Preparation And Application Of Cell Membrane Biomimetic Proteolipid Nanoprobes

BackgroundCancer is the primary factor threatening human life and health all over the world.The realization of early diagnosis is of great significance to prolong the survival time of patients.At present,among various diagnostic methods,optical imaging has the advantages of high sensitivity,high spatial resolution and fast acquisition.The second near infrared window(NIR-?,1000 nm-1700 nm)is a new optical window emerged in recent years,which has the characteristics of higher resolution,higher penetration depth and higher signal-to-noise ratio,as compared with NIR-I.The development of imaging probes with good imaging performance,great targeting and excellent biocompatibility is a key problem to promote the development of NIR-? optical imaging.Indocyanine green(ICG)is the first optical imaging probe approved by the Food and Drug Administration(FDA)for human use.The emission wavelength of ICG in the near infrared region is about 800 nm.The fluorescence emission tail can extend to the NIR-?,indicating that ICG has the potential of imaging in the NIR-? region.However,ICG has some disadvantages,such as low brightness,easy self-aggregation,fluorescence quenching,short half-life and so on,which limits its application in NIR-?fluorescence imaging.Liposome is a common organic nano-carrier,which has the advantages of low toxicity and great biocompatibility.Based on the amphiphilic nature of ICG,there are two forms of liposomal loading ICG.ICG could be loaded into the water-soluble core or phospholipid bilayer shell.In this study,ICG lipid nano-probe(LIPO@ICG)was constructed by loading on the shell.By controlling the amount of ICG,the self-aggregation effect was avoided and the cumulative fluorescence was enhanced.In addition,we plan to use cell membrane bionic technology to extract natural cell membrane and wrap it on the surface of LIPO@ICG.Based on the characteristics of different cells,functional imaging probes with tumor targeting and long circulation in vivo were prepared.Research content:In this study,we designed and synthesized a series of LIPO@ICG,to screen the optimal material synthesis ratio for fluorescence enhancement.We prepared biomimetic probes for tumor targeting functional analysis and material safety evaluation in vitro and in vivo,and the erythrocyte membrane biomimetic probes were used for functional analysis of vascular imaging.The neutrophil membrane modified biomimetic probes were prepared for functional analysis of inflammatory model imaging.The main contents are summarized as follows:Part I:Preparation of fluorescence-enhanced ICG lipid nanoprobe(LIPO@ICG)In this work,LIPO@ICG was synthesized by filming-rehydration method.ICG was dissolved in anhydrous methanol,mixed with lipids,and then blown into film in nitrogen and then hydrated to synthesize liposomes.A series of LIPO@ICG with different molar ratios of LIPO to ICG(25:1-1000:1)were synthesized.The particle size distribution of different proportions of LIPO@ICG was analyzed by dynamic light scattering.The NIR-? fluorescence intensity of LIPO@ICG was observed by NIR-?fluorescence imaging system.The optimal proportion of LIPO@ICG was selected and the change of fluorescence intensity within 60 days under different solvent conditions were detected.We conclude that the particle size of LIPO@ICG was about 200 nm,when the molar ratio of LIPO and ICG is 25:1-50:1.When the molar ratio LIPO:ICG was reduced,the size of LIPO@ICG was about 100 nm.Based on NIR-? fluorescence imaging,the optimal proportion of molar ratio was 250:1 while the drug loading of LIPO@ICG reached 95%.Compared with free ICG,the fluorescence signalof LIPO@ICG was significantly increased by 18.5 times.The signal of LIPO@ICG also remained at 62.07%of the fresh samples after being stored in 0.01M PBS at 4? for 60 days.Part II:Preparation and characterization of tumor membrane biomimetic probes(TLIPO@ICG)The membrane of SW1990 was modified onto the surface of the particles by mechanical extrusion.The tumor cell membrane biomimetic particles(TLIPO@ICG)were prepared characterized.The morphology of TLIPO@ICG was observed by transmission electron microscope,and the size,zeta potential,dispersion coefficient as well as the stability of TLIPO@ICG were analyzed by dynamic light scattering.The protein expression on TLIPO@ICG was detected by polyacrylamide gel electrophoresis(SDS-PAGE).We use circular dichroism spectrum to detect the secondary structure of protein on TLIPO@ICG.The spectral properties of TLIPO@ICG are explained by ultraviolet and fluorescence spectrometer,and the drug leakage of TLIPO@ICG was explored according to the results of dialysis.The experimental results reveal that TLIPO@ICG was uniform,the particle size of free liposome,LIPO@ICG and TLIPO@ICG was 122 nm,123 nm and 115 nm.From the results of SDS-PAGE and circular dichroism,we draw a conclusion that the tumor cell membrane protein was successfully modified to the surface of the particles and the secondary structure did not change.The absorption spectra characterized as TLIPO@ICG and LIPO@ICG had a redshift,the spectra of the pyrolyzed materials coincide with free ICG.The fluorescence emission spectra revealed that the fluorescence intensity of TLIPO@ICG and LIPO@ICG were increased obviously.Compared with free ICG,the fluorescence intensity of TLIPO@ICG in NIR-? region was increased 1.25-1.60 times and 12.80-16.79 times in NIR-? region.Finally,there was no significant change in the particle size of TLIPO@ICG stored in different solvents after storing for 7 days,and the drug leakage was less than 10%.Part III:TLIPO@ICG is used for tumor NIR-? fluorescence imaging.Next,the homologous targeting of TLIPO@ICG was studied.The uptake of TLIPO@ICG in different cell lines was detected by flow cytometry.The differences of TLIPO@ICG and LIPO@ICG uptake by SW1990,RAW264.7 and primary bone marrow macrophage were explored by laser confocal microscope.The mouse models of subcutaneous and orthotopic pancreatic cancer were established,and the targeting imaging performance of TLIPO@ICG in NIR-1 and NIR-? was analyzed.The biosafety of TLIPO@ICG was evaluated by hemolysis,cytotoxicity and side effects in vivo.The fluorescence of TLIPO@ICG in SW1990 cells was 1.99-9.27 times higher than that of the other cell lines.The uptake of TLIPO@ICG by SW1990 cells was 1.86 times higher than that of LIPO@ICG.On the contrary,the uptake of TLIPO@ICG by RAW264.7 macrophages was only 49.8%of that of LIPO@ICG,a similar phenomenon was observed in primary mouse bone marrow macrophages,indicating that TLIPO@ICG has immune biocompatibility.TLIPO@ICG has outstanding tumor targeting in both subcutaneous and orthotopic pancreatic cancer mouse models.The highest enrichment time in the tumor site was 12 hours after tail vein injection.The imaging effect of NIR-? window was significantly better than that of NIR-?,and the signal-to-noise ratio is significantly improved.The boundary of tumor tissue can be seen clearly by NIR-?imaging.The results of hemolysis,cytotoxicity and biochemical indexes in vivo showed that TLIPO@ICG had great biological safety.Part IV:The preparation and application of erythrocyte membrane biomimetic ICG-loaded nanoprobe(RLIPO@ICG)Similarly,on the basis of the preparation of LIPO@ICG,erythrocyte membrane biomimetic particles(RLIPO@ICG)were prepared by mechanical extrusion.The size distribution,zeta potential and polydispersion coefficient of RLIPO@ICG were detected by dynamic light scattering.The protein expression of RLIPO@ICG was analyzed by SDS-PAGE.The NIR-? fluorescence imaging ability of RLIPO@ICG,LIPO@ICG and free ICG on vascular was explored.The imaging data were semi-quantitatively quantitatived by Image J;the half-life of RLIPO@ICG in vivo was evaluated according to the blood fluorescence brightness.In summary,the particle size of free liposome,LIPO@ICG and RLIPO@ICG was about 110 nm.SDS-PAGE revealed that the band of RLIPO@ICG was consistent with that of erythrocyte membrane protein.The NIR-? fluorescence imaging of blood vessels demonstrated that RLIPO@ICG could prolong the circulation time.The blood half-life isthat the RLIPO@ICG circulation time in vivo was 13.2 times longer than that of free ICG.The erythrocyte membrane modified nanoparticles can significantly increase the half-life of the nanoprobe.Part V:The preparation and application of neutrophil membrane biomimetic ICG-loaded nanoprobe(NLIPO@ICG)In this chapter,we prepared neutrophil membrane modified biomimetic particles(NLIPO@ICG).The size distribution,Zeta potential and protein band of NLIPO@ICG were detcted in the first step.Next,the gouty ankle model was constructed.The particle size of free liposome,LIPO@ICG,and NLIPO@ICG was 118 nm,126 nm and 113nm.The synthetic particles are uniform.The band of NLIPO@ICG was consistent with that of neutrophil cell membrane protein.This part of the study reveals that the nanoparticles modified with neutrophil cell membrane have been successfully prepared and are expected to be used in the targeted imaging experiment of inflammatory sites.In summary,ICG liposomes with high fluorescence intensity were successfully prepared.A series of ICG biomimetic liposomes modified by cell membrane were synthesized.The homologous targeting of tumor cell membrane biomimetic probe was discussed in the mouse model of pancreatic cancer.By comparing the imaging effects of NIR-? and NIR-?,it was confirmed that TLIPO@ICG can be used for NIR-? fluorescence imaging of tumor.In this study,the erythrocyte membrane modified biomimetic nanoprobe exhibited the characteristic of long cycle,which can significantly improve the circulation time of fluorescence imaging probe in vivo.