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A microfluidic approach to synthesize lipid-polymer hybrid nanoparticles for siRNA delivery

Rosario Milazzo

A microfluidic approach to synthesize lipid-polymer hybrid nanoparticles for siRNA delivery.

Rel. Gianluca Ciardelli, Olivia Merkel, Benjamin Winkeljann. Politecnico di Torino, Corso di laurea magistrale in Ingegneria Biomedica, 2022

Abstract:

In the last years, two decades after its discovery, siRNA has finally completed its path from bench to bedside and its use is now expected to be extended to a growing number of diseases. Nevertheless, siRNA application is still limited by the barriers it has to face in the human body, hindering its efficacy. Several strategies have been investigated to overcome these barriers and many are still in a developmental stage: polymer and lipid based formulations are two of the main approaches currently used to effectively shield and deliver nucleic acids to targeted cells. However, both of them still have weaknesses, such as toxicity or instability, limiting the high potential of siRNA therapies. One of the latest strategies for siRNA delivery, currently under investigation, is the formulation of lipid-polymer hybrid nanoparticles, merging the mechanical stability of a polymeric core and the advantages of a lipid shell. In this work, PEI-PCL-PEI triblock copolymer and siRNA were used for the synthesis of micelleplexes which were subsequently coated with a layer of cationic lipids. The method of synthesis established in this work is based on the use of a microfluidic device, which allowed us to fabricate reproducible nanoparticles with the desired physical properties such as a diameter of approximately 150 nm, a polydispersity below 0.2 and a positive Zeta Potential. Comparable results were not obtained with the traditional nanoprecipitation synthesis approach that was used. After investigating different parameters, in particular N/P and flow rate ratios, and evaluating their influence on nanoparticles, two formulations emerged for further testing of their physical and biological properties. In the following, those are referred to as DOTAP 7 and DOTAP 14, while the uncoated micelleplexes from which hybrid nanoparticles were obtained are named Polyplexes 7 (PP7) and Polyplexes 14 (PP14). Size, PDI and Zeta Potential characterizations, were followed first by evaluation of encapsulation efficiency with SYBR Gold assay, and then by investigation in vitro in the H1299 cell line. Uptake experiments showed a higher ability of DOTAP 14 to deliver siRNA into the cells than DOTAP 7, but no improvements in comparison to the uncoated polyplexes. Also, regarding the toxicity, the coating did not improve PP 14 viability, while it did for N/P 7 formulations. TEM and confocal imaging confirmed the formation of the expected core-shell morphology and provided details about nanoparticles size and aggregation not detected by DLS, clarifying the different behaviour of formulations in vitro. Unfortunately, the gene knockdown efficacy remained poor for all the samples tested. The addition of endosomal disruptive compounds, highlighted a potential problem with endosomal escape. Investigating endosomal trafficking with imaging techniques may be helpful to prove this hypothesis and to eventually enable further optimization to improve knockdown efficacy of the formulations developed here. The use of ionizable lipids for the coating, thanks to their characteristic to be ionized in acidic pH conditions, may induce a stronger proton sponge effect once the nanoparticles are inside the endosomes, therefore improving endosomal escape and intracellular siRNA release. In addition, elastic modulus measurements with AFM may help to explain the difference in uptake results between the two formulations, since mechanical rigidity is considered to influence cell membrane penetration ability through clathrin mediated endocytosis,

Relatori: Gianluca Ciardelli, Olivia Merkel, Benjamin Winkeljann
Anno accademico: 2021/22
Tipo di pubblicazione: Elettronica
Numero di pagine: 62
Informazioni aggiuntive: Tesi secretata. Fulltext non presente
Soggetti:
Corso di laurea: Corso di laurea magistrale in Ingegneria Biomedica
Classe di laurea: Nuovo ordinamento > Laurea magistrale > LM-21 - INGEGNERIA BIOMEDICA
Ente in cotutela: Ludwig-Maximilians-Universität München (GERMANIA)
Aziende collaboratrici: Ludwig-Maximilians-Universitat Munchen
URI: http://webthesis.biblio.polito.it/id/eprint/23807
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