Preparation Of Porcine Glucagon-Like Peptide-2Microspheres And Its Intestinal Therapeutic Effects In Mice And Piglet

Porcine glucagon-like peptide-2(pGLP-2) can promote the growth of mucosa and repair the damage of intestinal specifically. It provides a new forward to ameliorate piglet weaning stress. But its rapidly degration by the proteolytic enzyme dipeptidyl peptidase-IV (DPP-IV) limits its further application. So it is very important to investigate the prolong half-time strategies.This experiment encapsulated porcine glucagon-like peptide-2(pGLP-2) by poly (D,L-lactic-co-glycolic acid)(PLGA) to form PLGA/pGLP-2microsphere drug delivery system. The drug delivery system prolonged the half-time of pGLP-2and showed great intestinal therapeutic effects in mice and piglet.Experiment1:preparation of pGLP-2/PLGA microsphere drug delivery system.This study compared the difference between W/O/W method and S/O/W method, investigated the effects of concentration of polyvinyl alcohol (PVA), concentration of PLGA, stir rate, theory drug loading, the volume ratio between dichloromethane and dimethyl sulfoxide on microsphere characteristics. The effects of addition of polyethylene glycol6000(PEG6000) in organic phase and addition of NaCl in aqueous phase on the drug release were also studied in this experiment. The results showed:(1) The EE was similar between microsphere prepared by W/O/W method and S/O/W method. But microsphere prepared by W/O/W showed larger size, higher burst release, larger wide particle size distribution and much more hole in surface compared to microsphere prepared by S/O/W method.(2) Prepared microsphere by S/O/W method and optimized theory drug loading as1%, stiring rate as2000rpm, PVA concentration as2%, PLGA concentration as100mg/mL, the ratio of DCM and DMSO as40. Under the above process, EE reached72%, burst release decreased to16.62%and particle size remain at37.5μm.(3) Addition of10%PEG increased the particle size and accelerated the drug release, but showed a high burst release. Addition of5%NaCl in aqueous phase decreased the particle size and burst release, but showed limited effects on the drug release. When addition of both PEG and NaCl, the microsphere exhibited accelerated drug release and showed litter increase in burst release.Brief summary:pGLP-2/PLGA microsphere was prepared with72%EE,16.62%burst release,37.5μm diameter after process optimization. The addition of NaCl and PEG accelerate the drug release and make microsphere released stablely at least9days in vitro.Experiment2:Therapeutics effects of pGLP-2/PLGA microsphere in murine model of ulcerative colitisThirty health BALB/C mice were randomly allocated to Control Group, Dextran sulfate sodium Group (DSS Group) and Microsphere Group (MS Group). Mice in the DSS treatment groups received4%DSS for successive9days and were intraperitoneally injected with10mg pGLP-2microspheres once on day0(dissolved in0.3mL saline, MS Group), and0.3mL saline once on day0(DSS Group). Mice in control group received normal water and were intraperitoneally injected with0.3mL saline on day0(Control Group). At day10, all mice were sacrificed after weighing. The length and weight of small intestinal and colon were measured immediately. The blood was collected for measuring DAO, duodenum and colon were collected for tissue section and isolation RNA. The results showed:(1) Mice treated with DSS significantly decreased body weight, colonic length, the height of villus and the ratio of villus and crypt in colon compared to Control Group. One injection of pGLP-2microsphere significantly reversed body weight loss, increased colonic length and small bowel weight compared to DSS Group. Colonic weight in mice of MS Group was significantly increased when compared to Control Group. (2) Treatment of DSS significantly increased the inflammation. One injection of pGLP-2microsphere markedly decreased the colonic damage score and the expression of IFN-y, TNF-a, IL-6, IL-10, and the expression of IFN-y as compared to DSS Group.(3) Treatment of DSS significantly damage the intestinal barrier function. One injection of pGLP-2microsphere markedly decreased plasma DAO, colonic MPO and the expression of Claudin-1in colon, increased the expression of Occludin and ZO-1in colon as compared to DSS Group. Brief summary:one injection of pGLP-2/PLGA microsphere reversed the body weight loss, increased colonic length and barrier function, decreased inflammation in mice with acute DSS colitis.Experiment3:therapeutics effects of pGLP-2/PLGA microsphere in LPS-challenged pigletsEighteen weaning piglets were randomly allocated to Control Group, Lipopolysaccharide Group (LPS Group), MS Group.0.2mL saline was intraperitoneally injected to mice of Control Group on day1and day8. LPS Group received intraperitoneally injection of same dose saline on day1and LPS at dose of100μg/kg body weight on day8. MS Group received intraperitoneally injection of100mg pGLP-2microsphere prepared with theory drug loading as2%on day1and LPS at dose of100μg/kg body weight on day8. Three hours after LPS injection, all piglets were sacrificed. Plasma was collected for measuring of IL-6, DAO, et al. Duodenum, jejunum and ileum was collected for tissue section and isolation RNA. The results showed:(1) Injection of LPS significantly decreased villus height and villus/cyrpt ratio in duodenum and jejunum as compared to Control Group. MS Group markedly increased villus/cyrpt ratio in duodenum compared to LPS Group. The villus height and villus/cyrpt ratio of ileum in MS Group were significantly increased when compared to both LPS Group and Control Group.(2) Injection of LPS significantly increased IL-6, IgA, DAO in plasma in LPS Group but there were no difference with MS Group.(3) LPS Group significantly decreased the expression of Occludin and increased the expression of Claudin-1, IL-8, TNF-α in jejunum as compared to Control Group. MS Group markedly increased the expression of ZO-1in duodenum and decreased the expression of IL-6as compared to LPS Group.Brief summary:one injection of pGLP-2/PLGA microsphere significantly improved intestinal barrier function and ameliorated the inflammation induced by LPS.Conclusion:pGLP-2/PLGA microsphere was prepared with72%EE,16.62%burst release,37.5μm diameter by S/O/W method. It released stablely at least9days in vitro. One injection of microsphere ameliorated the inflammation induced by DSS in mice and by LPS in weaning piglets.