A hypoxia-responsive prodrug liposome and preparation and application thereof

Abstract

The application belongs to the field of liposome drug delivery, and relates to a hypoxia-responsive prodrug liposome and a preparation and application thereof. The liposome contains a hypoxia-responsive prodrug and a photosensitizer, wherein the mass ratio between the hypoxia-responsive prodrug and the photosensitizer is 10:1-1:10; the hypoxia-responsive prodrug is a poorly soluble antitumor drug modified by a polyphenol. The hypoxia-responsive liposome prepared by the application has uniform particle size, high encapsulation efficiency and high drug loading capacity, good stability, significantly prolonged drug in-vivo circulation time and tumor site accumulation, and significantly improved antitumor effect. At the same time, the co-loaded liposome maintains fluorescence quenching in blood circulation, and specifically restores active oxygen capacity under the action of high glutathione in the tumor site, solving the problem of traditional photosensitizer phototoxicity.

Description

Technical Field

[0001] This invention belongs to the field of liposome drug delivery, and relates to a hypoxia-responsive prodrug liposome and its preparation and application. Background Technology

[0002] Hypoxia is a prominent feature of most solid tumors, primarily caused by the imbalance between the rapid oxygen consumption of invasive tumor cell proliferation and insufficient oxygen supply. Targeting the specific hypoxic microenvironment of tumor sites, a series of hypoxia-activated prodrugs have been developed by researchers. Hypoxia-activated prodrugs (HAPs) are a class of prodrugs that are non-toxic or have low toxicity and can be selectively activated in hypoxic tumor regions to become cytotoxic antitumor drugs. Although some progress has been made in HAP research, only a limited number of HAPs have entered clinical trials, and no products have yet been approved by the FDA for cancer treatment. In contrast, prodrugs containing hypoxia-responsive linkages appear to have greater potential for clinical translation. Furthermore, due to the heterogeneity of tumor hypoxia, the activation efficiency of prodrugs is low, leading to unsatisfactory therapeutic effects. To address this issue, some studies have combined photodynamic therapy with hypoxia-activated prodrugs. Photodynamic therapy's oxygen consumption exacerbates the hypoxic microenvironment at the tumor site, thereby promoting the activation of hypoxia-responsive prodrugs to exert their antitumor effects. However, hypoxia-responsive prodrugs and photosensitizers differ in their physicochemical and pharmacokinetic properties, and achieving efficient simultaneous delivery of hypoxia-responsive prodrugs and photosensitizers remains a challenge.

[0003] Liposomes are considered a promising nanocarrier, capable of simultaneously delivering multiple drugs and coordinating their pharmacokinetic properties. Analysis of current marketed formulation preparation methods reveals that active drug delivery technology holds greater promise for clinical translation due to its advantages such as high encapsulation efficiency, high drug loading capacity, and good stability. To broaden the application scope of active drug delivery technology, researchers have proposed a strategy of modifying drug structures lacking the conditions for active drug delivery with weak acid or weak basic groups, thereby endowing the drugs with active drug delivery capabilities. This technology has been applied to various drugs, successfully preparing actively drug-loaded liposomes with high encapsulation efficiency and high drug loading capacity, significantly improving drug circulation time and antitumor efficacy in vivo. However, liposomes prepared based on this technology still cannot achieve specific drug release at the tumor site. Furthermore, single-drug therapy is insufficient to achieve ideal therapeutic effects. Therefore, there is an urgent need to develop multifunctional liposomes capable of intelligent drug release from the tumor microenvironment. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a hypoxia-responsive prodrug liposome and its preparation and application.

[0005] The technical solution adopted in this invention is as follows:

[0006] A hypoxia-responsive prodrug liposome, comprising a hypoxia-responsive prodrug and a photosensitizer, wherein the mass ratio of the hypoxia-responsive prodrug to the photosensitizer is 10:1 to 1:10; wherein the hypoxia-responsive prodrug is a poorly soluble antitumor drug modified with polyphenols.

[0007] The hypoxia-responsive prodrug is a polyphenol linked to a poorly soluble antitumor drug via a hypoxia-responsive linker chain; wherein the hypoxia-responsive linker chain is azobenzene.

[0008] The hypoxia-responsive prodrug is

[0009] The photosensitizer is pyrophyllite a, dihydroporphyrin E6, protoporphyrin, hematoporphyrin monomethyl ether, bamboo red fungus A, hypericin, chlorophyll derivatives or phthalocyanine derivatives.

[0010] The liposomes comprise phospholipids, cholesterol, PEGylated phospholipids, hypoxia-responsive prodrugs, photosensitizers, an inner aqueous phase solution, and an outer aqueous buffer solution; wherein the molar ratio of cholesterol to phospholipids is 5:95 to 45:55, the ratio of hypoxia-responsive prodrugs to total phospholipids (the sum of phospholipids, cholesterol, and PEGylated phospholipids) is 1:20 to 1:5 (w / w), and the ratio of photosensitizers to total phospholipids is 1:50 to 1:5 (w / w).

[0011] The phospholipid is a natural phospholipid and / or a synthetic phospholipid; the phospholipid is a natural phospholipid such as egg yolk lecithin (EPC), soybean phospholipid, sphingomyelin, hydrogenated soybean phospholipid (HSPC), or a synthetic phospholipid such as distearate phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), myristoyl phosphatidylcholine (DMPC), distearate phosphatidylglycerol (DSPG) or one or two thereof, preferably hydrogenated soybean phospholipid (HSPC) or distearate phosphatidylcholine (DSPC);

[0012] The PEGylated phospholipid is DSPE-mPEG. 1000 DSPE-mPEG 2000 DSPE-mPEG 5000 One or more of them, preferably DSPE-mPEG 2000 PEGylated phospholipids account for 0.1%-5% of the total phospholipids, preferably 0.5%-1%.

[0013] The internal aqueous phase solution is a metal ion salt solution, wherein the metal ion salt solution is a zinc gluconate solution, and the concentration of the metal ion salt solution in the system is 50-350 mM;

[0014] The external aqueous buffer salt solution is a buffer salt solution with or without EDTA.

[0015] A method for preparing the hypoxia-responsive prodrug liposomes described above:

[0016] (1) Dissolve phospholipids, cholesterol, and PEGylated phospholipids in an appropriate amount of organic solvent, remove the organic solvent by rotary evaporation under reduced pressure, and obtain a uniform film.

[0017] (2) Add the internal aqueous phase solution to the film obtained in step (1) to hydrate it, and reduce the particle size by ultrasonication or extrusion equipment to obtain a single-chamber liposome with uniform particle size.

[0018] (3) The blank liposomes obtained above were passed through a Sepharose CL-4B agarose gel and eluted with a buffer solution containing EDTA. The collected liposome eluent was then eluted again with a buffer solution without EDTA to obtain blank liposomes.

[0019] (4) The organic solvent solution of hypoxia-responsive polyphenol prodrug and photosensitizer is added to blank liposomes in a certain order and drug-liposome ratio and stirred and incubated above the liposome phase transition temperature to obtain drug-loaded liposomes. The remaining organic solvent is removed to obtain the final liposome product.

[0020] The organic solvent in step (1) is one or more of dichloromethane, trichloromethane, methanol or ethanol;

[0021] In step (2), the external aqueous phase of the buffer salt solution containing EDTA is a combination of 300 mM sucrose, 20 mM hydroxyethylpiperazine ethanesulfonic acid and 15 mM EDTA.

[0022] The external aqueous phase of the buffer salt solution consisted of a combination of 300 mM sucrose and 20 mM hydroxyethylpiperazine ethanesulfonic acid.

[0023] In step (4), the drug solution is obtained by dissolving the hypoxia-responsive prodrug in an organic solvent, wherein the organic solvent is ethanol, dimethyl sulfoxide (DMSO), methanol, acetonitrile, acetone, N,N-dimethylformamide (DMF), and the amount of organic solvent used accounts for 2%-50% (v / v) of the volume of the blank liposome.

[0024] The order of drug addition in step (4) is to add the photosensitizer solution first, followed by the hypoxia response pre-drug solution.

[0025] A method for preparing hypoxia-responsive prodrug liposomes, and the application of the liposomes in the preparation of antitumor drugs.

[0026] The main advantages of this invention are:

[0027] (1) The hypoxia-responsive liposomes of the present invention use metal ions to encapsulate hypoxia-responsive polyphenol prodrugs and photosensitizers in the aqueous phase of the liposomes via active drug delivery, which has the characteristics of high encapsulation efficiency and high drug loading.

[0028] (2) The hypoxia-responsive liposomes of the present invention have uniform particle size, good stability, are not easy to leak, have a simple preparation process, and are suitable for industrial scale-up production.

[0029] (3) The hypoxia-responsive liposomes prepared in this invention can promote drug release under the action of glutathione highly expressed at the tumor site. The released photosensitizer generates reactive oxygen species under laser irradiation, consumes oxygen, and further promotes the release of hypoxia-responsive prodrugs to synergistically exert anti-tumor effects.

[0030] (4) The hypoxia-responsive liposomes of the present invention can significantly prolong the half-life of the drug in plasma and increase the accumulation of the drug at the tumor site.

[0031] (5) The hypoxia-responsive liposomes of the present invention have excellent anti-tumor effects and good safety, and no obvious toxic side effects were produced in mice during treatment. Attached image description:

[0032] Figure 1 The mass spectrum and NMR spectrum of PPT-Azo-GA (PAG) in Example 1 of this invention are shown.

[0033] Figure 2 This is a graph showing the combined index (CI) and inhibition rate (Fa) of the prodrug PAG and dihydroporphyrin e6 on breast cancer 4T1 cells in Example 2 of the present invention.

[0034] Figure 3 This is an HPLC chromatogram showing the response and cleavage of the prodrug PAG in sodium dithionite solutions of different concentrations in Example 3 of the present invention.

[0035] Figure 4 This is the ultraviolet absorption spectrum of dihydroporphyrin E6 mixed with different metal ion salt solutions in Example 4 of the present invention.

[0036] Figure 5 The fluorescence spectrum of dihydroporphyrin E6 mixed with different metal ion salt solutions in Example 4 of this invention is shown.

[0037] Figure 6 This is a graph showing the effect of liposomes containing different metal ions on the encapsulation efficiency of dihydroporphyrin E6 in Example 5 of the present invention.

[0038] Figure 7 This is a diagram showing the storage stability and colloidal stability of the dual-drug-loaded liposomes of Example 8 of the present invention.

[0039] Figure 8 This is a confocal image of cellular uptake of the dual-drug-loaded liposomes in Example 9 of the present invention.

[0040] Figure 9 This is a graph showing the intracellular glutathione concentration in 4T1 cells after treatment with dual-drug-loaded liposomes in Example 10 of the present invention.

[0041] Figure 10 This is a confocal image of intracellular reactive oxygen species (ROS) generation and hypoxia induced by dual-drug-loaded liposomes in Example 11 of the present invention.

[0042] Figure 11 This is an in vitro cytotoxicity diagram of the dual-drug-loaded liposomes of Example 12 of the present invention.

[0043] Figure 12 This is a blood drug concentration-time curve of the dual drug-loaded liposomes of Example 13 of the present invention.

[0044] Figure 13 This is an in vitro tissue distribution diagram of the dual drug-loaded liposomes of Example 14 of the present invention.

[0045] Figure 14 This is a graph showing the changes in tumor volume and body weight in the anti-tumor experiment of dual-drug-loaded liposomes in Example 15 of the present invention.

[0046] Figure 15 This is a verification diagram of the dual-drug-loaded liposomes used in Example 16 of the present invention to alleviate the in vivo phototoxicity of photosensitizers. Detailed Implementation

[0047] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the invention is not limited to the scope of the embodiments described herein.

[0048] The liposomes of this invention actively load hypoxia-responsive polyphenol prodrugs and photosensitizers into liposomes in a synergistic ratio using metal ions. The prepared dual-drug-loaded liposomes have uniform particle size and are characterized by high encapsulation efficiency, high drug loading capacity, and good stability. This significantly prolongs the drug's circulation time in vivo, increases drug accumulation at the tumor site, and improves the anti-tumor effect. At the same time, the co-loaded liposomes maintain fluorescence quenching in the blood circulation and specifically restore the ability to generate reactive oxygen species under the action of high glutathione at the tumor site, thus solving the phototoxicity problem of traditional photosensitizers.

[0049] Example 1: Synthesis of a polyphenol-modified, hypoxia-responsive, azobenzene-linked podophyllotoxin prodrug (PPT-Azo-GA, PAG)

[0050] 4-Nitrobenzyl alcohol (3 g, 20 mmol) was mixed with 35 mL of 6 M sodium hydroxide solution. Zinc powder was then slowly added to the reaction mixture. The mixture was refluxed for 10 hours. After cooling to room temperature, it was filtered. The crude product was recrystallized in methanol to give an orange-yellow powder (1.52 g, 51% yield), namely the azobenzene (Azo) linkage. Gallic acid (GA) (10 g, 58.78 mmol) was dissolved in 150 mL of dry DMF. Imidazole (40 g, 586 mmol) and tert-butylmethylsilyl chloride (50 g, 331.74 mmol) were added sequentially. After stirring at room temperature for 24 hours, the reaction mixture was diluted with ethyl acetate, extracted three times with brine, and the solvent was evaporated to dryness to give a white solid (35.24 g, 98% yield). The obtained white solid (10 g, 15.95 mmol) was then dissolved in THF at room temperature. Then, a mixture of acetic acid and water (3:1) was added to the solution and stirred at room temperature for 24 hours. The reaction solution was poured into cold water, extracted with ethyl acetate, and concentrated under vacuum to obtain TBS-protected gallic acid (white solid, 7.89 g, yield 94%).

[0051] Podophyllotoxin (PPT) (24.2 mg, 0.12 mmol) and triethylamine were dissolved in dichloromethane, and the solution was cooled to 0°C. Then, 4-nitrochloroformate (24.1 mg, 0.12 mmol) and a catalytic amount of DMAP were slowly added dropwise to the reaction solution. The reaction was carried out at room temperature under nitrogen protection for 6 hours, monitored by TLC. After the reaction was complete, the reaction solution was washed three times with brine. The organic layer was collected and the solvent was removed by vacuum evaporation. Purification by silica gel column chromatography yielded a pale yellow solid (60.2 mg, 86.6%). The obtained product (50 mg, 0.086 mmol) and the linked azobenzene (41.7 mg, 0.172 mmol) were dissolved in DMF, and the solution was cooled to 0°C. Then, DMAP was added, and the reaction mixture was heated to 30°C and stirred overnight in the dark. After the reaction was complete, the reaction solution was diluted with dichloromethane and washed three times with brine. The organic layer was collected and evaporated to dryness. The crude product was purified by silica gel column chromatography to give an orange solid (48.2 mg, yield 83.1%).

[0052] TBS-protected gallic acid (30.2 mg, 0.059 mmol) was dissolved in dichloromethane. After cooling the solution to 0°C, EDCI (33.9 mg, 0.177 mmol), DMAP (21.6 mg, 0.177 mmol), and HOBT (23.9 mg, 0.177 mmol) were added sequentially, and the mixture was stirred for 2 h to activate the carboxylic acid. Then, the orange solid obtained above (40 mg, 0.059 mmol) was added, and the mixture was stirred at room temperature in the dark for 24 h. The orange solid (59.8 mg, 86.3% yield) was purified by silica gel column chromatography. The purified orange solid (50 mg, 0.042 mmol) was then dissolved in 20 mL of anhydrous tetrahydrofuran, and a tetrabutylammonium fluoride (TBAF, 48 mg, 0.188 mmol) solution was added dropwise at 0°C, and the reaction was continued for 2 h. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed three times with brine. The final product was purified by preparative liquid chromatography to obtain the final product (PPT-Azo-GA, PAG) (32 mg, yield 91.4%).

[0053] Mass spectrometry and proton nuclear magnetic resonance (NMR) were used. 1 The structure of the derivative was determined by ¹H-NMR using deuterated DMSO as the solvent, and the results are as follows: Figure 1 As shown, the results of the hydrogen nuclear magnetic resonance spectrum analysis are as follows:

[0054] PAG: 1 H NMR (400MHz, DMSO-d6), δ = 7.93-7.95 (d, J = 8.4Hz, 4H, Ar-H), 7.64-7.67 (dd, J = 3.60, 3.60Hz, 4H, Ar-H), 7.02 (s, 2H, Ar-H), 6.99(s,1H,Ar-H),6.61(s,1H,Ar-H),6.35(s,2H,Ar-H),6.03(s,2H,2-H),5.87-5.89(d,J=9.20Hz,1H,5-H),5.33-5.36(d ,J=14.00Hz,4H,-OCH2-Ph-),4.57-4.58(d,J=4.80Hz,1H,9-H),4.45-4.49(t,J=7.80Hz,1H,6α-H),4.20-4.25(t,J=9.40H z,1H,6β-H),3.63-3.64(d,J=7.60Hz,9H,7'-H,8'-H,9'-H),3.38-3.43(dd,J=4.80,4.40Hz,1H,8a-H),2.86(m,1H,5a-H).

[0055] Example 2: Screening for the synergistic ratio of hypoxia-responsive polyphenol prodrug (PAG) and dihydroporphyrin e6 (Ce6).

[0056] Log-phase 4T1 cells were seeded at 2000 cells per well in 96-well plates and cultured for 12 hours until cell attachment. The old culture medium was discarded, and a series of drug-containing media of different concentrations were added to the wells (three parallel wells per concentration). An equal volume of blank culture medium was added to the control wells. After 4 hours of incubation, the group containing the photosensitizer was irradiated with a 660nm laser (200mW / cm²). 2 After incubation for 44 hours, add 20 μL of MTT solution to each well and continue incubation for another 4 hours. After incubation, discard the old culture medium, add 200 μL of DMSO solution to each well, and then shake on a shaker for 10 minutes to fully dissolve the formazan. Measure the absorbance at 490 nm using a multi-mode microplate reader.

[0057] The combination index of PAG and Ce6 was calculated using the Chou-Talalay method. The formula for the combination index of the two drugs is:

[0058] CIx=(D)1 / (Dx)1+(D)2 / (Dx)2

[0059] Where (Dx)1 and (Dx)2 are the concentrations at which the two drugs, acting alone, produce an inhibition rate of x% in cells, and (D)1 and (D)2 are the concentrations at which the two drugs, acting in combination, produce an inhibition rate of x% in cells. CI = 1 indicates additive effect, CI < 1 indicates synergistic effect, and CI > 1 indicates antagonistic effect. Inhibition rate-combination index curves were plotted using CompuSyn software, as shown below. Figure 2 As shown.

[0060] Table 1. Screening for the synergistic ratio of PAG and Ce6 (quality ratio)

[0061]

[0062] Experimental results show that PAG and Ce6 have synergistic effects at the selected ratios. The CI value is the smallest (0.41) when PAG:Ce6 = 5:1, indicating the strongest synergistic effect. Therefore, PAG:Ce6 = 5:1 was chosen for subsequent experimental studies.

[0063] Example 3: Verification of the hypoxia response fragmentation of the prodrug PAG

[0064] Different concentrations of sodium dithionite (Na₂S₂O₄) solution were used to simulate hypoxic conditions, and acetonitrile:PBS = 1:1 (v / v, pH 7.4) was selected as the release medium. PAG was incubated for 2 hours with release media containing 0 mM, 5 mM, 10 mM, 25 mM, or 50 mM Na₂S₂O₄, respectively. Then, acetonitrile was added to the mixture. After centrifugation at 13000 rpm for 5 minutes, the samples were monitored by HPLC.

[0065] Experimental results are as follows Figure 3 As shown, the prodrug PAG can be converted into the parent drug PPT under hypoxic conditions, and the conversion rate increases with the increase of Na2S2O4 concentration.

[0066] Example 4: Verification of the coordination ability of metal ions with Ce6

[0067] PAG was dissolved in an organic solvent and then mixed with solutions of different metal ion salts, including copper acetate solution, copper sulfate solution, copper gluconate solution, zinc acetate solution, zinc sulfate solution, zinc gluconate solution, ferric chloride solution, ferric sulfate solution, ferric ammonium citrate solution, manganese sulfate solution, manganese gluconate solution, magnesium sulfate solution, magnesium chloride solution, magnesium gluconate solution, calcium acetate solution, calcium chloride solution, and calcium gluconate solution. The resulting mixture was then diluted with methanol to an appropriate factor, and the ultraviolet absorption and fluorescence spectra of Ce6 were measured.

[0068] Experimental results are as follows Figure 4 and 5 As shown, the UV absorption spectrum of Ce6 significantly shifted after mixing with solutions of copper, zinc, and iron ions, indicating the formation of coordination interactions. However, the UV absorption spectrum did not change significantly after mixing with solutions of manganese, magnesium, and calcium ions. Interestingly, the fluorescence intensity decreased significantly after mixing with manganese ion solution, suggesting a possible interaction between the two. Furthermore, the fluorescence intensity was completely quenched after coordination of copper ion solution with Ce6, which may severely affect the photodynamic therapy effect. Considering the above factors, a solution of zinc, iron, and manganese ion salts was chosen as the internal aqueous phase for the preparation of blank liposomes.

[0069] Example 5: Effect of different metal ions on the encapsulation efficiency of Ce6-actively loaded liposomes

[0070] HSPC, cholesterol, and DSPE-PEG 2000The liposomes were prepared by rotary evaporation under reduced pressure at a mass ratio of 85:5:10 in a 500 mL round-bottom flask, dissolved in chloroform, and formed into a film at 40 °C. 300 mM solutions of different metal ion salts were added, and the mixture was hydrated by rotary evaporation at 65 °C for 30 min. The liposomes were then passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm ten times each under nitrogen protection to obtain uniformly sized monocomplex liposomes. Metal ions in the aqueous phase of the monocomplex liposomes were removed using a Sepharose CL-4B column pre-equilibrated with a buffer solution of 300 mM sucrose, 20 mM HEPES, and 15 mM EDTA to obtain blank liposomes. Ce6 was dissolved in DMSO to prepare a 10 mg / mL stock solution. The drug-liposome DMSO solution was added dropwise to the blank liposomes at a drug-liposome ratio of 1:10 (w / w), and the mixture was stirred and incubated at 65 °C for 30 min. After incubation, the drug loading was stopped by ice bath, and the encapsulation efficiency of Ce6 by the liposomes was measured.

[0071] Experimental results are as follows Figure 6 As shown, only liposomes Ce6 with zinc ion salt solution as the inner aqueous phase have a high encapsulation efficiency. Therefore, liposomes with zinc ion salt solution were selected to further investigate the encapsulation efficiency of the prodrug PAG.

[0072] Example 6: Effect of different zinc ion salt solutions on the encapsulation efficiency of PAG active drug-loaded liposomes

[0073] HSPC, cholesterol, and DSPE-PEG 2000 The liposomes were prepared by rotary evaporation under reduced pressure at a mass ratio of 85:5:10 in a 500 mL round-bottom flask, dissolved in chloroform, and formed into a film. 300 mM solutions of different zinc ion salts were added, and the mixture was hydrated by rotary evaporation at 65 °C for 30 min. The liposomes were then passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm ten times each under nitrogen protection to obtain uniformly sized monocomplex liposomes. Zinc ions in the aqueous phase of the monocomplex liposomes were removed using a Sepharose CL-4B column pre-equilibrated with a buffer solution of 300 mM sucrose, 20 mM HEPES, and 15 mM EDTA to obtain blank liposomes. PAG was dissolved in DMSO to prepare a 10 mg / mL stock solution. The drug-liposome DMSO solution was added dropwise to the blank liposomes at a drug-liposome ratio of 1:10 (w / w), and the mixture was stirred and incubated at 65 °C for 30 min. After incubation, the drug loading was stopped by ice bath, and the encapsulation efficiency of the liposomes with PAG was measured.

[0074] Experimental results showed that all three types of zinc ion liposomes had good encapsulation effects on the prodrug PAG (encapsulation efficiency >90%). However, after encapsulating PAG, the particle size of zinc acetate liposomes increased from 114 nm to 202 nm, which may affect the clearance of liposomes in vivo. Considering the above factors, zinc gluconate was selected as the internal aqueous phase for the preparation of dual-drug-loaded liposomes.

[0075] Table 2. Particle size, polydispersity index, and encapsulation efficiency of PAG prodrugs encapsulated in liposomes with different zinc ions.

[0076]

[0077] Example 7: Effect of drug loading order on the encapsulation efficiency of PAG and Ce6 dual-drug liposomes

[0078] HSPC, cholesterol, and DSPE-PEG 2000 The liposomes were placed in a 500 mL round-bottom flask at a mass ratio of 85:5:10, dissolved in chloroform, and evaporated under reduced pressure at 40 °C to form a film. A 300 mM zinc gluconate aqueous solution was added, and the mixture was hydrated by rotation at 65 °C for 30 min. Under nitrogen protection, the liposomes were passed through polycarbonate membranes with pore sizes of 400 nm, 200 nm, and 100 nm ten times each to obtain uniform single-chamber liposomes. Zinc ions in the aqueous phase of the single-compartment liposome were removed using an agarose gel Sepharose CL-4B column pre-equilibrated with a buffer solution of 300 mM sucrose, 20 mM HEPES, and 15 mM EDTA to obtain blank liposomes. PAG and Ce6 were dissolved in DMSO to prepare stock solutions of 14 mg / ml and 2.8 mg / ml, respectively. DMSO solutions of the drugs were added dropwise to the blank liposomes simultaneously or sequentially at drug-liposome ratios of 1:7 and 1:35 (w / w). The mixture was stirred and incubated at 65 °C for 30 min. After incubation, drug loading was stopped by placing the liposomes on an ice bath, and the encapsulation efficiency of PAG and Ce6 in the liposomes was measured.

[0079] Experimental results showed that when PAG solution was added first, followed by Ce6 solution, or both drug solutions were added simultaneously, significant drug precipitation occurred after incubation, indicating that the drugs were not well encapsulated in the liposomes. However, when Ce6 solution was added first, followed by PAG solution, the resulting liposomes showed higher encapsulation rates for both drugs: 98.3±1.2% for PAG and 94.3±0.7% for Ce6. Therefore, this method was chosen for subsequent experiments.

[0080] Example 8: Stability study of dual-drug-loaded liposomes

[0081] Storage stability: The dual-drug-loaded liposomes prepared under the optimal formulation were stored at 4°C for one month, and samples were taken at set time points to determine the liposome particle size and polydispersity index.

[0082] Colloidal stability: The prepared dual-drug-loaded liposomes were mixed with PBS solution containing 10% FBS (pH 7.4) and incubated at 37°C. Samples were taken at set time points to determine the particle size and polydispersity index of the liposomes.

[0083] Experimental results are as follows Figure 7 As shown, the prepared dual-drug-loaded liposomes exhibit good physical and colloidal stability.

[0084] Example 9: In vitro cellular uptake of dual-drug-loaded liposomes

[0085] 4T1 cells were fed at a rate of 1×10 5 Cells were seeded at a density of 100 cells / well in 12-well plates and cultured for 24 hours. The old culture medium was then discarded and replaced with fresh medium containing Ce6 solution or PAG / Ce6 liposomes (equivalent Ce6 concentration of 1 μg / mL), and incubated at 37°C for 4 h, 8 h, and 12 h, respectively. After incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 minutes, and washed again with PBS. After washing, Hoechst staining was performed for 15 minutes, followed by three washes with PBS. The samples were then observed under a confocal microscope to monitor cell uptake.

[0086] Experimental results are as follows Figure 8 As shown, the free Ce6 solution reached uptake equilibrium in 4 hours. In contrast, the cellular uptake of PAG / Ce6 liposomes was time-dependent within the set time period, and the cellular uptake was higher than that of the free Ce6 solution after 12 hours of incubation.

[0087] Example 10: Investigation of glutathione concentration in 4T1 cells after treatment with dual-drug-loaded liposomes

[0088] 4T1 cells were seeded into 6-well plates (1×10⁻⁶ cells per well). 5 Cells were cultured at 100 cells / well for 24 hours. Then, cells were treated for 24 hours with a solution of free Ce6 diluted in fresh culture medium, PAG liposomes, Ce6 liposomes, or PAG / Ce6 liposomes (equivalent Ce6 concentration of 1 μg / mL). After incubation, cells were washed with PBS, centrifuged, and collected. Deproteinized reagent M solution was then added. Intracellular GSH concentration was measured according to the instructions of the glutathione assay kit (Beyotime, Shanghai, China).

[0089] Experimental results are as follows Figure 9 As shown, free Ce6 solution did not significantly alter intracellular GSH concentration compared to the control group. In contrast, PAG / Ce6 liposomes significantly reduced GSH levels in tumor cells, and were lower than those of PAG liposomes or Ce6 liposomes alone.

[0090] Example 11: Investigation on the induction of reactive oxygen species production and hypoxia exacerbation by dual-drug-loaded liposomes

[0091] 4T1 cells were fed at a rate of 1×10 5Cells were seeded at a density of 100 cells / well in 12-well plates and cultured for 24 hours. Then, the old culture medium was discarded, and Ce6 solution diluted with fresh medium or PAG / Ce6 liposomes (equivalent Ce6 concentration of 1 μg / mL) were added to each well, and the plates were incubated at 37°C for 4 hours. After incubation, the cells were washed three times with PBS and treated with ROS / hypoxia probes according to the ROS-ID hypoxia / oxidative stress assay kit (Enzo Life Sciences). After 30 minutes of incubation, the cells were washed again and irradiated with a 660nm laser (2 minutes, 100mW / cm²). 2 After the light exposure, the cells were washed with PBS and observed using a confocal microscope to detect the production of reactive oxygen species and hypoxia within the cells.

[0092] Experimental results are as follows Figure 10 As shown, PAG / Ce6 liposomes induce more reactive oxygen species production and exacerbate intracellular hypoxia, which is beneficial for synergistically promoting the specific cleavage of the prodrug PAG.

[0093] Example 12: In vitro cytotoxicity test of dual-drug-loaded liposomes

[0094] 4T1 cells were seeded at a density of 1000 cells per well in 96-well plates and cultured for 12 hours until cell attachment. The old culture medium was discarded, and 200 μL of culture medium containing different concentrations of the prodrug PAG liposomes, dihydroporphyrin e6 liposomes, and prodrug PAG / dihydroporphyrin e6 liposomes was added to each well. The control group received fresh culture medium without the drugs. After incubation for 4 hours, the group containing the photosensitizer was irradiated with laser (200 mW / cm²). 2 After incubation for 44 hours, 20 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for another 4 hours. After incubation, the old culture medium was discarded, and 200 μL of LDMSO solution was added to each well. The wells were then shaken for 10 minutes to fully dissolve the formazan. The absorbance was measured at 490 nm using a multi-functional microplate reader. The cell inhibition rate was calculated using a formula, and the IC50 for each group was calculated using GraphPadPrism 5.0. 50 value.

[0095] Table 3. Median lethal concentrations (ng / mL) of PAG liposomes, Ce6 liposomes, and PAG / Ce6 liposomes in 4T1 cells.

[0096]

[0097] Experimental results are as follows Figure 11 As shown in Table 3, PAG / Ce6 liposomes exhibit stronger cytotoxicity than PAG liposomes and Ce6 liposomes alone, and the calculated CI... 50The value of 0.16 indicates that the prodrug PAG and dihydroporphyrin e6 still have a strong synergistic effect after being prepared into liposomes.

[0098] Example 13: In vivo pharmacokinetic study of dual-drug-loaded liposomes

[0099] Healthy SD rats were randomly divided into two groups of five each. The rats were fasted for 12 hours prior to the experiment, and their weight was recorded. Podophyllotoxin and dihydroporphyrin e6 mixed solution, or the prodrug PAG / dihydroporphyrin e6 liposome, were administered to the rats via tail vein injection. The equivalent dose of podophyllotoxin injected into each rat was 2.5 mg / kg. Blood samples were collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after administration into heparinized EP tubes. The samples were centrifuged at 13000 rpm for 5 min, and the supernatant was stored at -20℃. The concentrations of podophyllotoxin and the prodrug PAG in rat plasma were determined using UPLC-MS / MS, and pharmacokinetic parameters were calculated using DAS2.0 software.

[0100] Table 4. Pharmacokinetic parameters of podophyllotoxin solution and prodrug PAG / dihydroporphyrin e6 liposomes

[0101]

[0102] Experimental results are as follows Figure 12 As shown in Table 4, the experimental results show that free podophyllotoxin solution is rapidly cleared from the blood, while the prepared dual-drug-loaded liposomes can significantly increase the drug's half-life and prolong its circulation time in the body.

[0103] Example 14: In vivo tissue distribution assay of dual-drug-loaded liposomes

[0104] 4T1 cells were digested with trypsin and subcutaneously in BALB / c mice. When the tumor volume reached 200-300 mm², the cells were inoculated into the tumor cells. 3 At approximately 10:00 AM, tumor-bearing mice were randomly divided into two groups of 12 mice each: (1) a mixed solution of podophyllotoxin and dihydroporphyrin e6 (podophyllotoxin 5 mg / kg); and (2) a prodrug PAG / dihydroporphyrin e6 liposome (equivalent to podophyllotoxin dose). Three mice were sacrificed 4, 8, 12 and 24 hours after administration via tail vein injection, respectively. Heart, liver, spleen, lung, kidney and tumor tissue were removed, rinsed with physiological saline, and the distribution of drugs in vivo was monitored using a small animal in vivo imaging system. The drug content in tumor tissue was determined using UPLC-MS / MS.

[0105] The results are as follows Figure 13 As shown in the figure, the results indicate that, compared to solutions, dual-drug-loaded liposomes accumulate more drug at the tumor site, a finding confirmed by quantitative analysis.

[0106] Example 15: In vivo pharmacodynamic study of dual-drug-loaded liposomes

[0107] 4T1 cells were digested with trypsin, centrifuged, and resuspended in sterile PBS. The cell suspension was then subcutaneously seeded into BALB / c mice. When the tumor volume was 100 mm², the cells were... 3 At approximately 10:00 AM, tumor-bearing mice were divided into 8 groups of 5 mice each, based on tumor size. Each group received a tail vein injection of saline, podophyllotoxin solution, dihydroporphyrin E6 solution (+), a mixed podophyllotoxin and dihydroporphyrin E6 solution (+), PAG liposomes, dihydroporphyrin E6 liposomes (+), PAG / dihydroporphyrin E6 liposomes, or PAG / dihydroporphyrin E6 liposomes (+). The equivalent dose of podophyllotoxin was 5 mg / kg, and the equivalent dose of dihydroporphyrin E6 was 2 mg / kg. Groups containing dihydroporphyrin E6 were irradiated with a 660 nm laser (200 mW / cm²) after administration. 2 (5 min), administered once every two days, for a total of four administrations. Tumor volume and body weight changes in mice were measured daily. Tumor volume = (tumor long axis × tumor short axis) 2 ) / 2.

[0108] The curves showing the changes in tumor volume and body weight in mice are as follows: Figure 14 As shown in the figure. The experimental results indicate that encapsulating the prodrug and dihydroporphyrin e6 in liposomes can improve the antitumor effect. The prodrug PAG / dihydroporphyrin e6 liposome (+) showed the best antitumor effect, with the smallest tumor weight after administration and no significant change in the body weight of mice during administration, indicating good safety.

[0109] Example 16: In vivo phototoxicity verification of dual-drug-loaded liposomes

[0110] The hair on the backs of healthy mice was removed, and then dihydroporphyrin e6 solution and the prodrug PAG / dihydroporphyrin e6 liposome were injected via the tail vein. Four hours after administration, the mice in the treatment group were irradiated with a 660nm laser (20mW / cm²). 2 (30 min), and 24 hours after administration, the skin of the mice was peeled off and immersed in paraformaldehyde solution for H&E staining.

[0111] Experimental results are as follows Figure 15 As shown, the dihydroporphyrin e6 solution group exhibited thickened dermal edema, with numerous lymphocytes and hair follicles appearing in the skin network, indicating skin inflammation. In contrast, skin sections from the prodrug PAG / dihydroporphyrin e6 liposome group showed no significant difference from those from normal mice.

Claims

1. A hypoxia-responsive prodrug liposome, characterized in that: Liposomes are prepared from phospholipids, cholesterol, PEGylated phospholipids, hypoxia-responsive prodrugs, photosensitizers, an inner aqueous phase solution, and an outer aqueous buffer solution. The molar ratio of cholesterol to phospholipids is 5:95–45:

55. The hypoxia-responsive prodrug and total phospholipids... w / w The ratio is 1:20-1:5, photosensitizer and total phospholipids. w / w The ratio is 1:50-1:5; the mass ratio between the hypoxia-responsive prodrug and the photosensitizer is 10:1-1:10; the hypoxia-responsive prodrug is a poorly soluble antitumor drug modified with polyphenols; The hypoxia-responsive prodrug is ; The photosensitizer is dihydroporphyrin E6; The total phospholipids are the sum of phospholipids, cholesterol, and PEGylated phospholipids; The external aqueous buffer salt solution is either an EDTA-containing buffer salt solution or an EDTA-free buffer salt solution.

2. The hypoxia-responsive prodrug liposome according to claim 1, characterized in that: The phospholipids are natural phospholipids and / or synthetic phospholipids; The PEGylated phospholipid is DSPE-mPEG. 1000 DSPE-mPEG 2000 DSPE-mPEG 5000 One or more of them; The internal aqueous phase solution is a metal ion salt solution, wherein the metal ion salt solution is a zinc gluconate solution, and the concentration of the metal ion salt solution in the system is 50-350 mM.

3. A method for preparing hypoxia-responsive prodrug liposomes according to claim 1, characterized in that: (1) Dissolve phospholipids, cholesterol, and PEGylated phospholipids in an appropriate amount of organic solvent, remove the organic solvent by rotary evaporation under reduced pressure, and obtain a uniform film; (2) Add the internal aqueous phase solution to the film obtained in step (1) to hydrate it, and reduce the particle size by ultrasonication or extrusion equipment to obtain a single-chamber liposome with uniform particle size; (3) The single-compartment liposomes obtained above were passed through a Sepharose CL-4B agarose gel and eluted with a buffer solution containing EDTA. The collected liposome eluent was then eluted again with a buffer solution without EDTA to obtain blank liposomes. (4) Mix the organic solvent solution of the hypoxia-responsive prodrug and photosensitizer with the blank liposomes and stir and incubate above the liposome phase transition temperature to obtain drug-loaded liposomes. Remove the remaining organic solvent to obtain the final liposome product.

4. The method for preparing hypoxia-responsive prodrug liposomes according to claim 3, characterized in that: The organic solvent in step (1) is one or more of dichloromethane, trichloromethane, methanol or ethanol; In step (4), the organic solvent is ethanol, dimethyl sulfoxide (DMSO), methanol, acetonitrile, acetone, or N,N-dimethylformamide (DMF). The amount of organic solvent used accounts for 2%-50% of the volume of the blank liposomes. v / v ).

5. The method for preparing hypoxia-responsive prodrug liposomes according to claim 4, characterized in that: In step (4), the order of drug addition is to add the photosensitizer solution first, followed by the hypoxia response pre-drug solution.

6. The application of the hypoxia-responsive prodrug liposome according to claim 1, characterized in that: The application of the liposomes in the preparation of antitumor drugs.

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