(F-219) Developing a Self-Assembling Peptide Hydrogel for Combination Drug and Gene Therapy Delivery in Glioblastoma Multiforme

Introduction:: Glioblastoma multiforme (GBM) is the most common and most aggressive malignant central nervous system tumor.¹ Thus far, barriers to effective GBM treatment have prevented significant advances in patient survival despite aggressive treatment, including surgical resection, radiation therapy, and chemotherapy.² Because treatment for GBM has not significantly improved in over 40 years, we hope that a new bioengineered strategy such as this codelivery system can change patient outcomes. Two main barriers to effective treatment include the blood brain barrier (BBB) preventing entry of systemic therapy into the brain, and the lack of conversion of Temozolomide (TMZ), the chemotherapeutic prodrug used in GBM treatment. To overcome these obstacles, our group has explored both de novo and previously established peptide sequences to act as local drug delivery vehicles for GBM treatment. Peptides are ideal for in vivo use because they tend to be both biocompatible, biodegradable, and are highly tunable. Drug-loaded peptide assemblies injected or implanted locally during surgery circumvent the BBB entirely and can provide sustained drug release over extended periods of time and protect the loaded drug from degradation. In this project, we aim to develop peptide hydrogels which can co-deliver TMZ and siRNA-peptide nanocomplexes previously developed in our lab.³ Additionally, we hope to tune our peptide sequences to improve the conversion of TMZ to its active form at the target site. We hypothesize that the local codelivery of these two therapies will create a synergistic anticancer effect to more efficiently treat both resectable and non-resectable GBM tumors.

Materials and Methods:: First, peptide sequences were designed based on sequences commonly found in literature, specifically the RADA16, EAK16, and multidomain ABA block peptides. De novo peptides are named ALK1, ALK2, ALK3, RAE, and RAEF, and peptides explored from literature include RADA16 (RADARADARADARADA), CASP-R2 (RRSLSLSLSLSLSLRR), CASP-R6 (RRRRRRSLSLSLSLSLSLRRRRRR), and CASP-K2 (KKSLSLSLSLSLSLKK). To explore self-assembly characteristics, peptides were prepared at three concentrations (1 mg/mL, 2 mg/mL, 5 mg/mL) and in three different salt solutions (double distilled water (ddH2O), 1x phosphate-buffered saline (PBS), and 100 mM NaCl). The structural and mechanical aspects of the resulting assemblies were explored after 24 hours of assembly. A combination of inversion tests, scanning electron microscopy (SEM), circular dichroism (CD), and linear and oscillatory rheology measurements were used to characterize the size, structure, and rigidity of the hydrogels. Loading and localization of siRNA-peptide nanocomplexes and TMZ was observed using fluorescent imaging. To measure release of therapeutics, loaded hydrogels were covered with a supernatant of serum-supplemented media which was collected and replaced daily. Concentration of siRNA, TMZ, and AIC (TMZ’s final conversion product) released each day was measured using UV-Vis spectroscopy. Finally, siRNA stability after release was explored via gel electrophoresis.

Results, Conclusions, and Discussions:: SEM imaging showed that peptide assemblies at lower concentrations were generally smaller and more globular, while peptide assemblies at higher concentrations formed crystallized hydrogel structures. Peptide assemblies in water alone were also more likely to form globular structures, while all peptide sequences examined were more likely to form hydrogels in salt solutions. The addition of salts also drastically affected viscosity of peptides. The viscosity of peptides assembled in water alone was very low (between 1-10 mPa*s) and largely shear-independent. After the addition of 1xPBS, peptide assembly viscosity at 1 mg/mL concentrations increased up to 1,000 mPa*s at low shear rates. At higher peptide concentrations where strong gelation was observed, viscosities were in the 10-200 Pa*s range at low shear rates. Secondary structure is less affected by concentration and salt makeup. All peptides explored exhibited moderate beta-sheet secondary structure (~20-50%) and little to no alpha-helix secondary structure. Strong hydrogel-formers were generally likely to have more beta-sheet structure while more globular assemblies showed a slight decrease in beta-sheet structure. This was expected based on literature showing that peptide sequences such as RADA16 are beta-sheet-based. Fluorescent imaging of loading showed homogeneous distribution of siRNA-peptide nanocomplexes within the strong gel-formers. Release of siRNA-peptide nanocomplexes was linear and reached ~100% release at 9 days. Additionally, released siRNA was revealed via gel electrophoresis to be intact at the 9-day mark, meaning the peptide hydrogels protect siRNA for at least 9 days in a serum environment. TMZ release from peptide hydrogels and subsequent conversion was measured over 4 days. With a ddH₂O supernatant, TMZ remained stable in its inactive form, while in a 1xPBS supernatant, TMZ was observed to convert to its active component rapidly and completely. The results from this study so far suggest that the peptide hydrogels explored are effective delivery platforms for both siRNA nanocomplexes and TMZ. Sustained release of intact siRNA will lead to silencing of target oncogenes, while the release and conversion of TMZ to its active form will synergistically cause cancer cell death. Future work will focus on codelivery and corelease of both therapeutics, as well as in-vitro and in-vivo studies.

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References (Optional): : 1. Ostrom QT, et al. Neuro Oncol. 2021. 2. Markert JM, et al. Mol Ther. 2009. 3. Pitz ME, et al. Macromol Biosci. 2021.