Poly(ethylene oxide) Grafted with Short Polyethylenimine Gives DNA

Feb 17, 2012 - Lanzhou Military Command Center for Disease Control and Prevention, Lanzhou, 730020, People's Republic of China. § Jiangsu Key ...
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Poly(ethylene oxide) Grafted with Short Polyethylenimine Gives DNA Polyplexes with Superior Colloidal Stability, Low Cytotoxicity, and Potent In Vitro Gene Transfection under Serum Conditions Meng Zheng,† Zhihong Zhong,‡ Lei Zhou,† Fenghua Meng,† Rui Peng,§ and Zhiyuan Zhong*,† †

Biomedical Polymers Laboratory and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering, and Materials Science, Soochow University, Suzhou, 215123, People's Republic of China ‡ Lanzhou Military Command Center for Disease Control and Prevention, Lanzhou, 730020, People's Republic of China § Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, 215123, People's Republic of China S Supporting Information *

ABSTRACT: Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80−245 nm) particles with moderate positive surface charges (+7.2 ∼ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications.

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INTRODUCTION In recent years, cationic polymers have emerged as versatile and promising nonviral gene delivery platforms, due to their several advantages such as ease of large scale production and handling, facile vector modifications, lack of specific immune response, and enabling repeated administration.1−4 In particular, polyethylenimine (PEI) with a unique combination of high charge density and proton sponge effect has been established as one of the most potent nonviral gene carriers in vitro and in vivo.5−7 It should be noted, nevertheless, that good transfection activity is only observed for high molecular weight PEIs, among which are currently widely applied nonviral transfection standards, 25 kDa branched PEI (referred to as 25 kDa PEI) and 22 kDa linear PEI.8,9 These high molecular weight PEI © 2012 American Chemical Society

reagents, however, show varying levels of cytotoxicities in vitro, as well as acute and long-term toxicity in vivo as a result of high molecular weight and excessive positive charge. Low molecular weight PEIs such as 1.8 kDa branched PEI (referred to as 1.8 kDa PEI) with favorable cytotoxicity profiles display, nevertheless, 100× lower transfection activity than 25 kDa PEI.10 To obtain nontoxic and efficient gene transfectants, tremendous efforts have been directed to the modifications of PEIs, which include hydrophobic modification of low molecular weight PEIs,11−19 coupling of low molecular weight PEIs to Received: December 16, 2011 Revised: February 16, 2012 Published: February 17, 2012 881

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pyridine hydrochloride salt, precipitation in cold diethyl ether, filtration, and drying in vacuo. Yield: 78%. 1H NMR (400 MHz, CDCl3): δ 8.30 and 7.41 (dd, Ar), 4.42 (t, -S-CH2-CH2-O-), 3.44− 3.82 (PEO main chain), 2.86 (t, -S-CH2-CH2-O-), 2.66 (t, -CH2-CH2S-), 1.87 (m, -CH2-CH2-S-). Synthesis of PEO-g-PEI. A DCM solution (20 mL) of PEO-g-NC (0.15 g, 0.017 mmol NC) was added dropwise into a DCM solution (10 mL) of 1.8 kDa PEI (1.25 g, 0.134 mmol, 8 mol excess) in 2 h under stirring at r.t. After a 1 d reaction, the solvent was removed by rotary evaporation. The product was isolated by dissolution of the residues in D.I. water, extensive dialysis against D.I. water (MWCO: 7000) for 2 d, and lyophilization. Yield: 72%. 1H NMR (400 MHz, CDCl3): δ 4.20 (t, -S-CH2-CH2-O-), 3.44−3.82 (PEO main chain), 3.38 (t, -COONHCH2-), 2.86 (t, -S-CH2-CH2-O-), 2.66 (t, -CH2-CH2S-), 2.43−2.95 (PEI), 1.87 (m, -CH2-CH2-S-). Acid−Base Titration. The buffer capability of PEO-g-PEI was determined by acid−base titration assays over a pH range from 2.0 to 11.0. Briefly, the polymer (0.1 mmol nitrogen atoms) was dissolved in 5 mL of 150 mM NaCl solution. The solution was brought to a starting pH of 2.0 with 0.1 M HCl and then was titrated with 0.1 M NaOH using a pH meter (DELTA 320). For comparison, 25 kDa PEI was also titrated in the same way. The buffer capacity, defined as the percentage of amine groups becoming protonated from pH 5.1 to 7.4, was calculated according to the following equation:

form hydrolytically or reductively degradable PEI polymers and networks,20−29 grafting low molecular weight PEIs onto biocompatible polymers (e.g., dextran, chitosan, and polycarbonate),30−33 modification of the periphery of high molecular weight PEI,34 and PEGylation of high molecular weight PEIs.35−37 It should be noted that for in vivo gene transfer, modification of the PEI vector with stealth polymers like poly(ethylene glycol) (PEG) is generally required to improve polyplex colloidal stability, decrease systemic toxicity, and prolong circulation time. Inadequate polyplex colloidal stability has been a significant challenge for cationic polymer-based gene delivery systems. The stealth effect of PEG while enhancing PEI polyplex colloidal stability usually results in diminished transfection potency in vitro as well as in vivo due to poor cellular uptake.38 Interestingly, we found that low molecular weight linear PEI-PEG-PEI triblock copolymers form partially shielded DNA complexes that exhibit better colloidal stability, low cytotoxicity, and enhanced transfection efficiency comparable to 25 kDa PEI standards.39 In this paper, we report on poly(ethylene oxide) grafted with 1.8 kDa PEI (PEO-g-PEI) copolymers for nontoxic and efficient in vitro gene transfer. PEO was designed as the main chain because it offers several exceptional functions including excellent water-solubility, biocompatibility, flexibility, and stealth effect. The presence of PEO would largely improve polyplex colloidal stability and biocompatibility. We have shown recently that functional PEO can be readily prepared with controlled functionality and molecular weight.40 The large number of 1.8 kDa PEI molecules grafting to PEO backbone would on one hand enhance DNA condensation and protection and on the other hand facilitate cellular uptake due to presence of partial PEI at the outer surface. Here, synthesis of PEO-g-PEI copolymers and colloidal stability and cytotoxicity, as well as transfection performances of PEO-g-PEI polyplexes, were investigated.

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buffer capacity(%) = 100(ΔVNaOH × 0.1 M)/N mol wherein ΔVNaOH is the volume of NaOH solution (0.1 M) required to bring the pH value of the polymer solution from 5.1 to 7.4, and N mol is the total moles of protonable amine groups in the tested polymer (0.1 mmol). Particle Size and ζ-Potential Measurements. The polyplexes were prepared at varying N/P ratios from 5/1 to 40/1 by adding a HEPES buffer solution (20 mM, pH 7.4) of polymer (600 μL, varying concentrations) to a HEPES buffer solution (20 mM, pH 7.4) of plasmid DNA (150 μL, 37.5 μg/mL), followed by vortexing for 5 s and incubating at r.t. for 30 min. The surface charge and average size of polyplexes were measured at 25 °C with a Zetasizer Nano ZS instrument (Malvern) equipped with a standard capillary electrophoresis cell and dynamic light scattering (DLS, 10 mW He−Ne laser, 633 nm wavelength), respectively. The measurements were performed in triplicate. Transmission Electron Microscopy. The morphologies of PEOg-PEI polyplexes were obtained using Tecnai G2F20 Transmission Electron Microscopy (TEM) operated at an accelerating voltage of 200 kV. The samples were prepared by dropping 10 μL of the polyplexes solution on the copper grid followed by staining with phosphotungstic acid. Gel Retardation Assays. The DNA binding ability of PEO-g-PEI was studied by agarose gel electrophoresis. The polymer/DNA complexes prepared as above at varying N/P ratios from 1/1 to 5/1 were electrophoresed through a 0.8% agarose gel containing ethidium bromide at 100 V in TAE solution (40 mM Tris−HCl, 1 v/v % acetic acid, and 1 mM EDTA). Colloidal Stability of Polyplexes. The colloidal stability of PEOg-PEI polyplexes formed at an N/P ratio of 10/1 was studied by DLS and turbidity assays in the presence of 150 mM NaCl and 10−20% fetal bovine serum (FBS), respectively. For DLS measurements, 53 μL of NaCl solution (1.0 M) was added into 300 μL of preformed polyplex dispersions to reach a final NaCl concentration of 150 mM. The hydrodynamic sizes of polyplexes were monitored in time. For turbidity assays, 80 μL of HEPES buffer solution containing 12.5% and 25% FBS was added into 20 μL of polyplex dispersions to yield a final FBS concentration of 10% and 20%, respectively. The samples were incubated overnight at 37 °C. The aggregation in terms of turbidity increase was quantified using a multifunctional microplate reader (Multiskan Flash, Thermo) by absorbance detection at 595 nm. MTT Assays. The cytotoxicity of PEO-g-PEI/DNA polyplexes was evaluated in 293T cells by MTT assays. In brief, 293T cells were seeded in a 96-well plate (6 × 103 cells/well) in 100 μL of DMEM medium containing 10% FBS for 1 d. PEO-g-PEI polyplex dispersions

EXPERIMENTAL SECTION

Materials. The 1.8 and 25 kDa branched polyethylenimines (1.8 kDa PEI and 25 kDa PEI, Sigma), 4-nitrophenyl chloroformate (4-NC, 97%, Alfa Aesar), 2-mercaptoethanol (Amresco), 2,2′-azobisisobutyronitrile (AIBN, Sinopharm Chemical Reagent), pyridine (99.5%, Sinopharm Chemical Reagent), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco), Western and IP lysis buffer (Byontime), Cy5 (Mirus), and Hoechst 33342 (Sigma) were used as received. Allyl-functionalized poly(ethylene oxide) (PEO) polymers, that is, PEO(13k)-g-14allyl, PEO(24k)-g-12allyl, and PEO(13k)-g28allyl (Table S1), was synthesized according to our previous report.40 Dichloromethane (DCM) and toluene were dried by refluxing over CaH2 and sodium wire, respectively, and distilled before use. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin, and trypsin were obtained from Invitrogen. Synthesis of PEO-g-OH. In a typical experiment, under a N2 atmosphere, to a toluene solution (14 mL) of PEO(13k)-g-14allyl (2.0 g, 2.11 mmol allyl group, Table S1, entry 1) were added mercaptoethanol (0.99 g, 12.6 mmol) and AIBN (2.76 g, 16.8 mmol). The mixture was stirred for 2 d at 70 °C. The polymer was isolated by precipitation in diethyl ether, filtration, and drying in vacuo. Yield: 85%. 1H NMR (400 MHz, CDCl3): δ 3.44−3.82 (PEO main chain), 3.54 (t, -S-CH2-CH2-OH), 2.72 (t, -S-CH2-CH2-OH), 2.61 (t, -CH2-CH2-S-), 1.87 (m, -CH2-CH2-S-). Synthesis of PEO-g-NC. Under a N2 atmosphere, to a solution of PEO(13k)-g-OH (0.25 g, 0.26 mmol OH) in toluene (10 mL) were added pyridine (0.17 g, 2.15 mmol) and 4-nitrophenyl chloroformate (0.34 g, 1.58 mmol). The mixture was stirred at room temperature (r.t.) for 16 h. The resulting product was isolated by filtering off 882

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Scheme 1. Synthesis of PEO-g-PEI Conjugatesa

Reagents and conditions: (i) 2-mercaptoethanol, AIBN, toluene, 70 °C, 48 h; (ii) 4-NC, pyridine, toluene, r.t., overnight; (iii) 1.8 kDa PEI, CH2Cl2, r.t., 24 h.

a

Figure 1. 1H NMR spectrum (400 MHz, CDCl3) of PEO(13k)-g-10PEI (Table 1, entry 1). × 104 cells/well) and maintained in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2 until 70% confluency. In a standard transfection experiment, the cells were rinsed with PBS and incubated with 100 μL of polyplex dispersions (1 μg of plasmid DNA per well) and 400 μL of culture medium with or without 10% serum for 4 h at 37 °C. Next, the polyplexes were removed, 500 μL of fresh culture medium containing 10% serum was added, and the cells were cultured for 2 d. The transfected cells were lysed with a lysis buffer and centrifuged to remove the lysis debris. The GFP expression level of cell lysates were analyzed using FLS920 fluorescence spectrometer at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The 25 kDa PEI/DNA formulation, prepared at an optimal N/P ratio of 10/1, was used as a reference. All the experiments were carried out in triplicate. Confocal Microscopy. The cellular uptake behaviors of PEO-gPEI polyplexes at an N/P ratio of 20/1 were studied in 293T cells with

were added to give varying N/P ratios from 10/1 to 40/1, and the cells were cultured for 4 h at 37 °C. Next, the polyplexes were removed, 200 μL of fresh culture medium containing 10% serum was added, and the cells were cultured for 2 d. The medium was replaced with 100 μL of fresh medium containing 100 μg of MTT and cells were further cultured for 4 h at 37 °C. The medium was aspirated, the MTTformazan generated by live cells was dissolved in 150 μL of DMSO, and the absorbance at a wavelength of 570 nm of each well was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 570 nm with control wells containing only cell culture medium. Data are presented as average ± SD (n = 4). In Vitro Gene Transfection. Transfection experiments were performed in 293T cells using the plasmid pEGFP as a reporter gene. Transfections were conducted using polyplexes formed at N/P ratios ranging from 10/1 to 30/1. The cells were plated in 24-well plates (6 883

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CLSM using Cy5-labeled DNA. The 25 kDa PEI polyplexes, at an N/ P ratio of 10/1, were used as a control. In brief, 293T cells were plated on coverslips in 24-well plates (cell density 6 × 104 cells/well) and maintained in DMEM supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2 for 1 d. A 100 μL aliquot of polyplex dispersions (1 μg of Cy-5 labeled plasmid DNA per well) were added. The cells were cultured for 4 h at 37 °C. The polyplexes were removed and the cells were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min. The nuclei were stained with 200 μL of Hoechst 33342 (20 μg/mL) for 15 min at r.t. The cells following rinsing three times with PBS were observed with a confocal laser scanning microscope (TCS SP5 Leica).

PEO(x)-g-yPEI, wherein x represents molecular weight of PEO and y the average number of grafted 1.8 kDa PEI. bNumber of 1.8 kDa PEI per PEO chain determined by 1H NMR. cWeight percent of total PEI in the graft copolymer calculated from 1H NMR. dDefined as the percentage of amine groups becoming protonated from pH 5.1 to 7.4.

RESULTS AND DISCUSSION Synthesis of PEO-g-PEI Graft Copolymers. PEO-g-PEI copolymers were prepared in three steps from allyl-functionalized PEO (PEO-g-allyl; Scheme 1). As our previous report,40 PEO-g-allyl polymers were obtained with tailored functionalities and molecular weights (Table S1). First, PEO-g-allyl copolymers were reacted with excess mercaptoethanol in the presence of AIBN at 70 °C for 2 d. 1H NMR revealed that resonances assignable to the allyl protons (δ 5.28 and 5.90) completely disappeared, whereas new signals attributable to both methylene protons next to the sulfide ether (δ 2.61 and 2.72) were clearly detected (Figure S1A). The integral ratio between signals at δ 2.61 and δ 3.44−3.82 (PEO methylene protons) was close to the theoretical value of 1:41, indicating quantitative transformation of allyl into hydroxyl groups. Then, the hydroxyl groups were activated using 4-nitrophenyl chloroformate in toluene at r.t. for 16 h. 1H NMR showed new signals at δ 4.42 and 7.41−8.30, attributable to the methylene protons next to the carbonate and aromatic protons of 4-nitrophenyl group, respectively (Figure S1B). The signals at δ 7.41 and 2.86 (methylene protons next to the sulfide ether) had an integral ratio close to 1:1, supporting controlled activation of PEO-g-OH. Finally, treatment of PEO-g-NC with excess 1.8 kDa PEI furnished freely water-soluble PEO-g-PEI copolymers. The free PEI was removed by extensive dialysis. 1H NMR showed besides signals at δ 2.43−2.95 assignable to PEI methylene protons and δ 3.44−3.82 owing to PEO methylene protons, signals at δ 3.38 and 4.20 attributable to PEI methylene protons neighboring to the urethane group and methylene protons next to the carbonate group, respectively (Figure 1). The average grafting number of 1.8 kDa PEI molecules per PEO chain could be estimated by comparing the integrals at δ 2.43−2.95 and 3.44−3.82. The results showed that three PEO(x)-g-yPEI copolymers, wherein x represents Mn of starting PEO and y average number of PEI molecules per PEO chain, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were obtained from PEO(13k)-g14allyl, PEO(24k)-g-12allyl, and PEO(13k)-g-28allyl, respectively (Table 1). It is hypothesized that the high transfection activity of PEI is associated with its intrinsic proton sponge effect, which facilitates endosomal escape of its DNA complexes.6 The acid−base titration assays (Figure S2) revealed that these PEOg-PEI copolymers had buffer capacities of 14.7−16.4%, similar to that of 25 kDa PEI (16.2%; Table 1), indicating that PEO-gPEI polyplexes are likely capable of escaping from endosomes. Biophysical Characterization of DNA Polyplexes. The plasmid DNA condensation abilities of PEO-g-PEI copolymers were studied using gel electrophoresis, dynamic light scattering (DLS), and zeta potential measurements. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI,

and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively (Figure 2). Interestingly, DLS measurements revealed that all PEO-g-PEI graft copolymers were able to condense DNA into compact nanoparticles (80−245 nm) at N/P ratios ranging from 5/1 to 40/1 (Figure 3A). Notably, the polyplex sizes decreased with increasing PEI graft density and decreasing PEO molecular weight, following an order of PEO(24k)-g-10PEI > PEO(13k)-g-10PEI > PEO(13k)-g-22PEI (Figure 3A). The sizes of PEO(24k)-g-10PEI polyplexes decreased from 245 to 153 nm when increasing N/P ratios from 5/1 to 40/1, while PEO(13k)-g-10PEI formed DNA polyplexes of approximately 130 nm at all N/P ratios. For PEO(13k)-g-22PEI, polyplex sizes first decreased from 120 to 90 nm when increasing N/P ratios from 5/1 to 20/1 and then had little change when further increasing N/P ratios. TEM images showed that PEO-g-PEI polyplexes had a globular morphology (Figure S3). Zeta potential assays showed that PEO-g-PEI polyplexes formed at N/P ratios ranging from 5/1 to 40/1 had moderate positive surface charges of +7.2 ∼ +24.1 mV (Figure 3B). The surface charges decreased with decreasing PEI graft density and increasing PEO molecular weight, following an order of PEO(13k)-g-22PEI > PEO(13k)-g-10PEI > PEO(24k)-g10PEI (Figure 3B). It is commonly observed that fine DNA condensates are obtained at high N/P ratios, i.e. at the expense of having free cationic polymer in solution. We assumed that PEO as a backbone would largely improve the water dispersibility and colloidal stability of DNA polyplexes.41 To prove this, change of polyplex sizes under physiological salt as well as 10−20% serum conditions was monitored in time. The results demonstrated that sizes of PEOg-PEI polyplexes formed at an N/P ratio of 10/1 did not change in the presence of 150 mM NaCl in 4 h (Figure 4A). In contrast, a drastic increase of polyplex sizes (to over 1000 nm in 1.5 h) was observed for 25 kDa PEI polyplexes at an N/P ratio of 10/1 under otherwise the same conditions (Figure 4A). The turbidity measurements showed that turbidity of 25 kDa PEI polyplexes following 16 h incubation with 10 or 20% serum increased significantly and the more serum the higher the turbidity (Figure 4B), suggesting fast serum-induced aggregation of PEI polyplexes. In comparison, little increase of turbidity was discerned for polyplexes of PEO-g-PEI copolymers, in particular, PEO(13k)-g-10PEI and PEO(24k)-g-10PEI (Figure 4B). It is evident, therefore, that PEO-g-PEI copolymers are able to effectively condense DNA, yielding nanosized polyplexes with superior colloidal stability under physiological salt and serum conditions. In Vitro Cytotoxicity and Transfection. The cytotoxicity of PEO-g-PEI polyplexes was evaluated in 293T cells by MTT assays. The 25 kDa PEI was used as a control. Interestingly, the results displayed that polyplexes of PEO(13k)-g-22PEI, PEO-

Table 1. Characteristics of PEO-g-PEI Conjugates entry

polymera

NPEIb

PEI (wt %)c

buffer capacity (%)d

1 2 3

PEO(13k)-g-10PEI PEO(24k)-g-10PEI PEO(13k)-g-22PEI

10 10 22

55.2 41.6 71.6

14.7 15.5 16.4

a

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Figure 2. Agarose gel electrophoresis of PEO-g-PEI polyplexes formed at N/P ratios ranging from 0/1 to 5/1: (A) PEO(13k)-g-10PEI; (B) PEO(24k)-g-10PEI; (C) PEO(13k)-g-22PEI.

Figure 4. Colloidal stability of PEO-g-PEI polyplexes formed at an N/ P ratio of 10/1: (A) change of polyplex sizes under 150 mM NaCl condition in time as determined by dynamic light scattering and (B) change of turbidity under 10% or 20% serum conditions as quantified by absorbance detection at 595 nm. The 25 kDa PEI at an N/P ratio of 10/1 was used as a control. Data are shown as mean ± SD (n = 3).

Figure 3. Average particle size (A) and ζ-potentials (B) of PEO-g-PEI polyplexes formed at N/P ratios ranging from 5/1 to 40/1 in HEPES buffer (20 mM, pH 7.4). Data are shown as mean ± SD (n = 3).

(13k)-g-10PEI, and PEO(24k)-g-10PEI induced low cytotoxic effect (cell viability > 80%) up to an N/P ratio of 20/1, 30/1, and 40/1, respectively (Figure 5). In comparison, significant toxicity with a cell viability of 47.8% was observed for 25 kDa PEI at an N/P ratio of 20/1. As a rule, the cytotoxicity of PEOg-PEI polyplexes decreased with increasing PEO molecular weights and decreasing PEI graft densities. PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI polyplexes were practically nontoxic up to an N/P ratio of 10/1, 20/1, and 30/ 1, respectively (Figure 5). The in vitro transfection activity of PEO-g-PEI copolymers was evaluated in 293T cells using pEGFP as a reporter gene at three different N/P ratios (i.e., 10/1, 20/1 and 30/1) in the presence or absence of 10% serum. The results showed that polyplexes of both PEO(13k)-g-10PEI and PEO(13k)-g-22PEI exhibited high levels of GFP expression under serum-free conditions (Figure 6A). The transfection efficiencies of PEO(13k)-g-10PEI polyplexes at N/P ratios of 20/1 and 30/ 1 were over 3-fold higher than that of 25 kDa PEI control. PEO(13k)-g-22PEI displayed lower transfection activity than PEO(13k)-g-10PEI, in which comparable level of GFP

expression to 25 kDa PEI control was observed for PEO(13k)-g-22PEI polyplexes at an N/P ratio of 10/1. In comparison, PEO(24k)-g-10PEI revealed the lowest transfection activity, likely because it forms relatively large and effectively shielded DNA polyplexes. Notably, under 10% serum conditions, PEO(13k)-g-10PEI polyplexes at N/P ratios of 20/1 and 30/1 showed more than 4-fold higher level of GFP expression than 25 kDa PEI control (Figure 6B). In addition, polyplexes of PEO(13k)-g-22PEI at an N/P ratio of 10/1 and PEO(24k)-g-10PEI at an N/P ratio of 30/1 showed transfection efficiencies similar to 25 kDa PEI control (Figure 6B). Notably, a comparison of absolute GFP expression showed that 25 kDa PEI polyplexes had about 50% decrease in transfection efficiency by 10% serum, while only approximately 30% decrease of transfection was observed for PEO(13k)-g-10PEI polyplexes under otherwise the same conditions. It should be noted that both polyplexes of PEO(13k)-g22PEI at an N/P ratio of 10/1 and PEO(13k)-g-10PEI at an N/ P ratio of 20/1, which showed potent transfection, were 885

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PEO-g-PEI copolymers. The biophysical characterization studies showed that PEO(13k)-g-10PEI polyplexes gave median polyplex sizes (126−131 nm) and positive surface charges (+18.8 ∼ +20.2 mV) at N/P ratios ranging from 10/1 to 30/1 as compared to those of PEO(13k)-g-22PEI and PEO(24k)-g-10PEI (Figure 3). These results indicated that besides buffer capacity, balanced DNA condensation ability is of particular importance for cationic polymers to achieve efficient gene transfection.42 The best polymeric carrier on one hand should be able to condense DNA into small particles, protecting DNA from degradation and facilitating cellular uptake, and on the other hand should not form too tight DNA complexes, allowing sufficient release of DNA inside cells. Cellular Uptake and Intracellular DNA Trafficking. To visualize the cellular uptake of PEO-g-PEI polyplexes and intracellular fate of DNA, transfection experiments were performed using Cy5-labeled DNA at an N/P ratio of 20/1 in 293T cells in the presence of 10% serum. The cell nuclei were stained with Hoechst 33242 (blue). Interestingly, confocal laser scanning microscope (CLSM) showed that all three PEOg-PEI polyplexes effectively delivered DNA (red) into the perinuclei region, as well as nuclei of 293T cells, following 4 h transfection (Figure 7). Moreover, stronger DNA fluorescence was observed in the nuclei of cells transfected with PEO(13k)g-10PEI polyplexes than those with polyplexes of PEO(24k)-g10PEI and PEO(13k)-g-22PEI, further confirming that PEO(13k)-g-10PEI has the best transfection activity. In comparison, the cells transfected with 25 kDa PEI control under otherwise the same conditions displayed obviously less DNA fluorescence in the cell nuclei. Notably, as shown in Figure 6, PEO(24k)-g10PEI polyplexes gave rise to lower gene expression compared to 25 kDa PEI controls. The discrepancies observed in intracellular DNA fluorescence and gene expression are likely because DNA can not readily be released from PEO(24k)-g10PEI polyplexes due to effective shielding with PEO. These results indicate that polyplexes of PEO-g-PEI copolymers at their optimal compositions can well overcome the intracellular barriers for nonviral gene delivery such as endosomal escape and nuclear entry, efficiently delivering DNA into the nuclei of cells to accomplish potent gene transfection.

Figure 5. Cytotoxicity of PEO-g-PEI polyplexes at N/P ratios ranging from 10/1 to 40/1 in 293T cells determined by MTT assays. The 25 kDa PEI was used as a control. Cell viabilities are shown as mean ± SD (n = 4).

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CONCLUSIONS We have demonstrated that PEO-g-PEI graft copolymers mediate nontoxic and highly potent in vitro gene transfection under serum-free as well as 10% serum conditions. PEO-g-PEI copolymers present several unique features as nonviral vectors: (i) in contrast to many reported PEI derivatives, they have excellent water solubility (important for water-dispersibility of resulting DNA complexes); (ii) they can effectively condense DNA into nanosized particles with moderate positive surface charges as a result of many short branched PEIs grafting onto PEO backbone; (iii) their DNA complexes have high colloidal stability under physiological salt as well as serum conditions due to presence of PEO; (iv) they exhibit low or no carrierassociated cytotoxicity as they are composed of low toxic short PEI and biocompatible PEO; (v) they can effectively transport DNA into the cell nuclei, resulting in high levels of gene expression (up to several times higher than that of 25 kDa PEI control) in the presence or absence of serum; and (vi) they have well-defined structures with controlled PEO molecular weights and PEI graft densities. Inspired by the results of this work, we are currently investigating the in vivo applications of therapeutic DNA/PEO(13k)-g-10PEI formulations for cancer

Figure 6. Relative GFP expression of PEO-g-PEI polyplexes in 293T cells at N/P ratios of 10/1, 20/1 and 30/1 in serum-free medium (A) and 10% serum medium (B). The 25 kDa PEI formulation at its optimal N/P ratio of 10/1 was used as a control, and its GFP expression was defined as 100%. Data are shown as mean ± SD (n = 3; student’s t-test, ***p < 0.001).

nontoxic (100% cell viability; Figure 5). PEO(24k)-g-10PEI polyplexes revealed also low cytotoxicity (93% cell viability) at an N/P ratio of 30/1 (Figure 5). Hence, it appears that grafting low molecular weight PEI to PEO is a potential approach to obtain nontoxic and efficient nonviral gene transfer agents. Interestingly, no matter with or without 10% serum, PEO(13k)-g-10PEI, though with a comparably weak buffer capacity, displayed the highest transfection activity among these three 886

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Figure 7. CLSM images of 293T cells transfected with Cy5-labeled pDNA polyplexes of PEO-g-PEI at an N/P ratio of 20/1 in the presence of 10% serum (1 μg DNA/well). The 25 kDa PEI at an N/P ratio of 10/1 was used as a control. Cells were incubated with Cy5-labeled pDNA polyplexes for 4 h. For each panel, images from left to right show Cy5-labeled pDNA (red), cell nuclei stained by Hoechst 33342 (blue), and overlays of all images. The bar represents 20 μm. (A) PEO(13k)-g-10PEI; (B) PEO(24k)-g-10PEI; (C) PEO(13k)-g-22PEI; and (D) 25 kDa PEI.

treatment. We are convinced that these PEO-g-PEI copolymers have great potential for the development of safe and efficient in vitro, as well as in vivo, nonviral gene transfer agents.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by National Natural Science Foundation of China (NSFC 20874070, 50803043, 50973078, 20974073, and 31070707), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Program of Innovative Research Team of Soochow University.

ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of PEO-g-allyl copolymers, 1H NMR spectra of PEO(13k)-g-OH and PEO(13k)-g-NC, acid− base titration curves of PEO-g-PEI, and TEM images of PEO-gPEI polyplexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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(1) Jeong, J. H.; Kim, S. W.; Park, T. G. Prog. Polym. Sci. 2007, 32, 1239−1274. (2) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259−302. (3) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Nat. Rev. Drug Discovery 2005, 4, 581−593. (4) Wong, S. Y.; Pelet, J. M.; Putnam, D. Prog. Polym. Sci. 2007, 32, 799−837. (5) Akinc, A.; Thomas, M.; Klibanov, A. M.; Langer, R. J. Gene Med. 2005, 7, 657−663.

AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +86-512-65880098. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 887

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