Repurposing of cyclophosphamide as a chemo‑immunotherapy agent in glioma using D8/RI‑VAP modified-chitosan nanoparticles

Brain tumors possess little immune-cell activity with a cold tumor microenvironment (TME), and do not respond effectively to therapeutic regimens because of the blood-brain barrier (BBB) and the blood-brain tumor barrier (BBTB). Here, we developed cyclophosphamide (CP)-loaded D8/RI-VAP modified chitosan (CS) nanoparticles (NPs) (D8/RI@CP‑CSNPs) as an efficient platform for chemo-immunotherapy of glioma. Physicochemical results confirmed that dual-targeted CP-loaded CSNPs exhibit efficient in vitro and in vivo targeting, cell uptake, and drug release profiles. Biodistribution studies demonstrated substantial accumulation of D8/RI@CP‑CSNPs with a high brain-to-vital organs ratio. Furthermore, the damage-associated molecular pattern (DAMP) molecules such as HMGB1 (2.66-fold), dendritic cell (DC) maturation markers CD80 (1.42-fold) and CD86 (2.69-fold), and TNF‑α (3.92-fold) were increased in tumor parenchyma, indicating strong immunogenic cell death (ICD) induction in tumor-bearing rats. Interestingly, the recruitment of CD8 + and CD4 + T cells to the tumor site proved successful ICD-DC-T cells activation axis following D8/RI@CP‑CSNPs administration via IP injection (4 times at 3-day intervals). In addition to lower weight loss in D8/RI@CP‑CSNPs administered glioma-bearing animals, Kaplan-Meier analysis demonstrated an enhanced more than a two-fold increase in median survival, compared to free drug and untreated groups (60, 31, and 26 days, respectively). Therefore, our dual-targeted CSNPs offer an optimized approach to transform the glioblastoma multiforme suppressive TME "cold" to an immunologically active "hot" state.