Heme-based multifunctional nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a heme-based multifunctional nano-drug and a preparation method and application thereof, and relates to the technical field of nano-drugs, and the heme-based multifunctional nano-drug comprises heme and hydrophobic molecules wrapped in the heme. The hydrophobic molecules are wrapped in the heme, so that the bioavailability of the hydrophobic molecules is improved, the hydrophobic molecules are slowly released at bone joints, and the retention time of the hydrophobic molecules in the bone joints is prolonged; meanwhile, the nanostructure formed by the heme has better lubrication property, biocompatibility and cartilage steady state improving effect, can promote cartilage cell proliferation, and can enhance the capability of resisting oxygen deficit and continuously worsening articular cavity internal environment of cartilage tissues through synergistic effect, so that the cartilage steady state is better maintained. Therefore, the multifunctional nano-drug provided by the invention has universality in treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drugs, and particularly relates to a heme-based multifunctional nano-drug and its preparation method and application. Background Art

[0002] Osteoarthritis is a disabling disease worldwide, imposing a huge economic burden on individuals and society.

[0003] Currently, the existing methods for relieving osteoarthritis in joint lubrication mainly include using components similar to synovial fluid in the joint cavity, such as hyaluronic acid, lubricin, or glucosamine, or using liposomes and drug-loaded nanoparticles for intra-articular injection or oral administration, aiming to improve the internal friction in the joint and increase the residence time of related substances in the joint cavity.

[0004] However, osteoarthritis is not a problem of a single tissue. Cartilage, subchondral bone, synovium, and immune cells in the joint cavity are all involved, which means that improving lubrication alone cannot effectively regulate osteoarthritis. The existing treatment methods have the following deficiencies: (1) Currently, many existing synovial fluids need to be injected multiple times in a short period, and their functions are single, which will affect the treatment effect of osteoarthritis; (2) The functions of liposomes are relatively single; (3) The construction of nanoparticles is complex and difficult to prepare; (4) When treating with multiple substances in combination, there is a problem of mutual interference, and adjustments need to be made to meet the requirements of different environments. Summary of the Invention

[0005] The main object of the present invention is to propose a heme-based multifunctional nano-drug and its preparation method and application, aiming to solve the problem of poor treatment effect of osteoarthritis in the existing technology.

[0006] To achieve the above object, the present invention proposes a heme-based multifunctional nano-drug, including heme and a hydrophobic molecule encapsulated inside the heme.

[0007] In one embodiment, the mass ratio of the heme to the hydrophobic molecule is 1:(0.5 - 1.5); and / or,

[0008] The hydrophobic molecule includes aloe-emodin, curcumin, or resveratrol.

[0009] The present invention also provides a preparation method of a heme-based multifunctional nano-drug, including the following steps:

[0010] S10. Mix an organic solution containing heme with a hydrophobic molecule to obtain a first mixed solution;

[0011] S20. Mix the first mixture with water for hydration so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecules, thereby obtaining the heme-based multifunctional nano-drug.

[0012] In one embodiment, in step S10, the organic solution containing heme is obtained by mixing heme with an organic solvent:

[0013] The organic solvent includes toluene and / or dimethyl sulfoxide; and / or,

[0014] 1 mg of heme is mixed with every 150 μL of the organic solvent.

[0015] In one embodiment, step S20 includes:

[0016] Mix the first mixture with water under ultrasonic mixing at a water bath temperature of 0 - 4 °C for hydration so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecules, thereby obtaining the heme-based multifunctional nano-drug.

[0017] In one embodiment, the volume ratio of the first mixture to the water is 1:30 - 3:100; and / or,

[0018] The time of ultrasonic mixing is 12 - 17 min; and / or,

[0019] The power of ultrasonic mixing is 300 - 350 W; and / or,

[0020] The diameter of the ultrasonic rod for ultrasonic mixing is 1.5 - 2 mm.

[0021] In one embodiment, step S20 includes:

[0022] S201. Mix the first mixture with water for hydration so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecules, thereby obtaining a second mixture;

[0023] S202. After removing the organic solvent from the second mixture, centrifuge it and take the supernatant for vacuum freeze-drying to obtain the heme-based multifunctional nano-drug.

[0024] In one embodiment, when the organic solvent is toluene and dimethyl sulfoxide, step S202 includes:

[0025] Volatilize the toluene in the second mixture, then remove dimethyl sulfoxide by dialysis to obtain a third mixture, centrifuge the third mixture, and take the supernatant for vacuum freeze-drying to obtain the heme-based multifunctional nano-drug.

[0026] In one embodiment, the cut-off molecular weight of the dialysis bag used in the dialysis method is 3500 Da; and / or,

[0027] the centrifugation time is 15 - 25 min; and / or,

[0028] the centrifugal force for the centrifugation is 3500 - 5000 × g.

[0029] The present invention also provides an application of the aforementioned heme-based multifunctional nanomedicine or a heme-based multifunctional nanomedicine prepared by the preparation method of the aforementioned heme-based multifunctional nanomedicine in the preparation of a medicament for treating osteoarthritis.

[0030] In the technical solution of the present invention, the hydrophobic molecule is encapsulated inside the heme, which improves the bioavailability, enables slow release at the bone joint, and prolongs the residence time of the hydrophobic molecule in the bone joint; meanwhile, the nanostructure formed by the heme has good lubricating properties, biocompatibility and the effect of enhancing cartilage homeostasis, and can promote chondrocyte proliferation; there can also be a synergistic effect between the hydrophobic drug and the heme to enhance the ability of cartilage tissue to resist hypoxia and the deteriorating intra-articular environment, thereby better maintaining cartilage homeostasis. Therefore, the multifunctional nanomedicine provided by the present invention has universality in the treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0032] Figure 1 It is the transmission electron microscopy image of HE in Comparative Example 1;

[0033] Figure 2 It is the transmission electron microscopy image of AE in Comparative Example 2;

[0034] Figure 3 It is the transmission electron microscopy image of the heme-based multifunctional nanomedicine AE@HE prepared in Example 1;

[0035] Figure 4 It is the particle size diagram of the heme-based multifunctional nanomedicine AE@HE prepared in Example 1;

[0036] Figure 5 It is the comparison result diagram of the stability of the nanomedicines in aqueous solution in Example 1, Comparative Example 1 and Comparative Example 2;

[0037] Figure 6 Release curve of the heme-based multifunctional nano-drug AE@HE prepared in Example 1;

[0038] Figure 7 Coefficient of friction (COF) curves of the nano-drug HE in Comparative Example 1 after different doping ratios in GelMA hydrogel;

[0039] Figure 8 Statistical chart of the coefficient of friction (COF) of the nano-drug HE prepared in Comparative Example 1 after different doping ratios in GelMA hydrogel;

[0040] Figure 9 Statistical chart of the CCK8 net absorbance of HE prepared in Comparative Example 1 against ATDC5 cell line;

[0041] Figure 10 Fluorescence image of calcein staining of HE prepared in Comparative Example 1 against ATDC5 cell line;

[0042] Figure 11 Statistical chart of the relative expression levels of cartilage-related genes (SOX-9, ACAN, COL2A1) and hemoglobin-related genes (α-globin, β-globin) after 8 weeks of treatment of the rat osteoarthritis model.

[0043] The realization, functional features and advantages of the objectives of the present invention will be further described in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Embodiments

[0044] To make the objectives, 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. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, or solution B, or a solution that satisfies both A and B simultaneously. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that those skilled in the art can achieve it. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0045] Currently, the existing joint lubrication methods mainly include using components similar to synovial fluid in the joint cavity, such as hyaluronic acid, lubricin, or glucosamine, or performing intra-articular injection or oral administration using liposomes and drug-loaded nanoparticles, with the overall aim of improving intra-articular friction and increasing the residence time of relevant substances in the joint cavity. However, osteoarthritis is not a problem of a single tissue. Cartilage, subchondral bone, synovium, and immune cells in the joint cavity are all involved, which means that improving lubrication alone cannot effectively regulate osteoarthritis. The existing treatment methods have the following deficiencies: (1) Currently, many existing joint synovial fluids require multiple injections in a short period, and their functions are single, which will affect the treatment effect of osteoarthritis; (2) The function of liposomes is relatively single; (3) The construction of nanoparticles is complex and difficult to prepare; (4) When treating with a combination of multiple substances, there is a problem of mutual interference, and adjustments need to be made to meet the requirements of different environments.

[0046] In view of this, the present invention proposes a multifunctional nano-drug based on heme, including heme and a hydrophobic molecule encapsulated inside the heme.

[0047] It should be noted that the hydrophobic molecule in the present invention refers to a small molecule substance rich in hydrophobic functional groups, which results in low solubility in aqueous solution, difficulty in forming a stable solution, and easy aggregation and precipitation.

[0048] The present invention creatively discovers that heme can promote the expression of cartilage genes, that is, it can promote the homeostasis of chondrocytes. Heme can synergistically and significantly enhance the homeostasis of chondrocytes with hydrophobic drugs used to treat osteoarthritis. This may be because heme, as a prosthetic group of hemoglobin, can effectively promote chondrocytes to secrete hemoglobin, so that avascular cartilage tissue can better resist hypoxia and the deteriorating intra-articular environment and maintain cartilage homeostasis.

[0049] In the technical solution of the present invention, a multifunctional nano-drug with heme as the core is provided. Among them, heme, as a natural substance, has good biocompatibility, and it can be used as a carrier to include a variety of hydrophobic molecules. The encapsulated structure improves the bioavailability, enables it to be slowly released at the bone joint, and extends the residence time of the hydrophobic molecule in the bone joint; at the same time, the nano-structure formed by heme has good lubrication properties, biocompatibility, and the effect of enhancing cartilage homeostasis, which can enhance joint lubrication performance. After heme is loaded with hydrophobic drugs, it can promote chondrocyte proliferation and can also enhance the ability of cartilage tissue to resist hypoxia and the deteriorating intra-articular environment through synergistic effects, thereby better maintaining cartilage homeostasis. Therefore, the multifunctional nano-drug provided by the present invention has universality for the treatment of osteoarthritis.

[0050] In some embodiments, the mass ratio of the heme to the hydrophobic molecule is 1:(0.5 - 1.5). It can be understood that the mass ratio of the heme to the hydrophobic molecule can be 1:0.5, 1:1 or 1:1.5. When the mass ratio is within the above range, the heme can better encapsulate the hydrophobic molecule, preventing it from being directly exposed to the joint cavity after injection, increasing its residence time in the joint cavity, and prolonging the action time of the hydrophobic molecule.

[0051] In some embodiments, the hydrophobic molecule includes aloe-emodin, curcumin or resveratrol. The above-mentioned hydrophobic macromolecules can be used to treat osteoarthritis and can be well encapsulated by heme to form a stable multifunctional nano-drug.

[0052] The present invention also provides a method for preparing a multifunctional nano-drug based on heme, comprising the following steps: S10. Mix an organic solution containing heme with a hydrophobic molecule to obtain a first mixture;

[0053] S20. Mix the first mixture with water for hydration, so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecule to obtain the multifunctional nano-drug based on heme.

[0054] In the technical solution of the present invention, heme is amphiphilic, that is, it has both hydrophilic and hydrophobic groups. When an organic solution containing heme is mixed with a hydrophobic molecule and a part of water is added for hydration, the amphiphilic property of heme can enable it to self-assemble into nanoparticles and encapsulate the hydrophobic molecule inside through hydrophobic interaction, thereby effectively loading the hydrophobic molecule to achieve drug sustained release. Through the above preparation steps, a multifunctional nano-drug with heme as the core can be obtained relatively quickly. This drug can load a variety of hydrophobic molecules to achieve sustained release treatment, and has good biocompatibility; its component heme can achieve a lubricating effect and maintain the cartilage morphology at the same time, which has universality for the treatment of osteoarthritis.

[0055] In some embodiments, in step S10, the organic solution containing heme is obtained by mixing heme with an organic solvent: the organic solvent includes toluene and / or dimethyl sulfoxide; and / or, 1 mg of heme is mixed with every 150 μL of the organic solvent. Mixing heme with an organic solvent can pre-dissolve and disperse heme in the organic solvent to form a homogeneous solution at the molecular level. Controlling the amounts of the organic solvent and heme within the above range can ensure good dispersion of heme in the organic solvent and prevent aggregation.

[0056] In some embodiments, step S20 includes: ultrasonically mixing the first mixture with water under a water bath condition of 0 to 4°C for hydration, so that the heme in the first mixture self-assembles and wraps the hydrophobic molecules to obtain the heme-based multifunctional nanodrug. Bathing at 0 to 4°C can ensure better stability of the hydrophobic drug and heme. Ultrasonic mixing can enable the rapid hydration and self-assembly of heme under the action of water, so that the formed multifunctional nanodrug is dispersed in the system in a smaller and more uniform form after being broken.

[0057] In some embodiments, the volume ratio of the first mixture to the water is 1:30 to 3:100. The volume ratio of the first mixture to water within the above range can ensure a relatively fast hydration rate of heme, self-assembling and wrapping hydrophobic molecules to form a multifunctional nanodrug in a short time.

[0058] In some embodiments, the time of the ultrasonic mixing is 12 to 17 min; and / or, the power of the ultrasonic mixing is 300 to 350 W; and / or, the diameter of the ultrasonic rod for the ultrasonic mixing is 1.5 to 2 mm. The time of the ultrasonic mixing can be 12 min, 15 min or 17 min, the power of the ultrasonic mixing can be 300 W, 330 W or 350 W, and the diameter of the ultrasonic rod can be 1.5 mm, 1.7 mm or 2 mm. Controlling the time, power and diameter of the ultrasonic rod of the ultrasonic mixing within the above range can ensure that the heme and the hydrophobic drug in the system are relatively evenly dispersed, and the two are in full contact, and a core-shell structured multifunctional nanodrug is rapidly formed under the induction of the hydrophobic interaction.

[0059] In some embodiments, step S20 includes: S201. Mixing the first mixture with water for hydration, so that the heme in the first mixture self-assembles and wraps the hydrophobic molecules to obtain a second mixture; S202. Removing the organic solvent from the second mixture and then centrifuging, taking the supernatant for vacuum freeze-drying to obtain the heme-based multifunctional nanodrug. It can be understood that after removing the organic solvent from the system, centrifuging and taking the supernatant for freeze-drying to prepare the heme-based multifunctional nanodrug has relatively uniform particles and high purity, which is beneficial for storage and controlling the dosage.

[0060] In some embodiments, the organic solvent is toluene and dimethyl sulfoxide, and step S202 includes: volatilizing the toluene in the second mixture, then removing the dimethyl sulfoxide by dialysis to obtain a third mixture, centrifuging the third mixture, taking the supernatant for vacuum freeze-drying to obtain the heme-based multifunctional nanodrug. Volatilization can quickly remove toluene, and dialysis can more thoroughly remove dimethyl sulfoxide, unstructured heme and hydrophobic molecules.

[0061] In some embodiments, the cut-off molecular weight of the dialysis bag used in the dialysis method is 3500 Da. Controlling the cut-off molecular weight of the dialysis bag within the above range can ensure a more thorough removal of dimethyl sulfoxide.

[0062] In some embodiments, the centrifugation time is 15 - 25 min; and / or, the centrifugal force for the centrifugation is 3500 - 5000 × g. Controlling both the centrifugation time and the centrifugal force within the above ranges can ensure a better centrifugation effect and ultimately less impurities in the multifunctional nano-drug.

[0063] The present invention also provides an application of the aforementioned heme-based multifunctional nano-drug or a heme-based multifunctional nano-drug prepared by the preparation method of the aforementioned heme-based multifunctional nano-drug in the preparation of a drug for treating osteoarthritis. Therefore, it has all the beneficial effects of the aforementioned heme-based multifunctional nano-drug or the preparation method of the aforementioned heme-based multifunctional nano-drug, which will not be elaborated one by one here.

[0064] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0065] Example 1

[0066] A heme-based multifunctional nano-drug, comprising the following steps:

[0067] Step 1, Mix 100 μL of toluene and 50 μL of DMSO evenly to prepare an organic solvent.

[0068] Step 2, Dissolve 1 mg of heme powder fully in the mixed organic solvent to prepare a heme solution.

[0069] Step 3, Dissolve 1 mg of aloe-emodin fully in the heme solution to prepare a precursor solution.

[0070] Step 4, Quickly add 5 mL of pure water to the precursor solution and perform ultrasonic treatment for 15 minutes under the condition of a 0 °C cold water bath, with a power of 325 W.

[0071] Step 5, After overnight evaporation of the ultrasonicated solution, perform dialysis using a 3500 Da dialysis bag.

[0072] Step 6, Centrifuge the liquid after dialysis at 4000 × g for 20 minutes, then take the supernatant for vacuum freeze-drying to obtain a heme multifunctional nano-drug with aloe-emodin encapsulated inside, denoted as AE@HE.

[0073] Example 2

[0074] The difference between Example 2 and Example 1 is that:

[0075] Replace the aloe-emodin in Step 3 with an equal mass of curcumin.

[0076] Comparative Example 1

[0077] Comparative Example 1 is different from Example 1 in that:

[0078] Do not perform Step 3, and replace the precursor solution in Step 4 with the hemin solution in Step 2;

[0079] The finally obtained nano-drug is denoted as HE.

[0080] Comparative Example 2

[0081] Comparative Example 2 is different from Example 1 in that:

[0082] Do not perform Step 2, and replace the hemin solution in Step 3 with the organic solvent in Step 1;

[0083] The finally obtained nano-drug is denoted as AE.

[0084] Performance Test

[0085] Characterize and test the performance of the hemin-based multifunctional nano-drug prepared in Example 1. The detection methods and results are as follows:

[0086] (1) Morphology analysis: Observe the products prepared in Comparative Example 1, Comparative Example 2 and Example 1 by transmission electron microscopy. The results are as shown in Figure 1 , Figure 2 and Figure 3 respectively.

[0087] As can be seen from Figure 2 , the hydrophobic molecules cannot form nanoparticles, but are crystalline, with too large size and irregular structure, which are toxic to joints. As can be seen from Figure 3 , the AE@HE prepared in Example 1 is a nano-particle with uniform size. Analyze its particle size. As shown in Figure 4 , its particle size is about 220 nm.

[0088] (2) Stability analysis: Take 10 mg of the products prepared in Comparative Example 1, Comparative Example 2 and Example 1 respectively, mix them with 1 mL of water, vortex for 1 min, and let stand. Continuously observe for 4 h to judge whether they are layered. The results are as shown in Figure 5 respectively.

[0089] As can be seen from Figure 5 , the stability of AE@HE is better than that of AE.

[0090] (3) Sustained-release effect analysis: Weigh 5 mg of the nanoparticles AE@HE prepared in Example 1 and co-incubate them with phosphate buffer solution (PBS) at 37 °C for a degradation and release experiment. Centrifuge and collect the supernatant every day to detect the concentration of the released drug. The results are as Figure 6 shown.

[0091] As can be Figure 6 seen, the nanoparticles AE@HE prepared in Example 1 can gradually release the loaded substances within 14 days.

[0092] (4) Lubrication effect analysis: Mix the heme nanoparticles HE in Comparative Example 1 with 10% methacrylated gelatin (GelMA) so that the concentration of HE in the system is 0%, 0.001%, 0.005%, 0.05%, and 0.25% in turn. Use 0.25% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a photoinitiator, irradiate the above system with 60 W ultraviolet light for 1 min to prepare a hydrogel test sample, and use a nano-force tester to test the cyclic friction coefficient within 300 s under a 2 N force; the results are as Figure 7 and Figure 8 shown.

[0093] As can be Figure 7 and Figure 8 seen, the heme shell can effectively improve the lubricating performance of the nano-drug on the bone joints.

[0094] (5) Proliferation promotion analysis: Seed the ATDC5 chondrocyte line in a 96-well plate at a concentration of 3000 cells / well, and divide it into the following groups according to the final concentration of heme in the medium: 0 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 1000 μg / mL. Set five parallel wells for each group. Use the CCK8 toxicity detection kit to detect the cell viability after culturing for 1 day, 2 days, and 3 days respectively, and at the same time use the live / dead staining kit to stain and observe the cell state under a fluorescence microscope. The results are as Figure 9 , Figure 10 .

[0095] As can be Figure 9 seen, HE at a concentration of 1 - 100 μg / mL can promote the proliferation of chondrocytes. As can be Figure 10 seen, the morphology of chondrocytes will not be changed under the stimulation of a certain concentration of HE.

[0096] (6) Analysis of chondrogenic genes and hemoglobin-related genes: Sodium iodoacetate was used to establish a rat osteoarthritis model. Sodium iodoacetate was injected into each leg of the rats at a concentration of 2 mg / 50 μL. The experiment was carried out two weeks later. The experiment was divided into a model group (OA), an emodin group (AE), a hemin group (Hemin), and a multifunctional nanodrug group (AE@Hemin). Each group was injected with 200 μg of the corresponding drug every two weeks, and the model group was injected with 50 μL of normal saline. After 8 weeks, samples were taken for tissue RNA extraction, and qPCR technology was used to compare the expression levels of cartilage-related genes (SOX-9, ACAN, COL2A1) and hemoglobin-related genes (α-globin, β-globin) in each group; the results are as Figure 11 shown.

[0097] Figure 11 The figure on the left to the right in the figure shows the expression results of five genes: SOX-9, ACAN, α-globin, β-globin, and COL2A1. It can be Figure 11 seen that HE, AE, and their synergistic effects can all improve osteoarthritis, and the synergistic effect is the best.

[0098] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A heme-based multifunctional nano-drug, characterized in that, It includes heme and a hydrophobic molecule encapsulated inside the heme.

2. The heme-based multifunctional nano-drug according to claim 1, characterized in that, The mass ratio of the heme to the hydrophobic molecule is 1:(0.5 - 1.5); and / or, The hydrophobic molecule includes aloe-emodin, curcumin or resveratrol.

3. A method for preparing the heme-based multifunctional nano-drug as described in claim 1 or 2, characterized in that, It includes the following steps: S10. Mix an organic solution containing heme with a hydrophobic molecule to obtain a first mixture; S20. Mix the first mixture with water for hydration, so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecule, to obtain the heme-based multifunctional nano-drug.

4. The preparation method of the heme-based multifunctional nano-drug according to claim 3, wherein In step S10, the organic solution containing heme is obtained by mixing heme with an organic solvent: The organic solvent includes toluene and / or dimethyl sulfoxide; and / or, 1 mg of heme is mixed with every 150 μL of the organic solvent.

5. The preparation method of the heme-based multifunctional nano-drug according to claim 3, wherein, Step S20 includes: Mix the first mixture with water under ultrasonic conditions in a water bath at 0 - 4°C for hydration, so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecule, to obtain the heme-based multifunctional nano-drug.

6. The preparation method of the heme-based multifunctional nano-drug according to claim 5, characterized in that, The volume ratio of the first mixture to the water is 1:30 - 3:100; and / or, The time of the ultrasonic mixing is 12 - 17 min; and / or, The power of the ultrasonic mixing is 300 - 350 W; and / or, The diameter of the ultrasonic rod for the ultrasonic mixing is 1.5 - 2 mm.

7. The preparation method of the heme-based multifunctional nano-drug according to claim 3, characterized in that, Step S20 includes: S201. Mix the first mixture with water for hydration, so that the heme in the first mixture self-assembles and encapsulates the hydrophobic molecule, to obtain a second mixture; S202. After removing the organic solvent from the second mixture, centrifuge it, and take the supernatant for vacuum freeze-drying to obtain the heme-based multifunctional nano-drug.

8. The preparation method of the heme-based multifunctional nano-drug according to claim 7, characterized in that, The organic solvent is toluene and dimethyl sulfoxide. Step S202 includes: Volatilize toluene in the second mixture, then remove dimethyl sulfoxide by dialysis to obtain a third mixture, centrifuge the third mixture, and take the supernatant for vacuum freeze-drying to obtain the heme-based multifunctional nano-drug.

9. The preparation method of the heme-based multifunctional nano-drug according to claim 8, characterized in that, The cut-off molecular weight of the dialysis bag used in the dialysis method is 3500 Da; and / or, The time of the centrifugation is 15 - 25 min; and / or, The centrifugal force of the centrifugation is 3500 - 5000×g.

10. Use of a heme-based multifunctional nano-drug as described in claim 1 or 2 or a heme-based multifunctional nano-drug prepared by the preparation method of the heme-based multifunctional nano-drug as described in any one of claims 3 to 9 in the preparation of a drug for treating osteoarthritis.

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