CN115040487B

CN115040487B - Chitinase-responsive ivermectin microsphere as well as preparation method and application thereof

Info

Publication number: CN115040487B
Application number: CN202210777675.1A
Authority: CN
Inventors: 袁厚群; 鲍光明; 林埴
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2023-04-25
Anticipated expiration: 2042-07-04
Also published as: CN115040487A

Abstract

The invention discloses a chitinase-responsive ivermectin microsphere as well as a preparation method and application thereof. The ivermectin microsphere is prepared by preparing a chitin solution from a mixed solution of sodium hydroxide and urea, preparing chitin microparticles by an emulsion gel method, and loading the chitin microparticles serving as a drug controlled release carrier. The drug loading rate of the ivermectin microsphere prepared by the invention is 522.1mg/g, and the accumulated release of the ivermectin microsphere is 28% under the condition of chitinase for 48 hours and is 8% higher than that of the ivermectin microsphere without enzyme. The ivermectin microsphere has the advantages of simple synthesis and preparation steps, low-cost and easily-obtained raw materials, good chitinase response and controlled release characteristics, and can be used for reducing side effects caused by leakage of the encapsulated ivermectin and other pesticide in an animal body, selectively pumping and releasing the ivermectin microsphere by chitinase in the insect body, killing insects, and optimizing reasonable administration of the pesticide.

Description

Chitinase-responsive ivermectin microsphere as well as preparation method and application thereof

Technical Field

The invention belongs to the field of ivermectin drug delivery systems, and particularly relates to a chitinase-responsive ivermectin microsphere, and a preparation method and application thereof.

Background

Animal parasitic diseases are main epidemic diseases due to the development of the current breeding industry in China, and are particularly important for preventing and treating the parasitic diseases, so that economic losses caused by the diseases can be recovered, and the development of regional breeding animal husbandry is promoted. Although ivermectin plays a very important role in preventing and controlling flies in livestock houses, the ivermectin can cause problems of poisoning of practitioners and cultured animals, causing animal-derived food safety and the like. Heretofore, ivermectin is available in the form of a pour-on formulation, an injection, a tablet, a dry suspension, a capsule, a lick and the like for clinical application. However, the half-life period of ivermectin is short, and long-acting sustained release preparations in the forms of emulsifiable concentrates, powder, microcapsules and the like are reported at present to maintain the action time and the effect of ivermectin, and although the effective blood concentration time can be prolonged, the ivermectin is actually repeatedly administered at low dose and is easy to generate drug resistance. Thus, the design and development of low-toxicity and low-pollution ivermectin drug-carrying systems is an urgent need for current green cultivation.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a chitinase-responsive ivermectin microsphere and a preparation method thereof, and the preparation method specifically adopts the following technical scheme:

a preparation method of chitinase-responsive ivermectin microspheres comprises the following steps:

(1) Mixing sodium hydroxide, urea and water, adding chitin, freezing at-80deg.C for 4 hr, thawing at 0deg.C under stirring, and repeating the above freezing and thawing process for 2-4 times to obtain chitin solution with concentration of 4wt%;

(2) Mixing liquid paraffin with span 80, dropwise adding chitin solution at 1200r/min and 0 ℃, then continuously stirring for 3h, heating to 25 ℃ and continuously stirring for 1h, then adjusting pH to be neutral, centrifugally collecting a lower solid product, washing with ethanol, and drying to obtain chitin particles;

(3) Dissolving ivermectin in methanol, adding chitin particles under the condition of 1000r/min, continuously stirring for 48 hours in a sealing mode, centrifuging to recover the lower-layer sediment, and drying to obtain the chitinase-responsive ivermectin microspheres.

Chitinase is a kind of glycoprotein enzyme widely existing in insect midgut, ecdysis glands and certain insect poison glands of flies and the like, and can hydrolyze Chitin (also called Chitin or Chitin, ct) in insect body walls and midgut. Because of physiological requirements such as molting and feeding, the fly needs to continuously synthesize and degrade chitin in the growth and development process, so that the normal operation of a chitin metabolic system is crucial to the normal growth and development of the fly. However, since mammals do not use chitin metabolism as an essential system for life activities, drugs targeting chitinase have the advantage of being harmless to humans and animals.

The invention mainly prepares the ivermectin microsphere by dissolving chitin by a sodium hydroxide urea system, preparing chitin particles with porous structures by an emulsion gel method, and then loading the antiparasitic agent ivermectin into the chitin particles. In the microsphere, pesticide with toxic and side effects is loaded into a chitin carrier with stable property and good biocompatibility. Since no chitinase exists in the intestinal tracts of mammals such as human beings, livestock and the like, even if a breeder or a cultured animal contacts and ingests the intelligent nano-drug, ivermectin encapsulated in the chitin carrier is not released to cause adverse effects; however, after the microspheres are ingested by insects such as flies, mosquitoes and the like, chitin on the outer layer of the microspheres can be degraded by chitinase in the insect bodies to release ivermectin, so that the purpose of selectively killing the insects such as flies, mosquitoes and the like without injuring people and livestock is achieved. The result shows that the drug loading rate of the ivermectin microsphere is 522.1mg/g, and the accumulated release of the ivermectin microsphere is 28% in 48 hours under the condition of chitinase and is 8% higher than that of the ivermectin microsphere without enzyme. The ivermectin microsphere has the advantages of simple synthesis and preparation steps, low-cost and easily-obtained raw materials, good chitinase response and controlled release characteristics, and can be used for reducing side effects caused by leakage of the encapsulated ivermectin and other pesticide in an animal body, selectively pumping and releasing the ivermectin microsphere by chitinase in the insect body, killing insects, and optimizing reasonable administration of the pesticide.

For the system for dissolving the chitin, the aqueous solution forms a relatively stable inclusion compound which is mutually combined with sodium hydroxide, urea, water and chitin, so that the chitin has good dispersibility, and the urea can be used as a host of the inclusion compound to prevent chitin molecules from being freely combined. The chitin is wrapped on the surface of urea, and simultaneously chain weight polymerization among the chitin is reduced, so that the dissolution of the chitin is promoted. Meanwhile, a stable hydrogen chain network can be formed for the chitin, urea and sodium hydroxide systems at low temperature. Through research on dissolution mechanism, it is found that under the condition of using NaOH, urea and chitin at low temperature, the solvent molecules can also form a complex structure (Inclusion complex structure, IC structure) with chitin molecules due to the fact that strong hydrogen bond action can break hydrogen bonds of the chitin to dissolve the chitin, so that the solution is more stable. The intrinsic effect of hydrogen bonding is relatively weak acting force, so that the chitin solution obtained by dissolving by using intermolecular acting force is relatively unstable, and the control of temperature is the most important attention in the process of synthetically preparing chitin microparticles. In the preparation of the chitin urea dissolving solution, if the environmental temperature variation difference or the solution is in normal temperature for a long time, the complex structure comprising the chitin urea and sodium hydroxide is damaged, and the damage is irreversible, so that a gel-like suspension liquid can be formed.

The use of span 80 as an emulsifier in the present invention is also particularly selected in combination with the features of the present invention, like sodium dodecyl sulfate is unsuitable for mixing with liquid paraffin, which is desirable for aqueous solutions, which is determined by its nature, whereas cetyltrimethylammonium bromide is prepared to form a emulsion, the properties of the droplets are observed under a microscope, and no obvious spherical chitin production is seen. For the emulsifier using the Tween type, the emulsifier shows the phenomena of colloidal aggregation and the like, and no spherical microspheres are generated.

Similarly, the inventors have also tried to investigate the influence of different pH concentrations on the appearance of chitin microparticles in terms of the regulation of the reaction pH. The inventors tried to adjust the pH selection to ph=5.3, 5.8, 6.3. Because the NaOH and the chitin urea solution are alkaline solutions, when the pH value is regulated, the added hydrochloric acid solution contains water, and the water content influences the change of the chitin solution to a certain extent. If too much water is added, the chitin will expand after water absorption, destroying intermolecular action formed by urea and NaOH, and simultaneously chitin molecule has amphipathy, easy to form oil-in-water emulsion, and increase viscosity of emulsion, and certain difficulty is caused to pH adjustment. However, the pH is not changed much for the morphology, and the pH is finally selected to be neutral when the pH is adjusted in consideration of the applicability of the drug in the living body.

Because chitin contains impurities such as proteins, minerals and the like, in order to remove the impurities, the chitin powder is subjected to purification treatment before use, and the specific process is as follows: chitin is soaked in 5wt% NaOH solution for overnight at room temperature, washed to neutrality, soaked in hydrochloric acid solution for one day at room temperature, washed to neutrality and dried.

Preferably, in step (1), the mass ratio of sodium hydroxide, urea and water is 11:4:85; preferably, the chitin solution is centrifuged at 0℃and 7600r/min for 20min to remove undissolved chitin.

Preferably, in step (2), the liquid paraffin: span 80: chitin solution = 100mL:2g:10mL.

Preferably, in step (2) and step (3), the centrifugation conditions are 10000r/min.

Preferably, in step (3), ivermectin: methanol: chitin microparticles = 100mg:10mL:100mg.

The beneficial effects of the invention are as follows: the drug loading rate of the ivermectin microsphere prepared by the invention is 522.1mg/g, and the accumulated release of the ivermectin microsphere is 28% under the condition of chitinase for 48 hours and is 8% higher than that of the ivermectin microsphere without enzyme. The ivermectin microsphere has the advantages of simple synthesis and preparation steps, low-cost and easily-obtained raw materials, good chitinase response and controlled release characteristics, and can be used for reducing side effects caused by leakage of the encapsulated ivermectin and other pesticide in an animal body, selectively pumping and releasing the ivermectin microsphere by chitinase in the insect body, killing insects, and optimizing reasonable administration of the pesticide.

Drawings

FIG. 1 is a flow chart showing the preparation of chitinase-responsive ivermectin microspheres;

FIG. 2 is a micrograph of ivermectin microspheres;

FIG. 3 is a transmission electron microscope image of ivermectin microspheres;

FIG. 4 shows a particle size distribution of ivermectin microspheres;

FIG. 5 shows the Fourier IR spectra of ivermectin microspheres, ivermectin and chitin;

FIG. 6 shows thermogravimetric curves of chitin microparticles and ivermectin microspheres;

FIG. 7 shows the release profile of ivermectin microspheres in chitinase-containing buffer and enzyme-free phosphate buffer.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention. The main test equipment used in the examples is shown in table 1; the test materials are shown in Table 2.

TABLE 1

TABLE 2

Example 1:

a preparation method of chitinase-responsive ivermectin microspheres is shown in figure 1, and comprises the following steps:

(1) Purifying chitin: since chitin contains impurities such as proteins, minerals, etc., to remove these impurities, the chitin is purified before use, first, the chitin is soaked in 5wt% naoh solution overnight at room temperature, washed to neutrality with distilled water, then soaked in 7% (volume ratio) hydrochloric acid solution for one day at room temperature, then washed to neutrality with distilled water, finally the chitin is dried in a vacuum oven and stored in a dryer for later use.

(2) Preparation of chitin microparticles: the preparation of chitin microparticles adopts emulsion gel method, and sodium hydroxide: urea: mixing water in a mass ratio of 11:4:85 to obtain a sodium hydroxide-containing urea aqueous solution, dispersing 4g of purified chitin in the solution, mixing and stirring, freezing the suspension in an ultralow temperature refrigerator at-80 ℃ for 4 hours, and then stirring and thawing under ice bath conditions, and circulating for 5 times to obtain a chitin solution with the concentration of 4 wt%. And (3) centrifuging the chitin solution in batches in a refrigerated centrifuge for 20 minutes, adjusting the temperature to 0 ℃, and adjusting the rotating speed to 7600r/min to remove insoluble chitin. Adding 100mL of liquid paraffin into a beaker, adding 2g of emulsifier span 80, regulating the stirring rotation speed to 1200r/min, slowly dropwise adding 10mL of treated 4wt% chitin solution into the liquid paraffin in a stirring state, controlling the temperature of the ice bath to be 0 ℃ and continuously stirring for 3h, heating the ice bath to 25 ℃ and continuously stirring for 1h, finally dropwise adding a proper amount of hydrochloric acid until the mixed solution is neutral under the condition of maintaining the original stirring state, centrifuging, regulating the rotation speed to 10000r/min, centrifuging to separate the liquid paraffin, collecting a solid product at the lower layer, washing with ethanol for 5 times to remove excessive span 80 and the liquid paraffin, and putting the initial product into a vacuum drying oven at 37 ℃ for drying to obtain chitin particles.

(3) Chitin microparticle loaded ivermectin (preparation of ivermectin microsphere)

Taking 100mg of ivermectin as a raw material, dissolving in 10mL of methanol, stirring in a mechanical stirrer, regulating the rotating speed to 1000r/min, adding 100mg of dried chitin particles, hermetically stirring for 48h, centrifuging, regulating the rotating speed to 10000r/min, filtering supernatant, recovering the lower-layer precipitate, loading the lower-layer chitin to the ivermectin particles, and drying in a vacuum drying oven at 37 ℃. And calculating the concentration of the supernatant according to the concentration of the standard curve, and finally calculating the amount of ivermectin contained in the lower sediment according to a formula.

Example 2:

structural characterization of chitinase-responsive ivermectin microspheres prepared in example 1:

(1) As shown in FIG. 2, the microscopic image of ivermectin microsphere shows that the product has uniform particles, good dispersibility and regular spherical shape, and the particle size is about 1 μm.

(2) The transmission electron microscope image of the ivermectin microsphere is shown in figure 3, the ivermectin microsphere is in four regular indefinite shapes under an electron microscope, the area is clearly displayed, the surface is smooth, and the grain size is about 2 mu m after comparison.

(3) The particle size distribution diagram of the ivermectin microspheres is shown in fig. 4, the average particle size of the ivermectin microspheres prepared by the emulsion gel method is 1.38 μm, and the average particle size corresponds to the result observed by an electron microscope.

(4) The Fourier infrared spectrum of the ivermectin microsphere is shown in figure 5, and the ivermectin microsphere is compared with the ivermectin according to figure 5The main absorption peak of chitin is 1658.48cm as shown in infrared spectrogram of chitin and chitin ^-1 C=O stretching vibration, peak of chitin is 1552.41cm respectively ^-1 And 1374.997cm ^-1 Bending of N-H and telescoping of C-N, respectively. Through the loading of ivermectin, the main absorption peak 1647.64cm of chitin is reserved in IVM-Ct ^-1 、1546.12cm ^-1 、1377.54cm ^-1 . Ivermectin at 1727.424cm ^-1 The C=O stretching vibration absorption peak is covered, and meanwhile, the ivermectin is 1675.83cm ^-1 The absorption peak of the stretching vibration characteristic of the unsaturated olefin also disappeared, however, at 1451.79cm ^-1 The new absorption peak is the vibration of substituted benzene ring skeleton, is the infrared absorption characteristic peak of ivermectin, and shows that the ivermectin is coated by chitin, and no other obvious medicine characteristic absorption peak is found.

(5) The thermal weight curves of the chitin microparticles and the ivermectin microspheres are shown in figure 6, and the overall weight loss of the ivermectin microspheres and the ivermectin loaded microspheres is 67.75% and 90.41% respectively. As can be seen from the thermogravimetric curve, the chitin particles have obvious weight loss phenomenon at 100-200 ℃, which can be caused by residual solvent ethanol in the process of preparing the particles, and the drying time is possibly too short. The main decomposition temperature of the ivermectin particles is 300-400 ℃, the weight loss ratio is 50.33%, and the ivermectin can be carbonized at the stage of 400-500 ℃. The chitin microparticles were shown to be ivermectin-loaded. Meanwhile, the catalyst is in a platform phase at 100-225 ℃, and no obvious weight loss phenomenon exists, which indicates that the solvent added during loading can be removed during drying, and no residue exists.

Example 3:

release performance study of chitinase-responsive ivermectin microspheres prepared in example 1:

by evaluating the drug release properties of ivermectin microspheres in vitro, the test uses the control of chitinase to evaluate the drug release properties of the synthesized ivermectin microspheres using this variable. From fig. 7, it can be seen that the ivermectin microspheres are significantly affected by the release of chitinase response, and the difference of the overall cumulative dissolution rate is about 8% in the group containing chitinase after 48 hours as compared with the group not containing chitinase. Meanwhile, the accumulated dissolution rate of the drug release in the whole system is lower, the accumulated dissolution rate of the drug after 12 hours reaches 26% after the treatment of the chitinase, the accumulated dissolution rate of the drug after 12 hours is increased by 2% and the group without the chitinase is added, the accumulated dissolution rate after 12 hours is 20.6%, and the release of the drug after 48 hours is not too much, so that the release of the drug is still in progress, the ivermectin microsphere has a certain slow release effect, and the release time can be further prolonged. For ivermectin microspheres, the dispersion is single, and the effect on the exertion of chitinase in 2 hours is not obvious. The main reason may be that the ivermectin drug is contained on the surface of the ivermectin microsphere, which results in a rapid release in the solvent upon addition to the release system, which is controlled by the diffusion mechanism. The ivermectin medicine on the surface diffuses, takes precedence, and the effect of chitinase is covered, and the accumulated release of the medicine is about 9%. After 2 hours, the effect of chitinase is shown, chitin on the surface of the ivermectin microsphere is hydrolyzed, and the ivermectin loaded in the interior is released, so that the dissolution speed of the ivermectin in the ivermectin microsphere is increased due to chitinase reaction, the integral accumulated dissolution is increased, and the chitin is insoluble in a buffer solution for the group which does not contain chitinase, and meanwhile, the water swelling property of the chitin exists, so that the ivermectin is further slowly released due to the transformation phase. When the enzyme reaction is carried out for 10 hours, the enzyme content in the system is reduced or the enzyme is inactivated, and the like, and the accumulated dissolution rate is slow, so that the ivermectin is slowly released.

The present invention is not limited to the above embodiments, but is merely preferred embodiments of the present invention, and the present invention should be construed as being limited to the above embodiments as long as the technical effects of the present invention are achieved by the same means. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.

Claims

1. The preparation method of the chitinase-responsive ivermectin microsphere is characterized by comprising the following steps of:

2. The preparation method according to claim 1, wherein in the step (1), the chitin is further purified before use, and the specific process is as follows: chitin is soaked in 5wt% NaOH solution for overnight at room temperature, washed to neutrality, soaked in hydrochloric acid solution for one day at room temperature, washed to neutrality and dried.

3. The method according to claim 1, wherein in the step (1), the mass ratio of sodium hydroxide, urea and water is 11:4:85.

4. The method according to claim 1, wherein in the step (1), the chitin solution is centrifuged at 0℃and 7600r/min for 20min to remove insoluble chitin.

5. The method according to claim 1, wherein in the step (2), the liquid paraffin: span 80: chitin solution = 100mL:2g:10mL.

6. The method according to claim 1, wherein in both the step (2) and the step (3), the centrifugation conditions are 10000r/min.

7. The method of claim 1, wherein in step (3), ivermectin: methanol: chitin microparticles = 100mg:10mL:100mg.

8. A chitinase-responsive ivermectin microsphere obtainable by a process according to any one of claims 1 to 7.

9. Use of the chitinase-responsive ivermectin microsphere of claim 8 for the preparation of pesticides.