexes of 4 arm PEO-b-DMAEMA stars with plasmid DNA encoding green fluorescent protein in HEPES buffer. The authors had concluded that the maximum in polyplex size may have been caused by the sudden rearrangement of DNA structure. These observations could also explain the results obtained for the polyplexes of stars R3 and R4.

Figure 4. Patterns of electrophoretic mobility of plasmid DNA in the presence of star polymers (sample R1-R4, B1, B2, Table 1) at different N/P ratios.

pDNA by stars with random copolymer arms was not dependent on the content of DEGMA comonomer and was observed at an N/P = 4. For stars with block copolymer arms, the N/P ratio at which binding of the nucleic acid is complete was lower (N/P = 3) than for stars with random copolymer arms, suggesting a better binding of pDNA when amine groups are located next to each other. The higher amount of stars are needed for the total binding of pDNA than in the case of branched PEI (N/P = 2).9 The size of polyplexes is an important parameter for the preparation of nanocarriers for gene therapy. The general observed tendency is that transfection efficiency is inversely related to plasmid size.36 The sizes of star-pDNA complexes were dependent upon the change in the ratio of amine groups in polycationic star arms to phosphate groups in the nucleic acid. DLS measurements of star polyplexes that were performed in PBS buffer are shown in Figure 5. The sizes of studied polyplexes were compared with the sizes of pDNA complexes with branched polyethylenimine (PEI) (Mn = 25 000 g/mol). The value of Rh90 of naked pDNA before its complexation with stars was approximately 100 nm, with a monomodal but μ broad distribution 22 = 0.3. Γ̅

The size of polyplexes of the studied stars was found to be dependent on the DEGMA comonomer content and the architecture of star arms. The Rh90 of stars decreased to values lower than the Rh90 of naked pDNA at high N/P ratios.

Figure 6. Zeta potentials of the studied polyplexes at different N/P ratios in PBS at pH = 7.4 (the lines are guides for the eye). The zeta potential of naked DNA in PBS solution with concentration of 0.2 mg/mL was −70.3 mV. 3281


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Biomacromolecules The remaining star polyplexes, independent of the DEGMA content and monomers distribution in star arms, did not show a maximum in size; with the increase of the N/P ratio, the hydrodynamic radii of polyplexes decreased. For the stars with a higher content of DMAEMA (samples R3, B1, B2, Table 1), N/P = 6 is the limit required for the creation of polyplexes with dimensions smaller than naked pDNA. This limit should ensure the easy delivery of genetic material into a cell. Block copolymer star B2, with a similar molar mass as star R3 but a lower DEGMA content, created polyplexes with the smallest size from all obtained pairings. Unexpectedly, the branched PEI with the same pDNA used in star polyplexes formed complexes independent of N/P ratio, with sizes in the range of 300−400 nm. It should be emphasized that PEI polyplexes were prepared using a procedure identical to polyplexes of studied star polymers. It is known that the positively charged surface of a nanocarrier interacts electrostatically with the anionic cell surface and facilities cellular uptake.22,37 The zeta potentials of polyplexes were measured in PBS buffer (Figure 6) at different N/P ratios and compared with PEI−pDNA complexes. The zeta potential of naked pDNA in PBS solution with a concentration of 0.2 mg/mL was −70.3 mV. For all star polyplexes in the range of investigated N/P ratios, zeta potentials exhibited higher values than the naked pDNA. The positive values of zeta potential were observed for polyplexes at an N/P ratio of complete DNA complexation (N/P = 4 for R1-pDNA−R4-pDNA and N/P = 3 for B1pDNA, B2-pDNA), which was in agreement with gel electrophoresis (Figure 4). The zeta potential increased with an increase of N/P ratio and stabilized above N/P > 6. The excess of star polymer slightly affects both the size (Figure 5) and zeta potential of the polyplex (Figure 6). Generally, the zeta potential values of all polyplexes of DMAEMA and DEGMA star copolymers are lower than those obtained for the polyplexes made of homopolymer DMAEMA stars described in our previous paper.9 These lower zeta potential values can be explained by the shielding effect of methacrylates of oligo(ethylene glycols), which are known to reduce the positive charge of complexes between the star and pDNA.28 The polyplexes of DNA with commercially available branched polyethylenimine possess values of zeta potential similar to those of star polyplexes at N/P ratios below 8. For N/P > 8, zeta potentials of PEI-pDNA are lower than the values for all prepared star polyplexes. The cryo-TEM images (Figure 7) showed that tangled pDNA particles are surrounded by stars (little spots in images), but the structure of the polyplexes is irregular and loosely packed. No compact nanoparticles are observed. The structure of polyplexes is much less dense than in the case of PDMAEMA homopolymer stars,9 which could be caused by the uncharged DEGMA units (inert for nucleic acid) present in the star arms. The sizes of polyplexes from cryo-TEM images are in the range of 100−200 nm, which are comparable with those obtained by DLS measurements (Figure 5). Also it has to be noted that the cryo-TEM pictures were taken at N/P = 16; at this ratio the uncomplexed star polymers are also present in the sample, which can be seen in the images. The formation of star polyplexes with pDNA is schematically shown in Scheme 2. Cytotoxicity and Transfection Efficiency Studies. It is well-known that poly(ethylene oxide) is biocompatible and

Figure 7. Cryogenic transmission electron microscopy images obtained from a PBS solution of star-pDNA at N/P = 16. (A) B1pDNA, (B) B2-pDNA, (C) R1-pDNA, (D) R2-pDNA, (E) R3-pDNA, and (F) R4-pDNA (the white circles are just to guide the eye).

Scheme 2. Preparation of Polyplexes of Stars with Nucleic Acid

nontoxic.11,22,24 The main advantage of introducing DEGMA segments into the star arms should be a lowering of the cytotoxicity of the polycationic stars and their polyplexes in comparison to homopolymer PDMAEMA stars. The effect of DEGMA content in star arms on the viability of HT-1080 fibrosarcoma cells was investigated. The results are plotted in Figure 8, with branched polyethylenimine used for comparison. The viability of the HT-1080 cells was assessed using an alamarBlue reduction test assay. The use of all obtained stars leads to cell viability that is significantly higher than branched PEI over the entire range of investigated concentrations. The incorporation of DEGMA segments into the arms of the stars decreased the cytotoxicity substantially in comparison with PDMAEMA stars.9 The star 3282


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at N/P ≥ 16 was higher than that for polyplexes consisting of the homopolymer DMAEMA star, as has been previously published.9 This is in an agreement with our assumption and the previous studies of other groups, which have shown that the introduction of PEG segments into a star polymer decreases the cytotoxicity of polymers used for gene transfection.11,22 The in vitro gene transfection efficiency in HT-1080 cells was studied using prepared complexes with secreted Metrida luciferase as a reporter gene driven by CMV promoter. The gene transfection efficiency was estimated by the overall proper protein production (luciferase) by transfected cells. It was assumed that only living cells manufactured proteins at the time of measurement. The transfection efficiency, visible as luciferase activity, was evaluated indirectly as an average of three luminescence measurements. This assay was performed using a constant amount of plasmid DNA complexed with stars R1R4, B1 and B2 at different N/P ratios. The branched PEI polyplexes served as a control. The results of the transfection experiments are summarized in Figure 10. The efficiency of transfection related to the number of cells in the well was placed in the Supporting Information (Figure S1).

Figure 8. Effect of star (R1-R4, B1 and B2, Table 1) and branched PEI concentrations on HT-1080 cell viability (the lines through data points are guides for the eye).

with a random distribution of DMAEMA and DEGMA and with the highest content of DEGMA units (sample R4, Table 1) provided the highest cell viability for the complete polymer concentration range. However, the stars with block structured arms, even with 21 mol % of DEGMA, were more toxic than stars with a random distribution of arms, suggesting that the content of DEGMA creating the outer shell of the star is insufficient to prevent the necrosis of cells. All obtained polyplexes were less toxic than the polyplex of PEI-pDNA for all investigated N/P ratios (Figure 9).

Figure 10. Efficiency of HT-1080 cells transfection with polyplexes at different N/P ratios. The results are shown as overall luminescence values.

As shown in Figure 10, the transfection efficiency increased with the N/P ratio, and the highest transfection efficiencies for all star polyplexes were obtained at N/P = 32. Our results for branched PEI indicate the highest luciferase activity at ratios of 6 and 8 followed by its reduction in activity at higher N/P values, which correspond well with data reported in the literature.22 The highest gene expression levels were demonstrated by polyplexes made of random copolymer stars with the highest DEGMA content (sample R4-pDNA with 40 mol % of DEGMA) while maintaining almost 100% cell viability at N/P = 16 and 32 (Figure 9). When comparing the highest gene expression levels occurring at the N/P = 32 (Figure 10) with the smallest sizes of polyplexes measured at this ratio (Figure 5), it can be concluded that the size reduction of DNA, to a size smaller than the uncondensed naked nucleic acid, promotes a high transfection efficiency.

Figure 9. Toxicity of polyplexes for the transfection conditions (N/P ratio). The results are shown as a percentage of the control, where untreated cells constituted 100%.

Cytotoxicity was dependent on the architecture of the star forming polyplex. The polyplexes made of random star copolymers (R1-R4-pDNA) were slightly less toxic than those comprised of block star copolymers (B1-pDNA and B2-pDNA). The increased content of DEGMA in the arms of random structures decreased the cytotoxicity with all studied N/P ratios. For all polyplexes examined in this work, the cell viability 3283


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copolymer and resulted in the more efficient release of DNA inside the cell. Finally, the presence of DEGMA units in the star arms is the most important factor for enhancing cell viability and transfection efficiency when using such polymeric systems in gene therapy applications.

The overall protein production by transfected HT-1080 cells at N/P = 32 was almost 5 times greater for both R3-pDNA and R4-pDNA (30 and 40 mol % of DEGMA, respectively) than for polyplexes made with the homopolymer DMAEMA star of similar molar mass, for the same N/P value and the same cell line.9 The polyplexes formed from PDMAEMA-b-PDEGMA showed lower transfection efficiencies than polyplexes made of P(DMAEMA-ran-PDEGMA) for both N/P 16 and 32. The comparison of polyplexes of PDMAEMA-b-PDEGMA stars with polyplexes made of homopolymer DMAEMA stars9 also revealed a higher luciferase activity for copolymer stars. The Patrickios11 and Neoh22 groups, which synthesized star polymers with arms of DMAEMA and oligo(ethylene glycol) methacrylate as the second monomer,11,22 used more hydrophilic methacrylic monomers. The Patrickios group used hexa(ethylene glycol) methyl ether methacrylate with 6 ethylene glycol units per monomer,11 while the Neoh group used oligo(ethylene glycol) ethyl ether methacrylate with 3 ethylene glycol units.22 Our results for transfection are in opposition to the results presented by Patrickios group,11 in which block star polyplexes showed a higher transfection efficiency than random star polyplexes. This suggests that the method of star synthesis (“arm first” or “core first”), the structure of the star, the number of arms and their lengths are relevant in transfection experiments, and some of the results still cannot be directly correlated. In this work, we showed that the use of less hydrophilic DEGMA, with only 2 ethylene glycol units, as the second monomer in DMAEMA stars leads to a strong enhancement of polyplex transfection efficiencies and increases cell viability when compared with polyplexes made of homopolymer DMAEMA stars. The highest transfection levels that correlated with a high viability of cells were obtained for polyplexes of star polymers containing arms of DMAEMA and DEGMA of random composition, with DEGMA content in the range of 30 to 40 mol %.

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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.biomac.5b00948. The efficiency of HT-1080 cells transfection with star and PEI polyplexes related to the number of cells in the well at different N/P ratios (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]; Tel: +48 322716077; Fax: +48 322712969. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Polish National Science Center, contract No. DEC-2011/01/B/ST5/05982.

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REFERENCES

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CONCLUSIONS It was shown that nonviral plasmid DNA carriers based on DMAEMA and DEGMA star copolymers are promising transfection agents. For this purpose, star polymers with 28 arms made of random and block DMAEMA-co-DEGMA copolymer arms were synthesized via “core first” approach, using ATRP. The obtained copolymer stars were pH-sensitive in aqueous solutions. The block star copolymers completely complexed the pDNA to form polyplexes at lower N/P ratios than random star copolymers. The results indicate that the sizes of the polyplexes made of stars with block copolymer arms are smaller than those with random copolymer arms at N/P ratios of up to 16. Stars effectively interact with DNA by condensing it to half the size of naked DNA forming polyplexes of rather loose structure, as revealed by cryo-TEM. The results justify the conclusion that the introduction of DEGMA into the arms of the star, independent of the monomers distribution, significantly decreases cytotoxicity in comparison with polyplexes made of DMAEMA homopolymer stars9 for the studied range of N/P ratios. The transfection efficiency of HT-1080 cells obtained by polyplexes increases with the N/P ratio. Based on the correlation between the loose structure of the polyplexes and the relatively high transfection efficiency, it could be concluded that the shielding effect of the DEGMA in the random star arm is more pronounced than in the block star 3284


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