Spherical Gold–Cockle Shell Calcium Carbonate Nanoparticles: Fabrication, Characterization, and Cytotoxicity for Biomedical Use

Abstract

We report a simple, eco‑friendly synthesis of spherical gold‑conjugated cockle‑shell‑derived calcium carbonate nanoparticles (Au‑CSCaCO₃NPs) and assess their physicochemical properties and biocompatibility. Gold nanoparticles were produced by citrate reduction and coupled with aragonite CaCO₃ extracted from Anadara granosa shells via a dodecyl dimethyl betaine‑mediated precipitation. The resulting hybrid particles are 35 ± 16 nm in diameter, possess a negative surface charge (−16.4 mV), and display a characteristic LSPR peak at 530 nm. Cytotoxicity assays on MCF‑7 and NIH‑3T3 cells revealed >70 % inhibition of cancer cells at 100 µg mL⁻¹ while sparing fibroblasts, indicating selective biocompatibility. These findings suggest Au‑CSCaCO₃NPs as a promising platform for targeted drug delivery and imaging.

Background

Monodisperse nanoparticles are critical for advanced biomedical applications such as drug delivery, imaging, and biosensing. Gold nanoparticles (AuNPs) provide tunable optical properties, while biogenic calcium carbonate, especially the aragonite polymorph from cockle shells, offers excellent biocompatibility and high surface area. Conventional synthesis of CaCO₃ often yields non‑biogenic calcite/vaterite mixtures, unsuitable for clinical use. Our approach combines a scalable, cost‑effective gold reduction protocol with a dodecyl dimethyl betaine‑mediated precipitation of cockle‑shell CaCO₃, yielding a hybrid nanomaterial that retains the advantageous properties of both components.

Methods

Materials

Gold(III) chloride trihydrate, trisodium citrate, dodecyl dimethyl betaine (BS‑12), indocyanine green (ICG), and standard cell culture reagents were purchased from commercial suppliers. Fresh cockle shells were sourced locally, washed, dried, ground to <90 µm powder, and used directly for CaCO₃ synthesis.

Synthesis of AuNPs

Gold(III) chloride (0.1 % w/v) was reduced with 1 % trisodium citrate at 100 °C for 15 min, yielding a brilliant red colloid. The reaction follows: 2 HAuCl₄ + 3 C₆H₈O₇ → 2 Au + 3 C₃H₆O₅ + 8 HCl + 3 CO₂.

Preparation of Cockle‑Shell CaCO₃ Nanoparticles (CSCaCO₃NPs)

2 g of shell powder was dispersed in 50 mL deionized water with 0.5 mL BS‑12, stirred at 1000 rpm, 50 °C for 135 min. The precipitate was filtered, washed, dried, and milled for 5 days at 200 rpm to obtain monodisperse spherical CaCO₃ particles.

Conjugation to Form Au‑CSCaCO₃NPs

0.2 g CSCaCO₃NPs and 5 mg ICG were mixed with 20 mL AuNP colloid (pH 7). The suspension was sonicated (20 min) and stirred (200 rpm) for 3 days, then ultracentrifuged at 10,000 rpm to recover the conjugate, which was washed and dried.

Characterization

• Transmission Electron Microscopy (TEM): 19–51 nm spherical particles, mean 35 ± 16 nm.
• Field Emission SEM & EDX: morphology, elemental composition (C 65 %, O 13.5 %, Ca 0.02 %, Cu 17.6 %, Au 3.9 %).
• Fourier‑Transform IR: peaks at 1455, 1059, 854, 706 cm⁻¹ indicating carboxylate and aragonite signatures.
• UV‑Vis: LSPR peak at 530 nm.
• Zeta Potential: −16.4 ± 3.8 mV, PdI < 0.5.

Cell Culture & Cytotoxicity

MCF‑7 (breast adenocarcinoma) and NIH‑3T3 (mouse fibroblast) cells were cultured in DMEM with 10 % FBS. Cells were seeded in 96‑well plates (5 × 10³ cells/well) and exposed to Au‑CSCaCO₃NPs (25–100 µg mL⁻¹) for 24, 48, and 72 h. Cell viability was measured by MTT assay (570 nm).

Results and Discussion

Physicochemical Properties

The hybrid nanoparticles are uniformly spherical, with a narrow size distribution and strong negative surface charge, indicating good colloidal stability. The LSPR peak shift confirms successful gold conjugation.

Cytotoxicity

Au‑CSCaCO₃NPs inhibited >70 % proliferation of MCF‑7 cells at 100 µg mL⁻¹ while reducing NIH‑3T3 viability by only ~40 %. IC₅₀ values were 25–50 µg mL⁻¹ for cancer cells and >80 µg mL⁻¹ for fibroblasts, demonstrating selective cytotoxicity.

Conclusions

We have established a scalable, environmentally friendly route to produce spherical Au‑conjugated cockle‑shell CaCO₃ nanoparticles with controlled size, excellent stability, and selective anticancer activity. These hybrids are promising candidates for targeted drug delivery, imaging, and theranostic applications.