Method for regulating and controlling polarization of macrophages by using melanin nanoparticles

Abstract

The invention provides a method for regulating and controlling polarization of macrophages by using melanin nanoparticles. According to the application, the autophagy behavior of the macrophages is activated through the natural melanin nanoparticles to adjust the polarization of the macrophages, and the activation of cell autophagy is utilized to promote the transformation of the macrophages to M2 type macrophages, so that the occurrence and progress of inflammation are inhibited. The invention further provides application of the melanin nano-particles in preparation of immunoregulation and control drugs.

Description

Technical Field

[0001] The present application relates to the field of biomaterials, and specifically to a method for regulating macrophage polarization using melanin nanoparticles and its application. Background Art

[0002] The human immune system plays a crucial role in tissue metabolism and disease progression. Among the various immune cells of the immune system, macrophages occupy a key position. Macrophages were the first phagocytes discovered and are the body's immune effector cells (Kaufmann SHE, Nat. Immunol.). As an important component of the innate immune system, macrophages can recruit other immune cells to the site of infection, engulf and eliminate foreign pathogens, and activate the complement system and adaptive immunity. Among the immune cells involved in defense and homeostasis, macrophages play a key mediating role in development, disease (including cancer, infection, and inflammation), and tissue regeneration and remodeling (Yona S et al., Immunity). Macrophages can be divided into pro-inflammatory M1 macrophages based on their polarization function, which can produce pro-inflammatory cytokines such as IL-1β and TNF-α, and anti-inflammatory M2 macrophages, which produce cytokines such as IL-4 and IL-10 that play a significant role in clearing inflammation.

[0003] Autophagy is a lysosomal-mediated degradation process in which cells digest their own components (e.g., animal cells) to maintain normal intracellular physiological activities and homeostasis, thereby playing a key role in maintaining homeostasis in the cellular microenvironment. Autophagy is generally considered a survival response mechanism used by organisms to protect the internal and external cellular environment from damage when external nutrients are deficient or cells are damaged by external sources. Autophagy also plays a key role in the polarization of macrophages.

[0004] Biomaterials can promote the replacement, repair, and regeneration of damaged or diseased human tissues and organs, thereby restoring or enhancing their physiological functions. Biomaterials with immunomodulatory capabilities can regulate both innate and adaptive immunity and hold great promise in the prevention and treatment of a variety of diseases. Melanin is a readily available and inexpensive biopolymer with advantages such as biocompatibility, biodegradability, scavenging properties, metal chelation, and electronic conductivity. This biomaterial itself can be used as a nanocarrier or medium for various biomedical applications, such as imaging, controlled drug delivery, bioengineering and bioelectronics, antioxidant applications, and therapy.

[0005] Therefore, new uses of melanin remain to be developed. Summary of the Invention

[0006] Based on the current state of the art and after repeated exploration, the inventors of this application have developed a method for regulating macrophage polarization using melanin nanoparticles. In addition, this application also provides related applications of melanin nanoparticles.

[0007] Specifically, this application provides the following technical solutions:

[0008] In a first aspect, the present application provides a method for activating macrophage autophagy, comprising: contacting a melanin nanoparticle solution with macrophages.

[0009] In a second aspect, the present application provides a method for regulating macrophage polarization, comprising: contacting a melanin nanoparticle solution with macrophages.

[0010] In a third aspect, the present application provides the use of melanin nanoparticles in activating macrophage autophagy or regulating macrophage polarization.

[0011] In a fourth aspect, the present application provides the use of melanin nanoparticles in the preparation of drugs for preventing or treating diseases related to immune regulation.

[0012] The melanin nanoparticles used in this application are natural nanoparticles that are non-cytotoxic and well dispersed in the solution. They regulate macrophage polarization through cell autophagy, and the regulation of macrophage polarization by melanin nanoparticles is manifested in reducing the polarization of M1 macrophages and increasing the polarization of M2 macrophages, which can inhibit the occurrence and progression of inflammation, and thus can be further used to prepare immune-regulating drugs.

[0013] It can be seen that the present application regulates the polarization of macrophages by activating cell autophagy behavior through natural melanin nanoparticles, utilizes the activation of cell autophagy to promote the transformation of macrophages into M2 macrophages, inhibits the occurrence and progression of inflammation, and is further used to prepare immune regulatory drugs.

[0014] In other words, this application has discovered the pathways and mechanisms of immune regulation by melanin nanoparticles from a new perspective, providing a theoretical basis for the preparation of related drugs for immune regulation. It can be applied to a variety of diseases related to immune regulation, including but not limited to inflammatory diseases, cardiovascular diseases, craniofacial defects, etc., and has opened up new uses for melanin nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a transmission electron microscopy image of the melanin nanoparticles prepared in Example 1.

[0016] Figure 2Figure 2 shows the live / dead staining results of macrophages treated with various concentrations of melanin nanoparticles for one and three days, as measured by the live / dead staining method in Example 2. Live cells are shown in green, and dead cells in red. Treatment with various concentrations of melanin nanoparticles showed no toxicity to macrophages, with 0-50 μg / mL melanin nanoparticles having no effect on macrophage viability.

[0017] Figure 3 This figure shows the cell proliferation results of macrophages treated with different concentrations of melanin nanoparticles for one and three days, as measured by the CCK8 assay in Example 2. The bars above the different treatment days correspond, from left to right, to different melanin nanoparticle concentrations: 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. The ordinate represents the OD value. Treatment with melanin nanoparticles at concentrations of 0-100 μg / mL had essentially no significant effect on macrophage proliferation, while treatment with 200 μg / mL of melanin nanoparticles did affect macrophage proliferation.

[0018] Figure 4 The graph shows the immunofluorescence staining results for the M1 macrophage surface marker CD86 and the M2 macrophage surface marker CD206 after treatment of macrophages with different concentrations of melanin nanoparticles for 24 hours in Example 3. Melanin nanoparticles at concentrations ranging from 12.5 to 50 μg / mL promoted the expression of the M2 surface marker CD206 in M1 macrophages, with 25 μg / mL of melanin nanoparticles showing the strongest ability to promote the expression of the M2 surface marker CD206 in M1 macrophages.

[0019] Figure 5 The graph shows the results of flow cytometry analysis of the macrophage markers CD86, a surface marker of F4 / 80+M1 macrophages, and CD206, a surface marker of F4 / 80+M2 macrophages, after treatment of macrophages with different concentrations of melanin nanoparticles for 24 hours in Example 3. Melanin nanoparticles at concentrations ranging from 12.5 to 50 μg / mL promoted the expression of CD206, a surface marker of M2, in M1 macrophages, with 25 μg / mL of melanin nanoparticles showing the strongest ability to promote CD206, a surface marker of M2, in M1 macrophages.

[0020] Figure 6The graph shows the expression of macrophage markers CD86, CD206, iNOS, and Arg-1 in macrophages after 24 hours of treatment with different concentrations of melanin nanoparticles as described in Example 3. Melanin nanoparticles at concentrations ranging from 12.5 to 50 μg / mL promoted the expression of M2 surface markers CD206 and Arg-1 in M1 macrophages and reduced the expression of M1 surface markers CD86 and iNOS. Melanin nanoparticles at concentrations of 25 μg / mL showed the strongest ability to promote CD206 and Arg-1 expression.

[0021] Figure 7 This figure shows the results of immunofluorescence staining using mito-Red + Lyso-Green to detect the expression of the mitochondrial marker mito-Tracker and the lysosomal marker Lyso-Tracker in Example 3. The 25 μg / mL melanin nanoparticle group can promote the colocalization of mitochondria and lysosomes.

[0022] Figure 8 This figure shows the results of immunofluorescence staining using mito-Red + Lyso-Green to detect the expression of the mitochondrial marker mito-Tracker and the autophagy marker LC3 in Example 3. The 25 μg / mL melanin nanoparticle group can promote the colocalization of mitochondria and the autophagy marker LC3. DETAILED DESCRIPTION

[0023] In the present application, "melanin nanoparticles" include melanin nanoparticles derived from natural sources or artificially synthesized melanin nanoparticles, preferably melanin nanoparticles from natural sources, such as melanin nanoparticles derived from cuttlefish ink, melanin nanoparticles derived from animal skin and hair, melanin nanoparticles derived from fungal colonies, etc.

[0024] In the present application, “contacting melanin nanoparticles with macrophages” includes contacting in vivo or in vitro.

[0025] In this application, “macrophage polarization” refers to the process of macrophage differentiation into different phenotypes and functions, that is, differentiation into M1 macrophages that can produce pro-inflammatory cytokines such as IL-1β and TNF-α, or M2 macrophages that can produce anti-inflammatory cytokines such as IL-4 and IL-10.

[0026] In this application, "macrophage autophagy" refers to the process by which macrophages digest and degrade their own components, mediated by lysosomes. Autophagy plays an important role in macrophage polarization. When autophagy interferes with macrophage polarization, autophagy in macrophages becomes dysfunctional and promotes polarization toward the M1 phenotype. Activation of autophagy promotes M2 macrophage polarization, thereby attenuating the inflammatory response.

[0027] In this application, "immune regulation-related diseases" include but are not limited to: inflammatory diseases, cardiovascular diseases, craniofacial defects, tumors, etc.

[0028] Specifically, this application provides the following technical solutions:

[0029] In a first aspect, the present application provides a method for activating macrophage autophagy, comprising: contacting a melanin nanoparticle solution with macrophages.

[0030] In a second aspect, the present application provides a method for regulating macrophage polarization, comprising: contacting a melanin nanoparticle solution with macrophages.

[0031] In a specific embodiment, the concentration of the melanin nanoparticle solution is 12.5 μg / mL to 200 μg / mL.

[0032] In a specific embodiment, the concentration of the melanin nanoparticle solution is 12.5 μg / mL to 100 μg / mL.

[0033] In a specific embodiment, the concentration of the melanin nanoparticle solution is 12.5 μg / mL to 50 μg / mL.

[0034] In a specific embodiment, the concentration of the melanin nanoparticle solution is 12.5 μg / mL to 25 μg / mL.

[0035] In a specific embodiment, the concentration of the melanin nanoparticle solution is 25 μg / mL.

[0036] In a specific embodiment, the regulating macrophage polarization is to reduce the polarization of M1 macrophages and increase the polarization of M2 macrophages.

[0037] In specific embodiments, the melanin nanoparticles are derived from natural sources or are artificially synthesized.

[0038] In specific embodiments, the melanin nanoparticles are derived from cuttlefish ink, from animal skin and hair, or from fungi.

[0039] In a specific embodiment, the melanin nanoparticles are prepared by the following method: obtaining ink from cuttlefish ink sac; centrifuging at a low speed (e.g., 1000 r / min to 5000 r / min, preferably 2000 r / min) at 0°C to 8°C, preferably 4°C for 3 to 10 minutes, preferably 5 minutes, and discarding the supernatant; centrifuging at a high speed (e.g., 10000 r / min to 15000 r / min, preferably 12000 r / min) at 0°C to 8°C, preferably 4°C for 8 to 15 minutes, preferably 10 minutes, and discarding the supernatant to obtain melanin nanoparticles.

[0040] In a specific embodiment, the preparation of the melanin nanoparticles further comprises freeze-drying the obtained melanin nanoparticles to obtain melanin nanoparticle powder.

[0041] In a specific embodiment, the preparation of the melanin nanoparticles further comprises using deionized water to prepare the obtained melanin nanoparticle powder into a melanin nanoparticle solution.

[0042] In a third aspect, the present application provides the use of melanin nanoparticles in activating macrophage autophagy or regulating macrophage polarization.

[0043] In addition, the present application also provides the use of melanin nanoparticles in the preparation of drugs for preventing or treating diseases related to immune regulation.

[0044] In specific embodiments, the melanin nanoparticles are derived from natural sources or are artificially synthesized.

[0045] In a specific embodiment, the melanin nanoparticles are derived from cuttlefish ink.

[0046] In a specific embodiment, the disease related to immune regulation is selected from one or more of the following: inflammatory diseases, cardiovascular diseases, craniofacial defects and tumors.

[0047] Example

[0048] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Any modification or substitution of the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention shall fall within the scope of the present invention.

[0049] Unless otherwise specified, the reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0050] Example 1 Preparation of melanin nanoparticles

[0051] This embodiment provides a method for preparing melanin nanoparticles.

[0052] First, fresh cuttlefish were dissected to obtain the ink sac, and ink was extracted from the dissected ink sac.

[0053] Then, the mixture was centrifuged at 2000 r / min at 4°C for 5 minutes, the supernatant was discarded to remove large particles, and the mixture was further centrifuged at 12000 r / min at 4°C for 10 minutes, the supernatant was discarded to obtain melanin nanoparticles.

[0054] Finally, the melanin nanoparticles were washed three times with deionized water and freeze-dried in vacuum to obtain melanin nanoparticle powder.

[0055] The obtained melanin nanoparticle powder was tested using a transmission electron microscope and confirmed that the obtained particle diameter was about 200 nm. Figure 1 shown.

[0056] Example 2 Effect of melanin nanoparticles on macrophage activity

[0057] In this example, macrophages were extracted, and the melanin nanoparticles prepared in Example 1 were formulated into different concentrations to test the effect of the melanin nanoparticles on the activity of macrophages.

[0058] 1) Extraction of macrophages

[0059] 6-8 week old C57BL / 6 male mice (animal certificate number: SCXK (Beijing) 2019-0010, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were killed by cervical dislocation and immersed in 75% alcohol. Mouse bone marrow-derived macrophages were obtained by bone marrow flushing as follows.

[0060] Under sterile conditions, the skin of the mouse lower limbs was excised, and the tibia and femur were exposed. Soft tissues, including muscle and fascia, were removed. The femur and tibia were then flushed with a mixture of 100 U / ml penicillin and 0.1 mg / ml streptomycin in phosphate-buffered saline (PBS, purchased from Jiangsu Mag Biotechnology Co., Ltd.; the penicillin and streptomycin mixture was purchased from Cytiva). The epiphyses were then trimmed, and the bone marrow cavity was repeatedly flushed with serum-free α-MEM medium (purchased from Gibco) using a 1 mL sterile syringe until the bone marrow cavity was free of blood clots. The serum-free α-MEM medium containing bone marrow cells was transferred to a 15 mL centrifuge tube, and the cells were pipetted and mixed thoroughly. The tube was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in complete α-MEM medium (purchased from Gibco) containing 10% FBS, seeded into T25 culture flasks, and incubated in a 37°C, 5% CO2 incubator for 6-8 h. Collect the unattached cells from the culture flask, which are mouse bone marrow-derived macrophages (BMDM). After centrifugation at 1000 rpm for 5 minutes, discard the supernatant and resuspend the cells in 1640 complete medium (M-CSF purchased from PeproTech, 10% FBS and 1640 medium purchased from Gibco) supplemented with 10% FBS and 10 ng / mL M-CSF for subsequent experiments.

[0061] 2) Preparation of melanin nanoparticle solution

[0062] The melanin nanoparticle powder prepared in Example 1 was prepared with sterile deionized water to different concentrations, namely 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL.

[0063] 3) Preparation of Macrophage Suspension

[0064] The macrophages extracted above were cultured at a rate of 5×10 5 The cells were seeded at a density of 100 μg / well in a 96-well plate and cultured at 37°C and 5% CO2 for 7 days, with half the medium replaced every two days. The cells were then induced with 1640 complete medium containing 100 ng / mL LPS for 24 hours.

[0065] 4) Effect of melanin nanoparticles on macrophage viability

[0066] The prepared macrophage suspension was divided into 6 groups. Melanin nanoparticles with a concentration of 20 mg / mL were diluted to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL of melanin nanoparticle solutions by 1640 complete culture medium, and then directly added into 96-well plates. The cells were cultured at 37°C and 5% CO2 for 1 day and 3 days. The live / dead staining method (for specific methods, please refer to Wu C, Shen Z, Lu Y, Sun F, Shi H. p53 Promotes Ferroptosis inMacrophages Treated with Fe3O4 Nanoparticles. ACS Appl Mater Interfaces. 2022; 14(38): 42791-42803) was used to detect the effect of melanin nanoparticles on macrophage viability. The CCK8 method (for specific methods, please refer to Sun X, Liang Y, Wang Y, Sun C, Wang X. Bisdemethoxycurcumin, a curcumin inderivative, ameliorates adjuvant-induced arthritis by suppressing inflammatory reactions and macrophage migration. Chem Biol Interact. 2024; 387: 110822) Detect the effect of melanin nanoparticles on macrophage proliferation.

[0067] An equal amount of PBS (ie, 0 μg / mL) was added as a blank control.

[0068] The experimental data were processed by GraphPad Prism9.5 software. The effects of melanin nanoparticles on macrophage viability (cytotoxicity results) are shown in Figure 2 , the effects of melanin nanoparticles on macrophage proliferation Figure 3 .

[0069] The experimental results showed that different concentrations of melanin nanoparticles had no effect on the activity of macrophages, and the cell viability was highest when the concentration of melanin nanoparticles was 25 μg / mL.

[0070] It can be seen that melanin nanoparticles have no toxicity to macrophages and cell proliferation is not affected.

[0071] Example 3 Effect of melanin nanoparticles on macrophage polarization

[0072] In this example, macrophages were extracted, and the melanin nanoparticles prepared in Example 1 were formulated into different concentrations to test the effect of the melanin nanoparticles on macrophage polarization.

[0073] 1) Extraction of macrophages

[0074] The method for extracting macrophages is as described in Example 2.

[0075] 2) Preparation of melanin nanoparticle solution

[0076] The melanin nanoparticle powder prepared in Example 1 was prepared with sterile deionized water to different concentrations, namely 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL.

[0077] 3) Induction of M1 macrophages

[0078] The macrophage suspension prepared above was cultured at a volume of 5×10 6 The cells were seeded at a density of 100 μg / well in a 24-well plate and cultured at 37°C and 5% CO2 for 7 days, with half the medium replaced every two days. The cells were then induced with 1640 complete medium containing 100 ng / mL LPS for 24 hours.

[0079] 4) The prepared macrophage suspension was divided into 6 groups, and the melanin nanoparticles with a concentration of 20 mg / mL were diluted to 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL of melanin nanoparticle solutions by 1640 complete medium and directly added to a 24-well plate. After 24 hours, the surface markers CD86 and CD206 of M1 macrophages and M2 macrophages were detected by immunofluorescence staining; the expression of macrophage markers CD86, CD206 and CD206 on F4 / 80+M1 macrophages and F4 / 80+M2 macrophages were detected by flow cytometry; the expression of macrophage markers CD86, CD206, iNOS and Arg-1 was detected by qPCR to detect the polarization of macrophages.

[0080] 4) A 25 μg / mL melanin nanoparticle solution was added to macrophages seeded in a 24-well plate. After 24 hours, immunofluorescence staining was performed using mito-Red + Lyso-Green to detect the expression of the mitochondrial marker mito-Tracker + lysosomal marker Lyso-Tracker, and the mitochondrial marker mito-Tracker + autophagy marker LC3 to detect the occurrence of autophagy.

[0081] The experimental data were processed by GraphPad Prism9.5 software. Figures 4 to 8

[0082] The experimental results showed that after the addition of melanin nanoparticles, the immunofluorescence intensity of M1 macrophage lysosomes in macrophages increased compared with the control group (the group with equal amount of PBS added). At the same time, under the stimulation of melanin nanoparticles at a concentration of 25 μg / mL, the immunofluorescence intensity of macrophage lysosomes was stronger than that of other groups.

[0083] It can be seen that melanin nanoparticles promote the autophagy of macrophages.

[0084] The present application has been described in detail above using general instructions and specific implementation plans, and these modifications or improvements made on the basis of the present application without departing from the spirit of the present application are all within the scope of protection required by the present application.

Claims

1. A method for regulating macrophage polarization using melanin nanoparticles, comprising: contacting the melanin nanoparticle solution with macrophages; The concentration of the melanin nanoparticle solution is 12.5 μg / mL to 200 μg / mL, preferably 25 μg / mL. 2 . The method of claim 1 , wherein the regulating macrophage polarization is to reduce the polarization of M1 macrophages and increase the polarization of M2 macrophages.

3. The method of claim 2, wherein the regulating macrophage polarization is achieved by activating macrophage autophagy.

4. The method according to any one of claims 1 to 3, wherein the melanin nanoparticles are derived from natural sources or are artificially synthesized; Preferably, the melanin nanoparticles are derived from cuttlefish ink, animal skin and hair, or fungi.

5. The method of claim 4, wherein the melanin nanoparticles are prepared by the following method: Obtain ink from cuttlefish ink sacs; Centrifuge at 0°C to 8°C, preferably 4°C, at a low speed (e.g., 1000 r / min to 5000 r / min, preferably 2000 r / min) for 3 minutes to 10 minutes, preferably 5 minutes, and discard the supernatant; Centrifuge at 0°C to 8°C, preferably 4°C, at high speed (e.g., 10,000 r / min to 15,000 r / min, preferably 12,000 r / min) for 8 to 15 minutes, preferably 10 minutes, discard the supernatant, and obtain melanin nanoparticles. 6 . The method according to claim 5 , wherein the preparation of the melanin nanoparticles further comprises freeze-drying the obtained melanin nanoparticles to obtain melanin nanoparticle powder. 7 . The method according to claim 6 , wherein the preparation of the melanin nanoparticles further comprises preparing the obtained melanin nanoparticle powder into a melanin nanoparticle solution using deionized water.

8. Application of melanin nanoparticles in activating macrophage autophagy or regulating macrophage polarization; Preferably, the melanin nanoparticles are derived from natural sources or artificially synthesized; More preferably, the melanin nanoparticles are derived from cuttlefish ink, animal skin and hair, or fungi.

9. Use of melanin nanoparticles in the preparation of drugs for preventing or treating diseases related to immune regulation; Preferably, the melanin nanoparticles are derived from natural sources or artificially synthesized; More preferably, the melanin nanoparticles are derived from cuttlefish ink, animal skin and hair, or fungi.

10. The use according to claim 9, wherein the disease related to immune regulation is selected from one or more of the following: inflammatory diseases, cardiovascular diseases, craniofacial defects and tumors.

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