Wound healing difficulty is one of the major complications of diabetes mellitus which makes the healing process slower and treatment more expensive. Chitosan has been studied regarding its ability to accelerate healing and tested in the treatment of skin lesions with impaired healing as diabetic ones. Fifty-four Wistar female rats were divided randomly in three groups with 18 animals each, which were subdivided into three subgroups (n = 6) corresponding to periods of post-operative evaluation (7, 14 and 21 days). All subjects underwent experimental induction of diabetes by single intraperitoneal application of alloxan with a dose of 120mg/kg. After the diabetic state was confirmed, the study proceeded to general anesthesia for the manufacture of skin wounds in the dorsal region of the animals. The control group (GC) received saline on the wounds. The GA group was treated with oil-based essential fatty acids and the GQ wounds were treated with 2% chitosan gel characterized and standardized for this study. Macroscopic, histological and histomorphometric analyses were performed to monitor the healing process of wounds. The healing of wounds treated with chitosan was faster than the healing in the other groups at 14 and 21 days postoperatively, and the control group presented the lowest percentage of wound contraction. Statistical difference was found at 14 days between GQ and GC in the macroscopic analysis (p=0,02). Histologically there was an advance in the GA and in the GQ reeptelization compared to GC at 7 and 14 days, as well as the presence of polymorphonuclear cells in GQ at seven days and mononuclear cells in the other groups in this period. Fibroblasts and collagen were showed to be predominant in all groups during the period of 21 days characterizing the final phase of healing. The methodology used to prepare a 2% chitosan gel produced a drug with technical qualities that are suitable for topical use. This treatment was satisfactory as it accelerated the healing of injuries when compared to animals treated with saline and essential fatty acids oil. The proposed protocol becomes a promising and economically viable alternative for the treatment of diabetic wounds.

Alginate (ALG) is a natural polymer used in a wide range of applications. Cerium is a sustainable corrosion inhibitor for many metal substrates. In this study, ALG/Ce3+ hydrogel microparticles are presented as an innovative cerium(III) reservoir system for the smart release of cerium ions. The active corrosion inhibition capacity of the ALG/Ce3+ complex was investigated. Tests using ALG/Ce3+ macrogels (beads) and electrochemical experiments showed that, in saline media, ALG/Ce3+ hydrogel was able to release Ce3+, which was subsequently exchanged with Al3+, Fe2+ and Fe3+ and, thus slowing the corrosion process of AA2024 (aluminum alloy) and AISI1020 (carbon steel) substrates, respectively. It is suggested that the presence of metal ions originated from the corrosion process triggers the cerium release. The corrosion protection of ALG/Ce3+ of the aluminum alloy was confirmed by polarization curves, electrochemical impedance spectroscopy (EIS) and scanning vibrating electrode technique (SVET) and the protection of the carbon steel was demonstrated by EIS experiments. To the best of our knowledge, this is the first time that an alginate-based complex has been reported as an ion exchange corrosion-inhibiting system for metal substrates.

Fragaria vesca L. (F. vesca), popularly known as wild strawberry, is a plant from the Rosaceae family, found in temperate and subtropical areas of the northern hemisphere. F. vesca leaves have been shown to have antiseptic, emollient, and dermatological protection properties, due to the presence of bioactive compounds, such as flavonoids, phenolic acids, ellagitannins, and proanthocyanidins. In this study, a F. vesca extract was obtained by an optimized extraction process, and was characterized by HPLC, ROS scavenging activity, cytotoxicity assays in HaCaT cells, and tyrosinase inhibitory activity determination. The most active extract was then incorporated in a hydrogel with hydroxyethylcellulose at 2% (w/w), which was characterized at the physicochemical, stability, cytotoxicity, and ROS scavenging activity levels to evaluate its quality, safety, and efficacy. In vivo studies, human repeat insult patch testing, and an assay to determine their antioxidant efficacy, were also performed. The results showed that the Fragaria vesca extracts had antioxidant activity and that the F. vesca extract-based hydrogel exhibited cutaneous compatibility, acceptability and antioxidant efficacy, being stable, and suitable for topical application.

keletal muscle has good regenerative capacity, but the extent of muscle injury and the developed fibrosis might prevent complete regeneration. The<jats:italic>in vivo</jats:italic>application of human mesenchymal stem cells (HMSCs) of the umbilical cord and the conditioned media (CM) where the HMSCs were cultured and expanded, associated with different vehicles to induce muscle regeneration, was evaluated in a rat myectomy model. Two commercially available vehicles and a spherical hydrogel developed by our research group were used. The treated groups obtained interesting results in terms of muscle regeneration, both in the histological and in the functional assessments. A less evident scar tissue, demonstrated by collagen type I quantification, was present in the muscles treated with HMSCs or their CM. In terms of the histological evaluation performed by ISO 10993-6 scoring, it was observed that HMSCs apparently have a long-term negative effect, since the groups treated with CM presented better scores. CM could be considered an alternative to the<jats:italic>in vivo</jats:italic>transplantation of these cells, as it can benefit from the local tissue response to secreted molecules with similar results in terms of muscular regeneration. Searching for an optimal vehicle might be the key point in the future of skeletal muscle tissue engineering.

Polyvinyl alcohol (PVA) hydrogels have been widely studied for cartilage replacement due to their biocompatibility, chemical stability, and ability to be modified such that they approximate natural tissue behavior. Additionally, they may also be used with advantages as local drug delivery systems. However, their properties are not yet the most adequate for such applications. This work aimed to develop new PVA-based hydrogels for this purpose, displaying improved tribomechanical properties with the ability to control the release of diclofenac (DFN). Four types of PVA-based hydrogels were prepared via freeze-thawing: PVA, PVA/PAA (by polyacrylic acid (PAA) addition), PVA/PAA+PEG (by polyethylene glycol (PEG) immersion), and PVA/PAA+PEG+A (by annealing). Their morphology, water uptake, mechanical and rheological properties, wettability, friction coefficient, and drug release behavior were accessed. The irritability of the best-performing material was investigated. The results showed that the PAA addition increased the swelling and drug release amount. PEG immersion led to a more compact structure and significantly improved the material’s tribomechanical performance. The annealing treatment led to the material with the most suitable properties: besides presenting a low friction coefficient, it further enhanced the mechanical properties and ensured a controlled DFN release for at least 3 days. Moreover, it did not reveal irritability potential for biological tissues.

Chronic wound treatment accounts for a substantial percentage of the medical expenses worldwide. Improving and developing novel wound care systems can potentially help to handle this problem. Wound dressings loaded with antiseptics may be an important tool for wound care, as they inhibit bacterial growth at the wound site. The goal of the present work was to investigate the potential of using casein hydrogel dressings loaded with two antiseptic drugs, Octiset® or polyhexanide, to treat chronic wounds. Casein-based hydrogels are inexpensive and have several properties that make them suitable for biomedical applications. Two types of casein were used: casein sodium salt and acid casein, with the formulations being labelled CS and C, respectively. The hydrogels were characterised with respect to their physical properties (swelling capacity, water content, morphology, mechanical resistance, and stability), before and after sterilisation, and they showed adequate values for the intended application. The hydrogels of both formulations were able to sustain controlled drug-release for, at least, 48 h. They were demonstrated to be non-irritant, highly haemocompatible, and non-cytotoxic, and revealed good antimicrobial properties against Staphylococcus aureus and Pseudomonas aeruginosa. Steam-heat sterilisation did not compromise the material’s properties. The in vivo performance of C hydrogel loaded with Octiset® was evaluated in a case study with a dog. The efficient recovery of the wounds confirms its potential as an alternative for wound treatment. To our knowledge, this is the first time that wound dressings loaded with Octiset®, one of the most efficient drugs for wound treatment, were prepared and tested.

Inflammatory skin diseases, such as atopic dermatitis and psoriasis, can significantly impact patients' quality of life due to symptoms caused by an overactive immune response. Clobetasol, a potent corticosteroid drug, is commonly used to relieve these symptoms by reducing inflammation and suppressing immune responses. However, prolonged use can lead to adverse effects such as skin atrophy, hypopigmentation and Cushing-like syndrome. To tackle these issues, nanotechnology has been extensively used to enhance drug strength, stability, and controlled release, offering a higher therapeutic efficacy and safety. Hence, in this work, clobetasol-loaded poly lactic-co-glycolic acid (PLGA) nanoparticles were developed for the topical treatment of inflammatory skin diseases. Nanoparticles were prepared by nanoprecipitation using PLGA (polymer), polyvinyl alcohol (PVA, stabilizer), acetone (solvent), and clobetasol (drug). Particle size, polydispersity index (PDI), and zeta potential (ZP) were determined using a Zetasizer Nano ZS apparatus. The resultant optimized nanoparticles exhibited an average particle size of 232 ± 20 nm, PDI of 0.18 ± 0.06, and a slightly negative surface charge (- 6.94 ± 2.55 mV), as expected due to the ester terminal of the PLGA. The incorporation of clobetasol did not lead to a significant alteration in these characteristics. The current data shows that it was possible to develop small (size range between 100 and 400 nm) and homogeneous (PDI below 0.3) PLGA nanoparticles,which are properties predictive of optimal topical drug absorption. Thermosensitive hydrogels were successfully formulated using the cold method for the incorporation of clobetasol-loaded nanoparticles. The selected hydrogel exhibited an average gelation temperature (32.33 ± 0.58ºC) identical to skin temperature. In vitro studies revealed controlled release of the clobetasol-loaded nanoparticles, as well as the clobetasol nanoparticles incorporated hydrogel, the latter with a lower cumulative percentage. Therefore, the clobetasol-loaded nanoparticles and the clobetasol-nanoparticles incorporated hydrogel presented a controlled release, which is advantageous for minimizing the clobetasol side effects, constituting a potential innovative therapeutic approach for the treatment of inflammatory skin diseases.

Atualmente, nenhum penso utilizado clinicamente em feridas crónicas, mais especificamente em queimaduras, providencia adesão, hidratação, força mecânica semelhante à matriz extracelular natural (MEC) da pele e capacidade de ser removido sem causar trauma. Os hidrogéis surgem como uma alternativa promissora devido à sua capacidade de manter um ambiente húmido no local da ferida e, ao mesmo tempo, absorver o exsudato, às suas propriedades mecânicas versáteis e à competência de proteção contra infeções bacterianas. Os hidrogéis estímulo responsivos trazem a possibilidade de controlar a degradação dos hidrogéis, sem causar dor ao doente, colmatando uma das maiores desvantagens dos pensos tradicionais. Os hidrogéis foto responsivos são uma excelente opção devido às características inócuas da luz visível para os tecidos humanos. Utilizou-se a reação de click para sintetizar os hidrogéis, ao fazer reagir o polietilenoglicol funcionalizado com a polietilenoimina (PEI), resultando em uma ligação estéreo especifica de β-aminoacrilato facilmente clivável por oxigénio singleto (1O2). A caracterização química dos precursores foi realizada por ressonância magnética nuclear de protão (1H NMR) e espetroscopia de transformada de Fourier no infravermelho, no modo de refletância total atenuada (ATR-FTIR). O impacto do peso molecular da PEI nas propriedades dos hidrogéis foi determinado. Os hidrogéis apresentam uma capacidade de inchaço entre 139% e 404% o seu peso inicial, dependendo do peso molecular da PEI e da percentagem em massa utilizado na síntese dos hidrogéis. A caracterização das propriedades mecânicas revelou uma resistência à compressão máxima de 139.3 kPa. O ensaio de adesão revelou que os hidrogéis aderem à superfície do tecido com uma força média entre 1.6 e 5.2 N/cm2. A morfologia de superfície e a capacidade de eliminação de ROS pelo hidrogel foi também analisada. Esta dissertação propõe um mecanismo promissor para facilitar a degradação dos hidrogéis inspirado na terapia fotodinâmica. O mecanismo baseia-se na incorporação de um fotossensibilizador (PS) dentro da matriz polimérica do hidrogel. Após irradiação com um comprimento de onda adequado, o PS gera 1O2 capaz de degradar as ligações β-aminoacrilato presentes na estrutura do hidrogel. O sucesso do mecanismo proposto foi investigado quimicamente por ATR-FTIR. As propriedades físicas dos hidrogéis após irradiação foram avaliadas através da análise das propriedades mecânicas, da morfologia de superfície, da geração de 1O2 in vitro e através de testes de degradação por ação da luz e do pH. Uma perda visível das propriedades do hidrogel ocorre após irradiação, confirmando a capacidade promissora de dissolução in situ do hidrogel. Estudos de citocompatibilidade foram efetuados nos hidrogéis com e sem irradiação, destacando a boa citocompatibilidade e segurança dos hidrogéis sintetizados com PEI1.8k e 30% wt. após serem irradiados.

A dermatite alérgica de contato (DCA), é uma manifestação clínica da sensibilização da pele, e a doença ocupacional mais prevalente em humanos, com uma taxa de prevalência de 27% na Europa e entre 15-20% na população em geral. Esta elevada prevalência, aliada ao número crescente de sensibilizantes cutâneos a que a humanidade está exposta e à ausência de estratégias terapêuticas eficazes, não só resulta num elevado custo para o sistema de saúde como afeta fortemente a qualidade de vida dos doentes, destacando a relevância e a falta de desenvolvimento de novas e efetivas estratégias preventivas.A identificação de potenciais sensibilizantes cutâneos tem se baseado fortemente em métodos in vivo, embora grandes esforços de investigação estão a ser feitos para o desenvolvimento de novas metodologias de abordagem (NAMs). De fato, testes em animais na União Europeia encontram-se proibidos desde 2004 para produtos cosméticos (“testing ban”). Consequentemente, o desenvolvimento de NAM para sensibilização da pele foi incentivado e esforços consideráveis foram feitos. Os NAM disponíveis foram desenvolvidos com base no “Adverse Outcome Pathway” (AOP) da sensibilização da pele, que fornece a base mecanicista para a integração de informações relacionadas à sensibilização da pele. Os principais eventos na AOP incluem a ligação covalente de um produto químico às proteínas da pele, ativação de queratinócitos, ativação de células dendríticas e ativação e proliferação de células T, levando à sensibilização da pele. Por exemplo, está bem estabelecido que a conjugação químico-peptídeo representa o evento de iniciação molecular (MIE) na sensibilização da pele e este MIE tem sido explorado com sucesso no desenvolvimento de ensaios de reatividade peptídica in chemico, com o Direct Peptide Reactivity Assay (DPRA) sendo validado como uma ferramenta de triagem para perigo de sensibilização da pele, bem como uma diretriz de teste da OCDE. Este teste baseia-se no uso de cromatografia líquida de alta performance/método de deteção ultravioleta para quantificar a conjugação químico-peptídeo por meio da medição da depleção de dois peptídeos sintéticos contendo resíduos de lisina ou cisteína, sendo trabalhoso e demorado. Para melhorar o rendimento, sensibilidade e a precisão do ensaio, o capítulo 2 da tese consiste no desenvolvimento de um ensaio espectrofotométrico para o potencial de sensibilização da pele baseado na medição do MIE, que é uma alternativa mais barata, rápida, simples e acessível para o DPRA, fornecendo resultados comparáveis. O mesmo foi novamente utilizado no capítulo seguinte da tese.Deste modo, o capítulo 3 teve como objetivo divulgar moléculas capazes de evitar o “AOP” por alérgenos cutâneos humanos padrão, usando NAM para risco de sensibilização cutânea, em particular um teste desenvolvido internamente, a maturação (in vitro) de células THP-1 analisados pela regulação positiva das moléculas co estimuladoras CD86 e CD54 e pela expressão de genes dependentes de NRF2 em queratinócitos. Usando esses NAMs, a condição de lisina e N-acetilcisteína (NAC+LYS) foi a mais eficiente na mitigação de todos os principais eventos evocados por alérgenos cutâneos DNFB e MI e, portanto, selecionados para serem formulados e testados in vivo. Assim, dando um passo à frente, o efeito preventivo de um hidrogel contendo NAC mais LYS (desenvolvido no capítulo 4) foi o ensaio do linfonodo local murino (LLNA: BrdU-FCM), previamente aprovado pela OCDE para esse fim. Os resultados alcançados demonstraram que o hidrogel formulado com NAC mais LYS pode diminuir o índice de estimulação (SI) desencadeado por fortes alérgenos cutâneos e, consequentemente, a magnitude da sensibilização cutânea. Adicionalmente, a pré-aplicação do hidrogel reduziu significativamente o inchaço, espessura e inflamação da orelha do ratinho e as análises histológicas, realizadas em colaboração com o serviço de Anatomia Patológica do CHUC, também confirmaram o efeito protetor do hidrogel.Finalmente, o capítulo 4 descreve o desenvolvimento e caracterização de um novo hidrogel contendo os ingredientes ativos NAC mais LYS sob as premissas de qualidade e segurança desde o projeto. Dois planos fatoriais foram estabelecidos visando a otimização do hidrogel em termos de propriedades mecânicas e reológicas. Estudos de liberação e permeação in vitro suportaram e confirmaram o efeito de barreira na superfície da pele. Para além disso, o hidrogel selecionado demonstrou ser seguro sem causar irritação ou sensibilização da pele humana.De modo geral, o trabalho experimental desenvolvido nesta tese permitiu identificar moléculas capazes de bloquear/atenuar cada um dos eventos-chave da sensibilização da pele do AOP, dando um forte contributo para o campo da dermatite alérgica de contacto, uma patologia sem cura e a forma mais comum de imunotoxicidade nos humanos.

A poloxamer 407 (P407)—Casein hydrogel was chosen to carry polycaprolactone nanoparticles carrying terbinafine (PCL-TBH-NP). In this study, terbinafine hydrochloride (TBH) was encapsulated into polycaprolactone (PCL) nanoparticles, which were further incorporated into a poloxamercasein hydrogel in a different addition order to evaluate the effect of gel formation. Nanoparticles were prepared by the nanoprecipitation technique and characterized by evaluating their physicochemical characteristics and morphology. The nanoparticles had a mean diameter of 196.7 0.7 nm, PDI of 0.07, negative potential (0.713 mV), high encapsulation efficiency (>98%), and did not show cytotoxic effects in primary human keratinocytes. PCL-NP modulated terbinafine was released in artificial sweat. Rheological properties were analyzed by temperature sweep tests at different addition orders of nanoparticles into hydrogel formation. The rheological behavior of nanohybrid hydrogels showed the influence of TBH-PCL nanoparticles addition in the mechanical properties of the hydrogel and a long-term release of the nanoparticles from it.