Preparation and evaluation of mucus-penetrating polymeric micelles containing insulin for pulmonary delivery

Abstract

The present invention discloses the preparation and evaluation of mucus-penetrating polymeric micelles containing insulin for pulmonary delivery.

Description

Preparation and evaluation of mucus-penetrating polymer micelles containing insulin for pulmonary delivery

[0001] The present invention relates to the preparation and evaluation of mucus-penetrating polymer micelles containing insulin for pulmonary delivery.

[0002] Pulmonary delivery of proteins or peptides could potentially improve the quality of life of patients who regularly receive needle-based injections. However, for drugs to be effective in the lungs, three major physiological barriers must be overcome. First, protein drugs must cross the mucus layer that protects the epithelial surface in the lungs. Second, they must cross the pulmonary epithelial layer and enter the bloodstream. Third, the drug must avoid phagocytosis by macrophages. These first two barriers currently represent significant barriers to the bioavailability and therapeutic efficacy of protein drugs in the lungs. Therefore, to address the mucus barrier, we sought to develop a carrier with enhanced mucus permeability that prevents adhesion to mucus and rapid mucus clearance mechanisms. Soluplus ®) and poly(ethylene glycol, PEG) and lipid (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE) were conjugated to prepare polymeric micelles (see Fig. 1). Coating the micelles with PEG reduces the adsorption of mucin (the main protein of mucus) and promotes the movement of particles within the mucus. Cell penetrating peptides (CPPs), also known as protein transduction domains, are short positively charged peptides with a length of 5 to 30 amino acids that can penetrate biological membranes and deliver various substances into cells. Among various CPPs, arginine-rich CPPs can be internalized into biological membranes quite well due to their high electrostatic interaction with the negatively charged cell membrane. Therefore, in this study, a polymeric micelle with enhanced cell permeability was prepared by conjugating a cell-penetrating peptide, oligoarginine (arginine-9, R9), to DPPE-PEG, a polyethylene glycol-based lipid.

[0003] [Prior Art Literature]

[0004] [Non-patent literature]

[0005] (Non-patent Document 1) Osman, N., Kaneko, K., Carini, V., & Saleem, I. (2018). Carriers for the targeted delivery of aerosolized macromolecules for pulmonary pathologies. Expert Opinion on Drug Delivery, 15(8), 821-834.

[0006] The purpose of the present invention is to produce and evaluate insulin-encapsulated polymeric micelles using an amphiphilic polymer to incorporate insulin into micelles and a polyethylene glycol-containing lipid or a polyethylene glycol-containing lipid conjugated with a cell-penetrating peptide for the purpose of promoting mucus penetration and cell penetration.

[0007] Specifically, the present invention uses an amphiphilic polymer, Solplus, to incorporate insulin into micelles. ® Polymer micelles can be manufactured and evaluated using DPPE-PEG and DPPE-PEG-CPP combined materials for the purpose of enhancing mucus permeation and cell permeation.

[0008] In order to solve the above problem, the present invention discloses the following means.

[0009] In one aspect, the present invention provides a method for preparing a mucus-penetrating polymer micelle containing insulin, comprising the steps of (S1) dissolving insulin to prepare a pH-adjusted insulin solution; (S2) dissolving an amphiphilic polymer and a polyethylene glycol-containing lipid or a polyethylene glycol-containing lipid conjugated with a cell-penetrating peptide; (S3) adding the insulin solution to the solution of (S2) and mixing; and (S4) ultrafiltration of the mixed solution of (S3) to remove aggregated particles.

[0010] In the case of the method for manufacturing insulin-containing micelles according to the present invention, the amphiphilic polymer Soluplus ® There is an advantage in that by utilizing DPPE-PEG and DPPE-PEG-CPP, monodispersed micelles having a size of less than 100 nm can be manufactured.

[0011] In the method for manufacturing insulin-containing micelles according to the present invention, it was confirmed that micelles manufactured using PEG had a fast diffusion rate in mucus derived from pig lungs or calu-3 cells.

[0012] During the nebulization process using a nebulizer, the formulation exhibited aerosol dynamics similar to distilled water, and micelles maintained their size without breaking during the nebulization process. Furthermore, evaluation using NGI confirmed that the micellar formulation containing DPPE-PEG-CPP exhibited the best aerosolization performance.

[0013] The results of the MTT assay using DCP and A549 cells confirmed that the micellar formulation did not affect the lung function of animals and did not exhibit cytotoxicity.

[0014] It was confirmed that the blood sugar-lowering effect lasted for a long time when administered in a micellar formulation containing insulin in an animal model induced with diabetes.

[0015] The effects of the present invention are not limited to the effects mentioned above, and various effects may be included within a range apparent to those skilled in the art from the contents described below.

[0016] Figure 1 shows a schematic diagram of the preparation of a polymer micelle containing insulin.

[0017] Figure 2 illustrates the mechanism of conjugating a cell-penetrating peptide, oligoarginine (arginine-9, R9), to a polyethylene glycol-lipid, DPPE-PEG.

[0018] Figure 3 shows the process for manufacturing polymer micelles.

[0019] Figure 4 shows (A) the size and polydispersity index of polymer micelles, (B) a transmission electron microscope image, and (C) the encapsulation efficiency.

[0020] Figure 5 shows (A) the zeta potential results and (B) the CD spectrum analysis results of an insulin solution and polymer micelles.

[0021] Figure 6 shows (A) the trajectories of micelles in mucus and (B) the mean square displacement (MSD) values ​​over time.

[0022] Figure 7 is a fluorescence microscopic photograph of mucus derived from Calu-3 cells.

[0023] Figure 8 shows (A) a Z-stack image of the diffusion of micelle particles (red) in Calu-3 cell mucus (green), and (B) the average fluorescence intensity of each micelle formulation over time (in (B), * indicates a point significantly different from SOL, p < 0.05, and the Kruskal-Wallis test was performed for comparison).

[0024] Figure 9 shows the results of (A) cellular uptake experiments using Cau-3 cells and A549 cells, and (B) the average fluorescence intensity (* in (B) indicates a point that is significantly different from SOL, and p < 0.05 was used for comparison using the Kruskal-Wallis test).

[0025] Figure 10 shows a photograph of a nebulizer and a schematic diagram of the mechanism by which micelle particles are generated.

[0026] Figure 11 shows the spray flow of micelle particles through a nebulizer.

[0027] Figure 12 shows the particle size distribution before and after spraying micelle particles through a nebulizer.

[0028] Figure 13 shows the fine particle fraction (FPF) values ​​of insulin solutions and micelle particles based on the effective ingredient delivered at 3.3 μm or less.

[0029] Figure 14 shows the insulin ratio delivered to each stage of NGI.

[0030] Figure 15 shows the DCP equipment.

[0031] Figure 16 shows the results of measuring tidal volume (VT), specific airway resistance (sRaw), and enhanced pause (Pehn) values.

[0032] Figure 17 shows the results of evaluating the viability of A549 cells after treatment with an insulin solution and each micelle formulation at an insulin concentration of 0.01-1.00 mg / ml for 24 hours (mean ± standard deviation, n=3).

[0033] Figure 18 shows the results of confirming the blood sugar lowering effect of insulin in a diabetic animal model (mean ± standard deviation, n=4).

[0034] Hereinafter, the present specification will be described in more detail.

[0035] To explain this more specifically, the terms used in this specification are selected from widely used, general terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of engineers working in the field, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should not be defined simply as names of terms, but rather based on the meanings of the terms and the overall content of the present invention.

[0036] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0037] Numerical ranges are inclusive of the numbers defined in the ranges above. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if that lower numerical limitation were explicitly stated. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if that higher numerical limitation were explicitly stated. Every numerical limitation given throughout this specification will include every better numerical range within that broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0038] The following descriptions and embodiments disclosed in the present invention may also be applied to other descriptions and embodiments. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0039] Expressions such as “comprising” as used herein should be understood as open-ended terms implying the possibility of including other embodiments, unless specifically stated otherwise in the phrase or sentence in which the expression is included.

[0040] Hereinafter, the present invention will be described in detail.

[0041] Method for producing mucus-permeable polymer micelles containing insulin

[0042] The present invention provides a method for producing a mucus-permeable polymer micelle containing insulin as follows.

[0043] Specifically, the present invention provides a method for preparing a mucus-penetrating polymer micelle containing insulin, comprising the steps of (S1) preparing an insulin solution having an adjusted pH by dissolving insulin; (S2) dissolving an amphiphilic polymer and a polyethylene glycol-containing lipid or a polyethylene glycol-containing lipid conjugated with a cell-penetrating peptide; (S3) adding the insulin solution to the solution of (S2) and mixing; and (S4) ultrafiltration of the mixed solution of (S3) and removal of aggregated particles.

[0044] In the present invention, the amphiphilic polymer may be at least one selected from the group consisting of polyvinyl caprolactam (PCL)-polyvinylacetate (PVA)-polyethylene glycol (PEG) graft copolymer, low-saponification polyvinyl alcohol, polyacrylamidomethylpropanesulfonic acid (Poly(AMPS)), polyvinyl pyrrolidone, polyvinyl pyrrolidone / vinyl acetate copolymer, and polyvinyl pyrrolidone / alkyl acrylate copolymer, and specifically, may be a polyvinyl caprolactam (PCL)-polyvinylacetate (PVA)-polyethylene glycol (PEG) graft copolymer, but is not limited thereto.

[0045] Specifically, the polyvinyl caprolactam (PCL)-polyvinyl acetate (PVA)-polyethylene glycol (PEG) graft copolymer is commercially available under the name Solplus. ® It is an amphiphilic polymer sold as a polymer represented by the following chemical formula 1.

[0046] [Chemical Formula 1]

[0047]

[0048] At this time, in the chemical formula 1, l is 333 to 518, m is 263 to 410, and n is 321 to 488.

[0049] In the present invention, the polyvinyl caprolactam (PCL)-polyvinyl acetate (PVA)-polyethylene glycol (PEG) graft copolymer, Solplus ® At room temperature, it is in a sol state, but when the temperature rises, a phase transition to a gel form can occur.

[0050] In the present invention, the polyvinyl caprolactam (PCL)-polyvinyl acetate (PVA)-polyethylene glycol (PEG) graft copolymer, Solplus ® The molecular weight may be 1000 to 140000 g / mol, or 90000 to 140000 g / mol, and the average molecular weight may be about 118000 g / mol.

[0051] In the present invention, the term "polyethylene glycol lipid" means a lipid comprising polyethylene glycol (PEG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyloleoylphosphatidylethanolamine (POPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) are conjugated.

[0052] In the present invention, the polyethylene glycolated lipid is DSPE-PEG1000, DMPC-PEG1000, DOPC-PEG1000, DPPC-PEG1000, POPC-PEG1000, DOPE-PEG1000, DMPE-PEG1000, POPE-PEG1000, DPPE-PEG1000, DSPE-PEG2000, DMPC-PEG2000, DOPC-PEG2000, DPPC-PEG2000, POPC-PEG2000, DOPE-PEG2000, DMPE-PEG2000, POPE-PEG2000, DPPE-PEG2000, DSPE-PEG5000, DMPC-PEG5000, DOPC-PEG5000, DPPC-PEG5000, POPC-PEG5000, DOPE-PEG5000, DMPE-PEG5000, POPE-PEG5000 and may be at least one selected from the group consisting of DPPE-PEG5000, specifically, may be any one of DPPE-PEG2000 or DPPE-PEG5000, but is not limited thereto.

[0053] In the present invention, the term “cell penetrating peptide” means a short positively charged peptide with a length of 5 to 30 amino acids that can penetrate biological membranes and deliver various substances into cells.

[0054] In the present invention, the term “polyethylene glycol lipid conjugated with a cell-penetrating peptide” means a polyethylene glycol lipid defined above to which a cell-penetrating peptide is conjugated.

[0055] Here, as a method for conjugating a cell-penetrating peptide to a polyethylene glycol lipid, an aminohexanoic acid linker and R9 (AhxR9) were utilized in DPPE-PEG-SC (1,2-dipalmitoyl-sn-glycero-3-phophoethanolamine-PEG-Succinimidyl ester), and DPPE-PEG-R9 was finally synthesized (see Fig. 2).

[0056] In the present invention, the cell penetrating peptide may be at least one selected from the group consisting of oligoarginine (R9), TAT, R8, DPV3, DPV6, penetratin, pVEX, MPG, MAP, transportan, Bip4, C105Y, and Melittin, but is not limited thereto.

[0057] In the present invention, the polyethylene glycol lipid to which the cell penetrating peptide is conjugated may be DPPE-PEG2000 or DPPE-PEG5000 to which oligoarginine is conjugated, but is not limited thereto.

[0058] In the present invention, the step of dissolving the (S2) amphiphilic polymer and the polyethylene glycol-containing lipid or the polyethylene glycol-containing lipid conjugated with the cell-penetrating peptide may be a step of dissolving by adding at a weight ratio of 1:0.5 to 1.5, specifically 1:1, but is not limited thereto.

[0059] In the present invention, the mixed solution of (S3) may contain insulin, an amphiphilic polymer, and a polyethylene glycol-containing lipid or a cell-penetrating peptide-conjugated polyethylene glycol-containing lipid in a weight ratio of 1:0.5 to 1.5:0.5 to 1.5, specifically, 1:1:1, but is not limited thereto.

[0060] In the present invention, the step (S3) may be a step of adding the insulin solution to the (S2) solution dropwise and mixing by vortexing for 1 to 3 hours, but is not limited thereto.

[0061] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0062] Example

[0063] Example 1. Preparation of polymer micelles using insulin, DPPE-PEG, and DPPE-PEG-CPP

[0064] Prepare an insulin solution (5 mg / ml) by dissolving 10 mg of insulin in 2 mL of 10 mM HCl (pH 2.0) and adjusting the pH to 8.0 with 1.0 N NaOH. Then, add Solplus. ® (Content: 10 mg) and DPPE-PEG (wherein the content of DPPE-PEG(2.0k) is 10 mg, the content of DPPE-PEG(2.0k) is 10 mg, and the manufacturer of DPPE-PEG(2.0k) and DPPE-PEG(5.0k) is Biopharma PEG (Watertown, MA, USA), or DPPE-PEG-R9 (wherein the content of DPPE-PEG(2.0k)-R9 is 10 mg, and the content of DPPE-PEG(5.0k)-R9 is 10 mg, and DPPE-PEG(2.0k)-R9 and DPPE-PEG(5.0k)-R9 were manufactured through direct synthesis. The synthesis mechanism is shown in Fig. 2.) are dissolved in 2.0 mL of distilled water, and then added dropwise to the previously prepared insulin solution and mixed by vortexing for 2 hours. Amicon ® After performing ultrafiltration using a tube (10 kda, cutoff), the final polymer micelle solution is prepared by removing the aggregated particles using a 0.22 μm syringe filter (see Fig. 3).

[0065] ComponentSOLSP2kSP5kSPC2kSPC5kInsulin (INS)10 mg10 mg10 mg10 mg10 mgDPPE-PEG(2.0k)-10 mg---DPPE-PEG(5.0k)--10 mg--DPPE-PEG(2.0k)-R9---10 mg-DPPE-PEG(5.0k)-R9----10 mgPVCL-PVA-PEG (Soluplus ® )10 mg10 mg10 mg10 mg10 mg

[0066] (In Table 1 above, SOL is Soluplus, which is an amphiphilic polymer of insulin ® It means contained in micelle, and SP2k is insulin mixed with DPPE-PEG (2.0k), a polyethylene glycol lipid, and Soluplus, an amphiphilic polymer. ® It means contained in micelles, and SP5k is insulin mixed with DPPE-PEG (5.0k), a polyethylene glycol lipid, and Soluplus, an amphiphilic polymer. ® Meaning contained in micelles, SPC2k is a polyethylene glycol lipid conjugated with oligoarginine (R9), a cell penetrating peptide, DPPE-PEG(2.0k)-R9, and an amphiphilic polymer, Soluplus. ® It means contained in micelle, and SPC5k is a polyethylene glycol lipid conjugated with oligoarginine (R9), a cell penetrating peptide, and DPPE-PEG(5.0k)-R9, an amphiphilic polymer, and Soluplus. ® (meaning contained in micelles)

[0067] Hereinafter, the present invention will be described in more detail using experimental examples. These experimental examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by them.

[0068] Experimental example.

[0069] Experimental Example 1. Size and polydispersity index of insulin-loaded polymer micelles, transmission electron microscope (TEM) images, and encapsulation efficiency (EE) calculations.

[0070] Evaluation method

[0071] The particle size and polydispersity index (PDI) of insulin-loaded polymeric micelles were measured using a dynamic light scattering (DLS) device (Litesizer 500, Anton Paar, Graz, Austria), and the results are shown in Fig. 4 (A). The size measurement angle was automatically selected between side scattering (90°) and back scattering (175°).

[0072] And, in order to examine the insulin-loaded polymer micelles in more detail, TEM analysis was performed. The TME analysis images were analyzed using a transmission electron microscope (TEM, JEM-2100 Plus, JEOL, Tokyo, Japan), and the results are shown in (B) of Fig. 4.

[0073] In addition, in order to confirm the encapsulation efficiency of insulin encapsulated in polymer micelles, a portion of the insulin-loaded polymer micelles prepared according to Example 1 was accurately weighed, completely dissolved in 10 mL of DMSO (Dimethyl Sulfoxide, Sigma Aldrich), diluted 10-fold with the mobile phase, and then filtered through a 0.45 μm syringe filter. 10 μL of the filtrate was injected into a high-performance liquid chromatography (Agilent 1260 Infinity) for quantification. The analysis conditions of the high-performance liquid chromatography used for this experiment are as follows, and the encapsulation efficiency (%) of the drug was obtained using the following equation 1. The results are shown in (C) of Fig. 4.

[0074] [Formula 1]

[0075] Drug encapsulation efficiency (%) = (drug encapsulation amount (mg) / drug administration amount (mg)) X 100

[0076] (HPLC analysis conditions)

[0077] -HPLC system: Agilent 1260 infinity

[0078] -Column: YMC-Pack C8 (150 * 4.6 mm)

[0079] -Mobile phase A: Distilled water with 0.1% TFA (trifluoroacetic acid)

[0080] -Mobile phase B: Acetonitrile

[0081] -Flow rate: 1.0 ml / min

[0082] 0 min: Mobile phase A (71.0%), Mobile phase B (29.0%)

[0083] 2-3 min: Mobile phase A (64.0%), Mobile phase B (36.0%)

[0084] 6 min: Mobile phase A (71.0%), Mobile phase B (29.0%)

[0085] -Column temperature: 25℃

[0086] -UV detector: 210 nm

[0087] Evaluation results

[0088] Figure 4 (A) shows the results of the size and polydispersity index of the micelles. Referring to Figure 4 (A), it can be confirmed that the insulin-loaded polymer micelles manufactured according to the example were manufactured in a monodisperse form of 100 nm or less.

[0089] Fig. 4 (B) shows a transmission electron microscope (TEM) image of the micelle. Referring to Fig. 4 (B), it can be confirmed from the TEM image that spherical micelles of 100 nm or less were well manufactured.

[0090] Figure 4 (C) shows the encapsulation efficiency of the micelle. Referring to Figure 4 (C), it was confirmed that as DPPE-PEG and DPPE-PEG-CPP were added, the size of the micelle increased and the encapsulation efficiency improved, but no difference in size or encapsulation efficiency was observed according to the molecular weight of PEG (2.0 k and 5.0 k).

[0091] Experimental Example 2. Zeta potential and circular dichroism (CD) spectroscopy results of insulin solution and insulin-loaded polymer micelles.

[0092] Evaluation method

[0093] Zeta potential was measured five times by electrophoretic light scattering at 25°C and an electric field of 16 V / cm for the insulin solution and the insulin-loaded polymer micelles prepared according to Example 1, and the average value was calculated, and the results are shown in (A) of Fig. 5.

[0094] Circular dichroism spectroscopy was performed using a Chirascan Circular Dichroism Spectrometer equipped with a Peltier temperature controller. Spectra were recorded between 260 nm and 180 nm using a cuvette with a path length of 2 mm, and the results are shown in Fig. 5 (B).

[0095] Evaluation results

[0096] Referring to (A) of Fig. 5, the insulin solution exhibited a negative zeta potential value, whereas the insulin loaded into the micelle exhibited a zeta potential value close to neutral, confirming that the insulin was well encapsulated in the micelle.

[0097] In addition, referring to (B) of Fig. 5, the CD spectrum analysis results showed that the CD signals of all polymer micelles containing insulin were identical to the CD signals of the insulin solution, confirming that there was no change in the secondary structure or physiological activity of insulin during the process of manufacturing micelles.

[0098] Experimental Example 3. Observation of the diffusion effect of micelles in mucus derived from pig lungs.

[0099] Evaluation method

[0100] For each polymer micelle, the movement characteristics in porcine lung mucus were comparatively evaluated using the multiple particle tracking (MPT) method.

[0101] Specifically, 5 μl of micelles loaded with rhodamine instead of insulin were added to 150 μl of porcine lung mucus, vortexed for 10 minutes, and 50 μl of the mucus was sprayed onto a slide glass, followed by observation under a cone-shaped microscope. The mean-squared displacement (MSD) value was obtained using Equation 2 below, and the results are shown in Fig. 6.

[0102] [Formula 2]

[0103] MSD = Δr 2 (τ) = [x(t + τ)-x(τ)] 2 + [y(t + τ)-y(τ)] 2

[0104] (In the above equation 2, τ is the lag time between two points of the particle, and x(t) or y(t) represents the x and y positions of the particle at time t.)

[0105] Evaluation results

[0106] Referring to Figure 6, Solplus ®The behavior of micelles (SOL) containing only PEG showed a very restricted behavior, whereas the micelle formulations containing PEG (SP2k, SP5k, SPC2k, and SPC5k) traveled a longer distance and were found to diffuse well.

[0107] Mean square displacement (MSD) is the square of the distance traveled by a particle in a given time interval, and is proportional to the particle's diffusion rate. A larger MSD value indicates faster particle movement, and Solplus ® The formulations of micelles containing PEG (SP2k, SP5k, SPC2k, and SPC5k) showed significantly larger MSD values ​​than micelles manufactured alone (SOL). As a result, Soluplus ® While micellar formulations containing only PEG exhibited limited movement within mucus, micellar formulations containing PEG (SP2k, SP5k, SPC2k, and SPC5k) were observed to travel significantly longer distances. The PEG chain and neutral zeta potential values ​​are thought to minimize the affinity between mucus components, resulting in higher diffusion rates and enhanced mucus permeation.

[0108] Experimental Example 4. Observation of the diffusion effect of micelles in mucus derived from human non-small cell lung cancer cell line Calu-3.

[0109] Evaluation method

[0110] To evaluate the degree of mucin expression in the mucus layer derived from Calu-3 cells cultured for 14 to 17 days, staining was performed using Alexa-Fluor-488-labeled wheat germ agglutinin dye. Specifically, as shown in Figure 7, we examined whether mucin was expressed on the cell surface layer after 14 days of culture using the air-liquid-interface culture (AIC) method. Calu-3 cells cultured for 14 days were treated with prewarmed HBSS for 30 minutes, and then treated with 10 μl of rhodamine-labeled micelles. And after culturing at 37℃ for 1, 2, and 4 hours, it was washed three times with cold HBSS, fixed with 4% PFA (paraformaldehyde) for 10 minutes, stained with 50 μl of Alexa Fluor 488-labeled wheat germ allutinin (10 μg / ml) for 10 minutes, and observed under a confocal microscope, as shown in Fig. 8.

[0111] Evaluation results

[0112] The behavior of rhodamine-labeled micelle (red) particles is shown according to the treatment time in the fluorescence microscopic observation images of Fig. 8(A). After 4 hours of treatment with each micelle particle, it can be confirmed that the green fluorescence (mucus layer) is significantly covered by the strong red fluorescent particles in SP2k and SPC2k compared to the SOL particles. As explained in the MPT results (Experimental Example 3), SP2k and SPC2k show better diffusion in mucus than SOL micelle particles.

[0113] To quantify this, the average fluorescence intensity is shown in (B) of Fig. 8, and it was confirmed that SP2k and SPC2k showed significantly higher fluorescence intensity.

[0114] Experimental Example 5. Confirmation of the Cellular Uptake Effect of Rhodamine-Labeled Micelles

[0115] Evaluation method

[0116] Rhodamine (yellow)-loaded micelles were prepared and treated on calu-3 cells and a549 cells for 4 hours, respectively. Then, the nuclei of the cells were stained using Hoechest staining reagent (blue) to confirm the cellular uptake effect of Rhodamine (yellow)-loaded micelles.

[0117] At this time, Rhodamine (yellow)-loaded micelles were prepared in the same manner as described in Example 1, except that the final concentration of Rhodamine (yellow) was set to 5 μM instead of insulin.

[0118] Evaluation results

[0119] Referring to (A) of Fig. 9, the SPC2k micelle formulation manufactured using DPPE-PEG-CPP bound to CPP showed enhanced cellular uptake, and the average fluorescence intensity is shown in (B) of Fig. 9 to quantify this.

[0120] Referring to (B) of Fig. 9, SPC2k showed significantly stronger fluorescence intensity than SOL and SP2k micelles, and it is thought that the tendency of cellular uptake was enhanced due to the effect of CPP.

[0121] Experimental Example 6. Confirmation of the robustness of micellar particles through a nebulizer and particle atomization behavior using PIV.

[0122] Evaluation method

[0123] Figure 10 shows a photograph of a nebulizer used for atomization of micelles and a schematic diagram of the mechanism using ultrasonication as the atomization process.

[0124] To determine whether particles were well dispersed in the air during the atomization process using a nebulizer, distilled water and micelle solutions were evaluated using a particle image velocimetry (PIV) system, and the results are shown in Fig. 11. A high-speed camera (HAS-D71M, Ditect Corporation, Tokyo, Japan) and data processing software (Flownizer 2D, Ditect Corporation, Tokyo, Japan) were utilized in the PIV system.

[0125] In addition, a light scattering device (Litesizer 500, Anton Paar, Graz, Austria) was used to evaluate the particle size distribution before and after spraying micelle particles through a nebulizer, and the results are shown in Fig. 12.

[0126] Evaluation results

[0127] Referring to Figure 11, no significant difference was found in the spraying process of distilled water and micelle solution, and no difference was found between micelle formulations.

[0128] Referring to Figure 12, no significant change in the size of the micelle particles was observed after the nebulization process using the nebulizer. Therefore, it is believed that the robustness is maintained by maintaining the micelle size even after nebulization using the ultrasonication method.

[0129] Experimental Example 7. Aerosolization Performance Evaluation Using a Next Generation Impactor (NGI)

[0130] Evaluation method

[0131] To evaluate the aerosolization performance of micellar formulations, an evaluation was conducted using a next-generation impactor. The method for measuring the effective particle amount of the active ingredient delivered to the bronchial tubes was described in the United States Pharmacopeia (USP, Volume 28, Section <601> ) was applied. However, the nebulizer was Pari Velox shown in Fig. 10. ® A nebulizer was used, and the flow rate was 15 L / min until all 1 mL of the micellar solution was sprayed. The aerosolization performance was evaluated by calculating the amount of active ingredient that reached the bronchial tubes and deeper lung regions from stage 3 of the NGI and below, and the results are shown in Table 2 below. The fine particle fraction (FPF), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD) values ​​were presented as aerosolization evaluation indices.

[0132] Evaluation results

[0133] Referring to Table 2, when the FPF value was based on the active ingredient delivered at 5.39 μm or less (Stage 3 or less), there was no significant difference between the insulin solution (INS solution) and the remaining micellar formulations. However, when the FPF value was based on the active ingredient delivered at 3.3 μm or less (Stage 4 or less), it was confirmed that the FPF values ​​of the SP2k and SPC2k formulations were significantly higher than those of the INS solution. The results are shown in Fig. 13 (the FPF (%) of each formulation is shown when the FPF value was set to 3.3 μm or less, and the values ​​are the mean ± standard deviation, n is 3, * indicates a point that is significantly different from the INS solution, p < 0.05, and the Kruskal-Wallis test was performed to compare). This is because Solplus in the micellar formulation ®It is thought that the inclusion of DPPE-PEG and DPPE-PEG-CPP strengthens the structural rigidity of the micelles, allowing insulin to be delivered intact to higher stages, resulting in high FPF values. Figure 14 shows the amount of insulin deposited in each stage of NGI as a %. It was confirmed that the insulin solution (INS solution) was largely deposited in the front stages (S2 to S4), while the micelle solutions (SOL, SP2k, and SPC2k) were largely deposited in the rear stages (S4 to MOC).

[0134] ComponentINS solutionSOLSP2kSPC2kFPF < 5.39 μm(%)91.33 ± 4.0395.74 ± 0.7796.51 ± 0.7395.45 ± 1.04FPF < 3.3 μm(%)64.32 ± 5.6277.82 ± 1.8283.24 ± 5.6685.95 ± 3.46MMAD (μm)5.58 ± 1.484.36 ± 1.333.64 ± 1.113.38 ± 0.99GSD1.57 ± 0.021.75 ± 0.061.85 ± 0.051.77 ± 0.08

[0135] Experimental Example 8. Lung function assessment using double chamber plethysmography (DCP) equipment

[0136] Evaluation method

[0137] To determine whether there were any abnormalities in lung function when administering the micellar formulation via a nebulizer, pulmonary function was evaluated using a DCP device (see Fig. 15). Tidal volume (VT), specific airway resistance (sRaw), and enhanced pause (Pehn) values ​​were measured as evaluation indices. SD rats (8 weeks old, male) were administered 1 mL of normal saline, insulin solution (INS solution), and each micellar formulation via a nebulizer, and immediately thereafter evaluated using the DCP device, and the results are shown in Fig. 16.

[0138] Evaluation results

[0139] Referring to Figure 16, no formulation showed significant differences from normal saline in any of the three indices (VT, sRaw, and Pehn). Therefore, it is believed that the micellar formulation does not affect the lung function of animals.

[0140] Experimental Example 9. MTT Assay Evaluation Using A549 Cells

[0141] Evaluation method

[0142] Cytotoxicity evaluation (cell viability evaluation) of A549 cells was performed using the MTT assay. Specifically, A549 cells were seeded at a density of 20,000 cells per well in 96-well plates and cultured for 24 hours at 37°C and 5% CO2. The medium was removed, and the cells were treated with an insulin solution and each micelle formulation prepared according to Example 1 at an insulin concentration of 0.01–1.00 mg / mL for 24 hours (n = 3). The medium was then removed, and 100 μL of thiazolyl blue tetrazolium bromide (MTT) solution (0.5 mg / mL) was added, followed by incubation for 4 hours. Afterwards, the medium was removed, 100 μL of DMSO was added, and the plate was shaken at 200 rpm for 10 minutes on a rotary shaker (N-BIOTEK, NB-101S, Bucheon, Korea). Absorbance was measured at 540 nm using a microplate reader (Spectra Max ID3, Molecular Devices, San Jose, CA, USA). All data were processed and analyzed using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA, USA).

[0143] Evaluation results

[0144] Referring to Figure 17, when treated with insulin at concentrations ranging from 0.01 mg / ml to 1.00 mg / ml for 24 hours, a survival rate of over 90% was observed. Therefore, the micellar formulation demonstrates no issues with biocompatibility with alveolar A549 cells.

[0145] Experimental Example 10. Test to confirm the blood sugar-lowering effect of micellar formulations in a diabetic rat model.

[0146] Evaluation method

[0147] To induce diabetes, a single intraperitoneal injection of 60 mg / kg streptozotocin was administered to animals (SD rats, 8 weeks old, male). Normal saline and insulin solution (INS solution) were administered to the diabetic animals by subcutaneous (SC) injection and intratracheal instillation (ITI). Each micellar formulation prepared according to Example 1 was also administered directly into the lungs by ITI. The insulin concentration was 2 IU / kg in all cases.

[0148] Evaluation results

[0149] Referring to Figure 18, insulin solution and micellar formulation were administered to diabetic animals at a concentration of 2 IU / kg via subcutaneous injection or intratracheal infusion. As a result, when SP2k and SPC2k formulations were administered, they showed a significant blood glucose lowering effect compared to the insulin solution (subcutaneous injection or intratracheal infusion) and PM formulation. It is expected that the insulin solution and SOL formulation have restricted movement in the mucus layer and are therefore removed by mucociliary clearance. Therefore, it is thought that PEGylated SP2k and SPC2k quickly penetrate the mucus layer due to the PEG chain and quickly reach the cell membrane, thereby avoiding mucociliary clearance. However, since there was no significant difference between the SP2k and SPC2k formulations, it is thought that the mucus permeation rate is a more important absorption rate-determining step than the cell membrane permeation rate in insulin absorption.

[0150] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the relevant technical field that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. (S1) A step of preparing an insulin solution with adjusted pH by dissolving insulin; (S2) a step of dissolving a polyethylene glycol-hydrated lipid conjugated with an amphiphilic polymer or a cell-penetrating peptide; (S3) a step of adding the insulin solution to the (S2) solution and mixing; and (S4) A step of removing aggregated particles after ultra-filtration of the mixed solution of (S3); A method for producing a mucus-permeable polymer micelle comprising insulin.

2. A method for producing a mucus-permeating polymer micelle including insulin, characterized in that in the first paragraph, the amphiphilic polymer is at least one selected from the group consisting of a polyvinyl caprolactam (PCL)-polyvinylacetate (PVA)-polyethylene glycol (PEG) graft copolymer, a low-saponification polyvinyl alcohol, polyacrylamidomethylpropanesulfonic acid (Poly(AMPS)), polyvinyl pyrrolidone, a polyvinyl pyrrolidone / vinyl acetate copolymer, and a polyvinyl pyrrolidone / alkyl acrylate copolymer.

3. In the first paragraph, the polyethylene glycol lipid is DSPE-PEG1000, DMPC-PEG1000, DOPC-PEG1000, DPPC-PEG1000, POPC-PEG1000, DOPE-PEG1000, DMPE-PEG1000, POPE-PEG1000, DPPE-PEG1000, DSPE-PEG2000, DMPC-PEG2000, DOPC-PEG2000, DPPC-PEG2000, POPC-PEG2000, DOPE-PEG2000, DMPE-PEG2000, POPE-PEG2000, DPPE-PEG2000, DSPE-PEG5000, DMPC-PEG5000, DOPC-PEG5000, DPPC-PEG5000, POPC-PEG5000, DOPE-PEG5000, DMPE-PEG5000, A method for producing a mucus-permeating polymer micelle containing insulin, characterized in that at least one selected from the group consisting of POPE-PEG5000 and DPPE-PEG5000.

4. A method for producing a mucus-penetrating polymer micelle containing insulin, characterized in that in the first paragraph, the cell-penetrating peptide is at least one selected from the group consisting of oligoarginine (R9), TAT, R8, DPV3, DPV6, penetratin, pVEX, MPG, MAP, transportan, Bip4, C105Y, and Melittin.

5. A method for producing a mucus-permeable polymer micelle containing insulin, characterized in that in the first paragraph, (S3) is a step of adding the insulin solution to the (S2) solution dropwise one by one and mixing by vortexing for 1 to 3 hours.

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