WO2023019700A1

WO2023019700A1 - Co targeted delivery system, and construction method therefor and application thereof

Info

Publication number: WO2023019700A1
Application number: PCT/CN2021/122605
Authority: WO
Inventors: 季兴跃; 柯亨特; 闵庆强; 倪子惠
Original assignee: 苏州大学
Priority date: 2021-08-18
Filing date: 2021-10-08
Publication date: 2023-02-23
Also published as: CN113559071B; CN113559071A

Abstract

A CO targeted delivery system, and a construction method therefor and an application thereof, relating to the technical field of nanomaterial CO targeted delivery. Nanoparticles comprise a carbon monoxide prodrug and an oxalate ester or a polymer thereof, and are prepared by means of the following method: dissolving the oxalate ester or the polymer thereof and the carbon monoxide prodrug in dichloromethane, then adding an emulsifying agent and performing ultrasonic treatment, and performing distillation under reduced pressure to obtain the nanoparticles. In order to avoid the defect of poor transmittance of light to a body tissue, the chemical energy is used to excite Photo-CORM to release CO, H₂O₂ reacts with differently substituted oxalate esters to generate a high-energy intermediate peroxide oxalate ester in an excited state, and the high-energy intermediate is transferred to Photo-CORM by means of the chemical energy to excite Photo-CORM to enter the excited state so as to release CO, thereby finally achieving the purpose of targeted delivery.

Description

A CO targeted delivery system and its construction method and application

technical field

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

Background technique

For a long time, carbon monoxide (CO) has been recognized as a toxic gas, which can bind to heme in the hemoglobin structure with an affinity 200 times that of oxygen, thereby blocking the oxygen supply function of hemoglobin. Endogenous carbon monoxide (CO) is produced from heme through the catalysis of heme oxygenase. Over the past two to three decades, carbon monoxide (CO) has proven to be one of the most important gas messenger small molecules in the body. Like nitric oxide (NO) and hydrogen sulfide (H ₂ S), CO plays an important physiological regulatory role in mammals. According to reports, carbon monoxide (CO) has shown good therapeutic effects in antibacterial, anti-inflammatory, anti-tumor, cardiovascular and cerebrovascular diseases, organ transplantation and preservation, etc. These findings make carbon monoxide (CO) have broad clinical application prospects. The US FDA has approved the clinical experimental research of CO gas (ClinicalTrials.gov: NCT03799874, NCT01214187, etc.). However, there are great drawbacks in using gas as a clinical delivery method of CO: (1) administration in the form of inhalation can only be carried out in a hospital, and it is inconvenient for patients to carry it. (2) The dose of CO gas is difficult to control, and the way of administration depends heavily on whether the patient has sound lung function. (3) The release of CO gas is uncontrollable, and the resulting off-target effects cannot be underestimated. Therefore, the safe and controllable delivery of CO drugs is an urgent bottleneck for the clinical transformation of CO.

The prodrug strategy is a very effective way to solve the problem of safe drug delivery. However, unlike the development of traditional small molecule prodrugs, the development of CO prodrugs is a very challenging subject. First, CO is a gas molecule and is chemically very inert. Second, CO has a very simple structure and lacks corresponding functional groups for chemical derivatization. Therefore, traditional prodrug strategies are difficult to apply to the development of CO prodrugs. The reported CO prodrugs mainly include heavy metal CO complexes and organic CO prodrugs (Figure 13).

Although heavy metal-CO complexes (CORM-S1~3) have made important contributions to the study of the physiological effects of CO, such CO prodrugs are difficult to be used in clinical development due to the inherent toxicity of heavy metals. Another type of CO prodrug is a heavy metal-free organic small molecule CO prodrug, which can be divided into two types that require photoactivation (Photo-CORM, Figure 13) and non-photoactivation (Org-CORM, Figure 13). . Due to inherent defects such as poor light penetration into body tissues, Photo-CORM CO prodrugs are also difficult to be applied clinically. The heavy metal-free and light-activated CO prodrug Org-CORM utilizes the intramolecular DielsAlder cycloaddition reaction to release CO under physiological conditions. There are also some inherent problems in the drug: as the CO release is not targeted (it does not require any stimulus activation, once it is dissolved in aqueous solution, it will start to release CO), and there is a Michael addition receptor (cyclopentadiene) in the prodrug structure. enone skeleton), there is a risk of toxicity. Therefore, there is an urgent need to develop CO prodrugs with novel release mechanisms to address these bottlenecks.

Contents of the invention

In order to solve the above technical problems, the present invention provides a CO targeted delivery system and its construction method and application. In order to avoid the defect of poor penetration of light to body tissues, the present invention uses chemical energy to stimulate Photo-CORM to release CO, H ₂ O ₂ reacts with different substituted oxalate esters, and will generate a high-energy intermediate peroxidation in an excited state Oxalate, the high-energy intermediate, transfers chemical energy to Photo-CORM, which stimulates the latter to an excited state to release CO, and finally achieves the purpose of targeted delivery.

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

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

Wherein, Y=O or S; X=O, S or N;

R _1- R ₄ are independently alkane, alkoxy, amino, halogen, cyano, carboxyl, alkylthio, alkoxyformyl or carbamoyl;

R ₅ is aryl, heteroaryl or alkyl.

In one embodiment of the present invention, the structure of described oxalate polymer is shown in formula (2), (3):

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

In one embodiment of the present invention, the structure of the oxalate is shown in formula (4):

Wherein R ₁ is aryl or alkane group, R ₂ is aryl or alkane group.

In one embodiment of the present invention, the nanoparticles are prepared by the following method: in an organic solvent, mix the oxalate compound and the carbon monoxide prodrug, add an emulsifier, mix well, and distill under reduced pressure 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 present invention, the concentration of the oxalate compound is 40-1000 μM; the concentration of the carbon monoxide prodrug is 5-100 μM.

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

In one embodiment of the present 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.

The above technical solution of the present invention has the following advantages compared with the prior art:

In order to avoid the defect of poor penetration of light to body tissues, the present invention uses chemical energy to excite Photo-CORM to release CO, and H ₂ O ₂ reacts with different substituted oxalate compounds to generate a high-energy intermediate in an excited state. Oxidation of oxalate, a high-energy intermediate, transfers chemical energy to Photo-CORM, which excites the latter to an excited state to release CO. Since the half-life of the high-energy intermediate peroxalate is very short, in order to ensure the efficiency of energy transfer, it is necessary to use some means of drug carriers, such as nanoparticles, to co-deliver the two to the target site. On the one hand, the CO prodrug of the present invention avoids the problem of heavy metal toxicity; Tumor or inflammatory cells with high hydrogen expression are targeted to release carbon monoxide.

Description of drawings

In order to make the content of the present invention more easily understood, the present invention will be described in further detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

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

Fig. 2 is the ultraviolet absorption spectra of nanoparticles A at different time points in Test Example 1 of the present invention.

Fig. 3 is the NMR spectrum of the degradation product of 1-S in Test Example 1 of the present invention.

Fig. 4 is the ultraviolet absorption spectra of nanoparticles B at different time points in Test Example 2 of the present invention.

Fig. 5 is the NMR spectrum of the degradation product of 1-S in Test Example 2 of the present invention.

Fig. 6 is a schematic diagram of the results of carbon monoxide detected by myoglobin in the nanoparticle A of Example 1 in Test Example 3 of the present invention.

Fig. 7 is a study of the CO release kinetics of the nanoparticles prepared in Examples 5-Example 7 of the present invention and the nanoparticles obtained in Comparative Examples 1-5.

Fig. 8 is the molecular structural formulas 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 specific results of CO release 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 is a graph showing the results of H ₂ O ₂ content versus CO release in Test Example 8 of the present invention.

Fig. 13 is the result of the cytotoxicity experiment of Test Example 9 of the present invention.

Figure 14 and Figure 15 are the experimental results of intracellular CO imaging in Test Example 10 of the present invention.

Fig. 16 is the experimental result of the uptake of nanoparticles by cells in 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 ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

Example 1

1. The synthetic method of oxalate polymer:

Under nitrogen atmosphere, 4-hydroxybenzyl alcohol (16mmol) and 1,8-octanediol (2.4mmol) were dissolved in anhydrous tetrahydrofuran (10mL), and then triethylamine (40mmol ), add and close, and stir for 5 minutes. The above mixture was slowly added dropwise to oxalyl chloride (dissolved in 20 mL of anhydrous tetrahydrofuran) at 0° C., closed, the reaction was returned 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 obtain a crude product, and the polymer was obtained by using dichloromethane/n-hexane (1: 1) Precipitate and purify oxalate polymer A, wherein n=20-50. The NMR data of oxalate polymer A are: ¹ H NMR (400MHz, CDCl ₃ ) 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. The synthetic method of thiocarbonyl carbon monoxide prodrug 1-S

Under nitrogen protection, add 3-hydroxy-2-phenyl-4H-benzo[g]chromen-4-one (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-necked reaction flask together, after adding toluene Heated to 120°C and refluxed for 4 hours, the color gradually deepened, and the reaction process was monitored by a thin-layer chromatography plate. After the reaction was completed, the toluene was spin-dried, and column chromatography (petroleum ether: ethyl acetate = 10:1) was used for separation and purification to obtain the corresponding The carbon monoxide prodrug 1-S. ¹ H NMR (400MHz, CDCl ₃ )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); ¹³ C NMR (150MHz, CDCl ₃ ) 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. Carbon monoxide targeted delivery system, i.e. preparation method of chemical energy nanoparticles

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 1-S (0.5 mg) in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37° C. to remove dichloromethane to obtain a uniform and clear liquid, nanoparticle A.

Example 2

1. The synthesis of oxalate polymer is the same as in Example 1.

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 2-S

Under the protection of nitrogen, add 3-hydroxyflavone (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, and column chromatography (petroleum ether: ethyl acetate = 10:1) was used for separation and purification to obtain the corresponding carbon monoxide prodrug 2-S. ¹ H NMR (400MHz, CDCl ₃ ) 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); ¹³ C NMR(150MHz,CDCl ₃ )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 nanoparticles B

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 2-S (0.5 mg) in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (dissolve PVA solid in pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in phosphate buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nano grain B.

Example 3

1. The synthesis of oxalate polymer is the same as in Example 1.

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 3-S

Under the protection of nitrogen, add 3-hydroxy-4H-chromen-4-one (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene, heat to 120°C and reflux for 4 hours, the color gradually deepens , monitor the reaction process by thin-layer chromatographic plate, spin dry toluene after the reaction, use column chromatography (petroleum ether: ethyl acetate = 10:1) to separate and purify to obtain the corresponding carbon monoxide prodrug 3-S. ¹ H NMR (400MHz, CDCl ₃ ) 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); ¹³ C NMR(150MHz,CDCl ₃ )δ190.0,151.0,149.2,133.4,133.0,129.0,128.5,126.1, 119.0.

3. Carbon monoxide targeted delivery system, i.e. preparation method of chemical energy nanoparticles C

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 3-S (0.5 mg) in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles c.

Example 4

1. The synthesis of oxalate polymer is the same as in Example 1.

2. The synthesis of thiocarbonyl carbon monoxide prodrug 2-S is the same as in Example 2.

3. Preparation method of carbon monoxide targeted delivery system, i.e. chemical energy nanoparticles D

Dissolve oxalate polymer (2.24 mg) and carbon monoxide prodrug 2-S (0.16 mg) in 250 μL of dichloromethane, add 5 mL of 4 mg/mL human serum albumin, and ultrasonically emulsify for 10 min using an ultrasonic cell disruptor. Dichloromethane was removed by concentration under reduced pressure at 37 °C to obtain a homogeneous clear liquid, nanoparticles D.

The carbon monoxide targeted delivery system in Examples 5-7, that is, the preparation of nanoparticles is the same as in Example 1, the difference is that the carbon monoxide prodrugs in Examples 5-7 are 1-S, 2-S, and 3-S, respectively, and the carbon monoxide prodrugs are The number of drug moles is n=1.97 μmol, and the prepared nanoparticles are respectively E-1, E-2, and E-3.

Example 8-12

The preparation of nanoparticles in Examples 8-12 is the same as in Example 1, the difference is that the carbon monoxide prodrugs in Examples 8-12 are all 2-S, and the carbon monoxide prodrugs are all 2-S and the amount of oxalate polymer A 0.1mg/7.0mg; 0.3mg/7.0mg; 0.5mg/7.0mg; 0.8mg/7.0mg; 1.0mg/7.0mg. The prepared nanomaterials are respectively F-1, F-2, F-3, F-4 and F-5.

Example 13

1. Wherein oxalate B is:

Dissolve oxalate B (1 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, Nanoparticle H.

Example 14

1. Preparation of oxalate 1:

Under nitrogen atmosphere, dissolve p-cresol (1.05mL, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) into a 50mL double-necked flask, and add oxalyl chloride dropwise at 0°C Monoethyl ester (2.1mL, 2.0 equivalents), after addition, continue to react for 10 minutes, monitor the reaction by thin-layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify by column chromatography (PE:EA=20:1) to obtain Compound 1 (2.04g), yield 98%. ¹ H NMR (300MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ 159.5, 158.1, 150.1, 134.4, 129.6, 121.3, 61.4, 21.15(s), 14.7.

3. Preparation method of carbon monoxide targeted delivery system, i.e. chemical energy nanoparticles:

Dissolve oxalate 1 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, Nanoparticles 1.

Example 15

1. Preparation of oxalate 2:

Under nitrogen atmosphere, 3,4,5-trimethoxyphenol (1.83g, 1.0 equivalent) and triethylamine (2.8mL, 2.0 equivalent) were dissolved in dichloromethane (20mL) and placed in a 50mL two-necked flask, at 0 Add monoethyl oxalyl chloride (2.1mL, 2.0 equivalents) dropwise at ℃, continue to react for 10 minutes after the addition, and monitor the reaction by thin-layer chromatography. : 1) separation and purification to obtain compound 2 (2.69g), yield 95%. ¹ H NMR (500MHz, CDCl ₃ ) δ6.44(s,2H), 4.18(s,2H), 3.83(s,6H), 3.71(s,3H), 1.25(s,3H); ¹³ C NMR( 125MHz, CDCl ₃ ) δ159.5, 158.1, 155.7, 148.8, 135.7, 99.8, 61.5, 60.7, 56.8, 14.7.

Dissolve oxalate 2 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, Nanoparticles 2.

Example 16

1. Preparation of oxalate 3:

Under nitrogen atmosphere, dissolve p-cyanophenol (1.18g, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) into a 50mL double-necked flask, and add oxalyl chloride dropwise at 0°C Monoethyl ester (2.1mL, 2.0 equivalents), after addition, continue to react for 10 minutes, monitor the reaction by thin-layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify by column chromatography (PE:EA=20:1) to obtain Compound 3 (2.01 g), yield 92%. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ159.5, 158.1, 156.6, 133.2, 123.8, 118.9, 109.9, 61.4, 14.7.

Dissolve oxalate 3 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified with an ultrasonic cell disruptor for 10 min, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 3.

Example 17

1. Preparation of oxalate 4:

Under nitrogen atmosphere, dissolve p-methoxyphenol (1.24g, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) into a 50mL two-necked bottle, and add grass Acyl chloride monoethyl ester (2.1mL, 2.0 equivalents), continue to react for 10 minutes after addition, monitor the reaction by thin-layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify by column chromatography (PE:EA=20:1) Compound 4 (2.15 g) was obtained with a yield of 96%. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ 159.5, 158.1, 156.8, 145.1, 123.1, 114.6, 61.4, 56.1, 14.7.

Dissolve oxalate 4 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 4.

Example 18

1. Preparation of oxalate 5:

Under a nitrogen atmosphere, dissolve p-trifluoromethylphenol (1.63g, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) in a 50mL two-necked flask, and add dropwise at 0°C Oxalyl chloride monoethyl ester (2.1mL, 2.0 equivalents), after addition, continue to react for 10 minutes, monitor the reaction by thin layer chromatography, after the reaction is completed, spin to dry the solvent, and separate by column chromatography (PE:EA=20:1) Compound 5 (2.38 g) was obtained after purification with a yield of 91%. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ159.5, 158.1, 154.3, 129.3(q, J _CF ＝32.4Hz), 127.4(q, J _CF ＝3.7Hz), 123.6(q, J _CF ＝268.1Hz), 121.7, 61.4, 14.7.

Dissolve oxalate 5 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 5.

Example 19

1. Preparation of oxalate 6:

Under nitrogen atmosphere, dissolve p-fluorophenol (1.12g, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) into a 50mL double-necked flask, and add oxalyl chloride mono Ethyl ester (2.1mL, 2.0 equivalents), after the addition, continue to react for 10 minutes, monitor the reaction by thin layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify the compound by column chromatography (PE:EA=20:1) 6 (1.91 g), yield 90%. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ 161.2, 159.5, 159.2, 158.1, 148.6 (d, J _CF = 3.7 Hz), 121.48 (d, J _CF = 7.6 Hz), 115.4 (d, J _CF = 20.0 Hz), 61.4, 14.6.

Dissolve oxalate 6 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 6.

Example 20

1. Preparation of oxalate 7:

Under nitrogen atmosphere, dissolve p-chlorophenol (1.28g, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) into a 50mL double-necked flask, and add oxalyl chloride mono Ethyl ester (2.1mL, 2.0 equivalents), after the addition, continue to react for 10 minutes, monitor the reaction by thin layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify the compound by column chromatography (PE:EA=20:1) 7 (2.05g), yield 90%. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ159.5, 158.1, 150.2, 131.8, 129.7, 122.2, 61.4, 14.7.

Dissolve oxalate 7 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified with an ultrasonic cell disruptor for 10 min, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 7.

Example 21

1. Preparation of oxalate 8:

Under nitrogen atmosphere, dissolve p-heptylphenol (1.92g, 1.0eq) and triethylamine (2.8mL, 2.0eq) in dichloromethane (20mL) into a 50mL double-necked flask, and add oxalyl chloride dropwise at 0°C Monoethyl ester (2.1mL, 2.0 equivalents), after addition, continue to react for 10 minutes, monitor the reaction by thin-layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify by column chromatography (PE:EA=20:1) to obtain Compound 8 (2.48g), yield 85%. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz, CDCl ₃ ) 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.

Dissolve oxalate 8 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 8.

Example 22

1. Preparation of oxalate 9:

Under nitrogen atmosphere, 3,4,5-trimethoxyphenol (1.83g, 4.0 equivalents) and triethylamine (2.8mL, 4.0 equivalents) were dissolved in dichloromethane (20mL) and placed in a 50mL two-necked flask, at 0 Oxalyl chloride (227 μL, 1.0 equivalent) was added dropwise at ℃, and the reaction was continued for 10 minutes after addition, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the solvent was spin-dried and separated by column chromatography (PE:EA=20:1). Compound 9 (971 mg) was obtained after purification with a yield of 92%. ¹ H NMR(500MHz, CDCl ₃ )δ6.44(s,4H),3.83(s,12H),3.71(s,6H); ¹³ C NMR(125MHz,CDCl ₃ )δ155.7,155.1,148.8,135.7,99.8 ,60.7),56.8.

Dissolve oxalate 9 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 9.

Example 23

1. Preparation of oxalate 10:

Under nitrogen atmosphere, dissolve p-methoxyphenol (1.05mL, 4.0eq) and triethylamine (2.8mL, 4.0eq) in dichloromethane (20mL) into a 50mL double-necked bottle, and add grass Acid chloride (227 μ L, 1.0 equivalent), after addition, continue to react for 10 minutes, monitor the reaction by thin layer chromatography, after the reaction is completed, spin to dry the solvent, and separate and purify by column chromatography (PE:EA=20:1) to obtain compound 10 ( 740mg), yield 98%. ¹ H NMR (500MHz, CDCl ₃ ) δ7.21 (d, J = 15.0Hz, 4H), 6.99 (d, J = 15.0Hz, 4H), 3.79 (s, 6H); ¹³ C NMR (125MHz, CDCl ₃ )δ156.8, 155.1, 145.1, 123.1, 114.6, 56.1.

Dissolve oxalate 10 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 10.

Example 24

1. Preparation of oxalate 11:

Under nitrogen atmosphere, dissolve p-trifluoromethylphenol (1.63g, 4.0eq) and triethylamine (2.8mL, 4.0eq) in dichloromethane (20mL) in a 50mL double-necked flask, and add dropwise at 0°C Oxalyl chloride (227 μL, 1.0 equiv.) was added and continued to react for 10 minutes. The reaction was monitored by thin-layer chromatography. After the reaction was completed, the solvent was spin-dried and purified by column chromatography (PE:EA=20:1) to obtain compound 11 (832 mg), yield 88%. ¹ H NMR (500MHz, CDCl ₃ ) δ7.53-7.45 (m, 2H), 7.14-7.08 (m, 2H); ¹³ C NMR (125 MHz, CDCl ₃ ) δ 155.1, 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).

Dissolve oxalate 11 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 11.

Example 25

1. Wherein the oxalate is:

Dissolve oxalate 12 (4 mg), carbon monoxide prodrug 2-S (0.5 mg), PLGA (lactic acid-hydroxylactic acid copolymer) (7 mg) in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (Polyvinyl alcohol solid was dissolved in phosphate buffer solution with pH=7.4 to prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5%), ultrasonically emulsified for 10 min using an ultrasonic cell disruptor, and concentrated under reduced pressure at 37°C to remove dichloromethane to give a homogeneous clear liquid, nanoparticles 12.

Example 26

1. wherein the synthetic method of oxalate polymer is identical with embodiment 1.

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 21

Under the protection of nitrogen, add 21-O (1.0 equivalent) and Lawson's reagent (0.6 equivalent) into a double-necked reaction flask, add toluene, heat to 120 °C and reflux for 4 hours, the color gradually deepens, and the reaction process is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 21 was obtained. ¹ H NMR(500MHz, CDCl ₃ )δ7.71-7.57(m,2H),7.39(d,J=9.0Hz,4H),7.32-7.17(m,3H); ¹³ C NMR(125MHz,CDCl ₃ ) 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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 21 (0.5 mg) together in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles twenty one.

Example 27

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 22

Under the protection of nitrogen, add 22-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) together into a double-necked reaction flask, add toluene and heat to 120°C for 4 hours under reflux, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 22 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 22 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (polyvinyl alcohol solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles twenty two.

Example 28

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 23

Under the protection of nitrogen, add 23-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene, heat to 120 °C and reflux for 4 hours, the color gradually deepens, and the reaction process is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 23 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 23 (0.5 mg) together in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles twenty three.

Example 29

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 24

Under the protection of nitrogen, add 24-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene, heat to 120°C and reflux for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried and separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the corresponding carbon monoxide prodrug 24. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz, CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 24 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles twenty four.

Example 30

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 25

Under the protection of nitrogen, add 25-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) together into a double-necked reaction flask, add toluene, heat to 120°C and reflux for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 25 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) 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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 25 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 25.

Example 31

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 26

Under the protection of nitrogen, add 26-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 26 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) δ7.93-7.67 (m, 2H), 7.57-7.39 (m, 3H), 7.30-7.08 (m, 2H), 6.88-6.75 (m, 1H); ¹³ C NMR (125MHz, CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 26 (0.5 mg) together in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 26.

Example 32

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 27

Under the protection of nitrogen, add 27-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) together into a double-neck reaction flask, add toluene, heat to 120°C and reflux for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 27 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 27 (0.5 mg) together in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 27.

Example 33

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 28

Under the protection of nitrogen, add 28-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) together into a double-neck reaction flask, add toluene, heat to 120°C and reflux for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 28 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )δ190.3,154.7,153.0,148.6,146.9,145.8,132.3,130.2,130.0,129.3,128.6, 124.5.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 28 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 28.

Example 34

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 29

Under the protection of nitrogen, add 29-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, and the corresponding carbon monoxide prodrug 29 was obtained by column chromatography (petroleum ether: ethyl acetate = 10:1) for separation and purification. ¹ H NMR (500MHz, CDCl ₃ ) 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, 3.1 Hz,1H),6.92-6.81(m,1H); ¹³ C NMR(125MHz,CDCl ₃ )δ205.0,152.9,150.4,147.7,146.7,138.2,131.2,128.1,128.0,125.1,119.9,117.6.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 29 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 29.

Example 35

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 30

Under the protection of nitrogen, add 30-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-neck reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, and the corresponding carbon monoxide prodrug 30 was obtained by column chromatography (petroleum ether: ethyl acetate = 10:1) for separation and purification. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 30 (0.5 mg) together in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 30.

Example 36

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 31

Under the protection of nitrogen, add 31-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) together into a double-necked reaction flask, add toluene and heat to 120°C for 4 hours under reflux, the color gradually deepens, and the reaction progress is monitored by a thin-layer chromatography plate After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 31 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) δ208.1, 154.6, 153.1, 144.3, 132.5, 128.4, 127.4, 124.9, 117.5 ,12.8.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 31 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 31.

Example 37

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 32

Under the protection of nitrogen, add 32-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-necked reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 32 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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).; ¹³ C NMR (125MHz, CDCl ₃ ) 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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 32 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 32.

Example 38

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 33

Under the protection of nitrogen, add 33-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-necked reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction process is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 33 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 33 (0.5 mg) in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 33.

Example 39

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 34

Under the protection of nitrogen, add 34-O (1.0 equivalent) and Lawson’s reagent (0.6 equivalent) into a double-necked reaction flask, add toluene, heat to 120 °C and reflux for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 34 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 34 (0.5 mg) together in 500 μL of dichloromethane, and add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 34.

Example 40

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 35

Under the protection of nitrogen, add 35-O (1.0 equivalent) and lithium hydroxide (3.0 equivalent) together into a double-necked reaction flask, add a mixed solvent of tetrahydrofuran and water, stir at room temperature, monitor the reaction process through a thin-layer chromatography plate, and the reaction is complete Extract with dichloromethane and saturated brine, dry the organic phase with anhydrous sodium sulfate and concentrate under reduced pressure, use column chromatography (petroleum ether:ethyl acetate=2:1) to separate and purify to obtain the corresponding Carbon monoxide prodrugs 35 . ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR (125MHz, CDCl ₃ ) 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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 35 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (solid polyvinyl alcohol dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 35.

Example 41

2. The synthetic method of thiocarbonyl carbon monoxide prodrug 36

Under the protection of nitrogen, add 36-O (1.0 equivalent) and Lawson's reagent (0.6 equivalent) into a double-neck reaction flask, add toluene and heat to 120 ° C for 4 hours, the color gradually deepens, and the reaction progress is monitored by thin-layer chromatography. After the reaction, the toluene was spin-dried, separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1), and the corresponding carbon monoxide prodrug 36 was obtained. ¹ H NMR (500MHz, CDCl ₃ ) 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); ¹³ C NMR(125MHz,CDCl ₃ )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.

Dissolve oxalate polymer A (7 mg) and carbon monoxide prodrug 36 (0.5 mg) together in 500 μL of dichloromethane, add 3 mL of polyvinyl alcohol (PVA) solution (PVA solid dissolved in phosphoric acid at pH = 7.4 Prepare a polyvinyl alcohol (PVA) solution with a mass concentration of 5% in salt buffer solution), use an ultrasonic cell disruptor to ultrasonically emulsify for 10 minutes, and concentrate under reduced pressure at 37°C to remove dichloromethane to obtain a uniform and clear liquid, nanoparticles 36.

Comparative example 1-5

The preparation of nanoparticles in Comparative Examples 1-5 is the same as in Example 1, the difference is that the carbon monoxide prodrugs in Comparative Examples 1-5 are 4-S, 5-S, 6, 7, and 8 respectively, and the structures are shown in FIG. 8 . Wherein the carbon monoxide prodrug moles are n=1.97 μmol, the prodrug concentration: 40 μM, and the oxalate polymer concentration: 434 μM. The prepared nanoparticles are respectively G-4, G-5, G-6, G-7, G-8.

test case

Test Example 1 UV Absorption Experiment of Nanoparticle A of Carbon Monoxide Prodrug 1-S

Nanoparticles A prepared in Example 1 were dissolved in phosphate buffer at pH=7.4, and then H ₂ O ₂ at a concentration of 1 mM was added, and the ultraviolet absorption at different time points was detected. The results are shown in Figure 2.

It can be seen from Figure 2 that the UV absorption experiment of nanoparticles A shows that when 1mM H ₂ O ₂ is added, the absorbance of the absorption peaks around 380nm and 500nm decreases significantly with the increase of time, indicating that the degradation of 1-S and the release of CO in After 1h, the basic reaction was completed, and the absorbance tended to be stable. In addition, the present invention also separates and obtains the degraded product of 1-S, and the NMR data are shown in Figure 3 and Figure 5, indicating that 1-S releases CO in an excited state.

Test example 2 Ultraviolet absorption experiment of nanoparticle B of carbon monoxide prodrug 2-S

Nanoparticles B in Example 2 were dissolved in phosphate buffer at pH=7.4, and the ultraviolet absorption at different time points was detected when H ₂ O ₂ was added at a concentration of 1 mM. The results are shown in Figure 4.

It can be seen from Figure 4 that the UV absorption experiment of nanoparticles B shows that when 1mM H ₂ O ₂ is added, the absorbance of the absorption peaks around 380nm and 500nm decreases significantly with the increase of time, indicating that the degradation of 2-S and the release of CO in After 1h, the basic reaction was completed, and the absorbance tended to be stable.

Test Example 3 Myoglobin Detection of Carbon Monoxide (CO) Generation

The nanoparticle A prepared in Example 1, solution volume: 120 mL, H ₂ O ₂ : 10 mM, myoglobin concentration 0.5 mg/mL, sodium dithionite 22 mg/mL. The specific experimental operation is:

(1) Dissolve myoglobin in PBS (phosphate buffered saline with pH=7.4), and use nitrogen blowing under ultrasonic conditions to carry out deoxygenation operation to prepare deoxygenated myoglobin, and add 1 mL of 22 mg/mL sodium dithionite for later use .

(2) Dilute the prepared nanoparticles A to 120 mL with PBS solution.

(3) Add 10mM H ₂ O ₂ into (2), and use a double-ended needle to introduce gas into the solution of (1), and stir the reaction of (2) at 4°C for 4h, then put it into Store overnight in a 4°C refrigerator. Control group: the same volume of PBS (phosphate buffer saline with pH=7.4) was used instead of hydrogen peroxide, and other conditions were the same. The results are shown in Figure 6. It can be seen from the figure that compared with the control group, the absorption peak at 550nm observed in the experimental group has changed into a double peak, which further confirms the release of CO.

Test Example 4 Study on CO Release Kinetics in Different Carbon Monoxide Prodrugs

The nanoparticles E-1 prepared in Example 5-7, E-2, E-3 and the nanoparticles G-4, G-5, G-6, G-7 prepared in Comparative Example 1-5 , G-8. H ₂ O ₂ concentration: 1.0 mM (5 μL), solution volume: 50 mL. Nanoparticles E-1, E-2, E-3, G-4, G-6, G-7, G-8 were diluted with PBS solution into 50mL reaction vials, and added at a concentration of 1mM at 37°C After the detector started to detect H ₂ O ₂ , each experiment was repeated three times, and the average value was taken. The experimental results are shown in Figure 7.

It can be seen from Figure 7 that the present invention uses different carbon monoxide prodrugs with the same molar number to detect CO release through the detector respectively. The present invention finds that only nanoparticles E-1, E-2, and E-3 can release CO under this condition. , the release efficiency of nanoparticles E-2 is the best, and the release rate is also the fastest. The corresponding oxygen-substituted derivatives did not release CO significantly. The present invention finds that the energy-donating nanoparticle prodrug prepared by using the oxygen-substituted derivative is easy to precipitate in the system, which may cause the generated chemical energy to be difficult to transfer to the prodrug. In order to overcome the shortcoming of prodrug precipitation of oxygen-substituted derivatives, the present invention adds PLGA (lactic acid-hydroxylactic acid copolymer) as an auxiliary support for nanoparticles, and the mass ratio of PLGA to prodrug is 10:1. Experiments have shown that the nanoparticles prepared by adding the oxygen-substituted derivatives to PLGA have no drug precipitation, and successfully released 30ppm of CO after 1h under the condition of 1mM H ₂ O ₂ . Increasing the amount of oxalate polymer A provides more chemical energy in order to improve the release efficiency of 1-S, although the release efficiency of prodrug 1-S is slightly improved, but the increase is not obvious.

Test Example 5 Effect of the amount of oxalate polymer A on CO release

Nanoparticles F-1, F-2, F-3, F-4, F-5 prepared in Examples 8-12, H ₂ O ₂ concentration: 1.0 mM (5 μL), solution volume: 50 mL. Dilute F-1, F-2, F-3, F-4, and F-5 with PBS solution into 50mL reaction bottles respectively, and at 37°C, add 1mM H ₂ O ₂ and then the detector starts to detect , each experiment was repeated three times, and the average value was taken. The experimental results are shown in Figure 9.

The experimental results show that the ratio of 0.5mg/7.0mg has the best release effect, but the release of CO is not linearly related to the increase of the mass of the prodrug, which indicates that the quantitative chemical energy may have an effect on the energy supply with the increase of the mass of the prodrug. Insufficient. When we continued to increase the dosage of the prodrug, the release of CO decreased slightly.

Test example 6 Specificity of CO release

In order to determine the response specificity of chemical energy nanoparticles to hydrogen peroxide, some reactive oxygen species and some endogenous small molecules such as t-BuO, HO, H ₂ O ₂ , OCl ^- , GSH, and Cys were screened. In Example 10, the specificity experiment of the response of nanoparticle F3 to hydrogen peroxide was prepared. The specific steps were as follows: Dilute nanoparticle B into a 50mL reaction bottle with PBS solution, and add t-BuO, t-BuO, and After HO, H ₂ O ₂ , NaOCl, GSH, Cys, the detector started to detect. Each experiment was repeated three times, and the average value was taken. The experimental results are shown in Fig. 10, and it can be seen from the figure that the nanoparticle of the present invention is about 30 times more responsive to hydrogen peroxide than other active oxygen species, and has a good specific response, which is highly expressed to hydrogen peroxide. The targeted delivery of CO to tumor cells and inflammatory cells creates favorable conditions.

Test Example 7 Effect of pH on CO Release Rate

Dilute the nanoparticles B prepared in Example 10 into a 50mL reaction bottle with PBS solution, respectively at pH=7.4 and pH=6.5, at a temperature of 37°C, add _H2O2 with a concentration of 1mM, _and then the detector starts to detect , each experiment was repeated three times, and the average value was taken. It was found that when the pH=6.5, the release rate of the prodrug slowed down, and the release rate decreased slightly. The experimental results are shown in Figure 11.

Test example 8 Effect of H ₂ O ₂ content on CO release

In order to investigate the effect of the content of H ₂ O ₂ on the release of CO, the nanoparticle F-3 prepared in Example 10 was set to be subjected to different concentrations of H ₂ O ₂ at concentrations of 1.0 mM, 500 μM, 300 μM and 100 μM. Test and observe changes in its release with a carbon monoxide detector. The experimental results are shown in Figure 12. It can be seen from the figure that when the concentration of H ₂ O ₂ is reduced to 500 μM or even 300 μM, the release amount does not decrease significantly, which may be because the concentration of H ₂ O ₂ at this time is different from that of oxalate polymer A. When the concentration is close to one equivalent, it can still fully react and provide enough chemical energy. However, the release time will be different, which may be caused by the higher the concentration of H ₂ O ₂ , the faster the reaction rate. However, when the concentration of H ₂ O ₂ drops to 100 μM, the content of H ₂ O ₂ at this time is equivalent to 1/4 of the content of oxalate polymer A, so they cannot fully react to provide sufficient chemical energy, resulting in a decrease in release to the former About 40% of several groups.

Test Example 9 Cytotoxicity Experiment

4T1/B16F10/HEK293 cells (5× ¹⁰⁴ cells/mL) in the logarithmic growth phase were inoculated into 96-well plates, and after adherence, they were divided into 2-S/P@PVA group and 2-S/PLGA@PVA group , P@PVA group and Free 2-S group, each group of medicinal solution was diluted with medium to 1.875, 3.75, 7.5, 15, 30, 60 μg/ml (based on 2-S, the concentration of polymer was 14 times) For each concentration, 4 replicate wells were set up. After administration, incubate at 37°C for 12 hours, discard the drug solution, add 150 μL of MTT solution (0.5 mg/mL) to each well, incubate at 37°C for 4 hours, discard the supernatant, add 100 μL of DMSO to each well, and shake for 10 minutes. The blue-purple formazan crystals were dissolved, and the OD value at the wavelength of 490nm was measured by a microplate reader, and the cell viability was calculated (cell viability%=OD _{administration group} /OD _{control group} ×100%). The test structure is shown in Figure 13. The 2-S/P@PVA nanoparticle group has obvious cytotoxicity, and the two control groups have less cytotoxicity than the experimental group. From the comparison of the three cell lines, HEK293 belongs to normal cells , the content of hydrogen peroxide is lower, and the toxicity to cells will also be reduced accordingly. For tumor cells 4T1 and B16F10 cells, due to the higher hydrogen peroxide, the experimental group has greater cytotoxicity, so the carbon monoxide nanoparticles have Good targeting, less toxic to normal cells.

Test Example 10 Intracellular CO Imaging Experiment

4T1 cells (1× ¹⁰⁵ cells/mL) in the logarithmic growth phase were inoculated into 24-well plates and divided into Control group, 2-S/P@PVA group, 2-S/PLGA@PVA group, and P@PVA group and Free 2-S group, each group set 3 replicate holes. After the cells adhered to the wall, the CO probe NR-PdA (2μM) was first given to incubate for 30min, and washed twice with PBS. It was 14 times) the drug solution, incubated at 37°C for 1 hour, discarded the drug solution, washed 2 times with PBS, added Hoechst dye to stain the cell nuclei for 5 minutes, washed 2 times with PBS, added fresh PBS solution, and placed under a laser confocal microscope Observe the production of CO. The test results are shown in Figures 14 and 15. By using the CO probe for intracellular CO imaging detection, the red fluorescence of the probe appeared in the 2-S/P@PVA group, indicating that CO can indeed be released in the cell, while the fluorescence intensity of the control group relative to the experimental group can be ignored Negative, so none of them can release CO.

Test Example 11 Uptake of Nanoparticles by Cells

4T1 cells (1×10 ⁵ cells/mL) in the logarithmic growth phase were inoculated on 6-well plates, and divided into 2-S/P@PVA group, 2-S/PLGA@PVA group and Free 2-S group. Set up 3 replicate wells. After the cells adhered to the wall, each group was given a drug solution with a concentration of 30 μg/ml (calculated as 2-S), incubated at 37°C for 3, 6, and 12 hours, discarded the supernatant, washed twice with PBS, and digested with 0.25% trypsin Collect the cells, add 1ml PBS to resuspend after centrifugation, and count the cells in each well with a cell counting plate. Subsequently, the cells were disrupted with an ultrasonic cell disruptor, centrifuged at 12,000 rpm for 5 min, and the supernatant was taken. DMSO was added to the cell lysate to extract 2-S, and the ingested 2-S was quantified by the standard curve method with a microplate reader.

Cypate was used as a fluorescent marker to observe the uptake of nanoparticles by cells. First, use EDC and NHS to activate the carboxyl group of Cypate molecule, and then connect it to PVA through esterification reaction to prepare Cypate-PVA-wrapped 2-S/P@Cy-PVA nanoparticles. 4T1 cells (1×10 ⁵ cells/mL) in the logarithmic growth phase were inoculated into 24-well plates, and after the cells adhered to the wall, 15 μg/ml (calculated as 2-S) of 2-S/P@Cy-PVA nano At 3, 6, and 12 hours after administration, discard the drug solution, wash 2 times with PBS, add Hoechst dye to stain the nucleus for 5 minutes, wash 2 times with PBS, add fresh PBS solution, and observe the nanoparticles under a fluorescent microscope Colocalization with cells.

Cypate was used as a fluorescent marker to observe the uptake of nanoparticles by cells. First, use EDC and NHS to activate the carboxyl group of Cypate molecule, and then connect it to PVA through esterification reaction to prepare Cypate-PVA-wrapped 2-S/PLGA@Cy-PVA nanoparticles. 4T1 cells (1×10 ⁵ cells/mL) in the logarithmic growth phase were seeded in 24-well plates, and after the cells adhered to the wall, 15 μg/ml (calculated as 2-S) and 2-S/PLGA@Cy-PVA For nanoparticles, 3, 6, and 12 hours after administration, discard the drug solution, wash 2 times with PBS, add Hoechst dye to stain the cell nucleus for 5 minutes, wash 2 times with PBS, add fresh PBS solution, and observe the nanoparticles under a fluorescence microscope. Colocalization of granules and cells.

Through the cell uptake experiment (as shown in Figure 16), it can be found that the content in the cells of the 2-S/PLGA@Cy-PVA nanoparticle group and the Free 2-S group continued to increase at 3 hours, 6 hours, and 12 hours, respectively. However, the increase in the experimental group was not obvious. This is because the nanoparticles in the experimental group 2-S/P@PVA will undergo a chemical reaction when encountering hydrogen peroxide after entering the cells. The cells then also release CO without breaking down.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims

A CO targeted delivery system, characterized in that the targeted delivery system is a nanoparticle formed by a carbon monoxide prodrug and an oxalate compound; the oxalate compound includes an oxalate and/or an oxalate polymer .
The CO targeted delivery system according to claim 1, wherein the structure of the carbon monoxide prodrug is as shown in formula (1):

in,

Y=O or S; X=O, S or N;

R 1 -R 4 are independently alkane, alkoxy, amino, halogen, cyano, carboxyl, alkylthio, alkoxyformyl or carbamoyl;

R 5 is aryl, heteroaryl or alkyl.
The CO targeted delivery system according to claim 1, wherein the structure of the oxalate polymer is shown in formulas (2) and (3):

Wherein, n is an integer of 20-200, and m=an integer of 10-200.
The CO targeted delivery system according to claim 1, wherein the structure of the oxalate is shown in formula (4):

Wherein, R 1 is aryl or alkane group, R 2 is aryl or alkane group.
The CO targeted delivery system according to claim 1, wherein the nanoparticles are prepared by the following method: in an organic solvent, the oxalate compound is mixed with the carbon monoxide prodrug, and an emulsifier is added Mix evenly, and distill under reduced pressure to obtain the nanoparticles.
The CO targeted delivery system according to claim 5, wherein the organic solvent is one or more of ethyl acetate, diethyl ether and chloroform.
The CO targeted delivery system according to claim 5, wherein the mass ratio of the oxalate compound to the carbon monoxide prodrug is 0.1:1-70:1.
The CO targeted delivery system according to claim 5, wherein the molar ratio of the emulsifier to the oxalate compound is 0.1:1-20:1.
The CO targeted delivery system according to claim 5, wherein the emulsifier is polyvinyl alcohol solution or/and human serum albumin solution.
The CO targeted delivery system according to claim 9, wherein the mass concentration of the polyvinyl alcohol solution is 0.5%-5%, and the mass concentration of the human serum albumin solution is 2-10 mg/mL.