CN115212316B - Insulin-loaded ZIF-8 and response type insulin administration system, preparation method and application thereof - Google Patents

Insulin-loaded ZIF-8 and response type insulin administration system, preparation method and application thereof Download PDF

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CN115212316B
CN115212316B CN202210683657.7A CN202210683657A CN115212316B CN 115212316 B CN115212316 B CN 115212316B CN 202210683657 A CN202210683657 A CN 202210683657A CN 115212316 B CN115212316 B CN 115212316B
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张鹏
刘莹
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Shenyang Pharmaceutical University
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Abstract

An insulin-loaded ZIF-8 and a response type insulin administration system, a preparation method and application thereof belong to the field of biological medicine materials. The ZIF-8 loaded with insulin takes ZIF-8 as a carrier and is prepared by a one-pot method. The encapsulation efficiency is higher by adjusting the preparation process. Also provided are responsive insulin delivery systems comprising insulin-loaded ZIF-8, glucose oxidase, a gel matrix and an adjunct. The gel matrix of the system selects poloxamer with good biocompatibility and degradability. Responsive insulin delivery systems are made by the "cold melt method". The use of the above-described responsive insulin delivery system in the hypoglycemic effect was also investigated. The gel matrix can slow down the release speed of the medicine and prolong the action time of the medicine.

Description

Insulin-loaded ZIF-8 and response type insulin administration system, preparation method and application thereof
Technical Field
The invention belongs to the technical field of biomedical materials, relates to an insulin-loaded ZIF-8 and a response type insulin administration system thereof, a preparation method and application thereof, and in particular relates to a synergistic application of a zeolite-based imidazole acid ester skeleton-8 and temperature-sensitive hydrogel in the aspect of intelligent slow release of insulin.
Background
Diabetes is a serious chronic disease, has great influence on life of individuals, families and society, insulin is suitable for treatment of various types of diabetes, but the injection dosage is always a troublesome problem, the excessive dosage even endangers the life of patients, and the insulin injection quantity is regulated and controlled reasonably by monitoring blood sugar changes in clinic; however, the steps are cumbersome, resulting in poor patient compliance, so intelligent and effective treatment protocols are currently urgently needed to control blood glucose levels.
The glucose intelligent response drug delivery system can adjust insulin release amount through in vivo blood sugar condition, the common glucose intelligent response drug delivery system is usually glucose oxidase and phenylboronic acid, when phenylboronic acid is used as a response group, the preparation process is generally complex, an organic solvent is often used, so that the activity of insulin cannot be ensured, so that the research of taking glucose oxidase as a response switch is increased successively, the existing research can combine the glucose oxidase with zeolite-based imidazole acid ester skeleton-8 (ZIF-8), although good biocompatibility can be realized, after glucose oxidase is combined with acid-sensitive drug-carrying zeolite-based imidazole acid ester skeleton-8 (ZIF-8), the insulin release amount can be adjusted according to the glucose concentration, but glucose in normal tissues is consumed, the drug release speed cannot be controlled, and the drug release speed has a certain influence on the therapeutic effect. Currently, multiple-response drug delivery systems have entered a new stage of development, driven by the fields of material chemistry and pharmaceutical research.
Wherein the zeolite-based imidazole acid ester skeleton-8 is formed by self-assembly of imidazole groups and Zn ions, and the specific principle is as follows: n atoms on Zn ion coordination bonding imidazole groups form ZnN 4 clusters with regular tetrahedral structures, 4 or 6 ZnN 4 clusters form ZIF-8 crystal nuclei, and the crystal nuclei are combined together through organism connection and gradually grow into ZIF-8 crystals. The preparation method is a plurality of methods, such as a solvothermal method, a liquid phase diffusion method, a template method, an ultrasonic wave or microwave method and the like.
The temperature responsive in situ gel system is composed of temperature sensitive polymers which exist in liquid form below a low critical gelation temperature) and form gel when the ambient temperature reaches or exceeds LCGT, and the transformation of the sol-gel can not only meet the injectability of the preparation, but also reduce the drug release speed.
Poloxamer is a common matrix of temperature-sensitive hydrogel, and is a high molecular polymer composed of polyethylene oxide (PEO) and polypropylene oxide (PPO), when the concentration of the high molecular polymer reaches critical micelle in aqueous solution, the high molecular polymer forms micelle composed of PEO chains forming a hydrophilic shell and PPO chains forming a hydrophobic core, and the high molecular polymer is in a liquid state at low temperature, has fluidity and injectability, and is convenient to administer. When the temperature of the acting part is higher than the gelation temperature, the micelles are further entangled and piled up, the system is converted from a solution state to a semisolid gel state, and the acting time of the medicine can be prolonged.
Disclosure of Invention
Aiming at the problems of the existing glucose intelligent response drug delivery system, the invention provides an insulin-loaded ZIF-8, a response type insulin drug delivery system, a preparation method and application thereof. The response type insulin administration system formed by wrapping the ZIF-8 loaded with insulin and glucose oxidase in the temperature sensitive hydrogel has wide development prospect.
The first object of the present invention is to prepare an insulin-loaded zeolite-based imidazolate skeleton-8 (insulin-loaded ZIF-8).
The second object of the present invention is to prepare a responsive insulin delivery system of temperature sensitive hydrogel which simultaneously encapsulates glucose oxidase and insulin-loaded zeolite-based imidazolate backbone-8 (insulin-loaded ZIF-8).
A third object of the present invention is to investigate the use of the above-mentioned responsive insulin delivery system for the hypoglycemic effect.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the ZIF-8 loaded with insulin takes ZIF-8 as a carrier, the insulin is loaded, the encapsulation efficiency is 57% -75%, and the drug loading rate is 14% -27%.
The particle size of the ZIF-8 loaded with insulin is 80-500 nm, the particle size distribution is uniform, and the polydispersity index PDI is less than 0.2.
The invention relates to a preparation method of insulin-loaded ZIF-8, which is a one-pot preparation method and specifically comprises the following steps:
(1) Weighing zinc salt and organic imidazole, and stirring and dissolving the zinc salt and the organic imidazole by taking water as a solvent to obtain a zinc salt solution and an organic imidazole solution respectively;
(2) Adding insulin into the organic imidazole solution and uniformly stirring to obtain an organic imidazole solution containing insulin; wherein, according to the mass ratio, insulin: organic imidazole= (2-5): (50-200); more preferably 4:100;
(3) Mixing a zinc salt solution and an organic imidazole solution containing insulin to obtain a mixed solution; wherein, according to the mass ratio, zinc salt: organoimidazole=1: (2-8); more preferably 1:4;
stirring the mixed solution for reaction until the precipitation amount is not changed, and obtaining suspension after the reaction is completed;
(4) And (3) carrying out solid-liquid separation on the suspension, washing the precipitate with water, and drying to obtain the insulin-loaded zeolite-based imidazole acid ester skeleton-8.
In the step (1), preferably, the mass concentration of zinc salt in the zinc salt solution is 1.7-5.0 mg/mL; preferably, the mass concentration of the organic imidazole in the organic imidazole solution is 3.3-40 mg/mL.
In the step (1), the organic imidazole is preferably 2-methylimidazole, and the zinc salt is preferably zinc nitrate.
In the step (1), the stirring time is preferably 10-20 min.
In the step (3), preferably, the zinc salt solution is added dropwise to the insulin-containing organic imidazole solution at a dropping rate of 0.5 to 1mL/s.
In the step (3), preferably, the stirring speed is 300-500 r/min, and the stirring time is 0.5-3 h.
In the step (4), the solid-liquid separation is preferably centrifugal separation, more preferably centrifugation at 7000r/min for 10 to 15min.
In the step (4), the drying is preferably freeze drying.
The invention relates to a response type insulin administration system, which comprises ZIF-8 loaded with insulin, glucose oxidase, a gel matrix and auxiliary materials.
Further, in ZIF-8 insulin loaded with insulin in a mass ratio: glucose oxidase: gel matrix: auxiliary material= (0.2-2.0): (1-5): (230-290): (5-20).
The gel matrix is preferably poloxamer, more preferably a mixture of poloxamer 407 and poloxamer 188, and most preferably, the poloxamer 407 is prepared by the following weight ratio: poloxamer 188 = 22:3. wherein, in poloxamer 188, PEO accounts for 80% by mass, and in poloxamer 407, PEO accounts for 70% by mass.
The auxiliary material is preferably hydroxypropyl methylcellulose.
The preparation method of the responsive insulin delivery system is a cold dissolution method, and comprises the following steps:
Weighing ZIF-8 loaded with insulin, glucose oxidase, temperature-sensitive hydrogel solution and auxiliary material solution according to the material proportion of the response type insulin administration system, stirring by taking water as a solvent until the temperature-sensitive hydrogel is uniformly dispersed, and placing at 1-5 ℃ until the temperature-sensitive hydrogel is fully swelled, thereby obtaining the response type insulin administration system.
Preferably, the temperature-sensitive hydrogel solution is poloxamer water solution with the mass concentration of 1-25%.
Preferably, the auxiliary material solution is an auxiliary material solution with the mass concentration of 1-5%.
The time for the sufficient swelling is preferably 12 to 24 hours.
The responsive insulin delivery system of the present invention is useful in the preparation of hypoglycemic agents.
The responsive insulin delivery system of the present invention is useful in the preparation of injectable hypoglycemic agents.
The invention has the following advantages:
(1) The invention provides a pH-sensitive zeolite-based imidazole acid ester skeleton-8, which has uniform particle size distribution, a polydispersity index (PDI) of less than 0.2 and good physical stability after insulin is loaded. And because the mechanism of wrapping insulin by the zeolite-based imidazole acid ester skeleton-8 is the same as biomimetic mineralization, the local concentration of metal cations and organic ligands is increased by the biomacromolecule through intermolecular hydrogen bond and hydrophobic interaction, the formation of the zeolite-based imidazole acid ester skeleton-8 around the biomacromolecule is facilitated, and meanwhile, the medicine is wrapped in the skeleton, so that the method has the advantages of protecting the insulin activity and releasing the medicine in a responsive manner.
(2) According to the invention, poloxamer with good biocompatibility and degradability is selected as a gel matrix, the gel matrix and auxiliary materials form temperature-sensitive hydrogel, and a response type insulin administration system is successfully prepared by a cold dissolution method. The prepared response type insulin administration system has dual response properties of glucose and temperature, in-vitro release experiments and in-vivo blood sugar reducing effect research results prove that the response type insulin administration system can be used for intelligently regulating and controlling the release of insulin, has a large application prospect in the aspect of diabetes treatment, is mild in preparation condition, safe and green in raw materials and pollution-free, ZIF-8 is used for preventing the insulin structure from being damaged by virtue of a firm shell, so that the bioactivity of the insulin is maintained, excellent slow release effect is provided for the preparation by adding the temperature-sensitive hydrogel, in-vivo and in-vitro experiments prove that the administration system is sensitive to glucose, and meanwhile, the action time of insulin in-vivo can be prolonged.
(3) The auxiliary material hydroxypropyl methylcellulose adopted by the invention belongs to polysaccharide derivatives, and generates a sol-gel conversion process due to the interaction of hydrogen bonds or Van der Waals forces in polymer chains and the like.
(4) The temperature sensitive hydrogel is used as a drug carrier and can be loaded with various drugs, and the ZIF-8 loaded with insulin and glucose oxidase are simultaneously wrapped by the temperature sensitive hydrogel to prepare the responsive insulin administration system. On one hand, the insulin release amount can be regulated and controlled according to the blood sugar level in the body to avoid the risk of hypoglycemia, on the other hand, the temperature-sensitive hydrogel can slow down the drug release speed and prolong the drug action time, and meanwhile, the mobility of the temperature-sensitive hydrogel at room temperature meets the requirement of a subcutaneous injection administration mode.
Drawings
FIG. 1 is a transmission electron microscope image of zeolite-based imidazolate skeleton-8 before and after insulin loading in example 2 of the present invention, A: blank zeolite-based imidazolate framework-8, b: insulin-loaded zeolite-based imidazolate backbone-8.
FIG. 2 is an in vitro stability of the insulin-loaded zeolite-based imidazolate framework-8 of example 3 of the present invention.
Fig. 3 is a graph of the behavior of the MOF-based responsive insulin delivery system of example 5 of the present invention in different environments, a: at room temperature, B:37 ℃.
Fig. 4 is an illustration of the injectability at room temperature of the MOF-based responsive insulin delivery system of example 5 of the present invention.
FIG. 5 is an in vitro release profile of MOF-based responsive insulin delivery systems of example 6 of the present invention in various release media.
FIG. 6 is a graph showing the in vivo modulation of blood glucose levels by MOF-based responsive insulin delivery systems of example 7 of the present invention.
Detailed Description
The foregoing of the invention is illustrated in detail by the following specific examples, which are not intended to limit the invention thereto.
Example 1
Respectively weighing 100mg of 2-methylimidazole and 25mg of zinc nitrate hexahydrate (the mass ratio is 4:1), respectively dissolving in 10mL of water, and stirring at room temperature for 15min to obtain a zinc nitrate hexahydrate solution and a 2-methylimidazole solution;
Weighing 4mg of insulin to dissolve in the 2-methylimidazole solution, and stirring for 15min to obtain insulin-containing 2-methylimidazole solution; mixing insulin-containing 2-methylimidazole solution and zinc nitrate hexahydrate solution in different adding modes and adding sequences, stirring at 400r/min for 1h, centrifuging at 7000rpm for 15min, collecting precipitate, washing with water for three times, and freeze-drying to obtain insulin-loaded zeolite-based imidazole acid ester skeleton-8.
The effect of different order and mode of addition on the particle size, PDI and yield of the insulin-loaded zeolite-based imidazolate framework-8 was determined and the results are shown in Table 1. After changing the mixing sequence of the 2-methylimidazole solution containing insulin and the zinc nitrate hexahydrate solution, the particle size is found to be obviously changed; to improve the yield and maintain the uniformly distributed insulin-loaded zeolite-based imidazole acid ester skeleton-8, the final mixing mode was determined to be that zinc nitrate hexahydrate solution was added dropwise to the insulin-containing 2-methylimidazole solution.
TABLE 1
Example 2
Preparation of insulin/blank-loaded zeolite-based imidazolate backbone-8
Respectively weighing 100mg of 2-methylimidazole and 25mg of zinc nitrate hexahydrate in a proper amount (the mass ratio is 4:1), respectively dissolving in 10mL of water, and stirring at room temperature for 15min to obtain a zinc nitrate hexahydrate solution and a 2-methylimidazole solution;
weighing 4mg of insulin to dissolve in the 2-methylimidazole solution, and stirring for 15min to obtain insulin-containing 2-methylimidazole solution;
Dropwise adding a zinc nitrate hexahydrate solution into a 2-methylimidazole solution containing insulin, stirring at 400r/min for 1h, centrifuging at 7000rpm for 15min, collecting precipitate, washing with water for three times, and freeze-drying to obtain the zeolite-based imidazole acid ester skeleton-8 loaded with insulin. The empty Bai Feidan-yl imidazole acid ester skeleton-8 is prepared by the same preparation process under the condition of no insulin addition.
Diluting the blank/insulin-loaded zeolite-based imidazole acid ester skeleton-8 to 70 mug/mL with water respectively, dripping on a copper mesh with a carbon film, settling for a period of time, removing excessive liquid, airing, placing on a sample frame, feeding into a sample chamber, observing the form of the sample frame, and photographing. As shown in FIG.1, both of the particles are rhombic dodecahedron, the particles are uniformly dispersed, the particle size of the blank ZIF-8 is 130nm, the particle size of the insulin-loaded ZIF-8 is 270nm, and the loading of the medicine has little influence on the morphology of the ZIF-8, wherein the increase of the particle size is possibly related to the larger spatial structure of the insulin itself.
Example 3
In vitro stability of insulin-loaded zeolite-based imidazolate backbone-8
The zeolite-based imidazole acid ester skeleton-8 loaded with insulin was placed in an environment of 4 ℃ and room temperature respectively, and the particle size and PDI of the formulations were measured at 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60h respectively to examine the in vitro stability of the formulations. As shown in figure 2, the particle size and PDI of the drug-loaded preparation are not obviously changed when the drug-loaded preparation is stored for 60 hours at the temperature of 4 ℃ and the room temperature, and the good stability can be maintained.
Example 4
A method of preparing a responsive insulin delivery system comprising the steps of:
Weighing poloxamer 407, poloxamer 188 and 20mg hydroxypropyl methylcellulose with different masses, adding 1mL deionized water into a penicillin bottle, adding 4.7mg zeolite-based imidazole acid ester skeleton-8 loaded with insulin (1 mg calculated by insulin) and glucose oxidase (3 mg), stirring until the matrix is uniformly dispersed, and standing overnight at 4 ℃ to fully swell to obtain uniformly distributed milky white liquid, namely the responsive insulin administration system. The gel temperature (T sol-gel) and gel time (T) were determined by the tube inversion method. The results are shown in Table 2, in which the gelation temperature decreased with increasing mass of poloxamer 407 and increased with increasing mass of poloxamer 188 under the same conditions, without regular changes in gelation time.
TABLE 2
Example 5
Preparation of responsive insulin delivery system
220Mg of poloxamer 407, 30mg of poloxamer 188 and 20mg of hydroxypropyl methylcellulose are weighed into a penicillin bottle, 1mL of deionized water is added, 4.7mg of zeolite-based imidazole acid ester skeleton-8 (1 mg according to insulin) loaded with insulin and glucose oxidase (3 mg) are additionally added, the mixture is stirred until the matrix is uniformly dispersed, and the mixture is placed in a refrigerator at 4 ℃ for overnight to fully swell, so that uniformly distributed milky white liquid is obtained, and the insulin delivery system is a responsive insulin delivery system.
The behavior of the responsive insulin delivery system in different environments is shown in fig. 3, which is fluid in a room temperature environment and semi-solid in a 37 ℃ environment.
For injectability, the gel solution was added to a disposable insulin syringe at room temperature (25 ℃) to remove air bubbles and observe whether the gel solution passed smoothly through the syringe needle. As shown in fig. 4, the gel solution can be easily passed through the 29G needle in 20 seconds at room temperature.
Example 6
In vitro release experiments in responsive insulin delivery systems
Taking out in vitro drug release behaviors of temperature-sensitive gel preparation in phosphate buffer solution (pH 7.4) containing different glucose concentrations by adopting a non-membrane method, precisely measuring 1mL of the preparation respectively, placing the preparation in a sample bottle, adding 15mL of release medium after gelation, placing the preparation in a constant-temperature oscillator at 37 ℃ at the oscillation speed of 100 r.min -1, taking out 2mL of release solution respectively at 0.5, 1, 2,3, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96 and 108h, and supplementing the release medium with the same quantity and the same temperature. The released liquid was filtered, and the insulin concentration was measured by HPLC to calculate the cumulative released amount.
As can be seen from FIG. 5, the gel preparation has glucose sensitive property, and the accumulated release amount of insulin increases with increasing concentration of glucose, and the release time is as long as 96 hours.
Example 7
In vivo hypoglycemic effect study of responsive insulin administration system
The intraperitoneal injection of streptozotocin was used to establish a diabetic rat model, which was randomly divided into five groups (n=5), (1) model group, (2) ZIF-8@gel group, (3) insulin solution group, (4) ins@zif-8+gox group, (5) ins@zif-8/GOx-Gel group, and 5 healthy rats were randomly selected as blank control group (6) Healthy group. The administration was subcutaneous injection after grouping at a dose of 4U.kg -1, where the model group and the placebo group were injected with equal volumes of PBS. Immediately after blood glucose levels of each group of rats were measured by a blood glucose meter before administration, the administration was subcutaneously performed, blood was collected from the tail veins of 2, 4, 6, 8, 10, 12, and 24 hours after administration, and blood glucose levels were measured by a blood glucose meter.
As can be seen from fig. 6, in the case of no gel wrapping, the ins@zif-8+gox group is normal in blood sugar after 2 hours of injection, and the normal blood sugar level is maintained for about 3 hours, while the insulin solution group is rapidly lowered in blood sugar after injection, and the blood sugar is rapidly raised to hyperglycemia at 3 hours, the above results indicate that the responsive insulin administration system has a remarkable slow release effect, maintains the normal blood sugar time to be 3.5 times that of the insulin solution group, is 2.5 times that of the ins@zif-8+gox group, and can avoid the occurrence of hypoglycemia.
Comparative example 1
A preparation method of ZIF-8 loaded with insulin adopts an 'immersion method' method to load insulin, namely firstly synthesizing blank ZIF-8 and immersing in insulin solution. The drug loading rate of insulin is 3.24%, and the encapsulation efficiency is 63.14%.
As insulin belongs to macromolecular drugs, compared with small molecular drugs, insulin is not easy to enter a cavity through a gap of ZIF-8, and the preparation method is more suitable for loading the ZIF-8 on insulin.
Comparative example 2
A preparation method of insulin-loaded ZIF-8 is the same as in example 1, except that insulin is added into zinc nitrate solution and then 2-methylimidazole solution is added, so that insulin cannot be completely dissolved in zinc nitrate solution and cannot be effectively loaded.
Comparative example 3
A preparation method of the insulin-loaded ZIF-8 is the same as that of example 1, except that the stirring rate is 1000r/min, precipitation is generated in the stirring synthesis process, which indicates that the insulin-loaded ZIF-8 obtained at this time has larger particle size and is in micron order.

Claims (4)

1. A responsive insulin delivery system comprising insulin-loaded ZIF-8, glucose oxidase, a gel matrix and an excipient; the ZIF-8 loaded with insulin takes the ZIF-8 as a carrier, the insulin is loaded, and the ZIF-8 loaded with insulin comprises the following components in percentage by mass: glucose oxidase: gel matrix: auxiliary material= (0.2-2.0): (1-5): (230-290): (5-20);
the ZIF-8 loaded with insulin is prepared by a one-pot method and comprises the following steps:
(1) Weighing zinc salt and organic imidazole, and stirring and dissolving the zinc salt and the organic imidazole by taking water as a solvent to obtain a zinc salt solution and an organic imidazole solution respectively;
(2) Adding insulin into the organic imidazole solution and uniformly stirring to obtain an organic imidazole solution containing insulin; wherein, according to the mass ratio, insulin: organic imidazole= (2-5): (50-200);
(3) Mixing a zinc salt solution and an organic imidazole solution containing insulin to obtain a mixed solution; wherein, according to the mass ratio, zinc salt: organoimidazole=1: (2-8);
Stirring the mixed solution for reaction until the precipitation amount is not changed any more, and obtaining suspension after the reaction is completed;
(4) Carrying out solid-liquid separation on the suspension, washing the precipitate with water, and drying to obtain an insulin-loaded zeolite-based imidazole acid ester skeleton-8;
Wherein the zinc salt is zinc nitrate hexahydrate, and the mass concentration is 1.7-5.0 mg/mL; the organic imidazole is 2-methylimidazole, and the mass concentration is 3.3-40 mg/mL; the gel matrix is a mixture of poloxamer 407 and poloxamer 188; the auxiliary material is hydroxypropyl methyl cellulose; in the step (3), the zinc salt solution is dripped into an organic imidazole solution containing insulin, and the dripping speed is 0.5-1 mL/s; the stirring speed is 300-500 r/min, and the stirring time is 0.5-3 h;
the responsive insulin delivery system is prepared by a cold dissolution method, and specifically comprises the following steps:
Weighing a mixture of poloxamer 407 and poloxamer 188 and hydroxypropyl methyl cellulose into a penicillin bottle, adding deionized water, adding the zeolite-based imidazole acid ester skeleton-8 loaded with insulin and glucose oxidase, stirring until the matrix is uniformly dispersed, and standing at 1-5 ℃ to enable the matrix to be fully swelled, so as to obtain uniformly distributed milky white liquid, namely the responsive insulin administration system.
2. The responsive insulin delivery system of claim 1, wherein the insulin loaded ZIF-8 has an encapsulation efficiency of 57% -75% and a drug loading of 14% -27%;
the particle size of the ZIF-8 loaded with insulin is 80-500 nm, the particle size distribution is uniform, and the polydispersity index PDI is less than 0.2.
3. The responsive insulin delivery system as claimed in claim 1, wherein in the step (4), the solid-liquid separation is a centrifugal separation, and the centrifugal separation is performed at 7000r/min for 10-15 min.
4. Use of the responsive insulin delivery system as claimed in claim 1 for the manufacture of a hypoglycemic agent.
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