Amphiphilic polymer ly0016, and synthesis and use thereof

Abstract

An amphiphilic polymer LY0016, which has the chemical structure as shown in the following formula (I), a method for preparing same, and the use thereof in the preparation of a pharmaceutical preparation comprising a drug active molecule. The drug active molecule comprises, but is not limited to, a small molecule compound, a peptide analogue, a polypeptide or an antigen, a protein, an antibody and an immunologic adjuvant.

Description

An amphiphilic polymer LY0016, its synthesis and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefits and priority of the following Chinese invention patent application, the entire contents of which are hereby incorporated by reference into this document in their entirety: Patent application No. 202310714478.X filed with the State Intellectual Property Office of China on June 16, 2023. Technical Field

[0003] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to an amphiphilic polymer LY0016, and its synthesis and application. Background Art

[0004] Polymer micelle drug-loaded preparations are a relatively new type of drug preparation developed in recent years. The most basic material for forming polymer micelle drug-loaded preparations is amphiphilic polymers.

[0005] Amphiphilic polymers are the main excipient molecules for forming polymer micelle drug-loaded preparations. Their structure is mainly composed of hydrophilic blocks and hydrophobic blocks that are chemically covalently linked. In the aqueous phase or biological fluid environment (such as blood, lymph, etc.), the hydrophilic blocks are located in the outer corona region of the polymer micelle structure; the hydrophobic blocks assemble inward to form the inner core region of the micelle. Drug molecules are combined with amphiphilic copolymers based on their physicochemical properties (including but not limited to hydrophobic properties, charge properties, structural characteristics, etc.) through physical interactions (such as electrostatics, hydrogen bonds, van der Waals forces, hydrophobic forces, etc.) to form stable polymer micelle drug-loaded preparations.

[0006] Successfully assembled polymer micelle drug-loaded preparations generally have a size of less than 30 nanometers. The distribution and metabolic behavior of drugs in the body will be correspondingly improved due to the properties of polymer micelles. Common effects include but are not limited to enhanced stability, sustained release, reduced toxicity, changes in organ distribution of drugs, and regulation of the interaction between drugs and their ligands.

[0007] In the applicant's prior application, the methods and applications of studying the interaction between the amphiphilic polymer PEG-DSPE and biological macromolecules with higher-order structures at the molecular level were explored. As the applicant further deepened the research on the structural characteristics and functional relationship of the polymer micelle drug delivery system, a new amphiphilic polymer was designed and synthesized, which exhibited better drug delivery performance and function.

[0008] Based on this, the present invention is proposed.

[0009] Summary of the Invention

[0010] The present invention first relates to an amphiphilic polymer LY0016, the chemical structure of which is shown in the following formula:

[0011] In the formula, n is an integer of 35-55, preferably, n is an integer of 40-50, and most preferably, n=45.

[0012] The present invention also relates to a method for preparing the amphiphilic polymer LY0016, characterized in that the synthesis route is shown in the following reaction formula:

[0013] The specific steps are:

[0014] (1) Synthesis of Intermediate 1

[0015] Polyethylene glycol and PNC (p-nitrophenyl chloroformate) are used as substrates. DCM (dichloromethane) and TEA (triethylamine) are mixed under inert gas protection and stirred at room temperature for 1 to 3 hours. After the reaction is completed, the product is filtered, concentrated, and washed in sequence. Intermediate 1 is obtained by vacuum filtration.

[0016] Preferably, polyethylene glycol (molecular weight 2000): PNC (p-nitrophenyl chloroformate) = 1:2 (molar ratio); the polymer dispersity of the polyethylene glycol raw material is ≤1.05;

[0017] (2) Synthesis of Intermediate 2

[0018] Intermediate 1, L-tert-butyl glutamate as substrate, TEA (triethylamine) and DMF (dimethylformamide) are mixed under inert gas protection, stirred at room temperature for 15-20 hours, and after the reaction is complete, washed with water, extracted with the organic phase, concentrated, washed with the organic phase, and filtered under reduced pressure to obtain intermediate 2;

[0019] Preferably, intermediate 1: L-tert-butyl glutamate = 1:2 (molar ratio);

[0020] (3) Synthesis of Intermediate 3

[0021] Intermediate 2, distearoylglycerol, DMAP (4-dimethylaminopyridine) as substrate, HATU as condensing agent, DCM (dichloromethane) as solvent, after mixing at room temperature, stirring at room temperature for 4-6 hours, after the reaction is complete, washed with acid water, extracted with an organic solvent, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by suction;

[0022] The product was purified by silica gel column chromatography and the fractions were collected and concentrated to dryness to obtain intermediate 3;

[0023] Preferably, intermediate 2: distearoylglycerol:DMAP=1:1.5:2.5 (molar ratio); silica gel column chromatography purification using gradient elution, the eluent gradient is DCM:MeOH=50-20:1;

[0024] (4) Synthesis of product LY0016

[0025] Intermediate 3 is the substrate, TFA (trifluoroacetic acid, CF3COOH) is the cleavage agent, and DCM is the solvent. After mixing, stir at room temperature for 4 to 6 hours. After the reaction is complete, concentrate at 35°C, add DCM to the concentrate to dissolve it, and adjust the pH to alkaline.

[0026] After separating the organic phase, saturated brine was added and stirring was continued for 15 min. The organic phase was washed with 0.5N hydrochloric acid and saturated brine, and dried over anhydrous sodium sulfate;

[0027] After concentrating under reduced pressure, MTBE was added, and the mixture was heated to 40°C and stirred to dissolve. The mixture was then slowly cooled to 10°C for crystallization. After filtration, the mixture was rinsed with MTBE and dried to obtain the final product LY0016.

[0028] The present invention also relates to the following applications of the amphiphilic polymer LY0016:

[0029] (1) preparing drug-loaded micelles loaded with small molecule drugs, wherein the small molecule drugs are small molecule compounds with a hydrophobic core, including but not limited to small molecule compounds with an anthracycline core, paclitaxel and paclitaxel derivatives, and vinca alkaloid compounds;

[0030] Preferably, the small molecule drug is selected from paclitaxel, docetaxel, doxorubicin, topotecan, posaconazole, tacrolimus, amphotericin B, mepartricin, TLR4 agonist, and MPLA;

[0031] (2) preparing a drug-loaded complex loaded with a polypeptide or polypeptide analog;

[0032] Preferably, the polypeptide analog is selected from bortezomib, carfilzomib, cyclosporine, caspofungin, and anidulafungin;

[0033] Preferably, the polypeptide is a polypeptide with a length of less than 150 amino acids, including but not limited to insulin, GLP-1 and analogs thereof;

[0034] (3) Co-assembly with a protein having a higher-order structure to prepare a LY0016-protein assembly, wherein the dissociation constant (Kd value) of the protein and the amphiphilic polymer LY0016 after assembly is less than 10 -6 M;

[0035] Preferably, the protein is: cytokine, antibody;

[0036] More preferably, the cytokines are from the interleukin family, colony stimulating factor family, erythropoietin family, and growth hormone family, and the antibodies are from the IgG antibody and the IgA antibody.

[0037] (4) preparing a composite vaccine, wherein the composite vaccine comprises: an amphiphilic polymer LY0016 as a backbone carrier, an antigenic polypeptide, and an immune adjuvant;

[0038] Preferably, the immune adjuvant is MPLA;

[0039] Preferably, the antigen polypeptide is a recombinant antigen polypeptide with a length of less than 200 amino acids;

[0040] More preferably, the antigen polypeptide is a recombinant antigen polypeptide with a length of 8 to 150 amino acids;

[0041] Most preferably, the antigen polypeptide is a recombinant antigen polypeptide with a length of 20 to 80 amino acids.

[0042] The present invention also relates to a pharmaceutical preparation comprising the amphiphilic polymer LY0016, wherein the pharmaceutical preparation comprises:

[0043] (1) 50% by mass or more of the amphiphilic polymer LY0016, preferably 70% by mass or more;

[0044] (2) pharmaceutically active molecules, including but not limited to small molecule compounds, peptide analogs, polypeptides or antigens, proteins, antibodies, and immune adjuvants;

[0045] Preferably, the small molecule drug is a small molecule compound with a hydrophobic core, the polypeptide or antigen is a recombinant antigen polypeptide with a length of less than 200 amino acids, the protein is a cytokine or an antibody, and the immune adjuvant is MPLA;

[0046] Preferably, the pharmaceutical preparation further comprises:

[0047] (3) Other necessary pharmaceutical excipients, including but not limited to pH buffers, lyoprotectants, stabilizers, and excipients.

[0048] Preferably, the pharmaceutical preparation is an intravenous administration type pharmaceutical preparation or an intramuscular injection or subcutaneous administration type pharmaceutical preparation.

[0049] The beneficial effects of the present invention are:

[0050] (1) The physicochemical properties of amphiphilic polymer materials are combined with the folding process of protein higher-order structures in structural biology and molecular biology to design and synthesize new amphiphilic polymers. The new LY0016 polymer has structural units that can form more hydrogen bonds / salt bonds / hydrophobic interactions with drug molecules. It can aggregate into micelles as drug carriers, or form assembly complexes with proteins with typical higher-order structures by functioning like molecular chaperones, which can be used in pharmaceutical preparations.

[0051] (2) LY0016 polymer has good biosafety;

[0052] (3) LY0016 polymer can be used as a backbone molecule to form micelle carriers for the development of new formulations of small molecule drugs, peptide derivatives, and polypeptide drugs;

[0053] (4) LY0016 polymer molecules have the function of molecular chaperones and can assemble with protein drugs with higher structures with high affinity to form high-affinity protein assemblies, providing a new formulation for protein drugs;

[0054] (5) LY0016 polymer can be loaded with recombinant peptide antigens and TLR agonists simultaneously to prepare composite vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1. Chemical synthesis route of amphiphilic polymer LY0016.

[0056] Figure 2. NMR spectrum of intermediate 217K02.

[0057] Figure 3. NMR spectrum of product LY0016.

[0058] Figure 4. DLS test results of amphiphilic polymer LY0016 empty micelles and PEG2000-DSPE empty micelles (HOAC refers to acetic acid solvent).

[0059] Figure 5. Transmission electron microscopy (NEGATIVE STAIN) images of empty micelles of amphiphilic polymer LY0016 and PEG2000-DSPE (scale bar is 100 nm, HOAC refers to acetic acid solvent).

[0060] Figure 6. Toxicity of the amphiphilic polymer LY0016 to cells. Figure 6A, human cell line 293T; Figure 6B, human tumor cell line A549 (the abscissa in the figure, Micelle Concentration, is the concentration of the amphiphilic polymer LY0016).

[0061] Figure 7. Detection results of amphiphilic polymer LY0016-loaded micelles with poorly soluble chemical drugs. 6A, docetaxel-loaded micelle solution is a clear and transparent solution; 6B, transmission electron microscopy (negatively stained) photograph of docetaxel-loaded micelles (scale bar in the figure is 100 nm); 6C, posaconazole-loaded micelle solution is a clear and transparent solution; 6D, tacrolimus-loaded micelle solution is a clear and transparent solution.

[0062] Figure 8. Structural detection of amphiphilic polymer LY0016-assembled peptide derivative compounds. 8A, LY0016-bortezomib assembly (transmission electron microscopy, negatively stained sample, scale bar in the figure is 100 nm); 8B, LY0016-carfilzomib assembly (transmission electron microscopy, negatively stained sample, scale bar in the figure is 100 nm).

[0063] Figure 9. Activity test of LY0016-carfilzomib assembly (cytotoxicity assay).

[0064] Figure 10. Physical and chemical properties of the LY0016-IL2 protein assembly. 10A. DLS test results show that the particle size distribution of the assembly is highly concentrated at 14 nm. 10B. Electrophoresis (silver staining) detection of the assembly loading efficiency shows that when the protein concentration is diluted to 5 ng, the LY0016 polymer and IL2 protein can still be tightly assembled without leakage of free protein.

[0065] Figure 11. Activity detection of LY0016-IL2 protein assembly (CTLL-2 activation assay).

[0066] FIG12 . LY0016-MPLA-MUC1-40 composite vaccine enhances the expression of co-stimulatory factors in DCs.

[0067] Figure 13. LY0016-MPLA-MUC1-40 composite vaccine enables DCs to secrete more immune activation-related cytokines.

[0068] Figure 14. LY0016-MPLA-MUC1-40 composite vaccine enhances the antigen presentation efficiency of DCs.

[0069] Figure 15. The LY0016-MPLA-MUC1-40 composite vaccine can efficiently induce antigen-specific CTLs and antigen-specific IgG. DETAILED DESCRIPTION

[0070] ELISA reagents and ELISA kits were purchased from Invitrogen, specifically: mouse TNF-α (88-7324-88), mouse IL-2 (88-7024-88), mouse IL-12 (88-7121-88)

[0071] ELISPOT reagent: Mouse interferon-gamma enzyme-linked immunosorbent assay kit (551083) was purchased from BD Biosciences

[0072] Cytokine antibodies: anti-mouse CD80, anti-mouse CD86 were purchased from BioLegend

[0073] The sequence of the MUC1-40 antigen is shown in SEQ ID NO.1.

[0074] SEQ ID NO.3: PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA

[0075] The basic repeat sequence of the MUC1 protein is shown in SEQ ID NO.4.

[0076] SEQ ID NO.4:PDTRPAPGSTAPPAHGVTSA

[0077] The sequence of the MUC1-9 antigen is shown in SEQ ID NO.1.

[0078] SEQ ID NO. 5: STAPPAHGV.

[0079] PEG2000-DSPE (CAS: 147867-65-0): purchased from LIPOID (catalog number: 29232000), full name:

[0080] N-(Carbonyl-methoxypolyethylene glycol-2000)-1,2-distaroyl-sn-glycero-3-phosphoethanolamine,sodium salt

[0081] MPLA: purchased from SIGMA (1246298-63-4);

[0082] The compounds, solvents, and catalysts used in the synthesis of LY0016 were of chromatographic grade. Other raw materials were of analytical grade unless otherwise specified.

[0083] Mouse Model:

[0084] The MUC1 transgenic mouse model was constructed independently: the MUC1 transgenic mouse model contains all the genetic information of human MUC1. Compared with wild-type mice that have only 34% sequence similarity to human MUC1, it can fully simulate clinical pathological characteristics and correctly evaluate the role of the immune system in tumor treatment.

[0085] Example 1. Chemical synthesis of amphiphilic copolymer LY0016

[0086] The synthesis of LY0016 amphiphilic copolymer material was carried out according to the following synthetic route:

[0087] The process control of the specific synthesis process is as follows (unless otherwise specified, the raw materials used are analytically pure):

[0088] 1. Synthesis of intermediate 10117

[0089] 0.1 mol of polyethylene glycol (molecular weight 2000), 0.2 mol of p-nitrophenyl chloroformate (PNC), 0.2 mol of triethylamine (TEA), and 1 L of dichloromethane (DCM) were added. After the addition was completed, the mixture was mixed at 5°C for 10-15 minutes under inert gas protection. The mixture was then warmed to room temperature and stirred for 1-3 hours. The reaction process was monitored during the reaction. After the reaction was completed, the insoluble matter was removed by filtration. The filtrate was concentrated to dryness and washed with MTBE (methyl tert-butyl ether) and THF (tetrahydrofuran) in sequence. After filtration under reduced pressure, 203 g of a white solid (10117) was obtained with a molecular weight of 2177, a yield of 93.7%, and a purity of >98%.

[0090] Among them, the polymer dispersity of polyethylene glycol raw materials should be ≤1.05;

[0091] 2. Synthesis of intermediate 217K01

[0092] 92.34 mmol of the intermediate 10117 compound synthesized in the previous step, 184.7 mmol of L-tert-butyl glutamate, 230 mmol of TEA (triethylamine), and 2 L of DMF (dimethylformamide) were added. After the addition was completed, the mixture was protected by inert gas, mixed, and stirred at room temperature for 15-20 hours. After the reaction was completed as monitored by TLC, 1 L of saturated citric acid aqueous solution was added, and DCM was extracted twice. The organic phases were combined and concentrated to dryness, washed with DCM and MTBE, and the organic solvent was removed by filtration under reduced pressure to obtain 210.8 g of a white solid (217K01) with a molecular weight of 2130, a yield of 99%, and a purity of >98%.

[0093] 3. Synthesis of intermediate 217K02

[0094] 89.69 mmol of the intermediate 217K01 compound synthesized in the previous step, 134.5 mmol of 14303 (distearoyl glycerol), 179.4 mmol of HATU condensing agent, 224.2 mmol of DMAP (4-dimethylaminopyridine), and 2 L of DCM (dichloromethane) were mixed at room temperature and stirred at room temperature for 4-6 hours. After TLC monitoring, 1 L of 0.5N aqueous hydrochloric acid and 1 L of DCM were added. After separation, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (mobile elution: DCM / MeOH = 50:1 to 20:1). The product fractions were collected and concentrated to dryness to obtain 200 g of a light yellow solid with a molecular weight of 2837, a yield of 78%, and a purity of >98%. 1 The H NMR (0243-17-217K02-P1, CDCl3) spectrum is shown in Figure 2.

[0095] 4. Synthesis of product LY0016

[0096] The intermediate 217K02 compound 39.17mmol synthesized in the previous step, TFA 170ml, DCM 230ml, after adding the materials, mix and dissolve, stir at room temperature (20-25 ℃) for 4-6h, monitor the reaction of the raw materials by TLC, and concentrate at 35 ℃; add DCM 800-1000ml to the concentrate until it is completely dissolved, add saturated sodium bicarbonate solution to adjust the pH to alkaline; after separating the organic phase, add 1L saturated brine to the organic phase and continue stirring for 15min; wash the organic phase with 0.5N hydrochloric acid and saturated brine, dry with anhydrous sodium sulfate, concentrate under reduced pressure, add 2.8L MTBE to the concentrated residue, heat to 40 ℃, stir and dissolve, then slowly cool to 10 ℃, and a large amount of white solid precipitates; after filtration, rinse the filter cake with MTBE and dry to obtain 100g of white product with a yield of 92%. The final product LY0016 1 The H NMR (0243-25-LY0016-P1, CDCl3) spectrum is shown in Figure 3.

[0097] After testing, the quality parameters of the finished product LY0016 polymer under this synthetic process route are: appearance is white or off-white solid, moisture ≤0.5%, total impurities ≤2.0%, and solvent residue ≤1%.

[0098] Example 2: Preparation of empty micelles using LY0016 as a micelle backbone molecule and property evaluation

[0099] In this example, a direct dissolution method using solvents (of various properties) was used to prepare empty micelle samples with LY0016 as the skeleton, and their physicochemical properties were investigated.

[0100] 1. The preparation process of LY0016 empty micelles is as follows:

[0101] (1) The LY0016 dry powder prepared in Example 1 was dissolved in double-distilled water or pH 6 acetic acid solution to prepare an empty micelle solution with a concentration of 20 mg / ml;

[0102] (2) The stock solution was diluted to 4 mg / ml with PBS buffer to obtain an empty micelle dilution solution.

[0103] 2. The preparation process of the reference empty micelle sample is as follows:

[0104] (1) PEG2000-DSPE powder was dissolved in double-distilled water or pH 6 acetic acid solution to prepare an empty micelle solution with a concentration of 20 mg / ml;

[0105] (2) Also dilute to 4 mg / ml using PBS buffer to obtain PEG2000-DSPE empty micelle dilution solution.

[0106] 3. Use dynamic light scattering (DLS) to detect the properties of empty micelles

[0107] 20 μl of the sample was added to a quartz cell and placed in a dynamic light scattering instrument. Each sample was tested for 10 cycles, each cycle was 20 seconds, and the experiment was repeated three times in parallel to obtain the average value of the particle size.

[0108] The DLS test results are shown in Figure 4. The results show that

[0109] (1) In different solvents, the DLS detection results (particle size) of PEG2000-DSPE empty micelles are all around 2 nm (this result is quite different from the result of electron microscopy, and the reason for the difference is the lack of contrast of the solvent itself); while the detection results (particle size) of LY0016 are all around 10 nm;

[0110] (2) After diluting the empty micelles with PBS, the DLS detection results of the PEG2000-DSPE empty micelles (particle size) were 12-14 nm; while the detection results of LY0016 (particle size) were 15-17 nm;

[0111] 4. Transmission electron microscopy to detect the properties of empty micelles

[0112] Take the 20 mg / ml LY0016 and PEG2000-DSPE empty micelles (water or pH 6 acetic acid solvent) prepared in steps 1 and 2 and dilute them with pure water to a concentration of 1 mg / ml. Use the negative staining method to prepare samples, and observe the morphology of the empty micelles using transmission electron microscopy (TEM).

[0113] The result is shown in Figure 5.

[0114] (1) The particle size of LY0016 empty micelles in water or acetic acid is not much different (about 15 nm, the scale in the figure is 100 nm), but its distribution is different; the particle size is basically consistent with the particle size calculated by dynamic light scattering;

[0115] (2) The particle size of PEG2000-DSPE empty micelles is basically consistent with the particle size measured by dynamic light scattering instrument;

[0116] 5. Toxicity test of LY0016 polymer

[0117] 5.1. Investigation of the toxicity of LY0016 polymer on human normal cell line (293T) and human tumor cell line (A549)

[0118] A549 / 293T cells were seeded in 96-well plates at 5000 cells / well. After cell attachment, complete medium containing different concentrations of LY0016 was added and cultured for 48 h. The medium was discarded, and 100 μL MTT solution (0.5 mg / ml, prepared in PBS) was added to each well and incubated at 37°C for 4 h. The MTT solution was discarded, and 100 μL DMSO solution was added to each well. The absorbance at 570 nm was detected using a microplate reader.

[0119] The experimental results are shown in Figure 6. The results show that:

[0120] The toxicity of LY0016 to the normal human cell line 293T and the human tumor cell line A549: When the LY0016 concentration reached 1000μM, the median lethal concentration was still not reached.

[0121] In summary, the new material LY0016 has good safety.

[0122] Example 3: Preparation of poorly soluble drug-loaded micelles using LY0016 as a micelle backbone molecule

[0123] 1.1. LY0016-Docetaxel micelles

[0124] Docetaxel, CAS: 114977-28-5, molecular formula: C43H53NO14, molecular weight: 807.88, solubility: almost insoluble in water, readily soluble in anhydrous ethanol. Since docetaxel is a poorly soluble drug, the lipid membrane hydration method was used to prepare drug-loaded micelles. The specific steps are as follows:

[0125] (1) LY0016 (200 mg) and docetaxel (10 mg) were dissolved in 2 ml of ethanol.

[0126] (2) Under inert gas protection, the ethanol solvent was removed by rotary evaporation under reduced pressure to form a drug-containing lipid film;

[0127] (3) Add 1.5 ml of 10% sucrose solvent to hydrate the lipid membrane. After it is completely dissolved into a clear solution, filter it through a 0.22 μm filter membrane to obtain the drug-loaded micelle solution (the drug-loaded micelle is a colorless clear solution, see Figure 7A).

[0128] 1.2. Physical and chemical properties of LY0016-docetaxel micelles

[0129] The drug-loaded micelles were diluted to a concentration of 1 mg / ml, and negative staining was used to prepare the samples. The morphology of the empty micelles was observed using transmission electron microscopy (TEM). The results, shown in Figure 7B, show that the drug-loaded micelles were uniformly distributed, with a particle size of approximately 15 nm (scale bar in the figure represents 100 nm).

[0130] 2.1. LY0016-Posaconazole micelles

[0131] Posaconazole, CAS: 171228-49-2, molecular formula: C37H42F2N8O4, molecular weight: 700.78, solubility: chloroform (slightly soluble), methanol (slightly soluble).

[0132] The mass ratio of LY0016 to posaconazole was 20:1. The same method as that used to prepare docetaxel-loaded micelles was used to prepare LY0016-psaconazole-loaded micelles by film-forming hydration method (the loaded micelles were colorless and clear solutions, see Figure 7C).

[0133] 2.2. Physical and chemical properties of LY0016-Posaconazole loaded micelles

[0134] The drug-loaded micelles were diluted to a concentration of 1 mg / ml, and samples were prepared using the negative staining method. Transmission electron microscopy (TEM) was used to observe the morphology of the empty micelles, which showed that the drug-loaded micelles also had a uniform size distribution, with a particle size of approximately 15 nm.

[0135] 3.1. LY0016-Tacrolimus Micelles

[0136] Tacrolimus, CAS: 104987-11-3, molecular formula: C44H69NO12, molecular weight: 804.02, solubility: almost insoluble in water, easily soluble in anhydrous ethanol, soluble in dichloromethane.

[0137] The mass ratio of LY0016 to tacrolimus was 20:1. The same method as that for preparing docetaxel-loaded micelles was used to prepare LY0016-tacrolimus-loaded micelles by film-forming hydration method (the loaded micelles were colorless and clear solutions, see Figure 7D).

[0138] 3.2. Physical and Chemical Properties of LY0016-Tacrolimus-Loaded Micelle

[0139] The drug-loaded micelles were diluted to a concentration of 1 mg / ml, and samples were prepared using the negative staining method. Transmission electron microscopy (TEM) was used to observe the morphology of the empty micelles, which showed that the drug-loaded micelles also had a uniform size distribution, with a particle size of approximately 15 nm.

[0140] In summary, drug-loaded micelles prepared with LY0016 polymer as the skeleton can be loaded with a variety of poorly soluble drugs, and drug-loaded micelles with a uniform particle size of about 15 nm can be prepared.

[0141] Example 4: LY0016 as a backbone molecule for loading peptide drugs

[0142] 4.1. LY0016 Bortezomib / Carfilzomib Assembly

[0143] Bortezomib, CAS: 179324-69-7, molecular formula: C19H25BN4O4, molecular weight: 384.24, the structure of bortezomib compound is as follows, and the molecular structure is a peptide derivative compound;

[0144] Carfilzomib, CAS: 868540-17-4, molecular formula: C40H57N5O7, molecular weight: 719.92, the compound structure of carfilzomib is as follows, and the molecular structure is also a peptide derivative compound;

[0145] The preparation method of peptide derivative assembly using LY0016 is as follows:

[0146] Preparation of LY0016 bortezomib assembly

[0147] (1) Weigh 10 mg of bortezomib and 200 mg of LY0016, add 2 ml of ethanol, and heat at 50°C until the solution becomes clear.

[0148] (2) removing the solvent by distillation under reduced pressure to form a drug-containing lipid film;

[0149] (3) 5 ml of 5% mannitol solution was added to hydrate the sample to obtain LY0016 bortezomib assembly, and the sample was sterilized by filtering through a 0.22 μm filter membrane.

[0150] Preparation of LY0016 carfilzomib assembly

[0151] (1) Mix 2.7 mL of citric acid and 0.3 mL of ethanol;

[0152] (2) Add 200 mg of LY0016 and heat in a water bath at 45°C to dissolve;

[0153] (3) Add 10 mg of carfilzomib, stir and mix, and then add 10 μL of 1 M NaOH solution to obtain LY0016 carfilzomib assembly. The LY0016 carfilzomib assembly sample is sterilized by filtering through a 0.22 μm filter membrane.

[0154] 4.2. Physical and chemical properties of LY0016 bortezomib / carfilzomib assembly

[0155] The LY0016 bortezomib assembly was diluted to a concentration of 0.5 mg / ml, and the LY0016 carfilzomib assembly was diluted to a concentration of 1 mg / ml. Samples were prepared using a negative staining method, and the morphology of the drug-loaded micelles was observed using a transmission electron microscopy (TEM).

[0156] The results are shown in Figure 8. The negative staining electron microscopy results show that the drug-loaded micelles are also uniformly distributed in size, with a particle size of about 15 nm.

[0157] 4.3. Pharmacological Evaluation of LY0016 Carfilzomib Assembly

[0158] After the revived ARD cells were cultured for 2 generations, the CCK-8 test was performed. The specific steps are as follows:

[0159] (1) Collect tumor cells in the logarithmic growth phase and use culture medium to make 2×10 4 A single cell suspension of 100 μL (about 2 x 10 cells / mL) was inoculated into each well of a 96-well culture plate. 4 cells);

[0160] (2) Drugs were added simultaneously when cells were inoculated (carfilzomib group, LY0016 carfilzomib assembly group, and LY0016 empty micelle group). Culture medium containing different concentrations of drugs was added to the drug-treated groups, 100 μL per well.

[0161] Set the dose group as,

[0162] Carfilzomib group, the dosage was 0.5nM, 1nM, 2nM, 4nM, 8nM, 16nM, and 32nM respectively;

[0163] In the LY0016 carfilzomib assembly group, the dosages of carfilzomib were 0.5 nM, 1 nM, 2 nM, 4 nM, 8 nM, 16 nM, and 32 nM;

[0164] The LY0016 empty micelle group was only given empty micelles at the corresponding carrier concentration;

[0165] (3) After culturing at 37°C in a 5% CO2 incubator for 24 hours, 20 μL of CCK-8 solution was added to each well and the cells were cultured for another 3 hours. The absorbance A450 was measured at a wavelength of 450 nm.

[0166] The results are shown in Figure 9.

[0167] (1) The IC50 value of carfilzomib for ARD cells is 7.852 nM;

[0168] (2) The IC50 value of LY0016 carfilzomib assembly against ARD cells was 15.01 nM;

[0169] LY0016 carfilzomib assembly still has high biological activity

[0170] Example 5: Preparation of protein assembly using LY0016 polymer as a backbone and activity evaluation

[0171] 5.1 Preparation of protein assemblies

[0172] Referring to the process disclosed in the applicant's prior application (PCT / CN2022 / 128021), LY0016-IL2 protein assembly and LY0016-G-CSF protein assembly were prepared. The specific steps are as follows:

[0173] (1) Prepare 5mM dilute hydrochloric acid solution with pH = 3.0-3.5

[0174] (2) Weigh a certain amount of IL2 protein powder or G-CSF protein powder, add it to the acetic acid solution, and mix well to obtain a protein solution;

[0175] (3) Add LY0016 dry powder to the above protein solution at a molar ratio of LY0016:protein = 15-30:1 and mix thoroughly;

[0176] (4) After heating at 40-60°C for 10-30 minutes, the sample was allowed to cool to room temperature;

[0177] (5) Adjust the pH value of the solution to 6-7, let it stand for 10-60 minutes, and filter the sample through a 0.22 μm filter membrane to obtain the LY0016 / IL2 protein assembly or the LY0016 / G-CSF protein assembly.

[0178] The IL-2 protein and G-CSF protein used were expressed and purified in E. coli system, and their amino acid sequences are shown in SEQ ID NO. 1 and 2, respectively:

[0179] SEQ ID NO.1(IL2):

[0180] SEQ ID NO. 2 (G-CSF):

[0181] 5.2. Physicochemical Properties of Protein Assemblies

[0182] (1) Assembly stability test

[0183] The two protein assemblies prepared in the above steps were left to stand at room temperature for one week. The assembly solutions remained clear and no insoluble matter was observed to precipitate, demonstrating that the stability of the assemblies was basically satisfactory.

[0184] (2) Detection of particle size and uniformity of the assembly

[0185] The particle size and structural uniformity of the assembly were detected using a DLS dynamic light scattering experiment. The test sample was diluted to 1 mg / ml with pure water, and the particle size was detected using a dynamic light scattering instrument. Each sample was tested for 10 cycles, each cycle lasting 20 seconds. The experiment was repeated three times in parallel to obtain the average particle size.

[0186] The results are shown in Figure 10A.

[0187] The LY0016-IL-2 assembly is a complex with uniform particle size, and the particle size detected by DLS is about 14 nm.

[0188] (3) Electrophoresis detection of the stability of LY0016-IL-2 assembly

[0189] The experimental method is as follows:

[0190] Preparation of 1× Tris-MOPS buffer: Add 9.086 g of Tris powder and 5.232 g of MOPS powder to a beaker, add deionized water to make up to 1 L, stir to dissolve at room temperature, and adjust the pH to 9.5.

[0191] Preparation of 2× loading buffer: Mix 50% 50 mM Tris buffer (pH=10.0) with 50% glycerol and store at -20°C.

[0192] Electrophoresis detection was performed using 7% Native-PAGE gel

[0193] Preparation of samples for electrophoresis detection: Samples of different concentrations were mixed with equal volumes of 2× loading buffer before loading and directly loaded for electrophoresis.

[0194] The electrophoresis conditions were as follows: voltage 100 V, current 300 mA, and electrophoresis time 50 ± 10 min.

[0195] After electrophoresis, the gel was removed and silver stained (Biyuntian Rapid Silver Staining Kit, P0017S).

[0196] The results are shown in Figure 10B. The silver staining results of protein electrophoresis show:

[0197] (1) Compared with the IL2 protein sample directly dissolved in 1% SDS (lanes 6-9), the IL2 protein in the LY0016-IL2 assembly was clearly in the form of compact aggregates (lanes 1-4);

[0198] (2) The band positions of the assembly and the monomer are very different, indicating that the particle size and charge of the assembly are significantly different;

[0199] (3) No IL-2 monomer band was detected in the assembly lane, indicating that all proteins participated in the formation of the assembly.

[0200] In summary, the LY0016-IL2 protein assembly has extremely high assembly efficiency.

[0201] 5.3. Biological Activity Detection of LY0016-IL2 Protein Assembly

[0202] This example detects the activity of the LY0016-IL-2 assembly to verify the effect of IL-2 on IL-2 activity after forming a complex with LY0016.

[0203] The bioactivity of the LY0016-IL-2 assembly was investigated using the CTLL-2 / MTT cell proliferation assay. The assay method was similar to that in the applicant's prior application (PCT / CN2022 / 128021). Furthermore, the PEG2000-DSPE / IL2 protein assembly prepared in the applicant's prior application was used as a positive control.

[0204] The activity detection steps are as follows:

[0205] (1) Dilute the LY0016-IL-2 assembly and positive control drug to appropriate concentrations with basal culture medium;

[0206] (2) Preparation of CTLL-2 cell suspension: Take the CTLL-2 cells that have been revived and cultured in complete medium and are growing vigorously. Wash the cells three times with 1640 culture medium without other components, and then resuspend them in basal medium to make 6×10 5 / ml concentration of cell suspension;

[0207] (3) Adding samples: Prepare a flat-bottom 96-well plate and perform a gradient dilution of the test sample and the positive control drug with basal medium to obtain at least 8 concentrations with a difference of 2-fold or more. Prepare 2 to 3 wells for each concentration and add 50 μl of sample to each well.

[0208] (4) Cell inoculation: Add 50 μl of cell suspension to each well, mix well, and place in a 37°C cell culture incubator with 5% CO2 for 18–24 hours;

[0209] (5) Add 20 μl of MTT solution to each well and continue incubating in the incubator for 4 to 6 hours.

[0210] (6) Add 150 μl of 15% SDS solution to each well, mix thoroughly, and continue incubating in the incubator for 18 to 24 hours.

[0211] (7) Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 570 nm and the IL-2 activity of the sample

[0212] The experimental results are shown in Figure 11.

[0213] The IL-2 activity of the LY0016-IL-2 assembly was calculated to be 6.6×10 6 IU / mg, which is basically consistent with the activity of the positive control (IL2 protein standard prepared by the applicant).

[0214] In summary, the LY0016-IL2 protein assembly does not reduce the biological activity of the target protein after protein assembly.

[0215] Example 6: LY0016 polymer as an immune adjuvant carrier and preparation of (recombinant protein) vaccine and activity evaluation

[0216] LY0016 enhances the co-stimulatory level of the immune adjuvant MPLA (TLR4 receptor agonist)

[0217] Monophosphoryl lipid A (MPLA) is a natural agonist of toll-like receptor 4 (TLR4). It is commonly used as an immune adjuvant, and its use in vaccines can enhance B- and T-cell-mediated immunity.

[0218] This experiment investigated the changes in the MPLA co-stimulatory effect of LY0016-MPLA composite micelles. The specific experiments are as follows

[0219] 1. Preparation of LY0016-MPLA composite micelles

[0220] LY0016-MPLA composite micelles were prepared using a film-forming hydration method (referring to the method in WO2016 / 062227A1). The specific steps are as follows:

[0221] (1) LY0016 was dissolved in chloroform, and MPLA was dissolved in a chloroform-methanol mixed solvent (chloroform:methanol v / v = 2:1);

[0222] (2) The organic phase from step 1) was mixed at a mass ratio of LY0016 to MPLA of 100:1, and the organic solvent was removed by rotary evaporation under reduced pressure;

[0223] (3) Add an appropriate amount of double-distilled water to hydrate and dissolve the lipid membrane, and filter through a 0.22 μm filter membrane to obtain a LY0016-MPLA complex micelle solution with an MPLA content of 0.1 mg / ml.

[0224] 2. Obtaining and culturing mouse bone marrow-derived DCs (dendritic cells)

[0225] (1) 6-8 week old C57BL / 6 mice were dissected to obtain long bones and tibiae. The bone marrow was blown out and lysed with red blood cell lysis buffer (eBioscience 00-4300-54) for 1 min. The red blood cells were washed twice with PBS to obtain a mouse bone marrow cell suspension.

[0226] (2) 2×10 6 Cells / dish were seeded in 90 mm culture dishes and cultured in complete medium (RPMI 1640 containing 10% FBS and 50 μM mercaptoethanol) plus 20 ng / ml mGM-CSF. On the third day of culture, 10 ml of complete medium (containing 20 ng / ml mGM-CSF) was added;

[0227] (3) On the sixth day of culture, the non-adherent cells were collected as immature BMDCs (imDCs). The purity of DCs was above 80-90% as determined by flow cytometry.

[0228] 3. Effects of LY0016-MPLA composite micelles on co-stimulatory molecules CD80 and CD86 on the surface of DCs cells

[0229] (1) DCs cells prepared in step 2, 1×10 6 / well, inoculated in 24-well plates, and the drug-dosing groups were as follows:

[0230] LY0016-MPLA composite micelles, MPLA concentration is 100ng / ml, LY0016 concentration is 10μg / ml,

[0231] MPLA single administration group, MPLA concentration was also 100ng / ml,

[0232] LY0016 empty micelle control group, LY0016 concentration 10μg / ml,

[0233] LPS control group, LPS concentration was 100 ng / ml;

[0234] (2) After 16 hours of treatment, the expression levels of CD80 and CD86 were detected by flow cytometry;

[0235] The results are shown in Figure 12.

[0236] (1) The positive control LPS can significantly enhance the expression of CD80 and CD86 on the surface of DCs;

[0237] (2) Empty LY0016 micelles had almost no effect on the expression of CD80 and CD86 on the surface of DCs cells, and the activation effect of free MPLA on CD80 and CD86 on the surface of DCs cells was weaker than that of LPS;

[0238] (3) LY0016-MPLA composite micelles have the highest stimulation intensity on the expression of CD80 and CD86 on the surface of DCs cells;

[0239] The above results indicate that LY0016-MPLA composite micelles can significantly enhance the immunostimulatory effect of TLR4 receptor agonists.

[0240] 4. LY0016-MPLA composite micelles enhance the secretion of immune activation-related cytokines TNF-α, IL-12, and IL-2 by antigen-presenting cells

[0241] (1) DCs prepared in step 2 were 10 6 / well were seeded in a round-bottom 96-well plate, and the drug-dosing groups were as follows:

[0242] MPLA single-dose group, MPLA concentration 100ng / ml,

[0243] LY0016 empty micelle control group, LY0016 concentration 10 μg / ml;

[0244] LPS control group, LPS concentration 100 ng / ml;

[0245] LY0016-MPLA complex micelle group, MPLA concentration 100 ng / ml, LY0016 concentration 10 μg / ml;

[0246] (2) After 24 h of cell treatment, the culture supernatant was obtained by centrifugation and the extracellular secreted cytokines IL-12, IL-2, and TNF-α were detected by ELISA (mouse TNF-α (88-7324-88), mouse IL-2 (88-7024-88), and mouse IL-12 (88-7121-88)).

[0247] The results are shown in Figure 13.

[0248] (1) Empty micelles cannot activate DCs to secrete more TNF-α, IL-12, and IL-2;

[0249] (2) The levels of three cytokines secreted by activated DCs in the MPLA-treated group were significantly lower than those in the LY0016-MPLA composite micelle group and the LPS-positive control group;

[0250] (3) LY0016-MPLA composite micelles can efficiently stimulate DCs to secrete three cytokines.

[0251] 5. LY0016-MPLA composite micelles significantly enhance the antigen presentation ability of DCs

[0252] (1) The DCs prepared in step 2 of this example were 5 x 10 5 / well were inoculated in 96-well plates, and the drug-dosing groups were as follows:

[0253] MPLA alone administration + OVA antigen group, MPLA concentration 100 ng / ml, OVA antigen concentration 1 mg / ml;

[0254] LY0016 empty micelle control group, LY0016 concentration 10ug / ml, OVA antigen concentration 1mg / ml;

[0255] LPS control group, LPS concentration 100 ng / ml, OVA antigen concentration 1 mg / ml;

[0256] LY0016-MPLA complex micelle group, MPLA concentration 100 ng / ml, LY0016 concentration 10 μg / ml, OVA antigen concentration 1 mg / ml;

[0257] In the above-mentioned drug administration group, the amino acid sequence of the OVA antigen used was: SGLEQLESIINFEKL, and the solvent of the OVA antigen was: double distilled water

[0258] (2) After 48 h of cell treatment, the cells were harvested by centrifugation and the pMHC I complex on the surface of DC cells was detected by flow cytometry (kit catalog number: anti-mouse OVA257-264 (SIINFEKL) peptide bound to H-2Kb (17-5743-80)) to investigate the efficiency of antigen presentation through MHC I molecules.

[0259] The results are shown in Figure 14.

[0260] (1) LY0016 empty micelles had no significant effect on the OVA antigen presentation level of DCs;

[0261] (2) LY0016-MPLA composite micelles can significantly enhance the presentation of OVA antigen by DC cells, and the activation level is comparable to that of the LPS group;

[0262] This indicates that LY0016-MPLA composite micelles significantly enhanced the MHC class I antigen presentation of full-length OVA antigen.

[0263] 6. The composite vaccine with LY0016 as the backbone efficiently activates antigen-specific CTL (ELISPOT) and specific IgG (ELISA)

[0264] The preparation process of the composite vaccine with LY0016 and as the backbone is as follows (refer to the method in WO2016 / 062227A1):

[0265] (1) LY0016 was dissolved in chloroform, MPLA was dissolved in a chloroform-methanol mixed solvent (chloroform:methanol v / v=2:1), and MUC1-40 antigen (antigen sequence information PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA) was dissolved in methanol;

[0266] (2) The organic phases from step 1) were mixed in a mass ratio of LY0016:MPLA:MUC1 antigen of 100:1:10, and the organic solvent was removed by rotary evaporation under reduced pressure;

[0267] (3) Add an appropriate amount of 5% sucrose solution to hydrate and dissolve the lipid film, and filter through a 0.22 μm filter membrane to obtain a composite vaccine solution with LY0016 as the backbone. In the composite vaccine, the content of MPLA is 0.1 mg / ml, the content of OVA antigen is 1 mg / ml, and the content of LY0016 polymer is 10 mg / ml;

[0268] The preparation of the composite vaccine with PEG2000-DSPE as the backbone also refers to the method in WO2016 / 062227A1.

[0269] (1) PEG2000-DSPE was dissolved in chloroform, MPLA was dissolved in a chloroform-methanol mixed solvent (chloroform:methanol v / v=2:1), and MUC1-40 antigen (antigen sequence information PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA) was dissolved in methanol;

[0270] (2) The organic phase from step 1) was mixed in a mass ratio of PEG2000-DSPE:MPLA:MUC1 antigen of 100:1:10, and the organic solvent was removed by rotary evaporation under reduced pressure;

[0271] (3) Add an appropriate amount of 5% sucrose solution to hydrate and dissolve the lipid film, and filter through a 0.22 μm filter membrane to prepare a composite vaccine solution with PEG2000-DSPE (PEG-PE) as the backbone. In the composite vaccine, the content of MPLA is 0.1 mg / ml, the content of OVA antigen is 1 mg / ml, and the content of PEG2000-DSPE polymer is 10 mg / ml;

[0272] Preparation of MUC1 transgenic C57BL / 6 mice:

[0273] (1) Construction of a recombinant plasmid containing the human MUC1 target gene;

[0274] (2) The vector plasmid containing the complete genetic information of human MUC1, including 40 identical tandem repeat sequences (the amino acid sequence of each repeat sequence is: PDTRPAPGSTAPPAHGVTSA) and a length of 3786 bp, was double-digested with restriction endonucleases NheI and XhoI to obtain a MUC1 target gene fragment with sticky ends;

[0275] (3) Perform the same double enzyme digestion treatment on the eukaryotic expression plasmid pcDNA3.1. Use T4 ligase to insert the human MUC1 target gene into the multiple cloning site of the pcDNA3.1 plasmid, thereby constructing the pcDNA3.1-MUC1 recombinant plasmid;

[0276] (4) The pcDNA3.1-MUC1 recombinant plasmid was microinjected into mouse fertilized eggs to obtain human MUC1 transgenic C57BL / 6 mice.

[0277] Breeding and confirmation of MUC1 transgenic C57BL / 6 mice:

[0278] (1) The prepared heterozygous MUC1 transgenic C57BL / 6 mice were paired, and the MUC1 gene level, MUC1 gene transcription level, and MUC1 protein expression level of the offspring were identified;

[0279] (2) Individuals that were identified as MUC1 positive and with relatively high expression levels were selected and paired until all offspring mice were identified as positive, thereby obtaining homozygous MUC1 transgenic C57BL / 6 mice.

[0280] The steps of single / multiple immunization method are as follows:

[0281] (1) MUC1 transgenic C57BL / 6 mice were injected with 100 μl of LY0016-based micelle vaccine solution at the tail heel. The corresponding dosage for each mouse was: MPLA 5 μg / mouse, MUC1 antigen 50 μg / mouse. Mice in the multiple immunization group were given two more booster immunizations on the 7th and 14th days after the first immunization.

[0282] (2) On the 7th day after administration, mice in the single immunization group were sacrificed and their inguinal lymph nodes were harvested; mice in the multiple immunization group were sacrificed on the 7th day after the last administration, and their blood and spleen were collected;

[0283] The ELISPOT assay steps are as follows:

[0284] (1) Pre-incubate anti-mouse IFNγ antibody in an ELISPOT plate at a concentration of 5 μg / ml and incubate overnight at 4°C;

[0285] (2) The next day, the pre-applied antibody was discarded and blocking solution (RPMI1640 culture medium containing 10% fetal bovine serum, 1% penicillin-streptomycin antibody, and β-mercaptoethanol) was added for blocking at room temperature for 2 h.

[0286] (3) Grind and sieve the sampled mouse inguinal lymph nodes or spleen to obtain a single cell suspension (spleen cells also need to be lysed);

[0287] (4) Discard the blocking solution, add culture medium containing 40 μg / ml MUC1-9 (STAPPAHGV) short peptide, and then add the above single cell suspension at a volume ratio of 1:1 until the cell volume reaches 2x10 6 Each well was incubated at 37°C for 48 h.

[0288] (5) Discard the culture medium and add deionized water to lyse the cells, 5 min each time, for a total of 2 times;

[0289] (6) After washing with PBST, add biotin-labeled anti-mouse IFNγ antibody and incubate at room temperature for 2 h;

[0290] (6) After washing with PBST solution, add streptavidin-HRP labeled with horseradish peroxidase and incubate at room temperature for 1 h;

[0291] (7) Wash with PBS and add AEC substrate for color development. Terminate the reaction with deionized water and use a luciferase spot analyzer CTL analyzer LLC for scanning and counting analysis.

[0292] The results are shown in Figure 15. The experimental results show that

[0293] The combined vaccine efficiently activated antigen-specific CTLs in MUC1 transgenic mice, both in the lymph nodes after a single immunization (Figure 15A) and in the spleens of mice immunized multiple times (Figure 15B). Furthermore, the activation effect was comparable to that of a combined vaccine of the same formulation prepared with PEG2000-DSPE excipient.

[0294] The ELISA experimental steps are as follows:

[0295] (1) After multiple immunizations, blood samples from mice were placed on ice for 2 hours, centrifuged, and serum was collected. The MUC1-40 specific IgG level was detected by ELISA.

[0296] (2) Use 100ug / ml MUC1-40 polypeptide (antigen sequence

[0297] PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA) solution was applied to the plate overnight at 4°C and the plate was washed the next day with PBST containing 0.5% Tween-20;

[0298] (2) Block with PBST containing 1% BSA at room temperature for 1 h, and then wash with PBST;

[0299] (3) Mouse serum was diluted with PBST-BSA at a ratio of 1:10 1 ~10 5 The cells were diluted in a gradient ratio and added to the sample wells, incubated at room temperature for 2 h, and washed with PBST;

[0300] (4) After adding the secondary antibody HRP-linked goat anti-mouse IgG (CW0102S), incubate at room temperature for 1 h and wash with PBST;

[0301] (5) TMB (eBioscience CA, 00-4201-56) was used for color development for 10 min, and then a stop solution (1 M H2SO4) was added to terminate the reaction. The absorbance was measured at 450 nm.

[0302] The experimental results are shown in Figure 15C.

[0303] After multiple immunizations, in a transgenic immune-tolerant animal model, the LY0016 compound vaccine can efficiently activate antigen-specific IgG, and the activation effect is comparable to that of the compound vaccine with the same prescription prepared using PEG2000-DSPE excipients.

[0304] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the scope of protection of the present invention.

Claims

1. An amphiphilic polymer LY0016, whose chemical structure is shown in the following formula In the formula, n is an integer of 35-55, preferably, n is an integer of 40-50, and most preferably, n=45.

2. The method for preparing the amphiphilic polymer LY0016 according to claim 1, characterized in that: The synthetic route is shown in the following reaction formula The specific steps are: (1) Synthesis of intermediate 1 Polyethylene glycol and PNC (p-nitrophenyl chloroformate) are used as substrates, mixed under the protection of inert gas in the environment of DCM (dichloromethane) and TEA (triethylamine), and reacted for 1 to 3 hours at room temperature with stirring. After the reaction is completed, the mixture is filtered, concentrated, and washed in sequence, and filtered under reduced pressure to obtain intermediate 1; Preferably, polyethylene glycol (molecular weight 2000): PNC (p-nitrophenyl chloroformate) = 1:2 (molar ratio); the polymer dispersity of the polyethylene glycol raw material should be ≤1.05; (2) Synthesis of Intermediate 2 Intermediate 1, L-tert-butyl glutamate is used as a substrate, TEA (triethylamine) and DMF (dimethylformamide) are mixed under the protection of an inert gas, and stirred at room temperature for 15-20 hours. After the reaction is complete, the mixture is washed with water, extracted with an organic phase, concentrated, and washed with an organic phase, and filtered under reduced pressure to obtain intermediate 2; Preferably, intermediate 1: L-tert-butyl glutamate = 1:2 (molar ratio); (3) Synthesis of Intermediate 3 Intermediate 2, distearoylglycerol, DMAP (4-dimethylaminopyridine) as substrate, HATU as condensation agent, DCM (dichloromethane) as solvent, after mixing at room temperature, stirring at room temperature for 4-6 hours, after the reaction is complete, washing with acid water, extracting with organic solvent, washing with saturated brine, drying with anhydrous sodium sulfate, and then filtering and concentrating; The product fractions were collected and purified by silica gel column chromatography, and concentrated and dried to obtain intermediate 3; Preferably, intermediate 2: distearoylglycerol:DMAP=1:1.5:2.5 (molar ratio); silica gel column chromatography purification using gradient elution, the eluent gradient is DCM:MeOH=50-20:1; (4) Synthesis of product LY0016 Intermediate 3 is the substrate, TFA is the cleavage agent, and DCM is the solvent. After mixing, stir at room temperature for 4 to 6 hours. After the reaction is complete, concentrate at 35°C, add DCM to the concentrate to dissolve, and adjust the pH to alkaline; After separating the organic phase, saturated brine was added and stirring was continued for 15 min. The organic phase was washed with 0.5N hydrochloric acid and saturated brine, and dried over anhydrous sodium sulfate. After concentrating under reduced pressure, MTBE was added, heated to 40°C and stirred to dissolve, then slowly cooled to 10°C, crystallized, filtered, rinsed with MTBE, and dried to obtain the final product LY0016.

3. The following applications of the amphiphilic polymer LY0016 according to claim 1: (1) preparing drug-loaded micelles loaded with small molecule drugs, wherein the small molecule drugs are small molecule compounds with a hydrophobic core, including but not limited to small molecule compounds with an anthracycline core, paclitaxel and paclitaxel derivatives, and vinca alkaloid compounds; Preferably, the small molecule drug is selected from paclitaxel, docetaxel, doxorubicin, topotecan, posaconazole, tacrolimus, amphotericin B, mepartricin, TLR4 agonist, and MPLA; (2) preparing a drug-loaded complex loaded with a polypeptide or polypeptide analog; Preferably, the polypeptide analog is selected from bortezomib, carfilzomib, cyclosporine, caspofungin, anidulafungin; Preferably, the polypeptide is a polypeptide with a length of less than 150 amino acids, insulin, GLP-1 and its analogs; (3) Co-assembling with a protein having a higher-order structure to prepare a LY0016-protein assembly, wherein the dissociation constant (Kd value) of the protein and the amphiphilic polymer LY0016 after assembly is less than 10 -6 M; Preferably, the protein is: cytokine, antibody; More preferably, the cytokines are from the interleukin family, colony stimulating factor family, erythropoietin family, and growth hormone family, and the antibodies are from the IgG type and IgA type. (4) preparing a composite vaccine, wherein the composite vaccine comprises: The amphiphilic polymer LY0016 as a backbone carrier, antigenic peptide, and immune adjuvant; Preferably, the immune adjuvant is MPLA; Preferably, the antigen polypeptide is a recombinant antigen polypeptide with a length of less than 200 amino acids; More preferably, the antigen polypeptide is a recombinant antigen polypeptide with a length of 10 to 150 amino acids; Most preferably, the antigen polypeptide is a recombinant antigen polypeptide with a length of 20 to 80 amino acids.

4. A pharmaceutical preparation comprising the amphiphilic polymer LY0016 according to claim 1, wherein the pharmaceutical preparation comprises: (1) 50% by weight or more of the amphiphilic polymer LY0016, preferably 70% by weight or more; (2) pharmaceutically active molecules, including but not limited to small molecule compounds, peptide analogs, polypeptides or antigens, proteins, antibodies, and immune adjuvants; Preferably, the small molecule drug is a small molecule compound with a hydrophobic core, the polypeptide or antigen is a recombinant antigen polypeptide with a length of less than 200 amino acids, the protein is a cytokine or an antibody, and the immune adjuvant is MPLA.

5. The pharmaceutical preparation according to claim 4, characterized in that The pharmaceutical preparation further comprises: (3) Other necessary pharmaceutical excipients, including but not limited to pH buffers, lyoprotectants, stabilizers, and excipients.

6. The pharmaceutical preparation according to claim 4 or 5, characterized in that The pharmaceutical preparation is an intravenous administration type pharmaceutical preparation or an intramuscular injection or subcutaneous administration type pharmaceutical preparation.

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