CN113384557A

CN113384557A - Core-shell ratio controllable colon-targeted gel microsphere and preparation and application thereof

Info

Publication number: CN113384557A
Application number: CN202110656529.9A
Authority: CN
Inventors: 肖杰; 王浩楠; 陆午辉
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-09-14
Anticipated expiration: 2041-06-11
Also published as: CN113384557B

Abstract

The invention provides a colon-targeted gel microsphere with a controllable core-shell ratio, and a preparation method and application thereof. The gel microsphere sequentially comprises a core layer, an inner shell layer and an outer shell layer from inside to outside; wherein the core layer comprises a prolamin and an active; the inner shell layer comprises curdlan; the shell layer comprises chitosan, a cross-linking agent and a suspending agent; the inner shell layer and the core layer form core-shell structure fog drops through a coaxial electrostatic spraying technology, and in the outer shell layer solution, gel polysaccharide and a cross-linking agent of the inner shell layer of the core-shell structure fog drops are cross-linked and generate electrostatic layer-by-layer adsorption with chitosan, so that the colon-targeted gel microspheres are prepared. The gel microsphere has the particle size of 100-600 mu m, the core-shell ratio of 0.5-0.9, the encapsulation rate of water-soluble active substances of 60 percent and the encapsulation rate of alcohol-soluble active substances of 90 percent, the minimum release amount of 10 percent and 25 percent in 1h and 3h in stomach simulation liquid and small intestine simulation liquid, and the gel microsphere can be efficiently delivered and targeted to the colon.

Description

Core-shell ratio controllable colon-targeted gel microsphere and preparation and application thereof

Technical Field

The invention belongs to the technical field of preparation of colon delivery systems. More particularly, relates to a colon-targeted gel microsphere with controllable core-shell ratio, and a preparation method and an application thereof.

Background

The colon diseases are divided into inflammatory colon diseases, ischemic colon diseases, neoplastic colon diseases and the like, and the current treatment methods for colon-related diseases are limited and mainly comprise oral administration and rectal administration, wherein the rectal administration causes discomfort to patients and has limited administration range, the medicines cannot reach the upper part of the colon, and the treatment effect on inflammation occurring in the caecum and the upper part of the colon is very little. In order to make the drug reach the colon smoothly and release the drug and improve the action efficiency in oral administration, colon-targeted delivery of the drug material is a current research hotspot.

The gel microspheres as a colon targeted delivery material have the delivery advantages of both gel and microspheres, the gel has a porous structure, active substances can be adsorbed in pores, the release of the active substances in the upper digestive tract is reduced, so that the targeting efficiency is improved, the size and the characteristics of the microsphere structure are favorable for the application of the microsphere structure in the field of oral delivery, and discomfort of oral cavity and intestinal tract is not easily caused. Different technologies can be used for preparing gel microspheres with different structural characteristics, the multilayer core-shell structure has stronger delivery advantages than a single-layer core-shell structure, the multilayer core-shell structure can delay the release time of an active substance in a body and reduce the release of the active substance in the upper digestive tract of the body, and the gel microspheres with controllable core-shell ratio can regulate the release position and the sustained release time of the active substance in the body by regulating the core-shell ratio, so that the gel microspheres have stronger control effect and wider application value.

At present, electrostatic layer-by-layer self-assembly and electrostatic spraying are common gel microsphere preparation methods, the electrostatic layer-by-layer self-assembly utilizes electrostatic interaction and ionic crosslinking among materials to form a multilayer structure, but the preparation process needs multiple crosslinking, the reaction time is long, and the process is complicated; electrostatic spraying forms charged fog drops under the action of high-voltage static electricity, and then balls are formed through crosslinking, but the prepared microspheres have simple structures, and lack the regulation and control performance of the structures of the microspheres in the adaptability to the complex environment of the gastrointestinal tract. Patent CN109464423A discloses calcium alginate-chitosan microspheres and a preparation method thereof, and drug-loaded calcium alginate-chitosan microspheres and a preparation method thereof, wherein calcium alginate-chitosan microspheres prepared by electrostatic spraying only have a single-layer core-shell structure, the core-shell ratio is uncontrollable, and the highest encapsulation rate of naringenin is only 72.9%. The patent CN106702597A discloses a core-shell structured nanofiber membrane, and a preparation method and application thereof, the nanofiber membrane prepared by using coaxial electrospinning has a single-layer core-shell structure and an uncontrollable core-shell ratio, although the used wall material is a polysaccharide material, a plurality of polymers are added to promote membrane formation, and multi-step preparation is required, the operation is complicated, and the application of the nanofiber membrane in an oral delivery system is limited by the structure of the fiber membrane.

Therefore, the development of an oral colon drug delivery system with a multilayer core-shell structure, controllable core-shell ratio, high active substance encapsulation efficiency, high delivery efficiency and effective targeting is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a double-shell colon-targeted gel microsphere with controllable core-shell ratio, which is prepared by a one-step method.

The invention aims to provide a colon-targeted gel microsphere with controllable core-shell ratio.

Another object of the present invention is to provide a method for preparing the colon targeting gel microsphere.

The invention further aims to provide application of the gel microspheres in preparation of or as an oral colon targeted medicament.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a colon-targeted gel microsphere with a controllable core-shell ratio, which sequentially comprises a core layer, an inner shell layer and an outer shell layer from inside to outside; wherein the core layer comprises a prolamin and an active; the inner shell layer comprises curdlan; the shell layer comprises chitosan, a cross-linking agent and a suspending agent;

the inner shell layer and the core layer form core-shell structure fog drops through a coaxial electrostatic spraying technology, and in the outer shell layer solution, gel polysaccharide and a cross-linking agent of the inner shell layer of the core-shell structure fog drops are cross-linked and generate electrostatic layer-by-layer adsorption with chitosan, so that the colon-targeted gel microspheres are prepared.

The gel microsphere can reduce the release of drugs in stomach and small intestine, the hydrophobic interaction between the gliadin and active substance in the core layer can fix and enrich the active substance to the maximum, the gel characteristic polysaccharide of the inner shell layer plays a role in protecting the active substance through the gelation and reduces burst release, the gel characteristic polysaccharide is crosslinked with a crosslinking agent and forms a layer-by-layer coated delivery system through electrostatic interaction with chitosan, the chitosan in the outer shell layer can limit the swelling of gel and greatly improve the encapsulation efficiency of the active substance, the microsphere structure is favorable for oral delivery, the problems of less absorption, quick clearing and incapability of targeting of the existing active substance are solved, the release of the active substance before colon is reduced, the release time is prolonged and the action efficiency is improved.

In some preferred embodiments, the concentration of the prolamine in the core layer is 1 to 5 wt%, as in embodiments 1 to 10. Wherein, the prolamin is dissolved in 60-90% (v/v) acetic acid water solution or 60-90% (v/v) ethanol water solution to prepare a nuclear layer solution.

Further preferably, as in examples 1-2, the concentration of the prolamine in the core layer is 1-3 wt%.

In some preferred embodiments, the prolamin protein comprises zein, kafirin, Setarin, Rice gliadin, Triticum, hordein, Avena. The available prolamins are not limited to the above listed classes, as long as they have anti-solvent precipitation ability, and can be used in the colon targeting gel microspheres of the present invention.

In some preferred embodiments, the concentration of curdlan in the inner shell layer is 1 to 5 wt%, as in embodiments 1 to 10. Wherein the curdlan is dissolved in water to prepare an inner shell solution.

Further preferably, the concentration of curdlan in the inner shell layer is 2 to 3 wt% as in examples 1 to 2.

In some preferred embodiments, the curdlan comprises pectin, sodium alginate, konjac glucomannan, hyaluronic acid, carrageenan, and gellan gum, as in embodiments 1-10. The gel polysaccharide that may be used is not limited to the above listed kinds as long as the gel polysaccharide having a crosslinking function can be used in the colon targeting gel microsphere of the present invention.

In some preferred embodiments, as in embodiments 1-10, the chitosan concentration in the outer shell layer is 0.5-2 wt%; the concentration of the cross-linking agent is 1-3 wt%; the concentration of the suspending agent is 1-3 wt%. Wherein, the chitosan, the cross-linking agent and the suspending agent are dissolved in 1-2% (v/v) acetic acid water solution to prepare a shell layer solution.

Further preferably, as in examples 1-2, the concentration of chitosan in the outer shell layer is 1 wt%; the concentration of the cross-linking agent is 2-3 wt%; the concentration of the suspending agent is 3 wt%.

In some preferred embodiments, as in embodiments 1-10, the cross-linking agent comprises a potassium salt, a magnesium salt, a calcium salt, an iron salt, glutaraldehyde, genipin; the suspending agent comprises Tween 20, Tween 60, Tween 80, span 20, span 60 and glycerol. The actually usable crosslinking agent is not limited to the above listed kinds, and a general crosslinking agent having physical crosslinking and chemical crosslinking ability may be used in the colon targeting gel microsphere of the present invention.

In some preferred embodiments, as in examples 1-10, the active is a drug or active ingredient for treating and/or preventing colonic disease, including colitis-treating active ingredients such as curcumin, resveratrol, quercetin, apigenin, capsaicin, soy isoflavones, beta-carotene, hesperetin, oleuropein, carnosic acid, rosmarinic acid, and the like. The drugs or active ingredients that can be added for the treatment of colonic diseases are not limited to the above listed categories.

The invention also provides a preparation method of the gel microsphere, which comprises the following steps:

s1, preparing a core layer solution from cereal prolamin and an active substance; preparing the gel characteristic into an inner shell layer solution; preparing chitosan, a cross-linking agent and a suspending agent into a shell solution;

s2, respectively placing the nuclear layer solution and the inner shell layer solution in two sample injectors of a coaxial electrostatic spraying device, and forming core-shell structure fog drops through coaxial electrostatic spraying; allowing core-shell structure fog drops to enter the outer shell solution, and stirring and reacting at 200-700 rpm to obtain the colon targeted gel microspheres;

wherein the sample injection rate of the nuclear layer solution is 0.5 mL/h; the sample introduction rate of the inner shell layer solution is 1-2 mL/h; the voltage of the coaxial electrostatic spraying is 10-15 kV; the distance between the coaxial electrostatic spray nozzle and the surface of the shell layer solution is 100-150 mm; the ambient temperature is 20-30 ℃; the environmental humidity is 30-70%.

According to the method, a core-shell structure is prepared by adopting a coaxial electrostatic spraying device, a core layer solution is placed in a core layer sample injector, an inner shell layer solution is placed in the core layer sample injector, the inner shell layer solution and the core layer solution form core-shell structure fog drops through a coaxial electrostatic spraying technology, an outer shell layer solution is placed in a bottom receiving container, the core-shell structure fog drops enter the outer shell layer solution, inner shell layer gel characteristic polysaccharide is crosslinked with a crosslinking agent in the outer shell layer solution and generates electrostatic layer-by-layer adsorption with chitosan, colon-targeted gel microspheres are prepared in one step, and the core-shell ratio of the gel microspheres is regulated and controlled by regulating the sample injection rate, voltage and receiving distance of the core-shell solution, so that the colon-targeted gel microspheres with controllable core-shell ratio are obtained.

The particle size of the gel microsphere prepared by the method is 100-600 mu m, the core-shell ratio is 0.5-0.9, the encapsulation rate of the water-soluble active substance is 40-60%, the encapsulation rate of the alcohol-soluble active substance is 50-90%, the release amount in stomach simulated fluid and small intestine simulated fluid can be as low as 10% and 25% in 1h and 3h, the influence of gastrointestinal environment on the active substance is effectively reduced, and the slow release effect is good.

Therefore, the application of the gel microspheres in preparing or serving as oral colon targeted drugs is also within the protection scope of the invention.

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

(1) the gel microsphere of the invention forms a layer-by-layer coated delivery system through the hydrophobic interaction of the gliadin and active substances and the crosslinking action of the gel characteristic polysaccharide and a crosslinking agent, the electrostatic interaction of the chitosan and the gel characteristic polysaccharide forms a layer-by-layer coated delivery system, the gel characteristic polysaccharide of the inner shell layer and the chitosan of the outer shell layer are both enzyme-contact enteric-coated materials, the active substances are dissolved and reduced in the environment of stomach and small intestine and are gradually released under the action of colon intestinal flora to realize colon targeting and reduce the burst release phenomenon of the active substances in the colon, the gliadin in the core layer can maximally fix and enrich the active substances, the gel characteristic polysaccharide in the inner shell layer plays a protective effect and reduces the burst release through the gelation effect, the chitosan in the outer shell layer can limit the swelling of gel and greatly improve the encapsulation efficiency of the active substances, the microsphere structure is favorable for oral delivery, and the problems of low absorption rate, low content of the active substances and low cost of the existing active substances are solved, Fast clearance, no targeting, reduced release of active substance in the colon, prolonged release time and improved action efficiency.

(2) The microsphere prepared by adopting the coaxial electrostatic spraying technology has a core-shell structure with a controllable core-shell ratio, and the controllable core-shell ratio is 0.5-0.9; the gel microspheres can better load active matters, the encapsulation rate is higher, the encapsulation rate of the water-soluble active matters reaches 60%, and the encapsulation rate of the alcohol-soluble active matters reaches 90%.

(3) The release amount of the gel microsphere in stomach simulated fluid and small intestine simulated fluid can be as low as 10% and 25% in 1h and 3h, the influence of gastrointestinal tract environment on active substances is effectively reduced, and the sustained release effect is good.

(4) The materials used in the invention are all natural polymer materials, and have good biocompatibility.

(5) The coaxial electrostatic spraying preparation method adopted by the invention is different from the traditional preparation method of the gel microspheres, has the preparation advantages of one-step balling and simple operation, and is beneficial to the expanded production and application in the field of colon-targeted delivery controlled release of active substances.

Drawings

FIG. 1 is a flow chart of a one-step preparation method of a colon-targeted gel microsphere with controllable core-shell ratio and a structural schematic diagram of the microsphere;

FIG. 2 is a graph showing the structure verification of optical microscopy and fluorescence microscopy;

FIG. 3 is a microscopic structure of the microsphere prepared in example 1;

FIG. 4 is a micrograph showing the structure of microspheres prepared in example 2;

FIG. 5 is a micrograph of a microsphere prepared in example 4;

FIG. 6 is a micrograph of a microsphere prepared in example 6;

FIG. 7 is a view showing the state of the production process and the microscopic structure of the produced microspheres in comparative example 1;

FIG. 8 is a macroscopic view of comparative example 3 without centrifugation and with centrifugation;

FIG. 9 is a graph of the resistance of microspheres prepared in examples 1 and 2 and comparative example 4 to the cumulative release of free active in a model of gastrointestinal digestion;

FIG. 10 is a graph showing the cumulative release of free capsaicin in an in vitro colonic digestion model using microspheres prepared in example 1.

Detailed Description

The invention will be further described with reference to the drawings and the detailed description, which are not intended to limit the invention in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.

FIG. 1 is a flow chart and a structural schematic diagram of a one-step preparation method of a colon-targeted gel microsphere with controllable core-shell ratio.

The active encapsulation efficiency of the microspheres of the following examples and comparative examples was determined quantitatively by fluorescence spectroscopy, and the Encapsulation Efficiency (EE) was obtained by measuring the active content before and after extraction.

Taking capsaicin as an example:

scanning the emission wavelength (Em) of capsaicin solution in the wavelength range of 200-800nm under the conditions of an excitation slit of 5.0nm and an emission slit of 5.0nm to obtain the Em of the capsaicin which is 306.0nm, then scanning the excitation wavelength (Ex) by fixing the Em which is 306.0nm to obtain the optimal excitation wavelength which is 275.0nm, and determining the excitation wavelength and the emission wavelength of the capsaicin which are 275.0nm and 306.0 nm.

Capsaicin is weighed and taken as a solvent to prepare 1mg/mL capsaicin mother solution by taking 30% ethanol as a solvent, the mother solution is respectively diluted to prepare capsaicin solutions with the concentrations of 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 60 mu g/mL and 80 mu g/mL, and the capsaicin solutions are used for drawing a capsaicin standard curve.

The prepared suspension was centrifuged at 13000rpm for 30min to obtain 1mL of supernatant, which was: ethanol: ultrasonic assisted extraction with water (1:3:6) for 10min and membrane filtration to remove protein insoluble material, and measuring the content of free capsaicin under the above fluorescence conditions.

The Envelope Efficiency (EE) is calculated as follows:

other active matter (curcumin, oleuropein, apigenin, rosmarinic acid) encapsulation efficiency test method refers to capsaicin encapsulation efficiency determination method.

Example 1 preparation of Colon targeting gel microspheres loaded with alcohol-soluble capsaicin with controllable core/shell ratio (core/shell ratio of 0.60)

(1) Dissolving 1g of chitosan powder, 3g of calcium chloride and 3g of tween 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 1.5g of low-ester pectin powder and 1.5g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of capsaicin powder and 0.05g of zein powder in 4.6mL of 80% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 11kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 140mm, the temperature is 25 +/-0.5 ℃, and the humidity is 55 +/-5%;

(3) the prepared gel microspheres are stirred overnight at 400rpm to obtain stable gel microspheres.

An optical microscopic and fluorescence microscopic structure verification diagram of the capsaicin-zein/low-ester pectin-sodium alginate/chitosan microsphere prepared in the embodiment is shown in fig. 2, it can be seen that a core-shell structure of the microsphere can effectively verify the structural schematic diagram of fig. 1, fig. 3 is a microscopic structure diagram of the microsphere, the core-shell ratio of the microsphere prepared in the embodiment is 0.60, the average particle size is 336.83 μm, and the encapsulation rate is 76.84%.

Example 2 preparation of Colon targeting gel microspheres loaded with alcohol soluble curcumin and having controllable core-shell ratio (core-shell ratio of 0.58)

(1) Dissolving 1g of chitosan powder, 2g of calcium chloride and 3g of tween 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 2g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.05g of curcumin powder and 0.15g of zein powder in 4.6mL of 80% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 14kV, the sample injection rate of the inner shell layer is 2mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 120mm, the temperature is 24 +/-0.5 ℃, and the humidity is 60 +/-5%;

The microstructure of the curcumin-zein/sodium alginate/chitosan microsphere prepared in the embodiment is shown in fig. 4, the core-shell ratio of the microsphere is 0.58, the average particle size is 507.12 μm, and the encapsulation efficiency is 98.12%.

Example 3 preparation of Colon targeting gel microspheres loaded with Water soluble Oleuropein with controllable core/shell ratio (core/shell ratio of 0.86)

(1) Dissolving 2g of chitosan powder, 1g of calcium chloride and 1g of tween 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 4g of low-ester pectin powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.05g of oleuropein powder and 0.15g of kafirin powder in 4.6mL of 70% (v/v) acetic acid aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a core layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 15kV, the sample injection rate of the inner shell layer is 1mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 135mm, the temperature is 26 +/-0.5 ℃, and the humidity is 45 +/-5%;

The oleuropein-kafirin/low-ester pectin/chitosan microspheres prepared in this example have a core-shell ratio of 0.86, an average particle size of 286.94 μm, and an encapsulation efficiency of 51.77%.

Example 4 preparation of Colon targeting gel microspheres loaded with alcohol soluble capsaicin with controllable core/shell ratio (core/shell ratio of 0.84)

(1) Dissolving 1g of chitosan powder, 3g of calcium chloride and 3g of tween 20 in 94mL of 1% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 3g of low-ester pectin powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of capsaicin powder and 0.05g of zein powder in 4.6mL of 80% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 14kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 140mm, the temperature is 26 +/-0.5 ℃, and the humidity is 55 +/-5%;

Fig. 5 is a microscopic structure diagram of the capsaicin-zein/low-ester pectin/chitosan microspheres prepared in this example, where the core-shell ratio of the microspheres prepared in this example is 0.84, the average particle size is 560.21 μm, and the encapsulation efficiency is 86.19%.

Example 5 preparation of Colon targeting gel microspheres loaded with alcohol-soluble capsaicin with controllable core/shell ratio (core/shell ratio of 0.44)

(1) Dissolving 1g of chitosan powder, 3g of ferric chloride and 2g of tween 80 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 3g of low-ester pectin powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of capsaicin powder and 0.05g of zein powder in 4.6mL of 70% (v/v) ethanol water solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 11kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 130mm, the temperature is 25 +/-0.5 ℃, and the humidity is 50 +/-5%;

The capsaicin-zein/low-ester pectin/chitosan microspheres prepared by the embodiment have the core-shell ratio of 0.44, the average particle size of 247.77 mu m and the encapsulation rate of 83.51 percent

Example 6 preparation of Colon targeting gel microspheres loaded with alcohol-soluble capsaicin with controllable core/shell ratio (core/shell ratio of 0.74)

(1) Dissolving 1g of chitosan powder, 3g of potassium chloride and 3g of span 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 1.5g of low-ester pectin powder and 1.5g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g capsaicin powder and 0.05g hordein powder in 4.6mL 80% (v/v) ethanol water solution under vortex oscillation and 200W ultrasonic conditions to obtain a core layer solution;

Fig. 6 is a microscopic structure diagram of the capsaicin-hordein/low-ester pectin-sodium alginate/chitosan microspheres prepared in this example, wherein the core-shell ratio of the microspheres prepared in this example is 0.74, the average particle size is 235.05 μm, and the encapsulation efficiency is 87.12%.

Example 7 preparation of Colon targeting gel microspheres loaded with alcohol-soluble capsaicin with controllable core/shell ratio (core/shell ratio of 0.67)

(1) Dissolving 0.5g of chitosan powder, 2g of magnesium chloride and 3g of tween 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 2g of low-ester pectin powder and 2g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell solution; dissolving 0.075g of capsaicin powder and 0.05g of zein powder in 4.6mL of 80% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 11kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 140mm, the temperature is 27 +/-0.5 ℃, and the humidity is 50 +/-5%;

The core-shell ratio of the capsaicin-zein/low-ester pectin-sodium alginate/chitosan microspheres prepared by the embodiment is 0.67, the average particle size is 410.31 microns, and the encapsulation efficiency is 88.02%.

Example 8 preparation of Colon targeting gel microspheres loaded with alcohol-soluble capsaicin with controllable core/shell ratio (core/shell ratio of 0.85)

(1) Dissolving 1g of chitosan powder, 3g of calcium chloride and 3g of tween 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 1.5g of carrageenan powder and 1.5g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of capsaicin powder and 0.23g of prolamin powder in 4.6mL of 60% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 10kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 100mm, the temperature is 25 +/-0.5 ℃, and the humidity is 55 +/-5%;

The core-shell ratio of capsaicin-prolamin/low-ester pectin-sodium alginate/chitosan microspheres prepared in the embodiment is 0.85, the average particle size is 608.6 microns, and the encapsulation efficiency is 77.91%.

Example 9 preparation of Colon targeting gel microspheres loaded with alcohol soluble apigenin and controllable core/shell ratio (core/shell ratio of 0.69)

(1) Dissolving 1g of chitosan powder, 3g of genipin and 3g of span 60 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 3g of konjac glucomannan powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of apigenin powder and 0.15g of gliadin powder in 4.6mL of 90% (v/v) ethanol water solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 10kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 150mm, the temperature is 30 +/-0.5 ℃, and the humidity is 65 +/-5%;

The core-shell ratio of the apigenin-gliadin/low-ester pectin-sodium alginate/chitosan microspheres prepared in the embodiment is 0.69, the average particle size is 335.1 μm, and the encapsulation efficiency is 81.01%.

Example 10 preparation of core/shell ratio controllable Colon targeting gel microspheres loaded with Water soluble Rosmarinic acid (core/shell ratio 0.88)

(1) Dissolving 2g of chitosan powder, 1g of glutaraldehyde and 1g of span-20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 5g of hyaluronic acid powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell solution; dissolving 0.05g of oleuropein powder and 0.15g of highland barley alcohol soluble protein powder in 4.6mL of 70% (v/v) acetic acid aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 15kV, the sample injection rate of the inner shell layer is 1mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 135mm, the temperature is 25 +/-0.5 ℃, and the humidity is 35 +/-5%;

The core-shell ratio of the rosmarinic acid-highland barley alcohol-soluble protein/low-ester pectin/chitosan microspheres prepared by the embodiment is 0.88, the average particle size is 726.9 μm, and the encapsulation rate is 48.84%.

Comparative example 1 preparation of gel microsphere with Chitosan concentration of 0.1 wt% (Low concentration wall Material)

(1) Dissolving 0.1g of chitosan powder, 2g of calcium chloride and 3g of tween-20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 1.5g of low-ester pectin powder and 1.5g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of capsaicin powder and 0.05g of zein powder in 4.6mL of 80% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

(2) the preparation was carried out with the coaxial electrostatic spray parameters set as follows: the voltage is 11kV, the sample injection rate of the inner shell layer is 1.5mL/h, the sample injection rate of the nuclear layer is 0.5mL/h, the distance between the coaxial electrostatic spray nozzle and the surface of the solution of the outer shell layer is 140mm, the temperature is 25 +/-0.5 ℃, and the humidity is 50 +/-5%;

(3) the prepared gel microspheres are stirred at 400rpm overnight to obtain the gel microspheres.

The microstructure of the capsaicin-zein/low-ester pectin-sodium alginate/chitosan microsphere prepared by the comparative example is shown in figure 7, the core-shell ratio is avoided, the average particle size is 624.76 micrometers, and the encapsulation rate is only 2.97%.

According to the comparative example, as the chitosan concentration is lower than 0.5 wt%, the swelling of the polysaccharide gel inside cannot be completely limited, so that a large amount of gel flocculent precipitates appear in the solution, and as the chitosan concentration is too low, a core-shell structure cannot be formed, the core-layer zein and the capsaicin are released into the solution along with the swelling of the gel, and the encapsulation efficiency is remarkably reduced. Comparative example 2 preparation of gel microsphere with Chitosan concentration 4 wt% (high concentration wall Material)

(1) Dissolving 4g of chitosan powder, 2g of calcium chloride and 3g of tween 20 in 94mL of 2% (v/v) acetic acid aqueous solution under the stirring condition of 600rpm to obtain a shell solution; dissolving 1.5g of low-ester pectin powder and 1.5g of sodium alginate powder in 97mL of tertiary water under the conditions of stirring at 600rpm and 200W of ultrasound to obtain an inner shell layer solution; dissolving 0.075g of capsaicin powder and 0.05g of zein powder in 4.6mL of 80% (v/v) ethanol aqueous solution under the conditions of vortex oscillation and 200W ultrasound to obtain a nuclear layer solution;

The core-shell ratio of the capsaicin-zein/low-ester pectin-sodium alginate/chitosan nanospheres prepared by the comparative example is 0.91, the average particle size is 891.1 micrometers, and the encapsulation rate is 77.11%.

When the concentration of the chitosan is higher than 3 wt%, the viscosity of the chitosan solution is increased, fog drops cannot be rapidly dispersed when reaching the receiving liquid, and the fog drops are extremely easy to float on the surface of the solution and finally accumulate to form a film, so that a stable and uniform microsphere structure is difficult to form. Comparative example 3 preparation of Colon-targeting gel nanospheres by antisolvent precipitation-electrostatic layer-by-layer self-assembly technique

(2) 5mL of the core layer solution was injected into 20mL of the inner layer solution in a strand at 500rpm, followed by stirring for 10min to obtain a nanoparticle suspension.

(3) The nanoparticle suspension obtained above was injected into 20mL of the outer shell solution in a strand at 500rpm, followed by stirring for 10min to obtain a colon targeting gel nanosphere suspension.

In the comparative example, stable colon-targeted gel microspheres cannot be prepared, a large amount of flocculation precipitation and irregular gel appear in the preparation process, and the gel is divided into 3 layers after centrifugation as shown in fig. 8, namely water, polysaccharide gel and protein precipitation respectively, which indicates that the traditional preparation method of colon-targeted gel particles is not suitable for preparing microspheres from the raw materials of the invention at high concentration. Comparative example 4 capsaicin-calcium alginate-chitosan microspheres were prepared according to the prior art [ CN201811428339.6 ]

(1) Preparing a sodium alginate solution by using water as a solvent, wherein the mass concentration of the sodium alginate is 1.5%;

(2) preparing a mixed solution of chitosan and calcium chloride by taking acetic acid as a solvent, wherein the concentration of the chitosan is 0.2%, and the concentration of the calcium chloride is 1%;

(3) spraying a mixed solution of capsaicin and sodium alginate into a mixed solution of chitosan and calcium chloride by electrostatic spraying at a spraying speed of 4.5 mL/h;

(4) and (4) centrifuging the solution in the step (3) at the speed of 5000 r/min, removing supernatant, washing for 3 times by using deionized water to obtain a drug-loaded calcium alginate-chitosan microsphere suspension, and freeze-drying to obtain the drug-loaded calcium alginate-chitosan microsphere.

Experimental example 1 study on resistance of colon-targeted gel microspheres to digestion in stomach and small intestine

1. Experimental methods

(1) Simulated Gastric Fluid (SGF) consisted of 0.11mM sodium deoxycholate, 34.2mM sodium chloride, 2mg/mL pepsin and 0.5% Tween 80 adjusted to pH 1.6. Simulated Intestinal Fluid (SIF) consisted of 3.90mM sodium taurocholate, 19.12mM maleic acid, 10mg/mL pancreatin and 0.5% Tween 80, pH adjusted to 7.4.

(2) 50mL of gastric fluid and small intestine fluid were incubated with the colon targeting gel microspheres (active content 1mg) obtained in examples 1-2 and comparative example 4 at 37 ℃ and 100rpm for 1h and 3h, respectively. Gastric and intestinal fluids were sampled at 1mL per 30min, replacing an equivalent amount of fresh simulant. The sample was centrifuged at 10000rpm for 10min to collect 1mL of supernatant, the collected supernatant was extracted with 30% (v/v) ethanol and the free active content was determined by fluorescence spectroscopy.

2. Results of the experiment

The results of the stomach and small intestine digestion of examples 1 and 2 and comparative example 4 are shown in fig. 9, and it can be seen from the figure that capsaicin and curcumin encapsulated by the microspheres of examples 1 and 2 are released to a certain extent in the digestion process of the stomach and small intestine, the released active substances are mainly part of the active substances which are dispersed to the outer shell layer and the surface outer shell layer along with ethanol in the preparation process, more active substances are enriched in the core of the cereal protein, the cumulative release amount of the active substances in the SGF is about 10%, the cumulative release amount in the SIF is lower than 35%, the capsaicin in comparative example 4 is uniformly dispersed in the whole microspheres, the gel microspheres swell in the digestion process, the capsaicin is continuously released due to no enrichment and protection of the core structure of the cereal protein, the final cumulative release amount of the SGF is up to 20%, and the cumulative release amount in the SIF is up to 70%.

Experimental example 2 in vitro colon digestion simulation study of colon-targeted gel microspheres

1. Experimental methods

(1) Simulated Colonic Fluid (SCF) consisted of 5 wt% SD rat fecal filtrate (PBS pH 5.5). And (3) performing adaptive intragastric administration on the SD rat for two weeks by using the dosage of 10mg/d gel microspheres so as to improve the specific degradation capability of the intestinal flora of the rat on the microsphere wall materials. Rat feces were freshly taken and immediately made into a 10 wt% suspension and placed in an anaerobic workstation for subsequent operations. The 10 wt% fecal suspension was filtered and diluted to give 5 wt% SD rat fecal filtrate (PBS pH 5.5).

(2) 50mL of colon juice was incubated with the colon targeting gel microspheres of example 1 (active content 1mg) at 37 ℃ for 24h at 100 rpm. 1mL of colon fluid was sampled every 1h for the first 4h and 1mL every 4h for the last 20h, replacing the same amount of fresh simulant. The sample was centrifuged at 10000rpm for 10min to collect 1mL of supernatant, the collected supernatant was extracted with 30% (v/v) ethanol and the free active content was determined by fluorescence spectroscopy.

2. Results of the experiment

The colonic digestion results of example 1 are shown in fig. 10, from which it can be seen that under the action of the intestinal flora, the microspheres start to slowly degrade and release capsaicin, with a gradual and then steep increase and finally a gradual trend, because of the smaller time contact area in the front, the microspheres degrade more slowly. After the microspheres swell, the microspheres begin to be rapidly degraded by flora and enzyme invasion and release part of tightly bound capsaicin, the final release trend tends to be flat because the tightly bound capsaicin also begins to be slowly released, and the 24h accumulated release amount in the colon environment is only close to 60 percent, so that the gel microspheres have better sustained and controlled release capacity and are beneficial to being applied to the colon delivery field.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The colon-targeted gel microsphere with the controllable core-shell ratio is characterized by comprising a core layer, an inner shell layer and an outer shell layer from inside to outside in sequence; wherein the core layer comprises a prolamin and an active; the inner shell layer comprises curdlan; the shell layer comprises chitosan, a cross-linking agent and a suspending agent;

2. The gel microsphere of claim 1, wherein the concentration of the prolamin in the core layer is 1-5 wt%.

3. The gel microsphere of claim 2, wherein the prolamin protein comprises zein, kafirin, Setarin, Rice, Triticum, hordein, or Avena.

4. The gel microsphere of claim 1, wherein the concentration of curdlan in the inner shell layer is 1-5 wt%.

5. The gel microsphere of claim 4, wherein the curdlan comprises pectin, sodium alginate, konjac glucomannan, hyaluronic acid, carrageenan, and gellan gum.

6. The gel microsphere of claim 1, wherein the concentration of chitosan in the outer shell layer is 0.5-2 wt%; the concentration of the cross-linking agent is 1-3 wt%; the concentration of the suspending agent is 1-3 wt%.

7. The gel microsphere of claim 6, wherein the cross-linking agent comprises potassium salt, magnesium salt, calcium salt, iron salt, glutaraldehyde, genipin; the suspending agent comprises Tween 20, Tween 60, Tween 80, span 20, span 60 and glycerol.

8. The gel microsphere of claim 1, wherein the active substance is a drug or an active ingredient for the treatment and/or prevention of colonic diseases.

9. The preparation method of the gel microsphere of any one of claims 1 to 8, which is characterized by comprising the following steps:

10. Use of the gel microspheres according to any one of claims 1 to 8 or the gel microspheres prepared by the method according to claim 9 in the preparation of or as an oral colon targeted drug.