Preparation And In Vitro/in Vivo Evaluation Of Insulin-loaded Nanoparticles Assembled By Polyethylene Glycol, Polycarprolactone And Polyethyleneimine

Background and objectiveAs one of the most effective and relatively safe drug in glycemic control, insulin has been playing an irreplaceable role in the treatment of diabetes. However, oral administration and other non-injection routes of insulin still face challenges because the above-mentioned mode of administrations have diverse disadvantages to overcome such as short half-life, low bioavailability, low intestinal permeation and low stability of insulin in the gastrointestinal tract, etc. For the reasons given above, 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. Therefore, in order to maximum the effectiveness of insulin and get satisfactory therapeutic effect simultaneously, it’s quite necessary to choose proper drug carriers and develop a new drug delivery system of insulin such as sustained and controlled delivery system.In the past couple of decades, various strategies have been developed to design a effective insulin delivery system. Among all these approaches, nanoparticulate systems which based on polymer materials with good biocompatibility and biodegradability tend to be an attractive technique for drug formulation and also a valid approach to overcome those drawbacks since they can offer loads of advantages. Firstly, polymeric delivery system based on nanoparticles are able to protect active agents from enzymatic degradation and improve their stability. Sencondly, the small size favors their absorption by the M cells of Peyer’s patches, a type of lymphatic island within the intestinal tract, which lead to higher drug delivery efficiency. Thirdly, they can increase target distribution, prolong time of blood cycle and enhance curative effect of drugs by preparing sustained or controlled release dosages. Moreover, side effects of model drug incline to resolve or fade away when the drug’s efficacy is brought into full play. Futhermore, the particulate systems are able to achieve the goal of implementing targeting drug delivery or site-specific drug delivery for encapsulated drugs by different fabrication methods. In addition, nano-carriers are capable of changing the membrane transport mechanisms to some extent, thus helping to improve the permeability of the model drug through biofilms and then enter cells to exert therapeutic effects.At present, natural polymer materials and biodegradable polymer materials are the two primary polymer materials applied in the nano drug delivery system. The natural polymers mainly are chitosan, gelatin, alginate, polysaccharide compounds, dextran and its derivatives, etc. When it comes to biodegradable polymer materials, they have evolved quickly for the past few years because of their properties of being non-toxic, biocompatible, biodegradable after hydrolysis or enzymolysis. Among them, poly carbonate, poly (amino acid), poly lactic acid (PLA), polylactic-co-glycolic acid(PLGA), polylactic acid-polyethylene glycol(PLA-PEG) and s-caprolactone are the most commonly used polymer materials. As a new drug delivery and controlled release device, these polymer materials appear to be very promising and have been widely studied in drug delivery system of polypeptides and proteins. For example, Chitosan-alginate (CS/ALG) nanoparticles, a type of polyelectrolyte complexation, were formulated by Piyasi Mukhopadhyay et al[9] A nine-hour hypoglycemic effect and an improved insulin-relative bioavailability (-8.11%) were observed apparently on diabetic rats models after oral administration at the dosage of 100 IU/kg. And it’s worth mentioning that the acute toxicity research showed nonexistence of systematic toxity of the polymeric nanoparticles in animal models. Futhermore, the mechanism of how the insulin-loaded polymeric nanoparticles function in vitro and in vivo is clarified by some researchers. For example, the process of uptake and the intracellular fate of the insulin-loaded PLGA nanoparticles were illuminated in the Caco-2 cells by Nathalie Reix et al, which showed that there existed a clathrin-medicated endocytosis of the insulin-loaded PLGA nanoparticles, and an intracellular route resulting in the basolateral exocytosis of the nanoparticles. Also, its in vivo researches on diabetic rats demonstrated that the nanoparticles could function well in intestinal conditions and proved to be a long-acting one. In addition, some new polymer materials that take on new functions and lead to new traits are always the focus of researches, for instance, amino poly(glycerol methacrylate)s (PGOHMAs) were synthesized by Xueyou Lu et al, by exploiting the physical actions between insulin and amino PGOHMAs, polyelectrolyte complexes(PECs) were then formed and the study proved that insulin release can be adjusted by altering the architecture and structure of amino PGOHMAs.Polyethylene glycol(PEG) and polycaprolactone(PCL) are the most commonly used polymeric carriers in our laboratory for the following reasons. PEG, approved internal consumption by FDA, is regarded as kind of polymer with stronger hydrophilicity, low toxicity, low immunogenicity and favorable biocompatibility, so it is often used for surface modification so as to improve the hydrophilicity of the polymer chain. Meanwhile, PCL was extensively used for drug delivery system in light of its excellent biodegradability and biocompatibility. As a consequence, by graft copolymerization or other physicochemical modification methods, we can develop a series of synthetic copolymers which can be applied in drug delivery system on the basis of PEG and PCL.In the design of the pentablock copolymer PEI-PCL-PEG-PCL-PEI, the introduction of copolymer PEI was conceived with the attention of utilizing the electrostatic interactions between the positively charged groups of PEI-PCL-PEG-PCL-PEI and negatively charged groups of anionic biomolecule such as insulin. Moreover, conjugation of the pentablock copolymer with PEG is a common method to reduce toxicity of PEI by means of shielding its positive charge. In addition, owing to the existence of both hydrophilic segments and hydrophobic segments, the pentablock copolymer PEI-PCL-PEG-PCL-PEI has the potential to be employed as amphiphilic copolymer to fabricate core-shell nanoparticulate system for the incorporation of insulin. In the end, from the perspective of getting a high bioavailability and an obvious hypoglycemic effect, insulin is used mainly as injection clinically at present and it’s essential to develop sustained and controlled-release drug delivery systems that are available for insulin injections, thus reducing dosing frequency and improving patient compliance. Considering the factors above, we chose to investigate the release effect of insulin-loaded nanoparticles based on PEI-PCL-PEG-PCL-PEI in vitro and in vivo on streptozotocin-induced diabetic rats following the method of subcutaneous injection, for the purpose of hoping to provide some suggestions for evaluation of new drugs and exploitation of new dosage forms.Methods1. Synthesis and characterization of the pentablock copolymers PEI-PCL-PEG-PCL-PEI of different molecular weightThe pentablock copolymer PEI-PCL-PEG-PCL-PEI was synthesized in three steps in total. In particular, a series of PCL-PEG-PCL triblock copolymers were synthesized by ring-opening polymerization utilizing PEG as initiator and Sn(Oct)2 as catalyst. Then triethylamine was used as catalyst to conjugate acryloyl chloride to the hydroxyl groups of PCL-PEG-PCL triblock copolymer in the second step. Finally, the pentablock copolymer PEI-PCL-PEG-PCL-PEI was synthesized by Michael addition reaction between the vinyl groups at the ends of acrylated PCL-PEG-PCL and primary amino groups of PEL The obtained copolymers were characterized by FT-IR and 1H-NMR and their critical aggregation concentration(CAC) were measured by established fluorescence technique with pyrene as extrinsic probe.’H-NMR spectra were recorded on Avance II 40 instrument operated at 400 MHz, which were performed to confirm the molecular weight and composition of the copolymers. FT-IR spectra were recorded using a Nicolet 6700 spectrometer(Thermo Fisher Scientific Inc., USA) in a range between 4000 and 400 cm"1, and each sample was mixed with KBr and then pressed into tablets for futher measurement.2. Preparetion and characterization of the insulin-loaded nanoparticles based on PEI-PCL-PEG-PCL-PEI of different molecular weightThe prime objective of this part was to ensure adequate loading of the protein while maintaining its stability and structural integrity during preparation. The insulin-loaded polymeric nanoparticles were prepared by a modified solvent evaporation method in consideration of the cationic nature of PEI-PCL-PEG-PCL-PEI which can be used to make ionic complexes with anionic biomolecule such as insulin. On the basis of the preliminary experiment, we mainly measured the encapsulation efficiency and drug loading capacity of insulin-loaded nanoparticles with different insulin-coplymer mass ratio of 20%,33% and 44% to confirm the optimal condition. Moreover, particle size, polydispersity index and particle morphology of the insulin-loaded nanoparticles prepared under the optimal condition which gave maximum insulin loading were characterized by dynamic light scattering(DLS) and transmittion electron microscope(TEM).3. In vitro/in vivo evaluation of insulin-loaded nanoparticles based on PEI-PCL-PEG-PCL-PEI of different molecular weight(1) To investigate the kinetics of insulin release from the insulin-loaded polymeric nanoparticles, the lyophilized powder was dispersed in the PBS solution(0.01 mol·1-1, pH=6.0) and incubated under constant stirring(80 r/min) at 37 ℃. At desired time intervals,1 mL of release media was taken out for futher drug concentration measurement and then repenlished with the equal volume of fresh media. The insulin concentration in the supernatant was measured by Coomassie brilliant blue G-250 method according to Bradford (1976). Finally, in determination of finding the most appropriate model to describe the in vitro release kinetics, excluding the impact of initial burst release, seven mathematical models were fitted to the raw release data from the selected time range to investigate the in vitro release kinetics. Accoring to the results of the analyses of the collected data, we can find the most appropriate model to describe the delivery of insulin from the prepared insulin-loaded polymeric nanoparticles.(2)The prime objective of this part is to evaluate the efficacy of the insulin-loaded polymeric nanoparticles in diabetic rat models. Different insulin-loaded polymeric nanoparticles based on PEI-PCL-PEG-PCL-PEI of different molecular weight were administered subcutaneously to overnight fasted diabetic rats. Aqueous free insulin, insulin injection preparation commercially available and blank copolymer solution were administered subcutaneously in another three groups. Blood samples from the tail vein of rats that collected at different time intervals ranging from 30min to 12h after administration were analyzed by glucose meter immediately. Then hypoglycemic effect was evaluated by the decrease of plasma glucose levels relative to the insulin-loaded polymeric nanoparticles prepared in this passage compared to three other groups mentioned above.Results1. Synthesis and characterization of the pentablock PE1-PCL-PEG-PCL-PE1 of different molecular weight(1) Pentablock copolymers PEI-PCL-PEG-PCL-PEI of three different molecular weight were synthesized successfully according to the characterization results of FT-IR and’H-NMR. Their molecular weights were 30255、30299 and 33735, respectively, which suggesting that calculated values cocincide with theoretical ones.(2) The critical aggregation concentration(CAC) of the pentablock copolymers PEI10K-PCL4K-PEG2K-PCL4K-PEI10K、PEI10K-PCL5K-PEG2K-PCL5K-PEI10K and PEI10K-PCL6K-PEG2K-PCL6K-PEI10K were 10.96×10-4 g/L、8.13×104 g/L and 6.17×10-4 g/L, respectively. Besides, with the increase in the hydrophobic segments, the CAC value decreased in turn, which was lower than the most commonly used surfactants. The result was consistent with the related literatures and it indicated that pentablock copolymers were capable of forming stable core-shell structure via self-assemble after dispersing into proper solvents.2. Preparetion and characterization of the insulin-loaded nanoparticles based on PEI-PCL-PEG-PCL-PEI of different molecular weightWhen copolymer PEI-PCL-PEG-PCL-PEI was utilized as drug carrier, the spherical nanoparticles prepared with insulin-coplymer mass ratio of 40% was found to be the optimal condition to give maximum insulin loading. When the insulin-loaded polymeric nanoparticles were prepared with insulin-coplymer mass ratio of 40%, the drug loading efficiency for INS-PEI10K-PCL4K-PEG2K-PCL4K-PEI1 OK-NPs,INS-PEI10K-PCL5K-PEG2K-PCL5K-PEI1 OK-NPs and INS-PEI10K-PCL6K-PEG2K-PCL6K-PEI1 OK-NPs were (18.63±0.07)%, (19.69±0.08)% and (19.96±0.13)%, respectively. The drug encapsulation efficiency were (57.23±0.25)%, (61.30±0.31)% and (62.35±0.51)%, respectively. The mean diameter of the above nanoparticles are (175.3±19.51) nm, (242.7±12.20) nm and (352.5±10.73) nm, respectively. The polydisperisity index(PDI) of the above nanoparticles were 0.415,0.783 and 0.694, respectively.4. In vitro/in vivo evaluation of insulin-loaded nanoparticles based on PEI-PCL-PEG-PCL-PEI of different molecular weight(1) The insulin-loaded polymeric nanoparticles based on PEI-PCL-PEG-PCL-PEI exhibited a good effect on the drug sustained-release in vitro, and burst release of insulin from the nanoparticles were evidently minimized for the reason that copolymer composition plays a vital role in controlling the drug release. INS-PEI10K-PCL5K-PEG2K-PCL5K-PEI10K-NPs proved to have the most obvious suatained-release behavior, which showed a more suastained insulin release pattern for 48 h. The in vitro release characteristics of INS-PEI-PCL-PEG-PCL-PEI-NPs investigated were in accord with Weibull model when subtracting out the impact of initial burst release in the first 6 hours. To sum up, the result demonstrated that the release of insulin from the polymeric nanoparticcles prepared in this passage is a log (time) dependent process.(2) In vivo experiment indicated that the insulin-loaded polymeric nanoparticles based on PEI-PCL-PEG-PCL-PEI administered subcutaneous injection were capable of decreasing the glucose level obviously on streptozotocin-induced diabetic rats, of which the INS-PEI 10K-PCL5K-PEG2K-PCL5K-PEI1OK-NPs exhibited a more significant hypoglycemic effect when compared to that of other insulin preparations. And its hypoglycemic effect can last about 12 h in the current study.ConclusionInsulin-loaded polymeric nanoparticles composed of PEG, PCL and PEI are demonstrated to be effective nano-carriers showing high insulin incorporation efficiency, a slow release effect with reduced initial burst release in vitro and also a stable hypoglycemic effect in vivo, all of which can avoid adverse reaction caused by the rapid release of insulin in a short period of time. As a consequence, the work we did could provide some references for the development of insulin preparations to a certain extent.