RESUMO
Lipoplexes containing a hyaluronic acid-dioleoylphosphatidylethanolamine (HA-DOPE) conjugate were designed to target the CD44 receptor on breast cancer cells. Cationic liposomes composed of a mixture of [2-(2,3-didodecyloxypropyl)hydroxyethyl]ammonium bromide (DE) and dioleoylphosphatidylethanolamine (DOPE) with or without HA-DOPE were prepared, characterized, and used to form a complex with plasmid DNA pCMV-luc. Lipoplexes displayed a negative zeta potential and a mean diameter between 250-300 nm. Cytotoxicity and transfection efficiency of the lipoplexes were determined on the MDA-MB-231and MCF-7 breast cancer cell lines. Cytotoxicity was not modified by the presence of HA-DOPE. However HA-DOPE increased the level of transfection on CD44-expressing MDA-MB-231 cells compared to the MCF-7 line, which expresses very low levels of CD44. The transfection on the MDA-MB-231 cells was highly inhibited by anti-CD44 Hermes-1 antibody but not by the nonspecific anti-ErbB2 antibody. In conclusion, cationic liposomes containing the HA-DOPE conjugate mediated good transfection on CD44 expressing cell lines in culture.
Assuntos
Neoplasias da Mama/terapia , Sistemas de Liberação de Medicamentos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Fosfatidiletanolaminas/metabolismo , Transfecção , Sobrevivência Celular , Feminino , Técnicas de Transferência de Genes , Humanos , Receptores de Hialuronatos/imunologia , Ácido Hialurônico/química , Lipossomos , Luciferases/metabolismo , Fosfatidiletanolaminas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Cationic hyaluronic acid (HA)-modified DOTAP/DOPE liposomes were designed for the targeted delivery of anti-telomerase siRNA to CD44 receptor-expressing lung cancer cells. DOTAP/DOPE liposomes modified with 1%-20% (w/w) HA-DOPE conjugate were obtained by the ethanol injection method. Their size was below 170 nm and they exhibited zeta potentials higher than +50 mV. Lipoplexes prepared at different +/-ratios with siRNA were in the range of 200 nm and below and their zeta potentials were strongly dependent on the degree of modification and the +/-charge ratio. The presence of HA did not compromise binding, protection of siRNA from degradation, and complex stabilities in serum but rather resulted in an improvement of these properties. Liposome cytotoxicity, investigated by the MTT assay and LDH release after treatment of CD44(+) A549 cells and CD44(-) Calu-3, was demonstrated only at high concentrations. However, the addition of siRNA to HA-modified liposomes prevented cytotoxic effects compared to all other formulations. As shown by flow cytometry, transfection of siRNA into A549 cells was markedly improved with HA-modified liposomes, but not into Calu-3 cells. Using a qPCR-TRAP assay to test telomerase activity, no difference was demonstrated in the efficiency between HA-modified and nonmodified preparations. Moreover, some reduction in telomerase activity was observed with liposomes alone, lipoplexes prepared with nonsense siRNA and lipofectamine, indicative for some direct inhibitory effect of the lipids and siRNA on the expression of this enzyme. HA-modified DOTAP/DOPE liposomes represent a suitable carrier system for siRNA since properties like binding or protection of siRNA are not altered. They display an improved stability in cell culture medium and a reduced cytotoxicity. Furthermore, these novel lipoplexes could successfully be targeted to CD44-expressing A549 cells opening interesting perspectives for the treatment of lung cancer.