Stable Cholesterol-Palmitic Acid Sterosomes as Smart Nanocarriers for pH-Sensitive Doxorubicin Delivery in Breast Cancer Therapy

Abstract

Background: Breast cancer remains one of the most prevalent and lethal malignancies worldwide. Although doxorubicin (DOX) is widely used as a first-line chemotherapeutic agent, its clinical utility is constrained by dose-limiting cardiotoxicity and systemic adverse effects. Nanoparticulate drug delivery systems have therefore attracted attention for improving DOX stability, biocompatibility, and tumor selectivity. In this study, we explored sterosomes-simple non-phospholipid nanocarriers composed of cholesterol and palmitic acid-as an alternative DOX delivery platform with pH-responsive properties. Methods: DOX-loaded sterosomes (DOX-STs) were prepared using cholesterol and palmitic acid to impart acid-sensitive behavior. The nanocarriers were systematically evaluated through particle characterization, physicochemical stability assessment, in vitro pH-dependent drug release, and cellular uptake studies. Furthermore, therapeutic efficacy and systemic safety were investigated in an MDA-MB-231 breast cancer xenograft mouse model. Results: DOX-STs exhibited particle sizes below 100 nm, high encapsulation efficiency, and excellent colloidal stability for 28 days. The sterosomes demonstrated accelerated DOX release under acidic conditions relative to physiological pH, consistent with their pH-responsive design. Enhanced cellular uptake was observed in both MCF-7 and MDA-MB-231 cells. In vivo, DOX-ST treatment resulted in significant tumor growth suppression and prolonged survival without notable body weight loss, indicating reduced systemic toxicity compared to free DOX. Conclusions: This study presents a simple sterosome-based nanocarrier system that achieves pH-responsive DOX release and enhanced antitumor efficacy while minimizing toxicity. These findings highlight the potential of sterosomes as a translatable nanomedicine platform for breast cancer therapy.

Keywords: breast cancer therapy; doxorubicin; non-phospholipid nanoparticle; pH sensitive drug release; sterosome.

Figure 1

Physicochemical characterization of DOX-STs. (A) Particle size distribution and (B) zeta potential profiles of DOX-STs measured by DLS at pH 7.4. Data represent the mean ± SD from three independently prepared samples (n = 3).

Figure 2

Stability evaluation of DOX-STs during storage. (A) Particle size (●) and PDI (▲), and (B) zeta potential of DOX-STs were measured over a 28-day period at 4 °C at pH 7.4. Data are presented as mean ± SD (n = 3).

Figure 3

Encapsulation efficiency (EE) of DOX in sterosomes (DOX-STs) during storage. EE was continuously monitored over 28 days at 4 °C at pH 7.4 to assess the stability of the formulation. Data are presented as mean ± SD (n = 3).

Figure 4

In vitro release kinetics of DOX from sterosomes (DOX-STs). Drug release experiments were conducted for 96 h in PBS (pH 7.4, 6.5, and 5.5) at 37 °C with gentle continuous agitation. All values are expressed as mean ± SD (n = 3).

Figure 5

In vitro cytotoxicity of free DOX and DOX-STs. Cell viability was assessed using the MTT assay after 24 h incubation in (A) HepG2, (B) MDA-MB-231, (C) A549, and (D) MCF-7 human tumor cells. Results are expressed as mean ± SD (n = 4). For DOX-ST samples, the indicated concentrations correspond to the encapsulated DOX (DOX-equivalent) concentration, calculated based on the measured encapsulation efficiency.

Figure 6

Cellular uptake of free DOX and DOX-STs in MCF-7 cells. Cells were treated with 5 μM of free DOX or DOX-STs, and intracellular localization was evaluated at multiple time points. (A) Fluorescence microscopy images at 4 h, (B) at 8 h, and (C) at 12 h. Nuclei were visualized using Hoechst 33342 (blue), and DOX fluorescence was detected in the red channel. Regions where blue and red signals overlapped appeared as a pinkish-purple merge. Scale bar = 50 μm. (D) Quantitative assessment of cellular uptake fluorescence intensity at 4, 8, and 12 h. Results are shown as mean ± SD (n = 3).

Figure 7

Cellular uptake of free DOX and DOX-STs in MDA-MB-231 cells. Cells were incubated with 5 μM of free DOX or DOX-STs, and the intracellular distribution was evaluated at different time intervals. (A) Fluorescence microscopy images after 4 h. (B) Images after 8 h. (C) Images after 12 h. Nuclei were visualized using Hoechst 33342 (blue), and DOX fluorescence was detected in the red channel. Regions where blue and red signals overlapped appeared as a pinkish-purple merge. Scale bar = 50 μm. (D) Quantification of fluorescence intensity demonstrating cellular uptake at 4, 8, and 12 h. Data are expressed as mean ± SD (n = 3).

Figure 8

In vivo anti-tumor efficacy of DOX and DOX-STs in a breast cancer xenograft model. (A) Relative tumor volume, (B) representative tumor images collected on Day 0 and Day 14 after treatment, (C) relative body weight of mice, and (D) survival curves in different treatment groups (PBS, ●; DOX, ▲; DOX-ST, ■). Yellow circles indicate tumor regions. Data are presented as mean ± SEM (n = 5).

Figure 9

Plasma concentration–time profiles of DOX following intravenous administration. DOX levels in plasma were quantified at predetermined time points after intravenous injection of free DOX (●) or DOX-STs (▲) in a breast cancer xenograft mouse model. Data are presented as mean ± SEM (n = 3).