RESUMEN
Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) - nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior - have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.
RESUMEN
In this work, we report a component-minimal spherical nucleic acid (SNA) from monodisperse DNA-polymer conjugates that can load and release nucleic acid therapeutics in a stimuli-responsive manner. We show that this vehicle assembles from only four strands, and conditional release of its antisense therapeutic cargo can be induced upon recognition of specific oligonucleotide triggers via strand displacement. The latter (triggers) may be a microRNA that offers additional synergistic therapy, in addition to the previously shown ability of the SNA to load hydrophobic drugs. The SNA is easy to prepare, has dynamic character, releases its cargo only upon the presence of both triggers, and can survive biological conditions while protecting its cargo. The gene silencing potency of the cargo was tested in live cells and shown to be suppressed when loaded in the SNA, and its activity was restored only upon release with the two triggers. This vehicle has the essential characteristics of versatility, ease of synthesis, low cost, highly responsive behavior, and ability to support combination therapies, making it a promising candidate for cell-selective drug delivery and clinical transition.
Asunto(s)
Portadores de Fármacos/química , Ácidos Nucleicos/química , Oligonucleótidos Antisentido/química , Polímeros/química , Carbocianinas/química , Transferencia Resonante de Energía de Fluorescencia , Silenciador del Gen , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Nanotecnología , Oligonucleótidos Antisentido/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismoRESUMEN
RNA has inherent therapeutic and structural properties that make it an important component of biologically-functional nanoparticles. Using DNA-amphiphiles as synthetic templates, we report the synthesis of two classes of RNA-amphiphiles that self-assemble into spherical nanoparticles in aqueous solution and show gene silencing activity.
Asunto(s)
Silenciador del Gen , Nanopartículas/química , ARN/síntesis química , ARN/genética , Células HeLa , Humanos , Estructura Molecular , ARN/químicaRESUMEN
We report a spherical nucleic acid (SNA) system for the delivery of BKM120, an anticancer drug for treatment of chronic lymphocytic leukemia (CLL). While promising for cancer treatment, this drug crosses the blood-brain barrier causing significant side-effects in patients. The DNA nanoparticle encapsulates BKM120 in high efficiency, and is unparalleled in its monodispersity, ease of synthesis and stability in different biological media and in serum. These DNA nanostructures demonstrate efficient uptake in human cervical cancer (HeLa) cells, and increased internalization of cargo. In vitro studies show that BKM120-loaded nanoparticles promote apoptosis in primary patient CLL lymphocytes, and act as sensitizers of other antitumor drugs, without causing non-specific inflammation. Evaluation of this drug delivery system in vivo shows long circulation times up to 24 hours, full body distribution, accumulation at tumor sites and minimal leakage through the blood-brain barrier. Our results demonstrate the great potential of these delivery vehicles as a general platform for chemotherapeutic drug delivery.
RESUMEN
Correction for 'Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown' by Johans J. Fakhoury, et al., Nanoscale, 2015, 7, 20625-20634.
RESUMEN
Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.
Asunto(s)
ADN sin Sentido , Técnicas de Silenciamiento del Gen/métodos , Silenciador del Gen , Polietileneimina , ARN Interferente Pequeño , Transfección/métodos , ADN sin Sentido/química , ADN sin Sentido/genética , ADN sin Sentido/farmacología , Células HeLa , Humanos , Polietileneimina/química , Polietileneimina/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
RNA interference (RNAi) is a powerful therapeutic strategy that induces gene silencing by targeting disease-causing mRNA and can lead to their removal through degradation pathways. The potential of RNAi is especially relevant in cancer therapy, as it can be designed to regulate the expression of genes involved in all stages of tumor development (initiation, growth, and metastasis). We have generated gene silencing 3D DNA prisms that integrate antisense oligonucleotide therapeutics at 1, 2, 4, and 6 positions. Synthesis of these structures is readily achieved and leads to the assembly of highly monodisperse and well-characterized structures. We have shown that antisense strands scaffolded on DNA cages can readily induce gene silencing in mammalian cells and maintain gene knockdown levels more effectively than single and double stranded controls through increased stability of bound antisense units.