CN118766870A - Triamcinolone acetonide sustained-release microspheres containing microreservoir structure and preparation method thereof - Google Patents

Abstract

The present invention discloses a triamcinolone acetonide drug sustained-release microsphere containing a microreservoir structure and a preparation method thereof. The microreservoir structure contains an inclusion agent, which is a cyclic natural oligosaccharide formed by connecting 6 (α), 7 (β) or 8 (γ) glucose monomers through α-1,4-glycosidic bonds. This structural design enables the microspheres to have a drug loading of at least 25% and an encapsulation rate of more than 95%, significantly improving the loading efficiency and stability of the drug. During the preparation process, the present invention adopts advanced shearing technology, and successfully generates microspheres with a microreservoir structure by applying shear force to a dispersed phase containing drugs and polymers in a continuous phase. The present invention not only improves the preparation efficiency of the microspheres, but also ensures the uniform distribution of the drug in the microspheres, thereby ensuring a good sustained-release effect of the drug.

Description

Triamcinolone acetonide sustained-release microspheres containing microreservoir structure and preparation method thereof

Technical Field

The invention relates to triamcinolone acetonide sustained-release microspheres containing a micro-reservoir structure and a preparation method thereof, belonging to the technical field of pharmaceutical preparations.

Background Art

Osteoarthritis is a common joint degenerative disease that may be caused by a variety of factors, including strain, obesity, trauma, excessive exercise, congenital joint abnormalities, joint deformities and genetic factors. This disease not only causes joint pain, deformity and dysfunction, but also may increase the risk of cardiovascular events, deep vein thrombosis of the lower limbs, hip fractures and all-cause mortality, seriously affecting the quality of life of patients.

Triamcinolone acetonide is a corticosteroid with anti-inflammatory and immunomodulatory properties. It binds to glucocorticoid receptors, blocks the release of arachidonic acid, and inhibits the synthesis of prostaglandins and leukotrienes, thereby slowing down, preventing or reversing tissue damage caused by inflammatory diseases. Compared with traditional oral anti-inflammatory drugs, intra-articular injection of triamcinolone acetonide can provide higher local drug concentrations and reduce systemic side effects. However, the rapid clearance of small molecule anti-inflammatory drugs in the joint cavity limits their therapeutic effect, requiring frequent administration and causing pain to patients.

Microsphere technology provides an effective solution by controlling drug release, reducing the frequency of administration, enhancing drug effects, and reducing side effects and toxicity. Microspheres can deliver targeted drugs, increase drug concentrations at the site of action, reduce systemic exposure, and protect drugs from degradation through physical barriers. Polylactic-co-glycolic acid (abbreviated as PLGA) microspheres are a commonly used drug delivery system that can degrade under physiological conditions, release drugs at a certain rate, and maintain blood drug concentrations. However, existing pharmaceutical preparation technologies are limited in microsphere particle size and drug loading, which affects drug action time and release control. For example, when using the traditional emulsification volatilization method, due to the poor solubility of triamcinolone acetonide in common solvents, it is unevenly dispersed in the organic phase, which not only reduces the drug loading of the microspheres, but may also cause drug precipitation during the sphering process. These problems limit the sustained-release effect of microspheres, making it difficult to control the drug release rate and time, and unable to meet the needs of clinical treatment.

To overcome these limitations, researchers are exploring a new type of sustained-release microspheres containing a microreservoir structure for triamcinolone acetonide and its preparation method. This method aims to increase the drug loading and production efficiency of the microspheres while achieving controllable drug release. By optimizing the particle size distribution of the microspheres, the performance stability of the microspheres can be enhanced, thus providing the possibility for large-scale production. Solving these technical problems is of great significance for improving the treatment effect and quality of life of patients with osteoarthritis.

Summary of the invention

The primary technical problem to be solved by the present invention is to provide a triamcinolone acetonide drug sustained-release microsphere containing a micro-reservoir structure.

Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned triamcinolone acetonide drug sustained-release microspheres.

In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

According to a first aspect of an embodiment of the present invention, a method for preparing triamcinolone acetonide sustained-release microspheres containing a microreservoir structure is provided, the steps of which are as follows:

(1) dissolving PLGA in dichloromethane to prepare a PLGA dichloromethane solution as dispersed phase A solution, wherein the PLGA concentration is 0.01 g/ml to 0.5 g/ml, and the weight ratio of glycolide in the PLGA is 10% to 30%, and the weight ratio of lactide is 70% to 90%. After dissolution, the dispersed phase A solution is prepared;

(2) dissolving triamcinolone acetonide in N-methylpyrrolidone at a concentration of 0.4 g/ml to 0.5 g/ml; after ultrasonic dissolution, adding a complexing agent, and dissolving the complexing agent to form a dispersed phase B solution;

(3) The dispersed phase B solution is transferred into the dispersed phase A solution and stirred by a homogenizer at a speed of 3000 to 5000 rpm to form a stable and uniform dispersed phase;

(4) dissolving polyvinyl alcohol and sodium chloride in injection water as a continuous phase, wherein the concentration of polyvinyl alcohol is 1% to 5%, and the concentration of sodium chloride is 1% to 5%;

(5) Shearing step: by mechanical stirring, membrane emulsification or microfluidics, the prepared dispersed phase is brought into contact with the continuous phase by pressure or shearing to form droplets; stirring is performed at a low speed of 180 to 230 rpm at a low temperature of 6 to 15°C, the organic solvent in the droplets is gradually reduced, and the PLGA skeleton of the drug is gradually precipitated and solidified to form microspheres;

(6) Turn off stirring, allow the microspheres to settle naturally, collect by suction filtration, wash with pure water, and collect;

(7) The collected microspheres are transferred to a container and freeze-dried to obtain the product.

Preferably, in step (1), the molecular weight of the PLGA is 30 KDa to 100 KDa.

Preferably, in step (2), the inclusion agent is a cyclic natural oligosaccharide composed of 6 (α), 7 (β) or 8 (γ) glucose monomers connected by α-1,4-glycosidic bonds.

Preferably, in step (2), the inclusion agent is a diacyl chloride-modified inclusion agent.

Preferably, in step (2), the inclusion agent is a succinate-β-inclusion agent or an adipate-γ-inclusion agent.

Preferably, in step (2), the inclusion agent accounts for 0.5%-2% of the dispersed phase B solution.

Preferably, in step (2), the amount of the inclusion agent added to the dispersed phase B solution is 0.25 to 0.5 g.

Preferably, in step (4), the molecular weight of the polyvinyl alcohol is 30KDa to 50KDa.

Preferably, in step (6), the weight ratio of the dispersed phase to the continuous phase is 1:30-1:100.

According to a second aspect of an embodiment of the present invention, there is provided a triamcinolone acetonide sustained-release microsphere containing a microreservoir structure, wherein the microreservoir structure contains an inclusion agent.

Preferably, the micro-reservoir structures are densely arranged.

Preferably, the inclusion agent is a cyclic natural oligosaccharide composed of 6 (α), 7 (β) or 8 (γ) glucose monomers connected by α-1,4-glycosidic bonds.

Preferably, the inclusion agent is a diacyl chloride-modified inclusion agent.

Preferably, the inclusion agent is a succinate-β-inclusion agent or an adipate-γ-inclusion agent.

Preferably, the amount of the inclusion agent is 0.5%-2%.

Preferably, the drug loading % of the triamcinolone acetonide sustained-release microspheres containing a microreservoir structure is greater than 25%; and the encapsulation rate % is greater than 95%.

According to a third aspect of an embodiment of the present invention, a triamcinolone acetonide sustained-release preparation is provided, comprising triamcinolone acetonide sustained-release microspheres having a microreservoir structure.

Compared with the prior art, the present invention realizes the extrusion and shearing of the continuous phase to the dispersed phase by adopting mechanical stirring, membrane emulsification and microfluidic technology. These process steps significantly improve the uniformity of triamcinolone acetonide in the microspheres, thereby increasing the drug loading and output. In addition, the present invention also realizes the controllability of drug sustained release, making the microsphere particle size distribution more concentrated and the particle size distribution narrower. These improvements not only improve the performance stability of the microspheres, but also provide the possibility for large-scale production. Through these innovative processes, the present invention can provide a more effective and stable drug sustained release solution for the clinical treatment of chronic inflammatory diseases such as osteoarthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of the structure of triamcinolone acetonide in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the structure of the inclusion agent in an embodiment of the present invention.

FIG. 3 is a schematic diagram of the structure of a modified inclusion agent-immobilized drug microreservoir in an embodiment of the present invention.

FIG. 4 is a schematic diagram of the structure modified by the inclusion agent in an embodiment of the present invention.

FIG. 5 is a reaction formula for modification of the inclusion agent in an embodiment of the present invention.

FIG. 6 is a schematic diagram of the structure of PLGA microspheres containing triamcinolone acetonide microreservoirs in an embodiment of the present invention.

FIG. 7 is an electron microscope photograph of microspheres without triamcinolone acetonide, with triamcinolone acetonide but without inclusion agent, and with triamcinolone acetonide and inclusion agent.

FIG8 is an electron microscope photograph of samples 0 to 5.

Figure 9 is an electron microscope photograph of sample 5 containing the drug sustained-release microspheres of triamcinolone acetonide microreservoir structure.

DETAILED DESCRIPTION

The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

Unless otherwise specified, the raw materials described in the following examples are conventional raw materials available in the market in this field; the methods described are conventional methods in this field; the equipment or instruments used are conventional equipment or instruments in this field; and the units described are all weight units.

Comparative Example 1 (Preparation of Sample 0 Blank Microspheres)

The method for preparing blank microspheres without inclusion compound and triamcinolone acetonide drug comprises the following steps:

(1) dissolving polylactic acid-co-glycolic acid (PLGA) in dichloromethane to prepare a PLGA dichloromethane solution as dispersed phase A solution, wherein the PLGA concentration is 0.25 g/ml; the molecular weight of PLGA is 30 KDa, and the weight ratio of glycolide in PLGA is 25% and the weight ratio of lactide is 75%. After dissolution, the dispersed phase A solution is prepared;

(3) The dispersed phase A solution was stirred by a homogenizer at a speed of 35000 rpm to form a stable and uniform dispersed phase;

(4) dissolving 900 g of polyvinyl alcohol and 900 g of sodium chloride in 30 L of injection water, wherein the molecular weight of polyvinyl alcohol is 30 KDa, as the continuous phase, wherein the concentration of polyvinyl alcohol is 3%, and the concentration of sodium chloride is 3%;

(5) Shearing step: Prepare microspheres by mechanical stirring, transfer the prepared dispersed phase to a dropping funnel, start stirring, control the speed to 300 rpm, drip the dispersed phase into the continuous phase, control the temperature of the continuous phase to 25°C, and use a stirring paddle to shear and form droplets; after the dispersed phase is finished dripping, continue stirring for 30 min, reduce the stirring speed to 180 rpm, and the organic solvent in the emulsion gradually evaporates;

(6) Turn off stirring, allow the microspheres to settle naturally, collect by suction filtration, wash three times with pure water, and collect;

(7) The collected microspheres were transferred to a vial and placed in a freeze dryer for freeze drying to obtain a product (sample 0).

Comparative Example 2 (Preparation of Sample 1)

The other parts are the same as Comparative Example 1, except that 4.5 g of triamcinolone acetonide is added.

FIG1 is a schematic diagram showing the structure of triamcinolone acetonide in an embodiment of the present invention; triamcinolone acetonide includes hydrophilic groups and lipophilic groups.

Example 1 (Preparation of Sample 2)

FIG2 is a schematic diagram of the structure of the inclusion agent in an embodiment of the present invention; the inclusion agent selected in the present invention is a cyclic natural oligosaccharide composed of 6 (α), 7 (β) or 8 (γ) glucose monomers connected by α-1,4-glycosidic bonds, and the molecular shape is like a donut that is wide at the top and narrow at the bottom, with a hydrophilic outer surface (hydrophilic shell) and a lipophilic central cavity (hydrophobic cavity);

Among the three hydroxyl groups on the outer surface of the inclusion agent, positions 2 and 3 contain secondary hydroxyl groups, and position 6 contains primary hydroxyl groups. Primary hydroxyl groups are relatively free to rotate, and the hydroxyl group at position 6 is the most basic, the hydroxyl group at position 2 is the most acidic, and the hydroxyl group at position 3 is the most difficult to approach. Therefore, electrophilic reagents will attack the hydroxyl group at position 6.

The following is a detailed description of the method for preparing triamcinolone acetonide sustained-release microspheres containing a microreservoir structure by mechanical stirring, wherein the steps are as follows:

(1) dissolving PLGA in dichloromethane to prepare a PLGA dichloromethane solution as dispersed phase A solution, wherein the PLGA concentration is 0.25 g/ml; the molecular weight of PLGA is 30 KDa, and the weight ratio of glycolide in PLGA is 25% and the weight ratio of lactide is 75%;

(2) Dissolve 4.5 g of triamcinolone acetonide in 10 ml of N-methylpyrrolidone at a concentration of 0.45 g/ml; after ultrasonic dissolution, add an inclusion agent (a cyclic natural oligosaccharide composed of 6 glucose monomers (α) connected by α-1,4-glycosidic bonds), the mass of the inclusion agent is 0.4 g, and the dissolved solution is used as dispersed phase B solution;

(3) The dispersed phase B solution was transferred into the dispersed phase A solution and stirred by a homogenizer at a speed of 35000 rpm to form a stable and uniform dispersed phase;

(4) dissolving 900 g of polyvinyl alcohol and 900 g of sodium chloride in 30 L of injection water, wherein the molecular weight of the polyvinyl alcohol is 30 KDa to 50 KDa, as the continuous phase, wherein the concentration of the polyvinyl alcohol is 3%, and the concentration of the sodium chloride is 3%;

(5) Shearing step: Prepare microspheres by mechanical stirring, transfer the prepared dispersed phase to a dropping funnel, start stirring, control the speed to 300 rpm, drip the dispersed phase into the continuous phase, control the temperature of the continuous phase to 25°C, and use a stirring paddle to shear to form droplets; continue stirring for 30 min after the dispersed phase is finished dripping, reduce the stirring speed to 180 rpm, and gradually evaporate the organic solvent in the emulsion;

(6) Turn off stirring, allow the microspheres to settle naturally, collect by suction filtration, wash three times with pure water, and collect;

(7) The collected microspheres were transferred to a vial and placed in a freeze dryer for freeze drying to obtain a product (sample 2).

Example 2 (Preparation of Sample 3)

The rest is the same as Example 1, except that: the inclusion agent in step (2) is a cyclic natural oligosaccharide composed of 7 glucose monomers (β) connected by α-1,4-glycosidic bonds.

Example 3 (Preparation of Sample 4)

The rest is the same as Example 1, except that: the inclusion agent in step (2) is a cyclic natural oligosaccharide composed of 8 glucose monomers (γ) connected by α-1,4-glycosidic bonds.

The following Examples 4 and 5 are triamcinolone acetonide drug sustained-release microspheres prepared by modifying the inclusion agent. The inclusion agent is modified by diacyl chloride, and the hydroxyl groups at the bottom of the inclusion agent are connected to form a bowl-shaped structure; as shown in FIG3 , it is a schematic diagram of the structure of the modified inclusion agent-immobilized drug microreservoir in the present invention; the drug and the inclusion agent are combined through the affinity of the surface group to form a microreservoir, which increases the solubility of the drug in the polymer and greatly improves the drug loading; at the same time, the addition of the inclusion agent realizes the controllable release of the drug;

In FIG3 , TA represents the drug triamcinolone acetonide, and modified CD represents the modified inclusion agent, wherein the drug includes a hydrophilic group and a hydrophobic group. FIG3 is a schematic diagram showing the effect of the drug and the inclusion agent, and the fixed inclusion agent of the drug;

Figure 4 is a schematic diagram of the structure of the inclusion agent modification in the embodiment of the present invention. Figure 5 is a reaction formula of the inclusion agent modification in the embodiment of the present invention; the hydroxyl groups on the outer surface of the inclusion agent are reduced by modification, which increases the lipophilicity of the inclusion agent, increases the solubility of the micro-reservoir in the organic phase, reduces precipitation, and further increases the concentration of the drug in the organic phase; in addition, the structure of the inclusion agent changes to a bowl shape, increases the combination of the drug and the inclusion agent, makes it less likely to leak out, improves the stability of the micro-reservoir, increases the drug loading capacity, and prolongs the drug release time;

FIG4 shows the modification process of the inclusion agent. After the modification, a network is formed at the bottom of the inclusion agent, thereby fixing the drug.

Example 4 (Preparation of Sample 5)

The method for preparing triamcinolone acetonide sustained-release microspheres containing an inclusion compound comprises the following steps:

(1) PLGA is dissolved in dichloromethane to prepare a dichloromethane solution of PLGA as dispersed phase A solution, and the PLGA concentration is 0.25 g/ml; the molecular weight of PLGA is 30 KDa, and the weight ratio of glycolide in PLGA is 25%, and the weight ratio of lactide in PLGA is 75%. After dissolution, the dispersed phase A solution is prepared;

(2) Preparation of succinate-β-inclusion agent

In a 3000mL reaction bottle, 50g of β-inclusion agent (β-CD, 1eq.) and tetrahydrofuran (1500mL, 30vol.) were added respectively, stirred and dissolved at room temperature, and triethylamine (31.2g, 7eq.) was added, the reaction solution was cooled to -5-5°C, succinyl chloride (20.5g, 3eq.) was slowly added dropwise, and after the addition was completed, the temperature was maintained at -5-5°C, and the reaction was carried out for 3h, and then the temperature was raised to 10-20°C and the reaction was carried out for 5h. After the reaction was completed, the mixture was concentrated and the solvent was removed to obtain a crude product, which was slurried and filtered with water to obtain a filter cake, which was then purified with ethanol and ethyl acetate, respectively, and dried to obtain 28.2g of succinate-β-inclusion agent with a yield of 46%;

Product Confirmation:

The carbonyl (C=O) peak at wave number 1730 cm ^-1 and the (CO) peak at 1138 cm ^-1 proved the presence of ester groups and proved the introduction of ester groups;

(3) dissolving 4.5 g of triamcinolone acetonide in 10 mL of N-methylpyrrolidone to a concentration of 0.45 g/ml; after ultrasonic dissolution, adding the succinate-β-inclusion agent prepared in step (2) in an amount of 0.4 g, and dissolving to form the dispersed phase B solution;

(4) Shearing step: Prepare microspheres by mechanical stirring, transfer the prepared dispersed phase to a dropping funnel, start stirring, control the speed to 300 rpm, drip the dispersed phase into the continuous phase, control the temperature of the continuous phase to 25°C, and use a stirring paddle to shear to form droplets; continue stirring for 30 min after the dispersed phase is finished dripping, reduce the stirring speed to 180 rpm, and gradually evaporate the organic solvent in the emulsion;

(5) Turn off stirring, allow the microspheres to settle naturally, collect by suction filtration, wash three times with pure water, and collect;

(6) The collected microspheres are transferred to a vial and placed in a freeze dryer for freeze drying to obtain a product (sample 5).

Example 5 (Preparation of Sample 6)

The rest is the same as Example 4, except that:

Step (2) is as follows:

Preparation of adipate-γ-inclusion agent

In a 3000mL reaction bottle, 40g of γ-clathrate (γ-CD, 1eq.) and tetrahydrofuran (1200mL, 30vol.) were added respectively, stirred to dissolve, and then triethylamine (28.2g, 9eq.) was added. The reaction solution was cooled to -5-5°C, and adipic acid chloride (22.7g, 4eq.) was slowly added dropwise. After the addition was completed, the temperature was maintained at 0-10°C, and the reaction was continued for 3h. The temperature was then raised to 10-20°C and the reaction was continued for 6h. After the reaction was completed, the mixture was concentrated and the solvent was removed to obtain a crude product. The mixture was slurried and filtered with water to obtain a filter cake, which was then purified with ethanol and ethyl acetate and dried to obtain 26.0g of adipic acid ester-γ-clathrate with a yield of 48%.

Product Confirmation:

The carbonyl (C=O) peak at wave number 1730 cm ^-1 and the (CO) peak at 1138 cm ^-1 proved the presence of ester groups and proved the introduction of ester groups;

(3) dissolving triamcinolone acetonide in N-methylpyrrolidone at a concentration of 0.45 g/ml; after ultrasonic dissolution, adding the hexamethylenediamine-γ-inclusion agent prepared in step (2) in an amount of 0.4 g, and dissolving to form the dispersed phase B solution;

The degradation process of PLGA, a high molecular weight polymer, can be used for sustained targeted drug release without the need for surgery. The overall physical properties of the polymer-drug mechanism can also be adjusted by adjusting parameters such as the PLGA molecular weight, the ratio of lactide to glycolide, and the drug concentration to achieve the desired dose and release interval. During the preparation of triamcinolone acetonide microspheres, the inventors optimized the PLGA molecular weight and structure to control drug release; in addition, during the preparation of microspheres, by adding inclusion agents, the drug solubility was increased, the drug loading was increased, and a new dense drug reservoir microsphere structure with sustained release function was prepared.

Figure 6 is a schematic diagram of the structure of PLGA microspheres containing microreservoirs in an embodiment of the present invention. The microspheres have PLGA as the skeleton with holes on the surface, and the drugs are coated in the microspheres, and the drugs are slowly released through the holes and the degradation of PLGA;

Sample 2: 75% DL-lactide/25% glycolide copolymer, 6 glucose ring inclusion agent (α);

Sample 3: 75% DL-lactide/25% glycolide copolymer, 7 glucose ring inclusion agents (β);

Sample 4: 75% DL-lactide/25% glycolide copolymer, 8 glucose ring inclusion agents (γ);

The microspheres prepared in the above examples were observed under a scanning electron microscope;

1. Electron microscope: Quanta400 FEG; power supply voltage: 220±20V, frequency 50Hz±0.5Hz; indoor temperature: 20℃±5℃; indoor humidity ≤75%; environmental alternating interference magnetic field intensity ≤5*10-6T; foundation amplitude ≤5um

2. Particle size: optical microscopy

Drug loading: Drug loading refers to the weight percentage of the drug contained in the microparticle preparation, i.e.

Drug loading = drug weight contained in microparticle preparation / total weight of microparticle preparation * 100%

Encapsulation efficiency = (encapsulated drug amount in microparticle preparation/total encapsulated and unencapsulated drug amount in microparticle preparation) * 100%

=(1-(unencapsulated drug amount in liquid medium/total encapsulated and unencapsulated drug amount in microparticle preparation)*100%)

3. There are obvious differences between the microspheres with and without the inclusion agent. In Figure 7, a is a microsphere without the inclusion agent and the drug (sample 0), and the surface of the microsphere is smooth; b is a microsphere with a 4.5g addition amount of triamcinolone acetonide and no inclusion agent. In the absence of the inclusion agent, the drug is poorly dissolved and cannot be encapsulated into the microsphere, and precipitates on the surface of the microsphere. The scanning image shows that there are rough particles on the surface of the microsphere, which are the precipitated drugs, thereby reducing the drug loading of the microsphere; c is a microsphere with a fixed addition amount of triamcinolone acetonide of 4.5g and a small amount of inclusion agent of 0.4g. Through the inclusion action, the drug is stored in the inclusion agent, which improves the solubility of the drug. It can also be seen from the scanning electron microscope that the surface of c is smoother and has a small amount of grooves compared with b microspheres, thereby proving the concept of micro-reservoir of drugs encapsulated by inclusion agents provided in the embodiments of the present invention;

FIG8 shows electron microscope images of microspheres of sample 0 to sample 5 from left to right;

The drug loading of the microspheres prepared in the above comparative example and Examples 1-3 was measured by HPLC method. The test results are shown in Table 1:

Table 1

name Particle size (μm) Drug loading% Encapsulation rate% Sample 0 20-45 0 0 Sample 1 20-45 10 85 Sample 2 20-45 20.3 95 Sample 3 20-45 25 99 Sample 4 20-45 18 94

In Table 1, sample 1 is the drug loading and microsphere related data of the sustained-release microspheres prepared without inclusion agents; samples 2-4 are the particle size, drug loading, and encapsulation efficiency of the microspheres prepared with inclusion agents of different structures; it can be seen from the data that without adding inclusion agents, the drug loading is low; and after adding inclusion agents of different structures, the drug loading of the microspheres is improved to varying degrees, among which, the drug loading of the microspheres of sample 3 is the highest, and the micro-reservoir structure formed by the affinity of the inclusion agent and the drug significantly improves the solubility of the drug and further increases the drug loading; after the inclusion agent is added, the drug encapsulation efficiency is significantly increased, among which, the encapsulation efficiency of sample 3 is the largest;

As shown in FIG9 , it is an electron microscope image of the sustained-release microspheres containing the inclusion agent of the present invention (Example 4). After 40 times magnification, the micro-reservoir structure of the microspheres can be seen.

Example 4-5: Preparation of triamcinolone acetonide sustained-release microspheres containing modified inclusion agent

Sample 5: 75% DL-lactide/25% glycolide copolymer, succinate-β-inclusion agent;

Sample 6: 75% DL-lactide/25% glycolide copolymer, adipate-γ-inclusion agent.

The microspheres prepared in Examples 4 and 5 were observed under a scanning electron microscope, and the results are shown in FIG8 : It can be seen from the figure that the microspheres have good appearance and morphology, and a narrow particle size distribution.

The drug loading of the microspheres prepared in Examples 4 and 5 was measured by HPLC. The test results are shown in Table 2:

Table 2

name Particle size (μm) Drug loading% Encapsulation rate% Sample 1 20-45 10 85 Sample 3 20-45 25 99 Sample 5 20-45 31.5 100 Sample 6 20-45 29.8 100

In Table 2, Sample 1 is the drug loading and microsphere related data of the sustained-release microspheres prepared without an inclusion agent; Sample 3 is the drug loading and microsphere related data of the microreservoir microspheres made of drugs encapsulated by a β-structure inclusion agent; Samples 5 and 6 are the drug loading and microsphere related data of the microreservoir microspheres made of drugs encapsulated by modified inclusion agents.

4. The content of the inclusion agent, i.e., the inclusion agent % = the weight of the inclusion agent contained in the microparticle preparation/the total weight of the microparticle preparation*100%, and the test method is as follows:

Liquid chromatography, chromatographic column C18 (5 μm×250 mm×4.6 mm), differential refractometer, column temperature: 30°C, mobile phase: acetonitrile: water = 70:30, flow rate: 1.1 mL/min.

Take a sample, weigh it accurately, dissolve it with a certain amount of dimethyl sulfoxide to make up the volume, take 10 μL, and test it according to the above method.

Table 3

name Particle size (μm) Drug loading% Encapsulation rate% Inclusion agent% Sample 0 20-45 0 0 0 Sample 1 20-45 10 85 0 Sample 2 20-45 20.3 95 1.6 Sample 3 20-45 25 99 1.8 Sample 4 20-45 18 94 1.5 Sample 5 20-45 31.5 100 2.8 Sample 6 20-45 29.8 100 2.6

From the data, it can be seen that without adding inclusion agents, no microreservoir structure is produced, resulting in low drug loading and low encapsulation efficiency; after adding inclusion agents, the drug loading of microspheres is increased, but the drug loading does not reach the ideal target value; after the inclusion agent is modified, microspheres are prepared, and it can be seen from the data of samples 5 and 6 that the drug loading is significantly increased, and after the inclusion agent is modified, a bowl-shaped structure is formed, which successfully encapsulates the drug, improves drug stability, increases drug inclusion concentration, and increases drug loading and encapsulation efficiency.

The solubility order of the unmodified inclusion agent in water is γ-cyclodextrin>α-cyclodextrin>β-cyclodextrin. Since the inclusion agent has a certain solubility in water, part of the inclusion agent is dissolved in water and lost during the preparation of drug-loaded microspheres, which affects the increase in the drug loading of the microspheres. After the inclusion agent is modified, the lipophilicity of the outer surface is increased, and its solubility in water is reduced, making it more soluble in the organic phase, reducing the loss during the preparation process. It can be seen from the inclusion agent content in Table 3 that the inclusion agent content in the microspheres prepared using the modified inclusion agent is significantly increased. At the same time, the drug loading and encapsulation efficiency of the microspheres are also improved, further proving that the modified inclusion agent forms a drug microreservoir in the microspheres.

Example 6: A method for preparing triamcinolone acetonide sustained-release microspheres by membrane emulsification technology, the steps are as follows:

(1) dissolving PLGA in dichloromethane to prepare a PLGA dichloromethane solution as dispersed phase A solution, wherein the PLGA concentration is 0.25 g/ml; the molecular weight of PLGA is 30 KDa, and the weight ratio of glycolide in PLGA is 25% and the weight ratio of lactide is 75%. After dissolution, the dispersed phase A solution is prepared;

(2) 4.5 g of triamcinolone acetonide was dissolved in 10 ml of N-methylpyrrolidone to a concentration of 0.45 g/ml; after ultrasonic dissolution, the succinate-β-inclusion agent prepared in Example 4 was added, the mass of the inclusion agent was 0.4 g, and the solution was used as dispersed phase B solution after dissolution.

(3) The dispersed phase B solution was transferred into the dispersed phase A solution and stirred by a homogenizer at a speed of 3500 rpm to form a stable and uniform dispersed phase;

(4) dissolving 900 g of polyvinyl alcohol and 900 g of sodium chloride in 30 L of injection water, wherein the molecular weight of polyvinyl alcohol is 30 KDa, as the continuous phase, wherein the concentration of polyvinyl alcohol is 3%, and the concentration of sodium chloride is 3%;

(5) After the SPG membrane is cleaned and leak-checked, the device is assembled and an SPG membrane with a pore size of 15 μm is selected;

(6) The dispersed phase is transferred to the liquid storage tank of the SPG membrane emulsifier, and is pressurized by _N2 to form droplets through the membrane pores on the SPG membrane, and then enters the low-speed stirred continuous phase PVA aqueous solution to form microsphere droplets;

(7) Stirring at low speed (180-230 rpm) at low temperature (6-15°C) causes the organic solvent in the droplets to gradually decrease, and the PLGA skeleton of the drug gradually precipitates and solidifies into microspheres;

(8) Turn off stirring, allow the microspheres to settle naturally, collect by suction, wash three times with pure water, and collect;

(9) The collected microspheres are transferred into a vial and placed in a freeze dryer for freeze drying.

Example 7: A method for preparing triamcinolone acetonide sustained-release microspheres by microfluidic technology, the steps are as follows:

(1) dissolving PLGA in dichloromethane to prepare a PLGA dichloromethane solution as dispersed phase A solution, wherein the PLGA concentration is 0.25 g/ml; the PLGA molecular weight is 30 KDa, wherein the weight ratio of glycolide is 25%, and the weight ratio of DL-lactide is 75%, and the solution is used as dispersed phase A solution after dissolution;

(2) Dissolve 4.5 g of triamcinolone acetonide in 10 ml of N-methylpyrrolidone to a concentration of 0.45 g/ml; after ultrasonic dissolution, add 0.4 g of the succinate-β-inclusion agent prepared in Example 4, and use the solution as dispersed phase B after dissolution;

(3) The dispersed phase B solution was transferred into the dispersed phase A solution and stirred by a homogenizer at a speed of 3500 rpm to form a stable and uniform dispersed phase;

(4) dissolving 900 g of polyvinyl alcohol and 900 g of sodium chloride in 30 L of injection water, wherein the molecular weight of polyvinyl alcohol is 30 KDa, as the continuous phase, wherein the concentration of polyvinyl alcohol is 3%, and the concentration of sodium chloride is 3%;

(5) Select a cross-shaped channel microfluidic chip with an inner diameter of 40 μm, clean it, and check for leaks before assembling the device; use a plunger pump to pressurize and control the flow rate of the continuous phase to 30 ml/min and the flow rate of the dispersed phase to 0.5 ml/min, and form droplets through the channel and enter the low-speed stirred continuous phase PVA aqueous solution;

(6) Stirring at low speed (180-230 rpm) at low temperature (6-15°C) causes the organic solvent in the droplets to gradually decrease, and the PLGA skeleton of the drug gradually precipitates and solidifies into microspheres;

(7) Turn off stirring, allow the microspheres to settle naturally, collect by suction filtration, wash three times with pure water, and collect;

(8) The collected microspheres are transferred into a vial and placed in a freeze dryer for freeze drying.

The drug loading and encapsulation efficiency of the microspheres prepared by mechanical stirring, membrane emulsification and microfluidics are shown in Table 4:

Table 4

It can be seen from Table 4 that the drug loading and encapsulation of microspheres prepared by different processes are not much different, and all three processes can produce triamcinolone acetonide sustained-release microspheres.

Application Example 1: Sustained drug release from microspheres containing multiple microreservoirs under physiological conditions

put

The sustained release of the microsphere drugs in Comparative Example 1 and Examples 1-7 was detected. 20 mg of the triamcinolone acetonide sustained-release microspheres in Comparative Example 1 and Examples 1-7 were suspended in 100 ml of phosphate buffered saline containing 0.5% sodium dodecyl sulfate maintained at 37° C. 0.5 ml of the medium was removed every day and replaced with an equal amount of fresh medium to maintain a constant volume. The in vitro release was determined by HPLC analysis. HPLC analysis was performed using C18 and a mobile phase with a flow rate of 1 ml/min to determine the microspheres.

The drug elution of the spheres within 7 days, the resulting drug release is shown in Table 5 below:

Table 5

As can be seen from Table 5, without the inclusion agent, the sustained-release microspheres prepared had a fast drug release rate, with a cumulative release of more than 50% within seven days; microspheres 2-6 with the inclusion agent added showed different degrees of extended drug release time, and sample 4 was a sustained-release microsphere prepared with a modified inclusion agent, with a cumulative drug release of only 10% within 7 days. The results verified the sustained release of drugs from the microreservoir, and the drug release cycle of the microreservoir in the knee joint can be selected.

In the triamcinolone acetonide sustained-release microspheres containing a microreservoir structure provided by an embodiment of the present invention, the microreservoir is composed of an inclusion agent and a drug. After the drug is dissolved, it is combined with the inclusion agent through affinity, and multiple microreservoirs are dispersed in a degradable polymer to form a stable dispersed phase; droplets are prepared by mechanical stirring, membrane emulsification and microfluidics, and then, the solvent is removed by solvent evaporation to form microspheres containing a drug-loaded microreservoir structure. The present invention develops a drug-loaded microsphere with a novel structure by constructing a drug microreservoir in the microsphere, which can increase the drug loading of the microsphere, while achieving controllable drug release, prolonging the drug release time, and achieving a significant effect of long-term action.

The drug sustained-release microspheres containing triamcinolone acetonide microreservoir structure and the preparation method provided by the embodiment of the present invention have the following advantages:

1) Increase drug loading: In the preparation of drug-loaded microspheres in other patents, the dispersed phase liquid is prepared by dissolving the PLGA copolymer in a solvent, and then suspending the insoluble triamcinolone acetonide micronized solid particles in the organic phase liquid; this makes the triamcinolone acetonide particles difficult to be coated by PLGA and easy to precipitate during the ball-forming process. The present invention adds a polar solvent N-methylpyrrolidone to dissolve the drug, then adds a complexing agent to produce a complexing effect with the drug, and then adds it to a dichloromethane solution containing PLGA, thereby improving the solubility, permeability, dispersion uniformity and physicochemical stability of the drug in dichloromethane, thereby increasing the concentration of the triamcinolone acetonide drug in the PLGA skeleton and further increasing the drug loading.

2) Improve the yield: When using the traditional emulsification method to prepare microspheres, triamcinolone acetonide is insoluble in dichloromethane and easily precipitates, resulting in a low yield of microspheres. The present invention improves the utilization of the drug in the preparation process by adding a complexing agent and N-methylpyrrolidone to triamcinolone acetonide, thereby improving the product yield and reducing the loss.

3) The drug sustained-release microspheres containing triamcinolone acetonide microreservoir structure provided in the embodiment of the present invention achieve controllable drug sustained-release.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims (10)

1. A preparation method of triamcinolone acetonide drug sustained release microsphere containing a micro-reservoir structure is characterized by comprising the following steps:

(1) PLGA is dissolved in methylene dichloride to prepare PLGA methylene dichloride solution as disperse phase A solution, and the concentration of PLGA is 0.01g/ml to 0.5g/ml; wherein, the weight ratio of glycolide is 10-30%, the weight ratio of lactide is 70-90%, and the glycolide is dissolved to be used as a disperse phase A solution;

(2) Dissolving triamcinolone acetonide in N-methyl pyrrolidone at a concentration of 0.4 g/ml-0.5 g/ml; after ultrasonic dissolution, adding an inclusion agent, and dissolving to obtain a disperse phase B solution;

(3) Transferring the solution of the disperse phase B into the solution of the disperse phase A, and stirring by a homogenizer with the rotating speed of 3000-5000 rpm to form a stable and uniform disperse phase;

(4) Dissolving polyvinyl alcohol and sodium chloride into injection water to be used as a continuous phase, wherein the concentration of the polyvinyl alcohol is 1-5% and the concentration of the sodium chloride is 1-5%;

(5) And (3) cutting: and (3) cutting: the prepared disperse phase is contacted with the continuous phase in a pressure or shearing mode through a mechanical stirring, membrane emulsification or microfluidic technology to form liquid drops; stirring at low speed of 180-230 rpm at low temperature of 6-15 ℃, gradually reducing organic solvent in emulsion drops, gradually precipitating PLGA skeleton of the drug, solidifying and forming microspheres; (6) Closing stirring to naturally settle the microspheres, filtering, collecting, washing with pure water, and collecting;

(7) Transferring the collected microspheres into a container, and freeze-drying to obtain the product.

2. The method for preparing the triamcinolone acetonide drug sustained release microsphere containing the micro-reservoir structure according to claim 1, which is characterized in that:

in the step (1), the molecular weight of the PLGA is between 30KDa and 100KDa.

3. The method for preparing the triamcinolone acetonide drug sustained release microsphere containing the micro-reservoir structure according to claim 1, which is characterized in that:

in the step (2), the inclusion agent is a cyclic natural oligosaccharide formed by connecting 6 (alpha), 7 (beta) or 8 (gamma) glucose monomers through alpha-1, 4-glycosidic bonds; or the inclusion agent is a cyclic natural oligosaccharide formed by connecting a functionalized glucose ring 1) or a functionalized glucose ring 2) through alpha-1, 4-glycosidic bonds.

4. The method for preparing the triamcinolone acetonide drug sustained release microsphere containing the micro-reservoir structure according to claim 1, which is characterized in that:

in the step (2), the inclusion agent is succinate-beta-inclusion agent or adipate-gamma-inclusion agent.

5. The method for preparing the triamcinolone acetonide drug sustained release microsphere containing the micro-reservoir structure according to claim 1, which is characterized in that:

In the step (2), the addition amount of the inclusion agent in the solution of the disperse phase B is 0.25-0.5 g.

6. The method for preparing the triamcinolone acetonide drug sustained release microsphere containing the micro-reservoir structure according to claim 1, which is characterized in that:

In the step (4), the molecular weight of the polyvinyl alcohol is 30 KDa-50 KDa; in the step (5), the mechanical stirring speed is 300-500rpm; the membrane holes of the membrane emulsification process are 10-20 mu m; the diameter of the cross-shaped channel of the microfluidic chip is 30-50 mu m; in the step (6), the weight ratio of the disperse phase to the continuous phase is 1:30-1:50.

7. A triamcinolone acetonide drug sustained release microsphere containing a micro-reservoir structure, prepared by the preparation method of any one of claims 1 to 6, characterized in that the micro-reservoir structure contains an inclusion agent; wherein the drug loading rate is more than 25%; the encapsulation efficiency is more than 95%.

8. The triamcinolone acetonide drug delivery microsphere containing micro-depot structure of claim 7, wherein the microsphere comprises:

The micro-reservoir structures are densely arranged; the inclusion agent is a cyclic natural oligosaccharide formed by connecting 6 (alpha), 7 (beta) or 8 (gamma) glucose monomers through alpha-1, 4-glycosidic bonds; or the inclusion agent is a cyclic natural oligosaccharide formed by connecting a functionalized glucose ring 1) or a functionalized glucose ring 2) through alpha-1, 4-glycosidic bonds.

9. The triamcinolone acetonide drug delivery microsphere containing micro-depot structure of claim 7, wherein the microsphere comprises:

The inclusion agent is succinate-beta-inclusion agent or adipate-gamma-inclusion agent.

10. A sustained release formulation of triamcinolone acetonide comprising a sustained release microsphere of triamcinolone acetonide having a micro-reservoir structure according to any one of claims 7 to 9.