De Novo Self-Assembling Peptides Mediate the Conversion of Temozolomide and Delivery of a Model Drug into Glioblastoma Multiforme Cells

Abstract

Glioblastoma multiforme (GBM) is the most aggressive central nervous system tumor, and standard treatment, including surgical resection, radiation, and chemotherapy, has not significantly improved patient outcomes over the last 20 years. Temozolomide (TMZ), the prodrug most commonly used to treat GBM, must pass the blood-brain barrier and requires a basic pH to convert to its active form. Due to these barriers, less than 30% of orally delivered TMZ reaches the central nervous system and becomes bioactive. In this work, we have developed a novel biomaterial delivery system to convert TMZ to its active form and that shows promise for intracellular TMZ delivery. Self-assembling peptides were characterized under several different assembly conditions and evaluated for TMZ loading and conversion. Both solvent and method of assembly were found to affect the supramolecular and secondary structure of peptide assemblies. Additionally, as peptides degraded in phosphate-buffered saline, TMZ was rapidly converted to its active form. This work demonstrates that peptide-based drug delivery systems can effectively create a local stimulus during drug delivery while remaining biocompatible. This principle could be used in many future biomedical applications in addition to cancer treatment, such as wound healing and regenerative medicine.

Keywords: glioblastoma multiforme; self-assembling peptides; temozolomide.

Figure 1

Peptides were prepared using film dehydration and then resuspended in either water or PBS. Samples were dried and sputter-coated with platinum before conducting SEM. All images were captured at a magnification of 10.0 k, and scale bars represent 5 µm.

Figure 2

Peptides were prepared using film dehydration and then resuspended in either (A) water or (B) PBS. Samples were examined at shear rates from 1–300 Hz, and viscosity was plotted using Rheocompass.

Figure 3

Peptides were prepared and loaded with TMZ using film dehydration and resuspended in either (A) water or (B) PBS. TMZ concentration was read using UV–vis spectrophotometry and plotted as encapsulation efficiency (%). Data are presented as mean ± SEM (N = 3).

Figure 4

Peptides were prepared and loaded with TMZ using film dehydration and resuspended in either water (A) or PBS (B). TMZ concentration was measured from 0–6 days using UV–vis spectrophotometry. Data are presented as mean ± SEM (N = 3), where * p < 0.05 and ** p < 0.01 compared to time 0 (one-way ANOVA with Dunnett’s multiple comparisons test).

Figure 5

Peptides were assembled through incubation in PBS. Circular dichroism spectra were recorded from 190–300 nm (A). Spectra were analyzed on DichroWeb [22,23] using the K2D method to estimate secondary structure (B).

Figure 6

Peptides were assembled through incubation in PBS. Samples were dried and sputter-coated with platinum before conducting SEM. All images are captured at a magnification of 10.0 k, and scale bars represent 5 µm.

Figure 7

Peptides were prepared via incubation in PBS. Samples were examined at shear rates from 1–300 Hz and viscosity plotted using Rheocompass.

Figure 8

Peptides were prepared and loaded with TMZ using incubation assembly in PBS. TMZ concentration was read using UV–vis spectrophotometry, and encapsulation efficiency (EE) was calculated (A). TMZ conversion was measured over from 6 days (B) using UV-vis spectrophotometry. Data are presented as mean ± SEM (N = 3).

Figure 9

Peptides were prepared using film dehydration and rehydrated in water. Peptide assemblies were incubated with cells at varying concentrations for 48 h, followed by viability analysis via MTS assay. Untreated cells were treated as 100% viable. Results are presented as mean ± SEM (N = 3).

Figure 10

Peptides were mixed with 60, 80, or 100 µM C6 and then prepared using film dehydration and rehydrated in water. Peptide assemblies were incubated with cells for 4 or 24 h, followed by evaluation of cellular uptake using (A) flow cytometry and (B) fluorescent microscopy. Untreated cells were used as a negative control (NC), and 100 µM of C6 was used as a positive control (PC). Results are presented as mean ± SEM (N = 3), where * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to NC (one-way ANOVA). Scale bar = 200 µm.