## 3 Results
### Gene Therapy and RNA Interference (RNAi)  
#### 3.3.1 siRNA-Loaded Nanoparticles  
RNA interference is a powerful gene-silencing technique that uses small interfering RNA (siRNA) molecules to suppress the expression of specific genes. However, free siRNA is unstable in the bloodstream due to rapid degradation by nucleases and elimination via glomerular filtration, resulting in low bioavailability and limited therapeutic potential [66]. To address this, siRNA-loaded nanoparticles have emerged as a promising delivery platform, protecting siRNA and enhancing its accumulation in target tissues. Among these, lipid-based nanoparticles are particularly effective, as they can fuse with cellular membranes and facilitate the release of siRNA directly into the cytoplasm. The progression of hepatocellular carcinoma is largely driven by angiogenesis, which results from the excessive secretion of VEGF by tumor cells within the tumor microenvironment [67]. Thus, reducing VEGF expression through siRNA could form the basis for a promising anti-HCC therapy. [PERSON] et al. developed a system that overcomes these barriers by encapsulating siRNA in a pH-sensitive calcium phosphate core coated with a lipid membrane. This structure protects the siRNA from enzymatic degradation and enables the release of the nucleic acid directly into the cytoplasm of tumor cells. To achieve efficient siRNA delivery and targeting within HCC cells, given that galactose derivatives possess strong targeting capabilities toward ASGPR on tumor cells, the team synthesized various galactoside derivatives and encapsulated siRNA/VEGF into lipid/calcium/phosphate (LCP) nanoparticles specifically targeted to ASGPR. Their findings illustrate that LCP nanoparticles decorated with phenyl \(\beta\)-D-galactoside (L4-LCPNPs) enabled highly efficient siRNA delivery to HCC cells, with significantly less uptake in normal hepatocytes. In vitro and in vivo, VEGF siRNA released by L4-LCPNPs successfully downregulated VEGF expression in HCC, producing a marked antiangiogenic effect within the tumor microenvironment. This led to substantial tumor regression and suggests that phenyl galactoside could serve as a promising HCC-targeting ligand for therapeutic siRNA delivery in the treatment of liver cancer. Nonetheless, limitations remain, including limited biodistribution analysis and lack of long-term safety data, which warrant further investigation before clinical translation [68]. Previous research has indicated that AFP, a common HCC marker, plays a crucial role in the proliferation and apoptosis of hepatocellular carcinoma cells. Elevated levels of AFP are associated with increased cell growth and survival, highlighting its potential as a therapeutic target in HCC management. Studies suggest that AFP may influence various signaling pathways that regulate these processes, thus contributing to cancer progression [69]. This relationship highlights the potential of siRNA in targeting and modulating the pathways involved in HCC progression, offering a novel approach to treatment. Research on polymeric nanoparticles loaded with siRNA against AFP has demonstrated significant anticancer effects in HCC cells. Epigallocatechin-3-gallate (EGCG), a potent polyphenol found in green tea, has shown notable anticancer properties by blocking tumor progression through mechanisms such as cell cycle arrest and induction of apoptosis in cancer cells. In this context, co-delivery systems that combine biodegradable PLGA nanoparticles with AFP-targeting siRNA and EGCG have shown enhanced cytotoxicity against liver cancer cells compared to either treatment alone, suggesting a strong synergistic effect. However, these strategies have only shown efficacy in vitro, lacking in vivo validation, and their underlying molecular mechanisms remain only partially understood [69]. Among siRNA delivery strategies for HCC, selenium nanoparticles (SeNPs) functionalized with HA and coated with polyethyleneimine (PEI) have gained attention due to their favorable targeting and delivery properties.