*Article* **Coating of SPIONs with a Cysteine-Decorated Copolyester: A Possible Novel Nanoplatform for Enzymatic Release**

**Jeovandro Maria Beltrame 1,\*, Brena Beatriz Pereira Ribeiro 1, Camila Guindani 2, Graziâni Candiotto 3, Karina Bettega Felipe 4, Rodrigo Lucas 5, Alexandre D'Agostini Zottis 6, Eduardo Isoppo 7, Claudia Sayer 1,\* and Pedro Henrique Hermes de Araújo <sup>1</sup>**


**Abstract:** Superparamagnetic iron oxide nanoparticles (SPIONs) have their use approved for the diagnosis/treatment of malignant tumors and can be metabolized by the organism. To prevent embolism caused by these nanoparticles, they need to be coated with biocompatible and non-cytotoxic materials. Here, we synthesized an unsaturated and biocompatible copolyester, poly (globalide*co*-ε-caprolactone) (PGlCL), and modified it with the amino acid cysteine (Cys) via a thiol-ene reaction (PGlCLCys). The Cys-modified copolymer presented reduced crystallinity and increased hydrophilicity in comparison to PGlCL, thus being used for the coating of SPIONS (SPION@PGlCLCys). Additionally, cysteine pendant groups at the particle's surface allowed the direct conjugation of (bio)molecules that establish specific interactions with tumor cells (MDA-MB 231). The conjugation of either folic acid (FA) or the anti-cancer drug methotrexate (MTX) was carried out directly on the amine groups of cysteine molecules present in the SPION@PGlCLCys surface (SPION@PGlCLCys\_FA and SPION@PGlCLCys\_MTX) by carbodiimide-mediated coupling, leading to the formation of amide bonds, with conjugation efficiencies of 62% for FA and 60% for MTX. Then, the release of MTX from the nanoparticle surface was evaluated using a protease at 37 ◦C in phosphate buffer pH~5.3. It was found that 45% of MTX conjugated to the SPIONs were released after 72 h. Cell viability was measured by MTT assay, and after 72 h, 25% reduction in cell viability of tumor cells was observed. Thus, after a successful conjugation and subsequent triggered release of MTX, we understand that SPION@PGlCLCys has a strong potential to be treated as a model nanoplatform for the development of treatments and diagnosis techniques (or theranostic applications) that can be less aggressive to patients.

**Keywords:** copolyester; SPION; cysteine; bioconjugation; and enzymatic release
