Surface-modified anti-angiogenic nanosassembly and method of making same

Abstract

The present application provides a kind of surface modified anti-angiogenic nano-assembly and its preparation method, the nano-assembly of the present application is modified grafting KK polypeptide by being grafted at the end of anti-VEGF functional polypeptide, enhance its electro-positive to promote penetration, while self-assembled with copper ion to form stable nanoparticles, the nanoparticles can effectively treat the inflammation reaction and vascular regeneration problem after eye alkali burn.Further by amide reaction access penetrating peptide and ROS scavenger TEMPO, by regulating ROS level, inhibit the activation of NF-κB signal pathway, so as to regulate the state of immune cells, clear oxidative stress, reduce tissue damage.The method of the present application is simple in operation, experimental condition is mild, and has higher ocular surface residence time and endocytosis efficiency, the obtained nanoparticles can be used for the treatment of eye alkali burn, has significant curative effect, especially suitable for the combined treatment of anti-angiogenesis and anti-oxidative stress, has extensive clinical application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanocolloids, in particular to a surface-modified anti-angiogenic nano-assembly and a preparation method thereof. BACKGROUND

[0002] Alkali burn of the eye is one of the common eye injuries, which is usually caused by contact of strong alkali substances (such as sodium hydroxide, potassium hydroxide, etc.) with the eye, resulting in a series of complex pathological reactions such as extensive damage to the eye tissue, local inflammation, and abnormal vascular regeneration. After the burn, not only will it lead to scarring of the cornea and loss of vision, but also often accompanied by overexpression of vascular endothelial growth factor (VEGF), which promotes abnormal vascular regeneration and further aggravates the inflammatory response, affecting the normal function of the eye. Therefore, controlling angiogenesis and inflammation after alkali burn of the eye has become an important research direction in clinical treatment.

[0003] At present, the common treatment methods for alkali burn of the eye include local medication, surgical treatment, and laser treatment, etc. Although traditional drug therapy can relieve symptoms, it often can only deal with local inflammation, and it is difficult to effectively inhibit the abnormal generation of blood vessels and the persistence of oxidative stress response. Especially in terms of anti-angiogenesis and anti-oxidation, the existing treatment methods often face problems such as poor targeting and limited efficacy. Therefore, developing new treatment strategies, especially comprehensive treatment methods that can simultaneously target angiogenesis, oxidative stress, and immune regulation, has important clinical significance.

[0004] With the development of nanomedicine, nanoparticles have gradually become an important part of new treatment strategies due to their excellent biocompatibility, targeting, and controllable drug release properties. In recent years, nanoparticles self-assembled by polypeptides and metal ions have shown wide application potential in the treatment of tumors, inflammation, and other diseases. As a natural biological macromolecule, polypeptides have good biodegradability and biocompatibility, and can target specific cells or tissues by adjusting their structure and function. Transition metal ions such as copper ions often combine with polypeptides to form stable ligands in the metabolic process in the body, and by changing their coordination structure, they can achieve targeted drug delivery and functional regulation.

[0005] HRH polypeptides have been widely studied and proven to have the effect of inhibiting VEGF, and have the potential to reduce the angiogenesis and inflammatory response after ocular alkali burn. HRH polypeptides can inhibit the binding of VEGF to its receptor by binding to VEGF, reduce the formation of new blood vessels, and effectively control the inflammatory response after burn. However, the penetration of HRH polypeptides in vivo is weak, which limits its therapeutic effect. In addition, polypeptide drugs are usually susceptible to degradation by proteases in the body, which makes their stability in the body poor, and the therapeutic effect is further affected. Therefore, how to improve the penetration, stability and targeting of HRH polypeptides has become the key to optimizing the therapeutic effect. SUMMARY

[0006] The purpose of the present application is to provide a HRH polypeptide-copper ion coordination nanoparticle with high in vivo stability and excellent cell entry efficiency, and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose, the present application provides a surface-modified anti-angiogenic nanoassemblies, the nanoassemblies are Cu@HRHKK-TEMPO-P; first, the functional peptide HRHKK is coordinated with copper ions to obtain Cu@HRHKK, and then the penetrating peptide CPP and the ROS scavenger TEMPO are modified by amide reaction.

[0008] The present application also provides a preparation method of the surface-modified anti-angiogenic nanoassemblies, comprising the following steps:

[0009] S1: dissolving the functional peptide HRH in ultrapure water, grafting the KK amino acid and centrifuging and drying to obtain the KK-modified functional peptide HRHKK;

[0010] S2: dissolving it in ultrapure water, adding copper chloride solution drop by drop, then stirring and reacting, centrifuging and washing, and drying to obtain the nanoassemblies Cu@HRHKK;

[0011] S3: dissolving Cu@HRHKK in ultrapure water, adding NHS and EDC for activation, and stirring for a period of time;

[0012] S4: adding penetrating peptide CPP and TEMPO, stirring and reacting, and then purifying by dialysis to obtain Cu@HRHKK-TEMPO-P solution;

[0013] S5: pre-cooling the purified Cu@HRHKK-TEMPO-P solution to-20℃ to-80℃, so that the water in the system rapidly crystallizes into ice;

[0014] S6: under the condition of-40℃ to-60℃, applying vacuum, directly converting solid water molecules into gaseous water vapor by sublimation and discharging, and removing residual water in the system; obtaining Cu@HRHKK-TEMPO-P in powder form.

[0015] Further, in S2, the ratio of functional peptide HRHKK to copper chloride solution is 10-12 mg:0.1-0.15 mL; the concentration of copper chloride is 1 mg / mL, and the reaction time is 8-10 h.

[0016] Further, in S3, the amount of Cu@HRHKK is 10 mg, and the amount of ultrapure water is 10 mL; the stirring time is 24 h; the amount of added NHS is 1.6 mg, and the amount of added EDC is 2.6 mg.

[0017] Further, in S4, the amount of added penetrating peptide CPP is 1 mg; the amount of added TEMPO as a ROS scavenger is 0.1 mg; the stirring time is 24 h; and the dialysis time is 24 h.

[0018] Further, the reaction temperature of S3 is 25℃.

[0019] Further, in S5, a protective agent is added to the solution before freezing, and the protective agent includes trehalose, mannitol or glucose.

[0020] Further, the powder-shaped Cu@HRHKK-TEMPO-P is dissolved in ultrapure water or a solution to present a colloidal state.

[0021] Further, in S1-S4, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is introduced as a buffer to maintain the PH of the reaction system between 7.2 and 7.4.

[0022] Further, in S1-S4, ultrasonic assistance is introduced to promote the uniform dispersion and sufficient contact of the functional peptide, copper ions and TEMPO and the like, thereby improving the controllability of the assembly process and the consistency of the product.

[0023] Compared with the prior art, the advantages of the present application are that:

[0024] 1. The nanoparticles of the present application are formed by a new strategy of coordination reaction of copper ions and HRH polypeptides, which successfully improves the stability and bioavailability of the polypeptides, avoids the rapid degradation of the polypeptides in the body, and significantly prolongs the half-life. By optimizing the structure of the nanoparticles, the stability is enhanced, which provides a new idea for the nanodrug delivery system of eye diseases.

[0025] 2. The HRH polypeptide-copper ion coordination nanoparticles of the present application effectively solve the problems of poor drug penetration and insufficient targeting in the treatment of eye alkali burns for the first time through nanoparticle design, and enhance the eye tissue repair function by utilizing the ROS scavenging effect and immune regulation mechanism, and also have potential application value in the field of tumor treatment by regulating the activity of immune cells.

[0026] 3、The nanoparticles prepared by the method have excellent water solubility and biocompatibility, can be distributed in the alkali burn tissue of the eye part efficiently and keep stable activity for a long time.

[0027] 4、The nanoparticle preparation method is simple and mild in operation, suitable for large-scale production, and has good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a synthesis process schematic diagram of Cu@HRHKK-TEMPO-P complex in the application;

[0029] Figure 2 It is a mass spectrum diagram of Cu@HRHKK in Example 2 of the application;

[0030] Figure 3 It is a transmission electron microscope image of Cu@HRHKK-TEMPO-P in Example 3 of the application;

[0031] Figure 4 It is a scanning electron microscope image of Cu@HRHKK-TEMPO-P in Example 4 of the application;

[0032] Figure 5 It is a surface potential distribution of Cu@HRHKK-TEMPO-P in Example 5 of the application;

[0033] Figure 6 It is a laser confocal image of phagocytosis efficiency of Cu@HRHKK-TEMPO-P cells in Example 6 of the application;

[0034] Figure 7 It is an ICP detection result of endocytosis efficiency of Cu@HRHKK-TEMPO-P in Example 7 of the application;

[0035] Figure 8 It is an in-vitro anti-angiogenic effect detection of Cu@HRHKK-TEMPO-P in Example 8 of the application;

[0036] Figure 9 It is a 3D cell uptake result of Cu@HRHKK-TEMPO-P in Example 9 of the application;

[0037] Figure 10 It is a cell streaking result of Cu@HRHKK-TEMPO-P in Example 10 of the application;

[0038] Figure 11 It is a confocal image of ROS scavenging effect of Cu@HRHKK-TEMPO-P on LPS induction in Example 11 of the application;

[0039] Figure 12 Flow cytometry image of ROS scavenging effect of Cu@HRHKK-TEMPO-P on LPS induction in Example 12 of the present application;

[0040] Figure 13 Confocal image of RAW typing of Cu@HRHKK-TEMPO-P on LPS induction in Example 13 of the present application;

[0041] Figure 14 Flow cytometry image of RAW typing of Cu@HRHKK-TEMPO-P on LPS induction in Example 14 of the present application;

[0042] Figure 15 Column chart of stability of Cu@HRHKK-TEMPO-P in different environments in Example 15 of the present application.

[0043] Figure 16 Performance stability analysis chart of the assembly in a peroxide / ROS-rich environment in Example 16 of the present application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be further described below.

[0045] Example 1:

[0046] As described in the background art above, although a variety of nanoparticles have been used for the treatment of ocular alkali burns at present, most of them have problems such as poor targeting, insufficient penetration, limited oxidative stress scavenging effect, etc.

[0047] In order to solve the above problems, as shown in Figure 1 KK polypeptide is a polypeptide with strong electropositivity, which can significantly enhance the cell penetration ability of HRH polypeptide, so that it can better distribute and play a role in the tissues of ocular alkali burns. This strategy not only improves the bioavailability of HRH polypeptide, but also effectively improves its endocytosis efficiency in targeted therapy. By adding protective means or enhancing the stability of HRH polypeptide structure, its biological stability in vivo can be significantly improved, the influence of enzymatic hydrolysis is reduced, and the therapeutic effect is prolonged.

[0048] In addition to solving the problems of vascular regeneration and inflammation, ocular alkali burns are often accompanied by severe oxidative stress. Oxidative stress is a process of cellular damage triggered by excessive reactive oxygen species (ROS). Excessive accumulation of ROS can cause cell membrane damage, protein denaturation, and DNA damage, etc., which can further exacerbate inflammation and repair disorders of ocular tissues. In order to alleviate this process, the present application uses TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) as a free radical scavenger. TEMPO reacts with excessive ROS to eliminate oxidative damage in the body and inhibit tissue damage caused by ROS. In addition, the removal of ROS is not only crucial to reducing tissue damage, but also can regulate immune responses, especially by regulating the activity of macrophages and dendritic cells, further enhancing the effect of immunotherapy. Recent studies have shown that oxidative stress has a significant impact on the immune system, and excessive accumulation of ROS can interfere with the normal function of immune cells. By removing ROS, the function of immune cells, especially macrophages and dendritic cells, which play a key role in immune responses, can be restored. For example, the removal of ROS can improve the polarization state of macrophages, prompting them to transform from pro-inflammatory to anti-inflammatory, thereby reducing the inflammatory response; at the same time, it is also helpful for the functional recovery of dendritic cells.

[0049] Therefore, by using TEMPO to remove ROS, the present application not only alleviates oxidative damage after ocular alkali burns, but also enhances immune responses and improves disease resistance by regulating immune cells. This multi-functional design makes the nanoparticles of the present application not only solve the problems of local angiogenesis and oxidative stress, but also improve the treatment effect through immune regulation, providing a more comprehensive solution for the treatment of ocular alkali burns.

[0050] Specifically, the present application proposes a preparation method of a surface-modified anti-angiogenic nano-assembly, the steps are as follows:

[0051] S1: Dissolve 50 mg of functional peptide in 10 mL of ultrapure water, graft 2 mg of KK amino acid, and centrifuge to dry to obtain KK-modified functional peptide HRHKK;

[0052] S2: Dissolve 50 mg of the above product in 10 mL of ultrapure water, add 0.25 mL of copper chloride solution with a concentration of 1 mg / mL dropwise, then stir for 24 h, centrifuge and wash, and dry to obtain nano-assembly Cu@HRHKK;

[0053] S3: Dissolve 50 mg of Cu@HRHKK in 10 mL of ultrapure water, add 1.6 mg of NHS and 2.6 mg of EDC for activation, and stir for 24 h;

[0054] S4: 1 mg of penetrating peptide CPP and 0.1 mg of TEMPO were added, and the solution was stirred for 24 h. After reaction, the Cu@HRHKK-TEMPO-P solution was purified by dialysis for 24 h;

[0055] S5: The purified Cu@HRHKK-TEMPO-P solution was added with a protective agent trehalose, and the amount of trehalose added was 4% of the solution. The system was pre-cooled to -20°C, so that the water in the system rapidly crystallized into ice;

[0056] S6: Under the condition of -40°C, vacuum was applied, and the vacuum degree was 0.2 mbar. Through the sublimation effect, the solid water molecules were directly converted into gaseous water vapor and discharged, and the residual water in the system was removed, so that the residual water content was less than 2%. The Cu@HRHKK-TEMPO-P was obtained in the form of powder.

[0057] After freeze-drying treatment, the obtained nano-assemblies were white or white-like powders, which had good redispersibility and could be dissolved in ultrapure water or buffer solution in a short time (usually less than 5 minutes) to restore the original colloidal state and maintain the original particle size distribution and functional characteristics. Compared with the liquid sample without freeze-drying, the freeze-dried sample could be stored stably at 2-8°C for more than 6-12 months, and at -20°C for more than 24 months, and was not easy to appear obvious precipitation or lose function in the repeated freezing and thawing process. This step not only significantly prolongs the shelf life of the nano-assemblies, but also improves the convenience of transportation and storage, and is particularly suitable for large-scale preparation, sub-packaging and clinical application. In summary, freeze-drying treatment as an optional process step of the present application, although not necessary, has important technical significance in improving product stability and realizing industrialization and popularization.

[0058] It should be noted that in the preparation method of the present application, the preparation method of S1-S4, an amphoteric ion buffer is introduced to maintain the stability of the system and improve the controllability of the reaction. In this embodiment, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is selected as the buffer, and MOPS, TES or TRIS, etc. can also be used as common amphoteric ion buffers. The optimal addition amount of 3% (w / v) HEPES is less than ±0.05, which can effectively ensure the coordination process of functional peptides and copper ions and the smooth progress of the subsequent amide reaction. In the specific operation process, the initial pH of the buffer is adjusted to 7.2-7.4 to approach the physiological environment. This range can not only avoid the hydrolysis and precipitation of copper ions under alkaline conditions, but also inhibit the destruction of the stability of the peptide chain in the over-acid environment. At the same time, the activity of TEMPO in the system can also be maintained stable under this condition, so as to realize the continuous effect of antioxidant and free radical scavenging function. The introduction of the buffer also enables the amide reaction to be carried out under relatively mild conditions, improves the bonding efficiency by 15-30%, and reduces the occurrence rate of side reactions, so as to obtain nanometer assemblies with higher purity and more uniform particle size distribution.

[0059] In addition, the presence of the buffer has a positive effect on the application performance of the final product in vitro or in vivo. Experimental results show that in the PBS (without buffer control) environment, the nanometer assemblies precipitate obviously after 24 hours, while in the 3% HEPES condition, the system still maintains good dispersibility and functional activity within 72 hours. This effect shows that the amphoteric ion buffer system can significantly improve the stability of the nanometer assemblies under physiological conditions and reduce the influence of environmental fluctuations on their performance. In summary, although this step is an optional measure, it is of great significance in improving the repeatability of the assembly reaction, the consistency of the product and the application effect of the final product, further highlighting the advanced nature and practical value of the technical scheme of the present application.

[0060] In this embodiment, ultrasonic assistance is introduced in the nanometer assembly process of the present application to promote the uniform dispersion and sufficient contact of functional peptides, copper ions and TEMPO, etc. components, so as to improve the controllability of the assembly process and the consistency of the product. Specifically, after the functional peptide solution, the copper ion solution and TEMPO are mixed uniformly, they can be placed in an ultrasonic device for intermittent or continuous ultrasonic treatment. The ultrasonic condition is preferably 50W. The treatment time can be adjusted according to the volume of the reaction system, which is 5 minutes in this embodiment. If necessary, intermittent operation (such as 2 minutes each time, with an interval of 30 seconds) can be used to avoid excessive heat generated by long-time ultrasonic treatment. In order to further reduce the adverse effects of local temperature rise, the system can be placed in an ice water bath or a low-temperature constant temperature bath to maintain the reaction temperature at 4-15°C, so as to ensure that the structure of the functional peptide is not damaged.

[0061] Under the above conditions, the cavitation effect and micro-jet effect generated by ultrasonic waves in the solution can significantly improve the diffusion rate of the solution components, break the state of local concentration unevenness, and make the coordination reaction between the functional peptide and copper ions more sufficient. At the same time, ultrasonic treatment can prevent the occurrence of large particle precipitation or local aggregation during assembly, thereby obtaining nanometer assemblies with narrower particle size distribution and more uniform morphology. Experiments show that the particle size distribution of the nanometer assemblies prepared without ultrasonic conditions is between 80-150 nm, while the particle size distribution of the products treated under the above ultrasonic conditions is stable at 60-80 nm, and the aggregation rate is significantly reduced. In addition, ultrasonic assistance can also accelerate the occurrence of amide reaction, increase the reaction rate by 20-30%, and reduce the generation of by-products. The final nanometer assemblies not only perform excellently in dispersity and uniformity, but also exhibit higher stability and repeatability in vitro and in vivo environments.

[0062] In this example, unless otherwise specified, all chemical reagents are commercially available and can be used directly without further purification. HUVEC cells are from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI-1640 medium (1640 medium, GIBCO, Invitrogen, Carlsbad, CA), fetal bovine serum (FBS, GIBCO), penicillin-streptomycin (HyClone, Thermo Scientific, Logan, UT), and trypsin 0.25% solution (HyClone) are purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. (Hangzhou, China). About 4-6 weeks old black mice are purchased from Shanghai Slac Laboratory Animal Center (Shanghai, China). The water used in all experiments with a resistivity higher than 18.2 MΩ·cm is purified by a laboratory water purification system (Cascada I, PALL, Beijing, China).

[0063] Example 2:

[0064] The different chiral assemblies obtained in Example 1 are dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL. As shown in Figure 2 ESI-MS is used to characterize the polypeptide HRHKK, and the main molecular ion peak and characteristic fragment ions thereof are obtained in positive ion mode, the molecular weight is accurately determined, and the amino acid sequence is confirmed. The detection results are highly consistent with the theoretical values, proving the purity and structural accuracy of the prepared polypeptide, ensuring its reliability and repeatability in subsequent functional research, drug design, and application development.

[0065] Example 3

[0066] The present application detects and characterizes the TEM of the synthetic polypeptide assembly Cu@HRHKK-TEMPO-P, as shown in Figure 3 The results show that the polypeptide can self-assemble into nanoparticles in aqueous solution, which are regular spherical and have good dispersibility. The particle size of the assembly after amide locking is narrower, and the PDI is reduced by ≥30%, indicating that the assembly structure is highly consistent, and the particle size distribution is concentrated and uniform, with an average size mainly concentrated between 150-200 nm, indicating that the polypeptide has stable nanostructure characteristics, providing a reliable foundation for subsequent functional research and application development.

[0067] Example 4

[0068] The present application detects and characterizes the TEM of the synthetic polypeptide assembly Cu@HRHKK-TEMPO-P, as shown in Figure 4 The results show that the polypeptide can self-assemble into nanoparticles in aqueous solution, which are regular spherical and have good dispersibility. The particle size of the assembly after amide locking is narrower, and the PDI is reduced by ≥30%, indicating that the assembly structure is highly consistent, and the particle size distribution is concentrated and uniform, with an average size mainly concentrated between 150-200 nm, indicating that the polypeptide has stable nanostructure characteristics, providing a reliable foundation for subsequent functional research and application development.

[0069] Example 5

[0070] The present application detects and characterizes the TEM of the synthetic polypeptide assembly Cu@HRHKK-TEMPO-P, as shown in Figure 5 The results show that the unmodified HRH assembly has a surface charge close to neutral, with limited stability; after introducing double lysine (KK) modification on the HRH molecule, the surface charge of the assembly is significantly positive. This change not only improves the dispersibility and stability of the particles, but also enhances the ability to interact with negatively charged biological membranes. Therefore, the positive charge feature makes it more conducive to penetrating the corneal barrier and effectively entering the lesion area, thereby improving the treatment efficiency.

[0071] Example 6

[0072] The present application uses laser confocal microscopy to observe and analyze the uptake of polypeptide assembly Cu@HRHKK-TEMPO-P by cells. In the experiment, after co-culturing the fluorescently labeled assembly with target cells for a certain period of time, fluorescence images under different excitation wavelengths are collected. The results show that a large number of obvious fluorescence signals can be seen in the cytoplasm, and the fluorescence intensity gradually increases with the extension of the culture time, proving that the assembly can efficiently enter the cell interior. As shown in Figure 6As shown, further image reconstruction and quantitative analysis showed that Cu@HRHKK-TEMPO-P was uniformly distributed in cells, and the cell entry efficiency was significantly better than that of the control group, indicating that it had good intracellular delivery capacity and application potential.

[0073] Example 7

[0074] The present application uses inductively coupled plasma mass spectrometry (ICP-MS) to quantitatively detect the ability of the cell phagocytic polypeptide assembly Cu@HRHKK-TEMPO-P. As shown in Figure 7 In the experiment, after co-culturing the cells with the copper-containing polypeptide assembly, the free assembly was removed by washing, and then the copper content was determined after digestion. The results showed that the copper content in the experimental group was significantly higher than that in the control group, and increased with the extension of the treatment time. This result shows that Cu@HRHKK-TEMPO-P can efficiently enter the cells and accumulate in the cells, verifying its strong cell entry efficiency and good delivery potential.

[0075] Example 8

[0076] The present application uses a microscope observation method to evaluate the effect of the polypeptide assembly Cu@HRHKK-TEMPO-P on the HUVEC three-dimensional cell sphere model. As shown in Figure 8 In the experiment, the treated and control cells were cultured under the same conditions, and the cell sphere morphology and volume changes were continuously monitored. The results showed that the control cell spheres gradually increased in size and had complete boundaries, indicating that they had strong proliferation and blood vessel-like structure formation ability; while the cell spheres treated with Cu@HRHKK-TEMPO-P significantly reduced in volume and had loose structure, and their growth was significantly inhibited. The comprehensive results show that this polypeptide assembly can effectively inhibit the proliferation activity of HUVEC cells, thereby hindering the process of vascular regeneration, and has potential anti-angiogenic application value.

[0077] Example 9

[0078] The present application uses a laser confocal microscope to observe the uptake of the polypeptide assembly Cu@HRHKK-TEMPO-P by the HUVEC three-dimensional cell sphere. As shown in Figure 9 In the experiment, the fluorescently labeled assembly was co-cultured with the HUVEC cell sphere, and after imaging scanning, obvious fluorescence signals were observed on the surface and inside of the cell sphere, indicating that the assembly could enter the three-dimensional cell structure smoothly. The image shows that the fluorescence is widely distributed, and accumulates in the intercellular space and inside the sphere, proving that the assembly has good uptake characteristics in the three-dimensional tissue environment. The results show that Cu@HRHKK-TEMPO-P can be effectively taken up in the 3D cell sphere, and has excellent tissue penetration and delivery potential.

[0079] Example 10

[0080] The present application observes the scratch migration of HUVEC cell spheres under the action of polypeptide assembly Cu@HRHKK-TEMPO-P by microscope. As shown in the figure, Figure 10 the cell spheres are inoculated in the culture plate and subjected to scratch treatment, and the treatment group and the control group are set respectively, and the closure speed of the scratch area and the extracellular extension growth are continuously monitored. The results show that the cell spheres in the control group rapidly expand along the edge of the scratch and gradually fill the gap, showing strong proliferation and migration ability; while the closure of the scratch area in the Cu@HRHKK-TEMPO-P treatment group is significantly delayed, and the extracellular extension growth is limited. Comprehensive analysis shows that the polypeptide assembly can significantly inhibit the proliferation and migration of HUVEC cells, suggesting that it has potential application value in anti-angiogenesis and related disease treatment.

[0081] Example 11

[0082] The present application detects the level of reactive oxygen species (ROS) in RAW cells by laser confocal microscope to evaluate the scavenging ability of polypeptide assembly Cu@HRHKK-TEMPO-P. As shown in the figure, Figure 11 in the experiment, RAW cells are stimulated by lipopolysaccharide (LPS), and the results show that strong green fluorescence signal appears in the control group cells, indicating that the ROS level is significantly increased; while after adding Cu@HRHKK-TEMPO-P treatment, the fluorescence intensity is significantly weakened, and the ROS expression is significantly reduced. The image analysis results further confirm that the assembly can effectively weaken the oxidative stress induced by LPS. In summary, Cu@HRHKK-TEMPO-P has excellent ROS scavenging effect, which helps to alleviate inflammation-related oxidative damage.

[0083] Example 12

[0084] The present application detects the level of reactive oxygen species (ROS) in RAW cells by flow cytometry to verify the antioxidant effect of polypeptide assembly Cu@HRHKK-TEMPO-P. As shown in the figure, Figure 12 in the experiment, RAW cells are stimulated by lipopolysaccharide (LPS), and the results show that the ROS positive rate of the control group cells is significantly increased, and the fluorescence intensity peak is shifted to the right as a whole, indicating that the cells are in oxidative stress state. In the Cu@HRHKK-TEMPO-P treatment group, the proportion of ROS positive cells is significantly decreased, and the fluorescence signal distribution falls back to the low level interval, indicating that the assembly can effectively inhibit the accumulation of ROS. The comprehensive results prove that Cu@HRHKK-TEMPO-P has excellent ROS scavenging ability, which provides experimental basis for its anti-inflammatory and related disease intervention.

[0085] Example 13

[0086] The present application uses a laser confocal microscope to detect the differentiation of RAW cells under LPS induction, and focuses on observing the expression level of surface marker CD80. Figure 13 As shown in the figure, the experimental results show that under the condition of LPS stimulation, the RAW cell surface appears obvious enhanced CD80 fluorescence signal, which prompts that its differentiation trend to pro-inflammatory phenotype is significant. And after adding the polypeptide assembly Cu@HRHKK-TEMPO-P, the CD80 fluorescence intensity is obviously weakened, and the proportion of CD80 positive cells is decreased, which indicates that the assembly can effectively inhibit the high expression of CD80 induced by LPS. In summary, Cu@HRHKK-TEMPO-P can significantly reduce the CD80 level of RAW cells, thereby playing a role in inhibiting inflammatory response.

[0087] Example 14

[0088] The present application uses flow cytometry to detect the differentiation state of RAW cells under LPS stimulation, and focuses on analyzing the expression level of surface differentiation marker CD80. Figure 14 As shown in the figure, the experimental results show that after LPS induction, the proportion of CD80 positive cells in the control group is significantly increased, and the fluorescence intensity distribution is overall right-shifted, which prompts that the cells are obviously differentiated to pro-inflammatory phenotype. And in the polypeptide assembly Cu@HRHKK-TEMPO-P treatment group, the fluorescence signal of CD80 is obviously weakened, and the proportion of positive cells is decreased, which is significantly different from the control group. The results show that the polypeptide assembly can effectively reduce the CD80 expression of RAW cells, thereby inhibiting the inflammatory differentiation process induced by LPS to a certain extent.

[0089] Example 15

[0090] The present application uses flow cytometry to detect the differentiation state of RAW cells under LPS stimulation, and focuses on analyzing the expression level of surface differentiation marker CD80. Figure 15 As shown in the figure, the experimental results show that after LPS induction, the proportion of CD80 positive cells in the control group is significantly increased, and the fluorescence intensity distribution is overall right-shifted, which prompts that the cells are obviously differentiated to pro-inflammatory phenotype. And in the polypeptide assembly Cu@HRHKK-TEMPO-P treatment group, the fluorescence signal of CD80 is obviously weakened, and the proportion of positive cells is decreased, which is significantly different from the control group. The results show that the polypeptide assembly can effectively reduce the CD80 expression of RAW cells, thereby inhibiting the inflammatory differentiation process induced by LPS to a certain extent.

[0091] Example 16

[0092] The present application uses flow cytometry to detect the differentiation state of RAW cells under LPS stimulation, and focuses on analyzing the expression level of surface differentiation marker CD80. Figure 16It is found by the function maintenance experiment that the function maintenance time of the assembly in the strong oxidation environment is prolonged by 2-3 times after the introduction of TEMPO, and the oxidation stress resistance of the system is effectively improved. The result unexpectedly solves the problem that the metal coordination system is easy to be inactivated, and significantly enhances the durability and adaptability of the assembly under complex physiological conditions, thereby providing a new design idea and technical support for the in-depth research of subsequent drug delivery and biomedical materials.

[0093] In conclusion, the experimental results show that only under the specific combination conditions of CPP + TEMPO + amide reaction + Cu2+ coordination, the all-round improvement effects of stable particle size, oxidation resistance, metal migration resistance and high delivery efficiency can be obtained.

[0094] The above is only the preferred embodiment of the present application, and does not limit the present application in any way. Any person skilled in the art can make any form of equivalent replacement, modification or change to the technical solutions and technical contents disclosed in the present application without departing from the scope of the technical solutions of the present application, and still belongs to the protection scope of the present application.

Claims

1. A surface-modified anti-angiogenic nanoassemblies, characterized in that, The nano-assembly is Cu@HRHKK-TEMPO-P; The Cu@HRHKK is obtained by coordination of the modified functional peptide HRHKK and copper ions, and the penetrating peptide CPP and ROS scavenger TEMPO are modified by amide reaction.

2. A method for preparing a surface-modified anti-angiogenic nanoassemblies, the nanoassemblies of claim 1, characterized in that, The method comprises the following steps: S1: dissolving the functional peptide HRH in ultrapure water, grafting the KK amino acid, centrifuging and drying to obtain the KK-modified functional peptide HRHKK; S2: dissolving the HRHKK in ultrapure water, adding the copper chloride solution drop by drop, then stirring and reacting, centrifuging and washing, and drying to obtain the nano-assembly Cu@HRHKK; S3: dissolving the Cu@HRHKK in ultrapure water, adding NHS and EDC for activation, and stirring for a period of time; S4: adding the penetrating peptide CPP and TEMPO, stirring and reacting, and then purifying by dialysis to obtain a Cu@HRHKK-TEMPO-P solution; S5: pre-cooling the purified Cu@HRHKK-TEMPO-P solution to -20℃ to -80℃, so that the water in the system rapidly crystallizes into ice; S6: applying vacuum at -40℃ to -60℃, directly converting the solid water molecules into gaseous water vapor by sublimation and discharging, and removing the residual water in the system; and obtaining the powder-like Cu@HRHKK-TEMPO-P.

3. The method of claim 2, wherein the surface-modified anti-angiogenic nanoassemblies are prepared by the method comprising the steps of: In S2, the ratio of the functional peptide HRHKK to the copper chloride solution is 10-12 mg:0.1-0.15 mL; the concentration of the copper chloride is 1 mg / mL, and the reaction time is 8-10 h.

4. The method of claim 2, wherein the surface-modified anti-angiogenic nanoassemblies are prepared by the method comprising the steps of: In S3, the amount of the Cu@HRHKK is 10 mg, the amount of the ultrapure water is 10 ml, the stirring time is 24 h, the amount of the added NHS is 1.6 mg, and the amount of the added EDC is 2.6 mg.

5. The method of claim 4, wherein the surface-modified anti-angiogenic nanoassemblies are prepared by the method comprising the steps of: In S4, the amount of the added penetrating peptide CPP is 1 mg, the amount of the added TEMPO is 0.1 mg as a ROS scavenger, the stirring time is 24 h, and the dialysis time is 24 h.

6. The method of claim 2, wherein the surface-modified anti-angiogenic nanoassemblies are prepared by the method comprising: The reaction temperature of S3 is 25℃.

7. The method of claim 2, wherein the surface-modified anti-angiogenic nanoassemblies are prepared by the method comprising the steps of: In S5, a protective agent is added to the solution before freezing, and the protective agent comprises trehalose, mannitol or glucose.

8. The method for preparing the surface-modified anti-angiogenic nanoassemblies according to claim 2, characterized in that, The powder-like Cu@HRHKK-TEMPO-P is dissolved in ultrapure water or a solution to present a colloidal state.

9. The method for preparing the surface-modified anti-angiogenic nanoassemblies according to claim 2, characterized in that, In S1-S4, HEPES, i.e., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, is introduced as a buffer to maintain the PH of the reaction system at 7.2-7.4.

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