Targeted Delivery of DNA Topoisomerase Inhibitor SN38 to Intracranial Tumors of Glioblastoma Using Sub-5 Ultrafine Iron Oxide Nanoparticles

Abstract

Effectively delivering therapeutics for treating brain tumors is hindered by the physical and biological barriers in the brain. Even with the compromised blood-brain barrier and highly angiogenic blood-tumor barrier seen in glioblastoma (GBM), most drugs, including nanomaterial-based formulations, hardly reach intracranial tumors. This work investigates sub-5 nm ultrafine iron oxide nanoparticles (uIONP) with 3.5 nm core diameter as a carrier for delivering DNA topoisomerase inhibitor 7-ethyl-10-hydroxyl camptothecin (SN38) to treat GBM. Given a higher surface-to-volume ratio, uIONP shows one- or three-folds higher SN38 loading efficiency (48.3 ± 6.1%, mg/mg Fe) than those with core sizes of 10 or 20 nm. SN38 encapsulated in the coating polymer exhibits pH sensitive release with <10% over 48 h at pH 7.4, but 86% at pH 5, thus being protected from converting to inactive glucuronide by UDP-glucuronosyltransferase 1A1. Conjugating α_v β₃ -integrin-targeted cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGD) as ligands, RGD-uIONP/SN38 demonstrates targeted cytotoxicity to α_v β₃ -integrin-overexpressed U87MG GBM cells with a half-maximal inhibitory concentration (IC₅₀ ) of 30.9 ± 2.2 nm. The efficacy study using an orthotopic mouse model of GBM reveals tumor-specific delivery of 11.5% injected RGD-uIONP/SN38 (10 mg Fe kg^-1 ), significantly prolonging the survival in mice by 41%, comparing to those treated with SN38 alone (p < 0.001).

Keywords: SN38; brain tumors; drug delivery; glioblastoma; iron oxide nanoparticles; theranostics; tumor integrin.

Figure 1.

An FTIR spectrum of PEG-b-AGE coated uIONP (A). TEM images of PEG-b-AGE polymer coated uIONP (averaged core diameter of 3.5 nm) before (B) and after (C) SN38 encapsulation (uIONP/SN38) showed the sustained mono-dispersion of uIONP/SN38. The averaged changes (N = 3) of hydrodynamic diameters (D) and zeta-potentials (E) of uIONP before and after the step-wised modifications, i.e., encapsulation of SN38 (red), conjugation of RGD ligands and labeling with NIR830 (blue and green). Incubating uIONP/SN38 in the buffer solutions of pH 5.0 and 7.4, mimicking the lysosomal and physiological pH, for 2, 4, 12, 24 and 48 hours showed a pH responsive SN38 release at pH 5 (F).

Figure 2.

Fluorescence images of α_vβ₃ integrin targeted NIR830-RGD-uIONP incubated with U87MG GBM cells with over-expression of α_vβ₃ integrin (A, F and K), U87MG cells co-cultured with RGD (B, G and L), MCF-7 breast cancer cells with low expression of α_vβ₃ integrin (C, H and M), and Raw264.7 macrophages with low expression of α_vβ₃ integrin (D, I and N). NIR830-RAD-uIONP, the non-targeted control, exhibited no cellular uptake by U87MG cells (E, J and O). The scale bar is 50 μm.

Figure 3.

The cytotoxicity of RGD-uIONP/SN38 (red) to U87MG GBM cells (A), MCF-7 breast cancer cells (B), Raw264.7 macrophages (C) and HEK293 embryonic kidney cells (D) measured by AlamarBlue assays in comparison to RAD-uIONP/SN38 (blue), SN38 without encapsulation (black) and vehicle RGD-uIONP (cyan) at 37 °C with SN38 concentrations ranging from 1 to 10⁴ nM or equivalent Fe concentrations for RGD-uIONP. Each measurement was repeated and averaged ((N = 3). HPLC elution profiles of human liver microsomes treated with RGD-uIONP/SN38 and SN38 without encapsulation (E).

Figure 4.

Ex vivo NIR imaging of the brain, kidney, spleen and liver of tumor bearing mice at 2 and 12 hours after i.v. injection of NIR830-RGD-uIONP/SN38 (A). Quantification of RGD-uIONP/SN38 accumulating in tumor-bearing brains (N = 3) based on Fe concentrations after subtraction of the Fe concentration in control mouse brains receiving no RGD-uIONP/SN38 (B). The statistical significance indicated the results of comparison with the control brains. Confocal fluorescence images of tumor tissues collected at 2 and 12 hours showing DAPI stained nuclei (C, G), immunofluorescence-stained tumor blood vessels (D, H) and NIR830-RGD-uIONP/SN38 (E, I), and their overlay (F, J). Pixel counts of NIR830-RGD-uIONP/SN38 over the distance from the CD31 stained tumor blood vessels (K). Immunofluorescence staining of mouse brain tumor tissues collected at 12 hours after i.v. injection showing the nuclei (L, Q), tumor blood vessels (M, R), NIR830-RGD-uIONP/SN38 (N, S), α_vβ₃ integrin (O), tumor associated macrophages (T) and the overlay (P, U). The scale bar is 100 μm. * p < 0.05, ** p < 0.01, ns: no significance.

Figure 5.

The scheme of the survival study for evaluating the efficacy of treatment using an intracranial mouse model of U87MG GBM (A). Contrast enhanced T₁-weighted brain MRI of mice from the HD_NP and CrEL_SN38 groups post treatment with the enhanced tumor region segmented (colored) using a segmentation algorithm (B). Kaplan-Meier survival curve of mice bearing U87MG GBM tumors (N = 6) in the treatment groups (C). Averaged survivals of different treatment groups (D). * p < 0.05 and *** p < 0.001.

Figure 6.

IHC analysis of EGFR and Ki-67 in the mouse brain tumor tissues collected from mice treated with high dose of RGD-uIONP/SN38 (A, D), CrEL_SN38 (B, E) and PBS (C, F). The scale bar is 100 μm.

Figure 7.

Confocal fluorescence images of EGFR (A, C and D) and Ki-67 (I, K and L) after immunofluorescence staining and NIR830-RGD-uIONP/SN38 (B and J) in tumor-bearing brain tissues of mice treated with high dose RGD-uIONP/SN38 (A to B and I to J), CrEL_SN38 (C and K) and PBS (D and L). The overlay of fluorescence images of NIR830-RGD-uIONP/SN38, EGFR and Ki-67 with DAPI stained nuclei (E to H and M to P). The scale bar is 100 μm.

Scheme 1.

Illustration of the preparation of RGD-uIONP/SN38 labeled with near infrared dye NIR830. The prepared NIR830-RGD-uIONP/SN38 with a 3.5 nm core diameter can readily reach the intracranial tumor in the orthotopic mouse model of GBM after extravasating from the leaky tumor blood vasculatures with loosened tight junctions to accumulate in the tumor tissue, but not in the healthy brain tissue with intact BBB.