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1.
Chem Pharm Bull (Tokyo) ; 68(2): 129-132, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009079

RESUMO

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.


Assuntos
DNA Antissenso/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Aminas/química , Animais , Cátions/química , DNA Antissenso/química , DNA Antissenso/genética , DNA Antissenso/farmacocinética , Dissulfetos/química , Inativação Gênica , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos ICR , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , Transfecção/métodos
2.
Chem Commun (Camb) ; 56(8): 1255-1258, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31898700

RESUMO

Here we demonstrated that the stiffness of cationized gelatin nanoparticles determined the efficiency of RNAi in myeloid leukemia cells when the particle size and surface charges were kept constant. The siRNA delivery system with an elastic modulus of 0.87 MPa showed the largest siRNA uptake and RNAi efficiency for hard-to-transfect suspension cells.


Assuntos
Gelatina/química , Leucemia Mieloide/patologia , Nanopartículas/química , Interferência de RNA , Cátions/química , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Humanos , Células K562 , Leucemia Mieloide/tratamento farmacológico , Tamanho da Partícula , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Propriedades de Superfície
3.
Chem Commun (Camb) ; 56(16): 2411-2414, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-31994560

RESUMO

Herein, polymerization-induced electrostatic self-assembly (PIESA) is conducted to mediate the self-assembly behavior of short interfering RNA (siRNA) for the first time. In PIESA, siRNA not only formed a simple electrostatic polyplex with positively charged polycations, but also facilitated directed self-assembly due to the molecular rigidity of siRNA, leading to appealing nanotubes.


Assuntos
RNA Interferente Pequeno/síntese química , Estrutura Molecular , Tamanho da Partícula , Polimerização , RNA Interferente Pequeno/química , Eletricidade Estática , Propriedades de Superfície
4.
J Nanobiotechnology ; 18(1): 15, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952530

RESUMO

BACKGROUND: The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers. RESULTS: For better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model. CONCLUSION: The designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.


Assuntos
Peptídeos Penetradores de Células/química , Elastina/química , Peptídeos/química , RNA Interferente Pequeno/química , Animais , Biopolímeros , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Luciferases/genética , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Imagem Óptica , RNA Interferente Pequeno/administração & dosagem , Receptores de Interleucina-4/metabolismo , Transfecção
5.
Chemistry ; 26(3): 685-690, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31693228

RESUMO

The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3'-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5'-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents.


Assuntos
Amidas/química , Nucleosídeos/química , Fosfatos/química , RNA de Cadeia Dupla/síntese química , RNA Interferente Pequeno/síntese química , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/química , Transfecção
6.
Chem Commun (Camb) ; 56(3): 466-469, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31828267

RESUMO

We herein report a new approach for RNA interference, so-called "build-up RNAi" approach, where single-strand circular RNAs with a photocleavable unit or disulfide moiety were used as siRNA precursors. The advantages of using these circular RNA formats for RNAi were presented in aspects of immunogenicity and cellular uptake.


Assuntos
Interferência de RNA , Precursores de RNA/química , RNA Interferente Pequeno/química , Apolipoproteínas B/antagonistas & inibidores , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Precursores de RNA/síntese química , Precursores de RNA/efeitos da radiação , RNA Interferente Pequeno/metabolismo , Raios Ultravioleta
7.
Carbohydr Polym ; 227: 115339, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31590870

RESUMO

Poor buffering capacity of chitosan (CS) results in insufficient intracellular gene release which poses the major barrier in gene delivery. Herein, we reconstructed pristine CS with propylamine (PA), (diethylamino) propylamine (DEAPA), and N, N-dimethyl- dipropylenetriamine (DMAMAPA) to obtain a series of alkylamine-chitosan (AA-CS). The introduction of multiple amino groups with rational ratios functionally enhance the buffering capacity of AA-CS, among which DMAPAPA-CS showed buffering capacity of 1.58 times that of chitosan. The reconstructed AA-CS functionally enhance the ability of gene binding and endosomal escape. It was observed that the DMAPAPA-CS/pDNA complexes exhibit a notable gene delivery efficiency, which promotes the functionalization of loaded pDNA. Importantly, the in vivo delivery assay reveals that the deep penetration issue can be resolved using DMAPAPA-CS gene delivery vector. Finally, the DMAPAPA-CS is applied to deliver the therapeutic p53 gene in A549 bearing mice, showing efficient therapeutic potential for cancer.


Assuntos
Aminas/administração & dosagem , Quitosana/administração & dosagem , DNA/administração & dosagem , Endossomos , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Proteína Supressora de Tumor p53/genética , Células A549 , Aminas/química , Aminas/farmacocinética , Animais , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Quitosana/farmacocinética , DNA/química , Endocitose , Eritrócitos/efeitos dos fármacos , Feminino , Células HEK293 , Hemólise/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética
8.
Crit Rev Ther Drug Carrier Syst ; 36(4): 305-371, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679190

RESUMO

Ovarian cancer (OC) has emerged as one of the leading causes of death in women due to the lack of early-stage diagnosis resulting in impairment and delay in treatment of malignancy, which raises the morality rate. Existing diagnostic (pelvic examination, CA125, and enzyme-linked immunosorbent assay) or therapeutic modalities (radiotherapy, abdominal pelvic radiation therapy, and chemotherapy) are insufficient to decrease the 5-year survival rate. Nanoparticles (NPs) have been extensively explored as probes for imaging or therapy of cancer. As an extension of this, probes have been designed to possess both imaging and therapeutic modality in a single molecule and this has emerged as the science of nanotheranostics. This review presents the existing diagnostic and therapeutic strategies in use for OC and discusses their loopholes that limit the prognosis of OC. The review presents a general description of important properties of nanostructures and the type of nanostructures that have been used as imaging/therapeutic probe in cancer. The state-of-the-art nanotheranostics probe for targeting OC is presented. Systematic and complete studies that can correlate the findings of researchers from different global areas are lacking. The current status of nanostructures in various phases of clinical trials and those approved by U.S. Food and Drug Administration (FDA) has been presented. No specific targeted theranostic probe for OC has yet been approved by the FDA. Here, the underlying reasons and the challenges faced for nanotheranostics of OC are discussed, along with its future prospects.


Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Epitelial do Ovário/diagnóstico , Carcinoma Epitelial do Ovário/terapia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/terapia , Nanomedicina Teranóstica/métodos , Animais , Antineoplásicos/química , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nanoscale ; 11(39): 18052-18064, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576876

RESUMO

Nanomaterials hold promise for the delivery of nucleic acids to facilitate gene therapy in cardiac diseases. However, as much of the in vivo study of nanomaterials was conducted via the "trial and error" method, the understanding of the nanomaterial-mediated delivery in cardiac tissue was limited to the gross efficiency in manipulating the gene expression while little was known about the delivery process and mechanism in particular at the cell level. In this study, small interfering RNA (siRNA) nanoparticles formulated with a polyamidoamine (PAMAM) nanomaterial were applied to the injured heart of zebrafish. The distribution of nanoparticles in cardiomyocytes, endothelial cells, macrophages and leukocytes was quantitatively analyzed with precision at the cell level by using transgenic models. Based on the distribution characteristics, gene silencing effects in a specific group of cells were analyzed to illustrate how siRNA nanoparticles could get potent gene silencing in different cells in vivo. The results elucidated the heterogeneous distribution of siRNA nanoparticles and how nanoparticles could be efficient despite the significant difference in cellular uptake efficiency in different cells. It demonstrated a paradigm and the need to decouple cellular processes to understand nanoparticle-mediated delivery in complex tissue and the investigation/methodology may lead to important information to guide the design of advanced targeted drug-delivery systems in heart.


Assuntos
Sistemas de Liberação de Medicamentos , Inativação Gênica , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Nanopartículas/química , RNA Interferente Pequeno , Animais , Miocárdio/citologia , Miócitos Cardíacos/citologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Peixe-Zebra
10.
Chem Commun (Camb) ; 55(77): 11619-11622, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31501844

RESUMO

Mesoporous organosilica nanoparticles (PHT-PMO) have been prepared from an octa-triethoxysilylated Zn phthalocyanine precursor. These PHT-PMO nanoparticles had no dark toxicity but high phototoxicity when irradiated at 650 nm, and remarkable near-infrared phototoxicity when excited at 760 and 810 nm. The PHT-PMO were then aminated to promote electrostatic complexation with siRNA. Transfection experiments were performed upon NIR irradiation and photochemical internalization was very efficient, leading to 65% luciferase extinction in MCF-7 cancer cells expressing stable luciferase.


Assuntos
Indóis/química , Nanopartículas/química , Compostos Organometálicos/química , Fotoquimioterapia/métodos , RNA Interferente Pequeno/química , Silanos/química , Sobrevivência Celular , Cetrimônio/química , Humanos , Raios Infravermelhos , Luciferases/genética , Células MCF-7 , Processos Fotoquímicos , Porosidade , RNA Interferente Pequeno/metabolismo , Eletricidade Estática , Propriedades de Superfície
11.
Nanoscale ; 11(36): 17041-17051, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31506653

RESUMO

Small interfering RNA (siRNA) is a promising tool for the treatment of skin disorders including skin squamous cell carcinoma (SCC). This article develops a topical formulation for the transdermal delivery of siRNA. The formulation is built on mesoporous silica nanoparticles (MSNPs) with a loading capacity of 1.4 µg of oligonucleotide per mg of MSNPs. Cell experiments are employed to study the functionality of the formulation including the cellular uptake, the qualitative and quantitative detection of specific gene biomarkers. The clinical potential of this system is examined by topically delivering siRNA targeting TGFßR-1 (TGFßR-1) to the SCC in a mouse xenograft model. In comparison to the controls, MSNPs containing TGFßR-1 siRNA show a 2-fold suppression of TGFßR-1.


Assuntos
Carcinoma de Células Escamosas , Sistemas de Liberação de Medicamentos , Nanopartículas , Oligonucleotídeos , RNA Interferente Pequeno , Dióxido de Silício , Neoplasias Cutâneas , Administração Cutânea , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Humanos , Camundongos , Camundongos SCID , Nanopartículas/química , Nanopartículas/uso terapêutico , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , Porosidade , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
ACS Appl Mater Interfaces ; 11(38): 34634-34644, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31475516

RESUMO

Intravenous (IV) route is the most commonly used drug-delivery approach. However, the targeting efficiency to tumor through IV delivery is usually less than 10%. To address this limitation, we report a new systemic delivery method utilizing injectable and quadruple-functional hydrogels to improve targeting efficiency through passive, active, and magnetic targeting, and hydrogel-controlled sustained release. The hydrogels consist of a folate/polyethylenimine-conjugated poly(organophosphazene) polymer, which encapsulates small interfering RNA (siRNA) and Au-Fe3O4 nanoparticles to form a nanocapsule (NC) structure by a simple mixing. The hydrogels are localized as a long-term "drug-release depot" after a single subcutaneous injection and sol-gel phase transition. NCs released from the hydrogels enter the circulatory systems and then target the tumor through enhanced permeability and retention/folate/magnetism triple-targeting, over the course of circulation, itself prolonged by the controlled release. In vivo experiments show that 12% of NCs are successfully delivered to the tumor, which is a considerable improvement compared to most results through IV delivery. The sustained targeting of gold to tumor enables two cycles of photothermal therapy, resulting in an enhanced silencing effect of siRNA and considerable reduction of tumor volume, which we are unable to achieve via simple intravenous injection.


Assuntos
Hidrogéis , Hipertermia Induzida , Neoplasias Experimentais , Fototerapia , Administração Intravenosa , Animais , Linhagem Celular Tumoral , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Feminino , Óxido Ferroso-Férrico/química , Óxido Ferroso-Férrico/farmacologia , Ouro/química , Ouro/farmacologia , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Molecules ; 24(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509944

RESUMO

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.


Assuntos
Anticorpos/farmacologia , Imunoconjugados/química , Oligonucleotídeos Antissenso/imunologia , RNA Interferente Pequeno/imunologia , Anticorpos/química , Anticorpos/imunologia , Azidas/química , Linhagem da Célula/efeitos dos fármacos , Reação de Cicloadição , Ciclo-Octanos/química , Sistemas de Liberação de Medicamentos , Endossomos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Oligonucleotídeos Antissenso/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Peptídeos/química , Peptídeos/farmacologia , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/química , Transglutaminases/química , Transglutaminases/imunologia , Transglutaminases/farmacologia
14.
Pharm Res ; 36(10): 142, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31376020

RESUMO

BACKGROUND: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. METHODS: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. RESULTS: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 µm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. CONCLUSION: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.


Assuntos
Dessecação/métodos , Lipídeos/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , RNA Interferente Pequeno/química , Administração por Inalação , Animais , Composição de Medicamentos , Excipientes/química , Inativação Gênica , Técnicas de Transferência de Genes , Manitol/química , Camundongos , Nanomedicina , Tamanho da Partícula , Pós , Células RAW 264.7 , RNA Interferente Pequeno/administração & dosagem , Solubilidade , Solventes/química
15.
Int J Pharm ; 569: 118585, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31376467

RESUMO

In this work, we implemented a supramolecular approach in order to combine photodynamic therapy (PDT) with gene therapy. We made use of a simple cationic guanidylated porphyrin (H2­PG) with the hypothesis that porphyrin aggregates should be capable of complexing siRNA through multivalent interactions and thus contribute to its intracellular delivery, while remaining active photosensitizers for PDT. The PDT effect of H2­PG was shown by incubating human breast cancer cells (MDA-MB-231) with H2­PG followed by light-irradiation at 405 nm. On the other hand, while siRNA do not enter cells alone, we showed, by fluorescence confocal microscopy and flow cytometry, that H2­PG promotes the internalization of Atto-488 siRNA. Finally, studying the combined PDT and delivery of siRNA directed against inhibitory apoptotic protein (IAP) family, we found an additive effect of the two therapies, thereby demonstrating that H2­PG is capable of acting both as a photosensitizer and supramolecular siRNA vector.


Assuntos
Inativação Gênica , Fotoquimioterapia , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Terapia Genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Fármacos Fotossensibilizantes/química , Porfirinas/química , RNA Interferente Pequeno/química
16.
Int J Pharm ; 569: 118606, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31415879

RESUMO

Lipid-based nanoparticles, a potential nonviral vector due to their good biocompatibility and biodegradability, have been extensively developed for the delivery of small interfering RNA (siRNA). We designed a unique pH-responsive lipid derivative, a dioleylphosphate-diethylenetriamine conjugate (DOP-DETA). DOP-DETA consists of a pH-responsive triamine and unsaturated fatty acids that accelerate membrane fusion. Our results showed that DOP-DETA-based liposomes (DL) efficiently delivered siRNA into the cytoplasm and induced RNA interference even at a low siRNA concentration. The knockdown efficiency of DL depended on the molar ratio of total DL lipids to siRNA. When siRNA was formulated with a sufficient amount of DL, it was efficiently taken up by cells and induced effective gene silencing. Time-lapse imaging showed that siRNA transfected with DL was rapidly internalized into the cells and uniformly dispersed in the cytoplasm within a few minites. The results also showed that DL induced sufficient change in surface charge to allow it to interact with the cell membrane and to allow for rapid endosomal escape. Uptake pathway and time-lapse imaging studies revealed that siRNA was delivered by DL into the cytoplasm, possibly through both macropinocytosis and membrane fusion. The present results emphasize that the modulation of surface charge on nanoparticles is crucial for each siRNA delivery process. Our results also suggest that DL is a potentially useful vector for inducing gene silencing with low-doses of siRNA.


Assuntos
RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Citoplasma/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Concentração de Íons de Hidrogênio , Lipídeos/administração & dosagem , Lipídeos/química , Lipossomos , Interferência de RNA , RNA Interferente Pequeno/química
17.
Adv Mater ; 31(41): e1902251, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31465135

RESUMO

T cells help regulate immunity, which makes them an important target for RNA therapies. While nanoparticles carrying RNA have been directed to T cells in vivo using protein- and aptamer-based targeting ligands, systemic delivery to T cells without targeting ligands remains challenging. Given that T cells endocytose lipoprotein particles and enveloped viruses, two natural systems with structures that can be similar to lipid nanoparticles (LNPs), it is hypothesized that LNPs devoid of targeting ligands can deliver RNA to T cells in vivo. To test this hypothesis, the delivery of siRNA to 9 cell types in vivo by 168 nanoparticles using a novel siGFP-based barcoding system and bioinformatics is quantified. It is found that nanomaterials containing conformationally constrained lipids form stable LNPs, herein named constrained lipid nanoparticles (cLNPs). cLNPs deliver siRNA and sgRNA to T cells at doses as low as 0.5 mg kg-1 and, unlike previously reported LNPs, do not preferentially target hepatocytes. Delivery occurs via a chemical composition-dependent, size-independent mechanism. These data suggest that the degree to which lipids are constrained alters nanoparticle targeting, and also suggest that natural lipid trafficking pathways can promote T cell delivery, offering an alternative to active targeting approaches.


Assuntos
Portadores de Fármacos/química , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismo , Animais , Ligantes , Camundongos , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética
18.
Nat Biotechnol ; 37(8): 884-894, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375812

RESUMO

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.


Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Huntingtina/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Animais , Proteína Huntingtina/genética , Camundongos , Mutação , RNA Mensageiro , RNA Interferente Pequeno/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-31298608

RESUMO

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N6-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N6-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.


Assuntos
Adenosina/análogos & derivados , Interferência de RNA/imunologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Adenosina/química , Sequência de Bases , Expressão Gênica , Células HeLa , Humanos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo
20.
Chem Commun (Camb) ; 55(62): 9112-9115, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298670

RESUMO

We designed novel 4'-C-guanidinocarbohydrazidomethyl-5-methyl uridine (GMU) modified small interfering RNA (siRNA) and evaluated its biophysical and biochemical properties. Incorporation of GMU units significantly increased the thermodynamic stability as well as the enzymatic stability against nucleases in human serum. A gene silencing experiment indicated that GMU modfied siRNA (siRNA6) resulted in ≈4.9-fold more efficient knockdown than unmodified siRNA.


Assuntos
Guanidina/química , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Guanidina/análogos & derivados , Modelos Moleculares , RNA Interferente Pequeno/genética , Ribonucleases/sangue , Ribonucleases/metabolismo , Termodinâmica
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