Objective To develop engineered bacterial membrane biomimetic nanoparticles,Angiopep-2 E.coli membrane(ANG-2 EM)@PDA-PEI-CpG(ANG-2 EM@PPC),for efficient targeted drug delivery in the treatment of glioma,and to provide theoretical and technical support for targeted glioma therapy.Methods The expression of inaX-N-angiopep-2 engineered bacteria was constructed in the laboratory,and ANG-2 EM was obtained through lysozyme treatment and ultrafiltration centrifugation.ANG-2 EM@PPC was prepared by ultrasonication of bacterial membranes.Western blotting,agarose gel electrophoresis,and transmission electron microscopy(TEM)were used to verify the preparation.Particle size and Zeta potential were measured to investigate the stability of ANG-2 EM@PPC.Regarding cell experiments,CCK-8 assay was performed to determine the effect of ANG-2 EM@PPC on the survival rate of neutrophils.A flow chamber model was designed and constructed,and the uptake efficiency of neutrophils was measured by flow cytometry to investigate the hitchhiking efficiency of ANG 2 EM@PPC on neutrophils in inflammatory environment.Neutrophil death patterns were characterized by fluorescence microscopy,and flow cytometry and Western blotting were performed to examine neutrophil apoptotic bodies and the proportion of apoptotic bodies produced.Regarding animal experiments,a mouse model of in situ glioma was established and the inflammatory environment of tumor tissue was verified.The tumor model mice were divided into three groups,including DiR group,EM@PPC group,and ANG-2 EM@PPC group(all n=3),which were injected with DiR,ANG-2 EM@PDA-PEI-CpG,and EM@PDA-PEI-CpG via the tail vein,respectively(all at 10 mg/kg).Fluorescence images of organs and the brain were used to examine the distribution of the three formulations in vivo and in the brain.The tumor model mice were further divided into PBS group,PDA group,PC group,PPC group,EM@PPC group,and ANG-2 EM@PPC group(all n=4),which were injected with PBS,PDA,PC,PPC,EM@PPC,and ANG-2 EM@PPC injected via the tail vein,respectively(all at 10 mg/kg).Imaging was performed in vivo to observe tumor regression,and the survival rate and body mass of mice were measured to evaluate in vivo pharmacodynamics.TUNEL staining(brain tissue)and HE staining(brain,heart,liver,spleen,lung and kidney tissues)were performed to evaluate the therapeutic effect.Results The results of TEM showed successful preparation of engineered bacterial membrane biomimetic nanoparticles,with PPC exhibiting a distinct shell-core structure and a shell thickness of about 8.2 nm.Due to the coating of ANG-2 EM,the shell thickness of ANG-2 EM@PPC increased to about 9.6 nm,with a clear bacterial membrane layer on the surface.Stability was maintained for at least one week.ANG-2 EM@PPC had no significant effect on the activity of neutrophils according to the findings from the CCK-8 assay.Flow cytometry showed that ANG-2 EM@PPC uptake is enhanced in activated neutrophils and hitchhiking on neutrophils was more efficient in the stationary state than that in the flowing condition.Compared with the EM@PPC group,the neutrophil hitchhiking ability of the ANG-2 EM@PPC group was enhanced(uptake efficiency 24.9%vs.31.1%).Fluorescence microscopy showed that ANG-2 EM@PPC changed the death pathway of neutrophils from neutrophil extracellular traps-osis(NETosis)to apoptosis.Western blot confirmed the production of neutrophil apoptotic bodies,and flow cytometry showed that the production rate was as high as 77.7%.Animal experiments showed that there was no significant difference in the distribution of engineered bacterial membrane biomimetic nanoparticles in the organs(heart,liver,spleen,lungs,and kidney)in the DiR group,the EM@PPC gropu,and the ANG-2 EM@PPC group(P＞0.05),but there was higher distribution in the brain tissue in EM@PPC and ANG-2 EM@PPC groups compared to the DiR group(P＜0.05).Engineered bacterial membrane biomimetic nanoparticles crossed the blood-brain barrier(BBB),and exhibited high affinity to and internalization by neutrophils located in brain tumors.Compared with PBS,PDA,PC,and PPC groups,the survival rate and body mass of mice in the EM@PPC group were improved,tumor fluorescence intensity was weakened,and apoptotic cells were increased.These trends were even more prominent in the ANG-2 EM@PPC group.No abnormality was found in the HE staining of any group.Conclusion An ANG-2 EM@PPC nanodelivery system with inflammation response characteristics was successfully prepared,capable of crossing BBB and targeting the tumor inflammatory microenvironment to improve the anti-glioma efficacy.This study provides a new drug delivery strategy for glioma treatment and offers a new idea for targeted drug delivery in the non-invasive inflammatory microenvironments in other central nervous system diseases.

Objective To evaluate the relationship between the changes in gray matter volume ( GMV) in different brain regions in the early postoperative period and the development of chronic pain after radical mastectomy. Methods Forty-four American Society of Anesthesiologists physical status Ⅰ or Ⅱpatients, aged 25-64 yr, scheduled for elective radical mastectomy, were divided into 3 groups according to the numeric rating scale ( NRS) score within 3 months after surgery: severe chronic pain group ( NRS score≥3, group SEP), slight chronic pain group (NRS score=1 or 2, group SLP) and no chronic pain group ( NRS score=0, group NP ) . All the patients underwent whole brain MRI scan within 7 days after surgery. MRI data processing and analysis were carried out using SPM8-based VBM software package and REST 1. 8 software. Results There were 17 cases in group SEP, 10 cases in group SLP and 17 cases in group NP. Compared with group NP, GMV in the right postcentral gyrus was significantly decreased, and GMV in the right superior frontal gyrus was increased in group SEP ( P＜0. 01) . Conclusion The changes in GMV in the right postcentral and superior frontal gyrus in the early postoperative period may be related to the development of chronic pain after radical mastectomy.

Objective To evaluate the relationship between the mechanism of spinal monocyte chemoattractant protein-1 (MCP-1)-mediated maintenance of chronic pathological pain and synaptic transmission in spinal dorsal horns of rats.Methods Female Sprague-Dawley rats,aged 2-3 weeks after birth,weighing 150-210 g,were studied.The experiment was performed in 2 parts.Experiment Ⅰ Eighteen Sprague-Dawley rats were randomly divided into 2 groups (n =9 each) on 7 days after intrathecal catheters were inserted:phosphate buffer solution (PBS) group and MCP-1 group.PBS 10 μl was intrathecally injected in group PBS,and PBS 10 μ1 containing 100 ng MCP-1 was intrathecally injected in group MCP-1.The mechanical pain threshold was measured at 30 and 60 min before intrathecal injection,and 30,60,90,120,150 and 180 min and 1,2 and 3 days after intrathecal injection.Experiment Ⅱ The transverse spinal cord slices were prepared,and substantia gelatinosa neurons were selected for whole-cell patch-clamp recording.Electrophysiological recording was performed at 1 h of incubation with artificial cerebrospinal fluid (ACSF) and immediately after adding MCP-1:for excitatory synaptic transmission recording,MCP-1 (final concentration 100 nmol/L),N-methyl-D-aspartate (NMDA,final concentration 100 μmol/L) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA,final concentration 20 μmol/L) were added to ACSF,and spontaneous excitatory postsynaptic currents (sEPSCs),AMPA receptors-mediated currents and NMDA receptors-mediated currents were recorded;for inhibitory synaptic transmission recording,MCP-1 (final concentration 100 nmol/L) and γ-aminobutyric acid (GABA,final concentration 1 mmol/L) were added to ACSF,and spontaneous inhibitory postsynaptic currents (sIPSCs) and GABA receptors-mediated currents were recorded.Results Compared with group PBS,the mechanical pain threshold was significantly decreased at 30 min-2 days after intrathecal injection in group MCP-1 (P＜0.01).Compared with those at 1 h of incubation with ACSF,the frequency and amplitude of sEPSCs were significantly increased,the amplitude of NMDA receptors-and AMPA receptors-mediated currents were increased,the frequency and amplitude of sIPSCs were decreased,and the amplitude of GABA receptors-mediated currents was decreased immediately after adding MCP-1 (P＜0.05).Conclusion MCP-1 enhances excitatory synaptic transmission through enhancing the function of NMDA and AMPA receptors in the posterior substantia gelatinosa neurons of the spinal cord;MCP-1 weakens inhibitory synaptic transmission through inhibiting GABA receptor function,which may be involved in MCP-l-mediated maintenance of chronic pathological pain in rats.