PREPARATION OF FITC FUNCTIONALIZED CELLULOSE NANOFIBRILS FOR FURTHER IN VITRO TUMOR UPTAKE STUDIES

Introduction Natural polymeric nanoparticles are favored for their size, high drug-loading capacity, bioavailability, non-toxicity, biocompatibility, and the possibility of versatile surface modifications and functionalization’s. For these reasons, nanocellulose has attracted interest as a potential drug nanocarrier. Labeling nanomaterials with fluorescent molecules has become a popular tool for in vitro autophagy monitoring, nanotoxicological studies, and the development of therapeutic applications. Among several markers, fluorescein isothiocyanate (FITC) stands out primarily for its biocompatibility and direct conjugation chemistry. Purpose This project aims to prepare a controlled-release system suitable for in vivo application based on a fluorescein-labeled polymeric nanocellulose matrix. Material and methods Cellulose nanofibrils (CNF) were produced from sugarcane stalks using a mechanical and chemical process. The nanocompound was FITC-functionalized in three steps, as previously described (Environ. Sci.: Nano, 2019, 6,1516). Briefly. Step 1: 15 mL of 3.7% solid CNF suspension was reacted with 250 mL of epichlorohydrin in the presence of 50 mL of 1.34 M NaOH, under mechanical stirring at 60°C for 2h. The epoxy functionalized CNF was centrifuged at 17,500 crf for 5 minutes and then washed with DI water 3 times. Step 2: The epoxy-functionalized CNF after 24 hours was adjusted to pH 12 with 1.34 M NaOH and reacted with 2.8 mL of NH4OH under mechanical stirring at 60°C for 2h. The amine-functionalized CNF were then centrifuged and washed as described above. Step 3: The amine-functionalized CNF were placed in a Na2B4O7 and NaCl buffer solution for 30 min. A water solution of FITC was prepared by adding 0.07g of FITC isothiocyanate in 50 mL of DI water and stirring for 30 min. 1.05g of NaCl and 2.3g of Na2B4O7 were added to the 1% solids CNF suspension, which was then stirred for 30 min. The FITC solution was then added to the CNF and stirred for 12 hours. Finally, the FITC-tagged materials were centrifuged at 17,500 crf for 5 minutes and washed 10 times with DI water. The final 10 wash waters were saved for fluorescence analysis on a Typhoon fluorescence imaging system. The final product was analyzed by FT-IR. Results FITC conjugation to CNF was confirmed by the presence of C=S stretching at 1,083 cm -1 . Final product washing step confirmed the absence of FITC by the fourth portion. Conclusion FITC-CNF was adequately prepared, and it is suitable for the next step involving the tumor cell behavior as uptake and toxicity.