| Nano drugs have great application potential in anti-tumor.However,the immunogenicity of the carriers used in nano drugs,the complexity of structural design,the uncontrollability of quality caused by the uncertainty of composition,and the low efficiency of nano drug delivery in vivo seriously restrict the efficacy of nano drugs and the possibility of clinical transformation,so that the development of nano drugs has entered a bottleneck period.How to efficiently transport the nanodrugs into tumors is an urgent problem to be solved.Particle size,as an important parameter,plays a key role in the in vivo delivery of nanodrugs.In order to solve the above problems,a zwitterionic EK9 peptide(KEKDCDEKE),coupling with chemotherapy drug doxorubicin(DOX)through acid-sensitive acyl hydrazone bond,was designed in this work.Then above conjugate self-assembled into nano-micelles with different particle size.The drug delivery processes and anti-tumor efficacy were studied.The main contents are described as follows:(1)Preparation and particle size control of drug-loaded micelles.The selfassembled monomer EK9-BMPH-DOX was synthesized and characterized by 1H NMR and FT-IR spectra.The EK9-BMPH-DOX dissolved in deionized water was added drop by drop to continuously stirred aqueous solution at pH 9 to self-assemble into the micelles.A series of drug-loaded micelles with different particle size were prepared by controlling the assembly concentration of EK9-BMPH-DOX.Three drugloaded micelles with different particle size,EK9D-30(30 nm),EK9D-60(60 nm)and EK9D-90(90 nm),were selected for the follow-up study.(2)Study on circulation of drug-loaded micelles.The critical micelle concentrations of EK9D-30,EK9D-60 and EK9D-90 were determined,and smaller critical micelle concentration could ensure the structural stability of the drug-loaded micelles.The anti-nonspecific adsorption performance of EK9 peptide-based drugloaded micelles was evaluated by detecting the change of particle size of the micelles in PBS,bovine serum protein and human fibrinogen solution with time.The antiimmune system recognition and clearance ability of the drug-loaded micelles was detected by co-culture with macrophages RAW 264.7.These properties play key roles in long-acting circulation of drug-loaded micelles in vivo.Besides,through drug clearance experiments in plasma,it was found that the long-acting circulation of drugloaded micelles was positively correlated with their particle size.(3)Study on tumor cell internalization and drug release of drug-loaded micelles.Zeta potential characterization results showed that the three EK9 peptide-based drugloaded micelles had similar isoelectric points,which were all about 6.6,close to the pH value of tumor microenvironment.In vitro drug release experiments results showed that the drug release behavior of EK9 peptide-based drug-loaded micelles was closely related to the particle size and external environment pH values.By investigating the endocytosis of three EK9 peptide-based drug-loaded micelles in glioma cells U87 at pH 6.7/pH 7.4,the ability of the nanodrugs entering cells and the nucleus,as well as the pathways of endocytosis,it was found that the ability of three drug-loaded micelles entering the cells and the nucleus was not only pH-dependent,but also closely related to the particle size.Smaller particle size was conducive to the endocytosis of nanodrugs,the release of DOX and its entry into the nucleus,which was consistent with in vitro drug release experiments results.The endocytosis pathways of EK9 peptide-based nanodrugs mainly depended on the particle size.(4)Study on accumulation and penetration of drug-loaded micelles at tumor sites.Fluorescent imaging system and tissue homogenization method were used to investigate the accumulation and permeability of three EK9 peptide-based drugloaded micelles in tumors,and to analyze the effects of particle size on the accumulation and permeability of the EK9 peptide-based drug-loaded micelles in tumor sites.The results showed that the smallest particle EK9D-30 had good tumor targeting ability,but weak retention ability.The retention ability of EK9D-90 in tumor was strong,but its penetration in tumors was weak.EK9D-60 could combine the advantages of the two,and had excellent behaviors of accumulation and retention in tumors.(5)Tumor inhibition efficacy of drug-loaded micelles.The ability of three EK9 peptide-based micelles to inhibit the proliferation of tumor cells under different pH conditions in vitro was measured,and the tumor inhibition experiments of EK9D-30 and EK9D-60 with stronger tumor targeting ability were carried out in vivo.The results in vitro and vivo showed that EK9 peptide-based drug-loaded micelles could effectively inhibit tumor growth under condition of reduced cytotoxicity to normal tissues,and EK9D-60 with intermediate particle size had the best tumor inhibition efficacy.Histopathological analysis showed that EK9 peptide-based drug-loaded micelles could induce the initiation of tumor cell apoptosis after reaching the tumor site,and had no toxic effects on other organs in nude mice.This work supports the design of zwitterionic peptide-based nanodrugs with simple structure,controllable quality and convenient clinical transformation. |
