Ultrasound-responsive gasotransmitter donor molecules, polymer preparation methods, and applications

Abstract

The present disclosure provides an ultrasonic-responsive gas transmitter donor molecule, a polymer preparation method and application, wherein the ultrasonic-responsive gas transmitter donor molecule has a structure as shown in formula (I): wherein X is O or NH, Y is O or S, and R is H or Ph, and the Ph and the benzene ring connected therewith form a fused ring structure (naphthalene ring structure). The ultrasonic-responsive gas transmitter donor polymer provided by the present disclosure has a structure as shown in formula (II): in formula (II), X is O or NH, Y is O or S, and R is H or Ph; a is any decimal number between 0 and 1; m is any integer between 3 and 20, and n is any integer between 5 and 50.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of gas transmitter donor, and particularly relates to an ultrasound-responsive gas transmitter donor molecule, a polymer preparation method and application, and more particularly relates to a carbon monoxide, hydrogen sulfide gas transmitter donor molecule, a polymer preparation method and application. BACKGROUND

[0002] Gas transmitter as an important endogenous molecule has been widely studied. Similar to nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) as gas transmitter have been found to have therapeutic potential in cancer, cardiovascular disease, inflammation and bacterial infection related diseases. However, direct gas inhalation needs to overcome the problems of high pressure storage, inability to accurately administer and non-specific distribution of toxicity. The current gas releasing molecule (GRM) may have spontaneous release and lack controllability. How to achieve controllable release of gas transmitter in deep lesion tissue and thus realize biological effect is a key problem that needs to be considered. In addition, the physiological function of gas transmitter has a high concentration dependence. Higher concentration has biological toxicity, and lower concentration cannot achieve biological effect. Therefore, in the design of gas transmitter donor, the stimulation method, tissue penetration, controllable release and biological safety should be fully considered.

[0003] Sonodynamic therapy (SDT) has become a promising treatment method due to its high tissue penetration, non-invasiveness, high focusing and high safety, and is widely used in anti-tumor and anti-bacterial applications. Ultrasound has also been designed for controllable release of chemotherapeutic drugs. The sonosensitizer material, as one of the three elements of SDT, largely determines the therapeutic effect of SDT. The sonosensitizer materials currently used are mainly organic sonosensitizer materials and inorganic sonosensitizer materials. The hydrophobicity of such materials causes inconvenience in biological application of the materials, and the low ROS quantum yield under ultrasound and non-degradability also limit the biological applicability of SDT to some extent. SUMMARY

[0004] In view of the above technical problems, the present disclosure provides an ultrasound-responsive gas transmitter donor molecule, a polymer preparation method and application, so as to at least partially solve the above technical problems.

[0005] As a first aspect of the present disclosure, an ultrasound-responsive gas transmitter donor molecule is provided, wherein the donor molecule has a structure as shown in formula (I):

[0006]

[0007] wherein X is O or NH, Y is O or S, and R is H or Ph, and the Ph and the benzene ring connected thereto form a fused ring structure.

[0008] As a second aspect of the present disclosure, an ultrasound-responsive gas transmitter donor polymer is provided, wherein the donor polymer has a structure shown in formula (II):

[0009]

[0010] In formula (II), X is O or NH, Y is O or S, R is H or Ph; a is any decimal number between 0 and 1; m is any integer between 3 and 20, and n is any integer between 5 and 50.

[0011] As a third aspect of the present disclosure, a method for preparing an ultrasound-responsive gas transmitter donor polymer is provided, comprising: performing a reversible addition-fragmentation chain transfer polymerization reaction on the donor molecule in the above embodiment with a hydrophilic monomer and a chain transfer agent to obtain the donor polymer in the above embodiment.

[0012] wherein the structure of the hydrophilic monomer is shown in formula (III):

[0013]

[0014] In formula (III), m is any integer between 3 and 20.

[0015] The structure of the chain transfer agent is shown in formula (IV):

[0016]

[0017] As a fourth aspect of the present disclosure, an ultrasound-responsive gas transmitter donor polymer is provided for use as an antibacterial and anti-inflammatory drug, wherein the antibacterial and anti-inflammatory drug comprises the donor polymer in the above embodiment.

[0018] According to the embodiments of the present disclosure, the ultrasound-responsive gas transmitter donor molecule, the polymer preparation method and the application provided by the present disclosure at least have one of the following beneficial effects:

[0019] (1) In the embodiments of the present disclosure, hydroxyflavone or hydroxyflavone thione is selected as the gas transmitter releasing unit through structural design, and the donor molecule formed thereby has ultrasound responsiveness and can release carbon monoxide (CO) or both carbon monoxide and hydrogen sulfide (CO and H2S) under ultrasonic stimulation.

[0020] (2) In the embodiments of the present disclosure, the donor molecule is prepared into a donor polymer by reversible addition-fragmentation chain transfer polymerization with a hydrophilic monomer and a chain transfer agent, so that the donor polymer also has ultrasound responsiveness and can controllably release a gaseous mediator (CO or CO and H2S) under ultrasound, realizing release at a deep tissue layer and avoiding the problem of poor treatment effect caused by premature release. Moreover, the donor polymer provided by the present disclosure also has good water solubility and stability, avoiding the problem of biological application caused by the addition of organic or inorganic sonosensitizer materials. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1A NMR hydrogen spectrum of the SHF donor molecule in Embodiment 1 of the present disclosure;

[0022] Figure 1B NMR carbon spectrum of the SHF donor molecule in Embodiment 1 of the present disclosure;

[0023] Figure 1C Mass spectrum of the SHF donor molecule in Embodiment 1 of the present disclosure;

[0024] Figure 1D High performance liquid chromatogram of the SHF donor molecule in Embodiment 1 of the present disclosure;

[0025] Figure 2 NMR hydrogen spectrum of the PSHF donor polymer in Embodiment 1 of the present disclosure;

[0026] Figure 3 Gel permeation chromatogram of the SHF donor molecule in Embodiment 1, Embodiment 3 and part of the donor polymers of the present disclosure;

[0027] Figure 4A Degradation mechanism diagram of the SHF donor molecule in Embodiment 1 of the present disclosure under ultrasound conditions;

[0028] Figure 4B Analysis diagram of the degradation product of the SHF donor molecule in Embodiment 1 of the present disclosure under ultrasound conditions;

[0029] Figure 5A UV absorption spectrum diagram of the PSHF donor polymer in Embodiment 1 of the present disclosure with increasing ultrasound time;

[0030] Figure 5B Effect diagram of the PSHF donor polymer in Embodiment 1 of the present disclosure generating singlet oxygen;

[0031] Figure 5C Comparison diagram of the PSHF in Embodiment 1 of the present disclosure releasing carbon monoxide under ultrasound or non-ultrasound conditions;

[0032] Figure 5DA graph of hydrogen sulfide release curve of PSHF in Example 1 of the present disclosure with the increase of ultrasonic time;

[0033] Figure 6A A graph of antibacterial results of PSHF donor polymers on Staphylococcus aureus under ultrasonic or non-ultrasonic conditions in Example 1 of the present disclosure;

[0034] Figure 6B A graph of biosafety evaluation of PSHF donor polymers on RAW264.7 macrophages in Example 1 of the present disclosure;

[0035] Figure 6C A graph of nitrite content detection of different polymers on LPS-induced RAW264.7 macrophages in Example 1 of the present disclosure;

[0036] Figure 6D A graph of TNF-α content detection of different polymers on LPS-induced RAW264.7 macrophages in Example 1 of the present disclosure. DETAILED DESCRIPTION

[0037] In order to make the objects, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below with specific examples.

[0038] The present disclosure takes advantage of the high tissue penetration, non-invasiveness, high focusing and safety of ultrasound, and aims at the problem that the hydrophobicity of existing sonosensitizer materials leads to poor biological application. The present disclosure provides an ultrasound-responsive gas mediator donor molecule, a polymer preparation method and application. Through structural design, the response rate, gas mediator release content and type of the donor polymer can be adjusted, which can achieve bacterial killing and cell inflammation inhibition.

[0039] Specifically, the present disclosure provides an ultrasound-responsive gas mediator donor molecule, wherein the donor molecule has a structure shown in formula (I):

[0040]

[0041] wherein X is O or NH, Y is O or S, and R is H or Ph. The Ph and the benzene ring connected thereto form a fused ring structure (such as a naphthalene ring structure).

[0042] In the embodiments of the present disclosure, hydroxyflavone or hydroxyflavone thione is selected as a gas mediator release unit through structural design. The donor molecule formed thereby has ultrasound responsiveness and can release carbon monoxide (CO) or carbon monoxide and hydrogen sulfide (CO and H2S) gas mediators under ultrasonic stimulation.

[0043] According to the embodiments of the present disclosure, the specific structural formula of the donor molecule is as follows:

[0044]

[0045] According to embodiments of this disclosure, an ultrasonically responsive gaseous neurotransmitter donor polymer is provided, wherein the donor polymer has the structure shown in formula (II):

[0046]

[0047] In formula (II), X is O or NH, Y is O or S, R is H or Ph; a is any decimal between 0 and 1; m is any integer between 3 and 20, and n is any integer between 5 and 50.

[0048] In the embodiments of this disclosure, the donor polymer is ultrasonically responsive and can controllably release gaseous neurotransmitters (CO or CO and H2S) under ultrasound, achieving deep tissue release and avoiding poor therapeutic effects and biosafety issues caused by premature release.

[0049] Specifically, when Y is S in the donor polymer, donor polymers with structures as shown in formulas (II-1) and (II-2) are obtained, which can release two gaseous mediators, carbon monoxide and hydrogen sulfide, in response to ultrasonic stimulation; wherein the structures of formulas (II-1) and (II-2) are as follows:

[0050]

[0051] In embodiments of this disclosure, when Y in the donor polymer is S, the 3-hydroxyflavone thione unit in the donor polymer is subjected to ultrasound to generate singlet oxygen during ultrasound treatment. 1 O2 oxidation causes ring-opening, releasing CO gaseous neurotransmitter. The ring-opening product is further hydrolyzed under ultrasound to release H2S gaseous neurotransmitter, which avoids the inconvenience of biological applications caused by adding additional organic / inorganic acoustic sensitizers.

[0052] Specifically, when Y is O in the donor polymer, donor polymers with structures as shown in formulas (II-3) and (II-4) are obtained, which can release carbon monoxide gaseous mediator in response to ultrasonic stimulation; wherein, the structures of formulas (II-3) and (II-4) are as follows:

[0053]

[0054] In embodiments of this disclosure, when Y in the donor polymer is O, the 3-hydroxyflavonoid unit in the donor polymer is subjected to ultrasound by singlet oxygen generated during ultrasound treatment. 1O2) oxidation occurs to release CO gas transmitter by ring-opening. The ultrasound-responsive CO-releasing co-delivery polymer material constructed in this embodiment can exist stably in an aqueous solution and avoids the problem of inconvenience in biological applications caused by the addition of organic / inorganic sonosensitizers.

[0055] According to an embodiment of the present disclosure, the molecular weight of the donor polymer is 7000-9000 Da.

[0056] According to an embodiment of the present disclosure, the donor polymer has water solubility, solving the problem that existing donor polymers are difficult to be used biologically due to hydrophobicity.

[0057] According to an embodiment of the present disclosure, a method for preparing an ultrasound-responsive gas transmitter donor polymer is also provided, comprising: performing reversible addition-fragmentation chain transfer polymerization of the donor molecule in the above embodiment with a hydrophilic monomer and a chain transfer agent to obtain the donor polymer; wherein the structure of the hydrophilic monomer is shown in formula (III): In formula (III), m is selected from any integer between 3 and 20; the structure of the chain transfer agent is shown in formula (IV):

[0058] In an embodiment of the present disclosure, the donor molecule is reversibly added-fragmented chain transfer polymerized with a hydrophilic monomer and a chain transfer agent to obtain a donor polymer. Through structural design, the ultrasound response rate, gas transmitter release content and type of the donor polymer material can be controlled, effective release of the gas transmitter is achieved, and then the killing of bacteria and the inhibition of cell inflammation can be achieved.

[0059] According to an embodiment of the present disclosure, the equivalent ratio of the hydrophilic monomer to the donor molecule is 5:1-20:1, which helps the reaction of the hydrophilic monomer and the donor molecule and makes the prepared donor polymer have a certain hydrophilicity.

[0060] According to an embodiment of the present disclosure, an ultrasound-responsive gas transmitter donor polymer is also provided for use as an antibacterial and anti-inflammatory drug, wherein the antibacterial and anti-inflammatory drug comprises the donor polymer in the above embodiment.

[0061] In order to make the technical solutions and advantages of the present disclosure clearer, the ultrasound-responsive gas transmitter release strategy involved in the technical solutions of the present disclosure is further described below in combination with specific embodiments and drawings. It should be noted that the described embodiments are only part of the embodiments of the present disclosure, not all the embodiments.

[0062] Embodiment 1:

[0063] The synthetic procedure of the release of CO / H2S donor molecule SHF is as follows:

[0064]

[0065] The specific method for preparing the donor molecule is as follows:

[0066] 1) Preparation of SHF-Boc

[0067] The raw material OHF-Boc was prepared according to previous reports (Angew. Chem. Int. Ed. 2021, 60, 13513-13520; doi.org / 10.1002 / anie.202104024).

[0068] For the preparation of SHF-Boc, the reaction raw materials OHF-Boc (1.0 g, 1.0 eq.) and Lawesson's reagent (1.26 g, 1.2 eq.) were added to a two-necked round-bottom flask, and after adding 60 mL of toluene, it was heated to 130°C and refluxed under argon for about 10 h. During the reaction, the solution color gradually deepened from red to red, and the reaction degree was monitored by thin layer chromatography (TLC). After the reaction was completed, the toluene was removed with a rotary evaporator, and then the product SHF-Boc was separated and purified by column chromatography (dichloromethane / petroleum ether).

[0069] 2) Preparation of SHF-NH2

[0070] SHF-Boc (1.0 g, 1.0 eq.) was weighed into a 250 mL round-bottom flask, and an appropriate amount of chloroform was added to dissolve the raw material. Then, trifluoroacetic acid (TFA, 2.2 g, 8.0 eq.) was added dropwise to the round-bottom flask, and the reaction was carried out at room temperature. The reaction degree was monitored by thin layer chromatography, and about 10 h of raw material SHF-Boc was substantially completely reacted. Then the reaction mixture was settled in anhydrous ether, and the collected precipitate was the reaction product SHF-NH2.

[0071] 3) Preparation of SHF donor molecule

[0072] SHF-NH2 (1.0 g, 1.0 eq.) and pentafluorophenyl methacrylate (0.97 g, 1.2 eq.) were added to a 250 mL round-bottom flask, and chloroform was added to dissolve the raw material. Triethylamine (TEA, 0.388 g, 1.2 eq.) was slowly added under stirring, and the reaction degree was monitored by thin layer chromatography, and the reaction was carried out at room temperature for about 24 h. After the reaction was completed, the solvent was removed with a rotary evaporator, extracted with dichloromethane, washed with saturated brine, dried with anhydrous sodium sulfate, and concentrated the filtrate. The crude product was further purified by column chromatography to obtain the red product SHF.

[0073] The obtained ultrasound-responsive monomer SHF structure and purity were characterized by nuclear magnetic hydrogen spectrum, carbon spectrum, mass spectrum and high performance liquid chromatography, and the test results are shown in Figures 1A-1D .

[0074] The synthesis principle of the donor polymer PSHF releasing CO and H2S gas mediators is as follows:

[0075]

[0076] The specific method for synthesizing the PSHF donor polymer is as follows:

[0077] SHF (19 mg, 1 eq.), hydrophilic monomer (OEGMA500, 250 mg, 10 eq.), azobisisobutyronitrile initiator (AIBN, 8.2 mg, 1 eq.) and chain transfer agent Ph-CTA (18 mg, 1 eq.) were dissolved in 0.3 mL of DMSO, and then added to a 2.0 mL polymerization sealing tube equipped with a magnet. The sealing tube was subjected to three freeze-degassing-thawing operations to remove oxygen in the sealing tube, and then sealed under vacuum. The sealing tube was subjected to polymerization reaction at 70°C for 8 h. After the reaction, the sealing tube was treated with liquid nitrogen and opened under low-temperature freezing. The polymerization reaction liquid was settled in diethyl ether, and subjected to three settling operations. After being sufficiently dried in a vacuum drying box, the final oil-like polymer PSHF was obtained.

[0078] The polymer PSHF was characterized by nuclear magnetic resonance hydrogen spectrum, and the results are shown in Figure 2 .

[0079] Example 2

[0080] The synthesis principle of the donor molecule OHF releasing CO gas mediators is as follows:

[0081]

[0082] The specific method for preparing the donor molecule is as follows:

[0083] 1) Preparation of OHF-NH2

[0084] OHF-Boc (1.0 g, 1.0 eq.) was weighed into a 250 mL round-bottom flask, and an appropriate amount of chloroform was added to dissolve the raw material. Then, trifluoroacetic acid (TFA, 2.3 g, 8.0 eq.) was added dropwise into the round-bottom flask, and the reaction was carried out at room temperature. The reaction degree was monitored by thin layer chromatography, and the raw material OHF-Boc was substantially completely reacted after about 10 h. Then, the reaction mixture was settled in anhydrous diethyl ether, and the collected precipitate was the reaction product OHF-NH2.

[0085] 2) Preparation of OHF

[0086] Into a 250 mL round bottom flask, OHF-NH2(1.0 g, 1.0 eq.) and pentafluorophenyl methacrylate (1.02 g, 1.2 eq.) were added and the starting materials were dissolved by adding chloroform. Triethylamine (TEA, 0.409 g, 1.2 eq.) was added slowly under stirring and the reaction was monitored by thin layer chromatography. The reaction was allowed to proceed for about 24 h at room temperature. After the reaction was completed, the solvent was removed by rotary evaporation and the residue was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The crude product was further purified by column chromatography to obtain the yellow product OHF.

[0087] The synthesis principle of the donor polymer POHF releasing CO gas transmitter is as follows:

[0088]

[0089] The specific method for synthesizing the POHF donor polymer is as follows:

[0090] OHF (18.3 mg, 1 eq.), OEGMA500 (250 mg, 10 eq.), AIBN (8.2 mg, 1 eq.) and chain transfer agent Ph-CTA (18 mg, 1 eq.) were dissolved in 0.3 mL of DMSO and added to a 2.0 mL polymerization seal tube equipped with a magnetic bar. The seal tube was subjected to three freeze-thaw-degassing-thaw operations to remove oxygen in the seal tube, and the seal tube was sealed under vacuum. The seal tube was subjected to polymerization reaction at 70°C for 8 h. After the reaction was completed, the seal tube was treated with liquid nitrogen and opened under low-temperature freezing. The polymerization reaction liquid was precipitated into ether, and the operation was repeated three times. After being sufficiently dried in a vacuum drying box, the final product was an oily polymer POHF.

[0091] The preparation steps of other related polymers such as PSFlav, POFlav, etc. are similar to those of the above-mentioned PSHF and POHF. In order to control the proportion of the hydrophilic end and the hydrophobic end to be basically consistent, the proportions of various donor molecules, hydrophilic monomers (OEGMA), AIBN and chain transfer agent Ph-CTA are kept consistent. The only difference is that the molecular weights of the donor molecules are different, so that the monomer mass is slightly different during actual polymerization. The gel permeation chromatography (GPC) curves of some polymers are shown in FIG. 1. Figure 3

[0092] Test Example 1

[0093] LC-MS study on the ultrasonic degradation process of SHF is as follows:

[0094] ​First, the DMF stock solution (5 mM) of SHF (Example 1) was prepared. 100 μM stock solution was taken in 10 mL mixed solvent (DMF / H2O = 1 / 1) to get 50 μM sample for sonication. Then, 2 mL of sonicated sample was taken from 10 mL sample at fixed time points, after removing DMF / H2O mixed solvent by concentration, 1 mL CH3CN / H2O mixed solvent was added for LC-MS test (CH3CN / H2O = 6 / 4), the sample was tested through 450 nm organic membrane. According to the LC-MS results, the possible degradation process and products were analyzed in combination with the predicted ultrasonic degradation process, and the specific analysis is shown in Figures 4A-4B

[0095] Figure 4A The degradation mechanism diagram of SHF donor molecule in Example 1 of the present disclosure under ultrasonic condition is shown.

[0096] As shown in Figure 4A , under ultrasonic (US) condition, oxygen in water can be converted into singlet oxygen ( 1 O2), which oxidizes the SHF donor molecule (A1) to open ring to release CO gas transmitter, the open ring product is hydrolyzed under ultrasonic to release H2S gas transmitter, and the corresponding degradation product C1 is obtained (as shown in Figure 4B

[0097] Test Example 2

[0098] Ultrasonic response of donor polymer PSHF for double release of CO / H2S

[0099] After obtaining the PSHF donor polymer in Example 1, its ultrasonic response, CO and H2S release were measured in pure water solvent under ultrasonic intensity 1.0 W / cm 2 , and the specific results are shown in Figures 5A-5D

[0100] Figure 5A The ultraviolet absorption spectrum of PSHF donor polymer in Example 1 of the present disclosure with increasing ultrasonic time is shown.

[0101] As shown in Figure 5A , with the increase of ultrasonic time, the ultraviolet absorption spectrum showed a gradual downward trend, indicating that 1 O2 produced in the ultrasonic process can change the structure of SHF.

[0102] ​​​Singlet oxygen detection: The generation of singlet oxygen was detected using SOSG (singlet fluorescent probe). The specific process was as follows: the solution of PSHF (50 μM) and SOSG (2.5 μM) was treated with ultrasound or non-ultrasound for 5 min, and then the fluorescence emission spectrum in the range of 515 nm-700 nm was recorded. The excitation wavelength was 504 nm.

[0103] Figure 5B This is a diagram illustrating the effect of the PSHF donor polymer generating singlet oxygen in Example 1 of this disclosure.

[0104] like Figure 5B As shown, no generation was detected in either H2O or PSHF donor polymer solutions under conditions without ultrasound. 1 O2 emits a fluorescent signal, while under ultrasonic conditions, H2O can produce... 1 O2 and no additional consumption in water (H2O) 1 O2 is a substance, so in the H2O+US curve 1 O2 intensity was highest in the PSHF group treated with ultrasound, while oxygen production in H2O was highest under ultrasound conditions. 1 O2 is consumed by PSHF, so the PSHF+US curve shows... 1 The fluorescence intensity of O2 was slightly lower than that of the H2O+US curve.

[0105] Carbon monoxide (CO) detection: CO release was detected and quantified using gas chromatography. 5 mL of 100 μM PSHF polymer was added to a 10 mL glass sample vial and sealed with a rubber stopper. The sample was then subjected to ultrasonic treatment at an intensity of 1.0 W / cm². 2 After processing for 15 min, 2.5 mL of headspace gas was aspirated using a syringe for gas chromatography-FID analysis.

[0106] Figure 5C This is a comparison diagram of the release of carbon monoxide by PSHF under ultrasonic and non-ultrasonic conditions in Embodiment 1 of this disclosure.

[0107] like Figure 5C As shown, under ultrasonic conditions, the gas chromatogram of PSHF exhibits a distinct CO signal peak.

[0108] Hydrogen sulfide detection: The method of methylene blue was used to detect H2S release, and PBS standard solutions of different concentrations (100 μΜ, 50 μΜ, 25 μΜ, 12.5 μΜ, 6.25 μΜ, 3.125 μΜ and 0 μΜ) of Na2S were prepared. 1 mL of standard solution of different concentrations was mixed with 0.2 mL of Zn(OAc)2(1% w / v) aqueous solution, and then 0.4 mL of FeCl3(30 mM, 1.2 M HCl solution) and 0.4 mL of N,N-dimethyl-1,4-phenylenediamine sulfate (20 mM, 7.2 M HCl) were added. The reaction was carried out in a 37°C constant temperature water bath for 30 min, and finally the absorption spectrum was scanned by ultraviolet-visible light absorption spectrum, and the absorption at 670 nm was recorded. The PBS solution (50 μΜ) of the polymer sample PSHF was prepared, 1 mL of the sample was mixed with 0.2 mL of Zn(OAc)2(1% w / v) aqueous solution, and then ultrasonic (1.0 W / cm 2 ) treatment was carried out for different time. Then, 0.4 mL of FeCl3(30 mM, 1.2 M HCl solution) and 0.4 mL of N,N-dimethyl-1,4-phenylenediamine sulfate (20 mM, 7.2 M HCl) were added to the mixed system with different ultrasonic time, and incubated in a 37°C constant temperature incubator for 30 min. Finally, the ultraviolet-visible light absorption spectrum was scanned and the absorption intensity at 670 nm was recorded, and the release curve of H2S was obtained by comparing with the standard curve.

[0109] Figure 5D The hydrogen sulfide release curve of PSHF in Example 1 of the present disclosure is shown as the ultrasonic time increases.

[0110] As Figure 5D shown, the concentration of H2S released by the PSHF donor polymer increases continuously with the increase of ultrasonic time.

[0111] Test Example 3

[0112] Study on the antibacterial / anti-inflammatory properties of the gas transmitter donor polymer PSHF in vitro

[0113] In vitro antibacterial performance test: The ability of PSHF material to kill S. aureus was tested by standard colony forming unit counting. 100 μL of PSHF and 50 μL of S. aureus (1×10 6 CFU / mL) were mixed and incubated in a 37°C shaker for 20 min, and then incubated for another 20 min after ultrasonic treatment. Finally, the mixed solution was diluted by 100 times, 20 μL of which was spread on the bacterial plate, and the colony counting was carried out after incubation in a 37°C incubator overnight. The specific test results are shown in Figure 6A .

[0114] Figure 6A Figure 1 shows the antibacterial results of PSHF donor polymer against S. aureus under ultrasound or non-ultrasound conditions in Example 1 of the present disclosure.

[0115] As shown in Figure 6A , the antibacterial results show that the antibacterial effect of PSHF against S. aureus also gradually becomes significant with the increase of its concentration.

[0116] In vitro anti-inflammatory performance test: the anti-inflammatory performance of different sample treatment conditions is mainly compared by testing the nitrite and TNF-a content produced in LPS-induced RAW264.7 macrophages.

[0117] First, the content of nitrite in the culture medium of RAW264.7 cells was detected by Griess reagent. Briefly, RAW264.7 macrophages were inoculated in a 24-well plate at a cell density of 10 5 cells per well and cultured in a cell incubator (37°C, 5% CO2) for 24 h. Then, the culture medium of RAW264.7 macrophages was replaced with culture medium containing PBS, LPB, L-NAME (1 mM), PSHF, POHF, PSFlav, POFlav (100 μM), and NaHS (20 μM), respectively, and LPS (1 μg / mL) was added to all groups except the PBS group. After incubation at 37°C for 4 h, the cells were treated with ultrasound for 5 min and then cultured for another 20 h. 50 μL of the cell supernatant was collected in a 96-well plate, 50 μL of Griess reagent A was added and incubated at 37°C for 10 min, then 50 μL of Griess reagent B was added and incubated for another 10 min, and the absorbance at 550 nm was measured by a microplate reader. The nitrite level produced in the cells was quantified according to the standard curve.

[0118] Figure 6B Figure 2 shows the biological safety evaluation of PSHF donor polymer against RAW264.7 macrophages in Example 1 of the present disclosure.

[0119] As shown in Figure 6B , with the increase of the concentration of PSHF donor polymer, the survival ability of RAW264.7 macrophages hardly changed, which means that PSHF of different concentrations showed good biological safety after co-culturing with RAW264.7 macrophages.

[0120] The TNF-a level of RAW264.7 cells under different treatment conditions was quantified by TNF-a ELISA detection kit under LPS induction. The cell supernatant (100 μL) after different treatments was collected, and the TNF-a concentration in each well was determined according to the operation manual, in which RAW264.7 macrophages without LPS treatment were used as negative control, and the specific test results are shown in Figures 6C-6Das shown.

[0121] Figure 6C Figure 4 is a graph showing the detection of nitrite content in LPS-induced RAW264.7 macrophages treated with different polymers in Example 1 of the present disclosure, Figure 6D Figure 5 is a graph showing the detection of TNF-α content in LPS-induced RAW264.7 macrophages treated with different polymers in Example 1 of the present disclosure.

[0122] As Figures 6C-6D shown, the test results show that under the action of ultrasound, the levels of nitrite and TNF-α in the inflammatory factors of LPS-induced RAW264.7 macrophages treated with PSHF, POHF, PSFlav, and POFlav (100 μM) are all reduced to different degrees.

[0123] The specific embodiments described above further illustrate the purposes, technical solutions, and beneficial effects of the present disclosure. It should be understood that the above description is only for specific embodiments of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. An ultrasonically responsive gaseous neurotransmitter donor polymer, characterized in that, The donor polymer has the structure shown in formula (II): Formula (II); In formula (II), X is O or NH, Y is O or S, R is H or Ph, and Ph forms a fused ring structure with the connected benzene ring; a is any decimal from 0 to 1; m is any integer from 3 to 20, and n is any integer from 5 to 50.

2. The donor polymer according to claim 1, characterized in that, When Y is S in the donor polymer, a donor polymer with the structure shown in formula (II-1) and formula (II-2) is obtained, which can release two gaseous mediators, carbon monoxide and hydrogen sulfide, in response to ultrasonic stimulation. Formula (II-1); Equation (II-2).

3. The donor polymer according to claim 1, characterized in that, When Y is O in the donor polymer, a donor polymer with the structure shown in formula (II-3) and formula (II-4) is obtained, which can release carbon monoxide gaseous mediator in response to ultrasonic stimulation. Formula (II-3); Equation (II-4).

4. The donor polymer according to any one of claims 1-3, characterized in that, The donor polymer has a molecular weight of 7000~9000 Da.

5. The donor polymer according to claim 4, characterized in that, The donor polymer is water-soluble.

6. A method for preparing an ultrasound-responsive gaseous transducer donor polymer, comprising: The donor molecule undergoes a reversible addition-fragmentation chain transfer polymerization reaction with a hydrophilic monomer and a chain transfer agent to obtain the donor polymer as described in any one of claims 1-5; The donor molecule has the structure shown in formula (I): Equation (I); Wherein, X is O or NH, Y is O or S, R is H or Ph, and Ph forms a fused ring structure with the connected benzene ring; The structure of the hydrophilic monomer is shown in formula (III): Formula (III); In equation (III), m is selected from any integer between 3 and 20; The structure of the chain transfer agent is shown in formula (IV): Formula (IV).

7. The method according to claim 6, characterized in that, The equivalence ratio of the hydrophilic monomer to the donor molecule is 5:1 to 20:

1.

8. The method according to claim 6, characterized in that, The structural formula of the donor molecule is as follows: Equation (I-1), Equation (I-2), Equation (I-3), Equation (I-4), Equation (I-5), Equation (I-6), Equation (I-7), Equation (I-8).

9. The application of an ultrasound-responsive gaseous neurotransmitter donor polymer as an antibacterial and anti-inflammatory drug, characterized in that, The antibacterial and anti-inflammatory drugs comprise the donor polymers according to any one of claims 1-5.

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