Development of polymeric nanoparticles with controlled drug release for dermal application

Controlled delivery of corticosteroids to the skin and hair follicle using nanoparticles may reduce their side effects and maximize treatment effectiveness. To assess the quality of nanoparticles and estimate their in vivo performance, in vitro drug release measurement is one of the most important methods. Dexamethasone-loaded polymeric nanoparticles should be prepared, which adhere well to the skin and release the drug slowly, in a controlled manner. Additionally, sebum-responsive nanoparticles should be prepared, which are able to penetrate deep into the hair follicle and release the drug triggered by their dissolution in sebum. The discriminative power and reproducibility of three in vitro drug release methods for nanoparticles, namely dialysis bags, Franz diffusion cells and an in situ drug release method using Sirius® inForm apparatus should be assessed. The investigated nanoparticles were nanocrystals, polymeric nanoparticles and lipid nanoparticles. Dexamethasone-loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was assessed ex vivo using excised human skin. Follicular penetration of nanoparticles was investigated ex vivo using pig ear skin. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3–0.7%) than ethyl cellulose nanoparticles (1.4–2.2%). By blending the two polymers (1:1), small (105 nm), positively charged (+37 mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. Ethyl cellulose dissolved fast in artificial sebum, whereas Eudragit® RS was insoluble. Artificial sebum increased the drug release from ethyl cellulose nanoparticles, whereas it reduced the drug release from Eudragit® RS nanoparticles indicating a sebum-responsive drug release from ethyl cellulose nanoparticles. The hair follicle penetration depth of Eudragit® RS (330 µm) and ethyl cellulose nanoparticles (380 µm) was comparable, but the fluorescence intensity inside the hair follicle was higher from Nile red-loaded ethyl cellulose nanoparticles compared to Eudragit® RS nanoparticles. In conclusion, the prepared nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and in the hair follicle to maximize treatment effectiveness. The comparison of the different in vitro drug release methods indicated that the methods differ in their discriminative power and reproducibility. The in situ measurement was a simple and fast method, but not adequately discriminating because of a too rapid drug dissolution/release. Franz diffusion cells and dialysis bags were in most cases discriminative for the different nanoparticles with the drug dissolution/release being in the order of nanocrystals > Eudragit® RS nanoparticles > lipid nanoparticles ≥ ethyl cellulose nanoparticles. However, drug release experiments with Franz diffusion cells had the highest reproducibility

Estudos de competição microbiana na pele e mecanismos de adesão microbiana a queratinócitos

A pele é um órgão importante nos mamíferos e desempenha um papel essencial na defesa do organismo humano, sendo a sua primeira barreira face ao ambiente externo. Os principais filos que fazem parte da microbiota de uma pele saudável são: Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%), sendo os géneros predominantes o Corynebacterium, Propionibacterium e o Staphylococcus. Quando ocorre disbiose (desequilíbrio entre a microbiota comensal e os microrganismos oportunistas), podem-se originar patologias da pele. A maioria dos distúrbios da pele, são principalmente inflamatórios e são tradicionalmente tratados com corticosteróides. No entanto, existem alguns efeitos colaterais relacionados ao seu uso, como efeitos cutâneos deletérios, anormalidades eletrolíticas, hipertensão, hiperglicemia, efeitos imunológicos e neuropsicológicos, entre outros. Uma prescrição a longo prazo também está associada à osteoporose. Na tentativa de reduzir esses efeitos colaterais, os probióticos foram sugeridos como coadjuvantes na terapia, via aplicação tópica. Em vista disso, o presente trabalho teve como objetivo realizar estudos de competição entre probióticos selecionados e bactérias patogénicas nas seguintes combinações: (Lactobacillus paracasei / Staphylococcus aureus; L. paracasei / Staphylococcus epidermidis; L. paracasei / Escherichia coli; L. paracasei / Pseudomonas aeruginosa); (Propioniferax innocua / E. coli; P. innocua / P. aeruginosa); (Bifidobacterium longum spp. infantis / S. aureus; B. longum spp. infantis / S. epidermidis; B. longum spp. infantis / E. coli; B. longum spp. infantis / P. aeruginosa). O probiótico que apresenta a melhor vantagem competitiva foi posteriormente testado em ensaios de cultura celular usando células HaCat (linha celular de queratinócitos da pele humana) para entender-se o probiótico (L. paracasei) foi capaz de impedir a adesão das bactérias patogénicas (S. aureus, S. epidermidis e P. aeruginosa). Os melhores resultados foram obtidos pela técnica de deslocamento face ao S. aureus, onde foi observada uma redução de 3.8 unidades logarítmicas. Estes resultados não coincidem com os obtidos pelos ensaios de competição de células no seu estado livre. As principais razões provavelmente são a acessibilidade das células no meio, a auto/coagregação das células e a estimulação pelo Quorum Sensing. Com o intuito de se compreender alguns dos mecanismos utilizados para impedir essa adesão, também foi realizado um ensaio de adesão das proteínas. A partir dos resultados obtidos, pode-se inferir que parte do processo de adesão implica a utilização de proteínas, embora não seja um mecanismo exclusivo.As major organ in mammals, and first barrier of defense against external environment, the skin plays an essential role in the defense of the human organism, harboring a characteristic microflora. The most commonly found phyla of bacteria in healthy skin are Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes (6.3%) in which the most prevalent genera are Corynebacterium, Propionibacterium and Staphylococcus. Dysbiosis occurs when there is an unbalance between the commensal microbiota and opportunistic microorganisms., which can lead to skin pathologies. The majority of skin disorders, which are mainly inflammatory, are traditionally treated with corticosteroids. However, there are some side effects linked to their use such as deleterious cutaneous effects, electrolyte abnormalities, hypertension, hyperglycemia, immunologic, and neuropsychologic effects, among others. The long-term prescription is also associated with osteoporosis. In an attempt to reduce those side effects, probiotics have been suggested as co-adjuvandants in the therapy, via topical application. In this context, the present work was aimed at performing competition studies between selected probiotics and pathogenic bacteria in the following combinations: (Lactobacillus paracasei/Staphylococcus aureus; L. paracasei/Staphylococcus epidermidis; L. paracasei/Escherichia coli; L. paracasei/Pseudomonas aeruginosa); (Propioniferax innocua/E. coli; P. innocua/P. aeruginosa); (Bifidobacterium longum spp. infantis/S. aureus; B. longum spp. infantis/S. epidermidis; B. longum spp. infantis/E. coli; B. longum spp. infantis/P. aeruginosa). The probiotic presenting the best competitive advantage was then further tested in cell culture assays using HaCat cells (keratinocyte cell line from human skin) to assess the probiotic (L. paracasei) preventive role in the adhesion of the pathogenic bacteria (S. aureus, S. epidermidis and P. aeruginosa). The best results were achieved with the displacement technique towards S. aureus, in which a reduction of 3.75 logarithmic units was observed. These results did not match those obtained by the planktonic cell competition assays. The main reasons probably being cell accessibility in the media, cell (s) self / coaggregation and the empowerment by quorum sensing. In order to gain some insight on the mechanisms used to prevent this adhesion, a protein adhesion assay was also conducted. From the results obtained it can inferred that part of the adhesion process uses proteins, although not being an exclusive mechanism

The effect of hexosamine biosynthetic pathway (HBP) activation on wound healing

Wound healing is a not completely unraveled complex interplay of cells, extracellular matrix (ECM) and mediators. As ineffective wound healing is an increasing global medical and economical problem, research is highly required to develop new therapeutic strategies. The ECM consists of glycosaminoglycans (GAG) which are produced in the intracellular, ubiquitous hexosamine biosynthetic pathway (HBP). Its end product, UDP-N-acetylglucosamine (UDP- GlcNAc), is required for posttranslational modification. This project investigates the role of HBP activation in wound healing in vitro. Therefore, we tested the modulation of GlcNAc, UDP-HexNAc and hyaluronic acid (HA) by GlcNAc supplementation in both immortalized and primary murine fibroblasts and keratinocytes. Then, wound healing assays were performed in these cell types. The second part of the project focuses on the effect of HBP activation on cell proliferation potential on primary murine keratinocytes performing colony formation assays with either immortalized (NIH3T3 cell) or primary murine fibroblasts as feeder layer. HBP activation by 50 mM GlcNAc supplementation caused only a mild increase of UDP- HexNAc and HA concentration in fibroblasts, yet a significant raise in keratinocytes. Interestingly, this led to a slowing effect in keratinocyte migration and consequently slowed wound closing (HaCaT cells: 80% of the wounds in the control group closed within 72 h vs. 45% of the tested group; primary keratinocytes: 80% vs. 50%). There was no significant dose- dependent effect for GlcNAc supplementation. As HA is known to accelerate wound healing, we evaluated the effect of substrates with a similar viscosity and showed this also for dextran, being therefore a possible therapeutical strategy for chronic wounds. The second part of the project demonstrated no significant effect of 50 mM GlcNAc or 50 mM glucose on proliferative potential in keratinocytes. Surprisingly, 50 mM mannose supplementation caused a significant reduction. In conclusion, HBP activation seems to affect wound healing in vitro negatively. However, my project underlines the significance of the HBP for effective cellular function including migration and proliferation, which are required for wound healing. In the future, more experiments should be performed to unravel its complete effect and to possibly identify targets for clinical application

Development of gellan gum fluid gel as modified release drug delivery systems

Gelation of polysaccharides under shear conditions results in the formation of a weak gel which is able to resist elastic mechanical deformation at small strains but will flow if subjected to higher strains. The resulting material, described in the literature as a fluid gel or a sheared gel, consists of gelled microparticles which can be formulated to collectively act in bulk, as pourable viscoelastic fluids whilst retaining true gel characteristics at the micro/nano level. The tuneable behaviour of these fluid gel systems makes them potentially useful in pharmaceutical applications. Fluid gels prepared from gellan gum are particularly attractive, due to its sensitivity to physiological fluids, unique rheological and physical properties, and current regulatory approval for use as a food additive and pharmaceutical excipient. Therefore, the aim of the present study was to investigate gellan gum fluid gels as a new modified release drug delivery platform. The formation and production of fluid gels using low acyl (LA) gellan, high acyl (HA) gellan and LA HA gellan blends was investigated and applied in three different dosage forms; a modified release oral liquid, a mucoadhesive nasal spray and a topical formulation. A modified release oral liquid was designed using a fluid gel prepared from LA gellan gum. It was demonstrated that 0.75 % w/w LA gellan gum fluid gel, containing ibuprofen as the drug, could be formulated to have a similar viscosity profile as a marketed oral ibuprofen liquid. Furthermore, due to the acid insolubility of gels prepared from LA gellan, no ibuprofen was released in stimulated gastric fluid. Subsequent release at pH 7.4 however, was affected by the duration of exposure and strength of the acidic pH used and a linear relationship between onset of release and the preceding duration of acid exposure was observed. Delayed release was a result of increasing gel stiffness, a consequence of the acidity of the initial release media and exposure time. A much faster release rate was measured when exposure time in acid was 10 min compared with 60 min. This study highlights the potential to design fluid gels that are tuned to have a specified stiffness at a particular pH and exposure time allowing the intelligent design oral liquids with specific modified release behaviour. The second part of this study was to prepare mucoadhesive nasal drug delivery systems to enhance the retention of the nasal spray dosage form in the nasal cavity. Several groups have investigated using LA gellan solution as a drug delivery vehicle but only limited research however, has been performed on HA gellan for this purpose, despite its properties being more conducive to mucoadhesion. HA gellan (even with low concentration 0.25 % w/w) produces highly elastic gels below 60 °C which make it difficult to spray using a mechanical spray device. To address this problem, fluid gels were prepared as these systems can behave as sprayable viscoelastic fluids. In this study the rheological behaviour was investigated and the mucoadhesion behaviour of fluid gels prepared from the two different types of gellan (HA and LA) and fluid gels prepared from a blend of LA HA gellan. The results demonstrated that by preparing fluid gels from a blend of LA HA gellan, the rheological properties were sufficient to spray through a standard nasal spray device. Moreover, the fluid gels significantly enhanced both HA and LA gellan mucoadhesion properties. In the final part of this thesis the topical application of gellan fluid gels was explored. A range of gellan fluid gel formulations were prepared containing diclofenac sodium for topical application. The rheological results showed that it was possible to produce a topical formulation with a viscosity and the mechanical strength similar to that of the commercially available Voltaren® gel using 1 % w/w of a 50:50 LA HA gellan blend. The permeation results highlighted that the penetration of diclofenac through procaine tissue is significantly increased by increasing gellan concentration and decreasing sodium ion concentration in the formulation

Wound healing strategies - hyaluronic acid (HA) and chitosan (CS) as wound dressing materials

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.A pele é o maior órgão do corpo humano, com função de proteção, regulação da temperatura corporal e suporte de vasos sanguíneos e nervos. Quando esta barreira é danificada, criando uma ferida, inicia-se um processo de cicatrização. Este é habitualmente dividido em quatro fases, que se sobrepõe. A primeira, a homeostase, visa o bloqueio imediato do sangramento da ferida, por mecanismos de vasoconstrição e coagulação. Subsequentemente, dá-se um processo inflamatório, com o recrutamento de neutrófilos, macrófagos e linfócitos para o local da agressão, seguindo-se uma fase de proliferação, com a formação de novo tecido de granulação, re-epitelização e angiogénese. Finalmente, ocorre a formação da cicatriz, na fase de remodelação. A sequência destes eventos determina a formação de uma ferida aguda ou crónica. As feridas agudas passam por todos os passos do processo no espaço de poucos dias, ou semanas, enquanto uma ferida crónica se prolonga no tempo, tendo muitas vezes fatores externos e internos associados. Entre estes, destacam-se a presença de infeção, doença crónica, como diabetes, imunossupressão e a idade. O tratamento das feridas crónicas, e a sua manutenção, assume custos elevados para os sistemas de saúde, com perspetivas de aumento devido ao envelhecimento da população. Assim, a emergência de tratamentos mais eficazes tem sido uma área de grande exploração científica nas últimas décadas. Os novos tratamentos para feridas atualmente em estudo têm em consideração todos os passos da cicatrização, assim como os mecanismos envolvidos. Entre os materiais usados, destacam-se os biopolímeros, pela sua biocompatibilidade e biodegradabilidade, assim como o baixo custo e natureza renovável. Pelo seu papel na cicatrização das feridas, o ácido hialurónico e o quitosano são dois dos biopolímeros mais usados. Desse modo, esta monografia pretende explorar a utilização destes polímeros como materiais para o tratamento de feridas, nas formulações atualmente em estudo.The skin is the largest organ of the human body, with the primary function of protection. When its normal anatomic structure and function is disrupted, a wound is formed. A phenomenon called wound healing is subsequently activated, normally divided into four phases, that occur in an overlapping manner. The first, hemostasis, aims at immediately protecting the injury site by blocking blood and fluid loss. Then, inflammatory agents are recruited, including neutrophils, lymphocytes, and macrophages, in the inflammatory phase. Proliferation comes next, where new granulation tissue is formed simultaneously with re-epithelialization, and angiogenesis, and, finally, tissue remodeling or scar formation occurs. This sequence of events, and the time in which they occur, determine the outcome of an acute or chronic wound. Chronic wounds often reoccur and are frequently associated with local or systemic factors, such as immunosuppression, chronic diseases, like diabetes, infection, and aging. In the last decades, the high cost of wound management and increasingly aging population prone to the development of chronic wounds, have driven the research for more effective wound dressings. There is a wide variety of wound dressings currently available, with distinct characteristics, and a set of parameters should inform the better choice for each patient, and different types of wound. The novel wound dressings currently being studied take into consideration the different phases of the wound healing process and the external factors that may impair it. Therefore, biopolymers such as hyaluronic acid and chitosan, have become popular as wound dressing materials, as they exhibit biocompatible and biodegradable properties, allied with a low cost and renewable nature. Hydrogels and films using these polymers, as well as delivery systems, namely microparticles, have been explored. This monograph aims at reviewing some of the recent advances in the field of wound dressings using hyaluronic acid and chitosan