Receptor-mediated gene delivery to HepG2 cells by ternary assemblies containing cationic liposomes and cationized asialoorosomucoid

Unilamellar cationic liposomes have been prepared from an equimolar mixture of 3beta[N',N'-dimethylaminopropane)-carbomoyl] cholesterol (Chol-T), a higher homologue of 3beta[N',N'-dimethylaminoethane)-carbomoyl] cholesterol (DC-Chol), and dioleoylphosphatidyl-ethanolamine. The DNA binding capabilities of Chol-T and Chol-T/DOPE liposomes have been demonstrated in lipid impregnated paper-DNA binding assays and gel retardation experiments, respectively. These liposomes have been combined with pRSVL plasmid DNA and N-ethyl-N'-(3-trimethylpropylammonium) carbodiimide iodide modified asialoorosomucoid (Me+ CDI urea-AOM) to generate ternary electrostatic assemblies intended for selective entry into cells displaying the galactose-specific lectin. This effect has been evaluated in the human hepatocellular carcinoma cell line HepG2 in which high levels of luciferase activity were achieved (up to 1.84 x 10(7) relative light units/mg protein) after transfection with complexes containing liposomes (1-3 microg), Me+CDI urea-AOM (2 microg), and DNA (0.5 microg) in 0.5 mL culture medium. Transfections conducted in the presence of free asialoorosomucoid afforded much lower luciferase activity (up to 1.5 x 10(5) relative light units/mg protein) confirming that DNA uptake was predominantly via asialoorosomucoid receptor-mediated endocytosis. We concluded therefore that modular complexes used in our study display the carbohydrate moiety of the glycoprotein component prominently, thus permitting interaction of terminal galactose units with their cognate receptors on the cell membrane.     

Functional genomics may allow accurate categorization of the benzimidazole fungicide benomyl: lack of ability to act via steroid-receptor-mediated mechanisms

Although benomyl and its metabolite carbendazim have been shown to adversely affect male reproduction, the mechanisms of action do not appear to involve the endocrine system. However, few studies have been conducted using currently proposed tests specifically focused on endocrine disruption. Here, potential estrogen- and androgen-mediated activity of benomyl was therefore investigated in vitro and in vivo. Benomyl and carbendazim proved negative for agonistic and antagonistic activity in reporter gene assays for the human estrogen receptor alpha and androgen receptor. In uterotrophic and Hershberger assays using Crj:CD(SD)IGS rats, benomyl (100, 300 or 1000 mg/kg/day, p.o., N = 6) did not exert agonistic effects. However, the highest dose decreased uterine weights in the uterotrophic assay, and decreased weights of some androgen-related tissues of castrated rats receiving a testosterone propionate (TP, 0.2 mg/kg) injection in the Hershberger assay; the effects were less severe than those with p,p'-DDE (100 mg/kg/day). When 4 mg/kg/day of TP was injected, decrease of organ weights due to benomyl was attenuated but still observed. Thus, its influence in some tissues was more potent than that of p,p'-DDE. Benomyl had no apparent effects on serum androgen levels. Microarray analysis of the gene expression profile in the ventral prostate of TP-injected castrated rats treated with benomyl indicated clear differences from the patterns observed with p,p'-DDE and flutamide. Taken together, these findings suggest the decreased organ weights observed in vivo to be caused by mechanisms that are not steroid-receptor-mediated, such as interfering with assembly of microtubules by benomyl. The study furthermore suggests that functional genomics may provide a reliable evidence for accurate categorization of test chemicals.     

Pharmacological characterisation of the goldfish somatostatin sst5 receptor

Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst1-5). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst5 receptors (gfsst5) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [125I]-LTT-SRIF-28 ([Leu8, DTrp22, 125I-Tyr25]SRIF-28) and [125I][Tyr10]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (pKd: 9.99-9.71; Bmax: 300-350 fmol mg-1). 5'-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5-57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [125I]LTT-SRIF-28 and [125I][Tyr10]cortistatin-14 correlated significantly (r2=0.97, n=20). Pharmacological profiles of human and mouse sst5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst5 profile (r2=0.52-0.78, n > or = b16). Functional expression of the gfsst5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [35S]GTPgammaS ([35S]guanosine 5'-O-(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies (r2=0.81-0.93, n=20), although relative potency (pEC50) was reduced compared to pKd values. Relative efficacy profiles of luciferase expression and [35S]GTPgammaS binding, were rather divergent (r2=0.48, n=20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2) acted as an antagonist on the effects of SRIF-14 (pKB=6.74 +/- 0.23) on stimulation of [35S]GTPgammaS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [35S]GTPgammaS binding suggesting coupling of the receptor to G(i)/G(o) proteins. In summary, the present studies demonstrate that the gfsst5 receptor has a similar pharmacological profile and transductional properties to mammalian sst5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.     

Receptor-Mediated Gene Delivery to HepG2 Cells by Ternary Assemblies Containing Cationic Liposomes and Cationized Asialoorosomucoid

A new cholesterol-carborane conjugate (BCH) has been synthesized as a potential targeting agent for boron neutron capture therapy (BNCT) of cancers. The compound is extremely water insoluble and was formulated in two liposomal formulations to determine if the compound could be adequately taken up by 9L rat glioma cells in cell culture. Several factors potentially affecting the cellular uptake were evaluated, such as concentration of BCH in the incubation medium, incubation time, cell confluence, and the addition of polyethylene glycol (PEG) phospholipids to the liposomal formulation. The studies indicated that the cellular uptakes of BCH in the conventional and PEG liposomal formulations were 49.1 and 45.9 μg boron/g cells, respectively. Therefore, this compound, formulated in both liposomal formulations, delivered sufficient levels of boron to cancer cells in vitro, indicating that BCH is a promising approach for use in BNCT. The uptake appeared to depend upon BCH concentration in the media as well as the confluence of the cells. The greater boron uptake by nonconfluent cells indicated that active growth of cells was a factor in the uptake of this compound.     

Low Concentrations of Chlorpromazine and Related Phenothiazines Stimulate Gene Transfer in HeLa Cells Via Receptor-Mediated Endocytosis

Chlorpromazine and related phenothiazine antipsychotic compounds at the low concentration of 10 -5 M stimulated luciferase pRSVL DNA uptake and expression in HeLa cells. On the other hand, chloroquine at a 10 -5 M was without effect at this low concentration. However, at the higher normally used concentration of 10 -4 M (100 μM), chloroquine strongly stimulated luciferase expression and activity. Unfortunately, at 10 -4 M, the phenothiazines were toxic to the cells and could not be tested at this concentration. Further experimental work was carried out to elucidate the mechanism of action of phenothiazines and chloroquine on DNA uptake and expression. Interaction of [3 H] pBR 322 DNA with chlorpromazine, perphenazine, and chloroquine was studied using these compounds as their free bases dissolved in chloroform, followed by their impregnation onto Whatman No. 1 filter paper discs. Both phenothiazines on filter paper discs bound [3 H] pBR 322 DNA to a far greater extent than chloroquine. The method of assay (free base) suggests that the major contribution to binding is through intercalation. A further possible assay for studying the interaction of phenothiazines and chloroquine made use of the ethidium bromide/calf thymus DNA intercalation method. Intercalated calf thymus (CT) DNA complexes with ethidium bromide (EB) were examined for possible dissociation into free DNA and EB on the addition of either chloroquine. SO 4 or chlorpromazine.HCl (soluble salts). Partial dissociation was observed with both compounds. Further experiments on the stability of pBR 322 DNA-polylysine complexes were also carried out using an alternative method of assay. Chloroquine (10 -2 - 10 -4 M) and chlorpromazine (10 -4 M) did not bring about a dissociation of [3 H] pBR 322 DNA-polylysine 200 complexes when reactions were studied by nitrocellulose filter assays to measure released double-stranded DNA. The results indicate that chlorpromazine and related phenothiazines stimulate luciferase DNA uptake expression at 10 -5 M. Chloroquine at this concentration had practically no effect on expression of luciferase activity. Further studies of chloroquine and chlorpromazine on their interaction with plasmid DNA as well as DNA-polylysine complexes are reported.     

活体荧光成像技术评价X射线对小鼠乳腺癌移植瘤的治疗效果

目的 建立荧光素酶标记的小鼠乳腺癌细胞爪垫淋巴结转移模型,监测肿瘤早期淋巴结转移,并用荧光成像评价X射线局部治疗效果。方法 将表达荧光素酶的小鼠乳腺癌细胞系4T1-Luc接种至裸鼠爪垫皮下,建立爪垫皮下淋巴结转移模型。通过裸鼠活体荧光成像系统连续观察肿瘤细胞在淋巴结中的转移情况,并将有早期淋巴转移的荷瘤裸鼠按随机数字表法分为对照组和治疗组,活体荧光成像系统观察治疗效果,HE染色观察评价病理形态学变化。结果 成功建立了小鼠乳腺癌淋巴结转移模型,爪垫原发灶肿瘤体积与荧光光子数呈正相关性（r=0.958,P<0.001）,在肿瘤接种的第24天,治疗组爪垫肿瘤和腘窝处肿瘤部位的荧光光子数较对照组呈显著性降低（t=32.58,P<0.05）;用荧光光子数计算抑瘤率高达85%以上。HE染色观察到治疗组较对照组移植瘤坏死明显。结论 运用活体生物发光成像技术能够动态、客观、灵敏、可视化地评估X射线对小鼠乳腺癌肿瘤的抑瘤效应。     

Location of molecules in layer-by-layer assembled microcapsules influences activity,cell delivery and susceptibility to enzyme degradation

Layer-by-layer assembled microcapsules have the potential to be versatile cell delivery systems incorporating multiple activities and functions. However, it is necessary to determine the influence that different capsule locations have on activity of bioactive molecules in order to optimise delivery and for generation of multifunctional capsules. In this study we examine the influence that locating the bioluminescent enzyme luciferase in different microcapsule locations has on activity in intact synthetic and biodegradable microcapsules before and after cell delivery as well as its susceptibility to protease degradation. We also examine the effect of microcapsule position on cell transfection with plasmid DNA. Based on the findings of experiments in this study we also demonstrate co-delivery of luciferase protein and plasmid DNA encoding a fluorescent protein from two different locations within the same microcapsule. Our studies confirm that, the core, subouter layer, and outer layer are optimal for cell delivery but these positions offer least protection from protease activity. By contrast middle layer molecules remain entangled with capsule layers preventing their release which is inefficient for cell delivery but this provides better protection from protease degradation. The findings of this study will enable more rationale layer-by-layer assembly of microcapsules containing biologically active molecules for cell delivery and aid in the generation of multifunctional microcapsules.     

Synthesis and characterization of mannosylated pegylated polyethylenimine as a carrier for siRNA

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45-13.3 PEG chains and 4.7-3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169 to 357 nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2h post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone.     

红色荧光蛋白和荧光素酶双报告基因转基因小鼠的建立

背景与目的:活体动物体内光学成像(optical in vivo imaging)主要采用生物发光与荧光两种技术。生物发光是用荧光素酶(luciferase,Luc)基因标记细胞或DNA,而荧光技术则采用荧光报告基团(GFP、RFP、Cyt及dyes等)进行标记,利用一套非常灵敏的光学检测仪器,能够直接监控活体生物体内的细胞活动和基因行为,生物发光成像具有高的灵敏度和特异性,同时生物发光信号可用于精确定量,而荧光成像具有方便、便宜、直观、标记靶点多样和易于被大多数研究人员接受的优点。本研究基于慢病毒介导的转基因方法制备红色荧光蛋白(red fluorescent protein,RFP)和Luc双报告基因转基因小鼠(即RL转基因小鼠),将这两种技术融为一体。方法:制备携带RFP和Luc基因(简写RL基因)的慢病毒,然后将携带RL基因的慢病毒注入小鼠单细胞受精卵卵周隙以感染受精卵,胚胎移植进假孕母鼠以获得仔鼠,应用小动物活体成像仪、体视荧光显微镜和PCR等在蛋白和DNA水平上筛选和鉴定,并获得RL转基因小鼠。结果:移植卵周隙注射有慢病毒的胚胎125枚给6只假孕母鼠,其中4只假孕母鼠怀孕,共生仔鼠20只;利用小动物活体成像仪检测RFP和Luc表达,在蛋白水平证实20只F0代中,3只高表达RFP和Luc;DNA水平检测证实,3只RFP和Luc阳性的小鼠基因组中确实整合有外源转基因RL,预示基因型鉴定结果很好验证了小动物活体成像仪筛选和鉴定结果。此外,RL转基因首建鼠基因组中整合的RL转基因可稳定遗传至下一代,并能正常表达。RL转基因小鼠主要脏器均可见红色荧光和Luc信号,但不同脏器间荧光和Luc强度有差异。结论:成功制备RL双报告基因转基因小鼠,为后续研究干细胞在肿瘤发生、发展和转移中的作用和造血重构等提供双报告基因标记的各种移植用供体细胞,并对此供体细胞及其在体内衍生的细胞进行灵敏的非损伤、实时可视化体内跟踪。