Nerve growth factor-basic fibroblast growth factor poly-lactide co-glycolid sustained-release microspheres and the small gap sleeve bridging technique to repair peripheral nerve injury

We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique.Multiple growth factors play a synergistic role in promoting the repair of peripheral nerve injury;as a result,in this study,we added basic fibroblast growth factors to the microspheres to further promote nerve regeneration.First,in an in vitro biomimetic microenvironment,we developed and used a drug screening biomimetic microfluidic chip to screen the optimal combination of nerve growth factor/basic fibroblast growth factor to promote the regeneration of Schwann cells.We found that 22.56 ng/mL nerve growth factor combined with 4.29 ng/mL basic fibroblast growth factor exhibited optimal effects on the proliferation of primary rat Schwann cells.The successfully prepared nerve growth factor-basic fibroblast growth factor-poly-lactide-co-glycolid sustained-release microspheres were used to treat rat sciatic nerve transection injury using the small gap sleeve bridge technique.Compared with epithelium sutures and small gap sleeve bridging alone,the small gap sleeve bridging technique combined with drug-free sustained-release microspheres has a stronger effect on rat sciatic nerve transfection injury repair at the structural and functional level.

Preparation of Sustained-release Silybin Microspheres by Spherical Crystallization Technique

Aim To improve the dissolution rate and bioavailability of silybin. Methods Sustained-release silybin microspheres were prepared by the spherical crystallization technique with soliddispersing and release-retarding polymers. A differential scanning calorimeter and an X-ray diffractometer were used to investigate the dispersion state of silybin in the microspheres. The shape, surface morphology, and internal structure of the microspheres were observed using a scanning electron microscope. Characterization of the microspheres, such as average diameter, size distribution and bulk density of the microspheres was investigated. Results The particle size of the microspheres was determined mainly by the agitation speed. The dissolution rate of silybin from microspheres was enhanced by increasing the amount of the dispersing agents, and sustained by the retarding agents. The release rate of microspheres was controlled by adjusting the combination ratio of the dispersing agents to the retarding agents. The resuits of X-ray diffraction and differential scanning calorimetry analysis indicated that silybin was highly dispersed in the microspheres in amorphous state. The release profiles and content did not change after a three-month accelerated stability test at 40 ℃ and 75% relative humidity. Conclusion Sustained-release silybin microspheres with a solid dispersion structure were prepared successfully in one step by a spherical crystallization technique combined with solid dispersion technique. The preparation process is simple, reproducible and inexpensive. The method is efficient for designing sustained-release microspheres with water-insoluble drugs.

Release performance and sustained-release efficacy of emamectin benzoate-loaded polylactic acid microspheres

High-performance liquid chromatography （HPLC） was employed to determine drug release rates based on emamectin benzoate concentrations in the medium. Release kinetics equations were used to fit the drug release behavior. The effects of particle size and release medium pH on the release rate were also investigated. The indoor toxicity of emamectin benzoate-loaded polylactic acid microspheres on the diamondback moth larva （Plutella xylostella） was studied to explore drug sustained-release performance. In acidic and neutral media, the drug release behavior of the microspheres was in accord with the first-order kinetics equation. Increasing the spray dosage of emamectin benzoate-loaded polylactic acid microspheres initially resulted in an equivalent insecticidal efficacy with the conventional emamectin benzoate microemulsion. However, the drug persistence period was four-fold longer than that observed using the conventional formulation. The developed emamectin benzoate-loaded polylactic acid microspheres showed dramatic sustained-release performance. A treatment threshold of greater than 35 mg mL-1 was established for an efficient accumulated release concentration of emamectin benzoate-loaded microspheres.

In vitro release of 1,3-bis(2-chloroethyl)-1-nitrosourea sustained-release microspheres and the distribution in rat brain tissues

BACKGROUND: The implantation of released chemotherapeutic drugs, which takes biodegradable polymer as vector, into the tumor site can get high concentration and release the drug for a long time, it can directly act on the tumor cells, and reduce the general toxicity. OBJECTIVE: To explore the in vitro and in vivo course of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sustained-release from BCNU-loaded polylactide (PLA) microspheres (MS) and location in rat brain tissue. DESIGN: A repetitive measurement. SETTING:Central Pharmacy, General Hospital of Chinese People’s Armed Police Forces. MATERIALS: Thirty male SD rats were used. PLA (Mr5000, batch number: KSL8377) was produced by Wako Pure Chemical Inc.,Ltd. (Japan); BCNU (batch number: 021121) by Tianjin Jinyao Amino Acid Co., Ltd.; BCNU-PLA-MS was prepared by the method of solvent evaporation and pressed into tablets (10 mg/tablet). High-performance liquid chromatography (HPLC) Agilent 1100 (USA); LS9800 liquid-scintillation radiometric apparatus (Beckman). Chromatographic conditions: Elite Hypersil ODS2 C18 chromatographic column (5 μm, 4.6 mm×150 mm); Mobile phase: methanol: water (50:50), flow rate was 1.0 mL per minute, wave length of ultraviolet detection was 237 nm, and the inlet amount of samples was 10 μL. METHODS: The experiments were carried out in the experimental animal center of the General Hospital of Chinese Armed Police from May 2004 to July 2005. ① In vitro BCNU-PLA-MS release test: BCNU-PLA-MS was prepared by the method of solvent evaporation, then placed in 0.1 mol/L phosphate buffered solution (PBS, pH 7.4, 37 ℃), part of MS were taken out at 1, 2, 3, 7, 10 and 15 days respectively, and the rest amount of BCNU in MS was determined by HPLC, then the curve of BCNU-PLA-MS release was drawn. ②In vivo BCNU-PLA-MS release and distribution test: The rats were anesthetized, then BCNU-PLA-MS were implanted to the site 1 mm inferior to the cortex of frontal lobe. Five rats were killed postoperatively at 4 hours, 1, 2, 3, 7 and 15 days, the residual MS was removed from the brain tissue. The rest amount of BCNU was determined with HLPC, and the curve of BCNU-PLA-MS release was drawn as compared with the amount of BCNU in the implanted tablets. Besides, brain tissues (1 g) at the implanted side and the contralateral one were obtained respectively, blood sample (0.5 mL) was also collected, 3H-BCNU was counted radioactively in radioactive liquid flash solution. The distributions of BCNU-PLA-MS in normal rat brain tissue and serum were detected. The analysis of variance was applied to compare the intergroup differences of the measurement data. MAIN OUTCOME MEASURES: ① Characteristics of BCNU-PLA-MS release in phosphate buffered solution (PBS) and rat brain tissue; ② Distributions of BCNU-PLA-MS in normal rat brain tissue and serum. RESULTS: ① Release of BCNU-PLA-MS in PBS and rat brain tissue: The BCNU released from BCNU-PLA-MS could be sustained for over 2 weeks both in PBS and brain tissue. In PBS, the released rate of BCNU was over 15% at 24 hours, nearly 50% at 72 hours and over 90% at 15 days. In brain tissue, the released rate was 8% at 4 hours, 16% at 24 hours, 60% at 72 hours, respectively, and BCNU could be sustained released for over 15 days. ② Distributions of BCNU-PLA-MS in normal rat brain tissue and serum: The concentrations of BCNU in the ipsilateral brain tissue were 6 to 70 times higher than those in the contralateral one. The concentrations of BCNU in the ipsilateral brain tissue were obviously higher than those in serum and contralateral brain tissue (F =103.47, P < 0.01). CONCLUSION: BCNU-PLA-MS can increase the drug concentration in targeted brain tissue, decrease that in the non-targeted brain tissue, reduce general toxic and side effects, and have good releasing function.

Effect of allelochemicals sustained-release microspheres on the ingestion, incorporation, and digestion abilities of Daphnia magna Straus

Allelochemicals sustained-release microspheres(ACs-SMs)exhibited great inhibition effect on algae,however,few studies have focused on ACs-SMs toxicity on invertebrate.In this study,the effects of single high-concentration ACs(15 mg/L,SH-ACs),repeated lowconcentration ACs(3×5 mg/L,RL-ACs)and ACs-SMs containing 15 mg/L ACs exposure on the ingestion,incorporation,and digestion of Daphniamagna Straus(DS)were investigated by stable isotope 15N labeling method.Meanwhile,the diversity and abundance of microflora in DS guts were determined by 16S rRNA genes and cloning methods.The results showed that SH-ACs exposure caused 50%and 33.3%death rates for newborn and adult DS,while RL-ACs exposure caused 10%death rate for newborn DS and no obvious effect on the activity of adult DS.And ACs-SMs exposure did not diminish the motility of both newborn and adult DS,indicating the lower acute toxicity of ACs-SMs.Furthermore,SH-ACs inhibited the ingestion(-6.45%),incorporation(-47.1%)and digestion(-53.8%)abilities of DS and reduced the microbial abundance(-27.7%)in DS guts.Compared with SH-ACs,RL-ACs showed relatively low impact on the ingestion(-3.23%),incorporation(-5.89%)and digestion(-23.9%)abilities of DS.Interestingly,ACs-SMs enhanced the ingestion(+9.68%),incorporation(+52.9%)and digestion(+51.3%)abilities of DS and increased the microbial abundance(+10.7%)in DS guts.Overall ACs and ACs-SMs reduced the diversity of microflora in DS guts.In conclusion,ACs-SMs can release ACs sustainably and prolong the sustained release time,which not only effectively reduce the toxicity of ACs,but also had positive effects on DS.

利福喷丁聚乳酸缓释微球对兔脂肪干细胞生长及增殖的影响

目的探讨不同浓度的利福喷丁聚乳酸缓释微球对兔脂肪干细胞生长及增殖的影响。方法取健康新西兰大白兔双侧腹股沟脂肪组织,经过消化、洗涤处理,接种于培养瓶中培养。隔日换液,观察细胞贴壁、生长、增殖情况以及细胞形态,每日计数并绘制生长曲线。用优化复乳法制得利福喷丁聚乳酸缓释微球,取第3代兔脂肪干细胞,分为5组,分别加入含有不同浓度的利福喷丁聚乳酸缓释微球的培养液进行培养（浓度梯度：97.6、121.9、146.3、170.7、195.1μg/m L）,采用CCK-8比色方法隔日检测细胞的增殖活性。结果兔脂肪干细胞在最初3~4 d生长较慢,之后进入快速增殖期,第8天到达峰值,呈漩涡状排列。经过成骨诱导15 d后,茜红素染色可见类圆形红染、均质、不透明的矿化结节。含利福喷丁药物浓度分别为40、50、60、70、80μg/m L的利福喷丁聚乳酸缓释微球溶液对兔脂肪干细胞生长、增殖的影响较小,各浓度间差异无统计学意义（P〉0.05）。结论利福喷丁聚乳酸缓释微球对兔脂肪干细胞生长、增殖的影响较小。

利福喷丁聚乳酸缓释微球对兔脊柱结核局部治疗作用的药效评价

目的探讨不同剂量利福喷丁聚乳酸缓释微球对兔脊柱结核局部治疗的效果。方法选取75只造模成功、感染程度近似的新西兰大白兔,随机分为A、B、C、D、E组,每组15只,行病灶清除术后在每组病灶清除部位放入不同剂量(A组3 mg、B组6 mg、C组12 mg、D组空白微球、E组15 mg)含有利福喷丁聚乳酸缓释微球的异体冻干松质骨,充分填塞缺损处。在术后不同时间段行大体肉眼观察、影像学、组织病理学、细菌学等检查。结果75只新西兰大白兔中6只术后发生下肢截瘫,75只均完成实验。X线检查示术后15、30 d各组均无明显的腰4、5椎体破坏及椎间隙改变。术后60 d时A组4只(30.7%)、B组4只(30.7%)、C组2只(14.2%)、D组5只(35.7%)腰5椎体上部有骨质破坏及腰4、5椎间隙改变。E组15只至术后8 w无明显影像学改变。病理及组织学检查示:A、B、C、D组中感染复发的新西兰大白兔椎体、椎旁软组织切片苏木素-伊红(HE)染色示有脓细胞、类上皮样细胞或坏死灶等形成,脓液培养显示有结核分枝杆菌生长。E组新西兰大白兔椎体及软组织HE染色可见较多成纤维细胞和淋巴细胞,椎旁软组织培养无结核杆菌生长。A、B、C、D组与E组新西兰大白兔X线检查与组织学检查感染复发差异有统计学意义(P<0.05)。结论 15 mg利福喷丁聚乳酸缓释微球为兔脊柱结核短期体内局部预防复发的最低有效治疗剂量。

聚己内酯载药微球的制备及释药性能研究

首次以利福喷丁为模型药物、聚己内酯为载体材料,制备了用于长效抑制骨结核生长的利福喷丁聚己内酯缓释微球,观察其理化特性和体外释放性能。采用O/W乳化溶剂挥发法制备载利福喷丁聚己内酯微球,系统考察了投药量、聚乙烯醇的浓度、乳化速度对微球形貌、粒径、载药量和包封率的影响。通过对制备工艺的优化,得到最佳制备条件是乳化速度300r·min-1,投药量20mg,聚乙烯醇的浓度2%。所制备的载利福喷丁聚己内酯微球圆整,表面有微孔,大小分布均匀,粒径分布较窄,平均粒径为(27.249±0.256)μm、载药量(3.098±0.011)%、包封率(34.078±0.123)%。实验结果表明:聚己内酯是负载利福喷丁的一种理想控释材料。

聚乳酸-羟基乙酸载利福喷丁微球的体外释放机制研究

目的:探讨聚乳酸-羟基乙酸载利福喷丁微球体外释放机制。方法:采用乳化-溶剂挥发法制备不同载药量的聚乳酸-羟基乙酸载利福喷丁微球,于PBS缓冲溶液(0.2 mol/L,pH7.4)中测定微球体外释放量,对释放曲线进行零级方程拟合、一级方程拟合和Higuchi方程拟合。结果:聚乳酸-羟基乙酸载利福喷丁微球的载药量分别为(8.04±0.29)%、(17.16±0.40)%和(23.93±0.48)%,差异有统计学意义(F=1 195.325,P=0.000)。微球呈球形,分散性良好,药物均匀地分布于载体基质中。载药微球在前2 d释放药物较快,累计释放药物量分别为(9.07±0.11)%、(13.33±0.04)%和(15.5±0.09)%,差异有统计学意义(F=4 414.474,P=0.000)。从第3天开始,释放药物速率减缓,总累计释放量分别为(72.10±0.26)%、(80.22±0.56)%和(78.60±0.63)%,差异有统计学意义(F=212.916,P=0.000)。在3种拟合方程中,均以Higuchi方程的相关系数更接近于1。结论:聚乳酸-羟基乙酸载利福喷丁微球体外释放呈突释和缓释两相释放,释放曲线更符合Higuchi方程释放规律。

体外构建新型骨组织工程三维复合体

体外实现兔脂肪干细胞(r ADSCs)、利福喷丁聚乳酸缓释微球及羟基磷灰石/β-磷酸三钙(HA/β-TCP)的三维复合,构建既能抗结核又能填补、促进骨缺损的新型骨组织工程复合体。以HA/β-TCP为支架材料,通过缓慢滴加细胞型方式,将复乳-溶媒挥发法制备的利福喷丁聚乳和定性分化诱导的r ADSCs(茜红染色表征)体外制得新型骨组织工程三维复合体,并对其进行显微结构及药物缓释表征。结果表明,载药利福喷丁聚乳酸缓释微球大部分分布在18~28μm,载药利福喷丁聚乳酸缓释微球在一定浓度下不影响r ADSCs的分化诱导,层状膜形分布的成骨诱导后r ADSCs牢固包绕利福喷丁聚乳酸缓释微球,分布在HA/β-TCP上,载药支架材料有着良好的药物缓释,体外维持最低抑菌浓度长达46 d。新型骨组织工程的三维骨组织工程复合体具有缓慢、持续的释药特性,且具有具有成骨活性。

抗结核骨组织工程复合体局部治疗兔脊柱结核的对比研究

目的探讨以羟基磷灰石与/β-磷酸三钙(hydroxyapatite and/β-tricalcium phosphate,HA/β-TCP)复合体材料及同种异体骨为支架材料构建的抗结核性骨组织工程复合体,评价两种不同抗结核骨组织复合体治疗兔脊柱结核的效果。方法取3月龄经造模成功后的新西兰大白兔36只,行病灶清除术,随机分为3组,每组12只,A组于骨缺损处植入利福喷丁微球-rADSCs/HA/β-TCP抗结核骨组织工程复合体,B组于骨缺损处植入利福喷丁微球-rADSCs/同种异体骨抗结核骨组织工程复合体,C组清创后未做任何处理。术后4、8、12周行影像学(DR)检查,术后第12周将实验动物处死,取标本,行大体观察、组织病理学观察骨缺损修复情况。结果大体观察发现A组骨缺损区被新生骨组织取代;B组骨缺损基本修复,周边可见大量骨组织形成;C组骨缺损处有少量骨组织形成,可见大量纤维组织覆盖。X线观察发现:4周时,A组骨缺损区可见少量骨痂形成,材料与周围骨组织紧密接触。B组骨缺损区可见骨痂形成,C组骨缺损区界限清晰,可见片状低密度影。8周时,A组骨缺损区明显缩小,材料吸收,边界稍模糊。B组骨缺损区可见片絮状高密度影,C组骨缺损区可见点状钙化影。12周时,A组骨缺损区材料基本吸收,B组骨缺损材料部分吸收,椎间隙部分融合,C组骨缺损区界线尚清,椎间隙破坏缺损。组织病理学检查发现:术后12周,A组材料吸收明显,可见大量纤维骨痂组织生成骨组织。B组可见部分同种异体骨残留,周边可见大量纤维骨痂组织生成骨组织。C组可见大量纤维组织形成。结论利福喷丁微球-rADSCs/HA/β-TCP构建的抗结核骨组织工程复合体具有良好的生物相容性,能够有效填充兔腰椎结核病灶清除术后的骨缺损。

Paclitaxel formulation with stable sustained-release behavior and its biological safety evaluation

Biological safety and stable sustained-release of the drug are two crucial issues involved in the formulation of paclitaxel.Focusing on these issues, by using the FDA approved polylactide as carrier material, soybean lecithin as surfactant and maltodextrin as thickener, paclitaxel loaded PLA microspheres were simply prepared by solvent evaporation, thus guaranteeing the biological safety. The introduction of maltodextrin as a thickener aided to a stable sustained-release of paclitaxel. Surface morphology, particle size, drug loading rate, encapsulation efficiency and in vitro drug release behavior were investigated.Biological safety evaluations such as acute toxicity, allergies, hemolysis, skin stimulation and genotoxicity test were also carried out. Results showed that the obtained microspheres were biocompatible and could release paclitaxel at a desirable constant rate.Therefore, the simply prepared paclitaxel formulation with good biological safety and desirable release behavior exhibited great potential of local injection of paclitaxel for the clinical use in the future.

新型利福喷丁-对氨甲基苯甲酸／海藻酸钙微球的制备与体外性能评价

目的制备利福喷丁-对氨甲基苯甲酸/海藻酸钙微球,并对其进行性能表征和评价。方法使用高压静电液滴发生装置制备微球,测定其粒径、溶胀率、载药量、包封率以及药物累计释放率。结果利福喷丁-对氨甲基苯甲酸/海藻酸钙微球平均粒径163.12μm,载药量为51.5%,包封率为89.3%,在生理盐水中70 h后溶胀率为19,120 h后药物累积释放率达75%。结论对氨甲基苯甲酸的引入减缓了海藻酸钙微球在生理盐水中的溶蚀,药物释放平稳,无明显突释现象。