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Archivio digitale delle tesi discusse presso l’Università di Pisa

Tesi etd-11242022-141143


Tipo di tesi
Tesi di laurea magistrale
Autore
VINCENZI, AGNESE
URN
etd-11242022-141143
Titolo
Sviluppo di micelle polimeriche inverse per il trasporto di farmaci idrofilici al sistema nervoso centrale
Dipartimento
BIOLOGIA
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Relatori
relatore Prof. Cecchini, Marco
relatore Dott.ssa Gagliardi, Mariacristina
Parole chiave
  • central nervous system
  • drug delivery
  • micelle polimeriche inverse
  • reverse polymeric micelles
  • sistema nervoso centrale
Data inizio appello
13/12/2022
Consultabilità
Non consultabile
Data di rilascio
13/12/2092
Riassunto
Polymeric Reversed Micelles (PRMs) are self-assembled nanostructures consisting in particle-shaped structures with a polar core and a hydrophobic shell, resulting from the spontaneous self-assembly of amphiphilic copolymers in an organic solvent. The core-shell architecture of PRMs provides an optimal allocation for hydrophilic compounds, including therapeutics.
PRMs are dynamic entities prone to aggregation and disaggregation phenomena, thus their structure should be stabilized to be used as drug delivery systems. Stabilization consists, e.g. in the crosslinking of external macromolecular end-bones which can be easily reversed in the in vivo environment to provide a trigger for drug release.
Drug nanocarriers can significantly improve the stability of encapsulated cargos, also prolonging circulating times and drug release kinetics, and promoting transport across biological barriers, e.g. the blood-brain barrier (BBB). Indeed, the BBB limits the delivery of systemically administered therapies to the brain, and poses a significant challenge to drug development efforts for such treatments.
The aim of this thesis is the development of crosslinked PRMs as nano-vectors that can be exploited for hydrophilic drug delivery to the CNS. Developed PRMs will be functionalized with a brain targeting peptide (Angiopep-2) able to bind the LRP1 receptor of the BBB endothelial cells, and promote BBB penetration of PRMs.
The study reports the production and characterization of bare and functionalized PRMs, both with Angiopep-2, and labeled with a fluorescent rhodamine-based dye.
A commercial amphiphilic copolymer methoxy polyethyleneglycol-block-poly(lactide-co-glycolide) (mPEG-b-PLGA) is used to prepare the PRMs, after its chemical functionalization with lipoic acid (crosslinkable unit), peptide linker, Angiopep-2, and fluorescent dye.
The determination of the CMC by Dynamic Light Scattering (DLS) analysis provides the setting up of the recipe for the production of PRMs. Evaluation of size, surface charge and stability under storage conditions (4°C and -20°C, respectively up to 120 and 45 days) is performed by DLS analysis and determination of the molecular weight by Static Light Scattering (SLS) analysis.
PRMs are administered to mouse primary fibroblasts and tested via cell proliferation assay on at 24 h and 48 h after treatment, to evaluate the effect of different PRM concentrations (5.0 mg/ml, 2.5 mg/ml, 1.2 mg/ml, 0.6 mg/ml).
The CMC value for the selected mPEG-b-PLGA copolymer results around 2.5 mg/ml. Thus, a suitable value of 3 mg/ml for the copolymer concentration is selected to obtain an optimized PRM synthesis recipe. The size of produced PRMs by DLS analysis results ranging from 70 to 130 nm and the surface charge from -34 to -12 mV.
Both characteristics indicate that produced PRMs are suitable for blood circulation and cellular endocytosis. The bare formulation does not undergo significant size changes during stability tests in storage conditions up to 120 and 45 days, respectively at 4°C and -20°C.
The calculated molecular weight of PRMs is around 8800 kDa, suggesting a micelle aggregation number equal to 650.
In vitro assays exhibit a high degree of cytocompatibility of the tested formulations, both 24 h and 48 h after treatment.
Furthermore, PRMs functionalized with the fluorescence dye are suitable for the study of the internalization mechanism.
In conclusion, the present study demonstrates that PRMs are valuable candidates as drug delivery system with targeting capabilities. A further analysis is required for their validation, including evaluation of cargo encapsulation efficiency, quantification of cargo release over the time in physiological conditions, the determination of the internalization mechanism, and in vivo tests.
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