Platycladus orientalis essential oil-loaded potassium-doped metal organic framework antibacterial nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses potassium-doped metal organic framework antibacterial nanoparticles loaded with platycladus orientalis essential oil as well as a preparation method and application of the potassium-doped metal organic framework antibacterial nanoparticles. The antibacterial nanoparticle comprises a potassium-doped metal organic framework material and platycladus orientalis essential oil encapsulated in the metal organic framework material, and the metal organic framework material is ZIF-8. The high-porosity structure of the ZIF-8 material is utilized to package and load the platycladus orientalis essential oil, so that the loss of the platycladus orientalis essential oil caused by instability and volatility can be reduced, and meanwhile, potassium ions capable of influencing bacterial physiology are doped, so that the growth of staphylococcus aureus and escherichia coli can be inhibited at relatively low concentration. Therefore, the POEO (at) K-ZIF-8 prepared by the preparation method disclosed by the invention can be applied to preparation of antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-medicines, and in particular relates to potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, and a preparation method and application thereof. Background Art

[0002] Platycladus orientalis essential oil (POEO) is an evergreen tree of the genus Platycladus in the Cupressaceae family. It is also known as cedar and yellow cypress. It is widely distributed in my country, has abundant resources, and has important economic value. Platycladus orientalis essential oil (POEO) has anti-insect, antibacterial, antioxidant, and anti-cancer effects, and will have good application prospects in the development of natural pesticides, preservatives, and health products. However, due to the poor water solubility, unstable composition, and volatile properties of POEO, its practical application is limited to some extent.

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

[0004] Potassium ions can inhibit bacterial growth by affecting bacterial metabolism and cell membranes, and have a certain antibacterial effect. Therefore, potassium doping ZIF-8 can improve its antibacterial properties. How to effectively encapsulate POEO and effectively load potassium ions is a technical problem to be solved by the present invention. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a potassium-doped metal organic framework antibacterial nanoparticle loaded with oriental arborvitae essential oil and its preparation method and application. The present invention utilizes the high porosity structure of ZIF-8 material to encapsulate and load POEO, and modifies it with potassium ions to improve the application limitations of POEO and enhance the antibacterial effect.

[0006] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0007] The invention provides potassium-doped metal organic framework antibacterial nanoparticles loaded with oriental arborvitae essential oil. The antibacterial nanoparticles comprise a potassium-doped metal organic framework material and the oriental arborvitae essential oil encapsulated in the metal organic framework material, wherein the metal organic framework material is ZIF-8.

[0008] The present invention also provides a method for preparing the potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, which is characterized by comprising the following steps:

[0009] (1) dispersing the Platycladus orientalis essential oil in a solvent to obtain a Platycladus orientalis 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 the mixture at room temperature; after the reaction, separating the solid from the liquid, collecting the precipitate, washing, and drying the precipitate to obtain metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, which are referred to 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 reacting at room temperature; after the reaction, solid-liquid separation is performed, and the precipitate is collected, washed, and dried to obtain potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, which are recorded as POEO@K-ZIF-8.

[0013] Preferably, in step (1) and step (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 hours, the drying temperature is 50-70° C., and the drying time is 8-12 hours.

[0016] Preferably, 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.

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

[0018] Preferably, in step (1), the mass ratio of the Platycladus orientalis 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 present invention also provides the use of the potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil in the preparation of antibacterial drugs.

[0021] Beneficial effects:

[0022] The present invention utilizes the high porosity structure of ZIF-8 material to encapsulate and load POEO, and modifies it with potassium ions to improve the application limitations of POEO. Experimental findings show that compared with POEO and POEO@ZIF-8, POEO@K-ZIF-8 not only solves the problem of POEO's volatility and low bioavailability, but also the potassium doped therein can affect bacterial metabolism and inhibit the growth of Staphylococcus aureus and Escherichia coli at relatively low concentrations. Therefore, the POEO@K-ZIF-8 prepared by the present invention can be used to prepare antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0027] Figure 5 The effect of different POEO dosage on POEO@ZIF-8 Zeta potential;

[0028] Figure 6 The effect of different POEO dosage on the drug loading of POEO@ZIF-8;

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

[0030] Figure 8 The effect of different potassium nitrate inputs on the Zeta potential of POEO@K-ZIF-8;

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

[0032] Fig.10 The effect of POEO@K-ZIF-8 prepared by the present invention on the survival rate of Staphylococcus aureus;

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

[0034] Fig.12This is the effect of POEO@K-ZIF-8 prepared in the present invention on the survival rate of Escherichia coli. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0037] The Platycladus orientalis essential oil in the embodiments of the present invention is derived from Sriver Textile Machinery Co., Ltd.

[0038] The preparation method of blank ZIF-8 nanoparticles of the present invention is as follows: 4.42g of 2-methylimidazole is dissolved in 8mL of methanol to obtain solution A, 200mg of zinc nitrate hexahydrate is dissolved in 0.8mL of methanol to obtain solution B, and the mixture is stirred and reacted for 2h at room temperature. After the reaction is completed, the reactant is centrifuged at a centrifuge speed of 12000rpm, a centrifugal temperature of 20°C, and a centrifugal time of 15min, the supernatant is discarded, the precipitate is collected, washed 3 times, and placed in a 60°C oven for drying for 12 hours to obtain ZIF-8 nanoparticles.

[0039] Example 1 Preparation of potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil

[0040] (1) Weigh 100 mg of Platycladus orientalis essential oil and disperse it in 0.8 mL of anhydrous methanol to obtain a Platycladus orientalis essential oil dispersion, dissolve 200 mg of zinc nitrate hexahydrate in the dispersion, and stir for 5 min;

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

[0042] (3) The two dispersions were mixed and stirred at room temperature for 2 h. After the reaction, the reactants were centrifuged at a speed of 12,000 rpm, a centrifugal temperature of 20° C., and a centrifugal time of 15 min. The supernatant was discarded, and the precipitate was collected and washed three times. The precipitate was placed in an oven at 60° C. and dried for 12 h to obtain 84.9 mg of metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, which were recorded as POEO@ZIF-8 nanoparticles.

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

[0044] The Malvern particle size analyzer was used to analyze and determine the particle size of POEO@ZIF-8 nanoparticles to be 242.77 nm; the particle size of POEO@K-ZIF-8 was 244.17 nm.

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

[0046] Take the prepared POEO@ZIF-8 powder, add 3mL of n-hexane, ultrasonicate for 30min, centrifuge, take the supernatant, repeat 3 times, filter the supernatant through a filter membrane, send it to GC-MS to determine the concentration, and use the standard curve of the main component of cypress essential oil (cedarwood alcohol) to determine the loading amount of POEO in POEO@ZIF-8. 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 is 7.14%.

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

[0048] like Figure 1 As shown, 3132cm -1 and 2926cm -1 The absorption peaks at 1362-1458cm are attributed to the stretching vibration peaks of methyl and CH in the imidazole ring; -1 and 1569cm -1 The absorption peaks observed in the range can be attributed to the entire imidazole ring and the stretching of C=N, 421 cm -1 The Zn-N bond absorption peak stretching vibration is unique to ZIF-8 materials, indicating that the ZIF-8 framework of both is intact and the loading process does not destroy the structure of ZIF-8 nanoparticles. -1 The weak absorption peak at 675.21 cm in the infrared spectrum of POEO@K-ZIF-8 corresponds to the stretching vibration absorption peak of hydroxyl (-OH) in POEO. -1The absorption peak that appears at is due to the vibration caused by the interaction between potassium ions and surrounding molecules or ions.

[0049] Example 2 Investigating 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, respectively, and the POEO dosage was 100 mg to synthesize POEO@ZIF-8.

[0051] 2-Methylimidazole and 100 mg POEO were dissolved in 8 mL and 0.8 mL 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, the reactants were centrifuged at a speed of 12,000 rpm, a temperature of 20°C, and a time of 15 min. The supernatant was discarded, and the precipitate was collected, washed three times, and dried in an oven at 60°C for 12 h to obtain POEO@ZIF-8 nanoparticles.

[0053] The Malvern particle size analyzer was used for analysis and determination. Figure 2 , Figure 3 .

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

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

[0056] Example 3 Investigating the effects of different POEO dosages on POEO@ZIF-8 nanoparticles

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

[0058] The Malvern particle size analyzer was used for analysis and determination. Figure 4 , Figure 5 .

[0059] like Figure 4 As shown, when the dosage is 25-100 mg, the prepared POEO@ZIF-8 particle size is appropriate and the dispersion is uniform.

[0060] like Figure 5 As shown in the figure, the Zeta potential of POEO@ZIF-8 prepared at a dosage of 25-125 mg is around 30 mV, and the dispersion system has good stability. In contrast, the Zeta potential reaches the highest when the dosage is 100 mg, and the stability is good.

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

[0062] Results Figure 6 .

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

[0064] Example 4 Investigating the effects of different POEO@ZIF-8 and potassium nitrate mass ratios on POEO@K-ZIF-8 nanoparticles

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

[0066] The Malvern particle size analyzer was used for analysis and determination. Figure 7 , Figure 8 .

[0067] like Figure 7 As shown, except for the mass ratio of 1:1, the particle sizes of POEO@K-ZIF-8 prepared with other mass ratios are basically the same and are evenly distributed in the solution system.

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

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

[0070] Using 5% DMSO solution as the solvent, POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution with a concentration of 1 mg / mL were prepared. Sterile water was used as the control group. The S. aureus bacterial solution in the logarithmic growth period was diluted 50 times with tryptic soy casein broth (TSB), and then mixed with 1 mg / mL POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution in a volume ratio of 1:1 and placed on a shaker (250 rpm, 37°C) for incubation for 1.5 h.

[0071] The co-incubated sample was diluted 20,000 times, and 100 μL was evenly spread on a solid agar plate. After 24 hours, the count was performed and compared with the blank control group to calculate the bacterial survival rate.

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

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

[0074] POEO@K-ZIF-8 solutions with concentrations of 0.1 mg / mL, 0.08 mg / mL, 0.06 mg / mL, 0.04 mg / mL, and 0.02 mg / mL were prepared with 5% DMSO, and sterile water was used as the control group. The S. aureus bacterial solution in the logarithmic growth period was diluted 50 times with tryptic soy beta broth (TSB) and mixed evenly with POEO@K-ZIF-8 solutions of different concentrations in a volume ratio of 1:1, and then placed in a shaker (250 rpm, 37°C) for incubation for 1.5 h.

[0075] The co-incubated sample was diluted 20,000 times, and 100 μL was evenly spread on a solid agar plate. After 24 hours, the count was performed and compared with the blank control group to calculate the bacterial survival rate.

[0076] like Fig.10 As shown, when the concentration is 0.1 mg / ml, the antibacterial effect is 89.34%, indicating that the minimum inhibitory concentration of POEO@K-ZIF-8 against Staphylococcus aureus is around 0.1 mg / mL.

[0077] Example 6 Investigation of the antibacterial effects of POEO, POEO@ZIF-8 and POEO@K-ZIF-8 on Escherichia coli

[0078] Using 5% DMSO solution as the solvent, POEO solution, ZIF-8 solution, POEO@ZIF-8 solution and POEO@K-ZIF-8 solution with a concentration of 1 mg / mL were prepared. Sterile water was used as the control group. The E. coli bacterial solution in the logarithmic growth period was diluted 50 times with LB liquid culture medium, and then mixed evenly with 1 mg / mL 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 on a shaker (250 rpm, 37°C) for incubation for 1.5 hours.

[0079] The co-incubated sample was diluted 20,000 times, and 100 μL was evenly spread on a solid agar plate. After 24 hours, the count was performed and compared with the blank control group to calculate the bacterial survival rate.

[0080] like Fig.11 As shown, compared with the blank control group, the inhibitory effect of POEO solution on E. coli is about 80%, the inhibitory effect of ZIF-8 solution on E. coli is about 69%, the inhibitory effect of POEO@ZIF-8 on E. coli is about 84%, and the inhibitory effect of POEO@K-ZIF-8 on E. coli is about 98%.

[0081] The present invention also studies the minimum inhibitory concentration of the prepared POEO@K-ZIF-8 against Escherichia coli.

[0082] POEO@K-ZIF-8 solutions with concentrations of 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, and 0.1 mg / mL were prepared with 5% DMSO, and sterile water was used as the control group. The E. coli bacterial solution in the logarithmic growth period was diluted 50 times with LB liquid culture medium and mixed evenly with POEO@K-ZIF-8 solutions of different concentrations in a volume ratio of 1:1, and then placed in a shaker (250 rpm, 37°C) for incubation for 1.5 hours.

[0083] The co-incubated sample was diluted 20,000 times, and 100 μL was evenly spread on a solid agar plate. After 24 hours, the count was performed and compared with the blank control group to calculate the bacterial survival rate.

[0084] like Fig.12 As shown, when the concentration is 0.3 mg / ml, the antibacterial effect is 89.34%, indicating that the minimum inhibitory concentration of POEO@K-ZIF-8 against Escherichia coli is around 0.3 mg / mL.

[0085] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A potassium-doped metal organic framework antibacterial nanoparticle loaded with Platycladus orientalis essential oil, characterized in that: The antibacterial nanoparticles include 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.

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: The following steps are involved: (1) dispersing the Platycladus orientalis essential oil in a solvent to obtain a Platycladus orientalis essential oil dispersion, dissolving zinc nitrate hexahydrate in the dispersion, and stirring; (2) dispersing 2-methylimidazole in a solvent to obtain a 2-methylimidazole dispersion; (3) mixing the two dispersions and stirring the mixture at room temperature; after the reaction, separating the solid from the liquid, collecting the precipitate, washing, and drying the precipitate to obtain metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, which are referred to as POEO@ZIF-8 nanoparticles; (4) dispersing the POEO@ZIF-8 nanoparticles obtained in step (3) and potassium nitrate in a solvent respectively, mixing and reacting at room temperature; after the reaction, solid-liquid separation is performed, and the precipitate is collected, washed, and dried to obtain potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil, which are recorded as POEO@K-ZIF-8.

3. The preparation method according to claim 2, characterized in that: In step (1) and step (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 the 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 Platycladus orientalis 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. Use of potassium-doped metal organic framework antibacterial nanoparticles loaded with Platycladus orientalis essential oil according to claim 1 in the preparation of antibacterial drugs.

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