Nanometer assembly capable of automatically releasing CO as well as preparation method and application of nanometer assembly

Abstract

The invention discloses a nano assembly capable of automatically releasing CO as well as a preparation method and application of the nano assembly, and belongs to the technical field of biomedical materials. The nano assembly comprises a co-assembly product prepared from a system comprising the following components through a hydrophilic and hydrophobic self-assembly forming method; (1) an amphiphilic block polymer A for a CO donor, which has a chemical structure as shown in formula (I) or (II) in the application; and (2) an amphiphilic block polymer B for a singlet oxygen donor, wherein the amphiphilic block polymer B has a chemical structure as shown in a formula (III) in the application. The CO donor and the singlet oxygen donor capable of releasing singlet oxygen at the temperature close to the human body temperature are combined into a whole, so that singlet oxygen can be released through the singlet oxygen donor in the human body to oxidize the CO donor to release CO, and triggering dependence on external stimulation such as illumination / ultrasonic waves is avoided; the spontaneous release of CO in a normal oxygen, hypoxia and dark human body environment is effectively achieved.

Description

Technical Field

[0001] The present application belongs to the technical field of biomedical materials, and in particular relates to a nanoassembly capable of self-releasing CO, a preparation method thereof, and an application thereof. Background Art

[0002] Carbon monoxide (CO) is an endogenous gas signal molecule with physiological functions such as inhibiting inflammatory response, promoting vasodilation, regulating oxidative stress and inhibiting cell apoptosis. It has shown important application value in the treatment of cardiovascular diseases, hypoxia and chronic inflammation. However, due to the strong affinity between CO and hemoglobin, direct inhalation can lead to high levels of carboxyhemoglobin and cause CO poisoning, making it difficult to achieve its physiological and medical effects. Therefore, designing and preparing exogenous CO donor molecules that can be used in vivo is of great significance for achieving its physiological and medical effects.

[0003] At present, the exogenous CO donor molecules (CORMs) developed and prepared in the relevant field that can be used in vivo still face many challenges in the process of clinical transformation. For example, although traditional metal-based CORMs can achieve effective CO release, the biological toxicity of their metal groups greatly limits their safety in the human body. For this reason, the relevant field has designed and prepared organic small molecule CORMs that rely on external stimuli such as light and ultrasound to trigger, but organic small molecule CORMs that rely on external stimuli to trigger face the problem of limited penetration depth, resulting in poor clinical effects in deep tissue treatment.

[0004] Therefore, there is an urgent need to develop and design new CO-releasing materials that are not triggered by external stimuli, so as to achieve the goal of stably releasing CO in deep tissues in the body while overcoming the metal toxicity problem of metal-based CORMs, thereby achieving safer and more effective treatment methods. Summary of the invention

[0005] The present application discloses a nanoassembly capable of self-releasing CO, a preparation method and application thereof, aiming to solve the technical problem that the existing organic small molecule CORMs that rely on external stimulation for triggering have unsatisfactory clinical effects in deep tissue treatment.

[0006] In order to achieve the above objectives, the technical solution adopted in this application is: The present application first discloses a nanoassembly capable of self-releasing CO, which comprises a co-assembly product prepared by a hydrophilic-hydrophobic self-assembly forming method from a component system comprising the following: (1) An amphiphilic block polymer A for CO donor, which has the chemical structure of the following formula (I) or (II): (I); (II); R1 is selected from one of the chemical structures of the following formula (rI) or (rII); m and n are both integers between 2 and 100; (rI); (rII); R2 is one of N and O; R3 is one of O, S and Se; (2) An amphiphilic block polymer B for singlet oxygen donor having the chemical structure of the following formula (III): (III) R4, R5, R6 and R7 are independently H or -CH3; a and b are both integers between 2 and 100.

[0007] According to the nanoassembly disclosed in the present application, the amphiphilic block polymer A is generated by polymerization reaction of a component system including a CO donor molecule and polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate; Wherein, the CO donor molecule has the following chemical structure (IV) or (V): (IV) (V).

[0008] According to the nanoassembly disclosed in the present application, the amphiphilic block polymer B is generated by a polymerization reaction of a component system comprising a functionalized hydroxypyridine monomer and polyethylene glycol-4-cyano-4-(phenylcarbonylthiothio) pentanoate, and a photosensitive oxidation reaction of the obtained reaction product with tetraphenylporphyrin; Wherein, the functionalized hydroxypyridine monomer has the following chemical structure (VI): (VI).

[0009] According to the nanoassembly disclosed in the present application, the molecular weights of the amphiphilic block polymer A and the amphiphilic block polymer B are both 200-35000 Da.

[0010] According to the nanoassembly disclosed in the present application, the particle size of the nanoassembly is 20-800 nm.

[0011] According to the nanoassembly disclosed in the present application, the hydrophilic-hydrophobic self-assembly forming method comprises nano-flash precipitation, slow water addition, ultrasound and thin film hydration method.

[0012] The present application further discloses a method for preparing the nanoassembly by nano flash precipitation method, which comprises: A step of dissolving the amphiphilic block polymer A and the amphiphilic block polymer B in a co-solvent to prepare an organic phase; and, a step of flash-precipitating the organic phase into an aqueous phase.

[0013] According to the preparation method disclosed in the present application, the co-solvent is selected from one of tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide or 1,4-dioxane.

[0014] According to the preparation method disclosed in the present application, the mass volume concentration of the amphiphilic block polymer A and the amphiphilic block polymer B dissolved in the co-solvent is 0.01-10 g / L.

[0015] The present application finally discloses the use of the nanoassembly described in the present application in preparing anti-inflammatory drugs.

[0016] Compared with the prior art, the advantages or beneficial effects of the present application include at least: The nanoassembly disclosed in the present application is hydrophilic and hydrophobic self-assembled by amphiphilic block polymer A and amphiphilic block polymer B of the chemical structure shown, and a CO donor is effectively combined with a singlet oxygen donor that can release singlet oxygen at a temperature close to human body temperature (37°C), so that singlet oxygen can be released by the singlet oxygen donor in the human body environment to oxidize the CO donor, so that the CO donor releases CO, effectively avoiding the triggering dependence on external stimuli such as light / ultrasound, and realizing the spontaneous release of CO in the human body environment of normoxia, hypoxia and darkness, and having excellent anti-inflammatory effect in deep tissue treatment in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 The hydrogen spectrum of the amphiphilic block polymer PEG SHF provided in the examples of the present application; Figure 2 The GPC spectrum of the amphiphilic block polymer PEG SHF provided in the examples of the present application; Figure 3 The hydrogen spectrum of the amphiphilic block polymer PEG-Py provided in the examples of the present application; Figure 4 The hydrogen spectrum of the amphiphilic block polymer PEG-Py EPO provided in the examples of the present application; Figure 5 GPC spectra of the amphiphilic block polymers PEG-Py and PEG-Py EPO provided in the examples of the present application; Figure 6 TEM image of the nanoassembly PEG-SHF@PyEPO provided in the examples of the present application; Figure 7 This is a hydrated particle size diagram of the nanoassembly PEG-SHF@PyEPO provided in the examples of the present application; Figure 8 The ultraviolet absorption spectrum of the aqueous solution of the nanoassembly PEG-SHF@PyEPO provided in the examples of the present application; Fig. 9 A fluorescence curve diagram of the aqueous solution of the nanoassembly PEG-SHF@PyEPO provided in the examples of the present application; Fig.10 This is a test chart of nitrite levels produced in cells after different treatments provided in the examples of the present application. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments described in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0020] In the following description of this specification, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. Among them, A and B can be singular or plural; the symbol " / " means "or".

[0021] In the following description of this specification, the term "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, "at least one of A, B or C", or "at least one of A, B and C" can mean any one of A, B, C, or A+B, or A+C, or B+C, or A+B+C, where A, B, C can be single or multiple, respectively.

[0022] In the following description of this specification, the order of serial numbers does not mean the order of execution. Some or all of the steps can be executed in parallel or one after the other. The execution order of each process should be determined by its function and internal logic, and does not constitute any limitation on the execution process of this embodiment.

[0023] In the following description of this specification, the numerical range should be understood to also specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is also included in this embodiment, and the upper and lower limits of the smaller range can be independently included or excluded in the range.

[0024] Unless otherwise specified, the technical / scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. Although this specification only describes preferred materials and methods, any similar or equivalent methods and materials may also be used in specific embodiments or test cases. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] In a first aspect, the embodiments of the present application provide a nanoassembly capable of self-releasing CO, which comprises a co-assembly product prepared by a hydrophilic-hydrophobic self-assembly forming method from a component system comprising the following: (1) An amphiphilic block polymer A for CO donor, which has the chemical structure of the following formula (I) or (II): (I); (II); R1 is selected from one of the chemical structures of the following formula (rI) or (rII); m and n both represent the degree of polymerization, and their values ​​are preferably integers between 2 and 100; (rI); (rII); R2 is one of N and O; R3 is one of O, S and Se; (2) An amphiphilic block polymer B for singlet oxygen donor having the chemical structure of the following formula (III): (III) R4, R5, R6 and R7 are independently H or -CH3; a and b both represent the degree of polymerization, and their values ​​are preferably integers between 2 and 100.

[0026] Among them, the embodiment of the present application uses the amphiphilic block polymer A of the chemical structure shown in formula (I)-(II) and the amphiphilic block polymer B of the chemical structure shown in formula (III) to perform hydrophilic and hydrophobic self-assembly to construct a nanoassembly, effectively combining a CO donor with a singlet oxygen donor that can release singlet oxygen in an environment close to human body temperature (37°C). Specifically, the amphiphilic block polymer A of the chemical structure shown in formula (I)-(II) can be used as a CO donor to oxidize and release CO under the action of singlet oxygen, and the amphiphilic block polymer B of the chemical structure shown in formula (III) is used as a singlet oxygen donor and can release singlet oxygen in an environment close to human body temperature (37°C). ℃) environment, thereby giving the prepared nanoassembly the ability to release singlet oxygen by oxidizing the CO donor to spontaneously release CO through the release of singlet oxygen by the singlet oxygen donor in the human body environment based on the synergistic effect of the amphiphilic block polymer A and the amphiphilic block polymer B, effectively avoiding the triggering dependence on external stimuli such as light / ultrasound, and being able to achieve spontaneous release of CO in human body environments such as normoxia, hypoxia and darkness, and having excellent anti-inflammatory effect in deep tissue treatment in vivo.

[0027] As an example of the nanoassembly described in the present application, the amphiphilic block polymer A is preferably generated by a polymerization reaction of a component system comprising a CO donor molecule and polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate (PEG-CAT); The CO donor molecule preferably has the following chemical structure (IV) or (V): (IV) (V).

[0028] Of course, R1 is suitably one of the chemical structures of the above formula (rI) or (rII); R2 is suitably one of N and O; R3 is suitably one of O, S and Se.

[0029] It should be noted that the double bond part in the chemical structure shown in the above formula (IV) to (V) is mainly used to prepare the amphiphilic block polymer A by polymerization methods such as reversible addition-fragmentation chain transfer or atom transfer radical. According to the chemical structure of the above formula (rI) or (rII), the CO monomer molecule can be further exemplified as: ; ; ;

[0030] Of course, R2 is suitably one of N and O; R3 is suitably one of O, S and Se.

[0031] Specifically, in the present embodiment, R2 is O and R3 is S as an example, and the CO donor molecule has the following chemical structure of formula (AI) or (AII): (AI) (AII)

[0032] Of course, according to the different substitution atoms described in R2 and R3, the CO donor molecule described in the present application has a corresponding chemical structure, which will not be described in detail in this specification.

[0033] As an example of the nanoassembly described in the present application, the amphiphilic block polymer B is generated by a polymerization reaction of a component system comprising a functionalized hydroxypyridine monomer and polyethylene glycol-4-cyano-4-(phenylcarbonylthiothio) pentanoate (PEG-CAT), and a photosensitive oxidation reaction of the obtained reaction product with tetraphenylporphyrin; Wherein, the functionalized hydroxypyridine monomer has the following chemical structure (VI): (VI).

[0034] Of course, R4, R5, R6 and R7 are suitably H or -CH3.

[0035] It should be noted that the double bond portion in the chemical structure shown in the above formula (VI) is mainly used to prepare the amphiphilic block polymer B by polymerization methods such as reversible addition-fragmentation chain transfer or atom transfer radical, and according to the different substitution atoms described by R4, R5, R6 and R7, the functionalized hydroxypyridine monomer described in the present application has a corresponding chemical structure, wherein, taking R4, R5, R6 and R7 as an example, the functionalized hydroxypyridine monomer has the following chemical structure of formula (BI): (BI) Of course, according to the different substitution atoms described in R4, R5, R6 and R7, the functionalized hydroxypyridine monomer described in the present application has a corresponding chemical structure, which will not be described in detail in this specification.

[0036] The embodiments of the present application do not specifically limit the specific preparation methods of the CO monomer molecules and the functionalized hydroxypyridine monomers described in the above technical scheme, and they can be prepared by various synthesis methods known in the art. For example, the present application can synthesize the CO monomer molecule (SHF) shown in the above formula (AI) through the following reaction process:

[0037] Based on the reaction process described above, the preparation process includes: Step 1: Disperse 2-hydroxyacetophenone in ethanol (EtOH) and add NaOH aqueous solution for stirring, then add an appropriate amount of trifluoroethanol (TBFE) for stirring reaction, then add H2O2 under ice bath conditions and stir overnight, use HCl to adjust the pH to 6.5, precipitate solid, filter and vacuum dry, wash the solid product with anhydrous ether, and vacuum dry to obtain the product OHF-Boc.

[0038] Step 2: Dissolve OHF-Boc and Lawesson's reagent in anhydrous toluene, replace nitrogen and heat to reflux. After the reaction is completed, cool to room temperature and collect the liquid by filtration. After removing toluene, purify by DCM column chromatography, collect the product, spin dry and vacuum dry to obtain the product SHF-Boc.

[0039] Step 3: After SHF-Boc is dissolved in dichloromethane (DCM), trifluoroacetic acid is added and stirred at room temperature. After the plate reaction is completed, the trifluoroacetic acid is then removed and washed with ether. The solid product is vacuum dried to obtain the product SHF-NH2.

[0040] Step 4: Disperse SHF-NH2 in chloroform and add pentafluorophenol active ester and triethylamine, stir and react at room temperature. After the reaction is completed, purify by DCM column chromatography, collect the product, spin dry and vacuum dry to obtain the product SHF.

[0041] At the same time, the functionalized hydroxypyridine monomer (Py) shown in the above formula (BI) can be synthesized by the reaction process shown below:

[0042] Based on the reaction process described above, the preparation process includes: Step 1: Dry a 250 mL flask with a magnetic dryer to remove water, add hydroxyethyl methacrylate and anhydrous dichloromethane (DCM). o Triethylamine was added at 40 °C and stirred for 10 min, followed by slow addition of an anhydrous DCM solution of bromoacetyl bromide and reaction overnight. The mixture was extracted with an excess of saturated NaHCO3 solution, washed with saturated brine, and dried over anhydrous sodium sulfate. After column chromatography, the solvent was removed and the mixture was dried under vacuum to obtain product A.

[0043] Step 2: Add product A and 2-hydroxy-3-methylpyridine to a 250 mL flask in sequence, and dissolve them in ultra-dry DMF. Add anhydrous K2CO3, replace argon, and react at room temperature. After the reaction is completed, remove DMF by vacuum distillation. Dissolve in DCM, wash with saturated NaCl solution, and dry with anhydrous Na2SO4. Chromatography on a DCM column. Collect the product and dry it in vacuum to obtain the functionalized hydroxypyridine monomer (Py).

[0044] It is understood by those skilled in the art that the preparation method of the CO monomer molecule and the functionalized hydroxypyridine monomer described above is only an example, and can also be synthesized by other routes. As long as the CO monomer molecule and the functionalized hydroxypyridine monomer with the chemical structure can be synthesized, this specification will not go into details one by one.

[0045] As an example of the nanoassembly described in the present application, the amphiphilic block polymer A and the amphiphilic block polymer B preferably have a molecular weight of 200-35000 Da, which can regulate the nanoassembly to have a suitable particle size, thereby achieving better delivery and enrichment in vivo.

[0046] As an example of the nanoassembly described in the present application, the particle size of the nanoassembly is preferably 20-800 nm, which is conducive to better delivery and enrichment into the body, thereby achieving better therapeutic effects.

[0047] As an example of the nano-assembly described in the present application, the hydrophilic-hydrophobic self-assembly forming method includes nano-flash precipitation, slow water addition, ultrasound and thin film hydration method. Among them, the embodiment of the present application preferably prepares the nano-assembly described in the present application by nano-flash precipitation method.

[0048] In a second aspect, the present application also provides a method for preparing the nanoassembly described in the above application, preferably comprising: A step of dissolving the amphiphilic block polymer A and the amphiphilic block polymer B in a co-solvent to prepare an organic phase; and, a step of flash-precipitating the organic phase into an aqueous phase.

[0049] The preparation method provided in the embodiment of the present application enables the amphiphilic block polymer A and the amphiphilic block polymer B to fully self-assemble and form a co-assembly product with a stable system, thereby effectively achieving spontaneous and stable release of CO in human body environments such as normoxia, hypoxia and darkness, and the preparation is simple and easy to operate, and has good prospects for large-scale production.

[0050] As an example of the nanoassembly described in the present application, the co-solvent refers to a solvent that can dissolve the amphiphilic block polymer A and the amphiphilic block polymer B at the same time, and may preferably be one of tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide or 1,4-dioxane.

[0051] As an example of the nanoassembly described in the present application, the mass volume concentration of the amphiphilic block polymer A and the amphiphilic block polymer B dissolved in the co-solvent is preferably 0.01 to 10 g / L. Among them, the embodiment of the present application can effectively regulate the assembly ratio of the amphiphilic block polymer A and the amphiphilic block polymer B by controlling the mass volume ratio concentration, thereby achieving regulation of the release rate and release concentration of CO.

[0052] In the third aspect, based on the nanoassembly described in the above text application, it has the ability to spontaneously release CO in the human body temperature (15-55 ° C) environment. The embodiment of the present application also provides the application of the nanoassembly described in the above text application. Specifically, the application of the nanoassembly described in the present application in the preparation of anti-inflammatory drugs. Among them, based on the mechanism that the nanoassembly described in the present application triggers the release of CO at a temperature close to human body temperature (37 ° C), the nanoassembly described in the present application can be used to prepare anti-inflammatory drugs. After spontaneously releasing CO in the human body environment of normoxia, hypoxia and darkness, it effectively inhibits inflammatory signaling pathways, reduces the production of inflammatory mediators, and enhances antioxidant defense mechanisms to achieve anti-inflammatory and cell protection effects.

[0053] The technical solution of the present application will be further described below in conjunction with specific embodiments.

[0054] Example 1 This example provides a synthesis of an amphiphilic block polymer PEG SHF for use as a CO donor.

[0055] S11: Synthesis of CO donor molecule SHF, the reaction process is as follows:

[0056] According to the reaction process described above, the specific preparation process includes: S111: Disperse 3.6 g of 2-hydroxyacetophenone (HPE) in 80 mL of EtOH, add 18 mL of 5 M NaOH aqueous solution, and stir for 30 min. Add 7.7 g of TBFE and stir for 5 h. o After adding 18 mL of 30% H2O2 and stirring overnight under ice bath, the pH was adjusted to 6.5 with 0.5 M HCl to precipitate solids, which were collected by suction filtration. o C vacuum drying, washing with anhydrous ether three times, and again at 30 o C and dried in vacuo to obtain the product OHF-Boc (yield 56%).

[0057] S112: Dissolve 3.5 g OHF-Boc and 1.75 g Lawesson's reagent in 100 mL anhydrous toluene, replace nitrogen and heat under reflux for 12 h. After the reaction is completed, cool to room temperature, collect the liquid by suction filtration, remove toluene, purify by DCM column chromatography, collect the product, spin dry and purify for 30 o C and dried in vacuo to obtain the product SHF-Boc (yield 81%).

[0058] S113: Dissolve 2.1 g SHF-Boc in 30 mL DCM, add 5 mL trifluoroacetic acid and stir at room temperature. After the reaction is complete, remove the trifluoroacetic acid and collect the solid. Wash the solid with ether and 30 mL DCM. o C and dried in vacuo to obtain the product SHF-NH2 (yield 93%).

[0059] S114: 1.5 g SHF-NH2 was dispersed in 20 mL chloroform, and then 1.1 g pentafluorophenol active ester and 0.44 g triethylamine were added and stirred at room temperature. After the reaction was completed, DCM column chromatography was performed to purify the product, and the product was successively spin-dried and 30 o C was dried in vacuo to obtain the CO donor molecule SHF (yield 86%).

[0060] S12: Synthesis of the amphiphilic block polymer PEG SHF for CO donor, the reaction process is as follows:

[0061] According to the reaction process described above, the specific preparation process includes: S121: 30 mg PEG-CTA and 100 mg SHF were dissolved in 400 μL dimethyl sulfoxide, and 2.1 mg azobisisobutyronitrile was added. The tube was sealed after three cycles of liquid nitrogen cooling, vacuuming, and thawing. After polymerization at 70 °C for 8 h, the reaction solution was precipitated into anhydrous ether. The solid product was heated for 30 min. o C and dried under vacuum to obtain the amphiphilic block polymer PEG SHF used as CO donor.

[0062] The amphiphilic block polymer PEG SHF was characterized by hydrogen spectrum and GPC, and the results were Figures 1 to 2 As shown. Among them, Figure 1 It is the hydrogen spectrum of the amphiphilic block polymer PEG SHF; Figure 2 This is the GPC spectrum of the amphiphilic block polymer PEG SHF.

[0063] according to Figure 1It can be seen that the degree of polymerization of the CO donor molecule in the constituent components of the amphiphilic block polymer PEG-SHF prepared above is 6; according to Figure 2 It can be seen that the number average molecular weight of the amphiphilic block polymer PEG-SHF prepared above is 5.9 kDa, and the polydispersity index is 1.09.

[0064] Example 2 This example provides a synthesis of an amphiphilic block polymer PEG-Py EPO for singlet oxygen donor.

[0065] S21: Synthesis of functionalized hydroxypyridine monomer Py, the reaction process is as follows:

[0066] According to the reaction process described above, the specific preparation process includes: S211: Dry the 250 mL flask and magnetic dryer to remove water, add 10 g of hydroxyethyl methacrylate and 100 mL of anhydrous DCM. o 11.7 g of triethylamine was added at 40 °C and stirred for 10 min. A solution of 18.5 g of bromoacetyl bromide in 20 mL of anhydrous DCM was added over a period of about 2 h and the reaction was allowed to proceed overnight. The reaction solution was then extracted with an excess of saturated NaHCO3 solution, washed with saturated brine, dried over anhydrous sodium sulfate, and chromatographed on a DCM column. The solvent and 30 o C was dried in vacuo to obtain product A (yield 63%).

[0067] S212: 6 g of product A and 2.37 g of 2-hydroxy-3-methylpyridine were added to a 250 mL flask and dissolved in 60 mL of ultra-dry DMF. Then 9.0 g of anhydrous K2CO3 was added to replace the argon gas and reacted at room temperature. After the reaction was completed, DMF was removed by vacuum distillation. Then, it was dissolved in DCM, washed with saturated NaCl solution and dried with anhydrous Na2SO4. DCM column chromatography was performed. The product was collected and 30 o C and dried under vacuum to obtain a functionalized hydroxypyridine monomer Py.

[0068] S22: Synthesis of an amphiphilic block polymer PEG-Py EPO for singlet oxygen donor, the reaction process is as follows:

[0069]

[0070] According to the reaction process described above, the specific preparation process includes: S221: Dissolve 41 mg PEG-CTA and 300 mg Py in 350 μL dimethylformamide, add 2.9 mg azobisisobutyronitrile, cool with liquid nitrogen, vacuumize, and thaw three times, seal the tube, and polymerize at 70 °C for 12 h. Precipitate the reaction solution in anhydrous ether, and the oily product is heated to 30 °C. o C vacuum drying to obtain the amphiphilic block polymer PEG-Py; S222: 100 mg of amphiphilic block polymer PEG-Py and 2 mg of tetraphenylporphyrin were dissolved in 5 mL of dichloromethane and irradiated at 420 nm for 1 h under oxygen flow. After concentration, the solid product was immersed in 40 mL of anhydrous ether and the solid product was subjected to 30 o C and dried in vacuo to obtain the amphiphilic block polymer PEG-Py EPO used as a singlet oxygen donor.

[0071] The amphiphilic block polymer PEG-Py and the amphiphilic block polymer PEG-Py EPO prepared above were characterized by hydrogen spectrum and GPC. The results were Figures 3 to 5 As shown. Among them, Figure 3 is the hydrogen spectrum of the amphiphilic block polymer PEG-Py; Figure 4 is the hydrogen spectrum of the amphiphilic block polymer PEG-Py EPO; Figure 5 GPC spectra of amphiphilic block polymers PEG-Py and PEG-PyEPO.

[0072] according to Figure 3 It can be seen that the degree of polymerization of Py in the constituent components of the amphiphilic block polymer PEG-Py prepared above is 56.

[0073] according to Figure 4 It can be seen that Py in the constituent components of the amphiphilic block polymer PEG-Py prepared above is completely converted into EPO.

[0074] according to Figure 5 It can be seen that the number average molecular weights of the amphiphilic block polymers PEG-Py and PEG-Py EPO prepared above are 11.0 kDa and 12.2 kDa, respectively, and the polydispersity indexes are 1.13 and 1.26, respectively.

[0075] Example 3 This embodiment provides a construction of a nanoassembly PEG-SHF@PyEPO, the steps comprising: S301: After 1 mg of amphiphilic block polymer PEG SHF and 9 mg of amphiphilic block polymer PEG-Py EPO were co-dissolved in 1 mL of tetrahydrofuran, they were flash-precipitated in pure water at room temperature with high-speed stirring (1000 rpm) for self-assembly, and the resulting assembly was placed in a dialysis bag (molecular weight cutoff 14000 Da) and dialyzed in ice water for 12 h to obtain the nanoassembly PEG-SHF@PyEPO.

[0076] In order to illustrate the structural properties of the nanoassembly prepared in the present application, the nanoassembly PEG-SHF@PyEPO was used as a sample for testing.

[0077] 1. Morphological and structural characterization 1.1 TEM characterization The nanoassembly PEG-SHF@PyEPO was characterized by transmission electron microscopy. Figure 6 As shown. Among them, Figure 6 TEM image of the nanoassembly PEG-SHF@PyEPO.

[0078] according to Figure 6 It can be seen that the above embodiments assemble to form nanomicelles with uniform appearance.

[0079] 1.2 Particle size analysis The hydration diameter of the nanoassembly PEG-SHF@PyEPO was measured by dynamic light scattering. Figure 7 As shown. Among them, Figure 7 This is the hydrated particle size diagram of the nanoassembly PEG-SHF@PyEPO.

[0080] according to Figure 7 It can be seen that the particle size distribution of the nanoassembly PEG-SHF@PyEPO prepared above is uniform, and the hydrated particle size is about 44 nm.

[0081] 2.CO release test The nanoassembly PEG-SHF@PyEPO aqueous solution was incubated at 37 °C for different time periods to test the changes in UV absorption. Figure 8 As shown. Among them, Figure 8 This is the UV absorption spectrum of the aqueous solution of the nanoassembly PEG-SHF@PyEPO.

[0082] A 10 μM 1-Ac fluorescent probe was incubated with the nanoassembly PEG-SHF@PyEPO aqueous solution at 37 °C to test the fluorescence changes of the probe at different times. The results were as follows: Fig. 9 As shown. Among them, Fig. 9 This is the fluorescence curve of the aqueous solution of the nanoassembly PEG-SHF@PyEPO.

[0083] according to Figures 8 to 9 It can be seen that the nanoassembly PEG-SHF@PyEPO can produce CO at a temperature close to that of the human body (37 °C).

[0084] 3. CO release for anti-inflammatory effects in macrophages RAW 264.7 macrophages were plated at 10 per well. 5 The cells were seeded in a 24-well plate at a density of 100 cells / mL and cultured in a cell culture incubator for 24 h. Then, the culture medium in RAW 264.7 macrophages was replaced with a culture medium containing PBS, L-NAME (1 mM), and PEG-SHF@PyEPO, and LPS (1 μg / mL) was added to each group. After 6 h of culture in a cell culture incubator, fresh culture medium was replaced and the cells were cultured for another 18 h. 50 μL of culture medium was taken and placed in a 96-well plate, and 50 μL of Griess reagent A solution (1% p-aminobenzenesulfonic acid) was added and incubated at 37 o C for 5 min, then add Griess reagent B solution (0.1% NED solution) and continue to culture for 5 min, and measure the absorbance at 540 nm with a microplate reader. According to the standard curve prepared by the prepared sodium nitrite standard solution, the nitrite level produced in the cells was quantitatively tested, and the results were Fig.10 As shown. Among them, Fig.10 A test chart of nitrite levels produced in cells after different treatments.

[0085] according to Fig.10 It can be seen that releasing CO through the nanoassembly PEG-SHF@PyEPO can effectively achieve anti-inflammatory effects.

[0086] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0087] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A nanoassembly capable of self-releasing CO, characterized in that: The invention comprises a co-assembly product prepared by a hydrophilic-hydrophobic self-assembly forming method from a component system comprising the following components; (1) An amphiphilic block polymer A for CO donor, which has the chemical structure of the following formula (I) or (II): (AND); (II); R1 is selected from one of the chemical structures of the following formula (rI) or (rII); m and n are both integers between 2 and 100; (find); (rII); R2 is one of N and O; R3 is one of O, S and Se; (2) An amphiphilic block polymer B for singlet oxygen donor having the chemical structure of the following formula (III): (III) R4, R5, R6 and R7 are independently H or -CH3; a and b are both integers between 2 and 100.

2. The nanoassembly according to claim 1, characterized in that: The amphiphilic block polymer A is generated by a polymerization reaction of a component system comprising a CO donor molecule and polyethylene glycol-4-cyano-4-(phenylcarbonylthiothiothio) pentanoate; Wherein, the CO donor molecule has the following chemical structure (IV) or (V): (IV) (V)。 3. The nanoassembly according to claim 1, characterized in that: The amphiphilic block polymer B is generated by polymerizing a component system comprising a functionalized hydroxypyridine monomer and polyethylene glycol-4-cyano-4-(phenylcarbonylthiothio) pentanoate, and subjecting the obtained reaction product to a photosensitive oxidation reaction with tetraphenylporphyrin; Wherein, the functionalized hydroxypyridine monomer has the following chemical structure (VI): (WE).

4. The nanoassembly according to claim 1, characterized in that: The molecular weights of the amphiphilic block polymer A and the amphiphilic block polymer B are both 200-35000 Da.

5. The nanoassembly according to claim 4, characterized in that: The particle size of the nanoassembly is 20-800 nm.

6. The nanoassembly according to claim 1, characterized in that: The hydrophilic and hydrophobic self-assembly forming method comprises nano-flash precipitation, slow water addition, ultrasound and thin film hydration method.

7. A method for preparing the nanoassembly according to any one of claims 1 to 6, characterized in that: The nano-flash precipitation method is used to prepare the shape, which specifically includes: A step of dissolving the amphiphilic block polymer A and the amphiphilic block polymer B in a co-solvent to prepare an organic phase; and, a step of flash-precipitating the organic phase into an aqueous phase.

8. The preparation method according to claim 7, characterized in that: The co-solvent is selected from one of tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide or 1,4-dioxane.

9. The preparation method according to claim 7, characterized in that: The mass volume concentration of the amphiphilic block polymer A and the amphiphilic block polymer B dissolved in the co-solvent is 0.01-10 g / L.

10. Use of the nanoassembly according to any one of claims 1 to 6 or the nanoassembly prepared by the preparation method according to any one of claims 7 to 9 in preparing anti-inflammatory drugs.

Images