UCST Polymeric Micelles-based Drug Delivery Systems For Hepatocellular Carcinoma Visualized Therapy

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world and 50%have occurred in China.The morbidity and mortality are increasing year by year,thereby posing a serious threat to public health.At present,the main clinical treatments for HCC include surgery,chemotherapy,radiotherapy and immunotherapy.Surgery is generally suitable for early-stage HCC patients and also prone to trauma.Radiotherapy and chemotherapy have serious side effects and drug resistance,while the efficacy of immunotherapy is relatively low.Thus,there is a great significance to seek a safe and efficient treatment method for HCC patients.Nano drug delivery systems(NDDSs)are considered to be an effective means to improve in vivo drug distribution which can reduce the toxic and side effects,and the HCC targeted therapy can also be achieved through the functional modification of carrier materials.However,the existing NDDSs still have a slow and incomplete drug release profile in the systemic circulation.Therefore,the key issue for improving the efficacy of HCC is to promote a rapid and complete drug release at the tumor site.The stimuli-responsive NDDS is referred that its structure can be changed under exogenous(electric field,magnetic field,ultrasound,temperature)or endogenous(p H,enzyme,redox)stimulation,thereby automatically adjusting drug releasing rate and achieving stimuli-responsive release profile.Among them,the temperature-sensitive polymeric micellar drug delivery system has become a research hotspot.On the one hand,drug that enriched at the tumor site can be released rapidly and completely from polymeric micelles under temperature stimulation to improve the drug efficacy.On the other hand,high temperature can kill tumor cells,realizing the tumor hyperthermia.In this study,we used the relevant characteristics of the upper critical solution temperature(UCST)polymeric micellar drug delivery system which combined with anti-tumor drugs,phototherapy,immunotherapy and other treatment methods to achieve the accurate visualized therapy of HCC guided by imaging technologies.The main contents are as follows:Part 1:The p(AAm-co-AN)with UCST of 43℃was successfully synthesized by free radical polymerization,and then chemically grafted PEG onto p(AAm-co-AN)to obtain PEG-p(AAm-co-AN).The SA-PEG-p(AAm-co-AN)was synthesized by chemically modifying with sialic acid.The prepared polymers could self-assemble to micelles in aqueous media with a critical micelle concentration(CMC)of 28.9μg/m L.Gadolinium-copper sulfide nanoparticles(Gd-CuS NPs)with both photothermal conversion effect and magnetic resonance/photoacoustic dual mode imaging function were synthesized by thermal decomposition method,with an average particle size of13.2±2.31 nm,a potential value of-18.0±3.32 m V and a relatively strong near-infrared absorption peak at 980 nm.The hydrophobic drug Doxorubicin(DOX)and Gd-CuS NPs were using as a model drug and photothermal imaging agents to prepare co-loaded polymeric micelles(SPDG)by dialysis method and emulsifying solvent evaporation method.The particle size of SPDG was 212.0±19.8 nm and showed regular spheres.SPDG exhibited a good loading capacity,the drug loading and the encapsulation efficiency of DOX were 3.54%and 77.4%,respectively.And the payload of Gd-CuS NPs was 6.61%.SPDG also had an obvious photothermal conversion effect under 980 nm laser irradiation,which showed a temperature-triggered drug release behavior and caused a quick and complete drug release profile.In addition,SPDG had an enhanced magnetic resonance T1 imaging signal and an obvious photoacoustic imaging signal in vitro with a good linear relationship as a function of SPDG concentration.Human normal hepatocyte LO2 and hepatoma cell HepG2 were used as model cells.The cell viabilities of blank micelles were both above 85%after treatment at body temperature and high temperature(43°C),which showed a good biosafety.Compared with non-sialic acid modified co-loaded polymeric micelles(PDG),SPDG showed a better uptake behavior in HepG2 cells.After blocking E-selectin overpressed on the surface of HepG2 cells with free sialic acid,the uptake ratio of SPDG by HepG2 cells was decreased,which confirmed that the active transport of SPDG in HCC cells was mediated by the specific binding of sialic acid to E-selectin.SPDG also exhibited a temperature-sensitive drug release characteristic in HCC cells.When HepG2 cells were irradiated by a 980 nm laser at 2 W/cm~2 for 5 min after uptaking SPDG,DOX could be released rapidly and entered the nucleus,causing an improved anti-tumor efficacy.Compared with the chemotherapy or photothermal therapy alone,the SPDG+NIR group could significantly inhibit the activity of HCC cells and induce the cell apoptosis.The expression of apoptotic protein caspase-3 was up-regulated and the anti-apoptotic protein bcl-2 was decreased,indicating that chemotherapy-photothermal had a synergistic inhibitory effect in HCC cells.The tumor-bearing nude mice model was established by subcutaneous injection of HepG2 cells.After intravenous injection,due to the sialic acid modified polymeric micelles could specifically bind to the E-selectin that overexpressed on the surface of tumor cells through sialic acid,leading to a huge enrichment at the tumor site with the highest accumulation at 24 h.After intravenous injection of SPDG,magnetic resonance/photoacoustic imaging signals were obviously observed at the tumor site,indicating that SPDG had good dual mode imaging ability in vivo and realized real-time imaging at tumor site.The dual mode imaging signal was strongest at 24 h after intravenous injection,magnetic resonance/photoacoustic imaging signals in SPDG group were 1.33 times and 1.12 times higher than that of PDG group,which further verified a better targeting ability of SPDG.When the tumor-bearing mice in the SPDG group irradiated with 980 nm laser at 2 W/cm~2 for 6 min,the local temperature of the tumor region could be increased to 43℃,promoting a rapid and complete drug release at the tumor site.SPDG+NIR group showed a significant tumor growth inhibitory effect,the tumor inhibition rate was as high as 91.7%with obviously necrosis and apoptosis in tumor region.During the observation period,except the free DOX group,the body weights of tumor-bearing mice in each group were relatively stable and there were no pathological damages occurred to the main organs,indicating that the developed SPDG had a good biosafety in vivo.Part 2:Small molecule targeted drugs combined with immunotherapy are considered to be one of the most effective methods for the treatment advanced HCC,which has been granted as a breakthrough therapy by FDA.Unfortunately,patients are proned to suffer from side effects,non-tumor targeted aggregation and low oral bioavailability after long-term using small molecule targeted drugs.In immunotherapy,due to the low antigenicity of tumor cells during immune escape,it is difficult for immune cells to recognize tumor antigens,leading to a low therapeutic efficacy.Aiming to the abovementioned problems,HCC-specific targeting polypeptide SP94 modified PEG-p(AAm-co-AN)co-loaded with lenvatinib and near-infrared second-region(NIR-Ⅱ)probe IR-1061-Acridine(IR-1061-Ac D)was developed in this study.The combination of small molecule targeted drugs,photothermal and photodynamic therapy could kill HCC cells while promoting the most immunogenic cell death(ICD)of tumor cells to enhance its immunogenticity,which activated the anti-tumor immune response in vivo.Further applying with anti-PD-1(a PD-1)antibody,its anti-tumor efficacy could also be promoted,realizing the combination therapeutic effect.SP94-PEG-p(AAm-co-AN)was synthesized by chemical modification of HCC targeting polypeptide SP94.The polymers could self-assemble into micelles in aqueous medium with CMC of 32.2μg/m L and showed temperature sensitive response with the UCST of 43°C.Using IR-1061 and acridine as raw materials,NIR-Ⅱprobe IR-1061-Ac D with photothermal and photodynamic activity was successfully synthesized.The structure of IR-1061-Ac D was confirmed by ~1H-NMR and MS.In addition,IR-1061-Ac D had strong absorption at 1075 nm and the fluorescence intensity at 1175 nm.SP94 modified Lenvatinib(LEN)and IR-1061-Ac D co-loaded polymeric micelles(SPLI)showed a regular and uniform spherical shape with a particle size of212.0±7.3 nm.SPLI also had good drug loading capacity,drug loading capacity and encapsulation efficiency of LEN was 7.68%and 84.5%,respectively,the payload of IR-1061-Ac D was 4.94%.SPLI showed good in vitro photothermal effect and photodynamic activity under 1064 nm laser irradiation.Meanwhile,SPLI exhibited an obvious temperature-responsive in vitro drug release behavior with a slow and incomplete drug release under physiological conditions,while a quick and complete release rate after laser irradiation.Compared with ICG-loaded polymeric micelles,SPLI had higher signal-to-noise ratio imaging and stronger penetration at 8 mm depth in the NIR-Ⅱwindow.AML-12 and H22 cells were used as cell model in vitro.After treated with the blank polymeric micelles with body temperature and high temperature(43°C),the cell survival rates were both higher than 90%,indicating a good biological safety.Compared with non-SP94 polypeptide-modified LEN and IR-1061-Ac D co-loaded polymeric micelles(PLI),SPLI showed higher targeting ability to tumor cells due to SP94.After H22 cells uptaking SPLI and irradiating by 1064 nm laser with 1 W/cm~2 for4 min,amount of singlet oxygen was produced in cells which showed an increasing production rate with the extention of irradiation time.SPLI also exhibited temperature-sensitive drug release characteristics at the cellular level,the drug could be release rapidly and completely after laser irradiation.The combined treatment of small molecule targeted drugs,photothermal and photodynamic therapy could significantly inhibit the activity of HCC cells and cause the maximum cell apoptosis rate,as well as effectively induced ICD of HCC cells.When the co-cultured system of H22 cells and immune cells,HCC cells with ICD could effectively promote the maturation and antigen presentation of dendritic cells(DCs),and the strongest immune responses of lymphocytes were stimulated.The subcutaneous and liver orthotopic tumor-bearing mice models were respectively established by subcutaneous injection of H22 cells in the hindlimb and liver tissue.NIR-Ⅱfluorescence imaging results showed that SPLI had more distribution and accumulation in tumor regions with the maximum accumulation at 24 h and 6 h after intravenous injection in subcutaneous tumor-bearing mice and liver orthotopic tumor-bearing mice.Intraoperative imaging result of liver orthotopic tumor-bearing mice clearly showed that tumor tissue could be completely resected without residual lesions.The temperature of the tumor site could increase to 43°C and thus triggering a rapid and complete drug release profile after receiving SPLI with 1064nm irradiation at 1 W/cm~2 for 12 min.In bilateral subcutaneous tumor-bearing mice,SPLI+NIR combined with a PD-1 antibody treatment could effectively inhibit the growth of the primary tumor and distant tumor due to the combined effect of small molecular targeted drugs,photothermal,photodynamic and immunotherapy.In addition,the primary tumor showed a higher degree of ICD and benefited for the infiltration of DCs,therefore significantly promoting the maturation of DCs and further activating the T cell immune response.The proportion of helper T cells(Th),cytotoxic T lymphocytes (CTLs),effector memory T cells(MTCs)and anti-tumor cytokines in distant tumor and spleen were all increased,while Tregs were decreased.These results proved that the combined treatment strategy could effectively regulate immune microenvironment and activate the systemic immune system in tumor-bearing mice,as well as inhibiting the occurrence of lung metastasis of HCC cells.Tumor-bearing mice maintained stable body weights during the observation period and there were no significant pathological damages in major organs,indicating a good biosafety in vivo.The developed UCST polymeric micellar drug delivery system in this study could achieve the targeted delivery and controllable drug release at the tumor site.The combination treatment regimen had an obvious tumor inhibition effect.Meanwhile,the real-time treatment process of drug delivery system could be accurately tracked with the help of imaging technologies,which might lay a foundation for the development of visualization-guided HCC accurate combination therapy.