Nose-to-Brain Delivery of Biomimetic Nanoparticles for Glioblastoma Targeted Therapy

Abstract

Glioblastoma (GBM) is an extremely aggressive form of brain cancer that remains challenging to treat, especially owing to the lack of effective targeting and drug delivery concerns. Due to its anatomical advantages, the nose-to-brain strategy is an interesting route for drug delivery. Nanoengineering has provided technological tools and innovative strategies to overcome biotechnological limitations, which is promising for improving the effectiveness of conventional therapies. Herein, we designed a biomimetic multifunctional nanostructure produced by polymeric poly(d,l-lactic-co-glycolic) acid (PLGA) core loaded with Temozolomide (TMZ) coated with cell membrane isolated from glioma cancer cells. The developed nanostructures (NP-MB) were fully characterized, and their biological performance was investigated extensively. The results indicate that NP-MB could control TMZ release and promote TMZ permeation in the ex vivo nasal porcine mucosa. The higher cytotoxicity of NP-MB in different glioma cell lines, particularly against U251 cells, reinforces their potential for homotypic targeting. The chicken chorioallantoic membrane assay revealed a tumor size reduction and antiangiogenic activity. In vivo biodistribution studies showed that NP-MB effectively reaches the brain following nasal administration. These findings suggest that NP-MB holds promise as a biomimetic nanoplatform for effective targeting and homotypic recognition in GBM therapy with high potential for clinical translation.

Keywords: PLGA-based nanoparticles; Temozolomide; biomimetic delivery systems; glioblastoma treatment; homotypic recognition; nanotechnology; nose-to-brain delivery.

Figure 1

Development and characterization of NP-MB. (A) Schematic representation of NP and NP-MB development. Created with BioRender.com. (B) Evaluation of nanosystems Blank NP (uncolored plots) and NP (blue plots) stability in terms of size (nm), PDI, and zeta potential (mV). Statistical one-way ANOVA analysis was applied to identify differences over time p < 0.05. (C) Morphological analysis of nanostructures. Representative images of blank NP, NP, isolated membrane (MB) and NP-MB were recorded using negative-staining transmission electron microscopy (TEM) JEM-2100-JEOL 200 with a LaB6 source operating at an acceleration voltage of 200 Kv.

Figure 2

Analysis of protein corona (PC) formation for blank NP, NP, and NP-MB in different media. (A) Schematic representation of the conduct PC study. Created with BioRender.com. (B) size (nm); PDI and zeta potential (mV). Statistical analysis using two-way ANOVA was applied to identify differences over time (p < 0.05).

Figure 3

TMZ release profile (%) from NP (dark blue) and NP-MB (pink) in a phosphate buffer with 0.1% ascorbic acid, pH 5.5. Data shows the average of six measurements (n = 6) and their standard deviation (SD). Statistical analysis using one-way ANOVA with Tukey’s comparisons was applied to identify differences between experimental groups (p < 0.05).

Figure 4

Ex vivo permeation study applying nasal porcine mucosa. (A) Schematic representation of ex vivo permeation study applying Franz diffusion cells. Created with BioRender.com. (B) Total TMZ permeated (ug/cm²) from free TMZ (gray), NP (blue), and NP-MB (pink) in a phosphate buffer with 0.1% ascorbic acid, pH 5.0. Statistical analysis using one-way ANOVA with Tukey’s comparisons was applied to identify differences between experimental groups (p < 0.05). Data show the average of six measurements (n = 3) and their standard deviation (SD).

Figure 5

Cell viability assay and cell death. (A) Comparison of different treatments: free TMZ, blank NP, NP, and NP-MB in different cell lines (HDFn nontumoral, U251, U87, and HCB151). Results represent the median ± SD of at least 3 independent assays (n = 3). The following controls were applied: cells incubated with complete medium, cells incubated with complete medium and the same volume of DMSO in the TMZ groups, and cells incubated with complete medium and the same volume of water in the blank NP, NP, and NP-MB groups. Differences p < 0.01 between applied treatment were considered statistically significant (***). (B) Cell death was determined by Annexin V FITC and Propidium Iodide staining and flow cytometry after 72 h of treatment with either free TMZ, blank NP, NP, and NP-MB. Results are expressed as the percentage of Annexin + and PI + cells (n = 3) in relation to the control. p-values above the bar describe the trend from recorded applied treatment.

Figure 6

NP and NP-MB (10¹⁰ particles/mL) internalization in HDFn, U251, U87 and HCB151 cells. (A) Internalization kinetics of NP (blue) and NP-MB (pink) using Flow Cytometry. Results express the geometric mean of the fluorescence intensity and represent the mean ± SD of three independent replicates. Differences p < 0.05 between the control and applied treatment were considered statistically significant p < 0.05 (**). (B) Images were recorded using a Zeiss LSM 900 laser-scanning confocal microscope following 4 h treatment with NP and NP-MB.

Figure 7

NP and NP-MB (10¹⁰ particles/mL) internalization mechanisms in U251 cells. (A) U251 cells were treated with different pharmacological endocytosis inhibitors amiloride (AMI), nystatin (NYS), nocodazole (NOC), dynasore (DYN), and dansyl-cadaverine (DAN) before incubation with NP and NP-MB (10¹⁰ particles/mL), for 4 h in the presence of the inhibitors. (B) Relative fluorescence intensity normalized by no inhibitor group. Results express the geometric mean of the fluorescence intensity and represent the mean ± SD of three independent replicates. Differences p < 0.01 between the control (no inhibitor) and applied treatment were considered statistically significant p < 0.05 (***).

Figure 8

In vivo analysis for NP and NP-MB biological performance. (A) % of tumor growth after different treatments. (B) Ex ovo quantification of blood vessels number with representative images acquired 3 days after applying the treatment. Results are expressed as mean – SD. One-way analysis of variance, followed by Tukey’s multiple comparisons was used for statistical analysis **(p < 0.05). (C) Fluorescence tomography of the brain was conducted with images captured at 30, 60, and 180 min following the intranasal administration of IR780-loaded NP and NP-MB (n = 3).