紫杉醇聚合物纳米囊泡的制备和体外释放研究

Objective To develop paclitaxol (PTX)-loaded polymersomes based on poly (ε-caprolactone)-block-poly (ethylene glycol)-block-poly (ε-caprolactone)(PCL-b-PEG-b-PCL, PCEP) amphiphilic triblock copolymers. Methods A series of PCEP copolymers was synthesized by ring-opening polymerization of ε-caprolactone initiated by PEG. The block copolymers were characterized by FT-IR、1H NMR and GPC. PTX-loaded polymersomes were prepared by thin-film and ultrasonic dispersion method and characterized in terms of morphology,particle size and size distribution, encapsulation efficiency and in vitro release. The effect of different hydrophilic and hydrophobic chain length on the drug-loading content, entrapment efficiency, size and size distribution and in vitro release were also investigated. Results The PTX-loaded polymersomes showed nanometer size and spherical morphology with core and shell. The sizes of PTX-loaded polymersomes increased with the increasing of the molecular weight of PCEP. The PTX-loaded polymersomes showed a continuous and steady release of PTX without initial burst release. The release rate increased with the increasing of hydrophilic PEG chain content and decreased with the increasing of hydrophobic PCL chain content. Conclusion For the first time, a novel PTX-loaded polymersomes was developed based on PCL-b-PEG-b-PCL amphiphilic triblock copolymers. The PTX-loaded polymersomes showed nanometer size, narrow size distribution and high drug encapsulation efficiency.These results indicate that PTX-loaded polymersomes could be a promising novel controlled release dosage form to increase therapeutic effect with decreased toxic and side effect of PTX.     

高效液相色谱法快速测定脑梗塞模型大鼠脑组织中多胺的含量

目的:建立一种简便、灵敏和快速分离测定大鼠脑组织中腐胺、精脒和精胺等多胺含量的高效液相色谱法。方法:采用苯甲酰氯作为衍生化试剂,以1,6-己二胺为内标,采用polygocyl(250×4.6 mm 5μm)C18色谱柱,流动相为甲醇-水(62:38 V/V),流速1.0 mL/min,检测波长229 nm。结果:本法线性范围是1～2 000/mL,回收率大于90%。日内和日间变异系数为1.5%～4.2%。最低定量浓度为1 nmol/mL。结论:该方法符合生物样品分析要求,为脑组织中多胺的含量测定提供了一种简便、准确、灵敏度高的分析方法。     

DC靶向适配体修饰的载铜绿假单胞菌DNA疫苗递送系统的构建及体外评价

构建了一种DC靶向适配体修饰的铜绿假单胞菌(Pseudomonas aeruginosa,PA)DNA疫苗递送系统。采用乙醇注入法制备阳离子脂质体,静电吸附法制备载pVAX1-OprF-VP22的阳离子脂质体(Lip-pOprF-VP22),探讨不同DOTAP/pDNA质量比的Lip-pOprF-VP22对pVAX1-OprF-VP22的包封效果、对DC2.4的细胞毒性及转染率,筛选最佳质量比的Lip-pOprF-VP22测定其粒径及Zeta电位;后插法制备DC靶向适配体修饰的载pVAX1-OprF-VP22的阳离子脂质体(Apt-Lip-pOprF-VP22),检测其转染DC2.4后OprF蛋白的表达量及对小鼠骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDCs)成熟的影响。结果表明,Lip-pOprF-VP22随着DOTAP/pDNA质量比增加包封率逐渐增加,当质量比为5∶1时即能很好的包封pVAX1-OprF-VP22;当Lip-pOprF-VP22作用于DC2.4 24 h或48 h后,不同质量比的Lip-pOprF-VP22对DC2.4的存活率均在80%以上;当DOTAP/pDNA质量比由2∶1增加到10∶1,转染率表现为先增加、后降低的趋势,其中DOTAP/pDNA质量比为4∶1、5∶1时转染率相对较高;当DOTAP/pDNA质量比为5∶1时,Lip-pOprF-VP22粒径为(171.67±1.27)nm,Zeta电位为(11.30±0.57)mV;Apt-Lip-pOprF-VP22转染DC2.4后可表达更多OprF蛋白且可明显促进BMDCs的成熟。     

紫杉醇聚合物纳米囊泡的制备和体外释放研究

目的 以聚己内酯-b-聚乙二醇-6-聚己内酯（PCEP）两亲性三嵌段共聚物为载体研制紫杉醇聚合物纳米囊泡.方法 以不同分子量的聚乙二醇（PEG）引发合成不同亲水段、疏水段链长的PCEP并进行FT-IR、1H NMR和GPC表征,以合成的嵌段聚合物PCEP为载体,通过薄膜-超声分散法制备紫杉醇聚合物纳米囊泡,用透射电子显微镜（TEM）表征其形态和构造,用粒度分析仪测定其粒径及分布,用高效液相色谱（HPLC）法测定其载药量及包封率,用透析袋法研究药物体外释放;同时,研究不同亲水链长、疏水链长对紫杉醇聚合物囊泡载药量、包封率、粒径及体外释放紫杉醇药物的影响.结果 研制的紫杉醇聚合物囊泡呈核-壳结构球形,粒径为纳米级,随着PCEP共聚物相对分子质量的增加而增大;紫杉醇聚合物囊泡体外释放无突释现象,能稳定缓慢释放紫杉醇,且释放速率随共聚物中亲水段PEG含量增加而增大,随疏水段PCL含量增大而减小.结论 以PCEP两亲性三嵌段共聚物为载体制备的紫杉醇聚合物纳米囊泡,其粒径小且分布均匀,包封率较高,有望成为一种用于提高紫杉醇的药效且降低不良反应的新的紫杉醇缓控释剂型.     

紫杉醇聚合物纳米囊泡的制备和体外释放研究

Objective To develop paclitaxol (PTX)-loaded polymersomes based on poly (ε-caprolactone)-block-poly (ethylene glycol)-block-poly (ε-caprolactone)(PCL-b-PEG-b-PCL, PCEP) amphiphilic triblock copolymers. Methods A series of PCEP copolymers was synthesized by ring-opening polymerization of ε-caprolactone initiated by PEG. The block copolymers were characterized by FT-IR、1H NMR and GPC. PTX-loaded polymersomes were prepared by thin-film and ultrasonic dispersion method and characterized in terms of morphology,particle size and size distribution, encapsulation efficiency and in vitro release. The effect of different hydrophilic and hydrophobic chain length on the drug-loading content, entrapment efficiency, size and size distribution and in vitro release were also investigated. Results The PTX-loaded polymersomes showed nanometer size and spherical morphology with core and shell. The sizes of PTX-loaded polymersomes increased with the increasing of the molecular weight of PCEP. The PTX-loaded polymersomes showed a continuous and steady release of PTX without initial burst release. The release rate increased with the increasing of hydrophilic PEG chain content and decreased with the increasing of hydrophobic PCL chain content. Conclusion For the first time, a novel PTX-loaded polymersomes was developed based on PCL-b-PEG-b-PCL amphiphilic triblock copolymers. The PTX-loaded polymersomes showed nanometer size, narrow size distribution and high drug encapsulation efficiency.These results indicate that PTX-loaded polymersomes could be a promising novel controlled release dosage form to increase therapeutic effect with decreased toxic and side effect of PTX.     

叶酸靶向紫杉醇聚合物纳米囊泡的制备及其抗肿瘤活性研究

 目的 制备新型叶酸靶向紫杉醇聚合物纳米囊泡,研究其理化性质、细胞毒性和体内抗肿瘤活性。方法 以两亲性三嵌段共聚物聚己内酯-b-聚乙二醇-b-聚己内酯（PCL-b-PEG-b-PCL）、甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺（mPEG2000-DSPE）、偶联叶酸的磷脂DSPE-PEG(2000) Folate共混物为载体材料,通过薄膜-超声分散法制备出叶酸靶向紫杉醇聚合物纳米囊泡并对其进行形态、粒径及其分布、Zeta电位、载药量及包封率、体外释放等表征,用CCK-8法研究了聚合物纳米囊泡对H1299肺癌细胞的细胞毒性,并评价其在BALB/c小鼠EMT-6乳腺癌模型中的抗肿瘤效果。结果 叶酸靶向紫杉醇聚合物纳米囊泡呈球形,具有明显的核-壳结构,粒径均匀。随着紫杉醇投药量的增加,纳米囊泡的粒径增大,包封率降低。载药30%的叶酸靶向紫杉醇聚合物纳米囊泡的平均粒径为311 nm,多分散系数为0.171,Zeta电位为-30.3 mV,包封率为91.16%。体外释放研究表明,载紫杉醇聚合物纳米囊泡基本无药物突释现象,具有缓释特性。细胞毒性研究表明,当紫杉醇浓度为25 μg·mL-1时,叶酸靶向紫杉醇聚合物纳米囊泡的细胞毒性低于相应浓度的紫杉醇/聚氧乙烯蓖麻油注射剂。体内抗肿瘤活性实验研究表明,叶酸靶向紫杉醇聚合物纳米囊泡对小鼠EMT-6乳腺癌具有明显抑制作用,具有与紫杉醇/聚氧乙烯蓖麻油注射剂相似的抑制肿瘤作用。结论 叶酸靶向紫杉醇聚合物纳米囊泡具有高载药量及包封率、均匀的粒径及分布、缓释特性、低毒和较好的抗肿瘤作用,是一种有潜力的紫杉醇缓控释新剂型用于提高紫杉醇的药效并且降低不良反应。     

犬血浆中氨氯地平和阿托伐他汀的LC-MS／MS测定

建立了LC-MS/MS法同时测定犬血浆中氨氯地平和阿托伐他汀的含量.用甲醇沉淀血样中的蛋白质.采用C18色谱柱,2 mmol/L醋酸胺溶液(含0.5%甲酸).甲醇(32:68)为流动相,以地西泮为内标,电喷雾离子化,正离子多反应监测,检测离子分别为m/z409.2→m/z 238.1(氨氯地平)、m/z 559.2→m/z 440.5(阿托伐他汀)和m/z285.1→m/z 193.1(地西泮).结果氨氯地平和阿托伐他汀在0.1～20ng/ml和0.2～50 ng/ml度范围内线性关系良好,定量限为0.1和0.2 ng/ml,方法回收率均大于94%,日内、日间RSD均小于9%.     

八宝素的组织分布和排泄研究

目的 研究八宝素(Hylotelephin)的组织分布和排泄,为临床试验提供依据.方法 用HPLC紫外检测法,以蒽为内标,苯甲酰氯为衍生剂,甲醇-水为流动相,测定静脉注射八宝素后生物样品中的药物含量.结果 Beagle犬静脉注射八宝素10.6 mg/kg后5 min药物已迅速分布到各组织;药后90 min大部分组织中药物含量明显低于5 min时的含量.药物主要分布在肾、肝和脾组织,其次在心、肺、肠等组织中.肾、肝和脾中的药物含量与相同时间的血浆药物浓度接近.大鼠静脉注射八宝素36 mg/kg后从尿、粪和胆汁(0～48 h)中排泄的原型药物分别占给药总量的88.45%,0.61%和1.08%;给药后3 h 67%药物已从体内排出.结论 八宝素在Beagle犬体内分布和排泄较快,药物主要分布在肾、肝、脾和血浆等组织中,主要以原型药物方式由尿液排泄.     

心复康含药血清对骨髓干细胞转录和分泌SDF-1α的影响

目的 研究心复康含药血清对骨髓干细胞（BMSCs）中间质源性细胞因子α（SDF-1α）mRNA表达和蛋白分泌的影响.方法 采用全骨髓培养法分离和扩增BMSCs,并用流式细胞仪进行鉴定.以定量PCR（q-PCR）和酶联免疫吸附测定法（ELISA）分别检测含药血清作用后BMSCs中SDF-1α mRNA表达和蛋白分泌的改变情况.结果 经心复康含药血清作用72 h后,SDF-1α mRNA的表达量明显增加,实验组约为对照组的200倍（P＜0.05）;SDF-1α蛋白分泌量增加近1倍（P＜0.05）,其中实验组为（277.561 1±15.651 8）pg/ml,对照组（153.107 1±14.765 1）pg/ml.结论 全骨髓培养法可获得高纯度的BMSCs,含药血清可明显促进SDF-1αmRNA的表达及其蛋白分泌.     

SLCO1B1/ApoE基因多态性与瑞舒伐他汀疗效及安全性的相关性研究

目的 探讨SLCO1B1与ApoE基因多态性对瑞舒伐他汀的降脂疗效和安全性的相关性.方法 采用纳米磁珠法提取全血基因组DNA,PCR-焦磷酸测序法检测SLCO1B1与ApoE基因多态性.152名受试者口服瑞舒伐他汀10 mg/d,通过检测用药前以及用药8周后的低密度脂蛋白胆固醇(LDL-C)水平,评价降脂疗效;通过随访用药期间肌痛的发生,评价肌病不良反应的发生频率.结果 152名受试者SLCO1B1 521T＞C基因型分布为TT 73.7％,TC 23.7％,CC 2.6％;ApoE基因型分布为ε3/ε3 65.8％,ε3/ε213.2％,ε4/ε3 21.0％,未见ε4/ε4、ε2/ε2、ε4/ε2基因型.瑞舒伐他汀治疗8周后,APOE ε3/ε3,ε3/ε3与ε4/ε3基因组比较,患者血浆中的LDL-C下降水平有统计学意义(P＜0.05);SLCO1B1 521T＞C 3种基因型发生肌痛的频率明显不同(P＜0.05).结论 SLCO1B1/ApoE基因多态性与瑞舒伐他汀疗效及安全性具有相关性,联合检测SLCO1B1与ApoE基因型有助于预测疗效和不良反应,实现瑞舒伐他汀的个体化用药.