RESUMO
MicroRNA dysregulation often results in the development and progression of cancer. miR-143 is ubiquitously expressed in most human and murine tissues but downregulated in many cancer types. This differential miRNA expression can be utilized for targeted cancer gene therapies. Multiple copies of the miR-143 complementary target sequence were inserted into the 3'UTR of plasmid vectors encoding either for different reporter genes or for the therapeutic gene TNFα. With these transgenes, we analyzed the miR-143-dependent gene expression in cancer cells and normal cells. Moreover, we investigated miR-143-regulated luciferase expression in an NMRI nude/HUH7 xenograft mouse model using a nonviral carrier system for in vivo transfections. We showed low and high levels of miR-143 in cancer cells and normal cells, respectively, leading to a differential gene expression of the reporters and the therapeutic TNFα. According to the miR-143 levels, the luciferase reporter gene expression was silenced in the mouse lungs but not in HUH7 tumors. Thus, we utilized the differential miR-143 expression in healthy and cancerous tissues to de-target the lung by specifically targeting the tumor in an in vivo HUH7 xenograft mouse model. The use of an miR-143-regulated therapeutic transgene may present a promising approach for cancer gene therapy.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Transgenes , Fator de Necrose Tumoral alfa/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Pulmão/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The aim of this study was to identify the causative factors of porcine ear necrosis syndrome (PENS) in 72 pigs, 5.5-10 weeks in age housed on nine farms. Biopsy samples of ear pinnae were collected from all piglets for bacteriology, histopathology and in situ hybridization for porcine circovirus type 2 (PCV2). At the same time, serum samples were taken for serological analysis and viral PCR, and feed was sampled for mycotoxin analysis. The initial lesion of PENS seemed to be a focal epidermal necrosis. Streptococci were isolated from 44 and staphylococci from 36 pinnae. PCV2 could not be detected by in situ hybridization or qPCR. Seven piglets were positive for porcine reproductive and respiratory syndrome virus, and one for Mycoplasma suis. One piglet had antibodies against Sarcoptes scabiei var. suis. No infectious agents were found in 15 samples. Positive virology and parasitology were often found alongside positive bacteriology. Deoxynivalenol, zearalenone and ergot alkaloids were detected in feed. The findings suggest that PENS is multifactorial in origin and that although infectious agents can be involved in the development of the syndrome they are not the exclusive triggering factor.
Assuntos
Otopatias/veterinária , Orelha/patologia , Necrose/veterinária , Doenças dos Suínos/etiologia , Doenças dos Suínos/patologia , Ração Animal/análise , Ração Animal/toxicidade , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Cromatografia Líquida de Alta Pressão/veterinária , Contagem de Colônia Microbiana/veterinária , Orelha/microbiologia , Orelha/parasitologia , Orelha/virologia , Otopatias/epidemiologia , Otopatias/etiologia , Otopatias/patologia , Micotoxinas/análise , Micotoxinas/toxicidade , Necrose/epidemiologia , Necrose/etiologia , Necrose/patologia , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
Aim of this paper was that to prepare biocompatible, polyaspartamide based copolymers containing spermine or spermine/hydrophobic side chains able to condense nucleic acids and to transfect mammalian cells. Copolymers were prepared starting from alpha,beta-poly-(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) and exploiting the reactive hydroxyl groups in the polymeric side chains by subsequent activation reactions to obtain PHEA-Spermine (PHEA-Spm) and PHEA-Spermine-Butyramide (PHEA-Spm-C(4)). Molecular, physico-chemical and biological characterization of copolymers and interpolyelectrolyte complexes with plasmid DNA was performed. Experimental results evidenced that these copolymers are able to form complexes with plasmid DNA already at low polycation/DNA weight ratio ranging from 0.75/1 to 2/1. Interpolyelectrolyte complexes with decreased size were obtained when increasing the polycation/DNA weight ratio, until nanosized dimensions were reached. Copolymers as well as complexes were not haemolytic and non toxic in vitro. In vitro cell transfection with PHEA derivatives showed good biocompatibility and high transfection efficiency (luciferase) in cancer cells in comparison with commercially available, but toxic transfection agents.
Assuntos
Técnicas de Transferência de Genes , Peptídeos/administração & dosagem , Espermina/síntese química , Espermina/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/administração & dosagem , DNA/metabolismo , Adutos de DNA/síntese química , Adutos de DNA/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Humanos , Luciferases/genética , Peptídeos/síntese química , Peptídeos/química , Peptídeos/toxicidade , Espermina/química , Transfecção/métodosRESUMO
Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS(2a)) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS(2a) has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.
Assuntos
DNA/administração & dosagem , Portadores de Fármacos/química , Endossomos , Técnicas de Transferência de Genes , Fármacos Fotossensibilizantes/farmacologia , Polímeros/química , Porfirinas/farmacologia , Linhagem Celular Tumoral , Dextranos/química , Endossomos/efeitos dos fármacos , Endossomos/efeitos da radiação , Corantes Fluorescentes , Humanos , Microscopia de Fluorescência , Fármacos Fotossensibilizantes/efeitos adversos , Porfirinas/efeitos adversos , TransfecçãoRESUMO
Tumour therapy with cyclophosphamide (CPA), an alkylating chemotherapeutic agent, has been associated with reduced tumour blood supply and antiangiogenic effects when applied in a continuous, low-dose metronomic schedule. Compared to conventional high-dose scheduling, metronomic CPA therapy exhibits antitumoural activity with reduced side effects. We have studied potential antiangiogenic properties of acrolein which is released from CPA after hydroxylation. Acrolein adducts were found in tumour cells and tumour endothelial cells of CPA-treated mice, suggesting an in vivo relevance of acrolein. In vitro, acrolein inhibited endothelial cell proliferation, endothelial cell migration and tube formation. Moreover, acrolein caused disassembly of the F-actin cytoskeleton and inhibition of alphavbeta3 integrin clustering at focal adhesions points in endothelial cells. Acrolein treatment modulated expression of thrombospondin-1 (TSP-1), an endogenous inhibitor of angiogenesis known to be linked to antiangiogenic effects of metronomic CPA therapy. Further on, acrolein treatment of primary endothelial cells modified NF-(kappa)B activity levels. This is the first study that points at an antiangiogenic activity of acrolein in metronomically scheduled CPA therapy.
Assuntos
Acroleína/farmacologia , Inibidores da Angiogênese/farmacologia , Ciclofosfamida/administração & dosagem , Ciclofosfamida/farmacologia , Acroleína/análise , Actinas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/patologia , Meios de Cultura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Esquema de Medicação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Integrina alfaVbeta3/metabolismo , Masculino , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Trombospondina 1/metabolismoRESUMO
A novel class of cationic hyperbranched polymers, containing branched oligoethylenimine (OEI 800 Da) as core, diacrylate esters as linkers and oligoamines as surface modification, was synthesized and evaluated regarding their structure-activity relationship as gene carriers. We show that pseudodendritic core characteristics as well as different surface modifications on the core influence DNA-binding ability, cytotoxicity and transfection efficiency. As most promising gene carrier, the pseudodendrimer HD O, that is, the OEI 800 Da core modified with hexane-1,6-diol diacrylate and surface-modified with OEI 800 Da, was identified. HD O exhibits efficient DNA-condensing ability to nanosized polyplexes (100-200 nm), low cytotoxicity, a degradation half-life of 3 days at 37 degrees C at physiological pH and in vitro reporter gene-expression levels similar to high molecular weight linear and branched polyethylenimines (PEIs) (LPEI and BPEI). In vivo studies in mice reveal that HD O/DNA polyplexes upon i.v. tail-vein injection have the potential for transfection of tumor tissue at levels comparable to that obtained with LPEI. Importantly, HD O was better tolerated than LPEI, while transgene expression was more tumor-specific and much lower in all other investigated organs, especially in the lung (15,000-fold lower compared with LPEI).
Assuntos
Dendrímeros/síntese química , Terapia Genética/métodos , Neoplasias/terapia , Transfecção/métodos , Acrilatos , Animais , Aziridinas , Células Cultivadas , Dendrímeros/química , Dendrímeros/farmacocinética , Ésteres , Feminino , Engenharia Genética , Fígado/enzimologia , Luciferases/análise , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Neoplasias/enzimologia , PolímerosRESUMO
Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Efeito Espectador , Hipóxia Celular , Ciclofosfamida/farmacologia , Citocromo P-450 CYP2B1/metabolismo , Pró-Fármacos/farmacologia , Animais , Antineoplásicos Alquilantes/metabolismo , Técnicas de Cultura de Células , Morte Celular/efeitos dos fármacos , Ciclofosfamida/metabolismo , Citocromo P-450 CYP2B1/genética , Difusão , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Humanos , Camundongos , Plasmídeos , Pró-Fármacos/metabolismo , Sefarose , Transfecção , Transgenes , Células Tumorais CultivadasRESUMO
Systemic application of surface-shielded transferrin-polyethylenimine/DNA complexes leads to predominant DNA uptake and gene expression in Neuro2a tumors in syngeneic A/J mice. Similarly, high expression levels were found in Huh-7 and HepG2 human tumor xenografts in SCID mice after systemic application of surface-shielded EGF-PEG-PEI/DNA complexes. Significant DNA uptake but low gene expression were found in the M-3 melanoma while no DNA uptake and no gene expression were found in KB, 518A2, A549, and SW480 xenograft tumor models. To elucidate the reasons for these differences, the tumors were analyzed for vascularization and infiltration of macrophages. Neuro2a, Huh-7, and HepG2 tumors are well vascularized, with a high density of partially immature blood vessels and low numbers of infiltrating macrophages. The M-3 melanoma is well vascularized correlating with significant DNA uptake, however, necrosis and intensive infiltration by macrophages lead to rapid degradation of DNA. In contrast, the KB, 518A2, A549, and SW480 tumors are poorly vascularized, correlating with undetectable DNA uptake and gene expression. Using two different vector systems the data indicate that gene delivery to tumors in vivo is affected by tissue-dependent factors. Uptake of DNA into the tumor depends on vascularization of the tumor, while necrosis and macrophage infiltration may facilitate degradation of the DNA.
Assuntos
Terapia Genética/métodos , Neoplasias/terapia , Transfecção/métodos , Transferrina/genética , Animais , DNA/metabolismo , Fator de Crescimento Epidérmico/genética , Expressão Gênica , Humanos , Injeções Intravenosas , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Camundongos SCID , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica , Polietilenoimina , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor "mediated uptake is less effected by excessive PEGylation.
Assuntos
DNA/química , Técnicas de Transferência de Genes , Polietilenoglicóis/química , Polietilenoimina , DNA/metabolismo , Portadores de Fármacos , Endocitose , Fator de Crescimento Epidérmico/química , Citometria de Fluxo , Genes Reporter , Humanos , Células Jurkat , Células KB , Ligantes , Luciferases/genética , Luciferases/metabolismo , Peso Molecular , Muromonab-CD3/química , Plasmídeos , Polietilenoimina/química , Propriedades de Superfície , Transfecção , Transferrina/químicaRESUMO
Inefficient nuclear delivery restricts transgene expression using polyelectrolyte DNA vectors. To increase transfer from the cytoplasm to the nucleus, we have covalently linked adenovirus hexon protein to polyethylenimine (PEI, 800 kDa). Activity of the conjugate was compared with PEI and PEI linked to albumin. Hexon-containing complexes gave 10-fold greater transgene expression in HepG2 cells than PEI/DNA or complexes containing albumin, without increasing cell uptake. Following cytoplasmic injection into Xenopus laevis oocytes, hexon-containing complexes showed reporter gene expression to be elevated by 10-fold compared with PEI/DNA. The ability of hexon to promote nuclear delivery of PEI/DNA nanoparticles was compared with that of classical nuclear localization sequences (NLS) by measuring transgene expression following intracytoplasmic microinjection of hexon-PEI/DNA complexes and NLS-albumin-PEI/DNA complexes in rat-1 fibroblasts. The resulting nuclear transfer efficiency was in the following order: hexon-PEI/DNA>NLS-albumin-PEI/DNA>PEI/DNA>DNA alone>albumin-PEI/DNA. The activities of both NLS-albumin-PEI and hexon-PEI were abolished by co-injection of wheat germ agglutinin, suggesting that both act by means of the nuclear pore complex (NPC); in contrast, excess free NLS-albumin abolished transgene expression with NLS-albumin-PEI/DNA, but only partially inhibited hexon-PEI/DNA. Nuclear transfer efficiency following cytoplasmic injection was dependent on DNA concentration for all materials, although hexon conjugates showed much better activity than NLS-albumin at low DNA doses (500-1000 plasmids/cell). Our data are consistent with hexon mediating nuclear delivery of plasmid complexes by means of the NPC, using mechanisms that are only partially dependent on the classical NLS import pathway. The hexon-mediated mechanism of nuclear import enables substantially better transgene expression, particularly when DNA concentrations in the cytoplasm are limiting.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Expressão Gênica , Terapia Genética/métodos , Plasmídeos/genética , Polietilenoimina/metabolismo , Transgenes/genética , Transporte Ativo do Núcleo Celular , Animais , Capsídeo/genética , Bovinos , Fibroblastos , Fluoresceína-5-Isotiocianato , Vetores Genéticos/genética , Humanos , Microinjeções , Microscopia Eletrônica , Sinais de Localização Nuclear , Oócitos/citologia , Oócitos/metabolismo , Coelhos , Ratos , Soroalbumina Bovina/análise , Soroalbumina Bovina/genética , Soroalbumina Bovina/metabolismo , Transfecção , Células Tumorais Cultivadas , Xenopus laevisRESUMO
Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI.DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI.DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Meliteno/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Bromodesoxiuridina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Endossomos/metabolismo , Endotélio Vascular/citologia , Eritrócitos/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Genes Reporter , Células HeLa , Humanos , Luciferases/metabolismo , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Mitose , Fótons , Plasmídeos/metabolismo , Polietilenoimina/química , Polietilenoimina/farmacologia , Estrutura Terciária de Proteína , Ratos , Espectrofotometria , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells.
Assuntos
Oligopeptídeos/fisiologia , RNA/metabolismo , Transfecção/métodos , Sequência de Aminoácidos , Animais , Linhagem Celular , Sistema Livre de Células/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Meliteno/química , Meliteno/genética , Microinjeções , Mitose , Dados de Sequência Molecular , Oligopeptídeos/genética , Biossíntese de Proteínas , RNA/genética , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Reticulócitos/química , Reticulócitos/metabolismo , Fatores de Tempo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.
Assuntos
Técnicas de Transferência de Genes , Adesão Celular , DNA/metabolismo , Endocitose , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Células K562 , Poliaminas , Polieletrólitos , Espectrometria de FluorescênciaRESUMO
We investigated the in vitro and in vivo properties of DNA/transferrin-polyethylenimine (800 kDa) complexes before and after covalent coupling of poly(ethylene glycol) (PEG). Upon incubation with plasma, the positively charged non-PEGylated DNA complexes form aggregates. Plasma proteins such as IgM, fibrinogen, fibronectin and complement C3 were found to bind to non-PEGylated DNA complexes. At DNA concentrations relevant for in vivo gene delivery a strong aggregation of erythrocytes was also observed. PEGylation of the complexes strongly reduces plasma protein binding and erythrocyte aggregation. Furthermore, PEGylated complex size was stabilized and had a reduced surface charge. Prolonged circulation in the blood of the PEGylated complexes was also observed when injected intravenously. In tumor bearing mice, application of non-PEGylated complexes through the tail vein resulted in reporter gene expression in tail and lung, but severe toxicity was observed in some mice. In contrast, PEGylated complexes mediated reporter gene transfer to the tumor without significant toxicity.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Neoplasias Experimentais/terapia , Polietilenoglicóis/administração & dosagem , Polietilenoimina , Transfecção/métodos , Animais , Fenômenos Biofísicos , Biofísica , Proteínas Sanguíneas/metabolismo , Linhagem Celular , DNA/metabolismo , Eritrócitos/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/metabolismo , Luciferases/genética , Camundongos , Camundongos Endogâmicos , Transplante de NeoplasiasRESUMO
DNA delivery systems for gene therapy applications have to be able to trigger the uptake of plasmid DNA into the nucleus. We have tested two types of non-viral vector systems, lipofection (cationic lipid-based, using Lipofectamine) and polyfection (cationic polymer-based, using glycerol enhanced transferrinfection), for their ability to transfect confluent, contact inhibited primary human fibroblasts. While both systems worked well with growing fibroblasts, polyfection was superior with confluent cells. A slight reduction in cell associated plasmid DNA was observed with resting cells, but it was similar for both types of complexes. Lipofectamine showed a prevalence for transfecting cycling cells as judged by costaining transfected cells with cell cycle markers. No such bias was observed when glycerol enhanced transferrinfection was used. Microinjection of plasmid DNA/polylysine complexes into the cytoplasm of fibroblasts resulted in a higher percentage of expressing cells than injection of plasmid DNA, offering an explanation for the higher transfection levels obtained with transferrinfection in non-growing cells.
Assuntos
Resinas de Troca de Cátion , Fibroblastos/metabolismo , Lipídeos , Polilisina/análogos & derivados , Transfecção/métodos , Transferrina/análogos & derivados , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Terapia Genética , Humanos , Luciferases , Microinjeções , Plasmídeos , Polilisina/metabolismoRESUMO
BACKGROUND: Efficient and target-specific in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological fluids and extracellular matrix, and binding to a broad variety of non-target cell types. METHODS: Polycation-based vectors, including adenovirus-enhanced transferrinfection (AVET) and transferrin-polyethylenimine (Tf-PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. RESULTS: Gene transfer after intratumoral application was 10-100 fold more efficient with Tf-PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf-PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. CONCLUSION: For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.
Assuntos
Vacinas Anticâncer/administração & dosagem , Terapia Genética , Vacinas de DNA/administração & dosagem , Adenoviridae/genética , Animais , Vacinas Anticâncer/química , Estabilidade de Medicamentos , Eletroquímica , Feminino , Expressão Gênica , Marcação de Genes , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos DBA , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Tamanho da Partícula , Poliaminas , Polieletrólitos , Transfecção , Vacinas de DNA/químicaRESUMO
Dioctadecylamidoglycylspermine (DOGS, Transfectam) is a cationic lipid able to interact with DNA to form complexes that mediate efficient gene transfer into various eukaryotic cells. The state of condensation of the plasmid changes with the medium composition. We therefore investigated to what extent the DNA condensation buffer influences the transfection efficiency of Transfectam/DNA particles. Our results show that in a variety of cell lines, a greater than 100-fold difference in luciferase gene expression is observed with Transfectam/DNA complexes at a +/- charge ratio of 0.75 depending on the conditions of complex formation. The best transfection conditions consisted of particles formed in RPMI medium, NaHCO3/Na2HPO4 or sodium citrate solutions. Mixing in a 150 mM sodium chloride solution (as recommended) resulted in lower gene expression. When the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was present in the DNA/cationic lipid formulation, the increase in reporter activity was also observed, although to a lower extent. Thus, choosing the optimal conditions for formulating DNA/lipid complexes considerably reduces the amount of lipid and DNA needed to obtain maximum gene transfer.
Assuntos
DNA , Glicina/análogos & derivados , Espermina/análogos & derivados , Transfecção , Animais , Linhagem Celular , Expressão Gênica , Humanos , Luciferases/genética , Células Tumorais CultivadasRESUMO
Under physiological salt concentration, plasmid DNA complexed with transferrin-conjugated or unmodified polyethylenimine (PEI, 800 kDa) forms huge (up to > 1000 nm) aggregates, unless the individual components are mixed at a highly positive nitrogen/phosphate (N/P) charge ratio. At low ionic strengths, however, small particles with an average size of 40 nm are formed over a broad range of N/P ratios. Interestingly, in transfection experiments these small particles result in a 10-fold (B16F10 cells) to more than 100-fold (Neuro2A cells, K562 cells) reduced luciferase gene expression efficiency in comparison to the large complexes formed in physiological salt solutions. Limited transport of the small particles to the cell surfaces is one possible reason for this effect. Application of the small particles in more concentrated form and over extended periods of time improves transfection activity. Reduced intracellular release may be another explanation for the decreased transfection efficiency; incubation with chloroquine or incorporation of the endosomolytic peptide INF5 into the small complexes enhances gene expression approximately 10-fold. Analysis of gene expression at the cellular level using a green fluorescence protein reporter gene and flow cytometry revealed that the differences in overall gene expression largely result from different intensities per expressing cell, while the difference in the percentage of expressing cells is less substantial.
Assuntos
Terapia Genética , Vetores Genéticos , Transfecção/métodos , Animais , Linhagem Celular , Cloroquina/farmacologia , DNA , Expressão Gênica/efeitos dos fármacos , Engenharia Genética , Tamanho da Partícula , Polietilenoimina , TransferrinaRESUMO
Receptor-mediated gene transfer is a promising gene delivery technique. It employs a DNA-binding polycation, such as polylysine, to compact plasmid DNA to a size that can be taken up by cells (<100-200 nm). To allow internalization by receptor-mediated endocytosis, cell binding ligands, such as asialoglycoproteins or galactose for hepatocytes, anti-CD3 and anti-CD5 for T-cells, and transferrin, have been covalently attached to polylysine. Intracellular barriers for successful gene transfer include release of DNA complexes from endosomes or lysosomes, nuclear import of DNA complexes, and disassembly of the DNA-polylysine particles. Release of particles from internal vesicles has been achieved by the addition of lysosomotropic agents or glycerol to the transfection medium, or by the incorporation of endosomolytic compounds, such as viruses or membrane active peptides. This technique has already been used to transfect certain organs in vivo, including liver and lung.
RESUMO
Recently the high transfection potential of the cationic polymer polyethylenimine (PEI) was described (Boussif O et al. Proc Natl Acad Sci USA 1995; 92: 7297-7301). To combine the promising DNA delivering activity of PEI with the concept of receptor-mediated gene delivery, cell-binding ligands (transferrin or antiCD3 antibody) were incorporated by covalent linkage to PEI. DNA complexes of PEI or ligand-PEI conjugates were tested for transfection of cultured neuroblastoma Neuro 2A cells, melanoma B16 or H225 cells, erythroid leukemic K562 cells and T cell leukemia Jurkat E6.1 cells. Depending on the cell line, incorporation of the cell-binding ligand resulted in an up to 1000-fold increased transfection efficiency. This activity depends on ligand-receptor interaction and was observed also at low PEI cation:DNA anion ratios where ligand-free PEI lacks efficiency. Depending on the cell-binding ligand, specific targeting (CD3 antibody, Jurkat cells) can be achieved. Gene transfer can be augmented by the addition of an endosome-destabilizing influenza peptide, but is not dependent on the presence of additional endosomolytic agents. Application of transferrin-PEI for the production of murine interleukin-2 in B16 cells resulted in exceptionally high secretion rates of 19 micrograms IL-2 protein per 10(6) cells per 24 h.