RESUMEN
Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood-brain/blood-tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM.
Asunto(s)
Apoptosis/genética , Diferenciación Celular/genética , Glioblastoma/genética , MicroARNs/metabolismo , Animales , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/fisiopatología , Humanos , Ratones , Ratones SCID , MicroARNs/administración & dosificación , MicroARNs/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Análisis de SupervivenciaRESUMEN
We report the development of the multiplexed nanoflare, a nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are spherical nucleic acid (SNA) gold nanoparticle (Au NP) conjugates consisting of densely packed and highly oriented oligonucleotide sequences, many of which are hybridized to a reporter with a distinct fluorophore label and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nanoflares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.
Asunto(s)
Colorantes Fluorescentes , Oro/química , Proteínas Inhibidoras de la Apoptosis/genética , Nanopartículas del Metal/química , ARN Mensajero/análisis , ARN Mensajero/genética , Citometría de Flujo , Humanos , Nanopartículas , Neoplasias/genética , Hibridación de Ácido Nucleico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Células Tumorales CultivadasRESUMEN
We report the development of a novel Scanometric MicroRNA (Scano-miR) platform for the detection of relatively low abundance miRNAs with high specificity and reproducibility. The Scano-miR system was able to detect 1 fM concentrations of miRNA in serum with single nucleotide mismatch specificity. Indeed, it provides increased sensitivity for miRNA targets compared to molecular fluorophore-based detection systems, where 88% of the low abundance miRNA targets could not be detected under identical conditions. The application of the Scano-miR platform to high density array formats demonstrates its utility for high throughput and multiplexed miRNA profiling from various biological samples. To assess the accuracy of the Scano-miR system, we analyzed the miRNA profiles of samples from men with prostate cancer (CaP), the most common noncutaneous malignancy and the second leading cause of cancer death among American men. The platform exhibits 98.8% accuracy when detecting deregulated miRNAs involved in CaP, which demonstrates its potential utility in profiling and identifying clinical and research biomarkers.
Asunto(s)
Oro/química , MicroARNs/sangre , Nanopartículas/química , Ácidos Nucleicos/química , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Neoplasias de la Próstata/sangre , Diseño de Equipo , Perfilación de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Sensibilidad y EspecificidadRESUMEN
Introduction: Tumor immunotherapy is designed to control malignancies through the host immune response but requires circumventing tumor-dysregulated immunomodulation through immunostimulation, relieving immunorepression, or a combination of both approaches. Here we designed and characterized cavrotolimod (formerly AST-008), an immunostimulatory spherical nucleic acid (SNA) compound targeting Toll-like receptor 9 (TLR9). We assessed the safety and pharmacodynamic (PD) properties of cavrotolimod in healthy participants in a first-in-human Phase 1 study under protocol AST-008-101 (NCT03086278; https://clinicaltrials.gov/ct2/show/NCT03086278). Methods: Healthy participants aged 18 to 40 years were enrolled to evaluate four dose levels of cavrotolimod across four cohorts. Each cohort included four participants, and all received a single subcutaneous dose of cavrotolimod. The dose levels were 5, 10, 12.5 and 18.8 µg/kg. Results and discussion: Cavrotolimod was well tolerated and elicited no serious adverse events or dose limiting toxicities at the doses tested. The results demonstrated that cavrotolimod is a potent innate immune activator, specifically stimulating Th1-type immune responses, and exhibits PD properties that may result in anti-tumor effects in patients with cancer. This study suggests that cavrotolimod is a promising clinical immunotherapy agent.
Asunto(s)
Neoplasias , Ácidos Nucleicos , Humanos , Ácidos Nucleicos/uso terapéutico , Receptor Toll-Like 9 , Voluntarios Sanos , Neoplasias/patología , Adyuvantes Inmunológicos/uso terapéuticoRESUMEN
We report a method for increasing the rate of target hybridization on DNA-functionalized surfaces using a short internal complement DNA (sicDNA) strand. The sicDNA causes up to a 5-fold increase in association rate by inducing a conformational change that extends the DNA away from the surface, making it more available to bind target nucleic acids. The sicDNA-induced kinetic enhancement is a general phenomenon that occurred with all sequences and surfaces investigated. Additionally, the process is selective and can be used in multicomponent systems to controllably and orthogonally "turn on" specific sequences by the addition of the appropriate sicDNA. Finally, we show that sicDNA is compatible with systems used in gene regulation, intracellular detection, and microarrays, suggesting several potential therapeutic, diagnostic, and bioinformatic applications.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico/métodos , Oro/química , Cinética , Nanopartículas del Metal/química , Propiedades de SuperficieRESUMEN
Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NP entry in HeLa cells. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NP entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species, as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles.
Asunto(s)
ADN/metabolismo , Endocitosis , Oligonucleótidos/química , Oligonucleótidos/farmacocinética , Receptores Depuradores/metabolismo , Animales , Transporte Biológico , ADN/química , Silenciador del Gen , Técnicas de Transferencia de Gen , Oro/química , Células HeLa , Humanos , Ratones , Nanopartículas/química , Poli I/farmacología , Polisacáridos/farmacología , ARN Interferente Pequeño/metabolismo , Receptores Depuradores/antagonistas & inhibidores , Albúmina Sérica Bovina/metabolismo , Gravedad Específica , Transferrina/metabolismoRESUMEN
Gold colloids have fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Animales , Medios de Contraste/química , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Humanos , Fármacos Fotosensibilizantes/química , ARN sin Sentido/metabolismoRESUMEN
The activation of T helper 17 signaling plays a critical role in psoriasis pathogenesis, and systemically-administered IL-17 inhibitors are highly effective therapy for moderate-to-severe disease. We generated topically-delivered gene-regulating nanoconstructs, comprised of spherically-arrayed antisense DNA (liposomal spherical nucleic acids [L-SNAs]), which are able to penetrate human skin to knock down cutaneous gene targets. Topically-applied L-SNAs targeting the gene encoding the mouse IL-17A receptor (Il17ra) reversed the development of psoriasis clinically, histologically, and transcriptionally in imiquimod-treated psoriasis-like mouse skin. Il17ra L-SNAs reduced the modified PASI by 74% versus controls and decreased epidermal thickness by 56%. Il17ra L-SNA reduced Il17ra protein expression by 75% and significantly decreased the mRNA expression of psoriasis markers, including Defb4, Il17c, S100a7, Pi3, Krt16, and Tnfa versus scrambled spherical nucleic acid (Scr SNA) controls. A human IL17RA L-SNA penetrates 3-dimensional cultures and normal human explants to knock down IL17RA mRNA by 63% and 66%, respectively. After topical application to psoriatic 3-dimensional rafts, anti-human IL17RA L-SNAs reduced the expression of IL17RA (by 72%) and the IL-17-induced genes IL17C (by 85%), DEFB4 (by 83%), TNFA (by 77%), and PI3 (by 65%) versus scrambled L-SNA and vehicle controls (all P < 0.001). Taken together, these data suggest that targeted suppression of IL17RA is a promising new topical treatment strategy for psoriasis.
Asunto(s)
ADN sin Sentido/administración & dosificación , Nanosferas/administración & dosificación , Psoriasis/tratamiento farmacológico , ARN Mensajero/efectos de los fármacos , Receptores de Interleucina-17/antagonistas & inhibidores , Administración Cutánea , Animales , Biomarcadores/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Imiquimod/inmunología , Queratinocitos , Liposomas , Ratones , Cultivo Primario de Células , Psoriasis/inducido químicamente , Psoriasis/diagnóstico , Psoriasis/inmunología , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/inmunología , Índice de Severidad de la Enfermedad , Piel/citología , Piel/efectos de los fármacos , Piel/inmunología , Piel/patologíaRESUMEN
Amine-functionalized polyvalent oligonucleotide gold nanoparticles (DNA-Au NPs) were derivatized with a cisplatin prodrug, and the resulting DNA-Au NP conjugates were used to internalize multiple platinum centers. A platinum(IV) complex, c,c,t-[Pt(NH(3))(2)Cl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)], was tethered to the surface of DNA-Au NPs through amide linkages. The platinum-tethered gold nanoparticles were taken into several cancer cells. The drop in intracellular pH facilitated reductive release of cisplatin from the prodrug, which then formed 1,2-d(GpG) intrastrand cross-links in the cell nuclei, as confirmed by an antibody specific for this adduct. The cytotoxicity of the platinum(IV) complex increases significantly in several cancer cell lines when the complex is attached to the surface of the DNA-Au NPs and in some instances exceeds that of cisplatin.
Asunto(s)
Portadores de Fármacos/química , Oro/química , Nanopartículas del Metal/química , Oligonucleótidos/química , Platino (Metal)/química , Línea Celular Tumoral , ADN/química , Humanos , Platino (Metal)/metabolismo , Platino (Metal)/farmacología , Profármacos/químicaRESUMEN
We report the development of a novel colorimetric nitrite and nitrate ion assay based upon gold nanoparticle probes functionalized with Griess reaction reagents. This assay takes advantage of the distance-dependent plasmonic properties of the gold nanoparticles and the ability of nitrite ion to facilitate the cross coupling of novel nanoparticle probes modified with aniline and naphthalene moieties. The assay works on the concept of a kinetic end point and can be triggered at the EPA limit for this ion in drinking water (highlighted in red, microM). This rapid and simple assay could be useful for on-site water quality monitoring.
Asunto(s)
Colorimetría/métodos , Nitratos/análisis , Nitritos/análisis , Contaminantes Químicos del Agua/análisis , Oro , Cinética , Nanopartículas del Metal/química , Técnicas de Sonda Molecular , Sondas Moleculares/químicaRESUMEN
We report the synthesis of high density lipoprotein (HDL) biomimetic nanoparticles capable of binding cholesterol. These structures use a gold nanoparticle core to template the assembly of a mixed phospholipid layer and the adsorption of apolipoprotein A-I. These synthesized structures have the general size and surface composition of natural HDL and, importantly, bind free cholesterol (K(d) = 4 nM). The determination of the K(d) for these particles, with respect to cholesterol complexation, provides a key starting and comparison point for measuring and evaluating the properties of subsequently developed synthetic versions of HDL.
Asunto(s)
Lipoproteínas HDL/química , Nanopartículas del Metal/química , Oro/química , Microesferas , Estereoisomerismo , Agua/químicaRESUMEN
We report the use of electroless gold deposition as a light scattering signal enhancer in a multiplexed, microarray-based scanometric immunoassay using gold nanoparticle probes. The use of gold development results in greater signal enhancement than the typical silver development, and multiple rounds of metal development were found to increase the resulting signal compared to one development. Using these conditions, the assay was capable of detecting 300 aM (approximately 9000 copies) of prostate specific antigen in buffer and 3 fM in 10% serum. Additionally, the highly selective detection of three protein cancer markers at low picomolar concentrations in buffer and 10% serum was demonstrated. The use of gold deposition may have significant utility in scanometric detection schemes and broader clinical and research applications.
Asunto(s)
Oro/química , Inmunoensayo/métodos , Nanopartículas del Metal/química , Proteínas/análisis , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/metabolismo , Gonadotropina Coriónica/análisis , Humanos , Masculino , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/sangre , Análisis por Matrices de Proteínas , alfa-Fetoproteínas/análisisRESUMEN
We report a new assay for human telomerase activity that relies on polyvalent oligonucleotide nanoparticle conjugates as diagnostic probes and amplification units. Gold nanoparticles functionalized with specific oligonucleotide sequences can efficiently capture telomerase enzymes and subsequently be elongated. Both the elongated and unmodified oligonucleotide sequences are simultaneously measured. The two strands not only serve as internal positive controls for each other but also provide a way of amplifying signal. At high concentrations, both elongated and unmodified strands exhibit measurable responses. At low telomerase concentrations (e.g., from 10 HeLa cells), elongated strands cannot be detected, but the unmodified sequences, which come from the same probe particles, can be detected because their concentration is higher, providing a novel form of amplification. This new assay rivals the sensitivity of the conventional PCR-based method of telomerase detection.
Asunto(s)
Nanopartículas del Metal/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/química , Telomerasa/metabolismo , Amplificación de Genes , Oro/química , Células HeLa , Humanos , Luz , Oligonucleótidos/genética , ARN/genética , Dispersión de Radiación , Sensibilidad y Especificidad , Telomerasa/análisis , Telomerasa/genéticaAsunto(s)
Colorimetría/métodos , Cobre/análisis , Nanopartículas del Metal/química , ADN/química , Oro/químicaRESUMEN
Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many "undruggable" oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)-based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)--an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas--were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.
Asunto(s)
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Nanopartículas/química , Ácidos Nucleicos/química , Interferencia de ARN , Animales , Apoptosis , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones SCID , Proteínas Musculares/metabolismo , Ácidos Nucleicos/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This communication reports the use of click chemistry to site-specifically conjugate bioluminescent Renilla luciferase proteins to gold nanoparticles (Au NPs) for sensing protease activity. The bioluminescent emission from luciferase was efficiently quenched by Au NPs, but significantly recovered after the proteolytic cleavage.