Profeta, Martina (2017) Smart design and in vitro testing of nanoparticles for microenvironmentally-triggered extracellular drug release. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: Smart design and in vitro testing of nanoparticles for microenvironmentally-triggered extracellular drug release
Creators:
CreatorsEmail
Profeta, Martinamartina.profeta@unina.it
Date: 10 December 2017
Number of Pages: 127
Institution: Università degli Studi di Napoli Federico II
Department: dep08
Dottorato: phd038
Ciclo di dottorato: 30
Coordinatore del Corso di dottorato:
nomeemail
Mensitieri, Giuseppemensitie@unina.it
Tutor:
nomeemail
Netti, Paolo AntonioUNSPECIFIED
Date: 10 December 2017
Number of Pages: 127
Keywords: Stimuli-responsive drug delivery, MMP-sesitive nanoparticles, spheroidal model, microtissues
Settori scientifico-disciplinari del MIUR: Area 09 - Ingegneria industriale e dell'informazione > ING-IND/34 - Bioingegneria industriale
Date Deposited: 08 Jan 2018 01:14
Last Modified: 22 Mar 2019 10:08
URI: http://www.fedoa.unina.it/id/eprint/12165

Collection description

In the field of nanotechnology, one of the most operative research areas is nanomedicine, which applies nanotechnology to highly specific medical interventions for the prevention, diagnosis and treatment of diseases. Currently, the major issue that nanomedicine needs to face is the smart design and production of nanoparticles (NPs) based drug delivery systems for cancer therapy. Highly efficient drug delivery based on nanoparticles could potentially reduce the drug dose needed to achieve therapeutic benefit, thus reducing the side effects associated with the systemic delivery of drugs, whit great benefit to the patient. Indeed, a site-specific delivery of the active compound can be obtained manipulating NP surface by attaching ligands, such as peptides, antibodies or aptamers. Moreover, both passive and active targeting of the drug can be easily obtained by manipulating NP size and surface characteristics. NPs can also control and sustain the release of a drug during transport to, or at, the site of localization, altering drug distribution and subsequent clearance. At present, a new family of nanovectors, defined as stimuli-responsive nanocarriers (SRNs), is emerging. The key point in their mechanism of action lay in the fact that a specific cellular or extracellular endogenous stimulus of chemical, biochemical, or physical origin can modify NP conformation thus promoting the release of the active agent in a specific biological environment [1] [2]. In particular, a large variety of enzymes, such as proteases, glucuronidase, or carboxylesterases can be used as biochemical triggers. Generally the proteases, that are extracellularly expressed, such as the matrix metalloproteases (MMPs), are up-regulated in tumour microenvironment and are responsible for the proteolysis of the extracellular matrix (ECM) and of the basement membranes along with tissue remodelling and metastasis invasion. Since that, they are commonly identified as biomarkers of malignant tissues [3]. In the light of these considerations, Chapter.1 points out a smart approach in NPs design that takes benefits from the MMPs over-expression at tumour site, in order to produce a stimuli-responsive nanocarrier that allows a site specific drug release. To this aim, we proposed the use of a novel nanoparticle able to carry safely doxorubicin (Dox) at tumour tissues, and to respond to MMP-2 enzyme. The produced NPs are made up of a biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) – block – PEG copolymer (namely PELGA), blended with a TAP (Tumour Activated Pro-drug) composed by a MMP-2-sensitive peptide bound to Dox at the C-terminus and to PLGA molecule at the N-terminus. These NPs are named PELGA-TAP NPs. The presence of the MMP-2 enzyme in situ, leads to the destruction of the bond between the peptide and the Dox, with the consequent diffusion and accumulation of the drug in the extracellular environment. This mechanism allows the drug delivery only in presence of an endogenous stimulus that comes from the very nature of the tumour tissue itself. Furthermore, the same NPs were prepared without the presence of the peptide sequence, as negative control, and were named PELGA-Dox. Spheroids of U87 (Human Glioma cells) and HDF (Human Dermal Fibroblast) cells were used as in vitro models of tumour and healthy tissue, respectively, to demonstrate NPs ability to “sense” the differences in the expression levels of endogenous MMP-2 enzymes [4]. Since the production process and effectiveness of PELGA-TAP and PELGA-Dox NPs was well established and consolidate, in Chapter.2 we tested them in a new three-dimensional microtissue (3D µTP) model, which is an in vitro tissue equivalent proposed by Brancato et al. [5]. They fabricate µTPs with the aim to replicate in vitro the composition and the functionalities of the tumour microenvironment. In this work they clearly show that µTPs better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the tumour microenvironment. In particular, the 3D model was developed using normal fibroblasts (NF) and human epithelial cell lines (MCF10), or cancer-activated fibroblasts (CAF) and human breast adenocarcinoma cells (MCF7), to produce healthy and cancer microtissues, respectively. In this scenario, PELGA-TAP and PELGA-Dox NPs were tested in terms of Dox release on these µTPs in order to further validate their efficacy and selective drug release in a more realistic in vitro model, which better resemble tumour microenvironment, closer to the in vivo conditions [6]. Moreover, Chapter.3 shows an upgrade of the PELGA-TAP NP presented above. The approach used for the production of the nanocarrier takes advantages from the layer by layer polymer deposition technique developed and optimized by Vecchione et al. [7]. This technique allows the production of a very stable nanocarrier able to load large amounts of hydrophobic drugs and prevents their systemic leakage. The delivery system we proposed is a crosslinked polyelectrolytes nanocapsule (NC) based on an oil-core and a matrix metalloproteases-2-sensitive shell. MMP-2 enzymes catalyse the disassembly of the NC, which is stabilized by a MMP-2-cleavable peptide sequence as cross-linker. Also in this case, the drug release occurs in a spatially-controlled fashion upon an endogenous stimulus coming from the very nature of the tumour itself. The same NC was also produced with a scrambled peptide sequence as negative control. These NCs were tested on a spheroidal in vitro model, in order to proof their selective shell destabilization and consequent stimuli-responsive drug release in tumour microenvironment. Spheroids of U87 and HDF were used as models of tumour and healthy tissue, respectively. Cell viability was evaluated by means of Alamar Blue Assay. Moreover, the selective disassembly of the NC shell was followed using confocal microscopy and colocalization analyses were also performed. Finally, in Chapter.4 preliminary studies aimed to point out the advantages of an extracellular drug delivery are presented.

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