RESUMEN
Hydrogels are used extensively as cell-culture scaffolds for both 2D and 3D cell cultures due to their biocompatibility and the ease in which their mechanical and biological properties can be tailored to mimic natural tissue. The challenge when working with hydrogel-based scaffolds is in their handling, as hydrogels that mimic e.g. brain tissue, are both fragile and brittle when prepared as thin (sub-mm) membranes. Here, we describe a method for facile handling of thin hydrogel cell culture scaffolds by molding them onto a polycaprolactone (PCL) mesh support attached to a commonly used Transwell set-up in which the original membrane has been removed. In addition to demonstrating the assembly of this set-up, we also show some applications for this type of biological membrane. A polyethylene glycol (PEG)-gelatin hydrogel supports cell adhesion, and the structures can be used for biological barrier models comprising either one or multiple hydrogel layers. Here, we demonstrate the formation of a tight layer of an epithelial cell model comprising MDCK cells cultured over 9 days by following the build-up of the transepithelial electrical resistances. Second, by integrating a pure PEG hydrogel into the PCL mesh, significant swelling is induced, which leads to the formation of a non-adherent biological scaffold with a large curvature that is useful for spheroid formation. In conclusion, we demonstrate the development of a handling platform for hydrogel cell culture scaffolds for easy integration with conventional measurement techniques and miniaturized organs-on-chip systems.
Asunto(s)
Materiales Biocompatibles , Hidrogeles , Hidrogeles/química , Materiales Biocompatibles/química , Técnicas de Cultivo de Célula , Técnicas de Cultivo Tridimensional de Células , Tecnología , Andamios del Tejido/química , Ingeniería de Tejidos/métodosRESUMEN
Nerve terminals within the tumor microenvironment as potential pain-mitigating targets for local infiltration analgesia is relatively less explored. In this study, we examine the role of key analgesics administered as local infiltration analgesia in a model of cancer-induced bone pain (CIBP). CIBP was induced by administration of allogenic MRMT1 breast cancer cells in the proximal tibia of rats, and tumor mass characterized using radiogram, micro-CT, and histological analysis. In vitro responsiveness to key analgesics δ-opioid receptor agonist (DOPr), Ca2+ channel and TRPV1 antagonists was assessed using ratiometric Ca2+ imaging in sensory neurons innervating the tumor site. Effectiveness of locally infiltrated analgesics administered independently or in combination was assessed by quantifying evoked limb withdrawal thresholds at two distinct sites for up to 14 days. CIBP animals demonstrated DOPr, N-, and L-type and TRPV1 expression in lumbar dorsal root ganglion neurons (DRG), comparable to controls. Evoked Ca2+ transients in DRG neurons from CIBP animals were significantly reduced in response to treatment with compounds targeting DOPr, N-, L-type Ca2+ channels and TRPV1 proteins. Behaviourally, evoked hyperalgesia at the tumor site was strongly mitigated by peritumoral injection of the DOPr agonist and T-type calcium antagonist, via its activity on bone afferents. Results from this study suggest that nerve terminals at tumor site could be utilized as targets for specific analgesics, using local infiltration analgesia.
Asunto(s)
Analgesia , Microambiente Tumoral , Ratas , Animales , Ratas Sprague-Dawley , Dolor/metabolismo , Hiperalgesia/metabolismo , Analgésicos Opioides/farmacología , Células Receptoras Sensoriales , Analgésicos/efectos adversos , Analgésicos/metabolismo , Ganglios Espinales/metabolismoRESUMEN
The use of gold nanoparticles (AuNps) in applications connected to the peripheral nervous system (PNS) holds much promise in terms of therapeutic and diagnostic strategies. Despite their extensive use, a clear understanding of their effects on neurons and glia in the PNS is lacking. In this study, we set out to examine the effects of AuNps on dorsal root ganglion (DRG) cells, and how such AuNp-exposed cells could in-turn affect neurite differentiation. DRG cultures were exposed to mono-dispersed spherical-shaped AuNps of diameter 24.3 ± 2.3, 109.2 ± 14.7 or 175 ± 19.2 nm at varying concentrations. Cellular uptake and viability were quantified using flow-cytometry. Neurite differentiation was quantified using neurite tracing analysis in PC-12 and DRG neurons exposed to conditioned media derived from AuNp-treated DRG cells. Both neurons and glia were found to internalize AuNps. DRG cell viability was significantly reduced upon treatment with higher concentration of 175 nm sized AuNps, while 24 nm and 109 nm sized AuNps had no effect. Further, conditioned media from AuNp-treated DRG cells produced comparable neurite outgrowth and neurite branching measurement as controls in PC-12 and DRG neurons. DRG cells were quite resilient to AuNp exposure in mild-moderate concentration. AuNp-exposed DRG cells, irrespective of size and concentration range tested, did not affect neuronal differentiation.
Asunto(s)
Ganglios Espinales/citología , Oro/administración & dosificación , Nanopartículas del Metal/administración & dosificación , Neuronas/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endocitosis , Oro/química , Nanopartículas del Metal/química , Neuronas/fisiología , Células PC12 , Tamaño de la Partícula , Ratas , Ratas Sprague-DawleyRESUMEN
Accessing the peripheral nervous system (PNS) by topically applied nanoparticles is a simple and novel approach with clinical applications in several PNS disorders. Skin is richly innervated by long peripheral axons that arise from cell bodies located distally within ganglia. In this study we attempt to target dorsal root ganglia (DRG) neurons, via their axons by topical application of lectin-functionalized gold nanoparticles (IB4-AuNP). In vitro, 140.2 ± 1.9 nm IB4-AuNP were found to bind both axons and cell bodies of DRG neurons, and AuNP applied at the axonal terminals were found to translocate to the cell bodies. Topical application of IB4-AuNP on rat hind-paw resulted in accumulation of three to fourfold higher AuNP in lumbar DRG than in contralateral control DRGs. Results from this study clearly suggest that topically applied nanoparticles with neurotropic targeting ligands can be utilized for delivering nanoparticles to neuronal cell bodies via axonal transport mechanisms.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Neuronas/química , Piel/metabolismo , Animales , Electrofisiología , Femenino , Ganglios Espinales/metabolismo , Microscopía Electrónica de Transmisión , Fibras Nerviosas/metabolismo , Ratas Sprague-DawleyRESUMEN
Microparticle shape, as a tunable design parameter, holds much promise for controlling drug-release kinetics from polymeric microparticulate systems. In this study we hypothesized that the intensity and duration of a local nerve block can be controlled by administration of bupivacaine-loaded stretch-induced anisotropic poly(lactic-co-glycolic acid) microparticles (MPs). MPs of size 27.3 ± 8.5 µm were synthesized by single emulsion method and subjected to controlled stretching force. The aspect ratio of the anisotropic-bupivacaine MPs was quantified, and bupivacaine release was measured in vitro. The anisotropic MPs were administered as local nerve block injections in rats, and the intensity and duration of local anesthesia was measured. Bupivacaine-loaded anisotropic MPs used in this study were ellipsoid in shape and exhibited increased surface pores in comparison to spherical MPs. Anisotropic MPs exhibited a higher rate of bupivacaine release in vitro, and showed significantly (P < 0.05) stronger sensory nerve blocking as compared to spherical bupivacaine MPs, even though the duration of the nerve block remained similar. This study demonstrates the utility of stretch-induced anisotropic MPs in controlling drug release profiles from polymeric MPs, under both in vitro and in vivo conditions. We show that shape, as a tunable design parameter, could play an important role in engineering drug-delivery systems.
RESUMEN
Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.
Asunto(s)
Muerte Celular/efectos de los fármacos , Naftoquinonas/farmacología , Neoplasias/patología , Línea Celular , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Homeostasis , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Naftoquinonas/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de Proteasoma/farmacología , Compuestos de Sulfhidrilo/metabolismo , Vacuolas/metabolismoRESUMEN
BACKGROUND: Transdermal particulate penetration across thick-skin, such as that of palms and sole, is particularly important for drug delivery for disorders such as small fiber neuropathies. Nanoparticle-based drug delivery across skin is believed to have much translational applications, but their penetration especially through thick-skin, is not clear. OBJECTIVE: This study specifically investigates the effectiveness of gold nanoparticles (AuNPs) for thick-skin penetration, especially across the stratum corneum (SC) as a function of particle size. METHODS: The thick-skinned hind-paw of rat was used to characterize depth and distribution of AuNPs of varying sizes, namely, 22±3, 105±11, and 186±20nm. Epidermal penetration of AuNPs was characterized both, in harvested skin from the hind-paw using a diffusion chamber, as well as in vivo. RESULTS: Harvested skin segments exposed to 22nm AuNPs for only 3h demonstrated higher penetration (p<0.05) as compared to the 105 and 186nm particles. In animal studies, hind-paw skin of adult rats exposed to AuNPs solution for the same time, demonstrated nanoparticles in blood on the 4th day, and histological analysis revealed AuNPs in epidermal layers just below the SC, with no apparent tissue response. CONCLUSION: We conclude that the thick-skin allows nanoparticle penetration and acts as a depot for release of AuNPs into circulation long after the initial exposure has ceased.
Asunto(s)
Epidermis/metabolismo , Oro/farmacocinética , Absorción Cutánea , Administración Cutánea , Animales , Oro/administración & dosificación , Miembro Posterior , Masculino , Nanopartículas del Metal/administración & dosificación , Modelos Animales , Tamaño de la Partícula , Permeabilidad , Ratas , Ratas Sprague-DawleyRESUMEN
Polyethylenimine (PEI), a widely used cationic polymeric vector with high transfection efficiency, was converted into nanoparticles by introducing ionic and covalent crosslinkers with varying proportion of 1,6-hexanebisphosphate (HP), adipic acid (AA) and 1,4-butane dialdehyde (BA) to obtain a small library of HP-PEI (HPP), AA-PEI (AAP) and BA-PEI (BAP) nanoparticles, respectively. Particles were characterized by spectroscopic technique as well as physicochemical parameters such as size, morphology, surface charge, effect of crosslinking on buffering capacity and DNA binding ability. The entire series of nanoparticles were compared for their cytoxicity and ability to deliver genes in various cell lines. Among various nanoparticles, AAP-3 nanoparticle/DNA complex exhibited higher transfection efficiency (1.5-7.8 folds) than the native PEI (25kDa) and commercially available transfection reagents, such as GenePorter, GenePorter2, Fugene and Superfect, with cell viability >85%. The highest cell viability was observed with BAP nanoparticles (>95%). Importantly, the transfection activity of nanoparticle/DNA complexes was preserved in the presence of serum. Transfection with GFP-siRNA inhibited expression of transfected GFP gene by approximately 81-92%. All nanoparticle types (HPP, AAP and BAP) required a comparable time for entry into cells and subsequent intracellular passage from the cytoplasm to the nucleus. Intravenous delivery of (99)Tc labeled BAP-2/DNA complex to female Balb/c mice revealed the presence of the complex in most of the organs with the highest retention in liver. In conclusion, HPP, AAP and BAP nanoparticles are safe for efficient gene delivery.