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1.
Eur J Pharm Biopharm ; 195: 114077, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37579889

RESUMO

RNA therapeutics represents a powerful strategy for diseases where other approaches have failed, especially given the recent successes of mRNA vaccines against the coronavirus disease 2019 (COVID-19) and small interfering (siRNA) therapeutics. However, further developments are still required to reduce toxicity, improve stability and biodistribution of mRNA-LNPs (lipid nanoparticles). Here, we show a rational combinatorial approach to select the best formulation based on a new cationic lipid molecule (IM21.7c), which includes an imidazolium polar head. The study allowed us to select the optimal 5 lipids composition for in vivo mRNA delivery. IM21.7c based mRNA-LNPs measuring less than 100 nm had high encapsulation efficiency, protected mRNA from degradation, and exhibited sustained release kinetics for effective in vitro transfection. Most interestingly the biodistribution was significantly different from other clinically approved LNPs, with increased targeting to the lung. Further studies are now required to expand the possible applications of these new molecules.


Assuntos
Lipídeos , Nanopartículas , Distribuição Tecidual , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transfecção , Cátions
2.
BMC Cancer ; 13: 338, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23835136

RESUMO

BACKGROUND: Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference. Survivin and cyclin B1, both involved in cell survival and proliferation and frequently deregulated in human cancers, are good candidate target genes for siRNA mediated therapeutics. METHODS: We used our newly developed sticky siRNA-based technology delivered with linear polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor development. RESULTS: We confirm that survivin and cyclin B1 downregulation through a RNA interference mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10 cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10 tumors as well as formation and dissemination of melanoma lung metastases. In addition, we highlight that inhibition of survivin expression increases the effect of doxorubicin on lung B16-F10 metastasis growth inhibition. CONCLUSION: PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs, represents a promising strategy for melanoma treatment.


Assuntos
Ciclina B1/metabolismo , Terapia Genética/métodos , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/secundário , Melanoma Experimental/metabolismo , RNA Interferente Pequeno/genética , Proteínas Repressoras/metabolismo , Animais , Western Blotting , Ciclina B1/genética , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Técnicas de Transferência de Genes , Proteínas Inibidoras de Apoptose/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/secundário , Camundongos , Camundongos Nus , Polietilenoimina , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Control Release ; 170(2): 183-90, 2013 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-23727288

RESUMO

RNA interference allows the design of new inhibitors that target deregulated pathways in cancer. However systemic delivery of siRNA for the treatment of solid tumors still remains an issue. In our study, in order to suppress the progression of lung cancer metastasis in mice, we developed sticky siRNA (ssiRNA) to inhibit survivin and cyclin B1, two candidates involved in cell survival and proliferation. We exploited the linear polyethylenimine (PEI) as potent non-viral carrier to efficiently deliver our inhibitors. As a proof of concept, we have chosen a very aggressive mammary adenocarcinoma model (TSA-Luc cells), which forms lung metastases upon systemic cell injection. We confirmed in vitro, that the ssiRNAs delivered with PEI are not only able to inhibit our target genes at the mRNA and protein levels, but are also able to block the cell cycle and cell proliferation through a mechanism of RNA interference. More importantly, we showed in vivo by luciferase dosage, bioimaging and tissue section, an inhibition of lung tumor metastases after systemic delivery of cyclin B1 and survivin ssiRNA complexed with PEI. Alternating treatment with cisplatin and ssiRNA/PEI showed an additive effect between the two anticancer drugs on lung tumor inhibition leading to a significant increase in animal survival. Moreover a promising window between activity (IC50) and toxicity (LD50), essential for therapeutic application, was observed. Our data show that systemic delivery of ssiRNA/PEI complexes targeting the cell cycle is a valuable strategy for the treatment of lung tumor metastasis and that it can be combined with chemotherapy.


Assuntos
Adenocarcinoma/terapia , Ciclina B1/genética , Proteínas Inibidoras de Apoptose/genética , Neoplasias Pulmonares/terapia , Neoplasias Mamárias Experimentais/terapia , RNA Interferente Pequeno/administração & dosagem , Proteínas Repressoras/genética , Adenocarcinoma/patologia , Animais , Antineoplásicos/uso terapêutico , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/uso terapêutico , Feminino , Dose Letal Mediana , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Polietilenoimina/química , RNA Interferente Pequeno/genética , Survivina
4.
Mol Ther Nucleic Acids ; 2: e89, 2013 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-23612115

RESUMO

RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ). This brain region contains the neural stem and progenitor cells populations that express the stem cell marker, SOX2. Temporally and spatially controlled Sox2 knockdown was achieved using the monocationic lipid vector, IC10. siRNA/IC10 complexes were stable over time and smaller (<40 nm) than jetSi complexes (≈400 nm). Immunocytochemistry showed that siRNA/IC10 complexes efficiently target both the progenitor and stem cell populations in the adult SVZ. Injection of the complexes into the lateral brain ventricle resulted in specific knockdown of Sox2 in the SVZ. Furthermore, IC10-mediated transient in vivo knockdown of Sox2-modulated expression of several genes implicated in NSC maintenance. Taken together, these data show that IC10 cationic lipid formulation can efficiently vectorize siRNA in a specific area of the adult mouse brain, achieving spatially and temporally defined loss of function.Molecular Therapy-Nucleic Acids (2013) 2, e89; doi:10.1038/mtna.2013.8; published online 23 April 2013.

5.
Mol Pharm ; 9(12): 3464-75, 2012 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-23148419

RESUMO

siRNAs are usually formulated with cationic polymers or lipids to form supramolecular particles capable of binding and crossing the negatively charged cell membrane. However, particles hardly diffuse through tissues when administered in vivo. We therefore are developing cationic siRNAs, composed of an antisense sequence annealed to an oligophosphospermine-conjugated sense strand. Cationic siRNAs have been previously shown to display gene silencing activity in human cell line (Nothisen et al. J. Am. Chem. Soc.2009). We have improved the synthesis, purification and characterization of oligospermine-oligoribonucleotide conjugates which provide cationic siRNAs with enhanced biological activity. We show data supporting their carrier-free intracellular delivery in a molecular, soluble state. Additional results on the relationship between global charge, uptake and silencing activity confirm the requirement for an overall positive charge of the conjugated siRNA in order to enter cells. Importantly, conjugated siRNAs made of natural phosphodiester nucleotides are protected from nuclease degradation by the oligophosphospermine moiety, operate through the RNAi mechanism and mediate specific gene silencing at submicromolar concentration in the presence of serum.


Assuntos
Sistemas de Liberação de Medicamentos , Inativação Gênica , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Espermina/metabolismo , Animais , Western Blotting , Citometria de Fluxo , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Luciferases/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espermina/química , Survivina , Células Tumorais Cultivadas
6.
Angew Chem Int Ed Engl ; 51(34): 8478-84, 2012 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-22829421

RESUMO

An amphiphilic dendrimer bearing a hydrophobic alkyl chain and hydrophilic poly(amidoamine) dendrons is able to combine the advantageous features of lipid and dendrimer vectors to deliver a heat shock protein 27 siRNA and produce potent gene silencing and anticancer activity in vitro and in vivo in a prostate cancer model. This dendrimer can be used alternatively for treating various diseases.


Assuntos
Dendrímeros/administração & dosagem , Dendrímeros/química , Inativação Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Dendrímeros/síntese química , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Choque Térmico HSP27/genética , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , RNA Interferente Pequeno/genética
7.
Bioconjug Chem ; 22(12): 2461-73, 2011 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-22053932

RESUMO

With the aim of developing dendrimer nanovectors with a precisely controlled architecture and flexible structure for DNA transfection, we designed PAMAM dendrimers bearing a triethanolamine (TEA) core, with branching units pointing away from the center to create void spaces, reduce steric congestion, and increase water accessibility for the benefit of DNA delivery. These dendrimers are shown to form stable nanoparticles with DNA, promote cell uptake mainly via macropinocytosis, and act as effective nanovectors for DNA transfection in vitro on epithelial and fibroblast cells and, most importantly, in vivo in the mouse thymus, an exceedingly challenging organ for immune gene therapy. Collectively, these results validate our rational design approach of structurally flexible dendrimers with a chemically defined structure as effective nanovectors for gene delivery, and demonstrate the potential of these dendrimers in intrathymus gene delivery for future applications in immune gene therapy.


Assuntos
DNA/administração & dosagem , Dendrímeros/química , Etanolaminas/química , Timo/metabolismo , Transfecção , Animais , DNA/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL
8.
Biotechnol Bioeng ; 108(10): 2477-87, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21520021

RESUMO

Peptides, highly diverse by their nature, are important biochemical and pharmaceutical tools: ligands for cellular receptors, transcription factors, immunosuppressants, vaccines, etc. As the majority of their targets are intracellular, peptides need to cross the plasma membrane and gain access to the cytoplasm. However, due to their physicochemical properties, most peptides need to be entrapped by a molecular vehicle to be able to reach the cytosol compartment. In this study, we present new biological tools to enhance intracellular peptides delivery. Based on electrostatic interactions, two complementary types of amphiphilic molecules have been designed as delivery vehicles. A diverse set of fluorescently labeled peptides have successfully been delivered. This opens the avenue for the use of peptides combined to delivery vehicles as therapeutic aids.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Peptídeos/química , Células HeLa , Humanos
9.
Nucleic Acids Res ; 38(7): e95, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20071749

RESUMO

Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3' end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes.


Assuntos
Sondas de Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Pareamento Incorreto de Bases , Primers do DNA , Hidrólise , Desnaturação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Espermina/química
10.
Hum Gene Ther ; 21(7): 795-805, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19947826

RESUMO

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.


Assuntos
Adenoviridae/genética , Terapia Genética , Vetores Genéticos/genética , Neoplasias da Próstata/terapia , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Masculino
11.
Hum Gene Ther ; 21(7): 807-13, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20001452

RESUMO

Prostate cancer is at present the most common malignancy in men in the Western world. When localized to the prostate, this disease can be treated by curative therapy such as surgery and radiotherapy. However, a substantial number of patients experience a recurrence, resulting in spreading of tumor cells to other parts of the body. In this advanced stage of the disease only palliative treatment is available. Therefore, there is a clear clinical need for new treatment modalities that can, on the one hand, enhance the cure rate of primary therapy for localized prostate cancer and, on the other hand, improve the treatment of metastasized disease. Gene therapy is now being explored in the clinic as a treatment option for the various stages of prostate cancer. Current clinical experiences are based predominantly on trials with adenoviral vectors. As the first of a trilogy of reviews on the state of the art and future prospects of gene therapy in prostate cancer, this review focuses on the clinical experiences and progress of adenovirus-mediated gene therapy for this disease.


Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Neoplasias da Próstata/terapia , Terapia Genética/tendências , Humanos , Masculino , Estadiamento de Neoplasias , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/cirurgia , Resultado do Tratamento
12.
J Am Chem Soc ; 131(37): 13338-46, 2009 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-19715309

RESUMO

The preparation of chloride (1(n)) and bromide (2(n)) derivatives of 1-methyl-3-[3,4-bis(alkoxy)benzyl]-4H-imidazolium with n = 6, 12, 16, 18 is described. The two series of salts possess a rich thermotropic mesomorphism, chain-length dependent. Thus, a lamellar smectic A phase, a bicontinuous cubic Ia3d phase, and a columnar hexagonal liquid crystalline mesophase are induced as a function of increasing chain length. The mesomorphic properties were studied by polarizing optical microscopy, differential scanning calorimetry, and X-ray diffraction, and with the support of dilatometry and molecular dynamics, models for the various supramolecular arrangements of the salts are proposed. Such cationic amphiphiles were expected to be candidate molecules to design a new delivery reagent for nucleic acid transfection, particularly for short interfering RNA (siRNA). The use of an RNA interference mechanism, by introduction into cells by transfection of chemically synthesized siRNAs, is a powerful method for gene silencing studies. To exploit the potential of these amphilic imidazolium salts, these molecules were formulated with cohelper lipids and tested for their efficacy to deliver active siRNAs. Our results show high transfection efficacy of our formulated compounds and high silencing efficiency with more than 80% inhibition of the targeted gene at 10 nM siRNA concentration. Taken together our results show the potency of amphiphilic imidazolium salts as a new generation of transfection reagents for RNA interference.


Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Imidazóis/química , Imidazóis/metabolismo , RNA Interferente Pequeno/metabolismo , Sais/química , Linhagem Celular , Portadores de Fármacos/síntese química , Humanos , Imidazóis/síntese química , Indicadores e Reagentes/síntese química , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Cristais Líquidos/química , Modelos Moleculares , Conformação Molecular , Transfecção
13.
Nucleic Acids Res ; 37(19): e130, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19696078

RESUMO

Most nucleic acid-based technologies rely upon sequence recognition between an oligonucleotide and its nucleic acid target. With the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, novel modified oligonucleotides named Zip nucleic acids (ZNAs) were recently developed. ZNAs are oligonucleotide-oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotide. It was demonstrated that the melting temperature of a hybridized ZNA is easily predictable and increases linearly with the length of the oligocation. Furthermore, ZNAs retain the ability to discriminate between a perfect match and a single base-pair-mismatched complementary sequence. Using quantitative PCR, we show here that ZNAs are specific and efficient primers displaying an outstanding affinity toward their genomic target. ZNAs are particularly efficient at low magnesium concentration, low primer concentrations and high annealing temperatures, allowing to improve the amplification in AT-rich sequences and potentially multiplex PCR applications. In reverse transcription experiments, ZNA gene-specific primers improve the yield of cDNA synthesis, thus increasing the accuracy of detection, especially for genes expressed at low levels. Our data suggest that ZNAs exhibit faster binding kinetics than standard and locked nucleic acid-containing primers, which could explain why their target recognition is better for rare targets.


Assuntos
Primers do DNA , Reação em Cadeia da Polimerase , Transcrição Reversa , Sequência Rica em At , Reação em Cadeia da Polimerase/normas
14.
Cytotechnology ; 56(1): 41-8, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19002840

RESUMO

Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery.

15.
Pharm Res ; 25(12): 2972-82, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18709489

RESUMO

PURPOSE: The success of nucleic acid therapies depends upon delivery vehicle's ability to selectively and efficiently deliver therapeutic nucleic acids to target organ with minimal toxicity. The cationic polymer polyethylenimine (PEI) has been widely used for nucleic acid delivery due to its versatility and efficiency. In particular, the last generation of linear PEI (L-PEI) is being more efficient in vivo than the first generation of branched PEI. This led to several clinical trials including phase II bladder cancer therapy and human immunodeficiency virus immunotherapy. When moving towards to the clinic, it is crucial to identify potential side-effects induced by the delivery vehicle. MATERIALS AND METHODS: For this purpose we have analyzed the production of pro-inflammatory cytokines [tumor necrosis factor-alpha, interferon (IFN)-gamma, interleukin (IL)-6, IL-12/IL-23, IFN-beta and IL-1beta] and hepatic enzyme levels (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase) in the blood serum of mice after systemic injection of DNA or siRNAs delivered with L-PEI. RESULTS: Our data show no major production of pro-inflammatory cytokines or hepatic enzymes after injection of DNA or oligonucleotides active for RNA interference (siRNAs or sticky siRNAs) complexed with L-PEI. Only a slight induction of IFN-gamma was measured after DNA delivery, which is probably induced by the CpG mediated response. CONCLUSION: Taken together our data highlight that linear polyethylenimine is a delivery reagent of choice for nucleic acid therapeutics.


Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes/efeitos adversos , Inflamação/induzido quimicamente , Polietilenoimina/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Citocinas/biossíntese , Feminino , Camundongos , Polietilenoimina/toxicidade , RNA Interferente Pequeno/toxicidade
16.
Biotechniques ; 44(7): Pvii-Pxi, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18540862

RESUMO

The ability to introduce antibodies to live cells opens new insights to a wide range of applications, such as protein intracellular trafficking studies, protein interference studies with blocking antibodies, and live immunolabeling or protein phosphorylation states studies. Apart from single-chain format variable (scFv) antibodies, DNA transfection of eukaryotic cells is rarely used to produce antibodies in situ, mainly due to inappropriate folding of the antibody in the cytoplasm. Thus, the development of dedicated carriers is needed since antibodies, which are large, unable to cross the plasma membrane and effective release of the antibody in the cytoplasm need to be overcome. We studied these two crucial steps using a dedicated delivery reagent in live cells and compared the results with immunocytochemistry experiments in fixed cells.


Assuntos
Anticorpos/administração & dosagem , Citoplasma/metabolismo , Lipídeos/administração & dosagem , Anticorpos/metabolismo , Portadores de Fármacos , Células HeLa , Humanos , Imuno-Histoquímica
17.
Proc Natl Acad Sci U S A ; 104(41): 16050-5, 2007 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-17913877

RESUMO

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery. We show here that making siRNAs "gene-like," via short complementary A(5-8)/T(5-8) 3' overhangs, increases complex stability, and hence RNase protection and gene silencing in vitro up to 10-fold. After decomplexation in the cytoplasm, sticky siRNA (ssiRNA) concatemers fall apart. ssiRNAs are therefore not inducing antiviral responses, as shown by the absence of IFN-beta production. Finally, transfection experiments in the mouse lung show that ssiRNA should be particularly suited to silencing with linear polyethylenimine in vivo.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Animais , Sequência de Bases , Linhagem Celular , Feminino , Corantes Fluorescentes , Genes Reporter , Técnicas Genéticas , Vetores Genéticos , Glicosaminoglicanos , Células HeLa , Humanos , Lipídeos , Luciferases/genética , Pulmão/metabolismo , Camundongos , Células NIH 3T3 , Polietilenoimina , RNA Interferente Pequeno/administração & dosagem , Ribonucleases , Transfecção
18.
J Control Release ; 114(3): 389-97, 2006 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-16887230

RESUMO

This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe. A precise pump allowed their passage through the chamber under a range of shear stresses. The binding of polyethyleneimine (PEI)- and histidylated polylysine (His)-polyplexes was carried out over 30 min by time-lapse video microscopy. At 10 microg pDNA/ml in 10% serum, we found that 360+/-80 PEI- and 250+/-50 His-polyplexes were bound per 1000 cells at a shear stress of 0.3-1 dyn/cm(2). This number dropped to approximately 100 at 2 dyn/cm(2). These polyplexes exhibited differences in their interactions with the cell membrane. Concerning PEI-polyplexes, there was a shear threshold effect allowing a maximum binding at 0.06 dyn/cm(2) and a higher binding reduction (77%) at 5 microg/ml pDNA in 100% serum. The polyplex binding was augmented by 300% with PEI bearing tetraglucose moiety. This set-up is potentially helpful to screen a wide array of endothelial cells ligands prior in vivo experiments.


Assuntos
DNA/administração & dosagem , Células Endoteliais/metabolismo , Técnicas de Transferência de Genes , Polímeros/administração & dosagem , Células Cultivadas , DNA/metabolismo , Humanos , Tamanho da Partícula , Plasmídeos
20.
Mol Ther ; 13(6): 1156-62, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16564229

RESUMO

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.


Assuntos
Carcinoma de Células Escamosas/terapia , Terapia Genética/métodos , Neoplasias de Cabeça e Pescoço/terapia , Mutação , Fotoquimioterapia/métodos , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma de Células Escamosas/genética , Caspase 3 , Caspases/metabolismo , Terapia Combinada , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Neoplasias de Cabeça e Pescoço/genética , Humanos , Luciferases/genética , Camundongos , Fármacos Fotossensibilizantes/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Polietilenoimina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
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