RESUMEN
Few studies reported the application of miRNA in bone regeneration. In this study, the expression of miR133a and miR133b in murine BMSCs was inhibited via antagomiR-133a/b and the osteogenic differentiation in murine BMSCs was evaluated. The RT-PCR, flow cytometry, cell counting kit-8, and annexin V-FITC/PI double staining assays were performed. Double knockdown miR133a and miR133b can promote BMSC osteogenic differentiation. At optimum N/P ration (15:1), the loading efficiency can reach over 90%. CTH-antagomiR-133a/b showed no cytotoxicity to BMSCs and diminished miR133a and miR133b expression in BMSCs. Furthermore, chitosan-based sustained delivery system can facilitate continuous dosing of antagomiR-133a/b, which enhanced calcium deposition and osteogenic specific gene expression in vitro. The new bone formation was enhanced after the sustained delivery system containing CTH-antagomiR-133a/b nanoparticles was used in mouse calvarial bone defect model. Our results demonstrate that CTH nanoparticles could facilitate continuous dosing of antagomiR133a/b, which can promote osteogenic differentiation.
Asunto(s)
Antagomirs , Regeneración Ósea/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , MicroARNs/antagonistas & inhibidores , Nanopartículas/química , Osteogénesis/efectos de los fármacos , Animales , Antagomirs/química , Antagomirs/farmacocinética , Antagomirs/farmacología , Células de la Médula Ósea , Regeneración Ósea/genética , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Preparaciones de Acción Retardada/farmacología , Técnicas de Silenciamiento del Gen , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Osteogénesis/genéticaRESUMEN
Despite the vital role miRNA-27a plays in driving the development and progress of liver cancer, miRNA-based inhibition therapy is hampered due to its undesired degradation and off-target effects. Herein, a multifunctional nanoparticle for noninvasive tracking of targeted delivery of anti-miR-27a oligonucleotides against liver cancer was constructed. Methods: Dual-fluorescent conjugates (QD-HA-PEI) were first fabricated through crosslinking hyaluronic acid (HA), polyethyleneimine (PEI) and near-infrared (NIR) fluorescent quantum dots (QDs) via a facile one-pot approach. Antisense oligonucleotide was then encapsulated by QD-HA-PEI to form anti-miR-27a/QD-HA-PEI via electrostatic interactions. Targeting, biodistribution, bioimaging, in vitro cytotoxicity and in vivo anti-tumor effects were evaluated and the underlying mechanism was studied. Results: The NIR fluorescence of anti-miR-27a/QD-HA-PEI could be employed to monitor CD44 receptor-targeted cellular uptake and tumor accumulation. Importantly, the intrinsic fluorescence of anti-miR-27a/QD-HA-PEI remained in the "ON" state in extracellular or blood environment, but switched to the "OFF" state in the intracellular environment, indicating pH-responsive oligonucleotide release. Furthermore, anti-miR-27a/QD-HA-PEI exhibited effective and selective anti-cancer effects in vitro and in vivo with fewer side effects via the direct down-regulation of oncogenic transcription factors FOXO1 and PPAR-γ. Conclusion: Our findings validate the dual-fluorescent nanoparticles as delivery vectors of therapeutic miRNA, capable of simultaneous tumor imaging and tracking of miRNA-based modulation therapy, thereby providing an efficient and safe approach for liver cancer theranostics.
Asunto(s)
Antagomirs/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , MicroARNs/antagonistas & inhibidores , Terapia Molecular Dirigida/métodos , Nanopartículas/administración & dosificación , Nanomedicina Teranóstica/métodos , Animales , Antagomirs/análisis , Antagomirs/farmacocinética , Antineoplásicos/análisis , Antineoplásicos/farmacocinética , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Hep G2 , Xenoinjertos , Humanos , Nanopartículas/análisis , Trasplante de Neoplasias , Imagen Óptica/métodos , Resultado del TratamientoRESUMEN
The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.