Dissertations / Theses on the topic 'Insulin carrier'

Actuellement, l'injection sous-cutanée d'insuline est le seul moyen pour les diabétiques de type 1 d'équilibrer leur glycémie. Les travaux de thèse entrent dans le cadre du projet ORAIL qui vise à développer un système de délivrance orale d'insuline basé sur la double encapsulation, et à valider l'efficacité et la sécurité de ce système in vitro dans des modèles d'épithélium intestinal, et in vivo chez le rat. Le vecteur pharmaceutique développé est composé d'une gélule contenant des nanoparticules (NPs) d'insuline formulées à partir d'acide (lactique-co-glycolique) par la méthode de double émulsion eau/huile/eau. Un premier objectif de la thèse a été d'évaluer chez le rat la gastrorésistance et l'entérosolubilité de la gélule sélectionnée pour l'encapsulation des NPs, par tomodensitométrie aux rayons X et par l'étude de la biodisponibilité de l'ibuprofène et de l'acétaminophène. Les résultats de ces travaux ont montré que la gélule est résistante en conditions gastriques et se dégrade au niveau de l'intestin. Un deuxième objectif a été de synthétiser des NPs d'insuline de taille croissante (100 à 800 nm), et d'évaluer l'internalisation de ces NPs et leur sécurité dans des cultures de cellules Caco-2, et dans des co-cultures de cellules Caco-2 et HT29-MTX. Les résultats de ces travaux ont montré que les NPs sont internalisées de manière dose et temps-dépendante, et que la taille de NPs permettant une internalisation optimale est de 400 nm après 4h d'incubation. Des études mécanistiques ont suggéré l'implication de mécanismes cavéoline-dépendants dans l'internalisation des NPs. Aucune toxicité des NPs n'a été observée quels que soient les paramètres étudiés (viabilité et mort cellulaire, augmentation de perméabilité, production de mucus, sécrétion de cytokines pro-inflammatoires). Dans une dernière partie de notre travail, nous avons montré que l'administration intraduodénale de NPs d'insuline de 200 et 400 nm à des rats diabétiques permettait une diminution significative de leur glycémie sans altération morphologique de leur paroi intestinale, données confortant nos résultats in vitro. Notre vecteur basé sur la double encapsulation semble donc être un système prometteur pour l'administration orale d'insuline. Le vecteur complet doit cependant être évalué in vivo chez le rat.

The importance of insulin delivery and action is best characterized in Type 2 Diabetes, a disease that is becoming a pandemic both nationally and globally. Obesity is a principal risk factor for Type 2 Diabetes, and adipocyte function abnormalities due to adipose hypertrophy and hyperplasia, have been linked to obesity. Numerous reports suggest that the intracellular and systemic consequences of adipocyte function abnormalities include adipocyte insulin resistance, enhanced production of free fatty acids, and production of inflammatory mediators. A hallmark of adipocyte insulin sensitivity is the stimulation of glucose transporter isoform 4 (GLUT4) trafficking events to promote glucose uptake. In the Type 2 diabetic and insulin resistant states the mechanism behind insulin-stimulated GLUT4 trafficking is compromised. Therefore, understanding the role of factors involved in glucose-uptake in adipose tissue is of great importance. Studies from our laboratory suggest an important role for the unconventional myosin, Myo1c, in promoting insulin-mediated glucose uptake in cultured adipocytes. Our observations suggest that depletion of Myo1c in cultured adipocytes results in a significant reduction in the ability of adipocytes to take up glucose following insulin treatment, suggesting Myo1c is required for insulin-mediated glucose uptake. A plausible mechanism by which Myo1c promotes glucose uptake in adipocytes has been suggested by further work from our laboratory in which expression of fluorescently-tagged Myo1c in cultured adipocytes induces significant membrane ruffling at the cell periphery, insulin-independent GLUT4 translocation to the cell periphery, and accumulation of GLUT4 in membrane ruffling regions. Taken together Myo1c seems to facilitate glucose uptake through remodeling of cortical actin. In the first part of this thesis I, in collaboration with others, uncovered a possible mechanism through which Myo1c regulates adipocyte membrane ruffling. Here we identified a novel protein complex in cultured adipocytes, comprising Myo1c and the mTOR binding partner, Rictor. Interestingly our studies in cultured adipocytes suggest that the Rictor-Myo1c complex is biochemically distinct from the Rictor-mTOR complex of mTORC2. Functionally, only depletion of Rictor but not Myo1c results in decreased Akt phosphorylation at serine 473, but depletion of either Rictor or Myo1c results in compromised cortical actin dynamic events. Furthermore we observed that whereas the overexpression of Myo1c in cultured adipocytes causes remarkable membrane ruffling, Rictor depletion in cells overexpressing Myo1c significantly reduces these ruffling events. Taken together our findings suggest that Myo1c, in conjunction with Rictor, modulates cortical actin remodeling events in cultured adipocytes. These findings have implications for GLUT4 trafficking as GLUT4 has been previously observed to accumulate in Myo1c-induced membrane ruffles prior to fusion with the plasma membrane. During our studies of adipocyte function we noticed that current siRNA electroporation methods present numerous limitations. To silence genes more effectively we employed a lentivirus-mediated shRNA delivery system, and to standardize this technology in cultured adipocytes we targeted Myo1c and MAP4K4. Using this technology we were able to achieve clear advantages over siRNA oligonucleotide electroporation techniques in stability and permanence of gene silencing. Furthermore we showed that the use of lentiviral vectors in cultured adipocytes did not affect insulin signaling or insulin-mediated glucose uptake events. Despite our inability to use lentiviral vectors to achieve gene silencing in mice we were able to achieve adipose tissue-specific gene silencing effects in mice following manipulation of the lentiviral conditional silencing vector, and then crossing resulting founders with aP2-Cre mice. Interestingly however, only founders from the MAP4K4 conditional shRNA vector, but not founders from the Myo1c conditional shRNA vector, showed gene knockdown, possibly due to position-effect variegation. Taken together, findings from these studies are important because they present an alternative means of achieving gene silencing in cultured adipocytes, with numerous advantages not offered by siRNA oligonucleotide electroporation methods. Furthermore, the in vivo, adipose tissue-specific RNAi studies offer a quick, inexpensive, and less technically challenging means of achieving adipose tissue-specific gene ablations relative to traditional gene knockout approaches.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. The pathogenesis of these diseases involves β-cell dysfunction and death. The primary function of β-cells is to tightly regulate the secretion, production, and storage of insulin in response to blood glucose levels. In order to manage insulin biosynthesis, β-cells have an elaborate endoplasmic reticulum (ER). The ER is an essential organelle for the proper processing and folding of proteins such as proinsulin. Proteins fold properly when the ER protein load balances with the ER folding capacity that handles this load. Disruption of this ER homeostasis by genetic and environmental stimuli leads to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. Upon ER stress, the unfolded protein response (UPR) is activated. The UPR is a signaling network that aims to alleviate ER stress and restore ER homeostasis promoting cell survival. Hence, the UPR allows β-cells to handle the physiological fluctuations of insulin demand. However upon severe unresolvable ER stress conditions such as during diabetes progression, the UPR switches to pathological outputs leading to β-cell dysfunction and apoptosis. Severe ER stress may also trigger inflammation and accumulating evidence suggests that inflammation also contributes to β-cell failure, but the mechanisms remain elusive. In this dissertation, we demonstrate that thioredoxin interacting protein (TXNIP) mediates ER stress induced β-cell inflammation and apoptosis. During a DNA microarray analysis to identify novel survival and death components of the UPR, we identified TXNIP as an interesting proapoptotic candidate as it has been linked to glucotoxicity in β-cells. During our detailed investigation, we discovered that TXNIP is selectively expressed in β-cells of the pancreas and is strongly induced by ER stress through the IRE1α and PERK-eIF2α arms of the UPR and specifically its transcription is regulated by activating transcription factor 5 (ATF5) and carbohydrate response element binding protein (ChREBP) transcription factors. As TXNIP has been shown to activate the Nod-like receptor protein 3 (NLRP3) inflammasome leading to the production of the inflammatory cytokine interleukin-1β (IL- 1β), we hypothesized that perhaps TXNIP has a role in IL-1β production under ER stress. We show that ER stress can induce IL-1β production and that IL-1β is capable of binding to IL-1 type 1 receptor (IL-1R1) on the surface of β-cells stimulating its own expression. More importantly, we demonstrate that TXNIP does indeed play a role in ER stress mediated IL-1β production through the NLRP3 inflammasome. Furthermore, we also confirmed that TXNIP is a mediator of β-cell apoptosis under ER stress partially through IL-1β signaling. Collectively, we provide significant novel findings that TXNIP is a component of the UPR, mediates IL-1β production and autostimulation, and induces cell death under ER stress in β-cells. It is becoming clear that TXNIP has a role in the pathogenesis of diabetes and is a link between ER stress, oxidative stress and inflammation. Understanding the molecular mechanisms involved in TXNIP expression, activity, and function as we do here will shed light on potential therapeutic strategies to tackle diabetes.

L'hypoxie intermittente (HI), induite par les apnées du sommeil, conduit à des altérations de la sensibilité à l'insuline et de l'homéostasie glucidique mais les mécanismes impliqués restent mal connus. L'objectif de ce travail était d'étudier les effets et les mécanismes sous jacents d'une exposition chronique à l'HI sur l'homéostasie glucidique. L'HI induit une résistance à l'insuline à la fois systémique et tissulaire, ainsi qu'une amélioration de la tolérance au glucose associée à une activation de l'AMPK musculaire. L'HI cause également des altérations du foie et du tissu adipeux associées à un changement du pattern d'expression des gènes dans ces tissus et à un risque accru de développement de pathologies vasculaires comme l'athérosclérose. Enfin, la délétion de PHD1, une des protéines régulatrices de HIF-1, entraîne une résistance à l'insuline associée une stéatose hépatique, faisant de HIF-1 une cible potentielle impliquée dans les altérations metaboliques induites par l'HI
Intermittent hypoxia (IH), induced by sleep apnea, leads to alterations in insulin sensitivity and glucose homeostasis but the mechanisms involved remains poorly understood. The objective of this work was to study the effects and the underlying mechanisms of chronic exposure to IH on glucose homeostasis. IH induces both systemic and tissue-specific insulin resistance , as well as improved glucose tolerance associated with an activation of muscle AMPK. IH also causes a change in the pattern of gene expression in liver and adipose tissue and an increased risk of vascular pathologies such as atherosclerosis development. Finally, the deletion of PHD1, a regulatory protein of HIF-1, leads to insulin resistance associated with hepatic steatosis, making HIF-1 a possible target involved in the metabolic changes induced by IH

碩士
國立雲林科技大學
化學工程與材料工程研究所
95
The aim of this research is to Preparation N,O-carboxymethyl chitosan (NOCC)which is derivative of chitosan. Insulin- N,O-carboxymethyl chitosan nanoparticle were prepared by ionic gelation of N,O- carboxymethyl chitosan and tripolyphosphate pentasodium(TPP). The NOCC/TPP mass ratio and insulin initial concentration were studied and how influence insulin release phenomena. The physicochemical properties of nanoparticles were determined by particle size, zeta potential analysis, transmission electron microscope, FTIR, SS-NMR and XRD. Release study was conducted by in vitro investigation to simulate intestinal fluid and gastric fluid at 37℃. Insulin release were analysed by RP-HPLC. The result shows particles size increased with increasing NOCC/TPP mass ratio. All nanoparticles prepared by ionic gelation which zeta potential was positive. Release rate were decreased with decreasing NOCC/TPP mass ratio and increased with decreasing insulin initial concentration. The release profiles were fitted very well by the Higuchi release model.

While a number of PEGylated proteins have been studied for injectable applications and reviewers have used this data to speculate possible oral delivery improvements, a detailed investigation of PEGylated insulin for oral delivery and the development of an optimized pH-sensitive carrier for PEGylated insulin conjugates had yet to be accomplished. In order to proceed with oral delivery study, improvements in yield, with respect to previous PEGylation methods were necessary to enable the completion of high throughput drug delivery studies. Subsequently, a reaction scheme for the covalent attachment of PEG to insulin using nitrophenyl carbonate-PEG was developed. It was demonstrated that this reaction occurred at a 1:1 ratio and was site specific at the B29Lys position. A P(MAA-g-EG) hydrogel carrier was developed to optimize loading and release behavior for PEGylated insulin. It was demonstrated that the density and length of polymer grafts affected both loading and release behavior of PEGylated insulin. The best performing grafted polymers had a 3:1 methacrylic acid: ethylene glycol (MAA:EG) ratio and achieved loading efficiencies from 96% to nearly 100%. With respect to release, polymer particles containing fewer, but longer grafts shown to release faster than polymers with shorter grafts with the same MAA:EG ratio. Finally, the effects of PEGylation on intestinal absorption was investigated using an intestinal epithelial model as well as a rat model. It was demonstrated that PEGylated insulin in the presence of P(MAA-g-EG) microparticles did not significantly alter the tight junctions over unmodified insulin. However, the conjugate permeabilities across the membrane were reduced. The pharmacological availability (PA) was then verified by injecting the insulin conjugates subcutaneously in fasted Sprague-Dawley rats. It was determined that PEG 1000 insulin (1KPI) had a PA roughly equivalent to insulin, while it was reduced by 59% for 2KPI and by 81% for 5KPI. The effectiveness of utilizing PEGylated insulin as an oral drug delivery candidate was evaluated with a closed loop intestinal study, in which PEGylated insulin or insulin in solution was delivered directly to the jejunum. It was shown that 1KPI and insulin performed identically; with a pharmacological availability of 0.56%. 2KPI, however improved the pharmacological availability of insulin by 2.8 times. These results demonstrate that PEGylation holds promise for improving the oral delivery of proteins.
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Diabetes mellitus is one of the most grave and lethal non-communicable diseases. Insulin is normally used to medicate diabetes. Due to bioavailability issues, the most regular route of administration is through injection, which may pose compliance problems to treatment. The oral administration thus appears as a suitable alternative, but with several important problems. Low stability of insulin in the gastrointestinal tract and low intestinal permeation are some of the issues. Encapsulation of insulin into polymer-based particles emerges as a plausible strategy. Different encapsulation approaches and polymers have been used in this regard. Polymers with different characteristics from natural or synthetic origin have been assessed to attain this goal, with natural polymers being preferable. Natural polymer such as tragacanth, an anionic polysaccharide gum, can be alternative polymeric carrier for physiologically important peptides and proteins like insulin. Characterisation of tragacanth was explored in the first stage of the study, for providing a foundation for possible applications. Rheological studies colloidal solution of tragacanth at pH 3, 5 or 7 were carried out by means of steady shear and small amplitude oscillatory measurements. From preliminary study, 0.5% tragacanth was selected as optimum colloidal solution and 0.2 mg/ml insulin was chosen as concentration for a model protein. Tragacanth mucoadhesivity was also analysed using an applicable rheological method and compared to chitosan, alginate and PVP. The particle size and zeta potential were measured by a zetasizer. Thermal properties of solutions were obtained using a differential scanning calorimetry. The solution exhibited shearthinning characteristics. The value of the storage modulus (G′) and the loss modulus (G″) increased with an increase in angular frequency (Ω). In all cases, loss modulus values were higher than storage values (G″ > G′) and viscous character was, therefore, dominant. Tragacanth and alginate showed a good mucoadhesion. Tragacanth upon dispersion created particles of a submicron size with z-average diameters (mean) ranging between roughly 431 and 581 nm, with a negative zeta potential (-7.98 to -11.92 mV). These properties were pH dependant resulting in acid gel formation at pH 3.5. Tragacanth has thus a potential to be used as an excipient for peptide/protein delivery. Since tragacanth has a promising result to be used as a carrier in protein/peptide delivery and needs a further application, in the second study, insulin microparticles were prepared by the inclusion of insulin into a tragacanth hydrogel followed by freeze drying. The effect of the pH and concentration relationship involving polyelectrolytes offering individual particle size and zeta potential was assessed by zetasizer and scanning electron microscopy (SEM). Insulin– tragacanth interactions were prepared at varying pH (3.7, 4.3, 4.6, or 6), and concentration (0.1, 0.5, or 1% w/w) to optimize the conditions for optimal delivery of insulin. The pI of insulin can vary from 5.5 to 6.4, based on its origin. The pH 4.3; 4.6 and 6 was selected because these pH is below pI of insulin. At a pH lower than its pI value, insulin will be mainly positively charged. This insulin characteristic could be utilised to facilitate insulin–biopolymer complexes through electrostatic attraction with tragacanth (negatively charged). Individual and smaller particles with z-average diameters approximately 601 ± 19 nm (mean ± S.D.), were acquired at pH 4.6 with 0.5% of tragacanth. The acid gelation test indicated that insulin could be entrapped in the physical hydrogel of tragacanth. DSC thermograms of insulin–tragacanth showed shifts on the same unloaded tragacanth peaks and proposed polyelectrolyte–protein interactions at a pH close to 4.3– 4.6. FTIR spectra of tragacanth–insulin complexes exhibited amide absorption bands featuring in the protein spectra and revealed the creation of a new chemical substance. In the previous stage, tragacanth microparticles seem to have potential functional characteristics for oral insulin delivery by creating a complex with insulin under defined conditions followed by freeze drying. Since freeze-drying is up to 30–50 times more expensive than spray-drying and to make the overall process more industrially applicable, spray drying method has been explored in the third research. A spray-drying process was utilized to create microparticles from insulin/tragacanth GDL acidified solutions. The complexation process was performed at two tragacanth concentrations (0.5; 1%w/w) and several pH values (3.7; 4.3; 4.6; or 6). The SEM analysis indicated that almost spherical or sub-spherical microparticles were created with a diameter of less than 10 μm. The in vitro insulin release of microparticles prepared at a pH 4.3 and 4.6 was substantially minimized in comparison to other pH indicating improved retention of insulin. The selection of complexation pH appears to have an impact on insulin release profile and be an important parameter in protecting against peptic digestion. This finding stem from a possible creation of an insulin/tragacanth complex and hydrogel system. The evaluation of the interaction between insulin and tragacanth at different pH values by ATR-Fourier transform infrared and differential scanning calorimetry analysis verified this hypothesis. This finding suggests that these microparticles may act as a potentially promising device for oral insulin delivery.

Indiana University-Purdue University Indianapolis (IUPUI)
Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.

Tese de Mestrado Integrado, Engenharia Biomédica e Biofísica (Engenharia Clínica e Instrumentação Médica), 2021, Universidade de Lisboa, Faculdade de Ciências
A prevalência de feridas crónicas representa um dos maiores problemas de saúde pública a nível global, em função dos longos períodos de cicatrização associados a tratamentos pouco eficazes. A cicatrização de feridas é um processo fisiológico dinâmico e organizado que permite a recuperação da integridade da pele após uma lesão. Assim, a interrupção deste processo está associada a um impacto negativo e significante na qualidade de vida dos pacientes. Pacientes com patologias como a diabetes e obesidade estão mais sujeitos ao aparecimento deste tipo de feridas, devido a condições inerentes de hiperglicemia e complicações cardiovasculares. A idade é também um fator de risco para este tipo de lesão. Ao longo das últimas décadas equipas de investigação têm-se dedicado à descoberta de novas terapias, sendo que a principal abordagem para este tipo de patologia consiste em eliminar o foco de inflamação e aplicar topicamente fatores de crescimentos exógenos. A insulina é um fator de crescimento com um preço reduzido e vastamente disponível no mercado, capaz de estimular a migração celular, acelerando o processo de cicatrização. Contudo, a eficácia da administração tópica de insulina torna-se reduzida devido à ação das protéases presentes no leito da ferida. Assim, torna-se fundamental desenvolver novos métodos eficazes e capazes de contrariar este efeito proteolítico, protegendo o fator de crescimento da degradação. A encapsulação da insulina em micropartículas é capaz de conferir estabilidade à proteína quando administrada na ferida, promovendo uma libertação controlada e uma maior adesão às superfícies mucosas. Estudos revelaram, também, que a administração tópica de células estaminais mesenquimais acelera o processo de cicatrização, promovendo a formação de novos vasos sanguíneos num processo designado angiogénese, e reduzindo a inflamação. Este tipo de células estaminais têm a capacidade de se autorrenovarem em diferentes linhagens celulares, secretar fatores de crescimento e outras biomoléculas. Para além disso, o secretoma destas células é capaz de promover a deposição de matriz extracelular e aumentar a estabilidade dos fatores de crescimento no leito da ferida. Por outro lado, os fatores de crescimento têm, também, influência ao nível da modulação do efeito destas células estaminais. Assim, a co-encapsulação de insulina em conjunto com células estaminais mesenquimais em micropartículas pode ser uma estratégia inovadora no campo da medicina regenerativa, com aplicação na regeneração de feridas crónicas. Relativamente à administração destes fatores terapêuticas, esta pode ser feita através de aplicação de hidrogéis, capazes de conferir proteção contra a degradação provocada pelo ambiente. O PVA é um polímero sintético solúvel em água frequentemente utilizado na produção de hidrogéis, que pode ser reticulado de modo a adquirir excelentes propriedades viscoelásticas. Os hidrogéis compostos por este polímero conferem à pele uma camada resistente ao stress provocado pelo processo de cicatrização, promovendo uma sensação de conforto quando são aplicados na ferida. Por outro lado, o alginato é um polímero natural conhecido pela sua capacidade de absorver os fluídos em excesso existentes nas feridas, mantendo a hidratação da pele. Também a glicerina é frequentemente utilizada em hidrogéis, funcionado como emoliente e conferindo estrutura ao mesmo. Assim, o primeiro objetivo deste trabalho é desenvolver um hidrogel com nanopartículas contendo insulina, através de ciclos de congelação-descongelação. Posteriormente, é necessário otimizar este hidrogel que deverá ser apto para aplicação tópica, promovendo uma sensação de conforto quando aplicado na ferida. Para isso, as suas propriedades reológicas, tais como a viscosidade, vão ser avaliadas de modo a garantir a obtenção de um sistema de administração de insulina apto para aplicação em feridas crónicas. Para proceder à caracterização e produção deste hidrogel foram utilizadas nanopartículas revestidas com quitosano previamente produzidas pelo grupo de investigação que foram depois incorporadas no hidrogel composto por PVA, alginato e glicerina. Note-se que as nanopartículas revestidas com quitosano foram apenas utilizadas para estudar as propriedades reológicas do hidrogel, de modo a otimizar a sua formulação. Nesta primeira fase foi possível estudar e compreender a influência das variáveis independentes (percentagem de quitosano, alginato e glicerina, e número de ciclos de congelação-descongelação) nas propriedades do sistema composto pelo hidrogel e nanopartículas. A variável com maior impacto sobre as características do hidrogel é a quantidade de alginato, que influencia propriedades como a viscosidade e o potencial zeta. Correlacionando estes fatores tornou-se, então, possível, determinar a formulação ideal para o hidrogel. Numa segunda fase do projeto, pretende-se desenvolver um sistema para co-encapsulação de insulina com células estaminais mesenquimais através da técnica de microfluídica. Neste caso, o objetivo é estudar a estrutura da insulina após encapsulação, para mais tarde adicionar à formulação as células estaminais mesenquimais. A microfluídica foi a técnica escolhida para este efeito, uma vez que permite uma rápida produção de micropartículas com um tamanho e forma bem definidos, bem como a encapsulação de células em simultâneo. Assim, produziu-se um microchip com uma estrutura em forma de “T” e utilizaram-se propulsores de seringas para administrar duas fases imiscíveis com um fluxo constante. A fase contínua corresponde a uma solução de alginato e insulina, enquanto a fase dispersa corresponde a uma solução lipídica auto-emulsionante. Neste contexto, as partículas produzidas têm dimensões entre 10-100 µm, pelo que são consideradas micropartículas. Com o objetivo de otimizar a produção de micropartículas, produziram-se microchips com entradas e saídas reforçados para evitar pequenas fugas de conteúdo provocadas pela textura oleosa da fase dispersa. Apesar de eficaz, a técnica de microfluídica é bastante minuciosa, na medida em que é necessário determinar os fluxos ideias para um cada dos fluídos, possibilitando a formação de partículas esféricas. Para efeitos de controlo, foram produzidas partículas com e sem insulina. Uma vez produzidas, as partículas foram congeladas e posteriormente liofilizadas, com e sem crioprotetor. A morfologia da superfície das micropartículas foi avaliada através da técnica de microscopia eletrónica de varredura, e foram também realizados ensaio de libertação durante um período de 48 horas para avaliar e eficácia do sistema. A insulina foi depois extraída das micropartículas e a sua estrutura foi avaliada por microscopia de fluorescência, dicroísmo circular, espetroscopia de infravermelho com transformada de Fourier, e ensaios de Tio Flavina-T. Os resultados obtidos com o ensaio de libertação revelaram uma libertação uniforme e controlada de insulina durante as 48 horas, permitindo concluir que esta libertação poderá manter-se mesmo após este período. Os resultados de dicroísmo circular e microscopia de fluorescência revelaram que a insulina manteve a sua estrutura após encapsulação, com presença de apenas pequenas alterações. Os resultados de espetroscopia de infravermelho com transformada de Fourier confirmaram que a estrutura da insulina foi conservada após encapsulação, e não foram detetadas novas interações da proteína com o alginato. Os resultados obtidos relativamente às partículas congeladas com e sem crioprotetor revelaram-se semelhantes, sem grandes diferenças a notar relativamente a esta variável. Os resultados obtidos com este trabalho sugerem que a insulina pode ser eficazmente encapsulada através da técnica de microfluídica. Assim, reúnem-se condições para aliar os resultados obtidos em ambas as etapas deste trabalho, sendo que o produto final seria um hidrogel cuja formulação foi otimizada, contendo micropartículas com insulina, obtidas com recurso à técnica de microfluídica. Futuramente, pretende-se desenvolver um microchip com maior complexidade que permita encapsular as células estaminais mesenquimais em simultâneo com a insulina. Assim, será possível avaliar a viabilidade das células após encapsulação, bem como estudar a citotoxicidade e bioatividade da formulação in vitro. O resultado final será uma plataforma multipotente para administração tópica de fatores de crescimentos células estaminais mesenquimais para a cicatrização de feridas crónicas.
The prevalence of chronic wounds is a challenging public health issue, due to long-time recovery and inefficacious treatments. Incidence of chronic wounds increase with age and pathologies such as diabetes and obesity are risk-factors. Insulin, a peptide hormone, is one of the cheapest growth factors available, being able to mitigate the compromised skin by triggering cell migration and proliferation, stimulating wound healing. Mesenchymal stem cells offer promising approaches for cell therapy because of their self-renewal capacities and multi-lineage differentiation. Besides, it was demonstrated that the presence of insulin improves MSCs function and survival, accelerating chronic wound healing. The co-encapsulation of mesenchymal stem cells and insulin in microparticles improves its stability in the wound area, provide adhesion to the mucosal surfaces, and preserve the sustained release. Therefore, this work started with the development and optimization of a hydrogel containing nanoparticles for topical insulin administration by freeze-thawing. The objective is to obtain a hydrogel with good rheological particles suitable for topical application. The second phase of the work consists of the development of a delivery system co-encapsulating insulin and mesenchymal stem cells by microfluidics. In this case, the aim is to evaluate the protein structure upon encapsulation to later encapsulate the stem cells. Microfluidics was the chosen technique for microparticle production because it allows the rapid generation of controlled size particles and simultaneous cell encapsulation. Once produced, microparticles were lyophilized with and without cryoprotectant. The surface morphology of the produced microparticles was evaluated using scanning electronic microscopy and the release profile of insulin was also evaluated. Insulin structure upon encapsulation was assessed by fluorescence microscopy, circular dichroism, Fourier transform infrared spectroscopy and thioflavin-T assay. The results obtained with circular dichroism, fluorescence microscopy, and thioflavin-T assay revealed that insulin structure was maintained upon encapsulation with minimal structural modifications. Moreover, the presence of cryoprotectant did not affect the results concerning insulin structure. Fourier transform infrared spectroscopy results showed confirm these results, and no interactions with alginate are evidenced. Finally, the results regarding the release profile of insulin revealed a sustained release during a period of 48 hours. The results obtained in both phases of this work suggest that the developed encapsulation technique can be applied to the production of microparticles co-encapsulating insulin and mesenchymal stem cells. Therefore, the next step consists of evaluate the mesenchymal stem cells viability upon encapsulation, and the cytotoxicity profile of the formulation, as well its bioactivity. Then, these microparticles can be incorporated into the optimized hydrogel, creating a delivery system suitable for topical administration, with wound healing applications.