Silver nanoparticle loaded mannose conjugated pda-epl modified mesoporous silica composite material, preparation method and application thereof

Abstract

The application discloses a preparation method of a composite material, which comprises the following steps: S1, obtaining mesoporous silica, adding dopamine, and allowing the dopamine to self-polymerize on the surface of the mesoporous silica; S2, adding a silver source into the reaction system of S1, and carrying out chelation reaction with the polydopamine; S3, adding polylysine into the reaction system of S2; and S4, adding mannose into the reaction system of S3, carrying out coupling reaction with the polydopamine-polylysine, and forming a silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica material. The loading of the polylysine can improve the in-vivo bioavailability and has a broad-spectrum antibacterial performance; the introduction of the mannose coupling can be coupled to the micro-nano material as a target recognition molecule, so that the toxicity generated by off-target is reduced. By utilizing the characteristics that mycobacterium tuberculosis surface highly expresses CD206 receptors, i.e., mannose receptors, and based on the bactericidal effect of silver nanoparticles and Schiff bases, an anti-tuberculosis system of the composite material is constructed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, a preparation method thereof and application thereof. BACKGROUND

[0002] Tuberculosis is a chronic and consumptive disease with strong infectivity, and nearly one-third of the world's population is infected with it. At present, the first-line drugs for treating tuberculosis in the clinic mainly include rifampicin, isoniazid and pyrazinamide. Research has found that long-term simultaneous use of various anti-tuberculosis drugs has given rise to drug-resistant strains, and in addition, long-term use of these anti-tuberculosis drugs can also produce toxic side effects on the human body. Therefore, in the face of this serious problem, it is urgent to develop new antibacterial agents for stopping tuberculosis and provide new ideas for clinical quality.

[0003] Research has found that nanomaterials are very promising in solving the above-mentioned problems in anti-tuberculosis treatment. At present, antibacterial materials mainly include polypeptides, polymers and metal nanoparticles, and silver nanoparticles have attracted the attention of researchers due to their low toxicity and large specific surface area. More and more research uses bacteria, fungi and plants to synthesize silver nanoparticles, but the synthesis is expensive, the morphology is uncontrollable, and the particles are prone to aggregation, resulting in a decrease in particle dispersion and weakening of the antibacterial performance.

[0004] In summary, it is particularly important to develop a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material that can reduce the toxic side effects of silver nanoparticles and reduce off-targeting of antibacterial agents. SUMMARY

[0005] Therefore, the present application aims to overcome the deficiencies of the prior art and provides a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, a preparation method thereof and application thereof, which not only has low cost and high biocompatibility, but also can shorten the treatment period of mycobacterium combination, and provides a new idea for killing tuberculosis bacteria or even drug-resistant tuberculosis bacteria in the clinic.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] The first object of the present application is to provide a preparation method of a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, comprising the following steps:

[0008] S1. Obtain mesoporous silica (SBA-15), and then add dopamine to make the dopamine self-polymerize on the surface of the mesoporous silica to form polydopamine (PDA) modified mesoporous silica;

[0009] S2. Adding a silver source to the reaction system of S1, and the silver source is chelated with the polydopamine to form silver nanoparticle loaded polydopamine modified mesoporous silica (Ag@SBA-15 / PDA);

[0010] S3. Adding polylysine (EPL) to the reaction system of S2 to form silver nanoparticle loaded polydopamine-polylysine modified mesoporous silica (Ag@SBA-15 / PDA-EPL);

[0011] S4. Adding mannose to the reaction system of S3 to form silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material (Ag@SBA-15 / Man-PDA-EPL) through a coupling reaction with polydopamine-polylysine.

[0012] The mesoporous silica can be commercially purchased or prepared by a conventional method in the prior art, for example, SBA-15 can be synthesized by a simple hydrothermal method. In step S2, the phenolic hydroxyl group on the polydopamine chelates silver ions of the silver source to form nanosilver, thereby obtaining silver nanoparticle loaded polydopamine modified mesoporous silica. In step S3, polylysine is added, and in step S4, mannose is added, and a silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material is synthesized by using the Schiff base reaction of mannose with polydopamine and lysine.

[0013] The mucus molecules secreted by the foot part of marine mussels are rich in L-dopa and lysine and can adhere to almost all organic and inorganic interfaces. It is worth mentioning that polylysine has been approved by FDA as a food preservative in 2004, and can be absorbed and utilized by the human body as an essential amino acid after being eaten. Dopamine can self-polymerize in a weak alkaline environment, adhere to the surface of mesoporous silica to form a thin film of polydopamine, and the phenolic hydroxyl group of PDA can also act as a reducing agent to reduce silver ions in situ on the film surface to form stable and dispersed spherical silver nanoparticles. The molecular skeleton of polylysine (EPL) is rich in -NH2, which can improve the water solubility of hydrophobic drugs and thus improve the in vivo bioavailability, and at the same time, it has broad-spectrum antibacterial properties.

[0014] Specifically, in step S1, after adding dopamine, ultrasonic method is used to make dopamine self-polymerize on the surface of mesoporous silica.

[0015] Specifically, the molar ratio of the dopamine to the silver source is 1:(0.5-1);

[0016] Specifically, the mass ratio of the mesoporous silica to the dopamine is 1:(0.3-0.6).

[0017] Specifically, in the S3 step, the reaction temperature is 20-60 DEG C, the reaction time is 6-12h; the pH of the solution is 6-8.

[0018] Specifically, the mass ratio of the silver nanoparticle loaded polydopamine modified mesoporous silica to the polylysine is 1:(0.2-0.6).

[0019] Specifically, the silver source is one of silver nitrate, silver fluoride, silver cyanide, silver chlorate, silver perchlorate, silver bicarbonate and the like.

[0020] Specifically, in the S4 step, the coupling reaction is carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS).

[0021] Preferably, the mass ratio of the mannose to the polylysine is 1:(0.2-0.5).

[0022] The second object of the present application is to provide a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material prepared by the above preparation method.

[0023] The third object of the present application is to provide an application of a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, the composite material prepared by the above method, and / or the composite material is used for killing drug-resistant Mycobacterium tuberculosis.

[0024] In the prior art, off-target of antibacterial agents leads to poor anti-tuberculosis effect is also an important problem in the aspect of anti-Mycobacterium tuberculosis. The present application finds that mannose can be combined with specific C1q class receptors (CD206) on the surface of Mycobacterium tuberculosis, and the -COOH of mannose can be coupled with the -NH2 of PDA and EPL through amidation reaction, so as to couple mannose to the surface of PDA-EPL modified mesoporous silica, and construct a silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material anti-tuberculosis system.

[0025] Due to the application of the above technical solutions, this invention has the following advantages compared with the prior art: This invention realizes the preparation of a silver nanoparticle-loaded mannose-coupled dopamine-polylysine-modified mesoporous silica composite material. The loading of polylysine can improve in vivo bioavailability and has broad-spectrum antibacterial properties. In addition, the introduction of mannose coupling can act as a targeted recognition molecule to couple to micro- and nanomaterials, reducing off-target toxicity and providing a new approach to solving the clinical problem of killing Mycobacterium tuberculosis, even drug-resistant Mycobacterium tuberculosis. Utilizing the characteristic of Mycobacterium tuberculosis highly expressing the CD206 receptor, i.e., the mannose receptor, on its surface, and based on the bactericidal effects of silver nanoparticles and Schiff bases, an anti-tuberculosis system of silver nanoparticle-loaded mannose-coupled polydopamine-polylysine-modified mesoporous silica composite material was constructed. Attached Figure Description

[0026] Figure 1 This is a synthetic route diagram for the modification of mesoporous silica with silver nanoparticles loaded with mannose and coupled with dopamine-polylysine according to the present invention.

[0027] Figure 2 This is a schematic diagram of the anti-nodulation mechanism of silver nanoparticles loaded with mannose and coupled with dopamine-polylysine to modify mesoporous silica according to the present invention.

[0028] Figure 3 Transmission electron microscope (TEM) image of silver nanoparticles loaded with mannose and coupled with dopamine-polylysine to modify mesoporous silica prepared in this invention.

[0029] Figure 4 (A) X-ray electron diffraction (XRD) and (B) Raman spectroscopy of each step in the preparation of silver nanoparticles loaded with mannose and coupled with dopamine-polylysine modified mesoporous silica according to the present invention.

[0030] Figure 5 The high-resolution X-ray surface photoelectron spectroscopy (XPS) of C1s in each step of the preparation of silver nanoparticles loaded with mannose and coupled with dopamine-polylysine modified mesoporous silica of the present invention.

[0031] Figure 6 Plates showing different Ag@SBA-15 / PDA and EPL contents co-cultured with Mycobacterium tuberculosis (H37Rv) according to this invention;

[0032] Figure 7 The figures show the minimum inhibitory concentrations (MICs) of Ag@SBA-15 / PDA, EPL, Ag@SBA-15 / PDA-EPL, and Ag@SBA-15 / Man-PDA-EPL against (A) Mycobacterium tuberculosis (H37Rv) and (B) multidrug-resistant tuberculosis (MDR), respectively. Detailed Implementation

[0033] The preferred embodiments of the present application will be described in detail below.

[0034] We optimized the ratio of Ag@SBA-15 / PDA and EPL to obtain the best combination and the better combination for anti-tuberculosis effect. When 0.2 mg EPL was added to co-culture with Mycobacterium tuberculosis (H37Rv), there was no inhibition effect on the growth of H37Rv; when 2 mg EPL was added to co-culture with H37Rv, there was little inhibition effect on the growth of H37Rv; when 1 mg Ag@SBA-15 / PDA was added to co-culture with H37Rv, there was no inhibition effect on the growth of H37Rv; when 2 mg Ag@SBA-15 / PDA was added to co-culture with H37Rv, there was inhibition effect on the growth of H37Rv, but there was still growth of bacteria; when 1 mg Ag@SBA-15 / PDA and 0.2 mg EPL were added, there was no growth of H37Rv on the plate; when 0.5 mg Ag@SBA-15 / PDA and 0.2 mg EPL were added, there was still no growth of H37Rv on the plate. Therefore, the best anti-tuberculosis ratio of Ag@SBA-15 / PDA and EPL was 5:2, and the silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica (Ag@SBA-15 / Man-PDA-EPL) material was synthesized according to the above ratio. Examples 1-4 were prepared, and several groups of parallel experiments were performed.

[0035] Example 1

[0036] The present embodiment provides a preparation method of a silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material, comprising the following steps:

[0037] S1. 50 mg of mesoporous silica (SBA-15) and 20 mg of dopamine were added to a pH 8.5 Tris-HCl (0.1 M) solution, and under ultrasonic conditions, the dopamine was self-polymerized on the surface of the mesoporous silica. After washing, centrifugation and drying, polydopamine (PDA) modified mesoporous silica (SBA-15 / PDA) powder was obtained;

[0038] S2. 40 mg of polydopamine (PDA) modified mesoporous silica obtained in S1 and 2 mL of silver nitrate (32 mM) were added to a pH 8.5 Tris-HCl (0.1 M) solution, and stirred for 2-6 h. Silver nitrate was chelated with polydopamine to generate silver nanoparticle loaded polydopamine modified mesoporous silica (Ag@SBA-15 / PDA). After washing, centrifugation and drying, powder was obtained;

[0039] S3. Add 50 mg of silver nanoparticle loaded polydopamine modified mesoporous silica (Ag@SBA-15 / PDA) and 20 mg of polylysine (EPL) obtained in S2 into a pH 7.0 Tris-HCl (0.1 M) solution, stir for 6-12 h, after washing, centrifugation, drying, obtain silver nanoparticle loaded polydopamine-polylysine modified mesoporous silica (Ag@SBA-15 / PDA-EPL) powder;

[0040] S4. In the presence of EDC / NHS, add 50 mg of silver nanoparticle loaded polydopamine-polylysine modified mesoporous silica (Ag@SBA-15 / PDA-EPL) obtained in S3 and 5 mg of mannose, and the polydopamine-polylysine is coupled to generate Schiff base, after washing, centrifugation, drying, obtain silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material (Ag@SBA-15 / Man-PDA-EPL) powder.

[0041] The second object of the present application is to provide a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, which is prepared by the above preparation method.

[0042] Example 2

[0043] The present embodiment provides a preparation method of a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, which is basically the same as that of Example 1, except that in S3 step, the amount of EPL added is 10 mg.

[0044] Example 3

[0045] The present embodiment provides a preparation method of a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, which is basically the same as that of Example 1, except that in S3 step, the amount of EPL added is 15 mg.

[0046] Example 4

[0047] The present embodiment provides a preparation method of a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material, which is basically the same as that of Example 1, except that in S3 step, the amount of EPL added is 30 mg.

[0048] We respectively selected Ag@SBA-15 / PDA, EPL, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL, and made a plurality of groups of characterization tests, and the characterization results will be described in detail as follows:

[0049] As Figure 3 Transmission electron microscopy (TEM) of silver nanoparticles loaded mannose conjugated dopamine-polylysine modified mesoporous silica prepared in the present application (Ag@SBA-15 / Man-PDA-EPL), TEM results show that Ag@SBA-15 / Man-PDA-EPL has regular mesoporous channels, and the surface forms granular silver nanoparticles.

[0050] Figure 4 X-ray electron diffraction (XRD) and Raman spectrum of each step of preparation of silver nanoparticles loaded mannose conjugated dopamine-polylysine modified mesoporous silica in the present application.

[0051] XRD as Figure 4 (A) results show that the four peaks of Ag@SBA-15 / PDA, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL at 38°, 44°, 64° and 77° positions represent the Bragg reflection (111), (200), (220) and (311) planes, consistent with Ag (JCPDS Card No. 04-0783), indicating the formation of cubic crystal type metal silver, and the crystal form of Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL is more obvious. Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL also have other peaks at 28°, 32°, 46°, 55°, 57° and 67°, representing (111), (200), (220), (311), (222) and (400) crystal planes, consistent with AgCl (JCPDS Card No. 06-0480), indicating the presence of AgCl particles;

[0052] Raman spectrum (Raman) (as Figure 4 (B)) results show that Ag@SBA-15 / PDA, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL all have two peaks at 1406 and 1580 cm -1 , which are the stretching and deformation peaks of aromatic rings, confirming the presence of PDA layer.

[0053] Figure 5X-ray photoelectron spectroscopy (XPS) was used to analyze the preparation of the silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica of the present application. The XPS results showed that the atomic percentage of Ag in Ag@SBA-15 / PDA, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL was 0.87, 1.22 and 0.59%, respectively, and the mass percentage was 5.02, 6.91 and 4.02%, respectively. The peaks at 284.90 and 288.80 eV in the C1s high-resolution spectrum of Ag@SBA-15 / PDA were C-C / C-H and C=O of PDA; the peaks at 284.70, 285.30 and 288.00 eV in the C1s high-resolution spectrum of Ag@SBA-15 / PDA-EPL were C-C / C-H, C-N and C=O, which proved the successful loading of EPL; the peaks at 284.80, 286.00 and 287.60 eV in the C1s high-resolution spectrum of Ag@SBA-15 / Man-PDA-EPL were C-C / C-H, C=N and C=O, which indicated that the amino groups on PDA and EPL reacted with the carbonyl groups on mannose to form Schiff base (C=N) Figure 5

[0054] Example 5

[0055] The present embodiment provides an application of a silver nanoparticle loaded mannose coupled dopamine-polylysine modified mesoporous silica composite material. The composite material prepared by the above-mentioned Examples 1-4 is used for detection of Mycobacterium tuberculosis, respectively.

[0056] Comparative Example

[0057] When testing the anti-tuberculosis effect, we selected several groups of comparative experiments, which were co-cultured with Mycobacterium tuberculosis by using different amounts of EPL and Ag@SBA-15 / PDA, and the co-culture results are shown in Figure 6

[0058] In the present application, the Alamar Blue indicator method was used to detect the minimum inhibitory concentration (MIC) of Ag@SBA-15 / PDA, EPL, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL on Mycobacterium tuberculosis (H37Rv) and clinically isolated multidrug-resistant tuberculosis (MDR). The minimum inhibitory concentration (MIC) of clinically isolated multidrug-resistant tuberculosis (MDR) on rifampicin and isoniazid was 64 and 32 μg / mL, respectively. According to the Figure 7 ​​The results show that the MICs of Ag@SBA-15 / PDA, EPL, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL against H37Rv are 128, >512, 128 and 64 μg / mL, respectively; the MICs of Ag@SBA-15 / PDA, EPL, Ag@SBA-15 / PDA-EPL and Ag@SBA-15 / Man-PDA-EPL against MDR are 128, >512, 32 and 16 μg / mL, respectively. According to the calculation of fractional inhibitory concentration index (FICI) (as shown in Table 1), the components of Ag@SBA-15 / PDA and EPL in Ag@SBA-15 / PDA-EPL may be synergistic or irrelevant against H37Rv; the components of Ag@SBA-15 / PDA and EPL in Ag@SBA-15 / PDA-EPL are synergistic against MDR. In addition, the MICs of Ag@SBA-15 / Man-PDA-EPL against H37Rv and MDR are lower than those of Ag@SBA-15 / PDA-EPL, indicating that the introduction of mannose can enhance the antibacterial effect of silver nanoparticle-loaded polydopamine-polylysine modified mesoporous silica on Mycobacterium tuberculosis, which may be due to the high expression of CD206 receptor on the surface of Mycobacterium tuberculosis, which is beneficial to the recognition and binding of Ag@SBA-15 / Man-PDA-EPL, or due to the bactericidal effect of the Schiff base generated by mannose, PDA and EPL on Mycobacterium tuberculosis.

[0059] Table 1. MICs of Ag@SBA-15 / PDA, EPL and Ag@SBA-15 / PDA-EPL against Mycobacterium tuberculosis H37Rv and MDR and the corresponding fractional inhibitory concentration index (FICI)

[0060]

[0061] The above merely provides the preferred embodiments of the present application, but should not be used to limit the protection scope of the present application; and the above description should be understood by those skilled in the relevant art and implemented accordingly, so that other equivalent changes or modifications completed without departing from the disclosed concept of the present application should be covered within the protection scope of the present application.

Claims

1. A preparation method of silver nanoparticle loaded mannose conjugated dopamine-polylysine modified mesoporous silica composite material, characterized in that, The method comprises the following steps: S1. Obtain mesoporous silica, add dopamine, and use ultrasonic method to make dopamine self-polymerize on the surface of mesoporous silica to form polydopamine modified mesoporous silica; S2. Add a silver source to the reaction system of S1, and the silver source is chelated with polydopamine to form silver nanoparticle loaded polydopamine modified mesoporous silica; S3. Add polylysine to the reaction system of S2 to form silver nanoparticle loaded polydopamine-polylysine modified mesoporous silica; S4. Add mannose to the reaction system of S3 to form silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material; the coupling reaction is carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride or N-hydroxysuccinimide, and the feeding mass ratio of the mannose to the polylysine is 1:(0.2-0.5); The feeding mass ratio of the silver nanoparticle loaded polydopamine modified mesoporous silica to the polylysine is 1:(0.2-0.6); the feeding molar ratio of the dopamine to the silver source is 1:(0.5-1); and the feeding mass ratio of the mesoporous silica to the dopamine is 1:(0.3-0.6).

2. The method of claim 1, wherein the composite material is prepared by a method comprising: In the step S3, the reaction temperature is 20-60 DEG C, the reaction time is 6-12 h, and the pH of the solution is 6-8.

3. The method of claim 1, wherein the composite material is prepared by a method comprising: The silver source is one of silver nitrate, silver chlorate and silver perchlorate.

4. A silver nanoparticle loaded mannose conjugated dopamine-polylysine modified mesoporous silica composite material, characterized in that, The silver nanoparticle loaded mannose coupled polydopamine-polylysine modified mesoporous silica composite material is prepared by the preparation method of any one of claims 1-3.

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