A carrier, targeted nanoparticle and method of preparation and use thereof

Abstract

The application belongs to the technical field of targeted drugs, and discloses a carrier, a targeted nanoparticle and a preparation method and application thereof. The carrier disclosed by the application comprises darpin grafted with a dye containing a terminal carboxyl group. The targeted nanoparticle comprises the carrier and a CO releasing molecule, and the carrier coats the CO releasing molecule to form a core-shell structure. The targeted nanoparticle is based on darpin, the grafting of the dye containing the terminal carboxyl group is performed to construct the carrier, and then the carrier coats the CO releasing molecule, wherein the carrier has a targeting effect, and the photothermal effect of the carrier can realize the NIR laser controlled CO releasing or free radical stimulation, and can also target the controllable CO releasing, so that the double response releasing of exogenous CO and endogenous CO is realized, and the application has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of targeted drugs, and particularly relates to a carrier, a targeted nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] CO is a gaseous messenger with abundant biological effects, and thus has therapeutic potential for many diseases. As a bioactive molecule, CO has a regulatory role in many physiological and pathological aspects, which is mainly because CO can bind to molecules such as Hb (hemoglobin) and respiratory chain complexes, thereby affecting their functions and triggering a cascade effect. The CO-regulated vascular tension effect can activate blood and remove blood stasis, the antioxidant effect can reduce oxidative stress and relieve inflammation, the anti-apoptotic effect can inhibit apoptosis of the mitochondrial pathway, and the pro-repair effect can promote tissue regeneration. Therefore, CO is an ideal disease treatment active agent.

[0003] Carbon monoxide (CO) gas therapy is an emerging medical technology that has been gradually applied to the treatment of various diseases, such as cardiovascular diseases, sepsis, shock, acute lung, kidney and liver injury, microbial infection and cancer. CO has anti-inflammatory effects and promotes tissue repair, and thus has theoretical therapeutic potential for RA (rheumatoid arthritis).

[0004] CO-releasing molecules (CORMs) refer to substances that can release CO gas, which can release CO gas in vivo or in vitro as a gas cylinder. Common CORMs include metal porphyrin complexes, organic compounds and mononuclear iron porphyrin compounds.

[0005] The reasons why CO and CO-releasing molecules (CORMs) have not been widely used include poor water solubility of CORMs and uncontrollable diffusion and diffusion of CO. RA is clinically manifested as invasive inflammation and cartilage damage in multiple joints such as hands and feet. In order to apply CO gas therapy to RA treatment, the targeting problem needs to be solved first, that is, to control the release of CO at the lesion site and avoid causing systemic poisoning. It is also important to evaluate the treatment effect in real time, and thus avoid under-treatment or over-treatment.

[0006] In addition, gaseous CO is difficult to store and transport, and its diffusion makes its concentration and location in the body uncontrollable, which can easily cause ineffective or poisoning risk. Therefore, CO gas therapy must rely on a specific platform to achieve precise release on demand.

[0007] Metal carbonyls are coordination complexes of transition metals (nickel, cobalt, ruthenium, vanadium, chromium, manganese and iron) with CO, which are CO reservoirs that can release CO under certain conditions (light, heat, pH and magnetism, etc.). Considering the safety of metal metabolites, manganese carbonyl and iron carbonyl are more suitable for biological applications, and among these compounds, dodecacarbonyltriferiron (Fe3(CO) 12 ) has the highest CO storage capacity and is therefore an ideal CO donor. However, these compounds have poor water solubility, so the transport carrier must solve the problem of biocompatibility, be responsive to external stimuli to trigger the release of CO from the metal carbonyl, and have targeting and diagnostic properties to achieve simultaneous treatment and monitoring to optimize the therapeutic effect. However, the existing carriers or CO drugs often cannot meet these requirements.

[0008] Therefore, there is an urgent need to provide a new drug for targeted control of CO release. SUMMARY

[0009] The present application aims to at least solve one of the above technical problems in the prior art.

[0010] To this end, the present application provides a targeted nanoparticle and a preparation method and application thereof. The targeted nanoparticle of the present application is based on TCZ (tocilizumab), which is grafted with a dye containing a terminal carboxyl group (such as croconic cyanine dye (Croc)) to construct a carrier, and then the carrier is coated with a CO releasing molecule (CORMs), wherein the carrier has a targeting effect and can target and controllably release CO under near-infrared (NIR) laser or free radical stimulation.

[0011] The first aspect of the present application provides a carrier.

[0012] The carrier comprises tocilizumab grafted with a dye containing a terminal carboxyl group.

[0013] Preferably, the dye containing a terminal carboxyl group comprises two or more terminal carboxyl groups.

[0014] Preferably, the dye containing a terminal carboxyl group comprises a croconic cyanine dye.

[0015] Preferably, the carrier is obtained by reacting the carboxyl groups at both ends of the croconic cyanine dye with the free amino groups of the tocilizumab (denoted as Croc-TCZ).

[0016] Preferably, the carrier has IL-6R targeting and photothermal effect.

[0017] The second aspect of the present application provides a targeted nanoparticle.

[0018] Specifically, the targeted nanoparticle comprises a carrier and a CO releasing molecule, and the carrier coats the CO releasing molecule to form a core-shell structure.

[0019] Preferably, the CO releasing molecule comprises a metal carbonyl compound.

[0020] Preferably, the metal carbonyl compound is selected from Fe, Mn, Re, Ru containing metal carbonyl compound; further preferably Fe, Mn containing metal carbonyl compound. Fe, Mn containing metal carbonyl compound has better biocompatibility.

[0021] Preferably, the CO releasing molecule is Fe3(CO) 12 or Mn2(CO) 10 . Fe3(CO) 12 not only has good biocompatibility, but also Fe element is an essential trace element for human body, further, Fe3(CO) 12 has more carbonyl groups, can release more CO under the same molecular quantity, and can improve the drug efficiency. Due to the particularity of the carrier used in the application, the CO releasing molecule of the application can be coated with the carrier through hydrophilic and hydrophobic interaction, therefore, the CO releasing molecule of the application does not need to be further modified with organic or inorganic groups. The drug delivery system in the prior art needs to modify the CO releasing molecule with organic and / or inorganic groups, so that the CO releasing molecule can be loaded on the carrier for targeted drug delivery. Moreover, due to the need to modify the CO releasing molecule with organic and / or inorganic groups in the prior art, the amount of CO provided by the CO releasing molecule per unit molar quantity is significantly reduced, which reduces the drug efficiency.

[0022] Preferably, the size of the targeted nanoparticle is 10-300 nm, further preferably 50-200 nm, and more preferably 100-150 nm.

[0023] Preferably, the mass ratio of the carrier to the CO releasing molecule is (1-32):4, and more preferably (12-32):4.

[0024] Preferably, the chemical formula of the targeted nanoparticle comprises Fe3(CO) 12 @Croc-TCZ, wherein Croc-TCZ represents the carrier. Fe3(CO) 12 @Croc-TCZ can achieve controllable release of CO, the photothermal effect of Croc-TCZ can achieve NIR laser controlled release of CO, and free radicals can also promote the release of CO. NIR is an exogenous stimulus, and free radicals are an endogenous stimulus. Therefore, the Fe3(CO) 12 @Croc-TCZ of the application can achieve exogenous / endogenous dual response release of CO, and has broad application prospects.

[0025] The third aspect of the present application provides a preparation method of a carrier.

[0026] A preparation method of a carrier, comprising the following steps:

[0027] Mixing a dye containing terminal carboxyl group with an imine substance and tocilizumab, and reacting to obtain the carrier.

[0028] Preferably, the preparation method comprises the following steps:

[0029] Mixing a dye containing terminal carboxyl group with an imine substance to obtain an activated dye containing terminal carboxyl group;

[0030] Reacting the activated dye containing terminal carboxyl group with tocilizumab to obtain the carrier.

[0031] Preferably, the imine substance comprises at least one of N-hydroxysulfosuccinimide (Sulfo-NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl), N,N'-dicyclohexyl carbodiimide (DCC), and N,N'-diisopropyl carbodiimide (DIC).

[0032] Preferably, the dye containing terminal carboxyl group is mixed with the imine substance and a solvent.

[0033] Preferably, the solvent is an alcohol solvent, such as anhydrous ethanol or propanol.

[0034] Preferably, the mass ratio of the dye containing terminal carboxyl group to the imine substance is 12:(0.5-2):(0.5-2), and further preferably 12:(0.8-1.5):(0.8-1.5).

[0035] Preferably, the dye containing terminal carboxyl group is mixed with the imine substance and the solvent under a protective atmosphere and in the dark, and is stirred in an ice bath to obtain the activated dye containing terminal carboxyl group.

[0036] Preferably, the mass ratio of the dye containing terminal carboxyl group to the solvent is 12g:(5-30)mL, and further preferably 12g:(8-12)mL.

[0037] Preferably, the protective atmosphere is a noble gas or nitrogen, such as argon.

[0038] Preferably, the ice bath stirring time is 1-5 hours, and further preferably 2-4 hours.

[0039] Preferably, the mass ratio of the activated dye containing terminal carboxyl group to tocilizumab is 0.1-0.5:(0.8-1.5), and more preferably 0.2-0.3:(1.0-1.4).

[0040] Preferably, the concentration of the activated carboxyl-terminated dye and the tocilizumab in the reaction system during the reaction is 200-300 μg / mL: 1.0-1.4 mg / mL, more preferably 200-300 μg / mL: 1.0-1.4 mg / mL.

[0041] Preferably, the reaction is carried out in an ice bath under stirring and light shielding. The carboxyl-terminated dye is connected to the free amino group of the TCZ through condensation reaction of carboxyl and amino.

[0042] Preferably, the reaction time is 10-13 hours, more preferably 11-12 hours.

[0043] Preferably, after the reaction, the mixture formed after the reaction is filtered through a filter membrane to remove excess carboxyl-terminated dye, the mixture is added into a PBS solution, a rotary evaporator is used to remove solvent, and finally the mixture is concentrated using an ultrafiltration centrifuge tube to obtain the carboxyl-terminated dye grafted tocilizumab.

[0044] Preferably, the filtration is carried out through filter membranes with pore sizes of 0.8-0.88 μm, 0.4-0.45 μm and 0.2-0.22 μm in sequence.

[0045] Preferably, the concentration using the ultrafiltration centrifuge tube is carried out at 1-4 ℃, at a rotation speed of 7000-7500 rpm, and for 5-10 minutes.

[0046] Preferably, after the reaction, the mixture formed after the reaction is filtered through filter membranes with pore sizes of 0.8-0.88 μm, 0.4-0.45 μm and 0.2-0.22 μm in sequence to remove excess carboxyl-terminated dye, 4-6 mL of PBS solution is added into the mixture, a rotary evaporator is used to remove solvent, and finally the mixture is concentrated using an ultrafiltration centrifuge tube (4 ℃, 7500 rpm, 10 min) to obtain the carboxyl-terminated dye grafted tocilizumab.

[0047] Preferably, the tocilizumab is added in the form of a tocilizumab solution, and the concentration of the tocilizumab solution is 15-25 mg / mL.

[0048] Preferably, the mass ratio of the carboxyl-terminated dye to the tocilizumab is 12:(0.5-20), more preferably 12:(2-15).

[0049] The fourth aspect of the present application provides a preparation method of a targeted nanoparticle.

[0050] The preparation method of the targeted nanoparticle comprises the following steps:

[0051] The carrier is mixed with the CO releasing molecule to obtain the targeting nanoparticle.

[0052] Preferably, the CO releasing molecule is added in the form of a CO releasing molecule solution to mix with the grafting carboxyl-terminated dye-containing darpavumab.

[0053] Preferably, the CO releasing molecule solution is obtained by mixing 1-8 mg of the CO releasing molecule with an organic solvent. The organic solvent is a common substance, such as tetrahydrofuran.

[0054] Preferably, the CO releasing molecule solution is added dropwise into the grafting carboxyl-terminated dye-containing darpavumab under ultrasonic conditions. After the dropwise addition is completed, the ultrasonic treatment is continued for 10-20 minutes. After the ultrasonic treatment is completed, argon is introduced into the mixture in a fume hood until the organic solvent is completely blown out.

[0055] Preferably, the mixture of the grafting carboxyl-terminated dye-containing darpavumab and the CO releasing molecule is sequentially filtered through 0.8-0.88 μm, 0.4-0.45 μm, and 0.2-0.22 μm filter membranes, and then concentrated by using an 8-10 KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the targeting nanoparticle.

[0056] Preferably, the mass ratio of the carrier to the CO releasing molecule is (1-32) : 4, and more preferably (12-32) : 4.

[0057] The preparation method of the present application is an ultrasonic self-assembly method.

[0058] The fifth aspect of the present application provides an application of the targeting nanoparticle.

[0059] A pharmaceutical composition comprises the above-mentioned targeting nanoparticle.

[0060] The above-mentioned targeting nanoparticle is used in the preparation of a drug for treating rheumatoid arthritis, laser-responsive release, M1 macrophages having targeting properties, and promoting the growth and proliferation of macrophages and / or chondrocytes.

[0061] Specifically, the above-mentioned targeting nanoparticle is used in the preparation of a drug for laser-responsive release of CO.

[0062] Preferably, the wavelength of the laser is 806-808 nm, and more preferably 808 nm. Under 808 nm laser irradiation, Fe3(CO) 12 @Croc-TCZ releases CO, and the amount of CO released is positively correlated with the laser power and the irradiation time. The higher the laser power, the faster the CO release, and the higher the amount of CO released in the same time.

[0063] Specifically, the application of the above-mentioned targeted nanoparticles in the preparation of a drug with targeting effect on M1 type macrophages. Incubate different cells for the same time, and Fe3(CO) 12 @The accumulation amount of Croc-TCZ in M1 type inflammatory macrophages is obviously more than that in hMSC; in M1 type inflammatory macrophages, Fe3(CO) 12 @The accumulation amount of Croc-TCZ is obviously more than that of Fe3(CO) 12 @Croc-PEG5K.

[0064] Specifically, the application of the above-mentioned targeted nanoparticles in the preparation of a drug for promoting the growth and proliferation of macrophages and / or chondrocytes.

[0065] Compared with the prior art, the application has the following beneficial effects:

[0066] (1) The targeted nanoparticles described in the application are based on TCZ (tocilizumab), and a dye containing a terminal carboxyl group (such as croconin cyanine dye (Croc)) is grafted to construct a carrier, and then a CO releasing molecule is coated by the carrier, wherein the carrier has a targeting effect, and the photo-thermal effect of the carrier can achieve NIR laser controlled CO release or free radical stimulation, and can also achieve targeted and controllable release of CO, so as to realize double-response release of CO from exogenous / endogenous sources, and has a wide application prospect.

[0067] (2) The application adopts a carbonyl metal compound Fe3(CO) 12 as a CO donor, and the central metal of Fe3(CO) 12 is Fe, and Fe is a trace element necessary for the human body. In addition, one Fe atom can coordinate 12 CO, and has very strong gas storage capacity.

[0068] (3) The application adopts TCZ as a carrier, which has good biocompatibility and targeting effect, and meanwhile, TCZ can increase the therapeutic effect of CO gas therapy as a clinical drug.

[0069] (4) The application connects an organic functional croconin cyanine dye Croc on TCZ to obtain Croc-TCZ, which has good photo-thermal effect and imaging capacity, and has a great clinical transformation prospect.

[0070] (5) The application of Fe3(CO) 12 and Croc-TCZ to form a targeted nanoparticle Fe3(CO) 12 @Croc-TCZ through simple hydrophobic interaction. The targeted nanoparticle has simple components, a conventional preparation process, good repeatability, high stability, and is suitable for batch and industrial production.

[0071] (6) The application of Fe3(CO) 12Croc-TCZ as a nano gas tank can release CO on demand under the action of NIR laser, which overcomes the technical difficulty of uncontrollable CO concentration caused by diffusion, and provides technical guidance for the popularization of CO gas treatment.

[0072] (7) The Fe3(CO) 12 Croc-TCZ has no obvious cytotoxicity, and has a cell proliferation effect on cells in (non) inflammatory state. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 is a schematic diagram of the inventive concept of the application;

[0074] Figure 2 is a preparation process schematic diagram of Croc-TCZ in embodiment 1 of the application;

[0075] Figure 3 is a targeted nanoparticle Fe3(CO) 12 Croc-TCZ preparation process schematic diagram;

[0076] Figure 4 is a targeted nanoparticle Fe3(CO) 12 TEM, DLS characterization diagram of Croc-TCZ, raw materials, Fe3(CO) 12 Zeta potential, ultraviolet-visible absorption spectrum, absorption light and concentration relationship curve diagram of Croc-TCZ;

[0077] Figure 5 is a targeted nanoparticle Fe3(CO) 12 Croc-TCZ in vitro under different concentrations, different light density light heating curve, and the light heating effect diagram of the mixed solution of reduced Hb;

[0078] Figure 6 is a targeted nanoparticle Fe3(CO) 12 Croc-TCZ has the effect of targeting M1 type inflammatory macrophages;

[0079] Figure 7 is a targeted nanoparticle Fe3(CO) 12 Croc-TCZ cell activity result diagram of macrophages and chondrocytes;

[0080] Figure 8 is a live and dead cell staining diagram of TCZ, Croc-TCZ and chondrocytes on macrophages;

[0081] Figure 9TCZ, Croc-TCZ and targeted nanoparticle Fe3(CO) 12 Live and dead cell staining plot of the effect of Croc-TCZ on chondrocytes. DETAILED DESCRIPTION

[0082] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0083] The raw materials, reagents or devices used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0084] Example 1: Preparation of carrier, targeted nanoparticle

[0085] A targeted nanoparticle, whose chemical formula is Fe3(CO) 12 @Croc-TCZ, wherein Croc-TCZ represents a carrier, and Fe3(CO) 12 forms a core-shell structure.

[0086] A preparation method of a targeted nanoparticle, comprising the following steps:

[0087] Croc (chalcophyllin cyanine dye, 12.0 mg, 24.00 μmol), Sulfo-NHS (N-hydroxysulfosuccinimide) (1.0 mg, 4.61 μmol), EDC-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (1.0 mg, 5.22 μmol) were dissolved in anhydrous ethanol (8 mL) under argon protection and in the dark, and stirred in an ice bath for 4 h to activate the carboxyl groups at both ends of Croc; then 20.0 mg of TCZ (tocilizumab) solution (concentration of 20 mg / mL) was added, and stirred in an ice bath in the dark for 12 h to link Croc to the free amino groups of TCZ through condensation reaction of carboxyl and amino groups. After the reaction was completed, the obtained mixture was sequentially passed through 0.88 μm, 0.45 μm and 0.22 μm filter membranes to remove excess Croc. Then 5 mL of PBS solution was added to the mixture obtained by removing excess Croc, and a rotary evaporator was used to remove ethanol. The mixture obtained by removing ethanol was concentrated using a 10 KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain compound Croc-TCZ (i.e. a carrier was prepared, denoted as Croc-TCZ solution);

[0088] The nanoparticle Fe3(CO) 12 @Croc-TCZ was successfully prepared by ultrasonic self-assembly method. Specifically, Fe3(CO)12 (4.0 mg, 7.9 mmol) was dissolved in THF (2 mL) and added dropwise into the Croc-TCZ solution under ultrasonic condition. The ultrasonic was continued for 15 min. After the ultrasonic was finished, the mixture was purged with nitrogen until the THF was completely blown out in the fume hood. Then the mixture was filtered through 0.88 μm, 0.45 μm and 0.22 μm filter membranes, respectively. The mixture after the filtration was concentrated by using a 10 KD ultrafiltration centrifuge tube (4 °C, 7500 rpm, 10 min) to obtain the targeted nanoparticles Fe3(CO) 12 @Croc-TCZ.

[0089] Example 2: Preparation of the carrier and the targeted nanoparticles

[0090] A targeted nanoparticle has a chemical formula of Fe3(CO) 12 @Croc-TCZ, wherein the Croc-TCZ represents a carrier, and the Fe3(CO) 12 forms a core-shell structure.

[0091] A method for preparing a targeted nanoparticle includes the following steps:

[0092] Croc (Cyanine dye, 12.0 mg, 24.00 μmol), Sulfo-NHS (N-hydroxysulfosuccinimide) (1.0 mg, 4.61 μmol), EDC-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (1.0 mg, 5.22 μmol), and a TCZ (Tocilizumab) solution 20.0 mg (concentration of 20 mg / mL) were dissolved in anhydrous ethanol (8 mL) under argon protection and light shielding, and stirred in an ice bath for 16 h. The Croc was connected to the free amino group of the TCZ by condensation reaction of carboxyl and amino. After the reaction was completed, the obtained mixture was filtered through 0.88 μm, 0.45 μm and 0.22 μm filter membranes, respectively, to remove the excess Croc. Then 4 mL of PBS solution was added to the mixture after the removal of the excess Croc. The ethanol was removed by using a rotary evaporator. The mixture after the removal of the ethanol was concentrated by using a 10 KD ultrafiltration centrifuge tube (4 °C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e. the carrier was prepared).

[0093] The nanoparticles Fe3(CO) 12 @Croc-TCZ were successfully prepared by the ultrasonic self-assembly method. Specifically, Fe3(CO) 12(4.0 mg, 7.9 mmol) was dissolved in THF (2 mL) and added dropwise into the Croc-TCZ solution under ultrasonic condition. The ultrasonic was continued for 15 min. After the ultrasonic was stopped, the mixture was purged with argon until the THF was completely blown out. Then the mixture was sequentially filtered through 0.88 μm, 0.45 μm and 0.22 μm filter membranes. The mixture obtained after the filtration was concentrated by using a 10 KD ultrafiltration centrifuge tube (4 °C, 7500 rpm, 10 min) to obtain the targeted nanoparticles Fe3(CO) 12 @Croc-TCZ.

[0094] Example 3: Preparation of the carrier and the targeted nanoparticles

[0095] A targeted nanoparticle has a chemical formula of Fe3(CO) 12 @Croc-TCZ, wherein Croc-TCZ represents a carrier, and Fe3(CO) 12 forms a core-shell structure.

[0096] A method for preparing a targeted nanoparticle includes the following steps:

[0097] Croc (Cyanine dye, 12.0 mg, 24.00 μmol), Sulfo-NHS (N-hydroxysulfosuccinimide) (1.0 mg, 4.61 μmol) and EDC-HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (1.0 mg, 5.22 μmol) were dissolved in anhydrous ethanol (8 mL) under argon protection and ice bath stirring for 4 h to activate the carboxyl groups at both ends of Croc. Then, a TCZ (Tocilizumab) solution (20.0 mg, 20 mg / mL) was added and stirred for 12 h under ice bath and light protection. The Croc was connected to the free amino group of TCZ through condensation reaction of carboxyl and amino groups. After the reaction was completed, the obtained mixture was sequentially filtered through 0.88 μm, 0.45 μm and 0.22 μm filter membranes to remove excess Croc. Then, 6 mL of PBS solution was added to the mixture obtained after the removal of excess Croc. The ethanol was removed by using a rotary evaporator. The mixture obtained after the removal of ethanol was concentrated by using a 10 KD ultrafiltration centrifuge tube (4 °C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e. the carrier was prepared);

[0098] The nanoparticles Fe3(CO) 12 @Croc-TCZ were successfully prepared by ultrasonic self-assembly method. Specifically, Fe3(CO) 12(4.0 mg, 7.9 mmol) was dissolved in THF (2 mL) and added dropwise into the Croc-TCZ solution under ultrasonic condition. The ultrasonic was continued for 15 min. After the ultrasonic was stopped, the mixture was purged with argon until the THF was completely blown out. Then the mixture was passed through 0.88 μm, 0.45 μm, and 0.22 μm filter membranes in sequence. The mixture after passing through the filter membranes was concentrated by using a 10 KD ultrafiltration centrifuge tube (4 °C, 7500 rpm, 10 min). The lower liquid after concentration was passed through 0.88 μm, 0.45 μm, and 0.22 μm filter membranes again to obtain the targeted nanoparticles Fe3(CO) 12 @Croc-TCZ.

[0099] Product effect test

[0100] After the successful preparation of the targeted nanoparticles Fe3(CO) 12 @Croc-TCZ, the photothermal effect of Fe3(CO) 12 @Croc-TCZ and the stimulation release of CO by NIR laser were explored.

[0101] Fe3(CO) 12 @Croc-TCZ solution (20 μM, 1.0 mL) was irradiated by 808 nm laser at different light densities (0.0-2.0 W / cm 2 ) for 10 min to obtain the photothermal heating curves of Fe3(CO) 12 @Croc-TCZ solution under different light density conditions. Similarly, Fe3(CO) 2 @Croc-TCZ solution (0-30 μM) was irradiated by 808 nm laser at a fixed light density of 1.0 W / cm 12 ) to obtain the photothermal heating curves of Fe3(CO) 12 @Croc-TCZ solution under different concentration conditions.

[0102] Then the stimulation release of CO by the photothermal effect was explored. Fe3(CO) 12 @Croc-TCZ solution (0-15 μM) was added with hemoglobin (Hb, 5 μM) and sodium dithionite (SDT, 1.6 mg) to obtain a mixed solution of Fe3(CO) 12 @Croc-TCZ and reduced Hb with a total volume of 1.0 mL. The mixed solution was irradiated by 808 nm laser at the same light density (1.0 W / cm 2 ) for 60 min. The ultraviolet-visible absorption spectrum of the mixed solution at different time points and the absorbance values at 420 nm and 432 nm (denoted as Abs 420 and Abs 432The CO release at different time points is calculated using the following formula.

[0103]

[0104] In the 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.

[0105] This invention also explored the targeting properties of the targeted nanoparticles. Two groups of macrophages (RAW264.7) and one group of human mesenchymal stem cells (hMSCs) were cultured. The two groups of RAW264.7 cells were first incubated with LPS (bacterial lipopolysaccharide) for 24 hours to induce inflammatory cells, and then two different nanoparticles, Fe3(CO), were used respectively. 12 @Croc-PEG5K(20μM, Fe3(CO)) 12 The preparation process of @Croc-PEG5K differs from that of Example 1 in that TCZ in Example 1 is replaced with PEG5K, and the step of activating the carboxyl group with N-hydroxythiosuccinimide and EDC-HCl is omitted. That is, Croc-TCZ is prepared first, and then Fe3(CO) is obtained. 12 @Croc-PEG5K) and Fe3(CO) 12 Incubation was performed using @Croc-TCZ (20 μM), while hMSCs were incubated directly using Fe3(CO). 12 Cells were incubated with @Croc-TCZ (20 μM) and fixed 2-6 h after incubation with nanoparticles. The changes in fluorescence intensity of intracellular nanoparticles over time and the differences between groups were observed using confocal microscopy.

[0106] This invention further investigated the cell proliferation-promoting effect of nanoparticles. Two groups of macrophages and two groups of chondrocytes were cultured and incubated for 24 hours with or without LPS. Different concentrations (0-20 μM) of TCZ, Croc-TCZ, and Fe3(CO) were then used. 12 Cells were incubated with @Croc-TCZ for 24 hours, and cell viability was assessed using a CCK-8 (cell counting reagent) kit. Similarly, cells from the above groups were cultured in laser confocal culture dishes and incubated with ±LPS for 24 hours, followed by incubation with the same concentration (10 μM) of TCZ, Croc-TCZ, and Fe3(CO). 12 Incubate with @Croc-TCZ, and after 24 hours perform live / dead staining and observe using a confocal microscope.

[0107] Figure 1 This is a schematic diagram of the inventive concept of the present invention; from Figure 1 It can be seen that the targeted nanoparticle design concept of the present invention is composed of dye, CO-releasing molecules (CORMs), and tocilizumab (TCZ), and its mechanism of action in treating diseases is given.

[0108] Figure 2 This is a schematic diagram of the preparation process of Croc-TCZ in Embodiment 1 of the present invention;

[0109] Figure 3 The targeted nanoparticles Fe3(CO) in Example 1 of this invention 12 Schematic diagram of the preparation process of Croc-TCZ;

[0110] Figure 4 This refers to Example 1, which targets Fe3(CO) nanoparticles. 12 TEM and DLS characterization images of Croc-TCZ, as well as the raw materials and Fe3(CO) 12 Zeta potential, UV-Vis absorption spectrum, and absorption light versus concentration curves of Croc-TCZ; Figure 4 Figure a in the diagram shows the Fe3(CO) nanoparticles from Example 1. 12 TEM characterization of Croc-TCZ; Figure 4 Figure b in the diagram shows the targeted Fe3(CO) nanoparticles in Example 1. 12 DLS characterization diagram of Croc-TCZ; Figure 4 Figure c in the diagram represents the CO donor Fe3(CO) in Example 1. 12 Near-infrared ketone cyanine dyes Croc and TCZ, intermediate product Croc-TCZ, and targeted nanoparticles Fe3(CO) 12 Croc-TCZ Zeta potential diagram; Figure 4 The d-graph in the figure represents the CO donor Fe3(CO) in Example 1. 12 Near-infrared ketone cyanine dye Croc, carrier Croc-TCZ, and targeted nanoparticles Fe3(CO) 12 Ultraviolet-Vis absorption spectrum characterization of Croc-TCZ; Figure 4 Figure e in the diagram represents the targeted Fe3(CO) nanoparticles in Example 1. 12 The standard curve of Croc-TCZ.

[0111] Figure 5 The targeted nanoparticles Fe3(CO) in Example 1 of this invention 12 Photothermal heating curves of Croc-TCZ at different concentrations and optical densities in vitro, and photothermal effect diagram of mixed solution with reduced Hb; Figure 5Figure a in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO) Figure 5 Figure c in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO) Figure 5 Figure c in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO)

[0112] Figure 6 Figure a in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO)

[0113] Figure 7 Figure a in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO) Figure 7 Figure a in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO) Figure 7 Figure a in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO)

[0114] Figure 8 Figure a in the figure is a targeted nanoparticle Fe3(CO)

[0115] Figure 9 Figure a in the figure is a targeted nanoparticle Fe3(CO) 12 Figure b in the figure is a targeted nanoparticle Fe3(CO)

[0116] Effect 1

[0117] From Figure 4 It can be seen that Fe3(CO) 12 The nanostructure of @Croc-TCZ is characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), Fe3(CO) 12 @Croc-TCZ presents a uniform spherical structure, with an average particle size of about 150 nm, and a black center in the middle Fe3(CO) 12, the outer coating material is Croc-TCZ, and the Fe3(CO) 12 @structure and preparation principle of Croc-TCZ, i.e. self-assembly induced by hydrophilic and hydrophobic interaction to form core-shell structure. Fe3(CO) 12 @The hydrated particle size of Croc-TCZ is about 150 nm, which is consistent with the TEM particle size.

[0118] The surface charge of nanoparticles was characterized by measuring the Zeta potential. The results showed that TCZ (0.733 mV) obtained Croc-TCZ (-2.99 mV) after connecting Croc (-23.9 mV) and coated Fe3(CO) 12 (-28.9 mV) obtained nanoparticles Fe3(CO) 12 @Croc-TCZ (-4.81 mV), and the Zeta potential of all of them was decreased. Fe3(CO) 12 @Croc-TCZ was characterized by UV-Vis absorption spectrum. Fe3(CO) 12 @Croc-TCZ has both Fe3(CO) 12 and the UV absorption characteristic peak of Croc-TCZ, indicating the successful assembly and combination of the two.

[0119] Since the mass ratio of Fe3(CO) 12 and Croc-TCZ remains unchanged when preparing Fe3(CO) 12 Croc-TCZ, the concentration of Fe3(CO) 12 @Croc-TCZ can be quantified according to the characteristic absorption peak of Croc-TCZ, with concentration as the abscissa and Abs 772 as the ordinate. Their linear relationship is Y = 0.09137X + 0.05042.

[0120] Effect 2

[0121] From Figure 5 it can be seen that when the concentration of Fe3(CO) 12 @Croc-TCZ is 20 μM, the higher the optical density of 808 nm laser (0-2.0 W / cm 2 ), the better the photothermal heating effect. Fe3(CO) 12 @Croc-TCZ. The photothermal effect of Fe3(CO) 12 @Croc-TCZ comes from near-infrared croconic cyanine dye Croc, which has strong absorption in the near-infrared region and therefore has significant photothermal heating effect. Fe3(CO) 12 @Croc-TCZ heating effect changes with the optical density of laser, indicating that the appropriate temperature can be selected by adjusting the concentration and laser parameters, which embodies Fe3(CO) 12The photothermal tunability of Croc-TCZ. Similarly, the optical density was fixed at 1.0 W / cm 2 Fe3(CO) 12 Croc-TCZ solutions all showed good photothermal effects, and the warming effect was proportional to the concentration of nanoparticles. Fe3(CO) 12 Croc-TCZ has good photothermal effect, which can control the photothermal warming effect by controlling the laser parameters and nanoparticle concentration, and then control the release of CO, which provides a good prerequisite for the controllable release of CO.

[0122] The optical density was 1.0 W / cm 2 Fe3(CO) 12 The CO release amount of Croc-TCZ changes with concentration and time. The results show that the CO release amount in the same time is proportional to the concentration of Fe3(CO) 12 Croc-TCZ concentration; the growth rate of CO release amount shows a similar law, that is, the greater the concentration, the greater the curve slope in the first 20 minutes, which indicates that under the same laser irradiation conditions, the greater the total amount of releasable CO in the solution, the faster the release. At the same time, it can be observed that with the extension of time, the release speed of CO decreases, and the curve tends to be flat, which shows that under the laser conditions, the nanoparticles can quickly respond in a short time and promote the release of CO through photothermal effect.

[0123] Effect 3

[0124] RAW264.7 macrophages were inoculated into 4 laser confocal culture dishes, hMSCs were inoculated into 2 culture dishes, and incubated at 37℃ for 24h, the culture medium was replaced and LPS (1 μg / ml) was added to the RAW264.7 cell dish to stimulate and induce M1 type inflammatory macrophages, and incubated at 37℃ for 24h. Two RAW264.7 cell culture dishes were replaced with culture medium containing Fe3(CO) 12 Croc-PEG5K (20 μM), and the remaining 2 RAW264.7 cell culture dishes and 2 hMSC cell culture dishes were replaced with culture medium containing Fe3(CO) 12 Croc-TCZ (20 μM), and after 3h and 6h of incubation, cell fixation and nuclear staining were performed using DAPI (4', 6-diamidino-2-phenylindole), and after PBS solution washing, laser confocal fluorescence microscope imaging was performed to obtain nanoparticle targeted M1 type inflammatory macrophage effect diagram. From Figure 6 It can be seen that in the same cells (M1), after the same concentration and incubation time treatment, Fe3(CO) 12Croc-TCZ targeted nanoparticles have higher fluorescence intensity in cells than Fe3(CO) 12 Croc-PEG5K; in different cells, Fe3(CO) 12 Croc-TCZ targeted nanoparticles have higher fluorescence intensity in M1 macrophages than in hMSCs. This indicates that Fe3(CO) 12 Croc-TCZ has the ability to target M1 inflammatory macrophages.

[0125] Effect 4

[0126] RAW264.7 macrophage / chondrocyte cells were seeded in 6 96-well plates and incubated at 37°C for 24 h. The culture medium was replaced and LPS (1 μg / ml) was added to induce stimulation in 3 of the 96-well plates. After incubation at 37°C for 24 h, the culture medium was replaced with PBS solution, and then the culture medium was replaced again after washing with PBS solution. The culture medium containing different concentrations of TCZ, Croc-TCZ, and Fe3(CO) 12 Croc-TCZ (0-20 μM) was incubated at 37°C for 24 h. The culture medium was replaced with PBS solution, and then the culture medium was replaced again after washing with PBS solution. The culture medium containing CCK-8 (10%) was incubated at 37°C for 1 h. The cell survival rate was determined by an enzyme marker instrument. From Figure 7 It can be seen that Fe3(CO) 12 Croc-TCZ has the effect of promoting the growth and proliferation of macrophages and chondrocytes.

[0127] Effect 5

[0128] RAW264.7 macrophage / chondrocyte cells were seeded in 8 glass-bottom laser confocal culture dishes and incubated at 37°C for 24 h. The culture medium was replaced and LPS (1 μg / ml) was added to induce stimulation in 4 of the dishes. The dishes were divided into ± LPS groups and incubated at 37°C for 24 h. The culture medium was replaced with normal culture medium (negative and positive controls) and culture medium containing TCZ, Croc-TCZ, and Fe3(CO) 12 Croc-TCZ (10 μM, TCZ group protein concentration consistent with the latter two groups) was incubated at 37°C for 24 h. The culture medium was replaced with PBS buffer solution, and then the culture medium was replaced again after washing with PBS solution. The cells were stained with Calcein-AM and PI, and then washed with PBS solution. Laser confocal fluorescence microscopy imaging was performed to obtain the dead and live staining images. The results are shown in Figure 8 and Figure 9 It can be seen from Figure 8 and Figure 9 that Fe3(CO) 12 Croc-TCZ has no obvious toxicity to macrophages and chondrocytes and does not increase the number of dead cells.

[0129] The Fe3(CO) 12 @Croc-TCZ of Example 2 has similar effect data as Example 1. Furthermore, within the scope of the present application, adjusting the process parameters in the preparation of the carrier, the targeted nanoparticles, such as the amount of raw materials, the targeted nanoparticles prepared also have similar effect of Fe3(CO) 12 @Croc-TCZ of Example 1. Moreover, within the scope of the present application, adjusting the process parameters in the preparation of the carrier, the targeted nanoparticles, such as the amount of raw materials, the targeted nanoparticles prepared also have similar effect of Fe3(CO) 12 @Croc-TCZ of Example 1. Moreover, within the scope of the present application, adjusting the process parameters in the preparation of the carrier, the targeted nanoparticles, such as the amount of raw materials, the targeted nanoparticles prepared also have similar effect of Fe3(CO)

Claims

1. A targeted nanoparticle, characterized in that, The targeting nanoparticle comprises a carrier and a CO releasing molecule, the carrier comprises darpomab grafted with croconic cyanine dye, the CO releasing molecule is selected from Fe, Mn containing metal carbonyl compound, and the carrier coats the CO releasing molecule to form a core-shell structure; wherein the carrier is obtained by reaction of carboxyl at both ends of croconic cyanine dye with free amino group of darpomab; the reaction for preparing the carrier comprises the following steps: mixing the dye containing terminal carboxyl with imine substance and darpomab, and reacting to obtain the carrier.

2. The targeted nanoparticle of claim 1, wherein, The CO releasing molecule is Fe3(CO) 12 or Mn2(CO) 10 .

3. The targeting nanoparticle according to any one of claims 1-2, wherein, The mass ratio of the carrier to the CO releasing molecule is (1-32):

4.

4. The method of producing a targeted nanoparticle according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Mixing the carrier with the CO releasing molecule to obtain the targeting nanoparticle.

5. A pharmaceutical composition, characterized by, The targeting nanoparticle of any one of claims 1-2.

6. Use of the targeting nanoparticle of any one of claims 1-2 in the preparation of a medicament for treating rheumatoid arthritis.

Images