The Development Of Phosphorylated Chitooligosaccharide-modified Liposomes And Its Application In The Treatment Of Osteosarcoma In Vivo And In Vitro

Osteosarcoma?OS?is a common primary malignant bone tumor in adolescents and young people.It is prone to lung metastasis and thus has poor prognosis.At present,the conventional treatment is neoadjuvant chemotherapy combined with surgical treatment.However,under neoadjuvant chemotherapy,drugs often lack tissue selectivity,which necessitates a large dose for treatment,and often associated with severe side effects to patients.In order to reduce the side effects or improve the efficacy of chemotherapy,it is necessary to develop a drug delivery system specifically targeting OS,reducing the drug distribution to normal tissues.In order to improve the concentration of chemotherapeutic drugs in bone and the targeting of OS,a phosphorylated chitooligosaccharide?PCS?-modified nanoliposome was designed by utilizing the special affinity between PCS and hydroxyapatite?HAp?.Doxorubicin?DOX?was used as a model drug.The physicochemical properties,cellular uptake and cytotoxicity,bone targeting and pharmacokinetics of PCS-modified liposomes were studied.It provides a new idea for the study of noval bone targeting nano-scale drug carriers.This paper mainly studies the following aspects:1.Synthesis and characterization of PCS-CholPCS may be a potential bone targeting molecule.In order to improve the efficiency of modification,we introduced hydrophobic Chol into PCS polymers.Firstly,we synthesized the amphiphilic graft polymer?PCS-Chol?by two steps.The first step was to synthesize CS-Chol copolymer from chitooligosaccharide?CS?and cholesteryl hemisuccinate?CHEMS?by condensation reaction of amino group and carboxyl group between them.In the second step,PCS-Chol copolymer was obtained by phosphorylation of sugar chain in CS-Chol with P₂O₅.Then,the chemical structures of the two polymers were characterized by FT-IR and ³¹P-NMR,and the degree of substitution?DS?of Chol and phosphoric acid groups were determined.Results showed that the DS of Chol on CS-Chol and PCS-Chol were almost similar?90.9%and 82.0%,respectively?,and the grafting ratio of phosphoric acid group on PCS-Chol was?3.9±0.4?%.2.Preparation and characterization of PCS-DOX-LOrdinary blank liposomes were prepared by reverse phase evaporation and then DOX was loaded by ammonium sulfate gradient method,forming DOX-loaded liposome?DOX-L?.After decoration by post-insertion of CS or PCS,CS-modified drug-loaded liposomes?CS-DOX-L?and PCS-modified drug-loaded liposomes?PCS-DOX-L?were obtained respectively.The results showed that the particle size of the PCS-DOX-L was?209.5±0.5?nm,a little smaller than that of CS-DOX-L?245.3±2.6 nm?,polydispersity index?PDI?was 0.215±0.01,and the encapsulation efficiency?EE?was?85.4±2.2?%.Under TEM,these liposomes were regular spheres.The in vitro release of the liposomes were investigated using PBS with different pH?7.4 and 5?as the release medium.The results showed that these liposomes had some characteristics of slow release and pH response.For PCS-L,the hemolysis towards red cells was lower than that of CS-L,reflecting better safety profile in vivo.3.Cellular uptake and cytotoxicity of PCS-DOX-LThe cytotoxicity of PCS-DOX-L on MG63 cells was investigated by MTT assay.The results showed that the cytotoxicity of them was time-dependent as well as concentration-dependent,but the toxicity of PCS-DOX-L was weaker than that of CS-DOX-L to a large extent at the same concentration.The uptake of PCS modified liposomes by MG63 cells was investigated by inverted fluorescence microscopy.The results showed that this kind of liposomes could carry DOX into the nucleus of cells,and the uptake process was time-dependent;that was,with the prolongation of incubation time,the cellular uptake increased.Compared with DOX solution,PCS-DOX-L exhibited a faster uptake rate at any same time point,while PCS-DOX-L had a slightly weaker intensity than CS-DOX-L.In addition,flow cytometry showed that the mean fluorescence intensity of CS-DOX-L was slightly higher than that of PCS-DOX-L at any same time,which was consistent with the results of inverted fluorescence microscopy.The reason might be related to the accelerated release of PCS-DOX-L under weak acidic conditions and the smaller zeta potential it carries,which affects its cytotoxicity and uptake to a certain extent.4.Tissue distribution of PCS-modified liposomeFirst,an OS model in nude mice was established by inoculating MG63 cells into the tibia of the right limb.Then,DiR was used as a fluorescent probe to investigate the bone targeting of different liposomes in OS mice.The DiR-labeled preparations were administered by tail vein injection.Results showed that CS-DiR-L and PCS-DiR-L had similar targeting ability to OS initially within 12 h.But the PCS-DiR-L lasted longer?for at least 24 h?at the tumor site,indicating its improved bone targeting ability than CS-DiR-L.This phenomenon was probably due to the phosphorylation of CS.5.Pharmacokinetics of PCS-modified liposomeA method for the determination of DOX in plasma by HPLC-MS/MS was established.The pharmacokinetics of PCS-DOX-L in rats were then studied.The results showed that the values of biological half-life(t_1/2)and mean residence time?MRT?of PCS-DOX-L were nearly the same as that of PEG-DOX-L and CS-DOX-L;while the area under curve?AUC?value of PCS-DOX-L was between the other two groups.These results indicate that PCS-DOX-L had characteristics of better sustained release,longer circulation and higher AUC in vivo.In conclusion,this study was the first to confirm that PCS was a good bone targeting molecule,and PCS modified liposomes have strong targeting ability to orthotopic OS sites in vivo.Moreover,the favorable characteristics in cell levels as well as pharmacokinetics provides useful evidence for the development of bone targeting drug carriers and OS targeted therapy in the future.