Heparin-Superparamagnetic Iron Oxide Nanoparticles for Theranostic Applications

Abstract

In this study, superparamagnetic iron oxide nanoparticles (SPIONs) were engineered with an organic coating composed of low molecular weight heparin (LMWH) and bovine serum albumin (BSA), providing heparin-based nanoparticle systems (LMWH@SPIONs). The purpose was to merge the properties of the heparin skeleton and an inorganic core to build up a targeted theranostic nanosystem, which was eventually enhanced by loading a chemotherapeutic agent. Iron oxide cores were prepared via the co-precipitation of iron salts in an alkaline environment and oleic acid (OA) capping. Dopamine (DA) was covalently linked to BSA and LMWH by amide linkages via carbodiimide coupling. The following ligand exchange reaction between the DA-BSA/DA-LMWH and OA was conducted in a biphasic system composed of water and hexane, affording LMWH@SPIONs stabilized in water by polystyrene sulfonate (PSS). Their size and morphology were investigated via dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The LMWH@SPIONs' cytotoxicity was tested, showing marginal or no toxicity for samples prepared with PSS at concentrations of 50 µg/mL. Their inhibitory activity on the heparanase enzyme was measured, showing an effective inhibition at concentrations comparable to G4000 (N-desulfo-N-acetyl heparin, a non-anticoagulant and antiheparanase heparin derivative; Roneparstat). The LMWH@SPION encapsulation of paclitaxel (PTX) enhanced the antitumor effect of this chemotherapeutic on breast cancer cells, likely due to an improved internalization of the nanoformulated drug with respect to the free molecule. Lastly, time-domain NMR (TD-NMR) experiments were conducted on LMWH@SPIONs obtaining relaxivity values within the same order of magnitude as currently used commercial contrast agents.

Keywords: dopamine; heparanase; heparin; metastasis; paclitaxel; superparamagnetic iron oxide nanoparticles (SPION); theranostic; toxicity.

Scheme 1

A schematic protocol of SPION and LMWH/BSA condensation (top) and SPION and LMWH condensation (down).

Figure 1

Total (A) and partial (B) HSQC spectra of purification intermediates of DA-LMWH (blue) superimposed on a heteronuclear multiple-bond correlation spectrum (HMBC, pink) and on a ¹³C spectrum (green) projection.

Figure 2

DOSY spectrum of DA-LMWH. The polymer protons align at 1.1 × 10⁻¹⁰ m² s⁻¹. The free EDC protons show a faster speed.

Figure 3

TEM images of SPION1 (A,B), SPION3 (C,D), and SPION4 (E,F).

Figure 4

Cell viability assay of HeLa cells incubated with SPION1, SPION2, SPION3, CTRL1, and CTRL2 at different concentrations (i.e., A, B, C, and D respectively correspond to 50, 25, 10, and 1 µg/mL). Note: **** p < 0.0001 compared to untreated sample, n = 4 (two-way ANOVA with Dunnett’s test).

Figure 5

Cell viability of 4T1 cells incubated with SPIONs and free PTX at different concentrations (i.e., A, B, and C respectively correspond to 50, 10, and 1 µg/mL of vector for NP samples and 6.25, 1.25, and 0.13 µg/mL of PTX for free PTX samples). Normalized vs. untreated sample. Note: **** p < 0.0001, ** p < 0.01 vs. untreated sample, n = 4 (two-way ANOVA with Dunnett’s test).

Figure 6

Percentage variations in 4T1 cell viability after incubation with nanoformulated PTX versus free PTX at different concentrations (i.e., A, B, C respectively correspond to 6.25, 1.25, and 0.13 µg/mL of PTX). Note: **** p < 0.0001, *** p < 0.001, * p < 0.05 vs. free PTX sample at the same drug concentration (two-way ANOVA with Dunnett’s test).

Figure 7

The LMWH@SPIONs SPION3 and SPION4 inhibit the HPSE-mediated cleavage of the synthetic HS pentasaccharide fondaparinux. Dose–response inhibition of the heparanase-mediated cleavage of fondaparinux (Arixtra) by SPION3 and SPION4 vs. G4000 (SST0001 = Roneparstat). Assay performed as described in Materials and Methods.

Figure 8

The LMWH@SPIONs SPION3 and SPION4 effectively inhibit the heparanase-mediated liberation of H35S fragments from basement membrane H35SPGs. The compounds were first incubated with recombinant heparanase for 30 min at 4 °C followed by incubation (6 h, 37 °C) with the sulfate-labeled ECM in the absence (Hpa, 200 ng/mL) and presence of SST0001 (10 μg/mL), SPION3 (200 μg/mL), or SPION4 (200 μg/mL). Sulfate-labeled degradation fragments released into the incubation medium were subjected to gel filtration on Sepharose 6B as described in the ‘Materials and Methods’. HS degradation fragments are eluted in fractions 18–33.

Figure 9

The R₁ (left) and R₂ (right) values of both samples, plotted against the effective Fe ion concentration. The linear fitting is shown, from which the slopes r₁ and r₂ are extracted. Note that the scale of the r₂ graph is 10× the scale of the r₁ graph, since the r₂ relaxation is more intense by one order of magnitude. Table 4 reports the relaxometric parameters of SPION samples.