RESUMO
PURPOSE: To optimize gene delivery of SLN-based gene vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN gene vectors. METHODS: Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-L-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis. RESULTS: Incorporation of TAT2 into SLN gene vectors induced an up to 100-fold sequence-dependent increase of gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN gene vectors via jet nebulization increased SLN-based gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used. CONCLUSIONS: Incorporation of TAT2 into SLN gene vectors is suitable to optimize gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.
Assuntos
Produtos do Gene tat/química , Vetores Genéticos/química , HIV-1/química , Lipídeos/química , Nanoestruturas , Aerossóis , Animais , Células Cultivadas , Produtos do Gene tat/genética , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Intubação Intratraqueal , Pneumopatias/terapia , Camundongos , Mucosa Respiratória/citologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of cytokines such as interleukin 6 (IL-6), IL-12 and tumour necrosis factor-alpha (TNF-alpha). Results were compared with previously obtained data when peritoneal mouse macrophages were used. SLN produced with stabilizers/surfactants such as poloxamer 188, sodium cholate, Lipoid S75, Tween 80, Poloxamine 908 and sodium dodecylsulfate were shown to be nontoxic towards RAW 264.7 cells. Cytokine production was reduced and stimulation, expressed in elevated cytokine levels, could not be found. Using cetylpyridinium chloride (CPC) as stabilizing surfactant, SLN became cytotoxic in a concentration-dependent manner. Not only were the viabilities reduced but also cytokine production. Cytotoxic effects of CPC stabilized SLN could be antagonized using cytochalasin B to block phagocytosis. D114-SLN produced with pharmaceutically accepted surfactants for intravenous injection (poloxamer 188, Lipoid S75, sodium cholate, Tween 80) were very well tolerated by the cells. Even sodium dodecylsulfate-stabilized D114-SLN did not exert toxic effects. Comparison of the RAW 264.7 data with previously obtained data from toxicity studies of D114-SLN towards peritoneal mouse macrophages showed similar results. This offers the possibility of using the RAW 264.7 cell line for cytotoxicity studies of colloidal drug carrier systems, rather than using laboratory animals as source of macrophages for these kinds of studies.
Assuntos
Citocinas/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Fagocitose , Triglicerídeos/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Lipossomos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanotecnologia , Tensoativos , Testes de Toxicidade/métodos , Triglicerídeos/químicaRESUMO
Cationic solid lipid nanoparticles (SLN) for gene transfer are formulated using the same cationic lipids as for liposomal transfection agents. To investigate the differences and similarities in structure and performance between SLN and liposomes, a SLN preparation (S1), its counterpart formulation without matrix lipid (L1), a commercially available liposomal preparation (DLTR)--all based on the cationic lipid DOTAP--and a liposomal formulation that additionally contained the helper lipid dioleoylphosphatidylethanolamine (DOPE) (Escort) were compared. Photon correlation spectroscopy (PCS) showed that the SLN were smaller in diameter than the corresponding liposomes (88 vs. 148 nm) and atomic force microscopy (AFM) supported the expected structural differences. Desoxy ribonuclein acid (DNA) binding differed only marginally. Surprisingly, reporter gene expression was comparable between all DOTAP based formulations (S1, L1, DLTR), surpassed only by the DOPE containing liposomes (Escort). In conclusion, cationic lipid composition seems to be more dominant for in vitro transfection performance than the kind of colloidal structure it is arranged in. Hence, cationic SLN extend the range of highly potent non-viral transfection agents by one with favourable and distinct technological properties. Further SLN optimisation should be facilitated by the accumulated knowledge about cationic lipids in liposomal formulations.
Assuntos
Coloides/química , Lipídeos/química , Lipossomos/química , Nanoestruturas/química , Transfecção/métodos , Animais , Células COS , Chlorocebus aethiops , DNA/administração & dosagem , Eletroforese em Gel de Ágar , Ácidos Graxos Monoinsaturados/química , Microscopia de Força Atômica , Plasmídeos , Compostos de Amônio Quaternário/química , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
This investigation is focused on the enhancement of in vitro transfection activity by optimizing cationic lipid and matrix lipid composition of solid lipid nanoparticles (SLN). For this purpose SLN were formulated by using two different matrix lipids and six different cationic detergents. These 12 formulations were tested for physical parameters such as particle size, zeta potential and DNA-binding capacity, and also for their biological properties such as cytotoxicity and in vitro transfection efficiency. The SLN were produced by hot high-pressure homogenization, all formulations were physically stable and showed a highly positive surface charge (+34 to +45 mV). In vitro cytotoxicity measurements on COS-1 cells revealed that cytotoxicity is strongly dependent on the cationic lipid used. SLN made from one-tailed cationic detergents were highly cytotoxic. In contrast the two-tailed cationic lipids were all well tolerated. Transfection activity seems to be determined by both the cationic lipid and the matrix lipid used. Here, the combination of cetylpalmitate and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride led to significantly higher transfection efficiencies than in all other tested combinations. These results indicate that well tolerated and highly efficient in vitro transfection could be achieved with SLN whenever selecting good combinations of two-tailed cationic lipids and matrix lipids.
Assuntos
Ácidos Graxos Monoinsaturados/química , Ácidos Graxos Monoinsaturados/farmacologia , Nanotecnologia/métodos , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Transfecção/métodos , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Química Farmacêutica , Chlorocebus aethiops , Lipídeos/química , Lipídeos/farmacologiaRESUMO
A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glycerides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the "gold standard" among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100 nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100 nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow antibody titre development. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.