Multi-arm polymer coupled prodrug, nano-micelle thereof, preparation method and application

Abstract

The invention discloses a multi-arm polymer coupled prodrug, a nano-micelle thereof, a preparation method and application, a hydrophobic drug is structurally modified by multi-arm polylactic acid to construct a prodrug, and a multi-arm polylactic acid prodrug is prepared. Specifically, the multi-arm polylactic acid is prepared by taking small molecules with different hydroxyl numbers as initiators and carrying out ring-opening polymerization reaction on the initiators and lactide; the multi-arm type polymer coupling prodrug is formed by performing carboxyl modification on multi-arm type polylactic acid through succinic anhydride and then performing esterification reaction on the multi-arm type polylactic acid and hydroxyl of a hydrophobic drug. Compared with a traditional single-arm polylactic acid polymer prodrug, the multi-arm polylactic acid prodrug has the unique space structure advantage, the encapsulation efficiency, the drug loading capacity and the stability of polymer nano-micelles are remarkably improved, and the multi-arm polylactic acid prodrug has the advantages of high permeability and the retention effect and high bioavailability. The enrichment of the polymer nano-micelle at a tumor part is effectively improved; and the systematic toxic and side effects of the polymer nano-micelle are reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical polymer materials, and particularly relates to a multi-arm polymer conjugated prodrug, a nanomicelle thereof, and a preparation method and application thereof. BACKGROUND

[0002] Nanomicelles have excellent drug delivery performance and can significantly improve the therapeutic index of loaded drugs, and have become a hot spot in tumor treatment research. Compared with free drugs, nanomicelles can achieve a higher degree of drug accumulation in tumor tissues through the enhanced permeability and retention effect (EPR), thereby significantly enhancing the therapeutic effect. However, due to the differences in chemical structure between the chemotherapeutic drug and the carrier material, they exhibit different physical and chemical properties, which in turn limits the drug encapsulation efficiency and drug loading capacity, and this problem is still a key difficulty in the optimization process of nanomicelle delivery systems.

[0003] The prodrug strategy enhances the structural compatibility of the drug with the carrier material through reasonable chemical modification of the drug, thereby significantly improving the encapsulation efficiency and drug loading capacity of the nanomicelle, and is considered to be an effective way to cope with the above challenges. In particular, by chemically modifying the drug with a polymer, a polymer conjugated prodrug system can be constructed to effectively improve the structural compatibility between the drug and the carrier material. This strategy not only helps to improve the drug loading rate, but also enhances the stability of the nanomicelle, thereby improving the solubility and bioavailability of the drug and improving the overall delivery efficiency. Although polymer modified prodrugs have advantages in many aspects, they still face some challenges.

[0004] Taking traditional single-arm polylactide (PLA) polymer prodrugs as an example, they have certain limitations in drug stability and drug loading capacity. The linear structure of the single-arm polylactide prodrug can lead to instability in the drug release process, thereby affecting the therapeutic effect. In contrast, multi-arm polylactide (PLA) prodrugs can usually provide superior stability due to their more complex branched structure and stronger intermolecular interactions, and effectively prolong the biological half-life of the drug. In addition, the mismatch between the drug and the carrier in terms of hydrophilicity / hydrophobicity often limits the loading efficiency, thereby affecting the overall therapeutic effect of the drug. Insufficient drug loading not only limits the therapeutic effect of the drug, but also can lead to uneven distribution of the drug in the body, further affecting the targeting and therapeutic effect.

[0005] This invention discloses a method for preparing and applying polymer-coupled prodrug-based nanomicelles. Employing a multi-arm polylactic acid (PLA) modification strategy overcomes the limitations of traditional single-arm structures, significantly improving drug loading efficiency, encapsulation efficiency, and stability. Through the permeation-retention effect (EPR effect), this system effectively promotes the efficient accumulation of polymer micelles at tumor sites, thereby improving therapeutic efficacy and reducing systemic toxicity. Simultaneously, the multi-arm PLA-modified prodrug strategy significantly optimizes tumor-targeted delivery systems and has broad application prospects. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to prepare and apply polymer nanomicelles for multi-arm polymer-coupled prodrugs.

[0007] The specific technical solution is as follows:

[0008] A multi-arm polymer coupling prodrug is prepared by structurally modifying a hydrophobic drug with multi-arm polylactic acid (PLA). The PLA is prepared by ring-opening polymerization of lactide with small molecules having different numbers of hydroxyl groups as initiators. After carboxyl modification with succinic anhydride, the PLA is then esterified or amidated with the hydroxyl or amino groups of the hydrophobic drug to form the multi-arm polymer coupling prodrug, the structural formula of which is shown below:

[0009]

[0010] In the formula, R1 represents the polymer group of polylactic acid with multiple arms, n represents the number of arms of the polymer group, and n is an integer from 2 to 5; R2 represents the drug group.

[0011] The multi-arm polymer-coupled prodrug may have one of the following structures:

[0012] 1) When the number of arms of the polymer n = 2, the structural formula is one of the following:

[0013] 2) When the number of arms n of the polymer is 4, the structural formula is one of the following:

[0014]

[0015] R3 is selected from one of the following:

[0016]

[0017] R4 is selected from the following structures:

[0018]

[0019] 3) When the number of arms of the polymer n = 5, the structural formula is one of the following:

[0020]

[0021]

[0022] R5 is selected from the following structures:

[0023]

[0024] Where m and i represent the degree of polymerization, both of which are positive integers. Further, the pharmaceutical group R2 is selected from one of the following:

[0025]

[0026]

[0027] A polymer-coupled prodrug nanomicelle includes the multi-arm polymer-coupled prodrug described in this invention and a polymer carrier material.

[0028] Furthermore, the polymer carrier material is selected from one or more of polyethylene glycol-polylactic acid and polyethylene glycol-polylactic acid-glycolic acid.

[0029] The method for preparing a polymer-coupled prodrug nanomicelle includes the following steps: dispersing the multi-arm polymer-coupled prodrug and polymer carrier material in an organic solvent and mixing them evenly to form an oil phase solution; adding the oil phase solution dropwise into water under continuous stirring, and after the addition is complete, stirring is continued for 5-30 minutes; removing the organic solvent by vacuum distillation, thereby obtaining an aqueous solution of the polymer nanomicelle of the polymer prodrug.

[0030] Furthermore, the volume ratio of the oil phase solution to water is 1:8-12.

[0031] Furthermore, the mass ratio of the multi-arm polymer-coupled prodrug to the polymer carrier material is 1:8-12.

[0032] Furthermore, the organic solvent is one or more of propanol, methanol, ethanol, THF, DMF, and DMSO.

[0033] The present invention also discloses the application of the polymer-coupled prodrug nanomicelles, wherein the polymer-coupled prodrug nanomicelles are used to load chemotherapeutic drugs for targeted delivery in vivo.

[0034] This invention utilizes multi-arm polylactic acid (PLA) to structurally modify hydrophobic drugs to construct prodrugs, thus preparing multi-arm PLA prodrugs. Specifically, multi-arm PLA uses small molecules with different numbers of hydroxyl groups as initiators, and undergoes ring-opening polymerization with lactide to obtain a polymer carrier. The polymer carrier is then modified with succinic anhydride to form carboxyl groups, followed by esterification with the hydroxyl groups of the hydrophobic drug, ultimately preparing the multi-arm PLA prodrug. Compared to traditional single-arm PLA polymer prodrugs, multi-arm PLA prodrugs possess unique spatial structural advantages, significantly enhancing the encapsulation efficiency, drug loading, and stability of polymer nanomicelles. Through enhanced permeability and retention effect (EPR effect), they effectively improve the enrichment of polymer nanomicelles at tumor sites and reduce their systemic toxicity. This invention relates to a method for preparing and applying polymer nanomicelles based on multi-arm polylactic acid-coupled prodrugs. It overcomes the limitations of traditional single-arm prodrug strategies, optimizes the polymer nanomicelle chemotherapy drug delivery system, and provides an effective solution to the problem of poor compatibility between traditional chemotherapy drugs and carrier materials due to differences in chemical structure. It has broad application prospects in tumor treatment.

[0035] The superior effects of this invention are as follows:

[0036] 1) The polymer nanomicelles of the present invention have excellent in vitro stability and can maintain their structural stability under different physiological conditions, which helps to improve drug release control and therapeutic effect.

[0037] 2) Through the efficient encapsulation of polymer nanomicelles and the high drug loading capacity, it is possible to ensure that the drug is effectively released in the body and achieves the therapeutic effect, avoiding drug waste and improving bioavailability.

[0038] 3) The polymer nanomicelles of the present invention have good biosafety, low toxicity and high biocompatibility, which can effectively reduce damage to normal tissues and improve the safety of clinical treatment when applied in vivo.

[0039] 4) The polymer nanomicelles of the present invention have high toxicity to tumor cells and can effectively kill tumor cells while targeting them, thus providing a stronger anti-tumor therapeutic effect.

[0040] 5) The polymer nanomicelles of the present invention have good cellular uptake capacity, can effectively enter tumor cells and release drugs, thereby improving drug targeting and therapeutic efficiency. Attached Figure Description

[0041] Figure 1 The synthesis route for MPLA@SN38;

[0042] Figure 2 The synthesis route for DPLA@SN38;

[0043] Figure 3 The synthesis route for TPLA@SN38;

[0044] Figure 4 TEM image of PP5T5N;

[0045] Figure 5 Schematic diagram of the particle size distribution of drug-loaded nanomicelles;

[0046] Figure 6 Schematic diagram of the stability of drug-loaded nanomicelles;

[0047] Figure 7 SN38 standard curve obtained by ultraviolet-spectrum spectrophotometer;

[0048] Figure 8 SN38 standard curve obtained by reversed-phase liquid chromatography;

[0049] Figure 9 Polymers with different arm numbers (molecular weight 5k) conjugated with prodrugs and PEG 5k -PLA 5k Drug release curves of the prepared nanomicelles in different environments;

[0050] Figure 10 Polymers with different arm numbers (molecular weight 2k) conjugated with prodrugs and PEG 5k -PLA 5k Drug release curves of the prepared nanomicelles in different environments;

[0051] Figure 11 Polymers with different arm numbers (molecular weight 1k) conjugated with prodrugs and PEG 5k -PLA 5k Drug release curves of the prepared nanomicelles in different environments;

[0052] Figure 12 Figure showing the cytotoxicity results of drug-loaded polymer nanomicelles against A549 cells;

[0053] Figure 13 Figure showing the cellular uptake of A549 by drug-loaded polymer nanomicelles. Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0055] Special note: The naming conventions for each modified product are as follows: polyethylene glycol monomethyl ether is abbreviated as mPEG; lactide is abbreviated as LA; and polyethylene glycol monomethyl ether-polylactide is named mPEG. a -PLA b Where 'a' represents the molecular weight of the hydrophilic portion mPEG and 'b' represents the molecular weight of the hydrophobic portion polylactide PLA; the triethylene glycol monomethyl ether-polylactide is named MPLA. a 'a' represents the molecular weight of polylactide; 1,3-propylene glycol-polylactide is named DPLA. a 'a' represents the molecular weight of polylactide; pentaerythritol-polylactide is named TPLA. a 'a' represents the molecular weight of polylactide; MPLA a -SA represents carboxylated single-arm polylactic acid; DPLA a -SA represents carboxylated two-arm polylactic acid; TPLA a -SA represents carboxylated four-armed polylactic acid; MPLA a @SN38 represents a single-arm polylactic acid-modified SN38 prodrug; DPLA a @SN38 represents a polylactic acid-modified SN38 prodrug in both arms; TPLA a @SN38 represents a four-arm polylactic acid modified SN38 prodrug.

[0056] PPcMdN is a polymer carrier for mPEG. c -PLA c Polymer micelles prepared from "single-arm polylactic acid modified SN38 prodrug", where c represents the polymer carrier mPEG. c -PLA c The molecular weights of the corresponding mPEG and PLA are both c, and d represents the molecular weight of the polymer in the "single-arm polylactic acid modified SN38 prodrug".

[0057] For example: PP5M5N represents "polymer carrier mPEG" 5k -PLA 5k "and MPLA" 5K Polymer micelles were prepared using "@SN38 prodrug" as a raw material. The nanomicelles prepared by polymer coupling of two-armed and four-armed prodrugs were named PPcDdN and PPcTdN, respectively.

[0058] For example: PP5D5N represents "polymer carrier mPEG" 5k -PLA 5k "and DPLA" 5K Polymer micelles prepared from "@SN38 prodrug".

[0059] PPcTdN represents "polymer carrier mPEG"5k -PLA 5k "and TPLA" 5K Polymer micelles prepared from "@SN38 prodrug".

[0060] Example 1: Single-arm polymer MPLA a Synthesis

[0061] MPLA 5k For example, the glassware and stir bar used in the reaction need to be dried beforehand. Under nitrogen protection, lactide (15.8 g, 110 mmol) and triethylene glycol monomethyl ether (0.509 g, 3.10 mmol) are added sequentially to a 250 mL double-necked flask containing 50 mL of anhydrous toluene. The double-necked flask is placed in a 150 °C oil bath for reflux reaction. Anhydrous conditions must be ensured during the reaction. Using a water separator based on the azeotropic principle of toluene and water, 30 mL of the toluene-water mixture is separated to remove any remaining water, leaving 20 mL of the reaction mixture in the double-necked flask. The oil bath temperature is lowered to 115 °C. 250 μL of stannous octoate is rapidly added dropwise to the reaction system as a catalyst, and the reaction is continued under reflux at 115 °C for 3 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and 150 mL of dichloromethane (DCM) and three drops of 0.1 M dilute hydrochloric acid solution were added to quench the reaction. The quenched reaction solution was washed with water several times until the upper aqueous phase changed from acidic to neutral, and then the organic phase was separated. The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated to obtain a transparent viscous crude product. Finally, the crude product was dissolved with a small amount of DCM, and then n-hexane was added dropwise while stirring, accompanied by the appearance of a white flocculent precipitate. After the addition was completed, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was dried in a vacuum drying oven to obtain the final product MPLA. 5K (12.9g, yield 86%). 1 H NMR(400MHz,Chloroform-d)δ5.27–5.08(m,71H),4.42–4.20(m,3H),3.71–3.52(m,10H),3 .39-3.36(s,3H),1.65–1.50(ddd,J=9.8,7.1,2.4Hz,217H),0.92–0.85(t,J=6.7Hz,13H).

[0062] Using the same ring-opening polymerization method, MPLA with different molecular weights was synthesized by controlling the amount of triethylene glycol monomethyl ether added and the feed ratio of lactide. 2K and MPLA 1K A series of high molecular weight polymers, with yields ranging from 80% to 95%.

[0063] Example 2: Two-arm polymer DPLA a Synthesis

[0064] With DPLA 5k For example, the glassware and stir bar used in the reaction need to be dried beforehand. Under nitrogen protection, lactide (15.8 g, 0.110 mol) and 1,3-propanediol (0.232 g, 3.05 mmol) are added sequentially to a 250 mL double-necked flask containing 50 mL of anhydrous toluene. The double-necked flask is placed in a 150 °C oil bath for reflux reaction. Anhydrous conditions must be ensured during the reaction. Using a water separator based on the azeotropic principle of toluene and water, 30 mL of the toluene-water mixture is separated to remove any remaining water, leaving 20 mL of the reaction mixture in the double-necked flask. The oil bath temperature is lowered to 115 °C. 250 μL of stannous octoate is rapidly added dropwise to the reaction system as a catalyst, and the reaction is continued under reflux at 115 °C for 3 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and 150 mL of dichloromethane (DCM) and three drops of 0.1 M dilute hydrochloric acid solution were added to quench the reaction. The quenched reaction solution was washed with water several times until the upper aqueous phase changed from acidic to neutral, and then the organic phase was separated. The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated to obtain a transparent, viscous crude product. Finally, the crude product was dissolved in a small amount of DCM, and then n-hexane was added dropwise while stirring, accompanied by the appearance of a white flocculent precipitate. After the addition was complete, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was dried in a vacuum drying oven to obtain the final product DPLA. 5K (12.7g, 84.7%). 1 H NMR(400MHz,Chloroform-d)δ5.27–5.08(m,79H),4.43-4.32(q,J=7.1Hz,2H),4.25-4.15(dt,J=7.7,4.0Hz,4H),1.65-1.50(ddd,J=9.9,7.0,2.4Hz,237H).

[0065] Using the same ring-opening polymerization method, DPLA with different molecular weights was synthesized by controlling the amount of 1,3-propanediol added and the feed ratio of lactide. 2K and DPLA 1K A series of high molecular weight polymers, with yields ranging from 80% to 95%.

[0066] Example 3: Four-arm polymer TPLA a Synthesis

[0067] With TPLA 5kFor example, the glassware and stir bar used in the reaction need to be dried beforehand. Under nitrogen protection, lactide (15.8 g, 0.110 mol) and pentaerythritol (0.420 g, 3.08 mmol) are added sequentially to a 250 mL double-necked flask containing 50 mL of anhydrous toluene. The double-necked flask is placed in a 150 °C oil bath for reflux reaction. Anhydrous conditions must be ensured during the reaction. Using a water separator based on the azeotropic principle of toluene and water, 30 mL of the toluene-water mixture is separated to remove any remaining water, leaving 20 mL of the reaction mixture in the double-necked flask. The oil bath temperature is lowered to 115 °C. 250 μL of stannous octoate is rapidly added dropwise to the reaction system as a catalyst, and the reaction is continued under reflux at 115 °C for 3 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and 150 mL of dichloromethane (DCM) and three drops of 0.1 M dilute hydrochloric acid solution were added to quench the reaction. The quenched reaction solution was washed with water several times until the upper aqueous phase changed from acidic to neutral, and then the organic phase was separated. The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated to obtain a transparent, viscous crude product. Finally, the crude product was dissolved in a small amount of DCM, and then n-hexane was added dropwise while stirring, accompanied by the appearance of a white flocculent precipitate. After the addition was complete, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was dried in a vacuum drying oven to obtain the final product TPLA. 5K (13.8g, yield 92%). 1 HNMR(400MHz,Chloroform-d)δ5.27–5.10(m,68H),4.42-4.32(p,J=7.3,6.9Hz,4H),4.62-4. 02(q,J=10.9Hz,8H),1.64–1.51(m,208H),1.34–1.18(m,26H),0.95-0.75(q,J=6.5Hz,19H).

[0068] Using the same ring-opening polymerization method, TPLA with different molecular weights was synthesized by controlling the amount of pentaerythritol added and the feed ratio of lactide. 2K and TPLA 1K A series of high molecular weight polymers, with yields ranging from 80% to 95%.

[0069] Example 4: Carboxylate modification of MPLA with single-arm polylactic acid a -SA

[0070] MPLA 5k Taking -SA as an example, under nitrogen protection, m-PLA is added sequentially to a 50mL two-necked flask containing 20mL of anhydrous dichloromethane. 5k5.00 g (10 mmol), succinic anhydride (0.45 g, 4.5 mmol), pyridine (0.15 g, 1.8 mmol), and 4-dimethylaminopyridine (0.021 g, 0.17 mmol) were placed in an oil bath at 45 °C and stirred under reflux for 3 days. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with 150 mL of dichloromethane and 50 mL of saturated sodium bicarbonate. The extracted solution was allowed to stand for at least 8 hours. The retained organic layer was then extracted again with 50 mL of citric acid aqueous solution (5% by mass), and the organic layer was retained. The organic layer was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and finally concentrated by rotary evaporation to remove dichloromethane, yielding a viscous white crude product. The obtained viscous white crude product was dissolved in 5 mL of dichloromethane. Then, while stirring, n-hexane was added dropwise (dichloromethane:n-hexane = 1:10, v / v), accompanied by the appearance of a white gelatinous precipitate. After the addition was complete, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was then dried in a vacuum drying oven to obtain the final product MPLA. 5K SA was a white solid (4.07 g, yield 81.4%). 1 H NMR(400MHz,Chloroform-d)δ5.28–5.06(m,71H),4.35–4.22(m,2H),3.73–3.51(m,11H),3.4 2–3.34(s,3H),2.76–2.64(dq,J=7.3,4.1Hz,4H),1.65–1.45(ddd,J=9.9,7.0,2.5Hz,218H).

[0071] Using the same modification method, MPLA with different molecular weights was synthesized by adding single-arm polylactic acid of different molecular weights. 2K -SA and MPLA 1K -SA series polymers, with yields ranging from 70% to 90%.

[0072] Example 5: Carboxylation modification of DPLA with two arms of polylactic acid a -SA

[0073] With DPLA 5k Taking SA as an example, under nitrogen protection, DPLA was added to a 50 mL two-necked flask containing 20 mL of anhydrous dichloromethane. 5kThe following compounds were added: 4.08 g (0.82 mmol), succinic anhydride (0.37 g, 3.7 mmol), pyridine (0.11 g, 1.4 mmol), and 4-dimethylaminopyridine (0.019 g, 0.016 mmol). The mixture was refluxed and stirred in an oil bath at 45°C for 5 days. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with 150 mL of dichloromethane and 50 mL of saturated sodium bicarbonate. The extracted solution was allowed to stand for at least 8 hours. The retained organic layer was then extracted again with 50 mL of 5% citric acid aqueous solution, and the organic layer was retained. The organic layer was washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, and finally concentrated by rotary evaporation to remove dichloromethane, yielding a viscous white crude product. The obtained viscous white crude product was dissolved in 5 mL of dichloromethane. Then, while stirring, n-hexane was added dropwise (dichloromethane:n-hexane = 1:10, v / v), accompanied by the appearance of a white gelatinous precipitate. After the addition was complete, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was then dried in a vacuum drying oven to obtain the final product DPLA. 5K SA was a white solid (3.16 g, yield 77.4%). 1 H NMR (400MHz, Chloroform-d) δ5.26–5.10 (m, J=17.4, 13.3, 6.5Hz, 80H), 4.28– 4.13(t,J=5.9Hz,4H),2.76–2.68(dt,J=5.6,3.5Hz,8H),1.66–1.52(m,234H).

[0074] Using the same modification method, DPLA with different molecular weights was synthesized by adding two-arm polylactic acid of different molecular weights. 2K -SA and DPLA 1K -SA series of polymers, with yields ranging from 75% to 90%.

[0075] Example 6: Carboxylate-modified TPLA with four-arm polylactic acid a -SA

[0076] With TPLA 1k Taking -SA as an example, under nitrogen protection, P4-PLA was added to a 50mL two-necked flask containing 20mL of anhydrous dichloromethane. 1kThe following compounds were added: 1.361 g (0.091 mmol), succinic anhydride (0.87 g, 8.7 mmol), pyridine (0.14 g, 1.7 mmol), and 4-dimethylaminopyridine (0.11 mg, 0.90 mmol). The mixture was refluxed in an oil bath at 45°C for 10 days with stirring. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with 150 mL of dichloromethane and 50 mL of saturated sodium bicarbonate. The extracted solution was allowed to stand for at least 8 hours. The retained organic layer was then extracted again with 50 mL of 5% citric acid aqueous solution, and the organic layer was retained. The organic layer was washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, and finally concentrated by rotary evaporation to remove dichloromethane, yielding a viscous white crude product. The obtained viscous white crude product was dissolved in 5 mL of dichloromethane. Then, while stirring, n-hexane was added dropwise (dichloromethane:n-hexane = 1:10, v / v), accompanied by the appearance of a white gelatinous precipitate. After the addition was completed, the mixture was allowed to stand for a period of time and the supernatant was discarded. The above process was repeated three times. The precipitate was placed in a vacuum drying oven and dried to obtain the final product TPLA1K-SA as a white solid (0.66 g, yield 48.5%). 1 H NMR (400MHz, Chloroform-d) δ5.24–5.11(m,21H),4.3-4.05(d,J=21.1Hz,8H),2.81–2.58(q,J=4.8,4.2Hz,16H),1.65–1.52(d,J=7.1Hz,68H).

[0077] Using the same modification method, TPLA with different molecular weights was synthesized by adding four-armed polylactic acid of different molecular weights. 5K -SA and TPLA 2K -SA series polymers, the yield of which is 45%-60%.

[0078] Example 7: Synthesis of a single-arm polymer prodrug, MPLA a @SN38

[0079] refer to Figure 1 The synthetic route, with MPLA 5k Taking @SN38 as an example, under nitrogen protection, MPLA was added to a 250mL two-necked flask containing 100mL of anhydrous dichloromethane. 5KSA (220.00 mg, 0.044 mmol), 7-ethyl-10-hydroxycamptothecin (0.029 g, 0.074 mmol), 1-ethyl-3-(3-dimethylaminopropyl)aminomethylene (18.00 mg, 0.12 mmol), and 4-dimethylaminopyridine (13 mg, 0.110 mmol) were added to an oil bath at 45°C and stirred under reflux for one day. The reaction was confirmed by TLC (dichloromethane:methanol = 20:1, v / v), and the reaction solution was then cooled to room temperature. After the reaction was complete, the reaction solution was cooled to room temperature and diluted with 150 mL of dichloromethane. The solution was then washed successively with 50 mL of citric acid aqueous solution (5%), 50 mL of saturated sodium bicarbonate solution, and saturated brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a transparent, viscous, pale yellow crude product. The crude product was separated by silica gel column chromatography (dichloromethane:methanol = 100:1, v / v). The purified product was collected and concentrated by rotary evaporation to remove dichloromethane, yielding a pale yellow oily substance. Finally, the obtained product was dried in a vacuum drying oven to obtain the final product MPLA. 5K @SN38 is a pale yellow solid (0.154 mg, yield 70%). 1 HNMR (400MHz, Chloroform-d) δ8.42-7.57(m,3H),5.26-5.08(m,64H),3.37(s,3H),1.56(ddd,J=12.3,6.5,2.2Hz,196H).

[0080] Using the same esterification reaction method, MPLA with different molecular weights was synthesized by adding single-arm polymers modified with carboxylic acids of different molecular weights. 2K @SN38 and MPLA 1K @SN38 series of polymer prodrugs, the yield of which is 50%-80%.

[0081] Example 8 Synthesis of a two-arm polymer prodrug DPLA a @SN38

[0082] refer to Figure 2 The synthetic route, with DPLA 5k Taking @SN38 as an example, under nitrogen protection, DPLA was added to a 250mL double-necked flask containing 100mL of anhydrous dichloromethane. 5KSA (310.00 mg, 0.062 mmol), 7-ethyl-10-hydroxycamptothecin (37.00 mg, 0.094 mmol), 1-ethyl-3-(3-dimethylaminopropyl)aminomethylene (32.00 mg, 0.21 mmol), and 4-dimethylaminopyridine (19 mg, 0.160 mmol) were stirred and refluxed in an oil bath at 45°C for 3 days. The reaction was confirmed by TLC (dichloromethane:methanol = 20:1, v / v), and the reaction solution was then cooled to room temperature. After the reaction was complete, the reaction solution was cooled to room temperature and diluted with 150 mL of dichloromethane. The solution was then washed successively with 50 mL of citric acid aqueous solution (5%), 50 mL of saturated sodium bicarbonate solution, and saturated brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a transparent, viscous, pale yellow crude product. The crude product was separated by silica gel column chromatography (dichloromethane:methanol = 100:1, v / v). The purified product was collected and concentrated by rotary evaporation to remove dichloromethane, yielding a pale yellow oily substance. Finally, the obtained product was dried in a vacuum drying oven to obtain the final product DPLA. 5K @SN38 was a pale yellow solid (0.112 mg, yield 36.1%). ¹H NMR (400 MHz, Chloroform-d) δ 8.39 (s, 2H), 7.88 (s, 4H), 7.61 (d, J = 8.7 Hz, 2H), 5.20–5.11 (m, 73H), 4.20 (td, J = 6.5, 1.9 Hz, 4H), 1.58–1.53 (m, 234H).

[0083] Using the same esterification reaction method, DPLA with different molecular weights was synthesized by adding two-arm polymers modified with carboxylic acids of different molecular weights. 2K @SN38 and DPLA 1K @SN38 series of polymer prodrugs, the yield of which is 20%-50%.

[0084] Example 9: Synthesis of a four-arm polymer prodrug TPLA a @SN38

[0085] refer to Figure 3 The synthetic route, with TPLA 1k Taking @SN38 as an example, under nitrogen protection, TPLA is added sequentially to a 250mL two-necked flask containing 100mL of anhydrous dichloromethane. 5kSA (160.00 mg, 0.1066 mmol), 7-ethyl-10-hydroxycamptothecin (83 mg, 0.2115 mmol), 1-ethyl-3-(3-dimethylaminopropyl)aminomethylene (132.5 mg, 0.8533 mmol), and 4-dimethylaminopyridine (29.5 mg, 0.2414 mmol) were stirred and refluxed in an oil bath at 45°C for 15 days. The reaction was confirmed to be complete by TLC (dichloromethane:methanol = 20:1, v / v), and the reaction solution was then cooled to room temperature. After the reaction was complete, the reaction solution was cooled to room temperature and diluted with 150 mL of dichloromethane. The solution was then washed successively with 50 mL of citric acid aqueous solution (5%), 50 mL of saturated sodium bicarbonate solution, and saturated brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain a transparent, viscous, pale yellow crude product. The crude product was separated by silica gel column chromatography (dichloromethane:methanol = 100:1, v / v). The purified product was collected and concentrated by rotary evaporation to remove dichloromethane, yielding a pale yellow oily substance. Finally, the obtained product was dried in a vacuum drying oven to obtain the final product TPLA. 5K @SN38 was a pale yellow solid (75.5 mg, yield 47.2%). ¹H NMR (400 MHz, Chloroform-d) δ 8.28 (d, J = 9.1 Hz, 2.93 H), 7.86 (s, 3.02 H), 7.72 (q, J = 2.9, 2.4 Hz, 3.57 H), 7.59 (d, J = 9.3 Hz, 3.21 H), 5.24–5.12 (m, 87 H), 4.16 (s, 8 H), 1.61–1.53 (m, 279 H).

[0086] Using the same esterification reaction method, TPLA with different molecular weights was synthesized by adding tetra-arm polymers modified with carboxylic acids of different molecular weights. 2K @SN38 and TPLA 1K @SN38 series of polymer prodrugs, the yield of which is 20%-50%.

[0087] Example 10: mPEG Carrier a -PLA b Synthesis

[0088] With mPEG 5K -PLA 5KFor example, the glassware and stir bar used in the reaction need to be dried beforehand. Under nitrogen protection, lactide (7.875 g, 0.0547 mol) and polyethylene glycol 5000 (7.5 g, 0.0015 mol) are added sequentially to a 250 mL double-necked flask containing 50 mL of anhydrous toluene. The double-necked flask is placed in a 150 °C oil bath for reflux reaction. Anhydrous conditions must be ensured during the reaction. Using a water separator based on the azeotropic principle of toluene and water, 30 mL of the toluene-water mixture is separated to remove any remaining water, leaving 20 mL of the reaction mixture in the double-necked flask. The oil bath temperature is lowered to 115 °C. 250 μL of stannous octoate is rapidly added dropwise to the reaction system as a catalyst, and the reaction is continued under reflux at 115 °C for 3 hours. After the reaction was complete, the reaction solution was cooled to room temperature, and 150 mL of dichloromethane (DCM) and three drops of 0.1 M dilute hydrochloric acid solution were added to quench the reaction. The quenched reaction solution was washed with water several times until the upper aqueous phase changed from acidic to neutral, and then the organic phase was separated. The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated to obtain a transparent viscous crude product. Finally, the crude product was dissolved with a small amount of DCM, and then n-hexane was added dropwise while stirring, accompanied by the appearance of a white flocculent precipitate. After the addition was completed, the mixture was allowed to stand for a period of time, and the supernatant was discarded. The above process was repeated three times. The precipitate was dried in a vacuum drying oven to obtain the final product PEG. 5K -PLA 5K (10.75g, yield 71.67%). 1 HNMR(400MHz,Chloroform-d)δ5.25-5.08(m,75H),3.80-3.42(s,520H),3.38-3.34(s,3H),1.64-1.49(m,230H).

[0089] Using the same ring-opening polymerization method, by changing the amount of polyethylene glycol with different molecular weights as initiators and adding corresponding amounts of lactide according to the required molecular weight of PLA, PEGs with different molecular weights were synthesized. 2k -PLA 2k and PEG 1k -PLA 1k Polymer carriers. For example, in the preparation of mPEG. 2k -PLA 2k Under nitrogen protection, lactide (7.875 g, 0.0547 mol) and polyethylene glycol 2000 (7.5 g, 0.0038 mol) were added sequentially to a 250 mL two-necked flask containing 50 mL of anhydrous toluene. The remaining steps were repeated under the same conditions as described above for mPEG. 5K -PLA 5K The preparation process.

[0090] Example 11 Synthesis of drug-loaded nanomicelles

[0091] Drug-loaded nanoparticles were prepared via a nanoprecipitation method. Taking drug-loaded nanomicelles PP5T5N as an example, and using the amphiphilic polymer PEG... 5K -PLA 5K The polymer prodrug encapsulated as a carrier is TPLA. 5K @SN38. The polymeric prodrug TPLA 5K @SN38 was dissolved in acetone to prepare a prodrug solution containing 2 μg / μL of free SN38 drug. Example 10 shows the synthesized PEG. 5K -PLA 5K The carrier was prepared as a 20 μg / μL acetone solution. 100 μL of each carrier were mixed in equal volumes to form the oil phase. 2 mL of water was placed in a sample vial to form the aqueous phase. The oil phase was added dropwise to the continuously stirred aqueous phase at an O / W ratio of 1:10 to form a nanoparticle solution. After the addition was complete, stirring was continued for approximately 20 min. Acetone was removed by vacuum distillation to obtain an aqueous solution of polymer nanoparticles with a concentration of 1 mg / mL.

[0092] Blank carrier micelles were prepared using the same method, but without encapsulating polymer prodrugs. Figure 4 The image shows a TEM image of PP5T5N. The uniform circular morphology of the nanomicelles indicates that the prepared polymer-coupled prodrug and the polymer carrier PEG-PLA formed a stable nanomicelle structure through self-assembly.

[0093] In addition, drug-loaded nanomicelles PP5M5N, PP5M2N, PP5M1N, PP5D5N, PP5D2N, PP5D1N, PP5T5N, PP5T2N and PP5T1N were prepared according to the methods of Examples 10 and 11, respectively.

[0094] Example 12: Study on the storage stability of nanomicelles

[0095] Different drug-loaded nanomicelle aqueous solutions with a concentration of 1 mg / mL, prepared according to the method of Example 11, were stored at room temperature and in a cool place. Samples were taken at 0, 1, 3, 5, and 7 days after preparation, and the changes in particle size and PDI were detected by DLS. Figure 5 and 6 As shown, the prepared polylactic acid modified prodrug nanomicelles exhibit good stability and can maintain their structural integrity for seven days. Figure 5 Specifically, the particle size distribution of nanomicelles constructed by coupling prodrugs and carriers with polylactic acid of different molecular weights and arm numbers is presented.

[0096] Figure 6The horizontal axis 0 / 1 / 3 / 5 / 7 represent the sampling and analysis results at 0 days, 1 day, 3 days, 5 days, and 7 days of preparation, respectively. MPLA5 represents TPLA. 5k @SN38 NPs and a carrier were prepared in an aqueous solution of nanomicelles according to the method in Example 11.

[0097] Example 13 Determination of drug-loaded nanomicelle encapsulation

[0098] After reviewing literature and performing full-wavelength scanning, it was determined that the drug loading and encapsulation efficiency of drug-loaded nanomicelles could be determined by measuring the absorbance at 378 nm using a UV-Vis spectrophotometer. A stock solution of 1 mg / mL was prepared by dissolving 7-ethyl-10-hydroxycamptothecin (SN38) in DMSO. Different volumes of this stock solution were then diluted with ultrapure water to a series of concentrations: 0 μg / mL, 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL. The absorbance (A) was measured at 378 nm using a UV-Vis spectrophotometer. A regression equation was established by fitting the absorbance (y) to the SN38 drug concentration (x), resulting in a standard curve. Figure 7 As shown.

[0099] The absorbance of the prepared drug-loaded nanogel was measured using a UV-spectrophotometer, and the drug loading in the polymer was calculated using a standard curve.

[0100] Drug loading DL (%) = Amount of drug contained in nanoparticles / (Amount of drug contained + Amount of PLA in polymer-coupled prodrug + Amount of carrier added) × 100%.

[0101] Encapsulation efficiency EE (%) = Amount of drug contained in nanoparticles / Total amount of drug input × 100%.

[0102] Table 1 shows the drug loading and encapsulation efficiency of polymer prodrug-loaded nanomicelles. The results indicate that, under the condition of consistent SN38 content in the prodrug, the amount of polymer-coupled prodrug required for preparing nanomicelles is significantly reduced for two-armed and four-armed polymers compared to single-armed polymer prodrugs of the same molecular weight. Further experimental data show that despite the significantly reduced prodrug loading, the drug loading of two-armed and four-armed polymers remains almost identical to that of single-armed polymers, and in some cases even surpasses that of single-armed polymers. Furthermore, two-armed and four-armed polymers also exhibit higher encapsulation efficiencies. These results demonstrate that two-armed and four-armed polymer structures can achieve comparable or higher drug loading and encapsulation efficiencies with lower prodrug loading amounts, thus optimizing the performance of the drug delivery system.

[0103] Table 1. Drug loading and encapsulation efficiency of drug-loaded nanomicelles

[0104]

[0105] Table 1 shows the prodrug dosage. For the same molecular weight, different numbers of arms result in different drug loading efficiencies. When the amount of SN38 in the prodrug is kept consistent (i.e., the molar amount of SN38 is fixed, assuming it is 4 mol), then a single-arm prodrug requires 4 mol to have 4 mol of SN38. For two arms, only 2 mol of polymer prodrug is needed, and so on. For four arms, only 1 mol of polymer prodrug is needed because each arm of a four-arm compound can be loaded with a drug.

[0106] Example 14: In vitro release of drug-loaded nanomicelles

[0107] The in vitro release of drug-loaded nanomicelles was determined by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Welch XB-C18 column, UV wavelength 378 nm, mobile phase 60% methanol, 40% water, column temperature 37 °C, and flow rate 1 mL / min. 7-Ethyl-10-hydroxycamptothecin (SN38) was dissolved in DMSO to prepare a stock solution of 1 mg / mL. Then, different volumes of the stock solution were diluted with ultrapure water to a series of concentrations: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, 0.78125 μg / mL, 0.390625 μg / mL, 0.1953125 μg / mL, 0.09765625 μg / mL, 0.048828125 μg / mL, 0.024414063 μg / mL, 0.012220703 μg / mL, 0.006103516 μg / mL, and 0 μg / mL. Linear regression was performed on the peak areas of pure SN38 detected by HPLC with respect to concentration using a series of concentration gradients, resulting in the standard curve of SN38: y = 42444x⁻¹(R₀). 2 =0.9998), such as Figure 8 As shown.

[0108] Take 1 mL of 120 U / mL esterase solution as the enzymatic group or 1 mL of pH 7.4 phosphate buffered saline (PBS) as the blank control group. Add 2 mL of 1 mg / mL drug-loaded nanomicelle aqueous solution to 5 mL EP tubes, vortex to mix thoroughly, and then transfer to dialysis bags with a molecular weight cutoff of 8000 Da. Secure both ends with dialysis clamps and immerse the dialysis bag in a centrifuge tube containing 20 mL of release medium (pH 7.4 phosphate buffered saline containing 0.4% Tweens 80). Place the centrifuge tubes in a constant temperature shaker, adjust the instrument parameters to 100 rpm and 37 °C, and take 1 mL of the release medium from the centrifuge tubes at time points of 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h, while simultaneously adding 1 mL of the original release medium solution. The 1 mL external liquid was filtered through a syringe filter. Under the conditions of UV wavelength of 378 nm, mobile phase of 60% CH3OH and 40% water, column temperature of 37 °C and flow rate of 1 mL / min, the SN38 content was determined by HPLC. The SN38 release rate at a specific time point was calculated by substituting the solution into the standard curve.

[0109] Drug release in vitro from drug-loaded nanomicelles is as follows: Figure 9 , 10 As shown in Figure 11, the experimental results indicate that, in the presence of esterase, the drug-loaded nanomicelles achieved 85% of their maximum release within 192 hours, while the control group (without esterase) released only 30% in the same time. Due to the difference in esterase activity between normal and tumor cells, this result demonstrates that drug-loaded nanomicelles can achieve optimal drug release under pathological conditions.

[0110] Example 15 Cytotoxicity Experiment of Drug-Loaded Nanomicelles

[0111] This invention uses the MTT assay to assess the biosafety of polymer carriers and the cytotoxic effect of polymer prodrug nanomicelles on tumor cells (preparation method see Example 11). A549 cells were seeded in 96-well plates (5000 cells per well) and cultured at 37°C for 24 h. After cell adhesion, 200 μL of DMEM medium was replaced in each well, followed by the addition of the prepared nanomicelle aqueous solution or free SN38. After 72 h of incubation, 20 μL of 5 mg / mL MTT reagent was added to each well, and incubation continued for 4 h. The medium was then aspirated, and 150 μL of DMSO was added. The absorbance was measured at 490 nm using a microplate reader. Untreated cells served as a positive control, and DMEM served as a negative control. The experimental results are as follows: Figure 12 As shown, this indicates the selected polymer carrier PEG. 5K -PLA 5KIt exhibits good biocompatibility, and the prepared polymer prodrug nanomicelles also have strong cytotoxicity against tumor cells.

[0112] Example 16 Cellular uptake experiment of drug-loaded nanomicelles

[0113] This invention uses coumarin 6 as a hydrophobic drug model, encapsulating it in micelles and investigating its uptake by cells in vitro using fluorescence microscopy. A549 cells were seeded into 12-well plates (50,000 cells per well) and cultured at 37°C for 24 h. Each well was then replaced with 1000 mL of LDM-MEM medium, and a series of nanomicelles encapsulating coumarin 6 and a polymer prodrug were added (ensuring a final concentration of 5 μg / mL for both coumarin 6 and the SN38 loaded in the prodrug). Cells were cultured for 2 h and 6 h, respectively. After incubation, the medium was removed, and the cells were washed three times with PBS. 1 mL of 4% paraformaldehyde was added to each well for fixation at 4°C for 30 min, followed by three more washes with PBS. Then, 1 mL of 5 μg / mL DAPI was added to each well for fixation at 37°C for 20 min to facilitate nucleus localization. Images were captured using a fluorescence microscope, and the results are shown below. Figure 13 As shown, DAPI (blue fluorescence) is used to label cell nuclei, and Coumarin-6 (coumarin-6, green fluorescent dye) is used as a fluorescently labeled probe. The Merge diagram shows the colocalization overlay of the fluorescence signals of the two. Figure 13 As the incubation time increased, the green fluorescence intensity of coumarin 6 increased, and the overlap with the blue fluorescence of the cell nucleus also increased, indicating that the prepared nanomicelles had good uptake in the cells.

[0114] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multi-armed polymeric conjugate prodrug, characterized in that, It is a multi-arm polylactic acid for structural modification of hydrophobic drugs, wherein the multi-arm polylactic acid is prepared by ring-opening polymerization of propylene lactone with small molecules with different hydroxyl numbers as initiators; the multi-arm polylactic acid is modified by succinic anhydride to form carboxyl groups, and then esterification or amidation reaction occurs between the carboxyl groups and hydroxyl groups or amino groups of hydrophobic drugs, thereby forming the multi-arm polymer conjugated prodrug, which has the following structural formula: In the formula, R1 represents a polymer group of the multi-arm polylactic acid, n represents the number of arms of the polymer group, and n is an integer from 2 to 5; R2 represents a drug group; The multi-arm polymer conjugated prodrug has one of the following structures: 1) when the number of arms of the polymer n = 2, the structural formula is one of the following: 2) when the number of arms of the polymer n = 4, the structural formula is one of the following: R3 is selected from one of the following: R4 is selected from the following structures: 3) when the number of arms of the polymer n = 5, the structural formula is one of the following: R5 is selected from the following structures: wherein m and i represent the degree of polymerization and are positive integers.

2. The multi-arm polymer conjugated prodrug of claim 1, wherein, The drug group R2 is selected from one of the following:

3. A polymeric conjugated prodrug nanomicelle, characterized in that, The application also relates to a multi-arm polymer conjugated prodrug and a polymer carrier material.

4. The polymeric conjugate prodrug nanomicelle of claim 3, wherein the polymer is a poly (ethylene glycol) -poly (propylene glycol) -poly (ethylene glycol) tri-block copolymer (PEG-PPG-PEG). The polymer carrier material is selected from one or more of polyethylene glycol-polylactic acid, polyethylene glycol-polylactic acid-glycolic acid.

5. The method for preparing polymer-coupled prodrug nanomicelles as described in claim 3, characterized in that, The application also relates to a preparation method of the polymer prodrug nanomicelles, which comprises the following steps: The multi-arm polymer conjugated prodrug and the polymer carrier material are dispersed in an organic solvent to form an oil phase solution; the oil phase solution is added dropwise into water under continuous stirring, and after the dropwise addition is completed, the stirring is continued for 5-30 min, and then the organic solvent is removed by distillation under reduced pressure, thereby obtaining the polymer prodrug nanomicelles.

6. The method for preparing polymer-coupled prodrug nanomicelles as described in claim 5, characterized in that, The volume ratio of the oil phase solution to water is 1:8-12.

7. The method for preparing polymer-coupled prodrug nanomicelles as described in claim 5, characterized in that, The mass ratio of the multi-arm polymer conjugated prodrug to the polymer carrier material is 1:8-12.

8. The method for preparing polymer-coupled prodrug nanomicelles as described in claim 5, characterized in that, The organic solvent is one or more of propanol, methanol, ethanol, THF, DMF and DMSO.

9. The use of a polymeric conjugated prodrug nanomicelle according to claim 3, wherein the drug is a hydrophobic drug. The application is the use of the polymer conjugated prodrug nanomicelles for loading chemotherapeutic drugs for targeted delivery in vivo.

Images