TPGS修饰脂质体对阿霉素抗肿瘤活性的增敏作用

目的 制备聚乙二醇1000维生素E琥珀酸酯(TPGS)修饰的阿霉素脂质体并考察其对阿霉素抗肿瘤活性的增敏作用。方法 用阳离子树脂吸附法测定阿霉素脂质体的包封率;MTT法测定对MCF-7和MCF-7/ADR的毒性;用荧光显微镜观察阿霉素的细胞摄取,并用HPLC测定细胞内的阿霉素含量。结果 TPGS修饰的阿霉素脂质体增加了MCF-7/ADR对阿霉素的摄取,并增强了对MCF-7和MCF-7/ADR细胞的毒性。结论 TPGS修饰脂质体能显著增强MCF-7和MCF-7/ADR对阿霉素的敏感性。     

PAMAM树状大分子对阿霉素在HepG 2细胞中的摄取动力学的影响

目的利用聚酰胺-胺(polyamidoamine,PAMAM)树枝状大分子包裹抗癌药物阿霉素,建立细胞内测定阿霉素浓度的HPLC检测方法,研究PAMAM对阿霉素在HepG 2细胞内的摄取行为的影响。方法将盐酸阿霉素脱去盐酸后利用疏水作用进入PAMAM内腔制备载药胶束,以柔红霉素为内标,粉碎细胞后提取细胞内的阿霉素进行浓度测定。测定细胞摄取、外排期间的药物浓度,绘制时间-浓度曲线,并计算动力学参数。结果 PAMAM包裹脱盐酸阿霉素后得到阿霉素的质量浓度为1.034g·L-1的溶液,成功建立胞内阿霉素的含量测定方法。细胞摄取动力学结果显示PAMAM使得阿霉素胞内达峰时间为1h,胞内浓度明显增高;消除动力学结果显示,k减小为原来的0.347倍,t1/2延长为原来的2.878倍,MRT增加为原来的2倍。结论经PAMAM包裹后阿霉素能够在HepG 2细胞内快速达峰,胞内浓度增加,消除速率减慢,滞留时间延长,有利于药物药效的发挥。     

载阿霉素介孔二氧化硅纳米粒的细胞毒性及细胞摄取

目的制备载阿霉素的介孔二氧化硅纳米粒(mesoporous silica nanoparticles,MSN),对其理化性质及细胞摄取行为进行初步研究。方法通过聚合法制备MSN,透射电镜表征纳米粒的形态,动态光散射粒径测定仪测定粒子的平均粒径及分布。紫外分光光度计测定阿霉素的负载行为,MTT比色分析法研究粒子的细胞毒性,激光共聚焦显微镜观察其人乳腺癌MCF-7细胞对载药粒子的摄取。结果纳米粒分布均一,平均粒径约70 nm(PDI<0.1),载药量质量分数达到20%。MCF-7细胞对载药粒子的摄取较快,空白纳米粒具有较低的细胞毒性。结论介孔二氧化硅纳米粒具有较高的药物载药量和良好的生物相容性,能较快地被对人乳腺癌MCF-7细胞摄取,有望成为一种新型的药物化疗载体。     

RGD肽修饰重组高密度脂蛋白载药纳米粒的制备和表征以及肿瘤细胞对其摄取作用

目的：制备和表征RGD肽修饰的重组高密度脂蛋白（RGD—rHDL）载药纳米粒,考察肿瘤细胞对其摄取作用。方法：合成RGD与载脂蛋白AI（ApoAI）的偶联物（RGD—ApoAI）,并以藤黄酸（GA）作为模型药物,采用薄膜分散法制得GA脂质体（LP—GA）,再将RGD—ApoAI与LP—GA共孵育,制备载有GA的RGD—rHDL纳米粒（RGD—rHDL—GA）;随后,对RGD—rHDL—GA进行表征,且采用荧光标记示踪法,通过RGD—rHDL-香豆素-6来考察人肝癌细胞HepG2对载药RGD—rHDL纳米粒的摄取作用。结果：制备的RGD—rHDL—GA呈现规则圆整的类球形,粒径分布均一[（110,70±3．25）nm],Zeta电位为（-39．21±0．10）mV,包封率为（92．20±0．28）％,载药量为（9．03±0．75）％,且其体外释药缓慢,稳定性良好;RGD—rHDL-香豆素-6的肿瘤细胞摄取率明显高于rHDL-香豆素-6。结论：rHDL经RGD修饰后可有效促进所载药物进入肿瘤细胞,提高其肿瘤靶向性。     

双配体修饰脂质聚合物杂化纳米粒的制备及细胞摄取研究

目的制备双修饰的主动心肌靶向的载荧光探针香豆素-6(C-6)的脂质-聚合物杂化纳米粒(LPHNs),并考察其细胞摄取。方法通过巯基化反应,合成导向化合物心肌细胞靶向肽-聚乙二醇-磷脂酰乙醇胺(PCM-PEG-DSPE)。选取C-6为荧光探针,采用自动组装法制备包载C-6的LPHNs,再通过插入法,用PCM和细胞穿膜肽(TAT)对C-6-LPHNs进行修饰。以心肌细胞(H9C2)为模型做体外细胞摄取实验。结果双修饰的C-6-LPHNs的平均粒径为74.5±4.2 nm,分散系数(PDI)为0.051±0.013,Zeta电位为-32.8±1.2 mV,包封率为87.04%±3.19%。双修饰的C-6-LPHNs对H9C2心肌细胞的摄取率明显高于未修饰的C-6-LPHNs。结论 LPHNs经双修饰后可促进H9C2心肌细胞的摄取。     

吗丙嗪和阿霉素合用对肿瘤细胞周期及DNA合成的影响

目的：观察吗丙嗪（Ｐｒｏ）和阿霉素（Ｄｏｘ）合用对肿瘤细胞周期及ＤＮＡ合成的影响，以探讨吗丙嗪增强阿霉素抗肿瘤作用之机制。方法：采用拒染法观察Ｐｒｏ，Ｄｏｘ和Ｐｒｏ＋Ｄｏｘ对小鼠体外艾氏腹水癌细胞（ＥＡＣ）的杀伤作用；采用标记前体物参入法和显微分光光度术观察两药合用对ＥＡＣ细胞周期及ＤＮＡ合成的影响。结果：Ｐｒｏ２１．４６，４２．９２和６４．３８μｍｏｌ·Ｌ－１增强Ｄｏｘ对ＥＡＣ细胞的杀伤作用；Ｐｒｏ（２１４．５９μｍｏｌ·Ｌ－１）和Ｄｏｘ（１８．０２μｍｏｌ·Ｌ－１）合用对ＥＡＣ细胞ＤＮＡ合成有明显的抑制作用；用药后４～８ｈＧ１期细胞增加，ＤＮＡ直方图左移，各用药组细胞分裂指数无明显差异。结论：Ｐｒｏ可增强Ｄｏｘ的抗肿瘤作用，其机理可能与Ｐｒｏ加强Ｄｏｘ对ＤＮＡ合成的抑制及对Ｇ１期细胞累积作用增强有关。     

华蟾素对耐阿霉素人乳腺癌细胞内阿霉素蓄积的影响

目的：测定华蟾素（cinobufacine,Cino）对耐阿霉素人乳腺癌细胞MCF-7/ADM内阿霉素（ADM）积累的影响,以探索Cino逆转MCF-7/ADM多药耐药（MDR）的可能机制。方法：MTT法检测CinoMDR的逆转作用,HPLC法检测Cino作用MCF-7/ADM后细胞内ADM的浓度的变化。结果：Cino15mg/L能增加MCF-7/ADM细胞对ADM的敏感性,使ADM的半数抑制浓度（IC50）由38.14mg/L降至12.93mg/L,显著增加MCF-7/ADM株细胞内ADM的浓度。结论：Cino能明显增加MCF-7/ADM株细胞内ADM的含量,部分逆转MCF-7/ADM的MDR。     

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。     

丹参酮ⅡA对阿霉素抑制肿瘤细胞生长作用的影响

目的：观察丹参酮ⅡA对阿霉素诱导2种肿瘤细胞（K562和Bel-7402细胞）死亡作用的影响。方法：MTT法检测细胞存活率,Hoechst33342染色从形态学上反映肿瘤细胞凋亡情况。结果：丹参酮ⅡA本身对2种肿瘤细胞均有抑制增殖、促凋亡的作用,不会干扰阿霉素诱导肿瘤细胞死亡的作用,甚至对其对K562细胞的促凋亡作用有协同效果。结论：丹参酮ⅡA对阿霉素抑制肿瘤细胞生长有协同作用,但对肿瘤细胞有一定的选择性。     

钆喷酸葡胺长循环脂质体的制备及其肿瘤细胞摄取能力研究

目的:制备用聚乙烯100硬脂基醚(Brij700)修饰的长循环钆喷酸葡胺脂质体,研究其被肿瘤细胞摄取的情况。方法:利用逆向蒸发法制备钆喷酸葡胺普通脂质体和长循环脂质体并进行质量检测;用磁共振造影仪(MRI)检测前列腺癌细胞(PC3)中摄取的钆喷酸葡胺的浓度,以SE-T1W信号值为指标比较肿瘤细胞对2种脂质体的摄取能力。结果:制备的钆喷酸葡胺普通脂质体和长循环脂质体平均粒径分别为824、363nm,包封率分别为63.16%、61.15%;PC3对2种脂质体摄取后的SE-T1W信号值分别为362、299;平均每个细胞的摄取数分别为2.12×10-4、2.70×10-4。结论:与钆喷酸葡胺普通脂质体比较,其长循环脂质体更利于肿瘤细胞的摄取。     

Starlike vs. Classic Macromolecular Prodrugs: Two Different Antibody-Targeted HPMA Copolymers of Doxorubicin Studied in Vitro and in Vivo as Potential Anticancer Drugs

Purpose. Two different monoclonal antibody-targeted HPMA copolymer-doxorubicin conjugates, classic and starlike, were synthesized to be used for site-specific cancer therapy. The anti-mouse Thy-1.2 (IgG3) and two anti-human CD71/A (IgG1) and CD71/B (IgG2a) monoclonal antibodies were used as targeting structures. Methods. Their binding and cytotoxic activity in vitro, body distribution, and anticancer activity in vivo were evaluated. Results. The results of flow cytometric analysis showed comparable binding of classic and starlike conjugates to the target cells. The in vitro cytotoxic effect was 10-fold higher if cancer cells were exposed to the starlike conjugate compared to the classic one. Biodistribution studies showed that the starlike conjugate remained in a relatively high concentration in blood, whereas the classic conjugate was found in a 6.5-times lower amount. In contrast to the low antitumor activity of free doxorubicin and nontargeted HPMA copolymer-doxorubicin conjugate, both anti-Thy-1.2 targeted conjugates (classic and starlike) cured all mice bearing T-cell lymphoma EL4. On the other hand, starlike conjugates containing anti-CD71/A or anti-CD71/B monoclonals as targeting structures were more effective against human colorectal cancer SW 620 than the classic one. Conclusions. We have shown that the starlike conjugates are more effective systems for targeted drug delivery and cancer treatment than classic conjugates.     

HPMA based macromolecular therapeutics: Internalization,intracellular pathway and cell death depend on the character of covalent bond between the drug and the peptidic spacer and also on spacer composition

Polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) have been tested as potential carrier for anticancer drug—doxorubicin (Dox). Two types of conjugates were synthesized: (a) conjugates containing Dox bound through an amidic bond to an oligopeptidic side-chain (usually GFLG) and (b) hydrolytically cleavable conjugates wherein Dox is bound to the polymeric carrier through a pH sensitive bond. The mechanism of action of both conjugates is different and reflects the diverse way and intensity of their intracellular accumulation. All conjugates containing doxorubicin bound via an amidic bond directly penetrate the plasma membrane and are detectable in all associated cellular membranes, i.e. membranes of the endocytic compartment, a nuclear membrane as well as membranes of Golgi and endoplasmic reticulum. We have never been able to detect released doxorubicin inside the nuclei of the treated cells. The cytotoxicity of these conjugates seems to be primarily caused by the damage of cellular membranes. Necrosis is the main mechanism of the cell death. Conjugates containing hydrolytically bound doxorubicin are internalized by endocytosis and fluid phase pinocytosis and doxorubicin is cleaved from the polymeric carrier at low pH in late endosomes and lysosomes. An apoptosis is the main mechanism of the cell death. The spacer influences the rate of the intracellular release of the drug rather than the rate of internalization.     

Positive correlation between decreased cellular uptake,NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines

From a wild type strain of Ehrlich ascites tumor (EAT_WT) sublines resistant to daunorubicin (EAT_DNM), etoposide (EAT_ETO), and cisplatinum (EAT_CIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i. p. with EAT cells. Adriamycin concentrations causing 50% inhibition of ^3H-thymidine (ICT) and ^3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EAT_WT — EAT_CIS — EAT_DNM — EAT_ETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICT_C, ICU_C), ICT_C and ICU_C still varied up to the 3.2 fold in EAT_CIS, EAT_DAM and EAT_ETO in comparison to EAT_WT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p<0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICT_C, and ICU_C, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthdaySupported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, FRG: Sonderforschungsbereich 102, and Landesamt für Forschung Nordrhein-Westfalen, Düsseldorf, FRG     

Influence of the surface coating on the cytotoxicity,genotoxicity and uptake of gold nanoparticles in human HepG2 cells

The toxicological profile of gold nanoparticles (AuNPs) remains controversial. Significant efforts to develop surface coatings to improve biocompatibility have been carried out. In vivo biodistribution studies have shown that the liver is a target for AuNPs accumulation. Therefore, we investigated the effects induced by ~20 nm spherical AuNPs (0–200 μM Au) with two surface coatings, citrate (Cit) compared with 11‐mercaptoundecanoic acid (11‐MUA), in human liver HepG2 cells. Cytotoxicity was evaluated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays after 24 to 72 h of incubation. DNA damage was assessed by the comet assay, 24 h after incubation with the capped AuNPs. Uptake and subcellular distribution of the tested AuNPs was evaluated by quantifying the gold intracellular content by graphite furnace atomic absorption spectrometry (GFAAS) and transmission electron microscopy (TEM), respectively. The obtained results indicate that both differently coated AuNPs did not induce significant cytotoxicity. An inverse concentration‐dependent increase in comet tail intensity and tail moment was observed in Cit‐AuNPs‐ but not in MUA‐AuNPs‐exposed cells. Both AuNPs were internalized in a concentration‐dependent manner. However, no differences were found in the extent of the internalization between the two types of NPs. Electron‐dense deposits of agglomerates of Cit‐ and MUA‐AuNPs were observed either inside endosomes or in the intercellular spaces. In spite of the absence of cytotoxicity, DNA damage was observed after exposure to the lower concentrations of Cit‐ but not to MUA‐AuNPs. Thus, our data supports the importance of the surface properties to increase the biocompatibility and safety of AuNPs. Copyright © 2013 John Wiley & Sons, Ltd.     

The mechanism for cellular uptake,storage and release of daunorubicin: Studies on fibroblasts in culture

The mechanism for uptake, storage and release of daunorubicin have been studied in cultured fibroblasts. Analysis by high performance liquid chromatography of cells incubated with daunorubicin revealed that the major part of the accumulated drug did not undergo metabolic transformation. Small amounts of daunorubicinol and aglycone were formed. [³H]-daunorubicin was used to study membrane fluxes of the drug under different conditions. Metabolic inhibitors enhanced the influx of [³H]-dauno-rubicin and, under certain conditions, also reduced its efflux, indicating that the cells have an active mechanism for the outward transport of the drug. The very high intracellular drug accumulation is due to trapping in nuclei and lysosomes. Cell fractionation techniques have been used to study drug trapping under various conditions. Nuclear storage of daunorubicin is probably due to binding to DNA. Metabolic inhibitors, as well as lowering the incubation temperature, reduced the lysosomal trapping, supporting the hypothesis that the low pH in these organelles is maintained by a proton pump and that the drug is trapped in the protonated form. A hypothesis is presented, which by combining the mechanisms for membrane transport and intracellular storage of daunorubicin, gives also an explanation for the observed differences in the cellular accumulation and subcellular distribution of daunorubicin and its 14-hydroxy derivative, doxorubicin. Daunorubicin is more lipophilic than doxorubicin and will therefore diffuse faster through the cell membrane. Assuming that both substances have the same affinity for the proposed active outward transport mechanism, this will lead to a higher steady-state level of daunorubicin in the cytosol and as a consequence to a higher lysosomal storage level if the drug in the lysosomes is in equilibrium with that in the cytosol. The similarity in nuclear storage capacity for the two substances can be explained by saturation of the available storage sites.     

Design,characterization, and in vitro cellular inhibition and uptake of optimized genistein-loaded NLC for the prevention of posterior capsular opacification using response surface methodology

This study was to design an innovative nanostructured lipid carrier (NLC) for drug delivery of genistein applied after cataract surgery for the prevention of posterior capsular opacification. NLC loaded with genistein (GEN-NLC) was produced with Compritol 888 ATO, Gelucire 44/14 and Miglyol 812N, stabilized by Solutol^® HS15 by melt emulsification method. A 2⁴ central composite design of 4 independent variables was performed for optimization. Effects of drug concentration, Gelucire 44/14 concentration in total solid lipid, liquid lipid concentration, and surfactant concentration on the mean particle size, polydispersity index, zeta potential and encapsulation efficiency were investigated. Analysis of variance (ANOVA) statistical test was used to assess the optimization. The optimized GEN-NLC showed a homogeneous particle size of 90.16 nm (with PI = 0.33) of negatively charged surface (−25.08 mv) and high encapsulation efficiency (91.14%). Particle morphology assessed by TEM revealed a spherical shape. DSC analyses confirmed that GEN was mostly entrapped in amorphous state. In vitro release experiments indicated a prolonged and controlled genistein release for 72 h. In vitro growth inhibition assay showed an effective growth inhibition of GEN-NLCs on human lens epithelial cells (HLECs). Preliminary cellular uptake test proved a enhanced penetration of genistein into HLECs when delivered in NLC.     

Polypeptide nanogels with hydrophobic moieties in the cross-linked ionic cores: synthesis,characterization and implications for anticancer drug delivery

Abstract

Polymer nanogels have gained considerable attention as a potential platform for drug delivery applications. Here we describe the design and synthesis of novel polypeptide-based nanogels with hydrophobic moieties in the cross-linked ionic cores. Diblock copolymer, poly(ethylene glycol)-b-poly(l-glutamic acid), hydrophobically modified with l-phenylalanine methyl ester moieties was used for controlled template synthesis of nanogels with small size (ca. 70?nm in diameter) and narrow particle size distribution. Steady-state and time-resolved fluorescence studies using coumarin C153 indicated the existence of hydrophobic domains in the ionic cores of the nanogels. Stable doxorubicin-loaded nanogels were prepared at high drug capacity (30 w/w%). We show that nanogels are enzymatically-degradable leading to accelerated drug release under simulated lysosomal acidic pH. Furthermore, we demonstrate that the nanogel-based formulation of doxorubicin is well tolerated and exhibit an improved antitumor activity compared to a free doxorubicin in an ovarian tumor xenograft mouse model. Our results signify the point to a potential of these biodegradable nanogels as attractive carriers for delivery of chemotherapeutics.     

The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells

The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.