Lactoferrin-based gene carrier for type 2 diabetes treatment

Abstract

The present invention relates to a gene delivery complex comprising: a biocompatible polymer backbone; and pegylated lactoferrin connected to the biocompatible polymer backbone by means of a covalent bond. The gene delivery complex is orally administered into an individual, can be absorbed in vivo by means of a lactoferrin receptor, and enables the in vivo delivery of a target gene and the expression thereof.

Claims

1. A gene delivery complex comprising: (a) a glycol chitosan; and (b) pegylated lactoferrin connected to the glycol chitosan by means of a covalent bond.

2. A gene carrier comprising: a vector comprising a target gene fragment to be delivered in vivo; and the gene delivery complex of claim 1.

3. The gene carrier of claim 2, wherein the target gene fragment comprises the sequence of SEQ ID No. 1.

4. The gene carrier of claim 2, wherein the gene carrier has a binding ratio of the vector to the gene delivery complex of 1:2 to 1:15.

5. The gene carrier of claim 2, wherein the gene carrier is orally administered.

6. A pharmaceutical composition comprising the gene carrier of claim 2 as an active ingredient.

7. The pharmaceutical composition of claim 6, wherein the gene carrier comprises the sequence of SEQ ID No. 1.

8. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is for oral administration.

9. A method for treatment of type 2 diabetes mellitus in a subject in need thereof, the method comprising: administering orally, to the subject, an effective amount of a composition comprising the gene carrier of claim 2 as an active ingredient, wherein the composition is for oral administration.

10. The method of claim 9, wherein the gene carrier comprises the sequence of SEQ ID No. 1.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1A illustrates a process of synthesizing gCS-PEG-hydrolyzed MAL by reacting glycol chitosan (gCS) with N-hydroxylsuccinimide polyethylene glycol maleimide (NHS-PEG-MAL), FIG. 1B illustrates a process of synthesizing thiolated LF (SH-Lf) by reacting a Traut's solution with lactoferrin (Lf), and FIG. 1C illustrates a process of synthesizing gCS-PEG-Lf by reacting gCS-PEG-hydrolyzed MAL with SH-Lf.

(2) FIG. 2A illustrates the attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) analysis results of gCS, NHS-PEG-MAL, and gCS-PEG-hydrolyzed MAL.

(3) FIG. 2B illustrates the ATR-FTIR analysis results of Lf and SH-Lf.

(4) FIG. 2C illustrates the ATR-FTIR analysis results of gCS-PEG-hydrolyzed MAL, SH-Lf, and gCS-PEG-Lf.

(5) FIG. 3A illustrates the results of observing cells by an optical microscope after treating Caco-2 cells with gCS-PEG-Lf, and FIGS. 3B and C illustrate the results of confirming the presence or absence of cytotoxicity with a Live and Dead Cell Kit and a cell counting kit-8 (CCK-8) after treating Caco-2 cells with gCS-PEG-Lf.

(6) FIG. 4 illustrates the results of confirming the presence or absence of endocytosis of gCS-PEG-Lf by a lactoferrin receptor through a transepithelial electrical resistance (TEER) experiment.

(7) FIG. 5 illustrates the results of performing a gel retardation experiment after reacting a GFP gene with gCS-PEG-Lf by modifying the binding ratio of the GFP gene to the gCS-PEG-Lf.

(8) FIG. 6A illustrates the results of measuring the Zeta potential of a reaction solution after reacting a GFP gene with gCS-PEG-Lf by modifying the binding ratio of the GFP gene to the gCS-PEG-Lf.

(9) FIG. 6B illustrates the results of measuring the Zeta size of a reaction solution after reacting a GFP gene with gCS-PEG-Lf by modifying the binding ratio of the GFP gene to the gCS-PEG-Lf.

(10) FIG. 7 illustrates the results of isolating each tissue and staining the tissue with hematoxylin & eosin after orally administering GFP/gCS-PEG-Lf to mice and the results of confirming the expression levels of GFP.

(11) FIG. 8 illustrates the results of confirming the expression levels of fibroblast growth factor 21 (FGF21) and insulin by isolating each tissue after orally administering FGF21/gCS-PEG-Lf to mice.

(12) FIG. 9A illustrates the results of measuring the blood levels of an FGF21 protein after administering FGF21/gCS-PEG orally or by intraperitoneal injection to/into mice.

(13) FIG. 9B illustrates the results of measuring blood levels of insulin after administering FGF21/gCS-PEG-Lf orally or by intraperitoneal injection to/into mice.

(14) FIG. 10 illustrates the results of confirming the GFP expression levels after treating Langerhans islets of rats with GFP/gCS-PEG-Lf.

(15) FIG. 11 illustrates the results of confirming whether type 2 diabetes mellitus is induced after supplying a high fat diet to mice by an insulin tolerance test (ITT).

(16) FIG. 12A illustrates the results of measuring the body weight over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(17) FIG. 12B illustrates the results of measuring the body weight over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(18) FIG. 12C illustrates the results of measuring the average feed intake over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(19) FIG. 13A illustrates the results of measuring the non-fasting blood glucose level over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(20) FIG. 13B illustrates the results of expressing the non-fasting blood glucose level by percentage over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(21) FIG. 13C illustrates the results of measuring the fasting blood glucose level over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(22) FIG. 13D illustrates the results of expressing the fasting blood glucose level by percentage over time while orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice.

(23) FIG. 14A illustrates the results of measuring the FGF21 levels in serum after orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice for a short period of time or a long period of time.

(24) FIG. 14B illustrates the results of measuring the insulin levels in serum after orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice for a short period of time or a long period of time.

(25) FIG. 15 illustrates the results of confirming the expression levels of FGF21 and insulin by isolating each tissue after orally administering FGF21/gCS-PEG-Lf to type 2 diabetes mellitus model mice for a short period of time or a long period of time.

MODES OF THE INVENTION

(26) Hereinafter, one or more specific exemplary embodiments will be described in more detail through Examples. However, these Examples are provided only for exemplarily explaining the one or more specific exemplary embodiments, and the scope of the present invention is not limited to these Examples.

Example 1: Synthesis of Glycol Chitosan-PEG-Lactoferrin

(27) 1-1. Synthesis of Glycol Chitosan-PEG-Lactoferrin

(28) After glycol chitosan (hereinafter, referred to as gCS) was dissolved in phosphate buffered saline (PBS; pH 8.0), N-hydroxylsuccinimide polyethylene glycol maleimide (NHS-PEG-MAL) was added thereto and the resulting mixture was reacted for 20 minutes. A reactant (gCS-PEG-hydrolyzed MAL) in the form of a powder was obtained by dialyzing the reaction solution for 12 hours or more and freeze-drying the dialysate.

(29) Ethylenediaminetetraacetic acid sodium salt (sodium EDTA) was dissolved in PBS (pH 8.0), and lactoferrin (hereinafter, referred to as Lf) and a Traut's solution were put into the resulting solution, and the resulting mixture was reacted for 1 hour. After the reaction, dialysis was performed in a refrigerator for 12 hours or more. The reactant powder (gCS-PEG-hydrolyzed MAL) was dissolved in PBS (pH 6.8), the dialyzed lactoferrin reactant (including SH-Lf) was added thereto, and the resulting mixture was reacted for 1 hour. Thereafter, dialysis was performed for 12 hours or more, the dialysate was freeze-dried, and then gCS-PEG-Lf in the form of a powder was obtained.

(30) FIG. 1 schematically illustrates the glycol chitosan-PEG-lactoferrin synthesis process.

(31) 1-2. Confirmation of Synthesized gCS-PEG-Lf

(32) The materials before the synthesis, gCS and NHS-PEG-MAL, and the synthesized materials gCS-PEG-hydrolyzed MAL and gCS-PEG-Lf were analyzed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR).

(33) As a result, as illustrated in FIG. 2A, it could be confirmed that in the synthesized material gCS-PEG-hydrolyzed MAL, peaks at 3382 cm.sup.−1 and 3300 cm.sup.−1 corresponding to gCS and peaks at 2875 cm.sup.−1 and 520 cm.sup.−1 corresponding to NHS-PEG-MAL were all present. The peak corresponding to COOH newly formed by a hydrolysis reaction of MAL could be confirmed at 1696 to 1729 cm.sup.−1.

(34) Further, as illustrated in FIG. 2B, it could be seen that in the thiolated Lf (SH-Lf), peaks (1630 cm.sup.−1, 1525 cm.sup.−1) corresponding to existing Lf were present, and a new peak (2550 cm.sup.−1) for S—H was formed.

(35) In addition, as illustrated in FIG. 2C, as a result of analyzing gCS-PEG-Lf, it could be seen that peaks (1630 cm.sup.−1, 1525 cm.sup.−1) corresponding to existing Lf were present, and gCS-PEG-Lf had a peak (1050 cm.sup.−1) corresponding to gCS-PEG-MAL.

Example 2: Confirmation of Cytotoxicity of gCS-PEG-Lf

(36) As a result of treating small intestine epithelium-derived cells Caco-2 cells with gCS-PEG-Lf at different concentrations (0, 1, 2.5, 3.5, 5, 7.5, 10, and 12.5 mg/ml), as illustrated in FIG. 3A, it could be confirmed by an optical microscope that as the concentration of gCS-PEG-Lf treatment was increased, Caco-2 cells were killed.

(37) Furthermore, the same experiment was performed using a Live and Dead Cell Kit (Sigma-Aldrich, USA). As a result, as illustrated in FIG. 3B, it could be seen that as the concentration of gCS-PEG-Lf was increased, red fluorescence was increased, indicating that the death of Caco-2 cells was increased.

(38) After Caco-2 cells were treated with gCS-PEG-Lf for 24 hours, cell viability was confirmed by a cell counting kit-8 (CCK-8). As a result, as illustrated in FIG. 3C, it could be quantitatively seen that as the concentration of gCS-PEG-Lf was increased, the viability of Caco-2 cells was decreased. Further, it was confirmed that the IC.sub.50 value of gCS-PEG-Lf was 3.7308 mg/ml.

Example 3: Confirmation of Endocytosis of gCS-PEG-Lf by Lactoferrin Receptor

(39) A monolayer was formed by culturing Caco-2 cells in a transwell plate with a pore size of 0.4 μm, and a transepithelial electrical resistance (TEER) experiment was performed.

(40) Cultured Caco-2 cells (2×10.sup.4 cells/insert) were divided into a control (Con), a gCS (1,000 μg/ml) treatment group, a gCS-PEG treatment group, a gCS-PEG-Lf (1,000 μg/ml) treatment group, and a Lf+gCS-PEG-Lf treatment group and TEER values over time were measured after the corresponding treatment was performed. The Lf+gCS-PEG-Lf treatment group was treated with gCS-PEG-Lf (1,000 μg/ml) after an Lf receptor was saturated with Lf (1,000 μg/ml) for 3 hours.

(41) As a result of measurement, as illustrated in FIG. 4, it could be confirmed that, compared to the treatment group with unbound Lf, the TEER value was reduced over time in the gCS-PEG-Lf treatment group. However, it could be seen that gCS-PEG-Lf was endocytosed by the Lf receptor by confirming that the TEER value was not decreased in the Lf+gCS-PEG-Lf treatment group.

Example 4: Confirmation of Binding Ratio of gCS-PEG-Lf and DNA

(42) 4-1. Gel Retardation Experiment

(43) A reaction was carried out for 30 minutes by varying the binding ratio of 5 μg of the GFP gene and gCS-PEG-Lf (0, 5, 10, 15, 20, 35, and 50 μg). After the completion of the reaction, the product was loaded onto an agarose gel and electrophoresed at 100 V for 40 minutes.

(44) As a result, as illustrated in FIG. 5, it could be confirmed that as the ratio of the GFP gene and the gCS-PEG-Lf was increased, the binding force of the GFP gene and the gCS-PEG-Lf was increased. Furthermore, it could be seen that when the binding ratio (mass ratio) of the GFP gene and the gCS-PEG-Lf was 4 or higher, the GFP gene was bound to the gCS-PEG-Lf in the most ideal manner.

(45) 4-2. Measurement of Zeta Size and Zeta Potential

(46) After carrying out a reaction for 30 minutes by varying the binding ratio of the GFP gene and the gCS-PEG-Lf, a 0.2 mg/ml reaction solution was put into a disposable cuvette and mixed well. The Zeta potential and Zeta size of the GFP/gCS-PEG-Lf solution were measured using a dynamic light scattering (DLS) device.

(47) As a result, as illustrated in the Zeta potential graph shown in FIG. 6A, it could be seen that a negative charge appeared due to a phosphoric acid group present in the GFP gene, a positive charge appeared due to gCS-PEG-Lf, and when the binding ratio of the GFP gene and the gCS-PEG-Lf was 1:5, a charge closest to 0 appeared. In addition, in the Zeta size graph shown in FIG. 6B, it was confirmed that when the binding ratio of the GFP gene and the gCS-PEG-Lf was 1:3 or 1:5, the smallest size was exhibited.

Example 5: Confirmation of Targeting of gCS-PEG-Lf by Oral Administration

(48) The gCS-PEG-Lf synthesized to treat type 2 diabetes mellitus is absorbed in vivo and delivered to tissues after oral administration. Since the gCS-PEG-Lf encounters an acidic pH environment, proteolytic enzymes, and the like in the stomach during the process of reaching each tissue, it was confirmed whether the gCS-PEG-Lf was stably delivered to tissues under such an environment.

(49) The GFP/gCS-PEG-Lf was orally administered at a concentration of 100 μg/500 μg per mouse, and the brain, the heart, the duodenum, the jejunum, the ileum, the kidneys, the liver, the pancreas, the spleen, and the lungs were isolated by sacrificing the mouse three days later. A tissue block was prepared by embedding the isolated tissues in an OCT compound, and the tissue block was frozen in a cryogenic refrigerator for 24 hours. Thereafter, the tissue block was subjected to cryosection, hematoxylin & eosin staining, and observed under an optical microscope. Further, the degree of GFP expression was confirmed by an optical microscope.

(50) As a result, as illustrated in FIG. 7, GFP fluorescence signals could be confirmed in each tissue when the GFP/gCS-PEG-Lf was orally administered, as compared to the control. This means that the GFP/gCS-PEG-Lf is stably delivered to each tissue.

Example 6: Comparison of Expression Levels of FGF21 and Insulin According to Dosage of FGF21 Gene

(51) The FGF21 gene of SEQ ID No. 1 was used by being inserted into the EcoRI and SalI sites of a pCMV6-XL5 plasmid vector (ORIGENE, 4.7 kb). FGF21/gCS-PEG-Lf (FGF21 gene:gCS-PEG-Lf=1:5) including the FGF21 gene (200 or 500 μg) was orally administered to a mouse, and the liver, the duodenum, the jejunum, the ileum, and the pancreas were isolated by sacrificing the mouse three days later. The isolated organs were fixed with paraffin and then cut to prepare a tissue section, which was then subjected to immunohistochemistry (IHC) staining with an FGF21 antibody and an insulin antibody. As a result, as illustrated in FIG. 8, it could be confirmed that the higher the oral dose of the FGF21 gene was, the higher the expression levels of the FGF21 protein and insulin were. Based on this result, the oral dose of the FGF21 gene to be used in in vivo experiments was determined.

Example 7: Comparison of Blood FGF21 and Insulin Concentrations According to Administration Method

(52) The dose of the FGF21 gene was set at 500 μg, and FGF21/gCS-PEG-Lf (FGF21 gene:gCS-PEG-Lf=1:5) was administered orally to or injected intraperitoneally (i.p) into 10-week old C57BL6J mice. Three days later, the mice were sacrificed, blood was collected through the abdominal vein, and only plasma was isolated by centrifugation. ELISA experiments for FGF21 and insulin were performed on the isolated plasma.

(53) As a result, as illustrated in FIG. 9A, it could be confirmed that when FGF21/gCS-PEG-Lf was intraperitoneally injected, the blood FGF21 protein concentration was higher than when FGF21/gCS-PEG-Lf was orally administered. However, there was no significant difference in the blood insulin concentration (FIG. 9B), and since this was an experiment performed on normal mice, it was considered that there was no significant difference in the blood insulin concentration due to the fact that FGF21 affecting the amount of insulin secreted in the type 2 diabetes mellitus model did not show this effect.

Example 8: Confirmation of Endocytosis of GFP/gCS-PEG-Lf by Beta Cells of Pancreas

(54) From rats, Langerhans islets of the pancreas were separated, purified and cultured. After 5 μg of GFP and 25 μg of gCS-PEG-Lf were mixed and reacted at room temperature for 30 minutes, the Langerhans islets were treated with the reactant (GFP/gCS-PEG-Lf) for 4 hours. Four hours later, the medium was replaced, and the Langerhans islets were further cultured for 48 hours. After completion of the culture, the presence or absence of GFP expression was observed under a fluorescence microscope.

(55) As a result, as illustrated in FIG. 10, it could be confirmed that GFP was expressed inside the Langerhans islets, which means that gCS-PEG-Lf was transferred into the Langerhans islets through endocytosis.

Example 9: Effect of Alleviating Type 2 Diabetes Mellitus by Administration of FGF21/gCS-PEG-Lf

(56) 9-1. Preparation of Type 2 Diabetes Mellitus Animal Model

(57) C57BL6J mice were classified into a normal diet group (Normal; n=5) and a high fat diet group (HFD; n=5), and fed the corresponding feed for 14 weeks. Thereafter, an insulin tolerance test (ITT) was performed in order to confirm whether type 2 diabetes mellitus was induced. After a 6-hour fast in both groups of mice, an insulin solution (0.75 U/kg) was injected intraperitoneally. After the injection, blood glucose levels were checked at a predetermined time interval.

(58) As a result, as illustrated in FIG. 11, it could be confirmed that the blood glucose level was higher at all times in the high fat diet group compared to the normal diet group. From these results, it could be determined that type 2 diabetes mellitus was induced in the high fat diet group.

(59) 9-2. Effect of Improving Blood Glucose Level and Body Weight by Administration of FGF21/gCS-PEG-Lf

(60) 500 μg of the FGF21 gene was used per mouse. FGF21/gCS-PEG-Lf was obtained by reacting 500 μg of the FGF21 gene and 2500 μg of gCS-PEG-Lf at room temperature for 30 minutes. HFD mice were divided into a control (HFD control), an FGF21 short-term administration group (HFD+FGF21 short term), and an FGF21 long-term administration group (HFD+FGF21 long term), and FGF21/gCS-PEG-Lf was orally administered once every four days. Before FGF21/gCS-PEG-Lf was orally administered, a sodium borate (SB) buffer was orally administered. FGF21/gCS-PEG-Lf was administered three times in total to the FGF21 short-term administration group, and nine times in total to the FGF21 long-term administration group. During the experiment, the body weight, food intake and non-fasting blood glucose level were measured once every two days, and the fasting blood glucose level was measured once every four days immediately before administration of FGF21/gCS-PEG-Lf.

(61) As a result, as illustrated in FIGS. 12A and 12B, it could be confirmed that as time elapsed, the body weights were decreased in the FGF21 administration groups (short term & long term). However, it could be seen that in the case of the FGF21 short-term administration group, the body weight was increased after the oral administration of FGF21/gCS-PEG-Lf was stopped. Meanwhile, there was no significant difference in food intake between the control and the FGF21 administration groups (short term & long term) (FIGS. 12C and 12D).

(62) Furthermore, as illustrated in FIGS. 13A to 13D, the blood glucose level also exhibited a trend similar to the body weight. Through this, it could be seen that the effect of reducing body weight and blood glucose level was due to the oral administration of FGF21/gCS-PEG-Lf, and it was confirmed that the administration of FGF21/gCS-PEG-Lf was effective for the treatment of type 2 diabetes mellitus.

(63) 9-3. Confirmation of Blood FGF21 and Insulin Levels by Administration of FGF21/gCS-PEG-Lf

(64) After the completion of the oral administration experiment of FGF21/gCS-PEG-Lf, FGF21 and insulin levels were measured through ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunospecific assay) by collecting blood from mice and isolating plasma.

(65) As a result, as illustrated in FIG. 14A, it could be seen that in the normal group to which a HFD was not supplied, the FGF21 concentration was shown to be about 0.5 ng/ml, and the control (HFD Con) and the FGF21 short-term administration group exhibited higher FGF21 concentrations than the normal group. However, it could be confirmed that the blood FGF21 concentration in the FGF21 long-term administration group was remarkably higher than those in the other experimental groups.

(66) Further, as illustrated in FIG. 14B, the blood insulin concentration also exhibited a trend similar to FGF21. The normal group exhibited the lowest concentration, and the control and the FGF21 short-term administration group showed higher concentrations. It could be seen that the insulin concentration was highest in the FGF21 long-term administration group.

(67) It is considered that the results of the FGF21 short-term administration group and the control were similar because the FGF21 gene was removed (clearance) when a long time had elapsed after oral administration of FGF21/gCS-PEG-Lf.

(68) Through the present experiment, it could be confirmed that the concentrations of blood FGF21 protein and insulin could be increased by orally administering FGF21/gCS-PEG-Lf.

(69) 9-4. Confirmation of Change in FGF21 and Insulin Expression Levels by Administration of FGF21/gCS-PEG-Lf

(70) After the completion of the oral administration experiment of FGF21/gCS-PEG-Lf, the brain, the duodenum, the jejunum, the ileum, the heart, the kidneys, the liver, the lungs, and the pancreas were isolated by sacrificing the mice in each experimental group. After the organs isolated according to the method in Example 5 were subjected to immunohistochemical staining, the organs were observed under a microscope.

(71) As a result, as illustrated in FIG. 15, it could be seen that the expression levels of FGF21 (green) and insulin (red) in the control (HFD Con) were very low, and the FGF21 short-term administration group exhibited results similar to those of the control. However, it could be confirmed that in the FGF21 long-term administration group, the expression levels of FGF21 and insulin were high. Through these results, it was confirmed that the expression of the FGF21 protein and insulin could be induced in each organ by orally administering FGF21/gCS-PEG-Lf.

(72) In the foregoing, the present invention has been examined mainly based on the preferred examples thereof. A person with ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed examples should be considered not from a restrictive viewpoint, but from an explanatory viewpoint. The scope of the present invention is defined by the claims rather than the above-described description, and it should be interpreted that all the differences within a scope equivalent thereto are included in the present invention.