Ismail Es

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Author(s):	Ismail Es Total Authors: 1
Document type:	Doctoral Thesis
Press:	Campinas, SP.
Institution:	Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Química
Defense date:	2020-08-12
Examining board members:	Lucimara Gaziola de la Torre; CamilIa Foged; Tiago Albertini Balbino; Marco Cardoso Marques; Rosiane Lopes da Cunha
Advisor:	Lucimara Gaziola de la Torre
Abstract
Lipid-based nanotherapeutics are a promising solution to overcome the current limitations of conventional drug/gene delivery systems. Despite their vast potential, their clinical applications are still limited due to technical impediments in their efficient synthesis. The demand for the development of effective technological platforms for high-throughput production of these compounds is rapidly increasing. In this context, microfluidics emerged as a robust technological tool in the continuous synthesis of lipid-based vector systems for drug/gene delivery. This Ph. D. thesis aims the technological development of microfluidic systems to incorporate small interfering RNA (siRNA) into cationic liposomes (CLs) with stealth properties and on-chip transfection of multicellular spheroids with siRNA nanotherapeutics. In the first part, CLs were electrostatically complexed with siRNA using bulk mixing to better understand how siRNA interacts with liposomes. After our dynamic light scattering (DLS) analysis, the optimum molar charge ratio (R±) was found to be 3.27. This value was verified by employing siRNA accessibility assay and gel electrophoresis. The multilamellarity of the liposomes was confirmed using Cryo-electron microscopy. A significant knockdown of luciferase activity on HeLa cells was obtained with no cytotoxic effect. In the second part, conventional and stealth CLs were produced in a high-throughput microfluidic platform (chaotic advection-based) and physicochemical, structural, morphological and biological aspects of these lipid-based vector systems were evaluated. SAXS and Cryo-TEM analyses confirmed the unilamellarity of both CLs and in vitro transfection studies showed that they could deliver pDNA to the cancer cells. In this step, we achieved 70 times higher productivity in CL production compared to microfluidics hydrodynamic flow-focusing process. Moreover, we successfully eliminated the formation of micelle in the microchannels, which was inevitable in hydrodynamic flow-focusing devices. We also employed centrifugal vacuum concentrator as an alternative distillation process to remove the excess of ethanol in the final liposomal formulation, keeping the unilamellar structure of CLs. In the third part, a diffusion-based microfluidic platform was used to synthesize siRNA-containing lipid-based nanotherapeutics with stealth properties as one-step (phospholipids and PEG polymer with siRNA) and two-step (pre-formed stealth CLs with siRNA) approaches. One-step and two-step approaches led to the synthesis of lipid nanocarriers with different physico-chemical, structural, and morphological properties. SAXS analysis confirmed that the insertion of PEG into the formulation had a different impact on the structural properties. In the last part, static and dynamic microfluidic cell culture systems were developed to produce spheroids using GPF-expressing HEK-293 cells. The spheroids were transfected using siRNA on the selected microdevice (static system). Produced spheroids were morphologically characterized using advanced microscopy techniques and transfection levels were determined by evaluating GFP intensity. The transfection studies showed that static microfluidic platforms were not effective to test drug/gene formulations. Hence, new strategies are proposed to overcome the limitation of static microdevices. This dissertation provides original contributions of major significance in the field of microfluidics and its application in nanomedicine (AU)

FAPESP's process:	15/14468-0 - MICROFLUIDIC SYSTEMS FOR INCORPORATION OF SMALL INTERFERING RNA (siRNA) IN CATIONIC LIPOSOMES AND FOR IN VITRO ANIMAL CELL TRANSFECTION TARGETING GENE THERAPY
Grantee:	Ismail Es
Support Opportunities:	Scholarships in Brazil - Doctorate

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