Self-assembly PH-sensitive Chitosan/Alginate Coated Polyelectrolyte Complexes For Oral Delivery Of Insulin

Objective The study aims to prepare a good biocompatibility,nontoxicity,mucoadhesive,sustained-release and pH-sensitive polyelectrolyte complexes,which shows a good application prospect applying in insulin oral administration delivery system.The multifunctional pH-sensitive polyelectrolyte complex,which was formed by positively charged chitosan coated nanoparticles and negatively charged alginate coated nanoparticles,self-assembly cross-linked by the electrostatic interaction.Due to the electrostatic interaction intensity of PEC varies with pH,the polyelectrolyte complex morphological structure changing applied the sustained or controlled release properties,which can be used in oral administration.Methods Diblock amphiphilic copolymer mPEG5000-b-PLGA was synthesized by ring opening polymerization,which was selected as nanoparticles matrix materials.The synthetic polymers were characterized by Fourier transform infrared spectroscopy(FT-IR),1H-NMR and Differential Scanning Calorimeter(DSC).In order to obtain oppositely charged nanoparticles,the insulin-loaded polymer nanoparticles were prepared by double emulsion method firstly,the chitosan and alginate were exploited to introduce positive and negative charges on nanoparticles surface,respectively.The positive charged chitosan coated nanoparticles(CS NPs)and the negative charged alginate coated nanoparticles(AL NPs)can be combined by electrostatic interaction among the carboxyl groups of alginate and the amine groups of chitosan,forming a self-assembly polyelectrolyte complex with crosslinking structure.The formula was optimized by Box-Behnken design and response surface methodology.The particles size,polydispersity index(PDI),zeta potential and morphology were measured by dynamic light scattering(DLS),electrophoretic light scattering(ELS)and transmission electron microscope(TEM),respectively.The encapsulation efficiency(EE)and loading capacity(LC)of two insulin-loaded nanoparticles were measured by BCA protein assay kit,and the cytotoxicity assay of polymer materials and nanoparticles were evaluated by cck-8 assay conducted on the colon adenocarcinoma(Caco-2)cell.To investigate the pH-sensitive property of PEC,the morphological and structure changing in different pH environment were observed by TEM,the in vitro insulin release profile of PEC were investigated by BCA method and the stability and bioactivity of the release insulin from PEC were evaluated by analysing the secondary and tertiary structures using circular dichroism(CD)spectroscopy.The streptozotocin-induced diabetic rat models were established to evaluate the hypoglycemic effects after oral administration insulin-loaded PEC.Results The 10 wt%monomethoxy polyethylene glycol-poly(lactic-co-glycolic acid)was successfully synthesized,ensuring the synthetic product were target polymers by FT-IR,lH-NMR and DSC,and the resulting copolymer had a weight average molecular weight(Mw)24800.The Box-Behnken design and response surface methodology is an effective and efficient method to optimize the formulation of nanoparticles.The optimized nanoparticles were good dispersed particle sizes(200-300 nm)with a spherical shape in deionized water observing by TEM.The EE,the LC,average particles size and zeta potentials of AL NPs were(83.61±0.38)%,(10.90±0.23)%,(260.1±17.1)nm and(-54.27±2.75)mV,and of CS NPs were(55.2±7.0)%,(4.9±0.7)%,(224.4±13.8)nm and(+13.7±1.6)mV,respectively.The cytotoxicity test resulted that both the polymer materials,blank CS NPs and blank AL NPs were non-toxicity,safety.The tight crosslinking structure of PEC under the acidic condition can protect the insulin from the release of drugs in the stomach,while the loose structure at pH 6.8 can promote the insulin releasing easily in intestinal tract due to the weak interaction force of polyelectrolyte complex.The PEC morphological structure results at different pH(pH=1.2,6.8,7.4)observing by TEM indicated that the tight crosslinking structure of PEC under the acidic condition can protect the insulin from the release of drugs in the stomach,while the loose structure under neutral environment can promote the insulin releasing easily in intestinal tract.The in vitro release profile results showed that the insulin-loaded PEC have a good controlled sustained release property,the CD results indicated that the secondary structure of insulin releasing from PEC was consistent with native insulin,preserving insulin structure and maintain activity.Comparing with subcutaneous injection insulin solution group and oral administration CS NPs,AL NPs groups,the subcutaneous injection PEC groups shows good sustained-release with good hypoglycemic effect,releasing time up to 12 hours,with 7.51%relative bioavailability.Conclusion The chitosan/alginate coated polyelectrolyte complexes process pH-sensitive property,with good EE,LC and controlled sustained release effects both in vitro and in vivo.Thus,the pre-prepared pH-sensitive polyelectrolyte complexes may be employed as a potential oral administration delivery system of insulin.