Study On Several Important Problems Of An Insulin-loaded Emulsion System (IES) For Buccal Spray Delivery

Diabetes mellitus (DM) is one of the main threats to human health in modern society. It is reported that there are more than 20,000,000 diabetics in china, and an upward tendency was found each year. There are about 200,000,000 diabetics throughout the world according to the information of IDF, and the number of the diabetics may reach 300,000,000 in 2025. As we know, insulin is the primary drug for DM, and injection therapy is always the major delivery method. Due to the great pain, discomfort and inconvenience brought by injection therapy, the study of non-injectable insulin as new drug has been the hotspot for the pharmaceutical researchers. It took Bio-inorganic Lab of Huazhong University of Science and technology (HUST) almost ten years to obtain an effective insulin-loaded emulsion system (IES) for buccal spray delivery. Based on the research results, deeper studies of the determination of Rh-insulin in IES by HPLC, the weak interactions between insulin and the functional molecules in IES, the microstructure of IES and so on were performed in this paper, and the main results are as follows:①Soybean lecithin and phenol intensely interfered in the determination of insulin with HPLC-UV in IES which was developed for buccal spray delivery. Thus, Electrospray Ionization Mass Spectrometry (ESI-MS) with ion trap detection was applied to screen extractants to remove soybean lecithin and phenol in IES. The results showed that n-pentanol was a suited extractant to meet the measurement requests because a large quantity of soybean lecithin and phenol were removed simultaneously, and there appeared no interferences brought by n-pentanol in insulin measurement. The analytical method was assessed by the criteria including linearity (Linearity Range: 1.05~7.34U/mL, Correlation Coefficient: 0.99936), accuracy (Recoveries: 98.1 ~102.5%), within-day precision (RSD: 0.5%, n=6), LOD (0.01U/mL), and LOQ (0.03 U/mL), and exhibited high sensitivity and specificity, and considerable accuracy and precision on the determination of insulin in IES. The application of this method had been successfully performed to study the storage conditions of IES, and the results indicated that insulin in IES would be stable at 4℃in light proof condition, which was significant for further pharmaceutical research. The physicochemical characterization of IES showed that the diameter, pH value, polydispersity, viscosity, refractive index of IES changed very little, and the basic properties of emulsion were maintained completely in one year.②The stable noncovalent complex between insulin and phenol was found by ESI-MS with ion trap detection, which confirmed that there lay weak interactions between insulin and phenol. According to the detected noncovalent complex, it is supposed that insulin dimer, trimer, tetramer and hexamer all could form the complexes with phenol. The effect of phenol on amplifying the ESI-MS signal intensity of insulin has been found during the detection of the complexes. When phenol was added to the insulin solution, the signal intensity of insulin was amplified by more than ten times overall, and it increased in a manner dependent on the molar ratio of phenol and insulin, despite having different pH values (pH2.0, pH3.0 and pH4.0) and different concentrations (3.9E-02, 6.6E-02 and 6.2E-01 mmol/mL) of insulin solution. The different enhancement effect of each ion peak of insulin was observed. Highly charged ion peaks got more amplification than lower charged ones, some of them even more than one hundred. Moreover, the signal-to-noise (S/N) ratio of insulin ion peaks fitted well. Better enhancement effect commenced when the molar ratio of phenol and insulin was about 1000:1(phenol: insulin), and the best effect appeared almost at the ratio of 5000:1(phenol: insulin). A possible mechanism named"proton transfer mechanism"was proposed to interpret the enhancement of insulin detection by phenol. Phenol as a proton"transmitter"could make the insulin obtain additional protons in the electrospray process, which leads to the increase of charged insulin. The noncovalent complex between insulin and tyrosine was also found by ESI-MS, while the molar ratio of insulin and tyrosine was 1:1. The confirmation of insulin-tyrosine complex was more significative to explain some bioprocesses.Additionally, the secondary structure of insulin was studied by CD and IR, and the results showed that the pH value and concentration of insulin solution could affect the second structure and the aggregation of insulin. Phenol and some phenolic compounds could also affect the second structure intensively, and they could be the allosteric agents for insulin. All these results verified the ESI-MS results.③The pseudo-ternary phase diagram of the similar system of IES, lecithin /propanediol/PBS, and drug-loaded system (insulin-load) were obtained by the titration methods of transmittance and conductance, and the main difference between the two phase diagrams was that the districts of W/O and O/W of the drug-loaded system were smaller than those of the lecithin/propanediol/PBS system. The phase diagram of drug-loaded system demonstrated that IES belongs to the O/W emulsion, and soybean lecithin could act as both the absorption enhancer of insulin for transmucosal delivery and oily phase for the emulsion system to keep the emulsion properties stable. The confirmation of the emulsion type of IES prepared the ground for the further study of IES microsturctrue.The particles distribution and profile of IES was studied by Dynamic Light Scattering Analyzer (DLSA) and Transmission Electron Microscope (TEM). A monodispersed, narrow size distribution was obtained, and the global particles in IES were distributed evenly throughout the whole system. The particle size ranged from 20 nm to 260 nm, 90% of the particles size is less than 115 nm. The average size is 67.5nm. So IES could be belonged to the microemulsion delivery system. There lies weak interactions between insulin and soybean lecithin studied by ESI-MS,CD,31P-NMR and TIRFM. Insulin could exist in both PBS (aqueous phase) and the particles formed by lecithin (either on the surfaces or in the center of the particles). The results of HPLC test showed that more than 30% insulin molecules could bind with the particles formed by lecithin. The possible microstructure of IES was proposed that there were 4 main vesicles in IES, namely the semi-closed vesicles (SCV), large unilamellar vesicles (LUV), multilamellar vesicles (MLV) and multivesicular vesicles (MVV). The possible microstructure could be used as references for the further studies of the mechanism of insulin transmucosal delivery and the continued improvement of insulin biopotency.④The binding of insulin and insulin receptor (IR) of HepG2 cells and cortical neurons was studied by fluorescence imaging of FITC-insulin. Insulin could bind instantly with IR at cell membranes, and enter the cells via internalization. IR could distribute on the somas and neurites (including dendrite and axon), and these insulin receptors of neurons are very important for insulin to perform its functions in brain. The fluorescence imaging also demonstrated that soybean lecithin could enhance the uptake of insulin through the sublingual mucosa. Insulin could penetrate the sublingual mucosa with the help of soybean lecithin in 60 min, but it could hardly do so without the help of soybean lecithin. The above results indicated that the fluorescence imaging could be a direct and easy method for studying the distribution and function of protein and peptide drugs in vivo.