CN112961082B - Drug delivery system combining vascular blocking agent and double-drug-loading bionic liposome - Google Patents

Abstract

The invention belongs to the technical field of medicine, relates to a drug delivery system combining a vascular blocker and a dual-drug-loading biomimetic liposome, and in particular relates to a platelet-membrane fusion co-loading photosensitizer and a hypoxia-activated prodrug biomimetic liposome Preparation of plastids, and their use in combination with vascular blocking agents in modulating tumor microenvironment and inhibiting tumor growth and metastasis. The platelet membrane fusion biomimetic liposome co-loaded with photosensitizer and hypoxia-activated prodrug is a liposome comprising platelet membrane, photosensitizer and hypoxia-activated prodrug. The molar ratio of photosensitizer to hypoxia-activated prodrug is 1:1 to 1:20, and the drug-to-lipid ratio of photosensitizer to total lipid is 1:50 to 500; the ratio of hypoxia-activated prodrug to total lipid is 1:50 to 500. The drug-to-lipid ratio is 1:2 to 50, and the double-drug-loaded liposome of the present invention is preferably a double-drug-loaded liposome of dinitrobenzamide mustard derivative and pyropheophorbide a. The drug delivery system of the invention can promote the activation of the hypoxia-activated prodrug, and realize the synergistic anti-tumor treatment of photodynamic therapy and hypoxia selective chemotherapy.

Description

A drug delivery system for a vascular blocker combined with a dual drug-loaded biomimetic liposome

Technical field:

The invention belongs to the technical field of medicine, relates to a drug delivery system combining a vascular blocker and a dual-drug-loading biomimetic liposome, and in particular relates to a platelet-membrane fusion co-loading photosensitizer and a hypoxia-activated prodrug biomimetic liposome Preparation of plastids, and their use in combination with vascular blocking agents in modulating tumor microenvironment and inhibiting tumor growth and metastasis.

Background technique:

Due to defective tumor vasculature and impaired lymphatic drainage, nanomedicines can accumulate in solid tumors through the enhanced permeability and retention effect (EPR effect). Although nanoformulations have been successful in preclinical animal tumor models, studies have shown that after systemic administration, only <0.7% of nanoformulations can be successfully delivered to tumor tissue for efficacy, which limits the traditional nanoformulations that only rely on the EPR effect. Application value and clinical translation. Currently, cell membrane-coated nano-biomimetic carriers can be used to improve the tumor delivery efficiency of drugs, among which platelets have received extensive attention due to their high biocompatibility, low toxicity, and their indispensable roles in hemorrhage and coagulation. Taking advantage of the interaction between P-selectin on the platelet membrane surface and the CD44 receptor overexpressed by tumor cells, platelet membrane-coated biomimetic nanoformulations have the potential to actively target tumor tissues, circulating tumor cells, and tumor-derived exosomes. Function. However, off-target effects caused by complex tumor heterogeneity greatly reduce the tumor delivery efficiency and therapeutic effect of biomimetic nanoformulations. In addition, the tumor distribution of nanoformulations mainly depends on the basic physiological characteristics of the tumor, such as the degree of tumor blood perfusion, total blood volume and blood flow rate, etc., all of which emphasize the importance of artificially remodeling the tumor microenvironment.

Vascular blockers are a class of drugs that rapidly and selectively destroy tumor blood vessels. Vascular blocking agents block solid by rapidly destroying tumor vascular endothelial cells, resulting in basement membrane exposure and increased tumor vascular permeability, inducing tumor site-specific bleeding, while increased leakage leads to vascular atrophy, vascular occlusion, and initiation of coagulation. Oxygen and nutrient supply to tumor, resulting in anti-tumor effect. However, the degree of tumor killing only by vascular blocking agents is limited. It is urgent to construct a combined system of vascular blocking agents and other therapies and to design a more reasonable dosing sequence and dosing interval to maximize the synergistic tumor-inhibiting effect.

Hypoxia-activated prodrugs are a class of prodrugs that are non-toxic or less toxic, and can be selectively activated in the hypoxic region of tumors to become anti-tumor drugs with cytotoxic activity. Due to the hypoxia-selective cytotoxicity of the prodrug and the heterogeneity of tumor hypoxia, the therapeutic effect of a single hypoxia-activated prodrug is often unsatisfactory. Studies have shown that photodynamic therapy and hypoxia-activated prodrugs are used in combination. Although photodynamic therapy can rapidly deplete tumor oxygen and cause acute hypoxia while generating reactive oxygen species after laser irradiation, tumor hypoxia increases over time. The condition is gradually relieved, so how to prolong the tumor hypoxia time and aggravate the tumor hypoxia degree to promote the activation of the hypoxia-activated prodrug remains to be studied.

Invention content:

In view of the above-mentioned defects of the prior art, the present invention intends to design and construct a double drug-loaded biomimetic liposome for platelet membrane fusion to co-load a hypoxia-activated prodrug and a photosensitizer, and to be administered in combination with a vascular blocking agent. Induced tumor hemorrhage and coagulation cascades artificially modulate the tumor microenvironment, with enhanced tumor EPR effects at the bleeding stage and tumor-specific targeting capabilities of platelet-membrane-fused biomimetic liposomes to achieve high tumor delivery efficiency. In addition, by combining the oxygen consumption of dual-drug-loaded biomimetic liposome photodynamic therapy and the blockade of oxygen supply of vascular blockers, a persistent and severe tumor hypoxic microenvironment is actively constructed, which promotes the activation of hypoxia-activated prodrugs and inhibits tumor growth. growth and transfer.

The object of the present invention is to provide a combined drug delivery system of a photosensitizer/hypoxic activated prodrug double-loaded biomimetic liposome in which a vascular blocking agent is fused with a platelet membrane.

The present invention realizes above-mentioned purpose through following technical scheme:

The invention provides a drug delivery system combining a vascular blocking agent and a double-drug-loading biomimetic liposome, comprising a vascular-blocking agent and a double-drug-loading biomimetic liposome fused with platelet membranes. Described vascular blocking agent is combretastatin A4 and its phosphate, combretastatin A1 and its phosphate, flavonoid-8-acetic acid, 5,6-dimethylxanthone-4-acetic acid, (5S)- 5-(acetamido)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cyclohepten-3-yl dihydrogen phosphate, etc. one or more, preferably combretastatin A4 phosphate CA4P. The sequential administration interval between the vascular blocking agent and the double-drug-loading biomimetic liposome is 1-12 hours, and preferably, the double-drug-loading biomimetic liposome is administered 3 hours after the vascular blocking agent is administered.

The double drug-loading liposome includes hypoxia-activated prodrug, photosensitizer, phospholipid, cholesterol, and PEGylated phospholipid, and the molar ratio of photosensitizer to hypoxia-activated prodrug is 1:1 to 1:20, preferably 1:1 to 1:20. 1:5 to 1:20, more preferably 1:10 to 1:20. The drug-to-lipid ratio of photosensitizer to total lipid is 1:50-500, preferably 1:100-200; the drug-to-lipid ratio of hypoxia-activated prodrug to total lipid is 1:2 to 50, preferably 1:5 ~20, more preferably 1:10, the total lipid is the sum of phospholipids, cholesterol and PEGylated phospholipids, and the amounts of the phospholipids, cholesterol and PEGylated phospholipids are all conventional amounts in the art.

Described hypoxia-activated prodrug is tirapazamine, (2-bromoethyl) ({[(2-bromoethyl)amino][(2-nitro-3-methylimidazol-4-yl) Methoxy]phosphoryl})amine, apazquinone, banoxadone, N-methylmitomycin, or 4-[3-2-nitro-1-imidazolyl-propylamino] One or more of -7-chloroquinoline hydrochloride, dinitrobenzamide mustard (PR104A) and its weakly basic derivatives, preferably a dinitrobenzamide mustard derivative whose base part is dimethylamino DMG-PR104A (DP); the photosensitizers are pyropheophorbide a, chlorphene e6, 5-aminolevulinic acid, protoporphyrin, hematoporphyrin monomethyl ether, rhodopsin A , one or more of hypericin, chlorophyll derivatives or phthalocyanine derivatives, preferably pyropheophorbide a (PPa).

The double-drug-loaded liposome is a double-drug-loaded liposome of dinitrobenzamide mustard derivative and pyropheophorbide a.

The invention provides a weak basic derivative of dinitrobenzamide mustard, a prodrug activated by hypoxia, which connects PR104A and a tertiary amine group through an ester bond, and the weak basic physical and chemical properties help liposomes to actively carry drugs , PR104A can be subsequently released by esterase hydrolysis in vivo. The tertiary amine groups include dimethylamino, N-methylpiperazinyl, piperidinyl, 4-(1-piperidinyl) piperidinyl, morpholinyl, tetrahydropyrrolyl or other tertiary amines group, preferably dimethylamino group as basic modification group, described dinitrobenzamide mustard weak basic derivative or its pharmaceutically acceptable salt, its general structural formula is:

The pharmaceutically acceptable salt is a salt formed by a weakly basic derivative of dinitrobenzamide mustard and a pharmaceutically acceptable inorganic acid or organic acid.

The invention provides the synthetic method of the dinitrobenzamide mustard derivative DMG-PR104A (DP) whose base part is dimethylamino:

Weigh N,N-dimethylglycine into an eggplant-shaped flask, add anhydrous acetonitrile to dissolve it, and add 2-(7-azabenzotriazole)-N,N,N',N'-tetra Methylurea hexafluorophosphate and N-methylmorpholine were dissolved in a small amount of anhydrous acetonitrile and added dropwise to the reaction flask respectively, and the reaction was carried out in an ice bath for 1-2 hours. PR104A was dissolved in a small amount of anhydrous acetonitrile and added dropwise to the reaction flask. The reaction was carried out under nitrogen protection at room temperature for 24 hours, and DP was separated and purified.

The invention provides a preparation method of photosensitizer/hypoxic activated prodrug double drug-loaded liposome, comprising the following steps:

(1) dissolving phospholipid, cholesterol, PEGylated phospholipid and photosensitizer in an organic solvent, removing the organic solvent to form a lipid film;

(2) adding a gradient substance, hydrating above the phase transition temperature, ultrasonically or extruding to prepare a photosensitizer single-loaded liposome;

(3) by dialysis, column chromatography or ultrafiltration means, replace the outer water phase of the liposome with a buffer to obtain a photosensitizer single-loaded liposome with a gradient of the inner and outer water phases of the liposome;

(4) dropwise adding the hypoxia-activated prodrug solution dissolved in an organic solvent, incubating and loading the drug at a certain temperature to obtain a photosensitizer/hypoxia-activated prodrug co-loaded liposome.

In the described step (1), the organic solvent is one or more of dichloromethane, chloroform and methanol.

In the described step (2), the gradient material is one or more of citrate solution, ammonium sulfate solution, sucrose octasulfate triethylammonium salt solution, and sulfobutyl ether cyclodextrin salt solution, preferably ammonium sulfate solution; the concentration of the gradient substance is 100-650mM; the phase transition temperature is 55-75°C.

In the described step (3), the buffer is physiological saline, isotonic sucrose, histidine, hydroxyethylpiperazine-ethylsulfonate (HEPES), morpholine-ethylsulfonate, tartrate, lemon One or more of acid salts and other pharmaceutically acceptable buffer substances, and the pH of the buffer agent is 5.0-7.0.

In the step (4), the organic solvent is one or more of methanol, ethanol, acetone, tetrahydrofuran, acetonitrile and DMSO.

The invention provides double drug-loaded biomimetic liposomes fused with platelet membranes. The platelet membrane is a mouse-derived platelet membrane extracted from rat blood. The platelet membrane extraction steps are as follows: taking rat blood into anti-antibody In the coagulation tube, the red blood cells were removed by centrifugation, and the upper layer of platelet-rich plasma was taken and added to citrate dextrose solution (ACD) for centrifugation. After resuspending in PBS, the platelets were prepared by hypotonic swelling or repeated freezing and thawing to prepare platelet membranes. The drug-loaded liposome is obtained by mixing the extracted platelet membrane and double drug-loading liposome (platelet:lipid is 1～10×10 ⁸ : 1 mg, preferably 2.5×10 ⁸ : 1 mg), and then extruded for multiple times through polymerization. Carbon ester film obtained.

Another object of the present invention is to provide the application of the combined drug delivery system of the vascular blocking agent and the dual drug-loaded biomimetic liposome in the preparation of anti-tumor or anti-tumor metastasis drugs.

Compared with the prior art, the beneficial effects of the present invention:

The advantages of the combined drug delivery system of the vascular blocking agent and the dual drug-loaded biomimetic liposomes of the present invention are: (1) the particle size of the dual-drug-loaded biomimetic liposomes is small and uniform (~100 nm), and the drug-to-lipid ratio and encapsulation (2) Vascular blockers can selectively destroy tumor blood vessels, which is conducive to the active regulation of tumor microenvironment; (3) Vascular blockade The combination and sequential administration of the drug and the dual drug-loaded biomimetic liposome is beneficial to improve the tumor accumulation of nano-drugs by means of the enhanced EPR effect and the specific biological targeting function of platelets; (4) Vascular blocking agent and dual drug-loaded biomimetic liposomes The combined sequential administration of plastids is beneficial to aggravate tumor hypoxia in both directions through photodynamic therapy oxygen consumption and vascular blockers to inhibit oxygen supply, promote the activation of hypoxia-activated prodrugs, and achieve synergistic anti-tumor effects of photodynamic therapy and hypoxia-selective chemotherapy (5) The persistent and severe tumor hypoxic microenvironment constructed by the combined sequential administration of vascular blockers and dual drug-loaded biomimetic liposomes is conducive to inducing large-scale necrosis in the central area of the tumor, reducing the density of tumor microvessels, and thereby inhibiting the tumor. transfer.

Description of drawings:

Figure 1 is the (A) structural formula (B) mass spectrum and (C) ¹ H NMR spectrum of the dinitrobenzamide mustard derivative DMG-PR104A (DP) whose base moiety is dimethylamino in Example 1 of the present invention .

2 is a schematic diagram of the preparation of the photosensitizer/hypoxia-activated prodrug double drug-loaded biomimetic liposome for platelet membrane fusion in Example 12 of the present invention.

3 is a graph showing the results of screening results for the fusion ratio of the platelet membrane and the double drug-loaded liposome membrane of the platelet membrane-fused photosensitizer/hypoxia-activated prodrug double drug-loaded biomimetic liposome in Example 12 of the present invention.

4 is the transmission electron microscope of the photosensitizer/hypoxia-activated prodrug double drug-loaded liposome of Example 3 of the present invention and the photosensitizer/hypoxia-activated prodrug double drug-loaded biomimetic liposome of Example 12 platelet membrane fusion picture.

5 is the characterization of the photosensitizer/hypoxia-activated prodrug double drug-loaded biomimetic liposome protein for platelet membrane fusion in Example 12 of the present invention (A) SDS-PAGE protein electrophoresis analysis (B) Western blot analysis of key proteins.

FIG. 6 is a FERT characterization diagram of the platelet membrane fusion of the platelet membrane of the platelet membrane fused photosensitizer/hypoxia-activated prodrug double-drug-loaded biomimetic liposome and the double-drug-loaded liposome of Example 12 of the present invention.

7 is a graph showing the colloidal stability of the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome for platelet membrane fusion in Example 13 of the present invention.

FIG. 8 is a graph showing the in vitro release of the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome fused to platelet membranes in Example 14 of the present invention.

FIG. 9 is a flow chart of the cellular uptake of the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome for platelet membrane fusion in Example 15 of the present invention (*** represents significant p<0.001).

Figure 10 is the photosensitizer/hypoxia-activated prodrug double drug-loaded biomimetic liposome for platelet membrane fusion according to Example 16 of the present invention (A) singlet oxygen detection diagram in vitro (B) in vitro oxygen consumption detection diagram.

Figure 11 is the cytotoxicity experiment of Example 17 of the present invention (A) Dinitrobenzamide mustard derivative DMG-PR104A (DP) normoxic/hypoxic cytotoxicity (B) platelet membrane fusion with the base moiety of dimethylamino A photosensitizer/hypoxia-activated prodrug dual-loaded biomimetic liposome cytotoxicity.

Figure 12 is the tumor vascular permeability investigation of the vascular blocking agent CA4P of Example 18 of the present invention (A) Quantitative graph of tumor Evans blue at different time points of administration (B) Evans blue of tumor and normal tissue after administration for 3 hours Quantitative plot.

Figure 13 is a quantitative diagram of the effect of the vascular blocking agent in Example 19 of the present invention combined with the dual drug-loaded biomimetic liposome drug delivery system on tumor hypoxia levels (*, **, *** represent significant p< 0.05, p<0.01, p<0.001).

Figure 14 is a blood concentration-time curve diagram of the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome for platelet membrane fusion in Example 20 of the present invention.

Figure 15 is the distribution diagram of the vascular blocking agent combined with the dual drug-loaded biomimetic liposome drug delivery system in Example 21 of the present invention (A) 4 hours (B) 24 hours in vitro.

Figure 16 is a graph of the tumor volume change in the in situ anti-tumor experiment of the vascular blocking agent and the dual drug-loaded biomimetic liposome combined drug delivery system in Example 22 of the present invention (n.s. represents no significant difference; **, *** represent significant p<0.01, p<0.001, respectively).

Fig. 17 is a graph showing the body weight change of mice in the in situ antitumor experiment in vivo with the combined administration system of the vascular blocking agent and the double-loaded biomimetic liposome according to Example 22 of the present invention.

Figure 18 is a statistical diagram of anti-tumor lung metastasis in in situ anti-tumor experiments of the vascular blocking agent and dual drug-loaded biomimetic liposome combination drug delivery system in Example 22 of the present invention (n.s. means no significant difference; * means significant p<0.05).

FIG. 19 is the CD31 blood vessel staining diagram of the tumor tissue section of the in situ anti-tumor experiment in the in vivo anti-tumor experiment of the combined drug delivery system of the vascular blocking agent and the double drug-loaded biomimetic liposome according to Example 22 of the present invention.

Detailed ways:

The present invention is further described below by way of examples, but the invention is not limited to the scope of the described examples.

Example 1: Synthesis of dinitrobenzamide mustard derivative DMG-PR104A(DP) whose base moiety is dimethylamino

N,N-Dimethylglycine (DMG, 69mg, 0.67mmol) was dissolved in anhydrous acetonitrile and incubated in a low temperature ice bath, 2-(7-azabenzotriazole)-N,N,N', N'-tetramethylurea hexafluorophosphate (320 mg, 0.84 mmol) and N-methylmorpholine (180 μL, 1.78 mmol) were dissolved in a small amount of anhydrous acetonitrile and added dropwise to the above solution, respectively, and reacted under ice bath for 1- 2 hours. PR104A (500 mg, 1 mmol) was dissolved in a small amount of anhydrous acetonitrile and added dropwise to the reaction flask. The reaction was carried out under nitrogen protection at room temperature for 24 hours, and DP was separated and purified.

The structure of DP is shown in Fig. 1(A), the mass spectrum for structure confirmation and ¹ H NMR spectrum are shown in Fig. 1(B) (C), the solvent selected for nuclear magnetic resonance is CDCl ₃ , and the analytical results are as follows:

HRMS (ESI) m/z: [M+H] ⁺ calcd for C ₁₈ H ₂₇ BrN ₅ O ₁₀ S, 584.066774; found, 584.065652. ¹ H NMR (600 MHz, CDCl ₃ ) δ 8.62 (d, J=2.8 Hz, 1H), 8.52(d, J＝2.8Hz, 1H), 7.96(s, 1H), 4.41(t, J＝5.0Hz, 2H), 4.37(t, J＝5.2Hz, 2H), 3.77( q, J=5.4Hz, 2H), 3.64–3.49(m, 6H), 3.41(s, 2H), 3.02(s, 3H), 2.51(s, 6H).

Example 2: In vitro cytotoxicity experiments to screen the optimal ratio of photosensitizer PPa and hypoxia-activated prodrug DP for synergy.

Cytotoxicity was assessed by MTT viability assay. 4T1 cells were seeded into 96-well plates at a density of 2500 cells per well and incubated overnight. After the cells adhered, the old culture medium was discarded, and serial dilutions containing ^PPa and DP were added to each well to treat the cells. The culture was continued for 20 hours to investigate the cytotoxicity. The Chou-Talalay method (also known as the median drug effect method) was used to quantitatively describe the synergistic effect of the combination of the two drugs by calculating the Combination Index (CI) value when the two drugs were combined with PPa and DP. , additive or antagonistic effects. The CI calculation formula is as follows, when CI<1, the two drugs produce synergistic effect; when CI=1, produce additive effect; when CI>1, produce antagonistic effect.

Among them, IC _50A and IC _50B respectively represent the dosage of drug A and drug B when the cells are combined to produce a 50% inhibition rate; IC _50AI and IC _50BI respectively represent the doses of drug A and drug B when the cells are used alone to produce a 50% inhibition rate. quantity.

Table 1. PPa and DP synergistic ratio screening

As shown in Table 1, both PPa and DP can produce synergy under the above-mentioned screening ratio. When the ratio of PPa:DP is 1:20, the synergistic effect is the strongest, and the CI value is 0.24. follow-up experiments.

Example 3: Preparation of photosensitizer/hypoxia-activated prodrug double drug-loaded liposomes

Add DSPC, cholesterol, DSPE-mPEG _2k into the eggplant-shaped bottle at the ratio of 3:1:0.05 (w/w), and add the photosensitizer PPa. The ratio of PPa and total lipid is 1:200. Use a small amount of The chloroform was dissolved, and the chloroform was evaporated under reduced pressure at 37°C to form a lipid film. Add 300mM ammonium sulfate solution, hydrate at 65°C for 20 minutes, and sonicate for 10 minutes to prepare photosensitizer single-loaded liposomes. By means of agarose gel column chromatography, the outer aqueous phase of the liposome was replaced with a mixed solution of 300 mM sucrose and HEPES at pH 6, and the ethanol-dissolved hypoxia-activated prodrug DP solution was added dropwise. The drug-to-lipid ratio was 1:10, incubated at 60°C for 20 minutes, and the drug loading was terminated in an ice bath to obtain photosensitizer/hypoxia-activated prodrug co-loaded liposomes L/DP&PPa, and the obtained liposome DP encapsulation efficiency 82.3%, and the encapsulation efficiency of PPa was 98.4%.

Example 4: This example is the same as Example 3, the difference is: the drug loading of DP is reduced during drug loading, that is, the drug-to-lipid ratio of DP and total lipid is 1:20, and the obtained liposome DP package The sealing rate was 87.6%.

Example 5: This example is the same as Example 3, the difference is: the drug loading of DP is increased during drug loading, that is, the drug-to-lipid ratio of DP and total lipid is 1:5, and the obtained liposome DP The encapsulation efficiency was 72.7%.

Example 6: This example is the same as Example 3, except that the active drug loading time is shortened, that is, the drug loading time is 10 minutes, and the obtained liposome DP encapsulation efficiency is 78.0%.

Example 7: This example is the same as Example 3, the difference is that the active drug loading time is prolonged, that is, the drug loading time is 40 minutes, and the obtained liposome DP encapsulation efficiency is 70.0%.

Example 8: This example is the same as Example 3, except that the active drug loading temperature is increased, that is, the drug loading temperature is 65°C, and the obtained liposome DP encapsulation efficiency is 77.8%.

Example 9: This example is the same as Example 3, except that the active drug loading temperature is lowered, that is, the drug loading temperature is 37°C, and the obtained liposome DP encapsulation efficiency is 38.4%.

Example 10: This example is the same as Example 3, except that the organic solvent for dissolving DP used for active drug loading was replaced, that is, the DP solution dissolved in DMSO was dropped in, and the obtained liposome DP was encapsulated The rate was 81.1%.

Example 11: This example is the same as Example 3, the difference is that the organic solvent for dissolving DP used for active drug loading was replaced, that is, the DP solution dissolved in acetonitrile was dropped in, and the obtained liposome DP was encapsulated The rate was 77.3%.

Example 12: Preparation of photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposomes for platelet membrane fusion

The schematic diagram of the preparation is shown in Figure 2. Rat blood was taken in an anticoagulation tube, centrifuged to remove red blood cells, the upper platelet-rich plasma was taken and added to citrate dextrose solution (ACD) for centrifugation. After resuspending platelets in PBS, platelet membranes were prepared by hypotonic swelling method, and mixed with double drug-loaded lipids. Plastid L/DP&PPa was mixed and extruded multiple times through polycarbonate membrane to prepare platelet membrane fusion photosensitizer/hypoxia-activated prodrug double drug-loaded biomimetic liposome PML/DP&PPa. Diameter and particle size distribution (respectively, 1×10 ⁸ , 2.5×10 ⁸ , 5×10 ⁸ , 7.5×10 ⁸ , 10×10 ⁸ platelets were extracted from platelet membrane and 1 mg lipid double drug-loaded liposome L /DP&PPa mix), as shown in Figure 3, the platelet:lipid mixing ratio is 2.5×10 ⁸ : 1mg. After multiple extrusions through the polycarbonate membrane, the particle size of the prepared preparation has little change and the PDI is less than 0.2. Therefore, This ratio was used to prepare the double drug-loaded biomimetic liposomes PML/DP&PPa with platelet membrane fusion in the follow-up.

As shown in Table 2, the double-drug-loaded biomimetic liposome fused with the double-drug-loaded liposome and the platelet membrane was successfully prepared, and the particle size was relatively uniform about 90-100 nm. As shown in Figure 4, the liposomes were observed to have a uniform spherical structure by electron microscopy. The encapsulation efficiency and drug-to-lipid ratio of DP and PPa in the liposomes were high, and the efficient co-loading of photosensitizers and hypoxia-activated prodrugs was achieved.

Table 2. Formulation characterization of L/DP&PPa and PML/DP&PPa

As shown in Figure 5, the results of SDS-PAGE showed that PML/DP&PPa had the same protein composition as the platelet membrane, and the results of Western blot showed that a series of key proteins of the platelet membrane were well preserved in PML/DP&PPa. In order to further prove that the platelet membrane and liposome are combined through membrane fusion, the liposomes were labeled with a pair of fluorescence resonance energy transfer (FRET) dyes DiO and DiI, and the labeled liposomes and platelet membrane proteins were labeled according to different masses. Compared with coextrusion, as shown in Fig. 6, as the amount of platelet membrane increased, the liposome FRET peaks (DiO excitation, DiI emission) decreased, while the donor (DiO) peak intensity increased, proving that the weakened FERT effect was caused by It is caused by the lengthening of the donor-acceptor distance on the original liposome membrane after the fusion of the platelet membrane and the liposome membrane. In conclusion, it is preferred to mix the platelet membrane with the double drug-loaded liposome (platelet: lipid 2.5×10 ⁸ : 1 mg), and then extrude it for several times to successfully prepare the biomimetic liposome through the fusion route of the polycarbonate membrane.

Example 13: Colloidal stability test of the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome for platelet membrane fusion.

The photosensitizer/hypoxia-activated prodrug co-loaded liposome L/DP&PPa prepared in Example 3 and the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome PML prepared in Example 12 were fused to the platelet membrane /DP&PPa (0.25 mg/mL) were incubated at 37°C in pH 7.4 PBS containing 10% FBS for 24 hours, and their particle size changes were determined by dynamic light scattering at predetermined time points.

As shown in Figure 7, the particle size of the two liposomes did not change significantly within 24 hours, showing good colloidal stability.

Example 14: In vitro release test of the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome for platelet membrane fusion.

The photosensitizer/hypoxia-activated prodrug co-loaded liposome L/DP&PPa prepared in Example 3 and the photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome PML prepared in Example 12 were fused to the platelet membrane Pipette /DP&PPa into a dialysis bag, use pH7.4 PBS containing 5% ethanol as the release medium, place it in a 37°C constant temperature shaker with a speed of 100rpm, take samples at a set time point, and measure by high performance liquid chromatography The amount of DP released.

As shown in Figure 8, PML/DP&PPa released about 39% DP at 12 hours, and L/DP&PPa released about 45% DP, which indicated that membrane fusion did not affect the sustained-release properties of liposome-encapsulated DP.

Example 15: Cellular uptake assay of platelet membrane-fused photosensitizer/hypoxia-activated prodrug dual-loaded biomimetic liposomes.

Mouse breast cancer cells 4T1 and mouse fibroblasts 3T3 were seeded in 12-well plates at a density of 1×10 ⁵ cells per well and incubated for 24 hours. PPa solution and L/DP&PPa prepared in Example 3, PML/DP&PPa and PML/DP&PPa prepared in Example 12 and a mixed solution of hyaluronic acid (HA) (PPa equivalent of 2 μM) were added respectively and incubated for 4 hours. After the incubation, the medicated medium was discarded, and cold PBS was added to wash three times. The cells were collected by trypsinization, and the cell uptake was quantitatively analyzed by flow cytometry.

As shown in Figure 9, the fluorescence intensity of 4T1 cells treated with PML/DP&PPa was 1.86-fold higher than that of 3T3 cells, while the fluorescence intensity of 4T1 cells treated with L/DP&PPa or free PPa was comparable to that of 3T3. The uptake efficiency of PML/DP&PPa was reduced by about 60% when co-incubated with the CD44-competing ligand HA. The above results prove that the biomimetic liposome fused with platelet membrane has selective targeting effect on 4T1 cells, mainly through the interaction between platelet P-selectin and the CD44 receptor on the surface of tumor cells.

Example 16: Detection of singlet oxygen generation and oxygen consumption in vitro by photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposomes for platelet membrane fusion.

Using ¹ O ₂ indicator DPBF to investigate the ability of each group to generate ¹ O ₂ under laser irradiation, in pure water control group, PPa (DMSO), PPa (water), L/DP&PPa prepared in Example 3 and Example DPBF solution was added to the PML/DP&PPa (PPa equivalent of 20 μg/mL) prepared in 12. Under the irradiation of a 660 nm laser (100 mW/cm ² ), the absorbance change at 410 nm of the solution was measured with a UV spectrophotometer every 10 seconds, DPBF A drop in absorbance indicates the production of reactive oxygen species. At the same time, after 5 minutes of laser irradiation, the dissolved oxygen concentration in each group of solutions was measured using a portable dissolved oxygen meter.

As shown in Figure 10(A), due to the poor water solubility of PPa, PPa mainly exists in the form of large particle aggregates in the aqueous solution, and the aggregation quenching makes it only produce less ¹ O ₂ . The ability of L/DP&PPa, PML/DP&PPa and PPa(DMSO) to generate ¹ O ₂ is comparable, indicating that the photosensitizer can be uniformly distributed on the liposome bilayer membrane by passive drug loading, and can effectively generate reactive oxygen species. And as shown in Figure 10(B), after 5 minutes of laser irradiation, both liposomes could effectively consume O ₂ in solution, providing hypoxic conditions for the activation of hypoxia-activated prodrugs.

Example 17: Photosensitizer/hypoxia-activated prodrug double-loaded biomimetic liposome cytotoxicity assay for platelet membrane fusion.

Cytotoxicity was assessed by MTT viability assay. 4T1 cells were seeded into 96-well plates at a density of 2500 cells per well and incubated overnight to allow them to adhere. Then, the cells were treated with serial dilutions of PPa solution, DP solution, mixed solution of PPa and DP, L/DP&PPa prepared in Example 3, PML/DP, PML/PPa and PML/DP&PPa prepared in Example 12, and incubated After 4 hours, the cells were irradiated with 660 nm laser light (100 mW/cm ² , 2 minutes), and then cultured for 20 hours to investigate the cytotoxicity. In addition, cells were cultured in normoxia (20%) or hypoxia (1%) for 12 hours after adhesion, and then treated with DP serial dilutions for 48 hours to further investigate the free DP solution in hypoxia and normoxia conditions. cytotoxicity against 4T1 cells.

As shown in Figure 11(A), the cytotoxicity of DP was enhanced under hypoxic conditions compared to normoxic culture conditions, demonstrating that DP can be effectively activated under hypoxic conditions, i.e., DP has excellent safety and low Oxygen-dependent toxicity. As shown in Figure 11(B), under 660 nm laser irradiation, the cell killing ability of the mixed solution of DP and PPa is stronger than that of the single-component solution, which proves that the two have synergistic cytotoxicity. In addition, compared with L/DP&PPa or free combined PPa+DP, PML/DP&PPa was the most cytotoxic, thus PML/DP&PPa was more selective for 4T1 cytotoxicity by virtue of the active uptake of PML/DP&PPa by 4T1 cells.

Example 18: Investigation of vascular blocking agent CA4P on tumor vascular permeability.

4T1 tumor cells (100 μL; 5×10 ⁶ ) were subcutaneously inoculated into female BALB/c mice as an ectopic tumor model, and 12 mice were randomly divided into 4 groups. When the tumor volume was approximately 300 ^mm3 , intraperitoneal injection (CA4P 100 mg/kg) or PBS was performed. Mice were sacrificed and hearts, livers, spleens, lungs, kidneys, tumors were collected 30 minutes after intravenous injection of Evans blue (40 mg/kg) at predetermined time points (1, 3, 7, 24 and 48 hours) post-dose Organs were collected, and the tumor tissue was homogenized with formamide as the medium to obtain the supernatant, and the absorbance of each group at 620 nm was measured. At the same time, the tumor and each organ tissue of mice taken out three hours after administration were frozen sectioned, and imaged and observed by confocal laser scanning microscope (CLSM).

As shown in Figure 12(A), after intraperitoneal injection of CA4P, the accumulation of Evanslan at the tumor site showed a trend of first increasing and then decreasing, and the largest accumulation of Evanslan in the tumor site was observed at 3 hours, which was attributed to the early tumor Vascular collapse induced tumor hemorrhage, enhanced tumor vascular permeability, and subsequent tumor Evans blue infiltration gradually decreased with vascular occlusion and activation of the coagulation cascade, returning to normal accumulation within 48 hours. Figure 12(B) demonstrates that CA4P administration only selectively destroys tumor blood vessels, but has no effect on normal tissues. The above results demonstrate that CA4P can use its initial inducing tumor hemorrhage stage to regulate the tumor microenvironment to amplify the EPR effect. Therefore, the subsequent mode of administration was the intravenous injection of double drug-loaded biomimetic liposomes 3 hours after the administration of the vascular blocking agent.

Example 19: Effect of vascular blocking agent combined with dual drug-loaded biomimetic liposome drug delivery system on tumor hypoxia level

The Hypoxyprobe ^™ Red549 kit was selected to assess hypoxia in different tissues. The construction of the mouse ectopic tumor model was the same as that in Example 18, and 12 mice were randomly divided into 4 groups. When the tumor size was about 300 ^mm3 , normal saline, CA4P solution, PML/DP&PPa, and CA4P+PML/DP&PPa (CA4P 100 mg/kg; PPa equivalent was 2 mg/kg) were administered, and the combined administration group CA4P was administered intraperitoneally 3 hours after intraperitoneal administration. The other preparations were injected into the tail vein, and 660 nm laser irradiation treatment (200 mW/cm ² , 5 minutes) was performed 24 hours after the intravenous administration. Subsequently, the mice were injected with pimonidazole hydrochloride (60 mg/kg) intraperitoneally, and the mice were sacrificed 1.5 hours after administration. Incubate overnight at 4°C. After washing 3 times with PBS, the sections were stained with DAPI and mounted, followed by CLSM imaging observation, and the fluorescence intensity was quantitatively analyzed using ImageJ software.

As shown in Figure 13, compared with the control saline group, the use of CA4P or PML/DP&PPa alone can observe an increase in tumor hypoxic fluorescence expression. The drug delivery system uses CA4P-induced vascular rupture to hinder the supply and recovery of tumor oxygen, while PPa-mediated photodynamic therapy further depletes tumor oxygen. Using a parallel strategy of "blocking and depletion" enables the combination therapy group to achieve the most severe tumor reduction. oxygen.

Example 20: Pharmacokinetic study of photosensitizer/hypoxia-activated prodrug dual-loaded biomimetic liposomes for platelet membrane fusion.

Male Sprague-Dawley rats (220-250 g) were administered free PPa solution, L/DP & PPa prepared in Example 3 and PML/DP & PPa prepared in Example 12 (PPa equivalent of 1 mg/kg) by tail vein injection. Rat fundus venous plexus blood was collected at predetermined time points (0.083, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours) after administration. plasma concentration.

As shown in Figure 14, L/DP&PP and PML/DP&PPa plotted similar pharmacokinetic profiles. The PPa solution was rapidly cleared from the blood, and its plasma clearance (CLz) was 11.75 times faster than that of the two liposomes, while the drug concentration-time of L/DP&PP and PML/DP&PPa liposomes was compared with the PPa solution The area under the curve (AUC) was 12.1-fold and 12.2-fold higher, respectively, indicating that both liposomes have long circulation characteristics.

Example 21: Tissue distribution study of photosensitizer/hypoxia-activated prodrug dual-loaded biomimetic liposomes for platelet membrane fusion.

The construction of the mouse ectopic tumor model was the same as that in Example 18, and 24 mice were randomly divided into 8 groups. When the tumor volume was about 300 mm ³ , PPa solution, PML/DP&PPa, CA4P+L/DP&PPa and CA4P+PML/DP&PPa (CA4P 100 mg/kg; PPa equivalent was 2 mg/kg) were administered, wherein the combined administration method was the same as the implementation Example 19. At 4 and 24 hours after administration, the mice were sacrificed and organs such as heart, liver, spleen, lung, kidney, tumor were collected, and in vitro fluorescence imaging was performed by a small animal imaging system.

As shown in Figure 15, the PPa solution had a non-selective systemic distribution and was rapidly cleared 24 hours after injection. In comparison, the other three groups of preparations accumulated more at the tumor site and the tumor accumulation was time-dependent. Among them, the tumor fluorescence signal in the CA4P+PML/DP&PPa group was the strongest, which proves that the administration of PML/DP&PPa 3 hours after CA4P administration can artificially adjust the tumor microenvironment by using vascular blockers. With the help of enhanced EPR effect and platelet-specific biological Targeting function to enhance tumor accumulation of nanomedicines.

Example 22: In situ anti-tumor and anti-metastasis experiments of vascular blocking agent combined with dual drug-loaded biomimetic liposome drug delivery system.

An orthotopic 4T1 tumor-bearing mouse model was established by injecting 4T1 cells (5×10 ⁵ ) into the mammary fat pad of BALB/c female mice. When the tumor size was about 100 ^mm3 , normal saline, CA4P+PPa+DP mixed solution, PML/DP&PPa, CA4P+PML/DP, CA4P+L/DP&PPa, and CA4P+PML/DP&PPa (CA4P 100 mg/kg; PPa equivalents were 2 mg/kg), wherein the combined administration mode of administration and laser irradiation settings are the same as those in Example 19. The administration time was the 0th, 3rd, 6th and 9th days, and the body weight and tumor growth of the mice were recorded every two days. The mice were sacrificed on the 12th day, and the lungs of orthotopic 4T1 tumor-bearing mice were fixed in Bouin's solution for 18 hours and stored in 70% ethanol, and the number of tumor metastases on the surface of the lung lobes was recorded. The tumor tissue was paraffin-embedded and sectioned for CD31 immunohistochemical staining.

As shown in Figure 16, the tumor growth in the normal saline control group was the fastest, and the administration of the mixed solution of CA4P, DP and PPa had limited tumor inhibitory effect due to poor tumor delivery efficiency. Compared with the mixed solution group, PML/DP&PPa, CA4P+PML/DP and CA4P+L/DP&PPa could inhibit tumor growth to a certain extent, but there was no significant difference in tumor inhibition effect among the three groups. The CA4P+PML/DP&PPa group showed the best anti-tumor effect. The anti-tumor effect of this combined drug delivery system is mainly through improving the delivery efficiency of drugs and tumor drugs and aggravating tumor hypoxia after starting hypoxia selective chemotherapy, and combining with photodynamic therapy to play synergistic anti-tumor effect tumor effect. There was no significant change in the body weight of the mice shown in Figure 17, which proves that the combined drug delivery system has better safety.

As shown in Figure 18, there was almost no obvious tumor lung metastasis in the CA4P+PML/DP&PPa treatment group. In order to further explore its anti-metastatic mechanism, as shown in Figure 19, in the CA4P+PML/DP&PPa group due to acute insufficiency of blood supply caused by vascular collapse and severe and prolonged tumor hypoxia, the largest central necrotic area was observed in the tumor section, and The intratumoral microvessel density was significantly reduced, indicating that the combined drug delivery system can effectively destroy tumor blood vessels and prevent tumor cells from spreading into the circulatory system, thereby effectively inhibiting tumor metastasis.

Claims (16)

1. The double-drug-loading bionic liposome is characterized in that the double-drug-loading bionic liposome is a photosensitizer/hypoxia-activated prodrug co-loaded liposome and comprises a hypoxia-activated prodrug, a photosensitizer, phospholipid, cholesterol and PEG (polyethylene glycol) phospholipid; the molar ratio of photosensitizer to hypoxia-activated prodrug is: 1:1-1:20, wherein the hypoxia-activated prodrug is a dinitrobenzene amide mustard alkalescent derivative, and the structural formula of the derivative is as follows:

；

the photosensitizer is pyropheophorbide a, protoporphyrin dimethyl ester, chlorin e6 and hematoporphyrin monomethyl ether.

2. The double drug-loaded liposome of claim 1, wherein the structural formula of the hypoxia activated prodrug is R

(ii) a The photosensitizer is pyropheophorbide a.

3. The double-drug-loaded liposome of claim 1, wherein the molar ratio of photosensitizer to hypoxia-activated prodrug is: 1:5-1:20.

4. The double-drug-loaded liposome of claim 1, wherein the molar ratio of photosensitizer to hypoxia-activated prodrug is: 1:10-1:20.

5. The double-drug-loaded liposome of any one of claims 1-4, wherein the drug-to-lipid ratio of photosensitizer to total lipid is 1: 50-500; the drug-to-lipid ratio of the hypoxia-activated prodrug to the phospholipid is 1: 2-50.

6. The double-drug-loaded liposome of any one of claims 1-4, wherein the drug-to-lipid ratio of photosensitizer to total lipid is 1: 200.

7. The double-drug-loaded liposome of any of claims 1-4, wherein the ratio of the hypoxia-activated prodrug to the phospholipid is 1: 10.

8. The method of preparing a double drug-loaded liposome of any of claims 1-7, comprising the steps of:

(1) dissolving phospholipid, cholesterol, PEG phospholipid and photosensitizer in an organic solvent, and removing the organic solvent to form a film;

(2) adding a gradient substance, hydrating, and preparing the photosensitizer single-carrier liposome by ultrasonic or extrusion;

(3) replacing the external water phase of the liposome by a buffer through dialysis, column chromatography or ultrafiltration to obtain photosensitizer single-carrier liposome with gradient internal and external water phases of the liposome;

(4) dripping the hypoxia activation prodrug solution dissolved by the organic solvent into the liposome, and incubating the mixture at a certain temperature to obtain the double-drug-loaded liposome.

9. The preparation method of the double drug-loaded liposome of claim 8, wherein the gradient substance is one or more of citrate solution, ammonium sulfate solution, sucrose octasulfate triethylammonium salt solution and sulfobutyl ether cyclodextrin salt solution, the concentration of the gradient substance is 100-650 mM, the buffer is one or more of normal saline, isotonic sucrose, histidine, hydroxyethylpiperazine-ethylsulfonate, morpholine-ethylsulfonate, tartrate, citrate and other pharmaceutically acceptable buffer substances, and the pH of the buffer is 5.0-7.0.

10. The platelet membrane fused double-drug-loaded bionic liposome is characterized by comprising a platelet membrane and the double-drug-loaded liposome of any one of claims 1 to 7, wherein the platelet membrane fused double-drug-loaded bionic liposome is obtained by uniformly mixing the extracted platelet membrane and the double-drug-loaded liposome and then extruding the mixture for multiple times through a polycarbonate membrane, wherein the ratio of platelets to lipid is 1-10 x 10 ⁸ :1 mg。

11. The platelet membrane-fused dual drug-loaded biomimetic liposome of claim 10, wherein the platelet to lipid ratio is 2.5 x 10 ⁸ :1 mg。

12. A drug delivery system combining a vascular blocking agent and a double-drug-loading bionic liposome is characterized by comprising the vascular blocking agent and the double-drug-loading bionic liposome fused with a platelet membrane, wherein the vascular blocking agent and the double-drug-loading bionic liposome are sequentially delivered at intervals; the double-drug-loaded bionic liposome is the platelet membrane fused double-drug-loaded liposome of claim 10 or 11.

13. The combination of a vascular blocking agent and a bi-drug loaded biomimetic liposome as claimed in claim 12, wherein the vascular blocking agent is one or more of combretastatin a4 and its phosphate, combretastatin a1 and its phosphate, flavone-8-acetic acid, 5, 6-dimethylxanthone-4-acetic acid, (5S) -5- (acetamido) -9,10, 11-trimethoxy-6, 7-dihydro-5H-dibenzo [ a, c ] cyclohepten-3-yl dihydrogen phosphate; the interval between the two drugs of the vascular blocking agent and the double-drug-loading bionic liposome is 1-12 hours.

14. The drug delivery system of claim 13, wherein the vascular blocking agent is combretastatin a4 or a phosphate salt thereof.

15. The combination of a vascular blocking agent and a dual drug-loaded biomimetic liposome as in claim 13 or 14, wherein the sequential administration interval of the vascular blocking agent and the dual drug-loaded biomimetic liposome is that the dual drug-loaded biomimetic liposome is administered 3 hours after the administration of the vascular blocking agent.

16. Use of a drug delivery system of a dual drug-loaded liposome according to any one of claims 1 to 7 or a platelet membrane-fused dual drug-loaded biomimetic liposome according to claim 10 or 11 or a combination of a vascular blocking agent according to any one of claims 12 to 14 and a dual drug-loaded biomimetic liposome for the preparation of a medicament for the treatment of tumors or tumor metastases.

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