Inhibition Of Vasculogenic Mimicry By Cu-Apa Micelle/in Situ Gel Complex

Tumor growth requires blood supply,and blocking angiogenesis can inhibit tumor growth.Tinib and monoclonal antibodie targeting vascular endothelial cells are currently the main anti-angiogenic drugs at present,but they usually lead to cellular hypoxia during treatment,which in turn promotes re-induction of tumor angiogenesis by hypoxic factors.Therefore,it is particularly important to find new strategies to block tumor blood supply.Vasculogenic Mimicry（VM）is a tubular network composed of tumor cells,which can directly connect with the host and provide blood supply to the tumor,and the formation of VM does not involve endothelial cells.Inhibition of VM genesis provides a new direction for effective tumor inhibition.Apatinib（Apa）,a small molecule anti-angiogenesis drug,is often used for VM inhibition.However,Apa alone has weak inhibition effect and is prone to drug resistance,and causes a variety of adverse reactions.Therefore,drug combination becomes an important choice for inhibiting VM.Studies have found that metal ions play an important role in inhibiting VM,and Apa combined with metal ions has the potential of synergistic anti-VM formation.In addition,drugs encapsulated in micelles can realize the targeted delivery of hydrophobic drugs,enhance the bioavailability of drugs,and reduce adverse reactions.Meanwhile,more and more studies on natural polysaccharide based in-situ gels have been carried out,and the introduction of polymer micelles into gel networks can play a continuous anti-VM role.Based on the above analysis,Apa was selected as the main drug for VM inhibition and complexed with Cu²⁺to form the complex Cu-Apa,which exerted the combined effect of Cu²⁺and Apa to inhibit VM.Meanwhile,the polymer micelle HA-Chol was prepared as Cu-Apa carrier,and then embedded in astragalus polysaccharide based in situ gel APs Gels to form Cu-Apa micelle/in situ gel complex.The inhibitory effect of the complex on VM in vivo and in vitro was explored to provide effective strategies for tumor VM inhibition.Chapter one is the construction and characterization of Cu-Apa and Cu-Apa/HA-Chol.Firstly,Cu²⁺was linked to pyridine structure in Apa drug by solvent evaporation method to form blue crystal.FT-IR confirmed that Cu²⁺was bridgable to the nitrogen atom of pyridine structure through sodium dicyandiamide,and the concentration of Apa in the complex was 30%by UV-vis spectrophotometry.Secondly,oligo-hyaluronic acid（oligo-HA）was chemically linked with Cholesterol（Chol）to form amphiphilic polymer HA-Chol,and the esterification was verified by FT-IR and¹H NMR.The critical micelle concentration of HA-Chol was measured to be 37μM by pyrene fluorescence probe.The particle size potentiometer was used to determine that Cu-Apa/HA-Chol drug-loaded micelles were negatively charged and the particle size was less than 370 nm.The encapsulation rate was more than 80%and the drug loading was up to 42.04%.Finally,the release rate of Cu-Apa/HA-Chol in vitro was between30%and 40%by dialysis,which satisfied the first-order kinetic release model.Chapter two is the preparation and characterization of APs Gels and their drug-loaded gels.The oxidation rate of APs was 91.69%by potentiometric titration of hydroxylamine hydrochloride.The C=O stretching vibration of APs at 1697 cm^-1 was measured by FT-IR,indicating that the oxidation was successful.Secondly,APs Gels gel was prepared by Schiff base reaction,and the characteristic binding peak of APs Gels gel at 1650 cm^-1 was detected by FT-IR.The transformation time of APs Gels gel was 77.33 s,the transformation temperature was 37℃and the swelling rate was142%by test tube inversion method.In addition,the dialysis method showed that APs Gels gel could degrade slowly in vitro.Finally,SEM showed that the APs Gels gel had a spongy porous structure with small pore size,and the drug-loaded micelles were attached between the pores and the skeleton of the gel,which proved that Cu-Apa/HA-Chol@APs Gels was successfully prepared.Chapter three is the study of VM inhibition in vitro.First,VM model was constructed in vitro,and Matrigel was used as cell matrix to co-culture with mouse melanoma cells（B16-F10）for 24 h.The B16-F10 cells formed a closed ring mesh structure under microscope,and the nested tumor tubes were observed by SEM.PAS staining confirmed that there were no endothelial cells in the VM structure,indicating that the VM model of B16-F10 cells was successfully constructed in vitro.Secondly,B16-F10 cells and MCF-7 cells were used for cell scratch and invasion test.The results showed that Cu-Apa/HA-Chol@APs Gels group had 31.55%inhibition rate on B16-F10 cells,which was 31.11%lower than Apa group.The inhibition rate of Cu-Apa/HA-Chol@APs Gels group on MCF-7 cells was 13.73%,which was 61.20%lower than that of Apa free group.In addition,Cu-Apa/HA-Chol@APs Gels reduced the number of angiogenesis and vascular nodes in VM of B16-F10 cells by 60.84%and52.08%compared with Apa.Finally,MTT assay showed that the inhibition rate of HA-Chol and APs Gels gel on B16-F10 cells and MCF-7 cells was less than 20%,indicating that the two materials had low toxicity and could be used as vectors for drug delivery,while the cytotoxicity of other drug groups showed dose-dependent enhancement.Within 24 h,the IC₅₀ of Cu-Apa/HA-Chol@APs Gels in B16-F10 cells was 13.93μM lower than that in Apa drug group.The IC₅₀ of Cu-Apa/HA-Chol@APs Gels in MCF-7 cells was 7.62μM lower than that of Apa drug group,indicating that Cu-Apa/HA-Chol@APs Gels had better inhibitory effect on tumor cells than free drug group.Cu-Apa/HA-Chol@APs Gels not only increased the effective concentration of Apa on tumor cells,but also inhibited VM by inhibiting the migration and invasion of tumor cells.Chapter four is the study of VM inhibition in vivo.Female C57BL/6 mice were selected to establish the axillary tumor model of B16-F10 mice.The changes of body weight and tumor volume of mice were observed and recorded by intratumoral injection of PBS,Apa and Cu-Apa/HA-Chol@APs Gels.The effects of different treatment groups on tumor tissues were observed by HE staining and PAS/CD34staining.After 14 d of continuous drug injection,the body weight and tumor volume of mice in the blank group increased significantly,while those in the Apa group and Cu-Apa/HA-Chol@APs Gels group showed normal performance with a small increase in body weight and tumor volume,and Cu-Apa/HA-Chol@APs Gels group had no such adverse effects.HE staining and PAS/CD34 staining showed that Cu-Apa/HA-Chol@APs Gels treatment significantly reduced VM formation.Cu-Apa/HA-Chol@APs Gels not only reduced the toxicity of Apa,but also inhibited VM better than Apa.In this study,Cu-Apa copper complex was successfully prepared,and low toxicity HA-Chol polymer and APs Gels gel were synthesized as Cu-Apa carrier to construct Cu-Apa/HA-Chol@APs Gels complex.In vivo and in vitro experiments demonstrated that HA-Chol and APs Gels gel loaded Cu-Apa had good anti-tumor VM effect.Cu-Apa/HA-Chol@APs Gels inhibits tumor growth by blocking the blood supply of tumor tissues for anti-tumor purposes,providing data support and therapeutic strategies for anti-tumor VM formation.