Obtenção e funcionalização de nanodiamantes para aplicação biológica
Data
2021-08-27
Autores
Guimarães, Carolina Ramos Hurtado [UNIFESP]
Orientadores
Tada, Dayane Batista [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Dentre as inúmeras nanopartículas que demonstram efeito antimicrobiano, os nanodiamantes (NDs) são materiais promissores para aplicação na área biomédica, devido à estabilidade coloidal, sendo considerados biocompatíveis. O presente trabalho visou a utilização de filme residual de diamante-CVD, crescidos por deposição química na fase vapor assistido por filamento quente, para a obtenção de suspensões aquosas de NDCVD por diferentes metodologias. Também foram empregadas técnicas de funcionalizações superficiais dos NDCVD, bem como de ND de detonação (NDD), para a conjugação de fotossensibilizadores (FSs), permitindo a avaliação da aplicação como materiais antimicrobianos por meio da terapia fotodinâmica antimicrobiana (aPDT). Dentre as metodologias empregadas para a obtenção dos NDCVD, um destaque foi dado à técnica de maceração seguida de cavitação ultrassônica, devido a obtenção de NDs com pequenos tamanhos e facilidade de operação do equipamento. Visando a funcionalização da superfície, foram realizadas hidrogenação por plasma de H2 (ND-HCVD) e fotofuncionalização com grupo carboxilado (ND-COOHCVD-(UV)), originando suspensões aquosas de diâmetro hidrodinâmico na faixa de 50-70 nm. Também foi realizada funcionalização não covalente com porfirina mTHPP, com potencial para aplicação em aPDT. Os estudos foram estendidos para a modificação superficial de ND de detonação, originando superfície carboxilada, aminada e hidroxilada a partir de metodologias com vantagens relacionadas à redução do tempo de conversão e eliminação de etapas adicionais, comparadas a processos já relatados. Nanoconjugados com os FSs mTHPP e PHOTOGEM® foram obtidos com sucesso, utilizando via química e enzimática para a formação de ligação covalente do tipo éster. A eficácia na obtenção dos NDs e funcionalização superficial, puderam ser comprovadas por diversas técnicas de caracterização, como: espalhamento de luz dinâmico, difração de raio-x, microscopia eletrônica de transmissão e espectroscopias: Raman, infravermelho, absorção e fluorescência. Também foram realizados ensaios de citotoxicidade em células não tumorais (MEF), avaliação da atividade antitumoral in vitro (B16F10-Nex2) e potencial antibacteriano dos NDs. Os estudos in vitro, não mostraram citotoxicidade a células MEF após etapas de purificações e funcionalização, no entanto o ND-HCVD, NDD-COOH, NDD-NH2 e o nanoconjugado NDCVD+mTHPP, demonstraram citotoxicidade a células tumorais. Para os ensaios de avaliação da atividade antibacteriana, uma maior ação foi observada com os NDCVD, frente à cultura de E. coli (20 %) em comparação à S. aureus (12 %) após 24 h de incubação. Para os NDD-COOH, resultados de inibição semelhantes foram obtidos nas mesmas condições. Na presença de NDD-mTHPP por 2 h de incubação, foi observada inibição de 56 % no crescimento de E. coli, após irradiação com luz de 660 nm. No entanto, após esse período ocorreu diminuição da atividade antibacteriana por consequência da desativação do ROS. Com os nanoconjugados obtidos por rota enzimática foram observados danos na parede bacteriana (E. coli), observados por microscopia eletrônica de varredura, após irradiação com luzes azul e vermelha. Concluiu-se que os NDs obtidos apresentam um foco promissor para pesquisas na área de funcionalização superficial para a conjugação com FS como uma estratégia para aplicação em aPDT.
Amongst the innumerous nanoparticles that have demonstrate antimicrobial activity, nanodiamonds (NDs) are promising materials for application in the biomedical area, due to their colloidal stability, being considered biocompatible. The present work used a residual film diamond-CVD sample, grown by hot filament chemical vapor deposition, to obtain aqueous suspensions of NDCVD by different methodologies. NDCVD surface functionalization techniques were also used, as well as detonation DN (NDD), for the conjugation with photosensitizers (FSs) aiming the application in antimicrobial photodynamic therapy (aPDT). Among the methodologies used to obtain NDCVD, a highlight was given to the technique of maceration followed by ultrasonic cavitation, due to the obtainment of NDs with small sizes and ease of operation of the equipment. Aiming at surface functionalization, plasma hydrogenation of H2 (ND-HCVD) and photofunctionalization with a carboxyl group (ND-COOHCVD-(UV)) were carried out, resulting in aqueous suspensions with a hydrodynamic diameter in the range of 50-70 nm. Functionalization was also performed through non-covalent bonding with porphyrin (FS) mTHPP with potential for application in aPDT. The studies were extended to the surface modification of detonation ND, originating a carboxylated, aminated and hydroxylated surface from methodologies with advantages related to the reduction of conversion time and elimination of additional steps, compared to previously reported processes. Compared with the processes described in the literature, the methods described herein showed drastic reduction of the conversion time, as well as elimination of additional steps. Nanoconjugates with FSs mTHPP and PHOTOGEM® were successfully obtained by using chemical and also enzymatic routes resulting in covalent ester bonds. Obtained NDs and surface functionalized ND were characterized by: dynamic light scattering, x-ray diffraction, transmission electron microscopy and spectroscopies: Raman, infrared, absorption and fluorescence emission. Cytotoxicity assays in non-tumor cells (MEF), in vitro evaluation of antitumoral activity (B16F10-Nex2 cells) and antibacterial potential of NDs were also performed. In vitro studies did not show cytotoxicity to MEF cells after the purification and functionalization steps, however ND-HCVD, NDCVD+mTHPP nanoconjugate showed cytotoxicity to tumor cells. Regarding antibacterial activity, it was possible to note a greater activity against the culture of E. coli (Gram -) (20 %) compared to S. aureus (Gram +) (12 %) after 24 h of incubation. For NDD-COOH, similar inhibition results were identified under the same conditions. In the presence of NDD-mTHPP for 2 h of incubation, a 56 % inhibition of E. coli growth was observed after irradiation with 660 nm light. After a period of 2 h there was a decrease in antibacterial activity as a result of ROS deactivation. With the nanoconjugates prepared by enzymatic route, qualitative antibacterial assays and analysis of the morphology of the bacterial culture by SEM were carried out, demonstrating the potential to inhibit the growth of E. coli by irradiation with blue and red lights, showing damage to the bacterial wall. It was concluded that the NDs developed herein can be considered a promising focus for research for application in aPDT.
Amongst the innumerous nanoparticles that have demonstrate antimicrobial activity, nanodiamonds (NDs) are promising materials for application in the biomedical area, due to their colloidal stability, being considered biocompatible. The present work used a residual film diamond-CVD sample, grown by hot filament chemical vapor deposition, to obtain aqueous suspensions of NDCVD by different methodologies. NDCVD surface functionalization techniques were also used, as well as detonation DN (NDD), for the conjugation with photosensitizers (FSs) aiming the application in antimicrobial photodynamic therapy (aPDT). Among the methodologies used to obtain NDCVD, a highlight was given to the technique of maceration followed by ultrasonic cavitation, due to the obtainment of NDs with small sizes and ease of operation of the equipment. Aiming at surface functionalization, plasma hydrogenation of H2 (ND-HCVD) and photofunctionalization with a carboxyl group (ND-COOHCVD-(UV)) were carried out, resulting in aqueous suspensions with a hydrodynamic diameter in the range of 50-70 nm. Functionalization was also performed through non-covalent bonding with porphyrin (FS) mTHPP with potential for application in aPDT. The studies were extended to the surface modification of detonation ND, originating a carboxylated, aminated and hydroxylated surface from methodologies with advantages related to the reduction of conversion time and elimination of additional steps, compared to previously reported processes. Compared with the processes described in the literature, the methods described herein showed drastic reduction of the conversion time, as well as elimination of additional steps. Nanoconjugates with FSs mTHPP and PHOTOGEM® were successfully obtained by using chemical and also enzymatic routes resulting in covalent ester bonds. Obtained NDs and surface functionalized ND were characterized by: dynamic light scattering, x-ray diffraction, transmission electron microscopy and spectroscopies: Raman, infrared, absorption and fluorescence emission. Cytotoxicity assays in non-tumor cells (MEF), in vitro evaluation of antitumoral activity (B16F10-Nex2 cells) and antibacterial potential of NDs were also performed. In vitro studies did not show cytotoxicity to MEF cells after the purification and functionalization steps, however ND-HCVD, NDCVD+mTHPP nanoconjugate showed cytotoxicity to tumor cells. Regarding antibacterial activity, it was possible to note a greater activity against the culture of E. coli (Gram -) (20 %) compared to S. aureus (Gram +) (12 %) after 24 h of incubation. For NDD-COOH, similar inhibition results were identified under the same conditions. In the presence of NDD-mTHPP for 2 h of incubation, a 56 % inhibition of E. coli growth was observed after irradiation with 660 nm light. After a period of 2 h there was a decrease in antibacterial activity as a result of ROS deactivation. With the nanoconjugates prepared by enzymatic route, qualitative antibacterial assays and analysis of the morphology of the bacterial culture by SEM were carried out, demonstrating the potential to inhibit the growth of E. coli by irradiation with blue and red lights, showing damage to the bacterial wall. It was concluded that the NDs developed herein can be considered a promising focus for research for application in aPDT.