Nano-drug and preparation method thereof

Abstract

The invention relates to the technical field of vascular therapy, in particular to a nano-drug and a preparation method thereof.The nano-drug comprises a nano-carrier, a therapeutic drug and cross-linked protein are loaded on the nano-carrier, the cross-linked protein is at least partially loaded on the surface of the nano-carrier, and the cross-linked protein is at least one of silk fibroin, sericin and silk fibroin peptide; the therapeutic drug is at least partially loaded in the nano-carrier. On one hand, cross-linked protein on the surface of the nano-carrier is promoted to be cross-linked with protein in a blood vessel by initiating the photosensitive cross-linking agent, and the nano-carrier is anchored in the blood vessel, so that the risk that nano-drug particles are eluted from the blood vessel wall is greatly reduced; on the other hand, cross-linking of cross-linked protein on the surface of the nano-carrier can be promoted by initiating the photosensitive cross-linking agent, so that the surface of the nano-carrier is gelatinized, the slow release speed of the nano-drug anchored in the blood vessel is more lasting, and further effective drug concentration can be maintained for a long time, restenosis of the blood vessel is inhibited, and the treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of vascular treatment, and in particular to a nano drug and a preparation method thereof. Background Art

[0002] Atherosclerosis is the main cause of coronary heart disease, cerebral infarction, and peripheral arterial disease, and a drug-eluting tube is a method for treating atherosclerotic diseases. It includes an inner tube, an outer tube, and a balloon. By opening micropores on the surface of the balloon, the therapeutic drug is delivered from the proximal end of the drug-eluting tube to the distal end of the balloon, forcing the balloon to expand and adhere to the blood vessel wall corresponding to the target lesion location. The therapeutic drug is delivered to the target lesion location through the micropores on the balloon, achieving healing and repair of the blood vessels and achieving the therapeutic effect. However, in the process of realizing the present invention, the applicant found that for existing drugs used in conjunction with drug-eluting tubes to treat atherosclerotic diseases, the drug particles are easily eluted after being delivered to the blood vessel wall, affecting the therapeutic effect. Summary of the Invention

[0003] The purpose of this application is to provide a nanomedicine and a preparation method thereof to solve the above-mentioned technical problems existing in the prior art, which mainly includes the following main contents:

[0004] In a first aspect, the present application provides a nanomedicine comprising a nanocarrier, wherein the nanocarrier is loaded with a therapeutic drug and a cross-linked protein, wherein the cross-linked protein is at least partially loaded on the surface of the nanocarrier, and the cross-linked protein is at least one of silk fibroin, sericin, and silk peptide, and the therapeutic drug is at least partially loaded inside the nanocarrier.

[0005] Furthermore, a photosensitive crosslinking agent is also included.

[0006] Furthermore, the photosensitive crosslinking agent is at least one of a naphthalimide compound, a hydrophilic derivative of naphthalimide, camphorquinone, a hydrophilic derivative of camphorquinone, riboflavin, riboflavin sodium phosphate, rose bengal, curcumin, and eosin Y.

[0007] Furthermore, in the nanomedicine, the mass percentage content of the photosensitive crosslinking agent is 1% to 99%.

[0008] Furthermore, the size of the nanocarrier is 10 nm to 2000 nm;

[0009] And / or, the size of the photosensitive crosslinking agent is 1 nm to 2000 nm.

[0010] Furthermore, it also includes a photosensitizer.

[0011] Furthermore, the mass ratio of the photosensitizer to the photosensitizer is 0.02 to 5:1.

[0012] Furthermore, the photosensitizer is at least one of diphenyliodonium hexafluorophosphate, ethyl p-dimethylaminobenzoate, potassium persulfate, sodium persulfate and ammonium persulfate.

[0013] Furthermore, the invention further comprises at least one of an active drug, a penetration enhancer, a cell-penetrating peptide, oxygen and an oxygen delivery substance, wherein the active drug is used to enhance the therapeutic effect of the therapeutic drug.

[0014] Furthermore, the carrier is polyester, poly (amino acid), copoly (ether-ester), polyalkylene oxalate, polyamide, poly (imino carbonate), polyorthoester, polyethylene oxide, polyamide ester, polyethylene oxide containing amide groups, poly (anhydride), polyphosphazene, polyhydroxy acid, poly α-hydroxy acid, trimethylene carbonate, polyorganophosphazene, polyesteramide, polyethylene oxide, polyester ether, polyphosphate, polyphosphite polyurethane, cyanoacrylate, poly (trimethylene carbonate), poly (imino carbonate), polyalkylene oxalate, polyvinyl pyrrolidone, polyvinyl alcohol, shellac, gum arabic, poly-N- (2-alkylpropyl) -methacrylamide At least one of amines, polyglycols, aliphatic polyesters, poly(orthoesters), poly(ester-amides), polyanhydrides, poloxamer, hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharides, gelatin, Tween, amphiphilic diblock or block copolymers, lecithin, phosphatidylethanolamine, phosphatidylserine, iodine-containing contrast agents, sucrose, trehalose, mannitol, lactose, glucose, maltose, proline, tryptophan, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, lysine, polyarginine, polypeptide dendrimers, albumin, silk fibroin, sericin, silk peptides, and casein.

[0015] The second aspect of the present application provides a method for preparing a nanodrug, which is used to prepare the above-mentioned nanodrug, comprising the following steps: dissolving the therapeutic drug in an organic solvent that is miscible with water as an organic phase, dissolving the cross-linked protein in water as an aqueous phase, dissolving the nanocarrier along with the therapeutic drug or the cross-linked protein, then mixing and stirring the organic phase and the aqueous phase, and then dialyzing to remove the organic solvent.

[0016] Compared with the prior art, the present invention has at least the following technical effects:

[0017] On the one hand, the present invention triggers a photosensitive cross-linking agent to promote the cross-linking of cross-linked proteins on the surface of the nanocarrier with proteins in the blood vessels, thereby anchoring the nanocarrier in the blood vessels, thereby greatly reducing the risk of elution of nano-drug particles from the blood vessel wall. On the other hand, the triggering of the photosensitive cross-linking agent also promotes the cross-linking of cross-linked proteins on the surface of the nanocarrier, allowing multiple independent cross-linked proteins on the surface of the nanocarrier to cross-link with each other, causing the surface of the nanocarrier to gel, thereby slowing down the release rate of the drug inside the nanocarrier, making the sustained release rate of the nano-drug anchored in the blood vessels more sustained, thereby maintaining an effective drug concentration for a long time, inhibiting restenosis of the blood vessels, and improving the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 It is a schematic diagram of the structure of the drug dilation tube used in the test of the present invention. DETAILED DESCRIPTION

[0020] The following description provides many different embodiments or examples for implementing different features of the present invention. The components and arrangements described in the following specific examples are only used to simplify the present invention and are only used as examples, not to limit the present invention.

[0021] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention for which protection is sought, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Atherosclerosis is a major cause of coronary heart disease, cerebral infarction, and peripheral arterial disease. Drug-eluting catheters are a treatment for atherosclerosis. They include an inner tube, an outer tube, and a balloon. Micropores are formed on the balloon surface, allowing the delivery of therapeutic drugs from the proximal end of the drug-eluting catheter to the distal end. After expansion, the balloon is forced to approach the vascular wall corresponding to the target lesion. The therapeutic drug is delivered to the target lesion through the micropores on the balloon. The therapeutic drug rapidly binds to collagen in the vascular wall, forming an in situ stent, achieving vascular healing and repair, and achieving a therapeutic effect. Existing drugs used with drug-eluting catheters to treat atherosclerosis, such as the micro-nano drug and drug delivery system disclosed in Chinese Patent Publication No. CN116726184A, comprise a carrier and an active drug loaded on the carrier. A carrier containing a hydrophilic segment is used to bind to calcified lesions, thereby improving the transfer efficiency and retention time of the drug at the lesion site. However, the carrier containing a hydrophilic segment has limited binding efficiency and effectiveness with calcified lesions and is easily eluted during the binding process, resulting in less-than-expected therapeutic effects. In order to solve the problem that the therapeutic effect is affected by the easy elution of nanomedicines, the present application provides a nanomedicine that can reduce the risk of nanomedicine elution, as shown in the following examples.

[0023] Example 1

[0024] An embodiment of the present application provides a nanomedicine, including a nanocarrier, on which a therapeutic drug and a cross-linked protein are loaded, wherein the cross-linked protein is at least partially loaded on the surface of the nanocarrier, the cross-linked protein is at least one of silk fibroin, sericin and silk peptide, and the therapeutic drug is at least partially loaded on the interior of the nanocarrier.

[0025] During use, the nanocarrier loaded with therapeutic drugs and cross-linked proteins can be mixed with a photosensitive cross-linker to obtain a mixed solution, which is then transported from the proximal end of the drug dilation tube to the distal balloon, forcing the balloon to expand and contact the vascular wall corresponding to the target lesion location. The mixed solution is transported to the target lesion location through the micropores on the balloon, allowing the nanocarrier and the photosensitive cross-linker to directly contact the vascular wall. At the same time, a laser is generated at the distal end of the drug dilation tube, and the photosensitive cross-linker is triggered by the laser. On the one hand, the cross-linked protein on the surface of the nanocarrier is cross-linked with the protein in the blood vessel by triggering the photosensitive cross-linker, thereby anchoring the nanocarrier. In the blood vessels, the risk of elution of nano-drug particles from the blood vessel wall is greatly reduced. On the other hand, due to the use of silk fibroin, sericin, and silk peptide as cross-linking proteins, the cross-linking proteins on the surface of the nanocarrier are promoted to cross-link by triggering a photosensitive cross-linking agent, so that multiple independent cross-linking proteins on the surface of the nanocarrier are cross-linked with each other, and the surface of the nanocarrier is gelled, thereby slowing down the release rate of drugs inside the nanocarrier, making the sustained release rate of nano-drugs anchored in the blood vessels more sustained, thereby maintaining effective drug concentrations for a long time, inhibiting restenosis of blood vessels, and improving treatment effects.

[0026] Specifically, the carrier can be selected from polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(imino carbonates), polyorthoesters, polyethylene oxides, polyamide esters, polyethylene oxides containing amide groups, poly(anhydrides), polyphosphazenes, polyhydroxy acids, poly-α-hydroxy acids, trimethylene carbonate, polyorganophosphazenes, polyesteramides, polyethylene oxides, polyester ethers, polyphosphates, polyphospholipid polyurethanes, cyanoacrylates, poly(trimethylene carbonate), poly(imino carbonates), polyalkylene oxalates, polyvinyl pyrrolidone, polyvinyl alcohol, shellac, gum arabic, poly-N-(2-alkylpropyl)-methacrylic acid At least one of amide, polyglycol, aliphatic polyester, poly(orthoester), poly(ester-amide), polyanhydride, poloxamer, hyaluronic acid, polyvinyl alcohol, polyvinyl pyrrolidone, polysaccharide, gelatin, Tween, amphiphilic diblock or block copolymer, lecithin, phosphatidylethanolamine, phosphatidylserine, iodine-containing contrast agent, sucrose, trehalose, mannitol, lactose, glucose, maltose, proline, tryptophan, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, lysine, polyarginine, polypeptide dendrimer, albumin, silk fibroin, sericin, silk peptide, and casein.

[0027] In some embodiments, nanomedicines can be obtained by physical crushing methods to obtain nanomedicines of preset sizes, and physical crushing methods include but are not limited to high-pressure homogenization, medium milling, microfluidization, etc.; nanomedicines can also induce drug molecules to aggregate or crystallize to form nanoscale drug particles, and preparation methods include but are not limited to nanoprecipitation, emulsification, supercritical fluid method, etc.; illustratively, the therapeutic drug can be dissolved in an organic solvent miscible with water as an organic phase, and the organic solvent can be selected from at least one of ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide; the cross-linked protein is dissolved in water as the aqueous phase, and the nanocarrier is dissolved along with the therapeutic drug or cross-linked protein, and then the organic phase and the aqueous phase are mixed and stirred, preferably, the aqueous phase is stirred, and the organic phase is added to the aqueous phase for mixing and stirring; and then the organic solvent is removed by dialysis.

[0028] Example 2

[0029] An embodiment of the present application provides a nanomedicine, including a nanocarrier, on which a therapeutic drug and a cross-linked protein are loaded, wherein the cross-linked protein is at least partially loaded on the surface of the nanocarrier, the cross-linked protein is at least one of silk fibroin, sericin and silk peptide, and the therapeutic drug is at least partially loaded on the interior of the nanocarrier.

[0030] Specifically, the photosensitive crosslinking agent may be at least one selected from the group consisting of naphthalimide compounds, hydrophilic derivatives of naphthalimide, camphorquinone, hydrophilic derivatives of camphorquinone, riboflavin, riboflavin sodium phosphate, rose bengal, curcumin, and eosin Y.

[0031] The higher the enrichment concentration of the photosensitive crosslinker in the nanomedicine, the higher the crosslinking efficiency under light excitation. To improve the crosslinking efficiency, in some embodiments, the mass percentage content of the photosensitive crosslinker in the nanomedicine can be controlled to be 1% to 99%; preferably, the mass percentage content of the photosensitive crosslinker is 1% to 99%; preferably, the mass percentage content of the photosensitive crosslinker is 20% to 90%; more preferably, the mass percentage content of the photosensitive crosslinker is 40% to 90%.

[0032] Specifically, the size of the nanocarrier is 10 nm to 2000 nm; the size of the photosensitive crosslinker is 1 nm to 2000 nm.

[0033] Example 3

[0034] This embodiment of the present application provides a nanomedicine, which is different from Example 2 in that the nanomedicine further includes a photosensitizer, and the photoinitiation efficiency is enhanced by adding the photosensitizer.

[0035] Specifically, the mass ratio of the photosensitizer to the photosensitizer is 0.02 to 5:1.

[0036] Specifically, the photosensitizer may be selected from at least one of diphenyliodonium hexafluorophosphate, ethyl p-dimethylaminobenzoate, potassium persulfate, sodium persulfate, and ammonium persulfate.

[0037] Example 4

[0038] The embodiment of the present application provides a nanomedicine, which is different from embodiment 2 or 3. The nanomedicine also includes an active drug, and the therapeutic effect of the therapeutic drug is enhanced by adding the active drug.

[0039] Specifically, the active drug can be selected from rapamycin or its derivatives, ABT-578, zotarolimus, everolimus, biolimus A9, deforolimus (also known as rapamycin-42 (dimethylphosphinate) (ridaforolimus)), temsirolimus, tacrolimus, pimcrolimus, nitric oxide synthase, C3 exoenzyme, RhoA inhibitor, tubulusin, A3 agonist, CB2 agonist, 17-AAG, Hsp90 antagonist, tyrphostins, cathepsin S inhibitor, paclitaxel or its derivatives, taxol, docetaxel, corticosteroids, glucocorticoids, dexamethasone, ceramide, dimethylsphingosine, ether-linked diglycerides, ether-linked phosphatidic acid, sphinganines, estrogens, taxol, taxol analogs (taxol analogs), actinomycin D, prostaglandins, vitamin A, probucol, batimastat, statins, trapidil, mitomycin C and cytochalasin B, anti-angiogenic agents, antibodies.

[0040] Example 5

[0041] An embodiment of the present application provides a nanodrug, which is different from Example 2, 3 or 4. The nanodrug also includes a permeation enhancer and / or a cell-penetrating peptide. The mass ratio of the permeation enhancer and / or cell-penetrating peptide to the carrier is preferably controlled at 0.01 to 1:1, thereby enhancing the transfer of the nanodrug to the vascular media and adventitia, allowing protein cross-linking to occur simultaneously in the vascular intima, media and adventitia, thereby improving the therapeutic effect and cross-linking efficiency.

[0042] Specifically, the penetration enhancer can be selected from at least one of fatty acids and derivatives thereof, oleic acid, lauric acid, monoolein, terpenoids, menthol, limonene, farnesol, alcohol compounds, ethanol, isopropyl alcohol, lauryl alcohol, myristyl alcohol, sodium lauryl sulfate, Tween 80, lecithin, azone, fatty alcohols, propylene glycol, N-methyl-2-pyrrolidone, 2-pyrrolidone, estradiol, progesterone, acyclovir, salicylic acid, dimethyl sulfoxide, decyl methyl sulfoxide, benzalkonium chloride, ethylenediaminetetraacetic acid, tetracaine, tetrahydropiperine, inositol, hydroxypropyl cyclodextrin, polylysine, betaine, and hydrolyzed chitin.

[0043] Specifically, the cell-penetrating peptide can be selected from at least one of cationic peptides, amphipathic peptides, hydrophobic peptides, polyarginine, polyarginine, TAT polypeptide, hLF, (RXR)4, deca-arginine (R10), nona-arginine (R9), and octa-arginine (R8).

[0044] Example 6

[0045] An embodiment of the present application provides a nanomedicine, which is different from Examples 2, 3, 4 or 5. The nanomedicine also includes oxygen and / or oxygen delivery substances. The mass ratio of oxygen and / or oxygen delivery substances to the carrier is preferably controlled at 0.02 to 0.8:1; the intensity of the cross-linking effect produced by the photosensitive cross-linker during light excitation depends in part on the oxygen concentration around the tissue. By adding oxygen and / or oxygen delivery substances, the oxygen concentration around the cross-linking environment is increased, thereby improving the cross-linking effect.

[0046] Specifically, the oxygen delivery substance can be selected from at least one of hemoglobin, myoglobin, earthworm hemoglobin, hemocyanin, and perfluorocarbons. Preferably, the oxygen delivery substance and the photosensitive crosslinker are co-assembled with a carrier to form nanoparticles. After the oxygen delivery substance is loaded with oxygen, it is loaded into the nanoparticles together with the photosensitive crosslinker, resulting in a higher oxygen loading capacity. Oxygen loading can be performed during nanoparticle preparation or during preparation of the nanoparticle solution during surgery.

[0047] Experimental Example 1

[0048] 1.256 mg of riboflavin sodium phosphate and 12.56 mg of dextran were weighed and dissolved in 1.256 mL of purified water, designated Solution 1. 2.512 mg of rapamycin and 5.024 mg of lecithin were weighed and dissolved in 1 mL of ethanol. The mixture was then added to 5 mL of a 1 mg / mL silk fibroin aqueous solution and rapidly stirred to form nanoparticles. Stirring was continued to evaporate the organic solvent, and the nanoparticles were then placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000 Mw and dialyzed for 12 hours to remove unentrapped drug. This was designated Solution 2. Solutions 1 and 2 were then mixed and freeze-dried to produce the nanoparticles of the present invention.

[0049] Experimental Example 2

[0050] 1.256 mg of riboflavin sodium phosphate, 0.1256 mg of potassium persulfate, and 12.56 mg of dextran were weighed and dissolved in 1.256 mL of purified water, designated Solution 1. 2.512 mg of rapamycin and 5.024 mg of lecithin were weighed and dissolved in 1 mL of ethanol. The mixture was then added to 5 mL of a 1 mg / mL silk fibroin aqueous solution and rapidly stirred to form nanoparticles. Stirring was continued to evaporate the organic solvent, and the nanoparticles were then placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000 Mw and dialyzed for 12 hours to remove unentrapped drug. This was designated Solution 2. Solutions 1 and 2 were then mixed and freeze-dried to produce the nanoparticles of the present invention.

[0051] Experimental Example 3

[0052] 1.256 mg of riboflavin sodium phosphate, 10 mg of hemoglobin, and 12.56 mg of dextran were weighed and dissolved in 5 mL of purified water. 1 mL of a 2.5 mg / mL phosphatidylcholine ethanol solution was added dropwise with constant stirring. Stirring was continued overnight to evaporate the organic solvent. The solution was then placed in a dialysis bag with a molecular weight cut-off of 8000 to 14000 Mw and dialyzed for 12 hours to remove unentrapped drug. Oxygen was then purged through the solution for 5 minutes at an oxygen flow rate of 100 mL / min. This was designated Solution 1. 2.512 mg of rapamycin and 5.024 mg of phosphatidylcholine were weighed and dissolved in 1 mL of ethanol. The solution was then added to 5 mL of a 1 mg / mL silk fibroin aqueous solution with rapid stirring to form nanoparticles. Stirring was continued to evaporate the organic solvent. The solution was then placed in a dialysis bag with a molecular weight cut-off of 8000 to 14000 Mw and dialyzed for 12 hours to remove unentrapped drug. This was designated Solution 2. Then, solution 1 and solution 2 are mixed and freeze-dried to obtain the nanomedicine of the present invention.

[0053] Comparative Example 1

[0054] 1.256 mg of riboflavin sodium phosphate and 12.56 mg of dextran were weighed and dissolved in 1.256 mL of purified water to form a 1 mg / mL solution, designated Solution 1. 2.512 mg of rapamycin and 5.024 mg of phosphatidylcholine were weighed and dissolved in 1 mL of ethanol. The solution was then added to 5 mL of water and rapidly stirred to form nanoparticles. Stirring was continued to evaporate the organic solvent, and the nanoparticles were then placed in a dialysis bag with a molecular weight cutoff of 8,000 to 14,000 Mw and dialyzed for 12 hours to remove unentrapped drug. This solution was designated Solution 2. Solutions 1 and 2 were then mixed and freeze-dried to produce a control nanoparticle.

[0055] Cross-linking effect test

[0056] Fresh ex vivo porcine blood vessels were selected, and the nanomedicines of the present invention obtained in Experimental Examples 1 to 3 and the control nanomedicine obtained in Comparative Example 1 were respectively subjected to crosslinking effect tests under the same introduction conditions. The specific testing method includes: blending the nanomedicine with a mixed solution of physiological saline and contrast agent to obtain a mixed solution, wherein the volume ratio of physiological saline to contrast agent is 3:1; cutting a fresh ex vivo porcine blood vessel with a diameter of about 3.3 mm and a length of 20 mm, inserting the porous balloon portion of the drug dilation tube into the fresh ex vivo porcine blood vessel, and the structure of the drug dilation tube is as shown in FIG. Figure 1As shown, the nanodrug cavity is connected to the pressure pump through the nanodrug inlet, the light inlet is connected to the expansion cavity, and the expansion cavity is located in the nanodrug cavity; the balloon pressure pump is pushed and pressurized to 6atm~14atm, so that the blended liquid is ejected from the porous balloon portion, the perfusion solution volume is 1mL, and a total of 1.256mg of photosensitive crosslinker and 2.512mg of rapamycin are delivered; then a balloon catheter containing an optical fiber component (optical fiber core diameter 400μm, emission wavelength 450nm) is inserted, the balloon is expanded to 1.3 times the initial diameter of the blood vessel, and then the laser is turned on and the balloon is withdrawn after irradiation for 1min; Experimental Examples 1 to 3 and Comparative Example 1 each make 6 blood vessels and then divide them into two groups. One group of blood vessels is immediately tested for drug content in the blood vessels, and the other group of blood vessels is connected to the peristaltic pump pipeline, and the blood vessels are flushed with 37℃ phosphate buffer at a flow rate of 80mL / min for 2h, and then the drug content in the blood vessels is measured. The test results are shown in Table 1.

[0057] Table 1 Nano drug cross-linking effect test data

[0058]

[0059] The test results show that in Experimental Examples 1-3, since the nanodrugs were loaded with silk fibroin on their surfaces, they could simultaneously crosslink with vascular proteins during the crosslinking process, anchoring the nanodrugs in the blood vessels. Therefore, they remained firmly anchored under blood flow, reducing the elution of the nanodrug particles from the vessel wall. In Comparative Example 1, however, since no silk fibroin was loaded, the nanodrugs could not be crosslinked and anchored in the vessel wall. Therefore, under blood flow, the elution rate of the nanodrug was significantly increased.

[0060] Vascular support test

[0061] Fresh ex vivo porcine blood vessels were selected, and the nanomedicines of the present invention obtained in Experimental Examples 1 to 3 and the control nanomedicine obtained in Comparative Example 1 were respectively tested for their vascular support effects under the same introduction conditions. The specific testing method includes: blending the nanomedicine with a mixed solution of physiological saline and contrast agent to obtain a mixed solution, wherein the volume ratio of physiological saline to contrast agent is 3:1; cutting a fresh ex vivo porcine blood vessel with a diameter of about 3.3 mm and a length of 20 mm, inserting the porous balloon portion of the drug dilation tube into the fresh ex vivo porcine blood vessel, and the structure of the drug dilation tube is as shown in FIG. Figure 1As shown, the nanodrug cavity is connected to the pressure pump through the nanodrug introduction port, and the light introduction port is connected to the expansion cavity, and the expansion cavity is located in the nanodrug cavity; the balloon pressure pump is pushed to increase the pressure to 6atm~14atm, so that the mixed liquid is ejected from the porous balloon part, the perfusion solution volume is 1mL, and a total of 1.256mg photosensitive crosslinker and 2.512mg rapamycin are delivered; then a balloon catheter containing an optical fiber component (optical fiber core diameter 400μm, emission wavelength 450nm) is inserted, the balloon is expanded to 1.3 times the initial diameter of the blood vessel, and then the laser is turned on and the balloon is withdrawn after irradiation for 1min; the blood vessel diameter before and after expansion is measured; and the resistance when it is compressed to 50% of the initial diameter is measured using a stent radial force tester using a flat plate method. The test results are shown in Table 2.

[0062] Table 2 Test data of nano drug vascular support effect

[0063] project Example 1 Example 2 Example 3 Comparative Example 1 Vessel diameter before dilation (mm) 4.0 4.0 4.0 4.0 Vascular diameter after dilation (mm) 5.0 5.2 5.2 4.8 Resistance when diameter is compressed by 50% (mN) 36 52 53 20

[0064] According to the test results, it can be seen that compared with Example 1, Comparative Example 1 has low cross-linking efficiency and poor vascular support. After the balloon is withdrawn, the blood vessels will easily retract, making it difficult to effectively maintain vascular patency. Example 2 adds potassium superphosphate, which can serve as an electron acceptor for photosensitizers, thereby increasing the free radical concentration. Therefore, the speed of photocrosslinking is improved. Compared with Example 1 and Comparative Example 1, Example 2 has a larger vascular lumen, and a larger vascular diameter after expansion. Furthermore, Example 3 adds hemoglobin oxygen-carrying substances, which increase the oxygen content in the blood vessels at the lesion site. Therefore, the rate of free radical and singlet oxygen generation is increased, thereby improving the efficiency of photocrosslinking. Therefore, compared with Example 1 and Comparative Example 1, Example 3 has a larger lumen after crosslinking, and better vascular maintenance.

[0065] Sustained release effect test

[0066] Healthy domestic pigs were selected as the experimental model to determine the vascular retention time of the nanomedicines of the present invention obtained in Experimental Examples 1 to 3 and the control nanomedicine obtained in Comparative Example 1. The specific operation method includes the following steps: blending the nano drug with a mixed solution of physiological saline and contrast agent to obtain a mixed solution, wherein the volume ratio of physiological saline to contrast agent is 3:1; cutting a fresh ex vivo porcine blood vessel with a diameter of about 3.3 mm and a length of 20 mm, and inserting the porous balloon portion of the drug dilation tube into the fresh ex vivo porcine blood vessel; pushing the balloon pressure pump to pressurize to 6 atm to 14 atm to eject the mixed solution from the porous balloon portion, and the perfusion solution volume is 1 mL, and a total of 1.256 mg of photosensitive crosslinker and 2.512 mg of rapamycin are delivered; then inserting a balloon catheter containing an optical fiber component, dilating the balloon to 1.3 times the initial diameter of the blood vessel, and then turning on the laser, irradiating for 1 minute, and then withdrawing the balloon; using liquid chromatography-mass spectrometry to test the drug concentration at the lesion site immediately after surgery, 7 days, 28 days, and 60 days, the results are shown in Table 3.

[0067] Table 3 Nano drug sustained release effect test data

[0068]

[0069]

[0070] The test results show that compared to Comparative Example 1, Comparative Examples 1-3 effectively and immediately transferred the drug to the interior of the blood vessel wall, with high drug concentrations and a long-lasting sustained release effect, effectively and persistently inhibiting the proliferation of vascular smooth muscle. Furthermore, due to the addition of potassium superphosphate in Example 2 and silk fibroin in Example 3, the surface of the nanoparticles was more efficiently gelled and cross-linked to the vascular tissue protein during light exposure, significantly reducing the elution rate and achieving a more sustained sustained release of the drug.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nanomedicine, characterized in that: The invention comprises a nanocarrier, on which therapeutic drugs and cross-linked proteins are loaded. The cross-linked proteins are at least partially loaded on the surface of the nanocarrier, and the cross-linked proteins are at least one of silk fibroin, sericin and silk peptide. The therapeutic drugs are at least partially loaded inside the nanocarrier.

2. The nanomedicine according to claim 1, wherein Also included are photosensitive crosslinkers.

3. The nanomedicine according to claim 2, characterized in that The photosensitive crosslinking agent is at least one of a naphthalimide compound, a hydrophilic derivative of naphthalimide, camphorquinone, a hydrophilic derivative of camphorquinone, riboflavin, riboflavin sodium phosphate, rose bengal, curcumin, and eosin Y.

4. The nanomedicine according to claim 2, wherein In the nano-medicine, the mass percentage content of the photosensitive cross-linking agent is 1% to 99%.

5. The nanomedicine according to claim 2, characterized in that The size of the nanocarrier is 10 nm to 2000 nm; And / or, the size of the photosensitive crosslinking agent is 1 nm to 2000 nm.

6. The nanomedicine according to any one of claims 2 to 5, characterized in that Also included are photosensitizers.

7. The nanomedicine according to claim 6, characterized in that The mass ratio of the photosensitizer to the photosensitizer is 0.02 to 5:1; And / or, the photosensitizer is at least one of diphenyliodonium hexafluorophosphate, ethyl p-dimethylaminobenzoate, potassium persulfate, sodium persulfate and ammonium persulfate.

8. The nanomedicine according to any one of claims 2 to 5, characterized in that Also included is at least one of an active drug, a penetration enhancer, a cell-penetrating peptide, oxygen, and an oxygen delivery substance, wherein the active drug is used to enhance the therapeutic effect of the therapeutic drug.

9. The nanomedicine according to any one of claims 2 to 5, characterized in that The carrier is poly(amino acid), copoly(ether-ester), polyalkylene oxalate, polyamide, poly(imino carbonate), polyorthoester, polyethylene oxide, polyamide ester, polyethylene oxide containing amide groups, poly(anhydride), polyphosphazene, polyhydroxy acid, poly-alpha hydroxy acid, trimethylene carbonate, polyorganophosphazene, polyesteramide, polyethylene oxide, polyester ether, polyphosphate, polyphosphoester polyurethane, cyanoacrylate, poly(trimethylene carbonate), poly(imino carbonate), polyalkylene oxalate, polyvinyl pyrrolidone, polyvinyl alcohol, shellac, gum arabic, poly-N-(2-alkylpropyl)-methacrylamide, polydimethylsiloxane, dimethyl ... At least one of an alcohol, an aliphatic polyester, a poly(orthoester), a poly(ester-amide), a polyanhydride, a poloxamer, a hyaluronic acid, a polyvinyl alcohol, a polyvinyl pyrrolidone, a polysaccharide, a gelatin, a Tween, an amphiphilic diblock or block copolymer, a lecithin, a phosphatidylethanolamine, a phosphatidylserine, an iodine-containing contrast agent, sucrose, trehalose, mannitol, lactose, glucose, maltose, proline, tryptophan, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, L-tyrosine, phenylalanine, arginine, lysine, polyarginine, a polypeptide dendrimer, albumin, silk fibroin, sericin, silk peptide, and casein.

10. A method for preparing a nanomedicine, for preparing the nanomedicine according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: dissolving the therapeutic drug in an organic solvent miscible with water as an organic phase, dissolving the cross-linked protein in water as an aqueous phase, dissolving the nanocarrier along with the therapeutic drug or the cross-linked protein, then mixing and stirring the organic phase and the aqueous phase, and then dialyzing to remove the organic solvent.

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