Polyamino acid nano-drug carrier for responsive removal of polyethylene glycol, preparation method and nano-drug thereof

Abstract

The invention provides a polyamino acid nano-drug carrier for responsive removal of polyethylene glycol, a preparation method and a nano-drug thereof. The nano-drug carrier is loaded with an active drug component, and after entering a body, the nano-drug carrier can be passively targeted and accumulated at a tumor tissue by virtue of a blood long circulation effect and an EPR effect (the EPR effect means that a solid tumor tissue has the characteristics of high permeability and retention compared with a normal tissue) of polyethylene glycol. An environment with meta-acidity and high-concentration matrix metalloproteinase is arranged around a tumor tissue, and after a drug enters the tumor tissue, a connecting part of PEG and a glutamic acid unit has a response effect on the change of pH or matrix metalloproteinase concentration in a microenvironment around the tumor tissue and is broken, so that a polyethylene glycol structure is automatically removed, and the drug delivery efficiency is improved. The medicine without the polyethylene glycol can be more efficiently endocytosed by tumor cells, so that active medicine components are released, and the tumor cells are effectively killed.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer nano drug carriers, and specifically relates to a polyamino acid nano drug carrier capable of responsively removing polyethylene glycol, a preparation method and a nano drug thereof. Background Art

[0002] Nowadays, tumors have become one of the most serious diseases threatening human health. Commonly used cancer treatment methods in clinical practice include chemotherapy, radiotherapy and surgical treatment. Among them, chemotherapy is different from surgical treatment and radiotherapy. It can achieve the integrity of the treatment of the human body. It works on the whole body through oral and intravenous administration. It has its unique features in eliminating the distant metastasis of a certain tumor or preventing recurrence. It is the most commonly used and important treatment approach in tumor treatment methods. However, the anti-tumor drugs used in clinical practice have many defects in application, such as: poor water solubility and stability, and large drug toxicity and side effects. In order to solve these problems, the drug can be combined with a nano drug carrier to improve the water solubility and stability of the drug, and to achieve controlled release of the drug, thereby reducing the toxic and side effects of the drug on normal tissues and giving full play to the efficacy of the drug.

[0003] However, studies have found that conjugating polyethylene glycol (PEG) to a given platform (i.e., "PEGylation") is a mature strategy for drug delivery systems that can be applied to systemic administration. PEGylation forms a hydration layer around nanoparticles, increasing the exclusion volume around the nanoparticles, thereby spatially preventing the nanoparticles from interacting with components such as proteins in the blood. At the same time, the flexibility of PEG provides considerable conformational freedom, thereby preventing foreign matter from penetrating into the PEG outer layer. PEGylation prevents protein adsorption of nanoparticles and increases their circulation time in the blood, thereby increasing drug accumulation at the target site by enhancing the permeation and retention effect (EPR effect).

[0004] However, due to the spatial repulsion between the PEG chain and the cell membrane, PEGylation will prevent nanoparticles from being internalized by tumor cells to a certain extent, especially for those drugs that need to cross the cell membrane to exert their effects, which inevitably reduces the bioavailability and therapeutic effect of nanomedicines. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a polyamino acid nano drug carrier that responsively removes polyethylene glycol, a preparation method and a nano drug thereof. The nano drug carrier can responsively remove PEG at the tumor site, enhance the cell endocytosis rate, and improve drug enrichment.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a polyamino acid nano drug carrier for responsive removal of polyethylene glycol, which has a structure shown in Formula I or Formula II:

[0008] Formula I; Formula II;

[0009] Wherein, in formula I, 40≤n1≤230, 1≤x1≤15, 100≤y1≤200;

[0010] In formula II, 40≤n2≤230, 1≤x2≤15, 100≤y2≤200;

[0011] R1 is a peptide segment responsive to matrix metalloproteinase 2, selected from any one of PLGLAG, GPLGBRG, GPLGBRGK, GPLGVRGC, GPLGBRGCA, PLGVR, PLGGBRG, GPLGBRGGG, CGPLGGBRGK, PRCGV, SGAVRWLLTA, and GPPGVVGEKGEQ;

[0012] or R1 is a peptide segment responsive to matrix metalloproteinase 9, selected from any one of SGKGPRQITA, GGKGPLGLPG, GPLGMWSRC, LGRMGLPG, LGPRSLSGK, SGAVRWLLTA, PLGLRSW, and PLGVRGK;

[0013] R2 is selected from any one of the following formulas A-1 to A-5:

[0014] Formula A-1; Formula A-2; Formula A-3;

[0015] Formula A-4; Formula A-5.

[0016] Preferably, 50≤n1≤150, 3≤x1≤10, and 120≤y1≤160.

[0017] Preferably, 40≤n2≤230, 1≤x2≤15, 100≤y2≤200.

[0018] Preferably, the polyamino acid nano drug carrier for responsive removal of polyethylene glycol shown in Formula II is selected from any one of the following Formulas II-1 to II-5:

[0019] Formula II-1; Formula II-2;

[0020] Formula II-3; Formula II-4; Formula II-5.

[0021] Preferably, the polyamino acid nano drug carrier for responsive removal of polyethylene glycol shown in Formula I responsively removes polyethylene glycol under the action of matrix metalloproteinase 2 or matrix metalloproteinase 9.

[0022] Preferably, the polyamino acid nano drug carrier for responsive removal of polyethylene glycol shown in Formula II responsively removes polyethylene glycol under the condition of pH < 6.8.

[0023] In a second aspect, the present invention provides an application of the above-mentioned polyamino acid nano-drug carrier capable of responsively removing polyethylene glycol in the preparation of anti-tumor nano-drugs.

[0024] In a third aspect, the present invention provides an anti-tumor nanomedicine, which is composed of an active drug component and a carrier;

[0025] The carrier is the nano drug carrier mentioned above.

[0026] Preferably, the active pharmaceutical ingredient comprises any one of cisplatin, irinotecan or Compretin A4.

[0027] Preferably, the molar ratio of the glutamic acid unit to the active pharmaceutical ingredient in the carrier is (1-6):1.

[0028] Preferably, the anti-tumor nanomedicine responsively removes PEG at the tumor.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The polyamino acid nano drug carrier with responsive polyethylene glycol removal shown in formula I or II provided by the present invention has polyethylene glycol grafted on the side chain of polyglutamic acid. When it is loaded with active drug components and enters the body, it will passively target and accumulate in the tumor tissue by virtue of the long blood circulation effect of polyethylene glycol and the EPR effect (EPR effect refers to the characteristics of high permeability and retention of solid tumor tissue relative to normal tissue). The environment around the tumor tissue is slightly acidic and has a high concentration of matrix metalloproteinases. After the drug enters the tumor tissue, the connection part between PEG and the glutamic acid unit responds to the change of pH or matrix metalloproteinase concentration in the microenvironment around the tumor tissue and breaks, so that the polyethylene glycol structure is automatically removed. The drug with polyethylene glycol removed will be more efficiently internalized by tumor cells, thereby releasing the active drug components and effectively killing tumor cells;

[0031] (2) The polyamino acid nano drug carrier with responsive removal of polyethylene glycol shown in Formula I or Formula II provided by the present invention has only polyglutamic acid on its main chain, and the PEG structure with responsive removal effect can be modified to the polymer side chain without adding additional amino acid units. In addition, the modification steps of PEG are simplified, the synthetic route is convenient, and the overall structure is relatively simple;

[0032] (3) The polyamino acid nano drug carrier with responsive removal of polyethylene glycol shown in Formula I or Formula II provided by the present invention can be used as a new type of nano drug delivery platform to load various active drug components (such as cisplatin, irinotecan or Compretin A4) through complexation, positive and negative electrical interactions, and covalent bonding, and has a large number of available drug-carrying groups and a high drug loading capacity, which can be above 20%.

[0033] (4) The polyamino acid nano-drug carrier for responsive removal of polyethylene glycol shown in Formula I or Formula II provided by the present invention has enzyme responsiveness and pH responsiveness, wherein the pH-responsive polyamino acid nano-drug carrier has a high degree of responsiveness, PEG removal is slow in an alkaline environment, and PEG can be removed quickly in a weakly acidic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the H-NMR spectrum of PLGLAG-mPEG prepared in Preparation Example 5;

[0035] Figure 2 This is the H-NMR spectrum of the pH-responsive polyethylene glycol prepared in Preparation Example 7;

[0036] Figure 3 This is the H-NMR spectrum of the enzyme-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 1;

[0037] Figure 4 This is the H-NMR spectrum of the pH-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 7;

[0038] Figure 5 This is the H-NMR spectrum of the pH-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 14;

[0039] Figure 6 This is the H-NMR spectrum of the pH-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 15;

[0040] Figure 7 This is a diagram showing the result of removing PEG from the enzyme-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 1;

[0041] Figure 8 This is a diagram showing the results of removing PEG from the pH-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 7;

[0042] Fig. 9 The dynamic light scattering diagram of the enzyme-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 1;

[0043] Fig.10 The dynamic light scattering diagram of the enzyme-responsive depolyethylene glycol drug loaded with irinotecan prepared in Application Example 7;

[0044] Fig.11 The dynamic light scattering diagram of the pH-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 9;

[0045] Fig.12 Results of simulated in vivo drug release of the enzyme-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 1 and the pH-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 9;

[0046] Fig.13 This is a diagram showing the results of simulating in vivo drug release of the irinotecan-loaded nanomedicine prepared in Application Example 7 by enzyme-responsive removal of polyethylene glycol;

[0047] Fig.14 This is a diagram showing the cytotoxicity test results of the enzyme-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 1;

[0048] Fig.15 This is a diagram showing the cytotoxicity test results of the pH-responsive depolyethylene glycol-removing drug loaded with cisplatin prepared in Application Example 9;

[0049] Fig.16 This is a diagram showing the cytotoxicity test results of the pH-responsive depolyethylene glycol-removed drug loaded with irinotecan prepared in Application Example 7;

[0050] Fig.17 Figure 2 shows the anti-tumor results of different groups. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] In view of the problem in the prior art that PEGylation of nanomedicines reduces the endocytosis efficiency of tumor cells and thus reduces the bioavailability of nanomedicines, the present invention provides a polyamino acid nanomedicine carrier for responsive removal of polyethylene glycol, which has a structure shown in Formula I or Formula II:

[0053] Formula I; Formula II;

[0054] In the present invention, n1 and n2 refer to the degree of polymerization of polyethylene glycol, x1 and x2 refer to the number of structural units of polyglutamic acid grafted with polyethylene glycol, and y1 and y2 refer to the number of structural units of polyglutamic acid not grafted with polyethylene glycol.

[0055] Wherein, in Formula I and Formula II, n1 and n2, x1 and x2, y1 and y2 are each independently selected from the following ranges (n, x, y are all integers): 40≤n1 (or n2)≤230, 1≤x1 (or x2)≤15, 100≤y1 (or y2)≤200, preferably: 50≤n1 (or n2)≤150, 3≤x1 (or x2)≤10, 120≤y1 (or y2)≤160, more preferably 80≤n1 (or n2)≤120, 5≤x1 (or x2)≤10, 130≤y1 (or y2)≤150.

[0056] In the present invention, the polyamino acid nano drug carrier with responsive polyethylene glycol removal shown in Formula I has polyethylene glycol that can respond to matrix metalloproteinases grafted on the side chain of polyglutamic acid, which has the effect of enzyme responsive polyethylene glycol removal. Since the enzymes highly expressed in tumors are matrix metalloproteinases 2 and matrix metalloproteinases 9, the polyethylene glycol that can respond to matrix metalloproteinases is: polyethylene glycol that can respond to matrix metalloproteinases 2 and matrix metalloproteinases 9. According to research, the nano drug carrier with responsive polyethylene glycol removal shown in Formula I can responsively remove polyethylene glycol under the action of high concentrations of matrix metalloproteinases 2 and matrix metalloproteinases 9.

[0057] In the present invention, in the above structural formula I, R1 is a peptide segment responsive to matrix metalloproteinase 2, selected from any one of PLGLAG, GPLGBRG, GPLGBRGK, GPLGVRGC, GPLGBRGCA, PLGVR, PLGGBRG, GPLGBRGGG, CGPLGGBRGK, PRCGV, SGAVRWLLTA, and GPPGVVGEKGEQ;

[0058] or

[0059] R1 is a peptide segment responsive to matrix metalloproteinase 9, selected from any one of SGKGPRQITA, GGKGPLGLPG, GPLGMWSRC, LGRMGLPG, LGPRSLSGK, SGAVRWLLTA, PLGLRSW, and PLGVRGK.

[0060] In the present invention, the polyamino acid nano drug carrier with responsive removal of polyethylene glycol shown in formula ⅠⅠ has polyethylene glycol that can respond to acidic environment grafted on the side chain of polyglutamic acid, which has the effect of pH responsive removal of polyethylene glycol. According to research, the polyamino acid nano drug carrier with responsive removal of polyethylene glycol shown in formula ⅠⅠ has a high degree of pH responsiveness, PEG removal is slow in alkaline environment, and PEG can be quickly removed in weakly acidic environment (such as pH < 6.8, such as 6.5).

[0061] In the present invention, in formula ⅠⅠ, R2 is selected from any one of the following formulas A-1 to A-5:

[0062] Formula A-1; Formula A-2; Formula A-3;

[0063] Formula A-4; Formula A-5.

[0064] The present invention also provides a method for preparing the polyamino acid nano drug carrier for responsive removal of polyethylene glycol as shown in the above formula I, comprising the following steps:

[0065] Under the action of a condensing agent and a catalyst, the compound represented by Formula 1 is mixed with the compound represented by Formula 2 to react, thereby obtaining a polyamino acid nano drug carrier for responsive removal of polyethylene glycol represented by Formula I;

[0066] Formula 1; Formula 2;

[0067] Among them, the value ranges of n1, x1, and y1 are as described in the relevant contents of the above technical solution and will not be repeated here.

[0068] In the present invention, the compound represented by Formula 1 is the main part of the above-mentioned nano drug carrier - polyglutamic acid. In the present invention, the compound represented by Formula 1 is obtained by reacting n-hexylamine and γ-benzylglutamic acid-N-intra-ring carboxylic anhydride (abbreviated as "BLG-NCA") in the presence of an organic solvent in an inert atmosphere. Among them, the organic solvent is preferably N,N-dimethylformamide. The temperature of the reaction is preferably 15~50°C, more preferably 20~40°C, and most preferably 25~35°C. The reaction time is preferably 2~7 days, more preferably 3~5 days, and most preferably 3 days. Among them, γ-benzylglutamic acid-N-intra-ring carboxylic anhydride can be prepared according to conventional methods.

[0069] In the present invention, the compound shown in Formula 2 is a PEG with enzyme response characteristics, which can be prepared according to conventional methods known to those skilled in the art. For example, it can be prepared according to the following method: in an inert atmosphere, amino-terminal polyethylene glycol monomethyl ether (mPEG-NH2) and an amino-protected peptide segment are subjected to a dehydration condensation reaction at room temperature for 10 to 24 hours, and then the amino-protecting group is removed to obtain the compound shown in Formula 2. The average molecular weight of the mPEG-NH2 is 2000 to 10000.

[0070] For example, taking the peptide segment PLGLAG as an example, in some embodiments of the present invention, the compound represented by Formula 2 is prepared according to the following method:

[0071] [P] Add the amino-terminal polyethylene glycol monomethyl ether (mPEG-NH2) and the amino-protected peptide PLGLAG to a round-bottom flask, add a solvent, preferably anhydrous N,N-dimethylformamide (DMF) to dissolve, then add 4-dimethylaminopyridine (DMAP), add N,N'-diisopropylcarbodiimide (DIC), and stir at room temperature under nitrogen protection for 10-24 hours, preferably 12-15 hours. After the reaction is completed, precipitate into ice ether, filter to obtain a solid, dissolve the solid with DMF and precipitate into ice ether, filter to obtain a solid, and vacuum dry the solid for 12-24 hours. After drying, anhydrous DMF was added to dissolve, piperidine was added, and the reaction was stirred for 30-60 minutes to remove the protection of the amino group, and the mixture was precipitated into ice ether, and the solid was filtered out, dissolved in dichloromethane and precipitated into ice ether, and the solid was filtered out, and vacuum dried for 12-24 hours to obtain the compound shown in formula 2, referred to as PLGLAG-mPEG. [ / P][P]

[0072] In the present invention, the compound represented by formula 2 including the remaining peptide segments can be prepared by referring to the above method, and to avoid complexity, they will not be described one by one.

[0073] [P] In the present invention, the condensing agent is selected from dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, (3H-1,2,3-triazolo[4,5- b] pyridine-3-oxy) tri-1-pyrrolidinyl hexafluorophosphate or 1H-benzotriazole-1-yloxy tripyrrolidinyl phosphonium hexafluorophosphate, preferably 1H-benzotriazole-1-yloxy tripyrrolidinyl phosphonium hexafluorophosphate; the catalyst is selected from any one or more of 4-pyrrolidinyl pyridine, 4-N,N-dimethylpyridine or 1-hydroxybenzotriazole, preferably 4-pyrrolidinyl pyridine. In the present invention, the molar ratio of the compound represented by Formula 2 to Formula 1 is (1-15):1, preferably 10:1. The molar ratio of the catalyst to the compound represented by Formula 1 is (2-50):1, preferably 30:1; the molar ratio of the condensing agent to the compound represented by Formula 1 is (10-50):1, preferably 20:1. The temperature of the mixed reaction is 15-30°C, preferably 20-25°C; the time of the mixed reaction is 10-30h, preferably 12-24h. [ / P][P]In some embodiments of the present invention, the mixture

[0074] In some embodiments of the present invention, after the mixed reaction is completed, it is preferred that the following steps are further included: precipitating the product in icy ether, filtering to obtain a solid, dissolving the solid with a solvent, preferably anhydrous DMF, and then precipitating the solid in icy ether, filtering to obtain a solid, and vacuum drying to obtain a final product.

[0075] In the present invention, the responsive polyethylene glycol-removing nano drug carrier shown in the above formula II has different preparation methods due to the difference in R2.

[0076] In some embodiments of the present invention, the compounds represented by formulas A-1 to A-5 are PEGs having pH responsive properties. For example, taking R2 as the structure represented by formula A-1 as an example, the corresponding PEGs having pH responsive properties can be prepared according to the following method:

[0077] 2,2,2-trifluoro-N-(2-methoxy-[1,3]-dioxolane-4-methylene)acetamide and polyethylene glycol monomethyl ether are added to a round-bottom flask, toluene is added to dissolve, p-toluenesulfonic acid and pyridine are added, refluxed and stirred at 110°C overnight, ether is used for precipitation, and solids are obtained by suction filtration. The solid product is added to tetrahydrofuran for dissolution, sodium hydroxide solution is added, and the reaction is stirred at room temperature for 12 hours. The tetrahydrofuran is removed by rotary evaporation, and the remaining solution is extracted with dichloromethane for 3 times, and the dichloromethane is dried with anhydrous magnesium sulfate, filtered, and then precipitated with diethyl ether to obtain a solid, which is dried to obtain PEG with pH response characteristics, referred to as pH-mPEG. Wherein, the average molecular weight of the polyethylene glycol monomethyl ether is 2000-10000.

[0078] In the present invention, for R2 having a structure represented by Formula A-2 to Formula A-5, the preparation method of the corresponding PEG having pH responsive properties can be carried out by referring to the method well known to those skilled in the art, and will not be described in detail to avoid complexity.

[0079] The polyamino acid nano drug carrier for responsive removal of polyethylene glycol shown in Formula I or Formula II provided by the present invention has only polyglutamic acid on its main chain, and the PEG structure with responsive removal effect can be modified to the polymer side chain without adding additional amino acid units. In addition, in the overall preparation process, the modification steps of PEG are simplified and the synthetic route is convenient.

[0080] The present invention also provides an application of the polyamino acid nano drug carrier for responsive removal of polyethylene glycol shown in the above formula I or formula II in the preparation of anti-tumor nano drugs.

[0081] The present invention also provides an anti-tumor nano drug, which can responsively remove polyethylene glycol, and is composed of an active drug component and a carrier. The carrier is the nano drug carrier involved in the above technical solution; the active drug component includes any one of cisplatin, irinotecan or Compretin A4. In the present invention, the molar ratio of glutamic acid unit to active drug component in the carrier is (1-6):1, preferably (2-3):1.

[0082] The polyamino acid nano drug carrier with responsive polyethylene glycol removal shown in formula I or formula II provided by the present invention can be used as a new nano drug delivery platform to load various active drug components through complexation, positive and negative electrical interactions, and covalent bonding, and has a large number of available drug-carrying groups. The drug loading capacity (measured by high-performance liquid chromatography or ICP-MS) is high, all above 20%. It can be seen that the polyamino acid nano drug carrier with responsive polyethylene glycol removal shown in formula I or formula II provided by the present invention has better universality.

[0083] For example, in the present invention, when the active drug component is cisplatin, the responsively polyethylene glycol-removing nanomedicine can be prepared according to the following method:

[0084] The carrier is mixed with the active drug component to react to form a drug; wherein the carrier is the drug carrier described in the above technical solution.

[0085] In the present invention, the above-mentioned mixed reaction is preferably carried out in an aqueous medium, and the mixed reaction is preferably carried out at a constant temperature of 37° C. for 60 to 80 hours, preferably 72 hours, and protected from light during the reaction.

[0086] In the present invention, after the mixed reaction is completed, it is preferred to further include: placing the obtained product into a dialysis bag and dialyzing it in pure water for 12 to 24 hours. After the dialysis is completed, freezing it at -20°C overnight and freeze drying it in a freeze dryer for 2 to 3 days. The molecular weight of the dialysis bag is 3500Da to 20000Da.

[0087] It should be noted that when the active drug component is irinotecan or Compretin A4, the preparation method thereof can be carried out according to methods well known to those skilled in the art, and will not be described in detail here to avoid complexity.

[0088] The nano drug with responsive removal of polyethylene glycol provided by the present invention uses the compound shown in formula I or formula II as a carrier. When it is loaded with active drug components and enters the body, it will passively target and accumulate in the tumor tissue by virtue of the long blood circulation effect of polyethylene glycol and the EPR effect (EPR effect refers to the characteristics of high permeability and retention of solid tumor tissue relative to normal tissue). The environment around the tumor tissue is acidic and has a high concentration of matrix metalloproteinases. After the drug enters the tumor tissue, the connection part between PEG and the glutamic acid unit responds to the pH or matrix metalloproteinase concentration changes in the microenvironment around the tumor tissue and breaks, so that the polyethylene glycol structure is automatically removed. The drug with polyethylene glycol removed will be more efficiently internalized by tumor cells, thereby releasing active drug components and effectively killing tumor cells.

[0089] In order to further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention are all common commercially available products.

[0090] Preparation Example 1 - Synthesis of γ-Benzylglutamic acid-N-intracyclic carboxylic anhydride (BLG-NCA)

[0091] Choose a three-necked flask of appropriate size to bake the bottle, and replace nitrogen three times to ensure anhydrous and oxygen-free conditions. Weigh 20g of γ-benzylglutamic acid (BLG), add and vacuum for 30min, add anhydrous tetrahydrofuran (THF), the reaction solvent, and stir in an oil bath at 58°C under nitrogen protection. Add 10g of triphosgene under protective conditions and react for 1.5h with nitrogen circulation. After the reaction is completed, turn on the nitrogen to evaporate the reaction solution to 50mL, slowly settle in 400mL of ice petroleum ether, and filter to obtain a solid. Dissolve it in ice ethyl acetate and pour it into a separatory funnel, wash it three times with ice water. Pour the upper liquid into a conical flask, add anhydrous magnesium sulfate, and dry it at -20°C for 12h. After drying, filter it with a sand core funnel to obtain the filtrate, and put the filtrate into an ampoule for vacuum drying to remove ethyl acetate to obtain a solid. Add 15 mL of anhydrous THF, heat at 40°C to dissolve completely, add 1 mL of anhydrous n-hexane each time until the precipitate cannot be dissolved back, then add 3 mL of anhydrous THF to dissolve the precipitate back. Cool down and crystallize, pour out the supernatant after crystallization is complete, and vacuum dry the remainder to obtain 14 g of product BLG-NCA with a yield of 70%.

[0092] Preparation Examples 2 to 4 - Preparation of Polyglutamic Acid with Different Contents

[0093] Preparation Example 2

[0094] Select an ampoule of suitable size for baking, and replace nitrogen three times to ensure anhydrous and oxygen-free conditions. Weigh 9.25g of BLG-NCA obtained in Preparation Example 1 and add it to the bottle, evacuate for 30 minutes, and add 1g of n-hexylamine / N,N-dimethylformamide (DMF) solution with a concentration of 0.02g / g in the glove box. Add 25mL of anhydrous DMF, a reaction solvent, and react at room temperature for 3 days, during which the carbon dioxide produced by the reaction in the bottle is removed. After the reaction is completed, slowly settle in 200mL of ice ether, filter to obtain a solid, wash the solid with ice ether twice, and vacuum dry. After drying, dissolve it in 70mL of 10 times the volume of trifluoroacetic acid, add 21mL of 33wt% hydrogen bromide acetic acid solution after complete dissolution, and stir the reaction at room temperature for 1.5h. After the reaction is completed, slowly settle in 800mL of ice ether, filter to obtain a solid, wash the solid with ether twice, and vacuum dry overnight. After drying, dissolve it with 200 mL of distilled water, and add 1 mol / L sodium hydroxide solution dropwise while stirring until it is completely dissolved, and the pH is 8. Then, add 1 mol / L hydrochloric acid solution dropwise while stirring until the product is completely precipitated, and the pH is 1. Filter to obtain a solid, wash the solid with pure water twice, freeze it at -20°C overnight, and put it into a freeze dryer for freeze drying for three days to obtain 6.2 g of the product polyglutamic acid, in which an average of 160 γ-benzylglutamic acids are polymerized on each n-hexylamine, and the yield is 67%.

[0095] Preparation Example 3

[0096] Select an ampoule of suitable size for baking, and replace nitrogen three times to ensure anhydrous and oxygen-free conditions. Weigh 5.78g of BLG-NCA obtained in Preparation Example 1 and add it to the bottle, evacuate for 30 minutes, and add 1g of n-hexylamine / N,N-dimethylformamide (DMF) solution with a concentration of 0.02g / g in the glove box. Add 25mL of anhydrous DMF, a reaction solvent, and react at room temperature for 3 days, during which the carbon dioxide produced by the reaction in the bottle is removed. After the reaction is completed, slowly settle in 200mL of ice ether, filter to obtain a solid, wash the solid with ice ether twice, and vacuum dry. After drying, dissolve it in 70mL of 10 times the volume of trifluoroacetic acid, add 21mL of 33wt% hydrogen bromide acetic acid solution after complete dissolution, and stir the reaction at room temperature for 1.5h. After the reaction is completed, slowly settle in 800mL of ice ether, filter to obtain a solid, wash the solid with ether twice, and vacuum dry overnight. After drying, dissolve it with 200 mL of distilled water, and add 1 mol / L sodium hydroxide solution dropwise while stirring until it is completely dissolved, and the pH is 8. Then, add 1 mol / L hydrochloric acid solution dropwise while stirring until the product is completely precipitated, and the pH is 1. Filter to obtain a solid, wash the solid with pure water twice, freeze it at -20°C overnight, and put it into a freeze dryer for freeze drying for three days to obtain 3.8 g of the product polyglutamic acid, with an average of 100 γ-benzylglutamic acids polymerized on each n-hexylamine, and a yield of 65.7%.

[0097] Preparation Example 4

[0098] Select an ampoule of suitable size for baking, and replace nitrogen three times to ensure anhydrous and oxygen-free conditions. Weigh 11.56g of BLG-NCA obtained in Preparation Example 1 and add it to the bottle, evacuate for 30 minutes, and add 1g of n-hexylamine / N,N-dimethylformamide (DMF) solution with a concentration of 0.02g / g in the glove box. Add 25mL of anhydrous DMF, a reaction solvent, and react at room temperature for 3 days, during which the carbon dioxide produced by the reaction in the bottle is removed. After the reaction is completed, slowly settle in 200mL of ice ether, filter to obtain a solid, wash the solid with ice ether twice, and vacuum dry. After drying, dissolve it in 70mL of trifluoroacetic acid with a volume of 10 times, add 21mL of 33wt% hydrogen bromide acetic acid solution after complete dissolution, and stir the reaction at room temperature for 1.5h. After the reaction is completed, slowly settle in 800mL of ice ether, filter to obtain a solid, wash the solid with ether twice, and vacuum dry overnight. After drying, dissolve it with 200 mL of distilled water, and add 1 mol / L sodium hydroxide solution dropwise while stirring until it is completely dissolved, and the pH is 9. Then add 1 mol / L hydrochloric acid solution dropwise while stirring until the product is completely precipitated, and the pH is 2. Filter to obtain a solid, wash the solid with pure water twice, freeze it at -20°C overnight, and put it into a freeze dryer for freeze drying for three days to obtain 7.9 g of the product polyglutamic acid, with an average of 200 γ-benzylglutamic acids polymerized on each n-hexylamine, and a yield of 68.3%.

[0099] Preparation Examples 5-6 - Preparation of mPEG-PLGLAG compounds with different molecular weights

[0100] Preparation Example 5

[0101] [P] Add 2.5 g of amino-terminated polyethylene glycol monomethyl ether (mPEG-NH2) with an average molecular weight of 5000 and 0.56 g of amino-protected peptide PLGLAG to a round-bottom flask, add 30 mL of anhydrous DMF to dissolve, then add 0.05 g of 4-dimethylaminopyridine (DMAP), add 0.12 g of N,N'-diisopropylcarbodiimide (DIC), and stir at room temperature for 12 hours under nitrogen protection. After the reaction, precipitate into ice ether, filter to obtain a solid, dissolve the solid in DMF and then precipitate into ice ether, filter to obtain a solid, and vacuum dry the solid for 12 hours. After drying, add 20 mL of anhydrous DMF to dissolve, add 5 mL of piperidine and stir to react for 30 minutes to remove the protection of the amino group, precipitate into ice ether, filter to obtain a solid, dissolve it in dichloromethane and then precipitate it into ice ether, filter to obtain a solid, and vacuum dry it for 12 hours to obtain PLGLAG-mPEG, the structure is as follows Figure 1 As shown, spare.

[0102] Preparation Example 6

[0103] The only difference from Preparation Example 5 is that the average molecular weight of polyethylene glycol monomethyl ether (mPEG-NH2) is 2000, and the other parameters and steps are consistent with Preparation Example 5.

[0104] Preparation Examples 7 to 9 - Preparation of Different pH Responsive Polyethylene Glycol Removal

[0105] Preparation Example 7

[0106] Take 1 g of 2,2,2-trifluoro-N-(2-methoxy-[1,3]-dioxolane-4-methylene)acetamide and 5.5 g of polyethylene glycol monomethyl ether with an average molecular weight of 5000 and add them to a round-bottom flask, add 20 mL of toluene to dissolve, then add 0.01 g of p-toluenesulfonic acid and 0.05 mL of pyridine, reflux and stir at 110°C to react overnight, precipitate with ether, filter to obtain a solid, add 50 mL of tetrahydrofuran to dissolve the solid product, add 50 mL of 2M sodium hydroxide solution, stir and react at room temperature for 12 hours, remove tetrahydrofuran by rotary evaporation, extract the remaining solution with dichloromethane three times, dry the dichloromethane with anhydrous magnesium sulfate, filter and precipitate with ether to obtain a solid, and obtain pH-responsive polyethylene glycol after drying. The structure is as follows Figure 2 As shown, spare.

[0107] Preparation Example 8

[0108] The only difference from Preparation Example 7 is that the average molecular weight of polyethylene glycol monomethyl ether (mPEG) is 2000, and the other parameters and steps are consistent with Preparation Example 8.

[0109] Preparation Example 9

[0110] 5g of polyethylene glycol monomethyl ether with an average molecular weight of 5000 and 0.14g of acryloyl chloride were reacted in 40mL of anhydrous dichloromethane solvent, 0.05g of triethylamine was added, and the reaction was carried out for 12h. After the reaction was completed, it was precipitated into ice ether, and the dichloromethane was redissolved and precipitated into ice ether again, and polyethylene glycol-acrylate was obtained by suction filtration. 4g of polyethylene glycol-acrylate was dissolved in 40mL of phosphate buffer solution at pH 8.0, 0.11g of 3-mercapto-1-propanol was added, and the reaction was stirred at room temperature for 6h. After the reaction was completed, it was loaded into a dialysis bag with a molecular weight cutoff of 2000Da and dialyzed with pure water at pH 8.0 for 24h. After freezing, it was placed in a freeze dryer and freeze-dried for 3 days to obtain polyethylene glycol with pH response.

[0111] Examples 1 to 6 - Preparation of enzyme-responsive polyethylene glycol-modified polyglutamic acid

[0112] Example 1

[0113] Weigh 4g of PLG obtained in Preparation Example 2 and add it to a round-bottom flask, add 10g of PLGLAG-mPEG obtained in Preparation Example 5, add 100mL of solvent anhydrous DMF to dissolve, then add 2.1g of 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, add 0.98g of 4-pyrrolidinopyridine, and react for 24h. After the reaction is completed, precipitate in ice ether, filter to obtain a solid, dissolve the solid in anhydrous DMF and precipitate in ice ether, filter to obtain a solid, and vacuum dry to obtain the final product, that is, enzyme-responsive polyethylene glycol-modified polyglutamic acid, with a structure such as Figure 3 As shown, on average, each polyglutamic acid is connected to ten responsive polyethylene glycols, that is, x=10, y=150.

[0114] Example 2

[0115] Compared with Example 1, the difference is that 4 g of PLGLAG-mPEG obtained in Preparation Example 6 is weighed, the amount of DMF used is 60 mL, and the other parameters and steps are consistent with Example 1.

[0116] Example 3

[0117] Weigh 4g of PLG obtained in Preparation Example 3 and add it to a round-bottom flask, add 9g of PLGLAG-mPEG obtained in Preparation Example 5, add 100mL of anhydrous DMF solvent to dissolve, then add 1.9g of 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, add 0.88g of 4-pyrrolidinopyridine, and react for 24h. After the reaction is completed, precipitate in ice ether, filter to obtain a solid, dissolve the solid in anhydrous DMF and precipitate in ice ether, filter to obtain a solid, and vacuum dry to obtain the final product, i.e., enzyme-responsive polyethylene glycol-modified polyglutamic acid, with an average of 6 responsive polyethylene glycols attached to each polyglutamic acid, i.e., x=6, y=94.

[0118] Example 4

[0119] Compared with Example 3, the difference is that 3.6 g of PLGLAG-mPEG obtained in Preparation Example 6 is weighed, the amount of DMF used is 60 mL, and the other parameters and steps are consistent with Example 3.

[0120] Example 5

[0121] Weigh 4g of PLG obtained in Preparation Example 4 and add it to a round-bottom flask, add 11g of PLGLAG-mPEG obtained in Preparation Example 5, add 100mL of anhydrous DMF solvent to dissolve, then add 2.3g of 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, add 1.1g of 4-pyrrolidinopyridine, and react for 24h. After the reaction is completed, precipitate in ice ether, filter to obtain a solid, dissolve the solid in anhydrous DMF and precipitate in ice ether, filter to obtain a solid, and vacuum dry to obtain the final product, i.e., enzyme-responsive polyethylene glycol-modified polyglutamic acid, with an average of 15 responsive polyethylene glycols attached to each polyglutamic acid, i.e., x=15, y=185.

[0122] Example 6

[0123] Compared with Example 5, the difference is that 4.5 g of PLGLAG-mPEG obtained in Preparation Example 6 is weighed, the amount of DMF used is 60 mL, and the other parameters and steps are consistent with Example 5.

[0124] Example 7-16 - Preparation of pH-responsive polyethylene glycol-modified polyglutamic acid

[0125] Example 7

[0126] Weigh 4 g of PLG obtained in Preparation Example 2 and add it to a round-bottom flask, add 10 g of pH-responsive polyethylene glycol obtained in Preparation Example 7, add 100 mL of anhydrous DMF to dissolve, then add 2.1 g of 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, add 0.98 g of 4-pyrrolidinopyridine, and react for 24 hours. After the reaction is completed, precipitate in 7 volumes of ether, filter to obtain a solid, wash the solid with ether twice, and vacuum dry to obtain the final product, i.e., pH-responsive polyethylene glycol-modified polyglutamic acid (PLG-pH-PEG), with the structure shown in Figure 4 As shown, on average, each polyglutamic acid is connected to ten responsive polyethylene glycols, that is, x=10, y=150.

[0127] Example 8

[0128] Compared with Example 7, the difference is that 4 g of the pH-responsive polyethylene glycol obtained in Preparation Example 8 is weighed, the amount of DMF used is 60 mL, and the other parameters and steps are consistent with Example 7.

[0129] Example 9

[0130] Select a round-bottom flask of appropriate size for baking, replace nitrogen three times, and anhydrous conditions. Weigh 4g of PLG obtained in Preparation Example 2 and add it to the flask, add 100mL of anhydrous DMF solvent, and stir to dissolve completely. Add 0.24g of 4-dimethylaminopyridine (DMAP), 0.5g of N,N'-diisopropylcarbodiimide (DIC), and 10g of pH-responsive polyethylene glycol obtained in Preparation Example 9, and react at room temperature for 24h under nitrogen protection. After the reaction is completed, it is precipitated into ice ether, and the solid is filtered out. The solid is re-dissolved in anhydrous DMF and then precipitated into ice ether. The solid is filtered out and vacuum dried for 12h to obtain the product pH-responsive polyethylene glycol-modified polyglutamic acid. On average, each polyglutamic acid is connected to 10 responsive polyethylene glycols, that is, x=10, y=150.

[0131] Example 10

[0132] 4 g of PLG obtained in Preparation Example 3 was weighed and added to a round-bottom flask, 9 g of the pH-responsive polyethylene glycol obtained in Preparation Example 7 was added, and the solvent anhydrous DMF was added to dissolve, and then 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate was added, and 4-pyrrolidinopyridine was added to react for 24 hours. After the reaction was completed, it was precipitated in 7 volumes of ether, and the solid was filtered out. The solid was washed twice with ether and vacuum dried to obtain the final product, i.e., pH-responsive polyethylene glycol-modified polyglutamic acid (PLG-pH-PEG), with an average of 5 responsive polyethylene glycols attached to each polyglutamic acid, i.e., x=6, y=94.

[0133] Embodiment 11

[0134] Compared with Example 10, the difference is that 3.6 g of the pH-responsive polyethylene glycol obtained in Preparation Example 8 is weighed, the amount of DMF used is 60 mL, and the other parameters and steps are consistent with Example 10.

[0135] Example 12

[0136] Weigh 4g of PLG obtained in Preparation Example 4 and add it to a round-bottom flask, add 15g of pH-responsive polyethylene glycol obtained in Preparation Example 7, add anhydrous DMF solvent to dissolve, then add 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate, add 4-pyrrolidinopyridine, and react for 24h. After the reaction is completed, precipitate in 7 volumes of ether, filter to obtain a solid, wash the solid twice with ether, and vacuum dry to obtain the final product, i.e., pH-responsive polyethylene glycol-modified polyglutamic acid (PLG-pH-PEG), with an average of 15 responsive polyethylene glycols attached to each polyglutamic acid, i.e., x=15, y=185.

[0137] Embodiment 13

[0138] Compared with Example 12, the difference is that 4.5 g of the pH-responsive polyethylene glycol obtained in Preparation Example 8 is weighed, the amount of DMF used is 60 mL, and the other parameters and steps are consistent with Example 12.

[0139] Embodiment 14

[0140] Weigh 6g of PLG obtained in Preparation Example 2 and add 50mL of anhydrous DMF solvent, stir and dissolve completely, add 0.6g of N-(tert-butyloxycarbonyl)ethanolamine, 0.14g of 4-dimethylaminopyridine (DMAP), and 0.57g of N,N'-diisopropylcarbodiimide (DIC), stir and react at room temperature for 8h, and settle in ether after the reaction. Filter to remove ether, re-dissolve the solid in DMF and then settle, filter to obtain solid, and vacuum dry for 12h to obtain solid product. Take 4g of the product in the previous step and add 40mL of trifluoroacetic acid to dissolve, stir and react at 30℃ for 1h, settle in ether after the reaction, re-dissolve the solid in DMF, and then put it into a 3500Da dialysis bag, dialyze with pure water for 2 days, and freeze-dry in a freeze dryer to obtain the product PLG-ethanolamine.

[0141] Weigh 2.7g of 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid and add 60mL of anhydrous dichloromethane to dissolve, slowly add 3.7g of oxalyl chloride, add 0.4mL of DMF, stir and react at room temperature for 8h, then drain, add 10g of polyethylene glycol monomethyl ether with an average molecular weight of 5000, add 60mL of anhydrous dichloromethane to dissolve, add 0.3mL of pyridine and stir and react at room temperature for 4h, add 60mL of saturated ammonium chloride solution to quench the reaction, separate the liquids, add anhydrous magnesium sulfate to the organic phase and dry it for 12h, filter to remove anhydrous magnesium sulfate, precipitate in ether, and dry the solid in vacuum for 12h to obtain the product 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid-PEG.

[0142] Weigh 0.5 g of PLG-ethanolamine and 1.2 g of 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid-PEG, add 20 mL of anhydrous DMF to dissolve, add 0.1 mL of triethylamine, stir and react at room temperature for 12 h, and after the reaction, precipitate in ether, filter to obtain a solid, wash the solid twice with ether, and vacuum dry to obtain the final product, i.e., pH-responsive polyethylene glycol-modified polyglutamic acid (PLG-pH-PEG), with the structure shown below: Figure 5 As shown, on average, each polyglutamic acid is connected to 10 responsive polyethylene glycols, that is, x=10, y=150.

[0143] Embodiment 15

[0144] Weigh 10g of amino-terminated polyethylene glycol monomethyl ether with an average molecular weight of 5000, add 80mL of anhydrous DMF to dissolve, add 4.8g of p-hydroxybenzaldehyde, add 0.8mL of triethylamine, stir and react at 45°C for 12h, and settle in ether after the reaction. Wash both sides of the solid with ether, and vacuum dry for 12h to obtain the product PEG-p-hydroxybenzaldehyde. Weigh 3g of PLG obtained in Preparation Example 2, add 60mL of anhydrous DMF to dissolve completely, add 7.2g of PEG-p-hydroxybenzaldehyde, 0.5g of 4-dimethylaminopyridine (DMAP), and 0.4g of N,N'-diisopropylcarbodiimide (DIC), stir and react at room temperature for 24h, and settle in ether after the reaction. Filter and obtain the solid, wash the solid twice with ether, and vacuum dry to obtain the final product, i.e., pH-responsive polyethylene glycol-modified polyglutamic acid (PLG-pH-PEG), with the structure as shown below: Figure 6 As shown, on average, each polyglutamic acid is connected to 10 responsive polyethylene glycols, that is, x=10, y=150.

[0145] Example 16

[0146] Weigh 5g of PLG obtained in Preparation Example 2, add 50mL of anhydrous DMF solvent, stir and dissolve completely, add 0.38g of tert-butyl carbazate, add 0.48g of 1-hydroxybenzotriazole (HOBT) and 0.9g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), stir and react at room temperature for 24h, and settle in ether after the reaction. Filter to obtain a solid, re-dissolve the solid in anhydrous DMF and then settle in ether, filter to obtain a solid, and vacuum dry for 12h. Take the dried solid, add trifluoroacetic acid, stir and react at room temperature for 1h, settle in ether after the reaction, filter to obtain a solid, and wash twice with ether. After the solid is dissolved in DMF, put it into a 3500Da dialysis bag, dialyze with pure water for 2 days, and put it into a freeze dryer to freeze-dry to obtain the product polyglutamic acid-hydrazide. Weigh 3g of polyglutamic acid-hydrazide, add 70mL of anhydrous DMF, stir and dissolve, add 6.9g of propionaldehyde-polyethylene glycol monomethyl ether with an average molecular weight of 5000, add 0.7mL of triethylamine, and stir and react at room temperature for 24h. After the reaction is completed, precipitate into ether, filter to obtain a solid, dissolve the solid in anhydrous DMF, precipitate into ether, filter to obtain a solid, and vacuum dry for 12h to obtain the final product, i.e., pH-responsive polyethylene glycol-modified polyglutamic acid (PLG-pH-PEG), with an average of 10 responsive polyethylene glycols attached to each polyglutamic acid, i.e., x=10, y=150.

[0147] Application Examples 1 to 8 - Drugs Removing Polyethylene Glycol by Enzyme Response

[0148] Application Example 1

[0149] Weigh 1 g of the enzyme-responsive polyethylene glycol-modified polyglutamic acid obtained in Example 1 and 0.5 g of CDDP, dissolve in pure water, place at 37°C for constant temperature shaking reaction for 72 hours, and protect from light during the reaction. After the reaction, put it into a 3500Da dialysis bag and dialyze it in pure water for 24 hours. After the dialysis, freeze it at -20°C overnight and freeze-dry it in a freeze dryer for 3 days to obtain an enzyme-responsive drug for removing polyethylene glycol loaded with cisplatin.

[0150] Application Examples 2~6

[0151] Weigh 1 g of enzyme-responsive polyethylene glycol-modified polyglutamic acid and CDDP (1.1 g, 0.6 g, 0.9 g, 0.5 g, 1.0 g, respectively) obtained in Examples 2 to 6, dissolve in pure water, place at 37 ° C for constant temperature shaking reaction for 72 hours, and protect from light during the reaction. After the reaction is completed, put it into a 3500Da dialysis bag and dialyze it in pure water for 24 hours. After the dialysis is completed, put it in a freezer at -20 ° C overnight, freeze-dry it in a freeze dryer for 3 days, and obtain an enzyme-responsive polyethylene glycol-removed nanodrug loaded with cisplatin.

[0152] Application Example 7

[0153] Take 1g of the enzyme-responsive polyethylene glycol-modified polyglutamic acid obtained in Example 1 and 1.2g of irinotecan glycine ester, dissolve in 20mL of anhydrous N, N-dimethylformamide, add 1.14g of 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate and 0.37g of 4-pyrrolidinopyridine, and stir the reaction at room temperature for 24h. After the reaction is completed, put it into a 3500Da dialysis bag and dialyze it with pure water for 24h. After the dialysis is completed, put it into a freeze dryer and freeze-dry it to obtain an enzyme-responsive nanodrug loaded with irinotecan and remove polyethylene glycol.

[0154] Application Example 8

[0155] Take 1g of the enzyme-responsive polyethylene glycol-modified polyglutamic acid obtained in Example 1 and 0.5g of Compruetin A4 and dissolve them in 20mL of anhydrous N,N-dimethylformamide, then add 0.4g of 2,4,6-trichlorobenzoyl chloride, 0.2g of 4-dimethylaminopyridine and 0.17g of triethylamine. After stirring and reacting at room temperature for 24h, put it into a 3500Da dialysis bag after the reaction is completed, and dialyze it with pure water for 24h. After the dialysis is completed, put it into a freeze dryer and freeze-dry it to obtain an enzyme-responsive nanomedicine loaded with Compruetin A4 that removes polyethylene glycol.

[0156] Application Examples 9-19-PH responsive removal of polyethylene glycol from drugs

[0157] Application Example 9

[0158] Weigh 1 g of the pH-responsive polyethylene glycol-modified polyglutamic acid obtained in Example 7 and 0.5 g of CDDP, dissolve in pure water, place at 37°C for constant temperature shaking reaction for 72 hours, and protect from light during the reaction. After the reaction, put it into a 3500Da dialysis bag and dialyze it in pure water for 24 hours. After the dialysis, freeze it at -20°C overnight and freeze-dry it in a freeze dryer for 3 days to obtain a pH-responsive drug loaded with cisplatin and depolyethylene glycol.

[0159] Application Examples 10~15

[0160] Weigh 1g of the pH-responsive polyethylene glycol-modified polyglutamic acid obtained in Examples 8 to 13 (1.1g, 0.5g, 0.6g, 0.9g, 0.5g, 1.0g, respectively) and dissolve in pure water, place at 37°C for constant temperature shaking reaction for 72h, and protect from light during the reaction. After the reaction is completed, put it into a 3500Da dialysis bag and dialyze it in pure water for 24h. After the dialysis is completed, put it in a freezer at -20°C overnight, freeze-dry it in a freeze dryer for 3 days, and obtain a pH-responsive cisplatin-loaded nanomedicine with polyethylene glycol removed.

[0161] Application Example 16

[0162] Take 1g of the pH-responsive polyethylene glycol-modified polyglutamic acid obtained in Example 7 and 1.2g of irinotecan glycine ester, dissolve in 20mL of anhydrous N,N-dimethylformamide, add 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate 1.14g and 4-pyrrolidinopyridine 0.37g, and stir the reaction at room temperature for 24h. After the reaction is completed, put it into a 3500Da dialysis bag and dialyze it with water for 24h. After the dialysis is completed, put it into a freeze dryer and freeze-dry it to obtain a pH-responsive nanodrug loaded with irinotecan and removed polyethylene glycol.

[0163] Application Example 17

[0164] Take 1g of the pH-responsive polyethylene glycol-modified polyglutamic acid obtained in Example 7 and 0.5g of Compruetin A4 and dissolve them in 20mL of anhydrous N,N-dimethylformamide, then add 0.4g of 2,4,6-trichlorobenzoyl chloride, 0.2g of 4-dimethylaminopyridine and 0.17g of triethylamine. After stirring and reacting at room temperature for 24h, put it into a 3500Da dialysis bag after the reaction is completed, and dialyze it with water for 24h. After the dialysis is completed, put it into a freeze dryer and freeze-dry it to obtain a pH-responsive nanodrug loaded with Compruetin A4 and depolyethylene glycol.

[0165] Drug loading test

[0166] The test method is as follows:

[0167] The platinum content of the responsively depolyethylene glycol-removed nanodrug loaded with cisplatin is measured by plasma mass spectrometry and then converted into the cisplatin content. The drug loading amount of the nanodrug is obtained by the drug loading formula: (mass of cisplatin in the nanodrug / mass of the nanodrug) × 100%.

[0168] The responsive depolyethylene glycol nanodrug loaded with irinotecan was added to a 0.1 mol / L sodium hydroxide solution and shaken at 37°C for 2 hours to hydrolyze irinotecan from the nanocarrier. The irinotecan content in the nanodrug was then measured by high performance liquid chromatography. The drug loading of the nanodrug was obtained by the drug loading formula: (mass of irinotecan in the nanodrug / mass of the nanodrug) × 100%. Chromatographic separation was performed using an Agilent 5TC-C18 (2) column (4.6 mm × 250 mm, 5 µm), maintained at 40°C, gradient elution, solvent A was 0.1% formic acid aqueous solution, solvent B was 0.1% formic acid aqueous solution and methanol (50 / 50, v / v), and the flow rate was 1 mL / min. The gradient elution program was set as follows: 0-2.0 min, 10%-70% B; 2.0-15.0 min, 70%-95% B; 15.0-16.0 min, 95% B; 16.0-16.1 min, 95%-10% B; 16.1-20.0 min, 10% B.

[0169] The responsive polyethylene glycol-removed nanodrug loaded with Compruetin A4 was added to a 0.1 mol / L sodium hydroxide solution and shaken at 37°C for 2 h to hydrolyze Compruetin A4 from the nanocarrier. The content of Compruetin A4 in the nanodrug was then measured by high performance liquid chromatography. The drug loading amount of the nanodrug was obtained by the drug loading formula: (mass of Compruetin A4 in the nanodrug / mass of the nanodrug) × 100%.

[0170] The test results are shown in Table 1:

[0171] Table 1

[0172] Performance Testing

[0173] PEG removal test

[0174] The enzyme-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 1 was dissolved in 25 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer solution, with a material concentration of 1 mg / mL, and then 2 µg / mL of matrix metalloproteinase 2 was added, the solution pH was 7.4, and the mixture was shaken at 37°C. 1.0 mL of the solution was sampled at 0.5 h, 1 h, 2 h, and 4 h. The PEG content was then detected by high performance liquid chromatography. The test results are shown in Figure 2. Figure 7 shown.

[0175] Take the pH-responsive polyethylene glycol-modified polyglutamic acid prepared in Example 7 and dissolve it in deionized water, adjust the pH to 6.5 or 8.0 with hydrochloric acid or sodium hydroxide, and the material concentration is 1 mg / mL, and shake at 37°C. For pH 6.5, 1.0 mL of solution is sampled at 0.5 h, 1 h, 2 h, and 4 h, and for pH 8.0, 1.0 mL of solution is sampled at 2 h, 4 h, 8 h, and 12 h. Then, the PEG content is detected by high performance liquid chromatography. The test results are as follows: Figure 8 shown.

[0176] Particle size characterization

[0177] The enzyme-responsive depolyethylene glycol-removing drug loaded with cisplatin prepared in Example 1, the enzyme-responsive depolyethylene glycol-removing drug loaded with irinotecan prepared in Example 7, and the pH-responsive depolyethylene glycol-removing drug loaded with cisplatin prepared in Example 9 were subjected to dynamic light scattering test, and the results were as follows: Figures 9-11 As shown, it can be seen that the drug provided by the present invention is a nanoscale drug.

[0178] Simulated in vivo drug release testing

[0179] 2 mg of the enzyme-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 1 and 2 mg of the pH-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 9 were dissolved in 2 mL of PBS buffer solution with a pH of 5.5, respectively, and loaded into a 3500Da dialysis bag. Then, the dialysis bag was placed in 28.0 mL of PBS buffer with a pH of 5.5 and oscillated at 37°C. 2 mL of the solution was sampled at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h and 96 h, respectively, and 2 mL of new PBS buffer with a pH of 5.5 was added. The platinum content was determined by inductively coupled plasma mass spectrometry (ICP-MS), and the release of cisplatin was calculated. The test results are as follows: Fig.12 shown.

[0180] The enzyme-responsive depolyethylene glycol nanodrug loaded with irinotecan prepared in Application Example 7 was added to rat plasma at a concentration of 200 µg / mL and shaken at 37°C. 100 µL of plasma samples were taken at 1, 2, 4, 8, 12, 24, 36, 48, and 72 hours respectively. The amount of released irinotecan was detected by high performance liquid chromatography. The test results are shown in Figure 2. Fig.13 shown.

[0181] Cytotoxicity assay

[0182] CT26 cells were inoculated in 180 μL of 1640 medium at a concentration of 7 × 10 3 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured for 12 hours to adhere to the wall. Then, 20 µL of different concentrations of the enzyme-responsive de-PEGylated drug loaded with cisplatin prepared in Application Example 1, the pH-responsive de-PEGylated drug loaded with irinotecan prepared in Application Example 7, and the pH-responsive de-PEGylated drug loaded with cisplatin prepared in Application Example 9 were added to each well. After another 48 hours of culture, the cell viability was analyzed using MTT, and the cell viability was measured using a Bio-Rad 680 microplate reader at a wavelength of 492 nm.

[0183] Among them, the cytotoxicity test results of the enzyme-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 1 are as follows: Fig.14 As shown, the cytotoxicity test results of the pH-responsive depolyethylene glycol drug loaded with cisplatin prepared in Application Example 9 are as follows Fig.15 The cytotoxicity test results of the pH-responsive depolyethylene glycol-removed drug loaded with irinotecan prepared in Application Example 7 are shown in Fig.16 shown.

[0184] Anti-tumor effect test

[0185] CT26 Mouse Colon Cancer:

[0186] A total of 30 female BALB / c mice aged 6-8 weeks were selected as experimental animals, with a body weight of 18-20 g. The experimental groups were: blank group, cisplatin-loaded enzyme-responsive depolyethylene glycol drug (tested with the drug obtained in Application Example 1) group, and cisplatin-loaded pH-responsive depolyethylene glycol drug (tested with the drug obtained in Application Example 9) group.

[0187] Mice were subcutaneously inoculated with 1×10 6 CT26 cells were used to establish a CT26 subcutaneous tumor model. When the tumor volume reached 100 mm 3 The mice were then randomly divided into three groups, with 10 mice in each group. The blank group was injected with normal saline via the tail vein on days 0, 3, 6, and 9, 200 μL each time. The two drug groups were injected with 6 mg / kg (in terms of cisplatin) via the tail vein four times on days 0, 3, 6, and 9, 200 μL each time. The tumor volume was recorded every other day. The test results are shown in Figure 2. Fig.17 shown.

[0188] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polyamino acid nano drug carrier for responsive removal of polyethylene glycol, characterized in that: It has the structure shown in Formula I or Formula II: Formula I; Formula II; Wherein, in formula I, 40≤n1≤230, 1≤x1≤15, 100≤y1≤200; In formula II, 40≤n2≤230, 1≤x2≤15, 100≤y2≤200; R1 is a peptide segment responsive to matrix metalloproteinase 2, selected from any one of PLGLAG, GPLGBRG, GPLGBRGK, GPLGVRGC, GPLGBRGCA, PLGVR, PLGGBRG, GPLGBRGGG, CGPLGGBRGK, PRCGV, SGAVRWLLTA, and GPPGVVGEKGEQ; or R1 is a peptide segment responsive to matrix metalloproteinase 9, selected from any one of SGKGPRQITA, GGKGPLGLPG, GPLGMWSRC, LGRMGLPG, LGPRSLSGK, SGAVRWLLTA, PLGLRSW, and PLGVRGK; R2 is selected from any one of the following formulas A-1 to A-5: Formula A-1; Formula A-2; Formula A-3; Formula A-4; Formula A-5.

2. The polyamino acid nano drug carrier for responsive removal of polyethylene glycol according to claim 1, characterized in that: 50≤n1≤150, 3≤x1≤10, 120≤y1≤160; 50≤n2≤150, 3≤x2≤10, 120≤y2≤160.

3. The polyamino acid nano drug carrier for responsive removal of polyethylene glycol according to claim 1 or 2, characterized in that: The nano drug carrier for responsive removal of polyethylene glycol shown in Formula II is selected from any one of the following Formulas II-1 to II-5: Formula II-1; Formula II-2; Formula II-3; Formula II-4; Formula II-5.

4. The polyamino acid nano drug carrier for responsive removal of polyethylene glycol according to any one of claims 1 to 3, characterized in that: The nano drug carrier for responsive removal of polyethylene glycol shown in formula I responsively removes polyethylene glycol under the action of matrix metalloproteinase 2 or matrix metalloproteinase 9.

5. The polyamino acid nano drug carrier for responsive removal of polyethylene glycol according to any one of claims 1 to 4, characterized in that: The polyamino acid nano drug carrier for responsive removal of polyethylene glycol shown in formula II responsively removes polyethylene glycol under the condition of pH < 6.

8.

6. Use of the polyamino acid nano-drug carrier capable of responsively removing polyethylene glycol according to any one of claims 1 to 5 in the preparation of anti-tumor nano-drugs.

7. A nano drug, characterized in that: It is composed of active drug components and carriers; The carrier is the nano drug carrier according to any one of claims 1 to 5.

8. The nano drug according to claim 7, characterized in that The active pharmaceutical ingredient includes any one of cisplatin, irinotecan or Compretin A4.

9. The nano drug according to claim 7 or 8, characterized in that: The molar ratio of the glutamic acid unit to the active drug component in the carrier is (1-6):

1.

10. The nanodrug according to any one of claims 7 to 9, characterized in that: The nanomedicine responsively removes PEG at the tumor.

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