*3.2. Preparation of SPIONs Coated with PGlCLcys by Co-Precipitation Method*

The preparation of SPIONs coated with PGlCLcys (SPION@PGlCLCys) was performed according to the co-precipitation approach with modifications [25,39]. The SPION@PGlCLCys exhibited an average particle diameter of 7.2 ± 2.2 nm (Figure 2A,D), determined by transmission electron microscopy (TEM-100 kV), and a monomodal distribution, as well as high crystallinity (Figure 2B) determined by selected area electron diffraction (SAED) technique. The average crystalline domains and core size were around 7.6 nm as estimated from the results of X-ray powder diffraction (XRPD) analysis (Figure 2C). The lattice parameter obtained after Rietvield refinement (a = 8.36154 Å) applied to the XRPD data (0.252 nm to (311)) is consistent with the value of 8.46 Å based on the (220) interplanar spacing of 0.299 nm obtained using SAED (Figures S3 and S4, see SM). Thus, the results agree with those of other studies [40] related to the spinel structure of Fe3O4 and standard measurements (a = 8.3940 Å) (for more details, see Table S4, in SM).

**Figure 2.** SPION@PGlCLCys (**A**) Low magnification TEM image; (**B**) SAED; (**C**) XRPD diffractogram with diffraction peaks indexed to the spinel iron oxide phase. (**D**) Particle size distribution of the magnetic nanoparticles obtained from the TEM images.

The superparamagnetic behavior of SPION@PGlCLCys observed by VSM analysis corroborates the core size below 15 nm [41] and the successful coating of the SPIONs, presenting a saturation magnetization of 60 emu·g−1, which is consistent with saturation magnetization values obtained for magnetite in literature [42,43] (for details, see Figure S7, in SM). The coating of the SPIONs by PGlCLCys was also evidenced by the FTIR spectra (Figure S5, in SM), indicated by vibration frequencies at 1736 cm−<sup>1</sup> (elongation –C=O), associated with a carboxyl group and elongation of the amine group at 1643 cm−<sup>1</sup> (–NH2).

TGA results (Figure S6) gives important information on the SPION@PGlCLCys composition. The thermogram shows a mass loss of 1.7%, related to water evaporation, followed by a mass loss of 13.3%, assigned to PGlCLCys decomposition, and finally the 85% that remained corresponds to the iron oxide presence. The proportion of Fe3O4:PGlCL used in the preparation of the SPION@PGlCLCys was 8:1 (*w*/*w*), which means theoretically there are 11.1% PGlCLCys and 88.9% Fe3O4 in SPION@PGlCLCys. Therefore, one can say that the experimental results is in accordance with the theoretical composition values.

SPION@PGlCLCys were resuspended in buffer solution (pH = 8.0, close to physiological conditions) and Dynamic Light Scattering (DLS) measurements were performed (see Figure S8, in SM). The intensity average particle diameter measured was 145 nm with a PDI of 0.2. This result shows that after being dispersed, SPION@PGlCLCys formed some aggregates, but the unimodal size distribution and PDI value of 0.2 indicate that such aggregates are stable. A possible cause for this aggregation is the small amount of PGlCLCys used to coat the SPIONs, which provides a very thin layer of polymer coating the magnetite core. On one hand, this has a very positive impact on the saturation magnetization of SPION@PGlCLCys (60 emu·g−1). On the other hand, such a thin layer does not avoid a strong interaction between the nuclei and provides some aggregation of SPIONs after the lyophilization process. However, it is also important to emphasize that hydrodynamic diameter of 145 nm with a PDI of 0.2, which is an acceptable size for drug delivery purposes. The SPION@PGlCLCys also showed a zeta potential value of −35.4 mV (pH = 8.0, close to physiological conditions), suggesting moderate colloidal stability [44]. Such negative value is much likely related to the presence of cysteine in the polymer, suggesting that in addition to developing the role of protecting the SPIONs core, PGlCLCys also helps in some extent to maintain some colloidal stability in the system. The isoelectric point of the amino acid cysteine is widely reported in literature as being 5.07 [45]. That is, in pH values above 5.07, cysteine structure is deprotonated, assuming an overall negative charge.

### *3.3. Conjugation of FA or MTX on the SPION@PGLCLCys, Enzymatic Release, and Cell Viability*

Since SPION@PGlCLCys is stable in aqueous medium, its surface was covalently conjugated with FA by carbodiimide approach, aiming to target the folate receptors in breast cancer. The amount of FA covalently bonded to the SPION@PGlCLCys was determined by UV–vis spectrometry, by using a FA calibration curve, dissolved in pH = 8.0 buffer at 286 nm (further details see Figure S10A, in SM). Considering NH2:COOH ratios of 1:1 (stoichiometric proportion) and 1:2 (excess of FA), the conjugation efficiency of FA on the SPIONs surface was 62% and 183.5%, respectively, relative to the available NH2 sites for conjugation. A conjugation efficiency higher than 100% is explained by the adsorption of the FA in excess on the surface of the SPION@PGlCLCys. For the release assays and cell viability assays, we chose to proceed with the samples synthesized using a NH2:COOH ratio of 1:1, containing approximately 8.27 μg FA/mg iron oxide.

DFT calculations were also performed in order to provide theoretical information on the hydrophilicity of PGlCLCys\_FA, which presented an estimated log PO/W value of 0.81, a much lower value than PGlCL (log PO/W = 5.53) and PGlCLCys (log PO/W = 3.54), indicating that the presence of FA makes SPION@PGlCLCys\_FA quite hydrophilic. Such hydrophilic characteristic must be highlighted, since the hydrophilization of nanoparticles is commonly reported as a strategy to increase their circulation time in the blood stream [46].

The cytotoxicity of SPION@PGlCLCys and SPION@PGlCLCys\_FA was evaluated by the MTT assay using L929 and MDA-MB 231 cell lines (for details, see Figures S11 and S12

in SM). Cell viability reached 100% for all tested samples, in the concentration range from 1.10−<sup>4</sup> ppm to 100 ppm, for 24 h and 72 h, and for both tested cell lines. In sequence, the release profile of conjugated FA on the surface of SPION@PGlCLCys\_FA was evaluated. This way, bromelain was used as a protease to simulate a possible release triggered via lysosomal protease at 37 ◦C and pH~5.3, similar to cellular pH, to verify the cleavage of the amide bond formed in the conjugation of FA to the surface of SPION@PGlCLCys (more details, see Figure S13, in SM). Results showed that protease cleaves approximately ~28% of the amide bonds within 24 h, and ~35% in 72 h. This is the main route of drug release, since for the assay at the same pH but without enzyme, there is no release.

Finally, the conjugation of the drug MTX on SPION@PGlCLCys was performed, and the conjugation efficiency was determined by the UV–vis method (calibration curves in Figure S10, SM, dissolved in pH = 8.0 buffer, at 303 nm). The results obtained for MTX conjugation efficiency are 60.3% (1:1 stoichiometric proportion) and 132.4% (1:2 excess of MTX), relative to the available NH2 sites for conjugation. A conjugation efficiency higher than 100% is explained by the adsorption of the MTX in excess on the surface of the SPION@PGlCLCys Again, for the release assays and cell viability assays, we chose to proceed with the samples synthesized using a NH2:COOH ratio of 1:1, which provided the highest conjugation efficiency, containing approximately 3.20 μg MTX/mg iron oxide.

Similar to FA, the enzymatic release assay for SPION@PGlCLCys\_MTX was carried out at lysosomal pH. The results (Figure 3A) showed that protease cleaves approximately ~38% of the amide bonds within 24 h, and ~45% in 72 h. This can be considered a very positive result, since other works in literature that investigated MTX encapsulation in polyesters nanoparticles (e.g., polycaprolactone) often report a release of around 40% in 72 h, or even less depending on how hydrophobic the polymer matrix is [47,48]. Therefore, it is important to emphasize that in our conjugate (SPION@PGlCLCys\_MTX), MTX is not only acting as a drug itself, but also as a molecule that has specific interactions for folate receptors, promoting specific targeting in tumor cells, forming the basis of our proposal of a multifunctional nanoplatform for cancer treatment.

**Figure 3.** SPION@PGlCLCys\_MTX: (**A**) drug delivery assay at lysosomal pH (pH 5.3) with or without protease (ENZ); (**B**) breast carcinoma-derived MDA-MB 231 cells viability after 72 h at different MTX concentrations.

Regarding the MTT cell viability assay, for 72 h incubation, a strong reduction in breast carcinoma-derived MDA-MB 231 cell viability was observed in free MTX for concentrations above 0.1 ug·mL−1. For the conjugates SPION@PGlCLCys\_MTX, this decrease was smoother reaching a reduction of approximately 20% at the highest evaluated concentration (Figure 3B).

The superior behavior of free MTX in comparison to SPION@PGlCLCys\_MTX in the cell viability assays was expected, since free MTX is immediately available to interact with the cells, while MTX attached to SPION@PGlCLCys releases about 45% of MTX in 72 h.

### **4. Conclusions**

In this work, we have successfully prepared SPIONs coated with a polyester modified with the amino acid cysteine (SPION@PGlCLCys). The cysteine molecules present in the SPIONs' coating can be used as anchoring points for the conjugation of a wide variety of molecules (e.g., peptides, antibodies, drugs, etc.). The main goal here was to prepare SPION@PGlCLCys and test their potential as a multifunctional nanoplatform for future developments to the traditional cancer treatments. In order to test such potential, we conjugated the SPION@PGlCLCys with two different molecules, separately, in order to understand their behavior when conjugated to SPION@PGlCLCys. FA was chosen due to its characteristic of stablishing specific interactions to folate receptors (overexpressed in tumor cells), as well as methotrexate, an anti-cancer drug that also has specific interaction for folate receptors and acts controlling the growth of the tumor. Both FA and MTX presented satisfactory conjugation efficiencies (above 65%). Release assays were performed in the presence of a protease, and MTX presented 45% release after 72 h, which is a very positive result in comparison to other works that studied the encapsulation and release of MTX from polymeric nanoparticles. MTT assay revealed that after 72 h, the conjugates are capable of reducing the tumor cell viability in about 20%, which is also noted as a very positive result, considering the small amount of MTX conjugated to SPION@PGlCLCys. The results obtained in this work can be seen as the first step for the development of a promising nanoplatform that can be easily modified and improved for future applications in less aggressive cancer treatments, allying targeting of tumor cells, controlled drug delivery, hyperthermia, and eventually diagnosis (theranostics).

**Supplementary Materials:** The following Supplementary Materials can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15031000/s1. Figure S1. Representative scheme of the reaction steps to obtain PGlCL and its modification with cysteine followed by coating of SPIONs. A) Enzymatic Ring-Opening Polymerization reaction (e-ROP). B) Modification reaction with the amino acid cysteine (Cys) via thiol-ene reaction to obtain PGlCLCys. C) Synthesis and stabilization of SPIONs with PGlCLCys. D) Conjugation of SPIONs with FA. E) Conjugation of SPIONs with MTX. Figure S2: TEM dark field image showing individual crystalline particles (DF-TEM) of SPION@PGlCLCys. Figure S3: Selected area electron diffraction (SAED) image showing indexed diffraction rings corresponding to magnetite crystallographic planes of SPION@PGlCLCys. Figure S4: SAED image with Indexed diffraction pattern of magnetite and simulated diffraction ring pattern matching the results for the SPION@PGlCLCys sample. Figure S5: FT-IR spectra for SPIONs, and modified copolymer (PGlCLCys) and after coating of magnetic nanoparticles (SPION@PGlCLCys). Figure S6: Thermogravimetric analysis of SPION@PGlCLCys. Figure S7: VSM analysis of SPION@PGlCLCys. Figure S8: DLS analysis of SPION@PGlCLCys. Figure S9: Zeta potential surface analysis of SPION@PGlCLCys (ζ = −35.4 mV) dispersed in buffer solution (pH = 8.0). Figure S10: UV-vis calibration curve for: A) folic acid (FA), and B) methotrexate (MTX) in buffer solution (pH = 8.0). Figure S11: MTT assay of SPION@PGlCLCys and SPION@PGlCLCys\_FA showing cells viability as a function of nanoparticles concentration (0.0001 to 100 μg·mL−1) for 24 h. All SPIONs tested at different concentrations did not exert difference (ANOVA) in relations to the control group n = 3. Figure S12: MTT assay of SPION@PGlCLCys and SPION@PGlCLCys\_FA showing cells viability as a function of nanoparticles concentration (0.0001 to 100 μg·mL−1) for 72 h. All SPIONs tested at different concentrations did not exert difference (ANOVA) in relations to the control group n = 3. Figure S13: FA assay at lysosomal pH (pH 5.3). Table S1: Thiol-ene reaction conversion calculated based on the consumption of the double bonds present in PGlCL chains, determined by 1H NMR. Table S2: Thermal properties of the polymers, determined by DSC. Table S3: Gibbs free energy calculated at 1 atm and 25 ◦C using DFT/B3LYP/6-31G\*\* with water and n-octanol solvents in SMD model. Table S4: Interplanar distance.

**Author Contributions:** J.M.B.: investigation, methodology, writing—original draft, writing—review and editing. B.B.P.R.: formal analysis, investigation, methodology, writing—original draft, writing review and editing. C.G.: conceptualization, writing—original draft, writing—review and editing, project administration, funding acquisition, supervision. G.C.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing. K.B.F.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing. R.L.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing. A.D.Z.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing. E.I.: formal analysis, investigation, methodology, writing—original draft, writing—review and editing. C.S.: conceptualization, project administration, funding acquisition, supervision. P.H.H.d.A.: conceptualization, project administration, funding acquisition, supervision. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq (project number 406078/2018-1), CAPES PRINT Program (project number 88887.310560/2018-00). C.G. and G.C. thank FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), process number E-26/201.911/2020, E-26/201.912/2020, E-26/200.627/2022 and E-26/210.391/2022. A.D.A.Z. is grateful to FAPESC (Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina), project number IFS2020201000004.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank the Central Laboratory of Electron Microscopy (LCME) at UFSC (TEM analysis) as well as the Laboratory for Characterization Magnetic Materials (LabCAM) for the VSM measurements. In addition, special thanks are due to the Laboratory of Nanotechnology (LINDEN-UFSC) at UFSC. The authors gratefully acknowledge the computational support of Núcleo Avançado de Computação de Alto Desempenho (NACAD/COPPE/UFRJ), Sistema Nacional de Processamento de Alto Desempenho (SINAPAD), Centro Nacional de Processamento de Alto Desempenho em São Paulo (CENAPAD-SP) and the Startup SMMOL (Rio de Janeiro, RJ, Brazil) for the support. We also would like to thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ, the researcher Marco Augusto Stimamiglio, and the laboratory Stem Cells Basic Biology Laboratory, Carlos Chagas Institute—FIOCRUZ/PR for their collaboration and availability of infrastructure.

**Conflicts of Interest:** The authors declare no conflict of interest.
