Dual-enzyme activated theranostic nanoparticles for image-guided glioblastoma therapy

Abstract

Matrix metalloproteinase-14 (MMP-14) and Cathepsin-B (Cat-B) are overexpressed in glioblastoma (GBM) and not normal brain, making them promising targets for prodrug activation. We investigated a novel combination therapy using two tumor-enzyme activatable theranostic nanoprobes (TNP): TNP-MMP-14, which disrupts the blood tumor barrier via MMP-14 activation, and TNP-Cat-B, which selectively targets GBM cells through Cat-B activation. We hypothesized that combining TNP-MMP-14 and TNP-Cat-B would enhance TNP tumor accumulation and therapeutic efficacy compared to TNP-Cat-B monotherapy. Thirty NSG mice with luciferase-expressing GBM39 tumors received either TNP-MMP-14 plus TNP-Cat-B, TNP-Cat-B only, or saline. Magnetic resonance imaging (MRI) was conducted pre- and post-treatment, with T2* relaxation times analyzed using a generalized linear model. Histopathological differences were assessed using Kruskal-Wallis and Mann-Whitney tests. A Bonferroni correction was applied to account for multiple comparisons. Combination therapy significantly reduced tumor T2* relaxation times (12.98 ± 4.20 ms) compared to TNP-Cat-B monotherapy (22.49 ± 3.95 ms, p < 0.001). The apoptotic marker caspase-3 was also significantly higher in the combination group (64.46 ± 23.43 vs. 15.93 ± 5.81, p < 0.001). These findings demonstrate the potential of dual-enzyme activatable nanoparticles to enhance GBM treatment by overcoming drug delivery barriers and improving therapeutic efficacy over monotherapy.

Keywords: Ferumoxytol; Glioblastoma; MRI; Nanoparticles; Theranostic.

Fig. 1

Composition and mechanism of action of theranostic nanoparticles (TNP). (A) TNP-MMP-14 is composed of an iron oxide core (blue sphere) an MMP-14 cleavable peptide linker (grey bar) and the vascular disrupting agent azademethylcolchicine (ICT3105; red triangle). After cleavage by MMP-14 (blue pacman symbol), azademethylcolchicine is released and activated to disrupt the endothelium of tumor microvessels. (B) TNP-Cat-B is composed of an iron oxide core (blue circle), a Cat-B cleavable peptide linker (grey bar) and the therapeutic drug monomethyl auristatin E (MMAE, yellow triangle). After cleavage by Cat-B (red pacman symbol) in the tumor tissue, the tubulin inhibitor MMAE is released, inhibits tubulin polymerization in tumor cells and causes tumor cell death. MMP-14: matrix metalloproteinase 14.

Fig. 2

Experimental design for assessing the therapeutic efficacy of dual enzyme activated theranostic nanoparticles in GBM xenograft mouse models. GBM39 cells were stereotaxically implanted into nude mice. Tumor growth was monitored using bioluminescence imaging (BLI) until a flux exceeding 1E + 9 photons/sec (p/s) was observed. Mice were randomized into three groups which were treated with intravenous injections of either TNP-MMP-14 plus TNP-Cat-B combination therapy group, TNP-Cat-B monotherapy group or PBS treated control group. All mice underwent MRI before as well as 24 h and 24 h after TNP or PBS treatment. Histology was obtained directly after the second MRI (n = 4 per group) and 10 days after the second MRI (n = 6 per group). GBM glioblastoma multiforme, TNP theranostic nanoparticle, MMP-14 matrix metalloproteinase-14, Cat-B cathepsin B, PBS phosphate-buffered saline.

Fig. 3

TNP design. Schematic representation of TNP-MMP-14 (A) and TNP-Cat-B (B) nanoparticles. These TNPs are conjugated with azademethycolchicine and MMAE, respectively, and engineered for enzyme-specific cleavage (MMP-14 and Cat-B respectively), enabling targeted drug delivery.

Fig. 4

TNP physicochemical characterization. (A) Representative TEM images of ferumoxytol (left), TNP-MMP-14 (center), and TNP-Cat-B (right) at × 10 and × 50 magnifications (10 nm and 50 nm scale bars). (B) Hydrodynamic diameter distributions of ferumoxytol, TNP-MMP-14, and TNP-Cat-B, measured by dynamic light scattering (DLS). (C) R1 relaxation rates for increasing concentrations of ferumoxytol (red), TNP-MMP-14 (green), and TNP-Cat-B (blue), and corresponding r1 relaxivities. (D) R2 relaxation rates for increasing concentrations of ferumoxytol (red), TNP-MMP-14 (green), and TNP-Cat-B (blue), and corresponding r2 relaxivities. (E) Zeta potential stability of TNP-MMP-14 and (F) TNP-Cat-B measured at pH levels 5.0, 7.0, and 8.0 over a period of 48 h. The stability is indicated by minimal fluctuations in zeta potential values across the observed pH conditions. (G) The mean particle size stability of TNP-MMP-14 and (H) TNP-Cat-B measured at pH levels 5.0, 7.0, and 8.0 over a period of 48 h. The stability is indicated by minimal fluctuations in mean particle size across the observed pH conditions. (I) GBM39 cells were treated with PBS, TNP-Cat-B, TNP-MMP-14, or TNP-Cat-B + TNP-MMP-14 for 24 h. Cell viability was assessed using the Cell Titer Blue assay, and fluorescence w the fluorescence intensity was measured at excitation/emission 560/590 nm. Data are presented as mean ± SD (n = 3 per group). Statistical significance was determined using ANOVA with p < 0.05 was considered significant.

Fig. 5

Differential expression of Cathepsin B and MMP-14 in GBM39 xenografts and normal brain tissue in NSG mice. (A) Confocal microscopy images of GBM39 tumors (upper row) and normal brain specimens (lower row), stained for Cathepsin B (red) and DAPI (blue). Merged images are shown on the right. Scale bar: 100 μm; magnification: × 20. (B) Quantification of Cathepsin B expression: Quantitative measures of Cathepsin B/DAPI fluorescence intensity in GBM39 tumors and normal brain. (C) Confocal microscopy images of GBM39 tumors (upper row) and normal brain specimens (lower row), stained for MMP-14 (green) and DAPI (blue). Merged images are shown on the right. Scale bar: 100 μm; magnification: × 20. (D) Quantitative measures of MMP-14/DAPI fluorescence intensity in GBM39 tumors and normal brain specimens. (E) Quantitative measures of MMP-14 expression in GBM39 and SVG p12 cell lines using RT-PCR assay (p = 0.001), GAPDH and 18 s served as endogenous control. (F) Quantitative measures of Cat-B expression in GBM39 and SVG p12 cell lines using RT-PCR assay (p = 0.02), GAPDH and 18 s served as endogenous control.

Fig. 6

In vivo bioluminescence imaging of firefly luciferase-expressing GBM in NSG mice. (A) Representative bioluminescence images at baseline and 10 days after treatment: Tumors treated with TNP-MMP-14 and TNP-Cat-B combination therapy group demonstrated reduced luminescence signal after 10 days. Tumors treated with TNP-Cat-B monotherapy group demonstrated no change in luminescence signal and PBS-treated control group demonstrated increased luminescence signal. (B) Corresponding quantitative analysis of bioluminescence flux of GBM39 tumors. Data are displayed as mean data of 6 mice per group and standard deviations. (C) Statistical model predictions of intergroup differences with 95% confidence intervals (CIs). Results indicate significantly reduced flux in combination therapy group (p = 0.003) compared to monotherapy and control groups, and a significant increase in flux in control group (p = 0.002) compared to combination therapy and monotherapy groups. All comparisons were adjusted using Bonferroni correction for multiple testing.

Fig. 7

MR Imaging of GBM-bearing mice before and after theranostic therapy. (A) Representative T2-weighted MR Images of GBM-bearing mice at baseline, 1 day post-treatment and 2 days post-treatment. Mice treated with TNP-MMP-14 and TNP-Cat-B combination therapy showed a more pronounced negative (dark) tumor T2 enhancement compared to mice treated with TNP-Cat-B monotherapy or PBS control group. Arrows show the tumor region. (B) Quantitative analysis of tumor T2* relaxation times at baseline as well as day 1 and 2 after intravenous treatment with TNPs or PBS. The combination therapy group showed significantly shortened T2* relaxation times post-treatment compared to baseline in both days. The monotherapy group demonstrated no significant change in tumor T2 signal on day 1 but a decrease on day 2 post-injection. The control group exhibited no significant changes in tumor signal over time. Data are presented as mean ± standard deviation (n = 10 mice per group). (C) Statistical model predictions of inter-group differences in tumor T2* relaxation times with 95% confidence intervals (CIs). Results indicate significantly reduced T2* relaxation times in the combination therapy group compared to the monotherapy and control groups, and modest reductions in the monotherapy group compared to control group. A Bonferroni correction was applied to all comparisons (p < 0.001).

Fig. 8

MR Imaging of GBM-bearing mice before and after treatment. (A) Representative T2-weighted MR images of GBM-bearing mice at baseline and 10 days post-treatment. Tumor volume changes were assessed in mice treated with combination therapy, monotherapy, or PBS (control group). At baseline, no significant difference in tumor volume was observed between groups. After treatment, the combination therapy group demonstrated a reduction in tumor volume, while the monotherapy and control groups exhibited tumor growth. Arrows indicate the tumor region. (B) Quantitative analysis of tumor volume mean change (cm³) across groups. The combination therapy group showed a significant reduction in tumor volume compared to the monotherapy (p = 0.01) and control groups (p = 0.001), indicating a superior therapeutic effect. The monotherapy group exhibited a moderate but significant increase in tumor volume (p = 0.05), whereas the control group showed the most pronounced tumor growth (p = 0.01). No significant difference (ns) was observed between the monotherapy and control groups. Data are presented as mean ± standard deviation (n = 6 mice per group).

Fig. 9

Prussian blue iron stains of GBM39 tumors after theranostic therapy. (A,C,E) Representative Prussian blue (PB) stained tumor sections from the combination therapy group, monotherapy group, and control group at 48 h after intravenous injections. Scale bar: 100 μm; magnification: × 20. (G) Quantitative analysis of Prussian blue-positive staining area. Box plots illustrate the proportion of positively stained areas relative to the total tumor area (%) for each group (n = 4 mice in each group). Statistical comparison of PB-positive staining between the combination therapy group and monotherapy group was performed using the Mann–Whitney U test. The control group showed no detectable PB staining and was excluded from statistical analysis. (B,D,F) Representative hematoxylin and eosin (H&E) stained tumor sections from the combination therapy, monotherapy, and control groups, respectively. We did not observe any significant intra-tumoral hemorrhage in response to TNP treatment.

Fig. 10

Immunofluorescence analysis of apoptosis in GBM39 tumors 10 days post-treatment: (A) Representative confocal micrographs showing Caspase-3 staining (red) and DAPI staining (blue) in tumors from combination therapy group, monotherapy group and control group. Merged images are presented on the right. Scale bar 50 μm, magnification × 40. (B) Corresponding quantitative measures of the fluorescence intensity of Caspase 3-positive staining. Data are displayed as mean data of 6 tumors per group and standard deviations.

Fig. 11

Vascular density analysis in GBM39 tumors 10 days post-treatment: (A) Representative confocal micrographs showing CD31 staining (green) and DAPI staining (blue) in tumors from combination therapy group, monotherapy group and control group. Merged images are presented on the right. Scale bar 50 μm, magnification × 40. (B) Corresponding quantitative measures of the fluorescence intensity of CD31-positive staining. Data are displayed as mean data of 6 tumors per group and standard deviations.

Fig. 12

Analysis of glioma-initiating cell (GIC) populations in GBM39 tumors 10 days post-treatment. (A) Representative confocal micrographs showing CD133 expression (green), DAPI staining (blue) and combined stain for combination therapy group, monotherapy group and control group. Scale bar 100 μm, magnification × 40. (B) Corresponding quantitative measures of the fluorescence intensity of CD31-positive staining. Data are displayed as mean data of 6 tumors per group and standard deviations.