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

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 gFe/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.


Characterization
The NMR spectra were measured using a Bruker 400 MHz BBI spectrometer with deuterated DMSO as a solvent at 25 °C. Size-exclusion chromatography (SEC) measurements were performed using an Agilent 1260 Infinity quaternary LC system consisting of an Agilent 1260 Infinity quaternary pump (G1311B), autosampler (G1329B), two PLGel 5m MIXED-C columns (kept at 25 °C) and a refractive index detector (G1362A). THF (with BHT as inhibitor) was used as an eluent at a flow rate of 1 mL/min. The molar masses were determined using Agilent narrow molecular mass distribution polystyrene standards in THF. The particle sizes were characterized by dynamic light scattering (DLS) using a Malvern Instruments Zetasizer Nano series instrument.
An equilibration time of 120 seconds was allowed prior to each reading, and measurements were done in triplicate.
Transmission Electron Microscopy (TEM) images were recorded using a JEOL JEM 1011 electron microscope, equipped with a W thermionic electron source and a 11Mp Orius CCD Camera (Gatan company, USA), with an acceleration voltage of 100 kV. The samples were prepared by placing a drop of the sample onto a carbon coated copper grid, which was then left to be dried at ambient condition before being subjected to the microscope.

An elemental analysis was carried out via Inductively Coupled Plasma (ICP) Optical Emission
Spectroscopy on a ThermoFisher CAP 6000 series. The samples were prepared by digesting 10 μL of the sample in 1.0 mL of aqua regia overnight, followed by dilution with Milli-Q water to 10 mL. PhotoLuminescent (PL) spectra were measured using a Cary500 eclipse spectrometer, and the excitation wavelength was fixed at 480 nm. To determine the concentration of released dyes after MHT, a calibration curve was constructed using fluorescein sodium salt solution in water with different dye concentration ranging from 0.75 to 10.5 M. For doxorubicin loading and release experiment, solutions of Doxo-Mal in H2O/DMF (50% volume) having concentration from 1.0 to 40.0 (µg/mL) was used for the calibration curve.
FT-IR spectra were measured on a Bruker vertex 70v Fourier transform infrared spectrometer.
SAR measurements were carried out by a commercially available DM100 Series (NanoScale Biomagnetics Corp.) device. 150 μL of the sample ([Fe] = 3.0-4.0 g·L -1 ) was added to a cornical vial and exposed to an AC magnetic field at different field amplitudes and frequencies. The 4 measurement for each samples were performed in triplicate. SAR values were calculated according to the equation in which C is the specific heat capacity of the solvent (Cwater = 4185 J·L -1 ·K -1 , Cglycerol 81% = 2660J·L -1 ·K -1 ) 3-4 and m is the concentration (g·L -1 of Fe) of magnetic material in solution. As the measurements were carried out under non-adiabatic conditions, the slope of the curve was measured by taking into account only the first few seconds of the curve.

Synthesis of polymer having activated ester and PEG pendant (P(NSMA-co-PEGMA))
The copolymerization of PEGMA (Mw = 950 g·mol -1 ) and NSMA was carried out by taking advantage of the Photo-ATRP process. In a typical procedure, PEGMA (Mw = 950 g·mol -1 ) (7.6 g) was weighed in a 40 mL glass vial, and 8 mL DMF solvent was added. The monomer was dissolved by means of magnetic stirring for 30 min. After that, NSMA (2.18 g) was added and the solution was stirred for further 15 minutes to completely dissolve the monomers. Subsequently

Synthesis of polymer ligand having furfuryl, catechol and PEG pendant (PEG-CF)
In a 40 mL glass vial equipped with a magnetic stirring bar, 2.37 g of P(NSMA-co-PEGMA) was weighted, followed by the addition of 11.9 mL of DMF. After the polymer was completely dissolved, 460 mg of dopamine hydrochloride was added and left dissolved with the assistance of magnetic stirring for ca. 10 minutes. Next, a mixture containing 300.0 L of furfuryl amine and 443.0 L of TEA was successively added. Afterwards, the vial was covered by aluminum foil and left to vigorously stirrer for 24h at ambient condition. This solution was loaded in a dialysis bag (regenerated cellulose, molecular weight cut off -MWCOof 1000 Da) and dialyzed against 2 liters of diluted HCl solution (0.01 M). After 24h, the dyalized media was replaced with deionized water and the dialysis was kept for a duration of 48h (water was changed after every 12h). The final dialysate, recovered by the membrane, was lyophilized to yield 1.8 g of a brown solid of PEG-CF which was later characterized by 1 H NMR.

Synthesis of dyes-functionalized polymer ligand via thermal labile Diels-Alder adduct (PEG-CFluo)
In an 8 mL vial, 160 mg of PEG-CF was dissolved in 1.6 mL of DMF to form a clear and brown solution. To this vial, 25 mg of fluorescein maleimide was added and the vial was covered with aluminum foil, followed by the vigorously shaking by means of an orbital shaker at ambient condition. After 6 days, the reaction was stopped by addition of cold Et2O in excess (10 folds by volume with respect to DMF) to induce the precipitation of resulting polymer (PEG-CFluo). The supernatant upon the centrifugation (3500 rpm, 10 min) was discarded and the precipitate was dissolved in THF (5 mL) and precipitated again in an excess amount of cold Et2O (30 mL). The dissolution/precipitation in THF/Et2O was repeated for two more times before the final polymer pellet was dried in a vacuum oven set at 30 °C for 72 h. The obtained PEG-CFluo (175.0 mg) was characterized by 1 H NMR.

Synthesis of polymer ligand having furfuryl, catechol and PEG azide or PEG carboxylic acid pendants (PEG-CF-N3/ PEG-CF-COOH )
The synthesis of PEG-CF-COOH was carried out in a similar procedure used for PEG-CF with a minor modification. Briefly 0.5 g of P(NSMA-co-PEGMA) was weighted in a 40 mL glass vial,

Solvothermal synthesis of Fe3O4 nanocubes
Iron oxide nanocubes IONCs (edge size 17 nm) were prepared by solvothermal method accordingly to the procedure reported in the patent. 5

Water transfer of IONCs using a direct ligand exchange procedure
Oleic acid capped IONCs were transferred into water using PEG-CF in a similar procedure reported in our previous study. 1 Briefly, 0.25 mL of IONCs in CHCl3 (10.0 gFe.L -1 , 17.0 nm ± 2 nm) was diluted with 1.75 mL CHCl3 containing 50.0 mg PEG-CF. Here, catechol/nm 2 ratio was aimed to be 30. TEA (100 µL, 28 equivalent to catechol) was successively added. The vial was sealed and vigorously shaken overnight. In the next step, an excess amount of hexane (5 folds by volume) was added to precipitate the particles as a pellet and the supernatant was discarded.
Afterwards, 2 mL of THF was added to solubilize the particles pellet, followed by the addition of hexane (5 fold by volume) in excess to precipitate the particles again. The supernatant was discarded after centrifugation, and the dark precipitate obtained was dried with N2 flow for 30 min.

mL of MiliQ water was then added to disperse the particles, forming a black and milky solution.
This solution showed a quick response to a magnet (0.3 T) indicating the formation of some clusters. The free ligand was removed by magnetic decantation for three times and the obtained solution was characterized by DLS and TEM.

Water transfer of IONCs using a two-step (post) ligand exchange procedure
In this procedure, IONCs were first transferred in water using TMAOH following a procedure reported elsewhere in literature. 6 Initially, 500 L of IONCs (10.0 gFe·L -1 , 17 nm ± 2 nm) was precipitated in 7.5 mL of acetone in an 8 mL glass vial. The pellet of IONCs was separated using centrifugation (15 min, 4500 rpm) while the supernatant was discarded. To this nanocubes pellet,

Dyes release experiment
In a 1 mL glass vial, 500 L of IONCs-PEG-CFluo-COOH (0.5 g·L -1 or 1.0 g·L -1 ) was added. This vial was exposed for 10 minutes to an AMF of 16 kA.m -1 field intensity and 110 kHz frequency using a BioMangetic DM100 device. The temperature during MHT was monitor by an optical thermal probe. After the MHT, entire solution was loaded to a centrifugal filtration device (MWCO = 100 kDa) and spinned for 30 min at 4000 rpm. The downstream ( 400 L) was collected and subjected to photoluminescence (PL) measurement while the residue fraction of IONCs-PEG-CFluo-1 on top (50 L) was diluted with 450 L of water prior to the next cycle of exposure to the AMF for additional 10 minutes. This cycle was repeated for 3 times in total. Meanwhile, a control nanocube sample at the same concentration was left at room temperature and treated in the same manner with the only difference that this sample was not exposed to MHT. Also in this case, the experiment was repeated three times.

Doxorubicin release experiment
The