טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKuplennik Nataliya
SubjectPolymeric Nanocarriers Surface-Decorated with Proteins as
Platform for Targeted Drug Delivery to the Central
Nervous System in Neurodegenerative
Diseases
DepartmentDepartment of Materials Science and Engineering
Supervisor Professor Alejandro Sosnik


Abstract

Drug delivery to the central nervous system (CNS) in general, and brain in particular, is very challenging due to presence of two physiological barriers in the brain that restrict free passage of the molecules from the blood circulation into it. Recently, a new mechanism of folate receptor alpha (FRα)-dependent exosome-mediated folate delivery into the brain parenchyma was described. This pathway relies on formation of FRα-folate complex and internalization of the complex by choroid plexus epithelial cells. We hypothesized that it might serve as a loophole for the targeting drugs to the CNS. In this framework, this Ph.D. project investigated the design and synthesis of polymeric nanoparticles (NPs) surface modified with a FRα-folic acid (FA) complex and assessed their ability to reach the CNS from the systemic circulation.

In this framework, NPs of functionalized-poly(ethylene glycol)-b-poly(epsilon-caprolactone) (PEG-b-PCL) copolymers were produced by a nanoprecipitation method. Hydrophilic PEG blocks migrated to the nanoparticle surface during formation and exposed the functional groups allowing further modification of the NPs with FRα and its complex with FA. The size of the NPs was in the 58-98 nm range, as measured by dynamic light scattering accompanied by relatively small polydispersity index values, indicating the monomodal (one size population) character of the obtained NPs. Next, we modified the surface of the NPs with the FRα-FA complex via three conjugation approaches. FRα conjugation yield ranged between 50-75%. Cell compatibility, cellular uptake and permeability of fluorescently-labeled FRα-conjugated NPs was evaluated in vitro on human choroid plexus epithelial primary cells (HCPEpiC), which are part of CNS, and Brown Norway rat choriocarcinoma cells/rat yolk sac (BN16) cells, which are not a part of CNS but can undergo polarization and form tight junctions in a similar way as HCPEpiC. Pristine and FRα-modified NPs showed good compatibility with both cells. In addition, FRα-modified NPs were internalized by both cell types to a greater extent than the unmodified counterparts. Moreover, in HCPEpiC monolayers, NPs surface-modified with the FRα and complexed to FA showed significantly higher apparent permeability coefficient values than the unmodified ones. Finally, the biodistribution of unmodified and FRα-FA-modified NPs following intravenous and intranasal administration was compared in mice. Conjugation of FRα-FA complex to the NP surface promoted higher accumulation in the brain following intravenous injection. In parallel, we assessed encapsulation of the active agent sepiapterin (SP) in the model pristine NPs by means of SP complexation and hydrophobization with 2,3,6-triacetyl-β-cyclodextrin (TAβCD). For this, SP/TAβCD complexes were produced by spray-drying of SP/TAβCD binary solutions. The encapsulation efficiency and drug loading of the SP as a spray-dried SP/TAβCD complex were significantly higher as compared to encapsulation of pristine SP. Moreover, the encapsulation of the complex sustained SP release with relatively low burst effect of 20%, confirming that spray-drying of SP/TAβCD solutions leads to the hydrophobization of the relatively hydrophilic SP molecule, enabling its encapsulation within PEG-b-PCL NPs.  Overall, results of this research highlight the promise of FRα-FA-modified NPs to serve as platform for the targeting of active molecules to the CNS from the systemic circulation.