Nanoparticles based on metal coordination polymer as well as preparation and application of nanoparticles

Abstract

The invention belongs to the technical field of medical materials, and discloses metal coordination polymer-based nanoparticles as well as preparation and application thereof. The preparation method comprises the following steps: 1) mixing polyvinylpyrrolidone and ferric salt in a solvent, adjusting the pH value to 8-9, dropwise adding an ethanol solution of curcumin, and continuously stirring after dropwise adding to obtain iron-based curcumin; (2) tannic acid and iron-based curcumin are subjected to a reaction, and tannic acid coordinated iron-based curcumin is obtained; and 3) in a reaction medium, carrying out a reaction on polylysine and tannic acid coordinated iron-based curcumin to obtain the metal coordination polymer nanoparticles. The method disclosed by the invention is simple, and the components of the nanoparticles are all natural materials, so that the nanoparticles have good biocompatibility, can be retained in biological tissues to play a role and do not cause other injuries. The nano particles disclosed by the invention have relatively good antioxidant, antibacterial and anti-inflammatory effects. The nanoparticles are used for preparing drugs for treating bacterial keratitis or wound infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a metal coordination polymer and its preparation and application. Background Technology

[0002] Bacterial and biofilm-induced ulcers are characterized by persistent infection, severe inflammation, and tissue damage. Current bactericidal strategies for ulcer wounds include photodynamic therapy, photothermal therapy, sonodynamic therapy, and single-atom nanozymes. While these methods can effectively kill bacteria and even improve biofilm damage, their application in clinical practice remains a significant challenge due to drawbacks such as external energy penetration, material toxicity, and the need for engineering modifications.

[0003] In infected wounds, capillary damage leads to insufficient oxygen supply and immunity, exacerbated bacterial infection, and local inflammation. High levels of reactive oxygen species (ROS) produced by immune cells in the wound activate nuclear factor κB and significantly increase the expression of inflammatory mediators interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), resulting in chronic inflammation. On the one hand, poor angiogenesis and bacterial infection in the skin or limbus may lead to hypoxia and hinder wound healing. On the other hand, prolonged wound healing time, impaired immunity, and a hyperglycemic environment make wounds more susceptible to infection. Therefore, after infection occurs, simultaneous eradication of bacteria and alleviation of inflammation are crucial.

[0004] Rapid sterilization can inhibit bacterial biofilm formation and improve tissue damage caused by oxidative stress. Oxidative stress exacerbates the inflammatory response, leading to corneal tissue damage. Eliminating oxidative stress while killing bacteria to reduce inflammation can promote the repair of infected wounds.

[0005] In current technologies, antibiotics are used to treat bacterial corneal ulcers. However, the presence of bacterial biofilms significantly enhances bacterial resistance to antibiotics, making infection difficult to eradicate. Furthermore, the high expression of various enzymes in the infected area, such as phosphatases, phospholipids, toxins, lipases, and proteases, leads to irreversible damage to the corneal tissue and reduced light transmittance. Anti-inflammatory hormones are typically used about a week after antibiotic treatment. However, these drugs also have significant side effects. For example, glucocorticoids can induce cataracts and ocular hypertension, while nonsteroidal anti-inflammatory drugs (NSAIDs) can ablate the cornea. Therefore, there is an urgent need for a drug or material with antibacterial, antioxidant, and anti-inflammatory effects that does not induce resistance, to achieve effective treatment for refractory corneal ulcers. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a metal coordination polymer nanoparticle based on its preparation and application. Based on iron ions as the coordination center, this invention sequentially adds curcumin, a hydrophobic substance with a polyphenol structure, and tannic acid, a hydrophilic substance, to form negatively charged nanoparticles through coordination with ferric ions via the polyphenol or diketone structure. Finally, a layer of polylysine is coated onto the outside of the nanoparticles. The synergistic effect of the components in the metal coordination polymer nanoparticles of this invention results in a product with excellent antioxidant, anti-inflammatory, and antibacterial effects. The metal coordination polymer nanoparticles of this invention are used in corneal tissue engineering, particularly for the preparation of drugs or formulations for treating bacterial keratitis.

[0007] This invention is achieved through the following technical solution: A method for preparing metal-coordinated polymer nanoparticles includes the following steps: 1) Under stirring conditions, polyvinylpyrrolidone and iron salt are mixed in a solvent, the pH is adjusted to 8-9, and an ethanol solution of curcumin is added dropwise. After the addition is complete, stirring is continued for 6-12 hours to obtain iron-based curcumin. The iron salt is ferric chloride with or without water of crystallization. The solvent is an ethanol solution with a volume fraction of 95%-100%. The molar ratio of curcumin to iron salt is 2.6-3.2:1. The molar ratio of polyvinylpyrrolidone to iron salt is (4~4.5):1; the average molecular weight of polyvinylpyrrolidone is 9000~11000; the ethanol solution of curcumin is obtained by dissolving curcumin in ethanol, and its concentration is 1~3 mg / mL; the stirring speed is 800~1000 rpm; before adding the ethanol solution of curcumin, the stirring speed is adjusted to 1400~1600 rpm; 2) Tannic acid is reacted with iron-based curcumin to obtain tannic acid-coordinated iron-based curcumin; the mass ratio of iron-based curcumin to tannic acid is (7.5~8.5): 5; 3) In the reaction medium, polylysine is reacted with tannic acid-coordinated iron-based curcumin to obtain metal-coordinated polymer nanoparticles; the mass ratio of polylysine to tannic acid-coordinated iron-based curcumin is (2~5):1.

[0008] The iron-based curcumin was obtained by the following method: S1. Under stirring conditions, mix the ethanol solution of polyvinylpyrrolidone with the ethanol solution of iron salt, and adjust the pH to 8-9; increase the stirring speed, add the ethanol solution of curcumin dropwise, and continue stirring for 6-12 hours after the addition is complete. Dialyze with water to obtain iron-based curcumin; the mass-to-volume ratio of polyvinylpyrrolidone to ethanol in the ethanol solution of polyvinylpyrrolidone is 0.8 g: (70-100) mL; the ethanol is a 95%-100% ethanol solution by volume; the concentration of the ethanol solution of iron salt is 0.3-0.6 mg / mL, which is obtained by dissolving iron salt in a 95%-100% ethanol solution by volume.

[0009] The pH adjustment refers to using a 1M Tris-HCl solution. After adding the Tris-HCl solution, the mixture is stirred at a constant speed for 25-35 minutes.

[0010] The dialysis membrane is a 3500 Da dialysis membrane. The dialysis time is 2-3 days, and the water is changed 3-4 times a day during the dialysis process.

[0011] All operations after adding curcumin must be carried out in the dark, as curcumin is easily decomposed when exposed to light.

[0012] The specific preparation steps for tannic acid-coordinated iron-based curcumin (Fe-Cur-TA) in step 2) are as follows: In step 1), iron-based curcumin is used in solution form. The pH of the iron-based curcumin solution is adjusted to 8-9, and then it is stirred and reacted with tannic acid aqueous solution. After dialysis, tannic acid-coordinated iron-based curcumin is obtained. The concentration of the iron-based curcumin solution is 0.1~0.3 mg / mL; the mass-to-volume ratio of tannic acid to water in the tannic acid aqueous solution is 5 mg:(1~2) mL; the stirring reaction conditions are 1400~1600 rpm for 1.5~2.5 h. The mass ratio of tannic acid to iron-based curcumin is (7.5~8.5):5. The pH adjustment refers to adjustment using 1M Tris-HCl solution.

[0013] The dialysis membrane is a 3500 Da dialysis membrane. The dialysis time is 1-2 days. The reaction and dialysis must be carried out in a dark environment.

[0014] The molecular weight of the polylysine mentioned in step 3) is 2000-5000.

[0015] The specific steps of step 3) are as follows: In step 2), tannic acid-coordinated iron-based curcumin is used in solution form. The polylysine aqueous solution is stirred and reacted with the tannic acid-coordinated iron-based curcumin solution to obtain metal coordination polymer nanoparticles.

[0016] The mass-to-volume ratio of polylysine to water in the polylysine aqueous solution is 10 mg: (1~2) mL. The concentration of the tannic acid-coordinated iron-based curcumin solution is 0.1~0.3 mg / mL.

[0017] The stirring reaction is carried out at a speed of 1400~1600 rpm for 1.5~2.5 hours.

[0018] After the reaction was completed, the sample was dialyzed in water (7000 Da).

[0019] The retention solution after dialysis contains metal coordination polymer nanoparticles.

[0020] The metal coordination polymer nanoparticles of the present invention are used in corneal tissue engineering, particularly in the preparation of drugs for treating bacterial keratitis.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The raw materials of the present invention are derived from natural substances, which are easy to obtain. The preparation method has the advantages of simple circuit and convenient operation, low reagent toxicity and easy product preservation. (2) The components of the metal coordination polymer nanoparticles of this invention are all natural materials, which have good biocompatibility and can remain in biological tissues to play a role without causing other damage.

[0022] (3) The metal coordination polymer nanoparticles of the present invention have a high efficiency of ROS scavenging ability, which can quickly remove reactive oxygen species in the lesion site, reduce the damage caused by oxidative stress, and have broad application prospects. (4) The metal coordination polymer nanoparticles of the present invention have a unique bactericidal effect and have a strong bactericidal ability against various infectious diseases caused by Escherichia coli and Staphylococcus aureus. Attached Figure Description

[0023] Figure 1 This diagram illustrates the synthetic route of iron-based curcumin, tannic acid-coordinated iron curcumin, and polylysine-modified nanoparticles in this invention. In this diagram, a represents the preparation reaction of Fe-Cur (iron-based curcumin); b represents the preparation reaction of Fe-Cur-TA (tannic acid-coordinated iron curcumin); and c represents the preparation reaction of Fe-Cur-TA@PL nanoparticles (metal-coordinated polymer nanoparticles), where PL represents polylysine. Figure 2 The DLS particle size (left) and Zeta potential (right) of iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), and metal coordination polymer nanoparticles (FCTP) in Example 1 are shown. Figure 3The diagram shows the free radical scavenging effects of iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), and metal coordination polymer nanoparticles (FCTP) in Example 1. Figure 4 The images show the biocompatibility of the metal coordination polymer nanoparticles (FCTP) in Example 1; left image: L929 cells, right image: RCEC cells; Figure 5 The diagram shows the antibacterial performance of iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), and metal coordination polymer nanoparticles (FCTP) in Example 1. Figure 6 The therapeutic effect of nanoparticle-loaded microparticles on rabbit corneal ulcers. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. The raw materials and reagents used in the following examples are all commercially available.

[0025] Figure 1 This is a schematic diagram of the synthesis route of iron-based curcumin, tannic acid-coordinated iron curcumin, and polylysine-modified nanoparticles in this invention. Among them, a is a schematic diagram of the preparation reaction of Fe-Cur (iron-based curcumin); b is a schematic diagram of the preparation reaction of Fe-Cur-TA (tannic acid-coordinated iron curcumin); and c is a schematic diagram of the preparation reaction of Fe-Cur-TA@PL nanoparticles (metal coordination polymer nanoparticles).

[0026] Example 1 (1) At room temperature and under stirring at 900 rpm, 0.8 g of polyvinylpyrrolidone (molecular weight 10000) was dissolved in 80 mL of ethanol (95%). After dissolution for 10 min, and after the polyvinylpyrrolidone was completely dissolved, 10 mL of ferric chloride hexahydrate-ethanol solution (0.5 mg / mL) was slowly added to the solution, and 1.5 mL of 1M Tris-HCl solution was quickly added to adjust the pH to 8-9. The stirring speed was increased to 1500 rpm and kept stirring for 30 min. Curcumin-ethanol solution (2 mg / mL) was slowly added dropwise (at a rate of 20-50 drops / min) to the above reaction solution and stirred for 12 h. After the above reaction is completed, the solution is placed in deionized water and dialyzed at a water change frequency of 3 times a day (3500 Da). After dialysis for 2 days, the obtained product, iron-based curcumin, is collected. Iron-based curcumin exists in the form of nanoparticles in the retention solution and is stored in a refrigerator at 4 degrees Celsius. Because curcumin decomposes when exposed to light, the entire preparation process must be kept away from light.

[0027] (2) Take 40 mL of the iron-based curcumin solution prepared above (concentration 0.2 mg / mL, solvent is water), stir at 500 rpm for 10 min, add 0.2 mL of 1 M Tris-HCl buffer solution, adjust the pH to 8-9, dissolve 5 mg of tannic acid in 1 mL of deionized water, and dissolve at 500 rpm for 10 min. After the tannic acid is completely dissolved, add 1 mL of tannic acid solution to the iron-based curcumin nanoparticle solution, stir at 1500 rpm for 2 h, and dialyze in deionized water (3500 Da) after the reaction is completed. After dialysis for 1 day, collect the obtained product tannic acid coordinated iron-based curcumin. Tannic acid coordinated iron-based curcumin exists in the form of nanoparticles in the retention solution and is stored in a refrigerator at 4 degrees Celsius.

[0028] (3) Dissolve 20 mg of polylysine (molecular weight 4000) in 2 mL of deionized water at 500 rpm for 10 min to obtain a polylysine solution. Add 1 mL of polylysine solution to 20 mL of tannic acid-coordinated iron-based curcumin solution (0.2 mg / mL, solvent is water), stir at 1500 rpm for 2 h, and after the reaction is completed, dialyze in deionized water (7000 Da). After dialyzing for 1 day, collect the obtained metal coordination polymer nanoparticles and store them in a refrigerator at 4 degrees Celsius.

[0029] Iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), and metal coordination polymer nanoparticles (FCTP) were diluted at different ratios to test the particle size, zeta potential, antioxidant and antibacterial properties of the nanoparticles, as well as to treat a rabbit corneal ulcer model.

[0030] Performance testing: (1) After balancing the solutions of iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), and metal coordination polymer nanoparticles (FCTP), the solutions were centrifuged at 10,000 rpm for 40 min. The centrifuged solutions were then filtered through a 0.22 μm aqueous membrane, and DLS and Zeta potential were measured using a multi-angle particle size and high-sensitivity Zeta potential analyzer. The results are shown in […]. Figure 2 Repeat 3-4 times for all groups.

[0031] (2) Iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), and metal coordination polymer nanoparticles (FCTP) solutions were diluted and used to scavenge hydroxyl radicals, DPPH radicals, and ABTS radicals at the same concentration of 0.03 mg / mL. The scavenging efficiency of the free radicals was obtained. The scavenging effect was tested over time at this concentration, and the results are shown in […]. Figure 3 Repeat 3-4 times for all groups.

[0032] (3) The obtained FCTP nanoparticle solution was diluted with basal medium (DMEM high glucose medium) to 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL for L929 cells / RCEC cells. After one day of co-culture, cell viability and proliferation were tested using CCK-8 reagent. The results are shown in the figure. Figure 4 .

[0033] (4) Dilute the Escherichia coli / Staphylococcus aureus prepared overnight to 1*10. 6 CFU / mL was mixed with a nanoparticle solution (iron-based curcumin (FC), tannic acid-coordinated iron curcumin (FCT), metal coordination polymer nanoparticles (FCTP) solution, and PL as polylysine) diluted 1:1 with LB medium at a volume ratio and incubated for 3 h. 20 μL of the incubated suspension was plated and incubated at 37°C for 24 h. Colony counting was then performed, and the results are shown below. Figure 5 .

[0034] (5) Take 1*10g of Staphylococcus aureus that has been shaken overnight. 9 20 μL of FCTP eye drops (CFU / mL) was applied to the corneal lesion (7 mm in diameter) for 5 minutes, repeated twice with a 5-minute interval. The corneal ulcer model was successfully established the next day. Experimental groups were set up: levofloxacin eye drops group, FCTP eye drops group (FCTP particle concentration: 200 μg / mL), FCTP microneedles 100 group (FCTP particle concentration: 100 μg / mL), FCTP microneedles 200 group (FCTP particle concentration: 200 μg / mL), and a model group (no treatment). The treatment effects were compared as follows: Figure 6 .

[0035] The iron-based curcumin component of this invention is formed by coordination between the metal and the diketone structure in the curcumin molecule. The tannic acid added subsequently, due to its abundant catechol structure, easily coordinates with ferric ions in an alkaline environment to form nanoparticles with a negative potential. Finally, polylysine is added and encapsulates the surface of the nanoparticles through electrostatic interaction.

[0036] The coordination polymer nanoparticles based on natural small molecules of curcumin and tannic acid in this invention have good biocompatibility. The combined use of curcumin and tannic acid gives the nanoparticles extremely strong antioxidant and anti-inflammatory capabilities, effectively scavenging various free radicals. The coating of polylysine enhances the overall antibacterial properties, and it has strong bactericidal ability against Escherichia coli and Staphylococcus aureus. For diseases such as bacterial keratitis, it can achieve both rapid antibacterial action and effective anti-inflammatory and antioxidant effects, preventing the further development of inflammation and promoting corneal tissue regeneration and wound healing.

[0037] The above specific embodiments are all feasible implementations of the present invention, and further describe in detail the purpose, technical solution and beneficial effects of the present invention. However, the implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for preparing metal-coordinated polymer nanoparticles, characterized in that: Includes the following steps: 1) Under stirring conditions, polyvinylpyrrolidone and iron salt are mixed in a solvent, the pH is adjusted to 8-9, and an ethanol solution of curcumin is added dropwise. After the addition is complete, stirring is continued for 6-12 hours to obtain iron-based curcumin. The iron salt is ferric chloride with or without water of crystallization. The solvent is an ethanol solution with a volume fraction of 95%-100%. The molar ratio of curcumin to iron salt is 2.6-3.2:

1. The molar ratio of polyvinylpyrrolidone to iron salt is (4~4.5):1; the average molecular weight of polyvinylpyrrolidone is 9000~11000; before adding the ethanol solution of curcumin, adjust the stirring speed to 1400~1600 rpm. 2) Tannic acid is reacted with iron-based curcumin to obtain tannic acid-coordinated iron-based curcumin; the mass ratio of iron-based curcumin to tannic acid is (7.5~8.5): 5; 3) In the reaction medium, polylysine is reacted with tannic acid-coordinated iron-based curcumin to obtain metal-coordinated polymer nanoparticles; the mass ratio of polylysine to tannic acid-coordinated iron-based curcumin is (2~5):

1.

2. The preparation method of metal-coordinated polymer nanoparticles according to claim 1, characterized in that: The curcumin ethanol solution mentioned in step 1) refers to the curcumin solution obtained by dissolving curcumin in ethanol, with a concentration of 1~3 mg / mL; The iron-based curcumin described in step 1) is obtained by the following method: under stirring at 800-1000 rpm, an ethanol solution of polyvinylpyrrolidone is mixed with an ethanol solution of iron salt, and the pH is adjusted to 8-9; the stirring speed is increased to 1400-1600 rpm, and an ethanol solution of curcumin is added dropwise. After the addition is complete, stirring is continued for 6-12 hours, and the iron-based curcumin is obtained by water dialysis. The mass-to-volume ratio of polyvinylpyrrolidone to ethanol in the polyvinylpyrrolidone ethanol solution is 0.8 g: (70~100) mL; the ethanol is a 95%~100% ethanol solution by volume; the concentration of the ethanol solution of the iron salt is 0.3~0.6 mg / mL, which is obtained by dissolving the iron salt in a 95%~100% ethanol solution by volume.

3. The method for preparing metal coordination polymer nanoparticles according to claim 2, characterized in that: The pH adjustment refers to using a 1M Tris-HCl solution. After adding the Tris-HCl solution, the mixture is stirred at a constant speed for 25-35 minutes. The dialysis membrane is a 3500 Da dialysis membrane.

4. The method for preparing metal coordination polymer nanoparticles according to claim 1, characterized in that: The specific preparation steps of tannic acid-coordinated iron-based curcumin in step 2) are as follows: In step 1), the iron-based curcumin is used in solution form. The pH of the iron-based curcumin solution is adjusted to 8-9, and then it is stirred and reacted with tannic acid aqueous solution. After dialysis, tannic acid-coordinated iron-based curcumin is obtained. The stirring reaction conditions are 1400~1600 rpm for 1.5~2.5 h.

5. The preparation method of metal-coordinated polymer nanoparticles according to claim 4, characterized in that: The concentration of the iron-based curcumin solution is 0.1~0.3 mg / mL; the mass-to-volume ratio of tannic acid to water in the tannic acid aqueous solution is 5 mg: (1~2) mL; The pH adjustment refers to adjustment using a 1M Tris-HCl solution; The dialysis membrane is a 3500 Da dialysis membrane.

6. The method for preparing metal coordination polymer nanoparticles according to claim 1, characterized in that: The molecular weight of the polylysine mentioned in step 3) is 2000-5000; The specific steps of step 3) are as follows: In step 2), tannic acid-coordinated iron-based curcumin is used in solution form. The polylysine aqueous solution is stirred and reacted with the tannic acid-coordinated iron-based curcumin solution to obtain metal coordination polymer nanoparticles. The stirring reaction conditions are 1400~1600 rpm for 1.5~2.5 h.

7. The method for preparing metal coordination polymer nanoparticles according to claim 6, characterized in that: The mass-to-volume ratio of polylysine to water in the polylysine aqueous solution is 10 mg: (1~2) mL; the concentration of the tannic acid-coordinated iron-based curcumin solution is 0.1~0.3 mg / mL; After the reaction was completed, the sample was dialyzed in water using a 7000 Da dialysis membrane.

8. A metal coordination polymer nanoparticle obtained by the preparation method according to any one of claims 1 to 7.

9. The application of the metal coordination polymer nanoparticles according to claim 1, characterized in that: The metal-coordinated polymer nanoparticles are used to prepare drugs for treating bacterial keratitis or wound infections.

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