KR102394596B1 - external stimulus insulin smart drug delivery system - Google Patents

Abstract

The present invention provides an insulin drug delivery system comprising a polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin, which is self-regulated by external stimuli such as glucose concentration, pH, temperature or wavelength In this regard, the insulin drug delivery system according to the present invention has the advantage of being able to easily control drug release without difficulty and inconvenience of biosensor or blood sugar check.

Description

Insulin smart drug delivery system that responds to the outside {external stimulation insulin smart drug delivery system}

The present invention relates to a polyhedral oligomeric silsesquioxane (POSS)-based insulin drug delivery system. It relates to an insulin smart drug delivery system that can self-regulate.

Diabetes is a group of metabolic diseases caused by the failure of blood sugar level control mechanisms. As of 2014, 387 million people worldwide had diabetes, and it is estimated that this number will increase to 592 million by 2035. Traditional treatments for diabetics often require blood sugar monitoring and insulin injections to maintain normal blood sugar. However, such self-administration is associated with pain and often inadequate glycemic control. Inadequate glycemic control increases the risk of diabetic complications, including amputation, blindness, and kidney failure. Hypoglycemia can also result in behavioral and cognitive impairment, seizures, loss of consciousness, coma, brain damage, or death.

On the other hand, although it is not a fundamental treatment method for type 1 diabetes, the best treatment is to check blood sugar using a biosensor and directly administer an appropriate amount of a blood sugar lowering agent such as insulin to the diabetic patient. However, the method of administration through injection causes a situation such as a sudden drop in blood sugar level, endangering the life of the patient. In addition, in the case of injection, it is painful, and it is not good for patient compliance to receive multiple injections per day. In order to solve this problem, studies on insulin administration methods are being actively conducted at home and abroad. Representative examples include aerosol inhalation (pulmonary delivery), transdermal delivery using microneedles, and oral delivery. However, these methods have problems in that insulin is denatured, loss is large in the course of administration, and bioavailability is low. Recently, a self-regulated glucose-responsive insulin delivery system can maintain a normal blood glucose concentration by releasing a drug in response to a change in blood sugar and self-responding to a change in blood sugar for a long time when administered once.

Examples of glucose-responsive moieties include glucose oxidase (GOx), phenylboronic acid (PBA), and glucose binding protein (GBP). In the case of enzymes and proteins, there are difficulties in handling and loss due to oxidation in air, and PBA and its derivatives exhibit high affinity with cis-diol compounds through chemical reversible boronate group formation. It confers promising potential for glucose-sensitive drug delivery in However, there is a problem that PBA is formed only under alkaline conditions of pH 8.5 or higher, and in order to induce a stable reaction even in a neutral state, an amino group is added to the boric acid group to protect the boric acid-polyol bond from the nucleophilic attack of water molecules, but the boric acid group Functional groups, such as amino groups, have a problem of lowering the yield when synthesizing other functional materials due to their high reactivity.

On the other hand, Patent Document 1 discloses a hybrid nanoprobe comprising POSS functionalized with PBA and a dye. has a limitation in the glycemic control, and because it merely serves as a sensing role of glucose, it has a limitation in that spontaneous glucose control or quantitative control according to the presence of glucose is difficult.

(1) Republic of Korea Patent Publication No. 10-2019-0024686 (2019.03.08.)

Accordingly, in order to overcome the problems and/or limitations of the existing technology as described above, the present invention includes nanopores under physiological conditions, and is used as a core part of micelles for the following reasons (i) to (v). We provide an organic-inorganic hybrid smart drug delivery system containing POSS, which, by assembling inorganic molecules of POSS with boronic acid, reacts sensitively to glucose under human pH conditions and releases insulin in an exchange reaction manner.

(i) The cubic structure of POSS has a three-dimensional spherical shape, contains 8 alkyl groups, is dissolved in an organic solution, and it is easy to synthesize and assemble with organic substances.

(ii) The phenyl group of phenylboronic minimizes the overlap of benzene groups due to the steric hindrance of POSS. react in yield.

(iii) enables stable boric acid-polyol bonding in the neutral state.

(iv) POSS loads the protein insulin drug to give physicochemical stability to minimize the denaturation of the protein drug.

(v) The loading efficiency of hydrophobic drugs is high.

More specifically, it provides a new smart insulin delivery system that responds to glucose including insulin-loaded polysilsesquoxane (POSS).

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to one embodiment of the present invention, there is provided an insulin drug delivery system comprising polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin.

According to one aspect, there is provided a method for controlling insulin release through an insulin drug carrier that is self-regulated by external stimuli such as glucose concentration, pH, temperature or wavelength, wherein the insulin drug carrier is functionalized with aminophenylboronic acid (APBA) A method for controlling insulin release comprising polyhedral oligomeric silsesquioxane (POSS) and insulin can be provided.

According to one side, in the method for preparing an insulin drug carrier comprising polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin,

a) synthesizing polyhedral oligomeric silsesquioxane (POSS-APBA) functionalized with aminophenylboronic acid by mixing polyhedral oligomeric silsesquioxane (POSS) and aminophenylboronic acid (APBA);

b) reacting with insulin and polyethylene glycol to prepare polyethylene glycol-insulin (PEG-Insulin); and

c) mixing the POSS-APBA with PEG-Insulin;

It provides a method for producing an insulin drug delivery system comprising a.

Since the insulin drug delivery system according to an aspect of the present invention is characterized by self-regulation by external stimuli such as glucose concentration, pH, temperature or wavelength, there are disadvantages to the existing blood sugar control (the inconvenience of repeated blood sugar check and the pancreatic area). It can be expected to have an effect that can compensate for pain in the form of insulin injection). In addition, the insulin is rapidly released even at a low glucose concentration, and thus the response sensitivity is excellent. As the glucose concentration increases, the release amount of the insulin drug also increases, which has great functionality as an insulin smart drug delivery system.

In addition, by binding APBA to POSS, it exhibits a characteristic of releasing insulin through binding to glucose in various pH ranges (pH 3 to 10), confirming the biocompatibility of the insulin drug delivery system according to the present invention, and confirming the drug By elucidating the reaction mechanism during the preparation of the delivery system, it has the advantage that glucose self-regulation and quantitative control can be effectively performed while at the same time being able to analyze the glucose in the body.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 (a) is a view showing the synthesis method of POSS-APBA, PEG-modified Insulin and POSS-APBA@Insulin.
FIG. 1(b) is a diagram illustrating a process in which POSS-APBA@Insulin nanoparticles are generated by reacting with glucose and releasing insulin from POSS-APBA@Insulin nanoparticles.
2 is a view showing FT-IR spectra of POSS, APBA, POSS-APBA, POSS-APBA@Insulin modified nanoparticles (NPs, nanoparticles).
3 is a view showing the results of a glucose-responsive release experiment (in vitro) of POSS-APBA@Insulin particles.
Fig. 4(a) shows the change of zeta potential according to pH, Fig. 4(b) shows the zeta size, and Fig. 4(c) shows the Electro-Osmosis plot.
5 is a view showing the size and shape of POSS-APBA@Insulin NPs before (A) / after (B, C, D) reaction with glucose.
6 is a diagram showing cell viability, morphology, and immunoblotting results when POSS-APBA and POSS-APBA@Insulin were treated with respect to HeLa, HDF and HUVE cells.
7 is a view showing Y Data of POSS-APBA@Insulin according to the presence or absence of glucose for each pH.
8 is a diagram showing circular dichroism spectroscopy (CD) spectra of insulin, PEG-Insulin, and POSS-APBA@Insulin.

Hereinafter, the present invention will be specifically described.

According to one embodiment of the present invention, there is provided an insulin drug delivery system comprising polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin.

Polyhedral oligomeric silsesquioxane (POSS) is a cage-shaped molecule (R ₈ Si ₈ O ₁₂ ) with a silicon-oxygen bond and a vertex group (R: hydrogen, alkyl, alkene, aryl, arylene, etc.) and has a size of 1 to 3 nm. Polyhedral oligomeric silsesquioxane (POSS), as a nanomaterial, can easily immobilize a biometric device material, and can increase the number of binding sites reacting with a material to be detected due to its high surface area.

Polyhedral oligomeric silsesquioxane (POSS) functionalized with APBA may be expressed as "POSS-APBA" in the present specification, and may be represented by the following Chemical Formula 1 as an example.

[Formula 1]

POSS-(CH ₂ ) _n -APBA (n = 1 to 50)

In addition, in Formula 1, an optimized structure as an insulin drug carrier can be prepared by adjusting the length of n, and in this case, n may be in the range of 1 to 50, specifically 3 to 30, more Specifically, it may be 5 to 15 days.

In addition, polyhedral oligomeric silsesquioxane (POSS) functionalized with APBA may be typically represented by the following Chemical Formula 2 as an example.

[Formula 2]

On the other hand, the polyhedral oligomeric silsesquioxane (POSS) functionalized with APBA may be prepared by reacting POSS substituted with [2-(3,4-epoxycyclohexyl)ethyl]-heptisobutyl with APBA. POSS-APBA is an organic/inorganic hybrid material made by reacting polyhedral oligomeric silsesquioxane (POSS), an inorganic nanomaterial, and 3-aminophenylboronic acid (APBA), an organic material. It has excellent physical properties.

Insulin drug delivery system according to an embodiment of the present invention may have a structure including a core and a shell, and more specifically, a core including polyhedral oligomeric silsesquioxane (POSS) and aminophenylboronic acid (APBA) and a shell comprising insulin.

In addition, the insulin drug delivery system according to one aspect of the present invention has a structure of POSS-APBA@Insulin, and can release insulin and bind to glucose in the presence of glucose, and specifically, APBA contained in POSS-APBA@Insulin reacts with glucose and releases the insulin it was bound to, a reaction in which insulin and glucose are exchanged can occur. Furthermore, the POSS-APBA@Insulin has a different reaction rate depending on the concentration of glucose, and as the concentration increases, the amount of insulin released increases in direct proportion to it. Accordingly, in the in vivo assay, not only the detection of glucose but also the management of diabetes through binding to glucose and release of insulin can be effectively handled.

According to an embodiment of the present invention, there is provided a method for controlling insulin release through an insulin drug carrier that is self-regulated by external stimuli such as glucose concentration, pH, temperature or wavelength, wherein the insulin drug carrier comprises aminophenylboronic acid (APBA). ) provides a method for controlling insulin release comprising a polyhedral oligomeric silsesquioxane (POSS) functionalized with (POSS) and insulin.

In particular, the insulin drug delivery system used as the method for controlling insulin release exhibits a property of releasing insulin through binding to glucose in a pH range of 3 to 10.

As such, in the method for controlling insulin release according to an embodiment of the present invention, the response of the insulin drug carrier including POSS-APBA@Insulin can be controlled by external stimuli such as pH, temperature or wavelength as well as glucose concentration. Bar, there is an advantage in that it is possible to more effectively control blood sugar in diabetic patients, and the change of POSS-APBA@Insulin according to the change of each condition will be described in detail in Examples below.

On the other hand, according to an embodiment of the present invention, in the method for preparing an insulin drug delivery system comprising polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin,

a) synthesizing polyhedral oligomeric silsesquioxane (POSS-APBA) functionalized with aminophenylboronic acid by mixing polyhedral oligomeric silsesquioxane (POSS) and aminophenylboronic acid (APBA);

b) reacting with insulin and polyethylene glycol to prepare polyethylene glycol-insulin (PEG-Insulin); and

c) mixing the POSS-APBA with PEG-Insulin;

It provides a method for producing an insulin drug delivery system comprising a.

In step a), polyhedral oligomeric silsesquioxane (POSS) and aminophenylboronic acid (APBA) are mixed in an organic solvent, and polyhedral oligomeric silses functionalized with aminophenylboronic acid by adding hydrazine monohydrate and ammonia solution It may be to synthesize quioxane (POSS-APBA).

Here, as an example of the organic solvent used in step a), tetrahydrofuran (THF) may be mentioned, and the functionalized polyhedral oligomeric silsesquioxane (POSS-APBA) synthesized in step a). Silver can be synthesized by reacting POSS substituted with [2-(3,4-epoxycyclohexyl)ethyl]-heptisobutyl with aminophenylboronic acid (APBA).

On the other hand, in step b) of preparing polyethylene glycol-insulin (PEG-Insulin), insulin is dissolved in hydrochloric acid (HCl) and reacted with polyethylene glycol in dimethyl sulfoxide (DMSO), polyethylene glycol-insulin (PEG-Insulin) ) may be manufactured.

In this case, the polyethylene glycol may be directly bonded to the α chain (α helix) and the β chain (β sheet) of insulin to form polyethylene glycol-insulin (PEG-Insulin).

Furthermore, when mixing the POSS-APBA prepared in step a) and the PEG-Insulin prepared in step b) (step c)), a catalyst such as Na(CN)BH ₃ may be added and mixed.

Meanwhile, when POSS-APBA and PEG-Insulin are mixed in step c), the POSS-APBA and PEG-Insulin may react in a 1:1 ratio to form POSS-APBA@Insulin. The thus formed POSS-APBA@Insulin reacts with glucose and APBA to release insulin, and as a result, glucose control in the body becomes possible. By controlling the reaction of POSS-APBA and PEG-Insulin, It has the advantage of regulating the formation of insulin and finally facilitating the self-regulation of glucose in the body.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all modifications, equivalents and substitutes for the embodiments are included in the scope of the rights.

The terms used in the examples are used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, 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 the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

In addition, in the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

In addition, in describing the components of the embodiment, terms such as first, second, A, B, (a), (b), etc. may be used. These terms are only for distinguishing the elements from other elements, and the essence, order, or order of the elements are not limited by the terms.

Components included in one embodiment and components having a common function will be described using the same names in other embodiments. Unless otherwise stated, a description described in one embodiment may be applied to another embodiment, and a detailed description in the overlapping range will be omitted.

- Examples

1. Conduct of experiment

(1) Synthesis of POSS-APBA

[2-(3,4-epoxycyclohexyl)ethyl]-heptaisobutyl-substituted POSS (Polysilsesquoxane) 10 mg and APBA (3-AminoPhenyl Boronic Acid) 1.67 mg were placed in 8 ml of THF solvent, and then an oil bath At 50 °C, stirred for 3 hours to make a homogeneous solution.

Thereafter, 50 μL of each of hydrazine monohydrate and ammonia solution were slowly added, and the reaction was further carried out for 1 hour, followed by cooling at room temperature.

Finally, the THF solvent of the synthesized POSS-APBA solution was removed and dried at 45° C. in a vacuum oven for 24 hours to make a powder state. The prepared POSS-APBA powder was stored at room temperature in a desiccator.

The schematic synthesis process is shown in Fig. 1(a).

(2) Synthesis of PEG-Insulin

To form diol in insulin, 8 mg of insulin and 100 mg of PEG were dissolved in 10 ml of DMSO, 0.1 to 0.2% of sodium cyanoborohydride (Na(CN)BH ₃ ) catalyst was added to DMSO, followed by stirring for 1 hour. By stabilizing at room temperature for one hour, PEG-Insulin was prepared.

A schematic manufacturing method is shown in Fig. 1(a).

(3) Preparation of POSS-APBA-Insulin

11 mg of pre-synthesized POSS-APBA powder and PEG-Insulin solution were stirred for 2 hours and then stabilized for 30 minutes. After the stabilization process was completed, DMSO, the solvent of the solution, was changed to distilled water through dialysis, and the stabilized solution was transferred to a dialysis tube of MWCO (100-500D), and then the tube was immersed in a water bath filled with distilled water to proceed with dialysis. At this time, in order to increase the efficiency of dialysis, the distilled water in the water tank was replaced with fresh distilled water every 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours. Finally, after dialysis was completed, the POSS-APBA-Insulin solution was refrigerated at 3° C. (FIG. 1(a)).

(5) Insulin releasing

An extracorporeal insulin release study was conducted, and 400 ml of PBS was preferentially preheated to 36.5 degrees in an oil bath to make the experimental conditions similar to the in vivo environment. It was placed in a dialysis tube and stabilized at room temperature. The experiment was carried out by immersing the dialysis tube in a PBS solution in a bath that is similar to the body environment. In the non-glucose state without added sugar, 1.5 ml was collected once every 10 minutes for 1 hour, and a total of 6 samples were collected. After that, 400 mg, 600 mg, and 800 mg of sugar were added to PBS to set the sugar concentration in PBS to various sugar concentration conditions such as 1 mg/ml, 1.25 mg/ml, and 2 mg/ml, so that even under each sugar concentration condition, 1 Insulin release amount was compared for different glucose concentrations by collecting 1.5 ml once every 10 minutes for a period of time. At this time, when collecting all 1.5 ml, in order to keep the amount and concentration of the solution constant, 1.5 ml of the same sugar concentration solution was added to PBS at a time to maintain the experimental conditions perfectly, and an insulin release experiment was carried out. The insulin release sample thus collected was measured in a wavelength range of 200-700 nm using a UV-vis device, and then the absorbance at a wavelength of 275 nm was analyzed to determine the amount of insulin released.

(6) Cell cytotoxicity test of POSS-APBA and POSS-APBA@Insulin

In order to apply the drug delivery system to the cells, using HeLa, human dermal fibroblast (HDF) and human umbilical vein endothelial (HUVE) cells, cytotoxicity experiments of POSS-APBA and POSS-APBA@Insulin nanoparticles were performed. HeLa, HDF and HUVE cells were treated with various concentrations of POSS-APBA and POSS-APBA@Insulin for 24 hours, and the results were shown ( FIG. 6 ).

2. Analysis and Results

(1) Synthetic crystallization and structural analysis of samples

Fourier-transform infrared (FT-IR) spectral analysis was performed in Fourier-transform infrared spectroscopy (FT-IR, A Perkin ElmerSpectrum II) for synthetic crystallization and structural analysis of the sample.

Sample preparation was performed by the KBr method, and sampling was performed by mixing KBr powder and the sample in an approximately 100:1 ratio, mixing the sample with an agate mortar so that the sample was well dispersed, and then applying pressure using a pellet mold kit to prepare pellets and sampled. The sample was analyzed in the range of 4000 ~ 400 cm ^-1 to confirm the Si-O-Si bonding peak unique to POSS, the amine peak and the boronic group peak unique to APBA, and then the POSS-APBA functional group peak synthesized to verify the POSS-APBA synthesis. was compared and confirmed.

The analysis result for this is shown in FIG. 2 below.

In the FT-IR spectrum of Fig. 2(a), the peak at 1000-1300 cm ^-1 represents the stretching mode of Si-O-Si and is considered a characteristic peak of POSS. The POSS peak or CN stretch peak at 1240 cm ^-1 is due to the CO stretching vibration of the epoxide, and the peak intensity in the POSS-APBA spectrum is weaker than the intensity in the spectrum of POSS. The new peak at 1470 cm ^-1 in the POSS-APBA spectrum is due to the CNC, CN stretching, CC stretching (phenyl ring), and BO stretching of APBA, and this peak does not exist in POSS. The band at 3350 cm ^-1 is due to the NH stretching of the secondary amine of POSS-APBA.

In addition, the broad peak generated by OH stretching at 3300 cm ^-1 in the POSS-APBA spectrum is due to the epoxide ring opening. The presence of the α-chain protein insulin in POSS-APBA@Insulin was characterized by the characteristic protein absorption band peak, Amide I (at about 1660 cm ⁻¹ ) by C = O stretching vibration of the peptide group, and NH according to the CN stretching vibration It shows an Amide II band (located at about 1546 cm ⁻¹ ) by bending (see FIG. 2(b) ). It can be seen that the amino acid I and II peaks (about 1660, 1544 cm ^-1 ) of insulin disappear as insulin is removed after the addition of glucose. It is confirmed at 1035 cm ^-1 . Therefore, it can be confirmed that a new POSS-APBA-Glucose is formed.

(2) Insulin releasing (releasing) experiment analysis

After the experiment as in the method described in '(5) Insulin releasing' , POSS-APBA@Insulin modified particles were incubated in PBS buffer (pH 7.5, 37 ℃) to confirm the insulin releasing kinetics. As shown in Fig. 3(d), 1 mg/ml (1 g/L, 100 mg/dl) shows a normoglycemic concentration, and typical diabetes has a glucose concentration of 2 mg/ml or more.

3 (a) and (c) show the results of confirming the insulin release behavior while giving a concentration difference at 1 hour intervals. It can be seen that drug release occurs not only at a high concentration of glucose but also at a low concentration of 1 mg/ml.

On the other hand, POSS-APBA@Insulin exhibits fast response behavior such as no insulin release when no sugar is injected, insulin release within 10 minutes after sugar injection, and stable equilibrium after 30 minutes after injection. looks like This is thought to have contributed to widening the space through which sugar can approach the reactive molecule, boronic acid, under the influence of the bulky property of POSS.

3(b) shows the release amount of the insulin drug according to the sugar concentration, and it was confirmed that y = 185.83x + 30.89 is directly proportional, and the R ² value is 0.9792, which is close to 1. These results indicate that POSS-APBA@Insulin can be used as a material with great potential as a smart drug delivery system.

(3) Characteristic change analysis according to pH

As shown in Fig. 4(a) and Table 1 below, the zeta potential value of POSS-APBA significantly increased from 8.23 mV to -64.97 mV as the pH value increased from 3.1 to 10.0. The surface of APBA protonated at low pH is deprotonated past the isoelectric point (Ip) near pH 3.7, and the phenylboronic acid group on the surface is changed to negative conversion. Thereafter, in a neutral state (pH 7.4), the zeta potential is -40.69 mV, indicating the high stability of colloidal dispersions. It can be confirmed that this phenomenon also shows the same trend in POSS-APBA@Insulin nanoparticles. POSS-APBA@Insulin nanoparticles decreased from 31.44 mV to -73.14 mV with an increase from pH 3.1 to pH 10.0. It can be seen that it represents a difference in value greater than that of POSS-APBA, and exhibits higher colloidal stability. The isoelectric point of the POSS-APBA@Insulin nanoparticles is pH 4.6 and shows a value of -58.42 mV in a neutral state. In general, PBA pKA 8.6 or lower has trigonal -B(OH) ₂ , but above PBA pKA has tetrahedral -B(OH) ₃ , and is known to form a stable complex with polyol molecules. PBA alone is difficult to use in controlled release systems at physiological pH. To solve this problem, the APBA and POSS-APBA@Insulin nanoparticles prepared in the present invention exhibit a high dispersion state in a neutral state, and have a stable negative charge with boronic acid, so they can stably selectively bind to glucose, enabling a fast reaction. I think to do it.

Figure 4(b) shows the Zeta size change of the particles according to pH 3.0, 7.5, 10.0 of the POSS-APBA nanoparticles, POSS-APBA@Insulin nanoparticles, and POSS-APBA@glucose nanoparticles. POSS-APBA has a large particle size in the neutral state, but POSS-APBA@Insulin has a smaller particle size as the pH increases.

This phenomenon also shows the same trend and size in POSS-APBA@glucose, which is thought to be a phenomenon seen while boronic acid has high dispersion stability (more than ±30mV) at pH 3, 7, and 10 due to diol binding with glucose or insulin. do.

Figure 4(c) shows the Electro-Osmosis plot according to the pH change of POSS-APBA and POSS-APBA@Insulin. When a voltage is applied to the suspension, the particles move with speed due to the potential difference. As the pH increases, it can be seen that the mobility of POSS-APBA is improved by 10 times or more, and in POSS-APBA@Insulin, mobility is improved by about 2 times or more.

(4) Cell cytotoxicity test results of POSS-APBA and POSS-APBA@Insulin

As shown in Fig. 6 (a), (b) and (c), POSS-APBA and POSS-APBA@Insulin nanoparticles using HeLa, HDF and HUVE cells were subjected to cytotoxicity experiments, POSS-APBA and POSS- It was confirmed that APBA@Insulin nanoparticles did not show toxicity up to a concentration of 640 μg/ml. In addition, as shown in FIG. 6(d), it was confirmed that there was no change in morphology up to a concentration of 640 μg/ml.

Furthermore, when confirming intracellular apoptosis signals, cleaved caspase 3 and caspase 9 were not increased by POSS-APBA and POSS-APBA@Insulin at a concentration of 640 μg/ml in HeLa cells, HDF cells and HUVE cells. was confirmed (Fig. 6(e)).

These results indicate that POSS-APBA and POSS-APBA@Insulin nanoparticles can be applied to cells (biocompatibility).

(5) Insulin activity analysis according to the preparation of POSS-APBA and POSS-APBA@Insulin

Since it is most important to maintain the original structure of insulin in the preparation of nanoparticles, the secondary structure of PEG-Insulin and POSS-APBA@Insulin nanoparticles was studied using circular dichroism spectroscopy (CD). and the existence of α-chain (α-helix) and β-chain (β-sheet) secondary structures, characteristic of insulin, were confirmed in PEG-Insulin and POSS-APBA@Insulin nanoparticles. And, it was verified that the secondary structure of insulin was not denatured and the activity was maintained (FIG. 8).

8 shows the CD spectra of insulin, PEG-Insulin and POSS-APBA@Insulin, and the red line of FIG. 8 analyzing the secondary structure of insulin shows α-chain and β-chain peaks. This indicates the activity of insulin used as a control. Both the blue line and the green line indicate characteristic peaks of the insulin secondary structure, and the overall shape of the lines is very similar to the red line of insulin. This suggests that the secondary structure of insulin is not denatured during the preparation of PEG-Insulin and POSS-APBA@Insulin, and that the activity of insulin is maintained even after the preparation of nanoparticles.

Table 2 below shows the compositions (%) of α-chain, β-chain, and random coil in the secondary structure of insulin, and it can be seen that the compositions of α-chain, β-chain and random coil are all very similar. This is evidence indicating that the secondary structure of insulin is not denatured upon binding to PEG or POSS-APBA. As a result, when PEG-Insulin or POSS-APBA@Insulin is prepared, the secondary structure of insulin is not affected and activity is also maintained. indicates that it will be

Human Insulin PEG-Insulin POSS-APBA@Insulin % Helix 29.1 27.1 27.6 % Beta 16.5 16.5 23.7 % Turn 23.8 23.3 20.5 % Random 30.5 33.1 28.1 % Total 100 100 100

As described above, although the embodiments have been described with reference to the limited drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques are performed in an order different from the described method, and/or the described components of the system, structure, apparatus, circuit, etc. are combined or combined in a different form than the described method, or other components Or substituted or substituted by equivalents may achieve an appropriate result.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (16)

A polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin in a 1:1 molecular ratio,
The polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) is represented by the following Chemical Formula 2, Insulin drug delivery system.
[Formula 2]

(R = an alkyl group having 1 to 50 carbon atoms)
delete The method of claim 1,
wherein the polyhedral oligomeric silsesquioxane (POSS) is POSS substituted with [2-(3,4-epoxycyclohexyl)ethyl]-heptisobutyl.
According to claim 1,
The insulin drug carrier comprises a core comprising polyhedral oligomeric silsesquioxane (POSS) and a shell comprising aminophenylboronic acid (APBA) and insulin.
The method of claim 1,
The insulin drug carrier, characterized in that in the presence of glucose, releasing insulin and binding to glucose, the insulin drug carrier.
delete A method for controlling the release of insulin in animals other than humans through the insulin drug delivery system according to claim 1, which is self-regulated by an external stimulus, the method comprising:
The insulin drug delivery system includes polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin in a 1:1 molecular ratio,
The polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) is represented by the following Chemical Formula 2, a method for controlling insulin release.
[Formula 2]

(R = an alkyl group having 1 to 50 carbon atoms)
8. The method of claim 7,
The method for controlling insulin release, wherein the external stimulus comprises one or more stimuli selected from the group consisting of concentration, pH, temperature and wavelength of glucose.
8. The method of claim 7,
The method for controlling insulin release, wherein the insulin drug carrier releases insulin through binding to glucose in a pH range of 3 to 10.
In the method for preparing the insulin drug delivery system according to claim 1, comprising the polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) and insulin in a 1:1 molecular ratio,
a) synthesizing polyhedral oligomeric silsesquioxane (POSS-APBA) functionalized with aminophenylboronic acid by mixing polyhedral oligomeric silsesquioxane (POSS) and aminophenylboronic acid (APBA);
b) reacting with insulin and polyethylene glycol to prepare polyethylene glycol-insulin (PEG-Insulin); and
c) mixing the POSS-APBA with PEG-Insulin;
including,
The polyhedral oligomeric silsesquioxane (POSS) functionalized with aminophenylboronic acid (APBA) is represented by the following Chemical Formula 2, a method for producing an insulin drug delivery system.
[Formula 2]

(R = an alkyl group having 1 to 50 carbon atoms)
11. The method of claim 10,
In step a), polyhedral oligomeric silsesquioxane (POSS) and aminophenylboronic acid (APBA) are mixed in an organic solvent, and polyhedral oligomeric silses functionalized with aminophenylboronic acid by adding hydrazine monohydrate and ammonia solution A method of synthesizing quioxane (POSS-APBA), a method for producing an insulin drug delivery system.
11. The method of claim 10,
In step a), the functionalized polyhedral oligomeric silsesquioxane ( POSS-APBA) is synthesized, the method for producing an insulin drug delivery system.
11. The method of claim 10,
In step b), insulin is dissolved in hydrochloric acid (HCl) and reacted with polyethylene glycol in dimethyl sulfoxide (DMSO) to prepare polyethylene glycol-insulin (PEG-Insulin).
11. The method of claim 10,
In step b), the method for producing an insulin drug delivery system, characterized in that the polyethylene glycol is attached to the α chain and the β chain of insulin.
11. The method of claim 10,
In step c), POSS-APBA and PEG-Insulin are mixed and reacted in a molecular ratio of 1:1.
11. The method of claim 10,
The organic solvent used in step a) is tetrahydrofuran (THF),
In step c), POSS-APBA and PEG-Insulin are mixed in the presence of a Na(CN)BH ₃ catalyst.

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