Targeted Delivery of Doxorubicin Using Biocompatible Chitosan Nanobubbles and Ultrasound

Abstract

Ultrasound‑guided nanobubble (NB) delivery has emerged as a noninvasive strategy for site‑specific drug transport. Chitosan‑based NBs offer superior biocompatibility and a high loading capacity for chemotherapeutics. In this study we fabricated doxorubicin hydrochloride (DOX)‑loaded chitosan NBs, quantified their size, zeta potential, encapsulation efficiency (EE = 54.18 %) and drug‑release profile, and evaluated their imaging performance, biosafety, and therapeutic efficacy in MCF‑7 breast cancer cells. DOX‑NBs released 46 % of their payload within 5 h of ultrasound (US) exposure versus 9 % without US, and delivered significantly higher intracellular DOX levels and apoptosis rates than free DOX. These results demonstrate that biocompatible chitosan NBs are a promising platform for ultrasound‑mediated, targeted doxorubicin delivery.

Background

Chemotherapy remains the cornerstone of malignant tumor treatment, yet systemic toxicity limits the therapeutic index of agents such as doxorubicin. Localized drug delivery can increase the concentration at the tumor site while sparing healthy tissues. Ultrasound‑targeted micro‑ or nano‑bubble destruction (UTN/MD) provides a noninvasive, image‑guided means of enhancing permeability and drug uptake. Conventional microbubbles are too large to extravasate efficiently, whereas nanosized bubbles can traverse the capillary wall and reach tumor parenchyma more effectively.

Existing NB formulations often rely on synthetic lipids or polymers that raise biocompatibility concerns. Chitosan, a naturally derived, biodegradable polysaccharide, possesses low immunogenicity, antibacterial activity, and intrinsic antitumor properties. Coupled with lecithin and palmitic acid—both FDA‑approved, low‑toxicity surfactants—chitosan NBs can be engineered to be safe and highly functional.

Materials and Methods

Materials

Perfluoropropane (C₃F₈) served as the gas core, while medium‑molecular‑weight chitosan (100–300 kDa) formed the shell. Epikuron 200 (soy lecithin), palmitic acid, and Pluronic F68 were added to enhance stability and reduce aggregation. Doxorubicin hydrochloride (Sigma‑Aldrich) was the model chemotherapeutic.

Cell Line

MCF‑7 human breast carcinoma cells (ATCC) were cultured in DMEM + 10 % FBS at 37 °C, 5 % CO₂.

Preparation of DOX‑Loaded Chitosan NBs

DOX (1 mg mL⁻¹) was mixed with chitosan solution, vortexed, and incubated at 65 °C for 1 h. Separately, a palmitic acid–Epikuron 200 mixture was homogenized and filled into 1.5 mL tubes, which were then replaced with C₃F₈ and oscillated for 120 s. The resulting NBs were combined with the DOX‑chitosan solution in an ice bath, chilled at –4 °C for 30 min, and then stabilized with Pluronic F68. Free DOX was removed by 30 kDa dialysis.

Physical Characterization

Size and zeta potential were measured by dynamic light scattering (DLS) and a Delsa Nano C analyzer. Morphology was visualized with fluorescence microscopy (×100 oil immersion) and transmission electron microscopy (TEM) after uranyl acetate staining.

Stability

NBs were stored at 4 °C (48 h) or room temperature (6 h) and re‑evaluated by DLS and optical microscopy. Stability in human serum (Seronorm™) was assessed after 6 h incubation at 25 °C.

DOX‑Loading Capacity

A UV‑Vis standard curve (480 nm) quantified encapsulated DOX. Encapsulation efficiency (EE) was calculated as EE = (A/B) × 100 % (A = DOX in NBs, B = initial DOX). Freeze‑drying and weighing yielded a loading capacity of 64.12 mg DOX g⁻¹ NB.

Ultrasound‑Mediated DOX Release

DOX release was monitored by dialysis (cutoff 12,000–14,000 Da) at 37 °C with or without US (1.0 W cm⁻², 20 kHz, 40 s). Samples were collected hourly for 24 h and quantified by UV‑Vis.

In Vitro Ultrasound Imaging

Using a GE LOGIQ E9 scanner, we recorded time‑intensity curves at MI 0.10, depth 4.5 cm, and 60‑s clips. Decibel values were plotted to assess contrast stability.

Cytotoxicity of Empty NBs

MCF‑7 cells were exposed to varying NB concentrations (0–30 %) with US at 0.5 W cm⁻² (30 s) or 1.0 W cm⁻² (30 s). Cell viability was measured 24 h later with a CCK‑8 assay (450 nm absorbance).

Intracellular DOX Uptake

Cells were treated with DOX‑NBs (20 %) or free DOX (equivalent concentration) for 1 h, with or without US (0.5 W cm⁻², 30/60 s). Post‑wash, cells were analyzed by flow cytometry (red fluorescence gate, 10,000 events).

Cell Proliferation and Apoptosis

After 24 h treatment, viability was assessed by CCK‑8. Apoptosis after 6 h was quantified by Annexin V‑APC staining and flow cytometry.

Statistical Analysis

Data represent mean ± SD from three independent experiments (p < 0.05). Analyses were performed with SPSS 18.0.

Results

Physico‑Chemical Characterization

DOX‑NBs exhibited a uniform spherical morphology (Fig. 1). DLS revealed an average diameter of 641 nm (P.I. 0.256) and a highly positive zeta potential of +67.1 mV, ensuring colloidal stability.

Stability and Loading Efficiency

NBs remained stable for 48 h at 4 °C; slight size increase occurred after 6 h at 25 °C in PBS or serum. Encapsulation efficiency was 54.18 % with a loading capacity of 64.12 mg g⁻¹.

DOX Release Profile

US exposure accelerated DOX release: 46.5 % released after 5 h versus 9.3 % without US. After 24 h, ~80 % of the drug was released under US, whereas only 19.4 % released without.

Ultrasound Imaging Stability

Time‑intensity curves (Fig. 5) confirmed sustained contrast enhancement and slow attenuation, indicating that DOX‑NBs provide reliable imaging under clinical US settings.

Biosafety of Empty NBs

Cell viability remained >80 % at 30 % NB concentration with 0.5 W cm⁻², 30 s US. Higher intensity or prolonged exposure modestly reduced viability, confirming low cytotoxicity of the NB formulation.

Enhanced DOX Delivery by US

Flow cytometry showed markedly higher intracellular DOX fluorescence in cells treated with DOX‑NBs + US compared to free DOX (p < 0.01). Longer US pulses (60 s) further increased uptake.

Improved Anti‑Cancer Efficacy

Viability assays revealed 21.0 % survival for DOX‑NBs alone, dropping to 3.1 % with 30 s US and 2.2 % with 60 s US. Free DOX alone yielded 6.4 % survival, while free DOX + US achieved 4.1 %. Annexin V analysis showed 45.7 % apoptosis with DOX‑NBs + US versus 4.4 % with free DOX.

Discussion

Breast cancer remains a leading cause of female cancer mortality. Doxorubicin’s cardiotoxicity necessitates targeted delivery systems. Chitosan‑based NBs, coated with Pluronic F68, combine favorable biocompatibility with efficient drug loading and US‑triggered release. Compared with lipid microbubbles or extracorporeal shock wave approaches, US‑guided chitosan NBs offer superior imaging capability, lower systemic toxicity, and enhanced tumor cell uptake via sonoporation.

Our data confirm that US irradiation promotes rapid DOX release from NBs and markedly enhances intracellular delivery and apoptosis. The high zeta potential ensures colloidal stability, while the natural origin of chitosan mitigates rapid clearance and immune activation.

Conclusions

We successfully engineered biocompatible chitosan nanobubbles that load doxorubicin efficiently, maintain ultrasound contrast, and demonstrate low intrinsic toxicity. Ultrasound stimulation triggers rapid drug release and significantly improves therapeutic efficacy in breast cancer cells, underscoring the potential of this platform for targeted, noninvasive chemotherapy.

Abbreviations

CCK‑8

Cell Counting Kit‑8

DMEM

Dulbecco’s Modified Eagle Medium

DOX

Doxorubicin hydrochloride

EE

Encapsulation Efficiency

NBs

Nanobubbles

US

Ultrasound