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Synthesis of Cationic Polylactides with Tunable Charge Densities as Nanocarriers for Effective Gene Delivery Charles H. Jones,† Chih-Kuang Chen,† Ming Jiang, Lei Fang, Chong Cheng,* and Blaine A. Pfeifer* Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, New York 14260-4200, United States ABSTRACT: Well-defined cationic polylactides (CPLAs) with tertiary amine groups were synthesized by thiol−ene click functionalization of an allyl-functionalized polylactide to yield polymers with tunable charge densities. CPLAs have not previously been utilized in the context of DNA delivery. Thus, plasmid DNA (pDNA) encoding luciferase was delivered to two physiologically distinct cell lines (macrophage and fibroblast) via formation of CPLA/pDNA polyplexes by electrostatic interaction. The formulated polyplexes demonstrated high levels of transfection with low levels of cytotoxicity when compared to a positive control. Biophysical characterization of charge densities at various CPLA/pDNA weight ratios revealed a positive correlation between surface charge and gene delivery. Overall, these results help to elucidate the influence of polyplex charge and size upon the delivery of nucleic acid and support future gene delivery applications using this next-generation biomaterial. KEYWORDS: CPLA, polylactic acid, gene therapy, gene delivery, DNA

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INTRODUCTION Gene therapy is the delivery of exogenous nucleic acid to influence protein expression patterns for the treatment and curing of any number of acquired diseases.1 For gene-based therapeutics to be implemented clinically, a safe and efficient delivery system must also be developed. Recombinant viruses are the most researched vectors for this purpose and embody the vast majority of clinical trials.2 Despite heavy usage predicated on their high transfection efficiency, viral vectors are limited by biosafety, toxicity, immunogenicity, and gene capacity.3,4 As a result, a myriad of nonviral delivery systems have been studied as potential alternatives. Some of these include polypeptides, polysaccharides, dendrimers, lipids, cationic polymers, and inorganic nanoparticles that are safer, easier to manufacture, and more cost-effective.5 However, clinical implementation of alternatives has been hindered due to suboptimal delivery efficiency in vivo in comparison to viralbased vectors. The efficacy of viral vectors stems from natural selection of particular serotypes that can successfully cross the numerous barriers associated with extra- and intracellular gene delivery. Of the alternatives, cationic polymers are prime candidates for addressing the aforementioned hurdles due to improvements in polymer chemistry which allow for the insertion of particular functionalities that will result in not only improved gene delivery but also superior biocompatibility, enhanced biodegradability, and lower toxicity.6,7 A variety of cationic polymers have been studied as potential delivery systems, such as poly(ethyleneimine) (PEI), poly(2(dimethylamino)ethyl methacrylate) (PDMAEMA), poly(Llysine) (PLL), and poly(β-amino esters) (PBAEs).8,9 In general, cationic polymers facilitate the formation of polymer−gene polyplexes through electrostatic interaction. © 2013 American Chemical Society

The polymeric scaffolds of polyplexes improve gene delivery by initially protecting the genetic payload from nuclease degradation and aiding endosomal escape. Endosomal escape is facilitated by cationic polymers with amine groups possessing pKa values between 5 and 7 via the “proton sponge” effect,10 resulting in increased transfection but often at the cost of significant toxicity.11 For example, PEI is highly toxic and dosages of only 10 μg/mL or less can be used safely.12,13 Concern has arisen pertaining to long-term toxicity because most polymers do not degrade readily under normal biological aqueous conditions.14,15 To combat these concerns, several low-toxic, readily degradable polymers have been synthesized and applied to gene delivery applications, among which poly(βamino esters) have received the most attention due to remarkable degradability that can allow for repeated administration of gene-based therapies.16,17 This class of polymer further highlights the most important characteristics that a polymer system must possess to be clinically significant: high transfection efficiency coupled with safe degradation. Recently, we reported the synthesis of a novel degradable polymer, cationic polylactide (CPLA), for delivery of siRNA to prostate cancer cells.18 Significant gene silencing of IL-8 is presumably due to the high complexation ability and small welldefined structure (50 nm) of CPLA/siRNA complexes. CPLAs also exhibited considerable hydrolytic degradability and very low toxicity. It was observed that the degradation rate of CPLA depends on the amine mol % and the surrounding temperature, Received: Revised: Accepted: Published: 1138

November 21, 2012 January 14, 2013 January 22, 2013 January 22, 2013 dx.doi.org/10.1021/mp300666s | Mol. Pharmaceutics 2013, 10, 1138−1145

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Figure 1. Synthesis of CPLA via ROP and thiol−ene functionalization.

versity at Buffalo, SUNY) and maintained in DMEM supplemented with 10% bovine calf serum. Both cell lines were housed in T75 flasks and cultured at 37 °C/5% CO2. To determine the transfection efficacy of our DNA delivery system, we employed a luciferase reporter gene driven by a Cytomegalovirus promoter within plasmid pCMV-Luc (Elim Biopharmaceuticals). The plasmid was transformed into and prepared from an Escherichia coli cloning host (GeneHogs, Invitrogen) prior to being used in the experiments outlined below. Synthesis and Analysis of CPLA-26 and CPLA-54. Starting materials 4-dimethylaminopyridine (DMAP; 99+%) and L-lactide (L-LA) (98%) were purchased from SigmaAldrich, and 2,2′-dimethoxy-2-phenylacetophenone (DMPA; 98%) was purchased from Acros Organics. Dichloromethane (DCM; HPLC), acetone (HPLC), and benzyl alcohol (BnOH; HPLC) were purchased from Fisher Chemical. 2(Diethylamino)ethanethiol hydrochloride (DEAET, 98+%) was purchased from Amfinecom Inc. DCM was dried by distillation over CaH2. LA was recrystallized from dry ethyl acetate four times prior to being used. Allyl-functionalized LA monomer 1 was prepared through the method reported previously.19,20 All other chemicals were used without further purification. As illustrated in Figure 1, allyl-functional PLA (2) was synthesized according to previous methodologies.18,19 Briefly, 1 (1,440 mg; 10 mmol), LA (1,700 mg; 10 mmol), and DCM (16.3 mL) were added to a 25 mL reaction flask with a magnetic stirring bar under nitrogen atmosphere. Upon reaching a solution temperature of 35 °C, BnOH (21.6 mg, 0.2 mmol; in 0.5 mL DCM) and DMAP (97.7 mg, 0.8 mmol; in 0.5 mL of DCM) were added to initiate the polymerization. Synthesis was allowed to continue for 3 weeks at 35 °C, before being manually stopped at a comonomer conversion of ∼80%. Next, allyl-functionalized 2 was separated and purified by precipitation in cold methanol (50 mL). As determined by 1H NMR (Varian INOVA-500 maintained at 25 °C with tetramethylsilane (TMS) as an internal reference), comonomer conversion was calculated based on the resonance intensities of the CH3 protons of remaining comonomers at 1.67−1.73 ppm relative to the CH3 protons of the resulting polymer at 1.49−1.61 ppm. 1H NMR (500 MHz, CDCl3, ppm) of 2: δ 1.49−1.61 (br m, CH3 units from LA and

indicating the degradation time frame can be designed and tailored. In addition, CPLA was synthesized by living ringopening polymerization (ROP) and thiol−ene click functionalization allowing for the creation of a well-defined structure with tunable charge density and low polydispersity index (PDI). Cationic charge densities were controlled by grafting different amounts of tertiary amines on degradable polylactide backbones. To further establish the applicability of CPLA for gene delivery purposes, additional studies are needed to test the delivery of other classes of nucleic acids, such as plasmid DNA. Success in these studies would further support the utility of CPLA as a promising new gene delivery biomaterial. In this study, we synthesized two novel CPLA polymers that have different charge densities by controlling thiol−ene functionalization conditions. Initial DNA binding characterization and optimal polymer to pDNA weight ratio determination was carried out using CPLA-26 (number indicating amine mol % relative to PLA backbone repeat units). Subsequently, the experimentally determined optimal weight ratio was used to investigate differences of gene delivery and cytotoxicity between two charge densities in two physiologically distinct cell lines. Using this methodology, we observed a significant correlation between polymer zetapotential and transfection efficiency. With one exception, polymers of both charge densities demonstrated comparable or better transfection efficiency when compared to positive controls while also exhibiting virtually no cytotoxicity. Interestingly, optimal performance occurred in our murine macrophage cell line, which may highlight potential clinical immunological applicability.

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EXPERIMENTAL SECTION Cell Lines and Reporter Plasmid. A RAW264.7 (murine macrophage) cell line was kindly provided by Dr. Terry Connell (Department of Microbiology and Immunology, University at Buffalo, SUNY). The cell line was maintained in medium prepared as follows: 50 mL of fetal bovine serum (heat inactivated), 5 mL of 1 M HEPES buffer, 5 mL of 100 mM MEM sodium pyruvate, 5 mL of penicillin/streptomycin solution, and 1.25 g of D-(+)-glucose were added to 500 mL of RPMI-1640 and filter sterilized. An NIH3T3 (murine fibroblast) cell line was provided by Dr. Stelios Andreadis (Department of Chemical and Biological Engineering, Uni1139

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First, working dilutions of CPLA (CPLA-26, CPLA-54) were prepared in water to a final concentration of 1 mg/mL. Depending on the desired weight ratio, various amounts of CPLA stock solution were mixed with pCMV-Luc in water, gently vortexed, and incubated for 30 min to form the CPLA/ pCMV-Luc complex. Taking 128:1 CPLA-26 as an example, 512 μL of CPLA-26 stock solution was mixed with pCMV-Luc (final concentration of 4 μg/mL) to a final volume of 1 mL. Characterization of Polyplexes. Using CPLA/pDNA polyplexes prepared as described above, zeta-potential and effective diameter were measured by dynamic light scattering (DLS) on a nano-ZS90 (Malvern, Inc.). All data points resulted from measurements of three independently formulated polyplexes. Additionally, polyplex samples were prepared by the dropwise addition of 10 μL of an aqueous CPLA-54/pDNA (weight ratio 128:1) solution on a carbon-coated copper grid, followed by drying in a vacuum oven. Dried polyplexes were treated with volatile ruthenium tetroxide vapors overnight prior to conducting transmission electron microscopy (TEM, JEOL 2010 microscope). Cellular Toxicity. Cytotoxicity of CPLA/pDNA polyplexes was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)diphenyltetrazolium bromide) colorimetric assay. RAW264.7 and NIH3T3 cells were seeded in 96-well polystyrene plates at densities of 50 × 104 and 1.2 × 104 cells/well, respectively. After incubation for 24 h, medium was replaced with that containing various weight ratios of CPLA/pDNA polyplexes (100 ng of DNA/well). Following another 24 h incubation, cells were incubated with MTT solution (5 mg/mL), added at 10% v/v, for 3 h at 37 °C/5% CO2. Medium plus MTT solution then was aspirated, and the formazan reaction products were dissolved in DMSO and shaken for 1 h. The optical density of the formazan solution was read on a Synergy 4 MultiMode Microplate Reader (BioTek Instruments, Inc.) at 570 nm with 630 nm serving as the reference wavelength. The results are shown as a percentage of untreated cells with 100% viability for three independent experiments. Gene Delivery Assay. For gene delivery experiments, both cell lines were seeded in two different types of 96-well plates (four plates in total) at the densities stated above in 100 μL of medium/well and allowed 24 h for attachment. A tissue culturetreated, flat-bottom, sterile, white, polystyrene 96-well plate was used for luciferase assays; whereas, BCA assays were conducted in tissue culture-treated, sterile, polystyrene 96-well plates. Each plate was carried out as a duplicate of the other. Following the 24 h incubation, the medium was removed and replaced with 70 μL of growth medium plus 30 μL of CPLA/ pDNA polyplexes (total volume of 100 μL) and incubated for an additional 24 h. Fugene 6 (Promega) was included as a positive control and complexed to pDNA (100 ng/well) according to the manufacturer’s instructions. After a second 24 h incubation (48 h after initial seeding), plates were analyzed for luciferase expression using the Bright Glo assay (Promega) and protein content using the Micro BCA Protein Assay Kit (Pierce) according to each manufacturer’s instructions. Gene delivery was calculated by normalizing luciferase expression by protein content for each well/plate. Results derive from six replicates and two independent experiments. Serum Inhibition to Gene Delivery. To determine if serum inhibited gene delivery efficiency of CPLA/pDNA polyplexes, RAW264.7 cells were incubated with polyplexes in RPMI-1640 medium with and without 10% FBS for 24 h. Gene delivery was determined as described above.

1), 2.66−2.73 (br m, CH2CHCH2 units from 1), 5.14−5.23 (br m, CHCH3 units from LA; CHCH3, CHCH2CHCH2, and CH2CHCH2 units from 1), 5.77−5.79 (m, CH2CH CH2 units from 1), 7.33−7.39 (m, Ar−H from BnOH). MnNMR = 14.5 kDa; MnGPC = 21.9 kDa; PDIGPC = 1.12. Mole fraction of 1 was 54% based upon the 1H NMR resonance intensities of 1H from units of 1 at 5.77−5.79 ppm relative to 4H from units of 1 and 2H from units of LA at 5.14−5.23 ppm. CPLA-26 and CPLA-54 were synthesized by a similar procedure, so the detailed synthesis of CPLA-26 will be described as a representative example. In a 10 mL flask, 2 (200 mg), DEAET (57.3 mg), and photoinitiator DMPA (34.8 mg) were dissolved in CDCl3 (5 mL), resulting in the molar ratio of [allyl of 2]0:[SH of DEAET]0:[DMPA]0 = 1:0.5:0.2. The freeze−pump−thaw procedure was conducted for three cycles to deoxygenate the solution. Then, the thiol−ene reaction was induced by UV irradiation (λmax = 365 nm) for 30 min. Subsequently, to remove the unreacted DEAET and DMPA, dialysis of the resulting solution was conducted against acetone for 10 days. Following dialysis, the solution was completely dried by vacuum to give CPLA-26 at 90% yield. 1 H NMR (500 MHz, CDCl3, ppm) of CPLA-26: δ 1.39− 1.43 (br m, (CH3CH2)2NH+Cl− from amine-functionalized units), 1.49−1.61 (br m, CH3 units from LA, 1, and aminefunctionalized units), 1.76−1.79 (br m, CH2CH2CH2SCH2 from amine-functionalized units), 2.00−2.10 (br m, CH2CH2CH2SCH2 from amine-functionalized units), 2.49− 3.23 (br m, CH2CHCH2 units from 1; CH2CH2CH2SCH2 and SCH2CH2NH+Cl−(CH2CH3)2 from amine-functionalized units), 5.14−5.20 (br m, CHCH3 units from LA; CHCH3, CHCH2CHCH2, and CH2CHCH2 units from 1; and CHCH3 and CHCH2CH2CH2SCH2 from amine-functionalized units), 5.77−5.79 (br m, CH2CHCH2 units from 1), 7.33− 7.39 (m, Ar−H from BnOH). MnNMR = 18.5 kDa; MnGPC = 22.9 kDa; PDIGPC = 1.37. 1H NMR (500 MHz, CDCl3, ppm) of CPLA-54: δ 1.17−1.26 (br m, (CH3CH2)2NH+Cl− from amine-functionalized units), 1.49−1.61 (br m, CH3 units from LA and amine-functionalized units), 1.74−1.77 (br m, CH2CH2CH2SCH2 from amine-functionalized units), 2.00− 2.08 (br m, CH2CH2CH2SCH2 from amine-functionalized units), 2.49−3.20 (br m CH 2 CH 2 CH 2 SCH 2 and SCH 2 CH 2NH +Cl −(CH 2CH 3) 2 from amine-functionalized units), 5.14−5.20 (br m, CHCH3 units from LA; CHCH3 and CHCH2CH2CH2SCH2 from amine-functionalized units), 7.33−7.39 (m, Ar−H from BnOH). MnNMR = 22.8 kDa; MnGPC = 16.4 kDa; PDIGPC = 1.35. Gel permeation chromatography (GPC) analysis was conducted with a Viscotek system equipped with a VE-3580 refractive index detector, a VE 1122 pump, and two mixed-bed organic columns (PAS-103 M and PAS-105M). Dimethylformamide (DMF; HPLC) containing 0.1 M LiBr was used as the mobile phase with a flow rate of 0.5 mL/min at 57 °C. Results were calibrated against narrowly dispersed linear polystyrene standards purchased from Varian. DNA Gel-Shift Assay. Plasmid DNA (900 ng/sample) was mixed by gentle vortexing with CPLA-26 at various ratios in sterile water. After 15 min incubation at room temperature, the mixtures were loaded on a 0.8% agarose gel and electrophoresed at 80 V for 30 min. Gels were visualized under UV after electrophoresis. Preparation of CPLA/pDNA Polyplexes. Using various weight ratios of polymer to pDNA, polyplexes were prepared by electrostatic interaction between CPLA and pCMV-Luc. 1140

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RESULTS AND DISCUSSION

Synthesis and Characterization of CPLA Variants. In this study, CPLAs with different charge densities were synthesized by ring-opening polymerization and thiol−ene click functionalization. CPLA-26 and CPLA-54 were synthesized using BnOH as the initiator and DMAP as the organocatalyst; ROP of monomer 1 and LA was conducted with the following conditions: [1]0:[LA]0:[BnOH]0:[DMAP]0 = 50:50:1:4; 35 °C, 21 days, in DCM with an ∼80% overall conversion of comonomers. The resulting allyl-functionalized 2 was characterized by 1H NMR and GPC. Based on the resonance intensities of the allyl CH protons (CHCH2) from the monomer unit of 1 at 5.77−5.79 ppm and the CH protons from both comonomers and the allyl CH2 protons (CH CH2) from the monomer unit of 1 at 5.14−5.23 ppm, a molar fraction of 54% for the monomer unit of 1 in polymer 2 was determined. Comparing the resonance intensities of the CH protons from the comonomer units and the allyl CH2 protons (CHCH2) from monomer unit of 1 at 5.14−5.23 ppm with the resonance intensities of the C6H5 protons from BnOH at 7.35 ppm, the number-average degree of polymerization (DPn) of 91 for 2 was calculated. Based on the GPC analysis of 2, Mn of 21.9 kDa and PDI of 1.12 relative to linear polystyrene were determined. We further introduced tertiary amine functionalities on a well-defined allyl-functionalized 2 using a thiol−ene click reaction. The UV-induced thiol−ene reactions of the allylfunctionalized 2 with DEAET were carrried out in CDCl3 under UV irradiation for 30 min at room temperature, using DMPA as the photoinitiator. CPLA-26 and CPLA-54, denoting 26 and 54 mol % of amine functionalities grafted to the PLA backbone repeat units, can be successfully synthesized by changing the [ene]0:[SH]0:[DMPA]0 feed ratio (Table 1).

Figure 2. 500 MHz 1H NMR spectra of CPLA-26 (a) and CPLA-54 (b) in CDCl3.

Table 1. Synthesis of CPLAs via Thiol−Ene Functionalization of Allyl-Functional PLA 2 entry

[ene]0:[SH]0: [DMPA]0a

mol % of amineb

MnNMRb (kDa)

PDIGPCc

CPLA-26 CPLA-54

1:0.5:0.2 1:3:0.4

26 54

18.5 22.8

1.37 1.35

a All of the reactions were carried out in CDCl3 under UV irradiation (λmax = 365 nm) for 30 min; the precursor polymer 2 (MnNMR = 14.5 kDa, PDIGPC = 1.12) had 54 mol % of allyl functionality. bDetermined by 1H NMR spectroscopy. cRelative to linear polystyrenes.

The mole fraction of the tertiary amine-based unit of CPLA26 (MnNMR = 18.5 kDa) was determined by the comparison of the resonance intensities of allyl CH protons (CHCH2) from the monomer unit of 1 (at 5.78 ppm) and the methyl protons of the tertiary amine-based unit (at 1.39−1.43 ppm) (Figure 2a). For CPLA-54 (MnNMR = 22.8 kDa), all the alkene groups of 2 were completely converted to tertiary amine functionalities, confirmed by the complete disappearance of allyl CH protons (CHCH2) from the monomer unit of 1 (at 5.78 ppm) (Figure 2b). As before, MnGPC and PDI of CPLA-26 and CPLA-54 can be obtained by GPC (Figure 3). CPLA-26 (MnGPC = 22.9 kDa, PDIGPC = 1.37) and CPLA-54 (MnGPC = 16.4 kDa, PDIGPC = 1.35) were verified. The CPLAs exhibit ready degradability as previously observed (data not shown).18 Furthermore, the low PDI of the CPLA polymers is a reflection of molecular homogeneity (with 1 referring to completely homogeneous) and is the result

Figure 3. GPC curves of allyl-functionalized 2 and CPLAs.

of using the living ROP synthesis method. This well-defined structure will be used to screen optimal CPLA charge densities and/or molecular weights for plasmid DNA delivery without concern of artifacts arising due to broad molecular weight and charge distributions. Polyplex Formation and Plasmid DNA Transfection. To investigate the relationship between cationic charge density of CPLA and in vitro gene delivery, we formed polyplexes using the synthesized polymers and luciferase-encoding plasmid DNA 1141

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protein, was approximately double the signal obtained with the positive control (Fugene). Curiously, nearly complete pDNA retardation was observed for 32:1 CPLA-26/pDNA using the gel-shift assay, yet this complex ratio failed to match the gene delivery capability of the 128:1 complex ratio. As such, the 128:1 ratio was selected as the basis for the studies described below. To further study the gene delivery properties of CPLA, additional cell lines and charge densities were tested with 128:1 polyplexes. An NIH3T3 cell line was selected for gene delivery using 128:1 CPLA-26 on the basis of being physiologically distinct from RAW264.7 (i.e., fibroblast vs macrophage). In order to observe the effect of increased charge density, CPLA54 was selected in comparison. When testing gene delivery efficiencies of both charge densities in both cell lines at a 128:1 CPLA/pDNA weight ratio, Figure 5b indicates a positive correlation between gene delivery and charge density. CPLA-54 performed the best of the two charge densities across both cell types. In RAW264.7, CPLA-54 performed approximately two times better than CPLA-26 and four times better than the Fugene positive control with a value of 26 ± 5.2 lum/μg protein. However, CPLA gene delivery as a whole was significantly repressed in NIH3T3 cells. With this cell line, CPLA-26/pDNA complexes performed less efficiently than the positive control; whereas, the difference in pDNA delivery between CPLA-54 and Fugene was statistically insignificant. The distinctive gene delivery profiles across the two cell lines may translate to future studies involving immunomodulatory treatments which require specific gene delivery to similar antigen presenting cells.21 Cytotoxicity of CPLA Polyplexes. Positive evaluation of the cytotoxicity associated with degardable cationic polymers is a critical parameter for determining future biomedical and clinical applications. Thus, CPLA/pDNA polyplexes were examined for their cytotoxicity on both RAW264.7 and NIH3T3 cells using an MTT assay (Figure 6). The viability

and subsequently incubated these polyplexes with cultured RAW264.7 and NIH3T3 cells in serum-containing medium for 24 h. After the 24 h incubation, luciferase activity and total protein levels were measured using a Bright Glo assay kit and a micro BCA assay coupled with a 96-well plate reader. To normalize data, luciferase expression was divided by total protein levels. Initial weight ratio determination and all in vitro gene delivery optimization was carried out using CPLA-26 as the test case. Agarose gel retardation assays were utilized to determine the minimum dosage of CPLA-26 required to interact completely with DNA. In Figure 4, CPLA-26 retards pCMV-Luc at a 32:1

Figure 4. Agarose gel electrophoresis of CPLA/pDNA polyplexes. Lane assignments are as follows: (1) molecular weight marker; (2) pCMV-Luc DNA; (3−8) CPLA-26/pCMV-Luc polyplexes at polymer/pDNA ratios of (3) 4:1, (4) 8:1, (5) 16:1, (6) 32:1, (7) 64:1, (8) 128:1.

CPLA/pDNA weight ratio, but minor additional binding can be observed at higher ratios (lanes 7−8). Next, all weight ratios (2:1 to 128:1) were screened for gene delivery using a murine macrophage line (RAW264.7). Cells treated with CPLA-26 polyplexes demonstrated increasing efficiency of gene delivery with increasing weight ratio (Figure 5a), with maximum gene delivery observed with 128:1 CPLA-26/pDNA polyplexes. The maximum gene delivery of CPLA-26, 14.96 ± 0.77 lum/μg

Figure 5. Gene delivery studies of (a) RAW264.7 transfection using CPLA-26 at various polymer/pDNA weight ratios. (b) CPLA-26 and CPLA-54 transfection comparison at 128:1 polymer/pDNA weight ratio in RAW264.7 and NIH3T3. Delivery is measured in luminescence units normalized by total protein (luminescence/μg protein, y-axis). Error bars represent standard deviation resulting from three independent experiments. *Statistically significant (95% confidence) when compared to respective Fugene control. **Statistically significant (95% confidence) when compared to opposing charge density. 1142

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Figure 6. MTT assay in which RAW264.7 (a) and NIH3T3 (b) cells were incubated for 24 h with different concentrations of CPLA-26 and CPLA54. Error bars represent standard deviation resulting from three independent experiments.

Figure 7. Zeta-potential measurements of CPLA-26 (a) and CPLA-54 (b) polyplexes at various polymer/pDNA weight ratios. (c) Effective particle diameter comparison between polymers. (d) TEM image of CPLA-54/pDNA polyplexes. Error bars represent standard deviation resulting from three independent experiments. **Statistically significant (95% confidence) when compared to opposing charge density.

of both cell lines treated with polyplexes was estimated as >80% regardless of the CPLA/pDNA ratio. Results for CPLA-26 showed no sign of cytotoxicity with viabilities at ∼100%, even at 200 and 300 μg/mL dosages. In contrast, CPLA-54 demonstrated minor cytotoxcity (∼80%) for RAW264.7 and NIH3T3 at 200 μg/mL and 300 μg/mL, respectively. Dosages used for maximum transfection (∼100 μg/mL) demonstrated no cytotoxcity for either charge density. Despite minor cytotoxicity at higher dosages, our results are favorable in comparison to standards such as PEI, which boasts a maximum safe dosage of only 10 μg/mL. These results suggest that CPLAs have low toxicity over a wide range of CPLA/pDNA ratios and charge densities, and therefore show potential as nontoxic transfection materials. Biophysical Characterization of CPLA/pDNA Polyplexes. Generally for cationic polymer systems, efficient gene delivery is strongly associated with net surface charge and effective particle diameter. To evaluate such biophysical

properties, the CPLA/pDNA polyplexes were prepared as described above at various weight ratios and measured by dynamic light scattering (DLS). As shown in Figure 7a, CPLA26 shows a positive trend in relation to increasing polymer to pDNA weight ratio, with a maximum of 3.27 ± 3.27 mV. In relation to the gel shift assay, the weight ratios that correspond with major pDNA complexion all have positive zeta potential values. Interestingly, the CPLA-26 weight ratio that is responsible for maximum gene delivery efficiency is 0.1 ± 0.2 mV. In contrast, the 128:1 CPLA-54/pDNA weight ratio tested for gene delivery demonstrated the maximum zeta potential (Figure 7b; 12.7 ± 0.7 mV) of those assessed for charge density. The increased zeta potential of CPLA-54 in relation to CPLA-26 is due to the presence of additional tertiary amine groups grafted to the polylactide backbone. Additionally, the net positive surface charge of 128:1 CPLA-54 can prevent the polyplex from aggregating.22 1143

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Previous research with cationic polymers for gene delivery has shown that efficient transfection requires the formation of small, positively charged complexes.23,24 However, arbitrarily increasing charge density can lead to unwanted cytotoxicity and thus there exists an optimal charge density for each polymer of interest. The tunable and controllable synthesis of CPLAs offers the ability to balance surface charge, efficient gene delivery, and low cytotoxicity. To be useful as universal transfection agents, cationic polymers must be able to self-assemble with plasmid DNA into complexes small enough to enter cells through receptor mediated endocytosis. The size requirement for receptor mediated endocytosis varies, but for most cell types particle size is on the order of 200 nm.25 However, through pinocytosis cells can take up particles greater than 500 nm.26 In addition, phagocytic cell lines can accommodate substantially larger sizes (∼10 μm).27 To this end, average effective diameter of polyplexes at each weight ratio was measured (Figure 7c). A general positive trend can be observed for both CPLA-26 and CPLA-54 with increasing polymer/pDNA weight ratio. The diameters that correspond to maximum transfection efficiency are 330 ± 31 nm and 341 ± 16 nm for CPLA-26 and CPLA-54, respectively. The sizes of the 128:1 CPLA/pDNA polyplex can help explain the stark contrast in gene delivery efficiency trends of RAW264.7 and NIH3T3. The reduction of gene delivery in NIH3T3 can be attributed to polyplex sizes that are too large to readily be endocytosed. This theory is supported by previous gene expression results where nanoparticles with average sizes of 64 nm were comparable to PEI complexes.28 Inversely, the significant increase in gene delivery relative to Fugene of both charge densities in the RAW264.7 cell line can be explained by increased uptake by a combination of phagocytosis (absent in NIH3T3), endocytosis, and pinocytosis.29 Similarly, further distinction of gene delivery between charge densities confirms the previous findings that interactions between negatively charged cell surfaces of macrophages and cationic particles lead to increased phagocytosis and subsequent gene delivery.27,30−32 Transmission electron microscopy was utilized to verify the spontanteous formation of CPLA/pDNA polyplexes. The charge density and weight ratio corresponding to the highest resulting gene delivery was selected for observation (128:1 CPLA-54/pDNA weight ratio). The diameter observed by TEM was approximately 200 nm (Figure 7d). The difference of observed particle size from DLS and TEM can be explained by the methodolgy used to prepare TEM samples. TEM samples require dehydration and immobilization on a solid support, which has been reported to cause structural distortions compared to the solvent swollen state.33 Furthurmore, DLS is prone to errors, due in part to the curve fitting requirement to estimate the average distribution of particle sizes. Conversely, the difference can be attributed to minor aggregation between two or more polyplexes. In Vitro Serum Compatibility. For CPLA/pDNA polyplexes to be relevant for in vivo applications, gene delivery efficiency must not be biased by the presence of extracellular protein. Generally, negatively charged serum proteins can bind to positively charged complexes and limit in vivo gene delivery efficiency. To address these concerns, previous efforts have successfully utilized the inclusion of polyanions with cationic complexes to mitigate serum inhibition during cell transfection.33 In this study, polyplexes were seeded as described above, with RAW264.7 in RPMI-1640 with and without serum. Figure 8 shows no observable correlation between gene delivery

Figure 8. Serum inhibition study of gene delivery using CPLA-26. Error bars represent standard deviation resulting from three independent experiments.

and serum presence. This result suggests that the polyplexes offer structural characteristics, such as strong polymer−pDNA charge−charge interactions or pDNA shielding, that would withstand reduced gene delivery due to the presence of serum.

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CONCLUSION Well-defined cationic polylactides with tunable charge densities synthesized by tandem living ROP and click functionalization are emerging as a promising class of degradable gene delivery biomaterials. We investigated CPLA/pDNA biophysical properties, transfection activity, and cytotoxicity using two physiologically distinct cell lines. Results indicate that this class of tunable CPLA polymers is free of serum inhibition with low cytotoxicity and cell-specific transfection activity, which depends to a certain degree on polyplex charge but significantly on polyplex size. To our knowledge, this is the first report of CPLA in the context of gene delivery of plasmid DNA. Additionally, this is the first study that investigates CPLA’s ability to transfect more efficiently in an immunorelevant cell line.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 716-645-1198. Fax: 716-645-3822. E-mail: ccheng8@ buffalo.edu, blainepf@buffalo.edu. Author Contributions †

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thankfully acknowledge the NSF (CBET-1019227 and -1133737) and a SUNYBuffalo Schomburg fellowship (C.H.J.) for financial support.

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