Chitosan Derivatives Grafted Nanoparticles For Oral Insulin Delivery And Tumor Targeting

Chitosan derivative graft nanoparticles including chitosan hydrochloride graft nanoparticles(CHNPs),trimethylated chitosan graft nanoparticles(TMCNPs), carboxylation chitosan graft nanoparticles(CCNPs) and carboxymethyl chitosan graft nanoparticles(CMCNPs) were synthesized and investigated as peroral delivery systems for biomacromolecular drugs.With insulin as a model drug,drug loading,in vitro release,in vivo hypoglycemic effect and absorption mechanism in the GI treact were studied.6-coumarin labeled CCNPs were also evaluated as a potential candidate in tumor targeting.1 Preparation and characterization of chitosan derivative graft nanoparticlesNanoparticles were prepared by graft polymerization of methyl methacrylate (MMA) and chitosan derivatives including chitosan hydrochloride(CH), trimethylated chitosan(TMC),carboxylation chitosan(CC) and carboxymethyl chitosan(CMC) in aqueous solution.During polymerzation,amphiphilic graft polymers assembled in the solution to form core-shell type nanoparticles,which possessed hydrophobic cores and hydrophilic shell layers on their surfaces,resulting in an excellent aqueous dispersion.Structure of nanoparticles was characterized by dissolubility,FT-IR,¹H-NMR and X-ray.Particle size and zeta potential were determined;morphology was investigated and stability of nanoparticles in the GI tract and during storage and freeze drying process was evaluated.The nanoparticles varied from 100 to 300 nm in size.CHNPs and TMCNPs bore positive zeta potentials while CCNPs and CMCNPs showed negative zeta potentials.The CCNPs assembled in the gastric fluid while was well dispersed in the intestinal fluid.The CCNPs and CMCNPs were stable in the GI tract and during storage or freeze-drying.2 Insulin loaded nanopartielesThe encapsulation efficiencies,loading capacities,in vitro release,particle diameters,zeta potentials and morphologies of insulin loaded nanoparticles were investigated.Endogenous fluorescence,extrinsic fluorescence,circular dichroism(CD) and double labeled fluorescence were used to characterize the conformational structure of insulin when loaded in nanoparticles.Results demonstrated that the encapsulation efficiencies of TMCNPs and CCNPs were above 80%and their loading capacities surpassed 10%.Insulin was mainly located in the shell of the nanoparticles via hydrogen bonding,electrostatic interaction and Vanderwolf force.Morphologies, diameters and zeta potentials did not significantly alter after insulin loading.Insulin release from these nanoparticles exhibited pH-sensitivity.Hypoglycemic effects following oral administration of insulin-CCNPs in normal rats,diabetic rats and Beagle dogs were investigated.At 100 u·kg^-1 and 50 u·kg^-1,oral administration of insulin-CCNPs led to a significant blood glucose depression of 20-40%within 12 h,achieving a relative pharmacological bioavailability of 9.7%(25 u·kg^-1).Blood glucose level of diabetic rats decreased to below 80%following oral administration of CCNPs within 6-16 h,and reached the maximum hypoglycemic effect at 16 h(35%of the initial level).Oral administration of insulin-CCNPs in Beagle dogs at a dose of 16.5 u·kg^-1 also exhibited remarkable hypoglycemic effect.The potential of the CCNPs on enzyme inhibition,opening of the epithelial tight junctions,and absorption enhancement at the Peyer's patches were evaluated. Results showed that the activity of trypsin and pepsin decreased to 25%and 20%in the presence of the CCNPs,respectively.Transient and reversible opening of the tight juntions was achieved following treatment of the CCNPs and uptake of 6-coumarin labeled CCNPs at Peyer's patch was enhanced correspondingly.Nanoparticles loaded with FITC-insulin or phycocyanin(PC) were used for the assessment of oral absorption mechanism.The fluorescence spectrum of trypsin treated FITC-insulin was monitored and the in vivo distributions of FITC-insulin and FITC-insulin loaded CCNPs following oral administration in SD rats were investigated.The ultraviolet and fluorescence spectra of trypsin treated PC and chymotrypsin PC were measured.Results showed that fluorescence of FITC-insulin was intensified following exposure to trypsin.Strong fluorescence was detected in the liver and spleen following oral administration of FITC-insulin and FITC-insulin loaded CCNPs.After incubation with trypsin and chymotrypsin,ultraviolet as well as fluorescent intensities of PC were diminished,and positive correlation was determined between protein concentrations and ultraviolet absorption as well as fluorescent intensity.3 CCNPs loaded bilaminated film as oral delivery vehiclesA novel smart drug delivery system(NP-Film^CH) consisting of CCNPs and bilaminated films was developed,the films composed of the mucoadhesive chitosan-EDTA hydrogel layer(CH-EDTA) and the hydrophobic ethylcellulose layer.The CH-EDTA was characterized by morphology,viscosity,rheology and in vitro muco-adhesive force.In vitro release in different media was also performed. Results showed that the chitosan-EDTA hydrogel possessed typical network structure, where nanoparticles could be well-dispersed.Chitosan-EDTA hydrogel film showed a 2-8 fold increase in the muco-adhesive force as compared to the chitosan chloride film,and it was especially pronounced in the ileum.Nanoparticles were slowly released from the film at pH 1.2 while easily released at pH 7.4.Another novel polymeric composite carrier consisting of CCNPs and bilaminated films which were composed of the mucoadhesive alginate-Ca²⁺ hydrogel and the hydrophobic ethylcellulose layer was developed,which might be a promising drug carrier.Morphology of nanoparticles in the bilaminated films was examined using fluorescence microscopy and scanning electronic microscopy.Swelling, muco-adhesion and nanoparticle release was studied,and morphology of calcein loaded nanoparticles in the films was also examined.At low ionic strength,the alginate hydrogel film possessed high swelling ratios and vice-versa at high ionic strength.Swelling ratios were high at pH 5.8 and 8.0,while low at pH 1.7.In vitro muco-adhesive force of the alginate film was 6000 N-m^-2.Slow release of the CCNPs was achived in 0.01 mol·l^-1 HCl with a total release amount of 10%within 2h.Fast dissolution was observed in pH 6.5 and pH 7.4 PBS.Large quantities of pores were present in the alginate film,where nanoparticles could be well distributed.4 Distribution of coumarin-labeled nanoparticles in tumorsCCNPs were labeled with lipophilic fluorescent 6-coumarin,and their distributions in H1299 and HEK293 cells were compared.Besides,tissue distribution, in vivo optical imaging and in vitro macrophage uptake was investigated.Results showed that coumarin-labeled CCNPs could be uptaken by H1299 cells rather than HEK293 cells.Nanoparticles were concentrated in tumor tissues with low distribution in lungs,livers,kidneys and spleen,which indicated tumor-targeting properties.5 BiocompatibilityBlood compatibility of the nanoparticles was evaluated in terms of hemolysis, kinetic thrombus time,platelet adhesion.Tissue compatibility was assessed with respect to intestinal biochemical damage,in vivo implantation and tissue damage. Hemolysis ratios of the CCNPs,CMCNPs and TMCNPS were below 5%,which met the requirements of biomedical materials.The CCNPs showed prolonged thrombus time compared to silicated glass;CCNPs and CMCNPs exhibited prolonged recalcification time of 40%.Morphological deformation and activation of the platelets were not detected upon treatment with CHNPs,CCNPs,CHNPs,and chitosan hydrogel films.Lactate dehydrogenase leakage in the small intesetine of SD rats was minimal following exposure to CCNPs and NP-film^CH,indicating integrity of the intestinal epithelia.No damage towards the muscle was detected after implantation of chitosan-EDTA hydrogel film,CCNPs or CMCNPs,either.