Effect And Mechanism Of Doxil And CL-LIP Targeting Macrophages On Treatment And Recurrence Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the malignant tumors with the highest cause of death and increasing incidence in China.Although the diagnosis and treatment levels of liver cancer have increased in recent years,the incidence rate is still on the rise.TACE is widely used for patients with advanced HCC,while anti-cancer drug resistance and pharmacokinetics and adverse effects restricts its application.Additionally,patients with early-stage HCC usually perform surgery with a high recurrence of 50% in 3 years.Considering the lack of effective therapeutic treatments of advanced HCC and its recurrence,therapeutic resistance and tumor recurrence has been a hotspot of HCC research.Effective antitumour therapeutic strategies for preventing and treating late recurrence are urgent to discover.Small molecule anti-cancer drugs are commonly used for HCC in clinical.However,chemotherapeutic drugs have adverse effects on liver function and hepatic artery system.And multi-organ toxicity and drug resistance further limit their clinical application.With the development of nanotechnology,nanoparticles loaded with chemotherapeutic drugs have been developed playing a role of " reduce toxicity and increase efficacy" for chemotherapeutic drugs.Liposomes,one of nanoparticles,have become the most widely used nanocarriers in clinical because of their high biocompatibility,good pharmacokinetic properties.Characteristics of liposomes determined their final biological distribution after they enter the body.For liposomes with a diameter of 50-100 nm,they can penetrate and accumulate in tumor tissues depending on the enhanced permeability and retention effect.For larger particles(> 150 nm),especially those attached to conditioning proteins,they will be recognized and captured by mononuclear phagocytic system.The isolation and removal effect of MPS on liposomes greatly reduces the efficiency of drug delivery.Liver is one of the main organs of MPS.Kupffer cells in hepatic sinusoids,accounting for 80-90% of the total number of permanent macrophages in human tissues,are the main macrophages in the liver.As we all known,HCC is an inflammatory-related disease.Macrophages,as the main component of inflammatory microenvironment,play a critical role in the occurrence and development of HCC.Macrophages could promote malignant transformation of HCC by promoting angiogenesis,inducing epithelial-mesenchymal transition(EMT)and creating immunosuppressive environment.Lots of clinical researches showed that high density infiltrated of macrophages in tumor closely related to the poor prognosis of HCC patients.Macrophages also involved in the initiation of HCC.Studies have shown that tumor necrosis factor-?(TNF-?)secreted by macrophages could promote self-renewal of hepatic progenitor cell(HPC)and induce malignant transformation of HPC to cancer stem cells by up-regulating the expression of stem-related genes,which promoting tumorigenesis.As mentioned above,macrophages in inflammatory microenvironment are not only an important inducement to promote the tumorigenesis and development of HCC,but also an important obstacle in HCC therapy since their large number and strong phagocytosis ability and their impaired effects on targeted delivery of nanoparticles to the liver.Therefore,it is believed that targeting macrophages in the liver may effectively inhibit the occurrence of HCC and improve the therapeutic effect.Based on the fact that macrophages have the characteristics of capture and phagocytosis of liposomes and more than 80% reside in the liver and macrophages play an important role in the occurrence and development of HCC.Liposomes targeting macrophages containing doxorubicin and clodronate were prepared.Characteristics of liposomes were observed and then liposomes were administered at different stages of primary HCC model in rats to figure out wether they had preventive and therapeutic effects or not.With local injection of liposomse into the liver,not noly chemotherapeutic drugs could enrich in liver,macrophages could also release the chemotherapeutic drugs and inhibit the occurrence of tumor by its death.It is expected that the results of the study are expected to form a new strategy for the treatment of HCC and anti-postoperative recurrence.Part ?: Preparation of doxorubicin liposomes and clodronate liposome and their related functions.In order to achieve the enrichment and sustained release of chemotherapeutic drugs in liver by targeting macrophages,we prepared Doxil and CL-LIP by thin film hydration and analysised their characterics.Flow fluorescence quantitative method and High-Performance Liquid Chromatography-Mass Spectrometry(HPLC-MS)was used to detect the ability of macrophage ingesting Doxil in vitro and in vivo.Hepatic enrichment and sustained release of liver under the action of CL-LIP also detected.Doxil with particle size of about 100 nm,180nm,350 nm and CL-LIP were prepared successfully.We evaluated the stability of different size particles and found that the stability of 350 nm particle was lower than 100 nm or 180 nm particles.Phagocytosis test in NR8383 cell and rat peritoneal macrophages showed that the larger the particle size of Doxil,the more easily to be swallowed,vivo experiments of macrophages in rat liver further confirmed the result.Considering the stability of Doxil and phagocytosis of macrophage,we chose 180 nm Doxil for subsequent experiments.In order to determine the safe dose of Doxil administered in rats,we initially observed the toxicity of Doxil to rats and determined the three doses of 1mg/kg,2mg/kg and 4mg/kg for part ? and part ?.Next,we chose 2mg/kg Doxil to be injected into the rat through the spleen.HPLC-MS method detected that the concentration of Doxil in liver was much higher than that in heart,lung and kidney.Data in our study showed that CL-LIP had killing effect on NR8383 cell and rat peritoneal macrophages in a dose-dependent manner in a certain concentration range,and immunohistochemical results also showed that CL-LIP could deplete macrophages in liver mostly in 48 hours.The serum concentration of doxorubicin in rats administrated Doxil combined with CL-LIP could be maintained higher and longer than Doxil alone.Those results suggested that Doxil with180 nm size had a better stability,and it could be enriched in macrophages and sustainted release combined with CL-LIP in liver.Part ?: Therapeutic effect of Doxil combined with CL-LIP on the development HCC in rats.In order to investigate the therapeutic effect of Doxil combined with CL-LIP on advanced HCC,we initially screened the effective dose of Doxil in inhibiting the development of rat liver cancer(2mg/kg)and constructed advanced HCC rat model induced by DEN for 14 weeks and treated them with saline(Control),clodronate liposome(CL-LIP),free doxorubicin(DOX-free),doxorubicin liposome(Doxil),clodronate liposome combined with clodronate liposome(Doxil+CL-LIP).At the end of the administration,the tumor size and number and liver functions were observed.Results showed that tumor volume was reduced and the number was decreased,and the liver damage caused by DEN was alleviated.The results of HPLC-MS confirmed that Doxil could be enriched in liver cancer rats,further confirming the results of the part ? of Doxil enrichment in normal rat liver.The above results indicate that Doxil could inhibit the development of liver cancer by liver enrichment and combined with CL-LIP administration.Next,we explored its mechanism.IHC staining showed that combined administration could effectively remove macrophages in liver.After the death of macrophages,the released chemotherapeutic drugs could kill the surrounding tumor cells which comforted by the results of Tunnel staining indicating that the combined administration induced an increase in tumor cell apoptosis.The expression of M2 macrophages increased during DEN-induced liver cancer,and co-culture of hepatoma cells with M2 macrophages promoted their proliferation.TNF-a expression was down-regulated in the liver of DEN and DEN+CL-LIP rats.Considering that Doxil and CL-LIP can be swallowed by macrophages(the first part has been confirmed)and CL-LIP has specific killing effect on macrophages,M2 macrophages could be cleared and subsquently reduce the secretion of TNF-a which improving the tumor microenvironment.Additionally,chemotherapeutic drugs was released after the death of macrophages,which could kill tumor cells and improve the therapeutic effect on HCC.Part ?: Preventive effect of Doxil combined with CL-LIP on the tumorgenesis of HCC.In order to investigate the role of Doxil combined with CL-LIP in the occurrence/recurrence of HCC,we established a rat model of HCC induced by DEN for 8 weeks.Rats induced by DEN were randomly divided into four groups and treated with clodronate liposome(CL-LIP),free doxorubicin(DOX-free),doxorubicin liposome(Doxil),clodronate liposome combined with clodronate liposome(Doxil+CL-LIP).We investigated the tumor incidence rate and liver function of rats in each group,HE and immunohistochemical staining were used to evaluate the pathological structure of liver,and the depletion of macrophages and the activation of HPC in liver were accessed by immunohistochemical staining.The sensitivity of normal hepatocytes(BRL-3A cell)and HPC(WB-F344 cell)to Doxil was evaluated by CCK8 and flow cytometry-based apoptosis detection.Finally,we discussed the mechanism of co-administration Doxil and CL-LIP.The combination of Doxil and CL-LIP could significantly inhibit the occurrence of liver cancer in rats and improve liver function related indicators.Doxil combined with CL-LIP could effectively remove macrophages from the liver,inhibit HPCs activation and proliferation.In addition,the concentration of TNF? in the serum of the co-administered group was significantly decreased,and the expression of p-STAT3 was down-regulated indicating that STAT3 pathway was inhibited.These results suggested that Doxil combined with CL-LIP could effectively target macrophages,slowly release doxorubicin,and inhibit the proliferation of HPCs,which further inhibit the occurrence/recurrence of HCC.Its mechanism may be related to the clearance of macrophages and decreased secretion of TNF?,which further inhibited the activation of STAT3 pathway.Results of this study were expected to provide a new strategy for the treatment of recurrence of HCC.