RESUMO
Mucoadhesive thermogels were developed by crosslinking poly(n-isopropylacrylamide) based polymers with chitosan and incorporating disulfide bridges, capable of releasing cysteamine upon interaction with mucin, for the treatment of cystinosis. Through crosslinking with chitosan and incorporating varying concentrations of the disulfide monomer into the polymer backbone, the extent of how mucoadhesive the developed thermogels were could be controlled. Through disulfide bridging with mucin, the thermogels released 6 to 10 µg of the conjugate model 2-mercaptopyridine over five days. Utilizing chitosan as the crosslinker, the developed thermogels were shown to degrade to a statistically higher extent following incubation with lysozyme, the highest concentration tear enzyme, by gravimetric and rheologic analysis. The developed thermogels were extensively tested in vivo utilizing a rat model in which materials were applied directly to the corneal surface and a rabbit model in which thermogels were applied to the inferior fornix. With the developed models, there was no adverse reactions or visual discomfort incurred following application of the thermogels. It has been demonstrated that the thermogels produced can be applied to the inferior fornix and release the stable conjugated payload over several days. The developed thermogel was designed to improve upon the current clinical treatment options for ocular cystinosis which are acidic topical formulations that require reapplication multiple times a day.
Assuntos
Quitosana , Cistinose , Ratos , Animais , Coelhos , Polímeros , Géis , Dissulfetos , MucinasRESUMO
Thermo-gels based on chitosan crosslinked poly(N-isopropylacrylamide) were developed as alternatives to conventional eye drops for the sustained release of ketotifen fumarate in the treatment of allergic conjunctivitis. The thermo-gelling properties of the base polymer were altered prior to crosslinking with chitosan by incorporation of the hydrophilic and hydrophobic comonomers acrylic acid and methyl methacrylate respectively. Varying amounts of chitosan were incorporated by ionic interaction to produce polyelectrolyte complexes or by carbodiimide chemistry to produce covalently crosslinked networks. The lower critical solution temperature of all the chitosan crosslinked thermo-gels produced was below the surface temperature of the eye. All the chitosan crosslinked thermo-gels were found to have greater than 80% equilibrium water contents following gelation. The method and amount of chitosan incorporation allowed for tailor-ability of material rheologic properties, with full degradation occurring over a one-to-four-day period, and tailorable rates of release of 40-60% of the loaded allergy medication ketotifen fumarate. The chitosan crosslinked thermo-gels were demonstrated to be nontoxic both in vitro and in vivo. It was demonstrated that the synthesized materials could be applied to the inferior fornix of eye, sustaining a multiple day release of ketotifen fumarate, as an alternative to conventional eyedrops. STATEMENT OF SIGNIFICANCE: Topical eyedrops are the main treatment modality for anterior ocular conditions. However, due to the natural clearance mechanisms of the eye, topical eyedrops are well established to be largely ineffective as a method of drug delivery. Herein, we investigate a method of altering thermo-gel properties of an n-isopropylacrylamide based polymer to enable the incorporation of greater amounts of chitosan by different methods of crosslinking. By controlling the synthesis parameters, final material properties can be tailored to impart ideal spreading, retention on the eye, and the rate of degradation and drug release over several days. This work also focuses on studying the rheological properties of the chitosan crosslinked thermo-gels which has not been investigated previously.
Assuntos
Quitosana , Hidrogéis , Acrilamidas , Acrilatos , Quitosana/química , Hidrogéis/química , Cetotifeno , Metacrilatos , Metilmetacrilato , Soluções Oftálmicas , Polímeros , TemperaturaRESUMO
Polymeric drug carriers are widely used for providing temporal and/or spatial control of drug delivery, with corticosteroids being one class of drugs that have benefitted from their use for the treatment of inflammatory-mediated conditions. However, these polymer-based systems often have limited drug-loading capacity, suboptimal release kinetics, and/or promote adverse inflammatory responses. This manuscript investigates and describes a strategy for achieving controlled delivery of corticosteroids, based on a discovery that low molecular weight corticosteroid dimers can be processed into drug delivery implant materials using a broad range of established fabrication methods, without the use of polymers or excipients. These implants undergo surface erosion, achieving tightly controlled and reproducible drug release kinetics in vitro. As an example, when used as ocular implants in rats, a dexamethasone dimer implant is shown to effectively inhibit inflammation induced by lipopolysaccharide. In a rabbit model, dexamethasone dimer intravitreal implants demonstrate predictable pharmacokinetics and significantly extend drug release duration and efficacy (>6 months) compared to a leading commercial polymeric dexamethasone-releasing implant.
Assuntos
Corticosteroides/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Dexametasona/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Corticosteroides/química , Corticosteroides/farmacocinética , Animais , Células Cultivadas , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Dexametasona/química , Dexametasona/farmacocinética , Dimerização , Modelos Animais de Doenças , Implantes de Medicamento , Liberação Controlada de Fármacos , Polímeros/química , Coelhos , Ratos , Uveíte/metabolismo , Uveíte/prevenção & controleRESUMO
Vascular leak is a key driver of organ injury in diseases, and strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the angiopoietin-1 (ANG1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANG1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANG1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α. We refer to this new fusion protein biologic as Hepta-ANG1, which forms a stable heptamer and induces Tie2 phosphorylation in cultured cells, and in the lung following intravenous injection of mice. Injection of Hepta-ANG1 ameliorates vascular endothelial growth factor- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability. The new Hepta-ANG1 fusion is easy to produce and displays remarkable stability with high multimericity that can potently activate Tie2. It could be a new candidate ANG1 mimetic therapy for treatments of inflammatory vascular leak, such as acute respiratory distress syndrome and sepsis.
Assuntos
Angiopoietina-1 , Proteína de Ligação ao Complemento C4b , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Recombinantes de Fusão , Doenças Vasculares/tratamento farmacológico , Angiopoietina-1/biossíntese , Angiopoietina-1/genética , Angiopoietina-1/farmacologia , Animais , Proteína de Ligação ao Complemento C4b/biossíntese , Proteína de Ligação ao Complemento C4b/genética , Proteína de Ligação ao Complemento C4b/farmacologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Sepse/tratamento farmacológico , Sepse/metabolismo , Sepse/patologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
Purpose: The combined action of the activating protein-2 (AP-2) transcription factors, AP-2α and AP-2ß, is important in early retinal development, specifically in the formation of horizontal cells. However, in previous studies, it was not possible to analyze postnatal development and function of additional retinal subtypes. Methods: We used a double conditional deletion of AP-2α and AP-2ß from the retina to further examine the combinatory role of these genes in retinal cell patterning and function in postnatal adult mice as measured by Voronoi domain area and nearest-neighbor distance spatial analyses and ERGs, respectively. Results: Conditional deletion of both AP-2α and AP-2ß from the retina resulted in a variety of abnormalities, including the absence of horizontal cells, defects in the photoreceptor ribbons in which synapses failed to form, along with evidence of aberrant amacrine cell arrangement. Although no significant changes in amacrine cell population numbers were observed in the double mutants, significant irregularities in the mosaic patterning of amacrine cells was observed as demonstrated by both Voronoi domain areas and nearest-neighbor distances analyses. These changes were further accompanied by an alteration in the retinal response to light as recorded by ERGs. In particular, in the double-mutant mice lacking AP-2α and AP-2ß, the b-wave amplitude, representative of interneuron signal processing, was significantly reduced compared with control littermates. Conclusions: Together these findings demonstrate the requirement for both AP-2α and AP-2ß in proper amacrine mosaic patterning and a normal functional light response in the retina.
Assuntos
Células Amácrinas/metabolismo , Animais Recém-Nascidos , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Retina/metabolismo , Deleção de Sequência , Fator de Transcrição AP-2/genética , Células Amácrinas/ultraestrutura , Animais , Sequência de Bases , Contagem de Células , Eletrorretinografia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Modelos Animais , Retina/ultraestrutura , Fator de Transcrição AP-2/biossínteseRESUMO
Tubedown (Tbdn; Naa15), a subunit of the N-terminal acetyltransferase NatA, complexes with the c-Src substrate Cortactin and supports adult retinal homeostasis through regulation of vascular permeability. Here we investigate the role of Tbdn expression on signaling components of retinal endothelial permeability to understand how Tbdn regulates the vasculature and supports retinal homeostasis. Tbdn knockdown-induced hyperpermeability to Albumin in retinal endothelial cells was associated with an increase in the levels of activation of the Src family kinases (SFK) c-Src, Fyn and Lyn and phospho-Cortactin (Tyr421). The knockdown of Cortactin expression reduced Tbdn knockdown-induced permeability to Albumin and the levels of activated SFK. Inhibition of SFK in retinal endothelial cells decreased Tbdn knockdown-induced permeability to Albumin and phospho-Cortactin (Tyr421) levels. Retinal lesions of endothelial-specific Tbdn knockdown mice, with tissue thickening, fibrovascular growth, and hyperpermeable vessels displayed an increase in the levels of activated c-Src. Moreover, the retinal lesions of patients with proliferative diabetic retinopathy (PDR) associated with a loss of Tbdn expression and hyperpermeability to Albumin displayed increased levels of activated SFK in retinal blood vessels. Taken together, these results implicate Tbdn as an important regulator of retinal endothelial permeability and homeostasis by modulating a signaling pathway involving c-Src and Cortactin.