Method and application of oxygen-tolerant photoredox catalytic triggering of ONOO- release

Abstract

The present invention discloses an oxygen-resistant photo-redox catalytically triggered ONOO ‑ The invention relates to the field of photoredox catalysis technology. The invention adopts amphiphilic block polymer and photocatalyst molecules to self-assemble to prepare nanoassemblies. The photocatalyst molecules in the nanoassemblies can act as photoredox catalysts to activate NO donor molecules in the amphiphilic block polymer to release NO under light conditions, and can also act as type I photosensitizers to generate O2 under light conditions. ·‑ , while NO and O2 ·‑ Further reaction produces ONOO ‑ , used to kill Gram-positive / negative bacteria, fungi and resistant Gram-negative bacteria.

Description

Technical field:

[0001] The present invention relates to the technical field of photoredox catalysis, and in particular to an oxygen-tolerant photoredox catalytic triggering ONOO - Release methods and applications. Background technology:

[0002] In organisms, ONOO - (Peroxynitrite anion) is mainly produced from NO and O2 in the body ·- Spontaneous reaction formation is closely related to many diseases and plays a key role in many physiological and pathological processes. - As for the donor, it is difficult to produce two short-lived active species at the same time. Therefore, the development of new ONOO - Donors remain challenging. A variety of ONOO - Donors, including the commercially available SIN-1, which spontaneously releases ONOO under physiological conditions - To further improve ONOO - The stability of the donor reduces ONOO - To prevent premature leakage and side effects, researchers developed UV- and enzyme-responsive ONOO - However, currently ONOOs that respond to near-infrared light with deep tissue penetration and low phototoxicity are - There are few studies on donors.

[0003] Photoredox catalysis plays an important role in the fields of organic compound synthesis. However, it is very difficult to develop photocatalysts with near-infrared absorption and non-metallic properties, and to realize photoredox catalysis under normoxic conditions. In particular, it is still very challenging to realize photoredox catalysis under complex physiological conditions without adding any oxygen-scavenging reagents. In the early research process, the inventors adopted a photoredox strategy to activate NO donor molecules absorbed in the ultraviolet region by red light under hypoxic conditions, thereby realizing the release of NO. In addition, the reducing species generated by the photocatalytic process consume the locally generated active oxygen, reduce the local oxygen content, and realize an oxygen-resistant photoredox catalytic NO release system. Summary of the invention:

[0004] In order to overcome the shortcomings of the prior art, the present invention provides a NO donor molecule and a photocatalyst molecule (photosensitizer), wherein the NO donor molecule can be prepared by active polymerization to prepare an amphiphilic block polymer, and the photocatalyst is further loaded in a nanoassembly by self-assembly, and the prepared nanoassembly generates ONOO under light conditions. - , used to kill Gram-positive / negative bacteria, fungi and resistant Gram-negative bacteria.

[0005] One of the purposes of the present invention is to provide a NO donor molecule having the structure of Formula I and Formula II:

[0006]

[0007] In Formula I and Formula II, R1 is selected from one of the structures of Formulas 1 to 9;

[0008]

[0009] In formulae 1 to 9, R2 and R3 are selected from one of hydrogen, nitro, methoxy, trifluoromethyl and cyano, and n is selected from any integer between 1 and 5.

[0010] Taking R2=NO2, R3=H as an example, the structure of NO donor molecule is as follows:

[0011]

[0012] The NO donor molecule of the present invention can also be called NO donor molecule, and the N-nitrosamine moiety in its structure is the NO releasing part. The double bond part of formula I and formula II can be prepared into amphiphilic block polymers by polymerization methods such as reversible addition-fragmentation chain transfer polymerization and atom transfer radical polymerization, but is not limited to the above polymerization methods.

[0013] The second object of the present invention is to provide a method for preparing an amphiphilic block polymer, wherein the amphiphilic block polymer is prepared by controllable polymerization of the NO donor molecule.

[0014] The third object of the present invention is to provide an amphiphilic block polymer having the structure of formula III and formula IV:

[0015]

[0016] In formula III and formula IV, R1 is selected from one of the structures of formulae 1 to 9, m is selected from any integer between 2 and 150, and n is selected from any integer between 2 and 100.

[0017] Preferably, the molecular weight of the amphiphilic block polymer is 200-35000 Da.

[0018] The length of the hydrophilic segment in the embodiments of the present invention can be arbitrarily selected, such as the degree of polymerization m of polyethylene glycol (PEG) is 2 to 150; the degree of polymerization n of the hydrophobic block (NO donor unit) is 2 to 100, which can be appropriately changed according to the conditions. For the present invention, the degrees of polymerization m and n are not critical, as long as they have no adverse effect on the present invention.

[0019] A fourth object of the present invention is to provide a photocatalyst having a structure of Formula V:

[0020]

[0021] Wherein, X is sulfur or selenium.

[0022] The amphiphilic block polymer and the photocatalyst molecules are self-assembled to prepare a nano-assembly with a particle size of 20-800 nm, which is an oxygen-resistant near-infrared light redox catalytic triggering ONOO - Release the platform.

[0023] A fifth object of the present invention is to provide a nano-assembly, which is prepared by self-assembly of the amphiphilic block polymer and the photocatalyst.

[0024] The sixth object of the present invention is to provide a method for preparing the nano-assembly, including a slow water addition method, a nano-flash precipitation method, an ultrasonic method, a thin film hydration method, and the like.

[0025] In a preferred embodiment, the nanoassembly can be prepared by the following method: dissolving the amphiphilic block polymer and the photocatalyst molecule in an organic solvent (including but not limited to tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane, etc.) as a co-solvent, adding them into water at a certain temperature and stirring speed, removing the organic solvent by dialysis, and obtaining a nanoassembly loaded with photocatalyst molecules.

[0026] In the present invention, the nanoassembly can release NO and O2 simultaneously under light conditions. ·- and ONOO - There is no clear restriction on the illumination conditions, as long as they are within the absorption wavelength range of the photocatalyst molecules. There is no clear restriction on the illumination intensity, as long as the illumination conditions can excite the photocatalyst molecules and have no adverse effect on the release properties.

[0027] The seventh object of the present invention is to provide the nanoassembly in the light redox catalytic triggering ONOO - Release application.

[0028] The eighth object of the present invention is to provide the use of the nanoassembly in the preparation of antibacterial agents and antitumor drugs. The nanoassembly generates ONOO under light conditions. - , can kill Gram-positive / negative bacteria, fungi and resistant Gram-negative bacteria.

[0029] The beneficial effects of the present invention are as follows: the photocatalyst in the nanoassembly prepared by the present invention can act as a photocatalyst under light conditions to catalyze the NO donor molecules to release NO, that is, the excited photocatalyst transfers electrons to the NO donor molecules in the amphiphilic block polymer, thereby releasing NO; on the other hand, the photocatalyst can also act as a type I photosensitizer to generate O2 under light conditions. ·- , indirectly consumes oxygen, thereby releasing NO under normoxic conditions, and the generated NO and O2 ·- Can further react to produce ONOO - . Description of the drawings:

[0030] Figure 1 is the H NMR spectrum of the NO donor molecule in Example 1;

[0031] Figure 2 The NMR hydrogen spectrum, carbon spectrum and mass spectrum of the photocatalyst in Example 2;

[0032] Figure 3 The NMR hydrogen spectra and GPC analysis results of PNO and PNH in Example 3;

[0033] Figure 4 The particle size and SEM image of the nanoassembly in Example 4;

[0034] Figure 5 The nanoassembly in Example 5 before and after illumination (a) NO, (b) O2 ·- ,(c)ONOO - the release of

[0035] Figure 6 The antibacterial effect of the nanoassembly in Example 6 is (a) Staphylococcus aureus, (b) Escherichia coli, (c) ciprofloxacin-resistant Pseudomonas aeruginosa, and (d) fungi;

[0036] Figure 7 This is a crystal violet stained photo of the nanoassembly used to disperse the biofilm in Example 7. Specific implementation method:

[0037] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0038] Example 1: Synthesis of NO donor molecules

[0039]

[0040] 0.4 g of NBDNH was dissolved in a mixture of glacial acetic acid and tetrahydrofuran (30 mL, v / v = 1:1), and then sodium nitrite (NaNO2, 985 mg) was slowly added, and the resulting mixture was stirred at room temperature for 5 h. The solvent was removed by concentration under reduced pressure, and purified by column chromatography to obtain a yellow solid product NBDNO with a yield of 51.6%.

[0041]

[0042] The NBDNO (200 mg, 0.8 mmol) and 2-isocyanatoethyl methacrylate (184 mg, 1.19 mmol) obtained above were dissolved in 10 mL of tetrahydrofuran, and then the catalyst DTTL (100 μL) was added. The mixture was reacted at room temperature overnight and purified by column chromatography to obtain a yellow solid product. The structural characterization spectrum is shown in FIG. Figure 1 shown.

[0043] Example 2: Synthesis of photocatalyst

[0044]

[0045] Compound 3 (1.41 g, 7.03 mmol) and compound 4 (1.2 g, 2.34 mmol) were dissolved in 50 mL of 2,2,2-trifluoroethanol, and the resulting mixture was stirred at 80° C. for 2 h. The solvent was removed by concentration under reduced pressure, and purified by column chromatography to obtain 0.72 mg of a dark blue solid product, NBSe-OH, with a yield of 65%.

[0046] NBSe-OH (0.2 g, 0.42 mmol) was dissolved in 15 mL DMF, and 50 μL DBTL was added, followed by ethyl isocyanate (84 mg, 0.632 mmol), and the resulting mixture was stirred at room temperature overnight. The solvent was removed by concentration under reduced pressure, and purified by column chromatography to obtain 195 mg of dark blue solid product NBSe with a yield of 76%. Its structural characterization spectrum is shown in Figure 2 shown.

[0047] Example 3: Synthesis of amphiphilic block polymers

[0048]

[0049] The NO donor monomer molecule (80 mg, 20 equivalents), V70 initiator (0.6 mg, 0.2 equivalents) and PEO-based macroRAFT agent (22 mg, 1 equivalent, synthesis method refer to Macromolecules, 2008, 41, 12) prepared in Example 1 were dissolved in 0.3 mL of dimethyl sulfoxide and then added to a polymerization reaction tube. The reaction tube was degassed by repeating the freeze-degassing-thawing cycle three times and sealed under vacuum. The polymer reaction solution was reacted at 60°C for 10 hours, the polymerization reaction was quenched with liquid nitrogen, and the solution was precipitated into a mixed solution of excess ether and methanol (40 mL / 5 mL). The above dissolution-precipitation cycle was repeated three times, and then the filter cake was collected by filtration and dried in a vacuum oven at room temperature overnight to obtain a yellow solid product PNO. Its control polymer PNH was synthesized by the same method, and the structural characterization spectrum and GPC molecular weight distribution diagram are shown as follows Figure 3 shown.

[0050] Example 4: Synthesis of Nanoassemblies

[0051] 1 mg of the amphiphilic block polymer prepared in Example 3 and 200 μmol of the photocatalyst prepared in Example 2 were dissolved in 1 mL of dimethyl sulfoxide, and flash-precipitated in water under high-speed stirring at room temperature. The assembly after flash precipitation was placed in a dialysis bag (14000 Da), and dialyzed in water at room temperature for 12 h to obtain a nano-assembly NBSe@PNO, the hydrated particle size and transmission electron microscopy image of which are shown in FIG. Figure 4 The assembly without NO-releasing motif is denoted as NBSe@PNH, and its preparation method is the same as above.

[0052] Example 5: Photoredox catalysis triggering NO and O2 ·- and ONOO - release

[0053] NO release test:

[0054] The NO donor molecule prepared in Example 1 and the photocatalyst prepared in Example 2 were dissolved in DMSO, and then the resulting mixed solution was irradiated with light at a wavelength of 700 nm and a light intensity of 40 mW / cm 2 , test the changes in the UV-visible light absorption spectrum, and calculate the NO release amount according to the following formula:

[0055] A1=c0l(ε1 A x A )+ε1 B x B

[0056] A2=c0l(ε2 A x A )+ε2 B xB

[0057]

[0058]

[0059] Where A1 and A2 are the absorbances of NBDNO and NBDNH at wavelengths λ1 and λ2 respectively; ε1 A , ε2 A , ε1 B , ε2 B are the molar extinction coefficients of NBDNO and NBDNH at wavelengths of λ1 and λ2, respectively; c0 is the initial concentration of NBDNO, l is the optical path length (=1 cm); x A and x B is the molar content of NBDNO and NBDNH at any irradiation time point. The NO release of the nanoassembly prepared in Example 4 was tested and calculated using the same method.

[0060] O2 ·- Release test:

[0061] O2 was detected using the commercial fluorescent probe DHR 123 ·- The fluorescence emission spectra were recorded under different conditions.

[0062] ONOO - Release test:

[0063] ONOO was tested by high performance liquid chromatography (HPLC) using L-tyrosine as a probe molecule - After release, the aqueous dispersion of the assembly and L-tyrosine (L-tyr, 1 mM, 2% DMF) was treated with light and then ultrafiltrated and centrifuged. The filtrate was then freeze-dried and analyzed by HPLC.

[0064] Figure 5 (a) NO, (b) O2 of the nanoassembly before and after illumination ·- ,(c)ONOO - release situation.

[0065] The results show that the nanoassembly of the present invention can produce NO and O2 under light conditions. ·- , and further react to form ONOO - .

[0066] Example 6: Photoredox catalysis triggering ONOO - Released for use against bacteria and fungi

[0067] Antibacterial test: For each bacterial strain, 1-2 individual colonies were inoculated into fresh tryptone soy broth (TSB) medium and incubated at 37°C for 15 h. Then, 50 μL of bacterial suspension was added to 5 mL of fresh TSB and incubated at 37°C until the logarithmic growth phase (OD 600 =0.5-0.7). The bacteria were collected by centrifugation, washed twice with sterile PBS and centrifuged (10000 rpm, 5 min, 4°C), and the bacterial concentration was adjusted to 1.5×10 6 CFU / mL. Different assemblies (100 μL) were added to a 96-well plate, and 50 μL of bacterial suspension was added to each well. The 96-well plate was incubated at 37°C for 20 min and then exposed to light or not. The 96-well plate was further incubated at 37°C for 30 min and diluted 100 times with sterile PBS. The bacterial suspension (20 μL) was placed on a TSB agar plate and incubated at 37°C for 15 h. The number of bacterial colonies was recorded. Each experiment was repeated 3 times independently.

[0068] Antifungal test: The fungus was inoculated into yeast extract peptone dextrose medium (YPD) and incubated at 30°C for 15 h (200 rpm). The fungal cells were then collected by centrifugation and diluted to 2 × 10 5 CFU / mL was used as the working suspension. Different assemblies were added to a 96-well plate. 50 μL of fungal cell suspension was added to each well, and then the 96-well plate was incubated at 30°C for 20 min, and was illuminated or not. Then, the 96-well plate was further incubated at 30°C for 30 min and diluted 100 times with sterile PBS. The fungal cell suspension (20 μL) was cultured on a YPD agar plate and incubated at 30°C for 24 h before counting the number of fungi. Each experiment was repeated three times independently.

[0069] Figure 6 Release of ONOO for photoredox catalysis - Antibacterial effects against (a) Staphylococcus aureus; (b) Escherichia coli; (c) ciprofloxacin-resistant Pseudomonas aeruginosa; and (d) fungi.

[0070] The results show that the present invention releases ONOO through photoredox catalysis. - , which can effectively kill Gram-positive bacteria, Gram-negative bacteria, resistant Gram-negative bacteria and fungi.

[0071] Example 7: Photoredox catalysis triggering ONOO - Release for dispersing drug-resistant biofilms

[0072] Biofilm culture: 500 μL TSB medium and 100 μL bacterial suspension (10 8CFU / mL), the 24-well plate was placed in a 37°C incubator for 24 hours, and then the old culture medium was replaced with fresh TSB culture medium, and the culture medium was incubated for another 24 hours before further anti-biofilm experiments. Different assemblies were added to the cultured biofilms, and the biofilms were incubated at 37°C for 1 hour, and then the biofilms were illuminated or not. The biofilms were incubated at 37°C for another 30 minutes and washed twice with PBS. After adding 0.5mL of crystal violet staining solution and incubating for 20 minutes, the plates were then washed twice with PBS, and 500μL of pure ethanol was added and the OD of the homogenized suspension was measured using a microplate reader. 550 Quantification of biofilm amount.

[0073] Figure 7 Release of ONOO for photoredox catalysis - Removal and killing of ciprofloxacin-resistant Pseudomonas aeruginosa biofilm.

[0074] The results show that the present invention releases ONOO through photoredox catalysis. - , which can effectively remove bacterial biofilm and kill bacteria.

[0075] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A nanoassembly, characterized in that: The nanoassembly is self-assembled from an amphiphilic block polymer and a photocatalyst; The amphiphilic block polymer has the structure of formula III and formula IV: In formula III and formula IV, R1 is selected from one of the structures of formulas 1 to 9, m is selected from any integer between 2 and 150, and n is selected from any integer between 2 and 100; In formulas 1 to 9, R2 and R3 are selected from one of hydrogen, nitro, methoxy, trifluoromethyl, and cyano, and n is selected from any integer between 1 and 5; The photocatalyst molecule has a structure of formula V: Wherein, X is sulfur or selenium.

2. The nanoassembly according to claim 1, characterized in that: The molecular weight of the amphiphilic block polymer is 200-35000 Da.

3. The nanoassembly according to claim 1, characterized in that: The particle size of the nano-assembly is 20-800 nm.

4. A method for preparing the nanoassembly according to any one of claims 1 to 3, characterized in that: Including slow water addition method, nano flash precipitation method, ultrasonic method and thin film hydration method.

5. The nanoassembly according to any one of claims 1 to 3 triggers ONOO in photoredox catalysis - Release application.

6. Use of the nanoassembly according to any one of claims 1 to 3 in the preparation of an antibacterial agent.

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