Nanoalginate Carriers via Inverse‑Micelle Synthesis: Doxorubicin Encapsulation and Cytotoxicity in Murine Breast Cancer Cells

Abstract

Cross‑linked alginate nanoparticles are an attractive platform for drug encapsulation and delivery. Here we report a simple inverse‑micelle protocol that loads water‑soluble doxorubicin (DOX) into a calcium‑cross‑linked alginate matrix, yielding NALG‑DOX particles with a hydrodynamic diameter of ~83 nm and a zeta potential of +7.2 mV. The resulting nanoalginate (NALG) particles were spherical and monodisperse, as confirmed by TEM. DOX release was controlled, with ~70 % retained after 4 h and ~90 % released by 24 h at physiological pH. Fluorescence imaging confirmed cellular uptake of DOX from NALG‑DOX in 4T1 murine breast cancer cells. Cytotoxicity assays (LIVE/DEAD and Alamar Blue) revealed an IC₅₀ of 0.45 µg/mL for NALG‑DOX versus 0.093 µg/mL for free DOX, while the empty NALG carrier showed no toxicity. These results demonstrate the potential of the inverse‑micelle method for producing biocompatible, drug‑loaded alginate nanoparticles.

Background

Encapsulation of chemotherapeutics offers advantages over systemic free drug administration, including prolonged circulation, protection from plasma proteins, and reduced systemic toxicity. Nanocarriers can exploit the enhanced permeability and retention (EPR) effect of solid tumors, enabling passive targeting when appropriately sized. While liposomes, polymeric particles, and lipid nanoparticles have entered clinical practice, many novel formulations face challenges in scalable preparation and reproducibility. Alginate, a naturally derived, biodegradable, and non‑immunogenic polysaccharide, can be cross‑linked by divalent cations (typically Ca²⁺) to form hydrogel nanoparticles. This study presents a rapid, room‑temperature inverse‑micelle synthesis that produces uniform alginate nanoparticles capable of encapsulating hydrophilic drugs like DOX.

Methods

Materials

Sodium alginate, calcium chloride dihydrate, cyclohexane, dodecylamine, and DOX hydrochloride were sourced from Sigma‑Aldrich and MedChem Express. All solutions were prepared in 18 MΩ water.

Preparation of Nanoalginate (NALG)

Alginate (15 mg/mL) was stirred for 30 min to ensure full dissolution. For DOX loading, the drug was dissolved in the same aqueous phase. Eight milliliters of cyclohexane were placed in a vial, and 80 µL of dodecylamine (solubilized in warm water) was added to generate the oil phase. The aqueous alginate/DOX solution (20 µL) was then introduced under vigorous stirring (1,200 rpm) for 20 min, followed by addition of 30 µL of 50 mM CaCl₂ to cross‑link the alginate within the inverse micelles. After 25 min, 2 mL of water was added to separate the aqueous phase, from which the nanoparticles were recovered by pipette.

Purification

The aqueous extract was sequentially filtered through 100 kDa and 10 kDa centrifugal units (Pall, Millipore) to remove aggregates and free drug, yielding a final volume of 1 mL of purified NALG or NALG‑DOX.

Characterization

Dynamic light scattering (Malvern Zetasizer) measured size (633 nm laser, 25 °C, 173° backscatter). Zeta potential was recorded immediately thereafter. Transmission electron microscopy (TEM) on a Tecnai F‑20 (4 kV) confirmed morphology. DOX content was quantified by its intrinsic fluorescence (excitation 470 nm, emission 550 nm) and compared to a standard curve.

Release Study

Dialysis (Slide‑A‑Lyzer MINI, 2 kDa MWCO) in PBS (pH 7.4) and citrate buffer (pH 5.5) assessed DOX release at 1, 2, 4, 24, 48, and 72 h. Fluorescence readings were used to calculate released DOX concentration.

Cell Culture and Treatment

4T1‑luc2‑GFP murine breast cancer cells (PerkinElmer) were cultured in RPMI 1640 + 10 % FBS + 1 % penicillin/streptomycin. Cells were seeded at 4,000 cells/well (96‑well plate) and treated after 24 h with NALG, free DOX, or NALG‑DOX at varying concentrations.

Fluorescence Imaging

After 48 h, cells were fixed with formalin, stained with NucBlue, and imaged on an EVOS FL Auto (ThermoFisher) using DAPI, GFP, and DOX channels. Images were processed with ImageJ.

Live/Dead Assay

At 72 h, cells were incubated with NucBlue Live and NucGreen Dead reagents (ThermoFisher), fixed, and imaged as above. Green fluorescence indicated dead cells.

Alamar Blue Viability Assay

Alamar Blue (10 µL per well in 100 µL media) was added at 72 h, incubated 1 h at 37 °C, and read on a Tecan Infinite M200 Pro (560/590 nm). Cell viability was expressed as a percentage relative to untreated controls.

Results and Discussion

Inverse‑Micelle Synthesis

The inverse‑micelle approach produced transparent aqueous colloids of NALG and NALG‑DOX. Cyclohexane served as the oil phase, while dodecylamine functioned as the surfactant to stabilize alginate chains within micelles. Calcium chloride cross‑linking formed the hydrogel network, and subsequent aqueous extraction yielded monodisperse particles.

Particle Characterization

DLS data showed NALG at 92.2 ± 4.2 nm (PDI 0.320 ± 0.063, ζ –15.0 ± 0.8 mV) and NALG‑DOX at 82.8 ± 3.6 nm (PDI 0.204 ± 0.044, ζ +7.2 ± 4.6 mV). TEM images confirmed spherical morphology with diameters matching DLS measurements. DOX loading was ~7 % (≈4 µg/mL), indicating moderate encapsulation efficiency typical for polymeric carriers.

Release Kinetics

In PBS (pH 7.4), ~70 % of DOX remained after 4 h, with ~90 % released by 24 h. At pH 5.5, release accelerated, reflecting the acidic tumor microenvironment and intracellular compartments, and enabling a pH‑responsive drug release profile.

Cellular Uptake

Fluorescence microscopy after 48 h revealed DOX localized near the nucleus in both free and NALG‑DOX treatments. Encapsulated DOX displayed a distinct perinuclear distribution, consistent with endocytic uptake and subsequent release in the acidic endosome.

Cytotoxicity

LIVE/DEAD imaging showed minimal cell death with NALG alone, whereas DOX and NALG‑DOX induced dose‑dependent cytotoxicity. Alamar Blue analysis yielded IC₅₀ values of 0.093 µg/mL for free DOX and 0.45 µg/mL for NALG‑DOX. The higher IC₅₀ for NALG‑DOX reflects the controlled release and reduced immediate drug availability, yet the encapsulated drug remains efficacious.

Conclusions

We have established a rapid, scalable inverse‑micelle method to generate ~90 nm alginate nanoparticles that efficiently encapsulate DOX. The resulting NALG‑DOX shows controlled release, reduced systemic toxicity, and comparable antitumor activity to free DOX in vitro. Future work will focus on optimizing encapsulation efficiency and in vivo performance.