| Hepatocellular carcinoma(HCC)is one of the most common malignant cancer with the third mortality rate among all types of cancer,and the five-year survival rate is only 18%.Surgical therapy remains the primary treatment for HCC patients.However,early diagnosis of HCC is still a great challenge.Most patients have developed advanced HCC or have occurred metastasis when the diagnosis was confirmed,which resulted in only about 20-30%of HCC patients were suitable for surgical treatment.Besides,the recurrence and metastasis rates of HCC are high due to the presence of circulating tumor cells(CTC)and the prognosis is still very limited.CTC are markers for HCC metastasis and have become one of the critical targets for HCC metastasis therapy.To improve the prognosis of patients with HCC,it is necessary to address the difficulties of early diagnosis,limited therapeutic effect and post-treatment recurrence and metastasis of HCC.Therefore,exploring new and effective strategies to improve the specificity of early diagnosis,improve the therapeutic effect,and enhance metastasis inhibition are research hotspots in the treatment of HCC,and it is crucial to improve the prognosis of HCC.Omni-directional consideration of various factors affecting the prognosis of HCC is an advanced approach for improving the diagnosis and treatment of HCC.The multi-mode combined novel diagnosis and treatment concept is necessary means to improve the prognosis of HCC.Nano-based drug delivery system(NDDS)provides promising strategies to accomplish the multi-mode combined novel diagnosis and treatment concept of HCC.Currently,many NDDS-based nanomedicines have been approved for clinical application,such as Doxil(?),Lipusu(?),Abraxane(?),and Genexol-PM(?),those significantly improved the effects of tcancer treatment.NDDS would bring incomparable prospects for optimizing the clinical prognosis of HCC patients.Liposomes are the most widely used nanocarriers for clinical cancer therapy with the advantages of high drug loading,easy target ligand modification,good biocompatibility,simple preparation process,and easy for industrial transformation.Up to now,14 kinds of liposomes based nanomedicine have been approved for clinical use.Liposomes have been well applied in cancer therapy.Therefore,liposome was chosen as the delivery carrier for the multi-mode combined novel diagnosis and treatment concept.Specific delivery of diagnostic agents or therapeutic agents to target cells or sites is a crucial strategy to improve the early diagnosis,combination therapy and metastasis inhibition of HCC.The NDDS-based specific-targeting approach could promote the specificity of diagnosis,the accuracy of cancer treatment and metastasis inhibition.In order to improve the diagnostic specificity and therapeutic accuracy of HCC,new targeting receptor with specific distribution in tumors,high expression even at the early stages of disease and not expressed in normal tissue cells is the key fator.Glypican-3(GPC3)is an oncofetal proteoglycan located on the cytomembrane,which is highly expressed in 70?100%of HCC cases,whereas it is rarely expressed in normal adult organs.The expression of GPC3 is independent of the tumor size and HCC stage,and is detectable even in the very early stage of HCC.Thus,GPC3,as a specific-targeting receptor of HCC,has been widely studied as diagnostic marker or new target for diagnosis and treatment of HCC.Therefore,GPC3 specific-targeting strategy might carry great potential to improve the early diagnosis,precise therapy and metastasis inhibition effect of HCC with broad clinical application prospects.In this study,in order to improve the specificity of early diagnosis,enhance therapeutic effect and inhibit metastasis on HCC,liposomes with good bioavailability and easy clinical transformation were selected as drug delivery carriers,GPC3 was selected as the specific targeting receptor.Two kinds of GPC3 specific-targeting multifunctional liposomes were designed and prepared to improve the specificity of early diagnosis and the efficacy of combined therapy,and effectively inhibit metastasis on HCC.First,GPC3 specific-targeting multifunctional liposomes co-loaded with sorafenib(SF)and IR780 was developed(GSI-Lip),which aimed to realize early diagnosis and precise chemo-photothermal therapy of HCC.Second,the multipoint co-striking strategy was innovatively proposed to eliminate primary tumor cells and associated-CTC,which aimed to enhance metastasis inhibition and improved therapeutic effect on HCC.GPC3 and vascular cell adhesion molecule 1(Vascular cell adhesion molecule 1,VCAM1)specific-targeting multifunctional liposomes co-loaded with SF and digitoxin(DT)was developed(GV-Lipo/SF/DT),which not only were expected to eliminate the primary tumor cells,but also were supposed to specific recognize and captured CTC,dissociate CTC clusters and prevent CTC-neutrophils cluster formation,simultaneously,thus inhibiting metastasis and enhancing antitumor effects on HCC.The main contents and results of this study are listed as follows:Part One.Study on GPC3 specific-targeting SF and IR780 co-loaded multifunctional liposomes for early diagnosis and chemo-photothermal therapy on HCCChapter 1.Selection of GPC3-specific targeting peptide,synthesis and characterization of targeting functional materials.GPC3 was highly expressed on cell membrane of HepG2 cells and H22 cells,and was low expressed on HL-7702 and liver parenchymal cells.Thus,HepG2 cells and H22 cells were used as the GPC3-positive cell models,whereas HL-7702 cells and liver parenchymal cells were used as the GPC3-negative cell models.GPC3-targeting peptides were optimized by analyzing the relative binding ability of the three GPC3-targeting peptides to different cells line using confocal laser scanning microscope(CLSM)and flow cytometer(FCM).The results showed that G12 targeting peptide showed the highest specific recognition ability to GPC3-positive cells.Therefore,G12 was selected as the GPC3-targeting peptide for further evaluation.The targeting functional material DSPE-PEG2000-G12 was successfully synthesized.Chapter 2.Establishment of determination methods for SF and IR780.Determination method of SF and IR780 were established by high performance liquid chromatography(HPLC)method and ultraviolet-visible spectrophotometer(UV-Vis),respectively.Both methods showed good linear relationships under determinated concentrations,and the precisions and recoveries could meet the requirements.Chapter 3.Preparation and preliminary evaluation of GPC3 specific-targeting SF and IR780 co-loaded multifunctional liposomes.The single-factor investigation method was used to optimize the formulation and preparation process parameters of GSI-Lip.According to the results of drug loading and encapsulation efficiency,the optimal formulation and process parameters were as follows:phospholipid concentration was set as 20 mg/mL,the ratio of drug to lipid(W/W)was 1:15,the ratio of phospholipid to cholesterol(W/W)was 8:1,hydration temperature was 60? and hydration time was 30 min,respectively.GSI-Lip comprised well-dispersed spheres with a particle size of 140.913.70 nm,Polydispersion index(PDI)of 0.191±0.008,and zeta potential of-19.03±0.30 mV,respectively.The stability test results indicated that GSI-Lip showed good plasma stability and storage stability.In vitro release profile showed that SF could be effectively released from GSI-Lip,and photpthermal therapy(PTT)accelerated the release of SF,which might be beneficial to enhance the synergistic therapy effect of chemo-photothermal therapy.Cellular uptake assay,competitive inhibition assay,endocytic pathway study,and in vivo near-infrared fluorescence(NIRF)imaging results indicated that GPC3-targeted liposomes showed specific GPC3-targeting ability in vitro and in vivo.Chapter 4.Evaluation of the early diagnosis ability of GPC3 specific-targeting IR780 loaded multifunctional liposomes.IR780 was used as NIRF imaging diagnostic agent.H22 cells tumor-bearing KM mice were used to evaluate the diagnosis ability of GPC3 specific-targeting IR780 loaded multifunctional liposomes(GI-Lip)in vivo.The NIRF imaging results demonstrated that the diagnostic specificity GI-Lip was significantly higher than folic acid(FA)receptor-targeting liposomes FI-Lip in vivo(p<0.01).To evaluate the sensitivity of GSI-Lip for HCC early diagnosis,a convincing evaluation method under different tumor-bearing cell numbers and different tumor growth time was established.The results indicated that GI-Lip could diagnose early tumor on 2 days after injection of 5×105 H22 cells(3.45±0.98 mm3),FI-Lip showed an early diagnostic ability on day 4 after injection of 1×106 H22 cells(32.90 ± 10.01 mm3),the diagnostic sensitivity of GI-Lip was increased by 8.54 times.Collectively,these results demonstrated the higher diagnostic sensitivity and specificity of GPC3-targeted liposomes versus FA receptor-targeted liposomes,and the great potential of GPC3-targeting liposomes for the ultra early diagnosis of HCC.Chapter 5.Antitumor efficacy evaluation of GPC3 specific-targeting SF and IR780 co-loaded multifunctional liposomes.MTT assay indicated that GSI-Lip provided an advantage in improving in vitro cytotoxicity versus SF and IR780 co-loaded liposomes(SI-Lip).Precise antitumor efficacy test results indicated that GSI-Lip displayed superior antitumor efficacy versus FA receptor-targeted liposomes FSI-Lip(p<0.01),with a 90.52%rate of tumor inhibition.Precise chemo-photothermal synergistic antitumor efficacy studies showed that GSI-Lip plus irradiation with the laser exhibited superior antitumor activity versus GSI-Lip without laser(p<0.01),and a rate of tumor inhibition as high as 94.93%.Thus,the combination of chemotherapy and PTT enhanced treatment effects and the precision against HCC,The results of the hemolysis test and H&E tissue section showed that GSI-Lip had good hemocompatibility and was no obvious systemic toxicity to a certain extent.Part Two.Study on multipoint co-striking SF and DT co-loaded multifunctional liposomes eliminate primary tumor cells and associated-circulating tumor cells for enhancing metastasis inhibition and therapeutic effects on HCC.Chapter 6.Preparation and preliminary evaluation of multipoint co-striking SF and DT co-loaded multifunctional liposomes.GV-Lipo/SF/DT comprised well-dispersed spheres with particle size of 148.77±5.88 nm,PDI of 0.221±0.019,and zeta potential of-11.20±0.46 mV,respectively.The encapsulation efficiency(EE%)of GV-Lipo/SF/DT was 84.38±4.56%for SF and 89.07±1.69%for DT,respectively.The drug loading(DL%)of GV-Lipo/SF/DT was(4.38±0.14)%for SF and(0.28±0.04)%for DT,respectively.The stability test results indicated that GV-Lipo/SF/DT showed good plasma stability and storage stability.In vitro release profile showed that SF and DT could be effectively released from GV-Lipo/SF/DT,respectively.Cellular uptake assay,competitive inhibition assay,and in vivo NIRF imaging results indicated GV-Lipo/SF/DT exhibited a better targeting capability than GPC3 or VC AMI single-targeting liposomes in vitro and in vivo,respectively.Determination method of SF and DT were established by full wavelength sweeping HPLC.The method showed good linear relationships under determined concentrations,and the precisions and recoveries could meet the requirements.Chapter 7.Evaluation of eliminating associated-CTC ability of multipoint co-striking SF and DT co-loaded multifunctional liposomes.GV-Lipo/SF/DT exhibited specific targeting allowing recognition and capture of CTC,dissociation of CTC clusters,and prevention of CTC-neutrophil cluster formation.The specific targeting ability of GV-Lipo/SF/DT to CTC was evaluated by mimicking the in vivo blood flow environment using a rotary viscometer.The results indicated that GV-Lipo/SF/DT could recognize and capture Hepa1-6 cells under a flow environment,GFP Hepa1-6-luc clusters increased tumor metastasis compared with single GFP Hepa1-6-luc,while DT could dissociate GFP Hepa1-6-luc clusters,leading to a reduction in HCC pulmonary metastasis.The intracellular Ca2+ concentration assay indicated that DT dissociates CTC clusters might by increasing the intracellular Ca2+concentration.The results of neutrophils co-cultured with GFP Hepa1-6-luc cells showed that the competitive binding of the anti-VC AM1 mAb to VCAM1 could prevent the formation of CTC-neutrophil clusters,GFP Hepa1-6-luc cells were used as CTC model to establish mouse model of CTC in vivo.FCM was used to investigate the CTC elimination ability of GV-Lipo/SF/DT in vivo.The results showed that GV-Lipo/SF/DT could recognize and capture CTC more specifically,thus inhibiting CTC more efficiently.Chapter 8.Antitumor efficacy study of multipoint co-striking SF and DT co-loaded multifunctional liposomes.MTT assay indicated that GV-Lipo/SF/DT showed better cytotoxicity to Hepa1-6 cells than Lipo/SF/DT.Cell cycle arrest was induced in the G1 phase after treatment with Lipo/SF/DT,which indicated that SF and DT had certain combined effect on producing cell cycle arrest.The tumor penetration ability of GV-Lipo/SF/DT in Hepa1-6 3D tumor spheres indicated that free DT,Lipo/DT and GV-Lipo/DT could conducive to the deeper penetration of tumor tissue under the experimental concentration compared with untreatment group due to the certain dissociation effect on the primary tumor cells mass of DT.GV-Lipo/SF/DT showed enhanced antitumor efficacy in both H22 cells tumor-bearing xenograft tumor model mice and GFP Hepa1-6-luc cells tumor-bearing orthotopic HCC model mice,with an inhibition rate of 90.28%in xenograft tumor model mice.The results of the haemolysis test,mouse body weight,and H&E staining of main organs showed that GV-Lipo/SF/DT exhibited good biocompatibility without gross toxicity.Chapter 9.Evaluation of inhibiting tumor recurrence and metastasis of multipoint co-striking SF and DT co-loaded multifunctional liposomes.The ability of GV-Lipo/SF/DT to inhibit tumor recurrence after surgical resection and pulmonary metastasis was evaluated in post-surgical H22 tumor-bearing KM mice Results indicated that GV-Lipo/SF/DT exhibited superior inhibitory recurrence effects.The inhibitory effect of GV-Lipo/SF/DT on pulmonary metastasis was evaluated in metastasis model mice via intravenous injection of H22 cells.The results showed that DT and anti-VCAM1 mAb could inhibit HCC metastasis to a certain extent,and GV-Lipo/SF/DT could effectively inhibit lung metastasis of HCC,with a metastasis inhibition efficiency of 95.42%.In summary,the omni-directional consideration of multi-mode combined novel diagnosis and treatment concept could improved the specificity of early diagnosis,enhanced therapeutic effects and inhibited metastasis effectively of HCC.GSI-Lip promoted early diagnosis andprecise therapy of HCC.We demonstrated that GSI-Lip is excellent targeted imaging agent with advantages for very earlydiagnosis of HCC.GSI-Lip could detect tumor as early as on day 2 after injection of 5×105 H22cells(3.45±0.98 mm3).Importantly,GSI-Lip showed superior synergistic chemo-photothermal antitumor effect in vivo.Overall,GSI-Lip might provide a new strategy for development of novel theranostics agent of HCC and showed great potential for clinical applications.GV-Lipo/SF/DT exhibited enhanced therapeutic effects on primary HCC tumors and effectively prevented HCC metastasis.GV-Lipo/SF/DT showed increased targeting efficiency resulting in recognition,capture and efficient CTC elimination in vivo,dissociating CTC clusters,blocking the formation of CTC-neutrophil clusters,simultaneously,thus effectively preventing HCC metastasis.Importantly,GV-Lipo/SF/DT showed superior antitumor effects on the xenograft tumor model mice and orthotopic HCC model mice in vivo with remarkable tumor suppression.Overall,multipoint co-striking strategy has opened a new possibility for omni-directional inhibition of tumor metastasis,which could be translated into a new treatment concept for various cancers in the clinic. |
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