Silver nanoparticles have recently received considerable attention among scientists due to their unique biological properties1. The production and characterization of these nanoparticles is essential part to research2. Various methods have been used to produce silver nanoparticles, including physical, chemical, and newly established bio-mimetics processes3. However, these approaches are ecologically hazardous (especially chemical methods) and cause the quick agglomeration of nanoparticles, leading to the formation of large particles devoid of mono-dispersity4. Silver nanoparticles have been used for cancer therapy as anticancer materials and in drug delivery5. The succession of tumors has been attributed to a series of alterations in some cells leading to a certain type of unusual gene expression6, 7. Ubiquitously targeting cells inside malignant cells due to the inability of certain drugs to disperse proficiently may not be reasonable, as this possibly endorses multiple-drug resistance, resulting from resistance to the chemotherapeutic treatment of cancer cells8, 9. Furthermore, the expulsion of the drugs inevitably reduces healing activity such that tumor cells rapidly build up resistance to various drugs10.Silver nanoparticles (AgNPs) have been used considerably in cancer drug delivery. Owing to their unique properties and antimicrobial characteristics, many studies have used AgNPs’ inactive drug targeting and passive drug targeting11. Many recent studies have utilized AgNPs as a smart candidate for the targeted and controlled delivery of many different therapeutic agents, including proteins, lipids, carbohydrates, or even genes12, 13, 14, 15. Rajawat et al.16 used silver nanoparticles for the targeted delivery of breast cancer treatment and concluded that tumor volumes decreased significantly, with similar results in the positive control. Drug release from such nanoparticles can be elicited using internal or external stimuli17, 18.

Green chemistry using plant extracts and microorganisms has been steadily developed as an effective alternative to the environmentally harmful processes of silver nanoparticle synthesis19. Moreover, using microorganisms such as fungi in silver nanoparticle production is a low-cost process that helps to avoid generating. Recently, Roy et al.20 reviewed the green synthesized silver nanoparticles and their antimicrobial properties and revealed numerous types of microorganisms that could be utilized to produce different particle sizes with different functionalities in terms of antimicrobial and anticancer activity. The current research presents the utilization of R. stolonifer, one of the easiest fungi to grow, for silver nanoparticle production and studies the properties of bio synthesized nanoparticles in potential anticancer therapy.

The use of fungi as reducing and stabilizing agents in the biogenic synthesis of silver nanoparticles is advantageous due to the production of large quantities of proteins, high yields, easy handling, and low toxicity of the residues23. Results of the present study indicate that green synthesized silver nanoparticles were spherical and 3Dranging in size between 5 and 50 nm. Similar results have been reported in other studies with varying particle sizes24, 25. Our findings for the structural characterization of nanoparticles are in accordance with Sadowski et al.26 and Maliszwaska et al.27, who utilized different microorganisms in the synthesis process. In a different study, Mukherjee et al.28 used the same methodology as used in this study and demonstrated an intense peak of 410 nm. Bio-reduction of silver nitrate (AgNO₃) to synthesize silver in nano size and stabilization by capping agents are the two processes for manufacturing silver nanoparticles. The biological particles from fungi could conceivably play a role in the stabilization of silver nanoparticles29, 30. Cytotoxicity results of the present study were evaluated by inhibiting the growth of EAC cell lines using the prepared AgNPs at different concentrations. The maximum reduction of the EAC cell line was observed at 8 μg/ml concentration with an inhibition of over 68.13%, which was significantly higher than the gold nanoparticles used in the study conducted by Li et al.31 against the same cell line. The cytotoxicity of biologically prepared AgNPs was determined using MTT; the results indicate that the AgNPs were competent to reduce the viability of the Dalton's Lymphoma Ascites cells in a dose-dependent manner; the higher the AgNPs’ concentration, the more is the reduction rate. These results confirm findings by Priya et al.32, who synthesized AgNPs using a chitosan-mediated approach and achieved dose-dependent inhibition. They concluded that silver nanoparticles caused the up-regulation of certain genes, leading to cell death via apoptosis. In this study, the effectiveness of the biosynthesized AgNPs on tumor cells was analyzed after six hours of treatment at concentrations of 500 nM and higher. Biologically prepared nanoparticles at 500 nM dramatically inhibited the viability and growth of Dalton's Lymphoma Ascites cells by more than 50%, which was chosen as the IC₅₀. The toxicity to the cells increased when exposed for a longer period of time, confirming gene alteration even in the still viable cells.

The utilization of microorganisms for metal nanoparticle synthesis is a comparatively modern innovation for a cleaner and cost-effective synthesis method. The easy scalability of such a method may help to reduce the costs of production of viable anticancer agents such as silver nanoparticles. Accordingly, this research has shown the green process for the production of AgNPs using R. stolonifer. Our study has shown maximum absorbance at 422 nm with UV-Visible absorption spectroscopy. The size and shape of the silver nanoparticles were revealed by Transmission Electron Microscope as 5 to 50 nm and spherical, respectively. The silver nanoparticles produced by the biological method have extensive applications, including cancer therapy. This study shows the activity of biologically synthesized silver nanoparticles on the inhibition of cancer cells.

AFM: Atomic Force Microscope

AgNPs: Silver Nanoparticles

DMEM: Dulbecco’s Modified Eagle’s Medium

EAC: Esophageal squamous-cell carcinoma

NCCS: National Centre for Cell Science

R. stolonifer: Rhizopus stolonifer

TEM: Transmission Electron Microscope

The authors would like to thank the collaboration between Lincoln University College, Selangor, Malaysia and the Universiti Sains Malaysia, Penang, Malaysia that made this work possible.

All authors contributed equally to this work.

None.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.