CD44-mediated self-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses CD44-mediated self-assembled nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biology. The CD44-mediated self-assembled nanoparticles are formed by self-assembling polylysine with a phenylboronic acid side chain and dopamine-modified hyaluronic acid through electrostatic interaction, and the side-chain phenylboronic acid of the polylysine is connected with the side-chain dopamine on the hyaluronic acid through a covalent bond. The surface of the nanoparticle is connected with a target protein binding unit through a linker, and the target protein binding unit is used for binding a target protein. The synthesis process is simple, the components are safe, the prepared CD44-mediated self-assembled nano material can efficiently degrade tumor cell extracellular proteins such as PD-L1 and HER2, the specificity is high, the degradation rate is high, and the CD44-mediated self-assembled nano material has a very good application prospect in the field of treatment of diseases such as tumors.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to CD44-mediated self-assembly nanoparticles and a preparation method and application thereof. Background Art

[0002] The main method currently used for targeted cancer therapy is to inhibit the activity of proteins that drive tumor growth through small molecule inhibitors or monoclonal antibodies. Small molecule inhibitors mainly work by occupying the binding pockets or active sites of specific proteins in the form of non-covalent bonds. However, there are still many proteins that lack hydrophobic pockets and active centers, making it difficult to develop small molecule inhibitors for the corresponding targets. According to literature reports, more than 80% of pathogenic proteins associated with human diseases cannot be targeted. Even with small molecule inhibitors with targeted functions, tumors can develop drug resistance by overexpressing target proteins or escaping the inhibitory effects of drugs through new mutations in target proteins.

[0003] As a development strategy to break through drug targets, a series of targeted protein degradation technologies (Targeted Protein Degradation, TPD) have gradually emerged to eliminate undruggable proteins. This technology can specifically identify and utilize the cell's own protein degradation pathway to degrade pathogenic target proteins. The most representative protein degradation technologies are Proteolysis Targeting Chimeras (PROTAC) and molecular glue technology. This type of technology greatly expands the range of druggable protein targets by ubiquitinating the target protein and degrading it by the proteasome. Secondly, unlike traditional inhibitors that can only inhibit some functions of the target protein, protein degradation technology can eliminate all functions of pathogenic proteins by degrading the target protein, while also minimizing potential drug resistance. However, this type of technology can only degrade intracellular proteins, and the proteasome has little degradation effect on certain macromolecular proteins or aggregates, extracellular proteins, and organelles. Therefore, this limits the application of PROTAC, and there is an urgent need for targeted protein degradation technology that can degrade a wider range of proteins.

[0004] New targeted protein degradation technologies are constantly developing, such as lysosome-targeting chimaeras (LYTACs), autophagosome-tethering compounds (ATTEC), autophagy-targeting chimeras (AUTOTAC), degradation antibody conjugates (DAC), etc. Lysosomes play an important role in regulating the homeostasis of intracellular and extracellular proteins. They decompose damaged aging proteins and organelles through endocytosis, phagocytosis, autophagy, etc., thereby removing harmful substances. Lysosome-induced protein degradation drugs can directly regulate protein levels in the body to achieve the purpose of treating diseases. Summary of the invention

[0005] In view of the problems of low specific recognition efficiency, slow degradation rate, difficult design, etc. existing in most current protein degradation technologies, the purpose of the present invention is to provide a CD44-mediated self-assembly nanomaterial and its preparation method and application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a CD44-mediated self-assembled nanoparticle, wherein the self-assembled nanoparticle is formed by self-assembly of polylysine with phenylboronic acid side chains and dopamine-modified hyaluronic acid through electrostatic interaction, the side chain phenylboronic acid of polylysine and the side chain dopamine on the hyaluronic acid are covalently bonded, and a target protein binding unit is connected to the surface of the nanoparticle through a linker for binding to the target protein.

[0007] Based on the above technical solution, further, the target protein binding unit is selected from small molecule ligands, polypeptides, antibodies, antibody Fab fragments, nucleic acid aptamers, glycopeptide polymers, and oligonucleotides that can specifically bind to the target protein; the antibodies include α-PD-L1 and IgG.

[0008] Based on the above technical solution, further, the mass ratio of the polylysine with phenylboronic acid side chain to the dopamine-modified hyaluronic acid is 1:0.01~1:100.

[0009] Based on the above technical solution, further, the linker includes a PEG chain, an alkyl chain and an Fc-III-4C-DBCO polypeptide, and the Fc-III-4C-DBCO polypeptide is obtained by reacting the Fc-III-4C polypeptide with DBCO-C6-NHS.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned CD44-mediated self-assembled nanoparticles, comprising the following steps: (1) activating the carboxyl group of 3-carboxyphenylboronic acid and subjecting it to an amidation reaction with polylysine, and removing the unreacted 3-carboxyphenylboronic acid by dialysis to obtain polylysine with a phenylboronic acid side chain; (2) activating the carboxyl group of hyaluronic acid and reacting it with dopamine for amidation, and removing the unreacted dopamine by dialysis to obtain dopamine-modified hyaluronic acid; (3) dissolving the Fc-III-4C polypeptide in an organic solvent, adding DBCO-C6-NHS, and stirring to perform an amidation reaction to obtain the Fc-III-4C-DBCO polypeptide; (4) adding dropwise the aqueous solution containing the dopamine-modified hyaluronic acid prepared in step (2) to the aqueous solution containing the polylysine with phenylboronic acid side chains prepared in step (1) under stirring to form nanoparticles through self-assembly, wherein the side chain phenylboronic acid of the polylysine is cross-linked with the side chain dopamine on the hyaluronic acid through covalent bonds; (5) The Fc-III-4C-DBCO polypeptide of step (3) is linked to the surface of the nanoparticles obtained in step (4) through a click chemistry reaction, the free Fc-III-4C-DBCO polypeptide is removed, and the target protein binding unit is added. The reaction is carried out at 1 to 5°C for 1 to 10 hours, and the free target protein binding unit is removed to obtain CD44-mediated self-assembled nanoparticles. Based on the above technical solution, further, the molar ratio of 3-carboxyphenylboronic acid to polylysine monomer in step (1) is 10:1~0.1:1.

[0011] Based on the above technical solution, further, the molecular weight range of the polylysine described in step (1) is 10000Da-20000 Da.

[0012] Based on the above technical solution, further, the 3-carboxyphenylboronic acid described in step (1) is activated in DMSO dissolved with EDC and NHS.

[0013] Based on the above technical solution, further, the preparation process of polylysine in step (1) is: N ε -Benzyloxycarbonyl-L-lysine reacts with triphosgene to obtain N ε -Benzyloxycarbonyl-L-lysine cyclic anhydride NCA is dissolved in N,N-dimethylformamide, and n-butylamine is added. The reaction is carried out at room temperature for 20 to 80 hours to obtain polylysine.

[0014] Based on the above technical solution, further, the step (1) described N ε The molar ratio of -benzyloxycarbonyl-L-lysine to triphosgene is 1:1~1:5; the molar ratio of n-butylamine to Nε The molar ratio of -benzyloxycarbonyl-L-lysine cyclic anhydride NCA is 1:10~1:200.

[0015] Based on the above technical solution, further, the molecular weight range of the hyaluronic acid described in step (2) is 80kDa-1500 kDa.

[0016] Based on the above technical solution, further, the molecular weight of the hyaluronic acid is 400 kDa.

[0017] Based on the above technical solution, further, the molar ratio of hyaluronic acid to dopamine in step (2) is 1:100 to 1:1000.

[0018] Based on the above technical solution, further, the hyaluronic acid described in step (2) is activated in a PBS buffer solution containing EDC and BtOH.

[0019] Based on the above technical solution, further, the molar ratio of the Fc-III-4C polypeptide to DBCO-C6-NHS in step (3) is 1:1-1:10.

[0020] Based on the above technical solution, further, the Fc-III-4C polypeptide structure described in step (3) is NH2-PEG2-CDCAWHLGELVWCTC-OH.

[0021] Based on the above technical solution, further, the organic solvent described in step (3) includes DMSO and DMF.

[0022] Based on the above technical solution, further, the mass ratio of the polylysine with phenylboronic acid side chain to the dopamine-modified hyaluronic acid in step (4) is 1:0.01-1:100.

[0023] Based on the above technical solution, further, the mass ratio of the Fc-III-4C-DBCO polypeptide to the nanoparticles in step (5) is 1:2-1:10.

[0024] Based on the above technical solution, further, the click chemistry reaction described in step (5) is completed by adding 1H-imidazole-1-sulfonyl azide hydrochloride, K2CO3, and CuSO4.

[0025] In a third aspect, the present invention provides the use of the above-mentioned CD44-mediated self-assembled nanoparticles in the preparation of drugs for treating tumors.

[0026] Based on the above technical scheme, further, the tumor includes melanoma, thyroid cancer, pancreatic cancer, skin cancer, lung cancer, gallbladder cancer, rhinitis cancer, gastric cancer, esophageal cancer, breast cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, rectal cancer and colorectal cancer.

[0027] Based on the above technical solution, further, the hyaluronic acid part in the CD44-mediated self-assembled nanoparticles can bind to the CD44 site on the cell surface to achieve the targeting function; the antibody part can bind to the target protein on the cell surface, thereby degrading the target protein.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The synthesis process of the invention is simple and the ingredients are safe. The prepared CD44-mediated self-assembled nanomaterial can efficiently degrade the extracellular protein PD-L1 of tumor cells, has strong specificity and a very fast degradation rate, and has very good application prospects in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0030] Figure 1 This is a diagram showing the reaction process of polylysine with phenylboronic acid side chains forming a covalent bond with hyaluronic acid-dopamine.

[0031] Figure 2 This is the GPC chart of poly-lysine (PLL).

[0032] Figure 3 It is a structural diagram of Fc-III-4C-DBCO polypeptide.

[0033] Figure 4 The graph shows the change in particle size after mixing PLL / HA in different ratios.

[0034] Figure 5 This is the change of ZETA potential after mixing PLL / HA in different ratios.

[0035] Figure 6 Zeta potential of PLL, PLL with phenylboronic acid side chain, HA, and HA-dopamine materials.

[0036] Figure 7 The SDS-PAGE gel images of PLL-HA-IgG nanoparticles and the filtrate of each ultrafiltration.

[0037] Figure 8 The graph shows the particle size changes of PLL-HA, PLL-HA coupled with Fc-III-4C-DBCO polypeptide, and PLL-HA-IgG nanoparticles after coupling with IgG.

[0038] Fig. 9 Transmission electron microscopy (TEM) images of the nanoparticles before coupling with IgG (a) and PLL-HA-IgG nanoparticles after coupling with IgG (b) (scale bar is 20 nm).

[0039] Fig.10 This is a graph showing the toxicity of PLL-HA nanoparticles to B16-F10 melanoma cells.

[0040] Fig.11 This is a graph showing the toxicity results of PLL-HA-α-PD-L1 nanoparticles on B16-F10 melanoma cells.

[0041] Fig.12 Co-localization of nanoparticles in subcellular organelles (lysosomes) in B16-F10 cell lines at different PLL / HA ratios (scale bar is 20 μm).

[0042] Fig.13 The PD-L1 protein content in B16-F10 cells was compared after the nanoparticles PLL-HA-α-PD-L1 and the blank control group / single polymer carrier PLL-HA / antibody α-PD-L1 / PLL-α-HA-IgG were incubated with B16-F10 for 24 hours.

[0043] Fig.14 The degradation ability of different concentrations of nanoparticles PLL-HA-α-PDL1 on PD-L1 protein in B16-F10 cells (whole protein analysis).

[0044] Fig.15 This graph shows the effect of incubation of nanoparticles PLL-HA-α-PD-L1 with B16-F10 cells for different time periods on the PD-L1 protein content in B16-F10 cells.

[0045] Fig.16 The degradation ability of nanoparticles PLL-HA-α-PD-L1 on the PD-L1 protein on the surface of B16-F10 cell membrane (membrane protein).

[0046] Fig.17 To analyze the degradation pathway of PD-L1 protein on the cell membrane surface by nanoparticles PLL-HA-α-PD-L1. DETAILED DESCRIPTION

[0047] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0048] Example 1 The preparation of CD44-mediated targeted protein degradation self-assembled nanomaterial PLL-HA-α-PD-L1 includes the following steps: Step 1: N ε -Benzyloxycarbonyl-L-lysine was dissolved in ethyl acetate (concentration 119 mM), triphosgene was added to the solution, N ε The molar ratio of -benzyloxycarbonyl-L-lysine to triphosgene was 1:3, and the reaction was carried out in a 90°C oil bath for 6 hours. After the reaction, the device was quickly placed in an ice bath to cool the reaction solution; the cooled solution was first washed twice with saturated brine, and then washed twice with 0.5% (w / v) NaHCO3 aqueous solution; after washing, anhydrous magnesium sulfate was added to the organic layer to remove water for 30 minutes, and after the removal of water, the magnesium sulfate was filtered out, and the filtrate was collected and concentrated by vacuum rotary evaporation, and n-hexane was added for precipitation. A large amount of white floccules were precipitated, which was placed in a -20°C refrigerator overnight to allow it to fully crystallize and precipitate; the solvent was removed by suction the next day to obtain N ε The crude product of -benzyloxycarbonyl-L-lysine cyclic anhydride NCA was dissolved in ethyl acetate, filtered to remove insoluble impurities, and the obtained filtrate was concentrated by rotary evaporation under reduced pressure, and n-hexane was added for precipitation. It was placed in a -20°C refrigerator overnight to allow it to fully crystallize, and this was repeated 5 times; after the last filtration to remove the solvent, a white solid was obtained, which was placed in a vacuum drying oven for drying to obtain a pure NCA product; Step 2: Dissolve the NCA prepared in the first step in N, N-dimethylformamide (concentration of 0.326 M), freeze and thaw three times under nitrogen protection, then add the prepared N, N-dimethylformamide solution of n-butylamine (the molar ratio of n-butylamine to NCA is 1:100) to the solution, react for 36 hours at room temperature, terminate the reaction, precipitate with ice ether, dry, and use M W The 3000 Da dialysis bag was first dialyzed against a 0.5 g / mL NaHCO3 solution to remove salt, then dialyzed against water for 3 days, and lyophilized to obtain poly-lysine (average molecular weight 12800 Da); Step 3: Dissolve 3-carboxyphenylboronic acid in DMSO (concentration of 39 mM), add DMSO solution of EDC and NHS (final concentration of 32.5 mM), stir for 1 hour to activate the carboxyl group, then dissolve the polylysine prepared in the second step in water (concentration of 391 mM), add 3-carboxyphenylboronic acid solution to the polylysine aqueous solution, the molar ratio of 3-carboxyphenylboronic acid to polylysine monomer is 2:1, stir at room temperature for 24 hours, and purify the mixture with M W The product was dialyzed for 3 days using a 1000 Da dialysis bag and freeze-dried to obtain a poly-lysine solid with phenylboronic acid side chains; Step 4: Dissolve hyaluronic acid (average molecular weight 40w Da) in PBS (0.01 M, pH 5.0) (concentration 0.25 mM), add EDC / BtOH DMSO solution (final concentration 66.3 mM), activate for 1 hour, add dopamine to the solution, the molar ratio of dopamine to hyaluronic acid is 1:375, react at room temperature for 12 hours, and load the mixture into M W The 3000 Da dialysis bag was dialyzed in PBS at pH 5.0 for 1 day and in deionized water for 2 days, and lyophilized to obtain hyaluronic acid-dopamine (HA-DA); Step 5: Dissolve the Fc-III-4C polypeptide (NH2-PEG2-CDCAWHLGELVWCTC-OH) in DMF (concentration: 1 mM), add DBCO-C6-NHS (EXTENSION, concentration: 3 mM), stir and react overnight at 30°C, and purify by preparative high performance liquid chromatography (HPLC) to obtain Fc-III-4C-DBCO; Step 6: dissolving the polylysine with phenylboronic acid side chains obtained in the third step in deionized water, and dissolving the hyaluronic acid-dopamine obtained in the fourth step in deionized water to prepare an aqueous solution; slowly adding the hyaluronic acid aqueous solution dropwise into the stirred polylysine aqueous solution, and the mass ratios of the polylysine with phenylboronic acid side chains and the hyaluronic acid-dopamine are set to 1:0.08, 1:0.16, 1:0.24, 1:0.32, 1:0.40, 1:0.48, 1:0.56, and 1:0.64, respectively, and the hyaluronic acid and the polylysine form nanoparticles PLL-HA through electrostatic interaction; Step 7: 1H-imidazole-1-sulfonyl azide hydrochloride (12.6 μM), K2CO3 (26.5 μM), and CuSO4·5H2O (0.126 μM) were added to the nanoparticles formed in the step 6 with polylysine with phenylboronic acid side chains and hyaluronic acid-dopamine in a mass ratio of 1:0.24, and the reaction was carried out for 4 h. W A 100 kDa ultrafiltration tube was used to remove free 1H-imidazole-1-sulfonyl azide hydrochloride and K2CO3 and CuSO4·5H2O; Step 8: Add Fc-III-4C-DBCO obtained in step 5 to the product obtained in step 7, the mass ratio of Fc-III-4C-DBCO to the product is 1:5.81, react overnight at 4°C, and use M W A 100 kDa ultrafiltration tube was used to remove free Fc-III-4C-DBCO peptide; Step 9: Add PD-L1 antibody α-PD-L1 (Leinco) to the product obtained in step 8, the mass ratio of PD-L1 antibody to product is 1:4, react at 4°C for 4h (Fc-III-4C polypeptide is linked to α-PD-L1 by affinity), and use M W The free PD-L1 antibody was removed by a 100 kDa ultrafiltration tube, and the obtained nanoparticles were PLL-HA-α-PD-L1 based on CD44-mediated targeted protein degradation.

[0049] Example 2 Preparation of CD44-mediated targeted protein degradation self-assembled nanomaterials PLL-HA-IgG The preparation method was the same as that of PLL-HA-α-PD-L1, except that human IgG (Acmec) was added after step 8, and the reaction was carried out at 4°C for 4 h. W The free IgG was removed by a 100 kDa ultrafiltration tube, and the obtained bifunctional nanoparticles were PLL-HA-IgG.

[0050] Example 3 SDS-PAGE characterization of nanoparticle PLL-HA-IgG The nanoparticles prepared in Example 2 were ultrafiltered, and 50 μl of PBS was added each time at 4°C using M W Ultrafiltration was performed 10 times with a 100 kD filter membrane, and the filtrate was collected each time. 5× protein loading buffer was added to the filtrate, mixed well, and then placed in a metal bath at 96°C for 6 min. After cooling, the sample was added to the SDS-PAGE gel wells, and the concentrated gel was run at 80 V, and the remaining gel was run at 120 V. Then, the sample was stained with Coomassie Brilliant Blue for 1 hour, and then decolorized in water overnight.

[0051] The results are as follows Figure 7 As shown, the first and tenth lanes are markers, the second lane is PLL-HA-IgG, the third to eighth lanes are the 1st, 2nd, 3rd, 6th, 8th, and 10th ultrafiltration filtrates, and the ninth lane is IgG, with a content of 20.32 μg, which is consistent with the content of PLL-HA-IgG. The results show that free IgG exists in the 1st to 8th filtrates, but no free IgG is present in the 10th filtrate.

[0052] Example 4 CCK-8 toxicity experiment of PLL-HA nanoparticles and PLL-HA-α-PD-L1 nanoparticles on B16-F10 melanoma cells 5k / 100 μL / well B16-F10 cells were plated in a 96-well plate. After overnight incubation, the culture medium was removed. Three groups of PBS / PLL-HA / PLL-HA-α-PD-L1 were set up. Sample solutions of PLL-HA / PLL-HA-α-PD-L1 with different concentration gradients were prepared. The concentration of PLL-HA was set to 0.0625x10 -1 mg / mL, 0.125x10 -1 mg / mL, 0.25x10 -1 mg / mL, 0.5x10 -1 mg / mL, 1x10 -1 mg / mL, 2x10 -1 mg / mL, 4x10 -1 mg / mL, three replicates for each concentration, added to a 96-well plate. The concentrations of PLL-HA-α-PD-L1 were set to 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 μg / mL, and four replicates for each concentration were added to a 96-well plate. Incubate at 37°C in an incubator for 24 hours. After incubation, discard the culture medium, prepare a 10% cck8 solution in the culture medium, add 100 μL per well to the three groups of PBS / PLL-HA / PLL-HA-α-PD-L1, and set up a cell-free 10% cck8 solution group (blank control) and incubate for 2 hours. Measure the absorbance at 450 nm with an enzyme reader. Calculate the cell survival rate, cell survival rate = [(experimental well-blank well) / (PBS well-blank well)]×100%.

[0053] Example 5 15w / well B16-F10 cells were plated in a glass-bottomed confocal culture dish. After overnight incubation, the culture medium was aspirated, washed three times with PBS, and four groups of PLL-HA were added at four ratios of 1:0.24 / 1:0.32 / 1:0.40 / 1:0.48, respectively; the concentrations of PLL were all 0.1 mg / mL, and the concentrations of HA were 0.024 / 0.032 / 0.040 / 0.048 mg / mL, respectively. After the four groups of samples were incubated with cells for 3h, the culture medium was discarded and washed three times with PBS. Lysosomal dye Lyso-Tracker Green (ThermoFisher) was added, incubated at 37℃ for 30 min, the culture medium was discarded, and washed three times with PBS. Nuclear dye Hoechst (ThermoFisher) was added, incubated at 37℃ for 15 min, the culture medium was discarded, washed three times with PBS, and PBS was added. The images were taken using a confocal microscope, with Lyso-Tracker Green excitation wavelength at 504 nm and emission wavelength at 511 nm. Hoechst excitation wavelength at 360 nm and emission wavelength at 460 nm.

[0054] Example 6 Degradation of cellular PD-L1 protein by CD44-mediated targeted protein degradation self-assembled nanomaterial PLL-HA-α-PD-L1 (whole protein analysis) In this example, mouse melanoma cells (B16-F10) were selected for the experiment, and Western Blot was used for detection.

[0055] 1. First, 12 w / well B16-F10 cells were plated in a 12-well plate. After overnight incubation, the cells were washed three times with PBS. Then, PBS (blank control) / PLL-HA / α-PD-L1 / PLL-HA-IgG / PLL-HA-α-PD-L1 were added to the plates, with a total of 5 groups. The concentration of α-PD-L1 was 6.7 nM, the concentration of PLL-HA was 4.1 ug / mL, and the concentrations of α-PD-L1 and PLL-HA in PLL-HA-α-PD-L1 were the same as those in the α-PD-L1 group and the PLL-HA group (the concentration of α-PD-L1 was 6.7 nM, and the concentration of PLL-HA was 4.1 ug / mL); the concentration of IgG in PLL-HA-IgG was 6.7 nM, and the concentration of PLL-HA was 4.1 ug / mL). After the 5 groups of samples were incubated with the cells for 24 h, the culture medium was discarded and the cells were washed three times with PBS. 120 ul of RIPA (containing 1% PMSF) was added to each of the cells and the cells were lysed on ice for 20 min. 3 Centrifuge at 4 °C for 15 min and collect the supernatant.

[0056] The supernatant of each group was measured by BCA protein quantification kit, and the protein concentration was adjusted to a consistent level using RIPA (containing 1% PMSF). 5× protein loading buffer was added, mixed evenly, and then placed in a metal bath at 96°C for 6 min.

[0057] After the protein sample is cooled, it is loaded into the SDS-PAGE gel well and run at 80V for 30 min, and then run at 120V for the entire process. The protein is transferred to the PVDF membrane using the sandwich method, with the conditions of 200 mA and 75 min. After the transfer, the PVDF membrane is blocked with 5% BSA for 1h to prevent subsequent nonspecific adsorption; the primary antibody uses PD-L1 antibody (Abcam) diluted 1:1000; the internal reference primary antibody uses GAPDH antibody (Affinity) diluted 1:3000. The primary antibody is incubated at 4°C overnight, and the secondary antibody uses HRP-labeled goat anti-rabbit antibody (Biyuntian) diluted 1:1000. The secondary antibody is incubated at room temperature for 1h for development.

[0058] The results are as follows Fig.13As shown, compared with PBS / PLL-HA / α-PD-L1 / PLL-HA-IgG, the total PD-L1 protein in the cells of the targeted protein degradation nanomaterial PLL-HA-α-PD-L1 group was significantly reduced, and the degradation effect was significant.

[0059] 2. Degradation of cellular PD-L1 protein by targeted protein degradation nanomaterial PLL-HA-α-PD-L1 at different concentrations The specific process was as described in Method 1. PLL-HA-α-PD-L1 was incubated with cells at concentrations of 0, 0.7, 1.3, 3.3, and 6.7 nM (calculated based on α-PD-L1) for 24 h and then lysed. The subsequent experiments were the same as Method 1.

[0060] The results are as follows Fig.14 As shown in the figure, as the concentration of PLL-HA-α-PD-L1 increased from 0.7 nM to 6.7 nM, the PD-L1 total protein in the cells was significantly reduced, and the higher the concentration, the less the protein content, indicating that the degradation of PD-L1 by PLL-HA-α-PD-L1 nanoparticles was concentration-dependent.

[0061] 3. Degradation of cellular PD-L1 protein by targeted protein degradation nanomaterial PLL-HA-α-PD-L1 at different time points The specific process was as described in Method 1. PLL-HA-α-PD-L1 was incubated with cells at a concentration of 6.7 nM (calculated as α-PD-L1) for 3 / 6 / 12 / 24 / 48 h and then lysed. The subsequent experiments were the same as Method 1.

[0062] The results are as follows Fig.15 As shown, after incubation with PLL-HA-α-PD-L1, the PD-L1 protein on the cell surface decreased rapidly at 3 hours and was significantly degraded at 48 hours.

[0063] Example 7 CD44-mediated targeted protein degradation self-assembled nanomaterial PLL-HA-α-PD-L1 degradation of PD-L1 protein on the cell membrane surface (membrane protein analysis) 1. Select mouse melanoma cells (B16-F10) for flow cytometry experiments. The specific process is as follows: 12w / well B16-F10 cells were plated in a 12-well plate, and the targeted protein degradation nanomaterial PLL-HA-α-PD-L1 (final concentration of α-PD-L1 was 6.7 nM) was incubated with the cells for 3 / 6 / 12 / 24 / 48h, respectively, with 3 replicates in each group. After incubation, the cells were washed three times with PBS, 200μl of trypsin was added for digestion for 3min, and an equal amount of culture medium was added to terminate the digestion, centrifuged at 4℃, 1000rpm for 3min, the supernatant was discarded, and the cells were resuspended with PBS. The cells were washed twice with PBS in the same way.

[0064] APC anti-mouse CD274 (B7-H1, PD-L1, BioLegend) was diluted 1:800, and 100 μl of APC anti-mouse dilution was added to each well to resuspend the cells. The cells were incubated at 4°C in the dark for 30 min, washed three times with PBS, and finally resuspended with 200 μl PBS. After filtering with a cell mesh, the cells can be loaded for detection. The excitation wavelength of APC is 633 nm, and the emission wavelength is 650 nm.

[0065] The results are as follows Fig.16 As shown, after incubation with PLL-HA-α-PD-L1, the PD-L1 membrane protein on the cell surface decreased rapidly and tended to stabilize within 3 hours.

[0066] 2. Path analysis of the degradation of PD-L1 protein on the cell membrane surface by targeted protein degradation nanomaterial PLL-HA-α-PD-L1 Leuproretinol (LPT) is a lysosomal protease inhibitor that inhibits the activity of hydrolases in lysosomes.

[0067] This experiment set up 4 groups, namely PBS / LPT / LPT+PLL-HA-α-PD-L1 / PLL-HA-α-PD-L1, among which the working concentration of LPT was 0.1 mg / mL, and the final concentration of PLL-HA-α-PD-L1 (α-PD-L1 was 6.7 nM). The 4 groups of materials were incubated with B16-F10 cells for 24 hours and then lysed. The subsequent experiments were the same as method 1.

[0068] The results are as follows Fig.17 As shown, the addition of protease inhibitor leuproretinin (LPT) significantly inhibited the degradation of PD-L1, indicating that PLL-HA-PD-L1 nanoparticles degrade PD-L1 protein through the lysosomal pathway.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CD44-mediated self-assembled nanoparticle, characterized in that: The self-assembled nanoparticles are formed by self-assembly of polylysine with phenylboronic acid side chains and dopamine-modified hyaluronic acid through electrostatic interaction. The side chain phenylboronic acid of polylysine is covalently linked to the side chain dopamine on the hyaluronic acid. A target protein binding unit is connected to the surface of the nanoparticle via a linker for binding to the target protein.

2. The CD44-mediated self-assembled nanoparticles according to claim 1, characterized in that The target protein binding unit is selected from a small molecule ligand, a polypeptide, an antibody, an antibody Fab fragment, a nucleic acid aptamer, a glycopeptide polymer, and an oligonucleotide that can specifically bind to the target protein; the antibodies include α-PD-L1 and IgG; the mass ratio of the polylysine with a phenylboronic acid side chain to the dopamine-modified hyaluronic acid is 1:0.01 to 1:100; the linker includes a PEG chain, an alkyl chain, and an Fc-III-4C-DBCO polypeptide, and the Fc-III-4C-DBCO polypeptide is obtained by reacting an Fc-III-4C polypeptide with DBCO-C6-NHS.

3. The method for preparing CD44-mediated self-assembled nanoparticles according to claim 1 or 2, characterized in that: The steps include: (1) Activating the carboxyl group of 3-carboxyphenylboronic acid and reacting it with polylysine for amidation, and removing the unreacted 3-carboxyphenylboronic acid by dialysis to obtain polylysine with a phenylboronic acid side chain; (2) Activating the carboxyl group of hyaluronic acid and reacting it with dopamine for amidation reaction, and removing the unreacted dopamine by dialysis to obtain dopamine-modified hyaluronic acid; (3) Dissolving the Fc-III-4C polypeptide in an organic solvent, adding DBCO-C6-NHS, and stirring to carry out an amidation reaction to obtain the Fc-III-4C-DBCO polypeptide; (4) adding dropwise the aqueous solution containing the dopamine-modified hyaluronic acid prepared in step (2) to the aqueous solution containing the polylysine with phenylboronic acid side chains prepared in step (1) under stirring, and self-assembling to form nanoparticles, wherein the side chain phenylboronic acid of the polylysine is cross-linked with the side chain dopamine on the hyaluronic acid through covalent bonds; (5) The Fc-III-4C-DBCO polypeptide of step (3) is linked to the surface of the nanoparticles obtained in step (4) through a click chemistry reaction, the free Fc-III-4C-DBCO polypeptide is removed, and the target protein binding unit is added. The reaction is carried out at 1-5°C for 1-10 hours, and the free target protein binding unit is removed to obtain CD44-mediated self-assembled nanoparticles.

4. The preparation method according to claim 3, characterized in that The molar ratio of 3-carboxyphenylboronic acid to polylysine monomer in step (1) is 10:1 to 0.1:1; the preparation process of polylysine is as follows: N ε -Benzyloxycarbonyl-L-lysine reacts with triphosgene to obtain N ε -Benzyloxycarbonyl-L-lysine cyclic anhydride NCA is dissolved in N,N-dimethylformamide, and n-butylamine is added. The reaction is carried out at room temperature for 20-80 hours to obtain polylysine.

5. The preparation method according to claim 4, characterized in that As described in step (1) N ε The molar ratio of -benzyloxycarbonyl-L-lysine to triphosgene is 1:1~1:5; the molar ratio of n-butylamine to N ε The molar ratio of -benzyloxycarbonyl-L-lysine cyclic anhydride NCA is 1:10~1:

200.

6. The preparation method according to claim 3, characterized in that The molar ratio of hyaluronic acid to dopamine in step (2) is 1:100 to 1:1000.

7. The preparation method according to claim 3, characterized in that The molar ratio of the Fc-III-4C polypeptide to DBCO-C6-NHS in step (3) is 1:1 to 1:10; the structure of the Fc-III-4C polypeptide is NH2-PEG2-CDCAWHLGELVWCTC-OH; and the organic solvent includes DMSO and DMF.

8. The preparation method according to claim 3, characterized in that The mass ratio of the polylysine with phenylboronic acid side chains to the dopamine-modified hyaluronic acid described in step (4) is 1:0.01 to 1:

100.

9. The preparation method according to claim 3, characterized in that The mass ratio of the Fc-III-4C-DBCO polypeptide to the nanoparticles in step (5) is 1:2 to 1:10; the click chemistry reaction is completed by adding 1H-imidazole-1-sulfonyl azide hydrochloride, K2CO3, and CuSO4.

10. Use of the CD44-mediated self-assembled nanoparticles according to claim 1 or 2 in the preparation of drugs for treating tumors.

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