RESUMEN
Bisphosphonates (BPs) are a class of bone resorptive drug with a high affinity for the hydroxyapatite structure of bone matrices that are used for the treatment of osteoporosis. However, clinical application is limited by a common toxicity, BP-related osteonecrosis of the jaw. There is emerging evidence that BPs possess anticancer potential, but exploitation of these antiproliferative properties is limited by their toxicities. We previously reported the utility of a cationic amphipathic fusogenic peptide, RALA, to traffic anionic nucleic acids into various cell types in the form of cationic nanoparticles. We hypothesized that complexation with RALA could similarly be used to conceal a BP's hydroxyapatite affinity, and to enhance bioavailability, thereby improving anticancer efficacy. Incubation of RALA with alendronate, etidronate, risedronate, or zoledronate provoked spontaneous electrostatic formation of cationic nanoparticles that did not exceed 100 nm in diameter and that were stable over a range of temperatures and for up to 6 h. The nanoparticles demonstrated a pH responsiveness, possibly indicative of a conformational change, that could facilitate release of the BP cargo in the endosomal environment. RALA/BP nanoparticles were more potent anticancer agents than their free BP counterparts in assays investigating the viability of PC3 prostate cancer and MDA-MB-231 breast cancer cells. Moreover, RALA complexation potentiated the tumor growth delay activity of alendronate in a PC3 xenograft model of prostate cancer. Taken together, these findings further validate the use of BPs as repurposed anticancer agents.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Difosfonatos/química , Nanopartículas/química , Péptidos/química , Péptidos/farmacología , Alendronato/química , Alendronato/farmacología , Alendronato/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Conservadores de la Densidad Ósea/química , Conservadores de la Densidad Ósea/farmacología , Conservadores de la Densidad Ósea/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Humanos , Imidazoles/química , Imidazoles/farmacología , Masculino , Ratones , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido ZoledrónicoRESUMEN
Microneedle technology provides the opportunity for the delivery of DNA therapeutics by a non-invasive, patient acceptable route. To deliver DNA successfully requires consideration of both extra and intracellular biological barriers. In this study we present a novel two tier platform; i) a peptide delivery system, termed RALA, that is able to wrap the DNA into nanoparticles, protect the DNA from degradation, enter cells, disrupt endosomes and deliver the DNA to the nucleus of cells ii) a microneedle (MN) patch that will house the nanoparticles within the polymer matrix, breach the skin's stratum corneum barrier and dissolve upon contact with skin interstitial fluid thus releasing the nanoparticles into the skin. Our data demonstrates that the RALA is essential for preventing DNA degradation within the poly(vinylpyrrolidone) (PVP) polymer matrix. In fact the RALA/DNA nanoparticles (NPs) retained functionality when in the MN arrays after 28days and over a range of temperatures. Furthermore the physical strength and structure of the MNs was not compromised when loaded with the NPs. Finally we demonstrated the effectiveness of our MN-NP platform in vitro and in vivo, with systemic gene expression in highly vascularised regions. Taken together this 'smart-system' technology could be applied to a wide range of genetic therapies.
Asunto(s)
Péptidos de Penetración Celular/química , ADN/administración & dosificación , Técnicas de Transferencia de Gen/instrumentación , Nanopartículas/química , Agujas , Plásmidos/administración & dosificación , Administración Cutánea , Animales , Línea Celular , Péptidos de Penetración Celular/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Femenino , Expresión Génica , Humanos , Ratones Endogámicos C57BL , Nanopartículas/metabolismo , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Povidona/química , Povidona/metabolismo , Piel/metabolismo , PorcinosRESUMEN
Designer biopolymers (DBPs) represent state of the art genetically engineered biomacromolecules designed to condense plasmid DNA, and overcome intra- and extra- cellular barriers to gene delivery. Three DBPs were synthesized, each with the tumor molecular targeting peptide-1 (TMTP-1) motif to specifically target metastases. Each DBP was complexed with a pEGFP-N1 reporter plasmid to permit physiochemical and biological assay analysis. Results indicated that two of the biopolymers (RMHT and RM3GT) effectively condensed pEGFP-N1 into cationic nanoparticles <100 nm and were capable of transfecting PC-3 metastatic prostate cancer cells. Conversely the anionic RMGT DBP nanoparticles could not transfect PC-3 cells. RMHT and RM3GT nanoparticles were stable in the presence of serum and protected the cargo from degradation. Additionally it was concluded that cell viability could recover post-transfection with these DBPs, which were less toxic than the commercially available transfection reagent Lipofectamine(®) 2000. With both DBPs, a higher transfection efficacy was observed in PC-3 cells than in the moderately metastatic, DU145, and normal, PNT2-C2, cell lines. Blocking of the TMTP-1 receptors inhibited gene transfer indicating internalization via this receptor. In conclusion RMHT and RM3GT are fully functional DBPs that address major obstacles to gene delivery and target metastatic cells expressing the TMTP-1 receptor.
Asunto(s)
Biopolímeros/administración & dosificación , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Nanopartículas/administración & dosificación , Oligopéptidos/metabolismo , Biopolímeros/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/química , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Nanopartículas/química , Plásmidos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismoRESUMEN
The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were <100nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.