Injectable Thermoresponsive Microparticle/Hydrogel System with Superparamagnetic Nanoparticles for Drug Release and Magnetic Hyperthermia Applications

Citation: Carrelo, H.; Escoval, A.R.; Vieira, T.; Jiménez-Rosado, M.; Silva, J.C.; Romero, A.; Soares, P.I.P.; Borges, J.P. Injectable Thermoresponsive Microparticle/Hydrogel System with Superparamagnetic Nanoparticles for Drug Release and Magnetic Hyperthermia Applications. Gels 2023,9, 982. https://doi.org/ 10.3390/gels9120982 Academic Editor: Kai Liu Received: 10 November 2023 Revised: 7 December 2023 Accepted: 11 December 2023 Published: 15 December 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). gels Article Injectable Thermoresponsive Microparticle/Hydrogel System with Superparamagnetic Nanoparticles for Drug Release and Magnetic Hyperthermia Applications Henrique Carrelo 1, AndréR. Escoval 1, Tânia Vieira 2, Mercedes Jiménez-Rosado 3, Jorge Carvalho Silva 2,* , Alberto Romero 4, Paula Isabel P. Soares 1,* and João Paulo Borges 1,* 1CENIMAT/i3N, Department of Materials Science, NOVA School of Science and Technology (FCT NOVA), Campus de Caparica, 2829-516 Caparica, Portugal; [email protected] (H.C.) 2 CENIMAT/i3N, Department of Physics, NOVA School of Science and Technology (FCT NOVA), Campus de Caparica, 2829-516 Caparica, Portugal 3Department of Applied Chemistry and Physics, Universidad de León, 24007 Leon, Spain 4Department of Chemical Engineering, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain; alromer[email protected] *Correspondence: [email protected] (J.C.S.); [email protected] (P.I.P.S.); [email protected] (J.P.B.) Abstract: Cancer is a disease that continues to greatly impact our society. Developing new and more personalized treatment options is crucial to decreasing the cancer burden. In this study, we combined magnetic polysaccharide microparticles with a Pluronic thermoresponsive hydrogel to develop a multifunctional, injectable drug delivery system (DDS) for magnetic hyperthermia applications. Gellan gum and alginate microparticles were loaded with superparamagnetic iron oxide nanoparticles (SPIONs) with and without coating. The magnetic microparticles’ registered temperature increases up to 4 ◦ C upon the application of an alternating magnetic field. These magnetic microparticles were mixed with drug-loaded microparticles, and, subsequently, this mixture was embedded within a Pluronic thermoresponsive hydrogel that is capable of being in the gel state at 37 ◦ C. The proposed DDS was capable of slowly releasing methylene blue, used as a model drug, for up to 9 days. The developed hydrogel/microparticle system had a smaller rate of drug release compared with microparticles alone. This system proved to be a potential thermoresponsive DDS suitable for magnetic hyperthermia applications, thus enabling a synergistic treatment for cancer. Keywords: hydrogels; Pluronic; microparticles; rheology 1. Introduction Cancer is still one of the deadliest diseases that affect humankind. Many types of therapies can be performed to treat cancer, such as immunotherapy, radiation therapy, hormone therapy, hyperthermia, and chemotherapy. Chemotherapy has many severe side effects that decrease the patient’s quality of life [ 1 , 2 ]. In situ drug delivery systems (DDSs) that can be placed in tumor areas (pre- or post-tumor removal surgery) might be a possible innovative treatment option [ 3 , 4 ]. A DDS that can be inserted in a tumorous area and progressively release necessary bioactive agents that can treat and/or even prevent cancer reoccurrence is a possibility that needs to be explored [ 4 ]. Hydrogels are good candidates for injectable in situ DDSs [ 5 – 8 ]. They can be composed of multiple types of polymers, including biocompatible and biodegradable ones. Hydrogels can be stimuliresponsive, where they can change their properties and behavior in response to external stimuli, including temperature, which are known as thermoresponsive hydrogels [ 9 ]. These have been used as injectable systems in the human body since they can form a stable matrix in vivo , serve as transport for bioactive agents, and also have the advantage of adjusting their shape to the injected area [ 10 ]. One viable option to control the release profile from Gels 2023,9, 982. https://doi.org/10.3390/gels9120982 https://www.mdpi.com/journal/gels Gels 2023,9, 982 2 of 20 a hydrogel’s DDS is using polymeric microparticles that can be embedded within the structure of a thermoresponsive hydrogel to regulate and prolong the release profile of the system for one or multiple drugs [10–13]. To improve cancer treatments, magnetic nanoparticles (mNPs) can also be used in DDSs, for example, as carriers of therapeutic agents or combined with polymeric microparticles using them as carriers [ 14 – 16 ]. Magnetic nanoparticles can be sensitive to alternating magnetic fields (AMFs), for example, superparamagnetic iron oxide nanoparticles (SPI- ONs). With the application of an AMF, mNPs are capable of increasing the temperature to ranges that lead to the apoptosis of tumorous cells (from around 41 ◦ C to 46 ◦ C) [ 17 ]. These can also be used as contrast agents in magnetic resonance imaging [ 18 ]. The combination of mNPs and polymeric structures has been conducted in previous studies [ 16 , 19 ]. Matos et al. [ 20 ] combined mNPs with nanofibrous membranes of cellulose acetate produced by an electrospinning technique. These membranes were able to increase the surrounding temperature upon the application of an AMF and exhibited no significant cytotoxicity. Regarding the encapsulation/adsorption of mNPs into polymeric microparticles, Alpdemir et al. [ 21 ] developed alginate microparticles with mNPs for sorafenib release in cancer treatments. The mNPs conferred hyperthermic capabilities to the polymeric microparticles, leading to an increase in temperature of 4 ◦ C. Xue et al. [ 22 ] encapsulated mNPs within alginate–chitosan microspheres for doxorubicin (DOX) release in breast cancer treatments, where the application of a remote AMF affected the DOX release, enabling the regulation of release profiles. For a more controlled drug release, thermoresponsive hydrogels can be used for transportation and as a protective barrier that can delay the drug release from drug-loaded microparticles [ 10 ]. In our previous work [ 23 ], we developed an injectable DDS composed of alginate/gellan gum microparticles within a Pluronic thermoresponsive hydrogel. The combination of mNPs and a similar microparticle/hydrogel system can be used as a possible injectable system for drug release and magnetic hyperthermia applications in possible cancer treatments. Previous work focused on the combination of Pluronic and SPIONs [ 24 ]. Other work focused on the combination of mNPs with microparticles [ 25 , 26 ], but the inclusion of microparticles and SPIONs into a thermoresponsive hydrogel for a DDS has, as far as we know, not been studied. In this work, we developed polymeric microparticles with superparamagnetic iron oxide nanoparticles (SPIONs) and then included them in a multifunctional DDS composed of a microparticle/hydrogel injectable system. We aimed to confer hyperthermic capabilities to this system by adsorbing SPIONs onto the microparticles’ surface and then using them in the injectable DDS. Thus, in this study, we developed microparticles loaded with SPIONs and microparticles that were loaded with a model drug (methylene blue (MB) [ 27 ]) separately and then combined the two batches. The two batches were mixed in a 1:1 ratio. This can be useful to alter drug and mNP quantities in future studies by altering this ratio. We used bare SPIONs and SPIONs coated with 3-aminopropyl triethoxylsilane (SPIAPTS). With this study, we developed a novel DDS that combines magnetic microparticles with thermoresponsive hydrogels and has the potential for magnetic hyperthermia applications, which makes it a potential candidate for future synergistic cancer treatments. 2. Results and Discussion 2.1. Introduction of mNPs to GG:Alg Microparticles Figure 1a,b depict a TEM analysis of mNPs. Using these images, it was found that the average size of the iron core of the SPIONs was 8.59 ± 2.03 nm (Figure 1c). For SPIAPTS, the diameter was 7.55 ± 1.37 nm (Figure 1d). A difference between the average and the d(0.5) was noticed; however, they were close in both types of nanoparticles. For the two types of nanoparticles, a one-way ANOVA revealed a statistically significant difference (F(1, 370) = [32.17], p= 2.86 × 10 −8 ). The smaller diameters obtained for the SPIAPTS might be related to the smaller aggregation that the APTES coating confers, thus allowing for a better view of the nanoparticles in TEM. Gels 2023,9, 982 3 of 20 Gels 2023, 9, x FOR PEER REVIEW 3 of 21 (F(1, 370) = [32.17], p = 2.86 × 10 −8 ). The smaller diameters obtained for the SPIAPTS might be related to the smaller aggregation that the APTES coating confers, thus allowing for a better view of the nanoparticles in TEM. Figure 1. TEM analysis of (a) SPIONs and (b) SPIAPTS and their respective distribution in (c,d). 2.2. Magnetic GG:Alg Microparticle Characterization The introduction of magnetic nanoparticles to GG:Alg microparticles was performed by adsorption, where the microparticles were submerged in aqueous suspensions containing mNPs at different concentrations. The microparticles without mNPs had a diameter of 150–200 µm [23,27]. Within the mNP suspensions, the microparticles swelled, and the nanoparticles settled onto the particles’ surface. Higher adsorption efficiencies and adsorption capacities were achieved with an mNP suspension concentration of 5 mg/mL (Figure 2). The functionalization of SPIONs with APTES did not appear to affect the adsorption efficiency. Since submersion in suspensions of 5 mg/mL resulted in higher adsorption efficiencies, for the rest of the study, the default suspension to produce GG:Alg with mNPs will be 5 mg/mL. Figure 1. TEM analysis of (a) SPIONs and (b) SPIAPTS and their respective distribution in (c,d). 2.2. Magnetic GG:Alg Microparticle Characterization The introduction of magnetic nanoparticles to GG:Alg microparticles was performed by adsorption, where the microparticles were submerged in aqueous suspensions containing mNPs at different concentrations. The microparticles without mNPs had a diameter of 150–200 µ m [ 23 , 27 ]. Within the mNP suspensions, the microparticles swelled, and the nanoparticles settled onto the particles’ surface. Higher adsorption efficiencies and adsorption capacities were achieved with an mNP suspension concentration of 5 mg/mL (Figure 2). The functionalization of SPIONs with APTES did not appear to affect the adsorption efficiency. Since submersion in suspensions of 5 mg/mL resulted in higher adsorption efficiencies, for the rest of the study, the default suspension to produce GG:Alg with mNPs will be 5 mg/mL. Figure 3a,b show SEM analyses of microparticles loaded with SPIONs. Their surface exhibited some agglomerations of the nanoparticles. With EDX, it is possible to see the distribution of iron throughout the surface of the microparticles. As can be observed in the image, iron is distributed throughout the surface of the microparticles, with higher contents of iron detected in certain zones on the surface, probably caused by nanoparticle clusters. The size of the GG:Alg microparticles was found to be unaltered after the adsorption of the nanoparticles (150–200 µ m), as can be observed in Figure 3c,d. The APTES coating appeared to have no effect on the SPION distribution on the surface of the microparticles. Gels 2023,9, 982 4 of 20 Gels 2023, 9, x FOR PEER REVIEW 4 of 21 Figure 2. Adsorption efficiency (a) and adsorption capacity of SPIONs (b) in microparticles with and without APTES coating and at different nanoparticle concentrations (1, 2.5, and 5 mg/mL). Levels with statistically significant differences are presented by *, ** and *** above the bars (ANOVA, p < 0.05). Figure 3a,b show SEM analyses of microparticles loaded with SPIONs. Their surface exhibited some agglomerations of the nanoparticles. With EDX, it is possible to see the distribution of iron throughout the surface of the microparticles. As can be observed in the image, iron is distributed throughout the surface of the microparticles, with higher contents of iron detected in certain zones on the surface, probably caused by nanoparticle clusters. The size of the GG:Alg microparticles was found to be unaltered after the adsorption of the nanoparticles (150–200 µm), as can be observed in Figure 3c,d. The APTES coating appeared to have no effect on the SPION distribution on the surface of the microparticles. Figure 2. Adsorption efficiency ( a ) and adsorption capacity of SPIONs ( b ) in microparticles with and without APTES coating and at different nanoparticle concentrations (1, 2.5, and 5 mg/mL). Levels with statistically significant differences are presented by *, ** and *** above the bars (ANOVA, p< 0.05). Gels 2023, 9, x FOR PEER REVIEW 4 of 21 Figure 2. Adsorption efficiency (a) and adsorption capacity of SPIONs (b) in microparticles with and without APTES coating and at different nanoparticle concentrations (1, 2.5, and 5 mg/mL). Levels with statistically significant differences are presented by *, ** and *** above the bars (ANOVA, p < 0.05). Figure 3a,b show SEM analyses of microparticles loaded with SPIONs. Their surface exhibited some agglomerations of the nanoparticles. With EDX, it is possible to see the distribution of iron throughout the surface of the microparticles. As can be observed in the image, iron is distributed throughout the surface of the microparticles, with higher contents of iron detected in certain zones on the surface, probably caused by nanoparticle clusters. The size of the GG:Alg microparticles was found to be unaltered after the adsorption of the nanoparticles (150–200 µm), as can be observed in Figure 3c,d. The APTES coating appeared to have no effect on the SPION distribution on the surface of the microparticles. Gels 2023, 9, x FOR PEER REVIEW 5 of 21 Figure 3. SEM analysis (a) and EDX analysis (b) of the surface of a microparticle loaded with SPI- ONs. In Figure (b), the reddish zones depict the presence of iron, and the green zones represent the presence of calcium. Microscopy analysis of (c) microparticles loaded with SPIONs and (d) microparticles loaded with SPIAPTS. Microparticles with and without mNPs (SPIONs and SPIAPTS) were analyzed by TGA to determine their thermal stability and the effect of SPION adsorption. In all analyzed samples (Figure 4a), the first thermal event occurred at around 70 °C, characterized by a slight decrease in mass. This was associated with a loss of water from the microparticles. At around 230 °C, all samples had a significant mass loss. This was attributed to the depolymerization of gellan gum and alginate [28–31]. Subsequently, there was a steadier decline in mass percentage up to 800 °C. The microparticles containing adsorbed nanoparticles exhibited less weight loss than the microparticles without nanoparticles. This is due to the fact that the inorganic part of the nanoparticles does not degrade at the tested temperature since they are inorganic [32]. The microparticles that were submerged in a suspension of 5 mg/mL of the mNPs exhibited lower weight loss than the ones that were submerged in suspensions of 2.5 and 1 mg/mL of the mNPs. This was correlated with the loading capacity of the microparticles (Figure 2b), where higher mNP loadings were obtained at 5 mg/mL, hence the lower weight loss. It was also observed that the microparticles loaded with SPIAPTS had a slightly lower mass loss in all concentrations, possibly associated with the silane coating that did not degrade. Regarding the FTIR results (Figure 4b), the GG:Alg microparticles without mNPs present bands around 3400 cm −1 and 3100 cm −1 , attributed to the O-H groups and the -C- H stretching of gellan gum, respectively [33]. At 1598 cm −1 , the band was attributed to - COO stretching (asymmetric), followed by -COOH stretching at 1417 cm −1 (symmetric) and C-O-C stretching at 1023 cm −1 of alginate [34]. The band of O-H stretching vibration modes at 1630 cm −1 might be overlapped with the -COO stretching band of gellan gum. The bands of the microparticles with SPIONs and SPIAPTS, made in suspensions of 5 mg/mL, were very similar to those of the microparticles without mNPs, except in the 700– 500 cm −1 region in the band at 560 cm −1 , which is attributed to the Fe-O stretching vibration mode. Microparticles submerged in suspensions of 5 mg/mL were analyzed since higher adsorption capacities were registered, thus more mNPs were adsorbed onto the GG:Alg surface. Regarding SPIAPTS, the bands around 1100 cm −1 and 800 cm −1 can be attributed to Si-O-Si and Si-O stretching vibrations, thus confirming the link between the hydroxyl group at the mNP surfaces and the alkoxysilane moieties [35,36]. XRD analysis (Figure 4c) confirms the presence of mNPs within the GG:Alg microparticles made in suspensions of 5 mg/mL. The magnetite peaks exhibit peaks at 2θ at 30.1, 35.5, 42.6, 53.6, 57.0, and 62.8, which can be assigned to diffraction of the (220), (311), (400), (422), (511), and (440) planes, respectively [37]. This is corroborated by Khalkhali et Figure 3. SEM analysis ( a ) and EDX analysis ( b ) of the surface of a microparticle loaded with SPIONs. In Figure ( b ), the reddish zones depict the presence of iron, and the green zones represent the presence of calcium. Microscopy analysis of ( c ) microparticles loaded with SPIONs and ( d ) microparticles loaded with SPIAPTS. Microparticles with and without mNPs (SPIONs and SPIAPTS) were analyzed by TGA to determine their thermal stability and the effect of SPION adsorption. In all analyzed samples (Figure 4a), the first thermal event occurred at around 70 ◦ C, characterized by a Gels 2023,9, 982 5 of 20 slight decrease in mass. This was associated with a loss of water from the microparticles. At around 230 ◦ C, all samples had a significant mass loss. This was attributed to the depolymerization of gellan gum and alginate [ 28 – 31 ]. Subsequently, there was a steadier decline in mass percentage up to 800 ◦ C. The microparticles containing adsorbed nanoparticles exhibited less weight loss than the microparticles without nanoparticles. This is due to the fact that the inorganic part of the nanoparticles does not degrade at the tested temperature since they are inorganic [ 32 ]. The microparticles that were submerged in a suspension of 5 mg/mL of the mNPs exhibited lower weight loss than the ones that were submerged in suspensions of 2.5 and 1 mg/mL of the mNPs. This was correlated with the loading capacity of the microparticles (Figure 2b), where higher mNP loadings were obtained at 5 mg/mL, hence the lower weight loss. It was also observed that the microparticles loaded with SPIAPTS had a slightly lower mass loss in all concentrations, possibly associated with the silane coating that did not degrade. Gels 2023, 9, x FOR PEER REVIEW 6 of 21 al. [38], who also registered multiple peaks for the naked SPIONs at 2θ = 18.25° (1 1 1), 30.06° (2 2 0), 35.63° (3 1 1), 43.48° (4 0 0), 53.78° (4 2 2), 57.33° (5 1 1), and 63.11° (4 4 0) peaks (with associated planes in brackets). For the two types of microparticles loaded with SPIONs and SPIAPTS, peaks at 30° (2 2 0), 35.5° (3 1 1), 57° (5 1 1), and 62.5° (4 4 0) were obtained, typical of iron oxide nanoparticles [38]. A peak with a significant width appeared at around 21°, which might have conflicted with the significant peak at 18.25° (1 1 1) of SPIONs [38]. This peak was attributed to alginate and gellan gum, and even though they have an amorphous structure, they affect the intensity and peaks in an XRD analysis, as shown by Mun et al. [39]. Figure 4. (a) Thermal gravimetry analysis of GG:Alg microparticles with and without mNPs with different concentrations; (b) Fourier-transform infrared spectroscopy of the microparticles with and without mNPs (SPIONs and SPIAPTS made with 5 mg/mL); (c) DRX of microparticle-loaded mNPs (SPIONs and SPIAPTS made with 5 mg/mL). 2.3. Swelling and Degradation Tests Microparticles with and without mNPs (microparticles submerged in suspensions of 5 mg/mL of mNPs) were submerged in PBS pH 6.5 (pH of tumor microenvironments), and their swelling was evaluated. All swelling indexes were similar at the end of 3 days, without significant differences caused by the mNPs (Figure 5a). Similarly, the presence of APTES at the surface of the SPIONs did not affect the microparticles’ swelling. After 3 days of swelling assays, the concentration of the mNPs in the PBS solution was measured to evaluate the number of mNPs released from the microparticles during the swelling. In PBS pH 6.5, after 3 days, GG:Alg/SPIONs had released 6.57 ±0.58 wt.% of SPIONs, and GG:Alg/SPIAPTS released 6.24 ± 1.27 wt.% of SPIAPTS into the PBS solution. Therefore, the percentage in weight of mNPs released after swelling was similar for both non-coated and coated mNPs. Regarding degradation in PBS pH 6.5, the microparticles with mNPs appeared to have greater mass loss than the microparticles without mNPs (Figure 5b). We Figure 4. ( a ) Thermal gravimetry analysis of GG:Alg microparticles with and without mNPs with different concentrations; ( b ) Fourier-transform infrared spectroscopy of the microparticles with and without mNPs (SPIONs and SPIAPTS made with 5 mg/mL); ( c ) DRX of microparticle-loaded mNPs (SPIONs and SPIAPTS made with 5 mg/mL). Regarding the FTIR results (Figure 4b), the GG:Alg microparticles without mNPs present bands around 3400 cm −1 and 3100 cm −1 , attributed to the O–H groups and the –C–H stretching of gellan gum, respectively [ 33 ]. At 1598 cm −1 , the band was attributed to –COO stretching (asymmetric), followed by –COOH stretching at 1417 cm −1 (symmetric) and C–O–C stretching at 1023 cm −1 of alginate [ 34 ]. The band of O–H stretching vibration modes at 1630 cm −1 might be overlapped with the –COO stretching band of gellan gum. The bands of the microparticles with SPIONs and SPIAPTS, made in suspensions of 5 mg/mL, were very similar to those of the microparticles without mNPs, except in the 700–500 cm −1 region in the band at 560 cm −1 , which is attributed to the Fe–O stretching vibration mode. Microparticles submerged in suspensions of 5 mg/mL were analyzed Gels 2023,9, 982 6 of 20 since higher adsorption capacities were registered, thus more mNPs were adsorbed onto the GG:Alg surface. Regarding SPIAPTS, the bands around 1100 cm −1 and 800 cm −1 can be attributed to Si–O–Si and Si–O stretching vibrations, thus confirming the link between the hydroxyl group at the mNP surfaces and the alkoxysilane moieties [35,36]. XRD analysis (Figure 4c) confirms the presence of mNPs within the GG:Alg microparticles made in suspensions of 5 mg/mL. The magnetite peaks exhibit peaks at 2 θ at 30.1, 35.5, 42.6, 53.6, 57.0, and 62.8, which can be assigned to diffraction of the (220), (311), (400), (422), (511), and (440) planes, respectively [ 37 ]. This is corroborated by Khalkhali et al. [ 38 ], who also registered multiple peaks for the naked SPIONs at 2 θ = 18.25 ◦ (1 1 1), 30.06◦(2 2 0) , 35.63 ◦ (3 1 1), 43.48 ◦ (4 0 0), 53.78 ◦ (4 2 2), 57.33 ◦ (5 1 1), and 63.11◦(4 4 0) peaks (with associated planes in brackets). For the two types of microparticles loaded with SPIONs and SPIAPTS, peaks at 30 ◦ (2 2 0), 35.5 ◦ (3 1 1), 57 ◦ (5 1 1), and 62.5 ◦ (4 4 0) were obtained, typical of iron oxide nanoparticles [ 38 ]. A peak with a significant width appeared at around 21 ◦ , which might have conflicted with the significant peak at 18.25 ◦ (1 1 1) of SPIONs [ 38 ]. This peak was attributed to alginate and gellan gum, and even though they have an amorphous structure, they affect the intensity and peaks in an XRD analysis, as shown by Mun et al. [39]. 2.3. Swelling and Degradation Tests Microparticles with and without mNPs (microparticles submerged in suspensions of 5 mg/mL of mNPs) were submerged in PBS pH 6.5 (pH of tumor microenvironments), and their swelling was evaluated. All swelling indexes were similar at the end of 3 days, without significant differences caused by the mNPs (Figure 5a). Similarly, the presence of APTES at the surface of the SPIONs did not affect the microparticles’ swelling. After 3 days of swelling assays, the concentration of the mNPs in the PBS solution was measured to evaluate the number of mNPs released from the microparticles during the swelling. In PBS pH 6.5, after 3 days, GG:Alg/SPIONs had released 6.57 ± 0.58 wt.% of SPIONs, and GG:Alg/SPIAPTS released 6.24 ± 1.27 wt.% of SPIAPTS into the PBS solution. Therefore, the percentage in weight of mNPs released after swelling was similar for both non-coated and coated mNPs. Regarding degradation in PBS pH 6.5, the microparticles with mNPs appeared to have greater mass loss than the microparticles without mNPs (Figure 5b). We attribute this to the conjugation between the degradation of the gellan gum/alginate structure and the release of mNPs from the microparticles; thus, a greater mass loss was observed. Gels 2023, 9, x FOR PEER REVIEW 7 of 21 attribute this to the conjugation between the degradation of the gellan gum/alginate structure and the release of mNPs from the microparticles; thus, a greater mass loss was observed. Figure 5. (a) Swelling ratio in PBS (pH 6.5) with and without mNPs (microparticles submerged in suspensions of 5 mg/mL of mNPs); (b) Degradation (mass loss) of microparticles with and without mNPs. 2.4. Cytotoxicity Assays For the cytotoxicity assays, the ISO standard 10993-5:2009 [40] was followed using Vero cells with the extract method. Figure 6 shows that, regarding cellular viability, the GG:Alg microparticles without mNPs had no cytotoxicity. Likewise, the microparticles with SPIONs did not present cytotoxicity, and the different amounts of SPIONs within the microparticles did not affect cellular viability. Similarly, the microparticles with SPIAPTS were not cytotoxic, although they presented slightly lower cellular viability compared to the others. Other studies also studied the effect that polysaccharide microparticles with iron oxide nanoparticles had on different types of cells. Alpdemir et al. [21] found that alginate microparticles with SPIONs also had no negative effects on HEPG2 liver cancer and L929 fibroblast cell lines. However, in their study, they dispersed SPIONs in a precrosslinked alginate solution with an initial concentration of 5 mg/mL of the SPIONs in the alginate solution. In this study, the mNPs were adsorbed to the microparticles after their crosslinking in CaCl 2 . Before conjugating the GG:Alg microparticles with and without mNPs into a thermoresponsive hydrogel, it is fundamental to understand if they have a benign or harmful impact on the surrounding cells. Thus, cytotoxicity assays were performed, which are displayed in Figure 6. No cytotoxicity was found for the microparticles with and without mNPs. Magnetic microparticles submerged in suspensions with different concentrations of mNPs were tested and showed no significant differences between them. Although viable, the microparticles with SPIAPTS had lower cell viability than the ones with SPIONs. Cytotoxicity depends on many factors, such as the type of cell and the type of coating. For example, Alpdemir et al. [21] found that alginate microparticles containing SPIONs had no negative effects on L929 fibroblast and HEPG2 liver cancer cell lines. t-tests were performed, comparing each sample to a control. No significant differences were found, only with a degree of confidence of 95%, with the exception of the SPIAPTS at 2.5 mg/mL (t(2) = 0.55, p = 0.04). This significant difference did not alter the previously mentioned conclusions. In our study, with the assurance that the microparticles loaded with mNPs were noncytotoxic, we proceeded to incorporate them within the Pluronic system mixed with methylene blue (MB)-loaded microparticles. In future studies, cytotoxicity assays will be performed using cancer lines, for example, breast cancer cell lines [41]. Figure 5. ( a ) Swelling ratio in PBS (pH 6.5) with and without mNPs (microparticles submerged in suspensions of 5 mg/mL of mNPs); ( b ) Degradation (mass loss) of microparticles with and without mNPs. 2.4. Cytotoxicity Assays For the cytotoxicity assays, the ISO standard 10993-5:2009 [ 40 ] was followed using Vero cells with the extract method. Figure 6shows that, regarding cellular viability, the Gels 2023,9, 982 7 of 20 GG:Alg microparticles without mNPs had no cytotoxicity. Likewise, the microparticles with SPIONs did not present cytotoxicity, and the different amounts of SPIONs within the microparticles did not affect cellular viability. Similarly, the microparticles with SPIAPTS were not cytotoxic, although they presented slightly lower cellular viability compared to the others. Other studies also studied the effect that polysaccharide microparticles with iron oxide nanoparticles had on different types of cells. Alpdemir et al. [ 21 ] found that alginate microparticles with SPIONs also had no negative effects on HEPG2 liver cancer and L929 fibroblast cell lines. However, in their study, they dispersed SPIONs in a precrosslinked alginate solution with an initial concentration of 5 mg/mL of the SPIONs in the alginate solution. In this study, the mNPs were adsorbed to the microparticles after their crosslinking in CaCl2. Gels 2023, 9, x FOR PEER REVIEW 8 of 21 Figure 6. Vero cell viability (%) after 48 h of indirect exposure to the developed microparticles with and without mNPs (GG:Alg). Microparticles that were submerged in different concentrations of mNPs (1, 2.5, and 5 mg/mL) were analyzed. Data are expressed as the mean ± standard deviation of at least four experiments. C− is the negative control (no medium alterations), and C+ is the positive control (medium with 10 µL of DMSO). Levels with statistically significant differences are presented by *; ** and *** above the bars (ANOVA, p < 0.05). No cytotoxicity assays were performed on the Pluronic hydrogel containing microparticles. In this study, the Pluronic hydrogel was not crosslinked, and hydrogel formation is dependent on concentration and temperature. Pluronic F127 is approved by the Food and Drug Administration (FDA) of the United States of America [42]. For example, for dental pump stem cells, Pluronic F127 was found to be non-cytotoxic [43]. Regarding breast cancer cells, in triple-negative epithelial breast cancer cell lines, a Pluronic-based hydrogel combined with hyaluronic acid was found to be non-cytotoxic [44], and only with the introduction of nanoparticles did the cytotoxicity increase. Another possibility is the mixture of Pluronic F127 and chitosan with tripolyphosphate crosslinking, as performed by García Couce et al. [45]. The addition of chitosan increased the gel time compared to the gel alone. 2.5. Development of a Thermoresponsive Hydrogel with Magnetic Microparticles The optimized GG:Alg microparticles with SPIONs and APTES were mixed with plain GG:Alg microparticles at a ratio of 1:1 (wt.%). This system was analyzed via rheological and magnetic hyperthermia tests. For the release tests, plain microparticles were loaded with MB, as was previously stated. In a previous study [23], different mixtures of Pluronic F127 and Pluronic F68 were analyzed with different concentrations of GG:Alg microparticles. The system must be able to be injectable at 20 °C (operating room temperature) and be in the gel phase at 37 °C. It was found that a Pluronic mixture with 17 wt.% of Pluronic F27 and 3 wt.% of Pluronic F68 with 2 and 5 wt.% concentrations of GG:Alg Figure 6. Vero cell viability (%) after 48 h of indirect exposure to the developed microparticles with and without mNPs (GG:Alg). Microparticles that were submerged in different concentrations of mNPs (1, 2.5, and 5 mg/mL) were analyzed. Data are expressed as the mean ± standard deviation of at least four experiments. C − is the negative control (no medium alterations), and C+ is the positive control (medium with 10 µ L of DMSO). Levels with statistically significant differences are presented by *; ** and *** above the bars (ANOVA, p< 0.05). Before conjugating the GG:Alg microparticles with and without mNPs into a thermoresponsive hydrogel, it is fundamental to understand if they have a benign or harmful impact on the surrounding cells. Thus, cytotoxicity assays were performed, which are displayed in Figure 6. No cytotoxicity was found for the microparticles with and without mNPs. Magnetic microparticles submerged in suspensions with different concentrations of mNPs were tested and showed no significant differences between them. Although viable, the microparticles with SPIAPTS had lower cell viability than the ones with SPIONs. Cytotoxicity depends on many factors, such as the type of cell and the type of coating. For example, Alpdemir et al. [ 21 ] found that alginate microparticles containing SPIONs had no negative effects on L929 fibroblast and HEPG2 liver cancer cell lines. t-tests were performed, Gels 2023,9, 982 8 of 20 comparing each sample to a control. No significant differences were found, only with a degree of confidence of 95%, with the exception of the SPIAPTS at 2.5 mg/mL (t(2) = 0.55, p= 0.04). This significant difference did not alter the previously mentioned conclusions. In our study, with the assurance that the microparticles loaded with mNPs were non-cytotoxic, we proceeded to incorporate them within the Pluronic system mixed with methylene blue (MB)-loaded microparticles. In future studies, cytotoxicity assays will be performed using cancer lines, for example, breast cancer cell lines [41]. No cytotoxicity assays were performed on the Pluronic hydrogel containing microparticles. In this study, the Pluronic hydrogel was not crosslinked, and hydrogel formation is dependent on concentration and temperature. Pluronic F127 is approved by the Food and Drug Administration (FDA) of the United States of America [ 42 ]. For example, for dental pump stem cells, Pluronic F127 was found to be non-cytotoxic [ 43 ]. Regarding breast cancer cells, in triple-negative epithelial breast cancer cell lines, a Pluronic-based hydrogel combined with hyaluronic acid was found to be non-cytotoxic [ 44 ], and only with the introduction of nanoparticles did the cytotoxicity increase. Another possibility is the mixture of Pluronic F127 and chitosan with tripolyphosphate crosslinking, as performed by García Couce et al. [ 45 ]. The addition of chitosan increased the gel time compared to the gel alone. 2.5. Development of a Thermoresponsive Hydrogel with Magnetic Microparticles The optimized GG:Alg microparticles with SPIONs and APTES were mixed with plain GG:Alg microparticles at a ratio of 1:1 (wt.%). This system was analyzed via rheological and magnetic hyperthermia tests. For the release tests, plain microparticles were loaded with MB, as was previously stated. In a previous study [ 23 ], different mixtures of Pluronic F127 and Pluronic F68 were analyzed with different concentrations of GG:Alg microparticles. The system must be able to be injectable at 20 ◦ C (operating room temperature) and be in the gel phase at 37 ◦ C. It was found that a Pluronic mixture with 17 wt.% of Pluronic F27 and 3 wt.% of Pluronic F68 with 2 and 5 wt.% concentrations of GG:Alg microparticles meets the desired criteria. In this present study, we used a Pluronic aqueous solution with 17 wt.%:3 wt.% of F127:F68 and 2 and 5 wt.% of GG:Alg microparticles, where half were loaded with MB and the other with mNPs. 2.5.1. Rheological Tests All systems had viscous behavior at a lower temperature since the Pluronic was in the sol state, with G 00 > G 0 (Figure 7a). With a temperature increase, a sol-gel transition occurred due to micellization of the PEO-PPO-PEO structure, typical of Pluronic systems [ 46 ]. The Pluronic system without microparticles had a sol-gel transition at 32–34 ◦ C. With the addition of the microparticles at 5 w/v%, the transition temperature of the microparticle/Pluronic system decreased to around 29–32 ◦ C. The transition temperature was not affected by the presence of mNPs (coated or not). At a 2 w/v% concentration of microparticles with mNPs, there was no significant sol-gel temperature decrease. All systems were in the sol state at 21 ◦ C and in the gel state at 37 ◦ C. The decrease in sol-gel temperature might be attributed to the absorption of water from the microparticles, increasing the Pluronic concentration and leading to a decrease in the sol-gel temperature. These results are in agreement with our previous study with similar microparticles and similar Pluronic hydrogels [ 23 ]. With similar microparticle concentrations (2 and 5 w/v%) but without the adsorbed mNPs, similar transition temperatures were obtained. In this study, we observed that the mNPs did not alter the sol-gel transition. Different microparticle concentrations can be used to maintain the injectability of the system by altering the Pluronic F127:F68 ratio [23]. Gels 2023,9, 982 9 of 20 Gels 2023, 9, x FOR PEER REVIEW 10 of 21 Figure 7. (a) Temperature ramp in oscillation for Pluronic systems (F127:F68—17:3) with and without microparticles. The samples with just GG:Alg refer to samples without mNPs, and the samples with GG:Alg/SPIONs and GG:Alg/SPIAPTS refer to systems with mNPs (a ratio of 1:1 of GG:Alg and GG:Alg/mNPs); (b) frequency sweep tests at 37 °C for similar systems (with 5 w/v% of microparticles with and without mNPs); (c) tg(δ) of the analyzed systems in (b), where a line on 0.2 was put to mark the threshold between soft gel and non-soft gel; (d) flow curves at 21 °C of a similar system with 5 w/v% of microparticles within the Pluronic with and without mNPs). Figure 8 shows images of the proposed system with 2 w/v% of microparticles. The system is in the sol state at 21 °C (Figure 8a) and in the gel state at 37 °C (Figure 8b) according to the obtained moduli in Figure 7a. Figure 7. ( a ) Temperature ramp in oscillation for Pluronic systems (F127:F68—17:3) with and without microparticles. The samples with just GG:Alg refer to samples without mNPs, and the samples with GG:Alg/SPIONs and GG:Alg/SPIAPTS refer to systems with mNPs (a ratio of 1:1 of GG:Alg and GG:Alg/mNPs); ( b ) frequency sweep tests at 37 ◦ C for similar systems (with 5 w/v% of microparticles with and without mNPs); ( c ) tg( δ ) of the analyzed systems in ( b ), where a line on 0.2 was put to mark the threshold between soft gel and non-soft gel; ( d ) flow curves at 21 ◦ C of a similar system with 5w/v% of microparticles within the Pluronic with and without mNPs). Regarding frequency sweeps at 37 ◦ (Figure 7b), all analyzed systems were in the gel state, with G 0 > G 00 with a positive slope with frequency. The presence of SPIONs and SPIAPTS affected the G 0 and G 00 . Analyzing the tg( δ ) (G 00 /G 0 ) (Figure 7c), it was observed that, along the studied angular frequencies, at 37 ◦ C, the Pluronic system with microparticles (with and without mNPs) had values of tg( δ ) between 0.7 and 0.8. These values reveal that the gel had high values of G 00 , although G 0 was predominant. This indicates that the Pluronic system with microparticles at 37 ◦ C was a weak gel with a weak elastic structure [47]. An increase in the moduli was detected with the introduction of mNPs, with the microparticles with SPIAPTS having the higher moduli. Microparticles within the Pluronic solution might act as reinforcements, leading to an increase in the resistance to the flow of the system [ 48 ]. Other interactions between the mNPs and Pluronic can also be at play. Previous studies used Pluronic F127 to functionalize iron oxide mNPs, but with previous coating agents such as oleic acid, where the hydrophobic PPO block interacted with the oleic coating of the SPIONs [ 24 ]. For example, Jain et al. [ 49 ] in FTIR studies in systems with SPIONs with and without oleic acid coating and with Pluronic showed that without oleic acid, no interaction occurred with Pluronic. While, with the oleic layer, bonding was observed in the FTIR spectra. The increased viscosity that was observed with SPIAPTS might be an interaction between the hydrophilic portions of Pluronic and the amine groups Gels 2023,9, 982 16 of 20 where ms0 is the mass of the nanoparticles in the initial suspension, msf is the remaining mass of the nanoparticles in the suspension after swelling of the microparticles, mencap.mNPs is the mass of the adsorbed nanoparticles in the microparticles, and mGG:Alg is the weight of the microparticles. Four replicas were used in each assay. 4.6. Swelling (Sw) and Degradation For the swelling studies, microparticles with mNPs, previously prepared in a 5 mg/mL suspension, were submerged in PBS solution (pH 6.5). At different times, the microparticles were removed from the PBS solution and weighed. At equilibrium, the concentration of the mNPs in the PBS solution was analyzed using the previously mentioned 10-phenanthroline method [ 14 , 58 ] to evaluate the nanoparticles’ concentration released during swelling. Swelling in weight (Sw) was calculated as the following equation describes: Sw=mtw −m0 m0 (3) where m tw is the mass of the microparticles at different times after swelling, and m0 is the initial mass of the dried microparticles. For the analysis of degradation, different batches of microparticles with and without mNPs were submerged in PBS solution (pH 6.5), and, at different times, they were removed, filtered, rinsed with water, dried, and weighed. 4.7. Rheological Analysis The sol-gel transition of the microparticle/Pluronic system was rheologically characterized using an AR2000 stress-controlled oscillatory rheometer (TA Instruments, New Castle, DE, USA). The analyses were performed using a plate–plate smooth geometry (dia: 40 mm) with a selected gap of 2 mm. This gap was chosen to accommodate the size of the microparticles. Firstly, strain sweep tests were performed to determine the linear viscoelastic range. From these, a strain of 2% was selected for the rest of the tests. Temperature ramps were performed between 15 and 45 ◦ C at a constant frequency of 1 Hz. Frequency sweep tests were carried out between 0.5 and 100 Hz at 37 ◦ C (human body temperature). Finally, flow curves were performed at 21 ◦ C (operating room temperature) in a shear rate range of 1–100 s−1. At least 3 replicates of each sample were made. 4.8. Cytotoxicity Tests Cytotoxicity tests for the microparticles with and without SPIONs/SPIAPTS were performed according to the ISO 10993 standard [ 59 ]. The extract method was used with a Vero cell line provided by the American Type Culture Collection (ATCC CCL-81). The extract was produced at a concentration of 5 mg/mL. Each sample was placed in 1 mL of complete culture medium, DMEM (Dulbecco’s modified Eagle’s medium with 1.0 g/L glucose, stable glutamine, and sodium pyruvate, Biowest #L0066) supplemented with penicillin (100 U/mL), streptomycin (100 µ g/mL) (Invitrogen, Carlsbad, CA, USA, #15140122), and 10% FBS (fetal bovine serum, S. America origin, Biowest, #S1810) at 37 ◦ C for 48 h. The Vero cells were seeded in 96-well plates at a density of 30 000 cells/cm2. After 24 h, the medium was replaced by the extract and its dilutions (2.5 mg/mL and 1 mg/mL). A negative control corresponded to cells fed with a complete cell culture medium, and a positive control corresponded to cells cultured in a cytotoxic environment caused by the supplementation of the culture medium with 10% dimethyl sulfoxide (DMSO). After 48 h of incubation, the medium was removed from each well and replaced with a resazurin solution containing 50% of the complete medium and 50% of a 0.04 mg/mL resazurin solution in phosphate-buffered saline (PBS). After 3 h of incubation at 37 ◦ C and 5%, the absorbance was measured at 570 and 600 nm. Cell viability is given as a percentage of viable cells in the samples tested relative to the negative control. Gels 2023,9, 982 17 of 20 4.9. Magnetic Hyperthermia Assays Magnetic hyperthermia measurements were obtained with a NanoScale Biomagnetics D5 Series device (Spain) working at a frequency of 388.5 kHz and a magnetic field intensity of 300 Gauss for 10 min. At least 3 replicates were conducted in each sample, and all samples contained an equal amount of mNPs. Aqueous suspensions of SPIONs (5 mg/mL), aqueous suspensions of SPIAPTS (5 mg/mL), microparticles loaded with mNPs, and magnetic microparticles within Pluronic (17:3) hydrogel in the gel state were analyzed. The specific absorption rate (SAR) indicates the heating efficiency of mNPs through energy absorption with the application of an alternating magnetic field [54] (Equation (4)). SAR (W/g)=CNP·mFe +Cl·ml mFe ·dT dt max (4) In Equation (4), (dT/dt)_max is the maximum derivative of the temperature curve, C_NP is the specific heat of Fe 3 O 4 , Cl is the specific heat of the liquid, mFe is the iron mass, and ml is the mass of the liquid. 4.10. Drug Release Studies To characterize the release profiles of the microparticle/hydrogel composite system with and without mNPs, we used a 1:1 ratio (in mass) of the magnetic microparticles and MB-loaded microparticles. Within a donor–recipient made from a permeable membrane, Pluronic hydrogel composite systems with 2 w/w% of microparticles were introduced. The recipients of the microparticle/Pluronic were submerged in 10 mL of PBS solutions with pH 6.5 and pH 7.4 at 37 ◦ C. The PBS solutions were kept at 37 ◦ C with orbital agitation. At regular periods (0, 0.25, 1, 3, 6, 24, 48, 72, 144, 192, 240, and 312 h), 2 mL of the PBS was retrieved and replaced with fresh PBS. The 2 mL was then analyzed by UV–VIS spectroscopy using a calibration curve to determine the concentration of MB at each time. Five replicates were made. The results from the release assays were analyzed using mathematical models. Similarly to our previous work [ 10 ], we used a modified Korsmeyer–Peppas (KP) model (Equation (5)) and a modified Peppas–Sahlin (PS) model (Equation (6)) using the DDSolver program [24]. Qt=kt−Tlagn(5) Qt=k1t−Tlagm+k2t−Tlag2m(6) In Equations (5) and (6), Q t is the drug concentration released at each time (t), kis a structural factor, nis related to the release mechanism, and T lag is the lag time before drug release [ 58 ]. The modified Peppas–Sahlin model (PS) uses two constants, k 1 and k 2 , of which k1correlates with Fickian diffusion and k2with case II transport [60]. 4.11. Statistical Analysis In different tests, a statistical study was carried out. For this, at least 3 replicates of each sample and assay were performed. A single-factor analysis of variance (ANOVA) was carried out. Then, a series of statistical parameters were calculated, including the mean and standard deviation. In addition, a mean comparison test was performed to detect significant differences (a confidence level of 95%, p< 0.05). Author Contributions: Conceptualization, H.C., A.R., P.I.P.S. and J.P.B.; methodology, H.C.; software, H.C., A.R.E. and T.V.; validation, H.C., A.R., P.I.P.S., J.P.B. and J.C.S.; formal analysis, H.C.; investigation, H.C.; data curation, H.C.; writing—original draft preparation, H.C.; writing—review and editing, all; visualization, H.C.; supervision, A.R., P.I.P.S. and J.P.B. All authors have read and agreed to the published version of the manuscript. Gels 2023,9, 982 18 of 20 Funding: This work is funded by National Funds through the FCT—Fundação para a Ciência e a Tecnologia, I.P., under the scope of the projects UID/50025/2020-2023 and LA/0037/2020 of the Associate Laboratory Institute of Nanostructures, Nanomodelling and Nanofabrication-i3N. This study was also financially supported by MCIN/AEI/10.13039/501100011033///FEDER, UE, through project PID2021-124294OB-C21. H.C. acknowledges FCT for the PhD grant with reference SFRH/BD/144986/2019. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Conflicts of Interest: The authors declare no conflict of interest. References 1. Bosio, V.E.; Cacicedo, M.L.; Calvignac, B.; León, I.; Beuvier, T.; Boury, F.; Castro, G.R. Synthesis and characterization of CaCO 3 –biopolymer hybrid nanoporous microparticles for controlled release of doxorubicin. Colloids Surf. B Biointerfaces 2014 ,123, 158–169. [CrossRef] [PubMed] 2. Sun, X.; Liu, C.; Omer, A.; Yang, L.-Y.; Ouyang, X.-K. Dual-layered pH-sensitive alginate/chitosan/kappa-carrageenan microbeads for colon-targeted release of 5-fluorouracil. Int. J. Biol. Macromol. 2019,132, 487–494. [CrossRef] [PubMed] 3. Zhang, Y.; Jiang, C. Postoperative cancer treatments: In-situ delivery system designed on demand. J. Control. Release 2021 ,330, 554–564. [CrossRef] 4. Fakhari, A.; Subramony, J.A. Engineered in-situ depot-forming hydrogels for intratumoral drug delivery. J. Control. Release 2015 , 220, 465–475. [CrossRef] 5. Balakrishnan, B.; Jayakrishnan, A. Injectable Hydrogels for Biomedical Applications. In Injectable Hydrogels for Regenerative Engineering; Nair, L.S., Ed.; Imperial College Press: London, UK, 2016; pp. 33–94. [CrossRef] 6. Moura, M.; Gil, M.; Figueiredo, M. Cisplatin delivery systems based on different drug encapsulation techniques. Eur. Polym. J. 2019,113, 357–364. [CrossRef] 7. Pontremoli, C.; Boffito, M.; Laurano, R.; Iviglia, G.; Torre, E.; Cassinelli, C.; Morra, M.; Ciardelli, G.; Vitale-Brovarone, C.; Fiorilli, S. Mesoporous Bioactive Glasses Incorporated into an Injectable Thermosensitive Hydrogel for Sustained Co-Release of Sr 2+ Ions and N-Acetylcysteine. Pharmaceutics 2022,14, 1890. [CrossRef] 8. Young, S.; Wong, M.; Tabata, Y.; Mikos, A.G. Gelatin as a delivery vehicle for the controlled release of bioactive molecules. J. Control. Release 2005,109, 257–274. [CrossRef] 9. Shymborska, Y.; Budkowski, A.; Raczkowska, J.; Donchak, V.; Melnyk, Y.; Vasiichuk, V.; Stetsyshyn, Y. Switching it Up: The Promise of Stimuli-Responsive Polymer Systems in Biomedical Science. Chem. Rec. 2023, e202300217. [CrossRef] 10. Carrêlo, H.; Soares, P.I.P.; Borges, J.P.; Cidade, M.T. Injectable composite systems based on microparticles in hydrogels for bioactive cargo controlled delivery. Gels 2021,7, 147. [CrossRef] 11. Yu, H.; Ning, N.; Meng, X.; Chittasupho, C.; Jiang, L.; Zhao, Y. Sequential Drug Delivery in Targeted Cancer Therapy. Pharmaceutics 2022,14, 573. [CrossRef] [PubMed] 12. Naghizadeh, Z.; Karkhaneh, A.; Nokhbatolfoghahaei, H.; Farzad-Mohajeri, S.; Rezai-Rad, M.; Dehghan, M.M.; Aminishakib, P.; Khojasteh, A. Cartilage regeneration with dual-drug-releasing injectable hydrogel/microparticle system: In vitro and in vivo study. J. Cell. Physiol. 2021,236, 2194–2204. [CrossRef] [PubMed] 13. Min, Q.; Liu, J.; Yu, X.; Zhang, Y.; Wu, J.; Wan, Y. Sequential delivery of dual growth factors from injectable chitosan-based composite hydrogels. Mar. Drugs 2019,17, 365. [CrossRef] [PubMed] 14. Desai, K.G.H. Polymeric drug delivery systems for intraoral site-specific chemoprevention of oral cancer. J. Biomed. Mater. Res.—Part B Appl. Biomater. 2018,106, 1383–1413. [CrossRef] [PubMed] 15. Wang, A.Z.; Langer, R.; Farokhzad, O.C. Nanoparticle Delivery of Cancer Drugs. Annu. Rev. Med. 2012,63, 185–198. [CrossRef] 16. Schneider, M.G.M.; Martín, M.J.; Otarola, J.; Vakarelska, E.; Simeonov, V.; Lassalle, V.; Nedyalkova, M. Biomedical Applications of Iron Oxide Nanoparticles: Current Insights Progress and Perspectives. Pharmaceutics 2022,14, 204. [CrossRef] 17. Musielak, M.; Piotrowski, I.; Suchorska, W.M. Superparamagnetic iron oxide nanoparticles (SPIONs) as a multifunctional tool in various cancer therapies. Rep. Pract. Oncol. Radiother. 2019,24, 307–314. [CrossRef] 18. Neuwelt, A.; Sidhu, N.; Hu, C.A.A.; Mlady, G.; Eberhardt, S.C.; Sillerud, L.O. Iron-based superparamagnetic nanoparticle contrast agents for MRI of infection and inflammation. Am. J. Roentgenol. 2015,204, W302–W313. [CrossRef] 19. Finotelli, P.V.; Da Silva, D.; Sola-Penna, M.; Rossi, A.M.; Farina, M.; Andrade, L.R.; Takeuchi, A.Y.; Rocha-Leão, M.H. Microcapsules of alginate/chitosan containing magnetic nanoparticles for controlled release of insulin. Colloids Surf. B Biointerfaces 2010 ,81, 206–211. [CrossRef] 20. Matos, R.J.R.; Chaparro, C.I.P.; Silva, J.C.; Valente, M.A.; Borges, J.P.; Soares, P.I.P. Electrospun composite cellulose acetate/iron oxide nanoparticles non-woven membranes for magnetic hyperthermia applications. Carbohydr. Polym. 2018 ,198, 9–16. [CrossRef] 21. Alpdemir, ¸S.; Vural, T.; Kara, G.; Bayram, C.; Haberal, E.; Denkba¸s, E.B. Magnetically responsive, sorafenib loaded alginate microspheres for hepatocellular carcinoma treatment. IET Nanobiotechnol. 2020,14, 617–622. [CrossRef] Gels 2023,9, 982 19 of 20 22. Xue, W.; Liu, X.-L.; Ma, H.; Xie, W.; Huang, S.; Wen, H.; Jing, G.; Zhao, L.; Liang, X.-J.; Fan, H.M. AMF responsive DOX-loaded magnetic microspheres: Transmembrane drug release mechanism and multimodality postsurgical treatment of breast cancer. J. Mater. Chem. B 2018,6, 2289–2303. [CrossRef] 23. Carrêlo, H.; Escoval, A.R.; Soares, P.I.P.; Borges, J.P.; Cidade, M.T. Injectable Composite Systems of Gellan Gum:Alginate Microparticles in Pluronic Hydrogels for Bioactive Cargo Controlled Delivery: Optimization of Hydrogel Composition based on Rheological Behavior. Fluids 2022,7, 375. [CrossRef] 24. Vu-Quang, H.; Vinding, M.S.; Nielsen, T.; Ullisch, M.G.; Nielsen, N.C.; Nguyen, D.-T.; Kjems, J. Pluronic F127-folate coated super paramagenic iron oxide nanoparticles as contrast agent for cancer diagnosis in magnetic resonance imaging. Polymers 2019 ,11, 743. [CrossRef] 25. Rodríguez-López, J.; Shum, H.C.; Elvira, L.; de Espinosa, F.M.; Weitz, D.A. Fabrication and manipulation of polymeric magnetic particles with magnetorheological fluid. J. Magn. Magn. Mater. 2013,326, 220–224. [CrossRef] 26. Li, N.; Zhao, L.; Qi, L.; Li, Z.; Luan, Y. Polymer assembly: Promising carriers as co-delivery systems for cancer therapy. Prog. Polym. Sci. 2016,58, 1–26. [CrossRef] 27. Carrêlo, H.; Cidade, M.T.; Borges, J.P.; Soares, P. Gellan gum/alginate microparticles as drug delivery vehicles: DOE production optimization and drug delivery. Pharmaceuticals 2023,16, 1029. [CrossRef] [PubMed] 28. Halim, N.F.A.; Majid, S.R.; Arof, A.K.; Kajzar, F.; Pawlicka, A. Gellan Gum-LiI gel polymer electrolytes. Mol. Cryst. Liq. Cryst. 2012,554, 232–238. [CrossRef] 29. Khampieng, T.; Aramwit, P.; Supaphol, P. Silk sericin loaded alginate nanoparticles: Preparation and anti-inflammatory efficacy. Int. J. Biol. Macromol. 2015,80, 636–643. [CrossRef] 30. Noor, I.; Majid, S.; Arof, A.; Djurado, D.; Neto, S.C.; Pawlicka, A. Characteristics of gellan gum–LiCF 3 SO 3 polymer electrolytes. Solid State Ionics 2012,225, 649–653. [CrossRef] 31. Patel, N.; Lalwani, D.; Gollmer, S.; Injeti, E.; Sari, Y.; Nesamony, J. Development and evaluation of a calcium alginate based oral ceftriaxone sodium formulation. Prog. Biomater. 2016,5, 117–133. [CrossRef] 32. Soares, P.I.P.; Lochte, F.; Echeverria, C.; Pereira, L.C.J.; Coutinho, J.T.; Ferreira, I.M.M.; Novo, C.M.M.; Borges, J.P.M.R. Thermal and magnetic properties of iron oxide colloids: Influence of surfactants. Nanotechnology 2015,26, 425704. [CrossRef] [PubMed] 33. Kloster, G.A.; Muraca, D.; Londoño, O.M.; Pirota, K.R.; Mosiewicki, M.A.; Marcovich, N.E. Alginate based nanocomposites with magnetic properties. Compos. Part A: Appl. Sci. Manuf. 2020,135, 105936. [CrossRef] 34. Jana, S.; Das, A.; Nayak, A.K.; Sen, K.K.; Basu, S.K. Aceclofenac-loaded unsaturated esterified alginate/gellan gum microspheres: In vitro and in vivo assessment. Int. J. Biol. Macromol. 2013,57, 129–137. [CrossRef] [PubMed] 35. Villa, S.; Riani, P.; Locardi, F.; Canepa, F. Functionalization of Fe 3 O 4 NPs by silanization: Use of amine (APTES) and thiol (MPTMS) silanes and their physical characterization. Materials 2016,9, 826. [CrossRef] [PubMed] 36. Stankovi´c, A.; Mihailovi´c, J.; Mirkovi´c, M.; Radovi´c, M.; Milanovi´c, Z.; Ognjanovi´c, M.; Jankovi´c, D.; Anti´c, B.; Mijovi´c, M.; Vranješ-Ðuri´c, S.; et al. Aminosilanized flower-structured superparamagnetic iron oxide nanoparticles coupled to 131I-labeled CC49 antibody for combined radionuclide and hyperthermia therapy of cancer. Int. J. Pharm. 2020,587, 119628. [CrossRef] 37. Shukla, S.; Jadaun, A.; Arora, V.; Sinha, R.K.; Biyani, N.; Jain, V. In vitro toxicity assessment of chitosan oligosaccharide coated iron oxide nanoparticles. Toxicol. Rep. 2015,2, 27–39. [CrossRef] 38. Khalkhali, M.; Rostamizadeh, K.; Sadighian, S.; Khoeini, F.; Naghibi, M.; Hamidi, M. The impact of polymer coatings on magnetite nanoparticles performance as MRI contrast agents: A comparative study. DARU J. Pharm. Sci. 2015,23, 45. [CrossRef] 39. Mun, S.; Kim, H.C.; Yadave, M.; Kim, J. Graphene oxide–gellan gum–sodium alginate nanocomposites: Synthesis, characterization, and mechanical behavior. Compos. Interfaces 2015,22, 249–263. [CrossRef] 40. ISO 10993-5:2009; Biological Evaluation of Medical Devices Part 5: Tests for In Vitro Cytotoxicity. International Organization for Standardization: Geneva, Switzerland, 2009. 41. Amjadi, S.; Hamishehkar, H.; Ghorbani, M. A novel smart PEGylated gelatin nanoparticle for co-delivery of doxorubicin and betanin: A strategy for enhancing the therapeutic efficacy of chemotherapy. Mater. Sci. Eng. C Mater. Biol. Appl. 2019 ,97, 833–841. [CrossRef] 42. Nie, S.; Hsiao, W.L.W. Thermoreversible Pluronic ® F127-based hydrogel containing liposomes for the controlled delivery of paclitaxel: In vitro drug release, cell cytotoxicity, and uptake studies. Int. J. Nanomed. 2011,6, 151–166. [CrossRef] 43. Diniz, I.M.A.; Chen, C.; Xu, X.; Ansari, S.; Zadeh, H.H.; Marques, M.M.; Shi, S.; Moshaverinia, A. Pluronic F-127 hydrogel as a promising scaffold for encapsulation of dental-derived mesenchymal stem cells. J. Mater. Sci. Mater. Med. 2015 ,26, 153. [CrossRef] [PubMed] 44. de Melo Santana, B.; Pieretti, J.C.; Gomes, R.N.; Cerchiaro, G.; Seabra, A.B. Cytotoxicity towards Breast Cancer Cells of Pluronic F-127/Hyaluronic Acid Hydrogel Containing Nitric Oxide Donor and Silica Nanoparticles Loaded with Cisplatin. Pharmaceutics 2022,14, 2837. [CrossRef] [PubMed] 45. García-Couce, J.; Tomás, M.; Fuentes, G.; Que, I.; Almirall, A.; Cruz, L.J. Chitosan/Pluronic F127 Thermosensitive Hydrogel as an Injectable Dexamethasone Delivery Carrier. Gels 2022,8, 44. [CrossRef] [PubMed] 46. Pragatheeswaran, A.M.; Chen, S.B.; Chen, C.-F.; Chen, B.-H. Micellization and gelation of PEO-PPO-PEO binary mixture with non-identical PPO block lengths in aqueous solution. Polymer 2014,55, 5284–5291. [CrossRef] 47. Sánchez-Cid, P.; Jiménez-Rosado, M.; Rubio-Valle, J.F.; Romero, A.; Ostos, F.J.; Benhnia, M.R.-E.; Perez-Puyana, V. Biocompatible and Thermoresistant Hydrogels Based on Collagen and Chitosan. Polymers 2022,14, 272. [CrossRef] Gels 2023,9, 982 20 of 20 48. Gonçalves, L.C.; Seabra, A.B.; Pelegrino, M.T.; de Araujo, D.R.; Bernardes, J.S.; Haddad, P.S. Superparamagnetic iron oxide nanoparticles dispersed in Pluronic F127 hydrogel: Potential uses in topical applications. RSC Adv. 2017 ,7, 14496–14503. [CrossRef] 49. Jain, T.K.; Morales, M.A.; Sahoo, S.K.; Leslie-Pelecky, D.L.; Labhasetwar, V. Iron oxide nanoparticles for sustained delivery of anticancer agents. Mol. Pharm. 2005,2, 194–205. [CrossRef] [PubMed] 50. Chandra, S.; Mehta, S.; Nigam, S.; Bahadur, D. Dendritic magnetite nanocarriers for drug delivery applications. New J. Chem. 2010,34, 648–655. [CrossRef] 51. Ota, S.; Takemura, Y. Characterization of Néel and Brownian Relaxations Isolated from Complex Dynamics Influenced by Dipole Interactions in Magnetic Nanoparticles. J. Phys. Chem. C 2019,123, 28859–28866. [CrossRef] 52. Liu, X.; Wang, Y.; Zhu, W.; Li, G.; Ma, X.; Zhang, Y.; Chen, S.; Tiwari, S.; Shi, K.; Zhang, S.; et al. Comprehensive understanding of magnetic hyperthermia for improving antitumor therapeutic efficacy. Theranostics 2020,10, 3793–3815. [CrossRef] 53. Gonçalves, A.; Almeida, F.V.; Borges, J.P.; Soares, P.I.P. Incorporation of dual-stimuli responsive microgels in nanofibrous membranes for cancer treatment by magnetic hyperthermia. Gels 2021,7, 28. [CrossRef] 54. Monks, P.; Wychowaniec, J.K.; McKiernan, E.; Clerkin, S.; Crean, J.; Rodriguez, B.J.; Reynaud, E.G.; Heise, A.; Brougham, D.F. Spatiotemporally Resolved Heat Dissipation in 3D Patterned Magnetically Responsive Hydrogels. Small 2021 ,17, e2004452. [CrossRef] 55. Heo, J.Y.; Noh, J.H.; Park, S.H.; Ji, Y.B.; Ju, H.J.; Kim, D.Y.; Lee, B.; Kim, M.S. An injectable click-crosslinked hydrogel that prolongs dexamethasone release from dexamethasone-loaded microspheres. Pharmaceutics 2019,11, 438. [CrossRef] 56. Soares, P.I.; Laia, C.A.; Carvalho, A.; Pereira, L.C.; Coutinho, J.T.; Ferreira, I.M.; Novo, C.M.; Borges, J.P. Iron oxide nanoparticles stabilized with a bilayer of oleic acid for magnetic hyperthermia and MRI applications. Appl. Surf. Sci. 2016 ,383, 240–247. [CrossRef] 57. Liu, Y.; Li, Y.; Li, X.-M.; He, T. Kinetics of (3-aminopropyl)triethoxylsilane (APTES) silanization of superparamagnetic iron oxide nanoparticles. Langmuir 2013,29, 15275–15282. [CrossRef] 58. Zhang, Y.; Huo, M.; Zhou, J.; Zou, A.; Li, W.; Yao, C.; Xie, S. DDSolver: An add-in program for modeling and comparison of drug dissolution profiles. AAPS J. 2010,12, 263–271. [CrossRef] 59. ISO 10993-1:2018; Biological Evaluation of Medical Devices Part 1: Evaluation and Testing within a Risk Management Process. International Organization for Standardization: Geneva, Switzerland, 2018. 60. Arifin, D.Y.; Lee, L.Y.; Wang, C.-H. Mathematical modeling and simulation of drug release from microspheres: Implications to drug delivery systems. Adv. Drug Deliv. Rev. 2006,58, 1274–1325. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.