Keratin composite nanofibrous anti-tumor drug delivery system

Abstract

Tissue engineering is an emerging interdisciplinary field that applies the principles of biology and engineering to the development of viable substitutes that restore, maintain, or improve the function of human tissues and organs. The crucial step in the whole tissue engineering process is the selection of proper scaffolds. The present study is to investigate a keratin composite biocompatible material for scaffolds with certain functional purpose, e.g., biodegradable implant, drug delivery, anti-tumor therapy. The present work started with transforming wool into the functional protein biomaterials. In order to achieve this objective, isoelectric precipitation process was introduced to produce the targeted bio-functional keratin polypeptides. Wool fibers were hydrolyzed and adjusted to predetermined pH values. The results suggested that keratin polypeptides with different amino acids compositions could be tailored from wool hydrolyzed solution. Keratin particles at nanometer-scale were collected successfully. The chemical structure of keratin polypeptides endured the same as the pristine wool and the crystal structure of keratin polypeptides became more amorphous. Secondly, the prepared keratin polypeptides were applied into biofunctional tissue engineering scaffolds system. In order to achieve the keratin composite nanofibers, keratin polypeptides were blended with Poly (L-lactic acid) (PLLA) organic solution with an "ethanol replacement" process and electrospun into nanofibers. The results indicated that the nanofibrous composite membrane possessed ultrafine and homogenous structure without keratin aggregation. The Fourier transform infrared spectrometry (FT-IR) revealed the existence of keratin in the composite fibers. The thermal property of the composite membrane was more stable than pure keratin. XRD spectra revealed a crystal transformation of keratin after the process of electrospinning. The biocompatibility was examined by seeding cells on the surface of the composite nanofibers. Compared to pure PLLA nanofibers, keratin/PLLA fibrous membrane showed an enhanced impact on cells viability and affinity. Thirdly, in order to investigate both the chemical and physical change during the in vitro degradation process, the degradability of keratin/PLLA nanofibers was discussed through several aspects. The chemical change was mainly reflected by the loss of keratin from the nanofibers as a function of degradation time. The physical change was mainly indicated by the decrease of thermal-stability of keratin/PLLA nanofibers. The rate of degradation was positively related to the amount of keratin peptides added into the composite nanofibers comparing with pure PLLA nanofibers. Fourthly, in order to explore the drug delivery profile of keratin/PLLA nanofibers as a controlled release scaffolds system, 5-fluorouracil (5-FU) was loaded into the nanofibers as a drug model. The results proved the controlled release effect of 5-FU/keratin/PLLA nanofibers and the release periods of 5-FU were prolonged to 120 hours. The X-ray diffraction (XRD)analysis revealed that 5-FU dispersed uniformly within the nanofibers at a molecular level and electro-charges interactions were observed between 5-FU and keratin. The chemical characterization indicated 5-FU kept its original chemical structure within the nanofibers. Fifthly, in order to investigate the pH-sensitivity of 5-FU/keratin/PLLA nanofibers, two pH-valued environments, i.e. pH 6.0 and pH 7.4 were introduced. A full-factorial design with two factors at two-levels was employed to optimize fiber fabrication factors to achieve desirable controlled release performance of the composite nanofibers in different pH environments. The results indicated that electrospinning voltage of 15 KV and weight ratio of keratin at 10 wt. % can enhance the pH-sensitivity of 5-FU/keratin/PLLA nanofibers. HCT-116 cells line was utilized to examine the anti-tumor effect 5-FU/keratin/PLLA nanofibers. In 120 hours, 5-FU/keratin/PLLA significantly inhibited the proliferation rate of tumor cells comparing to 5-FU/PLLA (p<0.01). Finally, to address the issue of infection during antitumor operation, a regenerated antimicrobial peptide (AMP) Attacin2 was introduced into the above composite fibers. The results indicated that after adding the antimicrobial peptides into the membrane, the nanofibrous scaffolds demonstrated antibacterial function. Further, the antitumor effect could be observed by adding Attacin2 into the keratin/PLLA nanofibers, especially after 24 hours observation. The results verified the feasibility of combining both chemical antitumor drugs and biological antimicrobial peptides together, suggesting the further direction of applying nanofibers in the realm of antitumor therapy. In conclusion, the keratin composite nanofibrous anti-tumor drug delivery system including keratin/PLLA, 5-FU/keratin/PLLA, Attacin2/keratin/PLLA and Attacin2/5-FU/keratin/PLLA have been developed successfully by electrospinning technology. The composite controlled release system can be utilized for potential biomedical application, e.g., anti-tumor therapy.