Nanoparticle-based topical ophthalmic formulation for sustained release of stereoisomeric dipeptide prodrugs of ganciclovir

Poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) of Val-Val dipeptide monoester prodrugs of ganciclovir (GCV) including L-Val-L-Val-GCV (LLGCV), L-Val-D-Val-GCV (LDGCV) and D-Val-L-Val-GCV (DLGCV) were formulated and dispersed in thermosensitive PLGA-PEG-PLGA polymer gel for the treatment of herpes simplex virus type 1 (HSV-1)-induced viral corneal keratitis. Nanoparticles containing prodrugs of GCV were prepared by a double-emulsion solvent evaporation technique using various PLGA polymers with different drug/polymer ratios. Nanoparticles were characterized with respect to particle size, entrapment efficiency, polydispersity, drug loading, surface morphology, zeta potential and crystallinity. Prodrugs-loaded NP were incorporated into in situ gelling system. These formulations were examined for in vitro release and cytotoxicity. The results of optimized entrapment efficiencies of LLGCV-, LDGCV- and DLGCV-loaded NP are of 38.7?±?2.0%, 41.8?±?1.9%, and 45.3?±?2.2%; drug loadings 3.87?±?0.20%, 2.79?±?0.13% and 3.02?±?0.15%; yield 85.2?±?3.0%, 86.9?±?4.6% and 76.9?±?2.1%; particle sizes 116.6?±?4.5, 143.0?±?3.8 and 134.1?±?5.2?nm; and zeta potential ?15.0?±?4.96, ?13.8?±?5.26 and ?13.9?±?5.14?mV, respectively. Cytotoxicity studies suggested that all the formulations are non-toxic. In vitro release of prodrugs from NP showed a biphasic release pattern with an initial burst phase followed by a sustained phase. Such burst effect was completely eliminated when NP were suspended in thermosensitive gels with near zero-order release kinetics. Prodrugs-loaded PLGA NP dispersed in thermosensitive gels can thus serve as a promising drug delivery system for the treatment of anterior eye diseases.     

回顾性分析本院用更昔洛韦与阿昔洛韦治疗单纯疱疹病毒性角膜炎的疗效

目的 回顾性分析本院用更昔洛韦与阿昔洛韦治疗单纯疱疹病毒性角膜炎的疗效.方法 收集近年在本院门诊就诊的100例(106眼)单纯疱疹病毒性角膜炎患者,其中48例用更昔洛韦眼用凝胶;37例用阿昔洛韦眼液.比较两者疗效.结果 更昔洛韦组与阿昔洛韦组总有效率分别为91.7%,75.7%,显示更昔洛韦眼用凝胶的有效率好于阿昔洛韦眼液(P＜0.05).结论 更昔洛韦眼用凝胶治疗单纯疱疹病毒性角膜炎的效果较好.     

Uptake of FITC-chitosan nanoparticles by A549 cells

Purpose. The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. Methods. Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. Results. Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having K_m and V_max of 3.84 M and 58.14 g/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K⁺ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37°C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37°C. Conclusions. Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.     

Mitoxantrone-loaded superparamagnetic iron oxide nanoparticles as drug carriers for cancer therapy: Uptake and toxicity in primary human tubular epithelial cells

Superparamagnetic iron oxide nanoparticles (SPIONs) are in use for many clinical diagnostic and experimental therapeutic applications, for example, for targeted drug delivery. To analyze the cellular responses to mitoxantrone-carrying SPIONs (SPION-MTO), and to the drug released from SPIONs, we used an in vitro system that allows comparison of primary human cells with different endocytotic capacities, namely, epithelial cells from proximal and distal parts of the nephron. SPIONs were selectively and rapidly internalized by proximal tubular cells with high endocytotic potential, but not by distal tubular cells. Uptake did not affect cell viability or morphology. In both cell types, free MTO (10–100?nM) induced double-strand DNA breaks and senescence, cell hypertrophy and reduced cell proliferation. However, cadherin-mediated cell–cell adhesion, cytoskeletal structures or polarity of the cells were not affected. Interestingly, a comparable response was also observed upon treatment with SPION-MTO and was independent of uptake of the particles. The effect of SPION-MTO on cells which did not internalize particles was primarily related to the release of MTO from drug-coated particles upon incubation in serum-containing cell growth medium. In conclusion, we show that whereas the uptake of SPIONs does not affect cellular functions or viability, the toxicity of drug-loaded SPIONs depends essentially on the type of drug bound to nanoparticles. Due to the relatively low systemic toxicity of MTO, the effects of MTO-SPIONs on human tubular cells were moderate, but they may become clinically relevant when more nephrotoxic drugs are bound to SPIONs.     

A549细胞对单硬脂酸甘油酯固体脂质纳米粒的摄取作用A549细胞对单硬脂酸甘油酯固体脂质纳米粒的摄取作用

目的考察单硬脂酸甘油酯固体脂质纳米粒(monostearin solid lipid nanoparticles，MSLN)经PEG2000修饰后，对A549细胞摄取MSLN及J774A1细胞吞噬MSLN的影响。方法采用溶剂扩散法制备MSLN，测定其粒径和zeta电位；以罗丹明B(Rhodamine B)为荧光标记物，研究A549细胞对MSLN的摄取作用和J774A1细胞对MSLN的吞噬作用。结果MSLN的细胞毒性较低，A549细胞对MSLN的摄取可快速接近饱和，其摄取百分率与MSLN在细胞外的浓度呈负相关。结论MSLN经PEG2000修饰，可显著抑制J774A1细胞对MSLN的吞噬，但可增加A549细胞对MSLN的摄取。     

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC₅₀ of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.     

Effect of pulmonary surfactant on the dissolution,stability and uptake of zinc oxide nanowires by human respiratory epithelial cells

Inhaled nanoparticles (NPs) have high-deposition rates in the alveolar region of the lung but the effects of pulmonary surfactant (PS) on nanoparticle bioreactivity are unclear. Here, the impact of PS on the stability and dissolution of ZnO nanowires (ZnONWs) was investigated, and linked with their bioreactivity in vitro with human alveolar epithelial type 1-like cells (TT1). Pre-incubation of ZnONWs with Curosurf® (a natural porcine PS) decreased their dissolution at acidic pH, through the formation of a phospholipid corona. Confocal live cell microscopy confirmed that Curosurf® lowered intracellular dissolution, thus delaying the onset of cell death compared to bare ZnONWs. Despite reducing dissolution, Curosurf® significantly increased the uptake of ZnONWs within TT1 cells, ultimately increasing their toxicity after 24?h. Although serum improved ZnONW dispersion in suspension similar to Curosurf®, it had no effect on ZnONW internalization and toxicity, indicating a unique role of PS in promoting particle uptake. In the absence of PS, ZnONW length had no effect on dissolution kinetics or degree of cellular toxicity, indicating a less important role of length in determining ZnONW bioreactivity. This work provides unique findings on the effects of PS on the stability and toxicity of ZnONWs, which could be important in the study of pulmonary toxicity and epithelial-endothelial translocation of nanoparticles in general.     

高效液相色谱法-二极管阵列检测器考察炎琥宁在更昔洛韦葡萄糖注射液中的配伍稳定性

目的考察室温（20±1）℃下,注射用炎琥宁与更昔洛韦葡萄糖注射液配伍的稳定性,为临床合理用药提供依据。方法采用反相高效液相色谱法-二极管阵列检测器同时测定炎琥宁与更昔洛韦配伍后0-6 h内的含量变化,并观察配伍液的外观及pH值。结果6 h内混合液外观、pH及含量均无明显变化。结论在室温（20±1）℃条件下,注射用炎琥宁与更昔洛韦葡萄糖注射液6 h内可以配伍使用。     

LC-MS/MS法测定缬更昔洛韦/更昔洛韦血浆浓度及在药动学研究中的应用

目的建立LC-MS/MS法同时测定人血浆中缬更昔洛韦(VGCV)和更昔洛韦(GCV)的浓度。研究中国肾移植患者服用VGCV后,VGCV和GCV在体内的药动学特征。方法50μL血浆加入含内标乙腈150μL沉淀,离心后取上清液进样。色谱柱为Agilent Eclipse Plus C(18)柱(50 mm×2.1 mm,3.5μm),流动相为甲醇-纯水(含2 mmol·L^-1甲酸铵/0.02%甲酸),采用梯度洗脱的方法,流速0.25 mL·min^-1。采用ESI正离子方式以多反应监测定量,检测离子对分别为m/z 355.3→152.1(VGCV)和m/z 256.4→152.1(GCV)。检测40例肾移植患者口服VGCV片达稳态后VGCV及GCV浓度,并计算药动学参数。结果VGCV在0.0048~0.95 mg·L^-1和GCV在0.048~9.6 mg·L^-1内线性良好(r>0.9999)。VGCV和GCV提取回收率分别为92.74%~97.83%和92.12%~102.12%,基质效应分别为106.97%~107.76%和102.45%~107.04%。VGCV的日内及日间RSD<15%。肾移植患者服用450 mg与900 mg VGCV后,GCV的AUC0→24h分别为(28.40±8.35)和(60.67±17.50)mg·h·L^-1,峰浓度ρ(max)分别为(4.24±1.07)和(8.64±1.65)mg·L^-1。结论本研究所建立的方法具有快速、准确、灵敏和专属性强等优点,适用于VGCV及GCV的药动学研究。     

Uptake of plasmid-loaded nanoparticles in breast cancer cells and effect on Plk1 expression

The development of nucleic acid–based drugs for cancer therapeutic application has shown promising results in the past. However the delivery of these drugs to target cells is one problem which remains to be resolved. Nanoparticles have been described as promising strategies to deliver drugs into target cells. Human serum albumin (HSA) nanoparticles conjugated to trastuzumab for a cell type–specific targeting of human epidermal growth factor receptor 2 (HER2)-overexpressing cells were developed with incorporated expression plasmids for small hairpin RNAs (shRNAs) targeting polo-like kinase 1 (Plk1). Plk1 is a promising target for such an approach because it is overexpressed in all known cancer types and is a negative prognostic factor. Receptor-mediated uptake of the trastuzumab-modified nanoparticles into HER2-positive cells could be observed leading to reduced Plk1 expression. Taken together, HSA nanoparticles represent promising tools to deliver expression plasmids for shRNAs into target cells and should be further evaluated with regard to a therapeutic application of RNA interference in cancer therapy.     

Uptake and cytotoxicity of chitosan nanoparticles in human liver cells

Despite extensive research into the biomedical and pharmaceutical applications of nanoparticles, and the liver being the main detoxifying organ in the human body, there are limited studies which delineate the hepatotoxicity of nanoparticles. This paper reports on the biological interactions between liver cells and chitosan nanoparticles, which have been widely recognised as biocompatible. Using the MTT assay, human liver cells were shown to tolerate up to 4 h of exposure to 0.5% w/v of chitosan nanoparticles (18 ± 1 nm, 7.5 ± 1.0 mV in culture medium). At nanoparticle concentrations above 0.5% w/v, cell membrane integrity was compromised as evidenced by leakage of alanine transaminase into the extracellular milieu, and there was a dose-dependent increase in CYP3A4 enzyme activity. Uptake of chitosan nanoparticles into the cell nucleus was observed by confocal microscopic analysis after 4 h exposure with 1% w/v of chitosan nanoparticles. Electron micrographs further suggest necrotic or autophagic cell death, possibly caused by cell membrane damage and resultant enzyme leakage.     

格尔德霉素体内外抑制单纯疱疹病毒l型的复制

格尔德霉素(GA)是一种抗生素,它的作用靶点是热休克蛋白Hsp90 N末端的ATP/ADP结合位点.在我们对抗病毒的抗生素筛选中,发现格尔德霉素显著抗单纯疱疹病毒1型(HSV一1).体外在Vero细胞内GA显著抑制HSv-1的复制,IC50为0,093μmol/L,GA对Vero细胞的毒性CC50为350μmol/L,治疗指数可达3763.HSV-1腹腔(ip)感染1h后腹腔(ip)注射GA(0.093～0.37mg/kg)可以将存活率增加到67%～100%,皮下(sc)给药(0.37mg/kg)的存活率为43.8%,都明显高于生理盐水对照组(ipP＜0.001.scP＜0.05).GA对小白鼠的急性LD50为15.5mg/kg(ip).格尔德霉素不影响病毒的吸附、穿人.由于GA在体内外都能抑制单纯疱疹病毒1型,GA可以成为新的抗单纯疱疹病毒感染的药物.     

格尔德霉素体内外抑制单纯疱疹病毒1型的复制

格尔德霉素 ( GA)是一种抗生素 ,它的作用靶点是热休克蛋白 Hsp90 N末端的 ATP/ ADP结合位点。在我们对抗病毒的抗生素筛选中 ,发现格尔德霉素显著抗单纯疱疹病毒 1型 ( HSV- 1)。体外在 Vero细胞内 GA显著抑制 HSV- 1的复制 ,IC50 为0 .0 93μmol/ L,GA对 Vero细胞的毒性 CC50 为 35 0 μmol/ L,治疗指数可达 376 3。HSV- 1腹腔 ( ip)感染 1h后腹腔 ( ip)注射 GA( 0 .0 93～ 0 .37mg/ kg)可以将存活率增加到 6 7%～ 10 0 % ,皮下 ( sc)给药 ( 0 .37mg/ kg)的存活率为 4 3.8% ,都明显高于生理盐水对照组 ( ipP<0 .0 0 1,sc P<0 .0 5 )。GA对小白鼠的急性 L D50 为 15 .5 mg/ kg( ip)。格尔德霉素不影响病毒的吸附、穿入。由于 GA在体内外都能抑制单纯疱疹病毒 1型 ,GA可以成为新的抗单纯疱疹病毒感染的药物     

Uptake of Chitosan and Associated Insulin in Caco-2 Cell Monolayers: A Comparison Between Chitosan Molecules and Chitosan Nanoparticles

PURPOSE: To evaluate the uptake of chitosan molecules (fCS) and nanoparticles (fNP), and their ability to mediate insulin transport in Caco-2 cell monolayers. METHODS: Cell-associated fCS and fNP were evaluated by fluorometry, trypan blue quenching, and confocal microscopy using FITC-labeled chitosan. Chitosan-mediated transport of FITC-labeled insulin was studied in Caco-2 cell monolayers cultured on permeable inserts. RESULTS: Caco-2 cells showed twofold higher association with fNP than fCS after 2-h incubation with 1 mg/ml samples. fNP uptake was a saturable (Km 1.04 mg/ml; Vmax 74.15 microg/mg/h), concentration- and temperature-dependent process that was inhibited by coadministered chlorpromazine. fCS uptake was temperature dependent, but was less sensitive to concentration and was inhibited by filipin. Postuptake quenching with 100 microg/ml of trypan blue suggests a significant amount of intracellular fNP, although the bulk of fCS was extracellular. Internalized fNP were located by confocal microscopy at 15 microm from the apical membrane, but there was no apparent breaching of the basal membrane. This might explain the failure of the nanoparticles to mediate significant insulin transport across the Caco-2 cell monolayer. CONCLUSIONS: Formulation of chitosan into nanoparticles transforms its extracellular interactions with the Caco-2 cells to one of cellular internalization via clathrin-mediated endocytosis.     

Detection of levodopa, dopamine and its metabolites in rat striatum dialysates following peripheral administration of L-DOPA prodrugs by mean of HPLC-EC

A high performance liquid chromatography (HPLC) method was developed to detected simultaneously L-dihydroxyphenylalanine (L-DOPA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum dilaysates following oral administration of L-DOPA or its prodrugs. The chromatographic system uses a reversed-phase C18 column with electrochemical detection at +0.30 V. Mobile phase consisted of 0.05 M citric acid, sodium EDTA 50 microM, sodium octylsulphonate 0.4 nM at pH of 2.9 and 8% methanol (v/v) at a flow rate of 1 ml/min. The calibration curves were linear over the concentration range of 10nm to 100 microM and the lower limits of detections were 125 fmol for L-DOPA, 50 fmol for DOPAC, 250 fmol for DA and 150 fmol for HVA at signal noise to ratio of 3. The repeatability (or intra-day precision), expressed by the relative standard deviation, were better than 4%. The construction of microdialysis probes has been described. The in vitro relative recoveries of each microdialysis probe were evaluated and the results show that they are similar and reproducible for all the analytes with CVs from 1 to 4%. The HPLC-EC method was applied to detect the extracellular levels of L-DOPA, DA, DOPAC and HVA in the striatum dialysates of freely moving rats after oral administration of six new potential L-DOPA prodrugs.     

Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells

Due to their physicochemical characteristics, metal oxide nanoparticles (NPs) interact differently with cells compared to larger particles or soluble metals. Oxidative stress and cellular metal uptake were quantified in rat type II alveolar epithelial cells in culture exposed to three different NPs: manganese(II,III) oxide nanoparticles (Mn₃O₄-NPs), the soluble manganese sulfate (Mn-salt) at corresponding equivalent doses, titanium dioxide (TiO₂-NPs) and cerium dioxide nanoparticles (CeO₂-NPs). In the presence of reactive oxygen species an increased apoptosis rate was hypothesized. Oxidative stress was assessed by detection of fluorescently labeled reactive oxygen species and by measuring intracellular oxidized glutathione. Catalytic activity was determined by measuring catalyst-dependent oxidation of thiols (DTT-assay) in a cell free environment. Inductively coupled plasma mass spectrometry was used to quantify cellular metal uptake. Apoptosis rate was determined assessing the activity of caspase-3 and by fluorescence microscopic quantification of apoptotic nuclei. Reactive oxygen species were mainly generated in cells treated with Mn₃O₄-NPs. Only Mn₃O₄-NPs oxidized intracellular glutathione. Catalytic activity could be exclusively shown for Mn₃O₄-NPs. Cellular metal uptake was similar for all particles, whereas Mn-salt could hardly be detected within the cell. Apoptosis was induced by both, Mn₃O₄-NPs and Mn-salt. The combination of catalytic activity and capability of passing the cell membrane contributes to the toxicity of Mn₃O₄-NPs. Apoptosis of samples treated with Mn-salt is triggered by different, potentially extracellular mechanisms.     

Cytotoxicity and apoptotic signalling cascade induced by chelidonine-loaded PLGA nanoparticles in HepG2 cells in vitro and bioavailability of nano-chelidonine in mice in vivo

Poor oral bioavailability of chelidonine, a bio-active ingredient of Chelidonium majus, showing anti-cancer potentials against cancer cells with multidrug resistance, makes its optimal use rather limited. To address this problem, we encapsulated chelidonine in biodegradable poly(lactide-co-glycolide) (PLGA) polymers and evaluated nano-chelidonine's (NCs) anti-cancer efficacy vis-à-vis free chelidonine (FC) against HepG2 cells and also evaluated its bioavailability in mice. Physicochemical characteristics indicated that stable spherical NC were formed in nanometer size range (123 ± 1.15 nm) with good yield (86.34 ± 1.91%), better encapsulation efficiency (82.6 ± 0.574%), negative surface charge (−19.6 ± 2.48 mV) and ability of prolonged and sustained release of chelidonine. Fourier transform infrared analysis revealed that NC resembled similar peaks as that of FC suggesting effective encapsulation in PLGA. NC exhibited rapid cellular uptake and stronger apoptotic effect (∼46.6% reduced IC₅₀ value) than FC, blocking HepG2 cells at G2/M phase. p53, cyclin-D1, Bax, Bcl-2, cytochrome c, Apaf-1, caspase-9 and caspase-3 expressions also corroborated well to suggest greater anticancer potentials of NC. Our in vivo studies demonstrated NC to be more bio-available than FC and showed a better tissue distribution profile without inducing any toxicity (100 mg/kg bw) in mice. Unlike FC, NC could permeate into brain tissue, indicating thereby NC's better potentials for use in therapeutic oncology.     

UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells

The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5–25 mJ/cm²). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, and mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), 15-LOX-2, and the epidermal and platelet forms of 12-LOX. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase was found to be constitutively active, and its activity increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress.     

Novel flavonoid-based biodegradable nanoparticles for effective oral delivery of etoposide by P-glycoprotein modulation: an in vitro,ex vivo and in vivo investigations

Abstract

A receptor level interaction of etoposide with P-glycoprotein (P-gp) and subsequent intestinal efflux has an adverse effect on its oral absorption. The present work is aimed to enhance the bioavailability of etoposide by co-administering it with quercetin (a P-gp inhibitor) in dual-loaded polymeric nanoparticle formulation. Poly-lactic-co-glycolic acid (PLGA) nanoparticles were optimized for various parameters like o/w phase volume ratio, poly-vinyl alcohol concentration, PLGA concentration and sonication time. The cytotoxicity studies (MTT assay) revealed a 9- and 11-fold decrease in the IC 50 values for etoposide-loaded nanoparticles (ENP) and etoposide?+?quercetin dual-loaded nanoparticles (EQNP) when compared to that of free etoposide, respectively, and the results were further supported by florescent-activated cell sorter studies. The confocal imaging of the intestinal sections treated with ENP and EQNP containing fluorescent probe (rhodamine) showed the superiority of the EQNP to permeate deeper. Furthermore, pharmacokinetic studies on rats revealed that EQNP exhibited a 2.4-fold increase in bioavailability of etoposide than ENP with no quercetin. The developed loaded nanoparticles have the high potential to enhance the bioavailability of the etoposide and sensitize the resistant cells.     

Effect of aphidicolin on DNA synthesis in HSV-1 infected and uninfected Vero cells

The effect of aphidicolin on DNA synthesis in herpes simplex virus type 1 (HSV-1) infected and uninfected Vero cells was determined by isodensity banding of [³²P]-labelled DNA. A 50% inhibition of HSV-1 DNA synthesis was observed at 0.07 μM aphidicolin while 2.1 and 1.3 μM were required to inhibit the cellular DNA synthesis to 50% in infected and uninfected Vero cells, respectively. When the viral DNA synthesis was totally inhibited by 10 μM aphidicolin, the cellular DNA synthesis was inhibited to about 90% in both infected and uninfected cells. Aphidicolin inhibited the cellular DNA synthesis in HSV-1 infected and uninfected Vero cells remaining in the presence of 250 μM foscarnet to the same extent as the DNA synthesis in the absence of foscarnet.