| In recent years,the incidence of cancer has been increasing,and it has become a serious disease threatening health.However,most antitumor drugs have poor water solubility,high toxicity and serious adverse reactions,which limit their clinical application.How to realize better treatment of tumor,increase targeted drug delivery,reduce drug toxicity and side effects has become a research hotspot.With the rapid growth of biochemistry,material chemistry and nanotechnology,more and more excellent carrier materials are used for the delivery of antitumor drugs,among which serum albumin,as a carrier of natural origin,has good biocompatibility,Targetability and long half-life.And because of its unique structural characteristics,some physiological substances,such as fatty acids,can be inserted into the cavities of different domains of serum albumin or non-covalently bound to proteins through hydrophobic and electrostatic interactions,so as to be transmitted in vivo by means of serum albumin.Studies have shown that stearic acid has five stable binding sites with albumin.At the same time,the 34-position free sulfhydryl group in the albumin structure has high reactivity,can click reaction with maleimide group to achieve accurate covalent bonding,which has the characteristics of high efficiency and high selectivity,mild reaction conditions,and acts an important role in albumin modification.Here,we synthesized three small molecule prodrugs of paclitaxel,paclitaxel-stearic acid(PTX-SA),paclitaxel-octadecanedioic acid(PTX-ODDA)and paclitaxel-maleimidohexanoyl hydrazone(PTX-EMCH).Structure of the prodrugs respectively introduced stearic acid,octadecanedioic acid and maleimide group that could spontaneously bind to albumin noncovalently or covalently in vitro.Delivery of drugs was realized by a "hitchhiking" way,which increased the water solubility of drugs and exerted the advantages of albumin carriers.The preparation method was mild and simple,did not change the conformation of albumin,did not affect its activity,and could better play the targeting of the formulation.Among them,an acidsensitive acylhydrazone bond was introduced into PTX-EMCH to respond the tumor acidic microenvironment,which was planned to achieve better anti-tumor efficacy.The research mainly included the following four parts:1.Synthesis and characterization of paclitaxel(PTX)prodrugs containing albuminbinding functional groupThe 2’-OH of PTX has good reactivity and is often used as a reactive site for structural modification to prepare PTX prodrugs.PTX-SA and PTX-ODDA was synthesized by one-step esterification reaction of the 2’-OH of PTX with the-COOH in fatty acid.The synthesis of PTXEMCH used levulinic acid(LEA)as a linker,and its carboxyl group underwent esterification with the 2’-OH of PTX to synthesize paclitaxel-levulinic acid(PTX-LEA),and its carbonyl group reacted with the hydrazine bond of 6-Maleimidohexanehydrazide Trifluoroacetate(EMCH TFA)to form acylhydrazone bond,and finally PTX-EMCH was obtained.Finally,the structure of the synthesized PTX small molecule prodrug was characterized by 1H-NMR and MS,and its purity was verified by HPLC.2.Preparation and characterization of albumin bound PTX prodrugsThe binding ability of PTX prodrug to HSA was investigated.Abraxane? as a comparative preparation,the physicochemical properties of albumin conjugates(drug loading,particle size,potential,surface morphology,stability),changes of albumin spatial conformation,and the drug release behavior of albumin conjugates in vitro were investigated.Surface plasmon resonance(SPR)measured the affinity of PTX-SA to HSA was stronger than that of PTX.Micro Total Mercapto Assay Kit measured that there was 0.77±0.05 free sulfhydryl in HSA,which could realized covalent binding with PTX-EMCH.The successful synthesis of PTX-EMCH-HS A was characterized by IR chromatography.Based on the drug loading and encapsulation efficiency,the ratio of HSA to PTX-SA or PTX-ODDA was screened to be 1:5.The drug loadings of prodrugs were PTX-SA 5.57%±0.22%and PTX-ODDA 7.56%±0.3%,respectively.When the ratio HSA:PTX-EMCH=1:2,the drug loading of PTX-EMCH was 1.37%±0.1%,and the drug loading molar ratio of PTX-EMCH to HSA was 0.80±0.06.The particle size and Zeta potential of the preparations were detected by Malvern particle size analyzer.Surface morphology of the preparations were determined by transmission electron microscope.The particle sizes of PTXODDA/HSA,PTX-SA/HSA and Abraxane? was similar,and the PDI was less than 0.3.The average particle size of PTX-EMCH-HS A was 19.51 nm,the particle size distribution was wider,and the PDI was 0.45,which was closer to albumin state.The Zeta potentials of PTXODDA/HSA,PTX-SA/HSA and Abraxane? were similar,and the potential of PTX-EMCHHSA was closer to that of HSA.The results of transmission electron microscopy were basically consistent with the above particle size results.The stability results showed that the particle size of PTX-ODDA/HSA,PTX-SA/HSA and PTX-EMCH-HSA showed no significant change after being placed at 25℃ and 4℃ for 96 h,indicating that the preparation was relatively stable,while the particle size of Abraxane? increased after being placed at 4℃ for 96 h,indicating that its stability was poor at 4℃.The CD results showed that the secondary structure of albumin in the preparation was well preserved.The in vitro release results showed that PTX-EMCH-HSA exhibited acid sensitivity in releasing PTX-LEA in pH 5.5 PBS buffer(containing 0.5%Tween 80).The release behavior of Abraxane? in pH 5.5 and pH 7.4 buffers was consistent.The ester bonds of PTX-ODDA and PTX-SA were relatively stable,and the release of PTX was less than 20%in pH 5.5 and pH 7.4 buffers.3.In vitro antitumor evaluation of albumin bound PTX prodrugsB16F10 cells and MCF-7 cells were selected as cell models,and the cytotoxicity of each free drug and albumin preparation was investigated by MTT method.Fluorescence microscopy and flow cytometry were used to investigate the uptake behavior of the conjugates by the two cells.The active targeting of HSA was explored.The ability of albumin conjugates to induce apoptosis was verified using AnnexinV-FITC/PI apoptosis kit.A cell cycle detection kit was used to detect cell cycle arresting effect of the preparation.The experimental results showed that albumin preparations had higher cytotoxicity under certain drug concentrations.The in vitro uptake of albumin preparations was higher than that of free C6,and HSA showed active targeting effect in MCF-7 cells,but less obvious in B16F10 cells.PTX-EMCH-HSA and Abraxane? had similar apoptosis-inducing effects on both cells.PTX-ODDA/HSA and PTX-SA/HSA showed a higher apoptotic effect in B16F10 and a lower apoptotic effect than Abraxane? in MCF-7.The results of cell cycle experiments showed that albumin preparations could arrest the cell cycle in G2/M phase,and PTX-EMCH-HSA and Abraxane? had stronger effect on cell arrest than PTX-ODDA/HSA and PTX-SA/HSA.4.In vivo antitumor activity evaluation of albumin bound PTX prodrugsA Kunming mice model of B16F10 melanoma was established.The formulation was administered by tail vein injection.Dir was encapsulated in albumin conjugates as a fluorescent dye,resulting in Dir-modified formulations Dir@PTX-SA/HSA,Dir@PTX-ODDA/HSA and Dir@PTX-EMCH-HSA.In vivo and in vitro tissues were imaged by animal in vivo imaging technology to evaluate the targeting effect and distribution of albumin conjugates in vivo.The in vivo antitumor effects of PTX-SA/HSA,PTX-ODDA/HSA and PTX-EMCH-HSA were evaluated with Taxol and Abraxane? as comparative preparations.The antitumor effect and safety of the preparation were investigated by using tumor volume and body weight changes as indicators.After treatment,each organ and tumor of the mice were dissected for H&E staining,and the toxicity of the preparation to each organ and the destructive effect on the tumor were observed.The results of in vivo imaging showed that,compared with free Dir,the three albumin preparations could target the tumor site in a passive or active manner to achieve tumor targeting.The drug treatment groups had better antitumor efficacy,and the antitumor efficacy of albumin formulation group was stronger than that of Taxol.At a certain dose,PTX-EMCH-HSA and Abraxane? had similar antitumor effects,while PTX-ODDA/HSA and PTX-SA/HSA had slightly weaker effects.The mice in the Taxol-treated group lost a slight weight,while the weight of the mice treated with albumin formulation remained stable,indicating that the preparation with albumin as a carrier had less toxic and side effects.The results of H&E staining showed that the administration group did not show obvious organ toxicity,but showed the destructive effect on tumor tissue. |
