Construction Of Nerve Cell Membrane Display Nanomaterial And Its Application In The Treatment Of Bacteria Infection

Objective:Using cell membrane display technology to modify gold nanocage,construct nerve nanocapsule(U87MG@GNC)system and study its antibacterial and analgesic effects,providing a new strategy for the treatment of necrotizing infection caused by Streptococcus pyogenes.Materials and methods:GNCs were prepared by electrochemical substitution method,its absorption peak is about 800nm.Neuroglioma cells(U87MG)were pretreated with capsaicin(CPS)to make U87MG cell membrane highly express transient receptor potential cation channel subfamily V member 1(TRPV1).The membranes were disrupted by ultrasound and obtained by differential centrifugation.To coat U87MG membranes onto the surface of GNCs,we mixed U87MG membranes with GNCs.The mixture was iteratively extruded through a 200 nm membrane by using a mini extruder.Next measure the representation of U87MG@GNC,including hydrodynamic particle size,Zeta potential and UV-visible absorption spectra.The morphology of GNCs and U87MG@GNC was observed by TEM.The stability of the two nanoparticles was evaluated in PBS solution.The photothermal properties of the two solutions were evaluated by comparing the solution temperature rising conditions under different NIR intensity and different NIR time.Coomassie bright blue and Western Blot were used to detect the surface protein expression.Next,the analgesic ability of the material was tested.The area of hemolytic ring was measured after GNCs and U87MG@GNC treatment.Two kinds of material were co-cultured with mouse DRG,and the supernatant of Streptococcus pyogenes was added into the medium.Calcium ion influx at different time points was detected by calcein staining.Fluo-4/AM staining was used for quantification.The spontaneous pain behavior,thermal sensitivity and mechanical pain sensitivity of mice were detected by injecting different materials and bacterial suspensions into the plantar of mice.Then the antimicrobial ability of the material was tested.DRG was stimulated by different materials and bacterial supernatant,and we use ELISA to detect the secretion of CGRP in culture medium.Different materials and bacterial suspension were injected into the air pouch on the back of mice,then we use flow cytometry to detect the recruitment of neutrophils.Different materials were injected subcutaneously into the back of mice and irradiated with near-infrared light with different intensity and different duration.The photothermal properties of the materials in vivo were detected by infrared thermal imager.A mouse model of necrotizing fasciitis was established,and then flow cytometry was used to detect neutrophils recruitment in different materials exposed to near-infrared light for 15 minutes or without irradiation.Next,build the mice model of necrotizing fasciitis,illuminate near-infrared light 12 hours after subcutaneous injection of Streptococcus pyogenes and different materials,neutrophil recruitment was detected by flow cytometry 6 hours and 18 hours after injection,skin specimen sections were obtained 6 hours after injection for H&E and immunofluorescence staining,take routine blood tests 24 hours after injection,photographic record four and seven days lesions healing situation,and 7 days’ skin samples was taken to coat plate then did colony counting.CGRP secretion in skin explants after 6 hours of treatment with different materials and suppurative streptococcus infection was detected by ELISA.Results and discussion:The hydrodynamic particle size of GNCs is about 130nm,and the particle size of U87MG@GNC is about 150nm,which is increased compared with that before coating.The Zeta potential of GNCs is about-26mV,and the Zeta potential of U87MG@GNC is about-15mV.UV-visible absorption spectra showed that both GNCs and U87MG@GNC had characteristic absorption peak at 800nm.TEM results showed that both GNCs and U87MG@GNC were cubic nanoparticles,and the thin film structure with a thickness of about 5nm could be seen on the U87MG@GNC surface.The stability of GNCs and U87MG@GNC in PBS solution was good.Both GNCs and U87MG@GNC have excellent photothermal properties.Under NIR,the rate of solution temperature rise is positively correlated with the irradiation intensity.Western Blot analysis showed the glioma cell membrane surface specific receptors were retained.The above results indicate that U87MG@GNC was successfully constructed.The Oxford Cup experiment showed that the hemolytic ring formed by U87MG@GNC had a smaller area.Calcein staining and Fluo-4/AM staining indicated that U87MG@GNC could inhibit calcium ion influx induced by bacterial supernatant stimulation.The behavior analysis of mice demonstrated that U87MG@GNC can reduce the spontaneous pain behavior,reduce the sensitivity of heat and mechanical pain.The above results indicate that U87MG@GNC has analgesic function.ELISA results showed that U87MG@GNC could reduce the secretion of CGRP in DRG stimulated by bacterial supernatant.The results of flow cytometry in mouse back air pouch demonstrated that U87MG@GNC could promote neutrophils recruitment.In mouse model of necrotizing fasciitis,U87MG@GNC showed better neutrophils recruitment ability under NIR.The above results indicate that U87MG@GNC has antibacterial function.Flow cytometry at 6 and 18 hours after the injection of bacteria and materials showed that U87MG@GNC with NIR could recruit more neutrophils.Similar results were obtained by H&E and immunofluorescence staining of skin samples.Routine blood tests showed that U87MG@GNC with NIR also suppressed systemic inflammation.The healing of skin lesions for 4 and 7 days in the U87MG@GNC with NIR group was significantly better than that in the other groups,and the bacteria colonies in this group were also less after the 7 days’skin specimens were coated on the plate.The results of ELISA demonstrated that U87MG@GNC with NIR could inhibit the secretion of CGRP in skin explants.The above results indicate that U87MG@GNC has an ideal therapeutic effect on necrotizing fasciitis.Conclusion:It was found that U87MG@GNCs have been successfully constructed,they have good biocompatibility,stable structure and excellent photothermal property.They have antibacterial and analgesic ability,are effective in treating necrotizing infection caused by Streptococcus pyogenes.