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1.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360752

RESUMO

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.


Assuntos
Quitosana , Enzimas Imobilizadas , Teste de Materiais , Nanopartículas/química , Fenilalanina Hidroxilase , Quitosana/química , Quitosana/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/farmacologia
2.
Pharmaceutics ; 13(3)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806405

RESUMO

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.

3.
Eur J Pharm Sci ; 163: 105853, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33865976

RESUMO

Biopharmaceutics Classification System (BCS) class II and IV drugs may be formulated as supersaturating drug delivery systems (e.g., amorphous solid dispersions [ASDs]) that can generate a supersaturated drug solution during gastrointestinal (GI) transit. The mechanisms that contribute to increased bioavailability are generally attributed to the increased solubility of the amorphous form, but another mechanism with significant contributions to the improved bioavailability have been recently identified. This mechanism consists on the formation of colloidal species and may further improve the bioavailability several fold beyond that of the amorphous drug alone. These colloidal species occur when the concentration of drug generated in solution exceeds the amorphous solubility during dissolution, resulting in a liquid-liquid phase separation (LLPS). For the appearance of LLPS, the crystallization kinetics needs to be slow relatively to the dissolution process. This work intended to implement an analytical methodology to understand the ability of a drug to form colloidal species in a biorelevant dissolution media. This screening tool was therefore focused on following the colloidal formation and crystallization kinetics of itraconazole (ITZ; model drug from BSC class II) in the presence of hydroxypropyl methylcellulose (HPMC-AS L and HPMC-AS M, which are HPMC-AS with varying ratios of succinoyl:acetyl groups), using a laser diffraction-based methodology. The ability of ITZ to form colloids by a solvent-shift approach was compared with the actual colloidal formation of ITZ amorphous solid dispersions produced by spray-drying. Results indicate that regardless of the used methodology, colloids of ITZ can be detected and monitored. The extension of colloid generation showed to be correlated with the ASD disintegration/dissolution rate, i.e, polymers with faster wettability kinetics led to faster ASD disintegration and colloidal formation. As conclusion, this study showed that laser diffraction could give complementary information about colloidal formation and ASD dissolution profile, showing to be an excellent screening strategy to be applied in the early stage development of amorphous solid dispersions.


Assuntos
Lasers , Polímeros , Cristalização , Derivados da Hipromelose , Solubilidade
4.
Int J Pharm ; 516(1-2): 231-246, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27864069

RESUMO

Associating protein with nanoparticles is an interesting strategy to improve their bioavailability and biological activity. Solid lipid nanoparticles (SLN) have been sought as carriers for therapeutic proteins transport to the lung epithelium. Nevertheless, because of their low inertia, nanoparticles intended for pulmonary application usually escape from lung deposition. To overcome this problem, the production of spray-dried powders containing nanoparticles has been recently reported. Herein we developed new hybrid microencapsulated SLN for pulmonary administration, containing a model protein (papain, PAP). PAP was adsorbed onto glyceryl dibehenate and glyceryl tristearate SLN. Physical characterization using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) confirmed the interaction between PAP and SLN corroborating that the protein was efficiently adsorbed at SLN's surface. PAP adsorption onto SLN (PAP-SLN) slightly increased particle size, while decreasing the SLN negative surface charge. The adsorption process followed a Freundlich type of adsorption isotherm. Nanoformulations were then spray-dried, originating spherical microparticles with suitable aerodynamic characteristics. Full characterization of microparticles was performed using scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and isothermal titration calorimetry (ITC). PAP was released from dry powders in a higher extent when compared with non spray-dried SLN. Nevertheless, protein stability was kept throughout microsphere production, as assessed by SDS-PAGE.


Assuntos
Sistemas de Liberação de Medicamentos , Lipídeos/química , Nanopartículas , Papaína/administração & dosagem , Adsorção , Varredura Diferencial de Calorimetria , Portadores de Fármacos/química , Composição de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/química , Pulmão/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microesferas , Papaína/farmacocinética , Tamanho da Partícula , Pós , Espectroscopia de Infravermelho com Transformada de Fourier , Triglicerídeos/química
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