Preparation and application of a scaffold material carrying curcumin metal ion chelate liposomes

Abstract

This invention discloses a method for preparing and applying a scaffold material carrying curcumin-metal ion chelate liposomes. The method involves chelating curcumin with metal ions to form liposomes, using these liposomes as the osteogenic-antibacterial active ingredient, mixing them with polymers polylactic-co-glycolic acid copolymer and polycaprolactone, and then preparing a novel antibacterial bone regeneration scaffold using electrospinning technology. The method of this invention is simple and easy to operate; the resulting scaffold exhibits good bone regeneration-promoting, anti-inflammatory, and antibacterial activities; this invention can prepare biodegradable composite scaffold materials with mechanical strength, biocompatibility, and drug-loading capacity, and can be applied in the field of bone tissue regeneration.

Description

Technical Field

[0001] This invention relates to a method for preparing a scaffold material and its application, and more particularly to a method for preparing a scaffold material carrying curcumin metal ion chelate liposomes and its application. Background Technology

[0002] Curcumin is a yellow crystalline hydrophobic polyphenol isolated from turmeric (Curcuma longum). Curcumin is used to treat a variety of chronic diseases, including cardiovascular, neurodegenerative, respiratory, pulmonary, autoimmune, metabolic, and other types of illnesses. The preventative and therapeutic effects of curcumin are attributed to its pleiotropic pharmacological properties. Accumulated evidence suggests that curcumin possesses anti-inflammatory, antioxidant, wound-healing, hypoglycemic, and antibacterial properties.

[0003] In recent decades, complexes of curcumin with various metals have been synthesized, giving them stronger biological efficacy than curcumin itself. Generally, curcumin-metal chelates can reduce the toxicity of metals. Complexes chelated with antibacterial metal ions exhibit excellent antibacterial properties without reducing the osteogenic activity of curcumin itself, and can be used in osteogenic and antibacterial dual-function scaffolds.

[0004] Successful bone regeneration requires coordination of three key elements: cells, scaffolds, and growth factors. Biomaterial-based scaffolds are crucial platforms for supporting cell adhesion and growth factor delivery. Various methods for fabricating scaffolds have been investigated. Among them, electrospinning is renowned for its ability to create fibers and porous structures similar to the natural extracellular matrix. Other advantages of electrospinning include its ability to create relatively large surface-to-volume ratios; its control over fiber dimensions from the microscale to the nanoscale; and its versatility in material selection.

[0005] Biomaterial implantation is one of the main methods for treating bone defects in clinical practice, and many strategies have been developed to manufacture bone substitutes with ideal mechanical strength and osteogenic properties. However, implant-related infections and inflammation are serious complications that directly hinder the function and effectiveness of the implants. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a method for preparing a scaffold material carrying curcumin metal ion chelate liposomes that reduces the body's inflammatory response and improves osteogenic effects;

[0007] A second objective of this invention is to provide the application of the scaffold material prepared above, which carries curcumin metal ion chelate liposomes, in the field of bone tissue regeneration.

[0008] Technical solution: The preparation method of the scaffold material carrying curcumin metal ion chelate liposomes according to the present invention includes the following steps:

[0009] (1) Curcumin and metal chloride are mixed and dissolved in a solvent, the pH of the solution is adjusted to 8.0-9.0, and then stirred under cooling and reflux conditions. After cooling, the solid precipitate is separated, washed, and dried to obtain powdered curcumin metal chelate.

[0010] (2) Curcumin metal chelate, lecithin and cholesterol are mixed and dissolved in a solvent. The dissolved solution is added dropwise to PBS solution and reacted under heating conditions. After completion, the heating temperature is maintained and the mixture is allowed to stand to evaporate the organic solvent to obtain curcumin metal chelate liposome solution. After the organic solvent evaporates, the liposome solution is freeze-dried to obtain curcumin metal chelate liposomes.

[0011] (3) Dissolve curcumin metal chelate liposomes in an organic solvent; dissolve polylactic acid-glycolic acid copolymer particles and polycaprolactone particles in an organic solvent, and then add them to the curcumin metal chelate liposome solution to obtain a mixture;

[0012] (4) Electrospinning the mixture yields a biodegradable composite scaffold carrying curcumin metal chelate liposomes.

[0013] The pH must be adjusted within the above range to successfully prepare curcumin metal chelates; otherwise, it may not be possible to successfully prepare curcumin metal chelates.

[0014] In step (1), the metal chloride is zinc chloride, calcium chloride or copper chloride; the molar ratio of curcumin to metal chloride is 4:1-1:1.

[0015] The curcumin-metal chelate can only be successfully prepared if the molar ratio of curcumin to metal chloride is adjusted to within the above range. If it is not within the range, the curcumin-metal chelate may not be successfully prepared.

[0016] In step (1), the solvent is water / methanol, and the preferred volume ratio is 20:80-40:60w / w.

[0017] The curcumin metal chelate can only be successfully prepared if the volume ratio of water to methanol is adjusted within the above range. If it is not within the range, the curcumin metal chelate may not be successfully prepared.

[0018] In step (2), the heating temperature is 50℃-60℃; the stirring time is 0.5-1.5h; the rotation speed is 300-500r / min; the standing time is 0.5-1.5h; and the dropping speed is 1-2ml / min.

[0019] To obtain curcumin metal chelate liposome particles with excellent particle size and dispersion coefficient, the heating temperature, stirring time, rotation speed, settling time, and dropping speed must be adjusted within the above range. If they are not within this range, the particle size and dispersion coefficient of the obtained curcumin metal chelate liposome particles will not meet the experimental requirements.

[0020] In step (2), the mass ratio of lecithin to cholesterol is 20:1-2:1; the mass ratio of curcumin metal chelate to the sum of the masses of lecithin and cholesterol is 1:1.1-1:5.

[0021] To obtain curcumin metal chelate liposome particles with excellent particle size and dispersion coefficient, the mass ratio of lecithin to cholesterol and the mass ratio of curcumin metal chelate to the sum of the masses of lecithin and cholesterol must be adjusted to within the above-mentioned range. If they are not within this range, the particle size and dispersion coefficient of the obtained curcumin metal chelate liposome particles will not meet the experimental requirements.

[0022] In step (3), the mass of the curcumin metal chelate liposome particles is 1%-10% of the total mass of polylactic acid-hydroxyacetic acid copolymer and polycaprolactone; the mass ratio of polylactic acid-hydroxyacetic acid copolymer and polycaprolactone is 10:1-1:10.

[0023] To obtain a scaffold material with excellent mechanical properties through electrospinning, the mass of curcumin metal chelate liposome particles and the mass ratio of polylactic acid-glycolic acid copolymer to polycaprolactone must be adjusted to within the above-mentioned range. If they are not within the range, it is impossible to obtain a scaffold material with excellent mechanical properties through electrospinning.

[0024] In step (3), the organic solvent is hexafluoroisopropanol or trifluoroethanol.

[0025] The above-mentioned scaffold material carrying curcumin metal ion chelate liposomes is used in the field of bone tissue regeneration.

[0026] Invention Principle: This invention chelates curcumin with metal ions to form liposomes, which are then used as osteogenic and antibacterial active ingredients. These liposomes are mixed with polymers polylactic-glycolic acid copolymer and polycaprolactone, and a novel antibacterial bone regeneration scaffold is prepared using electrospinning technology. This addresses the problem of implantable scaffold-related infections, reduces the body's inflammatory response, and improves osteogenic efficacy. The invention chelates curcumin with metal ions to enhance its antibacterial properties by introducing metal ions. Highly drug-carrying nanoliposomes encapsulate the curcumin-metal ion chelate to improve drug utilization and reduce cytotoxicity. The polylactic-glycolic acid copolymer and polycaprolactone are blended with the prepared nanoliposomes, and a scaffold loaded with the curcumin-metal ion chelate liposomes is prepared by electrospinning. This process endows the scaffold with osteogenic and antibacterial functions to address the complex osteogenic microenvironment under infectious conditions.

[0027] Beneficial effects: Compared with the prior art, the present invention achieves the following significant effects: (1) The scaffold has good bone regeneration promotion, anti-inflammatory and antibacterial activities. (2) The method is simple and easy to operate. (3) It can prepare biodegradable composite scaffold materials with mechanical strength, biocompatibility and drug loading capacity, and apply them to the field of bone tissue regeneration. Attached Figure Description

[0028] Figure 1 This is a scanning electron microscope image of the support structure of Embodiment 1 of the present invention;

[0029] Figure 2 The infrared spectrum of the bracket in Embodiment 1 of the present invention;

[0030] Figure 3 The elastic modulus and fracture stress diagram of the bracket in Embodiment 1 of the present invention;

[0031] Figure 4 This is a CCK-8 experimental diagram of the stent in Embodiment 1 of the present invention;

[0032] Figure 5 This is a scanning electron microscope image of the scaffold and Escherichia coli after co-culturing for 24 hours in Example 1 of the present invention. Detailed Implementation

[0033] The present invention will now be described in further detail.

[0034] Example 1

[0035] (1) Accurately weigh 4 mmol of curcumin and 1 mmol of zinc chloride, and add them to 30 ml of a water / methanol solution, wherein the volume ratio of water to methanol is 20:80 w / w; adjust the pH of the solution to 8.0 using ammonia water, and stir the mixture under reflux at 50 °C for 20 min. After cooling, separate the solid precipitate by vacuum filtration, wash it several times with water and a small amount of methanol, and obtain powdered curcumin metal chelate after vacuum drying.

[0036] (2) Accurately weigh 19.09 mg of curcumin metal chelate, 20 mg of lecithin, and 1 mg of cholesterol using an analytical balance. Dissolve them in 1 ml of anhydrous ethanol and sonicate until completely dissolved. Use a peristaltic pump to add the dissolved solution dropwise to 20 ml of PBS solution at a rate of 1 ml / min. Stir the mixture in a 50°C water bath for 0.5 h at a speed of 300 r / min. Maintain the water bath temperature and let it stand for 0.5 h to evaporate the ethanol, thus obtaining a curcumin metal chelate liposome solution. After the ethanol has evaporated, place the liposome solution in an ice-water bath and sonicate it. Then transfer it to a -80°C freezer and freeze-dry it under vacuum to obtain the curcumin metal chelate liposomes.

[0037] (3) Accurately weigh 0.011 g of dried curcumin metal chelate liposome particles using an analytical balance, dissolve them in 2 ml of hexafluoroisopropanol, and sonicate until completely dissolved. Then, accurately weigh 1 g of PLGA and 0.1 g of PCL particles using an analytical balance, add 2 ml of hexafluoroisopropanol, and stir at room temperature until PLGA and PCL are completely dissolved. After complete dissolution, add the curcumin metal chelate liposome solution and continue stirring until evenly mixed.

[0038] (4) Finally, the mixture is transferred to a syringe for electrospinning to obtain a biodegradable composite scaffold carrying curcumin metal chelate liposomes.

[0039] This study used FE-SEM to observe the surface morphology of the electrospun scaffold, such as Figure 1 As shown, PP, PP / Cur, and PP / Cur-Zn all exhibit no obvious beading and have uniform fiber thickness distribution, ensuring sufficient mechanical properties of the scaffold. Furthermore, the high porosity of the scaffold facilitates the exchange of oxygen and nutrients between cells and the external environment, thereby promoting cell adhesion and proliferation, and promoting osteoblast differentiation. Compared to PP, the addition of nanoliposomes increases the surface roughness and surface area of ​​PP / Cur-Zn, which is also beneficial for cell adhesion.

[0040] To detect the major functional groups of the stent, this study performed infrared spectroscopy analysis on the stent. Figure 2 Infrared spectra of PP, PP / Cur, and PP / Cur-Zn are shown. Spectra located in the 3000–2800 cm⁻¹ region were found in all PLGA / PCL scaffolds. -1The absorption peak originates from the CH stretching vibration on the saturated carbon in PLGA and PCL; it is located at 1757 cm⁻¹. -1 and 1727cm -1 Typical absorption peaks originate from the free ester functional group (-COOR) of PLGA and the conjugated ester group (-OCOR) of PCL, respectively.

[0041] like Figure 3 As shown, the average Young's modulus of PP was 11.12 ± 1.25 MPa, and the fracture stress was 0.71 ± 0.1 MPa; the average Young's modulus of PP / Cur was 19.02 ± 1.1 MPa, and the fracture stress was 1.58 ± 0.19 MPa; the average Young's modulus of PP / Cur-Zn was 19.5 ± 1.1 MPa, and the fracture stress was 1.44 ± 0.16 MPa. Compared with PP, the elastic modulus and fracture stress of PP / Cur-Zn were both improved. This indicates that the addition of liposomes can improve the mechanical properties of the scaffold, which may be due to the formation of hydrogen bonds between lecithin in the liposome membrane and the hydroxyl groups in PLGA and PCL.

[0042] like Figure 4 As shown, after co-culturing with the scaffold, the number of cells on all three scaffold groups showed a stable increase, indicating that the prepared scaffold materials had no obvious cytotoxicity and promoted cell proliferation, demonstrating good cell compatibility. Starting from day three, compared with PP, the OD values ​​of both the PP / Cur and PP / Cur-Zn groups were significantly increased, indicating that the introduction of Cur and Cur-Zn can promote osteoblast proliferation. At day seven, compared with PP / Cur, the OD value of the PP / Cur-Zn group was higher, indicating that chelation with zinc ions can enhance Cur's ability to promote osteoblast proliferation.

[0043] In this study, PP, PP / Cur, and PP / Cur-Zn were co-cultured with *E. coli* for 24 hours, and the bacterial distribution on the scaffold surface was observed using SEM. The morphology and quantity of bacteria adhering to the scaffold surface were observed using SEM to understand the antibacterial effect of the scaffold. Figure 5 As shown, columnar E. coli were observed with a high density on PP. A high density of E. coli was also observed on PP / Cur, while the number of E. coli observed on PP / Cur-Zn was very small, and the bacteria exhibited deformed morphology and cell membrane damage. This indicates that PP / Cur-Zn can effectively reduce bacterial adhesion and biofilm formation, demonstrating excellent antibacterial effects.

[0044] Example 2

[0045] (1) Accurately weigh 1 mmol curcumin and 1 mmol calcium chloride, and add them to 30 ml of a water / methanol solution, wherein the volume ratio of water to methanol is 40:60 w / w; adjust the pH of the solution to 9.0 using ammonia water, and stir the mixture under reflux at 60 °C for 30 min. After cooling, separate the solid precipitate by vacuum filtration, wash it several times with water and a small amount of methanol, and obtain powdered curcumin metal chelate after vacuum drying.

[0046] (2) Accurately weigh 0.6 mg of curcumin metal chelate, 2 mg of lecithin, and 1 mg of cholesterol using an analytical balance. Dissolve them in 2 ml of anhydrous ethanol and sonicate until completely dissolved. Use a peristaltic pump to add the dissolved solution dropwise to 40 ml of PBS solution at a rate of 2 ml / min. Stir the mixture in a 60°C water bath for 1.5 h at a speed of 500 r / min. Maintain the water bath temperature and let it stand for 1.5 h to evaporate the ethanol, thus obtaining a curcumin metal chelate liposome solution. After the ethanol has evaporated, place the liposome solution in an ice-water bath and sonicate it. Then transfer it to a -80°C freezer and freeze-dry it under vacuum to obtain the curcumin metal chelate liposomes.

[0047] (3) Accurately weigh 0.11 g of dried curcumin metal chelate liposome particles using an analytical balance, dissolve them in 4 ml of trifluoroethanol, and sonicate until completely dissolved. Then, accurately weigh 0.1 g of PLGA and 1 g of PCL particles using an analytical balance, add 4 ml of trifluoroethanol, and stir at room temperature until PLGA and PCL are completely dissolved. After complete dissolution, add the curcumin metal chelate liposome solution and continue stirring until evenly mixed.

[0048] (4) Finally, the mixture is transferred to a syringe for electrospinning to obtain a biodegradable composite scaffold carrying curcumin metal chelate liposomes.

[0049] Example 3

[0050] (1) Accurately weigh 2 mmol of curcumin and 1 mmol of copper chloride, and add them to 30 ml of a water / methanol solution, wherein the volume ratio of water to methanol is 30:70 w / w; adjust the pH of the solution to 9.0 with ammonia water, and stir the mixture under reflux at 50 °C for 30 min. After cooling, separate the solid precipitate by vacuum filtration, wash it several times with water and a small amount of methanol, and obtain powdered curcumin metal chelate after vacuum drying.

[0051] (2) Accurately weigh 10 mg of curcumin metal chelate, 20 mg of lecithin, and 5 mg of cholesterol using an analytical balance. Dissolve them in 1 ml of anhydrous ethanol and sonicate until completely dissolved. Use a peristaltic pump to add the dissolved solution dropwise to 20 ml of PBS solution at a rate of 1 ml / min. Stir the reaction in a 50°C water bath for 1 h. After the reaction is complete, stop stirring and maintain the above water bath temperature for 1 h to allow the ethanol to evaporate, thus obtaining a curcumin metal chelate liposome solution. After the ethanol has evaporated, place the liposome solution in an ice-water bath and sonicate it. Then transfer it to a -80°C freezer and freeze-dry it under vacuum to obtain curcumin metal chelate liposomes.

[0052] (3) Accurately weigh 0.08 g of dried curcumin metal chelate liposome particles using an analytical balance, dissolve them in 2 ml of hexafluoroisopropanol, and sonicate until completely dissolved. Then, accurately weigh 0.2 g of PLGA and 0.8 g of PCL particles using an analytical balance, add 3 ml of trifluoroethanol, and stir at room temperature until PLGA and PCL are completely dissolved. After complete dissolution, add the curcumin metal chelate liposome solution and continue stirring until evenly mixed.

[0053] (4) Transfer the mixture to a syringe for electrospinning to obtain a biodegradable composite scaffold carrying curcumin metal chelate liposomes.

[0054] Comparative Example 1

[0055] The difference between Comparative Example 1 and Example 1 is that the amount of curcumin was 1 mmol and the amount of zinc chloride was 2 mmol, while all other conditions were the same. The CCK-8 assay results of Comparative Example 1 were worse than those of Example 1, indicating that Example 1 had a better effect on promoting osteoblast proliferation than Comparative Example 1.

[0056] Comparative Example 2

[0057] The difference between Comparative Example 2 and Example 1 is that the mass of curcumin metal chelate was 2 mg, the mass of lecithin was 10 mg, and the mass of cholesterol was 10 mg; all other conditions were the same. The CCK-8 assay results of Comparative Example 2 were worse than those of Example 1, indicating that Example 1 had a better effect on promoting osteoblast proliferation than Comparative Example 2.

[0058] Comparative Example 3

[0059] The difference between Comparative Example 3 and Example 1 was that the water bath temperature was 45°C, the stirring time was 2 hours, the stirring speed was 600 r / min, and the settling time was 2 hours; all other conditions were the same. The CCK-8 experiment results of Comparative Example 3 were worse than those of Example 1, indicating that Example 1 had a better effect on promoting osteoblast proliferation than Comparative Example 3.

[0060] Comparative Example 4

[0061] The difference between Comparative Example 4 and Example 1 is that the mass of the curcumin metal chelate liposomes was 0.15 g, while all other conditions were the same. The CCK-8 assay results of Comparative Example 4 were worse than those of Example 1, indicating that Example 1 had a better effect on promoting osteoblast proliferation than Comparative Example 4.

[0062] Comparative Example 5

[0063] The difference between Comparative Example 5 and Example 1 is that the mass of PLGA is 1.5g and the mass of PCL is 0.1g, while all other conditions are the same. The elastic modulus and fracture stress of Comparative Example 5 are worse than those of Example 1, indicating that Example 1 has better mechanical properties than Comparative Example 5.

Claims

1. A method for preparing a scaffold material carrying curcumin metal ion chelate liposomes, characterized in that, Includes the following steps: (1) Curcumin and metal chloride are mixed and dissolved in a solvent, the pH of the solution is adjusted to 8.0-9.0, and then stirred under cooling reflux. After cooling, the solid precipitate is separated, washed, and dried to obtain powdered curcumin metal chelate; the solvent is water / methanol with a volume ratio of 20:80-40:60w / w. (2) Curcumin metal chelate, lecithin and cholesterol are mixed and dissolved in a solvent. The dissolved solution is added dropwise to PBS solution and reacted under heating conditions. After completion, the heating temperature is maintained and the mixture is allowed to stand to evaporate the organic solvent, thereby obtaining a curcumin metal chelate liposome solution. After the organic solvent evaporates, the liposome solution is freeze-dried to obtain curcumin metal chelate liposomes. The heating temperature is 50℃-60℃; the stirring time is 0.5-1.5h; the rotation speed is 300-500 r / min; the standing time is 0.5-1.5h; the dropping rate is 1-2ml / min; the mass ratio of lecithin to cholesterol is 20:1-2:1; the mass ratio of curcumin metal chelate to the sum of the masses of lecithin and cholesterol is 1:1.1-1:

5. (3) Dissolve curcumin metal chelate liposomes in an organic solvent; dissolve polylactic acid-glycolic acid copolymer particles and polycaprolactone particles in an organic solvent, and then add them to the curcumin metal chelate liposome solution to obtain a mixture; (4) Electrospinning the mixture yields a biodegradable composite scaffold carrying curcumin metal chelate liposomes.

2. The method for preparing the scaffold material carrying curcumin metal ion chelate liposomes according to claim 1, characterized in that, The molar ratio of curcumin to metal chloride in step (1) is 4:1 to 1:

1.

3. The method for preparing the scaffold material carrying curcumin metal ion chelate liposomes according to claim 1, characterized in that, In step (3), the mass of the curcumin metal chelate liposome particles is 1%-10% of the total mass of polylactic acid-hydroxyacetic acid copolymer and polycaprolactone.

4. The method for preparing the scaffold material carrying curcumin metal ion chelate liposomes according to claim 1, characterized in that, In step (3), the organic solvent is hexafluoroisopropanol or trifluoroethanol.

5. The method for preparing the scaffold material carrying curcumin metal ion chelate liposomes according to claim 1, characterized in that, In step (3), the mass ratio of the polylactic acid-hydroxyacetic acid copolymer to polycaprolactone is 10:1 to 1:

10.

6. The method for preparing the scaffold material carrying curcumin metal ion chelate liposomes according to claim 1, characterized in that, In step (1), the metal chloride is zinc chloride, calcium chloride or copper chloride.

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