Studies On Self-assembled Micelles Based On Glycyrrhetinic Acid Modification For Targeted Drug Delivery

Chemotherapy still plays an important role in the clinic treatment of hepatocellular carcinoma(HCC)in present time.However,the application of conventional chemotherapy in HCC treatment always lead to severe side-effects due to the non-specific biodistribution of chemotherapeutic drugs.Moreover,the repeated doses of chemotherapeutics also result in the occurrence of multidrug resistence(MDR)and weaken the therapeutic efficacy of drug.Nano drug delivery system(NDDS),developed from nanotechnology for targeted drug delivery,has become a promising candidate in pharmaceutical field.NDDS,with nano-sized matrix loading therapeutic agents,could selectively deliver the agents to the liver lesions via the nanocarrier,reduce the non-specific drug biodistribution and improve anti-tumor effect.Also,the nanocarrier could protect the encapsulated drugs from degradation and overcome the MDR.which is useful for the continuous treatment of HCC.Polymeric self-assembled micelles are employed to achieve efficient drug delivery as drug carriers,which could improve the solubility of drugs,alter in vivo drug biodistribution,improve the bioavailability and control drug release.Chemical conjugation of liver-targeting ligand glycyrrhetinic acid(GA)in the design of polymeric self-assembled micelles could achieve active targeting of micelles and further improve the drug delivery efficiency.In the present study.two novel kinds of liver-targeted polymeric drug carrier were constructed through two different design strategies,aiming to realize active liver-targeted drug delivery and give an insight into the role of glycyrrhetinic acid modification in the drug delivery behaviors of self-assembled micelles.The first design strategy chose O-carboxymethyl chitosan(OCMC)as a hydrophilic matrix and firstly coupled cholic acid(CA)on the polymer backbone to develop a novel amphiphilic polymer CMCA,then glycyrrhetinic acid was conjugated to the CMCA backbone via the C-3 hydroxyl group using succinic anhydride as a chemical linker,finally the polymer was alkalified to obtain the glycyrrhetinic acid decorated CMCA(GA-CMCA).Quercetin(QC)as a model drug was incorporated into the CMCA and GA-CMCA polymer to fabricate the drug-loaded micelles.The physicochemical properties,in vitro drug release,in vitro antitumor capability and in vivo pharmacokinetic studies of QC/CMCA and QC/GA-CMCA were evaluated comparably.On the other hand,an antitumor agent doxorubicin(DOX)was used as a model drug to verify the drug loading capability of CMCA and GA-CMCA,and the physicochemical properties,in vitro cellular uptake,in vivo pharmacokinetics and tissue biodistribution as well as in vitro and vivo antitumor efficacy were investigated.In the second strategy,low molecular weight heparin(LMWH)was chosen as a hydrophilic polymer and GA was firstly coupled to the backbone of aminated LMWH via the C-3 hydroxyl group using succinic anhydride as a chemical linker to develop a novel amphiphilic polymer LMWH-GA,then a hydrophilic liver-targeted ligand lactobionic acid(LA)was conjugated to LMWH-GA to obtain the dual-ligand modified LMWH(LA-LMWH-GA).This design method takes advantage of the hydrophobicity of GA to construct self-assembled micelles,and DOX as a model drug was loaded into the LMWH-GA and LA-LMWH－GA micelles.The effect of hydrophilic LA modification on the micelles construction and drug delivery behaviors was evaluated through the investigation of the physicochemical properties,drug loading and release,in vitro cellular uptake and antitumor capability as well as the in vivo in vivo pharmacokinetics and biodistribution.By comparing the two design strategies,the role of glycyrrhetinic acid modification in the construction and drug delivery behaviors of self-assembled micelles could be highlighted and explored,providing an useful reference for the design of GA-modified self-assembled drug delivery system for efficient liver targeting.The main research contents and results in this study are summarized as follows:1.OCMC with 17 KDa molecular weight was obtained using hydrochloric acid degradation method and used as a hydrophilic polymer to develop two novel kinds of amphiphilic polymer CMCAand GA-CMCA.1H NMR,FT-IR and XRD confirmed the chemical structure of polymer,and UV was utilized to measure the degree of substitution(DS)of CA in CMCA and the DS of GA in GA-CMCA.Self-assembled micelles were prepared via sonication,and their morphology was observed under TEM.Particle size,Zeta potential and critical micelle concentration(CMC)were also characterized.Cell cytotoxicity of blank micelles were studied to evaluate their safety as drug carrier.The results showed that the synthesized CMCA and GA-CMCA polymers could self-assemble into spherical particles with uniform size.The average particle size of CMCA micelles was 110～257 nm,and their Zeta potential were around-20 mV and CMC values of 0.028～0.079 mg/ml.With the increase of DS of CA,the particle size and CMC values decreased while no significant differences in their Zeta potentials.CMCA with CA DS of 8.2%was chosen for further GA modification,and the obtained GA-CMCA demonstrated a slight increase in particle size and CMC values while a slight decline in their Zeta potentials.GA-CMCA with DS of GA of 6.5%was used for following studies due to its relatively higher GA DS.Sulforhodamine B(SRB)results showed that both CMCA and GA-CMCA had low cytotoxicity,and only at high concentration(>0.25 mg/ml),the cell proliferation inhibition effect of GA-CMCA was not negligible.2.Quercetin(QC)as a model drug was incorporated into the CMCA and GA-CMCA micelles by a modified sonication-dialysis method.At the same drug/carrier feeding ratio,GA-CMCA showed stronger drug entrapment ability than CMCA,and the prepared QC/GA-CMCA showed relatively smaller particle size and narrower particle size distribution.For example,at 1:5 drug/carrier feeding ratio,the entrapment efficiency(EE)and drug loading amount(DL)of GA-CMCA to QC could up to 56.94%and 9.69%.and the average particle size and polydipersity of the corresponding QC/GA-CMCA were 185.5 nm and 0.130,respectively;However,the EE and DL of QC/CMCA at 1:5 drug/carrier feeding ratio were only 47.03%and 8.32%,and the average particle size and polydipersity were 211.4 nm and 0.171.For both QC/CMCA and QC/GA-CMCA.release of QC revealed a sustained,delayed and pH responsive behavior.When the environmental pH dropped from 7.4 to 6.5,the QC release rate and cumulative drug release amount from the two kinds of drug-loaded micelles were obviously increased,especially for QC/GA-CMCA micelles.When the pH value of the release medium was reduced from 7.4 to 6.5,When the pH value of release media was further declined to 5.7,the drug release rate and cumulative release amount further increased for QC/CMCA micelles,while the QC/GA-CMCA micelles were precipitated obviously in the release media,while encapsulated QC was also precipitated along with the polymer matrix,resulting in delayed drug release manner without burst release behavior.To explore the differences of QC/CMCA and QC/GA-CMCA micelles in sensitivity to the environmental pH decline,further study were carried out to investigate the Zeta potential changes of two kinds of QC-loaded micelles at different pH values,and the results showed that the Zeta potential of QC/CMCA and QC/GA-CMCA decreased with the decrease of pH values,and the number for QC/GA-CMCA decreased significantly faster than that of QC/CMCA.The in vitro antitumor effects were evaluated in HepG2 cells and the results showed QC/GA-CMCA micelles could induce higher cytotoxicity and cell apoptosis compared to QC/CMCA micelles and DOX solution.In addition,pharmacokinetics study in rats showed that QC/CMCA and QC/GA-CMCA could significantly prolong the residence time of QC in blood circulation and lower the drug clearance rate,the area under QC concentration-time curve(AUC0-∞)of QC/CMCA and QC/GA-CMCA were 4.9 and 7.4 folds,respectively,higher than that of QC solution.3.DOX was used as another model drug to verify the drug loading capability and drug delivery efficiency of CMCA and GA-CMCA.DOX-loaded micelles were prepared by dialysis method at different drug/carrier feeding ratio.With the increase of drug/carrier feeding ratio,the DL increased while EE decreased for both DOX/CMCA and DOX/GA-CMCA micelles.It’s noteworthy that the EE of DOX/CMCA dropped more obvious as the drug/carrier feeding ratio increased compared to DOX/GA-CMCA.For example,at 3:10 drug/carrier feeding ratio,DOX/GA-CMCA had an EE of 71.25%while the figure for DOX/CMCA was only 63.41%,revealing a better DOX loading capability of GA-CMCA than that of CMCA.This phenomenon indicated that GA would be involved in the formation of hydrophobic inner cores in the self-assembly of DOX-loaded micelles,which could strengthen the hydrophobic force of inner cores and enhance the encapsulation of hydrophobic drug.Considering the EE and DL at the same time,DOX-loaded micelles developed from 1:5 drug/carrier feeding ratio were chosen for the following evaluation.The two kinds of DOX-loaded micelles had an average particle size of around 200 nm and a negative Zeta potential around-17 mV.In vitro cellular uptake study showed that DOX/CMCA and DOX/GA-CMCA micelles could significantly increase the uptake of DOX in drug-resistant HepG2/ADR cells,especially for DOX/GA-CMCA micelles.Competitive inhibition experiment in HepG2/ADR cells revealed that the uptake of DOX/GA-CMCA was significantly inhibited in the presence of free GA,indicating that the internalization of DOX/GA-CMCA in HepG2/ADR cells was related to GA-mediated endocytosis.However,the uptake efficiency of two kinds of DOX-loaded micelles in HepG2 cells was lower than that for DOX solution,which indirectly revealed that the DOX-loaded micelles could protect the encapsulated DOX and reverse the MDR in HepG2/ADR cells,and facilitate the accumulation of DOX in cells.Intracellular DOX distribution results revealed that free DOX showed quick accumulation in the nucleus of HepG2 cells and the fluorescence signal enhanced with the incubation time.However,treatment of free DOX in HepG2/ADR cells resulted in lower and punctate fluorescence in the cytoplasm during the incubation time and no obvious increase of fluorescence intensity between 2 h and 6 h incubation time.Treatment of DOX/CMCA and DOX/GA-CMCA micelles lead to similar intracellular DOX distribution behaviors both in HepG2 and HepG2/ADR cells:the DOX-loaded micelles firstly showed fluorescence accumulation throughout the cytoplasm and then distributed to the nucleus with the increase of incubation time.This result indicated that the DOX-loaded micelles had a delayed and sustained drug release behavior after internalization by HepG2 and HepG2/ADR cells.4.The in vitro antitumor capability and the potential to reverse MDR of tumor cells of DOX-loaded micelles was investigated in HepG2 and HepG2/ADR cells comparably.The results showed the DOX/CMCA and DOX/GA-CMCA micelles could partially reverse the MDR of HepG2/ADR cells due to the protective effect of the hydrophobic cores to the encapsulated DOX,and the reverse resistance index is 1.45 and 1.87.respectively.By contrast,DOX solution demonstrated the highest inhibitory effect on HepG2 cell proliferation,while the cytotoxic effect decreased after the encapsulation of DOX into CMCA and GA-CMCA micelles.The delayed DOX release from DOX-loaded micelles in cells could contribute to this result.5.In vivo pharmacokinetics study and tissue distribution of DOX/CMCA and DOX/GA-CMCA micelles were evaluated in Wistar rats and Kunming mice,respectively,compared to DOX solution.The results showed both DOX/CMCA and DOX/GA-CMCA micelles could significantly prolong the residence time of DOX in the blood circulation of rats.Compared to DOX/CMCA,DOX/GA-CMCA demonstrated better performance in prolonging the blood residence time of DOX.The AUC0-)value of DOX/GA-CMCA was 3.1 times higher than that of DOX/CMCA,while the clearance rate(CL)was only 1/3 of the DOX/CMCA group.In vivo biodistribution results showed that the distribution of DOX/GA-CMCA in liver was obviously higher than DOX solution group and DOX/CMCA group,and at the same time the distribution of DOX/GA-CMCA micelles in the liver was significantly higher than that in other tissues.Quantitative targeting analysis results showed that the DOX/GA-(CMCA micelles were strongly targeted to liver.Using DOX solution as control group,the relative uptake efficiency Re values of liver,spleen,heart,lung and kidney in 24 h were 3.50,1.04.0.18,0.04 and 0.69 respectively for DOX/GA-CMCA micelles.This means that GA-CMCA encapsulation could effectively improve the DOX accumulation in liver while reduce the non-targeted DOX distribution in other tissues especially in heart and lung,which has important clinical significance to reduce the adverse effects of chemotherapeutics and improve the compliance of patients.6.The in vivo antitumor effects of DOX/CMCA and DOX/GA-CMCA were evaluated in H-22 tumor bearing mice,and blank saline and DOX solution were used as control.Intravenous administration of DOX-loaded micelles especially for DOX/GA-CMCA micelles could effectively inhibit H-22 tumor growth and reduce the toxicity of DOX.In addition,blank GA-CMCA also showed some antitumor efficacy in vivo,which was related to the anti-tumor activity of free glycyrrhetinic acid.7.In the first design strategy,we firstly synthetized the amphiphilic polymer CMCA and further explored the effect of GA modification on the drug delivery behavior.Differently,in the second strategy,we made use of the hydrophobicity of GA to construct the self-assembled micelles,and further to investigate the effect of hydrophilic LA modification on the drug loading and delivery of micelles.This design method firstly synthetized amphiphilic LMWH-GA using 4.5 KDa LMWH as hydrophilic polymer and further conjugated LA to the polymer backbone to develop dual-ligand modified LA-LMWH-GA.1H NMR,FT-IR and elemental analysis were utilized to verify the chemical structure and quantify the DS of GA and LA in the polymer.Blank self-assembled micelles were prepared by sonication and their morphology was observed by TEM.Particle size,Zeta potential and CMC values were also characterized.The results showed both LMWH-GA and LA-LMWH-GA could self-assembled into micelles with average size of 109～204 nm,CMC values of 0.010～0.057 mg/ml and apparent negative Zeta potential around-35 mV.LMWH-GA with GA DS of 7.47 and 8.94 were chosen for further LA modification and obtained LA-LMWH-GA have a LA DS of 6.32 and 4.32 respectively.Compared with LMWH-GA,the corresponding LA-LMWH-GA saw an increase in the particle size and CMC values while showed an obvious decline in Zeta potential(-22～-26 mV).This study explored the relationship between the apparent molecular weight and size of the prepared polymeric micelles and the gel permeation chromatography(GPC)results showed the determined apparent molecular weight of polymeric micelles increased with the increase of particle size.Hemolysis test showed that hemolytic cytotoxicity of LMWH-GA increased as the increase of GA DS,and showed an obvious concentration-dependent manner.A slight decline in hemolytic cytotoxicity was observed after LA modification on the LMWH-GA polymer.For all experimental concentration,LMWH-GA with GA DS of 7.47 and the corresponding LA-LMWH-GA all showed limited hemolytic cytotoxicity,not higher than 5%,therefore this LMWH-GA and LA-LMWH-GA could be used as a safe carrier in intravenous administration.In addition,SRB results showed that both the two polymers had no obvious cytotoxic effect on HepG2 and HepG2/ADR cells,and they could be safely applied in the field of drug delivery.8.Using DOX as a model drug,we explored the characteristics of LMWH-GA micelles as a drug carrier for liver targeting and the effect of further hydrophilic LA modification on the drug delivery behavior of micelles.DOX-loaded micelles were prepared by a simple dialysis method at different drug/carrier feeding ratio,and had an average particle size of 77～164 nm and Zeta potential of-17～29 mV,which all showed an obvious decline in comparison with the blank micelles.At the same drug/carrier feeding ratio,LMWH-GA showed higher DOX loading capacity than that of LA-LMWH-GA.For example,at 3:10 drug/carrier feeding ratio,the EE of DOX/LMWH-GA could up to 70.3%while the figure for DOX/LA-LMWH-GA was only 58.44%,revealing that LA modification reduced the encapsulation ability of polymer to hydrophobic DOX.At 1:10 drug/carrier feeding ratio,LMWH-GA and LA-LMWH-GA showed similar EE to DOX,and therefore DOX-loaded self-assembled micelles at this drug/carrier feeding ratio were chosen for subsequent evaluation except for cellular uptake and cytotoxicity studies which need to investigate the effect of drug/carrier feeding ratio on their cellular performance.The DOX release from the two kinds of DOX-loaded micelles all demonstrated delayed,sustained and pH-responsive manner:the drug release rate and cumulative release amount at pH=5.0 media was significantly higher than those in pH=7.4 media,which was beneficial to the cell killing efficacy enhancement of micelles in the acidic tumor site.In vitro cellular uptake results showed I)DOX/LMWH-GA and DOX/LA-LMWH-GA could significantly improve the uptake of DOX in HepG2/ADR cells,while DOX/LA-LMWH-GA with dual-ligand modification did not show the synergistic effect and enhanced cellular uptake,the uptake of DOX/LA-LMWH-GA was less than DOX/LMWH-GA.Competitive inhibition study showed that the uptake of DOX/LMWH-GA was related to the GA-mediated endocytosis,while both GA-mediated and LA-mediated endocytosis were all involved in the DOX/LA-LMWH-GA uptake although the effect of GA-mediated endocytosis was weakened.Uptake pathway inhibition study showed that the internalization of DOX/LMWH-GA mainly depend on the micropinocytosis and caveolae-mediated endocytosis,while the effect of these two uptake pathways were obviously weakened in the uptake of DOX/LA-LMWH-GA,which might be the reason why decreased cellular uptake of DOX/LA-LMWH-GA was observed compared to DOX/LMWH-GA.In addition,to highlight the role of drug carrier in drug delivery process,the cellular uptake behaviors of DOX/LMWH-GA and DOX/LA-LMWH-GA developed from 1:5 and 1:10 drug/carrier feeding ratio were investigated comparably,and the results showed the DOX-loaded micelles from 1:10 drug/carrier feeding ratio had a higher cellular uptake efficiency than those from 1:5 drug/carrier feeding ratio in both HepG2 and HepG2/ADR cells.SRB results revealed that the encapsulation of DOX into LMWH-GA and LA-LMWH-GA could significantly improve the cell killing efficacy of DOX for HepG2/ADR cells,and DOX/LMWH-GA had higher cytotoxicity.However,DOX solution demonstrated the strongest cytotoxic effect,and the difference of DOX efficacy in two cell lines revealed that the DOX-loaded micelles could protect the encapsulated DOX and reverse the MDR in HepG2/ADR cells,facilitating the accumulation of intracellular DOX and enhanced cell killing efficacy.In addition,the DOX-loaded nanoparticles from 1/10 feeding ratio resulted to be less cytotoxic in both two cells at lower experimental DOX concentration while higher cytotoxicity was observed at higher experimental DOX concentration compared to those from 1/5 feeding ratio.9.The in vivo pharmacokinetic studies in Wistar rats showed that DOX-loaded micelles especially for DOX/LMWH-GA could efficiently improve the stability of DOX in vivo and prolong the residence time of DOX in blood circulation.The AUC0-∞ values of DOX/LMWH-GA and DOX/LA-LMWH-GA after intravenous administration were 22.3 and 10.8 folds,respectively,higher than that of QC solution group.Furthermore,the mean residence time(MRT)of DOX/LMWH-GA and DOX/LA-LMWH-GA were 8.2 times and 6.5 times as much as that of the DOX solution group.In vivo biodistribution study in Kunming mice showed the in vivo distribution of DOX could be altered significantly through entrapment,especially for DOX/LMWH-GA micelles.Comparatively,two kinds of DOX-loaded micelles especially for DOX/LMWH-GA could efficiently improve the DOX accumulation in liver while reduce the non-targeted DOX distribution in other tissues especially in heart and lung,which could reduce the side effects of chemotherapeutic drugs.For DOX/LMWH-GA micelles,the relative uptake efficiency Re values of liver,heart and lung in 24 h were 3.23,0.10 and 0.05 respectively.However,DOX/LA-LMWH-GA with further LA modification did not show better liver targeting efficiency,and the Re value in liver was only 0.59 of the number for DOX/LMWH-GA while the DOX distribution in spleen,heart and lung were slightly higher than those for DOX/LMWH-GA group.The possible reason for this result was the effect of hydrophilic LA modification on the polymer drug encapsulation capability and GA-mediated endocytosis,as well as the rapid blood clearance of DOX/LA-LMWH-GA in vivo.In summary,two novel kinds of GA-modified self-assembled micelles were constructed through two different design methods in this study.Both two kinds of micelles could efficiently encapsulate hydrophobic drug and improve the drug accumulation in liver via active targeting,which could enhance the therapeutic efficacy of chemotherapeutics and reduce the adverse effects due to the non-targeted drug distribution.In addition,the effects of two different GA modification methods on the drug delivery behaviors of self-assembled micelles are very useful for the research of GA-mediated liver-targeted self-assembled drug delivery system and promote the exploration of efficient and rational liver-targeted nanoformulations.