RESUMO
Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischaemia, inflammation and infection costing $7.5 billion/year in the U.S.A. alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA [poly(D,L-lactic-co-glycolic acid)] microparticles that provides a sustained release of bioactive insulin for >20 days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring healing. Using heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04 mg insulin/cm(2) every 3 days for 9 days have faster closure, a higher rate of disintegration of dead tissue and decreased oxidative stress. In addition, in insulin-treated wounds, the pattern of neutrophil inflammatory response suggests faster clearing of the burned dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibres organized more like a basket weave (normal skin) than aligned and cross-linked (scar tissue). In summary, application of ASD-containing insulin-loaded PLGA particles on burns every 3 days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.
Assuntos
Alginatos/química , Bandagens , Queimaduras/tratamento farmacológico , Portadores de Fármacos , Insulina Regular Humana/administração & dosagem , Ácido Láctico/química , Ácido Poliglicólico/química , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Administração Cutânea , Animais , Queimaduras/metabolismo , Queimaduras/patologia , Queimaduras/fisiopatologia , Química Farmacêutica , Cicatriz/metabolismo , Cicatriz/patologia , Cicatriz/prevenção & controle , Colágeno/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/prevenção & controle , Mediadores da Inflamação/metabolismo , Insulina Regular Humana/química , Neovascularização Fisiológica/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Pele/irrigação sanguínea , Pele/metabolismo , Pele/patologia , Solubilidade , Fatores de TempoRESUMO
Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.
Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Influenza Humana/prevenção & controle , Neuraminidase/genética , Vacinas Sintéticas/genética , RNARESUMO
Wound healing is a natural process involving several signaling molecules and cell types over a significant period of time. Although current dressings help to protect the wound from debris or infection, they do little in accelerating the healing process. Insulin has been shown to stimulate the healing of damaged skin. We have developed an alginate sponge dressing (ASD) that forms a hydrogel capable of providing a moist and protective healing environment. By incorporating insulin-loaded poly(d,l-lactide-co-glycolide) (PLGA) microparticles into ASD, we successfully stabilized and released insulin for up to 21 days. Insulin release and water absorption and transfer through the ASD were influenced by altering the levels of poly(ethylene glycol) (PEG) in the dressing matrix. Bioactivity of released insulin can be maintained for at least 10 days, demonstrated using a human keratinocyte migration assay. Results showed that insulin-loaded PLGA microparticles, embedded within PEG-ASD, functioned as an effective long-term delivery platform for bioactive insulin.
Assuntos
Alginatos/química , Insulina/metabolismo , Polietilenoglicóis/química , Movimento Celular , Células Cultivadas , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Insulina/química , Secreção de Insulina , Queratinócitos/citologia , Queratinócitos/metabolismo , Cinética , Ácido Láctico/química , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de Superfície , Água/químicaRESUMO
Silicone elastomers have proven to be useful implantable release matrices for hydrophobic drugs. However, their utility for the release of hydrophilic materials is less well developed and, even with the addition of polar excipients such as poly(ethylene oxide) (PEO), burst release profiles are often observed-achieving longer term release is more challenging. We report that linoleic acid, initially used to solubilize polar, cationic nicotine in silicone precursors, additionally acted to change the internal morphology of resulting silicone+PEO elastomers. The unexpected consequence of this change was a change in the distribution of hydrophilic domains of PEO/drug within the silicone and the ability to control the rate of release of the drug in vitro. The relationship between excipients, silicone morphology, and release profile is examined.
Assuntos
Preparações Farmacêuticas/química , Química Farmacêutica , Implantes de Medicamento , Excipientes , Ácido Linoleico/química , Microscopia Confocal , Microscopia Eletrônica de Varredura , Nicotina/administração & dosagem , Nicotina/química , Polietilenoglicóis/química , Elastômeros de Silicone , Silicones/química , Espectrofotometria Ultravioleta , TensoativosRESUMO
Lipase Candida rugosa was entrapped in silicone rubber via condensation-cure room temperature vulcanization of silanol-terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, to give a highly active silicone-enzyme elastomer. The effect on enzyme activity of addition of water and hydrophilic polymeric moieties based on poly(ethylene oxide) 2 was examined, as were crosslinker concentration, enzyme concentration, and elastomer thickness. It was demonstrated that lipase is most active in silicone elastomers and more active in silicone oils than simple hydrocarbons. Crosslink density in these elastomers was not an important factor in the reactivity of the rubber. However, the addition of hydrophilic species prior to elastomer formation decreased the efficiency both of the dispersion of the enzyme and the resulting activity of the elastomer. This effect could be moderated by prior exposure of the lipase to silicone oil. Thus, hydrophobic silicones play a protective/activating role for lipase.
Assuntos
Materiais Biocompatíveis/química , Lipase/química , Elastômeros de Silicone/química , Materiais Biocompatíveis/análise , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/análise , Enzimas Imobilizadas/química , Lipase/análise , Teste de Materiais , Elastômeros de Silicone/análiseRESUMO
Poly (lactic-co-glycolic acid) (PLGA) copolymers have been extensively used in cancer research. PLGA can be chemically engineered for conjugation or encapsulation of drugs in a particle formulation. We reported that oseltamivir phosphate (OP) treatment of human pancreatic tumor-bearing mice disrupted the tumor vasculature with daily injections. Here, the controlled release of OP from a biodegradable PLGA cylinder (PLGA-OP) implanted at tumor site was investigated for its role in limiting tumor neovascularization, growth, and metastasis. PLGA-OP cylinders over 30 days in vitro indicated 20%-25% release profiles within 48 hours followed by a continuous metronomic low dose release of 30%-50% OP for an additional 16 days. All OP was released by day 30. Surgically implanted PLGA-OP containing 20 mg OP and blank PLGA cylinders at the tumor site of heterotopic xenografts of human pancreatic PANC1 tumors in RAGxCγ double mutant mice impeded tumor neovascularization, growth rate, and spread to the liver and lungs compared with the untreated cohort. Xenograft tumors from PLGA and PLGA-OP-treated cohorts expressed significant higher levels of human E-cadherin with concomitant reduced N-cadherin and host CD31(+) endothelial cells compared with the untreated cohort. These results clearly indicate that OP delivered from PLGA cylinders surgically implanted at the site of the solid tumor show promise as an effective treatment therapy for cancer.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Carcinoma/tratamento farmacológico , Portadores de Fármacos , Neoplasias Hepáticas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Neovascularização Patológica , Oseltamivir/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Polietilenoglicóis/química , Implantes Absorvíveis , Inibidores da Angiogênese/química , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Carcinoma/irrigação sanguínea , Carcinoma/metabolismo , Carcinoma/secundário , Linhagem Celular Tumoral , Química Farmacêutica , Implantes de Medicamento , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos NOD , Camundongos Knockout , Oseltamivir/química , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Solubilidade , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lipase is more reactive in silicone oil or silicone elastomers than in hydrocarbons, and can be formulated into convenient, active, immobilized enzyme packages.
Assuntos
Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Silicones/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Lipase/química , Octanos/química , Elastômeros de Silicone/química , Óleos de Silicone/química , Siloxanas/química , Água/químicaRESUMO
Skin is a dynamic and complex organ that relies on the interaction of different cell types, biomacromolecules and signaling molecules. Injury triggers a cascade of events designed to quickly restore skin integrity. Depending on the size and severity of the wound, extensive physiological and metabolic changes can occur, resulting in impaired wound healing and increased morbidity resulting in higher rates of death. While wound dressings provide a temporary barrier, they are inherently incapable of significantly restoring metabolic upsets, post-burn insulin resistance, and impaired wound healing in patients with extensive burns. Exogenous insulin application has therefore been investigated as a potential therapeutic intervention for nearly a century to improve wound recovery. This review will highlight the important achievements that demonstrate insulin's ability to stimulate cellular migration and burn wound recovery, as well as providing a perspective on future therapeutic applications and research directions.
Assuntos
Queimaduras/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Cicatrização/fisiologia , Animais , Queimaduras/metabolismo , Movimento Celular , Humanos , Insulina/fisiologia , Resistência à Insulina , Transporte Proteico/fisiologia , Receptor de Insulina/fisiologiaRESUMO
One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk-alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk-alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk-alginate ratio and the concentration of crosslinker--a characteristic that is very hard to accomplish in current hydrogels. The hydrogel and the presented results represents key steps on the way of creating artificial stem cell niche, opening up new paths in regenerative medicine.
Assuntos
Alginatos/química , Células-Tronco Embrionárias/citologia , Hidrogéis , Seda/química , Transplante de Células-Tronco , Alicerces Teciduais , Animais , Adesão Celular , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , RatosRESUMO
Skin damaged by heat, radiation, or chemical exposure is difficult to treat and slow to heal. Indeed full restoration of the tissue is difficult to obtain. Sub-dermal insulin injection was recently shown to stimulate wound healing of the skin by accelerating wound closure, stimulating angiogenesis and inducing a regenerative process of healing. We have developed a topical delivery vehicle that is capable of releasing therapeutic levels of bioactive insulin for several weeks with the potential to stimulate and sustain healing. By encapsulating the crystalline form of insulin within poly(d,l-lactide-co-glycolide) microspheres, we succeeded in stabilizing and then releasing bioactive insulin for up to 25 days. To measure bioactivity we used Rat L6 myofibroblasts, stimulated them with this slow release insulin and determined activation of the receptors on the cell surface by quantifying AKT phosphorylation. There was only a minor and gradual decrease in AKT phosphorylation over time. To determine whether the slow release insulin could stimulate keratinocyte migration, wounding was simulated by scratching confluent cultures of human keratinocytes (HaCaT). Coverage of the scratch "wounds" was significantly faster in the presence of insulin released from microspheres than in the insulin-free control. Extended and sustained topical delivery of active insulin from a stable protein crystal-based reservoir shows promise in promoting tissue healing.