RESUMO
Polycationic polymers like poly(ethylene imine)s (PEIs) are extensively explored for the nonviral transfer of DNA or small RNAs (siRNAs). To enhance biocompatibility and alter pharmacokinetic properties, hyperbranched PEI was recently grafted with the nonligand oligosaccharides maltose or maltotriose at various degrees in a systematic study to yield (oligo-)maltose PEIs (OM-PEIs). In this paper, we investigate the in vivo biocompatibility and efficacy of a whole set of (OM-)PEIs and the corresponding (OM-)PEI-based DNA or siRNA complexes upon systemic (intravenous, i.v.) administration in mice. We determine the overall survival and animal welfare, hepatotoxicity, immune stimulation, erythrocyte aggregation, and the efficacy of DNA delivery in vivo. Higher-degree oligomaltose-grafting of PEI substantially decreases weight loss, abolishes lethality upon repeated treatment with the free polymers or with complexes, and abrogates hepatotoxicity, as determined by serum levels of liver enzymes. Immunostimulatory effects (TNF-α, IFN-γ) and erythrocyte aggregation are mainly observed upon treatment with partially maltotriose-grafted PEI or PEI-based complexes and are largely abolished upon higher-degree grafting. In vivo transfection experiments in mice bearing subcutaneous (s.c.) tumor xenografts reveal a strong dependence of reporter gene expression in a given organ on the mode of complex administration (i.v. vs intraperitoneal injection) and the OM-PEI architecture, with high-level maltose-grafted PEI (PEI-(2-Mal)) being most efficient for DNA delivery. We conclude that distinct differences between different patterns of maltose- or maltotriose-grafting are observed with regard to both biocompatibility and in vivo efficacy and identify optimal oligomaltose-PEIs for therapeutic applications.
Assuntos
Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , DNA/administração & dosagem , DNA/genética , Iminas/administração & dosagem , Iminas/química , Maltose/administração & dosagem , Maltose/química , Polietilenos/administração & dosagem , Polietilenos/química , Animais , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Melanoma Experimental , Camundongos , Polímeros/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Taxa de Sobrevida , Transfecção/métodos , Trissacarídeos/química , Redução de PesoRESUMO
Overexpression of members of the HER/erbB transmembrane tyrosine kinase family like HER2/erbB2/neu is associated with various cancers. Some heterodimers, especially HER2/HER3 heterodimers, are particularly potent inducers of oncogenic signaling. Still, from a clinical viewpoint their inhibition has yielded only moderate success so far, despite promising data from cell cultures. This suggests acquired resistance upon inhibitor therapy as one putative issue, requiring further studies in cell culture also aiming at rational combination therapies. In this paper, we demonstrate in ovarian carcinoma cells that the RNAi-mediated single knockdown of HER2 or HER3 leads to the rapid counter-upregulation of the respective other HER family member, thus providing a rational basis for combinatorial inhibition. Concomitantly, combined knockdown of HER2/HER3 exerts stronger anti-tumor effects as compared to single inhibition. In a tumor cell line xenograft mouse model, therapeutic intervention with nanoscale complexes based on polyethylenimine (PEI) for siRNA delivery, again reveals HER3 upregulation upon HER2 single knockdown and a therapeutic benefit from combination therapy. On the mechanistic side, we demonstrate that HER2 knockdown or inhibition reduces miR-143 levels with subsequent de-repression of HER3 expression, and validates HER3 as a direct target of miR-143. HER3 knockdown or inhibition, in turn, increases HER2 expression through the upregulation of the transcriptional regulator SATB1. These counter-upregulation processes of HER family members are thus based on distinct molecular mechanisms and may provide the basis for the rational combination of inhibitors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Regulação para Cima , Animais , Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA interference (RNAi) is a naturally occurring, powerful mechanism for gene silencing, based on the cleavage of a given target mRNA. It relies on small interfering RNAs (siRNAs) in the cell. Being similar in structure, microRNAs (miRNAs) are important regulators of gene expression which mainly act by blocking mRNA translation. In cancer, certain miRNAs have been found to be pathologically downregulated. The therapeutic application of siRNAs or miRNAs for the induction of RNAi or miRNA replacement, respectively, relies on their efficient delivery through a non-viral formulation. Complexation of siRNAs/miRNAs in polymeric nanoparticles based on polyethylenimines (PEIs) offers protection against degradation, delivery to the target site, cellular uptake, and intracellular release. This chapter provides protocols for therapeutic gene silencing and miRNA replacement therapy, based on PEI complexes for in vitro and in vivo use.
Assuntos
Sistemas de Liberação de Medicamentos , MicroRNAs/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Nanopartículas/administração & dosagem , Neoplasias Ovarianas/patologia , Polímeros/administração & dosagem , Polímeros/química , RNA Mensageiro/genética , RNA Interferente Pequeno/genéticaRESUMO
RNA interference (RNAi) is a promising strategy to inhibit the expression of pathologically relevant genes, which show aberrant (over-)expression, e.g. in tumors or other pathologies. The induction of RNAi relies on small interfering RNAs (siRNAs), which trigger the specific mRNA degradation. Their instability and poor delivery into target tissues including the lung, however, so far severely limits the therapeutic use of siRNAs and requires the development of nanoscale delivery systems. Polyethylenimines (PEIs) are synthetic polymers, which are able to form non-covalent complexes with siRNAs. These nanoscale complexes ('nanoplexes') allow the protection of siRNAs from nucleolytic degradation, their efficient cellular uptake through endocytosis and intracellular release through the 'proton sponge effect'. Chemical modifications of PEIs as well as the coupling of cell/tissue-specific ligands are promising approaches to increase the biocompatibility, specificity and efficacy of PEI-based nanoparticles. This review article gives a comprehensive overview of pre-clinical in vivo studies on the PEI-mediated delivery of therapeutic siRNAs in various animal models. It discusses the chemical properties of PEIs and PEI modifications, and their influences on siRNA knockdown efficacy, on adverse effects of the polymer or the nanoplex and on siRNA biodistribution in vivo. Beyond systemic application, PEI-based complexation allows the local siRNA application to the lung. Biodistribution studies demonstrate cellular uptake of PEI-complexed, but not of naked siRNAs in the lung with little systemic availability of the siRNAs, indicating the usefulness of this approach for the targeting of genes, which are pathologically relevant in lung tumors or lung metastases. Taken together, (i) PEI and PEI derivatives may represent an efficient delivery platform for siRNAs, (ii) siRNA-mediated induction of RNAi is a promising approach for the knockdown of pathologically relevant genes, and (iii) when sufficiently addressing biocompatibility issues, the locoregional delivery of PEI/siRNA complexes may become an attractive therapeutic strategy for the treatment of lung diseases with little systemic side effects.
Assuntos
Portadores de Fármacos/química , Marcação de Genes/métodos , Pulmão/metabolismo , Polietilenoimina/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Humanos , Pulmão/patologia , Nanopartículas , RNA Interferente Pequeno/genéticaRESUMO
PURPOSE: While the benefits of ascorbic acid (vitamin C, ascorbate) as an essential nutrient are well established, its effects on tumor cells and in tumor treatment are controversial. In particular, conflicting data exist whether ascorbate may increase the cytotoxic effects of antineoplastic drugs or may rather exert adverse effects on drug sensitivity during cancer treatment. Findings are further obscured regarding the distinction between ascorbate and dehydroascorbate (DHA). Thus, the purpose of this study was to evaluate and directly compare the cytotoxic efficacy of ascorbate compared to DHA, and to analyse if ascorbate at pharmacological concentrations affects the efficacy of antineoplastic agents in prostate carcinoma cells. METHODS: We directly compare the effects of ascorbate (supplied as 'Pascorbin solution for injection') and DHA on tumor cell viability, and determine IC(50) values for various cell lines. At concentrations well below the IC(50), ascorbate effects on cell proliferation and cell cycle are analysed. We furthermore determine changes in cellular sensitivity towards various cytostatic drugs upon pre-treatment of cells with ascorbate. RESULTS: We demonstrate higher therapeutic efficacy of ascorbate over DHA in various cell lines, independent of cell line-specific differences in ascorbate sensitivity, and identify the extracellular generation of H(2)O(2) as critical mechanism of ascorbate action. We furthermore show that, in addition to pro-apoptotic effects described previously, ascorbate treatment already at concentrations well below the IC(50) exerts anti-proliferative effects on tumor cells. Those are based on interference with the cell cycle, namely by inducing a G(0)/G(1) arrest. Pre-treatment of tumor cells with ascorbate leads to increased cellular sensitivity towards Docetaxel, Epirubicin, Irinotecan and 5-FU, but not towards Oxaliplatin and Vinorelbin. For Docetaxel and 5-FU, a linear correlation between this sensitizing effect and the ascorbate dosage is observed. CONCLUSIONS: The redox-active form of vitamin C, ascorbate, shows therapeutic efficacy in tumor cells. These antitumor effects of ascorbate are mainly based on its extracellular action and, in addition to the induction of apoptosis, also include an anti-proliferative effect by inducing cell cycle arrest. Furthermore, ascorbate treatment specifically enhances the cytostatic potency of certain chemotherapeutics, which implicates therapeutic benefit during tumor treatment.