CN113456803B - PH and alpha-amylase dual-response insulin-loaded hydrogel system and preparation - Google Patents

PH and alpha-amylase dual-response insulin-loaded hydrogel system and preparation Download PDF

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CN113456803B
CN113456803B CN202110591295.4A CN202110591295A CN113456803B CN 113456803 B CN113456803 B CN 113456803B CN 202110591295 A CN202110591295 A CN 202110591295A CN 113456803 B CN113456803 B CN 113456803B
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管骁
陈雅琼
宋洪东
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Abstract

The invention relates to a pH and alpha-amylase dual-response hydrogel system loaded with insulin and a preparation method thereof. Compared with the prior art, the hydrogel system of the invention forms dual stimulation-responsive release of pH and alpha-amylase of insulin, and simultaneously has the characteristic of adjustable gradient release of insulin.

Description

PH and alpha-amylase dual-response insulin-loaded hydrogel system and preparation
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a pH and alpha-amylase dual-response insulin-loaded hydrogel system and a preparation method thereof.
Background
Diabetes has become a metabolic disease that most affects human health because of its high incidence and its high secondary effects. At present, subcutaneous insulin and analogues thereof are the most effective administration mode for type I diabetes and later-stage type II diabetes, but considering the long course of disease and the lifetime characteristics of diabetes, the inconvenience of long-term injection administration and the pain and side effects brought to patients cause the compliance and compliance of patients to be extremely poor, thereby affecting the treatment effect and the life quality of patients. Since the intestinal absorption of insulin into the portal vein and liver more closely mimics the normal physiological secretory pathway of insulin than subcutaneous injection, oral administration, which is typically a non-invasive route of administration, is an important attractive direction of development. One of the major obstacles to oral administration of insulin is the strong damaging effects of the acid and pepsin environment in the stomach on insulin. Insulin encapsulation and delivery systems are considered as promising approaches to solve this problem, while allowing a controlled release of insulin in the intestinal tract. In addition, the dose of insulin injection or other oral drugs for diabetes treatment, which is not matched with the blood sugar rise rate after food intake, can lead to unstable blood sugar of patients, increase the risk of hypoglycemia, and become one of the obstacles of oral hypoglycemic drugs. Therefore, the development of safe and effective controlled-release insulin delivery systems is an urgent and very significant research.
Currently, environmentally sensitive materials are widely used in the research of encapsulation and delivery systems for insulin due to the rapid response to environmental changes and the triggered release pattern. Chinese patent publication No. CN102908627A discloses a pH-sensitive nanoparticle for oral insulin delivery, which is composed of a pH-sensitive polymer, a hydrophobic substance, an internal stabilizer, an external stabilizer content, and insulin, and shows good pH sensitivity, and the blood glucose content of diabetic rats is significantly reduced after oral administration of the nanoparticle, but the embedding material adopted in the patent is an artificially synthesized high-molecular polymer, which has a complicated preparation process, many introduced chemical substances, poor degradability, and potential safety hazard, and thus has a great limitation on the application in the field of oral insulin.
In order to overcome the defects of the synthetic high molecular polymer, natural polymers such as natural polysaccharide become excellent materials for embedding insulin due to good biocompatibility, easy degradation, no toxicity, hydrophilicity and easy gel formation. Sodium alginate is a typical polysaccharide, and a large number of free carboxyl groups exist in the molecular structure, so that the sodium alginate shrinks in the acidic environment of the stomach and swells in the near-neutral environment of the intestinal tract, and the characteristic makes the sodium alginate an ideal natural pH sensitive material for encapsulated protein and polypeptide drugs, and can avoid the triggered release of the encapsulated protein and polypeptide drugs in the severe environment of the stomach and the intestinal tract. However, the encapsulation and protection effect of pure sodium alginate hydrogel to low molecular weight and hydrophilic insulin is unsatisfactory due to the loose network structure. To overcome this drawback, complex hydrogels formed from alginate and other natural polymers have become an attractive trend in recent years. The paper (REACT FUNCT POLYM, SCI-US,2017,120,20) describes an oral insulin pH-sensitive hydrogel sphere prepared by compounding sodium alginate and carrageenan, which achieves protection of insulin from the gastric acid environment at pH1.2 and swelling at pH 7.4, achieving progressive release of insulin in the intestinal tract. However, the encapsulation efficiency of the hydrogel to insulin is low and is lower than 30%, and the problem that the hydrogel has single response to pH, namely the hypoglycemia risk of a diabetic patient after taking the hydrogel is still not solved.
Therefore, there is still a need to further develop safe and effective oral insulin loading systems.
Disclosure of Invention
The invention aims to provide a pH and alpha-amylase dual-response insulin-loaded hydrogel system and a preparation method thereof.
The purpose of the invention is realized by the following technical scheme:
a pH and alpha-amylase dual-response hydrogel system (English full name is Insulin-loaded hydrogel-porous starch with high-amylose starch hydrogel, hereinafter called INA-MS/HA for short) loaded with Insulin comprises sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high-amylose starch film and Insulin, wherein the gelatinization-retrogradation high-amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, channels and cavities are formed in the porous starch, and the Insulin is distributed in the sodium alginate hydrogel and the channels and the cavities of the porous starch. Insulin regulates the uptake, utilization and storage of glucose, amino acids and fatty acids by cells and inhibits the breakdown of glycogen, proteins and fats.
The porous starch is derived from type A starch, and the type A starch is derived from one or more of corn, waxy corn or rice.
The thickness of the gelatinized-retrograded high amylose film is increased along with the increase of the concentration of the gelatinized high amylose solution, and the starch concentration of the high amylose solution is 0.1-10% (w/v).
The high amylose in the gelatinized-retrograded high amylose film means that the amylose content is >30%.
In the hydrogel system, the mass ratio of the sodium alginate to the porous starch to the insulin is (10-1000): 0.1-10): 1-50, and the use amount of the gelatinization-retrogradation high amylose film is 0.03-0.3 times, preferably 0.03-0.12 times of that of the porous starch.
The embedding rate of the hydrogel system is 87.27-88.61%.
The preparation method of the hydrogel system comprises the following steps:
(a) Mixing the porous starch slurry and the insulin mother liquor, stirring, standing, pre-freezing, and freeze-drying to form insulin-loaded porous starch powder;
(b) Uniformly mixing the insulin-loaded porous starch powder obtained in the step (a) with a gelatinized high amylose solution, pre-freezing and retrograding, and then carrying out freeze drying to obtain the insulin-loaded porous starch powder covered with a gelatinized-retrograded high amylose corn starch film;
(c) Dispersing the porous starch powder which is covered with the gelatinized-regenerated high-amylose corn starch film and is loaded with insulin and is obtained in the step (b) in water, and uniformly mixing the porous starch powder with a sodium alginate solution to form a mixed solution;
(d) Dropwise adding the mixed solution obtained in the step (c) into CaCl 2 And (3) gelation occurs in the solution, and the hydrogel system loaded with the insulin is obtained.
In the step (a), the porous starch is prepared by a physical method, a chemical method, an enzymatic hydrolysis method or a synergistic method.
In the step (a), the porous starch is preferably prepared by an amylase hydrolysis method, and specifically comprises the following steps: starch was added to PBS buffer to form a starch slurry, and α -amylase was mixed with the starch slurry (the amount of α -amylase used was 0.5 IU/mg starch), reacted in a water bath at 37 ℃ for 6 hours, and then the resulting suspension was put into a test tube and centrifuged, and the resulting precipitate was washed with excess anhydrous ethanol repeatedly 3 times and vacuum-dried at a temperature of 40 ℃ overnight.
In the step (a), the porous starch is preferably prepared by a chemical acidolysis method, and specifically comprises the following steps: adding corn starch into 2M HCl solution to form starch slurry, carrying out water bath stirring reaction at 45 ℃ for 20 hours, taking out, carrying out suction filtration, washing with water to be neutral, and carrying out vacuum drying at 40 ℃ overnight.
In the step (a), the porous starch is preferably prepared by a physical ultrasonic method, and specifically comprises the following steps: starch slurry was prepared by adding starch (on a dry basis) to distilled water, which was then sonicated with dual frequency ultrasound at 20kHz, +25kHz for 40min at 30 ℃, rinsed three times with distilled water, and vacuum dried overnight at 40 ℃.
In the step (a), the concentration of the porous starch in the porous starch slurry is 0.01-1g/mL.
In the step (a), the concentration of insulin in the insulin mother liquor is 1-100mg/mL.
In the step (a), the preparation process of the insulin mother liquor comprises the following specific steps: insulin was dissolved in 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution and using 1M NaOH solution to not degrade insulin by acid.
In step (a), the insulin is naturally-occurring insulin, recombinant insulin, insulin analog or insulin derivative. Insulin may be insulin from any suitable species, such as human, porcine, bovine, canine, ovine, etc., and insulin with various degrees of life activity is commercially available.
In the step (a), the insulin is porcine insulin. Porcine insulin is preferably a glycosylated, double-stranded polypeptide chain comprising 51 amino acids and having a molecular weight of 5777 daltons, the alpha and beta chains being linked by two interchain disulfide bonds, the alpha chain comprising one intrachain disulfide bond. In a preferred embodiment, the biological activity of porcine insulin is in the range of 27-28IU/mg.
In the step (a), the pre-freezing temperature is-10 to-50 ℃, preferably-20 ℃, the pre-freezing time is 22 to 26 hours, preferably 24 hours, the freeze-drying temperature is-70 to-90 ℃, preferably-80 ℃, the freeze-drying time is 22 to 26, preferably 24 hours, and the porous starch powder loaded with insulin is formed.
In the step (b), the pre-freezing temperature is-10 to-50 ℃, preferably-20 ℃, the pre-freezing time is 22 to 26 hours, preferably 24 hours, the freeze-drying temperature is-70 to-90 ℃, preferably-80 ℃, and the freeze-drying time is 22 to 26, preferably 24 hours.
In step (b), the concentration of high amylose in the gelatinized high amylose solution is 1 to 10% (w/v), preferably 1 to 4% (w/v). The thickness of the gelatinized-retrograded high amylose film is positively correlated with the concentration of the high amylose solution, and the thicker the high amylose film. Under the action of alpha-amylase in intestinal tracts, the gelatinization-retrogradation high amylose films with different thicknesses have different digestion rates, the thicker the film is, the slower the digestion rate is, and the slower the insulin release rate is, and the thickness of the gelatinization-retrogradation high amylose film is adjusted by adjusting the concentration of a high amylose solution, so that the stepwise response release of insulin to the alpha-amylase is realized.
In the step (b), the preparation process of the gelatinized high amylose starch solution is specifically as follows: heating the high amylose corn starch solution to boiling point, gelatinizing for 15min to form gel, cooling to room temperature.
In the step (c), the concentration of the sodium alginate in the sodium alginate solution is 0.001-0.05g/mL.
In step (d), the CaCl 2 CaCl in solution 2 The concentration of (B) is 0.005-0.1g/mL, preferably 0.02g/mL.
The invention utilizes the protonation of carboxyl on sodium alginate under the acidic condition and the deprotonation characteristic under the near-neutral condition to realize the response of a hydrogel system to pH, and simultaneously, the porous starch can be gradually hydrolyzed under the action of alpha-amylase to realize the alpha-amylase response release of insulin, specifically, the sodium alginate hydrogel is shrunk in a gastric acid environment to protect the insulin so that the insulin can be released only in a small amount in the gastric acid environment, and can swell and release the insulin in the near-neutral environment of small intestine, and the gelatinized-regenerated high amylose starch film can be digested in the presence of the alpha-amylase so as to accelerate the release of the insulin in the small intestine.
The hydrogel system of the invention adds a gelatinization-regeneration high amylose starch film on the surface of porous starch, the thicker the high amylose starch film is, the slower the rate of digestion by alpha-amylase is, the slower the insulin in the porous starch is released, i.e. when the alpha-amylase exists in the environment, the thicker the alpha-amylase HAs on the INA-MS/HA hydrogel is, the slower the insulin is released. The hydrogel system can realize the dual stimulation responsive release of the pH value of the insulin and the alpha-amylase, and has the characteristic of adjustable gradient release of the insulin, so that the hydrogel system has great practical application significance for pertinently adjusting the postprandial blood sugar and reducing the risk of hypoglycemia after the foods with different blood sugar indexes are ingested.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention realizes the gradient response release of insulin to alpha-amylase by covering the surface of the porous starch with a gelatinization-retrogradation high amylose starch film, and the release rate of the insulin can be adjusted by adjusting the concentration of the high amylose starch solution, so that the insulin can be pertinently administrated after food with different blood glucose indexes is ingested, the change of postprandial blood glucose is stabilized, and the hypoglycemia risk of hyperglycemia patients after the medicine is taken is reduced.
2) Compared with the existing natural polymer pH response system loaded with insulin, the invention increases the response of alpha-amylase and realizes the simultaneous response release of the pH of the insulin and the alpha-amylase. After a human body ingests food, the body can regulate feedback secretion of alpha-amylase, and the insulin alpha-amylase is released in a stepwise response manner, so that the postprandial blood sugar after the food is ingested can be regulated in a targeted manner.
3) Compared with the existing environment-sensitive material for artificially synthesizing the polymer, the invention adopts sodium alginate and starch as pH and alpha-amylase response materials respectively, the sodium alginate and the starch are natural polysaccharide substances, the preparation is not needed, the sodium alginate and the starch are in a hydrogel state by adopting a gel method, and other raw materials are natural substances.
Drawings
FIG. 1 is a schematic diagram of the structure and the preparation process of a hydrogel system loaded with insulin;
FIG. 2 is an SEM image of the insulin-loaded porous corn starch powder covered with different thicknesses of gelatinized-retrograded high amylose corn starch films of example 1, example 2, example 3 and comparative example 1;
FIG. 3 is a CLSM image of the insulin loaded hydrogel systems prepared in example 1, example 2, example 3, comparative example 1 and comparative example 2;
FIG. 4 is a graph comparing the swelling behavior of the insulin-loaded hydrogel systems prepared in example 1, example 2, example 3, comparative example 1, and comparative example 2;
FIG. 5 is a graph comparing the release process of insulin from the insulin loaded hydrogel systems prepared in example 1, example 2, example 3, comparative example 1 and comparative example 2;
FIG. 6 is a graph comparing the in vitro digestion rates of three starches.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
A pH and alpha-amylase dual-response insulin-loaded hydrogel system comprises sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high amylose starch film and insulin, wherein the gelatinization-retrogradation high amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, pore channels and cavities exist in the porous starch, and the insulin is distributed in the sodium alginate hydrogel and the pore channels and the cavities of the porous starch and is prepared by adopting the following steps, as shown in figure 1:
(1) 40mg of porcine insulin (with a biological activity ranging from 27 to 28 IU/mg) was dissolved in 2mL of 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution to give an insulin stock solution with an insulin concentration of 20 mg/mL.
(2) The porous corn starch is obtained by the following steps: corn starch (MS) was added to PBS buffer to form a starch slurry, and α -amylase was mixed with the starch slurry (the amount of α -amylase used was 0.5 IU/mg starch), reacted in a water bath at 37 ℃ for 6 hours, and then the resulting suspension was put into a test tube and centrifuged, and the precipitate was washed 3 times with excess anhydrous ethanol and vacuum-dried at 40 ℃ overnight. Then the porous corn starch is taken and dissolved in distilled water to obtain porous corn starch slurry with the concentration of 0.2 g/mL.
(3) Mixing 1mL of insulin mother liquor with 7mL of porous corn starch slurry, stirring, standing, pre-freezing at-20 deg.C overnight, and freeze-drying at-80 deg.C for 24h (vacuum degree of 80 Pa) to obtain porous corn starch powder loaded with insulin.
(4) The insulin-loaded porous corn starch powder was mixed with a gelatinized 1% (w/v, specifically in g/100mL, expressed in "%" for simplicity) high amylose corn starch solution (the amount of the high amylose starch solution was 3 mL/g of porous starch, and the gelatinized high amylose corn starch solution was prepared by heating the high amylose corn starch solution to the boiling point and gelatinizing for 15min to exhibit a gel state, then cooling to room temperature, and the same applies), pre-freezing at-20 ℃ overnight and freeze-drying at-80 ℃ for 24h (vacuum degree of 80 Pa) to prepare an insulin-loaded porous corn starch powder covered with a gelatinized-regenerated high amylose corn starch film, and SEM images of the powder are shown as c and d in FIG. 2, wherein c is an SEM image at 2.00K magnification and d is an SEM image at 5.00K magnification, and it can be seen that the surface of the porous corn starch particles was covered with different degrees.
(5) The porous insulin-loaded corn starch powder covered with the gelatinized-regenerated high amylose corn starch film is dispersed in 8mL of distilled water and uniformly stirred with 8mL of a sodium alginate solution (sodium alginate solution is obtained by directly dissolving sodium alginate in water, the same applies below) with the concentration of 0.02g/mL to form a mixed solution.
(6) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.02g/mL 2 Forming a sodium alginate-covered gelatinization-retrogradation high amylose corn starch film-porous corn starch hydrogel system named INA-MS/HA loaded with insulin in the solution 1％ The mass ratio of the porous corn starch, the insulin and the sodium alginate is 1400mg, 1600mg, and the mass of the gelatinization-retrogradation high amylose film is as followsThe content of CaCl is 42mg 2 The residual 1.38mg of insulin in the solution, with an entrapment rate of 88.61%, is summarized in Table 1. Wherein the embedding rate is the amount of total insulin added minus CaCl 2 The ratio of the amount of insulin in the filtrate to the amount of total insulin added (the same applies below) is specifically calculated as:
Figure BDA0003089659580000071
the INA-MS/HA 1％ The CLSM map of (1) is shown in FIG. 3, in which rhodamine staining, FITC staining, rhodamine and FITC overlap staining are performed from the first row to the third row, respectively, and when starch and protein coexist, FITC (green) will preferentially stain starch, while RhoB (red) will preferentially stain protein, and it can be seen that INA-MS/HA 1％ The starch and the insulin are really present in the porous starch, and the insulin is in a non-uniform distribution state from the view point of a dyed distribution area, which indicates that the insulin enters into the pore canal and the cavity of the porous starch besides being distributed in the alginate hydrogel among the porous starch particles.
The INA-MS/HA 1％ The swelling behavior in an environment simulating gastric fluid (SGF, pH 1.2) and in an environment simulating intestinal fluid (SIF, pH 7.4) is shown in FIG. 4, and it can be seen that INA-MS/HA 1％ Shriveling in simulated gastric fluid, swelling in simulated intestinal fluid, and gradually disintegrating and dissolving after 20 min.
The INA-MS/HA 1％ Insulin release behavior in simulated gastric fluid (SGF, pH1.2, without alpha-amylase) and simulated intestinal fluid (SIF, pH 7.4, divided into both with and without alpha-amylase) is shown in FIG. 5, where INA-MS/HA can be seen 1％ The cumulative insulin release in SGF over 2 hours was about 8.1% (INA-MS/HA since the release conditions in the SGF region are the same 1％ The two curves of (a) are substantially coincident in the SGF region, the same applies below); INA-MS/HA 1％ In the next SIF stage, the release of insulin showed a significant gradient of slow release in the absence of alpha-amylase, with a cumulative release of 66% insulin over 2h (i.e., 240min on the axis of the graph) and with alpha-amylase additionIn the case of the enzyme, the release rate was further increased, with a cumulative insulin release of 84% over 2h (i.e., 240min on the axis of the graph) and 93.88% over 4h (i.e., 360min on the axis of the graph), i.e., INA-MS/HA in the simulated intestinal fluid environment 1％ Almost complete release within 4h indicates INA-MS/HA 1％ Has dual response to pH and alpha-amylase.
The INA-MS/HA 1％ The rate of insulin release in (a) can be explained by the in vitro digestibility of the porous corn starch contained in the hydrogel, as shown in FIG. 6 (i.e., maize stage in the figure), which shows that the digestibility of porous corn starch is directly correlated with time.
Example 2
A pH and alpha-amylase dual-response insulin-loaded hydrogel system comprises sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high amylose starch film and insulin, wherein the gelatinization-retrogradation high amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, pore channels and cavities exist in the porous starch, and the insulin is distributed in the sodium alginate hydrogel and the pore channels and the cavities of the porous starch and is prepared by adopting the following steps, as shown in figure 1:
(1) 40mg of porcine insulin (with a biological activity ranging from 27 to 28 IU/mg) was dissolved in 2mL of 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution to give an insulin stock solution with an insulin concentration of 20 mg/mL.
(2) The porous corn starch is obtained by the following steps: corn starch (MS) was added to PBS buffer to form a starch slurry, and α -amylase was mixed with the starch slurry (the amount of α -amylase used was 0.5 IU/mg starch), reacted in a water bath at 37 ℃ for 6 hours, and then the resulting suspension was put into a test tube and centrifuged, and the precipitate was washed 3 times with excess anhydrous ethanol and vacuum-dried at 40 ℃ overnight. Then the porous corn starch is taken and dissolved in distilled water to obtain porous corn starch slurry with the concentration of 0.2 g/mL.
(3) Mixing 1mL of insulin mother liquor with 7mL of porous corn starch slurry, stirring, standing, pre-freezing at-20 deg.C overnight, and freeze-drying at-80 deg.C for 24h (vacuum degree of 80 Pa) to obtain porous corn starch powder loaded with insulin.
(4) The insulin-loaded porous corn starch powder was uniformly mixed with a gelatinized 2% (w/v) high amylose corn starch solution (the amount of the high amylose starch solution was measured per gram of porous starch) and pre-frozen at-20 ℃ overnight for retrogradation and freeze-dried at-80 ℃ for 24 hours (vacuum degree of 80 Pa) to produce an insulin-loaded porous corn starch powder covered with a gelatinized-retrograded high amylose corn starch film, and SEM images of the powder are shown as e and f in fig. 2, where e is an SEM image at a magnification of 2.00K and f is an SEM image at a magnification of 5.00K, and it was found that the pores on the surface of the porous corn starch particles were covered to different degrees.
(5) The porous corn starch powder which is covered with the gelatinized-regenerated high amylose corn starch film and is loaded with insulin is taken to be dispersed in 8mL of distilled water and is evenly stirred with 8mL of sodium alginate solution with the concentration of 0.02g/mL to form mixed solution.
(6) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.02g/mL 2 Forming a sodium alginate-covered gelatinization-retrogradation high amylose corn starch film-porous corn starch hydrogel system which is named INA-MS/HA and loaded with insulin in the solution 2％ The mass ratio of the porous corn starch, the insulin and the sodium alginate is 1400mg, 1600mg, the mass of the gelatinization-retrogradation high amylose film is 84mg, and the mass of CaCl is 2 The solution contained 1.75mg of insulin remaining, and the encapsulation efficiency was 87.30%, which are summarized in Table 1.
The INA-MS/HA 2％ The CLSM map of (1) is shown in FIG. 3, in which rhodamine staining, FITC staining, rhodamine and FITC overlap staining are performed from the first row to the third row, respectively, and when starch and protein coexist, FITC (green) will preferentially stain starch, while RhoB (red) will preferentially stain protein, and it can be seen that INA-MS/HA 2％ Starch and insulin are indeed present in the porous starch, and the insulin is in a non-uniform distribution state from the view point of a dyed distribution area, which indicates that the insulin enters the pore channels of the porous starch and the pores of the porous starch in addition to the alginate hydrogel distributed among the porous starch particlesIn the cavity, and insulin is more retained inside the porous starch.
The INA-MS/HA 2％ The swelling behavior in an environment simulating gastric fluid (SGF, pH1.2, without alpha-amylase) and in an environment simulating intestinal fluid (SIF, pH 7.4) is shown in FIG. 4, where INA-MS/HA can be seen 2％ Shriveling in simulated gastric fluid, swelling in simulated intestinal fluid, and gradually disintegrating and dissolving after 20 min.
The INA-MS/HA 2％ Insulin release behavior in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.4, divided into alpha-amylase plus and alpha-amylase minus) is shown in FIG. 5, where INA-MS/HA can be seen 2％ Cumulative insulin release in SGF for 2 hours was about 6.3%; INA-MS/HA 2％ In the following SIF stage, the insulin release exhibited a significantly graded, retarded release in the absence of alpha-amylase, with a cumulative insulin release of 64% over 2h (i.e., 240min on the scale of the graph), and with the addition of alpha-amylase, the release rate was further increased, with a cumulative insulin release of 74% over 2h (i.e., 240min on the scale of the graph) and a cumulative insulin release of 80.01% over 4h (i.e., 360min on the scale of the graph), indicating INA-MS/HA 2％ Has dual responses to pH and alpha-amylase.
The INA-MS/HA 2％ The rate of insulin release in (a) can be explained by the in vitro digestibility of the porous corn starch contained in the hydrogel, as shown in FIG. 6 (i.e., maize stage in the figure), which shows that the digestibility of porous corn starch is directly correlated with time.
Example 3
A pH and alpha-amylase dual-response insulin-loaded hydrogel system comprises sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high amylose starch film and insulin, wherein the gelatinization-retrogradation high amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, pore channels and cavities exist in the porous starch, and the insulin is distributed in the sodium alginate hydrogel and the pore channels and the cavities of the porous starch and is prepared by adopting the following steps, as shown in figure 1:
(1) 40mg of porcine insulin (with a biological activity ranging from 27 to 28 IU/mg) was dissolved in 2mL of 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution to give an insulin stock solution with an insulin concentration of 20 mg/mL.
(2) The porous corn starch is obtained by the following steps: corn starch (MS) was added to PBS buffer to form a starch slurry, and α -amylase was mixed with the starch slurry (the amount of α -amylase used was 0.5 IU/mg starch), reacted in a water bath at 37 ℃ for 6 hours, and then the resulting suspension was put into a test tube and centrifuged, and the precipitate was washed 3 times with excess absolute ethanol and vacuum-dried at a temperature of 40 ℃ overnight. Then dissolving the porous corn starch in distilled water to obtain porous corn starch slurry with the concentration of 0.2 g/mL.
(3) Mixing 1mL of insulin mother liquor with 7mL of porous corn starch slurry, stirring, standing, pre-freezing at-20 deg.C overnight, and freeze-drying at-80 deg.C for 24h (vacuum degree of 80 Pa) to obtain porous corn starch powder loaded with insulin.
(4) The insulin-loaded porous corn starch powder was uniformly mixed with a gelatinized 4% (w/v) high amylose corn starch solution (the amount of the high amylose starch solution was measured per gram of porous starch) and pre-frozen at-20 ℃ overnight for retrogradation and freeze-dried at-80 ℃ for 24 hours (vacuum degree of 80 Pa) to produce an insulin-loaded porous corn starch powder covered with a gelatinized-retrograded high amylose corn starch film, and SEM images of the powder are shown in fig. 2 as g and h, where g is an SEM image at a magnification of 2.00K and f is an SEM image at a magnification of 5.00K, and it was found that the pores on the surface of the porous corn starch particles were covered to different degrees.
(5) The porous corn starch powder which is covered with the gelatinized-regenerated high amylose corn starch film and is loaded with insulin is taken to be dispersed in 8mL of distilled water and is evenly stirred with 8mL of sodium alginate solution with the concentration of 0.02g/mL to form mixed solution.
(6) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.02g/mL 2 Forming sodium alginate loaded with insulin in solution, covering with gelatinizing-retrogradation high amylose corn starch film-porous jadeRice starch hydrogel system named INA-MS/HA 4％ The mass ratio of the porous corn starch, the insulin and the sodium alginate is 1400mg, namely, 20mg 2 The residual 1.47mg of insulin in the solution, with an entrapment rate of 87.27%, is summarized in Table 1.
The INA-MS/HA 4％ The CLSM map of (A) is shown in FIG. 3, in which rhodamine staining, FITC staining, rhodamine and FITC overlap staining are performed from the first line to the third line, respectively, and when starch and protein coexist, FITC (green) will stain starch preferentially and RhoB (red) will stain protein preferentially, and it can be seen that INA-MS/HA 4％ The starch and the insulin are indeed present in the porous starch, and the insulin is in a non-uniform distribution state from the view point of a dyed distribution area, which indicates that the insulin enters into the pore canal and the cavity of the porous starch besides being distributed in the alginate hydrogel among the porous starch particles, and the insulin is more retained in the porous starch.
The INA-MS/HA 4％ The swelling behavior in an environment simulating gastric fluid (SGF, pH 1.2) and in an environment simulating intestinal fluid (SIF, pH 7.4) is shown in FIG. 4, and it can be seen that INA-MS/HA 4％ Shriveling in simulated gastric fluid, swelling in simulated intestinal fluid, and gradually disintegrating and dissolving after 20 min.
The INA-MS/HA 4％ Insulin release behavior in simulated gastric fluid (SGF, pH1.2, without alpha-amylase) and simulated intestinal fluid (SIF, pH 7.4, divided into both with and without alpha-amylase) is shown in FIG. 5, where INA-MS/HA can be seen 4％ The cumulative insulin release in SGF over 2 hours was about 6.2%; INA-MS/HA 4％ In the following SIF stage, the release of insulin exhibited a significantly graded, retarded release in the absence of alpha-amylase, with a cumulative insulin release of 56% over 2h (i.e., 240min on the scale of the graph), and with the addition of alpha-amylase, the release rate was further increased, with a cumulative insulin release of 59% over 2h (i.e., 240min on the scale of the graph) and a cumulative insulin release of 62.65% over 4h (i.e., 360min on the scale of the graph), indicating INA-MS/HA 4％ For pH and alpha-amylaseWith a double response.
The INA-MS/HA 4％ The rate of insulin release in (a) can be explained by the in vitro digestibility of the porous corn starch contained in the hydrogel, as shown in FIG. 6 (i.e., maize stage in the figure), which shows that the digestibility of porous corn starch is directly correlated with time. Fig. 6 can illustrate that the cumulative release rate of insulin increases with increasing starch digestion rate.
Comparative example 1
The sodium alginate-porous starch hydrogel embedded with insulin is prepared by the following steps:
(1) 40mg of porcine insulin (with a biological activity ranging from 27 to 28 IU/mg) was dissolved in 2mL of 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution to give an insulin stock solution with an insulin concentration of 20 mg/mL.
(2) The porous corn starch is obtained by the following steps: corn starch (MS) was added to PBS buffer to form a starch slurry, and α -amylase was mixed with the starch slurry (the amount of α -amylase used was 0.5 IU/mg starch), reacted in a water bath at 37 ℃ for 6 hours, and then the resulting suspension was put into a test tube and centrifuged, and the precipitate was washed 3 times with excess anhydrous ethanol and vacuum-dried at 40 ℃ overnight. Then the porous corn starch is taken and dissolved in distilled water to obtain porous corn starch slurry with the concentration of 0.2 g/mL.
(3) Mixing 1mL of insulin mother liquor with 7mL of porous corn starch slurry, stirring, standing, pre-freezing at-20 deg.C overnight, and freeze-drying at-80 deg.C for 24h (vacuum degree of 80 Pa) to obtain porous corn starch powder loaded with insulin.
(4) The porous corn starch powder loaded with the insulin is uniformly mixed with distilled water (the amount of the distilled water is 3mL per gram of the porous starch), pre-frozen at-20 ℃ for overnight retrogradation and freeze-dried at-80 ℃ for 24h (the vacuum degree is 80 Pa) to prepare the porous corn starch powder loaded with the insulin without the gelatinization-retrogradation high amylose starch film coverage, wherein SEM images of the powder are shown as a and b in figure 2, wherein the a image is an SEM image under the magnification of 2.00K, and the b image is an SEM image under the magnification of 5.00K, so that the surface of the porous corn starch particle loaded with the insulin without the gelatinization-retrogradation high amylose starch film coverage is clear, the surface of the porous corn starch particle loaded with the insulin covered with the gelatinization-retrogradation high amylose starch film is covered with different degrees, and the surface holes of the porous corn starch particle are covered with the larger degree and the thickness of the film is thicker along with the increase of the concentration of the gelatinization high amylose starch.
(5) The porous corn starch powder which is not covered by the gelatinization-retrogradation high amylose corn starch film and is loaded with insulin is taken to be dispersed in 8mL of distilled water and is evenly stirred with 8mL of sodium alginate solution with the concentration of 0.02g/mL to form mixed solution.
(6) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.02g/mL 2 Forming sodium alginate-porous starch hydrogel embedded with insulin in solution, named INA-MS/HA 0％ The mass ratio of the porous corn starch, the insulin and the sodium alginate is 1400mg 2 The residual 1.85mg of insulin in the solution, with an entrapment rate of 86.30%, is summarized in Table 1.
The INA-MS/HA 0％ The CLSM map of (1) is shown in FIG. 3, in which rhodamine staining, FITC staining, rhodamine and FITC overlap staining are performed from the first row to the third row, respectively, and when starch and protein coexist, FITC (green) will preferentially stain starch, while RhoB (red) will preferentially stain protein, and it can be seen that INA-MS/HA 0％ The starch and the insulin are indeed present, and the insulin is in a non-uniform distribution state from the view point of the dyed distribution area, which indicates that the insulin enters the pore canal and the cavity of the porous starch besides being distributed in the alginate hydrogel between the porous starch particles. The INA-MS/HA 0％ The swelling behavior in an environment simulating gastric fluid (SGF, pH 1.2) and in an environment simulating intestinal fluid (SIF, pH 7.4) is shown in FIG. 4, and it can be seen that INA-MS/HA 0％ Shrinkage in simulated gastric fluid, swelling in simulated intestinal fluid, and a lesser degree of shrinkage and swelling. Figure 3 illustrates that the hydrogel system of the present invention has an excellent pH response, which allows the patient to take insulin orally.
The INA-MS/HA 0％ Insulin release behavior in simulated gastric fluid (SGF, pH1.2, without alpha-amylase) and simulated intestinal fluid (SIF, pH 7.4, divided into both with and without alpha-amylase) is shown in FIG. 5, where INA-MS/HA can be seen 0％ The cumulative insulin release at 2 hours in SGF was about 6.6%; INA-MS/HA 0％ In the next SIF stage, the release of insulin exhibited a significantly graded, retarded release in the absence of alpha-amylase, with cumulative release of insulin exceeding 77.59% over 2h (i.e., 240min on the scale of the graph), whereas with alpha-amylase, the release rate was further increased, with cumulative release of insulin exceeding 96% over 2h (i.e., 240min on the scale of the graph) and 98.39% over 4h (i.e., 360min on the scale of the graph). In FIG. 5, INA-MS/HA 1％ ，INA-MS/HA 2％ ，INA-MS/HA 4％ The maximum cumulative insulin release at 2 hours in SGF is only about 8%, which means that the hydrogel system of the present invention can better protect insulin from low pH and pepsin in gastric juice. In the following SIF stage, INA-MS/HA in the presence of alpha-amylase 1％ ，INA-MS/HA 2％ ，INA-MS/HA 4％ The cumulative insulin release within 2h is less than 80%, which proves that the hydrogel system increases the alpha-starch triggered release characteristic and is characterized by amylase gradient response release, and the adjustable insulin release rate of the hydrogel has important significance for the smooth regulation of postprandial blood sugar at matched insulin release rate after foods with different glycemic indexes are ingested.
TABLE 1 list of embedding rates of hydrogel systems obtained in examples 1, 2, 3 and comparative example 1
Figure BDA0003089659580000131
(in the table, different letters a, b, c in the same column indicate significant differences (p < 0.05))
Comparative example 2
An insulin-embedded sodium alginate hydrogel (hereinafter referred to as INA) is prepared by the following steps:
(1) 40mg of porcine insulin (with a biological activity ranging from 27 to 28 IU/mg) was dissolved in 2mL of 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution to give an insulin stock solution with an insulin concentration of 20 mg/mL.
(3) 1mL of insulin mother liquor and 7mL of distilled water are mixed and stirred, 8mL of sodium alginate solution with the concentration of 0.02g/mL is added and stirred uniformly to form a mixed solution.
(4) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.02g/mL 2 Forming a sodium alginate and insulin embedding and transmitting system in the solution, wherein the sodium alginate and insulin embedding and transmitting system is named INA, the mass ratio of insulin to sodium alginate is 20mg 2 The residual amount of insulin in the solution was 8.17mg, and the entrapment rate was 64.14%, which is summarized in Table 1. As can be seen in Table 1, the encapsulation efficiency of insulin in the hydrogel system of the present invention increased from about 64% to over 86% compared to INA, i.e., porous starch affected the encapsulation efficiency of insulin, whereas INA-MS/HA 1％ 、INA-MS/HA 2％ 、INA-MS/HA 4％ And INA-MS/HA 0％ There was no significant difference in the embedding rate between them, i.e., the presence of the high amylose corn starch film had essentially no effect on the embedding rate.
The swelling behavior of the INA in an environment simulating gastric fluid (SGF, pH 1.2) and in an environment simulating intestinal fluid (SIF, pH 7.4) is shown in FIG. 4, and it can be seen that the INA shrinks in the simulated gastric fluid and swells in the simulated intestinal fluid, but the shrinkage and swelling degree are large. Figure 4 illustrates that the hydrogel system of the present invention has an excellent pH response, which allows the patient to take insulin orally.
The CLSM map of the INA is shown in fig. 3, and it can be seen that starch and insulin are indeed present in the INA, and from the stained distribution area, insulin is distributed more uniformly, indicating that the starch content is low and insulin is widely spread on the starch surface. The figures in fig. 3 illustrate that as the thickness of the high amylose corn starch film increases, the retention of insulin in the porous starch channels and cavities increases and the insulin distribution in the matrix of the sodium alginate hydrogel decreases, which should be due to the coverage of the porous starch surface with a gelatinised-retrograded high amylose corn starch film, which impedes the efflux of insulin.
Example 4
A pH and alpha-amylase dual-response insulin-loaded hydrogel system comprises sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high amylose starch film and insulin, wherein the gelatinization-retrogradation high amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, pore channels and cavities exist in the porous starch, and the insulin is distributed in the sodium alginate hydrogel and the pore channels and the cavities of the porous starch and is prepared by adopting the following steps, as shown in figure 1:
(1) 10mg of recombinant insulin was dissolved in 10mL of 0.01M HCl solution, and the pH was adjusted to 7.5 using 1M NaOH solution to obtain an insulin mother liquor having an insulin concentration of 1 mg/mL.
(2) The porous corn starch is obtained by the following steps: adding corn starch into 2M HCl solution to form starch slurry, carrying out water bath stirring reaction at 45 ℃ for 20 hours, taking out, carrying out suction filtration, washing with water to be neutral, and carrying out vacuum drying at 40 ℃ overnight. Then dissolving the porous corn starch in distilled water to obtain porous corn starch slurry with the concentration of 0.01 g/mL.
(3) Mixing 10mL of insulin mother liquor with 100mL of porous corn starch slurry, stirring, standing, pre-freezing at-50 ℃ for 22h, and freeze-drying at-90 ℃ for 22h (vacuum degree of 80 Pa) to form porous corn starch powder loaded with insulin.
(4) The amount of the porous corn starch powder loaded with insulin and the high amylose solution of the gelatinized 5 percent high amylose corn starch solution is weighed according to 3mL per gram of porous starch, and the preparation process of the gelatinized high amylose corn starch solution is specifically as follows: heating the high amylose corn starch solution to boiling point, gelatinizing for 15min to form gel, cooling to room temperature, mixing uniformly, pre-freezing at-50 deg.C for 22h, and freeze-drying at-90 deg.C for 22h (vacuum degree of 80 Pa) to obtain porous insulin-loaded corn starch powder covered with gelatinized-regenerated high amylose corn starch film.
(5) The porous insulin-loaded corn starch powder covered with the gelatinized-regenerated high amylose corn starch film is dispersed in 100mL of distilled water and uniformly stirred with 100mL of a sodium alginate solution (sodium alginate solution is obtained by directly dissolving sodium alginate in water, the same applies below) with the concentration of 0.001g/mL to form a mixed solution.
(6) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.005g/mL 2 Forming a sodium alginate-covered gelatinization-retrogradation high amylose corn starch film-porous corn starch hydrogel system loaded with insulin in the solution, wherein the mass ratio of the porous corn starch, the insulin and the sodium alginate is 1000mg.
Example 5
A pH and alpha-amylase dual-response insulin-loaded hydrogel system comprises sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high amylose starch film and insulin, wherein the gelatinization-retrogradation high amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, pore channels and cavities exist in the porous starch, and the insulin is distributed in the sodium alginate hydrogel and the pore channels and the cavities of the porous starch and is prepared by adopting the following steps, as shown in figure 1:
(1) 1000mg of insulin derivative was dissolved in 10mL of 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution to give an insulin stock solution with an insulin concentration of 100mg/mL.
(2) The porous corn starch is obtained by the following steps: a5% (w/w) starch slurry was prepared by adding 5g of starch (on a dry basis) to 95g of distilled water, sonicated at 30 ℃ for 40min with dual frequency, 20kHz +25kHz, then rinsed three times with distilled water, and vacuum dried at 40 ℃ overnight. Then dissolving the porous corn starch in distilled water to obtain porous corn starch slurry with the concentration of 1g/mL.
(3) Mixing 1mL of insulin mother liquor with 10mL of porous corn starch slurry, stirring, standing, pre-freezing at-10 ℃ for 26h, and freeze-drying at-70 ℃ for 26h (vacuum degree of 80 Pa) to form porous corn starch powder loaded with insulin.
(4) The amount of the porous corn starch powder loaded with insulin and the high amylose starch solution of the gelatinized 10 percent high amylose corn starch solution is weighed according to 3mL per gram of porous starch, and the preparation process of the gelatinized high amylose corn starch solution is specifically as follows: heating the high amylose corn starch solution to boiling point, gelatinizing for 15min to form gel, cooling to room temperature, mixing uniformly, pre-freezing at-10 deg.C for 26h, and freeze-drying at-70 deg.C for 26h (vacuum degree of 80 Pa) to obtain porous insulin-loaded corn starch powder covered with gelatinized-regenerated high amylose corn starch film.
(5) The porous corn starch powder loaded with insulin covered with the gelatinized-retrogradation high amylose corn starch film is dispersed in 10mL of distilled water and uniformly stirred with 10mL of a sodium alginate solution (sodium alginate solution obtained by directly dissolving sodium alginate in water, the same applies below) with the concentration of 0.05g/mL to form a mixed solution.
(6) At room temperature, the mixed solution is dripped into CaCl with the concentration of 0.1g/mL 2 Forming a sodium alginate-covered gelatinization-retrogradation high amylose corn starch membrane-porous corn starch hydrogel system loaded with insulin in the solution, wherein the mass ratio of the porous corn starch, the insulin and the sodium alginate is 10000 mg.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (9)

1. The hydrogel system loaded with insulin and having double responses of pH and alpha-amylase is characterized by comprising sodium alginate hydrogel serving as a matrix, porous starch, a gelatinization-retrogradation high amylose starch film and insulin, wherein the gelatinization-retrogradation high amylose starch film is covered on the outer surface of the porous starch, the sodium alginate hydrogel is wrapped outside the porous starch, pore channels and cavities exist in the porous starch, and the insulin is distributed in the sodium alginate hydrogel and the pore channels and the cavities of the porous starch; the high amylose in the gelatinized-regenerated high amylose film is that the content of amylose is more than 30 percent, the porous starch is derived from A-type starch, the porous starch is prepared by a physical method, a chemical method, an enzymatic hydrolysis method or a synergistic method,
the enzymolysis method, namely the amylase hydrolysis method, comprises the following steps: starch is added to PBS buffer to form a starch slurry, and alpha-amylase is mixed with the starch slurry at 37 o C water bath for 6 hours, then placing the obtained suspension into a test tube for centrifugation, repeatedly washing the obtained precipitate with excess absolute ethyl alcohol for 3 times, and performing reaction at 40 DEG o Drying in vacuum at the temperature of C overnight;
the chemical method, namely the chemical acidolysis method, comprises the following steps: adding corn starch into 2M HCl solution to form starch slurry, reacting in water bath at 45 deg.C under stirring for 20 hr, taking out, vacuum filtering, washing with water to neutrality, and standing at 40 deg.C o Drying in vacuum at the temperature of C overnight;
the physical method, namely the physical ultrasonic method, comprises the following steps: starch slurry was prepared by adding starch to distilled water, treated with 20kHz +25kHz dual frequency ultrasound for 40min at 30 ℃, rinsed three times with distilled water, and washed at 40 ℃ o Dry under vacuum at temperature C overnight.
2. The pH and alpha-amylase dual-responsive insulin-loaded hydrogel system of claim 1, wherein the amount of said gelatinized-retrograded high amylose starch film is 0.03 to 0.3 times the amount of porous starch.
3. A method for preparing an insulin-loaded hydrogel system according to claim 1 or 2, comprising the steps of:
(a) Mixing the porous starch slurry and the insulin mother liquor, stirring, standing, pre-freezing, and freeze-drying to form insulin-loaded porous starch powder;
(b) Uniformly mixing the insulin-loaded porous starch powder obtained in the step (a) with a gelatinized high amylose solution, pre-freezing and retrograding, and then carrying out freeze drying to obtain the insulin-loaded porous starch powder covered with a gelatinized-retrograded high amylose corn starch film;
(c) Dispersing the porous starch powder which is covered with the gelatinized-regenerated high-amylose corn starch film and is loaded with insulin and is obtained in the step (b) in water, and uniformly mixing the porous starch powder with a sodium alginate solution to form a mixed solution;
(d) Dropwise adding the mixed solution obtained in the step (c) into CaCl 2 And (3) gelatinizing in the solution to obtain the hydrogel system loaded with the insulin.
4. The method of claim 3, wherein in step (a), the concentration of porous starch in the porous starch slurry is 0.01-1g/mL;
in the step (a), the concentration of insulin in the insulin mother liquor is 1-100mg/mL.
5. The method for preparing the insulin-loaded hydrogel system according to claim 3, wherein the insulin mother liquor is prepared by the following steps in step (a): insulin was dissolved in 0.01M HCl solution and the pH was adjusted to 7.5 using 1M NaOH solution.
6. The method of claim 3, wherein in step (a), the insulin is selected from the group consisting of naturally occurring insulin, recombinant insulin, insulin analogs and insulin derivatives.
7. The method of claim 3, wherein the pre-freezing temperature in step (a) is at a temperature of-10 ~ -50 o And C, pre-freezing for 22 to 26h, and carrying out freeze drying at the temperature of-70 to-90 ℃ for 22 to 26h.
8. The method for preparing the hydrogel system loaded with insulin according to claim 3, wherein in the step (b), the pre-freezing temperature is from-10 to-50% o C, pre-freezing for 22 to 26h, wherein the temperature of freeze drying is-70 to-90 ℃, and the time of freeze drying is 22 to 26h;
in the step (b), the concentration of the high amylose in the gelatinized high amylose solution is 1 to 10% (w/v).
9. The method of claim 3, wherein in step (c), the concentration of sodium alginate in the sodium alginate solution is 0.001-0.05g/mL.
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