Nano-hydrogel for resisting multi-drug-resistant acinetobacter baumannii as well as preparation method and application of nano-hydrogel

Abstract

The invention discloses a nano hydrogel for resisting multi-drug-resistant acinetobacter baumannii and a preparation method and application thereof.The preparation is prepared from lavender essential oil, a surfactant, a cosurfactant, an oil phase and a water phase through a low-energy emulsification method, the particle size of the obtained hydrogel ranges from 1 nm to 100 nm, the dispersity index pdI is smaller than 0.3, the potential absolute value is smaller than 30 mV, and the pH value of the hydrogel ranges from 7-8. High-speed centrifugation is achieved, and the property is stable. The antibacterial effect of the nano-hydrogel on acinetobacter baumannii is superior to that of a lavender oil-water solution, meanwhile, the lavender nano-hydrogel can accelerate skin wound healing, reduce the bacterial colonization amount of a mouse skin wound part, damage bacterial cell membranes and improve the inhibition capacity of a biological membrane, and the nano-hydrogel has a remarkable in-vivo and in-vitro antibacterial synergistic effect. The invention has a remarkable effect on multi-drug-resistant bacteria, especially acinetobacter baumannii, and has a great clinical application value and a wide market prospect on treatment and eradication of infection of the acinetobacter baumannii, especially skin wound infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a nano hydrogel resistant to multidrug-resistant Acinetobacter baumannii and a preparation method thereof, and also to an application of the nano hydrogel in preparing an external preparation for skin wound infection caused by Acinetobacter baumannii. Background Art

[0002] Acinetobacter baumannii, also known as Acinetobacter baumannii, is a bacteria that causes infections caused by Acinetobacter baumannii. The speed of antibiotic development is far behind the speed at which bacteria develop drug resistance. Currently, there is a shortage of antibiotics that can be effectively used in clinical practice, and as its drug resistance continues to increase, it is very likely that there will be no drugs available in the future.

[0003] At present, bacterial resistance has become a huge challenge facing global public health, seriously threatening population health and sustainable development. There is an urgent need to develop new antibacterial prevention and control methods that replace antibiotics. Acinetobacter baumanii (commonly known as AB bacteria) is widely present in the natural environment. It is one of the conditional pathogens of humans and has long been an important pathogen of nosocomial infections. It mainly causes respiratory tract infections (54.2%), and can also cause sepsis, urinary tract infections, secondary meningitis, etc. The bacteria are highly resistant (the resistance rate to carbapenems exceeds 50%, ranking first in the "Extremely High Hazard" group of the "Catalogue of Key Bacterial Pathogens in 2024"), highly pathogenic, strong growth ability, strong colonization ability, and a wide range of transmission routes. It has become one of the most important pathogens of traumatic infections, and therefore the demand for alternative antibiotic prevention and control methods is particularly urgent.

[0004] As a widely used natural plant, essential oils have a long tradition of application in the fields of pharmacology, agricultural chemistry, nutrition and daily chemicals. Essential oils are often used in aromatherapy or phytotherapy, and most of them can also be used as antibacterial agents. Lavender (Lavandula aspic L.) is a common aromatic shrub belonging to the genus Lavandula in the family Lamiaceae. Its effective part, lavender essential oil, has a long history of treating mental disorders and anxiety and has been recognized by many international organizations. Studies have shown that lavender essential oil mainly contains nearly 20 compounds such as linalyl acetate, linalool, caryophyllene, trans-β-ocimene, myrcene, and terpineol. Among them, the main active ingredients are linalyl acetate and linalool, which have significant antibacterial activity against Escherichia coli, Staphylococcus aureus and Candida albicans. In addition, the composition and content of different lavender essential oils will directly affect the antibacterial pharmacological effects of essential oils. Lin Pengfei and others used the microdilution method to explore the inhibitory activity of three kinds of lavender essential oils against a variety of fungi and bacteria. Among them, the essential oil of Lavender lavender had the best activity, which had good effects on fungi such as Trichophyton mentagrophytes, Trichophyton rubrum, and Microsporum canis; Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The related activity was positively correlated with the content of linalyl acetate, linalool, and 1,8-cineole in the essential oil samples. Although lavender essential oil also showed good inhibitory activity against Acinetobacter baumannii, Candida albicans, cotton wilt pathogen, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus strains, and vancomycin-resistant Enterococcus bacteria. Although the above reports reveal that lavender essential oil has a significant inhibitory effect on a variety of pathogenic microorganisms, especially in the prevention and treatment of related diseases caused by drug-resistant bacteria, it shows good application prospects, but because its active ingredients are mainly unsaturated carbon chain structures (mainly monoterpenes and sesquiterpenes), it is also easily affected by factors such as light, thermal radiation, humidity, oxygen, etc., which volatilizes and oxidizes, resulting in poor stability, short retention time at the administration site, and low bioavailability, which greatly limits its clinical application as an antibacterial agent. Therefore, it is particularly important to overcome the above-mentioned shortcomings of lavender essential oil through pharmaceutics and nanotechnology for the practical application of lavender essential oil as an effective antibacterial agent.

[0005] Nanogels are hydrogel nanoparticles with a 3D cross-linked structure in the range of 1-1000nm formed by a physical or chemical cross-linked network. Hydrophilic or hydrophobic drugs are encapsulated in the nanogel by the 3D cross-linked structure, which can prevent the degradation of drugs during delivery or blood circulation and reduce toxic side effects on the human body. It can be used as a drug delivery carrier, which has the characteristics of high biocompatibility of hydrogels and similarity to extracellular matrix, as well as the advantages of large specific surface area of ​​nanoparticles and strong sustained and controlled release ability. At present, there are many methods for preparing micro-nanogels, including mechanical crushing method, template method, microfluidic preparation method, self-assembly method, particle cross-linking method, etc., and the selection methods are different for different application requirements. The particle cross-linking method usually uses the internal chemical energy of the raw materials to disperse and form nano-scale particles. It has low production energy consumption, mild application conditions, and high operational feasibility. It is suitable for active ingredients with poor stability such as plant essential oils. Using the particle cross-linking method, lavender essential oil is loaded in gel excipients at the nanoscale, which can not only improve its physical and chemical stability and dispersibility, but also improve its permeability. As a topical preparation for skin application, the nanogel preparation is expected to enhance the antibacterial activity of lavender essential oil against Acinetobacter baumannii and promote skin wound healing.

[0006] Based on the above reasons, the present invention uses the previously owned patented technology to develop lavender oil nanogel (LNG), hoping to obtain an antibacterial effect higher than that of lavender oil aqueous solution. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a nano-hydrogel against multidrug-resistant Acinetobacter baumannii; a second object of the present invention is to provide a method for preparing the nano-hydrogel against multidrug-resistant Acinetobacter baumannii; a third object of the present invention is to provide an application of the nano-hydrogel against multidrug-resistant Acinetobacter baumannii in the preparation of an external preparation for skin wound infection caused by Acinetobacter baumannii.

[0008] In order to achieve the above object, the present invention provides the following technical solutions and preparation methods:

[0009] 1. A nanohydrogel for resisting multidrug-resistant Acinetobacter baumannii, wherein the nanohydrogel mainly comprises the following components: lavender essential oil, a surfactant, an oil phase, a thickener and a cross-linking agent.

[0010] The preferred components of the present invention are as follows: lavender oil at a concentration of 0.1-12%, Tween 80 at a concentration of 12-27%, oil phase IPM at a concentration of 0.1-3%, thickener triethanolamine at a concentration of 0.4-3%, and crosslinking agent CMC-Na at a concentration of 0.1-0.5%.

[0011] Preferably, the particle size of the nano hydrogel is distributed between 1-100 nm, and the absolute value of the Zeta potential is less than 30 mV.

[0012] Preferably, the pH value is 4-7.

[0013] 2. A method for preparing the nano hydrogel against multidrug-resistant Acinetobacter baumannii, comprising the following steps:

[0014] (1) Accurately weigh the prescribed amount of lavender essential oil and surfactant, and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0015] (2) Add the prescribed amount of oil phase to (1) and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0016] (3) adding 50% of the prescribed amount of water, and adding dropwise to the prescription (2) under magnetic stirring at 50-100 rpm to form a smoked nanoemulsion;

[0017] (4) Add the prescribed amount of cross-linking agent to (3), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 minutes;

[0018] (5) Add the prescribed amount of thickener to (4), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 minutes;

[0019] (6) Slowly add sterile pure water dropwise to the final system in (5) while stirring;

[0020] (7) The nano-hydrogel is prepared by standing the mixture at room temperature and away from light for 24 hours, and then sterilizing and packaging the mixture.

[0021] 3. Use of the nano-hydrogel against multi-drug resistant Acinetobacter baumannii in the preparation of an external preparation against skin wound infection caused by Acinetobacter baumannii.

[0022] The beneficial effects of the present invention are as follows: the present invention successfully designs and develops a multi-drug resistant Acinetobacter baumannii nano-hydrogel, namely, a lavender nano-hydrogel. Through in vitro antibacterial experiments on Acinetobacter baumannii and in vivo wound healing experiments, it is found that the antibacterial effect of the lavender nano-hydrogel is better than that of the lavender oil aqueous solution. At the same time, the lavender nano-hydrogel can accelerate skin wound healing, reduce the amount of bacterial colonization at the skin wound site of mice, destroy bacterial cell membranes, and improve the inhibitory ability of biofilms. The lavender nano-hydrogel can be widely used in skin wound infections caused by multi-drug resistant Acinetobacter baumannii. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0024] Figure 1 The effect of the dosage of main excipients on the particle size and potential of the preparation.

[0025] Figure 2 The effect of different formulations on the stability of the preparations.

[0026] Figure 3 The effect of the order of adding raw materials and auxiliary materials on the particle size and potential of the preparation.

[0027] Figure 4 The effect of the order of adding raw materials and excipients on the stability of the preparation.

[0028] Figure 5 The effect of different drug loadings on the particle size and potential of the preparations.

[0029] Figure 6 The effect of changes in drug loading on the stability of the preparation.

[0030] Figure 7 This is the morphology of the nanohydrogel under transmission electron microscopy (AD, 30k, 80k, 150k, 150K times, scale bars are 0.5μm, 200nm, 100nm, 34nm respectively).

[0031] Figure 8 The morphology of nanogels in different views (different view AD) under a scanning electron microscope at 40K.

[0032] Fig. 9 Nanohydrogel characteristics determined for rheological analysis.

[0033] Fig.10 Nanohydrogel characteristics were determined by Fourier transform infrared spectroscopy and thermogravimetric analysis (A) DSC, (B) TG (C) FITR.

[0034] Fig.11 The results of lavender essential oil nanohydrogel irritation and toxicity on mouse skin.

[0035] Fig.12 MIC and MBC of lavender essential oil nanohydrogels with different concentrations against Acinetobacter baumannii 17978.

[0036] Fig.13 MIC and MBC of lavender essential oil nanohydrogels with different concentrations against Acinetobacter baumannii LAC-4.

[0037] Fig.14 The bactericidal kinetic results of lavender essential oil nano-hydrogel with the same concentration on 17978.

[0038] Fig.15 The mean wound healing area change.

[0039] Fig.16 Changes in bacterial colonization.

[0040] Fig.17 This is the wound healing status of mouse infection model after intervention with lavender essential oil nanohydrogel.

[0041] Fig.18 Crystal violet staining results of biofilm inhibition and clearance after lavender essential oil nanohydrogel intervention.

[0042] Fig.19 Confocal laser scanning microscopy results of biofilm formation inhibition.

[0043] Fig. 20 Confocal laser scanning microscopy results for clearing of biofilm formation.

[0044] Fig.21 The effect of lavender essential oil nanohydrogel on the DNA leakage of Acinetobacter baumannii.

[0045] Fig. 22 The effect of lavender essential oil nanohydrogel on protein leakage of Acinetobacter baumannii.

[0046] Fig.23 The effect of lavender essential oil nanohydrogel on potassium ion leakage of Acinetobacter baumannii.

[0047] Fig.24 The effect of lavender essential oil nanohydrogel on the bacterial morphology of Acinetobacter baumannii. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0049] Reagents used in the present invention: lavender oil was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., Tween 80 was purchased from Shanghai Sinopharm, isopropyl myristate (IPM) was purchased from Beijing Fengli Co., Ltd., triethanolamine was purchased from Sinopharm Group, and sodium carboxymethyl cellulose (Carboxyl methyl Cellulose-Na, CMC-Na) was purchased from BASF;

[0050] Standard strain: Acinetobacter baumannii (ATCC17978, USA); Experimental animals: SPF BALB / c mice, female, 6-8 weeks old, weight 18-22 g (Beijing Huafukang Biotechnology Co., Ltd.). Animal experiments were reviewed by the Experimental Animal Welfare Ethics Review Committee of the Army Medical University and were in compliance with animal ethics and animal welfare requirements.

[0051] Example 1. Optimization experiment of lavender oil nanohydrogel

[0052] 1. Optimization of the prescription of lavender gel preparation

[0053] (1) Accurately weigh the prescribed amount of lavender essential oil and surfactant, and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0054] (2) Add the prescribed amount of oil phase to (1) and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0055] (3) adding 50% of the prescribed amount of water, and adding dropwise to the prescription (2) under magnetic stirring at 50-100 rpm to form a smoked nanoemulsion;

[0056] (4) Add the prescribed amount of cross-linking agent to (3), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 minutes;

[0057] (5) Add the prescribed amount of thickener to (4), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 minutes;

[0058] (6) Slowly add sterile pure water dropwise to (5) while stirring until the system mass is 10 g;

[0059] (7) The mixture is allowed to stand for 24 hours at room temperature and away from light in (6) to prepare the lavender essential oil nano-hydrogel, which can then be sterilized and packaged.

[0060] Centrifugal stability study: Take the above preparation samples, centrifuge them at 4°C and 3000 rpm for 15 min, let them stand for 5 min, and observe whether the samples have stratification.

[0061] Particle size and Zeta potential investigation: Take 100 μL of lavender essential oil nanoformulation and dilute it with 2 mL of sterile pure water to ensure that the counting rate of the sample nanogel particles is around 250 Kcps to avoid multiple scattering. Use a potentiometric particle size analyzer to measure the particle size and Zeta potential of the nanogel particles.

[0062] Based on the preliminary experiments of the research group and this study, on the basis of determining the formulation process, the main excipients of the preparation (Tween 80 addition amount, triethanolamine addition amount, CMC-Na concentration) were investigated with centrifugal stability, particle size and potential as indicators, and the stability of the preparations prepared under different formulations was compared. The specific addition amounts of raw materials and excipients are shown in Table 1.

[0063] Table 1 Optimization parameters of different formulations

[0064]

[0065] In the preparation process of nano hydrogel preparations, the selection and dosage of excipient matrix have a significant impact on the stability of the preparation. Based on the existing prescription, this study systematically investigated the amount of Tween 80 added, the amount of triethanolamine added, the type and concentration of CMC-Na, see Figure 1 , Figure 2 and Table 2. The results showed that the particle size distribution and potential distribution of samples No. F2 and F5 were more concentrated, and the emulsion presented by No. F5 was milky white, uniform and without stratification, which was consistent with the nanohydrogel reported in the literature (Dong Qinwei, Li Qiaoqiao, Cui Yuanlu, et al., Preparation of lactoferrin-modified nanogel pellets and study of drug transport into the brain); the emulsions presented by No. F2, F3, and F7 were relatively thin, but no stratification was observed; while the samples No. F1, F4, F6, and F8 had stratification, and the preparations were unstable.

[0066] Therefore, based on the above analysis, the F5 formula was determined, that is, the amount of the main excipients added was determined to be: 1.8g Tween 80, 0.2g triethanolamine, 2g 0.5% CMC-Na. In addition, isopropyl myristate was confirmed to have little effect on nano-hydrogels by preliminary experiments, so the amount of 0.3g in the basic formula was used.

[0067] Table 2 Effect of different formulations on the particle size and potential of the preparations (X±SD)

[0068]

[0069] 2. Preparation process optimization

[0070] Based on the preliminary experiments of the research group and this study, the basic preparation method of the particle cross-linking method was determined as follows: in a 50mL dry beaker, accurately add 0.9g lavender essential oil, 1.8g Tween 80, and 0.3g isopropyl myristate in sequence, stir at a constant speed until fully mixed, slowly add water while stirring, and when the system reaches a suitable state, add 2g of 0.5% CMC-Na, mix evenly, then add 0.2g of triethanolamine, and slowly add sterile pure water while stirring until the system volume is 10g. Let it stand at room temperature and away from light for 24 hours to prepare lavender essential oil nanohydrogel.

[0071] The particle cross-linking method is simple to operate. In the preliminary experiment, it was found that the parameters such as stirring rate and standing time had little effect on the particle size and potential of the preparation samples. However, the order in which the raw materials and auxiliary materials are added to the system may affect the quality of the preparation. Based on the above method, different processes for the order of adding raw materials and auxiliary materials are designed as shown in Table 3.

[0072] Table 3 Experimental design table of different formulation process optimization parameters

[0073]

[0074]

[0075] In the process of preparing lavender essential oil nanohydrogel by particle cross-linking method, the order of adding raw materials and auxiliary materials may have a certain influence on the stability, particle size and Zeta potential of the preparation. The influence of different adding order of raw materials and auxiliary materials on particle size and potential can be seen in Figure 3 As shown in Table 4, from the particle size distribution, it can be seen that the preparation No. T1 is more concentrated in the size range of 10 to 100 nm, and the particle density in the size range of 1000 to 10000 nm is almost invisible, showing good monodispersity as a whole; the performance of samples No. T2 and T4 is slightly worse, with a small amount of density distribution in the size range of 1000 to 10000 nm; while the nanoparticles No. T3, T5, and T6 are scattered, and the particle size differences are obvious. From the potential distribution, the potential of the preparation No. T1 is concentrated between -100 and 0 mV, and is significantly greater than 0 mV. The high absolute value of the negative potential indicates that the preparation has good stability, which is beneficial to product storage and clinical application; No. T3, T4, and T5 are closer to 0 mV, indicating slightly poor stability; while the potential of samples No. T2 and T5 are scattered. The effect of different addition orders of raw materials and excipients on centrifugal stability can be seen in Figure 4 It can be found that there is almost no stratification of the preparations in the order of addition of T1, T2, and T3, while there is a slight stratification phenomenon in the samples of T4, T5, and T6.

[0076] Therefore, after comprehensive evaluation and consideration, the T1 process was selected: that is, after adding lavender essential oil, Tween 80, and isopropyl myristate, CMC-Na and triethanolamine were added in sequence.

[0077] Table 4 Data table of the effects of different processes on the particle size and potential of the preparation (X±SD)

[0078]

[0079] 3. Optimization of drug loading in formulations

[0080] On the basis of determining the preparation process and the main auxiliary material components and dosage, the drug loading of the preparation was investigated with centrifugal stability, particle size distribution and Zeta potential as indicators, and the effect of drug loading changes on the pharmaceutics characteristics was compared. The specific preparation method is: in a 50mL clean dry beaker, add different masses of lavender essential oil (0, 0.3, 0.6, 0.9, 1.2, 1.5g, corresponding to numbers L1 to L6), add 1.8g Tween 80, 0.3g isopropyl myristate, stir until fully mixed, slowly add sterile pure water during the period, add 2g of 0.5% CMC-Na when the system reaches a suitable state, mix evenly, add 0.2g triethanolamine, slowly add sterile pure water while stirring until the system volume is 10g, and store at room temperature.

[0081] In order to obtain nanohydrogel preparations with higher drug loading, this study investigated the effect of different lavender essential oil loadings on nanohydrogels and examined the centrifugal stability, particle size and Zeta potential of the preparations. Figure 5 As shown in Table 5, the particle size distribution indicates that the size of the L5 nanoparticles (corresponding to 12% drug loading) is relatively concentrated, and the potential distribution also indicates that the potential stability of the five samples is good. Figure 6 As shown, samples No. L1 to L6 showed no obvious stratification, indicating good stability. Therefore, considering multiple factors, the drug loading of lavender essential oil in the nanoformulation was determined to be 12%.

[0082] Table 5 Effect of drug loading of different preparations on particle size and potential (X±SD)

[0083]

[0084] Example 2. Characterization of lavender oil nanohydrogel

[0085] The lavender oil nano-hydrogel was prepared by the optimized method according to Example 1, and the specific operation steps are as follows:

[0086] (1) Accurately weigh 1.2 g of lavender essential oil and 1.8 g of Tween 80 and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0087] (2) Add 0.3 g of isopropyl myristate (IPM) to (1) and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0088] (3) adding 50% of the prescribed amount of water, and adding dropwise to the prescription (2) under magnetic stirring at 50-100 rpm to form a smoked nanoemulsion;

[0089] (4) Add 2 g of 0.5% sodium carboxymethyl cellulose (CMC-Na) to (3), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0090] (5) Add 0.2 g of triethanolamine to (4), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 min;

[0091] (6) Slowly add sterile pure water dropwise to (5) while stirring until the system mass is 10 g;

[0092] (7) The lavender essential oil nano-hydrogel (LNG) is prepared by standing at room temperature and in the dark for 24 hours in (6), and then sterilized and packaged.

[0093] Transmission electron microscopy observation: After determining the formulation and process, the prepared nanohydrogel was diluted 100 times with ultrapure water, 5 μL was taken on the electron microscope copper grid, and it was allowed to stand for 10 minutes. The excess water was carefully absorbed with filter paper, and 10 μL of 1% phosphotungstic acid was added for staining for 1 minute. After that, the excess water was absorbed with filter paper again, and the image was observed and collected under Tecnai 10TEM.

[0094] Scanning electron microscope observation: the prepared nano-hydrogel was diluted 100 times with ultrapure water, pre-fixed with 4% glutaraldehyde and post-fixed with 1% osmium acid, dehydrated with gradient concentration ethanol, replaced with pure tert-butanol, and finally vacuum freeze-dried and gold-sprayed before being placed under SEM for observation and recording; the prepared nano-hydrogel was diluted 100 times with ultrapure water, and the particle size and particle size distribution, potential and particle size distribution of the nano-hydrogel were determined using a laser particle size analyzer, Nano ZS90 potential and particle size analyzer.

[0095] After determining the preparation process and formula, a series of pharmaceutical characterization studies were conducted on the lavender essential oil nano-hydrogel prepared in this way. Among them, the TEM observation results are shown in Figure 7 At 150k times, the diameter of the representative gel nanoparticles was about 34nm. At 30k, 80k, and 150k times, it was clearly visible that the nanoparticles were spherical or quasi-spherical entities. The particle diameter remained within a certain range and the dispersion state was good, indicating that the particle cross-linking method had good feasibility, and the preparation process and formulation optimization were correct and reasonable. SEM observation results are shown in Figure 8 The conclusion is consistent with the above. After the process and prescription are optimized, the gel nanoparticles are basically spherical, and the particle size distribution is relatively concentrated. The particle size distribution of the nanogel preparation is mainly in the range of 0 to 100nm, and is most concentrated at 30 to 40nm; the Zeta potential is about -20 to -60mV.

[0096] Example 3. Stability test of lavender oil nanohydrogel

[0097] The frequency scanning measurement method is as follows: take 2 mL of the nanohydrogel sample prepared in Example 2 and place it on the flat plate mold of the rheometer, use the CP50-1 probe, select the oscillation scanning mode, set the measurement temperature to 37°C, set the strain force parameter (Gamma strain force parameter, γ) to 1%, and set the frequency scanning range to 100% to 0.1% rad / s.

[0098] The method for determining the linear viscoelastic region is as follows: take 2 mL of the nanohydrogel sample prepared in Example 2 and place it on the flat plate mold of the rheometer, use the CP50-1 probe, select the amplitude scanning mode, set the measurement temperature to 37°C, set the γ parameter to 0.1% to 300%, and fix the frequency to 1 Hz to determine the linear viscoelastic region of the sample.

[0099] The lavender essential oil, blank nanohydrogel (prepared according to the method of Example 2, but without lavender essential oil), and lavender essential oil nanohydrogel prepared in Example 2 were subjected to thermogravimetric analysis using a differential thermal and gravimetric analyzer, using inert gas nitrogen as carrier gas, a flow rate of 50 mL / min, a temperature range of 27 to 203°C, and a heating rate of 10°C / min. The differential thermal and gravimetric curves were plotted. A 4 cm -1 Fourier transform infrared spectroscopy was performed on lavender essential oil, blank nanohydrogel, and lavender essential oil nanohydrogel, and the scanning range was determined to be 400 cm -1 Up to 4000cm -1 , draw its infrared spectrum.

[0100] The results of the frequency sweep carried out using the rheometer are shown in Fig. 9 , A, it can be found that at an oscillation frequency of 0.1 to 10 Hz, the G′ of the lavender nanohydrogel preparation is always greater than G″, indicating that under the measurement of this oscillation frequency, the elasticity of the sample is always greater than the viscosity, showing an obvious gel structure, which also shows that the structure of the nanohydrogel preparation is relatively stable. Amplitude scanning measurement can help understand the linear viscoelastic region of the gel preparation. The amplitude scanning results carried out using a rheometer are shown in Fig. 9 , B, It can be found that the nano hydrogel preparation can be divided into two stages in the deformation process. The first stage is when the shear stress γ is less than 0.25%, the storage modulus G′> loss modulus G″, the elasticity of the gel preparation sample is stronger than the viscosity, showing a gel structure; the second stage is when the shear stress γ is between 0.25% and 100%, the storage modulus gradually decreases to less than the loss modulus, and when G′ and G″ are equal, it indicates that the gel structure is destroyed. At this time, the viscosity is greater than the elasticity, showing fluid properties.

[0101] In order to evaluate the thermal stability differences among lavender essential oil, blank nanohydrogel, and lavender essential oil nanohydrogel, and to characterize the formation of nanoformulations, thermogravimetric analysis was performed on the above samples. The results are shown in Fig.10, as shown in A and B. Referring to the analysis of the thermal gravimetric study in the existing research literature, it can be found that the first stage is the temperature condition below about 100°C, which corresponds to the rapid evaporation of free water or bound water in the three groups of samples and the carbonization of organic matter, which is the main thermal decomposition stage of the samples. Among them, the lavender essential oil sample in the free state is rich in oil esters and unsaturated organic matter, and the mass loss rate is the largest; the three-dimensional network structure in the blank nanohydrogel sample is tight, the molecular interaction force is significant, the excipient matrix has good thermal stability, and the mass loss rate is the smallest; the drug-loaded nanohydrogel sample is slightly weaker than the blank nanohydrogel, but still maintains a relatively outstanding thermal stability. The second stage is 100°C to 200°C, which corresponds to the plateau period after carbonization. In summary, the thermal gravimetric curve of the lavender essential oil nanohydrogel is similar to that of the blank nanohydrogel, and the weight loss rate is significantly lower than that of the lavender essential oil, which not only indicates the actual formation of the nanoformulation, but also suggests the superior stability of the preparation.

[0102] Fourier transform infrared spectroscopy is a commonly used characterization method for nanocarriers. The formation of the preparation can often be judged by the reduction, displacement or disappearance of the characteristic absorption peak. This study used this spectrum to compare the absorption characteristics of the infrared curves of the drug before and after it was made into nanoparticles and loaded with drugs. The experimental results are as follows Fig.10 As shown in C, in the comparison between the lavender essential oil group and the nano-hydrogel group before and after preparation, the lavender essential oil at 1640 cm -1 (corresponding to carbon-carbon double bond), 2095.3cm -1 (corresponding to carbon-carbon triple bond), 2922.5cm -1 The characteristic peak intensity at 2095.3cm-1 (corresponding to the carbon-hydrogen bond) decreased or disappeared, indicating that lavender essential oil and nano-hydrogel excipients formed a nano-preparation together; in the comparison between the blank nano-hydrogel and nano-hydrogel groups before and after drug loading, the blank nano-hydrogel had a peak intensity of 2095.3cm-1. -1 (corresponding to carbon-carbon triple bond), 3446cm -1 The intensity of the characteristic peak at (corresponding to the hydrogen-oxygen bond) is significantly reduced, indicating that lavender essential oil has been successfully encapsulated or combined in the nanohydrogel.

[0103] Example 4. Safety testing of lavender oil nanohydrogel

[0104] The mice were randomly divided into 3 groups, namely, the control group of Acinetobacter baumannii trauma infection, the 60mg / mL lavender essential oil group, and the 60mg / mL lavender essential oil nanohydrogel group. The back of the mice was depilated, and the depilated skin was carefully checked before administration. The experiment was conducted after confirming that there was no damage. 50μL / mouse was administered. During the experiment, the drug was administered every day for 9 consecutive days. Every two days, the skin of the mice at the application site was observed for erythema and edema. After the experiment, the liver and kidney function of the mice was detected. The main indicators were creatinine (Creatinine, Cr), alanine aminotransferase (Alanine Aminotransferase, ALT), aspartate aminotransferase (Aspartate Aminotransferase, AST), alkaline phosphatase (Alkaline Phosphatase, ALP), albumin (Albumin, Alb), bilirubin (Total Bilirubin, TBIL), and lactate dehydrogenase (LacticDehydrogenase, LDH).

[0105] The results of total lavender essential oil nanohydrogel on mouse skin irritation are shown in Fig.11 . Compared with the control group, the lavender essential oil nano-hydrogel showed no significant changes after 9 days of continuous administration (no skin irritation toxicity such as edema and erythema occurred), while the lavender essential oil showed slight redness and swelling on the 5th day after intervention, suggesting possible skin irritation at high doses. In further studies on liver and kidney toxicity, the results (see Table 6) showed that compared with the control group, lavender essential oil and its nano-hydrogel did not show significant statistical differences (all P>0.05), suggesting that lavender essential oil and nano-hydrogel preparations have little effect on the safety of liver and kidney function.

[0106] Table 6 Effects of lavender essential oil nanohydrogel on liver and kidney function toxicity in mice (X±SD)

[0107]

[0108] Example 5. Study on the in vitro antibacterial synergistic effect of LNG

[0109] Prepare tryptone soy agar and tryptone soy broth with deionized water, sterilize and cool at 121℃ for 20min, and prepare solid and liquid culture media. Use an inoculation loop to dip a loop of bacterial liquid in a sterile environment and divide and mark on the solid agar culture medium, and keep it at a constant temperature of 37℃ overnight. Pick a single colony after recovery and place it in a shake bottle containing 10mL of liquid culture medium, and culture it on a shaker at a constant temperature of 37℃ and 220rpm for 4-6h. The activated colony is ready for use.

[0110] For Acinetobacter baumannii strain ATCC17978, the MIC and MBC detection methods are as follows: dilute the activated colonies with OD600 nm = 1.0 by 1000 times. In a 96-well plate, 180 μL of diluted bacterial solution and 20 μL of the above-mentioned drugs at different concentrations were added to the lavender essential oil nanohydrogel group, lavender essential oil group (1.5, 2.25, 3, 4.5, 6, 9, 12 mg / mL) and blank gel group. Only 200 μL of culture medium was added to the negative culture medium control group. After adding the sample, the culture was cultured at 37°C for 24 hours, and the OD600 nm was detected. The bacterial solution was visually observed to be free of turbidity, and the difference between its absorbance value and the background value of the negative culture medium control group was less than 0.05, that is, the corresponding minimum concentration was obtained, which was the MIC. 2 μL of the samples from each group were taken and dropped into the solid culture medium plate, and cultured at 37°C for 24 hours. If there was no bacterial growth, the corresponding final concentration was the MBC.

[0111] Adjust the activated bacterial solution to OD 600nm =1.0, then diluted 10 3 times. Dilute lavender oil aqueous solution (LS), lavender oil nanohydrogel (LNG), and blank nanohydrogel (BNG) to a final concentration of 6, 3, and 1.5 mg / mL, respectively. Pipette 1.8 mL of AB bacterial solution into an EP tube, add 0.2 mL of the above-mentioned liquids of different concentrations, put it into an incubator, and culture at 37°C for 1 min, 5 min, 15 min, 30 min, 60 min, 90 min, 120 min, 240 min, 360 min, and 1080 min. Take 10 μL of sample from each well and dilute it continuously with TSB liquid culture medium at 0, 10, and 10 2 , 10 3 , 10 4 , 10 5 , 10 6 2 μL of the culture medium was spotted on 6 zones of TSA nutrient agar medium and cultured at 37°C for 24 h. The total number of colonies in the culture dish was determined and its logarithmic growth curve was drawn.

[0112] Based on the results of preliminary experiments, the detection methods of MIC and MBC of Acinetobacter baumannii strain LAC-4 were basically the same as above, but the concentrations of the lavender essential oil group and lavender essential oil nanohydrogel group were adjusted to 0.14, 0.28, 0.56, 1.125, 2.25, and 4.5 mg / mL.

[0113] The antibacterial effect of lavender essential oil nanohydrogel on Acinetobacter baumannii 17978 strain is as follows: The MIC results of lavender essential oil nanohydrogel on Acinetobacter baumannii 17978 are shown in Fig.12, A. The study found that lavender essential oil nanohydrogel at a dose of 1.5 mg / mL can produce an antibacterial effect (P<0.01), and 6 mg / mL lavender essential oil nanohydrogel is MIC (P<0.001). The study also found that at a dose of 1.5 mg / mL, the antibacterial effect of the drug-loaded nanohydrogel was still slightly better than that of 4.5 mg / mL lavender essential oil, and the antibacterial effect was increased by 3 times. It was not until the MIC dose condition that the absorbance between the two groups was almost consistent. The results of the MBC determination are shown in Fig.12 , B, It can be seen that when the concentration of lavender essential oil in the nanohydrogel is ≥3mg / mL, no plaques are observed, and almost all bacteria are killed. The MBC of the drug-loaded nanohydrogel is half of that of free lavender essential oil; while the blank gel control group all showed obvious bacterial plaques, which was consistent with the Acinetobacter baumannii control group.

[0114] The results of the minimum inhibition and minimum bactericidal tests showed that lavender essential oil nanohydrogel had significant inhibitory and killing effects on Acinetobacter baumannii, and the effect in horizontal comparison was better than that of lavender essential oil.

[0115] The MIC results of lavender essential oil nanohydrogel against Acinetobacter baumannii LAC-4 strain are shown in Fig.13 , A. The study found that lavender essential oil nano-hydrogel at a dose of 0.14 mg / mL can produce an antibacterial effect (P<0.01), and 0.56 mg / mL lavender essential oil nano-hydrogel is MIC (P<0.001). Horizontal comparison found that there is a 4 to 6 times difference in antibacterial effect compared with lavender essential oil. MBC test results are shown in Fig.12 , B, it can be seen that when the concentration of lavender essential oil in the nanohydrogel is ≥0.56mg / mL, no plaque is observed, and almost all bacteria are killed. The MBC of free lavender essential oil is 2.25mg / mL, and the minimum bactericidal dose of the drug-loaded nanohydrogel is about 1 / 4 of it. The results of the minimum inhibitory and minimum bactericidal tests show that lavender essential oil nanohydrogel also has a significant inhibitory and killing effect on Acinetobacter baumannii LAC-4 strain, and the effect is also better than lavender essential oil at the same concentration.

[0116] The results of the bactericidal kinetic curve showed that 6mg / mL LNG could kill all AB within 90 minutes, while LS could kill all AB within 120 minutes. 3mg / mL LNG only needed 60 minutes to reduce the bacterial viability by 99%, and killed all AB within 240 minutes; but LS needed 120 minutes to reduce the bacterial viability by 90%, and could not kill all bacteria. 1.5mg / mL LNG still had a certain antibacterial effect within the early 6 hours, while LS did not ( Fig.14 ).

[0117] Example 6. LNG promotes wound healing after traumatic infection

[0118] (1) Experimental groups

[0119] 40 mice were randomly divided into 6 groups, namely: ①AB Infected, ②6mg / mL LS, ③6mg / mLLNG, ④3mg / mL LS, ⑤3mg / mL LNG, ⑤BNG group, with 6 mice in each group of ①⑥ and 7 mice in each group of ②③④⑤.

[0120] (2) Establishment of trauma infection model and drug administration regimen

[0121] After 40 mice were anesthetized with intraperitoneal injection of pentobarbital, the backs were depilated, 15 mm × 15 mm of skin was cut to form a wound, and the wound was immediately infected with 100 μL of bacterial solution with OD600nm=1.5. The mice were fixed for 30 seconds and then returned to the cage; the second infection was performed 8 hours after the first infection; after the second infection, the drug was administered twice a day, 50 μL each time, for 14 consecutive days.

[0122] (3) Determination of wound healing area and bacterial colonization

[0123] Wound healing area: On days 1, 3, 5, 7 and 14 after infection, the wound area of ​​all mice was measured with a ruler and photographed. The time of scab formation, scab shedding and complete recovery of the wound site was observed and recorded every day.

[0124] Bacterial colonization amount: On days 1, 3, 5, 7 and 14 after infection, the backs of all mice were scratched 20 times with a clean sterile cotton swab, and the swabs were quickly dipped into an EP tube containing 1 mL of sterile saline. The bacterial solution was then diluted 0, 10, 102, 103, 104, 105, and 106 times with sterile saline, and 2 μL of each dilution was dropped onto a TSA plate, cultured in a 37°C constant temperature incubator for 24 h, and the colonies were counted. After 7 days of administration, one mouse in each group was randomly killed, and the wound skin was taken for HE staining.

[0125] The effect of LNG on wound healing after infection can be evaluated by the healing speed and healing process. In the calculation of the healing rate, the wound area on the first day was used as the denominator, and the actual healing area of ​​the mouse was used as the numerator to calculate the percentage of the unhealed wound area, thereby obtaining the wound percentage. Fig.15As shown. After each group of drugs were applied to wounds infected with Acinetobacter baumannii, on the 3rd day: both the high-concentration (6mg / ml) and low-concentration (3mg / ml) LNG groups reduced the wound area, while the wound area of ​​the LS group still expanded. On the 5th day: the wound healing rate of the high-concentration LNG group was 25.87%, while that of the LS was only 8.18%. The wound healing rate of the LNG group was 3.07 times higher than that of the LS group; the wound healing rate of the low-concentration LNG group was 25.82%, while that of the LS was only 13.4%. The wound healing rate of the LNG group was 1.88 times higher than that of the LS group. After 14 days, the wound healing rate of the LNG group was close to 100%, but that of the LS group was approximately 82%. The results show that the LNG group can increase the wound healing rate. Compared with the low-concentration LS group, the wound healing rate of the high-concentration LS group was slower. It is preliminarily speculated that the high-concentration LS has a certain skin toxicity, which needs to be proven by relevant experiments in the later stage. The effect of LNG in promoting wound healing after infection can be evaluated by the number of bacteria in the wound, and the results are shown in the figure below. Fig.16 As shown. After each group of drugs were applied to wounds infected with Acinetobacter baumannii, on the 5th and 7th days, the number of bacteria in the high-concentration and low-concentration LNG groups decreased more than that in the LS group (P<0.0001, ****). The results showed that at the same concentration, compared with its aqueous solution LS, LNG had a rapid and obvious effect in clearing bacterial colonization.

[0126] The effect of lavender essential oil nanohydrogel on removing Acinetobacter baumannii and promoting wound healing after infection Fig.17 , A. The results showed that compared with the control group of Acinetobacter baumannii, lavender essential oil nanohydrogels at doses of 3mg / mL and 6mg / mL could accelerate the reduction of wound area in a dose-dependent manner. Horizontal comparison showed that the efficacy of drug-loaded nanohydrogels was slightly better than that of lavender essential oil at the same dose.

[0127] In normal skin samples, there are three layers of structure: epidermis, dermis, and subcutaneous tissue, which are closely arranged and have clear boundaries. Fig.17 , B. The results show that the skin continuity is good in the high-concentration and low-concentration lavender essential oil nanohydrogel groups, and the wounds show a mild inflammatory reaction, with few inflammatory cells scattered. The high-concentration group can see three clear different levels of structure. The study found that the granulation tissue in the high-concentration lavender essential oil group was more obvious. It was preliminarily speculated that lavender essential oil has a certain skin toxicity, and nano-hydrogel has the effect of reducing skin toxicity. The mouse skin in the blank gel control group is similar to that in the positive bacterial infection group, with no normal tissue structure and a large number of inflammatory cells gathered. In summary, after the intervention of lavender essential oil nano-hydrogel, the mouse infection model showed better wound protection and healing effects.

[0128] Example 7. Study on the effect of LNG on Acinetobacter baumannii biofilm

[0129] The activated colonies with OD600 nm = 1.0 were diluted 100 times, and the diluted culture medium was added with 0.3% sucrose liquid culture medium. 1.8 mL of bacterial solution was added to a 24-well plate, and 3 mg / mL lavender essential oil nanohydrogel, lavender essential oil, and 0.2 mL of PBS solution were added to each plate, and cultured at 37°C for 48 hours. After discarding the supernatant, rinsed three times with sterile 0.9% NaCl solution. After air-drying at room temperature, 200 μL of 4% formaldehyde solution was added to each well for fixation for 30 minutes. After discarding the solution, air-dried at room temperature, rinsed repeatedly, and air-dried again. 200 μL of 0.1% crystal violet solution was added to each well, and after standing for 10 minutes, it was rinsed with sterile 0.9% NaCl solution until colorless. After drying, 200 μL of 95% ethanol was added to each well to fully dissolve, and 100 μL was transferred to a 96-well plate, and OD600 nm was detected to quantify the inhibitory ability of biofilm formation. The preliminary experiment determined that 6 mg / mL lavender essential oil nanohydrogel, lavender essential oil, and 0.2 mL PBS solution were used for intervention for 4 hours according to the above method, and OD600 nm was detected to quantify the removal ability of biofilm formation. Take the control or the inhibition and removal of protein sample biofilm samples after intervention, discard the upper culture medium, and wash three times with sterile saline. Stain with 30 μM PI and 6 μM SYTO-9 oil, 25 ° C, 15 min. The bacterial activity was determined at 488 nm and 561 nm wavelengths by CLSM imaging. Image J software was used to calculate the bacterial death and survival ratio. SEM can observe the changes of Acinetobacter baumannii after intervention with lavender essential oil nanohydrogel under microscopic conditions, and determine the effect of lavender essential oil and its nanohydrogel preparation on bacterial morphology. The scanning electron microscopy experiment set up four groups: positive bacterial control, blank gel control, 3 mg / mL lavender essential oil, and 3 mg / mL lavender essence nanohydrogel. Pick up a single colony in 10 mL of tryptone soy broth culture medium, shake horizontally for 4 h at 37 ° C and 200 rpm, and adjust the concentration of the activated bacterial solution to OD600 nm = 1.0. Add the above intervention drugs to the bacterial solution to the target drug concentration, shake horizontally for 16 h, and adjust the bacterial solution concentration to OD600 nm = 1.0 again for use.

[0130] The results of the biofilm inhibition and removal abilities are shown in Fig.18 , Fig.19 , Fig. 20 Biofilm inhibition results Fig.18 , A confirmed that the amount of biofilm in the 3mg / mL lavender essential oil nanohydrogel group was significantly lower than that in the Acinetobacter baumannii control group (P<0.001). The above results show that 3mg / mL lavender essential oil nanohydrogel has a certain inhibitory effect on the biofilm of Acinetobacter baumannii, and the horizontal comparison shows that its effect is better than lavender essential oil. The three-dimensional imaging of CLSM confirmed the above results from an intuitive perspective. The results are shown in Fig.19 Biofilm Removal Results Fig.18 As shown in Figure B, the 6 mg / mL lavender essential oil nanohydrogel group showed a clearing effect on the biofilm of Acinetobacter baumannii (P<0.001). Horizontal comparison found that the 6 mg / mL lavender essential oil nanohydrogel had a better clearing effect on the biofilm of Acinetobacter baumannii than the same dose of free drug. The three-dimensional imaging of CLSM also confirmed the above results from an intuitive perspective. The results are shown in Fig. 20 .

[0131] Example 8. DNA, protein and potassium ion leakage detection

[0132] The activated colonies with OD600 nm = 1.0 were diluted 1000 times, and 1.8 mL of bacterial solution was added to a 24-well plate. 3 mg / mL and 6 mg / mL of lavender essential oil nanohydrogel, lavender essential oil and 0.2 mL of PBS solution were added, and cultured at 37 ° C. 200 μL of bacterial solution from each group was taken at 1 h, 2 h, and 4 h, centrifuged at 6000 g for 5 min, 5 μL of supernatant was taken, and 200 μL of physiological saline was added. OD260 nm and OD280 nm were measured with the NanoDrop nucleic acid protein instrument. The leakage of DNA and protein was calculated according to the internal program of the instrument. Another bacterial supernatant was diluted 3 times and measured according to the instructions of the potassium ion kit of Nanjing Jiancheng Biotechnology Co., Ltd.

[0133] The results of DNA leakage measurement were as follows Fig.21 As shown, at 1 hour of action, the DNA leakage of the 3mg / mL and 6mg / mL drug-loaded nanoformulation groups was significantly higher than that of the blank nanohydrogel group (P<0.05 and P<0.001, respectively); horizontal comparison further found that compared with 6mg / mL lavender essential oil, the leakage of the drug-loaded nanohydrogel at the same dose also showed a very significant increase (P<0.001). After 2 hours of action, the leakage caused by free drugs and drug-loaded nanoformulations at both doses was higher than that of the corresponding blank nanohydrogel (P<0.01, P<0.001, P<0.001, P<0.001, respectively), and the leakage of the lavender essential oil nanohydrogel group was higher than that of the lavender essential oil group at the same dose (P<0.01 and P<0.001, respectively). The above trend continued to be maintained at 4 hours of action.

[0134] The results of protein leakage determination were as follows Fig. 22As shown in the figure, after 1 hour of action, the protein leakage caused by free drugs and drug-loaded nanoformulations at two doses was higher than that of the blank nanohydrogel group (P<0.05, P<0.001, P<0.05, P<0.001, respectively), and this trend was still maintained at 2 hours and 4 hours of action, and even the leakage of the preparation group continued to rise. Comparison of free drugs and drug-loaded nanoformulations at the same dose also showed significant differences.

[0135] Potassium ion leakage can also be used to evaluate the integrity of the biofilm of Acinetobacter baumannii. Fig.23 As shown in the figure, at 1h of culture, the potassium ion concentration of the 3mg / mL lavender essential oil nanohydrogel group was significantly higher than the corresponding concentration of the Acinetobacter baumannii control group (P<0.001), and it remained constant over time. And at the three time points of 1h, 2h, and 4h, there were very significant statistical differences between the free drug and the drug-loaded nanoformulations of the same dose (all P<0.001). The above results show that lavender essential oil nanohydrogel can act on the cell membrane and can promote the leakage of DNA, protein and potassium ions in the membrane compared with lavender essential oil.

[0136] Example 9. Observation of bacterial morphology using a scanning electron microscope

[0137] SEM can observe the effect of lavender essential oil and its nanohydrogel on the morphology of Acinetobacter baumannii under microscopic conditions. Fig.24 As shown in the figure, it can be found that the bacteria after the intervention of blank nanohydrogel are similar to the morphology of normal bacteria, showing spherical, quasi-spherical or hemispherical entities, and often form aggregated growth; after the intervention of 3mg / mL lavender essential oil nanohydrogel, the aggregated growth of bacteria is inhibited, and they show obvious rods, and the biofilm has obvious holes or linear ruptures, and the structure changes significantly. And the biofilm destruction ability is stronger than that of free drugs at the same dose.

[0138] In summary, the MIC and MBC of lavender essential oil nanogel against Acinetobacter baumannii strain ATCC17978 were 6 mg / mL and 3 mg / mL, respectively. Compared with lavender essential oil, the drug-loaded nanogel at the same dose can significantly promote wound healing. At the same time, the antibacterial effect of the preparation on Acinetobacter baumannii strain LAC-4 was more significant. Lavender essential oil nanogel has a more significant inhibitory and clearing effect on Acinetobacter baumannii biofilm, and can significantly increase the leakage of DNA nucleic acid, protein and potassium ions. At the same time, after the intervention of 3 mg / mL lavender essential oil nanogel, the bacterial biofilm showed obvious holes or linear ruptures, and the structure changed significantly, which was stronger than the biofilm destruction ability of free drugs at the same dose. Lavender essential oil nanogel did not cause skin irritation toxicity such as edema and erythema on the skin of normal mice, and there was no liver and kidney function toxicity signal.

[0139] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A nanohydrogel against multidrug-resistant Acinetobacter baumannii, characterized in that: The nano hydrogel mainly comprises the following components: lavender essential oil, a surfactant, an oil phase, a thickener and a cross-linking agent.

2. The nano hydrogel according to claim 1, characterized in that The components in weight portions thereof include: lavender oil with a concentration of 0.1-12%, a surfactant Tween 80 with a concentration of 12-27%, an oil phase IPM with a concentration of 0.1-3%, a thickener triethanolamine with a concentration of 0.4-3%, and a cross-linking agent CMC-Na with a concentration of 0.1-0.5%.

3. The nano hydrogel according to claim 2, characterized in that The particle size of the nano hydrogel is distributed between 1-100 nm, and the absolute value of the Zeta potential is less than 30 mV.

4. The nano hydrogel according to claim 2, characterized in that The pH value of the nano hydrogel is 4-7.

5. A method for preparing the nano hydrogel against multidrug-resistant Acinetobacter baumannii according to claim 2, characterized in that: The following steps are included: (1) Accurately weigh the prescribed amount of lavender essential oil and surfactant, and stir with a magnetic stirrer at 50-100 rpm for 5 min; (2) Add the prescribed amount of oil phase to (1) and stir with a magnetic stirrer at 50-100 rpm for 5 min; (3) adding 50% of the prescribed amount of water, and adding dropwise to the prescription (2) under magnetic stirring at 50-100 rpm to form a smoked nanoemulsion; (4) Add the prescribed amount of cross-linking agent to (3), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 minutes; (5) Add the prescribed amount of thickener to (4), mix well, and stir with a magnetic stirrer at 50-100 rpm for 5 minutes; (6) Slowly add sterile pure water dropwise to the final system in (5) while stirring; (7) The nano-hydrogel is prepared by standing the mixture at room temperature and away from light for 24 hours, and then sterilizing and packaging the mixture.

6. Use of the nano-hydrogel against multi-drug resistant Acinetobacter baumannii according to any one of claims 1 to 4 in the preparation of an external preparation against skin wound infection caused by Acinetobacter baumannii.

Images