Full text
| |
| Author(s): |
Bruna Gregatti de Carvalho
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Química |
| Defense date: | 2023-03-22 |
| Examining board members: |
Lucimara Gaziola de la Torre;
Angela Maria Moraes;
Rosiane Lopes da Cunha;
Marlus Chorilli;
Micaela Tamara Vitor
|
| Advisor: | Sang Won Han; Lucimara Gaziola de la Torre |
| Abstract | |
Gene therapy requires the transfer of genetic materials (DNA and RNAs) in vivo or ex vivo into target cells; one of its most significant challenges is the development of strategies able to improve the low levels of gene transfer and expression. The gene expression level depends primarily on the efficiency of delivering these materials into cells, which requires specific delivery systems, such as non-viral vectors and gene-activated biomaterials (microgels and scaffolds based on microgels). In this context, this works aimed to synthesize gene-activated biomaterials through incorporating lipid-based non-viral vectors. Microgels were obtained via droplet-based microfluidics, in which it is possible to control the size, polydispersity and shape. This present work was divided into three different strategies of incorporation: (A) encapsulation, (B) surface coating, and (C) hybrid process: encapsulation and surface coating. As the first strategy (A), hybrid microgels, alginate (ALG) /silk fibroin (SF), ALG / chondroitin sulfate (CS), and ALG/SF/CS were synthesized and encapsulated with nanoparticles (NPs) with different hydrophobicities. In the presence of SF, microgels obtained high loading efficiency of hydrophobic NPs (83-98 %); on the other hand, the presence of CS ensures the encapsulation of NPs with more hydrophilic character (85 %), improving its kinetic release. In the second approach (B), functional microgels for gene delivery and in vitro cell culture were developed by combining microfluidics and layer-by-layer techniques. The layered hybrid microgel, ALG/CS, consisting of polylysine (PLL), polyallylamine hydrochloride (PAH), and CS, (ALG/CS) PLL/PAH/CS, allowed cell adhesion and proliferation. The gene transfer of plasmid DNA (pDNA-eGFP) was also verified after the surface coating with lipid-based non-viral vectors, LP-pDNA, (ALG/CS) PLL/PAH/CS/LP-pDNA. The last strategy (C) consisted of the development of microporous scaffolds based on gelatin methacryloyl (GelMA) to incorporate mRNA-loaded lipid-based non-viral vectors (mRNA-eGFP LP). The intracellular delivery of mRNA was achieved by releasing the nanocomplexes, mRNA-LP, from the scaffolds and by the cellular infiltration, adhesion, and proliferation within the micropores of mRNA-activated scaffolds. Moreover, in this study, two hybrid lipid-based non-viral vectors were developed and modified to adapt them to the polymeric matrices: (i) a pseudo-ternary composed of cationic liposome (CL), pDNA, and CS, (CL-pDNA)-CS, and (ii) cationic lipid–polymer hybrid nanoparticles (cLPHNPs). Here, the colloidal properties and stability of non-viral vectors were extensively studied and reported. Thus, this presented research contributed to the development of innovative strategies to design gene-activated biomaterials acting in droplet microfluidics, layer-by-layer, nanobiotechnology, gene delivery (pDNA and mRNA), and tissue engineering (AU) | |
| FAPESP's process: | 18/18523-3 - Polymeric microparticle synthesis via droplet microfluidics for sustained release of non-viral vectors applied to gene therapy |
| Grantee: | Bruna Gregatti de Carvalho |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |