Glutamic acid-N-carboxyl-anhydride monomer containing ethyl nitrate group, polymer of glutamic acid-N-carboxyl-anhydride monomer and application of glutamic acid-N-carboxyl-anhydride monomer as nitric oxide donor

Abstract

The invention relates to a glutamic acid-N-carboxyl-anhydride monomer containing an ethyl nitrate group, a polymer of the glutamic acid-N-carboxyl-anhydride monomer containing the ethyl nitrate group, and an application of the glutamic acid-N-carboxyl-anhydride monomer containing the ethyl nitrate group and the polymer of the glutamic acid-N-carboxyl-anhydride monomer containing the ethyl nitrate group as a nitric oxide donor. The polyethylene glycol block-containing copolymer can be independently polymerized or copolymerized with other amino acid-N-carboxyl-anhydride monomers to form an NO donor polymer, and the polyethylene glycol block-containing copolymer can form a nano structure through self-assembly so as to efficiently load a hydrophobic drug. The drug can be accurately delivered to tumor tissues or cardiovascular and cerebrovascular disease parts in a targeted manner, and the controllable release of NO and / or loaded drugs is realized, so that the effect of inhibiting tumors and cardiovascular and cerebrovascular diseases is achieved. Structural formula of glutamic acid-N-carboxyl-intracyclic anhydride monomer containing ethyl nitrate group is as shown in formula (I): # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of heterocyclic compounds, and particularly relates to a glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, its polymer, and their application as a nitric oxide donor. Background Art

[0002] Nitric oxide (NO) is a gaseous neurotransmitter widely present in the human body, which has functions such as regulating vasodilation, immune response, inflammation, neurotransmission and other important functions, and also plays multiple functions during tumor treatment. First, nitric oxide (NO) is one of the endothelium-derived relaxing factors, and the discovery of this factor won the Nobel Prize in Physiology, so its research also started in the field of cardiovascular and cerebrovascular diseases. Secondly, in tumor treatment, the concentration of NO has a decisive impact on the treatment effect: low-concentration NO can promote tumor growth (Nat. Rev. Cancer. 2006, 6: 521), while high-concentration NO shows anti-tumor effects through multiple mechanisms such as DNA / mitochondrial damage, enhanced apoptosis, inhibition of tumor metastasis and hypoxia, etc. (Adv. Therap. 2018, 1: 1800084). At the same time, studies have shown that NO can react with reactive oxygen species (ROS) to generate peroxynitrite anion (ONOO - ) and other reactive nitrogen species (RNS) (Nitric Oxide, 2013, 30: 26 - 35). Compared with ROS, RNS shows stronger biocidal activity because it can exacerbate the overall damage by initiating free radical peroxidation. However, due to the limited concentration of endogenous NO, it is difficult to meet the treatment requirements, and exogenous NO supplementation must be relied on to achieve this. Studies have shown that various types of small molecule compounds, including organic ethyl nitrate, nitrite, inorganic metal nitrosyl complexes, S-nitrosothiol (SNO), furanone and nitrobenzene, etc., can be used as NO donors. However, traditional small molecule drugs have problems such as poor water solubility, easy metabolism, and lack of targeting, and it is difficult to reach the high concentration required for treatment, and the treatment effect is often not good. Therefore, how to achieve the effective concentration of NO and accurately deliver it to the diseased tissue has important research significance.

[0003] It is reported in the literature that NO donors with a nitrate ester structure can effectively release NO in the presence of glutathione (GSH), so people have tried to introduce the nitrate ester structure into the polymer chain. For example, Hien T et al. (J. Polym. Sci., Polym.

[0004] Chem., 2014, 52: 2099 - 2103) first synthesized an amphiphilic block copolymer P(OEGMA)-b-P(VBC-co-ST) by reversible addition-fragmentation chain transfer polymerization, and then used silver nitrate (AgNO 3)Replace the chlorine side groups therein with nitrate groups to form a polymer with nitrate ester groups on the side chain, and self-assemble it into micelles with a size of 30 nm as a delivery carrier for NO. Deng et al. (Biomaterials, 2018, 187: 55-65) combined the NO donor S-nitrosothiol (SNO) with α-cyclodextrin (α-CD) to synthesize the glutathione (GSH)-sensitive NO carrier prodrug α-CD-NO. Then, the prodrug was loaded into the PEG-b-PMPC block copolymer through the host-guest interaction between α-CD and PEG, and self-assembled with the photosensitizer chlorin e6 (Ce6) to form the NO prodrug nanoparticles α-CD-Ce6-NO NPs. These nanoparticles can specifically release NO in the reducing environment of GSH, significantly relieve hypoxia in tumor tissues, and be used in combination with Ce6 for anti-tumor therapy. The results show that they have a significant synergistic effect.

[0005] However, the existing methods still face multiple challenges. First, for some loading methods of certain NO donors, such as physical encapsulation, host-guest interaction, etc., problems such as leakage, instability, and poor controllability are often encountered. Especially when combined with other drugs, more complex problems may be caused, limiting their practical applications. Second, in the common method of introducing nitrate ester groups, the general monomer is first polymerized to ensure that the polymer contains specific reactive groups such as carboxyl (-COOH), hydroxyl (-OH), and amino (-NH 2 ) etc. Then, the nitrate ester group is introduced through chemical reactions such as amidation or esterification. This method usually has low efficiency, complex processes, is prone to side reactions, and it is difficult to precisely control the content of functional groups. Therefore, even though the NO donors obtained by these methods show certain curative effects, they will face many difficulties in mass production and promotion. Moreover, the polymers currently used to bond or load nitrate esters are mainly polyester polymers, and there are few reports on polyamino acids. SUMMARY OF THE INVENTION

[0006] The technical problem to be solved by the present invention is to provide, in view of the deficiencies in the prior art, a glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, its polymer, and their application as a nitric oxide donor. The glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group can be used to prepare a polyamino acid containing a nitrate ester group through ring-opening polymerization. As a nitric oxide donor, it can respond to GSH and release NO, has high NO loading and release capabilities, and this polyamino acid has the ability to form stable nano-assemblies, can efficiently load drugs, and can release drugs in a GSH-responsive manner after reaching the lesion site. These characteristics make this polymer have broad application prospects in the field of drug delivery.

[0007] To achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0008] A glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, and its structural formula is shown as the following formula (Ⅰ):

[0009]

[0010] The present invention also includes a preparation method of the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, comprising the following steps:

[0011] 1) 2-Bromoethanol and silver nitrate are dissolved in solvent 1 for a substitution reaction, and after-treatment is carried out to obtain 2-hydroxyethyl nitrate;

[0012] 2) The 2-hydroxyethyl nitrate obtained in step 1), Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine (DMAP), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) are dissolved in solvent 2 for an esterification reaction, and after-treatment is carried out to obtain Boc-L-glutamic acid-1-tert-butyl ester-5-nitrooxyethyl ester;

[0013] 3) The Boc-L-glutamic acid-1-tert-butyl ester-5-nitrooxyethyl ester obtained in step 2) is subjected to a deprotection reaction with a hydrochloric acid 1,4-dioxane solution, and after-treatment is carried out to obtain L-glutamic acid-5-nitrooxyethyl ester;

[0014] 4) The L-glutamic acid-5-nitrooxyethyl ester obtained in step 3) is subjected to a ring-forming reaction with triphosgene in solvent 3, and after-treatment is carried out to obtain the glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group.

[0015] The reaction route is as shown below:

[0016]

[0017] According to the above solution, the molar ratio of 2-bromoethanol to silver nitrate in step 1) is 1:1 to 4.

[0018] According to the above solution, the solvent 1 in step 1) is acetonitrile, and the mass-volume ratio of 2-bromoethanol to solvent 1 is 1 g:12.5 to 25 mL.

[0019] According to the above solution, the substitution reaction conditions in step 1) are: reacting at 70 to 80 °C for 12 to 25 h under light-shielded conditions.

[0020] According to the above scheme, the post-treatment steps of step 1) include: adding saturated NaCl brine, filtering the precipitate, rotary evaporating to remove acetonitrile, extracting (the organic solvent used for extraction is dichloromethane, chloroform, ethyl acetate), adding a desiccant for drying (the reagent used for drying is anhydrous magnesium sulfate, anhydrous sodium sulfate, and the drying time is 4 - 24 h), suction filtering, and rotary evaporating to remove the solvent.

[0021] According to the above scheme, the molar ratio of 2-hydroxyethyl nitrate to Boc-L-glutamic acid 1-tert-butyl ester, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in step 2) is 1 - 1.2:1:0.1 - 0.2:1.8 - 2.5.

[0022] According to the above scheme, the solvent 2 in step 2) is dichloromethane, and the mass-volume ratio of 2-hydroxyethyl nitrate to solvent 2 is 1 g:30 - 50 mL.

[0023] According to the above scheme, the esterification reaction conditions in step 2) are: reacting at 5 - 40 °C for 20 - 30 h.

[0024] According to the above scheme, the post-treatment steps of step 2) include: washing the organic phase successively with water, hydrochloric acid solution (concentration 0.1 - 0.8 mol / L), and saturated NaHCO 3 solution, adding a desiccant for drying (the reagent used for drying is anhydrous magnesium sulfate, anhydrous sodium sulfate, and the drying time is 4 - 24 h), filtering, and rotary evaporating to remove the solvent.

[0025] According to the above scheme, the molar ratio of Boc-L-glutamic acid 1-tert-butyl ester-5-nitrooxyethyl ester to hydrochloric acid in the 1,4-dioxane hydrochloric acid solution in step 3) is 1:3 - 7.

[0026] According to the above scheme, the deprotection reaction conditions in step 3) are: reacting at 5 - 40 °C for 9 - 12 h.

[0027] According to the above scheme, the post-treatment steps of step 3) include: precipitating with anhydrous ether, suction filtering and then drying in vacuum (the drying temperature is 20 - 50 °C).

[0028] According to the above scheme, the molar ratio of L-glutamic acid 5-nitrooxyethyl ester to triphosgene in step 4) is 1:0.40 - 1.

[0029] According to the above scheme, the solvent 3 in step 4) is one of tetrahydrofuran, dichloromethane, ethyl acetate, N,N-dimethylformamide, preferably tetrahydrofuran, and the mass-volume ratio of L-glutamic acid 5-nitrooxyethyl ester to solvent 3 is 0.04 - 0.08 g / mL.

[0030] According to the above scheme, the conditions for the ring-forming reaction in step 4) are: under an inert atmosphere, reacting at 50-70 °C for 0.2-1 h.

[0031] According to the above scheme, the post-treatment steps after step 4) include: rotary evaporation and concentration (the temperature of rotary evaporation is 20-40 °C), sedimentation (the solvent used for sedimentation is n-hexane or petroleum ether), filtration, washing, adding a desiccant (the reagent used for drying is anhydrous magnesium sulfate or anhydrous sodium sulfate, and the drying time is 4-24 h) for drying, filtration, rotary evaporation, and finally vacuum drying.

[0032] The present invention also includes a biodegradable polymer obtained by polymerizing alone or copolymerizing the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group with other amino acid-N-carboxy-cyclic anhydride monomers, and its chemical structural formula is as follows:

[0033]

[0034] Among them, R is a polymerization initiator residue, L is a repeating unit corresponding to other amino acid-N-carboxy-cyclic anhydride monomers, m is the degree of polymerization of the glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, n is an integer greater than or equal to 0, and is the degree of polymerization of other amino acid-N-carboxy-cyclic anhydride monomers.

[0035] The molecular weight of the above biodegradable polymer is 2000-20000.

[0036] The present invention also provides a preparation method of the above biodegradable polymer: using a primary amine initiator to initiate the glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, or initiating a mixture of the monomer and other amino acid-N-carboxy-cyclic anhydride monomers, and carrying out ring-opening polymerization to obtain it.

[0037] The polymerization initiator is a compound or polymer containing a primary amine functional group, preferably one of methoxypolyethylene glycol amine (Mn = 2000-10000), n-hexylamine, and benzylamine.

[0038] The other amino acid-N-carboxy-cyclic anhydride monomer is one of L-glutamic acid-γ-benzyl ester-N-carboxy-anhydride, L-phenylalanine-N-carboxy-cyclic anhydride, N-carbobenzoxy-L-lysine-N-carboxy-cyclic anhydride, or methionine-N-carboxy-cyclic anhydride, and its structural formula is as follows:

[0039]

[0040] Specifically, the preparation method of the biodegradable polymer is as follows:

[0041] Using N,N-dimethylformamide (DMF) as a solvent, the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group is initiated by an initiator and undergoes ring-opening copolymerization under the protection of a nitrogen atmosphere at room temperature to obtain an amphiphilic block copolymer; or the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group and other amino acid-N-carboxy-cyclic anhydride monomers are initiated by an initiator and undergo ring-opening copolymerization under the protection of a nitrogen atmosphere at room temperature to obtain a random copolymer.

[0042] Preferably, the ring-opening copolymerization reaction time is 72 to 84 h.

[0043] The present invention also includes nanoparticles formed by self-assembly of the above biodegradable polymers.

[0044] Preferably, the method for preparing the nanoparticles adopts the oil-in-water (O / W) emulsion method, and the specific steps are as follows: First, dissolve the above biodegradable polymer in a solvent (such as toluene), and then add it to a buffer solution or pure water with an appropriate pH value under ultrasonic conditions to form polymer micelles, and finally remove the solvent by evaporation to obtain nanoparticles.

[0045] The above polymer micelles have high stability in PBS solution (pH = 7.4).

[0046] The polymer micelles obtained by the above technical scheme can encapsulate hydrophobic drugs through hydrophobic interaction, and the hydrophobic drugs are selected from, but not limited to: doxorubicin, paclitaxel, docetaxel, camptothecin, vincristine, chlorin e6 (Ce6), statins, etc.

[0047] The encapsulation method is to dissolve the above biodegradable polymer in a solvent (such as toluene), dissolve the hydrophobic drug in an appropriate solvent, mix the two, then add it to a buffer solution or pure water with an appropriate pH value under ultrasonic conditions, then remove the solvent by evaporation, and finally dialyze to remove other solvents and unencapsulated drugs to obtain polymer micelles encapsulating hydrophobic drugs. The polymer micelles obtained by the present invention can significantly improve the therapeutic effects of treating tumors and cardiovascular and cerebrovascular diseases as a drug delivery system.

[0048] The present invention includes the application of the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, its polymer, and self-assembled nanoparticles in the aspect of drug carriers.

[0049] And the application of the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, its polymer, and self-assembled nanoparticles as NO donors.

[0050] And the application of the above-mentioned glutamic acid-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group, its polymer, and self-assembled nanoparticles in the preparation of anti-tumor drugs and cardiovascular and cerebrovascular drugs.

[0051] The glutamate-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group in the present invention can be used to prepare a polyamino acid containing an ethyl nitrate group through a ring-opening polymerization reaction. As a nitric oxide donor, it can respond to GSH and release NO, having high NO loading and release capabilities. Moreover, this polyamino acid has the ability to form stable nanoassemblies, can efficiently load drugs, and can release drugs in a GSH-responsive manner after reaching the lesion site. These characteristics make this polymer have broad application prospects in the field of drug delivery.

[0052] The beneficial effects of the present invention are as follows:

[0053] 1. The present invention designs and synthesizes a glutamate-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group. This monomer contains a NO donor ethyl nitrate group unit. By using a small molecule compound or polymer with a primary amine at the end as an initiator for ring-opening polymerization, a variety of biodegradable functional polymers with high NO donor contents can be obtained. This polymer forms nano-micelles with high NO donor contents through self-assembly and can further efficiently load hydrophobic drugs. These nano-micelles can accurately target and deliver the NO donor and / or drug to tumor tissues or cardiovascular and cerebrovascular lesion sites and achieve effective and controllable release, thereby achieving the effect of inhibiting tumors or cardiovascular and cerebrovascular diseases.

[0054] 2. The raw materials used in the present invention to prepare the glutamate-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group include Boc-L-glutamic acid-1-tert-butyl ester, silver nitrate, 2-bromoethanol, and triphosgene. These raw materials are rich in sources, the synthesis conditions are mild, the reaction process is simple, the polymerization molecular weight is controllable, and the by-products are mainly gases, water, and a small amount of salts, which are easy to separate and purify. Therefore, it is convenient to realize large-scale production and popularization and application.

[0055] 3. The polymerization monomer of the biodegradable polymer in the present invention is an amino acid-N-carboxy-cyclic anhydride monomer. Such monomers can be polymerized at room temperature, have a very high polymerization efficiency, and the by-product is only carbon dioxide, which is easy to separate and purify. Therefore, it is convenient for application promotion and mass production.

[0056] 4. The glutamate-N-carboxy-cyclic anhydride monomer containing an ethyl nitrate group prepared in the present invention can be copolymerized with other amino acid-N-carboxy-cyclic anhydride monomers to introduce other functional groups such as -COOH, -NH 2 、-SH, endowing the polymerization product and the self-assembled nano-micelles with corresponding functions, realizing the synergistic effect between NO and drugs and other functions, and expanding their application scope in the biomedical field.

[0057] 5. The types of drugs that can be loaded by the biodegradable polymer prepared by the present invention are almost unlimited, as long as the drugs have a certain degree of hydrophobicity. Small molecule anti-cancer drugs such as paclitaxel or doxorubicin can be loaded, as well as small molecule anti-inflammatory drugs and cardiovascular drugs. Fluorescent and nuclear magnetic resonance reagents for imaging and diagnostic purposes can also be loaded, realizing multiple uses on one platform. The method for drug loading is a self-assembly process in PBS solution and pure water, which can be achieved through simple room temperature dissolution, room temperature ultrasonic evaporation, and freeze-drying processes commonly accepted in the pharmaceutical industry. The operation steps are simple, which is conducive to the promotion and application of the products of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the infrared spectrum of HLG-NCA prepared in Example 1 of the present invention;

[0059] Figure 2 is the 1H NMR spectrum of HLG-NCA prepared in Example 1;

[0060] Figure 3 is the 13C NMR spectrum of HLG-NCA prepared in Example 1;

[0061] Figure 4 is the 1H NMR spectrum of MPEG-PHLG prepared in Example 2 15 ;

[0062] Figure 5 is the 1H NMR spectrum of MPEG-P(HLG 15 -co-BLG 15 ) prepared in Example 2;

[0063] Figure 6 is the micelle size distribution diagram of the polymer micelle solution obtained in Example 3;

[0064] Figure 7 is the comparison diagram of the polymer micelle size in the polymer micelle solution obtained in Example 3 at different times;

[0065] Figure 8 is the comparison diagram of the NO release concentration of MPEG-PHLG 15 polymer micelles in the presence and absence of GSH in Example 3;

[0066] Figure 9 is the ultraviolet absorption spectrum diagram of the mixed solution of MPEG-PHLG 15 @Ce6 polymer micelles and DPBF irradiated by a 660 nm laser for different times in Example 3;

[0067] Figure 10 is MPEG-PHLG in Example 315 @Ce6 polymer micelle ultraviolet-visible absorption spectrograms of the supernatant of the experimental group and the control group before and after ultraviolet light irradiation in the in vitro peroxynitrite release experiment;

[0068] Figure 11 For MPEG-PHLG in Example 3 15 @Ce6 polymer micelle hemolysis experiment comparison chart of the hemolysis percentage of each sample in the experimental group;

[0069] Figure 12 For MPEG-PHLG in Example 3 15 @Ce6 polymer micelle intracellular ROS generation experiment inverted fluorescence microscope test charts of the control group and the experimental group;

[0070] Figure 13 For MPEG-PHLG in Example 3 15 @Ce6 polymer micelle intracellular NO generation experiment inverted fluorescence microscope test charts of the control group and the experimental group;

[0071] Figure 14 For MPEG-PHLG with different concentrations in Example 3 15 Polymer micelle solution cell viability comparison chart in L929 and 4T1 cell tests;

[0072] Figure 15 For MPEG-PHLG in Example 3 15 @Ce6 polymer micelle cytotoxicity experiment four-group cell viability comparison chart. Detailed implementation manners

[0073] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to examples and the accompanying drawings.

[0074] Example 1

[0075] A glutamic acid-N-carboxy-cyclic anhydride monomer (HLG-NCA) containing an ethyl nitrate group, and its synthesis route

[0076] is as follows:

[0077]

[0078] The specific preparation steps are as follows:

[0079] 1) Weigh 6.5 g of compound 1 (52 mmol) under light-shielded conditions, dissolve it in 100 mL of acetonitrile, and add 26 g of AgNO 3(0.153 mol), stirred and reacted for 24 hours under light avoidance at 75 °C. Then, the reactants were added to 50 mL of saturated brine, and the brine was titrated to check if there was still AgCl precipitation. After stirring for 1 hour, the precipitate was filtered through a sintered glass funnel. Acetonitrile was removed by rotary evaporation. Then, it was extracted 6 times with 25 mL of ethyl acetate, and the upper organic layer was collected. Anhydrous magnesium sulfate was added, and it was dried in the refrigerator freezer layer overnight. Magnesium sulfate was removed by suction filtration, and 5 g of product compound 2 was obtained by rotary evaporation, with a yield of 90%;

[0080] 2) Dissolve 3.9 g of compound 2 (36 mmol) prepared above and 10 g of compound 3 (33 mmol) in 150 mL of dichloromethane. Under ice bath conditions, 0.38 g of DMAP (3.1 mmol) and 12 g of EDCI (63 mmol) were added successively. After stirring for 5 minutes, the reaction system was heated to room temperature and stirred for another 24 h. After the reaction, the organic phase was washed twice with 30 mL of water, 30 mL of 0.1 mol / L hydrochloric acid solution, and 30 mL of saturated NaHCO 3 aqueous solution in sequence. The collected organic phase was dried with anhydrous magnesium sulfate for 4 h, filtered to collect the filtrate, and 10.69 g of yellow oily product compound 4 was obtained after rotary evaporation, with a yield of 82.5%;

[0081] 3) Dissolve 10.69 g of compound 4 (27.3 mmol) obtained above in 45 mL of hydrochloric acid 1,4 - dioxane solution (0.18 mol), stir and react at room temperature for 9 h. After the reaction, the organic phase was precipitated with 500 mL of ice - ether, filtered by suction, and dried in vacuo to obtain 5.3 g of white solid compound 5, with a yield of 71.1 g%;

[0082] 4) Take a 250 - mL three - necked flask, bake the flask three times to remove the residual water vapor in the flask. Vacuum the flask. After the flask cooled to room temperature, stop vacuuming. Add 70 mL of anhydrous tetrahydrofuran with a syringe. Add 3 g of compound 5 (11 mmol) obtained above, stir until the system becomes a turbid liquid. Then, fill the flask with nitrogen, adjust the gas flow rate to produce 1 - 2 bubbles per second. Place the flask in an oil bath pot, set the oil bath pot temperature to 50 - 55 °C for heating and stirring. Then add 1.368 g of triphosgene (4.6 mmol). Stop heating after the system gradually becomes a yellow clear solution, continue to fill with nitrogen for 30 min, then concentrate by rotary evaporation to about 30 mL. Pour the obtained solution into 300 mL of ice - n - hexane for precipitation, let it stand at - 20 °C for 2 hours, discard the supernatant to obtain a yellow viscous liquid crude product. Then dissolve the crude product in 120 mL of ice - ethyl acetate stored at - 20 °C, transfer it to a separatory funnel, and use saturated NaHCO 3Wash with aqueous solution and pure aqueous solution three times, discard the lower aqueous phase, transfer the upper organic phase to a conical flask, add anhydrous magnesium sulfate and dry overnight, filter, transfer the filtrate to a round-bottom flask with a side arm, connect to a vacuum pump and dry under vacuum for half a day to remove the solvent, obtaining 1.3 g of a yellow viscous liquid product, compound 6, with a yield of 45%.

[0083] The infrared spectrum of the HLG-NCA prepared in this example is as shown in Figure 1 shown, IR(KBr): 1855, 1785, 1738, 1633, 1281 cm -1 .

[0084] The 1H NMR spectrum of the HLG-NCA prepared in this example is as shown in Figure 2 shown, 1 1H NMR(500 MHz, CDCl 3 ) δ 6.76(s, 1H), 4.70(t, J = 4.6 Hz, 2H), 4.48 - 4.45(m, 1H), 4.43(q, J = 4.3 Hz, 2H), 2.62(t, J = 7.2 Hz, 2H), 2.31(dq, J = 14.4, 6.5 Hz, 1H), 2.18(dq, J = 14.1, 6.9 Hz, 1H).

[0085] The 13C NMR spectrum of the HLG-NCA prepared in this example is as shown in Figure 3 shown, 13 13C NMR(126 MHz, CDCl 3 ) δ 172.15, 169.52, 152.18, 70.28, 60.80, 56.70, 29.19, 26.61.

[0086] Example 2

[0087] Using the HLG-NCA prepared in Example 1 as the raw material, biodegradable polymers MPEG-PHLG and MPEG-P(HLG-co-BLG) were prepared. The ring-opening copolymerization route diagrams of the two polymers are as follows:

[0088]

[0089] The synthesis method of monomer BLG-NCA in the above roadmap is as follows: Take a 500 mL flask, bake the flask three times to remove the residual water vapor in the flask, evacuate the flask, and stop evacuating after the flask cools to room temperature. Inject 150 mL of anhydrous tetrahydrofuran, add 15 g of L-glutamic acid-γ-benzyl ester (63.2 mmol), stir until the system becomes a turbid liquid, then fill the flask with nitrogen, adjust the gas flow rate to produce 1-2 bubbles per second, place the flask in an oil bath, set the oil bath temperature to 50-55 °C and heat with stirring, then add 7.817 g of triphosgene (26.3 mmol). Stop heating after the system gradually turns into a yellow clear solution, continue to fill with nitrogen for 30 min, then rotary evaporate and concentrate to about 30 mL. Pour the obtained solution into 300 mL of n-hexane at about 4 °C for precipitation, let it stand at -20 °C for 2 hours, discard the supernatant, and obtain a white powder solid crude product. Then dissolve the crude product in 200 mL of ice-cold ethyl acetate stored at -20 °C, transfer it to a separatory funnel, wash it three times with saturated NaHCO 3 aqueous solution and water stored at 4 °C, discard the lower aqueous phase, transfer the upper organic phase to a conical flask, add anhydrous magnesium sulfate and dry overnight, filter, transfer the filtrate to a round-bottom flask with a side neck, connect a vacuum pump and dry under vacuum for half a day to remove the solvent, and obtain 12.3 g of white solid powder with a yield of 73%.

[0090] Polymer MPEG-PHLG 15 Synthesis: Take a polymerization flask, add a magnetic stirrer, bake the flask three times, take 0.725 g of mPEG-NH 2 (Mn = 5000, 145 μmol) and add it to the polymerization flask. Evacuate under a 90 °C oil bath for 2 h. Dissolve 0.57 g of HLG-NCA (2.17 mmol) prepared in Example 1 in 11.4 mL of dry DMF under a nitrogen atmosphere, then inject it into the polymerization flask through a syringe. Continuously stir the mixture at 25 °C for 3 days. After the reaction is completed, rotary evaporate until it becomes viscous, then dissolve it in chloroform, and precipitate it with anhydrous ether at about -20 °C. Filter the solid and dry it under vacuum at 40 °C to constant weight. The NMR results show that its molecular weight is 8200, and its structure is labeled as MPEG-PHLG 15 . The 1H NMR spectrum is shown in Figure 4 .

[0091] Polymer MPEG-PHLG 25 Synthesis: Take a polymerization flask, add a magnetic stirrer, bake the flask three times, take 0.435 g of mPEG-NH 2(Mn = 5000, 87 μmol) was added to a polymerization flask, and the flask was evacuated under a vacuum at 90 °C for 2 h. Under a nitrogen atmosphere, 0.57 g of HLG-NCA (2.17 mmol) prepared in Example 1 was dissolved in 11.4 mL of dry DMF, and then injected into the polymerization flask through a syringe. The mixture was continuously stirred and reacted at 25 °C for 3 days. After the reaction, it was rotary evaporated to a viscous state, then dissolved in chloroform, and then precipitated with anhydrous ether at about -20 °C. The solid was filtered and vacuum dried at 40 °C to a constant weight. The NMR results showed that its molecular weight was 9600.

[0092] Polymer MPEG-PHLG 40 Synthesis: Take a polymerization flask, add a magnetic stirrer, and bake the flask three times. Take 0.272 g of mPEG-NH 2 (Mn = 5000, 54.4 μmol) was added to the polymerization flask, and the flask was evacuated under a vacuum at 90 °C for 2 h. Under a nitrogen atmosphere, 0.57 g of HLG-NCA (2.17 mmol) prepared in Example 1 was dissolved in 11.4 mL of dry DMF, and then injected into the polymerization flask through a syringe. The mixture was continuously stirred and reacted at 25 °C for 3 days. After the reaction, it was rotary evaporated to a viscous state, then dissolved in chloroform, and then precipitated with anhydrous ether at about -20 °C. The solid was filtered and vacuum dried at 40 °C to a constant weight. The NMR results showed that its molecular weight was 12600.

[0093] Polymer MPEG-P(HLG 15 -co-BLG 15 ) Synthesis: Take a polymerization flask, add a magnetic stirrer, and bake the flask three times. Take 0.725 g of PEG-NH 2 (Mn = 5000, 145 μmol) was added to the polymerization flask, and the flask was evacuated under a vacuum at 90 °C for 2 h. After cooling, 0.57 g of L-glutamic acid γ-benzyl ester-N-carboxyanhydride (BLG-NCA, 2.17 mmol) was added. Under a nitrogen atmosphere, 0.57 g of HLG-NCA (2.17 mmol) prepared in Example 1 was dissolved in 11.4 mL of dry DMF, and then injected into the polymerization flask through a syringe. The mixture was continuously stirred and reacted at 25 °C for 3 days, rotary evaporated to a viscous state, then dissolved in chloroform, and then precipitated with anhydrous ether at about -20 °C. The solid was filtered and vacuum dried at 40 °C to a constant weight. The NMR results showed that its molecular weight was 11100, and its 1H NMR spectrum is shown in the appendix Figure 5 .

[0094] The DP (degree of polymerization) and molecular weight data of the 4 copolymers obtained in this example are shown in Table 1.

[0095] Table 1

[0096]

[0097] DP and M in Table 1 n1 Adopted 1 Obtained by \(^1\)H NMR test, M n2 Determined by GPC method with DMF as the eluent.

[0098] Example 3

[0099] Polymer MPEG-PHLG 15 Performance test

[0100] Preparation of MPEG-PHLG by oil-in-water (O / W) emulsion method 15 Polymer micelles:

[0101] Add the MPEG-PHLG prepared in Example 2 15 to toluene for dissolution to obtain a polymer toluene solution with a concentration of 40 mg / mL. Under ultrasonic conditions, add 0.1 mL of the polymer toluene solution to 4 mL of PBS buffer solution (10 mM, pH 7.4), ultrasonically disperse for 20 minutes, then let it stand at room temperature overnight to volatilize toluene, and then filter with a microporous filter membrane (pore size 0.225 μm) to obtain a polymer micelle solution (1 mg / mL).

[0102] The average particle size of the polymer micelles measured by dynamic light scattering is 126.6 nm, the micelle size distribution index is 0.2148, and the micelle size distribution diagram is as Figure 6 shown.

[0103] Test the stability of the particle size of the above polymer micelle solution under PBS (pH 7.4) conditions: Continuously for three days, take 1 mL of the prepared polymer micelle solution (1 mg / mL) every 24 h, and detect the change of the polymer micelle particle size by dynamic light scattering. The comparison diagram of the polymer micelle particle size at different times is as Figure 7 shown. It can be seen that the polymer micelles at 0 h, 24 h, 48 h, and 72 h have no obvious changes, proving that the micelles have good stability.

[0104] MPEG-PHLG 15 In vitro NO release experiment of polymer micelles:

[0105] The in vitro release experiment of NO was carried out at 37 °C. Centrifuge and concentrate 4 mL of the MPEG-PHLG 15 polymer micelle solution prepared by the above method to 1 mL to obtain a MPEG-PHLG 15The polymeric micelle solution was divided into two groups. One group was added with GSH (the concentration of GSH in the formed solution was 10 mM, used to simulate the GSH environment in tumor cells), and the other group was not added with GSH as a control. It was placed in a constant temperature shaker at 37 °C. Every once in a while, 50 μL of the sample was taken, and the NO release amount of the polymeric micelles was measured using a Griess kit. MPEG-PHLG 15 The comparison chart of the NO release concentration of the polymeric micelles in two media is as Figure 8 shown. It can be seen that in the presence of GSH, the polymeric micelles gradually release a large amount of NO. At 24 h, the released NO concentration can reach 30 μM, and after about 48 h, the release is completed and tends to be stable. While in the case of not adding GSH, the concentration of NO released by the polymeric micelles is very low.

[0106] MPEG-PHLG 15 Encapsulation of Ce6 by polymeric micelles:

[0107] 200 μL of the DMSO solution of Ce6 (5 mg / mL) (calculated according to the dosage ratio of 25 wt% of the polymer) was added to 100 μL of the toluene solution of the polymer MPEG-PHLG 15 (40 mg / mL) to obtain a mixed solution of the polymer and Ce6. Then, under ultrasonic conditions, the obtained polymeric mixed solution was all added to 4 mL of PBS buffer solution (10 mM, pH 7.4), ultrasonically treated for 20 minutes, and then left standing overnight to volatilize toluene. DMSO and unencapsulated Ce6 were removed by dialysis to obtain the MPEG-PHLG 15 @Ce6 polymeric micelle solution. The drug loading capacity (DLC) and encapsulation efficiency (DLE) of the MPEG-PHLG 15 @Ce6 polymeric micelles were measured by an ultraviolet spectrophotometer. The calculation formulas are as follows:

[0108] Drug loading capacity (wt%) = (mass of loaded drug / mass of polymer) × 100%

[0109] Encapsulation efficiency (%) = (mass of loaded drug / total amount of drug input) × 100%

[0110] The test results are shown in Table 2.

[0111] Table 2

[0112]

[0113] When the theoretical drug loading capacity (i.e., the mass ratio of Ce6 to the polymer) was 25 wt%, the encapsulation efficiency of the MPEG-PHLG 15 polymeric micelles for Ce6 was about 60.3%, indicating that the polymeric micelles had good drug loading ability.

[0114] MPEG-PHLG 15 In vitro ROS release experiment of @Ce6 polymer micelles:

[0115] Determination of MPEG-PHLG using the singlet oxygen-indicating fluorescent probe 1,3-Diphenylisobenzofuran (DPBF) 15 @Ce6 polymer micelles under 660nm light irradiation, the concentration of ROS generated. Specifically, 1mL DPBF (25μM) and 1mL MPEG-PHLG 15 The mixed solution obtained after the co-dissolution of @Ce6 polymer micelle solution (Ce6 concentration 10μM) was exposed to 660nm laser irradiation, and the absorbance change of the mixed solution was measured every 5s using a UV spectrophotometer. The results are shown in Figure 9 As shown, it can be seen that under 660nm laser irradiation, with the increase of illumination time, the absorption peak of DPBF around 410nm gradually decreases, which indicates that Ce6 in the polymer micelles produces active oxygen after being activated by light, and then oxidizes DPBF.

[0116] MPEG-PHLG 15 @Ce6 polymer micelles in vitro peroxynitrite release experiment:

[0117] To detect MPEG-PHLG 15 @Ce6 The cascade reaction of NO released in the presence of GSH and Ce6 light-triggered ROS can generate peroxynitrite anion (ONOO-) - a major reactive nitrogen (RNS). The probe molecule dihydrorhodamine 123 (DHR) was used. The detection principle is that DHR can be specifically oxidized by ONOO- to generate rhodamine 123 (RH), whose characteristic absorption wavelength is 505nm. Prepare MPEG-PHLG according to the above method. 15 @Ce6 polymer micelle solution (Ce6 concentration 151μg / mL, polymer concentration 1mg / mL), diluted with PBS buffer (10mM, pH=7.4) to MPEG-PHLG with Ce6 concentration of 15μg / mL 15 @Ce6 polymer micelle solution, adding DHR, GSH to MPEG-PHLG 15 @Ce6 polymer micelle solution was made into 0.1mM and 10mM concentrations in the solution, which were set as the experimental group (denoted as MPEG-PHLG 15@Ce6 + GSH + DHR); The control groups were set as follows: (1) PBS buffer (10 mM, pH = 7.4) with only an equal amount of DHR added (denoted as DHR); (2) PBS buffer (10 mM, pH = 7.4) with only an equal amount of DHR and GSH added (GSH + DHR); (3) micellar solution with only an equal amount of DHR added and no GSH added (denoted as PEG-PHLG 15 @Ce6 + DHR). After the prepared experimental and control group samples were placed in a 37 °C constant temperature shaking incubator and shaken at a speed of 120 revolutions per minute for 6 h, they were exposed to 660 nm laser irradiation for 2 minutes. The ultraviolet-visible absorption spectra of the colored supernatant before and after laser irradiation were compared and analyzed. As Figure 10 shown, after shaking for 6 h in the presence of GSH and then irradiating with light, the experimental group (MPEG-PHLG15@Ce6 + GSH + DHR) had a strong absorption peak of the reaction product RH of ONOO− and DHR at 505 nm. On the contrary, there was no significant difference in the spectra of the control groups (GSH + DHR, PEG-PHLG 15 @Ce6 + DHR). The results confirmed the formation of NO and ONOO− and their cascade reaction under the experimental conditions.

[0118] MPEG-PHLG 15 Hemolysis experiment of MPEG-PHLG

[0119] The blood compatibility of MPEG-PHLG 15 was determined by a hemolysis experiment. 1 mL of rabbit blood was taken and heparin sodium was added to prevent blood coagulation. After dilution with PBS buffer and centrifugation to remove the supernatant, the obtained red blood cells were washed several times and then diluted with PBS buffer (10 mM, pH = 7.4) for standby. In the positive control group, 0.2 mL of diluted blood was mixed with 0.8 mL of pure water, and the absorbance at a wavelength of 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to ensure that the absorbance was between 0.5 and 0.55. In the negative control group, 0.2 mL of diluted blood was mixed with 0.8 mL of PBS buffer (10 mM, pH = 7.4). The prepared MPEG-PHLG 15 @Ce6 polymeric micelle solution (Ce6 concentration 151 μg / mL, polymer concentration 1 mg / mL) was diluted with PBS buffer (10 mM, pH = 7.4) so that the Ce6 concentrations in the MPEG-PHLG 15 @Ce6 polymeric micelles were 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL respectively. 0.8 mL of each was taken, and then 0.2 mL of diluted blood was added to obtain the experimental groups. After the samples were left standing for 3 h and centrifuged to precipitate the red blood cells, the supernatant was taken and the absorbance at 540 nm was measured using an ELISA reader. The hemolysis percentage was calculated using the following formula:

[0120]

[0121] where OD S is the absorbance of the experimental group, OD P is the absorbance of the positive control group, and OD N is the absorbance of the negative control group.

[0122] As Figure 11 shown is the comparison chart of the hemolysis percentage of each sample in the experimental group. The small figure is the photo of each sample in the experimental group, the positive control group, and the negative control group after standing and centrifugation. It can be seen that even when the concentration of Ce6 in the MPEG-PHLG 15 @Ce6 polymer micelles is as high as 20 μg / mL, its hemolysis rate is still within the safe range of 5%, which can prove that this polymer drug delivery system has good blood compatibility.

[0123] MPEG-PHLG 15 @Ce6 polymer micelle intracellular ROS generation experiment:

[0124] The release of intracellular ROS was studied using the ROS probe (DCFH-DA) under a fluorescence microscope. DCFH-DA reacts with ROS in cells to generate fluorescent DCF. Detecting the fluorescence of DCF can know the level of reactive oxygen species in cells. Specifically, 4T1 cells were seeded in a 12-well plate, with 1×10 5 cells per well, and cultured in 1640 medium containing 10% serum for 24 h at 37 °C under 5% carbon dioxide. 5 mg of Ce6 was dissolved in 1 mL of DMSO, and then diluted to 6 μg / mL with 1640 medium containing 10% serum, and 1 mL was added to co-incubate with the cells for 2 h, which was set as the control group (denoted as Free Ce6); the prepared MPEG-PHLG 15 @Ce6 polymer micelle solution (Ce6 concentration 151 μg / mL, polymer concentration 1 mg / mL) was diluted to a Ce6 concentration of 6 μg / mL with 1640 medium containing 10% serum, and 1 mL was added to co-incubate with the cells for 2 h, which was set as the experimental group (denoted as MPEG-PHLG@Ce6). Subsequently, the well plate was irradiated with 660 nm laser for 2 minutes, then washed 3 times with PBS buffer (10 mM, pH = 7.4), 1 mL of DCFH-DA medium solution (diluted 1:1000 with serum-free 1640 medium, and the 1640 medium was purchased from Beyotime Biotechnology Co., Ltd.) was added to incubate the cells for 30 minutes. Subsequently, the DCFH-DA medium solution was removed, washed 3 times with PBS buffer, and then observed with an inverted fluorescence microscope. As Figure 12The inverted fluorescence microscope test images of the control group and the experimental group are shown. It can be clearly seen that under the irradiation of 660 nm laser, green fluorescence generated by the reaction of ROS and the probe can be observed in both the control group and the experimental group cells, and more obvious green fluorescence can be observed in the experimental group, proving that compared with free Ce6, MPEG-PHLG 15 @Ce6 polymer micelles can be more endocytosed by cells and can release ROS intracellularly under laser irradiation.

[0125] MPEG-PHLG 15 Intracellular NO generation experiment of MPEG-PHLG@Ce6 polymer micelles:

[0126] The release of intracellular NO was studied by using a NO fluorescence probe (DAF-FM DA) under a fluorescence microscope. Specifically, A549 cells were seeded in a 12-well plate, with 1×10 5 cells per well, and cultured in 1640 medium containing 10% serum for 24 h at 37 °C under 5% carbon dioxide. The MPEG-PHLG 15 polymer micelle solution concentrated to 20 mg / mL was diluted to 1 mg / mL with 1640 medium containing 10% serum and co-incubated with A549 cells for 4 h to obtain the experimental group (denoted as MPEG-PHLG), and the control group was A549 cells growing normally for 4 h (denoted as Control). After washing the experimental group and the control group with PBS buffer (10 mM, pH = 7.4) three times, the cells were incubated with 500 μL DAF-FM DA solution (diluted 1:1500 with DAF-FM DA diluent, and the diluent was purchased from Beyotime Biotechnology Co., Ltd.) for 30 minutes, then the DAF-FM DA solution was removed, the cells were washed with PBS buffer (10 mM, pH = 7.4) three times, and then observed with an inverted fluorescence microscope. As Figure 13 shown are the inverted fluorescence microscope test images of the control group and the experimental group. Obvious green fluorescence generated by the reaction of NO and the probe can be observed in the experimental group cells, while almost no fluorescence can be observed in the control group, proving that MPEG-PHLG 15 @Ce6 polymer micelles can respond and release NO in A549 cells with overexpressed GSH, and the polymer MPEG-PHLG 15 is an effective NO donor.

[0127] MPEG-PHLG 15 In vitro cell safety experiment of polymer micelles:

[0128] The cytotoxicity of MPEG-PHLG 15 polymer micelles against L929 and 4T1 cells was detected by the MTT method:

[0129] 4T1 and L929 cells were seeded in 96-well plates at a density of approximately 8×10 3 cells / well and cultured in 1640 and DMEM media containing 10% serum at 37°C under a 5% CO 2 atmosphere for 24 h. The medium was removed, and the prepared MPEG-PHLG 15 polymeric micelle solution was concentrated to a concentration of 20 mg / mL and then diluted to different concentrations with 1640 and DMEM media containing 10% serum to obtain MPEG-PHLG 15 polymeric micelle solutions with concentrations of 0.32 μg / mL, 1.6 μg / mL, 8 μg / mL, 40 μg / mL, 200 μg / mL, and 1000 μg / mL, respectively. Three wells were used as parallels for each concentration, and 100 μL of the above polymeric micelle solution was added to each well as the medium. After continued incubation for 24 h, the medium was removed, and 110 μL of MTT assay solution medium [weigh 50 mg of MTT powder and dissolve it in 10 mL of PBS buffer solution (10 mM, pH = 7.4), filter it using a 0.22 μm filter membrane to remove bacteria in the solution before use, and prepare the MTT medium solution at a ratio of MTT:medium = 1:10] was added to each well. After continued incubation for 4 h, 100 μL of DMSO was added to each well, and then the absorbance value at 570 nm was measured using a microplate reader.

[0130] As Figure 14 shown is the comparison chart of cell viability tested for the two types of cells in MPEG-PHLG 15 polymeric micelle solutions with different concentrations. According to the experimental results, L929 cells showed a relatively high cell viability in MPEG-PHLG 15 polymeric micelle solutions with different concentrations. Even when the concentration of the polymeric micelles was as high as 1000 μg / mL, the viability of L929 cells was still higher than 100%, indicating that MPEG-PHLG 15 micelles have good biosafety for normal L929 cells. In contrast, the viability of 4T1 cells decreased with increasing MPEG-PHLG 15The increase in micelle concentration gradually decreased and finally dropped to 84.6%, indicating that the polymeric micelles had certain cytotoxicity to tumor 4T1 cells at high concentrations. Under the same experimental conditions, L929 cells showed higher cell viability than 4T1 cells, especially in the presence of higher concentrations of the polymer. This phenomenon can be attributed to the relatively low GSH concentration in L929 cells, the low NO concentration produced by the polymer, and the low concentration of NO contributing to cell growth. While the GSH concentration in 4T1 cells was relatively high, the reduction of GSH promoted the release of NO in the polymer, and the excessive NO concentration produced cytotoxicity, thus inhibiting the growth of 4T1 cells. Based on the above experimental results, it can be inferred that MPEG-PHLG15 has good biosafety for normal cells and is also applicable to the treatment of tumors or other cardiovascular and cerebrovascular diseases caused by the lesions of cells with high GSH expression.

[0131] MPEG-PHLG 15 Cytotoxicity experiment (MTT) of @Ce6 polymeric micelles:

[0132] Detect the cytotoxicity of MPEG-PHLG 15 @Ce6 polymeric micelles to 4T1 cells:

[0133] The specific experimental method is similar to the above. The prepared MPEG-PHLG 15 @Ce6 polymeric micelle solution (Ce6 concentration 151 μg / mL, polymer concentration 1 mg / mL) was diluted to different concentrations with 1640 medium containing 10% serum. The Ce6 concentrations in the diluted polymeric micelle solutions were 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL respectively, as the experimental groups; 5 mg Ce6 was dissolved in 1 mL DMSO and then diluted to different concentration gradients with 1640 medium containing 10% serum. The Ce6 concentrations in the solutions were 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL respectively, as the control groups. 4T1 cells were seeded in 96-well plates at a density of approximately 8×10 3 cells / well and incubated at 37°C with 5% CO 2Under an atmosphere, the cells were cultured in 1640 medium containing 10% serum for 24 h. Then the medium was removed, and the media of the experimental group and the control group containing different Ce6 concentrations were added. After continuing to incubate for 24 h, each group was further divided into a light irradiation group and a non-light irradiation group. The light irradiation group was irradiated with 660 nm laser for 5 min and then continued to incubate for 30 min, while the non-light irradiation group was not irradiated with laser and continued to incubate for 30 min. The medium was removed, and 110 μL of MTT detection solution medium (prepared in the same way as the safety experiment) was added to each well. After continuing to incubate for 4 h, 100 μL of DMSO was added to each well, and then the absorbance value at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0134] As Figure 15 shown is the comparison chart of the cell survival rates of four groups. In the figure, Free Ce6 is the control group without laser irradiation, Free Ce6+Laser is the control group with laser irradiation, NPs-NO is the experimental group without laser irradiation, and NPs-NO+Laser is the experimental group with laser irradiation. It can be seen from the comparison that without the action of laser irradiation, the cell killing abilities of the experimental group and the control group are both very limited and the difference is not significant, indicating that light irradiation is a necessary condition for generating cytotoxicity. Under the action of laser irradiation, at the same Ce6 concentration, MPEG-PHLG 15 @Ce6 polymer micelles showed more significant cytotoxicity than free Ce6, indicating that NO released by the NO donor polymer micelles reacted with the photolysis products of Ce6 to generate peroxynitrite with stronger cell killing ability, thus enhancing the therapeutic effect of photodynamic therapy.

Claims

1. A glutamic acid-N-carboxyl-intracyclic anhydride monomer containing an ethyl nitrate group, characterized in that: Its structural formula is shown in the following formula (I):

2. A method for preparing the glutamic acid-N-carboxyl-intracyclic anhydride monomer containing an ethyl nitrate group according to claim 1, characterized in that: The following steps are involved: 1) dissolving 2-bromoethanol and silver nitrate in solvent 1 to carry out substitution reaction, and post-treating to obtain 2-hydroxyethyl nitrate; 2) dissolving the 2-hydroxyethyl nitrate obtained in step 1) with Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in solvent 2 for esterification reaction, and post-treating to obtain Boc-L-glutamic acid-1-tert-butyl ester-5-nitrate ethanol ester; 3) subjecting the Boc-L-glutamic acid-1-tert-butyl ester-5-nitrate ethanol ester obtained in step 2) to a deprotection reaction with a hydrochloric acid 1,4-dioxane solution, and post-treating to obtain L-glutamic acid-5-nitrate ethanol ester; 4) performing a cyclization reaction between the L-glutamic acid-5-nitrate ethanol ester obtained in step 3) and triphosgene in solvent 3, The glutamic acid-N-carboxyl-intracyclic anhydride monomer containing ethyl nitrate group is obtained by post-treatment.

3. The method for preparing the glutamic acid-N-carboxyl-intracyclic anhydride monomer containing ethyl nitrate group according to claim 2, characterized in that: The solvent 1 in step 1) is acetonitrile, and the mass volume ratio of the 2-bromoethanol to the solvent 1 is 1 g: 12.5-25 mL; the substitution reaction conditions in step 1) are: reacting at 70-80° C. for 12-25 h under light-proof conditions; the molar ratio of the 2-hydroxyethyl nitrate to Boc-L-glutamic acid-1-tert-butyl ester, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in step 2) is 1-1.2: 1: 0.1-0.2: 1.8-2.5; the solvent 2 in step 2) is dichloromethane, and the mass volume ratio of the 2-hydroxyethyl nitrate to the solvent 2 is 1 g: 30-50 mL; the esterification reaction conditions in step 2) are: reacting at 5-40° C. for 20-30 h.

4. The method for preparing the glutamic acid-N-carboxyl-intracyclic anhydride monomer containing ethyl nitrate group according to claim 2, characterized in that: In step 3), the molar ratio of Boc-L-glutamic acid-1-tert-butyl ester-5-nitrate ethanol ester to hydrochloric acid in the hydrochloric acid 1,4-dioxane solution is 1:3-7; in step 3), the deprotection reaction conditions are: reacting at 5-40°C for 9-12h; in step 4), the molar ratio of L-glutamic acid-5-nitrate ethanol ester to triphosgene is 1:0.40-1; in step 4), the solvent 3 is one of tetrahydrofuran, dichloromethane, ethyl acetate, and N,N-dimethylformamide, and the mass volume ratio of L-glutamic acid-5-nitrate ethanol ester to solvent 3 is 0.04-0.08g / mL; in step 4), the cyclization reaction conditions are: reacting at 50-70°C for 0.2-1h under an inert atmosphere.

5. A biodegradable polymer obtained by polymerizing the glutamic acid-N-carboxyl-intracyclic anhydride monomer containing ethyl nitrate group as claimed in claim 1 alone or copolymerizing it with other amino acid-N-carboxyl-intracyclic anhydride monomers, characterized in that: Its chemical structure is shown below: Wherein, R is a polymerization initiator residue, L is a repeating unit corresponding to other amino acid-N-carboxyl-cyclic anhydride monomers, m is the degree of polymerization of the glutamic acid-N-carboxyl-cyclic anhydride monomers containing ethyl nitrate groups, and n is an integer greater than or equal to 0, which is the degree of polymerization of other amino acid-N-carboxyl-cyclic anhydride monomers.

6. The biodegradable polymer according to claim 5, characterized in that The glutamic acid-N-carboxyl-intracyclic anhydride monomer containing an ethyl nitrate group is initiated by a primary amine initiator, or a mixture of the monomer and other amino acid-N-carboxyl-intracyclic anhydride monomers is initiated to perform ring-opening polymerization to obtain the product; The primary amine initiator is one of amino-terminated polyethylene glycol monomethyl ether, n-hexylamine or benzylamine; The amino acid-N-carboxyl-intracyclic anhydride monomer is one of L-glutamic acid-γ-benzyl ester-N-carboxyl-intracyclic carboxylic anhydride, L-phenylalanine-N-carboxyl-intracyclic carboxylic anhydride, N-benzyloxycarbonyl-L-lysine-N-carboxyl-intracyclic carboxylic anhydride or methionine-N-carboxyl-intracyclic carboxylic anhydride, and its structural formula is as follows:

7. Nanoparticles formed by self-assembly of the biodegradable polymer according to claim 5 or 6.

8. Use of the glutamic acid-N-carboxyl-cyclic anhydride monomer containing ethyl nitrate group as claimed in claim 1 in drug carrier, NO donor and preparation of anti-tumor drugs and cardiovascular and cerebrovascular drugs.

9. Use of the biodegradable polymer according to claim 5 or 6 in drug carriers, NO donors and in the preparation of anti-tumor drugs and cardiovascular and cerebrovascular drugs.

10. Use of the nanoparticles according to claim 7 in drug carriers, NO donors, and in the preparation of anti-tumor drugs and cardiovascular and cerebrovascular drugs.

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