DOSE-DEPENDENT EFFECT OF HEPARIN-FUNCTIONALIZED GRAPHENE OXIDE: IMPLICATIONS FOR ITS POTENTIAL ANTI-HERPETIC USE

Resumo

INTRODUCTION: Graphene oxide (GO) is a promising nanomaterial in translationalmedicine, especially as a vehicle for controlled drug delivery. Its functionalization with heparin(GO@Heparin) has shown antiviral potential, particularly against herpesviruses, due to itsstructural similarity to heparan sulfate, a viral entry receptor. However, evaluating its safetyand toxicity is essential for therapeutic use. OBJECTIVE: To assess the safety profile andbiological effects of GO, GO@Heparin 5.0%, and heparin in VERO cells, focusing on cellviability and oxidative stress, after acute (24h) and chronic (72h) exposure. MATERIALSAND METHODS: This is an in vitro experimental study using VERO cells treated withdifferent concentrations of GO, GO@Heparin 5.0%, and heparin. The following assays wereperformed: MTT (cell viability), PicoGreen (cell death via double-stranded DNAquantification), and nitrite determination (as a marker of oxidative stress). Statistical analysiswas conducted using one-way ANOVA followed by Dunnett’s post hoc test, with the aid ofGraphPad Prism software. RESULTS: After 24 hours of exposure, GO and GO@Heparinstimulated cell proliferation at concentrations ranging from 3.9 to 125 µg/mL (GO) and up to62.5 µg/mL (GO@Heparin), with a dose-dependent increase in nitrite levels (p<0.0001).Heparin significantly promoted cell proliferation at all tested concentrations without a notableincrease in nitrite production, indicating a safe bio-stimulatory effect. After 72 hours, the dualnature of the nanomaterials became evident: GO and GO@Heparin maintained theirproliferative effects at low concentrations but showed significant cytotoxicity at 250 µg/mL(p<0.0001), with increased cell death and reduced DNA detection. Heparin, on the other hand,continued to promote proliferation, with only a slight increase in oxidative stress at the lowestdoses. CONCLUSION: GO and GO@Heparin demonstrated beneficial effects at lowconcentrations and toxicity at higher doses, especially after 72 hours. Functionalization withheparin did not completely eliminate toxicity at elevated concentrations but preserved part ofthe proliferative effect observed with free heparin, suggesting that the conjugation retains itsbioactivity. This is particularly relevant in the antiviral context, given the potential ofGO@Heparin as a nanocarrier with anti-herpetic activity provided that a safe therapeuticwindow is respected.