Preparation method of liposome with pH responsive membrane fusion effect and application thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a preparation method of a liposome with a pH response type membrane fusion effect and application of the liposome. The pyridium betaine-terminated lipid molecule provided by the application can be quickly and reversibly protonated in a weak acid microenvironment of a tumor, the surface charge of the liposome is changed from negative to positive, and the precise switch of the membrane fusion behavior is realized; the self-adaptive fusion liposome constructed based on the pyridium betaine-terminated lipid molecule successfully converts the non-specific toxicity risk of the traditional fusion liposome into a space-limited intelligent response, remains stable and inert in blood circulation (pH=7.4), and specifically activates the membrane fusion at a tumor site, realizes direct cytoplasmic delivery of a drug and avoids lysosome degradation; the STING nano agonist constructed by loading the nano composite of a STING pathway agonist and a divalent metal ion on the platform exhibits a strong pH-dependent anti-tumor immune activation effect in vitro and in vivo, and significantly inhibits tumor growth while the systemic toxicity is extremely low.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing liposomes with pH-responsive membrane fusion effect and their application. Background Technology

[0002] Membrane fusion-dependent drug delivery strategies have shown significant potential in cancer treatment due to their ability to bypass lysosomal capture and achieve direct cytoplasmic drug transport. These strategies typically rely on fusion liposomes with optimized lipid compositions, which bind to negatively charged cell membranes through electrostatic interactions, inducing membrane fusion and releasing their contents into the cytoplasm, thereby improving drug bioavailability and therapeutic efficacy. However, existing fusion systems often lack spatial selectivity after systemic administration, readily undergoing non-specific membrane fusion with non-target cells, leading to drug release in normal tissues and posing a risk of systemic toxicity, severely limiting their clinical translation.

[0003] To enhance the targeting of fusion behavior, researchers have developed surface modification strategies based on cleavable shielding groups. These strategies utilize tumor microenvironment characteristics (such as pH, enzyme activity, and reactive oxygen species levels) or external stimuli (such as light) to trigger the dissociation of shielding groups, thereby achieving tumor-specific activation of fusion activity. Although these strategies improve selectivity to some extent, they still have significant limitations: First, their sensitivity to microenvironment signals is limited, making it difficult to accurately distinguish between normal and tumor tissues; second, the regulatory process is mostly unidirectional and irreversible, meaning that once activated, it cannot be reversed. If activated liposomes re-permeate into the bloodstream due to tumor stroma hypertension or other reasons, they may still fuse with non-target cells, posing a safety risk.

[0004] Therefore, developing a novel intelligent liposome system that can respond rapidly, reversibly, and sensitively to the tumor microenvironment and achieve spatially confined fusion activity has become an urgent need to improve the safety and efficacy of cancer nanotherapy. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one object of the present invention is to provide a pyridine betaine-terminated lipid molecule.

[0006] A second objective of this invention is to provide a method for preparing such pyridine betaine-terminated lipid molecules.

[0007] The third objective of this invention is to provide an adaptive fusion liposome.

[0008] The fourth objective of this invention is to provide a method for preparing such adaptive fusion liposomes.

[0009] The fifth objective of this invention is to provide a STING nano-agonist.

[0010] The sixth objective of this invention is to provide an application of adaptive fusion liposomes or STING nano-agonists.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a pyridine betaine-terminated lipid molecule having the configuration shown in formula (I): Equation (Ⅰ); In equation (Ⅰ), R is selected from C 14 -C 18 A chain of amino hydrocarbons with saturated or unsaturated fatty acyl groups substituted; X is selected from hydrogen, alkyl or halogen; n is any integer selected from 1 to 3.

[0012] In some embodiments of the present invention, the pyridine betaine-terminated lipid molecule is: ; It is named 2-(4-((2,3-distearyloxy)propyl)carbamoyl)pyridin-1-onth-1-yl)acetate, abbreviated as DSPCPA.

[0013] A second aspect of the present invention provides a method for preparing the pyridine betaine-terminated lipid molecule described in the first aspect of the present invention, comprising the following steps: A1, C 14 -C 18 Saturated or unsaturated fatty acids are esterified with tert-butyl (2,3-dihydroxypropyl) carbamate in a halocarbon solvent under the action of an acylation catalyst and a condensing agent to obtain 3-((tert-butyloxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester. A2. 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester is reacted with an excess of strong organic acid in a halocarbon solvent to remove the tert-butoxycarbonyl group, thereby obtaining 3-aminopropane-1,2-dimethyldifatty acid ester. A3. 3-Aminopropane-1,2-dimethyldifatty acid ester and X-substituted isonicotinic acid are subjected to an amidation reaction in a polar aprotic solvent in the presence of a condensing agent, an activating agent and an organic base to obtain 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester. A4. 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester and n-membered halocarboxylic acid are subjected to quaternization reaction in a nitrile solvent to obtain the pyridine betaine-terminated lipid molecule. Wherein, X and n are defined as described in the first aspect of the present invention.

[0014] In some embodiments of the present invention, in step A1, the C 14 -C 18 The molar ratio of saturated or unsaturated fatty acids to tert-butyl (2,3-dihydroxypropyl) carbamate is (1.8-2.8):1.

[0015] In some preferred embodiments of the present invention, in step A1, the C 14 -C 18 The molar ratio of saturated or unsaturated fatty acids to tert-butyl (2,3-dihydroxypropyl) carbamate is (2.0-2.5):1.

[0016] In some embodiments of the present invention, in step A1, the C 14 -C 18 Saturated or unsaturated fatty acids include stearic acid.

[0017] In some embodiments of the present invention, in step A1, the ratio of the acylation catalyst, condensing agent, halocarbon solvent and tert-butyl (2,3-dihydroxypropyl) carbamate is: (0.15-0.25) mmol : (1.8-2.8) mmol : (15-25) mL : 1 mmol.

[0018] In some preferred embodiments of the present invention, in step A1, the ratio of the acylation catalyst, condensing agent, halocarbon solvent and tert-butyl (2,3-dihydroxypropyl) carbamate is: (0.18-0.22) mmol: (2.0-2.5) mmol: (18-22) mL: 1 mmol.

[0019] In some embodiments of the present invention, in step A1, the acylation catalyst is selected from one or more of 4-dimethylaminopyridine (DMAP), pyridine (Py), and triethylamine (TEA).

[0020] In some preferred embodiments of the present invention, in step A1, the acylation catalyst is DMAP.

[0021] In some embodiments of the present invention, in step A1, the condensing agent is selected from one or more of diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl).

[0022] In some preferred embodiments of the present invention, in step A1, the condensing agent is DIC.

[0023] In some embodiments of the present invention, in step A1, the halocarbon solvent is selected from one or more of dichloromethane, chloroform, and 1,2-dichloroethane.

[0024] In some preferred embodiments of the present invention, in step A1, the halogenated hydrocarbon solvent is dichloromethane.

[0025] In some embodiments of the present invention, in step A1, the esterification reaction is carried out at a temperature of 20-30°C for a time of 20-30 hours.

[0026] In some preferred embodiments of the present invention, in step A1, the esterification reaction is carried out at a temperature of 20-25°C for a time of 20-25 hours.

[0027] In some embodiments of the present invention, in step A1, the esterification reaction is carried out under an inert atmosphere; the inert atmosphere includes nitrogen.

[0028] In some embodiments of the present invention, after the esterification reaction is completed in step A1, the solid phase is further separated and collected, the acylation catalyst is washed away with 0.8-1.2 mol / L HCl, the organic layer is collected, and the organic layer is dried with anhydrous sodium sulfate and concentrated under vacuum to obtain the 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester.

[0029] In some embodiments of the present invention, in step A2, the molar ratio of the 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester to the strong organic acid is 1:(2-3).

[0030] In some preferred embodiments of the present invention, in step A2, the molar ratio of the 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester to the strong organic acid is 1:(2.2-2.8).

[0031] In some embodiments of the present invention, in step A2, the strong organic acid includes trifluoroacetic acid.

[0032] In some embodiments of the present invention, in step A2, the halocarbon solvent is selected from one or more of dichloromethane, chloroform, and 1,2-dichloroethane.

[0033] In some preferred embodiments of the present invention, in step A2, the halogenated hydrocarbon solvent is dichloromethane.

[0034] In some embodiments of the present invention, in step A2, the reaction temperature is 20-30°C and the time is 4.5-7.5 h.

[0035] In some preferred embodiments of the present invention, in step A2, the reaction temperature is 20-25°C and the time is 5-7 hours.

[0036] In some embodiments of the present invention, step A2 further includes rotary evaporation to remove the haloalkane solvent after the reaction is completed, precipitating the obtained residue in a dichloromethane / diethyl ether mixed solution, collecting the precipitate, and vacuum drying to obtain the 3-aminopropane-1,2-dimethyldifatty acid ester.

[0037] In some embodiments of the present invention, in step A3, the molar ratio of the 3-aminopropane-1,2-dimethyldifatty acid ester to the X-substituted isonicotinic acid is (0.8-1.2):1.

[0038] In some preferred embodiments of the present invention, in step A3, the molar ratio of the 3-aminopropane-1,2-dimethyldifatty acid ester to the X-substituted isonicotinic acid is (0.9-1.1):1.

[0039] In some embodiments of the present invention, in step A3, the ratio of the condensing reagent, activating reagent, organic base, polar aprotic solvent and 3-aminopropane-1,2-dimethyldifatty acid ester is (0.8-1.2) mmol: (0.8-1.2) mmol: (0.8-1.2) mmol: (20-30) mL: 1 mmol.

[0040] In some preferred embodiments of the present invention, in step A3, the ratio of the condensing reagent, activating reagent, organic base, polar aprotic solvent and 3-aminopropane-1,2-dimethyldifatty acid ester is (0.9-1.1) mmol: (0.9-1.1) mmol: (0.9-1.1) mmol: (22-27) mL: 1 mmol.

[0041] In some embodiments of the present invention, in step A3, the condensing agent is selected from one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-1-yl-oxytripyrrolidinephosphide hexafluorophosphate (PyBOP), and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU).

[0042] In some preferred embodiments of the present invention, in step A3, the condensation reagent is HATU.

[0043] In some embodiments of the present invention, in step A3, the activating agent is selected from one or more of 1-hydroxy-7-azobenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), and N-hydroxysuccinimide (NHS).

[0044] In some preferred embodiments of the present invention, in step A3, the activating agent is HOAt.

[0045] In some embodiments of the present invention, in step A3, the organic base is selected from one or more of N,N-diisopropylethylamine (DIEA), triethylamine (TEA), and 2,4,6-trimethylpyridine.

[0046] In some preferred embodiments of the present invention, in step A3, the organic base is DIEA.

[0047] In some embodiments of the present invention, in step A3, the polar aprotic solvent is selected from one or more of N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMAc).

[0048] In some preferred embodiments of the present invention, in step A3, the polar aprotic solvent is DMF.

[0049] In some embodiments of the present invention, in step A3, the amidation reaction is carried out at a temperature of 52-78°C for 3-5 hours.

[0050] In some preferred embodiments of the present invention, in step A3, the amidation reaction is carried out at a temperature of 60-70°C for a time of 3.5-4.5 h.

[0051] In some embodiments of the present invention, step A3 specifically includes: under an inert atmosphere and at 0°C, adding X-substituted isonicotinic acid, a condensing agent, an activating agent, and an organic base sequentially to an anhydrous polar aprotic solvent containing 3-aminopropane-1,2-distearate to carry out an amidation reaction to obtain the 3-(isonicotinamide)propane-1,2-dimethyldifatty acid ester.

[0052] In some embodiments of the present invention, after the amidation reaction is completed in step A3, ethyl acetate is added and the mixture is washed with saturated sodium chloride solution to remove water-soluble byproducts. The resulting organic layer is dried with anhydrous sodium sulfate and filtered. The crude product is concentrated under reduced pressure and purified by silica gel column chromatography to obtain the 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester.

[0053] In some embodiments of the present invention, in step A4, the molar ratio of the 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester to the n-membered halocarboxylic acid is 1:(2.5-3.5).

[0054] In some preferred embodiments of the present invention, in step A4, the molar ratio of the 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester to the n-membered halocarboxylic acid is 1:(2.7-3.3).

[0055] In some embodiments of the present invention, in step A4, the temperature of the quaternization reaction is 68-102°C and the time is 20-30 h.

[0056] In some preferred embodiments of the present invention, in step A4, the temperature of the quaternization reaction is 80-90°C and the time is 20-25 h.

[0057] In some embodiments of the present invention, in step A4, the nitrile solvent includes acetonitrile.

[0058] In some embodiments of the present invention, in step A4, the quaternization reaction is carried out under an inert atmosphere; the inert atmosphere includes nitrogen.

[0059] In some embodiments of the present invention, after the quaternization reaction is completed in step A4, the process further includes cooling and concentrating under reduced pressure to obtain a crude product, and then purifying it by recrystallization with a nitrile solvent to obtain the pyridine betaine-terminated lipid molecules.

[0060] A third aspect of the present invention provides an adaptive fusion liposome comprising a pyridine betaine-terminated lipid molecule as described in the first aspect of the present invention, as well as a neutral lipid and a polyethylene glycol-functionalized lipid.

[0061] In some embodiments of the present invention, the molar ratio of the pyridine betaine-terminated lipid molecule, the neutral lipid, and the polyethylene glycol functionalized lipid is (2-14): (5-37): 1.

[0062] In some preferred embodiments of the present invention, the molar ratio of the pyridine betaine-terminated lipid molecule, the neutral lipid, and the polyethylene glycol functionalized lipid is (2-4): (15-17): 1.

[0063] In some embodiments of the present invention, the pyridine betaine-terminated lipid molecule is 2-(4-((2,3-distearyloxy)propyl)carbamoyl)pyridin-1-onth-1-yl)acetate (DSPCPA).

[0064] In some embodiments of the present invention, the neutral lipid molecule is selected from one or more of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC).

[0065] In some preferred embodiments of the present invention, the neutral lipid molecule is DOPE.

[0066] In some embodiments of the present invention, the polyethylene glycol functionalized lipid comprises 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-methoxy (DSPE-PEG).

[0067] A fourth aspect of the present invention provides a method for preparing the adaptive fusion liposomes described in the third aspect of the present invention, comprising the following steps: B1. Pyridine betaine-terminated lipid molecules, neutral lipids, and polyethylene glycol-functionalized lipids were dissolved in a chlorinated hydrocarbon solvent, and the solvent was removed to obtain a lipid film. B2. The lipid film is subjected to hydration treatment to obtain the adaptive fusion liposome.

[0068] In some embodiments of the present invention, the chlorinated hydrocarbon solvent is selected from one or more of dichloromethane, chloroform, methanol, and ethanol.

[0069] In some preferred embodiments of the present invention, the chlorinated hydrocarbon solvent is chloroform.

[0070] In some embodiments of the invention, the hydration treatment includes hydrating the lipid film with sterile deionized water or phosphate-buffered saline (PBS).

[0071] In some embodiments of the present invention, after the hydration treatment is completed, the process further includes dialysis purification of the resulting mixture by placing it under sterile deionized water through a dialysis membrane with a molecular weight cutoff of 1600-2400 Da.

[0072] The fifth aspect of the present invention provides a STING nano-agonist having a core-shell structure; the core is a nanocomposite of the STING pathway agonist and a divalent metal ion; and the shell is the adaptive fusion liposome described in the third aspect of the present invention.

[0073] In some embodiments of the present invention, the molar ratio of the STING pathway agonist to the divalent metal ion is 1:(80-120).

[0074] In some preferred embodiments of the present invention, the molar ratio of the STING pathway agonist to the divalent metal ion is 1:(90-110).

[0075] In some embodiments of the present invention, the STING pathway agonist is selected from one of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic diguanosine monophosphate (c-di-GMP), cyclic diadenosine monophosphate (c-di-AMP), and MSA-2; the divalent metal ion includes Mn 2+ .

[0076] In some preferred embodiments of the present invention, the STING pathway agonist is cGAMP. In some embodiments of the present invention, the particle size of the STING nano-agonist is 150-250 nm.

[0077] In some preferred embodiments of the present invention, the particle size of the STING nano-agonist is 180-220 nm.

[0078] In some embodiments of the present invention, the STING nano-agonist is prepared by a method comprising the following steps: C1. The STING pathway agonist and divalent metal ions are coordinated and self-assembled in a solvent to form a nanocomposite, which is then purified to obtain a nanocomposite dispersion. C2. Using a thin-film hydration method, pyridine betaine-terminated lipid molecules, neutral lipids, and polyethylene glycol-functionalized lipids are dissolved in a chlorinated hydrocarbon solvent, and the solvent is removed to form a lipid film. The lipid film is then hydrated and purified using the nanocomposite dispersion to obtain the STING nano-agonist.

[0079] In some embodiments of the present invention, in step C1, the solvent includes methanol.

[0080] In some embodiments of the present invention, in step C1, the purification includes dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 800-1200 Da.

[0081] In some embodiments of the present invention, in step C2, the purification includes dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 1600-2400 Da.

[0082] The sixth aspect of the present invention provides the use of the adaptive fusion liposomes described in the third aspect of the present invention, or the STING nano-agonist described in the fifth aspect of the present invention, in the preparation of antitumor nanomedicines.

[0083] Compared with the prior art, the beneficial effects of the present invention are: The pyridinium betaine-terminated lipid molecules provided by this invention exhibit rapid and reversible protonation of the pyridinium betaine head group in the weakly acidic tumor microenvironment, driving the liposome surface charge to change from negative to positive, thus achieving precise switching of membrane fusion behavior. Based on this, an adaptive fusion liposome is constructed, successfully transforming the non-specific toxicity risk of traditional fusion liposomes into a spatially confined intelligent response. It remains stable and inert in blood circulation (pH=7.4), while specifically activating membrane fusion at the tumor site, achieving direct cytoplasmic drug delivery and avoiding lysosomal degradation. A core-shell STING nano-agonist constructed by loading a nanocomposite of a STING pathway agonist and divalent metal ions onto this platform exhibits a strong pH-dependent anti-tumor immune activation effect both in vivo and in vitro, significantly inhibiting tumor growth, while exhibiting extremely low systemic toxicity. This delivery system based on adaptive fusion liposomes has advantages such as high spatial selectivity, excellent transport efficiency, simple operation, and strong universality, showing broad clinical potential in biomedical applications beyond tumor therapy. Attached Figure Description

[0084] Figure 1 The 1H NMR spectrum (a) and mass spectrum (b) of 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldistearate in Example 1 are shown. Figure 2 The 1H NMR spectrum (a) and mass spectrum (b) of 3-(isonicotinamide)propane-1,2-dimethyldistearate in Example 1 are shown. Figure 3 The 1H NMR spectrum (a) and mass spectrum (b) of the pyridine betaine-terminated lipid molecule in Example 1 are shown. Figure 4 A schematic diagram of the pH-dependent hydration behavior of DSPCPA; Figure 5 The experimental results validate the pH-dependent hydration mechanism of DSPCPA. Figure 6 Cellular behavior of adaptive fusion liposomes with different labels at pH 7.4 and 6.0; Figure 7 For pH=7.4 and 6.0 conditions with DiD-SENDFUL FITC / DiD-unSENDFUL FITC Confocal laser scanning microscope image of co-incubated HeLa cells; Figure 8 Analysis of the uptake of DiD-unSENDFUL(a) and DiD-SENDFUL(b) by HeLa cells pretreated with membrane fusion inhibitors or endocytosis inhibitors at pH 7.4 and 6.0. Figure 9To adapt the pH-responsive cell internalization transition behavior of fused liposomes; Figure 10 The results of a study on the pH-responsive reversibility of adaptive fusion liposome internalization transition; Figure 11 Results of a study on the pH-dependent lysosomal escape ability of adaptive fusion liposomes; Figure 12 cGMn, SENDFUL cGMn and unSENDFUL cGMn Transmission electron microscopy images under different pH conditions; Figure 13 4T1 cells were subjected to different concentrations of cGMn(a) and unSENDFUL at different pH conditions. cGMn (b) SENDFUL cGMn (c) and SENDFUL(d) cell viability after treatment; Figure 14 4T1 cells were subjected to different pH conditions via cGMn and unSENDFUL cGMn and SENDFUL cGMn Processed live / dead stained fluorescence images; Figure 15 4T1 cells were subjected to different pH conditions via cGMn and unSENDFUL cGMn and SENDFUL cGMn Secretion levels of IFNβ(a) and TNFα(b) after treatment; Figure 16 A schematic diagram of the experimental timeline for the in vivo biodistribution study of the 4T1 tumor model (a), including injections of DiD-cGMn and DiD-unSENDFUL. cGMn and DiD-SENDFUL cGMn In vivo fluorescence images of mice (b) and quantitative analysis results of the mean fluorescence intensity of the tumor region in the in vivo fluorescence images (c); Figure 17 The average tumor growth kinetic curves after different treatments (a), the survival curves of 4T1-Luc tumor-bearing mice (b), and in vivo bioluminescence imaging of 4T1-Luc tumor growth in mice (c) are shown. Figure 18 The levels of IFNβ(a) and TNFα(b) cytokines in mouse tumors after different treatments; Figure 19 MHCII+ mature dendritic cells (a) and CD4+ in isolated tumor tissues of mice after different treatments. + T cells (b), CD8 + T cells (c), MHCII +Macrophages (d), CD80 + Macrophages (e), IFNγ + NK cells (f) and Foxp3 + The proportion of regulatory T cells (g); Figure 20 The results show the levels of alanine aminotransferase (a), aspartate aminotransferase (b), total bilirubin (c), alkaline phosphatase (d), creatinine (e), urea (f), creatine kinase isoenzyme MB (g), and lactate dehydrogenase (h) in the serum of tumor-bearing mice after different treatments. Detailed Implementation

[0085] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0086] Example 1 This embodiment prepares a pyridine betaine-terminated lipid molecule (DSPCPA) using the following steps: A11. Stearic acid (0.65 g, 2.3 mmol), tert-butyl(2,3-dihydroxypropyl)carbamate (0.19 g, 1.0 mmol), 4-dimethylaminopyridine (25 mg, 0.2 mmol), and diisopropylcarbodiimide (0.439 g, 2.3 mmol) were dissolved in dichloromethane (20 mL). The mixture was stirred at room temperature under nitrogen protection for 24 h. After the reaction was completed, the resulting mixture was filtered to remove the precipitate. 4-Dimethylaminopyridine was removed by washing with 1 mol / L HCl. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under vacuum. The resulting white solid product was 3-((tert-butyloxycarbonyl)amino)propane-1,2-dimethyldistearate, with a yield of 70%. A21. 3-((tert-Butoxycarbonyl)amino)propane-1,2-dimethyl distearate was dissolved in dichloromethane, and trifluoroacetic acid (5 mL) was added. The reaction was carried out at room temperature for 6 h. After the reaction was completed, dichloromethane was removed by rotary evaporation. The residue was precipitated in a dichloromethane / diethyl ether mixed solution. The white precipitate was collected and dried under vacuum. The resulting white solid product was 3-aminopropane-1,2-dimethyl distearate, with a yield of 80%. A31. Under a nitrogen atmosphere and at 0°C, isonicotinic acid (48.09 mg, 0.39 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (148.59 mg, 0.39 mmol), 1-hydroxy-7-azobenzotriazole (53 mg, 0.39 mmol), and N,N-diisopropylethylamine (100 mg, 0.78 mmol) were added sequentially to an anhydrous N,N-dimethylformamide (25 mL) containing 3-aminopropane-1,2-distearate (200 mg, 0.39 mmol), and stirred at 65°C for 4 h. After the reaction was complete, ethyl acetate (50 mL) was added, and the mixture was washed twice (50 mL each time) with saturated sodium chloride solution to remove water-soluble byproducts. The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was then analyzed by silica gel column chromatography using petroleum ether / ethyl acetate (1:1) Purification was performed using a gradient elution system (1, v / v) to obtain a white powdery product, which is 3-(isonicotinamide)propane-1,2-dimethyl distearate, with a yield of 65%. A41. Under a nitrogen atmosphere, 3-(isonicotinamide)propane-1,2-dimethyl distearate (200 mg, 0.27 mmol) and bromoacetic acid (114 mg, 0.82 mmol) were added to acetonitrile and stirred at 85 °C for 24 h to quaternize the pyridine group. After the reaction was completed, the mixture was cooled and concentrated under reduced pressure to obtain a pale yellow powdery crude product. The crude product was purified by recrystallization from acetonitrile, and the resulting white powdery product was the pyridine betaine-terminated lipid molecule with a yield of 60%.

[0087] 1. The intermediate and final products in Example 1 were characterized by proton NMR spectroscopy, carbon NMR spectroscopy, and high-resolution mass spectrometry: Figure 1 The 1H NMR spectrum (a) and mass spectrum (b) of 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyl distearate in Example 1 are shown. The solvent used to dissolve the sample was deuterated dimethyl sulfoxide (d6-DMSO). The 1H NMR and high-resolution mass spectrometry characterization data of 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyl distearate are as follows: 1H NMR (600 MHz, DMSO) δ 7.90 (s, 2H), 5.18 (td, J=6.3, 3.3 Hz, 1H), 4.32 (dd, J=12.1, 3.3 Hz, 1H), 4.04 (dd, J=12.2, 6.6 Hz, 1H), 3.17-2.98 (m,2H), 2.38-2.25 (m, 4H), 1.50 (d, J=15.4 Hz, 4H), 1.23 (s, 56H), 0.84 (t, J=7.0 Hz, 6H). ESI-MS: m / z [M+H] + Calculated for C 39 H 77 NO4 + : 634.5925; found: 624.5923. Figure 2 The 1H NMR spectrum (a) and mass spectrum (b) of 3-(isonicotinamide)propane-1,2-dimethyldistearate in Example 1 are shown. The solvent used to dissolve the sample was deuterated chloroform. The 1H NMR and high-resolution mass spectrometry characterization data of 3-(isonicotinamide)propane-1,2-dimethyldistearate are as follows: 1 H NMR (600 MHz, CDCl3) δ 8.77 (d, J=5.2 Hz, 2H), 7.70 (d, J=5.1 Hz, 2H), 6.96 (s, 1H), 5.32-5.16 (m, 1H), 4.37-4.22 (m, 2H), 3.69 (q, J=6.0 Hz,2H), 2.35 (td, J=7.5, 1.5 Hz, 4H), 1.62 (qd, J=7.4, 2.4 Hz, 4H), 1.24 (d, J=7.9 Hz, 56H), 0.88 (t, J=7.0 Hz, 6H). ESI-MS: m / z [M+H] + Calculated for C 45 H 81 N2O5 + : 729.6140; found: 729.6139. Figure 3The images (a) and (b) show the 1H NMR spectrum and mass spectrum of the pyridine betaine-terminated lipid molecules in Example 1. The solvent used to dissolve the sample was deuterated chloroform. The 1H NMR and high-resolution mass spectrometry characterization data of the pyridine betaine-terminated lipid molecules are as follows: 1 H NMR (600 MHz, CDCl3, ppm): 9.56 (s, 1H), 9.32-8.95 (m, 2H), 8.79 (d, J=5.3 Hz, 2H), 5.39-5.32 (m, 1H), 4.67 (s, 2H), 4.34 (dd, J=12.1, 3.5 Hz, 1H), 4.20 (dd, J=12.1, 6.3 Hz, 1H), 3.79-3.73 (m, 1H), 3.68-3.64 (m, 1H), 2.37-2.30 (m, 4H), 1.58 (q, J=6.6 Hz, 4H), 1.29-1.22 (m, 56H), 0.88 (t, J=7.0Hz, 6H). ESI-MS: m / z [M+H] + Calculated for C 47 H 83 N2O7 + : 787.6197; found: 787.6195. The above data indicate that pyridine betaine-terminated lipid molecules with the target structure were successfully prepared in Example 1.

[0088] Figure 4 This is a schematic diagram of the pH-dependent hydration behavior of DSPCPA, by Figure 4 It is known that the hydration / protonation process of DSPCPA is reversible and pH-regulated; when pH > 7.0, the hydrophilic head group of DSPCPA combines with a water molecule to form a DSPCPA-H2O complex. In this state, the head group retains the characteristics of a zwitterion, and the whole is electrically neutral or weakly negatively charged; when pH < 7.0, the same DSPCPA head group combines with a water molecule to form a DSPCPA-H2O complex. + Combined, it is transformed into DSPCPA-H3O + Complex, H3O + The proton (H) + The charge transfers to the basic sites of the head group, resulting in the head group carrying a net positive charge, i.e., DSPCPA-H3O. + The sudden increase in the binding energy of the complex is due to the enhanced positive charge of the pyridine nitrogen atom and the enhanced negative charge of the carboxylic acid oxygen atom under acidic conditions, which together drive the formation of high-strength hydrogen bonds.

[0089] 2. Experimental verification of the pH-dependent hydration mechanism of the DSPCPA prepared in Example 1, including: 1) Hydrated DSPCPA (DSPCPA-H2O complex) was equilibrated in deionized water at different pH values ​​(6.0, 6.4, 6.8, 7.2, 7.4) for 15 min, then freeze-dried and redissolved in deuterated chloroform. Nuclear magnetic resonance spectroscopy (NMR) was used to collect data at 25°C using a 600 MHz NMR spectrometer. 1 H NMR spectrum; 2) To evaluate the response kinetics, hydrated DSPCPA was treated in an environment with pH 6.5 at a time gradient (0-8 min) and detected using the same NMR analysis method; 3) The change in zeta potential of DSPCPA under different pH conditions was determined using dynamic light scattering (DLS) technology. Figure 5 The experimental results validate the pH-dependent hydration mechanism of DSPCPA, among which, Figure 5 (a) shows the zeta potential of DSPCP under different pH conditions. It can be seen that the surface charge of DSPCP is neutral (+0.7mV) at physiological pH (7.4), but becomes strongly positive (+26.9mV) under acidic conditions (pH=5.0), which directly proves the protonation-induced hydration. Figure 5 (b) shows the segmented proton NMR spectra of hydrated DSPCP after exposure to deionized water at different pH conditions for 15 min. Figure 5 (c) shows the segmented proton NMR spectra of hydrated DSPCPA after exposure to deionized water at pH 6.5 for different times. It can be seen that the resonance peak of hydrated DSPCPA migrates from 2.10 ppm (peak a) at pH 7.4 to 1.70 ppm (peak b) at pH 6.0. Moreover, this protonation behavior can be completed rapidly within 2 min after exposure to pH 6.5 environment. This indicates that DSPCPA can be rapidly hydrated in acidic water environment, which lays a solid foundation for sensitive pH-dependent regulation of liposome surface charge to manage its membrane fusion behavior.

[0090] Example 2 This embodiment prepares an adaptive fusion liposome, and the steps are as follows: B11. Dissolve DOPE, DSPCPA and DSPE-PEG prepared in Example 1 in chloroform to obtain a stock solution of 1 mg / mL. B12. Mix the stock solutions in a 10 mL round-bottom flask with a molar ratio of DOPE, DSPCPA and DSPE-PEG of 85 / 10 / 5. Remove chloroform by rotary evaporation at 100 rpm. Place the flask in a vacuum drying oven overnight to obtain a lipid film. B21. The dried lipid membrane was hydrated with sterile deionized water, and the resulting mixture was purified by dialysis under sterile deionized water through a dialysis membrane with a molecular weight cutoff of 2000 Da to obtain adaptive fusion liposomes.

[0091] 1. Optimize the optimal ratio of lipid components in adaptive fusion liposomes: First, the fusion behavior of adaptive fusion liposomes with cell membranes was visualized using confocal laser scanning microscopy (CLSM). Then, the membrane fusion efficiency was precisely quantified using flow cytometry (FCM) to determine the optimal ratio of each lipid component. In both CLSM visualization and FCM quantitative analysis, a series of adaptive fusion liposomes were prepared using the thin-film hydration method. The molar ratios of the lipid components DOPE, DSPCPA, and DSPE-PEG were 95 / 0 / 5, 85 / 10 / 5, 75 / 20 / 5, 65 / 30 / 5, 55 / 40 / 5, 45 / 50 / 5, 35 / 60 / 5, and 25 / 70 / 5, respectively. The specific steps are as follows: CLSM Visualization Observation: 1) Following the above liposome preparation method, 1 mol% of DiD dye was added to the total lipid components to obtain DiD-labeled adaptive fusion liposomes; 2) Arrange HeLa cells at a density of 8 × 10⁸ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 6-well plates pre-filled with sterile round coverslips. 2 mL of DMEM medium was added to each well, and the plates were incubated at 37°C for 24 h. The medium was then replaced with 1 mL of DMEM medium containing different DiD-labeled adaptive fusion liposomes (55 µg / mL), and co-incubated for another 2 h. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, stained with TRITC-phalloidin and DAPI, and finally photographed on a CLSM.

[0092] Quantitative analysis of FCM: 1) BODIPY FL-DHPE labeled adaptive fusion liposomes were prepared using the same thin-film hydration method as observed in CLSM visualization, wherein the amount of BODIPY FL-DHPE added was 2 mol% of the total lipid content. 2) Mix HeLa cells at a density of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells per well in 12-well plates, with 1 mL of DMEM medium per well. After incubation at 37°C for 24 h, the medium was replaced with 0.5 mL of DMEM medium containing different BODIPY FL-DHPE labeled adaptive fusion liposomes (55 µg / mL), and co-incubated for another 2 h. After incubation, the supernatant was removed, and the cells were washed three times with PBS, digested with trypsin, and collected. Finally, flow cytometry was used to detect and analyze the cells at a laser excitation wavelength of 488 nm and an emission wavelength of 690 nm.

[0093] Figure 6 Cellular behavior of adaptive fusion liposomes with different labels under pH 7.4 and 6.0 conditions, where, Figure 6 (a) shows CLSM images of different DiD-labeled adaptive fusion liposomes after treatment at pH 7.4 and 6.0. Cells treated with DiD-labeled adaptive fusion liposomes with a molar ratio of DOPE, DSPCPA, and DSPE-PEG of 85 / 10 / 5 showed a significant DiD signal (red) localized along the HeLa cell edge at pH 6.0, while this signal diffused into the cell interior at pH 7.4. Other DiD-labeled adaptive fusion liposomes did not show significant differences in pH-dependent internalization patterns. This phenomenon suggests that the formulation with a molar ratio of DOPE, DSPCPA, and DSPE-PEG of 85 / 10 / 5 may achieve cellular uptake through membrane fusion under acidic conditions, while endocytosis is the primary mechanism under physiological conditions. Figure 6 (b) shows the membrane fusion efficiency of different BODIPY FL-DHPE labeled adaptive fusion liposomes on HeLa cells at pH 7.4 and 6.0. It can be seen that the BODIPY FL-DHPE labeled adaptive fusion liposome with a molar ratio of DOPE, DSPCPA and DSPE-PEG of 85 / 10 / 5 can achieve a higher membrane fusion efficiency at pH 6.0, but the efficiency is relatively low at pH 7.4. Other BODIPY FL-DHPE labeled adaptive fusion liposomes have similar membrane fusion efficiencies under these two pH conditions.

[0094] Based on the above results, the molar ratio of lipid components DOPE, DSPCPA, and DSPE-PEG (85 / 10 / 5) was determined as the final formulation of adaptive membrane fusion liposomes and used in subsequent studies. As a control, lipid components DMPC, DOTAP, and DSPE-PEG with a molar ratio of 76 / 19 / 5 were selected as the final formulation of traditional non-adaptive membrane fusion liposomes and used in subsequent experiments.

[0095] 2. Investigating the pH-dependent cellular uptake behavior of adaptive fusion liposomes: Prepared dual-labeled adaptive fusion liposomes and adaptive fusion liposomes with FITC fluorescent dye loaded in their lumen and lipid layer labeled with DiD, denoted as DiD-SENDFUL FITC / DiD-unSENDFUL FITC The steps are as follows: 1) FITC was dissolved in deionized water to prepare a 0.1 mg / mL solution, which was then filtered through a 0.22 μm filter membrane for sterilization; subsequently, DiD-SENDFUL was prepared using the membrane hydration method. FITC With DiD-unSENDFUL FITC A lipid membrane was formed, and DiD (1 mol% of the total lipid content) was added during the membrane formation process. The lipid membrane was hydrated with FITC solution, and the resulting mixture was purified by dialysis under sterile deionized water through a dialysis membrane with a molecular weight cutoff of 2000 Da to obtain dual-labeled liposomes DiD-SENDFUL. FITC With DiD-unSENDFUL FITC ; 2) HeLa cells were treated according to the aforementioned CLSM experimental method at pH 7.4 and 6.0, respectively, and DiD-SENDFUL was used. FITC Or DiD-unSENDFU LFITC Co-incubate at (55µg / mL) for 2 h, then perform fixation, staining, and CLSM imaging analysis.

[0096] Figure 7 For pH=7.4 and 6.0 conditions with DiD-SENDFUL FITC / DiD-unSENDFUL FITC Confocal laser scanning microscopy images of co-incubated HeLa cells, by Figure 7 It can be seen that DiD-SENDFUL FITC The treated cells exhibited drastically different internalization patterns under different pH conditions: at pH 7.4, endocytosis was the dominant pathway, characterized by high overlap between DiD and FITC signals within the cell; however, when the environment changed to pH 6.0, the internalization mode shifted to membrane fusion, with DiD signals (red) localizing to the outer cell membrane while FITC signals (green) diffused into the cytoplasm; in contrast, DiD-unSENDFUL FITC Under different pH conditions, it exhibits typical membrane fusion characteristics, that is, DiD signal (located on the membrane) and FITC signal (located in the cytoplasm) are always separated in the cell.

[0097] 3. Investigating the intracellular delivery mechanism of adaptive fusion liposomes: Pharmacological inhibition studies were conducted using membrane fusion inhibitors and endocytosis inhibitors, including Z-Phe-Phe-Phe-OH, chlorpromazine, amiloride, nystatin, and methyl-β-cyclodextrin, following the steps outlined below: 1) Prepare DiD-labeled adaptive fusion liposomes, denoted as DiD-SENDFUL, and non-adaptive fusion liposomes as controls, denoted as DiD-unSENDFUL; 2) Under pH conditions of 6.0 and 7.4, HeLa cells were introduced at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 cells / well in 12-well plates, with 1 mL of DMEM medium per well, and cultured at 37°C for 24 h. Cells were then pretreated for 30 min with DMEM containing different inhibitors (Z-Phe-Phe-Phe-OH 100 μg / mL, chlorpromazine 10 μg / mL, amiloride 100 μg / mL, nystatin 15 μg / mL, or methyl-β-cyclodextrin 6 μg / mL). After pretreatment, the DMEM medium was replaced with 55 µg / mL DiD-SENDFUL / DiD-unSENDFUL medium, and incubated for another 2 h. Subsequently, the cells were washed with PBS, digested with trypsin, and detected by flow cytometry at an excitation wavelength of 638 nm and an emission wavelength of 670 nm to quantify the mean fluorescence intensity (MFI), which was then normalized according to the following formula: MFI (normalized) = MFI (experimental group) - MFI (negative control) / MFI (positive control) - MFI (negative control) × 100%.

[0098] Figure 8 The uptake of DiD-unSENDFUL(a) and DiD-SENDFUL(b) by HeLa cells pretreated with membrane fusion inhibitors or endocytosis inhibitors at pH 7.4 and 6.0 was analyzed. Statistical analysis was performed using one-way ANOVA combined with Fisher's LSD test. ,Depend on Figure 8 It was found that DiD-unSENDFUL was significantly inhibited only by Z-Phe-Phe-Phe-OH at both pH 7.4 and 6.0 (inhibition rate > 56%), indicating that it maintains its membrane fusion-based memory pathway under different pH conditions. For DiD-SENDFUL, its cellular uptake was inhibited only by Z-Phe-Phe-Phe-OH at pH 6.0, but when the environment changed to pH 7.4, both chlorpromazine and Z-Phe-Phe-Phe-OH significantly inhibited its memory transport, indicating that there are essential differences in its cellular transport mechanism under different pH conditions.

[0099] 4. Investigating the pH-responsive intracellular transformation behavior of adaptive fused liposomes: The membrane fusion behavior of adaptive fusion liposomes under different pH conditions was observed using confocal laser scanning microscopy, and the membrane fusion efficiency was quantitatively analyzed using flow cytometry. The steps are as follows: 1) Prepare DiD-labeled adaptive fusion liposomes DiD-SENDFUL and traditional DiD-labeled non-adaptive fusion liposomes DiD-unSENDFUL. Under pH conditions of 6.0-7.4 (interval of 0.2 pH units), HeLa cells were co-incubated with culture medium containing 55 µg / mL DiD-SENDFUL / DiD-unSENDFUL. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, stained with TRITC-phalloidin and DAPI, and finally observed on CLSM. 2) Prepare BODIPY FL-DHPE labeled adaptive fusion liposomes and conventional non-adaptive fusion liposomes, denoted as BODIPY FL-DHPE-SENDFUL and BODIPY FL-DHPE-unSENDFUL, respectively; process according to CLSM method and co-incubate with HeLa cells. After incubation, remove supernatant, wash cells three times with PBS, collect by trypsin digestion, and finally detect and analyze by flow cytometry. 3) Adaptive fusion liposomes SENDFUL were prepared and analyzed using differential scanning calorimetry (DSC). The pre-prepared samples were equilibrated in deionized water with a pH gradient (6.0-7.4, 0.2 unit intervals). Thermal scanning was performed at a heating rate of 10℃ / min within a temperature range of -20 to 80℃, and the characteristic phase transition temperature (T0) between the gel and liquid crystal states was recorded under each pH condition. m ); 4) SENDFUL and unSENDFUL were prepared and equilibrated by dialysis in buffer solutions with pH values ​​of 6.0-7.4 (0.2 pH units apart) to prepare liposome solutions under different pH conditions. Subsequently, their zeta potentials were measured using dynamic light scattering technology.

[0100] Figure 9 To adapt the pH-responsive cell internalization transition behavior of fused liposomes, wherein, Figure 9 (a) shows CLSM images of HeLa cells co-cultured with DiD-SENDFUL / DiD-unSENDFUL under different pH conditions. Figure 9(b) shows the Pearson correlation coefficient between the green and red fluorescence signals calculated using ImageJ software based on the CLSM image in (a). It can be seen that when the pH decreases from 7.4 to 6.0, the confocal image shows that the colocalization of the DiD signal (red) and the cell membrane (green) gradually increases, which proves that pH precisely regulates the membrane fusion behavior of DiD-unSENDFUL. DiD-unSENDFUL interacts with cells in a membrane fusion manner under all pH conditions. The Pearson correlation coefficient analysis further reinforces this trend. Figure 9 (c) shows the membrane fusion efficiency of HeLa cells co-incubated with BODIPY FL-DHPE-SENDFUL / unSENDFUL under different pH conditions. It can be seen that the fusion efficiency of BODIPY FL-DHPE-SENDFUL gradually increased from 32.5% at pH=7.4 to 81.1% at pH=6.0, while the fusion efficiency of BODIPY FL-DHPE-unSENDFUL changed very little throughout the pH range, highlighting the specific response of the adaptive fusion liposome in vivo in vivo mechanism to the acidic environment. Figure 9 In Figure (d), the Zeta potential of SENDFUL and unSENDFUL changes with pH. It can be seen that due to the protonation of pyridinium betaine on the surface, the potential of SENDFUL changes from negative to positive, while unSENDFUL does not exhibit this phenomenon. Figure 9 (e) shows the change in phase transition temperature of SENDFUL with pH. It can be seen that the phase transition temperature of SENDFUL is 12-13℃, and the phase transition temperature of liposomes does not change significantly in the pH range of 6.0-7.4, ruling out the influence of lipid bilayer phase transition on the internalization mechanism. That is, the pH-dependent membrane fusion behavior of SENDFUL should be attributed to the change in surface charge rather than the lipid phase transition. These results consistently indicate that electrostatic interaction is the main cause of membrane fusion transition under acidic conditions, consistent with previous reports.

[0101] 5. To investigate the reversibility of the pH response to the internalization of adaptive fused liposomes in cells, the steps are as follows: 1) Preparation of DiD-SENDFUL, BODIPY FL-DHPE-SENDFUL, and SENDFUL; 2) DiD-SENDFUL was dialyzed alternately between pH 6.0 and 7.4 for a total of three complete cycles. After each dialysis, the liposomes were co-incubated with HeLa cells at a concentration of 55 µg / mL. After incubation, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, stained with TRITC-phalloidin and DAPI, and finally photographed on CLSM. 3) BODIPY FL-DHPE-SENDFUL was processed according to the CLSM method and co-incubated with HeLa cells. After incubation, the supernatant was removed, the cells were washed three times with PBS, digested with trypsin and collected, and finally analyzed by flow cytometry. 4) SENDFUL was subjected to alternating dialysis treatment between pH 6.0 and 7.4 for a total of three complete cycles. After each dialysis, its zeta potential was measured using dynamic light scattering technology.

[0102] Figure 10 The results of the study on the pH-responsive reversibility of adaptive fusion liposome internalization transition were presented, among which... Figure 10 Image (a) shows a CLSM image of HeLa cells co-incubated with DiD-SENDFUL after alternating dialysis treatment. Figure 10 (b) shows the membrane fusion efficiency of HeLa cells co-incubated with BODIPY FL-DHPE-SENDFUL after alternating dialysis treatment. Figure 10 (c) in the figure represents the Zeta potential of SENDFUL after alternating dialysis treatment. It can be seen that within three cycles, the DiD fluorescence signal (red) and the cell membrane (green) consistently colocalize under pH=6.0 conditions, while the degree of overlap decreases significantly under pH=7.4 conditions. The membrane fusion efficiency measured by flow cytometry also confirms this cyclic membrane fusion switching behavior. The Zeta potential analysis further clarifies that the reversible surface charge transition originates from the reversible conformational change of the pyridinium betaine molecule, which is the main reason for the cyclic membrane fusion switching behavior, rather than other factors such as hydrodynamic size or polydispersity index.

[0103] 6. Investigating the pH-dependent lysosomal escape ability of adaptive fusion liposomes: Membrane fusion-mediated internalization can directly release the load into the cytoplasm, thereby avoiding lysosomal capture. The steps are as follows: Prepare a FITC-labeled SENDFUL, denoted as SENDFUL. FITC SENDFUL FITC HeLa cells were incubated at a concentration of 55 µg / mL in a medium containing a pH gradient (6.0–7.4, with 0.2 pH units in between), and their lysosomal transport was then observed using a confocal microscope.

[0104] Figure 11 The results of the study on the pH-dependent lysosomal escape ability of adaptive fusion liposomes, among which, Figure 11 (a) shows the intracellular SENDFUL values ​​in HeLa cells under different pH conditions. FITC CLSM images of lysosomal escape Figure 11(b) shows the Pearson correlation coefficient between the green and purple fluorescence signals calculated using ImageJ software based on the CLSM image in (a). It can be seen that as pH decreases, the overlap between the FITC signal (green) and the lysosomal signal (purple) is significantly reduced, indicating that the acidic tumor microenvironment can induce SENDFUL to exert a membrane fusion effect, directly delivering its internal load to the cytoplasm, thereby avoiding lysosomal degradation. In summary, SENDFUL can reversibly turn its membrane fusion behavior on / off in both the tumor microenvironment (pH 6.5) and the physiological environment (pH 7.4), and is expected to achieve precise localization of cytoplasmic delivery in the acidic tumor microenvironment.

[0105] Example 3 This embodiment prepares a STING nano-agonist, and the steps are as follows: C11. Dissolve cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and MnCl2 in methanol to prepare stock solutions of 300 µmol / L and 100 mmol / L, respectively; under vigorous stirring, dissolve cGAMP and MnCl2 in methanol according to the following concentrations. 2+ The molar ratio of MnCl2 stock solution to cGAMP stock solution was 1:100. After sonication for 1 min, the mixture was stirred at room temperature for 30 min. The resulting product was purified by dialysis in sterile deionized water using a dialysis membrane with a molecular weight cutoff of 1000 Da to obtain cGAMP / MnCl2 stock solution. 2+ Dispersion of nanocomposite (denoted as cGMn); C21. Dissolve DOPE, DSPCPA, and DSPE-PEG separately in chloroform to obtain stock solutions of 1 mg / mL. Mix the stock solutions in a 10 mL round-bottom flask at a molar ratio of 85 / 10 / 5 for DOPE, DSPCPA, and DSPE-PEG. Remove the chloroform by rotary evaporation at 100 rpm. Place the flask in a vacuum drying oven overnight to obtain a lipid film. Hydrate the lipid film using cGMn dispersion. Dialyze the resulting mixture using a dialysis membrane with a molecular weight cutoff of 2000 Da in sterile deionized water to obtain the STING nano-agonist, denoted as SENDFUL. cGMn Store at 4℃ for later use.

[0106] Non-adaptive fusion liposomes loaded with cGMn were prepared using the same method as described above, the only difference being that in step C21, the lipid components were DMPC, DOTAP, and DSPE-PEG in a molar ratio of 76 / 19 / 5, denoted as unSENDFUL. cGMn .

[0107] 1. Regarding cGMn and SENDFUL cGMn and unSENDFUL cGMn Physicochemical characterization: Figure 12 cGMn, SENDFUL cGMn and unSENDFUL cGMn Transmission electron microscopy images under different pH conditions, Table 1 shows cGMn and SENDFUL. cGMn unSENDFUL cGMn The average particle size and zeta potential of SENDFUL and unSENDFUL are determined by... Figure 12 As shown in Table 1, compared to the homogeneous form of a single cGMn kernel, SENDFUL cGMn The spherical particles exhibiting a distinct core-shell structure showed an average particle size increasing from 101.7 nm to 201.2 nm, and a zeta potential changing from -16.8 mV to -4.6 mV, further confirming the core-shell structure. cGMn Successful preparation.

[0108] Table 1, cGMn, SENDFUL cGMn unSENDFUL cGMn Physicochemical properties of SENDFUL and unSENDFUL

[0109] 2. Regarding cGMn and SENDFUL cGMn and unSENDFUL cGMn In vitro cytotoxicity tests were conducted: 4T1 cells were loaded at a rate of 1×10⁴ cells per well. 4 The cells were seeded at a density of 100 μL of culture medium per well in 96-well plates and incubated at 37°C for 24 h. cGMn and unSENDFUL were then treated with RPMI 1640 medium at pH 7.4 or 6.5, respectively. cGMn and SENDFUL cGMn The solutions were equilibrated by dialyzing and then diluted to a cGAMP concentration gradient (200, 150, 100, 50, 12.5, 6.25, 3.12 μmol / L), with SENDFUL as a control at concentrations ranging from 0 to 1.0 mg / mL (intervals of 0.2 mg / mL). The cell culture medium was then replaced with 100 μL of different treatment groups (PBS, cGAMP, SENDFUL, SENDFUL). cGMn or unSENDFUL cGMnThe cells were incubated in RPMI 1640 medium (pH 6.5 or 7.4) with the above-mentioned cGAMP concentration gradient. After 12 h of incubation, the medium was removed, and 30 μL of MTT solution (3 mg / mL dissolved in PBS) was added to each well. The cells were incubated for another 4 h to allow the viable cells to form formazan crystals. After removing the medium containing MTT, 150 μL of DMSO was added to dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader, and the cell viability was calculated using the formula: Cell viability = (sample absorbance / blank control absorbance) × 100%.

[0110] Figure 13 4T1 cells were subjected to different concentrations of cGMn(a) and unSENDFUL at different pH conditions. cGMn (b) SENDFUL cGMn (c) Cell viability after SENDFUL(d) treatment. Table 2 shows the IC50 values ​​obtained by fitting a four-parameter logistic model to the cell viability data and the logarithm of cGMn concentration. 50 Value, by Figure 13 As shown in Table 2, SENDFUL cGMn The cytotoxicity against 4T1 cells was significantly higher at pH 6.5 than at pH 7.4, with a lower half-maximal inhibitory concentration (IC50). 50 The concentration dropped sharply from 331.5 µmol / L (pH=7.4) to 123.7 µmol / L (pH=6.5). Considering that the blank SENDFUL had little effect on cell viability, SENDFUL... cGMn This pH-dependent cytotoxicity should be attributed to its encapsulated cGMn. In contrast, free cGMn and unSENDFUL cGMn The lack of significant difference in toxicity under these two pH conditions highlights the key role of SENDFUL in modulating drug activity through switching membrane fusion behavior.

[0111] Table 2. IC50 obtained by fitting a four-parameter logistic model to cell viability data and the logarithm of cGMn concentration. 50 value

[0112] 3. Regarding SENDFUL cGMn The impact on tumor cell viability was assessed. 4T1 cells were loaded at a rate of 1×10⁴ cells per well. 5 The cells were seeded at a density of 100 µL of medium per well in 24-well plates and incubated at 37°C for 24 h. cGMn and SENDFUL were then controlled using RPMI 1640 medium at pH 7.4 or 6.5, respectively. cGMn or unSENDFUL cGMnThe solution was dialyzed to equilibrate, and then the cGAMP concentration was adjusted to 125 μmol / L. The cell culture medium was replaced with 100 µL of different treatment groups (PBS, cGAMP, SENDFUL). cGMn or unSENDFUL cGMn Cells were incubated in RPMI 1640 medium (cGAMP concentration 125 μmol / L; pH = 6.5 or 7.4) for 24 h. After discarding the medium, 250 µL of Calcein AM / PI detection working solution was added to each well, and the cells were incubated at 37 °C in the dark for 30 min. After incubation, cells were observed using an inverted fluorescence microscope (Calcein AM: excitation / emission wavelength 494 / 517 nm; PI: excitation / emission wavelength 535 / 617 nm). Figure 14 4T1 cells were subjected to different pH conditions via cGMn and unSENDFUL cGMn and SENDFUL cGMn Images of processed live / dead cells stained with fluorescence, where live cells are labeled with calcein and dead cells are labeled with propidium iodide (PI). Figure 14 It can be seen that under pH=6.5 conditions, after SENDFUL cGMn The treated cells showed a more pronounced propidium iodide fluorescence signal (red), indicating enhanced cell death; this phenomenon was weaker at pH 7.4. This is consistent with MTT-based cell viability results; however, cGMn and unSENDFUL cGMn No such pH-dependent differences in cell death were observed in any of the treatment groups.

[0113] The above in vitro studies collectively elucidate the effects of the STING nano-agonist SENDFUL cGMn The feasibility of selectively killing cancer cells in the acidic environment of tumors could potentially minimize damage to normal tissues.

[0114] 4. Regarding SENDFUL cGMn The in vitro immunostimulatory activity was evaluated. The same cell seeding and drug treatment methods as the live / dead staining assay were used, briefly described as follows: 4T1 cells were seeded at a density of 5 × 10³ cells per well in 24-well plates. After culturing for 24 hours, the cells were replaced with different treatment groups (PBS, cGMn, SENDFUL) at pH 7.4 or 6.5. cGMn or unSENDFUL cGMn The cells were incubated in a culture medium containing 75 µmol / L cGAMP. After 24 hours of co-incubation, the cell supernatant was collected, and the secretion levels of IFN-β and TNF-α were detected according to the ELISA kit instructions.

[0115] Figure 15 4T1 cells were subjected to different pH conditions via cGMn and unSENDFUL cGMn and SENDFUL cGMn The secretion levels of IFNβ(a) and TNFα(b) after treatment were analyzed, and the statistical analysis was performed using a one-tailed t-test. ,Depend on Figure 15 It can be seen that, compared with the pH=7.4 condition, the SENDFUL test at pH=6.5 showed better results. cGMn The treated cells showed significantly increased levels of IFNβ and TNFα secretion, compared to cGMn and unSENDFUL. cGMn There were no significant differences in cytokine secretion between the two pH conditions, thus confirming the key regulatory role of pH responsiveness in STING pathway activation.

[0116] 5. Explore SENDFUL cGMn Biological distribution characteristics: By subcutaneously injecting 4T1 cells (1×10⁻⁶) into female Balb / c mice (6 weeks old) 6 A subcutaneous xenograft tumor model was established. Ten days after inoculation, the tumor-bearing mice were randomly divided into groups and intravenously injected with DiD-labeled cGMn and unSENDFUL, respectively. cGMn and SENDFUL cGMn These are respectively denoted as DiD-cGMn and DiD-unSENDFUL. cGMn and DiD-SENDFUL cGMn In vivo fluorescence imaging was performed using an in vivo imaging system (IVIS) at 1 h, 3 h, 6 h and 24 h after drug administration.

[0117] Figure 16 A schematic diagram of the experimental timeline for the in vivo biodistribution study of the 4T1 tumor model (a), including injections of DiD-cGMn and DiD-unSENDFUL. cGMn and DiD-SENDFUL cGMn The results of in vivo fluorescence images of mice (b) and quantitative analysis of the mean fluorescence intensity of the tumor region in the in vivo fluorescence images (c) are shown. Statistical analysis was performed using one-way ANOVA combined with Fisher's LSD test. Therefore, it can be seen that DiD-unSENDFUL cGMn It rapidly accumulates in the liver within 1 hour after administration and continues to accumulate for at least 24 hours; while DiD-SENDFUL cGMn It preferentially accumulates in tumor tissue at all time points, and quantitative analysis of DiD signal intensity in the tumor region (white dashed box) also verified this result.

[0118] 6. Explore SENDFUL cGMn Antitumor activity in 4T1-Luc tumor-bearing mice: Six-week-old female Balb / c mice were subcutaneously inoculated with 4T1-Luc cells (1×10⁻⁶) in the left chest. 6 Five days after inoculation, tumor-bearing mice were randomly divided into four groups and intravenously injected with PBS, cGMn, and SENDFUL, respectively. cGMn and unSENDFUL cGMn (100 µL per animal, cGAMP concentration 200 µmol / L), administered once every 3 days for a total of 5 times; tumor volume and body weight were monitored regularly during treatment to assess efficacy. The tumor volume was calculated using the formula: V = (W 2 ×L) / 2 (V: tumor volume, W: shortest diameter, L: longest diameter); In addition, 24 hours after each administration, 100µL of D-fluorescein potassium salt solution (30mg / mL, dissolved in PBS) was injected intraperitoneally; After anesthesia, in vivo bioluminescence imaging was performed using a quantitative imaging system to dynamically assess tumor growth by detecting tumor fluorescence signals. Mice were sacrificed the day after the fifth treatment, and tumor tissue was collected. After mechanical homogenization and centrifugation to separate the cell pellet from the supernatant, the secretion levels of related cytokines such as IFNβ and TNFα in the supernatant were quantitatively detected using an ELISA kit. The cell pellet was resuspended in 4% paraformaldehyde fixative containing 1% bovine serum albumin to prepare a single-cell suspension for comprehensive immunophenotypic analysis. The following antibody combinations were used for staining: FITC-labeled anti-mouse CD4, PE-labeled anti-mouse CD8a, APC-labeled anti-mouse CD3, and PE / Cyanine7-labeled anti-mouse CD45 to detect T cell activation status; FITC-labeled anti-mouse CD4, PE-labeled anti-mouse FOXP3, PerCP / Cyanine5.5-labeled anti-mouse CD25, APC-labeled anti-mouse CD3, and PE / Cyanine7-labeled anti-mouse CD45 to detect T cell activation status. Mouse CD45 was used to assess regulatory T cells (Tregs); FITC-labeled anti-mouse IFNγ, PE-labeled anti-mouse TNFα, PerCP / Cyanine5.5-labeled anti-mouse NK-1.1, APC-labeled anti-mouse CD49b, and PE / Cyanine7-labeled anti-mouse CD45 were used to detect the activation status of natural killer (NK) cells; FITC-labeled anti-mouse I-Ad, PE-labeled anti-mouse CD80, PerCP / Cyanine5.5-labeled anti-mouse CD11b, APC-labeled anti-mouse F4 / 80, and PE / Cyanine7-labeled anti-mouse CD45 were used to analyze M1 macrophages; FITC-labeled anti-mouse I-Ad, APC-labeled anti-mouse CD11c, and PE / Cyanine7-labeled anti-mouse CD45 were used to assess the activation status of dendritic cells (DCs).

[0119] Figure 17 The figures show the mean tumor growth kinetics curves after different treatments (a), the survival curves of 4T1-Luc tumor-bearing mice (b), and in vivo bioluminescence imaging of 4T1-Luc tumor growth in mice (c). Statistical analysis in (b) was performed using two-way ANOVA combined with Fisher's LSD test, while statistical analysis in (c) was performed using the Log-rank (Mantel-Cox) test. It can be seen that after 15 days of administration, SENDFUL cGMn The tumor growth inhibition rate in the group reached 60.1%, which was significantly better than that in the cGMn group (27.1%) and unSENDFUL. cGMn Group (43.6%), this potent tumor-suppressive effect was validated by bioluminescence monitoring, SENDFUL cGMn The tumor signal in the treatment group mice remained lower than that in the other groups.

[0120] Figure 18 The levels of IFNβ(a) and TNFα(b) in mouse tumors after different treatments were analyzed using a one-tailed t-test. It can be seen that SENDFUL cGMn The expression levels of IFNβ and TNFα in the tumor tissues of mice in the treatment group were significantly higher than those in the control group, indicating that they mediate tumor suppression through the STING pathway. This STING activation will reshape the immunosuppressive tumor microenvironment.

[0121] Figure 19 MHCII in isolated tumor tissues of mice after different treatments + Mature dendritic cells (a), CD4 + T cells (b), CD8 + T cells (c), MHCII + Macrophages (d), CD80 + Macrophages (e), IFNγ + NK cells (f) and Foxp3 + The proportion of regulatory T cells (g) was analyzed using a one-tailed t-test. It can be seen that SENDFUL cGMn The proportion of MHCII+ mature dendritic cells in the tumor tissue of mice in the treatment group was significantly higher than that in the control group, indicating that T cell-based adaptive immunity was activated. Simultaneously, [the text abruptly shifts to a seemingly unrelated topic:] ...and cGMn and unSENDFUL cGMn Compared to the treatment group, SENDFUL cGMn M1 phenotype macrophages (F4 / 80) in tumor tissue of mice in the treatment group + MHCII +and F4 / 80 + CD80 + Cell population and IFNγ + A higher proportion of NK cells indicates that innate anti-tumor immunity is activated, and SENDFUL cGMn Treatment can also inhibit Foxp3 + Regulatory T cells. In summary, SENDFUL cGMn The therapy promotes the activation of immune effector cells and remodels the immunosuppressive tumor microenvironment by activating the STING pathway, thereby achieving synergistic tumor suppression.

[0122] 7. Regarding SENDFUL cGMn The in vivo safety was assessed. Collected via PBS, cGMn, and SENDFUL cGMn and unSENDFUL cGMn Serum samples from 4T1-Luc tumor-bearing mice were used to detect biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), alkaline phosphatase (ALP), creatinine (CREAT), urea (UREA), creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH), to assess changes in important physiological parameters related to liver and kidney function. Simultaneously, major organs such as the heart, liver, spleen, lungs, and kidneys were collected, fixed in 4% paraformaldehyde, dehydrated, embedded, sectioned, stained with hematoxylin and eosin (H&E), and analyzed using scanning slide microscopy.

[0123] Figure 20 The results of detecting the levels of alanine aminotransferase (a), aspartate aminotransferase (b), total bilirubin (c), alkaline phosphatase (d), creatinine (e), urea (f), creatine kinase isoenzyme MB (g), and lactate dehydrogenase (h) in the serum of tumor-bearing mice after different treatments were presented. Statistical analysis was performed using a one-tailed t-test. It can be seen that cGMn and unSENDFUL cGMn All treatments consistently caused significant increases in certain indicators, suggesting a potential for acute toxicity. SENDFUL cGMn No significant changes were observed in any of the indicators after treatment compared to the control group, indicating that it did not induce systemic toxicity. These results are consistent with in vitro cytotoxicity studies.

[0124] The above results indicate that the STING nano-agonist SENDFUL provided by this invention... cGMn It exhibits excellent biosafety and did not cause significant biotoxicity, thus providing a key basis for its clinical translation.

Claims

1. A pyridine betaine-terminated lipid molecule, characterized in that, Its structure is shown in equation (Ⅰ): Equation (Ⅰ); In equation (Ⅰ), R is selected from C 14 -C 18 A chain of amino hydrocarbons with saturated or unsaturated fatty acyl groups substituted; X is selected from hydrogen, alkyl or halogen; n is any integer selected from 1 to 3.

2. The method for preparing the pyridine betaine-terminated lipid molecule according to claim 1, characterized in that, Includes the following steps: A1, C 14 -C 18 Saturated or unsaturated fatty acids are esterified with tert-butyl (2,3-dihydroxypropyl) carbamate in a halocarbon solvent under the action of an acylation catalyst and a condensing agent to obtain 3-((tert-butyloxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester. A2. 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester is reacted with a strong organic acid in a halocarbon solvent to remove the tert-butoxycarbonyl group, thereby obtaining 3-aminopropane-1,2-dimethyldifatty acid ester. A3. 3-Aminopropane-1,2-dimethyldifatty acid ester and X-substituted isonicotinic acid are subjected to an amidation reaction in a polar aprotic solvent in the presence of a condensing agent, an activating agent and an organic base to obtain 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester. A4. 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester and n-membered halocarboxylic acid are subjected to quaternization reaction in a nitrile solvent to obtain the pyridine betaine-terminated lipid molecule. The definitions of X and n are as described in claim 1.

3. The method for preparing pyridine betaine-terminated lipid molecules according to claim 2, characterized in that, In step A1, the C 14 -C 18 The molar ratio of saturated or unsaturated fatty acids to tert-butyl (2,3-dihydroxypropyl) carbamate is (1.8-2.8):1; And / or, in step A2, the molar ratio of the 3-((tert-butoxycarbonyl)amino)propane-1,2-dimethyldifatty acid ester to the strong organic acid is 1:(2-3); And / or, in step A3, the molar ratio of the 3-aminopropane-1,2-dimethyldifatty acid ester to the X-substituted isonicotinic acid is (0.8-1.2):1; And / or, in step A4, the molar ratio of the 3-(X-substituted isonicotinamide)propane-1,2-dimethyldifatty acid ester to the n-membered halocarboxylic acid is 1:(2.5-3.5); The definitions of X and n are as described in claim 1 or 2.

4. The method for preparing pyridine betaine-terminated lipid molecules according to claim 2, characterized in that, In step A1, the esterification reaction is carried out at a temperature of 20-30°C for 20-30 hours. And / or, in step A2, the reaction temperature is 20-30°C and the time is 4.5-9.5 h; And / or, in step A3, the amidation reaction is carried out at a temperature of 52-78°C for 3-5 hours; And / or, in step A4, the quaternization reaction is carried out at a temperature of 68-102°C for a time of 20-30 h.

5. An adaptive fusion liposome, characterized in that, It includes the following lipid components: the pyridine betaine-terminated lipid molecule as described in claim 1, as well as neutral lipids and polyethylene glycol-functionalized lipids.

6. The adaptive fusion liposome according to claim 5, characterized in that, The molar ratio of the pyridine betaine-terminated lipid molecules, neutral lipids, and polyethylene glycol-functionalized lipids is (2-14): (5-37):

1.

7. The method for preparing adaptive fusion liposomes according to claim 5 or 6, characterized in that, Includes the following steps: B1. Pyridine betaine-terminated lipid molecules, neutral lipids, and polyethylene glycol-functionalized lipids were dissolved in a chlorinated hydrocarbon solvent, and the solvent was removed to obtain a lipid film. B2. The lipid film is subjected to hydration treatment to obtain the adaptive fusion liposome.

8. A STING nano-agonist, characterized in that, The STING nano-agonist has a core-shell structure; the core is a nanocomposite of the STING pathway agonist and divalent metal ions; and the shell is the adaptive fusion liposome as described in claim 5 or 6.

9. The STING nano-agonist according to claim 8, characterized in that, The STING pathway agonist is selected from one of cyclic guanosine monophosphate (cGMP)-adenosine monophosphate (cGMP), cyclic diadenosine monophosphate (cDAMP), and MSA-2; the divalent metal ion includes Mn. 2+ .

10. The use of the adaptive fusion liposomes of claim 5 or 6, or the STING nano-agonist of claim 8 or 9, in the preparation of antitumor nanomedicines.

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