CN113559071A

CN113559071A - CO targeted delivery system and construction method and application thereof

Info

Publication number: CN113559071A
Application number: CN202110950869.2A
Authority: CN
Inventors: 季兴跃; 柯亨特; 闵庆强; 倪子惠
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-10-29
Anticipated expiration: 2041-08-18
Also published as: WO2023019700A1; CN113559071B

Abstract

The invention discloses a CO targeted delivery system and a construction method and application thereof, belonging to the technical field of CO targeted delivery of nano materials. The nanoparticle comprises a carbon monoxide prodrug and oxalate or a polymer thereof, and is prepared by the following method: dissolving oxalate or polymer thereof and carbon monoxide prodrug in dichloromethane, then adding emulsifier and carrying out ultrasonic treatment, and carrying out reduced pressure distillation to obtain the nanoparticles. In order to overcome the defect of poor light penetrability on body tissues, the invention utilizes chemical energy to excite Photo-CORM to release CO and H₂O₂The oxalate reacts with oxalate with different substitution states to generate a high-energy intermediate peroxyoxalate in an excited state, the high-energy intermediate is transferred to Photo-CORM through chemical energy, the Photo-CORM is excited to the excited state to release CO, and finally the aim of targeted delivery is fulfilled.

Description

CO targeted delivery system and construction method and application thereof

Technical Field

The invention relates to the technical field of CO targeted delivery, in particular to a CO targeted delivery system and a construction method and application thereof.

Background

Carbon monoxide (CO) has long been recognized as a toxic gas that binds hemoglobin in hemoglobin structure with an affinity of 200 times that of oxygen, thereby blocking the oxygen supply function of hemoglobin. Endogenous carbon monoxide (CO) is catalytically produced from heme by heme oxygenase. Over the past two thirty years, carbon monoxide (CO) has proven to be one of the very important gas messenger small molecules in the body. Such as Nitric Oxide (NO) and hydrogen sulfide (H)₂S), CO plays an important physiological regulatory role in the body of mammals. Carbon monoxide (CO) is reported to show good treatment effects in resisting bacteria, inflammation, tumors, cardiovascular and cerebrovascular diseases, organ transplantation, preservation and the like, and the findings make carbon monoxide (CO) have wide clinical application prospects. Clinical laboratory studies of CO gas have been approved by the FDA in the United states (clinical trials. gov: NCT03799874, NCT01214187, etc.). However, there are significant drawbacks to the way in which gas is used as a clinical delivery of CO: (1) administration in the form of inhalation can only be carried out in hospitals, and patients are inconvenient to carry. (2) The dosage of CO gas is difficult to control and the mode of administration depends heavily on whether the patient has a sound pulmonary function. (3) The CO gas release is not controllable, and the off-target effect brought by the CO gas release is not inconsiderable. Therefore, safe and controllable delivery of CO drugs is a bottleneck problem to be solved urgently in clinical transformation of CO.

The prodrug strategy is a very effective approach to address the safe delivery of drugs. However, unlike traditional small molecule prodrug development, the development of CO prodrugs is a very challenging issue. First, CO is a gas molecule and is chemically very inert. Secondly, CO has a very simple structure and lacks corresponding functional groups for chemical derivatization. Therefore, conventional prodrug strategies are poorly suited for CO prodrug development. Reported CO prodrugs mainly included heavy metal CO complexes and organic CO prodrugs (fig. 13).

Although the heavy metal-CO complex (CORM-S1-3) makes an important contribution to the research of CO physiological action, the CO prodrug is difficult to be used for clinical development due to the inherent toxicity problem of heavy metals. Another class of CO prodrugs are heavy metal-free small organic molecule CO prodrugs, which can be divided into two classes requiring Photo-activation (Photo-CORM, fig. 13) and non-Photo-activation (Org-CORM, fig. 13). Due to the inherent defects of poor light penetration to body tissues and the like, the Photo-CORM CO prodrug is difficult to be applied to clinic. Heavy metal-free, light-activated-free CO prodrugs Org-CORM release CO under physiological conditions using intramolecular DielsAlder cycloaddition, which, while circumventing the disadvantages of heavy metal toxicity and the need for light activation, have some inherent problems: for example, CO release is not targeted (no stimulation is required for activation, CO release begins once dissolved in aqueous solution), and there is a risk of toxicity due to the presence of a michael addition acceptor (cyclopentadienone backbone) in the prodrug structure. Therefore, there is an urgent need to develop CO prodrugs with completely new release mechanisms to solve these bottleneck problems.

Disclosure of Invention

In order to solve the technical problems, the invention provides a CO targeted delivery system and a construction method and application thereof. In order to overcome the defect of poor light penetrability on body tissues, the invention utilizes chemical energy to excite Photo-CORM to release CO and H₂O₂Reacting with oxalate with different substitution to generate a high-energy intermediate peroxyoxalate in excited state, wherein the high-energy intermediate is transferred to Photo-CORM by chemical energy to excite the latter to excited state and releaseCO, ultimately achieving the goal of targeted delivery.

A CO targeted delivery system, which is a nanoparticle formed by a carbon monoxide prodrug and an oxalate compound; the oxalate compound comprises oxalate and/or oxalate polymer.

In one embodiment of the present invention, the carbon monoxide prodrug has the structure shown in formula (1):

wherein Y ═ O or S; x is O, S or N;

R_1-R₄independently is an alkyl, alkoxy, amino, halogen, cyano, carboxy, alkylthio, carboalkoxy or carboalkoxy group;

R₅is aryl, heteroaryl or alkyl.

In one embodiment of the present invention, the oxalate polymer has a structure represented by formulas (2) and (3):

wherein n is an integer of 20-200, and m is an integer of 10-200.

In one embodiment of the present invention, the oxalate ester has a structure represented by formula (4):

wherein R is₁Is aryl or alkyl, R₂Is aryl or alkyl.

In one embodiment of the present invention, the nanoparticle is prepared by the following method: and (3) mixing the oxalate compound and the carbon monoxide prodrug in an organic solvent, adding an emulsifier, uniformly mixing, and carrying out reduced pressure distillation to obtain the nanoparticles.

In one embodiment of the present invention, the organic solvent is one or more of ethyl acetate, diethyl ether and chloroform.

In one embodiment of the present invention, the mass ratio of the oxalate compound to the carbon monoxide prodrug is 0.1: 1-70: 1.

in one embodiment of the present invention, the molar ratio of the emulsifier to the oxalate compound is 0.1: 1-20: 1.

in one embodiment of the invention, the concentration of the oxalate ester compound is 40-1000 μ M; the concentration of the carbon monoxide prodrug is 5-100 mu M.

In one embodiment of the invention, the emulsifier is a polyvinyl alcohol solution or/and a human serum albumin solution.

In one embodiment of the invention, the mass concentration of the polyvinyl alcohol solution is 0.5% -5%; the mass concentration of the human serum albumin solution is 2-10 mg/mL.

Compared with the prior art, the technical scheme of the invention has the following advantages:

in order to overcome the defect of poor light penetrability on body tissues, the invention utilizes chemical energy to excite Photo-CORM to release CO and H₂O₂Reacting with oxalate compounds with different substitution states to generate a high-energy intermediate peroxyoxalate in an excited state, wherein the high-energy intermediate is transferred to Photo-CORM through chemical energy, and the Photo-CORM is excited to the excited state to release CO. Because the half-life of the high-energy intermediate peroxyoxalate is very short, in order to guarantee the efficiency of energy transfer, some means of drug carriers, such as nanoparticles and the like, are needed to be used for co-delivering the high-energy intermediate peroxyoxalate and the target site. The CO prodrug provided by the invention avoids the problem of heavy metal toxicity on one hand, overcomes the defect that light is difficult to penetrate skin and tissues on the other hand because no external light source is used, and can release carbon monoxide in a tumor or inflammatory cell with high hydrogen peroxide expression in a targeted manner through atopic response to hydrogen peroxide.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

Fig. 1 is a schematic diagram of the principle of the present invention.

FIG. 2 is a graph of the UV absorption spectra of nanoparticles A of the invention at different time points in test example 1.

FIG. 3 is a nuclear magnetic spectrum of a degradation product of 1-S in test example 1 of the present invention.

FIG. 4 is a graph of the UV absorption spectrum of nanoparticles B of the invention at different time points in test example 2.

FIG. 5 is a nuclear magnetic spectrum of a degradation product of 1-S in test example 2 of the present invention.

FIG. 6 is a schematic diagram showing the results of detecting carbon monoxide by myoglobin in test example 3 of nanoparticle A in test example 1.

FIG. 7 is a study of CO release kinetics of nanoparticles prepared in examples 5 to 7 of the present invention and nanoparticles obtained in comparative examples 1 to 5.

FIG. 8 is a molecular structural formula of 4-S, 5-S, 6, 7, 8 in examples 1-S, 2-S, 3-S of the present invention and comparative examples 1-5.

FIG. 9 is a graph showing the effect of the amount of oxalate polymer A on CO release in test example 5 of the present invention.

FIG. 10 is a graph showing the results of CO release specificity in test example 6 of the present invention.

FIG. 11 is a graph showing the effect of pH on CO release rate in test example 7 of the present invention.

FIG. 12 shows a graph H in test example 8 of the present invention₂O₂Results plot of content versus CO release.

FIG. 13 shows the results of the cytotoxicity test in test example 9 of the present invention.

FIGS. 14 and 15 show the results of intracellular CO imaging experiments in test example 10 of the present invention.

FIG. 16 shows the results of the nanoparticle uptake assay by the cells of test example 11 of the present invention.

FIG. 17 is a molecular structure diagram of heavy metal CO complexes and organic CO prodrugs in the art.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

1. The synthesis method of the oxalate polymer comprises the following steps:

4-hydroxybenzyl alcohol (16mmol) and 1, 8-octanediol (2.4mmol) were dissolved in anhydrous tetrahydrofuran (10mL) under a nitrogen atmosphere, and triethylamine (40mmol) was added dropwise to the mixture at 0 ℃ to add dropwise, followed by stirring for 5 min. The mixture was slowly added dropwise to oxalyl chloride (dissolved in 20mL anhydrous tetrahydrofuran) at 0 deg.C, added shut, the reaction allowed to return to room temperature, and the reaction was stirred for 12 h. Quenched with saturated sodium chloride solution and extracted with ethyl acetate (50mL × 3), the organic phase was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to give a crude product, which was purified by precipitation of oxalate polymer a using dichloromethane/n-hexane (1:1), where n is 20-50. The nuclear magnetic data of the oxalate polymer A are as follows:¹H NMR(400MHz,CDCl₃)δ7.54-7.45(m,2H),7.24-7.16(m,2H),5.40-5.31(m,2H),4.40-4.26(m,4H),1.82-1.66(m,4H),1.47-1.28(m,8H)。

2. synthesis method of thiocarbonyl carbon monoxide prodrug 1-S

Under the protection of nitrogen, 3-hydroxy-2-phenyl-4H-benzo [ g)]Adding chromene-4-one (1.0 equivalent) and a Lawson reagent (0.6 equivalent) into a double-neck reaction bottle, adding toluene, heating to 120 ℃, refluxing for 4 hours, gradually deepening the color, monitoring the reaction process by using a thin-layer chromatography plate, spin-drying the toluene after the reaction is finished, and separating and purifying by using column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain the corresponding carbon monoxide prodrug 1-S.¹H NMR(400MHz,CDCl₃)δ9.14(s,1H),8.61(s,1H),8.48(d,J＝6.8Hz,2H),8.11(d,J＝11.3Hz,2H),7.94(d,J＝8.3Hz,1H),7.68-7.47(m,5H)；¹³C NMR(150MHz,CDCl₃)δ189.4,147.3,145.3,141.9,135.6,131.2,131.0,129.7,129.4,129.2,128.9,128.8,127.2,126.5,126.3,114.7.

3. Preparation method of carbon monoxide targeted delivery system namely chemical energy nanoparticles

Oxalate polymer a (7mg) and carbon monoxide prodrug 1-S (0.5mg) were dissolved in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer at pH 7.4 to prepare a 5% by mass polyvinyl alcohol solution) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to give a uniform clear liquid, nanoparticle a.

Example 2

1. The synthesis of oxalate polymer was the same as in example 1.

2. Synthesis method of thiocarbonyl carbon monoxide prodrug 2-S

Under the protection of nitrogen, 3-hydroxyflavone (1.0 equivalent) and a Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ and refluxed for 4 hours, the color is gradually deepened, the reaction process is monitored through a thin-layer chromatography plate, the toluene is dried by spinning after the reaction is finished, and the corresponding carbon monoxide prodrug 2-S is obtained by separation and purification through column chromatography (petroleum ether: ethyl acetate: 10: 1).¹H NMR(400MHz,CDCl₃)δ8.71(s,1H),8.57(d,J＝8.1Hz,1H),8.38(d,J＝7.1Hz,2H),7.71(t,J＝7.4Hz,1H),7.64(d,J＝8.3Hz,1H),7.59-7.51(m,3H),7.46(t,J＝7.5Hz,1H)；¹³C NMR(150MHz,CDCl₃)δ188.2,150.5,146.3,141.4,133.3,131.0,130.9,128.9,128.8,128.1,125.9,118.5。

3. Preparation method of chemical energy nanoparticle B

Oxalate polymer a (7mg) and carbon monoxide prodrug 2-S (0.5mg) were dissolved in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles B.

Example 3

1. The synthesis of oxalate polymer was the same as in example 1.

2. Synthesis method of thiocarbonyl carbon monoxide prodrug 3-S

Under the protection of nitrogen, 3-hydroxy-4H-chromen-4-one (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, then the mixture is heated to 120 ℃ for refluxing for 4 hours, the color is gradually deepened, the reaction process is monitored through a thin-layer chromatography plate, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 3-S is obtained.¹H NMR(400MHz,CDCl₃)δ8.60(d,J＝8.3Hz,1H),8.06(s,1H),7.71(t,J＝8.3,7.2Hz,1H),7.67(s,1H),7.57(d,J＝8.5Hz,1H),7.48(t,J＝7.6Hz,1H)；¹³C NMR(150MHz,CDCl₃)δ190.0,151.0,149.2,133.4,133.0,129.0,128.5,126.1,119.0。

3. Preparation method of carbon monoxide targeted delivery system, namely chemical energy nanoparticle C

Oxalate polymer a (7mg) and carbon monoxide prodrug 3-S (0.5mg) were dissolved in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles C.

Example 4

1. The synthesis of oxalate polymer was the same as in example 1.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

3. Preparation method of carbon monoxide targeted delivery system namely chemical energy nanoparticle D

Oxalate polymer (2.24mg) and carbon monoxide prodrug 2-S (0.16mg) were dissolved in 250. mu.L of dichloromethane, 5mL of 4mg/mL human serum albumin was added, sonicated using an ultrasonic cell disruptor for 10min, and the dichloromethane was removed by concentration at 37 ℃ under reduced pressure to give a homogeneous clear liquid, nanoparticle D.

The carbon monoxide targeted delivery system, namely nanoparticles in examples 5-7 were prepared as in example 1, except that the carbon monoxide prodrugs in examples 5-7 were 1-S, 2-S, and 3-S, respectively, wherein the molar numbers of the carbon monoxide prodrugs were all 1.97 μmol, and the nanoparticles prepared were E-1, E-2, and E-3, respectively.

Examples 8 to 12

Nanoparticles of examples 8-12 were prepared as in example 1, except that the carbon monoxide prodrugs of examples 8-12 were all 2-S, and the carbon monoxide prodrugs were all 2-S and oxalate polymer A at 0.1mg/7.0mg, respectively; 0.3mg/7.0 mg; 0.5mg/7.0 mg; 0.8mg/7.0 mg; 1.0mg/7.0 mg. The prepared nano materials are respectively F-1, F-2, F-3, F-4 and F-5.

Example 13

1. Wherein the oxalate B is:

2. the synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate B (1mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer solution at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles H.

Example 14

1. Preparation of oxalate 1:

p-methylphenol (1.05mL,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after the addition, the reaction was monitored by thin layer chromatography, the solvent was spun off, and compound 1(2.04g) was isolated and purified by column chromatography (PE: EA ═ 20:1) in 98% yield.¹H NMR(300MHz,CDCl₃)δ7.21(d,J＝8.6Hz,1H),7.07(d,J＝8.4Hz,1H),4.44(q,J＝7.1Hz,1H),2.35(s,2H),1.43(t,J＝7.1Hz,2H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,150.1,134.4,129.6,121.3,61.4,21.15(s),14.7。

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

3. The preparation method of the carbon monoxide targeted delivery system, namely the chemical energy nanoparticles comprises the following steps:

oxalate 1(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 1.

Example 15

1. Preparation of oxalate 2:

3,4, 5-trimethoxyphenol (1.83g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under nitrogen in a 50mL two-necked flask and oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 deg.C, followed by addition of oxalyl chlorideAfter the reaction was completed, the reaction was continued for 10 minutes, followed by monitoring the reaction by thin layer chromatography, after completion of the reaction, the solvent was dried by spinning, and the compound 2(2.69g) was isolated and purified by column chromatography (PE: EA ═ 20:1) with a yield of 95%.¹H NMR(500MHz,CDCl₃)δ6.44(s,2H),4.18(s,2H),3.83(s,6H),3.71(s,3H),1.25(s,3H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,155.7,148.8,135.7,99.8,61.5,60.7,56.8,14.7.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

oxalate 2(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer solution at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 2.

Example 16

1. Preparation of oxalate 3:

p-cyanophenol (1.18g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after addition, the reaction was monitored by thin layer chromatography, the solvent was spun off, and compound 3(2.01g) was isolated and purified by column chromatography (PE: EA ═ 20:1) in 92% yield.¹HNMR(500MHz,CDCl₃)δ7.89(d,J＝14.8Hz,2H),7.38(d,J＝15.0Hz,2H),4.18(q,J＝11.8Hz,2H),1.25(t,J＝11.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,156.6,133.2,123.8,118.9,109.9,61.4,14.7.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

oxalate 3(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 3.

Example 17

1. Preparation of oxalate 4:

p-methoxyphenol (1.24g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after addition, the reaction was monitored by thin layer chromatography, the solvent was spun off, and compound 4(2.15g) was isolated and purified by column chromatography (PE: EA ═ 20:1) in 96% yield.¹HNMR(500MHz,CDCl₃)δ7.21(d,J＝15.0Hz,2H),6.99(d,J＝15.0Hz,2H),4.18(q,J＝11.8Hz,2H),3.79(s,3H),1.25(t,J＝11.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,156.8,145.1,123.1,114.6,61.4,56.1,14.7。

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

oxalate 4(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 4.

Example 18

1. Preparation of oxalate 5:

p-trifluoromethylphenol (1.63g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after the addition, the reaction was monitored by thin layer chromatography, the solvent was spun off, and compound 5(2.38g) was isolated and purified by column chromatography (PE: EA ═ 20:1) in 91% yield.¹H NMR(500MHz,CDCl₃)δ7.50(d,J＝15.0Hz,2H),7.11(d,J＝15.0Hz,2H),4.18(q,J＝11.8Hz,2H),1.25(t,J＝11.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,154.3,129.3(q,J_C-F＝32.4Hz),127.4(q,J_C-F＝3.7Hz),123.6(q,J_C-F＝268.1Hz),121.7,61.4,14.7.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 5(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer solution at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 5.

Example 19

1. Preparation of oxalate 6:

p-fluorophenol (1.12g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes, the reaction was monitored by thin layer chromatography, the solvent was spun off, and compound 6(1.91g) was isolated and purified by column chromatography (PE: EA ═ 20:1) with a yield of 90%.¹H NMR(500MHz,CDCl₃)δ7.18-7.17(m,2H),7.16-7.14(m,2H),4.18(q,J＝11.8Hz,2H),1.25(t,J＝11.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ161.2,159.5,159.2,158.1,148.6(d,J_C-F＝3.7Hz),121.48(d,J_C-F＝7.6Hz),115.4(d,J_C-F＝20.0Hz),61.4,14.6.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 6(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 6.

Example 20

1. Preparation of oxalate ester 7:

p-chlorophenol (1.28g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under nitrogen in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after addition, the reaction was monitored by thin layer chromatography, the solvent was spun dry and the solution was fractionated by column chromatography (PE: EA ═ 20:1)Isolation and purification gave compound 7(2.05g) in 90% yield.¹H NMR(500MHz,CDCl₃)δ7.41(d,J＝15.2Hz,2H),7.35(d,J＝15.2Hz,2H),4.18(q,J＝11.8Hz,2H),1.25(t,J＝11.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,150.2,131.8,129.7,122.2,61.4,14.7.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 7(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer solution at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 7.

Example 21

1. Preparation of oxalate ester 8:

p-heptylphenol (1.92g,1.0 eq) and triethylamine (2.8mL,2.0 eq) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride monoethyl ester (2.1mL,2.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes, the reaction was monitored by thin layer chromatography, the solvent was spun off, and compound 8(2.48g) was isolated and purified by column chromatography (PE: EA ═ 20:1) in 85% yield.¹H NMR(500MHz,CDCl₃)δ7.16(d,J＝15.2Hz,2H),7.12(d,J＝15.2Hz,2H),4.16(q,J＝11.8Hz,2H),2.63(t,J＝15.7Hz,2H),1.63(tt,J＝30.7,15.2Hz,2H),1.30-1.21(m,12H),0.95-0.83(m,3H)；¹³C NMR(125MHz,CDCl₃)δ159.5,158.1,150.4,138.5,128.1,122.2,61.4,36.3,31.7,30.1,28.9,28.9,23.2,14.7,14.0.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 8(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 8.

Example 22

1. Preparation of oxalate 9:

3,4, 5-trimethoxyphenol (1.83g,4.0 equiv.) and triethylamine (2.8mL,4.0 equiv.) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride (227 μ L,1.0 equiv.) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after the addition, the reaction was monitored by thin layer chromatography, the solvent was dried by spinning, and compound 9(971mg) was isolated and purified by column chromatography (PE: EA ═ 20:1) with a yield of 92%.¹H NMR(500MHz,CDCl₃)δ6.44(s,4H),3.83(s,12H),3.71(s,6H)；¹³C NMR(125MHz,CDCl₃)δ155.7,155.1,148.8,135.7,99.8,60.7),56.8.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 9(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-co-hydroxy lactic acid) (7mg) were dissolved together in 500. mu.L of dichloromethane, and 3mL of polyvinyl alcohol was added

(PVA) solution (polyvinyl alcohol solid is dissolved in phosphate buffer solution with pH 7.4 to prepare polyvinyl alcohol (PVA) solution with mass concentration of 5%), ultrasonic emulsification is carried out for 10min by using an ultrasonic cell disruptor, and dichloromethane is removed by decompression and concentration at 37 ℃ to obtain uniform and clear liquid, namely nanoparticles 9.

Example 23

1. Preparation of oxalate 10:

p-methoxyphenol (1.05mL,4.0 eq) and triethylamine (2.8mL,4.0 eq) were dissolved in dichloromethane (20mL) under a nitrogen atmosphere in a 50mL two-necked flask, oxalyl chloride (227 μ L,1.0 eq) was added dropwise at 0 ℃, the reaction was continued for 10 minutes after addition, the reaction was monitored by thin layer chromatography, the solvent was spun off after completion, and compound 10(740mg) was isolated and purified by column chromatography (PE: EA ═ 20:1) in 98% yield.¹H NMR(500MHz,CDCl₃)δ7.21(d,J＝15.0Hz,4H),6.99(d,J＝15.0Hz,4H),3.79(s,6H)；¹³C NMR(125MHz,CDCl₃)δ156.8,155.1,145.1,123.1,114.6,56.1.

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 10(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 10.

Example 24

1. Preparation of oxalate ester 11:

para-trifluoromethylphenol (1.63g,4.0 equiv.) and triethylamine (2.8mL,4.0 equiv.) were dissolved in dichloromethane (20mL) under nitrogen and placed in a 50mL two-necked flaskAfter oxalyl chloride (227 μ L,1.0 eq) was added dropwise at 0 ℃ in a flask, the reaction was continued for 10 minutes, the reaction was monitored by thin layer chromatography, the solvent was dried by spinning, and the compound 11(832mg) was isolated and purified by column chromatography (PE: EA ═ 20:1) with 88% yield.¹H NMR(500MHz,CDCl₃)δ7.53-7.45(m,2H),7.14-7.08(m,2H)；¹³C NMR(125MHz,CDCl₃)δ155.1,154.3,129.3(q,J＝32.4Hz),127.4(q,J＝3.7Hz),123.6(q,J＝268.1Hz),121.7(d,J＝1.5Hz).

2. The synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 11(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphate buffer at pH 7.4 to prepare 5% by mass polyvinyl alcohol (PVA) solution) was added, ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 11.

Example 25

1. Wherein the oxalate is:

2. the synthesis of the thiocarbonylCO prodrug 2-S is the same as in example 2.

Oxalate 12(4mg) and carbon monoxide prodrug 2-S (0.5mg), PLGA (lactic acid-hydroxy lactic acid copolymer) (7mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%) was added, and ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 12.

Example 26

1. The method for synthesizing the oxalate polymer is the same as that in example 1.

2. Synthesis of thiocarbonyl carbon monoxide prodrug 21

Under the protection of nitrogen, 21-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and the corresponding carbon monoxide prodrug 21 is obtained by separation and purification through column chromatography (petroleum ether: ethyl acetate: 10: 1).¹H NMR(500MHz,CDCl₃)δ7.71-7.57(m,2H),7.39(d,J＝9.0Hz,4H),7.32-7.17(m,3H)；¹³C NMR(125MHz,CDCl₃)δ198.9,174.8,137.4,135.8,134.5,131.2,130.8,130.6,130.3,128.7,128.7,127.6,127.1.

Oxalate polymer a (7mg) and carbon monoxide prodrug 21(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of a polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 21.

Example 27

2. Synthesis of thiocarbonyl carbon monoxide prodrug 22

Under the protection of nitrogen, 22-O (1.0 equivalent) and LaoAdding the Sen reagent (0.6 equivalent) into a double-neck reaction bottle, adding toluene, heating to 120 ℃, refluxing for 4h, gradually deepening the color, monitoring the reaction process by a thin-layer chromatography plate, spin-drying the toluene after the reaction is finished, and separating and purifying by using column chromatography (petroleum ether: ethyl acetate: 10:1) to obtain the corresponding carbon monoxide prodrug 22.¹H NMR(500MHz,CDCl₃)δ7.77-7.67(m,2H),7.57-7.49(m,3H),7.42(td,J＝15.0,3.0Hz,1H),7.12(dd,J＝14.5,3.0Hz,1H),6.81-6.68(m,2H),3.36(s,3H)；¹³C NMR(125MHz,CDCl₃)δ178.3,154.2,140.3,138.8,135.7,132.9,129.5,129.3,128.0,127.7,127.0,124.4,117.1,40.3.

Oxalate polymer a (7mg) and carbon monoxide prodrug 22(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 22.

Example 28

2. Synthesis of thiocarbonyl carbon monoxide prodrug 23

Under the protection of nitrogen, adding 23-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) into a double-neck reaction bottle, adding toluene, heating to 120 ℃ for refluxing for 4 hours, gradually darkening the color, monitoring the reaction process through a thin-layer chromatography plate, spin-drying the toluene after the reaction is finished, and separating and purifying by using column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain the corresponding carbon monoxide prodrug 23.¹H NMR(500MHz,CDCl₃)δ7.77(ddd,J＝10.2,5.8,2.9Hz,2H),7.51–7.42(m,3H),7.11(dd,J＝14.9,3.0Hz,1H),6.63(d,J＝3.1Hz,1H),3.77(s,3H)；¹³C NMR(125MHz,CDCl₃)δ208.3,156.1,147.7,147.6,146.7,132.3,130.5,130.2,129.3,128.6,124.7,119.6,110.8,56.1.

Oxalate polymer a (7mg) and carbon monoxide prodrug 23(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of a polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 23.

Example 29

2. Synthesis of thiocarbonyl carbon monoxide prodrug 24

Under the protection of nitrogen, 24-O (1.0 equivalent) and a Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and the corresponding carbon monoxide prodrug 24 is obtained by separation and purification through column chromatography (petroleum ether: ethyl acetate: 10: 1).¹H NMR(500MHz,CDCl₃)δ7.83-7.69(m,2H),7.55-7.44(m,3H),7.42-7.40(m,2H),6.95(d,J＝2.1Hz,1H),2.42(s,3H)；¹³C NMR(125MHz,CDCl₃)δ208.3,151.2,147.7,146.7,137.7,135.4,132.3,130.2,129.3,128.6,127.7,125.1,117.0,21.2.

Oxalate polymer a (7mg) and carbon monoxide prodrug 24(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 24.

Example 30

2. Synthesis of thiocarbonyl carbon monoxide prodrug 25

Under the protection of nitrogen, 25-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 25 is obtained.¹H NMR(500MHz,CDCl₃)δ7.85-7.74(m,3H),7.71(d,J＝3.1Hz,1H),7.52-7.43(m,3H),6.96(d,J＝15.0Hz,1H)；¹³C NMR(125MHz,CDCl₃)δ208.3,159.0,147.7,146.7,141.4,132.3,130.2,129.3,128.6,128.6,124.4,120.5,118.8,108.6.

Oxalate polymer a (7mg) and carbon monoxide prodrug 25(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 25.

Example 31

2. Synthesis of thiocarbonyl carbon monoxide prodrug 26

Under the protection of nitrogen, 26-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 26 is obtained.¹H NMR(500MHz,CDCl₃)δ7.93-7.67(m,2H),7.57-7.39(m,3H),7.30-7.08(m,2H),6.88-6.75(m,1H)；¹³C NMR(125MHz,CDCl₃)δ188.9,154.3,147.6,145.5,134.4,132.3,130.2,130.1,129.3,128.6,126.2,126.0,116.1.

Oxalate polymer a (7mg) and carbon monoxide prodrug 26(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 26.

Example 32

2. Synthesis of thiocarbonyl carbon monoxide prodrug 27

Adding 27-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) into a double-neck reaction bottle under the protection of nitrogen, adding toluene, heating to 120 ℃, refluxing for 4h, gradually deepening the color, monitoring the reaction process by a thin-layer chromatography plate, spin-drying the toluene after the reaction is finished, and separating and purifying by using column chromatography (petroleum ether: ethyl acetate: 10:1) to obtain the corresponding mono-OOxidized carbon prodrug 27.¹H NMR(500MHz,CDCl₃)δ7.77-7.6(m,2H),7.53-7.41(m,3H),6.94(d,J＝2.0Hz,2H),6.48-6.34(m,1H),2.45(s,3H)；¹³C NMR(125MHz,CDCl₃)δ205.0,155.5,147.7,146.7,146.4,133.8,132.3,130.2,129.3,128.6,123.8,117.4,116.2,16.7.

Oxalate polymer a (7mg) and carbon monoxide prodrug 27(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 27.

Example 33

2. Synthesis of thiocarbonyl carbon monoxide prodrug 28

Under the protection of nitrogen, 28-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 28 is obtained.¹H NMR(500MHz,CDCl₃)δ8.23(dd,J＝14.7,3.2Hz,1H),8.16(dd,J＝15.0,3.1Hz,1H),7.97(t,J＝14.9Hz,1H),7.82-7.72(m,2H),7.53-7.42(m,3H)；¹³C NMR(125MHz,CDCl₃)δ190.3,154.7,153.0,148.6,146.9,145.8,132.3,130.2,130.0,129.3,128.6,124.5.

Oxalate polymer a (7mg) and carbon monoxide prodrug 28(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 28.

Example 34

2. Synthesis of thiocarbonyl carbon monoxide prodrug 29

Under the protection of nitrogen, 29-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 29 is obtained.¹H NMR(500MHz,CDCl₃)δ8.52(d,J＝15.0Hz,2H),7.55-7.43(m,1H),7.42-7.33(m,3H),7.05(dd,J＝15.0,3.1Hz,1H),6.92-6.81(m,1H)；¹³C NMR(125MHz,CDCl₃)δ205.0,152.9,150.4,147.7,146.7,138.2,131.2,128.1,128.0,125.1,119.9,117.6.

Oxalate polymer a (7mg) and carbon monoxide prodrug 29(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of a polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 29.

Example 35

2. Synthesis of thiocarbonyl carbon monoxide prodrug 30

Under the protection of nitrogen, 30-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 30 is obtained.¹H NMR(500MHz,CDCl₃)δ7.48(t,J＝7.5,1.5Hz,1H),7.36(d,J＝7.5,1.4Hz,1H),7.05(d,J＝7.5,1.4Hz,1H),6.88(t,J＝7.5,1.4Hz,1H),4.27-4.04(m,2H),3.53-3.34(m,1H),2.83-2.63(m,2H),1.90-1.75(m,2H),1.65-1.54(m,1H),1.24-1.16(m,2H),1.14-1.04(m,1H)；¹³C NMR(125MHz,CDCl₃)δ205.7,153.0,146.1,141.3,131.6,128.1,127.8,125.0,117.5,36.6,30.3,25.9,25.2.

Oxalate polymer a (7mg) and carbon monoxide prodrug 30(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 30.

Example 36

2. Synthesis of thiocarbonyl carbon monoxide prodrug 31

Under the protection of nitrogen, 31-O is added(1.0 equivalent) and a Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle, toluene is added, the mixture is heated to 120 ℃ and refluxed for 4 hours, the color gradually deepens, the reaction process is monitored through a thin-layer chromatography plate, the toluene is dried by spinning after the reaction is finished, and the corresponding carbon monoxide prodrug 31 is obtained by separation and purification through column chromatography (petroleum ether: ethyl acetate: 10: 1).¹H NMR(500MHz,CDCl₃)δ7.48(t,J＝14.9,3.0Hz,1H),7.36(d,J＝15.0,3.1Hz,1H),7.05(d,J＝15.0,3.1Hz,1H),6.88(t,J＝14.8,3.1Hz,1H),1.79(s,3H)；¹³C NMR(125MHz,CDCl₃)δ208.1,154.6,153.1,144.3,132.5,128.4,127.4,124.9,117.5,12.8.

Oxalate polymer a (7mg) and carbon monoxide prodrug 31(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of a polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 31.

Example 37

2. Synthesis of thiocarbonyl carbon monoxide prodrug 32

Under the protection of nitrogen, 32-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and column chromatography (petroleum ether: ethyl acetate: 10:1) is used for separation and purification, so that the corresponding carbon monoxide prodrug 32 is obtained.¹H NMR(500MHz,CDCl₃)δ7.59(d,J＝15.0Hz,2H),7.48(t,J＝14.9,3.0Hz,1H),7.42-7.28(m,3H),7.05(d,J＝15.0,3.1Hz,1H),6.88(t,J＝14.8,3.1Hz,1H),1.49(s,9H).；¹³C NMR(125MHz,CDCl₃)δ205.0,154.5,152.9,147.7,146.7,142.2,132.2,131.2,130.2,128.1,128.0,125.1,119.2,117.6,80.9,28.3.

Oxalate polymer a (7mg) and carbon monoxide prodrug 32(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 32.

Example 38

2. Synthesis of thiocarbonyl carbon monoxide prodrug 33

Under the protection of nitrogen, 33-O (1.0 equivalent) and a Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and the corresponding carbon monoxide prodrug 33 is obtained by separation and purification through column chromatography (petroleum ether: ethyl acetate: 10: 1).¹H NMR(500MHz,CDCl₃)δ7.48(t,J＝14.9,3.0Hz,1H),7.36(d,J＝15.0,3.1Hz,1H),7.12-7.02(m,3H),6.88(t,J＝14.8,3.1Hz,1H),6.29-6.16(m,2H)；¹³C NMR(125MHz,CDCl₃)δ205.0,152.9,151.9,147.7,146.7,131.2,129.7,128.1,128.0,125.1,122.8,117.6,115.6.

Oxalate polymer a (7mg) and carbon monoxide prodrug 33(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 33.

Example 39

2. Synthesis of thiocarbonyl carbon monoxide prodrug 34

Under the protection of nitrogen, 34-O (1.0 equivalent) and Lawson reagent (0.6 equivalent) are added into a double-neck reaction bottle together, toluene is added, the mixture is heated to 120 ℃ for reflux for 4 hours, the color is gradually deepened, a thin-layer chromatography plate is used for monitoring the reaction process, the toluene is dried by spinning after the reaction is finished, and the corresponding carbon monoxide prodrug 34 is obtained by separation and purification through column chromatography (petroleum ether: ethyl acetate: 10: 1).¹H NMR(500MHz,CDCl₃)δ7.71-7.62(m,2H),7.48(t,J＝14.9,3.0Hz,1H),7.41-7.32(m,3H),7.05(d,J＝15.0,3.1Hz,1H),6.88(t,J＝14.8,3.1Hz,1H),3.90(s,3H)；¹³C NMR(125MHz,CDCl₃)δ205.0,167.3,152.9,147.7,146.7,133.4,132.4,131.5,131.2,128.1,128.0,127.9,125.1,117.6,52.1.

Oxalate polymer a (7mg) and carbon monoxide prodrug 34(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticle 34.

Example 40

2. Synthesis of thiocarbonyl carbon monoxide prodrug 35

Under the protection of nitrogen, 35-O (1.0 equivalent) and lithium hydroxide (3.0 equivalent) were added together into a two-necked reaction flask, a mixed solvent of tetrahydrofuran and water was added thereto, the mixture was stirred at room temperature, the progress of the reaction was monitored by a thin layer chromatography plate, after the completion of the reaction, dichloromethane and saturated brine were used for extraction, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and separation and purification were carried out by column chromatography (petroleum ether: ethyl acetate ═ 2:1) to obtain the corresponding carbon monoxide prodrug 35.¹H NMR(500MHz,CDCl₃)δ7.83(d,J＝15.0Hz,2H),7.54–7.42(m,3H),7.36(d,J＝15.0,3.1Hz,1H),7.05(d,J＝15.0,3.1Hz,1H),6.88(t,J＝14.8,3.1Hz,1H)；¹³C NMR(125MHz,CDCl₃)δ205.0,168.8,152.9,147.7,146.7,138.0,131.7,131.2,128.9,128.1,128.0,127.8,125.1,117.6.

Oxalate polymer a (7mg) and carbon monoxide prodrug 35(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 35.

EXAMPLE 41

2. Synthesis of thiocarbonyl carbon monoxide prodrug 36

36-O (1.0 equiv.) and Lawson's reagent (0.6 equiv.) were added together under nitrogen to the double neckAdding toluene into a reaction bottle, heating to 120 ℃, refluxing for 4h, gradually darkening the color, monitoring the reaction process through a thin-layer chromatography plate, spinning off the toluene after the reaction is finished, and separating and purifying by using column chromatography (petroleum ether: ethyl acetate: 10:1) to obtain the corresponding carbon monoxide prodrug 36.¹H NMR(500MHz,CDCl₃)δ7.54–7.44(m,3H),7.36(d,J＝15.0,3.1Hz,1H),7.05(d,J＝15.0,3.1Hz,1H),6.88(t,J＝14.8,3.1Hz,1H),6.68–6.62(m,2H)；¹³C NMR(125MHz,CDCl₃)δ205.0,160.4,152.9,147.7,146.7,131.2,131.0,128.1,128.0,125.1,122.0,117.6,115.8.

Oxalate polymer a (7mg) and carbon monoxide prodrug 36(0.5mg) were dissolved together in 500 μ L of dichloromethane, 3mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid was dissolved in phosphate buffer solution at pH 7.4 to prepare a 5% by mass polyvinyl alcohol (PVA) solution) was added, and then ultrasonic emulsification was performed using an ultrasonic cell disruptor for 10min, and dichloromethane was removed by concentration under reduced pressure at 37 ℃ to obtain a uniform clear liquid, nanoparticles 36.

Comparative examples 1 to 5

The nanoparticles of comparative examples 1-5 were prepared as in example 1, except that the prodrugs of carbon monoxide of comparative examples 1-5 were 4-S, 5-S, 6, 7, and 8, respectively, and the structures are shown in FIG. 8. Wherein the molar number of the carbon monoxide prodrug is n ═ 1.97 mu mol, and the prodrug concentration: 40 μ Μ, oxalate polymer concentration: 434 μm. The prepared nanoparticles are G-4, G-5, G-6, G-7 and G-8 respectively.

Test example

Test example 1 UV absorption experiment of nanoparticles A of carbon monoxide prodrug 1-S

Nanoparticle a prepared in example 1 was dissolved in phosphate buffer at pH 7.4, and then 1mM H was added thereto₂O₂And detecting the ultraviolet absorption at different time points. The results are shown in FIG. 2.

As can be seen from FIG. 2, the UV absorption experiment of nanoparticle A shows that when 1mM H is added₂O₂When the absorbance of the absorption peaks at about 380nm and 500nm increased significantly with timeAnd (4) reduction, which indicates that the degradation of 1-S and the release of CO are basically completed in 1h, and the absorbance tends to be stable. In addition, the invention also separates the degraded product of 1-S, and the nuclear magnetic data are shown in figure 3 and figure 5, which shows that 1-S releases CO in the form of excited state.

Test example 2 UV absorption experiment of nanoparticles B of carbon monoxide prodrug 2-S

Nanoparticle B from example 2 was dissolved in phosphate buffer at pH 7.4 and H was added₂O₂UV absorption was measured at different time points at a concentration of 1 mM. The results are shown in FIG. 4.

As can be seen from FIG. 4, the UV absorption experiment of nanoparticle B showed that when 1mM H was added₂O₂In the process, the absorbance of the absorption peaks at about 380nm and 500nm is obviously reduced along with the increase of time, which shows that the degradation of 2-S and the release of CO are basically completed in 1 hour, and the absorbance tends to be stable.

Test example 3 detection of carbon monoxide (CO) production by Myoglobin

The nanoparticle a prepared in example 1, volume of solution: 120mL, H₂O₂: 10mM, myoglobin concentration 0.5mg/mL, sodium dithionite 22 mg/mL. The specific experimental operation is as follows:

(1) myoglobin was dissolved in PBS (phosphate buffered saline pH 7.4), deoxygenated myoglobin was prepared by deoxygenation using nitrogen sparge under sonication, and 1mL of 22mg/mL sodium dithionite was added for use.

(2) The prepared nanoparticle a was diluted to 120mL with PBS solution.

(3) 10mM of H₂O₂Was added to (2), and a gas was introduced into the solution of (1) using a double-ended needle, and (2) was reacted at 4 ℃ with stirring for 4 hours, and then placed in a refrigerator at 4 ℃ overnight. Control group: the hydrogen peroxide was replaced by an equal volume of PBS (phosphate buffered saline at pH 7.4) under the same conditions. The results are shown in FIG. 6. As can be seen from the figure, the absorption peak at 550nm observed in the experimental group was significantly changed to a double peak, compared to the control group, and further, the release of CO was confirmed.

Test example 4 study of CO Release kinetics from different carbon monoxide prodrugs

The nanoparticles E-1, E-2, E-3 prepared in examples 5-7 and the nanoparticles G-4, G-5, G-6, G-7, G-8 prepared in comparative examples 1-5 were used. H₂O₂Concentration: 1.0mM (5. mu.L), solution volume: 50 mL. Diluting the nanoparticles E-1, E-2, E-3, G-4, G-6, G-7 and G-8 with PBS solution to 50mL reaction bottles respectively, and adding 1mM H at 37 deg.C₂O₂The post detector begins detection, each set of experiments is repeated three times, and the average value is taken. The results of the experiment are shown in FIG. 7.

As can be seen from FIG. 7, the present invention uses the same mole number of different carbon monoxide prodrugs to perform CO release detection by a detector, and the present invention finds that only the nanoparticles E-1, E-2, E-3 can release CO under the condition, the release efficiency of the nanoparticles E-2 is the best, and the release rate is the fastest. No significant CO evolution was seen for the corresponding oxygen substituted derivatives. The invention finds that the energy-supplying nanoparticle prodrug prepared by using the oxygen-substituted derivative is easy to precipitate in a system, which can cause that the generated chemical energy is difficult to transfer to the prodrug. In order to overcome the defect that the oxygen-substituted derivative prodrug is separated out, PLGA (lactic acid-hydroxy lactic acid copolymer) is added as an auxiliary support of the nanoparticles, and the mass ratio of the PLGA to the prodrug is 10: 1. Experiments prove that the nanoparticles prepared by adding the oxygen-substituted derivative into PLGA have no drug precipitation and are in 1mM H₂O₂Under the condition, 30ppm of CO is successfully released after 1 hour. Increasing the amount of oxalate polymer A provides more chemical energy to hopefully increase the release efficiency of 1-S, although the release efficiency of prodrug 1-S is slightly increased, but the increase is not significant.

Test example 5 Effect of the amount of oxalate Polymer A on CO evolution

The nanoparticles F-1, F-2, F-3, F-4, F-5, H prepared in examples 8-12 were used₂O₂Concentration: 1.0mM (5. mu.L), solution volume: 50 mL. F-1, F-2, F-3, F-4 and F-5 were diluted with PBS solution to 50mL reaction bottles, respectively, and 1mM H was added thereto at 37 deg.C₂O₂The post detector begins detection, each set of experiments is repeated three times, and the average value is taken. The results of the experiment are shown in FIG. 9.

The experimental results show that the 0.5mg/7.0mg ratio has the best release effect, but the release of CO and the increase of the prodrug mass are not linear, which indicates that the quantitative chemical energy may not be supplied enough with the energy along with the increase of the prodrug mass. When we continued to increase the amount of prodrug, the amount of CO released decreased slightly.

Test example 6 specificity of CO Release

In order to determine the response specificity of the chemical energy nanoparticles to hydrogen peroxide, some active oxygen and some endogenous small molecules such as t-BuO, HO and H are screened₂O₂、OCl^-GSH and Cys, the specific steps of the experiment on the response specificity of the nanoparticle F3 prepared in example 10 to hydrogen peroxide are as follows: diluting the nanoparticles B with PBS solution to 50mL reaction bottle, and respectively adding 1mM t-BuO, HO and H at 37 deg.C₂O₂After the detector starts to detect, the NaOCl, GSH and Cys, each group of experiments are repeated three times, and an average value is taken. The experimental result is shown in fig. 10, and it can be known from the figure that the responsiveness of the nanoparticle obtained by the invention to hydrogen peroxide is about 30 times of that of other active oxygen, and the nanoparticle has a good specific response, so that a good condition is created for targeted delivery of CO to tumor cells and inflammatory cells with high hydrogen peroxide expression.

Test example 7 Effect of pH on CO Release Rate

The nanoparticles B prepared in example 10 were diluted with PBS solution into 50mL reaction vials, and 1mM concentration of H was added at 37 ℃ and pH 7.4 and pH 6.5, respectively₂O₂The post detector begins detection, each set of experiments is repeated three times, and the average value is taken. And it was found that at pH 6.5, the release rate of the prodrug was slowed and the release rate was slightly decreased. The results of the experiment are shown in FIG. 11.

Test example 8H₂O₂Effect of content on CO Release

To investigate H₂O₂The influence of the content of (b) on CO release was set to set the concentration of H in the groups of 1.0mM, 500. mu.M, 300. mu.M and 100. mu.M of the nanoparticle F-3 prepared in example 10 at different concentrations₂O₂The test was performed and the change in its release was observed by a carbon monoxide detector.The results are shown in FIG. 12, from which it can be seen that H is the product of₂O₂When the concentration is reduced to 500. mu.M or even 300. mu.M, the release amount is not significantly reduced, probably because of H₂O₂The concentration is close to one equivalent of the concentration of the oxalate polymer A, and the reaction can still be fully performed to provide enough chemical energy. But the release times may differ because of H₂O₂The higher the concentration of (b), the faster the reaction rate. When H is present₂O₂H at the time when the concentration was decreased to 100. mu.M₂O₂The amount of oxalate polymer a was equivalent to 1/4, so they did not react sufficiently to provide sufficient chemical energy, resulting in a reduction in release to around 40% of the first groups.

Test example 9 cytotoxicity test

4T1/B16F10/HEK293 cells (5X 10) in logarithmic growth phase are taken⁴one/mL) was inoculated in a 96-well plate, and after adherence, the solutions were divided into 2-S/P @ PVA group, 2-S/PLGA @ PVA group, P @ PVA group and Free 2-S group, and each group of solutions was diluted with a medium to 1.875, 3.75, 7.5, 15, 30, 60. mu.g/mL (2-S, 14 times the concentration of the polymer) solutions, each concentration being 4 duplicate wells. After administration, the cells were incubated at 37 ℃ for 12 hours, the drug solution was discarded, 150. mu. LMTT solution (0.5mg/mL) was added to each well, the cells were incubated at 37 ℃ for 4 hours, the supernatant was discarded, 100. mu.L of DMSO was added to each well, the mixture was shaken for 10min to dissolve blue-violet formazan crystals, and the OD value at a wavelength of 490nm was measured with a microplate reader to calculate the cell viability (% cell viability ═ OD_{Administration set}/OD_{Control group}X 100%). The test structure is shown in fig. 13, the 2-S/P @ PVA nanoparticle group has obvious cytotoxicity, the cytotoxicity of the two control groups is lower than that of the experimental group, and from comparison of three cell lines, the HEK293 belongs to a normal cell, the hydrogen peroxide content is lower, the toxicity to the cell is correspondingly reduced, and the carbon monoxide nanoparticle has good targeting property and lower toxicity to the normal cell because of higher hydrogen peroxide content and higher cytotoxicity to the tumor cells 4T1 and B16F 10.

Test example 10 intracellular CO imaging experiment

4T1 cells (1X 10) were harvested in logarithmic growth phase⁵one/mL) was inoculated in a 24-well plate, and divided into a Control group, a 2-S/P @ PVA group, a 2-S/PLGA @ PVA group, a P @ PVA group, and a Free 2-S group, each group having 3 multiple wells. After the cells are attached to the wall, a CO probe NR-PdA (2 mu M) is firstly given for incubation for 30min, PBS is washed for 2 times, a Control group is given with a culture medium, the rest groups are respectively given with liquid medicine with the concentration of 30 mu g/ml (calculated by 2-S, the concentration of a polymer is 14 times of that), incubation is carried out for 1h at 37 ℃, the liquid medicine is discarded, the Hoechst dye is added after the PBS is washed for 2 times for staining the cell nucleus for 5min, then the PBS is washed for 2 times, a fresh PBS solution is added, and the generation of CO is observed under a laser confocal microscope. The test results are shown in fig. 14 and 15. By using the CO probe for intracellular CO imaging detection, the 2-S/P @ PVA group showed red fluorescence of the probe, indicating that CO can be released in cells, while the fluorescence intensity of the control group relative to the experimental group is negligible, so that none of them can release CO.

Test example 11 uptake assay of nanoparticles by cells

4T1 cells (1X 10) were harvested in logarithmic growth phase⁵one/mL) was inoculated in 6-well plates, divided into 2-S/P @ PVA group, 2-S/PLGA @ PVA group and Free 2-S group, each group having 3 multiple wells. After the cells are attached to the wall, liquid medicine with the concentration of 30 mug/ml (calculated by 2-S) is respectively given to each group, the cells are incubated for 3, 6 and 12 hours at 37 ℃, the supernatant is discarded, PBS is washed for 2 times, the cells are digested and collected by 0.25 percent pancreatin, 1ml of PBS is added for re-suspension after centrifugation, and the cells of each hole are counted by using a cell counting plate. Subsequently, the cells were disrupted by an ultrasonic cell disrupter, centrifuged at 12000rpm for 5min, the supernatant was collected, DMSO was added to the cell lysate to extract 2-S, and the amount of 2-S taken up was determined by a standard curve method using a microplate reader.

Cypate is used as a fluorescent marker to observe the uptake condition of the cells to the nanoparticles. EDC and NHS are firstly used for activating carboxyl of a Cypate molecule, and then the carboxyl is connected to PVA through esterification reaction, so as to prepare the Cypate-PVA coated 2-S/P @ Cy-PVA nanoparticle. 4T1 cells (1X 10) were harvested in logarithmic growth phase⁵one/mL) is inoculated on a 24-well plate, after cells are attached to the wall, 15 mu g/mL (calculated by 2-S) of 2-S/P @ Cy-PVA nanoparticles are given, 3, 6 and 12 hours after the administration, the liquid medicine is discarded, the Hoechst dye is added to dye cell nuclei for 5min after PBS is washed for 2 times, then the PBS is washed for 2 times, the fresh PBS solution is added, and the nanoparticles are observed under a fluorescence microscopeCo-localization of cells.

Cypate is used as a fluorescent marker to observe the uptake condition of the cells to the nanoparticles. EDC and NHS are firstly used for activating carboxyl of a Cypate molecule, and then the carboxyl is connected to PVA through esterification reaction, so as to prepare the Cypate-PVA coated 2-S/PLGA @ Cy-PVA nanoparticle. 4T1 cells (1X 10) were harvested in logarithmic growth phase⁵one/mL) is inoculated on a 24-well plate, after cells are attached to the wall, 15 mu g/mL (calculated by 2-S) and 2-S/PLGA @ Cy-PVA nanoparticles are given, after 3, 6 and 12 hours after the administration, liquid medicine is discarded, the Hoechst dye is added for staining cell nucleus for 5min after PBS is washed for 2 times, then the PBS is washed for 2 times, fresh PBS solution is added, and the co-localization condition of the nanoparticles and the cells is observed under a fluorescence microscope.

Through a cell uptake experiment (as shown in fig. 16), it can be found that the contents of the 2-S/PLGA @ Cy-PVA nanoparticle group and the Free 2-S group in cells are continuously increased and the increase of the experimental group is not obvious after 3 hours, 6 hours, 12 hours, respectively, because the nanoparticles 2-S/P @ PVA in the experimental group are subjected to a chemical reaction when encountering hydrogen peroxide after entering the cells, the nanoparticles are continuously taken up by the cells, but the taken nanoparticles are continuously cracked and release CO after entering the cells.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A CO targeted delivery system is characterized in that the targeted delivery system is a nanoparticle formed by a carbon monoxide prodrug and an oxalate compound; the oxalate compound comprises oxalate and/or oxalate polymer.

2. The CO targeted delivery system of claim 1, wherein the carbon monoxide prodrug has a structure represented by formula (1):

wherein the content of the first and second substances,

y ═ O or S; x is O, S or N;

R₁-R₄independently is an alkyl, alkoxy, amino, halogen, cyano, carboxy, alkylthio, carboalkoxy or carboalkoxy group;

R₅is aryl, heteroaryl or alkyl.

3. The CO targeted delivery system according to claim 1, wherein the oxalate polymer has a structure represented by formula (2) or (3):

wherein n is an integer of 20-200, and m is an integer of 10-200.

4. The targeted CO delivery system of claim 1, wherein the oxalate ester has the structure shown in formula (4):

wherein R is₁Is aryl or alkyl, R₂Is aryl or alkyl.

5. The CO targeted delivery system of claim 1, wherein the nanoparticle is prepared by the following method: and (3) mixing the oxalate compound and the carbon monoxide prodrug in an organic solvent, adding an emulsifier, uniformly mixing, and carrying out reduced pressure distillation to obtain the nanoparticles.

6. The CO targeted delivery system of claim 5, wherein the organic solvent is one or more of ethyl acetate, diethyl ether, and chloroform.

7. The CO targeted delivery system of claim 5, wherein the mass ratio of the oxalate compound to the carbon monoxide prodrug is 0.1: 1-70: 1.

8. the CO targeted delivery system of claim 5, wherein the molar ratio of the emulsifier to the oxalate compound is 0.1: 1-20: 1.

9. the CO targeted delivery system of claim 5, wherein the emulsifier is a polyvinyl alcohol solution or/and a human serum albumin solution.

10. The CO targeted delivery system of claim 9, wherein the polyvinyl alcohol solution has a mass concentration of 0.5% -5% and the human serum albumin solution has a mass concentration of 2-10 mg/mL.