Polymeric microparticle synthesis via droplet microfluidics for sustained release of non-viral vectors applied to gene therapy

• Grant number: 18/18523-3
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): December 01, 2018
• Effective date (End): February 28, 2023
• Field of knowledge: Engineering - Chemical Engineering
• Principal Investigator: Lucimara Gaziola de la Torre
• Grantee: Bruna Gregatti de Carvalho
• Host Institution: Faculdade de Engenharia Química (FEQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 15/20206-8 - Modulation of monocytes, macrophages and pericytes by the colony stimulating factor genes to treat murine limb ischemia, AP.TEM
• Associated scholarship(s): 21/07057-4 - Gelatin methacryloyl (GelMA) microporous annealed particle (MAP) scaffold for non-viral vector delivery, BE.EP.DD

Abstract

This research project aims to develop multifunctional systems to release nanovectors for co-delivery of nucleic acids into cells. In this project, two important aspects will be explored for gene therapy improvement: (i) the development of nanovectors for co-delivery of plasmid DNA (pDNA) and small interfering (siRNA-silencer); and (ii) the synthesis of hybrid micro-carrier system composed of alginate/silk fibroin and alginate/chondroitin sulphate. Then, multifunctional systems will be designed using micro-carriers to sustained release of co-delivery nanovectors (i and ii). Gene therapy requires the transfer of genetic materials in vivo or ex vivo into target cells; being one of its greatest challenges the development of strategies able to improve the low levels of gene transfer and expression. In this context, the study of simultaneous and combined delivery (codelivery) of these therapeutic agents (pDNA and siRNA), which is still scarce in literature, can provide more expressive results to gene therapy. The gene expression level depends primarily on the efficiency of delivery of these materials into cells, which requires specific delivery systems, such as viral and non-viral vectors. The micro- carriers' synthesis and the encapsulation process of non-viral vectors' will be obtained by droplet microfluidics, which is a promising tool for synthesis of such structures; wherein it is possible to control size, polydispersity, shape, and internal structure. The sustained release implies less frequent in vivo administration when compared to conventional methods which may increase the patient's response to treatment. In the first stage of this project, hybrid microparticles will be synthesized in microchannels and characterized with respect to their morphological and physicochemical properties. Secondly, non-viral vectors as well as cationic liposomes (LCs) will be modified and adapted to the micro-carriers environment. Thus, these LCs will be used as standard vectors for the following nanocarriers development to codelivery of gene materials. In this step, LCs encapsulation efficiency and kinetic release will be verified in microcarrier system. Finally, the best non-viral vectors containing siRNA/pDNA and the best hybrid microparticles will form the multifunctional system, which will be used for transfection assay of HeLa Reporter Cells expressing Green Fluorescent Protein (GFP). Transfection efficiency will be determined by measuring GFP and RFP expression levels using fluorescence microscopy and flow cytometry. This project has collaboration with the Center for Cellular and Molecular Therapy (CTCMol) of the Federal University of São Paulo (UNIFESP), more specifically with the research group coordinated by Prof. Dr. Sang Won Han, who has expertise in the field of gene therapy. At the end of this project, it is expected with the creation of multifunctional systems to contribute to development of innovative strategies for sustained release. This project will act into the fields of droplet-microfluidics, microencapsulation, nanobiotechnology, sustained release, and gene therapy. (AU)

Scientific publications

(7)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

AICH, ANUPAM; LAMARRE, YANN; SACOMANI, DANIEL PEREIRA; KASHIMA, SIMONE; COVAS, DIMAS TADEU; DE LA TORRE, LUCIMARA GAZIOLA. Microfluidics in Sickle Cell Disease Research: State of the Art and a Perspective Beyond the Flow Problem. FRONTIERS IN MOLECULAR BIOSCIENCES, v. 7, MAR 8 2021. (18/18523-3, 15/23469-0)

CARVALHO, BRUNA G.; GARCIA, BIANCA B. M.; MALFATTI-GASPERINI, ANTONIO A.; HAN, SANG W.; DE LA TORRE, LUCIMARA G.. Hybrid polymer/lipid vesicle synthesis: Association between cationic liposomes and lipoplexes with chondroitin sulfate. COLLOIDS AND SURFACES B-BIOINTERFACES, v. 210, FEB 2022. (15/20206-8, 18/18523-3, 18/19537-8)

CINEL, VICTOR DAL POSOLO; TAKETA, THIAGO BEZERRA; DE CARVALHO, BRUNA GREGATTI; DE LA TORRE, LUCIMARA GAZIOLA; DE MELLO, LUCAS RODRIGUES; DA SILVA, EMERSON RODRIGO; HAN, SANG WON. Microfluidic encapsulation of nanoparticles in alginate microgels gelled via competitive ligand exchange crosslinking. Biopolymers, v. 112, n. 7, JUL 2021. (18/19537-8, 18/18523-3, 19/19719-1, 18/06635-1, 15/20206-8)

DE CARVALHO, BRUNA GREGATTI; TAKETA, THIAGO BEZERRA; MORENO GARCIA, BIANCA BONETTO; HAN, SANG WON; DE LA TORRE, LUCIMARA GAZIOLA. Hybrid microgels produced via droplet microfluidics for sustainable delivery of hydrophobic and hydrophilic model nanocarriers. Materials Science & Engineering C-Materials for Biological Applications, v. 118, JAN 2021. (18/19537-8, 15/20206-8, 18/18523-3)

CARVALHO, BRUNA G.; VIT, FRANCIELE F.; CARVALHO, HERNANDES F.; HAN, SANG W.; DE LA TORRE, LUCIMARA G.. Layer-by-Layer Biomimetic Microgels for 3D Cell Culture and Nonviral Gene Delivery. Biomacromolecules, DEC 2021. (18/18523-3, 15/20206-8, 18/19537-8, 17/20341-8)

CARVALHO, BRUNA G.; CECCATO, BRUNO T.; MICHELON, MARIANO; HAN, SANG W.; DE LA TORRE, LUCIMARA G.. dvanced Microfluidic Technologies for Lipid Nano-Microsystems from Synthesis to Biological Applicatio. HARMACEUTIC, v. 14, n. 1, JAN 2022. (15/20206-8, 18/18523-3, 18/19537-8)

CARVALHO, BRUNA G.; VIT, FRANCIELE F.; CARVALHO, HERNANDES F.; HAN, SANG W.; DE LA TORRE, LUCIMARA G.. Recent advances in co-delivery nanosystems for synergistic action in cancer treatment. JOURNAL OF MATERIALS CHEMISTRY B, v. 9, n. 5, p. 1208-1237, FEB 7 2021. (18/18523-3, 18/19537-8, 15/20206-8)