Cationic liposome preparation for broad-spectrum long-acting fluorescence labeling of cell membrane as well as preparation method and application of cationic liposome preparation

Abstract

The invention discloses a cationic liposome preparation of a broad-spectrum long-acting fluorescence labeled cell membrane as well as a preparation method and application of the cationic liposome preparation. The preparation raw materials of the cationic liposome preparation comprise the following components: lipophilic long-chain fluorescent molecules, neutral phospholipid, electropositive phospholipid, sterol and an organic solvent, wherein the lipophilic long-chain fluorescent molecule is prepared from at least one of a long-chain carbonyanine dye, QzST-1 and QzST-2. According to the liposome preparation prepared by adopting the formula, lipophilic long-chain fluorescent molecules can be effectively fused on a cell membrane in a broad spectrum manner through a cationic liposome transfection mechanism, the liposome preparation has excellent cell membrane targeting property, high dyeing efficiency and good stability, bright fluorescence signals can still be observed on the cell membrane after 12 hours after labeling, and the liposome preparation can be used for detecting the cell membrane. The problems of non-uniform dyeing and short tracing time of commercial cell membrane fluorescent probes in the market are solved, and the fluorescent probe has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cationic liposome preparation for broad-spectrum and long-acting fluorescent labeling of cell membranes, and a preparation method and application thereof. Background Art

[0002] The cell membrane is a phospholipid bilayer embedded with proteins. In addition to its basic function as a cell barrier, it also plays a key role in various cellular processes such as material exchange, signal transduction, and intercellular communication. It is highly correlated with many physiological and pathological processes. Therefore, efficient and stable cell membrane fluorescent labeling is crucial for cell membrane-based research.

[0003] Over the past few years, a series of commercial cell membrane fluorescent probes have appeared on the market, such as the long-chain carbocyanine dye DiO, the long-chain carbocyanine dye DiI, the long-chain carbocyanine dye DiD, and the long-chain carbocyanine dye DiR. These lipophilic carbocyanine dyes can penetrate the cell membrane through their lipophilic long hydrocarbon chains and diffuse laterally within the cell membrane, thereby staining the entire cell membrane. However, these dyes have certain limitations in terms of staining uniformity and the duration of cell membrane tracing. They usually show obvious leakage into the cytoplasm within 2 hours, which makes it difficult to meet the needs of long-term research.

[0004] In view of this, there is an urgent need to find a cell membrane fluorescent labeling reagent that has high cell membrane staining efficiency, uniform staining, is not easily quenched, and can track for a long time. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the prior art. To this end, the present invention provides a cationic liposome formulation for broad-spectrum, long-lasting fluorescent labeling of cell membranes. This liposome formulation exhibits excellent cell membrane targeting, high staining efficiency, and good stability. A bright fluorescent signal can still be observed on the cell membrane 12 hours after labeling, and no leakage into the cytoplasm occurs, suggesting promising application prospects.

[0006] The present invention also provides a method for preparing a cationic liposome preparation for broad-spectrum and long-acting fluorescent labeling of cell membranes.

[0007] The invention also provides a freeze-dried powder.

[0008] The present invention also proposes the use of the above-mentioned cationic liposome preparation for broad-spectrum and long-acting fluorescent labeling of cell membranes and its preparation method or lyophilized powder in the preparation of cell membrane fluorescence imaging or targeting-related products.

[0009] In a first aspect, the present invention provides a cationic liposome preparation for broad-spectrum and long-lasting fluorescent labeling of cell membranes, the raw materials for its preparation include the following components:

[0010] Lipophilic long-chain fluorescent molecules, neutral phospholipids, positively charged phospholipids, sterols and organic solvents;

[0011] Wherein, the lipophilic long-chain fluorescent molecule includes at least one of long-chain carbocyanine dye, QzST-1, and QzST-2;

[0012] The QzST-1 is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof;

[0013] The QzST-2 is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof;

[0014]

[0015] The liposome preparation according to the embodiment of the present invention has at least the following beneficial effects:

[0016] (1) The liposome preparation prepared by the formulation of the present invention has excellent cell membrane targeting, high staining efficiency and good stability. A relatively bright fluorescent signal can still be observed on the cell membrane 12 hours after labeling, and will not leak into the cytoplasm, thus solving the problems of uneven staining and short tracing time of commercial cell membrane fluorescent probes on the market.

[0017] (2) The liposome preparation of the present invention has a broad spectrum of cells. Whether it is normal cells or cancer cells, their cell membranes can be effectively labeled, and the fluorescent signal is bright and stable.

[0018] (3) The lipophilic long-chain fluorescent molecules QzST-1 and QzST-2 designed and synthesized in the present invention have a thickness closer to that of the cell membrane phospholipid bilayer. When used as dye molecules for cationic liposome preparations, they have better dyeing uniformity and stability than other dyes. They not only have high dyeing efficiency, but are also not easily quenched and can track for a long time.

[0019] In some embodiments of the present invention, the pharmaceutically acceptable salt refers to a salt of a free acid or base of a compound represented herein [such as a compound of formula (I) or formula (II)] that is non-toxic, biotolerable, or otherwise biologically suitable for administration to a subject.

[0020] In some embodiments of the present invention, the pharmaceutically acceptable salt is selected from the group consisting of disodium salt, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate (including trifluoroacetate), propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, oxalate, malonate, succinate, suberate, sebacate, fumarate , maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propanesulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate and mandelate.

[0021] In some embodiments of the present invention, the QzST-1 is a compound with the following chemical formula:

[0022]

[0023] In some embodiments of the present invention, the QzST-2 is a compound with the chemical formula shown below:

[0024]

[0025] In some embodiments of the present invention, the long-chain carbocyanine dye includes at least one of long-chain carbocyanine dye DiA, long-chain carbocyanine dye DiO, long-chain carbocyanine dye DiI, long-chain carbocyanine dye DiD, long-chain carbocyanine dye DiS, and long-chain carbocyanine dye DiR.

[0026] In some embodiments of the present invention, the neutral phospholipids include at least one of soy lecithin, hydrogenated soy lecithin, egg yolk lecithin, hydrogenated egg yolk lecithin, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dihexanoylphosphatidylcholine, dinonanoylphosphatidylcholine, diphytylphosphatidylcholine, lysophosphatidylcholine, and ditricosadiynylphosphatidylcholine.

[0027] In some embodiments of the present invention, the positively charged phospholipids include at least one of (2,3-dioleyloxypropyl)trimethylammonium chloride, 1,2-dioleoyloxy-3-(dimethylamino)propane, 1,2-dioctadecenoxy-3-methylammonium propane chloride, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, 1,2-dioleyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, SM-102, and DLin-M-C3-DMA.

[0028] In some embodiments of the present invention, the sterol includes at least one of cholesterol, lanosterol, sitosterol, stigmasterol, and ergosterol.

[0029] In some embodiments of the present invention, the organic solvent includes at least one of methanol, ethanol, dichloromethane, and chloroform.

[0030] In some embodiments of the present invention, the preparation raw materials include the following components, calculated by weight: 0.01 to 1 parts of lipophilic long-chain fluorescent molecules, 0.4 to 40 parts of neutral phospholipids, 0.04 to 4 parts of positively charged phospholipids, 0.2 to 20 parts of sterols and 500 to 5000 parts of solvents.

[0031] In some preferred embodiments of the present invention, the preparation raw materials include the following components, calculated by weight: 0.01-0.5 parts of lipophilic long-chain fluorescent molecules, 0.4-20 parts of neutral phospholipids, 0.04-2 parts of positively charged phospholipids, 0.2-10 parts of sterols and 500-2000 parts of solvents.

[0032] In some preferred embodiments of the present invention, the preparation raw materials include the following components, calculated by weight: 0.1-0.5 parts of lipophilic long-chain fluorescent molecules, 5-15 parts of neutral phospholipids, 0.5-3 parts of positively charged phospholipids, 1-10 parts of sterols and 800-1500 parts of solvents.

[0033] In some embodiments of the present invention, the prepared raw materials further include a working solution.

[0034] In some embodiments of the present invention, the working fluid includes one of ultrapure water, deionized water, sodium chloride solution, and phosphate buffer solution.

[0035] In some embodiments of the present invention, the working solution has an amount of 500 to 5000 parts by weight. For example, the working solution has an amount of 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 2500, 3000, 3500, 4000, 4500, 5000, etc.

[0036] The second aspect of the present invention provides a method for preparing the cationic liposome preparation for broad-spectrum and long-lasting fluorescent cell membrane labeling according to the first aspect, comprising:

[0037] S1, mixing the lipophilic long-chain fluorescent molecule, the neutral phospholipid, the positively charged phospholipid, the sterol and the organic solvent to obtain an organic phase;

[0038] S2. performing a rotary evaporation treatment on the organic phase, and then adding the working solution for hydration treatment to obtain a hydrated solution;

[0039] S3, using the hydrated solution as a raw material to prepare liposomes.

[0040] The preparation method according to the embodiment of the present invention has at least the following beneficial effects: the preparation method of the liposome preparation of the present invention is simple, low-cost, and the raw materials are easily available, and is suitable for industrial production.

[0041] In some embodiments of the present invention, the temperature of the rotary evaporation treatment is 45-65°C.

[0042] In some embodiments of the present invention, the temperature of the hydration treatment is 45-65°C.

[0043] In some embodiments of the present invention, the hydration treatment comprises: adding the working solution to the product after the rotary evaporation treatment, placing it in a constant temperature water bath at 45-65° C., and rotating it under normal pressure until the product is completely hydrated.

[0044] In some embodiments of the present invention, the preparing of liposomes using the hydration solution as a raw material comprises subjecting the hydration solution to ultrasonication, homogenization or extrusion treatment.

[0045] The third aspect of the present invention provides a freeze-dried powder, the raw materials of which include the cationic liposome preparation for broad-spectrum and long-lasting fluorescent cell membrane labeling according to any one of the second aspects and a freeze-drying protective agent.

[0046] The freeze-dried powder according to the embodiments of the present invention has at least the following beneficial effects:

[0047] The freeze-dried powder of the present invention has good stability and still has excellent staining uniformity and efficient cell membrane targeting labeling effect after reconstitution, which solves the problem of short storage period of liposome preparations and is conducive to the industrial production, storage and transportation of the product, thereby realizing the commercialization of the product.

[0048] In some embodiments of the present invention, the lyoprotectant comprises at least one of glucose, mannose, trehalose, sucrose, lactose, maltose, maltotriose, sorbitol, inositol, and mannitol.

[0049] In some preferred embodiments of the present invention, the lyoprotectant includes at least one of mannose, trehalose, and sucrose.

[0050] In some embodiments of the present invention, the mass ratio of the neutral phospholipid to the lyoprotectant in the cationic liposome preparation for broad-spectrum and long-lasting fluorescent labeling of cell membranes is 10:1 to 1:10.

[0051] A fourth aspect of the present invention provides the use of the cationic liposome preparation for broad-spectrum, long-lasting fluorescent labeling of cell membranes as described in any one of the first aspect, the preparation method as described in any one of the second aspect, or the lyophilized powder as described in any one of the third aspect in any of the following:

[0052] a) preparing a cell membrane fluorescence imaging reagent;

[0053] b) preparing a cell membrane fluorescence staining kit;

[0054] c) preparing a cell membrane targeting reagent.

[0055] Other features and advantages of the present invention will be set forth in the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0057] Figure 1 This is a confocal imaging image of the cell membrane of the DiO-LNP dye, DiI-LNP dye, DiD-LNP dye, Cur-LNP dye, and Cy5.5-LNP dye of the present invention with high efficiency fluorescence labeling.

[0058] Figure 2 This is a confocal imaging image of the cell membrane efficiently fluorescently labeled by the QzST-1-LNP dye and the QzST-2-LNP dye of the present invention.

[0059] Figure 3 This is a confocal imaging image of a cell membrane efficiently fluorescently labeled with the SM-LNP dye or PN-LNP dye of the present invention.

[0060] Figure 4 This is a comparison of confocal imaging of fluorescently labeled cell membranes using the DiD-LNP dye of the present invention and free DiD dye (control group).

[0061] Figure 5 This is a comparison of confocal imaging of cell membranes fluorescently labeled with the QzST-1-LNP dye of the present invention and free QzST-1 dye (control group).

[0062] Figure 6 These are the confocal imaging results of fluorescently labeling the cell membranes of different cells with the QzST-1-LNP dye of the present invention.

[0063] Figure 7 These are the confocal imaging results of fluorescently labeling the cell membrane of HT29 cells with different concentrations of QzST-1-LNP dye of the present invention.

[0064] Figure 8 These are the confocal imaging results of fluorescently labeling the cell membrane of HT29 cells with the QzST-1-LNP dye after being stored at low temperature and away from light for different periods of time.

[0065] Figure 9 The confocal imaging results of the fluorescent labeling of the cell membrane of HT29 cells by the QzST-1-LNP dye of the present invention and its lyophilized powder complex solution are shown.

[0066] Figure 10 These are the confocal imaging results of fluorescently labeled cell membranes of HT29 cells after reconstitution of the High-FD group freeze-dried powder stored at low temperature and in the dark for different periods of time.

[0067] Figure 11 These are the confocal imaging results of fluorescently labeled cell membranes of HT29 cells after the High-FD-RF solution of the present invention was stored at low temperature and in the dark for different periods of time.

[0068] Figure 12 These are the physical characteristics of the QzST-1-LNP solution, High-FD lyophilized powder, and High-FD reconstituted solution of the present invention after storage at low temperature and in the dark for different periods of time, where a is the particle size, b is the Zeta potential, and c is the PDI. DETAILED DESCRIPTION

[0069] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0070] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0071] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0072] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0073] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0074] In an embodiment of the present invention, the lipophilic long-chain carbocyanine dye DiO was purchased from Biyuntian with the item number C1038; soybean lecithin was purchased from Aladdin with the item number L105732; (2,3-dioleylpropyl)trimethylammonium chloride was purchased from Aladdin with the item number D292990; cholesterol was purchased from Aladdin with the item number C104029; SM-102 was purchased from Shanghai Baili with the item number BP-25499; DLin-M-C3-DMA was purchased from Aiweituo with the item number O02006.

[0075] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0076] Example 1: DiO-LNP cationic liposome preparation and preparation method thereof

[0077] This embodiment provides a cationic liposome preparation DiO-LNP loaded with a lipophilic long-chain carbocyanine dye, the preparation raw materials of which include:

[0078] 0.2 g of lipophilic long-chain carbocyanine dye DiO, 8 g of soybean lecithin, 0.8 g of (2,3-dioleylpropyl)trimethylammonium chloride, 4 g of cholesterol, 1000 mL of methanol and 1000 mL of ultrapure water.

[0079] The cationic liposome preparation DiO-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0080] S1, 0.2 g of lipophilic long-chain carbocyanine dye DiO, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0081] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0082] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0083] S4. The hydrated solution is extruded through a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size is uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic long-chain carbocyanine dye DiO (denoted as DiO-LNP group).

[0084] Example 2: DiI-LNP cationic liposome preparation and preparation method thereof

[0085] This embodiment provides a cationic liposome preparation DiI-LNP loaded with a lipophilic long-chain carbocyanine dye, the preparation raw materials of which include:

[0086] 0.2 g of lipophilic long-chain carbocyanine dye DiI, 8 g of soybean lecithin, 0.8 g of (2,3-dioleylpropyl)trimethylammonium chloride, 4 g of cholesterol, 1000 mL of methanol and 1000 mL of ultrapure water.

[0087] The cationic liposome preparation DiI-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0088] S1, 0.2 g of lipophilic long-chain carbocyanine dye DiI, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0089] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0090] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0091] S4. The hydrated solution is extruded through a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size is uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic long-chain carbocyanine dye DiI (denoted as DiI-LNP group).

[0092] Example 3: DiD-LNP cationic liposome preparation and preparation method thereof

[0093] This embodiment provides a cationic liposome preparation DiD-LNP loaded with a lipophilic long-chain carbocyanine dye, the preparation raw materials of which include:

[0094] 0.2 g lipophilic long-chain carbocyanine dye DiD, 8 g soybean lecithin, 0.8 g (2,3-dioleylpropyl)trimethylammonium chloride, 4 g cholesterol, 1000 mL methanol and 1000 mL ultrapure water.

[0095] The cationic liposome preparation DiD-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0096] S1, 0.2 g of lipophilic long-chain carbocyanine dye DiD, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0097] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0098] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0099] S4. The hydrated solution is extruded through a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size is uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic long-chain carbocyanine dye DiD (denoted as DiD-LNP group).

[0100] Example 4: QzST-1-LNP cationic liposome preparation 1 and its preparation method

[0101] In this example, a lipophilic long-chain fluorescent molecule QzST-1, whose molecular size is closer to the thickness of the cell membrane phospholipid bilayer, was designed and used as a dye to prepare a cationic liposome formulation QzST-1-LNP loaded with the lipophilic fluorescent molecule QzST-1, wherein the lipophilic fluorescent molecule QzST-1 is a compound with the chemical formula shown below:

[0102]

[0103] The synthetic route of the lipophilic long-chain fluorescent molecule QzST-1 is as follows:

[0104]

[0105] Synthesis of S2: At 0°C, di-tert-butyl dicarbonate ((Boc)2O, 27.1 g, 124.1 mmol) was slowly added dropwise to a solution of S1 (20 g, 56.4 mmol) and triethylamine (12.6 g, 124.1 mmol) in methanol (150 mL). The mixture was maintained at this temperature for 0.5 hours and then refluxed for 12 hours. After the reaction was complete, the solvent was removed by rotary evaporation and then purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 15:1) to obtain product S2 (27.8 g, 89% yield). S2: 1 H NMR (400MHz, DMSO-d6) δ [ppm] = 9.32 (s, 2H), 7.38 (d, J = 8.5Hz, 4H), 7.32 (d, J = 8.9Hz, 2H), 6.94 (d, J = 8.9Hz, 4H), 6.73 (d, J = 9.0Hz, 2H), 1.45 (s, 18H); 13 C NMR (101MHz, DMSO-d6) δ [ppm] = 153.0, 147.5, 141.1, 135.81, 132.0, 125.6, 122.1, 119.7, 111.9, 79.1, 28.3; ESI-HRMS: m / z calcdfor [M+H] + C 28 H 33 BrN3O4 + ,554.1649;found 554.1623.

[0106] Synthesis of S3: Under nitrogen protection, a solution of S2 (27.8g, 50.2mmol), bipyralidoboric acid pinacol ester (B2pin2, 19.1g, 75.3mmol), Pd(dppf)Cl2 (3.7g, 5.0mmol) and potassium acetate (14.8g, 150.6mmol) in 1,4-dioxane (150mL) was heated to 100°C and reacted for 18 hours. After the reaction was completed, 100mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100mL). The organic phases were combined, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and then purified by chromatographic column (SiO2, petroleum ether: ethyl acetate = 10:1) to obtain the product S3 (17.4g, yield 95%). S3: 1HNMR(400MHz,Chloroform-d)δ[ppm]=7.61(d,J=8.6Hz,2H),7.25(d,J=8.6Hz,4H),7 .03(d,J=8.8Hz,4H),6.94(d,J=8.5Hz,2H),6.43(s,2H),1.51(s,18H),1.32(s,12H); 13 C NMR (101MHz, Chloroform-d) δ [ppm] = 152.9, 150.7, 142.5, 135.8, 134.1, 126.0, 120.2, 119.9, 114.1, 83.5, 80.6, 28.4, 24.9; ESI-HRMS: m / z calcd for [M+H] + C 34 H 45 BN3O6 + ,602.3396;found 602.3361.

[0107] Synthesis of S4: Under nitrogen protection, a toluene / methanol / water (250 mL, V:V:V=8:1:1) solution of S3 (27.8 g, 46.2 mmol), 1-bromo-4-iodobenzene (78.4 g, 277.2 mmol), Pd(PPh3)4 (5.3 g, 4.6 mmol) and potassium carbonate (51.1 g, 369.6 mmol) was heated to 90°C for 12 hours. After the reaction was completed, 100 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and the product S4 (6.4 g, 45% yield) was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 10:1). S4: 1 13C NMR (101MHz, Chloroform-d) δ [ppm] = 153.0, 147.1, 142.7, 139.6, 133.8, 132.7, 131.8, 128.1, 127.4, 125.5, 122.1, 120.7, 112.0, 80.5, 28.4; ESI-HRMS: m / z calcd for[M+H] + C 34 H 37BrN3O4 + ,630.1962;found 630.1917.

[0108] Synthesis of S5: Add 75 mL of trifluoroacetic acid to a 150 mL dichloromethane solution of S4 (16.3 g, 25.9 mmol) and stir at room temperature for 12 hours. After the reaction is completed, remove the solvent using a rotary evaporator to obtain a residual liquid. The residual liquid is first dissolved in 100 mL of methanol, then a saturated sodium carbonate solution is added and the pH is adjusted to 7-8. Stir for 30 minutes to precipitate a solid. The precipitated solid is washed with water for the third time and dried in vacuo to obtain the product S5 (9.4 g, yield 84%). S5: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.50 (d, J = 8.2Hz, 2H), 7.40 (d, J = 8.2Hz, 2H ),7.33(d,J=8.4Hz,2H),7.07–6.81(m,6H),6.64(d,J=8.2Hz,4H),3.58(s,4H); 13 C NMR (101MHz, Chloroform-d) δ [ppm] = 149.0, 142.9, 140.0, 139.0, 131.8, 130.6, 128.0, 127.3, 120.4, 119.6, 116.3, 112.0; ESI-HRMS: m / z calcd for [M+H]+C 24 H 21 BrN3 + ,430.0914;found430.0896.

[0109] Synthesis of S6: Tert-butyl nitrite (t-BuONO, 4.9 g, 47.9 mmol) was slowly added dropwise to a solution of S5 (9.4 g, 21.8 mmol) in acetonitrile (150 mL) at 0°C. The mixture was maintained at this temperature for 0.5 hours. Trimethylsilyl azide (TMS-N3, 5.5 g, 47.9 mmol) was then added and stirred at room temperature for 12 hours. After the reaction, the solvent was removed by rotary evaporation, and the product S6 (9.5 g, 90% yield) was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1). S6: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.54 (d, J = 8.5Hz, 2H), 7.55–7.30 (m, 4H), 7.22-6.99 (m, 6H), 6.95 (d, J = 8.9Hz, 4H); 13C NMR (101MHz, Chloroform-d) δ [ppm] = 147.2, 144.5, 139.5, 134.9, 134.2, 132.0, 128.3, 127.9, 125.8, 123.5, 121.2, 120.2; ESI-HRMS: m / z calcd for [M+H] + C 24 H 17 BrN7 + ,482.0651;found 482.0674.

[0110] Synthesis of S7: Under nitrogen protection, a dichloromethane (150 mL) solution of S6 (9.5 g, 19.6 mmol), L2 (11.8 g, 49 mmol), Cu(MeCN)4PF6 (16.1 g, 43.1 mmol) and 2,6-dimethylpyridine (14.8 g, 150.6 mmol) was heated to 40 ° C and reacted for 18 hours. After the reaction was completed, 100 mL of dichloromethane was added to the reaction solution, and then washed for the third time with saturated EDTA (150 mL) solution, and then 100 mL of water was added to extract and separate the organic phase. The solvent of the organic phase was then removed by rotary evaporation, and the product S7 (13.8 g, yield 73%) was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:1). S7: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.89 (d, J = 58.2Hz, 2H), 7.63 (d, J = 8.5Hz, 4H), 7.56 (d, J = 8.5Hz, 2H), 7.51 (d, J = 8.6Hz, 2H), 7.45 (d,J=8.5Hz,2H),7.26(d,J=8.4Hz,4H),7.20(d,J=8.4Hz,2H),4.59(s,4H),3.79–3.52(m,10H),2.61(t,J=6.8Hz,4H),1.47(s,18H); 13 C NMR (101 MHz, Chloroform- d )δ[ppm]=172.1,155.4,147.5,146.2,145.7,139.1,135.9,132.3,132.0,128.4,128.2 ,125.2,124.7,121.9,121.5,120.9,80.4,51.7,43.9,42.8,33.7,28.5; ESI-HRMS:m / z calcd for[M+H] + C 48 H 55BrN9O8 + ,964.3352;found 964.3345.

[0111] Synthesis of S8: Under nitrogen protection, a solution of S7 (13.8g, 14.3mmol), bipyralidoboric acid pinacol ester (B2pin2, 5.4g, 21.5mmol), Pd(dppf)Cl2 (1.0g, 1.4mmol) and potassium acetate (4.2g, 42.9mmol) in 1,4-dioxane (150mL) was heated to 100°C and reacted for 18 hours. After the reaction was completed, 100mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100mL). The organic phases were combined, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and then purified by chromatographic column (SiO2, petroleum ether: ethyl acetate = 1:3) to obtain the product S8 (13.5g, yield 93%). S8: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.97 (s, 1H), 7.89 (d, J = 8.1Hz, 2H), 7.79–7.47 (m, 8H), 7.42– 7.11(m,7H),4.60(s,4H),3.78–3.51(m,10H),2.62(t,J=7.2Hz,4H),1.48(s,18H),1.26(s,12H); 13 C NMR (101MHz, Chloroform-d) δ [ppm] = 172.2, 155.5, 147.6, 146.2, 142.9, 137.1, 135.5, 132.2, 129.9, 128. 6,126.2,125.3,124.7,124.4,122.0,121.0,84.0,80.5,51.8,44.0,42.8,33.8,28.6,25.0; ESI-HRMS:m / z calcd for[M+H] + C 54 H 67 BN9O 10 + ,1012.5099; found 1012.5119.

[0112] Synthesis of S9: Under nitrogen protection, a toluene / methanol / water (150 mL, V:V = 4:1) solution of S8 (13.5 g, 13.2 mmol), 4,7-bis(5-iodothiophen-2-yl)benzo[c][1,2,5]thiadiazole (2.9 g, 5.3 mmol), Pd(PPh3)4 (0.61 g, 0.53 mmol) and potassium carbonate (2.6 g, 13.3 mmol) was heated to 90 ° C for 12 hours. After the reaction was completed, 100 mL of water was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50 mL). The organic phases were combined, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and then purified by column chromatography (SiO2, dichloromethane: ethyl acetate = 1:1) to obtain the product S9 (5.6 g, yield 51%). S9: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 8.14 (d, J = 3.8Hz, 2H), 7.97 (s, 2H), 7.92 (s, 2H), 7.79 (d, J = 8.1Hz, 6H), 7.73–7.53 (m, 16H), 7.47 (d,J=3.9Hz,2H),7.28(d,J=8.5Hz,8H),7.23(d,J=8.1Hz,4H),4.59(s,8H),3.81–3.49(m,20H),2.62(t,J=7.0Hz,8H),1.47(s,36H); 13 C NMR (101MHz, Chloroform-d) δ [ppm] = 172.1, 155.4, 152.6, 147.5, 146.0, 145.7, 145.1, 139.5, 138.7, 136.3, 133.1, 132.2, 128.7 ,128.1,127.2,126.2,125.7,125.4,125.3,124.6,124.2,121.9,120.9,80.4,51.7,43.9,42.8,33.7,28.5; ESI-HRMS:m / zcalcd for[M+2H] 2+ / 2C 110 H 116 N 20 O 16 S3 2+ ,1034.9031; found 1034.9016; m / z calcd for[2M+3H] 3+ / 3C 220 H 231 N 40 O 32 S6 3+,1379.8696; found 1379.8667.

[0113] Synthesis of S10: Under nitrogen, a 100 mL acetonitrile solution of S9 (5.6 g, 2.7 mmol) and potassium trimethylsilanol (TMSOK, 2.8 g, 21.6 mmol) was reacted at room temperature for 3 hours. After the reaction, the precipitated white precipitate was filtered, washed three times with acetonitrile (5 mL), and then acidified in a saturated citric acid solution for 15 minutes. The product S10 (3.5 g, 65% yield) was then filtered, washed with water, and dried under vacuum. 1 H NMR (400MHz, DMSO-d6) δ [ppm] = 11.60 (s, 4H), 8.60 (s, 4H), 8.35–7.99 (m, 4H), 7.97 –7.61(m,22H),7.35–7.06(m,12H),4.52(s,8H),3.61–3.23(m,16H),1.40(s,36H); 13 C NMR (101MHz, DMSO-d6) δ [ppm] = 174.9, 173.1, 171.5, 154.7, 154.2, 151.5, 146.7, 145.7, 145.3, 144.2, 138.3, 137.9, 13 4.6,132.3,132.2,132.0,127.7,126.8,125.6,124.6,121.6,121.3,120.9,79.4,43.2,43.0,33.5,28.2; ESI-HRMS:m / z calcd for[M+2H] 2+ / 2C 106 H 108 N 20 O 16 S3 2+ ,1006.8718; found 1006.8688.

[0114] Synthesis of QzST-1: Add 25 mL of trifluoroacetic acid to a 50 mL dichloromethane solution of S10 (3.5 g, 1.8 mmol) and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator and dry in vacuo to obtain the product QzST-1 (2.9 g, 79% yield). 1HNMR(400MHz,DMSO-d6)δ[ppm]=9.17(s,8H),8.85(s,4H),8.30–8.20(m,4H),7.93–7.77(m,22H),7.37–7.25(m,12H),4.41(s,8H),3.25(s,8H),2.70(t,J=7.2Hz,8H); 13 C NMR(101MHz,DMSO-d6)δ[ppm]=171.8,158.6(q,J=35.4Hz),151.8,147.2,145.9,144.8,139.5,138.8,137.9,135.2,132.5,131.6,129.0,128.1,127.2,126.1,125.7,125.3,124.9,124.7,123.8,122.0,42.3,41.3,30.5;ESI-HRMS:m / z calcd for[M] 4+ / 4C 86 H 78 N 20 O8S3 4+ ,403.8871;found 403.8857;m / z calcd for[M-H] 3+ / 3C 86 H 77 N 20 O8S3 3+ ,538.1804;found 538.1789;m / z calcd for[M-2H] 2+ / 2C 86 H 76 N 20 O8S3 2+ ,806.2653;found 806.2631;m / z calcd for[2M-4H] 4+ / 4C 172 H 152 N 40 O 16 S6 4+ ,806.5161;found806.5138;m / z calcd for[2M-5H] 3+ / 3C 172 H 151 N 40 O 16 S6 3+ ,1075.3536;found 1075.3506。

[0115] The raw materials for preparing the above-mentioned cationic liposome preparation QzST-1-LNP include:

[0116] 0.2 g lipophilic long-chain fluorescent molecule QzST-1, 8 g soybean lecithin, 0.8 g (2,3-dioleylpropyl)trimethylammonium chloride, 4 g cholesterol, 1000 mL methanol and 1000 mL ultrapure water.

[0117] The cationic liposome preparation QzST-1-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0118] S1, 0.2 g of lipophilic long-chain fluorescent molecule QzST-1, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0119] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0120] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0121] S4. The hydrated solution was extruded using a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size was uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic fluorescent molecule QzST-1 (denoted as QzST-1-LNP group).

[0122] Example 5: QzST-1-LNP cationic liposome preparation 2 and its preparation method

[0123] This example uses the lipophilic long-chain fluorescent molecule QzST-1 as a dye and provides another cationic liposome preparation SM-LNP loaded with the lipophilic fluorescent molecule QzST-1. The structure and preparation route of the lipophilic fluorescent molecule QzST-1 can be found in Example 4.

[0124] The raw materials for preparing the above-mentioned cationic liposome preparation SM-LNP include:

[0125] 0.2 g lipophilic long-chain fluorescent molecule QzST-1, 16 g distearoylphosphatidylcholine, 3 g SM-102, 6 g cholesterol, 1000 mL methanol and 1000 mL ultrapure water.

[0126] The cationic liposome preparation QzST-1-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0127] S1, 0.2 g of lipophilic long-chain fluorescent molecule QzST-1, 16 g of distearoylphosphatidylcholine, 3 g of SM-102, 6 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0128] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0129] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0130] S4. The hydrated solution was extruded through a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size was uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic fluorescent molecule QzST-1 (denoted as SM-LNP group).

[0131] Example 6: QzST-1-LNP cationic liposome preparation 3 and its preparation method

[0132] This example uses the lipophilic long-chain fluorescent molecule QzST-1 as a dye and provides another cationic liposome preparation PN-LNP loaded with the lipophilic fluorescent molecule QzST-1. The structure and preparation route of the lipophilic fluorescent molecule QzST-1 can be found in Example 4.

[0133] The raw materials for preparing the above-mentioned cationic liposome preparation PN-LNP include:

[0134] 0.2 g lipophilic long-chain fluorescent molecule QzST-1, 12 g dipalmitoylphosphatidylcholine, 2 g DLin-M-C3-DMA, 4 g cholesterol, 1000 mL methanol and 1000 mL ultrapure water.

[0135] The cationic liposome preparation QzST-1-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0136] S1, 0.2 g of lipophilic fluorescent molecule QzST-1, 12 g of dipalmitoylphosphatidylcholine, 2 g of DLin-M-C3-DMA, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0137] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0138] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0139] S4. The hydrated solution was extruded through a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size was uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic fluorescent molecule QzST-1 (denoted as PN-LNP group).

[0140] Example 7: QzST-2-LNP cationic liposome preparation and preparation method thereof

[0141] In this example, a lipophilic long-chain fluorescent molecule QzST-2, whose molecular size is closer to the thickness of the cell membrane phospholipid bilayer, was designed and used as a dye to prepare a cationic liposome formulation QzST-2-LNP loaded with the lipophilic fluorescent molecule QzST-2, wherein the lipophilic fluorescent molecule QzST-2 is a compound with the chemical formula shown below:

[0142]

[0143] The synthetic route of the lipophilic long-chain fluorescent molecule QzST-2 is as follows:

[0144]

[0145] Synthesis of Y1: Tert-butyl nitrite (t-BuONO, 3.7 g, 36.2 mmol) was slowly added dropwise to a solution of S1 (10.7 g, 30.2 mmol) in acetonitrile (150 mL) at 0°C. The mixture was maintained at this temperature for 0.5 hours. Trimethylsilyl azide (TMS-N3, 4.2 g, 36.2 mmol) was then added and stirred at room temperature for 12 hours. After the reaction, the solvent was removed by rotary evaporation, and the product Y1 (10.4 g, 85% yield) was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 100:1). Y1: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.33 (d, J = 8.8Hz, 2H), 7.04 (d, J = 8.9Hz, 4H), 7.01–6.75 (m, 6H); 13 C NMR (101MHz, Chloroform-d) δ [ppm] = 146.6, 144.2, 134.9, 132.4, 125.6, 124.7, 120.1, 115.2; ESI-HRMS: m / z calcd for [M+H] + C 18 H 13 BrN7+ ,406.0410;found 406.0363.

[0146] Synthesis of Y2: Under nitrogen protection, a dichloromethane (150 mL) solution of Y1 (10.4 g, 25.7 mmol), L2 (15.5 g, 64.2 mmol), Cu(MeCN)4PF6 (21.1 g, 56.5 mmol) and 2,6-dimethylpyridine (1.1 g, 10.28 mmol) was heated to 40°C for 18 hours. After the reaction was completed, 100 mL of dichloromethane was added to the reaction solution, and then washed for the third time with a saturated EDTA (150 mL) solution. Then, 100 mL of water was added and the organic phase was extracted and separated. The solvent of the organic phase was then removed by a rotary evaporator and purified by a chromatographic column (SiO2, petroleum ether: ethyl acetate = 1:1) to obtain the product Y2 (17.4 g, yield 76%). Y2: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.88 (d, J = 58.1Hz, 2H), 7.61 (d, J = 8.5Hz, 4H), 7.43 (d, J = 8.7Hz, 2H), 7.21 (d,J=8.4Hz,4H),7.03(d,J=8.8Hz,2H),4.58(s,4H),3.76–3.53(m,10H),2.61(t,J=7.1Hz,4H),1.47(s,18H); 13 C NMR(101MHz,Chloroform-d)δ[ppm]=172.1,155.4,147.2,145.8,145.7,132.9,132.4 ,126.5,124.6,122.0,120.9,117.2,80.5,51.8,43.9,42.8,33.7,28.5; ESI-HRMS:m / z calcd for[M+H] + C 42 H 51 BrN9O8 + ,888.3039;found 888.3037.

[0147] Synthesis of Y3: Under nitrogen protection, a solution of Y2 (17.4 g, 19.5 mmol), bipyralidoboric acid pinacol ester (B2pin2, 7.4 g, 29.3 mmol), Pd (dppf) Cl2 (1.4 g, 1.9 mmol) and potassium acetate (5.7 g, 58.5 mmol) in 1,4-dioxane (150 mL) was heated to 100 ° C and reacted for 18 hours. After the reaction was completed, 100 mL of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (100 mL). The organic phases were combined, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and then purified by chromatographic column (SiO2, petroleum ether: ethyl acetate = 1:2) to obtain product Y3 (17.4 g, yield 95%). Y3: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 7.90 (d, J = 59.4Hz, 2H), 7.77 (d, J = 8.1Hz, 2H), 7.63 (d, J = 8.4Hz, 4H), 7.25 (d, J = 8 .4Hz,4H),7.13(d,J=7.9Hz,2H),4.60(s,4H),3.75–3.56(m,10H),2.62(t,J=7.0Hz,4H),1.48(s,18H),1.36(s,12H); 13 C NMR(101MHz,Chloroform-d)δ[ppm]=172.1,155.4,149.2,147.3,136.3,132.4,131.1,128.8,1 25.0,123.4,121.8,120.9,83.9,80.4,51.7,43.8,42.7,33.7,28.4,24.9;ESI-HRMS:m / zcalcd for[M+H] + C 48 H 63 BN9O 10 + ,936.4786;found 936.4791.

[0148] Synthesis of Y4: Under nitrogen protection, a solution of Y3 (17.4 g, 18.5 mmol), 4,7-bis(5-iodothien-2-yl)benzo[c][1,2,5]thiadiazole (4.1 g, 7.4 mmol), Pd(PPh3)4 (0.86 g, 0.74 mmol) and potassium carbonate (2.6 g, 18.5 mmol) in toluene / methanol / water (150 mL, V:V = 4:1) was heated to 90°C for 12 hours. After the reaction was completed, 100 mL of water was added to the reaction solution, and the mixture was extracted three times with dichloromethane (50 mL). The organic phases were combined, washed once with saturated sodium chloride, and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation, and the product Y4 (6.4 g, 45% yield) was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:3). Y4: 1 H NMR (400MHz, Chloroform-d) δ [ppm] = 8.13 (d, J = 3.9 Hz, 2H), 7.99–7.94 (m, 2H), 7.90 (s, 2H), 7.82 (s, 1H), 7.73–7.59 (m, 12H), 7.55 (d, J = 8.6H z,1H),7.39(d,J=4.0Hz,2H),7.32–7.25(m,8H),7.18(d,J=8.3Hz,4H),4.59(s,8H),3.75–3.55(m,20H),2.61(t,J=7.2Hz,8H),1.47(s,36H); 13 C NMR (101MHz, Chloroform-d) δ [ppm] = 172.1, 152.6, 147.3, 146.1, 145.8, 144.8, 138.5, 132.3, 128.7, 128.0, 12 7.1,125.7,125.3,125.1,124.8,124.6,123.9,121.9,120.9,80.4,51.8,43.9,42.8,33.6,28.5; ESI-HRMS:m / z calcd for[M+2H] 2+ / 2C 98 H 108 N 20 O 16 S3 2+ ,958.8718;found958.8723.

[0149] Synthesis of Y5: Under nitrogen, Y4 (6.4 g, 3.3 mmol) and potassium trimethylsilanol (TMSOK, 3.4 g, 26.6 mmol) in 100 mL of acetonitrile were reacted at room temperature for 3 hours. After the reaction, the precipitated white precipitate was filtered, washed three times with acetonitrile (5 mL), and then acidified in saturated citric acid solution for 15 minutes. The product was then filtered, washed with water, and dried under vacuum to obtain product Y5 (4.5 g, 73% yield). Y5: 1 H NMR (400MHz, DMSO-d6) δ [ppm] = 11.89 (s, 4H), 8.62 (s, 4H), 8.18 (d, J = 3.8Hz, 2H), 8.13 (s, 2H), 7.88 (d, J = 8.5Hz, 8H), 7.76 (d, J = 8.2Hz, 4H),7.62(d,J=3.8Hz,2H),7.28(d,J=8.4Hz,8H),7.18(d,J=8.3Hz,4H),4.53(s,8H),3.47(t,J=7.3Hz,8H),3.34(s,8H),1.41(s,36H); 13 C NMR (101MHz, DMSO-d6) δ [ppm] = 175.0, 173.4, 171.8, 154.9, 152.0, 147.0, 146.9, 146.4, 145.9, 145.5, 14 4.9,137.8,132.4,129.3,127.1,125.1,125.0,122.0,121.6,79.7,43.3,42.0,33.8,28.5; ESI-HRMS:m / z calcd for[M-BOC+3H] 2+ / 2C 89 H 92 N 20 O 14 S3 2+ ,880.8143; found 880.8128; m / z calcd for[M+2H] 2+ / 2C 94 H 100 N 20 O 16 S3 2+ ,930.8405; found 930.8394; m / z calcd for[M+H] + C 94 H 99 N 20 O 16 S3 + ,1860.6738; found 1860.6707.

[0150] Synthesis of QzST-2: Add 25 mL of trifluoroacetic acid to a 50 mL dichloromethane solution of Y5 (4.5 g, 2.4 mmol) and stir at room temperature for 12 hours. After the reaction is complete, remove the solvent using a rotary evaporator and dry in vacuo to obtain the product QzST-2 (3.9 g, 85% yield). 1 H NMR (400MHz, DMSO-d6) δ [ppm] = 9.26 (s, 8H), 8.85 (s, 4H), 8.27–8.09 (m, 4H), 7.87 (d, J = 8.7Hz, 8H), 7.80 (d, J = 8.3Hz, 4H), 7 .66(d,J=3.9Hz,2H),7.33(d,J=8.7Hz,8H),7.22(d,J=8.3Hz,4H),4.40(s,8H),3.25(t,J=4Hz,8H),2.70(t,J=7.2Hz,8H); 13 C NMR (101MHz, DMSO-d6) δ [ppm] = 171.8, 158.45 (q, J = 33.3Hz), 151.7, 147.0, 146.0, 144.7, 139.4, 137.5, 131.7,129.4,128.9,127.0,125.6,125.1,124.8,124.5,123.7,122.0,42.2,41.2,30.4; ESI-HRMS:m / z calcd for[M] 4+ / 4C 74 H 70 N 20 O8S3 4+ ,365.6206; found365.6194; m / z calcd for[MH] 3+ / 3C 74 H 69 N 20 O8S3 3+ ,487.1584; found 487.1581; m / z calcd for[M-2H] 2+ / 2C 74 H 68 N 20 O8S3 2+ ,730.2340; found730.2321; m / z calcd for[2M-4H] 4+ / 4C 148 H 136 N 40 O 16 S6 4+,730.4848; found 730.4824; m / zcalcd for[2M-5H] 3+ / 3C 148 H 135 N 40 O 16 S6 3+ ,973.9785;found 973.9744.

[0151] The raw materials for preparing the above-mentioned cationic liposome preparation QzST-2-LNP include:

[0152] 0.2 g lipophilic long-chain fluorescent molecule QzST-2, 8 g soybean lecithin, 0.8 g (2,3-dioleylpropyl)trimethylammonium chloride, 4 g cholesterol, 1000 mL methanol and 1000 mL ultrapure water.

[0153] The cationic liposome preparation QzST-2-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0154] S1, 0.2 g of lipophilic fluorescent molecule QzST-2, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0155] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0156] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0157] S4. The hydrated solution is extruded using a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size is uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic fluorescent molecule QzST-2 (denoted as QzST-2-LNP group).

[0158] Comparative Example 1: Cationic liposomes loaded with lipophilic short-chain dye curcumin

[0159] This comparative example provides a cationic liposome preparation Cur-LNP loaded with a lipophilic short-chain dye curcumin, the preparation raw materials of which include:

[0160] 0.2 g of lipophilic short-chain dye curcumin, 8 g of soybean lecithin, 0.8 g of (2,3-dioleylpropyl)trimethylammonium chloride, 4 g of cholesterol, 1000 mL of methanol and 1000 mL of ultrapure water.

[0161] The cationic liposome preparation Cur-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0162] S1, 0.2 g of lipophilic short-chain dye curcumin, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0163] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0164] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0165] S4. The hydrated solution was extruded using a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size was uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic short-chain dye curcumin (denoted as Cur-LNP group).

[0166] Comparative Example 2: Cationic liposomes loaded with lipophilic short-chain dye Cy5.5

[0167] This comparative example provides a cationic liposome preparation Cy5.5-LNP loaded with a lipophilic short-chain dye Cy5.5, the preparation raw materials of which include:

[0168] 0.2 g of lipophilic short-chain dye Cy5.5, 8 g of soybean lecithin, 0.8 g of (2,3-dioleylpropyl)trimethylammonium chloride, 4 g of cholesterol, 1000 mL of methanol and 1000 mL of ultrapure water.

[0169] The cationic liposome preparation Cy5.5-LNP is prepared by a thin film hydration method, which specifically includes the following steps:

[0170] S1, 0.2 g of lipophilic short-chain dye Cy5.5, 8 g of soybean lecithin, 0.8 g of (2,3-dioleyloxypropyl)trimethylammonium chloride, 4 g of cholesterol and 1000 mL of methanol were mixed to obtain an organic phase;

[0171] S2. Add the organic phase to a round-bottom flask, place it in a 55°C constant temperature water bath and remove the solvent by rotary evaporation under reduced pressure, so that the lipid forms a thin film at the bottom of the round-bottom flask;

[0172] S3. Add 1000 mL of ultrapure water to the round-bottom flask, place it in a 55°C constant temperature water bath and rotate under normal pressure until the film is completely hydrated to obtain a hydration solution;

[0173] S4. Extrude the above hydrated solution using a liposome extruder with a 0.2 μM pore size filter membrane until the liposome particle size is uniform, thereby obtaining a cationic liposome solution loaded with the lipophilic short-chain dye Cy5.5 (denoted as Cy5.5-LNP group).

[0174] Test example 1: High-efficiency fluorescent labeling cell membrane test

[0175] This test example conducted a high-efficiency fluorescent labeling cell membrane test on the cationic liposome solutions prepared in Examples 1 to 7 and Comparative Examples 1 to 2. The specific method is as follows:

[0176] (1) Preparation of working solution: PBS powder was added to the cationic liposome solutions prepared in Examples 1 to 7 and Comparative Examples 1 to 2 until the final concentration of each PBS was 10 mM, and then diluted with blank culture medium four times the original volume to obtain the dye working solution.

[0177] (2) High-efficiency fluorescence labeling: This test takes HT29 cells as an example. HT29 cells in the logarithmic growth phase were cultured and seeded in confocal culture dishes. 1×10 6 After the cells were completely attached overnight, the original culture medium was aspirated and the cells were incubated with the above-mentioned dye working solution in the dark for 1 hour. Then, the cells were rinsed twice with blank culture medium to remove excess dye working solution, and the cells were observed using a laser confocal microscope.

[0178] Among them, the excitation wavelength of the long-chain carbocyanine dye DiO is 485nm, and the emission wavelength is 520nm; the excitation wavelength of the long-chain carbocyanine dye DiI is 519nm, and the emission wavelength is 570nm; the excitation wavelength of the long-chain carbocyanine dye DiD is 646nm, and the emission wavelength is 663nm; the excitation wavelength of the lipophilic long-chain fluorescent molecules QzST-1 and QzST-2 is 514nm, and the emission wavelength is 690nm; the excitation wavelength of the short-chain dye curcumin is 405nm, and the emission wavelength is 530nm; the excitation wavelength of the short-chain dye Cy5.5 is 658nm, and the emission wavelength is 695nm.

[0179] Figure 1The results of efficient fluorescent labeling of cell membranes by DiO-LNP dyes, DiI-LNP dyes, DiD-LNP dyes, Cur-LNP dyes, and Cy5.5-LNP dyes are shown. It can be observed that only the cell membranes of cells fluorescently labeled with DiO-LNP dyes, DiI-LNP dyes, or DiD-LNP dyes are fluorescently labeled, and the fluorescence on the membranes is complete, bright, and uniform, with strong fluorescent staining; whereas the cell membranes and cytoplasm of cells fluorescently labeled with Cur-LNP dyes or Cy5.5-LNP dyes are fluorescently labeled. This is because the lipophilic short-chain dyes cannot be stabilized on the cell membrane for a long time after being transfected onto the cell membrane by cationic liposomes, and easily pass through the cell membrane and enter the interior of the cell. This indicates that the cationic liposome preparation provided by the present invention, which carries a lipophilic long-chain carbocyanine dye, can be used as a general, efficient, and uniformly dyed cell membrane fluorescent labeling dye.

[0180] Figure 2 The results of the high-efficiency fluorescent labeling of cell membranes by QzST-1-LNP dye and QzST-2-LNP dye are shown, showing that only the cell membranes of cells fluorescently labeled with QzST-1-LNP dye or QzST-2-LNP dye are fluorescently labeled and the fluorescence on their membranes is complete, bright, and uniform with strong fluorescent staining. This shows that the cationic liposome formulation for fluorescently labeling cell membranes provided by the present invention is versatile, and its versatility lies in that the formulation is also applicable to the novel lipophilic long-chain fluorescent molecules QzST-1 and QzST-2 synthesized by the present invention based on the thickness of the cell membrane phospholipid bilayer and the cationic liposome transfection principle. In view of the fact that the molecular size of the lipophilic long-chain fluorescent molecule QzST-1 is closer to the thickness of the cell membrane phospholipid bilayer, the preferred preparation is a cationic liposome formulation loaded with the lipophilic long-chain fluorescent molecule QzST-1.

[0181] Figure 3 The results of the highly efficient fluorescent cell membrane labeling test using SM-LNP dyes or PN-LNP dyes are presented, demonstrating that only the cell membranes of cells fluorescently labeled with SM-LNP dyes or PN-LNP dyes are fluorescently labeled, and the fluorescence on the membranes is complete, bright, and uniform. This demonstrates the versatility of the cationic liposome formulation for fluorescent cell membrane labeling provided by the present invention, particularly in that cationic liposomes prepared from neutral phospholipids such as distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine, as well as electropositive phospholipids such as SM-102 and DLin-M-C3-DMA, also exhibit highly efficient cell membrane fluorescent labeling.

[0182] Test Example 2: Long-lasting fluorescent labeling of cell membranes

[0183] This test example tested the long-term fluorescent labeling of cell membranes using the cationic liposome solutions prepared in Examples 1 to 7 and Comparative Examples 1 to 2. The specific method is as follows:

[0184] (1) Preparation of working solution: Add PBS powder to the cationic liposome solution to a final PBS concentration of 10 mM, and dilute with four times the original volume of blank culture medium to obtain a liposome dye working solution. The control group of this test case used a free dye solution (containing only dye molecules without phospholipids and other components) at the same concentration, and the solvent was blank culture medium.

[0185] (2) Long-term fluorescent labeling: The cell line selected and cultured in this test example is HT29 cells. HT29 cells in the logarithmic growth phase were cultured and selected and seeded in confocal culture dishes, with 1×10 6 After the cells were completely attached overnight, they were incubated with liposome dye working solution or control dye working solution (containing only dye molecules) of the same concentration in the dark for 1 hour. Then, the cells were rinsed twice with blank culture medium to remove excess dye working solution. After continuing to culture for 0 hours, 2 hours, 4 hours, 6 hours, and 12 hours, the cells were observed using a laser confocal microscope.

[0186] Figure 4 The results of long-term fluorescence labeling with DiD-LNP dye and its control dye are presented. The results show that cells fluorescently labeled with DiD-LNP dye only have their cell membranes fluorescently labeled, and the fluorescence on the membrane is bright, stable, and intense. Within 12 hours after staining, the membrane fluorescence does not leak into the cytoplasm. Cells labeled with free DiD dye have slightly less stable membrane fluorescence, with the membrane fluorescence showing significant leakage into the cytoplasm within 2 hours of staining and completely losing the membrane fluorescence labeling after 12 hours. This indicates that the cationic liposome preparation provided by the present invention, which carries the lipophilic long-chain carbocyanine dye DiD, can be used as a long-term cell membrane fluorescent labeling dye.

[0187] Figure 5The long-term fluorescence labeling results of QzST-1-LNP dye and its control group dye are shown, showing that only the cell membrane of the cells fluorescently labeled with QzST-1-LNP dye is fluorescently labeled and the fluorescence on its membrane is bright, stable and strong fluorescence staining. Within 12 hours after staining, the fluorescence on its membrane will not leak into the cytoplasm, and the complete and stable labeling time on the cell membrane exceeds 6 hours; the stability of the fluorescence on the membrane of the cells labeled with free fluorescent molecule QzST-1 dye is lower than that of the former. The fluorescence on its membrane is distributed in a dotted manner and shows obvious leakage to the cytoplasm within 4 hours after staining, and the fluorescence labeling on the membrane is completely lost after 12 hours. It can be seen that the cationic liposome preparation containing the lipophilic long-chain fluorescent molecule QzST-1 provided by the present invention can be used as a long-term cell membrane fluorescent labeling dye that can be traced for a long time. And the lipophilic long-chain fluorescent molecule QzST-1 designed and synthesized by the present invention, whose molecular size is closer to the thickness of the cell membrane phospholipid bilayer, is the preferred dye component among the lipophilic long-chain fluorescent molecules involved in the prescription of the present invention.

[0188] Test Example 3: Broad-spectrum Fluorescence Labeling Cell Membrane Test

[0189] This test example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 above as an example to test the effect of the cationic liposomes on broad-spectrum fluorescent labeling of cell membranes. The specific method is as follows:

[0190] (1) Preparation of working solution: Add PBS dry powder to the above QzST-1-LNP group preparation until the final PBS concentration is 10 mM (the concentration of the fluorescent molecule QzST-1 is 100 μM), and dilute with four corresponding blank culture media to obtain the QzST-1-LNP dye working solution of this test example, in which the concentration of the fluorescent molecule QzST-1 is 20 μM, and set aside.

[0191] (2) Broad-spectrum fluorescence labeling: To test whether the prepared liposome solution can broadly fluorescently label cell membranes, different cell lines including cancer cells and normal cells were selected and cultured in this test example. The cancer cells included HT29 cells, MCF7 cells, and SKOV3 cells, and the normal cells included Hacat cells. The specific experimental steps are as follows:

[0192] HT29 cells, MCF7 cells, SKOV3 cells and Hacat cells in the logarithmic growth phase were cultured and selected, and seeded in different confocal culture dishes, with 1×10 cells seeded in each dish. 6 After the cells were completely attached overnight, the original culture medium was aspirated and the cells were incubated with QzST-1-LNP dye working solution in the dark for 1 hour. Then, the cells were rinsed twice with blank culture medium to remove excess QzST-1-LNP dye working solution, and the cells were observed using a laser confocal microscope.

[0193] Figure 6 The confocal imaging results of QzST-1-LNP dye fluorescently labeling the cell membranes of different cells were demonstrated, showing that only the cell membranes of both cancer cells and normal cells were fluorescently labeled, and the fluorescence on the membranes was complete, bright, and uniformly strong fluorescent staining, indicating that the cationic preparation provided by the present invention, which carries the lipophilic long-chain fluorescent molecule QzST-1, can be used as a broad-spectrum cell membrane fluorescent labeling dye with uniform staining.

[0194] Detection Example 4: High-efficiency fluorescent labeling cell membrane detection at different working concentrations

[0195] This test example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 above as an example to test the effect of cationic liposomes at different working concentrations on efficient fluorescent labeling of cell membranes. The specific method is as follows:

[0196] (1) Preparation of working solution: PBS dry powder was added to the QzST-1-LNP group preparation until the final PBS concentration was 10 mM (the concentration of the fluorescent molecule QzST-1 was 100 μM), and then gradiently diluted with blank culture medium to obtain a series of QzST-1-LNP dye working solutions, in which the working concentrations of the fluorescent molecule QzST-1 were 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, 1.56 μM, and 0.78 μM, respectively.

[0197] (2) High-efficiency fluorescence labeling: This test uses HT29 cells as an example. HT29 cells in the logarithmic growth phase were cultured and seeded in confocal culture dishes, with 1×10 cells seeded per dish. 6 After the cells were completely attached overnight, the original culture medium was aspirated and the cells were incubated with different concentrations of QzST-1-LNP dye working solution in the dark for 1 hour. Then, the cells were rinsed twice with blank culture medium to remove excess QzST-1-LNP dye working solution, and the cells were observed using a laser confocal microscope.

[0198] Figure 7Confocal imaging of HT29 cell membranes fluorescently labeled with QzST-1-LNP dye at varying concentrations demonstrates that the fluorescent molecule QzST-1 in the QzST-1-LNP dye working solution can produce strong, bright, and uniform fluorescence staining of the cell membrane at concentrations as low as 1.56 μM, with good staining even at 0.78 μM. Considering the recommended concentration of 1 to 30 μM for commercial free DiD dyes by Shanghai Bio-Technology Co., Ltd., the cationic liposome formulation provided by the present invention exhibits excellent staining effects even at relatively low concentrations, making it suitable as a highly efficient, uniformly staining cell membrane fluorescent labeling dye.

[0199] Test Example 5: Fluorescently labeled cell membrane stability test

[0200] This example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 above as an example to detect the stability of the fluorescently labeled cell membrane of QzST-1-LNP. The specific method is as follows:

[0201] (1) Preparation of working solution: The liposome solution prepared above was placed in a 4°C refrigerator for low-temperature storage in the dark, and removed on days 3, 7, and 14 after storage. PBS dry powder was added to a portion of the QzST-1-LNP group solution until the final PBS concentration was 10 mM (the concentration of the fluorescent molecule QzST-1 was 100 μM), and then diluted with blank culture medium to obtain the QzST-1-LNP dye working solution of this example, wherein the concentration of the fluorescent molecule QzST-1 was 20 μM.

[0202] (2) Stability test: In order to test whether the prepared liposome solution can be stored stably for a long time, HT29 cells were selected and cultured. The specific experimental steps are as follows:

[0203] HT29 cells in the logarithmic growth phase were cultured and seeded in confocal culture dishes, with 1×10 6 After the cells were completely attached overnight, they were incubated with QzST-1-LNP dye working solution in the dark for 1 h. Then, the cells were rinsed twice with blank culture medium to remove excess QzST-1-LNP dye working solution, and the cells were observed using a laser confocal microscope.

[0204] Figure 8The results of the QzST-1-LNP fluorescently labeled cell membrane stability test are presented. It can be observed that in all groups of fluorescently labeled cells, only the cell membranes were fluorescently labeled, and the fluorescence on the membranes was bright, complete, and strong fluorescent staining. This indicates that there is no significant difference in the ability of the QzST-1-LNP dye to fluorescently label cell membranes when stored at low temperature and in the dark on days 3, 7, and 14. The cationic liposome preparation of the present invention for broad-spectrum, long-lasting fluorescent cell membrane labeling has good stability.

[0205] Application Example 1:

[0206] This example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 as an example to prepare a liposome freeze-dried powder without adding trehalose (denoted as Non-FD). The specific preparation method is as follows:

[0207] 1000 mL of the cationic liposome preparation loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 was obtained, stirred evenly, pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer to obtain liposome freeze-dried powder Non-FD without added trehalose.

[0208] The obtained liposome freeze-dried powder was placed in a 4°C refrigerator for storage in the dark for future use.

[0209] Application Example 2:

[0210] In this example, the cationic liposomes loaded with the lipophilic long-chain fluorescent molecule QzST-1 (QzST-1-LNP) prepared in Example 4 above were used as an example to prepare a liposome freeze-dried powder to which a low content of trehalose (denoted as Low-FD) was added. The specific preparation method is as follows:

[0211] The cationic liposome preparation loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 was obtained, 1.6 g of trehalose was added per 1000 mL, stirred evenly, pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer to obtain a low-trehalose liposome freeze-dried powder Low-FD.

[0212] The obtained liposome freeze-dried powder was placed in a 4°C refrigerator for storage in the dark for future use.

[0213] Application Example 3:

[0214] In this example, the cationic liposomes loaded with the lipophilic long-chain fluorescent molecule QzST-1 (QzST-1-LNP) prepared in Example 4 above were used as an example to prepare a lyophilized liposome powder to which a medium content of trehalose (denoted as Medium-FD) was added. The specific preparation method is as follows:

[0215] The cationic liposome preparation loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 was obtained, 8 g of trehalose was added per 1000 mL, stirred evenly, pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer to obtain a liposome freeze-dried powder with a medium trehalose content, Medium-FD.

[0216] The obtained liposome freeze-dried powder was placed in a 4°C refrigerator for storage in the dark for future use.

[0217] Application Example 4:

[0218] This example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 as an example to prepare a liposome freeze-dried powder to which a high content of trehalose (denoted as High-FD) is added. The specific preparation method is as follows:

[0219] The cationic liposome preparation loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 was obtained, 40 g of trehalose was added to every 1000 mL, stirred evenly, placed in a -80°C refrigerator for pre-freezing, and then placed in a freeze dryer for freeze drying to obtain a liposome freeze-dried powder with a high trehalose content, High-FD.

[0220] The obtained liposome freeze-dried powder was placed in a 4°C refrigerator for storage in the dark for future use.

[0221] Application Example 5:

[0222] This example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 as an example to prepare a liposome lyophilized powder to which mannose (denoted as GLT-FD) is added. The specific preparation method is as follows:

[0223] The cationic liposome preparation loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 was obtained, 40 g of mannose was added per 1000 mL, stirred evenly, pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer to obtain a liposome freeze-dried powder GLT-FD with a high mannose content.

[0224] The obtained liposome freeze-dried powder was placed in a 4°C refrigerator for storage in the dark for future use.

[0225] Application Example 6:

[0226] This example uses the cationic liposomes (QzST-1-LNP) loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 as an example to prepare a liposome freeze-dried powder to which sucrose (denoted as ZT-FD) is added. The specific preparation method is as follows:

[0227] The cationic liposome preparation loaded with the lipophilic long-chain fluorescent molecule QzST-1 prepared in Example 4 was obtained, 40 g of sucrose was added to every 1000 mL, stirred evenly, pre-frozen in a -80°C refrigerator, and then freeze-dried in a freeze dryer to obtain a high-sucrose content liposome freeze-dried powder ZT-FD.

[0228] The obtained liposome freeze-dried powder was placed in a 4°C refrigerator for storage in the dark for future use.

[0229] Application Test Example 1: Zeta Potential, Particle Size Distribution, and Polydispersity Index Testing

[0230] In this test example, the zeta potential, particle size distribution and polydispersity index (PDI) of the liposome freeze-dried powder prepared in the above application examples and the QzST-1-LNP liposome solution prepared in Example 4 were tested. The specific test methods are as follows:

[0231] For the lyophilized powder, the liposome lyophilized powder was reconstituted in a 10 mM PBS solution to simulate a physiological environment three days after preparation. For the QzST-1-LNP liposome solution, PBS powder was added to the liposome solution until the final PBS concentration reached 10 mM. The concentration of the fluorescent molecule QzST-1 was 100 μM. To physically characterize each liposome preparation, the zeta potential, particle size, and PDI of each sample were measured using a Malvern particle size analyzer. The results are shown in Table 1.

[0232] Table 1:

[0233] Group Zeta potential (mV) Particle size (d.nm) PDI QzST-1-LNP 6.54±0.62 120.10±3.29 0.12±0.02 Non-FD 6.22±0.64 5775.00±430.40 1.00±0.00 Low-FD 7.03±0.70 1939.33±289.23 1.00±0.00 Medium-FD 7.20±0.86 363.53±19.98 0.91±0.03 High-FD 6.25±0.44 119.37±3.50 0.16±0.01 GLT-FD 6.81±0.58 1384.33±102.89 0.75±0.01 ZT-FD 6.93±0.75 938.60±104.51 0.52±0.13

[0234] The results showed that the potential of the liposomes in the QzST-1-LNP group was around 6mV under a simulated physiological environment, the average particle size was around 120nm, and the PDI was around 0.1, indicating that the prepared liposome solution was still well dispersed and had uniform particle size after being placed in the dark at low temperature for three days. In addition, by comparing the Zeta potential, particle size distribution, and PDI of the re-dissolved liposomes in the Non-FD group, Low-FD group, Medium-FD group, and High-FD group, it can be confirmed that the further addition of trehalose helps to improve the uniformity of particle size compared to low-content trehalose. Similarly, under high concentration addition, the particle size of the re-dissolved liposomes in the GLT-FD group and the ZT-FD group were both higher than 900nm, and the PDI were both higher than 0.5. The dispersion effect was not as good as trehalose, so it can be confirmed that trehalose is the preferred formulation of the liposome freeze-drying protectant.

[0235] Application test example 2: High-efficiency fluorescent labeling of cell membrane after reconstitution of lyophilized powder

[0236] In this test example, the liposome freeze-dried powders prepared in the above application examples 1 to 4 and the QzST-1-LNP liposome solution prepared in Example 4 were re-dissolved and fluorescently labeled on the cell membrane. The specific method is as follows:

[0237] (1) Preparation of working solution: Add PBS dry powder to the above QzST-1-LNP group preparation until the final PBS concentration is 10mM (the concentration of the fluorescent molecule QzST-1 is 100μM), and dilute with four times the original volume of blank culture medium to obtain QzST-1-LNP dye working solution. In addition, the liposome freeze-dried powder of the Non-FD group, Low-FD group, Medium-FD group and High-FD group was re-dissolved with blank culture medium to obtain the Non-FD dye working solution, Low-FD dye working solution, Medium-FD dye working solution and High-FD dye working solution of this test example. The final concentration of the fluorescent molecule QzST-1 in the above four groups of dye working solutions was 20μM.

[0238] (2) High-efficiency fluorescent labeling: In order to test whether the prepared lyophilized liposome powder still has the ability to stably fluorescently label cell membranes after reconstitution, HT29 cells were selected and cultured. The specific experimental steps are as follows:

[0239] HT29 cells in the logarithmic growth phase were cultured and seeded in confocal culture dishes, with 1×10 6After the cells were completely attached overnight, they were incubated in the dark with QzST-1-LNP dye working solution, Non-FD dye working solution, Low-FD dye working solution, Medium-FD dye working solution and High-FD dye working solution for 1 h, then rinsed the cells twice with blank culture medium to remove excess dye working solution, and observed the cells with a laser confocal microscope.

[0240] Figure 9 The results of efficient fluorescent labeling of cell membranes by QzST-1-LNP dye working solution, Non-FD dye working solution, Low-FD dye working solution, Medium-FD dye working solution and High-FD dye working solution are demonstrated. It can be observed that the cells fluorescently labeled with QzST-1-LNP (denoted as LNP) dye and High-FD dye have complete, bright and strong fluorescence staining on their membranes; while the cells labeled with Non-FD dye, Low-FD dye and Medium-FD dye have intermittent fluorescence segments on their membranes, indicating that under freeze-dried storage conditions, the liposome lyophilized powder prepared with the preferred High-FD formulation group of the present invention still has stable fluorescence labeling cell membrane capability after reconstitution, thereby solving the problem of short storage period of liposome preparations, facilitating the industrial production, storage and transportation of the product, and thus realizing the commercialization of the product.

[0241] Application test example 3: Lyophilized powder fluorescent labeling cell membrane stability test

[0242] This test example uses the High-FD lyophilized powder prepared in Example 4 as an example to test the stability of fluorescently labeled cell membranes. The specific method is as follows:

[0243] (1) Preparation of working solution: The liposome lyophilized powder prepared above was stored in a refrigerator at 4°C in the dark and removed on days 3, 7, and 14 after storage. The High-FD lyophilized powder was reconstituted with blank culture medium to prepare a 20 μM working solution of the fluorescent molecule QzST-1 for later use.

[0244] (2) Fluorescence labeling stability test: In order to test whether the prepared liposome freeze-dried powder can be stored stably for a long time, the cell line selected and cultured in this test example is HT29 cells. The specific experimental steps are as follows:

[0245] HT29 cells in the logarithmic growth phase were cultured and seeded in confocal culture dishes, with 1×10 6 After the cells were completely attached overnight, the original culture medium was aspirated and the cells were incubated with High-FD dye working solution in the dark for 1 hour. Then, the cells were rinsed twice with blank culture medium to remove excess High-FD dye working solution, and the cells were observed using a laser confocal microscope.

[0246] Figure 10 The confocal imaging results of fluorescent labeling of the cell membrane of HT29 cells after reconstitution of the High-FD group freeze-dried powder after different periods of storage at low temperature and in the dark are shown. It can be observed that only the cell membrane of the fluorescently labeled cells in all groups is fluorescently labeled, and the fluorescence on the membrane is bright, complete and strong fluorescent staining, indicating that there is no obvious difference in the ability of High-FD freeze-dried powder to fluorescently label the cell membrane after reconstitution after storage at low temperature and in the dark on the 3rd, 7th and 14th days.

[0247] Application test example 4: Lyophilized powder reconstitution solution fluorescence labeled cell membrane stability test

[0248] This test example uses the High-FD lyophilized powder prepared in Example 4 as an example to test the stability of fluorescently labeled cell membranes in the lyophilized powder reconstituted solution. The specific method is as follows:

[0249] (1) Preparation of working solution: The lyophilized liposome powder prepared above was reconstituted with blank culture medium to obtain a High-FD reconstituted solution (denoted as High-FD-RF, in which the concentration of the fluorescent molecule QzST-1 was 100 μM). The High-FD-RF was placed in a 4°C refrigerator for low-temperature storage in the dark. On the 3rd, 7th, and 14th days after storage, it was taken out and diluted with four times the original volume of blank culture medium to prepare a High-FD reconstituted solution dye working solution with a concentration of the fluorescent molecule QzST-1 of 20 μM for use.

[0250] (2) Fluorescence labeling stability test: In order to test whether the prepared liposome lyophilized powder reconstitution solution can be stored stably for a long time, the cell line selected and cultured in this test example is HT29 cells. The specific experimental steps are as follows:

[0251] HT29 cells in the logarithmic growth phase were cultured and seeded in confocal culture dishes, with 1×10 6 After the cells were completely attached overnight, the original culture medium was aspirated and the cells were incubated with High-FD resolution dye working solution in the dark for 1 hour. Then, the cells were rinsed twice with blank culture medium to remove excess High-FD resolution dye working solution, and the cells were observed using a laser confocal microscope.

[0252] Figure 11 The confocal imaging results of fluorescent labeling of the cell membrane of HT29 cells after High-FD-RF was stored at low temperature and in the dark for different periods of time were shown. It can be observed that only the cell membrane of the fluorescently labeled cells in all groups was fluorescently labeled, and the fluorescence on the membrane was bright, complete and strong fluorescent staining. This shows that the High-FD reconstituted solution obtained after the High-FD lyophilized powder was reconstituted had no significant difference in the ability to fluorescently label the cell membrane on the 3rd, 7th and 14th days of storage at low temperature and in the dark, and has excellent stability.

[0253] Application Test Example 5: Zeta potential, particle size, and PDI testing of liposome solutions, lyophilized powders, and their reconstituted solutions

[0254] The preparations used in this test example were the QzST-1-LNP solution and the High-FD lyophilized powder prepared using Example 4. Three groups of samples were further prepared: a QzST-1-LNP solution group, a High-FD lyophilized powder group, and a High-FD reconstituted solution group. Their zeta potential, particle size, and PDI were measured as follows:

[0255] (1) Preparation of working solution: The prepared High-FD lyophilized powder was reconstituted with 10mM PBS solution to simulate physiological environment to obtain High-FD reconstituted solution (denoted as High-FD-RF, in which the concentration of the fluorescent molecule QzST-1 was 100μM). The prepared QzST-1-LNP solution, High-FD lyophilized powder, and High-FD-RF reconstituted solution were all placed in a 4°C refrigerator for low-temperature and light-proof storage, and were taken out on the 3rd, 7th, and 14th days after storage. The QzST-1-LNP solution group was added with PBS dry powder until the final PBS concentration was 10mM, the High-FD lyophilized powder group was reconstituted with 10mM PBS solution to simulate physiological environment, and the High-FD-RF reconstituted solution group was not subjected to additional treatment. The final concentration of the fluorescent molecule QzST-1 was 100μM.

[0256] (2) Zeta potential, particle size and PDI detection: In order to physically characterize the liposome preparations of each group, the zeta potential, particle size and PDI of each group of samples were measured using a Malvern particle size analyzer.

[0257] The test results are as follows Figure 12 As shown in the results, the Zeta potential, particle size distribution and PDI of the QzST-1-LNP solution group, High-FD lyophilized powder group and High-FD reconstituted solution group did not change significantly within 14 days, indicating that the prepared cationic liposomes loaded with the lipophilic long-chain fluorescent molecule QzST-1 and its lyophilized powder and lyophilized powder reconstituted product still have good dispersion and uniform particle size at least within 14 days when stored at low temperature and away from light, which is conducive to the industrial production, storage and transportation of the product.

[0258] In summary, the present invention provides a cationic liposome preparation for long-term fluorescence labeling of cell membranes and its preparation method and application. The liposome preparation prepared by the present invention has a broad spectrum of cell labeling, and both normal cells and cancer cells can be effectively labeled. Secondly, the liposome preparation of the present invention has excellent cell membrane labeling specificity. During the labeling process, only the cell membrane is fluorescently labeled, and the fluorescence on the membrane is bright, stable, and strong fluorescent staining. In addition, the liposome preparation of the present invention has good stability and can be long-term labeled. A strong fluorescent signal can still be observed on the cell membrane 12 hours after labeling, and it will not leak into the cytoplasm, and has a good application prospect.

[0259] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A cationic liposome preparation for broad-spectrum and long-lasting fluorescent labeling of cell membranes, characterized in that: The raw materials for preparation include the following components: Lipophilic long-chain fluorescent molecules, neutral phospholipids, positively charged phospholipids, sterols and organic solvents; Wherein, the lipophilic long-chain fluorescent molecule includes at least one of long-chain carbocyanine dye, QzST-1, and QzST-2; The QzST-1 is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; The QzST-2 is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof; 2. The cationic liposome preparation according to claim 1, wherein The long-chain carbocyanine dye includes at least one of long-chain carbocyanine dye DiA, long-chain carbocyanine dye DiO, long-chain carbocyanine dye DiI, long-chain carbocyanine dye DiD, long-chain carbocyanine dye DiS, and long-chain carbocyanine dye DiR.

3. The cationic liposome preparation according to claim 1, wherein The neutral phospholipids include at least one of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, hydrogenated egg yolk lecithin, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dihexanoylphosphatidylcholine, dinonanoylphosphatidylcholine, diphytylphosphatidylcholine, lysophosphatidylcholine, and ditricosadiynylphosphatidylcholine; And / or, the electropositive phospholipids include at least one of (2,3-dioleyloxypropyl)trimethylammonium chloride, 1,2-dioleoyloxy-3-(dimethylamino)propane, 1,2-dioctadecenyloxy-3-methylammonium propane chloride, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, 1,2-dioleyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, SM-102, and DLin-M-C3-DMA; and / or, the sterol comprises at least one of cholesterol, lanosterol, sitosterol, stigmasterol, and ergosterol; And / or, the organic solvent includes at least one of methanol, ethanol, dichloromethane, and chloroform.

4. The cationic liposome preparation according to claim 1, wherein The preparation raw materials include the following components in parts by weight: 0.01 to 1 parts of lipophilic long-chain fluorescent molecules, 0.4 to 40 parts of neutral phospholipids, 0.04 to 4 parts of electropositive phospholipids, 0.2 to 20 parts of sterols and 500 to 5000 parts of solvents.

5. The cationic liposome preparation according to any one of claims 1 to 4, characterized in that The preparation raw materials also include working liquid; Preferably, the working fluid comprises one of ultrapure water, deionized water, sodium chloride solution, and phosphate buffer solution; Preferably, the working liquid has an amount of 500 to 5000 parts by weight.

6. The method for preparing the cationic liposome preparation for broad-spectrum and long-lasting fluorescent labeling of cell membranes according to claim 5, characterized in that: include: S1, mixing the lipophilic long-chain fluorescent molecule, the neutral phospholipid, the positively charged phospholipid, the sterol and the organic solvent to obtain an organic phase; S2. performing a rotary evaporation treatment on the organic phase, and then adding the working solution for hydration treatment to obtain a hydrated solution; S3, using the hydrated solution as a raw material to prepare liposomes.

7. The preparation method according to claim 6, characterized in that The temperature of the rotary evaporation treatment is 45 to 65°C; and / or, the temperature of the hydration treatment is 45-65° C.; And / or, the preparing of liposomes using the hydration solution as a raw material comprises sonicating, homogenizing or extruding the hydration solution.

8. A freeze-dried powder, characterized in that The preparation raw materials comprise the cationic liposome preparation for broad-spectrum and long-acting fluorescent labeling of cell membranes according to any one of claims 1 to 5 and a freeze-drying protective agent.

9. The freeze-dried powder according to claim 8, characterized in that The freeze-drying protective agent includes at least one of glucose, mannose, trehalose, sucrose, lactose, maltose, maltotriose, sorbitol, inositol, and mannitol; And / or, the mass ratio of the neutral phospholipid in the cationic liposome preparation for broad-spectrum and long-acting fluorescent labeling of cell membranes to the lyoprotectant is 10:1 to 1:

10.

10. Use of the cationic liposome preparation for broad-spectrum, long-lasting fluorescent labeling of cell membranes according to any one of claims 1 to 5, the preparation method according to any one of claims 6 to 7, or the lyophilized powder according to any one of claims 8 to 9 in any of the following: a) preparing a cell membrane fluorescence imaging reagent; b) preparing a cell membrane fluorescence staining kit; c) preparing a cell membrane targeting reagent.

Images