RESUMO
Photodynamic therapy (PDT) is an established anticancer treatment that combines the use of a photosensitiser (PS) and a light source of a specific wavelength for the generation of reactive oxygen species (ROS) that are toxic to the tumour cells. Foscan® (mTHPC) is a clinically-approved chlorin used for the PDT treatment of advanced head and neck, prostate and pancreatic cancers but is characterized by being photochemically unstable and associated with prolonged skin photosensitivity. Herein, we report the synthesis of new 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins, having the meso-tetra(3-hydroxyphenyl)macrocycle core of mTHPC, by exploring the [8πâ¯+â¯2π] cycloaddition of a meso-tetra(3-hydroxyphenyl)porphyrin derivative with diazafulvenium methides. These chlorins have photochemical properties similar to Foscan® but are much more photostable. Among the novel compounds, two chlorins with a hydroxymethyl group and its azide derivative present in the 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused system, are promising photodynamic agents with activity in the 100â¯nM range against triple-negative breast cancer cells and, in the case of azidomethyl chlorin, a safer phototherapeutic index compared to Foscan®.
Assuntos
Neoplasias Pancreáticas , Fotoquimioterapia , Porfirinas , Masculino , Humanos , Porfirinas/farmacologia , PiridinasAssuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Doenças do Sistema Nervoso Central/tratamento farmacológico , Excipientes/farmacologia , Neuropatias Amiloides Familiares/complicações , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/patologia , Benzoxazóis/química , Benzoxazóis/farmacologia , Doenças do Sistema Nervoso Central/complicações , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/patologia , Excipientes/química , Humanos , Mutação , Placa Amiloide/tratamento farmacológico , Placa Amiloide/genética , Placa Amiloide/patologia , Pré-Albumina/genéticaRESUMO
BACKGROUND: Site-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). METHODS: The Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. RESULTS: The FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. CONCLUSIONS: The prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. GENERAL SIGNIFICANCE: This study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.
Assuntos
Corantes Fluorescentes/química , Magnetismo , Nanopartículas/química , Receptores da Transferrina/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Imageamento por Ressonância Magnética , Pontos Quânticos , Espectrometria de Fluorescência , Transferrina/químicaRESUMO
Glioblastoma (GBM) is a deadly and therapy resistant malignant brain tumour, characterized by an aggressive and diffuse growth pattern, which prevents complete surgical resection. Despite advances in the identification of genomic and molecular alterations that fuel the tumour, average patient survival post-diagnosis remains very low (â¼14.6-months). In addition to being highly heterogeneous, GBM tumour cells exhibit high adaptive capacity to targeted molecular therapies owing to an established network of signalling cascades with functional redundancy, which provides them with robust compensatory survival mechanisms. Here, we investigated whether a multimodal strategy combining multitargeted tyrosine kinase inhibitors (MTKIs) and microRNA (miRNA) modulation could overcome the signalling pathway redundancy in GBM and, hence, promote tumour cell death. By performing a high-throughput screening, we identified a myriad of miRNAs, including those belonging to the miR-302-3p/372-3p/373-3p/520-3p family, which coordinately act with the MTKI sunitinib to decrease GBM cell viability. Two members of this family, hsa-miRNA-302a-3p and hsa-miRNA-520 b, were found to modulate the expression of receptor tyrosine kinase mediators (including AKT1, PIK3CA and SOS1) in U87 and DBTRG human GBM cells. Importantly, administration of mimics of these miRNAs with sunitinib or axitinib resulted in decreased tumour cell proliferation and enhanced cell death, whereas no significant effect was observed when coupling miRNA modulation with temozolomide, the first-line drug for GBM therapy. Overall, our results provide evidence that combining the 'horizontal' inhibition of signalling pathways promoted by MTKIs with the 'vertical' inhibition of the downstream signalling cascade promoted by hsa-miR-302a-3p and hsa-miR-520 b constitutes a promising approach towards GBM treatment.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , MicroRNAs/genética , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Terapia Combinada , Predisposição Genética para Doença , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , MicroRNAs/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , TransfecçãoRESUMO
A novel synthetic approach to bis(indolyl)methanes has been established. Our one-pot synthetic strategy based on two consecutive hetero-Diels-Alder cycloaddition reactions of electrophilic conjugated nitrosoalkenes with indoles was extended to a range of new 1-hydroxyiminomethyl-bis(indolyl)methanes. Furthermore, a similar and broad range approach was applied to the synthesis of previously unknown 1-hydrazonomethyl-bis(indolyl)methanes. The biological evaluation of the new bis(indolyl)methanes as anti-cancer agents was investigated.
Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Metano/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reação de Cicloadição , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/síntese química , Indóis/química , Metano/análogos & derivados , Metano/química , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The design and synthesis of a novel bis-furan scaffold tailored for high efficiency at inhibiting transthyretin amyloid formation is reported. In vitro results show that the discovered compounds are more efficient inhibitors of amyloid formation than tafamidis, a drug currently used in the treatment of familial amyloid polyneuropathy (FAP), despite their lower molecular weight and lipophilicity. Moreover, ex vivo experiments with the strongest inhibitor in the series, conducted in human blood plasma from normal and FAP Val30Met-transthyretin carriers, disclose remarkable affinity and selectivity profiles. The promises and challenges facing further development of this compound are discussed under the light of increasing evidence implicating transthyretin stability as a key factor not only in transthyretin amyloidoses and several associated co-morbidities, but also in Alzheimer's disease.
Assuntos
Amiloide/química , Desenho de Fármacos , Furanos/química , Furanos/farmacologia , Pré-Albumina/química , Amiloide/metabolismo , Furanos/metabolismo , Células Hep G2 , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Pré-Albumina/metabolismo , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacosRESUMO
BACKGROUND: Overexpression of transferrin receptors (TfRs), which are responsible for the intracellular uptake of ferric transferrin (Tf), has been described in various cancers. Although molecular biology methods allow the identification of different types of receptors in cancer cells, they do not provide features about TfRs internalization, quantification and distribution on cell surface. This information can, however, be accessed by fluorescence techniques. In this work, the quantum dots (QDs)' unique properties were explored to strengthen our understanding of TfRs in cancer cells. METHODS: QDs were conjugated to Tf by covalent coupling and QDs-(Tf) bioconjugates were applied to quantify and evaluate the distribution of TfRs in two human glioblastoma cells lines, U87 and DBTRG-05MG, and also in HeLa cells by using flow cytometry and confocal microscopy. RESULTS: HeLa and DBTRG-05MG cells showed practically the same TfR labeling profile by QDs-(Tf), while U87 cells were less labeled by bioconjugates. Furthermore, inhibition studies demonstrated that QDs-(Tf) were able to label cells with high specificity. CONCLUSIONS: HeLa and DBTRG-05MG cells presented a similar and a higher amount of TfR than U87 cells. Moreover, DBTRG-05MG cells are more efficient in recycling the TfR than the other two cells types. GENERAL SIGNIFICANCE: This is the first study about TfRs in human glioblastoma cells using QDs. This new fluorescent tool can contribute to our understanding of the cancer cell biology and can help in the development of new therapies targeting these receptors.
Assuntos
Neoplasias Encefálicas/química , Glioblastoma/química , Pontos Quânticos , Receptores da Transferrina/análise , Corantes Fluorescentes , Células HeLa , Humanos , Microscopia ConfocalRESUMO
Malignant brain tumors, including glioblastoma (GBM), are among the most lethal human cancers, due to their tremendous invasive capacity and limited therapeutic options. Despite remarkable advances in cancer theranostics, which resulted in significant improvement of clinical outcomes, GBM relapse is very frequent and patient survival remains under one year. The elucidation of the role of abnormally-expressed miRNAs in different steps of GBM pathogenesis and in tumor resistance to therapy paved the way for the development of new miRNA-based therapeutic approaches targeting this disease, aiming at increasing specific tumor cell killing and, ultimately, cancer eradication. Here, we demonstrate that intravenously-administered chlorotoxin (CTX)-coupled (targeted) stable nucleic acid lipid particle (SNALP)-formulated anti-miR-21 oligonucleotides accumulate preferentially within brain tumors and promote efficient miR-21 silencing, which results in increased mRNA and protein levels of its target RhoB, while showing no signs of systemic immunogenicity. Decreased tumor cell proliferation and tumor size, as well as enhanced apoptosis activation and, to a lesser extent, improvement of animal survival, were also observed in GBM-bearing mice upon systemic delivery of targeted nanoparticle-formulated anti-miR-21 oligonucleotides and exposure to the tyrosine kinase inhibitor sunitinib. Overall, our results provide evidence that CTX-coupled SNALPs are a reliable and efficient system for systemic delivery of anti-miRNA oligonucleotides. Moreover, although further studies are still necessary to demonstrate a therapeutic benefit in a clinical context, our findings suggest that miRNA modulation by the targeted nanoparticles combined with anti-angiogenic chemotherapy may hold promise as an attractive approach towards GBM treatment.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Neoplasias Encefálicas/terapia , Portadores de Fármacos , Glioblastoma/terapia , Indóis/administração & dosagem , MicroRNAs/genética , Nanopartículas , Oligonucleotídeos Antissenso/genética , Inibidores de Proteínas Quinases/administração & dosagem , Pirróis/administração & dosagem , Terapêutica com RNAi/métodos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica , Quimioterapia Adjuvante , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Indóis/química , Indóis/metabolismo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Nanomedicina , Oligonucleotídeos Antissenso/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Pirróis/química , Pirróis/metabolismo , Venenos de Escorpião/metabolismo , Sunitinibe , Tecnologia Farmacêutica/métodos , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Proteína rhoB de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/metabolismoRESUMO
Glioblastoma (GBM) is among the most lethal human cancers, being generally characterized by rapid diffuse and infiltrative growth and high level of cellular heterogeneity associated with therapeutic resistance. Despite remarkable advances in cancer theranostics, which resulted in significant improvement of clinical outcomes, patient survival remains under one year. In recent years, considerable progress has been made in understanding the role of small non-coding RNAs, designated microRNAs, in the pathogenesis of GBM. Indeed, microRNAs were found to play a critical role in multiple steps of the tumorigenic process, including cellular proliferation, apoptosis evasion, invasion, angiogenesis, and stemness. Moreover, the modulation of microRNA expression, using either antisense oligonucleotides or precursor/mimic sequences, revealed a tremendous potential for application in GBM-targeted therapeutic approaches, either per se or in combination with chemo- and/or radiotherapy. In this manuscript, we review the regulatory role of microRNAs in key cellular processes underlying GBM tumorigenesis, including migration and invasion, apoptosis evasion, angiogenesis and GBM stem-like cell proliferation/differentiation, and discuss the current knowledge on their potential as diagnostic, prognostic and predictive biomarkers in this disease. We also address the latest advances in microRNA-based therapeutic approaches for GBM, by summarizing the major achievements in in vitro and pre-clinical studies. The trends identified by these studies are highlighted in order to provide new prospects for future developments towards the successful treatment of GBM.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , MicroRNAs/metabolismo , Humanos , MicroRNAs/genéticaRESUMO
Gene knockdown has emerged as an important tool for cancer gene therapy as well as for viral infections and dominantly inherited genetic disorders. The generation of suitable siRNA delivery systems poses some challenges, namely, to avoid nuclease degradation, to surpass the cytoplasmic membrane, and to release the nucleic acids into the cytosol. Aiming at evaluating the ability of thermoresponsive block copolymers formed by units of N-isopropylacrylamide and of (3-acrylamidopropyl)trimethylammonium chloride to efficiently deliver siRNAs, an extensive study was performed with four different copolymers using a human fibrosarcoma cell line as cell model. The silencing ability and cytotoxicity of the generated copolymer-based siRNA delivery systems were found to be dependent on the cloud point of the polymer, which corresponds to the transition temperature at which the aggregation or precipitation of the polymer molecules becomes thermodynamically more favorable than their solubilization. In the present study, a system capable of delivering siRNAs efficiently, specifically and without presenting relevant cytotoxicity, even in the presence of serum, was developed. Confocal fluorescence experiments showed that the ability of the generated systems to silence the target gene is related to some extent to nucleic acid internalization, being also dependent on polymer/siRNA dissociation at 37 °C. Thus, a delicate balance between nucleic acid internalization and intracellular release must be met in order to reach an ideal knockdown efficiency. The special features and potential for manipulation of the N-isopropylacrylamide-based copolymers make them suitable materials for the design and synthesis of new and promising siRNA delivery systems.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Carcinoma de Células Escamosas/radioterapia , Proliferação de Células/efeitos da radiação , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/radioterapia , Lutécio/uso terapêutico , Radioimunoterapia , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células/efeitos dos fármacos , Cetuximab , Receptores ErbB/imunologia , Feminino , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Lutécio/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Panitumumabe , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy, nanoparticle-mediated miR-21 silencing constitutes a promising multimodal therapeutic approach towards GBM.Molecular Therapy-Nucleic Acids (2013) 2, e100; doi:10.1038/mtna.2013.30; published online 18 June 2013.
RESUMO
The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, a new cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating sequence and the NLS of the S4(13)-PV peptide influence the competence of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells.
Assuntos
Peptídeos Penetradores de Células/química , Western Blotting , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Citometria de Fluxo , Terapia Genética/métodos , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Lipossomos/química , Survivina , Transfecção/métodosRESUMO
Highly malignant glioblastoma (GBM) is characterized by high genetic heterogeneity and infiltrative brain invasion patterns, and aberrant miRNA expression has been associated with hallmark malignant properties of GBM. The lack of effective GBM treatment options prompted us to investigate whether miRNAs would constitute promising therapeutic targets toward the generation of a gene therapy approach with clinical significance for this disease. Here, we show that microRNA-21 (miR-21) is upregulated and microRNA-128 (miR-128) is downregulated in mouse and human GBM samples, a finding that is corroborated by analysis of a large set of human GBM data from The Cancer Genome Atlas. Moreover, we demonstrate that oligonucleotide-mediated miR-21 silencing in U87 human GBM cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Cell exposure to pifithrin, an inhibitor of p53 transcriptional activity, reduced the caspase activity associated with decreased miR-21 expression. Finally, we demonstrate for the first time that miR-21 silencing enhances the antitumoral effect of the tyrosine kinase inhibitor sunitinib, whereas no therapeutic benefit is observed when coupling miR-21 silencing with the first-line drug temozolomide. Overall, our results provide evidence that miR-21 is uniformly overexpressed in GBM and constitutes a highly promising target for multimodal therapeutic approaches toward GBM.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Indóis/administração & dosagem , MicroRNAs/genética , Pirróis/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Animais , Proteínas Reguladoras de Apoptose/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Camundongos , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Sunitinibe , Regulação para CimaRESUMO
Glioblastoma (GBM) is a highly heterogeneous type of tumor characterized by genomic and signaling abnormalities affecting pathways involved in control of cell fate, including tumor-suppressor- and growth factor-regulated pathways. An aberrant miRNA expression has been observed in GBM, being associated with impaired cellular functions resulting in malignant transformation, proliferation and invasion. Here, we demonstrate for the first time that platelet-derived growth factor-B (PDGF-B), a potent angiogenic growth factor involved in GBM development and progression, promotes downregulation of pro-oncogenic (miR-21) and anti-oncogenic (miR-128) miRNAs, as well as upregulation/downregulation of several miRNAs involved in GBM pathology. Retrovirally mediated overexpression of PDGF-B in U87 human GBM cells or their prolonged exposure, as well as that of F98 rat glioma cells to this ligand, resulted in decreased miR-21 and miR-128 levels, which was associated with increased cell proliferation. Furthermore, siRNA-mediated PDGF-B silencing led to increased levels of miR-21 and miR-128, while miRNA modulation through overexpression of miR-21 did not alter the levels of PDGF-B. Finally, we demonstrate that modulation of tumor suppressors PTEN and p53 in U87 cells does not affect the decrease in miR-21 levels associated with PDGF-B overexpression. Overall, our findings suggest that, besides its role in inducing GBM tumorigenesis, PDGF-B may enhance tumor proliferation by modulating the expression of oncomiRs and tumor suppressor miRNAs in U87 human GBM cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-sis/genética , Interferência de RNA , Ratos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The increasing knowledge on the genetic basis of disease provides a platform for the development of promising gene-targeted therapies that can be applied to numerous pathological conditions, including cancer. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or for the repair and regulation of specific genes. However, despite the huge pharmacological potential of these molecules, their application remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. The discovery of a class of small peptides, the so-called cell-penetrating peptides (CPPs), which are able to very efficiently cross cell membranes through a mechanism that is independent of membrane receptors or transporters and avoids lysosomal enzymatic degradation, has been enthusiastically considered of key interest to improve noninvasive cellular delivery of therapeutic molecules. A large number of CPPs have been applied successfully to mediate the intracellular delivery of nucleic acids, including the S4(13)PV peptide for which interactions with membranes and resulting biological effects are illustrated in this chapter. Here, we provide a description of the experimental procedures for the preparation of CPP-based nucleic acid complexes and assessment of their formation, the selection of those protocols leading to the most efficient complexes, the biophysical characterization of CPP membrane interactions, and the evaluation of the biological and cytotoxic activity of the complexes.
Assuntos
Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Sequência de Aminoácidos , Apoptose , Varredura Diferencial de Calorimetria , Caspases/metabolismo , Polarização de Fluorescência , Genes Reporter , Células HeLa , Humanos , Lipossomos/química , Luciferases/biossíntese , Luciferases/genética , Dados de Sequência Molecular , Oligonucleotídeos/genética , Tamanho da Partícula , Propriedades de Superfície , TransfecçãoRESUMO
The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.