Multifunctional Biomimetic Mineralization Of Bacterial Surface Based On Microfluidics

Bacteria play an important role in many fields such as environmental engineering and biomedicine,yet they face survival dilemmas in extreme environments.In nature,organisms have the ability to synthesize biominerals with fine morphology and complex structure,such as the formation of bone and teeth.Most bacteria do not have the ability to biomineralize themselves,and researchers have proposed to modify and modify bacteria through biomimetic mineralization methods,thus improving their ability to tolerate adverse environments and play an active role in various fields.Zeolitic imidazolate framework-8（ZIF-8）is a metal-organic framework compound consisting of transition metal ions Zn²⁺and 2-methylimidazole as bridging units,with a stable zeolite topology and good biocompatibility,which can be used as a bionic mineralized shell for bacteria to help them resist adverse environments.Microfluidics refers to the precise control of fluids on the micro and nanoscale,which is a potential method to achieve efficient biomimetic mineralization.Escherichia coli（E.coli）is the model bacterium commonly used in various studies.Escherichia coli Nissle 1917（Ec N）has been used as a probiotic for more than 100 years and is widely used in the treatment of gastrointestinal diseases.It has shown that Ec N can target tumor tissues and modulate host immune response.There are few related studies on Ec N in oral tumors,and the complex environment of oral tumors may affect the survival and function of Ec N.Objective:This experiment was completed with microfluidics for bacterial surface biomimetic mineralization.We explored the tolerance of biomineralized E.coli（E.coli@ZIF-8）to extreme environments such as nutrient deficiency and UV light.We preliminarily investigated the effect of encapsulated E.coli Nissle 1917（Ec N@ZIF-8）on human tongue squamous carcinoma Cal-27 cells.Methods:（1）Microfluidics-based biomimetic mineralization of E.coli,a genetically engineered E.coli strain that expresses green fluorescent protein（GFP）,and the probiotics Ec N was accomplished using 2-methylimidazole and zinc acetate dihydrate.（2）The structure and morphology of E.coli@ZIF-8 were characterized by X-ray photoelectron spectroscopy（XPS）,X-ray diffraction（XRD）,scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,and fourier transform infrared spectroscopy（FT-IR）.Confocal laser scanning microscopy（CLSM）and flow cytometry were used for the co-localization analysis of the encapsulated E.coli with GFP.（3）E.coli@ZIF-8 were placed in a nutrient-deficient environment for 7 days,and then the growth curve of the bacteria was measured after recovery.E.coli@ZIF-8 was exposed to UV irradiation,and the FDA/PI double-staining method was performed to detect live/dead cells.Intracellular reactive oxygen species（ROS）were labeled using a DCFH-DA fluorescent probe to detect changes in ROS production after UV irradiation.（4）The CCK-8 assay was taken to detect the effect of the biomimetic mineralized shell ZIF-8 on the growth of Cal-27 cells.Biomimetic mineralization of Ec N was performed,and the effect of Ec N and Ec N@ZIF-8 on Cal-27 cells was detected using the CCK-8 assay.Results:（1）In this experiment,a microfluidic device powered by a microinjection pump and supplemented with ultrasound was used to complete the biomimetic mineralization of E.coli,genetically engineered E.coli expressing GFP,and Ec N,respectively.（2）The results of XPS,XRD and EDS show that E.coli@ZIF-8 is consistent with the characteristic peak of ZIF-8,with 16.9%zinc element.The results of SEM,TEM and Mapping show that a uniform and consistent shell is formed on the surface of the biomineralized E.coli and that zinc element is evenly distributed on the surface of E.coli@ZIF-8.The results of FT-IR indicated that E.coli and ZIF-8 were cross-linked by hydrogen bonds.The results of CLSM confirmed that E.coli and ZIF-8 co-localized well,and flow cytometry results showed that the bacterial encapsulation rate was as high as 86.7%.（3）E.coli@ZIF-8 tolerated the extreme environment of lack of nutrients and could recover the growth activity after removing the shell.E.coli@ZIF-8 has the ability to tolerate UV irradiation and the biomineralized bacteria produce less ROS after UV irradiation.（4）The effect of ZIF-8 on the growth of Cal-27 cells was low.Ec N and Ec N@ZIF-8 showed some inhibitory effects on human tongue squamous carcinoma Cal-27 cells.Ec N@ZIF-8 showed better inhibition of Cal-27 cells at the same bacterial concentration.Conclusions:In this experiment,we completed the biomimetic mineralization of bacterial surface based on microfluidics,which improved the tolerance of bacteria to extreme conditions of nutrient deficiency and UV light.The biomimetic mineralization can enhance the inhibition of Ec N on human tongue squamous carcinoma Cal-27 cells,and the biomineralized probiotics have potential applications in therapy of oral tumor.