Carrier, targeting nanoparticle and preparation method and application thereof

Abstract

The invention belongs to the technical field of targeted drugs, and discloses a carrier, a targeted nano-particle and a preparation method and application of the targeted nano-particle. The carrier disclosed by the invention comprises tolbuzumab grafted with a dye containing a terminal carboxyl group. The targeting nanoparticle comprises a carrier and CO release molecules, and the carrier coats the CO release molecules to form a core-shell structure. According to the targeted nano-particle, a carrier is constructed by grafting a dye containing a terminal carboxyl group on the basis of tolbuzumab, then CO release molecules are coated by the carrier, the carrier has a targeting effect, and CO release regulated by NIR laser or CO release in a targeted and controllable manner under free radical stimulation can be realized by virtue of the photothermal effect of the carrier, so that CO release in an exogenous / endogenous dual response manner is realized, and CO release in a targeted and controllable manner is realized. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted drugs, and in particular relates to a carrier, targeted nanoparticles, and a preparation method and application thereof. Background Art

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

[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 disease, sepsis, shock, acute lung, kidney and liver damage, microbial infection and cancer. CO has anti-inflammatory effects and promotes tissue repair. Therefore, it has theoretical therapeutic potential for RA (rheumatoid arthritis).

[0004] CO-releasing molecules (CORMs) refer to substances that can release CO gas. They can be used as gas cylinders to release CO gas inside or outside the body. 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 the poor water solubility of CORMs and the diffusivity and uncontrollability of CO. The clinical manifestations of RA are invasive inflammation and cartilage damage in multiple joints such as hands and feet. If CO gas therapy is to be applied to the treatment of RA, the first thing to do is to solve the problem of targeting and control the release of CO at the lesion site to avoid systemic poisoning. At the same time, it is also important to evaluate the treatment effect in real time to avoid under-treatment or over-treatment.

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

[0007] Metal carbonyls are complexes formed by transition metals (nickel, cobalt, ruthenium, vanadium, chromium, manganese and iron) and CO. They are CO storage tanks that can release CO under specific conditions (light, heat, pH and magnetism, etc.). Considering the safety of metal metabolites, carbonyl manganese and carbonyl iron are more suitable for biological applications. Among these compounds, triiron dodecacarbonyl (Fe 3 (CO) 12 ) has the highest gas 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 and be responsive to external stimuli to trigger the release of CO from the carbonyl metal. In addition, it must also be targeted and diagnostic, so that treatment and monitoring can be carried out simultaneously to optimize the therapeutic effect. However, the carriers or CO drugs in the prior art often fail to meet these requirements.

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

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

[0010] To this end, the present invention proposes a targeted nanoparticle and a preparation method and application thereof. The targeted nanoparticle of the present invention is based on TCZ (tocilizumab), and a dye containing a terminal carboxyl group (such as crocinyl cyanine dye (Croc)) is grafted to construct a carrier, and then the carrier is coated with CO releasing molecules (CORMs), wherein the carrier has a targeting effect and can release CO in a targeted and controllable manner under the stimulation of near infrared (NIR) laser or free radicals.

[0011] A first aspect of the present invention provides a vector.

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

[0013] Preferably, the dye containing terminal carboxyl groups includes more than two terminal carboxyl groups.

[0014] Preferably, the terminal carboxyl group-containing dye comprises cremonate cyanine dye.

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

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

[0017] A second aspect of the present invention provides a targeting nanoparticle.

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

[0019] Preferably, the CO-releasing molecules comprise metal carbonyl compounds.

[0020] Preferably, the metal carbonyl compound is selected from metal carbonyl compounds containing Fe, Mn, Re, and Ru, and more preferably metal carbonyl compounds of Fe and Mn. Metal carbonyl compounds of Fe and Mn have better biocompatibility.

[0021] Preferably, the CO releasing molecule is Fe 3 (CO) 12 or Mn 2 (CO) 10 .Fe 3 (CO) 12 Not only does it have good biocompatibility, but Fe is also an essential trace element for the human body. 3 (CO) 12 With more carbonyl groups, more CO can be released under the same number of molecules, and the drug delivery efficiency can also be improved. Due to the particularity of the carrier used in the present invention, the CO releasing molecules of the present invention can be coated with the carrier through hydrophilic and hydrophobic interactions. Therefore, the present invention does not need to further modify the CO releasing molecules with organic or inorganic groups. However, the drug delivery system in the prior art requires the CO releasing molecules to be modified with organic and / or inorganic groups so that the CO releasing molecules can be loaded on the carrier for targeted drug delivery, and since the CO releasing molecules need to be modified with organic and / or inorganic groups in the prior art, the amount of CO provided by the unit molar amount of the CO releasing molecules is significantly reduced, which reduces the drug delivery efficiency.

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

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

[0024] Preferably, the chemical formula of the targeting nanoparticle includes Fe 3 (CO) 12 @Croc-TCZ, where Croc-TCZ represents a carrier. Fe 3 (CO) 12@Croc-TCZ can realize the controlled release of CO. The photothermal effect of Croc-TCZ can realize the CO release regulated by NIR laser. Meanwhile, free radicals can also promote the release of CO. NIR is an exogenous stimulus, while free radicals are an endogenous stimulus. 3 (CO) 12 @Croc-TCZ can achieve exogenous / endogenous dual response to release CO and has broad application prospects.

[0025] The third aspect of the present invention provides a method for preparing a carrier.

[0026] A method for preparing a carrier comprises the following steps:

[0027] The dye containing a terminal carboxyl group is mixed with an imine substance and tocilizumab to react and obtain the carrier.

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

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

[0030] The activated terminal carboxyl-containing dye is reacted with tocilizumab to obtain the carrier.

[0031] Preferably, the imide substance includes at least one of N-hydroxysulfosuccinimide (Sulfo-NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl), N,N'-dicyclohexylcarbodiimide (DCC), and N,N'-diisopropylcarbodiimide (DIC).

[0032] Preferably, the dye containing terminal carboxyl groups is mixed with an 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 terminal carboxyl group-containing dye to the imide substance is 12:(0.5-2):(0.5-2), and more preferably 12:(0.8-1.5):(0.8-1.5).

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

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

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

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

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

[0040] Preferably, during the reaction, the concentrations of the activated terminal carboxyl-containing dye and tocilizumab in the reaction system are 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 under the condition of stirring in an ice bath and in the dark. The dye containing a terminal carboxyl group is connected to the free amino group of TCZ through the condensation reaction of the carboxyl group and the amino group.

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

[0043] Preferably, after the reaction is completed, the mixed solution formed after the reaction is filtered through a filter membrane to remove excess terminal carboxyl-containing dye, PBS solution (phosphate buffer solution) is added to the mixed solution, the solvent is removed using a rotary evaporator, and finally the mixed solution is concentrated using an ultrafiltration centrifuge tube to obtain tocilizumab grafted with a terminal carboxyl-containing dye.

[0044] Preferably, the filtration is performed sequentially through filter membranes of 0.8-0.88 μm, 0.4-0.45 μm, and 0.2-0.22 μm.

[0045] Preferably, the conditions for concentration in the ultrafiltration centrifuge tube are 1-4°C, rotation speed 7000-7500 rpm, and centrifugation for 5-10 minutes.

[0046] Preferably, after the reaction is completed, the mixed solution formed after the reaction is filtered through 0.8-0.88 μm, 0.4-0.45 μm, and 0.2-0.22 μm filter membranes in sequence to remove excess carboxyl-terminated dye, 4-6 mL PBS solution is added to the mixed solution, and the solvent is removed using a rotary evaporator. Finally, the mixed solution is concentrated using an 8-10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain tocilizumab grafted with a carboxyl-terminated dye.

[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 terminal carboxyl-containing dye to tocilizumab is 12:(0.5-20), more preferably 12:(2-15).

[0049] The fourth aspect of the present invention provides a method for preparing targeted nanoparticles.

[0050] A method for preparing targeted nanoparticles comprises the following steps:

[0051] The carrier is mixed with CO-releasing molecules to prepare the targeted nanoparticles.

[0052] Preferably, the CO releasing molecule is added in the form of a CO releasing molecule solution and mixed with the tocilizumab grafted with a dye containing a terminal carboxyl group.

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

[0054] Preferably, the CO releasing molecule solution is added dropwise to the tocilizumab grafted with a terminal carboxyl dye under ultrasonic conditions. After the addition is completed, ultrasonication is continued for 10-20 minutes. After the ultrasonication is completed, argon gas is introduced into the mixture in a fume hood until the organic solvent is completely blown out.

[0055] Preferably, after the tocilizumab grafted with a dye containing a terminal carboxyl group is mixed with the CO releasing molecule, the formed mixture is filtered through 0.8-0.88 μm, 0.4-0.45 μm, and 0.2-0.22 μm filter membranes in sequence, and then concentrated using an 8-10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the targeted nanoparticles.

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

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

[0058] The fifth aspect of the present invention provides a use of targeted nanoparticles.

[0059] A pharmaceutical composition comprises the above-mentioned targeted nanoparticles.

[0060] The targeted nanoparticles are used in the preparation of drugs for treating rheumatoid arthritis, laser-responsive release, targeting M1 macrophages, and promoting the growth and proliferation of macrophages and / or chondrocytes.

[0061] Specifically, the above-mentioned targeted nanoparticles are used in the preparation of drugs capable of releasing CO in response to laser.

[0062] Preferably, the wavelength of the laser is 806-808 nm, more preferably 808 nm. Under 808 nm laser irradiation, Fe 3 (CO) 12 @Croc-TCZ releases CO, and the amount of CO released is positively correlated with the laser power and irradiation time. The higher the laser power, the faster the CO is released, and the higher the amount of CO released in the same time.

[0063] Specifically, the above-mentioned targeted nanoparticles are used in the preparation of drugs that are targeted to M1 macrophages. 3 (CO) 12 @Croc-TCZ accumulation in M1 inflammatory macrophages was significantly higher than that in hMSCs; in M1 inflammatory macrophages, Fe 3 (CO) 12 @Croc-TCZ accumulation ratio Fe 3 (CO) 12 @Croc-PEG 5 K is obviously more.

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

[0065] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0066] (1) The targeted nanoparticles of the present invention are based on TCZ (tocilizumab), and a dye containing a terminal carboxyl group (such as crocinyl cyanine dye (Croc)) is grafted to construct a carrier, and then the CO release molecule is coated with the carrier, wherein the carrier has a targeting effect. The photothermal effect of the carrier can realize the CO release regulated by NIR laser or the targeted and controllable release of CO under the stimulation of free radicals, thereby realizing the exogenous / endogenous dual response release of CO, which has broad application prospects.

[0067] (2) The present invention uses a carbonyl metal compound Fe 3 (CO) 12 As a CO donor, Fe 3 (CO) 12 The central metal of CO is Fe, which is an essential trace element for the human body. In addition, one Fe atom can cooperate with 12 CO, which has a very strong gas storage capacity.

[0068] (3) The present invention uses TCZ as a carrier, which has good biocompatibility and targeting. At the same time, TCZ, as a clinical drug, can increase the therapeutic effect of CO gas therapy.

[0069] (4) The present invention connects the organic functional crotonyl cyanine dye Croc to TCZ to obtain Croc-TCZ, which has good photothermal effect and imaging ability and has great prospects for clinical transformation.

[0070] (5) Fe of the present invention 3 (CO) 12 The targeted Fe nanoparticles were combined with Croc-TCZ through simple hydrophilic and hydrophobic interactions. 3 (CO) 12 @Croc-TCZ. The targeted nanoparticles have simple components, conventional preparation process, good repeatability, high stability, and are suitable for batch and industrial production.

[0071] (6) Fe of the present invention 3 (CO) 12 @Croc-TCZ is a nano gas storage tank that can release CO on demand under the action of NIR laser, overcoming the technical difficulty of uncontrollable concentration of CO caused by its diffusion, and providing technical guidance for the popularization of CO gas therapy.

[0072] (7) Fe of the present invention 3 (CO) 12 @Croc-TCZ has no obvious cytotoxicity and has a proliferative effect on cells under (non) inflammatory conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is a schematic diagram of the inventive concept of the present invention;

[0074] Figure 2 is a schematic diagram of the preparation process of Croc-TCZ in Example 1 of the present invention;

[0075] Figure 3 The targeted nanoparticle Fe in Example 1 of the present invention 3 (CO) 12 Schematic diagram of the preparation process of Croc-TCZ;

[0076] Figure 4 Example 1 Targeted Nanoparticle Fe 3 (CO) 12 TEM and DLS characterization of Croc-TCZ and raw materials, Fe 3 (CO) 12 Zeta potential, UV-visible absorption spectrum, and absorption-concentration relationship graph of Croc-TCZ;

[0077] Figure 5 The targeted nanoparticle Fe in Example 1 of the present invention 3 (CO) 12Photothermal temperature rise curves of Croc-TCZ at different concentrations and optical densities in vitro, and the photothermal effect diagram of the mixed solution with reduced Hb;

[0078] Figure 6 The targeted nanoparticle Fe in Example 1 of the present invention 3 (CO) 12 Croc-TCZ has the effect of targeting M1 inflammatory macrophages;

[0079] Figure 7 TCZ, Croc-TCZ and targeted nanoparticles Fe 3 (CO) 12 Croc-TCZ on the cell activity of macrophages and chondrocytes;

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

[0081] Fig. 9 TCZ, Croc-TCZ and targeted nanoparticles Fe 3 (CO) 12 Live-dead cell staining of the effect of Croc-TCZ on chondrocytes. DETAILED DESCRIPTION

[0082] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

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

[0084] Example 1: Preparation of carrier and targeted nanoparticles

[0085] A targeted nanoparticle with a chemical formula of Fe 3 (CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier, and Fe is coated by the carrier. 3 (CO) 12 Form a core-shell structure.

[0086] A method for preparing targeted nanoparticles comprises the following steps:

[0087] Croc (croc acid 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 mixed and dissolved in anhydrous ethanol (8 mL), protected by argon, protected from light, and stirred in an ice bath for 4 h to activate the carboxyl groups at both ends of Croc; then 20.0 mg (concentration of 20 mg / mL) of TCZ (tocilizumab) solution was added, and stirred in an ice bath in the dark for 12 h to connect Croc to the free amino group of TCZ through the condensation reaction of the carboxyl group and the amino group. After the reaction, the obtained mixture was filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes in turn to remove excess Croc, and the mixture obtained after removing excess Croc was added with 5 mL The PBS solution was used to remove ethanol using a rotary evaporator, and the resulting mixed solution was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e., the obtained carrier, recorded as Croc-TCZ solution);

[0088] Nanoparticles Fe were successfully prepared by ultrasonic self-assembly 3 (CO) 12 @Croc-TCZ, specifically, in a protective atmosphere (argon) and light-proof conditions, Fe 3 (CO) 12 (4.0 mg, 7.9 mmol) was dissolved in THF (tetrahydrofuran 2 mL), and added dropwise to the Croc-TCZ solution under ultrasonic conditions, and ultrasonication was continued for 15 min. After the ultrasonication, nitrogen was introduced into the mixed solution in a fume hood until the THF was completely blown out. Then, the mixed solution was passed through 0.88 μm, 0.45 μm, and 0.22 μm filter membranes in sequence. The mixed solution obtained after passing through the filter membrane was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain targeted nanoparticles Fe 3 (CO) 12 @Croc-TCZ.

[0089] Example 2: Preparation of carriers and targeted nanoparticles

[0090] A targeted nanoparticle with a chemical formula of Fe 3 (CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier, and Fe is coated by the carrier. 3 (CO) 12 Form a core-shell structure.

[0091] A method for preparing targeted nanoparticles comprises the following steps:

[0092] Croc (croc acid 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), TCZ (tocilizumab) solution 20.0 mg (concentration of 20 mg / mL) were mixed and dissolved in anhydrous ethanol (8 mL), protected by argon, protected from light, and stirred in an ice bath for 16 h. Croc was connected to the free amino group of TCZ through the condensation reaction of carboxyl and amino groups. After the reaction, the obtained mixture was filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes in sequence to remove excess Croc. The mixture obtained after removing excess Croc was added with 4 mL The PBS solution was used to remove ethanol using a rotary evaporator, and the resulting mixed solution was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e., the obtained carrier);

[0093] Nanoparticles Fe were successfully prepared by ultrasonic self-assembly 3 (CO) 12 @Croc-TCZ, specifically, in a protective atmosphere (argon) and light-proof conditions, Fe 3 (CO) 12 (4.0 mg, 7.9 mmol) was dissolved in THF (tetrahydrofuran 2 mL), and added dropwise to the Croc-TCZ solution under ultrasonic conditions, and ultrasonication was continued for 15 min. After the ultrasonication, argon gas was introduced into the mixed solution in a fume hood until the THF was completely blown out, and then the mixed solution was passed through 0.88 μm, 0.45 μm, and 0.22 μm filter membranes in sequence. The mixed solution obtained after passing through the filter membrane was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain targeted nanoparticles Fe 3 (CO) 12 @Croc-TCZ.

[0094] Example 3: Preparation of carriers and targeted nanoparticles

[0095] A targeted nanoparticle with a chemical formula of Fe 3 (CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier, and Fe is coated by the carrier. 3 (CO) 12 Form a core-shell structure.

[0096] A method for preparing targeted nanoparticles comprises the following steps:

[0097] Croc (croc acid 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 mixed and dissolved in anhydrous ethanol (8 mL), protected by argon, protected from light, and stirred in an ice bath for 4 h to activate the carboxyl groups at both ends of Croc; then 20.0 mg (concentration of 20 mg / mL) of TCZ (tocilizumab) solution was added, and stirred in an ice bath in the dark for 12 h to connect Croc to the free amino group of TCZ through the condensation reaction of the carboxyl group and the amino group. After the reaction, the obtained mixture was filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes in turn to remove excess Croc. The mixture obtained after removing excess Croc was added with 6 mL The PBS solution was used to remove ethanol using a rotary evaporator, and the resulting mixed solution was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e., the obtained carrier);

[0098] Nanoparticles Fe were successfully prepared by ultrasonic self-assembly 3 (CO) 12 @Croc-TCZ, specifically, in a protective atmosphere (argon) and light-proof conditions, Fe 3 (CO) 12 (4.0 mg, 7.9 mmol) was dissolved in THF (tetrahydrofuran 2 mL), and added dropwise to the Croc-TCZ solution under ultrasonic conditions, and the ultrasonication was continued for 15 min. After the ultrasonication, argon gas was introduced into the mixed solution in a fume hood until the THF was completely blown out. Then, the mixed solution was passed through 0.88 μm, 0.45 μm, and 0.22 μm filter membranes in sequence. The mixed solution obtained after passing through the filter membrane was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min). The lower layer of the concentrated liquid was repeatedly passed through 0.88 μm, 0.45 μm, and 0.22 μm filter membranes to obtain targeted nanoparticles Fe 3 (CO) 12 @Croc-TCZ.

[0099] Product effect testing

[0100] After successfully preparing targeted nanoparticles Fe 3 (CO) 12 @Croc-TCZ, the present invention explores Fe 3 (CO) 12 @Croc-The photothermal effect of TCZ and the stimulated release of CO by NIR laser.

[0101] Take Fe 3(CO) 12 @Croc-TCZ solution (20 μM, 1.0 mL) was irradiated with 808 nm laser at different light densities (0.0-2.0 W / cm 2 ) for 10 min, and Fe 3 (CO) 12 @Croc-TCZ solution photothermal heating curve; similarly, fixed at 1.0W / cm 2 Optical density, irradiation with different concentrations of Fe 3 (CO) 12 @Croc-TCZ solution (0~30μM), obtained Fe 3 (CO) 12 @Photothermal heating curve of Croc-TCZ solution.

[0102] Then, the photothermal effect on the release of CO was investigated. 3 (CO) 12 @Croc-TCZ solution (0-15 μM), hemoglobin (Hb, 5 μM) and sodium dithionite (SDT, 1.6 mg), the total volume of the mixed solution was 1.0 mL, and argon was continuously introduced to remove air to obtain Fe 3 (CO) 12 The mixed solution of Croc-TCZ and reduced Hb was irradiated with 808 nm laser at the same optical density (1.0 W / cm 2 ) for 60 min, and the UV-visible absorption spectra of the mixed solution at different time points and the absorbance values ​​at 420 nm and 432 nm (denoted as Abs 420 and Abs 432 ). The CO release at different time points was calculated according to the following formula.

[0103]

[0104] In the formula: C co is the release concentration of CO; C Hb is the concentration of Hb; Abs 420 Abs is the absorbance of the mixed solution at 420 nm; 432 is the absorbance of the mixed solution at 432 nm.

[0105] The present invention also explores the targeting of targeted nanoparticles. Two groups of macrophages (RAW264.7) and one group of human mesenchymal stem cells (hMSC) were cultured in the experiment. The two groups of RAW264.7 were first incubated with LPS (bacterial lipopolysaccharide) for 24 hours, and then induced into inflammatory cells and then treated with two different nanoparticles, namely Fe 3 (CO) 12@Croc-PEG5K (20 μM, Fe 3 (CO) 12 The preparation process of @Croc-PEG5K is different 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-hydroxysulfosuccinimide and EDC-HCl is omitted, that is, Croc-TCZ is prepared first, and then Fe 3 (CO) 12 @Croc-PEG5K) and Fe 3 (CO) 12 @Croc-TCZ (20 μM) was used for incubation, and hMSCs were directly treated with Fe 3 (CO) 12 The cells were incubated with @Croc-TCZ (20 μM). The three groups of cells were fixed 2-6 h after nanoparticle incubation, and confocal microscopy was used to observe the changes in the fluorescence intensity of the intracellular nanoparticles over time and the differences between the groups.

[0106] The present invention further explored the effect of nanoparticles on cell proliferation. Two groups of macrophages and two groups of chondrocytes were cultured in the experiment. After incubation for 24 hours with or without LPS, different concentrations (0-20 μM) of TCZ, Croc-TCZ and Fe 3 (CO) 12 @Croc-TCZ for incubation, and CCK-8 (cell counting reagent) kit was used to detect cell activity after 24 hours. Similarly, the cells in the above groups were cultured in laser confocal culture dishes, ±LPS incubated for 24 hours, and the same concentration (10μM) of TCZ, Croc-TCZ and Fe 3 (CO) 12 @Croc-TCZ was used for incubation, and live-dead staining was performed 24 hours later and photographed using a confocal microscope.

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

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

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

[0110] Figure 4 Example 1 Targeted Nanoparticle Fe 3 (CO) 12 TEM and DLS characterization of Croc-TCZ and raw materials, Fe 3 (CO) 12 Zeta potential, UV-visible absorption spectrum, and absorption-concentration relationship graph of Croc-TCZ; Figure 4 Figure a is the nanoparticle Fe in Example 1 3 (CO) 12 TEM characterization image of Croc-TCZ; Figure 4 Figure b is the targeted nanoparticle Fe in Example 1 3 (CO) 12 DLS characterization of Croc-TCZ; Figure 4 Figure c in Figure 1 is the CO donor Fe 3 (CO) 12 , near-infrared crotonyl cyanine dyes Croc, TCZ, intermediates Croc-TCZ and targeted nanoparticles Fe 3 (CO) 12 Zeta potential diagram of Croc-TCZ; Figure 4 Figure d in Example 1 shows the CO donor Fe 3 (CO) 12 , near-infrared crotonyl cyanine dye Croc, carrier Croc-TCZ and targeted nanoparticles Fe 3 (CO) 12 UV-visible absorption spectrum characterization of Croc-TCZ; Figure 4 Figure e in Example 1 shows the targeted nanoparticle Fe 3 (CO) 12 Standard curve graph of Croc-TCZ.

[0111] Figure 5 The targeted nanoparticle Fe in Example 1 of the present invention 3 (CO) 12 Photothermal temperature rise curves of Croc-TCZ at different concentrations and optical densities in vitro, and the photothermal effect diagram of the mixed solution with reduced Hb; Figure 5 Figure a is the targeted nanoparticle Fe in Example 1 3 (CO) 12 Photothermal heating curves of Croc-TCZ at different concentrations in vitro; Figure 5 Figure b is the targeted nanoparticle Fe in Example 1 3 (CO) 12 Photothermal heating curves of Croc-TCZ at different light densities in vitro; Figure 5Figure c is the targeted nanoparticle Fe in Example 1 3 (CO) 12 Photothermal effect diagram of the mixed solution of Croc-TCZ and reduced Hb.

[0112] Figure 6 The targeted nanoparticle Fe in Example 1 of the present invention 3 (CO) 12 Croc-TCZ has the effect of targeting M1 inflammatory macrophages;

[0113] Figure 7 TCZ, Croc-TCZ and targeted nanoparticles Fe 3 (CO) 12 Croc-TCZ on the cell activity of macrophages and chondrocytes; Figure 7 Figure a shows the TCZ, Croc-TCZ and targeted nanoparticles Fe in Example 1. 3 (CO) 12 Croc-TCZ on macrophage cell activity results; Figure 7 Figure b shows the TCZ, Croc-TCZ and targeted nanoparticles Fe in Example 1. 3 (CO) 12 Croc-TCZ on the cell activity of chondrocytes.

[0114] Figure 8 is the live-dead cell staining diagram of the effects of TCZ, Croc-TCZ, and chondrocytes on macrophages;

[0115] Fig. 9 TCZ, Croc-TCZ and targeted nanoparticles Fe 3 (CO) 12 Live-dead cell staining of the effect of Croc-TCZ on chondrocytes.

[0116] Effect 1

[0117] from Figure 4 It can be seen that Fe 3 (CO) 12 The nanostructure of @Croc-TCZ was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). 3 (CO) 12 @Croc-TCZ presents a uniform spherical structure with an average particle size of about 150nm. The black center in the middle is Fe 3 (CO) 12 The outer coating material is Croc-TCZ, which verifies Fe 3 (CO) 12@Croc-TCZ structure and preparation principle, namely, self-assembly triggered by hydrophilic and hydrophobic forces to form a core-shell structure. 3 (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 the nanoparticles was characterized by measuring the Zeta potential. The results showed that TCZ (0.733 mV) was connected to Croc (-23.9 mV) to obtain Croc-TCZ (-2.99 mV) and the Fe-coated 3 (CO) 12 (-28.9mV) to obtain nanoparticles Fe 3 (CO) 12 @Croc-TCZ (-4.81mV), the Zeta potential decreased. 3 (CO) 12 The optical properties of @Croc-TCZ were characterized by UV-visible absorption spectroscopy. 3 (CO) 12 @Croc-TCZ also has Fe 3 (CO) 12 The characteristic peaks of ultraviolet absorption of Croc-TCZ indicate the successful assembly and combination of the two.

[0119] Due to the preparation of Fe 3 (CO) 12 @Croc-TCZ, Fe 3 (CO) 12 The mass ratio of Fe and Croc-TCZ remains unchanged, so the Fe 3 (CO) 12 @Croc-TCZ was quantified, with concentration as the horizontal axis and Abs as the 772 is the vertical coordinate, and the linear relationship between the two is: Y=0.09137X+0.05042.

[0120] Effect 2

[0121] from Figure 5 It can be seen that when Fe 3 (CO) 12 @When the concentration of 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. Fe 3 (CO) 12The photothermal effect of @Croc-TCZ comes from the near-infrared crotonyl cyanine dye Croc, which has a significant photothermal warming effect due to its strong absorption in the near-infrared region. 3 (CO) 12 @Croc-TCZThe heating effect changes with the optical density of the laser, indicating that the appropriate temperature can be selected by adjusting the concentration and laser parameters, which reflects the Fe 3 (CO) 12 @Croc-TCZ’s photothermal tunability. Similarly, the light density is fixed at 1.0W / cm 2 , different concentrations of Fe 3 (CO) 12 @Croc-TCZ solutions all showed good photothermal effect, and the heating effect was proportional to the concentration of nanoparticles. 3 (CO) 12 @Croc-TCZ has a good photothermal effect. The photothermal heating effect can be controlled by controlling the laser parameters and the concentration of nanoparticles, thereby controlling the release of CO, which provides a good prerequisite for the controllable release of CO.

[0122] Optical density: 1.0W / cm 2 Under the same conditions, Fe was measured by Hb method. 3 (CO) 12 @Croc-TCZ CO release changes with concentration and time. The results clearly show that the CO release in the same time is similar to Fe 3 (CO) 12 @Croc-TCZ concentration is positively correlated; the growth rate of CO release shows a similar pattern. The higher the concentration in the first 20 minutes, the greater the slope of the curve, which means that under the same laser illumination conditions, the greater the total amount of CO that can be released in the solution, the faster the release. At the same time, it can also be observed that as time goes on, the rate of CO release decreases and the curve tends to be flat, which shows that under laser conditions, nanoparticles can respond quickly in a short time and promote the release of CO through photothermal effect.

[0123] Effect 3

[0124] RAW264.7 macrophages were inoculated into 4 glass-bottom laser confocal culture dishes, and hMSCs were inoculated into 2 culture dishes. They were incubated at 37°C for 24 h. The culture medium was replaced and LPS (1 μg / ml) was added to the RAW264.7 cell dishes to induce stimulation to obtain M1 inflammatory macrophages. The culture medium was replaced with Fe-containing medium in 2 RAW264.7 cell dishes to induce stimulation to obtain M1 inflammatory macrophages. 3 (CO) 12@Croc-PEG5K (20 μM) culture medium, and replace the remaining 2 RAW264.7 cell culture dishes and 2 hMSC cell culture dishes with Fe 3 (CO) 12 @Croc-TCZ (20μM) culture medium, the cells were fixed after incubation for 3h and 6h, and the nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). After washing with PBS solution, the cells were imaged with a laser confocal fluorescence microscope to obtain the effect of nanoparticles targeting M1 inflammatory macrophages. Figure 6 It can be seen that in the same cell type (M1), after treatment with the same concentration and incubation time, Fe 3 (CO) 12 The fluorescence intensity of @Croc-TCZ targeted nanoparticles in cells is higher than that of Fe 3 (CO) 12 @Croc-PEG5K; In different cells, Fe 3 (CO) 12 @Croc-TCZ targeted nanoparticles have a higher fluorescence intensity in M1 macrophages than in hMSCs. 3 (CO) 12 @Croc-TCZ has the ability to target M1 inflammatory macrophages.

[0125] Effect 4

[0126] RAW264.7 macrophages / chondrocytes were seeded into six 96-well plates and incubated at 37°C for 24 h. The culture medium was replaced and LPS (1 μg / ml) was added to three of the 96-well plates for stimulation and induction. The plates were incubated at 37°C for 24 h. The culture medium containing different concentrations of TCZ, Croc-TCZ and Fe 3 (CO) 12 @Croc-TCZ (0~20μM) culture medium, incubate at 37℃ for 24h, wash with PBS solution and replace the culture medium, incubate at 37℃ for 24h, replace serum-free DMEM culture medium containing CCK-8 (10%), incubate at 37℃ for 1h, and measure the cell survival rate by microplate reader. Figure 7 It can be seen that Fe 3 (CO) 12 @Croc-TCZ has the effect of promoting the growth and proliferation of macrophages and chondrocytes.

[0127] Effect 5

[0128] RAW264.7 macrophages / chondrocytes were inoculated into 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 4 of the dishes for stimulation and induction. The dishes were divided into two groups: ±LPS and incubated at 37°C for 24 h. The 4 dishes in each group were replaced with normal culture medium (negative and positive controls) and culture medium containing TCZ, Croc-TCZ and Fe 3 (CO) 12 @Croc-TCZ (10μM, TCZ histone concentration is the same as the latter two groups) culture medium, incubated at 37℃ for 24h, washed with PBS buffer and replaced with culture medium, incubated at 37℃ for 24h, stained with Calcein-AM and propidium iodide (PI), washed with PBS solution and used for laser confocal fluorescence microscopy imaging to obtain its live and dead staining images. The results are shown in Figure 8 and Fig. 9 As shown, from Figure 8 and Fig. 9 It can be seen that Fe 3 (CO) 12 @Croc-TCZ has no obvious toxicity to macrophages and chondrocytes and does not increase the number of dead cells.

[0129] The Fe prepared in Example 1 is given above. 3 (CO) 12 @Croc-TCZ effect data, Fe prepared in Example 2 3 (CO) 12 The effect data of @Croc-TCZ are similar to those in Example 1. Moreover, within the scope of the present invention, the process parameters in the preparation process of the carrier and the targeted nanoparticles, such as the amount of raw materials, are adjusted, and the prepared targeted nanoparticles also have similar effects as those in Example 1. 3 (CO) 12 @Croc - The effect of TCZ.

Claims

1. A carrier, It is characterized in that It includes tocilizumab grafted with a dye containing a terminal carboxyl group.

2. The vector according to claim 1, It is characterized in that The terminal carboxyl group-containing dye includes cremonate cyanine dye.

3. The vector according to claim 2, It is characterized in that The carrier is obtained by reacting the carboxyl groups at both ends of the cremonate cyanine dye with the free amino groups of tocilizumab.

4. A method for preparing a carrier according to any one of claims 1 to 3, It is characterized in that The following steps are involved: The dye containing a terminal carboxyl group is mixed with an imine substance and tocilizumab to react and obtain the carrier.

5. A targeted nanoparticle, It is characterized in that The invention comprises the carrier according to any one of claims 1 to 3 and a CO-releasing molecule, wherein the carrier covers the CO-releasing molecule to form a core-shell structure.

6. The targeted nanoparticle according to claim 5, It is characterized in that The CO-releasing molecules include metal carbonyl compounds.

7. The targeted nanoparticle according to claim 5, It is characterized in that The CO releasing molecule is Fe 3 (CO) 12 or Mn 2 (CO) 10 .

8. The targeted nanoparticle according to any one of claims 5 to 7, It is characterized in that The mass ratio of the carrier to the CO releasing molecules is (1-32):

4.

9. A method for preparing the targeted nanoparticles according to any one of claims 5 to 8, It is characterized in that The following steps are involved: The carrier is mixed with CO-releasing molecules to prepare the targeted nanoparticles.

10. A pharmaceutical composition, It is characterized in that The targeting nanoparticle comprises the targeting nanoparticle described in any one of claims 5 to 8.

11. Use of the targeted nanoparticles according to any one of claims 5 to 8 in the preparation of a drug for treating rheumatoid arthritis, laser-responsive release, targeting M1 macrophages, and promoting the growth and proliferation of macrophages and / or chondrocytes.

Images