RESUMEN
Abnormal scarring is a consequence of dysregulation in the wound healing process, with limited options for effective and noninvasive therapies. Given the ability of spherical nucleic acids (SNAs) to penetrate skin and regulate gene expression within, we investigated whether gold-core SNAs (AuSNAs) and liposome-core SNAs (LSNAs) bearing antisense oligonucleotides targeting transforming growth factor beta 1 (TGF-ß1) can function as a topical therapy for scarring. Importantly, both SNA constructs appreciably downregulated TGF-ß1 protein expression in primary hypertrophic and keloid scar fibroblasts in vitro. In vivo, topically applied AuSNAs and LSNAs downregulated TGF-ß1 protein expression levels and improved scar histology as determined by the scar elevation index. These data underscore the potential of SNAs as a localized, self-manageable treatment for skin-related diseases and disorders that are driven by increased gene expression.
RESUMEN
RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Nanoconjugados/administración & dosificación , ARN Interferente Pequeño/genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos Alquilantes/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Femenino , Fluorescencia , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Ratones SCID , Nanoconjugados/química , Interferencia de ARN , Temozolomida , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Micelas , Ácidos Nucleicos/química , Temperatura , Supervivencia Celular , Células HEK293 , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Small-sized (â¼65 nm) doxorubicin (Dox)-loaded polymeric nanoparticles (PNPs) were modified with oligonucleotides to form colloidally stable Dox-loaded polymeric spherical nucleic acid (Dox-PSNA) nanostructures in biological media. The nucleic acid shell facilitates the cellular uptake of Dox-PSNA, which results in in vitro cytotoxicity against SKOV3 cancer cells.
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Antibióticos Antineoplásicos/farmacología , ADN/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Nanopartículas/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Polímeros/química , Antibióticos Antineoplásicos/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Femenino , Humanos , Nanopartículas/química , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
Emerging evidence indicates that long noncoding RNAs (lncRNAs) are actively involved in a number of developmental and tumorigenic processes. Here, the authors describe the first successful use of spherical nucleic acids as an effective nanoparticle platform for regulating lncRNAs in cells; specifically, for the targeted knockdown of the nuclear-retained metastasis associated lung adenocarcinoma transcript 1 (Malat1), a key oncogenic lncRNA involved in metastasis of several cancers. Utilizing the liposomal spherical nucleic acid (LSNA) constructs, the authors first explored the delivery of antisense oligonucleotides to the nucleus. A dose-dependent inhibition of Malat1 upon LSNA treatment as well as the consequent up-regulation of tumor suppressor messenger RNA associated with Malat1 knockdown are shown. These findings reveal the biologic and therapeutic potential of a LSNA-based antisense strategy in targeting disease-associated, nuclear-retained lncRNAs.
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Liposomas/metabolismo , Ácidos Nucleicos/metabolismo , Células A549 , Núcleo Celular , Humanos , Oligonucleótidos Fosforotioatos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
We report a strategy for creating a new class of protein transfection materials composed of a functional protein core chemically modified with a dense shell of oligonucleotides. These materials retain the native structure and catalytic ability of the hydrolytic enzyme ß-galactosidase, which serves as the protein core, despite the functionalization of its surface with â¼25 DNA strands. The covalent attachment of a shell of oligonucleotides to the surface of ß-galactosidase enhances its cellular uptake of by up to â¼280-fold and allows for the use of working concentrations as low as 100 pM enzyme. DNA-functionalized ß-galactosidase retains its ability to catalyze the hydrolysis of ß-glycosidic linkages once endocytosed, whereas equal concentrations of protein show little to no intracellular catalytic activity.
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ADN/química , beta-Galactosidasa/administración & dosificación , Catálisis , Línea Celular , Humanos , Espectrofotometría Ultravioleta , beta-Galactosidasa/químicaRESUMEN
Limiting the precise study of the biochemical impact of whole molecule extracellular matrix (ECM) proteins on stem cell differentiation is the lack of 3D in vitro models that can accommodate many different types of ECM. Here we sought to generate such a system while maintaining consistent mechanical properties and supporting stem cell survival. To this end, we used native chemical ligation to cross-link poly(ethylene glycol) macromonomers under mild conditions while entrapping ECM proteins (termed ECM composites) and stem cells. Sufficiently low concentrations of ECM were used to maintain constant storage moduli and pore size. Viability of stem cells in composites was maintained over multiple weeks. ECM of composites encompassed stem cells and directed the formation of distinct structures dependent on ECM type. Thus, we introduce a powerful approach to study the biochemical impact of multiple ECM proteins (either alone or in combination) on stem cell behavior.
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Proteínas de la Matriz Extracelular/química , Hidrogeles/química , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Mesenquimatosas/fisiología , Animales , Técnicas de Cultivo de Célula , Supervivencia Celular , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Medios de Cultivo , Humanos , Ratones , Microscopía Fluorescente , Polietilenglicoles/química , Porosidad , Termogravimetría , Sustancias Viscoelásticas/química , ViscosidadRESUMEN
The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts(1-4). More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation(5-7). Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease. Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo(8-15). We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system(16, 17), we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo. To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.