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Tesi etd-09162018-205831


Thesis type
Tesi di laurea magistrale LM5
Author
TREMOLANTI, CHIARA
URN
etd-09162018-205831
Title
Synthesis and characterization of PLGA nanoparticles tailored to gene delivery: assessing the endosomal escape
Struttura
FARMACIA
Corso di studi
CHIMICA E TECNOLOGIA FARMACEUTICHE
Commissione
relatore Prof.ssa Da Pozzo, Eleonora
relatore Dott. Salvadori, Piero A.
relatore Dott. Signore, Giovanni
Parole chiave
  • endosomal escape
  • nanoparticles
  • gene delivery
  • PLGA
Data inizio appello
03/10/2018;
Consultabilità
parziale
Data di rilascio
03/10/2021
Riassunto analitico
The use of poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to perform targeted delivery of therapeutics to cells has the potential of recasting the treatment of many diseases and their intracellular fate is critical for successful therapy. One of the major challenges is controlling where the encapsulated drug is trafficked once the NP is taken up into the cell and which are the parameters that affect it. Actually, NPs after internalization are entrapped in endosomes and subsequently degraded by specific enzymes in lysosomes. A proper engineering of NPs would facilitate the endosomal escape and ensure cytosolic delivery of the therapeutics, especially for payloads that are susceptible to lysosomal degradation, such as DNA, siRNA, or miRNA. These payloads have huge potential as alternative to viral gene therapy or in treatment of gene-associated human diseases, such as cancer. In all these cases, nuclear or cytosolic delivery of the payload is a key requirement to achieve a therapeutic activity, and engineered NPs could represent the key to promote such translocation.
This thesis work was focused on developing a NP-based tool able to address this issue. First objective was developing two PLGA-based nanoparticles formulations that have the potential to disrupt the endosomal vesicles thanks to the presence of the cationic polymer polyethyleneimine (PEI). The formulations were optimized in terms of stability and synthesis efficiency, and characterized by measuring size, surface zeta potential and encapsulation efficiency. Preliminary in vitro evaluations were performed to asses NPs toxicity and uptake.
Before proceeding to test the ambitious and experimentally challenging DNA delivery inside cells, NPs were loaded with a small fluorescent molecule, fluorescein-glycine (FGLY). To establish the ability of NPs to escape the endo-lysosomal compartment, we easily evaluated the intracellular fate of FGLY loaded-NPs by means of fluorescence confocal microscopy. Colocalization analysis results supported the evidence that NPs formulated with PEI can partially escape the endosomes in the cell and carry their payload outside endocytic vesicles. Nanoparticles were further engineered with a second endosomal escape agent, CM18 peptide. Although results from FGLY delivery demonstrate the potential use of these two kinds of nanoparticles to do endosomal escape, transfection experiment performed using DNA-loaded NPs has not been successful. A reasonable hypothesis can be related to the huge dimension of pDNA or the insufficient amount of released DNA into the cytosol.
In conclusion, a promising result of this thesis work is the development of two nanostructurated systems based on PLGA and PEI polymers, which has been demonstrated able to encapsulate hydrophilic molecules such as a plasmid and a small drug. In addition, these carriers are able to transport the payload inside cells and allow their escape from the endosomes. This aspect is particularly promising with the aim of intracellular delivery of therapeutics that have to be released inside the cytosol to perform their action.
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