| Background and objectiveAs an essential drug in glycemic control,basal insulin therapy is commonly used to treat diabetes.However,it has several limitations such as poor permeability,short plasma half-life,and rapid enzymatic degradation in the gastrointestinal tract.The low bioavailability via oral administrationor other non-injection routes of administration leading to the limited clinical applications of insulin.Multiple daily injection of insulin remains to be the traditional option for the treatment of insulin-dependent diabetic patients in clinical practice which exposes patients to compliance problems and large fluctuations between peak and trough plasma concentration.Therefore,in order to get satisfactory therapeutic effect of insulin,it is quite necessary to choose proper drug carriers and to develop a new drug delivery system such as sustained and controlled delivery system.In this study,a kind of pH-sensitive amphiphilic triblock polymer was designed to be used as a drug carrier for insulin.Moreover,its physicochemical properties,performance on drug loading and release,and hypoglycemic effects in vivo were explored,respectively,which could provide some references for developing sustained and controlled delivery system of insulin.Methods1.The triblock copolymers with different molecular weights were synthesized by combination of ring opening polymerization(ROP)and atom transfer radical polymerization(ATRP).The obtained copolymers were characterized by Fourier-transform Infrared(FT-IR)spectroscopy as well as 1H-NMR and their critical aggregation concentration(CAC)were measured by established fluorescence technique with pyrene as an extrinsic probe.2.The insulin-loaded polymeric nanoparticles were prepared by nanoprecipitation techniques in consideration of the cationic nature of obtained copolymers which can be used to make ionic complexes with anionic biomolecule such as insulin.Moreover,particle size,polydispersity index and particle morphology of the insulin-loaded polymeric nanoparticles were characterized by dynamic light scattering(DLS)and transmittion electron microscope(TEM).3.The encapsulation efficiency(EE%)and drug loading(DL%)capacity of insulin-loaded nanoparticles with different insulin-coplymer mass ratio of 20%,50%and 70%were measured to confirm the optimal condition.To investigate the kinetics of insulin release from the insulin-loaded polymeric nanoparticles,the in vitro release were detected by BCA Protein Assay Kit and seven mathematical models were fitted to the raw release data from the selected time range.4.The cytotoxicity assay of polymeric nanoparticles was studyed on mouse fibroblast cells(L929)by MTT method.5.The streptozotocin-induced diabetic rats models were established to evaluate the hypoglycemic effects of the insulin-loaded polymeric nanoparticles.Results1.The amphiphilic triblock copolymers confirmed by FT-IRand 1H-NMR were constructed by methoxy poly(ethyleneglycol)-poly(?-caprolactone)-poly(N,N-diethylamino-2-ethylmethacrylate)(mPEG-PCL-PDEAEMA).According to the characterization results,their molecular weights were proved to coincide with the theoretical ones,suggesting that the synthetic method we used was stable and controllable.The critical aggregation concentration(CAC)of the triblock copolymers were 3-6 orders of magnitude lower than the most commonly used surfactants,which showed that triblock copolymers were capable of forming stable core-shell structure.2.The particle size and morphology of polymeric nanoparticles,via self-assemble after dispersing into proper solvents,were apparently changed with varied pH values,which indicated the pH sensitive of triblock copolymers.In addition,the mean diameter of the insulin-loaded polymeric nanoparticles,spherical with uniform particle size,was obviously increased as a result of drug loading.3.When copolymer mPEG-PCL-PDEAEMA was utilized as a drug carrier,the spherical nanoparticles prepared with insulin-coplymer mass ratio of 90%was found higher EE%and DL%than the other three kinds of mass ratios,which had maximal EE%of 81.99±1.77%and maximal DL%of 42.46±0.53%,respectively.The insulin-loaded polymeric nanoparticles based on mPEG-PCL-PDEAEMA were proved to have the most obvious suatained-release behavior,which was sensitive on the pH values of release medium.The results proved that the release of insulin-loaded polymeric nanoparticlesin weak acid environment(pH 6.0)was much faster than that of nanoparticles in neutral environment(pH 7.4).What's more,both release mechanisms were fitted to Weibull model,which showed that the insulin-loaded polymeric nanoparticles have a good effect on the drug sustained-release.Besides,the drug release process included Fick's diffusion and matrix-eroded procedure was found in pH 7.4 while only Fick's diffusion was found in pH 6.0.All results showed that the burst release of insulin from the nanoparticles were evidently minimized owing to the increase of hydrophobic segments.4.The drug-loaded nanocarriers were proved as nontoxic to L929 cells when the concentration was no higher than 0.4mg/ml.5.The hypoglycemic effects study revealed that the insulin-loaded polymeric nanoparticles,displaying a certain degree of sustained-release characteristics in vivo,significantly exhibited prolonged hypoglycemic action in diabetic ratscompared with free drug.The insulin-loaded polymeric nanoparticles based on mPEG114-PCL86-PDEAEMA53 and mPEG114-PCL114-PDEAEMA53 played a continue role in controlling blood glucose levels in diabetic rats for 12h and 48h,while only 4h for free insulin.ConclusionThe insulin-loaded polymeric nanoparticles based on mPEG-PCL-PDEAEMA are demonstrated to be effective nano-carriers showing high insulin incorporation efficiency,obvious sustained release character in vitro and prolonged hypoglycemicaction in vivo.Thus,the pH-sensitive amphiphilic triblock copolymer mPEG-PCL-PDEAEMA has promising potential to be a sustained-release carrier for insulin. |
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