A pH-responsive switchable photothermal / catalytic nanomedicine and its preparation method

Abstract

The present invention discloses a pH-responsive switchable photothermal / catalytic nanodrug and its preparation method. Using amino-functionalized mesoporous silica as the main body, ABTS is loaded through electrostatic interaction, and then the pores are blocked by the hydrazone bond formed by the reaction of the aldehyde group of oxidized hyaluronic acid with the amino group. Finally, metal ions Fe(III) are coordinated to obtain the target product. The nanodrug of the present invention: in normal tissues, the hyaluronic acid on the surface effectively blocks the pores, preventing the leakage of ABTS, and the nanodrug is always in the "off" state without photothermal / catalytic effects; under the weak acid microenvironment conditions of bacterial infection, the hydrazone bond of the nanodrug undergoes hydrolysis and cleavage, and the released ABTS undergoes an oxidation-reduction reaction with Fe(III) to produce the oxidized photothermal conversion agent ABTS ·+ for photothermal therapy. At the same time, the reduced Fe(II) reacts with hydrogen peroxide to produce hydroxyl radicals through the Fenton reaction, realizing the synergistic antibacterial therapy of pH-responsive switchable photothermal / chemodynamic therapy.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and specifically relates to a method for preparing a pH-responsive switch-type photothermal / catalytic nanomedicine. Background Art

[0002] Infectious diseases caused by bacteria pose a serious threat to human health, and the emergence of drug-resistant bacteria has greatly limited traditional therapies that rely on antibiotics to kill bacteria. Therefore, the development of new and highly effective antimicrobial agents to curb the spread of bacterial resistance and the increase in mortality has become a scientific problem that needs to be urgently addressed in the current global public health field. In recent years, the emergence of new antimicrobial therapies such as photodynamic therapy (PDT), photothermal therapy (PTT) and chemodynamic therapy (CDT) has attracted widespread attention in the antibacterial field due to their broad-spectrum antimicrobial activity and low resistance to drug resistance.

[0003] Among them, photothermal antibacterial is a new method that uses photothermal agents with photothermal conversion properties to convert near-infrared light into local heat energy, denature nucleic acids and proteins, and destroy the selective permeability of membranes to achieve antibacterial effects. It has the advantages of safety, non-invasiveness, and high spatiotemporal selectivity, and shows great potential in antibacterial treatment and diagnosis. However, despite the important progress made in the photothermal antibacterial strategy, there are still some key issues: (1) Most photothermal agents are non-specific and are always in the "on" state during the treatment process, which has side effects on normal tissues and severely limits further clinical applications; (2) Due to bacterial resistance and the protective effect of biofilms, a single PTT treatment method is often difficult to achieve satisfactory antibacterial effects.

[0004] Therefore, developing new, efficient and low-toxic photothermal antibacterial strategies and integrating the advantages of different treatment methods are of great significance for promoting the further development and clinical application of photothermal antibacterial drugs. Summary of the invention

[0005] Based on the special weak acid microenvironment of the bacterial infection site, the present invention designs and synthesizes a pH-responsive switch-type photothermal / catalytic nanomedicine, which solves the nonspecificity in the photothermal treatment process and achieves efficient and low-toxic treatment of bacterial infection through the synergy of photothermal and chemical kinetics.

[0006] In order to achieve the purpose, the present invention adopts the following technical scheme:

[0007] A preparation method of a pH-responsive switchable photothermal / catalytic nanomedicine, characterized in that: the pH-responsive switchable photothermal / catalytic nanomedicine uses amino-functionalized mesoporous silica as the main body, and loads the photothermal conversion agent 2,2′-hydrazinobis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt ABTS through electrostatic interaction. Then, the pores are blocked by the acylhydrazone bond formed by the reaction of the aldehyde group of oxidized hyaluronic acid with the amino group. Finally, metal ions Fe(III) are coordinated to obtain the target product, denoted as AMSN-NH 2 @oxHA / Fe 3+ (abbreviated as AMHF). The nanomedicine of the present invention: in normal tissues, the hyaluronic acid on the surface effectively blocks the pores, preventing the leakage of ABTS, and the nanomedicine is always in the "off" state without photothermal / catalytic effects; in the weakly acidic (pH less than 5.5) microenvironment of bacterial infection, the acylhydrazone bond of the nanomedicine undergoes hydrolysis and cleavage, and the released ABTS undergoes an oxidation-reduction reaction with Fe(III) to produce the oxidized photothermal conversion agent ABTS ·+ for photothermal therapy. At the same time, the reduced Fe(II) reacts with hydrogen peroxide to produce hydroxyl radicals through the Fenton reaction, realizing the synergistic therapy of pH-responsive "switchable" photothermal / chemodynamic therapy.

[0008] Furthermore, the preparation method of the pH-responsive switchable photothermal / catalytic nanomedicine of the present invention includes the following steps:

[0009] Step 1: Prepare amino-functionalized mesoporous silica containing a template agent by the grafting method, and then remove the template agent by the extraction method to obtain amino-functionalized mesoporous silica (denoted as MSN-NH 2 );

[0010] Step 2: Dissolve the photothermal conversion agent ABTS in deionized water, and then add the amino-functionalized mesoporous silica (MSN-NH 2 ) prepared in Step 1, and stir in the dark at room temperature for 12 - 24 h to obtain nanoparticles loaded with the photothermal conversion agent (denoted as AM).

[0011] Step 3: Oxidatively modify hyaluronic acid (HA) to obtain oxidized hyaluronic acid (denoted as oxHA); add the AM obtained in Step 2 to a Tris-HCl buffer solution containing oxidized hyaluronic acid, and stir in the dark at room temperature for 12 - 24 h to obtain nanoparticles coated with oxidized hyaluronic acid (denoted as AMH).

[0012] Step 4: Add the AMH obtained in Step 3 to a Tris-HCl buffer solution containing ferric salt, and stir in the dark at room temperature for 1 - 2 h to obtain Fe(III)-coordinated nanoparticles, which are the target product pH-responsive switchable photothermal / catalytic nanomedicine (AMHF).

[0013] Further, in Step 1, the preparation steps of the aminated mesoporous silica are as follows: Using cetyltrimethylammonium bromide (CTAB) as a template agent, tetraethyl orthosilicate (TEOS) as a silicon source, ammonia water as a basic catalyst, and ethanol and water as a dispersion solution, reacting at room temperature for 6 - 15 h to obtain MSN / CTAB; then dissolving MSN / CTAB in an organic solvent, heating and refluxing at 85 °C for 2 - 4 h, slowly dropping 3-aminopropyltriethoxysilane (APTES) drop by drop, and continuing to reflux for 2 - 4 h to obtain MSN-NH 2 / CTAB; finally, extracting MSN-NH 2 / CTAB with a hydrochloric acid / ethanol system or an ammonium nitrate / ethanol system in a Soxhlet extractor at 95 °C for 24 - 48 h to remove the CTAB template agent and obtain aminated mesoporous silica (MSN-NH 2 ).

[0014] Further, in Step 2, the mass ratio of ABTS to the MSN-NH 2 is 1 - 4:4.

[0015] Further, in Step 3, the preparation steps of the oxHA are as follows: Weigh HA and dissolve it in deionized water, mix it with sodium periodate (NaIO 4 ) according to a mass ratio of 1 - 10:1, stir in the dark at room temperature for 12 - 24 h, and obtain oxHA through dialysis and freeze-drying.

[0016] Further, in Steps 3 and 4, the pH of the Tris-HCl buffer solution is 8.5.

[0017] Further, in Step 3, the mass ratio of the oxHA to AM is 1 - 2:1.

[0018] Further, in Step 4, the mass ratio of the ferric salt to AMH is 1 - 5:20.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. Using aminated mesoporous silica as a carrier to load ABTS, and utilizing the large specific surface area of mesoporous silica and the electrostatic interaction between the two to achieve efficient loading of ABTS. When the mass ratio of MSN-NH 2 :ABTS is 2:1, the drug loading rate and drug loading amount are 71.1% and 26.2% respectively.

[0021] 2. Function of pH-responsive switch: Since normal tissues are weakly alkaline, hyaluronic acid on the surface in normal tissues effectively blocks the pores, preventing the leakage of ABTS. The nano-drug is always in the "off" state and has no photothermal / catalytic effect. After reaching the diseased tissues, it changes from the "off" state to the "on" state in the weak acid microenvironment, generating photothermal / catalytic effects. This pH-responsive switch photothermal / catalytic therapy can not only improve the antibacterial efficacy but also reduce the side effects on normal tissues.

[0022] 3. Achieve efficient synergy of CDT / PTT: On the one hand, the reactive oxygen species (ROS) generated by CDT can damage the integrity of the bacterial cell membrane and increase the permeability of the bacterial membrane, thereby enhancing its sensitivity to photothermal therapy. On the other hand, the high temperature caused by PTT can significantly promote the rate of the catalytic reaction, thus improving its catalytic ability. Brief Description of the Drawings

[0023] Figure 1 It is the transmission electron microscope image of AMHF prepared in Example 1 of the present invention.

[0024] Figure 2 It is the XPS full scan spectrum of AMHF prepared in Example 1 of the present invention.

[0025] Figure 3 It is the ultraviolet-visible light spectrum of AMHF prepared in Example 1 of the present invention under different concentrations and pH values.

[0026] Figure 4 It is the temperature change curve of AMHF prepared in Example 1 of the present invention under different treatment conditions (pH, concentration).

[0027] Figure 5 It is the plate coating bactericidal performance test of AMHF prepared in Example 1 of the present invention. Detailed Embodiments

[0028] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1

[0030] This example prepares a pH-responsive switchable photothermal / catalytic nano-drug according to the following steps:

[0031] (1) Preparation of mesoporous silica

[0032] Weigh 0.5 g of CTAB and add it to a mixed solution of 0.9 mL of ammonia water, 80 mL of deionized water and 60 mL of absolute ethanol. Stir mechanically for 30 min to completely dissolve CTAB. Slowly add 0.9 mL of tetraethyl orthosilicate (TEOS) drop by drop to the continuously stirred mixed solution. After reacting for 15 h, centrifuge and wash the sample three times with absolute ethanol, and then dry it at 70 °C in a vacuum oven to obtain MSN / CTAB.

[0033] (2) Preparation of amino-functionalized mesoporous silica

[0034] Dissolve MSN / CTAB (150 mg) in 100 mg of isopropanol (IPA). After heating under reflux at 85 °C for 2 h, slowly add 0.3 mL of 3-aminopropyltriethoxysilane (APTES) drop by drop, and continue refluxing for 2 h. Finally, centrifuge and wash the sample three times with absolute ethanol, and then dry it under vacuum at 70 °C to obtain MSN-NH 2 / CTAB.

[0035] Dissolve MSN-NH 2 / CTAB in a mixture of ethanol and hydrochloric acid (V EtOH :V HCl = 10:1) and perform Soxhlet extraction at 95 °C for 48 h to remove the CTAB template agent. Finally, centrifuge and wash the sample three times with absolute ethanol, and dry it under vacuum at 70 °C to obtain the white solid powder MSN-NH 2 .

[0036] (3) Loading ABTS on amino-functionalized mesoporous silica

[0037] Weigh 250 mg of MSN-NH 2 and add it to 25 mL of an ABTS aqueous solution with a concentration of 5 mg / mL. Stir in the dark at room temperature for 24 h. Centrifuge and wash the sample, and dry it under vacuum at 70 °C to obtain the green solid powder AM.

[0038] (4) Preparation of oxidized hyaluronic acid

[0039] Weigh 0.2 g of HA (10 - 100 KDa) and dissolve it in 20 mL of deionized water. After adding 104 mg of NaIO 4 , stir in the dark at room temperature for 24 h. Finally, place the mixed solution in a dialysis bag with a molecular weight cut-off of 3500 Da and purify it with an ice-water mixture, changing the dialysis fluid every 2 h. After 8 h, freeze-dry the purified solution to obtain the white solid powder oxHA.

[0040] Weigh AM (100 mg) and add it to 20 mL of Tris-HCl buffer solution of oxHA with a concentration of 5 mg / mL. Keep the pH value of the solution at 8.5 and stir it in the dark at room temperature for 24 h. Centrifuge and wash the sample, and dry it in vacuum at 70 °C to obtain AMH.

[0041] (5) Oxidized hyaluronic acid-coated amino-functionalized mesoporous silica

[0042] Weigh AMH (25 mg) and add it to 0.5 mL of Tris-HCl buffer solution of FeCl 3 with a concentration of 5 mg / mL. Keep the pH value of the solution at 8.5 and stir it in the dark at room temperature for 1 h. Centrifuge and wash the sample, and dry it in vacuum at 70 °C to obtain yellow solid powder AMHF.

[0043] The following characterization tests were carried out on the nano-drugs obtained in this example:

[0044] (1) Transmission electron microscopy (TEM)

[0045] As Figure 1 shown, the TEM test results show that after surface modification, the morphology of the nano-drug AMHF changes little, the particle size is about 500 nm, while the outer layer becomes significantly thicker and there is an obvious boundary line, proving that oxHA can form a thin film to coat the surface and block the pores of MSN-NH 2 well.

[0046] (2) X-ray photoelectron spectroscopy (XPS)

[0047] As Figure 2 shown, the characteristic peaks of Si 2p and Si 2s, C 1s, N 1s, O 1s, S2p, Fe 2p are observed in the XPS total spectrum of the nano-drug AMHF, confirming that the nano-drug MSN-NH 2 has been loaded with ABTS, coated with oxHA and coordinated with Fe 3+ .

[0048] (3) pH response of AMHF

[0049] Prepare AMHF solutions with different concentrations of 0.5 mg / mL and 1 mg / mL with deionized water respectively, and adjust the pH values of the solutions to 7.4, 5.0, 2.0 with hydrochloric acid respectively. After the solutions are stable, observe the color changes of the solutions, and centrifuge to take the supernatant and record the absorbance changes at different pH values and different concentrations with an ultraviolet-visible spectrophotometer.

[0050] As Figure 3 shown, affected by the pH value and concentration, ABTS ·+The absorbance is in the range of 700 - 900 nm. The absorbance increases as the pH value decreases and also increases as the concentration increases.

[0051] (4) Photothermal performance of AMHF

[0052] Prepare AMHF suspensions with concentrations of 1 and 2 mg / mL using deionized water, and then adjust the pH values of the solutions to 7.4, 5.0, and 2.0 respectively with hydrochloric acid. Take 1.5 mL of AMHF with different concentrations and different pH values and place them in a quartz cell, and irradiate with an 808 nm laser with a power density of 1.2 W / cm 2 for 10 min. The test results are as Figure 4 shown, confirming that the concentration and pH value are important factors affecting the heating rate and threshold of the sample.

[0053] (5) Antibacterial performance

[0054] In a laminar flow hood, Escherichia coli cultured in agar medium for about 12 h is washed with normal saline, the solution is aspirated into a centrifuge tube, and the bacterial solution concentration is diluted to 3×10 8 CFU / mL. Mix 200 μL of the original bacterial solution with 500 μL of normal saline, H 2 O 2 , nano - drug AMHF (2 mg / mL), nano - drug AMHF (2 mg / mL) and H 2 O 2 (1 mM) pH = 7.4 mixture, and nano - drug AMHF (2 mg / mL) and H 2 O 2 (1 mM) pH = 5.0 mixture, and make up the volume to 1 mL with normal saline. The total solution is incubated in a shaker for 4 h; the parallel group is exposed to an 808 nm (1.2 W·cm -2 ) laser for 10 min and then incubated in a shaker for 4 h. Take 100 μL of the resulting bacterial suspension and spread it on an LB agar plate, and culture it at 37 °C for 12 h. The number of viable bacteria is determined by the conventional plate counting method. See Figure 5 , AMHF shows good antibacterial performance with a pH - responsive switch.

[0055] Example 2

[0056] This example verifies the influence of different mass ratios of MSN - NH 2 to ABTS on the drug - loading efficiency and drug - loading capacity. Specifically, prepare the pH - responsive switch - type photothermal / catalytic nano - drug according to the same steps as in Example 1, and set the dosages of ABTS aqueous solution in step (3) to 12.5 mL, 25 mL, 37.5 mL, and 50 mL, so that MSN - NH 2The mass ratios to ABTS are 4:1, 4:2, 4:3, and 4:4 respectively.

[0057] The drug loading efficiency and drug loading amount of the obtained AMHF are shown in Table 1.

[0058] Table 1, MSN-NH 2 Effect of different mass ratios of ABTS on drug loading performance

[0059] <![CDATA[MSN-NH 2 Mass ratio with ABTS]]> Drug loading efficiency (%) Drug loading amount (%) 4：1 78.3％ 16.4％ 4：2 71.1％ 26.2％ 4：3 53.1％ 28.5％ 4：4 49.5％ 33.1％

[0060] The absorbance at 340 nm was measured using an ultraviolet-visible spectrophotometer, and then the drug loading efficiency and drug loading amount were calculated based on the standard curve of ABTS. The drug loading amount refers to the amount of ABTS loaded per unit weight of the carrier, and the drug loading efficiency refers to the mass of ABTS bound to the carrier divided by the total mass of ABTS of the input drug.

[0061] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Preparation method of pH-responsive switchable photothermal / catalytic nano-drug, Characterized in that: The pH-responsive switchable photothermal / catalytic nano-drug uses amino-functionalized mesoporous silica as a carrier, electrostatically loads 2,2′-hydrazino-bis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS), and then seals the pores through the acylhydrazone bond formed by the reaction of the aldehyde group and amino group of oxidized hyaluronic acid, and finally coordinates metal ions Fe(III) to obtain the target product.

2. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 1, Characterized in that, It includes the following steps: Step 1: Prepare amino-functionalized mesoporous silica containing a template agent by the grafting method, and then remove the template agent by extraction method to obtain amino-functionalized mesoporous silica; Step 2: Dissolve the photothermal conversion agent ABTS in deionized water, then add the amino-functionalized mesoporous silica prepared in Step 1, and stir in the dark at room temperature for 12 - 24 h to obtain nanoparticles loaded with the photothermal conversion agent; Step 3: Oxidatively modify hyaluronic acid to obtain oxidized hyaluronic acid; add the nanoparticles loaded with the photothermal conversion agent obtained in Step 2 to the Tris-HCl buffer solution containing oxidized hyaluronic acid, and stir in the dark at room temperature for 12 - 24 h to obtain nanoparticles coated with oxidized hyaluronic acid; Step 4: Add the nanoparticles coated with oxidized hyaluronic acid obtained in Step 3 to the Tris-HCl buffer solution containing ferric salt, and stir in the dark at room temperature for 1 - 2 h to obtain Fe(III)-coordinated nanoparticles, which are the target product pH-responsive switchable photothermal / catalytic nano-drug.

3. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 2, Characterized in that, In Step 1, the preparation steps of the aminated mesoporous silica are as follows: Using cetyltrimethylammonium bromide as a surfactant as a template agent, tetraethyl orthosilicate as a silicon source, ammonia water as a basic catalyst, and ethanol and water as a dispersion solution, reacting at room temperature for 6 - 15 h to obtain MSN / CTAB; then dissolving MSN / CTAB in an organic solvent, heating and refluxing at 85 °C for 2 - 4 h, slowly dropwise adding 3-aminopropyltriethoxysilane, and continuing to reflux for 2 - 4 h to obtain MSN-NH 2 / CTAB; finally, extracting MSN-NH 2 / CTAB with a hydrochloric acid / ethanol system or an ammonium nitrate / ethanol system by Soxhlet extraction at 95 °C for 24 - 48 h to remove the CTAB template agent and obtain the aminated mesoporous silica.

4. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 2, Characterized in that, In Step 2, the mass ratio of ABTS to the amino-functionalized mesoporous silica is 1 - 4:

4.

5. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 2, Characterized in that, In Step 3, the preparation steps of the oxidized hyaluronic acid are: weigh hyaluronic acid and dissolve it in deionized water, mix it with sodium periodate at a mass ratio of 1 - 10:1, stir in the dark at room temperature for 12 - 24 h, and obtain oxidized hyaluronic acid through dialysis and freeze-drying.

6. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 2, Characterized in that, In Steps 3 and 4, the pH of the Tris-HCl buffer solution is 8.

5.

7. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 2, Characterized in that, In Step 3, the mass ratio of the oxidized hyaluronic acid to the nanoparticles loaded with the photothermal conversion agent is 1 - 2:

1.

8. The preparation method of the pH-responsive switchable photothermal / catalytic nano-drug according to claim 2, Characterized in that, In Step 4, the mass ratio of the ferric salt to the nanoparticles coated with oxidized hyaluronic acid is 1 - 5:

20.

9. A pH-responsive switchable photothermal / catalytic nanodrug prepared by the preparation method according to any one of claims 1 to 8.

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