Use of Biotinylated Chitosan for Substrate-Mediated Gene Delivery

Article pubs.acs.org/bc

Use of Biotinylated Chitosan for Substrate-Mediated Gene Delivery Wei-Wen Hu,*,† Wun-Jheng Syu,† Wen-Yih Chen,†,‡ Ruoh-Chyu Ruaan,†,§ Yu-Che Cheng,§,∥ Chih-Cheng Chien,§,∥,⊥,# Chuan Li,¶ Chih-Ang Chung,¶ and Chia-Wen Tsao¶ †

Department of Chemical and Materials Engineering, ‡Center for Dynamical Biomarkers and Translational Medicine, §Institute of Biomedical Engineering, and ¶Department of Mechanical Engineering, National Central University, Jhongli City, Taiwan ∥ Department of Medical Research, Cathay General Hospital, Taipei, Taiwan ⊥ School of Medicine, Fu Jen Catholic University, Taipei, Taiwan # Department of Anesthesiology, Sijhih Cathay General Hospital, Sijhih City, Taipei, Taiwan ABSTRACT: To improve transfection efficiency of nonviral vectors, biotinylated chitosan was applied to complex with DNA in different N/P ratios. The morphologies and the sizes of formed nanoparticles were suitable for cell uptake. The biotinylation decreased the surface charges of nanoparticles and hence reduced the cytotoxicity. The loading capacities of chitosan were slightly decreased with the increase of biotinylation, but most of the DNA molecules were still complexed. Using different avidin-coated surfaces, the interaction between biotinylated nanoparticles to the substrate may be manipulated. The in vitro transfection results demonstrated that biotinylated nanoparticles may be bound to avidin coated surfaces, and the transfection efficiencies were thus increased. Through regulating the N/P ratio, biotinylation levels, and surface avidin, the gene delivery can be optimized. Compared to the nonmodified chitosan, biotinylated nanoparticles on biomaterial surfaces can increase their chances to contact adhered cells. This spatially controlled gene delivery improved the gene transfer efficiency of nonviral vectors and could be broadly applied to different biomaterial scaffolds for tissue engineering applications.

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INTRODUCTION Regenerative gene therapy is a promising strategy for tissue engineering in which therapeutic genes can be delivered to express biological signals to guide tissue repair.1 Both viral and nonviral vectors can be used for gene transfer. Viral vectors have been applied due to their excellent transduction efficiency. However, the risks of immunogenic response, host reaction, and insertion-caused mutation restrict their application for clinical treatment.2 In contrast, nonviral vectors, such as cationic polymer and liposome, are synthetic polymer materials which may encapsulate or complex DNA for transportation through the cell membrane. Such vehicles are desired because of their relative safety, low immunogenicity and toxicity, ease of administration and manufacture, as well as lack of DNA size limitation.3 Chitosan is a polysaccharide material derived from crustacean shells and composed of N-acetyl-D-glucosamine and D-glucosamine monomers linked by β-(1,4) glycosidic bonds. It has been broadly applied for pharmaceutical and drug delivery because of its nontoxicity and tissue compatibility.4−7 In addition, there are abundant amines in chitosan chains because it is synthesized by deacetylation of chitin. Therefore, chitosan can condense DNA and is a suitable carrier for gene delivery. It also has been reported that chitosan can be applied for siRNA delivery for gene silencing application.8,9 In addition, chitosan is one of few agents which can be administrated through the oral route for gene transfer.10 Compared to other frequently used © 2012 American Chemical Society

polycationic vectors, such as poly(L-lysine) (PLL) and polyethyleneimine (PEI), chitosan has a relatively low charge density.11 This property not only enhances the biocompatibility of chitosan, but also reduces its gene transfer ability.12,13 Different strategies have been developed to improve the transfection efficiency of chitosan. For example, chitosan has been grafted with PEI,14,15 PLL,16 or spermine 17 to increase its amine density; however, the grafted pendants may increase the toxicity. Because amino groups of chitosan can be easily reacted for conjugation, different modifications have been developed to improve gene delivery. Cell penetrating peptides (CPP) have been conjugated to chitosan to improve gene internalization efficiency.18−20 PEGylation and folate modification have been applied to chitosan to improve the water solubility and the specificity of transportation to tumor cells.21 Chitosan has also been modified with nucleus localization signal (NLS) to increase its nucleus transportation.22,23 Furthermore, chitosan has been biotinylated to bind different ligands on the outer layer of a polymer/DNA complex for targeting delivery.24 These results suggested that bioconjugation is accessible to improve and control gene delivery pathway of chitosan. Substrate-mediated delivery is a useful way to enhance gene transfer efficiency by concentrating gene vehicles on bioReceived: March 13, 2012 Revised: June 27, 2012 Published: July 9, 2012 1587

dx.doi.org/10.1021/bc300121y | Bioconjugate Chem. 2012, 23, 1587−1599

Bioconjugate Chemistry

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SulfoNHS-LC-Biotin (Pierce, Rockford, IL, USA) was applied to biotinylate chitosan molecules. Different molar ratios of SulfoNHS-LC-Biotin to amine groups of chitosan from 0.01 to 1 were used for modification, which were denoted as B0.01 to B1 in the following description. First, SulfoNHS-LC-Biotin was dissolved in PBS, and then was reacted with chitosan for 3 h at room temperature. Unreacted SulfoNHS-LC-Biotin molecules were removed by dialysis, and the level of biotinylation was quantified by HABA (2-(4-hydroxyazobenzene) benzoic acid, HABA) assay.26 Briefly, the HABA solution was prepared by adding 6 μmol of HABA and 5 mg of avidin to 10 mL of PBS, which had an absorption at wavelength of 500 nm.33 This absorption decreased proportionally when biotin was added due to high affinity of biotin to avidin. The changes of OD 500 nm before and after adding biotinylated chitosan were compared to determine the grafted biotin on chitosan molecules. The conjugation efficiencies were defined as the molar ratios of SulfoNHS-LC-Biotin molecules added in the reaction to the biotin moieties grafted on chitosan molecules. Physical Properties of Chitosan-DNA Complexes. To determine the zeta potentials and the size distribution of nanoparticles, dynamic light scattering (DLS) analysis was performed using Zetasizer Nano ZS (Malvern, Worcestershire, UK). The size distribution was measured by photon correlation spectroscopy at a 173° scattering angle. Three measurements were performed for each sample to confirm that the record was reliable, and the mean hydrodynamic diameter was generated by cumulative analysis. The zeta potential was performed by laser Doppler anemometry. The samples were placed in a disposable capillary cell, and the electrophoretic mobility was measured to determine the zeta potential of nanoparticles. Complexation Efficiency of Chitosan to DNA. The loading capacity of chitosan to DNA was analyed by ethidium bromide (EtBr) displacement assay and gel retardation assay. About EtBr displacement assay, DNA was dissolved in 20 mM sodium acetate buffer (pH = 5.0) with final concentration of 0.05 mg/mL and was mixed with EtBr (DNA base:EtBr (mol) = 200:1). The EtBr/DNA solution was mixed with equal volume of biotinylated chitosan and then was vortexed for 10 s. After 30 min incubation at room temperature, 100 μL samples were placed in 96-well microplates and measured by fluorescence spectroscopy (Synergy H1m, Biotek, Winooski, VT, USA). The excitation and emission wavelengths were 511 and 603 nm, respectively. For gel retardation assay, nanoparticles were loaded in 1% agarose gels to perform electrophoresis at 100 V for 40 min and were stained by EtBr. Integrity Assay of DNA/Chitosan Nanoparticles. The protection efficiency of chitosan to complexed DNA was evaluated by deoxyribonuclease I (DNase I) digestion assay. Nanoparticles (20 μL) were mixed with 5 μL of DNase I (Sigma-Aldrich, St. Louis, MO, USA) and were incubated at 37 °C for 30 min. To terminate the activity of DNase I, 10 μL of 0.2 M EDTA was added and was heated to 68 °C for 15 min. Furthermore, to release the complexed DNA from nanoparticles, 1 μL of chitosanase (Merck, Germany) was added to nanoparticles at 37 °C for 1 h. Finally, the sample was loaded to 1% agarose for electrophoresis analysis. In Vitro Studies for Transfection. Both pCMV-β and pEGFP-C3 were delivered in this study. Chitosan with different levels of biotinylation was used to complex with 0.8 μg of pCMV-β DNA in different N/P ratios for gene delivery. Human embryonic kidney cell line (HEK-293T) was used to evaluate gene transfer efficiency. Nanoparticles were loaded to

materials. For example, physical adhesion or chemical conjugation has been applied to localize or immobilize viral vectors on material surfaces.25,26 These strategies demonstrated that gene delivery can be controlled to in situ transduce cells on scaffolds. Because adhered cells can easily contact gene vectors on material surfaces, gene transfer may thus be improved. Consequently, different studies of substrate-mediated delivery have been performed to chitosan. For example, chitosan has been coated on hydroxyapatite nanoparticles to increase its precipitation to the substrate for gene delivery.27 Layer-by-layer assembly has also been applied to incorporate chitosan/DNA nanoparticles into multilayers with hyaluronic acid (HA) on biomaterial surfaces.28 Such polyelectrolyte multilayer may further transfect mesenchymal stem cells for differentiation.29 In addition, chitosan/DNA nanoparticles have been directly coated on stent surfaces for localizing and prolonging transgene expression in vivo.30 These results demonstrate that spatially controlled delivery should improve transfection efficiency of chitosan. The biotin−avidin interaction is known to be the strongest noncovalent bond.31 They are highly specific, commercially available, and can be easily linked to different molecules by conjugation. Consequently, we would like to combine bioconjugation and substrate-mediate delivery to improve the transfection ability of chitosan. Chitosan was biotinylated before complexing with DNA, and the formed nanoparticles were immobilized on biomaterial surfaces using biotin−avidin interaction. Although biotin has high specificity to avidin for spatial control, too strong biotin−avidin interaction may retard gene delivery.32 Therefore, chitosan molecules were biotinylated at different levels to adjust the density of binding ligands. These biotin-modified chitosan molecules were complexed with DNA as nanoparticles in different ratios, and then were immobilized on surfaces coated with different amounts of avidin. By changing the ratio of biotin and avidin moieties on nanoparticles and material surfaces, respectively, the binding forces could be delicately adjusted to stably maintain gene vehicles for cell internalization.

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MATERIALS AND METHODS Plasmid DNA Preparation. Plasmid DNA, pCMV-βand pEGFP-C3 (Clontech, Mountain View, CA, USA), were used as reporter genes for in vitro study. They were cloned and amplified in Escherichia coli DH5α. The amplified plasmid DNA was isolated by sodium hydroxide and then was precipitated by PEG purification. Finally, restriction enzyme mapping, polymerase chain reaction (PCR) detection, and DNA sequencing were performed to determine the quality of purified plasmid DNA. Preparation of Chitosan-DNA Complexes. Chitosans with molecular weight of 10 kDa was purchased from Charming & Beauty Co. (Taiwan). Chitosan and DNA were both dissolved in 20 mM sodium acetate buffer, and the final pH 5.0 was adjusted by 0.2 N NaOH. To control the charge ratio between chitosan and DNA, chitosan was used to complex with DNA in different ratios of amino groups to phosphate groups (N/P ratios). The concentration of DNA was fixed to 0.05 g/L, and the concentrations of chitosan varied according to the required N/P ratios. Equal volumes of both solutions were mixed together and vortexed for 10 s, and were then incubated at room temperature for 30 min before experiments. Biotinylation of Chitosan. To immobilize chitosan vehicles on material surfaces using biotin−avidin interaction, 1588



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96-well multiplates for 2 h before cell seeding. The transfection medium was DMEM with 10% FBS (pH = 6.5), which was replaced by normal culture medium (DMEM with 10% FBS, pH = 7.0) after 20 h.34 The transgene efficiency was evaluated after 48 h of transfection. For pCMV-β delivery, the gene expression was quantified by the activity of β-galactosidase. Medium was removed and 40 μL of 0.25 M Tris-HCl was added following freeze/thaw cycles to lyze cells. Then, 20 μL of cell lysis was reacted with ortho-nitrophenyl-β-D-galactopyranoside (ONPG) for substrate analysis. To normalize the ONPG results, the other 20 μL of sample was used for protein quantification by BCA kit (Pierce, Rockford, IL, USA). The expression of pEGFP-C3 was determined by fluorescent microscopy (Eclipse Ti−U, Nikon, Japan). Preparation of Avidin-Coated Surfaces. Avidin (Pierce) was dissolved in PBS in different concentrations, and was added in 96-well ELISA plates (Corning, Lowell, MA, USA) at volume of 100 μL per well. After overnight incubation at 4 °C, well surfaces were washed by PBS and then were used to immobilize biotinylated nanoparticles. To determine levels of avidin coated on the surfaces, alkaline phosphatase (biotin-AP; Pierce) was used to illustrate the avidin binding sites. Botin-AP was diluted in PBS and was added to the plates at volume of 100 μL/well for 1 h. After three washes in PBST, the surfaces were developed by 100 μL of p-nitrophenyl phosphate (pNPP) substrate for 20 min before being read spectrophotometrically at OD 405 nm. Cytotoxicity and Relative Viability Analysis of Biotinylated Nanoparticles. To investigate the extent to which nonviral vectors affected cell physiology during transfection, 3(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase (LDH) assays were performed to determine relative cell viability and relative cytotoxicity, respectively. After 48 h of transfection, 20 μL of CellTiter 96 AQueous kit (Promega, Madison, WI, USA) was added per well and incubated at 37 °C for 1 h, then read spectrophotometrically at wavelength of 490 nm. The LDH assay was performed by LDH kit (CytoTox 96 Nonradioactive Cytotoxicity assay, Promega), then the sample analyzed spectrophotometrically at a wavelength of 490 nm.

Figure 1. Biotinylation of chitosan using different concentrations of SulfoNHS-LC-biotin. Chitosan was biotinylated in different mole ratios of SulfoNHS-LC-biotin to amine moieties of chitosan. The conjugation efficiency (line) was defined as the number of moles of SulfoNHS-LC-biotin to the grafted biotin moieties on chitosan molecules, which was measured by HABA assay. The level of biotinylation was also compared, which was the average number of biotin moieties grafted per chitosan molecule (bar).

of chitosan/DNA complexes due to there being more chitosan molecules per nanoparticle (Figure 2a). Except DNA, which has negative zeta potential due to its phosphate groups, all chitosan/DNA groups demonstrated positive surface charge and that the zeta potentials were between 20 and 35 mV, indicating that these nanoparticles should be able to be applied for gene delivery. The zeta potential of nanoparticles decreased with the increasing level of biotinylation, suggesting that the conjugation of biotin moieties reduced amine groups of chitosan molecules, thus leading to declining positive charge of nanoparticle surfaces (Figure 2b). Interestingly, the B0 group was always the group with highest zeta potential except for the N/P ratio of 30 (Figure 2b). This may be because biotin moieties shielded the charge interaction between chitosan and DNA, and hence, more chitosan molecules were required to adhere on the surface of nanoparticles, which resulted in higher zeta potentials of biotinylated chitosan groups than that of the nonmodified group (B0) at N/P = 30. The size distribution was determined by DLS analysis. Naked DNA had a diameter of 67.0 nm, and all of the nanoparticles demonstrated larger diameters than that of the naked DNA. For the nonmodified group (B0), nanoparticle sizes increased with the increase of N/P ratio (Figure 2c). It may be because using more chitosan increased the amount of chitosan molecules per nanoparticle, hence enlarging the nanoparticles. However, this trend was not significant for the biotinylated chitosan groups. Most of the particle sizes were between 66.8 and 109.8 nm. Only the B1 group at N/P ratio of 2 exhibited an unusually large particle size (578 nm), which also demonstrated a cloudy appearance. It was consistent with its weak zeta potential that aggregation may occur without forming stable nanoparticles. To confirm the size distribution analysis by DLS, scanning electron microscopy (SEM) was also performed. Different chitosan groups from B0 to B1 were used to complex with DNA at N/P ratio of 10 (Figure 3). The SEM result suggested that the sizes of the B0, B0.01, B0.03, B0.1, B0.3, and B1 groups were in the range 50−90 nm, 70−80 nm, 40−100 nm, 40−100 nm, 70−100 nm, and 70−90 nm, respectively. These results were consistent with the results of DLS analysis (B0, 88.9 nm;

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RESULTS Biotinylation of Chitosan. To specifically immobilize chitosan/DNA nanoparticles on material surfaces, biotin moieties were conjugated to amine groups of chitosan using biotinylation reagent, SulfoNHS-LC-biotin. Different molar ratios of SulfoNHS-LC-Biotin to amine groups of chitosan from 0.01 to 1 were used to control the level of biotinylation, and were denoted as B0.01 to B1. The conjugation efficiencies were evaluated by HABA assays (Figure 1). Although the conjugation efficiencies decreased with the increase of the concentrations of Sulfo-NHS-LC-biotin (from 57% to 12%), the levels of biotinylation increased with the number of moles of Sulfo-NHS-LC-biotin (from 0.302 to 5.230 biotin moieties per chitosan molecules). These results suggest that the molar ratio of biotin to chitosan could be adjusted by simply controlling the concentration of biotinylation reagent. Physical Properties of Biotinylated Chitosan/DNA Complex Nanoparticles. Chitosan with different levels of biotinylation was used to complex with DNA in different N/P ratios. Their zeta potentials and sizes were analyzed to determine the surface charges and the diameter distribution, respectively. Increasing the N/P ratio raised the zeta potential 1589



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Figure 2. Dynamic light scattering assay for determining the surface charges and size distributions of nanoparticles. The zeta potentials of chitosan/ DNA nanoparticles were compared in different (a) N/P ratios and (b) biotinylations. (c) Their sizes were also determined. The control group DNA was naked DNA only.

Figure 3. Scanning electron microscopy images of biotinylated chitosan/DNA nanoaprticles. Chitosans with different biotinylations were used to complex with DNA in N/P ratio of 10. The nanoparticles formed were illustrated by SEM to determine their morphologies and size distribution. The scale bar in each picture was 500 nm.

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Figure 4. Complexation efficiency of biotinylated chitosan. DNA was stained by EtBr before complexing with chitosan. The intensity of fluorescence was compared before and after nanoparticle formation to determine the complexation efficiency of chitosan at different (a) N/P ratios and (b) biotinylation. (c) Chitosan with different biotinylation was complexed with DNA at different N/P ratios, and was loaded to agarose gel for electrophoresis analysis.

This suggested that high biotinylation may retard the electrostatic affinity of chitosan to DNA, and this phenomenon was more obvious when the chitosan molecules were insufficient. Gel retardation assay was also analyzed to confirm the results of chitosan complexation efficiencies (Figure 4c). Chitosan/ DNA nanoparticles were formed and loaded to agarose gels to perform electrophoresis. Because the negatively charged DNA was shielded by chitosan, only unloaded DNA may move down in the electrical field and be separated from the complex. Therefore, nanoparticles formed from chitosan/DNA complex were retained in the loading wells. For the nonmodified B0 group, there was some free DNA at N/P ratio of 2, suggesting that chitosan molecules were insufficient to complex DNA. In contrast, DNA was completely loaded when N/P ratio was higher than 2. The bands in the well even moved in the opposite direction to the free DNA when N/P ratio was larger than 10. This should be because free chitosan molecules may become entangled with nanoparticles so that their positive charges eventually moved DNA/chitosan complexes to the cathode. The results of the B0.01 to B0.1 groups were similar to those of the B0 group except that some released DNA was found at N/P ratio of 5, suggesting that biotinylation should reduce the charge density, which further reduced the stability of complex to maintain DNA in the well. The diffusion of nanoparticles to the cathode occurred only at N/P ratio of 30 for the B0.1 group. It should be because the increasing

B0.01, 76.7 nm; B0.03, 78.5 nm; B0.1, 87.0 nm; B0.3, 76.0 nm; and B1, 67.5 nm) (Figure 2c). It suggested that DLS results were reliable. In addition, chitosan with or without biotinylation can complex with DNA to display a spherical appearance, suggesting that nanoparticle formation should be accessible after biotin modification. Effect of Biotinylation on the Complexation Efficiency of Chitosan to DNA. Ethidium bromide (EtBr) is a compound which can intercalate between the base pairs of the DNA double helix, and the DNA-EtBr complex shows high fluorescence activity.35 When DNA was bound by the cationic polymer, EtBr would be expelled from the DNA-EtBr complex.36,37 Therefore, the DNA loaded by chitosan led to a decrease in fluorescence. The complexation rates of nanoparticles varied with the N/P ratios and the levels of biotinylation. Higher N/P ratio groups had more chitosan to adsorb DNA, and thus caused better complexation efficiency (Figure 4a). Most of the groups had lower complexation rates when the N/P ratio was 2, and there were almost no significant differences in loading capacities at N/P ratios higher than 5, indicating that the chitosan molecules at this ratio should be saturated for complex formation. On the other hand, the biotinylation used up positive amines and hence reduced the attracting force to DNA, causing lower complexation efficiency (Figure 4b). Most of the complexation rates can be higher than 65% except the groups of N/P = 2 and biotinylation ratios of 0.3 and 1, which only had complexation rates less than 20%. 1591



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Figure 5. Protection Assay of Biotinylated Chitosan to DNA. Chitosan with biotinylation of (a) B0; (b) B0.01; (c) B0.03; (d) B0.1; (e) B0.3; (f) B1 was complexed with DNA in various N/P ratios. Different treatments were performed before electrophoresis analysis: (A) nontreatment; (B) nanoparticles treated with DNase I for 30 min and then inactivated; (C) nanoparticles treated with chitosanase; (D) nanoparticles treated with DNase I for 30 min, inactivated, and then treated with chitosanase. The labels at the right sides of the gels were to indicate DNA in open-circular (O), linear (L), or supercoiled (S) forms.

the extent to which the complexed DNA may be attacked.39,40 Chitosan molecules with different levels of biotinylation were complexed with DNA at various N/P ratios. These nanoparticles were treated four different ways and then were analyzed by electrophoresis (Figure 5). Nanoparticles in the A treatment were the nontreated groups; the B treatment was the nanoparticles treated with DNase I for 30 min and then inactivated. The C treatment was the nanoparticles only treated with chitosanase, and the D treatment was the nanoparticles treated with DNase I for 30 min, which were inactivated and then treated with chitosanase. The A treatment was used to illustrate nonloaded free DNA. The B treatment was used to determine if DNase I may destroy the construction of nanoparticles. The C treatment was used to release the loaded

biotinylation decreased the charge density, and thus, the driving forces of positive chitosan molecules to move nanoparticles to the cathode were reduced. As the level of biotinylation increased to B0.3, there was no diffusion of nanoparticles to the cathode, and some free DNA was found when the N/P ratio was less than 30. The loading efficiency of the B1 group further declined so that free DNA was found even when the N/ P ratio was 30. Protection Assay of Botinylated Chitosan to DNA. DNA loaded to chitosan carrier can be shielded from enzymatic digestion.38 However, because biotinylation used up amine groups of chitosan, it probably affected the protection ability of nanoparticles for in vivo application. Therefore, a protection assessment was applied using DNase I digestion to determine 1592



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DNA, and the D treatment was used to confirm the integrity of the loaded DNA after the DNase I treatment. For the nonmodified B0 group, only DNA was released at the N/P ratio of 2 (Figure 5a). The B treatment demonstrated that only the released free DNA was digested by DNase I, whereas the brightness of the DNA/chitosan complex staying in the loading well was almost identical to it in the A treatment. The D treatment results indicated that DNase I digestion made the composition of DNA within the nanoparticles a little different from DNA directly released by chitosanase in the C treatment. The band of supercoiled DNA was slightly reduced, but the band of the open circular DNA was increased. However, the total amounts of these two bands in these two treatments were almost equal, and linear DNA was not found in all N/P ratios. Compared to open circular DNA, supercoiled DNA has a complicated conformation, which may inhibit chitosan from completely warping DNA around. Therefore, DNase I may react with the unprotected part of the DNA/ chitosan complex to relax DNA to an open circular form. However, the conformation of circular DNA is looser than in the supercoiled form, which made the adsorption of chitosan easier than with supercoiled DNA, and thus the protection of chitosan was able to avoid further digestion. There was a similar trend of the B0.01 group to the nonmodified chitosan (Figure 5b). Only N/P ratio less than 10 displayed free DNA release, which was digested by DNase I, and the biotinylated chitosan may protect circular plasmid DNA from DNase I digestion. However, the integrity of nanoparticles became reduced when the biotinylation increased. The bands of supercoiled DNA disappeared after DNase I treatment for B0.03−B1 groups, which was seen along with the appearance of linear DNA bands (Figure 5c−f). These results suggest that increasing biotinylation may reduce the electrostatic forces between DNA and chitosan; the wrapped polycation was not strong enough to protect DNA from enzymatic digestion. Biocompatibility of Biotinylated Chitosan/DNA Nanoparticles. To ensure that the biotinylation does not negatively affect the physiology of the tranfected cells, it is necessary to determine the biocompatibilities of nanoparticles. Two assessments, LDH and MTS assays, were applied to evaluate cytotoxicity and cell viability, respectively. For the LDH assay, there were no obvious trends of cytotoxicity for different N/P ratios and biotinylations; all of the nanoparticles exhibited relative cytotoxicities between 70% and 130% (Figure 6a). In contrast, the Lipofectamine group caused severe cell death that the relative cytoxicity was 2.5 times more than that of the untreated group. These results suggested that chitosan was a biocompatible material which is suitable for gene delivery application, and the biotinylation did not increase cytotoxicity to the transfected cells. In the MTS assay, the cells treated with naked DNA were used as a control group, for which the viability was defined as 100%. The absorbance of experimental groups at 490 nm was compared to that of the control group to determine the relative viability, and the Lipofectamine 2000 transfection was used as the positive group (Figure 6b). For the nonmodified B0 group, the cellular viabilities decreased with the increasing N/P ratios. This should be because the increase of chitosan molecules increased positive charges, which interfered with cell physiology through the interaction/disruption of cellular organelles including mitochondria.41 Similar trends were found at the B0.01 and B0.03 groups, suggesting that the low level of

Figure 6. Biocompatibility of biotinylated nanoparticles. Nanoparticles formed by chitosan with different biotinylation in different N/P ratios were applied to transfect HEK 293T cells for 2 days. (a) The relative cytotoxicity of nanoparticles was evaluated by the LDH assay. (b) The relative viability of trasnfected cells was determined by the MTS assay. These spectrometric results were normalized by comparing with the untreated group. Lipofectamine 2000 (Lipo) was used as the positive control in both analyses.

biotinylation did not affect the cellular bioactivity. However, the viabilities of the high N/P ratios were improved when the level of biotinylation was higher than B0.03. There were no significant differences of bioactivity between different N/P ratios for the B0.3 and B1 groups; almost all of the viabilities were close to 100%. Our zeta potential results have demonstrated that the biotinylation used up amines and thus decreased the positive charges (Figure 2b). Therefore, the interference of charge with organelles may thus be reduced, which rescues cells from the cytotoxicity of the polycation. Transfection Efficiency of Biotinylated Chitosan/DNA Nanoparticles. Chitosan with different levels of biotinylation was complexed with DNA at different N/P ratios to examine the transfection ability of these nanoparticles on tissue culture polystyrene (TCPS) (Figure 7a). For the nonmodified B0 group, the N/P ratio of 5 exhibited highest LacZ gene expression. When the level of biotinylation increased to B0.1, the N/P ratio of the best transfection shifted to 10. The zeta potential results indicated that the positive charges were reduced with the increase of biotinylation (Figure 2b), and thus the electrostatic forces between DNA and chitosan were weakened so that more chitosan molecules were required to increase the charge density for transfection. In addition, the increasing biotinylation improved transfection ability of nanoparticles (Figure 7a). It should be relative to the increasing viability that biotinylation may reduce the damage caused by 1593



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Figure 7. In vitro transfection efficiency of biotinylated chitosan/DNA nanoparticles. Nanoparticles formed by chitosan with different biotinylation at different N/P ratios were applied to transfect HEK 293T cells for 2 days on (a) TCPS or surfaces coated with avidin solution of concentrations of (b) 0.0001 mg/mL, (c) 0.001 mg/mL, (d) 0.01 mg/mL, (e) 0.1 mg/mL, and (f) 1 mg/mL. The expression of β-galactosidase was determined by ONPG substrate, which was normalized by total protein amount. Naked DNA (DNA) only was used as the negative control group, and the Lipofectamine 2000 (Lipo) delivery was used as the positive control group. (g) Coated avidin on multiple plates was analyzed by biotin-AP labeling, which was developed by substrate pNPP and was read spectrophotometrically at OD405.

the positive charges and thus enhance the cellular activity, which facilitated the expression of the transgene (Figure 6b). However, when the biotinylation increased to B1, the transfection efficiency was reduced. The complexation experiments demonstrated that the loading capacity of DNA at the

B1 group was significantly lower than that of the other groups, which may thus reduce the transfection efficiency (Figure 4b). In Vitro Transfection Regulated by Biotin/Avidin Interaction. Because the biotinylated nanoparticles demonstrated a successful transfection, these groups were applied to 1594



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Figure 8. Transfection efficiencies of biotinylated chitosan on avidin-coated surfaces. Nanoparticles formed by biotinylated chitosan (B0.1, N/P = 10, square) and nonmodified chitosan (B0, N/P = 5, diamond) were used to transfect HEK 293T cells on surfaces coated with different concentrations of avidin. (a) Plasmid pCMV-β was delivered, and the expression of β-galactosidase was determined by ONPG substrate, which was normalized by total protein amount. The Lipofectamine 2000 (Lipo) delivery was used as the positive control group (triangle). (b) Plasmid pEGFPC3 was delivered and the expression of eGFP was evaluated by fluorescent microscopy (scale bar = 200 μm).

the binding affinity of nanoparticles to the surfaces was thus reduced, which may further enhance the uptake of cell to gene. As the avidin coating increased to 0.01 mg/mL, the transfection results were not as good as those at the 0.001 mg/mL avidin-coated surface (Figure 7d), which may be because the high density of avidin provided more biotin binding sites, thus hindering nanoparticle release for transfection. Interestingly, the transfection of the B1 group was better than that at surfaces with lower avidin coating. Because the B1 groups had a loose interaction between DNA and biotinylated chitosan, DNA may release from nanoparticles and complex with free biotinylated chitosan molecules in the solution for cell transfection, which thus rescued the difficulty due to too-strong biotin−avidin interaction. When the avidin coating kept increasing to 0.1 mg/mL, the high affinity of avidin to biotinylated nanoparticles obstructed gene transfer so that the transfection efficiencies were negatively affected (Figure 7e). The trend was more obvious when the coating concentration increased to 1 mg/mL so that almost all of the groups exhibited an extremely low level of transgene expression (Figure 7f). The transfection results on different surfaces indicated that biotinylation indeed enhanced the transfection efficiency, and the best formula was at B0.1 with N/P of 10 (Figure 7). To systemically determine the effect of biotinylation to gene delivery, the transfection efficiencies of biotinylated nanoparticles, nonmodified nanoparticles, and the positive group (Lipofectamine 2000) were compared on different avidincoated surfaces (Figure 8a). The results demonstrated that biotinylated groups always have better transfection than nonmodified groups, which may result in higher bioactivity of biotinylated chitosan (Figure 6b). The best transfection result of biotinylated nanoparticles was at 0.001 g/mL of avidin coating concentration in which the expressed β-galactosidase resulted in 12 μmol of ONPG converted per mg of protein, which was almost 3 times more than that of the nonmodified

avidin-modified surfaces to determine if biotin−avidin interaction may facilitate substrate-mediated gene delivery. To regulate the binding forces of avidin to biotinylated chitosan/ DNA nanoparticles, different concentrations of avidin were used for coating on ELISA plates from 0.0001 to 1 mg/mL. Biotin-AP was used to evaluate the binding sites of coated avidin on the plates. The absorbance of 405 nm revealed that the surface-coated avidin was increased with increasing concentrations of avidin (Figure 7g). For the lowest avidincoated surface (0.0001 mg/mL), the transfection efficiencies were slightly reduced in comparison to the results at TCPS only (Figure 7a,b). Because the ELISA plates were not suitable for cell attachment and growth, the tough environment may negatively affect cell transfection. In addition, the best N/P ratios were shifted from 5 to 10 for B0.01 and B0.03 groups compared to that in TCPS (Figure 7a,b). It may be because nanoparticles with N/P ratio of 10 had more biotin moieties than that of 5, and thus more biotinylated particles were maintained on surfaces with even a low level of coated avidin to facilitate transfection. When the level of avidin coating increased to 0.001 mg/mL, the transfection efficiencies were highly improved (Figure 7c). The ONPG result of the best transfection group (B0.1) was 11.97 μmol/mg of protein, which was double that at TCPS (5.31 μmol of ONPG/mg of protein) (Figure 7a,c), suggesting that biotin modification may increase retention of nanoparticles on the substrate and thus improve transfection efficiencies. In addition, the B0.3 group exhibited a reduced transfection at the N/P ratio of 10, while expression was improved when the N/P ratio increased to 20. This should be because B0.3 had more biotin moieties, and thus the binding affinities became large, which retarded the cell uptake. In contrast, when the N/P ratio shifted to 20, more free biotinylated chitosan molecules existed in the system which may compete with biotinylated nanoparticles for the binding sites of surface avidin. Consequently, 1595



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1.6 μg. These results suggest that our biotinylation method could significantly enhance gene delivery compared to the conventional method, so the drawback of low transfection efficiency of chitosan should thus be improved.

group, suggesting that specifically immobilized nanoparticles may increase the chance to contact adhered cells and enhance gene delivery. However, higher coated avidin caused stronger binding affinity, which further diminished the transfection ability of biotinylated nanoparticles. The transfection efficiencies of nonmodified nanoparticles were also reduced when they were applied to surfaces with high concentration of coated avidin (0.1 and 1 mg/mL), which may result in nonspecific binding of a high level of surface protein. Although the transfection of both biotinylated and nonmodified nanoparticles were highly affected by the surfaces, there was no difference to the Lipofectamine group. This should contribute to their different transfection mechanisms in which Lipofectamine 2000 is a liposomal transfection reagent, while chitosan/ DNA complexes are nanoparticles fabricated by electrostatic forces. Therefore, liposomes may be released from the substrate even if partial molecules of it are adsorbed on the surface. The integrity of the liposome may recover easily and thus still have the ability for transfection. Another plasmid DNA, pEGFP-C3, encoding enhanced green fluorescent protein (eGFP) was also applied to compare their delivery by modified and nonmodified nanoparticles on different avidin-coated surfaces and TCPS (Figure 8b). The green fluorescence expression of transfected cells illustrated similar trends to the pCMV-β results, suggesting that our developed method should be able to be applied to different plasmid DNA. Previous experiments were based on 0.8 μg of pCMV-β delivery. To investigate the extent to which the biotinylation method may improve transfection, different dosages of DNA were applied for cell transfection. The expression of βgalactosidase was dose dependent on the amount of DNA, and the expressions were saturated when the DNA amounts were 0.8 and 1.6 μg for the nonmodified and biotinylated nanoparticles, respectively (Figure 9). Compared to the

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DISCUSSION Chitosan is a frequently used nonviral vector. Compared to other polycationic vectors, chitosan exhibits better biocompatibility with lower transfection ability. Therefore, in this study we developed substrate-mediated gene delivery to enhance the delivery efficiency of chitosan. Because the surface charge densities of nanoparticles may affect the efficiencies of gene delivery,42 DLS analysis was performed to determine the zeta potential and the size distribution of biotinylated chitosan/DNA complex. Previous studies have indicated that the size of the nanoparticles may significantly determine their transfection ability because of the process of endocytosis.43 For example, a diameter of PLL/DNA complex less than 100 nm is suitable for gene delivery.44 Regarding chitosan, the appropriate size and surface charge for transfection vary with studies; however, all of them indicate that chitosan/DNA nanoparticles with sizes between 50 and 200 nm and zeta potentials between 15 and 37 mV should be suitable for gene delivery.38,45,46 In our study, the surface charges were increased with the N/P ratio and decreased with the level of biotinylation (Figure 2a,b). Except for the B1 group with N/P of 2, all of the biotinylated nanoparticles demonstrate a positive zeta potential between 20 and 35 mV. In addition, the sizes of formed nanoparticles were between 66.8 and 109.8 nm. These results suggest that our developed biotinyalted nanoparticles are proper for gene transfer. To determine if the loading capacities of chitosan to DNA are affected by biotinylation, two experiments have been performed: the fluorescent dye displacement assay and the gel retardation assay. Compared to the results of the fluorescent dye displacement, relatively low complexation efficiencies were demonstrated in the gel retardation experiment (Figure 4). The EtBr assay is based on reduction of fluorescence due to the dye exclusion from DNA during complex formation. In contrast, gel retardation is to apply the resistance of the agarose gel during electrophoresis. Because the biotinylation may reduce the interaction between chitosan and DNA, DNA may be extracted in the electrical field from loosely composed nanoparticles in which the positive charges were not strong enough to maintain DNA within the loading wells. The protection abilities of chitosan with and without modification to DNA were analyzed by DNase I digestion assay. Our results demonstrate that the complexed DNA in nonmodified chitosan was partially digested so that the intensity of the supercoiled form was reduced, while that of the open circular form was increased (Figure 5a). The damage caused by enzyme attack increases with increase of biotinylation (Figure 5c−f). Chitosan with molecular weight of 10 kDa was applied in this study because lower molecular weight usually demonstrated better transfection ability and thus was frequently used for gene delivery application.47−49 However, some studies have indicated that the protection ability of chitosan to DNA may be reduced with decreasing molecular weight. Huang et al. indicated that chitosan may completely shield DNA when its molecular weight greater than 48 kDa.39 Their results demonstrated that chitosan with molecular weight of 17 kDa was partially degraded by DNase I, and was completely degraded for chitosan with molecular weight of 10 kDa.

Figure 9. Dosage effect on transfection efficiencies of biotinylated chitosan on avidin-coated surfaces. Nanoparticles formed by biotinylated chitosan with different amount of DNA (B0.1 and N/P = 10) were used to transfect HEK 293T cells on surfaces coated with avidin solutions of 0.001 mg/mL for 2 days (square). The expression of β-galactosidase was determined by ONPG substrate, which was normalized by total protein amount. The corresponding transfection results were compared with that of nonmodified chitosan (B0, N/P = 5) on TCPS (diamond).

nonmodified nanoparticle (B0, N/P = 5 at TCPS), which may only achieve the expression of 3 μmol of ONPG/mg of protein, biotinylated nanoparticles (B0.1, N/P = 10 at 0.001 mg/mL avidin) demonstrated an excellent transfection efficiency so that the expression level was almost 6 times higher than that of the nonmodified group when the DNA was 1596



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4c). These results suggested that biotinylation should loosen the chitosan/DNA complex and thus improve the transduction efficiencies. By controlling the coating of avidin as well as the N/P ratio and the biotinylation levels of chitosan/DNA complex, we may regulate the affinity of surfaces to nanoparticles. Through appropriate ratio, the transfection efficiency was significantly improved (Figure 7c). The optimal N/P ratio was increased with the increase of biotinylation. In this study, we did not remove free biotinylated chitosan, because a DNA/polycation complex only is not sufficient for gene delivery. Sabine Boeckle et al. have compared the transfection ability between purified and nonpurified PEI/DNA complexes.60 Their results indicated that the gene delivery of purified nanoparticles was inefficient; however, the transfection can be rescued by providing free PEI. It may be because the free PEI molecules aid in releasing polyplexes from the endosomes.61 Excess free polycations may induce a proton sponge effect to facilitate endosomal escape and gene expression.62 In addition, our results indicated that free biotinylated chitosan may occupy binding sites and hence regulate the binding affinity between nanoparticles and biomaterials.

Douglas et al. also suggested that chitosan with molecular weight of 10 kDa may not protect DNA from enzymatic attack.50 Therefore, it is not surprising that complexed DNA may not be perfectly shielded in our results, and biotinylation may further reduce the protection ability of chitosan. The biocompatibilities of nanoparticles were evaluated by MTS and LDH assay. Our results suggest that the level of LDH was low in all groups, suggesting that the cytotoxicity should not an important concern in this study (Figure 6a). For nonmodified chitosan, the MTS results of transfected cells was reduced with the increase of the N/P ratio (Figure 6b). However, the biotinylation may compensate the decrease of bioactivity at high N/P ratios. This may be because biotinylation diminished amine groups and thus weakened the charge density of chitosan molecules. Similar results have been found for biotinylated PEI which was bioconjugated with avidin for gene delivery.51 Compared to nonmodified PEI, the cytotoxicity of biotinylated PEI demonstrated lower cytotoxicity due to lower surface charges. These results suggest that reducing positive charges of polycations should be beneficial to cell survival rates. To enhance gene delivery of chitosan, we developed chitosan biotinylation to tether nanoparticles on biomaterial surfaces for substrate mediated delivery. Previous studies suggest that gene vehicles may thus be concentrated to facilitate gene transfer.52,53 For example, DNA has been complexed with cationic polymers or lipids and was fixed on surface-coated fetal bovine serum (FBS), which demonstrated better transfection efficiency than bolus delivery.54 However, nonspecific binding forces may lead DNA to diffuse from target sites and elicit unwanted systemic transfection and immune response.55 Our previous studies suggested that the biotin−avidin interaction for viral vector immobilization may not only enhance cell transduction efficiency, but also spatially control gene delivery at the target sites.26,56 Therefore, in this study we intended to apply this strategy to improve the poor transfection ability of chitosan. Comparing the transfection ability of nanoparticles on TCPS, biotinylation reduced the charge density of chitosan, and thus, more chitosan molecules were necessary for transfection (Figure 7a). Interestingly, biotinylated chitosan demonstrated superior transfection efficiency. This may be because the biotinylation reduced the positive charges of chitosan. Decreasing the charge density of the polycations may reduce their damage to organelles.41 Other studies also indicated that the stable interaction between chitosan and DNA may preclude the translation of the transgene.45 The transfection efficacy of chitosans is governed by the interaction strength between the chitosan vector and the DNA in the polyplex.57 Alatorre-Meda et al. have compared the transfection ability of chitosan with different molecular weights and assembly conditions.58 Their results suggested that the weaker DNA−chitosan binding resulted in higher transfection efficiency. Incorporating anionic polymers, such as alginate, is another frequently used strategy to mediate the stability of the complex so the negative charged polyanion may compete with DNA and the electrostatic interaction between DNA and chitosan should thus be regulated.50 About chitosan modification, histidine has been applied to modify chitosan, and the complex with DNA demonstrated an improvement to enhance endosomal escape, which eventually improved the gene delivery.59 Our gel retardation experiment indicated that the biotinylation treatment may weaken the interaction between DNA and chitosan, so that loaded DNA may thus escape from chitosan (Figure

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CONCLUSION In this study, we improved the transfection efficiency of nonviral vectors using substrate-mediated delivery. Biotinylation of chitosan can successfully complex with DNA and the formed nanoparticles have proper surface charges and size for gene delivery. The biotinylated nanoparticles provide good complexation efficiency and may protect DNA to a certain extent. In addition, the cytotoxicity of chitosan may be reduced by the biotinylation, which further increases the cell activity and transgene expression. Through appropriate biotin−avidin interaction, the transfection efficiency can be highly increased. These results suggest that this gene delivery may improve the transfection ability of nonviral vectors, and this approach should be able to be applied to various biomaterial surfaces for tissue engineering applications.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +886 3 422 7151 Ex 34243. Fax: +886 3 422 5258. Email address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the grant of NSC 98-2218-E008-009 from the National Science Council of Taiwan, and 99CGH-NCU-A2 from the National Central University and Cathy General Hospital Joint Research Center.

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Use of Biotinylated Chitosan for Substrate-Mediated Gene Delivery

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