Performance evaluation of Cryo Laser Phoresis technique as biophysical method to promote diclofenac sodium cutaneous perfusion

No sentido de alterar e/ou melhorar a penetração de substâncias na pele, diversas estratégias têm sido desenvolvidas, variando desde a aplicação de novos veículos e ativos encapsulados, até equipamentos que atuam por métodos biofísicos. Uma das técnicas de absorção física, atualmente conhecida como Crio Laser Forese (CLF), consiste em um aparato que emite radiação sobre moléculas polares ou apolares da substância ativa, tornando sua penetração mais rápida, se comparada à administração tópica comum. O objetivo deste trabalho foi avaliar a eficácia de um método que propõe aumentar a permeação cutânea do diclofenaco de sódio incorporado a um gel, por meio do uso da CLF. A influência sobre a permeação foi avaliada ex vivo, utilizando célula de Franz e pele humana obtida de cirurgia plástica. Os resultados foram balizados mediante aplicação de métodos estatísticos e análise exploratória de dados: clusters, k-means e Análise por Componentes Principais. Os resultados demonstraram aumento na concentração do diclofenaco de sódio na derme com o uso do laser. Em todas as amostras (com ou sem aplicação de laser), observou-se, uma quantidade de diclofenaco de sódio na superfície da pele e que não houve passagem de ativo para o líquido do receptor, sugerindo que o diclofenaco de sódio não foi absorvido. Estes resultados indicam que CLF usada sob as condições descritas neste estudo é capaz de aumentar a penetração do diclofenaco de sódio e sua retenção em camadas mais profundas da peleAiming to alter and/or improve permeation of active compounds in the skin, many strategies have been developed, including biophysical methods. One of the physical absorption techniques, currently known as Cryo Laser Phoresis (CLP), consists of an apparatus that emits radiation on polar or nonpolar molecules of the active substance, resulting in faster penetration when in comparison to the standard topical application. The goal of this work was to evaluate the efficacy of a method that proposes to increase cutaneous permeation of diclofenac sodium by using CLP technique. The influence on permeation was evaluated ex vivo, using Franz cell and human skin obtained from cosmetic surgery. The results were evaluated using statistical methods and data exploratory analysis: clusters, k-means and Principal Component Analysis. The results showed a larger increase in the concentration of diclofenac sodium in the dermis with the use of laser. In all samples (with or without laser application) it was observed that skin surface showed an amount of diclofenac sodium and that there was no active passage to the receptor liquid, suggesting that diclofenac sodium was not absorbed. These results indicate that CLP, when used under the conditions described in this study, is able to increase diclofenac sodium penetration and its retention into deeper layers

Performance Evaluation Of Cryo Laser Phoresis Technique As Biophysical Method To Promote Diclofenac Sodium Cutaneous Perfusion

Aiming to alter and/or improve permeation of active compounds in the skin, many strategies have been developed, including biophysical methods. One of the physical absorption techniques, currently known as Cryo Laser Phoresis (CLP), consists of an apparatus that emits radiation on polar or nonpolar molecules of the active substance, resulting in faster penetration when in comparison to the standard topical application. The goal of this work was to evaluate the efficacy of a method that proposes to increase cutaneous permeation of diclofenac sodium by using CLP technique. The influence on permeation was evaluated ex vivo, using Franz cell and human skin obtained from cosmetic surgery. The results were evaluated using statistical methods and data exploratory analysis: clusters, k-means and Principal Component Analysis. The results showed a larger increase in the concentration of diclofenac sodium in the dermis with the use of laser. In all samples (with or without laser application) it was observed that skin surface showed an amount of diclofenac sodium and that there was no active passage to the receptor liquid, suggesting that diclofenac sodium was not absorbed. These results indicate that CLP, when used under the conditions described in this study, is able to increase diclofenac sodium penetration and its retention into deeper layers.521697

Dicationic Alkylammonium Bromide Gemini Surfactants. Membrane Perturbation and Skin Irritation

Dicationic alkylammonium bromide gemini surfactants represent a class of amphiphiles potentially effective as skin permeation enhancers. However, only a limited number of studies has been dedicated to the evaluation of the respective cytotoxicity, and none directed to skin irritation endpoints. Supported on a cell viability study, the cytotoxicity of gemini surfactants of variable tail and spacer length was assessed. For this purpose, keratinocyte cells from human skin (NCTC 2544 cell line), frequently used as a model for skin irritation, were employed. The impact of the different gemini surfactants on the permeability and morphology of model vesicles was additionally investigated by measuring the leakage of calcein fluorescent dye and analyzing the NMR spectra of 31P, respectively. Detail on the interaction of gemini molecules with model membranes was also provided by a systematic differential scanning calorimetry (DSC) and molecular dynamics (MD) simulation. An irreversible impact on the viability of the NCTC 2544 cell line was observed for gemini concentrations higher than 25 mM, while no cytotoxicity was found for any of the surfactants in a concentration range up to 10 mM. A higher cytotoxicity was also found for gemini surfactants presenting longer spacer and shorter tails. The same trend was obtained in the calorimetric and permeability studies, with the gemini of longest spacer promoting the highest degree of membrane destabilization. Additional structural and dynamical characterization of the various systems, obtained by 31P NMR and MD, provide some insight on the relationship between the architecture of gemini surfactants and the respective perturbation mechanism

Carrier-mediated dermal delivery for prevention or treatment of skin disorders

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2013The effects of solar ultraviolet radiation that reaches the Earth surface can be beneficial but also harmful depending on the circumstances. The recent search for new chemical entities against photo-induced skin disorders has received particular focus on phytocompounds investigation. The use of natural compounds, working through different pathways in the cell, in both skin care products and therapeutic purposes may be an effective approach for reducing UV-B generated reactive oxygen species (ROS) mediated photoaging and photocarcinogenesis. In this work, the role of two active molecules is presented and discussed: tretinoin (the acid form of vitamin A) and lycopene (a carotenoid without vitamin A activity) in the context of photoaging and photocarcinogenesis, respectively. Tretinoin is involved in the control of cell differentiation and proliferation in several tissues, particularly in the skin epithelium. It has been demonstrated that photoaging can be ameliorated by topical retinoids, such as tretinoin, and long term effects of their topical application on the skin can be the reduction and redistribution of epidermal melanin, improved ultrastructural characteristics of the epidermis, increased anchoring fibrils, increased deposition of papillary dermal collagen, and increased vascularity in the papillary dermis. On the other hand, the anticancer activity of lycopene has already been demonstrated in both in vitro and in vivo different tumour models. The mechanisms underlying the inhibitory effects of lycopene on carcinogenesis could involve ROS scavenging, upregulation of detoxification systems, interference with cell proliferation, induction of gap-junctional communication, inhibition of cell cycle progression and modulation of signal transduction pathways. Dermal delivery after topical application of actives has gained increased interest and development due to the lower risk of systemic side effects. In particular, for antioxidants skin delivery, the search for a new delivery system that, simultaneously, preserves the antioxidant stability and enhances its deposition on the skin, opened a new chapter in drug delivery design. Since epidermal lipids are predominantly found within the penetration skin barrier (stratum corneum, SC), topically applied lipid nanocarriers, allowing lipid interaction between the outermost layer of SC and the carrier, appear promising. In fact, nanocarriers, and among these advanced drug delivery systems, liposomes and cyclodextrins, have successfully enhanced the clinical efficiency of several drugs. Liposomes are typically hollow spheres surrounded by a lipid bilayer. More recently, specially designed carriers have claimed the ability to cross the skin intact and deliver the loaded drugs into the systemic circulation, being at the same time responsible for the percutaneous absorption of the drug within the skin. These carriers were firstly introduced as transfersomes®*, and this denomination as well as deformable vesicles, were used to differentiate them from the more conventional liposomes. The highly flexible membranes are the result of combining into a single structure phospholipids and an edge-active component (surfactant) in order to give to transfersomes the necessary deformability to move spontaneously into the skin, delivering the associated drugs dermal or systemically. Cyclodextrins are cyclic watersoluble, non-reducing and macrocycle carbohydrate polymers. Some derivatives, such as β- and methylated-β-cyclodextrins are usually used for topical formulations. The aim of this thesis was to use cyclodextrins and transfersomes, or their combination, as delivery systems for tretinoin and lycopene, and further investigate the resulting systems behavior in in vitro and in vivo conditions. Tretinoin was incorporated in transfersomes (tretinoin-in-dimethyl-β-cyclodextrin-inultradeformable vesicles and tretinoin-in-ultradeformable vesicles) in order to fully characterize these new formulations, and further study its dermal delivery. The need of an appropriate source of lycopene led to the development of a simple and fast process for the extraction of lycopene from tomato. The incorporation of this lycopene-rich-extract in vesicular nanocarriers suitable for topical application was also performed and reported on this thesis. Lycopene was described as the antioxidant most quickly depleted in the skin upon exposure to solar radiation, and consequently, might play a role for protecting skin against UV radiation. To test this hypothesis, the main effects of UV-B irradiation on human keratinocytes (HaCaT cells line) after lycopene exposure were studied. Regarding the methods used, the tretinoin-cyclodextrin complex was prepared by the kneading process and transfersomes resulted from the combination of soybean phosphatidylcholine and Tween® The topical delivery of the tested formulations involved different studies, such as the release and permeation profiles (tape stripping); skin penetration (fluorescence analysis) and induced electrical changes in skin barrier properties. The in vitro toxicity was also achieved. 80 to make 10 % - 20 % lipid suspension. The resulting suspension was brought to the final mean vesicles size of approximately 100-150 nm, by sequential filtration. In the case of lycopene, an extraction process from dried tomatoes was previously carried for 30 min at room temperature, protected from light. The extracts were analyzed by high-performance liquid chromatography and mass spectroscopy and incorporated in deformable vesicles. The physicochemical characterization of the vesicular formulations was based on the evaluation of the following parameters: mean particle size and polydispersity index measured by photon correlation spectroscopy and atomic force microscopy; zeta potential determined by laser-doppler anemometry; phospholipid concentration determined by an enzymaticcolorimetric test; and drug assay by an adequate high performance liquid chromatography method. The irritation potential of the developed tretinoin formulation was evaluated in vivo using a modified Draize test and compared to a commercially available tretinoin semisolid form. The in vivo therapeutic effect of lycopene topical formulations was tested in mice, using the anthralin-induced ear swelling model, being anthralin topical application responsible for the generation of reactive oxygen species in the skin. Thiazolyl blue tetrazolium bromide (MTT) assay was performed in order to select the UV-B irradiation protocol specific conditions. After lycopene exposure and irradiation experiments, HaCaT cells were analyzed in order to achieve information on the following parameters: genotoxicity/clastogenicity by determining the cell cycle by flow cytometry (FCM) and analyzing apoptosis biomarkers and gene expression by real time – polymerase chain reaction quantification; apoptosis by performing the Annexin V assay by FCM; and oxidative stress damage by ROS quantification by FCM. Results of tretinoin-loaded transfersomes revealed that the incorporation efficiency was high for all formulations tested. However, the tretinoin vesicular formulation with 20 % lipid concentration presented better drug stabilization, attending to the higher chemical stability achieved. Tretinoin-loaded deformable vesicles applied on the skin were able to decrease the skin resistance, suggesting their ability to induce barrier disruption, as theoretically expected. It was also observed by fluorescence microscopy that these vesicles were mainly concentrated in the SC and epidermis layers. Permeation studies showed that no tretinoin is measurable in the receptor phase. In fact, drug retention in the SC and in epidermis/dermis are indicative of topical delivery, whereas the drug quantification in the receptor phase is indicative of transdermal delivery. These results showed that tretinoin-in-transfersomes has a dermal instead of a transdermal delivery, which serves the therapeutic purpose since tretinoin receptors, such as RAR-γ, are mainly located in epidermis. Finally, tretinoin-in-transfersomes formulation was not toxic in in vitro and in vivo conditions at least at 5 × 10 -3 mg/mL and 0.5 mg/mL of tretinoin, respectively. Regarding lycopene extraction, the little number of steps involved in the extraction process may have improved the yields of lycopene in the extract and exerted a beneficial role in the stability of this carotenoid. The vesicular formulations presented a good incorporation efficiency of lycopene in deformable vesicles (transfersomes and ethosomes). Cytotoxic data demonstrated that extracted lycopene and pure (commercial) lycopene present similar performances of low cytotoxicity, and were similarly affected by environmental conditions. The vesicular formulations containing extracted lycopene were able to inhibit the edema formation in vivo on anthralininduced ear swelling model relative to untreated controls, and without a statistically significant difference relative to the control treated with a model drug. ix In vitro photocarcinogenesis studies with UV-B irradiated keratinocytes revealed that lycopene decreased the irradiated cells viability according to MTT and Annexin V results. It was also observed the cell cycle arrest (at G0/ G1 phase and a higher apoptotic Sub- G1 peak), and a higher ROS level. In summary, according to the obtained results, an effective dermal delivery of actives could be achieved by the use of a carrier-mediated transport. Some aspects regarding the elucidation of drug biological activity behind this delivery system were also successfully explored.Os efeitos da radiação solar ultravioleta (UV) que atinge a superfície da Terra podem ser benéficos, mas também prejudiciais, dependendo das circunstâncias. Nos últimos anos, a pesquisa de novas moléculas com actividade na prevenção e/ou tratamento de lesões cutâneas, associadas a uma excessiva exposição à radiação solar, tem focado com especial interesse os fitocompostos. A utilização de compostos naturais que atuem através de diferentes vias celulares, em produtos terapêuticos e de cuidados da pele, poderá ser uma abordagem eficaz ao nível da redução de espécies reativas de oxigénio geradas pela radiação UV-B e envolvidas nos processos de fotoenvelhecimento e fotocarcinogénese. Neste trabalho, é apresentado e discutido o papel de duas moléculas ativas, a tretinoína (forma ácida da vitamina A) e o licopeno (carotenóide sem atividade provitamina A) no contexto do fotoenvelhecimento e da fotocarcinogénese, respetivamente. A tretinoína está envolvida no controlo da diferenciação celular e proliferação em vários tecidos, especialmente no tecido epitelial da pele. Tem sido demonstrado que o fotoenvelhecimento pode ser tratado com retinóides tópicos, como é o caso da tretinoína, e que os seus efeitos a longo prazo, ao nível da pele, passam pela redução e redistribuição da melanina epidérmica, melhoria ultra-estrutural da epiderme, aumento das fibrilhas de ancoragem, aumento da deposição de colagénio da derme papilar e aumento da vascularização na derme papilar. Por outro lado, a atividade anticancerígena do licopeno foi já demonstrada em estudos in vitro e, in vivo, em diferentes modelos tumorais. Os mecanismos subjacentes aos efeitos inibitórios do licopeno ao nível da carcinogénese poderão envolver a captura de espécies reativas de oxigénio, a supra-regulação de sistemas de desintoxicação, a interferência com a proliferação celular, a indução da comunicação ao nível das junções intercelulares, a inibição da progressão do ciclo celular e a modulação de vias de transdução de sinal. A veiculação dérmica de moléculas ativas constitui um tema que tem captado cada vez mais interesse e crescente investigação, uma vez que o risco de efeitos colaterais sistémicos é mais reduzido. Particularmente, no que se refere à veiculação de antioxidantes para a pele, a procura de um novo sistema de veiculação que mantenha a estabilidade da molécula antioxidante e, simultaneamente, aumente a sua deposição na pele, abriu um novo capítulo na área de veiculação de fármacos. Uma vez que os lípidos epidérmicos predominam ao nível da barreira de penetração da pele (estrato córneo, EC), os nanotransportadores lipídicos administrados topicamente, que permitam a interação dos lípidos entre a camada mais externa do EC e o transportador, parecem ser uma abordagem promissora. De facto, a utilização de nanotransportadores, tais como lipossomas e ciclodextrinas, tem sido bem-sucedida ao nível da eficácia clínica de vários fármacos. Os lipossomas são tipicamente vesículas rodeadas por uma bicamada lipídica. Mais recentemente, foram concebidos e desenvolvidos lipossomas deformáveis capazes de atravessar a pele intata, promovendo a absorção percutânea de moléculas ativas e a sua veiculação para a circulação sanguínea. Para os diferenciar dos lipossomas convencionais, foram nomeados de vesículas deformáveis, tendo sido inicialmente introduzidos com a designação de transfersomas*. As membranas altamente flexíveis destas vesículas são o resultado da combinação numa única estrutura de fosfolípidos e um tensioativo, que contribui para a sua deformabilidade e capacidade de se moverem espontaneamente através da pele, libertando as moléculas a eles associadas dérmica e sistemicamente. As ciclodextrinas são polímeros cíclicos de hidratos de carbono nãoredutores e solúveis em água. Alguns derivados, tais como β-ciclodextrinas e ciclodextrinas β-metiladas, têm sido geralmente utilizados em formulações tópicas. Esta dissertação teve como objetivo a utilização de ciclodextrinas e transfersomas, ou a sua combinação, como sistemas de veiculação de tretinoína e licopeno e, posteriormente, o estudo in vitro e in vivo das formulações desenvolvidas. A tretinoína foi incorporada em transfersomas (tretinoína-dimetil-β-ciclodextrinatransfersomas e tretinoína-transfersomas), posteriormente caracterizadas e usadas nos estudos de aplicação tópica. A necessidade de obter licopeno de forma economicamente viável, relativamente à oferta do mercado, conduziu ao desenvolvimento de um processo simples e rápido de extração de licopeno a partir do tomate. Posteriormente, esse extrato rico em licopeno foi incorporado em nanotransportadores vesiculares adequados à aplicação tópica. O licopeno tem sido descrito com um dos antioxidantes mais rapidamente eliminados na pele após a exposição à radiação solar, podendo por isso desempenhar um papel de proteção contra a radiação UV. Para testar esta hipótese, foram feitos estudos sobre os principais efeitos da radiação UV-B em queratinócitos humanos (linha celular HaCaT) previamente expostos ao licopeno. Relativamente aos métodos utilizados, a complexação da tretinoína com ciclodextrinas foi obtida por malaxagem e os transfersomas foram preparados através da adição de fosfatidilcolina de soja ao Tween® 80, de modo a obter suspensões com concentrações lipídicas de 10 % a 20 %. A suspensão resultante foi filtrada até obter vesículas com um diâmetro médio de 100-150 nm. O licopeno foi obtido previamente por um processo de extração a partir de tomate seco, durante 30 minutos, à temperatura ambiente e sob proteção da luz. Os extratos de licopeno foram analisados por cromatografia líquida de alta eficiência e por espectroscopia de massa e incorporados em transfersomas e etossomas. A caracterização físico-química das formulações vesiculares foi baseada na avaliação dos seguintes parâmetros: diâmetro médio das vesículas e índice de polidispersão, medidos por espectroscopia de correlação fotónica e microscopia de força atómica; potencial zeta, determinado por anemometria laser-doppler; concentração de fosfolípidos, determinada por um ensaio enzimático-colorimétrico e doseamento do fármaco por cromatografia líquida de alta eficiência. A aplicação tópica das formulações desenvolvidas envolveu diversos estudos, nomeadamente, os perfis de cedência e permeação, penetração na pele e alterações induzidas ao nível da barreira cutânea. A toxicidade das formulações finais foi testada in vitro. O potencial de irritação da formulação vesicular de tretinoína foi avaliado in vivo, utilizando o teste de Draize modificado, e comparado com uma forma tópica comercializada de tretinoína. Em relação às formulações vesiculares de licopeno, foi avaliado o efeito terapêutico in vivo, utilizando o modelo de inflamação aguda cutânea, induzido pela formação de espécies reativas de oxigénio após a aplicação de antralina na orelha de ratinho. As condições de irradiação dos queratinócitos por UV-B, previamente expostos ao licopeno, foram determinadas através da realização do ensaio MTT. Após a exposição ao licopeno e irradiação UV-B, as células foram analisadas a fim de se obter informação sobre: genotoxicidade / clastogenicidade, através da análise do ciclo celular e de biomarcadores de apoptose e expressão genética, por citometria de fluxo e quantificação da reação da transcriptase reversa em tempo real, respetivamente; apoptose, através do ensaio da anexina V; danos causados pelo stresse oxidativo, através da quantificação de substâncias reativas de oxigénio por citometria de fluxo. Os resultados referentes à formulação tretinoína-transfersomas revelaram que a eficácia de incorporação foi elevada para todas as condições testadas. No entanto, a formulação tretinoína-transfersomas com 20 % de concentração lipídica apresentou maior estabilidade química. De acordo com os resultados dos estudos de libertação tópica da formulação tretinoínatransfersomas, as vesículas deformáveis diminuíram a resistência elétrica da pele, sugerindo a sua capacidade para induzir a rutura da barreira cutânea, tal como teoricamente seria esperado. Além disso, através da microscopia de fluorescência, foi possível observar uma maior concentração destas vesículas ao nível do EC e epiderme. No entanto, a tretinoína não foi detetada na fase recetora nos estudos de permeação. A maior concentração de fármaco a nível do EC, epiderme e derme é indicativa de veiculação dérmica, ao passo que a presença de fármaco na fase recetora é indicativa de veiculação transdérmica. Deste modo, os resultados demonstraram que se obteve uma veiculação dérmica com a formulação tretinoína-transfersomas, a qual serve o propósito terapêutico, uma vez que os recetores da tretinoína, tais como RAR-γ, estão localizados sobretudo na epiderme. Finalmente, esta formulação não revelou toxicidade em condições in vitro e in vivo, pelo menos, a 5 x 10-3 mg/mL e 0,5 mg/mL de tretinoína, respetivamente. Em relação à extração do licopeno, o número reduzido de passos envolvidos neste processo pode ter melhorado o rendimento e exercido um papel benéfico na estabilidade deste carotenóide. Os resultados experimentais mostraram ainda uma incorporação eficiente do licopeno nas vesículas deformáveis. Além disso, tanto o licopeno extraído como o comercializado apresentaram valores reduzidos e semelhantes de citotoxicidade. As formulações vesiculares de licopeno extraído, posteriormente testadas in vivo, usando o modelo de inflamação aguda em orelha de ratinho induzida por antralina, promoveram a redução do edema tal como o controlo tratado com um fármaco modelo. Nos estudos in vitro de fotocarcinogénese com queratinócitos irradiados com UV-B, foi possível observar que o licopeno reduziu a viabilidade destas células de acordo com os resultados obtidos nos ensaios de MTT e anexina V. Foi também observada a paragem do ciclo celular (na fase G0/G1 e um pico mais alto Sub-G1 apoptótico) e, ainda, um aumento do nível de espécies reativas de oxigénio. Em suma, e de acordo com os resultados obtidos, a veiculação dérmica de fármacos pode ser conseguida com sucesso através da utilização de um nanotransportador. Foram ainda identificados, com sucesso, alguns aspetos relacionados com a atividade biológica das moléculas estudadas associadas a este tipo de transporte.Fundação para a Ciência e a Tecnologia (FCT, SFRH/BPD/48853/2008 e projeto PEst-OE/SAU/UI4013/2011

Novel Delivery Systems For Iron Replenishment

Iron is an integral part of hemoglobin and essential for the production of red blood cells. Iron deficiency and the resulting anemia are major nutritional deficiency disorders. The majority of patient populations suffering from iron deficiency anemia (IDA) are women of child bearing age and children of all ages. Iron deficiency is a complication of various other chronic conditions. Oral iron salts or colloidal parenteral iron formulations are treatment options for iron replenishment since several decades, but they are associated with severe side effects along with other patient noncompliance issues. Transdermal delivery of iron could be a potential alternative to treat iron deficiency due to safety and offers more acceptability. Since conventional iron formulations are not suitable for transdermal delivery, quest for an ideal iron compound resulted in identification of soluble Ferric Pyrophosphate (FPP), which was demonstrated to be very stable and safe for parenteral administration. Passive delivery of FPP was not successful due to its high molecular weight (745 Da) and low lipid solubility. Transdermal delivery of FPP using chemical permeation enhancers, iontophoresis, microneedle pretreatment and combination of these techniques were evaluated and proved to be successful in delivering iron across the skin. When iontophoresis was combined with microneedle pretreatment, adequate iron could be delivered in anemic rat models to reverse the iron deficiency. Further, a safe and patient friendly iron delivery system was developed by incorporating FPP in soluble microneedles. In vitro and in vivo studies were carried out to evaluate the FPP release and dermal kinetic profile of the iron from the soluble microneedles. Safety and toxicity of FPP in human skin cell lines was also investigated. The feasibility of transdermal delivery of Iron-dextran was also evaluated. Passive delivery of iron dextran is impossible due to its high molecular weight. Microneedle assisted delivery of iron dextran was investigated and soluble microneedle system with iron dextran was developed. Overall, the results of the project suggest that transdermal delivery of iron could be a potential alternate to treat IDA. Iron replenishment via transdermal route is likely to be more effective and safer than the conventional routes of administration

Therapeutic deep eutectic systems as promising new tools in the anticancer battle

Cancer remains a major health problem worldwide, with colorectal cancer (CRC) being the third most incident and the second most lethal. Inflammation has been highly associated with cancer development and maintenance, therefore, the reduction of the inflammatory microenvironment represents a promising therapeutic strategy. Deep eutectic systems (DES) are based on the combination of different components which together, at a certain molar ratio, present a deep decrease in their melting point compared with the individual compounds. When an active pharmaceutical ingredient is part of a DES it is designated by therapeutic deep eutectic system (THEDES). New THEDES combining terpenes with anticancer properties, such as safranal, menthol and linalool, with NSAIDs, like ibuprofen, ketoprofen and flurbiprofen were produced. To evaluate THEDES therapeutic potential activity, their physico-chemical properties, bioavailability and bioactivity, were explored using an integrative approach to determine their anti-CRC activity. Our results show that Safranal:Ibuprofen (3:1), Safranal:Ibuprofen (4:1) and Menthol:Ibuprofen (3:1) present promising therapeutic activity towards CRC cells due to a selective cytotoxic action towards cancer cells. Menthol:Ibuprofen (3:1) anti proliferative action seems to be related with cell membrane disruption, reduction of the inflammation through the reduction of ROS production, and induction of apoptosis via caspase-3. On the other hand, Safranal:Ibuprofen (3:1) and Safranal:Ibuprofen (4:1) seem to prevent tumour cells expansion only through the induction of apoptosis via caspase-3. These systems also present an increasement in Ibuprofen permeability, with Menthol:Ibuprofen (3:1) increasing also Ibuprofen solubility and the overall bioavailability.O cancro continua a ser um dos maiores problemas de saúde em todo o mundo, sendo que o cancro colorretal é o terceiro mais incidente e o segundo mais letal. A inflamação está altamente associada ao desenvolvimento e persistência do cancro, por esse motivo, a redução do microambiente inflamatório representa uma estratégia terapêutica promissora. Sistemas eutéticos, em inglês, Deep eutectic systems (DES) são sistemas baseados na combinação de diferentes componentes que em conjunto e a um determinado rácio molar apresentam uma grande diminuição do ponto de fusão em comparação com os compostos individuais. Quando um dos componentes do DES é um ingrediente farmacêutico ativo ele é designado de sistemas eutéticos terapêuticos, therapeutic deep eutectic system (THEDES). Novos THEDES a conjugar terpenos com propriedades anticancerígenas, como mentol, safranal e linalool, com anti-inflamatórios não esteroides, como ibuprofeno, cetoprofen e flurbiprofeno. Para avaliar o potencial terapêutico dos THEDES, as suas propriedades físico- -químicas, biodisponibilidade e bioatividade foram exploradas ao longo da tese como uma análise integrativa para determinar as suas atividades contra o cancro colorretal. Os nossos resultados demonstram que o Safranal:Ibuprofeno (3:1), Safranal:Ibuprofeno (4:1) e Mentol:Ibuprofeno (3:1) apresentam uma atividade terapêutica promissora contra as células do cancro colorretal devido a uma ação citotóxica seletiva contra as células cancerígenas. A ação do Mentol:Ibuprofeno (3:1) como anti-proliferativo contra as células cancerígenas aparenta estar relacionada com a disrupção da membrana celular, redução da inflamação através da redução da produção de ROS, e através da indução da apoptose via caspase-3. Por outro lado, Safranal:Ibuprofeno (3:1) e Safranal:Ibuprofeno (4:1) aparentam prevenir a expansão das células tumorais apenas através da indução da apoptose via caspase-3. Adicionalmente, estes sistemas também apresentam um aumento da permeabilidade do Ibuprofeno, com o sistema Mentol:Ibuprofeno (3:1) aumentando também a solubilidade do Ibuprofeno e a sua biodisponibilidade no geral

Novel Amphiphilogels: Potential as Topical Drug and Vaccine Delivery Vehicles.

Purpose: To characterise amphiphilogels (gels where both solid and liquid phases are amphiphilic and investigate their potential as topical drug and vaccine delivery vehicles. Methods: Amphiphilogels were prepared by mixing and heating the components (nonionic surfactants) at 60°C to form a clear isotropic sol phase; this was then cooled and set to a semisolid. The gels were characterised with respect to their microstructures, gelation temperatures (Tg), and flow properties. Some small hydrophobic drugs were incorporated into the gels, and the release of such drugs from the gels, through a semi- permeable membrane, measured in vitro with the use of diffusion cells. The permeation of a model protein into full-thickness pig skin in vitro using the diffusion cells was investigated; as was the immune responses elicited following topical application of this model protein antigen when formulated into the gels. In vivo skin irritation studies evaluated the irritation potential of the gels following single and repeated applications. Results: The gels are smooth, opaque, thermoreversible semisolids with a lifetime greater than two years. The microstructure consists of a 3-D network of interconnected gelator tubule clusters, or fibres, which immobilises the continuous phase. Within the tubular aggregates, the gelator molecules seem to be arranged in lamellae. Increasing gelator concentration results in a denser microstructural network, higher viscosity, and higher Tg. Three drugs were solubilised at concentrations up to 20% w/w in some amphiphilogeis. The drugs could also be incorporated into the gels by simply mixing the drug and gel; this method enabled a greater release rate of drugs from the gels. An aqueous phase (containing dissolved solute) could be incorporated within the more hydrophilic amphiphilogeis (to form water-in-gel systems) with little compromise to the gel stability. Hydrophilic molecules, e.g. proteins and vaccines, can thus be incorporated into the gels. The permeation of protein from water-in-gel systems into the epidermal layers of the skin was demonstrated in vitro. Immunisation studies showed no differences between the immune responses generated to a model antigen administered topically within the gels or when dissolved in aqueous solution. Skin irritation studies showed that the amphiphilogeis caused very little perturbation to the skin. Conclusions: This research indicates that the amphiphilogeis have the potential to be developed into topical drug delivery vehicles

Ionic Liquids and Deep Eutectic Solvents for Application in Pharmaceutics

Dear Colleagues, In recent years, functional coating technology has attracted increased attention due to its effective potential for improved engineered materials. Growing demand for new materials with synergistic properties pushed research toward a new field to obtain innovative and smart coatings with functional capabilities that greatly differ from the conventional ones. In such a context, the expression of “functional coatings” has acquired specific relevance. This Special Issue will assess cutting-edge developments in this research area for the improvement and growth of actual performance, industrial scale-up, and marketability of functional coatings. This Special Issue is useful for researchers who are approaching this application context to improve their knowledge, with the aim of providing valuable scientific support for new research paths concerning functional surface engineering design and tailoring. Prof. Luigi Calabrese, Prof. Edoardo Proverbio Guest Editor

Micro-RNA family that modulates fibrosis and uses thereof

The present invention relates to the identification of a microRNA family, designated miR-29a-c, that is a key regulator of fibrosis in cardiac tissue. The inventors show that members of the miR-29 family are down-regulated in the heart tissue in response to stress, and are up-regulated in heart tissue of mice that are resistant to both stress and fibrosis. Also provided are methods of modulating expression and activity of the miR-29 family of miRNAs as a treatment for fibrotic disease, including cardiac hypertrophy, skeletal muscle fibrosis other fibrosis related diseases and collagen loss-related disease.Board of Regents, University of Texas Syste