Nanoparticles of cisplatin augment drug accumulations and inhibit multidrug resistance transporters in human glioblastoma cells

BackgroundCisplatin (CSP) is a potent anticancer drug widely used in treating glioblastoma multiforme (GBM). However, CSP's clinical efficacy in GBM contrasted with low therapeutic ratio, toxicity, and multidrug resistance (MDR). Therefore, we have developed a system for the active targeting of cisplatin in GBM via cisplatin loaded polymeric nanoplatforms (CSP-NPs).MethodsCSP-NPs were prepared by modified double emulsion and nanoprecipitation techniques. The physiochemical characterizations of CSP-NPs were performed using zeta sizer, scanning electron microscopy (SEM), drug release kinetics, and drug content analysis. Cytotoxicity, induction of apoptosis, and cell cycle-specific activity of CSP-NPs in human GBM cell lines were evaluated by MTT assay, fluorescent microscopy, and flow cytometry. Intracellular drug uptake was gauged by fluorescent imaging and flow cytometry. The potential of CSP-NPs to inhibit MDR transporters were assessed by flow cytometry-based drug efflux assays.ResultsCSP-NPs have smooth surface properties with discrete particle size with required zeta potential, polydispersity index, drug entrapment efficiency, and drug content. CSP-NPs has demonstrated an ‘initial burst effect’ followed by sustained drug release properties. CSP-NPs imparted dose and time-dependent cytotoxicity and triggered apoptosis in human GBM cells. Interestingly, CSP-NPs significantly increased uptake, internalization, and accumulations of anticancer drugs. Moreover, CSP-NPs significantly reversed the MDR transporters (ABCB1 and ABCG2) in human GBM cells.ConclusionThe nanoparticulate system of cisplatin seems to has a promising potential for active targeting of cisplatin as an effective and specific therapeutic for human GBM, thus eliminating current chemotherapy's limitations.     

纳米羟基磷灰石人工心脏机械瓣膜细胞相容性的研究

目的探讨利用脉冲激光沉积（PLD）方法在人工心脏机械瓣膜上沉积的纳米相羟基磷灰石（HA）薄膜的组织相容性,为人工心脏瓣膜的构建提供理想的支架薄膜材料。方法将传代的人血管内皮细胞制成细胞悬液接种在HA材料上,置含5％（V／V）CO2、37℃的恒温培养箱内静态培养3周。分别在培养3、7、14和21d后取出部分材料,用2．5％戊二醛固定,乙醇梯度脱水,临界点干燥、喷金,于扫描电子显微镜（SEM）下观察细胞在材料上的附着情况。用四氮唑盐比色（MTT）法测定血管内皮细胞与材料复合培养的增殖能力。结果扫描电镜观察复合培养,见HA材料的表面有细胞附着且数目逐渐增多,到第21天时可见细胞融合成片,并将材料表面覆盖,部分区域有细胞外基质形成。MTT法评价材料对血管内皮细胞的细胞毒性,见细胞增殖未受到明显影响,生长良好。结论HA对血管内皮细胞的增殖和分化功能无明显影响,对细胞无明显毒性,具有良好的生物相容性,可以作为心脏瓣膜材料安全应用。     

Individualized cancer chemotherapy integrating drug sensitivity tests, pathological profile analysis and computational coordination - an effective strategy to improve clinical treatment

BACKGROUND: Most current cancer chemotherapy is unsatisfactory. There is a trend towards changing the norm for drug selection; one approach is to seek individualized cancer chemotherapy (ICC). METHODS AND RESULTS: ICC is an approach to maximizing the efficacy of chemotherapy and reducing its adverse effects to a minimum. It involves choosing anticancer drugs through the following critical steps: (i) performing drug sensitivity tests in vivo and/or in vitro; (ii) analyzing pathogenic information from morphology, histology and bioinformatics, so that targeted therapy can be offered to disrupt the escalating tumorigenic molecules and pathways; (iii) introducing mathematical and computational systems to assist in improving the quality of decision-making. CONCLUSION: Increasing clinical evidence indicates that drug sensitivity tests, pathological profile analyses and computational coordination are ways to improve therapeutic quality. In future, each patient should have his own unique chemotherapy protocol.     

Relationships among tumor burden, tumor size, and the changing concentrations of fibrin degradation products and fibrinolytic factors in the pleural effusions of rabbits with VX2 lung tumors

The VX2 tumor is derived from a papilloma virus-induced rabbit epithelial cell line. If VX2 tumor cells (trapped in a plasma clot) are introduced intravenously into NZW rabbits, the cells lodge in the lung capillary bed and produce tumors. Independently of the tumor burden (ie, the total tumor weight per rabbit), approximately 15% of rabbits with VX2 lung tumors accumulate an effusion in the interpleural space and this pleural effusion contains products of hemostasis. We hypothesized that these products were of intra-tumoral origin and that they changed in concentration as tumor burden increased. Interrelationships among lung-, tumor-weights, and pleural effusion volumes, and the concentrations of fibrinolytic factors, their catabolic products, and other proteins of pleural effusions were measured in rabbits with a wide range of tumor burdens. Positive correlations between tumor burden and total lung weight and between pleural effusion volume and net lung weight suggested that interstitial fluid from the stroma of tumors passed directly into the extravascular space of the lung(s) and into the interpleural space(s). Analyses of pleural effusions indicated that plasminogen-, alpha(2)-antiplasmin-, and plasminogen activator inhibitor-1-related proteins, urokinase-like- and tissue-plasminogen activator activities, and vascular endothelial growth factor increased in concentration up to a tumor burden of approximately 20-25 g. Plasmin activity and intact fibrinogen were absent. The concentration of fibrin(ogen) degradation products did not change significantly up to a tumor burden of approximately 25 g but increased substantially as tumor burdens exceeded 25 g. In conclusion, interstitial fluid from tumors enters the extravascular space of the host and may accumulate with fluid from non-tumor sources as a pleural effusion. The concentrations of fibrinolytic factors and their products in pleural effusions reflect the tumor burden of the rabbit. Conceivably, the components of a malignant effusion contain much information about the extent of tumor growth.     

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray

OBJECTIVES: To establish a modified microarray method for detecting HBV gene mutations in the clinic. DESIGN AND METHODS: Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. RESULTS: HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. CONCLUSIONS: An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.     

Establishment of a methodology for investigating protectants against ethanol-induced hepatotoxicity

Ethanol-induced liver injury has been extensively reported in clinic, but still lacks an efficient in vitro platform for investigating its hepatotoxicity and protectants. This study aimed to establish a methodology on the culture conditions regarding the sealability against evaporation of ethanol, culture medium and 2D/3D culture of hepatocytes. Based on the experimental findings, it was indicated that the ethanol evaporation from culture plates was a severe problem reducing its toxicity in hepatocyte. According to the detected ethanol toxic response marked by reduced cell viability, 3D cultured hepatocytes in gel entrapment were suggested to be better than 2D hepatocyte in monolayer, but the cultures in either William’s Medium E or DMEM exhibited comparable sensitivity to ethanol toxicity. Subsequently, 3D cultured hepatocytes with Parafilm sealing were systematically illustrated to well reflect the ethanol-induced lipid accumulation, reactive oxygen species/malondialdehyde generation, glutathione depletion and cytochrome 2E1 induction. Finally, such hepatocyte models were proposed as a platform for screening of herbal component against ethanol hepatotoxicity. Nano-silibinin, for the first time, found to perform significant protection against ethanol-induced hepatotoxicity while silibinin in normal particles could not inhibit such toxicity. This protection of nano-silibinin might relate to its improved bioavailability compared to normal insoluble silibinin and could act as an anti-oxidative and anti-steatosis agent against ethanol-induced hepatotoxicity.     

AMPA/kainate receptor-mediated up-regulation of GABAA receptor delta subunit mRNA expression in cultured rat cerebellar granule cells is dependent on NMDA receptor activation

We have studied the effects of AMPA/kainate receptor agonists on GABA(A) receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 microM) and high K(+) (25 mM) dramatically up-regulated delta subunit mRNA expression to 500-700% of that in control cells grown in low K(+) (5 mM). KA or high K(+) had no effect on the expression of the other major GABA(A) receptor subunits alpha1, alpha6, beta2, beta3 or gamma2. Up-regulation of delta mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced delta mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced delta up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to delta mRNA up-regulation. In CGCs, delta subunit is a component of extrasynaptic alpha6betadelta receptors that mediate tonic inhibition. Up-regulation of delta during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.     

奥沙利铂长循环纳米脂质体对结肠癌细胞株体外作用研究

目的 检测奥沙利铂长循环纳米脂质体药物样品对SW480结肠癌细胞株的毒性影响,并验证其长效性和缓控释作用.方法 乙醇注入法制备出奥沙利铂长循环纳米脂质体药物样品,MTT实验检测其对SW480细胞株作用时效性与抑制率,并与标准品组对照分析.结果 阳性对照组的IC50=70.96μg/mL,36h内抑制率随着奥沙利铂浓度的增加迅速上升而达到顶峰.药物样品组的IC50=85.39μg/mL,72h内抑制率随着药物样品浓度的增加而稳步上升.结论 奥沙利铂长循环纳米脂质体药物样品IC50=85.39μg/mL,抑制率随着浓度的增加而上升且增势稳定,100μg/mL以上组别的抑制率在72h后均达到90％以上,具有长效性,缓释稳定性,其生物利用度更优.     

The neuroprotective effects of intraperitoneal injection of hydrogen in rabbits with cardiac arrest

Objective

The purpose of this study was to investigate the neuroprotective effects of intraperitoneal injection of hydrogen (H₂) in rabbits with cardiac arrest (CA).

Methods

A rabbit model of CA was established by the delivery of alternating current between the esophagus and chest wall to induce ventricular fibrillation. Before CA, the animals were randomly divided into four groups: a sham group (no CA), a CA group, a CA + low dose (10 ml/kg) H₂ group (CA + H₂ group 1), and a CA + high dose (20 ml/kg) H₂ group (CA + H₂ group 2). In the first experiment, animals were observed for 72 h after the restoration of spontaneous circulation (ROSC). The neurological scores were assessed at 24, 48 and 72 h after ROSC. The rabbits that survived until 72 h were sacrificed using an overdose of anesthetic, and the brain tissues were collected and Nissl-stained to observe nerve cell damage in the hippocampal CA1 area. In addition, TUNEL assay was performed to detect apoptosis. In the second experiment, animals were observed for 6 h after ROSC. Blood samples and brain hippocampal tissues were collected, and differences in oxidative stress indicators were compared among the four groups.

Results

Intraperitoneal injection of H₂ improved the 72-h survival rate and neurological scores, reduced neuronal injury and inhibited neuronal apoptosis. Intraperitoneal injection of H₂ reduced oxidative stress indicators in the plasma and hippocampal tissues and enhanced antioxidant enzyme activity. No significant difference was observed between the two CA groups treated with different doses of H₂.

Conclusions

Intraperitoneal injection of H₂ is a novel hydrogen administration method and can reduce cerebral ischemia-reperfusion injury and improve the prognosis of cardiopulmonary cerebral resuscitation in a rabbit model of CA.     

Image analysis.

In rat hepatocytes in primary culture incubated with nitro blue tetrazolium, formazan content was increased by addition of t-butyl hydroperoxide, a potent oxidant, in a dose-related manner, but not by addition of valinomycin, which kills hepatocytes through mitochondrial damage. This increment after t-butyl hydroperoxide addition was not seen in hepatotyctes preincubated with deferoxamine mesylate, a ferric iron chelator which inhibits radical formation. Liver perfusion with nitro blue tetrazolium and t-butyl hydroperoxide in rats produced formazan deposition faintly on the surface of hepatocytes throughout the liver and prominently in the cytoplasm of some hepatocytes, which was attenuated when performed following deferoxamine mesylate perfusion. When liver perfusion with nitro blue tetrazolium was performed in carbon tetrachloride-intoxicated rats, formazan deposition appeared diffusely in hepatocytes in the centrilobular areas. Similar deposition was also observed on the surface and in the cytoplasm of hepatocytes in the periportal and mid-zonal areas in rats undergoing post-ischaemic reperfusion. Liver perfusion with nitro blue tetrazolium can detect in situ oxidative stress in hepatocytes and may be a useful tool for studying the role of lipid peroxidation in rat liver injury.