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。     

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     

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.     

用免疫磁性微球从骨髓中分离癌细胞

用物理吸附结合化学键共价结合的方法,将抗人膀胱癌单克隆抗体连接到预先制备的聚苯乙烯磁性微球表面,构建了能特异地与靶细胞结合并赋予其以磁响应性的免疫磁性微球(immunomagnetic microspheres,IMMS)。间接免疫荧光检测和IMMS与细胞的结合实验证明,所构建的IMMS可有效地和靶细胞结合。研究了聚苯乙烯磁性微球和抗体反应的质量比、IMMS与靶细胞比例对IMMS与细胞结合效果的影响。用IMMS从动物骨髓中分离癌细胞的初步实验表明,IMMS可有效清除癌细胞,而骨髓细胞仅有很少量的损失。     

阿霉素白蛋白微球冻干制剂体外释放及稳定性研究

阿霉素白蛋白微球冻干制剂是用于肝动脉栓塞治疗肝癌的新制剂.它由缓释和速释两部分组成.与白蛋白微球相结合的阿霉素为缓释部分,未与白蛋白微球结合的游离阿霉素为速释部分.当用于肝动脉栓塞时,速释部分迅速释放到肝组织发挥作用,缓释部分逐步释放药物,保持其组织的药物浓度.同时,白蛋白微球本身有阻断肝动脉供血使肿瘤组织坏死的治疗作用.本文着重介绍该制剂的体外释放及稳定性研究结果.     

抗癌药物白蛋白微球的研究进展

为提高抗癌药物对癌细胞或组织的靶向性,增强疗效,降低其全身毒副作用,以不同材料作载体的抗癌药物微球相继研制成功:不可生物降解的乙基纤维素微球,可生物降解的白蛋白微球、淀粉微球、明胶微球、聚乳酸微球,以及近年来问世的磁性微球、毫微球等。白蛋白微球以其良好的生物相容性和可降解性被广泛用于抗癌药靶向给药系统。 白蛋白微球最初用于动物及人的肺部扫描和循环系统研究,自1974年首次作为抗癌药物载体以来,相继用作诊断试剂及靶向给药、化学栓塞治疗的载体。1 抗癌药物动脉栓塞白蛋白微球 肿瘤动脉栓塞疗法是将抗癌药物制剂选择性注入支配     

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。     

Nanoparticle formulation of 11-keto-β-boswellic acid (KBA): anti-inflammatory activity and in vivo pharmacokinetics

Context: The oleo-gum-resin of Boswellia serrata Roxb. (Burseraceae) is widely used for the treatment of inflammatory diseases such as osteoarthritis, rheumatoid arthritis and cancer. Anti-inflammatory activity of 11-keto-β-boswellic acid (KBA) is impeded by poor oral bioavailability due to its high lipid solubility, rapid phase-1 metabolism and poor intestinal permeability.

Objective: This study developed a poly-dl-lactide-co-glycolide-based nanoparticle formulation of KBA to improve its oral bioavailability and in vivo anti-inflammatory activity.

Materials and methods: KBA was isolated from the oleo-gum resin of B. serrata, and its nanoparticle formulation (KBA-NPs) was prepared by the emulsion–diffusion–evaporation method. Oral bioavailability of KBA and KBA-NPs was studied at 50?mg/kg p.o. dose in Sprague–Dawley rats, and further evaluated for in vivo anti-inflammatory activity in carrageenan-induced rat paw oedema assay at the same dose level.

Results: The prepared KBA-NPs had a particle size of 152.6?nm with polydispersity index of 0.194, 79.7% entrapment efficiency and a cumulative 61.5% release of KBA from KBA-NPs, at 72?h. KBA-NPs showed 60.8% inhibition of rat paw oedema at 5?h as compared to 34.9% as that of KBA. The results of oral bioavailability study and in vivo anti-inflammatory activity showed 7- and 1.7-fold increase in bioavailability and anti-inflammatory activity, respectively, of KBA in KBA-NPs as compared to KBA alone.

Conclusion: The results of improved oral bioavailability and in vivo anti-inflammatory activity of KBA-NPs suggested successful development of KBA nanoparticle formulation.     

In vitro and in vivo evaluation of macromolecular prodrug GC-FUA based nanoparticle for hepatocellular carcinoma chemotherapy

A novel type of macromolecular prodrug delivery system is reported in this research. The N-galactosylated-chitosan-5-fluorouracil acetic acid conjugate (GC-FUA) based nanoparticle delivery system was evaluated in vitro and in vivo. Biocompatibility of GC-FUA-NPs was screened by BSA adsorption test and hemolysis activity examination in vitro. Cytotoxicity and cellular uptake study in HepG2 and A549 cells demonstrated that compared to free 5-Fu, the GC-FUA-NPs play great function in killing cancer cells for the cell endocytosis mediated by asialoglycoprotein receptor (ASGPR), which overexpresses on the cell surface. Pharmacokinetics study further illustrated that the drug-loaded nanoparticles has a much longer half-time than free 5-Fu in blood circulation in Sprague–Dawley (SD) rats. Tissue distribution was investigated in Kunming mice, and the result showed that the GC-FUA-NPs have a long circulation effect. The obtained data suggested that GC-FUA-NP is a very promising drug delivery system for efficient treatment of hepatocellular carcinoma.     

Three-dimensional hydrogel constructs for exposing cells to nanoparticles

In evaluating nanoparticle risks to human health, there is often a disconnect between results obtained from in vitro toxicology studies and those from in vivo activity, prompting the need for improved methods to rapidly assess the hazards of engineered nanomaterials. In vitro studies of nanoparticle toxicology often rely on high doses and short exposure periods due to the difficulty of maintaining monolayer cell cultures over extended time periods as well as the difficulty of maintaining nanoparticle dispersions within the culture environment. In this work, tissue-engineered constructs are investigated as a platform for providing doses of nanoparticles over different exposure periods to cells within a three-dimensional environment that can be tuned to mimic in vivo conditions. Uptake of quantum dots (QDs) by model neural cells was first investigated in a high-dose exposure scenario, resulting in a strong concentration-dependent uptake of carboxyl-functionalised QDs. Poly(ethylene glycol) hydrogel scaffolds with varying mesh sizes were then investigated for their ability to support cell survival and proliferation. Cells were co-encapsulated with carboxyl-functionalised poly(ethylene glycol)-coated QDs at a lower dose than is typical for monolayer cultures. Although the QDs leach from the hydrogel within 24 h, they are also incorporated by cells within the scaffold, enabling the use of these constructs in future studies of cell behaviour and function.     

Design of a Multifunctional PLGA Nanoparticulate Drug Delivery System: Evaluation of its Physicochemical Properties and Anticancer Activity to Malignant Cancer Cells

Purpose  Several individual approaches were combined to fabricate a novel nanoparticulate drug delivery system to achieve targeting and anticancer effects in various malignant cancer cells. Methods  Doxorubicin was conjugated to Poly(lactic-co-glycolic acid) (PLGA), which was formulated into nanoparticle via solvent-diffusion method. The surface of the nanoparticles was subsequently linked with Poly(ethylene glycol) (PEG) and Arg-Gly-Asp (RGD) peptide to realize both passive and active targeting functions. The multifunctional nanoparticles were then tested against several malignant cancer cell lines. Results  The conjugation increased loading efficiency of doxorubicin to PLGA nanoparticles (the encapsulation efficiency was over 85%) and alleviated the drug burst release effect substantially. The drug was released from the polymeric matrix in a sustained release manner over a period of 12 days. The resultant nanoparticles were spherically uniform and well-dispersed. The nanoparticle targeting ability was proven through strong affinity to various integrin-expressing cancer cells, and much less affinity to the low integrin expression cancer cells. The nanoparticles also showed high efficacy in inducing apoptosis in specific malignant cancer cell. Conclusion  The developed multifunctional nanoparticles hold potential to treat malignant integrin-expressing cancers.     

Effect of farmorubicin both free and associated with poly(butylcyanoacrylate) nanoparticles on phagocytic and NK activity of peritoneal exudate cells from tumor-bearing mice

The effect of Epirubicin (farmorubicin, FR), either free or associated with poly(butylcyanoacrylate) nanoparticles (PBCN) upon the phagocytic and natural killer (NK) activity of peritoneal exudate cells (PECs) harvested from Lewis lung carcinoma (LLC)-bearing-mice was investigated. Phagocytic and NK activity were tested 72 and 96 h, respectively after the last four intraperitoneal (i.p.) injections of the tested compounds have been administered to the mice. Phagocytic activity was evaluated in vitro by phagocytic index and ingestion capacity using a phagocytic assay. NK activity was evaluated in a direct cytotoxic test, in which PECs were used as effector cells while human erythroleukemic K-562 cells were used as target cells. The phagocytic activity of PECs, harvested from tumor-bearing mice, was stimulated after treatment with FR free, FR associated with polymer nanoparticles and with unloaded PBCN. The NK activity of PECs was strongly stimulated by unloaded PBCN. FR both free and encapsulated into the polymer matrix during the polymerization of n-butylcyanoacrylate (n-BCA) stimulated the NK activity of PECs, while FR adsorbed onto nanoparticles restrained it. These results suggest that the association of FR with nanoparticles modifies selectively its immunomodulating ability without producing any significant immunological disturbances. The toxicity of some of FR polymer forms towards PECs, displaying NK activity, probably comes from the enhanced local drug concentration on the membrane surface of the immune cells. However, it is insufficient to preclude the use of nanoparticles as drug delivery system.     

Cytotoxic,genotoxic and the hemolytic effect of titanium dioxide (TiO2) nanoparticles on human erythrocyte and lymphocyte cells in vitro

With the increasing clinical use of titanium dioxide (TiO₂) nanoparticles, a better understanding of their safety in the blood stream is required. The present study evaluates the toxic effect of commercially available TiO₂ nanoparticles (~100 nm) using a battery of cytotoxic, genotoxic, hemolytic and morphological parameters. The cytotoxic effects of TiO₂ nanoparticles in human lymphocyte cells were studied with respect to membrane damage, mitochondrial function, metabolic activity and lysosomal membrane stability. Genotoxicity in lymphocyte cells was quantitated using a comet assay. The mode of cell death (apoptosis/necrosis) was evaluated using PI/Annexin V staining. TiO₂ nanoparticles were also evaluated for their hemolytic properties, osmotic fragility and interaction with hemoglobin. Human erythrocyte cells were studied for morphological alterations using atomic force microscopy (AFM). Results suggest that the particles could induce a significant reduction in mitochondrial dehydrogenase activity in human lymphocyte cells. Membrane integrity remained unaffected by nanoparticle treatment. DNA damage and apoptosis were induced by TiO₂ nanoparticles in a dose‐dependent manner. A study on human erythrocyte cells revealed a hemolytic property of TiO₂ nanoparticles characterized by spherocytosis and echinocytosis. Spectral analysis revealed a hemoglobin TiO₂ nanoparticle interaction. Our in vitro study results suggest that commercially available blood contacting nanoparticles (TiO₂ nanoparticle) should be carefully evaluated for their toxic potential. Copyright © 2013 John Wiley & Sons, Ltd.     

载阿霉素透明质酸聚合物纳米粒的构建及其体外性质研究

目的 合成透明质酸（HA）接枝单油酸甘油酯（GMO）两亲性聚合物HGO,并研究其所制备载阿霉素（DOX）纳米粒的理化性质及体外抗肿瘤效果。方法 HA与GMO通过酯化反应制得载体聚合物HGO,通过核磁共振波谱法及红外光谱法对其进行结构表征;采用芘荧光探针法测定聚合物临界聚集浓度（CAC）。采用透析法制备聚合物HGO载阿霉素（DOX@HGO）纳米粒,并对其进行粒径分布、Zeta电位及微观形态的表征;通过检测其在不同离子强度、不同pH条件下的粒径变化考察纳米粒的体外稳定性;考察DOX@HGO纳米粒在不同pH条件下的体外释放行为;CCK-8法考察DOX@HGO纳米粒对MDA-MB-231细胞的体外抑瘤效果;并通过荧光显微镜研究MDA-MB-231细胞对DOX溶液、DOX@HGO纳米粒的摄取能力,以及HA预处理对DOX@HGO纳米粒摄取的影响。结果 成功制得两亲性聚合物HGO,聚合物HGO中GMO的取代度为15.8%,CAC为0.023 mg·mL^-1。DOX@HGO纳米粒呈规则的球形,平均粒径为（130.800±1.709）nm,平均电位为（-32.600±0.153）mV,包封率和载药量分别为（98.65±0.74）%和（33.03±0.17）%,在不同离子强度下、模拟胃肠液中表现出良好的稳定性;DOX@HGO纳米粒的体外释放表现出pH依赖性。体外抗肿瘤活性实验表明,DOX@HGO纳米粒对MDA-MB-231细胞的生长具有较好的抑制作用;与DOX溶液比较,DOX@HGO纳米粒显著增加肿瘤细胞对于DOX的摄取（P＜0.05） ,HA预处理显著减少肿瘤细胞对DOX@HGO的摄取（P＜0.05）。结论 所构建的DOX@HGO纳米粒具有良好的理化性质,并且具有一定的pH敏感性及靶向抗肿瘤细胞的能力,是具有应用潜力的药物载体。     

NIST gold nanoparticle reference materials do not induce oxidative DNA damage

Abstract

One primary challenge in nanotoxicology studies is the lack of well-characterised nanoparticle reference materials which could be used as positive or negative nanoparticle controls. The National Institute of Standards and Technology (NIST) has developed three gold nanoparticle (AuNP) reference materials (10, 30 and 60 nm). The genotoxicity of these nanoparticles was tested using HepG2 cells and calf-thymus DNA. DNA damage was assessed based on the specific and sensitive measurement of four oxidatively-modified DNA lesions (8-hydroxy-2´-deoxyguanosine, 8-hydroxy-2´-deoxyadenosine, (5´S)-8,5´-cyclo-2´-deoxyadenosine and (5´R)-8,5´-cyclo-2´-deoxyadenosine) using liquid chromatography/tandem mass spectrometry. Significantly elevated, dose-dependent DNA damage was not detected at concentrations up to 0.2 μg/ml, and free radicals were not detected using electron paramagnetic resonance spectroscopy. These data suggest that the NIST AuNPs could potentially serve as suitable negative-control nanoparticle reference materials for in vitro and in vivo genotoxicity studies. NIST AuNPs thus hold substantial promise for improving the reproducibility and reliability of nanoparticle genotoxicity studies.     

Photobiological and Ultrastructural Studies of Nanoparticles of Poly(lactic-co-glycolic acid)-Containing Bacteriochlorophyll-a as a Photosensitizer Useful for PDT Treatment

The interaction of polymeric nanoparticles formulated from the biodegradable polymer poly(DL-lactide-co-glycolide) loaded with bacteriochlorophyll-a was studied in homogeneous solution and in vitro in the presence of a macrophage cell line (P388-D1-ATCC). Photodynamic therapy (PDT) activity after different laser doses also was investigated. Scanning electron microscopy analysis of cell phagocyte nanoparticles showed that after 30 min of incubation most of the nanoparticles are in a clear adhesion process to the cell surface. The majority of nanoparticles became phagocytic after 2 hr of incubation time. After laser irradiation of the dye-containing system a total photodamage by nanoparticle phagocyte cells was observed and the cell survival was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Our results indicate that polymeric nanoparticles work as an efficient drug delivery system for PDT drugs. This approach can be widely used for many other hydrophobic photosensitizers with higher aggregation tendency in neoplastic cell treatment.