Delivery of drugs into brain tumors using multicomponent silica nanoparticles

Abstract

Glioblastomas are highly lethal cancers defined by resistance to conventional therapies and rapid recurrence. While new brain tumor cell-specific drugs are continuously becoming available, efficient drug delivery to brain tumors remains a limiting factor. We developed a multicomponent nanoparticle, consisting of an iron oxide core and a mesoporous silica shell that can effectively deliver drugs across the blood-brain barrier into glioma cells. When exposed to alternating low-power radiofrequency (RF) fields, the nanoparticle's mechanical tumbling releases the entrapped drug molecules from the pores of the silica shell. After directing the nanoparticle to target the near-perivascular regions and altered endothelium of the brain tumor via fibronectin-targeting ligands, rapid drug release from the nanoparticles is triggered by RF facilitating wide distribution of drug delivery across the blood-brain tumor interface.

Fig. 1

Illustration of therapeutic strategy. (a) The nanoparticle, termed (Fe@MSN), is comprised of an iron core surrounded by a drug-loaded a mesoporous silica shell. (b) Drug release from the Fe@MSN nanoparticles is triggered by an external low-power radiofrequency (RF) field at frequencies of about 50 kHz, which makes the nanoparticle to vibrate giving kinetic energy to the drug molecules to escape the pores of the silica shell. (c) The therapeutic strategy consists of vascular targeting of the nanoparticle to the endothelium of glioma sites, and RF-triggered release of drug cargo from Fe@MSN nanoparticles resulting in effective drug delivery across the BBB to glioma cells.

Fig. 2

Characterization of the nanoparticles. (a) TEM image of the Fe@MSN nanoparticle. (b) Elemental analysis of the composition of the Fe@MSN nanoparticle was performed using ICP-OES. (c) FT-IR spectrum of the nanoparticle. (d) The number of peptides on each Fe@MSN nanoparticle was measured using a Bio-Rad DC protein assay. (e) The zeta potential of the DOX-loaded Fe@MSN nanoparticle was measured in 1 M KCl before and after conjugation with the targeting peptide CREKA using a Malvern Zeta Potential Analyzer. (f) Size distribution of the starting iron oxide core and the final Fe@MSN nanoparticles obtained by DLS. (g) The drug cargo of the Fe@MSN nanoparticles is shown. (h) Drug loading into the particles demonstrated good stability in PBS with 10% FBS at 37 °C.

Fig. 3

In vitro evaluation of radiofrequency (RF)-triggered drug release from Fe@MSN nanoparticles. (a) The release of DOX was triggered from Fe@MSN particles using an RF field at different frequencies (1, 20, 50 and 380 kHz; n = 4). (b) The percent released drug of the nanoparticle’s cargo is shown upon application of the RF field at 50 kHz for 30 min (c) Effect of elevated temperature on the drug release from Fe@MSN particles with an incubation time of 60 min (n = 4; unpaired t-test P < 0.0001). (d) Drug release from Fe@MSN at different particle concentration under an RF field at 50 kHz. (e) Drug release from Fe@MSN particles at different depths in the RF source (RF field: 50 kHz). (f) FT-IR spectra of free unmodified DOX and DOX released from Fe@MSN nanoparticle.

Fig. 4

Targeting of CREKA-targeted Fe@MSN nanoparticles to brain tumors. The deposition of CREKA-targeted DOX-loaded Fe@MSN particles in GBM was evaluated in mice bearing orthotopic CNS-1 brain tumors. (a) The accumulation of targeted Fe@MSN particles in brain tumors is shown after 1, 3 and 8 h from i.v. administration at a dose of 5 mg DOX per kg b.w. (n = 5 mice in each group). (b) The accumulation of targeted Fe@MSN in brain tumors was compared to non-targeted Fe@MSN or free DOX using a dose of 5 mg kg⁻¹ DOX for all formulations. Grouped analysis ANOVA; correct for multiple comparisons using the Holm–Sidak method. P values: * <0.01, **** <0.0001. (c) The accumulation of targeted Fe@MSN in liver and spleen is shown after 8 h from i.v. administration at a dose of 5 mg kg⁻¹ DOX (n = 5 mice in each group).

Fig. 5

Histological evaluation of the anticancer effect of RF-triggered drug release from the Fe@MSN nanoparticles in vivo. Histological analysis was performed 3 h after the animals were treated with a single dose of DOX-loaded Fe@MSN particles. (a) The degree and topology of fibronectin was assessed in the orthotopic CNS-1 model in mice (20× magnification; green: glioma cells; red: fibronectin; purple: endothelial cells). Microdistribution of Fe@MSN particles was visualized by staining iron with Prussian blue. (b, c) Using the fluorescence properties of DOX, fluorescence microscopy shows the widespread distribution of DOX molecules (purple: DOX) after a 60 min application of RF (10× magnification). The distribution of DOX molecules is shown (b) without or (c) with RF. (d) The percent of DOX-stained cells relative to the total number of glioma cells was measured by counting the total number of glioma cells (GFP-GL261) and DOX-stained nuclei in two histological sections per tumor (n = 4 mice in each group). We also drew ROIs to distinguish the periphery from the core of the tumor (unpaired t-test; P values: *0.034, *0.011 and *0.044).

Fig. 6

Macroscopic ex vivo evaluation of the therapeutic efficacy of the Fe@MSN treatment. (a) Using the orthotopic CNS-1 model in mice, photographs of brains show the treatment response of the RF-triggered release of DOX in animals treated with Fe@MSN. All animals were euthanized 48 h after treatment with DOX-loaded Fe@MSN particles at a dose of 5 mg kg⁻¹ DOX. Animals were perfused and whole brains were excised and photographed from the top. (b) After euthanasia, tumors were excised and their size was measured (n = 4 mice in each group; unpaired t-test; P value *0.011).

Fig. 7

Evaluation of therapeutic efficacy of Fe@MSN treatments in vivo. (a) Various formulations were i.v. injected in mice bearing orthotopic GL261 brain tumor on day 6, 7 and 9 after tumor inoculation. Treatments included free TMZ, DOX, and DOX-loaded Fe@MSN (5 mg kg⁻¹). In the case of treatments combined with the RF field, animals were exposed for 60 min to the RF field (5 mT, 50 kHz). The response to treatment was monitored using longitudinal bioluminescence imaging (BLI). Quantification of the whole head BLI light emission is shown (n = 7 mice in each group).

Fig. 8

Survival curves in the orthotopic CNS-1 glioma model in mice. The survival time of animals treated with DOX-loaded nanoparticles (+RF) was compared to that of animals treated with free unmodified DOX and the untreated group (n = 8 mice in each group). Each formulation was administered at a dose of 5 mg DOX per kg of body weight. Treatments were systemically administered via a tail vein injection twice at days 5 and 7 after tumor inoculation (arrows). Statistical significance was determined using the log-rank (Mantel-Cox) test.