An amphiphilic polymer-drug conjugate, and a preparation method and application thereof

Abstract

The application discloses an amphiphilic polymer-drug conjugate and a preparation method and application thereof, the conjugate is formed by covalent combination of an amphiphilic polymer and an amino-containing drug through an amide bond, the amphiphilic polymer is prepared by amidation reaction of poly(maleic anhydride-alt-1-octadecene) and methoxypolyethylene glycol amine, and the amino-containing drug is preferably doxorubicin or ampicillin. The conjugate self-assembles to form spherical micelles with a particle size of 30-50 nm in an aqueous medium, has a pH response release characteristic, the drug release rate under an acidic condition is significantly higher than that under a neutral condition, and has bacteriostatic activity on escherichia coli. The polymer-drug conjugate of the application significantly improves the solubility and stability of a poorly soluble drug, realizes pH controlled release, and has a simple and controllable preparation method, good biocompatibility and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine delivery technology, specifically relating to an amphiphilic polymer-drug conjugate, its preparation method, and its application. Background Technology

[0002] Poorly soluble drugs face challenges in clinical application due to low solubility, poor bioavailability, and instability. These drugs, due to their strong hydrophobicity, high molecular weight, or stable crystal form, exhibit low dissolution rates and poor bioavailability, severely limiting their clinical application. They also present safety and tolerability issues. Doxorubicin (DOX) is a broad-spectrum anthracycline antitumor drug effective against various tumors, including breast cancer, lymphoma, and ovarian cancer. However, its water solubility is extremely low (only 0.51 mg / mL at pH 7.4), requiring the use of organic solvents such as polysorbate 80 for intravenous injection, which can easily lead to cardiotoxicity, allergic reactions, and other side effects. Furthermore, DOX is prone to photolysis and hydrolysis in aqueous solutions, resulting in poor stability. Ampicillin (AMP) is a broad-spectrum β-lactam antibiotic with antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it has poor water solubility, exhibiting very low solubility in pure water, ethanol, and methanol, with an oral bioavailability of only 30%-50%. Furthermore, the β-lactam ring of AMP is sensitive to acids and bases, and is easily degraded under conditions of pH < 5.5 or pH > 8, resulting in poor formulation stability. To improve the solubility of poorly soluble drugs, researchers have developed various strategies, such as reducing particle size, salt formation, cyclodextrin inclusion, liposomes, and polymer micelles. Among these, amphiphilic polymer carriers have attracted much attention due to their advantages such as self-assembly to form micelles, improved drug solubility, and extended cycle time. However, most existing technologies use physical encapsulation methods to load drugs, which suffer from problems such as low drug loading, easy drug leakage, and insufficient stability. Chemical bonding (covalent coupling) strategies directly link drugs to the polymer backbone, which can significantly improve drug loading stability and controlled release performance. However, efficient coupling methods for different drugs still need to be explored, especially for drug molecules with different structures and reactivity. Achieving high drug loading and high stability coupling remains a technical challenge. Summary of the Invention

[0003] To overcome the shortcomings of poorly soluble drugs in the prior art, such as low solubility, poor stability, and insufficient bioavailability, the present invention aims to provide an amphiphilic polymer-drug conjugate, its preparation method, and its application.

[0004] The method for preparing amphiphilic polymer-drug conjugates provided by the present invention involves linking the amino group of the drug molecule with the carboxyl group of the polymer side chain (formed by ring opening of acid anhydride) through amidation.

[0005] The specific technical solution is as follows: A method for preparing an amphiphilic polymer-drug conjugate, wherein the conjugate is prepared by a segmented pH control method, comprising first activating the carboxyl groups of the side chains of the amphiphilic polymer under acidic conditions of pH=3.0-6.5, and then covalently binding the activated carboxyl groups with an amino-containing drug through amide bonds under conditions of pH=6.5-8.5. The amphiphilic polymer was prepared by amidation reaction of poly(maleic anhydride-alt-1-octadecene) (PMAO) and methoxy polyethylene glycolamine (mPEG-NH2).

[0006] Specifically, the steps include the following: 1) Poly(maleic anhydride-alt-1-octadecene) and methoxy polyethylene glycolamine were placed in an organic solvent and subjected to an amidation reaction in the presence of a condensing agent. After the reaction was completed, the mixture was purified and dried to obtain the amphiphilic polymer PMAO-PEG. 2) Dissolve the amphiphilic polymer PMAO-PEG obtained in step 1) in an organic solvent, adjust the pH of the solution to 3.0-6.5 with acid or base to activate the carboxyl groups on the side chains of the amphiphilic polymer, add a condensing agent, stir to activate the reaction, and convert the carboxyl groups into highly reactive intermediates. 3) Dissolve or disperse the amino-containing target drug in a solvent and add it to the activation system in step 2). Adjust the pH to 6.5-8.5 with an alkaline solution (such as sodium hydroxide solution) or buffer solution to promote the nucleophilic substitution reaction. Stir the reaction under light protection (for photosensitized drugs) and inert gas protection (optional) to allow the amino group of the drug to undergo amidation reaction with the activated carboxyl group on the amphiphilic polymer to form a stable covalent bond. 4) After the reaction is complete, the amphiphilic polymer-drug conjugate is purified and dried. The purification method can be dialysis or gel column chromatography. If dialysis is used, the reaction solution should be appropriately diluted and transferred to a dialysis bag with a suitable molecular weight cutoff (MWCO). Dialyze in pure water or buffer at a low temperature (e.g., 4°C) for 10 to 30 hours, during which the external dialysis solution needs to be changed multiple times. The molecular weight cutoff of the dialysis bag should be selected according to the molecular weight of the conjugate, preferably in the range of 2000 to 13000 Da.

[0007] Further, in step 1), the molar ratio of poly(maleic anhydride-alt-1-octadecene) to methoxy polyethylene glycol amine is 1:0.1-1.2. By adjusting this ratio, the hydrophilic-hydrophobic balance of the polymer can be optimized, thereby affecting its self-assembly behavior and drug loading capacity. The number average molecular weight of mPEG-NH2 is preferably 1000-20000 Da, more preferably 1000-10000 Da, and most preferably 2000 Da. The organic solvent in step 1) is dichloromethane, trichloromethane, methanol, ethanol, or N,N-dimethylformamide, preferably anhydrous dichloromethane. The condensing agent in step 1) is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide. The reaction time in step 1) is 10-40 hours, preferably 20-30 hours.

[0008] Further, the organic solvent in step 2) is dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) or N-methylpyrrolidone (NMP). The organic solvent must be able to dissolve the polymer well and have no adverse effect on subsequent reactions. The condensing agent in step 2) is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the carboxyl group in the amphiphilic polymer is 1.2-3:1, preferably 2:1, and the molar ratio of N-hydroxythiosuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1-5:1, preferably 1.1-2:1. The activation time in step 2) is 5-60 minutes, preferably 30 minutes.

[0009] Further, in step 3), the amino-containing target drug is doxorubicin, ampicillin, or a pharmaceutically acceptable salt thereof; the molar ratio of the amino-containing target drug to the amphiphilic polymer is 1:1-8, preferably 1:1-4; the reaction time is 10-40 hours, and optimization has shown that a better coupling efficiency can be obtained by reacting for 24 hours.

[0010] Preferably, adjusting the solution pH to 4.0-5.5 in step 2) can effectively promote the formation of active ester intermediates by carboxyl groups and condensing agents, while avoiding degradation of polymers or drugs in an overly acidic environment; adjusting the pH to 6.5-7.5 in step 3) is beneficial for the deprotonation of amino groups to enhance their nucleophilicity, and can also ensure the stability of reaction intermediates and the structural integrity of drugs.

[0011] An amphiphilic polymer-drug conjugate prepared by the above preparation method.

[0012] A nanomicelle formulation is formed by the self-assembly of the above-mentioned amphiphilic polymer-drug conjugate in an aqueous medium, and its particle size is 30-50 nm.

[0013] Application of an amphiphilic polymer-drug conjugate in the preparation of antitumor drugs or antibacterial drugs.

[0014] A pH-responsive drug delivery system for loading and delivering other amino-containing active molecules, comprising the aforementioned amphiphilic polymer-drug conjugate, wherein the drug release rate is higher under acidic conditions than under neutral conditions.

[0015] This invention provides an amphiphilic polymer-drug conjugate, the core of which lies in the use of poly(maleic anhydride-alt-1-octadecene) and methoxy polyethylene glycolamine via an amidation reaction to form a block copolymer carrier with a well-defined amphiphilic structure, and the optimization of hydrophilic-hydrophobic balance by controlling the molar ratio of the two components. This carrier covalently binds to amino-containing, poorly soluble drugs (such as doxorubicin and ampicillin) via amide bonds, unlike traditional physical encapsulation methods, forming a well-defined polymer-drug conjugate. The preparation method employs a unique segmented pH control strategy—first activating the carboxyl groups under acidic conditions, then carrying out the coupling reaction under near-neutral conditions. This mild process effectively protects the integrity of the drug structure. The resulting conjugate spontaneously assembles in an aqueous medium to form spherical micelles with uniform particle size and good monodispersity (PDI ≤ 0.4), and AFM-IR confirms that the drug and polymer are covalently linked and uniformly distributed within the micelles. This system exhibits significant pH-responsive release characteristics, with a drug release rate significantly higher under acidic conditions than in a neutral environment. Simultaneously, the critical micelle concentration decreases after coupling with a hydrophobic drug (from 0.005 mg / mL to 0.002 mg / mL), which is beneficial for in vivo stability and long-term circulation. This technology has been successfully applied to two structurally different, poorly soluble drugs, doxorubicin and ampicillin, demonstrating its versatility and providing a novel delivery platform for poorly soluble amino-containing drugs that combines structural stability, pH responsiveness, and preservation of biological activity.

[0016] The beneficial effects of this invention are as follows: 1) Covalent coupling enhances stability: By covalently linking the drug to the polymer through amide bonds, the problem of drug leakage in physical encapsulation methods is avoided, and the physicochemical stability of the drug delivery system is significantly improved. 2) Significantly improves drug solubility: The hydrophilic segments of the amphiphilic polymer enable hydrophobic drugs to be dispersed in water, forming stable nanomicelles, which effectively improves the apparent solubility of poorly soluble drugs. 3) Controllable drug loading: By adjusting the drug feed ratio and reaction conditions, the drug loading can be controlled. Doxorubicin can reach a drug loading of 47 mol%, and ampicillin can reach a drug loading of 29 mol%. 4) pH-responsive release: The amide bond can be hydrolyzed and broken under acidic conditions, giving the conjugate a pH-responsive release characteristic. P1M5-DOX has a cumulative release rate of 52.6% after 48 hours at pH 5.0, while it is only 23.1% at pH 7.4; P1M5-AMP has a cumulative release rate of 60-70% after 48 hours at pH 2.0, while it is about 50% at pH 6.8. 5) Good physical stability: The particle size change of the coupling micelles is <20% after 30 days of storage at 0℃ and <30% after 7 days of storage at room temperature, demonstrating good physical stability; 6) Decreased critical micelle concentration: After polymer coupling with hydrophobic drugs, the CMC decreased from 0.005 mg / mL to 0.002 mg / mL, which is beneficial to the stability of micelle structure in vivo and prolongs blood circulation time; 7) Retention of bioactivity: P1M5-DOX has cytotoxic activity against HeLa cells and P1M5-AMP has antibacterial activity against Escherichia coli, proving that the drug activity is retained; 8) The preparation method is simple and controllable: both steps of the reaction are carried out under mild conditions, the operation is simple, the reaction conditions are easy to control, and it is suitable for large-scale production. Attached Figure Description

[0017] Figure 1 This is the proton NMR spectrum of the amphiphilic polymer P1M5.

[0018] Figure 2 This is the proton NMR spectrum of P1M5-DOX.

[0019] Figure 3 This is the proton NMR spectrum of P1M5-AMP.

[0020] Figure 4 Transmission electron micrographs of P1M5-DOX and P1M5-AMP.

[0021] Figure 5 AFM topography and AFM-IR absorption images of P1M5-DOX and P1M5-AMP.

[0022] Figure 6 The diagram shows the particle size variation of P1M5-DOX and P1M5-AMP.

[0023] Figure 7 The in vitro release curves of P1M5-DOX and P1M5-AMP under different pH conditions are shown.

[0024] Figure 8 The results of the toxicity experiment of P1M5-DOX on HeLa cells.

[0025] Figure 9 The results of the antibacterial experiment of P1M5-AMP on Escherichia coli are shown. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the reagents, instruments, etc., used are all commercially available conventional products. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] Example 1: Synthesis of the amphiphilic polymer P1M5 Weigh 0.4 mmol of PMAO (average molecular weight ~5000 based on monomer units) into a 100 mL round-bottom flask, add 10 mL of anhydrous dichloromethane (DCM) to dissolve, and stir magnetically until completely dissolved. Then, add 0.8 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 0.88 mmol of N-hydroxythiosuccinimide (NHS) sequentially to the reaction system, and stir at room temperature for 30 min to activate the carboxyl groups. Add 0.08 mmol of mPEG-NH2 (MW 2000), and simultaneously add an appropriate amount of triethylamine to neutralize the hydrochloric acid generated in the reaction, shifting the reaction equilibrium towards the product. Then react at room temperature for 24 h. After the reaction was completed, the reaction solution was distilled under reduced pressure to remove dichloromethane. 250 μL LDMSO was added as a co-solvent to the remaining orange-red residue after reduced pressure distillation, followed by redissolution with 10 mL of water. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed against pure water for 24 h (6 water changes) to remove unreacted mPEG-NH2, condensing agents, and other small molecule byproducts. The dialysate was freeze-dried to obtain a white, fluffy solid, P1M5. ¹H NMR (CDCl3) δ (ppm): 0.8-1.5 (PMAO chain alkyl protons), 3.5-3.7 (PEG chain -CH2CH2O- protons). The calculated PEG / C... 18 The actual molar ratio was 0.274, which is basically consistent with the theoretical value of 0.2. Figure 1 ).

[0028] Example 2: Synthesis of P1M5-DOX First, doxorubicin hydrochloride (70 mg, 0.1 mmol) was dissolved in 2 mL of dimethyl sulfoxide (DMSO), followed by the addition of triethylamine (28 μL, 0.2 mmol). The mixture was stirred at room temperature in the dark for 2 hours to remove the hydrochloric acid, yielding a DMSO solution of free doxorubicin (DOX) for later use. Then, 90 mg of P1M5 (containing 0.2 mmol of carboxyl groups) was weighed using an electronic analytical balance and dissolved in 5 mL of DMSO. The pH was adjusted to 4.5 with 0.1 M HCl. EDC·HCl (76.7 mg, 0.4 mmol) and NHS (50.7 mg, 0.44 mmol) were added, and the mixture was stirred at room temperature for 30 minutes to activate the carboxyl groups. The dehydrochlorinated doxorubicin was then added, and the pH was adjusted to 7.0 with 0.1 M NaOH. The mixture was stirred at room temperature in the dark for 24 hours.

[0029] The reaction solution was diluted with pure water to 50 mL and transferred to a dialysis bag with a molecular weight cutoff of 3 kDa. Dialysis was performed at 4°C for 24 hours with the dialysate replaced every 4-6 hours (5-6 times in total) to thoroughly remove DMSO, unreacted drug, and small molecule byproducts. After dialysis, the liquid in the bag was collected and freeze-dried (-45°C, 15 bar) for 24 hours to obtain a red, fluffy, flocculent solid, which is the P1M5-DOX conjugate. This conjugate was stored at -20°C in the dark for later use.

[0030] ¹H NMR (DMSO-d6) δ (ppm): 0.8–1.5 (PMAO chain alkyl protons), 3.5–3.7 (PEG chain protons), 7.5–8.2 (DOX aromatic protons). Based on the integral area ratios of δ 1.0–1.3 and δ 7.7–7.9, the DOX loading was calculated to be 47 mol%. Figure 2 ).

[0031] Example 3: Synthesis of P1M5-AMP 90 mg of P1M5 (containing 0.2 mmol of carboxyl group) was weighed and dissolved in 5 mL of DMSO. The pH of the reaction mixture was adjusted to 4.5 with 0.1 M dilute hydrochloric acid. Then, EDC·HCl (76.7 mg, 0.4 mmol) and NHS (50.7 mg, 0.44 mmol) were added, and the mixture was activated for 30 min. Ampicillin (35 mg, 0.1 mmol) was added, and the pH was adjusted to 7.0 with 0.1 M NaOH. The reaction was carried out at room temperature for 24 h.

[0032] The purification steps were the same as in Example 2, yielding a white solid, P1M5-AMP.

[0033] ¹H NMR (DMSO-d6) δ (ppm): 0.8–1.5 (PMAO chain alkyl protons), 3.5–3.7 (PEG chain protons), 7.2–7.5 (AMP benzene ring protons), 8.1–8.3 (AMP β-lactam ring NH protons). Based on the integral area ratios of δ 1.0–1.3 and δ 7.2–7.5, the AMP loading was calculated to be 29 mol%. Figure 3 ).

[0034] Example 4: Optimization of Reaction Conditions (1) pH optimization With a fixed feed ratio of COOH:DOX = 4:1 and a reaction time of 24 h, the effects of pH during the activation stage and pH during the reaction stage on the drug loading were investigated. Other conditions were the same as in Example 2. The results are shown in Table 1.

[0035] Table 1 Effect of pH on DOX loading

[0036] The following conclusions can be drawn from Table 1. ① The optimal pH for the activation phase is 4.0-5.5. The highest drug loading (41 mol% and 40 mol% respectively) was observed at activation pH 4.5 and 5.0, indicating that weakly acidic conditions are favorable for the effective activation of carboxyl groups by EDC / NHS.

[0037] The drug loading decreases when the pH is too low (4.0) or too high (5.5-6.0), indicating that pH has a significant impact on activation efficiency.

[0038] ② The optimal pH for the reaction stage is 6.5-7.5. The highest drug loading (37.5 mol%) was observed at pH 7.0, indicating that near-neutral conditions are most favorable for nucleophilic attack of amino groups, promoting amide bond formation.

[0039] The drug loading was low at pH 6.0 (28 mol%), possibly due to the high degree of amino protonation and weakened nucleophilicity; the drug loading also decreased at pH 8.0 (32 mol%), possibly because the drug or intermediate is unstable under alkaline conditions.

[0040] ③ The necessity of a segmented pH control strategy The optimal pH for the activation phase (4.5-5.0) is significantly different from the optimal pH for the reaction phase (7.0), indicating that a single pH condition cannot simultaneously optimize the activation and coupling reactions.

[0041] This verifies the rationality of the segmented pH control method (first acidic activation, then near-neutral coupling), which is the key innovation of this preparation method.

[0042] ④ pH sensitivity of drug loading The drug loading showed a trend of first increasing and then decreasing with pH, ​​indicating that pH is a key control parameter for this coupling reaction and needs to be precisely controlled to obtain a high drug loading.

[0043] (2) Optimization of feed ratio The activation pH was fixed at 4.5, the reaction pH at 7.0, and the reaction time at 24 h. The effect of the COOH:DOX molar ratio on the drug loading was investigated. Results: A 1:1 ratio resulted in the highest drug loading (54 mol%), but precipitation occurred after purification; a 2:1 ratio resulted in a drug loading of 47 mol% and a clear solution; a 4:1 ratio resulted in a drug loading of 37.5 mol% and a clear solution. Considering both drug utilization and loading capacity, and given that the differences in drug loading were not significant, a COOH:DOX ratio of 4:1 was chosen as the optimal ratio, which can greatly reduce costs when preparing the same amount of polymer-drug conjugate.

[0044] (3) Optimization of reaction time The optimal pH (4.5 for the activation phase and 7.0 for the reaction phase) and feed ratio (COOH:DOX = 4:1) were fixed, and the effect of reaction time was investigated. The drug loadings at 12 h, 24 h, and 36 h were 22 mol%, 37.5 mol%, and 35 mol%, respectively, with 24 h selected as the optimal reaction time. The reason for choosing 24 hours as the optimal reaction time is that there is a competitive balance between the forward and side reactions in this coupling reaction. In the initial stage of the reaction (within 12 hours), the nucleophilic substitution of the active ester intermediate activated by EDC / NHS with the amino group of the drug has not yet fully occurred, resulting in incomplete coupling. By 24 hours, most of the active esters have been converted into stable amide bonds, the forward reaction is nearing completion, and side reactions (such as hydrolysis of the active ester or breakage of existing amide bonds) have not yet become dominant, at which point the drug loading reaches its peak. After 24 hours, residual moisture or acidic / alkaline environments in the system may induce amide bond hydrolysis, and the drug or polymer may degrade during long-term reactions, leading to a decrease in drug loading. Furthermore, the fact that the pH of the reaction system tended to stabilize after 24 hours also confirmed that the reaction was basically completed. Therefore, 24 hours is the optimal balance point for achieving high drug loading.

[0045] (4) Optimization of EDC usage With the optimal pH (4.5 for the activation phase and 7.0 for the reaction phase) fixed, the feed ratio (COOH:DOX = 4:1) and reaction time (24 h) were investigated to examine the effect of the EDC:COOH molar ratio. The drug loadings at ratios of 1.2:1, 1.5:1, 2:1, and 3:1 were 26 mol%, 30 mol%, 37.5 mol%, and 31.5 mol%, respectively. The optimal EDC:COOH ratio was selected as 2:1. This is because EDC, as a carboxyl activator, first reacts with the carboxyl groups of the polymer side chain to form an unstable O-acylisourea intermediate. This intermediate is converted into a semi-stable NHS ester in the presence of NHS, which then undergoes nucleophilic substitution with the amino group of the drug to form an amide bond. When the amount of EDC is insufficient (e.g., EDC:COOH = 1.2:1), the carboxyl groups are not fully activated, resulting in a low amount of active intermediates and a low drug loading. As the EDC ratio increases to 2:1, the activation efficiency reaches its optimum, the active intermediates are fully generated, and the drug loading reaches its peak (37.5 mol%). However, further increasing the EDC ratio to 3:1 may trigger side reactions, such as non-specific cross-linking with already formed amide bonds, or promoting intramolecular rearrangement, hydrolysis, and other deactivation processes in the active intermediates, thus reducing coupling efficiency. Therefore, EDC:COOH = 2:1 is the optimal ratio to balance activation efficiency and side reactions.

[0046] Example 5: Characterization of the properties of nanomicelles P1M5, P1M5-DOX, and P1M5-AMP were prepared into 0.5 mg / mL aqueous solutions for characterization.

[0047] (1) Dynamic light scattering (DLS) The results of the measurements are shown in Table 2.

[0048] Table 2 Particle size, PDI, and Zeta potential of polymers and couplings

[0049] (2) Transmission electron microscopy (TEM) High-contrast transmission electron microscopy (TEM) revealed that both P1M5-DOX and P1M5-AMP conjugates exhibited spherical micelle structures with particle sizes of 20-40 nm, which is largely consistent with the DLS results. Figure 4 A is a transmission electron microscope (TEM) image of P1M5-DOX, and B is a TEM image of P1M5-AMP.

[0050] (3) AFM-IR In P1M5-DOX, the characteristic DOX peak (1730 cm⁻¹) is spatially co-located with the polymer amide peak (1650 cm⁻¹) and the PEG peak (1100 cm⁻¹); in P1M5-AMP, the amide bond (1650 cm⁻¹) and the PEG peak (1100 cm⁻¹) have the same spatial distribution, confirming that the drug is covalently linked and uniformly distributed through amide bonds. Figure 5 A and B are AFM topography images of P1M5-DOX and P1M5-AMP, respectively. C and D are AFM topography images of P1M5-DOX and P1M5-AMP at a fixed wavenumber of 1730 cm⁻¹, respectively. -1 1650cm -1 1100cm -1 and 1650cm -1 1100cm -1 (Superposition of AFM-IR absorption images at the location).

[0051] Example 6: Storage stability study of polymer-drug conjugate P1M5-DOX and P1M5-AMP were dissolved in PBS buffer (pH 7.4) to prepare solutions of 0.5 mg / mL, and sonicated for 5 minutes to ensure uniform dispersion. The samples were then stored in sealed containers protected from light at 0°C (ice bath) and at room temperature (25°C), respectively.

[0052] Samples were taken on day 1 (initial), day 7, day 18, and day 30, and the particle size distribution was determined using a Malvern Zetasizer NanoZS90 particle size analyzer. Three parallel measurements were performed, and the average particle size and polydispersity index (PDI) were recorded. The physical stability was then investigated. The results are as follows: Figure 6 As shown, A and B represent the particle size changes of P1M5-DOX stored at 0 ℃ and room temperature, respectively; C and D represent the particle size changes of P1M5-AMP stored at 0 ℃ and room temperature, respectively.

[0053] Example 7: Determination of Critical Micelle Concentration The critical micelle concentration (CMC) was determined using the pyrene fluorescent probe method. The results showed that the CMC of P1M5 was approximately 0.005 mg / mL, while the CMCs of both P1M5-DOX and P1M5-AMP were approximately 0.002 mg / mL. The decrease in CMC after coupling with hydrophobic drugs is beneficial to the thermodynamic stability of micelles in vivo, allowing them to remain in vivo for a longer period and prolonging their circulation time.

[0054] Example 8: Study on in vitro release behavior (1) Release of P1M5-DOX Release experiments were conducted at pH 7.4 and pH 5.0. The cumulative release rate after 48 hours was 23.1% at pH 7.4 and 52.6% at pH 5.0. The release rate was significantly increased under acidic conditions, exhibiting a biphasic release characteristic. Figure 7 In the figure, A represents the release curves of P1M5-DOX at pH 7.4 and 5.0.

[0055] (2) Release of P1M5-AMP Release experiments were conducted at pH 2.0 and pH 6.8. The cumulative release rate after 48 hours was 60-70% at pH 2.0 and approximately 50% at pH 6.8. Release was faster under acidic conditions, validating the pH-responsive characteristics. Figure 7 In Figure B, the release curves of P1M5-AMP at pH 6.8 and 2.0 are shown.

[0056] Example 9: Cytotoxicity assay of P1M5-DOX The cytotoxicity against HeLa cells was assessed using the CCK-8 assay. Three groups were set up: P1M5, DOX, and P1M5-DOX, with DOX concentration gradients of 0 μg / mL, 0.85 μg / mL, 4.25 μg / mL, 8.5 μg / mL, 17 μg / mL, and 42.5 μg / mL (corresponding to polymer concentrations of 0 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL), and incubated for 24 h and 48 h, respectively.

[0057] result( Figure 8 The cell viability of the blank polymer (P1M5) group was ~100%, with no cytotoxicity (CG group refers to the positive control group, i.e., "bacterial solution + culture medium, no treatment" group); the cell viability of the free DOX group was the lowest; the cytotoxicity of the P1M5-DOX group was weaker than that of free DOX at low concentrations, and the killing effect was close to that of free DOX at high concentrations (equivalent to DOX 42.5 μg / mL); the cytotoxicity of the P1M5-DOX group incubated for 48 h was stronger than that of the 24 h group, demonstrating a sustained-release and long-lasting effect.

[0058] Example 10: Antibacterial activity experiment of P1M5-AMP The antibacterial activity against *Escherichia coli* (DH5α) was assessed using the microbroth dilution method. Five groups were set up: P1M5, PBS, P1M5-AMP, AMP, and culture medium. The AMP concentration was 6.5 μg / mL. OD was measured after incubation at 37℃ for 24 h. 600 .

[0059] result( Figure 9 P1M5-AMP group OD 600Significantly reduced, exhibiting antibacterial activity; AMP group OD 600 The lowest concentration and strongest antibacterial activity were observed in the P1M5 group and the PBS group, respectively; the polymer showed no significant antibacterial activity.

Claims

1. A method for preparing an amphiphilic polymer-drug conjugate, characterized in that, The conjugates were prepared using a segmented pH-controlled method, which involved first activating the carboxyl groups of the amphiphilic polymer side chains under acidic conditions of pH 3.0-6.5, and then covalently binding the activated carboxyl groups with an amino-containing drug via amide bonds under conditions of pH 6.5-8.

5. The amphiphilic polymer is prepared by amidation reaction of poly(maleic anhydride-alt-1-octadecene) and methoxy polyethylene glycolamine.

2. The method for preparing an amphiphilic polymer-drug conjugate as described in claim 1, characterized in that, Specifically, the steps include the following: 1) Poly(maleic anhydride-alt-1-octadecene) and methoxy polyethylene glycolamine were placed in an organic solvent and subjected to an amidation reaction in the presence of a condensing agent. After the reaction was completed, the mixture was purified and dried to obtain the amphiphilic polymer PMAO-PEG. 2) Dissolve the amphiphilic polymer PMAO-PEG obtained in step 1) in an organic solvent, adjust the pH of the solution to 3.0-6.5 to activate the carboxyl groups on the side chains of the amphiphilic polymer, add a condensing agent, stir to activate the reaction, and convert the carboxyl groups into highly reactive intermediates. 3) Dissolve or disperse the amino-containing target drug in a solvent and add it to the activation system in step 2). Adjust the pH to 6.5-8.5 to promote the nucleophilic substitution reaction. Stir the reaction to allow the amino group of the drug to undergo amidation reaction with the activated carboxyl group on the amphiphilic polymer to form a stable covalent bond. 4) After the reaction is complete, the amphiphilic polymer-drug conjugate is obtained by purification and drying.

3. The method for preparing an amphiphilic polymer-drug conjugate as described in claim 2, characterized in that, In step 1), the molar ratio of poly(maleic anhydride-alt-1-octadecene) to methoxy polyethylene glycol amine is 1:0.1-1.2; the organic solvent in step 1) is dichloromethane, chloroform, methanol, ethanol, or N,N-dimethylformamide; the condensing agent in step 1) is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide; and the reaction time in step 1) is 10-40 hours.

4. The method for preparing an amphiphilic polymer-drug conjugate as described in claim 3, characterized in that, The organic solvent in step 2) is dimethyl sulfoxide, N,N-dimethylformamide, or N-methylpyrrolidone; the condensing agent in step 2) is a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide, wherein the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the carboxyl group in the amphiphilic polymer is 1.2-3:1, the molar ratio of N-hydroxythiosuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1-5:1, and the activation time in step 2) is 5-60 minutes.

5. The method for preparing an amphiphilic polymer-drug conjugate as described in claim 4, characterized in that, In step 3), the amino-containing target drug is doxorubicin, ampicillin, or a pharmaceutically acceptable salt thereof; the molar ratio of the amino-containing target drug to the amphiphilic polymer is 1:1-8; and the reaction time is 10-40 hours.

6. The method for preparing an amphiphilic polymer-drug conjugate as described in claim 2, characterized in that, In step 2), adjust the pH of the solution to 4.0-5.5; in step 3), adjust the pH to 6.5-7.

5.

7. An amphiphilic polymer-drug conjugate prepared by the preparation method described in claims 1-6.

8. A nanomicelle formulation, characterized in that, The amphiphilic polymer-drug conjugate described in claim 7 is self-assembled in an aqueous medium, and its particle size is 30-50 nm.

9. The use of the amphiphilic polymer-drug conjugate as described in claim 7 in the preparation of antitumor drugs or antibacterial drugs.

10. A pH-responsive drug delivery system, characterized in that, The product comprises the amphiphilic polymer-drug conjugate of claim 7, wherein the drug release rate is higher under acidic conditions than under neutral conditions.

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