Imaging And Treatment Of Hepatocellular Carcinoma With IRGD Peptide-Mediated Dual-Modal Imaging Loaded Nanoparticles

PART ? PREPARATION OF IRGD PEPTIDE-MEDIATED PHASE-CHANGE LIPID NANOPARTICLES AND SOME PERFORMANCE TESTSObjective To prepare a new type of molecular probe,which is a liposomal nanoparticle(iRGD-ICG-10-HCPT-PFP-NPs)equipped with iRGD homing membrane penetrating peptide.ICG,10-HCPT and PFP were encapsulated to detect their average particle size,potential,entrapment efficiency and drug loading,morphology under confocal laser scanning microscope,morphology under transmission electron microscope,as well as in vitro phase transition,photoacoustic and ultrasonic imaging.Methods We used thin-film hydration and secondary ultrasonic emulsification to prepare molecular probe iRGD-ICG-10-HCPT-PFP-NPs,and used a laser particle size instrument to detect the particle size and potential of the prepared nanoparticles,while confocal microscopy and transmission electron microscopy were used to detect their morphology.The encapsulation efficiency and drug loading were detected by high performance liquid chromatography.Photoacoustic imaging as well as ultrasound imaging of nanoparticles was performed in vitro,while they were irradiated with a LIFU instrument to produce phase transitions and observed.Results Nanoparticle iRGD-ICG-10-HCPT-PFP-NPs were finally prepared,and their appearance was emerald green emulsion with fluidity.Observation under a common microscope showed regular morphology without agglomeration,small size,average particle size around 298.4 ±10.42 nm,average zeta potential of and-35.4 ± 6.23 m V,regular spherical shape under transmission electron microscope,and dense PFP wrapped in the middle.The standard curve was prepared by HPLC.The encapsulation was(45.53 ± 2.31)% and the drug loading was(4.79 ±0.45)%.Nanoparticles are able to produce phase transitions under the excitation of the LIFU instrument,and are able to achieve enhanced ultrasound imaging,while under changes in ICG concentration,photoacoustic signals are generated to achieve photoacoustic imaging.Conclusion We successfully prepared a novel nanoscale molecular probe iRGD-ICG-10-HCPT-PFP-NPs for ultrasound molecular imaging,which has small particle size,is located at the nanoscale level,regular morphology and good dispersity,is equipped with ICG as a light absorber,and 10-HCPT as a chemotherapeutic drug,which can realize ultrasound and photoacoustic dual-modality imaging.PART ? TARGETING AND KILLING EFFECT OF IRGD PEPTIDE-MEDIATED DRUG-LOADED PHASE CHANGE NANOPARTICLES ON HEPATOCELLULAR CARCINOMA CELLSObjective To co-incubate the prepared nanoparticle iRGD-ICG-10-HCPT-PFP-NPs with hepatoma cells SK-Hep1 and irradiate them with LIFU instrument according to different groups to detect the killing effect on hepatoma cells and pave the way for subsequent in vivo experiments.Methods In order to detect the targeting of nanoparticles to hepatoma cells,the prepared nanoparticles were divided into targeted group iRGD-ICG-10-HCPT-PFP-NPs and non-targeted group ICG-10-HCPT-PFP-NPs,and the two groups of nanoparticles were co-incubated with hepatoma cells SK-Hep1,respectively,and the binding of the two groups of nanoparticles to the cells was observed under a confocal laser scanning microscope,so as to determine the targeting of nanoparticles to hepatoma cells.Flow cytometry was also used to detect the intracellular fluorescence content of the targeted non-targeted group,respectively,so as to also determine the targeting ability of nanoparticles.In order to examine the penetration effect of nanoparticles on tumor cells,we constructed 3D multicellular tumor spheres,simulated the in vivo cellular environment,and co-incubated the two groups of nanoparticles to examine their penetration effect on 3D tumor spheres under a confocal microscope.In order to examine the killing effect of nanoparticles on tumor cells,we used AM/PI double staining to divide the cells into six groups according to different treatment factors: control group,LIFU group,iRGD-ICG-10-HCPT-PFP-NPs group,ICG-10-HCPT-PFP-NPs group,ICG-10-HCPT-PFP-NPs+LIFU group,and iRGD-ICG-PFP-10-HCPT-NPgroup+LIFU,and finally observed the fluorescence of each group under a confocal laser scanning microscope.In order to examine the anti-proliferative ability of nanoparticles on tumor cells,we used the CCK-8 method and divided the cells into 10 groups according to different treatment factors: 1 control group,2 non-peptide drug-free NPs group(ICG-PFP-NPs group),3 non-peptide drug-free NPs group(ICG-10-HCPT-PFP-NPs),4 peptide drug-free NPs group(iRGD-ICG-PFP-NPs group),5 peptide drug-free NPs group(iRGD-ICG-10-HCPT-PFP-NPs group),6LIFU group,7 non-peptide drug-free NPs+LIFU group(ICG-PFP-NPs+LIFU group),8 non-peptide drug-free NPs+LIFU group(ICG-10-HCPT-PFP+LIFU group),and 9 peptide drug-free NPs + LIFU group(iRGD-ICG-PFP-NPs + LIFU group).Finally,the OD value of each group was measured by a microplate reader to calculate the anti-proliferation rate.In order to detect the pro-apoptotic effect of nanoparticles on tumor cells,we used flow cytometry to detect apoptosis,using the same grouping as CCK-8 experiment,and finally detected the apoptosis rate of each group.Results In the targeted experiment,the red fluorescence emitted by iRGD-ICG-10-HCPT-PFP-NPs in the targeted group surrounded the blue fluorescence emitted by the nucleus,suggesting its targeting to hepatoma cells,while the blue fluorescence emitted by the nucleus was only seen by ICG-10-HCPT-PFP-NPs in the non-targeted group,and the red fluorescence emitted by the nanoparticles was not obvious.The same result was observed in the normal hepatocytes group.A similar situation was found in experiments where intracellular fluorescence intensity was measured by flow cytometry,with the targeted group having significantly higher intracellular fluorescence intensity than the other two groups.By co-incubating two sets of nanoparticles with 3D multicellular tumor spheres,the penetration depth of iRGD-ICG-10-HCPT-PFP-NPs was twice that of ICG-10-HCPT-PFP-NPs.In the AM/PI double staining method,the iRGD-ICG-PFP-10-HCPT-NPs group + LIFU had the highest red fluorescence,suggesting the strongest lethality to tumor cells.In CCK-8,the cell survival rate of the iRGD-ICG-PFP-10-HCPT-NPs group + LIFU was the lowest,and the difference was statistically significant(P < 0.01),and in the flow cytometry apoptosis assay,the pro-apoptotic rate of the iRGD-ICG-PFP-10-HCPT-NPs group+LIFU was the highest.Conclusion iRGD-ICG-PFP-10-HCPT-NPs have targeting ability to tumor cells,and have penetration effect to tumor cells mediated by iRGD peptide.At the same time,they can kill tumor cells under the irradiation of LIFU instrument,with anti-proliferation and pro-apoptosis effects,which lays a foundation for in vivo therapy.PART ? IMAGING AND THERAPEUTIC EFFECTS OF IRGD PEPTIDE-MEDIATED DRUG-LOADED PHASE CHANGE NANOPARTICLES IN A LIVING ANIMAL MODELObjective To evaluate the diagnostic ability of nanoparticles in vivo by injecting them into the body of established subcutaneous xenografts and performing in vivo fluorescence imaging,in vivo photoacoustic and ultrasound imaging.At the same time,LIFU irradiation was used to treat tumor-bearing tumors,and the therapeutic effect and in vivo safety were evaluated.Methods First,the subcutaneous xenograft of hepatocellular carcinoma cell line SK-Hep1 was used to establish a living tumor model.The prepared nanoparticles were divided into targeted group iRGD-ICG-10-HCPT-PFP-NPs and non-targeted group ICG-10-HCPT-PFP-NPs,which were injected into tumor-bearing mice by tail vein injection,respectively.In order to verify the targeting of nanoparticles in vivo,a small animal in vivo fluorometer was used to detect,and the in vivo fluorescence signals were collected before and 1 h,3 h,6 h,and 24 h after nanoparticle injection,and after 24 h,the rats were sacrificed to separate the heart,liver,spleen,lung,kidney,and subcutaneous tumors for fluorescence signal acquisition,and finally the in vivo fluorescence signal intensity of the tumor area at each time point was analyzed.In order to verify the photoacoustic imaging performance and targeting of nanoparticles in vivo,nanoparticles were also divided into targeted group iRGD-ICG-10-HCPT-PFP-NPs and non-targeted group ICG-10-HCPT-PFP-NPs,which entered the body through the tail vein injection,and the photoacoustic signals of the tumor area were collected before and 1 h,3 h,6 h,and 24 h after injection of nanoparticles with a small animal photoacoustic imager,and the photoacoustic signal intensities were analyzed.In order to test the performance of nanoparticles in ultrasound imaging in vivo and the effect on the imaging effect of enhanced ultrasound,they were still grouped according to the targeted group iRGD-ICG-10-HCPT-PFP-NPs and the non-targeted group ICG-10-HCPT-PFP-NPs,which entered the body through the tail vein injection,respectively,and ultrasound B-Mode and enhanced ultrasound CEUS images were collected,followed by images 1 hour later,and irradiated with a LIFU instrument(pulse mode(2son,2soff),power of 3 w/cm2,focal length of 1.5 cm),and ultrasound B-Mode and enhanced ultrasound CEUS images were collected again.In order to verify the tumor therapeutic effect of nanoparticles on tumor-bearing tumors,they were divided into 10 groups according to different treatment methods: 1 control group,2 non-peptide drug-free NPs group(ICG-PFP-NPs group),3 non-peptide drug-free NPs group(ICG-10-HCPT-PFP-NPs),4 peptide drug-free NPs group(iRGD-ICG-PFP-NPs group),5 peptide drug-free NPs group(iRGD-ICG-10-HCPT-PFP-NPs group),6 LIFU group,7 non-peptide drug-free NPs + LIFU group(ICG-PFP-NPs + LIFU group),8 non-peptide drug-free NPs + LIFU group(ICG-10-HCPT-PFP + LIFU group),9 peptide drug-free NPs + LIFU group(iRGD-ICG-PFP-NPs + LIFU group)Among them,the control and LIFU groups were only given PBS injection,and 1 hour after injection,the LIFU-treated group was irradiated in a pulse mode(2son,2soff)with parameters of power of 3 w/cm2 and focal length of 1.5 cm once daily for a total of 10 days,and pictures of the tumor site were taken daily.At the end of the treatment,each group was sacrificed,the tumors were isolated,pathological tissue sections were performed,and H&E staining,PCNA immunohistochemical staining,and TUNEL staining were performed to assess the therapeutic effect,anti-proliferative effect,and pro-apoptotic effect of each group on solid tumors after injection of different nanoparticles.At the same time,the heart,liver,spleen,lung and kidney sections of each group were dissected and stained with H&E to assess the biosafety.Results In the small animal in vivo fluorescence imaging experiment,the nanoparticles injected in the targeted group showed a strong fluorescence signal in the liver at 1 h,and a small amount of fluorescence signal was detected at the blood vessels between the liver and the subcutaneous tumor,while in the non-targeted group,only the fluorescence signal appeared in the liver,and no significant fluorescence signal was observed in the other sites.At 3 h after injection,it was found in the targeting group that the fluorescence signal in the tumor area became different.However,in the non-targeted group,only the liver region still had fluorescence signal,and the tumor region did not have a significant fluorescence signal.At 6 hours after injection,the fluorescence signal in the tumor area continued to increase in the targeted group.In the non-targeted group,the fluorescence signal could still be observed only in the liver region.After 24 hours,the fluorescence signal was still observed in the tumor area of the targeted group,and no fluorescence signal was observed in this area of the non-targeted group.Twenty-four hours later,the viscera and tumor masses were separated from the two groups after sacrifice,and the detected fluorescence signals were consistent with those in vivo,that is,in the targeted group,the fluorescence signals of the tumors were weak,in the non-targeted group,the fluorescence signals of the liver were even,and the tumors had no fluorescence signals.In small animal photoacoustic imaging experiments,we found similar results.No significant photoacoustic signal was detected in the tumor area of both groups before injection of nanoparticles.At 1 h after injection,a small amount of photoacoustic signal began to appear in the group given iRGD-ICG-10-HCPT-PFP-NPs injection,while no significant photoacoustic signal was detected in the group given ICG-10-HCPT-PFP-NPs injection.Over time,after 3 h,the photoacoustic signal gradually increased in the targeted group,and only a small amount of photoacoustic signal was detected at the edge of the tumor area in the non-targeted group.After 6 hours,the photoacoustic signal remained in the targeted group,and a small amount of photoacoustic signal appeared in the non-targeted group.After 24 hours,there were still some photoacoustic signals in the targeted group,while only a very small amount of photoacoustic signals could be detected in the non-targeted group.It is confirmed by counting the intensity of photoacoustic signal at each time point in the tumor area.At the same time,two groups were given ultrasonic detection,and the following results were obtained.No significant enhanced ultrasound signal was detected in the tumor area of both groups before nanoparticle injection.At 1 hour after injection,no significant enhanced ultrasound signal was still detected in the tumor area of both groups.After irradiation with two groups of LIFU,more enhanced ultrasound signals appeared in the iRGD-ICG-10-HCPT-PFP-NPs injection group,while no significant enhanced ultrasound signals were detected in the ICG-10-HCPT-PFP-NPs injection group.Through the treatment of tumor-bearing mice,it was found that the iRGD-ICG-10-HCPT-PFP-NPs + LIFU group had the highest overall tumor inhibition rate,and the difference was statistically significant(P < 0.05).In addition,more karyolysis and nuclear fragmentation were observed in the H&E sections,PCNA staining was observed,the anti-proliferation index was the highest,TUNEL staining was performed,and the apoptotic index was the highest,while it had little effect on the body weight.The H&E staining of viscera showed no significant abnormality,suggesting better biosafety.Conclusion The iRGD-ICG-PFP-10-HCPT-NPs prepared by us have the ability to target subcutaneous xenografts of hepatocellular carcinoma in vivo and can perform in vivo fluorescence imaging and photoacoustic imaging,while enhancing ultrasound imaging under LIFU irradiation.And it has a good therapeutic effect on tumors and good biosafety,realizing the demand of integrated diagnosis and treatment.