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This study aims to establish a time-resolved fluorescence immunochromatography method for simultaneous determination of human papillomavirus (HPV) type 16 E6 and L1 protein concentrations. The amount of lanthanide microsphere-labeled antibodies, the concentration of coated antibodies, and the reaction time were optimized, and then a test strip for the simultaneous determination of the protein concentrations was prepared. The performance of the detection method was evaluated based on the concordance of the results from clinical practice. The optimal conditions were 8 μg and 10 μg of HPV16 L1 and E6-labeled antibodies, respectively, 1.5 mg/mL coated antibodies, and reaction for 10 min. The detection with the established method for L1 and E6 proteins showed the linear ranges of 5-320 ng/mL and 2-64 ng/mL and the lowest limits of detection of 1.78 ng/mL and 1.09 ng/mL, respectively. There was no cross reaction with human immunodeficiency virus (HIV), treponema pallidum (TP), or HPV18 E6 and L1 proteins. The average recovery rate of the established method was between 97% and 107%. The test strip prepared in this study showed the sensitivity, specificity, and diagnostic accuracy of 97.46%, 90.57%, and 95.32%, respectively, in distinguishing patients with cervical cancer and precancerous lesions from healthy subjects, with the area under the curve (AUC) of 0.980 1 and 95% Confidence Interval (CI) of 0.956 5 to 1.000 0. The time-resolved fluorescence immunochromatography combined with the test strips prepared in this study showed high sensitivity, high accuracy, simple operation, and rapid reaction in the quantitation of HPV16 E6 and L1 proteins. It thus can be used as an auxiliary method for the diagnosis and early screening of cervical cancer and precancerous lesions and the assessment of disease course.

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., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Office of Journal of Sichuan University (Medical Sciences).)