Magnetite Nanocluster-Based Theranostic Agents for T2‑Weighted MRI and pH‑Responsive Doxorubicin Delivery

Abstract

Multifunctional nanostructures that merge anticancer drugs with inorganic nanocrystals are emerging as powerful platforms for tumour diagnosis and therapy. In this study, we fabricated magnetite (Fe₃O₄) nanoclusters (NCs) by combining an oil‑in‑water microemulsion and a layer‑by‑layer (LBL) assembly. The Fe₃O₄ NCs were first assembled from oleic‑acid‑capped Fe₃O₄ nanoparticles (NPs). Sequential adsorption of poly(allylamine hydrochloride) (PAH), poly(sodium 4‑styrenesulfonate) (PSS), and doxorubicin hydrochloride (DOX) yielded Fe₃O₄ NC/PAH/PSS/DOX hybrid nanostructures. These carriers exhibit pH‑responsive DOX release, enhanced cytotoxicity against A549 lung‑carcinoma cells, and superior T₂-weighted MRI contrast (r₂ = 651 mM⁻¹ s⁻¹). Fluorescent DOX emission (λ_ex = 490 nm) facilitates cellular imaging. Our work establishes a robust Fe₃O₄-based theranostic platform that integrates MRI, fluorescence imaging, and controlled drug delivery.

Introduction

Recent advances in multifunctional drug delivery systems have enabled simultaneous imaging and therapy, particularly through stimuli‑responsive designs that exploit tumour microenvironmental cues such as acidity (pH ≈ 6.5–6.8). Magnetite nanoparticles (Fe₃O₄) are especially attractive for biomedical applications due to their superparamagnetism, biocompatibility, and low cytotoxicity. However, single Fe₃O₄ NPs often suffer from low magnetic responsiveness and limited drug loading. Assembly of Fe₃O₄ NPs into nanoclusters (NCs) amplifies collective magnetic effects, enhancing T₂ relaxivity and enabling higher drug payloads when combined with polyelectrolyte multilayers. Despite these advantages, few studies have reported Fe₃O₄ NCs functionalized with PAH/PSS for pH‑triggered drug release.

Here, we report a scalable fabrication route for Fe₃O₄ NC/PAH/PSS/DOX hybrid nanostructures, combining a microemulsion self‑assembly and LBL electrostatic adsorption. The resulting carriers deliver DOX with controlled, pH‑dependent release, demonstrate strong T₂ MRI contrast, and efficiently kill A549 cells in vitro.

Materials and Methods

Materials

FeCl₃·6H₂O (99.99 %), FeCl₂·4H₂O (99.99 %), oleic acid (OA, 90 %), 1‑octadecene (ODE, 90 %), sodium oleate (NaOA), ethanol, hexane, cyclohexane, isopropanol, sodium dodecyl benzene sulfonate (SDBS), ammonium fluoride (NH₄F), NaOH, DMSO, and ammonia were purchased from Alfa Aesar and Sinopharm. PAH (MW 15 kDa), PSS (MW 70 kDa), and doxorubicin hydrochloride (DOX, > 98 %) were sourced from Sigma‑Aldrich and Shanghai Sangon Biotech. APMI 1640 medium and fetal bovine serum (FBS) were obtained from Hyclone. All reagents were used without further purification.

Preparation of Ferric Oleate

FeCl₃·6H₂O (2.59 g), NaOA (14.6 g), ethanol (32 mL), H₂O (24 mL), and hexane (56 mL) were mixed in a 150 mL flask and refluxed at 70 °C for 4 h to form a transparent ferric oleate solution. The oil layer was separated, hexane removed by rotary evaporation at 70 °C, and the residue stored under vacuum.

Synthesis of Fe₃O₄ NPs

Ferric oleate (7.2 g), OA (1.28 mL), and ODE (50 mL) were heated to 300 °C for 40 min under Ar; the mixture was then cooled and oxidized in air for >12 h. Nanocrystals were precipitated with isopropanol, washed twice with ethanol–water (1:1 v/v), and dispersed in 200 mL cyclohexane.

Preparation of Fe₃O₄ NCs

200 µL of Fe₃O₄ NPs in cyclohexane was added to 4 mL aqueous SDBS (14 mg). Ultrasonic treatment (5 min × 4) formed a solid‑in‑oil‑in‑water emulsion. Stirring at room temperature for 6 h evaporated cyclohexane, allowing self‑assembly into quasi‑spherical NCs. The product was washed three times with deionized water.

Preparation of Fe₃O₄ NC/PAH/PSS/DOX Hybrid Nanostructures

Fe₃O₄ NCs (3 mL) were first coated with PAH (10 g/L, 4 mM NaCl) by dropwise addition and stirring for 24 h; excess PAH was removed by centrifugation. The PAH‑coated NCs were then coated with PSS (10 g/L, 4 mM NaCl) under identical conditions, followed by centrifugation. DOX (5 mg/mL) was added (60 µL) to the Fe₃O₄ NC/PAH/PSS solution (3 mL) and stirred for 24 h in the dark. The final Fe₃O₄ NC/PAH/PSS/DOX hybrid nanostructures were isolated by centrifugation.

MRI Measurements

Hybrid nanostructures were dispersed in 1.2 mL agarose (1 % w/v) at Fe concentrations ranging from 0 to 0.065 mM. Imaging was performed on an 11.7 T Bruker micro‑MRI system (TR = 300 ms, TE = 4.5 ms, 128 × 128 matrix, 1.2 mm slice, 2.0 × 2.0 cm FOV, 2 averages).

Cellular Uptake and MR Imaging

A549 cells were incubated with hybrid nanostructures (Fe = 0–13.5 µM) for 2 h, washed, and imaged by T₂-weighted MRI.

Standard Curve of DOX

DOX fluorescence (λ_ex = 490 nm) was measured for concentrations 0–0.03 mg/mL. The linear regression gave Y = 447.44 + 69,745 X (R² = 0.9992).

DOX Loading and Release

Loading efficiency was calculated from the difference between initial DOX mass (W₀) and the mass in the supernatant (W_s) after centrifugation. Cumulative release was monitored in PBS (pH 5.0 or 7.4) at 37 °C using a dialysis bag, sampling 100 µL every 1–3 h.

In Vitro Cytotoxicity

A549 cells were seeded in 96‑well plates (8 × 10³ cells/well) and treated with free DOX, Fe₃O₄ NC/PAH/PSS, or Fe₃O₄ NC/PAH/PSS/DOX (0.1–2.0 µM DOX equivalents). After 24 h, MTT (5 mg/mL) was added, incubated 4 h, and absorbance measured at 490 nm.

Characterization

Transmission electron microscopy (TEM, 200 kV) assessed morphology; dynamic light scattering (DLS) measured hydrodynamic size and ζ‑potential; UV–vis spectroscopy determined DOX loading; fluorescence spectroscopy monitored DOX release; ICP‑AES quantified Fe content.

Results and Discussion

Nanostructure Morphology and Size

TEM images revealed that Fe₃O₄ NCs are quasi‑spherical aggregates (~57 nm, DLS) formed by self‑assembly of 15‑nm primary Fe₃O₄ NPs. Layer deposition (PAH, PSS, DOX) was confirmed by ζ‑potential shifts: −19.7 mV (NC), +32 mV (NC/PAH), −34 mV (NC/PAH/PSS), +1.9 mV (NC/PAH/PSS/DOX). The final hybrid nanostructures exhibited a monodisperse size of ~84 nm (DLS).

Enhanced T₂ MRI Contrast

Transverse relaxivity (r₂) was 651.38 mM⁻¹s⁻¹, surpassing many commercial agents. Linear 1/T₂ versus Fe concentration confirmed efficient T₂ contrast.

DOX Loading and pH‑Responsive Release

UV–vis and fluorescence spectra confirmed DOX adsorption. The loading efficiency reached 24.4 %. In PBS (pH 7.4) only ~20 wt% of DOX was released over 30 h, whereas at pH 5.0 ~80 wt% was released within 15 h, demonstrating strong pH responsiveness driven by protonation of DOX and reduced electrostatic binding.

Cellular Uptake and Cytotoxicity

Confocal microscopy showed time‑dependent internalization of DOX‑loaded hybrids, with strong red fluorescence after 24 h. MTT assays revealed negligible cytotoxicity of Fe₃O₄ NC/PAH/PSS alone (cell viability > 85 % at 2 µM). Free DOX and DOX‑loaded hybrids exhibited dose‑dependent killing of A549 cells (IC₅₀ ≈ 1–2 µM). Enhanced cytotoxicity of hybrids is attributed to sustained, endosomal pH‑triggered DOX release.

Cellular MRI

Fe₃O₄ NC/PAH/PSS/DOX hybrids internalized into A549 cells and produced significant T₂ signal reduction, confirming their utility as cellular MRI contrast agents.

Conclusion

We have engineered Fe₃O₄ NC/PAH/PSS/DOX hybrid nanostructures that combine high T₂ MRI contrast, pH‑triggered DOX release, and potent antitumour activity in vitro. Their modular design allows further functionalization (e.g., targeting ligands) for in vivo applications, positioning them as versatile theranostic platforms for cancer imaging and therapy.