A thujaplicin-loaded potassium-doped metal-organic framework antibacterial nanoparticle, and a preparation method and application thereof

Abstract

The application discloses a kind of potassium-doped metal organic framework antibacterial nanoparticles loaded with thujaplicin oil and a preparation method and application thereof.The antibacterial nanoparticles of the application include potassium-doped metal organic framework material and thujaplicin oil encapsulated in the metal organic framework material, wherein the metal organic framework material is ZIF-8.The application encapsulates and loads thujaplicin oil by using the high-pore structure of ZIF-8 material, which can reduce the loss of thujaplicin oil caused by instability and volatility, and the incorporation of potassium ions, which can affect the physiology of bacteria, can inhibit the growth of Staphylococcus aureus and Escherichia coli at a relatively low concentration.Therefore, the POEO@K-ZIF-8 prepared by the application can be applied to the preparation of antibacterial drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine, and particularly relates to a potassium-doped metal organic framework antibacterial nanoparticle loaded with Platycladus orientalis essential oil and a preparation method and application thereof. BACKGROUND

[0002] Platycladus orientalis is a coniferous evergreen tree, also known as Chinese arborvitae or yellow cypress, which is widely distributed in China and has important economic value. Platycladus orientalis essential oil (POEO) has the effects of insect resistance, antibiosis, oxidation resistance, anticancer and the like, and will have good application prospects in the development of natural insecticides, preservatives and health products. However, the practical application of POEO is limited to some extent due to its poor water solubility, unstable composition and easy volatilization.

[0003] Zeolitic Imidazolate Framework-8 (ZIF-8) is a kind of metal organic framework material (MOFs), which can be used as an ideal carrier for preparing antibacterial materials due to its good biocompatibility, simple synthesis, high porosity, large loading capacity, easy modification and antibacterial activity.

[0004] Potassium ions can inhibit the growth of bacteria by affecting bacterial metabolism and cell membranes, and therefore have a certain antibacterial effect. Therefore, potassium-doped ZIF-8 can improve its antibacterial performance. How to effectively encapsulate POEO and load potassium ions is a technical problem to be solved by the application. SUMMARY

[0005] The application aims to provide a potassium-doped metal organic framework antibacterial nanoparticle loaded with Platycladus orientalis essential oil and a preparation method and application thereof. The application encapsulates and loads POEO by using the high-porosity structure of ZIF-8 material, and modifies it with potassium ions to improve the application limitations of POEO and improve the antibacterial effect.

[0006] TECHNICAL SOLUTION

[0007] The application provides a potassium-doped metal organic framework antibacterial nanoparticle loaded with Platycladus orientalis essential oil, which comprises a potassium-doped metal organic framework material and Platycladus orientalis essential oil encapsulated in the metal organic framework material, wherein the metal organic framework material is ZIF-8.

[0008] The application further provides a preparation method of the above-mentioned potassium-doped metal organic framework antibacterial nanoparticle loaded with Platycladus orientalis essential oil, characterized by comprising the following steps:

[0009] (1) dispersing the Chinese arborvitae essential oil in a solvent to obtain a Chinese arborvitae essential oil dispersion, dissolving zinc nitrate hexahydrate in the dispersion, and stirring;

[0010] (2) dispersing 2-methylimidazole in a solvent to obtain a 2-methylimidazole dispersion;

[0011] (3) mixing the two dispersions and stirring at room temperature to react; after the reaction is completed, solid-liquid separation is performed, the precipitate is collected, and washing and drying are performed to obtain metal-organic framework antibacterial nanoparticles loaded with Chinese arborvitae essential oil, denoted as POEO@ZIF-8 nanoparticles;

[0012] (4) dispersing the POEO@ZIF-8 nanoparticles obtained in step (3) and potassium nitrate in a solvent respectively, mixing, and stirring at room temperature to react; after the reaction is completed, solid-liquid separation is performed, the precipitate is collected, and washing and drying are performed to obtain potassium-doped metal-organic framework antibacterial nanoparticles loaded with Chinese arborvitae essential oil, denoted as POEO@K-ZIF-8.

[0013] Preferably, in steps (1) and (2), the solvent is anhydrous methanol.

[0014] Preferably, in step (4), the solvent is a 30%-50% ethanol solution.

[0015] Preferably, in step (3), the stirring time is 2-3 h, the drying temperature is 50-70 DEG C, and the drying time is 8-12 h.

[0016] Preferably, in step (4), the stirring time is 2-3 h, the drying temperature is 50-70 DEG C, and the drying time is 8-12 h.

[0017] Preferably, the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:60-100.

[0018] Preferably, in step (1), the mass ratio of Chinese arborvitae essential oil to zinc nitrate hexahydrate is 1:1.6-8.

[0019] Preferably, in step (4), the mass ratio of potassium nitrate to POEO@ZIF-8 is 1:4-6.

[0020] The application also provides a use of the above-mentioned potassium-doped metal-organic framework antibacterial nanoparticles loaded with Chinese arborvitae essential oil in the preparation of antibacterial drugs.

[0021] Advantages:

[0022] The application utilizes the high pore structure of ZIF-8 material to encapsulate and load POEO, and is modified with potassium ions to improve the application limitation of POEO. Experiments show that: compared with POEO and POEO@ZIF-8, POEO@K-ZIF-8 not only solves the problems of easy volatilization and low bioavailability of POEO, but also the doped potassium can affect bacterial metabolism and can inhibit the growth of Staphylococcus aureus and Escherichia coli at a relatively low concentration. Therefore, the POEO@K-ZIF-8 prepared by the application can be applied to the preparation of antibacterial drugs. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The infrared spectrum of the POEO@ZIF-8 and POEO@K-ZIF-8 nanomaterials prepared by the application;

[0024] Figure 2 The influence of different reactant ratios on the particle size of POEO@ZIF-8;

[0025] Figure 3 The influence of different reactant ratios on the zeta potential of POEO@ZIF-8;

[0026] Figure 4 The influence of different POEO dosages on the particle size of POEO@ZIF-8;

[0027] Figure 5 The influence of different POEO dosages on the zeta potential of POEO@ZIF-8;

[0028] Figure 6 The influence of different POEO dosages on the drug loading capacity of POEO@ZIF-8;

[0029] Figure 7 The influence of different potassium nitrate dosages on the particle size of POEO@K-ZIF-8;

[0030] Figure 8 The influence of different potassium nitrate dosages on the zeta potential of POEO@K-ZIF-8;

[0031] Figure 9 The influence of each component of POEO@K-ZIF-8 on the survival rate of Staphylococcus aureus;

[0032] Figure 10 The influence of POEO@K-ZIF-8 prepared by the application on the survival rate of Staphylococcus aureus;

[0033] Figure 11 The influence of each component of POEO@K-ZIF-8 on the survival rate of Escherichia coli;

[0034] Figure 12Effect of POEO@K-ZIF-8 prepared in the application on survival rate of E. coli. DETAILED DESCRIPTION

[0035] The technical solutions of the application will be described in detail below through specific examples, but the protection scope of the application is not limited to the examples.

[0036] In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available products unless otherwise specified.

[0037] The cypress essential oil in the examples of the application is from Sreevarth Spinning Mills Limited.

[0038] The preparation method of the blank ZIF-8 nanoparticles in the application is as follows: 4.42 g of 2-methylimidazole is dissolved in 8 mL of methanol to obtain solution A, 200 mg of zinc nitrate hexahydrate is dissolved in 0.8 mL of methanol to obtain solution B, and the two solutions are mixed and stirred at room temperature for 2 h. After the reaction is completed, the reaction product is centrifuged at a speed of 12000 rpm and a temperature of 20℃ for 15 min, the supernatant is discarded, the precipitate is collected and washed for 3 times, and then dried in a 60℃ oven for 12 h to obtain ZIF-8 nanoparticles.

[0039] Preparation of potassium-doped metal-organic framework antibacterial nanoparticles loaded with cypress essential oil

[0040] (1) 100 mg of cypress essential oil is dispersed in 0.8 mL of anhydrous methanol to obtain a cypress essential oil dispersion, and 200 mg of zinc nitrate hexahydrate is dissolved in the dispersion and stirred for 5 min;

[0041] (2) 4.42 g of 2-methylimidazole is dispersed in 8 mL of anhydrous methanol to obtain a 2-methylimidazole dispersion;

[0042] (3) The two dispersions are mixed and stirred at room temperature for 2 h; after the reaction is completed, the reaction product is centrifuged at a speed of 12000 rpm and a temperature of 20℃ for 15 min, the supernatant is discarded, the precipitate is collected and washed for 3 times, and then dried in a 60℃ oven for 12 h to obtain 84.9 mg of metal-organic framework antibacterial nanoparticles loaded with cypress essential oil, which is recorded as POEO@ZIF-8 nanoparticles;

[0043] (4) 18 mg POEO@ZIF-8 nanoparticles and 3 mg potassium nitrate were dispersed in 2 mL of 30% ethanol solution, respectively, and stirred at room temperature for 2 h after mixing; after the reaction was completed, the reaction was centrifuged at 12000 rpm, the centrifuge temperature was 20℃, the centrifugation time was 15 min, the supernatant was discarded, the precipitate was collected and washed 3 times, and dried in a 60℃ oven for 12 h to obtain 19.2 mg of cypress essential oil loaded potassium doped metal organic framework antibacterial nanoparticles, recorded as POEO@K-ZIF-8.

[0044] The particle size of POEO@ZIF-8 nanoparticles was 242.77 nm, and the particle size of POEO@K-ZIF-8 was 244.17 nm, which were analyzed and determined by a Malvern particle size analyzer.

[0045] The potential of POEO@ZIF-8 was 34.07 mV, and the potential of POEO@K-ZIF-8 was 23.77 mV.

[0046] The prepared POEO@ZIF-8 powder was taken, 3 mL of n-hexane was added, and ultrasonic was performed for 30 min, then centrifugation was performed, the supernatant was taken, and the above steps were repeated 3 times. The supernatant was filtered by a filter membrane and sent to GC-MS for determination of concentration. The loading amount of POEO in POEO@ZIF-8 was determined by using the standard curve of the main component (cypressol) of cypress essential oil. The drug loading amount = (the mass of POEO loaded into POEO@ZIF-8 / the total mass of POEO@ZIF-8) * 100%. The drug loading amount of POEO@ZIF-8 was 7.14%.

[0047] Figure 1 The infrared spectra of blank ZIF-8, POEO@ZIF-8 and POEO@K-ZIF-8.

[0048] As shown in Figure 1 , the absorption peaks at 3132 cm -1 and 2926 cm -1 belong to the stretching vibration peaks of methyl and imidazole ring C-H, respectively; the absorption peaks observed in the range of 1362-1458 cm -1 and 1569 cm -1 belong to the stretching of the entire imidazole ring and C=N, respectively, and 421 cm -1 is the Zn-N bond absorption peak stretching vibration specific to ZIF-8 material, indicating that the ZIF-8 framework of both is complete, and the loading process does not destroy the structure of ZIF-8 nanoparticles, while a weak absorption peak appears at 3207.88 cm -1 in the POEO@ZIF-8 infrared spectrum, which corresponds to the stretching vibration absorption peak of hydroxyl (-OH) in POEO, and 675.21 cm -1The absorption peak that appears is caused by the vibration resulting from the interaction between potassium ions and surrounding molecules or ions.

[0049] Example 2 investigated the effect of different molar ratios of 2-methylimidazole to zinc nitrate hexahydrate on POEO@ZIF-8 nanoparticles.

[0050] The molar ratio of 2-methylimidazole to zinc nitrate hexahydrate was adjusted to 20:1, 40:1, 60:1, 80:1, 100:1, and 150:1, and the dosage of POEO was 100 mg to synthesize POEO@ZIF-8.

[0051] 2-Methylimidazole and 100 mg POEO were dissolved in 8 mL and 0.8 mL of methanol, respectively, to obtain solutions A and B. 200 mg zinc nitrate hexahydrate was dissolved in solution B, stirred for 5 min, and then mixed with solution A. The mixture was stirred and reacted at room temperature for 2 h.

[0052] After the reaction was completed, the reactants were centrifuged at 12,000 rpm, 20°C, and 15 min. The supernatant was discarded, the precipitate was collected, washed three times, and dried in a 60°C oven for 12 h to obtain POEO@ZIF-8 nanoparticles.

[0053] The particle size distribution was analyzed using a Malvern particle size analyzer, and the results are shown in the figure. Figure 2 , Figure 3 .

[0054] like Figure 2 As shown, the particle size of POEO@ZIF-8 prepared when the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is between 1:20 and 100 is appropriate, and the aggregation index comparison shows that each group of POEO@ZIF-8 is uniformly dispersed in the system.

[0055] like Figure 3 As shown, when the molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is between 1:60 and 100, the POEO@ZIF-8Zeta potential prepared is above 20 mV, and the dispersion system has good stability. Moreover, when the ratio of the two is 1:80, the Zeta potential reaches the highest level, indicating good stability.

[0056] Example 3 investigated the effect of different POEO dosages on POEO@ZIF-8 nanoparticles.

[0057] The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole was 1:80, the amount of zinc nitrate hexahydrate added was 200 mg, and the dosage of POEO was adjusted to 25 mg, 50 mg, 75 mg, 100 mg, and 125 mg respectively. The preparation steps were the same as in Example 1.

[0058] The particle size distribution was analyzed using a Malvern particle size analyzer, and the results are shown in the figure. Figure 4 , Figure 5 .

[0059] like Figure 4 As shown, when the dosage is 25-100mg, the POEO@ZIF-8 particles prepared have a suitable particle size and are evenly dispersed.

[0060] like Figure 5 As shown, the zeta potentials of POEO@ZIF-8 prepared with dosages of 25-125 mg were all around 30 mV, indicating good stability of the dispersion system. In contrast, the zeta potential reached its highest value at a dosage of 100 mg, demonstrating good stability.

[0061] Take POEO@ZIF-8 powder prepared with different dosages, add 3 mL of n-hexane, sonicate for 30 min, centrifuge, take the supernatant, repeat 3 times, filter the supernatant through a filter membrane, and send it to GC-MS to determine the concentration. Use the standard curve of the main component of arborvitae essential oil (arborvitae alcohol) to determine the loading of POEO in POEO@ZIF-8. The loading amount = (mass of POEO loaded into POEO@ZIF-8 / total mass of POEO@ZIF-8) * 100%.

[0062] See results Figure 6 .

[0063] like Figure 6 As shown, with the increase of POEO dosage, the POEO loading in POEO@ZIF-8 also increases. When the POEO dosage is 125mg, the POEO loading in POEO@ZIF-8 reaches 11.06%.

[0064] Example 4 investigated the effect of different POEO@ZIF-8 to potassium nitrate mass ratios on POEO@K-ZIF-8 nanoparticles.

[0065] The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole was 1:80, the amount of zinc nitrate hexahydrate added was 200 mg, the amount of POEO was 100 mg, and the mass ratio of POEO@ZIF-8 to potassium nitrate was adjusted to 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1. The preparation steps were the same as in Example 1.

[0066] The particle size distribution was analyzed using a Malvern particle size analyzer, and the results are shown in the figure. Figure 7 , Figure 8 .

[0067] like Figure 7 As shown, except for a mass ratio of 1:1, the POEO@K-ZIF-8 particles prepared by other mass ratios have basically the same size and are uniformly distributed in the solution system.

[0068] As Figure 8 shown, the absolute value of Zeta potential is higher when the mass ratio of POEO@ZIF-8 to potassium nitrate is 4:1 and 6:1, indicating that the prepared POEO@K-ZIF-8 has higher stability.

[0069] Example 5 investigates the antibacterial effect of POEO, POEO@ZIF-8 and POEO@K-ZIF-8 on Staphylococcus aureus

[0070] A 5% DMSO solution was used as a solvent to prepare POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution with a concentration of 1 mg / mL. Sterile water was used as a control group. The S.aureus bacterial solution in the logarithmic growth phase was diluted 50 times with tryptone soya broth liquid medium (TSB), and then mixed with 1 mg / mL of POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution at a volume ratio of 1:1. The mixture was placed in a shaking bed (250 rpm, 37°C) for 1.5 h.

[0071] After co-incubation, the sample was diluted 20,000 times, and 100 μL was uniformly coated on a solid agar plate. After 24 h, the number of colonies was counted and compared with the blank control group to calculate the survival rate of bacteria.

[0072] As Figure 9 shown, compared with the blank control group, the inhibition effect of POEO solution on S.aureus is about 80%, the inhibition effect of ZIF-8 solution on S.aureus is about 69%, the inhibition effect of POEO@ZIF-8 on S.aureus is about 84%, and the inhibition effect of POEO@K-ZIF-8 on S.aureus is about 98%.

[0073] The present application also studies the minimum inhibitory concentration of the prepared POEO@K-ZIF-8 on Staphylococcus aureus.

[0074] A 5% DMSO solution was used as a solvent to prepare POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution with a concentration of 1 mg / mL. Sterile water was used as a control group. The S.aureus bacterial solution in the logarithmic growth phase was diluted 50 times with tryptone soya broth liquid medium (TSB), and then mixed with 1 mg / mL of POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution at a volume ratio of 1:1. The mixture was placed in a shaking bed (250 rpm, 37°C) for 1.5 h.

[0075] The sample after co-incubation is diluted 20,000 times, 100 muL is uniformly coated on a solid agar plate, and the number of bacteria is counted after 24 hours, compared with the blank control group, and the survival rate of bacteria is calculated.

[0076] As shown in Figure 10 , when the concentration is 0.1 mg / ml, the antibacterial effect is 89.34%, which indicates that the minimum inhibitory concentration of POEO@K-ZIF-8 to Staphylococcus aureus is about 0.1 mg / mL.

[0077] Example 6 investigates the antibacterial effect of POEO, POEO@ZIF-8 and POEO@K-ZIF-8 on E. coli

[0078] A 5% DMSO solution is used as a solvent to prepare POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution with a concentration of 1 mg / mL, and sterile water is used as a control group. The E. coli bacterial solution in the logarithmic growth phase is diluted 50 times with LB liquid medium, and then mixed with 1 mg / mL of POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution in a volume ratio of 1:1, and then placed in a shaking bed (250 rpm, 37 DEG C) for co-incubation for 1.5 hours.

[0079] The sample after co-incubation is diluted 20,000 times, 100 muL is uniformly coated on a solid agar plate, and the number of bacteria is counted after 24 hours, compared with the blank control group, and the survival rate of bacteria is calculated.

[0080] As shown in Figure 11 , the inhibition effect of POEO@K-ZIF-8 on E. coli is about 98%.

[0081] The present application also studies the minimum inhibitory concentration of the prepared POEO@K-ZIF-8 to E. coli.

[0082] A 5% DMSO solution is used as a solvent to prepare POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution with a concentration of 1 mg / mL, and sterile water is used as a control group. The E. coli bacterial solution in the logarithmic growth phase is diluted 50 times with LB liquid medium, and then mixed with 1 mg / mL of POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution in a volume ratio of 1:1, and then placed in a shaking bed (250 rpm, 37 DEG C) for co-incubation for 1.5 hours.

[0083] The sample after co-incubation is diluted 20,000 times, 100 muL is uniformly coated on a solid agar plate, and the number of bacteria is counted after 24 hours, compared with the blank control group, and the survival rate of bacteria is calculated.

[0084] AsFigure 12 As shown, the antibacterial effect is 89.34% when the concentration is 0.3 mg / ml, indicating that the minimum inhibitory concentration of POEO@K-ZIF-8 on E. coli is about 0.3 mg / mL.

[0085] As mentioned above, although the present application has been illustrated and described with reference to specific preferred embodiments, it is not intended to be limited to the details shown, since various modifications and substitutions can be made without departing from the spirit of the application as defined in the appended claims.

Claims

1. A potassium-doped metal-organic framework antibacterial nanoparticle loaded with cypress essential oil, characterized in that, The antibacterial nanoparticles include a potassium-doped metal-organic framework material and arborvitae essential oil encapsulated in the potassium-doped metal-organic framework material, wherein the metal-organic framework material is ZIF-8.

2. The method for preparing potassium-doped metal-organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil according to claim 1, characterized in that, Includes the following steps: (1) Disperse arborvitae essential oil in a solvent to obtain arborvitae essential oil dispersion, dissolve zinc nitrate hexahydrate in the dispersion, and stir; (2) Disperse 2-methylimidazole in a solvent to obtain a 2-methylimidazole dispersion; (3) Mix the two dispersions and stir at room temperature; after the reaction is complete, separate the solid and liquid, collect the precipitate, wash and dry it to obtain metal-organic framework antibacterial nanoparticles loaded with cypress essential oil, which are denoted as POEO@ZIF-8 nanoparticles. (4) Disperse the POEO@ZIF-8 nanoparticles obtained in step (3) and potassium nitrate in a solvent, mix and stir at room temperature; after the reaction is completed, separate the solid and liquid, collect the precipitate, wash and dry to obtain potassium-doped metal-organic framework antibacterial nanoparticles loaded with cypress essential oil, denoted as POEO@K-ZIF-8.

3. The preparation method according to claim 2, characterized in that, In steps (1) and (2), the solvent is anhydrous methanol.

4. The preparation method according to claim 2, characterized in that, In step (4), the solvent is a 30%-50% ethanol solution.

5. The preparation method according to claim 2, characterized in that, In step (3), the stirring time is 2-3 hours, the drying temperature is 50-70°C, and the drying time is 8-12 hours.

6. The preparation method according to claim 2, characterized in that, In step (4), the stirring time is 2-3 hours, the drying temperature is 50-70°C, and the drying time is 8-12 hours.

7. The preparation method according to claim 2, characterized in that, The molar ratio of zinc nitrate hexahydrate to 2-methylimidazole is 1:60~100.

8. The preparation method according to claim 2, characterized in that, In step (1), the mass ratio of the arborvitae essential oil to zinc nitrate hexahydrate is 1:1.6-8.

9. The preparation method according to claim 2, characterized in that, In step (4), the mass ratio of potassium nitrate to POEO@ZIF-8 is 1:4-6.

10. The application of the potassium-doped metal-organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil as described in claim 1 in the preparation of antibacterial drugs, characterized in that, The bacteria in question are Staphylococcus aureus and Escherichia coli.

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