A preparation method of tumor-targeting pH / reduction-sensitive polymersomes

Abstract

This invention discloses a method for preparing tumor-targeting pH / reduction-sensitive polymer vesicles, relating to the fields of biomaterials and biomedicine. This invention prepares RGD-functionalized tumor-targeting cationic block copolymer RGD-PEG-PLys(N3) and pH-responsive anionic polymer PLYs(ss-DBCO / CAA), and uses RGD-PEG-PLys(N3) and PLYs(ss-DBCO / CAA) to prepare polymer vesicles exhibiting both pH and reduction responsiveness.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and biomedicine, specifically to a method for preparing tumor-targeting pH / reduction-sensitive polymer vesicles. Background Technology

[0002] Currently, researchers have developed various nanocarriers for tumor treatment, such as liposomes, polymers, inorganic nanoparticles, and viral vectors. Generally, the physical binding of carriers and drugs occurs through hydrophobic interactions or electrostatic interactions. However, nanosystems formed through self-assembly via physical forces exhibit poor stability in complex physiological environments, potentially leading to premature leakage and insufficient drug delivery to the target site, or even adverse effects on other organs. Furthermore, chemically bonded drug delivery suffers from incomplete or delayed drug release, or compromised drug activity. Summary of the Invention

[0003] To address the problems of existing technologies, this invention provides a method for preparing tumor-targeting pH / reduction-sensitive polymer vesicles.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] A method for preparing tumor-targeting pH / reduction-sensitive polymer vesicles includes the following steps:

[0006] 1. Polymer Synthesis

[0007] (1) Synthesis of Plys(ss-DBCO / CAA)

[0008] 1) Synthesis of Plys

[0009] ① In an inert gas environment, trifluoroacetyl-L-lysine intracyclic anhydride (Lys(TFA)-NCA) powder was dissolved in anhydrous N,N-dimethylformamide (DMF) to obtain a Lys(TFA)-NCA solution. Subsequently, a dichloromethane (DCM) solution of n-butylamine was added to the Lys(TFA)-NCA solution to initiate a ring-opening polymerization reaction. The reaction system was stirred in an oil bath at 25℃-30℃ for 48-72 hours. The reaction solution was collected as a precipitate in pre-cooled diethyl ether and then vacuum dried to obtain a white solid Plys(TFA).

[0010] ② Dissolve Plys(TFA) in a methanol solution containing NaOH, then place the reaction system in an oil bath at 25-30℃ and stir for 12-24 hours to remove the TFA protecting group. Transfer the reaction solution to a dialysis bag for dialysis, and then freeze-dry the dialyzed sample to obtain a white solid Plys.

[0011]

[0012] The molar ratio of n-butylamine to Lys(TFA)-NCA is 1:90; the concentration of the Lys(TFA)-NCA solution is 0.1-0.25 mg / mL, preferably 0.25 mg / mL; the volume ratio of n-butylamine to DCM in the n-butylamine DCM solution is 1:9; and the ratio of Lys(TFA)-NCA to the methanol solution containing NaOH is 0.37-5 mmol:5-20 mL, preferably 3.7 mmol:5 mL.

[0013] Based on the above technical solutions, preferably, the inert gas is argon or nitrogen.

[0014] Based on the above technical solutions, preferably, the concentration of NaOH in the NaOH-containing methanol solution is 1-2M, and more preferably 1M.

[0015] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da, and more preferably 3500 Da.

[0016] Based on the above technical solution, preferably, the dialysis involves first dialysis in an HCl solution, followed by dialysis in ultrapure water, with a dialysis time of 18-72 hours, including 9-36 hours of dialysis in the HCl solution and 9-36 hours of dialysis in ultrapure water; the concentration of the HCl solution is 0.01M. For example, dialysis can be performed three times, each time in 0.01M HCl solution and ultrapure water, with each dialysis session lasting 3-12 hours.

[0017] 2) Synthesis of Plys(ss-DBCO)

[0018] Dibenzocyclooctyn-disulfide-carboxylic acid (DBCO-ss-COOH) was dissolved in DMF, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added. The reaction system was then stirred in an oil bath at 25-30°C for 3-5 hours to activate the carboxyl groups, yielding a carboxyl-activated reaction solution. Plys solid was dissolved in Hepes buffer and added to the carboxyl-activated reaction solution. The reaction system was then stirred in an oil bath at 25-30°C for 12-24 hours. The reaction solution was then transferred to a dialysis bag for dialysis, and the dialyzed sample was freeze-dried to obtain a white Plys(ss-DBCO) solid.

[0019]

[0020] The molar ratio of DBCO-ss-COOH to EDC·HCl and NHS is 1:1.2-1.5:1.2-1.5, preferably 1:1.5:1.5; the ratio of DBCO-ss-COOH to DMF is 9.8 μmol:4.0 mL; the concentration of Plys in Hepes buffer is 5.0-10.0 mg / mL, preferably 9.0 mg / mL; and the molar ratio of DBCO-ss-COOH to Plys is 5:1.

[0021] Based on the above technical solutions, preferably, the pH of the Hepes buffer solution is 8.0-9.0, and more preferably 8.4.

[0022] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da, and more preferably 3500 Da.

[0023] Based on the above technical solutions, preferably, the dialysis is performed in ultrapure water for 9-36 hours, for example, dialysis is performed three times in ultrapure water, with each dialysis session lasting 3-12 hours.

[0024] 3) Synthesis of Plys(ss-DBCO / CAA)

[0025] PLYs(ss-DBCO) was dissolved in NaHCO3 buffer, and then cis-aconitine (CAA) was added. The reaction system was stirred at 0-4℃ for 2-4 hours. Subsequently, the reaction solution was transferred to an ultrafiltration tube and centrifuged to remove unreacted CAA. The solution was then freeze-dried to obtain a white solid of PLYs(ss-DBCO / CAA).

[0026]

[0027] The concentration of Plys(ss-DBCO) in NaHCO3 buffer is 0.5-1 mg / mL, preferably 1 mg / mL; the molar ratio of Plys(ss-DBCO) to CAA is 1:10-1:50, preferably 1:10.

[0028] Based on the above technical solutions, preferably, the concentration of the NaHCO3 buffer solution is 0.1-0.5M, more preferably 0.1M; the pH of the NaHCO3 buffer solution is 9.0-10.0, more preferably 10.0.

[0029] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the ultrafiltration tube is 3000-5000 Da, for example, the ultrafiltration tube (MWCO 3000 Da) is centrifuged three times.

[0030] (2) Synthesis of RGD-PEG-PLys(N3)

[0031] 1) Synthesis of RGD-PEG-NH2

[0032] ① Dissolve arginine-glycine-aspartic acid polypeptide (cRGDfk) in NaHCO3 buffer, then add tert-butyl-imine-polyethylene glycol-active ester (NHS-PEG-NH-BOC), and stir the reaction in an oil bath at 25-30℃ for 3-5 hours. Transfer the reaction solution to an ultrafiltration tube and centrifuge to remove unreacted cRGDfk. Then freeze-dry the sample to obtain RGD-PEG-NH-BOC white solid.

[0033] ② Dissolve the lyophilized RGD-PEG-NH-BOC in HCl solution and stir in an oil bath at 25-30℃ for 12-24 hours to remove the protective BOC groups. Transfer the reaction solution to a dialysis bag for dialysis, and then lyophilize the dialyzed sample to obtain a white solid RGD-PEG-NH2.

[0034]

[0035] The concentration of cRGDfk in NaHCO3 buffer is 0.5-1 mg / mL, preferably 1 mg / mL; the molar ratio of NHS-PEG-NH-BOC to cRGDfk is 1:2-1:5, preferably 1:3; the ratio of NHS-PEG-NH-BOC to HCl solution is 40.0 μmol:10-20 mL, preferably 40.0 μmol:10 mL.

[0036] Based on the above technical solutions, preferably, the concentration of the NaHCO3 buffer solution is 0.1-0.5M, more preferably 0.1M; the pH of the NaHCO3 buffer solution is 9.0-10.0, more preferably 10.0.

[0037] Based on the above technical solutions, preferably, the weight-average molecular weight of the NHS-PEG-NH-BOC is 5000 Da.

[0038] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the ultrafiltration tube is 3000-5000 Da, preferably 3000 Da, for example, by centrifuging three times in an ultrafiltration tube (MWCO 3000 Da).

[0039] Based on the above technical solutions, preferably, the concentration of the HCl solution is 1-2M, and more preferably 1M.

[0040] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da, and more preferably 3500 Da.

[0041] Based on the above technical solutions, preferably, the dialysis is performed in ultrapure water for 9-36 hours, for example, dialysis is performed three times in ultrapure water, with each dialysis session lasting 3-12 hours.

[0042] 2) Synthesis of RGD-PEG-PLys

[0043] ① First, the obtained RGD-PEG-NH2 is dried: RGD-PEG-NH2 is dissolved in a small amount of DCM, excess benzene is added and mixed evenly, frozen and dried under reduced pressure with cold hydrazine, and then transferred to an anhydrous and oxygen-free environment to obtain the dried RGD-PEG-NH2.

[0044] ② In an inert gas environment, the dried RGD-PEG-NH2 was dissolved in DMF to obtain a DMF solution of RGD-PEG-NH2. In the same inert gas environment, Lys(TFA)-NCA was dissolved in DMF to obtain a DMF solution of Lys(TFA)-NCA. The RGD-PEG-NH2 and Lys(TFA)-NCA DMF solutions were then mixed, and the reaction system was stirred in an oil bath at 25-30℃ for 48-72 hours. The reaction solution was collected as a precipitate in pre-cooled diethyl ether and dried under vacuum to obtain a white solid, RGD-PEG-PLys(TFA).

[0045] ③ Dissolve RGD-PEG-PLys (TFA) in a methanol solution containing NaOH. Place the reaction system in an oil bath at 25-30℃ and stir for 12-24 hours to remove the TFA protecting group. Transfer the reaction solution to a dialysis bag for dialysis. Then freeze-dry the dialyzed sample to obtain a white solid RGD-PEG-PLys.

[0046]

[0047] The ratio of RGD-PEG-NH2 to a small amount of DCM and excess benzene is 3.9 μmol: 1-2 mL: 8-16 mL, preferably 3.9 μmol: 1 mL: 8 mL; the ratio of RGD-PEG-NH2 to DMF in the RGD-PEG-NH2 DMF solution is 3.9 μmol: 4-8 mL, preferably 3.9 μmol: 4 mL; the ratio of Lys(TFA)-NCA to DMF in the Lys(TFA)-NCA DMF solution is 353.3 μmol: 4-8 mL, preferably 353.3 μmol: 4 mL; the molar ratio of Lys(TFA)-NCA to RGD-PEG-NH2 is 90:1; the ratio of Lys(TFA)-NCA to a methanol solution containing NaOH is 353.3 μmol: 5-20 mL, preferably 353.3 μmol: 5 mL.

[0048] Based on the above technical solutions, preferably, the inert gas is argon or nitrogen.

[0049] Based on the above technical solutions, preferably, the concentration of NaOH in the NaOH-containing methanol solution is 1-2M, and more preferably 1M.

[0050] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the dialysis bag is 3000-5000 Da, and more preferably 3500 Da.

[0051] Based on the above technical solution, preferably, the dialysis involves first dialysis in an HCl solution, followed by dialysis in ultrapure water, with a dialysis time of 18-72 hours, including 9-36 hours of dialysis in the HCl solution and 9-36 hours of dialysis in ultrapure water; the concentration of the HCl solution is 0.01M. For example, dialysis can be performed three times, each time in 0.01M HCl solution and ultrapure water, with each dialysis session lasting 3-12 hours.

[0052] 3) Synthesis of RGD-PEG-PLys(N3)

[0053] RGD-PEG-PLys was dissolved in NaHCO3 buffer, and active ester polyethylene glycol azide (NHS-PEG4-N3) was added. The reaction system was placed in an oil bath at 25-30℃ and stirred for 4-12 hours. The reaction solution was transferred to an ultrafiltration tube and centrifuged to remove unreacted NHS-PEG4-N3. The sample was then freeze-dried to obtain RGD-PEG-PLys(N3) as a white solid.

[0054]

[0055] The concentration of RGD-PEG-Plys in NaHCO3 buffer is 0.5-1 mg / mL, preferably 1 mg / mL; the molar ratio of RGD-PEG-Plys to NHS-PEG4-N3 is 1:5.

[0056] Based on the above technical solutions, preferably, the concentration of the NaHCO3 buffer solution is 0.1-0.5M, more preferably 0.1M; the pH of the NaHCO3 buffer solution is 9.0-10.0, more preferably 10.0.

[0057] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the ultrafiltration tube is 3000-5000 Da, preferably 3000 Da; for example, the ultrafiltration tube (MWCO 3000 Da) is centrifuged three times.

[0058] 2. Preparation of polymer vesicles

[0059] The cationic block copolymer RGD-PEG-PLys(N3) (hereinafter referred to as RPL(N3)) and the anionic polymer PLYs(ss-DBCO / CAA) (hereinafter referred to as L(ss-D / C)) first recombine in a buffer solution via electrostatic interaction. The -N3 on the RGD-PEG-PLys backbone and the -DBCO on the PLYs backbone undergo a copper-free click reaction, thereby achieving bonding of the anionic / cationic block copolymer chains and forming a structurally stable vesicle structure RPL-ss-LC with an aqueous cavity. The specific preparation method is as follows:

[0060] (1) Dissolve RPL(N3) and L(ss-D / C) in Hepes buffer to obtain RPL(N3) solution and L(ss-D / C) solution respectively.

[0061] (2) Subsequently, the above RPL(N3) solution and L(ss-D / C) solution were added to Hepes buffer solution at the same time. After stirring at 25-30°C for 24-48 hours, the unreacted polymer molecules were removed by centrifugation using an ultrafiltration tube to obtain polymer vesicles RPL-ss-LC.

[0062] The concentrations of both the RPL(N3) solution and the L(ss-D / C) solution are 0.5 mg / mL, and the volume ratio of the RPL(N3) solution, the L(ss-D / C) solution to the Hepes buffer is 1:1:10-20, preferably 1:1:20.

[0063] Based on the above technical solutions, preferably, the pH of the Hepes buffer solution is 7.4 and the concentration is 10mM.

[0064] Based on the above technical solutions, preferably, the molecular weight cutoff (MWCO) of the ultrafiltration tube is 20,000-50,000 Da, and more preferably 30,000 Da.

[0065] Based on the above technical solution, preferably, the obtained polymer vesicle solution is concentrated, for example, until the concentration of polymer vesicle RPL-ss-LC is 1 mg / mL.

[0066] The present invention also relates to protecting tumor-targeting pH / reduction-sensitive polymeric vesicles prepared by the above method, wherein the resulting polymeric vesicles can be used to deliver drugs, such as proteins.

[0067] Polymer vesicles are hollow spheres formed by the self-assembly of amphiphilic block copolymers in water. They can encapsulate target drugs within their cavities, and their sizes typically range from tens of nanometers to tens of micrometers. Due to their structural specificity and functionalizability, dense polymer vesicles can not only stably encapsulate drugs and prevent premature leakage, but also maintain the integrity of the drug's structure. By designing polymer structures, nanodelivery systems can possess intelligent stimulus-responsive functions, such as stimulus responsiveness related to physiological signals: the slightly acidic environment (pH < 6) of tumor tissue or intracellular lysosomes, and the reducing environment (2-10 mM GSH) in the cytoplasm. Therefore, polymer vesicles can significantly improve therapeutic efficacy by precisely controlling the transport and release of therapeutic drugs.

[0068] This invention prepares RGD-functionalized tumor-targeting cationic block copolymer RGD-PEG-PLys(N3) and pH-responsive anionic polymer PLYs(ss-DBCO / CAA). In Hepes buffer, polymer vesicles with drug-loadable cavities are formed sequentially through electrostatic interactions and click reactions of RGD-PEG-PLys(N3) and PLYs(ss-DBCO / CAA). Due to the interaction between the RGD ligands on the polymer vesicle surface and the α-coating surface of tumor cells... ν Due to the specific affinity of β3 integrin, polymeric vesicles accumulate at the tumor site and undergo endocytosis mediated by RGD. Following endocytosis, the polymeric vesicles entering the acidic intracellular endosomes experience charge reversal due to decarboxylation of the anionic polymer PLYs (ss-DBCO / CAA), exposing a large number of positively charged amino groups. Simultaneously, the electrostatic neutralization force with the cationic block copolymer RGD-PEG-PLys disappears, leading to an increase in the positive charge density of the cationic polymer chains PLYs. Therefore, the high-density positively charged polymeric vesicles disrupt the anionic endosome membrane and translocate to the cytoplasm. Finally, in response to extremely high levels of glutathione (GSH) in the cytoplasm, the polymeric vesicles cleave disulfide bonds between polylysine fragments, causing vesicle disintegration and releasing the intracellular drug.

[0069] The beneficial effects of this invention are:

[0070] (1) Through 1 H-NMR analysis verified the structures of RPL(N3), L(ss-D / C), and their intermediates, proving that RPL(N3) and L(ss-D / C) were successfully synthesized.

[0071] (2) Polymer vesicles RPL-ss-LC were successfully prepared with a hydrated particle size of about 128.6 nm and a zeta potential of about +3.9 mV.

[0072] (3) Polymer vesicle RPL-ss-LC has a low rate of erythrocyte hemolysis and cytotoxicity.

[0073] (4) The uptake efficiency of polymer vesicle cells modified by RGD is increased by about 4.2 times.

[0074] (5) The zeta potential of RPL-ss-LC in an acidic environment (pH 5.0) increased from +3.9mV to +6.1mV; LDH activity assays proved that polymer vesicles disrupted biofilms under pH 5.0 conditions.

[0075] (6) Deassembly behavior of RPL-ss-LC in a reducing environment (GSH: 10mM) (particle size increased to more than 2μm).

[0076] (7) RPL-ss-LC can prevent drug leakage in acidic environments and can release drugs rapidly in reducing environments. Attached Figure Description

[0077] Figure 1 1H NMR spectra of PLys(ss-DBCO / CAA) and its intermediates in D2O.

[0078] Figure 2 1H NMR spectra of RGD-PEG-PLys(N3) and its intermediates in D2O.

[0079] Figure 3 Particle size distribution of polymer vesicle RPL-ss-LC.

[0080] Figure 4 Particle size change of RPL-ss-LC after incubation in PBS buffer for 168 hours.

[0081] Figure 5 Red blood cell hemolysis rate of RPL-ss-LC polymer vesicles.

[0082] Figure 6 Cell survival rate of U87 cells after incubation of RPL-ss-LC polymer vesicles for 48 hours.

[0083] Figure 7Uptake of RNase A, RNase@PL-ss-LC, and RNase@RPL-ss-LC by U87 cells.

[0084] Figure 8 Uptake efficiency of U87 cells after incubation with RNase A, RNase@PL-ss-LC, or RNase@RPL-ss-LC for 4 hours and 24 hours.

[0085] Figure 9 Changes in zeta potential of empty vector vesicles RPL-ss-LC and protein polymer vesicles RNase@RPL-ss-LC after incubation at pH 7.4 or pH 5.0 for 12 hours.

[0086] Figure 10 LDH activity of PL-ss-LC and RPL-ss-LC after incubation with U87 cells at pH 7.4 or 5.0.

[0087] Figure 11 Particle size change of RPL-ss-LC after incubation at pH 5.0 and GSH for 2 hours.

[0088] Figure 12 Cumulative release curve of RNase A in RNase@RPL-ss-LC polymer vesicles after treatment with pH 5.0 and GSH.

[0089] Figure 13 Cell survival rate of U87 cells after incubation with RNase A, RNase@PL-ss-LC or RNase@RPL-ss-LC for 48 hours. Detailed Implementation

[0090] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0091] Example 1

[0092] (1) Synthesis of Plys(ss-DBCO / CAA)

[0093] 1) Synthesis of Plys

[0094] In an Ar glove box environment, Lys(TFA)-NCA powder (1 g, 3.7 mmol) was dissolved in anhydrous DMF (4.0 mL). Then, n-butylamine (41.5 μmol) diluted 10 times with DCM was added to the Lys(TFA)-NCA solution to initiate a ring-opening polymerization reaction. The reaction was stirred in an oil bath at 25 °C for 48 hours. The reaction solution was collected as a precipitate in pre-cooled diethyl ether and vacuum dried to obtain a white solid, PLYs(TFA). Plys (TFA) was dissolved in 5 mL of methanol solution containing 1 M NaOH. The reaction was then stirred in a 30 °C oil bath for 12 hours to remove the TFA protecting group. The reaction solution was transferred to a MWCO 3500 Da dialysis bag and dialyzed first in 0.01 M HCl, then in ultrapure water. This dialyzing was repeated three times in both 0.01 M HCl and ultrapure water, with each dialyzing session lasting 6 hours. The dialyzed sample was then freeze-dried to obtain a white Plys solid.

[0095]

[0096] 2) Synthesis of Plys(ss-DBCO)

[0097] 4.6 mg (9.8 μmol) of DBCO-ss-COOH was dissolved in 4.0 mL of DMF, and 1.5 molar amounts of EDC·HCl (2.8 mg) and NHS (1.7 mg) were added. The reaction was then stirred in an oil bath at 25 °C for 4 hours to activate the carboxyl groups. 18.0 mg of Plys (2.0 μmol) solid was dissolved in 2 mL of Hepes buffer (pH 8.4, 10 mM) and added to the above carboxyl-activated reaction solution. The reaction was stirred in an oil bath at 25 °C for 24 hours. The reaction solution was then transferred to a MWCO 3500 Da dialysis bag and dialyzed three times in ultrapure water for 6 hours each time. The dialyzed sample was then freeze-dried to obtain a white solid Plys(ss-DBCO).

[0098]

[0099] 3) Synthesis of Plys(ss-DBCO / CAA)

[0100] Plys(ss-DBCO) (20 mg) was dissolved in 20 mL of NaHCO3 buffer (0.1 M, pH 10.0) at a concentration of 1 mg / mL. Then, 10 molar amounts of CAA were added. The reaction mixture was stirred in an ice bath at 0°C for 4 hours. Subsequently, the reaction solution was transferred to an ultrafiltration tube (MWCO 3000 Da) and centrifuged three times to remove unreacted CAA. The solution was then freeze-dried to obtain a white solid of Plys(ss-DBCO / CAA).

[0101]

[0102] (2) Synthesis of RGD-PEG-PLys(N3)

[0103] 1) Synthesis of RGD-PEG-NH2

[0104] cRGDfk (Shanghai Jietai Biotechnology Co., Ltd.) (72.6 mg, 120.0 μmol) was dissolved in NaHCO3 buffer (0.1 M, pH 10.0) at a concentration of 1 mg / mL. Then, BOC-NH-PEG-NHS (Mw 5000 Da, 200 mg, 40.0 μmol) was added. The reaction was carried out in an oil bath at 25 °C and stirred for 4 hours. The reaction solution was transferred to an ultrafiltration tube (MWCO 3000 Da) and centrifuged three times to remove unreacted cRGDfk. The sample was then freeze-dried to obtain RGD-PEG-NH-BOC white solid. The lyophilized RGD-PEG-NH-BOC was dissolved in 10 mL of 1 M HCl and stirred in an oil bath at 25 °C for 12 hours to remove the protective BOC groups. The reaction solution was transferred to a MWCO 3500 Da dialysis bag and dialyzed three times in ultrapure water, each time for 6 hours. The dialyzed sample was then lyophilized to obtain a white solid RGD-PEG-NH2.

[0105]

[0106] 2) Synthesis of RGD-PEG-PLys

[0107] First, the obtained RGD-PEG-NH2 was dried: RGD-PEG-NH2 (22.0 mg, 3.9 μmol) was dissolved in a small amount of DCM (1 mL), and excess benzene (8 mL) was added and mixed thoroughly. The mixture was then frozen and dried under reduced pressure with hydrazine, and subsequently transferred to an anhydrous and oxygen-free glove box. Under Ar conditions in the glove box, Lys(TFA)-NCA (94.7 mg, 353.3 μmol) was dissolved in 4 mL of DMF, and then RGD-PEG-NH2 dissolved in 4 mL of DMF was added. The reaction was stirred in an oil bath at 25 °C for 48 hours. The reaction solution was precipitated in pre-cooled diethyl ether and collected, and then dried under vacuum to obtain a white solid RGD-PEG-PLys(TFA). RGD-PEG-PLys (TFA) was dissolved in 5 mL of methanol solution containing 1 M NaOH. The reaction was carried out in a 30 °C oil bath with stirring for 12 hours to remove the TFA protecting group. The reaction solution was transferred to a MWCO 3500 Da dialysis bag and dialyzed first in 0.01 M HCl, then in ultrapure water. Dialysis was repeated three times in both 0.01 M HCl and ultrapure water, with each dialyze lasting 6 hours. The dialyzed sample was then freeze-dried to obtain a white solid of RGD-PEG-PLys.

[0108]

[0109] 3) Synthesis of RGD-PEG-PLys(N3)

[0110] RGD-PEG-PLys (20.0 mg, 1.4 μmol) was dissolved in 20 mL of NaHCO3 buffer (0.1 M, pH 10.0), and 5 times the molar volume of NHS-PEG4-N3 (6.8 μmol, 2.6 mg) was added. The reaction was carried out in an oil bath at 25 °C with stirring for 4 hours. The reaction solution was transferred to an ultrafiltration tube (MWCO 3000 Da) and centrifuged three times to remove unreacted NHS-PEG4-N3. The sample was then freeze-dried to obtain RGD-PEG-PLys(N3) as a white solid.

[0111]

[0112] (3) Preparation of polymer vesicles

[0113] The cationic block copolymer RGD-PEG-PLys(N3) (hereinafter referred to as RPL(N3)) and the anionic polymer PLYs(ss-DBCO / CAA) (hereinafter referred to as L(ss-D / C)) first recombine in a buffer solution via electrostatic interaction. The -N3 on the RGD-PEG-PLys backbone and the -DBCO on the PLYs backbone undergo a copper-free click reaction, thereby achieving bonding between the anionic and cationic block copolymer chains and forming a structurally stable vesicle structure RPL-ss-LC with an aqueous cavity. Specifically, RPL(N3) and L(ss-D / C) are dissolved in Hepes buffer (pH 7.4, 10 mM) to obtain RPL(N3) solution and L(ss-D / C) solution, both with a concentration of 0.5 mg / mL. Subsequently, 0.5 mL of the above RPL(N3) solution and L(ss-D / C) solution were simultaneously added to 10 mL of Hepes buffer (pH 7.4, 10 mM). After stirring at room temperature for 24 hours, unreacted polymer molecules were removed by ultrafiltration (MWCO 30000 Da) and concentrated to a RPL-ss-LC polymer vesicle concentration of 1 mg / mL.

[0114] (4) Preparation of protein-loaded polymer vesicles

[0115] RNase A was used as a model protein to prepare protein-loaded polymer vesicles RNase@RPL-ss-LC: RNase A (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) was dissolved in 10 mL of Hepes buffer (pH 7.4, 10 mM) to obtain RNase A solutions with final concentrations of 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. RPL(N3) and L(ss-D / C) were dissolved in Hepes buffer (pH 7.4, 10 mM) to obtain RPL(N3) and L(ss-D / C) solutions, respectively, with a concentration of 0.5 mg / mL. Subsequently, 0.5 mL of RPL(N3) solution and L(ss-D / C) solution were added to the above RNase A solutions, stirred at room temperature for 24 hours, and then centrifuged through an ultrafiltration tube (MWCO 30000 Da) to remove unreacted polymer molecules and unloaded RNase A, and concentrated to a final volume of 1 mL.

[0116] Example 2

[0117] The structures of the cationic block copolymer RPL(N3) and the anionic polymer L(ss-D / C) prepared in Example 1 were verified. Subsequently, the physicochemical properties and biocompatibility of the empty carrier vesicles RPL-ss-LC and the protein-loaded vesicles RNase@RPL-ss-LC were investigated, and their cellular uptake capacity, pH responsiveness, reduction responsiveness, and cytotoxicity were verified. The results are as follows:

[0118] (1) 1H NMR spectra of Plys(ss-DBCO / CAA)

[0119] pass 1 The structures of Plys(ss-DBCO / CAA) and intermediate products were verified by 1H-NMR. Figure 1 PLYs shown 1 ¹H-NMR analysis revealed that the degree of polymerization of Lys was 71.0, calculated based on the integrated area of ​​the proton peak of the terminal methyl group (-CH₃) of n-butylamine (chemical shift: 0.8 ppm) and the proton peak of the methylene group (-CH₂-) of Lys (chemical shift: 1.3-1.9 ppm). The grafting degree of DBCO was calculated to be 4.3, based on the integrated area of ​​the proton peak of diphenylcyclooctynylene (chemical shift: 7.0-7.5 ppm) and the proton peak of the methylene group (-CH₂-) of Lys (chemical shift: 1.3-1.9 ppm). After grafting CAA, the peak appearing at chemical shifts of 5.8-6.0 ppm represents the methylene (-CH₂-) proton peak of aconitine; * indicates the olefin (C=CH₂) proton peak of itacamide (chemical shift: 5.5-7.0 ppm). These results indicate that PLYs(ss-DBCO / CAA) was successfully synthesized.

[0120] (2) 1H NMR spectrum of RGD-PEG-PLys(N3)

[0121] pass 1 The structures of RGD-PEG-PLys(N3) and its intermediates were verified by H-NMR. Figure 2As shown, the reaction efficiency of cRGDfk was calculated to be 78.0% based on the integrated area of ​​the methylene (-CH2-) proton peak (chemical shift: 3.7 ppm) in PEG and the benzyl (-C6H5) proton peak (chemical shift: 7.0-7.5 ppm) in cRGDfk. The degree of polymerization of Lys in RGD-PEG-PLys was calculated to be approximately 71.3 based on the integrated area of ​​the methylene (-CH2-) proton peak (chemical shift: 3.7 ppm) in PEG and the methylene (-CH2-) proton peak (chemical shift: 1.3-1.9 ppm) in Lys. After grafting NHS-PEG4-N3, the grafting degree of NHS-PEG4-N3 was calculated to be 4.2 based on the integrated area of ​​the methylene (-CH2-) proton peak (chemical shift: 1.3-1.9 ppm) next to the amide bond and the methylene (-CH2-) proton peak in PEG.

[0122] (3) Characterization of particle size and potential of polymer vesicles

[0123] The physicochemical properties of polymer vesicles were characterized using a nanoparticle size analyzer, such as... Figure 3 As shown, the hydrated particle size of the polymer vesicle RPL-ss-LC is 128.6±1.8nm, the PDI is 0.20±0.01, and the particle size distribution range is relatively narrow. The zeta potential is +3.9±0.3mV.

[0124] (4) Stability of polymer vesicles

[0125] The particle size stability of polymer vesicles was tested using PBS buffer to simulate a physiological environment. Furthermore, to investigate the important role of anion / cation polymer chain bonding in the structural stability of the RPL-ss-LC polymer vesicles, electrostatically composite RPL / LC polymer vesicles were also prepared. The specific preparation process was as follows: RGD-PEG-PLys (hereinafter referred to as RPL) and Plys(CAA) (hereinafter referred to as LC) were dissolved separately in Hepes buffer (pH 7.4, 10 mM), each at a concentration of 0.5 mg / mL. Subsequently, 0.5 mL of the RPL Hepes solution and 0.5 mL of the LC Hepes solution were simultaneously added to 10 mL of Hepes buffer (pH 7.4, 10 mM). After stirring at room temperature for 24 hours, unbound polymer molecules were removed by centrifugation using an ultrafiltration tube (MWCO 30000 Da), and the concentration of the RPL / LC polymer vesicles was concentrated to 1 mg / mL.

[0126] The particle size variation of RPL / LC polymer vesicles and RPL-ss-LC polymer vesicles was tested. Figure 4As shown, after incubation in PBS buffer at pH 7.4 for 168 hours, the particle size of RPL-ss-LC polymer vesicles remained almost unchanged. In stark contrast, the particle size of uncrosslinked RPL / LC polymer vesicles increased to 1995.3 ± 774.7 nm after incubation in PBS buffer at pH 7.4 for 168 hours. These results demonstrate that the anionic / cationic crosslinked polymers significantly improve the stability of the vesicle structure, maintaining a stable particle size even after incubation for up to one week in a physiological environment.

[0127] (5) Biocompatibility of polymer vesicles

[0128] The biocompatibility of polymer vesicles was assessed by detecting their blood compatibility and cytotoxicity.

[0129] The hemocompatibility of empty vector polymer vesicle RPL-ss-LC was evaluated by detecting the erythrocyte hemolysis rate. 1000g of sterile defibrinated sheep blood was centrifuged for 3 minutes to collect the erythrocyte pellet. The erythrocytes were resuspended in PBS and washed repeatedly before centrifugation until the supernatant was clear and colorless. Subsequently, the erythrocytes were resuspended in PBS and diluted to a concentration of 2% (v / v). 200 μL of the erythrocyte suspension was mixed with an equal volume of RPL-ss-LC (2.0 mg / mL) and Tween 20 (2.0 mg / mL) dissolved in PBS. The final erythrocyte concentration was 1% (v / v), and the final concentrations of RPL-ss-LC and Tween 20 were 1.0 mg / mL. Samples were placed in a 37°C constant-temperature shaker. 200 μL of sample was collected at 1 hour, 2 hours, and 12 hours, and centrifuged at 1000g for 3 minutes to settle red blood cells. 100 μL of the supernatant was collected into a 96-well plate, and the absorbance of the released hemoglobin at 540 nm was detected using a multi-functional plate reader. PBS and distilled water were used as negative and positive controls, respectively. Each test was performed three times. The hemolysis rate (%) was calculated using the formula: Hemolysis rate (%) = (OD0.05) / (OD0.05) 样品 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 )×100%. For example... Figure 5 As shown, even when RPL-ss-LC polymer vesicles were incubated with red blood cells at a concentration of 1.0 mg / mL for 12 hours, the red blood cell hemolysis rate remained at an extremely low level of 3.9%, which significantly improved blood compatibility compared to 0.2% Tween 20 (74.9%).

[0130] The cytotoxicity of empty carrier polymer vesicles RPL-ss-LC against U87 cells (glioma cells) was determined by the MTT assay. U87 cells were cultured at 1 × 10⁶ cells per well. 4Cells were seeded at a density of 10% in 96-well plates and cultured in DMEM medium containing 10% FBS. After incubation at 37°C and 5% CO2 for 24 hours, the medium was replaced with 100 μL of fresh medium containing different concentrations of RPL-SS-LC. After incubation at 37°C and 5% CO2 for 48 hours, the medium containing the sample was discarded, and the cells were washed twice with 100 μL of PBS. Subsequently, 100 μL of fresh medium containing MTT (0.5 mg / mL) was added to each well, and the cells were incubated at 37°C and 5% CO2 for 4 hours. Finally, the medium containing MTT was discarded, and 100 μL of DMSO was added and shaken to dissolve the formazan crystals. The absorbance at 570 nm and 630 nm (background) was measured using a multi-functional plate reader. Cells without sample treatment served as the control group, with three replicates per group. Cell viability was calculated using the formula: Cell viability (%) = (OD) / (Cells with different concentrations of RPL-SS-LC). 样品(570nm) -OD 样品(630nm) ) / (OD 对照(570nm) -OD 对照(630nm) )×100%. The result is as follows: Figure 6 As shown, when the concentration of RPL-ss-LC polymer vesicles was 200 μg / mL, the cell viability remained at 83.3% after incubation with cells for 48 hours, demonstrating good cell compatibility.

[0131] (6) Cellular uptake experiment of protein-loaded polymer vesicles

[0132] The uptake of the protein-loaded RNase A by U87 cells was observed using laser confocal microscopy. First, RNase A was labeled with Alexa Fluor 647NHS, and protein-loaded polymer vesicles RNase@RPL-ss-LC and RNase@PL-ss-LC were prepared. U87 cells were then loaded with 1×10⁻⁶ cells. 5 Cells were seeded at a density of [number] cells / mL in confocal culture dishes and cultured in DMEM medium containing 10% FBS. After incubation at 37°C and 5% CO2 for 24 hours, the medium was replaced with 2 mL of fresh medium containing RNase A, RNase@PL-ss-LC, or RNase@RPL-ss-LC (RNase A concentration: 50 μg / mL). After incubation at 37°C and 5% CO2 for 12 hours, the medium containing the sample was discarded, and the cells were washed twice with PBS. Subsequently, 2 mL of fresh medium containing Hoechst 33342 (1 μL / mL) was added to each well to stain the cell nuclei. After incubation at 37°C and 5% CO2 for 15 minutes, the cells were washed with PBS and observed under a laser confocal microscope. Figure 7As shown in the CLSM images, the fluorescence signal of RNase A in the unsupported RNase A experimental group was extremely weak, while significant fluorescence signals were observed in U87 cells of all experimental groups with polymer vesicles encapsulating RNase A. Compared to the RNase@PL-ss-LC experimental group without the RGD outer layer, the RGD-modified RNase@RPL-ss-LC experimental group showed a more significant RNase A fluorescence signal.

[0133] To further quantify the uptake efficiency of RNase A by U87 cells, Alexa Fluor647-labeled RNase A, RNase@PL-ss-LC, and RNase@RPL-ss-LC were prepared according to the above method. U87 cells were then subjected to a 1×10⁻⁶ ppm... 5 Cells were seeded at a density of 100 cells / mL in six-well plates and cultured in DMEM medium containing 10% FBS. After incubation at 37°C and 5% CO2 for 24 hours, the medium was replaced with 2 mL of fresh medium containing RNase A, RNase@PL-ss-LC, or RNase@RPL-ss-LC (RNase A concentration: 50 μg / mL). After incubation at 37°C and 5% CO2 for 4 hours or 24 hours, respectively, the medium containing the sample was discarded, and the cells were washed with PBS, digested with trypsin, and collected by pipetting in DMEM. Finally, the cells were resuspended in 300 μL of PBS, and the Alexa Fluor 647 signal intensity in the cells was detected by flow cytometry. In addition, the uptake capacity of U87 cells for polymer vesicles at different time points was quantified by FCM, and the results are shown below. Figure 8 As shown, after 4 hours of incubation with polymer vesicles, the uptake of RNase@RPL-ss-LC by cells was approximately 1.3 times higher than that of RNase@PL-ss-LC. However, after an incubation period of 24 hours, the uptake of RNase@RPL-ss-LC by cells was approximately 4.2 times higher than that of RNase@PL-ss-LC. In conclusion, RGD-functionalized polymer vesicles can significantly enhance the uptake of loaded proteins by glioma cells.

[0134] (7) pH responsiveness of polymer vesicles

[0135] The zeta potential changes of empty carrier polymer vesicles RPL-ss-LC and protein-carrying polymer vesicles RNase@RPL-ss-LC in physiological environment (pH 7.4) or lysosomal environment (pH 5.0) were measured using a nanoparticle size analyzer. The results are as follows: Figure 9As shown, the zeta potential of the carrier polymer vesicle RPL-ss-LC in PBS at pH 7.4 was +3.9 ± 0.3 mV, while after incubation for 12 hours in an acidic lysosomal environment (pH 5.0 PBS), the zeta potential increased to +6.1 ± 0.2 mV. The zeta potential of the protein-loaded polymer vesicle RNase@RPL-ss-LC in PBS at pH 7.4 was +1.0 ± 0.1 mV, while after incubation for 12 hours in PBS at pH 5.0, the zeta potential increased to +7.2 ± 0.3 mV. These results indicate that both empty carrier vesicles and protein-loaded vesicles exhibited an increase in positive charge, mainly attributed to the charge reversal of the anionic polymer chain L(ss-D / C) to a cationic polymer chain. Simultaneously, the amino groups of RPL(N3) lost their original anionic neutralizing charge, exposing a large number of amino groups, leading to a significant increase in the surface charge of the polymer vesicles.

[0136] (8) Lactate dehydrogenase (LDH) activity

[0137] The lysosomal escape ability of polymer vesicles was investigated by testing the LDH activity of cells treated with PL-ss-LC and RPL-ss-LC polymer vesicles at pH 7.4 and pH 5.0, respectively. U87 cells were cultured at 1 × 10⁻⁶ cells per well. 4 Samples were seeded at a density of [number] cells / well in 96-well plates and cultured in DMEM containing 10% FBS for 24 hours (37°C, 5% CO2). Simultaneously, the prepared PL-ss-LC and RPL-ss-LC were dissolved at a carrier concentration of 1 mg / mL in PBS buffer (pH 7.4 or pH 5.0, 200 μL, 10 mM) and incubated at room temperature for 12 hours. The 96-well plates were then removed, the original culture medium was discarded, and 100 μL of sample was added to each well. The plates were immediately transferred to 4°C and incubated for 2 hours. The supernatant was then collected, and LDH activity was measured according to the LDH kit instructions. The positive control group consisted of 0.2% Tween 20 solution, and the negative control group consisted of PBS (pH 7.4 or pH 5.0, 10 mM). All operations were performed at low temperatures to ensure sample integrity. LDH activity was calculated using the formula: LDH activity (%) = (OD[number] / [number]) * [ ... 样品 -OD 阴性对照 ) / (OD 阳性对照 -OD 阴性对照 ).like Figure 10As shown, the polymer vesicles of PL-ss-LC and RPL-ss-LC had negligible disruptive effect on the U87 cell membrane at pH 7.4, with LDH contents of approximately 1.2% and 9.5%, respectively, confirming that the cell membrane retained a high degree of integrity. In stark contrast, the LDH contents of the PL-ss-LC and RPL-ss-LC experimental groups increased to 14.8% and 17.7%, respectively, at pH 5.0, indicating that biomembrane damage led to a significant leakage of intracellular LDH into the extracellular space.

[0138] (9) Reduction responsiveness of polymer vesicles

[0139] Polymer vesicles were incubated sequentially at pH 5.0 and 10 mM GSH for 2 hours, and their particle size was measured by DLS. Figure 11 As shown, after incubation in PBS at pH 5.0 for 2 hours, the particle size of RPL-ss-LC increased from 105.8 ± 0.7 nm to 183.1 ± 2.7 nm. This is presumably due to the decarboxylation behavior of the polymer, which leads to the loss of electrostatic attraction between polymer chains, and the electrostatic repulsion resulting in a decrease in structural compactness and a slight increase in particle size. However, after the addition of GSH, the particle size of RPL-ss-LC increased to over 2 μm, demonstrating that the polymer vesicles had completely dissociated in response to the reducing environment.

[0140] To investigate the reduction-responsive drug release behavior of protein-loaded polymer vesicles, RNase A was used as a model protein, and RNase A labeled with Alexa Fluor 647 was prepared. Alexa Fluor 647 and RNase Alexa Fluor 647 @RPL-ss-LC polymer vesicles were analyzed, and the release of RNase A was measured under pH 5.0 and GSH conditions. Results are as follows: Figure 12 As shown, after 10 hours of treatment with protein polymer vesicle solution at pH 5.0, the release of RNase A was still less than 10%. However, after 1 hour of treatment with 10 mM GSH, the release of RNase A rapidly increased to 58.7%, and after 10 hours of treatment, the release increased to 98.7%.

[0141] (10) Protein-loaded polymer vesicle cytotoxicity assay

[0142] The cytotoxicity of RNase A, RNase@PL-ss-LC, and RNase@RPL-ss-LC to U87 cells was determined by the MTT assay, and the results are as follows: Figure 13As shown, after 48 hours of incubation with U87 cells using RNase A, RNase@PL-ss-LC, and RNase@RPL-ss-LC, the cell viability of the RNase A group remained at 96.1%, showing almost no effect, even at a concentration of 200 μg / mL. Significant cytotoxicity was observed in both the RNase@PL-ss-LC and RNase@RPL-ss-LC groups at different concentrations, with cytotoxicity increasing with increasing vesicle concentration. Furthermore, at an RNase A concentration of 200 μg / mL, the cell viability of the RNase@RPL-ss-LC group was only 32.4%, significantly lower than the cell viability of the RNase@PL-ss-LC group at the same concentration (47.0%).

Claims

1. A method for preparing tumor-targeting pH / reduction-sensitive polymer vesicles, characterized in that, Includes the following steps: (a) Polymer Synthesis (1) Synthesis of PLYs(ss-DBCO / CAA) 1) Synthesis of Plys ① In an inert gas atmosphere, Lys(TFA)-NCA powder was dissolved in anhydrous N,N-dimethylformamide (DMF) to obtain a Lys(TFA)-NCA solution. Subsequently, a dichloromethane solution of n-butylamine was added to the above Lys(TFA)-NCA solution, and the mixture was stirred in an oil bath at 25°C-30°C for 48-72 hours. The reaction solution was precipitated with diethyl ether and then dried under vacuum to obtain a white solid PLYs(TFA); wherein, Lys(TFA)-NCA is a trifluoroacetyl-L-lysine intracyclic anhydride. ② Dissolve Plys(TFA) in a methanol solution containing NaOH, stir in an oil bath at 25-30°C for 12-24 hours, dialyze the reaction solution, and then freeze-dry the dialyzed sample to obtain a white solid Plys. 2) Synthesis of Plys(ss-DBCO) DBCO-ss-COOH was dissolved in N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added. The mixture was stirred in an oil bath at 25-30°C for 3-5 hours to obtain a carboxyl-activated reaction solution. Plys solid was dissolved in Hepes buffer and added to the above carboxyl-activated reaction solution. The mixture was stirred in an oil bath at 25-30°C for 12-24 hours. The reaction solution was dialyzed, and the dialyzed sample was then freeze-dried to obtain Plys(ss-DBCO) white solid. 3) Synthesis of Plys(ss-DBCO / CAA) PLYs(ss-DBCO) was dissolved in NaHCO3 buffer, and then CAA was added. The mixture was stirred at 0-4°C for 2-4 hours. The reaction solution was centrifuged and freeze-dried to obtain a white solid PLYs(ss-DBCO / CAA), where CAA is cis-aconitine. (2) Synthesis of RGD-PEG-PLys(N3) 1) Synthesis of RGD-PEG-NH2 ① Dissolve cRGDfk in NaHCO3 buffer, then add NHS-PEG-NH-BOC, stir in an oil bath at 25-30°C for 3-5 hours, centrifuge the reaction solution, freeze dry to obtain RGD-PEG-NH-BOC white solid; ② Dissolve RGD-PEG-NH-BOC in HCl solution and stir in an oil bath at 25-30°C for 12-24 hours. Dialyze the reaction solution and then freeze-dry the dialyzed sample to obtain RGD-PEG-NH2 white solid. 2) Synthesis of RGD-PEG-PLys ① Drying treatment of RGD-PEG-NH2: Dissolve RGD-PEG-NH2 in dichloromethane, add benzene and mix well, freeze and dry under reduced pressure with cold hydrazine, and then transfer to an anhydrous and oxygen-free environment to obtain dried RGD-PEG-NH2; ② In an inert gas atmosphere, the dried RGD-PEG-NH2 was dissolved in N,N-dimethylformamide to obtain an N,N-dimethylformamide solution of RGD-PEG-NH2; in an inert gas atmosphere, Lys(TFA)-NCA was dissolved in N,N-dimethylformamide to obtain an N,N-dimethylformamide solution of Lys(TFA)-NCA; then the N,N-dimethylformamide solutions of RGD-PEG-NH2 and Lys(TFA)-NCA were mixed and stirred in an oil bath at 25-30°C for 48-72 hours. The reaction solution was precipitated with diethyl ether and then dried under vacuum to obtain a white solid RGD-PEG-PLys(TFA). ③ Dissolve RGD-PEG-PLys (TFA) in a methanol solution containing NaOH, stir in an oil bath at 25-30°C for 12-24 hours, dialyze the reaction solution, and then freeze-dry the dialyzed sample to obtain RGD-PEG-PLys white solid; 3) Synthesis of RGD-PEG-PLys(N3) Dissolve RGD-PEG-PLys in NaHCO3 buffer, add NHS-PEG4-N3, stir in an oil bath at 25-30°C for 4-12 hours, centrifuge the reaction solution, and then freeze-dry the sample to obtain RGD-PEG-PLys(N3) white solid. (II) Preparation of polymer vesicles (1) Dissolve RGD-PEG-PLys(N3) and PLYs(ss-DBCO / CAA) in Hepes buffer to obtain RGD-PEG-PLys(N3) solution and PLYs(ss-DBCO / CAA) solution respectively; (2) Add the above RGD-PEG-PLys(N3) solution and Plys(ss-DBCO / CAA) solution to Hepes buffer at the same time, stir at 25-30°C for 24-48 hours, and then centrifuge to obtain polymer vesicles; In the synthesis of PLYs, the molar ratio of n-butylamine to Lys(TFA)-NCA is 1:90; In the synthesis of PLYs(ss-DBCO), the molar ratio of DBCO-ss-COOH to EDC·HCl and NHS is 1:1.2-1.5:1.2-1.5; the molar ratio of DBCO-ss-COOH to PLYs is 5:

1. In the synthesis of PLYs(ss-DBCO / CAA), the molar ratio of PLYs(ss-DBCO) to CAA is 1:10-1:

50. In the synthesis of RGD-PEG-NH2, the molar ratio of NHS-PEG-NH-BOC to cRGDfk is 1:2-1:5; In the synthesis of RGD-PEG-PLys, the molar ratio of Lys(TFA)-NCA to RGD-PEG-NH2 is 90:1; In the synthesis of RGD-PEG-PLys(N3), the molar ratio of RGD-PEG-PLys to NHS-PEG4-N3 is 1:

5.

2. The preparation method according to claim 1, characterized in that, In the synthesis of PLYs, the concentration of the Lys(TFA)-NCA solution is 0.1-0.25 mg / mL; the volume ratio of n-butylamine to dichloromethane in the n-butylamine dichloromethane solution is 1:9; and the ratio of Lys(TFA)-NCA to a methanol solution containing NaOH is 0.37-5 mmol: 5-20 mL.

3. The preparation method according to claim 1, characterized in that, In the synthesis of PLYs(ss-DBCO), the ratio of DBCO-ss-COOH to DMF is 9.8 µmol: 4.0 mL; the concentration of PLYs in Hepes buffer is 5.0-10.0 mg / mL.

4. The preparation method according to claim 1, characterized in that, In the synthesis of RGD-PEG-NH2, the concentration of cRGDfk in NaHCO3 buffer is 0.5-1 mg / mL; the ratio of NHS-PEG-NH-BOC to HCl solution is 40.0 μmol: 10-20 mL.

5. The preparation method according to claim 1, characterized in that, In the synthesis of RGD-PEG-PLys, the ratio of RGD-PEG-NH2 to dichloromethane and benzene is 3.9 μmol: 1-2 mL: 8-16 mL; the ratio of RGD-PEG-NH2 to N,N-dimethylformamide in the N,N-dimethylformamide solution is 3.9 μmol: 4-8 mL; the ratio of Lys(TFA)-NCA to DMF in the N,N-dimethylformamide solution is 353.3 μmol: 4-8 mL; and the ratio of Lys(TFA)-NCA to a methanol solution containing NaOH is 353.3 μmol: 5-20 mL.

6. The preparation method according to claim 1, characterized in that, In the synthesis of RGD-PEG-PLys(N3), the concentration of RGD-PEG-PLys in NaHCO3 buffer is 0.5-1 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step (ii) preparation of polymer vesicles, the concentrations of the RGD-PEG-PLys(N3) solution and the Plys(ss-DBCO / CAA) solution are both 0.5 mg / mL, and the volume ratio of the RGD-PEG-PLys(N3) solution, the Plys(ss-DBCO / CAA) solution to the Hepes buffer is 1:1:10-20; the pH of the Hepes buffer is 7.

4.

8. The preparation method according to claim 1, characterized in that, In the synthesis of the polymer, the inert gas is argon or nitrogen; the concentration of NaOH in the methanol solution containing NaOH is 1-2 M; the pH of the Hepes buffer is 8.0-9.0; the concentration of the NaHCO3 buffer is 0.1-0.5 M, and the pH is 9.0-10.0; the weight-average molecular weight of the NHS-PEG-NH-BOC is 5000 Da; the concentration of the HCl solution is 1-2 M; and the molecular weight cutoff of the dialysis bag used for dialysis is 3000-5000 Da.

9. The preparation method according to claim 1 or 8, characterized in that, In the synthesis of PLYs and RGD-PEG-PLys, the dialysis is performed by first dialysis in HCl solution for 9-36 hours, and then dialysis in ultrapure water for 9-36 hours. In the synthesis of PLYs(ss-DBCO) and RGD-PEG-NH2, the dialysis was performed by dialyzing in ultrapure water for 9-36 hours.

10. Tumor-targeting pH / reduction-sensitive polymer vesicles prepared by the method of any one of claims 1-9.

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