RESUMO
The therapeutic use of small interfering RNAs (siRNAs) as gene regulation agents has been limited by their poor stability and delivery. Although arranging siRNAs into a spherical nucleic acid (SNA) architecture to form siRNA-SNAs increases their stability and uptake, prototypical siRNA-SNAs consist of a hybridized architecture that causes guide strand dissociation from passenger strands, which limits the delivery of active siRNA duplexes. In this study, a new SNA design that directly attaches both siRNA strands to the SNA core through a single hairpin-shaped molecule to prevent guide strand dissociation is introduced and investigated. This hairpin-like architecture increases the number of siRNA duplexes that can be loaded onto an SNA by 4-fold compared to the original hybridized siRNA-SNA architecture. As a result, the hairpin-like siRNA-SNAs exhibit a 6-fold longer half-life in serum and decreased cytotoxicity. In addition, the hairpin-like siRNA-SNA produces more durable gene knockdown than the hybridized siRNA-SNA. This study shows how the chemistry used to immobilize siRNA on nanoparticles can markedly enhance biological function, and it establishes the hairpin-like architecture as a next-generation SNA construct that will be useful in life science and medical research.
Assuntos
Ácidos Nucleicos Imobilizados/farmacologia , RNA Interferente Pequeno/farmacologia , Linhagem Celular Tumoral , Inativação Gênica/efeitos dos fármacos , Ouro/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/toxicidade , Sequências Repetidas Invertidas , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Hibridização de Ácido Nucleico , RNA Interferente Pequeno/química , RNA Interferente Pequeno/toxicidadeRESUMO
The present study investigated the potential subchronic toxicity of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) in mice. The test reagent was administered once-daily by intravenous injection for 4 weeks at 0, 100, 200, or 300 mg/kg/day doses. Additional recovery groups (vehicle control and high dose groups) were observed for a 2-week recovery period. During the test period, mortality, clinical signs, body weight, food consumption, ophthalmology, urinalysis, hematology, serum biochemistry, gross pathology, organ weight, and histopathology were examined. An increase in the percentages of basophil and large unstained cells was observed in the 200 and 300 mg/kg/day groups of both sexes. In addition, the absolute and relative weights of the spleen were higher in males given 300 mg/kg/day relative to the concurrent controls. However, these findings were considered of no toxicological significance because the changes were minimal, were not accompanied by other relevant results (eg, correlating microscopic changes), and were not observed at the end of the 2-week recovery period indicating recovery of the findings. Based on the results, SAMiRNA-AREG did not cause treatment-related adverse effects at dose levels of up to 300 mg/kg/day in mice after 4-week repeated intravenous doses. Under these conditions, the no-observed-adverse-effect level of the SAMiRNA-AREG was ≥300 mg/kg/day in both sexes and no target organs were identified.
Assuntos
Anfirregulina/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Anfirregulina/toxicidade , Animais , Feminino , Injeções Intravenosas , Masculino , Camundongos Endogâmicos ICR , Micelas , Nanopartículas/toxicidade , Nível de Efeito Adverso não Observado , RNA Interferente Pequeno/toxicidade , Testes de Toxicidade SubagudaRESUMO
Small interfering RNA (siRNA) is a clinically approved therapeutic modality, which has attracted widespread attention not only from basic research but also from pharmaceutical industry. As siRNA can theoretically modulate any disease-related gene's expression, plenty of siRNA therapeutic pipelines have been established by tens of biotechnology companies. The drug performance of siRNA heavily depends on the sequence, the chemical modification, and the delivery of siRNA. Here, we describe the rational design protocol of siRNA, and provide some modification patterns that can enhance siRNA's stability and reduce its off-target effect. Also, the delivery method based on N-acetylgalactosamine (GalNAc)-siRNA conjugate that is widely employed to develop therapeutic regimens for liver-related diseases is also recapitulated.
Assuntos
Acetilgalactosamina/metabolismo , Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Hepatopatias/terapia , Fígado/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Terapêutica com RNAi , Acetilgalactosamina/toxicidade , Animais , Técnicas de Transferência de Genes/efeitos adversos , Células HEK293 , Humanos , Hepatopatias/genética , Hepatopatias/metabolismo , Camundongos Endogâmicos C57BL , Pré-Albumina/genética , Pré-Albumina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/toxicidade , Terapêutica com RNAi/efeitos adversos , Projetos de Pesquisa , Testes de Toxicidade , Fluxo de TrabalhoRESUMO
Extracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano-therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.
Assuntos
Vesículas Extracelulares/metabolismo , Técnicas de Transferência de Genes , Leite/toxicidade , RNA Interferente Pequeno/toxicidade , Terapêutica com RNAi , Animais , Bovinos , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário , Vesículas Extracelulares/genética , Vesículas Extracelulares/imunologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Leite/imunologia , Leite/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Medição de Risco , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Testes de Toxicidade Aguda , Peixe-Zebra/embriologiaRESUMO
The anomalous high expression of matrix metalloproteinase 9 (MMP-9) is one important factor that impedes diabetic wound healing. Therefore, inhibition of MMP-9 expression in a diabetic wound could be a feasible method to promote wound healing. In this study, we studied the possibility of self-therapy using wound dressings that contain bacterial cellulose-hyperbranched cationic polysaccharide (BC-HCP) derivatives that encapsulate siRNA (BC-HCP/siMMP-9) and have controlled release properties. Herein, we used four HCPs (Gly-DMAPA, Gly-D4, Amyp-DMAPA, Amyp-D4) as gene carriers. Our results showed that all HCP derivatives were minimally toxic to cells in vitro, while the cationic properties of HCP could be used as a complexation agent for MMP-9 siRNA (siMMP-9). Upon exposure to bacterial cellulose (BC), the BC slowly released HCP/siMMP-9. The released siMMP-9 effectively reduced the gene expression and protein levels of MMP-9 in a human immortalized epithelial cell line (HaCAT) and in diabetic rat wounds. Inhibition of MMP-9 in the wounds of diabetic rats resulted in a significant enhancement of wound healing, suggesting that the BC-HCP/siMMP-9 composite dressing could be used as a safe and effective dressing to promote wound healing in diabetic rats. STATEMENT OF SIGNIFICANCE: In this work, we evaluated the possibility of using bacterial cellulose-hyperbranched cationic polysaccharide derivatives (BC-HCP) as a self-therapeutic wound dressing with siRNA encapsulated and controlled release properties. Our results showed that the BC-HCP/siMMP-9 composite dressing slowly released HCP/siMMP-9. The released siMMP-9 effectively reduced the gene expression and protein level of MMP-9 in human immortalized epithelial cell line and in the wound of diabetic rats. The BC-HCP/siMMP-9 composite dressing promoted diabetic wound healing by the unique nanostructure of BC and by releasing siMMP-9 for specific MMP-9 inhibition. Therefore, it could be used as a safe and effective dressing to promote wound healing in diabetic rats. This is the first evidence on the study of using BC as a dressing composite by encapsulating HCP/siRNA complexes for efficient RNAi gene silencing for better wound healing in diabetic rats.
Assuntos
Bandagens , Celulose/farmacologia , Dendrímeros/farmacologia , Diabetes Mellitus Experimental/fisiopatologia , RNA Interferente Pequeno/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Celulose/toxicidade , Dendrímeros/toxicidade , Células HaCaT , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , RNA Interferente Pequeno/toxicidade , Ratos Sprague-DawleyRESUMO
Diabetes mellitus is a chronic metabolic disorder characterized by insulin deficiency and impaired glucose metabolism. Overexpression of cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) plays an important role in high gluconeogenesis in patients with diabetes type II. Using RNA interference technology for silencing CRTC2 gene expression could be helpful in controlling the level of blood glucose and gluconeogenesis. In this study, we designed a siRNA delivery platform comprising blended chitosan lactate (CT) and polyethylene glycol (PEG) conjugated with glycyrrhetinic acid (GA) for controlling gluconeogenesis. The nanoparticles showed spherical and smooth surface with ~ 189-nm size and + 5.1 zeta potential. Targeted nanoparticles were efficiently stable in serum and different levels of heparin media over 48 h. The gene knockdown efficiency of nanoparticles was comparable to Lipofectamine®, while they had no significant in vitro and in vivo toxicity. The in vivo therapeutic efficacy of targeted nanoparticles was also confirmed by reduced amount of fasting blood sugar in diabetic rat models. Furthermore, the nanoparticles were mostly accumulated in the liver after 2 h indicating the significant targeting ability of the prepared nanoparticles. Therefore, CT/PEG-GA nanoparticles can be considered as a potential candidate for targeted delivery of siRNA into hepatocytes in order to regulate gluconeogenesis in diabetes.
Assuntos
Quitosana/administração & dosagem , Gluconeogênese/efeitos dos fármacos , Ácido Glicirretínico/administração & dosagem , Ácido Láctico/administração & dosagem , Fígado/efeitos dos fármacos , Nanopartículas/administração & dosagem , Polietilenoglicóis/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fatores de Transcrição/genética , Animais , Quitosana/farmacocinética , Quitosana/toxicidade , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/terapia , Expressão Gênica , Ácido Glicirretínico/farmacocinética , Ácido Glicirretínico/toxicidade , Células Hep G2 , Humanos , Ácido Láctico/farmacocinética , Ácido Láctico/toxicidade , Fígado/metabolismo , Fígado/patologia , Masculino , Nanopartículas/toxicidade , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/toxicidade , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/toxicidade , Ratos Wistar , Distribuição TecidualRESUMO
Inhibition of p53 has been shown to be an efficient strategy for ameliorating kidney ischemia-reperfusion (I/R) injury in experimental models. The therapeutic value of p53 siRNA-based inhibition for I/R in renal transplantation is currently being evaluated in clinical studies. While the major rationale for these studies is the suppression of proapoptotic properties, there are more equally important injury response pathways regulated by p53. A p53-dependent pathway shown to be crucial for renal long-term outcome is cellular senescence. In this study, we tested the hypothesis that p53 siRNA reduces I/R-induced senescence and thereby improves kidney outcome. By comparing the impact of different treatment durations in a mouse model of renal I/R, we found that repetitive administration of p53 siRNA during the first 14 days after I/R reduced the senescence load and ameliorated the postischemic phenotype. Prolonged application of p53 siRNA over a 26-day period after I/R, however, did not provide any additional benefit for senescence reduction but reversed some of the renoprotective effects of the early treatment. These data suggest a time-dependent role of p53 activity supporting the current therapeutic concept of a short-term inhibition, while advocating against a prolonged treatment after I/R.
Assuntos
Injúria Renal Aguda/terapia , Senescência Celular , Túbulos Renais Proximais/metabolismo , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi , Traumatismo por Reperfusão/terapia , Proteína Supressora de Tumor p53/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Apoptose , Modelos Animais de Doenças , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/toxicidade , Terapêutica com RNAi/efeitos adversos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fatores de Tempo , Proteína Supressora de Tumor p53/genéticaRESUMO
Subcortical band heterotopia (SBH), also known as doublecortex syndrome, is a malformation of cortical development resulting from mutations in the doublecortin gene (DCX). It is characterized by a lack of migration of cortical neurons that accumulate in the white matter forming a heterotopic band. Patients with SBH may present mild to moderate intellectual disability as well as epilepsy. The SBH condition can be modeled in rats by in utero knockdown (KD) of Dcx. The affected cells form an SBH reminiscent of that observed in human patients and the animals develop a chronic epileptic condition in adulthood. Here, we investigated if the presence of a SBH is sufficient to induce cognitive impairment in juvenile Dcx-KD rats, before the onset of epilepsy. Using a wide range of behavioral tests, we found that the presence of SBH did not appear to affect motor control or somatosensory processing. In addition, cognitive abilities such as learning, short-term and long-term memory, were normal in pre-epileptic Dcx-KD rats. We suggest that the SBH presence is not sufficient to impair these behavioral functions.
Assuntos
Comportamento Animal , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/psicologia , Cognição , Modelos Animais de Doenças , Epilepsia/genética , Deficiência Intelectual/genética , Animais , Ansiedade/genética , Doenças Assintomáticas , Movimento Celular , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/complicações , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/embriologia , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/genética , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Eletroporação , Comportamento Exploratório , Substância Cinzenta/anormalidades , Substância Cinzenta/embriologia , Aprendizagem , Aprendizagem em Labirinto , Memória , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mosaicismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/toxicidade , Ratos , Teste de Desempenho do Rota-Rod , Sensação , Substância Branca/anormalidades , Substância Branca/embriologiaRESUMO
Activated autophagy has been intensively observed in cerebrovascular diseases, including focal cerebral ischemia injury, but its molecular mechanisms remain unclear. TOM7, which is a component of the protein translocase of the outer mitochondrial membrane (TOM) complex, may modulate assembly of the TOM complex. However, an understanding of how TOM7 affects cerebral ischemia injury is limited. In this study, we demonstrate that the expression of TOM7 is up-regulated after a photothrombotic cerebral ischemic model in rats, peaking at 3 days. In addition, TOM7 knockdown may aggravate cerebral ischemic injury and inhibit autophagy after ischemic stroke. Mechanically, TOM7 may regulate autophagy through the PINK1/Beclin1 pathway after cerebral ischemia injury. These results demonstrate that TOM7 silencing may aggravate cerebral ischemia injury through inhibiting PINK1/Beclin1 pathway- mediated autophagy.
Assuntos
Autofagia , Proteína Beclina-1/metabolismo , Isquemia Encefálica/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Quinases/metabolismo , Análise de Variância , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Masculino , Microscopia Eletrônica de Transmissão , Proteínas de Transporte da Membrana Mitocondrial/genética , Exame Neurológico , RNA Interferente Pequeno/toxicidade , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Fatores de Tempo , Regulação para Cima/genéticaRESUMO
Many small-interfering (si)RNAs are toxic to cancer cells through a 6mer seed sequence (positions 2-7 of the guide strand). Here we performed an siRNA screen with all 4096 6mer seeds revealing a preference for guanine in positions 1 and 2 and a high overall G or C content in the seed of the most toxic siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3'UTRs. The master tumor suppressor miRNA miR-34a-5p is toxic through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during evolution most miRNAs evolved to avoid guanine at the 5' end of the 6mer seed sequence of the guide strand. In contrast, for certain tumor-suppressive miRNAs the guide strand contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.
Assuntos
Linhagem Celular Tumoral/efeitos dos fármacos , MicroRNAs/toxicidade , RNA Interferente Pequeno/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Marcação de Genes , Genes Essenciais/efeitos dos fármacos , Guanina , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Regiões não TraduzidasRESUMO
Stimuli-responsive polycations have been developed for improved nucleic acid transfection and enhanced therapeutic efficacy. The most reported mechanisms for controlled release of siRNA are based on polyelectrolyte exchange reactions in the cytoplasm and the degradation of polycations initiated by specific triggers. However, the degradation strategy has not always been sufficient due to unsatisfactory kinetics and binding of cationic fragments to siRNA, which limits the gene silencing effect. In this study, a new strategy that combines degradation and charge reversal is proposed. Methods: We prepared a polycation (CrossPPA) by crosslinking of phenylboronic acid (PBA)-grafted 1.8k PEI with alginate. It was compared with 25k PEI, 1.8k PEI and 1.8k PEI-PBA on siRNA encapsulation, ATP-responsive behavior and mechanism, cytotoxicity, cell uptake, siRNA transfection, in vivo biodistribution and in vivo anti-tumor efficacy. The in vitro and in vivo experiments were performed on 4T1 murine breast cancer cells and 4T1 tumor model separately. Results: The crosslinking strategy obviously improve the siRNA loading ability of 1.8k PEI. We validated that intracellular levels of ATP could trigger CrossPPA disassembly and charge reversal, which resulted in efficient and rapid siRNA release due to electrostatic repulsion. Besides, CrossPPA/siRNA showed strong cell uptake in 4T1 cells compared with 1.8k PEI/siRNA. Notably, the cytotoxicity of CrossPPA was pretty low, which was owing to its biodegradability. Furthermore, the crosslinked polyplexes significantly enhanced siRNA transfection and improved tumor accumulation. The high gene silencing ability of CrossPPA polyplex led to strong anti-tumor efficacy when using Bcl2-targeted siRNA. Conclusion: These results indicated that the ATP-triggered disassembly and charge reversal strategy provided a new way for developing stimuli-responsive siRNA carriers and showed potential for nucleic acid delivery in the treatment of cancer.
Assuntos
Trifosfato de Adenosina/metabolismo , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Neoplasias Mamárias Animais/terapia , RNA Interferente Pequeno/administração & dosagem , Doenças dos Roedores/terapia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Inativação Gênica , Camundongos , Terapia de Alvo Molecular/métodos , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/toxicidade , Transfecção/métodos , Resultado do TratamentoRESUMO
Short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are the most clinically advanced oligonucleotide-based platforms. A number of N-acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNAs), also referred to as RNA interference (RNAi) therapeutics, are currently in various stages of development, though none is yet approved. While the safety of ASOs has been the subject of extensive review, the nonclinical safety profiles of GalNAc-siRNAs have not been reported. With the exception of sequence differences that confer target RNA specificity, GalNAc-siRNAs are largely chemically uniform, containing limited number of phosphorothioate linkages, and 2'-O-methyl and 2'-deoxy-2'-fluoro ribose modifications. Here, we present the outcomes of short-term (3-5 week) rat and monkey weekly repeat-dose toxicology studies of six Enhanced Stabilization Chemistry GalNAc-siRNAs currently in clinical development. In nonclinical studies at supratherapeutic doses, these molecules share similar safety signals, with histologic findings in the organ of pharmacodynamic effect (liver), the organ of elimination (kidney), and the reticuloendothelial system (lymph nodes). The majority of these changes are nonadverse, partially to completely reversible, correlate well with pharmacokinetic parameters and tissue distribution, and often reflect drug accumulation. Furthermore, all GalNAc-siRNAs tested to date have been negative in genotoxicity and safety pharmacology studies.
Assuntos
Acetilgalactosamina/toxicidade , Aberrações Cromossômicas/induzido quimicamente , Fígado/efeitos dos fármacos , RNA Interferente Pequeno/toxicidade , Acetilgalactosamina/química , Acetilgalactosamina/farmacologia , Animais , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Fígado/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Macaca fascicularis , Testes de Mutagenicidade , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Ratos Sprague-Dawley , Especificidade da Espécie , Testes de Toxicidade SubagudaRESUMO
The development of an efficient small interfering RNA (siRNA) delivery system has held scientists interest since the discovery of the RNA interference mechanism (RNAi). This strategy gives hope for the treatment of many severe diseases. Herein, we developed hybrid nanovectors able to deliver siRNA to triple negative breast cancer cells. The nanovectors are based on PEGylated superparamagnetic iron oxide nanoparticles (SPION) functionalized with gH625 peptide, chitosan and poly-l-arginine. Every component has a key role and specific function: SPION is the core scaffolding the nanovector; PEG participates in the colloidal stability and the immune stealthiness; gH625 peptide promotes the nanovector internalization into cancer cells; cationic polymers provide the siRNA protection and favor siRNA endosomal escape and delivery to cytosol. The formulation was optimized by varying the amount of each compound. The efficacy of the siRNA retention and protection were investigated in the presence of high concentration of serum. Optimized nanovectors show a high uptake by MDA-MB-231 cells. The resulting down regulation of GFP expression was 73⯱â¯3% with our nanovector compared to 59⯱â¯8% obtained with the siRNA-Oligofectamine™ complex in the same conditions.
Assuntos
Técnicas de Transferência de Genes , Peptídeos/química , RNA Interferente Pequeno/administração & dosagem , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/terapia , Proteínas do Envelope Viral/química , Linhagem Celular Tumoral , Quitosana , Citosol/metabolismo , Composição de Medicamentos , Feminino , Vetores Genéticos , Humanos , Ferro , Nanopartículas Metálicas , Modelos Moleculares , Polietilenoglicóis , RNA Interferente Pequeno/toxicidade , TransfecçãoRESUMO
ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a key enzyme to cleave the amyloid precursor protein to develop Alzheimer's disease (AD). Reducing BACE1 expression in central neuron through RNA interference technology shows great promise to overcome AD. However, to obtain an efficient and neurons-specific delivery of siRNA against BACE1 through systemic administration remains challenging. Here, we design and prepare siRNA nano-carriers based on PEGylated poly(2-(N,N-dimethylamino) ethyl methacrylate) (PEG-PDMAEMA) modified with both the CGN peptide for blood-brain barrier (BBB) penetration and the Tet1 peptide for neuron-specific binding. The nanocomplexes CT/siRNA, composed of CGN-PEG-PDMAEMA and Tet1-PEG-PDMAEMA at a weight ratio of 1:1, display a good stability in the blood and do not lead to hemolysis at N/Pâ¯=â¯10. The internalization of nanocomplexes in neuron cells relies on clathrin-mediated endocytosis and micropinocytosis, while caveolae-mediated endocytosis plays a major role in entrance of CT/siRNA into cerebral capillary endothelial cell bEnd.3. The nanocomplexes successfully escape from lysosomes and enter in the cytoplasm of the neuron cells, inducing effective gene silence (about 50% decrease in BACE1 mRNA levels) and reversing Aß25-35 oligomer-induced synaptic injury. After caudal vein injection in mice, CT/siRNA display higher brain accumulation than unmodified nanocomplexes (brain drug targeting indexâ¯=â¯2.62), and colocalize with neurons or locate nearby. In APP/PS1 transgenic mice, the nanocomplexes significantly decrease BACE1 mRNA and the amyloid plaques, suppress phosphorylated tau protein levels, as well as promote hippocampal neurogenesis. Noticeably, administration of the nanocomplexes restores the cognitive performance of the AD transgenic mice to the level of wild-type control without significant adverse effects on myelination. Our results demonstrate the CT/siRNA nanocomplexes capable of specifically directing BACE1 siRNA to brain neurons with great potential for AD therapy.
Assuntos
Doença de Alzheimer/terapia , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Encéfalo/metabolismo , Nanopartículas , RNA Interferente Pequeno/administração & dosagem , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Transtornos Cognitivos/genética , Transtornos Cognitivos/terapia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Endocitose/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Neurônios/metabolismo , Placa Amiloide/metabolismo , Polímeros/química , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/toxicidadeRESUMO
Further quantitative understanding of the biological effects and mechanisms involved in cellular and intracellular delivery of nucleic acid materials is required to produce clinical applications of gene therapy. Several modeling approaches have been used in this field; however, a comprehensive approach that integrates all the key pharmacological issues into a holistic framework that is applicable for in vivo conditions is still lacking. This contribution presents a pharmacokinetic/pharmacodynamic model-based control study of non-viral siRNA delivery describing the dynamics of the delivery process and takes into account the main multi-objective optimization issues such as efficacy and toxicity, as well as the effect of uncertainty in cell doubling time. The methodology developed in this work is used to predict the optimal dosage injection rate and optimal intracellular exposure of siRNAs in order to improve the pharmacological effects before cell division occurs. The present analysis successfully provides quantitative predictions of non-viral siRNA activity paving the path for further experimental work to probe more efficient delivery systems.
Assuntos
Produtos Biológicos/farmacologia , Produtos Biológicos/farmacocinética , Terapia Genética/métodos , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/farmacocinética , Produtos Biológicos/administração & dosagem , Produtos Biológicos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inativação Gênica , Terapia Genética/efeitos adversos , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Humanos , Modelos Estatísticos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/toxicidadeRESUMO
A superior biocompatible spherical nucleic acid (SNA) conjugate was fabricated by grafting siRNA onto the surface of a core composed of a spherical DNA nanostructure that we have termed a DNA nanoclew (DC). After uptake by cultured cancer cells, SNA nanoparticles release engrafted siRNAs by cleavage of the intracellular Dicer enzyme. Moreover, in vitro experiments reveal that such SNAs demonstrate potent gene knockdown at both mRNA and protein levels, while with negligible cytotoxicity.
Assuntos
DNA/química , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Silenciamento de Genes/métodos , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Inativação Gênica , Células HeLa , Humanos , Nanopartículas/toxicidade , Hibridização de Ácido Nucleico , Tamanho da Partícula , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/toxicidade , Ribonuclease III/químicaRESUMO
Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.
Assuntos
Acetilgalactosamina/química , Fígado/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/toxicidade , Acetilgalactosamina/toxicidade , Animais , Fígado/metabolismo , Masculino , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
In this paper, the starch was firstly modified by quaternary reagent to obtain cationic starch. Then self-assembled folate-biotin-quaternized starch nanoparticles were prepared by a one-pot synthesis via N,N'-dicyclohexylcarbodiimide/N-hydroxysuccinimide/4-dimethylaminopyridine-mediated esterification reaction. The physicochemical properties of the prepared folate-biotin-quaternized starch nanoparticles were characterized. The average diameter of folate-biotin-quaternized starch nanoparticles was 109 nm with polydispersity index of 0.183 and zeta potential of 28.59 mV. The folate-biotin-quaternized starch nanoparticles were used as co-carrier of siRNA and doxorubicin with satisfactory drug loading capacity (6.98%) and encapsulation efficiency (69.66 %), and siRNA could be efficiently encapsulated at 40/1 weight ratio of doxorubicin/folate-biotin-quaternized starch nanoparticles to siRNA. The folate-biotin-quaternized starch nanoparticles could effectively protect siRNA from degradation of serum RNAase for up to 48 h. The release characteristics of doxorubicin and siRNA from folate-biotin-quaternized starch nanoparticles were studied in different pH environment and the release behaviors of two drugs were all pH sensitive. The folate-biotin-quaternized starch nanoparticles as a potential co-carrier of anticancer agents and gene drugs was expected to achieve future practical application in vitro and in vivo.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Biotina/análogos & derivados , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Ácido Fólico/análogos & derivados , RNA Interferente Pequeno/administração & dosagem , Amido/análogos & derivados , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidade , Biotina/toxicidade , Doxorrubicina/química , Doxorrubicina/toxicidade , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Ácido Fólico/toxicidade , Hemólise/efeitos dos fármacos , Teste de Materiais , Modelos Moleculares , Nanopartículas/química , Nanopartículas/toxicidade , Nanopartículas/ultraestrutura , RNA Interferente Pequeno/química , RNA Interferente Pequeno/toxicidade , Ratos , Amido/toxicidadeRESUMO
siRNA-loaded nanoparticles (NPs) administered systemically can overcome the poor stability and rapid elimination of free double-stranded RNA in circulation, resulting in increased tumor accumulation and efficacy. siRNA against osteopontin (siOPN), a protein involved in breast cancer development, was encapsulated in poly(D,L-lactic-co-glycolic acid) NPs by a double emulsion solvent diffusion (DESD) technique. We also compared the effect of polyethylenimine (PEI) molecular weight (800 Da and 25 kDa), used as the counter-ion for siRNA complexation, on the physicochemical properties of the NPs, cytotoxicity, and cellular uptake. NPs prepared by the DESD technique were obtained at the desired size (â¼170 nm) using both types of PEIs, and were characterized with a neutral surface charge, high encapsulation yield (up to â¼60%), siOPN concentration of 5.6-8.4 µg/mg, stability in physiologic conditions in vitro and in vivo, and long-term shelf-life stability (> 3 years). The NPs prepared using both PEIs exhibited no cytotoxicity in primary smooth muscle culture, and no detrimental effect on mice liver enzymes following their IV administration. Following cellular uptake and biodistribution studies, the therapeutic potential of the NPs was demonstrated by a significant decrease of tumor progression and size in an ectopic xenograft model of mammary carcinoma in mice.
Assuntos
Emulsões/química , Ácido Láctico/química , Neoplasias Mamárias Experimentais/terapia , Nanopartículas/química , Ácido Poliglicólico/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/toxicidade , Solventes/química , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Difusão , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Peso Molecular , Nanopartículas/ultraestrutura , Osteopontina/metabolismo , Tamanho da Partícula , Polietilenoimina/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Soro , Eletricidade Estática , Distribuição Tecidual/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epidemiological studies of childhood leukemia survivors reveal an alarmingly high incidence of chronic health disabilities after treatment, therefore, more specific therapies need to be developed. Polo-like kinase 1 (Plk1) is a key player in mitosis and a target for drug development as it is upregulated in multiple cancer types. Small molecules targeting Plk1 are mainly ATP-competitors and, therefore, are known to elicit side effects due to lack of specificity. RNA interference (RNAi) is known for its high catalytic activity and target selectivity; however, the biggest barrier for its introduction into clinical use is its delivery. RNAi prodrugs are modified, self-delivering short interfering Ribonucleic Neutrals (siRNNs), cleaved by cytoplasmic enzymes into short interfering Ribonucleic Acids (siRNAs) once inside cells. In this study we aimed to investigate the potential of siRNNs as therapeutic tools in T-acute lymphoblastic leukemia (T-ALL) using T-ALL cell lines and patient-derived samples. We demonstrate for the first time that RNAi prodrugs (siRNNs) targeting Plk1, can enter pediatric T-ALL patient cells without a transfection reagent and induce Plk1 knockdown on both protein and mRNA levels resulting in G2/M-arrest and apoptosis. We also show that siRNNs targeting Plk1 generate less toxicity in normal cells compared to the small molecule Plk1 inhibitor, BI6727, suggesting a potentially good therapeutic index.