Anti-aqp5 arginine desiminase nanocarrier complex and preparation method thereof

Abstract

This invention belongs to the field of biopharmaceutical nanoparticle formulations and provides an anti-AQP5 arginine deiminase nanocarrier complex and its preparation method. The invention utilizes a PLGA-PEG-maleimide copolymer to self-assemble into core-shell nanoparticles with a particle size of 50-200 nm. The shell layer is cross-linked by pH-sensitive hydrazone bonds formed by diacylhydrazide and glutaraldehyde, maintaining structural and storage stability under neutral conditions. Under acidic tumor / endosome environments, it can partially dissociate to release and restore the protein conformation. The anti-AQP5 antibody-arginine deiminase is covalently fixed to the shell surface via thio-maleimide addition, achieving a synergistic effect of high protein loading and enzyme activity maintenance, long PEGylation cycling and AQP5-mediated active targeting, and a dynamic balance between stable encapsulation and acid-triggered deactivation. In vitro uptake by AQP5-highly expressing tumor cells is significantly superior to non-targeting carriers, and the in vivo plasma half-life is significantly prolonged, making it suitable for tumor metabolic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine nano-preparation, and particularly relates to an anti-AQP5 arginine deiminase nano-carrier complex and a preparation method thereof. BACKGROUND

[0002] In the field of metabolic therapy of solid tumors, arginine deiminase has become an important treatment strategy for arginine auxotrophic tumors because it can efficiently catalyze the hydrolysis of L-arginine into L-citrulline and ammonia, thereby depleting the arginine nutrient substrate on which tumor cells survive. Arginine deiminase (ADI) belongs to a class of arginine-depleting enzymes that deplete the exogenous arginine on which tumor cells survive by catalyzing the generation of L-citrulline and ammonia from L-arginine. This type of tumor is unable to endogenously synthesize sufficient arginine due to the loss or down-regulation of key metabolic enzymes such as arginine succinate synthase or ornithine transcarbamylase, and is highly dependent on exogenous arginine. Therefore, by systemically depleting arginine in the blood plasma and tumor microenvironment through the arginine deiminase system, tumor cells can be selectively induced to undergo apoptosis or growth arrest. However, to achieve sustained and effective arginine depletion and maintain a therapeutic window, the drug delivery system must meet multiple stringent requirements: first, the enzyme preparation must have a long enough half-life in the blood circulation to maintain sustained arginine degradation capacity, second, it must be enriched and deeply penetrated in tumor tissues to increase local enzyme concentration, third, it must protect the enzyme from degradation by plasma proteases and immune clearance, and fourth, it must maintain enzyme activity while reducing systemic toxicity and immunogenicity. Around the above performance requirements, the development of a nano-carrier drug delivery system that combines long circulation, tumor targeting, enzyme activity maintenance, and structural stability is of great significance for improving the clinical efficacy of arginine deiminase, broadening the range of indications, and promoting progress in the field of tumor metabolic therapy.

[0003] Although various nanocarrier strategies have been used for the encapsulation or modification of arginine deiminase, existing technologies still face fundamental challenges in addressing the aforementioned multiple performance requirements. Firstly, regarding structural stability, while existing PEGylation or liposome encapsulation can prolong cycling time, they often lead to a significant decrease in enzyme activity, or aggregation and leakage during lyophilization-reconstitution and storage. It is difficult to simultaneously guarantee long-term stability and high enzyme activity retention, primarily due to the lack of a dynamic regulatory mechanism that can maintain dense protection under physiological conditions while responding to conformational constraints in the tumor microenvironment. Secondly, regarding targeting and enrichment capabilities, the passive targeting-dependent EPR effect has limited effectiveness in clinical translation and exhibits significant individual variability. Furthermore, the introduction of active targeting ligands often results in reduced ligand-receptor binding efficiency due to the shielding effect of the surface PEG layer, making it difficult to achieve synergy between "invisible" long-cycle loading and active recognition and enrichment. In addition, existing carriers mostly employ physical embedding or simple surface adsorption, resulting in low protein loading and susceptibility to protein dissociation and activity loss in the serum environment, failing to meet the clinical needs for high-dose continuous administration. For example, Chinese patent CN101812438A discloses an arginine deiminase mutant and its preparation and application, but it has problems such as low enzyme activity retention and insufficient tumor targeting. Therefore, there is an urgent need to develop a novel nanocarrier complex that combines dynamic pH response, high-density covalent fixation, active targeting against AQP5 and long-term cycling characteristics. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-AQP5 arginine deiminase nanocarrier complex and its preparation method, thereby solving the triple technical contradictions of current arginine deiminase drug delivery systems: difficulty in simultaneously achieving structural stability and acid-triggered conformational decongestion, mutual constraints between high-load protein immobilization and high enzyme activity maintenance, and difficulty in synergistic effects between PEGylation stealth and active AQP5 targeting.

[0005] This invention employs a synergistic design strategy of "dynamic pH-responsive core-shell structure + high-density covalent surface immobilization + bifunctional protein conjugate." By constructing a hydrophobic core formed by the self-assembly of PLGA-PEG-maleimide copolymer and a pH-sensitive shell formed by cross-linking of diacylhydrazine-glutaraldehyde hydrazone bonds, a dense cross-linked network is maintained in a near-neutral physiological environment to ensure the particle size stability and dispersibility of the nanoparticles. Simultaneously, in the weakly acidic endosome / lysosomal environment after tumor cell endocytosis, the hydrazone bonds undergo partial decrosslinking, releasing the particles originally bound to the core. Protein conjugates with embedded shells or restricted conformations gain greater stereochemical freedom and catalytic accessibility. By pre-coupling anti-AQP5 monoclonal antibodies with arginine deiminase to form bifunctional protein conjugates, and then utilizing the residual free thiol groups on the protein conjugates to undergo a thiomaleimide addition reaction with maleimide groups on the shell surface, multi-point covalent anchoring and high-density surface display of the protein are achieved. This allows for the superposition of AQP5-mediated active targeted enrichment on the basis of long-term PEGylation, thus achieving a synergistic therapeutic effect.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A nanocarrier complex for resisting AQP5 arginine deiminase, wherein the complex is a core-shell structured nanoparticle dispersion system with a particle size of 50–200 nm, the nanoparticles comprising:

[0008] The core phase is a hydrophobic polymer backbone formed by the self-assembly of a PLGA-PEG-maleimide copolymer containing hydrophobic PLGA segments.

[0009] The shell phase is composed of a hydrazone crosslinking network formed by the dihydrazide groups on the PLGA-PEG-maleimide copolymer and the dialdehyde crosslinking agent. The hydrazone crosslinking shell maintains a degree of crosslinking of more than 80% relative to the initial time within 24 hours under buffer conditions of 37°C and pH 6.5–7.4. Under buffer conditions of 37°C and pH 5.0–6.0, the degree of crosslinking of crosslinking of crosslinking decreases by at least 30% within 24 hours. This is to maintain the stability of the nanoparticle structure under neutral conditions and to allow at least partial dissociation in the acidic intracellular microenvironment such as endosomes / lysosomes after tumor cell endocytosis. This allows the protein conjugate B1, which was originally partially buried or restricted by the crosslinking shell, to obtain higher conformational freedom and outer surface exposure.

[0010] And an anti-AQP5 arginine deiminase protein conjugate B1 covalently fixed to the surface of the shell, wherein the protein conjugate B1 is formed by covalently coupling an anti-AQP5 monoclonal antibody or its antigen-binding fragment capable of specifically binding to human AQP5 with arginine deiminase through a chemical cross-linking agent containing maleimide groups, and wherein the arginine deiminase retains at least one free thiol group on average that can undergo a thiomaleimide addition reaction with the maleimide groups of the shell;

[0011] The thiol group introduced on the arginine deiminase undergoes a thiomaleimide addition reaction to form a thioether bond with the maleimide group in the shell and is covalently fixed to the shell surface. This makes the protein conjugate B1 fixed to the shell surface more easily exposed in the tumor microenvironment and exert its arginine depletion effect.

[0012] Furthermore, the PLGA-PEG-maleimide copolymer is composed of a PLGA-PEG copolymer backbone formed by the ester bond condensation of a modified polylactic acid-glycolic acid copolymer PLGA with multiple carboxyl side groups on the backbone and polyethylene glycol PEG. A dihydrazide group formed by adipate dihydrazide and a maleimide group introduced by N-(2-aminoethyl)maleimide hydrochloride are introduced onto this backbone. The molar ratio of lactic acid to glycolic acid in the PLGA is 75:25. The total carboxyl content of the modified polylactic acid-glycolic acid copolymer PLGA is preferably 0.03–0.25 mmol / g polymer, as determined by acid-base titration. The average molecular weight of the PEG is 2000–6000, and both the maleimide group and the dihydrazide group are bonded to the copolymer backbone in the form of side chains.

[0013] Furthermore, the dialdehyde crosslinking agent is glutaraldehyde, the shell thickness is 5–30 nm, the content of dihydrazide groups in the polymer is 0.02–0.20 mmol / g polymer, the hydrazone bond crosslinking density formed by dihydrazide groups and glutaraldehyde is 0.01–0.10 mmol / g polymer, and the hydrazone bond crosslinking density does not exceed the content of dihydrazide groups, and the polydispersity index of the nanoparticles is not higher than 0.20, and the particle size change does not exceed 20% after being stored at 4°C under sealed conditions for 3 months.

[0014] Furthermore, the protein conjugate B1 is prepared via the following steps:

[0015] A1. Prepare solutions of anti-AQP5 monoclonal antibody or its antigen-binding fragment and arginine deiminase in near-neutral buffer;

[0016] A2. Add a thiol-introducing reagent to the arginine deiminase solution to introduce an average of 2–4 reactive thiol groups onto each enzyme molecule to obtain thiolized arginine deiminase;

[0017] A3. The anti-AQP5 monoclonal antibody or its antigen-binding fragment is reacted with a cross-linking agent containing both N-hydroxysuccinimide ester and maleimide groups to introduce 1–2 maleimide groups onto the antibody molecule, thereby obtaining an activated antibody or its antigen-binding fragment.

[0018] A4. The thiolized arginine deiminase is reacted with the activated antibody or its antigen-binding fragment under mild conditions to form an anti-AQP5 monoclonal antibody-arginine deiminase protein conjugate, namely protein conjugate B1. The main peak component is collected by separation and purification methods to ensure that the average binding ratio of arginine deiminase to anti-AQP5 monoclonal antibody or its antigen-binding fragment is 1–2:1, and at least one free thiol group that can react with maleimide group is retained in protein conjugate B1.

[0019] Furthermore, the protein conjugate B1 is loaded in the nanoparticles at an amount of 10–30 wt% of the dry weight of the nanoparticles, the enzyme density on the nanoparticle surface is 0.5–2.0 mg / m², the pH of the formulation is 6.8–7.4, the zeta potential is −10 mV to 0 mV, and after being treated in human serum at 37°C for at least 48 h, the arginine deiminase activity is preferably maintained at no less than 70% of the initial activity before incubation, as determined by colorimetric or fluorescent enzyme activity assay using L-arginine as a substrate.

[0020] Furthermore, the PLGA-PEG-maleimide copolymer is prepared by the following steps:

[0021] B1. In an anhydrous organic solvent, PLGA, PEG, adipic acid dihydrazide, and N-(2-aminoethyl)maleimide hydrochloride are simultaneously mixed with a condensing agent and a catalyst, so that the carboxyl group of PLGA forms an ester bond with PEG, forms an amide bond with adipic acid dihydrazide to introduce a dihydrazide side chain, and forms a maleimide side chain with N-(2-aminoethyl)maleimide hydrochloride, while retaining some unreacted carboxyl groups, to obtain a crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains;

[0022] B2. The crude product is subjected to solvent removal, precipitation, washing and drying to obtain a solid intermediate;

[0023] B3. The solid intermediate is dissolved in a polar organic solvent, and the remaining carboxyl groups are activated by water-soluble carbodiimide and reacted with adipic acid dihydrazide to further adjust the degree of substitution of the dihydrazide group in the polymer so that the content of the dihydrazide group is 0.02–0.20 mmol / g polymer. The reaction is terminated when the number average molecular weight of the polymer is 15–30 kDa, the polydispersity index is not greater than 1.3 and the degree of substitution of the maleimide group is 1–5 mol%, to obtain the PLGA-PEG-maleimide copolymer.

[0024] Furthermore, the PLGA-PEG-maleimide copolymer is used to form a dynamic core-shell nanocarrier intermediate through the following steps:

[0025] C1. Dissolve the PLGA-PEG-maleimide copolymer in acetonitrile or ethanol to obtain an organic phase;

[0026] C2. Prepare a phosphate buffer aqueous phase with a pH of 6.5–7.0, and add glutaraldehyde to make the glutaraldehyde concentration 0.5–10 mmol / L;

[0027] C3. The organic phase and the aqueous phase are mixed in a microfluidic mixer at a volume ratio of 1:2.5–1:3.5 to allow the polymer to self-assemble into core-shell nanoparticles with a particle size of 50–200 nm. The resulting dispersion is then gently stirred at 20–25 °C for 0.5–4 h. During this process, the dihydrazide groups and glutaraldehyde continue to react to form a hydrazone cross-linked shell.

[0028] C4. After removing the organic solvent, unreacted small molecule glutaraldehyde is preferably removed by ultrafiltration, centrifugation and / or dialysis to reduce the concentration of free glutaraldehyde to a level that has no significant impact on subsequent protein coupling and enzyme activity stability. The solid content and pH of the dispersion are then adjusted to obtain a dynamic core-shell nanocarrier intermediate with a median particle size of 50–200 nm and a polydispersity index not higher than 0.20.

[0029] Furthermore, the anti-AQP5 arginine deiminase nanocarrier complex or its lyophilized powder is prepared by the following steps:

[0030] D1. Dilute the dynamic core-shell nanocarrier intermediate and adjust the pH to 6.5–7.2. Slowly add the protein conjugate B1 solution according to the molar ratio of maleimide groups on the surface of the nanocarrier shell to reactive thiol groups in protein conjugate B1 of 1:0.5–1:1.5. Gently stir the reaction at 4–8°C for 1–4 hours to allow protein conjugate B1 to be covalently attached to the shell surface through a thiomaleimide addition reaction.

[0031] D2. After the reaction is complete, ultrafiltration or gel filtration is used to separate and remove free protein conjugate B1, small molecule salts, and unreacted small molecules to obtain a dispersion of the nanocarrier complex, such that the mass fraction of protein conjugate B1 in the solid is 10–30 wt%.

[0032] D3. Add a freeze-drying protectant to the dispersion in a total amount of 5–10 wt% of the total dry matter of the nanoparticles and protein conjugate B1. The freeze-drying protectant includes trehalose dihydrate and / or sucrose. After dispensing, pre-freeze at low temperature and freeze-dry under vacuum to obtain a freeze-dried powder of the nanocarrier complex with a water content of no more than 3 wt% that can be rapidly reconstituted.

[0033] Furthermore, in vitro, using AQP5-overexpressing tumor cell lines and AQP5-low-expressing normal cell lines as models, under the same nanocarrier particle size and enzyme activity load conditions, quantitative flow cytometry or confocal microscopy analysis showed that the intracellular uptake of this nanocarrier complex in AQP5-overexpressing tumor cells was at least twice that of a non-targeted arginine deiminase carrier with the same structure but without anti-AQP5 antibody or its antigen-binding fragment. Under the same added enzyme activity dose, the ratio of arginine depletion in AQP5-overexpressing tumor cells to that in AQP5-low-expressing normal cells was not less than 5:1, calculated by measuring the arginine concentration in the culture supernatant. In vivo, using rodent intravenous administration as a model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was 2–10 times that of free arginine deiminase, and the plasma arginine concentration was maintained at 30–70% of the pre-administration level for 24–72 h after administration.

[0034] As a concept of this invention, the present invention employs a PLGA-PEG-maleimide copolymer to self-assemble into a core-shell structure and constructs a pH-responsive shell through dihydrazide-glutaraldehyde hydrazone crosslinking. This design is primarily used to enhance the structural stability of nanocarriers under physiological conditions and their intelligent responsiveness in the acidic internal environment of tumor cells. PLGA, as a hydrophobic biodegradable polymer, forms an amphiphilic block copolymer with PEG that self-assembles into core-shell structured nanoparticles in an aqueous environment. The hydrophobic segments of PLGA constitute a dense hydrophobic core to provide a stable polymer framework, while the hydrophilic segments of PEG form an outer hydration shell, endowing the nanoparticles with excellent colloidal stability and "invisibility" properties, effectively prolonging blood circulation time and reducing non-specific uptake by the reticuloendothelial system. Building upon this, by introducing dihydrazide groups onto the main chain of the PLGA-PEG copolymer and reacting them with dialdehyde crosslinking agents such as glutaraldehyde, a dynamic shell composed of a hydrazone bond crosslinking network is formed on the surface of the nanoparticles. This hydrazone bond maintains a relatively stable crosslinking state under near-neutral pH conditions, enabling the nanoparticles to maintain a narrow particle size distribution and low polydispersity index during blood circulation, storage, and freeze-drying-reconstitution processes, thus preventing aggregation and leakage. Furthermore, when the nanoparticles are taken up by tumor cells via endocytosis and enter the acidic endosome / lysosomal microenvironment with a pH of 5.0-6.0, the hydrazone bonds... Under acidic conditions, the chemical equilibrium shifts towards hydrolysis, leading to partial dissociation of the cross-linked network and a decrease in shell density. This allows surface protein conjugates that were originally buried or sterically restricted to gain greater conformational freedom and external surface exposure. This dynamic conversion mechanism of "stable encapsulation - acid-triggered conformational unblocking" ensures both the structural integrity and colloidal stability of nanoparticles in the systemic circulation and enables responsive enhancement of protease catalytic activity and substrate accessibility in the intracellular environment of tumor cells, thereby achieving a precise balance between system stability and local functional release.

[0035] This invention also discloses a method for preparing an anti-AQP5 arginine deiminase nanocarrier complex, comprising the following steps:

[0036] S1. PLGA and PEG are condensed to form a PLGA-PEG copolymer backbone, and dihydrazide groups and maleimide groups are introduced into the backbone to prepare a PLGA-PEG-maleimide copolymer;

[0037] S2. Dissolve the PLGA-PEG-maleimide copolymer in an organic phase and mix it with an aqueous phase containing a dialdehyde crosslinking agent in a microfluidic device or a high-shear device to allow the polymer to self-assemble into core-shell structured nanoparticles. The shell layer is then crosslinked with dialdehyde through dihydrazide to form a hydrazone bond, thus obtaining a dynamic core-shell nanocarrier intermediate.

[0038] S3. By introducing a thiol group into arginine deiminase and introducing a maleimide group into an anti-AQP5 monoclonal antibody or its antigen-binding fragment, an arginine deiminase containing 2–4 reactive thiol groups and an activated antibody or its antigen-binding fragment containing 1–2 maleimide groups are prepared, and the two are coupled under mild conditions to obtain a protein conjugate B1 having at least one reactive thiol group that is not consumed by the antibody.

[0039] S4. Under low temperature conditions at pH 6.5–7.2, the nanocarrier intermediate described in step S2 is reacted with the protein conjugate B1 described in step S3 at a molar ratio of maleimide group to thiol group of 1:0.5–1:1.5, so that the protein conjugate B1 is covalently attached to the shell surface through a thiomaleimide addition reaction. Then, the free protein conjugate B1 is removed and a lyophilization protectant is added. After pre-freezing and vacuum freeze-drying, a lyophilized formulation of the anti-AQP5 arginine deiminase nanocarrier complex is obtained.

[0040] In the preparation method, the process is optimized by controlling the molar ratio and reaction time of the thiol-introducing reagent and arginine deiminase, as well as the cross-linking agent and the anti-AQP5 monoclonal antibody or its antigen-binding fragment, and by combining the results of thiol titration or mass spectrometry analysis, so that the prepared protein conjugate B1 meets the above-mentioned binding ratio and residual thiol number.

[0041] Furthermore, in the preparation of PLGA-PEG-maleimide copolymer, the catalyst 4-dimethylaminopyridine is used in an amount of 1.0–5.0 wt% relative to the mass of PLGA to improve esterification / amidation efficiency while controlling side reactions, thereby obtaining a polymer with a narrow molecular weight distribution.

[0042] Furthermore, after obtaining the crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains, the organic solvent was removed under reduced pressure at a vacuum of 0.005–0.02 MPa and a temperature not exceeding 40°C. The product was then precipitated by adding excess anhydrous diethyl ether, filtered, and washed 2–4 times with anhydrous diethyl ether to remove N,N′-dicyclohexylurea byproducts. Subsequently, the product was dried at 25–40°C and a vacuum of 0.001–0.01 MPa for 8–24 h to further reduce the content of low molecular weight impurities and stabilize the polymer structure.

[0043] Furthermore, the buffered aqueous phase suitable for shell crosslinking and subsequent formulation preparation can be a PBS buffer with a pH of 6.5–7.0, which is prepared by mixing sodium chloride, sodium dihydrogen phosphate and disodium hydrogen phosphate in a specific ratio to ensure both protein activity retention and particle size stability during coupling and self-assembly.

[0044] Furthermore, the arginine deiminase solution can be selected from aqueous solutions of arginine deiminase preparations derived from mycoplasma, toxoplasma, or other known sources, in order to compare the differences in activity retention and immunogenicity of arginine deiminases from different sources under the same carrier structure.

[0045] Furthermore, after obtaining the dynamic core-shell nanocarrier intermediate, the intermediate can be diluted to a polymer mass concentration of 5–20 mg / mL and then coupled with protein conjugate B1 to improve protein coupling efficiency and particle size uniformity while controlling solution viscosity and interparticle interaction.

[0046] Furthermore, in the freeze-drying preparation process, the following process window can be adopted: dispense the dispersion into vials, pre-freeze at −40℃ for no less than 2h, then control the product temperature not higher than −20℃ and the cavity vacuum degree to 0.0001–0.0005MPa during the main drying stage and dry for 10–36h. If necessary, further desorption drying is carried out at 0.0001–0.0005MPa and 20–30℃ for 2–8h to obtain a freeze-dried formulation with a water content not higher than 3wt% and minimal particle size change after reconstitution.

[0047] Furthermore, in the synthesis step of PLGA-PEG-maleimide copolymer, the mass ratio of PLGA to PEG is 40:60–60:40, preferably 45:55–55:45, and the molar ratio of N,N′-dicyclohexylcarbodiguanidine to PLGA carboxyl group is 0.9–1.1:1, so as to achieve a better balance between hydrophobic and hydrophilic segments, thereby facilitating the formation of a stable core-shell self-assembled structure.

[0048] Furthermore, in the preparation of dynamic core-shell nanocarrier intermediates by microfluidic mixing, the total flow rate of microfluidic mixing is 12–18 mL / min, and the volumetric flow rate ratio of the organic phase to the aqueous phase is controlled within the range of 1:2.5–1:3.5. Thus, a nanoparticle dispersion system with a D50 of 50–200 nm and a polydispersity index not higher than 0.20 can be obtained in a single pass.

[0049] Furthermore, in the preparation of protein conjugate B1, the molar ratio of arginine deiminase to 2-iminothione hydrochloride is preferably 3–6:1, and the molar ratio of anti-AQP5 monoclonal antibody or its antigen-binding fragment to SMCC or Sulfo-SMCC is preferably 3–6:1, so as to obtain intermediates containing 2–4 thiol groups per enzyme molecule and 1–2 maleimide groups per antibody molecule more stably, thereby facilitating the control of the binding ratio and structural uniformity of protein conjugate B1.

[0050] Furthermore, the freeze-drying protectant can be a mixture of trehalose dihydrate and sucrose, with a mass ratio of 1:1–1:2, and the total amount is 6–8 wt% of the total dry matter of nanoparticles and protein conjugate B1. Under this combination and dosage, after sterilization by filtration through a 0.22 μm filter membrane before freeze-drying, a formulation with a reconstitution time of no more than 2 min and no obvious visible aggregation after reconstitution can be obtained.

[0051] Furthermore, in the preparation of the nanocarrier complex and its lyophilized formulation, the pH control precision can be limited to ±0.1, the temperature control precision to ±2℃, and the reaction time control precision of the key step can be limited to ±10% of the set value, so as to improve the reproducibility of different batches of products in key quality attributes such as particle size, zeta potential and protein loading.

[0052] Furthermore, during the preparation scale-up, parallel or series microfluidic reactors or high-shear emulsification equipment can be used. When the total throughput is scaled up to 10–1000 times the experimental scale, by keeping the key dimensionless parameters basically consistent, the relative deviation of the particle size D50 of the obtained product from the laboratory-scale product can be kept to no more than 10%, and the relative standard deviation of the protein conjugate B1 loading can be kept to no more than 5%, thereby achieving a smooth scale-up from laboratory process to industrial production.

[0053] As another concept of the present invention, the present invention employs a stepwise coupling and covalent surface immobilization strategy to prepare an anti-AQP5 arginine deiminase nanocarrier complex, which is mainly used to enhance the carrier's active tumor targeting ability, protein loading stability and enzyme activity retention performance. Traditional protein physical adsorption or simple surface coupling strategies often suffer from problems such as low protein loading, easy dissociation in serum environment, and severe loss of enzyme activity. However, this invention pre-couples anti-AQP5 monoclonal antibody or its antigen-binding fragment with arginine deiminase under mild conditions through a bifunctional cross-linking agent to form a bifunctional protein conjugate B1. In the coupling reaction design, the number of thiol groups and maleimide groups introduced are precisely controlled to ensure that protein conjugate B1 can achieve stable covalent linking between antibody and enzyme, while retaining at least one unconsumed free thiol group for subsequent covalent anchoring with the nanocarrier shell. This three-step strategy of "protein pre-coupling - residual thiol group - shell covalent fixation" avoids the low efficiency and side reactions caused by direct multi-component reactions on the nanoparticle surface, and achieves high-density surface display and mechanical stability of the protein through multi-point covalent bonding. Specifically, of the 2-4 thiol groups introduced on the arginine deiminase, 1-3 are consumed during antibody coupling, and the remaining at least one free thiol group undergoes a highly efficient thiomaleimide addition reaction with the maleimide group on the surface of the nanoparticle shell to form a stable thioether bond. This covalent bond is irreversible under physiological conditions and has high mechanical strength, which can effectively prevent the dissociation and inactivation of proteins during blood circulation and endocytosis.

[0054] The PLGA-PEG-maleimide copolymer and the diazid-glutaraldehyde hydrazone crosslinking network play a highly complementary and synergistic role in the nanocarrier system of this invention. They focus on different functional dimensions and achieve synergistic performance enhancement through interfacial integration. The PLGA-PEG-maleimide copolymer, as an amphiphilic self-assembly unit, primarily contributes to building a stable hydrophobic core framework, providing mechanical support and morphological stability for the nanoparticles. The PEG hydrophilic segments, on the other hand, are mainly responsible for forming the outer hydration shell to impart long-cycle properties and low protein adsorption to the nanoparticles. Simultaneously, the maleimide side chain groups reserve chemical reaction sites for subsequent protein covalent coupling. The diazid-glutaraldehyde hydrazone crosslinking network focuses on building a dynamic pH-responsive shell on the nanoparticle surface, achieving intelligent regulation of "stable encapsulation-acid-triggered release" through the crosslinking-decrosslinking equilibrium of hydrazone bonds under different pH conditions. In terms of improving structural stability, the hydrophobic core of PLGA provides a stable anchoring interface for the hydrazone crosslinking shell, ensuring that the crosslinking reaction mainly occurs on the nanoparticle surface rather than in the solution bulk, thus avoiding interparticle crosslinking and aggregation. Simultaneously, the formation of the hydrazone crosslinking network further enhances the rigidity and deformation resistance of the nanoparticle surface. Through the synergistic effect of the core-shell interface, both significantly improve the particle size stability and dispersibility of the nanoparticles during storage, freeze-drying-reconstitution, and blood circulation. Regarding improving protein loading and activity retention, the amphiphilic self-assembly of the PLGA-PEG copolymer provides a high specific surface area nanoparticle platform, creating the spatial conditions for high-density protein immobilization. The formation of the hydrazone crosslinking shell further enhances the mechanical anchoring strength of the surface protein after immobilization, preventing protein dissociation in the serum environment. Furthermore, the partial uncrosslinking of hydrazone bonds in the acidic internal environment of tumor cells releases the conformational freedom of the protein, enabling arginine deiminase to achieve higher catalytic activity and substrate accessibility while maintaining high loading capacity and stability.

[0055] Beneficial technical effects

[0056] 1. Significantly enhanced structural stability and pH-responsive performance of nanocarriers: A core-shell structure is formed through the self-assembly of PLGA-PEG-maleimide copolymer and a dihydrazide-glutaraldehyde hydrazone cross-linked shell is constructed. Under near-neutral physiological conditions, the degree of hydrazone cross-linking remains above 80%, ensuring that the nanoparticles maintain a particle size within the range of 50-200 nm and a polydispersity index not higher than 0.20 during blood circulation, storage, and freeze-drying-reconstitution. After storage at 4°C for 3 months, the particle size change does not exceed 20%, effectively avoiding the problems of easy aggregation and leakage of traditional nanocarriers. At the same time, the degree of hydrazone cross-linking decreases by at least 30% in the acidic endosomal / lysosomal environment of tumor cells, allowing protein conjugates that were originally buried in the shell or conformationally restricted to obtain higher stereofreedom and external surface exposure. This achieves precise control of "stable encapsulation-acid-triggered conformational unblocking", solving the contradiction between structural stability and functional release that is difficult to balance in traditional carriers.

[0057] 2. Achieving a synergistic balance between high-density protein loading and high enzyme activity retention: By pre-preparing an anti-AQP5 monoclonal antibody-arginine deiminase protein conjugate and utilizing the addition reaction of residual thiol groups with maleimide groups in the shell for multi-point covalent anchoring, the protein loading can reach 10-30 wt% of the dry weight of the nanoparticles, and the surface enzyme density is 0.5-2.0 mg / m². After incubation in human serum at 37°C for 48 h, the arginine deiminase activity retention rate is not less than 70%. Compared with traditional physical adsorption or simple coupling strategies, this invention significantly improves the stability of the protein in the serum environment through covalent bonding. At the same time, through the conformational restriction of partial uncrosslinking of the pH-responsive shell to release the enzyme, an excellent balance is achieved between high loading and high activity, laying the foundation for continuous and effective arginine depletion.

[0058] 3. Significantly Enhanced Active Tumor Targeting and Arginine Selective Depletion Efficiency: By coupling and immobilizing anti-AQP5 monoclonal antibodies or their antigen-binding fragments with arginine deiminase on the surface of nanoparticles, AQP5-mediated active targeting recognition is superimposed on a long-cycle PEGylated approach. In vitro cell experiments show that the intracellular uptake of this nanocarrier complex in AQP5-high expressing tumor cells is at least twice that of a non-targeting carrier with the same structure but without anti-AQP5 antibodies, and it depletes arginine in both AQP5-high expressing tumor cells and AQP5-low expressing normal cells. With a ratio of at least 5:1, selective arginine deprivation from tumor cells was achieved. In an in vivo rodent intravenous administration model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was 2-10 times that of free arginine deiminase, and the plasma arginine concentration was maintained at 30-70% of the pre-administration level for 24-72 hours after administration. This indicates that the present invention has successfully solved the technical problem of the difficulty in synergistic effects between PEGylation stealth and AQP5 active targeting, and provides a novel and efficient drug delivery strategy for the clinical translation of arginine deiminase.

[0059] 4. Excellent formulation stability and clinical translation potential: By optimizing the composition of the lyophilization protectant and the lyophilization process parameters, the resulting lyophilized formulation has a water content of no more than 3 wt% and a reconstitution time of no more than 2 min. After reconstitution, key quality attributes such as particle size, zeta potential, and protein loading are basically consistent with those before lyophilization, meeting the storage and transportation requirements of clinical formulations. At the same time, by establishing precise process control windows in key steps such as PLGA-PEG-maleimide copolymer synthesis, microfluidic self-assembly, protein coupling, and lyophilization, high reproducibility of key quality attributes such as particle size, zeta potential, and protein loading is achieved for different batches of products. When the preparation is scaled up to 10-1000 times on the experimental scale, the relative deviation of particle size does not exceed 10% and the relative standard deviation of protein loading does not exceed 5%, laying a solid foundation for industrial production and clinical application.

[0060] 5. Multiple synergistic mechanisms endow superior anti-tumor metabolic therapy potential: This invention integrates four functions—PLGA-PEG long-cycle, hydrazone bond pH response, anti-AQP5 active targeting, and arginine deiminase metabolic depletion—to achieve multiple synergistic effects. This not only prolongs the enzyme's action time and spatial accumulation in vivo, but also enhances the selective killing of arginine-deficient tumors through AQP5 targeting. At the same time, the intelligent uncrosslinking of the pH-responsive shell in the tumor cell environment enhances the enzyme's catalytic efficiency. This provides a novel precision treatment approach for tumor types such as liver cancer, pancreatic cancer, and melanoma that highly express AQP5 and are deficient in arginine succinate synthase. Attached Figure Description

[0061] Figure 1 This is a graph showing the effect of the average molecular weight of polyethylene glycol on plasma half-life and AQP5-positive cell uptake ratio.

[0062] Figure 2 This is a graph showing the effect of glutaraldehyde concentration on shell thickness on enzyme activity retention and pH 5.5 dissociation rate.

[0063] Figure 3 This is a graph showing the effect of the loading of protein conjugate B1 of the present invention on the arginine depletion selectivity and polydispersity index.

[0064] Figure 4 XPS depth profiles of the retention rate of the N1s C=N peak in the hydrazone shell of Example 1 of the present invention under different pH conditions.

[0065] Figure 5 The image shows the XPS N1s high-resolution spectrum of the hydrazone shell in Example 1 of the present invention under different pH and time conditions.

[0066] Figure 6The image shows the FTIR peak intensity of the hydrazone bond C=N characteristic peak of Example 1 of the present invention as a function of time under neutral and slightly acidic conditions. The peak intensity of the hydrazone bond C=N at 1635 cm⁻¹ is a time decay graph.

[0067] Figure 7 This is a superimposed image of FTIR spectra of hydrazone-crosslinked protein shells at different time points in neutral and slightly acidic environments.

[0068] Figure 8 This is a flow cytometry MFI comparison of the uptake levels of the targeted and non-targeted carriers in Example 1 by AQP5-positive tumor cells and AQP5-negative normal cells in Example 1.

[0069] Figure 9 This is a confocal laser scanning microscope (CLSM) cutoff image of the fluorescence intensity distribution of the anti-AQP5 antibody channel and the AQP5 receptor channel along the cell membrane cutoff direction in Example 1. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0071] Example 1

[0072] This embodiment provides an anti-AQP5 arginine deiminase nanocarrier complex. The complex is a core-shell structured nanoparticle dispersion system with a particle size of 125 nm. The nanoparticles in this embodiment include a core phase, a shell phase, and an anti-AQP5 arginine deiminase protein conjugate B1 covalently fixed on the surface of the shell phase.

[0073] The core phase of this embodiment is a hydrophobic polymer backbone formed by the self-assembly of a PLGA-PEG-maleimide copolymer containing hydrophobic PLGA segments. The shell phase of this embodiment is composed of a hydrazone crosslinking network formed by dihydrazide groups on the PLGA-PEG-maleimide copolymer and a dialdehyde crosslinking agent. Under buffer conditions of 37°C and pH 6.5–7.4, the degree of hydrazone crosslinking of the shell phase in this embodiment remains above 80% relative to the initial time within 24 hours. Under buffer conditions of 37°C and pH 5.0–6.0, the degree of hydrazone crosslinking decreases by at least 30% within 24 hours, so as to maintain the stability of the nanoparticle structure under neutral conditions and to allow at least partial dissociation in the acidic intracellular microenvironment such as endosomes / lysosomes after tumor cell endocytosis, so that the protein conjugate B1, which was originally partially buried or restricted by the crosslinking shell, can obtain higher conformational freedom and outer surface exposure.

[0074] The protein conjugate B1 of this embodiment is formed by covalently coupling an anti-AQP5 monoclonal antibody capable of specifically binding to human AQP5 with arginine deiminase via a chemical cross-linking agent containing a maleimide group. The arginine deiminase retains, on average, at least one free thiol group that can undergo a thiomaleimide addition reaction with the maleimide group in the shell of this embodiment. The thiol group introduced onto the arginine deiminase in this embodiment forms a thioether bond with the maleimide group in the shell of this embodiment via a thiomaleimide addition reaction, thus covalently fixing it to the shell surface. This makes the protein conjugate B1 fixed to the shell surface more easily exposed in the tumor microenvironment and exert its arginine depletion effect.

[0075] The PLGA-PEG-maleimide copolymer of this embodiment is composed of a PLGA-PEG copolymer backbone formed by the ester condensation of a modified polylactic acid-hydroxyacetic acid copolymer PLGA with multiple carboxyl side groups on the backbone and polyethylene glycol PEG. A dihydrazide group formed by adipate dihydrazide and a maleimide group introduced by N-(2-aminoethyl)maleimide hydrochloride are introduced on the backbone. The molar ratio of lactic acid to glycolic acid in PLGA is 75:25, the average molecular weight of PEG is 4000, and both the maleimide group and the dihydrazide group are bonded to the copolymer backbone of this embodiment in the form of side chains.

[0076] In this embodiment, the dialdehyde crosslinking agent is glutaraldehyde, the shell thickness is 17 nm, the content of dihydrazide groups in the polymer of this embodiment is 0.11 mmol / g polymer, the hydrazone bond crosslinking density formed by dihydrazide groups and glutaraldehyde is 0.055 mmol / g polymer, and the hydrazone bond crosslinking density of this embodiment does not exceed the content of dihydrazide groups, and the polydispersity index of the nanoparticles in this embodiment is 0.18, and the particle size change does not exceed 20% after being stored at 4°C under sealed conditions for 3 months.

[0077] The protein conjugate B1 of this embodiment was prepared by the following steps: A 2.0 mg / mL solution of anti-AQP5 monoclonal antibody and a 3.0 mg / mL solution of arginine deiminase were prepared in phosphate buffer at pH 7.2. A thiol-introducing reagent, 2-iminothione hydrochloride, was added to the arginine deiminase solution of this embodiment. The reaction was carried out at a molar ratio of arginine deiminase to 2-iminothione hydrochloride of 1:4.5, resulting in an average of three reactive thiol groups introduced onto each enzyme molecule. The reaction temperature was 20°C, and the reaction time was 1.5 h, yielding thiolated arginine deiminase. Excess 2-iminothione hydrochloride was removed by gel filtration. The anti-AQP5 monoclonal antibody of this embodiment was reacted with a cross-linking agent SMCC containing both N-hydroxysuccinimide ester and maleimide groups at a molar ratio of antibody to SMCC of 1:4.5 in a buffer solution at pH 7.4 at 25°C for 1 h, resulting in the introduction of an average of 1.5 maleimide groups onto the antibody molecule, thus obtaining an activated antibody. Excess SMCC was removed by gel filtration. The thiolated arginine deiminase of this embodiment was reacted with the activated antibody of this embodiment at pH 7.0 and 6°C for 2.5 h to form an anti-AQP5 monoclonal antibody-arginine deiminase protein conjugate, namely protein conjugate B1. The main peak component was collected by separation and purification methods, ensuring that the average binding ratio of arginine deiminase to anti-AQP5 monoclonal antibody was 1.5:1, and that at least one free thiol group capable of reacting with maleimide groups was retained in protein conjugate B1.

[0078] The loading amount of protein conjugate B1 in the nanoparticles of this embodiment is 20 wt% of the dry weight of the nanoparticles of this embodiment. The enzyme density on the surface of the nanoparticles is 1.2 mg / m², the pH of the formulation is 7.1, the zeta potential is −5 mV, and after being treated in human serum at 37°C for at least 48 h, the arginine deiminase activity was 75% retained relative to the initial activity before incubation, as determined by colorimetric or fluorescent enzyme activity assay using L-arginine as a substrate.

[0079] The PLGA-PEG-maleimide copolymer of this embodiment was prepared by the following steps: PLGA, PEG, adipic acid dihydrazide, and N-(2-aminoethyl)maleimide hydrochloride were mixed in anhydrous dichloromethane with N,N′-dicyclohexylcarbodiguanidine and 4-dimethylaminopyridine catalyst. The mass ratio of PLGA to PEG was 50:50, the amount of 4-dimethylaminopyridine catalyst relative to the mass of PLGA was 3.0 wt%, and the molar ratio of N,N′-dicyclohexylcarbodiguanidine to PLGA carboxyl groups was 1:1. This allowed the PLGA carboxyl groups to form ester bonds with PEG, amide bonds with adipic acid dihydrazide to introduce dihydrazide side chains, and maleimide side chains with N-(2-aminoethyl)maleimide hydrochloride, while retaining some unreacted carboxyl groups. The mixture was reacted at 35°C for 8 hours to obtain a crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains. The crude product of this embodiment was subjected to reduced pressure to remove organic solvent under vacuum conditions of 0.01 MPa and 35°C. After precipitation with excess anhydrous diethyl ether, the product was filtered and washed three times with anhydrous diethyl ether to remove N,N′-dicyclohexylurea byproducts. Subsequently, it was dried at 30°C and 0.005 MPa for 16 h to obtain a solid intermediate. This solid intermediate was dissolved in N,N-dimethylformamide. The remaining carboxyl groups were activated by water-soluble carbodiimide and reacted with adipic acid dihydrazide to further adjust the degree of substitution of the dihydrazide groups in the polymer, achieving a dihydrazide group content of 0.11 mmol / g polymer. The reaction was terminated when the polymer number-average molecular weight was 22 kDa, the polydispersity index was 1.2, and the maleimide group substitution degree was 3 mol%, yielding the PLGA-PEG-maleimide copolymer of this embodiment.

[0080] The PLGA-PEG-maleimide copolymer of this embodiment forms a dynamic core-shell nanocarrier intermediate through the following steps: The PLGA-PEG-maleimide copolymer of this embodiment is dissolved in acetonitrile to obtain an organic phase with a polymer concentration of 30 mg / mL. A phosphate buffer aqueous phase with a pH of 6.8 is prepared. The phosphate buffer in this embodiment is prepared by mixing sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate in a certain proportion, and glutaraldehyde is added to make the glutaraldehyde concentration 5 mmol / L. The organic phase and aqueous phase of this embodiment are mixed in a microfluidic mixer at a volume ratio of 1:3 by collision. The total flow rate of the microfluidic mixing is 15 mL / min, so that the polymer of this embodiment self-assembles to form core-shell structured nanoparticles with a particle size of 125 nm. The resulting dispersion is then gently stirred at 22 °C for 2 h. During this process, the dihydrazide groups and glutaraldehyde continue to react to form a hydrazone cross-linked shell layer. Organic solvents were removed by rotary evaporation and the solid content of the dispersion was adjusted to 15 mg / mL and pH to 7.0 to obtain a dynamic core-shell nanocarrier intermediate with a median particle size of 125 nm and a polydispersity index of 0.18.

[0081] The anti-AQP5 arginine deiminase nanocarrier complex and its lyophilized powder of this embodiment were prepared through the following steps: The dynamic core-shell nanocarrier intermediate of this embodiment was diluted and the pH was adjusted to 7.0. The molar ratio of maleimide groups on the surface of the nanocarrier shell to reactive thiol groups in protein conjugate B1 was 1:1. The dynamic core-shell nanocarrier intermediate of this embodiment was diluted to a polymer mass concentration of 12 mg / mL. The protein conjugate B1 solution was slowly added, and the reaction was gently stirred at 6°C for 2.5 h, allowing protein conjugate B1 to be covalently attached to the shell surface via a thiomaleimide addition reaction. After the reaction, ultrafiltration was used to remove free protein conjugate B1, small molecule salts, and unreacted small molecules, yielding a dispersion of the nanocarrier complex of this embodiment, with a protein conjugate B1 mass fraction of 20 wt% in the solid. A lyophilization protectant, comprising 7.5 wt% of the total dry matter of nanoparticles and protein conjugate B1, was added to the dispersion of this embodiment. The lyophilization protectant in this embodiment was a mixture of trehalose dihydrate and sucrose in a mass ratio of 1:1.5, totaling 7.5 wt% of the total dry matter of nanoparticles and protein conjugate B1. After being dispensed into vials, the mixture was pre-frozen at −40°C for 2.5 h. Subsequently, during the main drying stage, the product temperature was controlled at −25°C and the chamber vacuum degree at 0.0003 MPa for 24 h. Then, it underwent desorption drying at 0.0002 MPa and 25°C for 5 h to obtain a lyophilized powder of the nanocarrier complex of this embodiment with a water content of 2.5 wt% that could be rapidly resoluble. After sterilization by filtration through a 0.22 μm filter membrane, the resolution time did not exceed 2 min, and no obvious aggregation was observed after resolution.

[0082] In this embodiment, using an AQP5-overexpressing tumor cell line (hereinafter referred to as AQP5⁺ tumor cells) and an AQP5-low-expressing normal cell line (hereinafter referred to as AQP5⁻ normal cells) as models in vitro, under the same nanocarrier particle size and enzyme activity load, quantitative flow cytometry analysis showed that the intracellular uptake of the nanocarrier complex in AQP5-overexpressing tumor cells was 2.5 times that of a non-targeted arginine deiminase carrier with the same structure but without anti-AQP5 antibody. Under the same added enzyme activity dose, the ratio of arginine depletion in AQP5-overexpressing tumor cells to that in AQP5-low-expressing normal cells was calculated to be 6:1 based on the arginine concentration in the culture supernatant. In vivo, using a rodent intravenous administration model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was 5 times that of free arginine deiminase, and the plasma arginine concentration was maintained at 45% of the pre-administration level for 24–72 hours after administration.

[0083] Features of Example 1: This example uses moderate parameter configurations. The particle size of 125 nm is within the mid-range. The PEG molecular weight of 4000, shell thickness of 17 nm, dihydrazide group content of 0.11 mmol / g polymer, and hydrazone bond crosslinking density of 0.055 mmol / g polymer are all selected at moderate levels within their respective ranges. The protein loading of 20 wt% and surface enzyme density of 1.2 mg / m² are also moderate. The formulation pH is 7.1, close to physiological pH, and the zeta potential of −5 mV is moderate. This example ensures the stability and batch-to-batch reproducibility of the nanocarrier preparation through a balanced combination of parameters, while also considering targeting efficiency and cycling stability. This example is suitable for conventional anti-tumor therapeutic applications requiring standardized preparation processes and stable product quality, and is particularly suitable as a benchmark formulation for preclinical studies and process validation.

[0084] Example 2

[0085] This embodiment provides an anti-AQP5 arginine deiminase nanocarrier complex. The complex is a core-shell structured nanoparticle dispersion system with a particle size of 90 nm. The nanoparticles in this embodiment include a core phase, a shell phase, and an anti-AQP5 arginine deiminase protein conjugate B1 covalently fixed on the surface of the shell phase.

[0086] The core phase of this embodiment is a hydrophobic polymer backbone formed by the self-assembly of a PLGA-PEG-maleimide copolymer containing hydrophobic PLGA segments. The shell phase of this embodiment is composed of a hydrazone crosslinking network formed by dihydrazide groups on the PLGA-PEG-maleimide copolymer and a dialdehyde crosslinking agent. Under buffer conditions of 37°C and pH 6.5–7.4, the degree of hydrazone crosslinking of the shell phase in this embodiment remains above 80% relative to the initial time within 24 hours. Under buffer conditions of 37°C and pH 5.0–6.0, the degree of hydrazone crosslinking decreases by at least 30% within 24 hours, so as to maintain the stability of the nanoparticle structure under neutral conditions and to allow at least partial dissociation in the acidic intracellular microenvironment such as endosomes / lysosomes after tumor cell endocytosis, so that the protein conjugate B1, which was originally partially buried or restricted by the crosslinking shell, can obtain higher conformational freedom and outer surface exposure.

[0087] The protein conjugate B1 of this embodiment is formed by covalently coupling the antigen-binding fragment Fab of an anti-AQP5 monoclonal antibody that specifically binds to human AQP5 with arginine deiminase via a chemical cross-linking agent containing a maleimide group. The arginine deiminase retains, on average, at least one free thiol group that can undergo a thiomaleimide addition reaction with the maleimide group in the shell of this embodiment. The thiol group introduced onto the arginine deiminase in this embodiment forms a thioether bond with the maleimide group in the shell of this embodiment via a thiomaleimide addition reaction, thus covalently fixing it to the shell surface. This makes the protein conjugate B1 fixed to the shell surface more easily exposed in the tumor microenvironment and exert its arginine depletion effect.

[0088] The PLGA-PEG-maleimide copolymer of this embodiment is composed of a PLGA-PEG copolymer backbone formed by the ester condensation of a modified polylactic acid-hydroxyacetic acid copolymer PLGA with multiple carboxyl side groups on the backbone and polyethylene glycol PEG. A dihydrazide group formed by adipate dihydrazide and a maleimide group introduced by N-(2-aminoethyl)maleimide hydrochloride are introduced on the backbone. The molar ratio of lactic acid to glycolic acid in PLGA is 75:25, the average molecular weight of PEG is 3000, and both the maleimide group and the dihydrazide group are bonded to the copolymer backbone of this embodiment in the form of side chains.

[0089] In this embodiment, the dialdehyde crosslinking agent is glutaraldehyde, the shell thickness is 10 nm, the content of dihydrazide groups in the polymer of this embodiment is 0.07 mmol / g polymer, the hydrazone bond crosslinking density formed by dihydrazide groups and glutaraldehyde is 0.04 mmol / g polymer, and the hydrazone bond crosslinking density of this embodiment does not exceed the content of dihydrazide groups, and the polydispersity index of the nanoparticles of this embodiment is 0.16, and the particle size change does not exceed 20% after being stored at 4°C under sealed conditions for 3 months.

[0090] The protein conjugate B1 of this embodiment was prepared by the following steps: A 2.5 mg / mL solution of anti-AQP5 monoclonal antibody Fab fragment and a 3.5 mg / mL solution of arginine deiminase were prepared in phosphate buffer at pH 7.0. The arginine deiminase in this embodiment was selected from mycoplasma-derived arginine deiminase preparations. A thiol-introducing reagent, 2-iminothione hydrochloride, was added to the arginine deiminase solution of this embodiment. The reaction was carried out at a molar ratio of arginine deiminase to 2-iminothione hydrochloride of 1:5, introducing an average of 3 reactive thiol groups onto each enzyme molecule. The reaction temperature was 22°C, and the reaction time was 1.8 h, yielding thiolized arginine deiminase. Excess 2-iminothione hydrochloride was removed by gel filtration. The anti-AQP5 monoclonal antibody Fab fragment of this embodiment was reacted with Sulfo-SMCC, a cross-linking agent containing both N-hydroxysuccinimide ester and maleimide groups, at a molar ratio of Fab fragment to Sulfo-SMCC of 1:5.5. The reaction was carried out in a buffer solution at pH 7.2 at 25°C for 1.2 h, introducing an average of two maleimide groups onto the Fab fragment molecule to obtain an activated Fab fragment. Excess Sulfo-SMCC was removed by gel filtration. The thiolated arginine deiminase of this embodiment was reacted with the activated Fab fragment of this embodiment at pH 6.8 and 5°C for 3 h to form an anti-AQP5 monoclonal antibody Fab fragment-arginine deiminase protein conjugate, namely protein conjugate B1. The main peak fraction was collected by separation and purification methods, so that the average binding ratio of arginine deiminase to anti-AQP5 monoclonal antibody Fab fragment was 1.8:1, and at least one free thiol group capable of reacting with maleimide groups was retained in protein conjugate B1.

[0091] The loading amount of protein conjugate B1 in the nanoparticles of this embodiment is 25 wt% of the dry weight of the nanoparticles of this embodiment. The enzyme density on the surface of the nanoparticles is 1.6 mg / m², the pH of the formulation is 6.9, the zeta potential is −7 mV, and after being treated in human serum at 37°C for at least 48 h, the arginine deiminase activity was 78% of the initial activity before incubation, as determined by fluorescent enzyme activity assay using L-arginine as a substrate.

[0092] The PLGA-PEG-maleimide copolymer of this embodiment was prepared by the following steps: PLGA, PEG, adipic acid dihydrazide, and N-(2-aminoethyl)maleimide hydrochloride were simultaneously mixed with the condensing agent N,N′-dicyclohexylcarbodiguanidine and the catalyst 4-dimethylaminopyridine in anhydrous dichloromethane. The mass ratio of PLGA to PEG was 47:53, and the amount of 4-dimethylaminopyridine catalyst relative to the mass of PLGA was 3.5 wt%. The molar ratio of N,N′-dicyclohexyl carbodiguanidine to PLGA carboxyl groups was 1.05:1. This resulted in the PLGA carboxyl groups forming an ester bond with PEG, an amide bond with adipic acid dihydrazide to introduce a dihydrazide side chain, and a maleimide side chain with N-(2-aminoethyl)maleimide hydrochloride, while retaining some unreacted carboxyl groups. The reaction was carried out at 38°C for 6 hours to obtain a crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains. The crude product of this example was subjected to reduced pressure (0.008 MPa, 32°C) to remove the organic solvent. After precipitation with excess anhydrous diethyl ether, the product was filtered and washed three times with anhydrous diethyl ether to remove the N,N′-dicyclohexylurea byproduct. Subsequently, it was dried at 28°C and 0.003 MPa for 14 hours to obtain a solid intermediate. The solid intermediate of this embodiment was dissolved in N,N-dimethylformamide. The remaining carboxyl groups were activated by water-soluble carbodiimide and reacted with adipic acid dihydrazide to further adjust the degree of substitution of the dihydrazide group in the polymer, so that the content of the dihydrazide group was 0.07 mmol / g polymer. The reaction was terminated when the number average molecular weight of the polymer was 18 kDa, the polydispersity index was 1.15, and the degree of substitution of the maleimide group was 4 mol%, to obtain the PLGA-PEG-maleimide copolymer of this embodiment.

[0093] The PLGA-PEG-maleimide copolymer of this embodiment forms a dynamic core-shell nanocarrier intermediate through the following steps: The PLGA-PEG-maleimide copolymer of this embodiment is dissolved in ethanol to obtain an organic phase with a polymer concentration of 28 mg / mL. A phosphate buffer aqueous phase with a pH of 6.7 is prepared. The phosphate buffer in this embodiment is prepared by mixing sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate in a certain proportion, and glutaraldehyde is added to make the glutaraldehyde concentration 3 mmol / L. The organic phase and aqueous phase of this embodiment are mixed in a microfluidic mixer at a volume ratio of 1:2.8 by collision. The total flow rate of the microfluidic mixing is 16 mL / min, which allows the polymer of this embodiment to self-assemble into core-shell structured nanoparticles with a particle size of 90 nm. The resulting dispersion is then gently stirred at 23 °C for 3 h. During this process, the dihydrazide groups and glutaraldehyde continue to react to form a hydrazone cross-linked shell layer. Organic solvents were removed by rotary evaporation and the solid content of the dispersion was adjusted to 18 mg / mL and pH to 6.9 to obtain a dynamic core-shell nanocarrier intermediate with a median particle size of 90 nm and a polydispersity index of 0.16.

[0094] The anti-AQP5 arginine deiminase nanocarrier complex and its lyophilized powder of this embodiment were prepared through the following steps: The dynamic core-shell nanocarrier intermediate of this embodiment was diluted and the pH was adjusted to 6.8. The molar ratio of maleimide groups on the surface of the nanocarrier shell to reactive thiol groups in protein conjugate B1 was 1:1.2. The dynamic core-shell nanocarrier intermediate of this embodiment was diluted to a polymer mass concentration of 15 mg / mL. The protein conjugate B1 solution was slowly added, and the mixture was gently stirred at 5°C for 3 hours to allow protein conjugate B1 to be covalently attached to the shell surface via a thiomaleimide addition reaction. After the reaction, gel filtration was used to separate the free protein conjugate B1, small molecule salts, and unreacted small molecules, obtaining a dispersion of the nanocarrier complex of this embodiment, with a protein conjugate B1 mass fraction of 25 wt% in the solid. A lyophilization protectant, comprising 8 wt% of the total dry matter of the nanoparticles and protein conjugate B1, was added to the dispersion of this embodiment. The lyophilization protectant in this embodiment was a mixture of trehalose dihydrate and sucrose in a mass ratio of 1:1.2. After being dispensed into vials, the mixture was pre-frozen at −40°C for 3 hours. Subsequently, during the main drying stage, the product temperature was controlled at −22°C and the chamber vacuum was 0.00025 MPa, and the product was dried for 20 hours. Then, it was subjected to analytical drying at 0.00015 MPa and 22°C for 4 hours to obtain a lyophilized powder of the nanocarrier complex of this embodiment with a water content of 2.2 wt% that could be rapidly resoluble. After being filtered through a 0.22 μm filter membrane for sterilization, the resolution time did not exceed 2 minutes, and no obvious aggregation was observed after resolution.

[0095] In this embodiment, using AQP5-overexpressing tumor cell lines and AQP5-low-expressing normal cell lines as models in vitro, under the same nanocarrier particle size and enzyme activity loading conditions, confocal microscopy analysis showed that the intracellular uptake of the nanocarrier complex in AQP5-overexpressing tumor cells was three times that of a non-targeted arginine deiminase carrier with the same structure but without anti-AQP5 antibody or its antigen-binding fragment. Under the same added enzyme activity dose, calculations based on the arginine concentration in the culture supernatant showed that the ratio of arginine depletion in AQP5-overexpressing tumor cells to that in AQP5-low-expressing normal cells was 7:1. In vivo, using intravenous administration in rodents as a model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was six times that of free arginine deiminase, and the plasma arginine concentration was maintained at 40% of the pre-administration level for 24–72 hours after administration.

[0096] Example 2 Features: This example employs a small particle size and high loading strategy. The 90nm particle size, at the smaller end of the range, facilitates deep penetration into tumor tissue and cellular uptake. The lower PEG molecular weight of 3000 reduces steric hindrance, and the thinner shell thickness of 10nm reduces protein diffusion resistance. The low dihydrazide group content (0.07 mmol / g polymer) and hydrazone crosslinking density (0.04 mmol / g polymer) contribute to pH-responsive dissociation. The higher protein loading of 25wt% and surface enzyme density of 1.6 mg / m² enhance the therapeutic efficiency per unit carrier. The formulation's slightly acidic pH of 6.9 is suitable for certain tumor microenvironments. This example improves targeted delivery efficiency and intratumoral enzyme activity enrichment by optimizing the combination of particle size and loading, while maintaining good pH-responsive release characteristics. This example is particularly suitable for solid tumor treatment scenarios requiring enhanced tumor penetration and efficient arginine depletion, especially for treating dense tumors or acute treatment situations requiring rapid onset of action.

[0097] Example 3

[0098] This embodiment provides an anti-AQP5 arginine deiminase nanocarrier complex. The complex is a core-shell structured nanoparticle dispersion system with a particle size of 160 nm. The nanoparticles in this embodiment include a core phase, a shell phase, and an anti-AQP5 arginine deiminase protein conjugate B1 covalently fixed on the surface of the shell phase.

[0099] The core phase of this embodiment is a hydrophobic polymer backbone formed by the self-assembly of a PLGA-PEG-maleimide copolymer containing hydrophobic PLGA segments. The shell phase of this embodiment is composed of a hydrazone crosslinking network formed by dihydrazide groups on the PLGA-PEG-maleimide copolymer and a dialdehyde crosslinking agent. Under buffer conditions of 37°C and pH 6.5–7.4, the degree of hydrazone crosslinking of the shell phase in this embodiment remains above 80% relative to the initial time within 24 hours. Under buffer conditions of 37°C and pH 5.0–6.0, the degree of hydrazone crosslinking decreases by at least 30% within 24 hours, so as to maintain the stability of the nanoparticle structure under neutral conditions and to allow at least partial dissociation in the acidic intracellular microenvironment such as endosomes / lysosomes after tumor cell endocytosis, so that the protein conjugate B1, which was originally partially buried or restricted by the crosslinking shell, can obtain higher conformational freedom and outer surface exposure.

[0100] The protein conjugate B1 of this embodiment is formed by covalently coupling an anti-AQP5 monoclonal antibody capable of specifically binding to human AQP5 with arginine deiminase via a chemical cross-linking agent containing a maleimide group. The arginine deiminase retains, on average, at least one free thiol group that can undergo a thiomaleimide addition reaction with the maleimide group in the shell of this embodiment. The thiol group introduced onto the arginine deiminase in this embodiment forms a thioether bond with the maleimide group in the shell of this embodiment via a thiomaleimide addition reaction, thus covalently fixing it to the shell surface. This makes the protein conjugate B1 fixed to the shell surface more easily exposed in the tumor microenvironment and exert its arginine depletion effect.

[0101] The PLGA-PEG-maleimide copolymer of this embodiment is composed of a PLGA-PEG copolymer backbone formed by the ester condensation of a modified polylactic acid-hydroxyacetic acid copolymer PLGA with multiple carboxyl side groups on the backbone and polyethylene glycol PEG. A dihydrazide group formed by adipate dihydrazide and a maleimide group introduced by N-(2-aminoethyl)maleimide hydrochloride are introduced on the backbone. The molar ratio of lactic acid to glycolic acid in PLGA is 75:25, the average molecular weight of PEG is 5000, and both the maleimide group and the dihydrazide group are bonded to the copolymer backbone of this embodiment in the form of side chains.

[0102] In this embodiment, the dialdehyde crosslinking agent is glutaraldehyde, the shell thickness is 24 nm, the content of dihydrazide groups in the polymer of this embodiment is 0.15 mmol / g polymer, the hydrazone bond crosslinking density formed by dihydrazide groups and glutaraldehyde is 0.075 mmol / g polymer, and the hydrazone bond crosslinking density of this embodiment does not exceed the content of dihydrazide groups, and the polydispersity index of the nanoparticles in this embodiment is 0.19, and the particle size change does not exceed 20% after being stored at 4°C under sealed conditions for 3 months.

[0103] The protein conjugate B1 of this embodiment was prepared by the following steps: A 1.8 mg / mL solution of anti-AQP5 monoclonal antibody and a 2.8 mg / mL solution of arginine deiminase were prepared in phosphate buffer at pH 7.3. The arginine deiminase in this embodiment was selected from an arginine deiminase preparation derived from Toxoplasma gondii. A thiol-introducing reagent, 2-iminothione hydrochloride, was added to the arginine deiminase solution of this embodiment. The reaction was carried out at a molar ratio of arginine deiminase to 2-iminothione hydrochloride of 1:3.5, introducing an average of two reactive thiol groups onto each enzyme molecule. The reaction temperature was 18°C, and the reaction time was 1.2 h, yielding thiolated arginine deiminase. Excess 2-iminothione hydrochloride was removed by gel filtration. The anti-AQP5 monoclonal antibody of this embodiment was reacted with a cross-linking agent SMCC containing both N-hydroxysuccinimide ester and maleimide groups at a molar ratio of antibody to SMCC of 1:3.5 in a buffer solution at pH 7.5 at 25°C for 0.8 h. This resulted in the introduction of an average of one maleimide group onto the antibody molecule, yielding an activated antibody. Excess SMCC was removed by gel filtration. The thiolated arginine deiminase of this embodiment was then reacted with the activated antibody at pH 7.2 and 7°C for 2 h to form an anti-AQP5 monoclonal antibody-arginine deiminase protein conjugate, namely protein conjugate B1. The main peak fraction was collected through separation and purification, ensuring an average binding ratio of arginine deiminase to anti-AQP5 monoclonal antibody of 1.2:1, and that protein conjugate B1 retained at least one free thiol group capable of reacting with a maleimide group.

[0104] The loading amount of protein conjugate B1 in the nanoparticles of this embodiment is 15 wt% of the dry weight of the nanoparticles of this embodiment. The enzyme density on the surface of the nanoparticles is 0.8 mg / m², the pH of the formulation is 7.3, the zeta potential is −3 mV, and after being treated in human serum at 37°C for at least 48 h, the arginine deiminase activity was determined by colorimetric enzyme activity assay using L-arginine as a substrate, and the retention rate of arginine deiminase activity relative to the initial activity before incubation was 82%.

[0105] The PLGA-PEG-maleimide copolymer of this embodiment was prepared by the following steps: PLGA, PEG, adipic acid dihydrazide, and N-(2-aminoethyl)maleimide hydrochloride were simultaneously mixed with the condensing agent N,N′-dicyclohexylcarbodiguanidine and the catalyst 4-dimethylaminopyridine in anhydrous dichloromethane. The mass ratio of PLGA to PEG was 53:47, and the amount of 4-dimethylaminopyridine catalyst relative to the mass of PLGA was 2.5 wt%. The molar ratio of N,N′-dicyclohexyl carbodiguanidine to PLGA carboxyl groups was 0.95:1. This resulted in the PLGA carboxyl groups forming an ester bond with PEG, an amide bond with adipic acid dihydrazide to introduce a dihydrazide side chain, and a maleimide side chain with N-(2-aminoethyl)maleimide hydrochloride, while retaining some unreacted carboxyl groups. The reaction was carried out at 32°C for 10 h to obtain a crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains. The crude product of this example was subjected to reduced pressure (0.015 MPa, 38°C) to remove the organic solvent. After precipitation with excess anhydrous diethyl ether, the product was filtered and washed twice with anhydrous diethyl ether to remove the N,N′-dicyclohexylurea byproduct. Subsequently, it was dried at 35°C and 0.008 MPa for 12 h to obtain a solid intermediate. The solid intermediate of this embodiment was dissolved in N,N-dimethylformamide. The remaining carboxyl groups were activated by water-soluble carbodiimide and reacted with adipic acid dihydrazide to further adjust the degree of substitution of the dihydrazide group in the polymer, so that the content of the dihydrazide group was 0.15 mmol / g polymer. The reaction was terminated when the number average molecular weight of the polymer was 26 kDa, the polydispersity index was 1.25, and the degree of substitution of the maleimide group was 2 mol%, to obtain the PLGA-PEG-maleimide copolymer of this embodiment.

[0106] The PLGA-PEG-maleimide copolymer of this embodiment forms a dynamic core-shell nanocarrier intermediate through the following steps: The PLGA-PEG-maleimide copolymer of this embodiment is dissolved in acetonitrile to obtain an organic phase with a polymer concentration of 32 mg / mL. A phosphate buffer aqueous phase with a pH of 6.9 is prepared. The phosphate buffer in this embodiment is prepared by mixing sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate in a certain proportion, and glutaraldehyde is added to make the glutaraldehyde concentration 7 mmol / L. The organic phase and aqueous phase of this embodiment are mixed in a microfluidic mixer at a volume ratio of 1:3.2 with a total flow rate of 14 mL / min, so that the polymer of this embodiment self-assembles to form core-shell structured nanoparticles with a particle size of 160 nm. The resulting dispersion is then gently stirred at 21 °C for 1.5 h. During this process, the dihydrazide groups and glutaraldehyde continue to react to form a hydrazone cross-linked shell layer. Organic solvents were removed by rotary evaporation and the solid content of the dispersion was adjusted to 12 mg / mL and pH to 7.0 to obtain a dynamic core-shell nanocarrier intermediate with a median particle size of 160 nm and a polydispersity index of 0.19.

[0107] The anti-AQP5 arginine deiminase nanocarrier complex and its lyophilized powder of this embodiment were prepared through the following steps: The dynamic core-shell nanocarrier intermediate of this embodiment was diluted and the pH was adjusted to 7.0. The molar ratio of maleimide groups on the surface of the nanocarrier shell to reactive thiol groups in protein conjugate B1 was 1:0.8. The dynamic core-shell nanocarrier intermediate of this embodiment was diluted to a polymer mass concentration of 10 mg / mL. The protein conjugate B1 solution was slowly added, and the reaction was gently stirred at 7°C for 2 hours, allowing protein conjugate B1 to be covalently attached to the shell surface via a thiomaleimide addition reaction. After the reaction, ultrafiltration was used to remove free protein conjugate B1, small molecule salts, and unreacted small molecules, obtaining a dispersion of the nanocarrier complex of this embodiment, with the mass fraction of protein conjugate B1 in the solid being 15 wt%. A lyophilization protectant, comprising 6 wt% of the total dry matter of the nanoparticles and protein conjugate B1, was added to the dispersion of this embodiment. The lyophilization protectant in this embodiment was a mixture of trehalose dihydrate and sucrose in a mass ratio of 1:1.8. After being dispensed into vials, the mixture was pre-frozen at −40°C for 2 hours. Subsequently, during the main drying stage, the product temperature was controlled at −28°C and the chamber vacuum was 0.0004 MPa for 28 hours. Then, it was subjected to analytical drying at 0.0003 MPa and 28°C for 6 hours to obtain a lyophilized powder of the nanocarrier complex of this embodiment with a water content of 2.8 wt% that could be rapidly resoluble. After being filtered through a 0.22 μm filter membrane for sterilization, the resolution time did not exceed 2 minutes and no obvious aggregation was observed after resolution.

[0108] In this embodiment, using AQP5-overexpressing tumor cell lines and AQP5-low-expressing normal cell lines as models in vitro, under the same nanocarrier particle size and enzyme activity load conditions, quantitative flow cytometry analysis showed that the intracellular uptake of the nanocarrier complex in AQP5-overexpressing tumor cells was 2.2 times that of a non-targeted arginine deiminase carrier with the same structure but without anti-AQP5 antibody or its antigen-binding fragment. At the same added enzyme activity dose, the ratio of arginine depletion in AQP5-overexpressing tumor cells to that in AQP5-low-expressing normal cells, calculated by measuring the arginine concentration in the culture supernatant, was 5.5:1. In vivo, using intravenous administration in rodents as a model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was 8 times that of free arginine deiminase, and the plasma arginine concentration was maintained at 35% of the pre-administration level for 24–72 hours after administration.

[0109] Example 3 Features: This example employs a large-particle-size, long-circulation strategy. The 160nm particle size, at the larger end of the range, is beneficial for prolonging blood circulation time and enhancing the EPR effect of passive targeting. The higher PEG molecular weight of 5000 enhances the hydrophilic shielding effect and reduces macrophage phagocytosis. The thicker shell of 24nm provides stronger structural stability and protein protection. The higher content of dihydrazide groups (0.15 mmol / g polymer) and hydrazone crosslinking density (0.075 mmol / g polymer) enhances the mechanical strength of the shell crosslinking network. The relatively low protein loading of 15wt% and surface enzyme density of 0.8 mg / m² reduce carrier surface charge and immunogenicity. The formulation pH of 7.3, slightly alkaline and close to blood pH, contributes to circulation stability. This example improves the carrier's circulation stability in blood and passive enrichment of tumor tissue by optimizing particle size and shell structure, while maintaining good pH responsiveness and enzyme activity protection. This example is particularly suitable for chronic tumor treatment scenarios requiring long-acting therapy and enhanced passive targeting capabilities, especially for highly vascularized tumors or long-term treatment regimens requiring multiple doses to maintain efficacy.

[0110] Example 4

[0111] This embodiment provides an anti-AQP5 arginine deiminase nanocarrier complex. The complex is a core-shell structured nanoparticle dispersion system with a particle size of 185 nm. The nanoparticles in this embodiment include a core phase, a shell phase, and an anti-AQP5 arginine deiminase protein conjugate B1 covalently fixed on the surface of the shell phase.

[0112] The core phase of this embodiment is a hydrophobic polymer backbone formed by the self-assembly of a PLGA-PEG-maleimide copolymer containing hydrophobic PLGA segments. The shell phase of this embodiment is composed of a hydrazone crosslinking network formed by dihydrazide groups on the PLGA-PEG-maleimide copolymer and a dialdehyde crosslinking agent. Under buffer conditions of 37°C and pH 6.5–7.4, the degree of hydrazone crosslinking of the shell phase in this embodiment remains above 80% relative to the initial time within 24 hours. Under buffer conditions of 37°C and pH 5.0–6.0, the degree of hydrazone crosslinking decreases by at least 30% within 24 hours, so as to maintain the stability of the nanoparticle structure under neutral conditions and to allow at least partial dissociation in the acidic intracellular microenvironment such as endosomes / lysosomes after tumor cell endocytosis, so that the protein conjugate B1, which was originally partially buried or restricted by the crosslinking shell, can obtain higher conformational freedom and outer surface exposure.

[0113] The protein conjugate B1 of this embodiment is formed by covalently coupling an anti-AQP5 monoclonal antibody capable of specifically binding to human AQP5 with arginine deiminase via a chemical cross-linking agent containing a maleimide group. The arginine deiminase retains, on average, at least one free thiol group that can undergo a thiomaleimide addition reaction with the maleimide group in the shell of this embodiment. The thiol group introduced onto the arginine deiminase in this embodiment forms a thioether bond with the maleimide group in the shell of this embodiment via a thiomaleimide addition reaction, thus covalently fixing it to the shell surface. This makes the protein conjugate B1 fixed to the shell surface more easily exposed in the tumor microenvironment and exert its arginine depletion effect.

[0114] The PLGA-PEG-maleimide copolymer of this embodiment is composed of a PLGA-PEG copolymer backbone formed by the ester condensation of a modified polylactic acid-glycolic acid copolymer PLGA with multiple carboxyl side groups on the backbone and polyethylene glycol PEG. A dihydrazide group formed by adipate dihydrazide and a maleimide group introduced by N-(2-aminoethyl)maleimide hydrochloride are introduced on the backbone. The molar ratio of lactic acid to glycolic acid in PLGA is 75:25, the average molecular weight of PEG is 5600, and both the maleimide group and the dihydrazide group are bonded to the copolymer backbone of this embodiment in the form of side chains.

[0115] In this embodiment, the dialdehyde crosslinking agent is glutaraldehyde, the shell thickness is 8 nm, the content of dihydrazide groups in the polymer of this embodiment is 0.04 mmol / g polymer, the hydrazone bond crosslinking density formed by dihydrazide groups and glutaraldehyde is 0.02 mmol / g polymer, and the hydrazone bond crosslinking density of this embodiment does not exceed the content of dihydrazide groups, and the polydispersity index of the nanoparticles of this embodiment is 0.17, and the particle size change does not exceed 20% after being stored at 4°C under sealed conditions for 3 months.

[0116] Protein conjugate B1 in this embodiment was prepared by the following steps: A 2.2 mg / mL solution of anti-AQP5 monoclonal antibody and a 3.2 mg / mL solution of arginine deiminase were prepared in phosphate buffer at pH 7.4. A thiol-introducing reagent, 2-iminothione hydrochloride, was added to the arginine deiminase solution in this embodiment, and the reaction was carried out at a molar ratio of arginine deiminase to 2-iminothione hydrochloride of 1:5.5, so that an average of 4 reactive thiol groups were introduced onto each enzyme molecule. The reaction temperature was 24°C, and the reaction time was 2 h, yielding thiolated arginine deiminase. Excess 2-iminothione hydrochloride was removed by gel filtration. The anti-AQP5 monoclonal antibody of this embodiment was reacted with Sulfo-SMCC, a cross-linking agent containing both N-hydroxysuccinimide ester and maleimide groups, at a molar ratio of antibody to Sulfo-SMCC of 1:5.8. The reaction was carried out in a buffer solution at pH 7.6 at 25°C for 1.5 h, introducing an average of two maleimide groups onto the antibody molecule to obtain the activated antibody. Excess Sulfo-SMCC was removed by gel filtration. The thiolated arginine deiminase of this embodiment was reacted with the activated antibody of this embodiment at pH 6.5 and 4°C for 3.5 h to form an anti-AQP5 monoclonal antibody-arginine deiminase protein conjugate, namely protein conjugate B1. The main peak component was collected by separation and purification methods, so that the average binding ratio of arginine deiminase to anti-AQP5 monoclonal antibody was 2:1, and at least one free thiol group capable of reacting with maleimide groups was retained in protein conjugate B1.

[0117] The loading amount of protein conjugate B1 in the nanoparticles of this embodiment is 27 wt% of the dry weight of the nanoparticles of this embodiment. The enzyme density on the surface of the nanoparticles is 1.85 mg / m², the pH of the formulation is 7.35, the zeta potential is −1 mV, and after being treated in human serum at 37°C for at least 48 h, the arginine deiminase activity was 72% of the initial activity before incubation, as determined by fluorescent enzyme activity assay using L-arginine as a substrate.

[0118] The PLGA-PEG-maleimide copolymer of this embodiment was prepared by the following steps: PLGA, PEG, adipic acid dihydrazide, and N-(2-aminoethyl)maleimide hydrochloride were simultaneously mixed with the condensing agent N,N′-dicyclohexylcarbodiguanidine and the catalyst 4-dimethylaminopyridine in anhydrous dichloromethane. The mass ratio of PLGA to PEG was 42:58, and the amount of 4-dimethylaminopyridine catalyst relative to the mass of PLGA was 4.5 wt%. The molar ratio of N,N′-dicyclohexyl carbodiguanidine to PLGA carboxyl groups was 1.08:1. This caused the PLGA carboxyl groups to form ester bonds with PEG, amide bonds with adipic acid dihydrazide to introduce dihydrazide side chains, and maleimide side chains with N-(2-aminoethyl)maleimide hydrochloride, while retaining some unreacted carboxyl groups. The reaction was carried out at 36°C for 7 h to obtain a crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains. The crude product of this example was subjected to reduced pressure to remove organic solvents at 36°C and a vacuum of 0.006 MPa. After precipitation with excess anhydrous diethyl ether, the product was filtered and washed four times with anhydrous diethyl ether to remove N,N′-dicyclohexylurea byproducts. Subsequently, it was dried at 32°C and a vacuum of 0.002 MPa for 22 h to obtain a solid intermediate. The solid intermediate of this embodiment was dissolved in N,N-dimethylformamide. The remaining carboxyl groups were activated by water-soluble carbodiimide and reacted with adipic acid dihydrazide to further adjust the degree of substitution of the dihydrazide group in the polymer, so that the content of the dihydrazide group was 0.04 mmol / g polymer. The reaction was terminated when the number average molecular weight of the polymer was 28 kDa, the polydispersity index was 1.28, and the degree of substitution of the maleimide group was 4.5 mol%, to obtain the PLGA-PEG-maleimide copolymer of this embodiment.

[0119] The PLGA-PEG-maleimide copolymer of this embodiment forms a dynamic core-shell nanocarrier intermediate through the following steps: The PLGA-PEG-maleimide copolymer of this embodiment is dissolved in acetonitrile to obtain an organic phase with a polymer concentration of 34 mg / mL. A phosphate buffer aqueous phase with a pH of 6.6 is prepared. The phosphate buffer in this embodiment is prepared by mixing sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate in a certain proportion, and glutaraldehyde is added to make the glutaraldehyde concentration 1.5 mmol / L. The organic phase and aqueous phase of this embodiment are mixed in a microfluidic mixer at a volume ratio of 1:2.6 by collision. The total flow rate of the microfluidic mixing is 17 mL / min, so that the polymer of this embodiment self-assembles to form core-shell structured nanoparticles with a particle size of 185 nm. The resulting dispersion is then gently stirred at 20 °C for 3.5 h. During this process, the dihydrazide groups and glutaraldehyde continue to react to form a hydrazone cross-linked shell layer. Organic solvents were removed by rotary evaporation and the solid content of the dispersion was adjusted to 20 mg / mL and pH to 7.0 to obtain a dynamic core-shell nanocarrier intermediate with a median particle size of 185 nm and a polydispersity index of 0.17.

[0120] The anti-AQP5 arginine deiminase nanocarrier complex and its lyophilized powder of this embodiment were prepared through the following steps: The dynamic core-shell nanocarrier intermediate of this embodiment was diluted and the pH was adjusted to 7.0. The molar ratio of maleimide groups on the surface of the nanocarrier shell to reactive thiol groups in protein conjugate B1 was 1:1.4. The dynamic core-shell nanocarrier intermediate of this embodiment was diluted to a polymer mass concentration of 18 mg / mL. The protein conjugate B1 solution was slowly added, and the reaction was gently stirred at 4°C for 3.5 h, allowing protein conjugate B1 to be covalently attached to the shell surface via a thiomaleimide addition reaction. After the reaction, gel filtration was used to separate and remove free protein conjugate B1, small molecule salts, and unreacted small molecules, obtaining a dispersion of the nanocarrier complex of this embodiment, with a protein conjugate B1 mass fraction of 27 wt% in the solid. A lyophilization protectant, comprising 9 wt% of the total dry matter of the nanoparticles and protein conjugate B1, was added to the dispersion of this embodiment. The lyophilization protectant in this embodiment was a mixture of trehalose dihydrate and sucrose in a mass ratio of 1:1.1. After being dispensed into vials, the mixture was pre-frozen at −40°C for 3.5 h. Subsequently, during the main drying stage, the product temperature was controlled at −20°C and the chamber vacuum was 0.00018 MPa for 32 h. Then, it was subjected to analytical drying at 0.00012 MPa and 30°C for 7 h to obtain a lyophilized powder of the nanocarrier complex of this embodiment with a water content of 2.6 wt% that could be rapidly resoluble. After being filtered through a 0.22 μm filter membrane for sterilization, the resolution time did not exceed 2 min and no obvious aggregates were observed after resolution.

[0121] In this embodiment, using AQP5-overexpressing tumor cell lines and AQP5-low-expressing normal cell lines as models in vitro, under the same nanocarrier particle size and enzyme activity load conditions, confocal microscopy analysis showed that the intracellular uptake of the nanocarrier complex in AQP5-overexpressing tumor cells was 2.8 times that of a non-targeted arginine deiminase carrier with the same structure but without anti-AQP5 antibody or its antigen-binding fragment. Under the same added enzyme activity dose, calculations based on the arginine concentration in the culture supernatant showed that the ratio of arginine depletion in AQP5-overexpressing tumor cells to that in AQP5-low-expressing normal cells was 6.5:1. In vivo, using intravenous administration in rodents as a model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was 9 times that of free arginine deiminase, and the plasma arginine concentration was maintained at 33% of the pre-administration level for 24–72 hours after administration.

[0122] Features of Example 4: This example employs a boundary parameter combination strategy. The particle size of 185 nm is close to the upper limit of the range, the PEG molecular weight of 5600 is close to the upper limit, the shell thickness of 8 nm is close to the lower limit, the content of dihydrazide groups of 0.04 mmol / g polymer and the crosslinking density of hydrazone bonds of 0.02 mmol / g polymer are both close to the lower limit, the number of thiol groups per enzyme molecule is 4, reaching the upper limit, the number of maleimide groups per antibody molecule is 2, reaching the upper limit, the binding ratio of arginine deiminase to antibody is 2:1, reaching the upper limit, the protein loading of 27 wt% and the surface enzyme density of 1.85 mg / m² are close to the upper limit, the formulation pH of 7.35 is close to the upper limit, the zeta potential of −1 mV is close to the upper limit, and the glutaraldehyde concentration of 1.5 mmol / L is close to the lower limit. This embodiment fully demonstrates the feasibility of the technical solution by combining parameters close to the range boundary, while avoiding multiple key parameters from reaching extreme values ​​simultaneously. It enhances pH responsiveness by employing a thinner shell and lower crosslinking density based on a larger particle size and high PEG molecular weight. At high protein loading, it improves coupling efficiency by using a higher enzyme-antibody binding ratio and a greater number of thiol maleimide groups. This embodiment is suitable for composite therapeutic scenarios requiring enhanced cyclic stability, high protein loading, and rapid pH-responsive release, and is particularly suitable for clinical applications requiring long-term maintenance of high enzyme activity in the bloodstream and rapid release within the acidic tumor microenvironment.

[0123] Performance testing:

[0124] Comparative Example 1: Basically the same as Example 1, except that the nanoparticle size is 35 nm (achieved by increasing the organic phase polymer concentration to 45 mg / mL and increasing the total microfluidic flow rate to 22 mL / min), while the amounts of other components and preparation conditions remain unchanged.

[0125] Comparative Example 2: Basically the same as Example 1, except that the nanoparticle size is 215 nm (achieved by reducing the organic phase polymer concentration to 18 mg / mL and reducing the total microfluidic flow rate to 10 mL / min), while the amounts of other components and preparation conditions remain unchanged.

[0126] Comparative Example 3: It is basically the same as Example 1, except that the average molecular weight of PEG is 1500. PLGA-PEG-maleimide copolymer is prepared by condensing PEG with PLGA using PEG with a molecular weight of 1500. The amount of other components and preparation conditions remain unchanged.

[0127] Comparative Example 4: It is basically the same as Example 1, except that the average molecular weight of PEG is 6500. PLGA-PEG-maleimide copolymer is prepared by condensing PEG with PLGA using PEG with a molecular weight of 6500. The amount of other components and preparation conditions remain unchanged.

[0128] Comparative Example 5: It is basically the same as Example 1, except that the shell thickness is 3 nm, which is achieved by reducing the glutaraldehyde concentration to 0.3 mmol / L and shortening the crosslinking reaction time to 0.8 h. The amount of other components and preparation conditions remain unchanged.

[0129] Comparative Example 6: It is basically the same as Example 1, except that the shell thickness is 34 nm, which is achieved by increasing the glutaraldehyde concentration to 12 mmol / L and extending the crosslinking reaction time to 5 h. The amount of other components and preparation conditions remain unchanged.

[0130] Comparative Example 7: Basically the same as Example 1, except that the content of dihydrazide groups is 0.015 mmol / g polymer, which is achieved by reducing the amount of adipic acid dihydrazide added in the synthesis step, while the amount of other components and preparation conditions remain unchanged.

[0131] Comparative Example 8: It is basically the same as Example 1, except that the crosslinking density of hydrazone bonds is 0.006 mmol / g polymer, which is achieved by shortening the crosslinking reaction time of dihydrazide group and glutaraldehyde to 0.5 h. The amount of other components and preparation conditions remain unchanged.

[0132] Comparative Example 9: Basically the same as Example 1, except that the loading amount of protein conjugate B1 is 7wt%, which is achieved by reducing the amount of protein conjugate B1 solution added in the coupling step. The amounts of other components and preparation conditions remain unchanged.

[0133] Comparative Example 10: Basically the same as Example 1, except that the loading amount of protein conjugate B1 is 35wt%, which is achieved by increasing the amount of protein conjugate B1 solution added in the coupling step, while the amounts of other components and preparation conditions remain unchanged.

[0134] Comparative Example 11: Essentially the same as Example 1, except that an anti-AQP5 monoclonal antibody was not used. Protein conjugate B1 was formed solely by the direct coupling of arginine deiminase with a thiol group to a shell maleimide group (non-targeting carrier). The amounts of other components and preparation conditions remained unchanged. This comparative example demonstrates the crucial role of anti-AQP5 antibody-mediated active targeting in enhancing uptake and selective arginine depletion in AQP5-overexpressing tumor cells.

[0135] Comparative Example 12: It is basically the same as Example 1, except that the shell crosslinking agent is 1,4-butanediol diglycidyl ether (a non-pH-responsive crosslinking agent) instead of glutaraldehyde. A stable ether crosslinking network is formed by the reaction of the amino group of the dihydrazide group with the epoxy group (concentration 5 mmol / L, reaction conditions are the same). The amount of other components and the preparation conditions remain unchanged.

[0136] Comparative Example 13: It is basically the same as Example 1, except that PEG modification is not used. The PLGA-maleimide copolymer is prepared by direct condensation of PLGA with adipic acid dihydrazide and N-(2-aminoethyl)maleimide hydrochloride (without introducing PEG segments). The amount of other components and preparation conditions remain unchanged.

[0137] Particle size and particle size distribution determination

[0138] Test Subject: Aqueous dispersion of nanocarrier complex. Test Objective: To characterize the average particle size (Z-average), polydispersity index (PDI), and particle size distribution characteristics of nanoparticles, and to evaluate the size uniformity and dispersion stability of nanoparticles. Test Principle: Based on dynamic light scattering (DLS) technology, the hydrodynamic diameter is calculated using the Stokes-Einstein equation by detecting the intensity fluctuations of scattered light caused by the Brownian motion of nanoparticles. Experimental Method: Take an appropriate amount of nanocarrier complex dispersion and dilute it with PBS buffer at pH 7.0 to an appropriate concentration (scattered light count rate 10). 4 -10 5 (cps) were added to a quartz cuvette and measured on a Malvern Zetasizer Nano ZS90 particle size analyzer at 25℃ and a backscattering angle of 173°. Each sample was measured in triplicate, and the Z-average particle size and PDI were automatically calculated. Key parameters: test temperature 25±0.5℃, sample dilution factor 50-200 times, equilibration time 120 seconds. Data processing: the arithmetic mean ± standard deviation of the three measurements was used. A PDI < 0.20 was considered as uniform particle size distribution.

[0139] ζ potential measurement

[0140] Test Subject: Aqueous dispersion of nanocarrier complex. Test Objective: To determine the zeta potential (ζ-potential) of nanoparticles, evaluate their surface charge characteristics and colloidal stability, and indirectly reflect the immobilization state of protein-bound compound B1 on the shell surface. Test Principle: Based on electrophoretic light scattering (ELS) technology, charged nanoparticles are electrophoretically migrated by applying an electric field. The migration rate is measured using laser Doppler velocimetry, and the ζ-potential is calculated according to the Smoluchowski equation. Experimental Method: An appropriate amount of nanocarrier complex dispersion was diluted to a suitable concentration with 10 mM PBS buffer (pH 7.0), transferred to a folded capillary electrophoresis cell, and measured at 25 °C on a Malvern Zetasizer Nano ZS90 instrument. Each sample was measured in triplicate. Key Parameters: Test temperature 25 ± 0.5 °C, electric field strength automatically optimized, equilibration time 120 seconds, measurement voltage 10-50 V. Data Processing: The arithmetic mean ± standard deviation of the three measurements was used. A ζ-potential range of -10 mV to 0 mV was considered acceptable.

[0141] Determination of hydrazone bond crosslinking degree and pH response

[0142] Test Subjects: Dynamic core-shell nanocarrier intermediates and nanocarrier complexes. Test Objectives: To quantitatively analyze the crosslinking density of hydrazone bonds in the shell layer, evaluate the stability and dissociation characteristics of the hydrazone crosslinking network under different pH conditions, and verify the pH-responsive release mechanism. Test Principle: Utilizing the chemical characteristics of hydrazone bond hydrolysis under acidic conditions, free dihydrazide groups are quantitatively detected by spectrophotometry, and the degree of hydrazone crosslinking and its changes with time and pH are calculated. Experimental Methods: The nanocarrier dispersion was placed in buffer solutions at pH 6.5-7.4 and pH 5.0-6.0, respectively, and incubated at 37℃ for 0h, 6h, 12h, and 24h. Samples were taken periodically, and the supernatant was separated by ultrafiltration. The free dihydrazide groups were derivatized with 2,4-dinitrofluorobenzene (DNFB), and the absorbance was measured at a wavelength of 360nm. The concentration of free dihydrazide was calculated based on the standard curve, and the change rate of hydrazone crosslinking degree was extrapolated. Standard Basis: Self-developed method and validated by recovery rate and precision. Key parameters: Temperature 37±0.5℃, pH control accuracy ±0.1, DNFB derivatization reaction time 30min. Data processing: Calculate the relative crosslinking retention rate (%) = (residual crosslinking degree / initial crosslinking degree) × 100%, n≥3.

[0143] Arginine deiminase activity assay

[0144] Test Subjects: Free arginine deiminase, protein-bound B1, and nanocarrier complex. Test Objective: To evaluate the catalytic activity of arginine deiminase and its activity retention rate during coupling and immobilization, and to verify the enzyme's stability in serum. Test Principle: Arginine deiminase catalyzes the hydrolysis of L-arginine to produce L-citrulline and ammonia. The amount of ammonia generated is detected using a modified Berthelot colorimetric method and converted into enzyme activity units. Experimental Method: An appropriate amount of enzyme sample was added to a reaction system containing 50 mM Tris-HCl (pH 7.4) and 50 mM L-arginine substrate. The reaction was carried out at 37°C for 10 min. The reaction was terminated by adding phenol-hypochlorite chromogenic reagent and color development was performed at 60°C for 15 min. The absorbance was measured at a wavelength of 625 nm, and the amount of ammonia generated was calculated based on the ammonium chloride standard curve. Serum Stability Test: The nanocarrier complex was incubated with human serum (volume ratio 1:1) at 37°C for 48 h, and enzyme activity was measured periodically. Standard Basis: Refer to published methods for the determination of arginine deiminase activity. Key Parameters: Reaction temperature 37±0.5℃, pH 7.4±0.1, substrate concentration 50mM, reaction time 10min. Data Processing: Enzyme activity units (U / mg protein) = (ammonia production / reaction time / protein amount), activity retention rate (%) = (enzyme activity after treatment / initial enzyme activity) × 100%, n=3.

[0145] In vitro cell uptake and targeting evaluation

[0146] Test Subjects: Nanocarrier complex, non-targeted control carrier, and free protein. Test Objective: To quantitatively and qualitatively analyze the uptake efficiency of the nanocarrier in AQP5-high expressing tumor cells and AQP5-low expressing normal cells using flow cytometry and confocal laser scanning microscopy (CLSM), and to evaluate the active targeting characteristics mediated by anti-AQP5 antibodies. Experimental Methods: The nanocarrier complex was labeled with a fluorescent probe (e.g., FITC or Rhodamine B) and co-incubated with AQP5-high expressing cell lines (e.g., human lung adenocarcinoma A549 cells) and AQP5-low expressing cell lines (e.g., human normal lung epithelial BEAS-2B cells) at 37°C for 2-4 h. After washing with PBS, fluorescence intensity was detected using a BD FACSCalibur flow cytometer (excitation light 488 nm, emission light 525 nm). The mean fluorescence intensity (MFI) of 10,000 cells was calculated, and the uptake ratio was determined. Parallel samples were fixed with 4% paraformaldehyde and stained with DAPI. The intracellular localization and co-localization of fluorescence signals were observed under a confocal laser microscope. Standard Basis: Refer to standard operating procedures for cell biology. Key Parameters: Incubation temperature 37℃, CO2 concentration 5%, incubation time 2-4h, sample concentration equivalent enzyme activity 10-50μg / mL. Data Processing: Targeting index = (AQP5⁺ cell MFI) / (AQP5⁻ cell MFI), n=3.

[0147] In vivo pharmacokinetics and evaluation of arginine depletion

[0148] Test subjects: Nanocarrier complex, free arginine deiminase. Test objectives: To evaluate the plasma pharmacokinetic characteristics (half-life, clearance, volume of distribution) of the nanocarrier in a rodent model, as well as the duration of maintaining low plasma arginine concentrations, and to verify the long-circulating properties and arginine depletion capacity of the carrier. Experimental Methods: Healthy BALB / c mice or SD rats were injected via tail vein with either a nanocarrier complex or free enzyme (at an equivalent enzyme activity dose). Blood samples were collected from the orbital cavity at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h post-administration (anticoagulated with heparin sodium). Plasma was separated, and arginine deiminase concentration was determined using ELISA (detecting binding with anti-arginine deiminase polyclonal antibody). Concentration-time curves were plotted, and a two-compartment model was fitted using WinNonlin software to calculate pharmacokinetic parameters. Simultaneously, plasma L-arginine concentration was determined using HPLC-MS / MS (pre-column derivatization with phthalaldehyde, C18 column separation, and fluorescence detection). The change in arginine concentration over time and the percentage of depletion were calculated. Key Parameters: Animal weight 18-22 g (mice) or 180-220 g (rats); administered dose equivalent to enzyme activity 5-10 U / kg; blood volume ≤100 μL per administration. Data processing: Calculate t1 / 2 (half-life), AUC (area under the curve), CL (clearance), Vd (volume of distribution), and duration of arginine depletion, n=6.

[0149] Figure 1 This figure shows the effect of the average molecular weight of polyethylene glycol on plasma half-life and AQP5 positive cell uptake ratio. The fixed parameters were: PLGA to PEG mass ratio of 50:50, dihydrazide group content of 0.11 mmol·g⁻¹ polymer, maleimide group substitution degree of 3 mol%, glutaraldehyde concentration of 5 mmol·L⁻¹, organic phase polymer concentration of 30 mg·mL⁻¹, microfluidic total flow rate of 15 mL·min⁻¹, organic phase to aqueous phase volume ratio of 1:3, crosslinking reaction time of 2 h, reaction temperature of 22℃, and protein conjugate B1 loading of 20 wt%. The variable parameter was the average molecular weight of polyethylene glycol from 1500 to 6500. When the molecular weight of polyethylene glycol is between 3500 and 4500, the plasma half-life and the uptake ratio of AQP5 positive cells reach a balance range simultaneously, with a half-life of 18.2 to 19.2 h and an uptake ratio of 2.4 to 2.6 times. When the molecular weight is below 2000, the hydrophilic shielding effect is insufficient, resulting in a half-life of less than 14.2 h. Although the uptake ratio remains at 2.3 times, the long-circulation ability is weakened. When the molecular weight is above 5500, although the half-life is extended to more than 24.8 h, the steric hindrance masks the antibody ligand, causing the uptake ratio to drop to less than 2.0 times. This proves that a moderate molecular weight can take into account both the long-circulation stealth effect and the active targeting binding ability.

[0150] Figure 2 This diagram illustrates the effect of glutaraldehyde concentration on shell thickness on enzyme activity retention and pH 5.5 dissociation rate. Fixed parameters included a PLGA to PEG mass ratio of 50:50, average polyethylene glycol molecular weight of 4000, dihydrazide group content of 0.11 mmol·g⁻¹ polymer, maleimide group substitution degree of 3 mol%, organic phase polymer concentration of 30 mg·mL⁻¹, microfluidic total flow rate of 15 mL·min⁻¹, organic phase to aqueous phase volume ratio of 1:3, crosslinking reaction time of 2 h, reaction temperature of 22℃, and protein conjugate B1 loading of 20 wt%. Variations were achieved by increasing glutaraldehyde concentration from 0.3 mmol·L⁻¹ to 12 mmol·L⁻¹, corresponding to shell thicknesses from 3 nm to 34 nm. When the shell thickness is 15 to 25 nm, the enzyme activity retention rate and pH 5.5 dissociation rate reach the optimal balance, with an enzyme activity retention rate of 75 to 80% and a dissociation rate of 28 to 38%, achieving synergistic optimization of protection and release. When the thickness is less than 5 nm, the cross-linked network is too thin, resulting in a decrease in enzyme activity retention rate to 58%. Although the dissociation rate increases to 58%, the protein protection effect is insufficient. When the thickness is greater than 30 nm, although the enzyme activity retention rate increases to 81 to 82%, the excessively thick cross-linked network hinders acid-triggered dissociation, causing the dissociation rate to drop below 25%. This indicates that an appropriate shell thickness is the structural basis for achieving a dynamic balance between stable encapsulation and acid-responsive release.

[0151] Figure 3 This is a graph showing the effect of the loading of protein conjugate B1 on the arginine depletion selectivity and polydispersity index of the present invention. The fixed parameters are: nanoparticle size 125 nm, average molecular weight of polyethylene glycol 4000, shell thickness 17 nm, dihydrazide group content 0.11 mmol·g⁻¹ polymer, hydrazone bond crosslinking density 0.055 mmol·g⁻¹ polymer, and formulation pH 7.1. The variable parameters are the loading of protein conjugate B1 from 7 wt% to 35 wt%. When the protein loading is 18 to 26 wt%, the optimal combination of arginine depletion selectivity and particle size uniformity is achieved, with a depletion selectivity of 5.8 to 6.3 and a polydispersity index of 0.18 to 0.21, demonstrating the dual advantages of therapeutic efficiency and formulation stability. When the loading is less than 10 wt%, the insufficient enzyme activity per unit carrier leads to a depletion selectivity of only 4.5. Although the polydispersity index is maintained at 0.18, the therapeutic efficiency is low. When the loading is greater than 30 wt%, although the depletion selectivity increases slightly to 6.4, the polydispersity index increases to 0.24, and the negative value of the zeta potential increases to -7.8 to -8.8 mV, indicating that high protein loading leads to an increase in surface charge and a decrease in colloidal stability. This verifies that an appropriate loading can balance the arginine depletion capacity and the dispersion stability of nanoparticles.

[0152] Figure 4This is an XPS depth profile of the N1s C=N peak retention rate of the hydrazone shell under different pH conditions in Example 1 of the present invention. The characterization method is X-ray photoelectron spectroscopy (XPS) N1s elemental depth profile. The sample is the shell structure of the hydrazone crosslinked protein coating system after being treated in pH 7.0 and pH 5.5 buffer solutions for 24 h. The fixed parameters are: initial hydrazone crosslinking degree of about 80% or more, shell thickness of about 20 nm, constant protein type and crosslinking agent ratio, and the same incident angle and sputtering rate used for testing. The variable parameters are: sputtering depth from 0 nm to 17 nm and external pH condition from 7.0 to 5.5. The results showed that the retention rate of the C=N peak in the range of 0 to 17 nm was consistently between 88% and 100% at pH 7.0, while the retention rate at the corresponding depth was only 52% to 70% at pH 5.5. This indicates that the hydrazone bond network is structurally stable throughout the entire shell under neutral conditions, while at least 30% of the hydrazone bonds break at the same depth under acidic conditions. This proves that the shell remains intact at physiological pH but can be selectively unlocked in a slightly acidic environment.

[0153] Figure 5 The XPS N1s high-resolution spectra of the hydrazone shell in Example 1 of this invention under different pH and time conditions are shown. The characterization method is high-resolution X-ray photoelectron spectroscopy (XPS) N1s fitting analysis. The sample is the surface chemical state of the hydrazone cross-linked protein shell after initial incubation at 0 h and after incubation at pH 7.0 and pH 5.5 for 24 h, respectively. The fixed parameters are constant XPS test flux and energy resolution, elemental correction using C 1s 284.8 eV, and initial hydrazone C=N and free –NH2 content prepared with the same formula. The changing parameters are external pH conditions changed from 7.0 to 5.5 and incubation time extended from 0 h to 24 h. The results showed that the C=N peak near 398.5 eV retained about 80% at pH 7.0 for 24 h, while only about 50% was retained at pH 5.5. Meanwhile, the free –NH2 peak from 399.5 to 400.0 eV was significantly enhanced in the acidic sample after 24 h, accompanied by a slight increase in the high binding energy component near 401 eV. This indicates that the acidic environment induces hydrazone bond cleavage to release protein amino groups, while neutral conditions only cause slight relaxation. This confirms that the hydrazone bond has controllable cleavage behavior in the target pH window and that the changes in chemical sites are consistent with the shell unlocking mechanism.

[0154] Figure 6This is a time decay graph of the 1635 cm⁻¹ hydrazone C=N characteristic peak under neutral and slightly acidic conditions, obtained by FTIR analysis of Fourier transform infrared spectroscopy (FTIR) peak intensity over time in Example 1 of this invention. The characterization method was Fourier transform infrared spectroscopy (FTIR) peak-fixed integral analysis. The samples were dried samples of hydrazone cross-linked protein shells obtained by the same preparation process, which were treated in pH 7.0 and pH 5.5 buffer solutions for 0, 2, 4, 8, 12, and 24 h, respectively. The fixed parameters were constant film thickness and formulation composition, the same number of scans and resolution for infrared testing, and normalization of peak intensity to the initial 1635 cm⁻¹ hydrazone C=N peak at 0 h. The changing parameters were the change of external pH conditions from 7.0 to 5.5 and the extension of treatment time from 0 h to 24 h. The results showed that at pH 7.0, the C=N peak still maintained a relative intensity of about 88% after 24 h, while at pH 5.5, it rapidly decreased to about 50% within 24 h, and showed a significant accelerated decline in the 2 to 8 h range. This indicates that the hydrazone bond network was basically stable under neutral conditions within the observation period, while under slightly acidic conditions, the hydrazone bonds underwent stepwise dissociation and completed more than half of the bond breakage within 24 h. This proves that the hydrazone bond breakage kinetics of the system matches the expected pH-triggered release time window.

[0155] Figure 7 This is a superimposed image of FTIR spectra of hydrazone-crosslinked protein shells at different time points in neutral and slightly acidic environments. The characterization method was Fourier transform infrared spectroscopy (FTIR) full-spectrum scanning and comparison of absorption peaks of key functional groups. The samples were dried samples of hydrazone-crosslinked protein shells prepared with the same formulation after being treated in pH 7.0 and pH 5.5 buffer solutions for 0 h and 24 h. The fixed parameters were that the sample thickness was consistent with the formulation, the spectrum was normalized to the overall absorption of the fingerprint region, and the test optical path was the same as the instrument parameters. The changing parameters were that the external pH conditions changed from 7.0 to 5.5 and the treatment time changed from 0 h to 24 h. The results showed that in the pH 7.0 sample, the 1635 cm⁻¹ hydrazone C=N peak decreased only slightly after 24 h, while the 1730 cm⁻¹ C=O peak and the 3300 cm⁻¹ –NH2 peak increased only slightly. In the pH 5.5 sample, the 1635 cm⁻¹ hydrazone C=N peak decreased significantly after 24 h, while the 1730 cm⁻¹ carbonyl peak and the 3300 cm⁻¹ hydrazide –NH2 stretching vibration peak were significantly enhanced, indicating that more aldehyde groups and free amino sites were generated after the hydrazone bond broke. This result is highly consistent with the decrease in the area of ​​the C=N peak and the enhancement of the –NH2 component in XPS, systematically proving that the hydrazone shell is stable in a neutral environment but is effectively unlocked and exposes the protein active sites in a slightly acidic environment.

[0156] Figure 8This is a flow cytometry MFI comparison of the uptake levels of AQP5-positive tumor cells and AQP5-negative normal cells by the targeted and non-targeted carriers of Example 1 in Example 1. Fixed parameters included: FITC channel selected for flow cytometry detection; 10,000 events per cell group; fixed gating strategy of forward and side scattering to exclude debris and aggregates; uniform laser power and voltage; fixed incubation time of 37°C for 2 hours; and uniform carrier concentration of fluorescently labeled protein. Variable parameters included: cell type (AQP5-positive tumor cells and AQP5-negative normal cells) and treatment method (targeted and non-targeted carriers of Example 1). The results showed that the MFI of the targeted vector in Example 1 in AQP5-positive tumor cells was approximately 2500±300, which was significantly higher than that of the non-targeted vector in Example 1 (1000±120), an increase of about 2.5 times. In AQP5-negative normal cells, the uptake of the targeted vector in Example 1 and the non-targeted vector in Example 1 was close, with MFIs of approximately 880±110 and 800±100, respectively, showing only slight differences. This indicates that the targeted vector in Example 1 exhibits a significant active targeted uptake advantage in AQP5-high expression cells, while maintaining a limited uptake level in AQP5-low expression normal cells. This demonstrates that surface modification mediated by anti-AQP5 antibody and PEG stealth design can achieve selective enrichment of tumor cells without excessively increasing non-specific uptake of normal cells under the same dosing conditions.

[0157] Figure 9This is a confocal laser scanning microscope (CLSM) cutoff image of the fluorescence intensity distribution of the anti-AQP5 antibody channel and the AQP5 receptor channel along the cell membrane cutoff direction in Example 1. The fixed parameters are: the microscope objective is a 63× oil immersion lens with a numerical aperture greater than 1.3; the scanning laser wavelength corresponds to the detection filter; the red channel is used for the anti-AQP5 antibody signal and the green channel is used for the AQP5 receptor signal; the pixel size is uniformly 0.2 µm; the cutoff length is fixed from 0 to 10 µm; and the cell incubation conditions and carrier dosage are consistent with those of the flow cytometry experiment. The variable parameters are the continuous variation of the cutoff distance from 0 to 10 µm along different spatial positions of the cell membrane and the fluorescence intensity of the corresponding red anti-AQP5 antibody signal and green AQP5 receptor signal. The results showed that the red and green channels formed multiple overlapping peaks near 2.0 µm, 5.0 µm, and 8.0 µm. The peak heights of the two channels were close, the overall curves were smooth and continuous, and the intensity change trends were highly consistent throughout the entire distance. The fitting yielded a Manders coefficient with a mean greater than 0.8 and a Pearson correlation coefficient greater than 0.75. This indicates that the anti-AQP5 antibody in Example 1 was mainly distributed in the high-density region of the AQP5 receptor and had significant spatial overlap with the receptor on the cell membrane plane. This proves that the antibody-modified layer was not completely masked by PEG but was positioned on the surface of the nanoshell with an appropriate degree of exposure, enabling it to specifically bind to AQP5 on the membrane. From the perspective of spatial co-localization, this supports the enhanced uptake effect on AQP5-positive cells observed in the flow cytometry results.

[0158] As can be seen from the performance of the examples and comparative examples in Table 1, Examples 1-4 all meet the requirements of the claims in terms of key parameters such as particle size, dispersibility, surface potential, and protein loading. Furthermore, the four examples achieved optimized performance balance through different parameter combinations, with overall performance significantly superior to the comparative examples. Comparative Example 1, due to its excessively small particle size (35nm), resulted in a significantly increased polydispersity index (0.28), decreased protein loading (12.5wt%), reduced enzyme activity retention (58%), and a shortened plasma half-life to 8.2h, indicating that particle size below the lower limit of the claims impairs the stability and cycling ability of the carrier. Comparative Example 2, due to its excessively large particle size (215nm), although exhibiting a longer half-life (24.5h), had a cell uptake ratio of only 1.5 times and a decrease in arginine depletion selectivity to 3.5, demonstrating that exceeding the upper limit of the particle size weakens tumor penetration and cell internalization efficiency. Comparative Examples 3 and 4, due to excessively low or high PEG molecular weight, resulted in insufficient cycling time (11.5h) or inhibited target binding (uptake ratio 1.6 times), respectively, verifying the rationality of the PEG molecular weight range. Comparative Examples 5 and 6 showed reduced enzyme activity retention rates of 48% and 55%, respectively, due to shell thickness deviations, and impaired pH responsiveness, indicating that shell thickness is crucial for enzyme activity protection and acid-triggered release. Comparative Examples 7 and 8 exhibited decreased structural stability (PDI ≥ 0.26) and reduced enzyme activity retention rates (50-52%) due to insufficient cross-linking, demonstrating that the content of dihydrazide groups and hydrazone cross-linking density must meet minimum requirements to maintain shell integrity. Comparative Examples 9 and 10 showed insufficient therapeutic efficiency (exhaustion selectivity 3.2) or poor dispersibility (PDI 0.24, ζ potential -8.8 mV) due to protein loading deviations, validating the rationality of the loading window. Comparative Example 11 showed a sharp drop in targeted uptake ratio to 1.0-fold and selectivity to 1.5 due to the absence of anti-AQP5 antibody, fully demonstrating the crucial role of antibody-mediated active targeting. Comparative Example 12, despite maintaining high enzyme activity (76%) due to the use of a non-pH-responsive crosslinking agent, had a plasma half-life of only 12.5 h, indicating that the acid-sensitive nature of hydrazone bonds is indispensable for achieving intracellular environment-triggered release and maintaining cyclic stability. Comparative Example 13, lacking PEGylation modification, experienced a sharp drop in plasma half-life to 6.8 h, verifying the necessity of the PEG stealth effect for extending circulation time. The comprehensive data show that the technical solution of this invention, through precise control of core parameters such as particle size, PEG molecular weight, shell thickness, crosslinking density, and protein loading, combined with the innovative design of anti-AQP5 antibody targeting and pH-responsive hydrazone bond crosslinking, successfully achieved synergistic optimization of "long circulation + active targeting + acid-triggered release + high enzyme activity maintenance," significantly outperforming comparative examples that deviated from the scope of the claims or lacked key technical features, demonstrating outstanding technical advantages and application value.

[0159] Table 1. Performance comparison data of the embodiments and comparative examples.

[0160]

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A nanocarrier complex for resisting AQP5 arginine deiminase, characterized in that, The composite is a core-shell structured nanoparticle dispersion system with a particle size of 50–200 nm, wherein the nanoparticles include: The core phase is a hydrophobic polymer backbone formed by the self-assembly of a PLGA-PEG-maleimide copolymer containing hydrophobic PLGA segments. The shell phase is composed of a hydrazone crosslinking network formed by the dihydrazide groups on the PLGA-PEG-maleimide copolymer and the dialdehyde crosslinking agent. The hydrazone crosslinking shell maintains a degree of crosslinking of more than 80% relative to the initial time within 24 hours under buffer conditions of 37°C and pH 6.5–7.

4. Under buffer conditions of 37°C and pH 5.0–6.0, the degree of crosslinking of crosslinking of crosslinking decreases by at least 30% within 24 hours. This is to maintain the stability of the nanoparticle structure under neutral conditions and to allow at least partial dissociation in the acidic intracellular microenvironment such as endosomes / lysosomes after tumor cell endocytosis. This allows the protein conjugate B1, which was originally partially buried or restricted by the crosslinking shell, to obtain higher conformational freedom and outer surface exposure. And an anti-AQP5 arginine deiminase protein conjugate B1 covalently fixed to the surface of the shell, wherein the protein conjugate B1 is formed by covalently coupling an anti-AQP5 monoclonal antibody or its antigen-binding fragment capable of specifically binding to human AQP5 with arginine deiminase through a chemical cross-linking agent containing maleimide groups, and wherein the arginine deiminase retains at least one free thiol group on average that can undergo a thiomaleimide addition reaction with the maleimide groups of the shell; The thiol group introduced on the arginine deiminase undergoes a thiomaleimide addition reaction to form a thioether bond with the maleimide group in the shell and is covalently fixed to the shell surface. This makes the protein conjugate B1 fixed to the shell surface more easily exposed in the tumor microenvironment and exert its arginine depletion effect.

2. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The PLGA-PEG-maleimide copolymer is composed of a PLGA-PEG copolymer backbone formed by the ester condensation of a modified polylactic acid-hydroxyacetic acid copolymer PLGA with multiple carboxyl side groups on the backbone and polyethylene glycol PEG. A dihydrazide group formed by adipate dihydrazide and a maleimide group introduced by N-(2-aminoethyl)maleimide hydrochloride are introduced on the backbone. The molar ratio of lactic acid to glycolic acid in PLGA is 75:25, the average molecular weight of PEG is 2000–6000, and both the maleimide group and the dihydrazide group are bonded to the copolymer backbone in the form of side chains.

3. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The dialdehyde crosslinking agent is glutaraldehyde, the shell thickness is 5–30 nm, the content of dihydrazide groups in the polymer is 0.02–0.20 mmol / g polymer, the hydrazone bond crosslinking density formed by dihydrazide groups and glutaraldehyde is 0.01–0.10 mmol / g polymer, and the hydrazone bond crosslinking density does not exceed the content of dihydrazide groups, and the polydispersity index of the nanoparticles is not higher than 0.20, and the particle size change does not exceed 20% after being stored at 4°C under sealed conditions for 3 months.

4. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The protein conjugate B1 is prepared by the following steps: A1. Prepare solutions of anti-AQP5 monoclonal antibody or its antigen-binding fragment and arginine deiminase in near-neutral buffer; A2. Add a thiol-introducing reagent to the arginine deiminase solution to introduce an average of 2–4 reactive thiol groups onto each enzyme molecule to obtain thiolized arginine deiminase; A3. The anti-AQP5 monoclonal antibody or its antigen-binding fragment is reacted with a cross-linking agent containing both N-hydroxysuccinimide ester and maleimide groups to introduce 1–2 maleimide groups onto the antibody molecule, thereby obtaining an activated antibody or its antigen-binding fragment. A4. The thiolized arginine deiminase is reacted with the activated antibody or its antigen-binding fragment under mild conditions to form an anti-AQP5 monoclonal antibody-arginine deiminase protein conjugate, namely protein conjugate B1. The main peak component is collected by separation and purification methods to ensure that the average binding ratio of arginine deiminase to anti-AQP5 monoclonal antibody or its antigen-binding fragment is 1–2:1, and at least one free thiol group that can react with maleimide group is retained in protein conjugate B1.

5. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The loading amount of the protein conjugate B1 in the nanoparticles is 10–30 wt% of the dry weight of the nanoparticles, the enzyme density on the nanoparticle surface is 0.5–2.0 mg / m², the pH of the formulation is 6.8–7.4, the zeta potential is −10 mV to 0 mV, and after being treated in human serum at 37°C for at least 48 h, the arginine deiminase activity is preferably not less than 70% of the initial activity before incubation, as determined by colorimetric or fluorescent enzyme activity assay using L-arginine as a substrate.

6. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The PLGA-PEG-maleimide copolymer is prepared by the following steps: B1. In an anhydrous organic solvent, PLGA, PEG, adipic acid dihydrazide, and N-(2-aminoethyl)maleimide hydrochloride are simultaneously mixed with a condensing agent and a catalyst, so that the carboxyl group of PLGA forms an ester bond with PEG, forms an amide bond with adipic acid dihydrazide to introduce a dihydrazide side chain, and forms a maleimide side chain with N-(2-aminoethyl)maleimide hydrochloride, while retaining some unreacted carboxyl groups, to obtain a crude product containing PLGA-PEG ester bonds, dihydrazide side chains, and maleimide side chains; B2. The crude product is subjected to solvent removal, precipitation, washing and drying to obtain a solid intermediate; B3. The solid intermediate is dissolved in a polar organic solvent, and the remaining carboxyl groups are activated by water-soluble carbodiimide and reacted with adipic acid dihydrazide to further adjust the degree of substitution of the dihydrazide group in the polymer so that the content of the dihydrazide group is 0.02–0.20 mmol / g polymer. The reaction is terminated when the number average molecular weight of the polymer is 15–30 kDa, the polydispersity index is not greater than 1.3 and the degree of substitution of the maleimide group is 1–5 mol%, to obtain the PLGA-PEG-maleimide copolymer.

7. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The PLGA-PEG-maleimide copolymer is used to form a dynamic core-shell nanocarrier intermediate through the following steps: C1. Dissolve the PLGA-PEG-maleimide copolymer in acetonitrile or ethanol to obtain an organic phase; C2. Prepare a phosphate buffer aqueous phase with a pH of 6.5–7.0, and add glutaraldehyde to make the glutaraldehyde concentration 0.5–10 mmol / L; C3. The organic phase and the aqueous phase are mixed in a microfluidic mixer at a volume ratio of 1:2.5–1:3.5 to allow the polymer to self-assemble into core-shell nanoparticles with a particle size of 50–200 nm. The resulting dispersion is then gently stirred at 20–25 °C for 0.5–4 h. During this process, the dihydrazide groups and glutaraldehyde continue to react to form a hydrazone cross-linked shell. C4. After removing the organic solvent and adjusting the solid content and pH of the dispersion, a dynamic core-shell nanocarrier intermediate with a median particle size of 50–200 nm and a polydispersity index not higher than 0.20 was obtained.

8. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, The anti-AQP5 arginine deiminase nanocarrier complex or its lyophilized powder was prepared by the following steps: D1. Dilute the dynamic core-shell nanocarrier intermediate and adjust the pH to 6.5–7.

2. Slowly add the protein conjugate B1 solution according to the molar ratio of maleimide groups on the surface of the nanocarrier shell to reactive thiol groups in protein conjugate B1 of 1:0.5–1:1.

5. Gently stir the reaction at 4–8°C for 1–4 hours to allow protein conjugate B1 to be covalently attached to the shell surface through a thiomaleimide addition reaction. D2. After the reaction is complete, ultrafiltration or gel filtration is used to separate and remove free protein conjugate B1, small molecule salts, and unreacted small molecules to obtain a dispersion of the nanocarrier complex, such that the mass fraction of protein conjugate B1 in the solid is 10–30 wt%. D3. Add a freeze-drying protectant to the dispersion in a total amount of 5–10 wt% of the total dry matter of the nanoparticles and protein conjugate B1. The freeze-drying protectant includes trehalose dihydrate and / or sucrose. After dispensing, pre-freeze at low temperature and freeze-dry under vacuum to obtain a freeze-dried powder of the nanocarrier complex with a water content of no more than 3 wt% that can be rapidly reconstituted.

9. The anti-AQP5 arginine deiminase nanocarrier complex according to claim 1, characterized in that, In vitro, using AQP5-overexpressing tumor cell lines and AQP5-low-expressing normal cell lines as models, under the same nanocarrier particle size and enzyme activity load conditions, quantitative flow cytometry or confocal microscopy analysis showed that the intracellular uptake of this nanocarrier complex in AQP5-overexpressing tumor cells was at least twice that of a non-targeting arginine deiminase carrier with the same structure but without anti-AQP5 antibody or its antigen-binding fragment. Under the same added enzyme activity dose, the ratio of arginine depletion in AQP5-high expressing tumor cells to that in AQP5-low expressing normal cells was not less than 5:1, calculated by measuring the arginine concentration in the culture supernatant. In vivo, using rodent intravenous administration as a model, plasma pharmacokinetic analysis showed that the apparent half-life in plasma after intravenous administration was 2–10 times that of free arginine deiminase, and the plasma arginine concentration was maintained at 30–70% of the pre-administration level for 24–72 hours after administration.

10. A method for preparing an anti-AQP5 arginine deiminase nanocarrier complex as described in any one of claims 1-9, characterized in that, Includes the following steps: S1. PLGA and PEG are condensed to form a PLGA-PEG copolymer backbone, and dihydrazide groups and maleimide groups are introduced into the backbone to prepare a PLGA-PEG-maleimide copolymer; S2. Dissolve the PLGA-PEG-maleimide copolymer in an organic phase and mix it with an aqueous phase containing a dialdehyde crosslinking agent in a microfluidic device or a high-shear device to allow the polymer to self-assemble into core-shell structured nanoparticles. The shell layer is then crosslinked with dialdehyde through dihydrazide to form a hydrazone bond, thus obtaining a dynamic core-shell nanocarrier intermediate. S3. By introducing a thiol group into arginine deiminase and introducing a maleimide group into an anti-AQP5 monoclonal antibody or its antigen-binding fragment, an arginine deiminase containing 2–4 reactive thiol groups and an activated antibody or its antigen-binding fragment containing 1–2 maleimide groups are prepared, and the two are coupled under mild conditions to obtain a protein conjugate B1 having at least one reactive thiol group that is not consumed by the antibody. S4. Under low temperature conditions at pH 6.5–7.2, the nanocarrier intermediate described in step S2 is reacted with the protein conjugate B1 described in step S3 at a molar ratio of maleimide group to thiol group of 1:0.5–1:1.5, so that the protein conjugate B1 is covalently attached to the shell surface through a thiomaleimide addition reaction. Then, the free protein conjugate B1 is removed and a lyophilization protectant is added. After pre-freezing and vacuum freeze-drying, a lyophilized formulation of the anti-AQP5 arginine deiminase nanocarrier complex is obtained. In the preparation method, the process is optimized by controlling the molar ratio and reaction time of the thiol-introducing reagent and arginine deiminase, as well as the cross-linking agent and the anti-AQP5 monoclonal antibody or its antigen-binding fragment, and by combining the results of thiol titration or mass spectrometry analysis, so that the prepared protein conjugate B1 meets the above-mentioned binding ratio and residual thiol number.

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