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Projeto de investigação
Retinal erythropoietin distribution and neuroprotective effect in a nanoparticulate drug delivery system after subconjunctival and topical adminstration in an animal glaucoma model
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Retinal erythropoietin distribution and neuroprotective effect in a nanoparticulate drug delivery system after subconjunctival and topical administration in an animal glaucoma model
Publication . Silva, Beatriz Rosa Fernandes Duarte da; Delgado, Esmeralda Sofia da Costa; São Braz, Berta Maria Fernandes Ferreira; Gonçalves, Lídia Maria Diogo
ABSTRACT - Glaucoma is a neurodegenerative ocular disease with substantial impact in public health, as it causes retinal ganglion cells (RGC) degeneration and irreversible blindness. Neuroprotective strategies have been a focus of research in glaucoma, and the use of erythropoietin and its recombinant forms, like epoetin beta (EPOβ), have shown anti-apoptotic effects on RGC. We aimed to create a nanoformulation carrying EPOβ, adequate for topical ocular administration, that could provide neuroprotection to the retina in cases of glaucoma, with absent or residual secondary effects and the advantage of promoting patients’ compliance to the treatment. Therefore, chitosan and hyaluronic acid (CS/HA) nanoparticles for topical ocular deliver of epoetin beta (EPOβ) were developed, aiming to deliver EPOβ to the retina in a sustained profile. Firstly, in vitro and ex vivo studies of the physicochemical stability, cytotoxicity, release and permeation profiles and mucoadhesive strength of the CS/HA-EPOβ nanoparticles were performed. These nanoparticles allowed EPOβ permeation through ocular membranes in ex vivo assays, with no in vitro cytotoxicity. Afterwards, healthy Wistar Hannover rats were used for subconjunctival and topical administrations of CS/HA-EPOβ nanoparticles, to assess the formulation’s local and systemic influence, its biological tolerance and safety, its effect in retinal electrophysiology, and EPOβ’s distribution in ocular tissues. A sustained EPOβ delivery to the retina was observed using both routes of administration, with no side-effects. Finally, we explored the topical ocular delivery of CS/HA-EPOβ nanoparticles using a rat glaucoma model. We assessed retinal morphological and physiological changes in response to the nanoformulation applied through topical ocular route, using electroretinography and histological evaluation, which comprised immunofluorescence, hematoxylin and eosin staining and apoptosis assessment (cleaved caspase-3). Topical ocular administration of CS/HA EPOβ nanoparticles in glaucomatous rats allowed EPOβ permeation, including by transcorneal pathway, reaching the inner retina. Improvements in retinal electrical activity and thickness, and in apoptosis reduction occurred earlier and more significantly in the treatment group. In conclusion, EPOβ reached the retina, where its neuroprotective action was observed, thus demonstrating the feasibility of topical administration of neuroprotective agents in nanoformulations, targeting the posterior ocular segment. Results were promising and contribute to the development of new therapeutic strategies to preserve the visual acuity of patients with glaucoma or other neurodegenerative ocular diseases
Chitosan and hyaluronic acid nanoparticles as vehicles of epoetin beta for subconjunctival ocular delivery
Publication . Silva, Beatriz; Gonçalves, Lídia M.; Ferreira São Braz, Berta; Delgado, Esmeralda
Neuroprotection in glaucoma using epoetin beta (EPOβ) has yielded promising results. Our team has developed chitosan-hyaluronic acid nanoparticles (CS/HA) designed to carry EPOβ into the ocular globe, improving the drug’s mucoadhesion and retention time on the ocular surface to increase its bioavailability. In the present in vivo study, we explored the possibility of delivering EPOβ to the eye through subconjunctival administration of chitosan-hyaluronic acid-EPOβ (CS/HA-EPOβ) nanoparticles. Healthy Wistar Hannover rats (n = 21) were split into 7 groups and underwent complete ophthalmological examinations, including electroretinography and microhematocrit evaluations before and after the subconjunctival administrations. CS/HA-EPOβ nanoparticles were administered to the right eye (OD), and the contralateral eye (OS) served as control. At selected timepoints, animals from each group (n = 3) were euthanized, and both eyes were enucleated for histological evaluation (immunofluorescence and HE). No adverse ocular signs, no changes in the microhematocrits (≈45%), and no deviations in the electroretinographies in both photopic and scotopic exams were observed after the administrations (p < 0.05). Intraocular pressure remained in the physiological range during the assays (11–22 mmHg). EPOβ was detected in the retina by immunofluorescence 12 h after the subconjunctival administration and remained detectable until day 21. We concluded that CS/HA nanoparticles could efficiently deliver EPOβ into the retina, and this alternative was considered biologically safe. This nanoformulation could be a promising tool for treating retinopathies, namely optic nerve degeneration associated with glaucoma.
Antioxidant-Loaded Mucoadhesive Nanoparticles for Eye Drug Delivery: A New Strategy to Reduce Oxidative Stress
Publication . Cordeiro, Sandra; Silva, Beatriz; Martins, Ana; Ribeiro, Helena; Gonçalves, Lídia; Marto, Joana
There are several approaches to treat ocular diseases, which can be invasive or non-invasive. Within the non-invasive, new pharmaceutical strategies based on nanotechnology and mucoadhesive polymers are emerging methodologies, which aim to reach an efficient treatment of eye diseases. The aim of this work was the development of novel chitosan/hyaluronic acid nanoparticle systems with mucoadhesive properties, intended to encapsulate antioxidant molecules (e.g., crocin) aiming to reduce eye oxidative stress and, consequently, ocular disease. An ultraviolet (UV) absorber molecule, actinoquinol, was also added to the nanoparticles, to further decrease oxidative stress. The developed nanoparticles were characterized and the results showed a mean particle size lower than 400 nm, polydispersity index of 0.220 ± 0.034, positive zeta potential, and high yield. The nanoparticles were also characterized in terms of pH, osmolality, and viscosity. Mucoadhesion studies involving the determination of zeta potential, viscosity, and tackiness, showed a strong interaction between the nanoparticles and mucin. In vitro release studies using synthetic membranes in Franz diffusion cells were conducted to unravel the drug release kinetic profile. Ex vitro studies using pig eye scleras in Franz diffusion cells were performed to evaluate the permeation of the nanoparticles. Furthermore, in vitro assays using the ARPE-19 (adult retinal pigment epithelium) cell line showed that the nanoparticles can efficiently decrease oxidative stress and showed low cytotoxicity. Thus, the developed chitosan/hyaluronic acid nanoparticles are a promising system for the delivery of antioxidants to the eye, by increasing their residence time and controlling their delivery.
New nanoparticles for topical ocular delivery of erythropoietin
Publication . Silva, Beatriz; Marto, Joana; Braz, Berta São; Delgado, Esmeralda; Almeida, António José; Gonçalves, Lídia
Erythropoietin (EPO) is known for its neuroprotective and neuroregenerative properties. EPO topical ocular administration has not been tested yet and its bioavailability could be improved by mucoadhesive hydrogels. Thus, this study aimed to develop and evaluate a chitosan (CS) and hyaluronic acid (HA) nanoparticulate system for topical ocular delivery of EPO. Nanoparticles were prepared by ionotropic gelation using six different HAs (HA1-HA6), and characterized by size, zeta potential (ZP), polydispersity index (Pdi), cytotoxicity and mucoadhesion. Encapsulation efficiency and drug loading capacity were also determined. Ex vivo permeation was tested using fresh porcine corneas, scleras and conjunctivas. The permeated EPO was quantified by ELISA, and its presence in the membranes was confirmed by immunohistochemistry. Nanoparticles (NPs) presented size ≤300 nm, ZP around +30 mV and low Pdi (0.167–0.539) at a 1:1 CS:HA mass ratio. The most suitable HA was HA6 (300 kDa – Eye), which had the best mucoadhesive properties. CS/HA6-EPO nanoformulation permeated more rapidly through porcine conjunctiva, followed by sclera and thirdly by cornea, as assessed by immunohistochemistry. All formulations were noncytotoxic on ARPE-19 and HaCaT cell lines, as evaluated by metabolic and membrane integrity tests. In conclusion, CS/HA6-EPO NPs could be a promising formulation for increasing EPO ocular bioavailability by enhancing its retention time and permeation through the different ocular membranes.
Topical ocular delivery of nanoparticles with epoetin beta in Wistar Hannover rats
Publication . Silva, Beatriz; Gonçalves, Lídia; Braz, Berta São; Delgado, Esmeralda
Topical instillation of drugs targeting the posterior ocular segment is an expanding area of research. Chitosan and hyaluronic acid have remarkable mucoadhesive properties and potentially enhance pre-corneal retention time after topical instillation. Bearing this in mind, we explored the possibility of delivering epoetin beta (EPOβ) to the posterior segment of the eye in a chitosan-hyaluronic acid (CS/HA-EPOβ) nanoparticulate system using the topical route of administration. Complete ophthalmological examinations, electroretinography and microhematocrit evaluations were performed in Wistar Hannover (WH) rats, before and after topical administration of nanoparticles. The right eye received CS/HA-EPOβ and the left eye received only empty nanocarriers (control). Animals were split into 6 groups and at designated timepoints, all animals from each group (n = 3) were euthanized and both eyes enucleated. Retinal morphology and EPOβ ocular distribution were assessed, respectively, through hematoxylin and eosin (HE) and immunofluorescence staining. After topical administration, no adverse ocular signs were noted and no significant changes either in microhematocrits nor in electroretinographies were detected. During the study, intraocular pressure (IOP) was always kept within physiological range bilaterally. No histological changes were detected in any of the ocular globes. Immunofluorescence enabled the identification of EPOβ in the retina 12 h after the administration, its presence still being detectable at day 21. In conclusion, CS/HA nanoparticles could efficiently deliver EPOβ to the retina of WH rats after topical instillation, being considered biologically safe. Topical administration of this nanoformulation could be a valuable tool for retinal neuroprotection, decreasing risks associated with more invasive routes of administration, being cost effective and also increasing long-term patients’ compliance.
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SFRH/BD/130476/2017