Protamine-DOPAC conjugate as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: by taking 3, 4-dihydroxyphenylacetic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as raw materials, carrying out pre-activation reaction in an organic solvent, and adding 1-hydroxybenzotriazole to carry out activation reaction, so as to obtain a reaction product; slowly adding the reaction product into a protamine buffer solution, carrying out amidation coupling reaction, and purifying and drying to obtain the protamine-DOPAC conjugate. According to the protamine-DOPAC conjugate, the residence time of the exosome in a uterine cavity can be prolonged, and the action efficiency of the exosome in an endometrium can be improved. The protamine-DOPAC conjugate provided by the invention has an application prospect in the drug delivery direction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to protamine-DOPAC conjugate, its preparation method and application. BACKGROUND

[0002] Protamine is a basic protein, mainly existing in mature sperm cell nuclei of fish (such as frog fish, squat fish, herring, etc.) as nucleoprotein combined with DNA. Protamine is a cationic protein with high affinity to cell membranes, so it can be used as a drug carrier to improve the cell uptake efficiency of drugs. However, protamine is difficult to dissolve in organic solvents, so it is a technical challenge to couple protamine with other compounds.

[0003] 3,4-dihydroxyphenylacetic acid (DOPAC) belongs to ortho-diphenol / catechol small molecules, which has excellent antioxidant, anti-inflammatory and cell protection effects, can scavenge free radicals, inhibit lipid peroxidation and regulate cell antioxidant enzyme expression; coupling it to high polymers or proteins can endow the high polymers or proteins with adhesion, antioxidant and coordination functions, so it can be used in the fields of biological adhesion material and functional carrier construction.

[0004] In the amide coupling reaction of proteins and small molecules, carbodiimide (EDC) system is usually used to activate carboxyl groups in water phase, so that they react with amino groups on the protein to form amide bonds. However, the O-acylisourea intermediate generated by EDC is extremely unstable in water phase and is easily hydrolyzed or rearranged into irreversible N-acylisourea by-products, resulting in low coupling efficiency and the need to use excessive EDC, which increases the burden of post-processing. In addition, in order to improve the selectivity and efficiency of the reaction, NHS, HOBt or HOAt and other activators are usually used to generate more stable active esters, but the residues of these small molecules in the product may affect the biocompatibility and increase the difficulty of purification. Finally, DOPAC contains an ortho-diphenol structure that is easily oxidized, which is easily oxidized to quinone in the presence of air or metal ions, and the quinone further non-specifically crosslinks with the amino or sulfhydryl groups in the protein, resulting in non-uniform products and even loss of activity.

[0005] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0006] In view of the above deficiencies of the prior art, the purpose of the present application is to provide protamine-DOPAC conjugate, its preparation method and application, aiming to solve the problems of unstable activation intermediate EDC, residual organic small molecules and easy oxidation of DOPAC in the prior art.

[0007] The technical scheme of the present application is as follows: In a first aspect of the present application, a preparation method of protamine-DOPAC conjugate is provided, comprising the following steps: (1) pre-activating 3,4-dihydroxyphenylacetic acid (DOPAC) and 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) in a solvent; adding 1-hydroxybenzotriazole (HOBt) to activate the reaction to obtain a reaction product; (2) adding the reaction product into a buffer solution of protamine to perform an amidation reaction to obtain a crude product; (3) purifying and drying the crude product to obtain the protamine-DOPAC conjugate.

[0008] Optionally, in step (1), the solvent is an organic solvent or water.

[0009] Preferably, the organic solvent is at least one of dimethyl sulfoxide (DMSO), N,N- dimethylformamide (DMF), acetonitrile (MeCN), tetrahydrofuran (THF), and methyltetrahydrofuran (MeTHF).

[0010] Optionally, the molar ratio of 3,4-dihydroxyphenylacetic acid to 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide is 1:(1.2-3.0). Preferably, the molar ratio of 3,4- dihydroxyphenylacetic acid to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 1:(1.5- 2.8).

[0011] Optionally, the molar ratio of 3,4-dihydroxyphenylacetic acid to 1-hydroxybenzotriazole is 1:(1.0-1.5). Preferably, the molar ratio of 3,4-dihydroxyphenylacetic acid to 1- hydroxybenzotriazole is 1:1.

[0012] Optionally, the final volume fraction of the organic solvent is 1-10%. Preferably, the final volume fraction of the organic solvent is 1-8%.

[0013] Optionally, in step (1), the reaction time of pre-activation is 5-30 min. Preferably, the reaction time of pre-activation is 5-15 min.

[0014] Optionally, in step (2), the amidation reaction of adding the reaction product into a buffer solution of protamine specifically includes: slowly adding the reaction product into the buffer solution of protamine, and the slow adding rate is 0.02-0.1 mL / s. Preferably, the slow adding mode is dropwise or injection pump adding.

[0015] Optionally, in step (2), the reaction time of the amidation reaction is 6-16 h; the reaction temperature is 4-25℃; and the pH value of the reaction system is 5.0-7.5. Preferably, to inhibit the oxidation of DOPAC, the reaction temperature is controlled at 4-15℃; and more preferably, to improve the reaction rate, the reaction temperature is controlled at 20-25℃. Preferably, the pH value of the reaction system is 6.5-7.2.

[0016] Optionally, in step (3), the purification method comprises at least one of dialysis, ultrafiltration or gel filtration. Preferably, when the dialysis method is used for purification, the crude product is transferred into a dialysis bag with a molecular weight cut-off of 1 kDa, and dialyzed in pure water for not less than 24 h, and the water is changed for not less than 5 times. Preferably, when the ultrafiltration method is used for purification, an ultrafiltration membrane with a molecular weight cut-off range of 3-10 kDa can be used. More preferably, the ultrafiltration and gel filtration methods are combined to realize scale-up purification.

[0017] In a second aspect of the present application, a protamine-DOPAC conjugate prepared by the above preparation method is provided.

[0018] In a third aspect of the present application, a protamine-DOPAC conjugate prepared by the above preparation method, or the application of the above protamine-DOPAC conjugate in drug delivery is provided.

[0019] Beneficial effects: The present application provides a protamine-DOPAC conjugate, a preparation method and application thereof. Compared with the prior art, the present application has the following advantages: (1) Improved coupling efficiency and product uniformity: By using the method of first pre-activating DOPAC with EDC and then adding HOBt after 5-10 minutes, the stable formation of active ester is promoted, and the amidation reaction of carboxylic acid and protein amino group is more sufficient, thereby significantly improving the coupling efficiency and improving the product uniformity.

[0020] (2) Reduced irreversible by-products and small molecule residues: By accurately controlling the amount of EDC and 1-hydroxybenzotriazole, the rearrangement reaction of O-acylisourea intermediate to N-acylisourea is effectively inhibited, and the generation of irreversible by-products is reduced. In combination with the subsequent purification step, the residual small molecules can be efficiently removed, so that the obtained conjugate has high purity and good biocompatibility.

[0021] (3) Inhibition of oxidation reaction of DOPAC: By adjusting the time, temperature and pH value of the reaction system of DOPAC and EDC, the side reaction of the o-diphenol structure being oxidized to quinone can be effectively inhibited.

[0022] (4) reducing the damage of organic solvents to proteins and maintaining the functionality of proteins: in order to ensure the dissolution and activation of small molecules, organic solvents are introduced in the reaction, and the final volume fraction of organic solvents in the reaction system is controlled to ensure the dissolution and activation of DOPAC, while avoiding the changes in protein conformation, aggregation or loss of activity caused by organic solvents, so as to maintain the structural and functional integrity of protamine.

[0023] Obtaining stable freeze-dried products: the protamine-DOPAC conjugate prepared by the present application forms a freeze-dried powder after freeze-drying, which can be stored for a long time under dry and light-proof conditions, has good dispersibility and redissolvability, and is convenient for dosage form development, industrialization and quality control. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The reaction equation diagram for preparing the protamine-DOPAC conjugate of the present application.

[0025] Figure 2 The process flow diagram for preparing the protamine-DOPAC conjugate in Example 1 of the present application.

[0026] Figure 3 The ultraviolet-visible absorption spectrum of the protamine-DOPAC conjugate (material, red line) prepared in Example 1 of the present application, protamine (PS, blue line) and 3,4-dihydroxyphenylacetic acid (DOPAC, gray line).

[0027] Figure 4 The nuclear magnetic hydrogen spectrum of protamine.

[0028] Figure 5 The nuclear magnetic hydrogen spectrum of 3,4-dihydroxyphenylacetic acid.

[0029] Figure 6 The nuclear magnetic hydrogen spectrum of the protamine-DOPAC conjugate prepared in Example 1 of the present application.

[0030] Figure 7 The experimental result diagram of the protamine-DOPAC conjugate increasing the retention of exosomes in the uterine cavity.

[0031] Figure 8 The experimental result diagram of the protamine-DOPAC conjugate increasing the retention of exosomes in the uterine cavity, thereby promoting the repair of endometrium. DETAILED DESCRIPTION

[0032] The present application provides a protamine-DOPAC conjugate and a preparation method and application thereof, in order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0033] In the following examples, the test methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0034] In the process of coupling protamine and DOPAC by using carbodiimide coupling (EDC) system and HOBt / HOAt, there are the following technical obstacles: first, the activated intermediate is unstable: the O-acyl isourea intermediate generated by EDC is easily hydrolyzed or rearranged into irreversible N-acylurea in aqueous phase, resulting in low coupling efficiency, and often requiring excessive use of EDC, which increases the difficulty of post-processing. Second, organic solvent residue and purification difficulty: to improve the coupling efficiency, HOBt / HOAt is often used in excess, but the residue of such small molecules in the product will affect the biocompatibility and increase the burden of purification and quality control. Third, the oxidation of ortho-diphenol easily leads to non-specific crosslinking: DOPAC and other ortho-diphenols are easily oxidized to quinone in the presence of air or metal ions, and the quinone can non-specifically add to the amine or sulfhydryl of the protein, resulting in uneven product, discoloration or loss of activity. Fourth, solvent-protein stability contradiction: in order to activate small molecules, organic solvents are needed, but organic solvents (even low percentage) can cause changes in protein conformation, aggregation or activity decline, which is more difficult to control in scale-up. Finally, lack of systematic process parameters and scale-up scheme: the existing technology is mostly research process, and there is a lack of a set of systematic process parameters covering dissolution, activation, dropwise addition, pH / temperature control, antioxidant, purification and freeze-drying, etc. to support the reproducible scale-up production and quality consistency control.

[0035] Based on this, the embodiment of the present application provides a preparation method of protamine-DOPAC conjugate, comprising the following steps: (1) pre-activating 3,4-dihydroxyphenylacetic acid (DOPAC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in a solvent; adding 1-hydroxybenzotriazole (HOBt) to carry out an activation reaction to obtain a reaction product; (2) adding the reaction product to a buffer solution of protamine to carry out an amidation reaction to obtain a crude product; (3) purifying and drying the crude product to obtain the protamine-DOPAC conjugate.

[0036] The present application adopts EDC to pre-activate DOPAC, and then adds HOBt to form a stable active ester, so as to improve coupling efficiency and product uniformity, reduce irreversible by-products and small molecule residues, and inhibit DOPAC oxidation. Subsequently, the active ester solution is added dropwise into a fish sperm protein aqueous phase dissolved in PBS (or fed at a constant speed by using a syringe pump) at a controllable speed, and the amidation coupling is completed under the conditions of controlling pH, temperature and reaction time. After the reaction, small molecule residues are removed by using a combination of dialysis, ultrafiltration, gel filtration and other purification methods, and finally, a stable powder product is obtained by quick freezing and freeze-drying Figure 2 The fish sperm protein-DOPAC conjugate prepared by the above method can maintain the functionality of fish sperm protein, and form a freeze-dried powder which can be long-term stored and is convenient for dosage form development, so as to facilitate scale-up production and quality control. The fish sperm protein-DOPAC conjugate reaction equation of the present application is shown in Figure 1 , wherein is fish sperm protein.

[0037] HOBt can convert the unstable O-acyl isourea intermediate into a more stable and more easily attacked by amine nucleophilic "active ester" form, so as to inhibit the rearrangement of the O-acyl isourea intermediate to N-acyl urea, and improve the effective conversion rate of amidation.

[0038] In some embodiments, the solvent is an organic solvent or water.

[0039] Since the chemical activity of EDC is still effective in the aqueous phase, the activation efficiency is closely related to pH (usually weak acid buffer is used for carboxylic acid activation to promote the formation of O-acyl isourea intermediate). Therefore, MES, acetate and other buffer systems can provide a stable pH environment in the aqueous phase to support short-time activation. Preferably, the solvent is an organic solvent. In the organic solvent, DOPAC can be better dissolved to promote the EDC activation reaction.

[0040] Preferably, the organic solvent is at least one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (MeCN), tetrahydrofuran (THF), and methyltetrahydrofuran (MeTHF).

[0041] Since DMSO, DMF and MeCN are all polar organic solvents, specifically: DMSO (containing a sulfur-oxygen group) has strong polarity and can form a hydrogen bond acceptor, DMF (N,N-dimethylformamide) contains an amide group which has good solubility for solutes, and MeCN (acetonitrile) has high polarity and good volatility. Therefore, the organic solvent can dissolve DOPAC and EDC and promote the activation reaction in the organic phase.

[0042] In some embodiments, the molar ratio of the 3,4-dihydroxyphenylacetic acid to the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:(1.2-3.0). Preferably, the molar ratio of the 3,4-dihydroxyphenylacetic acid to the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:(1.5-2.8).

[0043] In some embodiments, the molar ratio of the 3,4-dihydroxyphenylacetic acid to the 1-hydroxybenzotriazole is 1:(1.0-1.5). Preferably, the molar ratio of the 3,4-dihydroxyphenylacetic acid to the 1-hydroxybenzotriazole is 1:1.

[0044] In some embodiments, the final volume fraction of the organic solvent is 1-10%. Preferably, the final volume fraction of the organic solvent is 1-8%.

[0045] In some embodiments, in step (1), the reaction time of the pre-activation is 5-30 min. Preferably, the reaction time of the pre-activation is 5-15 min.

[0046] In some embodiments, in step (2), the amidation reaction is carried out by slowly adding the reaction product into the buffered solution of protamine, and the slow adding rate is 0.02-0.1 mL / s. Preferably, the slow adding is carried out by dropwise adding or injection pump adding.

[0047] In some embodiments, in step (2), the reaction time of the amidation reaction is 6-16 h; the reaction temperature is 4-25°C; and the pH value of the reaction system is 5.0-7.5. Preferably, to inhibit the oxidation of DOPAC, the reaction temperature is controlled at 4-15°C; and more preferably, to improve the reaction rate, the reaction temperature is controlled at 20-25°C. Preferably, the pH value of the reaction system is 6.5-7.2.

[0048] In some embodiments, in step (3), the purification method comprises at least one of dialysis, ultrafiltration and gel filtration. Preferably, when the dialysis method is used for purification, the crude product is transferred into a dialysis bag with a molecular weight cut-off of 1 kDa, and dialysis is carried out in pure water for not less than 24 h, and the water is changed for not less than 5 times. Preferably, when the ultrafiltration method is used for purification, an ultrafiltration membrane with a molecular weight cut-off range of 3-10 kDa can be used. More preferably, the ultrafiltration and gel filtration methods are combined to realize scale-up purification.

[0049] In some embodiments, ascorbic acid, EDTA or inert atmosphere (such as N2) can be added to inhibit the oxidation of catechol during the activation and / or coupling stage.

[0050] Ascorbic acid maintains the ortho-diphenol by direct chemical reduction, EDTA reduces oxidation catalysis by removing metal catalytic factors, and inert atmosphere reduces the driving force of oxidation; these measures can reduce the conversion of DOPAC to quinone in structure, thereby maintaining the coupling form of DOPAC and reducing non-specific side reactions.

[0051] In some embodiments, the protamine can also be partially protected (e.g., PEGylated) before selective coupling to control the site distribution.

[0052] In the following examples, the Periplaneta americana exosome (PA-EVs) refers to a nanoscale membrane-encapsulated vesicle isolated from Periplaneta americana.

[0053] The application will be further described in the following specific examples.

[0054] Example 1 This example provides a protamine-DOPAC conjugate and a preparation method thereof, and the specific steps are as follows and Figure 2 as shown: S1, take 200 mg protamine (purchased from Sigma, product number P0675) and dissolve it in 10 mL PBS buffer (pH 6.8-7.4), and stir magnetically at room temperature until completely dissolved.

[0055] S2, take 29 mg EDC·HCl and dissolve it in 1.0 mL DMSO, then add 9.0 mg 3,4-dihydroxyphenylacetic acid (DOPAC) solid, and stir at room temperature for 5-10 min to generate an O-acylisourea intermediate; then add 21.5 mg HOBt and continue stirring for 5-10 min to obtain a HOBt ester.

[0056] S3, add the HOBt ester solution in step S2 to the protamine PBS solution dropwise at a constant rate (10-30 min) under light shielding conditions, while maintaining slow stirring.

[0057] S4, continue to react at room temperature for 12 h after the completion of the dropwise addition.

[0058] S5, after the reaction is completed, remove the precipitate by low-speed centrifugation, and transfer it to a dialysis bag (MWCO 1 kDa) for dialysis in pure water for 24 h (5-6 times of water change).

[0059] S6, measure the protein content (A280) after dialysis, analyze the degree of coupling by TNBS method or UV-visible absorption spectrum, and detect the residues (EDU, HOBt and DMSO, etc. small molecules) by HPLC or LC-MS.

[0060] S7, the product after dialysis is divided into freeze-drying bottles, pre-frozen at -80℃ for 2 h, and then freeze-dried in a freeze dryer for 24 h to obtain a freeze-dried powder, which is protamine-DOPAC conjugate. The protamine-DOPAC conjugate is stored in a dry and dark condition.

[0061] The protamine-DOPAC conjugate is characterized by infrared spectrum and nuclear magnetic resonance hydrogen spectrum. As shown in Figure 3 , in the ultraviolet-visible absorption spectrum, the protamine-DOPAC conjugate (material, red line) has characteristic peaks of protamine (PS, blue line) and 3,4-dihydroxyphenylacetic acid (DOPAC, gray line) at the same time; as shown in Figures 4-6 , in the nuclear magnetic hydrogen spectrum, the protamine-DOPAC conjugate has characteristic peaks of protamine and DOPAC at the same time. The above results show that the protamine-DOPAC conjugate is successfully synthesized.

[0062] Example 2 This example verifies the protamine-DOPAC conjugate prepared in Example 1, which can increase the residence time of exosomes in the uterine cavity. The specific steps are as follows: 1. DiD staining and purification of Periplaneta americana exosomes (PA-EVs) (1) Take out the Periplaneta americana exosomes (PA-EVs), quantitate according to the protein amount (BCA method), and adjust to 0.5-1.0 mg / mL for standby.

[0063] (2) Take 100 μg of protein of the above PA-EVs and add PBS (total volume is 100 μL).

[0064] (3) Add DiD stock solution (purchased from Biyun Tian Company, product number C1995S) to make the final concentration 5 mg / ml, mix gently, and incubate at room temperature for 15-30 min.

[0065] (4) After incubation, centrifuge at 100,000 x g at 4℃ for 70 min, discard the supernatant, and obtain the precipitate. Resuspend the precipitate in PBS solution, repeat the washing for 2 times to remove free DiD, and finally resuspend and quantitate to obtain the labeled PA-EVs sample.

[0066] 2. Sample processing (1) Preparation of PA-EVs (differential / ultracentrifugation method): Take xx g of Periplaneta americana extract and resuspend in 4°C 1x PBS. Remove large particles by centrifugation in the following order: 300xg for 10 min, 2,000xg for 20 min, and 10,000xg for 30 min, all at 4°C. Filter the supernatant (0.22 µm).

[0067] Ultracentrifugation to precipitate PA-EVs: 100,000xg for 70 min at 4°C. Discard the supernatant. Wash and repeat ultracentrifugation once with a large volume of 4°C PBS. Resuspend in xx ml of PBS solution. Obtain PA-EVs suspension and store at -80°C.

[0068] (2) Preparation of PA-EVs+PS (protamine sulfate) complex: Under sterile conditions, prepare the PS stock solution. Mix PA-EVs and PS at a volume ratio of 1:1. Then gently mix and incubate for 15-30 min (room temperature or 4°C).

[0069] Detect changes in particle size and potential to confirm PA-EVs+PS binding. Remove free PS by dialysis. Obtain PA-EVs+PS complex for subsequent experiments or storage.

[0070] (3) Preparation of protamine-DOPAC conjugate (PA-EVs+material) Carboxyl-containing material is activated with EDC (0.05 M) / NHS (0.02 M) in 0.1 M MES (pH 5.5) for 15 min. After washing with buffer to remove free reagents, co-incubate with PA-EVs-containing PBS (pH 7.4, 10-200 µg / mL) for 1-4 h (4°C or room temperature). Block residual sites with ethanolamine and wash with PBS to obtain protamine-DOPAC conjugate.

[0071] 3. Animal administration (1) Select 32 female C57BL / 6 mice aged 6-8 weeks (randomly divided into 4 groups, 8 mice per group). Anesthetize the mice by injecting sodium pentobarbital.

[0072] (2) Keep the mouse body temperature and lay a sterile operating table. Expose the mouse's abdomen, make a small sterile incision, and expose the bilateral mouse uterine horns.

[0073] (3) Using a 10-20 μL Hamilton syringe, slowly inject the sample (PA-EVs, PA-EVs+PS or PA-EVs+material) into the uterine cavity, inject the sample into both uterine horns, 10-20 μL per uterine horn, and the total injection amount of each mouse is 20-40 μg of protein. After injection, keep the needle for a few seconds to prevent backflow of the sample.

[0074] (4) Close the incision and perform postoperative observation and warming, and give analgesic drugs according to the condition of the mouse.

[0075] 4. In vivo fluorescence imaging (IVIS) detection (1) Before imaging, the mouse is anesthetized by pentobarbital injection, placed on a constant temperature table, positioned consistently, and the abdominal hair is shaved to reduce scattering.

[0076] (2) Adjust the instrument parameters of the animal imager, and perform fluorescence imaging on the sample after staining with Did, set the excitation wavelength to 640 nm, set the emission filter to 680 nm, and the exposure time is 1-5 s.

[0077] (3) Each mouse is imaged under the same settings at 24 h, 48 h, 72 h and 120 h after injection of the sample; if necessary, early time points (1 h, 6 h, 12 h) can be added to obtain complete fluorescence kinetic curves. Avoid external light interference and fluorescence crosstalk during shooting.

[0078] 5. Experimental results As shown in Figure 7 , the IVIS in vivo imaging results show that protamine-DOPAC conjugates can prolong the residence time of exosomes in the uterine cavity.

[0079] Example 3 This example verifies that the protamine-DOPAC conjugate prepared in Example 1 can enhance the residence time of Periplaneta americana exosomes in the uterine cavity, as follows: 1. Animal handling Select female C57BL / 6 mice 6-8 weeks old, weighing 18-20 g, purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd.; raise in a SPF level mouse breeding environment, with a breeding temperature of 22±1℃, free water and food. Two consecutive cycles of vaginal smear detection were performed 7 days before the experiment, and mice in the third estrus were selected for modeling to ensure that the endometrial thickness was in a relatively stable state.

[0080] 2. Establishment of thin endometrial model Anesthesia and surgical exposure: After the mouse was anesthetized with sodium pentobarbital, the abdominal hair was shaved and disinfected with iodophor, and a 1 cm incision was made in the middle of the abdomen to expose the uterine horn.

[0081] Chemical injury: After clamping the uterine horn at both ends with a hemostatic clamp, a 25 G injection needle was inserted gently about 30° parallel to the uterine horn, and 50 μL of 95% ethanol was injected, retaining for 40 seconds. Then it was flushed twice with sterile saline, sutured the incision, and a thin endometrial model was established.

[0082] 3. Preparation of PA-EVs-protamine-DOPAC conjugate (PA-EVs+material) sample (1) Collect fresh Periplaneta americana body fluid or culture supernatant.

[0083] (2) The above sample was subjected to continuous centrifugation (300 x g for 10 min, 2000 x g for 20 min), and the precipitate was discarded, and the supernatant was retained to remove cells and large particle impurities. The supernatant was subjected to 100,000 x g ultracentrifugation for 70 min (4°C), and the supernatant was discarded, and the precipitate was collected as the crude exosome product.

[0084] (3) The crude exosome product was resuspended in PBS, and then subjected to 100,000 x g ultracentrifugation for 70 min (4°C) for further purification, and the purified exosome precipitate was obtained.

[0085] (4) The purified exosome precipitate was resuspended in 100 μL PBS, and the protein concentration was quantified by BCA method to obtain Periplaneta americana exosomes for use.

[0086] (5) The exosomes prepared in step (4) (7 mg / ml) were mixed with an equal concentration of protamine-DOPAC conjugate solution (7 mg / ml) at a volume ratio of 1:1, and incubated in 1 x PBS buffer at pH 7.4 for 1 hour.

[0087] 4. Mouse uterine cavity administration scheme Time node: 7 days after the establishment of a thin endometrial model; Administration method: Repeat the model operation process, expose the uterine horn, and slowly inject 20 μL of sample (PBS, PA-EVs, or PA-EVs+material) into both uterine horns using a Hamilton microsyringe. Keep the needle in place for 1 min after injection to prevent backflow. Suture the incision and recover after the operation. The mice were given routine analgesia and warming care after the operation.

[0088] Among them, the sham operation group (Sham group) only underwent anesthesia and laparotomy treatment without injecting drugs, and the rest of the operations were the same.

[0089] 5. Histological evaluation (1) Sampling and euthanasia Sample collection was performed on day 7 post-dose according to the experimental design. Prior to sample collection, animals were anesthetized by intraperitoneal injection of sodium pentobarbital 50 mg / kg (deep anesthesia). After confirming the absence of reflexes, animals were sacrificed by cervical dislocation. Immediately after sacrifice, the uterus was exposed by laparotomy and the entire uterine body with both horns (entirely removed) was excised from the uterine body to the uterine horn and placed in a pre-prepared 10-fold volume of 4% paraformaldehyde, rinsed briefly at room temperature to remove blood fragments and then fixed at 4°C.

[0090] (2) Fixation conditions Uterine tissue was fixed in 4% PFA for 24 h (± 2 h) at 4°C. The volume of fixative should be at least 10 times the volume of the tissue. After fixation, residual fixative was removed by washing twice in flowing PBS (pH 7.4) for 10 min each.

[0091] (3) Tissue dehydration, clearing and paraffin embedding (routine paraffin procedure) Gradual alcohol dehydration (room temperature, light shaking on a shaker): 70% ethanol for 30 min; 80% ethanol for 30 min; 95% ethanol for 30 min; 100% ethanol for 2 x 30 min (each time, new liquid was added). Xylene / xylene substitute clearing: xylene for 2 x 15 min (operation in a fume hood). Paraffin infiltration: 60°C paraffin for 2 x 45-60 min. The tissue was removed and placed in a paraffin mold in the correct orientation for transverse sections (sections containing the uterine lumen and endometrium). The tissue was cooled and solidified into a paraffin block.

[0092] (4) Sectioning and pre-treatment before mounting The paraffin block was sectioned into 10 pm thick sections using a microtome. Three non-adjacent levels (intervals of > 50 pm) were taken from each specimen, each with 3 consecutive sections (for H&E, Masson, reserved). This means that at least 9 sections per animal were obtained for evaluation. The sections were mounted on positively charged glass slides and left at room temperature until they were fixed. They were then dried in an oven at 40°C overnight or at 60°C for 1 h to ensure adhesion.

[0093] (5) H&E staining (evaluation of endometrial thickness, gland number) Dewaxing of paraffin sections: xylene for 2 x 5 min.

[0094] Rehydration from thick to thin: 100% ethanol for 2 x 3 min, followed by 95% ethanol for 2 min, followed by 80% ethanol for 2 min, followed by 70% ethanol for 2 min, followed by distilled water for 2 min.

[0095] Mayer hematoxylin stain 5 min (or Harris hematoxylin 5 min), tap water rinse 2 min. Acid alcohol differentiation (1% HC1 in 70% ethanol) fast dip 1-3 s (watch color). Immediately rinse to baseline with water. Alkaline bluing (0.2% NH4OH or tap water pH adjusted to alkaline) 30 s, water rinse 1 min. Eosin Y (0.5%) stain 1-2 min, running water rinse 30 s. Dehydrate: stepwise dehydration through 70%, 80%, 95%, 100% ethanol (2 min each), 2 x 3-5 min xylene. Finally, mount with neutral resin (neutral resin mounting medium or neutral resin mounting gel), air dry and observe.

[0096] 6) Masson's trichrome staining (to assess fibrosis area) Classical Masson's trichrome is used (collagen is stained blue / green, myofibrils and cytoplasm are red).

[0097] a. Rehydrate sections from deparaffinization same as for H&E (same as above).

[0098] b. Weigert's iron hematoxylin counterstain (nuclear stain): Weigert's iron hematoxylin 10 min, tap water rinse 5 min.

[0099] c. Biebrich scarlet-acid fuchsin solution 5-10 min (stains red cytoplasm and myofibrils).

[0100] d. Phosphomolybdic / phosphotungstic acid treatment 5-10 min (rinses away red and makes collagen stainable blue).

[0101] e. Aniline blue stain 5-10 min (stains collagen).

[0102] f. 1% acetic acid solution rinse 1-2 min to fix color.

[0103] g. Dehydrate (quickly): stepwise dehydration in 70%, 80%, 95%, 100% ethanol (1-2 min each), 2 x 3-5 min xylene.

[0104] h. Mount with neutral resin, air dry.

[0105] (7) Microscopy and standardized photography Images were acquired using an optical microscope or a panoramic slide scanner (10×, 20×, or 40× objectives recommended). For quantification consistency, the same magnification and exposure parameters were selected for each slide.

[0106] At least three representative fields of view were taken from each slice (one region in the middle of the uterus and one region on each side), and each field of view image was saved in TIFF format for subsequent analysis.

[0107] (8) Quantitative and statistical methods for data a. Endometrial thickness measurement (H&E): In the transverse image (including the uterine cavity), a reference line is drawn along the endometrial-myometrial junction, and the vertical distance from the basal layer to the luminal epithelium is measured. For each slice, measurements are taken three times at three standard locations (median, left, and right), for a total of nine measurements; the average value is taken as the endometrial thickness of that specimen. Each group should contain at least three animals (n≥6 recommended). Finally, the mean ± SD of the endometrial thickness for each group is calculated, and appropriate statistical tests (such as t-test or ANOVA) are used to compare differences between groups.

[0108] b. Glandular Count (H&E): The number of glands in cross-sections was counted using ROIs of fixed width (e.g., a 1 mm band along the endometrium). Glands were identified by intact epithelial rings or lumens. Three ROIs (median and lateral) were selected for each slice. The number of glands was recorded and the gland density (numbers / mm) was calculated for each ROI. The average value of multiple slices for each specimen was taken, and the differences between groups were statistically analyzed.

[0109] c. Quantitative analysis of fibrosis area (Masson): Using ImageJ / Fiji: Open the Masson-stained color image and set the ROI to the entire endometrial region (either manually drawn or automatically identified based on the uterine cavity boundary). Use the "Color Deconvolution" plugin or directly based on a color threshold to separate the blue (collagen) region; adjust the threshold to accurately cover collagen staining. Calculate the collagen-positive area and the total ROI area; fibrosis percentage = (collagen-positive area / total ROI area) × 100%. Take values ​​from 3 representative fields for each slide, at least 3 slides per animal, and finally calculate the average value for each animal and compare the differences between groups.

[0110] 6. Experimental Results like Figure 8 As shown, in a mouse model of thin endometrium: (1) Endometrial thickness: The endometrium in the PBS group was thinner and the glands were reduced; the recovery in the PA-EVs group was limited; while the endometrial thickness in the PA-EVs+ material group increased significantly, almost recovering to the level of the Sham group.

[0111] (2) Gland number: The gland number of PA-EVs+material group was significantly higher than that of other groups, suggesting that functional gland regeneration was good.

[0112] (3) Masson staining (fibrosis): A large number of blue collagen deposition was observed in the PBS group; while the collagen deposition of the PA-EVs+material group was significantly reduced. Quantitative analysis showed that the fibrosis area of the PA-EVs+material group was similar to that of the Sham group.

[0113] The above histological indexes collectively indicate that the PA-EVs+material group not only inhibits fibrosis, but also promotes the reconstruction of the functional layer.

[0114] The above results show that the protamine-DOPAC conjugate significantly increases the retention time of PA-EVs (Periplaneta americana exosomes) in the uterine cavity, promotes the increase of endometrial thickness and the recovery of gland number, and significantly reduces the collagen fiber area, achieving the promoting effect on the repair of the endometrium.

[0115] In summary, the present application provides a preparation method of protamine-DOPAC conjugate, and the prepared protamine-DOPAC conjugate is suitable for the construction of drug delivery system and functional biomaterials.

[0116] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A method for preparing a protamine-DOPAC conjugate, characterized by, The method comprises the following steps: (1) pre-activating 3,4-dihydroxyphenylacetic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a solvent; adding 1-hydroxybenzotriazole to perform an activation reaction to obtain a reaction product; (2) adding the reaction product into a buffer solution of protamine to perform an amidation reaction to obtain a crude product; (3) purifying and drying the crude product to obtain the protamine-DOPAC conjugate.

2. The production method according to claim 1, characterized by, In step (1), the solvent is an organic solvent or water.

3. The production method according to claim 2, characterized by, The organic solvent is at least one of dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and methyltetrahydrofuran.

4. The method of claim 1, wherein, The molar ratio of 3,4-dihydroxyphenylacetic acid to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is 1:(1.2-3.0).

5. The preparation method according to claim 1, characterized in that, The molar ratio of 3,4-dihydroxyphenylacetic acid to 1-hydroxybenzotriazole is 1:(1.0-1.5).

6. The method of claim 1, wherein, In step (2), the amidation reaction of the reaction product in the buffer solution of protamine specifically comprises: slowly adding the reaction product into the buffer solution of protamine, and the slow adding rate is 0.02-0.1 mL / s.

7. The preparation method according to claim 1, characterized in that, In step (2), the reaction time of the amidation reaction is 6-16 h; the reaction temperature is 4-25°C; and the pH value of the reaction system is 5.0-7.

5.

8. The method of claim 1, wherein, In step (3), the purification method comprises at least one of dialysis, ultrafiltration, and gel filtration.

9. A protamine-DOPAC conjugate prepared by the preparation method of any one of claims 1-8.

10. The protamine-DOPAC conjugate prepared by the preparation method of any one of claims 1-8, or the protamine-DOPAC conjugate of claim 9, in the application of drug delivery.

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