EHOOPrAHH^ECKAa XHMH3, 2015, moM 41, № 5, c. 627-632
SYNTHESIS AND CHARACTERIZATION OF POLYSIALIC ACID/CARBOXYMETHYL CHITOSAN HYDROGEL WITH POTENTIAL FOR DRUG DELIVERY © 2015 J. R. Wu#, X. B. Zhan, Z. Y. Zheng, and H. T. Zhang
Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology,
Jiangnan University, Wuxi, 214122 China Received September 10, 2014; in final form March 3, 2015
A novel hydrogel was prepared from polysialic acid (PSA) and carboxymethyl chitosan (CMCS) using glut-araldehyde as the cross-linking agent. The resulting PSA—CMCS hydrogel exhibited pH sensitivity, in which the swelling ratio under acidic conditions was higher than those under neutral or alkaline conditions. The swelling ratio of PSA—CMCS hydrogel at equilibrium depended on the medium pH, the cross-linking agent concentration, and the ratio of PSA to CMCS (w/w). Bovine serum albumin (BSA) and 5-fluorouracil (5-FU) were used as model drugs to prepare hydrogel delivery systems. The loading efficiencies of the hydrogel for BSA and 5-FU were 26.25 and 36.74%, respectively. Release behaviors of BSA and 5-FU were influenced by the pH. MTT assays confirmed that PSA—CMCS hydrogel has no cytotoxicity toward the NIH-3T3 cell line; in fact, the 100% aqueous extract of the PSA—CMCS hydrogel enhanced cell growth. These results suggest that PSA—CMCS hydrogel may be a promising pH-sensitive delivery system, especially for hydro-phobic chemicals.
Keywords: polysialic acid, carboxymethyl chitosan, hydrogel, drug delivery.
Polysialic acid (PSA) is a negatively charged homopolymer of a-2,8-linked sialic acids (N-acytyl-neuraminic acid) found at the N-glycan terminal of neural cell adhesion molecule (NCAM) . PSA can be easily produced in large-scale setups by fermentation with Escherichia coli  and presents great potential in biomedical applications. PSA exhibits prospective use as a biomaterial because of its biodegradability, biocompatibility, and non-immunogenicity . Certain pathogenic bacteria, such as Neisseria meningitis, E. coli K1, Haemophilus ducreyi, and Pasteurella heamolytica, can synthesize PSA, and the polysaccharide can help these pathogens escape from mammalian immunological recognition and killing through mimicking the host's cellular oligosaccharides . Based on this observation, Gregoriadis et al.  developed a polysialy-lation method for modifying therapeutic proteins to improve protein pharmacokinetics and stability. Several groups have employed this technology to modify catalase , asparaginase [7, 8], insulin , CuZn-su-peroxide dismutase , and butyrylcholinesterase
Abbreviations: BSA, bovine serum albumin; CMCS, carboxymethyl chitosan; GA, glutaraldehyde; 5-FU, 5-fluorouracil; PSA, polysialic acid; RGR, relative growth rate. # Corresponding author (phone +86 (510) 8591-8299; e-mail: email@example.com).
. Some of these proteins have been used in clinical studies, and their functions were observed to be similar to those of PEGylation. PSA may also be employed in nerve-cell tissue engineering because of the similarity of its structure to NCAM [12-14].
Functional groups, such as amino, hydroxyl, and carboxyl groups, can react with other macromolecules to produce nanoparticles, hydrogels, and microcapsules for drug delivery. In 2009, Bezuglov et al.  mixed PSA with insulin, interferon, and rhG-CSF to produce nanoparticles and found that PSA-insulin nanoparticles could prolong hypoglycemic ability. In 2011, Bader et al.  used PSA and decylamine to produce micelles that could encapsulate hydrophobic cyclosporin by self-assembly. Similarly, a PSA-epiru-bicin conjugate prolonged the half-life of epirubicin in vivo with low cytotoxicity . In 2012, Zhang et al.  prepared PSA-N-trimethyl chitosan nanoparticles that exhibited excellent slow-release characteristics for methotrexate. Wilson et al.  employed PSA and polycaprolactone to prepare a self-assembled micelle for encapsulation of cyclosporin.
PSA, as a carrier in drug delivery, may improve pharmacokinetics and stability as well as anti-immu-nogenicity for therapeutic drugs . Hydrogels are an important formulation in the development of new drug delivery technologies. In this work, PSA and car-
Fig. 1. Influence of weight ratio of PSA to CMCS on the pH-sensitivity of hydrogel: mpsA : mCMCS = 1 • 10 (■), 1 : 4 (•), 1 : 2 (A), 1 : 1(Y), 5 : 2 (♦).
at Og el
Fig. 2. Influence of glutaraldehyde on the swelling ratio of PSA—CMCS hydrogel (the mpsA : mCMCS was set as 1 : 1, pH 7.4, 40 mM PBS).
boxymethyl chitosan (CMCS) are used to synthesize a novel functional hydrogel that may potentially be used as a drug carrier.
RESULTS AND DICUSSION
Chitosan and its derivatives are sensitive to pH because of their characteristic chemical structure. As shown in Fig. 1, PSA—CMCS hydrogel with various weight ratios showed different swelling ratios in media of different pH. The swelling ratio of the hydrogel was the highest at pH 1.2, and no linear dependence in the concentrations of PSA and CMCS were observed. This finding may be explained by protonation of the amino group (—NH2) in CMCS, which increases the electrostatic repulsion and hydrophilicity of the hy-drogel. At pH 3.4, CMCS was close to its isoelectric point, and the swelling ratio reached minimal levels.
In this situation, the charges of —COO- and —N H+ were equivalent, and electrostatic interactions reduced the free ions. Ionic bonding between the amino and carboxyl groups could also lead to the compact structure of the PSA—CMCS hydrogel.
When the pH was increased, the swelling ratio was improved. The swelling ratio of the hydrogel under pH 7.4 reached about 40% for the case of mPSA : mCMCS = = 1 : 10 and mPSA : mCMCS = 1 : 4, which indicates that relatively high amounts of CMCS render the hydrogel more liable to pH shift. Less protonated —NH2 and more hydrophobic —COOH led to the loose structure of the hydrogel. At pH beyond 8.0, the swelling ratio of the PSA—CMCS hydrogel slightly decreased. In all cases, however, the hydrogel with more CMCS achieved slightly higher swelling ratios.
Glutaraldehyde (GA) was used as the cross-linking agent, and its concentration influenced the hydrogel
property . As shown in Fig. 2, the swelling ratio increased and then decreased as the amount of cross-linking agent increased. When the GA dose was 0.05% (w/w), the hydrogel did not form readily and the gelling time was long. When the GA dose was increased to 0.1%, the polymers formed a three-dimensional (3D) network and absorbed water, leading to a higher swelling ratio. At 0.15% GA dose, the highest swelling ratio was obtained. When the cross-linking agent was increased further, the connection points in the network could be improved, and the distances between these connections could decrease, leading to smaller pores in the hydrogel network. The resulting swelling ratio decreased with less water adsorption. The optimal dose of GA was thus determined as 0.15%.
A colorless and transparent hydrogel was prepared using PSA and CMCS. In the scanning electron microscopy (SEM) images shown in Fig. 3, the PSA— CMCS hydrogel was interconnected and showed a 3D porous structure. The distribution of pores with various sizes was heterogeneous. As a potential biomaterial, the interconnections of the pore structure could favor water and nutrient transportation. Adsorption of water into the pores would also endow the hydrogel with a high swelling rate.
When analyzed by IR spectroscopy, the spectra of chitosan (CS) and CMCS showed no significant difference (Fig. 4). In CMCS, an absorbance peak at 1608 cm—1, which indicates carboxyl groups, was observed. The absorbance of—NH in CMCS at 1427 and 1334 cm—1 showed a slight shift compared with those of chitosan. Peaks at 1334—1600 cm—1 indicate the characteristic absorbance of —NH2 group of CMCS. With regard to PSA, peaks at 1684 and 1402 cm—1 reflect the absorbance of stretching vibrations of —COOH. For the PSA—CMCS hydrogel, peaks of—NH2 vibrations
Fig. 3. Morphology and scanning emission microscopy of PSA—CMCS hydrogel.
at 1334-1600 cm-1 weakened. Peaks at 1684 and 1402 cm-1, which indicate the -COOH of PSA, also
weakened. The absorbance peak of -N H+ • COO- appeared at around 1620 cm-1. At 1420 cm-1, the peak indicating -COO- symmetrical stretching vibrations was enhanced, which illustrates the interaction of PSA and CMCS in the gelling process.
In a typical digestion process, drugs stay in the stomach for about 1 h and then move to the intestinal tract, where they stay for some time. In this work, we attempted to simulate the gastric juice (HCl at pH 1.2) and body fluid (PBS at pH 7.4) as simulative intestinal juices for in vitro testing of the loading and release behaviors of bovine serum albumin (BSA) in the hydrogel. Result
4000 3500 3000 2500 2000 1500 1000 500 Wave number, cm-1
Fig. 4. Infrared spectroscopy of PSA—CMCS hydrogel.
showed that the drug-loading rate is 26.25 ± 0.82% (n = = 3). In terms of drug-release rate, the release rate of BSA from the hydrogel under acidic conditions occurred faster than that under neutral conditions (Fig. 5). At pH 1.2, the release rate of BSA occurred rapidly during initial stages and reached 75.78% after 8 h of incubation, which is double that at pH 7.4. At later stages, the BSA release rate stabilized to 85.86% at 24 h, likely because -NH2 in the hydrogel may have become pro-
tonated and the repulsive effect of -N H+ improved the pore size of the hydrogel, leading to the release of BSA. Under neutral conditions, only a small number of amino groups are protonated, and hydrogen bonding between amino and carboxyl groups may be en-
0 5 10 15 20 25 Ti
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.