Supramolecular assembly as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine and nano delivery, and particularly relates to a supramolecular assembly as well as a preparation method and application thereof. The supramolecular assembly constructed by the invention is based on mediation of a polyphenol molecular adhesive, the polyphenol molecular adhesive can generate relatively strong interaction with a target protein, so that the surface of the target protein is modified with a catechol group, and the catechol group on the surface of the target protein can be combined with a functional polypeptide modified with a phenylboronic acid group; dynamic chemical bonds are formed through catechol and phenylboronic acid, a compound beneficial to endocytosis of cells is formed, and therefore the binding problem of cationic polypeptide and target protein is solved. According to the invention, the functional polypeptide modified with phenylboronic acid is constructed by utilizing the principle, and a nano-scale supramolecular assembly is formed by virtue of multiple interaction synergistic driving of the polyphenol molecular adhesive and the target protein, so that an efficient, universal and safe new scheme is provided for intracellular delivery of protein drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanodelivery technology, specifically relating to a supramolecular assembly, its preparation method, and its application. Background Technology

[0002] Compared to traditional small-molecule drugs, protein drugs exhibit higher activity, better safety, stronger specificity, and more significant therapeutic effects. However, proteins themselves have large molecular weights, low surface charge, and strong hydrophilicity, typically preventing them from crossing cell membranes to enter cells. Therefore, existing protein drugs primarily exert their effects extracellularly by modulating receptor molecules in blood, interstitial fluid, or on cell membranes. However, approximately two-thirds of human proteins reside intracellularly, offering a richer pool of potential therapeutic targets.

[0003] To deliver functional proteins into cells for cell biology regulation or disease treatment, various physical delivery methods and delivery vectors have been developed. Among these, peptides, as delivery vectors, are low-cost to synthesize, easily chemically modified, and biocompatible, showing great promise for intracellular delivery of nucleic acids or proteins. However, the limited number of binding sites on the protein surface and its relatively large molecular weight hinder the formation of stable complexes with delivery vectors. Especially for positively charged proteins, their positive surface potential leads to electrostatic repulsion with positively charged peptide vectors, thus preventing the binding of proteins to cationic peptides and hindering the formation of complexes easily endocytosed by cells.

[0004] To address these challenges, researchers have enhanced the binding affinity between proteins and delivery vectors through genetic engineering or chemical conjugation with ligands. For example, proteins are fused with anionic green fluorescent protein tags, peptides, or nucleic acids to create anionicly charged proteins, also known as "supercharged proteins," ensuring effective binding with cationic lipids, polymers, or nanoparticles. However, these methods require covalent or chemical modification of the protein, resulting in high synthesis costs, reduced protein bioactivity, and unforeseen safety issues.

[0005] In summary, how to fully bind cationic peptides to natural proteins is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a supramolecular assembly, its preparation method, and its application. The supramolecular assembly provided by this invention solves the problem that cationic polypeptide carriers and proteins cannot fully bind to form stable complexes.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a supramolecular assembly comprising the following components co-assembled: a functional polypeptide modified with phenylboronic acid groups, a polyphenolic binder, and a target protein; wherein the functional polypeptide modified with phenylboronic acid groups contains a membrane-penetrating sequence, the membrane-penetrating sequence being an amino acid sequence rich in arginine or histidine, and the membrane-penetrating sequence is shown in any one of SEQ ID NO. 1 to 6: SEQ ID NO. 1: B(OH)2-FFGPLGLA-G-RRRR-NH2; SEQ ID NO. 2: B(OH)2-RRRR-GPLGLAG-FF-NH2; SEQ ID NO. 3: B(OH)2-FFGPLGLA-G-KKKK-NH2; SEQ ID NO. 4: B(OH)2-KKKK-GPLGLAG-FF-NH2; SEQ ID NO. 5: B(OH)2-FFGPLGLA-G-RKRK-NH2; SEQ ID NO. 6: B(OH)2-RKRK-GPLGLAG-FF-NH2.

[0008] Preferably, the polyphenol molecular binder includes one or more of tannic acid, baicalin, curcumin, and catechin.

[0009] Preferably, the molar ratio of the functional polypeptide modified with phenylboronic acid groups to the target protein is 28-32:1.

[0010] Preferably, the target protein includes one or more of bovine serum albumin, horseradish peroxidase, ribonuclease A, trypsin, and superoxide dismutase.

[0011] Preferably, the molar ratio of the polyphenol molecular binder to the target protein is no higher than 10:1.

[0012] Preferably, the particle size of the supramolecular assembly is 50~200 nm.

[0013] The present invention also provides a method for preparing the supramolecular assembly described above, comprising the following steps: (1) The target protein and polyphenol molecular binder were incubated in a buffer solution to obtain a polyphenol protein complex; (2) The polyphenol protein complex and the functional polypeptide modified with phenylboronic acid groups are mixed and assembled to obtain the supramolecular assembly.

[0014] Preferably, the pH value of the buffer solution is 7.0 to 8.5; and the incubation time is 5 to 30 minutes.

[0015] Preferably, the assembly temperature is 25~37℃ and the assembly time is 10~60 minutes.

[0016] The present invention also provides the application of the supramolecular assemblies described in the above-described scheme or the supramolecular assemblies prepared by the above-described scheme in intracellular protein delivery.

[0017] This invention provides a supramolecular assembly. The key obstacle preventing the binding of proteins to cationic peptides lies in the positively charged groups on the protein surface, including imidazole (histidine residues), amino (lysine residues), and guanidino (arginine residues). The supramolecular assembly provided by this invention can bind to both cations and anions on the protein surface, thereby solving the problem of cationic peptide carriers failing to fully bind with proteins to form stable complexes, and enabling universal delivery of a variety of proteins.

[0018] The supramolecular assembly constructed in this invention is mediated by a polyphenol molecular binder. This binder contains both phenolic hydroxyl groups and hydrophobic aromatic rings, enabling strong interactions with the target protein. This allows the target protein surface to be modified with catechol groups. These catechol groups can then bind to functional peptides (cationic peptides) modified with phenylboronic acid groups. Through the formation of dynamic chemical bonds between catechol and phenylboronic acid, a complex is formed that facilitates endocytosis, thus solving the problem of binding catechol peptides to the target protein. Utilizing this principle, this invention constructs functional peptides modified with phenylboronic acid. Through a polyphenol molecular binder, these peptides interact with the target protein via multiple interactions (non-covalent interactions and dynamic covalent bonds, specifically including hydrogen bonds, hydrophobic interactions, and the crucial borate ester bonds) to form a nanoscale supramolecular assembly. This provides a highly efficient, universal, and safe new approach for intracellular delivery of protein drugs.

[0019] This invention also provides a method for preparing the supramolecular assembly described above. The preparation method provided by this invention is simple in steps, convenient in operation, safe, and highly stable, making it suitable for large-scale applications.

[0020] This invention also provides the application of the supramolecular assemblies described in the above-described schemes or the supramolecular assemblies prepared by the above-described schemes in intracellular protein delivery. The supramolecular assemblies provided by this invention have high intracellular protein delivery efficiency, can efficiently overcome cell membrane barriers, promote endosome escape, are effective for proteins of different molecular weights and isoelectric points, and can maintain the biological activity of proteins while having low toxicity to cells and good biocompatibility.

[0021] Using the supramolecular assembly of this invention, target proteins were delivered in HepG2, A549, 293E, and Cos7 cell lines, respectively. Experimental results show that the supramolecular assembly provided by this invention has the following advantages: high intracellular protein delivery efficiency; activity assays using proteins such as HRP showed that the proteins delivered into cells by this invention retained their biological activity; delivery of cytotoxic proteins such as RNase A demonstrated that this invention delivered toxic proteins to cancer cells such as HepG2, exhibiting significant cytotoxicity and achieving a therapeutic effect of killing cancer cells; cytotoxicity experiments showed that the supramolecular assembly provided by this invention has low cytotoxicity, with cell survival exceeding 80% under optimal conditions, exhibiting good biocompatibility; the supramolecular assembly provided by this invention has high delivery efficiency during intracellular delivery, low preparation cost, and low material toxicity, effectively and safely delivering target proteins into the cytoplasm without requiring chemical modification of the target protein. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The fluorescence characterization results of different polyphenol molecular binders forming complexes with target proteins through non-covalent bonds are shown below; (a) is the fluorescence characterization result corresponding to polyphenol molecular binder A1, (b) is the fluorescence characterization result corresponding to polyphenol molecular binder A2, (c) is the fluorescence characterization result corresponding to polyphenol molecular binder A3, and (d) is the fluorescence characterization result corresponding to polyphenol molecular binder A4. Figure 2 A comparison of the efficiency of supramolecular assemblies in delivering bovine serum albumin into HepG2 cells; Figure 3 The graph shows the results of the isothermal calorimetric titration of polyphenol molecular binder A1 with bovine serum albumin. Figure 4 The circular dichroism spectrum of polyphenol molecular binder A1 and bovine serum albumin; Figure 5 The graph shows the efficiency of polyphenol molecular binder A1 in delivering bovine serum albumin into HepG2 cells by functional peptides P1-P6 modified with phenylboronic acid groups; where (a) is a fluorescence microscope image and (b) is a bar chart analysis of the average fluorescence intensity in (a). Figure 6The graph shows the toxicity detection of the complex formed by polyphenol molecular binder A1, functional polypeptide P1 modified with phenylboronic acid groups, and target protein in HepG2 cells. Figure 7 Potential characterization diagram of the complex formed by polyphenol molecular binder A1, functional polypeptide P1 modified with phenylboronic acid group and target protein; Figure 8 Dynamic light scattering characterization of the complex formed by polyphenol molecular binder A1, functional polypeptide P1 modified with phenylboronic acid groups, and target protein; Figure 9 This diagram illustrates the cytotoxicity of supramolecular assemblies delivering ribonuclease A into HepG2 cells. Figure 10 This diagram illustrates the cytotoxicity of supramolecular assemblies delivering trypsin into HepG2 cells. Figure 11 The diagram shows the effect of supramolecular assemblies delivering horseradish peroxidase into HepG2 cells; (a) is a diagram of the mechanism of action of HRP, (b) is a fluorescence microscope image, (c) is a bar chart analysis of the average fluorescence intensity of HRP in (b), and (d) is a comparison diagram of catalase activity detection. Figure 12 The image shows the effect of supramolecular assemblies delivering peroxidase into HepG2 cells; (a) is a fluorescence microscope image, and (b) is the corresponding bar chart. Figure 13 The image shows the effect of supramolecular assemblies delivering superoxide dismutase into HepG2 cells; (a) is a fluorescence microscope image, and (b) is the corresponding bar chart. Detailed Implementation

[0024] This invention provides a supramolecular assembly comprising the following components co-assembled: a functional polypeptide modified with phenylboronic acid groups, a polyphenolic binder, and a target protein; wherein the functional polypeptide modified with phenylboronic acid groups contains a membrane-penetrating sequence, the membrane-penetrating sequence being an amino acid sequence rich in arginine or histidine, and the membrane-penetrating sequence is shown in any one of SEQ ID NO. 1 to 6: SEQ ID NO. 1: B(OH)2-FFGPLGLA-G-RRRR-NH2; SEQ ID NO. 2: B(OH)2-RRRR-GPLGLAG-FF-NH2; SEQ ID NO. 3: B(OH)2-FFGPLGLA-G-KKKK-NH2; SEQ ID NO. 4: B(OH)2-KKKK-GPLGLAG-FF-NH2; SEQ ID NO. 5: B(OH)2-FFGPLGLA-G-RKRK-NH2; SEQ ID NO. 6: B(OH)2-RKRK-GPLGLAG-FF-NH2.

[0025] In this invention, the polyphenol molecular binder preferably includes one or more of tannic acid, baicalin, curcumin and catechin.

[0026] In this invention, the molar ratio of the functional polypeptide modified with phenylboronic acid groups to the target protein is preferably 28-32:1, more preferably 30:1.

[0027] In this invention, the target protein preferably includes one or more of bovine serum albumin (BSA), horseradish peroxidase (HRP), ribonuclease A (RNase A), trypsin, catalase (CAT), and superoxide dismutase (SOD).

[0028] In this invention, the molar ratio of the polyphenol molecular binder to the target protein is preferably no higher than 10:1, more preferably 1 to 10:1, and even more preferably 3 to 8:1.

[0029] In this invention, the particle size of the supramolecular assembly is preferably 50-200 nm, more preferably 100-150 nm, and even more preferably 120 nm.

[0030] The present invention also provides a method for preparing the supramolecular assembly described above, comprising the following steps: (1) The target protein and polyphenol molecular binder were incubated in a buffer solution to obtain a polyphenol protein complex; (2) The polyphenol protein complex and the functional polypeptide modified with phenylboronic acid groups are mixed and assembled to obtain the supramolecular assembly.

[0031] This invention involves incubating a target protein and a polyphenol molecular binder in a buffer solution to obtain a polyphenol-protein complex. In this invention, the pH of the buffer solution is preferably 7.0 to 8.5, more preferably 8.

[0032] In this invention, the incubation temperature is preferably room temperature (25°C); the incubation time is preferably 5 to 240 minutes, more preferably 10 to 60 minutes, and even more preferably 30 minutes.

[0033] After obtaining the polyphenol-protein complex, the present invention mixes the polyphenol-protein complex with a functional polypeptide modified with a phenylboronic acid group and assembles them to obtain the supramolecular assembly. In the present invention, the assembly temperature is preferably 25~37℃, more preferably 27~35℃, and even more preferably 32℃, and the assembly time is preferably 10~60 minutes, more preferably 20~50 minutes, and even more preferably 30~40 minutes.

[0034] The present invention also provides the application of the supramolecular assemblies described in the above-described scheme or the supramolecular assemblies prepared by the above-described scheme in intracellular protein delivery.

[0035] The supramolecular assemblies provided by this invention can be used for intracellular protein delivery, including the preparation of intracellular protein delivery drugs, which can efficiently overcome cell membrane barriers, promote endosome escape, and maintain the biological activity of target proteins.

[0036] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0037] Example 1 A general preparation method for polyphenol molecular binder-mediated supramolecular assemblies is described in the following steps: The polyphenol molecular binder was mixed with the target protein, blown evenly, and incubated at room temperature (25°C) for 30 min in a buffer solution with a pH of 7.0-8.5 to obtain a polyphenol protein complex solution. Then, the functional peptide modified with phenylboronic acid groups was mixed with the polyphenol protein complex solution, blown evenly, and incubated at room temperature (25°C) for 30 min for assembly to obtain a supramolecular assembly.

[0038] Example 2 Fluorescence characterization was performed on the non-covalent complexes formed between different polyphenolic binders (tannic acid, catechol, curcumin, and baicalin, denoted as A1-A4) and the target protein (bovine serum albumin). The specific steps were as follows: The fluorescence spectrum of the target protein was detected using a fluorometer with excitation and emission wavelengths of 280 nm and 300-400 nm, respectively. The polyphenolic binder aqueous solution was added dropwise to the target protein solution at different molar ratios (0:1, 0.5:1, 1:1, 2:1, 4:1, 8:1, and 10:1), thoroughly mixed, and incubated at room temperature (25°C) for 1 hour to ensure complete mixing. The mixture was then continuously diluted with deionized water at different molar ratios until a final concentration of 1 mL was achieved. After stabilizing for 1 minute, the fluorescence spectrum of the diluted mixture was examined. Bovine serum albumin (BSA) was used as the model protein. The final concentration of BSA in the mixed solution was 3 μM during the test. The results are as follows: Figure 1 As shown.

[0039] according to Figure 1 It can be seen that as the molar ratio of polyphenol molecular binder to target protein increases, the fluorescence of target protein is significantly quenched. This is because the addition of polyphenol molecular binder changes the microenvironment of amino acid residues (such as tryptophan) inside the target protein, thereby causing fluorescence quenching. This indicates that polyphenol molecular binder, such as tryptophan, interacts with the target protein.

[0040] Example 3 The efficiency of delivering bovine serum albumin into HepG2 cells using the supramolecular assembly prepared in Example 1 was tested. Bovine serum albumin labeled with green fluorescent FITC (BSA-FITC) was used as the model protein. The delivery efficiency of the polyphenol molecular binder-mediated supramolecular assembly was evaluated on HepG2 cells by detecting intracellular fluorescence.

[0041] The specific steps are as follows: HepG2 cells were seeded into 24-well plates and incubated overnight. When the cell density reached more than 80%, protein delivery testing was started. Following the same method as the supramolecular assembly prepared in Example 1, BSA-FITC was thoroughly mixed with polyphenols and functional peptides, respectively. The concentration of BSA-FITC was 0.17 μM, the molar ratio of functional peptide P1 (membrane-penetrating sequence as shown in SEQ ID NO. 1) to BSA-FITC was 30:1, and the molar ratio of polyphenol binder to BSA-FITC was 1:1, 5:1, 10:1, or 20:1, respectively. The cells were incubated at room temperature for 30 min. The cell culture medium was removed, and the cells were washed twice with PBS. The cells were then added to a culture medium solution containing the protein complex and incubated at 37°C for 4 hours. After the cells were treated with the culture medium solution containing the protein complex, the culture medium was removed, and the cells were washed twice with PBS. The cells were then trypsinized and collected, centrifuged, and resuspended in PBS. The intracellular green fluorescence intensity was then detected using flow cytometry, and the average fluorescence intensity of HepG2 cells was quantitatively analyzed. The test results are shown below. Figure 2 As shown.

[0042] according to Figure 2 It can be seen that, under the same conditions, A1 exhibits the strongest fluorescence intensity among the four polyphenolic binders, which may be attributed to the greater number of phenolic hydroxyl groups in the individual molecule of A1. Secondly, the average fluorescence intensity increases with increasing polyphenolic binder dosage, reaching its highest intensity at a polyphenolic binder to BSA molar ratio of 10:1. When the polyphenolic binder is excessive, the delivery efficiency decreases. This may be because, on the one hand, excessive polyphenolic binder forms a larger complex with the target protein, hindering cellular uptake; on the other hand, excessive polyphenolic binder binds to borate peptides, reducing the number of unbound borate peptides within the complex system. The binding of excessive polyphenolic binder to phenylboronic acid can also inhibit endocytosis of the complex. The dosage of the polyphenolic binder in this invention, within a certain range, can achieve optimal intracellular protein delivery efficiency. Subsequently, this invention uses A1 for testing, maintaining a molar ratio of A1 to the target protein of 10:1.

[0043] Example 4 To further investigate the driving force behind the formation of a complex between polyphenol molecular binders and proteins, the endothermic and exothermic behavior of the interaction between polyphenol molecular binder A1 and bovine serum albumin (BSA) was tested using isothermal titration calorimetry (ITC). The specific steps were as follows: Measurements were taken in a 200 μL dish. BSA and A1 were dissolved separately in 10 mM PBS buffer (pH 7.0). The 10 mM A1 solution was added dropwise to the 200 μM BSA solution in 2 μL increments, with a 2-minute interval between each addition to allow the solution to reach equilibrium. The A1 solution was then added dropwise to PBS buffer without BSA to measure the ITC data, serving as a control to deduct the heat of dilution of A1 in solution. The obtained ITC data represents the total heat generated per second during the detection period. The test results are as follows: Figure 3 As shown.

[0044] according to Figure 3 As can be seen from the isothermal titration calorimetric data, the interaction between A1 and bovine serum albumin is an exothermic reaction, which indicates that hydrogen bonds play a dominant role in the formation of the complex.

[0045] Example 5 Circular dichroism (CD) chromatography was used to detect changes in the secondary structure of the target protein during the formation of a complex with a polyphenol binder. The instrument used was a MOS-450 (Bio-Logic) circular dichroism spectroscopy unit. The specific procedure was as follows: the polyphenol binder and bovine serum albumin (BSA) were mixed at a molar ratio of 10:1, thoroughly mixed, and incubated in an aqueous solution at room temperature for 1 hour to obtain a 50 μL stock solution. This stock solution was then diluted to 1 mL with deionized water before detection. The final concentration of BSA was 3 μM, and the final concentration of the polyphenol binder was 30 μM. Simultaneously, the CD spectrum of a protein solution without the polyphenol binder was measured as a control. The test results are as follows: Figure 4 As shown.

[0046] according to Figure 4 It can be seen that, under the experimental concentration and molar ratio conditions, the addition of A1 hardly altered the secondary structure of bovine serum albumin. This is crucial for the protein to maintain its spatial structure and perform its subsequent biological functions after release from the complex. Therefore, under the experimental conditions, the addition of natural polyphenol molecular binders did not disrupt the protein's spatial structure and could maintain its biological activity.

[0047] Example 6 The efficiency of polyphenol molecular binder A1 in delivering bovine serum albumin into HepG2 cells using functional peptides modified with phenylboronic acid—cationic peptides P1-P6 (transmembrane sequences shown in SEQ ID NO. 1-6, respectively)—was tested. The specific steps were as follows: HepG2 cells were seeded in 24-well plates and incubated overnight. When the cell density reached over 80%, the protein delivery test was initiated. A1 was mixed with BSA-FITC at room temperature for 1 h (BSA-FITC concentration: 0.17 μM), and then incubated with P1-P6 at room temperature for 30 min, with a molar ratio of P1-P6 to BSA-FITC of 30:1. Remove the cell culture medium, wash twice with PBS, add the cells to a culture medium solution containing protein complexes, and incubate at 37°C for 4 hours. After treating the cells with the protein complex-containing culture medium solution, remove the culture medium, wash the cells twice with PBS, trypsinize and collect the cells, centrifuge, resuspend in PBS, and detect the intensity of green fluorescence in the cells using flow cytometry. Observe the fluorescence intensity and distribution in the cells using an inverted fluorescence microscope, and quantitatively analyze the mean fluorescence intensity of HepG2 cells. The test results are as follows: Figure 5 As shown.

[0048] according to Figure 5 It can be seen that the delivery efficiency of BSA-FITC by P1~P6 was significantly improved under the mediation of polyphenol molecular binder A1. Inverted fluorescence images of the four complexes P1 / A1 / BSA-FITC, P2 / A1 / BSA-FITC, P3 / A1 / BSA-FITC, and P6 / A1 / BSA-FITC showed strong green fluorescence in most cells, with uniform distribution within the cells. This indicates that the complex formed by the borate peptide / polyphenol molecular binder / protein crossed the cell membrane surface, entered the cell interior, and achieved endosome escape, distributing uniformly within the cell. Subsequent tests used the functional peptide P1.

[0049] Example 7 The toxicity of the complex formed by the polyphenol binder A1, the functional peptide P1 modified with phenylboronic acid groups, and the target protein in HepG2 cells was detected. The specific steps were as follows: HepG2 cells were pre-seeded in 96-well plates and cultured overnight. The culture medium was removed, and 100 μL of serum-free medium containing the complex A1 / P1 / BSA was added, and the cells were incubated for 4 hours. The culture medium was then removed, replaced with 10% serum-containing medium, and cultured for another 20 hours. Cell viability was assessed according to the standard MTT assay. Five replicates were performed for each test group. The test results are shown below. Figure 6 As shown.

[0050] according to Figure 6As can be seen, under the protein delivery test concentration conditions, the cell survival rate of HepG2 cells treated with the MTT assay material A1 / P1 / BSA complex was higher than 90%, indicating that the supramolecular assembly prepared in this invention has low cytotoxicity and does not produce significant toxicity to cells during protein delivery, thus exhibiting good biocompatibility.

[0051] Example 8 The potential and particle size of the complex formed by the polyphenol binder A1, the functional peptide P1 modified with phenylboronic acid groups, and the target protein BSA were characterized. The specific steps were as follows: A1 was mixed with the protein and incubated at room temperature for 1 h. Then, P1 was added and incubation continued at room temperature for 30 min. After dilution with 1 mL of deionized water, the size distribution and surface potential of the nanoparticles in the solution were detected using a nanoforce spectrophotometer. The final concentration of BSA was 3 μM, the molar ratio of polyphenol binder to protein was 10:1, and the molar ratio of functional peptide to protein was 30:1. The test results are as follows: Figures 7-8 As shown.

[0052] according to Figures 7-8 It can be seen that the Zeta potentials of BSA / A1 and BSA / P1 are -17.86 mV and 5.21 mV, respectively, while the Zeta potential of the BSA / A1 / P1 complex is 15.66 mV. This indicates that the catechol-boronic acid ester complex between catechol-BSA and P1 changes the Zeta potential of the polyphenol molecular binder-BSA nanoparticles from negative to positive. Simultaneously, the addition of P1 increases the particle size of the complex, ranging from 100 to 200 nm.

[0053] Example 9 The cytotoxicity of supramolecular assemblies delivering toxic proteins ribonuclease A and trypsin into HepG2 cells was assessed. The specific steps were as follows: HepG2 cells were seeded in 96-well cell culture plates overnight. Different concentrations of ribonuclease (final concentrations of 2.5 μM, 5 μM, 10 μM, 15 μM, and 20 μM) or trypsin (final concentration of 20 μM) were thoroughly mixed with P1 and polyphenol binder A1 (the molar ratio of polyphenol binder to protein was 10:1, and the molar ratio of functional peptide P1 to protein was 30:1). Serum-free medium was added, and the mixture was vortexed. After incubation at room temperature for 30 minutes, serum-free medium was added to the mixture. The cell culture medium was removed, and the cells were washed once with PBS. A culture medium solution containing the protein complex was added, and the cells were incubated at 37°C for 6 hours. The culture medium was removed, and medium containing 10% serum was added. The cells were cultured for another 42 hours. Subsequently, the cell viability after delivery of ribonuclease or trypsin was detected using the standard MTT assay. Each test was repeated with 5 samples to reduce error. Test results are as follows: Figures 9-10 As shown.

[0054] according to Figures 9-10 It can be seen that the cell viability of HepG2 cells after delivery of different concentrations of ribonuclease decreased significantly with increasing ribonuclease concentration, reaching approximately 15% at a concentration of 20 μM. In contrast, under the same experimental conditions, cells treated with ribonucleic acid alone showed no significant cytotoxicity, with a cell viability close to 100%. These results indicate that the supramolecular assembly prepared in this invention can efficiently deliver toxic proteins into cells, exhibiting significant cytotoxicity against cancer cells.

[0055] Example 10 The efficacy of supramolecular assemblies in delivering horseradish peroxidase into HepG2 cells was investigated. The specific steps were as follows: HepG2 cells were seeded into 24-well cell culture plates. When the HepG2 cell density reached 80% or higher, the protein delivery assay began. HRP was thoroughly mixed with P1 and A1 (final HRP concentration 3.06 μM, A1:HRP molar ratio 10:1, P1:HRP molar ratio 30:1), and 100 μL of serum-free medium was added. The mixture was vortexed and incubated at room temperature for 30 minutes. Serum-free medium was then added to the mixture. The cell culture medium was removed, and the cells were washed twice with PBS. A culture medium solution containing the protein complex was added, and the cells were incubated at 37°C for 4 hours. The culture medium was removed, and the cells were washed three times with PBS. A PBS solution containing the enzyme substrate Amplex Red (50 μM) and hydrogen peroxide (500 μM) was added. After incubating at room temperature for 30 minutes, the substrate solution was removed, and the cells were washed three more times with PBS. The fluorescence intensity and distribution within the cells were observed using an inverted fluorescence microscope. The test results are as follows: Figure 11 As shown.

[0056] according to Figure 11 The image shows the observation of intracellular red fluorescence in HepG2 cells delivered with HRP using the colorless substrate Amplex Red. Cells delivered with HRP by the supramolecular assembly prepared in this invention exhibit obvious red fluorescence, indicating that the supramolecular assembly prepared in this invention can efficiently deliver HRP into cells while maintaining enzyme activity.

[0057] Example 11 The efficacy of supramolecular assemblies in delivering catalase into HepG2 cells was assessed. The specific steps were as follows: HepG2 cells were cultured in 24-well plates and incubated with the complex (final concentration of CAT-RB: 3.06 μM, molar ratio of A1 to CAT-RB: 10:1, molar ratio of P1 to CAT-RB: 30:1) for 4 hours. The cells were then washed three times with PBS and imaged using an inverted fluorescence microscope. For enzyme activity assays, HeLa cells were cultured in 48-well plates and treated with the same concentration of the complex for 4 hours. CAT activity in the treated cells was measured using a catalase activity assay kit. The test results are as follows: Figure 12 As shown.

[0058] according to Figure 12 It can be seen that the supramolecular assembly effectively delivers catalase into HepG2 cells. Catalase (CAT) is a major enzymatic antioxidant that protects cells from reactive oxygen species (ROS). The catalase labeled with red dye can be effectively delivered into the cells and can effectively degrade H2O2. Therefore, the supramolecular assembly prepared in this invention can efficiently deliver catalase into cells and maintain the enzyme's activity.

[0059] Example 12 The efficacy of supramolecular assemblies in delivering superoxide dismutase (SOD) to HepG2 cells was assessed using the following steps: Cells were cultured in 24-well plates and incubated with the complex (final SOD-FITC concentration 3.06 μM, A1 to SOD-FITC molar ratio 10:1, P1 to SOD-FITC molar ratio 30:1) for 4 hours. Cells were then washed with PBS and analyzed by flow cytometry, followed by treatment with 800 μL of the same concentration of the complex for 4 hours. Cells were then washed three times with PBS and imaged using an inverted fluorescence microscope. Free SOD-FITC was used as a negative control, and P1 / SOD-FITC was used as a positive control, according to the manufacturer's protocol.

[0060] according to Figure 13 It can be seen that superoxide dismutase is also a major enzymatic antioxidant that protects cells from reactive oxygen species. FITC-labeled superoxide dismutase can be effectively delivered into cells and also effectively downregulates ROS in cells caused by ROS2. These results clearly demonstrate that the supramolecular assembly of the present invention can deliver bioactive proteins into living cells and maintain robustness in terms of protein activity.

[0061] As can be seen from the above embodiments, the supramolecular assembly provided by the present invention solves the problem that cationic polypeptide carriers and proteins cannot fully bind to form a stable complex.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A supramolecular assembly, characterized in that, The following components were co-assembled to obtain the product: Functional peptides modified with phenylboronic acid groups, polyphenol molecular binders, and target proteins; The functional polypeptide modified with phenylboronic acid groups contains a membrane-penetrating sequence, which is an amino acid sequence rich in arginine or histidine, as shown in any one of SEQ ID NO. 1 to 6: SEQ ID NO. 1: B(OH)2-FFGPLGLA-G-RRRR-NH2; SEQ ID NO. 2: B(OH)2-RRRR-GPLGLAG-FF-NH2; SEQ ID NO. 3: B(OH)2-FFGPLGLA-G-KKKK-NH2; SEQ ID NO. 4: B(OH)2-KKKK-GPLGLAG-FF-NH2; SEQ ID NO. 5: B(OH)2-FFGPLGLA-G-RKRK-NH2; SEQ ID NO. 6: B(OH)2-RKRK-GPLGLAG-FF-NH2.

2. The supramolecular assembly according to claim 1, characterized in that, The polyphenol molecular binder includes one or more of tannic acid, baicalin, curcumin, and catechin.

3. The supramolecular assembly according to claim 1, characterized in that, The molar ratio of the functional polypeptide modified with phenylboronic acid groups to the target protein is 28~32:

1.

4. The supramolecular assembly according to claim 1, characterized in that, The target protein includes one or more of bovine serum albumin, horseradish peroxidase, ribonuclease A, trypsin, and superoxide dismutase.

5. The supramolecular assembly according to claim 1, characterized in that, The molar ratio of the polyphenol molecular binder to the target protein is no higher than 10:

1.

6. The supramolecular assembly according to claim 1, characterized in that, The particle size of the supramolecular assembly is 50~200 nm.

7. A method for preparing the supramolecular assembly according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) The target protein and polyphenol molecular binder were incubated in a buffer solution to obtain a polyphenol protein complex; (2) The polyphenol protein complex and the functional polypeptide modified with phenylboronic acid groups are mixed and assembled to obtain the supramolecular assembly.

8. The preparation method according to claim 7, characterized in that, The pH of the buffer solution is 7.0 to 8.5, and the incubation time is 5 to 30 minutes.

9. The preparation method according to claim 7, characterized in that, The assembly temperature is 25~37℃, and the assembly time is 10~60 minutes.

10. The use of the supramolecular assembly according to any one of claims 1 to 6 or the supramolecular assembly obtained by the preparation method according to any one of claims 7 to 9 in intracellular protein delivery.

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