Chit-nanoEGCG Synthesis and Characterization

The Chit-nanoEGCG were synthesized by ionic gelation with TPP ions and the nanoparticles were characterized using TEM and dynamic light scattering. TEM micrograph of the CS-NPs was shown in Fig. S1A. The Chit-nanoEGCG showed a spherical shape with a particle size of 16–30 nm. A representative diagram of the size distribution of Chit-nanoEGCG by dynamic light scattering is shown in Fig. S1B. The sample was analyzed in triplicate to yield the average particle size (Z-Average) at a constant temperature of 25 °C. Z-Average of Chit-nanoEGCG NPs was 219.7 ± 1.96 diameter nanometer (d. nm) and the polydispersity index (PDI) of NPs, which indicates the size distribution, was 0.4 in average. A nanoparticle system with PDI value in range of 0.1–0.4 indicated that the system has moderately disperse distribution. It is observed that the zeta potential value of the prepared Chit-nanoEGCG was found to be − 51.5 ± 5.91 mV which indicated that NPs are moderately to highly dispersed (more than + 30 mV or less than − 30 mV) with moderate to high stability and the conductivity was 0.26 milliSiemens/cm at a constant temperature of 25 °C (Fig. S1C). Chit-nanoEGCG showed the encapsulation efficiency from 32.50 ± 7.44 to 69.00 ± 2.92%.

Cell Viability, Apoptosis, and Assessment of Efficacy of Chit-nanoEGCG

The treatment of HepG2 cells with Chit-nanoEGCG, native EGCG, and cisplatin caused dose-dependent loss of cell viability after 48 h (Fig. 1A). The mean concentration which inhibited 50% of cell growth (IC50) was detected by averaging the individual results from three repeated experiments. The IC50 doses of chit-nanoEGCG, native EGCG, and cisplatin in HepG2 cells were 340.0, 228.7, and 515.8 µg/ml, respectively. FITC-Annexin V/PI analysis was performed on HepG2 cells after treatment with a 60% of IC50 dose of Chit-nanoEGCG, native EGCG, and cisplatin for 48 h. Figure 1B shows highly significant apoptosis in the Chit-nanoEGCG group (early and late apoptosis are 18.92 ± 0.54 and 25.03 ± 0.63, respectively) when compared to the control group (early and late apoptosis are 0.34 ± 0.07and 0.25 ± 0.04, respectively) (p < 0.001).

Fig. 1

Cytotoxic effect of Chit-nanoEGCG on HepG2 cancer cells after 48 h post-treatment; A HepG2 cell viability and IC50 after treatment with varying concentrations of Chit-nanoEGCG, EGCG, cisplatin, and chitosan NPs by MTT assay. B The percentages of viable, apoptotic, and necrotic cells in control and treated HepG2 cells with Chit-nanoEGCG, EGCG, and cisplatin as shown by flow cytometry. Data are expressed as mean ± SD values from at least 3 independent experiments

Flow Cytometric Analysis

In HepG2 cells, Chit-nanoEGCG treatment significantly upregulated the expression of apoptotic proteins including P53 (Fig. 2A), Bax (Fig. 2B), caspase-3 (Fig. 2E), caspase-9 (Fig. 2F), and PARP (Fig. 2G), compared to the untreated cells. In contrast, compared to native EGCG treatment, cisplatin treatment, and the untreated cells, the antiapoptotic protein Bcl-2 (Fig. 2C) was significantly downregulated after Chit-nanoEGCG treatment.

Fig. 2

Apoptotic proteins expression of P53 (A), Bax (B), Bcl-2 (C), Bax/Bcl-2 ratio (D), caspase-3 (E), caspase-9 (F), and PARP (G) proteins expression in control and treated HepG2 cells with Chit-nanoEGCG, EGCG, and cisplatin using flow cytometric analysis. Data are expressed as mean ± SD values from at least 3 independent experiments. *, **, and *** indicate statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the Chit-nanoEGCG–treated group

RT-qPCRApoptotic Genes Expression

RT-qPCR shows a significant increase in P53 (Fig. 3A) and Bax (Fig. 3B) apoptotic genes after Chit-nanoEGCG treatment when compared with native EGCG treatment, cisplatin group, or control group in HepG2 cells. In contrast, the antiapoptotic protein Bcl-2 was significantly decreased after treatment with Chit-nanoEGCG when compared with native EGCG treatment, cisplatin treatment, and control group in HepG2 cells as shown in Fig. 3C.

Fig. 3

Apoptotic genes expression of P53 (A), Bax (B), and Bcl-2 (C) in control and treated HepG2 cells with Chit-nanoEGCG, EGCG, and cisplatin using RT-qPCR. Data are expressed as mean ± SD values from at least 3 independent experiments. ** and *** indicate statistical significance at p < 0.01 and p < 0.001, respectively, compared to the Chit-nanoEGCG–treated group

Transcription Genes Expression

The current results demonstrated a significant decrease in OCT4 (Fig. 4A) and SOX2 (Fig. 4B) expression, while an insignificant expression in NANOG (Fig. 4C) transcription genes after Chit-nanoEGCG treatment was observed when compared with control groups.

Fig. 4

Transcription gene expression of OCT4 (A), SOX2 (B), and NANOG (C) in control and treated HepG2 cells with Chit-nanoEGCG, EGCG, and cisplatin using RT-qPCR. Data are expressed as mean ± SD values from at least 3 independent experiments. *, **, and *** indicate statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the Chit-nanoEGCG–treated group

Receptor and Transporter Genes Expression

When comparing the Chit-nanoEGCG treatment to the control, RT-qPCR reveals a substantial rise in CD95 (Fig. 5B) but a significant decrease in CD133 (Fig. 5C), NOTCH1 (Fig. 5D), c-MET (Fig. 5E), and Ezrin (Fig. 5F). However, there was no significance in the expression of CD44 (Fig. 5A) and ABCG2 (Fig. 5G) genes.

Fig. 5

Receptor and transporter gene expression of CD44 (A), CD95 (B), CD133 (C), NOTCH1 (D), c-MET (E), Ezrin (F), and ABCG2 (G) in control and treated HepG2 cells with Chit-nanoEGCG, EGCG, and cisplatin using RT-qPCR. Data are expressed as mean ± SD values from at least 3 independent experiments. *, **, and *** indicate statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the Chit-nanoEGCG–treated group

Oncogenes Expression

When HepG2 cells were treated with Chit-nanoEGCG, native EGCG, and cisplatin or left untreated, the expressions of the oncogenes mTOR (Fig. 6A), PI3K (Fig. 6B), RALA (Fig. 6C), and BMI (Fig. 6A) were significantly decreased after Chit-nanoEGCG treatment when compared with control groups.

Fig. 6

Oncogene expression of mTOR (A), PI3K (B), RALA (C), and BMI (D) in control and treated HepG2 cells with Chit-nanoEGCG, EGCG, and cisplatin using RT-qPCR. Data are expressed as mean ± SD values from at least 3 independent experiments. * and *** indicate statistical significance at p < 0.05 and p < 0.001, respectively, compared to the chit-nanoEGCG–treated group

Hepatocellular carcinoma is one of the most serious malignancies worldwide with a high morbidity and mortality. A passible approach for managing cancer is chemoprevention employing naturally occurring phytochemicals that may inhibit the cancer initiation and progression. However, their bioavailability for medication restricts their application. In this study, we evaluated the efficacy of delivering EGCG to human HCC cells (HepG2) in an in vitro condition using chitosan nanoparticles. Several factors influenced our opinion to use EGCG and HepG2 cells in this study. This includes liver cancer as one of the most common malignancies in both men and women worldwide. Furthermore, in numerous cell culture and preclinical investigations, EGCG has demonstrated impressive chemopreventive potential (Li et al. 2023). The current study investigated the oncostatic influence of Chit-nanoEGCG nanoparticles on HepG2 hepatocellular carcinoma cells. The results demonstrated that the Chit-nanoEGCG induced apoptosis and suppressed the proliferation and the growth of cancer cells. Further proof that the Chit-nanoEGCG increased the expression of genes involved in apoptosis and decreased the expression of genes involved in proliferation is provided by the current investigation. This effect might be attributed to the targeting ability of the EGCG-loaded nanoparticles and the cellular uptake of EGCG by cancer cells. The cellular uptake of Chit-nanoEGCG needs further investigations. In another study, low doses of small nano-EGCG that activate the AMPK signaling pathway significantly suppressed the invasion, colony formation, and proliferation of human lung cancer cells (Chen et al. 2020).

In the current study, ionic gelation with TPP was used to encapsulate EGCG in chitosan to produce Chit-nanoEGCG. Zeta potential and TEM imaging were used to characterize the prepared Chit-nanoEGCG which showed small, highly stable, and spherical nanoparticles, with quite accurate matching between the zeta and TEM image values. The present findings agree with recent work that ionic gelation-prepared chitosan-TPP nanoparticles enhance the antioxidant activities of astaxanthin in both in vitro and in vivo models (Kim et al. 2022).

The main goal of cancer therapy is to induce apoptosis in cancer cells while limiting concurrent death in normal cells. In numerous cancer preclinical models, numerous reports have shown that EGCG has broad anticancer activities on cancer cell growth (Bonsignore et al. 2021). In agreement with our results, previous studies demonstrated a significantly increased anticancer effect of EGCG encapsulated in CSNPs than native EGCG, indicating that nanoformulations increased the stability and bioavailability of EGCG leading to better clinical results. HepG2 cells remained viable regardless of the CS-NP concentration. These findings support earlier research showing that, after 48 h of cell exposure, CS-NPs with a diameter of up to 150 nm had no less than 10% harmful effect on human liver cancer cells (hepG2) (Loutfy et al. 2016). Having the vehicle be inactive to the cells indicated that cell viability reduction is due to the active compound in the Chit-nanoEGCG and not the vehicle itself (Mohammed et al. 2017). Therefore, before drawing a clear conclusion and beginning its biological application, a more quantitative investigation at the molecular and protein levels is needed to verify the influence of chitosan size and duration on genotoxic effect.

In this study, Chit-nanoEGCG showed dose-dependent loss of cell viability and inhibited cell growth at 48-h posttreatment with low IC50 doses compared with native EGCG. The anticancer effect exerted by EGCG on HepG2 cells seems to be achieved by inhibiting cell growth and proliferation as evidenced in Fig. 2. Decreasing HepG2 cell viability by EGCG agrees with other previous findings in different cancer cells, including cervical carcinoma cells (Zhu et al. 2019), lung cancer cells (Jiang et al. 2018), and breast cancer cells (Chen et al. 2018).

There is a lack of studies describing the effect of EGCG on HCCs, compared to other cancers. Our data demonstrated that HepG2 cells treated with Chit-nanoEGCG showed a significant increase in the apoptotic protein expression (P53, Bax, caspase-3, caspase-9, and PARP) with a concomitant decrease in the anti-apoptotic Bcl-2 proteins when compared with groups treated with natural EGCG or control group, and these results are confirmed by qRT-PCR analysis. So, a second possible anticancer mechanism of EGCG in HepG2 cells may be through the increased expression of apoptotic genes. P53 plays an essential part in cancer concerning cell cycle arrest and apoptosis. The fundamental role of p53 is its ability to induce apoptosis (Neamatallah et al. 2014). P53 performs its function by suppression of antiapoptotic Bcl-2 family proteins and upregulation of apoptotic proteins Bax and caspase-3 (Öhlinger et al. 2020). Upregulation of P53 increases the Bax/Bcl-2 ratio, which is an indicator of apoptosis in cancer cells (Pan et al. 2020). P53 can directly interact with Bax and stimulates the release of cytochrome C that aid in apoptosis. Bax is a protein that is involved in the intrinsic apoptotic pathway as a complex with Bak protein. This protein complex is inserted into the mitochondrial membrane and activates the caspase-3 signaling cascade, which leads to the subsequent release of cytochrome c and ATP from the mitochondrial membrane into the cytosol and finally apoptosis (Chi et al. 2014). Poly (ADP-ribose) polymerase (PARPs) induces cell death through the caspase-independent pathway parthanatos (Peter et al. 2015). BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. So, a third possible anticancer mechanism of EGCG is via inhibition of the glycolytic pathway by suppression of Bcl2 (Wang et al. 2015).

The HCC development is a complicated process characterized by the activation of numerous signaling pathways regulated by specific genes, which contributes to carcinogenesis in a complementary manner. OCT4 and SOX2 are crucial transcriptional regulators that keep cancer stem cells pluripotent and capable of self-renewal; their overexpression may lead to the development of a variety of malignancies (Kim et al. 2015). The current study showed a marked decrease in the expression of OCT4 and SOX2 genes in cells treated with Chit-nanoEGCG confirming a previous study that chemotherapy inhibited OCT4 and SOX2 overexpression in human neuroblastoma (Yang et al. 2012). These results associated with an insignificant change in the expression of the transcription gene, NANOG, in cells treated with Chit-nanoEGCG.

The preservation of pluripotency in embryonic stem cells depends on numerous factors, including the transcription factors OCT4, SOX2, and NANOG (Saunders et al. 2013). SOX2 and NANOG also maintained cancer stem cells (CSCs) in solid tumors (Rady et al. 2018). As surface biomarkers for cancer cells that are resistant to chemotherapeutic drugs, CD44 and CD133 have both been identified (Liu et al. 2013). The results showed downregulation in CD44 and CD133 expression in HepG2 cells after treatment with Chit-nanoEGCG. By enlisting a large number of pro-apoptotic factors to create the death-inducing signaling complex, the surface receptor CD95 causes apoptosis. The NOTCH signaling pathway controlled stem cell self-renewal and differentiation. Although the function of the NOTCH signaling pathway in HCC is still unclear, NOTCH1 was found to be essential for maintaining the self-renewal of cancer-initiating cells in various malignancies (Sikandar et al. 2010).

This study showed that NOTCH1 expression was downregulated after treatment with Chit-nanoEGCG. The activation of the proto-oncogene mesenchymal-epithelial transition (c-MET) signaling results in invasion and metastasis of cancer and also drug resistance (Kim and Kim 2017). Many studies showed that c-MET is highly expressed in different types of soft and solid cancers. Inhibition of c-MET expression has shown promising results in the inhibition of cancer cell growth (Frampton et al. 2015). Additionally, there is a strong correlation between the overexpression of the Ezrin gene and the development of portal vein thrombosis, invasion, and recurrence of HCC (Kang et al. 2010). According to the results of this study, Chit-nanoEGCG can stop the expression of Ezrin, a downstream effector molecule of Rho (the Ras homolog family), which stops the progression and metastasis of HCC (Ruan et al. 2013). The results showed the downregulated expression of ABCG2 after treatment with Chit-nanoEGCG. ABCG2 overexpression was associated with increased drug efflux, chemo-resistance, and invasion of the cell (Nomura et al. 2015).

This study showed that Chit-nanoEGCG could inhibit the expression of mTOR, PI3K, RALA, and BMI oncogenes which play a pivotal role in HCC, leading to inhibiting the HCC progression and metastasis (Almatroodi et al. 2020). Another possible anticancer mechanism of EGCG in HepG2 cells may be via the DNA methyltransferase. Many tumor suppressor genes are inactivated by hypermethylation in many tumors including hepatocellular carcinoma (El-Bendary et al. 2020). EGCG inhibits the DNA methyltransferase of tumor cells causing an epigenetic reactivation of genes silenced during carcinogenesis (Negri et al. 2018). Figure 7 provides a mechanistic summary of the effects of Chit-nanoEGCG on HepG2 cells in the current study.

Fig. 7

An illustrative diagram summarized mechanisms of how Chit-nanoEGCG affected proliferation and apoptosis of HepG2 cells. In comparison with control and native EGCG, the following alterations occurred when HepG2 cells are treated with Chit-nanoEGCG: (1) reduced mRNA transcription levels of OCT4, SOX2, and NANOG transcription factors; (2) downregulation of oncogenes; mTOR, PI3K, RALA, and BMI at mRNA transcription levels; (3) transcription levels of transporter and receptor genes, where CD95 is upregulated while CD133, NOTCH1, c-MET, Ezrin, and CD44 are downregulated; (4) the variation of apoptotic markers at transcription levels, through which Bcl-2 is downregulated and P53 and Bax are upregulated; (5) the modulations of apoptotic protein expression levels, wherein Bcl-2 is lowering and P53, PARP, Caspase-3, Caspase-9, and Bax are elevated