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1.
Int J Pharm ; 596: 120291, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33524527

RESUMO

Lack of selectivity together with severe side effects in conventional cancer treatment have afforded the development of new strategies based on gene therapy. Nowadays, gene therapy is employed through both viral and non-viral vectors. In spite of the high transfection activity of viral vectors, some drawbacks have pointed out to non-viral vectors as a safer alternative. To overcome low efficiency as well as other issues associated with the use of non-viral vectors, complexes formed by lipids and polymers with DNA, named lipoplexes and polyplexes respectively, have been modified in order to improve its features. Suitability of cancer gene therapy also requires the capacity to distinguish between normal and tumoral cells. This requirement has been solved by the addition of specific ligands that enable receptor binding and subsequent endocytosis. In this article we review the most recent approaches in structure modification of non-viral vectors through different methods comprising conjugation, addition of helper lipids or changes in design and synthesis as well as the strategy based on exploiting receptors that are usually overexpressed in malignancies. Such improvements confer specificity, efficient gene delivery, condensation, protection of DNA and low levels of toxicity avoiding off-target effects which turn into a potential tool to treat cancer.


Assuntos
Técnicas de Transferência de Genes , Neoplasias , Genes Neoplásicos , Terapia Genética , Vetores Genéticos , Humanos , Neoplasias/genética , Neoplasias/terapia , Transfecção
2.
Chem Commun (Camb) ; 52(66): 10117-20, 2016 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-27387975

RESUMO

The convergent preparation of Janus molecular nanoparticles by thiourea-"clicking" of α,α'-trehalose halves has been implemented; the strategy allows access to macrocyclic derivatives with seggregated cationic and lipophilic domains that in the presence of DNA undergo pH-dependent self-assembly into lamellar superstructures, as established by electrochemical, structural (SAXS), microscopical (TEM) and computational techniques, that mediate transfection in vitro and in vivo.


Assuntos
Química Click/métodos , DNA/química , Nanopartículas/química , Oligossacarídeos/química , Trealose/química , Animais , Células COS , Chlorocebus aethiops , DNA/metabolismo , Concentração de Íons de Hidrogênio , Nanopartículas/metabolismo , Oligossacarídeos/metabolismo , Espalhamento a Baixo Ângulo , Trealose/metabolismo , Difração de Raios X
3.
Gene Ther ; 10(1): 5-14, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12525832

RESUMO

A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 microg/microg DNA) showed a 16-fold higher transfection activity in HepG2 cells than non-targeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AF-complexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 microg/mcirog DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liver-specific vector described here may be useful for the delivery of clinically important genes to this organ.


Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Hepatócitos/metabolismo , Transfecção/métodos , Animais , Receptor de Asialoglicoproteína/metabolismo , Assialoglicoproteínas , Ácidos Graxos Monoinsaturados , Feminino , Fetuínas , Corantes Fluorescentes , Expressão Gênica , Células HeLa , Humanos , Injeções Intravenosas , Lipossomos , Luciferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Protaminas , Compostos de Amônio Quaternário , Células Tumorais Cultivadas , alfa-Fetoproteínas
5.
Biochim Biophys Acta ; 1561(2): 209-21, 2002 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-11997121

RESUMO

Cationic liposomes and the complexes they form with DNA (lipoplexes) constitute the most promising alternative to the use of viral vectors for gene therapy. One of the limitations to their application in vivo, however, is the inhibition of gene delivery by serum. In a previous study, we demonstrated that transferrin (Tf)-lipoplexes were superior to plain lipoplexes in transfecting HeLa cells in the presence of high concentrations of serum. With the goal of obtaining efficient gene expression in vivo, we evaluated the efficacy of Tf-lipoplexes (containing DOTAP and cholesterol) in transfecting primary hepatocytes and adipocytes in the presence of high serum concentrations. The association of transferrin with cationic liposomes increased luciferase expression compared to plain lipoplexes in primary cells as well as in HepG2 and 3T3-L1 differentiated adipocytes. The complexes were not cytotoxic and were highly effective in protecting DNA from attack by DNase I. An efficient and reliable method was developed to prepare lipoplexes containing both Tf and protamine sulfate, where the latter was mixed with transferrin, followed by the addition of cationic liposomes and DNA. The resulting protamine-Tf-lipoplexes increased significantly the levels of gene expression in cultured cells and in various tissues in mice following i.v. administration.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Células 3T3 , Animais , Cátions , Sobrevivência Celular , Etídio , Expressão Gênica , Terapia Genética , Humanos , Lipossomos , Camundongos , Plasmídeos/metabolismo , Protaminas/metabolismo , Transfecção , Transferrina , Células Tumorais Cultivadas
6.
Biochim Biophys Acta ; 1463(2): 333-42, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10675511

RESUMO

Cationic lipids are being used increasingly as reagents for gene delivery both in vitro and in vivo. One of the limitations to the application of cationic lipid-DNA complexes (lipoplexes) in vivo is the inhibition of gene delivery by serum. In this study, we have shown that transferrin (Tf)-lipoplexes, which had transferrin adsorbed at their surface via electrostatic interactions, are much more effective than plain lipoplexes in transfecting cells in the presence of relatively high concentrations (up to 60%) of fetal bovine serum (FBS). Serum even enhanced transfection by Tf-lipoplexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/dioleoylphosphatidylethanolamine (DOPE)/pCMVLacZ at high lipid/DNA (+/-) charge ratios, and inhibited lipofection for those with low charge ratios when they were added to the cells immediately after the preparation of complexes. The effect of serum on lipofection was dose-dependent. Preincubation of the complexes at 20 degrees C for 6 h led to serum resistance, even for the negatively charged transferrin-lipoplexes. A similar tendency was observed for DOTAP/cholesterol and DOTAP/DOPE/cholesterol liposomes. The percentage of cells transfected, measured by beta-galactosidase expression, also increased with the serum concentration. Cell viability was not affected significantly when the cells were incubated with the complexes for 4 h at 37 degrees C, followed by a 48-h incubation. Our findings extend the scope of previous studies where transferrin-lipoplexes were used to introduce DNA into cells, rendering these complexes and their future derivatives potential alternatives to viral vectors for gene delivery in vivo.


Assuntos
Transfecção/métodos , Transferrina , beta-Galactosidase/genética , Animais , Sangue , Bovinos , Colesterol , Meios de Cultura , Citomegalovirus , Portadores de Fármacos , Ácidos Graxos Monoinsaturados , Corantes Fluorescentes , Técnicas de Transferência de Genes , Vetores Genéticos , Células HeLa , Humanos , Lipossomos , Fosfatidiletanolaminas , Compostos de Amônio Quaternário , beta-Galactosidase/análise
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