Luteolin-loaded mesoporous polydopamine nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of nano-particles, and particularly provides a preparation method of luteolin-loaded mesoporous polydopamine nano-particles, which mainly comprises the following steps: adding mesoporous polydopamine nano-particles into a luteolin solution, stirring until luteolin is adsorbed onto mesoporous polydopamine, centrifuging, washing, and drying to obtain luteolin-loaded mesoporous polydopamine nano-particles. The luteolin-loaded mesoporous polydopamine nano-particles are obtained by adding the luteolin into the mesoporous polydopamine nano-particles; according to the luteolin-loaded mesoporous polydopamine nano-particles, the photo-thermal sterilization effect of the mesoporous polydopamine nano-particles is combined with the antibacterial effect of luteolin, so that the illumination sterilization effect at low temperature is achieved, and the luteolin-loaded mesoporous polydopamine nano-particles have an excellent antibacterial rate on staphylococcus aureus and escherichia coli.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoparticles, and in particular to a mesoporous polydopamine nanoparticle loaded with luteolin, and a preparation method and application thereof. Background Art

[0002] Bacterial infection is a common clinical problem. The use of antibiotics is the main way to treat bacterial infections. Due to the increase in the use of antibiotics worldwide, drug-resistant bacteria have emerged, especially infections caused by multidrug-resistant bacteria, which will lead to increased mortality.

[0003] Photothermal therapy (PTT) has the advantages of being remote, minimally invasive, non-surgical, and deep in tissue, and is gaining more and more attention from researchers. Under the action of near-infrared light, the photothermal agent generates local high temperature, thereby destroying the bacterial cell membrane and internal proteins, causing irreversible damage to the bacteria and leading to bacterial death; compared with antibiotics, photothermal therapy does not induce bacterial resistance, so PTT is considered a promising antibacterial strategy.

[0004] Mesoporous polydopamine has been found by many researchers to have good photothermal conversion efficiency under 808nm near-infrared laser irradiation, and is an excellent photothermal agent. However, high temperature can damage normal cells / tissues around the wound. At the same time, excessive reactive oxygen species (ROS) may be produced during the photothermal process, thereby disrupting the redox balance, impairing immune function, and hindering infection clearance and tissue repair. Therefore, how to reduce the damage of high temperature to surrounding healthy host cells and tissues during PTT and effectively kill bacteria remains an unresolved problem. Summary of the invention

[0005] The object of the present invention is to provide a mesoporous polydopamine nanoparticle loaded with luteolin and a preparation method and application thereof. Luteolin is a naturally occurring flavonoid and is the main chemical component of Platycodon grandiflorum. As a member of the flavonoids, luteolin has multiple biological activities such as anti-inflammatory, antibacterial, and antioxidant. However, the inventors found that due to the poor water solubility of luteolin, its clinical application was affected. Based on this, the inventors considered using π-π conjugation to adsorb luteolin onto mesoporous polydopamine nanoparticles to obtain mesoporous polydopamine nanoparticles loaded with luteolin. In the process of photothermal sterilization, combined with the antibacterial effect of luteolin, a better antibacterial effect can be achieved at low temperature, which effectively solves the problem of damage to surrounding healthy host cells and tissues during the PTT process in the prior art.

[0006] The embodiments of the present invention are achieved through the following technical solutions:

[0007] A method for preparing mesoporous polydopamine nanoparticles loaded with luteolin mainly comprises the following steps: adding mesoporous polydopamine nanoparticles into a luteolin solution, stirring until luteolin is adsorbed on the mesoporous polydopamine, centrifuging, washing, and obtaining the mesoporous polydopamine nanoparticles loaded with luteolin.

[0008] Specifically, the steps include:

[0009] S1. Preparation of mesoporous polydopamine nanoparticles: Dissolve block polyether F-127 and 1,3,5-trimethylbenzene in an organic solvent (a mixture of water and ethanol), and stir for a period of time (preferably, 30-60 min) to obtain a mixture;

[0010] The tris(hydroxymethyl)aminomethane) solution was added to the above mixture, and then dopamine hydrochloride was added, and the mixture was stirred at room temperature (24 hours). After centrifugation and washing, the mixture was subjected to ultrasonic treatment to obtain mesoporous polydopamine nanoparticles.

[0011] Furthermore, the mass ratio of the block polyether F-127, 1,3,5-trimethylbenzene and dopamine hydrochloride is 5-10:5-10:1; preferably 6:6:1.

[0012] Furthermore, the organic solvent is a mixture of water and ethanol, and the volume ratio of water to ethanol is 1-1.5:1.

[0013] Furthermore, the content of tris(hydroxymethyl)aminomethane in the tris(hydroxymethyl)aminomethane solution is not less than 90%.

[0014] Furthermore, during the centrifugal separation, the centrifugal separation is performed at 10000-15000 rpm / min for 10-20 min; during the washing, the washing is performed with ethanol and acetone, and the volume ratio of ethanol and acetone is 2:1; during the ultrasonic treatment, the ultrasonic time is 0.5-1 h, and the ultrasonic washing is performed at least twice.

[0015] S2. Preparing a luteolin solution: dissolving luteolin powder in an organic solution (such as methanol) to obtain a luteolin solution;

[0016] Furthermore, the mass ratio of the luteolin powder to the volume of the organic solution is 1 mg: 1-3 mL.

[0017] S3. Load

[0018] Mesoporous polydopamine nanoparticles and luteolin solution are dispersed in an organic solution (such as methanol). After stirring for a period of time (24 hours), the luteolin solution is gradually enriched in the mesoporous polydopamine nanoparticles through π-π accumulation and hydrophilic-hydrophobic interaction. The drug-loaded nanoparticles are collected by centrifugation, which are mesoporous polydopamine nanoparticles loaded with luteolin.

[0019] Furthermore, the mass ratio of the mesoporous polydopamine nanoparticles to the volume ratio of the luteolin solution is 1 mg: 1-3 mL.

[0020] The present invention also provides a mesoporous polydopamine nanoparticle loaded with luteolin, which is prepared by the above preparation method.

[0021] The present invention also provides an application of luteolin-loaded mesoporous polydopamine nanoparticles in the preparation of antibacterial drugs.

[0022] The technical solution of the embodiment of the present invention has at least the following advantages and beneficial effects:

[0023] The mesoporous polydopamine nanoparticles loaded with luteolin of the present invention utilize the photothermal sterilization effect of the mesoporous polydopamine nanoparticles and combine them with the antibacterial effect of luteolin to achieve a light sterilization effect at low temperatures, and have excellent antibacterial rates against Staphylococcus aureus and Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a particle size distribution diagram of the mesoporous polydopamine nanoparticles prepared in Example 1;

[0026] Figure 2 This is a particle size diagram of the mesoporous polydopamine nanoparticles prepared in Example 1;

[0027] Figure 3 Zeta potential diagram of the mesoporous polydopamine nanoparticles prepared in Example 1;

[0028] Figure 4 This is a Tyndall effect diagram of the mesoporous polydopamine nanoparticles prepared in Example 1;

[0029] Figure 5 This is an appearance picture of the luteolin-loaded mesoporous polydopamine nanoparticles prepared in Example 1 dispersed in a PBS solution;

[0030] Figure 6 This is a transmission electron microscopy image of the mesoporous polydopamine nanoparticles prepared in Example 1;

[0031] Figure 7 This is a transmission electron microscopy image of the luteolin-loaded mesoporous polydopamine nanoparticles prepared in Example 1;

[0032] Figure 8 is a standard curve of absorbance of the luteolin solution prepared in Example 1;

[0033] Fig. 9 The light-induced temperature rise curves of mesoporous polydopamine nanoparticles with different concentrations;

[0034] Fig.10 The light-induced temperature rise curves of mesoporous polydopamine nanoparticles loaded with luteolin at different concentrations;

[0035] Fig.11 This is the cooling diagram of 100 μg / mL mesoporous polydopamine nanoparticles after heating and then removing the light;

[0036] Fig.12 This is the cooling diagram of the mesoporous polydopamine nanoparticles loaded with luteolin at a concentration of 100 μg / mL after heating and then removing the light;

[0037] Fig.13 Thermal imaging of mesoporous polydopamine nanoparticles and mesoporous polydopamine nanoparticles loaded with luteolin during illumination;

[0038] Fig.14 This is a plate count effect diagram of different samples against Staphylococcus aureus;

[0039] Fig.15 The colony counts of different samples against Staphylococcus aureus are shown;

[0040] Fig.16 The antibacterial rate diagram of different samples against Staphylococcus aureus;

[0041] Fig.17 This is a plate count effect diagram of different samples against Escherichia coli;

[0042] Fig.18 This is the colony count diagram of different samples against Escherichia coli;

[0043] Fig.19 This is the antibacterial rate diagram of different samples against Escherichia coli. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The following is a detailed description of a luteolin-loaded mesoporous polydopamine nanoparticle and a preparation method and application thereof provided in an embodiment of the present invention.

[0046] Example 1

[0047] A method for preparing luteolin-loaded mesoporous polydopamine nanoparticles comprises the following steps:

[0048] S1. Preparation of mesoporous polydopamine nanoparticles: 360 mg of block polyether F-127 and 360 mg of 1,3,5-trimethylbenzene were dissolved in a mixture of water and ethanol (wherein the volumes of water and ethanol in the mixture were 65 mL and 60 mL, respectively), and the mixture was stirred for 30 min to obtain a mixture; 90 mg of tris(hydroxymethyl)aminomethane solution dissolved in 10 mL of water was added to the above mixture, and then 60 mg of dopamine hydrochloride was added, and stirred at room temperature for 24 h; then, centrifuged at 12000 rpm / min for 15 min, and after centrifugation, washed with ethanol and acetone in a volume ratio of 2:1, and ultrasonically treated for 0.5 h, and ultrasonically washed three times in total; thus, mesoporous polydopamine nanoparticles were obtained;

[0049] S2. Prepare luteolin solution: dissolve 1 mg of luteolin powder in 1 mL of methanol to obtain a luteolin solution;

[0050] S3. Loading: Take 1 mg of the mesoporous polydopamine nanoparticles obtained in S1 and 1 mL of the luteolin solution obtained in S2, disperse them in methanol, stir for 24 hours to load the drug, and the luteolin solution is gradually enriched in the mesoporous polydopamine nanoparticles through π-π accumulation and hydrophilic and hydrophobic interactions. The drug-loaded nanoparticles are collected by centrifugation to obtain mesoporous polydopamine nanoparticles loaded with luteolin.

[0051] This embodiment also provides an application of luteolin-loaded mesoporous polydopamine nanoparticles in antibacterial applications.

[0052] Example 2

[0053] The difference between this embodiment and embodiment 1 is that: S1. Preparation of mesoporous polydopamine nanoparticles: 350 mg of block polyether F-127 and 350 mg of 1,3,5-trimethylbenzene are dissolved in a mixture of water and ethanol (wherein the volumes of water and ethanol in the mixture are 65 mL and 60 mL, respectively), and the mixture is stirred for 30 min to obtain a mixture; 90 mg of tris(hydroxymethyl)aminomethane solution dissolved in 10 mL of water is added to the above mixture, and then 50 mg of dopamine hydrochloride is added, and stirred at room temperature for 24 h; then, centrifuged at 14000 rpm / min for 20 min, and after centrifugation, washed with ethanol and acetone in a volume ratio of 2:1, and ultrasonically treated for 0.5 h, and ultrasonically washed three times in total; mesoporous polydopamine nanoparticles are obtained.

[0054] Example 3

[0055] The difference between this embodiment and embodiment 1 is that: S1. Preparation of mesoporous polydopamine nanoparticles: 400 mg of block polyether F-127 and 400 mg of 1,3,5-trimethylbenzene are dissolved in a mixture of water and ethanol (wherein the volumes of water and ethanol in the mixture are 75 mL and 60 mL, respectively), and the mixture is stirred for 30 min to obtain a mixture; 95 mg of tris(hydroxymethyl)aminomethane solution dissolved in 10 mL of water is added to the above mixture, and then 60 mg of dopamine hydrochloride is added, and stirred at room temperature for 24 h; then, centrifuged at 10000 rpm / min for 20 min, and after centrifugation, washed with ethanol and acetone in a volume ratio of 2:1, and ultrasonically treated for 0.5 h, and ultrasonically washed three times in total; mesoporous polydopamine nanoparticles are obtained.

[0056] Experimental Example 1

[0057] The mesoporous polydopamine nanoparticles obtained in S1 in Example 1 were tested as follows:

[0058] The particle size distribution, particle size and Zeta potential of mesoporous polydopamine particles were measured using a laser particle size analyzer. Figure 1 , Figure 2 , Figure 3 As shown;

[0059] The mesoporous polydopamine nanoparticle solution was irradiated with laser to observe its Tyndall effect. Figure 4 As shown;

[0060] Mesoporous polydopamine nanoparticles were photographed using a transmission electron microscope. Figure 5 As shown;

[0061] Depend on Figure 1 , Figure 2 , Figure 3The results showed that the particle size of mesoporous polydopamine nanoparticles measured by laser particle size analyzer was about 250nm, the potential was close to -0.7mV, and the particle size distribution was good; Figure 4 The results show that the prepared mesoporous polydopamine nanoparticles have obvious Tyndall effect in liquid; Figure 5 The results show that the particle size of the prepared mesoporous polydopamine nanoparticles is about 140nm, the surface of the nanoparticles has many channels and the surface is relatively rough. Compared with the results of transmission electron microscopy, the particle size of the nanoparticles measured by the laser particle size analyzer is larger. The possible reason is that the mesoporous polydopamine nanoparticles themselves have greater viscosity and the nanoparticles are easy to adhere to each other. The above results show that the present invention successfully prepares mesoporous polydopamine nanoparticles.

[0062] Experimental Example 2

[0063] After diluting the luteolin solution obtained in S2 of Example 1, different concentrations of luteolin solutions were tested by ultraviolet spectrophotometer, and the absorbance at 350 nm was used to draw a standard curve. The results are as follows: Figure 6 shown by Figure 6 It can be seen that the formula of the obtained standard curve is y = 0.0773x-0.026, R 2 =0.9992. Wherein, x is the concentration of the luteolin solution, and y is the absorbance value at 350 nm. It can be seen that there is a good correlation between the concentration and absorbance of the luteolin solution.

[0064] Experimental Example 3

[0065] The mesoporous polydopamine nanoparticles loaded with luteolin obtained in S3 of Example 1 were dispersed in a PBS solution, and the appearance was as follows. Figure 7 The mesoporous polydopamine nanoparticles loaded with luteolin were photographed using a transmission electron microscope. The results are shown in Figure 8 shown.

[0066] Depend on Figure 7 The results showed that the mesoporous polydopamine nanoparticles loaded with luteolin had good dispersion in PBS, making the whole solution appear black.

[0067] Depend on Figure 5 , Figure 8 The results showed that after drug loading, the particle size of the mesoporous polydopamine nanoparticles increased slightly, the pores became fewer, and the surface became smoother, proving that the mesoporous polydopamine nanoparticles successfully loaded luteolin.

[0068] The absorbance of the supernatant was tested by UV spectrophotometer. The supernatant was diluted 100 times and the measured absorbance was 0.201. The absorbance y value was substituted into Figure 6In the standard curve (the functional relationship between the concentration of luteolin solution and the absorbance value) y = 0.0773x-0.026, the x value of the luteolin solution concentration is 2.936μg / mL, and the free luteolin mass in the supernatant is 2.936μg / mL*100*1mL=293.6μg=0.2936mg; drug loading rate (%) = (total mass of luteolin-free mass of luteolin) / mass of mesoporous polydopamine nanoparticles = (1mg-0.2936mg) / 1mg, and the drug loading is 70.64%. It can be seen that this experimental example further proves that the mesoporous polydopamine nanoparticles prepared by the present invention successfully load luteolin, and its drug loading performance is ideal.

[0069] Experimental Example 4-Photothermal Performance Test of Nanoparticles

[0070] The mesoporous polydopamine nanoparticles prepared in Example 1 and the mesoporous polydopamine nanoparticles loaded with luteolin were prepared into different concentrations, and then the temperature changes were recorded under irradiation with 808 nm laser light; wherein the different concentrations were: 0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 300 μg / mL.

[0071] The above different samples were irradiated under 808nm laser light for 10 minutes, and their temperatures were recorded every 10 seconds to draw temperature rise curves. The light temperature rise curves of mesoporous polydopamine nanoparticles with different concentrations and mesoporous polydopamine nanoparticles loaded with luteolin with different concentrations were as follows: Fig. 9 , Fig.10 shown.

[0072] After 10 minutes of irradiation, the laser irradiation was stopped, and the temperature changes of 100 μg / mL mesoporous polydopamine nanoparticles and luteolin-loaded mesoporous polydopamine nanoparticles were recorded within 5 minutes (i.e., 600s-900s). The results are as follows: Fig.11 , Fig.12 shown.

[0073] At the same time, thermal images were taken every minute for three substances: PBS solution, 100 μg / mL mesoporous polydopamine nanoparticles dispersed in PBS solution, and 100 μg / mL mesoporous polydopamine nanoparticles loaded with luteolin dispersed in PBS solution. The results are as follows: Fig.13 shown.

[0074] Depend on Fig. 9 , Fig.10The results show that the mesoporous polydopamine nanoparticles prepared in Example 1 of the present invention have good photothermal effects, and the good photothermal conversion performance of the carrier mesoporous polydopamine nanoparticles is still retained after loading luteolin. Under the same illumination conditions, as the concentration of the nanoparticles increases, the temperature change of the nanoparticles also gradually increases, indicating that the temperature change of the nanoparticles is concentration-dependent; Fig.11 , Fig.12 The results showed that after 10 minutes of laser irradiation, the mesoporous polydopamine nanoparticles could gradually recover their temperature, and the loading of luteolin would not affect the performance of the mesoporous polydopamine nanoparticles. Fig.13 The results showed that the temperature of pure PBS remained basically unchanged after ten minutes of laser irradiation; however, after adding mesoporous polydopamine nanoparticles and mesoporous polydopamine nanoparticles loaded with luteolin, the thermal imaging of the solution showed obvious changes, further demonstrating that both nanoparticles have good photothermal conversion properties.

[0075] Experimental Example 5-In vitro antibacterial performance test of nanoparticles

[0076] 500 μL of different samples, 200 μg / ml, were mixed with 500 μL of Staphylococcus aureus or Escherichia coli bacterial solution (10 6 CFU / mL), incubated at 37°C for 24 hours, taken out, irradiated with near-infrared light for 10 minutes, then diluted 1000 times and 100 μL was spread on an agar plate and placed in an incubator; taken out after 18 hours and colony counted; the above experiment was repeated 3 times. The antibacterial rate and antibacterial effect of different samples were calculated. The specific results are shown in Figure 14-19 .

[0077] The different samples are: MPDA, MPDA-Lut, MPDA+NIR, MPDA-Lut+NIR; among them, MPDA is mesoporous polydopamine nanoparticles, MPDA-Lut is mesoporous polydopamine nanoparticles loaded with luteolin; MPDA+NIR is mesoporous polydopamine nanoparticles irradiated under near-infrared light for 10 minutes, and MPDA-Lut+NIR is mesoporous polydopamine nanoparticles loaded with luteolin irradiated under near-infrared light for 10 minutes.

[0078] The inhibition rate was calculated using the following formula: inhibition rate = (number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group.

[0079] The antibacterial results of different samples against Staphylococcus aureus are shown in Fig.14 , 15 As shown in Fig.16 The antibacterial results of Escherichia coli are shown in Fig.17 , 18 As shown in Fig.19shown.

[0080] Depend on Figure 14-19 The results show that mesoporous polydopamine nanoparticles loaded with luteolin have the best antibacterial effect under laser irradiation conditions. The antibacterial rate against Staphylococcus aureus can reach more than 90%, and the antibacterial rate against Escherichia coli can basically reach 80%. Compared with the traditional methods, the antibacterial rate of mesoporous polydopamine against Staphylococcus aureus and Escherichia coli has been significantly improved.

[0081] In summary, the mesoporous polydopamine nanoparticles loaded with luteolin of the present invention utilize the photothermal sterilization effect of mesoporous polydopamine nanoparticles and combine them with the antibacterial effect of luteolin to achieve light sterilization effect at low temperature, and have excellent antibacterial rates against Staphylococcus aureus and Escherichia coli.

[0082] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing mesoporous polydopamine nanoparticles loaded with luteolin, characterized in that: The following steps are involved: adding mesoporous polydopamine nanoparticles to the luteolin solution and stirring until the luteolin is adsorbed onto the mesoporous polydopamine; After centrifugation and washing, mesoporous polydopamine nanoparticles loaded with luteolin were obtained.

2. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 1, characterized in that: The preparation method of the mesoporous polydopamine nanoparticles is: (1) dissolving block polyether F-127 and 1,3,5-trimethylbenzene in an organic solvent, and stirring for a period of time to obtain a mixture; (2) Adding the tris(hydroxymethyl)aminomethane solution to the mixture obtained in step (1), and then adding dopamine hydrochloride, stirring and mixing at room temperature, centrifuging, washing, and then ultrasonically treating to obtain mesoporous polydopamine nanoparticles.

3. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 2, characterized in that: The mass ratio of the block polyether F-127, 1,3,5-trimethylbenzene and dopamine hydrochloride is 5-10:5-10:

1.

4. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 2, characterized in that: In step (2), during centrifugation, the mixture is centrifuged at 10000-15000 rpm / min for 10-20 min; during washing, the mixture is washed with ethanol and acetone in a volume ratio of 2:1; during ultrasonic treatment, the ultrasonic time is 0.5-1 h, and ultrasonic washing is performed at least twice.

5. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 1, characterized in that: The preparation method of the luteolin solution is: dissolving luteolin powder into an organic solution to obtain the luteolin solution.

6. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 5, characterized in that: The mass ratio of the luteolin powder to the volume of the organic solution is 1 mg: 1-3 mL.

7. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 1, characterized in that: Mesoporous polydopamine nanoparticles and luteolin solution are dispersed in an organic solution and loaded with drugs after stirring for a period of time. The luteolin solution is gradually enriched in the mesoporous polydopamine nanoparticles through π-π accumulation and hydrophilic-hydrophobic interaction. The drug-loaded nanoparticles are collected by centrifugation, which are mesoporous polydopamine nanoparticles loaded with luteolin.

8. The method for preparing luteolin-loaded mesoporous polydopamine nanoparticles according to claim 7, characterized in that: The mass ratio of the mesoporous polydopamine nanoparticles to the volume ratio of the luteolin solution is 1 mg: 1-3 mL.

9. A mesoporous polydopamine nanoparticle loaded with luteolin, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of mesoporous polydopamine nanoparticles loaded with luteolin prepared by the preparation method according to any one of claims 1 to 8 in the preparation of antibacterial drugs.

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