FePO4 Nanoparticles: Biocompatible Carriers for Doxorubicin Delivery, RNA Stabilization, and Enhanced Anticancer Activity

Abstract

FePO₄ nanoparticles combine biocompatibility, high bioavailability, magnetic responsiveness, and superior sensory properties, making them ideal for food fortification, biomedical imaging, and drug delivery. In this study, we engineered FePO₄ as a carrier for the anticancer agent doxorubicin (DOX), achieving an optimal drug loading of 26.81 ± 1.0 %. The resulting FePO₄-DOX particles exhibited uniform size distribution, concentration‑dependent biocompatibility (>70 % viability up to 80 µg/mL), and a dramatic 10‑fold increase in cytotoxicity compared to free DOX, with enhanced selectivity for cancer cells. Moreover, FePO₄ nanoparticles stabilized RNA and preserved mRNA translation activity, underscoring their potential as multifunctional delivery platforms for therapeutic nucleic acids. These findings highlight the versatility of FePO₄ particles for safe drug and RNA delivery, and their promise in food fortification applications.

Introduction

Among inorganic nanomaterials, iron‑based nanoparticles (Fe‑NPs) stand out for their magnetic properties, endogenous metabolism, and FDA‑approved applications in contrast imaging, drug delivery, and hyperthermia. FePO₄ nanoparticles, in particular, exhibit excellent biocompatibility, high oral bioavailability, and minimal organoleptic impact, making them attractive for iron‑deficiency anemia prevention through food fortification. Their uptake in intestinal cells occurs primarily via divalent metal transporter‑1 (DMT‑1), ensuring efficient absorption. Beyond imaging, FePO₄ offers a chemically simple, scalable synthesis route that yields monodisperse particles with controlled surface charge, enabling robust drug loading and nucleic acid stabilization. This work explores FePO₄ as a drug‑delivery vehicle for DOX and as a stabilizer for RNA, aiming to demonstrate a single platform that addresses both therapeutic and nutritional needs.

Results and Discussion

FePO₄ Nanoparticle Synthesis, Characterization, and Biocompatibility

FePO₄ was synthesized via a straightforward co‑precipitation of ammonium phosphate and iron(III) nitrate, followed by dispersion in DSPE‑PEG‑COOH to prevent aggregation. Dynamic light scattering revealed a hydrodynamic diameter of 175 ± 5 nm with a polydispersity index of 0.15 ± 0.01, indicating excellent size homogeneity. Zeta potential measurements showed a negative surface charge of −19.1 ± 8 mV, which reduces protein opsonization and prolongs circulation time. FTIR spectra confirmed the presence of PO₄³⁻ groups and absence of precursor residues, verifying product purity. MTT assays on mouse osteosarcoma K7M2 and fibroblast NIH/3T3 cells demonstrated concentration‑dependent viability, with >70 % cell survival up to 80 µg/mL, confirming the biocompatibility of FePO₄ NPs within a therapeutically relevant window.

FePO4 Nanoparticles: Biocompatible Carriers for Doxorubicin Delivery, RNA Stabilization, and Enhanced Anticancer Activity

Doxorubicin Loading and Enhanced Cytotoxicity of FePO₄-DOX

DOX was incorporated via co‑incubation with Fe(NO₃)₃ followed by precipitation with (NH₄)₃PO₄, yielding a loading efficiency of 26.81 ± 1.0 %. The Fe³⁺–DOX complex forms during this step, as confirmed by a color shift from yellow to brown and unchanged fluorescence emission (590 nm). Hydrodynamic size remained 187 ± 7 nm, while the surface charge shifted to −8.89 ± 5 mV, reflecting the cationic nature of the bound DOX. Cytotoxicity assays revealed a 10‑fold reduction in IC₅₀ values compared to free DOX (NIH/3T3: 0.248 µM vs. 2.61 µM; K7M2: 0.107 µM vs. 1.01 µM). Importantly, the FePO₄-DOX nanoparticles maintained >70 % viability at the equivalent FePO₄ concentrations, indicating that the enhanced cytotoxicity arises from Fe³⁺-DOX complexation rather than nanoparticle toxicity. The selective killing of cancer cells over fibroblasts was also amplified, with a 19 % difference in viability at 0.1 µM DOX equivalent.

Cellular Internalization of FePO₄-DOX Nanoparticles

Confocal microscopy of K7M2 cells revealed negligible uptake at 0.5–1 h, followed by pronounced intracellular fluorescence at 3 h, comparable to free DOX. The time‑dependent internalization profile demonstrates that FePO₄-DOX nanoparticles can effectively deliver DOX into cancer cells within a clinically relevant timeframe, while the magnetic properties of FePO₄ enable potential theranostic applications.

RNA Stabilization and Functional Expression

FePO₄ nanoparticles protect model RNA from hydrolysis and nucleases, as evidenced by intact bands on RNA agarose gel electrophoresis (RAGE) after incubation in water and serum. In contrast, copper and carbon nanotube nanoparticles accelerated degradation. Functional assays using luciferase‑encoding mRNA demonstrated that FePO₄ preserved translation efficiency, delivering a two‑fold increase in luminescence relative to control, whereas copper nanoparticles reduced expression. These results underscore FePO₄ as a robust RNA stabilizer that maintains biological activity, positioning it as a candidate for mRNA‑based therapeutics and vaccines.

Conclusion

FePO₄ nanoparticles, synthesized via a simple co‑precipitation and PEGylation strategy, form monodisperse particles (~175 nm) with a stable negative surface charge. They exhibit high biocompatibility (>70 % viability up to 80 µg/mL) and efficient DOX loading (26.8 %). FePO₄-DOX nanoparticles deliver enhanced cytotoxicity (10‑fold IC₅₀ reduction) and increased selectivity for cancer cells. The particles also stabilize RNA against hydrolysis and preserve mRNA translation activity, validating their dual role as drug and nucleic acid carriers. These attributes make FePO₄ nanoparticles a compelling platform for therapeutic delivery and for fortifying foods with iron and bioactive compounds.

Methods

Synthesis and Characterization of FePO₄ Nanoparticles

FePO₄ nanoparticles were produced by mixing equimolar solutions of ammonium phosphate (16 mg/mL) and iron(III) nitrate (8 mg/mL) under constant stirring, followed by centrifugation and washing to remove residual precursors. The resulting precipitate was dispersed in DSPE‑PEG‑COOH (10 % w/w) to generate stable nanoparticles. Size and zeta potential were measured by DLS; chemical identity was confirmed by FTIR.

Doxorubicin Loading

DOX (100 µg) was added to Fe(NO₃)₃ or (NH₄)₃PO₄ solutions before precipitation to achieve complexation with Fe³⁺. The resulting FePO₄-DOX particles were washed thrice and quantified by fluorescence spectroscopy (excitation 490 nm, emission 595 nm). Loading efficiency was calculated as ((DOX in NPs – DOX in supernatant)/initial DOX) × 100.

Biocompatibility and Cytotoxicity Assays

Mouse osteosarcoma K7M2 and fibroblast NIH/3T3 cells were seeded at 10,000 cells/well in 96‑well plates, treated with FePO₄ or FePO₄-DOX (20–600 µg/mL) for 48 h, and viability assessed by MTT (550 nm). Cell lines were also exposed to free DOX (0.05–5 µM) for comparative cytotoxicity.

Cellular Internalization

For confocal imaging, K7M2 cells (12,000 cells/well) were treated with 5 µg/mL DOX or FePO₄-DOX for 0.5, 1, and 3 h, fixed, stained with DAPI, and imaged on a Zeiss LSM‑700. DOX fluorescence (560 nm) was used to track intracellular uptake.

RNA Stability and Expression

Torula yeast RNA (1 mg/mL) was incubated with 20 µg/mL of Cu, FePO₄, Ag, CNT, or ZnO nanoparticles at 37 °C and monitored over time by RAGE. For serum stability, RNA was incubated in 10 % FBS/DMEM. Luciferase‑encoding mRNA was translated in rabbit reticulocyte lysate with or without 20 µg/mL FePO₄ or Cu nanoparticles, and luminescence was measured with a Biotek Synergy H1 reader.