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THE PREPARATION AND CHARACTERIZATION OF HA-MEDIATED CELL-PENETRATING PEPTIDE-MODIFIED 10-HCPT-LOADED PHASE-TRANSFORMATION LIPID NANOPARTICLESObjectives To prepare a novel multifunction ultrasound probe----HA-mediated Cell-penetrating Peptide-modified 10-HCPT-loaded Phase-transformation Lipid Nanoparticles(HA/CPPs-10-HCPT-NPs)and test their particle size,zeta potential,morphology,drug-loading efficiency,encapsulation efficiency and explore the condition of phase transformation caused by temperature and ultrasound.Methods HA/CPPs-10-HCPT-NPs were prepared using thin-film dispersion,ultrasound emulsification and electrostatic effect;particle size and zeta potential were measured using Malvern dynamic light scattering analyzer;drug-loading efficiency and encapsulation efficiency were tested by high performance liquid chromatograph(HPLC);under the heating plate heating condition,the condition of phase transformation caused by temperature was explored;added into the agar gel phantom,HA/CPPs-10-HCPT-NPs suffered low-intensity focused ultrasound irradiation to explore the condition of acoustic droplet vaporization(ADV).Results The appearance of HA/CPPs-10-HCPT-NPs was milky white,their mean particle size and zeta potential were(284.2±13.3)nm,-(16.55±1.50)m V,respectively,HA/CPPs-10-HCPT-NPs were dot and good dispersity detected using optical microscope and confocal laser scanning microscope(CLSM);transmission electron microscope(TEM)found that HA/CPPs-10-HCPT-NPs were a spherical morphology;the drug-loading efficiency and encapsulation efficiency were(48.10±3.13)% and(5.23±0.34)%,respectively;the temperature of phase transformation caused by temperature was about 45?;the optimal condition of ADV caused by LIFU was 2.4 w/cm2 3min.Conclusion We successfully prepared a novel multifunctional ultrasound probe----HA/CPPs-10-HCPT-NPs,which had uniform size,regular morphology and good dispersity.HA/CPPs-10-HCPT-NPs had good phase-transformation property under a certain condition(such as temperature,ultrasound irradiation and so on).PART II THE EXPERIMENTAL STUDY OF HA/CPPS-10-HCPT-NPS COMBINED WITH LIFU FOR KILLING LIVER CANCER SMMC-7721 CELLSObjectives To evaluate the effect of HA/CPPs-10-HCPT-NPs combined with LIFU irradiation for killing SMMC-7721.To lay the foundation for further animal experiment.Methods The expression of CD44 on the surface of SMMC-7721 cell membrane was detected using western-blotting;to observe the target binding ability of HA/CPPs-10-HCPT-NPs and SMMC-7721,SMMC-7721 cells were divided into three groups: HA/CPPs-10-HCPT-NPs,CPPs-10-HCPT-NPs and HA/CPPs-10-HCPT-NPs + HAase group,the target binding was detected using CLSM;to evaluate the penetrate ability of HA/CPPs-10-HCPT-NPs in vitro,three-dimensional(3D)multicellular tumor spheroid(MCTS)model was structured to mimic the tumor microenvironment of solid tumor in vivo and divided into target group(HA/CPPs-10-HCPT-NPs)and non-target group(CPPs-10-HCPT-NPs),the penetration of Di I-HA/CPPs-10-HCPT-NPs was observed using CLSM;in anti-proliferation experiment,SMMC-7721 cells were divided into 12 groups: control,free10-HCPT,HA/CPPs-10-HCPT-NPs,CPPs-10-HCPT-NPs,HA/10-HCPT-NPs,10-HCPT-NPs,LIFU,10-HCPT+LIFU,HA/CPPs-10-HCPT-NPs+LIFU,CPPs-10-HCPT-NPs+LIFU,HA/10-HCPT-NPs+LIFU,10-HCPT-NPs+LIFU,the cell survival rate of each group was measured using CCK-8;in apoptosis experiment,the cell apoptosis rate of each group was measured using flow cytometry(FCM).Results Cell membrane of SMMC-7721 cell overexpressed the CD44;we observed that HA/CPPs-10-HCPT-NPs could closely adhere to SMMC-7721 cells and penetrate cells through the cell membrane,however,in the CPPs-10-HCPT-NPs group,NPs rarely adhered to SMMC-7721 cells,meanwhile,there were no or few NPs adhereing to SMMC-7721 cells in HA/CPPs-10-HCPT-NPs + HAase group;in 3D MCTS model,we observed that lots of HA/CPPs-10-HCPT-NPs clustered around the 3D MCTS and which penetrated into the cell sphere,while in HA/10-HCPT-NPs group,there were few NPs adhering to 3D MCTS,the penetrating depth of HA/CPPs-10-HCPT-NPs was 27.14?m,while it was 9.83?m in HA/10-HCPT-NPs group,the former was 2.76-fold thicker than the latter;in HA/CPPs-10-HCPT-NPs + LIFU group,cell survival rate of SMMC-7721 was the lowest and the apoptosis rate was the highest in all groups.Conclusion Under HA-mediation,HA/CPPs-10-HCPT-NPs could specially adhere to SMMC-7721 cell membrane overexpressing CD44,under CPPs-mediation,HA/CPPs-10-HCPT-NPs could penetrate into cell through cell membrane,realized that HA/CPPs-10-HCPT-NPs precise target at cell-level,HA/CPPs-10-HCPT-NPs combined with LIFU irradiation had a significant effect of killing SMMC-7721 cells.PART III THE EXPERIMENTAL STUDY OF HA-MEDIATED CELL-PENETRATING PEPTIDE-MODIFIED 10-HCPT-LOADED PHASE-TRANSFORMATION LIPID NANOPARTICLES COMBINED WITH LOW-INTENSITY FOCUSED ULTRASOUND FOR PRECISION THERANSOTICS AGAINST HEPATOCELLULAR CARCINOMAObjectives To evaluate the effect of HA/CPPs-10-HCPT-NPs combined with LIFU for theranostics against subcutaneous tumor of the liver cancer.Methods To evaluate the ability of HA/CPPs-10-HCPT-NPs target to liver cancer in nude mice,nude mice bearing SMMC-7721 cell tumor xenografts were divide into HA/CPPs-10-HCPT-NPs and CPPs-10-HCPT-NPs group,Di R-labled nanoparticles were injected into nude mice though the tail vein,real-time imaging was conducted by a small-animal in vivo fluorescence imaging system at 4 h point and 24 h point after injection,after 24 h injection,nude mice were killed,the tumor,heart,liver,spleen,lung and kidney were extracted the mice were killed,and the tumor and organs extracted for additional ex vivo fluorescence imaging to assess the change in fluorescence intensity;To further verify the target property of HA/CPPs-10-HCPT-NPs,Di I-HA/CPPs-10-HCPT-NPs and Di I-CPPs-10-HCPT-NPs were injected into nude mice via the tail vein,after 1 h,the mice were killed,the tumor were extracted,snap-freeze sectioned,the distribution of NPs was observed using CLSM;To further verify the target and enchanced ultrasound imaging properties of HA/CPPs-10-HCPT-NPs,nude mice divided into HA/CPPs-10-HCPT-NPs+LIFU,CPPs-10-HCPT-NPs+LIFU,HA/CPPs-10-HCPT-NPs group,the first two groups were irradiated by LIFU after injecting relative NPs,the third group did not treat using LIFU,ultrasonograms of tumor pre-and post-LIFU irradiation were observed to evaluate the enhanced ultrasound imaging of NPs irradiated by LIFU,the echo intensity of the images was calculated using an ultrasound image analyzer;to evaluate the anti-tumor therapeutic effect of combined application of HA/CPPs-10-HCPT-NPs with LIFU in mice bearing tumor xenografts,the tumor-bearing mice were distributed to 12 groups at random: control,free 10-HCPT,HA/CPPs-10-HCPT-NPs,CPPs-10-HCPT-NPs,HA/10-HCPT-NPs,10-HCPT-NPs,LIFU,10-HCPT+LIFU,HA/CPPs-10-HCPT-NPs+LIFU,CPPs-10-HCPT-NPs+LIFU,HA/10-HCPT-NPs+LIFU,10-HCPT-NPs+LIFU group,in control group and only LIFU irradiation group,nude mice were injected normal saline,the different formulations of 10-HCPT were injected at the same 10-HCPT dose of 4mg/kg through the vena caudalis once every 2 days,10-HCPT treatment started 20 days and ended 30 days after injection into SMMC-7721 cells,LIFU irradiation was carried out for 5 min at 1 h after injection of the 10-HCPT formulation,after 31 days,all mice were killed and tumor xenografts excised,the weight and size of tumor xenografts were measured and used to calculate tumor inhibition rate and tumor volume,tumor was fixed in 4% polyformaldehyde and sent to H&E,PCNA and TUNEL staining,proliferation index(PI)and apoptosis index(AI)of each group were calculated.Results At 4 h and 24 h time point,fluorescence signal at tumor site in HA/CPPs-10-HCPT-NPs group was observed,but in CPPs-10-HCPT-NPs group,we did not observe fluorescence signal,after 24 h injection,fluorescence intensity in HA/CPPs-10-HCPT-NPs group was stronger than that in CPPs-10-HCPT-NPs group;a scattered distribution of “red dot” fluorescence signals in the tumor tissue frozen sections of the targeted group was observed using CLSM,whereas few were seen in the non-targeted group;efore NPs injection,the ultrasonograms of tumors in each groups showed regular low echo,in the HA/CPPs-10-HCPT-NPs+LIFU group,the focal spot in tumor showed a strong echo signal after LIFU irradiation,whereas no obvious ultrasound enhancement was observed in the whole tumor site in the CPPs-10-HCPT-NPs+LIFU group,a high echo signal was also not observed in the HA/CPPs-10-HCPT-NPs without LIFU group,analyses of ultrasound intensity showed that the echo intensity was markedly stronger after LIFU irradiation than before LIFU irradiation in the HA/CPPs-10-HCPT-NPs+LIFU group,the difference was statistically significant;in the experiment of evaluation of therapeutic effect of HA/CPPs-10-HCPT-NPs combined with LIFU irradiation on tumor xenografts,inhibition rate in HA/CPPs-10-HCPT-NPs+LIFU group was the highest,the tumor volume was the smallest;H&E staining of tumor tissue showed the largest number of lysed cell membranes and nuclear fragmentation were observed in the HA/CPPs-10-HCPT-NPs +LIFU group,PCNA staining showed that PI in HA/CPPs-10-HCPT-NPs +LIFU group was the lowest,TUNEL staining showed that AI in HA/CPPs-10-HCPT-NPs +LIFU group was the highest,the difference was statistically significant.Conclusion HA/CPPs-10-HCPT-NPs could target specially tumor xenograft of nude mice bearing SMMC-7721 cell and penetrate extracellular matrix and cell membrane barrier to realize targeting more and deeper tumor cell,HA/CPPs-10-HCPT-NPs combined with LIFU irradiation could enhance ultrasound imaging at tumor site and significantly inhibit growth of liver cancer.Hence,HA/CPPs-10-HCPT-NPs combined with LIFU irradiation was expected to be a new strategy of precision theranositcs against HCC. |
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