| Objective:Liver cancer is one of the most common cancers with high morbidity and mortality in the world.There are three main pathological types of HCC,namely hepatocellular type,cholangiocarcinoma type and mixed type[1].The incidence of HCC is on the rise worldwide,and 80%-90%of HCC develops from cirrhosis.Because there are no obvious symptoms in the early stage,most patients have reached the advanced stage when they are diagnosed,which is one of the reasons for their poor prognosis[2].According to the latest guidelines of the European Association for the Study of the Liver(EASL),surgical resection and percutaneous thermal ablation are the first-line treatment for early HCC in patients without portal vein invasion and extrahepatic metastasis[3].However,HCC after ablation is prone to metastasis and postoperative recurrence,which brings great difficulties to disease treatment.The main purpose of thermal ablation therapy is to destroy the tumor completely by local heating and form a safe edge of at least 10 mm around the edge of the tumor[4].According to the American Association for the Study of Liver Disease(AASLD)guidelines,ablation is recommended for a single tumor smaller than 2 cm[5].Thermal ablation therapy causes protein degeneration of tumor cells by increasing the temperature of the lesion and surrounding tissues,resulting in coagulation necrosis[6].However,the efficacy of ablation therapy is related to many factors,such as tumor size,location,blood supply around the lesion and the equipment used,which is also closely related to the technology of the operator.These are the main factors affecting tumor recurrence after ablation.In addition,the ’ thermal deposition effect’,also known as the ’ cooling effect’,is an important factor leading to incomplete ablation and postoperative recurrence[7].When the lesion is close to the blood vessel,the blood flow will take away part of the heat generated by ablation,so that the ablation effect is significantly reduced.After ablation,a large number of tumor stem cells remain in the surrounding area of necrosis,which directly leads to postoperative tumor recurrence[8].In order to reduce the high recurrence rate caused by incomplete ablation,experts have been looking for new methods.Ablation therapy combined with other drug adjuvant therapy is one of the important means[9].However,the general small molecule drugs used in clinical practice at this stage have many limitations,such as poor water solubility,low selectivity,and poor stability in vivo.Necrosis avid agents(NAAs)can selectively accumulate in necrotic tissues and are expected to become a supplementary treatment after tumor ablation.However,their clinical application is limited by phototoxicity,intolerance in vivo and other potential side effects[10].Hypericin(Hyp)is the extract of Hypericum perforatum,which has been considered as a kind of NAAs,owning a strong affinity for necrotic tissue[11].However,Hyp is almost insoluble in water and most non-polar solvents,and aggregates in organs rich in mononuclear macrophage systems for a long time,which greatly limits its application in organisms[12].In order to realize the clinical transformation of Hyp,further exploration is needed.At present,it is urgent to find a safe and effective carrier to transport Hyp into organisms and exert its necrosis affinity function.The development of biocompatible nanotechnology has had a great impact on cancer diagnosis and targeted drug delivery.Nanomaterials have been widely used as drug carriers to deliver drugs with diagnostic and therapeutic effects to targets while reducing systemic side effects after administration[13].In this study,the necrotic area after ablation was used as the target,and the necrotic affinity substance Hyp coupled with radionuclide 131I was encapsulated in polymer nanoparticles.The residual tumor cells around the necrotic area are continuously killed by β-rays released by radionuclide 131I,which plays a supplementary therapeutic effect after ablation and effectively reduces the damage to healthy tissues.The killing sites include all levels of cells,especially DNA in the nucleus.The nano-micelles are expected to play an important role in the diagnosis and treatment of recurrence liver cancer after ablation.Methods:1.Balanced 1 mg Iodogen and dissolved in 1 ml acetone solution to make Iodogen solution.150 μl of Iodogen solution was sucked out and added into 2 ml EP tube.The acetone in the tube was dried by nitrogen to make Iodogen tube.0.5 mg hypericin was dissolved in 500 μl acetone solution and transferred to the Iodogen tube.Then 500 μl Na131 I solution was added and the reaction was carried out at room temperature for 30 min to obtain 131I-Hyp solution.The labeling rate and in vitro stability of 131I-Hyp were detected by γ counter.20 mg PEG-PCL was dissolved in 561 μl acetone to prepare PEG-PCL solution.In turn,1012 μl acetone,488 μl PEG-PCL solution and 1000 μl Hyp-131 I solution were added to the glass bottle,shocked away from light at 45℃,and 1500 μl saline was added during the process.The acetone was fully volatilized overnight in a ventilated kitchen,and the supernatant was extracted after centrifugation(10000 rpm,3 min)the next day.The PNP@(131I-Hyp)nano-micelles aqueous solution was obtained after filtration with a 220 nm filter membrane.The encapsulation efficiency of PNP@(131I-Hyp)was detected by ultraviolet spectrophotometer.The hydrodynamic diameter,dispersion index and surface charge of PNP@(131I-Hyp)were detected by Malvern particle size analyzer.The morphology of PNP@(131I-Hyp)was detected by Cryo-TEM.The fluorescence characteristics of PNP@(131I-Hyp)in vivo and in vitro were detected by fluorescence imaging system.The synthesized PNP@(131I-Hyp)was stored in the dark for 28 days,and the above indicators were re-tested to evaluate its stability in vitro and simulated physiological environment.2.SK-hep-1 hepatoma cells were inoculated into a small petri dish at 20,000/well until the cells were 100%confluent.The dry ice rod was in contact with the center of the bottom of the petri dish for 30 s,and the contact area was about 0.5 cm2.DAPI(1 μl),PNP@(131I-Hyp)(100 μl)and PBS(400 μl)were added to the culture dish,respectively,and incubated at 37℃ for 10 min in the dark.The necrosis affinity of nano-micelles was observed by fluorescence microscopy.SK-hep-1 hepatoma cells were cultured in 75 cm2 culture flasks until they were completely covered.All cells were collected and counted,and then divided into experimental group and control group on average.The experimental group was induced by 60℃ water bath,and the control group was given 37℃ water bath.100 μl PNP@(131I-Hyp)and 1 ml PBS solution were added respectively,and then incubated for 10 min at room temperature.Centrifugation(3000 rpm,5 min)was used to separate the cell precipitate and supernatant,and the radioactive count rate of the two groups of cell precipitate was detected by γ counter.The cytotoxicity test(CCK8 method)and hemolysis test were performed using gradient concentrations of nanomaterials to evaluate the in vitro cytotoxicity and blood compatibility of PNP@(131I-Hyp).The SK-hep-1 cell suspension 100 μl(containing 5×106 cells)and matrix gel 100 μl were fully mixed and injected subcutaneously into the right thigh back of 4-week-old male nude mice.When the volume of the tumor exceeded 200 mm3,the long axis of the tumor was punctured with a microwave ablation needle,with a power of 3 W and ablation time of 8 s to make a residual tumor model after microwave ablation.The animal model was successfully established by magnetic resonance scanning and paraffin pathological section.Six nude mice with residual tumor model were randomly divided into experimental group and control group(3 mice in each group).The experimental group was injected with 100 μL PNP@(131I-Hyp)with radioactivity of 0.1 mCi by tail vein,and the control group was injected with the same amount of saline.SPECT scanning was performed at 1 h,2 h,8 h,12 h,24 h,48 h,72 h and 96 h after administration to observe the concentration of radionuclides.Six nude mice with residual tumor model were randomly divided into experimental group and control group(3 mice in each group).The experimental group was injected with 100 μl PNP@(131I-Hyp)containing 2.5 μg hypericin by tail vein,and the control group was injected with 100 μl saline by tail vein.At 0.5 h,1 h,2 h,4 h,8 h,24 h,48 h,72 h,120 h,168 h,216 h and 264 h after administration,fluorescence imaging was performed on a small animal in vivo imaging system to observe the distribution of PNP@(131I-Hyp).Three tumor-bearing nude mice were injected intravenously with 100μl nano-micelles(containing 2.5 μg Hyp and 0.1 mCi 131I).Five days later,the mice were sacrificed and subcutaneous tumors,heart,liver,spleen,lung,kidney,brain and pancreas were collected.The distribution of nanoparticles in different organs ex vitro was observed by fluorescence imaging.At the same time,the radioactivity of different tissues and organs was detected by γ counter and weighed.After that,the subcutaneous tumor was made into frozen sections for autoradiography,and the radioactivity intensity of different regions of the tumor tissue was compared.Eighteen nude mice with residual tumor model after ablation were randomly divided into 3 groups,with 6 mice in each group.The experimental group was injected with 100 μl PNP@(131I-Hyp)(containing 2.5 μg Hyp and 0.1 mCi 131I)through the tail vein,the blank group was injected with 100 μl Hyp-NP(containing 2.5 μg Hyp),and the control group was injected with 100 μl saline.Tumor size and body weight of nude mice were measured every day to evaluate the therapeutic effect of PNP@(131I-Hyp).The heart,liver,spleen,lung,kidney,brain and intestinal tissues of the experimental group and the control group were taken after the nude mice were sacrificed.The paraffin sections were made and HE staining was performed to evaluate the biosafety of PNP@(131I-Hyp).Twelve SD rats were randomly divided into 2 groups with 6 rats in each group.The experimental group was injected with PNP@(131I-Hyp)(131I concentration was 5 mCi/kg,Hyp concentration was 125.5μg/kg)through the tail vein of rats,and the control group was injected with an equal volume of saline through the tail vein.After 14 days,blood was collected from the tail vein of rats and WBC,RBC,HGB,PLT,ALT,AST,ALB and Cr were detected to evaluate the effect of PNP@(131I-Hyp)on blood routine and biochemistry in rats.Results:1.131I was successfully coupled with Hyp by Iodogen oxidation and had good stability in vitro.PNP@(131I-Hyp)was successfully synthesized by solvent evaporation method,and the encapsulation efficiency of 131I-Hyp was 85.32%.2.DLS analysis showed that the hydrodynamic diameter of PNP@(131I-Hyp)was 45.93 ± 0.58 nm,the dispersion index was 0.19±0.01,and the zeta potential was-23.6±4.96 mV.The PNP@(131I-Hyp)was uniform spherical with an average diameter of 33.07 ± 3.94 nm.In vitro fluorescence imaging showed that the PEG-PCL shell of the nanoparticles degraded in the cytoplasmic environment,and 131I-Hyp with excellent fluorescence characteristics was released.3.PNP@(131I-Hyp)has good stability in vitro,and the content of hypericin released within 4 weeks was 6.2%.After cultured in RPMI 1640 medium and rabbit whole blood for 72 hours,the content of hypericin in nanomaterials did not change significantly,indicating the stability of PNP@(131I-Hyp)in physiological environment was good.4.The cell cold necrosis model and heat necrosis model experiments showed that PNP@(131I-Hyp)had a significant affinity for necrotic cells,mainly deposited in the cytoplasm of necrotic cells.5.The hemolysis rate of different concentrations of PNP@(131I-Hyp)was lower than 5%,indicating that PNP@(131I-Hyp)had good compatibility with blood.6.The cytotoxicity experiment showed that the inhibitory ability of PNP@(131I-Hyp)to liver cancer cells in vitro mainly came from the β-rays released by 131I and showed a dose-dependent mode.The median inhibitory concentration was 0.86 mCi/mL,indicating that PNP@(131I-Hyp)had a good killing effect on SK-hep-1 human liver cancer cells in vitro.7.The residual tumor model after ablation of hepatocellular carcinoma xenografts in nude mice was successfully established and confirmed by MR imaging and pathology.8.PNP@(131I-Hyp)was injected through the tail vein of nude mice,and then the whole body SPECT scan was performed.Radionuclides were concentrated in the subcutaneous tumor area from 2 h to 48 h after injection.9.After intravenous injection of PNP@(131I-Hyp)into the tail of mice,the distribution of PNP@(131I-Hyp)in nude mice was monitored by in vivo imaging system.The fluorescence signal began to gather in subcutaneous tumors at 1 h after injection,and the tumor fluorescence signal intensity was the highest at 120 h after administration.Subsequently,the fluorescence signal gradually weakened and declined to the baseline level 264 h after administration,which confirmed that PNP@(131I-Hyp)has dual performance of fluorescence imaging and radionuclide imaging.10.The distribution of PNP@(131I-Hyp)in different tissues and organs ex vitro was analyzed by gamma counting and fluorescence imaging.The results showed that PNP@(131I-Hyp)mainly concentrated in subcutaneous tumors 5 days after intravenous injection.11.Autoradiography of frozen sections of tumor tissue showed that the highly radioactive area was consistent with the necrotic area stained by HE.When the fluorescence microscope focused on the edge of necrotic area,the red fluorescence of hypericin co-located with necrotic cells,which further verified the necrotic affinity of PNP@(131I-Hyp)in subcutaneous tumors and the stability of coupling between Hyp and 131I.12.After the drug was administered via tail vein,the antitumor effect was verified by comparing the subcutaneous tumor volume and the body weight of nude mice in each group.The results showed that single injection of PNP@(131I-Hyp)significantly inhibited tumor growth,while injection of the same volume of saline or Hyp-NP could not inhibit tumor growth.The change trend of body weight of the three groups of experimental mice is consistent,and there is no obvious statistical difference.13.There was no significant difference in HE staining sections of nude mice between experimental group and control group.After intravenous injection of PNP@(131I-Hyp)in SD rats,there was no significant change in blood routine and blood biochemical indexes between the experimental group and the control group,and the results were within the reference range of healthy rats.The results showed that PNP@(131I-Hyp)was safe for intravenous injection at the current dose and had no obvious toxic and side effects.Conclusion:1.Nano-micelles loaded with hypericin coupled with 131I were successfully synthesized by solution evaporation method,and the synthesis process was simple and feasible.The synthesized PNP@(131I-Hyp)has the characteristics of high labeling efficiency,high encapsulation efficiency and high stability,which provides a reliable guarantee for subsequent experiments.2.PNP@(131I-Hyp)has a significant targeting effect on necrotic cells and is mainly deposited in the cytoplasm of necrotic cells;Nano-micelles have good killing effect on SK-hep-1 human hepatoma cells in vitro and have good compatibility with blood.3.Necrosis model of subcutaneous hepatocellular carcinoma after ablation was successfully established in nude mice.PNP@(131I-Hyp)mainly concentrates in subcutaneous tumors of nude mice after intravenous injection,and has dual imaging properties of fluorescence and radionuclide imaging.Nano-micelles can obviously inhibit tumor growth while playing a multi-modal imaging monitoring role,and have no obvious toxic and side effects on experimental animals. |
