Synthesis and cell activity of novel galactosylated chitosan as a gene carrier

Colloids and Surfaces B: Biointerfaces 70 (2009) 181–186

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Synthesis and cell activity of novel galactosylated chitosan as a gene carrier Baofeng Song a,1 , Wei Zhang a , Rong Peng b,1 , Jie Huang b , Ting Nie a , Ying Li a,∗ , Qing Jiang a , Rong Gao b,∗∗ a b

College of Chemistry, Sichuan University, Chengdu 610064, PR China College of Life Science, Sichuan University, Chengdu 610064, PR China

a r t i c l e

i n f o

Article history: Received 19 July 2008 Received in revised form 10 December 2008 Accepted 11 December 2008 Available online 24 December 2008 Keywords: Chitosan Galactose Non-viral vector DNA Gene delivery

a b s t r a c t It is important for gene carrier to transport DNA into target cells. Although viral vectors are very efficient gene-transfer vehicles, significant drawbacks limit their applications. Chitosan (CS) has been researched widely as a non-viral vector. However, the low cell specificity and low transfection efficiency of chitosan need to be overcome. In order to conquer the drawback of chitosan, the present paper is concerned with the synthesis of novel galactosylated chitosan (GC) through etherization of chitosan and galactose in THF using BF3 ·OEt2 as promoter. The final product was characterized and confirmed by FT-IR and 1 H NMR. The degree of O-substitution (DS) of chitosan by galactose was measured to be 10.38% using anthrone-sulfuric acid colorimetric method. The mean particle diameter and average zeta potential of the GC/DNA complex were 350 nm and +22.1 mV, respectively. The GC/DNA nanoparticle was tested to transfect HEK293 cells, and the viability of HEK293 cells was not affected by the GC/DNA nanoparticle compared to that of the control. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved.

1. Introduction Gene therapy has recently received increasing interest due to the treatment of a wide range of diseases, both inherited and acquired diseases [1]. Recently, the key research of gene therapy is to search effective and safe vector systems. The main systems for gene delivery are both viral and non-viral vectors. Although viral vectors show high transfection efficiency, many drawbacks limit their applications, such as non-specificity, immunogenicity to the target cells and degradation by enzymes [2]. Non-viral vectors for gene therapy are preferred as safer alternatives to viral vectors. They have many advantages including safety, stability and lower immunogenicity [3]. Currently, the two major types of non-viral gene delivery vectors are cationic liposomes and cationic polymers [4,5]. Cationic liposomes have potential as a gene delivery vector. However, their applications are limited to local delivery due to low stability and rapid degradation in the body [6,7]. Cationic polymers have been used to deliver DNA both in vitro and in vivo in terms of biocompatibility, low cytotoxicity and cost-effective [8]. As a natural cationic polymer, chitosan has been widely employed in gene delivery due to its biocompatibility and biodegradability [9]. Chitosan protonated in acidic condition can form complex nanoparticles with anionic DNA by electrostatic interaction [10] and protect

∗ Corresponding author. Tel.: +86 28 85413601; fax: +86 28 85413601. ∗∗ Corresponding author. Tel.: +86 28 85416856; fax: +86 28 85471599. E-mail addresses: profl[email protected] (Y. Li), [email protected] (R. Gao). 1 These authors equally contributed to this research.

it against nuclease degradation [11]. However, chitosan as a gene vector has still some disadvantages such as relative inefficiency and low specificity [12]. The basic definition of gene therapy is the transfer of genetic materials to specific cells. That induces the development of cellspecific targeting carriers in gene therapy. In the body, the liver is an attractive target tissue for gene therapy due to its large size and metabolic capacity [13]. The research of hepatocyte-targeting gene delivery is much more attractive. Since Gref mentioned that galactose-modified oligosaccharides showed high affinity for asialoglycoprotein receptors in hepatocyte [14], a number of synthetic approaches for galactosylate compounds have been reported. Chung et al. [15] synthesized galactosylated chitosan through the covalent coupling of lactobionic acid with chitosan. Park et al. [16,17] reported that galactosylated chitosan-graft-dextran (GCD) and chitosan-graft-PEG (GCP) were synthesized and characterized as hepatocyte-targeting gene carriers, respectively. The complexes were only transfected with cells having asialoglycoprotein (ASGR). Chitosan-O-PEG-galactose as a targeting ligand for glycoprotein receptor was prepared through several steps [14]. In this study, we attempted the synthesis of a novel galactosylated chitosan (GC) in order to improve the solubility and specificity of chitosan to target cells. Subsequently, the CS/DNA and GC/DNA nanoparticles were prepared, with an average diameter of 381 nm and 350 nm, and average zeta potential of +11.1 mV and +22.1 mV, respectively. Then, we studied cell viability after treatment of HEK293 cells with GC. The results showed that the novel galactosylated chitosan may be used as a potential gene delivery system.

0927-7765/$ – see front matter. Crown Copyright © 2008 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.12.018

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B. Song et al. / Colloids and Surfaces B: Biointerfaces 70 (2009) 181–186

2. Experimental

and the specific viscosity is defined as

2.1. Materials

sp = nr − 1

Chitosan (Mv = 48 kDa, degree of deacetylation 90%) was supplied from Nanjing Weikang Biotechnology Co., Ltd. (Nanjing, China). Dialysis tube (molecular weight cut-off 8–14 kDa) was obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). Other chemicals were purchased from Aldrich and Acros Chemical Companies. All the solvents were properly purified before use.

Galactose-g-chitosan (GC) was prepared [14,18,19] as in Fig. 1. Chitosan (0.5 g, 3.03 mmol) and d-galactose (0.75 g, 4.17 mmol) were dissolved in 50 mL dry THF, and then 5.24 mL BF3 ·OEt2 was added under argon atmosphere. The solution was stirred at 60 ◦ C for 24 h in argon and concentrated by rotary evaporator. The condensed liquid was poured into anhydrous methanol under magnetic stirring, and the obtained precipitate was washed several times with anhydrous methanol. The resulting product was dialyzed in deionized water for 3 days. The solution was evaporated with rotary evaporator to give 0.65 g of a light yellow filmy material. 2.3. Measurement of FT-IR and 1 H NMR FT-IR spectra were measured using Shimadzu FTIR-4200 spectrometer as KBr pellets or thin films on KBr plates. 1 H NMR spectra were recorded on a Bruker DRX 400 spectrometer (400 MHz). 2.4. Determination of galactose content The anthrone-sulfuric acid colorimetric assay was used to determine the content of galactose in GC [14,20]. Anthrone was dissolved in 80% sulfuric acid to prepare anthrone-sulfuric acid reagent before use. The calibration curve was constructed based on the various galactose concentrations and their corresponding absorbance determined by spectrophotometer at 628 nm. The concentration of galactose graft onto chitosan was calculated from the calibration curve via its absorbance at 628 nm. The amount of galactose grafted onto GC and the degree of substitution (DS) of chitosan by galactose was calculated by the following equations: Gal (w/w)

 measured galactose weight in the sample 

DS =

sample weight

× 100%

Gal (w/w)/M1 × 100% (100 − Gal (w/w))/M2

where M1 is the molecular weight of galactose (180 g/mol), and M2 is the average molecular weight of chitosan monomer (165 g/mol) according to the degree of deacetylation of chitosan. 2.5. Determination of intrinsic viscosity Viscosity is also important characteristic to consider properties of polymer. Intrinsic viscosity of GC and chitosan in 0.5 mol/L HOAc/0.5 mol/L NaOAc were measured using an Ubbelohde viscometer in a water bath at 25 ± 0.01 ◦ C in triplicate. The capillary diameter used was 0.4 mm. The intrinsic viscosity [] determined by r and sp . The relative viscosity is defined as r =

t to

[] =

[2(sp − ln r )]1/2 C

where C is the concentration of tested solution.

2.2. Preparation of galactose-g-chitosan

=

where to is the flow time of solvent and t is the flow time of tested solution. The intrinsic viscosity was calculated by the following equation:

2.6. Preparation of the GC/DNA nanoparticles GC and chitosan were dissolved in a 1% (v/v, pH 5.5) aqueous acetic acid solution, and they were then filtered through a 0.22 ␮m membrane to wide off bacteria, respectively. The ion-crosslinking method was used to prepare chitosan nanoparticles. To prepare GC/VRMFat-1 nanoparticle solution, GC and plasmid VRMFat-1 solution containing appropriate concentration of triphenylphosphine (TPP) were heated on a water bath of 65–70 ◦ C for 10 min and were then mixed adequately with stirring. The ratio of GC to plasmid VRMFat-1 was 30:1 (w/w). The CS/VRMFat-1 nanoparticle solution was prepared by the same procedures described as above. 2.7. Morphology, size and zeta potential measurements The morphology of GC/VRMFat-1 nanoparticle was observed using a transmission electron microscope (TEM; JEOL, Japan). The particle size, zeta potential and polydispersity (size distribution) of GC/VRMFat-1 nanoparticles were measured using a 3000HS/IHPL Zetasizer. The size of nanoparticles formulated at different parameters was statistically analyzed as the mean values of three measurements ± the standard deviation (S.D.). 2.8. Interaction between GC and plasmid VRMFat-1 The interaction between GC and plasmid VRMFat-1 was studied using analysis of the electrophoretic mobility of DNA within a 0.7% agarose gel containing ethidium bromide. And the N/P ratio was 10/1, 20/1 and 30/1. Bands were visualized by UV light and photographed. 2.9. In vitro cell transfection The human embryonic kidney (HEK293) cells, obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (CBTCCCAS), were washed with RPMI1640 standard growth medium, and the cells were collected by centrifugation for 5 min at 800–1000 rpm. Cells were cultured in standard growth medium (DMEM supplemented with 10% fetal calf serum and 100 ␮g/mL ampicillin–strephomycin) at 37 ◦ C and 5% CO2 until the confluency reached over 80%. Thereafter, DMEM was used to adjust the number of cells and cells were plated in 12-well plates at 1 × 105 cells/mL. The cells were incubated with the CS/VRMFat-1 nanoparticle and GC/VRMFat-1 nanoparticle at 37 ◦ C in 5% CO2 incubator, respectively. At different time point (24 h, 48 h and 72 h) after transfection, the growth situation of cells was observed with microscope and photographed. The cells were collected, and then centrifuged for 5 min at 1000 rpm and thoroughly washed with 0.9% NaCl. The precipitate was extracted with chloroform/methanol (2:1, v/v) containing 0.2% acetic acid. The chloroform phase containing fatty acid of cells was collected.

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Fig. 1. The synthesis method of GC.

2.10. Fatty acid analysis The collected fatty acids were esterified using BF3 ·OEt2 /CH3 OH (1:4, v/v) in hexane and heated at 100 ◦ C for 1 h under argon atmosphere [21]. Fatty acid methyl esters were analyzed by gas chromatography–mass spectrometry (GC–MS, Shimadzu GCMS QP 2010). 3. Results and discussion 3.1. Synthesis and characterization of GC Fig. 1 shows the synthesis method of galactose-g-chitosan (GC), in which galactose moieties were grafted onto chitosan molecule by etherification of chitosan and galactose in THF using BF3 ·OEt2 as promoter. Thus a useful one-pot synthesis which is more convenient than the usual multistep reaction, is important for the preparation of chitosan derivatives. The heterogeneous media may avoid the self-condensation of galactose which was not protected [18]. However, due to the heterogeneous media, the degree of substitute of galactose was not high. The degree of O-substitution of chitosan by galactose was measured to be 10.38% using anthronesulfuric acid colorimetric method. Fig. 2 shows the FT-IR spectra of chitosan (a) and GC (b). Chitosan spectrum exhibits the absorption bands (cm−1 ) at 3441 (O–H stretch overlapped with N–H stretch), 2875 (C–H stretch), 1645 (amide I band, C–O stretch of acetyl group), 1599 (amide II band,

Fig. 2. The spectra of infrared spectroscopy of chitosan (a) and GC (b).

N–H stretch), 1327 (amide III), 1158 (anti-symmetric stretching of the C–O–C) and 1094 and 1029 (skeletal vibrations involving the C–O stretching) [22,23]. Compared with chitosan, the peaks of amides I and II of GC appeared at 1633 and 1528. Compared with those of chitosan, the peaks of amides I and II of GC showed bathochromic shift. The above information indicated that the conformation of chitosan changed after reaction with galactose [24]. Fig. 3 shows the 1 H NMR spectra of chitosan (a) and GC (b). The proton assignment of chitosan is as follows (D2 O/CF3 COOD, ppm) 4.8 (H1), 3.5–3.8 (H3, H4, H5), 3.56 (H6), 3.1 (H2), 2.0 (NHCOCH3 ). Compared with chitosan, the 1 H NMR spectrum of GC (D2 O, ppm) showed a new peak at 3.44, which was assigned to H6 of GC. The others of GC were assigned to 4.6 (H1), 3.4–3.8 (H3, H4, H5, H2 , H3 , H4 , H5 , H6 ), 3.0 (H2), 1.9 (NHCOCH3 ). All above confirmed that galactose was successfully reacted with chitosan by etherization. 3.2. The viscosity characterization of CS and GC solutions Due to strong intermolecular hydrogen bonding of amino and hydroxyl groups, chitosan is not dissolved in neutral water. In addition, the intrinsic viscosity [] can estimate the polymer-solvent interaction parameter. So [] of CS and GC were measured in acetate buffer (aqueous 0.5 mol/L HOAc/0.5 mol/L NaOAc) using an Ubbelohde viscometer in a water bath at 25 ± 0.01 ◦ C. Table 1 shows the r , sp and [] of CS and GC, respectively. [] of CS is 3.55 dL/g, and [] of GC is 2.60 dL/g. These could demonstrate that chitosan was degraded in the reaction process.

Fig. 3. The spectra of 1 H NMR of chitosan (a) and GC (b).

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B. Song et al. / Colloids and Surfaces B: Biointerfaces 70 (2009) 181–186 Table 1 The r , sp and [] of CS and GC. Sample name

r

sp

[] (dL/g)

CS GC

1.03 1.05

0.03 0.05

3.55 2.60

Fig. 5. TEM image of GC/VRMFat-1 complexes (80,000×).

3.3. Characterization of GC/VRMFat-1 nanoparticles

Fig. 4. Electrophoretic mobility analysis of GC/VRMFat-1 nanoparticles. Nanaoparticles were incubated with different N/P mass ratios. All samples were run in a 0.7% agarose gel and stained with ethidium bromide. Lane 1: nanoparticles (N/P 30); lane 2: nanoparticles (N/P 20); lane 3: nanoparticles (N/P 10); lane 4: ␭DNA marker.

The formation of complexes between GC and VRMFat-1 was confirmed by gel retardation assay (Fig. 4). Free DNA plasmid alone was shown on the fourth lane. When GC was mixed with DNA, electrostatic interactions drove the formation of complexes between GC and DNA. The migration of DNA in the agarose gel was retarded because of the charge neutralization and increase in the molecular size of the complexes. Besides, the figure also shows that the addition of N/P mass ratio played an important role in making structure of complex more compact (lanes 1–3 of Fig. 4).

Fig. 6. HEK293 cells were transfected with CS/VRMFat-1 (a) and GC/VRMFat-1 (b) (N/P mass ratio of 30), and cell viability was observed under microscope 24 h (1), 48 h (2) and 72 h (3) after transfection, respectively.

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Table 2 The particle characterization of CS/VRMFat-1 and GC/VRMFat-1 (N/P mass ratio 30).

Fig. 7. The GCMS spectra of fatty acids of transfection cells. In the figure (a) plamitic acid and (b) linoleic acid.

Sample name

T (◦ C)

Particle diameter (nm)

Zeta potential (mV)

CS/VRMFat-1 GC/VRMFat-1

25 25

381 350

11.1 22.1

the zeta potential values, being positive or negative, depend on the N/P ratios. While N/P mass ratio was 20:1, the zeta potential of GC/VRMFat-1 nanoparticles was −7.25 mV. When the value of GC to DNA mass ratio was 30:1, the zeta potential of GC/VRMFat-1 nanoparticles increased to +22.1 mV. 3.4. Cell viability

Fig. 5 shows TEM image of GC/VRMFat-1 complexes at N/P mass ratio 30. In addition, the image indicates that the complexes had well-formed spherical shape. The subsequent particle characterization demonstrated that the particle size of the nanoparticles was for both CS/VRMFat-1 and GC/VRMFat-1 complexes in the nanometer range (Table 2), and the mean particle diameter for GC/VRMFat1 was 350 nm. The measurement of the zeta potential is a useful method for evaluating GC binding DNA. In order to successfully facilitate uptake by the cell, the positive surface charge of polyplexes is necessary for binding to anionic cell surfaces [24]. In general,

HEK293 cells were transfected by CS/VRMFat-1 and GC/VRMFat1, respectively. The growth situation of cells was observed and recorded with microscope after 24 h, 48 h and 72 h (Fig. 6). At 24 h and 48 h, the cells had no death. After 72 h, only a few cells had started dying and desquamating. The above showed that cytotoxicity of transfection material was very low. After HEK293 cells were transfected by CS/VRMFat-1 and GC/VRMFat-1, fatty acids of cells were measured by gas chromatography mass spectrometry (GCMS) (Fig. 7). Statistical analysis indicates that as transfection time increased, saturated fatty acid (palmitic acid) and omega-6 (n-6) polyunsaturated fatty acid (linoleic acid) percentages gradually decreased in the transfected groups (CS/VRMFat-1 and GC/VRMFat-1 group) (P < 0.05), while omega-3 (n-3) polyunsaturated fatty acid (docosahexaenoic acid, DHA) percentage increased in the transfected cells (P < 0.05) (Fig. 8). These results indicated the transcription and expression of MFat-1 gene in the eukaryotic cells was successful. That provides the basis for the study of transcription expression in vivo. The result shows that VRMFat-1 was successfully transfected into HEK293 cells by GC and CS, respectively. 4. Conclusions A novel GC was synthesized. The chemical structure of the product was characterized by FT-IR and 1 H NMR. The hydrophilic property of GC conquered the insolubilization of chitosan in neutral water. In this research, GC showed great ability to form a complex with DNA and proper physicochemical properties for a gene carrier. Compared with CS/VRMFat-1, the average zeta potential of GC/VRMFat-1 complexes was higher positive. Our future work will focus on researching hepatocyte culture and transfection activity in vivo as a novel hepatocyte-targeting gene carrier. Acknowledgements This work was supported by the Foundation of Education Ministry of China (No. 20070610053) and Key project of Sichuan Province (No. 2006Z06-010). We thank the excellent analysis service of fatty acids provided by Analytical & Testing Center of Sichuan University. References

Fig. 8. With increase of transfection time, the change of palmitic acid (a), linoleic acid (b) and DHA (c) percentages of the transfected groups (CS/VRMFat-1 and GC/VRMFat-1 group) were obvious in comparison with those of the blank control on 24 h, 48 h and 72 h after transfection (P < 0.05), respectively.

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