Controlled release of sunitinib in targeted cancer therapy: smart magnetically responsive hydrogels as restricted access materials

Abstract

The aim of the present work was the preparation of a magnetic hydrogel for guided release of sunitinib malate (SUM), an anticancer drug. Precipitation polymerization method has been employed in order to synthesize the hydrogel, using methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), glycidyl methacrylate (GMA) and 2,2′-azobisisobutyronitrile (AIBN) as monomer, cross-linker, pro-hydrophilic monomer and initiator, respectively. To confer magnetic responsiveness, particles of magnetite have been encapsulated during the polymerization process, followed by epoxide ring opening on the nanospheres' surface, in order to obtain a restricted access material (RAM) with low protein adsorption ability and improved biocompatibility. The successful introduction of magnetite has been confirmed through FT-IR and TG analyses, while protein adsorbing tests have been conducted to verify the RAM features. Furthermore, swelling properties have been evaluated before and after epoxide ring opening. Finally, in vitro tests have been performed to evaluate the release profile and cytotoxic effect on ARO, WRO, HeLa and MCF-7 cell lines.

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Introduction

Sunitinib (SUT) is a multi-targeted receptor tyrosine kinase (RTK) inhibitor that blocks vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), FMS-like tyrosine kinase-3 (FLT3), glial cell-line derived neurotrophic factor receptor (RET) and colony-stimulating factor 1 receptor.¹ SUT is approved by the FDA (Sutent®) as an anticancer drug, orally administered in the form of sunitinib malate (SUM), for the treatment of gastrointestinal stromal tumors (GIST),² advanced renal cell carcinoma (RCC)³ and progressive, well-differentiated pancreatic neuroendocrine tumors (pNET).⁴ SUT has also been used in a few clinical trials including a limited number of patients with progressive differentiated thyroid carcinoma (DTC) with encouraging results. Furthermore, multi-kinase inhibitors have been included in clinical practice guidelines for advanced metastatic thyroid cancer.⁵ Sunitinib indeed decreases the growth rate of various malignant cell lines and induces differentiation and apoptosis in diverse carcinomas, including thyroid cancer.^6–8

This proves its wide application in many kinds of tumoral diseases, due to its unspecific and unselective activity towards human tissues. For this reason, concentrating efforts in the study and development of new materials able to confer selectivity towards cancerous tissues to antineoplastic compounds is important. Cancer therapy is indeed always accompanied with adverse and undesirable systemic toxic effects. Furthermore, anticancer drugs suffer of low solubility in aqueous solution, narrow therapeutic index and limited penetration into cytoplasmic area.

The purpose of the present study was the development of a novel magnetic nanospherical hydrogel, in order to achieve a guidable and biocompatible system for targeted SUM release. Hydrogels are known to be, by definition, polymeric materials organized in hydrophilic networks capable of a great retention of water. Hydrophilic groups, such as –OH, –CONH–, –CONH₂ and –SO₃H, are responsible of their high affinity for aqueous and biological media. The water absorbing capability confers to the polymer a hydration degree up to 90%, without dissolution of the material.⁹ Hydrogels are extensively used as delivery systems for chemotherapeutic agents in cancer therapy,^10,11 often able of a stimuli-triggered release.^12,13 However, today, guidable devices towards the site of interest are of great importance, aiming to non-systemic effects and limiting cytotoxicity to cancer cells. For this reason, encapsulation of magnetite in hydrogel network is a good strategy to obtain a magnetic guidance.¹⁴

Polymers are advantageous for hydrogel synthesis, thanks to the possibility of manipulation of their physico-chemical properties. It is indeed possible to achieve micro- or nano-sized polymeric hydrogel, modifying the polymerization conditions.

Microgels and nanogels have been already reported as delivery vehicles in cancer therapy.^15,16 In particular nanogels are interesting nanoscale networks that provide several advantages inherent to their nanometric size.

Magnetically responsive micro- and nanogels have been previously prepared.^17,18 However, polymeric materials can strongly adsorb on their surface biological components, such as lipids and proteins.¹⁹ Different kinds of proteins compete in vivo with each other to surfaces causing the so-called Vroman effect.²⁰ These undesired interactions, occurring in biological fluids, are responsible of decreased biocompatibility and bioavailability, causing a reduction of therapeutic efficacy and possible thrombotic episodes.^21,22 To avoid the interference of these biological substances many strategies could be carried out.^23,24 Therefore, the purpose of this work was also the synthesis of a restricted access material (RAM) to prevent these non-specific interactions. RAMs are supports designed to limit the access into inner core only to small molecules, excluding biological macromolecules, thanks to a chemical barrier created on polymeric surface.²⁵ In order to achieve the RAM system, glycidyl methacrylate has been used for the insertion of hydrophilic moieties on polymers outer surface, responsible of the reduced adsorption of proteins.²⁶

Therefore, the purpose of the present work was the development of a hybrid material in which multiple characteristics co-exist in the same system. The magnetic responsiveness of nanogel is, indeed, combined with the restricted accessibility.

Thus in this paper the synthesis of magnetic nanospherical hydrogel as RAM for guided release of sunitinib malate is reported and the nanospheres obtained are characterized. Also in vitro toxicity of several cellular lines has been evaluated.

Results and discussion

Synthesis of the magnetic nanospheres

Magnetic nanospheres (MNs) have been synthesized through precipitation polymerization, i.e. a heterogeneous technique that leads to uniform beads, without adding surfactant agents.²⁷ The particles have been found to be regular in size and shape, with no other treatments (Fig. 1).

Fig. 1 Scanning electron micrograph of magnetic nanospheres.

In the polymerization environment monomers (methacrylic acid, MAA), cross-linkers (ethylene glycol dimethacrylate, EGDMA), pro-hydrophilic monomers (glycidyl methacrylate, GMA) and initiator (2,2′-azobisisobutyronitrile, AIBN) are present, dissolved in toluene/acetonitrile mixture. The magnetite particles (Fe₃O₄) are, instead, dispersed in this system. The first step of the process is the formation of radical oligomers by action of radical species originated from initiator molecules by UV photo-triggering (λ = 360 nm). Oligomers crosslink simultaneously surrounding the magnetite particles, serving as seeds for nanospheres formation. After the nucleation phase, the second step is represented by microgels growth that produces uniformly sized spheres (Fig. 2). In this second phase of the reaction, the number of particles are constant, while size is increasing,^27,28 continuously capturing oligomers and preventing any further nucleation.

Fig. 2 Schematic representation of magnetic nanospherical hydrogels synthesis.

The grown beads are, in the end, separated from the continuous phase by enthalpic or entropic precipitation, if repulsive interactions between polymer and solvent occur or positive interactions are avoided, respectively.²⁸

Dimensional characterisation of the magnetic nanospheres

Size, size distribution and morphology of MNs depend on monomers, cross-linkers and initiator concentrations. The mean diameter increases as the concentrations of monomer or initiator increase,^27,28 but it decreases as the concentration of cross-linker increases.²⁹

The ideal MAA : GMA : EGDMA ratio for the formation of nanospheres has been found to be 8 : 8 : 10, resulting in spherical nanoparticles with a mean diameter of 170 nm, in the dry-collapsed form (Table 1), as confirmed by scanning electron microscope image (Fig. 1). In addition, the dimensional data obtained from the analysis performed with the stereomicroscope agree with the SEM image information about the narrow size distribution (Table 1).

Table 1 Dimensional data. MNs size and size distribution of both dry-collapsed and hydrated-swollen MNs are expressed as mean diameter ± standard deviation and polydispersity index (P.I.)

Magnetic nanospheres	Mean diameter	Polydispersity index
Dry MNs (collapsed)	170 ± 11 nm	0.256
Hydrated MNs (swollen)	287 ± 15 nm	0.270

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Moreover, dimensional evaluation of hydrated MNs has been performed with dynamic light scattering, putting a small amount of MNs in a phosphate buffer saline solution at pH 7.4. In this case the mean diameter was 287 nm (Fig. 3). Data, collected in Table 1, show two different size for the two different states of MNs. This could be easily ascribed to the volume of water within the MNs in the swollen state, absent, instead, in the dry-collapsed form. In both cases polydispersity index is <0.3 and mean diameter is below 300 nm.

Fig. 3 Graph of size and size distribution obtained by DLS analysis.

The importance of obtaining monodisperse spherical nanoparticles for anticancer drugs delivery is validated by the concept of the enhanced permeability and retention (EPR) effect. EPR effect is described as the capability of cancer tissue of being selectively targeted by nanosized particles that are then retained in the site. The explanation of this effect rely on increased vascularization, defective permeability of endothelium and absence of lymphatic drainage in cancer tissue.³⁰

Composition of the magnetic nanospheres

Evaluation by FT-IR analyses has been performed in order to verify the incorporation of magnetite, comparing the FT-IR spectra of Fe₃O₄, magnetic hydrogel (synthesized in presence of magnetic particles) and of the same polymer synthesized in absence of magnetite. The Fe₃O₄ spectrum shows the typical band at 588 cm⁻¹ of the Fe–O bond (Fig. 4). The same band is observable in the magnetic polymer spectrum, confirming the incorporation of magnetite, while it is not present in the non-magnetic polymer spectrum.

Fig. 4 FT-IR spectra; (A) magnetite; (B) nanospheres synthesized in absence of magnetite; (C) nanospheres synthesized in presence of magnetite.

Thermogravimetric analyses (TGA) have been carried out at the range of 30 to 800 °C to determine the amount of magnetite encapsulated in MNs (Fig. 5). In case of magnetite weight loss around 3.5% was due to the physically adsorbed water molecules on the magnetic particles surface (Fig. 5 – black line).

Fig. 5 The TGA curves of () magnetite, () magnetic nanospheres, () non magnetic nanospheres.

The TGA curve of MNs showed a weight loss of 92.3% associated with the complete thermal decomposition of the polymeric material (Fig. 5 – red line). In fact, the TGA curve of non magnetic nanospheres showed a complete decomposition at 800 °C (Fig. 5 – blue line). The residual weight at 800 °C in the TGA curve of MNs could be easily ascribed to the presence of magnetite. The remaining weight at 800 °C was about 7.7% of the amount of MNs used for the experiment i.e. the percentage of magnetite encapsulated in MNs.

Magnetic properties

For the evaluation of magnetic properties of magnetite and magnetic hydrogel, the two materials have been exposed to a magnetic field (H) at 300 K using a VSM and the magnetization (M) has been recorded during the hysteresis loops. The graph of H versus M and a picture of the separation of nanospheres dispersed in water media using a magnet are shown in Fig. 6.

Fig. 6 Hysteresis loops with pictures of MNs separation in presence of a magnet.

As seen in Fig. 6, MNs have been found to be responsive to the magnetic field. The lower response, compared to magnetite, is supposed to be caused by the shielding of the Fe₃O₄ particles by the surrounding polymeric matrix.

The confirmed magnetic property of Fe₃O₄-encapsulated beads, together with the passive targeting of nanospheres based on EPR effect, is the other characteristic exploitable for selective drug delivery. It is, indeed, possible to guide the magnetic nanospheres, applying an appropriate magnetic field, towards a specific target, i.e. cancer site.

Protein adsorption measurement and swelling behavior

The Vroman effect is responsible for polymers surface suffering of unspecific adsorption of proteins. According to the Vroman sequence, the first proteins to be adsorbed are albumins, i.e. the smallest and most concentrated in biological fluids, that are then substituted by others proteins larger and less concentrated.²¹ For this reason, bovine serum albumin (BSA) has been chosen to perform the evaluation of proteins adsorption.

Data showed a reduced adsorption of BSA after GMA epoxide ring opening on the surface of the nanospheres, from 21.9 ± 1.0% to 3.0 ± 0.8% after the reaction. The same experiment has been conducted using polymeric nanospheres as control, synthesized in same condition of MNs but in absence of GMA. Also the control showed a similar behaviour to MNs with unreacted epoxide rings (see Table 2). These results demonstrated the importance of hydrophilic groups in decreasing albumin adsorption, thus increasing biocompatibility.

Table 2 Hydrophilic properties of MNs

Magnetic hydrogel	Water uptake (%)	BSA adsorption (%)
Control (MNs without GMA)	368.6 ± 1.1	16.2 ± 0.9
MNs before epoxide ring opening	343.1 ± 0.9	21.9 ± 1.0
MNs after epoxide ring opening	477.2 ± 1.1	3.0 ± 0.8

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The hydrophilic moieties are also fundamental in order to confer swelling ability to polymeric matrix in aqueous solutions. For the evaluation of swelling properties, small amounts of nanospheres have been immersed in a biological-like solution (a phosphate buffer saline solution at pH 7.4, PBS) and the water uptake was measured. As expected, the nanomaterial showed a water uptake of 343.1 ± 0.9% before the opening of epoxide ring and 477.2 ± 1.1% after the reaction. Furthermore, compared to the control, MNs showed also a higher water content, due to the higher number of hydrophilic groups. Data confirm a good swelling behaviour and epoxide opening improved hydrophilic property thanks to the new –OH groups present on the surface. All data are collected in Table 2.

Drug loading and in vitro release studies

The mechanism of loading SUM presumably involves physical diffusion of sunitinib into the matrix of MNs. The characteristics of drug and polymeric particles affected drug–polymer interactions, through H-bonding, electrostatic and other non-covalent interactions. In order to evaluate the ability of the nanoparticles to interact with SUM, the drug loading content (DLC) and the drug loading efficiency (DLE) were obtained following the procedure described in the experimental section. The DLC value calculated was 10.1 ± 0.7% while DLE was 56.5 ± 0.8%.

Furthermore in vitro release studies were performed by immersing aliquots of MNs/SUM in PBS pH 7.4 at 37 °C, simulating the physiological environment. The release profile of SUM from nanospherical hydrogels is shown in Fig. 7.

Fig. 7 Release profile of SUM from MNs.

In order to understand the mechanism involved in the in vitro release studies, drug release kinetics and mechanism of drug release from MNs have been investigated using zero order, first order and Korsmeyer–Peppas models.

The release data have been analyzed with zero order and first order kinetics.

Zero order kinetic eqn (1) explains the drug dissolution from various drug delivery systems, as well as from polymeric systems.

Q_t = Q₀ + K₀t (1)

where Q_t is the amount of drug in time t, Q₀ is the initial amount of drug in solution, usually Q₀ = 0.0 M, K₀ is the zero order rate constant (concentration/time) and t is the time. The zero order kinetic describes a constant drug release rate, independent of existing concentration.³¹

On the other hand, first order kinetic describes drug release dependent on concentration. The release rate is indeed proportional to the amount of drug still present within the system. A first order kinetic is expressed by eqn (2).

where Q_t is the amount of drug in time t, Q₀ is the amount of drug available at that time and K₁ is the first order rate constant.³¹

Data obtained from SUM release profile study fit better first order kinetic than zero order kinetic. This could be stated on the basis of the higher R² value (see Table 3).

Table 3 R² value for zero and first order kinetic, calculated from the SUM release curve

Kinetic order	R^2 value
Zero order kinetic	0.9105
First order kinetic	0.9978

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In order to understand the drug release mechanism, the Korsmeyer–Peppas model was used. The Korsmeyer–Peppas eqn (3) can be applied up to 60% of the total amount of drug released

In which M_t/M_∞ is the fraction of drug release in time t, K is the constant incorporating the structural characteristics of the polymer and n is the diffusion exponent, indicative of the mechanism of transport of the drug.³²

According to Ritger–Peppas considerations, for swellable spherical systems the diffusion exponent n could assume value n ≤ 0.43, indicating a Fickian diffusion of the drug through the polymer. For value of n in the range of 0.43 < n < 0.85 the mechanism is non-Fickian or anomalous diffusion, in which polymer relaxation and drug diffusion rates are comparable. When n ≥ 0.85 the mechanism is case II diffusion, where diffusion is rapid and relaxation is slow.³³

For SUM release from MNs, the value obtained is n = 0.63 clearly indicating that the mechanism involved is non-Fickian diffusion and both relaxation of polymer and drug diffusion participate in SUM release.

Citotoxicity studies

Due to the promising application of sunitinib in the treatment of thyroid cancer and to investigate if SUM-loaded magnetic nanospheres could be exploited as a drug carrier in targeted cancer therapy, viability experiments were primarily conducted on two different human thyroid tumor cell lines: the undifferentiated/anaplastic ARO cells and the well-differentiated follicular WRO cells (Fig. 8A and B). In both cell lines, MNs/SUM treatment was able to induce a significant cell death at all time points, with respect to the relative controls and to the day 0 (no treatment). MNs/SUM effect was even more dramatic than free sunitinib malate in WRO cells after 2 and 3 days of exposure (Fig. 8B), while it was not as efficacious in ARO cells (Fig. 8A), but this could be, most likely, ascribed to the higher proliferative turnover of ARO cells compared to that of WRO cells.

Fig. 8 ARO (A), WRO (B), HeLa (C) and MCF-7 (D) cells were treated with polymer (MNs), free SUM or MNs/SUM. The viability was determined as described in Materials and methods after 1, 2 and 3 days. Values represent the mean of four triplicate independent experiments and are reported as percentage of cell viability. The error bars indicate SD. ♦p < 0.01 vs. day 0; *p < 0.01 vs. the corresponding untreated controls.

MNs/SUM was also tested in HeLa (human cervix tumor) and MCF-7 (human breast cancer) cell lines, which have been reported to be sensible to SUT treatment.^34,35 Compared to controls, MCF-7 cells showed up to 30% growth retardation after 3 days of MNs/SUM treatment (Fig. 8D), while HeLa cells reached almost the 60% growth inhibition on the same day (Fig. 8C). As for ARO cells, the high proliferative rate of HeLa and MCF-7 cells might be responsible of the lower efficacy of MNs/SUM in suppressing the growth of these cell lines compared to WRO cells.

Also the controlled release of SUM from hydrogel can explain the difference when compared to the free drug. Exposition of cells to free SUM is indeed responsible for the rapid cell death. In fact, it is still able to induce cell death in these two cell lines, as observed in thyroid tumor cells.

It is worth to underline that, in all cell lines tested, polymers (MNs) alone did not show any significant effect at all time-points if compared to controls (Fig. 8). These results confirm previously published data reporting non detectable hydrogels toxicity on cell systems.³⁶

Experimental

Materials, cell lines and culture conditions

Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), glycidyl methacrylate (GMA), 2,2′-azobisisobutyronitrile (AIBN), sodium dihydrogen phosphate (NaH₂PO₄), disodium hydrogen phosphate (Na₂HPO4), iron(III) chloride hexahydrate (FeCl₃·6H₂O), iron(II) chloride tetrahydrate (FeCl₂·4H₂O), sodium hydroxide (NaOH), perchloric acid (HClO₄), bovine serum albumin (BSA), sunitinib malate (SUM) were provided by Sigma-Aldrich (Sigma Chemical Co., St Louis, MO, USA).

MAA was purified before use by distillation under reduced pressure and AIBN was purified by recrystallization from methanol.

All solvents were reagent grade or HPLC-grade and purchased from Carlo Erba reagents (Milan, Italy).

Human anaplastic ARO and follicular WRO thyroid tumor cells (gifts from F. Arturi and A. Belfiore, University of Magna Grecia, Catanzaro, Italy), were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with GlutaMAX™, containing 10% fetal bovine serum (FBS; Invitrogen). HeLa cervical adenocarcinoma cells and MCF-7 breast cancer cells were purchased from Interlab Cell Line Collection, ICLC, Genoa, Italy, and grown in modified Eagle's medium (MEM, Sigma-Aldrich, Milan, Italy) plus 10% fetal bovine serum (FBS) or Dulbecco's modified Eagle's/Ham's F-12 medium (1 : 1) (DMEM-F12) plus 5% FBS, respectively. All culture media were supplemented with 100 IU mL^−l penicillin, 100 mg mL^−l streptomycin, and 0.2 mM L-glutamine (all from Life Technologies, Monza, Italy). Cells were maintained as monolayer culture in a humidified incubator at 5% CO₂ and 37 °C.

Instrumentation

UV-Vis absorption measurements were obtained using a Jasco V-530 UV/Vis spectrometer.

IR spectra were recorded as KBr pellets on a Jasco FT-IR 4200.

The scanning electron microscopy (SEM) micrographs were obtained with a Jeol JSMT 300 A; the samples were made conductive by gold layer deposition on samples surfaces in a vacuum chamber. Particles size distribution range was calculated employing an image processing and analysis system, a Leica DMRB endowed with a LEICA Wild 3D stereomicroscope.

Particles size and distribution were determined by Dynamic Light Scattering (DLS) analyses using a 90 Plus particle size analyzer (Brookhaven Instruments Corporation, New York, NY USA), at 25.0 ± 0.1 °C by measuring the autocorrelation function at 90°. The laser was operating at 658 nm. The distribution size was directly obtained from the instrument fitting data by the inverse “Laplace transformation” method. The Polydispersity index (P.I.) was used as a measure of the width of size distribution. P.I. less than 0.3 indicates a homogenous population. Each sample was measured three times and the results are expressed as mean ± standard deviation.

The thermoanalytical measurements were performed on an automatic TG instrument (Netsch STA 490) under nitrogen flow of 40 cm³ min⁻¹ in the range of 30–800 °C at a rate of 10 °C min⁻¹.

Magnetization of the samples was measured as function of the applied magnetic field (H) with a 9600 VSM (LDJ, USA) superconducting quantum interference device (SQUID) magnetometer. The magnetic hysteresis loop graphs were obtained by changing H between +20 000 and −20 000 Oe, at 25 °C.

Preparation of magnetite particles

Magnetite (Fe₃O₄) particles were prepared according to Yang et al., exploiting the so-called coprecipitation method.³⁷

In 100 mL of water 0.01 mol of FeCl₂·4H₂O and 0.02 mol of FeCl₃·6H₂O were firstly dissolved together. The mixture was stirred vigorously until the complete dissolution. Afterwards the solution was purged with nitrogen gas and the temperature was increased to 80 °C. 40 mL of a NaOH solution (2 M) were then added into it. After 1 h a black solid appeared, confirming the completion of reaction. The precipitate magnetite was collected using an external magnetic field and washed several times, with water first and then ethanol, and finally dried under vacuum.

Synthesis of magnetic nanospheres (MNs)

In a 100 mL round bottom flask, 0.5 g of magnetite were suspended in a mixture of acetonitrile (20 mL) and toluene (20 mL) promoted by sonication for 10 min. 8 mmol of the functional monomer MAA, 8 mmol of GMA, 10 mmol of EGDMA and 100 mg of AIBN were added and the mixture was sonicated for other 10 min and purged with nitrogen. The flask was gently agitated (40 rpm) and the mixture was put under a 360 nm light for 24 h in order to initiate the photo-polymerization, keeping the temperature constant (4 ± 0.5 °C). At the end of the reaction, the particles were collected by filtration and washed with solvents in this exact order: ethanol, acetone and diethyl ether. In the end, the obtained nanospheres were dryed overnight, under vacuum, at 40 ± 0.5 °C.

Two different polymeric materials as controls have been prepared using same conditions, but in absence of magnetite or in absence of GMA.

Epoxide ring opening

400 mg of magnetic nanospheres (MNs) were put in a suitable round bottom flask, then 25 mL of a perchloric acid water solution (10% v/v) were added. The flask was kept under agitation (200 rpm) for 24 h at room temperature. Afterwards that time, at the completion of the reaction, the particles were recovered as previously described: they were filtered and washed again with ethanol, acetone and diethyl ether. Al last the powder was dried under vacuum overnight, at 40 ± 0.5 °C. The same treatment was performed for the two materials synthesized as controls.

Protein adsorption measurement

A BSA standard solution (1.2 mg mL⁻¹) was prepared in a 25 mM phosphate buffer saline solution at pH 7.4.

Three different sets of three experiments each were performed. In the first set 150 mg of MNs before epoxide ring opening were packed into 6.0 mL polypropylene SPE columns. The columns were attached with a stop cock and a reservoir at the bottom end and the top end, respectively. The columns were preconditioned before use by successive washing steps with water, HCl (0.07 M), water, methanol/water (50 : 50, v/v), water, and finally 25 mM phosphate buffer (pH 7.4). The adsorption test was performed by loading the cartridges with 2.0 mL of the prepared BSA standard solution. The second set was prepared in same conditions, except for MNs used after epoxide ring opening. In the third set, control with no GMA was used.

The amount of adsorbed protein after loading step was calculated by UV-Vis spectrophotometer at λ_max 290 nm.

Swelling behaviour

In order to evaluate the MNs swelling behaviour, dry aliquots (40–50 mg) of nanospheres before and after the epoxide rings opening were placed in 5 mL sintered glass filters (Ø10 mm; porosity, G3), weighed, and immersed in beakers containing phosphate buffer (PBS, pH 7.4), as the swelling media. After 24 h, the excess water was removed by percolation at atmospheric pressure. Then, the filters were secured with silicon stoppers in proper centrifuge test tubes, and centrifuged for 5 min at 2000 rpm and weighed at last. The weights of the filters were determined by immersing them in PBS and then centrifuged. The weights of swollen MNs were obtained by subtraction of the filters tare. The water content percentage (WR%) was calculated using the weights recorded with the following eqn (4):where W_s and W_d are weights of swollen and dried MNs, respectively.

The experiments were also performed on nanospheres synthesized in the same reaction conditions, but in the absence of GMA.

Experiment was carried out in triplicate.

Drug loading procedure

In order to determine the extent of drug that can be loaded into the MNs and the encapsulation efficiency, in a round bottom flask of 10 mL, 20 mg of SUM were dissolved in 2 mL of ethanol and sonicated for 2 min. Then 100 mg of MNs were added to the solution and soaked for 3 days in the dark under stirring at room temperature. Then MNs were put in a sintered glass filter and the excess of solvent, containing the amount of drug not loaded into the polymeric matrix, was removed by percolation at atmospheric pressure. MNs were finally dried under vacuum overnight.

The amount of SUM not loaded, contained in the excess of solvent, was measured by UV-Vis absorption at their maximum wavelength (λ_max = 430 nm).³⁸ Thus, the amount of SUM loaded was calculated by difference from the total amount.

The drug loading content (DLC) and the drug loading efficiency (DLE) were obtained using eqn (5) and (6), respectively,

in which W_dn, W_n and W_d represent the weight of drug within the nanoparticles, weight of dried nanoparticles and the total weight of drug used for the loading procedure, respectively.

In vitro release procedure

In a 15 mL Falcon centrifuge tube, 10 mg of MNs loaded with SUM were added to 10 mL of a 0.01 M phosphate buffer saline (PBS) solution (pH 7.4). Incubation were carried out with a shaking thermostated Dubnoff bath at 37 ± 0.5 °C. To evaluate the drug release profile, 2 mL of sample releasing media were withdrawn and replaced with other 2 mL of 0.01 M PBS solution (pH 7.4). Withdrawals were made at different intervals of time (1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h).

Analyses were performed with a UV-Vis spectrophotometer and the amounts of released SUM were calculated from the equation of the calibration curve of SUM, previously determined.

Experiments were repeated three times.

Cell viability

The effects on cell growth of the drug bearing hydrogel (MNs/SUM) has been tested on different cell lines by trypan blue exclusion assay. Cells were plated in triplicates in growing medium in 12-well plates, at about 30–40% confluence. After 6 h, cells were shifted to serum free media (SFM) for 24 h to synchronize the cells in the same cell cycle phase, thus avoiding growth differences among cells. Following starvation, cells were shifted back to growing medium and treated with 25 μg per well of MNs/SUM, corresponding to a concentration of about 10 μM of free SUM. As negative controls, the vehicle alone (DMSO) or MNs were added to the cells in the same amount used for MNs/SUM. After 1, 2 or 3 days, cells were harvested by trypsinization and incubated in a 0.5% trypan blue solution for 10 min at room temperature. Cell viability was determined microscopically by counting trypan blue negative cells using a hemacytometer (Burker, Brand, Germany).

Conclusions

A novel nanospheric hydrogel was successfully synthesized as restricted access material, to prevent proteins adsorption on surface. The material was obtained in high yield via precipitation polymerization and, from tests performed, we could state that albumin and the other proteins have no access to nanospheres surface. Therefore biocompatibility in blood environment is improved for such materials.

The encapsulation of magnetite in polymeric matrix contributes to targeted release of SUM, due to the magnetic responsiveness showed.

The efficacy of MNs as carriers for SUM has been proven on several cancer cell lines showing a good behaviour, especially in thyroid tumor cells, and opening to new possible applications of such antineoplastic drug.

Acknowledgements

The financial support of the Italian Minister of University and Research and University of Calabria is most gratefully acknowledged.

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Footnotes

† Ortensia Ilaria Parisi and Catia Morelli have contributed equally to the manuscript.

‡ Joint senior author.

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