Feather Keratin Based Antitumor-drug Delivery System:Design And In Vitro Evaluation

In recent years,the differences between cancer cell microenvironment and normal cell microenvironment have been used to design antitumor drug delivery systems（DDSs）in tumor chemotherapy,in order to improve the safety and therapeutic effect of chemotherapeutic drugs.Protein has attracted more and more interests as carrier materials,owing to its good biocompatibility,good biodegradability,and very low cytotoxicity.Here,feather keratin with desired thiol content was selected to fabricate anti-tumor DDSs with excellent tumor microenvironment triggered DOX delivery.First,keratin was used as a multifunctional cross-linking agent to fabricate a series of DOX-loaded keratin-hyaluronic acid microgels（DOX@C-FK/HA）:i)the amino groups in keratin combined with the carboxyl groups in hyaluronic acid（HA）by electrostatic interaction to achieve ionic gelation;ii)the thiol groups in keratin was then oxidized to form covalently crosslinked structure via disulfide bonds,endowing reduction stimuli responsiveness while improving the stability of the microgel.The results showed that the particle size of the microgel which was crosslinked after the drug loading was significantly smaller than the microgel that drug loading after crosslinked.Increasing the hyaluronic acid content,both the hydrodynamic diameter（D_h）and drug loading capacity（DLC）of the DOX@C-FK/HA decreased.Taking the optimized one,DOX@C-FK/HA₅-1,as an example,it could release about 60%of DOX in 60 h in pH 5.0+10 mM GSH solution mimicking the tumor intracellular microenvironment.The drug leakage was 24%in pH 7.4+10μM GSH solution mimicking the normal human body fluid.In view of the high drug leakage caused by the lower isoelectric point（pI）of keratin,the carboxyl groups in keratin was consumed by the amidation reaction with mPEG-NH₂.During the drug loading via hydrogen bonding,electrostatic interaction and hydrophobic interaction of doxorubicin with keratin,the complex self-assembled into DOX/Ker-PEG micelles.Then the core-crosslinked micelles（CCMs）pH and reduced dual-responsive were successfully synthesized by oxidative crosslinking of the thiol groups in keratin.The DOX/Ker-PEG CCMs released about 60%of DOX in85 h in pH 5.0+10 mM GSH.The drug leakage was 14%in pH 7.4+10μM GSH.MTT assay revealed that DOX/Ker-PEG CCMs have good cytotoxicity and can effectively inhibit the growth of HepG2 cells.Thirdly,the drug-protein conjugates via acid-labile hydrazone and reduction-cleavable disulfide linkages in series were designed as antitumor prodrug by modifying PEGylated keratin（PK）with a DOX derivative（M-Hy-D）.The polymer prodrug nanoparticles PK-SS-Hy-D NPs and C-PK/-SS-Hy-D NPs with significantly different composition were obtained in different solvents.Both polymer prodrug nanoparticles showed pH and reduction dual-triggered release.The PK-SS-Hy-D NPs with a drug content of 29.4%prepared in mixed solvent could release 42.8%of DOX in 107 h in pH 5.0+10 mM GSH,while the drug leakage was only 5.5%in pH 7.4+10μM GSH.Due to solubility,the C-PK/-SS-Hy-D NPs prepared in pH 7.4 PBS possessed a higher drug content of 45.8%and a faster pH and reduction dual-triggered DOX release,owing to a higher content of D-Hy-SS-Hy-D encapsulated in the polymer prodrug nanoparticles.Finally,drug-conjugation induced self-assembly technique was used to fabricate reductionresponsivePK-SS-Dmicelles,byconjugatingDOXonto mercaptopropionic acid-modified PK.The PK-SS-D micelles possessed a drug content of 20%and a D_h of 175 nm.The cumulative drug release from the PK-SS-D micelles reached 52%in a sustained release mode in pH 5.0+10 mM GSH within 10days.The drug leakage was 17%in the simulated normal body fluid.