An amide bond-linked polyethylene glycol-phospholipid lipid-forming material, its preparation method and applications

Abstract

The present invention discloses an amide bond-linked polyethylene glycol-phospholipid lipid-forming material with the following structural formula, its preparation method and application, belonging to the technical field of biomedical materials. The amide bond-linked polyethylene glycol-phospholipid lipid-forming material of the present invention is prepared from distearoyl phosphatidylethanolamine DSPE and PEG 2K -CDM through an acylation reaction. The amide bond-linked polyethylene glycol-phospholipid lipid-forming material of the present invention can be used to prepare nanoliposomes with the effect of alleviating tumor hypoxia. The liposomes prepared from the amide bond-linked polyethylene glycol-phospholipid lipid-forming material and biguanide cationic materials in the present invention can remove PEG in the tumor tissue matrix, promote the penetration of nanoparticles in tumors and the internalization of tumor cells, increase the drug content in tumor cells, and thus improve the drug treatment effect. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to an amide bond-linked polyethylene glycol-phospholipid lipid-forming material, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant cell proliferation and insufficient angiogenesis lead to tumor hypoxia, which is a common feature of rapidly growing solid tumors. It is worth noting that the adaptation of advanced tumors to hypoxia stress not only accelerates tumor invasion and metastasis, but also leads to treatment resistance and poor prognosis to oxygen-dependent therapies. Due to the limited generation of reactive oxygen species (ROS) under hypoxic conditions, photodynamic therapy (PDT) as a promising interventional therapy method has been severely challenged. Many strategies have been used to overcome the hypoxia limitation of PDT, such as direct delivery of oxygen (O2), catalytic decomposition of cellular hydrogen peroxide (H2O2) to produce oxygen, and photocatalytic water splitting to produce oxygen. However, due to limitations such as limited oxygen loading, O2 leakage, insufficient endogenous H2O2 levels, and low oxygen production rates, the treatment effect is not ideal. Generally speaking, the balance between oxygen supply and consumption determines the oxygen pressure level in tumors. In the case of insufficient angiogenesis and slow blood flow in tumor tissues, compared with directly increasing oxygen supply, reducing oxygen consumption may be a better strategy to eliminate tumor hypoxia. In particular, the oxidative phosphorylation (OXPHOS) metabolic pathway continuously consumes O2 to produce ATP to ensure cell survival. It is feasible to reduce the oxygen consumption of OXPHOS by inhibiting mitochondrial complexes in the mitochondrial electron transport chain (ETC) to achieve O2 conservation. In addition, the co-localization of OXPHOS inhibitors and photosensitizers (PSs) is crucial for O2-conserving PDT to avoid the non-uniform distribution of PSs and O2.

[0003] Metformin is a widely used small molecule drug for the treatment of type 2 diabetes. Studies have shown that metformin can effectively relieve tumor hypoxia by inhibiting mitochondrial complex I. However, the hydrophilic property of metformin leads to insufficient accumulation in tumors, and different physicochemical properties limit its co-delivery with other drugs. Nanocarriers can solve this problem, but complex designs and non-biodegradability limit their clinical translation. Therefore, it is promising to design a lipid-forming material that can act as both a drug carrier material and an anti-tumor drug, avoiding the use of excessive excipients.

[0004] The following conditions need to be met for nanoparticles to improve drug efficacy: 1) having an appropriate blood circulation time; 2) being able to enhance the enrichment of drugs in tumors; 3) being able to promote the internalization of drugs by tumor cells; 4) the drug molecules can be rapidly released after entering tumor cells. When preparing liposomes, phospholipid materials modified with polyethylene glycol (PEG) are usually added to prolong the blood circulation time of liposomes, improve the distribution of drugs in the body, and enhance the therapeutic effect. Although the PEG covering the surface of liposomes prolongs the blood circulation time of liposomes and increases the enrichment of nanoparticles in tumors, it also hinders the uptake of nano-drugs by tumor cells.

[0005] Research shows that tumor cells meet the nutrients and energy required for their rapid proliferation through glycolysis. The accumulation of lactic acid produced by glycolysis makes the pH value of tumor tissues lower than that of normal tissues and blood. The pH of the tumor tissue matrix is 6.5 - 6.8, which is not much different from the pH value of the normal physiological environment. This puts higher requirements on acid-sensitive chemical bonds. Summary of the Invention

[0006] Object of the Invention: The present invention provides a polyethylene glycol-phospholipid lipid-forming material connected by an amide bond, its preparation method and application. The liposomes prepared from the polyethylene glycol-phospholipid lipid-forming material connected by an amide bond and a biguanide cationic material can remove PEG in the tumor tissue matrix, promote the penetration of nanoparticles in tumors and the internalization of tumor cells, increase the drug content in tumor cells, and thus improve the drug treatment effect.

[0007] The first object of the present invention is to provide a polyethylene glycol-phospholipid lipid-forming material connected by an amide bond with the following structural formula:

[0008]

[0009] The above-mentioned polyethylene glycol-phospholipid lipid-forming material connected by an amide bond provided by the present invention has a good pH sensitivity, and its amide bond can be broken when the pH is less than 6.8.

[0010] The second object of the present invention is to provide a preparation method of the above-mentioned polyethylene glycol-phospholipid lipid-forming material connected by an amide bond. The polyethylene glycol-phospholipid lipid-forming material DSPE-ASlink-PEG connected by an amide bond is obtained by an acylation reaction of distearoyl phosphatidylethanolamine DSPE and PEG 2K -CDM. 2k .

[0011] A preferred embodiment of the present invention is that the PEG 2K-CDM is prepared by the following method: using 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid as a raw material, adding oxalyl chloride to obtain 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionyl chloride; using 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionyl chloride as a raw material, performing an esterification reaction with PEG 2K -OH to obtain PEG 2K -CDM.

[0012] Preferably, after mixing PEG 2K -CDM with DSPE, adding triethylamine, and heating and stirring for reaction, DSPE-ASlink-PEG 2k is obtained; the molar ratio of PEG 2K -CDM to DSPE is 1:1 to 2.

[0013] More preferably, the molar ratio of PEG 2K -CDM to DSPE is 1:1.

[0014] More preferably, the solvent for the reaction is chloroform; the reaction temperature is 45 to 50 °C; the reaction time is 16 to 24 h.

[0015] Preferably, the purification process of DSPE-ASlink-PEG 2k is: dropping the reaction solution into cold ether for precipitation, and then washing twice with cold ether.

[0016] The third object of the present invention is to provide the application of the above amide bond-linked polyethylene glycol-phospholipid lipid-forming material in the preparation of nanoliposomes with the effect of alleviating tumor hypoxia.

[0017] The fourth object of the present invention is to provide a nanoliposome with the effect of alleviating tumor hypoxia, which is prepared by the following method: N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine forms a cationic liposome with cholesterol, and inserts DSPE-ASlink-PEG 2K to prepare a nanoliposome with relatively stable blood circulation.

[0018] Preferably, the particle size of the nanoliposome is 100 to 250 nm. Under the condition of the same prescription ratio, by passing through membranes with different pore sizes, nanoparticles with different particle sizes can be prepared.

[0019] The nanoliposome in the tumor microenvironment, that is, at a pH less than 6.8, removes the PEG hydration layer, thereby promoting the penetration and cellular internalization of tumor tissues.

[0020] The fifth object of the present invention is to provide a preparation method of the above nanoliposome with the effect of alleviating tumor hypoxia, including the following steps:

[0021] Mix N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine (DOBG), cholesterol, and DSPE-ASlink-PEG 2K at a mass ratio of 10 - 40:4:2 - 5, mix them evenly, distill to obtain a lipid film, and hydrate the film by ultrasound to obtain a crude liposome solution; pass the crude liposome solution through a membrane to obtain the final nano-liposome solution.

[0022] The nano-liposomes prepared by the present invention as a drug carrier can load anti-tumor drugs such as T780. The above nano-liposomes have good pH sensitivity, can remove the PEG hydration layer in the tumor microenvironment, promote the penetration of nanoparticles in tumor tissues and the internalization of tumor cells, and increase the drug content in cells. When loading T780, due to the co-localization of N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine acting on mitochondrial complex I and T780 in mitochondria, the phototherapy effect can be enhanced.

[0023] The loaded drug T780 is obtained by the substitution reaction of IR780 and (3-aminopropyl) triphenylphosphonium bromide.

[0024] A preferred preparation method of the drug-loaded nano-liposomes of the present invention includes the following steps:

[0025] Dissolve DOBG (N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine), cholesterol, DSPE-ASlink-PEG2k, and T780 in 5 ml of chloroform respectively, at a mass ratio of 13:4:2:1, mix them evenly by ultrasound, and rotary evaporate at 40 °C to obtain a lipid film, and hydrate the film by ultrasound to obtain a crude liposome solution. Pass the crude liposome solution through a membrane to obtain the final liposome solution.

[0026] The N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine described in the present invention is a biguanide cationic lipid material with both carrier and medicinal properties, and its structural formula is as follows:

[0027]

[0028] The biguanide cationic lipid material provided by the present invention can not only act as a lipid-forming material for liposomes, but also has the functions of inhibiting mitochondrial complex I, alleviating tumor hypoxia and anti-tumor. When the nano-carrier prepared by it loads a photosensitizer, the photodynamic efficacy of the photosensitizer can be enhanced. Especially when loading a mitochondria-targeted photosensitizer, the effect of photodynamic therapy can be enhanced through its co-localization with the photosensitizer in mitochondria.

[0029] The biguanide cationic lipid material provided by the present invention forms salts with hydrochloric acid or trifluoroacetic acid.

[0030] A preferred embodiment of the present invention is that the N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine is prepared by the following method: N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine reacts with oleoyl chloride through an esterification reaction to obtain Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine, then the Boc group is removed to obtain N,N-dioleoyl hydroxyethyl-2-aminoethylamine, and finally N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine is obtained through a nucleophilic addition reaction.

[0031] Further preferably, N,N-dioleoyl hydroxyethyl-2-aminoethylamine and dicyandiamide are heated and stirred in a nitrogen atmosphere for reaction. After the reaction is completed, the solvent is removed by rotary evaporation and redissolved in dichloromethane, washed with a mixed solution of methanol and sodium chloride, and the solvent is removed by rotary evaporation to obtain the crude product of N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine; the molar ratio of N,N-dioleoyl hydroxyethyl-2-aminoethylamine to dicyandiamide is 1:1 to 4.

[0032] More preferably, the molar ratio of N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine to dicyandiamide is 1:3.5.

[0033] More preferably, the reaction temperature is 120 °C; the reaction time is 24 h.

[0034] Further preferably, the crude product of N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine is purified by silica gel column chromatography, and the purification process uses gradient elution with dichloromethane and methanol.

[0035] The present invention also provides a preparation method of the N,N-dioleoyl hydroxyethyl-2-aminoethylamine: Dissolve Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine in a mixed solution of dichloromethane and trifluoroacetic acid, stir and react at room temperature. After the reaction is completed, remove the solvent, redissolve in dichloromethane, wash with ultrapure water, and rotary evaporate the solvent to obtain an oily crude product; the oily crude product is purified by silica gel column chromatography, and the purification process uses gradient elution with dichloromethane and methanol.

[0036] Further preferably, the volume ratio of dichloromethane to trifluoroacetic acid is 1:0.25 to 1.

[0037] Even more preferably, the volume ratio of dichloromethane to trifluoroacetic acid is 1:1; the reaction time of Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine with trifluoroacetic acid is 6 h.

[0038] The present invention also provides a method for preparing the Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine: Mix N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine with DMAP and triethylamine, slowly add the oleoyl chloride solution, stir at room temperature. After the reaction is completed, terminate the reaction with a mixed solution of methanol and sodium chloride, collect the organic phase, wash it with a dilute sodium bicarbonate solution, and remove the organic solvent by rotary evaporation to obtain the crude product.

[0039] Further preferably, the reaction solvent is chloroform; the solvent of the oleoyl chloride solution is chloroform; the molar ratio of N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine, oleoyl chloride, DMAP and triethylamine is 1:2-5:0.1-1:2-5.

[0040] Furthermore, the molar ratio of N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine, oleoyl chloride, DMAP and triethylamine is 1:2:0.15:2; the reaction time of N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine, oleoyl chloride, DMAP and triethylamine is 16 h; the volume ratio of the methanol and sodium chloride solution is 1:1.

[0041] Beneficial effects:

[0042] (1) Simulating DSPE-PEG 2k , the present invention synthesizes DSPE-ASlink-PEG linked by an amide bond 2k . Compared with DSPE-PEG 2k , DSPE-ASlink-PEG 2k has good pH sensitivity, can degrade and remove PEG in tumor tissues, and promotes the internalization of nanoparticles.

[0043] (2) The nanoliposomes prepared from DSPE-ASlink-PEG 2k , N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine and cholesterol can remove the PEG hydration layer in tumor tissues, promote the penetration of tumor tissues and the internalization of tumor cells, and solve the problem of insufficient tumor accumulation caused by the strong hydrophilicity of metformin.

[0044] (3) After the above nanoliposomes encapsulate T780, the photodynamic therapy effect is synergistically enhanced by the oxidative respiratory chain inhibitor and the photosensitizer.

[0045] (4) The present invention synthesizes a biguanide-based cationic lipid material (N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine), which is both a carrier material and can relieve tumor hypoxia. Description of the drawings

[0046] Figure 11H NMR spectrum of N,N-dioleoyl-2-hydroxyethyl-2-aminoethylamine in Example 1;

[0047] Figure 2 1H NMR spectrum of N,N-dioleoyl-2-hydroxyethyl-2-biguanidoethylamine in Example 1;

[0048] Figure 3 1H NMR spectrum of DSPE-ASlink-PEG in Example 3 2k ;

[0049] Figure 4 Particle size distribution diagram of T780@ASDOBG;

[0050] Figure 5 Western blotting was used to detect the up-regulated expression level of p-AMPK protein, the down-regulated expression levels of HIF-1α protein and PD-L1 protein in cells after treatment with different drugs. Detailed implementation manners

[0051] The technical solutions of the present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited to the described examples.

[0052] In view of the strong water solubility and poor intratumoral accumulation of metformin, in order to solve the above technical problems, the present invention provides a preparation method of a biguanide-based cationic lipid material, which includes the following steps:

[0053] (1) Synthesis of Boc-protected N,N-dioleoyl-2-hydroxyethyl-2-aminoethylamine

[0054] N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine was mixed with DMAP and triethylamine, and then an oleoyl chloride solution was slowly added, stirred at room temperature. After the reaction was completed, the reaction was quenched with a mixed solution of methanol and sodium chloride, the organic phase was collected, washed with a dilute sodium bicarbonate solution, and the organic solvent was removed by rotary evaporation to obtain a crude product of Boc-protected N,N-dioleoyl-2-aminoethylamine.

[0055] (2) Synthesis of N,N-dioleoyl-2-hydroxyethyl-2-aminoethylamine

[0056] Boc-protected N,N-dioleoyl-2-aminoethylamine was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid, stirred at room temperature for reaction. After the reaction was completed, the solvent was removed, redissolved in dichloromethane, then washed with ultrapure water, and the solvent was removed by rotary evaporation to obtain an oily crude product. Purified by column chromatography (gradient elution with dichloromethane and methanol).

[0057] (3) Synthesis of N,N-dioleoyl-2-biguanidoethylamine.

[0058] N,N-dioleoyl hydroxyethyl-2-aminoethylamine and dicyandiamide were heated and stirred under a nitrogen atmosphere. After the reaction was completed, the solvent was removed by rotary evaporation and redissolved in dichloromethane. It was washed with a mixed solution of methanol and sodium chloride, and the solvent was removed by rotary evaporation to obtain the crude product of N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine. Purification by column chromatography (gradient elution with dichloromethane and methanol).

[0059] Example 1 Preparation of N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine

[0060]

[0061] (1) Synthesis of Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine

[0062] N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine (0.415 g, 1.67 mmol), DMAP (0.306 g, 0.25 mmol) and triethylamine (0.464 ml, 3.34 mmol) were added to 7 ml of chloroform and mixed. A solution of oleoyl chloride (1.05 g, 3.49 mmol) in 3 ml of chloroform was slowly added under ice bath conditions, and the mixture was stirred at room temperature for 16 h. After the reaction was completed, the reaction was quenched with 8 ml of a mixed solution of methanol and sodium chloride (volume ratio of methanol to sodium chloride was 1:1). The organic phase was collected, washed with 4 ml of dilute sodium bicarbonate solution, and the organic solvent was removed by rotary evaporation to obtain 1.3 g of a crude product in the form of a pale yellow oil.

[0063] (2) Synthesis of N,N-dioleoyl hydroxyethyl-2-aminoethylamine

[0064] Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine (1.3 g, 1.67 mmol) was dissolved in a mixed solution of 8 ml of dichloromethane and trifluoroacetic acid, and the reaction was stirred at room temperature for 6 h, where the volume ratio of dichloromethane to trifluoroacetic acid was 1:1. After the reaction was completed, the solvent was removed, redissolved in 10 ml of dichloromethane, washed with ultrapure water, and the solvent was removed by rotary evaporation to obtain an oily crude product. The crude product was purified by column chromatography with gradient elution of dichloromethane and methanol, and the elution program was 100:1, 50:1, 20:1, and finally 0.91 g of an oily product was obtained (yield: 60%). The NMR spectrum is shown in Figure 1 .

[0065] 1HNMR (300 MHz, CDCl3), δ 0.87 (t, 6H, CH3), δ 1.27 (m, 40H, CH2), δ 1.56 (m, 4H, CH2CH2C(=O)), δ 2.00 (m, 8H, CH2CH=CH), δ 2.29 (t, 4H, CH2C(=O)), δ 2.82 - 2.90 (t, 4H, CH2N), δ 3.04 (t, 2H, CH2NH), δ 4.14 (t, 4H, NCH2CH2O), δ 5.35 (m, 4H, CH=CH).

[0066] (3) Synthesis of N,N - dioleoyl - hydroxyethyl - 2 - biguanidoethylamine (DOBG)

[0067] Dissolve N,N - dioleoyl - hydroxyethyl - 2 - aminoethylamine (0.904 g, 1 mmol) in 5 ml of n - butanol, then add dicyandiamide (0.168 mg, 3.5 mmol), heat and stir the reaction at 120 °C under a nitrogen atmosphere for 24 h. After the reaction is completed, remove the solvent by rotary evaporation and redissolve it in 20 ml of dichloromethane. Wash it with a mixed solution of 10 ml of methanol and sodium chloride (the volume ratio of methanol to sodium chloride is 1:1), and remove the solvent by rotary evaporation to obtain the crude product of N,N - dioleoyl - hydroxyethyl - 2 - biguanidoethylamine. The crude product is purified by column chromatography with gradient elution of dichloromethane and methanol, and the elution program is 50:1, 30:1, 20:1, 10:1. Finally, 0.445 mg of a waxy product is obtained (yield 45%). The NMR spectrum is shown in Figure 2 .

[0068] 1 HNMR (300 MHz, CDCl3), δ 0.87 (t, 6H, CH3), δ 1.27 (m, 40H, CH2), δ 1.56 (m, 4H, CH2CH2C(=O)), δ 2.00 (m, 8H, CH2CH=CH), δ 2.29 (t, 4H, CH2C(=O)), δ 2.77 (t, 2H, CH2N), δ 2.86 (t, 4H, NCH2CH2O), δ 3.31 (t, 2H, CH2NH), δ 4.14 (t, 4H, NCH2CH2O), δ 5.35 (m, 4H, CH=CH), δ 6.5 - 7.2 is the signal peak of biguanide.

[0069] Example 2 Preparation of N,N - dioleoyl - hydroxyethyl - 2 - biguanidoethylamine

[0070] (1) Synthesis of Boc - protected N,N - dioleoyl - hydroxyethyl - 2 - aminoethylamine

[0071] N-Boc-N,N-(2-hydroxyethyl)ethylenediamine (0.415 g, 1.67 mmol), DMAP (0.47 g, 0.38 mmol) and triethylamine (0.7 ml, 5.01 mmol) were added to 7 ml of chloroform. A solution of oleoyl chloride (1.58 g, 5.24 mmol) in 3 ml of chloroform was slowly added under ice bath conditions. The mixture was stirred at room temperature for 16 h. After the reaction was completed, the reaction was quenched with an 8-ml mixed solution of methanol and sodium chloride (the volume ratio of methanol to sodium chloride solution was 1:1). The organic phase was collected, washed with 4 ml of dilute sodium bicarbonate solution, and the organic solvent was removed by rotary evaporation to obtain 1.6 g of a crude product in the form of a pale yellow oil.

[0072] (2) Synthesis of N,N-dioleoyl-2-aminoethylhydroxyethylamine

[0073] Boc-protected N,N-dioleoyl-2-aminoethylhydroxyethylamine (1.6 g, 2 mmol) was dissolved in a mixed solution of 6 ml of dichloromethane and trifluoroacetic acid. The mixture was stirred at room temperature for 6 h, where the volume ratio of dichloromethane to trifluoroacetic acid was 1:0.25. After the reaction was completed, the solvent was removed, redissolved in 10 ml of dichloromethane, washed with ultrapure water, and the solvent was removed by rotary evaporation to obtain a crude oily product. The crude product was purified by column chromatography with gradient elution of dichloromethane and methanol. The elution program was 100:1, 50:1, 20:1, and finally 0.91 g of an oily product was obtained (yield: 50%).

[0074] (3) Synthesis of N,N-dioleoyl-2-biguanidoethylamine (DOBG)

[0075] N,N-dioleoyl-2-aminoethylhydroxyethylamine (0.904 g, 1 mmol) was dissolved in 5 ml of n-butanol, and then dicyandiamide (0.096 g, 2 mmol) was added. The mixture was heated to 120 °C under a nitrogen atmosphere and stirred for 24 h. After the reaction was completed, the solvent was removed by rotary evaporation and redissolved in 20 ml of dichloromethane. It was washed with a 10-ml mixed solution of methanol and sodium chloride (the volume ratio of methanol to sodium chloride solution was 1:1), and the solvent was removed by rotary evaporation to obtain a crude product of N,N-dioleoyl-2-biguanidoethylamine. The crude product was purified by column chromatography with gradient elution of dichloromethane and methanol. The elution program was 50:1, 30:1, 20:1, 10:1, and finally 0.396 mg of a waxy product was obtained (yield: 40%).

[0076] Example 3 Preparation of Amide Bond-Linked Polyethylene Glycol-Phospholipid Lipid Material (DSPE-ASlink-PEG)

[0077]

[0078] Using 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid as a raw material, oxalyl chloride was added to obtain 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionyl chloride; using 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionyl chloride as a raw material, an esterification reaction was carried out with PEG 2K -OH to obtain PEG 2K -CDM. For the specific preparation method, see: Feng Bing, Research on the Enhancement of Tumor Chemotherapy-Immunotherapy Effects by a Nano Drug Delivery System Based on Oxaliplatin Prodrug [D]. University of Chinese Academy of Sciences (Shanghai Institute of Materia Medica, Chinese Academy of Sciences), 2019.

[0079] DSPE (0.374 g, 0.5 mmol) and PEG 2K -CDM (1 g, 0.5 mmol) were mixed in 10 ml of chloroform, then 0.07 ml of triethylamine was added, and the mixture was heated and stirred at 45 °C for 24 h. After the reaction was completed, the reaction solution was concentrated by vacuum drying, and then dropped into 40 ml of cold diethyl ether, and washed twice with 20 ml of cold diethyl ether to obtain 0.9 g of the target product (yield: 66%). The NMR spectrum is shown in Figure 3 . As shown in the 1H NMR spectrum, δ 0.87 is the methyl signal peak on DSPE, δ 3.4 is the methyl signal peak on PEG 2K -CDM, a labile hydrogen signal peak appears at δ 8.18 for the amide nitrogen, and a labile hydrogen signal peak of the carboxyl group after ring opening appears at δ 12. The above signal peaks prove the successful synthesis of DSPE-ASlink-PEG 2K .

[0080] Example 4 Preparation of DSPE-ASlink-PEG

[0081] DSPE (0.374 g, 0.5 mmol) and PEG 2K -CDM (0.5 g, 0.25 mmol) were mixed in 10 ml of chloroform, then 0.035 ml of triethylamine was added, and the mixture was heated and stirred at 50 °C for 18 h. After the reaction was completed, the reaction solution was concentrated by vacuum drying, and then dropped into 40 ml of cold diethyl ether, and washed twice with 20 ml of cold diethyl ether to obtain 0.43 mg of the target product (yield: 63%).

[0082] Example 5 Preparation of Drug-Loaded Nano Liposomes T780@ASDOBG

[0083] Respectively, 13 mg of DOBG (N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine), 4 mg of cholesterol, and 2 mg of DSPE-ASlink-PEG 2k, 1 mg of T780 was dissolved in 5 ml of chloroform, ultrasonically mixed evenly, and rotary evaporated at 40 °C to obtain a lipid film. The film was ultrasonically hydrated to obtain a crude liposome solution. The crude liposome solution was filtered through a 0.22 μm filter membrane to obtain the final liposome solution. Figure 4 It is the particle size distribution diagram of T780@ASDOBG. The particle size detected by DLS is 140 nm, and the particle size distribution is 0.136.

[0084] T780 was self-prepared. For the specific preparation method, see: Zhang Jing, Research on Mitochondria-Targeted Photo-Chemotherapy Nanoparticles for Enhancing Immunotherapy of Breast Cancer [D]. Shandong University, 2020.

[0085] Example 6 Detection of the Expression Levels of p-AMPK, HIF-α, and PD-L1

[0086] (1) Cell culture

[0087] Using 4T1 cells as the research object, the cryopreserved cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in RPMI1640 medium (Gibco, USA) at 37 °C and 5% CO2. When the cells grew to a high density, they were passaged.

[0088] (2) Detection of the Expression Levels of p-AMPK, HIF-α, and PD-L1

[0089] Digest the 4T1 cells in the logarithmic growth phase with 0.05% trypsin (Gibco, USA) and prepare a single-cell suspension with fresh medium. After cell counting, 2×10 5Cells were inoculated in 6-well plates at a density of [number of cells], incubated overnight in an incubator at 37°C and 5% CO2, the medium in the wells was discarded, and solutions of metformin diluted with fresh medium (administered concentration 26 μM), T780@AIDOBG liposomes (effective ingredient DOBG concentration 26 μM), and T780@ASDOBG liposomes (effective ingredient DOBG concentration 26 μM) were added. After incubation, 100 μl of lysis buffer (Shanghai Beyotime Biotechnology Co., Ltd.) was added to lyse the cells, samples from each group were collected, centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected. The protein concentration of each group was detected using a BCA protein assay kit (Shanghai Beyotime Biotechnology Co., Ltd.) and adjusted to be consistent. A certain amount (volume ratio of sample solution to loading buffer is 4:1) of SDS-PAGE protein loading buffer (Shanghai Beyotime Biotechnology Co., Ltd.) was added to the samples in each group, and boiled at 100°C for 5 minutes to fully denature the proteins. Then, electrophoresis, transfer membrane, blocking, primary antibody incubation (HIF-1α Rabbit mAb was purchased from Cell Signaling Technology, USA; Phospho-AMPKα (Thr172) Rabbit mAb was purchased from Cell Signaling Technology, USA; PD-L1 Rabbit pAb was purchased from Proteintech; β-Actin Rabbit mAb was purchased from Shanghai Beyotime Biotechnology Co., Ltd.), secondary antibody incubation (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.) were carried out in sequence. The proteins were stained using an ECL chemiluminescence kit (Shanghai Beyotime Biotechnology Co., Ltd.), and each protein band was detected using a gel imaging system.

[0090] Figure 5 For western blotting to detect the expression levels of upregulated p-AMPK protein, downregulated HIF-1α protein, and PD-L1 protein in cells after treatment with different drugs. The expression level of p-AMPK protein in the drug-administered groups was higher than that in the blank group (phosphate buffer PBS), and the expression level of PD-L1 protein in the drug-administered groups was lower than that in the blank group, indicating that DOBG can affect the glycosylation of PD-L1 protein by activating AMPK and thus reduce the expression level of PD-L1. The expression level of HIF-α protein in the drug-administered groups was lower than that in the blank group, indicating that DOBG can inhibit mitochondrial oxidative respiratory chain complex I, thereby inhibiting oxygen consumption, alleviating tumor hypoxia, and reducing the expression of HIF-α.

[0091] As a control, the present invention synthesized nanoparticles that cannot degrade and remove PEG in tumor tissues, designated as T780@AIDOBG. The preparation method of T780@AIDOBG is exactly the same as that of T780@ASDOBG, and only DSPE-ASlink-PEG 2K needs to be replaced with an equimolar amount of DSPE-AIlink-PEG 2K to obtain T780@AIDOBG.

[0092] DSPE-AIlink-PEG 2K was prepared as follows: DSPE (0.374 g, 0.5 mmol), PEG 2K -COOH (1 g, 0.5 mmol, purchased from Chongqing Yusai Medical Technology Co., Ltd.), EDCI (0.115 g, 0.6 mmol), and HOBT (0.081 g, 0.6 mmol) were dissolved in 10 ml of chloroform, heated and stirred at 45 °C for 24 h, then the solution was concentrated and dropped into 40 ml of cold ether, and washed twice with 20 ml of cold ether to obtain 887 mg of DSPE-AIlink-PEG2K (yield: 65%).

[0093] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes may be made in its form and details without departing from the spirit and scope of the present invention defined by the appended claims.

Claims

1. A nano-liposome with the effect of alleviating tumor hypoxia, characterized in that, The nano-liposomes are prepared by the following method: N,N-dioleoyl-2-hydroxyethyl-bis(2-guanidinoethyl)amine (DOBG) and cholesterol form cationic liposomes, and the lipid-forming material DSPE-ASlink-PEG linked by an amide bond is inserted to prepare nano-liposomes. 2K , and nano-liposomes are prepared. The DSPE-ASlink-PEG 2K has the following structural formula: The N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine is a biguanide-based cationic lipid material, and its structural formula is as follows:

2. The nano-liposome with the effect of alleviating tumor hypoxia according to claim 1, characterized in that, Composed of distearoyl phosphatidylethanolamine DSPE and PEG 2K -CDM obtains the polyethylene glycol-phospholipid lipid-forming material DSPE-ASlink-PEG linked by an amide bond through an acylation reaction 2k ; The PEG 2K -CDM is prepared by the following method: Using 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionic acid as a raw material, oxalyl chloride is added to obtain 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionyl chloride; using 2,5-dihydroxy-4-methyl-2,5-dioxo-3-furanpropionyl chloride as a raw material, it undergoes an esterification reaction with PEG 2K -OH to obtain PEG 2K -CDM.

3. The nano-liposome with the effect of alleviating tumor hypoxia according to claim 2, characterized in that, The PEG 2K -CDM and DSPE are mixed, then triethylamine is added, and the mixture is heated and stirred for reaction to obtain DSPE-ASlink-PEG 2k ; The PEG 2K -CDM and DSPE have a molar ratio of 1:1 to 2.

4. The nano-liposome with the effect of alleviating tumor hypoxia according to claim 3, characterized in that, The solvent for the reaction is chloroform; the reaction temperature is 45-50 °C; the reaction time is 16-24 h.

5. The nano-liposome with the effect of alleviating tumor hypoxia according to claim 1, characterized in that, The particle size of the nano-liposomes is 100-250 nm.

6. A method for preparing the nano-liposome with the function of alleviating tumor hypoxia as described in claim 1 or 5, characterized in that, It includes the following steps: Mix N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine DOBG, cholesterol and DSPE-ASlink-PEG 2K at a mass ratio of 10 to 40:4:2 to 5, mix evenly, distill to obtain a lipid film, and ultrasonically hydrate the film to obtain a crude liposome solution; pass the crude liposome solution through a membrane to obtain a final nano-liposome solution.

7. The preparation method according to claim 6, characterized in that, The N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine is prepared by the following method: N-tert-butoxycarbonyl-N,N-(2-hydroxyethyl)ethylenediamine reacts with oleoyl chloride through an esterification reaction to obtain Boc-protected N,N-dioleoyl hydroxyethyl-2-aminoethylamine, and then N,N-dioleoyl hydroxyethyl-2-aminoethylamine is obtained by removing Boc, and finally N,N-dioleoyl hydroxyethyl-2-biguanidoethylamine is obtained through a nucleophilic addition reaction.

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