Folic acid conjugated glycol chitosan micelles for targeted delivery of doxorubicin: preparation and preliminary evaluation in vitro

For folate receptor (FR) targeted anticancer therapy, novel folic acid (FA) conjugated cholesterol-modified glycol chitosan (FCHGC) micelles were synthesized and characterized by ¹H NMR, dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The degree of substitution was 1.4 FA groups and 7.7 cholesterol groups per 100 sugar residues of glycol chitosan. The critical aggregation concentration of FCHGC micelles in aqueous solution was 0.0169?mg/ml. The doxorubicin (DOX)-loaded FCHGC (DFCHGC) micelles were prepared by an emulsion/solvent evaporation method. The DFCHGC micelles were almost spherical in shape and their size increased from 282 to 320?nm with the DOX-loading content increasing from 4.53 to 11.4%. DOX released from DOX-loaded micelles displayed sustained release behavior. The targeted micelles encapsulated DOX showed significantly greater cytotoxicity against FR-positive HeLa cells than the nontargeted DOX-loaded micelles and free DOX. These results suggested that FCHGC micelles could be a potential carrier for targeted drug delivery.     

壳聚糖多孔支架制备新方法初探

利用NaCl作为致孔剂,采用干热交联壳聚糖与盐淅沥致孔法制备壳聚糖多孔支架,探索该支架制备方法的可行性和安全性.在研究中利用干热交联壳聚糖与盐淅沥致孔法制备了不同质量比(chitosan/NaCl)的壳聚糖多孔支架和冻干法制备的支架,并对所有支架的孔隙率、孔结构、力学性能等参数指标进行评价.结果表明,利用于热交联壳聚糖与盐淅沥致孔法制备过程中加入致孔剂(NaCl)越多,即NaCl/chitosan质量比越大,所构建多孔支架的平均孔径越大,孔隙率越高,抗拉性则下降.     

Tumor-derived microparticles in tumor immunology and immunotherapy

Microvesicles or microparticles, a type of cytoplasm membrane-derived extracellular vesicles, can be released by cancer cells or normal cell types. Alteration of F-actin cytoskeleton by various signals may lead to the cytoplasm membrane encapsulating cellular contents to form microparticles, which contain various messenger molecules, including enzymes, RNAs and even DNA fragments, and are released to extracellular space. The release of microparticles by tumor cells (T-MPs) is a very common event in tumor microenvironments. As a result, T-MPs not only influence tumor cell biology but also profoundly forge tumor immunology. Moreover, T-MPs can act as a natural vehicle that delivers therapeutic drugs to tumor cells and immune cells, thus, remodeling tumor microenvironments and resetting antitumor immune responses, thus, conferring T-MPs a potential role in tumor immunotherapies and tumor vaccines. In this review, we focus on the double-edged sword role of T-MPs in tumor immunology, specifically in TAMs and DCs, and emphasize the application of drug-packaging T-MPs in cancer patients. We aim to provide a new angle to understand immuno-oncology and new strategies for cancer immunotherapy.     

新型壳聚糖对血浆胆红素和细胞因子吸附性能的体外筛选初步研究

目的观察各类新型吸附剂对重型肝炎患者血浆中的胆红素和细胞因子的吸附性能。方法第一步,收集第一组重型肝炎患者的血浆各3ml,以8种不同的吸附剂(1#～3#为致孔剂浓度分别为3%、1%、5%的壳聚糖,4#为胺基化壳聚糖,5#为苯乙烯/二乙烯苯聚合物,6#为后交连苯乙烯/二乙烯苯聚合物,7#为壳聚糖-苯乙烯/二乙烯苯聚合物,8#为壳聚糖-后交连苯乙烯/二乙烯苯聚合物)各1ml进行吸附,检测对胆红素的吸附能力,做吸附剂筛选实验和吸附实验。第二步,用以上筛选出吸附率较好的两种吸附剂各1ml对第二组患者血浆(各3ml)进行胆红素和细胞因子的吸附实验。结果第一步实验显示胺基壳聚糖(4#)和苯乙烯/二乙烯苯聚合物(5#)有较好的吸附效果,4#吸附剂对总胆红素(TBiL)、直接胆红素(DBiL)、间接胆红素(IBiL)的吸附率分别为50.5%±3.4%、57.0%±11.3%、39.0%±7.2%;5#分别为30.1%±2.5%、32.6%±3.0%、27.6%±2.9%。第二步实验显示4#胺基化壳聚糖吸附后血浆中TBiL、DBiL、IBiL、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)水平下降比5#明显,有统计学意义(P<0.001)。结论胺基化壳聚糖(4#)体外吸附重型肝炎患者血浆中胆红素、细胞因子效果有明显优势。     

Self-aggregated nanoparticles of linoleic acid-modified glycol chitosan conjugate as delivery vehicles for paclitaxel: preparation,characterization and evaluation

A series of linoleic acid-modified glycol chitosan (LAGC) conjugates were synthesized and characterized by FTIR and ¹H NMR. The effect of the amount of linoleic acid (LA) on the physicochemical properties of LAGC conjugates was investigated. The mean diameters of three LAGC nanoparticles determined by dynamic light scattering ranged from 204 to 289 nm. The critical aggregation concentration values of LAGC conjugates in aqueous solution were 0.0148, 0.0348, and 0.0807 mg/ml, respectively. Paclitaxel (PTX) was physically loaded into the LAGC nanoparticles by a dialysis method. The drug loading content and encapsulation efficiency of PTX-loaded LAGC (PTX-LAGC) nanoparticles increased with an increasing ratio of the hydrophobic LA to hydrophilic glycol chitosan in the conjugates. PTX-LAGC nanoparticles were almost spherical in shape observed by transmission electron microscopy. In vitro release revealed that PTX release from the nanoparticles was reduced as the LA substitution degree of LAGC conjugates increased. Compared with the commercial formulation Taxol, PTX-LAGC-1 nanoparticles exhibited comparable cellular uptake and cytotoxicity against HepG2 cells in vitro. Importantly, PTX-LAGC-1 nanoparticles demonstrated the stronger antitumor efficacy against hepatic H22 tumor-bearing mice than Taxol (p < 0.05). Therefore, glycolipid-like LAGC nanoparticles had a potential as delivery vehicles for tumor therapy.     

Synthesis and in vitro evaluation of a novel thiolated chitosan

In order to achieve the same properties as chitosan-4-thio-butyl-amidine and to overcome at the same time its insufficient stability, the aim of this study was to evaluate the imidoester reaction of isopropyl-S-acetylthioacetimidate for the chemical modification of chitosan and to study the properties of the resulting chitosan-thioethylamidine (TEA) derivative. The thioalkylamidine substitute was introduced without the formation of N-substituted non-thiol products. The resulting conjugates exhibited 1.05+/-0.17% or 139.68+/-17.13 micromol immobilized free thiol groups per gram polymer and a total amount of reduced and oxidized thiol groups of 1.81+/-0.65% or 179.46+/-67.95 micromol/g polymer. By the immobilization of thiol groups mucoadhesion was strongly improved due to the formation of disulfide bonds with mucus glycoproteins. Chitosan-TEA was investigated regarding to its mucoadhesive properties via tensile studies and the rotating cylinder method. In tensile studies the total work of adhesion of chitosan-TEA was increased 3.3-fold in comparison to unmodified chitosan. Results from the rotating cylinder method showed an improvement ratio of 8.9 for chitosan-TEA compared with unmodified chitosan. In spite of the immobilization of thiol groups onto chitosan its swelling behavior in aqueous solutions was not significantly altered. Cumulative release studies out of matrix tablets comprising the chitosan-TEA and the model compound fluorescence labeled dextrane (FD(4)) demonstrated a controlled release over 3h with a trend toward a pseudo-zero-order kinetic. Because of these features the new chitosan thioamidine conjugate might represent a promising new polymeric excipient for various drug delivery systems.     

Cytochalasin B loaded nano/microparticles:preparation and in vitro drug release evaluation

INTRODUCTION　　 Controlled release drug delivery system can supply a pharmacologically activeagentata predetermined rate fora prolonged period of time.Itcan achieve high localdrug concentrations with lower systemic concentrations when itis placed at or n…     

In vitro and in vivo evaluation of the chitosan/Tur composite film for wound healing applications

We have developed tourmaline/chitosan (Tur/CS) composite films for wound healing applications. The characteristics of composite films were studied by optical microscope, infrared spectra and X-ray diffraction. Tur particles were uniformly distributed in the CS film and the crystal structure of CS was not remarkably changed except the decrease of crystallinity. The influence of Tur on wound healing applications was characterized by modulating Tur concentrations in the Tur/CS composite film prepared by loading Tur powder into CS matrix with different proportion (0, 1/40 and 1/10). Then L929 cells were co-cultured on the composite films to access the cytotoxicity in vitro. Tur concentrations strongly influenced cell process extension. Tur/CS composite film with 1/40 mass ratio could promote the cell adhesion and proliferation. Fewer and shorter processes were observed at high Tur density. When the composite films were transplanted on porcine full-thickness burn wounds, histological results demonstrated that the Tur/CS group with 1/40 mass ratio had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of dermis. Based on the observed facts these films can be tailored for their potential utilization in wound healing and skin tissue engineering applications.     

Nonionic polymer cross-linked chitosan hydrogel: preparation and bioevaluation

A straightforward method to prepare nonionic polymer (polyacrylamide, PAM) cross-linked chitosan hydrogel has been developed. The chitosan–polyacrylamide (CS–PAM) hydrogel could be quickly obtained by simply mixing of chitosan and polyacrylamide solutions under very benign condition (room temperature, <30?s). The cytotoxicity and hemocompatibility of the CS–PAM hydrogel were subsequently investigated. Cells retained normal morphology even when the concentration of CS–PAM hydrogel in culture system was as high as 640?μg?mL^?1, indicating the CS–PAM hydrogel has minimal cytotoxicity to A549 and HeLa cells. Meanwhile, no hemolysis was observed after incubating the CS–PAM hydrogel with red blood cells for 6?h, further suggesting excellent biocompatibility of the hydrogel. Scanning electron microscopy images illustrated that the CS–PAM hydrogel can absorb red blood cells through the interaction between the protonated amine groups on chitosan and the negative charged residues on red blood cell membranes. Given its low cost, simple preparation, and excellent biocompatibility, this CS–PAM hydrogel might be a potential biomaterial for future healthcare and biomedical applications.     

季铵盐壳聚糖影响A549细胞增殖及基质金属蛋白酶-9表达的研究

目的 研究季铵盐壳聚糖对A549细胞增殖能力、侵袭力的影响及机制.方法 使用含有不同浓度季铵盐壳聚糖的RPMI1640培养基培养肺癌A549细胞,采用台盼蓝拒染法检测A549细胞的存活率,MTT法检测季铵盐壳聚糖对A549细胞生长的抑制率,Western Blot检测基质金属蛋白酶基质金属蛋白酶9（MMP-9）的表达情况.结果 台盼蓝拒染实验显微镜下细胞计数显示,A549细胞的存活率大于95％,满足实验要求.MTT实验中随着季铵盐壳聚糖浓度的增高（分别为50,100,200 μg/mL）对A549细胞生长的抑制率的逐渐增强[分别为（27.57±0.037）％,（66.69±0.40）％,（81.55±0.72）％],MMP-9蛋白表达水平也随之降低,呈剂量依赖性,实验组与对照组比较有差异有统计学意义（P＜0.05）.结论 季铵盐壳聚糖能够抑制人非小细胞肺癌A549细胞的增殖,减少MMP-9的表达,进而降低肿瘤细胞的侵袭、转移能力,其作用效应呈剂量依赖性.     

N-氢化可的松琥珀酰基壳聚糖的制备及其体外性质的初步考察

目的 将氢化可的松与壳聚糖进行偶连,以改善其溶解性,延缓其释放。方法以琥珀酰基为连接臂将氢化可的松与壳聚糖进行偶连（N-氢化可的松琥珀酰基壳聚糖）,并通过红外及紫外光谱对偶连物的化学结构进行确证,分别采用溶解实验的方法及动态透析法考察偶连物的溶解性及体外释放特征。结果当氢化可的松琥珀酰基的取代度为15．5％时,N-氢化可的松琥珀酰基壳聚糖易溶于酸性水溶液,而在中性及弱碱性水溶液中形成凝胶;其体外释放速度缓慢,36h内的总释放量为5．8％。结论 壳聚糖作为氢化可的松的高分子载体,能明显改善其溶解性,延缓其释放。     

Cell outer membrane mimetic chitosan nanoparticles: preparation,characterization and cytotoxicity

A negatively charged copolymer poly (MPC-co-AMPS) of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-acrylamide-2-methyl propane sulfonic acid (AMPS) was designed and synthesized. Chitosan nanoparticles with cell outer membrane mimetic structure were prepared by electrostatic interaction between the sulfonic acid groups of poly (MPC-co-AMPS) and the protonated amino groups of chitosan. Effects of factors on influencing the particle size, distribution, and stability were investigated. The experimental results showed that cell membrane mimetic chitosan nanoparticles with controllable and homogeneous size ranged from 100 to 300 nm were prepared at the concentration of 0.1–2.0 mg/mL and the charge ratio of 0.5–1.1. Chitosan nanoparticles prepared can exist stably for more than 45 days when placed at 4° C and pH < 7.5. The cytotoxicity of the chitosan nanoparticles reduced significantly after surface modification with cell membrane mimetic structure, meeting the basic requirements of biomedical materials. The results suggest cell membrane mimetic chitosan nanoparticles prepared with polyanion and polycation obtain good biological compatibility and immune stealth ability, which has important academic significance and great application prospects.     

壳聚糖及其纳米粒子在组织工程中的应用

壳聚糖由于其生物学特性而受到广泛关注,纳米材料在生物医学领域中表现出良好的应用前景。对壳聚糖纳米粒子的制备方法和壳聚糖及其纳米粒子在组织工程中的应用进行了综述。     

壳聚糖增强型左旋聚乳酸与嗅鞘细胞的生物相容性

背景：既往研究表明壳聚糖和左旋聚乳酸制备的复合支架与一些细胞有着良好的生物相容性。 目的：观察壳聚糖增强型左旋聚乳酸支架与大鼠嗅鞘细胞的生物相容性。 方法：将出生1-3 d SD大鼠的嗅鞘细胞接种于壳聚糖增强型左旋聚乳酸膜上作为实验组,设置嗅鞘细胞与多聚赖氨酸联合培养为对照组,培养1,3,5,7 d进行细胞增殖力检测和免疫荧光抗体标记检测。 结果与结论：嗅鞘细胞可在壳聚糖增强型左旋聚乳酸膜上存活,细胞毒性评级为Ⅰ级。实验组细胞形态呈圆形和椭圆形,突起少,细胞聚集成团;接种1 d后,可见部分细胞团的外围细胞伸出短小突起,并渐向外周散开;接种第3天,见聚集成团的细胞扩散开来,大部分细胞生成突起,呈双极或三极,以双极为主;接种第5天,细胞突起明显伸长,形态仍以双极细胞和三极细胞为主,并可见扁圆细胞和不规则三角形细胞;接种第7天,细胞密度增加,突起伸展。对照组细胞形态与实验组具有类似特征。两组嗅鞘细胞数量、细胞周长和细胞面积差异无显著性意义(P > 0.05)。表明壳聚糖增强型左旋聚乳酸支架与嗅鞘细胞具有良好的生物相容性。     

转铁蛋白修饰共载阿霉素与蒽贝素脂质体的构建及其体外评价

目的 研究转铁蛋白修饰共载阿霉素(DOX)和蒽贝素的脂质体(TELP-DOX/EB)与乳腺癌细胞的亲和力和增殖抑制作用.方法 后插入法制备转铁蛋白修饰共载阿霉素和蒽贝素脂质体(TELP-DOX/EB).细胞摄取法检测细胞对普通脂质体(LP)和转铁蛋白修饰脂质体(TFLP)的摄取.将MCF-7细胞分为TELP-DOX/EB组、TELP-DOX组、TELP-EB组、LP-DOX/EB组和PBS组,MTT检测细胞的增殖.构建乳腺癌MCF-7细胞肿瘤球模型,激光共聚焦显微镜检测脂质体的肿瘤球穿透能力和肿瘤球的生长.结果 MCF-7细胞对TFLP的摄取效率是LP的3.2倍(P＜0.01);给药48 h后,TELP-DOX/EB对MCF-3细胞的抑制率分别是TELP-DOX、TELP-EB和LP-DOX/EB、的1.95倍、2.43倍和2.75倍(P＜0.01).TELP-DOX/EB具有良好的肿瘤球穿透作用和肿瘤球生长抑制作用(P＜0.01).结论 铁蛋白修饰共载阿霉素和蒽贝素脂质体是一种潜在高效的乳腺癌靶向给药系统.     

Synthesis and in vitro antimicrobial and antioxidant activities of quaternary ammonium chitosan modified with nisin

Nisin had been grafted onto quaternary ammonium chitosan (QCS) through an enzyme-catalyzed reaction to enhance its limited antimicrobial activity. QCS was synthesized by incorporating N-(3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) onto chitosan’s primary amine group. The modification had been confirmed by FT-IR and ¹H NMR spectroscopy. Degree of substitution (DS) of QCS–nisin could be controlled by adjusting the reaction conditions. The synthesized compounds were screened in vitro to evaluate their antimicrobial and antioxidant activities. The results suggested that QCS–nisin significantly suppressed the growth of both gram-positive bacteria and gram-negative bacteria; The antioxidant effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical and hydrogen peroxide (H₂O₂) proved to be enhanced with increasing DS and concentration. In addition, QCS–nisin showed excellent moisture absorption and retention properties; MTT assay exhibited that QCS–nisin revealed low cytotoxicity effects on cultured NIH-3T3 fibroblasts. These results suggest that QCS–nisin would appear to be a promising candidate for wound dressing application.     

壳聚糖抗肿瘤缓释药物的特征☆

背景：肿瘤严重危害人类的健康,应用有效的化学药物治疗具有重要的意义。 目的：分析壳聚糖载药缓释系统抗肿瘤的作用机制及治疗效果。 方法：分析壳聚糖抗肿瘤药物缓释材料释放药物机制以及抗肿瘤的作用机制,并分析壳聚糖及其衍生物负载多种抗肿瘤药物如阿霉素、表阿霉素、表柔比星、5-氟尿嘧啶、紫杉醇等的特征及抗肿瘤作用效果。 结果与结论：壳聚糖抗肿瘤缓释药物材料具有良好的药物缓释性能以及抗肿瘤性能,能够负载各种常用的抗肿瘤药物,可以控制药物释放速率,延长药物作用时间,维持有效的药物浓度,降低药物的毒副作用,并且对肿瘤及组织器官具有靶向性作用,明显提高了化疗药物对肿瘤的治疗作用。     

Effects of preparation methods on the bone formation potential of apatite-coated chitosan microspheres

To investigate the effects of preparation methods on the bone formation potential of apatite-coated chitosan microspheres, coacervate precipitation method and emulsion cross-linking method were chosen to prepare chitosan microspheres, and then apatite coatings were deposited using simulated body fluid. Rat bone marrow-derived mesenchymal stem cells (BMSCs) were seeded on these microspheres. Cell adhesion, proliferation, and differentiation potential were monitored. For in vivo analysis, some cell/microsphere constructs were implanted in the subcutaneous pockets of male Wistar rats. After 3, 6, 12 weeks, the samples were retrieved and stained with hematoxylin and eosin (HE). Some cell/microsphere constructs were implanted in the calvarial defects of rats. Micro-CT and HE analysis were performed to analyze the new bone formation. It was found that BMSCs on apatite-coated emulsion cross-linked microspheres (EM1) exhibited better proliferation and differentiation than cells on apatite-coated coacervate-precipitated microspheres. The in vivo results showed that no bone was observed in ectopic areas. While in calvarial defects, both histological slices and Micro-CT images demonstrated that a substantial amount of new bone was formed in the EM1/BMSCs construct. These data suggest that preparation methods do exert great influence on the in vitro cell behaviors and in vivo orthotopic bone regeneration of apatite-coated chitosan microspheres. Appropriate method should be considered when preparing chitosan microspheres for bone tissue engineering scaffold.     

壳聚糖转运针对TNFαsiRNA的可行性及其对于炎症因子抑制效应的初步研究

目的:初步探索壳聚糖/siRNA纳米颗粒应用于炎症性肠病的可行性.方法:应用直接混合法将壳聚糖溶液与siRNA照N/P比为60混合形成壳聚糖/siRNA纳米颗粒,采用Zetasizer粒度仪对纳米颗粒直径进行检测,扫描电镜观察其形态以及透射电镜观察其结构;将纳米粒与RNA酶孵育后采用2%琼脂糖凝胶电泳分析其保护作用;采用细菌脂多糖(lipopolysaccharides,LPS)对小鼠巨噬细胞系RAW264.7进行炎症因子诱导后进行体外实验,荧光显微镜观察其转染效率,采用针对TNFα的siRNA进行转染,TaqMan荧光定量PCR和ELISA对TNFα的表达进行定量分析;利用MTT方法对转染后的细胞进行活力测试.结果:壳聚糖/siRNA纳米粒形态较为均一,粒径在200 nm左右;壳聚糖能够与siRNA形成可逆性结合,保护siRNA不被酶降解长达120 min以上;该纳米粒能够高效率的实现对巨噬细胞的转染;纳米粒转染后对LPS诱导巨噬细胞分泌大量的TNFα起到显著的抑制作用,效率高达50%以上,且细胞活性没有明显改变.结论:壳聚糖/siRNA纳米粒系统能够有效的转染巨噬细胞,实现对炎症因子TNFα的抑制作用,为炎症性肠病的分子治疗提供了一定的参考价值.