Graphene Oxide–Antisense miR-21 Nanosystem Modulates Gene Expression and Suppresses Tumorigenesis in HepG2-Derived CAM Xenografts

Graphene oxide (GO) is a promising nanocarrier for the delivery of oligonucleotides. It offers a high loading capacity, efficient cellular uptake, and surface functionalization. MicroRNA-21 (miR-21) is a well-characterized oncomiR commonly overexpressed in hepatocellular carcinoma (HCC). In HCC, miR-21 contributes to tumor progression, inflammation, and angiogenesis. In a previous in vitro study, we showed that GO alone induces the upregulation of pro-inflammatory and tumor-related genes in HepG2 cells. However, conjugation with an antisense miR-21 (GO-antisense miRNA 21) reverses this effect, suggesting a potential therapeutic application. This study aims to evaluate the antitumor and anti-angiogenic efficacy of the GO-antisense miR-21 nanosystem in ovo using the chick embryo chorioallantoic membrane (CAM) model. Fertilized chicken eggs (n = 4 per group) were randomized into untreated, GO-treated, and GO–antisense miR-21-treated cohorts. A dose of 200 μL (GO 10.0 µg/mL: antisense miR-21 5.0 pmol/mL) was administered intratumorally. Tumor size, volume, and vascularization were monitored through stereomicroscopy and histological analysis. The expression of inflammatory and tumor-associated genes (IL-8, MCP-1, TIMP-2, ICAM-1 and NF-kB) was assessed by quantitative PCR. Given its prominent response, IL-8 protein expression was further analyzed via immunofluorescence. To evaluate tumor-specific delivery, FITC-labeled GO was tracked by confocal microscopy. Our data revealed that treatment with unfunctionalized graphene oxide (GO) unexpectedly promoted tumor vascularization and led to a significant increase in tumor weight. This was accompanied by upregulation of inflammatory markers. In contrast, GO-antisense miR-21 significantly reduced the tumor volume and vessel density. It also successfully downregulated all target genes. Confocal imaging demonstrated preferential accumulation of the nanosystem within the tumor mass. Our results highlight the dual anti-inflammatory and anti-angiogenic effects of GO-antisense miRNA 21 in ovo and support its potential as a targeted nanoplatform for HCC treatment.