NEW SRATEGIES FOR ANTIGEN AND PROTEIN DELIVERY USING NANOBIOMATERIALS

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Transfection of pulmonary cells by stable pDNA-polycationic hybrid nanostructured particles

Publication . Gaspar, Diana P.; Vital, Joana; Leiva, María C.; Gonçalves, Lídia M. D.; Taboada, Pablo; Remuñán-López, Carmen; Vítor, Jorge M. B.; Almeida, António J.

Aim: Cationically modified solid lipid nanoparticles (SLN) were investigated as plasmid DNA (pDNA) carriers and transfection agents for the pulmonary route. Materials & methods:pDNA-loaded SLN were produced using glyceryl dibehenate or tristearate as matrix lipids and chitosan as surface charge modifier, and encapsulated by spray-drying in mannitol and trehalose microspheres. Results: Nanoparticles of 200 nm, and zeta potential around +15 mV were produced. Electrophorectic analysis confirmed plasmid stability and integrity. The pDNA-loaded SLN were able to transfect the Calu-3 and A549 pulmonary cell lines, while showing low cytotoxicity. Microencapsulation of SLN yielded dry powders suitable for inhalation that protected pDNA from degradation. Conclusion: Microencapsulated SLN are a promising safe and effective carrier system for pulmonary gene delivery following pulmonary administration.

2019-02Journal article

Restricted access

In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme

Publication . Lino, Paulo Roque; Leandro, João; Amaro, Mariana; Gonçalves, Lídia; Leandro, Paula; Almeida, António José

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.

2021-03-04Journal article

Open access