SLNP-based CDK4- targeted nanotherapy against glioblastoma

Abstract

Introduction: Glioblastoma is a grade IV solid brain tumor and has a 15-month survival rate even after treatment. Glioblastoma development is heavily influenced by retinoblastoma protein (pRB) pathway changes. The blood-brain barrier, drug resistance, and severe toxicity of Temozolamide are key obstacles in treating glioblastoma. Innovative treatments targeting the pRB pathway with efficient delivery vehicles are required to treat glioblastoma.

Methods: For this purpose, a library of 691 plant extracts previously tested in vitro for anti-cancerous, anti inflammatory, and anti-proliferative characteristics was created after thorough literature investigations. Compounds were docked against pRB pathway protein ligands using molecular operating environment and chimera. Their nuclear structure and drug-like properties were predicted through Lipinski rule and density functional theory analysis. Physio-chemical characterizations of naked and drug-encapsulated SLNPs assessed size, stability, entrapment efficiency, and drug release rate. Anti-cancer potential of drug and drug- loaded SLNPs was evaluated using U87, U251, and HEK cell lines. Formulations were tested for cancer cell metastatic potential using cell migration assays.

Results: Silymarin (Sil) was identified as the most potent compound against CDK4, which was then encapsulated in stearic acid solid lipid nanoparticles (SLNP-Sil). Sil showed decreased cell viability 72 h after treatment against both U87 and U251 cell lines but had negligible cytotoxic effect on HEK-293. IC50 value of Sil was 155.14 µM for U87 and 195.93 µM for U251. Sil and SLNP-Sil effectively inhibited U87 and U251 cell migration 24 h after treatment.

Discussion: Our results indicated that Sil and SLNP-Sil are promising therapeutic approaches against glioblastoma and merit in vivo experimental verification using orthotropic xenograft mouse models against glioblastoma.

Keywords: blood brain barrier; glioblastoma; silymarin; solid lipid nanoparticles; temozolamide.

Figure 1

(I) Optimized geometries and charge distribution on (A) Silymarin, (B) Omtriptolid, (C) Belamide A, (D) Isomalyngamide A, and (E) Daphnoretin. (II) HOMO, LUMO, E_HOMO, E_LUMO, and band gap (ΔE) for (A) Silymarin, (B) Omtriptolid, (C) Belamide A, (D) Isomalyngamide A, and (E) Daphnoretin. (III) Molecular electrostatic potential surfaces (MESPs) of (A) Silymarin, (B) Omtriptolid, (C) Belamide A, (D) Isomalyngamide A, and (E) Daphnoretin.

Figure 2

(A) 3D analysis of interaction of Silymarin in binding pocket of CDK4 (left), 2D LigPlot showing interactions of Silymarin with residues of protein (right). (B) 3D analysis of interaction of TMZ in binding pocket of CDK4 (left). 2D LigPlot showing interactions of Silymarin with residues of protein (right).

Figure 3

SEM images of blank solid lipid nanoparticles (A, B), Silymarin- loaded solid lipid nanoparticles (C, D), and TMZ- loaded Solid lipid nanoparticles (E, F) at ×100,000, ×80,000, and ×50,000 with size ranging from 20 nm to 35 nm.

Figure 4

FTIR spectrum showing (A) blank solid lipid nanoparticles peaks, (B) Silymarin peaks, (C) Silymarin- encapsulated solid lipid nanoparticles peaks, (D) Temozolomide peaks, and (E) Temozolomide-encapsulated solid lipid nanoparticles. Characteristic peaks of Silymarin is shown on 3,419 cm⁻¹, 1,736 cm⁻¹, 947.98 cm⁻¹, and 723.79 cm⁻¹in SLNP-Sil, confirming Sil encapsulation in SLNPs. Characteristic peaks of TMZ is shown on 1,351.57 cm⁻¹, 1,106.75 cm⁻¹, 954.44 cm⁻¹, and 716.17 cm⁻¹in SLNP-TMZ, confirming TMZ encapsulation in SLNPs.

Figure 5

XRD patterns of (A) Silymarin, (B) Silymarin- encapsulated solid lipid nanoparticles, (C) Temozolomide, and (D) Temozolomide-encapsulated solid lipid nanoparticles. Both Silymarin and TMZ shows decrease in crystallinity when loaded in SLNP, as shown by reduction in crystalline peaks.

Figure 6

(I) Time- dependent Silymarin release from solid lipid nanoparticles observed at six different time points ranging from 30 min to 48 h and showing ~ 75% of drug release after 24 h (II) Line graphs show the cell viability of Silymarin and TMZ at different concentrations ranging from 0.01 µM to 1,000 µM at three different time points (24 h, 48 h, and 72 h) on (A) U87-MG, (B) U251-MG, and (C) HEK-293 cell lines.

Figure 7

Bar graphs shows the comparison between naked Silymarin and Silymarin- encapsulated in SLNPs, aswell as naked TMZ and TMZ- encapsulated in SLNPs and at different concentrations and 72-h time point, on U87-MG (A), U251-MG (B), and HEK-293 (C) cell lines. Results have been presented as mean ± SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001.

Figure 8

(I) Effect of (A) control (B) blank SLNPs (C) Silymarin, (D) SLNP-Sil, (E) TMZ, and (F) SLNP-TMZ on migration of U87 cells after 24 h. (II) Effect of (A) control (B) blank SLNPs (C) Silymarin, (D) SLNP- Sil, (E) TMZ, and (F) SLNP-TMZ on migration of U51 cells after 24 h. (III) Quantitative analysis of the percentage of wound healing in U87 (A) and U251 cells (B) after 24 h by untreated (control) and treated groups (Sil, TMZ, and their loaded nanoparticles). ImageJ software was used for this analysis. Migratory potential of both U87 and U251 cells is suppressed by Silymarin and Silymarin- loaded nanoparticles, whereas no hindrance in the migratory potential of the cells is observed when treated with naked SLNPs. Cells treated with TMZ and SLNP-TMZ showed very limited hindrance in cell migratory activities.