Study On Yeast Spores As Natural Carrier For Drug Delivery And Oral Vaccine

Yeast is a single-celled fungus,which plays an important role in genetic engineering and cell cycle studies.Recently,scientists try to use it for oral drug delivery.Sporulation happens when yeast cells face nutrient limitation.Spore wall has four layers,they are mannan,β-glucan,chitosan and dityrosine layer.Previous studies suggested that yeast spores could be used to absorb heavy metals from solution or serve as carriers for immobilization of enzymes.Wild-type spore wall can protect spore from low pH and enzymatic degradation.Previous work also shown that yeast spores could hold secretory proteins in the periplasmic space of the spore wall because their outer layers work as a diffusion barrier.Knocking out the essential genes which are responsible for producing the related wall of spores will get spores with different outer layers.For example,knocking out DIT1 gene will get deficient spores(dit1Δspores),which cannot form the outer walls of dityrosine.In this study,whether yeast spores could be used as natural carrier for drug delivery was examined.Synthetic short,single-stranded oligodeoxynucleotides(ODN)that contain CpG motifs can be internalized by immune cells,which interact with Toll-Like receptor 9(TLR9)and stimulate the innate and acquired immune responses.CpG ODN is widely used as immunologic adjuvant.However,the nuclease degradation and low cellular uptake of CpG ODN led to low bioavailability,which extremely limited its applications in immunotherapy.This research firstly checked the ability of spores as carrier for CpG ODN delivery.The results showed that negatively charged CpG ODN could be efficiently loaded onto the surface of positively charged dit1Δspores through electrostatic interactions.In addition,dit1Δspores had good biocompatibility.Dit1Δ/CpG ODN complex could stimulated more amount of cytokines from RAW264.7 macrophages compared with free CpG ODN.In conclusion,dit1Δspores could efficiently deliver CpG ODN into RAW264.7 and stimulate higher immunostimulatory effect.Oral administration of GLP-1(Glucagon-like peptide-1)has attracted many attentions in the treatment of diabetes.GLP-1 is a short peptide secreted by L cells.GLP-1 can increase insulin release and reduce blood sugar.However,one of the major limitations of native GLP-1 is its rapid degradation by dipeptidyl peptidase 4(DPP-4),leading to its short half-life in vivo.Yeast spores were used as carrier to protect GLP-1 from being digested by gastric and intestine juice.The results showed that GLP-1 was successfully expressed in yeast spores.After pre-treated with simulated gastric and intestine juice,spores with GLP-1 obviously enhanced insulin secretion from MIN6 cells compared with GLP-1.Using yeast spores as carrier,the anti-protease degradation ability and insulinotropic activity of GLP-1 were significantly enhanced.In summary,spores could enhance the activity of both peptide drug GLP-1 and nucleic acid drug CpG ODN.Our study will lay a foundation for further development of oral drugs.