单克隆抗体BDI-I导向的阿霉素白蛋白毫微球对人膀胱癌细胞的特异杀伤活性

用化学偶联法将抗人膀胱癌单克隆抗体分子偶联到阿霉素白蛋白毫微球上,构建了一个有靶向杀伤性的免疫毫微球,即:阿霉素白蛋白载单克隆抗体毫微球(ADR-NP-Ab)。改变阿霉素毫微球和单克隆抗体的反应分子比,确定了制备该免疫毫微球的最佳条件。经免疫荧光检测及显微照像分析证明,免疫毫微球可有效地和人膀胱癌细胞结合。体外杀伤试验表明,此免疫毫微球对靶细胞EJ有高度特异杀伤活性,而对无关的人直肠癌Lovo细胞则无明显作用。     

具有活性羧基末端的长循环脂质体的制备和分布

目的　研究长循环免疫脂质体(immunoliposomes,IML)的制备方法, 体外靶细胞杀伤活性和在小鼠体内的组织分布。方法　合成和纯化了1个带末端羧基的磷脂酰乙醇胺(PE)的聚乙二醇衍生物(DPPE-PEG3000-COOH),掺入脂质体中制成长循环脂质体;通过羧基活泼酯化,将膀胱癌单克隆抗体BDI-1或小鼠IgG共价连接到该脂质体表面制成免疫脂质体,体外肿瘤细胞杀伤实验检测载阿霉素免疫脂质体(ADM-BDI-1-IML)特异杀伤靶细胞的能力。用同位素氚示踪法测量免疫脂质体在小鼠的组织分布。结果　抗体在脂质体上的结合率可达30％。体外肿瘤细胞杀伤实验证明载阿霉素免疫脂质体有选择性杀伤靶细胞人膀胱癌细胞EJ的能力。和普通脂质体相比,免疫脂质体在血中的滞留时间明显延长,并减少了在肝、脾的聚集。结论　长循环免疫脂质体在血中有较长的滞留时间,在体外有特异寻靶活性,载阿霉素免疫脂质体有选择性杀伤靶细胞的活性,这些性质为其在体内主动寻靶和选择性杀伤靶肿瘤细胞提供了必要条件。     

抗体F(ab'')2片段靶向的载多柔比星免疫毫微粒的构建及其抗肝癌作用

目的：构建抗体F（ab')2片段靶向的载药免疫毫微粒；评价F（ab')2片段免疫毫微粒对肿瘤的特异结合性及杀伤作用。方法：采用异型双功能交联剂琥珀酰亚胺基－3－（2－吡啶二硫）丙酸酯（SPDP）将抗人肝癌单抗HAb18或对照抗体4E3的F（ab')2片段与多柔比星（ADR）人血清白蛋白毫微粒（ADR－HAS－NP）共价交联构建抗体F（ab')2片段靶向的载药免疫毫微粒（HAb18F（ab')2ADR-HSA-NP,4E3F（ab')2ADR-HSANP);采用玻片凝集试验，免疫荧光染色实验研究F（ab')2片段是否结合在毫微粒表面；采用花环形成实验，花环形成阻断实验，扫描电镜分别从光镜、电镜水平观察F（ab')2片段免疫毫微粒是否同人肝癌细胞株SMMC7721特异性结合；采用MTT比色分析法研究F（ab')2片段免疫毫微粒的体外细胞毒作用；采用皮下荷人肝癌裸鼠模型，经瘤体注射观察免疫毫微业的抗人肝癌作用。结果：F（ab')2片段免疫毫微粒圆球状，粒径1.2μm左右，免疫荧光染色阳性而ADR－HSA－NP为阴性，提示F（ab')2片段已偶联到ARD－HSA－NP表面；HAb18F（ab')2ADR－HSA－NP能有效结合于人肝癌细胞株SMMC－7721，该结合能被HAb18抗体的F（ab')2片段阻断，未见该免疫毫微粒与对照细胞（SW1116）有效结合，这些结果提示，HAb18F（ab')2ADR－HSA－NP能有效特异性结合人肝癌细胞。HAb18F（ab')2ADR-HSA-NP能有效杀伤人肝癌细胞株SMMC－7721，且呈剂量依赖性，而4E3F（ab')2ADR－HSA－NP无明显杀伤作用；经瘤体给药，HAb18F（ab')2ADR-HSA－NP能阻止肿瘤生长，其20d后的抑瘤率为67.5%，明显高于ADR－HSA－NP（55.1%)。结论：采用SPDP化学校联法能构建抗体F（ab')2片段靶向的载药免疫毫微粒；该免疫毫微粒对肿瘤具有良好的特异靶向结合性及杀伤作用。     

单抗导向阿霉素免疫毫微粒抗肝癌作用的机制

目的研究抗人肝癌单克隆抗体HAb18为导向载体的阿霉素(ADR)人体白蛋白(HSA)免疫毫微粒HAb18-ADR-HSA-NP抗肝癌作用的机制.方法利用激光共聚焦仪和透射电镜观察HAb18-ADR-HSA-NP在人肝癌细胞SMMC-7721中的内化作用,通过扫描电镜和透射电镜观察HAb18-ADR-HSA-NP对SMMC-7721多药耐药株(SMMC-7721/MDR+)的结合和内化现象,采用四甲基偶氮唑蓝比色法测定HAb18-ADR-HSA-NP对SMMC-7721及其耐药细胞的杀伤作用.结果HAb18-ADR-HSA-NP在SMMC-7721细胞中存在内化现象,且该内化与温度有关,具抗体特异性.同时,HAb18-ADR-HSA-NP在SMMC-7721/MDR+表面结合,并能内化;且其能增强SMMC-7721/MDR+对ADR杀伤的敏感性.结论HAb18-ADR-HSA-NP抗肝癌作用的机制是其内化释药杀伤机制.     

单抗导向阿霉素免疫毫微粒抗肝癌作用的机制

目的　研究抗人肝癌单克隆抗体HAb18为导向载体的阿霉素 (ADR )人体白蛋白 (HSA)免疫毫微粒HAb18 ADR HSA NP抗肝癌作用的机制。方法　利用激光共聚焦仪和透射电镜观察HAb18 ADR HSA NP在人肝癌细胞SMMC 772 1中的内化作用 ,通过扫描电镜和透射电镜观察HAb18 ADR HSA NP对SMMC 772 1多药耐药株 (SMMC 772 1/MDR+ )的结合和内化现象 ,采用四甲基偶氮唑蓝比色法测定HAb18 ADR HSA NP对SMMC 772 1及其耐药细胞的杀伤作用。结果　HAb18 ADR HSA NP在SMMC 772 1细胞中存在内化现象 ,且该内化与温度有关 ,具抗体特异性。同时 ,HAb18 ADR HSA NP在SMMC 772 1/MDR+ 表面结合 ,并能内化 ;且其能增强SMMC 772 1/MDR+ 对ADR杀伤的敏感性。结论　HAb18 ADR HSA NP抗肝癌作用的机制是其内化释药杀伤机制     

第三代载药免疫脂质体及体内外寻靶研究

目的　研究载阿霉素第三代免疫脂质体的制备及体内外寻靶、抑瘤效果。方法　设计将人膀胱癌单抗与聚乙二醇羧酸(PEG-COOH)端相联,使构成的脂质体既充分发挥PEG的保护功能,延长药物血循环时间,又使单抗伸展在外部充分发挥其寻靶作用,即第三代免疫脂质体(IML)。进而研究载抗癌药的免疫脂质体的制备方案,制备出阿霉素免疫脂质体(IML-ADM)使达到对药物高包封、高稳定,又不降低单抗活性的目的。以人膀胱癌靶细胞EJ和人直肠癌非靶细胞LOVO进行体外杀伤和体内肿瘤的抑瘤实验。结果　IML-ADM对EJ细胞和LOVO细胞杀伤,及对EJ细胞移植瘤体的抑制与对照组比较均有显著性差异。结论　证实脂质体载药以单抗制导达到主动靶向给药是可行的     

阿霉素磁性明胶微球的研究

报告了阿霉素磁性明胶微球（Ａｄｒ－ＭＧ－ｍｓ）的制备与性质，研究了超细氧化铁粒子的合成和磁性明胶微球（ＭＧ－ｍｓ）在狗体内的栓塞效果。阿霉素磁性明胶微球由２％阿霉素（Ａｄｒ）、６８％明胶和３０％的磁铁粒子组成，微球的平均粒径为２２μｍ。在体外实验中，药物释放速度证明微球有缓释的性质。磁铁粒子的平均粒径约为１０ｎｍ，磁性明胶微球与￣（９９ｍ）Ｔｃ标记磁性明胶微球通过导管分别输入狗的肝动脉内进行栓塞，照相和血管造影显示在未加外磁场时磁性明胶微球在左右肝叶分布几乎相等，而在１２００高斯的外磁场作用下，靶部位肝左叶的微球分布是肝右叶的２．２５倍，而甲状腺、脑、心脏的微球很微量，结果表明磁性明胶微球在外磁场作用下是一个很好的治疗肝癌的栓塞剂。     

磁性纳米粒阿霉素微球制备的初探

目的:制备靶向抗癌药物即磁性纳米粒阿霉素白蛋白微球.方法:以阿霉素(ADR)、人血清白蛋白(HSA)和纳米Fe3O4为材料,采用乳化高温固化法制备出磁性纳米粒阿霉素白蛋白微球,并利用Hrtem对其包裹结合性能进行了观察,同时采用HPLC法对其载药量进行测试.结果:有效载药量为2.35%、表观载药量为3.55%.结论:采用乳化高温固化法能制备出磁性纳米粒阿霉素白蛋白微球.     

磁性免疫微球在人血清白蛋白纯化中的应用

目的为了快速地从人血清中提纯人血清白蛋白，利用磁性免疫微球作为提取手段，再用间接酶联免疫法测定人血清白蛋白的回收率。方法将经过羧基修饰的聚苯乙烯微球作为载体，用EDC(碳化亚胺)活化微球表面的羧基，再将兔抗人血清白蛋白抗体包被于微球上，这种微球-抗体复合物能特异性地捕获人血清白蛋白，磁分离复合物后，通过将兔抗人血清白蛋白抗体作为捕获抗体，将酶联羊抗人血清白蛋白抗体作为检测抗体，建立起间接酶联免疫法，用于检测人血清中和磁性免疫微球上吸附人血清白蛋白的浓度，得到微球从人血清中提纯人血清白蛋白的回收率。结果第1次提纯的回收率为(86±4)%，重复利用微球2次，回收率分别为(69.0±0.6)%和(40.8±0.8)%，而提纯的人血清白蛋白的纯度为90%。结论以上结果表明，免疫磁性微球提纯人血清白蛋白的实验是有效的，为工业上大规模提纯人血清白蛋白提供了一条新的思路。     

MTT方法测定培养细胞抗药水平的评价

用ＭＴＴ方法测定了多药抗药细胞ＭＣＦ－７Ａｄｒ及其敏感细胞ＭＣＦ－７ＷＴ对阿霉素、长春新碱、秋水仙碱的化学敏感性。ＭＣＦ－７Ａｄｒ与ＭＣＦ－７ＷＴ相比，对三种药物有明显的抗药性。维拉帕米可逆转ＭＣＦ－７Ａｄｒ对阿霉素的抗药性。随选择药物撤除时间的延长，ＭＣＦ－７Ａｄｒ的抗药水平逐渐下降。Ｓｏｕｔｈｅｒｎ杂交显示ＭＣＦ－７Ａｄｒ细胞有典型的ＭＤＲ１基因扩增现象。用ＭＴＴ方法测定Ｓｗｉｓｓ３Ｔ３细胞对阿霉素、长春新碱、秋水仙碱的化学敏感性，同时测定时各亚克隆株间差异较小，而分批测定时，不同批次相差很大。结果说明，ＭＴＴ方法能有效地检测出抗药细胞株的相对抗性；并提示，应在同时用完全相同的条件进行杀伤试验与ＭＴＴ测试，以避免批次间差异。     

Mixed micellar nanoparticle of amphotericin b and poly styrene-block-poly ethylene oxide reduces nephrotoxicity but retains antifungal activity

Mixed micellar nanoparticle consisting of amphotericin B (AmB) and poly styrene-block-poly ethylene oxide (PS-block-PEO) was prepared by high pressure homogenizer. Nephrotoxicity of the nanoparticle was investigated along with antifungal activity and self-aggregation status of the drug in the nanoparticle. Nephrotoxicity was markedly reduced when AmB was intravenously administered to rats as mixed micellar nanoparticle with PS-block-PEO in terms of transmission electron microscopy of tubular cells and creatinine clearance. Antifungal activity of AmB was not altered when the drug was in the form of mixed micellar nanoparticle compared to both conventional formulation and AmB micelle treated by same procedure without PS-block-PEO. Self-aggregation status of AmB molecules revealed monomeric in the mixed micellar nanoparticle with PS-block-PEO up to the therapeutic level of the drug (1-3 mM). The reduced nephrotoxicity of AmB in mixed micellar nanoparticle may be associated with the existence of the drug as monomeric form in the nanoparticle. Based on our result, formulation of AmB as mixed micellar nanoparticle with PS-block-PEO may be a promising alternative for the treatment of fungal diseases in patients who are at risk of renal dysfunction.     

In vitro and in vivo anti-tumor activities of a gemcitabine derivative carried by nanoparticles

Gemcitabine (Gemzar(?)) is the first line treatment for pancreatic cancer and often used in combination therapy for non-small cell lung, ovarian, and metastatic breast cancers. Although extremely toxic to a variety of tumor cells in culture, the clinical outcome of gemcitabine treatment still needs improvement. In the present study, a new gemcitabine nanoparticle formulation was developed by incorporating a previously reported stearic acid amide derivative of gemcitabine into nanoparticles prepared from lecithin/glyceryl monostearate-in-water emulsions. The stearoyl gemcitabine nanoparticles were cytotoxic to tumor cells in culture, although it took a longer time for the gemcitabine in the nanoparticles to kill tumor cells than for free gemcitabine. In mice with pre-established model mouse or human tumors, the stearoyl gemcitabine nanoparticles were significantly more effective than free gemcitabine in controlling the tumor growth. PEGylation of the gemcitabine nanoparticles with polyethylene glycol (2000) prolonged the circulation of the nanoparticles in blood and increased the accumulation of the nanoparticles in tumor tissues (>6-fold), but the PEGylated and un-PEGylated gemcitabine nanoparticles showed similar anti-tumor activity in mice. Nevertheless, the nanoparticle formulation was critical for the stearoyl gemcitabine to show a strong anti-tumor activity. It is concluded that for the gemcitabine derivate-containing nanoparticles, cytotoxicity data in culture may not be used to predict their in vivo anti-tumor activity, and this novel gemcitabine nanoparticle formulation has the potential to improve the clinical outcome of gemcitabine treatment.     

毛蚶多糖的分离纯化和免疫活性测定

目的从毛蚶体内分离纯化得到毛蚶多糖精品（polysaccharide from Arca subcrenata Lischke．简称ASLP），分析其纯度和单糖组成，同时考察免疫活性。方法经除蛋白、DEAE-52和Sephadex-GS0柱层析得多糖精品。糖醇乙酸酯法测定单糖组成。体外脾淋巴细胞增殖测定ASLP的免疫活性。结果从毛蚶体内提取到一种均一的多糖组分，其单糖组成只有葡萄糖，ASLP能够显著地促进脾淋巴细胞的增殖。结论从毛蚶中分离纯化得到一种均一的多糖组分，具有显著的体外免疫活性。     

Chitosan-based zinc oxide nanoparticle for enhanced anticancer effect in cervical cancer: A physicochemical and biological perspective

In this study, chitosan-assembled zinc oxide nanoparticle (CZNP) was successfully prepared for evaluated for its anticancer efficacy against cervical cancer cells. The CZNP particles were nanosized and spherical in shape. The zinc oxide nanoparticle (ZNP) and CZNP showed significant cytotoxicity in cervical cancer cells in a concentration-dependent manner. Results showed that the enhanced cytotoxicity was mainly attributed to the reactive oxygen species (ROS) generation in the cancer cells. The apoptosis assay further revealed that apoptosis was the main reason behind the cell killing effect of the zinc oxide nanoparticles. The apoptosis was further confirmed by the nuclear chromatin assay. Live dead assay showed increased red fluorescent cell for CZNP treated cancer cells. Overall, metal oxide present in nanoparticulate dimensions will be advantageous in imparting the cytotoxicity to cervical cancer cell.     

叶酸受体介导的负载紫杉醇纳米药物输送系统的体外生物活性的研究

目的:叶酸受体介导的负载紫杉醇纳米药物输送系统的的体外生物活性研究。方法:应用激光共聚焦观察肝素-叶酸-紫杉醇纳米粒进入叶酸受体阳性表达KB细胞和叶酸受体阴性表达A549细胞的情况,考察纳米粒对靶细胞摄取情况;以原药紫杉醇为对照组,应用MTT法检测纳米粒子对叶酸受体阳性表达KB细胞的抗癌抑制活性;应用流式细胞仪对纳米粒子抗癌机制进行分析。结果:细胞摄取实验表明,肝素-叶酸-紫杉醇纳米药物是通过叶酸受体介导的内吞作用实现对靶细胞的特异性;与紫杉醇对比,纳米粒子的针对叶酸受体阳性细胞的抑制效果更好,半数抑制浓度(IC50)为0.06g/mL,MTT实验结果与细胞摄取实验结果一致;流式细胞仪检测表明肝素-叶酸-紫杉醇纳米药物也表现出与紫杉醇相似的抑制作用,即G2/M期均有所增长。结论:体外生物活性检测表明叶酸受体介导的负载紫杉醇的纳米药物输送系统有较好的靶向性,其应用前景值得期待。     

Tumoricidal effects of etoposide incorporated into solid lipid nanoparticles after intraperitoneal administration in Dalton's lymphoma bearing mice

The tumoricidal effects of etoposide incorporated into lipid nanoparticles after single-dose administration were investigated in Dalton's lymphoma ascites bearing mice. Etoposide and its nanoparticle formulations were administered intraperitoneally, and the cell cycle perturbation, cytogenetic damage, cell death (apoptosis), tumor regression, and animal survival were investigated as parameters of response with time. The tumor burden of mice treated with etoposide and its nanoparticle formulations decreased significantly (P < .001) compared with the initial up to 4 to 6 days, followed by an increase at later time intervals. Of the 3 different formulations, the survival time of mice was higher when treated with etoposide-loaded tripalmitin (ETP) nanoparticles, followed by etoposide-loaded glycerol monostearate (EGMS) (27.3%) and etoposide-loaded glycerol distearate (EGDS) (27.3%) compared with free etoposide. Cell cycle analysis revealed the hypodiploid peak (sub G0/G1 cell population) as well as G2 arrest in mice treated with etoposide and its nanoparticle formulations. The frequency of dead cells treated with the nanoparticle formulations remained high even after 8 days of treatment compared with free etoposide. The mice treated with nanoparticle formulations exhibited hypodiploid peaks and reduced S phase even 8 days after treatment, whereas the free etoposide-treated mice showed decrease in apoptosis after 3 days of treatment. The apoptotic frequency in cells 17 days after treatment was in the order of ETP > EGMS > EGDS > etoposide. The experimental results indicated that among the 3 nanoparticle formulations studied, the ETP nanoparticles showed greater and prolonged apoptotic induction properties, resulting in the higher increase in survival time of tumor bearing mice.     

Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium

An in vitro model of the human follicle associated epithelium (FAE) was characterized and the influence of nanoparticle properties on the transcellular transport across the in vitro model was investigated. The model was established by co-culturing Caco-2 and Raji cells, with Caco-2 cells alone as control. The conversion of Caco-2 cells to follicle associated epithelium (FAE) like cells was monitored by following the surface expression of β1-integrins (immunofluorescence) and nanoparticle transport (flow cytometry). The influence of the nanoparticle concentration at the apical side, temperature, size and surface properties of nanoparticles on transport was evaluated, as well as the influence of transport conditions. The conversion of Caco-2 cells into FAE-like cells occurred. The transport was concentration, temperature and size-dependent. Aminated nanoparticles were more efficiently transported than carboxylated nanoparticles, suggesting a role of nanoparticle surface functional groups and hydrophobicity, possibly leading to a different pattern of protein adsorption at their surface. In conclusion, this in vitro model is a promising tool to study the role of M cells in transintestinal nanoparticle transport, as well as to evaluate new drug delivery systems.     

Colloidal Dispersions for the Delivery of Acyclovir: A Comparative Study

This paper describes a comparative study on the performances of ethosomes and solid lipid nanoparticle as delivery systems for acyclovir. Ethosomes were spontaneously produced by dissolution of phosphatidylcholine and acyclovir in ethanol followed by addition of an aqueous buffer while solid lipid nanoparticle were produced by homogenization and ultrasonication. Both colloidal systems were morphologically characterized by cryo-transmission electron microscopy. The encapsulation efficiency was 94.2±2.8% for ethosomes and 53.2±0.2% for solid lipid nanoparticle. Concerning Z potential, both formulations are close to neutrality. The diffusion coefficients of the drug from ethosomes and solid lipid nanoparticle, determined by a Franz cell method, were 9.4 and 1.2-fold lower as compared to the free acyclovir in solution, thus evidencing the ability of both colloidal systems in enhancing the diffusion of the drug. The antiviral activity against HSV-1 of both systems was tested by plaque reduction assay in monolayer cultures of Vero cells. Data showed that no significant differences in the antiviral activity were observed by acyclovir in the free or loaded forms. Taken together these results, colloidal systems could be interesting to mediate the penetration of acyclovir within Vero cells.     

Synthesis of oligo(ethylenediamino)-beta-cyclodextrin modified gold nanoparticle as a DNA concentrator

A novel oligo(ethylenediamino)-beta-cyclodextrin-modified gold nanoparticle (OEA-CD-NP) was synthesized as a vector for DNA binding and comprehensively investigated by means of absorption and circular dichroism spectroscopies as well as transmission electron microscopy, and its plasmid transfection efficiency as a carrier into cultivated cells in vitro was also evaluated. Possessing many hydrophobic cavities at the outer space, OEA-CD-NP may have a capability of carrying biological and/or medicinal substrates into cells, which will make it potentially applicable in many fields of material science and biological technology. In contrast with OEA-CD-NP, the oligo(ethylenediamino)-lipoic amido-modified gold nanoparticle (OEA-L-NP) without CD was synthesized to investigate the interaction with DNA. The results showed that OEA-L-NPs could only weakly bind DNA.