The current results showed that MD, GA, and WPI at a ratio of 1:1:1 (w/w) was successfully used for the synthesis of Rg3-NPs when added to the extract at a ratio of 2:1 (polymer: extract). The synthesized Rg3-NPs showed sphere shape with good uniformity with an average size of 30 nm. The particle size reported in this study was different from the ginsenoside Rb1 synthesized by Dai et al. (2018) by the self-assembly method which showed a particle size of 78.9 nm (Dai et al. 2018). However, Hong et al. (2019) used novel ginsenoside-based liposomes to synthesize Rg3-NPs with an average size of 52.02 nm. The synthesized Rg3-NPs reported in our study showed ζ-potential of -5.58 mV. Similar negative ζ potential was also reported in the previous studies (Dai et al. 2018; Hong et al. 2019). ζ-potential is considered the critical factor in the evaluation of the stability of nanoparticle dispersion. The negative charge on Rg3NPs is attributed to the sugar residues on C-3 and C-20 of Rg3 (Dai et al. 2018) and the charge of carboxylate groups in WPI use in the synthesis of Rg3-NPs (Eratte et al. 2014; Hassan et al. 2021).
To achieve an effective treatment for the solid tumors, the anticancer drugs must be able to reach all viable cells within tumors at concentrations sufficient to cause death. Meanwhile, most solid tumors have a weak vascular network with unstable blood flow rates and large intercapillary space compared to normal tissue (Gavhane et al. 2011). Thus, the need for drug penetration to multiple layers of tissue may be a barrier to the effective treatment of solid tumors, and nanoparticles (NPs) have thus proven to have tremendous potential in cancer therapy. In the current study, we synthesized and characterized ginsenosides Rg3 nanoparticles (Rg3-NPs) and evaluated their antitumor properties in Ehrlich solid tumor model in mice compared to the normal ginsenosides Rg3. The induction of solid tumor was carried out by the injection of mice with 2.5 × 106 Ehrlich ascites cells as described by Mansour and Anis (2010); however, the selected dose of Rg3 and Rg3-NPs was based on the previous work of Khalil et al. (2008) and Liu et al. (2019).
The results indicated that injection of mice with Ehrlich ascites cells resulted in the development of tumor which induced a significant increase in serum AFP, TNF-α, MMP-9, and VEGF accompanied by a significant decrease in casp3. Additionally, the percentage of DNA damage and the level of MDA showed a significant increase in the tumor tissue accompanied by a significant decrease in the activity of antioxidant enzymes (GPx and SOD). AFP is a glycoprotein regarded as a characteristic tumor biomarker for screening and supervising malignant solid tumors. AFP promoted endothelial cell migration, invasion, and tumor formation, and decreased apoptosis (Abdel-Aziem et al. 2011; Chen et al. 2019b). The elevation of AFP in EST mice in the current study indicated the inflammatory response and agree with the previous reports (Ahmed et al. 2019; Choi and Kakar 2017; Aldubayan et al. 2019; Medhat et al. 2017) who reported a similar increase in the level of AFP in the EAC bearing mice. TNF-α is a central proinflammatory cytokine that can activate various mitogen-activated protein kinase kinases (MAP3Ks), regulating several genes involved in cell transformation, proliferation, and angiogenesis (Reuter et al. 2010). The increase in this cytokine in the serum in EST mice is similar to that occur in cancer patients who develop cachexia as a result of the increased production of TNF-α by the macrophages (Bachmann et al. 2008; Gueta et al. 2010) suggesting that TNF-α is playing an important role in the immune defense, homeostasis and inflammation (Warren et al. 2009). Moreover, the increase in TNF-α reflex increased in MMP-9 and VEGF since TNF-α can activate the endothelial/epithelial tyrosine kinase (Etk) which mediate activation of vascular endothelial growth factor (VEGF), resulting in enhanced angiogenesis (Dugan and Quick 2005). Angiogenesis is fast tumor growth and expansion by providing oxygen, nutrients, and paracrine stimuli to the tumor (Gu et al. 2013).
Additionally, TNF-α up-regulates the expression of matrix metalloproteinases-9 (MMP-9) as reported by Liu et al. (2015). MMP-9 is a significant protease that plays a vital role in tumor metastasis through extracellular matrix remodeling and membrane protein cleavage (Huang 2018). Metastasis is a complex process through which cancer cells spread from a primary site to form tumors at other distant parts of the body (Sun et al. 2017). The current results also showed that apoptotic Casp3 was increased in EST mice. Apoptosis is a genetically regulated form of cell death that plays an important role in eliminating infected, damaged, and other unwanted cells from the body (Fuchs and Steller 2011). Casp3 is a member of the cysteine protease family, which plays a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins. Lack of caspase-3 expression in a cancerous environment induces a functional deletion mutation in the Casp3 gene (Devarajan et al. 2002).
The results also revealed that EST mice showed a significant decrease in GPx and SOD and a significant increase in MDA and the percentage of DNA damage. These results indicated that the status of oxidative stress occurred in these animals. It is well known that the development of cancer is linked with the generation of free radicals resulting in lipid peroxidation and DNA damage, chromosomal aberration and mutations consequently the tissue damage and disorganization (Abdel-Wahhab et al. 2012; Abd Eldaim et al. 2019). Moreover, the uncontrolled growth and proliferation of abnormal cells during cancer progression is associated with a dysregulation of apoptosis (Hafez et al. 2015; Jan and Chaudhry 2019; Emami Nejad et al. 2021). When DNA damage, the p53 apoptosis arrests the cell cycle at G1 and G2 and activates DNA repair proteins (Benedict et al. 2018; Tiwari and Wilson 2019; Tousson et al. 2011); however, the activation of the Bax gene is occurring if the damage is irreparable resulting in apoptosis (Hassan et al. 2015; Haris et al. 1996; Giono and Manfredi 2006).
Oxidized and damaged DNA can stimulate genetic mutation increasing cell proliferation and eventually normal cells are converted into malignant tumor cells (Nourazarian et al. 2014). Comet assay is primarily measured the strand breaks of DNA, cross-linking sites, and incomplete excision repair in a single cell (Kang et al. 2013). Based on the results of the comet assay, EST showed a significantly high rate of all DNA damage parameters (tail parameters), which include the percent of DNA in the tail, tail moment, and olive tail moment compared to the control group, while EST mice treated with Rg3 or Rg3-NPs showed less DNA damage parameters compared to other treated groups and this effect was more pronounced in EST mice received Rg3-NPs (LD). The percentage of DNA in the tail is mainly related to DNA damage frequency because it represents the amount of migrated DNA from the nucleus (Dogan et al. 2011). Otherwise, tail moment evaluation reflects the percentage of migrated DNA to the tail multiplied by the tail length, where it has more accuracy and shows the intensity of DNA damage (Yahia et al. 2019). Olive tail moment results were correlated with other markers and it is also represented as a good index for DNA damage (Kumaravel and Jha 2006).
The current data indicated that administration of Rg3 either in the normal or nanoforms induced a potent antitumor effect in EST mice. Treatment with Rg3 decreased the tumor weight and size, TNF-α, MMP-9, VEGF, MDA, and the percentage of DNA damage; however, it induced a significant improvement in casp3, GPx, and SOD in EST mice. The improvement in these parameters due to Rg3 was more pronounced in the groups that received Rg3-NPs and the high dose was more effective than the low dose. These results suggested that the nano synthesized Rg3 enhanced the efficiency of the extract via the increase of bioavailability and solubility. In this concern, several attempts were conducted to synthesize Rg3-NPs to solve the challenges facing the application of Rg3 in cancer treatment. Previous studies reported that Rg3 nanoparticles can be synthesized using hydrogels, nanofibers (Cheng et al. 2013), PEG, chitosan, RGD peptide (Cheng et al. 2016; Sun et al. 2014) for the treatment of hypertrophic scar (HS) via the apoptosis of fibroblasts, inhibition of the inflammation and down-regulation the expression of VEGF (Zhao et al. 2020).
Several reports showed that Rg3 induced its anticancer activity through different mechanisms including the induction of apoptosis (Abdel-Wahahb et al. 2010; Li et al. 2015; Teng et al. 2017; Chen et al. 2019a), autophagy via up-regulation of autophagy-associated molecules (Zheng et al. 2017), the suppression of proliferation ( Sun et al. 2016; Li et al. 2016; Song et al. 2020), cell cycle arrest (Liang et al. 2021; Peng et al. 2019), metastasis (Nakhjavani et al. 2019; Huang et al. 2020) and angiogenesis (Nakhjavani et al. 2020; Li et al. 2018), immunomodulatory effects (Liu et al. 2019; Park et al. 2011), reducing multidrug resistance (Liu et al. 2018), sensitization to radiation (Nakhjavani et al. 2019; Wang et al. 2015), and encouraging the genotoxicity in cancer cells (Chen et al. 2020; Zhang et al. 2014).
In addition to the abovementioned mechanisms of Rg3, we propose other mechanisms which enhanced the antitumor properties of Rg3-NPs. In the current study, MD, WPI, and GA were utilized in the synthesis of Rg3-NPs. These agents are well known to have antioxidant activity. For instance, MD is a polysaccharide known to display antioxidant property (Wang et al. 2016; Zhong et al. 2019), radical scavenging activity (Wang et al. 2016), and may impose a synergistic effect and promotes the antioxidant property of Rg3 (Wang et al. 2016; Zhong et al. 2019). Generally, the antioxidant properties of MD able to reduce the risk of different diseases linked to oxidative stress including breast cancer, neurodegenerative disease, colitis, diabetes, obesity, and liver injury via three direct mechanisms which include antioxidant system regulation, oxidative stress-mediated signaling, and ROS scavenging pathways (Li et al. 2017). GA also contains branched polysaccharides chains which are potent antioxidants and effective in the reduction of toxicity (Kong et al. 2014). Additionally, it modulates the mRNA expression of different antioxidant genes (Ahmed et al. 2015) due to its high content of phenolics which are associated with the antioxidant than its scavenging activity (Mirghani et al. 2018). Furthermore, GA reduces the production of superoxide and MDA and elevated the level of GSH and TAC levels (Ali et al. 2020). These antioxidant activities are correlated to its amino acids content (Ali et al. 2020; Khalid et al. 2017) alongside its immune-modulatory and anti-inflammatory actions (Kamal 2018) through the diminishing of TNFα and C-reactive protein levels and the elevating of IL10, the anti-inflammatory cytokine (Ali et al. 2013; Ushida et al. 2011). Moreover, the antioxidant activity of WPI is another pathway in the protective role of Rg3-NPs through its high content of cysteine, β-lactoglobulin, and α-lactoglobulin and its ability to increase GSH which is accountable for the prevention of cell damage (Bayrama et al. 2009; Gad et al. 2011; Hassan et al. 2021; Mohammed et al. 2020; Rodzik et al. 2020; Kennedy et al. 2020).