Novel CD44-Targeted Albumin Nanoparticles: An Innovative Approach to Improve Breast Cancer Treatment

Abstract

This study introduces novel CD44-targeted and redox-responsive nanoparticles (FNPs), proposed as doxorubicin (DOX) delivery devices for breast cancer. A cationized and redox-responsive Human Serum Albumin derivative was synthesized by conjugating Human Serum Albumin with cystamine moieties and then ionically complexing it with HA. The suitability of FNPs for cancer therapy was assessed through physicochemical measurements of size distribution (mean diameter of 240 nm), shape, and zeta potential (15.4 mV). Nanoparticles possessed high DOX loading efficiency (90%) and were able to trigger the drug release under redox conditions of the tumor environment (55% release after 2 h incubation). The use of the carrier increased the cytotoxic effect of DOX by targeting the CD44 protein. It was shown that, upon loading, the cytotoxic effect of DOX was enhanced in relation to CD44 protein expression in both 2D and 3D models. DOX@FNPs significantly decrease cellular metabolism by reducing both oxygen consumption and extracellular acidification rates. Moreover, they decrease the expression of proteins involved in the oxidative phosphorylation pathway, consequently reducing cellular viability and motility, as well as breast cancer stem cells and spheroid formation, compared to free DOX. This new formulation could become pioneering in reducing chemoresistance phenomena and increasing the specificity of DOX in breast cancer patients.

Keywords: breast cancer; cationized albumin; hyaluronic acid; ionic nanoparticles; vectorization.

Figure 1

Preparation and characterization of Human Serum Albumin derivative (cysHSA), redox-responsive nanoparticle (FNPs), and doxorubicin-loaded FNPs (DOX@FNPs). (A) Schematic representation of cysHSA conjugate; (B) SDS page of HSA and cysHSA under not-reducing conditions; (C) determination of PI for Human Serum Albumin (HSA, orange) and cysHSA (blue) by zeta potential measurements; (D) formation of FNPs by ionic complexation of cysHSA (positive charge) and Hyaluronic acid (HA, negative charge)) at physiological pH; (E) representative TEM image of negatively stained FNPs; (F) schematic representation of DOX@FNPs; (G) DOX release profiles from DOX@FNPs at physiological (green) vs. redox (red) conditions.

Figure 2

The cell viability evaluation of FNPs and DOX@FNPs. (A) The cytotoxic effect of FNPs vehicle alone on non-cancerous cells MCF10A and h-TERT BJ1. Note that no significant effects were detected after treatment with high concentrations of FNPs. The effect of DOX and DOX@FNPs on non-cancerous cells (B) MCF10A and (C) h-TERT BJ1; One-way ANOVA, **** p-value <0.0001. (D,E) Panel (C) shows the mRNA levels of the glycoprotein CD44, while Panel (D) shows the protein expression of CD44. The data were obtained through bioinformatic analysis on www.cbioportal.org, accessed on 1 June 2024. (F–J) MTT viability assay performed on 2D cultures. It is possible to note the significance of the effect of DOX@FNPs, especially after 72 h of treatment. Two-way ANOVA. ns: not significant; * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001.

Figure 3

The metabolic profiles of breast cancer cell lines after treatment with DOX@FNPs. Panels (A–E) show the metabolic profiles of various cell lines obtained with the Seahorse Analyzer XFe96. OCR and ECAR were monitored in the presence of DOX@FNPs for 72 h. The bar graphs represent the levels of glycolysis, glycolytic reserve, and glycolytic capacity obtained from the ECAR analysis. Additionally, basal respiration levels, proton leak, ATP production, and maximal respiration were evaluated. One-way ANOVA, ns: not significant; * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. (F) Western Blotting analysis of the expression levels of subunits involved in mitochondrial respiration and oxidative phosphorylation after 72 h treatment with DOX@FNPs.

Figure 4

Invasiveness and stemness evaluation of breast cancer cell lines treated with DOX@FNPs. (A,B) Scratch assay on TNBC cells treated with DOX@FNPs for 24 h. After treatment, migratory capacity of the cells is significantly reduced. Scale bar 100 μm. One-way ANOVA, ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001. (C) Expression of CD44 transcript in 2D and 3D models of MCF7, T-47D, and MDA-MB-231 cells. Two-way ANOVA ** p-value < 0.01; *** p-value < 0.001. (D) Mammosphere formation efficiency of breast cancer cell lines treated with DOX 0.5 µM and DOX@FNPs 0.5 µM for 5 days. Two-way ANOVA, ns: not significant; * p value < 0.05; ** p-value < 0.01; **** p-value < 0.0001.

Figure 5

Three-dimensional spheroid analysis after treatment with DOX@FNPs. (A) Spheroid formation of all breast cancer cell lines treated with DOX and DOX@FNPs. Scale bar 50 μm. (B) The significant effect at 20 days demonstrated that DOX@FNPs 0.5 µM, compared to DOX, can significantly reduce spheroid formation in MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines. The spheroids generated from T-47D and MCF7 cell lines were completely disintegrated after 20 days of treatment with DOX@FNPs 0.5 µM. DOX vs. CTRL: One-way ANOVA, ## p-value < 0.01; ### p-value < 0.001; #### p-value < 0.0001. DOX@FNPs vs. CTRL: One-way ANOVA, * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001.