CN115845033A - Flexible sugar-sensitive gel microneedle patch containing bubble structure and preparation method thereof - Google Patents

Abstract

The invention discloses a tough sugar-sensitive gel microneedle patch containing air bubbles and a preparation method thereof. The microneedle patch consists of a substrate and microneedles arranged in an array on the substrate, wherein the bottom of the microneedles contains air bubbles. Both the substrate and the microneedles are composed of a tough material mixed with phenylboronic acid-modified ε-polylysine, polyvinyl alcohol, and insulin. The strength of the microneedle patch of the present invention meets the needs of piercing the skin. At the same time, the tough material and air bubble design make the microneedle bend under strong pressure instead of breaking the microneedle, which can prevent the microneedle from breaking in the human skin during improper use. the harm caused. In addition, the present invention can be obtained by the two-step method of "injection-drying", without polymerization, washing or freeze-thaw steps, and without adding enhancers, which can avoid the problems of microneedle patch deformation and insulin activity loss, and can reduce process costs , which is conducive to large-scale production; it also has blood sugar response performance, and can automatically adjust the release rate and release dose of insulin according to the actual blood sugar level.

Description

A kind of flexible sugar-sensitive gel microneedle patch containing bubble structure and preparation method thereof

technical field

The invention belongs to the technical field of functional polymers, and relates to a gel microneedle patch and a preparation method thereof, in particular to a tough sugar-sensitive gel microneedle patch with a bubble structure and a preparation method thereof.

Background technique

Diabetes is considered to be one of the most challenging public health problems in recent years. According to statistics from the International Diabetes Federation, the number of diabetic patients worldwide will reach 537 million in 2021. Supplementing insulin from the outside is one of the important components in the treatment of diabetes. At present, subcutaneous injection of insulin is a common method for diabetic patients to supplement insulin. However, the subcutaneous injection causes a strong sense of pain, and thus imposes a large burden on the patient.

Recently, glucose-sensitive gel microneedle patches containing insulin have received extensive attention from researchers. The principle is that the glucose-sensitive gel microneedle patch containing insulin is pierced into human skin, and the swelling of the gel microneedle induces insulin to diffuse from the inside of the microneedle to the subcutaneous tissue, and automatically releases an appropriate dose of insulin according to the actual blood sugar level, thereby Have a hypoglycemic effect. Since the microneedles are not long enough to reach the subcutaneous nerve, there is no pain.

At present, the technical difficulties of the sugar-sensitive gel microneedle patch lie in two aspects: the complicated preparation process and the brittleness of the microneedles. For example, Yu et al. reported a four-step process of "injection molding-polymerization-washing-drying" to prepare glucose-sensitive gel microneedle patches. The process flow is long, and the polymerization step can affect insulin activity, and the washing step can also cause Partial loss of insulin and deformation of the microneedle patch; in addition, the obtained microneedle patch will break when subjected to greater pressure, and the broken fragments can accumulate in the patient's body to induce long-term safety problems. Patent CN113197838 discloses a three-step process of “injection molding-freezing-thawing-drying” to prepare sugar-sensitive gel microneedle patches, which avoids the problems caused by polymerization and washing steps, and at the same time, the new freeze-thawing step can strengthen the microneedle. Strength; however, the freeze-thaw steps take a long time and require high process parameters (temperature, humidity, freeze-thaw times and time, etc.), which will increase the cost of large-scale production.

Contents of the invention

In order to solve the above problems, the present invention provides a tough sugar-sensitive gel microneedle patch containing air bubbles and a preparation method thereof. The flexible glucose-sensitive gel microneedle patch with bubble structure is composed of a base and microneedles arranged in an array on the base, wherein the bottom of the microneedles contains bubbles. The tough sugar-sensitive gel microneedle patch with bubble structure is composed of a tough material mixed with phenylboronic acid-modified ε-polylysine, polyvinyl alcohol and insulin. The strength of the tough sugar-sensitive gel microneedle patch with bubble structure of the present invention meets the needs of piercing the skin. At the same time, the design of the tough material and bubbles makes the microneedle bend under strong pressure instead of breaking the microneedle, which can avoid the occurrence of The problem of high brittleness and easy breakage of microneedles is mentioned in the background art, so as to eliminate the harm caused by microneedle breakage in human skin during improper use. In addition, the flexible sugar-sensitive gel microneedle patch with bubble structure of the present invention can be obtained by the two-step "injection-drying" method, which can avoid the three-step or even four-step loading preparation process in the prior art, without the need for polymerization, There is no need to add enhancers during washing or freeze-thawing steps, which can avoid the deformation of the microneedle patch and the loss of insulin activity. At the same time, it can reduce the process cost and facilitate large-scale production; the tough glucose-sensitive gel microneedle patch with bubble structure has blood glucose response It can automatically adjust the release rate and release dose of insulin according to the actual blood sugar level.

The technical scheme adopted in the present invention is as follows:

1. A tough glucose-sensitive gel microneedle patch with bubble structure

The microneedle patch consists of a substrate and microneedles arranged in an array on the substrate, wherein the bottom of the microneedles contains air bubbles; both the substrate and the microneedles are made of phenylboronic acid modified ε-polylysine, polyvinyl alcohol and It is made of tough materials mixed with insulin; the tough sugar-sensitive gel microneedle patch with bubble structure is prepared by two-step method of "injection-drying".

The microneedles of the microneedle patch are in the shape of a quadrangular pyramid, the height of the microneedles is 0.2-1 mm, and the side length of the bottom surface of the microneedles is 0.05-0.5 mm; the base is a cylinder, the height of the base is 0.01-2 mm, and the diameter of the base is 0.5-0.5 mm. 5cm; the array density of the microneedles on the substrate is 10-800 needles/(cm ² substrate).

Two, the synthetic method of phenylboronic acid modified epsilon-polylysine

1) Put 4-carboxy-3-fluorophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into dimethyl In sulfone, stir at room temperature at a stirring rate of 200-600rpm for 30-60min to obtain solution A;

2) Put ε-polylysine into water, stir at a stirring rate of 200-600rpm at room temperature for 5-15min, and obtain solution B;

3) Mix solution A and solution B, stir at room temperature at a stirring rate of 200-600rpm for 60-600min, and obtain mixture C;

4) Put the mixture C into a dialysis bag with a molecular weight cut-off of 500-3500Da, and place it in water for dialysis, change the dialysis water every 4-12 hours, the number of water changes is 6-10 times, and the volume of the dialysis water is 3-5 After the dialysis is completed, the mixture in the dialysis bag is freeze-dried to obtain phenylboronic acid-modified ε-polylysine.

In the step 1) and step 2), in parts by mass, 4-carboxy-3-fluorophenylboronic acid is 0.07-0.72 parts, 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride is 0.07-0.75 parts, N-hydroxysuccinimide is 0.04-0.45 parts, dimethyl sulfoxide is 10-100 parts, ε-polylysine is 1 part, water is 5- 10 servings.

The structural formula of the phenylboronic acid modified ε-polylysine is as follows:

The graft ratio of 4-carboxy-3-fluorophenylboronic acid is y/x.

3. A preparation method of a tough sugar-sensitive gel microneedle patch with a bubble structure

1) Add insulin into polyvinyl alcohol aqueous solution, mix and stir at room temperature at a stirring rate of 200-600rpm for 10-20min, then add phenylboronic acid modified ε-polylysine aqueous solution, at room temperature at room temperature at a stirring rate of 200-600rpm Continue to mix and stir for 30-120min, and record the obtained mixture as the precursor;

2) Add the precursor to the microneedle female mold, then place the microneedle female mold in a vacuum box with a pressure of 0.1 bar and store it at room temperature for 30 minutes, and then dry the precursor in a desiccator at room temperature for 12-24 hours to obtain a bubble-containing structure The toughness of the sugar-sensitive gel microneedle patch.

In the step 1), in terms of mass fraction, insulin is 1 part; polyvinyl alcohol aqueous solution is 40-60 parts, and the mass concentration is 5%-10%; phenylboronic acid modified ε-polylysine aqueous solution is 40-60 parts parts, the mass concentration is 5%-10%.

The tough sugar-sensitive gel microneedle patch with bubble structure in the present invention has enough strength to pierce the skin in normal use, and can meet the requirement of transdermal drug delivery. The precursor of the tough sugar-sensitive gel microneedle patch can spontaneously form the bubble structure at the bottom of the microneedle after drying. Part of the axial force becomes shear force; the tough glucose-sensitive gel microneedle patch with bubble structure is made of tough material, and the microneedle bends instead of breaking under the shear force caused by the bubble structure. Therefore, the ductile glucose-sensitive gel microneedle patch with bubble structure and the characteristics of both bubble structure and tough material can prevent the microneedles from breaking in the human body during actual use, thereby avoiding the damage caused by the accumulation of broken microneedle fragments in the body , so it is safe to use. The flexible sugar-sensitive gel microneedle patch with bubble structure contains phenylboronate bond cross-linking points and amino groups. The former partly breaks when blood sugar rises, which makes the gel microneedle network loose, and insulin is easier to pass through the microneedle. Diffusion to the outside of the microneedle; the latter amino group provides a positive charge, which creates an electrostatic interaction with the negatively charged insulin to avoid hypoglycemia problems caused by the rapid release of insulin.

Beneficial effects of the present invention:

1. The present invention adopts the two-step preparation process of "injection molding-drying", which eliminates multiple steps including polymerization, washing and freeze-thawing, and does not need to add enhancers, which can completely avoid the loss of insulin activity or loading capacity, Problems such as microneedle deformation and long time-consuming process steps simplify the process to the greatest extent and reduce material costs;

2. The bottom of the microneedle has a bubble structure, and the bubble can be used as an unstable point of the microneedle under strong pressure, causing the tough microneedle to bend rather than break, which can avoid the use safety problems caused by the broken fragments entering the patient's body;

3. The tough sugar-sensitive gel microneedle patch with bubble structure has the performance of piercing the skin, and also has blood sugar response performance, which can realize the function of automatically adjusting the insulin release rate based on blood sugar level, so it can maintain blood sugar stability and avoid subcutaneous The pain of insulin injections.

Description of drawings

Fig. 1 is the proton nuclear magnetic spectrum spectrogram of phenylboronic acid modified epsilon-polylysine;

Fig. 2 is the electron micrograph of the tough sugar-sensitive gel microneedle patch containing bubble structure;

Fig. 3 is the optical photo of the tough sugar-sensitive gel microneedle patch containing bubble structure;

Fig. 4 is the electron microscope picture after the tough sugar-sensitive gel microneedle with bubble structure is pasted under high pressure;

Fig. 5 is the piercing micropore photo of the tough sugar-sensitive gel microneedle with bubble structure attached to the rat skin;

Figure 6 is the in vitro insulin cumulative release diagram of the flexible glucose-sensitive gel microneedle patch containing bubble structure;

Fig. 7 is a blood sugar lowering diagram of the flexible glucose-sensitive gel microneedle patch with bubble structure.

Detailed ways

Below in conjunction with embodiment the present invention is described in more detail, but the present invention is not limited thereto, for those of ordinary skill in the art, under the premise of not departing from the principle of the present invention, some improvements and modifications can also be made, These improvements and modifications are also considered within the protection scope of the present invention. The content not described in detail in this specification belongs to the prior art known to those skilled in the art.

Embodiments of the present invention are as follows:

Example 1:

Put 0.3450g of 4-carboxy-3-fluorophenylboronic acid, 0.3599g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.2161g of N-hydroxysuccinimide into 10 g of dimethyl sulfoxide was stirred at room temperature at a stirring rate of 200 rpm for 30 min to obtain a solution A. 0.9998g ε-polylysine was put into 5g water, and stirred at room temperature at a stirring rate of 600rpm for 5min to obtain solution B. Mix solution A and solution B, and stir at room temperature at a stirring rate of 200 rpm for 600 min to obtain mixture C. Put the mixture C into a dialysis bag with a molecular weight cut-off of 1000Da, and place it in water for dialysis, change the dialysis water every 8 hours, the number of water changes is 10 times, and the volume of the dialysis water is 5 liters. The mixture was lyophilized to obtain ε-polylysine modified with phenylboronic acid.

The results of this implementation case are shown in Figure 1, which is the H NMR spectrum of phenylboronic acid modified ε-polylysine. After calculation, the grafting rate of 4-carboxy-3-fluorophenylboronic acid was 0.24.

Example 2:

Put 0.0720g of 4-carboxy-3-fluorophenylboronic acid, 0.0759g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.0452g of N-hydroxysuccinimide into 10 g of dimethyl sulfoxide was stirred at room temperature at a stirring rate of 200 rpm for 30 min to obtain a solution A. 0.9995g ε-polylysine was put into 5g water, and stirred at room temperature at a stirring rate of 600rpm for 5min to obtain solution B. Mix solution A and solution B, and stir at room temperature at a stirring rate of 200 rpm for 600 min to obtain mixture C. Put the mixture C into a dialysis bag with a molecular weight cut-off of 500 Da, and place it in water for dialysis, change the dialysis water once every 4 hours, the number of water changes is 6 times, and the volume of the dialysis water is 5 liters. The mixture was lyophilized to obtain ε-polylysine modified with phenylboronic acid.

Example 3:

Put 0.7161g of 4-carboxy-3-fluorophenylboronic acid, 0.7452g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.4483g of N-hydroxysuccinimide into 100 g of dimethyl sulfoxide was stirred at room temperature at a stirring speed of 600 rpm for 60 min to obtain a solution A. Add 1.0208g of ε-polylysine into 10g of water, stir at room temperature at a stirring rate of 200rpm for 15min, and obtain solution B. Mix solution A and solution B, and stir at room temperature at a stirring speed of 600 rpm for 60 min to obtain mixture C. Put the mixture C into a dialysis bag with a molecular weight cut-off of 3500Da, and place it in water for dialysis, change the dialysis water once every 12 hours, the number of water changes is 6 times, and the volume of the dialysis water is 3 liters. The mixture was lyophilized to obtain ε-polylysine modified with phenylboronic acid.

Example 4:

The ε-polylysine was modified with phenylboronic acid prepared in Example 1. Add 10.1 mg of insulin to 410 mg of 5% polyvinyl alcohol aqueous solution and mix and stir at room temperature at a stirring rate of 200 rpm for 20 min, then add 600 mg of 5% phenylboronic acid-modified ε-polylysine aqueous solution and stir at room temperature at a stirring rate of 200 rpm Continue to mix and stir for 120min, and the resulting mixture is called a precursor. The precursor was added to the female microneedle mold, placed in a vacuum box with a pressure of 0.1 bar and stored at room temperature for 30 minutes, and then the precursor was dried in a desiccator at room temperature for 24 hours. After drying, a tough glucose-sensitive gel microneedle patch with bubble structure was obtained.

The results of this implementation case are shown in Figures 2-5. Figure 2 is the electron microscope image of the tough sugar-sensitive gel microneedle patch with bubble structure. It can be observed that the microneedle arrays on the microneedle patch have the same appearance and sharp needle tips. Figure 3 is an optical photo of the tough glucose-sensitive gel microneedle patch with bubble structure. It can be seen that the bottom of the microneedle contains bubble structure. Figure 4 is the electron micrograph of the tough sugar-sensitive gel microneedle with bubble structure when it is subjected to high pressure, and part of the microneedle body is covered with the tough sugar-sensitive gel microneedle with bubble structure in Figure 4a after being subjected to high pressure Press into the air bubble at the bottom, and at the same time, the microneedle needle bends instead of breaking; Figure 4 b shows that the gel microneedle sticking of the prior art breaks after being subjected to high pressure, resulting in a large number of cracks. Fig. 5 is a photo of piercing micropores of tough sugar-sensitive gel microneedles with air bubble structure pasted on rat skin. The punctured micropores were dyed dark by trypan blue staining, and the array-like lattice formed by the microneedles successfully piercing the skin can be clearly seen. Fig. 6 is a diagram of the cumulative release of insulin in vitro of the flexible glucose-sensitive gel microneedle patch with bubble structure. The glucose concentration was designed to be 0mg/dL (control group), 100mg/dL (normal blood sugar simulation group) and 400mg/dL (hyperglycemia simulation group), and the release medium was designed to be a phosphate buffer solution (37°C, pH7. 4). With the increase of glucose concentration, the release of insulin from the flexible glucose-sensitive gel microneedle patch with bubble structure increases, that is, the flexible glucose-sensitive gel microneedle patch with bubble structure has glucose-responsive insulin release performance, which has It is beneficial to release insulin quickly in the state of hyperglycemia to quickly reduce blood sugar, and release less insulin in the state of normal blood sugar to avoid hypoglycemia. Fig. 7 is a blood sugar lowering diagram of the flexible glucose-sensitive gel microneedle patch with bubble structure. Diabetic rats were given subcutaneous injection of insulin (INS group) or the application of flexible glucose-sensitive gel microneedle patch with air bubble structure (GR-MNP group). Fast from 19:00 (-13h) of the previous day to 8:00 (0h) of the same day to simulate the behavior of diabetic patients without eating between dinner and breakfast. Then at 8:00 (0h) on the same day, a food simulated breakfast was given, and at 9:00 (1h) on the same day, the above two treatments were performed respectively. After subcutaneous injection of insulin, the blood sugar quickly returns from hyperglycemia (higher than 200mg/dL) to normal blood sugar range (50-200mg/dL), and then the second meal at 12:00 (4h) on the same day, that is, the blood sugar rises to hyperglycemia after simulated lunch , which means that diabetics need to inject insulin 2-4 times a day to cope with the life pattern of three meals a day. In contrast, the application of the flexible glucose-sensitive gel microneedle patch with air bubble structure did not increase blood sugar after simulating lunch, and the blood sugar increased slightly when simulating dinner at 17:00 (9h) of the same day, and then at 8:00 ( 24h) has been maintained in normal blood sugar. The simulated breakfast was performed after 8:00 (24h) the next day, and the blood sugar rose. This shows that the application of flexible glucose-sensitive gel microneedles with bubble structure can cope with the blood sugar changes in the simulated three meals a day mode, which is far superior to the current commonly used subcutaneous injection of insulin treatment.

Example 5:

The ε-polylysine was modified with phenylboronic acid prepared in Example 1. Add 10.0 mg of insulin to 590 mg of 10% polyvinyl alcohol aqueous solution and mix and stir at room temperature at a stirring rate of 600 rpm for 10 min, then add 400 mg of 10% phenylboronic acid-modified ε-polylysine aqueous solution and at room temperature at a stirring rate of 600 rpm Continue to mix and stir for 30 minutes, and the resulting mixture is called a precursor. The precursor was added to the female microneedle mold, placed in a vacuum box with a pressure of 0.1 bar and stored at room temperature for 30 minutes, and then the precursor was dried in a desiccator at room temperature for 24 hours. After drying, a tough glucose-sensitive gel microneedle patch with bubble structure was obtained.

Embodiment 6:

The ε-polylysine was modified with phenylboronic acid prepared in Example 2. Add 10.5 mg of insulin to 500 mg of 10% polyvinyl alcohol aqueous solution and mix and stir at room temperature at a stirring rate of 600 rpm for 10 min, then add 500 mg of 10% phenylboronic acid-modified ε-polylysine aqueous solution and at room temperature at a stirring rate of 600 rpm Continue to mix and stir for 30 minutes, and the resulting mixture is called a precursor. The precursor was added to the female microneedle mold, placed in a vacuum box with a pressure of 0.1 bar and stored at room temperature for 30 minutes, and then the precursor was dried in a desiccator at room temperature for 24 hours. After drying, a tough glucose-sensitive gel microneedle patch with bubble structure was obtained.

Embodiment 7:

The ε-polylysine was modified with phenylboronic acid prepared in Example 3. Add 10.0 mg of insulin to 400 mg of 10% polyvinyl alcohol aqueous solution and mix and stir at room temperature at a stirring rate of 600 rpm for 10 min, then add 600 mg of 10% phenylboronic acid-modified ε-polylysine aqueous solution and stir at room temperature at a stirring rate of 600 rpm Continue to mix and stir for 30 minutes, and the resulting mixture is called a precursor. The precursor was added to the microneedle female mold, placed in a vacuum box with a pressure of 0.1 bar and stored at room temperature for 30 minutes, and then the precursor was dried in a desiccator at room temperature for 12 hours. After drying, a tough glucose-sensitive gel microneedle patch with bubble structure was obtained.

Claims (6)

1. a kind of tough sugar-sensitive gel microneedle patch containing bubble structure, it is characterized in that, described microneedle patch is made up of substrate and the microneedle that is arrayed on substrate, wherein the bottom of microneedle comprises air bubble; Both the substrate and the microneedles are made of tough materials mixed with phenylboronic acid modified ε-polylysine, polyvinyl alcohol and insulin. 2. A kind of tough sugar-sensitive gel microneedle patch containing air bubbles according to claim 1, characterized in that, the microneedles of the microneedle patch are square pyramids, and the height of the microneedles is 0.2-1mm. The side length of the bottom surface of the microneedle is 0.05-0.5mm; the base is a cylinder, the height of the base is 0.01-2mm, and the diameter of the base is 0.5-5cm; the array density of the microneedles on the base is 10-800 needles/(cm ² base). 3. A kind of flexible sugar-sensitive gel microneedle patch containing bubble structure according to claim 1, characterized in that, the synthesis method of the phenylboronic acid modified ε-polylysine comprises the following steps: 1) Put 4-carboxy-3-fluorophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into dimethyl In sulfone, stir at room temperature at a stirring rate of 200-600rpm for 30-60min to obtain solution A; 2) Put ε-polylysine into water, stir at a stirring rate of 200-600rpm at room temperature for 5-15min, and obtain solution B; 3) Mix solution A and solution B, stir at room temperature at a stirring rate of 200-600rpm for 60-600min, and obtain mixture C; 4) Put the mixture C into a dialysis bag with a molecular weight cut-off of 500-3500Da, and place it in water for dialysis, change the dialysis water every 4-12 hours, the number of water changes is 6-10 times, and the volume of the dialysis water is 3-5 After the dialysis is completed, the mixture in the dialysis bag is freeze-dried to obtain phenylboronic acid-modified ε-polylysine. 4. A kind of flexible sugar-sensitive gel microneedle patch containing bubble structure according to claim 3, characterized in that, in the step 1) and step 2), in parts by mass, 4-carboxy-3 - Fluorophenylboronic acid is 0.07-0.72 parts, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.07-0.75 parts, N-hydroxysuccinimide is 0.04- 0.45 parts, 10-100 parts of dimethyl sulfoxide, 1 part of ε-polylysine, 5-10 parts of water. 5. The preparation method of a flexible sugar-sensitive gel microneedle patch containing bubble structure according to claim 1 or 2, characterized in that it comprises the following steps: 1) Add insulin into polyvinyl alcohol aqueous solution, mix and stir at room temperature at a stirring rate of 200-600rpm for 10-20min, then add phenylboronic acid modified ε-polylysine aqueous solution, at room temperature at room temperature at a stirring rate of 200-600rpm Continue to mix and stir for 30-120min, and record the obtained mixture as the precursor; 2) Add the precursor to the microneedle female mold, then place the microneedle female mold in a vacuum box with a pressure of 0.1 bar and store it at room temperature for 30 minutes, and then dry the precursor in a desiccator at room temperature for 12-24 hours to obtain a bubble-containing structure The toughness of the sugar-sensitive gel microneedle patch. 6. the preparation method of a kind of tough sugar-sensitive gel microneedle patch containing bubble structure according to claim 5, is characterized in that, in described step 1), by mass fraction, insulin is 1 part; Polyethylene The alcohol aqueous solution is 40-60 parts, and the mass concentration is 5%-10%; the phenylboronic acid modified ε-polylysine aqueous solution is 40-60 parts, and the mass concentration is 5%-10%.

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