Porous core-shell microsphere with temperature sensitivity and acid responsiveness and preparation method thereof
By preparing thermosensitive and acid-responsive porous core-shell microspheres, the problems of poor drug stability and permeability were solved, and efficient drug delivery and therapeutic effects in the treatment of COPD were achieved.
Patent Information
- Application Number
- CN202510892891.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-09-16
AI Technical Summary
Existing hormone drugs face the problems of poor drug stability and penetrability when treating chronic obstructive pulmonary disease (COPD), making it difficult to effectively deliver them to the site of bronchial inflammation, resulting in poor treatment effects.
Porous core-shell microspheres with thermosensitivity and acid responsiveness were prepared. The core of spherical gel particles was prepared by emulsification, and the shell material was wrapped by uniaxial electrospraying. The core was acid-responsive and the shell was thermosensitive. PLGA and PLGA-PEG composite materials were combined to promote drug release and penetration of the mucus barrier at body temperature.
The drug is quickly released at body temperature and penetrates the mucus barrier. The inner core quickly disintegrates and releases the drug under inflammatory conditions, which improves the drug delivery efficiency and therapeutic effect and relieves COPD symptoms.
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Figure CN120643528A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, in particular to porous core-shell microspheres with temperature sensitivity and acid responsiveness and a preparation method thereof. Background Art
[0002] Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by airflow limitation that is not fully reversible and is progressive. Its occurrence is related to the lungs' abnormal inflammatory response to harmful gases or particles. Beclomethasone dipropionate is a synthetic, potent topical glucocorticoid with anti-inflammatory and anti-allergic effects. Beclomethasone dipropionate is usually used in the form of an aerosol or cream and is commonly used to treat COPD. Ambroxol hydrochloride is a commonly used expectorant that can also increase the speed of ciliary beats in the airway ciliated epithelium, promote the discharge of sputum from the airways, and increase the amount of airway mucus secretion, making it easier to expel small amounts of sputum. These effects help improve the respiratory condition and quality of life of COPD patients.
[0003] However, the therapeutic effects of existing drugs for COPD are still unsatisfactory. The main reasons are: the delivery of hormone drugs to the site of bronchial inflammation faces difficulties in drug stability and drug penetration. In terms of drug stability, steroid hormones are easily hydrolyzed, oxidized or decomposed due to the destruction of ester bonds, ether bonds or non-covalent bonds between molecules in the warm, humid and enzyme-rich respiratory environment, resulting in structural destruction of hormones and loss of biological activity; in terms of drug penetration, the inherent viscosity resistance, adhesion and dragging effects and synergistic clearance mechanisms of the respiratory mucus layer hinder the penetration of hormones, making it difficult for them to reach the site of bronchial inflammation. In addition, in long-term maintenance treatment, differences in drug absorption efficiency in the respiratory tract, short duration of drug efficacy, immune system suppression and drug dependence also limit the anti-inflammatory effects of hormone drugs. Therefore, how to overcome the challenges of drug stability and penetration and effectively deliver anti-inflammatory drugs to the site of inflammation is the key to improving the treatment effect of COPD. Summary of the Invention
[0004] In order to solve the problems of poor drug stability and poor permeability faced by existing hormone drugs during the delivery process, the present invention provides a porous core-shell microsphere with temperature sensitivity and acid responsiveness and a preparation method thereof.
[0005] The porous core-shell microspheres with temperature sensitivity and acid responsiveness provided by the present invention are prepared as follows: S1. Hyaluronic acid and 3,3'-dithiobis(propionylhydrazide) were sequentially dissolved in 2-(N-morpholino)ethanesulfonic acid solution, followed by the addition of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride. The mixture was reacted at room temperature for 24 hours. The solution was dialyzed with deionized water and then freeze-dried to obtain hydrazide-modified hyaluronic acid, referred to as HA-TPH.
[0006] S2. Take another portion of hyaluronic acid and dissolve it in water. Add sodium periodate and stir the mixture at room temperature in the dark. Then dialyze and freeze-dry to obtain oxidatively modified hyaluronic acid, referred to as HA-CHO.
[0007] S3. Dissolve HA-TPH in water, and then add beclomethasone dipropionate to obtain solution A. Similarly, dissolve HA-CHO in water, and then add beclomethasone dipropionate to obtain solution B.
[0008] S4. Gel particles are prepared by an emulsification method: Solution A and Solution B are mixed and then added dropwise to vegetable oil. Stirring is continued to emulsify the mixture to obtain a suspension. Dichloromethane is added to the suspension to remove the vegetable oil, and the suspension is then freeze-dried to obtain spherical gel particles. The gel particles have a particle size range of 100-2000 nm.
[0009] S5. Dissolving the shell material in dichloromethane, and adding ambroxol hydrochloride and the gel particles prepared in step S4 to form an electrospray suspension. The shell material is a mixture of polylactic acid-co-glycolic acid (PLGA) and polylactic acid-co-glycolic acid-polyethylene glycol (PLGA-PEG).
[0010] S6. Using electrospray suspension as raw material, a uniaxial electrospray method was used to prepare porous core-shell structured microspheres in which the shell material wrapped the gel particles.
[0011] The device used in the uniaxial electrospray method comprises a syringe pump, a high-voltage power supply, a conductive needle and a collection tank; the syringe pump is connected to the needle through a liquid inlet tube, the needle is placed vertically with its liquid outlet facing downward, and a collection tank is provided below the needle for receiving droplets dripping from the needle; the collection tank is filled with a polyvinyl alcohol aqueous solution; the output end of the high-voltage power supply is connected to the needle; the electrospray suspension is continuously injected into the needle through the syringe pump, and when a Taylor cone is observed to form at the liquid outlet of the needle, the high-voltage power supply is turned on to apply electrostatic high voltage to the needle for electrospraying, and after the electrospray state is stable for at least 5 minutes, the dripping droplets are collected in the collection tank, and stirring is continued until the dichloromethane is completely volatilized to obtain core-shell structured microspheres.
[0012] During the entire preparation process, the ambient temperature and operating temperature were controlled to be no higher than 30°C.
[0013] Preferably, the inner diameter of the needle outlet is 0.4-0.8 mm, and the distance from the needle outlet to the liquid surface in the collection tank is 15-25 cm. The voltage applied to the needle by the high-voltage power supply is 12-18 kV, and the optimal voltage is 15 kV.
[0014] Preferably, the mass percentage concentration of the polyvinyl alcohol aqueous solution in the collection pool is 2-4%, and optimally 2%.
[0015] Preferably, in the electrospray suspension, the mass percentage concentration of the shell material is 5-20%, optimally 10%; the mass ratio of the gel particles to the shell material is 1:4; and the mass ratio of ambroxol hydrochloride to the shell material is 1:2.
[0016] Preferably, in the shell material, the mass proportion of PLGA-PEG is 20-80%, and the rest is PLGA, totaling 100%.
[0017] Preferably, the concentrations of HA-TPH and HA-CHO in solution A and solution B are the same, both being 1.5% by mass.
[0018] Compared with the prior art, the present invention is beneficial in that: The present invention prepares porous core-shell microspheres that mimic pollen for inhalation administration. Spherical gel particles prepared by an emulsification method serve as the core, which is then encapsulated in a shell material via a uniaxial electrospraying method to produce porous core-shell microspheres. The shell of the microspheres is thermosensitive, and the core is acid-responsive. The shell material is a thermosensitive composite of PLGA and PLGA-PEG. Under normal conditions, it forms a gel, but upon entry into the human body at a body temperature of 37°C, it transforms into a sol. The amphiphilic nature of PLGA-PEG promotes contact between the shell and mucus, facilitating the release of the mucolytic agent ambroxol hydrochloride and diluting sputum. This not only promotes sputum excretion and alleviates cough symptoms, but also penetrates the mucus barrier to expose the core, allowing it to reach lung macrophages. The core matrix is hydrazide- and aldehyde-modified hyaluronic acid, which exhibits good biocompatibility and macrophage targeting. When the inner core diameter is about 3 microns, the macrophage phagocytosis efficiency is the highest. After being phagocytosed, the inner core responds to the GSH and slightly acidic environment in the phagolysosome (the pH value of the internal environment of macrophages under inflammatory conditions is about 5.0-5.2), breaking the glycosidic bonds in the hyaluronic acid and releasing the drug beclomethasone dipropionate to stimulate the phenotypic transformation of macrophages, so that the cell function in the airway returns to normal, the airway returns to homeostasis, and the inflammation subsides, thereby achieving the purpose of treating COPD.
[0019] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is a graph showing the results of a study on the gelation time of hyaluronic acid at different concentrations.
[0021] Figure 2 This is the swelling test result of the hydrogel formed after cross-linking of HA-TPH solution and HA-CHO solution.
[0022] Figure 3 This is the experimental result of acid responsiveness of hydrogel.
[0023] Figure 4 SEM test images of gel particles obtained at different stirring speeds.
[0024] Figure 5 The graph shows the test results of gel particle hydration particle size obtained at different stirring speeds.
[0025] Figure 6 This is the result of the stability test of gel particles.
[0026] Figure 7 Figure 2 shows the acid response experimental results of gel particles.
[0027] Figure 8 Continuous dynamic monitoring diagram of the particle size of 1000nm and 20000nm gel particles at different pH values after 14 hours.
[0028] Figure 9 Schematic diagram of the apparatus used in the single-axis electrospray method. In the figure, the following numbers are used: 1-syringe pump, 2-high-voltage power supply, 3-needle, 4-collection reservoir, 5-liquid inlet tube, 6-bracket, 7-wire.
[0029] Figure 10 PLGA 10k and PLGA 4.5k -PEG 1.5k Microscopic morphology of core-shell microspheres prepared with different mixing ratios.
[0030] Figure 11 PLGA 10k and PLGA 4.5k -PEG 1.5k Analysis results of surface pore number, porosity, pore diameter, and pore size of core-shell microspheres prepared with different mixing ratios.
[0031] Figure 12 This is a test diagram of the temperature-sensitive performance of core-shell microspheres.
[0032] Figure 13 This is the experimental result of the effect of core-shell microsphere concentration on cytotoxicity.
[0033] Figure 14 This figure shows the results of testing the cell viability of co-incubated cells using a cell counting reagent.
[0034] Figure 15 Schematic diagram of the action mechanism of core-shell microspheres. DETAILED DESCRIPTION
[0035] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.
[0036] Example 1 The preparation method of hydrazide-modified hyaluronic acid (HA-TPH) is as follows: 400 mg of hyaluronic acid (HA) and 1700 mg of 3,3'-dithiobis(propionylhydrazide) (TPH) were sequentially dissolved in 100 ml of 2-(N-morpholino)ethanesulfonic acid (MES-hydrate) aqueous solution (MES concentration was 0.01 M, pH was 5.5). 35 mg of N-hydroxysuccinimide (NHS) and 288 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) were then added. The mixture was stirred at room temperature for 24 hours. The resulting solution was dialyzed against deionized water for 5 days and lyophilized to obtain hydrazide-modified hyaluronic acid (HA-TPH). The reaction principle is as follows: .
[0037] The preparation method of oxidatively modified hyaluronic acid (HA-CHO) is as follows: Dissolve 400 mg of HA in 500 ml of distilled water, and dissolve 214 mg of sodium periodate (NaIO4) in 3 ml of distilled water. Add the NaIO4 aqueous solution dropwise to the HA aqueous solution, and stir the reaction at room temperature in the dark for 24 hours. Then add 500 μL of ethylene glycol to terminate the unreacted NaIO4, and continue stirring for 1 hour. Dialyze with deionized water for 5 days and freeze-dry to obtain oxidatively modified hyaluronic acid (HA-CHO).
[0038]
[0039] Example 2 The spherical gel particles are prepared as follows: (1) HA-TPH and HA-CHO prepared in Example 1 were used to prepare aqueous solutions with mass percentage concentrations of 0.5%, 1%, 1.5%, 2%, and 3%, respectively. HA-TPH solution and HA-CHO solution of the same concentration were mixed in equal volume ratios and stirred at room temperature to form a hydrogel by dynamic Schiff base bonds. The gelation time was recorded and gelation photos were taken by the test tube inversion method. The appropriate concentrations of HA-TPH solution and HA-CHO solution were screened according to the gelation time. The experimental results are shown in FIG. Figure 1 It can be seen that 1.5% HA-TPH solution and HA-CHO solution can form gel in 49 s, which is conducive to the smooth implementation of the preparation process. Therefore, 1.5% HA-TPH solution and 1.5% HA-CHO solution were selected to prepare gel particles.
[0040] The hydrogel formed by cross-linking the 1.5% HA-TPH solution and HA-CHO solution in the above experiment was used for swelling test. Specific method: The hydrogel was freeze-dried (pre-frozen at -80℃ and then vacuum-dried for 12 hours) to remove moisture, weighed and recorded the initial mass, and then soaked in PBS buffer. The gel was taken out and weighed and recorded at 1, 2, 3, 4, 8, 12, 16, and 24 hours respectively. The ratio of the mass after soaking to the initial mass was the swelling rate. The test results are shown in Figure 2 . It can be seen that the hydrogel can increase its weight by 20 times within 24 hours, indicating that the hydrogel has good swelling properties. Then, PBS phosphate buffer with an initial pH of 7.4 was used as a simulation solution, and the pH of the solution was adjusted by adding HCl and NaOH to simulate the pH value in the cells under an inflammatory environment. The acid responsiveness of the hydrogel was measured under the conditions of normal extracellular fluid pH 7.4, mucus pH 6.7, and inflammatory environment pH 5.0 in macrophages. The measurement results are shown in Figure 3 The results showed that the G' of the hydrogel decreased with decreasing pH, which indicated that the hydrogel had a certain acid responsiveness, which was beneficial to the hydrolysis of the hydrogel and thus could effectively release the drug in an acidic environment.
[0041] (2) A 1.5% HA-TPH solution was prepared, and an amount of beclomethasone dipropionate equal to the mass of HA-TPH was added to obtain solution A. Similarly, a 1.5% HA-CHO solution was prepared, and an amount of beclomethasone dipropionate equal to the mass of HA-CHO was added to obtain solution B. The solution A and solution B were quickly mixed in equal volume ratios and added dropwise to the vegetable oil, and the mixture was stirred and emulsified to obtain a suspension. Dichloromethane was added to the suspension to wash the suspension to remove the vegetable oil, and then the suspension was freeze-dried to obtain spherical gel particles. The vegetable oil can be selected from peanut oil, soybean oil, linseed oil, castor oil, rapeseed oil, etc., and rapeseed oil was used in this embodiment.
[0042] During the emulsification process, the mixture was stirred by a magnetic stirrer at different stirring speeds of 1000 rpm, 2000 rpm, 3000 rpm, 5000 rpm, and 8000 rpm to study the effect of stirring speed on the gel particles. The obtained gel particles were subjected to SEM tests and hydrated particle size tests. The results are shown in Figure 4 and Figure 5 . It can be seen that the stirring speed of the emulsification process will affect the particle size of the gel particles. The greater the stirring speed, the smaller the particle size of the prepared gel particles; stirring speeds of 1000rpm, 2000rpm, 3000rpm, 5000rpm, and 8000rpm respectively prepare gel particles with average particle sizes of 2000, 1000, 400, 200, and 100nm. Therefore, in the present invention, gel particles with a particle size range of 100-2000nm can be successfully prepared by changing the stirring speed. The stability of gel particles of different particle sizes was further tested. The gel particles were added to water to form a gel particle suspension. 1 mL of gel particle suspension with a concentration of 50 μg / mL was taken and placed in a quartz particle size cuvette. The particle size of the gel particles in the suspension was measured using a laser particle size analyzer at different times. The test results are shown in FIG. Figure 6 It can be seen that, except for the gel particles with a size of 2000 nm, the particle sizes of the other smaller gel particles remained almost unchanged within 30 days, which indicates that the gel particles have good stability within 30 days under storage conditions.
[0043] The acid response performance of the gel particles was further tested: gel particles of different particle sizes were soaked in PBS phosphate buffer solutions of different pH values (5.0, 6.0, 7.4) and the particle size changes of the gel particles were tested at different times. The test results are shown in Figure 7 It can be seen that at the second hour, microspheres of different particle sizes can all achieve particle size increase in acidic pH 5.0 and pH 6.0 environments, and PDI increases, but at the eighth hour, the particle size decreases. Therefore, gel particles with particle sizes of 1000nm and 2000nm were selected to continuously monitor the changes in gel particle size within 14 hours. The results are shown in Figure 2. Figure 8 . The results show that under acidic conditions, the particle size shows a trend of first increasing and then decreasing. The main reason is that, at the beginning, the Schiff base bonds on the surface of the gel particles begin to break, the gel segments gradually become loose, and at the same time, water molecules enter, resulting in an increase in particle size under acidic conditions. As time goes by, the Schiff base bonds disintegrate in large quantities, it is difficult to maintain the spherical shape and gradually dissolve in water, resulting in a smaller particle size. Since the particle size of the core-shell structure microspheres prepared subsequently needs to be kept below 5 microns in order to reach below the bronchi, and studies have shown that phagocytes have a higher phagocytic efficiency for particles with a diameter of 1-3 microns. Therefore, in this embodiment, gel particles with a particle size of 1000nm are preferably used as the core.
[0044] Example 3 The preparation method of core-shell structure microspheres is as follows: The shell material was dissolved in dichloromethane, and ambroxol hydrochloride and the 1000 nm-sized gel particles prepared in Example 2 were added to form an electrospray suspension. The shell material was a mixture of poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid) (poly(ethylene glycol) glycol) (PLGA-PEG).
[0045] The single-axis electrospray method refers to a polymer melt or solution being simultaneously subjected to gravity, Coulomb force and liquid surface tension in a high-voltage electrostatic field. When the three forces reach equilibrium, a Taylor cone is formed, and the polymer liquid is atomized and broken into fine micro-nano droplets. Due to the difference in electric potential, the products are collected at a receiving device. The device used in the single-axis electrospray method of the present invention is as follows: Figure 9 As shown, it consists of a syringe pump 1, a high-voltage power supply 2, a conductive needle 3 and a collecting tank 4; the syringe pump 1 is connected to the needle 3 through a liquid inlet tube 5, the needle is placed vertically and fixed by a bracket 6 so that the liquid outlet of the needle is downward, and a collecting tank 4 is set under the needle to receive droplets dripping from the needle 3; the collecting tank is filled with a polyvinyl alcohol aqueous solution; the output end of the high-voltage power supply 2 is connected to a wire 7, and the other end of the wire 7 is connected to the needle; the electrospray suspension is continuously injected into the needle 3 through the syringe pump 1, and when the solution is observed to form a Taylor cone at the liquid outlet of the needle, the high-voltage power supply 2 is turned on to apply electrostatic high voltage to the needle 3 for electrospraying, and after the electrospray state is stable for 10 minutes, the dripping droplets are collected by the collecting tank 4, and stirring is continued until the dichloromethane is completely volatilized to obtain core-shell structured microspheres.
[0046] This example uses commercially available PLGA 10k and PLGA 4.5k -PEG 1.5k The subscript represents the polymer molecular weight, for example, 1.5k represents a molecular weight of 1500. In the early experimental process, PLGA was compared with 4.5k -PEG 1k and PLGA 4.5k -PEG 1.5k The temperature phase change properties of these two materials. The results show that PLGA 4.5k -PEG 1.5k It can begin to transform into sol at 37℃ close to the body temperature, and PLGA 4.5k -PEG 1k Phase transition occurs at 33°C, which is not conducive to the subsequent particles reaching the deep respiratory tract and will cause premature release of the drug. Therefore, PLGA is preferred in this embodiment. 4.5k -PEG 1.5k As one of the shell materials for preparing microspheres.
[0047] First, the concentration of the shell material was studied: PLGA 10k and PLGA 4.5k -PEG 1.5k A mixture of 2% and 4% (6:4) was used as the shell material. The shell material was dissolved in dichloromethane (DCM) to prepare solutions with different concentrations of 5 wt%, 10 wt%, 15 wt%, and 20 wt%. Core-shell microspheres were prepared using these shell solutions at different concentrations. The preparation process conditions were: an infusion pump flow rate of 2.0 mL / h, a needle outlet inner diameter of 0.6 mm, and a distance from the needle outlet to the liquid surface in the collection reservoir (i.e., the receiving distance) of 15 cm. A high-voltage power supply applied a voltage of 15 kV to the needle. After the electrospray stabilized, the microspheres were collected in a collection reservoir with a PVA concentration of 2% wt. The microspheres were then centrifuged at 10,000 rpm to remove surface PVA and other impurities, lyophilized to a powder, and stored in a vacuum desiccator. Based on the spheroidization and dispersibility during the preparation process, the optimal shell solution concentration was 10 wt%.
[0048] Furthermore, the PLGA in the shell material was studied under the condition that the optimal concentration of the shell solution was 10w%. 10k and PLGA 4.5k -PEG 1.5k The effect of the mixing ratio on the microspheres. 10k and PLGA 4.5k -PEG 1.5k When the total dosage remains unchanged, PLGA 10k and PLGA 4.5k -PEG 1.5k The mixing ratio was adjusted to 10:0, 8:2, 6:4, and 2:8 by mass, and other conditions remained unchanged. The micromorphology of the final prepared microspheres was shown in Figure 10 IMAGE J software was used to calculate and analyze the number of pores on the microsphere surface, porosity, pore diameter, and pore size. The analysis results are shown in Figure 11 It can be seen that the PLGA 10k and PLGA 4.5k -PEG 1.5k Microspheres with different porous structures can be prepared by mixing them in different proportions. 10k When the concentration is 100%, the surface is smooth and non-porous; 4.5k -PEG 1.5k As the ratio increases, the number of pores on the microsphere surface, porosity, pore diameter, and pore size all show an increasing trend. This shows that the appearance of the pore structure on the microsphere surface is due to the continuous movement of PLGA 4.5k -PEG 1.5kTherefore, the preparation method of the present invention can be used to regulate the PLGA 10k and PLGA 4.5k -PEG 1.5k The porosity of the core-shell microspheres can be adjusted by adjusting the ratio of
[0049] Thermosensitive properties test of core-shell microspheres: The parameters for preparing microspheres are: the particle size of the core gel particles is 1000nm, and the PLGA 10k and PLGA 4.5k -PEG 1.5k The mixture with a mass ratio of 6:4 was used as the shell material. The shell solution concentration (10 wt%), flow rate (2.0 mL / h), receiving distance (15 cm), voltage (15 kV), PVA concentration in the collection pool (2% wt), and stirring speed were 1000 rpm. The prepared microspheres were subjected to a heating test. The specific method was: the suspension containing the microspheres was dropped onto a silicon wafer, and the microspheres were heated under an infrared heating lamp. SEM images were taken at different times (10s, 20s, 45s, 60s) to observe the temperature-sensitive properties of the microspheres. The results are shown in Fig. Figure 12 It can be seen that the microspheres can completely break and release the core within 1 minute of heating.
[0050] Studies on the drug-to-carrier material ratios revealed that when the mass ratio of the shell material to ambroxol hydrochloride was 1:2, the drug loading capacity and encapsulation efficiency of ambroxol hydrochloride reached 20.78% and 77.85% respectively. When the mass ratio of beclomethasone dipropionate to the total mass of HA-TPH and HA-CHO was 1:2, the drug loading capacity and efficiency of beclomethasone dipropionate reached a maximum of 13.9% and 61.17% respectively.
[0051] The effect of the concentration of prepared core-shell microspheres on cytotoxicity was tested: human bronchial epithelial cells (BEAS-2B) were incubated with microspheres at different concentrations (0 μg / mL, 250 μg / mL, 500 μg / mL, 1.0 mg / mL, 2.5 mg / mL, and 5 mg / mL) for 24 h. Cells cultured in 12-well plates were incubated with 3 μM calcichlorotin and 30 μg / mL propidium iodide for 20 min for live-dead fluorescence staining. The cells were then photographed using an inverted fluorescence microscope. The experimental results are shown in Table 2. Figure 13 .
[0052] The cell viability of the co-incubated cells was tested using a cell counting reagent (CCK-8): BEAS-2B cells were co-incubated with microspheres at different concentrations (125 μg / mL, 250 μg / mL, 500 μg / mL, 1.0 mg / mL, and 2.5 mg / mL) for 24 hours; 10 μL of CCK-8 solution was added to each well and incubated at 37°C, 5% CO2, and 90% humidity for 0.5 to 4 hours; after the liquid in the well turned yellow, the absorbance was measured at 450 nm using a microplate reader. The experimental results are shown in Figure 2. Figure 14 .
[0053] Depend on Figure 13 and Figure 14 It can be seen that when the core-shell microspheres are used at a concentration of 2.5 mg / ml, the survival rate of human bronchial epithelial cells (BEAS-2B) can reach more than 85%, and they have good cell compatibility.
[0054] In summary, from the above examples and experimental test results, it can be concluded that the biomimetic pollen microspheres prepared by the present invention have a porous core-shell structure. The shell of the microsphere contains the drug ingredient ambroxol hydrochloride, and the shell material has temperature-sensitive properties; the core contains the drug ingredient beclomethasone dipropionate, and the matrix material (hydrogel) of the core has acid-responsive properties; the core-shell structure of the microsphere can realize the sequential release of different drugs in different environments, and the porous structure of the microsphere gives it good aerodynamic properties. The structural characteristics and mechanism of action of the microsphere are as follows Figure 15 As shown, when the microspheres enter the lungs by inhalation, the porous structure of the microspheres has good aerodynamic properties and can reach deeper into the lungs (bronchi, bronchioles, alveoli). The thermosensitive shell can dissolve in a human body environment of 37°C and quickly release ambroxol hydrochloride to avoid being cleared by the mucus and ciliated cells in the lungs, thereby improving the retention effect of the microspheres. The exposed core after the shell dissolves can be phagocytosed by the cells of the lungs and enter the cells to take effect. Since the pH value of the internal environment of macrophages under inflammatory conditions is about 5.0-5.2, the core can disintegrate faster in this environment, thereby accelerating the release of beclomethasone dipropionate, thereby achieving the purpose of treating COPD.
[0055] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness, characterized in that: The following steps are involved: S1. Dissolve hyaluronic acid and 3,3'-dithiobis(propionylhydrazide) in 2-(N-morpholino)ethanesulfonic acid solution, then add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and stir at room temperature for 24 hours. After the reaction, dialyze with deionized water and lyophilize to obtain hydrazide-modified hyaluronic acid, referred to as HA-TPH. S2. Dissolve another portion of hyaluronic acid in water, add sodium periodate, and stir to react at room temperature in the dark. Then, dialyze and freeze-dry to obtain oxidatively modified hyaluronic acid, referred to as HA-CHO. S3. Dissolve HA-TPH in water, and then add beclomethasone dipropionate to obtain solution A. Similarly, dissolve HA-CHO in another portion of water, and then add beclomethasone dipropionate to obtain solution B. S4, mixing solution A and solution B and adding dropwise to vegetable oil, stirring continuously for emulsification to obtain a suspension, adding dichloromethane to the suspension for washing to remove the vegetable oil, and then freeze-drying to obtain spherical gel particles; S5, dissolving the shell material in dichloromethane, then adding ambroxol hydrochloride and the gel particles obtained in step S4 to form an electrospray suspension; the shell material is a mixture of polylactic acid-co-glycolic acid and polylactic acid-co-glycolic acid polyethylene glycol; S6, using electrospray suspension combined with uniaxial electrospraying to prepare porous core-shell microspheres with gel particles wrapped by shell materials; During the entire preparation process, the ambient temperature and the operating temperature were controlled to be no higher than 30°C.
2. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 1, characterized in that: In step S6, the device used in the uniaxial electrospray method consists of a syringe pump, a high-voltage power supply, a conductive needle and a collection tank; the syringe pump is connected to the needle through a liquid inlet tube, the needle is placed vertically with its liquid outlet facing downward, and a collection tank is set under the needle to receive droplets dripping from the needle; the collection tank is filled with a polyvinyl alcohol aqueous solution; the output end of the high-voltage power supply is connected to the needle; the electrospray suspension is injected into the needle through the syringe pump, and when the solution is observed to form a Taylor cone at the liquid outlet of the needle, the high-voltage power supply is turned on to apply electrostatic high voltage to the needle for electrospraying, and after the electrospray state is stable for at least 5 minutes, the dripping droplets are collected in the collection tank, and the collection tank is continuously stirred until the dichloromethane is completely volatilized to obtain porous core-shell microspheres.
3. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 2, characterized in that: The inner diameter of the needle liquid outlet is 0.4-0.8 mm, and the distance from the needle liquid outlet to the liquid surface in the collection pool is 15-25 cm.
4. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 2, characterized in that: The voltage applied to the needle by the high-voltage power supply is 12-18 kV.
5. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 2, characterized in that: The mass percentage concentration of the polyvinyl alcohol aqueous solution in the collection pool is 2-4%.
6. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 1, characterized in that: In the electrospray suspension, the mass percentage concentration of the shell material is 5-20%, the mass ratio of the gel particles to the shell material is 1:4; and the mass ratio of ambroxol hydrochloride to the shell material is 1:
2.
7. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 6, characterized in that: In the shell material, the mass proportion of polylactic acid-glycolic acid polyethylene glycol copolymer is 20-80%, and the rest is polylactic acid-glycolic acid copolymer, totaling 100%.
8. The method for preparing porous core-shell microspheres with temperature sensitivity and acid responsiveness according to claim 1, characterized in that: In the solution A and the solution B, the concentrations of HA-TPH and HA-CHO are the same, both of which are 1.5% by mass, and the solution A and the solution B are mixed in an equal volume ratio.
9. A porous core-shell microsphere with temperature sensitivity and acid responsiveness, characterized in that: The method is prepared according to any one of claims 1 to 8.