盐酸表阿霉素纳米靶向制剂的缓释效应

背景：盐酸表阿霉素是一种广谱抗生素,目前临床使用的不足多为药物释放快、目标组织药物浓度低,静脉给药后广泛分布于体内各种组织器官,不良反应明显。 目的：针对盐酸表阿霉素临床应用的不足,制备盐酸表阿霉素纳米靶向注射制剂。 方法：以叶酸偶联牛血清白蛋白为载体,采用乳化-高压匀质法,制备盐酸表阿霉素纳米靶向注射制剂,以激光粒度分析仪测定纳米颗粒的粒径大小、粒径分布及Zeta电位,扫描电镜观察纳米颗粒的表面形态,高效液相色谱法分析白蛋白负载盐酸表阿霉素纳米制剂的包封率、载药量和释药性能。 结果与结论：制备的盐酸表阿霉素纳米粒外观呈均匀球型,粒径分布较窄,平均粒径为(157.73±     0.40) nm,平均 Zeta 电位为(-30.85±0.43) mV,载药量 22.78%,包封率可达96.24%。体外模拟释药结果表明药物释放曲线分为两个阶段,突释阶段微球释药量在24 h内达42.6%,缓释阶段纳米粒释药持续时间长,在112 h 时释药量达 84.1%,载药纳米粒的药物释放速率持续稳定。结果表明乳化结合高压匀质法制备的盐酸表阿霉素纳米靶向制剂粒径均匀,粒径范围分布窄,载药量和包封率高,具有一定的缓释作用。     

载异烟肼利福平聚乳酸纳米粒的制备及体外释药**

背景：微载体药物因具有靶向性、控释性、稳定性、更好的安全性备受关注。 目的：观察载异烟肼利福平两种抗结核药于同一聚乳酸纳米粒的给药系统及体外释放特性。 方法：采用改良的自乳化二元溶剂扩散法制备载异烟肼和利福平纳米粒,亚微粒径分析仪测定纳米粒粒径及分布,透射电镜观察其形态;高效液相色谱仪建立测定异烟肼、利福平的载药量和包封率;以磷酸盐缓冲液为释放介质,观察载异烟肼和利福平纳米粒的体外释药特性。 结果与结论：载利福平和异烟肼纳米粒表面完整光滑,无明显粘连现象,纳米粒均匀度好。亚微粒径分析仪测定纳米粒平均粒径80.4 nm。异烟肼载药量为(15.95±1.34)%,包封率为(5.01±0.17)%;利福平载药量为(4.66±0.97)%,包封率为(4.05±0.18)%。体外释药结果显示纳米粒的体外释药过程较平稳。突释期纳米粒中异烟肼释放度为15.22%,到3 d累积释放度可达95.6%;利福平释放度为9.26%,到3 d累积释放度可达90.3%。提示采用改良的自乳化二元溶剂扩散法制备载异烟肼和利福平纳米粒,所得载药纳米粒的粒径小且较均匀。纳米粒体外释药过程较平稳,无明显突释现象。关键词：聚乳酸;异烟肼;利福平;纳米粒;体外释药 doi:10.3969/j.issn.1673-8225.2012.16.014     

乳酸-羟基乙酸共聚物载药纳米粒的制备及体外释药性

背景：医用纳米粒作为药物传递的新型载体,目前已经成为医药领域研究的重点。 目的：构建以生物可降解材料乳酸-羟基乙酸共聚物为载体,负载抗肿瘤药物5-氟尿嘧啶的载药纳米粒。 方法：利用复乳-溶剂挥发法制备乳酸-羟基乙酸共聚物载药纳米粒。场发射扫描电子显微镜观察纳米粒表面形态;激光粒度分析仪测定粒径分布并计算成球率;紫外分光光度计测定5-氟尿嘧啶载药量、包封率,并对体外释药进行评估。 结果与结论：纳米粒呈球性,平均粒径为(186±14) nm,成球率、载药量和包封率分别为70.8%、6.6%、28.1%,体外释药有突释现象,24 h内5-氟尿嘧啶累积释药量达36.2%,10 d达83.6%。提示成功制备乳酸-羟基乙酸共聚物载药纳米粒,其具有缓释效应。     

表皮生长因子偶联牛血清白蛋白纳米粒荷载紫杉醇的制备及鉴定

背景:紫杉醇是临床常用的广谱抗肿瘤植物药,但其不良反应较多,因此迫切需要合适的载体以减小紫杉醇本身的毒性,同时达到更好的靶向性。目的:研究表皮生长因子偶联牛血清白蛋白纳米粒荷载紫杉醇的制备及其理化性质的鉴定。方法:采用超声乳化和溶剂挥发技术制备白蛋白纳米粒,通过化学交联试剂将表皮生长因子与白蛋白纳米粒偶联,制得表皮生长因子偶联白蛋白纳米粒,使用表皮生长因子偶联白蛋白纳米粒荷载125I标记紫杉醇,作为实验样品。在白蛋白纳米粒与表皮生长因子偶联白蛋白纳米粒中分别加入100,200,400,800 mg/L的125I标记紫杉醇,进行包封率和载药量检测;检测实验样品、荷载125I标记紫杉醇白蛋白纳米粒的药物释放率;检测实验样品、荷载125I标记紫杉醇白蛋白纳米粒与125I标记紫杉醇的室温稳定性及血清稳定性。结果与结论:荷载125I标记紫杉醇的白蛋白纳米粒组和实验样品组的包封率和载药量随着紫杉醇药量的增加,包封率呈现下降趋势,载药量呈现上升趋势。实验样品、荷载125I标记紫杉醇白蛋白纳米粒释药趋势基本相同,表现为包载药物缓慢释放。实验样品、荷载125I标记紫杉醇白蛋白纳米粒的室温稳定性与血清稳定性均高于125I标记紫杉醇(P0.05)。表明表皮生长因子偶联白蛋白纳米粒荷载紫杉醇具有良好的载药率、释药率和稳定性。     

磁性聚乳酸-羟基乙酸氧化苦参碱纳米粒的制备及其特性

目的研究磁性聚乳酸-羟基乙酸氧化苦参碱纳米粒(M-PLGA-OM-NP)的制备工艺,并对纳米粒子进行评价。方法运用复乳法制备M-PLGA-OM-NP,通过透射电子显微镜观察纳米粒形态,并对纳米粒的平均粒径、载药量、包封率、体外释药情况等进行评价。结果纳米粒外观呈规则球形,其平均粒径为146.5 nm,载药量为7.61%,包封率为44.8%。突释后至第72小时,纳米粒维持较稳定的释药速度,累积释放达52.9%。72～240 h,药物释放缓慢,累计释放约为16.6%,体外释放符合Ritger peppas方程lny=1.280 6+lnt。氧化苦参碱药性不受温度影响。结论获得了较满意的M-PLGA-OM-NP制备工艺,其过程简单,粒子性状符合要求。     

聚乳酸/聚乙二醇琥珀酸酯-姜黄素纳米粒的制备及体外评价*

背景：聚乳酸及其共聚物是一类具有良好生物相容性的可降解高分子材料,已被广泛用于可生物降解型药物缓释或靶向给药系统中。 目的：探索载药纳米粒制备条件对包封率和载药量的影响,确定最佳制备工艺条件。 方法：以维生素E1000聚乙二醇琥珀酸酯(TPGS)为乳化剂、姜黄素为模型药物、聚乳酸为载体材料,采用O/W型乳化-溶剂挥发法制备聚乳酸-姜黄素纳米粒,以包封率和载药量为主要指标,单因素实验探索影响两指标的主要因素,再正交试验设计优化制备工艺。 结果与结论：通过正交试验设计制备聚乳酸-姜黄素纳米粒的最佳工艺为：水油相比10∶1,聚合物浓度15 g/L,药物浓度3 g/L,乳化剂TPGS浓度0.03%。以此工艺制备的载药纳米粒外形圆整光滑,粒度分布较为均匀,平均粒径为167.5 nm,包封率为89.52%,载药量为13.72%,纳米粒前期突释不明显具有良好的缓释作用。该工艺稳定、简单可行,优化制备工艺得到的聚乳酸-姜黄素纳米粒粒径适中、包封率和载药量较高。     

载表柔比星的壳聚糖纳米粒的制备及其特性研究

目的 制备经聚乙二醇修饰的壳聚糖纳米粒(PEG/CS NP),并负载表柔比星(EPI),研究载表柔比星的壳聚糖纳米粒(PEG/CS-EPI NP)体外释药性能.方法 应用阴离子凝聚法制备PEG/CS-EPI NP,透射电镜观察纳米粒的形态特征,激光粒度分析仪测定粒径大小,紫外分光光度法测定纳米粒的载EPI量,动态透析法考察载EPI纳米粒的体外释放特性.结果 当壳聚糖与三聚磷酸钠质量比为6∶1,壳聚糖与EPI质量比为8∶1时,制备的PEG/CS-EPI NP呈圆形或椭圆形,分散性良好,平均粒径(322.1±14.4)nm,载EPI量为(13.9±1.1)%,包封率(74.2±1.8)%,72 h累积释药率达(82.0±2.1)%.结论 采用阴离子凝聚法制备的PEG/CS-EPI NP形状规则、粒度分布均匀,具有较高包封率和较好缓释性能.     

左旋多巴和姜黄素共递送protocells纳米粒的制备及体外评价

目的制备共载左旋多巴和姜黄素protocells纳米粒并进行体外评价。方法以介孔二氧化硅为内核,脂质双分子层为外膜,制备共载左旋多巴和姜黄素protocells纳米粒。使用激光粒度分析仪和透射电子显微镜对所制备纳米粒的形貌、粒径、多分散系数(PDI)和Zeta电势进行表征;采用高效液相色谱法对所制备纳米粒的载药量和包封率进行测定;采用透析袋法对所制备纳米粒的体外释放特性进行考察;应用粒径、Zeta电势、载药量等指标对所制备纳米粒的室温贮存稳定性进行评价。结果制备的载左旋多巴和姜黄素protocells纳米粒粒径分布均一性好、粒子表面呈电负性、平均粒径为(210.9±2.8)nm、PDI为(0.201±0.011)。其中左旋多巴的载药量为(20.28±0.43)%、包封率为(10.14±0.22)%;姜黄素的载药量为(1.97±0.01)%、包封率为(98.32±0.01)%。体外释放结果表明该纳米粒48 h姜黄素累计释放率为59.2%,且可有效阻止左旋多巴的泄漏,降低其在循环系统中的暴露量。稳定性结果表明左旋多巴和姜黄素在protocells纳米粒中稳定性良好。结论载左旋多巴和姜黄素的protocells纳米粒制备工艺简单,具有良好的理化性质、稳定性及所预期的释放性能。     

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.     

正交优化聚乳酸-羟基乙酸纳米粒的制备

背景:聚乳酸-羟基乙酸纳米粒或纳米微球用于制备生物降解型缓释或定向给药体系已经研究了近30年,是国内外研究的热点。该体系能够控制粒径大小、延缓药物降解、延长药物释放时间、靶向释放、降低药物毒性和刺激性等。目的:以紫杉醇为模型药物、聚乳酸-羟基乙酸为包裹材料,探索载药纳米粒的制备条件对粒径、包封率等的影响,确定最佳制备工艺条件。方法:采用乳化-溶剂挥发法制备聚乳酸-羟基乙酸纳米粒,以粒径、包封率和载药量等为观察指标,通过正交设计法优化纳米粒制备工艺条件。结果与结论:通过正交实验设计,优化了制备工艺条件,其最佳条件是超声乳化时间为15min,乳化剂浓度为1%,油水相比为1∶25,合成温度为25℃。在此条件下进行实验,制备出的载药纳米粒粒径为217.6nm,载药量1.79%,包封率85%。该制备工艺简单、稳定,优化制备条件,可制备出包封率高、粒径适宜的紫杉醇-聚乳酸-羟基乙酸纳米粒。     

Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function

Despite recent advances in biomaterial science, there is yet no culture system that supports long-term culture expansion of human adult hepatocytes, while preserving continued function. Previous studies suggested that acellular liver extracellular matrix (ECM), employed as a substrate, improved proliferation and function of liver cells. Here we investigated whether extracts prepared from acellular liver ECM (liver ECM extract, LEE), or from whole (fresh) liver tissue (liver tissue extract, LTE), could be combined with collagen Type I, hyaluronic acid (HA), or heparin-conjugated HA (HP) hydrogels to enhance survival and functional output of primary human hepatocytes. The liver-specific semi-synthetic ECMs (sECMs) were prepared by incorporating LEE or LTE into the gel matrices. Subsequently, primary human hepatocytes were maintained in sandwich-style hydrogel cultures for 4 weeks. Progressive increase in hepatocyte metabolism was observed in all HA and HP groups. Hepatocytes cultured in HA and HP hydrogels containing LEE or LTE synthesized and secreted steady levels of albumin and urea and sustained cytochrome p450-dependent drug metabolism of ethoxycoumarin. Collectively, these results indicate that customized HA hydrogels with liver-specific ECM components may be an efficient method for expansion human hepatocytes in vitro for cell therapy and drug and toxicology screening purposes.     

Culture of primary rat hepatocytes within porous chitosan scaffolds

Chitosan is considered to be a very promising biopolymer for various biomedical and pharmaceutical uses because of its nontoxic and biocompatible natures (Chandy T, Shama P. Biomater Artif Cells Artif Org 1990;18:1-24). In this study, we prepared porous chitosan scaffolds by lyophilization of chitosan solution. The scaffolds were modified with water-soluble polyanionic species such as alginate and heparin. The pore structures of these scaffolds were viewed via light and scanning electron microscopy. The scaffolds prepared have a high porosity of approximately 90% with mean pore sizes from 50 to 200 microm. They were used as substrates for hepatocytes culture. The cell attachment ratio was much higher than on monolayer membrane and hepatocytes exhibited a round cellular morphology with many microvilli evident on the surface of the cells. Metabolic activities of the cells were evaluated in terms of albumin secretion and urea synthesis. It was found that hepatocytes cultured on the modified scaffolds showed an increase in albumin secretion during the first 4 days and were more stable than those on monolayer membrane and nonmodified scaffolds. Therefore, primary rat hepatocytes cultured on modified scaffolds would be beneficial to liver assist device.     

Acellular liver matrix improves the survival and functions of isolated rat hepatocytes cultured in vitro

Alternative approaches to overcome the shortage of donors for liver transplantation may be the use of hepatocytes for bioartificial devices or transplantation. Therefore, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of hepatocytes represents a formidable challenge. Aim of this study was to obtain a liver homologous acellular matrix (HAM) able to support viability and metabolic functions of rat hepatocytes in primary culture. HAMs were prepared by sequential incubation of rat liver slices in deoxycholic acid and DNase solutions. Dispersed rat hepatocytes were obtained by collagenase digestion and mechanical disaggregation. Isolated hepatocytes were seeded on uncoated and collagen- or HAM-coated tissue culture plastic wells. Cultures were examined by scanning electron microscopy (SEM), and the viability of hepatocytes and their ability to produce albumin and urea were assessed. The viability of freshly dispersed hepatocytes was about 98%. Hepatocytes seeded on HAM exhibited a significantly higher viability and a markedly lower apoptotic rate than those grown on plastic or collagen. Accordingly, albumin and urea nitrogen productions were significantly higher in HAM-cultured hepatocytes. SEM showed that hepatocytes seeded on HAM displayed a clustered organization, and were well anchored to the matrix and morphologically stable. Taken together, these findings indicate that HAM strongly improves viability and functional activity of rat hepatocytes cultured in vitro.     

原代肝细胞的分离和移植

背景：肝细胞移植可以作为一种桥梁作用,帮助肝功能衰竭患者渡过肝衰期,并提高患者生存率和预后。 目的：采用Seglen改良的原位灌注探讨SD大鼠原代肝细胞分离的影响因素和方法的改进,同时分析原代肝细胞治疗急性肝功能衰竭大鼠的疗效。 方法：两步法分离大鼠肝细胞。D-氨基半乳糖诱导大鼠急性肝衰竭,24 h后分2组：细胞移植组经脾脏移植约2×107个肝细胞;对照组经脾脏注射0.4 mL Hank’s液。观察不同时间点大鼠的生存率和血清中转氨酶、总胆红素变化情况、脾内白蛋白分泌作用及脾内肝细胞分布情况。 结果与结论：大鼠肝细胞分离存活率达85%~95%。细胞移植组14 d存活率(75%)显著高于对照组组(30%)(P =0.01),且肝功能改善情况明显优于对照组。移植30 d后,脾内有肝细胞白蛋白绿色荧光信号;移植15 d后,可以看到肝细胞在脾脏红髓中簇集在一起并定植。结果说明胶原酶、pH值、灌注液、灌注方法等均是影响肝细胞分离存活率的因素;经脾脏移植的肝细胞能提高急性肝衰竭大鼠的生存率和改善肝功能。     

Isolation and characterization of inflammatory leukocytes from glomeruli in an in situ model of glomerulonephritis in the rat.

Inflammatory cell populations in glomerulonephritis (GN) are not well characterized. A method is reported for isolating leukocytes from glomeruli. GN was induced in rats by perfusing left kidneys (LKs) with cationized human IgG followed by intravenous rat anti-human IgG serum. Acute GN developed in LKs with proteinuria, deposition of human and rat IgG and C3, leukocyte infiltration, and capillary wall electron-dense deposits. Glomeruli (GL) isolated at 24 hours were digested with collagenase, trypsin, and DNase, and the resulting cells were as follows (mean +/- SEM): LK, 354 +/- 25/GL; RK, 214 +/- 32/GL. Cells were labeled with monoclonal antibody MRCOX1 (anti-rat leukocyte common [LC] antigen) followed by FITC F(ab')2 rabbit anti-mouse Ig: LK, 170 +/- 11 leukocytes/GL;RK, 8 +/- 2 leukocytes/GL (P less than 0.001). Isolated cells were sorted by flow cytometry to 98% pure LC+ cells with greater than 80% viability (Giemsa staining: 86% mononuclear cells, 14% neutrophils); the ultrastructure was that of maturing macrophages and neutrophils. This method quantitates leukocyte infiltration and provides leukocytes from nephritic glomeruli suitable for in vitro studies.     

C3A细胞与L-02永生化肝细胞低温保存条件下的生物学特性比较

背景：获得大量功能良好的肝细胞是生物人工肝的核心。探索出一种可靠的肝细胞低温保存方法进而构建一个肝细胞库是目前生物人工肝研究的热点。 目的：比较用UW液在4 ℃条件下保存已经进入Ⅲ期临床试验的C3A细胞与国内构建的永生化肝细胞株L-02细胞的生物学特性。 方法：贴壁培养C3A与L-02细胞,胰酶消化,制备成细胞悬液,UW液保存。4 ℃低温保存0,24,48及72 h后,采用流式细胞术分别测定细胞存活率与凋亡率,测定谷草转氨酶与乳酸脱氢酶释放、尿素合成功能及白蛋白分泌功能。 结果与结论：随低温保存时间延长,C3A与L-02细胞存活率呈下降的趋势,但C3A细胞的存活率明显高于L-02细胞(P < 0.01);细胞凋亡率呈上升趋势,但48 h后C3A细胞同L-02细胞无差异(P  > 0.05)。谷草转氨酶及乳酸脱氢酶释放呈现上升的趋势,但C3A细胞明显低于L-02细胞(P  < 0.01)。白蛋白分泌功能呈下降的趋势,但C3A细胞明显优于L-02细胞(P  < 0.01)。尿素合成功能呈下降的趋势,但是L-02细胞明显优于C3A细胞(P  < 0.01)。结果提示,UW液4 ℃保存C3A细胞与L-02细胞时间不易超过48 h。以C3A细胞为材料的人工肝可能更适用于肝功能衰竭合并低白蛋白血症,以L-02细胞为材料的人工肝更适用于肝功能衰竭合并肝性脑病。     

Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation

The combination of ex vivo gene transfer and a sufficient transplant model for hepatocytes may permit treatment of single enzyme-based metabolic liver diseases. Induction of replicative potential (priming) in hepatocyte cultures may enhance the efficiency of gene transfer under stable in vitro conditions. It is known that hepatocyte replication is increased in vivo after partial hepatectomy. We investigated the effect of partial hepatectomy prior to cell isolation on hepatocytes in vitro. Male Lewis rats served as donors. Hepatocytes were isolated by collagenase digestion from either intact livers or from livers 48 h after 70% hepatectomy (PH). Cells were seeded on collagen-coated culture dishes with hormone-supplemented culture media. Hepatocyte morphology, number, albumin secretion rate, and mono-ethyl-glycin-xylidid (MEGX)-biotransformation capacity were assessed on days 1, 3, and 5 in culture. PH significantly increased hepatocyte number and albumin secretion of cultured hepatocytes over the whole observation period. In contrast, MEGX-biotransformation capacity was significantly decreased. Morphology of cultured hepatocytes was not affected by PH prior to hepatocyte isolation. These results suggest a prolonged and complex response of hepatocytes to PH in vitro. Hepatocyte priming by PH is a promising approach toward stable cultures of proliferating hepatocytes and may provide a model for in vitro studies of hepatic regeneration mechanisms. Further research on hepatocyte priming toward an application in ex vivo gene transfer and hepatic tissue engineering seems justified.