Metal nanoparticle hybrid polymer vesicle, and preparation method and application thereof

Abstract

This invention discloses a metal nanoparticle hybrid polymeric vesicle, its preparation method, and its applications. The method includes the following steps: adding a block copolymer solution to a metal salt aqueous solution via a syringe pump, i.e., preparing a preliminary hybrid vesicle by solvent exchange; dialyzing the obtained preliminary hybrid vesicle to remove unloaded metal ions; and adding an oxidizing agent or a reducing agent to the dialyzed preliminary hybrid vesicle to obtain the metal nanoparticle hybrid polymeric vesicle. This invention is simple to operate and highly reproducible. The prepared hybrid polymeric vesicle can act as a nanomotor during drug delivery, increasing tumor penetration under the driving force. After intracellular degradation, the metal nanoparticles can also act as nanozymes to produce therapeutic effects. This hybrid polymeric vesicle has excellent drug delivery capabilities and nanozyme characteristics, showing promising applications in drug delivery systems and the preparation of tumor therapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a metal nanoparticle hybrid polymer vesicle, its preparation method, and its application. Background Technology

[0002] Cancer remains a major health problem threatening human life, and researchers are constantly developing new treatments, such as photothermal therapy, chemokinetic therapy, and sonodynamic therapy. These highly specific and manipulable novel treatments have brought new hope to cancer treatment. With the continuous development of nanomaterials science, innovations in biomedical materials have provided new ideas for novel therapies. Many new biomedical materials have already entered clinical use for tumor diagnosis and treatment. To achieve better therapeutic effects and reduce toxic side effects, nanomedicine delivery systems are often used for in vivo delivery of therapeutic agents, such as microspheres, liposomes, and hydrogels. Among them, polymeric vesicles are frequently used in nanomedicine delivery systems because of their unique hydrophilic lumen and hydrophobic membrane structure, which allows them to simultaneously load multiple hydrophilic / hydrophobic drugs and have high drug loading efficiency. Polymeric vesicles have significant advantages as drug carriers, not only in terms of the diversity of drugs they can load, including small molecule drugs, peptides / proteins, DNA / RNA, and nanoparticles, but also in their ability to be rapidly and efficiently endocytosed by cells, demonstrating strong drug delivery capabilities.

[0003] Metal nanoparticles are frequently used in the biomedical field due to their excellent optical and catalytic properties. However, their low selectivity and high cytotoxicity limit their application in biomedicine. To further leverage the biomedical value of metal nanoparticles, researchers often use drug delivery systems for in vivo delivery, reducing cytotoxicity through tumor-specific accumulation and tumor microenvironment responsiveness, thereby improving therapeutic efficacy. Furthermore, different functionalities can be achieved by regulating the distribution of metal nanoparticles within polymeric vesicles. Therefore, combining polymeric vesicles with metal nanoparticles to prepare novel hybrid drug delivery carriers can achieve both the excellent therapeutic effects of metals and address their cytotoxicity issues.

[0004] By combining metals and polymeric vesicles, certain functionalities can be endowed to the vesicles. For example, in the patent of Zhao Xubo et al. (A vesicular micelle metal oxide and its preparation method and application, patent number: 201910644293.X), polymer vesicles loaded with uniformly distributed metal oxides were prepared by the interaction of block copolymers and metal oxides, and used as a drug delivery system.

[0005] Compared to polymeric vesicles loaded with uniformly distributed metal nanoparticles, aggregating metal nanoparticles on one side of a polymeric vesicle can achieve new functions, while still retaining the inherent properties of the metal nanoparticles upon vesicle disintegration. How to prepare metal nanoparticle hybrid polymeric vesicles with metal nanoparticles aggregated on one side remains an unsolved technical problem. Summary of the Invention

[0006] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing metal nanoparticle hybrid polymer vesicles;

[0007] Another object of the present invention is to provide metal nanoparticle hybrid polymer vesicles prepared by the above preparation method;

[0008] Another object of the present invention is to provide the above-described preparation method and its application in metal nanoparticle hybrid polymer vesicles.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0010] A method for preparing metal nanoparticle hybrid polymer vesicles includes the following steps:

[0011] 1) The block copolymer solution was added to the metal salt aqueous solution by a syringe pump, thus preparing the pre-hybrid vesicles by solvent exchange method;

[0012] 2) Dialyze the prepared hybrid vesicles obtained in step 1) to remove unloaded metal ions;

[0013] 3) Add an oxidant or a reducing agent to the dialysis-prepared hybrid vesicles obtained in step 2) to obtain metal nanoparticle hybrid polymer vesicles;

[0014] The solute in the block copolymer solution described in step 1) is a block copolymer, and the solvent is an organic solvent.

[0015] Further, the block copolymer mentioned in step 1) is polyethylene glycol-b-polylactic acid (mPEG-b-PLA), polyethylene glycol-b-polycaprolactone (mPEG-b-PCL), polyethylene glycol-b-polystyrene (mPEG-b-PS), or polylactic acid-polyethylene glycol-polylactic acid (PLA-mPEG-PLA).

[0016] Further, the concentration of the block copolymer solution described in step 1) is 1 to 10 mg / mL; even further, it is 10 mg / mL.

[0017] Further, the organic solvent mentioned in step 1) includes at least one of tetrahydrofuran, 1,4-dioxane, methanol, ethanol, dichloromethane, chloroform, and methyl acetate; even further, the organic solvent mentioned in step 1) is tetrahydrofuran, 1,4-dioxane, or dichloromethane.

[0018] Further, the metal salt mentioned in step 1) includes at least one of manganese chloride (MnCl2), chloroauric acid (HAuCl4), and chloroplatinic acid (H2Cl6Pt).

[0019] Further, the concentration of the metal salt aqueous solution described in step 1) is 1–50 mg / mL; even further, it is 1–30 mg / mL.

[0020] Further, the volume ratio of the block copolymer solution to the metal salt aqueous solution in step 1) is 1:1 to 10.

[0021] Further, the conditions for adding via syringe pump in step 1) are: injection speed of 0.5 to 5 mL / h, and block copolymer solution temperature controlled at 25 to 60°C; even further, the injection speed for adding via syringe pump in step 1) is 1 to 3 mL / h.

[0022] Further, in step 2), the dialysis involves placing the prepared hybrid vesicles in a dialysis bag (MWCO = 3.5 kDa) and dialyzing for 12–48 hours, changing the water every 4 hours; even further, the prepared hybrid vesicles are placed in a dialysis bag (MWCO = 3.5 kDa) and dialyzed for 24–48 hours, changing the water every 4 hours.

[0023] Further, the oxidizing agent or reducing agent mentioned in step 3) is one of sodium hydroxide (NaOH), hydrogen peroxide (H2O2), sodium borohydride (NaBH4), and citric acid.

[0024] A metal nanoparticle hybrid polymer vesicle was obtained by the above preparation method.

[0025] Furthermore, the average particle size of the metal nanoparticle hybrid polymer vesicles is 50-500 nm, and the mass fraction of metal nanoparticles in the hybrid vesicles is 1-10 wt%.

[0026] The above-mentioned applications of metal nanoparticle hybrid polymer vesicles in constructing drug delivery systems.

[0027] The above-mentioned application of metal nanoparticle hybrid polymer vesicles in the preparation of tumor therapeutic drugs.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] (1) The preparation method of the present invention is simple to operate, highly reproducible, and has high versatility. It is applicable to most block copolymers that can be used to prepare polymeric vesicles. Since the block copolymers do not interact with metal ions or metal nanoparticles, the incompatibility between metal nanoparticles and polymeric blocks can be used to prepare polymeric vesicles with nanoparticles concentrated on one side.

[0030] (2) The metal nanoparticles of the present invention aggregate on one side of the polymer vesicle to form a metal nanoparticle hybrid polymer vesicle, which achieves new functions and also exerts the effects of the metal nanoparticles themselves when the vesicles disintegrate. For example, when manganese dioxide is aggregated on one side of the polymer vesicle, it can continuously catalyze the generation of oxygen from hydrogen peroxide, thereby generating a forward propulsion force, making the polymer vesicle a nanomotor with more efficient penetration ability in solid tumors. For another example, when ultra-small gold nanoparticles are loaded on one side of the polymer vesicle, gold can be effectively aggregated, possessing specific photothermal properties. It can generate a thermal gradient under near-infrared laser irradiation, propelling the vesicle forward and enhancing its tumor penetration ability. When the metal nanoparticles are released, they can also be used as nanoenzymes for tumor treatment. The hybrid vesicles prepared in this invention have strong drug delivery and tissue penetration capabilities because the metal nanoparticles are aggregated on one side of the polymer vesicle. When the free drug is still on the surface of the 3D cell sphere, the hybrid vesicle has already penetrated into the interior of the 3D cell sphere, significantly improving the tissue penetration ability.

[0031] (3) The hybrid vesicles prepared in this invention have nanoenzyme characteristics. The metal nanoparticles can catalyze the generation of reactive oxygen species (ROS) from endogenous H2O2 in tumors, and have a certain tumor killing ability. Real-time imaging and precise treatment can be achieved through the response characteristics of metal nanoparticles in the tumor microenvironment.

[0032] (4) The hybrid vesicles prepared in this invention can be used as drug delivery carriers, can load multiple drugs, achieve the combined effect of multiple treatment methods, and have a strong tumor treatment effect.

[0033] (5) The hybrid vesicles prepared by the present invention improve the biodistribution of metal nanoparticles in vivo, enhance their stability, and reduce biotoxicity, providing a new approach for further clinical translation. Attached Figure Description

[0034] Figure 1 The image shows a transmission electron microscope (TEM) image of the MnO2 hybrid vesicles prepared in Example 1, with the scale bar at 200 nm.

[0035] Figure 2 This is a graph showing the oxygen production of the MnO2 hybrid vesicles prepared in Example 1 within 300 seconds.

[0036] Figure 3The image shows a scanning electron microscope image of the Au NPs hybrid vesicles prepared in Example 2, with the scale bar at 100 nm.

[0037] Figure 4 The temperature change curve of the Au NPs hybrid vesicles prepared in Example 2 under 1064nm laser irradiation is shown.

[0038] Figure 5 The image shows a laser confocal image of the permeation of 3D multicellular spheres of Au NPs hybrid vesicles prepared in Example 3, with the scale bar at 200 μm.

[0039] Figure 6 This is a photograph of a solid tumor after treatment with the drug-loaded MnO2 hybrid vesicles prepared in Example 7. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0041] Example 1

[0042] Weigh out 5 mg of the block copolymer mPEG 2000 -b-PS 6000 Add the copolymer to a tetrahydrofuran solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL, and simultaneously prepare a 3 mL aqueous solution of manganese chloride with a concentration of 15 mg / mL. The block copolymer solution is then added to the manganese chloride aqueous solution using a syringe pump. Polymer vesicles are prepared by solvent exchange, with the following parameters: injection rate of 1 mL / h and block copolymer solution temperature of 45°C. The prepared hybrid vesicles are dialyzed (MWCO = 3.5 kDa) for 24 h (water changed every 4 h) to remove unloaded metal ions. The pH of the dialyzed hybrid vesicles is adjusted to 11 with NaOH, and the reaction is carried out in an open environment for 4 h. MnO2 hybrid vesicles are obtained through in-situ reduction. The transmission electron microscopy image of the MnO2 hybrid vesicles prepared in this example is shown below. Figure 1 As shown, the average particle size is approximately 220 nm. The mass fraction of MnO2 in the hybrid vesicles was approximately 3% after lyophilization and determination by inductively coupled plasma mass spectrometry.

[0043] Weigh out 5 mg of the block copolymer mPEG 2000 -b-PS 6000The copolymer was added to a tetrahydrofuran solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL. This block copolymer solution was then added to an ultrapure aqueous solution using a syringe pump. Polymer vesicles were prepared via solvent exchange, with the following parameters: injection rate of 1 mL / h and block copolymer solution temperature of 45℃. After dialysis (MWCO = 3.5 kDa) for 24 h (with water changed every 4 h), pure PEG-PS polymer vesicles were obtained.

[0044] The hybrid vesicles and pure PEG-PS polymeric vesicles (200 μg / mL) were placed in a 100 mM H₂O₂ solution, and the oxygen production was recorded using a dissolved oxygen analyzer. Figure 2 As shown, pure polymeric vesicles produce almost no oxygen, while the oxygen concentration in the hybrid vesicle group can reach up to 13.7 mg / L.

[0045] Example 2

[0046] Weigh out 5 mg of the block copolymer mPEG 5000 -b-PLA 12000 Add the copolymer to a 1,4-dioxane solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL. Simultaneously, prepare a 3 mL aqueous solution of chloroauric acid with a concentration of 10 mg / mL. Add the block copolymer solution to the chloroauric acid aqueous solution using a syringe pump. Prepare polymeric vesicles via solvent exchange, with the following parameters: injection rate of 2 mL / h and block copolymer solution temperature controlled at 45℃. Dialyze the prepared hybrid vesicles (MWCO = 3.5 kDa) for 24 h (water changed every 4 h) to remove unloaded metal ions. Adjust the pH of the dialyzed prepared hybrid vesicles to 11 with NaOH, allow the reaction to proceed openly for 4 h, and obtain Au NPs hybrid vesicles through in-situ reduction. Transmission electron microscopy (TEM) results are shown below. Figure 3 As shown.

[0047] Weigh out 5 mg of the block copolymer mPEG 5000 -b-PLA 12000 The copolymer was added to a 1,4-dioxane solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL. This block copolymer solution was then added to an ultrapure aqueous solution using a syringe pump. Polymer vesicles were prepared via solvent exchange, with the following parameters: injection rate of 2 mL / h and block copolymer solution temperature of 45℃. After dialysis (MWCO = 3.5 kDa) for 24 h (with water changed every 4 h), pure PEG-PLA vesicles were obtained.

[0048] The hybrid vesicles and pure PEG-PLA vesicles were diluted to 200 μg / mL and irradiated with a 1064 nm laser (1.8 W / cm²). -2 Record the temperature change over 5 minutes. Figure 4 As shown, pure polymer vesicles do not show significant temperature changes under laser irradiation, while AuNPs hybrid vesicles can heat up by about 22°C.

[0049] Example 3

[0050] Weigh 5 mg of block copolymer PEG 2000 -b-PCL 7000 100 μg of coumarin 6 (C6) was added to a tetrahydrofuran solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL. Simultaneously, a 1 mL aqueous solution of chloroauric acid with a concentration of 1 mg / mL was prepared. The block copolymer solution was added to the chloroauric acid aqueous solution using a syringe pump, and polymeric vesicles were prepared by solvent exchange, with the following parameters: injection rate of 1 mL / h and block copolymer solution temperature controlled at 25 °C. The prepared hybrid vesicles were dialyzed (MWCO = 3.5 kDa) for 48 h (water changed every 4 h) to remove unloaded metal ions. Sodium borohydride was added to the dialyzed prepared hybrid vesicles, and Au NPs hybrid vesicles were obtained through in-situ reduction.

[0051] Weigh 5 mg of block copolymer PEG 2000 -b-PCL 7000 100 μg of coumarin 6 (C6) was added to a tetrahydrofuran solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL. The block copolymer solution was then added to an ultrapure aqueous solution using a syringe pump. Polymer vesicles were prepared by solvent exchange, with the following parameters: injection rate of 1 mL / h and block copolymer solution temperature controlled at 25 °C. After dialysis (MWCO = 3.5 kDa) for 48 h (with water changed every 4 h), pure PEG-PCL vesicles were obtained.

[0052] 4T1 cells (purchased from Guangzhou Institute of Microbiology) were seeded in U-shaped 96-well plates at 5 × 10⁶ cells / well. 3 Cells / well were incubated for 48 hours to form 3D multicellular spheres. Coumarin 6 (C6)-labeled hybrid vesicles were co-incubated with the 3D multicellular spheres, and laser confocal micrographs of the infiltration of the 3D multicellular spheres were taken after 12 hours. Laser confocal micrographs of the infiltration of Au NPs hybrid vesicle cells into 3D multicellular spheres prepared in this example are shown below. Figure 5 As shown, after 12 hours, pure PEG-PCL vesicles were mainly distributed on the surface of cell spheres, while AuNPs hybrid vesicles were able to enter the interior of cell spheres and showed a significantly higher fluorescence signal than C6. This indicates that AuNPs hybrid vesicles can significantly improve the ability to penetrate 3D multicellular spheres and have a great advantage in the treatment of solid tumors.

[0053] Example 4

[0054] Weigh 10 mg of the block copolymer mPEG 5000 -b-PLA 12000 The block copolymer solution was prepared by adding it to a dichloromethane solution to obtain 1 mL of a 10 mg / mL solution, and simultaneously preparing 10 mL of a 1 mg / mL aqueous solution of chloroplatinic acid. The block copolymer solution was then added to the chloroplatinic acid solution using a syringe pump, and polymeric vesicles were prepared by solvent exchange, with the following parameters: injection rate of 3 mL / h and block copolymer solution temperature controlled at 45℃. The prepared hybrid vesicles were dialyzed (MWCO = 3.5 kDa) for 48 h (water changed every 4 h) to remove unloaded metal ions. Sodium borohydride was added to the dialyzed prepared hybrid vesicles, and Pt NPs hybrid vesicles were obtained through in-situ reduction.

[0055] Example 5

[0056] Weigh out 5 mg of the block copolymer mPEG 2000 -b-PCL 7000 The copolymer was added to a dichloromethane solution to prepare a 0.5 mL solution of block copolymer with a concentration of 10 mg / mL, and simultaneously a 0.5 mL aqueous solution of chloroauric acid with a concentration of 5 mg / mL was prepared. The block copolymer solution was added to the chloroauric acid aqueous solution using a syringe pump, and polymeric vesicles were prepared by solvent exchange method, with the following parameters: injection rate of 1 mL / h and block copolymer solution temperature controlled at 60℃. The prepared hybrid vesicles were dialyzed (MWCO = 3.5 kDa) for 48 h (with water changed every 4 h) to remove unloaded metal ions. Citric acid was added to the dialyzed prepared hybrid vesicles to obtain Au NPs hybrid vesicles.

[0057] Example 6

[0058] Weigh 1 mg of block copolymer PLA 6000 -mPEG 5000 -PLA 6000 A 1 mL block copolymer solution with a concentration of 1 mg / mL was prepared by adding it to a tetrahydrofuran solution. Simultaneously, a 10 mL aqueous solution of 30 mg / mL manganese chloride was prepared. The block copolymer solution was added to the manganese chloride aqueous solution using a syringe pump, and polymeric vesicles were prepared by solvent exchange. Specific parameters were: injection rate of 1 mL / h, and block copolymer solution temperature controlled at 25℃. The prepared hybrid vesicles were dialyzed (MWCO = 3.5 kDa) for 24 h (water changed every 4 h) to remove unloaded metal ions. The pH of the dialyzed prepared hybrid vesicles was adjusted to 11 with NaOH, and the reaction was carried out in an open environment for 4 h. MnO2 hybrid vesicles were obtained by in-situ reduction.

[0059] Example 7

[0060] Weigh out 5 mg of the block copolymer mPEG 2000 -b-PS 6000 400 μg of IR808 and 1 mg of doxorubicin (DOX) were added to a tetrahydrofuran solution to prepare a 0.5 mL block copolymer solution with a concentration of 10 mg / mL. Simultaneously, 3 mL of a 30 mg / mL manganese chloride aqueous solution was prepared. The block copolymer solution was added to the manganese chloride aqueous solution using a syringe pump, and polymeric vesicles were prepared by solvent exchange, with the following parameters: injection rate of 1 mL / h and block copolymer solution temperature controlled at 45℃. The prepared hybrid vesicles were dialyzed (MWCO = 3.5 kDa) for 24 h (water changed every 4 h) to remove unloaded metal ions. The pH of the dialyzed prepared hybrid vesicles was adjusted to 11 with NaOH, and the reaction was carried out in an open environment for 4 h. Drug-loaded MnO2 hybrid vesicles were obtained by in-situ reduction.

[0061] The obtained drug-loaded MnO2 hybrid vesicles were evaluated for their in vitro antitumor properties. Specifically, 4T1 cells (50 μL, 1 × 10⁻⁶ cells, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were subcutaneously injected into mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.). 6 A solid tumor model was constructed using 100 cells, and then 50 μL (1 mg / mL) of hybrid vesicles was injected into mice on days 0, 4, and 8. Eight hours after injection, mice were irradiated with laser (808 nm, 1.0 W / cm²). -2 After 16 days (5 min), the mice were euthanized and the solid tumors were removed. The tumors were compared with those in the control group (which received only saline) to observe changes in tumor size. Results are as follows: Figure 6 As shown, drug-loaded MnO2 hybrid vesicles can be combined with chemokinetics (MnO2), photodynamic therapy (IR808), and chemotherapy (DOX) to inhibit the growth of solid tumors, with significant therapeutic effects.

[0062] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing metal nanoparticle hybrid polymer vesicles, characterized in that: Includes the following steps: 1) The block copolymer solution was added to the metal salt aqueous solution by a syringe pump, thus preparing the pre-hybrid vesicles by solvent exchange method; 2) Dialyze the prepared hybrid vesicles obtained in step 1) to remove unloaded metal ions; 3) Add an oxidant or a reducing agent to the dialysis-prepared hybrid vesicles obtained in step 2) to obtain metal nanoparticle hybrid polymer vesicles; The solute in the block copolymer solution described in step 1) is a block copolymer, and the solvent is an organic solvent; The block copolymer mentioned in step 1) is polyethylene glycol- b -Polylactic acid, polyethylene glycol- b -Polycaprolactone, polyethylene glycol- b -Polystyrene or polylactic acid-polyethylene glycol-polylactic acid; The metal salt mentioned in step 1) is at least one of manganese chloride, chloroauric acid, and chloroplatinic acid; The concentration of the block copolymer solution mentioned in step 1) is 1–10 mg / mL; The concentration of the metal salt aqueous solution mentioned in step 1) is 1–50 mg / mL; The volume ratio of the block copolymer solution to the metal salt aqueous solution in step 1) is 1:(1~10). The oxidizing or reducing agent mentioned in step 3) is one of sodium hydroxide, hydrogen peroxide, sodium borohydride, and citric acid.

2. The method for preparing metal nanoparticle hybrid polymer vesicles according to claim 1, characterized in that: The organic solvent mentioned in step 1) is at least one of tetrahydrofuran, 1,4-dioxane, methanol, ethanol, dichloromethane, chloroform and methyl acetate.

3. The method for preparing metal nanoparticle hybrid polymer vesicles according to claim 1, characterized in that: The concentration of the block copolymer solution mentioned in step 1) is 10 mg / mL; The concentration of the metal salt aqueous solution mentioned in step 1) is 1–30 mg / mL.

4. The method for preparing metal nanoparticle hybrid polymer vesicles according to claim 1, characterized in that: In step 1), the injection rate via syringe pump is 0.5–5 mL / h, and the temperature of the block copolymer solution during injection is 25–60°C. Step 2) refers to dialysis in which the prepared hybrid vesicles are placed in a dialysis bag with MWCO = 3.5 kDa and dialyzed for 12 to 48 hours, with the water changed every 4 hours.

5. Hybrid vesicles obtained by the preparation method of metal nanoparticle hybrid polymer vesicles as described in any one of claims 1 to 4.

6. The application of the metal nanoparticle hybrid polymer vesicles according to claim 5 in the construction of drug delivery systems.

7. The application of the metal nanoparticle hybrid polymer vesicles according to claim 5 in the preparation of breast cancer therapeutic drugs.

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