Structure Modification Of Oligonucleotides And Peptides For Oral Delivery

Since biotechnology development over the past several decades,a growing number of biomacromolecules including peptides,proteins and nucleic acids,have been found potential and made available as therapeutic agents.However,biomacromolecules appear poor intestinal absorption,due to relatively high molecule weight and hydrophilic of biomacromolecules and their susceptibility to acid and enzymatic hydrolysis in the stomach and gastrointestinal(GI)Tract.Therefore,they are often administrated by injection route,oral delivery of them is still challenging.Structure modification is one important strategy to improve the effect of biomacromolecules via oral delivery.In our study,we focus on two kinds of specific small molecules.One is lipids,which are natural and amphiphilic,and they can naturally be absorbed in the gastrointestinal(GI)Tract,here we choose cholesterol,methyl cholate and four kinds of fatty acids with different chain length(stearic acid,palmitic acid,myristic acid and lauric acid).The other is dipeptides,as one of important substrates for Peptide Transporter 1(PEPT1),they can be recognized and transported by PEPT1 in the intestine.In previous study of our lab,a library of dipeptides was established based on the PEPT1.Dipeptides with high PEPT1 affinity were picked up after screening,from which,we choose Phe-Ala(AF),Pro-Leu(LP),Arg-Ile(IR)and Val-Lys(KV)in our study.CpG oligonucleotide(CpG ODN)representing oligonucleotides and Exenatide representing peptides are chosen to be investigated in our study.CpG ODN containing unmethylated CpG motif is toll-like receptor 9(TLR9)agonists,which can activate immune system,induce and enhance both the innate immune and adaptive immune responses.Therefore,they are widely studied in the treatment of infectious diseases,allergic disease and cancer.CpG 1826 was utilized in our study,which stimulates IL-6 expression of B cells and antibody production.Exenatide is a synthetic GLP-1(glucagon like peptide 1)agonist,consisting of 39 amino acids,which could lower blood glucose level similar with natural GLP-1.Exenatide is more stable than GLP-1 and utilized as a treatment for type 2 diabetes.These small molecule modifications for oral delivery were firstly investigated on CpG ODN.In the chapter 2,azide modifications of cholesterol,methyl cholate and four kinds of fatty acids were achieved,through different reaction routes.Then CuAAC reactions were performed between azide modified lipids and alkyne modified CpG ODN,six kinds of lipid modified CpG ODNs were obtained.In the chapter 3,in vitro activity and in vivo effect of lipid modified CpG ODNs and other four kinds of dipeptide modified CpG ODNs were evaluated.CpG ODNs were incubated with spleen cells from mice,then IL-6 levels in the supernate were measured to indicate the in vitro activity.The results suggested that lipid modifications slightly affected the activity but not significantly,except methyl cholate,and dipeptide modifications almost did not influence the activity of CpG ODN.Then the mice were immuned with antigen Ovalbumin(OVA)by intraperitoneal injection,together with oral delivery of CpG ODNs.Serum antibody levels were measured to determine the effect of modifications.Among the six lipid modifications,cholesterol,methyl cholate and myristic acid modifications did not improve the effect of CpG ODN via oral delivery,palmitic acid modification improved the effect to some degree but not significantly,stearic acid and lauric acid modifications significantly improved the effect,the latter is better.Interestingly,when we compared the effect of stearic acid modified CpG ODN with CpG ODN via tail vein injection,stearic acid modification also significantly improved the antibody expression,which may partially contribute to the improved effect via oral delivery.In addition,lauric acid modified CpG ODN was tested in CT-26 tumor-bearing mice,despite IL-6 expression level of spleen cells was enhanced,the tumor growth was not inhibited.The results suggest that CpG ODN monotherapy for treatment of tumor is difficulty.Among four dipeptide modifications,AF and LP modifications are more capable to significantly improve the effect of CpG ODN via oral delivery.In conclusion,four small molecules(stearic acid,lauric acid,AF and LP)belonging to two different types were found to improve the effect of CpG ODN via oral delivery,through in vitro and in vivo evalution.On the basis of the results of modified CpG ODN,whether small molecule modifications could improve the effect of Exenatide via oral delivery was investigated next.In consideration of Exenatide belonging to peptides,dipeptide modifications may maximally retain activity of Exenatide.Furthermore,dipeptide modifications can be directly introduced during solid phase synthesis of Exenatide,avoiding the separation and purification of other subsequent modifications.Therefore,AF and LP modifications were chosen,two kinds of dipeptide-substitution Exenatide were designed and obtained as HA-39 and HL-39 respectively.In vitro activity and in vivo effect of HA-39 and HL-39 were evaluated.GLP-1 agonist activity test showed that both AF and LP modifications affected the activity to some degree,the former affected more strongly.Then HA-39 and HL-39 were tested in mice with high blood glucose level.LP modification significantly improved the effect of Exenatide via oral delivery,but AF modification did not improve the effect.Further,the impact of HL-39 dose on the effect was surveyed.The results showed that the effect of high and middle dose were significantly better than that of low dose,but the effect of high dose was slightly better than that of middle dose,possibly due to the self-regulation of blood glucose.Gly-Sar known as PEPT1 substrate was employed to perform competition study.The results showed that Gly-Sar significantly reduced the effect of HL-39 via oral delivery,indicating PEPT1 participated in the intestinal absorption of HL-39.Through single-pass intestinal perfusion studies in SD rats,HL-39 was found to have improved absorption rate constant(K a)and effective permeability coefficients(Peff)compared with Exenatide,demonstrating that LP modification enhanced the intestinal absorption of Exenatide.In summary,lipid(six kinds)and dipeptide(four kinds)modified CpG ODNs were evaluated in vitro and in vivo.Stearic acid,lauric acid,AF and LP modifications were found to significantly improve the effect of CpG ODN via oral delivery.Further,AF and LP were chosen to modify Exenatide,in vivo study showed LP modificationsignificantly improved the effect of Exenatide via oral delivery.Thereby,we conclude that specific small molecule modification such as lipids and dipeptides could improve the effect of oligonucleotides and peptides via oral delivery,which provides a newchoice for oral delivery of biomarcomolecules.