Self-assembled co-prodrug nanomedicine, preparation method and application thereof

Abstract

The application discloses the technical field of biological medicine, and particularly discloses a self-assembled CO prodrug nanomedicine, a preparation method and application thereof, which comprises the following steps: step 1: under the action of a catalyst, mercaptansuccinic acid, ethylene glycol and polyethylene glycol are subjected to polycondensation reaction under an inert atmosphere to generate polythiol polyester; step 2: the polythiol polyester and a CO precursor are dissolved in a solvent, and under the condition of an inert atmosphere and stirring, poly-carbonylated polyester is generated; and step 3: after ultrasonic treatment, the poly-carbonylated polyester is self-assembled into stable nanoscale particles in deionized water, namely the self-assembled CO prodrug nanomedicine. The self-assembled CO prodrug nanomedicine, the preparation method and application thereof have the advantages that the drug is stable, has strong water solubility, good drug efficacy, and can improve the solubility, biocompatibility and half-life of the CO drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a self-assembled CO prodrug nanomedicine, a preparation method and applications thereof. BACKGROUND

[0002] Sepsis 3.0 is a newly defined clinical syndrome characterized by the imbalance of inflammatory response caused by the infection of bacteria, viruses, fungi and other pathogens in the body, leading to physiological and organ dysfunction. It affects millions of patients worldwide every year, with a mortality rate of more than 25%, and is still one of the main causes of death in intensive care units (ICU). It is worrying that 40% of sepsis patients are hospitalized again within 3 months after discharge, mainly due to residual inflammatory factors in the body. At present, the clinical treatment is still mainly symptomatic support treatment, mainly relying on antibiotics, antagonists and supportive care. This goal-oriented treatment has not reduced the morbidity and mortality of critically ill patients. The complex, dynamic and uncontrollable inflammatory response in the body poses a great challenge to the treatment of sepsis.

[0003] Under the condition of sepsis, the imbalance of inflammation is usually triggered by the overactivation of toll-like receptors (TLRs), which can recognize various inflammatory mediators such as lipopolysaccharide (LPS), reactive oxygen species (ROS) and free DNA (cfDNA), and induce the production and release of inflammatory cytokines and nitric oxide (NO). These circulating inflammatory mediators permanently cause systemic inflammation, leading to cell death, even organ failure and death. Based on this point of view, it is believed that reducing ROS levels, removing various inflammatory mediators, and inhibiting overactive immune responses are key to the treatment of sepsis.

[0004] With the development of sepsis, on the one hand, infection, damage-related signals and other factors trigger pyroptosis, further release a large amount of cytokines through plasma membrane rupture, leading to cell death and accelerating the development of sepsis. On the other hand, autophagy is inhibited, leading to an imbalance of inflammatory response, hindering the clearance of damaged cells, and damaging the morphology of cells, ultimately affecting the function of important organs. Therefore, another effective measure to improve sepsis is to inhibit or block pyroptosis and promote autophagy, which is beneficial to cell repair and survival and has a protective effect on tissue and organ damage. In the face of the complex physiological and pathological state of sepsis, it is urgent to develop drug treatment that can act on multiple targets at the same time to effectively manage sepsis.

[0005] In recent years, gas therapy has attracted extensive attention in the treatment of inflammation. Heme oxygenase-1 (HO-1) and its catalytic product carbon monoxide (CO) are one of the important endogenous protective mechanisms of cells under stress, which are involved in the regulation of oxidative stress, inflammation, autophagy, pyroptosis and other pathophysiological processes, and are involved in organ protection. As a signaling molecule, CO can trigger a series of cell protection mechanisms such as antioxidant stress, anti-inflammatory, inhibition of cell pyroptosis, and promotion of autophagy during stress and inflammation. In addition, carbon monoxide is a non-free radical group and does not react with cell membranes or any other molecules, and has no side effects at a safe dose, making it an ideal gas molecule for the treatment of sepsis. However, due to its inherent chemical activity, CO is a relatively stable molecule. It is mainly coordinated with metals with specific redox states, such as iron, manganese, ruthenium, and molybdenum, to synthesize CO precursor molecule drugs. However, these metal carbonyl compounds have poor water solubility, are easily oxidized, are easily degraded in vivo, have short half-lives, and have poor biological safety of heavy metals. SUMMARY

[0006] In view of the above-mentioned deficiencies existing at present, the present application provides a self-assembled CO prodrug nanomedicine, a preparation method and applications thereof, which has the advantages of high drug stability, strong water solubility, good drug efficacy, and can improve the solubility, biocompatibility, half-life and other advantages of CO drugs.

[0007] In order to achieve the above-mentioned purpose, the present application provides a preparation method of a self-assembled CO prodrug nanomedicine, comprising the following steps:

[0008] Step 1: under the action of a catalyst, mercaptosuccinic acid, ethylene glycol and polyethylene glycol are subjected to condensation reaction under an inert atmosphere to generate polythiol polyester;

[0009] Step 2: the polythiol polyester and the CO precursor are dissolved in a solvent, and under the conditions of an inert atmosphere and stirring, poly-carbonylated polyester with different degrees of polymerization is generated;

[0010] Step 3: after ultrasonic treatment of the poly-carbonylated polyester, stable nanoscale particles are self-assembled in deionized water, that is, a self-assembled CO prodrug nanomedicine.

[0011] It should be noted that the polyethylene glycol in the poly-carbonylated polyester molecule can enhance the water solubility of the poly-carbonylated polyester molecule, thereby enabling the self-assembly of the poly-carbonylated polyester molecule.

[0012] According to an aspect of the present application, in step 1, the condensation reaction temperature is 100-120℃, and the time is 8-12h.

[0013] According to one aspect of the present application, in step 1, the catalyst comprises p-toluenesulfonic acid and EDTA; the product of the polycondensation reaction is dissolved, precipitated, to obtain polythiol polyester.

[0014] According to one aspect of the present application, in step 2, the CO precursor is dinonylcarbonyl iron; the solvent is any one of tetrahydrofuran, dimethyl sulfoxide, ethanol.

[0015] According to one aspect of the present application, in step 2, the reaction temperature is 50-80℃, and the reaction time is 2-4h; after the reaction is completed, the solution becomes brown, the solution is cooled to-20℃, and poly-carbonylated polyester brown solid is obtained by precipitation, washing, and drying, and the obtained poly-carbonylated polyester is stored at-4℃.

[0016] According to one aspect of the present application, in steps 1 and 2, the inert atmosphere is any one of nitrogen, helium, and argon.

[0017] According to one aspect of the present application, in step 3, the temperature of ultrasonic treatment is 0-4℃, and the time is 5-10min.

[0018] Based on the same inventive concept, the present application also provides a self-assembled CO prodrug nanomedicine prepared by any one of the preparation methods.

[0019] Based on the same inventive concept, the present application also provides a use of the self-assembled CO prodrug nanomedicine prepared by any one of the preparation methods in preparing an inflammation gas therapy drug.

[0020] According to one aspect of the present application, the inflammation is sepsis.

[0021] The present application has the following beneficial effects:

[0022] (1) The present application synthesizes polythiol polyester by polycondensation reaction of mercaptosuccinic acid, ethylene glycol, and polyethylene glycol, and carries hydrophobic CO precursor molecules (dinonylcarbonyl iron), and then forms CO prodrug nanomedicine (Nano CO) by using hydrophilic and hydrophobic interaction self-assembly technology. Among them, any degree of polythiol polyester molecules can only polymerize one polyethylene glycol, which can appear at any position of the polythiol polyester molecule. Since polyethylene glycol has strong hydrophilicity, it can be used as a good solubilizer, and the hydrogen bond network produced after solvation matches well with the hydrogen bond network of surrounding water molecules, so the solubility of CO drug and the stability of hydrophobic drug can be improved. The excellent dispersion and stability of PEG drugs in vivo can enhance the in vivo circulation period, thereby improving the half-life of the drug, which provides greater flexibility and multifunctionality for the development of CO-based drugs.

[0023] (2) The CO prodrug nano drug (Nano CO) obtained by the application can release CO by reacting with hydrogen peroxide, and the release rate increases with the increase of the concentration of hydrogen peroxide.

[0024] (3) The CO prodrug nano drug (Nano CO) obtained by the application can effectively reduce the concentration of ·OH, ·O2 - and NO in the solution in a concentration-dependent manner, indicating that CO can effectively scavenge reactive oxygen species.

[0025] (4) The NO and TNF-α secreted by the macrophages intervened by the CO prodrug nano drug (Nano CO) obtained by the application are significantly lower than those secreted by the LPS-stimulated macrophages, indicating that the CO drug can effectively inhibit the inflammatory response in cells.

[0026] (5) After the intervention of the CO prodrug nano drug (Nano CO), the release amount of LDH shows a concentration-dependent decrease, indicating that the nano CO drug can effectively inhibit excessive pyroptosis of cells.

[0027] (6) After the treatment of Nano CO, the survival rate of mice reaches 80%, which can improve the survival rate of mice. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a synthesis schematic diagram of the self-assembled CO prodrug nano drug described in the application;

[0029] Figure 2 is a transmission electron microscope image of the CO prodrug nano drug prepared in Example 1 of the application;

[0030] Figure 3 is an ultraviolet absorption spectrum of the CO prodrug nano drug prepared in Example 1 of the application;

[0031] Figure 4 is a thermogravimetric analysis diagram of the CO prodrug nano drug prepared in Example 1 of the application;

[0032] Figure 5 is the release condition of the CO prodrug nano drug prepared in Example 1 of the application;

[0033] Figure 6 is an ultraviolet absorption spectrum of the CO prodrug nano drug prepared in Example 1 of the application for scavenging different ROS; wherein (a) is an ultraviolet absorption spectrum of the CO prodrug nano drug for scavenging ·OH; (b) is an ultraviolet absorption spectrum of the CO prodrug nano drug for scavenging ·O2 - ; (c) is an ultraviolet absorption spectrum of the CO prodrug nano drug for scavenging ·NO;

[0034] Figure 7HO-1 overexpression activation of the CO prodrug nanomedicine prepared in Example 1 of the present application;

[0035] Figure 8 Inhibition of NO expression induced by inflammation of the CO prodrug nanomedicine prepared in Example 1 of the present application;

[0036] Figure 9 Inhibition of TNF-α expression induced by inflammation of the CO prodrug nanomedicine prepared in Example 1 of the present application;

[0037] Figure 10 Inhibition of LDH release caused by pyroptosis of the CO prodrug nanomedicine prepared in Example 1 of the present application;

[0038] Figure 11 Survival rate of the CO prodrug nanomedicine prepared in Example 1 of the present application in the treatment of sepsis mice. DETAILED DESCRIPTION

[0039] In order to make the present application easier to understand, the present application will be further described below in combination with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without making creative efforts are within the scope of protection of the present application. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by those skilled in the art; unless otherwise specified, the raw materials and reagents involved in the present application can be purchased from the market or prepared by known methods.

[0040] In order to solve the technical problems in the background art, the present application provides a preparation method of a self-assembled CO prodrug nanomedicine, as shown in Figure 1 The preparation method comprises the following steps:

[0041] Step 1: under the action of a catalyst, mercaptosuccinic acid, ethylene glycol and polyethylene glycol are subjected to a condensation reaction under an inert atmosphere to generate a polythiol polyester; preferably, the condensation reaction is carried out at a temperature of 100-120℃ for 8-12h; the catalyst comprises p-toluenesulfonic acid and EDTA; preferably, the product of the condensation reaction is subjected to dissolution and precipitation to obtain the polythiol polyester; preferably, the inert atmosphere is any one of nitrogen, helium and argon;

[0042] It should be noted that the polythiol polyester provides a target for the CO prodrug molecule.

[0043] It should be noted that the dissolution of the product of the polycondensation reaction is specifically: selecting a solvent that is good in solubility to the polythiol polyester and is not good in solubility to the reactants or other by-products for dissolution, such as acetone.

[0044] It should be noted that the precipitation of the product of the polycondensation reaction after dissolution is specifically: selecting a solvent that is poor in solubility to the polythiol polyester for precipitation, such as diethyl ether.

[0045] It should be noted that PEG has strong hydrophilicity and can be used as a good solubilizer. The hydrogen bond network generated after solvation matches well with the hydrogen bond network of surrounding water molecules, which can improve the solubility of CO drugs and the stability of hydrophobic drugs. The excellent dispersibility and stability of PEG drugs in vivo can enhance the circulation period in vivo, thereby improving the half-life of the drugs.

[0046] Step 2: Dissolve the polythiol polyester and the CO precursor in a solvent, and generate a poly-carbonylated polyester under stirring in an inert atmosphere; preferably, the CO precursor is dinonyl iron carbonyl; preferably, the solvent is any one of tetrahydrofuran, dimethyl sulfoxide, and ethanol; preferably, the reaction temperature is 50-80℃, and the reaction time is 2-4h; preferably, after the reaction is completed, the solution becomes brown, the solution is cooled to-20℃, and the poly-carbonylated polyester brown solid is obtained through precipitation, washing, and drying, and the obtained poly-carbonylated polyester is stored at-4℃; preferably, the reagent used for washing is diethyl ether; preferably, the inert atmosphere is any one of nitrogen, helium, and argon;

[0047] It should be noted that the precipitation is specifically: selecting a solvent that is poor in solubility to the poly-carbonylated polyester for precipitation, such as n-hexane.

[0048] Step 3: After the poly-carbonylated polyester is treated by ultrasonic, it is self-assembled into stable nanoscale particles in deionized water, that is, a self-assembled CO prodrug nanomedicine. Preferably, the temperature for ultrasonic treatment is 0-4℃, and the time is 5-10min.

[0049] It should be noted that the reason why the poly-carbonylated polyester is self-assembled into stable nanoscale particles in deionized water after ultrasonic treatment is: the hydrophilic-hydrophobic interaction.

[0050] The following further illustrates with specific examples.

[0051] Example 1

[0052] A preparation method of a self-assembled CO prodrug nanomedicine:

[0053] (1) Synthesis of polythiol polyester. 42 mmol of p-toluenesulfonic acid and 0.27 mmol of EDTA were added to the reaction flask as catalysts, followed by 3.3 mmol of ethylene glycol, 3.3 mmol of mercaptosuccinic acid and 0.2 mmol of mPEG-OH (Mn = 4000). The reaction was further carried out at 120 °C under a nitrogen atmosphere for 8 h. The product was then dissolved in acetone and precipitated three times in diethyl ether to obtain polythiol polyester.

[0054] (2) Synthesis of polycarbonylated polyester. 50 mg of dinonyl iron carbonyl and 200 mg of polythiol polyester were dissolved in 50 mL of tetrahydrofuran and stirred under nitrogen atmosphere at 50 °C for 2 h. After the reaction was complete, the solution turned brown. The solution was cooled to -20 °C, and n-hexane was added to obtain a brown precipitate. The precipitate was washed with diethyl ether and dried to obtain a brown solid polycarbonylated polyester. Finally, the obtained polycarbonylated polyester was stored at -4 °C.

[0055] (3) Self-assembly to form stable nanoscale particles. The obtained polycarbonylated polyester was subjected to ultrasonic treatment at 0℃ for 10 min and then self-assembled into CO prodrug nanoparticles in deionized water.

[0056] The CO prodrug nanomedicine prepared above was analyzed by transmission electron microscopy, and the results are as follows: Figure 2 As shown, by Figure 2 It can be seen that the nano-sized CO drug is spherical, has good dispersibility, and its size distribution is between 100-150 nm. The ultraviolet absorption spectroscopy analysis of the CO prodrug nanodrug prepared above yielded the following results: Figure 3 As shown, by Figure 3 As can be seen from the ultraviolet absorption spectrum, the polycarbonylated polyester and dinonylcarbonyl iron have the same characteristic ultraviolet absorption peaks, indicating that dinonylcarbonyl iron was successfully attached to the polythiolated polyester to form the polycarbonylated polyester. Thermogravimetric analysis was performed on the CO prodrug nanomedicine prepared above, and the results are as follows: Figure 4 As shown, by Figure 4 The thermogravimetric analysis results show that the CO drug loading rate is 15%.

[0057] The CO prodrug nanomedicine prepared above was subjected to release kinetics evaluation. The specific experimental method is as follows: UV spectrophotometry was used to determine the conversion of hemoglobin (Hb) to carboxyhemoglobin (HbCO) to evaluate the released CO in PBS. The bovine hemoglobin (5.5 μM) was completely dissolved in PBS, and then reduced by adding sodium bisulfite (1.5 mg) under a nitrogen atmosphere. 5 mg / mL NanoCO (50 μL) and hydrogen peroxide (20 μM, 5 μM) were added to the above 4 mL solution, and the absorption spectrum of the solution at 375-475 nm was detected every 10 min. In order to eliminate the influencing factors and improve the accuracy, the conversion of Hb to HbCO was quantitatively determined by using two strong absorption bands of 410 nm and 430 nm belonging to HbCO and Hb respectively. The conversion rate (x) of Hb-to-HbCO and the concentration of released CO coordinated with Hb were calculated by using the following functions, and the release rate results are as follows Figure 5 . It can be seen from Figure 5 that the release kinetics shows that Nano CO reacts with hydrogen peroxide to release CO, and the release rate increases with the increase of the concentration of hydrogen peroxide.

[0058]

[0059] In the above formula, A 410nm is the absorbance at 410 nm; A 430nm is the absorbance at 430 nm; C CO is 5 mg / mL; C Hb is the concentration of released CO coordinated with Hb; and x is the conversion rate.

[0060] Example 2

[0061] Application of a self-assembled CO prodrug nanomedicine:

[0062] The CO prodrug nanomedicine prepared in Example 1 was interacted with different kinds of active oxygen probes to evaluate the ability of CO drug to scavenge active oxygen. The specific experimental scheme is as follows:

[0063] Scavenging ability of hydroxyl radical (·OH): 10 mM FeSO4 solution was reacted with 5 μL of 1% H2O2 to generate ·OH. Different concentrations of NanoCO drug were added to the above solution. Then, the ·OH detection working solution was added to the above mixed solution, and the absorption spectrum at 400-700 nm was recorded.

[0064] Scavenging ability of superoxide anion (·O2 - ): The mixture of xanthine (5 μL, 560 mm) and xanthine oxidase (5 μL, 1:10 dilution) generated ·O2 -The CO drug was added to the above solution at different concentrations. Then, the OH detection working solution was added to the above mixed solution, and the absorption spectrum of 400-700 nm was recorded.

[0065] Nitric oxide (NO) scavenging ability: 100 μL SNP (20 mM) as a NO donor was mixed with 100 μL of different concentrations of Nano CO in 200 μL PBS solution (0.2 M, pH 7.4) at 37°C with shaking. After the reaction, the nitric oxide detection kit was used to determine the ultraviolet spectrum of 400-700 nm.

[0066] The results of the above experiments are shown in Figure 6 : Nano CO can effectively reduce the concentration of OH, O2 - and NO in the solution, and show a concentration-dependent decrease, indicating that CO can effectively scavenge reactive oxygen species.

[0067] The CO prodrug nanodrug prepared in Example 1 was interacted with macrophages stimulated by LPS to evaluate its protective effect on cells. The specific experimental scheme is as follows: RAW 264.7 cells were seeded on the coverslips of a 96-well plate and adhered overnight in a growth medium (37°C, 5% CO2). 10 μg / mL LPS, 10 μg / mL LPS+200 μg / mL PSP and 10 μg / mL LPS+200 μg / mL Nano CO were added to each well to treat the cells for 24 h. The negative control group did not add LPS and drugs; the positive control group only added LPS. The supernatant was collected and detected using the hemin oxygenase-1 (HO-1) Elisa detection kit, nitric oxide (NO) detection kit and tumor necrosis factor (TNF-α) detection kit, respectively. The results are shown in Figure 7 、 8 , 9: After LPS stimulation, macrophages produce a large amount of HO-1 to resist inflammation, and after CO drug intervention, macrophages are further stimulated to secrete more HO-1 to resist the inflammatory response in cells. Further evaluation of the inflammatory response by secreting NO and TNF-α shows that the NO and TNF-α secreted by CO drug-intervened macrophages are significantly lower than those secreted by LPS-stimulated macrophages, indicating that CO drugs can effectively inhibit the inflammatory response in cells.

[0068] The CO prodrug nanodrug prepared in Example 1 was interacted with macrophages stimulated by LPS to evaluate its ability to inhibit excessive pyroptosis. The specific experimental method is as follows:

[0069] RAW 264.74h was first stimulated with LPS (100 ng / mL) to induce caspase-11 expression, then different concentrations of nano CO (200, 100, 50 μg / mL) were added for incubation for 24 h, and the cell culture supernatant was collected after stimulation to detect the release of LDH. The results are shown in Figure 10 Figure 2: LPS can induce pyroptosis to release LDH, and after the intervention of nano CO drugs, the release amount of LDH shows a concentration-dependent decrease. It shows that nano CO drugs can effectively inhibit excessive pyroptosis of cells.

[0070] The CO prodrug nano drug prepared in Example 1 was used to intervene in LPS-induced sepsis to evaluate its therapeutic effect on sepsis. Male C57BL / 6 mice were randomly divided into a control group (PBS), a positive group (LPS), an LPS+PSP group, and an LPS+Nano CO group (20 mice in each group). The C57BL / 6 mice were intraperitoneally injected with PBS, PSP (30 mg / kg), or Nano CO (30 mg / kg) at 6 and 3 h, respectively, and then intraperitoneally injected with 25 mg / kg LPS. The mortality of mice was recorded for one week. The results are shown in Figure 11 Figure 3: The survival rate of sepsis mice was only 30%, and after Nano CO treatment, the survival rate of mice reached 80%, which can improve the survival rate of mice.

[0071] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the scope of the disclosed technology, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for preparing a self-assembled CO prodrug nanomedicine, characterized in that, The method comprises the following steps: Step 1: under the action of a catalyst, mercaptosuccinic acid, ethylene glycol and polyethylene glycol are subjected to condensation polymerization under an inert atmosphere to generate polythiol polyester; wherein the catalyst is p-toluenesulfonic acid and EDTA; the condensation polymerization temperature is 100-120 DEG C, and the time is 8-12 h; Step 2: polythiol polyester and CO precursor dinonyl iron carbonyl are dissolved in tetrahydrofuran, and after the reaction is completed in an inert atmosphere, normal hexane is added to obtain a brown precipitate, the precipitate is washed with diethyl ether and dried to obtain poly-carbonylated polyester; wherein the reaction temperature is 50-80 DEG C, and the reaction time is 2-4 h; Step 3: after ultrasonic treatment, the poly-carbonylated polyester is self-assembled into stable nanoscale particles in deionized water, namely self-assembled CO prodrug nanomedicine; wherein the ultrasonic treatment temperature is 0-4 DEG C, and the time is 5-10 min.

2. The method for preparing self-assembled CO prodrug nanomedicine according to claim 1, characterized in that, In step 1, the product of the condensation polymerization is dissolved and precipitated to obtain polythiol polyester.

3. The method for preparing self-assembled CO prodrug nanomedicine according to claim 1, characterized in that, In step 1 and step 2, the inert atmosphere is any one of nitrogen, helium and argon.

4. Self-assembled CO prodrug nanomedicine prepared by the preparation method of any one of claims 1-3.

5. Use of the self-assembled CO prodrug nanomedicine prepared by the preparation method of any one of claims 1-3 or the self-assembled CO prodrug nanomedicine of claim 4 in the preparation of a sepsis gas therapy drug.

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