Nano material based on supramolecular polypeptide-drug coupled prodrug as well as preparation method and application of nano material

Abstract

The invention discloses a supramolecular polypeptide-drug coupled prodrug nano material for treating multiple myeloma as well as a preparation method and application of the supramolecular polypeptide-drug coupled prodrug nano material. According to the nano material, hyaluronic acid modified by beta-cyclodextrin is taken as a skeleton, and a melphalan-dexamethasone double-drug coupling compound coupled with an adamantane modified GFLG short peptide connexon is loaded through a supramolecular host-guest effect. The nano material has good stability and drug entrapment capacity under physiological conditions, and can realize quick-response drug release under the condition of simulating an enzymatic environment in myeloma cells. The nano material can realize efficient cellular uptake and specific drug activated release, so that proliferation of multiple myeloma cells is inhibited, and a new strategy for delivery of the multiple myeloma nano drug with a synergistic treatment effect is provided.

Description

Technical Field

[0001] This invention relates to the field of drug delivery system technology, specifically to a nanomaterial based on supramolecular peptide-drug conjugated prodrug, its preparation method, and its application. Background Technology

[0002] Multiple myeloma is a hematologic malignancy originating from the malignant clonal proliferation of plasma cells. Clinically, it often presents with bone marrow infiltration, osteolytic destruction, hypercalcemia, renal impairment, and a chronic inflammatory microenvironment. Currently, it remains difficult to cure and has a high relapse rate and mortality rate. Existing treatments include proteasome inhibitors, immunomodulators, monoclonal antibodies, CAR-T cell therapy, and bispecific antibodies. While these have significantly improved patient survival, high-dose chemotherapy combined with autologous hematopoietic stem cell transplantation remains one of the first-line treatment options for eligible transplant recipients. Among these, the alkylating agent melphalan and the glucocorticoid dexamethasone are still widely used.

[0003] However, the use of combined chemotherapy with melphalan and dexamethasone in clinical treatment has significant limitations:

[0004] (1) It lacks tumor tissue specificity and is easily distributed throughout the body, leading to systemic toxicity such as bone marrow suppression and immunosuppression;

[0005] (2) When the two drugs are used in combination in clinical practice, the pharmacokinetic parameters of the two drugs are significantly different in vivo, making it difficult to maintain the preset synergistic ratio at the myeloma lesion site;

[0006] (3) Traditional small molecule drugs lack specific activation mechanisms and are difficult to release on demand according to the differences in the tumor microenvironment.

[0007] To mitigate the toxicity associated with melphalan, studies have developed melphalan-based peptide-drug conjugate prodrugs, such as Melflufen, which utilize peptide linkers cleavable by aminopeptidase to enhance tumor uptake and promote intracellular release of melphalan. However, these products have been withdrawn from the market due to safety concerns and insufficient survival benefits, indicating that single small-molecule prodrug strategies still have platform limitations and a greater need for more robust and controllable delivery systems.

[0008] Nanomedicine delivery systems are considered an important direction for improving chemotherapy in multiple myeloma due to their advantages such as improving drug circulation time, enhancing accumulation in myeloma lesions, and reducing non-specific toxicity. Hyaluronic acid, a polysaccharide with good biocompatibility and degradability, can specifically bind to the CD44 receptor, which is highly expressed in various tumor cells, making it a commonly used framework for constructing actively targeted nanomedicines. On the other hand, reversible nanoassembly systems constructed using host-guest supramolecular recognition, such as β-cyclodextrin / adamantine, can achieve efficient loading and stimulus-responsive release without altering the drug core structure, providing a new platform for precision chemotherapy.

[0009] Therefore, it is necessary to provide a nanosystem based on the supramolecular framework of hyaluronic acid-β-cyclodextrin that enables efficient loading of peptide-drug conjugated prodrugs and tumor-specific activation, while precisely co-delivering melphalan and dexamethasone to improve the efficacy of multiple myeloma treatment and reduce systemic toxicity. Summary of the Invention

[0010] This invention overcomes the shortcomings of existing technologies by providing a nanomaterial based on supramolecular peptide-drug conjugated prodrugs, its preparation method, and applications. This material utilizes the strong affinity of hyaluronic acid for the CD44 receptor on the surface of multiple myeloma cells to achieve precise accumulation in lesions. Through prodrug molecule design and recognition, it ensures the co-delivery of melphalan and dexamethasone in a 1:1 ratio. The material releases the drug on demand under intracellular enzyme triggering, exhibiting significant synergistic and anti-inflammatory effects in the treatment of multiple myeloma. Clinical application holds promise for effectively alleviating bone destruction and increasing bone mineral density while powerfully inhibiting tumors, achieving precise diagnosis and treatment and bone protection. This invention addresses the following technical problems existing in current melphalan and dexamethasone combination chemotherapy: high drug toxicity, imbalanced drug ratios, and poor targeting.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a nanomaterial based on a supramolecular peptide-drug conjugated prodrug, the nanomaterial comprising a host polymer scaffold and a guest peptide-drug conjugated prodrug loaded thereon:

[0013] The polypeptide-drug conjugated prodrug molecule has the structure shown in formula (I), which is composed of an adamantane unit, a melphalan unit, an enzyme-responsive polypeptide linker GFLG, and a dexamethasone unit linked by covalent bonds; in formula (I), n is an integer from 1 to 6, and m is an integer from 1 to 5.

[0014]

[0015] Preferably, the polypeptide-drug conjugate prodrug molecule has the following structure:

[0016]

[0017] The main polymer scaffold is β-cyclodextrin-modified hyaluronic acid (HA-β-CD), which has the structure shown in formula (II);

[0018]

[0019] The host polymer scaffold and the guest dual-drug conjugate molecules achieve loading through host-guest interactions between β-cyclodextrin and adamantane units.

[0020] As a preferred option:

[0021] In the main polymer scaffold HA-β-CD, the molecular weight of hyaluronic acid is preferably 20-40 kDa, and more preferably 34 kDa;

[0022] The degree of substitution of the β-cyclodextrin on the hyaluronic acid backbone is preferably 15%-20%, more preferably 18%;

[0023] The nanomaterials are formed by self-assembly in an aqueous phase, and their particle size is preferably 80-100 nm.

[0024] Secondly, the present invention provides a method for preparing the above-mentioned nanomaterials, specifically comprising: synthesizing and preparing a peptide-drug conjugate prodrug, and then preparing an aqueous solution of HA-β-CD and an organic solvent solution of the peptide-drug conjugate prodrug respectively; under ultrasonic conditions, adding the peptide-drug conjugate prodrug solution dropwise into the HA-β-CD aqueous solution; and after the mixture is allowed to stand and dialyzed to remove the organic solvent, the nanomaterials are obtained.

[0025] Preferably, the preparation of the polypeptide-drug conjugate prodrug includes the following steps:

[0026] S1. According to the following reaction formula, melphalan (CAS No.: 148-82-3) is reacted with di-tert-butyl dicarbonate in the presence of a base catalyst to obtain the compound shown in formula (A1).

[0027]

[0028] S2. According to the following reaction formula, the compound shown in formula (A1) and the compound shown in formula (A2) are reacted in the presence of a condensing agent and a basic reagent under the action of a catalyst, so that the carboxyl group in formula (A1) and the hydroxyl group in formula (A2) undergo a condensation reaction to obtain the compound shown in formula (A3).

[0029]

[0030] S3. According to the following reaction formula, the compound shown in formula (A3) is subjected to an amino deprotection reaction in the presence of a deprotection reagent to convert it into a reactive free amino intermediate, namely the compound shown in formula (A4).

[0031]

[0032] S4. According to the following reaction formula, the compound shown in formula (B) is coupled with p-aminobenzyl alcohol in the presence of a condensing agent to form an amide bond between the carboxyl group in the compound shown in formula (B) and the amino group in p-aminobenzyl alcohol, so as to obtain the compound shown in formula (B1).

[0033]

[0034] S5. According to the following reaction formula, the compound shown in formula (B1) is reacted with p-nitrophenyl chloroformate in the presence of a basic reagent, so that the hydroxyl group in the compound shown in formula (B1) reacts with p-nitrophenyl chloroformate to form a carbonate bond, thereby obtaining the compound shown in formula (B2).

[0035]

[0036] S6. According to the following reaction formula, the compound shown in formula (B2) and the compound shown in formula (A4) are coupled in the presence of a basic reagent to form an urethane bond between the active carbonate group in the compound shown in formula (B2) and the amino group in the compound shown in formula (A4), thereby obtaining the compound shown in formula (B3).

[0037]

[0038] S7. According to the following reaction formula, dexamethasone is reacted with the compound shown in formula (D) in the presence of a basic reagent, so that the hydroxyl group in dexamethasone reacts with the compound shown in formula (D) to form an ester bond, and the compound shown in formula (C1) is obtained.

[0039]

[0040] S8. According to the following reaction formula, the compound shown in formula (B3) is deprotected by an amino protecting group in the presence of a deprotecting agent. The resulting product is then coupled with the compound shown in formula (C1) in the presence of a condensing agent, causing the amino group in the deprotected product (B3) to form an amide bond with the carboxyl group in formula (C1), yielding the compound shown in formula (I).

[0041]

[0042] In the compounds shown in formulas (A2), (A3), (A4), (B3), and (I), n is an integer from 1 to 6. In the compounds shown in formulas (D) and (I), m is an integer from 1 to 5.

[0043] As a preferred option:

[0044] The concentration of the HA-β-CD aqueous solution is preferably 0.1-1.0 mM;

[0045] The organic solvent is preferably dimethyl sulfoxide (DMSO).

[0046] This invention uses β-cyclodextrin-modified hyaluronic acid as the drug carrier backbone, which possesses good biosafety and biodegradability, meeting the prerequisite requirement that drug delivery materials must be safe. This invention uses peptide-drug conjugates as the main building blocks, which feature precise fixation of drug molar ratios and dual enzyme-responsive release. This offers the advantages of synergistic effects in dual-drug combination therapy, as well as reduced systemic toxicity and improved drug cycling stability. Through supramolecular host-guest recognition between the β-cyclodextrin units on the carrier backbone and the adamantane units on the peptide-drug conjugates, the molecules spontaneously assemble in an aqueous phase, resulting in nanomedicines with uniform particle size and spherical morphology. Through a CD44 receptor-mediated tumor-targeting enrichment and precise intracellular enzymatic activation mechanism, it provides potent inhibition of tumor growth, regulation of the inflammatory microenvironment, and significant alleviation of bone destruction, potentially improving the precision and overall clinical benefits of multiple myeloma treatment.

[0047] More preferably, the base catalyst in step S1 is selected from triethylamine, pyridine, and diisopropylethylamine.

[0048] More preferably, in step S2, the condensing agent is selected from carbodiimide condensing agents, preferably from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; the alkaline reagent is selected from amine bases, preferably from triethylamine, pyridine, or diisopropylethylamine; and the catalyst is selected from 4-dimethylaminopyridine, diisopropylethylamine, or N-methylimidazole.

[0049] More preferably, the deprotecting agent in the deprotection reaction in step S3 is selected from trifluoroacetic acid, hydrochloric acid, hydrobromic acid, or a combination thereof.

[0050] More preferably, in step S4, the condensing agent is selected from carbodiimide condensing agents, preferably from N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide, or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and the reaction is carried out in the presence of an auxiliary agent selected from 1-hydroxybenzotriazole or a derivative thereof.

[0051] More preferably, in step S4, the alkaline reagent is selected from organic bases, and more preferably from one or more of pyridine, triethylamine or N,N-diisopropylethylamine.

[0052] More preferably, in step S6, the alkaline reagent is selected from organic bases, and more preferably from one or more of N,N-diisopropylethylamine, triethylamine, pyridine or 4-dimethylaminopyridine.

[0053] More preferably, in step S7, the alkaline reagent is selected from organic bases, and more preferably from one or more of N,N-diisopropylethylamine, triethylamine, pyridine or 4-dimethylaminopyridine.

[0054] More preferably, in step S8, the deprotecting agent is selected from amine basic reagents, preferably piperidine; the condensing agent is selected from one or more of quinoline condensing agents, carbodiimide condensing agents, onium salt condensing agents or active ester condensing agents, preferably 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.

[0055] Thirdly, the present invention provides the application of the above-mentioned nanomaterials in the preparation of drugs for treating multiple myeloma.

[0056] The supramolecular peptide-drug conjugated prodrug nanomaterials have the effects of targeting multiple myeloma lesions, releasing drugs on demand, inhibiting tumor growth, regulating the inflammatory microenvironment, and alleviating bone destruction.

[0057] Compared with the prior art, the beneficial effects of the present invention are:

[0058] This invention discloses a supramolecular peptide-drug conjugated prodrug nanomaterial. This nanomaterial uses hyaluronic acid (HA-β-CD), which has good CD44 receptor targeting, as its material backbone. Simultaneously, it designs and synthesizes conjugated prodrug molecules containing adamantane units, GFLG enzyme-responsive peptides, and melphalan and dexamethasone dual-drug units. The peptide-drug conjugated prodrug is loaded onto the carrier backbone via host-guest recognition and prepared through self-assembly. This nanomaterial can achieve precise drug release under the triggering condition of overexpressed enzymes (cathepsin B and esterase) in multiple myeloma cells, ensuring a synergistic effect of the two drugs at a fixed 1:1 molar ratio, thereby effectively killing tumor cells. It also significantly alleviates bone destruction and regulates the tumor inflammatory microenvironment. Furthermore, the preparation steps of this supramolecular nanoprodrug are simple, the drug loading ratio is precisely controllable, and the encapsulation efficiency is high. It possesses the potential for both bone protection and therapeutic integration, and has broad application prospects in the precision treatment of hematological malignancies such as multiple myeloma. Attached Figure Description

[0059] Figure 1 The results are from the proton NMR spectrum of the peptide-drug conjugated prodrug molecule.

[0060] Figure 2 The results are from the carbon NMR spectrum of a peptide-drug conjugated prodrug molecule.

[0061] Figure 3 The results are high-resolution mass spectrometry measurements of peptide-drug conjugated prodrug molecules.

[0062] Figure 4 The image shows the transmission electron microscopy results of nanomaterials based on supramolecular peptide-drug conjugated prodrugs.

[0063] Figure 5 The kinetic curves for the release of dexamethasone from nanomaterials based on supramolecular peptide-drug conjugated prodrugs in the presence of cathepsin B and esterases are shown.

[0064] Figure 6 The figure shows the cytotoxicity results of nanomaterials based on supramolecular peptide-drug conjugated prodrugs and the control group on multiple myeloma cells MM1S. Detailed Implementation

[0065] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0066] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0067] Example 1: Preparation of nanomaterials based on supramolecular peptide-drug conjugated prodrugs

[0068] 1. Synthesis of peptide-drug conjugated prodrugs

[0069] (1) As shown in the following reaction formula, melphalan (0.98 g) and triethylamine (1.73 mL) were dissolved in methanol (50 mL), and di-tert-butyl dicarbonate (1.25 g) was added under stirring. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The resulting oily residue was washed with hydrochloric acid solution (concentration of 0.01 mol / L) and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate as eluent) to obtain the solid product, compound A1.

[0070]

[0071] (2) As shown in the following reaction formula, compound A1 (117 mg), DMAP (14.1 mg), 1-adamantaneethanol (52.1 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (110.6 mg), and triethylamine (58.8 mg) were dissolved in dichloromethane and mixed under ice bath stirring. The reaction was then continued to be stirred at room temperature for 24 hours. After the reaction was completed, the reaction solution was poured into water and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using petroleum ether / ethyl acetate (volume ratio 10:1) as eluent to obtain compound A3.

[0072]

[0073] (3) As shown in the following reaction formula, compound A3 (360 mg) was dissolved in anhydrous dichloromethane, trifluoroacetic acid was added, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain compound A4.

[0074]

[0075] (4) As shown in the following reaction formula, compound B (1080 mg), 1-hydroxybenzotriazole (475.7 mg), and N,N'-diisopropylcarbodiimide (444.2 mg) were dissolved in N,N-dimethylformamide under nitrogen protection and stirred for 5 minutes. 4-aminobenzyl alcohol (433.5 mg) dissolved in N,N-dimethylformamide was added to the mixture, and the mixture was stirred overnight under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove N,N-dimethylformamide. Dichloromethane was added, and a precipitate formed. After filtration, a yellow solid compound B1 was obtained.

[0076]

[0077] (5) As shown in the following reaction formula, compound B1 (450 mg) was dissolved in anhydrous tetrahydrofuran and cooled to 0°C under nitrogen protection. Pyridine (246.6 mg) was then added, followed by slow dropwise addition of anhydrous tetrahydrofuran solution containing 4-nitrophenyl chloroformate (630 mg). After the addition was complete, the reaction mixture was stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the resulting crude product was dissolved in dichloromethane and washed with 1 M potassium hydrogen sulfate aqueous solution. The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by rapid column chromatography using a dichloromethane / methanol gradient (volume ratio from 100:1 to 50:1) to give a white solid compound B2.

[0078]

[0079] (6) As shown in the following reaction formula, compound A4 (350 mg) was dissolved in tetrahydrofuran and cooled to 0°C under nitrogen protection. Then, N,N-diisopropylethylamine (569.9 mg) and compound 5 (390 mg) were added, and the reaction mixture was slowly heated to room temperature and stirred overnight. After the reaction was complete, the solvent was removed under reduced pressure, the residue was diluted with ethyl acetate, and washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography, eluting with a dichloromethane / methanol gradient (volume ratio from 100:1 to 50:1) to give a white solid compound B3.

[0080]

[0081] (7) As shown in the following reaction formula, dexamethasone (500 mg) was dissolved in a mixed solvent of N,N-dimethylformamide / N,N-dimethylacetamide (volume ratio 8:2, 10 mL), and stirred under nitrogen protection and an ice bath. Separately, succinic anhydride (192 mg) was dissolved in a mixed solvent of N,N-dimethylformamide / N,N-dimethylacetamide (volume ratio 8:2, 5 mL), and triethylamine (250 μL) was added. After stirring at 0°C for 3 hours, the reaction mixture was transferred to room temperature and stirred for another 36 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography, eluting with a gradient of ethyl acetate / methanol (volume ratio from 200:1 to 100:1) to give a white solid product, compound C1.

[0082]

[0083] (8) As shown in the following reaction formula, compound B3 was dissolved in N,N-dimethylformamide, and piperidine equivalent to 20% of the volume of N,N-dimethylformamide was added. The mixture was stirred under nitrogen protection for 24 hours. After the reaction was completed, N,N-dimethylformamide was removed by vacuum distillation, and n-hexane was added to precipitate the product. After filtration, a yellow solid was obtained. This solid was then dissolved in anhydrous dichloromethane with compound C1 (342.8 mg) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (344.2 mg). The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography. The residue was eluted with a dichloromethane / methanol gradient (volume ratio from 100:1 to 50:1) to obtain a white solid final product, namely the peptide-drug conjugate prodrug molecule.

[0084]

[0085] The final synthesized peptide-drug conjugate prodrug molecule structure was verified to be correct by characterization results of proton NMR spectroscopy, carbon NMR spectroscopy, and high-resolution mass spectrometry.

[0086] The specific 1H NMR spectrum results are as follows: 1 H NMR (600 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.12(s, 3H), 8.05 (s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.59 (s, 2H), 7.23 (s, 7H),7.05 (s, 2H), 6.64 (s, 2H), 6.23 (s, 1H), 6.01 (s, 1H), 5.41 (s, 1H), 5.16(s, 1H), 5.01 (s, 1H), 4.93 (s, 2H), 4.79 (s, 1H), 4.54 (s, 1H), 4.31 (s,1H), 4.13 (s, 1H), 4.04 (s, 2H), 3.87 (s, 2H), 3.69 (s, 8H), 3.59 (d, J =21.7 Hz, 1H), 3.48 (s, 1H), 3.06 (s, 1H), 2.87 (s, 2H), 2.76 (s, 2H), 2.59(s, 3H), 2.33 (s, 3H), 2.15 (s, 2H), 1.89 (s, 3H), 1.76 (s, 1H), 1.63 (s,7H), 1.52 (s, 2H), 1.48 (s, 3H), 1.44 (s, 4H), 1.37 (s, 2H), 1.24 (s, 3H), 1.03 (s, 1H), 0.89 (d, J = 22.9 Hz, 9H), 0.79 (s, 3H).

[0087] The specific carbon NMR spectrum results are as follows: 13C NMR (150 MHz, DMSO-d6) δ 205.26, 185.75,172.75, 172.56, 172.32, 171.58, 171.51, 169.36, 167.99, 167.54, 156.44,153.22, 145.45, 139.01, 138.17, 130.56, 129.67, 129.46, 129.13, 128.50,126.72, 125.77, 124.57, 119.27, 112.11, 102.31, 101.15, 90.92, 68.44, 65.76,61.12, 56.57, 55.39, 54.26, 52.61, 51.81, 48.49, 48.41, 43.75, 42.47, 42.29,42.25, 41.60, 41.11, 40.52, 37.80, 36.91, 36.12, 35.83, 34.11, 32.37, 31.70,30.73, 30.08, 29.09, 28.40, 27.75, 24.57, 23.49, 23.46, 23.42, 22.12, 16.72,15.58.

[0088] The specific high-resolution mass spectrometry test results are as follows: [C] 78 H 100 N7O 15 FCl2Na2] 2+ 754.8211 (calculated value), 754.8244 (measured value).

[0089] 2. Synthesis of the main polymer scaffold HA-β-CD

[0090] As shown in the following reaction formula, mono-6-deoxy-6-ethylenediamino-β-cyclodextrin (β-CD-NH2) was used as a starting material and coupled with hyaluronic acid. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (200.3 mg) and sodium N-hydroxythiosuccinimide sulfonate (113.4 mg) were added to a solution of hyaluronic acid (120 mg, molecular weight approximately 34,000) dissolved in phosphate buffer (0.1 M, pH 7.2), and the mixture was stirred at room temperature for 30 minutes. Subsequently, β-CD-NH2 (305 mg) dissolved in phosphate buffer was added, and the reaction was continued at room temperature for 24 hours. After the reaction was completed, the resulting reaction solution was placed in a dialysis bag and dialyzed against deionized water for 5 days. After lyophilization, a white powdery main polymer scaffold HA-β-CD was obtained.

[0091]

[0092] 3. Preparation of nanomaterials based on supramolecular peptide-drug conjugated prodrugs through supramolecular host-guest self-assembly

[0093] The host polymer scaffold HA-β-CD was dissolved in ultrapure water to prepare a stock solution with a concentration of 2 mM (calculated as β-cyclodextrin). Separately, a peptide-drug conjugation prodrug was dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 2 mM. For the preparation of nanomaterials based on supramolecular peptide-drug conjugation prodrugs, the above HA-β-CD stock solution (5 mL) was diluted 20-fold with water to achieve a concentration of 100 μM. Subsequently, under ultrasonic conditions, Ada-M-GFLG-D stock solution (0.25 mL) was slowly added dropwise to the diluted HA-β-CD solution over 5 minutes to obtain a colloidal solution. This mixture was allowed to stand overnight at room temperature to ensure complete assembly. Finally, the solution was dialyzed against water for 12 hours to remove trace amounts of dimethyl sulfoxide, and the resulting nanoparticle solution was stored at 4°C for later use.

[0094] The final nanoparticle structure was characterized by transmission electron microscopy. The test results showed that the nanomaterial exhibited a uniform spherical vesicle morphology with an average particle size of about 90 nm.

[0095] Example 2: Enzyme-responsive release of nanomaterials based on supramolecular peptide-drug conjugated prodrugs

[0096] Concentrated stock solutions of nanomaterials based on supramolecular peptide-drug conjugated prodrugs were prepared with ultrapure water and diluted to a final concentration of 100 μM with different buffers to evaluate their drug release behavior under different conditions. The tested conditions included: (i) pH 5.4 acidic buffer; (ii) pH 5.4 acidic buffer with the addition of cathepsin B (100 μg / mL); (iii) pH 5.4 acidic buffer with the addition of esterase (100 μg / mL); and (iv) pH 5.4 acidic buffer with the addition of both cathepsin B and esterase (100 μg / mL each). Subsequently, release media samples were collected at predetermined time points and analyzed using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: mobile phase was methanol-water; gradient elution program was: 0–9 min maintaining methanol:water = 70:30, 9–21 min linearly changing to 95:5, and 21–30 min returning to 70:30; detection wavelength was 254 nm. The enzymatic release kinetics of dexamethasone are shown below. Figure 5As shown, in PBS buffer at pH 5.4 or in the presence of only cathepsin B, the cumulative release rate of dexamethasone from nanomaterials based on supramolecular peptide-drug conjugated prodrugs was less than 10% within 72 hours, indicating that dexamethasone is difficult to release effectively in the absence of esterase or in the presence of only cathepsin B. When only esterase was added, dexamethasone was almost completely released within 72 hours, which is consistent with the structural characteristic of dexamethasone being linked to the peptide-drug conjugated prodrug molecule via a single ester bond. However, under the condition of co-existence of cathepsin B and esterase, the cumulative release rate of dexamethasone increased rapidly and reached near-complete release within 72 hours. The enzyme-responsive release kinetics of melphalan are shown below. Figure 6 As shown, melphalan release exhibits a significant dual-enzyme synergistic response. In the presence of only esterase or only cathepsin B, its cumulative release rate over 72 hours is less than 10%, indicating that either esterase or cathepsin B alone cannot effectively trigger melphalan release. Only when both cathepsin B and esterase are present does melphalan release initiate rapidly and reach near-complete release within 72 hours.

[0097] Example 3: Cytotoxicity experiment of nanomaterials based on supramolecular peptide-drug conjugated prodrugs against multiple myeloma cells MM1S.

[0098] Cytotoxicity of the samples was assessed using the CCK-8 assay. MM1S cells were seeded at a density of 1 × 10³ cells per well in 96-well plates and cultured for 24 hours to allow cell adhesion. Subsequently, the cells were treated with PBS, dexamethasone, melphalan, a physical mixture of dexamethasone and melphalan (DEX+MA), or nanomaterials based on supramolecular peptide-drug conjugated prodrugs, at concentrations ranging from 0.0001 to 100 µM (in 10-fold increments). After 48 hours of further culture, the culture medium was discarded and the wells were washed with PBS. 100 µL of DMEM solution containing 10% CCK-8 reagent was added to each well, and after incubation for 1 hour, the absorbance was measured at 450 nm.

[0099] Test results are as follows Figure 6 As shown, the nanomaterials based on supramolecular peptide-drug conjugated prodrugs exhibited significantly better in vitro cytotoxicity against MM1S cells than the other groups.

[0100] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing nanomaterials based on supramolecular peptide-drug conjugated prodrugs, characterized in that, The nanomaterials include: (1) HA-β-CD is a polymeric carrier whose main chain is hyaluronic acid and whose glycoside units are covalently grafted with β-cyclodextrin side chains; (2) A peptide-drug conjugated prodrug compound is loaded onto the HA-β-CD via host-guest interaction; the structure of the peptide-drug conjugated prodrug compound is shown in formula (I); The compound of formula (I) includes a Gly-Phe-Leu-Gly (GFLG) tetrapeptide linker group, a mefarin residue linked to one end of GFLG and a dexamethasone derivative residue linked to the other end of GFLG, and an adamantyl group is introduced into the mefarin portion. In formula (I), n is an integer from 1 to 6 and m is an integer from 1 to 5. The HA-β-CD and the compound of formula (I) self-assemble in an aqueous phase to form nanoparticles with a particle size of 50-200 nm.

2. The method for preparing nanomaterials based on supramolecular peptide-drug conjugated prodrugs according to claim 1, characterized in that, The compound of formula (I) is prepared by the following steps: (1) Reaction of melphalan with ditert-butyl dicarbonate in the presence of an alkaline catalyst yields the compound shown in formula (A1); (2) The compound shown in formula (A1) and the compound shown in formula (A2) are reacted in the presence of a condensing agent and a basic reagent under the action of a catalyst, so that the carboxyl group in formula (A1) and the hydroxyl group in formula (A2) undergo a condensation reaction to obtain the compound shown in formula (A3). (3) The compound shown in formula (A3) is subjected to an amino deprotection reaction in the presence of a deprotection reagent to convert it into a reactive free amino intermediate, namely the compound shown in formula (A4). (4) The compound shown in formula (B) is coupled with p-aminobenzyl alcohol in the presence of a condensing agent to form an amide bond between the carboxyl group in the compound shown in formula (B) and the amino group in p-aminobenzyl alcohol, to obtain the compound shown in formula (B1). (5) The compound shown in formula (B1) is reacted with p-nitrophenyl chloroformate in the presence of an alkaline reagent, so that the hydroxyl group in the compound shown in formula (B1) reacts with p-nitrophenyl chloroformate to form a carbonate bond, thereby obtaining the compound shown in formula (B2). (6) The compound shown in formula (B2) and the compound shown in formula (A4) are coupled in the presence of a basic reagent to form an urethane bond between the active carbonate group in the compound shown in formula (B2) and the amino group in the compound shown in formula (A4) to obtain the compound shown in formula (B3). (7) Dexamethasone (C) was reacted with the compound shown in formula (D) in the presence of a basic reagent to form an ester bond with the hydroxyl group in dexamethasone and the compound shown in formula (D) to obtain the compound shown in formula (C1). (8) The compound shown in formula (B3) is deprotected in the presence of a deprotecting agent, and then the resulting product is coupled with the compound shown in formula (C1) in the presence of a condensing agent to form an amide bond between the amino group in the deprotected product of formula (B3) and the carboxyl group in formula (C1) to obtain the compound shown in formula (I). The structures of the compounds shown in the above reaction steps and formulas (A1), (A2), (A3), (A4), (B), (B1), (B2), (B3), and (C1) are as follows: In the compounds shown in formulas (A2), (A3), (A4), (B3), and (I), n is an integer from 1 to 6; in the compounds shown in formulas (D) and (I), m is an integer from 1 to 5.

3. The preparation method according to claim 2, characterized in that, In step (1), the base catalyst is selected from triethylamine, pyridine, and diisopropylethylamine.

4. The preparation method according to claim 2, characterized in that, In step (2), the condensing agent is selected from carbodiimide condensing agents; the carbodiimide condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; the basic reagent is selected from amine bases; the amine base is one of triethylamine, pyridine, or diisopropylethylamine; the catalyst is one of 4-dimethylaminopyridine, diisopropylethylamine, or N-methylimidazole. In step (3), the deprotection reagent is selected from one or more of trifluoroacetic acid, hydrochloric acid, and hydrobromic acid.

5. The preparation method according to claim 2, characterized in that, In step (4), the condensing agent is selected from carbodiimide condensing agents, specifically N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide, or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the coupling reaction is carried out in the presence of an auxiliary agent selected from 1-hydroxybenzotriazole or its derivatives.

6. The preparation method according to claim 2, characterized in that, In step (5), the alkaline reagent is selected from organic bases, and the organic base is one or more of pyridine, triethylamine or N,N-diisopropylethylamine; In step (6), the alkaline reagent is selected from organic bases, and the organic base is one or more of N,N-diisopropylethylamine, triethylamine, pyridine or 4-dimethylaminopyridine.

7. The preparation method according to claim 2, characterized in that, In step (7), the alkaline reagent is selected from organic bases, and the organic base is one or more of N,N-diisopropylethylamine, triethylamine, pyridine or 4-dimethylaminopyridine; The dexamethasone derivative shown in formula (D) is a compound with a carboxyl group obtained by modifying dexamethasone with succinic anhydride or other cyclic anhydrides. In step (8), the deprotecting agent is selected from amine basic reagents, and the amine basic reagent is piperidine; the condensing agent is selected from one or more of quinoline condensing agents, carbodiimide condensing agents, onium salt condensing agents or active ester condensing agents, and the condensing agent is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline.

8. The preparation method according to claim 1, characterized in that, Specifically, the following steps are included: Prepare an aqueous solution of HA-β-CD with a concentration of 0.1-1.0 mM; under ultrasonic conditions, add a solution of a polypeptide-drug conjugate prodrug compound dissolved in an organic solvent dropwise to the HA-β-CD solution; allow to stand at room temperature and dialyze to remove the organic solvent to obtain a dispersion of the nanosystem.

9. A nanomaterial based on a supramolecular polypeptide-drug conjugated prodrug prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The nanomaterial uses β-cyclodextrin-modified hyaluronic acid as the drug carrier backbone, and the polypeptide-drug conjugate prodrug is loaded within the drug carrier backbone. The nanomaterial has a uniform particle size and a spherical morphology. The particle size of the nanomaterial is 90±3 nm.

10. The use of the nanomaterial of claim 9 in the preparation of a drug for treating multiple myeloma.

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