Traditional treatment methods for glioblastoma multiforme (GBM) including resection, radiation, and chemotherapy have been largely unsuccessful, with a current 5-year survival rate of 5.6%. In this project we examine the potential of nanosized self-assembling peptide hydrogels to locally deliver and convert temozolomide (TMZ), an FDA-approved pH-sensitive prodrug, for GBM treatment. The peptide hydrogel is designed to load TMZ into the hydrophobic regions of the hydrogels, and during hydrogel degradation in vivo, convert TMZ into its active form. Hydrogel characterization, drug loading and release cellular uptake, and viability are examined to determine the in vitro efficacy of this delivery method. A combination of dynamic light scattering (DLS), scanning electron microscopy (SEM), and circular dichroism (CD) are used to characterize size and structure of the hydrogels. High performance liquid chromatography and microcentrifuge dialysis are used to quantify drug loading and release from the hydrogels. Flow cytometry, fluorescent imaging, and cell viability assays are used to determine uptake and anti-cancer effects of the drug-loaded hydrogels on glioblastoma cells. Our results show high drug loading efficiency and uptake in drug-resistant T98G and non-resistant LN-18 glioblastoma cell lines using several of our tunable peptide formulations. CD has shown that all peptide formulations form mostly beta-sheet and random structures during self-assembly. SEM and DLS show that certain formulations are fairly consistent in size and structure; we are focused on refining preparation methods to further improve uniform size and structure for these formulations and those that exhibit promising drug loading and uptake. Using a pH-sensitive dye, we have shown that as the peptides degrade, the degradation products cause the local pH to become basic, confirming that more TMZ will convert more quickly as the hydrogels degrade. Preliminary cell viability studies have shown promising results for anti-cancer effects of the drug-loaded hydrogels. Future studies for the project will include further cell viability studies to confirm preliminary results with the most promising peptide formulations once uniform self-assembly has been achieved. Finally, this project will culminate in an in vivo study to confirm the overall anti-cancer effect of the drug-loaded peptide hydrogels in a tumor model of GBM. Acknowledgements: This research was supported in part by the National Science Foundation EPSCoR Program under NSF Award # OIA-1655740. Citation Format: Megan Pitz, Margaret Elpers, Arica Gregory, Alexandra Nukovic, Angela Alexander-Bryant. Self-assembling peptide hydrogel for delivery of therapeutically active temozolomide in glioblastoma treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1726.