A carbon monoxide controllable release nanoparticle, and a preparation method and application thereof

Abstract

The application discloses controllable release carbon monoxide nanoparticles and a preparation method and application thereof, and belongs to the technical field of nano drugs. The controllable release carbon monoxide nanoparticles provided by the application comprise triron dodecacarbonyl and Croc-PEG for coating the triron dodecacarbonyl; the Croc-PEG is a condensation product of Croc and MPEG-NH2. The nanoparticles provided by the application can effectively improve the carbon monoxide loading capacity, the biological safety of the nanoparticles is strong, and the carbon monoxide can be released in a controllable manner, so that efficient and low-toxicity CO gas therapy can be realized. The application further provides a preparation method and application of the nanoparticles.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a nanoparticle that can controllably release carbon monoxide, its preparation method, and its application. Background Technology

[0002] Carbon monoxide (CO) is a gaseous messenger molecule with a wealth of biological effects and therapeutic potential for many diseases. As a bioactive substance, CO plays a regulatory role in many physiological and pathological aspects, primarily because it can bind to respiratory chain complexes such as ferrohemoglobin (Hb), thereby affecting their function and triggering cascade effects. CO's vascular tone-regulating effect can improve blood circulation, its antioxidant effect can reduce oxidative stress and alleviate inflammation, its anti-apoptotic effect can inhibit mitochondrial apoptosis, and its repair-promoting effect can promote tissue regeneration. Therefore, CO is an ideal therapeutic agent. Due to the numerous effects of CO, carbon monoxide (CO) gas therapy has become an emerging medical technology and has been gradually applied to the treatment of various diseases, such as cardiovascular diseases, sepsis, shock, acute lung, kidney and liver injuries, microbial infections, and cancer.

[0003] Gaseous CO is difficult to store and transport. Its diffuse nature makes its concentration and location in the body uncontrollable, easily leading to ineffectiveness or the risk of poisoning. Furthermore, the efficacy of CO gas therapy depends on the concentration and location of CO in the body. Too low a concentration has no significant therapeutic effect, while too high a concentration poses a risk of poisoning. At the same time, CO can easily cause systemic toxicity when it diffuses aimlessly throughout the body. Therefore, the controlled and precise release of CO is crucial.

[0004] Therefore, CO gas therapy must rely on specific platforms to achieve precise, on-demand release. The construction of gas-generating platforms is crucial for the application of CO gas therapy, and nanomaterials, due to their diversity, structural variability, and functional versatility, have become an ideal choice. Currently, materials such as liposomes, hydrogels, peptides, proteins, and metal-organic frameworks are used in the construction of gas-generating nanoplatforms (GGNs). Based on specific GGNs, controlled CO release can be achieved by adjusting the structure, shape, size, and surface modification of nanoparticles. Simultaneously, GGNs themselves can achieve diverse functions, such as combination therapy, synergistic therapy, and image-guided therapy. The construction of GGNs provides valuable insights for the development and widespread adoption of CO gas therapy.

[0005] CO-releasing molecules (CORMs) are gas-producing nanoplatforms that can release CO gas. They can serve as gas reservoirs for releasing CO gas in vivo or in vitro. Common CORMs include metal porphyrin complexes, organic compounds, and mononuclear iron porphyrin compounds. Carbonyl metals are complexes formed between transition metals (nickel, cobalt, ruthenium, vanadium, chromium, manganese, and iron) and CO, which can release CO under specific conditions. Considering the safety of metal metabolites, manganese carbonyl and iron carbonyl are more suitable for biological applications. Among these compounds, dodecacarbonyltriferric (Fe3(CO)) is particularly suitable. 12 These compounds have the highest gas storage capacity, making them ideal CO donors. However, their water solubility, biocompatibility, and the controllability of CO release need to be improved. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a nanoparticle that can controllably release carbon monoxide, which can effectively improve the carbon monoxide loading, enhance the biocompatibility of the nanoparticle, and control the release of carbon monoxide, thereby achieving highly efficient and low-toxicity CO gas therapy.

[0007] The present invention also provides a method for preparing the above-mentioned nanoparticles.

[0008] This invention also provides applications of the aforementioned nanoparticles.

[0009] According to an embodiment of the first aspect of the present invention, nanoparticles (Fe3(CO)) that controllably release carbon monoxide are provided. 12 @Croc-PEG), the nanoparticles comprising dodecacarbonyltriferric (Fe3(CO)) 12 And Croc-PEG coated with the dodecyltriferric oxide, wherein the Croc-PEG is a condensation product of Croc (a ketone cyanine dye) and MPEG-NH2 (amino-terminated polyethylene glycol); the molecular formula of the Croc is:

[0010]

[0011] The nanoparticles according to embodiments of the present invention have at least the following beneficial effects:

[0012] (1)Fe3(CO) 12 It is a carbonyl metal with a high gas storage capacity, which can release CO under photothermal stimulation, and the metal element iron in it is an essential trace element for the human body. Fe3(CO) is used. 12 As a CO donor, it has high CO loading capacity, biosafety, and the potential for controlled CO release; however, its water solubility and biocompatibility are very poor.

[0013] Croc-PEG is a polymeric functional crocine cyanine dye exhibiting a structure with one hydrophilic end (provided by PEG) and one hydrophobic end. The hydrophobic structure can react with Fe3(CO). 12 With the hydrophilic structure facing outwards, a Croc-PEG coating of Fe3(CO) can be formed. 12 The core-shell structure solved the problem of Fe3(CO). 12 The invention addresses the issues of insolubility in water, poor biocompatibility, and short half-life. Experimental verification shows that the nanoparticles provided by this invention can exist stably in various biological media (water, PBS, culture medium, and serum); moreover, the nanoparticles exhibit stable assembly, and neither Fe3(CO)12 precipitation nor Croc-PEG release occurs during repeated centrifugation-resuspension operations.

[0014] (2) Croc-PEG mainly consists of two parts: Croc and PEG. Croc is the core of the dye and has pH-responsive fluorescence imaging and photoacoustic imaging capabilities. Since many diseases occur in slightly acidic environments, it can be used to monitor the disease microenvironment and also solves the problem of Fe3(CO). 12 The issue of controlled CO release. Specifically, Croc-PEG exhibits a significant photothermal heating effect under near-infrared (NIR) laser irradiation; Fe3(CO) 12 Under thermal action, CO can be released. Therefore, the nanoparticles provided by this invention can achieve the function of photothermally promoting CO release, and free radicals also promote CO release. Therefore, Croc-PEG is an ideal delivery carrier, and Fe3(CO) 12 When used together, they can achieve controlled release of CO.

[0015] (3) Croc-PEG as a carrier has good biodegradability and biocompatibility. It has a rich variety of functional groups, which makes it easy to modify and improve, and has great prospects for clinical translation.

[0016] In summary, the nanoparticles provided by this invention have the advantages of high loading capacity, strong biosafety, and controllable CO release, and are expected to achieve highly efficient and low-toxicity CO gas therapy.

[0017] According to some embodiments of the present invention, the molecular weight of MPEG-NH2 is 2k to 5k. When the molecular weight of MPEG-NH2 is 5k, Croc-PEG is referred to as Croc-PEG5K. Other molecular weights are abbreviated herein.

[0018] The molecular weight length of MPEG-NH2 and the encapsulation efficiency of dodecyltriferrite are positively correlated with the particle size of the nanoparticles. Within the aforementioned molecular weight range, nanoparticles with a particle size in the range of 100–200 nm, suitable for biological applications, can be prepared, exhibiting a high encapsulation efficiency of dodecyltriferrite. In actual production, the molecular weight of MPEG-NH2 used can be adjusted according to the required dodecyltriferrite loading.

[0019] According to some embodiments of the present invention, the molar ratio of the dodecyltriferric oxide to the Croc-PEG is 5 to 6:1. For example, it can be 5.5 to 5.6:1.

[0020] According to some embodiments of the present invention, the nanoparticles have a near-spherical structure.

[0021] According to some embodiments of the present invention, the average size of the nanoparticles is 80–110 nm. Specifically, it can be approximately 100 nm. Unless otherwise specified, the average size in this invention refers to the peak particle size obtained from a DLS spectrum.

[0022] According to some embodiments of the present invention, the nanoparticles exhibit dual responsiveness to near-infrared light and free radicals. That is, under the influence of either exogenous near-infrared light or internal free radicals, the release of carbon oxide can be promoted. This allows for the controllable release of carbon oxide through multiple pathways, overcoming the technical challenge of uncontrollable CO concentration due to its diffusivity, and providing technical guidance for the widespread application of CO gas therapy.

[0023] According to an embodiment of a second aspect of the present invention, a method for preparing the nanoparticles is provided, the method comprising the following steps:

[0024] S1. Disperse the dodecyltriferric oxide in THF to obtain dispersion A;

[0025] The Croc-PEG was dispersed in THF to obtain dispersion B;

[0026] S2. Add the dispersion A and dispersion B to water and mix;

[0027] S3. Blow protective gas into the mixture obtained in step S2, and then pass the mixture through a filter membrane to concentrate the filtrate by ultrafiltration.

[0028] The preparation method provided by the present invention has at least the following beneficial effects:

[0029] (1) Because Croc-PEG has the property of being hydrophilic at one end and hydrophobic at the other, it can encapsulate Fe3(CO) through hydrophilic-hydrophobic interactions. 12 Fe3(CO) nanoparticles with a core-shell structure were prepared. 12@Croc-PEG, these nanoparticles can release CO in a dual response of NIR (near-infrared) laser and free radical.

[0030] (2) Due to the Croc-PEG core Fe3(CO) 12 Due to their inherent hydrophilic and hydrophobic properties, the two can spontaneously assemble to form a core-shell structure in aqueous solution. Therefore, this invention can prepare nanoparticles with a core-shell structure through a simple nano-coprecipitation method. The raw materials used in this preparation method are simple, the preparation process is conventional, the repeatability is good, the stability is high, and it is suitable for batch and industrial production.

[0031] (3) In step S1 of the preparation method, THF is selected as the solvent, which has the ability to dissolve dodecacarbonyltriferric and Croc-PEG, and is also miscible with water; thus, the nanoparticles are easily prepared.

[0032] According to some embodiments of the present invention, in step S1, the source of the Croc-PEG includes external purchase or laboratory preparation.

[0033] According to some embodiments of the present invention, in step S1, the synthesis method of Croc-PEG (when the source is laboratory-made) includes the following steps:

[0034] D1. The reaction product of 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone (hereinafter referred to as the intermediate product) is reacted with crocoic acid to obtain crocoic acid cyanine dye (hereinafter referred to as Croc);

[0035] D2. Cause the ketone cyanine dye and MPEG-NH2 to undergo an amide condensation reaction.

[0036] According to some embodiments of the present invention, in step D1, the molar ratio of 4-carboxyphenylhydrazine hydrochloride to 3-methyl-2-butanone is 1:1.5 to 2. For example, it can be 1:1.76.

[0037] According to some embodiments of the present invention, in step D1, the reaction of the 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone further requires the addition of sodium acetate and acetic acid.

[0038] The molar ratio of 4-carboxyphenylhydrazine hydrochloride to sodium acetate is 1:2.5 to 3.0. For example, it can be approximately 1:2.75.

[0039] The molar ratio of 4-carboxyphenylhydrazine hydrochloride to acetic acid is 0.6–1 mmol / mL. For example, it can be 0.81–0.95 mmol / mL.

[0040] According to some embodiments of the present invention, in step D1, the reaction of the 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone is carried out under a protective atmosphere. Specifically, it can be carried out under a continuous argon flow.

[0041] According to some embodiments of the present invention, in step D1, the reaction of the 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone is carried out at a temperature of 100–150°C. Specifically, it can be about 130°C.

[0042] According to some embodiments of the present invention, in step D1, the reaction of the 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone lasts for 5 to 10 hours. For example, it can specifically be about 7 hours.

[0043] According to some embodiments of the present invention, in step D1, the reaction between the 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone is carried out under stirring. The stirring speed is not strictly limited, as long as mass transfer can be achieved.

[0044] According to some embodiments of the present invention, step D1 further includes purifying the intermediate product before reacting with the keto acid. The purification method includes extraction. The extractant used in the extraction includes dichloromethane. The O / A ratio of the extraction is approximately 1:3. The purification also includes removing the extractant from the resulting supported organic phase; specific removal methods include rotary evaporation and drying.

[0045] According to some embodiments of the present invention, in step D1, the intermediate product is orange in color; and / or is solid at room temperature (about 25°C).

[0046] According to some embodiments of the present invention, in step D1, the reaction of the 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone has a yield of ≥84%, specifically about 84.5%.

[0047] According to some embodiments of the present invention, in step D1, the molar ratio of the intermediate product to the keto acid is 1.8 to 2.2:1. For example, it can be 2.0 to 2.1:1.

[0048] According to some embodiments of the present invention, in step D1, the reaction between the intermediate product and the ketoacid uses a solvent comprising n-butanol and toluene. The volume ratio of n-butanol to toluene is 1:0.8 to 1.2. Specifically, it can be about 1:1. In this reaction step, the molar amount of the ketoacid and the volume ratio of the solvent are 0.3 to 0.5 mmol / mL (0.3 to 0.5 mmol / mL). Specifically, it can be 0.35 to 0.45 mmol / mL, and more specifically, about 0.4 mmol / mL.

[0049] According to some embodiments of the present invention, in step D1, the reaction between the intermediate product and the keto acid is carried out under a protective atmosphere. Specifically, it can be carried out under a continuous argon flow.

[0050] According to some embodiments of the present invention, in step D1, the reaction between the intermediate product and the keto acid is carried out at a temperature of 40–60°C. Specifically, it can be about 50°C.

[0051] According to some embodiments of the present invention, in step D1, the reaction between the intermediate product and the keto acid lasts for 15 to 25 hours. For example, it can specifically be about 20 hours.

[0052] According to some embodiments of the present invention, in step D1, the reaction between the intermediate product and the keto acid is carried out under stirring. The stirring speed is not strictly limited, as long as mass transfer can be achieved.

[0053] According to some embodiments of the present invention, step D1 further includes purifying the ketocyanine dye. The purification of the ketocyanine dye includes sequential cooling, solid-liquid separation, and drying. Cooling includes sequential cooling to room temperature and cooling-incubation. The cooling-incubation temperature is -25 to -10°C, specifically approximately -20°C. The cooling-incubation time is 8 to 15 hours, specifically approximately 10 hours. The cooling-incubation also includes the addition of diethyl ether.

[0054] According to some embodiments of the present invention, the color of the ketone cyanine dye is dark green; and / or, it is solid at room temperature.

[0055] According to some embodiments of the present invention, the reaction of the intermediate product and the keto acid yields ≥45%. Specifically, it can be about 48%.

[0056] Based on the structural formula of ketocyanine dye, it can be seen that ketocyanine dye consists of a central keto acid and symmetrical side chains, and has typical electron-donating-electron-withdrawing-electron-donating (DAD) structural characteristics.

[0057] Further analysis of the structural formula of the ketocyanine dye reveals that in step D2, the ketocyanine dye and polyethylene glycol undergo an amide condensation reaction to produce a polymer dye, and the amide condensation reaction requires relatively mild reaction conditions.

[0058] According to some embodiments of the present invention, in step D2, the molar ratio of the cyanine dye to polyethylene glycol is approximately 2:1. This allows for the reaction of approximately half of the carboxyl groups on the cyanine dye with polyethylene glycol, thereby preserving the hydrophobicity of the cyanine dye while introducing some hydrophilicity through the introduction of polyethylene glycol. The resulting Croc-PEG exhibits both hydrophilic and hydrophobic properties.

[0059] According to some embodiments of the present invention, in step D2, the amide condensation reaction further requires the addition of EDC.HCl and HOBT. The molar ratio of the ketone cyanine dye to the EDC.HCl is 1:3 to 4; specifically, it can be about 1:3.5. The molar ratio of the ketone cyanine dye to the HOBT is 1:3 to 4; specifically, it can be about 1:3.5.

[0060] According to some embodiments of the present invention, in step D2, the amide condensation reaction is carried out in a solvent. The solvent for the amide condensation reaction includes anhydrous DMF. In the amide condensation reaction, the ratio of the amount of the ketone cyanine dye to the volume of the solvent is 0.05 to 1 mmol / L. For example, it can be about 0.07 mmol / L.

[0061] According to some embodiments of the present invention, in step D2, the amide condensation reaction is carried out in a protective atmosphere, specifically in an argon gas flow.

[0062] According to some embodiments of the present invention, in step D2, the amide condensation reaction is carried out at a temperature of 30–40°C. Specifically, it can be about 35°C.

[0063] According to some embodiments of the present invention, in step D2, the amide condensation reaction lasts for 60 to 80 hours. For example, it can specifically be about 72 hours.

[0064] According to some embodiments of the present invention, step D2 further includes purifying the Croc-PEG. The purification procedure here is similar to the purification procedure for the ketocyanine in step D1.

[0065] According to some embodiments of the present invention, in step D2, the amide condensation reaction has a yield of ≥75%. Specifically, it can be about 77%.

[0066] According to some embodiments of the present invention, in step S1, the Croc-PEG is yellow-green in color; and / or, it is solid at room temperature.

[0067] According to some embodiments of the present invention, in step S1, the concentration of dispersion A is 3.5–4.5 mmol / mL. For example, it can be 3.9–4 mmol / mL.

[0068] According to some embodiments of the present invention, in step S1, the concentration of dispersion B is 0.5 to 1 mmol / mL. For example, it can be 0.7 to 0.75 mmol / mL.

[0069] According to some embodiments of the present invention, in step S2, the volume ratio of the dispersion A to the water is 1:8 to 9. For example, it can be approximately 1:8.5.

[0070] According to some embodiments of the present invention, in step S2, the mixing time is 25 to 35 minutes. Specifically, it can be approximately 30 minutes. The mixing is performed under ultrasonic conditions.

[0071] According to some embodiments of the present invention, step S2 includes first adding the dispersion B dropwise to water, and then adding the dispersion A dropwise to the resulting mixture. Croc-PEG is soluble in water, while dodecyltriferric iron is insoluble in water. This arrangement allows Croc-PEG and dodecyltriferric iron to fully contact and mix, improving the uniformity of assembly and encapsulation.

[0072] In step S2, the dropping rate of the dispersion B is not strictly limited, as long as it can be fully mixed.

[0073] In step S2, the dropping rate of dispersion A is 0.8–1.2 mL / min. Specifically, it can be approximately 1 mL / min. This allows sufficient time for the mixing and assembly of Croc-PEG and dodecyl iron(II)carbonyl.

[0074] Step S2 is performed under ultrasonic conditions.

[0075] According to some embodiments of the present invention, in step S3, the pore size of the filter membrane is approximately 0.22 μm. Specifically, it can be that the filter membranes are sequentially passed through membranes of 0.8 μm, 0.45 μm, and 0.22 μm. This reduces the difficulty of filtration and avoids excessive clogging of the filter membrane pores by insoluble substances.

[0076] According to some embodiments of the present invention, in step S3, the molecular cutoff weight of the ultrafiltration centrifuge tube used for ultrafiltration concentration is 10 kilodaltons.

[0077] Unless otherwise specified, the order of material addition in the preparation method is not limited, and in actual production, mixing, dissolution, or dispersion can be carried out directly.

[0078] According to an embodiment of a third aspect of the present invention, the nanoparticles are provided for use in the preparation of drugs for treating cardiovascular diseases, sepsis, shock, acute lung, kidney and liver injuries, microbial infections, arthritis and cancer.

[0079] Since the application employs all the technical solutions of the nanoparticles described in the above embodiments, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0080] Unless otherwise specified, the term "about" in this invention actually means that the error is allowed to be within ±2%, for example, about 100 is actually 100 ± 2% × 100.

[0081] Unless otherwise specified, "between" in this invention includes the number itself, for example, "between 2 and 3" includes the endpoint values ​​2 and 3.

[0082] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0083] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0084] Figure 1 This describes the mechanism of action of the nanoparticles obtained in Example 1 of this invention.

[0085] Figure 2 This is a schematic diagram of the preparation process in Embodiment 1 of the present invention.

[0086] Figure 3 These are TEM characterization images (a) of the nanoparticles obtained in Example 1 of this invention; DLS characterization image at 0 h (b); DLS characterization images from 0 to 72 h (c); Fe3(CO) 12 UV-Vis absorption spectra of Croc-PEG5K and nanoparticles (d); Fe3(CO) 12 Infrared spectral characterization of Croc-PEG5K and nanoparticles (e); Fe3(CO) nanoparticles 12 @Croc-PEG5K absorbance and concentration standard curve (f).

[0087] Figure 4 This is an apparent diagram of the dispersion and stability of the nanoparticles obtained in Example 1 of the present invention in different dispersion media.

[0088] Figure 5 This is a stability diagram of the nanoparticles obtained in Example 1 of the present invention during repeated centrifugation-resuspending operations.

[0089] Figure 6 The images show the photothermal effect of the nanoparticles obtained in Example 1 of this invention in vitro (a) and the photothermal effect of the mixed solution of nanoparticle K and reduced Hb (b).

[0090] Figure 7 The mixed solution of nanoparticles and reduced Hb obtained in Example 1 of this invention is at 808 nm (0.6 W / cm²). 2 (a) Ultraviolet-visible absorption spectrum within 0–60 min under laser irradiation; (b) Change in CO release over time under 808 nm laser irradiation at different optical densities; (c) Change in CO release over time under repeated laser “on-off” states.

[0091] Figure 8 The UV-Vis absorption spectra of the mixed solution of nanoparticles and reduced Hb obtained in Example 1 of this invention, after adding 5.0 μM H2O2, are shown in (a) within 0–60 min; the relationship between the CO release from the mixed solution containing different concentrations of H2O2 and time is shown in (b). Detailed Implementation

[0092] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0093] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0094] Material preparation example

[0095] This example demonstrates the preparation of a Croc-PEG5K, and the specific synthesis steps are as follows:

[0096] D1. 4-Carboxyphenylhydrazine hydrochloride (9.2 g, 48.8 mmol, CAS: 24589-77-3), 3-methyl-2-butanone (7.4 g, 86.0 mmol, CAS: 563-80-4) and sodium acetate (10.0 g, 122.0 mmol, CAS: 127-09-3) were mixed in acetic acid (50 mL). Argon gas was continuously introduced into the reaction system and the mixture was heated to 120 °C. After stirring for 7 h, stirring was stopped and the mixture was cooled to room temperature. The resulting reaction solution was extracted (dichloromethane / water = 1:3), and dried under vacuum after rotary evaporation to obtain an orange solid product, compound 1 (14.0 g, yield 84.3%).

[0097] Compound 1 (5.8 g, 28.6 mmol) and chlortetracycline (2.0 g, 14.0 mmol, CAS: 488-86-8) were mixed in a mixed solvent (n-butanol / toluene = 1:1, 30 mL). Argon gas was continuously introduced into the reaction system and the mixture was heated to 50 °C. After stirring for 20 h, stirring was stopped and the mixture was cooled to room temperature. A large amount of diethyl ether was added to the resulting reaction solution and the mixture was placed in a -20 °C refrigerator overnight. The precipitate was then centrifuged and vacuum dried to obtain a dark green solid product, Croc (chlortetracycline cyanine dye, 3.7 g, yield 46.8%).

[0098] D2. Croc (2.0 g, 4.0 mmol), MPEG5000-NH2 (10.0 g, 2.00 mmol, purchased from Shanghai Pengshuo Biotechnology, number average molecular weight approximately 5000), EDC.HCl (3.0 g, 14.7 mmol, CAS: 25952-53-8), and HOBT (2.0 g, 15.6 mmol, CAS: 2592-95-2) were mixed in anhydrous DMF (50 mL). Argon gas was continuously introduced into the reaction system and the mixture was heated to 35 °C. After stirring for 72 h, stirring was stopped and the mixture was cooled to room temperature. A large amount of diethyl ether was added to the resulting reaction solution and the mixture was placed in a -20 °C refrigerator overnight. The precipitate was then centrifuged, and the product was obtained by vacuum drying and freeze drying to obtain a yellow-green solid product, Croc-PEG5K (9.2 g, yield 76.8%).

[0099] The process for synthesizing Croc-PEG5K in this example is shown below:

[0100]

[0101] In this example, the product of step D1, ketone cyanine dye, was characterized by high-resolution mass spectrometry (HR-MS), 1H NMR, and 13C NMR. The results are as follows:

[0102] [M+H]+=511.15033(calcd for C29 H 23 N2O7+:511.14998);

[0103] 1 H NMR (600MHz, DMSO-d6) δ12.61(s,2H),8.05(d,3H),7.59(s,1H),.39–7.11(m,2H),6.05(s,2H),1.54(s,10H),1.11(d,2H);

[0104] 13 C NMR 150MHz, DMSO-d6)δ192.25,185.31,184.84,167.95,167.42,157.77,46.56,144.98,137.79,128.65,127.43,124.51,1 19.55,113.70,65.38,0.83,54.58,53.94,50.40,35.14,29.18,25.67,22.73,22.36,21.51,9.10,15.82,15.63,14.31.

[0105] The successful synthesis of Croc was confirmed by the molecular mass, proton NMR peak positions, and carbon NMR peak positions. The molecular mass, proton NMR peak positions, and carbon NMR peak positions in the mass spectrum also corresponded to those of Croc, indicating a successful synthesis.

[0106] Using a similar method, this example also characterized the Croc-PEG5K obtained in step D2. The specific results are as follows:

[0107] 1 ¹H NMR (600MHz, CDCl₃) δ 11.46 (s, 1H), 7.73 (m, 8H), 6.11 (t, 2H), 4.07–3.12 (m, 440H), 2.90–2.72 (m, 2H), 2.30 (s, 9H), 1.59–1.44 (m, 8H), 1.20 (d, H), 0.79 (d, 2H). Mass spectrometry results showed that the molecular weight of Croc-PEG₅K was around 5500, but the peak positions were not unique, indicating that Croc-PEG₅K was successfully synthesized. However, the molecular weight of the polymer MPEG₅₀₀-NH₂ is a range, therefore the molecular weight of the obtained Croc-PEG₅K is not unique either.

[0108] The successful synthesis of Croc-PEG5K was confirmed by its molecular mass and the position of the ¹H NMR peak. The molecular mass and ¹H NMR peak positions in the mass spectrum are consistent with those of Croc-PEG5K, indicating the successful synthesis of Croc-PEG5K.

[0109] Example 1

[0110] This example demonstrates the preparation of nanoparticles (Fe3(CO)). 12 @Croc-PEG5K), the specific preparation steps are as follows:

[0111] S1. Fe3(CO) 12 (12.0 mg, 23.8 mmol, CAS: 17685-52-8) was dispersed in THF (6 mL) to obtain dispersion A;

[0112] Croc-PEG5K (24.0 mg, 4.3 mmol, from the material preparation example) was dispersed in THF (6 mL) to obtain dispersion B;

[0113] S2. Under ultrasonic conditions, add dispersion B and dispersion A dropwise to ultrapure water (50 mL) and mix thoroughly. Continue ultrasonication for 30 min. The dropping rate is approximately 1 mL / min.

[0114] S3. After continuously introducing argon gas into the mixture obtained in step S2 to remove THF from the water, the mixture is squeezed through a membrane (passing through 0.8μm, 0.45μm and 0.22μm successively), and then the filtrate is concentrated by centrifugation using an ultrafiltration centrifuge tube (10KD) (products with a molecular weight of 10kD or higher are concentrated), finally obtaining the nanocomposite Fe3(CO). 12 @Croc-PEG5K.

[0115] The preparation process in this example is as follows: Figure 2 As shown.

[0116] Test Example 1

[0117] In this example, the nanoparticles obtained in Example 1 were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results showed that Fe3(CO) 12 @Croc-PEG5K exhibits a spherical structure with an average particle size of approximately 100 nm. TEM thickness contrast analysis reveals that the nanoparticles obtained in Example 1 possess a core-shell structure, with the black center being Fe3(CO). 12 The outer coating material is Croc-PEG5K, which also verifies Fe3(CO) 12 The preparation principle of @Croc-PEG5K is self-assembly induced by hydrophilic-hydrophobic interactions. (Fe3(CO)) 12The hydrated particle size of @Croc-PEG5K is approximately 100 nm, which is consistent with the TEM particle size. Furthermore, the hydrated particle size does not change significantly within 48 hours, indicating that it does not exhibit significant aggregation. Slight aggregation occurs at 72 hours, with a slight increase in particle size, but this slight aggregation can be reversed by ultrasound. Specific test results are as follows... Figure 3 As shown in (a), (b) and (c) in the figure.

[0118] This example also characterizes Fe3(CO). 12 The chemical structure and optical properties of @Croc-PEG5K were characterized using UV-Vis absorption spectroscopy and infrared spectroscopy. In the UV-Vis absorption spectroscopy, due to the preparation of Fe3(CO)... 12 @Croc-PEG5K, Fe3(CO) 12 Since the mass ratio of Fe3(CO) to Croc-PEG5K remains constant, the characteristic absorption peaks of Croc-PEG5K can be used to determine the relationship between Fe3(CO) and Croc-PEG5K. 12 Quantification of @Croc-PEG5K was performed, with concentration (μM) as the x-axis and Abs as the y-axis. 760 The vertical axis represents the coordinates, and the linear relationship between the two is: Y = 0.06016X + 0.05113. Infrared results show that Fe3(CO)... 12 @Croc-PEG5K also possesses Fe3(CO) 12 The UV absorption characteristic peaks and infrared functional group characteristic peaks of Croc-PEG5K indicate the successful assembly and binding of the two. Specific test results are as follows: Figure 3 As shown in (d), (e), and (f), where Figure 3 (b) shows the dispersion result for 0h.

[0119] Test Example 2

[0120] This example tests the dispersibility and stability of the nanoparticles obtained in Example 1. The specific method is as follows: Fe3(CO) 12 @Croc-PEG5K was dispersed in water, PBS (1X (1mM)), DMEM medium (stock solution), and fetal bovine serum (stock solution), and incubated at 4°C for 72 hours to observe whether it would deposit; Fe3(CO) was then added. 12 @Croc-PEG5K was repeatedly centrifuged and resuspended (dispersion medium: water). The color of the supernatant after centrifugation was observed to determine the free status of Croc-PEG5K, and the presence of precipitate in the resuspended solution was observed to determine the presence of Fe3(CO). 12 The precipitation condition.

[0121] The results showed that Fe3(CO) content increased in different media (water, PBS, DMEM, and fetal bovine serum). 12@Croc-PEG5K remains stable without significant changes and exhibits good biocompatibility. During multiple centrifugation-resuspending operations, neither the release of Croc-PEG5K nor the reaction of Fe3(CO) occurred. 12 The precipitation indicates that the two are stably combined.

[0122] The test results in this example are as follows: Figures 4-5 As shown. Among them, Figure 4 The time in the figure represents the settling time of the dispersion. Figure 5 The numerical designation in the text refers to the number of times the centrifugation-resuspending process has been repeated.

[0123] Test Example 3

[0124] (1) In this example, the photothermal properties of the nanoparticles obtained in Example 1 were tested (without Hb). The specific test method was as follows: Fe3(CO) was taken. 12 @Croc-PEG5K aqueous solution (30 μM, 1.0 mL), used an 808 nm laser at different optical densities (0.0–1.0 W / cm²). 2 Fe3(CO) was obtained under different optical density conditions after irradiation for 16 minutes. 12 Photothermal heating curve of @Croc-PEG5K solution. When Fe3(CO) 12 When the concentration of @Croc-PEG5K is 30 μM, the optical density of the 808 nm laser is higher (0~1.0 W / cm²). 2 The better the photothermal heating effect, the better. (Fe3(CO)) 12 The photothermal effect of @Croc-PEG5K originates from the near-infrared chlorine dye Croc-PEG5K, which exhibits a significant photothermal heating effect due to its strong absorption in the near-infrared region. Fe3(CO) 12 The heating effect of @Croc-PEG5K changes with the optical density of the laser, indicating that a suitable temperature can be selected by adjusting the concentration and laser parameters, reflecting the effect of Fe3(CO). 12 The photothermal tunability of @Croc-PEG5K. Specific test results are as follows: Figure 6 As shown in Figure (a).

[0125] (2) Exogenous laser promotes Fe3(CO) 12 @Croc-PEG5K CO release is measured using the Hb (hemoglobin) method, therefore it requires Fe3(CO)... 12 Hb and the reducing agent SDT (sodium dithionite) were added to the Croc-PEG5K solution. The addition of these substances may affect the photothermal heating effect of Croc-PEG5K; therefore, the photothermal effect of the mixed solution was investigated. Specifically, Fe3(CO) was taken... 12@Croc-PEG5K aqueous solution (30 μM), Hb (5 μM) and sodium dithionite (SDT, 1.6 mg) were added, the total volume of the mixed solution was 1.0 mL, and argon gas was continuously purged to remove air, yielding Fe3(CO). 12 A mixed solution of @Croc-PEG5K and reduced Hb was analyzed using an 808nm laser at different optical densities (0.0–1.0 W / cm²). 2 Irradiation for 16 minutes was performed to obtain the photothermal heating curves of the mixed solution under different optical densities. The results show that Fe3(CO) 12 Adding Hb to the @Croc-PEG5K solution weakens its photothermal heating effect, but the trend remains that higher optical density results in a better photothermal heating effect. (Fe3(CO)) 12 @Croc-PEG5K exhibits excellent photothermal effects, and the photothermal heating effect can be controlled by adjusting laser parameters, thereby controlling CO release. This provides a favorable prerequisite for the controllable release of CO. Specific test results are as follows... Figure 6 As shown in (b).

[0126] (3) This example also tested the CO release rate of the nanoparticles obtained in Example 1 under in vitro laser irradiation. The specific method was as follows: Fe3(CO) was taken. 12 @Croc-PEG5K aqueous solution (30 μM), hemoglobin (Hb, 5 μM) and sodium dithionite (SDT, 1.6 mg) were added, the total volume of the mixed solution was 1.0 mL, and argon gas was continuously purged to remove air, yielding Fe3(CO). 12 A mixed solution of @Croc-PEG5K and reduced Hb was analyzed using an 808nm laser at different optical densities (0.0–1.0 W / cm²). 2 Irradiation was performed, and the UV-Vis absorption spectra of the mixed solution at different time points and the absorbance values ​​at 420 nm and 432 nm were measured (Abs). 420 and Abs 432 The CO release at different time points is calculated using the following formula.

[0127]

[0128] In the above formula, C co The concentration of CO released; C Hb The concentration of Hb; Abs 420 Abs represents the absorbance of the mixed solution at 420 nm. 432 The absorbance of the mixed solution at 432 nm is given.

[0129] The mechanism of the above testing method is as follows: the characteristic absorption peak of reduced Hb is at 432 nm. After Hb binds with CO, it becomes carboxyhemoglobin (COHb), and its characteristic absorption peak blue-shifts to 420 nm. As CO is released, reduced Hb is gradually converted into COHb, the absorbance at 432 nm gradually decreases, and the absorbance at 420 nm gradually increases.

[0130] The results show that within the same time frame, the higher the laser light density, the faster the CO release rate and the greater the CO release amount. However, it is worth noting that as time continues indefinitely, the CO release amount will tend to converge to a uniform level, because the maximum CO release amount depends on Fe3(CO). 12 The concentration of @Croc-PEG5K can be controlled, but the laser can control the CO release rate, thus allowing control over the amount of CO released over a period of time. Specific test results are as follows... Figure 7 As shown in (a) and (b), Figure 7 In (a), the optical density is 0.6 W / cm². 2 .

[0131] To further verify the controllability of the CO release rate, the laser was alternately turned on and off, and the CO release amount was measured at different time points. The results showed that the CO release rate significantly increased when the laser was on and significantly decreased when the laser was off, further demonstrating the controllability of the photothermal effect on CO release. Specific test results are as follows... Figure 7 As shown in (c).

[0132] (4) This example also explores the controlled release of CO by free radicals. The specific test method is as follows: Fe3(CO) was taken. 12 @Croc-PEG5K solution (30 μM), hemoglobin (Hb, 5 μM) and sodium dithionite (SDT, 1.6 mg) were added, and the total volume of the mixed solution was 1.0 mL. Argon gas was continuously purged to remove air, yielding Fe3(CO). 12 A mixed solution of @Croc-PEG5K and reduced Hb was prepared by adding different concentrations of hydrogen peroxide (H2O2, 0.0–5.0 μM, without laser irradiation). The UV-Vis absorption spectra of the mixed solution and the absorbance values ​​at 420 nm and 432 nm were measured at different time points. 420 and Abs 432 ), and calculate the CO release at different time points according to Formula 1.

[0133] The results showed that the effect of H2O2 concentration on CO release followed a similar trend to that of Example 6, namely: the higher the H2O2 concentration, the faster the CO release rate, and the greater the amount of CO released in the same amount of time. It is also worth noting that as time continues indefinitely, the amount of CO released will tend to be uniform, because at the same concentration of Fe3(CO)... 12 The total amount of CO that can be released in the end is consistent with that of @Croc-PEG5K. Specific test results are as follows: Figure 8 As shown in (a) and (b), Figure 8 In (a), the H2O2 concentration is 5.0 μM.

[0134] Based on the above test results, the working mechanism of the nanoparticles provided by this invention is as follows: Fe3(CO) 12 As a stable source of CO, loading it into Croc-PEG5K is equivalent to forming a compressed gas, thereby constructing smart-response gas-to-gas (GGNs). Fe3(CO) 12 @Croc-PEG5K enables controlled CO release. The photothermal effect of Croc-PEG5K allows for NIR laser-regulated CO release, while free radicals also promote CO release. NIR is an exogenous stimulus, and free radicals are an endogenous stimulus; therefore, the Fe3(CO) design of this invention... 12 @Croc-PEG5K can achieve both extrinsic and intrinsic CO release. The specific mechanism is as follows: Figure 1 As shown.

[0135] Since the nanoparticles provided by this invention can safely and controllably release carbon monoxide, they are expected to have wide applications in the preparation of drugs for treating cardiovascular diseases, sepsis, shock, acute lung, kidney and liver injuries, microbial infections and cancer.

[0136] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nanoparticle capable of controllably releasing carbon monoxide, characterized in that, The nanoparticles comprise dodecyltriferric iron and Croc-PEG coated with the dodecyltriferric iron, wherein the Croc-PEG is a condensation product of Croc and MPEG-NH2; the molecular formula of the Croc is: 。 2. The nanoparticles according to claim 1, characterized in that, The molar ratio of the dodecyltriferric oxide to the Croc-PEG is 5~6:1; and / or the molecular weight of the MPEG-NH2 is 2k~5k.

3. The nanoparticles according to claim 1 or 2, characterized in that, The nanoparticles have a near-spherical structure; and / or, the average size of the nanoparticles is 80~110 nm.

4. A method for preparing nanoparticles as described in any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: S1. Disperse the dodecyltriferric oxide in THF to obtain dispersion A; The Croc-PEG was dispersed in THF to obtain dispersion B; S2. Add the dispersion A and dispersion B to water and mix; S3. Blow protective gas into the mixture obtained in step S2, and then pass the mixture through a filter membrane to concentrate the filtrate by ultrafiltration.

5. The preparation method according to claim 4, characterized in that, In step S1, the method for synthesizing Croc-PEG includes the following steps: D1. The reaction product of 4-carboxyphenylhydrazine hydrochloride and 3-methyl-2-butanone is reacted with a keto acid to obtain the Croc; D2. Cause the Croc and MPEG-NH2 to undergo an amide condensation reaction.

6. The preparation method according to claim 4, characterized in that, In step S1, the concentration of dispersion A is 3.5~4.5 mmol / mL; and / or, the concentration of dispersion B is 0.5~1 mmol / mL.

7. The preparation method according to claim 4, characterized in that, In step S2, the volume ratio of the dispersion A to the water is 1:8~9.

8. The preparation method according to claim 4, characterized in that, In step S2, the mixing time is 25~35 minutes.

9. The preparation method according to claim 4, characterized in that, In step S3, the pore size of the filter membrane is 0.22 μm; and / or, in step S3, the molecular cutoff weight of the ultrafiltration centrifuge tube used for ultrafiltration concentration is 10 kilodaltons.

10. The use of the nanoparticles as described in any one of claims 1 to 3 in the preparation of drugs for treating cardiovascular diseases, sepsis, shock, acute lung, kidney and liver injuries, microbial infections, arthritis and cancer.

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