ABSTRACT
We evaluated the 2G-NN16-carbosilane dendrimer activities in Th17 response as a potential therapy for Th17 deregulated pathologies. IL17A, IL17F, IL22, IL23 and other interleukins secreted by Th17 cells CD4+ cells were down regulated when cells were cultured in the presence of this dendrimer. Furthermore, IL17F and IL17A protein levels in splenocytes from mice pretreated with 2G-NN16 dendrimer in a Th17 induction mouse model were lower than those corresponding to PBS treated mice. Treatment of mice with 2G-NN16 inhibited the Th17 response causing much more pathogenicity as indicated by the increase in the number of Candida albicans colonies in the kidneys as compared to PBS-treated mice. All these results suggest a potential pharmacological application for this dendrimer in the therapy of Th17-mediated diseases.
Subject(s)
Dendrimers/pharmacology , Silanes/pharmacology , Th17 Cells/drug effects , Th17 Cells/immunology , Animals , Candida albicans/drug effects , Candida albicans/immunology , Cell Death/drug effects , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Survival/drug effects , Dendrimers/toxicity , Flow Cytometry , Gene Expression Regulation/drug effects , Humans , Interleukin-17/biosynthesis , Mice , Mice, Inbred BALB C , Receptors, CCR6/metabolism , Silanes/toxicity , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Spleen/microbiology , Th17 Cells/cytology , Th17 Cells/metabolism , Time FactorsABSTRACT
We studied changes in gene expression induced by the carbosilane dendrimer 2G-NN16 to evaluate their potential as a vehicle for gene therapy and as medication. Global gene expression profiles on CD8+ T lymphocytes reveal that ribosomal proteins are induced in the presence of 2G-NN16. IL17A and IL17F, the principal interleukins secreted by Tc17 cells, a subset of CD8+ T lymphocytes, were down-regulated when cultured in the presence of this dendrimer. Microarray results were confirmed by real time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). 2G-NN16 also showed a high potential for in vitro inhibition of Tc17 differentiation of CD8+ T lymphocytes in the presence of the Tc17 differentiation molecules IL6 and TGF-B1. These findings suggest that 2G-NN16 could facilitate drug delivery and may be used to treat inflammatory processes driven by Tc17 cells.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Cell Differentiation/drug effects , Dendrimers/pharmacology , Down-Regulation/drug effects , Silanes/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/chemistry , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Survival/drug effects , Cells, Cultured , Dendrimers/adverse effects , Dendrimers/chemistry , Gene Expression Profiling , Gene Transfer Techniques/adverse effects , Humans , Interleukin-17/genetics , Interleukin-17/metabolism , Nanoparticles/adverse effects , Nanoparticles/chemistry , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Silanes/adverse effects , Silanes/chemistry , Up-Regulation/drug effectsABSTRACT
The ability of dendrimer 2G-[Si{O(CH(2))(2)N(Me)(2) (+)(CH(2))(2)NMe(3) (+)(I(-))(2)}](8) (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection.
Subject(s)
Dendrimers/chemistry , Genetic Therapy , HIV-1/physiology , Transfection , Astrocytes/metabolism , Cell Line, Tumor , Dendrimers/toxicity , Dendritic Cells/immunology , Dendritic Cells/metabolism , Flow Cytometry , HIV Core Protein p24/genetics , HIV Core Protein p24/metabolism , HIV-1/genetics , Humans , Lymphocytes/immunology , Lymphocytes/metabolism , Macrophages/immunology , Macrophages/metabolism , Oligoribonucleotides, Antisense , RNA, Small Interfering , RNA, ViralABSTRACT
PURPOSE: The use of dendrimers for biomedical applications has emerged with promising results. 2G-NN16 is a carbosilane dendrimer with sixteen positive charges per molecule tested to be capable to bind and release antisense oligonucleotides (ODNs) and small interference RNA (siRNA) in vitro. In spite of low cytotoxicity observed for these dendrimers, little is known about cellular changes they produce in cells in general and in immune cells in particular. MATERIALS AND METHODS: Genomic technologies allow us to identify global gene expression profile changes in macrophages exposed to a non-toxic concentration (5 microM) of 2G-NN16, alone or complexed with a random siRNA (dendriplex). Results were confirmed by quantitative real-time RT-PCR. RESULTS: Exposing macrophages to this dendrimer or dendriplex causes multiple gene expression changes, but no specific action of random siRNA was detected. Pathway analysis of differentially expressed genes shows the altered functions to be immune response, proliferation and transcription regulation. Interleukin 17F (IL17F) was the most regulated gene. CONCLUSIONS: Global gene expression profiles are a highly sensitive method to measure the toxicity degree of a gene delivery vehicle. The strong repression of IL17F, IL23R and IL23A, all of which are involved in autoimmune disease, by this particular dendrimer suggests a potential pharmacological application.
