Clickable Polymer Ligand-Functionalized Iron Oxide Nanocubes: A Promising Nanoplatform for 'Local Hot Spots' Magnetically Triggered Drug Release

Abstract

Exploiting the local heat on the surface of magnetic nanoparticles (MNPs) upon exposure to an alternating magnetic field (AMF) to cleave thermal labile bonds represents an interesting approach in the context of remotely triggered drug delivery. Here, taking advantages of a simple and scalable two-step ligand exchange reaction, we have prepared iron oxide nanocubes (IONCs) functionalized with a novel multifunctional polymer ligand having multiple catechol moieties, furfuryl pendants, and polyethylene glycol (PEG) side chains. Catechol groups ensure a strong binding of the polymer ligands to the IONCs surface, while the PEG chains provide good colloidal stability to the polymer-coated IONCs. More importantly, furfuryl pendants on the polymer enable to click the molecules of interest (either maleimide-fluorescein or maleimide-doxorubicin) via a thermal labile Diels-Alder adduct. The resulting IONCs functionalized with a fluorescein/doxorubicin-conjugated polymer ligand exhibit good colloidal stability in buffer saline and serum solution along with outstanding heating performance in aqueous solution or even in viscous media (81% glycerol/water) when exposed to the AMF of clinical use. The release of conjugated bioactive molecules such as fluorescein and doxorubicin could be boosted by applying AMF conditions of clinical use (16 kAm^-1 and 110 kHz). It is remarkable that the magnetic hyperthermia-mediated release of the dye/drug falls in the concentration range 1.0-5.0 μM at an IONCs dose as low as 0.5 g_Fe/L and at no macroscopical temperature change. This local release effect makes this magnetic nanoplatform a potential tool for drug delivery with remote magnetic hyperthermia actuation and with a dose-independent action of MNPs.

Keywords: drug release; heat-mediated release; hot-spot effect; iron oxide nanoparticles; magnetic hyperthermia; multifunctional polymer.

Figure 1

Characterization of multidentate and functional polymers by ¹H NMR. ¹H NMR spectra (with the assignments of the characteristic peaks) of P(PEGMA-co-NSMA) (A); polymer precursor upon the reaction with NH₂-PEG-N₃, furfurylamine, and dopamine hydrochloride (PEG-CF-N₃) (B); and multifunctional PEGylated polymeric ligand (PEG-CDoxo-N₃) after the reaction between PEG-CF-N₃ and maleimide-derived doxorubicin (C); and (D) multifunctional PEGylated polymeric ligand (PEG-CFluo-COOH) after the reaction between PEG-CF-COOH and maleimide-derived fluorescein. The measurements were done using deuterated DMSO as the solvent.

Scheme 1. Representative Synthetic Approach To Prepare Multifunctional PEGylated Polymeric Ligands Using Activated Ester Methacrylate-Based Polymers and Diels–Alder Click Chemistry

Poly(polyethylene glycol methacrylate-co-N-succinimidyl methacrylate), P(PEGMA-co-NSMA), is used as a reactive precursor to introduce functional PEG, catechol, and furfuryl pendants by means of a one-pot aminolysis reaction, followed by the conjugation of biomolecules such as maleimide-derived fluorescein (Fluo) or maleimide-derived doxorubicin (Doxo) by Diels–Alder click chemistry.

Figure 2

Phase transfer of iron oxide nanocubes (IONCs) using a two-step ligand exchange. (A) Sketch represents the two-step phase transfer procedure involving first the transfer of IONCs from chloroform into water using tetramethylammonium hydroxide (TMAOH), followed by the postexchange in water of TMAOH with the developed ligands in basic solution, to yield physiologically stable IONCs. The dye/drug conjugated to the ligand platform via a thermal labile Diels–Alder adduct could be released by the local heat generated on the nanocube surface during MHT, as illustrated in the inset. FT-IR spectra of surface modification of IONCs for each step of the water transfer protocol (B) and in the extended region of interest from 1000 to 1900 cm^–1 (C). Dynamic light scattering (DLS) traces of water-soluble IONCs modified with TMAOH (green), PEG-CF (blue), PEG-CFluo-COOH (red), and PEG-CDoxo-N₃ (deep red) weighted by intensity (D). TEM images of IONCs functionalized with TMAOH (E), or with PEG-CF (F), or with PEG-CFluo-COOH (G), or with PEG-CDoxo-N₃ (H) deposited from water.

Figure 3

Stability of IONCs functionalized with multidentate and functional polymer ligands in physiological conditions. DLS traces of IONCs modified with PEG-CDoxo-N₃ (A) or with PEG-CF-N₃ (B) dispersed in complete cell culture media at 10% fetal bovine serum at day 0 and after 2, 5, and 8 days of storage at ambient conditions. The insets show the vials, as observed under visible light for the culture media (1) and for IONCs modified with either PEG-CDoxo-N₃ or PEG-CF-N₃ ligands, respectively (2).

Figure 4

Heating capability of IONCs in water and viscous media. (A) Specific absorption rate (SAR) values in water of IONCs having different surface ligands. (B,C) Comparison of the SAR value of IONCs functionalized with PEG-CFluo-COOH in water and viscous media (glycerol 81%) measured under the MHT conditions with respect to the biological limit (H·f < 5 × 10⁹ A·m^–1·s^–1). The values reported in panel 4B were measured at 110 kHz, with the field varied from 16 to 40 kA·m^–1.

Figure 5

Release of dye molecules by means of MHT-induced local (hot-spot) heat effect. Heating profiles of IONC-PEG-CDoxo-N₃ (A) and IONC-PEG-CFluo-COOH (D) solution in water at different Fe concentrations (0.5 and 1.0 g/L) and control solution (only water) under MHT (16 kA·m^–1 and 110 kHz) during the first 10 min of MHT. We observed that the maximum temperature was reached after 10 min; thus, further heating profiles are not shown. The comparison of the PL signal of Doxo (B) and fluorescein sodium salt (E) between the samples kept on bench and the one undergoing MHT (110 kHz, 16 kA·m^–1) at the Fe concentration of 0.5 gFe/L (at different durations of MHT). The normalized concentration of Doxo (C) and fluorescein sodium salt (F) released upon MHT (16 kA·m^–1 and 110 kHz) at Fe concentrations of 0.5 gFe/L.