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Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing

The main aim of this work was the development, characterization and in vitro and in vivo evaluation of different nanocarriers with specific nanoparticles for the treatment of melanoma and β-lapachone liposomes incorporated in biopolymer hydrogels for the healing of topical wounds. The first part of...

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Main Author: PALÁCIO, Sarah Brandão
Other Authors: MAGALHÃES, Nereide Stela Santos
Format: doctoralThesis
Language: por
Published: Universidade Federal de Pernambuco 2019
Subjects:
Nanopartículas
Melanoma
Biopolímero
Lipossomas
Cicatrização
Online Access: https://repositorio.ufpe.br/handle/123456789/29658
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spelling ir-123456789-296582019-03-13T05:04:42Z Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing PALÁCIO, Sarah Brandão MAGALHÃES, Nereide Stela Santos PONCHEL, Gilles http://lattes.cnpq.br/9995253092484803 http://lattes.cnpq.br/0375372674397504 Nanopartículas Melanoma Biopolímero Lipossomas Cicatrização The main aim of this work was the development, characterization and in vitro and in vivo evaluation of different nanocarriers with specific nanoparticles for the treatment of melanoma and β-lapachone liposomes incorporated in biopolymer hydrogels for the healing of topical wounds. The first part of this thesis presents a review of the literature and recent advances in the field of targeting mesenchymal circulating cells derived from melanomas. The main biomarkers of these cells have been reviewed to define the suitable characteristics of this nanocarriers. The experimental part of the work consisted of development of nanoparticles (non-spherical) by nanoprecipitation of copolymers derived from poly (γ-benzyl-L-glutamate). These nanoparticles, size between 20 and 100 nm and carrying a negative charge (-3 to -30 mV) were then combined with the MART-1 antibody, specific for the melanoma cell membrane, by biotin-streptavidin binding. The binding of the antibody on the surface of nanoparticles was evaluated by Western blot. The affinity of immuno-nanoparticles for melanoma cells (B16-GFP line) and for endothelial cells of human umbilical vein (HUVECs) was then evaluated in vitro by flow cytometry and, being intended for intravenous injection, it was important to evaluate the degree of activation of the complement system induced by the nanoparticles. The 2D immunoelectrophoresis technique used made it possible to conclude that the activation was limited and favourable to increase the blood circulation time of nanoparticles, after intravenous injection. The nanoparticles exhibited low cytotoxicity (MTT assay) against melanoma cells or endothelial cells. In terms of cellular uptake, the immuno-nanoparticles functionalized with MART1, a specific antibody for the recognition of the overexpressed antigen in melanoma cells, was increased by 40 to 50% compared to control. The second part of this thesis was dedicated to the development, characterization and in vivo evaluation of the wound healing activity of β-lapachone encapsulated in multilamellar liposomes and incorporated in a hydrogel of a biopolymer produced by the bacterium Zoogloea sp. These original formulations (β-lap-Lipo/ ZBP/HEC) had a pH and rheological behavior suitable for topical application, as well as the ability to slow the release of β-lapachone from the hydrogel. A detailed histopathological study of the wound healing activity was conducted in an in vivo model and showed that the biopolymer hydrogel was able to stimulate tissue repair, increasing the local cellularity, fibroblasts, cells inflammatory, blood vessels and the production of collagen fibrils during the proliferative phase of healing. In addition, the β-lap-Lipo/ZBP/HEC formulation promoted local angiogenesis and reduced inflammation of the wound, demonstrating the potential of this original formulation of β-lap-Lipo/ZBP/HEC in cutaneous lesions therapy. To conclude, the developed nanocarriers are interesting approachs for intercepting the circulating melanoma cells, while liposomal formulations combining with original biopolymers have an interesting potential for wound healing applications. CAPES O objetivo principal deste trabalho foi o desenvolvimento, caracterização e avaliação in vitro e in vivo de diferentes nanocarreadores: nanopartículas específicas para o tratamento de melanoma e lipossomas contendo β-lapachona incorporados em hidrogéis de biopolímero para a cicatrização de feridas tópicas. A primeira parte desta tese apresenta uma revisão da literatura e avanços recentes no campo do direcionamento de fármacos através do uso de nanocarreadores para a células circulantes mesenquimatosas derivadas de melanomas. Os principais biomarcadores presentes nestas células foram descritos com o objetivo de definir as características adequadas para o desenvolvimento de nanocarreadores. A parte experimental do trabalho consistiu no desenvolvimento de nanopartículas (não esféricas) por nanoprecipitação de copolímeros derivados de poli (γ-benzil-L-glutamato). Essas nanopartículas, tamanho entre 20 e 100 nm e carga negativa (-3 a -30 mV) foram então combinadas com o anticorpo MART-1, específico para a membrana das células do melanoma, pela ligação biotina-estreptavidina. A ligação do anticorpo na superfície das nanopartículas foi avaliada por Western blot. A afinidade das imuno-nanopartículas para células de melanoma (linha B16-GFP) e para células endoteliais de veia umbilical humana (HUVECs) foi então avaliada in vitro por citometria de fluxo e, sendo destinada a injeção intravenosa, era importante avaliar o grau de ativação do sistema complemento induzido pelas nanopartículas. A técnica utilizada de imunoeletroforese 2D permitiu concluir que a ativação foi limitada e favorável para aumentar o tempo de circulação sanguínea das nanopartículas, após a injeção intravenosa. As nanopartículas apresentaram baixa citotoxicidade (teste MTT) contra células de melanoma ou células endoteliais. Em termos de absorção celular, as imuno-nanopartículas funcionalizadas com MART1, um anticorpo específico para o reconhecimento do antígeno super-expresso em células de melanoma, foi aumentada em 40 a 50% em comparação com o controle. A segunda parte desta tese foi dedicada ao desenvolvimento, caracterização e avaliação in vivo da atividade de cicatrização da β-lapachona encapsulada em lipossomas multilamelares e incorporada em um hidrogel de um biopolímero produzido pela bactéria Zoogloea sp. Estas formulações originais (β-lap-Lipo/ZBP/HEC) apresentaram um pH e comportamento reológico adequados para aplicação tópica, bem como a capacidade de retardar a liberação de β-lapachona do hidrogel. Um estudo histopatológico detalhado da atividade de cicatrização de feridas foi conduzido em um modelo in vivo e mostrou que o hidrogel de biopolímero foi capaz de estimular o reparo tecidual, aumentando a celularidade local, fibroblastos, células inflamatórias, vasos sanguíneos e a produção de fibrilas de colágeno durante a fase proliferativa. Além disso, a formulação β-lap-Lipo/ZBP/HEC promoveu angiogênese local e reduziu a inflamação da ferida, demonstrando o potencial desta formulação original de β-lap-Lipo/ZBP/HEC na terapia de lesões cutâneas. Para concluir, os nanocarreadores desenvolvidos demonstraram ser abordagens interessantes para interceptar as células de melanoma circulantes, enquanto as formulações lipossomais combinada com biopolímero têm um potencial interessante para aplicações de cicatrização de feridas. 2019-03-12T19:00:19Z 2019-03-12T19:00:19Z 2017-12-01 doctoralThesis https://repositorio.ufpe.br/handle/123456789/29658 por embargoedAccess Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ Universidade Federal de Pernambuco UFPE Brasil Programa de Pos Graduacao em Nanotecnologia Farmaceutica
institution REPOSITORIO UFPE
collection REPOSITORIO UFPE
language por
topic Nanopartículas
Melanoma
Biopolímero
Lipossomas
Cicatrização
spellingShingle Nanopartículas
Melanoma
Biopolímero
Lipossomas
Cicatrização
PALÁCIO, Sarah Brandão
Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
description The main aim of this work was the development, characterization and in vitro and in vivo evaluation of different nanocarriers with specific nanoparticles for the treatment of melanoma and β-lapachone liposomes incorporated in biopolymer hydrogels for the healing of topical wounds. The first part of this thesis presents a review of the literature and recent advances in the field of targeting mesenchymal circulating cells derived from melanomas. The main biomarkers of these cells have been reviewed to define the suitable characteristics of this nanocarriers. The experimental part of the work consisted of development of nanoparticles (non-spherical) by nanoprecipitation of copolymers derived from poly (γ-benzyl-L-glutamate). These nanoparticles, size between 20 and 100 nm and carrying a negative charge (-3 to -30 mV) were then combined with the MART-1 antibody, specific for the melanoma cell membrane, by biotin-streptavidin binding. The binding of the antibody on the surface of nanoparticles was evaluated by Western blot. The affinity of immuno-nanoparticles for melanoma cells (B16-GFP line) and for endothelial cells of human umbilical vein (HUVECs) was then evaluated in vitro by flow cytometry and, being intended for intravenous injection, it was important to evaluate the degree of activation of the complement system induced by the nanoparticles. The 2D immunoelectrophoresis technique used made it possible to conclude that the activation was limited and favourable to increase the blood circulation time of nanoparticles, after intravenous injection. The nanoparticles exhibited low cytotoxicity (MTT assay) against melanoma cells or endothelial cells. In terms of cellular uptake, the immuno-nanoparticles functionalized with MART1, a specific antibody for the recognition of the overexpressed antigen in melanoma cells, was increased by 40 to 50% compared to control. The second part of this thesis was dedicated to the development, characterization and in vivo evaluation of the wound healing activity of β-lapachone encapsulated in multilamellar liposomes and incorporated in a hydrogel of a biopolymer produced by the bacterium Zoogloea sp. These original formulations (β-lap-Lipo/ ZBP/HEC) had a pH and rheological behavior suitable for topical application, as well as the ability to slow the release of β-lapachone from the hydrogel. A detailed histopathological study of the wound healing activity was conducted in an in vivo model and showed that the biopolymer hydrogel was able to stimulate tissue repair, increasing the local cellularity, fibroblasts, cells inflammatory, blood vessels and the production of collagen fibrils during the proliferative phase of healing. In addition, the β-lap-Lipo/ZBP/HEC formulation promoted local angiogenesis and reduced inflammation of the wound, demonstrating the potential of this original formulation of β-lap-Lipo/ZBP/HEC in cutaneous lesions therapy. To conclude, the developed nanocarriers are interesting approachs for intercepting the circulating melanoma cells, while liposomal formulations combining with original biopolymers have an interesting potential for wound healing applications.
author2 MAGALHÃES, Nereide Stela Santos
format doctoralThesis
author PALÁCIO, Sarah Brandão
author_sort PALÁCIO, Sarah Brandão
title Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
title_short Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
title_full Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
title_fullStr Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
title_full_unstemmed Development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
title_sort development and characterization of targeted mart-1-nanoparticles for melanoma treatment and β-lapachone-loaded liposomes in hydrogel for wound healing
publisher Universidade Federal de Pernambuco
publishDate 2019
url https://repositorio.ufpe.br/handle/123456789/29658
_version_ 1641987370436788224
score 13.657419

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