Effect of Proteins with Different Isoelectric Points on the Gene

May 16, 2013 - In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporat...
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Effect of Proteins with Different Isoelectric Points on the Gene Transfection Efficiency Mediated by Stearic Acid Grafted Chitosan Oligosaccharide Micelles Jingjing Yan,† Yong-Zhong Du,*,† Feng-Ying Chen,‡ Jian, You,† Hong Yuan,† and Fu-Qiang Hu*,† †

College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, People’s Republic of China Women Hospital, School of Medicine, Zhejiang University, 2 Xueshi Road, Hangzhou 310006, People’s Republic of China

‡

ABSTRACT: A stearic acid-grafted chitosan oligosaccharide (CSSA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection, and excellent cellular internalization, a CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with a different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into a CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/ BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However, referring to the transfection activity, the BSA of different isoelectric point value (pI) had a distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA(4.7)/DNA complexes which showed the highest transfection efficiency. On the contrary, CS-SA/BSA(9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA(4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes, and the study provided guidelines in the design of an efficient vector for DNA transfection. KEYWORDS: chitosan, micelle, bovine serum albumin, isoelectric point, gene release, transfection efficiency

1. INTRODUCTION Successful clinical application of gene therapy requires efficient and safe gene delivery vectors that deliver the therapeutic genes to a specific target tissue or organ.1,2 Viral vectors are very effective in achieving high transfection efficiency, but their application in the human body is often frustrated by safety problems, immunogenicity, low transgenic size, and high cost.3,4 Nonviral vectors are emerging as an alternative to the use of viral vectors with the potential to have a significant impact on clinical therapies.5 For biodegradability, biocompatibility, low immunogenicity, and possibility for large-scale preparation and further modification, a great number of materials have been synthesized and evaluated as a gene vector.6−8 However, the strategy to enhance transfection efficiency has been a topic of debate. Gene delivery is a complex process and should overcome several potential hurdles, such as efficient cellular entry,9 protection from nuclease degradation,10 endosomal escape,11 vector unpacking, and nuclear transport.12,13 Efforts to improve transfection efficiency need to be based on a detailed identification of rate-limiting barriers to the specific gene delivery system.14 Chitosan (CS), as a cationic natural © 2013 American Chemical Society

polysaccharide, can condense negatively charged DNA by ionic interactions and has been considered to be a good gene carrier candidate.15 However, the transfection efficiency is still not satisfied due to the strength of electrostatic interaction between CS and DNA which prevents their dissociation within cells. The general consensus is that a balance between sufficient DNA protection and intracellular DNA release from the complex is required for a chitosan-based gene delivery system.16,17 For this purpose, numerous efforts have been made, and several protein or amino acids have been adopted to enhance the transfection efficency of CS based gene delivery system. For example, one approach is to degradate CS by incorporating chitosanase, which would promote gene unpacking, consequently increasing gene expression.18 Another approach is to modify the internal structure of CS/DNA complexes by incorporating a negatively charged poly(gglutamic acid).19 However, we think the charge of protein or Received: Revised: Accepted: Published: 2568

December 28, 2012 April 30, 2013 May 16, 2013 May 16, 2013 dx.doi.org/10.1021/mp300732d | Mol. Pharmaceutics 2013, 10, 2568−2577

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dimethylaminopropyl) carbodiimide (EDC). CS (17.5 kDa; 1.0 g) was dissolved in 60 mL of superpurified water, and SA with EDC was dissolved in 40 mL of ethanol by sonicate treatment, followed by stirring in water bath at 60 °C for 30 min. The carboxyl groups of SA were activated by EDC. The activated SA was added into CS solution under vigorous stirring. After mixing the solutions, the reaction was conducted for another 4 h at 80 °C. The final reactant was dialyzed by employing dialysis membrane (molecular weight cutoff 7 kDa, Spectrum Laboratories, Laguna Hills, CA) against superpurified water for two days to remove the water-soluble byproducts, followed by freeze-drying. The lyophilized dialyzed product was then washed thrice with ethanol to remove unreacted SA. Finally, the product CS-SA was dispersed in deionized water and lyophilized. 2.2.2. Characterization of CS-SA. Substitute degrees of amino groups (SD %), defined as the number of SA groups per 100 amino groups of CS, were determined by the 2,4,6trinitrobenzene sulfonic acid (TNBS) method. A sample of 2.0 mL of CS-SA solution or CS solution with content of 0.1 mg/ mL was incubated with 2.0 mL of 4% NaHCO3 and 2.0 mL of 0.1% TNBS at 37 °C for 2 h. The UV absorbance at 344 nm of all the samples was determined by the UV spectrophotometer (TU-1800PC, Beijing Purkinje General Instrument Co., Ltd., China) after the addition of 2.0 mL of HCl (2 N). The amount of SA groups substituted was calculated from the UV absorbance of CS-SA and unmodified CS. The critical micelle concentration (CMC) of the CS-SA in deionized water was determined by pyrene fluorescence method using a fluorometer (F-2500, Hitachi Co., Japan). A serial of sample solutions with CS-SA concentration range from 1.0 mg/mL to 10−4 mg/mL were sonicated at room temperature for 30 min. The solutions contain pyrene (6.0 × 10−7 mol/L). The slit openings were set at 10 nm (excitation) and 2.5 nm (emission), and the excitation wavelength was 337 nm. The CMC of CS-SA was calculated by intensity ratio of the first highest energy bands to the third in the pyrene emission spectra. 2.3. Synthesis of BSA with Different Isoelectric Points. BSA of different pI was obtained by blocking carboxyl with methanol catalyzed by acetyl chloride. Briefly, 0.5 g of BSA was added into 63.0 mL of anhydrous methanol, followed by stirring at room temperature in a fume hood. Then 0.5 mL of acetyl chloride was added dropwise into the reaction system. After stirred for 10 min or 4 h at room temperature, the resulting product was purified by dialysis (molecular weight cutoff 7 kDa, Spectrum Laboratories, Laguna Hills, CA) against superpurified water for 2 days, followed by lyophilization. The isoelectric point is determined by the pH value of BSA solution at which the zeta-potential is approximately zero.24 Briefly, the above synthesized BSA samples were dissolved in a series of phosphate buffered solutions with different pH values, then the zeta potential was measured by dynamic light scattering (Zetasizer 3000HS, Malvern Instruments Ltd., UK) at a fixed protein concentration of 1 mg/mL. The solution pH values in these studies were chosen based on their relevance to the isoelectric point (pI) of the protein. 2.4. Preparation of CS-SA/DNA and CS-SA/BSA/DNA Nanoparticles. Plasmid DNA (2 μg) and BSA (with various WBSA/WCS‑SA ratios range from 0 to 0.5) were mixed vortically, followed by incubation for 5 min at room temperature. The CSSA/DNA nanoparticles were prepared by adding CS-SA micelles to BSA and DNA mixtures with desired amounts at various N/P ratios. The N/P ratios of chitosan or its derivative/

amino acids cannot be overlooked, which may also affect the strength of charge-based interactions between CS and DNA. In our previous study, a novel amphiphilic-grafted copolymer, that is, stearic acid-grafted chitosan copolymer (CS-SA), was applied to gene delivery.20 CS-SA could self-aggregate to form micelle in aqueous solution and condense DNA into nanoparticles easily, which exhibits good nuclease degradation protection ability and excellent internalization into cancer cells.21 Protamine could successfully improve the gene expression of CS-SA micelle by the formation of a novel virus-like vector.22 Also, effective antitumor gene therapy can be achieved using a polyethylenimine-conjugated stearic acid-gchitosan micelle based gene delivery system.23 However, the difficulty in polymer/DNA complexes dissociation should be overcome to obtain a high gene transfection efficiency. Bovine serum albumin (BSA) was chosen as a model protein to improve the CS-SA/DNA complexes dissociation and transfection efficiency. BSA as a naturally occurring protein maintains negative charges in a physiological environment. After carboxyl groups were blocked with methanol catalyzed by acetyl chloride, BSA of different pI was obtained, accompanied with charges altered. In this research, CS-SA micelles were taken as the gene vector, and BSA of different pI were incorporated to form CS-SA/BSA/DNA complexes. The trasfection capability was evaluated by inverted fluorescence microscope and flow cytometry. The cellular uptake and intracellular trafficking were observed by a confocal laser scanning microscope (CLSM). The study focused on the influence of three kinds of BSA of different pI on the gene delivery mediated by CS-SA micelles and investigated the possible mechanism in intracellular trafficking of CS-SA/BSA/ DNA complexes.

2. MATERIALS AND METHODS 2.1. Materials. Chitosan oligosaccharide (Mw = 17.5 kDa, 95 % deacetylated degree) was obtained by enzymatic degradation from chitosan (Mw = 450.0 kDa).20 Stearic acid was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2,4,6-trinitrobenzene sulfonic acid (TNBS), fluorescein isothiocyanate (FITC), and 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Pyrene was purchased from Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). Lipofectamine 2000 was supplied from Invitrogen Corporation (Carlsbad, CA, USA). DNase I, Dulbecco’s modified Eagle’s medium, and trypsin-ethylenediamine tetraacetic acid were purchased from Gibco BRL (Gaithersburg, MD). Fetal bovine serum was purchased from Sijiqing Biologic Co. Ltd. (Zhejiang, China). EGFP-C1 plasmid as the GFP gene, transformed in Escherichia coli DH5a, was donated from the First Affiliated Hospital of College of Medicine, Zhejiang University (Hangzhou, China). Tetramethylrhodamine (TAMRA) labeled DNA was purchased from Sangon Biotech (Shanghai) Co., Ltd. (China). The DNA was an antisense oligonucleotide LY2181308 (sequence: 5′-TGTGCTATTCTGTGAATT-3′). All other chemicals were of analytical grade and used without further purification. 2.2. Synthesis and Characterization of Stearic Acid Grafted Chitosan Copolymer. 2.2.1. Synthesis of CS-SA. Stearic acid (SA) grafted chitosan (CS) copolymer (CS-SA) was synthesized via the reaction of carboxyl groups of SA with primary amino groups of CS catalyzed by 1-ethyl-3-(32569

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80% confluence. Then, the media were replaced with fresh growth media containing CS-SA/DNA and CS-SA/BSA/DNA nanoparticles and incubated for 48 h, 72 h, or a longer time. For comparison, Lipofectamine 2000 at the weight ratio of 2 was used as the positive control. Untransfected cells and cells transfected with naked pDNA (2 μg/well) were used as a negative control. All transfection experiments were performed in triplicate. The cells were taken to an inverted fluorescence microscope (Leica DMI 4000B, Leica, Germany) for imaging. To quantitatively study the transfection efficiency, HEK293 cells were collected, and approximately 1.0 × 104 cells were counted and measured with a flow cytofluorometer (FC500MCL, Beckman Coulter, USA). 2.10. Cellular Uptake and Intracellular Trafficking. FITC-labeled CS-SA (FITC-CS-SA) was synthesized as the methods described in the literature.25 To remove the unconjugated FITC, the synthesized FITC-CS-SA was dialyzed in the dark against deionized water followed by freeze-drying. Test complexes of FITC-labeled CS-SA and TAMRD-labeled DNA were then prepared as described in Section 2.4 to track the internalization of complexes by CLSM. Cells were seeded on 12-well plates with a sterile coverslip at 1 × 105 cells/well. When cells have reached the desired confluence, the culture medium was removed, and the fresh medium containing fluorescence-labeled complexes was added. After incubation for an adequate time, cells were washed twice with the PBS and then treated with 50 nM Lysotracker (LysoTracker Blue DND22, Invitrogene, USA) for 30 min at 37 °C following the supplier’s protocol. The nucleus was stained with DAPI. Finally, the fixed cells were examined under a CLSM (IX81-FV1000, Olympus, Japan). 2.11. Statistical Analysis. Results were reported as means ± standard deviation. A paired t test was applied to assess the statistical difference among different groups. Differences were considered statistically significant when p < 0.05.

pDNA complexes were expressed as the molar ratios of amine group of chitosan or its derivative to phosphate group of DNA. Complexes were vortexed gently and then allowed to incubate for 30 min at 37 °C before use. 2.5. Measurement of Particle Size and Zeta Potential. The particle size and zeta potential of CS-SA micelles, CS-SA/ DNA, and CS-SA/BSA/DNA nanoparticles were measured by dynamic light scattering (Zetasizer 3000HS, Malvern Instruments Ltd., UK). Distilled water was used for preparation of the micelles with a final CS-SA concentration of 1 mg/mL. Complexes at different WBSA/WCS‑SA ratios at an N/P ratio of 10 were prepared. The volume was 2.0 mL for each sample with a final DNA concentration of 10 μg/mL. Each sample determination was done in triplicate. 2.6. TEM Observation. The morphology of CS-SA micelles, CS-SA/DNA, and CS-SA/BSA/DNA complex nanoparticles was examined by transmission electronic microscopy (TEM, STEREOSCAN, LEICA, England). N/P ratios were 10, and WBSA/WCS‑SA ratios were 0.1. A drop of nanoparticle dispersions was dropped onto a copper grid without any staining. The air-dried samples were then directly observed under TEM. 2.7. Agarose Gel Electrophoresis Experiments. A gel retardation assay was performed for the examination of the condensation ability of polymer with DNA by electrophoresis. CS-SA/DNA and CS-SA/BSA/DNA (pEGFP-C1, 1 μg) complexes were prepared at various WBSA/WCS‑SA ratios ranging from 0.1 to 0.5 by incubating at room temperature for 30 min. The final volume of the complexes was unified to 18 μL prior to loading. Electrophoresis was then carried out with a current of 120 V for 30 min in TAE buffer solution (40 mM Tris−HCl, 1% (v/v) acetic acid, 1 mM EDTA). DNA retardation was visualized by the staining of ethidium bromide with a UV lamp. 2.8. Cell Lines, Cell Culture, and MTT Assay. HEK293 (human embryonic kidney cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heatinactivated fetal bovine serum (FBS with 1% penicillin/ streptomycin). All cells were cultured under standard incubation conditions at 37 °C with 5% CO2. Evaluation of the in vitro cytotoxicity of CS-SA was carried out in HEK293 and compared with the cytotoxicity of Lipofectamine 2000 by MTT assay. Cells were seeded in 96-well plates at an initial density of 1 × 104 cells/well in 200 μL of DMEM complete medium. After 24 h growth, the original media were replaced with 200 μL of polymer containing fresh medium at concentrations ranging from 20 μg/mL to 400 μg/mL. After 48 h under standard incubation conditions, 20 μL of MTT reagent (an MTT stock solution of 5 mg/mL) solution was added to each well and incubated for an additional 4 h. The medium was then removed, and 200 μL of DMSO was added to each well to dissolve the formed purple formazan crystals. The plates were mildly shaken for 10 min to ensure the dissolution of formazan. The absorbance value was measured using a microplate reader (BioRad, model 680, USA). Three replicates were counted for each sample. The mean value was used as the final result. For the CS-SA/BSA/DNA with different isoelectric point groups, cells were treated with complexes containing 2 μg/well DNA (N/P = 10) and incubated at standard culture conditions and subjected to the MTT assay as described above. 2.9. GFP Gene Transfection. HEK293 cells were seeded in 24-well plates at a density of 1 × 105 cells per well in 1 mL DMEM and incubated for 24 h, yielding a cell density of about

3. RESULTS 3.1. Synthesis and Characterization of CS-SA. CS-SA was synthesized by the coupling reaction of SA and CS according to the previous reports.20 As shown in Table 1, the Table 1. Characteristics of Synthesized CS-SA

material

theoretical substitution of stearic acid (%)

degree of grafted SA (%)

CMC (μg/L)

size (nm)

zeta potential (mV)

CS-SA

20

7.4

81.0

36.4 ± 0.9

32.7 ± 1.2

actual substitution degree (SD) of stearic acid was measured by TNBS assay to 7.4%. CS-SA micelles were easily prepared by dispersing CS-SA in distilled water due to its inherent selfaggregation in an aqueous environment. The aggregation behavior of CS-SA in aqueous vehicle was investigated by fluorescence spectroscopy with pyrene as a fluorescent probe.26 From Table 1, the low CMC value (81.0 μg/L) meant that CSSA showed excellent self-assembly properties and could keep the core−shell structure even under highly diluted conditions. Size and zeta potential are two important factors which influence DNA compact ability, complex stability, and the efficiency of cell uptake and therefore influence the transfection efficiency of complexes. The size and zeta potential of CS-SA micelles formed in aqueous medium with concentration of 1.0 mg/mL were determined as 36.4 ± 0.9 nm and 32.7 ± 1.2 mV 2570

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(Table 1), respectively. Figure 1 shows the distribution of size and zeta potential of CS-SA micelle with a 1.0 mg/mL

Figure 1. Distribution for number average size and zeta potential of CS-SA micelles with a 1.0 mg/mL concentration.

concentration. It can be seen the CS-SA micelles had relatively a narrow distribution of size and zeta potential. 3.2. Synthesis and Characterization of BSA with Different Isoelectric Point Value. Bovine serum albumin (BSA) is one of the most widely studied proteins and is the most abundant protein in plasma. The isoelectric point value of BSA is 4.7.27 After reacting with methanol in the presence of acetyl chloride, the carboxyl was esterified, and the isoelectric point increased. BSA with different isoelectric point value was obtained by controlling the reaction time such as 10 min or 4 h. The isoelectric point is given by the pH value at which the zeta potential is approximately zero. At a pH near the isoelectric point, the protein molecules are usually quite unstable and tends to aggregate.24 It is worth mentioning that the protein concentration used in our experiments was fixed at 1.0 mg/mL. This concentration was found to be adequate to obtain reproducible data. The dependence of the zeta potential on pH is shown in Figure 2. As can be seen in Figure 2A, the zeta potential of BSA decreased from 13.7 mV at pH 1.5 to −4.8 mV at pH 7.2. For 10 min synthetic product, the zeta potential was 21.2 mV at pH 1.42 and −4.0 mV at pH 7.2 (Figure 2B). As shown in Figure 2C, the zeta potential of 4 h synthetic product decreased from 20.8 mV at pH 5.3 to −10.3 mV at pH 12.3. Zeta potential values were close to zero at pH 4.7 for BSA,27 at pH 6.0 for 10 min synthetic product, and at pH 9.3 for 4 h synthetic product. Those proteins hereafter were referred to as BSA(4.7), BSA(6.0), and BSA(9.3), respectively. 3.3. Preparation and Characteristics of Gene Delivery Complexes. For efficient cellular endocytosis and gene transfer, the complex of gene vector and DNA should be small and compact.28 Due to the positive charge, the CS-SA micelle was used to condense the DNA forming CS-SA/DNA and CS-SA/BSA/DNA complex nanoparticles. The sizes of both binary complexes and ternary complexes were determined by DLS. The number average sizes of CS-SA/DNA, CS-SA/ BSA(4.7)/DNA, CS-SA/BSA(6.0)/DNA, and CS-SA/ BSA(9.3)/DNA complexes were 74.8 ± 9.6 nm, 68.5 ± 8.1 nm, 61.6 ± 6.3 nm, and 90.3 ± 3.1 nm, respectively (WBSA/ WCS‑SA ratios were fixed as 0.1). It was found that binary complexes were appreciably larger than CS-SA/BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA complexes but smaller than CSSA/BSA(9.3)/DNA. Transmission electronic microscopy (TEM) was then used to examine the morphologies of micelles, binary and ternary complexes, the results are indicated

Figure 2. Zeta potential of BSA in phosphate buffers as a function of pH for the different ester forms of the protein: BSA(4.7) (A), BSA(6.0) (B), and BSA(9.3) (C).

in Figure 3a−e. It could be observed that the CS-SA micelles, CS-SA/DNA, CS-SA/BSA(4.7)/DNA, and CS-SA/BSA(6.0)/ DNA complexes were spherical particles, in addition to CS-SA/ BSA(9.3)/DNA complexes being an irregular shape. All of the sizes observed for TEM images corresponded to the results determined by DLS. The binding capacity of CS-SA with DNA prepared at various N/P ratios was evaluated using the gel retardation assay, and the results are shown in Figure 4a. When the N/P ratio of CS-SA to plasmid DNA reached 2, the migration of DNA was retarded completely. It suggested that when the weight ratio of CS-SA micelles to plasmid DNA was or above 2, stable complexes of CS-SA micelles and plasmid DNA were formed. Therefore, the preparation of test NPs was carried out using an N/P ratio of 2 in the subsequent experiment. Figure 4b shows the gel retardation result of ternary complexes incorporating three kinds of BSA with different WBSA/WCS‑SA ratios. No DNA release was observed even when the WBSA/WCS‑SA was increased to 0.5. It is clear that the CS-SA micelles have powerful DNA compactability even when protein was incorporated. 3.4. Gene Expression Level. To investigate the effect of BSA of different pI values on the gene transfection efficiency mediated by stearic acid grafted chitosan micelles, the in vitro transfection capability of CS-SA/BSA/DNA complexes with BSA was carried out by a green fluorescent protein assay. pEGFP-C1 was used as a reporter gene, and the cells were directly visualized by an inverted fluorescence microscope. The fluorescence images of green fluorescence protein expression of CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes on HEK293 are shown in Figure 5. Lipofectamine 2571

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Figure 3. Transmission electron microscopy images of CS-SA micelles, CS-SA/DNA and CS-SA/BSA(4.7)/DNA, CS-SA/BSA(6.0)/DNA, and CSSA/BSA(9.3)/DNA complex nanoparticles (WBSA/WCS‑SA ratio = 0.1).

Figure 4. (a) Gel retardation analyses of CS-SA/DNA complex nanoparticles prepared at different N/P ratios. (b) Gel retardation analyses of CSSA/BSA/DNA nanoparticles with three kinds of BSA prepared at different WBSA/WCS‑SA ratios.

analyzed by FACS 72 h after transfection, and the percentage of cells expressing the GFP was determined. As shown in Figure 6, the addition of BSA(4.7) at WBSA/WCS‑SA ratio of 0.05 and 0.1 increased significantly the transfection efficiency mediated by CS-SA (11.5%); particularly, the WBSA/WCS‑SA ratio of 0.1 was found to be optimal (25.2%), which approached that of Lipofectamine 2000/DNA (31.1%). For the BSA(6.0) at WBSA/ WCS‑SA ratio of 0.05 and 0.1, a slightly increase of transfection efficiency was observed. BSA(9.3) decreased the transfection efficiency at all WBSA/WCS‑SA ratios tested compared to CS-SA/ DNA binary complexes. Furthermore, all of the transfection results for BSA(4.7) except WBSA/WCS‑SA = 0.5 were higher than those of BSA(6.0) and BSA(9.3). This results could contribute the lower PI for BSA(4.7). When the WBSA/WCS‑SA =

2000 was used as the positive control. It showed that CS-SA/ BSA(4.7)/DNA and CS-SA/BSA(6.0)/DNA owned higher gene expression than CS-SA/DNA on HEK293 cells when the WBSA/WCS‑SA ratio was low (0.05 and 0.1). CS-SA/BSA(4.7)/ DNA complexes showed the best performance, especially at the WBSA/WCS‑SA ratio of 0.1. The transfection efficiency of CS-SA/ BSA(6.0)/DNA was just slightly increased. On the contrary, CS-SA/BSA(9.3)/DNA complexes had the lowest transfection efficiency, and the gene expression decreased obviously with an increase in the amount of BSA(9.3) incorporated. The fluorescence of HEK293 cells treated with naked plasmid DNA was too low to be detected. In order to directly compare the effect of proteins with different pI on the gene transfection efficiency, the cells were 2572

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Figure 5. Green fluorescent protein expression of transfected HEK293 cells observed by inverted microscopy. Cells were transfected in vitro using CS-SA/DNA and CS-SA/BSA/DNA complexes prepared at different WBSA/WCS‑SA ratios for 72 h, in which N/P is 10. Naked DNA and Lipofectamine 2000 were used as negative control and positive control.

were incubated with HEK293 cells for 4, 8, and 12 h, respectively. Complexes were internalized by most of cells at 4 h. An apparent fluorescence can be detected in the nucleus at 12 h. The percentage of cells that internalized FITC-labeled complexes and their fluorescence intensity were quantified by flow cytometry. As shown in Figure 7b and c, the cellular uptake was time-dependent in both binary and ternary complexes. Test complexes were found to be internalized in almost 100% of cells at 12 h. However, a noteworthy feature was that there were no significant differences between the percentage of fluorescent cells and their fluorescence intensity of CS-SA/DNA and CS-SA/BSA(4.7)DNA. It suggested that BSA(4.7) may not affect the DNA transport capacity of CS-SA. These observations indicated that the facilitating role of BSA(4.7) lies downstream of cellular uptake. To track complexes following their uptake, colocalization between FITC-CS-SA, TAMRA-DNA, and labeled lysosomes was assessed by confocal microscopy. As showed in Figure 8, CS-SA and DNA showed a generalized punctate pattern for CS-SA/DNA at 24 h. The merged images showed the apparent colocalization of test complexes with lysosomes, which showed that most of complexes were entrapped within the lysosomal vesicles. However, very little colocalization with lysosomes was observed for the group treated with CS-SA/BSA(4.7)/DNA complexes. CS-SA and DNA separated in the cytoplasm, and a large amount of DNA was transported into nucleus, indicating that CS-SA/BSA(4.7)/DNA complexes could disassemble for DNA dissociation and enter into the nucleus.

Figure 6. Percentages of transfected cells determined by FACS. Cells were transfected in vitro using CS-SA/DNA and CS-SA/BSA/DNA complexes for 72 h. The N/P was 10, and WBSA/WCS‑SA ratios were 0.05, 0.1, and 0.05. (n = 3, error bars represent s.d.). #p < 0.05 comparing CS-SA/BSA(4.7)/DNA (WBSA/WCS‑SA ratio was 0.01) to CS-SA/DNA.*p < 0.05 comparing CS-SA/BSA(4.7)/DNA (WBSA/ WCS‑SA ratio was 0.1) to each of all other groups except Lipofectamine 2000.

0.5, the sizes of ternary complexes became bigger (200−300 nm), and lower transfection efficiencies were obtained. Notice those values were lower than that of the binary complex. 3.5. Cellular Uptake. To investigate the possible mechanism about the promotion of transfection efficiency of CS-SA/BSA(4.7)/DNA complexes, a confocal laser scanning microscope (CLSM) was used to visualize the cellular uptake of CS-SA/DNA and CS-SA/BSA(4.7)/DNA labeled with FITC. Figure 7a showed the fluorescence images after the complexes

4. DISCUSSION Chitosan and its derivatives were studied to overcome several barriers during the process of gene transfer, such as high rate of cellular uptake, endosome escape, polyplex unpacking, and so forth.29 CS-SA has been considered as an attractive gene delivery vehicle for advantageous DNA package and protection, with excellent cell internalization ability. However, the 2573

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Figure 7. (a) Confocal images of HEK293 cells incubated with FITC-labeled CS-SA/DNA and CS-SA/BSA/DNA nanoparticles for 4, 8, and 12 h. Cell nucleus was stained by DAPI. (b) Percentages of cellular uptake of FITC-labeled CS/DNA, CS-SA/BSA/DNA nanoparticles analyzed by flow cytometry (n = 3). (C) Intracellular fluorescence intensities of CS/DNA, CS-SA/BSA/DNA nanoparticles determined by flow cytometry. NC: negative control (the group without any treatment).

achieved by virus vectors or other lipid nonviral transfection systems.4,31 Also, the cellular uptake was not inhibited by the incorporation of BSA(4.7). It is the specific spatial structure with multiple hydrophobic “minor cores” of our CS-SA micelle that obtains excellent internalization into cancer cells and accumulation in cytoplasm.21 BSA of diverse pI value had a different influence on the transfection efficiency of CS-SA/BSA/DNA complexes. In general, when compared with CS/DNA counterparts, appropriate amounts of BSA(4.7) and BSA(6.0) enhanced transfection efficiency, in which BSA(4.7) showed the optimal performance. On the contrary, BSA(9.3) inhibited the transfection efficiency. The endosome-lysosome organelles are distinct from other cellular organelles because of its low pH (pH 5.0−6.0) relative to the cytoplasm (pH = 7.4).32 Therefore, BSA(4.7) maintains a negative charge, and BSA(9.3) preserves a positive charge in both intracellular and extracellular environments. The charge of BSA(6.0) may shift from negative to positive in the pH range of 5−7.4. We hypothesized that it was the acid group of BSA owning negative charge that enhanced the gene transfection efficiency. The protonated acid group of BSA molecular acted as a proton reservoir and was able to modulate the charge density of the

difficulties of complex dissociation and DNA release due to the strong strength of the charge-based interaction inhibit gene transfection efficiency, thus limiting its application in gene therapy. In this study, we demonstrated that incorporating BSA with a overall negative charge enhanced the gene expression level. BSA, a naturally occurring and the most abundant protein in plasma, is characterized by a high content of the charged amino acids. It is an amphiphilic protein due to the presence of NH2 and COOH groups in its molecular structure. After reacting with methanol in the presence of acetyl chloride, the acid group of BSA was blocked, and the isoelectric point value (pI) increased. When environment pH is below the pI, proteins will be ionized with positive charge. Otherwise they will be ionized with negative charge. The charge of BSA and its methyl ester, namely, BSA(4.7), BSA(6.0), and BSA(9.3), will vary in the pH range of 5−7.4. The pKa value of CS-SA approaches 8.4.30 The ionized CSSA, DNA, and BSA can form nano polyelectrolyte complexes (CS-SA/DNA and CS-SA/BSA/DNA) by electrostatic interactions, and the incorporation of BSA of different pI will not affect the binding capacity of CS with DNA. The strong biomacromolecule compacting and transfer capacity cannot be 2574

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Figure 8. Images of the intracellular trafficking of CS-SA/DNA and CS-SA/BSA(4.7)/DNA observed by a Confocal laser scanning microscope: lysotracker labeled lysosome (blue), FITC-labeled CS-SA (green), TAMRA-labeled DNA (red), and the merged images, also nucleus staining with DAPI (blue). Scale bar = 10 μm.

Figure 9. Long-acting green fluorescent protein expression of transfected HEK293 cells observed by inverted microscopy. Cells were transfected in vitro using CS-SA/DNA and CS-SA/BSA/DNA nanoparticles for 2, 4, and 7 days. Lipofectamine 2000 was used as a positive control.

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polymer/DNA. The affinity between the DNA and chitosan was weakened, yielding complexes that dissociated more easily than complexes of the corresponding chitosan with high charged density. It was then explored by our intracellular trafficking experiment that incorporated BSA(4.7) can largely enhance the DNA release from complexes (Figure 8). In a word, without influenced the cellular uptake, our best prescription can achieve effective gene release behavior. In the long-acting green fluorescent protein expression experiment, we can see that in 2 and 4 days, ternary complexes with BSA(4.7) showed significantly better performance than binary complexes. When extending the expression time to 7 days (Figure 9), the gene expression level of binary complexes catches up with that of the ternary complex. Interestingly, the transfection efficency of both binary and ternary complexes exceeded the Lipofectamine 2000. It revealed that the contribution of BSA(4.7) to complex dissociation played an important role in early stages. The Lipofectamine-associated gene delivery system was prone to combine with serum components, which lead to poor transfection efficency. BSA is widespread in the blood. This indicates that CS-SA may be more effective and compatible for transfection in vivo than Lipofectamine. The cell viability of varying test samples was tested by MTT assay as shown in Figure 10a; CS-SA exhibit good cell viability, even though there was a slightly decrease in

viability when the cells were cultured with either binary or ternary complexes, whereas the cytotoxicity of Lipofectamine 2000 was found to be very high as well-known. Also, complexes with different BSA also showed high cell viability (Figure 10b). The application of CS-SA in gene therapy can benefit from efficient, long-acting therapeutic effects in lesions and low vector toxicity toward the normal tissues at the same time. Therefore CS-SA will prove to be a safer and efficient gene carrier in vivo.

5. CONCLUSIONS A CS-SA based gene delivery system incorporating BSA protein was developed in the study as an efficient vector for gene delivery. All of the CS-SA/DNA binary and CS-SA/BSA/DNA ternary complexes showed a good capability to form complexes. However, referring to transfection activity, the isoelectric point of BSA had obvious influence on the transfection efficiency of CS-SA/BSA/DNA complexes. BSA(4.7) and BSA(6.0) enhanced the transfection efficiency, while BSA(9.3) showed an inhibited effect. The acid groups of protein act as a proton reservoir, causing efficient intracellular complexes dissociation, which contribute to an excellent gene transfection. Moreover, the application of CS-SA can achieve an efficient and longacting gene delivery system.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 571 88208441. Fax: +86 571 88208439. E-mail [email protected] (F.-Q.H.); [email protected] (Y.Z.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate the financial support from the National Nature Science Foundation of China under Contract 81072583 and 81273442, Zhejiang Provincial Natural Science Foundation (LY12H30007), and Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents.

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REFERENCES

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