AAV9-Mimetic Peptides and Electroporation Synergistically Enhance Nanoparticle Transport through the Blood–Brain Barrier

Crossing the blood-brain barrier (BBB) remains a major hurdle in neurotherapeutics delivery. However, adeno-associated virus serotype 9 (AAV9) variants can cross from the bloodstream to accumulate in brain tissue, with their tropism facilitated by surface peptide loops. In this work, we examined the feasibility of grafting AAV9-based peptides (AAV.PHP.eB, AAV.X1, and AAV.CPP.16) onto nanoparticles (NPs) to increase transport through brain endothelial cells. We also evaluated the effects of combining the cell-mediated transcytosis strategy with nanosecond pulsed electric fields (nsPEFs). To test the importance of the AAV9-mimetic peptide structural presentation, we chemically conjugated linear peptides or recombinantly inserted peptides into external loops of a protein NP delivery scaffold (E2). E2 shares similar size, symmetry, and nanostructure features to AAV9 but is based on a non-viral source. Computational modeling, particle sizes, and circular dichroism data showed that NPs were folded and intact, even after peptide insertion. NP uptake by brain endothelial cells was 6.2- and 3.4-fold greater (after 4 h) for designs that integrated AAV.X1 or AAV.CPP.16 loop peptides into the NP, relative to chemical conjugation of their respective linear peptides; this highlights the importance of retaining the structural context of the AAV9-derived peptide loops. Using a transwell BBB assay which was optimized to be conducted with nsPEF, we showed that the highest transcellular passage of NPs through the endothelial monolayer was obtained by combining the dual delivery strategies of AAV9 peptide loop incorporation and treatment with nsPEFs. This study also determined the AAV.CPP.16 loop to be the most effective peptide, with E2 NP transport to the basolateral side to be almost twice that of the AAV.X1 peptide in E2 (with nsPEFs). These results support the integration of AAV9-mimetic peptides into biomolecules and drug delivery carriers to facilitate their passage through the BBB.