阅读说明：本技术 一种微马达载体及其制备方法与应用 (Micro motor carrier and preparation method and application thereof ) 是由 涂盈锋 刘坤 刘秋月 王双虎 杨佳蓉 于 2021-06-10 设计创作，主要内容包括：本发明属于微马达技术领域,公开了一种微马达载体及其制备方法与应用。所述微马达载体自内由外包括镁基微马达、Au包覆层、壳聚糖涂层、磷脂层和酯化淀粉层。所述微马达载体为自驱动微马达,并具有良好的结肠靶向特性,适用于作为药物尤其是多肽类和蛋白质类药物的口服给药载体,可提高结肠的接触机率和对肠粘膜的渗透能力,从而提高结肠部位对药物的摄入量和生物利用度。(The invention belongs to the technical field of micromotors, and discloses a micromotor carrier and a preparation method and application thereof. The micro-motor carrier comprises a magnesium-based micro-motor, an Au coating layer, a chitosan coating layer, a phospholipid layer and an esterified starch layer from inside to outside. The micro-motor carrier is a self-driven micro-motor, has good colon targeting property, is suitable for being used as an oral administration carrier of medicaments, particularly polypeptide and protein medicaments, and can improve the contact probability of the colon and the permeability of the colon to intestinal mucosa, thereby improving the intake and bioavailability of the medicaments at the colon part.)

1. A micro-motor carrier is characterized in that the micro-motor carrier comprises a magnesium-based micro-motor, a chitosan coating, a phospholipid layer and an esterified starch layer from inside to outside.

2. The micromotor carrier of claim 1, wherein the magnesium-based micromotor is Au-coated magnesium particles.

3. The micromotor carrier according to claim 2, wherein the particle size of the magnesium microparticles is 9 to 36 μm.

4. The micromotor carrier of claim 1, wherein the esterified starch has a degree of substitution of 1.5 to 2.5.

5. The micro-motor carrier of claim 1, wherein the esterified starch layer has a thickness of 50-80 μ ι η.

6. The micromotor carrier of claim 1, wherein said phospholipid layer comprises cholesterol and lecithin.

7. The method for preparing a micromotor carrier according to any one of claims 1 to 6, comprising the steps of:

sequentially coating chitosan and phospholipid on a magnesium-based micromotor, and coating by using esterified starch to obtain the micromotor carrier.

8. The method of manufacturing of claim 7, wherein the magnesium-based micromotor is manufactured by: and coating the magnesium particles by using the magnesium particles as a core and adopting an ion sputtering method to obtain the magnesium-based micromotor.

9. Use of the micromotor carrier of any one of claims 1 to 6 for carrying a drug.

10. The use according to claim 9, wherein the drug is a polypeptide drug and/or a protein drug.

Technical Field

The invention belongs to the technical field of micromotors, and particularly relates to a micromotor carrier and a preparation method and application thereof.

Background

Oral administration has the advantages of better compliance, simplicity and convenience, and is an ideal administration mode. However, during the process of the polypeptide biological macromolecule drug (such as insulin) passing through the digestive tract of human body, the harsh digestive environment in the stomach and intestine is easy to cause the destruction of the drug structure and the activity is damaged, so that the bioavailability is reduced. The traditional insulin preparation achieves the treatment effect mainly through a subcutaneous injection administration mode, has the defects of inconvenient administration, harm to skin and subcutaneous tissues caused by long-term injection, easy allergy and other adverse risks. Thus, how to improve insulin delivery in the intestinal tract and produce an effective glycemic control effect is critical to the success of oral insulin administration.

As part of the human digestive tract, the colon has less proteases, less macromolecular penetration resistance, and abundant lymphoid tissues, making it the best site for oral protein drug absorption. However, the traditional micro-nano drug preparation mainly moves in a passive diffusion mode, and only can perform brownian motion in body fluid, and the capacity of crossing cell and tissue barriers is relatively weak.

Compared with the traditional micro-nano drug carrier based on passive diffusion, the self-driven micro-nano motor can convert external energy into mechanical kinetic energy, so that autonomous movement is realized, and the micro-nano drug carrier has stronger initiative and selectivity. Therefore, it is desirable to provide a novel self-driven micromotor to improve the ability of proteinaceous drugs to cross the intestinal mucosa, increase active enrichment at the colonic site, and thereby achieve bioavailability for oral administration.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a micromotor carrier, a preparation method and an application thereof, wherein the micromotor carrier is a self-driven micromotor, has good colon targeting property, is suitable for being used as an oral administration carrier of medicaments, particularly polypeptide and protein medicaments, and can improve the contact probability of the colon and the permeability on intestinal mucosa so as to improve the intake and bioavailability of the medicaments at the colon part.

The invention provides a micro-motor carrier, which comprises a magnesium-based micro-motor, a chitosan coating, a phospholipid layer and an esterified starch layer from inside to outside.

The magnesium-based micromotor used by the invention is an asymmetric micromotor (namely, the micromotor has asymmetric structural characteristics, and the micromotor generally utilizes the asymmetry of the structure to carry out self-driving movement), and the magnesium-based micromotor forms a miniature galvanic cell which rapidly generates a large amount of hydrogen bubbles through rapid reaction with water when in a simulated tissue solution, so that the micromotor is favorably driven to rapidly move, the possibility of enhancing intestinal mucosa permeation is provided for the micromotor movement, and the delivery efficiency of carried objects is further improved. The negatively charged phospholipid layer and chitosan on the surface of the micro-motor can be coated on the surface of the micro-motor through electrostatic adsorption, so that the reliable packaging of a carried object is realized, the positive charge property of the surface of the micro-motor is reduced, the resistance of the micro-motor when penetrating through intestinal mucosa is favorably reduced, and the delivery efficiency is further improved. The coating formed by the esterified starch layer can effectively resist the degradation of gastric acid and pancreatic amylase, so that the micro-motor carrier can be degraded in the colon.

Preferably, the magnesium-based micromotor is Au-coated magnesium particles. The surface of the magnesium micro powder is modified and coated with Au, and the prepared magnesium-based asymmetric micro motor has better movement performance and can improve the delivery efficiency of carried objects.

More preferably, the particle size of the magnesium fine particles is 9 to 36 μm. As the core of the magnesium-based micromotor, the magnesium particles generate hydrogen bubbles through reaction with water, so as to push the micromotor to move rapidly, and the particle size has an important influence on the movement speed and time of the micromotor. By controlling the particle size of the magnesium micro powder, the invention ensures the uniformity of the movement of the micro motor on one hand and is beneficial to regulating and controlling the movement speed and the movement time of the micro motor on the other hand. When the particle size of the magnesium particles is less than 9 μm, the motor movement time is reduced, thereby affecting the overall movement distance of the micro-motor; when the particle size of the magnesium particles is larger than 36 μm, the movement rate of the micro motor is greatly hindered by the weight of the particle size of the magnesium particles, thereby affecting the movement performance of the micro motor.

Preferably, the thickness of the chitosan coating is 1-3 μm.

Preferably, the esterified starch has a Degree of Substitution (DS) of 1.5 to 2.5. Too low DS value (DS is less than 1.5) can reduce the film forming property and the film forming quality of the material, so that the film material can not effectively resist the degradation of gastric acid and pancreatic amylase and can not provide effective protection for the micro-motor carrier; too high a DS value (DS > 2.5) is detrimental to the degradation of the membrane in the lower digestive tract, especially in the colon, and thus to the release of the cargo. Wherein the Degree of Substitution (DS) has the meaning: representing the average number of hydroxyl groups derivatized per D-glucopyranosyl group, commonly referred to as Anhydroglucose (AGU) units, starch AGU has a maximum of 3 hydroxyl groups that can be substituted, so the DS has a maximum of 3.

Preferably, the phospholipid layer comprises cholesterol and lecithin.

More preferably, the mass ratio of cholesterol to lecithin in the phospholipid layer is 1: 1.

Preferably, the thickness of the esterified starch layer is 50-80 μm. When the thickness of the esterified starch layer is 50-80 μm, the ileum response time is 15-20min, and the esterified starch layer has good performance.

The invention also provides a preparation method of the micromotor carrier, which comprises the following steps:

sequentially coating chitosan and phospholipid on a magnesium-based micromotor, and coating by using esterified starch to obtain the micromotor carrier.

Preferably, the magnesium-based micromotor is prepared by the following steps: and coating the magnesium particles by using the magnesium particles as a core and adopting an ion sputtering method to obtain the magnesium-based micromotor.

More preferably, the parameters set in the ion sputtering method are as follows: the current is 25-35mA, and the treatment time is 2-4 min.

Preferably, the coating process parameters are as follows: the rotation speed of the fan is 1500 plus 1800rpm, the material temperature is 25-29 ℃, the air inlet temperature is 40-43 ℃, the fluidization pressure is 0.01-0.1MPa, and the atomization pressure is 0.01-0.1 MPa.

The invention also provides application of the micromotor carrier in carrying medicines. When the micro-motor carrier is used for carrying the medicine, the medicine and the chitosan can be mixed and then coated, so that the carrying of the medicine is realized.

Preferably, the medicament is a polypeptide medicament and/or a protein medicament. The micromotor carrier provided by the invention has good colon targeting property, can be used as an oral drug carrier, and is used for carrying conventional drug types which are difficult to treat through oral administration, such as polypeptide drugs, protein drugs and the like.

More preferably, the drug is insulin.

Compared with the prior art, the invention has the following beneficial effects:

the micro-motor carrier has colon targeting property, can be used as a drug carrier for carrying drugs, can resist the damage of the harsh environment of the digestive tract such as gastric acid, pepsin, pancreatin and the like to the drugs, and can improve the delivery efficiency of the drugs, particularly the macro-molecular drugs such as insulin and the like due to the self-driving property of the micro-motor.

Drawings

FIG. 1 shows the structural diagram (scale: 5 μm) of the drug-free micromotor carrier prepared in example 1;

FIG. 2 shows the movement of the drug-free micromotor vector prepared in example 1 (ruler: 125 μm), wherein A is the movement micrograph, B is the movement trace, and C is the movement parameter;

FIG. 3 shows a schematic of the process for preparing an insulin-loaded micromotor vehicle and the oral delivery of insulin;

FIG. 4 is a structural diagram (scale: 5 μm) showing an insulin-loaded micromotor carrier prepared in example 2;

FIG. 5 shows the movement of the insulin carrying micromotor vector prepared in example 2 (scale: 125 μm), wherein A is a movement micrograph, B is a movement trace, and C is a movement parameter;

FIG. 6 shows the movement of the insulin carrying micromotor vector prepared in example 3 (ruler: 125 μm), wherein A is a movement micrograph, B is a movement trace, and C is a movement parameter;

FIG. 7 shows a scanning electron microscope micrograph of an esterified starch film treated with a simulant (scale: 30 μm);

FIG. 8 shows a scanning electron microscope and energy dispersive X-ray spectrometer (ruler: 40 μm) spectrum of a micromotor carrier without the esterified starch coating treatment;

FIG. 9 shows a scanning electron microscope and energy dispersive X-ray spectrometer (ruler: 40 μm) spectrum of an esterified starch coated micromotor carrier;

FIG. 10 shows a scanning electron microscope and energy dispersive X-ray spectrometer (ruler: 100 μm) spectrum of a cross section of a micromotor carrier treated with an esterified starch coating;

FIG. 11 shows in vitro simulated drug release profiles of uncoated and coated micromotor mini-tablets;

FIG. 12 shows in vivo imaging results of animals with uncoated micromotor tablets and coated micromotor tablets taken orally;

FIG. 13 shows in vivo imaging results of animals with uncoated non-motor tablets and coated non-motor tablets orally administered;

FIG. 14 shows the in vivo blood glucose and insulin levels after administration to successfully molded SD rats.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1

This example provides an unloaded micromotor carrier (in the form of a microtablet) prepared by a process comprising the steps of:

(1) screening the magnesium particles to obtain magnesium powder particles with the particle size of 9-15 mu m; preparing the Au-coated micro motor by taking magnesium particles as a core and adopting an ion sputtering method (with the current of 25-35mA and the processing time of 2-4 min);

(2) coating the chitosan solution on the surface of the micro motor by adopting a spin coating technology, wherein the thickness of the chitosan coating is 1-3 mu m; coating the micromotor with 5 times diluted phospholipid solution (mixed by cholesterol and lecithin at a ratio of 1: 1) by spin coating to obtain phospholipid membrane with thickness of about 1-3 μm, tabletting, and coating the micromotor with esterified starch (DS value of 1.5-2.5) to obtain the micromotor carrier (shown in figure 1) with particle size of 15-20 μm.

As shown in FIG. 2, the prepared drug-free micromotor carrier has a movement speed of about 131.583 μm/s and a movement time of 1.5-2.5 min.

Example 2

This example provides an insulin-loaded micromotor vehicle (in the form of a mini-tablet) prepared according to the process scheme and schematic insulin delivery scheme shown in fig. 3, and comprising the following steps:

(1) screening the magnesium particles to obtain magnesium powder particles with the particle size of 9-15 mu m; preparing the Au-coated micro motor by taking magnesium particles as a core and adopting an ion sputtering method (with the current of 25-35mA and the processing time of 2-4 min);

(2) coating chitosan solution (concentration of insulin in the chitosan solution is 1 wt%) loaded with insulin on the surface of the micro-motor by adopting a spin coating technology, wherein the thickness of the chitosan coating is 1-3 mu m; coating the micromotor with 5 times diluted phospholipid solution (mixed by cholesterol and lecithin at a ratio of 1: 1) by spin coating to obtain phospholipid membrane with thickness of about 1-3 μm, tabletting, and coating with esterified starch (DS value of 1.5-2.5) to obtain micromotor carrier (shown in figure 4) with insulin particle size of 15-20 μm.

As shown in FIG. 5, the prepared micromotor carrier loaded with insulin has a movement speed of about 94.7563 μm/s and a movement time of 3.5-5.0 min.

Example 3

This example provides an insulin-loaded micromotor vehicle (in the form of a mini-tablet) prepared according to the process scheme and schematic insulin delivery scheme shown in fig. 3, and comprising the following steps:

(1) screening the magnesium particles to obtain magnesium powder particles with the particle size of 20-28 microns; preparing the Au-coated micro motor by taking magnesium particles as a core and adopting an ion sputtering method (with the current of 25-35mA and the processing time of 2-4 min);

(2) coating chitosan solution (concentration of insulin in the chitosan solution is 1 wt%) loaded with insulin on the surface of the micro-motor by adopting a spin coating technology, wherein the thickness of the chitosan coating is 1-3 mu m; coating the micromotor with 5 times diluted phospholipid solution (mixed by cholesterol and lecithin in a ratio of 1: 1) by a spin coating method, wherein the thickness of the phospholipid membrane is about 1-3 μm, tabletting, and coating the micromotor with esterified starch (DS value of 1.5-2.5) to obtain the micromotor carrier with insulin, wherein the particle size is 15-40 μm.

As shown in FIG. 6, the prepared micromotor carrier loaded with insulin in the simulated solution containing mucin has a movement speed of about 70.9 μm/s, a movement time of 2.5-3.5min and an effective mucosal penetration thickness of 1-2 mm.

Example 4

Construction of micromotor carriers (microtablets) and their properties

The degradation behavior of the esterified starch film (layer) in the digestive tract environment was determined using the insulin-loaded micro-motor vehicle prepared in example 3 as the material, and the test results are shown in fig. 7, where the prepared esterified starch film (layer) has good colon-responsive degradation characteristics.

The micrographs of the micromotor carriers (microtablets) before and after the coating treatment with the esterified starch were continuously measured, and the test results are shown in fig. 8-10, where fig. 8 is the scanning electron microscope and energy dispersive X-ray spectrometer spectrogram of the micromotor carriers (uncoated micromotor microtablets) without the coating treatment with the esterified starch, fig. 9 is the scanning electron microscope and energy dispersive X-ray spectrometer spectrogram of the micromotor carriers (coated micromotor microtablets) with the coating treatment with the esterified starch, and fig. 10 is the scanning electron microscope and energy dispersive X-ray spectrometer spectrogram of the cross-section of the micromotor carriers (coated micromotor microtablets) with the coating treatment with the esterified starch.

The uncoated micromotor mini-tablets and the coated micromotor mini-tablets are placed in a simulated digestive tract solution, and the insulin release behavior of the coated micromotor mini-tablets is measured, and the result is shown in fig. 11, compared with the uncoated micromotor mini-tablets, the coated micromotor mini-tablets have obviously longer drug release time which can be up to 24 hours and have good long-acting release property.

(II) evaluation of oral Effect

ICG (Indocyanine green) fluorescent dye was mixed with chitosan at a concentration of 2% to label the chitosan, and uncoated micromotor tablets (i.e., the micromotor tablets without the esterified starch coating treatment), coated micromotor tablets, uncoated non-motor tablets and coated non-motor tablets were prepared, respectively. After the rats were fasted and deprived of water for 24 hours, the tablets were separately administered by gavage, the rats were sacrificed at time points of 15min, 45min, 1.5h, 3h, and 5h for dissection, the stomach and intestinal tracts were removed, and the removed organs and animals were observed using a biopsy instrument.

The test results are shown in fig. 12-13, where the uncoated micromotor tablets were released in the stomach in large amounts, distributed rapidly throughout the intestinal tract, and essentially disappeared in vivo for about 5 h; the coated micromotor tablet is released when reaching the colon part within 3 hours, fluorescence is detected, and it can be seen from the figure that the coated pellets still have stronger fluorescence within 5 hours in the body, the duration is obviously prolonged compared with the uncoated pellets, and the coating material (esterified starch) is further proved to have good colon targeting property in the body. In addition, the motor tablet has excellent motion characteristics visually by comparing the non-motor tablet with the motor tablet, and the motion distance of the motor tablet is obviously greater than that of the non-motor tablet at the same time.

The SD rat successfully molded is subjected to normal saline intragastric administration, insulin intraperitoneal subcutaneous injection (5IU/kg), insulin intragastric administration group (75IU/kg), coated micromotor tablet (75IU/kg), uncoated micromotor tablet (75IU/kg), insulin-free coated micromotor tablet, insulin-free (uncoated) micromotor tablet and coated non-motor tablet (75IU/kg) oral administration treatment, blood sugar is measured by a glucometer at 0h, 1h, 2h, 4h, 6h, 8h and 10h respectively, animal whole blood samples at different time points after administration are obtained by blood sampling in the orbit, and the content of insulin is detected by an ELISA kit.

The results of the test are shown in FIG. 14, wherein A in FIG. 14 represents a line graph of blood glucose fluctuation of the rats after treatment, and B in FIG. 14 represents a line graph of serum insulin level of the rats after treatment. It can be seen from a in fig. 14 that only oral coated drug-loaded tablets and intraperitoneal injections of insulin have significant hypoglycemic effects, and that the hypoglycemic effects of oral coated drug-loaded tablets are more moderate and long-lasting. The blood sugar curve of the direct oral insulin shows that the oral insulin is inactivated after passing through the stomach and intestinal tract and loses the curative effect of reducing blood sugar, and the comparison with the curve of the oral coated drug-loaded tablet shows that the coating material can successfully resist the erosion of gastric juice and well protect the activity of the insulin; the blood sugar curves of the oral coated tablet without the micromotor and the oral coated blank tablet and the oral uncoated blank tablet are compared and analyzed, and the result shows that the blood sugar curve has no descending trend and no obvious change of blood sugar, so that other components in the micromotor carrier have no regulating effect on the blood sugar, and the influence of other materials except insulin on the blood sugar is eliminated. In fig. 14, B shows that the insulin level line graph corresponds to the blood glucose level line graph, and the change of the insulin content corresponding to the oral coated drug-loaded tablet is more gradual and lasting than the change of the insulin content injected into the abdominal cavity, which fully proves that the coated drug-loaded pellet can well maintain the activity of insulin and exert good blood glucose-lowering efficacy.

The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.