Nanoparticle for blocking SIRP alpha from recovering interlacing effect, preparation method and application

Abstract

The invention relates to the field of medicines, in particular to a nanoparticle for blocking SIRP alpha from recovering a cell burial effect as well as a preparation method and application of the nanoparticle. The nanoparticle comprises a polypeptide and a drug with an active oxygen scavenging function, the polypeptide is at least formed by connecting an SIRP alpha blocking peptide, a plaque response peptide and a plaque targeting peptide, and the drug is coated with the polypeptide. According to the scheme of the invention, the method is not limited to the treatment of a certain illness characteristic of the AS, but improves the pathological environment of AS continuous inflammation and oxidative stress disorder by improving the low-level interburial effect in the AS, removing apoptotic necrotic cells in the plaque and removing necrotic substances and inflammatory mediators in the plaque from the source. Therefore, the risks of plaque rupture and advanced thrombosis are reduced.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a nanoparticle capable of blocking SIRPα and restoring the cell thrombosis effect, a preparation method and an application thereof. Background Art

[0002] Atherosclerosis (AS) is an immune-mediated chronic inflammatory process characterized by the formation of subendothelial atherosclerotic plaques. The acute cardiovascular events caused by AS have become the main cause of death in humans.

[0003] There are a large number of apoptotic and necrotic cells in the necrotic core of atherosclerotic plaques. If the therapeutic drugs cannot effectively remove these necrotic cells, these cells will continue to release inflammatory mediators, leading to secondary expansion of the necrotic core in the plaque, and eventually forming unstable plaques, increasing the risk of AS rupture and bleeding. The regular removal of apoptotic cells helps maintain the body's homeostasis and protect tissues from the inflammatory contents of apoptotic cells. In order to clean up these cells, the body will initiate a procedure called "cell burial". Cell burial is a highly conservative process that induces phagocytes (macrophages or dendritic cells) to engulf and degrade dying cells by sending an "eat me" signal to them.

[0004] Phagocyte-mediated phagocytosis is regulated by both pro-phagocytic and anti-phagocytic signals on the surface of target cells. For example, macrophages can specifically recognize pro-phagocytic signals such as phosphatidylserine, cell adhesion molecule-3, and calreticulin on the surface of dying cells, which are then absorbed and degraded by macrophages. However, the phagocytic function of macrophages may also be inhibited by anti-phagocytic signals expressed on the cell surface. For example, CD47, which is highly expressed on the surface of tumor cells, interacts with SIRPα on macrophages to transmit a negative regulatory signal of "don't eat me", disguising itself as a normal cell and avoiding macrophage phagocytosis.

[0005] Studies have found that the level of macrophage-mediated efferocytosis in AS plaques is only about 1 / 20 of the physiological level, while the level of CD47-SIRPα is upregulated in AS, which is an important reason for the decreased ability of macrophages to clear apoptotic cells (ACs) in plaques. Based on the above research, Kojima et al. (Nature. 2016; 536(7614)) first tried to use CD47 monoclonal antibodies to treat AS in 2016. They found that injection of CD47 monoclonal antibodies can restore the efferocytosis effect in plaques to prevent the progression of lesions and improve the AS symptoms of mice. However, this treatment has significant side effects. Since CD47 is also widely expressed in normal cells, such as red blood cells and platelets, the use of CD47 monoclonal antibodies may also block the CD47 protein highly expressed on the surface of red blood cells, resulting in healthy cells that have lost their anti-phagocytic ability being mistakenly phagocytosed as diseased cells, causing side effects such as acute anemia and thrombocytopenia. Therefore, it is urgent to find an alternative to restore the efferocytosis effect in plaques without causing the above side effects.

[0006] In addition, the current mainstream choice of drugs for treating AS in clinical practice mainly focuses on lowering blood lipids, anti-inflammatory, anti-thrombotic, etc. However, the above-mentioned single class of drugs can only unilaterally regulate a certain pathological feature of AS, and it is difficult to achieve a synergistic effect in the same time and space; in addition, these drugs do not have imaging, sensing and other detection functions, and cannot monitor the development of AS in real time during the treatment process. Summary of the invention

[0007] The present invention aims to provide a nanoparticle, preparation method and application for blocking SIRPα to restore the efferocytosis effect. Through the scheme of the present application, it is no longer limited to treating a certain pathogenic feature of AS, but by improving the low-level efferocytosis effect in AS, clearing the apoptotic and necrotic cells in the plaque, and clearing the source of necrotic substances and inflammatory mediators in the plaque from the source, so as to improve the pathological environment of AS with persistent inflammation and oxidative stress disorder, thereby reducing the risk of plaque rupture and late thrombosis.

[0008] To achieve the above-mentioned purpose, the present invention adopts the following first basic technical scheme: a nanoparticle that blocks the SIRPα restoration cell burial effect, including a polypeptide and a drug with the function of scavenging reactive oxygen species, wherein the polypeptide is at least composed of a SIRPα blocking peptide, a plaque response peptide and a plaque targeting peptide connected together, and the polypeptide is coated on the outside of the drug.

[0009] In this scheme, since the polypeptide is formed by connecting SIRPα blocking peptide, plaque response peptide and plaque targeting peptide, the nanoparticles simultaneously have the functions of plaque targeting, plaque response drug release and SIRPα blocking.

[0010] The peptides in this scheme have a targeting function, so that the nanoparticles can be targeted to the atherosclerotic plaque site for effective accumulation. After the nanoparticles reach the plaque site, they can bind to SIRPα on the surface of macrophages in the plaque. The nanoparticles can not only block the CD47-SIRPα interaction and restore the "appetite" of macrophages, which is beneficial to the removal of ACs, but also remove the source of necrotic substances and inflammatory mediators in the plaque from the source, and will not affect the expression of CD47 in normal cells, avoiding side effects, and avoiding the risks of acute anemia and thrombocytopenia in the existing CD47 monoclonal antibody treatment of AS. At the same time, the nanoparticles gather at the plaque and can respond to drug release. The drug is released. Since the drug has the function of removing reactive oxygen, it can downregulate RhoA / Rho kinase by consuming reactive oxygen, thereby further enhancing the removal of ACs by macrophages.

[0011] The biggest feature and innovation of this scheme is that the nanoparticles have both targeting and responsive functions. The nanoparticles in this scheme use SIRPα blocking peptides to restore the function of phagocytes to clear plaque ACs, reduce the risk of secondary damage to the necrotic core, rupture and bleeding, and accelerate the process of inflammation resolution. At the same time, targeted release of drugs at the lesion site can remove reactive oxygen and enhance the clearance of ACs by macrophages. In summary, the nanoparticles used in this application to treat AS are no longer limited to treating a certain pathological feature of AS, but clear the source of necrotic substances and inflammatory mediators in the plaque from the source, providing a new idea for the treatment of atherosclerotic plaques.

[0012] To achieve the above-mentioned purpose, the present invention adopts the following second basic technical scheme: a nanoparticle that blocks the SIRPα restoration cell burial effect, including a polypeptide and a drug with imaging and contrast function, the polypeptide is at least composed of a SIRPα blocking peptide, a plaque response peptide and a plaque targeting peptide, and the polypeptide is coated on the outside of the drug.

[0013] The difference between this solution and the first basic technical solution is that the drug coated in the polypeptide is different. The drug coated in the polypeptide in this solution is a drug with imaging and contrast function. In this way, when the nanoparticles are used to treat atherosclerosis, the nanoparticles not only have the functions of targeting, plaque response drug release and SIRPα blocking, but also have the function of imaging and contrast after the drug is released, so that the prognosis of AS after treatment can be visually monitored. The nanoparticles can be used as a non-invasive plaque detection imaging tool to monitor the development of AS in real time during the treatment process.

[0014] Preferably, the first basic technical solution or the second basic solution is improved by one, wherein the drug is tetracarboxyphenyl manganese porphyrin.

[0015] Therefore, the drug selected was tetracarboxyphenyl manganese porphyrin (MnTCPP). The metal ligand in the drug can guide the reactive oxygen into it to undergo redox reaction. This coordination environment makes the manganese porphyrin have catalase (CAT) and peroxidase (POD)-like activity, which can clear the excess reactive oxygen in AS. The consumed reactive oxygen can further enhance the clearance of ACs by macrophages by downregulating the RhoA / Rho kinase pathway.

[0016] In addition, the manganese ions in MnTCPP have good near-infrared absorption and paramagnetic properties, making the nanoparticles a potential photoacoustic / magnetic resonance dual-modality imaging contrast agent for visual monitoring of the prognosis after AS treatment. In summary, this scheme innovatively uses the selective blocking of SIRPα combined with reactive oxygen scavenging to enhance the low-level burial effect in AS, remove apoptotic and necrotic cells in plaques, and improve the pathological environment of AS with persistent inflammation and oxidative stress disorders, thereby reducing the risk of plaque rupture and late thrombosis.

[0017] In addition, during the preparation of the nanoparticles in this scheme, after MnTCPP is mixed with the polypeptide solution, MnTCPP has strong hydrophobicity and can spontaneously enter the hydrophobic domain of the peptide chain to form the "core" of the nanoparticles, while the hydrophilic plaque targeting peptide (such as LyP-1 targeting peptide) forms the "shell" of the nanoparticles, thereby promoting the self-assembly process of the nanoparticles and ultimately forming stable spherical nanoparticles.

[0018] Preferably, the first basic technical solution or the second basic solution is improved by the second improvement, wherein the SIRPα blocking peptide is the D4-2 peptide, and the sequence of the D4-2 peptide is CYRYSAVYSIHPSWCG.

[0019] D4-2 peptide has the function of blocking SIRPα. In the prior art, D4-2 peptide is used to treat tumors and enhance the therapeutic effect of tumors, but D4-2 peptide has not been used to treat atherosclerotic diseases through "cell burial". Therefore, this scheme creatively uses D4-2 peptide to treat atherosclerotic diseases, so that SIRPα on the surface of macrophages in plaques is blocked, restoring the "appetite" of macrophages, which is beneficial to the clearance of ACs, thereby achieving the purpose of treating atherosclerotic diseases from the source.

[0020] Preferably, the third improvement is made to the first basic technical solution or the second basic solution, wherein the plaque response peptide is an MMP response peptide, and the sequence of the MMP response peptide is PVGLIGK.

[0021] Therefore, after the nanoparticles reach the plaque, the high concentration of matrix metalloenzymes in the plaque tissue and related cells cut the MMP response peptide sequence, thereby separating the SIRPα blocking peptide and releasing the drug.

[0022] Preferably, the first basic technical solution or the second basic solution is improved in that the plaque targeting peptide is LyP-1 peptide, and the sequence of LyP-1 peptide is CGNKRTRGC.

[0023] LyP-1 peptide also has a specific affinity for the receptor p32 protein highly expressed in macrophages, endothelial cells and foam cells within the plaque. It can not only improve the effective accumulation of nanoparticles in atherosclerotic plaques, but also avoid the nonspecific distribution of nanoparticles, further improving the biosafety of nanoparticles.

[0024] To achieve the above-mentioned purpose, the present invention adopts the following third basic technical scheme: application of a SIRPα blocking peptide in a drug for treating atherosclerosis, wherein the SIRPα blocking peptide is a D4-2 peptide, and the sequence of the D4-2 peptide is CYRYSAVYSIHPSWCG.

[0025] In this scheme, the D4-2 peptide is creatively used in drugs for the treatment of atherosclerosis, so that SIRPα on the surface of macrophages in the plaque is blocked, restoring the "appetite" of macrophages, blocking SIRPα and restoring the cell burial effect, which is beneficial to the clearance of ACs and achieves the purpose of treating atherosclerosis from the source.

[0026] In order to achieve the above-mentioned purpose, the present invention adopts the following fourth basic technical scheme: use of any of the above-mentioned nanoparticles that block SIRPα and restore the cell burial effect in drugs for treating atherosclerosis.

[0027] In this scheme, any of the above-mentioned nanoparticles is used in drugs for treating atherosclerosis, so that SIRPα on the surface of macrophages in the plaque is blocked, the "appetite" of macrophages is restored, and it is beneficial to the clearance of ACs, thereby achieving the purpose of treating atherosclerotic diseases from the source.

[0028] To achieve the above objectives, the present invention adopts the following fifth basic technical solution: the use of nanoparticles that block SIRPα and restore the cell burial effect in the second basic technical solution or one of the improved two as imaging contrast agents in the treatment of atherosclerotic diseases.

[0029] In this scheme, the nanoparticles have the function of imaging and contrast imaging, and are used as imaging contrast agents for the treatment of atherosclerotic diseases. The development process of AS can be monitored in real time during the treatment process.

[0030] To achieve the above object, the present invention adopts the following sixth basic technical solution: a method for preparing nanoparticles that block SIRPα and restore the cell burial effect, comprising the following steps: S1. Peptide-1 polypeptide was synthesized by standard solid phase peptide synthesis technology. The sequence of Peptide-1 polypeptide was CYRYSAVYSIHPSWCGPVGLIGKCGNKRTRGC; S2. The Peptide-1 aqueous solution and the tetracarboxyphenyl manganese porphyrin aqueous solution were mixed, magnetically stirred for 24 hours, and then ultrafiltered at a speed of 14000-16000 rpm for 20-40 minutes in a centrifuge instrument to prepare nanoparticles, which were then collected. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 TEM image of PTna-1, the scale bar is 100 nm.

[0032] Figure 2 FTIR spectra of MnTCPP, Peptide-1 and PTna-1.

[0033] Figure 3 This is the particle size distribution diagram of PTna-1 under different feed ratios.

[0034] Figure 4 hi in the figure shows the diameter and zeta potential of PTna-1, PTna-2 and PTna-3, n = 3.

[0035] Figure 5 Schematic diagram of HPLC tracing before and after incubation of PTna-1 and recombinant MMP-9 protein for 12 h or 24 h.

[0036] Figure 6 The drug release of PTna-1 at different time intervals in the presence or absence of MMP-9 protein is shown, n=3.

[0037] Figure 7 Schematic diagram of ESR spectral analysis of PBS, Peptide-1, MnTCPP and PTna-1 scavenging •OH.

[0038] Figure 8 For PBS, Peptide-1, MnTCPP and PTna-1 removal • O 2 - Schematic diagram of ESR spectrum analysis.

[0039] Fig. 9 The figure shows the remaining H2O2 after treatment with different concentrations of PTna-1 or MnTCPP, n=3.

[0040] Fig.10 Schematic diagram of measuring oxygen production after different treatments using a dissolved oxygen meter.

[0041] Fig.11The CLSM images of RDPP in Raw264.7 cells pretreated with H2O2 and treated with different methods are shown. The scale bar is 50 μm.

[0042] Fig.12 CLMS images of ROS generation in DCFH-DA-stained cells after different treatments are shown, with the scale bar being 50 μm.

[0043] Fig.13 Quantitative analysis of intracellular ROS generation after different treatments, n = 3; Fig.14 Flow cytometry results are shown; data are expressed as mean ± standard deviation, **P < 0.05, ***P < 0.01.

[0044] Fig.15 CLSM images of Raw264.7 cells co-incubated with PTna-1 or PTna-2 for 2, 4, and 8 h. The scale bar is 25 μm.

[0045] Fig.16 Flow cytometry results of Raw264.7 cells co-incubated with PTna-1 or PTna-2 for 2, 4, and 8 h.

[0046] Fig.17 The intracellular Mn content of Raw264.7 cells after incubation with PTna-1 or PTna-2 for 0.5, 1, and 4 h is shown.

[0047] Fig.18 In vitro fluorescence images.

[0048] Fig.19 In vitro fluorescence of major organs 6 hours after PTna-1 or PTna-2 injection.

[0049] Fig. 20 The localization of PTna-1 and PTna-2 in plaque tissue and the corresponding fluorescence intensity, n=3; data are expressed as mean±SD, ***P<0.01.

[0050] Fig.21 The co-localization of SIRPα and FITC-labeled D4-2 polypeptide or control polypeptide in Raw264.7 cells is shown.

[0051] Fig. 22 c shows the CLSM images of CD47-SIRPα interaction in cells after different treatments, the scale bar is 25 μm; Fig. 22 d shows the results of flow cytometry analysis.

[0052] Fig.23 Flow cytometry analysis results are shown.

[0053] Fig.24 Quantitative results of in vitro efferocytosis efficiency of macrophages after different treatments are shown; double-positive cells represent macrophages that have taken up ACs, n = 5.

[0054] Fig.25 The figure shows the CLSM images of Raw264.7 macrophages treated with serum-free medium, Anti-CD47, PTna-3, and PTna-1 to promote cell efferocytosis; macrophages and ACs were stained with Cell Tracker Green and CellTracker Green, respectively; the scale bar is 50 μm.

[0055] Fig.26 jm shows the release levels of IFN-γ, TGF-β, IL-6 and IL-10 in the cell supernatant after corresponding treatment, n=5.

[0056] Fig. 27 Results of flow cytometry analysis.

[0057] Fig.28 The quantitative results of in vitro efferocytosis efficiency of macrophages treated with serum-free medium, Peptide-1 or PTna-1 are shown, n=5.

[0058] Fig.29 p indicates the expression of RhoA and ROCK-1 proteins in each group detected by Western blot; Fig.29 q shows the relative gray value of quantitative analysis of RhoA and ROCK-1, n=3; data are expressed as mean±SD, **P<0.05, ***P<0.01.

[0059] Fig.30 The imaging ability of PTna-1 in vivo and in vitro is shown, where a is the in vitro PA value and PA image (inset) of different concentrations of PTna-1; b is the in vitro t1-weighted relaxation coefficient and image (inset) of different concentrations of PTna-1; e is the in vivo PA image; c is the corresponding PA signal intensity of the plaque area of ​​atherosclerotic mice before and 6 hours after PTna-1 injection; f is the in vivo MR image; d is the corresponding MR signal intensity of the plaque area of ​​atherosclerotic mice before and 6 hours after PTna-1 injection. Data are expressed as mean ± standard deviation. *** p<0.01.

[0060] Fig.31 The hysteresis loop of PTna-1 is shown.

[0061] Fig.32The in vivo anti-atherosclerotic effect of nanoparticles is shown; a is a schematic diagram of the treatment plan for atherosclerotic mice; b is a representative photo of ORO-stained aorta; c is a schematic diagram of the corresponding quantitative analysis after treatment with saline, anti-CD47, PTna-3, PTna-2 and PTna-1; d is a schematic diagram of HE staining image; e is a schematic diagram of the quantitative analysis of aortic sinus sections after treatment with saline, Anti-CD47, PTna-3, PTna-2, PTna-1; f is a schematic diagram of α-SMA staining image; g is a schematic diagram of saline, anti-CD47, PTna-3, PTna-2, PTna -1 treatment of aortic sinus section quantitative analysis; h is Masson staining image; i shows the quantitative analysis of aortic sinus section after treatment with saline, anti-CD47, PTna-3, PTna-2, PTna-1; j is ORO staining image; k shows the quantitative analysis of aortic root section after treatment with saline, anti-CD47, PTna-3, PTna-2 and PTna-1; l is DHE staining image; m shows the quantitative analysis of aortic root section after treatment with saline, anti-CD47, PTna-3, PTna-2, PTna-1, n=5, scale bar is 200μm. Data are expressed as mean ± standard deviation, **P<0.05, ***P<0.01. DETAILED DESCRIPTION

[0062] The following is further described in detail through specific implementation methods: This embodiment discloses a nanoparticle that blocks the SIRPα restoration thrombocytosis effect, including a polypeptide and a drug with the function of scavenging reactive oxygen species. The polypeptide is at least formed by connecting a SIRPα blocking peptide, a plaque response peptide and a plaque targeting peptide, and the polypeptide is coated on the outside of the drug.

[0063] Specifically, the drug in this embodiment is tetracarboxyphenyl manganese porphyrin (MnTCPP). The tetracarboxyphenyl manganese porphyrin in this embodiment has the function of removing excessive reactive oxygen species from plaques and synergistically enhancing macrophage burial, and the manganese ions in MnTCPP have good near-infrared absorption and paramagnetic properties, and also have the function of photoacoustic / magnetic resonance dual-modal imaging and contrast imaging, and visually monitor the prognosis after AS treatment. Of course, in other embodiments, the drug coated in the polypeptide can also be other types of drugs, all of which can have the function of removing reactive oxygen species, such as curcumin, astaxanthin, etc. In addition, in other embodiments, the drug coated in the polypeptide may not have the function of removing reactive oxygen species, but only have the function of imaging and contrast imaging, such as dihydrochlorin, IR780, etc.

[0064] The SIRPα blocking peptide is used to non-competitively bind to SIRPα and inhibit CD47-SIRPα binding. In this embodiment, the SIRPα blocking peptide is the D4-2 peptide, and the sequence of the D4-2 peptide is CYRYSAVYSIHPSWCG. Of course, in other embodiments, any peptide that can be used to non-competitively bind to SIRPα and inhibit CD47-SIRPα binding can be used.

[0065] The function of the plaque response peptide is that after binding to the plaque, the plaque response peptide responds and causes the polypeptide drug to be released. In this embodiment, the plaque response peptide is an MMP response peptide, and the sequence of the MMP response peptide is PVGLIGK. Of course, in other embodiments, any response peptide that can bind to the plaque and respond to cause the polypeptide to release the drug can be used, such as an MMP-2 response peptide with a peptide sequence of PLGVR, an MMP-2 response peptide with a peptide sequence of PLGLAG, and an MMP-2 / 9 response peptide with a peptide sequence of GGGPQGIWGQGK.

[0066] The plaque targeting peptide is used to target the nanoparticles to the plaque. The plaque targeting peptide in this embodiment is LyP-1 peptide, and the sequence of LyP-1 peptide is CGNKRTRGC. Of course, in other embodiments, any peptide that can play a targeting function can be used, such as a targeting peptide targeting neovascularization in plaques (sequence CRGD), a targeting peptide targeting collagen in plaques (sequence KLWVLPKGGGC), and a targeting peptide targeting macrophage scavenger receptors (sequence LSLERFRCLWSDAPAK).

[0067] Thus, the polypeptide in this embodiment is formed by connecting the above-mentioned D4-2 peptide, MMP response peptide and LyP-1 peptide. The polypeptide is based on the D4-2 peptide, and an MMP response peptide capable of responding to high levels of matrix metalloproteinase-9 (MMP-9) in plaques is introduced at its carboxyl end, and a short peptide LyP-1 with atherosclerotic plaque targeting ability is connected to the carboxyl end of the MMP response peptide. In this way, the sequence of the polypeptide on the final nanoparticle is CYRYSAVYSIHPSWCGPVGLIGKCGNKRTRGC, and the polypeptide is coated with tetracarboxylphenyl manganese porphyrin to form a spherical shape. The nanoparticle in this embodiment is named: PTna-1.

[0068] This embodiment discloses a method for preparing nanoparticles that block SIRPα and restore the cell burial effect, comprising the following steps: S1. Peptide-1 polypeptide was synthesized by standard solid phase peptide synthesis technology. Peptide-1 polypeptide was synthesized from the above-mentioned D4-2 peptide, MMP responsive peptide and LyP-1 peptide. The sequence of Peptide-1 polypeptide was CYRYSAVYSIHPSWCGPVGLIGKCGNKRTRGC. S2. Mix 1 ml of Peptide-1 aqueous solution (1 mg / ml) with 50µl of MnTCPP aqueous solution (5 mg / ml). After magnetic stirring for 24 h, ultrafiltration was performed at a speed of 15,000 rpm for 30 min in a centrifuge instrument, and the nanoparticles were collected twice. The molecular weight critical value of the ultrafiltration membrane is 10 kDa. In this step, in other embodiments, the MnTCPP aqueous solution can also be 100 or 200µl, the speed of the centrifuge instrument can also be 14,000 or 16,000 rpm, and the ultrafiltration time is 20min or 40min. In this embodiment, when MnTCPP is mixed with the polypeptide solution, the highly hydrophobic MnTCPP can also spontaneously enter the hydrophobic domain of the peptide chain to form the "core" of the nanoparticles, while the hydrophilic LyP-1 forms the "shell" of the nanoparticles, realizing the self-assembly of the nanoparticles and finally forming stable spherical nanoparticles.

[0069] In this embodiment, by applying SIRPα blocking peptide to the drug for treating atherosclerosis, it can bind to SIRPα on the surface of macrophages, non-competitively inhibit CD47-SIRPα interaction, and regulate the phagocytosis of ACs by macrophages, thereby achieving the effect of treating atherosclerosis. Therefore, this embodiment discloses the application of SIRPα blocking peptide in the drug for treating atherosclerosis.

[0070] In this embodiment, the nanoparticles are applied to drugs for treating atherosclerosis. The nanoparticles actively target and aggregate at the plaques under the mediation of LyP-1, and release the drug through the MMP response peptide response. The D4-2 peptide inhibits the binding of CD47-SIRPα. At the same time, MnTCPP has multiple effects such as scavenging reactive oxygen species to synergistically treat atherosclerotic plaques in the thoracic aorta of mice. Therefore, this embodiment discloses the application of nanoparticles that block the SIRPα recovery cell burial effect in drugs for treating atherosclerosis.

[0071] In addition, the nanoparticles in this embodiment have the function of imaging contrast, so the nanoparticles in this embodiment that block SIRPα and restore the cell thrombosis effect can be used as imaging contrast agents in the treatment of atherosclerotic diseases.

[0072] experiment The following series of experiments prove that the nanoparticles in this example are successfully prepared and have corresponding effects. In order to clarify the anti-atherosclerotic effect associated with the function of the peptide, we also synthesized nanoparticles of a control polypeptide without a plaque targeting motif (named PTna-2, with a polypeptide sequence of CYRYSAVYSIHPSWCGPVGLIGKARALPSQRSR) or a control polypeptide without a SIRP α binding motif (named PTna-3, with a sequence of YRVTPVFDFEDSWCGPVGLIGKCGNKRTRGC) 1. Successful verification experiment of nanoparticle preparation First, combine Figure 1 As shown, the nanoparticles PTna-1 prepared in this example were subjected to transmission electron microscopy (TEM) detection, which showed that the nanoparticles were nanoscale spherical.

[0073] Fourier transform infrared spectroscopy (FTIR) spectra of nanoparticles showed that the combination Figure 2 As shown, at 1010 cm −1 There is a characteristic peak at + The large heterocyclic vibration at 1660 cm −1 There is a peak at , which is related to -CONH in the peptide chain, indicating that there is efficient co-assembly between MnTCPP and peptide-1 polypeptide.

[0074] In addition, the inventors found that when the Peptide-1 polypeptide / MnTCPP feed ratio was 1:1, the combination Figure 3 As shown in the figure, the nanoparticles have a relatively ideal particle size distribution and MnTCPP encapsulation amount (49.50±0.003%). In addition, at this feed ratio, the encapsulation efficiency of the peptide (81.33±4.04%) is also satisfactory. These indicate that the nanoparticles have a high drug loading rate and a simple preparation process, which is conducive to clinical transformation.

[0075] Combination Figure 4 As shown in h, the DLS detection results show that the diameters of PTna1-3 are similar, which are (71.9±3.91), (75.6±11.51) and (81.9±9.29) nm respectively. Figure 4 As shown in Fig. i, the zeta potential of PTna-1 and PTna-3 is positive because the LyP-1 sequences are dispersed on the surface of these nanoparticles.

[0076] In addition, a standard cell counting kit (CCK-8) was used to detect the in vitro biosafety of different concentrations of nanoparticles (364 μg / mL, 182 μg / mL, 81 μg / mL). After Raw 264.7 macrophages and human umbilical vein endothelial cells (HUVECs) were incubated with nanoparticles PTna-1 for 24 h, it was found that the cell survival rate was greater than 85%, indicating that the nanoparticles of this example have low toxicity to normal tissues.

[0077] 2. Study on the response function of nanoparticles HPLC tracing showed that the binding Figure 5 As shown, after the nanoparticles were treated with recombinant MMP-9 protein (100 ng / ml of recombinant MMP-9 protein was added to the nanoparticle solution (364 μg / mL) for 12 or 24 hours), the initial peak of peptide-1 containing the MMP-9 cleavage motif decreased with the extension of incubation time, indicating that MMP-9 induced the disassembly of the peptide chain. In addition, the binding Figure 6 As shown, the drug release was detected by measuring the absorbance of the released drug with an ultraviolet spectrophotometer. The cumulative release curve showed that in the presence of MMP-9 protein, the release efficiency of PTna-1 increased rapidly with time. After 48 h, about 89.3% of MnTCPP was released.

[0078] It can be seen from this that nanoparticles have the function of drug responsive release.

[0079] 3. Verification of the Reactive Oxygen Scavenging Function of MnTCPP and Nanoparticles In this study, electron spin resonance (ESR) spectroscopy was used to analyze the removal of hydroxyl radicals (•OH) and superoxide anions (•O 2- ) ability.

[0080] In the •OH scavenging experiment, first, add 20 μL DMPO to 200 μL FeSO4 (5 mg / mL) solution; add 20 μL 30% hydrogen peroxide (H2O2) to the above solution and mix for 5 min to generate hydroxyl radicals (•OH). The experiment set up four groups, and then added 160 μL PBS, 160 μL Peptide-1 (164 μg / mL), 160 μL MnTCPP (200 μg / mL) or 160 μL MMP-9 pretreated (100 ng / mL) PTna-1 (364 μg / mL), and let the mixture react for 5 min. The remaining •OH was detected using an electron spin resonance spectrometer (ESR, Bruker A300-10 / 12 spectrometer, Billerica, MA). Figure 7As shown, after incubation with PTna-1 solution pretreated with MnTCPP and MMP-9 protein, the 1:2:2:1 quaternary ammonium salt signal of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) used to capture ·OH decreased, however, obvious characteristic signals were still observed in the control group and peptide-1 group. This indicates that MnTCPP and nanoparticle PTna-1 have the function of scavenging reactive oxygen species.

[0081] In •O 2− In the clearance experiment, first, 100 μL of xanthine oxidase (1 U / mL) reacted with 100 μL of xanthine (10 mM) to generate superoxide anions (•O 2- ), and then 20 μL DMPO was added. Four groups were set up in the experiment, and 160 μL PBS, 160 μL Peptide-1, 160 μL MnTCPP or 160 μL mmp-9 pretreated PTna-1 were added respectively. The reaction was carried out for 10 min, and the residual •O 2- . Combined Figure 8 As shown in the figure, after adding purine and xanthine oxygenase to the four groups of solutions, •O 2− The characteristic peak of the nanoparticles was significantly reduced after adding MnTCPP and PTna-1, which indicated that MnTCPP and nanoparticles PTna-1 had the function of scavenging active oxygen.

[0082] In addition, combined Fig. 9 As shown, different concentrations of MnTCPP and PTna-1 were mixed with H2O2 (H2O2 was 450µM, reaction time was 12 h), and the residual H2O2 content was determined by using a H2O2 assay kit (the residual H2O2 content was determined by reading with an ELISA reader). The ability of PTna-1 to remove H2O2 was evaluated. The inventors found that MnTCPP and PTna-1 removed H2O2 in a concentration-dependent manner. The increase in O2 concentration in the mixed solution of H2O2 and PTna-1 was detected using a portable dissolved oxygen meter, combined with Fig.10 As shown, the mixed solution of H2O2 and Peptide-1, or the solution of PTna-1 alone did not increase the O2 level. These all prove that PTna-1 retains the ability of MnTCPP to scavenge ROS.

[0083] In addition, the inventors stimulated macrophages by adding 1 μg / ml lipopolysaccharide (LPS) to explore the antioxidant effect of PTna-1 on cells. The production of intracellular O2 indirectly reflects the consumption of H2O2, and the experiment was evaluated using tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (RDPP) probe. The results showed that the fluorescence of the MnTCPP and PTna-1 groups was significantly reduced because the fluorescence was quenched after oxidation by O2 ( Fig.11 ).

[0084] In addition, compared with the control group and Peptide-1 group, the activated macrophages incubated with MnTCPP or PTna-1 showed relatively lower intracellular ROS levels, as indicated by the weak green fluorescence emitted by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Figure Fig.12 ). Consistent with the confocal fluorescence images, quantitative analysis based on flow cytometry confirmed the effective elimination of ROS by MnTCPP and PTna-1 ( Fig.13 and Fig.14 ).

[0085] Through the above experiments, it can be proved that MnTCPP and nanoparticles have the function of removing reactive oxygen species.

[0086] 4. Verification of the targeting ability of nanoparticles RAW264.7 cells were seeded in a confocal dish and incubated with LPS (1 μg / ml) for 24 h. Then, the old culture medium in the confocal dish was removed, and fresh culture medium containing DiI-labeled PTna-1 and DiI-labeled PTna-2 (both at a concentration of 364 μg / ml) was incubated with the cells for 2, 4, and 8 h. After LPS-treated macrophages were incubated with PTna-1 and PTna-2, respectively, the culture medium was removed, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and then stained with DAPI for 10 minutes, and then washed three times with PBS. The inventors observed the targeting of PTna-1 and PTna-2 by confocal laser microscopy (CLSM). Fig.15 As shown, the fluorescence increased with time, indicating that the uptake of PTna-1 and PTna-2 by LPS-treated macrophages was time-dependent. The fluorescence intensity values ​​of the PTna-1 group at 2, 4, and 8 h of incubation were greater than those of the PTna-2 group, indicating that macrophages had a greater uptake of nanoparticles containing the LyP-1 sequence.

[0087] Nanoparticle endocytosis was further assessed by flow cytometry analysis. Fig.16 As shown, the internalization rates of PTna-1 at 2, 4, and 8 h were 45.88%, 63.01%, and 80.04%, respectively, which were all higher than those of nanoparticles without LyP-1 motif, indicating that PTna-1 has good targeting properties.

[0088] Inductively coupled plasma mass spectrometry (ICP-MS) results showed that Fig.17 As shown, the intracellular Mn content was greater in the PTna-1 group, which confirmed that LyP-1 could also enhance the ability of nanoparticles to deliver MnTCPP.

[0089] The inventors evaluated the in vivo plaque targeting efficiency and biodistribution of the nanoparticles in the ApoE- / - mouse atherosclerosis model (the model is the model of the in vivo experiment below). Fig.18 As shown, in vivo fluorescence imaging technology showed that PTna-1 produced a stronger fluorescence signal than PTna-2 in atherosclerotic lesions, indicating better targeting efficiency. In addition, the inventors further prepared frozen sections of the aorta and found that PTna-1 aggregated more strongly in plaques than PTna-2 ( Fig.19 and Fig. 20 ).

[0090] These results suggest that PTna-1 is well targeted in vivo and contributes to the efficiency of efferocytic reactivation and ROS clearance.

[0091] 5. Verification of macrophage efferocytosis ability In order to verify that the macrocyclic D4-2 peptide has a high binding affinity with SIRPα on the surface of macrophages. The inventors first synthesized the D4-2 peptide labeled with fluorescein isothiocyanate (FITC) and incubated 364 μg / mL of D4-2 peptide with Raw264.7 macrophages for 6 hours. At the same time, a peptide (YRYTPVFDFEDSWCG) with relatively low affinity to SIRPα was used as a control. Fig.21 As shown, compared with the control group, the red fluorescence of SIRPα in the D4-2 group overlapped more strongly with the green fluorescence of the peptide, and the Pearson colocalization coefficient (PCC) of the D4-2 peptide was calculated to be 0.95.

[0092] The inhibitory effect of PTna on CD47-SIRPα was evaluated by immunofluorescence experiments. Macrophages pretreated with PTna-1 or PTna-3 were first incubated with recombinant CD47 protein labeled with the Fc region of human IgG1 for 24 hours. Then, the treated cells were incubated with 100 μl of PE-labeled anti-Fc antibody (PE-anti -Fc) at a concentration of 100 gg / mL, and the cells were collected for CLSM and flow cytometry analysis. Untreated cells (without protein and peptide) were used as negative controls to compare the blocking effect. Combined with Fig. 22 As shown in c, relatively high fluorescence was observed in cells treated with serum-free medium or PTna-3, which is due to the exposed SIRPα interacting with the recombinant CD47 protein, which then binds to PE-anti-Fc and emits fluorescence. However, the fluorescence of cells treated with PTna-1 decreased significantly. Fig. 22As shown in d, flow cytometry showed that the fluorescence intensity of the PTna-1 group was 0.52 times that of the control group, indicating that PTna-1 containing the D4-2 peptide could block the binding of CD47 to SIRPα.

[0093] Then, cells were treated with tumor necrosis factor-α (TNF-α) to establish an apoptosis model and perform cell burial experiments. Macrophages or ACs were treated with different treatments (control treatment, PTna-1 (364μg / ml), anti-CD47 antibody (10μg / ml) and PTna-3 (364μg / ml) respectively) and then co-incubated for 4 hours, and cell tracer staining was used to determine the ability of phagocytic cells to phagocytose ACs. Fig.23 and Fig.24 As shown, CLSM and flow cytometry showed that the number of macrophage-associated ACs (green and red positive) increased after treatment with PTNA-1 or anti-CD47 antibodies; however, PTNA-3 did not enhance phagocytic efficiency. Figure 23-Figure 25 As shown, it is noteworthy that PTna-1 exhibited the greatest degree of AC clearance, increasing from an average of 10.36% in the control group to 22.00%. These results demonstrate that PTna-1 maintains the ability of the D4-2 peptide to block SIRPα and activate macrophage efferocytosis.

[0094] Effective cell efferocytosis can also reduce inflammation and prevent secondary necrosis of diseased cells; therefore, we used ELISA detection kits to detect the secretion of various inflammatory factors after normalization of cell efferocytosis. Fig.26 As shown, the levels of pro-inflammatory factors IFN-γ and IL-6 in the cell supernatant of the anti-CD47 group and PTna-1 group were decreased, and the levels of anti-inflammatory factors IL-10 and TGF-β were increased.

[0095] In addition, previous studies have shown that excessive ROS may inhibit AC phagocytosis by activating the RhoA / Rho kinase-1 (ROCK-1) pathway. Therefore, PTna, which has the ability to scavenge ROS, can synergistically improve AC phagocytosis through its antioxidant effect. The inventors found that the average percentage of macrophage-associated ACs decreased to 7.36% after LPS stimulation, indicating that the phagocytic efficiency of macrophages was further reduced under oxidative stress. However, it is impressive that combined with Fig. 27 and Fig.28 As shown in the figure, the restorative effect of PTna-1 on cell phagocytosis is more obvious than that of Peptide-1, which indicates that in addition to binding to SIRP-α, PTna-1 also synergistically enhances phagocytosis by alleviating the oxidative stress state of macrophages. The inventors detected the expression of proteins related to the signaling pathway by western blot. The results showed that the combination Fig.29As shown, PTna-1 treatment decreased the protein expressions of RhoA and ROCK-1 compared with the control + LPS group and Peptide-1 + LPS group.

[0096] In summary, we demonstrated that PTna-1, through its efficient design, can regulate the efferocytic behavior of macrophages through multiple mechanisms.

[0097] 6. MnTCPP imaging verification Since MnTCPP has strong near-infrared absorption and magnetism, PTna-1 has the potential to be used as a contrast enhancer for PAI and MRI, providing real-time visual monitoring and diagnosis and treatment guidance for atherosclerotic plaques. To this end, the inventors conducted experimental verification. First, after full-spectrum scanning, the excitation wavelength of 720 nm was determined to be the optimal excitation wavelength for subsequent PAI experiments. The PA signal increased linearly in vitro with the increase of MnTCPP concentration (R2=0.986), as shown in Figure 2. Fig.30 As shown in a.

[0098] The effect of PTna-1 on PA performance was verified using an atherosclerosis model mouse (hereinafter referred to as the model). Fig.30 c and Fig.30 As shown in Figure e, no PA signal was detected in the plaque area before the injection of nanoparticles. 6 h after PTna-1 injection, obvious PA signals were observed in the plaques. This indicates that PTna-1 has great application prospects in PA imaging.

[0099] Magnetic resonance imaging has become the most widely used imaging modality in clinical practice due to its high spatial resolution and deep tissue penetration. Fig.31 As shown, the hysteresis loop of PTna-1 exhibits paramagnetism. T1-weighted images of PTna-1 were collected in vitro. The longitudinal relaxation time decreased with the increase of Mn concentration, and the longitudinal relaxation coefficient was determined to be 4.603 mM-1s-1( Fig.30 b). After in vivo application, the signal intensity of the plaque T1-weighted MR images was significantly enhanced 6 h after injection, and the signal intensity was 1.95 times that before injection ( Fig.30 d and Fig.30 f).

[0100] These results suggest that PTna-1 is a promising noninvasive imaging tool for plaque detection.

[0101] 7. In vivo validation Establishment of atherosclerosis model: ApoE- / - mice were fed a high cholesterol diet for 12 weeks to promote the development of advanced atherosclerotic plaques. These successfully modeled mice were then treated with different intravenous administrations (Anti-CD47 (7 mg / kg), PTna-3, PTna-2, PTna-1 administration, and the concentrations of MnTCPP and peptides in PTna-1, PTna-2, and PTna-3 were 2.8 mg / kg and 2.3 mg / kg, respectively) twice a week for 6 weeks (eg Fig.32 a).

[0102] After treatment, the aorta was separated longitudinally, the vascular lumen was observed, and the aortic atherosclerotic plaques were stained with Oil Red O (ORO). According to the percentage of ORO staining area in the vascular lumen, the plaque size of different treatment groups decreased to varying degrees, among which the reduction in the PTna-1 group was the most significant (9.8±3.0%) (e.g. Fig.32 b and 32c).

[0103] The inventors studied the regression of atherosclerotic plaques by histochemical analysis of aortic sinus sections. Hematoxylin & eosin (H&E) staining showed that combined with Fig.32 d and Fig.32 As shown in Figure e, the plaques in the control group were filled with a large number of decellularized, lipid-rich necrotic cores, while PTNA-1 treatment most significantly alleviated this pathological feature, reducing the area of ​​the necrotic core to 5.0%.

[0104] The proliferation of vascular smooth muscle cells (Vsmc) has been shown to contribute to plaque stability. Aortic tissue was stained for α-smooth muscle actin (α-SMA). Fig.32 f and Fig.32 As shown in g, the proliferation of VSMCs in the PTna-1 group was particularly significant (19.2±4.9%), and the generation of VSMCs increased by 1.77 times compared with the untreated control group.

[0105] Consistent with the proliferation of VSMCs, Masson staining also showed that collagen deposition around plaques in mice increased after different treatments, such as Fig.32As shown in Figures 32h and 32i, compared with the control group (37.8±7.0%), the collagen content in the anti-CD47 (50.8±6.8%), PTNA-3 (49.8±5.8%), and PTNA-2 (51.2±8.8%) groups increased significantly, which means that the thickness of the fibrous cap increased. In addition, the collagen production in the PTNA-1 (64.6±7.9%) group showed a further upward trend. Since collagen and VSMc within the plaque are considered to be closely related to the stability of the plaque, these results indicate that PTNA-1 treatment can effectively improve the stability of atherosclerotic plaques.

[0106] ORO staining was performed on aortic root sections of atherosclerotic mice. Both observation and quantitative analysis showed that the nanoparticles had a strong anti-atherosclerotic effect. Fig.32 j and Fig.32 As shown in k, the degree of lumen stenosis was significantly reduced from 37.8% in the control group to 24.8%, 17.6%, 17.4% and 9.6% in the anti-CD47 group, PTna-3 group, PTna-2 group and PTna-1 group, respectively, with the greatest reduction in the PTna-1 group. Dihydroethidium (DHE) is an oxidative stress probe that reacts with superoxide anions and emits red fluorescence. Fig.32 As shown in Figures 1 and 32m, the aortic root stained with DHE in the control group showed brighter fluorescence, showing red fluorescence due to the reaction of DHE with ·O2- to generate ethidium bromide. However, the ROS level in the PTna-1-treated group was the lowest, indicating that our multimodal combined treatment significantly attenuated oxidative stress.

[0107] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A nanoparticle that blocks SIRPα and restores the cell burial effect, characterized in that: It comprises a polypeptide and a drug with the function of removing active oxygen, wherein the polypeptide is formed by connecting at least a SIRPα blocking peptide, a plaque response peptide and a plaque targeting peptide, and the polypeptide is coated on the outside of the drug.

2. A nanoparticle that blocks SIRPα and restores the cell burial effect, characterized in that: The invention comprises a polypeptide and a drug with imaging and contrast function, wherein the polypeptide is formed by connecting at least a SIRPα blocking peptide, a plaque response peptide and a plaque targeting peptide, and the polypeptide is coated on the outside of the drug.

3. A nanoparticle for blocking SIRPα and restoring the cell efferocytosis effect according to claim 1 or 2, characterized in that: The drug is tetracarboxyphenyl manganese porphyrin.

4. A nanoparticle for blocking SIRPα and restoring the cell burial effect according to claim 1 or 2, characterized in that: The SIRPα blocking peptide is D4-2 peptide, and the sequence of D4-2 peptide is CYRYSAVYSIHPSWCG.

5. The nanoparticle for blocking SIRPα and restoring the cell efferocytosis effect according to claim 1 or 2, characterized in that: The plaque response peptide is an MMP response peptide, and the sequence of the MMP response peptide is PVGLIGK.

6. The nanoparticle for blocking SIRPα and restoring the cell efferocytosis effect according to claim 1 or 2, characterized in that: The plaque targeting peptide is LyP-1 peptide, and the sequence of the LyP-1 peptide is CGNKRTRGC.

7. Use of a SIRPα blocking peptide in a drug for treating atherosclerosis, characterized in that: The SIRPα blocking peptide is D4-2 peptide, and the sequence of D4-2 peptide is CYRYSAVYSIHPSWCG.

8. Use of the nanoparticles for blocking SIRPα and restoring the cell efferocytosis effect as claimed in any one of claims 1 to 6 in drugs for treating atherosclerosis.

9. Use of the nanoparticles for blocking SIRPα and restoring the cell efferocytosis effect as claimed in claim 2 or 3 as imaging contrast agents in the treatment of atherosclerotic diseases.

10. A method for preparing nanoparticles that block SIRPα and restore the cell burial effect, characterized in that: The following steps are involved: S1. Synthesize Peptide-1 polypeptide by standard solid phase peptide synthesis technology, wherein the sequence of Peptide-1 polypeptide is CYRYSAVYSIHPSWCGPVGLIGKCGNKRTRGC; S2. The Peptide-1 aqueous solution and the tetracarboxyphenyl manganese porphyrin aqueous solution were mixed, and after magnetic stirring for 24 hours, ultrafiltration was performed in a centrifuge at a speed of 14000-16000 rpm for 20-40 minutes to prepare nanoparticles, which were then collected.

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