NGB基因转染拮抗庆大霉素耳毒性作用的实验研究

目的验证阳离子脂质体介导脑红蛋白(neuroglobin,NGB)基因转染对庆大霉素致豚鼠耳毒性的保护作用。方法将ABR反应阈均不超过40dB SPL的120只健康花色豚鼠随机分为5组,每组24只:Ⅰ组:空白对照组;Ⅱ组:人工外淋巴液对照组(经左耳注入人工外淋巴液);Ⅲ组:人工外淋巴液实验组(经左耳注入人工外淋巴液后肌肉注射庆大霉素);Ⅳ组:空质粒转染组(经左耳注入空质粒pEGFP-C1后肌肉注射庆大霉素);Ⅴ组:NGB基因转染组(经左耳注入pEGFP-NGB后肌肉注射庆大霉素),庆大霉素均经后腿肌肉注射120mg.kg-1.d-1,共给药14天。停止给药后喂养14天,各组均行ABR检测,耳蜗基底膜铺片、免疫组化观察各组豚鼠耳蜗基底膜毛细胞形态学及NGB蛋白表达的变化。结果给药后Ⅰ组ABR反应阈平均为37.22dB SPL(左耳)和36.94dB SPL(右耳),Ⅱ组阈值平均为37.22dB SPL(左耳)和37.50dB SPL(右耳),Ⅲ组阈值平均为119.44dB SPL(左耳)和122.22dB SPL(右耳);Ⅳ组阈值平均为119.72dB SPL(左耳)和120.83dB SPL(右耳);Ⅴ组阈值平均为83.89dB SPL(左耳)和100.56dB SPL(右耳)。Ⅴ组ABR反应阈较Ⅰ组和Ⅱ组显著升高(P<0.05),较Ⅲ组和Ⅳ组显著降低(P<0.05)。Ⅴ组中手术耳ABR反应阈较非手术耳降低(P<0.05)。耳蜗基底膜铺片示Ⅰ组和Ⅱ组内外毛细胞排列整齐,无缺失,Ⅲ组和Ⅳ组内外毛细胞极少量残存,其中ABR阈值大于135dB SPL的豚鼠耳蜗毛细胞几乎消失殆尽,Ⅴ组毛细胞部分缺失,且主要是外毛细胞;免疫组织化学染色示Ⅴ组耳蜗毛细胞NGB蛋白表达量较其余各组均显著增高(P<0.05),其余各组几乎均未见明显阳性表达。结论本研究成功验证了阳离子脂质体介导NGB基因转染对庆大霉素致豚鼠耳毒性具有有效的保护作用。     

阳离子脂质体基因载体的研究进展

肿瘤基因治疗的最大挑战之一是研制安全有效的基因转染载体。基因治疗载体一般分为病毒性载体和非病毒性载体。由于病毒型载体存在安全性问题,目前非病毒性载体的研究越来越受到人们的重视。脂质体作为常用的非病毒性载体容易制备,安全性高,但亦存在质控要求高,体内基因导入效率较低,且无靶向性,使得其应用受到限制。pH敏脂质体、阳离子脂质体等大大提高了脂质体的转运效率,也为非病毒载体在临床上的应用开辟了广阔的前景,本文就阳离子脂质体作为基因载体的研究予以综述。     

Pharmaceutical emulsions: a new approach for gene therapy

The concept of gene therapy involves the experimental transfer of a therapeutic gene into an individual’s cells and tissues to replace an abnormal gene aiming to treat a disease, or to use the gene to treat a disease just like a medicine, improving the clinical status of a patient. The achievement of a foreigner nucleic acid into a population of cells requires its transfer to the target. Therefore, it is essential to create carriers (vectors) that transfer and protect the nucleic acid until it reaches the target. The obvious disadvantages of the use of viral vectors have directed the research for the development of a nonviral organized system such as emulsions. In fact, recently, there has been an increase of interest in its use in biotechnology as a nonviral vector for gene therapy. This review focuses on the progress of cationic emulsions and the improvement of the formulations, as a potential delivery system for gene therapy.     

Determination of cationic surfactants as the preservatives in an oral solution and a cosmetic product by capillary electrophoresis

In this study, a capillary electrophoresis method was developed for the determination of cationic surfactants, benzethonium and cetylpyridinium ions, which are commonly used as preservatives in various pharmaceutical and cosmetic products. Determination was performed in a fused-silica capillary using a mixed 75 mmol/L phosphoric acid and 50% acetonitrile electrolyte at pH 2.5. Analysis of benzethonium and cetylpyridinium ions was achieved in around 5 min. Repeatability in migration times (R.S.D.%) for benzethonium and cetylpyridinium ions were 0.3. The calibration curves were linear from 0.0125 to 0.400 mmol/L for benzethonium ions and from 0.025 to 0.400 mmol/L for cetylpyridinium ions. The minimum detection limits (signal-to-noise ratio = 3) are 1.47 and 4.30 μg/mL for benzethonium and cetylpyridinium ions, respectively. The method was applied to the analysis of benzethonium ion in a cosmetic product and cetylpyridinium ion in a mouthwash.     

Characteristics and biodistribution of soybean sterylglucoside and polyethylene glycol-modified cationic liposomes and their complexes with antisense oligodeoxynucleotide

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG₂ 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at ∼200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG₂ 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatoctyes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL-mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting.     

阳离子脂质体包裹的IL-12基因对小鼠黑色素瘤的治疗作用

目的:观察体内注射阳离子脂质体包裹的小鼠白细胞介素12(mIL-12)基因对小鼠黑色素细胞瘤的治疗作用.方法:C57BL/6小鼠在接种B16黑色素细胞后3、5、7、9 d分别给予lipofectin包裹的pCmIL-12质粒治疗,观察小鼠的肿瘤生长速度、生存期以及NK细胞活性的变化.结果:阳离子脂质体明显增强pCmIL-12质粒在体内的抗肿瘤生长活性,并延长荷瘤小鼠的存活期.此外,pCmIL-12/阳离子脂质体还能显著增强荷瘤小鼠的NK细胞活性.结论:阳离子脂质体介导的IL-12基因对小鼠黑色素瘤有明显的治疗效果.     

Reversal of multidrug resistance by a liposome-MDR1 ribozyme complex

Purpose: Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. We examined whether cationic liposome-mediated transfer of a ribozyme could reverse MDR. Methods: A ribozyme which cleaved codon 196 of MDR1 mRNA was constructed from synthetic oligonucleotides. The MDR1 ribozyme was mixed with N-(1-(2,3-dileoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (DOTAP) to form a liposomal complex. The complex was used to treat two P-glycoprotein-producing MDR cell lines: MCF-7/R human breast cancer cells resistant to doxorubicin and MOLT-3/TMQ₈₀₀ human ALL cells resistant to trimetrexate (TMQ). In order to investigate the differential sensitivity of these two cell lines to the liposome-ribozyme complex, cellular pharmacological studies including phase-contrast and confocal microscopic studies were performed. Results: Treatment with the liposome-ribozyme complex resulted in reversal of vincristine (VCR) resistance in MCF-7/R cells, but not in MOLT-3/TMQ₈₀₀ cells. In MCF-7/R cells the treatment resulted in decreases in MDR1 mRNA expression and P-glycoprotein production, whereas no changes in these parameters were seen in MOLT-3/TMQ₈₀₀ cells. Phase-contrast microscopy revealed that in MCF-7/R cells treatment with DOTAP led to the formation of cytoplasmic vacuoles, and treatment with latex beads resulted in the development of a shiny material in the cytoplasm. In contrast, in MOLT-3/TMQ₈₀₀ cells hardly any morphological changes occurred. Confocal microscopic imaging showed cytoplasmic fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads. In MOLT-3/TMQ₈₀₀ cells no fluorescence was detected. Treatment with cytochalasin B abolished fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads. These studies show that MCF-7/R cells have high endocytotic activity whereas MOLT-3/TMQ₈₀₀ cells have little activity. Conclusions: Endocytotic activity was correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme. Determination of endocytotic activity of target tumor cells may be predictive of efficacy of liposome-mediated gene transfer. Received: 18 June 1997 / Accepted: 8 October 1997     

纳米颗粒包裹CpG序列对猪副伤寒疫苗接种小鼠免疫应答的影响

本实验制备聚乙交酯丙交酯(PLG)纳米颗粒,比较了阳离子PLG纳米颗粒和脂质体作为包装分子包裹pUC18-CpG质粒对猪副伤寒疫苗接种小鼠体液IgG和特异抗体滴度、脾脏淋巴细胞增殖和白细胞介素-2诱生活性的影响。实验结果发现:与裸pUC18-CpG质粒接种小鼠比较,阳离子纳米颗粒包裹pUC18-CpG质粒能显著提高免疫小鼠IgG含量和特异抗沙门氏菌抗体滴度,增强淋巴细胞增殖活性及白细胞介素-2的诱生活性;同阳离子脂质体作为包装分子的细胞和体液免疫佐剂效应相似或较强。证明阳离子PLG纳米颗粒包装能显著提高裸CpG质粒的免疫增强活性。     

Lipoplex-mediated delivery of nucleic acids: factors affecting in vivo transfection

For the past 15 years cationic liposomes have routinely been utilised for the delivery of nucleic acids such as plasmids and oligodeoxynucleotides to cells in culture and in vivo. These reagents are commercially available or are formulated inhouse. However, particularly in cultured cells, toxicity remains a significant problem, and this is confirmed by several in vivo findings. In addition, these complexes exhibit an immunostimulation effect, a phenomenon that may either be harmful or beneficial. Furthermore, lipoplexes have been recently found to have a certain degree of selectivity for dividing vascular endothelial cells. The development of cationic lipids that are safe to use especially in vivo and possess enhanced transfection capabilities is an ongoing process. More research is needed to understand the basic biology behind lipofection, first at the cellular level, then at the multicellular (whole organism) level.Abbreviations CL Cationic liposome - CLNAC Cationic lipid–nucleic acid complex - CpG Cytosine-phosphate-guanine - DC-chol 3[N-(N,N-Dimethylaminoethan)-carbamoyl] cholesterol - DMRIE 3-Dimethyl-hydroxyethylammonium bromide - DOPE Dioleoylphosphatidylethanolamine - DOTAP N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate 1,2-dioleoyltrimethyl ammonium propane chloride - IFN Interferon - IL Interleukin - pDNA Plasmid DNA - PEI Polyethyleneimine - TNF Tumor necrosis factor - VEC Vascular endothelial cell     

Accuracy of calculated pH-dependent aqueous drug solubility

The aim of the present study was to investigate the extent to which the Henderson–Hasselbalch (HH) relationship can be used to predict the pH-dependent aqueous solubility of cationic drugs. The pH-dependent solubility for 25 amines, carrying a single positive charge, was determined with a small-scale shake flask method. Each sample was prepared as a suspension in 150 mM phosphate buffer. The pH-dependent solubility curves were obtained using at least 10 different pH values. The intrinsic solubility, the solubility at the pK_a and the solubility at pH values reflecting the pH of the bulk and acid microclimate in the human small intestine (pH 7.4 and 6.5, respectively) were determined for all compounds. The experimental study revealed a large diversity in slope, from −0.5 (celiprolol) to −8.6 (hydralazine) in the linear pH-dependent solubility interval, which is in sharp contrast to the slope of −1 assumed by the HH equation. In addition, a large variation in the range of solubility between the completely uncharged and completely charged drug species was observed. The range for disopyramide was only 1.1 log units, whereas that for amiodarone was greater than 6.3 log units, pointing at the compound specific response to counter-ion effects. In conclusion, the investigated cationic drugs displayed compound specific pH-dependent solubility profiles, indicating that that the HH equation in many cases will only give rough estimations of the pH-dependent solubility of drugs in divalent buffer systems.