联合包载阿霉素和siRNA阳离子脂质体的制备及体外评价

目的制备联合包载阿霉素和siRNA阳离子脂质体并进行体外评价。方法采用薄膜分散法制备载阿霉素阳离子脂质体(doxorubicin cationic liposomes,DCL),与siRNA静电复合得联合载药阳离子脂质体(liposome complexes,lipoplexes);动态激光散射法测定lipoplexes的粒径和zeta电位;透射电镜观察lipoplexes形态;超滤离心法测定lipoplexes中阿霉素和siRNA的包封率;琼脂糖凝胶阻滞实验考察lipoplexes中siRNA的结合能力;荧光显微镜观察MCF-7/ADR细胞对siRNA的摄取情况。结果所制备的lipoplexes外形圆整、分散均匀,当(2,3-二油氧基丙基)三甲基氯化铵与siRNA质量比为20时,其平均粒径为(125.7±2.7)nm,zeta电位为((45.8±1.5)m V,阿霉素包封率为(52.3±2.6)%,siRNA包封率为(88.1±1.8)%,且lipoplexes中siRNA可以紧密结合。与游离siRNA相比,lipoplexes可显著增加MCF-7/ADR细胞对siRNA的摄取。结论联合包载阿霉素和siRNA阳离子脂质体体外性质良好,能增加MCF-7/ADR细胞对siRNA的摄取,可用于小分子化疗药物和siRNA的共输送。     

钙调素拮抗剂O-(4-乙氧基)-丁基-小檗胺增强阿霉素杀伤MCF-7/ADR细胞的机制研究

目的研究钙调素拮抗剂0-4-乙氧基-丁基-小檗胺(EBB)增强阿霉素诱导乳腺癌多药耐药细胞系MCF-7/ADR细胞的杀伤作用及其相关机制。方法用MTT法测定阿霉素、EBB单独及联合用药对阿霉素杀伤乳腺癌多药耐药细胞系(MCF-7/ADR)及其亲代细胞系(MCF-7)的作用的IC50值,用不同浓度EBB处理MCF-7/ADR细胞后用FACS法分析EBB对阿霉素诱导细胞凋亡及对mdr1mRNA和P-gp蛋白水平表达的影响,通过激光共聚焦显微镜观察EBB处理前后及用EBB预处理24和48h后MCF-7/ADR和MCF-7细胞内阿霉素浓度的改变。结果MTT结果显示EBB对MCF-7和MCF-7/ADR都具有抗肿瘤活性;EBB还能协同提高阿霉素的细胞毒作用,MCF-7组两药相互作用指数(CDI)值为0.73,MCF-7/ADR组CDI值为0.49,其对耐药细胞的协同作用更为明显。随EBB剂量增加,低剂量阿霉素诱导MCF-7/ADR细胞凋亡增加而且P-gp蛋白表达水平逐渐下降,细胞内阿霉素浓度逐渐提高,而且用EBB预处理MCF-7/ADR细胞24和48h后细胞内阿霉素和罗丹明浓度也逐渐提高。结论EBB是有效的肿瘤细胞化疗药物,它不但能直接抑制P-gp功能还具有下调P-gp蛋白表达的作用,从而有效逆转MCF-7/ADR细胞的耐药现象,协同增强化疗药物对耐药细胞的杀伤作用。     

线粒体靶向DQA-DOX连接物的制备及其逆转肿瘤耐药活性研究

目的为寻找线粒体靶向的抗肿瘤药物,将阿霉素(doxorubicin,DOX)与地喹氯铵(dequalinium,DQA)连接形成DQADOX,观察DQA-DOX的抗肿瘤作用。方法合成DQA-DOX连接物,通过MTT、细胞摄取实验对DQA-DOX进行体外抗肿瘤活性研究。结果细胞毒性实验显示:与游离DOX相比,DQA-DOX对A549细胞的毒性比游离DOX明显低,但DQA-DOX对耐DOX细胞MCF-7/ADR细胞的毒性比游离DOX显著增强。共聚焦显微镜观察结果显示:MCF-7/ADR细胞给予游离DOX后,DOX在细胞浆和细胞核分布得很少;MCF-7/ADR细胞给予DQA-DOX后,DQA-DOX特异性地分布于线粒体。结论DQA-DOX具有对抗阿霉素耐药作用,有进一步研究的价值。     

蛇床子素对耐阿霉素人乳腺癌细胞耐药的逆转作用

目的:研究蛇床子素(Ost)对人乳腺癌细胞阿霉素耐药株多药耐药(MDR)的逆转作用及其机制。方法:采用MTT比色法测定药物的细胞毒性,高效液相-紫外检测法检测细胞内ADR浓度。结果:Ost对MCF-7及MCF-7/ADR细胞均有增殖抑制作用,IC50值分别为(58.1±2.3)μmol.L-1和(59±5)μmol.L-1;在5~15μmol.L-1范围内,Ost可以不同程度地增强ADR对MCF-7细胞杀伤作用,与ADR联合用药降低ADR对MCF-7/ADR的IC50值,显著增加MCF-7/ADR细胞内ADR的积累。结论:Ost对人乳腺癌细胞株MCF-7及其阿霉素耐药株MCF-7/ADR细胞均有增殖抑制作用,而且Ost通过明显增加MCF-7/ADR细胞内ADR的浓度,逆转其阿霉素耐药性。     

叶酸受体及二氢叶酸还原酶与人乳腺癌细胞MCF-7/ADR多药耐药的关系

目的以人乳腺癌多药耐药细胞系MCF-7/ADR及其敏感亲本系MCF-7为对象,探讨叶酸受体(FOLRα)及其下游基因二氢叶酸还原酶(DHFR)的表达与乳腺癌细胞多药耐药的关系。方法 MTT法测定阿霉素的细胞毒性作用及DHFR抑制剂对MCF-7/ADR细胞多药耐药的逆转作用;RT-PCR检测细胞FOLRα和DHFR mRNA的表达水平;免疫细胞化学检测FOLRα、P-gp的表达水平。结果 MCF-7细胞增殖速度快于MCF-7/ADR细胞,MCF-7细胞FOLRα mRNA转录水平较MCF-7/ADR细胞高,而MCF-7/ADR细胞DHFR mRNA较MCF-7细胞转录水平高;免疫细胞化学显示MCF-7细胞FOLRα的表达高于MCF-7/ADR细胞,而MCF-7/ADR细胞P-gp的表达较MCF-7细胞高;MCF-7/ADR对甲氨蝶啶无耐药性,甲氨蝶啶对MCF-7/ADR细胞多药耐药有逆转作用。结论 MCF-7/ADR细胞FOLRα的表达水平下调可能与细胞增殖水平有关,其下游基因DHFR表达水平与MCF-7/ADR细胞的MDR可能存在相关性。     

pH敏感多肽修饰载紫杉醇脂质体的制备及评价

目的制备p H敏感多肽修饰载紫杉醇脂质体(p HS-LP-PTX),并评价其体外性质。方法采用薄膜分散法制备PHS-LP-PTX,MTT实验考察脂质体对MCF-7细胞和Hep G2细胞的毒性,通过细胞摄取实验考察脂质体与肿瘤细胞的结合能力。结果所制备的p HS-LP-PTX在p H6.4时平均粒径为125.5±13.4 nm,Zeta电位为10.47±2.53 m V,24 h内具有良好的血清稳定性。MTT结果显示:p H6.4时,p HS-LP-PTX的细胞毒性优于各对照组。体外细胞摄取实验表明:p H6.4时,MCF-7细胞和Hep G2细胞对p HS-LP-PTX的摄取效率优于p H7.4时和普通脂质体。结论紫杉醇脂质体经过p H敏感多肽修饰后,能增强脂质体在酸性环境下的细胞穿透能力,是一种良好的p H敏感型肿瘤靶向给药系统。     

耐药人乳腺癌细胞中葡萄糖神经酰胺合成酶的表达及意义

目的:探讨葡萄糖神经酰胺合成酶(GCS)基因在人乳腺癌细胞株(MCF-7)和耐阿霉素人乳腺癌细胞株(MCF-7/ADR)中的表达及意义。方法:采用MTT法检测阿霉素对MCF-7/ADR和MCF-7的半数抑制浓度(IC50),用不同浓度的GCS抑制剂PDMP预处理MCF-7/ADR不同时间后检测IC50;运用流式细胞术检测MCF-7及PDMP预处理MCF-7/ADR前、后GCS蛋白的表达水平;应用实时荧光定量PCR法检测MCF-7及PDMP作用前、后MCF-7/ADR中GCS基因的表达(GCSN)水平。结果:阿霉素对MCF-7/ADR和MCF-7的IC50分别为(18.95±0.54)、(0.84±0.07)μg·mL-1;MCF-7/ADR对MCF-7的耐药倍数为22.69倍,PDMP作用后阿霉素对MCF-7/ADR的IC50下降至(5.63±0.58)μg·mL-1。MCF-7/ADR中GCS蛋白及GCSN的表达均高于MCF-7(P<0.01),PDMP使MCF-7/ADR中GCSN表达水平从0.0048±0.0017下降至0.0021±0.0004。结论:GCS可能参与肿瘤耐药的发生过程,并在MCF-7/ADR多药耐药中起重要作用。     

高胰岛素对 MCF-7/ADR 细胞化疗敏感性的影响

摘要:目的 分析高胰岛素对 MCF-7/ADR 细胞 P-糖蛋白（P-gp）表达和功能的影响, 并初步探讨胰岛素对 MCF-7/ADR 细胞化疗敏感性的影响。方法 用不同胰岛素浓度（0.001、 0.005、 0.01、 0.05、 0.10 μmol/L）的全细胞培 养基干预 MCF-7/ADR 细胞, 分别采用 Real-time PCR 法检测细胞中 P-gp mRNA 表达, Western Blot 法检测细胞中 P-gp 蛋白表达水平, 罗丹明 123 荧光实验测定 P-gp 的外排功能变化, MTT 法检测 MCF-7/ADR 细胞活性及化疗敏 感性。结果 0.10 μmol/L 的胰岛素可促进 MCF-7/ADR 细胞增殖, 0.05 及 0.10 μmol/L 浓度的胰岛素可增加 MCF-7/ ADR 细胞 P-gp mRNA 及蛋白水平表达, 并能增加 P-gp 的外排功能, 降低 MCF-7/ADR 细胞对表阿霉素的化疗敏感 性。结论 高浓度胰岛素可能通过促进 MCF-7/ADR 细胞 P-gp 的表达和功能, 增加乳腺癌细胞化疗耐药性, 从而降 低 MCF-7/ADR 细胞的化疗敏感性。     

两种新型西地那非同系物对MCF-7/ADR细胞对阿霉素耐药的影响

目的探讨两种新型西地那非同系物对人乳腺癌MCF-7/ADR细胞耐阿霉素的逆转作用。方法采用GENMED磷酸二酯酶5（PDE5）活性酶测定试剂盒检测两种同系物对5型磷酸二酯酶（PDE5）的抑制作用,用MTT法检测西地那非、西地那非同系物、阿霉素以及西地那非同系物与阿霉素联合作用对人乳腺癌耐药细胞MCF-7/ADR和敏感株MCF-7的抑制率及IC50值,运用Western blot检测P-gp蛋白的表达。结果两种同系物与西地那非相比具有较低的PDE5抑制活性;10μmol·L^-1两种西地那非同系物对MCF-7/ADR细胞耐药性的逆转倍数分别是6.36和5.58,20μmol·L^-1两种西地那非同系物对MCF-7/ADR细胞耐药性的逆转倍数分别是11.83和13.47;两种同系物作用对P-gp蛋白的表达无明显影响。结论两种新型西地那非同系物具有较低的PDE5抑制活性,一定浓度的西地那非同系物可显著逆转MCF-7/ADR细胞对阿霉素的耐药。     

介孔碳纳米粒的构建及化疗-光疗联合抗多药耐药肿瘤研究

目的:构建负载化疗药物并具有光热和光动力联合治疗效果的介孔碳纳米粒(MCNs),研究其体外抗多药耐药肿瘤的作用。方法:利用低浓度水热法制备MCNs,通过混酸超声法将MCNs表面羧基化制成MCNs-COOH(MCNC),对其形貌、表面性质等进行表征。利用吸附法构建负载阿霉素(ADR)的ADR/MCNC,通过紫外吸光度计算其载药量,利用透析法考察其释放特性。选用耐ADR人乳腺癌MCF-7/ADR细胞通过共聚焦激光显微镜观察ADR/MCNC的细胞摄取和定位,MTT法考察ADR/MCNC的细胞毒性,用流式细胞术测定NIR光照下细胞内活性氧自由基(ROS)水平。结果:所制备的MCNC的粒径约为90 nm,表面含有羧基,BET比表面积为541.62 m~2/g,孔容为0.34 cm~3/g,孔径分布约为2.5 nm,具有显著光热效应。ADR/MCNC的载药量为47.4%,具有pH/NIR响应性释放特性;NIR光照下能促进ADR的细胞摄取和核内蓄积,能诱导MCF-7/ADR细胞产生ROS,并对细胞有显著的抑制作用。结论:成功制得MCNs,此外ADR/MCNC具有抗多药耐药肿瘤的作用。     

泊洛沙姆188-PLGA纳米给药系统对抗耐药肿瘤的研究

目的 利用泊洛沙姆188对PLGA进行化学修饰,制备包载阿霉素的纳米粒,并评价纳米粒在人耐药乳腺癌细胞中的摄取能力及毒性。方法 通过EDC/NHS法合成泊洛沙姆188-PLGA,通过核磁共振对其结构进行表征并测定临界胶束浓度;通过纳米沉淀法制备包载阿霉素的纳米粒,通过粒度仪对纳米粒的粒径及分布进行分析,通过细胞摄取实验及细胞毒性实验对纳米粒的摄取效果及毒性进行评价。结果 成功合成了泊洛沙姆188-PLGA,并制备了粒径在140 nm左右的纳米粒,该纳米粒在人耐药乳腺癌细胞中有较好的摄取效果及较强的毒性。结论 泊洛沙姆188能够逆转耐药,增强耐药细胞对化疗药物的敏感程度。     

pH-sensitive micelles self-assembled from star-shaped TPGS copolymers with ortho ester linkages for enhanced MDR reversal and chemotherapy

TPGS approved by FDA can be used as a P-gp inhibitor to effectively reverse multi-drug resistance (MDR) and as an anticancer agent for synergistic antitumor effects. However, the comparatively high critical micelle concentration (CMC), low drug loading (DL) and poor tumor target limit its further clinical application. To overcome these drawbacks, the pH-sensitive star-shaped TPGS copolymers were successfully constructed via using pentaerythritol as the initial materials, ortho esters as the pH-triggered linkages and TPGS active-ester as the terminated MDR material. The amphiphilic star-shaped TPGS copolymers could self-assemble into free and doxorubicin (DOX)-loaded micelles at neutral aqueous solutions. The micelles exhibited the lower CMC (8.2 × 10⁻⁵ mg/ml), higher DL (10.8%) and long-term storage and circulation stability, and showed enhanced cellular uptake, apoptosis, cytotoxicity, and growth inhibition for in vitro MCF-7/ADR and/or MCF-7/ADR multicellular spheroids and in vivo MCF-7/ADR tumors via efficiently targeted drug release at tumoral intracellular pH (5.0), MDR reversal of TPGS, and synergistic effect of DOX and TPGS. Therefore, the pH-sensitive micelles self-assembled from star-shaped TPGS copolymers with ortho ester linkages are potentially useful to clinically transform for enhanced MDR cancer treatment.     

The efficacy of WGA modified daunorubicin anti-resistant liposomes in treatment of drug-resistant MCF-7 breast cancer

Background: Breast cancer is the most common malignancy and remains a leading cause of cancer-related deaths in female. Chemotherapy failure of breast cancer is mainly associated with multidrug resistance of cancer cells.

Purpose: The WGA modified daunorubicin anti-resistant liposomes were developed for circumventing the multidrug resistance and eliminating cancer cells.

Methods: WGA was modified on liposomal surface for increasing the intracellular uptake. Tetrandrine was inserted into the phospholipid bilayer for reversing cancer drug-resistance, and daunorubicin was encapsulated in liposomal aqueous core as an anticancer agent. Evaluations were performed on MCF-7 cells, MCF-7/ADR cells and xenografts of MCF-7/ADR cells.

Results: In vitro results showed that WGA modified daunorubicin anti-resistant liposomes exhibited suitable physicochemical properties, significantly increased intracellular uptake in both MCF-7 cells and MCF-7/ADR cells, and circumvented the multidrug resistance via inhibiting P-gp. In vivo results demonstrated that the targeting liposomes showed a long-circulatory effect in blood system, and could remarkably accumulate at the tumor location. The involved action mechanisms for the enhanced anticancer efficacy were activation of pro-apoptotic proteins (Bax and Bok), apoptotic enzymes (caspase 8, caspase 9 and caspase 3).

Conclusion: The established WGA modified daunorubicin anti-resistant liposomes could provide a potential strategy for treating resistant MCF-7 breast cancer.     

Naringenin Enhances the Anti-Tumor Effect of Doxorubicin Through Selectively Inhibiting the Activity of Multidrug Resistance-Associated Proteins but not P-glycoprotein

Purpose  Naringenin has shown paradoxical results to modulate the function of multidrug resistance-associated proteins (MRPs). The aim of this study is to interpret whether naringenin can reverse intrinsic and/or acquired resistance of cancer cells to chemotherapeutic agents. Methods  The effects of naringenin on the uptake, retention and cytotoxicity of doxorubicin were investigated in A549, MCF-7, HepG2 and MCF-7/DOX cells. Cellular efflux pathways modulated by naringenin were assessed with their specific substrates and inhibitors. The improved antitumor activity of doxorubicin in combination with naringenin was also investigated in vivo. Results  The IC₅₀ values of doxorubicin in combination with naringenin in A549 and MCF-7 cells were approximately 2-fold lower than that of doxorubicin alone. The increased sensitivity to doxorubicin by naringenin in HepG2 and MCF-7/DOX cells was not observed. Naringenin increased the cellular doxorubicin accumulation through inhibiting doxorubicin efflux in the cells expressing MRPs but not P-gp. In contrast to doxorubicin alone, doxorubicin in combination with naringenin enhanced antitumor activity in vivo with low systemic toxicity. Conclusion  Naringenin enhances antitumor effect of doxorubicin by selective modulating drug efflux pathways. Naringenin will be a useful adjunct to improve the effectiveness of chemotherapeutic agents in treatment of human cancers.     

Folate-modified poly(2-ethyl-2-oxazoline) as hydrophilic corona in polymeric micelles for enhanced intracellular doxorubicin delivery

The transmembrane transport of drug loaded micelles to intracellular compartment is quite crucial for efficient drug delivery. In the current study, we investigated the cellular internalization and anticancer activity of doxorubicin loaded micelles with folate modified stealthy PEOz corona. Folate-decorated micelles incorporating doxorubicin were characterized for particle size, degree of folate decoration, drug loading content and encapsulation efficiency, morphology, and surface charge. The targeting capability and cell viability were assessed using HeLa, KB, A549 and MCF-7/ADR cell lines. In vitro study clearly illustrated the folate receptor (FR) mediated targeting of FA modified micelles to FR-positive human HeLa, KB and MCF-7/ADR cells, while specific delivery to FR-negative A549 cells was not apparently increased at the same experimental conditions. Cytotoxicity assay showed 60% and 58% decrease in IC₅₀ values for HeLa and KB cells, while only a slight decrease for A549 cells, following treatment with folate modified formulations. The enhanced intracellular delivery of FA modified micelles in MCF-7/ADR cells was also observed. In vivo antitumor tests revealed DOX entrapped FA-PEOz-PCL micelles effectively inhibited the tumor growth and reduced the toxicity to mice compared with free DOX. The current study showed that the targeted nano-vector improved cytotoxicity of DOX and suggested that this novel PEOz endowed stealthy micelle system held great promise in tumor targeted therapy.     

Cellular uptake and cytotoxicity of solid lipid nanospheres (SLN) incorporating doxorubicin or paclitaxel

Solid Lipid Nanospheres (SLN) are colloidal therapeutic systems proposed for several administration routes and obtained by dispersing warm microemulsions in cold water. SLN as carriers of doxorubicin and paclitaxel have been previously studied. In this study, the cellular uptake of SLN and the cytotoxicity of doxorubicin and paclitaxel incorporated into SLN were investigated on two cell-lines, human promyelocytic leukemia (HL60) and human breast carcinoma (MCF-7). Cellular uptake of SLN was determined by incorporating 6-coumarin as fluorescent marker. The cellular uptake of fluorescent SLN was clearly evidenced by fluorescence microscopy. The cytotoxicity of doxorubicin incorporated in SLN was higher compared to the conventional doxorubicin solution, even at the lower concentrations. Paclitaxel in SLN was about 100-fold more effective than free paclitaxel on MCF-7 cells, while on HL60 cells a lower sensitivity was achieved with paclitaxel in SLN. Unloaded SLN had no cytotoxic effect on HL60 and MCF-7 cells.     

Pegylated phosphotidylethanolamine inhibiting P-glycoprotein expression and enhancing retention of doxorubicin in MCF7/ADR cells

The failure of the clinical treatment of cancer patients is often attributed to drug resistance of the tumor to chemotherapeutic agents. P-glycoprotein (P-gp) contributes to drug resistance via adenosine 5'-triphosphate (ATP)-dependent drug efflux pumps and is widely expressed in many human cancers. Up to date, a few of nanomaterials have shown the effects on P-gp function by different ways. To study the mechanism of the increased cytotoxicity of doxorubicin (DOX) by pegylated phosphotidylethanolamine (PEG-PE) in drug-resistant cancer cells, a series of in vitro cell assays were performed, including identification of P-gp function, quantitative studies on uptake and efflux of DOX, inhibitory effects of blank PEG-PE micelles on mRNA and protein levels of P-gp, and intracellular ATP content alteration. Finally, combining MDR-1 RNA interference (siRNA) with DOX encapsulated in PEG-PE micelles (M-DOX) to improve cytotoxicity of DOX was also studied. M-DOX showed fivefold lower the concentration that caused 50% killing tumor cell than that of free DOX in the P-gp-overexpressing MCF-7 breast cancer (MCF-7/ADR) cells. M-DOX enhanced the cellular uptake and retention of DOX in MCF-7/ADR cells. PEG-PE block molecules can inhibit P-gp expression through downregulating MDR-1 gene. Cytotoxicity of M-DOX was further improved by knocking down the MDR-1 gene using siRNA in the multidrug-resistant cells. We conclude that the increased cytotoxicity of DOX encapsulated in PEG-PE micelle is due to the reduced P-gp expression by PEG-PE block molecules, and accordingly enhancing the cellular accumulation of DOX. To overcome drug resistance of tumor cells, the combination of nanotechnology and biotechnology could be an effective strategy such as PEG-PE formed micelles and siRNA.     

The Effect of Fatty Acids and Analogues upon Intracellular Levels of Doxorubicin in Cells Displaying P-Glycoprotein Mediated Multidrug Resistance

Abstract

Multidrug resistance mediated by overexpression of P-glycoprotein (P-gp) is a major obstacle in the chemotherapeutic management of cancer. The objectives of the current work were to examine if fatty acids affect the intracellular transport and dynamics of doxorubicin in drug-resistant cancer cell lines, and to assess if such effects were mediated through modulation of P-gp efflux pump activity. Among the range of fatty acids tested in this study, eicosapentaenoic acid diester (EPADI) increased doxorubicin accumulation [A] to 137% and retention [R] to 212% in doxorubicin-resistant MCF-7/ADR breast carcinoma cells, and [A] to 147% and [R] to 163% in vinblastine-resistant KBV1 nasopharyngeal carcinoma cells. Consistent with EPADI-induced increases in intracellular doxorubicin concentrations, EPADI (10 μg/ml) sensitized MCF-7/ADR cells to the cytotoxic effects of doxorubicin (1 μg/ml) as assessed by MTT assay (viability < 50% of control), while EPADI itself displayed no cytotoxicity. The combination of EPADI (10 μg/ml) with verapamil (1 μM) resulted in a considerable increase in the [A] and [R] of the model P-gp substrate rhodamine-123 within drug-resistant cells compared to when either agent were used alone. KBV1 cells treated with combination of EPADI (10 μg/ml) and verapamil (1 μM) achieved 160% and 1120% greater [A] and [R] of rhodamine-123, respectively, compared to untreated cells. The P-gp modulatory effects of EPADI either alone, or as part of a combination with more potent inhibitors, should be further investigated.