The Feasibility Study Of Modified Angelica Sinensis Polysaccharide As Liver Cancer-targeted Drug Carrier

Liver cancer is one of the most common cancers worldwide,and the incidence in China is gradually increasing.In addition to surgical resection and liver transplantation,chemotherapy remains the most widely used therapeutic strategy.In order to overcome the limitations of chemotherapy,the construction of liver-cancer targeted delivery system has gradually attracted more and more attention,which could significantly improve the solubility of insoluble agents,and increase the accumulation as well as the biocompatibility of the anti-tumor agents,improving the anti-tumor efficiency meanwhile decreasing its toxicity.Among the materials preparing the nanoparticles,polysaccharides have been attracting more and more attention because of its wide-ranging sources,low toxicity,high biocompatibility and easy to be modified.Angelica sinensis,as a traditional Chinese medicinal and edible plant in our country,has been used for thousands of years.Angelica sinensis polysaccharide（ASP）is one of the most important components,which has been attracting more and more attention because of its excellent pharmacological activities.In addition,ASP has good water solubility,economical,easy to obtained and could be modified in different ways,which fulfills the requirement to be designed as the drug delivery systems.However,there are only few studies using ASP as anti-tumor drug carriers.Since there are different functional groups distributed on polysaccharides,the most commonly used way to design drug carriers using polysaccharides is introducing hydrophobic groups to synthesis the amphiphilic polymers,which could self-assemble into nanoparticles through the hydrophobic force in aqueous solution.The most commonly used groups includes liner small molecular hydrophobic groups（stearic acid,etc）,cyclic small molecular hydrophobic groups（cholesterol,deoxycholic acid,etc.）and high molecular polymers（PLGA,etc.）.Here,the authentic medical material Minxian Angelica sinensis were used as the raw material,ASP was first obtained by hot water extraction and ethanol precipitation method.Liner small molecular group stearic acid（SA）was first used as the hydrophobic group to modify ASP and the ASP-SA amphiphilic polymer was synthesized,it could self-assemble in water and accumulate in Hep G2 cells.Curcumin was used as the hydrophobic anti-tumor agents and was incapsulated into the nanoparticles.The micelles significantly improved the solubility of curcumin,as well as its anti-tumor efficiency towards Hep G2 cells and Hep G2 multi-cellular spheroids.Then,cyclic small molecular group deoxycholic acid（DOCA）were introduced and doxorubicin（DOX）was utilized as anti-tumor agents.The physicochemical properties,targeting ability and antitumor efficiency in vitro and in vivo of DOX/ASP-DOCA micelles were explored.It could be specifically recognized and internalized by the asialoglycoprotein（ASGPR）expressed on the surface of Hep G2 cells,increasing the concentration of the drug in liver cancer tissues,thereby improving the anti-tumor effect.In order to improve the stability of the micelles,simplify the preparing protocols and maintaining the structure of ASP,two different methods were used to prepared ASP modified PLGA nanoparticles to encapsulate the specific multi-target anti-liver cancer agents sorafenib（SOR）.It was found that the two kinds of ASP modified PLGA nanoparticles could still be enriched into the liver cancer cells and Hep G2 multicellular spheroids by specifically binding to ASGPR,thereby the anti-tumor effect of SOR was significantly enhanced compared to SOR/PLGA nanoparticles.To sum up,the ASP was purified and its natural liver-cancer targeted property was used to construct ASP-based carries in different ways,in order to explore the feasibility and the role of ASP in drug delivery.The main research contents of this study are summarized as follows:Part Ⅰ Extraction,purification of Angelica sinensis polysaccharide（ASP）and stearic acid modified ASP and itself application as self-assembled micellesIn this part,Minxian Angelica sinensis pieces were used as raw material,hot water extraction and ethanol precipitation method was used to obtain the water-soluble crude polysaccharide.After that,freezing-thaw method was introduced to remove the protein.Small molecular pigments and metal salts were removed by dialysis method.The other impurities such as oligosaccharides are further got rid of by the Sephadex G50 column.The total sugar content of ASP was around 90%.There were no absorption peaks at 260and 280 nm as shown by UV-vis spectrophotometry,indicating that there were no proteins and nucleic acids inside the polysaccharide.Moreover,ASP exhibited a single peak symmetrically distributed on HPGPC with an average molecular weight determined to be 80 k Da,demonstrating that ASP was a homogeneous polysaccharide.Here a safe,effective and simple method with low cost of extracting,separating and purifying ASP with uniform molecular weight and high purity was established.In this part,stearic acid（SA）was introduced as a hydrophobic group to modify ASP and ASP-SA amphiphilic polymer was synthesized,which could self-assemble in water to form polymeric micelles due to the hydrophobic force.The characterization of the ASP-SA polymer was investigated by FTIR,XRD and ¹H-NMR,which proved the successful synthesis of the polymer.The polymeric micelles were fabricated with dialysis method,and their particle size,Zeta potential and morphology characterization were respectively studied by DLS and TEM.The particle prepared by this method was spherical in shape with a size of 402±9.17 nm and a negative Zeta potential of-14.9±0.513 m V.Its critical micelle concentration（CMC）in aqueous solution is about 0.01mg/m L,indicating its good self-assemble behavior in aqueous solutions.Curcumin（CUR）was used as a model drug and encapsulated in the micelles.X-ray diffraction（XRD）was used to analysis the characterizations of free drug and the micelles before and after encapsulation.It was proved that the CUR was successfully loaded in the hydrophobic core.The in vitro release experiment indicated the CUR cumulative release was significantly lower than that of free drug with a p H-dependent manner.The light stability,thermal stability and storage stability were investigated by UV spectrophotometry.It was found that the stability of free CUR was significantly improved once incorporated into the ASP-SA micelles.The biocompatibility of the blank ASP-SA micelles was carried out with MTT assay with human cervical cancer Hela cells and human liver cancer Hep G2 cells.The results showed that the cell viability was still above 80%with a range of micelles concentrations.In addition,the cellular uptake of free CUR and CUR/ASP-SA was conducted with CLSM,the fluorescence intensity of CUR/ASP-SA group in Hep G2 cells was significantly stronger than that of Hela cells at the same time point.Encapsulation of CUR in ASP-SA micelles not only significantly increased the accumulation of CUR in Hep G2monolayer cells,and can also significantly improve the penetration depth of CUR into Hep G2 multicellular spheroids.Its cytotoxicity is also significantly stronger than that of free CUR.Combined with the previous study in our group,we speculate there might be a specific pathway for ASP-SA in the Hep G2 uptake.Part Ⅱ Deoxycholic acid modified ASP and its application as liver cancer-targeted self-assembled micellesIn last part,we speculate there might be a specific pathway for ASP-SA micelles in the Hep G2 uptake.In this part,the amphiphilic polymer ASP-DOCA was synthesized using the cyclic small molecule hydrophobic group deoxycholic acid（DOCA）as hydrophobic group.Its successful synthesis was proved by FTIR,¹H-NMR,XRD and DSC（differential scanning calorimetry）.The CMC value of the ASP-DOCA micelles was determined to be 0.005 mg/m L,indicating it could self-assemble into core-shell structure in aqueous solution with a relatively low concentration.Doxorubicin（DOX）was used as the model drug and incorporated into the ASP-DOCA micelles by dialysis.XRD and DSC analysis were carried out to verify the successful encapsulation of DOX.In addition,the characterization of the blank and drug-loaded micelles was further investigated by DLS,TEM and SEM.The results indicated that the particle size of the blank and drug loaded micelles were around 210～250 nm with a negative Zeta potential around-17 m V.Both TEM and SEM results confirmed that the particles were spherical in shape without aggregation.Compared with the rapid release of free DOX,the DOX in DOX/ASP-DOCA exhibited a controlled and p H-dependent release manner.The human liver cancer HepG2 cells with high ASGPR expression and human cervical Hela cells with low ASGPR expression were respectively selected as model cells.The deoxycholic acid modified dextran polymer micelles DEX-DOCA micelles were fabricated according to the protocol of ASP-DOCA micelles as negative contrast group.CLSM and flow cytometry were utilized to analysis the cellular uptake.The DOX/ASP-DOCA micelles witnessed a significantly stronger uptake by Hep G2 cells than that of DOX/DEX-DOCA micelles.At the same time,the cellular uptake of Hep G2cells was significantly inhibited once the cells were preincubated with NGA（the ASGPR antagonist）,verifying the ASP-DOCA micelles could be specifically and efficiently taken by Hep G2 cells through the ASGPR-mediated endocytic pathway.The in vivo targeting experiment was conducted with Hep G2-bearing nude mice and DIR loaded ASP-DOCA micelles.The fluorescence signals in tumors decreased obviously when the mice were simultaneously injected with ASGPR antagonist NGA.In addition,more DIR accumulation could be observed in ASP-DOCA micelles group than that of free DIR,indicating that ASP-DOCA micelles could specifically target Hep G2 cells with high ASGPR expression.The hemolysis test and cytotoxicity assay were further carried out to evaluate the biocompatibility of ASP-DOCA micelles.The hemolysis results verified that the blank micelles have no hemolysis effect.Moreover,the blank micelles not only have no growth inhibitory effect on Hep G2 cells and Hela cells but also did not show growth inhibitory effect on the multi-cellular Hep G2 tumor spheroids.Meanwhile,the in vivo experiment showed that the mice body weight change remained stable and there was no obvious toxicity on major organs of the blank micelles group.The in vitro and in vivo experiments confirmed the biocompatibility of the blank micelles.The MTT method was applied to evaluate the toxicity of DOX/ASP-DOCA micelles to Hep G2 cells and Hela cells.The result demonstrated that its toxicity was time and dose-dependent.Furthermore,the DOX/ASP-DOCA micelles exhibited a higher cytotoxicity and a lower IC₅₀ value compared with the DOX/DEX-DOCA group.Multi-cellular Hep G2 tumor spheroids were utilized to evaluate the toxicity of different DOX formulations.Similarly,DOX/ASP-DOCA micelles witnessed obvious inhibitory effect on tumor spheroids,which is close to free DOX.The in vivo pharmacodynamic evaluation was conducted using Hep G2 tumor-bearing nude mice.It was found that the growth inhibitory effect of DOX/ASP-DOCA micelles on Hep G2 xenografted tumors was significantly higher than those of free DOX and DOX/DEX-DOCA micelles.Taken together,the DOX/ASP-DOCA micelles could actively target the tumor cells through ASGPR-mediated endocytic pathway to complete the drug accumulation and exert a better anti-tumor effect.Part Ⅲ ASP modified PLGA nanoparticles and their application as liver cancer-targeted nano-drug carriersThe structure characterizations of polysaccharides could be kept from being destroyed once the polysaccharide-based nanoparticles were prepared by electronic absorption.In addition,when the polysaccharides were covalently attached to a polymer,less reaction sites were used compared with low molecular hydrophobic agents.In this part,two different methods were used to prepare the ASP-modified PLGA nanoparticles.（1）The first one is that preparing positively charged chitosan PLGA nanoparticles（Chi/PLGA）by emulsification solvent evaporation method using PVA as stabilizer,followed by being covered with negatively charged ASP to fabricate ASP/Chi/PLGA nanoparticles.The particle sizes and Zeta potential were measured by DLS.When the ASP concentration is fixed at 1 mg/m L,the particle size is 208.3±2.740 nm and the PDI is less than 0.1 with a negative Zeta potential of-13.8±0.361m V.The morphology characterized by TEM combined with potential changes proved the successful modification of ASP on the Chi/PLGA nanoparticles.（2）Another method is to synthesis the ASP-PLGA polymers via covalent bonds.Its successful synthesis was further characterized by FTIR and ¹H-NMR.The ASP-PLGA nanoparticles were fabricated by dialysis and their characterizations including size,Zeta potential and morphology were measured by DLS,TEM,SEM and AFM.It is shown that these two kinds of nanoparticles were spherical in shape and uniformly distributed.The clinical first-line anti-liver cancer agent sorafenib（SOR）was applied as the model drug,SOR-loaded SOR@ASP/Chi/PLGA and SOR/ASP-PLGA nanoparticles were respectively prepared by emulsification solvent evaporation method and dialysis method.The DLE and DLC were measured with different drug ratio.Furthermore,the drug release study was carried out using dialysis.It was proved that both ASP-modified nanoparticles exhibited sustainable release of SOR.The coumarin 6（C6）was used as fluorescence probe and encapsulated into the nanoparticles to investigate the uptake of ASP-PLGA and ASP/Chi/PLGA nanoparticles.ASGPR positive Hep G2 cells and ASGPR negative Hela cells were used in order to analyze the target ability of these two kinds of nanoparticles.The results showed that there were no obvious difference for Hela cells.However,the nanoparticles exhibited more accumulation in Hep G2 cells than PLGA nanoparticles and the fluorescence signals decreased significantly once Hep G2 cells were pretreated with ASGPR antagonist.In addition,the penetration depth of ASP-PLGA and ASP/Chi/PLGA nanoparticles towards Hep G2 multicellular spheroids is also deeper than that of PLGA nanoparticles,indicating these two kinds of nanoparticles could enter Hep G2 cells via ASGPR-mediated pathway.The MTT assay and hemolysis experiment together verified the good biocompatibility of the ASP/Chi/PLGA and ASP-PLGA nanoparticles.The cytotoxicity of SOR,SOR@PLGA,SOR@ASP/Chi/PLGA and SOR@ASP-PLGA nanoparticles on Hep G2 cells and Hela cells was studied by MTT method.The different SOR formulation exhibited cytotoxicity with a time-and dose-dependent manner.For Hep G2 cells,SOR@ASP/Chi/PLGA and SOR@ASP-PLGA showed significantly stronger cell growth inhibitory effects than SOR@PLGA,which could not been observed in Hela cells.Meanwhile,compared with SOR and SOR@PLGA nanoparticles,nanoparticles modified by ASP are more able to inhibit the growth of Hep G2 multicellular spheroids.This result is consistent with the results of cellular uptake,further confirming these two nanoparticles could specifically bind to the ASGPR highly expressed on the surface of Hep G2 cells,and could further been efficiently enriched in Hep G2 cells through the receptor-mediated endocytosis pathway.Considering that the drug release of the drug-loaded nanoparticles is not complete,SOR@ASP/Chi/PLGA and SOR@ASP-PLGA are expected to perform a stronger anti-tumor effect in practical applications.In summary,the ASP was purified and several different ASP-based nanocarriers were fabricated.In addition,three insoluble anti-tumor agents were encapsulated into the nanoparticles and their characterizations including physicochemical properties,drug loading capability,in vitro and in vivo uptake,liver cancer-targeted capability were investigated.The liver cancer-targeted ability of ASP was utilized to increase the drug concentration in cells,enhance the utilization of therapeutic effects of drugs,meanwhile reduce the toxicity and side effects of free drugs,providing some insights for the design of ASP-based drug delivery system for liver cancer therapy.