CN102652833B - Gastric targeted drug carrier and preparation method thereof - Google Patents

Abstract

The invention relates to the field of drug carrier preparation, in particular, the invention relates to a stomach-targeting drug carrier and a preparation method thereof. The raw materials of the stomach-targeting drug carrier of the present invention include: a drug-containing emulsion containing chitosan, chitosan quaternary ammonium salt, and sodium glycerophosphate; and a cross-linking agent for cross-linking the above-mentioned emulsion into microspheres; its preparation method includes Following steps: 1) preparation of drug-containing emulsion: use chitosan, the aqueous solution of chitosan quaternary ammonium salt and sodium glycerophosphate as water phase W, and make drug-containing emulsion with medicine and oil phase containing emulsifier; 2 ) heat up the drug-containing emulsion in step 1), stir, and cross-link the chitosan quaternary ammonium salt and sodium glycerophosphate contained in it to obtain a gel microsphere suspension, heat up and add a cross-linking agent, and the second cross-linking occurs to solidify the gel microspheres. The drug carrier of the invention has uniform and controllable particle size, high embedding rate and good dispersibility, and at the same time, meets the requirements of stomach-targeted drug delivery carrier.

Description

A gastric-targeted drug carrier and preparation method thereof

technical field

The invention relates to the field of drug carrier preparation, in particular, the invention relates to a stomach-targeting drug carrier and a preparation method thereof.

Background technique

Ruth Duncan，中均有报道。以壳聚糖及壳聚糖衍生物为材料进行修饰改造制备的壳聚糖季铵盐，是一种带正电荷的天然多糖，作为药物载体很容易粘附到细胞的表面。与壳聚糖相比，壳聚糖季铵盐不仅具有良好的亲水性和水溶性，而且季铵基团的引入大大增加了壳聚糖季铵盐的促黏膜渗透吸收性([4]Kotze A F，De Leeuw B J，Lueben H L，DeBoer A G，Verhoef J C，Junginger HE.Chitosans for enhanced delivery of therapeuticpeptides across intestinal epithelia：In vitro evaluation in Caco-2 cell monolayers.Int.J.Pharm，1997，159：243-263)。Murata等在1995年将壳聚糖季铵盐用于基因载体实验，可以有效地转染HepG2细胞(Murata JI，Ohya Y，Ouchi T.Design of quaternary chitosanconjugate having antennary galactose residues as a gene delivery tool.Carbohydr.Polym，1997，32(2)：105-109)。此外作为一种新型基因载体，Kean对壳聚糖季铵盐载体的细胞毒性和转染率都进行了研究。结果表明，与常用的阳离子载体聚赖氨酸相比，在所测试的所有浓度范围内，所有被测的壳聚糖季铵盐都没有表现出明显的细胞毒性。因此，壳聚糖和壳聚糖季铵盐混合材料的微球是一种带有正电荷的凝胶性微球，具有很好的pH敏感性、粘附性，而这些特性是胃部靶向载体所具备的特征，所以此类微球有望成为一种应用前景广阔的胃部靶向药物传输载体。Chitosan is a polysaccharide composed of glucosamine and N-acetylglucosamine copolymer, which is the product of deacetylation of chitin. Chitosan can be dissolved in dilute acid solution due to the free amino group on the molecular chain, so It has a wider range of uses than chitin. As a cationic biopolymer, chitosan has bioadhesion, biocompatibility and biodegradability, and its degradation products are non-toxic, non-immunogenic and non-carcinogenic. These unique properties of chitosan The properties make it a biomedical carrier with broad application prospects. In the literature Chitosan-cellulose composite membrane for affinity purification of biopolymers and immunoadsorption, Journal of Membrane Science, 2002, 197: 185-197. ofchitosan ethylcellulose complex microcapsule and its application in controlled release ofVitamin D ₂ , Biomaterials, 2002, 23: 4469-4473, Xin-Yuan Shi, Tian-Wei Tan,; Evaluation of the biological properties of soluble chitosan and chitosan microspheres of Journals, International 1997, 148: 231-240.

Ruth Duncan, reported in both. The chitosan quaternary ammonium salt prepared by modifying chitosan and chitosan derivatives is a positively charged natural polysaccharide, which can easily adhere to the surface of cells as a drug carrier. Compared with chitosan, chitosan quaternary ammonium salt not only has good hydrophilicity and water solubility, but also the introduction of quaternary ammonium groups greatly increases the mucous membrane permeation absorption of chitosan quaternary ammonium salt ([4]Kotze A F , De Leeuw B J, Lueben H L, DeBoer A G, Verhoef J C, Junginger HE. Chitosans for enhanced delivery of therapeutic peptides across intestinal epithelia: In vitro evaluation in Caco-2 cell monolayers. Int. J. Pharm, 1997, 159: 243-263 ). Murata etc. used chitosan quaternary ammonium salt in gene carrier experiments in 1995, which can effectively transfect HepG2 cells (Murata JI, Ohya Y, Ouchi T.Design of quaternary chitosanconjugate having antennanary galactose residues as a gene delivery tool.Carbohydr . Polym, 1997, 32(2): 105-109). In addition, as a new type of gene carrier, Kean studied the cytotoxicity and transfection rate of the chitosan quaternary ammonium salt carrier. The results showed that all tested chitosan quaternary ammonium salts showed no obvious cytotoxicity in all concentration ranges tested compared with the commonly used cationic carrier polylysine. Therefore, the microsphere of chitosan and chitosan quaternary ammonium salt mixed material is a kind of positively charged gel microsphere, which has good pH sensitivity and adhesion, and these characteristics are the target of stomach. Therefore, this type of microsphere is expected to become a stomach-targeted drug delivery carrier with broad application prospects.

Chitosan microspheres have excellent biocompatibility, bioadhesion and controlled drug release properties. Because chitosan microspheres contain more amino groups, they have the properties of polycations, so they can interact with negatively charged groups on the cell surface, and have non-specific adsorption with cells, which is beneficial for cells in chitosan microspheres. Surface adhesion has good cell compatibility; in addition, chitosan has high hydrophilicity, studies have shown (Sarah Nsereko, Mansoor Amiji, Loacallized delivery of paglitaxel in solid tumors from biodegradable chitin microparticle formulations, Biomaterials, 23( 2002) 2733-2731.), in diluted serum, hydrophilic materials can promote the adsorption and spreading of cells on the surface of materials more than hydrophobic materials, and the cytoskeleton of cells on hydrophilic materials is also more organized, and chitosan materials It has good water absorption properties and reaches saturation after 20 minutes of water absorption. The water swelling and porosity of this hydrophilic gel of chitosan is very similar to that of cell matrix, and it has good tissue compatibility. The positive charge of chitosan and the negatively charged mucosal surface form intermolecular attraction through electrostatic interaction, making it have adhesive properties. Another important characteristic of chitosan is that it can be used as a drug carrier to slow down drug release, reduce drug toxicity, and prolong drug efficacy (Radi Hejazi, Mansoor Amiji, Chitosan-based gastrointestinal delivery systems, Journal of Controlled Release, 89( 2003) 151-165.). For example, chitosan is used to embed insulin, but when chitosan reacts with a cross-linking agent, the non-specific adsorption between cells is lost due to the reaction between groups on its surface and aldehyde groups. The retention of polypeptide drugs in the three-dimensional network of cationic polymers is mainly accomplished through simple hydration and diffusion processes or ionic interactions between polymers and drug ions. Although chitosan quaternary ammonium hydrogel microspheres have good bioadhesiveness, they disintegrate rapidly in the acidic environment of the stomach as a gastric drug delivery carrier. In the acidic release medium (pH=5.0), due to Dissociation and dissolution of chitosan quaternary ammonium salt microspheres, the drug embedded in it is also rapidly released into the surrounding medium, and all the drug embedded in it is released within 1 hour, which does not achieve good drug sustained release performance, and the shell Although polysan has good pH sensitivity, it loses its targeting effect after reacting with cross-linking agents, so chitosan and chitosan quaternary ammonium salt alone cannot meet the requirements of stomach-targeted drug delivery carriers.

Contents of the invention

The purpose of the present invention is to provide a gastric targeting drug carrier.

Another object of the present invention is to provide a method for preparing a stomach-targeting drug carrier.

According to the stomach-targeting drug carrier of the present invention, the drug carrier raw material includes a drug-containing emulsion containing chitosan, chitosan quaternary ammonium salt, and sodium glycerophosphate; and a cross-linking agent for cross-linking the above-mentioned emulsion into microspheres .

According to the stomach-targeted drug carrier of the present invention, the mass ratio of the chitosan to the chitosan quaternary ammonium salt is 1: 0.1 to 10, the total mass of the chitosan and the chitosan quaternary ammonium salt to the sodium glycerophosphate The mass ratio of the chitosan quaternary ammonium salt is 1:1-10, preferably 1:0.5-2, wherein the degree of quaternization substitution of the chitosan quaternary ammonium salt is 10%-80%.

According to the stomach-targeted drug carrier of the present invention, the cross-linking agent is glutaraldehyde, genipin or epoxy chloride, wherein the molar ratio of the cross-linking agent to chitosan and chitosan quaternary ammonium salt amino group is 1 : 1-10.

The present invention also provides a method for preparing a stomach-targeting drug carrier, the method comprising the following steps:

1) Preparation of medicated emulsion:

Dissolve the water-soluble drug in the acetic acid aqueous solution of chitosan quaternary ammonium salt, chitosan and sodium glycerophosphate as the water phase W, and use the oily substance dissolved with the emulsifier as the oil phase O to obtain the W/O type containing medicated lotion;

or

Dissolve fat-soluble drugs in organic solvents as the internal oil phase O ₁ , use acetic acid aqueous solution dissolved with chitosan quaternary ammonium salt, chitosan and sodium glycerophosphate as the water phase W, and emulsify to form O ₁ /W type primary emulsion , and then use the oily substance dissolved with emulsifier as the external oil phase O ₂ , emulsify the external oil phase O ₂ and O ₁ /W type primary emulsion to obtain O ₁ /W/O ₂ type medicated emulsion;

2) Warm up the W/O type or O ₁ /W/O ₂ type drug-containing emulsion in step 1), so that the chitosan quaternary ammonium salt and sodium glycerophosphate contained in it are initially cross-linked to obtain gel microsphere suspension solution, continue to heat up and add a cross-linking agent, the second cross-linking occurs, the gel microspheres are solidified, washed and dried to obtain a stomach-targeting drug carrier.

According to the method for preparing stomach targeting drug carrier according to the present invention, it is characterized in that the mass ratio of chitosan and chitosan quaternary ammonium salt in the step 1) is 1: 0.1～10, preferably 1: 0.5～2 ; The mass ratio of the total mass of chitosan and chitosan quaternary ammonium salt to sodium glycerophosphate is 1:1-10; wherein the degree of quaternization substitution of chitosan quaternary ammonium salt is 10%-80%.

According to an embodiment of the present invention, a specific method for preparing a stomach-targeting drug carrier includes the following steps:

1) Prepare the drug-containing emulsion first: when the drug is a hydrophilic drug, the aqueous acetic acid solution dissolved with the hydrophilic drug, chitosan quaternary ammonium salt, chitosan and sodium glycerophosphate is used as the water phase (W), Use the oily substance dissolved with emulsifier as oil phase O, and prepare W/O type drug-containing emulsion through emulsification; when the drug is a lipophilic drug, first use the organic solvent dissolved with lipophilic drug as the internal oil phase (O ₁ ), using the aqueous solution of acetic acid dissolved with chitosan quaternary ammonium salt, chitosan and sodium glycerophosphate as the water phase (W), emulsified to make O ₁ /W type primary emulsion, and then the O ₁ /W Add type primary emulsion to the oily substance dissolved with emulsifier, and prepare O ₁ /W/O ₂ type drug-containing emulsion through emulsification;

2) The drug-containing emulsion is made into a drug carrier by a step-by-step solidification method: first, the emulsion is initially heated to cross-link the chitosan quaternary ammonium salt and sodium glycerophosphate to make a gel microsphere suspension, and then heating up, and adding a cross-linking agent to the gel microsphere suspension, and through a cross-linking reaction, the microspheres are completely solidified;

3) The above microspheres are washed, collected and dried to make a drug carrier.

According to the method for preparing the stomach-targeting drug carrier of the present invention, when the first step is solidified, sodium glycerophosphate (GP) is used as a cross-linking agent, and the consumption range of the cross-linking agent is chitosan and chitosan quaternary ammonium salt The mass ratio of the total mass to sodium glycerophosphate is 1:1～10; the crosslinking time is 0.5～2h, and the crosslinking temperature is 18～40°C, preferably 30°C～40°C, most preferably 36°C～38°C °C, the rotation speed of the oil phase during crosslinking is 150-700rpm. The second step cross-linking agent and consumption are as follows: cross-linking agent can be glutaraldehyde, genipin, epichlorohydrin etc., preferably use glutaraldehyde, can be the saturated toluene solution (GST) containing glutaraldehyde or For the aqueous solution containing glutaraldehyde, the dosage range of the cross-linking agent is that the molar ratio of the aldehyde group or epoxy group to the amino group of chitosan is 1:1-10; the cross-linking time is 0.5-5h, and the cross-linking temperature is 18 ~60°C, the preferred crosslinking temperature is 45°C~60°C, most preferably 48°C~50°C, the temperature program is used, the temperature rise rate is 2°C/min, and the oil phase rotation number during crosslinking is 150~700rpm.

According to the method for preparing stomach-targeting drug carrier of the present invention, the oily substance and water are immiscible, wherein the oil phase is selected from castor oil, olive oil, soybean oil or a mixture of liquid paraffin and petroleum ether, preferably liquid paraffin and petroleum ether The mixture of the two, the volume ratio of the two is preferably 1:2. The oily emulsifier must be dissolved in the oily substance used, sorbitan sesquioleate (Arlacel83), glyceryl ether polymer (such as PO-500, PO-310), polyoxyethylene hydrogenated castor oil, Sorbitan trioleate (Span 85), sorbitan monooleate (Span 80), sorbitan tristearate (Span 65), lipophilic-hydrophilic block Copolymer etc. The concentration of the emulsifier in the oil phase is 0.5-10 wt%.

According to the method for preparing stomach-targeting drug carriers of the present invention, the preparation method of the above-mentioned W/O emulsion is to first prepare a W/O type pre-emulsion, and then press the pre-emulsion through a microporous membrane under relatively high pressure, In order to prepare a W/O emulsion with uniform particle size, the microporous membrane is preferably a hydrophobic SPG membrane. Under optimized conditions, the diameter distribution coefficient of the microspheres is controlled within 20%, and the diameter can be freely within 0.1 to 10 microns. control.

According to the method for preparing the stomach-targeting drug carrier of the present invention, the lipophilic drug can be selected from Tagamet, ranitidine, rapamycin, etc., and when the lipophilic drug is prepared in the present invention, the internal oil phase O ₁ It is an organic solvent that can dissolve the drug, which is immiscible with water, preferably DMSO or chloroform, and an emulsifier can be added to the internal oil phase to increase the embedding rate when preparing the O ₁ /W type primary emulsion. The hydrophilic drug can be cimetidine, proglumide, amoxicillin or gentamicin sulfate. When rapamycin is administered systemically, it has been found in a large number of clinical trials that its side effects are nephrotoxicity and hepatotoxicity. , cholesterol, hyperlipidemia, thrombocytopenia and other symptoms, the drug carrier prepared by the invention can be targeted to the stomach for drug delivery, reducing side effects.

According to the method for preparing the stomach-targeting drug carrier of the present invention, in the above preparation, after the drug carrier is completely solidified, it is washed with petroleum ether, methanol, distilled water, etc. in sequence, and after supercritical extraction and drying, it is stored at low temperature; or Store the nanospheres directly in distilled water or cell culture medium at room temperature or low temperature.

According to the method for preparing stomach-targeting drug carrier of the present invention, due to the mixing of chitosan, chitosan quaternary ammonium salt and sodium glycerophosphate, the composite microspheres have both chitosan microspheres and chitosan quaternary ammonium salt microspheres. The respective advantages of the balls, such as the thermosensitivity and gelatinity of the quaternary ammonium salt microspheres due to the presence of sodium glycerophosphate, the spontaneous and stable fluorescence properties of the chitosan microspheres, etc., also endow the composite microspheres with new characteristics. . Specifically: (1) When chitosan quaternary ammonium salt microspheres are used as a drug carrier alone, due to their porosity and large pore size on the surface of the microspheres, small molecule drugs can easily flow out from the pores when they are embedded. , which greatly reduces the embedding rate of the drug, and if chitosan is added, glutaraldehyde crosslinking agent is added at a certain temperature, the aldehyde group of glutaraldehyde and the amino group on chitosan and chitosan quaternary ammonium salt The unquaternized amino group undergoes an aldol condensation reaction, and a certain network structure is formed on the surface of the previously formed gel microspheres, which greatly reduces the pore size of the gel microspheres and improves the embedding rate of the drug. (2) At the same time, due to the aldol condensation reaction of the aldehyde group of glutaraldehyde and the amino group on the chitosan and the non-quaternized amino group on the chitosan quaternary ammonium salt, a large conjugated double bond (-C= N-C=C-), can show strong and stable autofluorescence; at the same time, the nano-microspheres prepared by chitosan not only have good biocompatibility, but also have abundant functional groups (hydroxyl and amino groups) on the surface, It can be used for surface modification and modification; therefore, using the stable fluorescent properties of chitosan nano-microspheres and the unique advantages of chitosan, biological experiments at the cell level and animal level can be performed, and it is easy to accurately quantify, solving the problem of In the past, accurate quantification could not be performed due to unstable labeling or easy leakage of fluorescent substances that were difficult to label or embed.

According to the stomach targeting drug carrier and preparation method thereof of the present invention, by mixing chitosan and chitosan quaternary ammonium salt materials, and strictly controlling the ratio between the two and the selected chitosan quaternary ammonium salt The degree of quaternary ammonium has achieved that the prepared composite microsphere is a positively charged microsphere, and the prepared chitosan and chitosan quaternary ammonium salt composite microspheres are dispersed in PBS (pH=7.4), and the measured Its zeta potential is 20-28mV, so it can interact with negatively charged groups on the cell surface and undergo non-specific adsorption with cells, which is conducive to the adhesion of drug carriers on the cell surface. The residence time in the gastric mucosal tissue prolongs the absorption time of the drug, thereby greatly improving the bioavailability of the drug in the body. At the same time, due to the mixing of chitosan and chitosan quaternary ammonium salt materials, cross-linking agents such as glutaraldehyde are added at a certain temperature, and by controlling the time and temperature of the cross-linking reaction, an appropriate degree of cross-linking is achieved, so that The surface of the formed gel microspheres forms a certain network structure, which greatly reduces the pore size of the gel microspheres and improves the embedding rate of the drug, so that the composite microspheres can be used in the extremely acidic gastric physiological environment (pH=1.2). Under the condition of good sensitivity, the composite microspheres can be retained in the stomach for 24 hours without being degraded (see Figure 7). The pH sensitivity of the composite microspheres makes it possible to be used as a targeted drug carrier for acidic sites, such as as Targeted drug delivery carrier for gastric drugs. The drug carrier prepared by the above method has the properties of pH sensitivity, gastric adhesion and sustained drug release. In an environment of pH=1-3, it can maintain a good shape without being degraded by an acidic environment. It has good adhesion in the stomach, and the amount of carrier adhered in the stomach accounts for 40% to 90% of the total amount of oral carriers. After the drug is loaded, it can achieve the sustained release effect of the drug in the stomach, and the sustained release within 24 hours Drug, the release rate of the drug in the stomach is 0.06mg/h～0.12mg/h.

The invention also solves the inhomogeneous and uncontrollable particle size of chitosan and chitosan quaternary ammonium salt drug carriers prepared by the existing stirring emulsification method, low embedding rate and poor dispersibility, and at the same time, the prepared gel microspheres meet the requirements of Requirements for stomach-targeted drug delivery vehicles, namely adhesion, certain pH sensitivity and drug sustained release function. It is expected to use the uniformity of particle size, pH sensitivity, adhesion and other characteristics to achieve stable gastric targeted release of drugs, improve the local concentration of drugs in the stomach and avoid systemic side effects and other key issues.

Description of drawings

Fig. 1 is the schematic flow sheet of the drug carrier that mechanical stirring method prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 2 is the schematic flow sheet of the drug carrier of chitosan and chitosan quaternary ammonium salt mixed material prepared by fast membrane emulsification method of the present invention;

Fig. 3 is the optical micrograph of the drug carrier that embodiment 1 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 4 is the electron micrograph of the drug carrier that embodiment 1 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 5 is the optical micrograph of the drug carrier that embodiment 2 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 6 is the electron micrograph of the drug carrier that embodiment 2 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 7 is the electron micrograph of the drug carrier that embodiment 3 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 8 is the electron micrograph of the drug carrier that embodiment 4 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 9 is the particle size distribution of the drug carrier that embodiment 4 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 10 is the in vitro release curve of rapamycin in the drug carrier of chitosan and chitosan quaternary ammonium salt mixed material prepared by embodiment 4 membrane emulsification method;

Fig. 11 is the electron micrograph of the drug carrier that embodiment 5 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 12 is the electron micrograph of the drug carrier that embodiment 6 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 13 is the electron micrograph of the drug carrier that embodiment 7 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 14 is the electron micrograph of the drug carrier that embodiment 8 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 15 is the electron micrograph of the drug carrier that embodiment 9 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 16 is the electron micrograph of the drug carrier that embodiment 10 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 17 is the electron micrograph of the drug carrier that embodiment 11 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 18 is the electron micrograph of the drug carrier that embodiment 12 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 19 is the electron micrograph of the drug carrier that embodiment 13 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 20 is the electron micrograph of the drug carrier that embodiment 14 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 21 is the electron micrograph of the drug carrier that embodiment 15 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 22 is the electron micrograph of the drug carrier that embodiment 16 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 23 is the electron micrograph of the drug carrier that embodiment 17 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 24 is the electron micrograph of the drug carrier that embodiment 18 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 25 is the electron micrograph of the drug carrier that embodiment 19 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 26 is the electron micrograph of the drug carrier that embodiment 20 prepares chitosan and chitosan quaternary ammonium salt mixed material;

Fig. 27 is the adhesion rate of the drug carrier of chitosan and chitosan quaternary ammonium salt mixed material prepared in the stomach in different time in embodiment 4;

Fig. 28 is the adhesion time distribution diagram of the drug carrier in the stomach prepared by the chitosan and chitosan quaternary ammonium salt mixed material in Example 4.

Detailed ways

Example 1

Accurately weigh chitosan (molecular weight 50,000), chitosan quaternary ammonium salt (quaternary ammonium substitution degree 50%, molecular weight 50,000), sodium glycerophosphate and dissolve it in 1% acetic acid aqueous solution, and make it fully dissolve under magnetic stirring to obtain The acetic acid aqueous solution of chitosan, chitosan quaternary ammonium salt, sodium glycerophosphate, its total concentration is 2.5wt%, wherein the concentration of chitosan, chitosan quaternary ammonium salt is respectively 0.5wt%, the concentration of sodium glycerophosphate The mass ratio of chitosan and chitosan quaternary ammonium salt is 1:1, and the mass ratio of chitosan and chitosan quaternary ammonium salt to sodium glycerophosphate is 2:3. The solution was centrifuged at 8000 rpm to remove insoluble impurities, and the supernatant was retained as the aqueous phase for subsequent use. Take 400 μL of 10 mg/mL rapamycin solution O ₁ (rapamycin is dissolved in chloroform, and the concentration of emulsifier PO-500 is 1 wt%), add it to the water phase, stir mechanically at room temperature, the stirring condition is 1000rpm, 10min, O ₁ /W colostrum is formed; then the colostrum is slowly added dropwise into the mixed external oil phase O ₂ of liquid paraffin and petroleum ether (volume ratio is 1:2, emulsifier PO-500 concentration is 4wt%). Stir rapidly with a mechanical stirrer at 1000rpm for 10 minutes to obtain O ₁ /W/O ₂ double emulsion, solidify at 37°C for 1 hour; add a certain amount of glutaraldehyde-saturated toluene solution to the emulsion after 1 hour, of which The amount of glutaraldehyde is calculated according to the following ratio, that is, the molar ratio of the aldehyde group on the glutaraldehyde to the amino group on the chitosan is 1:1, and the temperature is programmed to rise at a rate of about 2°C/min until cross-linking at 50°C The reaction was carried out for 5 hours, and the stirring speed of the crosslinking reaction was 700 rpm. After the cross-linking reaction is over, centrifuge at 5000rpm, pour off the supernatant, separate chitosan and chitosan quaternary ammonium salt nano-microspheres from the oil phase, wash with petroleum ether, methanol, and distilled water successively, and store them in distilled water. The embedding rate of the drug carrier was detected by a microplate reader to be 80%, and it was completely released in the simulated gastric juice within 24 hours. Has good adhesion. The average particle size and particle size distribution of the carrier microspheres are measured by Zeta potential and particle size analyzer ZetaPlus. The average diameter of the microspheres in water is 1000.0 nanometers, and the CV value is 38.07%. The microscope picture is shown in Figure 3, and the electron microscope picture is shown in Figure 3 4.

Example 2

Accurately weigh chitosan (molecular weight 50,000), chitosan quaternary ammonium salt (quaternary ammonium substitution degree 50%, molecular weight 50,000), sodium glycerophosphate and dissolve it in 1% acetic acid aqueous solution, and make it fully dissolve under magnetic stirring to obtain The acetic acid aqueous solution of chitosan, chitosan quaternary ammonium salt, sodium glycerophosphate, its total concentration is 2.5wt%, wherein the concentration of chitosan, chitosan quaternary ammonium salt is respectively 0.5wt%, the concentration of sodium glycerophosphate The mass ratio of chitosan and chitosan quaternary ammonium salt is 1:1, and the mass ratio of chitosan and chitosan quaternary ammonium salt to sodium glycerophosphate is 2:3. This solution was centrifuged at 8000rpm to remove insoluble impurities, then added 20 wt% gentamicin sulfate (stomach medicine) of the total dry weight of chitosan and chitosan quaternary ammonium salt and fully dissolved for future use. Add the oil-soluble emulsifier PO-500 into the mixed oil phase (volume ratio of 1:2) of 60mL of liquid paraffin and sherwood oil, and its concentration is 4wt%, stir until completely dissolved as the oil phase; take 6.0g of water Phase and oil phase were mixed, mechanically stirred at 1000rpm for 30min to obtain a W/O emulsion; cured at 37°C for 1 hour, and after 1 hour, a certain amount of glutaraldehyde-saturated toluene solution was added to the emulsion, and the amount of glutaraldehyde was based on The ratio is calculated as follows, that is, the molar ratio of the aldehyde group on the glutaraldehyde to the amino group on the chitosan is 1:1, the temperature is programmed to 50° C. for cross-linking reaction for 5 hours, and the stirring rate of the cross-linking reaction is 700 rpm. After the cross-linking reaction is over, centrifuge at 5000rpm, pour off the supernatant, separate chitosan and chitosan quaternary ammonium salt nano-microspheres from the oil phase, wash with petroleum ether, methanol, and distilled water successively, and store them in distilled water. The embedding rate of the drug carrier was detected by a microplate reader to be 83%, and it was completely released in simulated gastric juice within 28 hours, with a release rate of about 0.09 mg/h. The surface potential of the microspheres was 23.29, and the gastric targeting effect was good. The average particle size and particle size distribution of the microspheres are measured by Zeta potential and particle size analyzer ZetaPlus. The average diameter of the microspheres in water is 1300.0 nanometers, and the C.V. value is 35.12%. The microscope image is shown in Figure 5, and the electron microscope image is shown in Figure 6.

Example 3

Accurately weigh chitosan (molecular weight 50,000), chitosan quaternary ammonium salt (quaternary ammonium substitution degree 70%, molecular weight 100,000), sodium glycerophosphate and dissolve it in 1% acetic acid aqueous solution, and make it fully dissolve under mechanical stirring to obtain The acetic acid aqueous solution of chitosan, chitosan quaternary ammonium salt, sodium glycerophosphate, its total concentration is 2.5wt%, wherein the concentration of chitosan, chitosan quaternary ammonium salt is respectively 0.5wt%, the concentration of sodium glycerophosphate The mass ratio of chitosan and chitosan quaternary ammonium salt is 1:1, and the mass ratio of chitosan and chitosan quaternary ammonium salt to sodium glycerophosphate is 2:3. The solution was centrifuged at 8000 rpm to remove insoluble impurities, and the supernatant was retained as the aqueous phase for subsequent use. Take 400 μL of 10 mg/mL rapamycin solution O ₁ (rapamycin is dissolved in chloroform, and the concentration of emulsifier PO-500 is 1 wt%), add it to the water phase, stir mechanically at room temperature, the stirring condition is 1000rpm, 10min, O1/W colostrum is formed. Add the oil-soluble emulsifier PO-500 into 60mL of the mixed oil phase of liquid paraffin and petroleum ether (volume ratio is 1:2), its concentration is 4wt%, stir until completely dissolved as the outer oil phase O ₂ , and then Colostrum is slowly added dropwise into the mixed external oil phase of liquid paraffin and petroleum ether. Homogeneous emulsification for 60s, 3 gears (14000rpm/min), to obtain O ₁ /W/O ₂ double emulsion, and curing at 37°C for 1 hour. After 1 hour, add a certain amount of glutaraldehyde-saturated toluene solution to the emulsion, wherein the amount of glutaraldehyde is calculated according to the following ratio, that is, the molar ratio of the aldehyde group on the glutaraldehyde to the amino group on the chitosan is 1:1 , temperature program, the heating rate is about 2 ° C / min, to 50 ° C cross-linking reaction for 5 hours, the stirring rate of the cross-linking reaction is 700 rpm. After the cross-linking reaction, centrifuge at 5000rpm, pour off the supernatant, separate chitosan and chitosan quaternary ammonium salt nano-microspheres from the oil phase, wash with petroleum ether, methanol, distilled water successively, and store them in distilled water. The embedding rate of the drug carrier was detected by a microplate reader to be 85%, and it was completely released within 24 hours in the simulated gastric juice, with a release rate of about 0.11mg/h. The surface potential of the microspheres was +30mV, and the gastric targeting effect was good, and The results in Example 1 are consistent. The average particle size and particle size distribution of the microspheres were measured by a Zeta potential and particle size analyzer ZetaPlus. The average diameter of the microspheres in water was 1500.0 nm, and the CV value was 34.28%. The scanning electron microscope photo is shown in FIG. 7 .

Example 4

The hydrophilic SPG glass membrane with a pore size of 2.8 microns was placed in a mixed oil phase of liquid paraffin and petroleum ether (volume ratio 1:2), soaked overnight or ultrasonicated for half an hour to fully infiltrate the membrane pores with the oil phase. Accurately weigh chitosan (molecular weight 50,000), chitosan quaternary ammonium salt (quaternary ammonium substitution degree 70%, molecular weight 50,000), sodium glycerophosphate, dissolve in 1% acetic acid aqueous solution, and make it fully dissolved under magnetic stirring Obtain the acetic acid aqueous solution of chitosan, chitosan quaternary ammonium salt, sodium glycerophosphate, as internal water phase, its total concentration is 2.5wt%, wherein the concentration of chitosan, chitosan quaternary ammonium salt is respectively 0.5wt% , the concentration of sodium glycerophosphate is 1.5wt%, wherein the mass ratio of chitosan and chitosan quaternary ammonium salt is 1: 1, and the mass ratio of chitosan and chitosan quaternary ammonium salt total mass to sodium glycerophosphate 2:3 The solution was centrifuged at 8000 rpm to remove insoluble impurities, and the supernatant was reserved as the aqueous phase for later use. Take 400 μL of 10 mg/mL rapamycin solution O ₁ (rapamycin is dissolved in chloroform, and the concentration of emulsifier PO-500 is 1 wt%), add it to the water phase and use a homogeneous emulsifier at the third gear (6000rpm) Emulsify for 1 min to form O ₁ /W type colostrum. Add the oil-soluble emulsifier PO-500 into 60mL of the mixed oil phase of liquid paraffin and petroleum ether (volume ratio is 1:2), its concentration is 4wt%, stir until completely dissolved as the outer oil phase O ₂ , and then The obtained O ₁ /W type colostrum is mixed with the external oil, and emulsified with a homogeneous emulsifier at the third gear (6000rpm) for 1 min to form an O ₁ /W/O ₂ pre-emulsion, and quickly pour it into a membrane emulsification device. Under the pressure of nitrogen gas, press it quickly through the SPG microporous membrane with uniform pore size to obtain an O ₁ /W/O ₂ type emulsion with relatively uniform particle size, and use the obtained emulsion as a pre-emulsion to press it again under the nitrogen pressure of 0.4MPa Microporous membrane, emulsified five times repeatedly, and finally obtained an O ₁ /W/O ₂ type emulsion with uniform particle size; after emulsification was completed, it was solidified at 37°C for 1 hour; after 1 hour, a certain amount of toluene saturated with glutaraldehyde was added to the emulsion Solution, wherein the amount of glutaraldehyde is calculated according to the following ratio, that is, the molar ratio of the aldehyde group on the glutaraldehyde to the amino group on the chitosan is 1: 1, the temperature is programmed, the temperature is programmed, and the heating rate is about 2 °C/min , to 50° C. for cross-linking reaction for 5 hours, and the stirring rate of the cross-linking reaction was 700 rpm. After the cross-linking reaction is finished, centrifuge at 5000rpm, pour off the supernatant and separate chitosan and chitosan quaternary ammonium salt nano-microspheres from the oil phase, wash with petroleum ether, methanol, distilled water successively, and wash them Store in distilled water. The average particle size and particle size distribution of microspheres are measured by Zeta potential and particle size analyzer ZetaPlus. The average diameter of microspheres in water is 1500.0 nanometers, and the CV value is 13.8%. The scanning electron microscope photo is shown in Figure 8, and the particle size distribution is as follows: The results shown in Figure 9 show that the prepared chitosan and chitosan quaternary ammonium salt nano-microspheres have a uniform particle size.

Among them, the entrapment of rapamycin drug reaches more than 90%, and the release of the drug is close to 100% within 24 hours. Adhesion on the gastric mucosa results in microspheres with more positive charges and fewer and smaller holes in the microspheres. Therefore, chitosan quaternary ammonium salts with a certain degree of substitution help to improve the embedding rate of drugs. Simultaneously reduce the release rate of the drug.

Example 5

The preparation method is the same as 4, except that the mass ratio of chitosan to chitosan quaternary ammonium salt is 1:0.1, and its Zeta potential is +9.2mV, and its electron microscope photo is shown in Figure 11.

Example 6

The preparation method is the same as 4, except that the mass ratio of chitosan to chitosan quaternary ammonium salt is 1:10, and its Zeta potential is +26.17mV, and its electron microscope photo is shown in Figure 12.

Example 7

The preparation method is the same as 4, except that the mass ratio of chitosan and the total mass of chitosan quaternary ammonium salt to sodium glycerophosphate is 1:1, and its Zeta potential is +21.32mV, and its electron micrograph is shown in Figure 13.

Example 8

The preparation method is the same as 4, except that the mass ratio of chitosan and the total mass of chitosan quaternary ammonium salt to sodium glycerophosphate is 1:10, and its Zeta potential is +19.18mV, and its electron microscope photo is shown in Figure 14.

Example 9

The preparation method is the same as 4, except that the quaternary ammonium substitution degree of the chitosan quaternary ammonium salt is 10%, and the Zeta potential of the prepared microsphere is +9.23mV, and its electron microscope photo is shown in FIG. 15 .

Example 10

The preparation method is the same as 4, except that the quaternary ammonium substitution degree of the chitosan quaternary ammonium salt is 80%, and the Zeta potential of the prepared microsphere is +28.24mV, and its electron microscope photo is shown in FIG. 16 .

Example 11

The preparation method is the same as 4, except that the molar ratio of cross-linking agent to chitosan and chitosan quaternary ammonium salt amino group is 1:10, and its Zeta potential is +24.32mV, and its electron microscope photo is shown in Figure 17.

Example 12

The preparation method is the same as 4, except that the crosslinking temperature in the first step is 18°C, and the crosslinking time is 0.5h. The zeta potential of the prepared microspheres is +23.21mV, and its electron micrograph is shown in Figure 18.

Example 13

The preparation method is the same as 4, except that the crosslinking temperature in the first step is 30°C, and the crosslinking time is 0.5h. The zeta potential of the prepared microspheres is +24.33mV, and its electron micrograph is shown in Figure 19.

Example 14

The preparation method is the same as 4, except that the crosslinking temperature in the first step is 36°C, and the crosslinking time is 0.5h. The Zeta potential of the prepared microspheres is +26.22mV, and its electron micrograph is shown in Figure 20.

Example 15

The preparation method is the same as 4, except that the mass ratio of chitosan and chitosan quaternary ammonium salt is 1: 0.5, the first step of crosslinking temperature is 40°C, and the crosslinking time is 0.5h, and the Zeta potential of the prepared microspheres is +20.34mV, and its electron microscope photo is shown in Figure 21.

Example 16

The preparation method is the same as 9, except that the mass ratio of chitosan and chitosan quaternary ammonium salt is 1:2, the crosslinking temperature in the first step of crosslinking is 38°C, and the first step of crosslinking time is 2h. The zeta potential of the ball is +20.10mV, and its electron microscope photo is shown in Figure 22.

Example 17

The preparation method is the same as 4, except that the second step crosslinking temperature is 18°C, and the second step crosslinking time is 0.5h, the Zeta potential of the prepared microspheres is +18.31mV, and its electron micrograph is shown in Figure 23.

Example 18

The preparation method is the same as 4, except that the second step crosslinking temperature is 45°C, and the second step crosslinking time is 0.5h, the Zeta potential of the prepared microspheres is +24.13mV, and its electron micrograph is shown in Figure 24.

Example 19

The preparation method is the same as 4, except that the second step crosslinking temperature is 48°C, and the second step crosslinking time is 0.5h, the Zeta potential of the prepared microspheres is +26.64mV, and its electron micrograph is shown in Figure 25.

Example 20

The preparation method is the same as 4, except that the second step crosslinking temperature is 60°C, and the second step crosslinking time is 0.5h, the Zeta potential of the prepared microspheres is +24.53mV, and its electron micrograph is shown in Figure 26.

Effect Example 1

The drug carrier prepared in Preparation Example 4 was subjected to a mouse gavage experiment to further investigate the adhesion properties of the carrier in the stomach. Before the experiment, the mice were divided into 8 groups, 8 mice in each group, fasted for 12 hours in advance, without water. The amount of gavage for each mouse was 400 μL (3.6mg/mL), and the stomach of each group of mice was taken out at the time period 0h, 1h, 3h, 4h, 6h, 8h, 12h, and 24h respectively, and PBS=7.4 (0.1 mol/mL), and the gastric tissues of 2 mice in each group were solidified, desugared and other steps were taken to make tissue slices, and the adhesion of the drug carrier in the stomach was qualitatively observed, and the carrier was visually observed. In terms of adhesion in the stomach, it was found through fluorescence detection that the carrier can adhere well to the gastric mucosa, and there is an obvious adhesion band on the gastric mucosa, which will facilitate the release of stomach-targeted drugs. The gastric tissues of the remaining 6 mice in each group were rinsed, digested with corresponding enzymes, centrifuged at 3000rpm, resuspended in PBS=7.4 (0.1mol/mL), and tested the gastric adhesion of the carrier with a microplate reader. Qualitative detection, the results are shown in Figure 28, after the mouse gavage experiment, the carrier can maintain a certain adhesion rate within 20 hours after entering the stomach, and the results are shown in Figure 27, indicating that the prepared microsphere carrier can The effect of targeted sustained release and controlled release of stomach drugs can be realized for a long time.

The remaining 19 groups of microsphere carriers obtained in the preparation examples were also subjected to the stomach adhesion effect experiment, and the results found that the adhesion effect and drug release rate in the stomach were similar to the results obtained in Example 4, and both could achieve gastric adhesion for a long time. The effect of targeted sustained release and controlled release of the drug shows that the method for preparing the microsphere carrier according to this patent is feasible, and the adhesion characteristics of the microsphere and the sustained release effect of the drug have good repeatability.

Claims (10)

1. A gastric targeting drug carrier, characterized in that the drug carrier raw materials include: Medicated emulsions containing chitosan, chitosan quaternary ammonium salt, sodium glycerophosphate; and A cross-linking agent for cross-linking the above-mentioned emulsion into microspheres; Wherein, the mass ratio of chitosan and chitosan quaternary ammonium salt is 1:0.1～10, and the mass ratio of the total mass of chitosan and chitosan quaternary ammonium salt to sodium glycerophosphate is 1:1～10 ; Wherein, the molar ratio of the aldehyde group or epoxy group of the crosslinking agent to the amino group of chitosan and chitosan quaternary ammonium salt is 1:1～10; Wherein, the method for preparing the stomach targeting drug carrier comprises the following steps: 1) Preparation of drug-containing emulsion: the aqueous phase W of chitosan, chitosan quaternary ammonium salt and sodium glycerophosphate is used as the water phase W, and the drug and the oil phase containing emulsifier are used to prepare the drug-containing emulsion; 2) Heat up the drug-containing emulsion in step 1) and stir to make the chitosan quaternary ammonium salt and sodium glycerophosphate contained in it undergo preliminary cross-linking to obtain a suspension of gel microspheres, heat up and add a cross-linking agent, and the second Secondary cross-linking to solidify the gel microspheres, washing and drying to obtain the stomach-targeting drug carrier. the 2. The stomach-targeting drug carrier according to claim 1, wherein the degree of quaternization substitution of the chitosan quaternary ammonium salt is 10% to 80%. the 3. The stomach-targeting drug carrier according to claim 1 or 2, characterized in that the mass ratio of chitosan to chitosan quaternary ammonium salt is 1:0.5-2. the 4. The stomach-targeting drug carrier according to claim 1, wherein the cross-linking agent is glutaraldehyde, genipin or epichlorohydrin. the 5. A method for preparing a gastric-targeted drug carrier, characterized in that the method comprises the following steps: 1) Preparation of drug-containing emulsion: the aqueous phase W of chitosan, chitosan quaternary ammonium salt and sodium glycerophosphate is used as the water phase W, and the drug and the oil phase containing emulsifier are used to prepare the drug-containing emulsion; 2) Heat up the drug-containing emulsion in step 1) and stir to make the chitosan quaternary ammonium salt and sodium glycerophosphate contained in it undergo preliminary cross-linking to obtain a suspension of gel microspheres, heat up and add a cross-linking agent, and the second Secondary cross-linking to solidify the gel microspheres, wash and dry to obtain the stomach targeting drug carrier; Wherein, the mass ratio of chitosan and chitosan quaternary ammonium salt in the step 1) is 1:0.1-10; the mass ratio of the total mass of chitosan and chitosan quaternary ammonium salt to sodium glycerophosphate 1:1-10; the molar ratio of the aldehyde group or epoxy group of the crosslinking agent in the step 2) to the amino group of chitosan and chitosan quaternary ammonium salt is 1:1-10. the 6 . The method for preparing a stomach-targeting drug carrier according to claim 5 , wherein the degree of quaternization substitution of the chitosan quaternary ammonium salt in the step 1) is 10% to 80%. the 7. The method for preparing stomach-targeting drug carrier according to claim 5, characterized in that, the cross-linking temperature of the preliminary cross-linking in the step 2) is 18-60°C, the cross-linking time is 0.5-5h, and the temperature is raised The cross-linking agent for the second cross-linking is glutaraldehyde, genipin or epichlorohydrin, the cross-linking temperature is 18-60°C, the cross-linking time is 0.5-5h, and the number of stirring speeds during cross-linking is 150 ~700rpm. the 8. The method for preparing a stomach-targeting drug carrier according to claim 7, characterized in that the cross-linking temperature of the step 2) for the primary cross-linking is 30°C to 40°C, and the second cross-linking temperature is 45°C ~60°C. the 9. The method for preparing a stomach-targeting drug carrier according to claim 8, characterized in that the cross-linking temperature of the step 2) for the primary cross-linking is 36°C to 38°C, and the second cross-linking temperature is 48°C ~50°C. the 10. The method for preparing a stomach-targeted drug carrier according to claim 5, wherein the emulsifier in step 1) is selected from sorbitan sesquioleate, glyceryl ether polymers, polyoxygen Ethylene hydrogenated castor oil, sorbitan trioleate, sorbitan monooleate, sorbitan tristearate or lipophilic-hydrophilic block copolymer, concentration of emulsifier in oil phase 0.5-10wt%. the

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