Antibody-Free Immunopeptide Nanoconjugates for Brain-Targeted Drug Delivery in Glioblastoma Multiforme

Abstract

Glioblastoma Multiforme (GBM) represents a significant clinical challenge among central nervous system tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P-12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P-12/AP-2/NCs exert their immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-sized PD-L1/PD-L2 antagonist peptide, Aurigene NP-12 (P-12). P-12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight β-poly(l-malic acid) (PMLA) polymer conjugated with P-12 and Angiopep-2 (AP-2) to yield P-12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3D tumor model efficacy using an in vitro BBB-Transwell spheroid-based model demonstrated successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cells' cytotoxicity on 3D tumor region death in a U87 GBM 3D spheroid model. AP-2/P-12/NCs are selectively inhibited in PD1/PDL1 interaction on T cells and the tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in vivo murine brain environment, rhodamine fluorophore-labeled AP-2/P-12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals postinjection with a prolonged bioavailability over unconjugated peptides. AP-2/P-12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases such as glioblastoma multiforme and beyond.