| Due to low stability and large molecular weight,bioacromolecular drugs still face various difficulties by oral administration.And,protecting drugs from damage,improving their mucus penetration and intestinal absorption,and controlling drug release rate to reach the physiological concentration in the blood are recognized as the main challenges in this field.With the development of nanotechnoloty,the nano-delivery systems,which can improve drugs’stability and intestinal absorption,have been widely developed in the oral delivery of biomacromolecular.However,to the best of our knowledge,there are still several points unaddressed for the bioacromolecular loaded nano-delivery systems:1)current researches are mainly concerned with mucus penetration or absorption mechanism of nano-delivery systems,influence of drug release rate on its in vivo absorption is still unclear;2)the oral nano-delivery systems could improve drug absorption by prolonging their residence time at the absorption site(mucoadhesive)or improving particles penetration across mucus gel layer to reach even the underlying epithelium(mucopenetrating).However,there are few systematic comparative studies on the role of mucoadhesive and mucopenetrating in the oral delivery of bioacromolecular based on the same nano-delivery system,thus their individual contribution and absorption mechanmism need elucidation;3)mucus penetration and intestinal cells targeting are two main strategies to improve insulin oral delivery efficiency.However,few studies are available regarding the effectiveness of combining these two strategies into one nano-delivery system.Firstly,the effect of biomacromolecular release rate on its in vitro and in vivo behavior was investigated when drug stability and mucus penetration were firstly overcame.Taking insulin as the model drug,n-butylcyanoacrylate(BCA)as the carrier,insulin-loaded poly(n-butylcyanoacrylate)nanoparticles(Ins/PBCA NPs)were prepared by self-polymerization and the release rate of insulin was controlled by adjusting the mass ratio of Ins/BCA.The Ins/PBCA NPs exhibited good dilution stability(60-160 nm,8 h)and enzyme stability(>17%,2 h)in the gastrointestinal fluids.Moreover,the Ins/PBCA NPs showed pH-dependent release characteristics,and insulin was not released in the gastric fluid with controlled release in intestine and its release rate was in the order of Ins/BCA=2/5>Ins/BCA=2/10>Ins/BCA=2/15(w/w).And the Ins/PBCA NPs with different release rate all showed excellent mucus penetration(>60%,10 min)and strong gastrointestinal retention(～70%,12 h).In vivo studies revealed,the Ins/BCA=2/10 or Ins/BCA=2/15 NPs showed stronger hypoglycemic effect than Ins/BCA=2/5 NPs in diabetic rats.And The Ins/BCA=2/10 NPs exhibited fast hypoglycemic effect,while the Ins/BCA=2/15 NPs showed sustained hypoglycemic effect,and overall no significant difference in pharmacological availability was found between them.In conclusion,Ins/PBCA NPs could overcome the oral barriers of insulin delivery and the release rate of insulin had a certain effect on its hypoglycemic effect,which could provide desired hypoglycemic effect by controlling insulin release rate.Then,in order to better understand the role of mucusadhesive and mucopenetrating ability in the oral delivery of insulin,taking Ins/PBCA NPs(Ins/BCA=2/10,w/w)as the core,chitosan(CS)and alginate(Alg)as the shell,the mucoadhesive(Ins/PBCA/CS)and mucopenetrating(Ins/PBCA/CS/Alg)nanoparticles were designed herein and their in vitro and in vivo behaviors as well as absorption enhancing mechanisms were further elucidated and compared.The prepared Ins/PBCA/CS and Ins/PBCA/CS/Alg NPs showed small particle size(150-250 nm),high insulin entrapment efficiency(>99%)with opposite surface charge,which was 27.55 mV for Ins/PBCA/CS NPs and-20.55 mV for Ins/PBCA/CS/Alg NPs.It was demonstrated that both the Ins/PBCA/CS and Ins/PBCA/CS/Alg NPs showed good stability and similar release profile in the gastrointestinal fluid,and the Ins/PBCA/CS NPs presented an enrichment in mucus(70%,10 min)while most of the Ins/PBCA/CS/Alg NPs penetrated through the mucus(80%,10 min).Uptake mechanisms studies revealed clathrin-and caveolae-mediated endocytosis were mainly involved in the intestinal transport of Ins/PBCA/CS NPs while caveolae-mediated endocytosis and macropinocytosis contributed to the absorption of Ins/PBCA/CS/Alg NPs,and especially,M cells favored the absorption of Ins/PBCA/CS NPs.In vivo studies revealed that the Ins/PBCA/CS/Alg NPs has a fast onset of action while the Ins/PBCA/CS NPs presented a sustained hypoglycemic effect in diabetic rats,but overall no significant difference in pharmacological availability was found between the Ins/PBCA/CS/Alg(8.80%)and Ins/PBCA/CS NPs(8.44%).To sum up,due to the dual function of mucus and different absorption mechanisms in intestine,the mucoadhesive nanoparticles(Ins/PBCA/CS)designed herein had a comparable effect in enhancing insulin oral delivery compared to the mucopenetrating nanoparticles(Ins/PBCA/CS/Alg),which provided a reference for the design of biomacromolecular loaded nano-delivery system.Finally,in order to effectively combine mucus penetration and intestinal cells targeting into one nano-delivery system and improve insulin oral delivery efficiency,the folic acid decorated virus-mimicking nanoparticles were designed and influence of folic acid graft ratio on the in vitro properties of insulin loaded nanoparticles and oral insulin absorption in rats was studied systemically.First of all,using folic acid as active ligand,different folic acid grafted chitosan copolymers(FA-CS)were synthesized and characterized.Thereafter,using Ins/PBCA NPs(Ins/BCA=2/10,w/w)as the core,the virus-mimicking nanoparticles(Ins/PBCA/FACS/HA)were fabricated by further coating of positively charged FA-CS copolymer and negatively charged hyaluronic acid(HA).All the nanoparticles showed small particle size(<300 nm),slight negative charge(-10 mV)and high insulin entrapment efficiency(>99%).Irrespective of the FA-CS graft ratio,all the nanoparticles showed good stability,similar insulin release behavior in the gastrointestinal fluid,excellent and similar penetration in mucus(>60%,10 min).The nanoparticles permeability in intestine was FA graft ratio and segment dependent,with FA graft ratio at/over 12.51%presenting better effect in the order of duodenum>jejunum≈ileum.Both mechanism studies and confocal microscopy observation demonstrated FA-mediated process was involved in the transport of Ins/PBCA/FACS/HA NPs.In vivo studies revealed hypoglycemic effect of the nanoparticles was FA-CS graft ratio dependent,but a saturation phenomenon was observed when FA graft ratio was at/over 12.51%.In conclusion,the folic acid decorated virus-mimicking nanoparticles presented improved insulin intestinal absorption,implying combining mucus penetration and active transcellular transport is an effective way to promote oral absorption of insulin,while the modification ratio of active ligand needs optimization. |
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