UC San Diego

Of all injury types, brain injuries are the most likely to result in permanent disabilities or even death. A key driver of the chronic symptoms of brain injuries is the persistent activation of microglia, or the resident immune cells of the central nervous system, which triggers prolonged neurodegeneration lasting from months to years. Thus, suppressing the sustained microglial activation presents a promising therapeutic strategy. This dissertation investigates a microglia-targeting peptide (MGP) conjugated to a generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer for the delivery of nucleic acid cargo. Successful peptide conjugation to PAMAM dendrimers via a heterobifunctional 2k polyethylene glycol (PEG) linker was achieved at a molar ratio of 1:9.6:4.8 (dendrimer: PEG-linker : peptide) which was validated by SDS-PAGE. Agarose gel electrophoresis identified that the required nitrogen-to-phosphate (N/P) ratio for full complexation of the cargo, plasmid DNA (pDNA), increases as the size of the targeting moiety increases with the peptide-conjugated dendrimers requiring a ratio of 2.5. Dynamic light scattering (DLS) confirmed that the complexes remain stable in physiological conditions at an N/P ratio greater than 3. This colloidal stability is attributed to the PEGylation effect, which maintained a complex size of < 200 nm necessary for traversing the blood-brain barrier (BBB) in brain injury applications. Furthermore, in vitro binding assays in BV2 microglial cells preliminarily demonstrated that MGP possess enhanced binding affinity compared to a scrambled peptide-conjugated dendrimer control. To evaluate the biological functionality of the system, transfection studies were performed in three cell lines: BV2, J774A.1, and HEK293T. The results demonstrated that peptide conjugation reduced transfection efficiency, potentially because conjugation partially consumed the positive charges of the dendrimer that are important for cellular uptake. Taken together, these findings validate the successful conjugation of a targeting peptide onto a PAMAM dendrimer platform for nucleic acid delivery and support its potential for microglia-directed therapeutic applications in brain injury.