RESUMO
In this paper, liposomes containing a lipopeptide bearing a ligand specifically recognized by neuropilin-1 (NRP-1) have been used to target a human breast cancer cell line overexpressing this receptor. The synthesis of this lipopeptide, C16-A7R, formed by the sequence of 7 amino acids ATWLPPR, linked to a palmitoyl fatty chain by an amide bond was described. After the characterisation of cationic liposomes formulated with the lipopeptide, the results obtained using various techniques showed that the lipopeptide-based liposomes were well accumulated in cells of the human breast cancer line MDA-MB-231 overexpressing NRP-1. Delivery of reporter genes expressing either beta-galactosidase (beta-gal) or green fluorescent protein (GFP) was selectively enhanced in these cells when compared with NRP-1-negative cells. In MDA-MB-231 cells, an increase by 250% in beta-gal activity was observed when delivered by lipopeptide-based liposomes compared to cationic liposomes alone.
Assuntos
DNA/administração & dosagem , DNA/genética , Lipossomos/química , Neuropilina-1/biossíntese , Neuropilina-1/genética , Peptídeos/química , Western Blotting , Divisão Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Cromatografia Líquida de Alta Pressão , Citomegalovirus/genética , Citometria de Fluxo , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Indicadores e Reagentes , Espectrometria de Massas , Microscopia de Fluorescência , Peptídeos/isolamento & purificação , Plasmídeos/genética , Sais de Tetrazólio , Tiazóis , Transfecção , beta-Galactosidase/genéticaRESUMO
We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3 beta(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3 beta(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.
Assuntos
Colesterol , Técnicas de Transferência de Genes , Lipossomos , Cátions , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Colesterol/análogos & derivados , Colesterol/química , Digoxigenina , Endocitose , Vetores Genéticos , Humanos , Imuno-Histoquímica , Lipossomos/química , Transfecção , Células Tumorais Cultivadas , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
1. Since the sodium phenylacetate (NaPa) was reported to enhance the inhibitory effect of carboxymethyl benzylamide dextran (CMDB) on the breast cancer growth, we performed the esterification of CMDB with NaPa to obtain a new drug carrying the characteristics of these two components. A new molecule, phenylacetate carboxymethyl benzylamide dextran, was named NaPaC. 2. We investigated in vitro and in vivo the effects of NaPaC on MCF-7ras cell growth as well as its apoptotic and antiangiogenic effects in comparison to NaPa and CMDB. In addition, we assessed in vitro the antiproliferative effects of these drugs on other breast cancer cells, including MDA-MB-231, MDA-MB-435 and MCF-7. 3. In vitro, NaPaC inhibited MCF-7ras cell proliferation by 40% at concentration lower than that of CMDB and NaPa (12 microM vs 73 microM and 10 mM). IC(50)s were 6 and 28 microM for NaPaC and CMDB, respectively. The similar results were obtained for three other breast cancer cell lines. NaPaC reduced the DNA replication and induced cell recruitment in G(0)/G(1) phase more efficiently than its components. Moreover, it induced a cell death at concentration 1000-fold lower than NaPa. 4. In vivo, CMDB (150 mg kg(-1)) and NaPa (40 mg kg(-1)) inhibited the MCF-7ras tumour growth by 37 and 57%, respectively, whereas NaPaC (15 mg kg(-1)) decreased tumour growth by 66% without toxicity. 5. NaPa or CMDB reduced the microvessel number in tumour by 50% after 7 weeks of treatment. NaPaC had the same effect after only 2 weeks. After 7 weeks, it generated a large necrosis area without detectable microvessels. In vitro, NaPaC inhibited human endothelial cell proliferation more efficiently than CMDB or NaPa. NaPaC interacts with vascular endothelial growth factor as observed by affinity electrophoresis. 6. NaPaC acts like NaPa and CMDB but in more potent manner than components used separately. Its antiproliferative, aponecrotic and anti-angiogenic actions make it a good candidate for a new anti-cancer drug.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Dextranos/farmacologia , Inibidores do Crescimento/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , Células 3T3 , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Meios de Cultivo Condicionados/farmacologia , Dextranos/química , Dextranos/uso terapêutico , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Inibidores do Crescimento/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Necrose , Fenilacetatos/farmacologia , Fenilacetatos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto/estatística & dados numéricosRESUMO
In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid ( Percot et al., 2004 ), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.
Assuntos
Colesterol/análogos & derivados , Portadores de Fármacos/química , RNA Interferente Pequeno , Fator A de Crescimento do Endotélio Vascular/genética , Cátions , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colesterol/síntese química , Colesterol/química , DNA/administração & dosagem , DNA/genética , Portadores de Fármacos/síntese química , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Inativação Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Lipossomos , Plasmídeos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The determination of chemiluminescent intensity of reporter gene expression in vivo is generally disturbed by the presence of hemoglobin. Current methods consist in using perfusion to eliminate blood from investigated tumors or organs. In this work we propose a simple method to overcome this difficulty. The method consists in establishing an absorbance-dependence plot of the ratio R% = phi/phi(0) between the chemiluminescent intensities measured when hemoglobin is present or absent. For every measurement of the luminescent intensity phi on sample containing blood, if the absorbance A of the hemoglobin is determined, it allows one to have the intensity ratio R% which in turn gives the corrected intensity phi(0) when the absorption by hemoglobin is eliminated. The method is particularly adapted for comparative measurements of transfection levels in tumors where perfusion cannot be easily performed.
Assuntos
Genes Reporter , Hemoglobinas/farmacologia , Luciferases/análise , Animais , Calibragem/normas , Genes Reporter/efeitos dos fármacos , Hemoglobinas/química , Lipossomos/metabolismo , Medições Luminescentes , Melanoma , Camundongos , Camundongos Nus , Neoplasias/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Espectrofotometria/métodos , Transfecção/métodos , Células Tumorais CultivadasRESUMO
Encapsulation of technetium-99m sestamibi ((99m)Tc-MIBI) in polyethyleneglycol-liposomes ((99m)Tc-MIBI-PEG-liposomes) could extend the duration of its circulation in blood and alter its biodistribution, enabling its concentration in tumours to be increased. An original method to encapsulate (99m)Tc-MIBI in PEG-liposomes is described. The (99m)Tc-MIBI-PEG-liposomes were compared with free (99m)Tc-MIBI with respect to (a) tumour availability (b) ability to distinguish between chemotherapy-sensitive and -resistant cells and (c) uptake ratio in tumour imaging. PEG-liposomal systems composed of distearoylphosphatidylcholine/cholesterol/PEG(2000)-distearoyl phosphatidylethanolamine and lissamine-rhodamine B-labelled liposomes were used. The encapsulation of (99m)Tc-MIBI in liposomes was achieved using the K(+) diffusion potential method. We compared the uptake of free versus encapsulated (99m)Tc-MIBI by sensitive and resistant erythroleukaemia (K562) and breast tumour (MCF-7ras) cells. To assess the internalisation of these liposomes into cells, rhodamine B-labelled PEG-liposomes were used and visualised by fluorescence microscopy. Biodistribution and imaging characteristics of encapsulated and free radiotracer were determined in rats and tumour-bearing nude mice. The efficiency of (99m)Tc-MIBI encapsulation in PEG-liposomes was 50+/-5%. Use of (99m)Tc-MIBI-PEG-liposomes did not impair the ability of this tracer to distinguish between chemotherapy-sensitive and -resistant tumour cells; the percentage of radioactivity accumulated in the sensitive K562 cells was 1.24+/-0.04%, as compared with 0.41+/-0.04% in the resistant K562 cells. One hour post injection in rats, PEG-liposomes showed a ten times higher activity in blood than free (99m)Tc-MIBI, whereas activity of free (99m)Tc-MIBI in kidneys and bladder was markedly higher than that of encapsulated (99m)Tc-MIBI, indicating faster clearance of the free radiotracer. In the (MCF7-ras)-bearing nude mice, PEG-liposome uptake in tumour was two times that of free (99m)Tc-MIBI. Summarising, the (99m)Tc-MIBI-PEG-liposomes demonstrated a longer blood circulation time, enabled distinction between chemotherapy-sensitive and -resistant cells and improved tumour to background contrast in in vivo imaging. (99m)Tc-MIBI-PEG-liposomes therefore show promising potential for tumour imaging.