Biomedical Materials Based On Collagen/Soy Protein Via Self-assembly And Their Structure And Properties

Yak skin collagen(UPYC)and soy protein isolate(SPI)are both natural biomacromolecule materials with abundant sources and low price.Among them,Yaks growing in high altitude,anoxic,pollution-free and extremely cold areas give yak skin collagen good antioxidant activity,which creates conditions for the safe use of yak skin collagen in organism.Arginine and glutamic,which are rich in soy protein isolate,can repair damaged genes and accelerate wound healing.However,the rapid degradation and poor stability of collagen,and the sensitivity of soy protein isolate to acid environment limit their application in biomedical materials.Therefore,a UPYC/SPI self-assembled biomaterial with stability in weak acid environment was developed via self-assembly.The research expands the application range of hydrophobic drug carrier and wound dressing film.It provides a theoretical basis for the development of functional materials of heterologous proteins.In this paper,UPYC and SPI were used as raw materials to self-assemble natural polymer micelles via hydrogen bonding and electrostatic interactions.Four fixation methods of amide bond crosslinking,radical coupling,O-benzoquinone addition crosslinking and disulfide bond crosslinking were investigated indetail.Then drug-loading properties of the UPYC/SPI self-assembled nanomicelles were studied using curcumin(CUR)as a drug model.Finally,the structure and properties of the UPYC/SPI self-assembled colloid films were researched.The feasibility of the UPYC/SPI self-assembled colloid films as wound dressing was discussed.The main content of the paper was as follows:Firstly,the new ultrasound-assisted lactic acid-pepsin hydrolysis method was used to extract UPYC from yak skin.In the extraction process,strong mechanical vibration wave and "cavitation effect" destoried the interaction between UPYC and its matrix.At the same time,the yak skin collagen had good quality.Lactic acid made yak skin collagen fibers loose enough and shorten the extraction time.Lactic acid had also certain antiseptic and bactericidal effect.Pepsin could remove the non-helical structure of yak skin collagen and reduce the risk of immune rejection of yak skin collagen.The optimum conditions of ultrasonic-assisted acid enzymatic method were as follows:At first,degreased yak hide was soaked in lactic acid solution with pH 2.0 for 24 h and control the liquor ratio of 15.Then,acid treated yak hide was soaked in 2.0%(dry hide)pepsin solution with pH 2.0 for 24 h and control the liquor ratio of 15 under pulsating ultrasound treatmented with 20 KHz ultrasonic power 200 W for 20 min.The extraction rate of UPYC could reach 82.33%.UPYC had the structural characteristics of type I collagen.The new ultrasound-assisted lactic acid-pepsin hydrolysis method shortened the extraction time of UPYC,which was economical and environmental friendly.Secondly,the novel UPYC/SPI spherical nanomicelles via self-assembly were developed.The UPYC/SPI spherical nanomicelles had three layers:the peptide chains of UPYC intertwined with the peptide chains of SPI,resulting in the repositioning hydrophobic groups on the peptide chains of UPYC and SPI to form the core,and the repositioning of hydrophilic groups to form the shell.There was a bright ring between the core and the outer layer,which might be active groups on the peptide chains of SPI and UPYC.These active groups anchored the peptide chains of UPYC around the peptide chains of SPI via noncovalent bond interactions.Both UPYC and SPI belong to amphiphilic random polyelectrolytes,the core-shell structure of the UPYC/SPI self-assembled nanomicelles was different from that of traditional block copolymers.Because hydrophobic and hydrophilic microdomains were dispersed randomly on the peptide chains of UPYC and SPI,the surface of UPYC/SPI self-assembled nanomicelles with core-shell structure was enriched with some hydrophilic regions and hydrophobic regions,which provided the surface of UPYC/SPI self-assembled nanomicelles with amphiphilic structure.During the formation of the UPYC/SPI nanomicelles,the UPYC peptides competed with the SPI peptides and were bound to the interior sites of the SPI nanomicelles.The peptides were bonded and in situ assembled by hydrogen bonding interactions among the active groups(carbonyl,amino and hydroxyl groups)of the SPI and UPYC,and electrostatic interactions between the partial amino groups of the collagen and carboxylic groups of the SPI.The hydrogen bonding and electrostatic interactions promoted the formation of stable UPYC/SPI nanomicelles.The formation of UPYC/SPI nanomicelles improved the stability of SPI in weak acidic environment.Thirdly,the structure of UPYC/SPI self-assembled nanomicelles was easily destroyed and lacked long-term stability.To solve these problems,four fixing strategies of amide bond cross-linking,radical cross-linking,O-benzoquinone addition cross-linking and disulfide bond cross-linking were proposed.The cross-linked UPYC/SPI self-assembled nanomicelles had good structural stability(dilution resistance,storage stability and thermal stability).Among them,the fixed effects of amide bond cross-linking and disulfide bond cross-linking were better than that of radical cross-linking and of O-benzoquinone addition cross-linking.Amide bond and disulfide bond were the key to maintain the structural stability of UPYC/SPI self-assembled micelles.Therefore,the UPYC/SPI self-assembled nanomicelles would be potential candidate as carriers of nutrients or drugs.Fourthly,the UPY C/SPI-CUR self-assembled nanocomposites with concentrated distribution of diameter and good dispersion were prepared using CUR as model drug and the UPYC/SPI self-assembled micelles fixed by amide bond cross-linking as carriers.The average diameter of UPYC/SPI-CUR nanocomposite was 377.5 nm,and its PDI was 0.229.The UPYC/SPI self-assembled nanomicelles could load some hydrophobic drug molecules in hydrophobic regions.The structure of CUR changed from crystalline to amorphous in the UPYC/SPI-CUR nanocomposites.Because the amorphous structure of CUR had larger specific heat capacity and internal energy,the UPYC/SPI nanomicelles could improve the water solubility,dispersion,oxidation resistance,stability and bioavailability of hydrophobic drug.The 24 h drug release rate reached to 90.38%in acid environment(SGF)and 55.58%in alkaline environment(SIF).The nanocomposites had strong performances of specific targeting and pH-responsive.Finally,the UPYC/SPI self-assembled colloid films with regular structure were prepared using the UPYC/SPI self-assembled micelles fixed by disulfide bond cross-linking as film-forming agent and glycerol as plasticizer.The compact cross-linked network structure was formed during the UPYC/SPI self-assembled micelles film-formation process.The compatibility between UPYC and SPI had been enhanced.The UPYC/SPI self-assembled colloid films had better mechanical properties,ultraviolet shielding qualities,visible light transmission qualities and thermal stability than that of blend film.The interactions between SPI and UPYC in the self-assembled colloid films were stronger than that of in the blend film,which could delay fragmentation in water and enhance the water resistance.The amphiphilic structure of UPYC/SPI self-assembled micelles promoted the affinity between the films and cells.There was no cytotoxicity to fibroblast L929 and osteoblast MT3C3.The self-assembled colloid films could promote cell adhesion,proliferation and differentiation.Therefore,the UPYC/SPI self-assembled colloid films represented potential candidates for wound dressing,masks,dressings for periodontal diseases and drug-loaded film,etc.In conclusion,the UPYC/SPI self-assembled biomedical materials had been developed.The self-assembled mechanism,structure and properties of new materials had been studied.New phenomena and mechanisms in the self-assembled process had been found.