Preparation of Electrospun Colon-specific Delivery System for Prebiotics and Bioactive Compound and Its Properties

Author:Wen Peng

Supervisor:zong min hua wu hong


Degree Year:2019





Bioactive compounds(BCs),such as proteins,polyphenols and flavonoids,possess many bioactivities,including anti-oxidant and anti-cancer activities,etc.However,the instability of BCs during food processing(like high temperature or organic solvent etc.)and low bioavailability when exposed to upper gastrointestinal tract(GIT)or underwent first-pass metabolism significantly compromised the envisioned benefits,and thus limiting their applications for colon cancer disease.Therefore,an effective colon-specific delivery system that can preserve bioactivity and achieve colon targeted release of BCs is desiderated to improve their stability and bioavailability.Compared to the traditional encapsulation techniques,electrospinning,a simple,mild and cost-effective nano-technology,has attracted great attention for the encapsulation of BCs.The key advantage of electrospinning process is the absence of heat,which is important for preserving the structure and thus achieving high encapsulation efficacy of the compounds.In particular,the core-sheath nanofibers can be fabricated by coaxial electrospinning,which proffer considerable merits including protecting the fragile BCs from the stress of upper GIT and achieving colon-specific release by selecting the sutiable core/shell materials.Nevertheless,there is no study about preparing polysaccharieds-based colon-specific delivery system for encapsulation of BCs by electrospinning,moreover,the effects of prebiotics on the colon targeted behavior of BCs and their activities have not yet been reported.Consequently,in this study,bovine serum albumin(BSA)was employed as protein model,the feasibility of using coaxial electrospinning to construct colon targeted deivery system for BSA was first explored;then,the applicabilities of the obtaiend system for functional protein(phycocyanin,PC)and small molecule(quercetin,Q)were further studied;Finally,the anti-cancer activity of prebiotics and BCs(PC or Q)-loaded electrospun fiber mat(EFM)on colon cancer cells and the underlying mechanism of action studies were investigated.The main research contents and results are as follows:(1)Preparation and charaterization of BSA-loaded electrospun colon-specific delivery systemFirstly,the BSA-loaded chitosan nanoparticle(BCNP)was prepared and the optimal conditions were chitosan(CS)concentration 3 mg/mL,CS:sodium tripolyphosphate(TPP)4:1,pH 5.3.TEM image revealed that the single BCNP had a spherical structure and the particle size was around 20 nm.Then,the core-sheath structured BSA-loaded EFM with good morphology was fabricated by co-axial electrospinning using the above obtained BCNP and polyvinyl alcohol(PVA)as the core layer and sodium alginate(SA)and polyoxyethylene(PEO)as the shell layer.The results of fourier transform infrared spectroscopy(FTIR),X-ray diffraction and thermogravimetric analysis suggested that there are interactions between different components of SA/PEO and CS/PVA fiber mats.The results of in vitro release study showed that approximately 18%of BSA was released in simulated gastric fluid(SGF)and simulated intestinal fluid(SIF),while 75%of BSA was released in simulated colorectal fluid(SCF),indicating that the release behavior of BSA was in a colon-specific way.The release kinetics analysis showed that the release of BSA in SGF and SIF was controlled by fickian diffusion,while the release of BSA in SCF followed a case II mechanism.The results of FTIR and circular dichroism demonstrated that there are no significant changes on the secondary structure of the encapsulated BSA during the nanoparticle and electrospinning process(P>0.05).(2)Preparation and charaterization of prebiotics and PC-loaded electrospun colon-specific delivery systemBased on the aforementioned study,the effect of obtained system on the activity of functional protein was further studied.The prebiotics and PC-loaded EFM was fabricated by co-axial electrospinning by adding the prebiotics(galactooligosaccharide,GOS)into the core layer.About 15%of PC was released in SGF and SIF,while around 82%of PC could be released in SCF over 20 h.In particular,in comparison with PC-loaded EFM,faster release of PC was achieved for GOS and PC-loaded EFM,indicating that the enhanced colon-specific property was achieved by the addition of prebiotics,as well as the growth stimultion effect on probiotics.Release kinectics study showed that in SGF and SIF,the PC release followed a fickian diffusion mechanism,and the PC release in SCF involved a super Case II transport mechanism,in which the degradation of the polymer matrix was dominant.CCK-8 results indicated that the combination of GOS and PC exerts a significant anti-proliferative effect on HCT116 cells when compared with GOS or PC alone(P<0.05),and the effect was in a dose-and time-dependent manner,as the IC50 values were 22.31、17.12 and 11.63 mg/mL after 24,48 and 72 h of incubation,respectively.Moreover,the EFM exhibited favorable biocompatibility to normal intestinal CCC-HIE-2 cells.The current study suggests that the GOS and PC-loaded EFM can be used for the colon targeted delivery of bioactive protein.(3)Preparation and charaterization of prebiotics and Q-loaded electrospun colon-specific delivery systemTo further investigate the applicability of obtaiend system for small molecules,herein,the core-sheath structured GOS and Q-loaded EFM was also prepared.The antioxidant activity study suggested that Q was effectively encapsulated in the fiber mat,as the DPPH scavenging concentration of 50%(DC50)of the GOS and Q-loaded EFM was 522.13μg/mL.Results of in vitro release study showed that approximately 20%of Q was released in SGF and SIF,and 73%of Q was released SCF.Similarly,compared to Q-loaded EFM,a faster release rate of Q was achieved for GOS and Q-loaded EFM.Cell viability study also showed that the inhibitory effect of GOS and Q-loaded EFM on Caco-2 cells was increased with concentration and incubation time,and the calculated IC50 values after 24,48 and 72 h of incubation were 3.52,2.08 and 1.51 mg/mL,respectively.No significant cytotoxic effect was observed for EFM treated the normal CCC-HIE-2 cells.The functional GOS and Q-loaded EFM indicated a potential application as a colon-specific controlled delivery system for bioactive compounds in cancer prevention.(4)Study on the action mechanism of loaded prebiotics and BCs on the inhibition of colon cancer cellsThe effect of prebiotics and BCs(PC or Q)-loaded EFM on cell cycle,apoptosis and the relavant protein expression were investigated.The results showed that the GOS and BCs(PC or Q)-loaded EFM exerts their inhibition activities on colon cancer cells by blocking cell cell cycle at G0/G1 phase involving the inhibition of cyclin D1 and CDK4 and the up-regulation of p21 expression,and inducing cell apoptosis as well,which is relevant to the decrease of Bcl-2/Bax,activation of caspase 3 and release of cytochrome c,and there is synergistic effect between GOS and BCs.This study will provide the theoretical base and scientific methods for the colon-targeted delivery and utility of macromolecular and small molecule bioactive compounds,in addition,it will offer the reference in developing electrospun targeted release system of other similar functional compounds.