载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

CoSalen immobilized to chitosan and its electrochemical behavior

A chitosan (CS) immobilized cobalt complex (CS–CoSalen) was prepared by immersing a CS film into the saturated aqueous solution of a cobalt(II) complex of N,N′-bis(salicylaldehyde)ethylenediimino (CoSalen). The CS–CoSalen was characterized, and it was found that CoSalen was immobilized onto CS through the coordination of the amino group of CS. The cobalt(II) ion in the CS–CoSalen has a five-coordination structure. Its vacant sixth coordination site was found to bind molecular oxygen reversibly. Electrochemical studies demonstrated that the CS–CoSalen is active for the catalysis of the electrochemical reduction of oxygen to hydrogen peroxide in an aqueous medium.     

Effects of Cricket Extract on Lipid Metabolism and Body Fat Content in High- Fat Diet Fed Rats

The effect of cricket extract on high‐fat diet fed rat was observed. It was shown that cricket extract prevented the increment of body weight by high‐fat diet. The extract also decreased the value of AST in liver. The most significant effect of the extract was shown on lipid metabolism. The contents of total lipid and total cholesterol in liver and feces were reduced by the extract on dose‐dependent. These statistically significant results were clear in 3% extract treated group (HFD3) while were slight in 1% extract treated group (HFD1). The same result was also shown in body fat content.     

Insulin-loaded alginate microspheres for oral delivery – Effect of polysaccharide reinforcement on physicochemical properties and release profile

Oral administration of insulin requires protein protection from degradation in the gastric environment and its absorption improvement in the intestinal tract. To achieve this objective several types of microspheres composed of alginate, chitosan and dextran sulphate have been prepared by ionotropic gelation. Parameters such as the mean particle size, swelling behaviour, insulin encapsulation efficiency, loading capacity and release profiles in simulated gastric and intestinal fluids have been compared for the systems developed. In this study, attempts have been made to increase insulin protection and to improve its release from microspheres by reinforcing the alginate matrix with chitosan and/or dextran sulphate. Dextran sulphate was able to avoid insulin release at pH 1.2, protecting the protein from the acidic environment and reducing the total insulin released at pH 6.8. This effect was explained by an interaction between the permanent negatively charged groups of dextran sulphate and insulin molecules.     

Antitumor effect of therapeutic HPV DNA vaccines with chitosan-based nanodelivery systems

Background

Cervical cancer is the second-most-common cause of malignancies in women worldwide, and the oncogenic activity of the human papilloma virus types (HPV) E7 protein has a crucial role in anogenital tumors. In this study, we have designed a therapeutic vaccine based on chitosan nanodelivery systems to deliver HPV-16 E7 DNA vaccine, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer. We have developed a Nano-chitosan (NCS) as a carrier system for intramuscular administration using a recombinant DNA vaccine expressing HPV-16 E7 (NCS-DNA E7 vaccine). NCS were characterized in vitro for their gene transfection ability.

Results

The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. In addition, NCS-DNA E7 vaccine induced the strongest E7-specific CD8+ T cell and interferon γ responses in C57BL/6 mice. Mice vaccinated with NCS-DNA E7 vaccine were able to generate potent protective and therapeutic antitumor effects against challenge with E7-expressing tumor cell line, TC-1.

Conclusions

The strong therapeutic effect induced by the Chitosan-based nanodelivery suggest that nanoparticles may be an efficient carrier to improve the immunogenicity of DNA vaccination upon intramuscular administration and the platform could be further exploited as a potential cancer vaccine candidate in humans.     

The release of active substances from selected carbohydrate biopolymer membranes

As a biopolymer application to control release systems is increasing at present, flat matrices (transdermal systems) should be highlighted. They constitute one of the most friendly form of drug administration to the patient. The present results concern investigations on the active substance release (ibuprofen and salicylic acid) from film matrices made from biopolymers: polylactid acid (PLA), dibutyrylchitin (DBC) and chitosan (CH). The amount of released active substance was examined with UV-VIS spectrophotometer. The release process was conducted in the medium of pH = 5.6 as the transdermal systems are applied to human skin surface of pH value approximating 5.6. Swelling of polymer samples was confirmed in the buffer of pH = 5.6 as well.The paper comprises the analysis of obtained release results according to the proposed two stage complex diffusion model.     

Synthesis and characterization of a brush-like copolymer of polylactide grafted onto chitosan

A brush-like poly(DL)-lactide grafted onto chitosan as the backbone was investigated. The graft copolymerization was carried out with triethylaluminum as catalyst in toluene at 70 degrees C. It was found that a greater lactide content in the feeding ratio results in a higher grafting percentage. FTIR spectrometry, (1)H NMR, DSC scanning, and wide-angle X-ray scattering, respectively, are used to characterize these branch copolymers. A copolymer has a definite melting point when the molar feeding ratio of lactide to chitosan is more than 10:1, and the deltaH of the copolymers increases with the feed ratio of lactide to chitosan in feeding.     

Synthesis and properties of chitosan hydrogels modified with heterocycles

Preparation and properties of chitosan modified with heterocycles in absence or presence of gluteraldehyde as a cross linker is described. New modified chitosan–heterocyclic hydrogels were prepared from chitosan and heterocyclic compounds such as N,N′-biisomaleimide, N,N′-biisophthalimide, and N,N′-phthalimidomaleimide via a crosslinking reaction. The new hydrogels chemical structure was characterized by spectral analysis (IR), X-ray diffraction, thermal gravimetric analysis (TGA), solubility, and swellability in water and different organic solvents. Evaluation of the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems was carried out and promising results were obtained.     

Miscibility study of chitosan/2-hydroxyethyl starch blends and evaluation of their effectiveness as drug sustained release hydrogels

Polymeric matrices of chitosan (CS), 2-hydroxyethyl starch (HES) and their blends prepared by solvent evaporation technique, have been tested as sustained release hydrogels of ropinirole drug. X-Ray diffraction (XRD), infrared spectroscopy (FT-IR) and viscometry measurements showed that the two polymers can form miscible blends. This miscibility is owed to formed hydrogen bonds taking place between the reactive groups of CS and HES and one glass transition is recorded in all blends. Neat polymers were used to prepare solid dispersion formulations with ropinirole drug. It was found that drug was released immediately within 15-30 min from HES while the release was slower from CS matrix. Completely different were the release rates from ropinirole with physical mixtures using neat polymers and their blends. Due to the different solubility and swelling behaviour of CS and HES the release rates showed a sustained profile from the blends containing high amounts of CS.