ETD

• (hPSCs) human pluripotent stem cells
• Bovine serum albumine (BSA)
• Control release
• Design of experiments (DoE)
• double emulsion solvent evaporation
• growth factors
• PLGA nanoparticles
• stem cell expansion

Human pluripotent stem cells (hPSCs) hold enormous promise for regenerative medicine, disease modelling, and drug discovery, but their expansion in vitro remains constrained by the instability of essential growth factors- particularly bFGF and Activin A- under conventional daily medium exchange protocols. This practice generates supraphysiological concentration peaks followed by rapid depletion- conditions far removed from the stable signalling environments that characterise stem cell niche in vivo. To address this limitation, this thesis investigates the development and optimization of PLGA-based polymeric nanoparticles as controlled delivery vehicles for bioactive proteins, using bovine serum albumin (BSA) as a model protein.
Nanoparticles were fabricated via double emulsion solvent evaporation method using ethyl acetate as the organic solvent. A Box-Behnken design of experiments (DoE) with four independent factors – polymer concentration, PVA concentration, BSA concentration, and outer water phase volume- was employed to systematically optimize particle size, polydispersity index (PDI), zeta potential and encapsulation efficiency. Statistical modelling revealed that polymer concentration was the dominant factor governing particle size, while PVA concentration was the primary determinant for both PDI and zeta potential. BSA concentration showed a stablished effect on particle size, consistent with its role as interfacial stabiliser, and its lack on influence on zeta potential was interpreted as evidence of successful encapsulation. The optimized formulations achieved mean particle size below 220 nm, PDI values below 0,25 and zeta potential more negative than -30 mV, meeting colloidal stability criteria for pharmaceutical applications.
The nanoparticles are designed for embedding within alginate hydrogel beads, creating a hierarchical dual-release system intended to provide sustained, physiologically relevant growth factor concentrations during stem cell expansion while physically preventing particles from direct cell contact. Characterization methods including DLS, BCA protein assay, SEM, cytotoxicity assay, internalization assay and drug release assay were used.