Magnetic Dehydrodipeptide-Based Self-Assembled Hydrogels for Theragnostic Applications

nanomaterials

Article Magnetic Dehydrodipeptide-Based Self-Assembled Hydrogels for Theragnostic Applications

André Carvalho 1, Juan Gallo 2 , David M. Pereira 3 , Patrícia Valentão 3 , Paula B. Andrade 3 , Loic Hilliou 4 , Paula M.T. Ferreira 1,*, Manuel Bañobre-López 2,* and José A. Martins 1,* 1 Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; [email protected] 2 Diagnostic Tools & Methods/Advanced (Magnetic) Theranostic Nanostructures Lab, International Iberian Nanotechnology Laboratory (INL), Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal; [email protected] 3 REQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, R. Jorge Viterbo Ferreira, n◦ 228, 4050-313 Porto, Portugal; [email protected] (D.M.P.); [email protected] (P.V.); [email protected] (P.B.A.) 4 Institute for Polymers and Composites/I3N, Department of Polymer Engineering, University of Minho, Campus de Azurém, 4800-058 Guimarães, Portugal; [email protected] * Correspondence: [email protected] (P.M.T.F.); [email protected] (M.B.-L.); [email protected] (J.A.M.)

Received: 5 March 2019; Accepted: 26 March 2019; Published: 3 April 2019

Abstract: Self-assembled peptide hydrogels have emerged in recent years as the new paradigm in biomaterials research. We have contributed to this field the development of hydrogels based on dehydrodipeptides N-capped with naproxen. The dehydrodipeptide hydrogels can be loaded with drugs, thus being potential nanocarriers for drug delivery. In this work novel dehydrodipeptides containing tyrosine and aspartic acid amino acid residues N-capped with naproxen and C-terminal dehydrophenylalanine were prepared and characterized. Superparamagnetic iron oxide nanoparticles (SPIONs) were incorporated into the dehydrodipeptide-based hydrogels and their effect on the self-assembly, structure and rheological and magnetic properties of the hydrogels was studied. Magnetic hydrogels, with incorporated SPIONs, displayed concentration-dependent T2-MRI contrast enhancement. Moreover, upon magnetic excitation (alternating magnetic field –AMF–) the SPIONs were able to generate a significant amount of heat. Hence, magnetic hyperthermia can be used as a remote trigger for release of drug cargos and SPIONs incorporated into the self-assembled dehydrodipeptide hydrogels.

Keywords: naproxen N-capped dehydrodipeptides; self-assembled hydrogels; SPION; magnetic hydrogels; MRI; magnetic hyperthermia; drug-delivery

1. Introduction Crystalline, single domain iron oxide nanoparticles, usually with core size between 4–18 nm, display superparamagnetism. Superparamagnetic iron oxide nanoparticles (SPIONs) are characterized by fully reversible magnetization curves, i.e., zero coercivity and remanance [1,2] Superparamagnetic properties, allied to amenable methodologies for controlled bottom up synthesis and biocompatibility make SPIONs ideal for biomedical applications—as contrast agents (CA) for magnetic resonance imaging (MRI) and magnetic hyperthermia (MH) [3]. MRI is the leading modality in clinical imaging: anatomical and functional imaging of soft tissues with high spatial-temporal resolution, non-invasive and considered safe owing to use of benign (non-ionizing) radiation, magnetic ﬁelds and radiofrequencies. The main limitation of MRI is low detection sensitivity—inherent to the nuclear

Nanomaterials 2019, 9, 541; doi:10.3390/nano9040541 www.mdpi.com/journal/nanomaterials Nanomaterials 2019, 9, 541 2 of 16 magnetic resonance (NMR) phenomenon [4]. Paramagnetic metal complexes (Gd3+, Mn2+, Fe3+, organic radicals) and magnetic nanoparticles (i.e., SPIONs) reduce selectively the relaxation times (T1,2) of the water protons of tissues in their vicinity—Contrast Agents. The efficacy of a CA is measured by the parameter relaxivity (r1,2)—enhancement of the relaxation rates (R1,2 = 1/T1,2) normalized to 1 mM concentration of paramagnetic species. SPIONs are preferentially used as T2 contrast agents for MRI due to a strong T2 effect and high ratio r2/r1 [5,6]. Size, shape and magnetic composition of the nanocrystal are the main factors that determine the magnetization saturation and the relaxivity of SPIONs [6]. Self-assembly and controlled aggregation of SPIONs is a strategy for development of smart responsive T2 CA [7]. Upon remote magnetic excitation (AMF) SPIONs generate heat [8,9]. A localized temperature rise above 45 ◦C induces cell apoptosis [10]. SPIONs is well established in biomedical research and is clinically approved for hyperthermia therapy of cancer [11,12]. Heat generation by SPIONs results from magnetic spin relaxation by Néel and Brownian relaxation mechanisms [13]. The efficacy of magnetic nanoparticles as heat generators is measured by the parameter specific absorption rate (SAR) (named also specific loss power, SLP) [2,9]. The SAR depends on several parameters related to the AMF excitation (frequency and amplitude), magnetic properties of the nanoparticles (density of nanoparticles, saturation magnetization and nanoparticle anisotropy) and environment properties such as medium viscosity and self-assembly of magnetic nanoparticles by dipole-dipole interactions [14,15]. Nanoparticles displaying high SAR can reach the target temperature at lower doses, minimizing potential toxicity to surrounding tissues. Obtaining nanoparticles with high values of SAR requires simultaneous optimization of the parameters that govern SAR [15,16]. Self-assembled peptide hydrogels have emerged in recent decades as the new paradigm in biomaterials research: high water content; soft materials with fully organic structure; intrinsic nontoxicity and biocompatibility and fibrillar nanostructure reminiscent of the extracellular matrix [17]. Importantly, the synthesis of hydrogelator peptides (di- and tripeptides) is straightforward and can be automatized by solid-phase synthesis. The properties of the hydrogels can be tuned by design and there is a great variety of possible hydrogelator structures using proteinogenic and non proteinogenic aminoacids [18,19]. Hydrogel assembly/disassembly can be instructed by enzymatic modification of hydrogelator molecules (hydrolysis, phosphorylation/dephosphorylation, etc.) and environmental stimuli (pH, ionic strength, etc.) [20,21]. The self-assembly of hydrogelator peptide molecules is mediated by an ensemble of weak non-covalent intermolecular interactions: hydrogen bonding, van der Walls and dispersion interactions, π–π stacking [22]. Our research group has recently described a series of self-assembled hydrogels based on dehydrodipeptides N-protected with the Non-Steroidal Anti-inflammatory Drug Naproxen (NSAID). The C-terminal dehydroaminoacid residue (dehydrophenylalanine, dehydroaminobutyric acid and dehydroalanine) is prone to make the peptide resistant to proteolysis and restrains the conformational freedom of the peptide [23–25]. The naproxen naphtalene bulky aromatic moiety is fundamental for self-assembly as it contributes with intermolecular π–π stacking interactions. The dehydrodipeptide hydrogels revealed an entangled fibrillar structure with nanometric/micrometric dimensions. Interestingly, drug molecules could be incorporated into the dehydrodipeptide-based hydrogels and were found, by FRET experiments, to be in close proximity, presumably associated with the hydrogelator fibers. We have previously proposed that dehydrodipeptide based-hydrogels are potential nanostructured drug delivery agents [24,25]. A remote (external) magnetic field (noninvasive, safe and tunable) is the ideal actuator for in vivo biomedical applications of magnetic materials: as CA for MRI, hyperthermia, drug delivery, etc. Hydrogels can be rendered magneto-responsive by the incorporation of magnetic nanoparticles (γ-Fe2O3, Fe3O4, CoFe2O4, etc.). Magnetic nanoparticles respond to magnetic actuation changing the micro-nanostructure of the hydrogels which in turn leads to modification of the hydrogel properties. Magnetic hydrogels, based on synthetic and natural polymers, have been extensively reported in the literature as potential agents for magnetic field-activated hyperthermia and drug delivery [26,27]. The reversible nature of self-assembly is prone to make supramolecular magnetic hydrogels intrinsically more susceptible to magnetic actuation than polymer-based hydrogels. Bing Xu Nanomaterials 2019, 9, 541 3 of 16 and co-workers demonstrated that a self-assembled peptide hydrogel, incorporating SPIONs coated with hydrogelator molecules, displays typical magnetorheological behavior, i.e., a gel-sol transition trigged by a non-uniform magnetic field [28]. More recently, Cienfuegos and co-workers reported that the nanostructure and rheological properties of a peptide-based supramolecular hydrogel containing iron nanoparticles can be substantially improved by performing the self-assembly in the presence of a magnetic field [29]. In this work, novel dehydrodipeptide hydrogelators with aspartic acid and tyrosine amino acid residues were synthesized and fully characterized. Dehydrodipeptide hydrogels incorporating SPIONs were prepared and characterized. The effect of SPIONs incorporation on the self-assembly and micro-nanostructure of the hydrogels and on their rheological and magnetic properties, as well as their efficiency as CA for MRI and heat source for magnetic hyperthermia, were studied. Preliminary data on the biological properties of the hydrogelators were collected as well.

2. Materials and Methods Synthesis—Hydrogelators 7 and 8 were prepared by synthetic methodologies developed in our laboratory and fully characterized by 1H and 13C NMR spectroscopy (NMR) and High Resolution Mass Spectrometry (HRM) [23–25]. Spectra were acquired on a Bruker Avance III 400 spectrometer, operating at 400.13 MHz and 100.62 MHz, for 1H and 13C NMR respectively. HRMS data were acquired from the mass spectrometry service of the University of Vigo, Spain. MS was acquired in a Thermo Finnigan LxQ (Linear Ion Trap, San Jose, CA, USA Mass Detector with Electro Spray Ionization (ESI). Hydrogel preparation—Dehydrodipeptides 7 and 8 were weighted into sample vials, water was added and the suspension was adjusted under magnetic stirring to circa pH 10 (pH meter) by addition of NaOH (1M). The resulting solutions were sonicated for 30 s and D-glucono-δ-lactone (GDL) was added under magnetic stirring (around 10 s). The solutions were left standing overnight at room temperature (20–25 ◦C). Hydrogels loaded with SPIONs were prepared following the same methodology. A SPIONs solution was added to the hydrogelator suspension in water, followed by pH adjustment to circa 10, sonication and the addition of GDL [25]. Rheology—Hydrogel formation kinetics and mechanical spectra were measured with a stress- controlled rotational rheometer (MCR300, Anton Paar, Graz, Austria). Liquid samples, prepared as described above, were loaded into the Couette geometry of the rheometer. The temperature was kept at 25 ◦C during testing. For measuring gel kinetics, a small amplitude oscillatory shear strain, with a frequency of 1 Hz and an amplitude varying from 0.0001% to 1%, depending on the SPIONs loading in samples, was applied for 60,000 s. After the kinetic experiments, mechanical spectra were recorded using the same strain amplitude as in the kinetics tests, and ramping the frequency from 100 Hz down to 0.01 Hz. Finally, a sweep in the strain amplitude was performed from 0.001% to 100% and both storage (G0) and loss (G00) shear moduli were measured. Transmission Electron Microscopy—TEM images were obtained on a JEOL (Tokyo, Japan) JEM-2100 microscope at an accelerating voltage of 200 kV. Samples for TEM were prepared as described for circular dichroism (CD) spectroscopy. A small volume of sample (10 µL) was placed on the top of a carbon-coated grid and left for 30 s. Excess water was removed using filter paper. UranyLess stain (10 µL) was added to the grid and left for 30 s. The grid was washed twice with water. CD spectroscopy—CD spectra were recorded under N2 on a Jasco (Tokyo, Japan) J815 CD spectrometer using solutions of hydrogelators 7 and 8 (0.02 wt%) without and with 10% SPIONs. Magnetization studies—Magnetization curves were recorded at 5 and 260 K as function of the applied magnetic field (up to ±2 T). Zero-field-cooled (ZFC, H = 0) and field-cooled (FC, H = 100 Oe) magnetization curves were recorded over the temperature range of 5–300 K. A superconducting quantum interference device magnetometer (SQUID-VSM, Quantum Design, San Diego, CA, USA) was used for all experiments. Magnetic Resonance Imaging—MRI was performed in a 3.0 T horizontal bore MR Solutions (Guildford, UK) Benchtop MRI system equipped with 48 G/cm actively shielded gradients. To image the samples, a 56 mm diameter quadrature birdcage coil was used in transmit/receive mode. Samples Nanomaterials 2019, 9, x FOR PEER REVIEW 4 of 16

Magnetic Resonance Imaging—MRI was performed in a 3.0 T horizontal bore MR Solutions (Guildford, UK) Benchtop MRI system equipped with 48 G/cm actively shielded gradients. To image Nanomaterials 2019, 9, 541 4 of 16 the samples, a 56 mm diameter quadrature birdcage coil was used in transmit/receive mode. Samples for phantom measurements, SPIONs in water and SPIONs incorporated into hydrogels 7 forand phantom 8 at different measurements, concentrations SPIONs (0–0.43 in mM water Fe), and were SPIONs prepared incorporated with a total into volume hydrogels of 2007 µLand in 8300at differentµL tubes. concentrations All MR images (0–0.43 of the mM phantoms Fe), were were prepared acquired with with a total an volumeimage matrix of 200 µ256L in × 300252,µ Lfield tubes. of Allview MR (FOV) images 60 × of 60 the mm, phantoms 3 slices with were a acquired slice thickness with an of image 1 mm matrixand 1 mm 256 ×slice252, gap. field For of T view2-weighted (FOV) 60imaging,× 60 mm, fast 3spin slices echo with (FSE) a slice sequences thickness with of the 1 mm following and 1 mm parameters slice gap. were For used:T2-weighted TE = 15 ms, imaging, TR = fast1500 spin ms, echoNA = (FSE)12, AT sequences = 24 m 48 withs. For the T1-weighted following imaging parameters a FSE were sequence used: TE with = 15TE ms, = 11 TR ms, = TR 1500 = 400 ms, NAms, NA = 12, = AT12 and = 24 AT= m 48 7 m s. 34 For s Twas1-weighted used. imaging a FSE sequence with TE = 11 ms, TR = 400 ms, NA =Hyperthernia 12 and AT = studies 7 m 34—SPIONs s was used. in water and SPIONs (25–35%) incorporated into hydrogels 7 and 8 wereHyperthernia studied as studies magnetic—SPIONs hyperthermia in water andeffector SPIONss using (25–35%) a nB nanoScale incorporated Biomagnetics into hydrogels (Zaragoza,7 and 8 wereSpain) studied DM-100 as instrument. magnetic hyperthermia effectors using a nB nanoScale Biomagnetics (Zaragoza, Spain) DM-100Biological instrument. studies—The toxicity of hydrogelator molecules 7 and 8 was evaluated in vitro towardsBiological macrophage studies—The cells toxicity(RAW 264.7 of hydrogelator cell line) an moleculesd towards7 theand human8 was evaluatednon-cancerin fibroblast vitro towards cell macrophageline MRC-5 using cells (RAWthe MTT 264.7 reduction cell line) assay. and towards The effect the of human hydrogelators non-cancer 7 and fibroblast 8 on the cell NO line levels MRC-5 on usingRAW 264.7 the MTT cells reductionchallenged assay. with LPS The [30] effect and of the hydrogelators ability of these7 and molecules8 on the to inhibit NO levels key enzymes on RAW 264.7in the cells inflammatory challenged withcascade, LPS [namely30] and the5-lipooxygenase, ability of these which molecules is involved to inhibit in key the enzymes synthesis in theof inflammatoryleukotrienes, cascade,and cyclooxygenase-2, namely 5-lipooxygenase, which is whichinvolved is involved in the insynthesis the synthesis of prostaglandins, of leukotrienes, was and cyclooxygenase-2,evaluated as well. whichThe LOX is involved inhibition in assay the synthesis [31] was of performed prostaglandins, with soybean was evaluated lipoxygenase. as well. The LOXCOX-1/COX-2 inhibition inhibition assay [31] wasassay performed was performed with soybean using a lipoxygenase. commercial screening The COX-1/COX-2 assay kit inhibition(Cayman assaychemical, was Ann performed Arbor, using MI, USA). a commercial screening assay kit (Cayman chemical, Ann Arbor, MI, USA).

3. Results and Discussion

3.1. Synthesis

Dehydrodipeptides naproxen tyrosyldehydrophenylalanine [Npx- L-Tyr--Tyr-Z-∆Phe-OHPhe-OH ( 7)])] and aspartyldehydrophenylalanine [Npx- LL-Asp--Asp-ZZ--∆Phe-OHPhe-OH ( (88)])] NN-protected-protected with with the non-steroidalnon-steroidal anti-inﬂammatoryanti-inflammatory drugdrug (NSAID) (NSAID) naproxen naproxen (Npx) (Npx) were preparedwere prepared using a solutionusing a strategysolution developed strategy bydeveloped the research by the group research (Scheme group1)[ (Scheme23–25]. 1) [23–25].

H N R HO R O O a) O HN O OCH3 N H CO OH H 3 NH O Fmoc O

R=Ph(4-OtBu), 1 R=Ph(4-OtBu), 3 t t R=CO2 Bu, 2 R=CO2 Bu, 4

OH b)

O OH O OCH O R O 3

t N R=Ph(4-O Bu) N H H NH c) NH

O 7 O O O

R=Ph(4-OtBu), 5 O OCH d) R=CO tBu, 6 CO HO 3 2 2 t R=CO2 Bu, N H NH

O O 8 Scheme 1. Synthesis of dehydrodipeptides N-conjugated with naproxen 7 and 8.. a) i.i. piperidine in

DMF, ii. naproxen chloride, NEt33; b) i. Boc Boc22O/DMAP,O/DMAP, ii. ii. TMG TMG (2%); (2%); c) c) i. i. TFA, TFA, ii. ii. NaOH NaOH d) d) TFA. TFA.

Dehydrodipeptides 77 andand 88 werewere obtained obtained in in moderate moderate yiel yieldd and and were were fully fully characterized characterized by 1 byH 1 13 andH and 13C bi-dimensionalC bi-dimensional NMR NMR techniques. techniques. The The Z Zster stereochemistryeochemistry of of the dehydrophenylalaninedehydrophenylalanine residue was established by NOE experiments.experiments. The dehydropheny dehydrophenylalaninelalanine residue (∆Phe) is prone to make the peptide recalcitrant to proteolysis and conﬁne the available conformational space [23,32]. The synthetic pathway developed for dehydrodipeptide Npx-L-Asp-Z-∆Phe-OH (8) allows either orthogonal deprotection of the C-terminal methyl ester or deprotection of the aspartyl side-chain Nanomaterials 2019, 9, 541 5 of 16

β-tert-Butyl ester. Orthogonal deprotection of the aspartyl side-chain β-tert-Butyl ester was deemed sufﬁcient to render the peptide water soluble for self-assembly studies.

3.2. Preparation of Hydrogels A ﬁne balance between hydrophilicity (and water solubility) and hydrophobicity is critical for peptide self-assembly [22]. Dehydrodipeptides 7 and 8 showed limited solubility in buffer solutions in the physiological pH range (6–8), but could be rendered soluble upon the adjustment of water dispersions (0.4–0.8 wt%; 4–8 mg/mL) to pH 10 with diluted NaOH (1 M). Gelation of dehydrodipeptides 7 and 8 was trigged by slow pH dropping (to pH 6–7) induced by hydrolysis of added GDL (0.3 wt%; 3 mg/mL). The minimal concentration required for peptide gelation, critical gelation concentration (cgc), was determined for dehydrodipeptides 7 and 8 as 0.4 wt% (4 mg/mL) (Figure S1). This value is of the same order of magnitude as that reported previously for other dehydrodipeptides [23–25]. We envisaged that incorporation of SPIONs into hydrogels 7 and 8 could endow them with magnetic properties. Water stabilized SPIONs with a polyacrylic acid corona (core size 8 nm; hydrodynamic diameter circa 100 nm) were used to minimize the likelihood of nanoparticle aggregation and precipitation (Figure S2 and Table SI) [1,2]. Hydrogels 7 and 8 (0.8 wt%) loaded with SPIONs (25–35% m/m; in relation to the hydrogelator weight) were prepared by the GDL method. A solution of SPIONs was added to suspensions of hydrogelators 7 and 8 in water and adjusted to pH 10, followed by addition of GDL. Hydrogels, homogeneous to visual inspection, were obtained in all cases, indicating that nanoparticle precipitation did not occur during gelation. Hydrogels were stable for extended time periods (Figure S3). The incorporation of SPIONs into the hydrogels resulted in slower gelation kinetics and weaker (less elastic) gels. Hydrogels 7 and 8, with SPIONs loadings in the range 25%–35%, could not be obtained at hydrogelator concentrations 0.4 wt% (cgc). A 0.8 wt% hydrogelator concentration was required for obtaining hydrogels 7 and 8 with 25–35% SPIONs. As the hyperthermia studies (see section on magnetic hyperthermia) required SPIONs in a concentration range of 25%–35%, all studies were carried out with both hydrogels at 0.8 wt% hydrogelator concentration.

3.3. TEM Studies The micro-nanostructure structure of hydrogels 7 and 8 was studied by transmission electron microscopy (TEM). Images were obtained with diluted solutions of hydrogelators 7 and 8 (0.02 wt%) prepared under the same set of conditions as those used for hydrogel preparation. Self-assembly of dehydrodipeptides 7 and 8 results in entangled fibril structures (Figure S4). Gelation is the result of water trapping by the fibril network. The fibers formed by self-assembly of dehydrodipeptide 7 display a thickness around 10–20 nm and its length varies from approximately 0.2 to 0.4 µm. Dehydrodipeptide 8 shows fibers with diameters around 10–20 nm and lengths between 70 nm and 0.8 µm. TEM images of hydrogels 7 and 8 with incorporated SPIONs could not be obtained.

3.4. Circular Dichroism CD spectroscopy is a valuable tool for evaluating the secondary structure, folding and binding properties of peptides and proteins [33]. The possibility of detecting chiral self-assembly of molecular units is especially relevant for the characterization of self-assembled peptide hydrogels [34]. CD spectra were acquired with solutions of hydrogelators 7 and 8 (0.02 wt%), without and with 10% SPIONs (Figure1). The CD spectrum of dehydrodipeptide 7 exhibits bands at around 200 and 220 nm (positive bands) and at 230 nm (broad negative band). The CD spectrum of dehydrodipeptide 8 shows a positive band below 200 nm and two negative bands at 210 nm and 230 nm. The positive broad band at around 280 nm, present in the CD spectra of both hydrogels, can be attributed to chiral self-assembly of the naphthalene moieties of the Npx group [23–25]. Addition of SPIONs results in broadening of the CD spectra of both hydrogels, although the general spectral features seem Nanomaterials 2019, 9, x FOR PEER REVIEW 6 of 16

15 Compound 8 (0.02 wt%) Compound 8 (0.02 wt%) with 10% SPION 10 Compound 7 (0.02 wt%) Compound 7 (0.02 wt%) WITH 10% SPION

0 Nanomaterials 2019, 9, 541 6 of 16

CD (mdeg) -5

-10 unchanged. Hence, incorporation of SPIONs seems not to change the structure of the self-assembled Nanomaterialspeptide nanoﬁbers 2019, 9, x FOR [29]. PEER REVIEW-15 6 of 16

200 220 240 260 280 300 320 340 15 Wavelength Compound (nm) 8 (0.02 wt%) Compound 8 (0.02 wt%) with 10% SPION Figure 1. CD spectra of solutions10 of dehydrodipeptides Compound 7 (0.027 and wt%) 8 (0.02 wt%) without and with 10% Compound 7 (0.02 wt%) WITH 10% SPION SPIONs. 5

The CD spectrum of dehydrodipeptide0 7 exhibits bands at around 200 and 220 nm (positive bands) and at 230 nm (broad negative band). The CD spectrum of dehydrodipeptide 8 shows a

CD (mdeg) -5 positive band below 200 nm and two negative bands at 210 nm and 230 nm. The positive broad band at around 280 nm, present-10 in the CD spectra of both hydrogels, can be attributed to chiral self-assembly of the naphthalene moieties of the Npx group [23–25]. Addition of SPIONs results in -15 broadening of the CD spectra of both hydrogels, although the general spectral features seem unchanged. Hence, incorporation200 of SPIONs 220 240 seems 260 not 280 to change 300 320 the struct 340 ure of the self-assembled Wavelength (nm) peptide nanofibers [29]. Figure 1. 1. CDCD spectra spectra of ofsolutions solutions of ofdehydrodipeptides dehydrodipeptides 7 and7 and 8 (0.028 (0.02 wt%) wt%) without without and with and with10% 3.5. RheologySPIONs.10% SPIONs. 3.5. Rheology TheThe CDstructural spectrum buil dupof dehydrodipeptide of hydrogel 7 (0.8 7wt%) exhibits without bands SPIONs at around and with 200 andSPIONs 220 (20–35%)nm (positive was followed by rotational rheometry. Incorporation of SPIONs into hydrogel 7 leads to longer gelation bands)The and structural at 230 buildupnm (broad of hydrogelnegative 7band(0.8). wt%) The without CD spectrum SPIONs of and dehydrodipeptide with SPIONs (20–35%) 8 shows was a half times, i.e. the time for attaining half of the equilibrium value for the gel elastic modulus G’ positivefollowed band by rotational below 200 rheometry. nm and two Incorporation negative band of SPIONss at 210 into nm hydrogeland 230 nm.7 leads The to positive longer gelationbroad band half (Figure S5). As a general trend, the gelation half time increases with the SPIONs content in the attimes, around i.e., the280 time nm, for present attaining in half the of CD the equilibriumspectra of both value hydrogels, for the gel elasticcan be modulus attributed G0 (Figure to chiral S5). hydrogel (inset to Figure S5). The mechanical spectra of equilibrated hydrogels 7 and 8 formulated self-assemblyAs a general trend, of the the naphthalene gelation half moieties time increases of the Npx with group the SPIONs [23–25]. content Addition in theof SPIONs hydrogel results (inset in to without and with SPIONs are presented in Figure 2. It is noteworthy that the rheological properties broadeningFigure S5). Theof the mechanical CD spectra spectra of ofboth equilibrated hydrogels, hydrogels although7 theand general8 formulated spectral without features and seem with of hydrogels 7 and 8 are not affected by the temperature up to 40 °C (see Figure S6). unchanged.SPIONs are presentedHence, incorporation in Figure2. Itof is SPIONs noteworthy seems that not the to rheologicalchange the propertiesstructure of of the hydrogels self-assembled7 and 8 peptideare not affectednanofibers by the[29]. temperature up to 40 ◦C (see Figure S6).

3.5. Rheology 105 105 The structural buildup of hydrogel 7 (0.8 wt%) without SPIONs and with SPIONs (20–35%) was followed by rotational rheometry. Incorporation of SPIONs into hydrogel 7 leads to longer gelation 104 104 half times, i.e. the time for attaining half of the equilibrium value for the gel elastic modulus G’ (Figure S5). As a general trend, the gelation half time increases with the SPIONs content in the 3 3 hydrogel (inset to Figure10 S5). The mechanical spectra10 of equilibrated hydrogels 7 and 8 formulated without and with SPIONs are presented in Figure 2. It is noteworthy0.8 that the rheological properties 2 2 of hydrogels 7 and10 8 are not affected by the temperature10 up 0 to 40 °C (see Figure S6).

G', G'' (Pa) G', G'' (Pa)

/ G / 0.4 1Hz 1 1 10 10 G 0.0 a) 0102030b) 5 5 % NP 10 0 10 0 10 10 -2 -1 0 1 2 10-2 10-1 100 101 102 10 10 10 10 10 Frequency (Hz) Frequency (Hz) 104 104

Figure 2. Mechanical spectra (solid symbols: G0; empty symbols: G”) of hydrogel 7 (a) and hydrogel Figure 2. Mechanical spectra (solid symbols: G′; empty symbols: G″) of hydrogel 7 (a) and hydrogel 8 8 (b) containing 0%3 NP (squares), 10% NP (circles) and3 30% NP (triangles). Inset: effect of SPIONs 10 10 content(b) containing (in %) on 0% the NP shear (squares), storage 10% moduli NP of (circles) hydrogels and7 30%(stars) NP and (triangl hydrogelses). Inset:8 (crosses) effect measured of SPIONs at

1 Hz, G , and rationalized by the corresponding value of neat0.8 gels, G . 1Hz 2 2 0 10 10 0

G', G'' (Pa) G', G'' (Pa) For hydrogel 7 formulated without SPIONs and all hydrogelsG / 0.4 8 a waiting time of 6.104 s with 1Hz 1 1 no mechanical excitation10 was applied before performing10 theG 0.0 frequency sweep, in order to avoid any strain-induced structure and gel elasticity. 0102030 a) % NP b) Hydrogels 8 was0 revealed to be signiﬁcantly more elastic0 (stronger gels) than hydrogels 7 (Table1). 10 10 -2 -1 0 1 2 10-2 10-1 100 101 102 10 10 10 10 10 Frequency (Hz) Frequency (Hz)

Figure 2. Mechanical spectra (solid symbols: G′; empty symbols: G″) of hydrogel 7 (a) and hydrogel 8 (b) containing 0% NP (squares), 10% NP (circles) and 30% NP (triangles). Inset: effect of SPIONs

Nanomaterials 2019, 9, 541 7 of 16

Table 1. Effect of SPION content in G0 and G” for hydrogels 7 and 8.

Hydrogel 7 Hydrogel 8 SPIONs % G0 (Pa) G00 (Pa) G0 (Pa) G00 (Pa) 0 39,300 3530 119,000 5520 10 7390 593 80,000 3400 20 1310 30 76,600 3300 30 122 13 43,500 1400

The minimum in the frequency dependence of the shear loss modulus G” is shifted to lower frequencies upon the addition of SPIONs, implying that the nanoparticles modulates the gel structure of both hydrogels. Hydrogels 7 were revealed to be more sensitive to the addition of SPIONs. This result is in accordance with the SPIONs-induced delayed structural buildup (Figure S5). Most importantly, the addition of SPIONs weakens gel elasticity-the shear storage modulus measured at 1 Hz (G1Hz) decreases with the increasing SPIONs content. The drop in gel elasticity upon the addition of SPIONs is more significant for hydrogels 7 comparing to hydrogels 8 (inset to Figure2). The strain dependence of the storage and loss moduli for hydrogels 8 (similar results were obtained for hydrogels 7) shows that at low strains both G0 and G00 moduli are constant, as expected for the linear regime of viscoelasticity. G00 goes through a maximum at large strain before dropping and crossing over with the storage modulus G0 (Figure S7). The local maximum in G00 in the strain regime, as G0 increases with increasing strain, reveals a hardening behavior that is the hallmark of many biological networks made by the assembly of fiber-like objects (inset to Figure S7) [35,36]. Strain hardening with concentration is also found in colloidal gels [37], gelatin gels [38] and some polysaccharide gels [39]. Considering a fiber-like structure of self-assembling units, which rule the elasticity of the gels over SPION-SPION contacts, the picture emerging from the rheological data is that the presence of SPIONs swells the mesh size of the structure and slows down the formation of elastically effective fiber-fiber contacts. Alternatively, the decrease of the gel elasticity with increasing SPIONs content could result from a stiffening effect of the SPIONs on the fibers without changing the mesh size [36].

3.6. Magnetic Properties The magnetic properties of both non-magnetic (without SPIONs) and magnetic (with SPIONs) hydrogels 7 and 8 (0.8 wt%) were evaluated by magnetic measurements in a superconducting quantum interference device (SQUID-VSM) magnetometer as a function of the temperature (5–260 K) and applied magnetic field (up to ±2 T) at 5 and 260 K. The hysteresis loop for hydrogel 8 (0.8 wt%) without SPIONs at 260 K confirms the expected diamagnetic behavior of the hydrogels. The paramagnetic behavior observed at 5 K was confirmed as arising from the sample holder (Figure S8). Hydrogels 7 and 8 (0.8 wt%) loaded with 10% SPIONs exhibit superparamagnetic behavior at 260 K indicated by negligible hysteresis and remnant magnetization at zero field of the hysteresis loop (Figure3). Superparamagnetism is typical of single domain magnetic nanoparticles, i.e., with diameter bellow the single-to-multi domain critical size (around 20–25 nm for magnetite) [40,41]. The decrease of the magnetization observed at high magnetic fields still shows a significant diamagnetic contribution (coming from the organic matrix) to the overall paramagnetism. At 5 K, a ferromagnetic-like contribution (coercive field, Hc = 210 Oe) is indicated by an open hysteresis loop due to the presence of a magnetically blocked state. The linear increase of the magnetization at high applied magnetic fields results from a dominant paramagnetic contribution. The ZFC-FC (H = 100 Oe) magnetization curves confirm the magnetic behavior observed in the hysteresis loops. The ZFC and FC (100 Oe) curves for hydrogel 8 without SPIONs follow a paramagnetic (Curie law) decay with temperature (Figure S8). The ZFC and FC curves for hydrogels 7 and 8 loaded with 10% SPIONs, indicate a magnetic transition from a magnetic blocked state (ferromagnetic-like behavior) to a superparamagnetic state above the blocking temperature (TB) (TB = 138 and 136 K for hydrogels 7 and 8 with 10% SPIONs, respectively). Nanomaterials 2019, 9, 541 8 of 16

The blocking temperature, defined as the maximum of the ZFC curve, indicates the temperature at which the thermal energy becomes comparable to the anisotropy energy barrier [42]. Interestingly, the divergence between the ZFC and the FC curves in the whole range of temperature, up to 300 K, is also indicative of strong magnetic dipolar interactions between the SPIONs in the gel network. In addition to the presence of a distribution of magnetic anisotropies in the sample, likely due to particle size polydispersity, large differences between TB and ZFC-FC irreversibility temperature (Tirr) indicate strong magnetic coupling between nanoparticles. In a closer inspection, we can observe that the magnetization monotonically increases with the temperature down to 2 K, further pointing to a strong interacting magnetite nanoparticle system and highlighting the presence of significant dipolar magnetic interactions. This magnetically strong interacting-like behavior differs from the collective behavior observed in other magnetic nanoparticles-based systems, such as magnetite nanoparticles arrays and magnetic zeolites, where both ZFC and FC magnetization curves decrease below TB [43,44]. The rheology experiments support the fibril nature of the hydrogels established by TEM and indicate that the SPIONs incorporated into the hydrogels are likely distributed into the water pools created by fiber entanglement. The alternative rheological view of the hydrogels, that the nanoparticles decorate the hydrogel fibers, seems unlikely given the expanded anionic polyacrylic acid corona surrounding the SPIONs. The magnetic results seem to support the rheological picture, pointing also to random distribution of the SPIONs inside the hydrogel matrix which allows the nanoparticles to interact strongly, rather than this magnetic coupling being induced by the fibrillar nano-structure of

Nanomaterialsthe hydrogels. 2019, 9, x FOR PEER REVIEW 8 of 16

5 K 0.004 A 260 K B 0.0006 FC 0.002

0.000 0.0004 0.002 M (emu)

M (emu) 0.001 -0.002 0.000 ZFC -0.001 M (emu) 0.0002 -0.002 -0.004 -0.8 -0.4 0.0 0.4 0.8 H (kOe)

-20 -10 0 10 20 0 50 100 150 200 250 H (kOe) Temperature (K) 0.010 5 K 260 K D C 0.0020 FC 0.005

0.0015

0.000 0.0050 M (emu)

M (emu) 0.0025 0.0010 0.0000 -0.005 -0.0025 ZFC M (emu) -0.0050 -0.8 -0.4 0.0 0.4 0.8 0.0005 H (kOe) -0.010 -20 -10 0 10 20 0 50 100 150 200 250 H (kOe) Temperature (K) Figure 3.3. HysteresisHysteresis loop loop at 5at and 5 and 260 K260 for K hydrogel for hydrogel7 (A) and 7 ( hydrogelA) and hydrogel8 (C) with 8 10% (C of) with incorporated 10% of incorporatedSPIONs. ZFC-FC SPIONs. (100 ZFC-FC Oe) magnetization (100 Oe) magnetization curves for hydrogel curves 7for(B hydrogel) and 8 (D 7) ( withB) and 10% 8 ( incorporatedD) with 10% incorporatedSPIONs. Insets: SPIONs. High magniﬁcationInsets: High magnification at low magnetic at low ﬁelds. magnetic fields. 3.7. Magnetic Resonance Imaging Superparamagnetism is typical of single domain magnetic nanoparticles, i.e., with diameter bellowSPIONs the single-to-multi selectively reduce domain the transversecritical size relaxation (around times20–25 (T nm2) of for the magnetite) water protons [40,41]. resulting The decreasein dark imagesof the magnetization in MRI under observedT2-weighted at high (T magnetic2w) sequences, fields still thus shows being a consideredsignificant diamagneticT2 contrast contribution (coming from the organic matrix) to the overall paramagnetism. At 5 K, a ferromagnetic-like contribution (coercive field, Hc = 210 Oe) is indicated by an open hysteresis loop due to the presence of a magnetically blocked state. The linear increase of the magnetization at high applied magnetic fields results from a dominant paramagnetic contribution. The ZFC-FC (H = 100 Oe) magnetization curves confirm the magnetic behavior observed in the hysteresis loops. The ZFC and FC (100 Oe) curves for hydrogel 8 without SPIONs follow a paramagnetic (Curie law) decay with temperature (Figure S8). The ZFC and FC curves for hydrogels 7 and 8 loaded with 10% SPIONs, indicate a magnetic transition from a magnetic blocked state (ferromagnetic-like behavior) to a superparamagnetic state above the blocking temperature (TB) (TB = 138 and 136 K for hydrogels 7 and 8 with 10% SPIONs, respectively). The blocking temperature, defined as the maximum of the ZFC curve, indicates the temperature at which the thermal energy becomes comparable to the anisotropy energy barrier [42]. Interestingly, the divergence between the ZFC and the FC curves in the whole range of temperature, up to 300 K, is also indicative of strong magnetic dipolar interactions between the SPIONs in the gel network. In addition to the presence of a distribution of magnetic anisotropies in the sample, likely due to particle size polydispersity, large differences between TB and ZFC-FC irreversibility temperature (Tirr) indicate strong magnetic coupling between nanoparticles. In a closer inspection, we can observe that the magnetization monotonically increases with the temperature down to 2 K, further pointing to a strong interacting magnetite nanoparticle system and highlighting the presence of significant dipolar magnetic interactions. This magnetically strong interacting-like behavior differs from the collective behavior observed in other magnetic nanoparticles-based systems, such as magnetite nanoparticles arrays and magnetic zeolites, where both ZFC and FC magnetization curves decrease below TB [43,44]. The rheology experiments support the fibril nature of the hydrogels established by TEM and indicate that the SPIONs

Nanomaterials 2019, 9, x FOR PEER REVIEW 9 of 16

incorporated into the hydrogels are likely distributed into the water pools created by fiber entanglement. The alternative rheological view of the hydrogels, that the nanoparticles decorate the hydrogel fibers, seems unlikely given the expanded anionic polyacrylic acid corona surrounding the SPIONs. The magnetic results seem to support the rheological picture, pointing also to random distribution of the SPIONs inside the hydrogel matrix which allows the nanoparticles to interact strongly, rather than this magnetic coupling being induced by the fibrillar nano-structure of the hydrogels.

3.7. Magnetic Resonance Imaging Nanomaterials 2019, 9, 541 9 of 16 SPIONs selectively reduce the transverse relaxation times (T2) of the water protons resulting in dark images in MRI under T2-weighted (T2w) sequences, thus being considered T2 contrast agents agents[6,45]. [However,6,45]. However, it has itbeen has beenalready already observed observed that that metal metal doping doping and and particle particle size size effectseffects (surface/volume(surface/volume ratio) ratio) can can originate predominant T1 or dualdual TT11/TT22 behaviorbehavior in in iron iron oxide based nanoparticles [[46,47].46,47]. The performance of the SPIONs as contrast agents for MRI, in bothboth aqueousaqueous solution andand incorporatedincorporated intointo hydrogelshydrogels7 7and and8 8,, was was evaluated evaluated by byT T1w1w-- andandT T2w2w-MRI imagesimages andand TT22 ◦ relaxation mapsmaps (25(25 °C,C, 3 T) (Figure4 4).).

◦ Figure 4.4. TT1w1w- andandT T2w2w-MRI-MRI phantomphantom images images and andT2 Trelaxation2 relaxation maps maps (25 (25C, 3°C, T): 3 ( AT):) SPIONs (A) SPIONs in water in (waterB) SPIONs (B) SPIONs incorporated incorporated into hydrogel into hydrogel7 (0.8 wt%); 7 (0.8 (C wt) SPIONs %); (C) incorporatedSPIONs incorporated into hydrogel into 8hydrogel(0.8 wt%). 8

The(0.8 Twt%).2-MRI The relaxation T2-MRI map relaxation was acquired map usingwas theacquired MEMS us (multi-echo-multi-spin)ing the MEMS (multi-echo-multi-spin) sequence. sequence. Figure4 4AA clearlyclearly showsshows aa concentration-dependent concentration-dependent darkdark effecteffect underunder aa TT22-MRI sequence for aqueous dispersionsdispersions ofof SPIONs.SPIONs. The The MRI contrast under TT1w1w sequence showed no significantsignificant differences between the different nanoparticlenanoparticle concentrationsconcentrations in relation toto thethe waterwater control.control. This confirmsconfirms the SPIONs used used in in this this study study as as being being TT2-MRI2-MRI enhancers. enhancers. The The efficacy efficacy of ofthe the SPIONs SPIONs as astransverse transverse paramagnetic paramagnetic water water relaxers relaxers was was eval evaluateduated by by the the parameter parameter transverse transverse relaxivity, rr22 (Table(Table2 2;; Figure Figure S9), S9), calculated calculated from from the theT T22-MRI-MRI mapsmaps atat 33T T and and room room temperature.

Table 2. ◦ 7 Table 2.SPIONs SPIONs transverse transverse relaxivity relaxivity (25 (25C, 3°C, T) in3 waterT) inand water following and following incorporation incorporation into hydrogel into and hydrogel 8 (0.8 wt%). hydrogel 7 and hydrogel 8 (0.8 wt%). SPIONs Incorporated SPIONs Incorporated SPIONs inSPIONs SPIONs in Water Incorporated into SPIONs Incorporated into into Hydrogel 7 into Hydrogel 8 Water Hydrogel 7 Hydrogel 8 −1 −1 ◦ r2 (mM s ) (25 C; 3 T) 122 30 44 r2 (mM−1 s−1) (25 122 30 44 °C; 3 T) −1 −1 The SPIONs in aqueous solution displays a considerable transverse relaxivity (r2 = 122 mM s at 3 T)The comparable SPIONs in to aqueous that exhibited solution by otherdisplays materials a considerable with similar transverse core size relaxivity and magnetic (r2 = composition122 mM−1s−1 −1 −1 (e.g.,at 3T) Feridex, comparabler2 = 120 to mM that sexhibited, 1.5 T) [by48 ].other The relaxivitymaterials of with the SPIONssimilar usedcore insize this and work magnetic is likely stillcomposition limited by (e.g., the smallFeridex, size r2 of= 120 the mM magnetic−1s−1, 1.5 core T)[48]. (~8 nm) The and relaxivity inherent of surfacethe SPIONsspin canting used ineffects this work [49]. is likelyIncorporation still limited of by the the SPIONs small size into of hydrogels the magnetic7 and core8 results (~8 nm in) up and to inherent a 4-fold reductionsurface spin of rcanting2 (r2 = −1 −1 −1 −1 30effects mM [49].s , 3 T, for hydrogel 7; r2 = 44 mM s , 3 T, for hydrogel 8) (Table2). The enhancement of the relaxationIncorporation rates ofof thethe water SPIONs protons into hydrogels brought about 7 and by 8 SPIONs results resultsin up to from a 4-fold the relative reduction translational of r2 (r2 = diffusion30 mM−1s−1 of, 3 theT, for water hydrogel molecules 7; r2 = 44 and mM magnetic−1s−1, 3 T, for nanoparticles, hydrogel 8) (Table reminiscent 2). Theof enhancement the outer-sphere of the Solomon-Bloembergen-Morganrelaxation rates of the water relaxation protons mechanismbrought about for paramagnetic by SPIONs metal results complexes from the [4,6 ].relative Hence, thetranslational transverse diffusion relaxivity ofr 2theis compromisedwater molecules by alland effects magnetic that restrictnanoparticles, water diffusion. reminiscent The of TEM the images show that the self-assembled hydrogels 7 and 8 are made of a network of entangled nanosized fibers. The rheology results suggest that the SPIONs incorporated into the hydrogels are likely distributed into the water pools created by fiber entanglement. This is further supported by the strong magnetic dipolar particle interaction deduced from the magnetic properties, as the magnetic particles would be confined in the aqueous regions delimited by the peptide fibers. Restricted water diffusion inside the hydrogel water compartments is very likely the cause of the observed low r2 relaxivities. Aggregation of the SPIONs in the aqueous hydrogel compartments is unlikely, owing to steric and electrostatic stabilization resulting from the nanoparticles bulky PAA anionic corona (Table SI) [2]. This dramatic reduction of r2 brings an increase of the r2/r1 ratio, which interestingly enables dual T1/T2-MRI performance. Dual CAs are gaining increasing attention, as they can be separately used Nanomaterials 2019, 9, x FOR PEER REVIEW 10 of 16

outer-sphere Solomon-Bloembergen-Morgan relaxation mechanism for paramagnetic metal complexes [4,6]. Hence, the transverse relaxivity r2 is compromised by all effects that restrict water diffusion. The TEM images show that the self-assembled hydrogels 7 and 8 are made of a network of entangled nanosized fibers. The rheology results suggest that the SPIONs incorporated into the hydrogels are likely distributed into the water pools created by fiber entanglement. This is further supported by the strong magnetic dipolar particle interaction deduced from the magnetic properties, as the magnetic particles would be confined in the aqueous regions delimited by the peptide fibers. Restricted water diffusion inside the hydrogel water compartments is very likely the cause of the observed low r2 relaxivities. Aggregation of the SPIONs in the aqueous hydrogel compartments is unlikely, owing to steric and electrostatic stabilization resulting from the nanoparticles bulky PAA anionic corona (Table SI)[2]. This dramatic reduction of r2 brings an Nanomaterialsincrease of 2019the, 9r,2 541/r1 ratio, which interestingly enables dual T1/T2-MRI performance. Dual CAs10 ofare 16 gaining increasing attention, as they can be separately used under both T1w and T2w imaging sequences and so contribute to identify interferences and approach a more accurate diagnosis of under both T and T imaging sequences and so contribute to identify interferences and approach disease. In addition1w to2w the observed darker contrast with increasing SPIONs concentration under a a more accurate diagnosis of disease. In addition to the observed darker contrast with increasing T2w sequence, phantom images of hydrogels 7 and 8 (Figure 4B,C, respectively) show a SPIONs concentration under a T2w sequence, phantom images of hydrogels 7 and 8 (Figure4B,C, concentration-dependent positive contrast when acquired with a T1w sequence, which significantly respectively) show a concentration-dependent positive contrast when acquired with a T1w sequence, becomes brighter as the particle concentration increases compared to the water control. which signiﬁcantly becomes brighter as the particle concentration increases compared to the water Disassembly of magnetic hydrogels 7 and 8 implanted in vivo could be monitored overtime. control. Disassembly of magnetic hydrogels 7 and 8 implanted in vivo could be monitored overtime. Hydrogel disassembly trigged by environmental/biochemical cues or remote triggers (magnetic Hydrogel disassembly trigged by environmental/biochemical cues or remote triggers (magnetic hyperthermia) could also report on the drug release and on the hydrogel environment. hyperthermia) could also report on the drug release and on the hydrogel environment.

3.8. Hyperthermia Studies The concentration of SPIONs deployed for thethe MRIMRI studiesstudies (0.01–0.06%)(0.01–0.06%) revealedrevealed unsuitableunsuitable (too low)low) forfor hyperthermiahyperthermia purposes.purposes. Hydrogels Hydrogels 7 and 8 were studied at 0.8 wt% concentrationconcentration to ensure gelation with SPIONsSPIONs inin thethe concentrationconcentration rangerange 25–35%,25–35%, asas previouslypreviously explained.explained. SPION concentrations in the range 25–35% were required for thethe hydrogelshydrogels undergoundergo gel-solutiongel-solution phase transition uponupon magneticmagnetic actuation actuation (H (H = 240= 240 G, G,f = f 869= 869 kHz). kHz). The The capacity capacity of theof the SPIONs SPIONs in water in water and incorporatedand incorporated into hydrogelsinto hydrogels7 and 78 and(0.8 8 wt%) (0.8 wt%) to transduce to transduce magnetic magnetic power power (H = 240(H = G, 240f = G, 869 f = kHz) 869 intokHz) heat into was heat evaluated was evaluated by the by SAR the parameter SAR parameter (Figure (Figure5, Table 5,3 Tableand Table 3 and SII). Table SII).

25% 25% 25 % 100 30% 70 30% 30 % A B 100 C 90 35% 35% 35 % 90 60 80 80

70 50 70 60 60

50 40 50 40

Temperature (ºC) 40 Temperature (ºC) Temperature (ºC) 30 30 30 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 Time (s) Time (s) Time (s) Figure 5.5. Magnetic hyperthermia curves for SPIONs (25–35%) in water ( A) and SPIONs (25–35%) incorporated intointo hydrogels hydrogels7 7(B ()B and) and8 (8C )(C (0.8) (0.8 wt%) wt%) upon upon magnetic magnetic actuation actuation (H = (H 240 = G, 240f = G, 869 f kHz).= 869 kHz). Table 3. SAR for SPIONS in water and hydrogel 7 and 8 (0.8 wt%) loaded with SPIONs (25–35% m/m).

The specific absorption rate SAR (W/g) parameterSAR was (w/g) calculated by the initial slope method SPIONs (%) from the magnetic hyperthermiaSPIONs curves in Water (Table 3, SPIONs Table inSII) Hydr. [14]. 7 SPIONs in Hydr. 8 25 357 161 347 Table 3. SAR for SPIONS in water and hydrogel 7 and 8 (0.8 wt%) loaded with SPIONs (25–35% 30 325 100 305 m/m). 35 227 65 240 SAR (w/g) SPIONs (%) The specific absorptionSPIONs rate SAR in Water (W/g) parameter SPIONs in was Hydr. calculated 7 SPIONs by the in initial Hydr. slope 8 method from the magnetic25 hyperthermia curves357 (Table3, Table SII) 161 [14]. 347 The SPIONs in water display a SAR value (300 W/g; H = 240 G, f = 869 kHz), within the range expected for Fe3O4 nanoparticles with a core size around 8 nm [50]. Whereas incorporation of SPIONs into hydrogels 7 leads to a very substantial reduction (over 50%) of their efficacy (SAR) as heat generators, hydrogel 8 has no effect on the SAR. The heating properties of SPIONs activated by an oscillating magnetic field result from Néel and Brownian relaxation of the magnetic moment of the nanoparticles [8,9]. The significant decrease of SAR for the SPIONs incorporated into the hydrogels 7 is likely to result from a reduction of the contribution of the Brownian relaxation mechanism to the overall relaxation. Néel relaxation, less effective than Brown relaxation for small magnetite nanoparticles, presumably becomes the dominating heat generating mechanism when the SPIONs are incorporated into hydrogel [8]. This suggests that the SPIONs incorporated into hydrogels 7 have less mobility than when incorporated into hydrogels 8. SPIONs incorporated into hydrogels 8 seem to be in a quasi aqueous environment. The rheological experiments showed that the elasticity of both hydrogels 7 Nanomaterials 2019, 9, x FOR PEER REVIEW 11 of 16

30 325 100 305 35 227 65 240

The SPIONs in water display a SAR value (300 W/g; H = 240 G, f = 869 kHz), within the range expected for Fe3O4 nanoparticles with a core size around 8 nm [50]. Whereas incorporation of SPIONs into hydrogels 7 leads to a very substantial reduction (over 50%) of their efficacy (SAR) as heat generators, hydrogel 8 has no effect on the SAR. The heating properties of SPIONs activated by an oscillating magnetic field result from Néel and Brownian relaxation of the magnetic moment of the nanoparticles [8,9]. The significant decrease of SAR for the SPIONs incorporated into the hydrogels 7 is likely to result from a reduction of the contribution of the Brownian relaxation mechanism to the overall relaxation. Néel relaxation, less effective than Brown relaxation for small magnetite nanoparticles, presumably becomes the dominating heat generating mechanism when the SPIONs are incorporated into hydrogel [8]. This suggests that the SPIONs incorporated into hydrogels 7 have less mobility than when incorporated into hydrogels 8. SPIONs incorporated into hydrogelsNanomaterials 82019 seem, 9, 541 to be in a quasi aqueous environment. The rheological experiments showed11 that of 16 the elasticity of both hydrogels 7 and 8 is reduced by incorporation of SPIONs, suggesting that the SPIONsand 8 is reducedswell the by mesh incorporation size of the of hydrogel SPIONs, fibril suggesting network. that As the this SPIONs effect swell is more the meshpronounced size of thefor hydrogel ﬁbril7 the network.hydrogel Aswater this pools effect are is likely more to pronounced be bigger for for hydrogel hydrogel 87. theThis hydrogel result is waterin accordance pools are alsolikely with to be the bigger higher for relaxivity hydrogel measur8. Thised result for SPIONs is in accordance incorporated also with in hydrogel the higher 8. Using relaxivity hydrogelator measured concentrationsfor SPIONs incorporated 0.8 wt% and in hydrogela SPIONs8 content. Using of hydrogelator 25%–35% magnetic concentrations excitation 0.8 wt%(H = and240 aG; SPIONs f = 869 kHz)content is ofstill 25%–35% efficient magnetic in inducing excitation a phase (H =transition 240 G; f =that 869 kHz)leads isto still hydrogel efﬁcient collapse. in inducing Hence, a phase the hydrogelstransition thatloaded leads with to hydrogel SPIONs collapse. can be Hence,potential the drug hydrogels carriers loaded for withmagnetically-triggered SPIONs can be potential drug delivery.drug carriers for magnetically-triggered drug delivery.

3.9. Biological Studies The NSAID properties of of naproxen naproxen are are well well establ establishedished in in the the clinical clinical practice. practice. Naproxen Naproxen is isa potenta potent non-selective non-selective inhibitor inhibitor of ofboth both COX-1 COX-1 and and COX-2 COX-2 enzymes enzymes [51]. [51 ].Hydrogelator Hydrogelator molecules molecules 7 and7 and 8 8areare unique unique molecular molecular entities, entities, but but can can also also be beconsidered considered naproxen naproxen conjugates conjugates with with a dehydrodipeptidea dehydrodipeptide [52]. [52 In]. Inthis this work work we we eval evaluateduated the the toxicity toxicity of ofhydrogelator hydrogelator molecules molecules 7 and7 and 8 in8 vitroin vitro towardstowards macrophage macrophage cells cells (RAW (RAW 264.7 264.7 cell cell line) line) and and also also their their potential anti-inﬂammatoryanti-inflammatory properties. As As shown shown in in Figure Figure 6,6 ,both both molecules molecules are are non-toxic non-toxic in in concentrations concentrations up up to to100 100 µM.µ M.In additionIn addition to macrophages, to macrophages, we also we evaluated also evaluated the toxicity the toxicity of the ofhydrogelators the hydrogelators towards towards the human the non-cancerhuman non-cancer fibroblast ﬁbroblast cell line cell MRC-5 line MRC-5 (Figure (Figure S10). S10). No Notoxicity toxicity has has been been observed observed in in this concentration range.

A B

5 l 5 ol 25 50 2 25 50 r 100 tro 100 6.25 12. n 6. 12.5 Cont Co

Figure 6. Viability ofof RAWRAW 264.7 264.7 cells cells incubated incubated with with hydrogelators hydrogelators7 ( A7 )(A and) and8 ( B8). (B Data). Data represent represent the ± themean meanstandard ± standard deviation deviation of the meanof the of mean three independentof three independent experiments, experiments, performed inperformed triplicate. in triplicate. As experimental model for the evaluation of anti-inflammatory activity, we have used RAW 264.7 cells challenged with the pro-inflammatory molecule lipopolysaccharide (LPS), a molecule As experimental model for the evaluation of anti-inflammatory activity, we have used RAW derived from Gram negative bacteria, which elicits a number of genetic, biochemical and morphological 264.7 cells challenged with the pro-inflammatory molecule lipopolysaccharide (LPS), a molecule changes in macrophages that result in a pro-inflammatory phenotype. Among the pro-inflammatory mediators that are upregulated, nitric oxide (NO) is the most widely used for screening proposes. Incubation of LPS-challenged RAW 264.7 cells with compounds 7 and 8 significantly decreased NO levels when compared with non-treated cells (Figure7). While both molecules displayed a concentration-dependent effect, compound 8 displayed higher potency, as it showed capacity to lower NO levels in cells in concentrations as low as 25 µM and resulted in over a 50% reduction at the highest concentration tested. In addition to the effect found towards NO levels, we were interested in evaluating the ability of these molecules to inhibit key enzymes in the inflammatory cascade, namely 5-lipooxygenase, which is involved in the synthesis of leukotrienes, and cyclooxygenase-2, which is involved in the synthesis of prostaglandins. In fact, dual inhibition COX/LOX has been proposed as a smart strategy to optimize the anti-inflammatory activity of NSAID inhibitors [53]. At the highest concentration that was shown to be devoid of toxicity, 100 µM, both hydrogelators 7 and 8 almost completely inhibited the activity of LOX (Figure8). Nanomaterials 2019, 9, x FOR PEER REVIEW 12 of 16 derived from Gram negative bacteria, which elicits a number of genetic, biochemical and morphological changes in macrophages that result in a pro-inflammatory phenotype. Among the pro-inflammatory mediators that are upregulated, nitric oxide (NO) is the most widely used for screening proposes. Incubation of LPS-challenged RAW 264.7 cells with compounds 7 and 8 significantly decreased NO levels when compared with non-treated cells (Figure 7). While both molecules displayed a concentration-dependent effect, compound 8 displayed higher potency, as it showed capacity to lower NO levels in cells in concentrations as low as 25 µM and resulted in over a 50% reduction at Nanomaterials 2019, 9, x FOR PEER REVIEW 12 of 16 the highest concentration tested. derived from Gram negative bacteria, which elicits a number of genetic, biochemical and morphological changes in macrophages that result in a pro-inflammatory phenotype. Among the pro-inflammatory mediators that are upregulated, nitric oxide (NO) is the most widely used for screening proposes. Incubation of LPS-challenged RAW 264.7 cells with compounds 7 and 8 significantly decreased NO levels when compared with non-treated cells (Figure 7). While both molecules displayed a concentration-dependent effect, compound 8 displayed higher potency, as it showed capacity to lower NO levels in cells in concentrations as low as 25 µM and resulted in over a 50% reduction at theNanomaterials highest 2019concentration, 9, 541 tested. 12 of 16

Figure 7. Effect of compounds 7 (A) and 8 (B) in the NO levels of LPS-challenged RAW 264.7 cells. Data represent the mean ± standard deviation of the mean of three independent experiments, performed in triplicate. * p < 0.05, *** p < 0.001, **** p < 0.0001.

In addition to the effect found towards NO levels, we were interested in evaluating the ability of these molecules to inhibit key enzymes in the inflammatory cascade, namely 5-lipooxygenase, which is involved in the synthesis of leukotrienes, and cyclooxygenase-2, which is involved in the synthesis of prostaglandins. In fact, dual inhibition COX/LOX has been proposed as a smart strategy to optimize the anti-inflammatory activity of NSAID inhibitors [53]. At the highest concentration that Figurewas shown 7. Effect to of be compounds devoid of7 ( Atoxicity,) and 8 ( B100) in theµM, NO both levels hydrogelators of LPS-challenged 7 and RAW 8 264.7almost cells. completely Data Figure 7. Effect of compounds 7 (A) and 8 (B) in the NO levels of LPS-challenged RAW 264.7 cells. inhibitedrepresent the theactivity mean of± LOXstandard (Figure deviation 8). of the mean of three independent experiments, performed in Data represent the mean ± standard deviation of the mean of three independent experiments, triplicate. * p < 0.05, *** p < 0.001, **** p < 0.0001. performed in triplicate. * p < 0.05, *** p < 0.001, **** p < 0.0001.

to optimizeActivity (%) the anti-inflammatory activity of NSAID inhibitors [53]. At the highest concentration that was shown to be devoid of toxicity, 100 µM, both hydrogelators 7 and 8 almost completely inhibited the activity of LOX (Figure 8).

l 6b tro H5 n o C Figure 8. Inhibitory effects of 7 and 8 towards 5-LOX activity. Data represents the mean ± standard Figure 8. Inhibitory effects of 7 and 8 towards 5-LOX activity. Data represents the mean ± standard deviation of the mean of three independent experiments, performed in triplicate. *** p < 0.001, deviation of the mean of three independent experiments, performed in triplicate. *** p < 0.001, **** p < **** p < 0.0001. 0.0001. The inhibitory activity of compounds 7 and 8 towards COX-1 and COX-2 enzymes was shown to beThe moderate inhibitory at the activity concentrations of compounds studied. 7 and It is 8 interestingtowards COX-1 to note and that COX-2 compounds enzymes7 andwas8 shownshow someto be moderate degreeActivity (%) of at selectivity the concentrations towards the studied. enzyme It is COX-2, interesting while to naproxen note that iscompounds non-selective 7 and [19 8]. show This issome particularly degree of important selectivity iftowards we consider the enzyme that inhibition COX-2, while of COX-1 naproxen is associated is non-selective with many [19]. This of the is particularly important if we consider that inhibition of COX-1 is associated with many of the side-effects of NSAIDs [54]. l 6b tro H5 n side-effects of NSAIDs [54]. o Compounds 7 and 8 show interesting anti-inflammatoryC properties, demonstrating that nontoxic hydrogelatorFigure 8. Inhibitory molecules effects can combine of 7 and a8 structuraltowards 5-LOX function activity. with Data intrinsic represents pharmacological the mean ± standard activity. deviation of the mean of three independent experiments, performed in triplicate. *** p < 0.001, **** p < 4. Conclusions 0.0001. In this work we demonstrate that SPIONs incorporated into dehydrodipeptide-based self-assembledThe inhibitory hydrogels activity retain of compounds their magnetic 7 and properties 8 towards despite COX-1 the and strong COX-2 diamagnetic enzymes contributionwas shown tofrom be themoderate organic at matrix the concentrations of the hydrogel. studied. Magnetic It ishydrogels interesting with to note a significant, that compounds SPIONs concentration-7 and 8 show somedependent, degree dualof selectivityT1/T2 MRI towards contrast the enzyme were obtained, COX-2, suggestingwhile naproxen that is the non-selective fate hydrogels [19].7 Thisand is8 particularlyimplanted in important vivo could if be we monitored consider by that MRI. inhibition Moreover, of upon COX-1 magnetic is associated excitation with with many an AMF of the side-effectsSPIONs generated of NSAIDs a significant [54]. amount of heat, sufficient to induce in vitro a gel to solution phase transition of hydrogels 7 and 8. Thus, we propose that MH could be used as a remote trigger for release of SPIONs and drug cargos incorporated into dehydrodipeptide hydrogels. Nontoxic hydrogelator molecules can combine a structural function with intrinsic pharmacological activity allowing combined hyperthermia with multi-drug therapy and imaging. The AMF-triggered release of SPIONs from the magnetic hydrogel could be used as a self-reporting mechanism (darker MRI contrast as consequence of the recovery of the original SPIONs transverse relaxivity, r2), constituting as a step forward towards image-guided drug delivery and the design of more efficient theragnostic platforms. Nanomaterials 2019, 9, 541 13 of 16

Supplementary Materials: The Supplementary Materials are available online at http://www.mdpi.com/2079- 4991/9/4/541/s1. Author Contributions: Conceptualization, J.A.M. and P.M.T.F.; methodology, D.M.P., M.B.-L., L.H., J.A.M. and P.M.T.F. investigation, A.C., J.G. and L.H.; writing—original draft preparation, D.M.P., J.A.M., L.H. and M.B.-L.; writing—review and editing, D.M.P., P.V. and P.B.A. supervision, J.A.M. and M.B.-L.; project administration, P.M.T.F. and J.A.M.; funding acquisition, D.M.P., P.V., P.B.A., M.B.-L., L.H., P.M.T.F. and J.A.M. Funding: This work is funded by National Funds through FCT-Portuguese Foundation for Science and Technology under the Project PTDC/QUI-QOR/29015/2017 and CQ/UM UID/QUI/00686/2013 and UID/QUI/0686/2016. The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Acknowledgments: This work is funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT-Portuguese Foundation for Science and Technology under the Project UID/CTM/50025/2013. L.H. acknowledges the support from FCT through Investigador FCT contract IF/00606/2014. MB-L also thanks the NORTE 2020 (2014–2020 North Portugal Regional Operational Programme) and the ERDF (European Regional Development Fund) under the Grant NORTE-45-2015-02, and also the National Foundation for Science and Technology (FCT) for support, under the scope of the projects PT-DZ/0004/2015 (Magnetic nanocomposite hydrogels from biopolymers as smart delivery systems) and 028052 (SELF-I, Self-reporting immunostimulating formulation for on-demand cancer therapy with realtime treatment response monitoring). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Kolen’Ko, Y.V.; Bañobre-López, M.; Rodríguez-Abreu, C.; Carbó-Argibay, E.; Sailsman, A.; Piñeiro- Redondo, Y.; Cerqueira, M.F.; Petrovykh, D.Y.; Kovnir, K.; Lebedev, O.I.; et al. Large-scale synthesis of

colloidal Fe3O4 nanoparticles exhibiting high heating efficiency in magnetic hyperthermia. J. Phys. Chem. C 2014, 118, 8691–8701. [CrossRef] 2. Piñeiro-Redondo, Y.; Bañobre-López, M.; Pardiñas-Blanco, I.; Goya, G.; López-Quintela, M.A.; Rivas, J. The influence of colloidal parameters on the specific power absorption of PAA-coated magnetite nanoparticles. Nanoscale Res. Lett. 2011, 6, 383. [CrossRef][PubMed] 3. Gao, J.; Gu, H.; Xu, B. Multifunctional Magnetic Nanoparticles: Design, Synthesis, and Biomedical Applications. Acc. Chem. Res. 2009, 42, 1097–1107. [CrossRef] 4. Caravan, P.; Ellison, J.J.; Mcmurry, T.J.; Lauffer, R.B. Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 1999, 99, 2293–2352. [CrossRef] 5. Laurent, S.; Bhujwalla, Z.M.; Muller, R.N.; Qin, J.; Jo, Y.S.; Mikhaylova, M.; Muhammed, M.; Roch, A. A High-Performance Magnetic Resonance ImagingT2 Contrast Agent. Adv. Mater. 2007, 19, 1874–1878. 6. Muller, R.N.; Port, M.; Vander Elst, L.; Forge, D.; Robic, C.; Laurent, S.; Roch, A. Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations, and Biological Applications. Chem. Rev. 2008, 108, 2064–2110. 7. Park, J.W.; Bae, K.H.; Kim, C.; Park, T.G. Clustered magnetite nanocrystals cross-linked with PEI for efficient siRNA delivery. Biomacromolecules 2011, 12, 457–465. [CrossRef] 8. Pineiro, Y.; Vargas-Osorio, Z.; Banobre-Lopez, M.; Kolen’Ko, Y.V.; Lopez-Quintela, M.A.; Rivas, J. Relevant parameters for magnetic hyperthermia in biological applications: Agglomeration, concentration, and viscosity. IEEE Trans. Magn. 2016, 52, 18–21. [CrossRef] 9. Ribeiro, C.; Cardoso, V.F.; Francesko, A.; Martins, P.; Bañobre-López, M.; Lanceros-Mendez, S. Advances in Magnetic Nanoparticles for Biomedical Applications. Adv. Healthc. Mater. 2017, 7, 1700845. 10. Hildebrandt, B.; Wust, P.; Ahlers, O.; Dieing, A.; Sreenivasa, G.; Kerner, T.; Felix, R.; Riess, H. The cellular and molecular basis of hyperthermia. Crit. Rev. Oncol. Hematol. 2002, 43, 33–56. [CrossRef] 11. Gneveckow, U.; Jordan, A.; Scholz, R.; Brüß, V.; Waldöfner, N.; Ricke, J.; Feussner, A.; Hildebrandt, B.; Rau, B.; Wust, P. Description and characterization of the novel hyperthermia- and thermoablation-system MFH®300F for clinical magnetic fluid hyperthermia. Med. Phys. 2004, 31, 1444–1451. [CrossRef] 12. Arruebo, M.; Vilaboa, N.; Sáez-Gutierrez, B.; Lambea, J.; Tres, A.; Valladares, M.; González-Fernández, Á. Assessment of the evolution of cancer treatment therapies. Cancers 2011, 3, 3279–3330. [CrossRef][PubMed] 13. Rosensweig, R.E. Heating magnetic fluid with alternating magnetic field. J. Magn. Magn. Mater. 2002, 252, 370–374. [CrossRef] Nanomaterials 2019, 9, 541 14 of 16

14. Kim, J.; Kim, I.-S.; Choi, J.; Jang, J.; Park, K.I.; Cheon, J.; Kim, J.-G.; Lee, J.-H.; Moon, S.H.; Noh, S. Exchange-coupled magnetic nanoparticles for efficient heat induction. Nat. Nanotechnol. 2011, 6, 418–422. 15. Nemati, Z.; Alonso, J.; Rodrigo, I.; Das, R.; Garaio, E.; García, J.Á.; Orue, I.; Phan, M.H.; Srikanth, H. Improving the Heating Efficiency of Iron Oxide Nanoparticles by Tuning Their Shape and Size. J. Phys. Chem. C 2018, 122, 2367–2381. [CrossRef] 16. Das, R.; Alonso, J.; Nemati Porshokouh, Z.; Kalappattil, V.; Torres, D.; Phan, M.H.; Garaio, E.; García, J.Á.; Sanchez Llamazares, J.L.; Srikanth, H. Tunable High Aspect Ratio Iron Oxide Nanorods for Enhanced Hyperthermia. J. Phys. Chem. C 2016, 120, 10086–10093. [CrossRef] 17. Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165–13307. [CrossRef] 18. Panda, J.J.; Mishra, A.; Basu, A.; Chauhan, V.S. Stimuli Responsive Self-Assembled Hydrogel of a Low Molecular Weight Free Dipeptide with Potential for Tunable Drug Delivery. Biomacromolecules 2008, 9, 2244–2250. [CrossRef][PubMed] 19. Li, J.; Kuang, Y.; Gao, Y.; Du, X.; Shi, J.; Xu, B. D-amino acids boost the selectivity and confer supramolecular hydrogels of a nonsteroidal anti-inflammatory drug (NSAID). J. Am. Chem. Soc. 2013, 135, 542–545. [CrossRef][PubMed] 20. Yang, Z.; Liang, G.; Xu, B. Enzymatic hydrogelation of small molecules. Acc. Chem. Res. 2008, 41, 315–326. [CrossRef] 21. Ehrbar, M.; Rizzi, S.C.; Schoenmakers, R.G.; San Miguel, B.; Hubbell, J.A.; Weber, F.E.; Lutoff, M.P. Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions. Biomacromolecules 2007, 8, 3000–3007. [CrossRef] 22. Fleming, S.; Ulijn, R.V. Design of nanostructures based on aromatic peptide amphiphiles. Chem. Soc. Rev. 2014, 43, 8150–8177. [CrossRef][PubMed] 23. Vilaça, H.; Pereira, G.; Castro, T.G.; Hermenegildo, B.F.; Shi, J.; Faria, T.Q.; Micaêlo, N.; Brito, R.M.M.; Xu, B.; Castanheira, E.M.S.; et al. New self-assembled supramolecular hydrogels based on dehydropeptides. J. Mater. Chem. B 2015, 3, 6355–6367. [CrossRef] 24. Vilaça, H.; Castro, T.; Costa, F.M.G.; Melle-Franco, M.; Hilliou, L.; Hamley, I.W.; Castanheira, E.M.S.; Martins, J.A.; Ferreira, P.M.T. Self-assembled RGD dehydropeptide hydrogels for drug delivery applications. J. Mater. Chem. B 2017, 5, 8607–8617. [CrossRef] 25. Vilaça, H.; Hortelão, A.C.L.; Castanheira, E.M.S.; Queiroz, M.J.R.P.; Hilliou, L.; Hamley, I.W.; Martins, J.A.; Ferreira, P.M.T. Dehydrodipeptide Hydrogelators Containing Naproxen N-Capped Tryptophan: Self-Assembly, Hydrogel Characterization, and Evaluation as Potential Drug Nanocarriers. Biomacromolecules 2015, 16, 3562–3573. [CrossRef] 26. Dionigi, C.; Lungaro, L.; Goranov, V.; Riminucci, A.; Piñeiro-Redondo, Y.; Bañobre-López, M.; Rivas, J.; Dediu, V. Smart magnetic poly(N-isopropylacrylamide) to control the release of bio-active molecules. J. Mater. Sci. Mater. Med. 2014, 25, 2365–2371. [CrossRef][PubMed] 27. Jaiswal, M.K.; De, M.; Chou, S.S.; Vasavada, S.; Bleher, R.; Prasad, P.V.; Bahadur, D.; Dravid, V.P. Thermoresponsive magnetic hydrogels as theranostic nanoconstructs. ACS Appl. Mater. Interfaces 2014, 6, 6237–6247. [CrossRef] 28. Yang, Z.; Gu, H.; Du, J.; Gao, J.; Zhang, B.; Zhang, X.; Xu, B. Self-assembled hybrid nanofibers confer a magnetorheological supramolecular hydrogel. Tetrahedron 2007, 63, 7349–7357. [CrossRef] 29. Contreras-Montoya, R.; Bonhome-Espinosa, A.B.; Orte, A.; Miguel, D.; Delgado-López, J.M.; Duran, J.D.G.; Cuerva, J.M.; Lopez-Lopez, M.T.; Álvarez de Cienfuegos, L. Iron nanoparticles-based supramolecular hydrogels to originate anisotropic hybrid materials with enhanced mechanical strength. Mater. Chem. Front. 2018, 2, 686–699. [CrossRef] 30. Pereira, D.M.; Correia-da-Silva, G.; Valentão, P.; Teixeira, N.; Andrade, P.B. Anti-inflammatory effect of unsaturated fatty acids and ergosta-7,22-dien-3-ol from Marthasterias glacialis: Prevention of CHOP-mediated ER-stress and NF-κB activation. PLoS ONE 2014, 9, e88341. [CrossRef] 31. Pereira, R.B.; Pinto, D.C.G.A.; Pereira, D.M.; Gomes, N.G.M.; Silva, A.M.S.; Andrade, P.B.; Valentão, P. UHPLC-MS/MS profiling of Aplysia depilans and assessment of its potential therapeutic use: Interference on iNOS expression in LPS-stimulated RAW 264.7 macrophages and caspase-mediated pro-apoptotic effect on SH-SY5Y cells. J. Funct. Foods 2017, 37, 164–175. [CrossRef] Nanomaterials 2019, 9, 541 15 of 16

32. Ramagopal, U.; Ramakumar, S.; Sahal, D.; Chauhan, V.S. De novo design and characterization of an apolar helical hairpin peptide at atomic resolution: Compaction mediated by weak interactions. Proc. Natl. Acad. Sci. USA 2001, 98, 870–874. [CrossRef][PubMed] 33. Greenfield, N.J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2007, 1, 2876–2890. [CrossRef] 34. Liu, M.; Zhang, L.; Wang, T. Supramolecular Chirality in Self-Assembled Systems. Chem. Rev. 2015, 115, 7304–7397. [CrossRef] 35. Pritchard, R.H.; Shery Huang, Y.Y.; Terentjev, E.M. Mechanics of biological networks: From the cell cytoskeleton to connective tissue. Soft Matter 2014, 10, 1864–1884. [CrossRef][PubMed] 36. Dobrynin, A.V.; Carrillo, J.M.Y. Universality in nonlinear elasticity of biological and polymeric networks and gels. Macromolecules 2011, 44, 140–146. [CrossRef] 37. Gisler, T.; Ball, R.C.; Weitz, D.A. Strain hardening of fractal colloidal gels. Phys. Rev. Lett. 1999, 82, 1064–1067. [CrossRef] 38. Hemar, Y.; Yang, Z.; Hilliou, L.; Williams, M.A.K.; Chaieb, S.; McGillivray, D.J.; Gilbert, E.P. Nonlinear Behavior of Gelatin Networks Reveals a Hierarchical Structure. Biomacromolecules 2015, 17, 590–600. 39. Hilliou, L.; Wilhelm, M.; Yamanoi, M.; Gonçalves, M.P. Structural and mechanical characterization of κ/ι-hybrid carrageenan gels in potassium salt using Fourier Transform rheology. Food Hydrocoll. 2009, 23, 2322–2330. [CrossRef] 40. Krishnan, K.M. Biomedical nanomagnetics: A spin through possibilities in imaging, diagnostics, and therapy. IEEE Trans. Magn. 2010, 46, 2523–2558. [CrossRef] 41. Mohapatra, J.; Mitra, A.; Bahadur, D.; Aslam, M. Surface controlled synthesis of MFe2O4 (M = Mn, Fe, Co, Ni and Zn) nanoparticles and their magnetic characteristics. CrystEngComm 2013, 15, 524–532. [CrossRef] 42. Cullity, B.D.; Graham, C.D. Introduction to Magnetic Materials, 2nd ed.; John Wiley & Sons: Hoboken, NJ, USA, 2008. 43. Martínez-Junza, V.; Fibikar, S.; De Cola, L.; Luppi, G.; Clemente-León, M. Manipulation and Orientation of Zeolite L by Using a Magnetic Field. ChemPlusChem 2014, 80, 62–67. 44. Knobel, M.; Nunes, W.C.; Winnischofer, H.; Rocha, T.C.R.; Socolovsky, L.M.; Mayorga, C.L.; Zanchet, D. Effects of magnetic interparticle coupling on the blocking temperature of ferromagnetic nanoparticle arrays. J. Non. Cryst. Solids 2007, 353, 743–747. [CrossRef] 45. Laurent, S.; Saei, A.A.; Behzadi, S.; Panahifar, A.; Mahmoudi, M. Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: Opportunities and challenges. Expert Opin. Drug Deliv. 2014, 11, 1449–1470. [CrossRef] 46. Bao, Y.; Sherwood, J.A.; Sun, Z. Magnetic iron oxide nanoparticles as: T 1 contrast agents for magnetic resonance imaging. J. Mater. Chem. C 2018, 6, 1280–1290. [CrossRef] 47. Guldris, N.; Argibay, B.; Kolen’ko, Y.V.; Carbó-Argibay, E.; Sobrino, T.; Campos, F.; Salonen, L.M.; Bañobre-López, M.; Castillo, J.; Rivas, J. Influence of the separation procedure on the properties of magnetic nanoparticles: Gaining in vitro stability and T1-T2 magnetic resonance imaging performance. J. Colloid Interface Sci. 2016, 472, 229–236. [CrossRef] 48. Thorek, D.L.J.; Chen, A.K.; Czupryna, J.; Tsourkas, A. Superparamagnetic iron oxide nanoparticle probes for molecular imaging. Ann. Biomed. Eng. 2006, 34, 23–38. [CrossRef] 49. Noh, S.H.; Na, W.; Jang, J.T.; Lee, J.H.; Lee, E.J.; Moon, S.H.; Lim, Y.; Shin, J.S.; Cheon, J. Nanoscale magnetism control via surface and exchange anisotropy for optimized ferrimagnetic hysteresis. Nano Lett. 2012, 12, 3716–3721. [CrossRef][PubMed] 50. Tada, M.; Kallumadil, M.; Abe, M.; Pankhurst, Q.A.; Southern, P.; Nakagawa, T. Suitability of commercial colloids for magnetic hyperthermia. J. Magn. Magn. Mater. 2009, 321, 1509–1513. 51. Duggan, K.C.; Walters, M.J.; Harp, J.M.; Kiefer, J.R.; Oates, A.; Marnett, L.J.; Duggan, K.C.; Walters, M.J.; Musee, J.; Harp, J.M.; et al. Molecular Basis for Cyclooxygenase Inhibition by the Non-steroidal Anti-inflammatory Drug Naproxen. J. Biol. Chem. 2010, 285, 34950–34959. [CrossRef][PubMed] 52. Li, J.; Kuang, Y.; Shi, J.; Gao, Y.; Zhou, J.; Xu, B. The conjugation of nonsteroidal anti-inflammatory drugs (NSAID) to small peptides for generating multifunctional supramolecular nanofibers/hydrogels. Beilstein J. Org. Chem. 2013, 9, 908–917. [CrossRef][PubMed] Nanomaterials 2019, 9, 541 16 of 16

53. Charlier, C.; Michaux, C. Dual inhibition of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) as a new strategy to provide safer non-steroidal anti-inﬂamatory drugs. Eur. J. Med. Chem. 2003, 38, 645–659. [CrossRef] 54. Vane, S.J.R.; Botting, J. Selective COX-2 Inhibitors: Pharmacology, Clinical Effects and Therapeutic Potential; Springer Science & Business Media: Berlin, Germany, 2012.

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