Carrier, targeted nanoparticle, method for preparing same, and use thereof

Abstract

The present invention relates to the field of targeted therapy technology, and particularly, to a carrier, a targeted nanoparticle, a method for preparing same, and use thereof. The carrier disclosed in the present invention comprises tocilizumab grafted with a terminal carboxyl group-containing dye. The targeted nanoparticle comprises a carrier and a CO-release molecule. The carrier encapsulates the CO-release molecule to form a core-shell structure. In the targeted nanoparticle based on tocilizumab, the carrier is constructed by grafting the terminal carboxyl group-containing dye, and the carrier encapsulates the CO-release molecule. The carrier possesses a targeting effect, and the photothermal effect of the carrier can achieve the regulated CO release by NIR laser or targeted and controllable CO release under free radical stimulation, thereby achieving exogenous / endogenous dual-response release of CO. The present invention features good application prospects.

Description

A carrier, targeted nanoparticles, and preparation method and application thereof Technical Field

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

[0002] CO is a gaseous messenger with a rich array of biological effects, thus holding therapeutic potential for numerous diseases. As a bioactive molecule, CO modulates numerous physiological and pathological processes, primarily due to its ability to bind to molecules such as iron-containing hemoglobin (Hb) and respiratory chain complexes, thereby affecting their function and initiating a cascade of effects. CO's vascular tone-regulating effects can invigorate blood circulation and remove stasis, its antioxidant properties can reduce oxidative stress and alleviate inflammation, its anti-apoptotic effects can inhibit mitochondrial apoptosis, and its pro-repair effects can promote tissue regeneration. Therefore, CO is an ideal therapeutic agent.

[0003] Carbon monoxide (CO) gas therapy is an emerging medical technology that has been gradually applied to treat a variety of diseases, such as cardiovascular disease, sepsis, shock, acute lung, kidney, and liver injury, microbial infections, 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) are substances that can release CO gas, acting 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 lack of widespread use of CO and CO-releasing molecules (CORMs) stems from their poor water solubility and the diffusivity and uncontrollability of CO. RA clinically manifests as aggressive inflammation and cartilage damage in multiple joints, including the hands and feet. Applying CO gas therapy to RA requires addressing the issue of targeting, controlling CO release at the lesion site to avoid systemic toxicity. Real-time evaluation of treatment efficacy is also crucial to avoid under- or overtreatment.

[0006] Furthermore, gaseous CO is difficult to store and transport, and its diffusivity makes its concentration and location in the body uncontrollable, easily leading to the risk of ineffectiveness or poisoning. Therefore, CO gas therapy must rely on a specific platform to achieve precise, on-demand release.

[0007] Metal carbonyls are complexes formed by transition metals (nickel, cobalt, ruthenium, vanadium, chromium, manganese and iron) with 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, manganese carbonyls and iron carbonyls are more suitable for biological applications. Among these compounds, triiron dodecacarbonyl (Fe3(CO) 12 ) have the highest gas storage capacity, making them ideal CO donors. However, these compounds have poor water solubility. Therefore, transport carriers must address biocompatibility issues and be responsive to external stimuli to trigger CO release from metal carbonyls. Furthermore, they must be both targeted and diagnostic, enabling simultaneous treatment and monitoring to optimize therapeutic efficacy. However, existing carriers or CO drugs often fail to meet these requirements.

[0008] Therefore, there is an urgent need to provide a new drug that targets and controls CO release.

[0009] Summary of the Invention

[0010] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art.

[0011] To this end, the present invention proposes a targeted nanoparticle, its preparation method, and application. The targeted nanoparticle is based on TCZ (tocilizumab), onto which a carboxyl-terminated dye (e.g., crotonyl cyanine dye (Croc)) is grafted to form a carrier. The carrier is then coated with CO-releasing molecules (CORMs). The carrier has a targeting effect, enabling targeted and controlled release of CO under near-infrared (NIR) laser or free radical stimulation.

[0012] A first aspect of the present invention provides a vector.

[0013] A carrier comprises tocilizumab grafted with a dye containing a terminal carboxyl group.

[0014] Preferably, the dye containing terminal carboxyl groups includes two or more terminal carboxyl groups.

[0015] Preferably, the dye containing a terminal carboxyl group comprises a crotonate cyanine dye.

[0016] Preferably, the carrier is obtained by reacting the carboxyl groups at both ends of crocinato cyanine dye with the free amino groups of tocilizumab (denoted as Croc-TCZ).

[0017] Preferably, the vector has IL-6R targeting and photothermal effect.

[0018] A second aspect of the present invention provides a targeting nanoparticle.

[0019] 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.

[0020] Preferably, the CO-releasing molecule comprises a metal carbonyl compound.

[0021] Preferably, the metal carbonyl compound is selected from metal carbonyl compounds containing Fe, Mn, Re, and Ru, and more preferably metal carbonyl compounds containing Fe and Mn. Metal carbonyl compounds containing Fe and Mn have better biocompatibility.

[0022] Preferably, the CO releasing molecule is Fe3(CO) 12 or Mn2(CO) 10 Fe3(CO) 12 Not only does it have good biocompatibility, but Fe is also an essential trace element for the human body. Furthermore, Fe3(CO) 12 With more carbonyl groups, more CO can be released with 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 require further modification of the CO-releasing molecules with organic or inorganic groups. However, the drug delivery system in the prior art requires modification of the CO-releasing molecules with organic and / or inorganic groups in order to load the CO-releasing molecules on the carrier for targeted drug delivery. Moreover, 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 CO-releasing molecules per unit mole is significantly reduced, which reduces the drug delivery efficiency.

[0023] Preferably, the particle size of the targeted nanoparticles is 10-300 nm, more preferably 50-200 nm, and even more preferably 100-150 nm.

[0024] Preferably, the mass ratio of the carrier to the CO-releasing molecules is (1-32):4, more preferably (12-32):4.

[0025] Preferably, the chemical formula of the targeting nanoparticles includes Fe3(CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier. Fe3(CO) 12 @Croc-TCZ can achieve controlled release of CO. The photothermal effect of Croc-TCZ can achieve NIR laser-regulated CO release. At the same time, free radicals can also promote CO release. NIR is an exogenous stimulus, while free radicals are an endogenous stimulus. Therefore, the Fe3(CO) 12@Croc-TCZ can achieve dual response of exogenous / endogenous CO release and has broad application prospects.

[0026] The third aspect of the present invention provides a method for preparing a carrier.

[0027] A method for preparing a carrier comprises the following steps:

[0028] The dye containing a terminal carboxyl group is mixed with an imine substance and tocilizumab, and the mixture is reacted to prepare the carrier.

[0029] Preferably, the preparation method comprises the following steps:

[0030] Mixing a terminal carboxyl group-containing dye with an imine substance to obtain an activated terminal carboxyl group-containing dye;

[0031] The activated terminal carboxyl-containing dye is reacted with tocilizumab to obtain the carrier.

[0032] Preferably, the imine 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).

[0033] Preferably, the dye containing terminal carboxyl groups is mixed with an imine substance and a solvent.

[0034] Preferably, the solvent is an alcohol solvent, such as anhydrous ethanol or propanol.

[0035] Preferably, the mass ratio of the terminal carboxyl group-containing dye to the imine substance is 12:(0.5-2):(0.5-2), and more preferably 12:(0.8-1.5):(0.8-1.5).

[0036] 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.

[0037] Preferably, the mass ratio of the terminal carboxyl group-containing dye to the solvent is 12 g:(5-30) mL, more preferably 12 g:(8-12) mL.

[0038] Preferably, the protective atmosphere is a rare gas or nitrogen, such as argon.

[0039] Preferably, the ice bath stirring time is 1-5 hours, more preferably 2-4 hours.

[0040] 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).

[0041] Preferably, during the reaction, the concentrations of the activated carboxyl-terminated 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.

[0042] Preferably, the reaction is carried out in an ice bath with stirring and in the dark. The carboxyl-terminated dye is linked to the free amino group of TCZ through a condensation reaction between the carboxyl group and the amino group.

[0043] Preferably, the reaction time is 10-13 hours, more preferably 11-12 hours.

[0044] Preferably, after the reaction is completed, the mixed solution formed after the reaction is filtered through a filter membrane to remove excess carboxyl-terminated 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 carboxyl-terminated dye.

[0045] 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.

[0046] Preferably, the conditions for concentration in the ultrafiltration centrifuge tube are 1-4° C., a rotation speed of 7000-7500 rpm, and centrifugation for 5-10 minutes.

[0047] 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 of 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.

[0048] Preferably, the tocilizumab is added in the form of a tocilizumab solution, and the concentration of the tocilizumab solution is 15-25 mg / mL.

[0049] Preferably, the mass ratio of the carboxyl-terminated dye to tocilizumab is 12:(0.5-20), more preferably 12:(2-15).

[0050] The fourth aspect of the present invention provides a method for preparing targeted nanoparticles.

[0051] A method for preparing targeted nanoparticles comprises the following steps:

[0052] The carrier is mixed with CO-releasing molecules to prepare the targeted nanoparticles.

[0053] 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.

[0054] Preferably, the CO2-releasing molecule solution is prepared by mixing 1-8 mg of CO2-releasing molecules with an organic solvent. The organic solvent is a common substance, such as tetrahydrofuran.

[0055] Preferably, the CO2-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.

[0056] Preferably, after the tocilizumab grafted with a dye containing a terminal carboxyl group is mixed with the CO-releasing molecule, the resulting 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.

[0057] Preferably, the mass ratio of the carrier to the CO-releasing molecules is (1-32):4, more preferably (12-32):4.

[0058] The preparation method of the invention is an ultrasonic self-assembly method.

[0059] The fifth aspect of the present invention provides a use of targeted nanoparticles.

[0060] A pharmaceutical composition comprising the above-mentioned targeted nanoparticles.

[0061] The targeted nanoparticles are used in the preparation of drugs for treating rheumatoid arthritis, releasing in response to laser, targeting M1 macrophages, and promoting the growth and proliferation of macrophages and / or chondrocytes.

[0062] Specifically, the above-mentioned targeted nanoparticles are used in the preparation of drugs that release CO in response to laser.

[0063] Preferably, the wavelength of the laser is 806-808 nm, 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 irradiation time. The higher the laser power, the faster the CO release, and the higher the amount of CO released in the same time.

[0064] Specifically, the above-mentioned targeted nanoparticles are used in the preparation of drugs that target M1 macrophages. Different cells are incubated for the same time, Fe3(CO) 12 The accumulation of @Croc-TCZ in M1 inflammatory macrophages was significantly higher than that in hMSCs; in M1 inflammatory macrophages, Fe3(CO) 12 @Croc-TCZ accumulation ratio Fe3(CO) 12 @Croc-PEG5K is obviously more.

[0065] Specifically, the above-mentioned targeted nanoparticles are used in the preparation of drugs for promoting the growth and proliferation of macrophages and / or chondrocytes.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (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 on the carrier. 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 free radical stimulation, thereby realizing the exogenous / endogenous dual response release of CO, which has broad application prospects.

[0068] (2) The present invention uses a carbonyl metal compound Fe3(CO) 12 As a CO donor, Fe3(CO) 12 The central metal of CO is Fe, an essential trace element for the human body. In addition, one Fe atom can combine with 12 CO atoms, giving it a very strong gas storage capacity.

[0069] (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.

[0070] (4) The present invention connects the organic functional crotonate cyanine dye Croc to TCZ to obtain Croc-TCZ, which has good photothermal effect and imaging ability and has great prospects for clinical transformation.

[0071] (5) Fe3(CO) of the present invention 12 and Croc-TCZ through simple hydrophilic and hydrophobic interactions to form targeted nanoparticles Fe3(CO) 12@Croc-TCZ. These targeted nanoparticles have simple components, a conventional preparation process, good reproducibility, and high stability, making them suitable for batch and industrial production.

[0072] (6) Fe3(CO) of the present invention 12 @Croc-TCZ, as a nano gas storage tank, can release CO on demand under the action of NIR laser, overcoming the technical difficulty of uncontrollable CO concentration caused by its diffusion, and providing technical guidance for the popularization of CO gas therapy.

[0073] (7) Fe3(CO) of the present invention 12 @Croc-TCZ has no obvious cytotoxicity and has a pro-proliferative effect on cells under (non-)inflammatory conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG1 is a schematic diagram of the inventive concept of the present invention;

[0075] FIG2 is a schematic diagram of the preparation process of Croc-TCZ in Example 1 of the present invention;

[0076] FIG3 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 of the present invention. 12 Schematic diagram of the preparation process of Croc-TCZ;

[0077] Figure 4 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 12 TEM and DLS characterization of Croc-TCZ and raw materials, Fe3(CO) 12 Zeta potential, UV-visible absorption spectrum, and absorption-concentration relationship graph of Croc-TCZ;

[0078] Figure 5 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 of the present invention. 12 Photothermal temperature rise curves of Croc-TCZ at different concentrations and optical densities in vitro, as well as the photothermal effect diagram of its mixed solution with reduced Hb;

[0079] FIG6 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 of the present invention. 12 Croc-TCZ has the effect of targeting M1 inflammatory macrophages;

[0080] Figure 7 is TCZ, Croc-TCZ and targeted nanoparticles Fe3(CO) 12 Croc-TCZ on the cell activity of macrophages and chondrocytes;

[0081] Figure 8 is a live-dead cell staining image of the effects of TCZ, Croc-TCZ, and chondrocytes on macrophages;

[0082] Figure 9 is a graph showing the relationship between TCZ, Croc-TCZ and targeted nanoparticles Fe3(CO) 12 Live-dead cell staining of the effect of Croc-TCZ on chondrocytes. DETAILED DESCRIPTION

[0083] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0084] 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.

[0085] Example 1: Preparation of carriers and targeted nanoparticles

[0086] A targeted nanoparticle with the chemical formula Fe3(CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier, and Fe3(CO) is coated by the carrier. 12 Form a core-shell structure.

[0087] A method for preparing targeted nanoparticles comprises the following steps:

[0088] Croc (crocinyl 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), protected by argon, shielded from light, and stirred on ice 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 the mixture was stirred on ice for 12 h in the dark 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 resulting 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 5 mL The PBS solution was used to remove ethanol using a rotary evaporator, and the resulting mixture was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e., the prepared carrier, recorded as Croc-TCZ solution);

[0089] Nanoparticles Fe3(CO) were successfully prepared by ultrasonic self-assembly 12 @Croc-TCZ, specifically, in a protective atmosphere (argon) and light-proof conditions, Fe3(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. The ultrasonication was continued for 15 min. After the ultrasonication, nitrogen was introduced into the mixture in a fume hood until the THF was completely blown out. The mixture was then filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes in sequence. The mixture obtained after passing through the membranes was concentrated using a 10KD ultrafiltration centrifuge tube (4 ° C, 7500 rpm, 10 min) to obtain targeted nanoparticles Fe3 (CO) 12 @Croc-TCZ.

[0090] Example 2: Preparation of carriers and targeted nanoparticles

[0091] A targeted nanoparticle with the chemical formula Fe3(CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier, and Fe3(CO) is coated by the carrier. 12 Form a core-shell structure.

[0092] A method for preparing targeted nanoparticles comprises the following steps:

[0093] 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), and 20.0 mg of TCZ (tocilizumab) solution (concentration of 20 mg / mL) were 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 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 sequence to remove excess Croc. The mixture obtained after removing excess Croc was added to 4 mL The PBS solution was used to remove ethanol using a rotary evaporator, and the resulting mixture was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e., the prepared vector);

[0094] Nanoparticles Fe3(CO) were successfully prepared by ultrasonic self-assembly 12 @Croc-TCZ, specifically, in a protective atmosphere (argon) and light-proof conditions, Fe3(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. The ultrasonication was continued for 15 min. After the ultrasonication, argon was introduced into the mixture in a fume hood until the THF was completely blown out. The mixture was then filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes in sequence. The mixture obtained after passing through the membranes was concentrated using a 10KD ultrafiltration centrifuge tube (4 ° C, 7500 rpm, 10 min) to obtain targeted nanoparticles Fe3 (CO) 12 @Croc-TCZ.

[0095] Example 3: Preparation of carriers and targeted nanoparticles

[0096] A targeted nanoparticle with the chemical formula Fe3(CO) 12 @Croc-TCZ, where Croc-TCZ represents the carrier, and Fe3(CO) is coated by the carrier. 12 Form a core-shell structure.

[0097] A method for preparing targeted nanoparticles comprises the following steps:

[0098] Croc (crocinyl 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), protected by argon, shielded from light, and stirred on ice 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 the mixture was shielded from light and stirred on ice for 12 h to connect Croc to the free amino group of TCZ through the condensation reaction of the carboxyl and amino groups. After the reaction, the resulting 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 to 6 mL The PBS solution was used to remove ethanol using a rotary evaporator, and the resulting mixture was concentrated using a 10KD ultrafiltration centrifuge tube (4°C, 7500 rpm, 10 min) to obtain the compound Croc-TCZ (i.e., the prepared vector);

[0099] Nanoparticles Fe3(CO) were successfully prepared by ultrasonic self-assembly 12 @Croc-TCZ, specifically, in a protective atmosphere (argon) and light-proof conditions, Fe3(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. The ultrasonication was continued for 15 min. After the ultrasonication, argon was introduced into the mixture in a fume hood until the THF was completely blown out. The mixture was then filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes in sequence. The mixture obtained after passing through the membranes was concentrated using a 10KD ultrafiltration centrifuge tube (4 ° C, 7500 rpm, 10 min). The lower layer of the concentrated liquid was repeatedly filtered through 0.88 μm, 0.45 μm, and 0.22 μm membranes to obtain targeted nanoparticles Fe3(CO 12 @Croc-TCZ.

[0100] Product effect testing

[0101] After successfully preparing targeted nanoparticles Fe3(CO) 12 @Croc-TCZ, the present invention explores the Fe3(CO) 12 @Croc-TCZ photothermal effect and NIR laser-stimulated CO release.

[0102] Take Fe3(CO) 12 Croc-TCZ solution (20 μM, 1.0 mL) was irradiated with 808 nm laser at different optical densities (0.0-2.0 W / cm 2 ) irradiated for 10 min, and Fe3(CO) was obtained under different optical density conditions. 12 @Croc-TCZ solution photothermal heating curve; similarly, fixed at 1.0W / cm 2 Optical density, irradiation with different concentrations of Fe3(CO) 12 @Croc-TCZ solution (0~30μM), Fe3(CO) 12 @Croc-TCZ solution photothermal heating curve.

[0103] Then the photothermal effect on the release of CO was investigated. 12 To a Croc-TCZ solution (0-15 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 was continuously introduced to remove air, and Fe3(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 432The CO release at different time points was calculated according to the following formula.

[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 explored the targeting properties 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 to induce them into inflammatory cells. Then, two different nanoparticles, namely Fe3(CO) 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-hydroxysulfosuccinimide and EDC-HCl is omitted, that is, Croc-TCZ is prepared first, and then Fe3(CO) 12 @Croc-PEG5K) and Fe3(CO) 12 @Croc-TCZ (20 μM) was used for incubation, while hMSCs were directly incubated with Fe3(CO 12 The cells were incubated with @Croc-TCZ (20 μM). The three groups of cells were fixed 2-6 hours 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 Fe3(CO) were used. 12 @Croc-TCZ incubation, 24 hours later using CCK-8 (cell counting reagent) kit to detect cell activity. Similarly, the above groups of cells were cultured in laser confocal microscopy culture dishes, ± LPS incubation for 24 hours, and then the same concentration (10μM) of TCZ, Croc-TCZ and Fe3(CO) 12 The cells were incubated with Croc-TCZ, and live-dead staining was performed 24 hours later, and the cells were photographed using a confocal microscope.

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

[0108] FIG2 is a schematic diagram of the preparation process of Croc-TCZ in Example 1 of the present invention;

[0109] FIG3 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 of the present invention. 12 Schematic diagram of the preparation process of Croc-TCZ;

[0110] Figure 4 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 12 TEM and DLS characterization of Croc-TCZ and raw materials, Fe3(CO) 12 Zeta potential, UV-visible absorption spectrum, and absorption light versus concentration curve of Croc-TCZ; Figure 4 a is a graph showing the relationship between the Fe3(CO) nanoparticles in Example 1. 12 TEM characterization of Croc-TCZ; Figure b in Figure 4 is the targeted nanoparticles Fe3(CO) in Example 1 12 DLS characterization of Croc-TCZ; Figure c in Figure 4 is the CO donor Fe3(CO) in Example 1 12 , near-infrared crotonate cyanine dyes Croc, TCZ, intermediate Croc-TCZ and targeted nanoparticles Fe3(CO) 12 Zeta potential diagram of Croc-TCZ; Figure 4 d is the CO donor Fe3(CO) in Example 1 12 , near-infrared crotonate cyanine dye Croc, carrier Croc-TCZ and targeting nanoparticles Fe3(CO) 12 Croc-TCZ UV-visible absorption spectrum characterization diagram; Figure e in Figure 4 is the targeted nanoparticles Fe3 (CO) in Example 1 12 Standard curve of Croc-TCZ.

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

[0112] FIG6 is a diagram of targeted nanoparticles Fe3(CO) in Example 1 of the present invention. 12 Croc-TCZ has the effect of targeting M1 inflammatory macrophages;

[0113] Figure 7 is TCZ, Croc-TCZ and targeted nanoparticles Fe3(CO) 12 Croc-TCZ on the cell activity of macrophages and chondrocytes; Figure 7 a is a graph showing the cell activity of TCZ, Croc-TCZ and targeted nanoparticles Fe3(CO3) in Example 1. 12 Croc-TCZ on macrophage cell activity results; Figure 7 b is TCZ, Croc-TCZ and targeted nanoparticles Fe3 (CO) in Example 1 12 Croc-TCZ shows the effect of chondrocyte cell activity.

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

[0115] Figure 9 is a graph showing the relationship between TCZ, Croc-TCZ and targeted nanoparticles Fe3(CO) 12 Live-dead cell staining of the effect of Croc-TCZ on chondrocytes.

[0116] Effect 1

[0117] As can be seen from Figure 4, Fe3(CO) 12 The nanostructure of @Croc-TCZ was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). 12 @Croc-TCZ presents a uniform spherical structure with an average particle size of about 150nm. The black center in the middle is Fe3(CO) 12 The outer coating material is Croc-TCZ, which verifies that Fe3(CO) 12 The structure and preparation principle of @Croc-TCZ is the self-assembly triggered by hydrophilic and hydrophobic forces to form a core-shell structure. 12 The hydrated particle size of @Croc-TCZ is approximately 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 significantly higher than that of Croc (-23.9 mV) to obtain Croc-TCZ (-2.99 mV) and Fe3(CO)-coated 12 (-28.9mV) to obtain nanoparticles Fe3(CO) 12 @Croc-TCZ (-4.81mV), the Zeta potential of Fe3(CO) 12 The optical properties of @Croc-TCZ were characterized by UV-visible absorption spectroscopy. 12 @Croc-TCZ also has Fe3(CO) 12 The characteristic UV absorption peaks of Croc-TCZ indicate the successful assembly and combination of the two.

[0119] Due to the preparation of Fe3(CO) 12 @Croc-TCZ, Fe3(CO) 12 The mass ratio of Croc-TCZ to Fe3(CO) remains unchanged, so the Fe3(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] As can be seen from Figure 5, when Fe3(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. Fe3(CO) 12 The photothermal effect of @Croc-TCZ comes from the near-infrared crotonate cyanine dye Croc, which has a significant photothermal heating effect due to its strong absorption in the near-infrared region. 12 @Croc-TCZ 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, reflecting the Fe3(CO) 12 @Croc-TCZ’s photothermal tunability. Similarly, the light density is fixed at 1.0W / cm 2 , different concentrations of Fe3(CO) 12 @Croc-TCZ solutions all showed good photothermal effect, and the heating effect was proportional to the concentration of nanoparticles. 12@Croc-TCZ has a good photothermal effect. The photothermal heating effect can be controlled by controlling the laser parameters and nanoparticle concentration, thereby controlling the release of CO, which provides a good prerequisite for the controlled release of CO.

[0122] Optical density is 1.0W / cm 2 Under the conditions, Fe3(CO) was measured by Hb method. 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 that of Fe3(CO) 12 The growth rate of CO release exhibited a similar pattern: the higher the concentration within the first 20 minutes, the steeper the curve slope. This indicates that under the same laser illumination conditions, the greater the total amount of releasable CO contained in the solution, the faster the release. Furthermore, it was observed that the rate of CO release decreased over time, and the curve flattened. This demonstrates that under laser illumination, the nanoparticles were able to respond quickly within a relatively short period of time, promoting CO release through the photothermal effect.

[0123] Effect 3

[0124] RAW264.7 macrophages were seeded into 4 glass-bottom laser confocal microscopy culture dishes, and hMSCs were seeded into 2 culture dishes. The culture medium was replaced and LPS (1 μg / ml) was added to the RAW264.7 cell dishes to induce M1 inflammatory macrophages. The culture medium was replaced with Fe3(CO)-containing medium in 2 RAW264.7 cell culture dishes. 12 @Croc-PEG5K (20μM) culture medium, and the remaining two RAW264.7 cell culture dishes and two hMSC cell culture dishes were replaced with Fe3(CO 12 @Croc-TCZ (20μM) culture medium, after incubation for 3h and 6h, the cells were fixed and the nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). After washing with PBS solution, the cells were imaged by laser confocal fluorescence microscopy to obtain the effect of nanoparticles targeting M1 inflammatory macrophages. As can be seen from Figure 6, in the same cell type (M1), after treatment with the same concentration and incubation time, Fe3(CO) 12 @Croc-TCZ targeted nanoparticles have a higher fluorescence intensity in cells than Fe3(CO) 12 @Croc-PEG5K; Fe3(CO) in different cells 12 @Croc-TCZ targeted nanoparticles have a higher fluorescence intensity in M1 macrophages than in hMSCs. 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 Fe3(CO) was replaced. 12 @Croc-TCZ (0-20 μM) culture medium was incubated at 37°C for 24 h, washed with PBS solution and replaced with culture medium, incubated at 37°C for 24 h, replaced with serum-free DMEM culture medium containing CCK-8 (10%), incubated at 37°C for 1 h, and the cell viability was determined by microplate reader. As can be seen from Figure 7, Fe3(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 seeded into 8 glass-bottomed laser confocal microscopy 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 cells 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 Fe3(CO)2, respectively. 12 The cells were incubated at 37°C for 24 hours with culture medium containing @Croc-TCZ (10 μM, the TCZ histone concentration was the same as the last two groups). After washing with PBS buffer, the culture medium was replaced and incubated at 37°C for 24 hours. The cells were stained with calcein-AM and propidium iodide (PI). After washing with PBS solution, the cells were imaged with a laser confocal fluorescence microscope to obtain the live-death staining images. The results are shown in Figures 8 and 9. As can be seen from Figures 8 and 9, 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) prepared in Example 1 is given above. 12 @Croc-TCZ effect data, Fe3(CO) prepared in Example 2 12 The effect data of @Croc-TCZ are similar to those in Example 1. Moreover, within the scope of the present invention, by adjusting the process parameters in the preparation process of the carrier and the targeted nanoparticles, such as the amount of raw materials, the prepared targeted nanoparticles also have similar effects as those of Fe3(CO) in Example 1. 12@Croc - The effect of TCZ.

Claims

1. A carrier, characterized in that, it comprises tocilizumab grafted with a dye containing a terminal carboxyl group.

2. The carrier according to claim 1, characterized in that, the dye containing a terminal carboxyl group comprises croconium cyanine dye.

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

4. A method for preparing the carrier according to any one of claims 1 - 3, characterized in that, it comprises the following steps: mixing the dye containing a terminal carboxyl group with an imine substance and tocilizumab, and reacting to obtain the carrier.

5. A targeted nanoparticle, characterized in that, it comprises the carrier according to any one of claims 1 - 3 and a CO releasing molecule, and the carrier coats the CO releasing molecule to form a core - shell structure.

6. The targeted nanoparticle according to claim 5, characterized in that, the CO releasing molecule comprises a metal carbonyl compound.

7. The targeted nanoparticle according to claim 5, 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 - 7, characterized in that, the mass ratio of the carrier to the CO releasing molecule is (1 - 32):

4.

9. A method for preparing the targeted nanoparticle according to any one of claims 5 - 8, characterized in that, it comprises the following steps: mixing the carrier with the CO releasing molecule to obtain the targeted nanoparticle.

10. A pharmaceutical composition, characterized in that, it comprises the targeted nanoparticle according to any one of claims 5 - 8.

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

Images