Biodegradable Poly(vinyl alcohol)-polyethylenimine Nanocomposites

Dec 6, 2011 - The transfection efficiency was calculated by quantifying the percent cells expressing green fluorescent protein (GFP) using flow cytome...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/Biomac

Biodegradable Poly(vinyl alcohol)-polyethylenimine Nanocomposites for Enhanced Gene Expression In Vitro and In Vivo Ritu Goyal,† Sushil K. Tripathi,† Esther Vazquez,§ Pradeep Kumar,†,* and Kailash C. Gupta*,†,‡ †

CSIR-Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi-110007, India CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow-226001, U.P., India § Department of Genetics and Microbiology, Autonomous University of Barcelona, Campus Universitari, Bellaterra 08193, Barcelona, Spain ‡

S Supporting Information *

ABSTRACT: Use of cationic polymers as nonviral gene vectors has several limitations such as low transfection efficiency, high toxicity, and inactivation by serum. In this study, varying amounts of low molecular weight branched polyethylenimine 1.8 kDa (bPEI 1.8) were introduced on to a neutral polymer, poly(vinyl alcohol) (PVA), to bring in cationic charge on the resulting PVA-PEI (PP) nanocomposites. We rationalized that by introducing bPEI 1.8, buffering and condensation properties of the proposed nanocomposites would result in improved gene transfer capability. A series of PVAPEI (PP) nanocomposites was synthesized using well-established epoxide chemistry and characterized by IR and NMR. Particle size of the PP/DNA complexes ranged between 120 to 135 nm, as determined by dynamic light scattering (DLS), and DNA retardation assay revealed efficient binding capability of PP nanocomposites to negatively charged nucleic acids. In vitro transfection of PP/DNA complexes in HEK293, HeLa, and CHO cells revealed that the best working formulation in the synthesized series, PP-3/DNA complex, displayed ∼2−50-fold higher transfection efficiency than bPEIs (1.8 and 25 kDa) and commercial transfection reagents. More importantly, the PP/DNA complexes were stable over a period of time, along with their superior transfection efficiency in the presence of serum compared to serum-free conditions, retaining the nontoxic property of low molecular weight bPEI. The in vivo administration of PP-3/DNA complex in Balb/c mice showed maximum gene expression in their spleen. The study demonstrates the potential of PP nanocomposites as promising nonviral gene vectors for in vivo applications.

1. INTRODUCTION Gene delivery is a multistep process that includes DNA condensation, cellular uptake, endosomal escape, DNA release into the cytoplasm, and transfer of DNA into the nucleus. The clinical applications of gene therapy depend mainly on the development of safe and efficient carriers for the delivery of genetic material. Viral vectors though have proven to be very efficient in gene delivery, their potential safety and immunogenicity concerns raise the risk for their clinical applications.1 Therefore, as an alternative to viral vectors, cationic polymers have been synthesized and used as gene delivery carriers. These vectors offer several advantages over the conventional viral vectors, which include safety, stability, large nucleic acid loading capacity, strong interactions with nucleic acids, protection of DNA from nucleases, and easy and largescale production.2−5 Different classes of polymers with amine functionalities, such as chitosan, poly(L-lysine), poly(amido amine) [PAMAM] dendrimers, and polyethylenimines (PEIs), have been reported to condense DNA into very small particles and initiate cellular uptake via endocytosis.6,7 However, low transfection efficiency, especially in the presence of serum, is one of the major limitations associated with nonviral vectors.8,9 Available reports have shown that gene expression is significantly inhibited in the presence of serum, when PEIs, © 2011 American Chemical Society

the most studied cationic polymers, have been used and thereby limiting their use in in vivo applications.8 For overcoming this problem, use of nonionic and hydrophilic polymers in conjugation with PEI is envisaged to improve the solubility and to prevent the agglomeration of polycation/DNA complexes in vivo.10,11 Simultaneously, these polymers have been tried to improve their complexation with DNA to provide protection of the complex against nucleases and to prevent interaction of the carrier substance with serum albumin.12 These efforts, however, did not improve the transfection properties to a significant level, and efforts are afoot to find alternative polymers.13,14 Poly(vinyl alcohol) (PVA) is a biocompatible and hydrophilic polymer, which is excreted with urine.15,16 Wittmar and co-workers were the first to modify its backbone by covalently attaching amine groups using carbonyldiimidazole.17 Subsequently, they studied the effect of electrostatic and hydrophobic interactions on DNA condensation and evaluated these complexes for gene transfection. However, these polyplexes required exogenous addition of chloroquine, an endosomolytic Received: August 19, 2011 Revised: November 29, 2011 Published: December 6, 2011 73

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

respectively. FTIR spectra of nanocomposites were recorded on a single beam Perkin-Elmer (Spectrum BX Series), with the following scan parameters: scan range, 4400−400 cm−1; number of scans, 16; resolution, 4.0 cm−1; interval, 1.0 cm−1; units, %T. Zetasizer Nano, ZS (Malvern Instruments, U.K.), was used to determine the particle size and zeta potential of PP nanocomposites and their DNA complexes. GFP (Green Fluorescent Protein) reporter gene expression was observed under Nikon Eclipse TE-2000-S inverted microscope, and the pattern in transfected cells was analyzed (excitation at 488 nm, emission at 509 nm) on NanoDrop ND-3300 spectrofluorometer (U.S.A.). The uptake and intracellular passage of tetramethylrhodamine (TMR) labeled PP nanocomposite/YOYO-1-labeled DNA complexes in CHO cells were examined under a LSM 510 Meta Confocal microscope (Axio Observer, Zeiss, Germany). 1H NMR spectra of PVA-chlorohydrin and PP nanocomposites were obtained from JEOL-DELTA2 400 spectrometer operating at 400 MHz using D2O as solvent. Chemical shifts (δ) are expressed in ppm. Transmission electron microscopy (TEM) of particles was carried out on FEI Tecnai 12 Twin TEM, operated at 60 kV, spot size 3 after complete gun alignment, and astigmatism correction. Cell Culture. Human embryonic kidney (HEK293), human cervical adenocarcinoma (HeLa), and chinese hamster ovary (CHO) cells were obtained from the Cell Repository Facility at National Centre for Cell Sciences, Pune, India. Cell cultures were maintained at 37 °C, 5% CO2−air in Dulbecco’s modified Eagle’s culture medium (DMEM; Sigma, U.S.A.), supplemented with 10% heat inactivated fetal bovine serum (GIBCO-BRL-Life Technologies, U.K.) and 1% antibiotic cocktail of streptomycin and penicillin. These cells were chosen based on their different origins and, hence, the transfection data generated, can be interpreted in the context of wider applicability. In addition, nonsynchronized cells were used since the data has been validated in in vivo conditions. Synthesis of PVA-PEI Nanocomposites (PP). PP nanocomposites were synthesized in two steps. In the first step, poly(vinyl alcohol) was converted into its chlorohydrin derivative (PVA-CH), which was reacted with varying amounts of bPEI 1.8, in the second step, to obtain PP nanocomposites. Synthesis of Poly(vinyl alcohol)-chlorohydrin (PVA-CH). Chlorohydrin of PVA was prepared following the standard procedure.22 Briefly, PVA (500 mg) was dissolved in water (5 mL) at 85 °C. After complete dissolution, dilute sulphuric acid (2 N, 0.7 mmol) was added followed by dropwise addition of epichlorohydrin (22.8 mmol). The reaction mixture was stirred for 24 h and then neutralized with a 5% aqueous solution of sodium hydrogen carbonate. The chlorohydrin derivative so obtained was precipitated with acetone, filtered, dissolved in water (10 mL) and dialyzed for 48 h with intermittent change (4× 12 h) of water. This was subsequently lyophilized to obtain PVA-CH (∼95% yield) as a white powder and characterized by FTIR and 1H NMR. Reaction of bPEI 1.8 with PVA-CH. PVA-CH (25 mg) was dissolved in dd water (10 mL) at 65 °C. To this solution, aqueous solutions of bPEI 1.8 (200 mg, 10 mg/mL for 1:8 weight ratio of PVACH/bPEI 1.8) and sodium hydroxide (4 M, 5 mL) were added sequentially, and the resulting reaction mixture was stirred for ∼20 h at 65 °C. Thereafter, the reaction mixture was poured into a dialysis bag (12 kDa cut off) and dialyzed against water for 48 h with intermittent change (4× 12 h) of water. The dialyzed solution was lyophilized to obtain PVA-PEI (PP) nanocomposite (∼87% yield) as a white powder. Similarly, PP nanocomposites with increasing amount of bPEI 1.8, namely, 1:10 (PP-2), 1:12 (PP-3), and 1:15 (PP-4) were prepared and obtained in ∼82−89% yield. These nanocomposites were then characterized by FTIR and 1H NMR. Preparation of PP/DNA Complexes. All PP/DNA complexes were freshly prepared at different w/w ratios prior to their use. Briefly, an aqueous solution of PP-1 (1 mg/mL) was added to 1 μL of pDNA (0.3 μg/μL) at various w/w ratios (1.66:1, 2.33:1, 3.33:1, 4:1, 5:1, and 6.66:1) or N/P ratios (1.28, 1.8, 2.58, 3.1, 3.87, and 5.16) in 5% dextrose (5 μL) and final volume was made up to 20 μL with water. The resulting complexes were gently vortexed, incubated at 25 ± 2 °C for 30 min and used in biophysical studies and transfection

agent, for escaping from the endosomes for meaningful transfection. In yet another study, the PVA backbone was modified by poly-L-lactide-co-glycolide (PLGA) using carbonyldiimidazole, and DNA was entrapped within the hydrophobic compartment of the resulting polymers.18 Thereafter, DNA released from these polymers got exposed to acidic degradation products of PLGA, namely, lactic and glycolic acids, which led to its acid-catalyzed cleavage.19 Further, these polymers could not provide adequate protection to bound pDNA from nuclease degradation for a considerable period of time.19 The results obtained by Wittmar et al.17 encouraged us to design an alternative strategy to modify the PVA backbone in such a way so as to overcome the limitations as reported by this research group. The aim of the present study was to develop superior transfection reagents by modifying the PVA backbone with a low molecular weight branched polyethylenimine 1.8 kDa (bPEI 1.8) using epoxide chemistry. The rationale of selecting low molecular weight bPEI for the surface modification of PVA is based on the findings of previous studies, wherein the former has been shown to possess enough buffering capacity and good DNA binding/release property along with its nontoxic nature.8,9 Conversely, bPEI 1.8, despite having good buffering capacity, is known to possess poor transfection efficiency.8,9 There are reports that transfection efficiency of PEIs is dependent on its molecular weight where transfection increases with increase in molecular weight.20,21 However, with increasing molecular weight, cytotoxicity also increases. Therefore, in this study, PVA was chosen as a template for grafting low molecular weight bPEI to increase the overall charge on the PVA backbone (this resulted in the enhancement of charge density without increasing the molecular weight of PEI and toxicity). It was envisaged that, on one hand, presence of PVA would provide the biodegradability and biocompatibility to the system, while on the other, bPEI 1.8 would bring in the buffering capacity and also provide useful positive charge to the resultant PVA-PEI (PP) nanocomposites required to bind nucleic acids. Further, hydrophilic properties of PVA would help in reducing the nonspecific interactions with serum proteins and improve the viability of the proposed system under in vivo conditions. Likewise, the attachment of low molecular weight bPEI would not only introduce enough positive charge into the nanocomposites that will help not only to condense DNA, but also improve the endosomal escape rate to release the complex into cytoplasm for nuclear localization. Using this strategy, a series of PP nanocomposites was prepared by varying the weight ratios of PVA and bPEI 1.8. The DNA complexes of PP nanocomposites were evaluated for DNA complexation efficiency, protection against DNase, intracellular uptake, knockdown efficiency, and in vitro, as well as in vivo gene delivery efficiency.

2. EXPERIMENTAL SECTION Materials. Branched polyethylenimine 1.8 and 25 kDa (bPEI 1.8 and 25), poly(vinyl alcohol) (PVA, 30 kDa, 87−90% hydrolyzed), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and high retention dialysis tubing (cut off 12 kDa) were obtained from Sigma-Aldrich Chemical Co., U.S.A. Linear polyethylenimine 2.5 kDa (lPEI 2.5) was obtained from Polysciences Inc. (U.S.A.). Bradford reagent was obtained from Bio-Rad Inc., U.S.A. Commercial transfection agents, namely, Superfect and Lipofectamine 2000, were purchased from Qiagen (France) and Invitrogen (U.S.A.), respectively. Plasmid purification kit and luciferase cell lysis reagent were purchased from Qiagen (France) and Promega (U.S.A.), 74

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

medium was aspirated, replaced with 200 μL growth medium (DMEM with 10% FBS), and the cells were further incubated for 36 h. Sequential Delivery of siRNA. In another experiment, GFP-specific siRNA (2.5 μM) was delivered using PP-3 nanocomposite as a carrier (the best working sample in terms of transfection). Cells were treated first with the best working concentration of PP-3/DNA complex for 3−4 h, washed with 1× PBS (100 μL) and treated with 2 μL GFPspecific siRNA (2.5 μM) using PP-3/siRNA formulation in 20 μL reaction mixture. PP-3/DNA complex alone was used as control. Similarly, pDNA and GFP-specific siRNA were delivered using Superfect for comparison. Flow Cytometric Analysis. The transfection efficiency was calculated by quantifying the percent cells expressing green fluorescent protein (GFP) using flow cytometry (FACS Caliber System from GUAVA, San Joes, CA). HEK293 cells (6 × 105 cells/well) were seeded in 24-well plates and incubated at 37 °C in 5% CO2 for 24 h in DMEM medium containing 10% FBS. PP/pDNA complexes at w/w ratios 2.33, 3.33, 4.0, and 5.0, bPEIs/pDNA polyplexes at w/w ratio 1.66 and commercial reagents, namely, Superfect and Lipofectamine/ DNA complexes, as per the manufacturers’ protocols, were prepared as described above. At the time of transfection, the medium in each well was replaced by 300 μL of DMEM without FBS, 100 μL of polymer/ pDNA complex containing 1.5 μg (0.3 μg/μL) of pEGFPDNA, and incubated the cells for 4 h at 37 °C. After 4 h, the medium was replaced by 1 mL of DMEM containing 10% FBS, and the cells were further incubated for 36 h. The cells were observed under a fluorescence microscope, then harvested separately from each well by the trypsin-EDTA treatment, and suspended in 200 μL of PBS (pH 7.4). Subsequently, the cells were transferred to flow cytometry cuvettes for analysis. The nontransfected cells were used as negative control with their fluorescence set to 1%. In order to investigate the effect of serum on the transfection efficiency, the assay was performed in the presence of serum (as described above) by taking 5, 10, 15, 20, 30, 40, and 50% serum containing medium and expression was quantified using flow cytometry for PP-3/DNA and bPEI 25/DNA complexes. Quantification of GFP Expression. Post-transfection, the GFP fluorescence intensity was observed under inverted fluorescent microscope at 10× magnification. Transfected cells were washed with 1× PBS (2 × 100 μL), treated with 50 μL lysis buffer (10 mM Tris, 1 mM EDTA and 0.5% SDS pH 7.4), and agitated for 45 min at 37 °C. GFP was estimated in 2 μL lysates spectrofluorometrically and corrected for background and autofluorescence in mock-treated cells. Total protein content in cell lysate from each well was estimated using Bradford reagent (BioRad) with BSA (Bangalore Genei, India) as a standard. The GFP fluorescence intensity was estimated in triplicate. Background was subtracted and fluorescence intensity expressed as arbitrary units/mg of protein. Heparin Competition Assay. To examine the DNA binding ability of the PP nanocomposites, heparin competition assay was carried out using gel electrophoresis. PP/DNA and bPEIs (1.8 and 25 kDa)/DNA complexes were prepared at their best w/w ratios, 4 and 1.66, respectively. Afterward, an aqueous solution of heparin (1 U/μL) was added by varying the amount from 0 to 10 U for the release of bound pDNA. The samples were then incubated for 30 min and electrophoresed (100 V, 1 h) on a 0.8% agarose gel, stained with ethidium bromide and visualized on a UV transilluminator using a Gel Documentation system. The amount of DNA released from the complexes after heparin treatment was estimated by densitometry. Likewise, the assay was repeated with PP-2 to PP-4 nanocomposites. DNase I Protection Assay. DNase I protection assay was performed to examine the ability of PP-3 nanocomposite to provide protection to bound DNA against DNase. For this study, we treated the native DNA and PP-3/DNA complex (at w/w ratio 4) with 1 μL DNase (1 U/μL in a buffer containing 100 mM Tris, 25 mM CaCl2) and incubated for 0.25, 0.5, 1, and 2 h. Samples taken up in 1× PBS served as control. After incubation, DNase I was inactivated by adding 5 μL of 100 mM ethylenediaminetetraacetic acid (EDTA) followed by incubation at 70 °C for 10 min. The pDNA was then released from the polyplexes by adding 10 μL heparin (1 U/μL) and incubated for 2 h at

experiments. Similarly, PP-2, PP-3, PP-4, and bPEI (1.8 and 25 kDa)/ pDNA polyplexes were prepared at the same w/w ratios. Particle Size and Zeta Potential Measurements. The particle size and zeta potential of the nanocomposites, suspended in water (1 mg/mL), and their DNA complexes, at their best working concentration (prepared as described above), were determined in Milli-Q water and DMEM containing 10% serum by dynamic light scattering (DLS) using Zetasizer Nano ZS. The data analysis was performed in automatic mode and the measured sizes and zeta potential (in triplicates) were presented as the average value of 20 and 30 runs, respectively. The average values of zeta potential were estimated by Smoluchowski approximation from the electrophoretic mobility.23 TEM analysis was carried out at 60 kV and images were recorded using a SIS MegaView II CCD camera. Gel Retardation Assay. DNA condensation ability of the PP nanocomposites and bPEIs (1.8 and 25 kDa) was assessed by electrophoretic mobility assay. Complex formation was induced at various w/w ratios (polymer/DNA: 0.5:1, 1:1, 1.66:1 and 2.33:1) and the final volume made up with 6× agarose gel loading dye (xylene cyanol) to 20 μL. The complexes were loaded on a 0.8% agarose gel with EtBr (0.1 μg/mL) and run with Tris-acetate (TAE) buffer at 100 V for 1 h. Retardation in DNA mobility caused by nanocomposites and bPEIs was observed under a UV transilluminator attached with a Gel Documentation system (Syngene, U.K.). Measurement of Intrinsic Buffering Capacity. The buffering capacity of bPEIs and PP nanocomposites was measured by performing an acid−base titration according to the previously described method.24 In brief, each sample (∼3 mg) was dissolved in 150 mM NaCl solution and made up to a final concentration of 0.1 mg/mL. The pH of the solutions was brought to 10 using 0.1 N NaOH and, subsequently, the resulting solutions were titrated to pH 3.0 with 50 μL aliquots of 0.1 N HCl. pH values of the solutions were measured after each addition of the aliquot and changes in proton concentration were calculated from the cumulative volumes of HCl added. The slope of the line in the plot for pH vs amount of HC1 consumed indicates the intrinsic buffering capability of the system. Cell Viability Assay. To examine the toxicity of the projected nanocomposites, MTT assay was performed spectrophotometrically at 540 nm post 36 h of transfection on HEK293, CHO, and HeLa cells.23 Briefly, HEK293 cells were plated in a 96-well plate in 200 μL of DMEM supplemented with 10% FBS and allowed to grow for 48 h. After removal of the medium, cells were treated with PP/DNA complexes and bPEIs/DNA polyplexes at w/w ratios of 1.0, 1.66, 2.33, 3.33, 4.0, and 5.0 (see preparation of PP/DNA complexes). Similarly, Superfect/DNA and Lipofectamine/DNA complexes were prepared following the manufacturers’ protocols and added gently to the cells. After 4 h, the medium was removed from each well and replaced by growth medium supplemented with 10% FBS. The plates were kept in an incubator (37 °C in a humidified 5% CO2 atmosphere) for 36 h, after which the cell viability was measured by adding fresh growth medium (100 μL) containing MTT (50 μg) to each well and incubating the cells for 4 h. Untreated cells served as control with 100% viability, and wells having MTT reagent only without cells were used as blank to calibrate the spectrophotometer to zero absorbance. All the experiments were carried out in triplicates. In Vitro Transfection. Delivery of Plasmid DNA. HEK293, HeLa, and CHO cells were seeded separately in 96-well plates 24 h prior to putting transfection assay. pEGFPDNA (plasmid encoding for enhanced green fluorescent protein; 0.3 μg) was complexed with PP-1 to PP-4 at weight ratios 1.66, 2.33, 3.33, 4.0, 5.0, and 6.66 and bPEIs at weight ratios 1.0, 1.66, 2.33, 3.33, 4.0, and 5.0. Similarly, DNA complexes were prepared with commercial reagents, namely, Superfect and Lipofectamine, as per the manufacturers’ protocols. The medium was removed from each well and the cells were washed once with 1× PBS (100 μL). Nanocomposite/DNA complexes and bPEIs/DNA polyplexes, diluted with serum-free DMEM (60 μL) made up to a final volume of 80 μL, were added gently onto the cells. In another set, DNA complexes were diluted with DMEM supplemented with 10% FBS and added to the cells. After 3 h of treatment, the transfection 75

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

Scheme 1. Schematic Presentation of Preparation of PVA-PEI (PP) Nanocompositesa

a

Inset: 1H-NMR spectra of (A) PVA and (B) PVA-PEI (PP-4) nanocomposites in D2O.

25 ± 2 °C followed by analysis on a 0.8% agarose gel and imaging on a Gel Documentation system (Syngene, U.S.A.). The amount of DNA released from the complexes was estimated by densitometry. Protein Adsorption Assay. bPEIs and PP-3 nanocomposites were complexed with pDNA at their best working w/w ratios of 1.66 and 4, respectively. Samples were incubated with standard bovine serum albumin (BSA) for 3 h at 25 ± 2 °C and final pDNA and BSA concentrations were adjusted to 3.0 μg/mL and 1.0 mg/mL, respectively. The samples (DNA complexes/BSA) were centrifuged at 14000g for 2 h at 4 °C. The supernatant was removed and the pellet was washed with Milli-Q water (10 μL) to remove unbound BSA. The pellet was suspended in Milli-Q water (10 μL), added loading buffer [0.2 M Tris-HCl pH 6.8, 10% w/v sodium dodecyl sulfate (SDS), 20% v/v glycerol, 0.05% w/v bromophenol blue, 10 mM dithriothreitol (10 μL)] and incubated at 100 °C for 10 min to extract bound BSA from the samples. Extracted solutions were analyzed on a 12% denaturing SDS-polyacrylamide gel (2 h at 25 mA) and stained with RAPID stain to visualize BSA that had been adsorbed on the samples. In Vivo Transgene Expression. Animals. In vivo studies were carried out using male Balb/c mice (6−7 weeks old, 25 ± 3 g) obtained from the animal breeding colony of the Indian Institute of Toxicology Research (IITR), Lucknow (India), and were acclimatized under standard laboratory conditions. The animals were cared for humanely, as per the guidelines laid down by the Animal Ethics Committee of the Institute. Transgene Expression. As a reporter technology, bioluminescence finds its greatest potential by helping in characterizing the enormous complexity of living systems. It provides 10- to 1000-fold higher assay sensitivity than fluorescent reporters such as GFP, and it can improve assay performance substantially when applied to complex biological samples. Therefore, we chose pGL3 as a reporter gene for our in vivo assays. PP-3/DNA, bPEI 25/DNA, and naked DNA complexes were prepared at w/w ratios of 4, 1.66, and 0, respectively, using 25 μg of pGL3 control vector, taking normal saline as a medium. A 100 μL aliquot of each complex was injected into Balb/c mice through the tail

vein using a syringe of 40 U (insulin syringe) with a needle size of 0.3 × 8 mm. At predetermined time periods after injection (3 and 7 d), the mice were sacrificed and liver, spleen, kidney, lung, heart, and brain were excised. The organs were rinsed with chilled normal saline and weighed, and 25% w/v homogenate was prepared in lysis buffer (Promega, U.S.A.) containing 1× protease inhibitor cocktail (Sigma, U.S.A.). After three cycles of freezing and thawing, the homogenates were centrifuged at 10000g for 10 min at 4 °C, and 100 μL of supernatant was used to measure luciferase activity, which was represented as RLU/mg of total protein. All the experiments were carried out in duplicate. Intracellular Trafficking. Zeiss LSM 510 inverted laser-scanning confocal microscope was used to image TMR-PP-3/YOYO-1-pDNA in CHO cells.25 For labeling, PP-3 (1 mL, 10 mg/mL in H2O) was allowed to react with tetramethylrhodamine isothiocyanate (TRITC; 21 μL, 10 mg/mL in DMF) overnight to block ∼1% of total amines. The solution was concentrated in a speed vac and unreacted/ hydrolyzed TRITC was removed by triturating with ethyl acetate (3 × 2 mL). For labeling of pDNA, YOYO-1 iodide (2 μL, 1 mM solution in DMSO) was added to pDNA (0.3 mg) and stirred for 2 h at 25 ± 1 °C in the dark and then stored at −20 °C. TMR-labeled PP-3 nanocomposite was complexed with YOYO-1 labeled pDNA at a w/w ratio of 4:1 and incubated with CHO cells in a 6-well plate at different time points (15, 30, 60, 120, and 240 min). After incubation, the cells were washed with 1× PBS (3 × 500 μL) and finally fixed with 4% paraformaldehyde solution for 10 min at 4 °C. The fixed cells were again washed with 1× PBS (2 × 500 μL) and counterstained with a blue nuclear dye, 4,6-diamino-2-phenylindole (DAPI). The cells were viewed under a confocal laser scanning microscope. Statistical Analysis. Statistical analysis wherever needed was carried out by one-way analysis of variance followed by Tukey’s test after ascertaining homogeneity of variance and normality of data. A value of P < 0.05 was considered statistically significant. JMP version 6.0.0, Statistical Discovery from SAS and R studio were used for 76

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

Table 1. Size and Zeta Potential Measurements of PP Nanocomposites and Their DNA Complexes in Water and Medium Containing 10% FBSa average particle size in nm ± SD (PDI)

sample ID

DNA loaded complexes (in H2O)

PP-1

131.1 ± 4.1 (0.237)

PP-2

124.7 ± 2.8 (0.21E)

PP-3

133.2 ± 3.2 (0.166)

PP-4

131.7 ± 3.1 (0.256)

bPEI 1.8

245.8t21.5 (0.4−29)

bPEI25

287.8 ± 2.9 (0.675)

a

zeta potential in mV = SD ratio of nanopar tide/DNA (w/w)

DNA loaded complexes (in DMEM)

nanocompo sites (in H2O)

DNA loaded complexes (in H2O)

DNA loaded complexes (in DMEM)

percent realized substitution of PEI on PVAas estimated using 1H NMR (attempted substitution; PVA/PEI)

30.5 ± 1.9 (0.562) 35.2 ± 3.8 (0.672) 34.5 ± 2.9 (0.721) 34.7 ± 3.7 (0.645) 156.3 ± 6.8 (0.582) 125.6 ± 2.9 (0.489)

12.7 ± 2.8

8.3 ± 1.9

−12.1 ± 2.4

4:1

10.6 (1:8)

15.2 ± 3.5

10.5 ± 1.6

−12.5 ± 2.3

4:1

12.9 (1:10)

16.5 ± 3.9

11.9 ± 2.6

−12.0 ± 2.1

4:1

13.7 (1:12)

17.7 ± 3.6

15.4 ± 1.9

−13.0 ± 2.3

4:1

17.3 (1:15)

22.7 ± 4.6

17.5 ± 3.7

−16.3 ± 3.1

1.66:1

31.8t2.9

21.4 ± 2.8

−13.6 ± 1.9

1.66:1

Estimation of percent substitution of bPEI 1.8 on PVA by 1H NMR.

analysis. A detailed statistical analysis for all the tested groups can be found in the Supporting Information of the article.

The splendor of selecting epichlorohydrin in the projected strategy, as a cross-linker, is that (i) it has an epoxide function, which can react with a wide variety of nucleophiles/electrophiles (here, OH groups of PVA to generate chlorohydrin), and (ii) on alkali-mediated generation of epoxide ring on the other end, primary and secondary amines of bPEI can react to form secondary and tertiary amines. Alternatively, the reaction of bPEI with epoxide results in the charge conversion from one form to another keeping the overall amines intact.27 The conjugation of bPEI on to PVA-CH was determined by 1H NMR taking D2O as a solvent (Scheme 1, inset). On increasing the concentration of bPEI 1.8, down the series, there is a gradual increase in the conjugation of bPEI on to PVA backbone (Table 1). In IR, the absorption band at 3369−3401 cm−1 confirmed the presence of NH2 and OH groups. Strong absorption band at 1066−1070 cm−1 was consistent with hydroxyl groups. Absorption band at 1452−1458 cm−1 was attributed to deformation mode of CH2 groups. Thus, the overall data confirmed the formation of PP nanocomposites. 3.1. Size and Zeta Potential Measurements. To determine the size and charge of the PP/pDNA complexes, these were characterized by DLS in water and medium containing 10% FBS. The size of the PP/pDNA complexes was found to be in nanometric range (120−135 nm), which decreased in medium (30−35 nm; Table 1). All the PP/pDNA complexes were prepared at w/w ratio of 4:1 and bPEIs/pDNA at w/w ratio 1.66:1. A decrease in size in the presence of serum might be due to absorption of water from the cationic surface by the anionic serum proteins leading to dehydration, which is in agreement with the previous studies.23,27 Zeta potential of the PP nanocomposites was found to be in the range of +12 to +17 mV in water. Concomitant with an increase in weight ratio of PEI in the nanocomposites (PP-1 to PP-4), a gradual increase in the positive character on the nanocomposites was observed (Table 1). Zeta potential of PP nanocomposites though remained less than bPEI 1.8 (due to incorporation of a neutral polymer), it was still sufficient to allow them to interact directly with inner surface of the endosome and promote its disruption, thereby, facilitating the release of nanocomposite/ pDNA complexes into the cytoplasm.28 A similar trend was observed, when these nanocomposites were complexed with pDNA, which might be due to neutralization of positive surface

3. RESULTS AND DISCUSSION In this study, we have prepared a series of transfection reagents by grafting low molecular weight bPEI 1.8 on to PVA using a simple epoxide chemistry. The rationale to develop the projected strategy was as follows: (i) the resulting nanocomposites (PP) should easily disperse in water at physiological pH, and their particle size should remain in the nano range, (ii) PP nanocomposites should possess enough buffering capacity to release the desired therapeutics into cytoplasm, (iii) once released into cytoplasm, these nanocomposites should efficiently carry DNA for its nuclear localization, and (iv) the PP nanocomposites should retain the properties of both the native polymers such as biocompatibility and cell viability. It is well studied that transfection efficiency of bPEIs is molecular weight dependent. While high molecular weight bPEIs have better gene transfer capability, low molecular weight PEIs offer poor transfection efficiency.25,26 Conversely, high molecular weight bPEIs suffer from charge-associated toxicity, while low molecular weight bPEIs are almost nontoxic to the cells in vitro and in vivo.25,26 Taking a cue from the above, we carefully selected low molecular weight cationic polymers (bPEI 1.8 and lPEI 2.5), which can easily get cleared via various secreting pathways from the body, for synthesizing PVA-based nanocomposites in two steps. In the first step, PVA was converted into its chlorohydrin derivative (PVA-CH) by reacting it with epichlorohydrin in an acidic medium at 85 °C. PVA-CH derivative was subsequently reacted with amines of bPEI 1.8/lPEI 2.5 via in situ generation of epoxide under alkaline conditions. This follows opening of epoxide ring for generation of a series of bPEI 1.8/lPEI 2.5-conjugated-PVA nanocomposites with varying amounts of PEIs. The resultant nanocomposites were tested for their in vitro transfection efficiency. The nanocomposites obtained from reaction of lPEI 2.5 did not show significant improvement in transfection properties over bPEI 25 (a gold standard, P > 0.05; data not shown), while the other analog, that is, bPEI 1.8 conjugated PVA, exhibited significantly (P < 0.05) improved transfection efficiency over bPEI 25 and selected commercial transfection reagents. Therefore, for further studies, we only used bPEI 1.8 conjugated PVA nanocomposites (Scheme 1). 77

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

the endosomes.31,32 The buffering capacity of the proposed nanocomposites (PP-1−PP-4) was determined by an acid−base titration method (Figure 3). A nonsignificant (P > 0.05)

charge by the negative phosphate backbone of DNA. In DMEM supplemented with 10% serum, the zeta potential became negative, which is in agreement with the findings of the earlier studies.29,30 The morphology and size of PP-3/DNA complex (the best working formulations in terms of transfection efficiency) was also examined using TEM. We observed average size of complex to be ∼58 nm (Figure 1). The comparatively larger

Figure 3. Buffering capacity of bPEIs, PVA, PVA-CH, and PP nanocomposites determined by performing acid−base titration from pH 10−3.

increase in the proton capturing tendency of the nanocomposites on increasing the amount of bPEI 1.8 in the series was observed. This may reflect the fact that, upon copolymerization, the number of amines increase and more amines are available for charge neutralization. Interestingly, for PP nanocomposites, volume of 0.1N HCl required was comparable to that of bPEI 25. These observations are indicative of improved transfection efficiency of PP/DNA complexes in comparison to bPEI 1.8/DNA complex. 3.4. Cytotoxicity Assay. Before carrying out transfection experiments, it is pertinent to evaluate the cell viability properties of the synthesized nanocomposites, which is a major area of concern for novel gene delivery system. Cytotoxicity of the cationic polymers arises due to the aggregation of these polymers on the cell surface impairing the important membrane function like protein kinase activity.33 To investigate the cytotoxicity of PP/DNA complexes, MTT assay was performed on three cell lines, namely, HEK293, HeLa, and CHO, and the data compared with the DNA complexes of bPEI 1.8, bPEI 25, Superfect, and Lipofectamine. Untreated cells were considered as positive control with their cell viability set as 100%. PP/DNA complexes exhibited significantly higher cell viability (93−98%, P < 0.05) compared to that observed with bPEI 25/DNA complex (∼55−60%). bPEI 1.8/DNA complex, on the other hand, displayed nonsignificant (P > 0.05; ∼5−10%) cytotoxicity in the tested cell lines in comparison to PP/DNA complexes (Figure 4).

Figure 1. Transmission electron microscopic image of PP-3/DNA complex.

sizes obtained in DLS may be due to the dry particle size in the former case (i.e., TEM) and hydrodynamic diameter in the latter (i.e., DLS).29 3.2. Binding Ability of Nanocomposite with pDNA. Having characterized these nanocomposites and their DNA complexes, gel retardation assay was carried out to examine the pDNA binding ability of the nanocomposites. Nanocomposite/ pDNA complexes were prepared at w/w ratios from 1.5 to 4.0, using 0.3 μg pDNA, incubated at 25 ± 2 °C for 30 min and the samples were run on an agarose gel. We observed that the mobility of pDNA was completely retarded between w/w ratios of 2−3 (Figure 2). It was observed that from PP-1 to PP-4, the amount of PP nanocomposites required to retard the mobility of a fixed amount of pDNA was found to be reduced. This decrease in the amount of the nanocomposites required from PP-1 to PP-4 might be due to increased percent incorporation of PEI in the nanocomposites, which is in accordance with the spectroscopic data and zeta potential measurements (Table 1). 3.3. Buffering Capacity and Endosomal Release. Buffering capacity of the polymer is one of the important properties that enables it to buffer the endosomes and helps in the rupture and subsequent release of the genetic material into the cytosol. The high transfection efficacy of bPEI/DNA polyplexes is attributed to their excellent buffering capacity in

Figure 2. DNA retardation assay of PP/DNA complexes. All the PP/DNA complexes retard the mobility of 300 ng of DNA at w/w ratio of 2−3. 78

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

Figure 4. Cell viability profile of PP/DNA complexes, bPEIs/DNA polyplexes, Lipofectamine/DNA and Superfect/DNA complexes in HEK293, HeLa, and CHO cells. Percent viability of cells is expressed relative to control cells. Each point represents the mean of three independent experiments performed in triplicates. βP < 0.05 vs bPEI 25, δP < 0.05 vs Lipofectamine, and #P < 0.05 vs Superfect.

Because cytotoxicity has been shown to be directly proportional to the molecular weight of PEIs,25,26 data presented in this study finds support from the previous studies where low molecular weight PEIs were found to possess negligible cytotoxicity.20,21 Among the commercial reagents tested, DNA complexes of Superfect and Lipofectamine exhibited ∼18−21 and ∼48−52% toxicity, respectively. Thus, the higher cell viability in PP/DNA complexes makes them potential candidates to be used as gene delivery vectors in vivo. 3.5. In Vitro Transfection. Following the observation that PP/DNA complexes are almost nontoxic to the cells, they were subsequently evaluated for their transfection efficiency on HEK293, HeLa, and CHO cells using pEGFP (plasmid containing reporter gene encoding for enhanced green fluorescent protein) in the presence and absence of serum. Transfection efficiency of the PP nanocomposite/DNA complexes was found to be cell line dependent, showing the highest transfection in HEK293 cells. All the nanocomposite/ DNA complexes showed higher transfection efficiencies (P < 0.05) compared to bPEI 1.8 and Lipofectamine/DNA complexes. Transfection efficiency initially increased with increasing w/w ratio and thereafter, decreased after attaining maxima. Among the synthesized nanocomposites, maximum transfection efficiency was observed with PP-3/DNA complex, which was ∼36-fold (P < 0.05) higher than the bPEI 1.8/DNA polyplex, ∼2.9-fold higher than bPEI 25/DNA polyplex, and ∼2.2−4-fold higher than Lipofectamine and Superfect/DNA complexes. Interestingly, transfection efficiency of the synthesized nanocomposite/DNA complexes in the presence of serum was found to be nonsignificantly higher, than that observed in the absence of serum. In the presence of serum (10%), EGFP expression, mediated by PP/DNA complexes, in all the three cell lines was found to be ∼49−51-fold higher (P < 0.05) than bPEI 1.8/DNA polyplex, ∼2.7−3.7-fold higher than bPEI 25/ DNA polyplex, ∼2.7−5.0-fold higher than Lipofectamine and Superfect/DNA complexes (Figure 5a−c). In conclusion, conjugation of bPEI 1.8 on to PVA backbone led to the synthesis of transfection reagents with considerably improved transfection efficiency than the native polymer, that is, bPEI 1.8. Quantification of EGFP expression by measuring the expression of GFP in cells was also carried out using

Figure 5. GFP fluorescence intensity in the absence and presence of serum in (a) HEK293, (b) CHO, and (c) HeLa cells, transfected with DNA complexes of PP, bPEIs, Superfect, and Lipofectamine. The transfection profiles show fluorescence intensity expressed in terms of arbitrary units/mg of total cellular protein. The data represent the mean of three independent experiments performed in triplicates. αP < 0.05 vs bPEI 1.8, βP < 0.05 vs bPEI 25, δP < 0.05 vs Lipofectamine, and # P < 0.05 vs Superfect.

fluorescence activated cell sorting (FACS). The analysis was performed on HEK293 cells in the presence of serum using enhanced green fluorescent protein (pEGFP) expressing vector. Nontransfected cells were used as negative control. We observed that the transfection efficiency reached up to ∼48 ± 2% for the best working PP-3/DNA complex at w/w ratio of 4:1, while only ∼2, 26, 29, and 14% GFP positive cells were scored for the other reagents like DNA complexes of bPEI 1.8, bPEI 25, Superfect, and Lipofectamine, respectively (Figure 6a). 79

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

∼48% knockdown by Superfect, as a delivery vector (Figure 7). The strong inhibition of GFP expression using PP-3/siRNA

Figure 6. (a) Percent transfection efficiency of PP/DNA complexes, bPEIs/DNA polyplexes, Superfect, and Lipofectamine/DNA complexes, determined by FACS at various w/w ratios; *P < 0.05 vs bPEI 1.8 and Lipofectamine; (b) dependence of transfection efficiency of PP-3 and bPEI 25/DNA complexes on increasing percentage of serum as determined by FACS.

Figure 7. PP-3 nanocomposite was examined for their ability to deliver siRNA in HEK293 cells by sequentially transfecting pEGFP DNA and GFP specific siRNA. The expression of GFP in cells was reduced by more than 80%, in comparison to Superfect/pGFPDNA/siRNA complex, which suppressed GFP expression by only 48%, as monitored by measuring fluorescence on a spectrofluorometer. All the experiments were performed at least thrice and error bars represent the standard deviation.

Cationic polymer-based nonviral vectors usually exhibit low transfection efficiency in the presence of serum that remains as one of the bottlenecks for their in vivo applications. Serum can cause aggregation of cationic polymer-DNA complexes and due to changes in the size, their uptake by endocytosis gets reduced, which, in turn, results in a decrease in the transfection efficiency of these complexes. To examine the influence of serum on the transfection efficiency of the PP/DNA complexes, we performed transfection assay with increasing concentration of serum (5−50%) in the transfection medium with PP-3/DNA complex (the best working formulation) and compared the results with that observed in bPEI 25/DNA polyplex. The results revealed marginal increase (P > 0.05) in percent transfected cells with increasing amount of serum (∼62 ± 4% transfected cells in 30% serum), which later started decreasing slowly. However, in the case of bPEI 25/DNA polyplex, the transfection efficiency decreased drastically (∼4 ± 2% transfected cells in 50% serum, P < 0.05; Figure 6b). The ability of PP-3/DNA complex to generate higher transfection efficiency in serum-containing medium enhances the potential of this vector for translational applications in vivo. The efficacy and versatility of PP-3 nanocomposite in transporting siRNA into HEK293 cells was also evaluated. We selected GFP as a target for gene knockdown, which has already been used to study RNAi (RNA interference).34,35 First, PP-3/EGFPDNA complex was transfected for 3 h and then PP3/siRNA was added. After 36 h of transfection, cells were scored for GFP expression. PP-3/siRNA was able to inhibit the GFP expression by ∼80% as compared to the mock control (cells transfected with PP-3/DNA complexes) in comparison to

complex further strengthens utility of the synthesized nanocomposite as a versatile carrier for nucleic acids (DNA and RNA) and, hence, can be used as an effective carrier of siRNA as well in mammalian cells. Biophysical properties of the nanocomposites examined earlier were found to be in correlation with the transfection data, namely, optimum binding and release of pDNA, buffering, and ability to protect pDNA from nucleases. An increase in transfection efficiency of the PP/DNA complexes in presence of serum suggests prevention of the serum-mediated aggregation of nanoparticles through stabilization as in the case of polysaccharide polymers.36 The extent of nonspecific interactions of the synthesized nanocomposites with serum components was further evaluated by protein adsorption assay. 3.6. Protein Adsorption Assay. To evaluate the nonspecific interaction of PP-3 nanocomposite with proteins, BSA adsorption assay was performed. Under in vivo conditions, adsorption of serum proteins is known to reduce the transfection efficiency of the complexed pDNA.8 To investigate the protein adsorption on to the exterior of the PP-3/DNA complex, it was incubated with BSA for 3 h. After washing off free BSA, amount of BSA adsorbed on to PP-3/DNA complex was compared with the amount of BSA adsorbed on to bPEIs 1.8 and 25/DNA polyplexes. BSA bands from bPEI 1.8/DNA (73% of the total BSA taken) and bPEI 25/DNA complexes (81% of the total BSA taken) after BSA incubation is 80

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

Conversely, highly stable complexes are less capable to release DNA inside a cell or release it too slowly, which again may be responsible for poor transfection efficiency. A vector system carrying a desired gene inside the cell must release it to facilitate efficient transfection.37 PP nanocomposites and bPEIs (1.8 and 25 kDa) were complexed with pDNA at their best working w/ w ratio of 4:1 and 1.66:1, respectively, with 0.3 μg of pDNA. The complexes, incubated with increasing units of heparin, were run on a 0.8% agarose gel. Quantification results through densitometry revealed that with 5 U of heparin, PP/DNA complexes released up to ∼73−78% of pDNA with ∼76% release in case of PP-3/DNA complex, while bPEI 25 and bPEI 1.8/DNA polyplexes released only ∼58−60% of pDNA (Figure

significantly more (∼7−8 folds) intense as compared to the band of BSA from the PP-3/DNA complex (10% of the total BSA taken; P < 0.05 vs bPEI 1.8 and bPEI 25; Figure 8). The

Figure 8. SDS-PAGE of amount of protein adsorbed on to surface of PP-3/DNA, bPEI 1.8, and bPEI 25/DNA complexes. bPEI 1.8, bPEI 25, and PP-3/DNA complexes were incubated with BSA for 3 h and unbound BSA was removed by washing and centrifugation. Bound BSA was removed from the particles and run on a SDS-PAGE. bPEI 1.8, bPEI 25, and PP-3 nanocomposites were also run in parallel without incubation with BSA. PP-3/DNA complex adsorbed ∼12− 14% of protein on their surface compared to the amount adsorbed on bPEIs/DNA polyplexes.

results indicate that PP-3/DNA complex adsorbed a significantly less amount of protein than bPEIs/DNA polyplexes, which might be due to the presence of a hydrophilic backbone of PVA in the nanocomposite that prevented interactions with protein, that is, reduction in nonspecific binding of proteins. The results are in agreement with the previous studies involving PEG/polysaccharides modified cationic vector systems.36 3.7. Intracellular Trafficking Using Confocal Laser Scanning Microscopy. After evaluating the actual percentage of transfected cells by flow cytometry, intracellular transport of dual labeled TMR-PP-3/YOYO-1-DNA complex was visualized in CHO cells by confocal laser scanning microscopy after fixing the cells at different time points (15, 30, 60, 120, and 240 min). It was observed that only few particles were seen inside the cell after 15 min of the exposure, while, after 120 min, sufficient number of labeled particles, DNA and dual labeled complex were observed inside the nucleus (Figure 9). The result shows that the nanocomposite itself along with the DNA is capable to enter inside the nucleus of a cell. 3.8. DNA Release Assay. After promising transfection activity displayed by PP/DNA complexes, it becomes pertinent to examine their DNA release efficiency. Release of pDNA from the complexes is one of the key parameters, which affects the ability of the particles to deliver a specific gene to be expressed correctly and efficiently in the cells. Usually, complexes, lacking in stability, dissociate too early to show good expression.

Figure 10. DNA release assay of bPEIs and PP/DNA complexes. Heparin, in increasing concentration, was added to 20 μL of complexes and incubated for 20 min at 25 ± 2 °C. The samples were run on a 0.8% agarose gel at 100 V for 45 min. Note that bPEIs/DNA polyplexes required more number of units to release the same amount of DNA as compared to PP nanocomposite/DNA complexes.

10). Further, on increasing the heparin concentration (10 U), the amount of pDNA released from PP-3/DNA complex was found to be ∼92%, while bPEI 25 and bPEI 1.8/DNA polyplexes released ∼67 and 70% of pDNA, respectively. This clearly indicates that binding of bPEIs to pDNA is quite strong, while PP nanocomposites form relatively loose complexes with pDNA and release it timely in the cellular milieu. 3.9. DNA Protection Assay. For an efficient gene expression, the interaction between delivery vector and pDNA should be strong enough, so that it can protect the bound DNA from degradation by the nucleases.38 To examine the ability of the nanocomposites to provide protection to bound DNA against nucleases, DNase I protection assay was

Figure 9. Confocal microscopic images of CHO cells treated with TMR-PP-3/YOYO-1-pDNA complex for 2 h showing the labeled particles in the nucleus. 81

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

Figure 11. DNase I protection assay. PP-3/DNA complex was treated with DNase I for different time intervals. The complexed DNA was released by treating the samples with heparin. The amount of DNA protected (%) after DNase I treatment was calculated as the relative integrated densitometry values (IDV), quantified, and normalized by that of pDNA values (without DNase I treatment) using Gel Documentation system (Syngene, U.K.). Note that PP-3 nanocomposite protected pDNA even after 2 h while naked DNA was degraded within 15 min in the presence of DNase I.

observation of getting the highest gene expression in the spleen is intriguing with poorly understood mechanism so far. Notwithstanding the above, it is tempting to speculate that the polymer-based vector may find suitable applications in spleen targeted gene delivery.

performed by incubating the PP-3/DNA complex with DNase I for different time points (15, 30, 60, and 120 min). Complete degradation of free pDNA (0.3 μg) was observed within 15 min, while the nanocomposite, PP-3, effectively protected complexed pDNA (Figure 11) and ∼86% DNA was found to be recovered after 30 min and ∼79% after 2 h. Thus, PP-3 nanocomposite protected DNA against DNase I for a considerable time period, thereby showing its potential for in vivo administration of pDNA. 3.10. In Vivo Transfection Efficiency. In vivo reporter gene transfection experiments were performed using PP-3 as a delivery vector (also used in in vitro studies), and the results were compared with that of bPEI 25 and naked DNA. In vivo transfection efficiency of PP-3/pDNA was examined in Balb/c male mice by measuring luciferase activity in all the vital organs of the organism, post three and seven days of intravenous injection. A nonsignificantly (P > 0.05) elevated luciferase activity was observed in spleen and heart of the exposed organisms after 3 days (data not shown). However, the maximum gene expression in terms of RLU/mg of the total protein was observed in spleen in all the groups after 7 days with expression being the highest for PP-3/DNA polyplex (P < 0.05 vs bPEI 25/DNA polyplex and naked DNA) and the lowest for naked DNA (Figure 12). The gene expression observed in spleen using bPEI 25/DNA polyplex was found to be nonsignificantly higher (P > 0.05) than naked DNA. Thus,

4. CONCLUSIONS A neutral biodegradable polymer, PVA, was uniquely modified by conjugating with bPEI 1.8 to introduce positive charge and subsequently made it an efficient gene carrier. The synthesized PP nanocomposite/DNA complexes while exhibiting improved transfection efficiencies also showed their enhanced efficacy in presence of serum compared to that observed in the absence of the same. The PP/DNA complexes were found as proven gene delivery agents in vivo with maximum gene expression observed in spleen. Taken together, the study raises promising potential of these nanocomposites as gene carrier for spleen targeted gene delivery.

■

ASSOCIATED CONTENT

S Supporting Information *

Statistical analysis data. This material is available free of charge via the Internet at http://pubs.acs.org..

■

AUTHOR INFORMATION Corresponding Author *Tel.: +91-522-2621856 (K.C.G.); +91-11-27662491 (P.K.). Fax: +91-522-2628227 (K.C.G.); +91-11-27667471 (P.K.). Email: [email protected] (K.C.G.); [email protected] (P.K.).

■

ACKNOWLEDGMENTS Financial support from CSIR Task Force Project, NWP035, Inhouse Project, EXP002, and Indo-Spanish Project is gratefully acknowledged. R.G. thanks UGC for the award of senior research fellowship to carry out this work. Authors are also thankful to Prof. Srikant Kukreti, Department of Chemistry, University of Delhi, Delhi, and Dr. K. Mitra (CDRI, Lucknow) for their help in NMR and TEM analysis, respectively. We also acknowledge Dr. S. Ramachandran (IGIB, Delhi) for his help in carrying out statistical analysis.

Figure 12. In vivo gene expression analysis using PP-3/DNA complex, bPEI 25/DNA polyplex and control (naked DNA) in Balb/c mice 7 days post-intravenous injection using pGL3 control vector as a reporter gene. *P < 0.05 vs bPEI 25/DNA polyplex and naked DNA in the spleen. Note a significant increase in luciferase activity in spleen and lung tissues of the exposed mice as compared to the controls.

■

REFERENCES

(1) Baum, C.; Kustikova, O.; Modlich, U.; Li, Z.; Fehse, B. Hum. Gene Ther. 2006, 17, 253−263. (2) Li, S.; Huang, L. Gene Ther. 2000, 7, 31−34.

82

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83

Biomacromolecules

Article

(3) Liu, F.; Shollenberger, L. M.; Huang, L. FASEB J. 2004, 18, 1779−1781. (4) Niidome, T.; Huang, L. Gene Ther. 2002, 9, 1647−1652. (5) Cho, Y. W.; Kim, J. D.; Park, K. J. Pharm. Pharmacol. 2003, 55, 721−734. (6) Duceppe, N.; Tabrizian, M. Expert Opin. Drug Delivery 2010, 7, 1191−1207. (7) Sun, X.; Zhang, N. Mini-Rev. Med. Chem. 2010, 10, 108−125. (8) Vicennati, P.; Giuliano, A.; Ortaggi, G.; Masotti, A. Curr. Med. Chem. 2008, 15, 2826−2839. (9) Sizovs, A.; McLendon, P. M.; Srinivasachari, S.; Reineke, T. M. Top. Curr. Chem. 2010, 296, 131−190. (10) Chew, S. A.; Michael, H. C.; Saraf, A.; Raphael, R. M.; Kasper, F. K.; Mikos, A. G. Biomacromolecules 2009, 10, 2436−2445. (11) Wang, Z. H.; Li, W. B.; Ma, J.; Tang, G. P.; Yang, W. T.; Xu, F. J. Macromolecules 2011, 44, 230−239. (12) Kichler, A. J. Gene Med. 2004, 6, S3−S10. (13) Jiang, X.; Lok, M. C.; Hennink, W. E. Bioconjugate Chem. 2007, 18, 2077−2084. (14) Su, W.; Wang, H.; Feng, J.; Luo, X.; Zhang, X.; Zhuo, R. J. Mater. Chem. 2011, 21, 6327−6336. (15) Chiellini, E.; Corti, A.; Antone, S.; Solaro, R. Prog. Polym. Sci. 2003, 28, 963−1014. (16) Matsumura, S.; Tomizawa, N.; Toki, A.; Nishikawa, K.; Toshima, K. Macromolecules 1999, 32, 7753−7761. (17) Wittmar, M.; Ellis, J. S.; Morell, F.; Unger, F.; Schumacher, J. C.; Roberts, C. J.; Tendler, S. J. B.; Davies, M. C.; Kissel, T. Bioconjugate Chem. 2005, 16, 1390−1398. (18) Oster, C. G.; Wittmar, M.; Bakowsky, U.; Kissel, T. J. Controlled Release 2006, 111, 371−381. (19) Evans, R. K.; Xu, Z.; Bohannon, K. E.; Wang, B.; Bruner, M. W.; Volkin, D. B. J. Pharm. Sci. 2000, 89, 76−87. (20) Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Biomed. Mater. Res. 1999, 45, 268−275. (21) Fischer, D.; Bieber, T.; Li, Y.; Elsasser, H. P.; Kissel, T. Pharm. Res. 1999, 16, 1273−1279. (22) Carlotti, S. J.; Beaune, O. G.; Schue, F. J. Appl. Polym. Sci. 2001, 81, 2868−2874. (23) Goyal, R.; Tripathi, S. K.; Tyagi, S.; Ram, K. R.; Ansari, K. M.; Shukla, Y.; Chowdhuri, D. K.; Kumar, P.; Gupta, K. C. Eur. J. Pharm. Biopharm. 2011, 79, 3−14. (24) Tseng, W. C.; Tang, C. H.; Fang, T. Y. J. Gene Med. 2004, 6, 895−905. (25) Lungwitz, U.; Breunig, M.; Blunk, T.; Göpferich, A. Eur. J. Pharm. Biopharm. 2005, 60, 247−266. (26) Neu, M.; Fischer, D.; Kissel, T. J. Gene Med. 2005, 7, 992−1009. (27) (a) Swami, A.; Kurupati, R.; Pathak, A.; Singh, Y.; Kumar, P.; Gupta, K. C. Biochem. Biophys. Res. Commun. 2007, 362, 835−841. (b) Goyal, R.; Tripathi, S. K.; Tyagi, S.; Sharma, A.; Ram, K. R.; Chowdhuri, D. K.; Shukla, Y.; Kumar, P.; Gupta, K. C. Nanomedicine 2011, DOI: 10.1016/j.nano.2011.06.001. (c) Tripathi, S. K.; Goyal, R.; Kumar, P.; Gupta, K. C. Nanomedicine 2011, DOI: 10.1016/ j.nano.2011.06.022. (28) Yang, Y.; Zhang, Z.; Chen, L.; Li, Y. Int. J. Pharm. 2010, 10, 191−197. (29) Patnaik, S.; Tripathi, S. K.; Goyal, R.; Arora, A.; Mitra, K.; Villaverde, A.; Vazquez, E.; Shukla, Y.; Kumar, P.; Gupta, K. C. Soft Matter 2011, 7, 6103−6112. (30) Patnaik, S.; Arif, M.; Pathak, A.; Kurupati, R.; Singh, Y.; Gupta, K. C. Nanomedicine 2010, 6, 344−354. (31) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297−7301. (32) Goyal, R.; Bansal, R.; Tyagi, S.; Shukla, Y.; Kumar, P.; Gupta, K. C. Mol. BioSyst. 2011, 7, 2055−2065. (33) Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug Delivery Rev. 2001, 53, 341−358. (34) Jeong, J. H.; Mok, H.; Oh, Y. K.; Park, T. G. Bioconjugate Chem. 2009, 20, 5−14.

(35) Akhtar, S.; Benter, F. I. J. Clin. Invest. 2007, 117, 3623−3632. (36) Pathak, A.; Kumar, P.; Chuttani, K.; Jain, S.; Mishra, A. K.; Vyas, S. P.; Gupta, K. C. ACS Nano 2009, 3, 1493−1505. (37) Ganesh, K. N.; Sastry, M. J. Ind. Inst. Sci. 2002, 82, 105−112. (38) Lechardeur, D.; Sohn, K. J.; Haardt, M.; Joshi, P. B.; Monck, M.; Graham, R. W.; Beatty, B.; Squire, J.; Brodovich, H. O.; Lukacs, G. L. Gene Ther. 1999, 6, 482−497.

83

dx.doi.org/10.1021/bm201157f | Biomacromolecules 2012, 13, 73−83