白蛋白纳米粒的制备及内耳跨膜给药的初步研究

目的 目前在临床上国内外尚无对内耳病局部用药的缓释剂,本研究旨在探讨能否将白蛋白纳米粒载体材料作为鼓室跨膜给药缓释剂.方法 采用去溶剂化法制备空白白蛋白纳米粒并进行系统表征和细胞毒性评价.为便于观察,选取一种红色荧光染料即罗丹明B（RhB）作为模型药物,以物理吸附方式与空白白蛋白纳米粒结合形成载药白蛋白纳米粒,测定其载药量、包封率及体外药物释放曲线,同时采用小动物活体成像技术观察其注入豚鼠听泡内跨圆窗膜转运扩散情况.结果 制备的白蛋白纳米粒为实心球形,表面光滑,平均粒径大小为476 nm,Zeta电位为15.4 mV.体外药物释放结果表明,该纳米粒具有缓释效果.经戊二醛交联固定的白蛋白纳米粒具有一定的细胞毒性;而经热变性处理的白蛋白纳米粒具有较好的细胞相容性.小动物活体成像实验可以看到RhB在听泡内滞留扩散,而后经解剖观察,证明白蛋白纳米粒可在圆窗膜表面附着并穿越圆窗膜实现跨膜向耳蜗内转运.结论 制备的白蛋白纳米粒结构完整,制备方法简单、无毒性,可以很好地包载药物并具有缓释功能,为进一步制备可注射跨圆窗膜定向缓释纳米凝胶奠定了坚实的基础.     

缓释抗菌微球复合型组织工程材料水凝胶的制备及性能评价

背景:氧化羟丙基甲基纤维素-透明质酸钠水凝胶体系具有良好的力学性能、生物相容性与降解性,可作为组织工程支架对受损组织起保护支撑作用,也可作为药物载体实现局部缓释作用.目的:制备缓释抗菌微球复合型组织工程材料水凝胶,研究其理化性能、生物学性能、骨诱导性能和抗菌性能.方法:以聚乳酸乙醇酸共聚物、壳聚糖和透明质酸钠为材料,通...     

花样结构白蛋白缓释载体的制备及在豚鼠内耳跨膜给药的应用

目的制备花样结构白蛋白(flower-shaped bovine serum albumin,FBSA)微纳材料作为载体进行内耳跨圆窗膜给药研究,为临床寻找新的药物缓释载体奠定基础。方法应用改良的去溶剂法制备FBSA微纳材料,并用荧光显微镜、电子显微镜、粒径分析仪等对其进行系统的表征。通过体外药物释放实验、MTT法来评估其细胞相容性和细胞毒性。通过小动物活体成像观察FBSA在豚鼠听泡内的扩散及在圆窗膜上的附着。结果蛋白基微纳米材料为放射状花样结构,大小约为50⁸0μm。空白FBSA微纳材料的zeta电位是-16.2 mV,其最高的载药量和包封率分别是21.4%和40.0%,具有缓释效果。通过L929细胞的毒性实验测试提示经热变性处理固定后的材料具有更低的毒性和更好的细胞相容性。小动物活体实验可见药物在内耳中扩散及在圆窗膜表面附着。结论成功构建FBSA微纳材料载体,在治疗内耳病的局部给药方面有着良好的应用前景。     

神经干细胞与透明质酸水凝胶材料生物相容性的研究

目的:评价神经干细胞与改性透明质酸水凝胶支架新材料的生物相容性,研究该透明质酸水凝胶支架作为中枢神经组织工程载体材料的可行性,为用组织工程及干细胞技术治疗中枢神经系统损伤提供基础。方法:以冷冻干燥法制备透明质酸水凝胶材料支架,通过化学接枝法将抗Nogo受体抗体(Anti-NogoR)和多聚赖氨酸(poly-l-lysine,PLL)分子接枝到水凝胶上对其改性,制成新的支架材料。体外培养胚胎13.5d大鼠前脑泡神经干细胞.将神经干细胞与生物支架共培养,通过扫描电镜观察透明质酸水凝胶的内部结构及神经干细胞在支架材料上的粘附与生长情况,通过细胞免疫组织化学技术观察神经干细胞在透明质酸水凝胶材料上的存活与分化情况。结果:制备的透明质酸水凝胶材料具有疏松的三维多孔结构,神经干细胞在支架材料上能够粘附并且有突起长出,生长良好。神经干细胞在支架材料上能够分化。结论:神经干细胞与经过改性的透明质酸水凝胶新材料有很好的生物相容性,能够在材料上存活分化。该新透明质酸水凝胶材料有望作为修复中枢神经损伤的组织工程载体。     

释放吲哚美辛的固定式宫内节育器--药物缓释制剂研制

①研究并制备一种棒状缓释制剂,这种棒状缓释制剂是以医用硅橡胶为载体,吲哚美辛为药物的混合物通过交联技术而制成.②棒状缓释制剂药物释放可控制,释药量达到一年.结果:①采用挤出成型法制备棒状缓释药芯具有生产效率高药芯精度符合要求的特点.②缓释药芯的释放率初期很大,然后快降低,逐步趋向稳定,说明着爆破效应;缓释药芯包覆硅橡胶簿膜则药物呈零级释放.③缓释药芯与包覆硅橡胶簿膜的药芯复配制成棒状缓释制剂,研究表明药物释放符合一级释放规律,有效释药量达到一年以上.④这种缓释制剂用于含铜的宫内节育器(IUD)之中,可有效减轻由于IUD引起经血量偏大的副反应.     

成骨生长肽壳聚糖-海藻酸钠微球的制备及体外检测

背景：成骨生长肽体外注射可以刺激外周血和骨髓细胞数增加,增加动物的骨量,加速骨折愈合,但因多肽不稳定性及注射应用不方便,限制了其临床应用。 目的：应用乳化交联法制备成骨生长肽壳聚糖-海藻酸钠缓释微球,并对其粒径、载药、体外释药、理化特性进行检测。 方法：以戊二醛作为交联剂,应用乳化交联法制备具有控制释放功能的负载成骨生长肽壳聚糖-海藻酸钠微球,显微镜及扫描电镜观察微球的形态和粒径;利用酶联免疫吸附实验动态检测成骨生长肽壳聚糖-海藻酸钠微球的载药率、包封率和缓释规律。 结果与结论：乳化交联法制备的壳聚糖-海藻酸钠微球,球形良好,球体表面有较多微孔,具有较高的包封率(>72%)。体外药物释放实验表明,成骨生长肽可以从壳聚糖-海藻酸钠微球中缓慢释放,整个释放过程可达49 d,累积释放率>85%。提示应用乳化交联法制备的负载成骨生长肽壳聚糖-海藻酸钠缓释微球,具有很好的控制释放成骨生长肽的能力。     

壳聚糖多孔明胶微球温敏凝胶载药体的建立与评价

目的寻找有效的跨圆窗膜载药体,制备多孔明胶微球结合壳聚糖/β-GP溶液的温敏特点,制备新型可注射、可降解、无毒副作用并具有缓释效应的药物载体,为后续动物实验提供研究基础。方法利用化学反应合成碳酸钙(CaCO_3)模板,采用W/O型乳化-固化法制备了明胶-碳酸钙复合微球,用EDTA处理除去碳酸钙,得到多孔的明胶微球。并以环境扫描电子显微镜对多孔明胶微球进行表征。利用壳聚糖与β-GP合成溶液物理混合形成壳聚糖多孔明胶微球温敏凝胶载药体。结果扫描电镜显示,明胶微球表面呈多孔结构,粒径在2~4um之间。壳聚糖/β-GP冻干后呈多孔网络状。壳聚糖/β-GP具有温敏性,且两者共混后温敏性不变。结论壳聚糖/β-GP溶液共混可制备成具有温敏效果的凝胶载药体,为跨膜药物转运的研究提供基础。     

生物医用水凝胶研究进展

该文综述了可注射水凝胶、互穿网络结构水凝胶、智能水凝胶以及纳米复合水凝胶等四种新型生物医用水凝胶的研究及应用进展。与传统水凝胶相比,新型水凝胶在结构、力学性能及应用方面具有明显的优势。该文重点介绍了各新型生物医用水凝胶的制备方法及在细胞及蛋白质传输、药物固载、组织工程等方面的应用。最后,针对应用过程中仍然存在的不足,对新型生物医用水凝胶的发展进行了展望,并提出未来可进一步研究的方向。     

羟丁基壳聚糖的生物相容性研究

目的研究新型温敏性生物材料羟丁基壳聚糖的生物安全性,初步探讨该材料植入体内有无毒副作用。方法将壳聚糖进行羟丁基化制备出温敏性水凝胶,选用小鼠成纤维细胞L929细胞,通过MTS法、倒置显微镜观察等方法进行体外细胞毒性试验。通过SD大鼠尾缘静脉浸提液注射,进行急性毒性试验,通过新西兰兔耳缘静脉浸提液注射及皮内浸提液注射,进行致热原反应试验及皮内刺激试验。结果羟丁基壳聚糖无细胞毒性,不引起试验动物体温升高,无皮肤刺激反应,急性毒性试验不引起试验动物死亡或者其它不适。结论羟丁基壳聚糖材料生物相容性好,是一种安全的生物材料,有望作为新型药物载体应用于临床。     

新型多功能抗肿瘤微球研究——Ⅰ.体外释药及定向栓塞

以明胶为原料、戊二醛为交联剂、疏水性高分子聚合物浓溶液为分散介质,制备出平均粒径60μ、表面亲水性好、可生物降解的明胶微球。该微球能同时浓集抗肿瘤药物及放射性同位素。体外药物释放结果表明,该抗癌药物—明胶微球有很好的药物缓释效果。同位素显像结果表明,~(131)Ⅰ标记的明胶微球经肝动脉插管灌注,可高选择性地定向进入肝动脉,并在肝区长期滞留,缓慢降解。     

Functionalizable hydrogel microparticles of tunable size and stiffness for soft-tissue filler applications

Particle size, stiffness and surface functionality are important in determining the injection site, safety and efficacy of injectable soft-tissue fillers. Methods to produce soft injectable biomaterials with controlled particle characteristics are therefore desirable. Here we report a method based on suspension photopolymerization and semi-interpenetrating network (semi-IPN) to synthesize soft, functionalizable, spherical hydrogel microparticles (MP) of independently tunable size and stiffness. MP were prepared using acrylated forms of polyethylene glycol (PEG), gelatin and hyaluronic acid. Semi-IPN MP of PEG-diacrylate and PEG were used to study the effect of process parameters on particle characteristics. The process parameters were systematically varied to produce MP with size ranging from 115 to 515 μm and stiffness ranging from 190 to 1600 Pa. In vitro studies showed that the MP thus prepared were cytocompatible. The ratio and identity of the polymers used to make the semi-IPN MP were varied to control their stiffness and to introduce amine groups for potential functionalization. Slow-release polymeric particles loaded with Rhodamine or dexamethasone were incorporated in the MP as a proof-of-principle of drug incorporation and release from the MP. This work has implications in preparing injectable biomaterials of natural or synthetic polymers for applications as soft-tissue fillers.     

Development of an injectable two-phase drug delivery system for sequential release of antiresorptive and osteogenic drugs

Unlike controlled release systems that deliver a single drug, dual or multidrug delivery systems with distinct release profiles are more likely to promote timely and effective tissue regeneration as they provide both temporally and concentration-dependent release of different molecules to mimic natural biological events. In this study, an injectable and biodegradable delivery system was developed to sequentially release an antiresorptive drug (clodronate) followed by an osteogenic agent (simvastatin) to treat bone disease. The injectable delivery system comprised simvastatin-loaded gelatin microspheres suspended in a viscous solution of carboxymethylcellulose (CMC) containing clodronate. Several factors (CMC concentration, glutaraldehyde concentration, simvastatin loading, and gelatin microsphere processing conditions) were investigated for their effects on drug release. Clodronate release was not affected by CMC concentration, with complete delivery within 12 hr, and simvastatin release could be modulated by cross-linking of the gelatin microspheres, loading, and washing conditions. Burst release of simvastatin was reduced from 70% to 6% in conjunction with sustained release for up to 3 weeks. The combined system showed early release of the antiresorptive clodronate sequentially followed by sustained delivery of the osteogenic simvastatin. This robust and flexible two-phase delivery system may prove useful for applications in which multiple drug delivery is desired.     

Delivery of dexamethasone, ascorbate, and growth factor (TGF beta-3) in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes

The aim of this study was to assess the efficacy of poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The p(NiPAAm-co-AAc) hydrogel itself without specific differentiation-inducing drugs was used as a control in order to determine the effects of these materials on chondrogenic differentiation. The level of cartilage associated extracellular matrix (ECM) proteins was examined by immunohistochemical staining for collagen type II as well as Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with chondrocytes and differentiation materials as an injectable delivery vehicle for use in neocartilage formation.     

Biodegradable in situ gel-forming controlled drug delivery system based on thermosensitive PCL–PEG–PCL hydrogel. Part 2: Sol–gel–sol transition and drug delivery behavior

In this work, a biodegradable and injectable in situ gel-forming controlled drug delivery system based on thermosensitive poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCEC) hydrogel was studied. The prepared PCEC hydrogel undergoes temperature-dependent sol–gel–sol transition, which is a flowing sol at ambient temperature and turns into a non-flowing gel at around physiological body temperature. Furthermore, the sol–gel phase transition mechanism was investigated using ¹³C-nuclear magnetic resonance imaging and a laser diffraction particle size analyzer. The in vitro release behaviors of several model drugs, including a hydrophilic small-molecule drug, a hydrophobic small-molecule drug and a macromolecular protein drug, from PCEC hydrogel were also investigated in detail. The results showed that the model drugs could be released from the PCEC hydrogel system over a sustained period. In addition, an anaesthesia assay was conducted using the tail flick latency (TFL) test to evaluate the in vivo controlled drug delivery effect of the PCEC hydrogel system. In the TFL assay, a lidocaine-loaded PCEC hydrogel produced significantly longer-lasting local anaesthetic effects compared with lidocaine aqueous solution at the same dose. Therefore, PCEC hydrogel is promising for use as an injectable local drug delivery system.     

Self-cross-linking biopolymers as injectable in situ forming biodegradable scaffolds

The injectable polymer scaffolds which are biocompatible and biodegradable are important biomaterials for tissue engineering and drug delivery. Hydrogels derived from natural proteins and polysaccharides are ideal scaffolds for tissue engineering since they resemble the extracellular matrices of the tissue comprised of various amino acids and sugar-based macromolecules. Here, we report a new class of hydrogels derived from oxidized alginate and gelatin. We show that periodate-oxidized sodium alginate having appropriate molecular weight and degree of oxidation rapidly cross-links proteins such as gelatin in the presence of small concentrations of sodium tetraborate (borax) to give injectable systems for tissue engineering, drug delivery and other medical applications. The rapid gelation in the presence of borax is attributed to the slightly alkaline pH of the medium as well as the ability of borax to complex with hydroxyl groups of polysaccharides. The effect of degree of oxidation and concentration of alginate dialdehyde, gelatin and borax on the speed of gelation was examined. As a general rule, the gelling time decreased with increase in concentration of oxidized alginate, gelatin and borax and increase in the degree of oxidation of alginate. Cross-linking parameters of the gel matrix were studied by swelling measurements and trinitrobenzene sulphonic acid (TNBS) assay. In general, the degree of cross-linking was found to increase with increase in the degree of oxidation of alginate, whereas the swelling ratio and the degree of swelling decreased. The gel was found to be biocompatible and biodegradable. The potential of the system as an injectable drug delivery vehicle and as a tissue-engineering scaffold is demonstrated by using primaquine as a model drug and by encapsulation of hepatocytes inside the gel matrix, respectively.     

Controlled release of sphingosine-1-phosphate agonist with gelatin hydrogels for macrophage recruitment

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by l-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo.     

pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan: In vitro characteristics and in vivo biocompatibility

In-situ forming hydrogels triggered by environmental stimuli have emerged as a promising injectable strategy targeted for various biomedical applications. However, several drawbacks associated with temperature-stimulated hydrogels have been reported. Employing a hydrophobically-modified chitosan (N-palmitoyl chitosan, NPCS), we developed a pH-triggered hydrogel system which showed a rapid nanostructure transformation within a narrow pH range (pH 6.5–7.0). NPCS in an aqueous environment was found to be a shear-thinning fluid and exhibited an instant recovery of its elastic properties after shear thinning, thereby making it an injectable material. Additionally, aqueous NPCS, an associating polyelectrolyte, can be rapidly transformed into hydrogel triggered simply by its environmental pH through a proper balance between charge repulsion and hydrophobic interaction. This in-situ hydrogel system was shown to be nontoxic. Subcutaneous injection of aqueous NPCS (pH 6.5) into a rat model resulted in rapid formation of a massive hydrogel at the location of injection. The implanted hydrogel was found to be degradable and was associated with an initial macrophage response which decreased with time as the degradation proceeded. These results suggested that the developed NPCS hydrogel may be used as an injectable drug/cell delivery system.     

Bioimaging of dexamethasone and TGF beta-1 and its biological activities of chondrogenic differentiation in hydrogel constructs

This study examined the efficacy of poly(NiPAAm-co-AAc) as an injectable drug delivery vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. The hydrogel constructs, which consisted of embedded cells co-encapsulating dexamethasone (Dex) and TGF beta-1 or unloaded Dex, were used as controls to determine the effects of Dex and TGF beta-1 on chondrogenic differentiation. The level of Dex and TGF beta-1 released was monitored using a bioimaging method. The amount of Dex released from hydrogel was faster than that of TGF beta-1. TGF beta-1 was present in hydrogel for more than 4 weeks after the injection. The level of the cartilage associated ECM proteins was examined by immunohistochemical staining for collagen type II as well as by Safranin-O and Alcian blue (GAG) staining. These results highlight the potential of a thermo-reversible hydrogel mixed with the chondrocytes and differentiation delivery material for applications in neocartilage formation.     

Cationized gelatin hydrogels mixed with plasmid DNA induce stronger and more sustained gene expression than atelocollagen at calvarial bone defects in vivo

Gene transduction of exogenous factors at local sites in vivo is a promising approach to promote regeneration of tissue defects owing to its simplicity and capacity for expression of a variety of genes. Gene transduction by viral vectors is highly efficient; however, there are safety concerns associated with viruses. As a method for nonviral gene transduction, plasmid DNA delivery is safer and simpler, but requires an efficient carrier substance. Here, we aimed to develop a simple, efficient method for bone regeneration by gene transduction and to identify optimal conditions for plasmid DNA delivery at bone defect sites. We focused on carrier substances and compared the efficiencies of two collagen derivatives, atelocollagen, and gelatin hydrogel, as substrates for plasmid DNA delivery in vivo. To assess the efficiencies of these substrates, we examined exogenous expression of green fluorescence protein (GFP) by fluorescence microscopy, polymerase chain reaction, and immunohistochemistry. GFP expression at the bone defect site was higher when gelatin hydrogel was used as a substrate to deliver plasmids than when atelocollagen was used. Moreover, the gelatin hydrogel was almost completely absorbed at the defect site, whereas some atelocollagen remained. When a plasmid harboring bone morphogenic protein 2 was delivered with the substrate to bony defect sites, more new bone formation was observed in the gelatin group than in the atelocollagen group. These results suggested that the gelatin hydrogel was more efficient than atelocollagen as a substrate for local gene delivery and may be a superior material for induction of bone regeneration.