Thermo-sensitive core-shell structure microsphere loaded with active component and preparation method of thermo-sensitive core-shell structure microsphere

Abstract

The invention provides a temperature-sensitive core-shell structure microsphere loaded with active ingredients and a preparation method of the temperature-sensitive core-shell structure microsphere. The preparation method comprises the following steps: uniformly mixing bioactive molecules with a sodium alginate solution, and cross-linking to form a gel microsphere core layer loaded with the bioactive molecules; and soaking the gel microsphere core layer in a PNIPAM prepolymer aqueous solution, pre-adsorbing, and carrying out ultraviolet irradiation crosslinking to obtain the active molecule loaded PNIPAM temperature-sensitive core-shell microsphere. The structure is controllable, the performance is stable, and the particle size is accurate and adjustable; the prepared microspheres are good in temperature-sensitive responsiveness, the PNIPAM shell layer endows the microspheres with the remarkable temperature-sensitive characteristic, SA and PNIPAM which are excellent in biocompatibility serve as main materials, safety is good, biocompatibility is good, the process is simple and convenient, and conditions are mild.

Description

Technical Field

[0001] This invention relates to the field of drug carrier technology, and in particular to a thermosensitive core-shell structured microsphere loaded with an active ingredient and its preparation method. Background Technology

[0002] Temperature-sensitive polymers, such as poly(N-isopropylacrylamide) (PNIPAM), exhibit great application potential in drug controlled release and tissue engineering due to their reversible hydrophilic-hydrophobic transition near a low critical solution temperature (LCST, approximately 32-34°C). Combining PNIPAM with biocompatible natural polymers (such as sodium alginate, SA) to construct core-shell microspheres enables temperature-responsive drug release while leveraging the good biocompatibility and mild ionogel conditions of SA to protect active molecules.

[0003] In existing technologies, temperature-sensitive microspheres are often prepared using a one-step method or simple blending and crosslinking. However, this method suffers from problems such as poor structural uniformity, low encapsulation efficiency of active molecules, and difficulty in balancing temperature-sensitive response performance with structural strength. In particular, improvements are still needed in how to precisely control the microsphere particle size, achieve stable construction of the core-shell structure, and ensure the activity of active molecules during the preparation process. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing core-shell structured microspheres loaded with active molecules that have controllable structure, high encapsulation efficiency, and good temperature-sensitive responsiveness.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients includes the following steps:

[0007] (1) Core layer preparation: The bioactive molecules are mixed evenly with sodium alginate solution to form a mixture; then the mixture is added dropwise to the crosslinking agent solution and allowed to stand for crosslinking for 30-45 minutes to form a gel microsphere core layer loaded with active molecules;

[0008] (2) Shell construction: N-isopropylacrylamide (NIPAM) monomer, crosslinking agent and photoinitiator were added to water to obtain a prepolymer aqueous solution; the gel microsphere core layer obtained in step (1) was washed and then immersed in the prepolymer aqueous solution, and pre-adsorbed for 3-12 hours under light-protected conditions, and then irradiated with ultraviolet light at a wavelength of 365 nm for 45 min to obtain PNIPAM thermosensitive core-shell microspheres loaded with active molecules; the ultraviolet light irradiation intensity was 10-40 mW / cm 2 .

[0009] Preferably, the bioactive molecule in step (1) is at least one of Centella asiatica extract (Centella asiatica glycoside 80%, purchased from Guangzhou Haoxiang Fine Chemicals), hyaluronic acid, and Leonurus japonicus extract (Leonurus japonicus extract (food grade) Leonurus japonicus alkaloid, purchased from Guanying Biotechnology).

[0010] Preferably, the concentration of the sodium alginate solution in step (1) is 0.5 w / v%-2.5 w / v.

[0011] Preferably, the mass ratio of the bioactive molecule to the sodium alginate solution in step (1) is 0.5-1:3-5.

[0012] Preferably, the concentration of the crosslinking agent solution in step (1) is 1.5 w / v% to 3.0 w / v%; the crosslinking agent is CaCl2 or FeCl3.

[0013] Preferably, the volume ratio of the mixture to the crosslinking agent solution in step (1) is 1:(0.005-10); the droplet addition is performed by microfluidic injection at a flow rate of 0.1 mL / min-50 mL / min.

[0014] Preferably, the crosslinking agent in step (2) is at least one of N,N'-methylenebisacrylamide (MBA), ethylene glycol methacrylate, and triglyceride methacrylate; and the photoinitiator is at least one of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (2659), 2-hydroxy-2-methyl-1-phenyl-1-propanone (1173), hydroxycyclohexylphenyl ketone (184), and isopropylthioxanthanone (ITX).

[0015] Preferably, in the prepolymer aqueous solution of step (2), the concentration of N-isopropylacrylamide monomer is 5-8%, the concentration of crosslinking agent is 0.1-10%, and the concentration of photoinitiator is 0.005-1%.

[0016] Preferably, the ratio of the gel microsphere core layer to the prepolymer aqueous solution in step (2) is 1:(0.01-2).

[0017] Another object of the present invention is to provide a thermosensitive core-shell structured microsphere prepared by the above method, wherein the particle size of the thermosensitive core-shell structured microsphere is 1.22 μm-1750 μm.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) Controllable structure and stable performance: The stepwise cross-linking method of "core first and shell later" is adopted. First, a stable calcium alginate gel core is formed to encapsulate the active molecules, and then a PNIPAM temperature-sensitive shell is constructed on its surface. This avoids the deactivation of the active molecules in the polymerization reaction, and the core-shell structure is clear and firm.

[0020] (2) Precisely adjustable particle size: By systematically studying and adjusting the concentrations of SA and CaCl2, linear and precise control of microsphere particle size can be achieved to meet the needs of different application scenarios for carrier size.

[0021] (3) Good temperature-sensitive response: The PNIPAM shell endows the microspheres with significant temperature-sensitive properties. A volume phase transition occurs near LCST, which can effectively regulate the release rate of internal active molecules and realize intelligent controlled release.

[0022] (4) High biocompatibility: It uses SA and PNIPAM, which have excellent biocompatibility, as the main materials, and has good safety and is suitable for the biomedical field.

[0023] (5) Simple process and mild conditions: The entire preparation process is carried out in the aqueous phase, and the cross-linking conditions are mild (ionic cross-linking and ultraviolet light initiation), which is conducive to maintaining the activity of bioactive molecules. Attached Figure Description

[0024] Figure 1 The images show the morphology of microspheres of different sizes prepared in Examples 1 and 4.

[0025] Figure 2 Comparison of Fourier transform infrared (FTIR) spectra of SA-PNIPAM thermosensitive core-shell microspheres loaded with Centella asiatica and Centella asiatica and sodium alginate in Example 1;

[0026] Figure 3 Comparison of release curves of microspheres from Example 1 and Comparative Example 1 at different temperatures. Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments.

[0028] Example 1

[0029] A method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients, comprising the following steps:

[0030] (1) Core layer preparation: Weigh 0.15g sodium alginate and dissolve it in 10mL distilled water, sonicate to dissolve, and prepare a 0.05w / v% SA solution; mix 0.03g Centella asiatica extract with sodium alginate solution to form a mixture; then add 5mL of the mixture to 50mL of 2.5w / v% CaCl2 aqueous solution at a height of 10cm above the surface of CaCl2 solution at a dropping rate of 1mL / min, and let it stand for cross-linking for 30 minutes to form a gel microsphere core layer loaded with active molecules;

[0031] (2) Shell construction: 6g of N-isopropylacrylamide monomer, 0.6g of MBA and 40μL of 0.1% (w / v) 2659 photoinitiator were added to water to obtain a prepolymer aqueous solution; the gel microsphere core layer obtained in step (1) was washed three times with distilled water until clean, then immersed in the prepolymer aqueous solution and allowed to stand in the dark for 6 hours for pre-adsorption. The microspheres were filtered out and then subjected to ultraviolet light with a wavelength of 365 nm (intensity 20 mW / cm²). 2 After irradiation for 30 minutes and polymerization, the mixture was filtered and thoroughly washed with distilled water to obtain SA-PNIPAM thermosensitive core-shell microspheres loaded with Centella asiatica.

[0032] The average particle size of the microspheres was measured to be approximately 1.3 μm. FTIR spectroscopy showed the presence of characteristic absorption peaks of SA, Centella asiatica extract, and PNIPAM, confirming the successful construction of the core-shell structure.

[0033] Examples 2-4

[0034] The SA concentration was varied (0.5 w / v%, 1 w / v%, and 2.5 w / v%, respectively), while the CaCl2 concentration was kept constant at 2.5 w / v and the PNIPAM concentration at 6%. Other steps were the same as in Example 1. The results showed a positive correlation between SA concentration and microsphere size, with the resulting microspheres having average particle sizes of 600 μm, 1000 μm, and 1320 μm, respectively.

[0035] Examples 5-7

[0036] With the SA concentration fixed at 1.5%, the CaCl2 concentration was varied (0.015%, 0.5%, and 3.0%, respectively), and the PNIPAM concentration was 6%, with other steps identical to those in Example 1. The resulting microspheres had average particle sizes of 1300 μm, 1.9 μm, and 1.3 μm, respectively. The results indicate that the CaCl2 concentration is negatively correlated with the microsphere particle size.

[0037] The morphology images of microspheres of different sizes prepared in Examples 1 and 4 are shown below. Figure 1 (a) is an SEM image of the small-sized (1.3 μm) microspheres prepared in Example 1, and (b) is a camera image of the large-sized (1320 μm) microspheres prepared in Example 4.

[0038] Example 1: Comparison of Fourier transform infrared (FTIR) spectra of SA-PNIPAM thermosensitive core-shell microspheres loaded with Centella asiatica and Centella asiatica and sodium alginate, as shown in the figure. Figure 2 Infrared spectroscopy analysis ( Figure 2This confirmed the successful preparation of the composite hydrogel and the interactions between its components. The spectra of the loaded sample showed significant changes compared to those of pure sodium alginate (SA) and Centella asiatica extract. After surface polymerization of PNIPAM, the ~2900 cm⁻¹... -1 The characteristic peak of CH bending vibration appeared nearby. ~3300 cm⁻¹ -1 The broad absorption peak at [value missing] was retained, which is a superposition peak of the stretching vibrations of OH in SA and NH in PNIPAM. However, its peak shape was significantly broadened and showed a multi-peak distribution, indicating that a dense hydrogen bond network was formed between PNIPAM, SA, and Centella asiatica extract in the system. The characteristic double peak of the SA carboxylate group (~1600 cm⁻¹) was also observed. -1 With ~1410 cm -1 A slight shift occurred after recombination, and ~1600 cm -1 The peak at this point corresponds to the PNIPAM amide I band (C=O stretching vibration, typically located at ~1650 cm⁻¹). -1 The overlapping and splitting of the groups directly proves that hydrogen bonding occurs between the carboxyl group of SA and the amide group of PNIPAM. Simultaneously, a new peak (~1630–1650 cm⁻¹) clearly belonging to the amide bond of PNIPAM appears in the spectrum. -1 Characteristic absorptions of C=O and glycosidic / ether bonds (~1070-1020 cm⁻¹) -1 (COC). Although the amount of Centella asiatica extract added was small, some of its characteristic peaks (e.g., ~3100 cm⁻¹) were still present. -1 ~1300 cm -1 and fingerprint area 500-600 cm -1 The peaks of the α-Callic acid overlapped with those of SA and PNIPAM, but the presence of these characteristic signals of Centella asiatica, combined with the aforementioned evidence of hydrogen bonding, together constituted evidence that the active ingredients of Centella asiatica were successfully introduced into the microspheres.

[0039] Comparative Example 1

[0040] Performing only steps 1-5, without PNIPAM shell grafting, yields pure calcium alginate / Centella asiatica microspheres. These microspheres are not thermosensitive, and their drug release behavior is not regulated by temperature.

[0041] Comparison of release curves of microspheres from Example 1 and Comparative Example 1 at different temperatures Figure 3 .Depend on Figure 3It can be seen that the sample prepared according to Example 1 accelerated the release of the active substance upon heating, while the comparative example did not have this effect. As shown in the figure, the release of the active substance in Example 1 was slow at room temperature before coating, but accelerated after heating to 40 °C. In contrast, the comparative example did not use PNIPAM coating; both the hydrogel microspheres and the drug were miscible with water. Therefore, the active molecules were rapidly released and then reached equilibrium. Furthermore, changes in ambient temperature had little effect on the release rate and amount of the active molecules, indicating that the sample was not thermosensitive.

[0042] The above embodiments demonstrate that the method of the present invention can successfully prepare core-shell structured microspheres with significant temperature-sensitive properties, and effectively control the microsphere particle size by adjusting process parameters, providing a new approach for the development of functional drug carriers.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients, characterized in that, Includes the following steps: (1) Core layer preparation: The bioactive molecules are mixed evenly with sodium alginate solution to form a mixture; then the mixture is added dropwise to the crosslinking agent solution and allowed to stand for crosslinking for 30-45 minutes to form a gel microsphere core layer loaded with active molecules; (2) Shell construction: N-isopropylacrylamide monomer, crosslinking agent and photoinitiator are added to water to obtain a prepolymer aqueous solution; the gel microsphere core layer obtained in step (1) is washed and then immersed in the prepolymer aqueous solution for pre-adsorption for 3-12 hours under light-protected conditions, and then irradiated with ultraviolet light at a wavelength of 365 nm for 0.5 min-24 h to obtain PNIPAM thermosensitive core-shell microspheres loaded with active molecules; the ultraviolet light irradiation intensity is 10-40 mW / cm 2 .

2. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, The bioactive molecule mentioned in step (1) is at least one of Centella asiatica extract, hyaluronic acid, and Leonurus japonicus extract.

3. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, The concentration of the sodium alginate solution in step (1) is 0.5 w / v%-2.5 w / v.

4. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, The mass ratio of the bioactive molecule to the sodium alginate solution in step (1) is 0.5-1:3-5.

5. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, The concentration of the crosslinking agent solution in step (1) is 1.5 w / v%~3.0 w / v%; the crosslinking agent is CaCl2 or FeCl3.

6. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, The volume ratio of the mixture to the crosslinking agent solution in step (1) is 1:(0.005-10); the droplet addition is performed by microfluidic injection at a flow rate of 0.1 mL / min-100 mL / min.

7. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, The crosslinking agent in step (2) is at least one of N,N'-methylenebisacrylamide, ethylene glycol methacrylate, and triglyceride methacrylate; the photoinitiator is at least one of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone, 2-hydroxy-2-methyl-1-phenyl-1-propanone, hydroxycyclohexylphenyl ketone, and isopropylthioxanthrone.

8. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, In step (2), the concentration of N-isopropylacrylamide monomer in the prepolymer aqueous solution is 5-8%, the concentration of crosslinking agent is 0.1-20%, and the concentration of photoinitiator is 0.005-1%.

9. The method for preparing thermosensitive core-shell structured microspheres loaded with active ingredients according to claim 1, characterized in that, In step (2), the ratio of the gel microsphere core layer to the prepolymer aqueous solution is 1:(0.01-2).

10. A thermosensitive core-shell structured microsphere prepared by the method of claim 1, characterized in that, The thermosensitive core-shell structured microspheres have a calcium alginate / bioactive molecule composite gel core and a PNIPAM cross-linked polymer shell covering the core, and the particle size of the thermosensitive core-shell structured microspheres is 1.22μm-1750μm.

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