Dextran–Peptide Hybrid for Efficient Gene Delivery - Langmuir (ACS

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Dextran−Peptide Hybrid for Efficient Gene Delivery Qiong Tang,† Bin Cao,† Xia Lei,† Bingbing Sun,‡ Yanqiao Zhang,§ and Gang Cheng*,† †

Department of Chemical and Biomolecular Engineering, University of Akron, Akron, Ohio 44325, United States Department of Medicine, University of California at Los Angeles, Los Angeles, California 90095, United States § Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio 44272, United States ‡

S Supporting Information *

ABSTRACT: Gene therapy has drawn significant interest in the past two decades since it provides a promising strategy to treat both genetic disorders and acquired diseases. However, the transfer of gene therapy to clinical applications is troubled with many difficulties, since many current systems are of toxicity, low transfection efficiency and low biodegradability. To address these challenges, we developed a dextran−peptide hybrid system as a safe and efficient vector for gene therapy and investigated the structure−function−cytotoxicity relationship of this dextran−peptide hybrid system. Dextrans (Dex10, Dex20, and Dex70) with different molecular weights (10, 20 and 70 kDa) were conjugated with a cationic peptide, R5H5, at various degrees of substitution. Gene expression and cytotoxicity mediated by this delivery system were evaluated against SKOV-3 human ovarian carcinoma cells and compared to 25 kDa branched poly(ethylenimine) (PEI). The results showed that Dex10−R5H5 and Dex20−R5H5 hybrids derived from low molecular weight dextrans induced higher gene expression and lower cytotoxicity than Dex70−R5H5 hybrid from higher molecular weight dextran. The best performance on gene expression was achieved by Dex10− R5H5 at 40% substitution of R5H5, which induced greater gene expression than PEI at a low N/P ratio of 5. Dex10−R5H5/ DNA complexes at 40% substitution of R5H5 also showed much higher cell viability (93%) than PEI/DNA (66%) at the same N/P ratio. These results indicate that the Dex−R5H5 hybrid with the low molecular weight of dextran and the high degree of substitution of R5H5 is a very promising material for safe and efficient gene therapy.

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transfection in vitro;16 however, many of these systems are of toxicity, low biodegradability and poor biocompatibility in vivo.17,18 Natural materials, such as cationic polysaccharides19 and peptides,20 have been studied as potential gene delivery carriers to avoid the chronic toxicity associated with carriers that are based on synthetic materials. Chitosan has been intensively studied as the gene delivery carrier due to its biocompatibility, but its transfection efficiency is still unsatisfactory and the solubility at the neutral condition is low.21,22 Peptide-based vectors composed of natural amino acids are compatible with biological environment, less cytotoxic and biodegradable. In addition, these vectors are adaptable to rational design since

INTRODUCTION

Gene therapy has drawn significant interest in the past two decades since it is a promising strategy for the treatment of genetic disorders1−3 or acquired diseases4,5 that are currently considered incurable. The success of gene therapy relies on safe and efficient gene delivery systems. These systems must have the following characteristics, DNA protection, cellular uptake, endosomal escape,6 and low toxicity,7,8 to overcome obstacles in gene therapy.9,10 So far, various delivery systems have been developed and they can be grouped into two categories: viral and nonviral vectors. Nonviral vectors are not as efficient as viral vectors but provide safer delivery strategies since they could avoid problems associated with viruses, including complexity of production, immunogenicity, and mutagenesis.11 Currently, the majority of nonviral vectors are made of synthetic materials, such as cationic lipids, polymers and dendrimers.12−15 Some of them can induce high gene © XXXX American Chemical Society

Received: March 7, 2014 Revised: April 16, 2014

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encoding a 5.2 kb firefly luciferase (pCMV-luc) was purchased from Elim Biopharmaceuticals (Hayward, CA). SKOV-3 cell line was purchased from ATCC (St. Cloud, MN) and cultured in DMEM medium supplemented with 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, GlutaMAX, and penicillin−streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Synthesis of Dextran−Peptide Hybrids. Firstly, methacrylatefunctionalized dextran (Dex-MA) was synthesized following a published procedure.34,35 Dextran (500 mg) was dissolved in DMSO (8 mL) in a stoppered 25 mL round-bottom flask under a nitrogen atmosphere. After the dissolution of DMAP (450 mg), a calculated amount of GMA was added. The solution was stirred at room temperature for 4 days, after which the reaction mixture was stopped by adding an equimolar amount of concentrated HCl to neutralize the DMAP. The reaction mixture was dialyzed against deionized (DI) water for 4 days at 4 °C. Then Dex-MA was lyophilized and analyzed with 1H NMR. The conjugation of Dex-MA and peptide R5H5 was carried out in DI water. R5H5 (25 mg) was dissolved in DI water (50 μL) and 200 mM TCEP (19.7 μL) was added to reduce disulfide bonds that may form between two peptides. The mixture was incubated at room temperature for 5 min, followed by the addition of 1 M NaOH (25 μL) to neutralize TCEP and trifluoroacetic acid (TFA) from peptide synthesis. In a separate tube, a calculated amount of Dex-MA was added, followed by the addition of DI water with 3fold weight of Dex-MA, and sonicated for 5 min. The Dex-MA solution was added to the peptide solution and the final pH of the mixture was adjusted to 8.0 by 1 M NaOH. The mixture was incubated at room temperature for 48 h under gentle shake. The Dex−R5H5 hybrid was purified with a Zeba spin desalting column (Thermo Fisher Scientific Inc., Rockford, IL) and analyzed by 1H NMR. The degree of substitution (DS) of R5H5, which is defined as the number of R5H5 peptide chains per 100 dextran glucopyranose residues, was calculated by the integration of the anomeric protons of dextran (5.0 ppm) and the integration of the protons from the imidazole ring of histidine (6.9 and 7.7 ppm). Preparation of Dex−R5H5/DNA Complexes. The Dex−R5H5 hybrid was dissolved in water to make a stock solution at 10 mg/mL. The DNA was prepared at a concentration of 1 mg/mL in nucleasefree water. The Dex−R5H5/DNA complexes were formed by mixing equal volume of Dex−R5H5 and DNA solutions at various N/P ratios and then incubated at room temperature for 20 min to allow the complete electrostatic interaction between peptide and DNA molecules. Here, the N/P ratio is a molar ratio of arginine residue (N) in the peptide to phosphate group (P) in the DNA molecule. The phosphate content of the DNA molecules was estimated based on the assumed 330 g/mol of the average molecular weight of the nucleotides, while the nitrogen content of the peptide molecule was estimated based on the number of arginine residues in each Dex− R5H5 hybrid. Size and Zeta-Potential Measurements. Dex−R5H5/DNA complexes were prepared as described above at different N/P ratios. The size and zeta-potential of Dex−R5H5/DNA complexes were measured by a Malvern Nano-ZS Zetasizer (UK). The size measurements were performed in disposable sizing cuvettes at a laser wavelength of 633 nm and a scattering angle of 173°, while the zeta-potential measurements were performed in disposable zetapotential cells. Before the measurement, Dex−R5H5 complexes were diluted 10 times with PBS and each measurement was repeated for three runs per sample at 25 °C. Gel Retardation Assay. Dex−R5H5/DNA complex solutions were prepared at N/P ratios from 0 to 30. An aliquot (5 μL, 0.5 μg DNA) of each solution was mixed with loading dye and loaded to an agarose gel (0.8%). The loaded gel was exposed to 100 V for 40 min in 0.5 × TBE buffer and was stained in ethidium bromide (0.5 μg/mL) for 30 min and destained with water for 15 min. Then the gel was visualized and documented using a UVP BioDoc-It imaging system (Upland, CA). In Vitro Gene Transfection and Expression. SKOV-3 cells were seeded at a density of 1 × 105 cells/mL onto a 24-well plate (0.5 mL/ well) and incubated for 24 h to reach 80−90% confluence. Then, old

amino acid building blocks with diverse properties provide us the freedom for developing multifunctional drug carriers with desired functions. For example, arginine23 residue which is positively charged in physiological environment has been used to condense negatively charged therapeutic DNA or RNA.24−27 Histidine residue is introduced into peptides to help DNA escape from endosome and thus to improve transfection efficiency.28,29 Our previous study demonstrated that integrated peptides with ligand peptide sequence could achieve targeted gene delivery to the specific cells.30 However, the size of the peptide has to be over a certain value to accommodate all desired functions and it will dramatically increase the cost of the peptide. In general, there are three major obstacles for the further development of natural polymer-based gene carriers. Firstly, all of reported gene drug vectors provide only part of required functions, such as high transfection efficiency, long blood circulation and effective targeting properties. Secondly, the gene transfection efficiency of natural polymer in general is low. Thirdly, drug loading efficiency of these natural polymerbased carriers is low, and thus optimal gene transfection is achieved at relatively high N/P ratios. Our research effort is focused on developing biodegradable, efficient, low toxic and integrated gene delivery systems. A better understanding on the structure−function correlation of natural materials is critical to design more effective gene delivery systems. In this work, dextrans with three different molecular weights (10, 20 and 70 kDa) were conjugated with an oligopeptide NH2-RRRRRHHHHHC-COOH (R5H5) at various degrees of substitution (DS). Dextran, a natural biodegradable polysaccharide, is widely used as polymeric carriers in drug delivery systems.31 The presence of a high amount of hydroxyl groups in dextran enables the efficient incorporation of other functional molecules into the polymer. Dextran conjugated with polyethylenimine (PEI), an efficient transfection reagent, was reported to reduce the toxicity of PEI and increase its stability in the presence of serum.32 Our previous study33 demonstrated that R5H5 could effectively condense nucleic acids binding via arginine residues and facilitate endosomal escape via histidine residues, so it was used here as a DNA loading block of the carrier. The utility of the dextran−peptide hybrids for gene condensation and gene delivery was investigated in a human ovarian carcinoma cell line SKOV-3, in comparison with that of 25 kDa branched PEI.

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MATERIALS AND METHODS

Materials. Dextran (Dex10, Mw 9−11 kDa), Dextran (Dex20, Mw 15−25 kDa), Dextran (Dex70, Mw 64−76 kDa), glycidyl methacrylate (GMA), 4-(dimethylamino)pyridine (DMAP), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and polyethylenimine (PEI, branched, Mw 25 kDa) were purchased from Sigma-Aldrich (St Louis, MO). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Chem-Impex International (Wood Dale, IL). The peptide, NH2-RRRRRHHHHHC-COOH (R5H5) was designed by us and synthesized at >95% purity by GenScript (Piscataway, NJ). Agarose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, nonessential amino acids, sodium pyruvate, GlutaMAX, and Trypsin-EDTA were all purchased from Life Technologies (Carlsbad, CA). Ethidium bromide and nuclease-free water were purchased from EMD Millipore (Billerica, MA). Tris/Borate/EDTA (TBE) was purchased from AMRESCO (Solon, OH). DNA loading dye was purchased from New England Biolabs (Ipswich, MA). Glo lysis buffer and luciferase assay system were purchased from Promega (Madison, WI). BCA protein assay kit was purchased from Thermo Scientific (Waltham, MA). Plasmid DNA B

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Scheme 1. Synthesis of Dex−R5H5

medium was replaced with 450 μL of fresh medium, and 50 μL of the complex solution (containing 2.5 μg DNA) was added into each well. After 4 h of incubation, the transfection medium was removed and replaced by 500 μL culture medium. Cells were incubated for another 68 h. After incubation, cells were washed with PBS and 0.2 mL of 1 × Glo lysis buffer was added to each well to lyse cells. After being incubated at room temperature for 15 min and subjected to two cycles of freezing (80 °C for 30 min) and thawing (room temperature), cell lysates were centrifuged at 14 000 rpm at 4 °C for 15 min to remove cell debris. The supernatant (20 μL) was transferred to a luminometer tube and mixed with 100 μL of luciferase assay reagent, and the luciferase activity was immediately measured for a 10 s read by a Berthold Lumat LB9507 luminometer (Germany). The relative light unit (RLU) reading from the luminometer was normalized by the protein content in the supernatant, which was determined by BCA protein assay. All the experiments were performed with four replicates. Cytotoxicity Assay. The cytotoxicity of Dex−R5H5/DNA complexes was evaluated against SKOV-3 cell line in a 96-well plate, following the procedure of Vybrant MTT Cell Proliferation Assay (Life Technology, Carlsbad, CA). SKOV-3 cells were seeded on a 96well plate and incubated for 24 h. Then the old medium was replaced with 90 μL of fresh culture medium and 10 μL of the complex solution (0.5 μg DNA) was added to each well. The cells were incubated at 37 °C for 4 h. Then the transfection medium was removed and replaced by 100 μL of culture medium, and cells were incubated for another 20 h. The medium was then replaced with 100 μL of the fresh medium and 10 μL of MTT stock solution (5 mg/mL in PBS), and cells were incubated at 37 °C for 4 h. Finally, the medium was removed and 150 μL of DMSO was added to each well to dissolve purple formazan crystals. The absorbance of DMSO supernatant was measured at 540 nm using a Tecan Infinite 200 microplate reader (Switzerland). The cytotoxicity test was performed in eight replicates of each sample. The cells treated with naked DNA were used as control and the cell viability was expressed as a percentage of the control. Statistical Analysis. Data were analyzed using single-factor analysis of variance (ANOVA) and expressed as mean ± standard deviation from 4 to 8 replicates. The comparison among groups was performed using Student’s t test. A p value less than 0.05 was considered statistically significant.

MA (Scheme 1). Then R5H5 containing a C-terminal cysteine residue was conjugated to Dex-MA through a very efficient thiol−acrylate Michael type reaction at slightly basic condition.36 To avoid the steric hindrance on the conjugation and enhance the charge density, cysteine residue was put at the Cterminal of the peptide. This approach does not require the modification of the peptide and it can be used to conjugate other peptide sequences with a terminal cysteine residue. The Dex−R5H5 hybrids were analyzed by 1H NMR (Figures S1− S5). The DS of R5H5 was calculated by integration of the anomeric protons of dextran (5.0 ppm) and the integration of protons from imidazole ring of histidine (6.9 and 7.7 ppm). Because of the overlap with solvent peak in some cases, integration of the anomeric proton peak is very difficult, so the resonance of six protons from dextran backbone (from 3.4 to 4.1 ppm) was used as a reference to verify the accuracy of the calculation. Peaks used for calculations have been assigned in the 1H NMR spectrum as shown in Figure S1. Five Dex−R5H5 hybrids with different molecular weights and DS, Dex10− R5H5(20%), Dex10−R5H5(40%), Dex20−R5H5(10%), Dex20−R5H5(20%) and Dex70−R5H5(10%), were obtained. Particle size and surface charge are two important factors that influence transfection efficiency both in vitro and in vivo. Different Dex−R5H5/DNA complexes were formed at the N/ P ratios from 1 to 30, and their size and surface charge were determined by dynamic light scattering (DLS). As shown in Table 1, the size of all Dex−R5H5/DNA complexes follows a similar trend, which increases at low N/P ratios and then decreases after that. This phenomenon has been observed in many similar systems.30,33,37 The size of carrier/DNA complexes is controlled by two factors, the binding ability of carrier to DNA and the surface charge of complexes, but two factors cannot be completely separated. At low N/P ratios (1 and 5), the electrostatic interaction was not strong enough to form compact complexes due to the low carrier concentration. At higher concentrations, more carrier molecules can condense DNA to form much smaller complexes. Also for particles with net surface charge, the repulsion force between particles prevents them from aggregating. During the transition between the negative and positive surface charge, particles tend to

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RESULTS AND DISCUSSION Dextrans with molecular weights of 10, 20 and 70 kDa were firstly functionalized with methacrylate via transesterification of the methacryloyl group from GMA to dextran, yielding DexC

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vectors was evaluated by the agarose electrophoresis. As shown in Figure 1, Dex20−R5H5/DNA and Dex10−R5H5/DNA

Table 1. Hydrodynamic Size and Zeta Potential of Dex10− R5H5(20%)/DNA, Dex10−R5H5(40%)/DNA, Dex20− R5H5(10%)/DNA, Dex20−R5H5(20%)/DNA, and Dex70− R5H5(10%)/DNA Complexes Carrier Dex10− R5H5(20%)

Dex10− R5H5(40%)

Dex20− R5H5(10%)

Dex20− R5H5(20%)

Dex70− R5H5(10%)

N/P

size (d, nm)

SD (±d, nm)

zeta (mV)

SD (±mV)

1 5 10 20 30 1 5 10 20 30 1 5 10 20 30 1 5 10 20 30 1 5 10 20 30

245 377 306 304 276 86 263 345 276 256 203 426 334 307 291 102 310 295 269 268 262 287 221 199 184

17 10 25 24 26 4 26 33 25 25 20 31 21 21 27 18 17 22 24 23 9 27 20 9 10

−10.2 5.2 6.0 6.5 6.9 −15.2 6.8 5.7 9.7 10.9 −6.2 0.3 4.2 5.3 5.3 −13.7 4.3 8.7 8.7 9.3 −0.1 2.0 3.0 2.9 3.8

1.1 0.6 0.5 0.1 0.5 0.1 0.8 0.2 0.4 0.3 0.5 0.7 0.6 0.7 0.7 0.4 0.2 0.7 0.2 0.6 0.3 0.6 0.5 0.6 0.2

Figure 1. Electrophoretic mobility of DNA in Dex10−R5H5(20%)/ DNA (A), Dex10−R5H5(40%)/DNA (B), Dex20−R5H5(10%)/ DNA (C), Dex20−R5H5(20%)/DNA (D), and Dex70−R5H5(10%)/DNA (E) complexes at N/P ratio of 0, 1, 5, 10, 20 and 30, respectively.

aggregate due to weaker repulsion forces. In this system, the transitional point of the particle surface charge appeared between N/P ratio 1 and 5. Among all tested N/P ratios of Dex−R5H5 vectors, smallest particles were achieved at the N/P ratio of 30. For the vector with Dex70, the particle size was 184 nm, which was much smaller than that of the vector system with Dex10 or Dex20. It indicates that Dex-R5H5 with higher molecular weight (70 kDa) dextran has better condensing ability than these with lower molecular weight dextrans (20 and 10 kDa). Table 1 shows that the zeta-potential of complexes increases dramatically from the N/P ratio of 1 to 5 and does not change much above the N/P ratio of 5. The zeta-potential of Dex70−R5H5/DNA complexes was lower than that of Dex20−R5H5/DNA or Dex10−R5H5/DNA complexes at the same N/P ratio. This phenomenon can be explained with the low DS (10%) of R5H5 in Dex70−R5H5. For Dex20−R5H5 or Dex10−R5H5 with different DS (10%−40%), the zetapotential of Dex−R5H5/DNA complexes at higher DS was higher than that of complexes at lower DS. It was reported that the net positive charge of carrier/nucleic acid complexes facilitates their cellular uptake because of the interaction of the negatively charged cell membrane and positively charged complexes. However, if the charge density of complexes is too high (>30 mV), it might induce cellular toxicity38 and reduce their blood circulation time and specificity to target cells due to the elevated nonspecific binding to serum protein and cells. The results show that Dex−R5H5 vesicles can efficiently condense DNA to the desired size and surface potential in vitro. Dex−R5H5/DNA complexes were formed at different N/P ratios (1 to 30), and the DNA binding ability of Dex−R5H5

complexes formed at the N/P ratio of 1 traveled slightly slower than the naked DNA across the gel, which indicated the incomplete retardation of DNA at the N/P ratio of 1, and the complete retardation of DNA was achieved at the N/P ratio of 5. For Dex70−R5H5(10%), the complete retardation of DNA was achieved even at the N/P ratio of 1, which indicated that Dex70−R5H5 had better condensing ability than Dex10− R5H5 and Dex20−R5H5 hybrids. This result agrees with the result observed above in the section of size and zeta-potential of Dex−R5H5/DNA complexes. To study whether Dex−R5H5 hybrids as gene carriers can enhance the transgene expression, in vitro transfection efficiency of five Dex−R5H5 hybrids were evaluated in a model cancer cell line, SKOV-3. For all Dex10−R5H5 and Dex20−R5H5 hybrids, high luciferase expression levels (Figure 2A) were achieved at the N/P ratio of 1, which are about 3 orders of magnitude greater than that of naked DNA. Besides, as the N/P ratio increased from 1 to 30, the luciferase expression level increased initially and then followed a decreasing trend. For the Dex10−R5H5 system, the highest expression level of Dex10− R5H5(20%) was achieved at the N/P of 5 (9.4 × 108 RLU/mg protein). The Dex10−R5H5(40%) induced 50% higher luciferase expression with a value of 7.3 × 109 RLU/mg protein than PEI/DNA at the same N/P ratio (5). The highest expression level of Dex10−R5H5(40%) was achieved at the N/ P ratio of 20 and it is 90% higher than PEI/DNA. The highest expression levels of Dex20−R5H5 at 10% and 20% DS were both achieved at the N/P of 10, 3.0 × 109 and 3.5 × 109 RLU/ D

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transcription of the luciferase gene carried by the plasmid DNA.40 Secondly, the higher zeta-potential of Dex−R5H5/ DNA complexes derived from low molecular weight dextrans leads to an increased interaction with cell surfaces and cellular uptake. Thirdly, Dex10−R5H5 and Dex20−R5H5 hybrids possess higher buffering capacity and subsequently lead to more effective lysosomal escape, because Dex10−R5H5 and Dex20− R5H5 with higher DS (10−40%) of R5H5 contains more imidazole groups compared to Dex70−R5H5 (10%). As discussed above, the low molecular weight backbone and high DS of R5H5 in the Dex−R5H5 system are favorable for gene transfection. Cytotoxicity of Dex−R5H5/DNA complexes was determined against SKOV-3 cells using the MTT assay, and compared to that of naked DNA as a negative control as well as PEI/DNA (N/P 5) as a positive control. The DNA concentration was 5 μg/mL for all samples. Dex10−R5H5/ DNA and Dex20−R5H5/DNA complexes lead to higher cell viability (above 80%) at N/P ratios between 1 and 10 and generally follow a decreasing trend as the N/P ratio increases from 1 to 30 (Figure 3A). In particular, Dex10−R5H5(40%)/ DNA complexes induced the lowest cytotoxicity among the five Dex−R5H5 systems and its cytotoxicity at all tested N/P ratios (1 to 30) was lower than that of 25 kDa PEI at the N/P ratio of 5. At the N/P ratio of 5, Dex10−R5H5(40%)/DNA complexes showed much higher cell viability (93%) than PEI/DNA complexes at the same N/P ratio. In contrast, as shown in Figure 3B, Dex70−R5H5(10%)/DNA complexes at the low N/ P range, from 1 to 15, induced relatively low cell viability, which was 60−70%. The results indicate that the Dex70 system is more toxic to cells than Dex10 and Dex20 systems. Similar results have been reported in other polycation systems,41−43 in which the cytotoxicity is as a function of the molecular weight. It should be noted that, the cytotoxicity (below 20%) of all five Dex−R5H5 hybrids/DNA complexes at the N/P of 5 is lower than that (34%) of PEI (25 kDa)/DNA complex at the same N/P ratio. We expect that Dex−R5H5 hybrids will have even lower in vivo toxicity than PEI. The further study is planned to investigate the in vivo transfection efficiency and cytotoxicity of Dex−R5H5 hybrids.

Figure 2. Luciferase expression levels induced by (A) Dex10− R5H5(20%)/DNA (right-slanted lines), Dex10−R5H5(40%)/DNA (left-slanted lines), Dex20−R5H5(10%)/DNA (crossed lines), and Dex20−R5H5(20%)/DNA (horizontal lines) and (B) Dex70−R5H5/ DNA in SKOV3 cell line. DNA and PEI−DNA complex at an N/P ratio of 5 are used as negative and positive controls, respectively. Error bars represent standard deviation of four replicates. *, **P < 0.05.

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CONCLUSIONS In this study, we designed and synthesized a polysaccharide− peptide (Dex−R5H5) hybrid platform for gene delivery. We also developed an understanding on how the molecular weight of the dextran backbone and the degree of substitution of cationic peptide side chains affect the transfection efficiency and cytotoxicity of Dex−R5H5 hybrids. All Dex−R5H5 hybrids showed strong DNA binding ability at low N/P ratios. Dex− R5H5 hybrids derived from low molecular weight dextrans (10 and 20 kDa) have greater gene transfection efficiency and lower cytotoxicity than that with high molecular weight dextran (70 kDa). The best performance on gene expression was achieved by Dex10−R5H5 at 40% DS, which induced the greater gene expression than that of PEI/DNA at the N/P ratio of 5, and it also had much higher cell viability (93%) than that of PEI/ DNA (66%). These results indicate that the Dex10−R5H5 hybrid at 40% DS may be a promising delivery system for safe and efficient gene therapy. Moreover, this dextran-based delivery system is designed as a versatile platform, which can be readily adapted to incorporate other functional groups to achieve dramatically improved therapeutic effects in the gene therapy.

mg protein, respectively, which are comparable to the luciferase expression (4.8 × 109 RLU/mg protein) induced by PEI/DNA at the N/P ratio of 5. Figure 2B shows that the Dex70− R5H5(10%)/DNA complexes induced relatively lower luciferase expression than other Dex−R5H5/DNA complexes derived from low molecular weight dextrans (20 and 10 kDa). The luciferase expression level of Dex70−R5H5(10%)/DNA complexes increased slightly as the N/P ratio increased from 1 to 15, and the luciferase expression level achieved at the N/P of 15 was 3 orders of magnitude lower than that of PEI/DNA. The low transfection efficiency of Dex70−R5H5(10%)/DNA complexes could be caused by its stronger DNA condensing ability, which leads to inefficient unpacking and release of condensed DNA.39 Our study demonstrated that Dex−R5H5 vector systems with low molecular weight dextrans (10 and 20 kDa) had greater gene transfection efficiency than that with higher molecular weight dextran. This increased efficiency could be attributed to three factors observed in this study. Firstly, the weaker DNA condensation of Dex10−R5H5 and Dex20−R5H5 hybrids could facilitate the disassembly of Dex− R5H5/DNA complexes in nuclei, which is critical for E

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Figure 3. Cell viability of SKOV3 cell line after incubation with (A) Dex10−R5H5(20%)/DNA (right-slanted lines), Dex10−R5H5(40%)/DNA (left-slanted lines), Dex20−R5H5(10%)/DNA (crossed lines), and Dex20−R5H5(20%)/DNA (horizontal lines) complexes and (B) Dex70−R5H5/DNA at different N/P ratios compared to DNA and PEI−DNA complex at an N/P ratio of 5. DNA concentration is 5 μg/mL. Error bars represent standard deviation of eight replicates. *, **P < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (G.C.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by US National Science Foundation (NSF) Grant DMR-12062923. REFERENCES

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