Manganese dioxide nanoparticles are designed as nano-immunomodulators. On this page, we describe the chemical modification process including the thiolation of bacterial surfaces, decoration of manganese dioxide nanospheres with Au nanoparticles and the link of nano-immunomodulators with bacteria.

Figure 8 shows the overall framework of the chemical modification of bacteria with nano-immunomodulators. Manganese dioxide nanospheres are used as nano-immunomodulators that can relieve the hypoxic microenvironment and oxidative stress of tumors.

Figure 8. Chemical modification of E coli Nissle 1917 with nano-immunomodulators.

There are great advantages to use manganese dioxide nanospheres as nano-immunomodulators because they have shown lots of biological functions that can directly regulate tumor growth.¹ Cancer cells often produce large amounts of H₂O and GSH for their metabolism and resistance to immunological killings.² With the peroxidase-like activity, MnO₂ nanospheres can effectively catalyze the in situ production of O₂ and relieve hypoxia by reacting with endogenous H₂O₂ to release Mn^2+,3 They can also react with GSH in tumor cells to generate GSSH and Mn^2+.4 Figure 9 shows the reactions. The ability of MnO₂ nanospheres to modulate TME allows them to enhance the antitumor immunotherapy. When the mission is completed and the tumor microenvironment (TME) goes back to normal pH value, engineered bacteria undergo clearance program.

Figure 9. Catalytic reactions of manganese dioxide nanoparticles in tumor microenvironment.

Bacteria then undergo surface thiolation through a simple one-step imidoester reaction by which primary amino groups on bacterial surface can be converted to free thiols under cytocompatible conditions.⁵ Briefly, collected bacterial cells are washed with PBS and then mixed with 2-iminothiophane solution. The mixture is incubated for 90min at room temperature. Then thiolated cells are washed with PBS to remove residual 2-iminothiolane. The quantity of thiol group on bacteria is determined with DTNB reagent (5,5-dithio-bis (2-nitrobenzoic acid, Ellman's reagent) based on the absorption at 412 nm. L-cysteine hydrochloride hydrate is used as the control for the quantification. DTNB reagent has favorable cytocompatibility and bacterial viability.

Manganese dioxide nanospheres are suspended in the solution of tetrachloroauric acid. The addition of a reducing reagent trisodium citrate deposits gold nanoparticles on surfaces of manganese dioxide nanospheres. Au nanoparticles are set for the link with thiol groups on bacterial surfaces.⁶

Manganese dioxide nanospheres carrying with Au nanoparticles are then mixed with thiolated bacteria. Those cells can rapidly bound with thiolated bacteria because of the high affinity of Au nanoparticles with the thiol groups.

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