Preparation of camptothecin-loaded targeting nanoparticles and their antitumor effects on hepatocellular carcinoma cell line H22

Abstract

Camptothecin (CPT) is an effective anticancer agent against various cancers but the clinical application is limited because of its poor water solubility, low bioavailability and severe toxic side effects. The aim of the present study was to evaluate the feasibility of using targeted NPs as a high-performance CPT delivery system that targets liver cancer cells through intravenous (i.v.) administration route. CPT was incorporated into biotin-F127-PLA or F127-PLA polymeric nanoparticles (NPs) by a dialysis method. The preparation of the targeting NPs was performed by conjugating biotin-F127-PLA NPs with anti-3A5 antibody. The antitumor effect of the CPT-loaded nanoparticles against H22 cells in vitro was determined using an MTT assay. Tissue distribution and tumor inhibition in vivo were also evaluated. Survivin mRNA expression was assessed by real-time polymerase chain reaction. Results showed that the targeted CPT NPs exhibited regular spherical shapes with a mean diameter of approximately 180?nm. In vitro release of the targeted CPT NPs exhibited an initial burst (40%) within 12?h, followed by a slow release. Cytotoxicity test against H22 cells indicated that the targeted CPT NPs exerted significant antitumor effects. Compared with free CPT and non-targeted CPT NPs, the targeted CPT NPs showed superior inhibition ratio against tumor in vivo, which may be associated with reduced survivin mRNA expression. The results suggested that the new targeted CPT NPs may be a promising injectable delivery system for cancer therapy.     

Attaching folic acid on gold nanoparticles using noncovalent interaction via different polyethylene glycol backbones and targeting of cancer cells

This article reports a simple one-step method of attaching folic acid (FA) to gold nanoparticles (AuNPs) and its fine tuning using different polyethylene glycol (PEG) backbones. PEG backbones used in this study are PEG-diamine with molecular weights 2000 (PAM2-2K) and 10,000 (PAM2-10K), PEG-tetramine with molecular weight 20,000 (PAM4-20K), and PEG-dithiol with molecular weight 2000 (PSH2-2K). The nanoconjugates were characterized with ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), thermogravimetric analysis, Fourier transform infrared spectroscopy, inductively coupled plasma analysis, and radioactivity measurement with a scintillation counter. Attachment and release profiles of FAs from the nanoconjugates are done using ³H-labelled FAs (³FA). The binding of ³FA follows the order Au-PAM4-20K > Au-PAM2-10K > Au-PAM2-2K > Au > Au-PSH2-2K, whereas its release profile follows the reverse order. Au-PAM4-20K-FA has been used for folate receptor (FR)–mediated targeting of AuNPs to cancer cells. Seven different cancer cell lines (SKOV-3, OVCAR-5, OV-202, OV-167, OPM-1, RPMI, and U266) were screened for expression of FRs. Among ovarian cancer cells, the expression pattern of FRs follows the order OV-167 > SKOV-3 > OV-202 > OVCAR-5, and multiple myeloma cell lines follow the order OPM-1 > U266 > RPMI. Intracellular uptakes of the nanoconjugates containing FA or no FA were monitored with digital optical photography and TEM. Quantitation of the internalization of nanoconjugates in different cell lines was determined by gold analysis with inductively coupled plasma. It is found that the uptake of the nanoconjugates correlates with FR expression. Maximum uptake is observed for OV-167, whereas it is minimum for OVCAR-5. TEM images of the cells treated with Au-PAM4-20K-FA confirm the endocytosis of the nanoconjugates. This study is an important step for targeted delivery of anticancer drugs as well as metal nanoparticles for targeted therapy, tumor imaging, and ablation exploiting the overexpression of FRs on cancer cells.     

Design of a bifunctional fusion protein for ovarian cancer drug delivery: single-chain anti-CA125 core-streptavidin fusion protein.

We have developed a universal ovarian cancer cell targeting vehicle that can deliver biotinylated therapeutic drugs. A single-chain antibody variable domain (scFv) that recognizes the CA125 antigen of ovarian cancer cells was fused with a core-streptavidin domain (core-streptavidin-VL-VH and VL-VH-core-streptavidin orientations) using recombinant DNA technology and then expressed in Escherichia coli using the T7 expression system. The bifunctional fusion protein (bfFp) was expressed in a shaker flask culture, extracted from the periplasmic soluble protein, and affinity purified using an IMAC column. The two distinct activities (biotin binding and anti-CA125) of the bfFp were demonstrated using ELISA, Western blot and confocal laser-scanning microscopy (CLSM). The ELISA method utilized human NIH OVCAR-3 cells along with biotinylated bovine serum albumin (B-BSA) or biotinylated liposomes, whereas, the Western blot involved probing with B-BSA. The CLSM study has shown specificity in binding to the OVCAR-3 cell-line. ELISA and Western blot studies have confirmed the bifunctional activity and specificity. In the presence of bfFp, there was enhanced binding of biotinylated antigen and liposome to OVCAR-3 cells. In contrast, the control EMT6 cells, which do not express the CA125 antigen, showed minimal binding of the bfFp. Consequently, bfFp based targeting of biotinylated therapeutic drugs, proteins, liposomes, or nanoparticles could be an alternative, convenient method to deliver effective therapy to ovarian cancer patients. Peritoneal infusion of the bfFp-therapeutic complex could also be effective in locally targeting the most common site of metastatic spread.     

Magnetic micelles as a potential platform for dual targeted drug delivery in cancer therapy

The magnetic nanomicelles as a potential platform for dual targeted (folate-mediated and magnetic-guided) drug delivery were developed to enhance the efficiency and veracity of drug delivering to tumor site. The magnetic nanocarriers were synthesized based on superparamagnetic iron oxide nanoparticles (SPIONs), biocompatible Pluronic F127 and poly(dl-lactic acid) (F127-PLA) copolymer chemically conjugated with tumor-targeting ligand-folic acid (FA) via a facile chemical conjugation method. Doxorubicin hydrochloride (DOX·HCl) was selected as a model anticancer drug to investigate the in vitro drug release and antiproliferative effect of tumor cells in vitro and in vivo in the presence or absence of an external magnetic filed (MF) with strength of 0.1T. The Alamar blue assay exhibited that these magnetic nanomicelles possessed remarkable cell-specific targeting in vitro. Additionally this smart system enabling folate receptor-mediated uptake into tumor cells, showed strong responsiveness to MF. The primary in vivo tumor model study, which was carried out in VX2 tumor-bearing male New Zealand white rabbits, demonstrated that the nanomicelles could be guided into tumor site more efficiently by application of MF, and further represented significant therapeutic efficiency to solid tumor.     

Three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin-biotin technology: Formulation development and in vitro anticancer activity

Despite recent advances in cancer therapy, many malignant tumors still lack effective treatment and the prognosis is very poor. Paclitaxel is a potential anticancer drug, but its use is limited by the facts that paclitaxel is a P-gp substrate and its aqueous solubility is poor. In this study, three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin-biotin technology was evaluated in vitro as a way of enhancing delivery of paclitaxel. Paclitaxel was incorporated both in biotinylated (BP) and non-biotinylated (LP) PEG-PLA nanoparticles by the interfacial deposition method. Small (mean size approximately 110nm), spherical and slightly negatively charged (-10mV) BP and LP nanoparticles achieving over 90% paclitaxel incorporation were obtained. The successful biotinylation of nanoparticles was confirmed in a novel streptavidin assay. BP nanoparticles were targeted in vitro to brain tumor (glioma) cells (BT4C) by three-step avidin-biotin technology using transferrin as the targeting ligand. The three-step targeting procedure increased the anti-tumoral activity of paclitaxel when compared to the commercial paclitaxel formulation Taxol((R)) and non-targeted BP and LP nanoparticles. These results indicate that the efficacy of paclitaxel against tumor cells can be increased by this three-step targeting method.     

PLGA-TPGS 负载α-TIF-siRNA 纳米粒对疱疹病毒1的抑制作用

目的：研究以 PLGA-TPGS 生物可降解材料为载体包载α-TIF-siRNA 的纳米粒对 HSV1的抑制作用。方法以 PLGA-TPGS 为载体,采用双乳蒸发法制备包载α-TIF-siRNA 的 PLGA-TPGS 纳米粒（命名为 PLGA-TPGS/α-TIF-siRNA NPs）,并对其进行表征,包括粒径大小、zeta 电位、包封率和释放率,用 MTT 法检测纳米粒对上皮细胞和HeLa 细胞的细胞毒作用,免疫荧光观察纳米粒在细胞内的释放,用空斑实验研究体外研究纳米粒对 HSV1病毒的抑制作用。结果 PLGA-TPGS/α-TIF-siRNA NPs 的粒径大小为（257±2.94）nm,zeta 电位为（-31.25±1.70）mV,siRNA的包封率为（56.23±3.68）％,纳米释放 siRNA 呈双相,即在96 h 释放达到50％,之后呈缓慢释放,用 MTT 法分析 PL-GA-TPGS/α-TIF-siRNA NPs 对原代角质形成细胞和 HeLa 细胞几乎无细胞毒性。荧光显微镜能观察纳米粒 siRNA 细胞内释放。PLGA-TPGS/α-TIF-siRNA NPs 能明显延长抑制感染 HeLa 细胞的 HSV1。结论 PLGA-TPGS 纳米粒可以作 siRNA 的载体。PLGA-TPGS/α-TIF-siRNA NPs 在体外对 HSV1病毒具有明显的抑制作用,可以成为治疗 HSV1-诱导的角膜炎在内的相关疾病的候选药物。     

Doxorubicin-conjugated D-glucosamine- and folate- bi-functionalised InP/ZnS quantum dots for cancer cells imaging and therapy

Nanoscaled quantum dots (QDs), with unique optical properties have been used for the development of theranostics. Here, InP/ZnS QDs were synthesised and functionalised with folate (QD-FA), D-glucosamine (QD-GA) or both (QD-FA-GA). The bi-functionalised QDs were further conjugated with doxorubicin (QD-FA-GA-DOX). Optimum Indium to fatty acid (In:MA) ratio was 1:3.5. Transmission electron microscopy (TEM) micrographs revealed spherical morphology for the QDs (11?nm). Energy-dispersive spectroscopy (EDS) spectrum confirmed the chemical composition of the QDs. MTT analysis in the OVCAR-3 cells treated with bare QDs, QD-FA, QD-GA, QD-FA-GA and QD-FA-GA-DOX (0.2?mg/mL of QDs) after 24?h indicated low toxicity for the bare QDs and functionalised QDs (about 80–90% cell viability). QD-FA-GA-DOX nanoparticles elicited toxicity in the cells. Cellular uptake of the engineered QDs were investigated in both folate receptor (FR)-positive OVCAR-3 cells and FR-negative A549 cells using fluorescence microscopy and FACS flow cytometry. The FA-functionalised QDs showed significantly higher uptake in the FR-positive OVCAR-3 cells, nonetheless the GA-functionalised QDs resulted in an indiscriminate uptake in both cell lines. In conclusion, our findings indicated that DOX-conjugated FA-armed QDs can be used as theranostics for simultaneous imaging and therapy of cancer.     

Gemcitabine cationic polymeric nanoparticles against ovarian cancer: formulation,characterization, and targeted drug delivery

This study focused on gemcitabine (GTB) delivery of cationic polymeric nanoparticles to treat ovarian cancer in order to promote effective localized delivery and drug retention during biological discharge. To begin, four GTB-loaded polymer nanoparticles were prepared: chitosan nanoparticles (CS-NPs), polysarcosin nanoparticles (PSar-NPs), poly-l-lysine & polysarcosin nanoparticles (PLL-PSar-NPs), and chitosan & polysarcosin nanoparticles (CS-PSar-NPs). Based on preliminary particle size, zeta potential, encapsulation efficiency, DSC, surface morphology, release profiling, and cellular internalization studies using rhodamine 123 and Nile red fluorescent markers, it was hypothesized that CS-PSar-NPs could be the best cationic formulation with strong biocompatibility and anticancer activity against the OVCAR-8 ovarian cancer cell line. To improve effective targeting, cellular penetration, and in vitro cytotoxicity, epidermal growth factor receptor variation III (EGFRvIII) is attached over all four polymeric nanoparticles. Confocal imaging revealed that EGFRvIII-conjugated cationic GTB polymeric nanoparticles had a greater cellular uptake and double internalization capabilities than unconjugated nanoparticles, as well as time-dependent cell entrance. GTB and EGFRvIII-conjugated polymer nanoparticles would have a stronger potential to infiltrate ovarian cancer cells during the first hour of incubation. According to TEM and FTIR findings, EGFRvIII conjugation across the non-target CS-PSar-NP surface was successful, making CS-PSar-NPS-EGFRvIII more target-specific and thus a safer drug delivery candidate for ovarian cancer treatment.

Highlights

• GTB loaded non-target CS-PSar-NPs & active targeted CS-PSar-NPs-EGFRvII developed.
• SEM, AFM, DSC, particle size, zeta potential, internalization performed for CS-PSar-NPs.
• MTT & CLSM study confirmed CS-PSar-NPS-EGFRvII was binding specific to OVCAR-8 cells
• Fabrication of EGFRvII over nanoparticles confirmed by TEM.
• CS-PSar-NPS-EGFRvII safer candidate for ovarian cancer.

     

紫杉醇Pluronic P105聚合物胶束的制备、表征与逆转肿瘤多药耐药性的体外研究

药物递送系统是克服肿瘤多药耐药性(MDR)的一种新策略。本文以聚合物胶束系统和难溶性药物紫杉醇(PTX)为研究对象，旨在制备一种新型的PTX给药系统，既能增溶难溶性药物，又具有克服肿瘤MDR的能力。以Pluronic P105为载体，采用固体分散-水化法制备PTX聚合物胶束，并以星点设计-效应面优化法进行处方优化。对其粒径、体外释放等性质进行表征后，以人耐药卵巢癌细胞SKOV-3/PTX为细胞模型，体外评价PTX聚合物胶束的细胞摄取及其逆转肿瘤细胞耐药性的作用。结果显示，聚合物胶束制剂的载药量约为1.1%、药物浓度约为700 μg·mL^-1、平均粒径约为24 nm。胶束制剂与普通制剂(Taxol)在6 h内的累积释放分别为45.4%和95.2%，前者具有较强的缓释作用；胶束制剂与Taxol对SKOV-3/PTX的IC₅₀值分别为1.14和5.11 μg·mL^-1，二者的耐药逆转指数(RRI)分别为9.65和2.15。胶束制剂可促进耐药细胞对P-糖蛋白(P-gp)底物(PTX或Rhodamine-123)的摄取。结果表明，Pluronic P105可有效增溶难溶性药物PTX，并形成具有较强缓释作用的纳米级聚合物胶束制剂，该制剂可显著提高PTX对人卵巢癌耐药细胞的细胞毒性，能逆转其耐药性。     

Degradable Poly(β-amino ester) nanoparticles for cancer cytoplasmic drug delivery

Fast cytoplasmic drug delivery can overcome cancer cells' drug resistance and thus have an enhanced therapeutic efficacy. Such a drug delivery regime requires drug carriers capable of entering cancer cells, localizing and rapidly releasing the drug into endosomes/lysosomes, and subsequently disrupting their membranes to release the drug into the cytosol. We herein report a low-toxic and degradable poly(β-amino ester)-graft-polyethylene glycol (BAE-PEG) co-polymer forming pH-responsive nanoparticles capable of cytoplasmic drug delivery. BAE-PEG was synthesized by condensation polymerization of diacrylate and piperazine in the presence of a PEG-diacrylate macromonomer. BAE-PEG with 2% or 5% PEG side chains formed micelles (nanoparticles) with diameters of about 100 nm. The BAE-PEG nanoparticles were shown to rapidly enter cancer cells, localize in their endosomes/lysosomes, and subsequently disrupt them to release the drugs into the cytosol. Camptothecin loaded in the nanoparticles had a higher cytotoxicity to SKOV-3 ovarian cancer cells than free camptothecin.     

川芎嗪联合mTOR抑制剂调控卵巢癌SKOV-3细胞增殖、侵袭迁移的实验研究

目的 研究川芎嗪联合mTOR抑制剂对卵巢癌SKOV-3细胞增殖、侵袭迁移的作用.方法 分别设置对照组、40μmol/L川芎嗪组、5μmol/L雷帕霉素组、40μmol/L川芎嗪联合5μmol/L雷帕霉素组,各组SKOV-3细胞给药后继续培养48 h后,MTT法考察各组SKOV-3细胞的增殖抑制率,Transwell实验...     

大蒜素对TRAIL诱导SKOV-3细胞凋亡影响的作用研究

目的研究大蒜素对肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导人卵巢癌细胞SKOV-3凋亡的作用机制。方法体外培养的人卵巢癌细胞SKOV-3,以大蒜素和重组人TRAIL蛋白单独及联合作用后MTT法检测细胞的生长抑制率;流式细胞仪检测大蒜素作用后细胞死亡受体DR4、DR5表达变化;Caspase活性检测试剂盒检测大蒜素作用后细胞Caspase-3、8活性变化。结果 （1）单独应用12.5、25、50、75mg/L的大蒜素、单独应用25、50、100、200ng/ml的TRAIL蛋白以及50mg/L的大蒜素联合100ng/ml的TRAIL蛋白处理SKOV-3细胞24、48、72h后,随着药物浓度和作用时间的增加,抑制率增高。各组抑制率与对照组比较,差异有统计学意义（P〈0.05）。（2）大蒜素作用48h后SKOV-3细胞TRAIL死亡受体DR4、DR5表达上调,DR4、DR5的FI值分别由用药前的1.78、1.94升高到2.27、2.58。用药前后DR4、DR5表达变化比较有统计学意义（P〈0.05）。（3）大蒜素作用48h后,SKOV-3细胞Caspase-3、8活性明显上调,Caspase-3、8活性的OD试验组/OD空白组的比值分别是对照组的2.49、2.08倍,用药前后Caspase-3、8活性变化比较差异有统计学意义（P〈0.05）。结论 （1）SKOV-3细胞是大蒜素和TRAIL敏感细胞株。（2）大蒜素可以增强TRAIL诱导细胞凋亡作用。（3）大蒜素上调死亡受体4、5的表达,提高Caspase-3、8活性是其增强TRAIL诱导肿瘤细胞凋亡的重要机制。     

Radioiodinated folic acid conjugates: evaluation of a valuable concept to improve tumor-to-background contrast

Folic acid radioconjugates can be used for targeting folate receptor positive (FR(+)) tumors. However, the high renal uptake of radiofolates is a drawback of this strategy, particularly with respect to a therapeutic application due to the risk of damage to the kidneys by particle radiation. The goal of this study was to develop and evaluate radioiodinated folate conjugates as a novel class of folate-based radiopharmaceuticals potentially suitable for therapeutic application. Two different folic acid conjugates, tyrosine-folate (1) and tyrosine-click-folate (3), were synthesized and radioiodinated using the Iodogen method resulting in [(125)I]-2 and [(125/131)I]-4. Both radiofolates were highly stable in mouse and human plasma. Determination of FR binding affinities using (3)H-folic acid and FR(+) KB tumor cells revealed affinities in the nanomolar range for 2 and 4. The cell uptake of [(125)I]-2 and [(125/131)I]-4 proved to be FR specific as it was blocked by the coincubation of folic acid. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro assays were employed for the determination of tumor cell viability upon exposure to [(131)I]-4. Compared to untreated control cells, significantly reduced cell viability was observed for FR(+) cancer cells (KB, IGROV-1, SKOV-3), while FR(-) cells (PC-3) were not affected. Biodistribution studies performed in tumor bearing nude mice showed the specific accumulation of both radiofolates in KB tumor xenografts ([(125)I]-2: 3.43 ± 0.28% ID/g; [(125)I]-4: 2.28 ± 0.46% ID/g, 4 h p.i.) and increasing tumor-to-kidney ratios over time. The further improvement of the tumor-to-background contrast was achieved by preinjection of the mice with pemetrexed allowing excellent imaging via single-photon emission computed tomography (SPECT/CT). These findings confirmed the hypothesis that the application of radioiodinated folate conjugates may be a valuable concept to improve tumor-to-background contrast. The inhibitory effect of [(131)I]-4 on FR(+) cancer cells in vitro indicates the potential of this class of radiofolates for therapeutic application.     

Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene

Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.     

Effects of chemotherapy and/or radiotherapy on survivin expression in ovarian cancer

Survivin, a member of apoptosis protein inhibitor family, is upregulated in various malignancies, especially in chemotherapy- and/or radiation-resistant cell lines. In this study, the correlation between the level of survivin expression and degree of apoptosis was investigated in three ovarian cancer lines (two chemoresistant cell lines SKOV-3 and OVCAR-3, as well as one chemosensitive cell line OV2008) treated with 5 microg/ml of cisdiamminedichloroplatinum (cisplatin, CDDP) for 24 h, 2 Gy of (60)Co irradiation, or 5 microg/ml CDDP for 3 h plus 2 Gy of (60)Co, respectively. We also evaluated the survivin mRNA abundance in patients with advanced ovarian cancers during CDDP treatment. In the ovarian cancer cell lines, survivin mRNA abundance and protein contents were significantly increased after the treatments while the apoptotic rates did not change in SKOV-3 and OVCAR-3. Moreover, in OVA2008 cells the expression of survivin decreased and the apoptotic rate significantly increased after CDDP and combined treatments. Survivin mRNA was not detectable in normal ovarian tissues and benign ovarian tumors. However, it was observed in the resected tumor specimens from 20 patients with advanced ovarian cancer. These results suggested that survivin may play an important role in the resistance to chemotherapy and radiotherapy in ovarian cancer cell lines and in the progression of ovarian tumors. Survivin may also provide a pivotal prognostic implication for epithelial ovarian carcinomas.     

白花蛇舌草注射液诱导SKOV-3细胞株凋亡的研究

目的研究白花蛇舌草注射液对体外培养条件下卵巢癌细胞SKOV-3细胞增殖的影响以及诱导其凋亡的情况。方法取对数生长期的SKOV-3细胞（细胞数为5X104^／mL）分别接种于不同细胞培养板上,试验组每孔加入200μL不同质量浓度的白花蛇舌草注射液,空白对照组加入等体积PRMI-1640培养液孵育,采用四甲基偶氮唑盐（MTF）比色法检测吸光度（A490值）并计算SKOV-3细胞增殖抑制率;Annexin—V—FITC／PI染色细胞后,用流式细胞仪技术检测空白对照组与试验组的细胞凋亡率,并置荧光显微镜下观察不同组细胞的凋亡形态变化。结果MTT法检测结果显示,随着白花蛇舌草注射液质量浓度和作用时间的增加,SKOV-3细胞增殖抑制率均明显增大,12mL／L的试验组培养48h的抑制率最高,达（35．67±2．16）％;流式细胞仪检测显示,白花蛇舌草注射液处理后可引起SKOV-3细胞凋亡率随着白花蛇舌草注射液质量浓度的增加呈升高趋势,且荧光镜下试验组细胞可呈现典型凋亡改变。结论白花蛇舌草注射液能诱导卵巢癌SKOV-3细胞凋亡。     

芹菜素丝素蛋白纳米粒的制备及其安全性、抗肿瘤活性研究

目的制备芹菜素丝素蛋白(API@SF)纳米粒,并评价其安全性和抗肿瘤活性。方法采用纳米沉淀法制备API@SF纳米粒,并对其形态、粒径、Zeta电位、载药量、体外释放等进行表征;采用溶血实验和HE染色法评价该纳米粒的安全性;采用MTT法评价该纳米粒对小鼠乳腺癌4T1细胞的抑制作用。结果本研究所制得的API@SF纳米粒呈类球形,粒径分布均匀,平均粒径为406.61 nm,多分散性指数为0.154,Zeta电位为-18.4 mV,平均载药量为5.20%。体外释放结果显示,该纳米粒在pH 5.0的释放介质中释放速率相对较快,在pH 7.4的释放介质中释放速率相对较慢。溶血实验和HE染色结果显示,该纳米粒具有良好的生物相容性。MTT实验结果显示,API@SF纳米粒对4T1细胞的抑制作用显著高于API原料药(P<0.05),其作用机制可能与提高细胞中活性氧水平有关。结论本研究成功制备了API@SF纳米粒,该纳米粒具有良好的安全性和抗肿瘤活性。     

Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.     

Apoptosis-inducing oxovanadium(IV) complexes of 1,10-phenanthroline against human ovarian cancer

In a systematic effort to identify a potent anticancer agent against human ovarian cancer, we synthesized 15 oxovanadium(IV) complexes, and examined their cytotoxic activity against human ovarian cancer cell lines PA-1, SKOV-3, ES-2 and OVCAR-3 using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyletetrazolium bromide]-based assay. The apoptosis-inducing ability of the oxovanadium compounds was evaluated by the two-color flow cytometric terminal deoxynucleotidyl transferase-based assay that labels 3'-hydroxyl ends of fragmented DNA (TUNEL) assay and confocal laser scanning microscopy. Notably, all eight oxovanadium complexes of 1,10 phenanthroline exhibited significant cytotoxicity and induced apoptosis within 24 h. The mono-chelated, VO(NO2-phen) and bis-chelated, VO(Me2-phen)2, VO(Cl-phen)2 and VO(NO2-phen)2 complexes were the most potent oxovanadium compounds, and killed target cancer cells at low micromolar concentrations. The marked differences in the cytotoxic activity of oxovanadium(IV) complexes containing different heterocyclic ancillary ligands suggest that the cytotoxic activity of these compounds is determined by the identity of the five-member bidentate ligands, as well as the nature of the substituents on the heterocyclic aromatic rings. Our results presented herein provide experimental evidence that oxovanadium compounds induce apoptosis in human ovarian cancer cells. The lead compounds, VO(Me2-phen)2 and VO(NO2-phen)2, may be useful in the treatment of ovarian cancer.     

The reversal of multidrug resistance in ovarian carcinoma cells by co-application of tariquidar and paclitaxel in transferrin-targeted polymeric micelles

In this study, a transferrin (Tf)-modified polyethylene glycol-phosphatidyl ethanolamine (PEG-PE)-based micellar delivery system containing paclitaxel (PTX) and tariquidar (TRQ), a potent third generation P-gp inhibitor, was prepared. The nanoformulation was evaluated by targeting efficiency, cellular association, cellular internalization pathway and cytotoxicity for reversal of PTX resistance on two multidrug resistant (MDR) ovarian carcinoma cell lines, SKOV-3TR and A2780-Adr. PTX and TRQ are both hydrophobic compounds. They were successfully encapsulated into the micellar structure containing vitamin E as the encapsulation enhancer. The Tf-targeted micelles were internalized mainly via clathrin-dependent endocytosis by both cell lines. For SKOV-3TR, additional mechanisms including caveolin-dependent endocytosis and macropinocytosis were found to play a significant role. The PTX cytotoxicity against the SKOV-3TR and A2780-Adr MDR cells was increased significantly in the presence of micellar encapsulation. However, unlike the A2780-Adr cell line, the Tf-targeting effect was significant on SKOV-3TR cells when co-administrated with TRQ. Penetration of the Tf-targeted micelles in a cancer cell spheroid culture was also investigated.