Dialysis‑Derived Tadpole and Sphere Hemin Nanoparticles: A 308‑Fold Solubility Boost for Iron Bioavailability

Abstract

Hemin, a highly bioavailable iron source, is limited by its negligible aqueous solubility. We report a simple dialysis‑based approach that yields two distinct hemin nanoparticle morphologies—tadpole‑shaped (≈200 nm head, 100 nm tail) and sphere‑shaped (50–100 nm). Both forms exhibit dramatically improved solubility, with sphere‑shaped particles achieving a 308‑fold increase at 25 °C compared to free hemin. The nanoparticles remain stable across acidic pH, high temperature, and ionic strength, and they display strong near‑infrared absorption, positioning them as promising candidates for food fortification, biomedical delivery, and photothermal therapies.

Introduction

Iron deficiency affects nearly 20 % of the global population, leading to anemia and reduced quality of life. While non‑heme iron from plant sources is readily available, its absorption is often suboptimal. Hemin—derived from hemoglobin and myoglobin—offers superior absorption (50–87 %) but suffers from hydrophobicity and poor solubility, hampering its practical use. Conventional strategies such as protein complexation, co‑crystallization with arginine, and cyclodextrin inclusion have improved solubility but involve complex procedures, limiting scalability.

Nanotechnology provides a compelling route to enhance the physicochemical properties of hydrophobic bioactives without altering sensory attributes. Nanoparticles can increase apparent solubility, improve bioavailability, and enable targeted delivery. Here, we develop a straightforward dialysis method to produce hemically distinct nanoparticles, evaluate their morphology, physicochemical stability, and solubility, and highlight their potential applications.

Materials and Methods

Materials

Hemin and 8–12 kDa dialysis membranes were sourced from Beijing Solarbio. Acetone (≥ 99.5 %) was obtained from Kant Chemical. All other reagents were analytical grade.

Preparation of Hemin Nanoparticles

Hemin (0.1 or 0.5 mg mL⁻¹) was dissolved in acetone acidified with 0.1 mL HCl. The solution was dialyzed against deionized water, with daily water changes, until freeze‑drying yielded nanoparticles. Parameters varied included hemin/water ratios (1:3, 1:5, 1:10, 1:50), temperature (4 °C or 25 °C), and dialysis duration (1–5 days).

Transmission Electron Microscopy (TEM)

Images were captured on a Hitachi 7700 TEM at 80 kV. Samples were drop‑cast onto carbon‑coated grids and freeze‑dried.

Dynamic Light Scattering (DLS)

Size, ζ‑potential, and PDI were measured with a Malvern Zetasizer Nano (0.05 % dilution in MilliQ water, 25 °C).

UV–Vis Spectroscopy

Absorption spectra (200–800 nm) were recorded on a TU‑1810 spectrophotometer. Concentrations were determined at 640 nm.

Solubility Assay

Supersaturated solutions of free hemin and nanoparticles were stirred at 25, 37, 60, and 80 °C (500 rpm, 30 min). Supernatants were centrifuged, diluted, and analyzed by UV–Vis.

Stability Tests

Nanoparticle suspensions (0.5 mg mL⁻¹) were exposed to pH 2–11, temperatures 25–80 °C, and NaCl (0–500 mM). Size, ζ‑potential, PDI, and turbidity were monitored overnight at 25 °C.

FTIR, Fluorescence, and XRD

FTIR spectra (400–4000 cm⁻¹) were collected with a Jasco Tensor 27. Fluorescence (excitation 402 nm, emission 300–600 nm) was measured on a Hitachi F‑7000. XRD patterns (2θ 4–40°) were obtained using a Bruker AXS D8 ADVANCE.

Results and Discussion

Formation and Morphology

At 0.5 mg mL⁻¹ hemin, sphere‑shaped nanoparticles (50–100 nm) emerged at a 1:10 hemin/water ratio after 3 days of dialysis. Higher ratios (1:50) produced rod‑like aggregates. In contrast, 0.1 mg mL⁻¹ hemin yielded tadpole‑shaped particles (≈200 nm head, 100 nm tail) under identical conditions. TEM confirmed monodisperse structures; DLS showed hydrodynamic diameters of 218 ± 6 nm (spheres) and 300 ± 8 nm (tadpoles). ζ‑potentials were −21 mV (spheres) and −11 mV (tadpoles), indicating good colloidal stability. PDI values (0.35–0.40) reflected narrow size distributions.

Optical Properties

UV–Vis spectra revealed a Soret band shift from 344 nm (free hemin) to 265 nm (nanoparticles), signifying enhanced π‑π conjugation. Both nanoparticle forms displayed strong near‑infrared absorption (≈660–770 nm), advantageous for photothermal and photoacoustic applications. Fluorescence emission peaked at 500 nm, higher than free hemin, likely due to increased solubility.

Solubility Enhancement

Free hemin solubility at 25 °C was 0.009 mg mL⁻¹. Sphere‑shaped nanoparticles reached 1.33 mg mL⁻¹, a 308‑fold increase. Tadpole‑shaped particles solubilized to 1.00 mg mL⁻¹. Solubility rose with temperature across all samples, confirming thermally activated dissolution.

Structural Confirmation

FTIR spectra displayed broadened N–H/O–H bands (3470 cm⁻¹) in nanoparticles, indicating stronger hydrogen bonding. XRD patterns of spheres retained the crystalline peaks of free hemin, whereas tadpoles showed reduced crystallinity (35.7 % vs. 56.7 % for free hemin), suggesting partial amorphization.

Stability

Nanoparticles remained stable under acidic pH (≤ 7) with minimal size and PDI changes. At alkaline pH (≥ 9), aggregation increased (> 400 nm). Thermal exposure up to 80 °C caused slight size growth but preserved colloidal integrity. High ionic strength (≥ 250 mM NaCl) led to dispersion destabilization.

Conclusions

We have successfully engineered tadpole‑ and sphere‑shaped hemin nanoparticles via a simple dialysis method, achieving a 308‑fold solubility enhancement at 25 °C. The particles demonstrate excellent acid and thermal stability and strong near‑infrared absorption, positioning them as versatile platforms for iron fortification, drug delivery, and photothermal therapies. Future research will focus on loading therapeutics and optimizing optothermal responses.