Infliximab microencapsulation: an innovative approach for intra-articular administration of biologics in the management of rheumatoid arthritis-in vitro evaluation

Microencapsulation of the therapeutical monoclonal antibody infliximab (INF) was investigated as an innovative approach to improve its stability and to achieve formulations with convenient features for intra-articular administration. Ultrasonic atomization (UA), a novel alternative to microencapsulate labile drugs, was compared with the conventional emulsion/evaporation method (Em/Ev) using biodegradable polymers, specifically Polyactive® 1000PEOT70PBT30 [poly(ethylene-oxide-terephthalate)/poly(butylene-terephthalate); PEOT-PBT] and its polymeric blends with poly-(D, L-lactide-co-glycolide) (PLGA) RG502 and RG503 (PEOT-PBT:PLGA; 65:35). Six different formulations of spherical core-shell microcapsules were successfully developed and characterized. The UA method achieved a significantly higher encapsulation efficiency (69.7-80.25%) than Em/Ev (17.3-23.0%). Mean particle size, strongly determined by the microencapsulation method and to a lesser extent by polymeric composition, ranged from 26.6 to 49.9 µm for UA and 1.5-2.1 µm for Em/Ev. All formulations demonstrated sustained INF release in vitro for up to 24 days, with release rates modulated by polymeric composition and microencapsulation technique. Both methods preserved INF biological activity, with microencapsulated INF showing higher efficacy than commercial formulations at comparable doses regarding bioactive tumor necrosis factor-alpha (TNF-α) neutralization according to WEHI-13VAR bioassay. Microparticles' biocompatibility and extensive internalization by THP-1-derived macrophages was demonstrated. Furthermore, high in vitro anti-inflammatory activity was achieved after treatment of THP-1 cells with INF-loaded microcapsules, significatively reducing in vitro production of TNF-α and interleucine-6 (Il-6)Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Iván Lamela-Gómez received funding from the “Axudas á etapa predoutoral da Xunta de Galicia, cofinanciadas polo programa operativo FSE Galicia 2014–2020” predoctoral grant program. Consellería de Cultura, Educación e ordenación universitaria, Xunta de Galicia, Spain. This research was partially funded by Consellería de Cultura, Educación e ordenación universitaria, Xunta de Galicia, Spain. Axudas para a consolidación e estructuración de unidades de investigación competitivas Modalidad A: Grupos de Referencia Competitiva (ED341C 2017/13). This research was also partially funded by the Fundação para a Ciência e a Tecnologia (FCT), Portugal (UID/DTP/04138/2019 and UIDB/04138/2020 to iMed.ULisboa), and principal investigator grants CEECIND/03143/2017 (L. M. Gonçalves)S

Development, Characterization, and In Vitro Evaluation of Resveratrol-Loaded Poly-(ε-caprolactone) Microcapsules Prepared by Ultrasonic Atomization for Intra-Articular Administration

Intra-articular administration of drugs to the joint in the treatment of joint disease has the potential to minimize the systemic bioavailability and the usual side-effects associated with oral drug administration. In this work, a drug delivery system is proposed to achieve an anti-inflammatory local effect using resveratrol (RSV). This study aims to develop microcapsules made of poly-(ε-caprolactone) (PCL) by ultrasonic atomization to preserve the antioxidant activity of RSV, to prevent its degradation and to suppress the inflammatory response in activated RAW 264.7 macrophages. An experimental design was performed to build a mathematical model that could estimate the effect of nozzle power and polymer concentration on particle size and encapsulation efficiency. RSV-loaded microcapsules showed adequate morphology, particle size, and loading efficiency properties. RSV formulations exhibited negligible cytotoxicity and an efficient amelioration of inflammatory responses, in terms of Nitric Oxide (NO), ROS (Reactive Oxygen Species), and lipid peroxidation in macrophages. Thus, RSV-loaded microcapsules merit consideration as a drug delivery system suitable for intra-articular administration in inflammatory disorders affecting the jointAcknowledgments: I.L.-G. is grateful to “Programa de axudas á etapa predoutoral da Xunta de Galicia (cofinanciadas por FSE Galicia 2014-2020), Consellería de Cultura, Educación e Ordenación Universitaria” program for doctoral fellowshipS

Proteomic analysis in morquio a cells treated with immobilized enzymatic replacement therapy on nanostructured lipid systems

[ENG]Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERTS

Intravitreal implants manufactured by supercritical foaming for treating retinal diseases

Chronic retinal diseases, such as age-related macular degeneration (AMD), are a major cause of global visual impairment. However, current treatment methods involving repetitive intravitreal injections pose financial and health burdens for patients. The development of controlled drug release systems, particularly for biological drugs, is still an unmet need in prolonging drug release within the vitreous chamber. To address this, green supercritical carbon dioxide (scCO2) foaming technology was employed to manufacture porous poly(lactic-co-glycolic acid) (PLGA)-based intravitreal implants loaded with dexamethasone. The desired implant dimensions were achieved through 3D printing of customised moulds. By varying the depressurisation rates during the foaming process, implants with different porosities and dexamethasone release rates were successfully obtained. These implants demonstrated controlled drug release for up to four months, surpassing the performance of previously developed implants. In view of the positive results obtained, a pilot study was conducted using the monoclonal antibody bevacizumab to explore the feasibility of this technology for preparing intraocular implants loaded with biologic drug molecules. Overall, this study presents a greener and more sustainable alternative to conventional implant manufacturing techniques, particularly suited for drugs that are susceptible to degradation under harsh conditions

Enzyme-Loaded Gel Core Nanostructured Lipid Carriers to Improve Treatment of Lysosomal Storage Diseases: Formulation and In Vitro Cellular Studies of Elosulfase Alfa-Loaded Systems

Mucopolysaccharidosis IVA (Morquio A) is a rare inherited metabolic disease caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS). Until now, treatments employed included hematopoietic stem cell transplantation and enzyme replacement therapy (ERT); the latter being the most commonly used to treat mucopolysaccharidoses, but with serious disadvantages due to rapid degradation and clearance. The purpose of this study was to develop and evaluate the potential of nanostructured lipid carriers (NLCs) by encapsulating elosulfase alfa and preserving its enzyme activity, leading to enhancement of its biological effect in chondrocyte cells. A pegylated elosulfase alfa-loaded NLC was characterized in terms of size, ζ potential, structural lipid composition (DSC and XRD), morphology (TEM microscopy), and stability in human plasma. The final formulation was freeze-dried by selecting the appropriate cryoprotective agent. Viability assays confirmed that NLCs were non-cytotoxic to human fibroblasts. Imaging techniques (confocal and TEM) were used to assess the cellular uptake of NLCs loaded with elosulfase alfa. This study provides evidence that the encapsulated drug exhibits enzyme activity inside the cells. Overall, this study provides a new approach regarding NLCs as a promising delivery system for the encapsulation of elosulfase alfa or other enzymes and the preservation of its activity and stability to be used in enzymatic replacement therapy (ERT).This research was funded by Xunta de Galicia, grant number GRC2013/015 and GPC2017/015S

Three-Dimensional Hybrid Mesoporous Scaffolds for Simvastatin Sustained Delivery with in Vitro Cell Compatibility

The development of scaffolds with suitable physicochemical and mechanical properties allowing for the structural regeneration of injured bone and recovery of the natural biological functionality is still a challenge in the tissue engineering field. Nanostructured materials with added theranostic abilities, together with an interconnected hierarchy of pores, offer the possibility to provide a new generation of bone implants. In this work, scaffolds with highly porous and resistant threedimensional structures have been successfully developed by homogeneously embedding mesoporous silica nanostructures in a bioactive matrix of chitosan/κ-carrageenan. Moreover, magnetite (Fe3O4) nanoparticles were also added to the mesoporous scaffold to include additional magnetic functionalities for diagnostic or therapeutic actions. The complete physicochemical characterization shows mesoporous materials with a wide range of interconnected pores, remarkable surface roughness, and large effective surface area, suitable for cell adhesion. In accordance to these properties, a simvastatin loading and release assay showed high loading capacities and sustained release over a long period of time. Together with a suitable resistance against degradation and biocompatible performance assessed by cell viability assays, these scaffolds show interesting features for delivering drugs with activity in bone regeneration processes.This work was supported by the European Commission (PANA project, Call H2020-NMP-2015-two-stage, grant 686009); and the Xunta de Galicia (GPC2017/015), and partially supported by the Consellería de Educacion Program ́ for the Development of Strategic Grouping in Materials AEMAT at the University of Santiago de Compostela under grant no. ED431E2018/08, Xunta de Galicia and Program for the Consolidation of Research Units of Competitive Reference GRC2017, grant no. ED431C 2017/22S

3D Printed Tacrolimus Rectal Formulations Ameliorate Colitis in an Experimental Animal Model of Inflammatory Bowel Disease

The aim of this study was to fabricate novel self-supporting tacrolimus suppositories using semisolid extrusion 3-dimensional printing (3DP) and to investigate their efficacy in an experimental model of inflammatory bowel disease. Blends of Gelucire 44/14 and coconut oil were employed as lipid excipients to obtain suppository formulations with self-emulsifying properties, which were then tested in a TNBS (2,4,6-trinitrobenzenesulfonic acid) induced rat colitis model. Disease activity was monitored using PET/CT medical imaging; maximum standardized uptake values (SUVmax), a measure of tissue radiotracer accumulation rate, together with body weight changes and histological assessments, were used as inflammatory indices to monitor treatment efficacy. Following tacrolimus treatment, a significant reduction in SUVmax was observed on days 7 and 10 in the rat colon sections compared to non-treated animals. Histological analysis using Nancy index confirmed disease remission. Moreover, statistical analysis showed a positive correlation (R2 = 71.48%) between SUVmax values and weight changes over time. Overall, this study demonstrates the effectiveness of 3D printed tacrolimus suppositories to ameliorate colitis and highlights the utility of non-invasive PET/CT imaging to evaluate new therapies in the preclinical areaThis research was funded by Xunta de Galicia grant number GRC2013/015 and GPC2017/015. A.F.-F. acknowledges the support of Instituto de Salud Carlos III (Juan Rodés research grant JR18/00014). P.A. acknowledges the support of RYC-2015/17430 (Ramón y Cajal). X.G.-O. acknowledges the financial support of the IDIS (Health Research Institute of Santiago de Compostela)S