RESUMEN
Histone deacetylases (HDACs) are known to be key enzymes in cancer development and progression through their modulation of chromatin structure and the expression and post-translational modification of numerous proteins. Aggressive dedifferentiated tumors, like glioblastoma, frequently overexpress HDACs, while HDAC inhibition can lead to cell cycle arrest, promote cellular differentiation and induce apoptosis. Although multiple HDAC inhibitors, such as quisinostat, are of interest in oncology due to their potent in vitro efficacy, their failure in the clinic as monotherapies against solid tumors has been attributed to poor delivery. Thus, we were motivated to develop quisinostat loaded poly(D,L-lactide)-b-methoxy poly(ethylene glycol) nanoparticles (NPs) to test their ability to treat orthotopic glioblastoma. In developing our NP formulation, we identified a novel, pH-driven approach for achieving over 9% (w/w) quisinostat loading. We show quisinostat-loaded NPs maintain drug potency in vitro and effectively slow tumor growth in vivo, leading to a prolonged survival compared to control mice.
Asunto(s)
Glioblastoma/tratamiento farmacológico , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/uso terapéutico , Polietilenglicoles/química , Animales , Sistemas de Liberación de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , RatonesRESUMEN
Glioblastoma (GBM) is an aggressive primary brain tumor. The rapid growth and the privileged provenance of the tumor within the brain contribute to its aggressivity and poor therapeutic targeting. A poor prognostic factor in glioblastoma is the deletion or mutation of the Nf1 gene. This gene codes for the protein neurofibromin, a tumor suppressor gene that is known to interact with the collapsin response mediator protein 2 (CRMP2). CRMP2 expression and elevated expression of nuclear phosphorylated CRMP2 have recently been implicated in cancer progression. The CRMP2-neurofibromin interaction protects CRMP2 from its phosphorylation by cyclin-dependent kinase 5 (Cdk5), an event linked to cancer progression. In three human glioblastoma cell lines (GL15, A172, and U87), we observed an inverse correlation between neurofibromin expression and CRMP2 phosphorylation levels. Glioblastoma cell proliferation was dependent on CRMP2 expression and phosphorylation by Cdk5 and glycogen synthase kinase 3 beta (GSK3ß). The CRMP2 phosphorylation inhibitor (S)-lacosamide reduces, in a concentration-dependent manner, glioblastoma cell proliferation and induced apoptosis in all three GBM cell lines tested. Since (S)-lacosamide is bioavailable in the brain, we tested its utility in an in vivo orthotopic model of GBM using GL261-LucNeo glioma cells. (S)-lacosamide decreased tumor size, as measured via in vivo bioluminescence imaging, by ~54% compared to vehicle control. Our results introduce CRMP2 expression and phosphorylation as a novel player in GBM proliferation and survival, which is enhanced by loss of Nf1.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioblastoma/metabolismo , Glioblastoma/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/patología , Proliferación Celular/efectos de los fármacos , Humanos , Lacosamida/farmacología , Ratones Endogámicos C57BL , Neurofibromina 1/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
Understanding of the mechanisms by which systemically administered nanoparticles achieve delivery across biological barriers remains incomplete, due in part to the challenge of tracking nanoparticle fate in the body. Here, we develop a new approach for "barcoding" nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) with bright, spectrally defined quantum dots (QDs) to enable direct, fluorescent detection of nanoparticle fate with subcellular resolution. We show that QD labeling does not affect major biophysical properties of nanoparticles or their interaction with cells and tissues. Live cell imaging enabled simultaneous visualization of the interaction of control and targeted nanoparticles with bEnd.3 cells in a flow chamber, providing direct evidence that surface modification of nanoparticles with the cell-penetrating peptide TAT increases their biophysical association with cell surfaces over very short time periods under convective current. We next developed this technique for quantitative biodistribution analysis in vivo. These studies demonstrate that nanoparticle surface modification with the cell penetrating peptide TAT facilitates brain-specific delivery that is restricted to brain vasculature. Although nanoparticle entry into the healthy brain parenchyma is minimal, with no evidence for movement of nanoparticles across the blood-brain barrier (BBB), we observed that nanoparticles are able to enter to the central nervous system (CNS) through regions of altered BBB permeability - for example, into circumventricular organs in the brain or leaky vasculature of late-stage intracranial tumors. In sum, these data demonstrate a new, multispectral approach for barcoding PLGA, which enables simultaneous, quantitative analysis of the fate of multiple nanoparticle formulations in vivo.
Asunto(s)
Encéfalo/metabolismo , Péptidos de Penetración Celular , Ácido Láctico , Nanopartículas , Ácido Poliglicólico , Animales , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Productos del Gen tat , Células HEK293 , Humanos , Ácido Láctico/administración & dosificación , Ácido Láctico/química , Ácido Láctico/farmacocinética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/administración & dosificación , Nanopartículas/química , Fenómenos Ópticos , Ácido Poliglicólico/administración & dosificación , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Distribución TisularRESUMEN
In this work, we sought to test how surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with peptide ligand alters the brain specific delivery of encapsulated molecules. For biodistribution studies, nanoparticles modified with rabies virus glycoprotein (RVG29) were loaded with small molecule drug surrogates and administered to healthy mice by lateral tail vein injection. Mice were perfused 2h after injection and major anatomical regions of the CNS were dissected (striatum, midbrain, cerebellum, hippocampus, cortex, olfactory bulb, brainstem, and cervical, thoracic, lumbar and sacral spinal cord). For functional studies, surface modified nanoparticles were loaded with the chemotherapeutic camptothecin (CPT) and administered to mice bearing intracranial GL261-Luc2 gliomas. Outcome measures included tumor growth, as measured by bioluminescent imaging, and median survival time. We observed that small molecule delivery from PLGA nanoparticles varied by as much as 150% for different tissue regions within the CNS. These differences were directly correlated to regional differences in cerebral blood volume. Although the presence of RVG29 enhanced apparent brain delivery for multiple small molecule payloads, we observed minimal evidence for targeting to muscle or spinal cord, which are the known sites for rabies virus entry into the CNS, and enhancements in brain delivery were not prolonged due to an apparent aqueous instability of the RVG29 ligand. Furthermore, we have identified concerning differences in apparent delivery kinetics as measured by different payloads: nanoparticle encapsulated DiR was observed to accumulate in the brain, whereas encapsulated Nile red was rapidly cleared. Although systemically administered CPT loaded nanoparticles slowed the growth of orthotopic brain tumors to prolong survival, the presence of RVG29 did not enhance therapeutic efficacy compared to control nanoparticles. These data are consistent with a model of delivery of hydrophobic small molecules to the brain that does not rely on internalization of polymer nanoparticles in target tissue. We discuss an important risk for discordance between biodistribution, as typically measured by drug surrogate, and therapeutic outcome, as determined by clinically relevant measurement of drug function in a disease model. These results pose critical considerations for the methods used to design and evaluate targeted drug delivery systems in vivo.
Asunto(s)
Camptotecina/administración & dosificación , Sistema Nervioso Central/metabolismo , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Animales , Barrera Hematoencefálica , Línea Celular Tumoral , Femenino , Glicoproteínas/administración & dosificación , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fragmentos de Péptidos/administración & dosificación , Distribución Tisular , Proteínas Virales/administración & dosificaciónRESUMEN
Effective treatment of glioblastoma multiforme remains a major clinical challenge, due in part to the difficulty of delivering chemotherapeutics across the blood-brain barrier. Systemically administered drugs are often poorly bioavailable in the brain, and drug efficacy within the central nervous system can be limited by peripheral toxicity. Here, we investigate the ability of systemically administered poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) to deliver hydrophobic payloads to intracranial glioma. Hydrophobic payload encapsulated within PLGA NPs accumulated at â¼10× higher levels in tumor compared to healthy brain. Tolerability of the chemotherapeutic camptothecin (CPT) was improved by encapsulation, enabling safe administration of up to 20mg/kg drug when encapsulated within NPs. Immunohistochemistry staining for γ-H2AFX, a marker for double-strand breaks, demonstrated higher levels of drug activity in tumors treated with CPT-loaded NPs compared to free drug. CPT-loaded NPs were effective in slowing the growth of intracranial GL261 tumors in immune competent C57 albino mice, providing a significant survival benefit compared to mice receiving saline, free CPT or low dose CPT NPs (median survival of 36.5 days compared to 28, 32, 33.5 days respectively). In sum, these data demonstrate the feasibility of treating intracranial glioma with systemically administered nanoparticles loaded with the otherwise ineffective chemotherapeutic CPT.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Camptotecina/administración & dosificación , Glioma/tratamiento farmacológico , Ácido Láctico/química , Ácido Poliglicólico/química , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/toxicidad , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/patología , Camptotecina/farmacología , Camptotecina/toxicidad , Portadores de Fármacos/química , Estudios de Factibilidad , Glioma/patología , Interacciones Hidrofóbicas e Hidrofílicas , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Nanopartículas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Tasa de Supervivencia , Factores de TiempoRESUMEN
Rat models have emerged as a common tool to study neuroinflammation to intracortical microelectrodes. While a number of studies have attempted to understand the factors resulting in neuroinflammation using rat models, a complete understanding of key mechanistic pathways remains elusive. Transgenic mouse models, however, could facilitate a deeper understanding of mechanistic pathways due to an ease of genetic alteration. Therefore, the goal of the present study is to compare neuroinflammation following microelectrode implantation between the rat and the mouse model. Our study suggests that subtle differences in the classic neuroinflammatory markers exist between the animal models at both two and sixteen weeks post implantation. Most notably, neuronal densities surrounding microelectrodes were significantly lower in the rat model at two weeks, while similar densities were observed between the animal models at sixteen weeks. Physiological differences between the species and slight alterations in surgical methods are likely key contributors to the observed differences. Moving forward, we propose that differences in the time course of neuroinflammation between the animal models should be considered when trying to understand and prevent intracortical microelectrode failure.
Asunto(s)
Encéfalo/inmunología , Electrodos Implantados/efectos adversos , Encefalitis/etiología , Animales , Astrocitos/inmunología , Modelos Animales de Enfermedad , Encefalitis/inmunología , Inflamación , Macrófagos/inmunología , Ratones , Microglía/inmunología , RatasRESUMEN
The cellular and molecular mechanisms by which neuroinflammatory pathways respond to and propagate the reactive tissue response to intracortical microelectrodes remain active areas of research. We previously demonstrated that both the mechanical mismatch between rigid implants and the much softer brain tissue, as well as oxidative stress, contribute to the neurodegenerative reactive tissue response to intracortical implants. In this study, we utilize physiologically responsive, mechanically adaptive polymer implants based on poly(vinyl alcohol) (PVA), with the capability to also locally administer the antioxidant curcumin. The goal of this study is to investigate if the combination of two independently effective mechanisms - softening of the implant and antioxidant release - leads to synergistic effects in vivo. Over the first 4weeks of the implantation, curcumin-releasing, mechanically adaptive implants were associated with higher neuron survival and a more stable blood-brain barrier at the implant-tissue interface than the neat PVA controls. 12weeks post-implantation, the benefits of the curcumin release were lost, and both sets of compliant materials (with and without curcumin) had no statistically significant differences in neuronal density distribution profiles. Overall, however, the curcumin-releasing softening polymer implants cause minimal implant-mediated neuroinflammation, and embody the new concept of localized drug delivery from mechanically adaptive intracortical implants.