ABSTRACT
Cationic liposomes can be designed and developed in order to be an efficient gene delivery system for mammalian cells. Dendritic cell (DC) vaccines can be used to treat cancer, as cationic liposomes can deliver tumor antigens to cells while cells remain active. However, most methods used for liposome production are not able to reproduce in large scale the physicochemical and biological properties of liposomes produced in laboratory scale. In this context, ethanol injection method achieved promising results, although requiring post-treatment for size reduction and/or to remove residual ethanol. Thus, the purpose of this study was to generate cationic liposomes suitable for gene therapies via ethanol injection method in only one step (VEI) and compared to those submitted to a size reduction processes by microfluidization (MFV). For this, the method to produce cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoylphosphatidylethanolamine (DOPE) was optimized using a statistical design approach. As a result, the size of VEI decreased from 290 nm to 110 nm and the polydispersity from 0.54 to 0.17. In the case of MFV, size decreased from 128 nm to 107 nm and polydispersity from 0.40 to 0.18. ST and MFV before and after optimization were also characterized in terms of morphology by transmission electron microscopy (TEM) and structure by differential scanning calorimetry (DSC). Finally, to show their potential in gene/immune therapies applications, DCs were stimulated by such liposomes. Cells internalized liposomes, increasing expression of the costimulatory molecule CD86 and inducing T lymphocyte proliferation.
Subject(s)
Ethanol/chemistry , Gene Transfer Techniques , Liposomes/chemistry , Animals , B7-2 Antigen/metabolism , Cations , Cell Proliferation , Dendritic Cells/immunology , Fatty Acids, Monounsaturated/chemistry , Genetic Therapy , Humans , Immunotherapy , Particle Size , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Quaternary Ammonium Compounds/chemistry , Surface Properties , T-Lymphocytes/cytologyABSTRACT
Immunotherapy of cancer aims to harness the immune system to detect and destroy cancer cells. To induce an immune response against cancer, activated dendritic cells (DCs) must present tumor antigens to T lymphocytes of patients. However, cancer patients' DCs are frequently defective, therefore, they are prone to induce rather tolerance than immune responses. In this context, loading tumor antigens into DCs and, at the same time, activating these cells, is a tempting goal within the field. Thus, we investigated the effects of cationic liposomes on the DCs differentiation/maturation, evaluating their surface phenotype and ability to stimulate T lymphocytes proliferation in vitro. The cationic liposomes composed by egg phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium propane and 1,2-dioleoylphosphatidylethanolamine (50/25/25% molar) were prepared by the thin film method followed by extrusion (65 nm, polydispersity of 0.13) and by the dehydration-rehydration method (95% of the population 107 nm, polydispersity of 0.52). The phenotypic analysis of dendritic cells and the analysis of T lymphocyte proliferation were performed by flow cytometry and showed that both cationic liposomes were incorporated and activated dendritic cells. Extruded liposomes were better incorporated and induced higher CD86 expression for dendritic cells than dehydrated-rehydrated vesicles. Furthermore, dendritic cells which internalized extruded liposomes also provided stronger T lymphocyte stimulation. Thus, cationic liposomes with a smaller size and polydispersity seem to be better incorporated by dendritic cells. Hence, these cationic liposomes could be used as a potential tool in further cancer immunotherapy strategies and contribute to new strategies in immunotherapy.
