A nanocomposite useful for the preparation of an anti-atherosclerotic medicament

Abstract

The present application relates to a kind of nanocomposite for preparing anti-atherosclerosis drug, belong to biomedical nanomaterial technical field.The present application discloses a kind of nanocomposite for preparing anti-atherosclerosis drug, the nanocomposite includes organic polymer carrier core material, and / or the composite material of modification material is loaded on the outer surface of organic polymer carrier core material;The organic polymer carrier core material includes material A and the material B loaded in material A inside, the modification material includes at least one of targeting material C, contrast agent D.This application also discloses an anti-atherosclerosis drug, the anti-atherosclerosis drug contains nanocomposite for preparing anti-atherosclerosis drug;After anti-atherosclerosis drug administration, under the action of near-infrared laser, at least one of mild photothermal effect, photodynamic therapy effect is generated to the target cell of atherosclerotic plaque site.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanomaterials technology and relates to a nanocomposite that can be used to prepare anti-atherosclerotic drugs. Background Technology

[0002] Atherosclerosis is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease, with an extremely high incidence rate. Furthermore, late-stage atherosclerotic plaques may rupture, leading to serious consequences such as myocardial infarction and cerebral infarction. Therefore, early treatment of atherosclerosis is crucial. Abnormal lipid metabolism and long-term chronic inflammation are important factors contributing to the occurrence and development of atherosclerosis.

[0003] Currently, nanoparticle-based anti-atherosclerosis therapies have attracted widespread attention, mainly including photodynamic therapy, photothermal therapy, and sonodynamic therapy. Photodynamic therapy and sonodynamic therapy operate on similar principles, using light or ultrasound to excite photosensitizer or sonosensitive nanoparticles accumulated at the atherosclerotic plaque site. The excited photosensitizers or sonosensitive nanoparticles generate reactive oxygen species that induce apoptosis in plaque-related foam cells, macrophages, or smooth muscle cells. Photothermal therapy primarily involves photothermal reagents accumulated at the plaque site being irradiated with near-infrared light, converting the absorbed light energy into heat, inducing necrosis of plaque-related cells. It is evident that all of these treatments reduce the number of foam cells and lower plaque lipid levels by inducing apoptosis or necrosis within the plaque, thereby inhibiting the progression of atherosclerosis. However, apoptosis itself is an independent risk factor for atherosclerosis; excessive apoptosis can expand the necrotic core, reduce plaque stability, and increase the risk of plaque rupture. Currently, photodynamic therapy, photothermal therapy, and sonodynamic therapy based on nanoparticles are still unable to control the level of plaque cell apoptosis. While existing treatments can reduce plaque lipid content and inhibit atherosclerosis, they may also trigger serious acute cardiovascular events. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned problems in existing technologies by proposing a nanocomposite containing an organic polymer carrier. This nanocomposite comprises an organic polymer carrier core material and / or a modified material loaded on the outer surface of the organic polymer carrier core material. Upon application, it can exert mild photothermal and / or photodynamic therapeutic effects. In the mild photothermal effect, it activates heat shock proteins with stress-protective functions without inducing significant cell apoptosis and death, increasing the ratio of anti-inflammatory macrophages to inflammatory macrophages in plaques, reducing plaque inflammation levels, and increasing plaque fibrin content. In the photodynamic therapeutic effect, it affects the expression of intracellular lipid metabolism-related genes and proteins, thereby regulating intracellular cholesterol levels and cholesterol reversal transport. This achieves the simultaneous inhibition of atherosclerosis and control of plaque cell apoptosis levels, improving plaque stability, ensuring safety, and enabling visualized treatment.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A nanocomposite that can be used to prepare anti-atherosclerotic drugs, the nanocomposite comprising an organic polymer carrier core material, and / or a composite material on which a modifying material is loaded on the outer surface of the organic polymer carrier core material;

[0007] The organic polymer carrier core material includes material A and material B loaded inside material A.

[0008] Material A includes at least one of liposomes, polymer micelles, polydopamine, polylactic acid-glycolic acid copolymer, and albumin nanospheres;

[0009] Material B is at least one of photosensitizer and sound-sensitizer;

[0010] The modified material includes at least one of the target material C and the contrast agent D;

[0011] The targeting material C has receptor recognition function and includes at least one of the following: ligand molecules of atherosclerotic cell pattern recognition receptor, antibody, and polypeptide.

[0012] The contrast agent D has at least one of the functions of enhancing photoacoustic imaging, photothermal imaging, magnetic resonance imaging, computed tomography imaging, ultrasound imaging, and fluorescence imaging signals.

[0013] The nanocomposite of the present invention includes an organic polymer carrier core material, which is composed of material A and material B loaded inside material A. The material B selected in the present invention is hydrophobic and can be encapsulated inside material A. The organic polymer carrier has a long in vivo circulation time and good biocompatibility.

[0014] The nanocomposite of the present invention may also have a modifying material coupled to the outer surface of the organic polymer carrier core material. The modifying material is one or two of the target material C and the contrast agent D. The target material C has a receptor recognition function, which is beneficial to quickly find the corresponding cells to act.

[0015] Contrast agent D can enhance the signals of photoacoustic imaging, photothermal imaging, magnetic resonance imaging, computed tomography imaging, ultrasound imaging, and fluorescence imaging, and can enhance the imaging of cells within atherosclerotic plaques.

[0016] Preferably, the hydrated particle size of the organic polymer carrier core material in the nanocomposite is 20-40 nm; the hydrated particle size of the nanocomposite on which the modifying material is loaded on the outer surface of the organic polymer carrier core material is 80-150 nm.

[0017] Preferably, the modification material is coupled to the outer surface of the organic polymer carrier core material by means of at least one of physical action and chemical reaction;

[0018] The chemical reaction includes, but is not limited to, at least one of the following: substitution reaction, addition reaction, elimination reaction, polymerization reaction, hydrolysis reaction, and esterification reaction.

[0019] Preferably, the loading of the modifying material is 0-100%, and more preferably 1-90%.

[0020] Preferably, when the material A is a polymer micelle, the polymer micelle includes at least one of block polymer micelles, polyelectrolyte copolymer micelles, non-covalent micelles, and graft copolymer micelles; wherein the block polymer micelles include at least two block copolymers.

[0021] More preferably, the material A includes at least one of DSPE-PEG-COOH, DSPE-PEG-FA, HS-PEG-COOH, HS-PEG-NH2, IR825@C18PMH-PEG, 4s-PLGA-PEG-NH2, PEG-PBLG, OH-PCL-PEG-NC, and BSA-OGC.

[0022] Preferably, when the material A is a liposome, the liposome includes at least one of neutral liposomes, negatively charged liposomes, and positively charged liposomes.

[0023] Preferably, when material B is a photosensitizer, it includes at least one of porphyrin derivatives, metal phthalocyanines, fused cyclic quinones, and indocyanine green photosensitizers;

[0024] The indocyanine green photosensitizer includes at least one of IR780, IR783, IR797, IR808, IR825, IR1064, and IR1080.

[0025] Preferably, when material B is a sound-sensitive agent, it includes at least one of porphyrins and their derivatives, xanthracene compounds, nonsteroidal anti-inflammatory drugs, quinolone antibacterial drugs, and phenothiazine compounds.

[0026] Preferably, the organic polymer carrier core material includes at least one of IR780-PEG nanomicelles, IR808-DSPE-PEG-COOH nanomicelles, DSPE-PEG-FA@DPP-BT nanomicelles, DSPE-mPEG5000@DPP–BDT nanomicelles, mPEG-b-PAsp(DA)@Cy / Ce6 nanomicelles, IR825@C18PMH-PEG-Ce6-Gd nanomicelles, PD / PTF@ICG nanomicelles, TPGS / dc-IR825 nanomicelles, and ICG / IR-1061PCL-PEG nanomicelles.

[0027] Preferably, the ligand molecules, antibodies, and peptides of the atherosclerotic cell pattern recognition receptor include at least one of phosphatidylserine, hyaluronic acid, hyaluronic acid salt, mannan, fucoidan, chitosan oligosaccharide, and yeast mannan.

[0028] The antibody includes at least one of CD68, F4 / 80, CD80, CD86, CD206, CD301, CD163, vascular cell adhesion molecule-1, and profibrin-1;

[0029] The polypeptide includes at least one of cRGD, Cyclic-M2pep(RY)Biotin peptide, macrophage-targeting peptide, M2 peptide, CTT2 peptide, and osteopontin active short peptide.

[0030] Preferably, the contrast agent includes at least one selected from iron oxides, gadolinium compounds, manganese compounds, iodine compounds, and barium compounds;

[0031] Gadolinium compounds include at least one of gadolinium oxide, gadolinium acetate, gadopentetate meglumine, gadoteric acid meglumine, gadodiamine, gadobemeglumine, gadoteryl alcohol, and gadobutrol.

[0032] Manganese compounds include at least one of manganese oxide, manganese dioxide, and manganese tetroxide;

[0033] Barium compounds include at least one of barium sulfate dry powder and barium sulfate suspension;

[0034] Iodine compounds include at least one of iohexol, iopromide, iodixanol, and ioflufenicol.

[0035] Preferably, when the nanocomposite is an organic polymer carrier core material, the preparation method of the nanocomposite includes: dissolving material A, material B, and triethylamine in a mass ratio of 1:(1-5):(1-5) in a solvent, mixing and stirring the mixture, transferring the reaction solution to a dialysis bag with a molecular weight cutoff of 1000-10000, dialyzing 3-12 times, and changing the dialysis solution every 1-12 hours; after dialysis, freezing-drying the liquid; then mixing it with the organic polymer carrier core material and dissolving it in methanol, adding the mixture dropwise to water, stirring at room temperature for 1-20 minutes, and evaporating and drying to obtain the final product.

[0036] Stabilizers may also be added to the above-mentioned raw materials. The stabilizers include at least one of polyethylene glycol, amino polyethylene glycol, carboxylated polyethylene glycol, phospholipid polyethylene glycol carboxyl group, dextran, carboxymethyl dextran, carboxymethyl chitosan, carboxymethyl starch, polyacrylic acid, polystyrene-b-polyacrylic acid, polymaleic acid, polylactic acid, polylactic-glycolic acid polyethyleneimine, polyethyleneamine, amino acids, polycaprolactone, and short-chain phospholipids.

[0037] Triethylamine acts as an organic base to adjust pH and activate the reaction process.

[0038] Preferably, the nanocomposite comprises an organic polymer carrier core material and a modifying material, wherein the modifying material is a targeting material with plaque cell recognition function;

[0039] The modifying material replaces the outer surface of the core material of the reactively coupled organic polymer carrier, and its loading is 1-100%, preferably 1-80%.

[0040] The preparation method of the nanocomposite includes:

[0041] An organic polymer carrier core material, a targeting material, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in a mass ratio of 1:(1-10):(0.1-5):(0.01-3) are dissolved in water and stirred at room temperature for 12-48 hours. The mixture is then transferred to a dialysis bag with a molecular weight cutoff of 1000-10000 Da and dialyzed 3-12 times, with the dialysate replaced every 1-12 hours. After dialysis, the liquid is freeze-dried. It is then mixed with the organic polymer carrier core material and dissolved in methanol. The mixture is then added dropwise to water and stirred at room temperature for 1-20 minutes. After evaporation and drying, the final product is obtained.

[0042] Preferably, the nanocomposite comprises an organic polymer carrier core material and a modifying material, wherein the modifying material is a contrast agent;

[0043] The contrast agent is coupled to the outer surface of the organic polymer carrier core material via a substitution reaction; its loading is 1–100%, preferably 1–90%;

[0044] The preparation method of the nanocomposite includes: dissolving an organic polymer carrier core material, a contrast agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in water at a mass ratio of 1:(0.1-5):(0.1-5):(0.1-10), stirring at room temperature for 12-48 hours, transferring the mixture to a dialysis bag with a molecular weight cutoff of 1000-10000 Da, using methanol as the dialysis solution, dialyzing 3-12 times, changing the dialysis solution every 1-12 hours; after dialysis, freeze-drying the liquid; then mixing it with the organic polymer carrier core material and dissolving it in methanol, adding the mixture dropwise to water, stirring at room temperature for 1-20 minutes, and evaporating and drying to obtain the final product.

[0045] Preferably, the nanocomposite comprises an organic polymer carrier core material and a modifying material;

[0046] The modified material is a targeting material and a contrast agent with plaque cell recognition function;

[0047] The target material and the contrast agent are sequentially coupled to the outer surface of the organic polymer carrier core material via a substitution reaction; the total loading is 1-100%, preferably 1-90%.

[0048] Preferably, the contrast agent is a contrast agent loaded on nanomicelles;

[0049] The targeting material is preferably a targeting material loaded on nanomicelles.

[0050] Preferably, the preparation method of the nanocomposite includes: dissolving an organic polymer carrier core, a targeting material, and a contrast agent in an organic solvent at a mass ratio of 1:(0.1-10):(0.01-5), dropping the mixture into water, stirring at room temperature, evaporating and drying to obtain the nanocomposite;

[0051] The volume ratio of the organic solvent to water is (1.01–20):1;

[0052] The evaporation and drying temperature is 20–40°C.

[0053] Preferably, the outer surface of the organic polymer carrier core can also be loaded with other functional modification materials.

[0054] An anti-atherosclerotic drug, wherein the anti-atherosclerotic drug contains a nanocomposite that can be used to prepare the anti-atherosclerotic drug.

[0055] Preferably, the concentration of the nanocomposite in the anti-atherosclerotic drug is 0.01–1000 μg / mL, more preferably 1–100 μg / mL.

[0056] Preferably, the anti-atherosclerotic drug can target target cells in atherosclerotic plaques;

[0057] The target cells include at least one of activated macrophages, activated endothelial cells, activated smooth muscle cells, and activated foam cells in atherosclerotic plaques.

[0058] Further preferably, the target cells contain a targeting peptide that can recognize foam cells in atherosclerotic plaques.

[0059] Preferably, the administration method of the anti-atherosclerotic drug includes at least one of systemic administration and intravascular interventional administration;

[0060] The dosage of the anti-atherosclerotic drug is 0.01–50 mg / kg, preferably 5–20 mg / kg.

[0061] Preferably, after administration, a near-infrared laser with a wavelength of 780–2526 nm is applied, with the laser wavelength preferably being 780–1100 nm, and more preferably 808 nm.

[0062] The penetration depth of the anti-atherosclerotic drug in the plaque tissue can reach 0.5-3 cm, preferably 0.8-2 cm, and more preferably 1-1.5 cm;

[0063] The laser power is 0.1–5 W / cm². 2 The time is 1–20 min, and the laser temperature is 38–50℃;

[0064] After the laser is applied, the temperature of the patch area is 38-50°C; preferably, the temperature of the patch area is 38-45°C.

[0065] Preferably, after the anti-atherosclerotic drug is administered to the atherosclerotic plaque site, it produces at least one of the following effects on the target cells of the atherosclerotic plaque site under the action of a near-infrared laser with a wavelength of 780-2526nm: mild photothermal effect and photodynamic therapy effect.

[0066] When a mild photothermal effect is generated, the nanocomposite activates heat shock proteins with stress protection in target cells without causing significant apoptosis and death of target cells; it also reduces the inflammation level at the site of atherosclerotic plaques, increases the fibrin content of atherosclerotic plaques, and improves the stability of atherosclerotic plaques.

[0067] When photodynamic therapy is generated, the nanocomposite affects the expression of lipid metabolism-related genes and proteins in the target cells, thereby regulating the cholesterol content and cholesterol reversal transport in the target cells and reducing lipid content.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] 1. The present invention can be used to prepare nanocomposites for anti-atherosclerotic drugs, comprising an organic polymer carrier core material, wherein photosensitizers and / or sonosensitizers in the organic polymer carrier core material are encapsulated inside the organic polymer nanocarrier to improve drug loading rate; loading hydrophobic drugs into hydrophilic carriers can improve in vivo delivery effect;

[0070] 2. The present invention can be used to prepare nanocomposites for anti-atherosclerotic drugs. The outer surface of the organic polymer carrier core material is modified with a targeting peptide material that can recognize foam cells in plaques, which has the ability to target atherosclerotic plaques and can improve the therapeutic effect on atherosclerosis.

[0071] 3. The nanocomposite of the present invention, which can be used to prepare anti-atherosclerotic drugs, is modified on the outer surface of an organic polymer carrier core material with a contrast agent that has at least one function of enhancing photoacoustic imaging, photothermal imaging, magnetic resonance imaging, computed tomography imaging, ultrasound imaging, and fluorescence imaging signal function, thereby enhancing the imaging of cells in atherosclerotic plaques and realizing the visualization of treatment.

[0072] 4. The present invention provides an anti-atherosclerotic drug prepared from a nanocomposite that can be used to prepare anti-atherosclerotic drugs. When used, by adjusting the laser power, material concentration, and irradiation time, under 808nm laser irradiation, the nanocomposite generates a mild photothermal effect that activates heat shock proteins with stress protection in cells without causing significant apoptosis and death. Furthermore, it reduces the inflammation level at the atherosclerotic plaque site, increases the fibrin content of the atherosclerotic plaque, and improves the stability of the atherosclerotic plaque.

[0073] 5. The present invention can be used to prepare anti-atherosclerotic drugs. When the anti-atherosclerotic drugs prepared by the nanocomposite are used, the mild phototherapy can affect the expression of intracellular lipid metabolism-related genes and proteins, thereby regulating the intracellular cholesterol content and cholesterol reversal transport, and thus exerting a therapeutic effect on atherosclerotic plaques.

[0074] 6. The nanocomposite of the present invention, which can be used to prepare anti-atherosclerotic drugs, has a near-infrared light response. When using anti-atherosclerotic drugs containing the nanocomposite, the penetration depth in tissues is deeper, which can improve the therapeutic effect of phototherapy on atherosclerotic plaques. Attached Figure Description

[0075] Figure 1 The particle size and zeta potential of the nanocomposites in Examples 1, 2, and 4 of this invention are shown.

[0076] Figure 2 The images show cell counts (a) and micrographs (b) of macrophage-derived foam cells in Example 1 of this invention.

[0077] Figure 3 Microscopic images (a), quantitative graph of lipid content (b), and graph of cholesterol ester content (c) of macrophage-derived foam cells before and after laser treatment in Example 2 of the present invention.

[0078] Figure 4 Images of H&E, Oil Red O, Masson, iNOS, and CD206 staining in cross-sectional sections of the aorta in Example 3 of this invention are shown in (a) (scale bar 400 μm), along with quantitative maps of plaque area (b), collagen fiber content (c), iNOS area (d), and CD206 area (e). Detailed Implementation

[0079] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0080] Unless otherwise specified, the materials used in this invention are commercially available products, and the methods used are conventional technical means.

[0081] Example 1

[0082] In this embodiment, the nanocomposite that can be used to prepare anti-atherosclerotic drugs is IR780-PEG nanomicelles, specifically HS-PEG-COOH coated with the photosensitizer IR780.

[0083] The preparation method includes: weighing 120 mg HS-PEG-COOH, 50 mg IR780 and 110 μL TEA and dissolving them in 50 mL methanol, and stirring magnetically at 30 °C for 24 h; after the reaction, the mixture is placed in a 2000 Da dialysis bag and dialyzed in methanol solution for 2 days, changing the dialysate 3 times a day, and recovering the liquid after dialysis; using a rotary evaporator, the IR780-PEG solution is evaporated at 35 °C to remove methanol and obtain IR780-PEG nanomicelles.

[0084] Figure 1 The values ​​represent the particle size and zeta potential of the nanocomposite in this embodiment. The hydrated particle size is 28 nm. According to the zeta potential, the system has good stability.

[0085] Example 2

[0086] In this embodiment, the nanocomposite that can be used to prepare anti-atherosclerotic drugs is IR780-Gd nanomicelles, wherein the contrast agent NH2-DOTA-Gd is coupled as a modifying material on the surface of an organic polymer carrier core material, and the organic polymer carrier core material is IR780-PEG; wherein the loading of the modifying material is 18.2%.

[0087] Preparation methods include:

[0088] (1) Weigh 26 mg of gadolinium acetate hexahydrate and 20 mg of amino-macrocyclic ligand-glycyrrhetinic acid (NH2-DOTA-GA) and dissolve them in 10 mL of deionized water. Stir magnetically at 40 °C for 16 h to obtain NH2-DOTA-Gd solution.

[0089] (2) Weigh 120 mg HS-PEG-COOH, 50 mg IR780 and 110 μL TEA and dissolve them in 50 mL methanol. Stir magnetically at 30 °C for 24 h. After the reaction is complete, put the mixture into a 2000 Da dialysis bag and dialyze it in methanol solution for 2 days. Change the dialysate 3 times a day. After the dialysis is complete, recover the liquid. Use a rotary evaporator to evaporate the IR780-PEG solution at 35 °C to remove methanol and obtain IR780-PEG nano micelles.

[0090] (3) Weigh 24mg of IR780-PEG, 18.4mg of EDC and 27.6mg of NHS from (2) and dissolve them in 4mL of deionized water. Stir magnetically for 20min at room temperature. Then, slowly add PBS buffer to the mixture to raise the pH of the reaction solution to 7.3±0.2.

[0091] (4) Add the NH2-DOTA-Gd solution from (1) to (3) and stir magnetically for 16 hours at room temperature; put the mixture into a 1000Da dialysis bag and dialyze in deionized water for 2 days, changing the dialysate 3 times a day. After dialysis, freeze-dry the IR780-PEG-Gd solution.

[0092] (5) Weigh 12mg of IR780-PEG in (2) and 12mg of IR780-PEG-Gd in (3) and dissolve them in 20mL of methanol. Slowly add the solution dropwise to 2mL of deionized water and stir magnetically for 10min at room temperature.

[0093] (6) Using a rotary evaporator, the solution in (5) was evaporated at 35°C to remove methanol and IR780-Gd nanomicelles were obtained.

[0094] Figure 1The values ​​represent the particle size and zeta potential of the nanocomposite in this embodiment. The hydrated particle size is 92 nm. The absolute value of the zeta potential is significantly lower than that in Example 1, indicating that the stability of the system has deteriorated.

[0095] Example 3

[0096] In this embodiment, the nanocomposite that can be used to prepare anti-atherosclerotic drugs is IR780-PEG-OPN nanomicelles, wherein the targeting material osteopontin active short peptide (OPN) is coupled as a modifying material on the surface of an organic polymer carrier core material, and the organic polymer carrier core material is IR780-PEG; wherein the loading of the modifying material is 2.1%.

[0097] (1) Weigh 120 mg HS-PEG-COOH, 50 mg IR780 and 110 μL TEA and dissolve them in 50 mL methanol. Stir magnetically at 30 °C for 24 h. After the reaction is complete, put the mixture into a 2000 Da dialysis bag and dialyze it in methanol solution for 2 days. Change the dialysate 3 times a day. After the dialysis is complete, recover the liquid. Use a rotary evaporator to evaporate the IR780-PEG solution at 35 °C to remove methanol and obtain IR780-PEG nano micelles.

[0098] (2) Weigh 12mg of IR780-PEG, 2.6mg of EDC and 1.2mg of NHS from (2) and dissolve them in 2mL of deionized water. Stir magnetically for 4h at room temperature.

[0099] (3) Weigh 20 mg of osteopontin active short peptide (OPN) and dissolve it in 1 mL of deionized water;

[0100] (4) Slowly add the solution in (3) to the solution in (2) and stir magnetically for 16 hours at room temperature; then, put the mixture into a 3500 Da dialysis bag and dialyze in deionized water for 3 days, changing the dialysate 3 times a day. After the dialysis is completed, the liquid is recovered and the solution is freeze-dried to obtain a solid.

[0101] (5) Weigh 12mg of IR780-PEG from (2) and 12mg of solid from (4) and dissolve them in 20mL of methanol. Slowly add the solution dropwise to 2mL of deionized water and stir magnetically for 10min at room temperature.

[0102] (6) Using a rotary evaporator, the solution in (4) was evaporated at 35°C to remove methanol and IR780-PEG-OPN nanomicelles were obtained.

[0103] Example 4

[0104] In this embodiment, the nanocomposite that can be used to prepare anti-atherosclerotic drugs is IR780-Gd-OPN nanomicelles, wherein the organic polymer carrier core material is IR780-PEG, and the modifiers coupled to the surface of the organic polymer carrier core material are the targeting material osteopontin active short peptide (OPN) and the contrast agent NH2-DOTA-Gd, with a loading of 10.2%.

[0105] Preparation methods include:

[0106] (1) Weigh 120 mg HS-PEG-COOH, 50 mg IR780 and 110 μL TEA and dissolve them in 50 mL methanol. Stir magnetically at 30 °C for 24 h. After the reaction is complete, put the mixture into a 2000 Da dialysis bag and dialyze it in methanol solution for 2 days. Change the dialysate 3 times a day. After the dialysis is complete, recover the liquid. Use a rotary evaporator to evaporate the IR780-PEG solution at 35 °C to remove methanol and obtain IR780-PEG nano micelles.

[0107] (2) Weigh 12mg of IR780-PEG, 2.6mg of EDC and 1.2mg of NHS from (1) and dissolve them in 2mL of deionized water. Stir magnetically for 4h at room temperature.

[0108] (3) Weigh 20 mg of osteopontin active short peptide (OPN) and dissolve it in 1 mL of deionized water;

[0109] (4) Slowly add the solution in (3) to the solution in (2) and stir magnetically for 16 hours at room temperature; then, put the mixture into a 3500Da dialysis bag and dialyze in deionized water for 3 days, changing the dialysate 3 times a day. After dialysis, freeze-dry the IR780-PEG-OPN solution.

[0110] (5) Weigh 26 mg of gadolinium acetate hexahydrate and 20 mg of amino-macrocyclic ligand-glycyrrhetinic acid (NH2-DOTA-GA) and dissolve them in 10 mL of deionized water. Stir magnetically at 40 °C for 16 h to obtain NH2-DOTA-Gd solution.

[0111] (6) Weigh 24mg of IR780-PEG, 18.4mg of EDC and 27.6mg of NHS from (1) and dissolve them in 4mL of deionized water. Stir magnetically for 20min at room temperature. Then, slowly add PBS buffer to the mixture to raise the pH of the reaction solution to 7.3±0.2.

[0112] (7) Add the NH2-DOTA-Gd solution from (5) to (6) and stir magnetically for 16 hours at room temperature; put the mixture into a 1000Da dialysis bag and dialyze in deionized water for 2 days, changing the dialysate 3 times a day. After dialysis, freeze-dry the IR780-PEG-Gd solution.

[0113] (8) Dissolve 12 mg (7) of freeze-dried IR780-PEG-Gd and 12 mg (4) of freeze-dried IR780-PEG-OPN in 20 mL of methanol, slowly drop the mixture into 2 mL of deionized water, and stir magnetically for 10 min at room temperature.

[0114] (9) Using a rotary evaporator, the solution in (8) was evaporated at 35°C to remove methanol and IR780-Gd-OPN nanomicelles were obtained.

[0115] Figure 1 In this embodiment, the particle size and potential of the nanocomposite are as follows: the hydrated particle size is 108 nm. According to the zeta potential, the system has good stability.

[0116] Application Example 1

[0117] Cell experiments were conducted using the anti-atherosclerotic drugs prepared from the nanocomposites used in Examples 2 and 4, which can be used to prepare anti-atherosclerotic drugs.

[0118] (1) Set up five groups of cell experiments, and add an equal amount (1 ml) of macrophage-derived foam cell preservation solution to each culture dish;

[0119] (2) Group 1: Control group, no nanocomposite was added to the macrophage-derived foam cell preservation solution;

[0120] Group 2: IR780-Gd group, with anti-atherosclerotic drugs added to macrophage-derived foam cell preservation solution, specifically the nanocomposite of Example 2 at a concentration of 50 μg / mL, and the diluent being DMEM culture medium containing 10% fetal bovine serum;

[0121] Group 3: IR780-Gd-OPN group, anti-atherosclerotic drugs were added to macrophage-derived foam cell preservation solution, specifically the nanocomposite of Example 4 at a concentration of 50 μg / mL, and the diluent was DMEM culture medium containing 10% fetal bovine serum.

[0122] Group 4: IR780-Gd-OPN+NAC group, with anti-atherosclerotic drugs added to macrophage-derived foam cell preservation medium, specifically the nanocomposite of Example 4 at a concentration of 50 μg / mL, diluted with DMEM culture medium containing 10% fetal bovine serum; and 1 ml of reactive oxygen species inhibitor (NAC) added.

[0123] Group 5: IR780-Gd-OPN+J2 group, with anti-atherosclerotic drugs added to macrophage-derived foam cell preservation medium, specifically the nanocomposite of Example 4 at a concentration of 50 μg / mL, diluted with DMEM culture medium containing 10% fetal bovine serum; and 1 ml of heat shock protein inhibitor (J2) added.

[0124] (3) The cell viability of the five groups of cells was tested by the CCK-8 method; the five groups were stained with live and dead cells respectively to observe whether the cells died.

[0125] (4) Apply 808nm laser light to each of the five groups, with a laser intensity of 1W / cm². 2 The irradiation time was 5 minutes; the cell viability of the five groups of cells after laser application was tested using the CCK-8 assay.

[0126] (5) Observe the survival of the five groups of cells after laser treatment.

[0127] The results are as follows Figure 2 As shown, according to Figure 2 (a) It can be seen that (*: p < 0.05) that after the laser was applied, the cell activity in groups 1 and 4 increased slightly. This is because the nanocomposite in group 4 produced a mild photothermal effect, which activated the heat shock protein in the cell that has a stress protection function, without causing obvious apoptosis and death of the cells. Group 1 was a blank control group, without the addition of materials, without photothermal and photodynamic effects, and without the generation of harmful stimuli such as heat, so the cells did not undergo obvious apoptosis and death.

[0128] Cell activity was reduced in groups 2, 3, and 5, with group 5 showing the most significant reduction. This was due to the presence of heat shock protein inhibitors in group 5, which prevented the activation of heat shock proteins. In contrast, the nanocomplexes in groups 2 and 3 could activate heat shock proteins with stress protection effects in cells to some extent, reducing cell apoptosis and death.

[0129] according to Figure 2 (b) It can be seen (scale bar is 25μm) that before the laser was applied, all cells in groups 1 to 3 were alive, while some dead cells appeared in groups 4 and 5, and the number of dead cells in both groups was similar. The NAC and J2 reagents added to groups 4 and 5 may have affected cell survival, or the presence of some dead cells may be due to unavoidable stimulation of cells during the operation.

[0130] After laser application, all cells in groups 1 to 3 remained viable. The number of dead cells increased in groups 4 and 5, but the increase in dead cells in group 4 was not significant, while the number of dead cells in group 5 increased considerably. This is because the reactive oxygen species inhibitor (NAC) added in group 4 inhibits the production of intracellular reactive oxygen species and reduces photodynamic effects, thus not affecting the activation of intracellular heat shock proteins. However, the presence of heat shock protein inhibitors in group 5 prevents the activation of intracellular heat shock proteins that have a stress-protective function, resulting in more cell damage.

[0131] Application Example 2

[0132] Cellular experiments were conducted using an anti-atherosclerotic drug prepared from a nanocomposite that can be used to prepare anti-atherosclerotic drugs.

[0133] (1) Set up four groups of cell experiments, and add an equal amount (1 ml) of macrophage-derived foam cell preservation solution to each culture dish;

[0134] (2) Group 1: Control group, no nanocomposite was added to the culture dish;

[0135] Group 2: IR780-Gd group, with anti-atherosclerotic drugs added to the culture dish, specifically the nanocomposite of Example 2 at a concentration of 50 μg / mL, and the diluent being DMEM culture medium containing 10% fetal bovine serum;

[0136] Group 3: IR780-Gd-OPN group, with anti-atherosclerotic drugs added to the culture dish, specifically the nanocomposite of Example 4 at a concentration of 50 μg / mL, and the diluent being DMEM culture medium containing 10% fetal bovine serum;

[0137] Group 4: IR780-Gd-OPN+NAC group, anti-atherosclerotic drugs were added to the culture dish, specifically the nanocomposite of Example 4 with a concentration of 50 μg / mL, and the diluent was DMEM culture medium containing 10% fetal bovine serum; then (1 ml) reactive oxygen species inhibitor (NAC) was added.

[0138] (3) The four groups of cells were stained using Oil Red O staining method;

[0139] (4) Apply 808nm laser light with a laser intensity of 1W / cm². 2 The irradiation time is 5 minutes;

[0140] Figure 3 (a) Microscopic images of macrophage-derived foam cells in each group before and after laser treatment (scale bar: 50 μm); Figure 3 (b) is a quantitative graph of lipid content in macrophage-derived foam cells in each group before and after laser treatment; Figure 3(c) Plots showing cholesterol ester content in macrophage-derived foam cells before and after laser treatment (*: p < 0.05; **: p < 0.01);

[0141] from Figure 3 As shown in (a), after the addition of nanocomplexes in groups 2 to 4, the lipid content in foam cells decreased, proving that the mild phototherapy mediated by nanocomplexes can regulate lipid metabolism in foam cells and reduce the lipid content in cells.

[0142] from Figure 3 As shown in (b), under the action of laser, the lipid content in foam cells of groups 2 to 4 decreased after the addition of nanocomposite. The lipid content of group 3, which added IR780-Gd-OPN, decreased the most. The reason why the lipid content of group 4, which added IR780-Gd-OPN+NAC, did not decrease much is that the addition of NAC inhibited the photodynamic effect and weakened the phototherapy effect. Therefore, the decrease in lipid content in cells of IR780-Gd-OPN+NAC group under laser irradiation was not as obvious as that of IR780-Gd-OPN group.

[0143] from Figure 3 As shown in (c), under the action of laser, the cholesterol ester content of all four groups of foam cells decreased. Among them, the group with IR780-Gd-OPN added in group 3 showed the most significant decrease, which proved that under mild photothermal conditions, the nanocomposite can affect the expression of lipid metabolism-related genes and proteins in macrophage-derived foam cells, thereby regulating the cholesterol content and cholesterol reverse transport in macrophage-derived foam cells. In group 4, the presence of reactive oxygen species inhibitor (NAC) affected the production of intracellular reactive oxygen species, inhibited photodynamic effects, and reduced the phototherapy effect.

[0144] Application Example 3

[0145] Animal experiments were conducted using an anti-atherosclerotic drug prepared from a nanocomposite that can be used to prepare anti-atherosclerotic drugs.

[0146] (1) Five groups of experiments were set up, and animal models were established according to methods known to those skilled in the art. Interventional drug administration was performed on the target sites of the five groups of mice with atherosclerosis. The drug dosage was the same for all five groups (10 mg / kg).

[0147] (2) Group 1: PBS group, the drug is sterile PBS solution;

[0148] Group 2: IR780-Gd group, the anti-atherosclerotic drug was the nanocomposite of Example 2 at a concentration of 50 μg / mL, and the diluent was sterile PBS solution;

[0149] Group 3: IR780-Gd+Laser group, the anti-atherosclerotic drug was the nanocomposite of Example 2 at a concentration of 50 μg / mL, the diluent was sterile PBS; and an 808 nm laser was applied with a laser intensity of 1 W / cm². 2 The irradiation time is 5 minutes;

[0150] Group 4: IR780-Gd-OPN group, the anti-atherosclerotic drug is the nanocomposite of Example 4 at a concentration of 50 μg / mL, and the diluent is sterile PBS solution;

[0151] Group 5: IR780-Gd-OPN+Laser group, the anti-atherosclerotic drug was the nanocomposite of Example 4 at a concentration of 50 μg / mL, the diluent was sterile PBS; and an 808 nm laser was applied with a laser intensity of 1 W / cm². 2 The irradiation time is 5 minutes;

[0152] (3) Aortic cross-sectional sections from each group were stained with H&E, Oil Red O, Masson's, iNOS, and CD206. Specific staining images are shown below. Figure 4 (a) H&E (hematoxylin-eosin staining) was used to characterize the tissue; Oil Red O was used to stain the lipids in the plaque to observe the size of the aortic plaque; Masson staining was used to stain the collagen fibers in the plaque to observe the stability of the plaque; iNOS (M1 macrophage marker) and CD206 (M2 macrophage marker) were used to observe the proportion of M1 and M2 macrophages in the plaque, respectively, reflecting the level of inflammation at the plaque site (M1 is pro-inflammatory, M2 is anti-inflammatory). Figure 4 (b) is a quantitative map of the patch area. Figure 4 (c) is a quantitative graph of collagen fiber content. Figure 4 (d) is a quantized map of the iNOS area. Figure 4 (e) is a quantized plot of the CD206 area; (*: p < 0.05; **: p < 0.01; ***: p < 0.001);

[0153] from Figure 4 As shown in (a, b), the lipid area of ​​plaque in group 5 decreased significantly, proving that under mild photothermal conditions, the nanocomposite can reduce the lipid content in the plaque and reduce the aortic plaque area.

[0154] from Figure 4 As shown in (a, c), the collagen fiber content in group 5 plaques increased significantly, proving that under mild photothermal conditions, the nanocomposite can improve plaque stability.

[0155] from Figure 4As shown in (a, d, e), the area of ​​iNOS in plaque group 5 was significantly reduced and the area of ​​CD206 was significantly increased, demonstrating that under mild photothermal conditions, the nanocomposite can reduce the proportion of M1 macrophages and increase the proportion of M2 macrophages in plaques, thereby reducing the level of inflammation at the plaque site and improving plaque stability.

[0156] Example 5

[0157] The nanocomposite in this embodiment is IR808-DSPE-PEG-COOH.

[0158] The preparation method includes: weighing 110 mg DSPE-PEG-COOH, 50 mg IR808 and 100 μL LTEA and dissolving them in 60 mL of methanol, and stirring magnetically at 30 °C for 24 h; after the reaction, the mixture is placed in a 3000 Da dialysis bag and dialyzed in methanol solution for 2 days, changing the dialysate 3 times a day, and recovering the liquid after dialysis; using a rotary evaporator, the solution is evaporated at 35 °C to remove methanol to obtain IR808-DSPE-PEG-COOH.

[0159] Example 6

[0160] The nanocomposite in this embodiment is IR808-DSPE-PEG-M2pep-Gd;

[0161] (1) Weigh 12 mg of IR808-DSPE-PEG-COOH, 2.6 mg of EDC and 1.2 mg of NHS from Example 5 and dissolve them in 2 mL of deionized water. Stir magnetically for 4 h at room temperature.

[0162] (2) Weigh 30 mg of M2 macrophage-targeting peptide (M2pep) and dissolve it in 1 mL of deionized water;

[0163] (3) Slowly add the solution in (2) to the solution in (1) and stir magnetically for 16 hours at room temperature; then, put the mixture into a 4500 Da dialysis bag and dialyze in deionized water for 2 days, changing the dialysate 3 times a day. After the dialysis is completed, the liquid is recovered and freeze-dried.

[0164] (4) Weigh 26 mg of gadolinium acetate hexahydrate and 20 mg of amino-macrocyclic ligand-glycyrrhetinic acid (NH2-DOTA-GA) and dissolve them in 10 mL of deionized water. Stir magnetically at 40 °C for 16 h to obtain NH2-DOTA-Gd solution.

[0165] (5) Weigh 24 mg of IR808-DSPE-PEG-COOH, 18.4 mg of EDC and 27.6 mg of NHS from Example 5 and dissolve them in 4 mL of deionized water. Stir magnetically for 20 min at room temperature. Then, slowly add PBS buffer to the mixture to raise the pH of the reaction solution to 7.3 ± 0.2.

[0166] (6) Add the NH2-DOTA-Gd solution from (4) to (5) and stir magnetically for 16 hours at room temperature; put the mixture into a 2000Da dialysis bag and dialyze in deionized water for 2 days, changing the dialysate 3 times a day. After dialysis, freeze-dry the IR808-DSPE-PEG-Gd solution.

[0167] (7) Weigh 12mg of IR808-DSPE-PEG-M2pep from (3) and 12mg of IR808-DSPE-PEG-Gd from (6) and dissolve them in 10mL of methanol. Slowly add the solution dropwise to 2mL of deionized water and stir magnetically for 10min at room temperature.

[0168] (8) Using a rotary evaporator, the solution in (7) was evaporated at 35°C to remove methanol and IR808-DSPE-PEG-M2pep-Gd was obtained.

[0169] In summary, the nanocomposite of the present invention, which can be used to prepare anti-atherosclerotic drugs, comprises an organic polymer carrier core material, and / or a modified material loaded on the outer surface of the core material with a core-shell structure. Under laser irradiation, it can exert mild photothermal and / or photodynamic therapeutic effects. In the mild photothermal effect, it activates heat shock proteins with stress-protective functions without causing significant cell apoptosis and death; it also reduces the inflammation level at atherosclerotic plaque sites, increases the fibrin content of atherosclerotic plaques, and improves the stability of atherosclerotic plaques. In the photodynamic therapeutic effect, it affects the expression of intracellular lipid metabolism-related genes and proteins, thereby regulating intracellular cholesterol content and cholesterol reverse transport, and reducing lipid content. It achieves the inhibition of atherosclerosis while controlling plaque cell apoptosis levels, ensuring safety; and it enables visualized treatment.

[0170] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. An anti-atherosclerotic drug, characterized in that, The anti-atherosclerotic drug contains a nanocomposite IR780-Gd-OPN that can be used to prepare the anti-atherosclerotic drug; The nanocomposite is a composite material on which a modifying material is loaded onto the outer surface of an organic polymer carrier core material; the modifying material includes a targeting material C and a contrast agent D. The organic polymer carrier core material is IR780-PEG, and the modification materials are the target material osteopontin active short peptide OPN and the contrast agent NH2-DOTA-Gd. After the anti-atherosclerotic drug is administered to the atherosclerotic plaque site, it produces at least one of the following effects on the target cells of the atherosclerotic plaque site under the action of near-infrared laser with a wavelength of 780~2526nm: mild photothermal effect and photodynamic therapy effect. When a mild photothermal effect is generated, the nanocomposite activates heat shock proteins with stress protection in target cells without causing apoptosis or death of target cells; it also reduces the inflammation level at the site of atherosclerotic plaques, increases the fibrin content of atherosclerotic plaques, and improves the stability of atherosclerotic plaques. When photodynamic therapy is generated, the nanocomposite affects the expression of lipid metabolism-related genes and proteins in the target cells, thereby regulating the cholesterol content and cholesterol reversal transport in the target cells and reducing lipid content.

2. The anti-atherosclerotic drug according to claim 1, characterized in that, The preparation method of the organic polymer carrier core material includes: Weigh 120 mg HS-PEG-COOH, 50 mg IR780 and 110 μL TEA and dissolve them in 50 mL methanol. Stir magnetically at 30 °C for 24 h. After the reaction is complete, put the mixture into a 2000 Da dialysis bag and dialyze it in methanol solution for 2 days, changing the dialysate 3 times a day. After dialysis, recover the liquid. Use a rotary evaporator to evaporate the IR780-PEG solution at 35 °C to remove methanol and obtain IR780-PEG nanomicelles.

3. The anti-atherosclerotic drug according to claim 1, characterized in that, The preparation method of the nanocomposite includes: (1) Weigh 120 mg HS-PEG-COOH, 50 mg IR780 and 110 µL TEA and dissolve them in 50 mL methanol. Stir magnetically at 30 °C for 24 h. After the reaction is complete, put the mixture into a 2000 Da dialysis bag and dialyze it in methanol solution for 2 days. Change the dialysate 3 times a day. After the dialysis is complete, recover the liquid. Use a rotary evaporator to evaporate the IR780-PEG solution at 35 °C to remove methanol and obtain IR780-PEG nano micelles. (2) Weigh 12mg of IR780-PEG, 2.6mg of EDC and 1.2mg of NHS from (1) and dissolve them in 2mL of deionized water. Stir magnetically for 4h at room temperature. (3) Weigh 20 mg of osteopontin active short peptide OPN and dissolve it in 1 mL of deionized water; (4) Slowly add the solution in (3) to the solution in (2) and stir magnetically for 16 hours at room temperature; then, put the mixture into a 3500Da dialysis bag and dialyze in deionized water for 3 days, changing the dialysate 3 times a day. After dialysis, freeze-dry the IR780-PEG-OPN solution. (5) Weigh 26 mg of gadolinium acetate hexahydrate and 20 mg of amino-macrocyclic ligand-glycyrrhetinic acid NH2-DOTA-GA and dissolve them in 10 mL of deionized water. Stir magnetically at 40 °C for 16 h to obtain NH2-DOTA-Gd solution. (6) Weigh 24mg of IR780-PEG, 18.4mg of EDC and 27.6mg of NHS from (1) and dissolve them in 4mL of deionized water. Stir magnetically for 20min at room temperature. Then, slowly add PBS buffer to the mixture to raise the pH of the reaction solution to 7.3±0.

2. (7) Add the NH2-DOTA-Gd solution from (5) to (6) and stir magnetically for 16 hours at room temperature; put the mixture into a 1000Da dialysis bag and dialyze in deionized water for 2 days, changing the dialysate 3 times a day. After dialysis, freeze-dry the IR780-PEG-Gd solution. (8) Dissolve 12 mg (7) of freeze-dried IR780-PEG-Gd and 12 mg (4) of freeze-dried IR780-PEG-OPN in 20 mL of methanol, slowly add the mixture to 2 mL of deionized water, and stir magnetically for 10 min at room temperature; (9) Using a rotary evaporator, the solution in (8) was evaporated at 35°C to remove methanol and IR780-Gd-OPN nanomicelles were obtained.

4. The anti-atherosclerotic drug according to claim 1, characterized in that, The concentration of the nanocomposite in the anti-atherosclerotic drug is 0.01~1000µg / mL.

5. The anti-atherosclerotic drug according to claim 1, characterized in that, The anti-atherosclerotic drug can target target cells in atherosclerotic plaques; The target cells include at least one of activated macrophages, activated endothelial cells, activated smooth muscle cells, and foam cells in atherosclerotic plaques.

6. The anti-atherosclerotic drug according to claim 1, characterized in that, The administration route of the anti-atherosclerotic drug includes at least one of systemic administration and intravascular interventional administration; the dosage of the anti-atherosclerotic drug is 0.01~100 mg / kg; After administration, a near-infrared laser with a wavelength of 780~2526nm is applied; The laser power is 0.1~5W / cm. 2 The time is 1~20min, and the laser temperature is 38~50℃; After the laser is applied, the temperature of the patch area is 38~50℃; The nanocomposite can penetrate 0.5 to 3 cm into the tissue at the plaque site.

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