Advanced Materials Research Vols. 466-467 (2012) pp 454-457 Online available since 2012/Feb/10 at www.scientific.net © (2012) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.466-467.454
Preparation of nanoparticles by crosslinking folate conjugated chitosan with vanillin and its characterization Min Zhou1, a, Puwang Li1, b*, Guang Wang1, c, Ziming Yang1, d, Zheng Peng1, e, Lingxue Kong2f, 1 2
Agricultural Product Processing Research Institute, CATAS, Zhanjiang 524001, P. R. China Centre for Material and Fibre Innovation, Institute for Technology and Research Innovation, Deakin University, Geelong Vic 3217 a
[email protected], b*
[email protected],
[email protected], f
[email protected],
[email protected]
Keywords: Folic acid, chitosan, vanillin, chemical modification
Abstract. Functionalized chitosan (CS) were widely used as drug delivery system in the chemotherapy of various disease. In this work, folate (FA) was conjugated into chitosan molecular as targeting ligand based on Schiff reaction between –NH2 group of CS and –COOH group of FA. And nanoparticles were made by emulsion method with vanillin novel cross-linking agent. The FA modified CS and its nanoparticles were characterized by Fourier transform spectroscopy (FT-IR), scanning electron microscope (SEM) and Zeta potential. SEM results confirmed the nanoparticles made from FA-CS conjugate were spherical in shape and were about 100 nm in size. Zeta potential analysis revealed that the nanoparticles were negatively charged with charge density of −7.73mV. Introduction Biocompatible and biodegradable polymeric nanoparticles have attracted great attention as potential transporter for the controlled-release and site-specific delivery of drug molecules[1]. CS is the only natural alkaline polysaccharide, which is obtained from the deacetylation of chitin. It can form nanoparticles or microparticles due to the existing of one amino group and two hydroxyl groups. CS nanoparticles are attractive drug carriers due to their good properties such as biocompatiblity, biodegradability, low toxicity and anti-infectional activity[2] . Therefore, CS nanoparticles were widely investigated as drug carrier for the delivery of anticancer drug. Several cross-linking agent have been used for the preparation of CS nanoparticles such as glutaraldehyde[3], formaldehyde[4] or sodium tripolyphosphate. However, glutaraldehyde and formaldehyde would give rise to health concerns and could cause undesirable side effects. The structure of CS nanoparticles made by crosslinking with TPP was not compact and its application was often limited by its burst release effect. In view of the above-mentioned disadvantages it is necessary to find a novel cross-linker for the preparation of CS nanoparticles. Vanillin, obtained from the vanilla bean, is often used as flavouring agent and food preservative, and is generally regarded as a safe substance. Moreover, vanillin is also used for other purposes such as drug preparation[5]. The amino groups in CS and the aldehyde groups in vanillin may occur Schiff base reaction and form a network structure which can replace poisonous cross-linkers to meet the controlled-release demand. Although controlled release has been solved by using vanillin as across-linking agent, the ideal drug delivery system must combine targeted delivery with controlled release so that the drug can delivered and released in a selective and discriminatory fashion[6,7]. Such a system not only minimizes the systemic toxicity, but enhances the efficacy of the drug which we transported. Thereby improve the quality of the patients’ life. With respect to targeted release, the primarily targeted agent is due to its significantly over-expressed on many cancerous cells, while limited expression on normal cells[8,9]. Besides, the presence of functional carboxylic groups on the FA molecules offers possibility for its coupling with the polymeric carriers such as CS.
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The aim of the current work is to prepare FA-CS nanoparticles drug carriers. The resultant FA-CS nanoparticles were characterized with FTIR and their physicochemical properties were investigated in terms of surface morphology, Zeta potential and particle size. Material and methods Materials. Chitosan (CS) derived from crab shell, was obtained from, the deacetylation degree was 90%. Folate (FA) was purchased from Sigma Chemistry (Beijing, China). All other solvent and reagents were of analytical grade and were used us received. Synthesis of FA-CS conjugates Chitosan (1g) was previously dissolved in 100 mL acetic acid solution at the concentration of 1%. FA (0.4g) was dissolved in anhydrous 30 mL DMSO and was activated by EDC (0.1g). Then, the mixture solution was dropped into the CS solution and were kept stirring for 6 h. After that, the resultant products were coagulated by adding 300 mL acetone, and the coagulation was purified twice with DMSO and distilled water. Finally, yellow colored FA-CS products were collected and freeze-dried at −50 °C for 24 h. Preparation of NPs FA-CS NPs were prepared by emulsion chemical cross-linking method. Briefly, FA-CS (0.1 g) was dissolved in 10 mL acetic acid solution (1%) as water phase. Liquid paraffin (50mL) containing 1% span 80 and 1% tween 80 were used as oil phase. The water phase was added dropwise into the oil phase by an injector under mechanical stirring (1000 rpm) and the system were kept stirring for about 30 min. Then vanillin dissolved in acetone was added slowly into the emulsion system under high speed stirring. Nanoparticles were formed spontaneously and were collected after washed with petroleum ether twice and acetone twice. The products were freeze-dried and kept in a desiccator. Characterization of FA-CS and its NPs FTIR spectrum was obtained by scanning samples from 4000 to 400 cm−1 on a FTIR analyzer (GX-1, Perkin Elmer, USA). Morphology of the nanoparticles was observed at 10 kV on a scanning electronic microscopy (SEM, S-4800, Hitachi, Japan). Particles size and Zeta potential of nanoparticles were analyzed on Nano-ZS (Malvern, UK). Result and discussion FT-IR analysis of FA-CS conjugates and its nanoparticles Fig. 1 shows the FT-IR spectrum of chitosan, folate, FA-CS, vanillin and FA-CS NPs. In the FT-IR spectrum of chitosan (Fig.1a), the absorption peak in 3428 cm−1 was due to the vibration of –OH and N-H, and the absorption peaks in 1646 and 1598 cm−1 belong to the vibration of amide Ⅰand amide Ⅱ groups. Fig.1b shows the FT-IR spectrum of folate, the strong absorption peaks in 3416,1694,1606 and 1458 cm−1 are corresponding to the vibration of N−H, C=O, benzene ring and C=N. Significant difference is observed between the spectrum of CS and FA-CS. It can be seen in the IR spectra of FA-CS, a new absorption peak appears in 1605 cm−1, which belongs to the benzene ring of FA. In addition, the absorption peak of amide at 1646 cm−1 of CS shifts to 1633 cm−1, which is overlapped with the absorption peak of a newly formed C−N bond. The FT-IR spectrum of FA-CS-NPs (Fig. 1d) shown the peak at 1641cm−1 corresponding to characteristic stretching vibration of C=N, which can be attributed to the Schiff base reaction between the aldehyde group of vanillin and amino group of chitosan. From this result, it is assumed that chitosan was cross-linked by vanillin successfully through Schiff base reaction.
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Fig. 1. FT-IR spectrum of (a)CS, (b) FA, (c) FA-CS conjugate and（d）FA-CS nanoparticles. Morphology of FA-CS nanoparticles Surface morphology and particle size distribution of FA-CS nanoparticles were investigated by SEM images (Fig. 2) and laser light scattering (DLS) technology (Fig. 3). It can be seen in Fig.3 that the nanoparticles are spherical in shape with mono-dispersity and the average size is around 100nm (Fig. 2). The average particle size of FA-CS nanoparticles obtained by DLS technology is 105.7nm (Fig. 3). which is consistent with the result obtained from SEM.
Fig. 2. SEM image of FA-CS-NPs. Zeta potential of FA-CS nanoparticles Zeta potential of FA-CS nanoparticles is narrowly distribution with an average Zeta potential of −7.73 mV (Fig. 4). pure CS nanoparticles have an average zeta potential of 50mV. The decrease of Zeta potential of vanillin cross-linked FA-CS nanoparticles has two reasons. Firstly, the modification of CS with FA leads to a dramatic decrease in Zeta potential ascribed to the partial substitution of –NH2 by FA during the process of coupling. Another reason is that the rest of the amino groups in modified chitosan are reacted with aldehyde groups in vanillin to form Schiff base.
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Conclusions FA was employed as targeting ligand and was successfully incorporated into CS for form FA-CS cognates. FA-CS nanoparticles were synthesized by crosslinking with vanillin. FA-CS nanoparticles were negatively charged and were smooth in surfaces with average particle size of 105.7 nm. FTIR results confirmed that the Cross-linking mechanism was based on Schiff base reaction. Such drug carrier could deliver drug into cancer cell precisely due to its FA ligand. Thus it is a promising biomaterial for the tumor targeted drug delivery. Acknowledgement The authors gratefully acknowledges the financial support from Structure Evolution of Epoxidized Natural Rubber under Complex Condition via the Rubber Research Institute at Chinese Academy of Tropical Agricultural Sciences. References [1]
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Intelligent System and Applied Material 10.4028/www.scientific.net/AMR.466-467
Preparation of Nanoparticles by Crosslinking Folate Conjugated Chitosan with Vanillin and its Characterization 10.4028/www.scientific.net/AMR.466-467.454
