CN110037989B - Self-cracking multifunctional liposome and application thereof - Google Patents

Self-cracking multifunctional liposome and application thereof Download PDF

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CN110037989B
CN110037989B CN201910337505.XA CN201910337505A CN110037989B CN 110037989 B CN110037989 B CN 110037989B CN 201910337505 A CN201910337505 A CN 201910337505A CN 110037989 B CN110037989 B CN 110037989B
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liposome
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CN110037989A (en
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房雷
徐方泽
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Southeast University
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Abstract

The invention relates to a self-cracking multifunctional liposome and application thereof, wherein the preparation method comprises the steps of sequentially introducing a nitric oxide donor, polyethylene glycol and superparamagnetic ferroferric oxide nanoparticles into an amino terminal of distearoyl phosphatidyl ethanolamine to obtain magnetic targeting phospholipid of an NO donor bridge chain; the NO donor bridge-chain type magnetic targeting phospholipid disclosed by the invention can be used as a main component and is matched with other auxiliary materials such as distearoyl phosphatidylcholine, cholesterol and the like to prepare a nano liposome; the liposome disclosed by the invention can wrap antitumor drugs such as adriamycin, and the obtained drug delivery system has good stability under physiological conditions, and meanwhile, the liposome can be enriched in a magnetic field area under the condition of an external magnetic field to realize magnetic targeting; the NO donor can be cracked to release NO molecules under the action of glutathione and other promoting factors, which is not only favorable for improving the antitumor activity and overcoming the tumor drug resistance, but also can cause the nano liposome to disintegrate and release the loaded drug, thereby improving the drug release efficiency of the drug delivery system and improving the tumor treatment effect.

Description

Self-cracking multifunctional liposome and application thereof
Technical Field
The invention relates to a self-splitting multifunctional liposome and application thereof, belonging to the field of tumor targeted therapy.
Background
Cancer is one of the most feared diseases currently afflicting humans, and seriously threatens human life and health. In recent decades, despite tremendous advances in medicine and biology, the therapeutic status of tumors has not improved significantly. The world health organization survey showed that cancer patients and death cases worldwide have been growing rapidly since 2012, with asian populations accounting for nearly half of the new population.
Surgery and chemotherapy are the main weapons for cancer patients to fight against diseases, unfortunately, the treatment effect of the chemotherapy drugs applied clinically at present is not satisfactory, and the main defects are poor selectivity and serious toxic and side effects. Therefore, the development of novel anticancer drugs with good tumor targeting and high safety is imminent.
The liposome carrier has a unique bilayer structure, and can be used for loading lipophilic substances and hydrophilic substances. The use of liposomes as drug carriers has many advantages, such as enhanced solubility of the encapsulated drug, protection from chemical or biological degradation of the drug during storage or administration, and biodegradability as well as non-toxic and non-hazardous properties of the phospholipid material. In the seventies of the twentieth century, the liposome system was first used as a carrier for transporting drugs by Rahman et al, and since then, a plurality of liposome drugs have been approved for clinical use, and liposomes have become an indispensable part of research and clinical application in the field of nanomedicine. However, long-term use of liposomes as drug delivery vehicles has also found some disadvantages: (1) active targeting is deficient, and the medicament cannot accurately reach a target part to play a role, so that the curative effect of the medicament is influenced, and toxic and side effects on other tissues and organs are caused; (2) the liposome carrier has no stimulation responsiveness, can not specifically release the drug at the focus part, generally has better stability, is not easy to release or has less release amount after loading the drug, and limits the further action of the drug; (3) the drug resistance of the tumor cannot be effectively overcome, the drug resistance mechanism of the tumor is various, and the traditional liposome cannot effectively overcome the drug resistance of the tumor, so that the advantages of a drug delivery system are fully exerted.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a self-cracking multifunctional liposome and application thereof, and aims to solve the problems in the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a self-splitting multifunctional liposome has the following structural formula:
Figure GDA0003080327840000021
as an improvement of the invention, the preparation method comprises the following steps: and (3) sequentially introducing a nitric oxide donor, polyethylene glycol and superparamagnetic ferroferric oxide nanoparticles into the amino terminal of the distearoyl phosphatidyl ethanolamine to obtain the magnetic targeting phospholipid of the NO donor bridge chain.
As an improvement of the invention, the preparation method comprises the following steps:
(1) synthesis of NO donor:
step 1: p-methyl thiophenol reacts with bromoacetic acid under alkaline condition to obtain a thioether intermediate 1;
step 2: oxidizing the intermediate 1 with hydrogen peroxide to obtain an intermediate 2;
and step 3: carrying out NBS bromination reaction on the intermediate 2 to obtain an intermediate 3;
and 4, step 4: the intermediate 3 reacts with fuming nitric acid to cyclize to obtain a furazan nitrogen oxide NO donor 4;
Figure GDA0003080327840000022
(2) synthesis of magnetically targeted phospholipids for NO donor bridge chain:
step 1: NO Donor 4 with azido substituted PEG2000Reacting to obtain an intermediate 5;
step 2: the intermediate 5 reacts with distearoyl phosphatidyl ethanolamine (DSPE) to obtain an intermediate 6;
and step 3: reacting the nano ferroferric oxide nano particles with the surfaces modified with amino with propargyl bromide to prepare an intermediate 7; carrying out click reaction on the intermediate 7 and the intermediate 6 to obtain the magnetic targeting phospholipid of the NO donor bridge chain;
Figure GDA0003080327840000031
as an improvement of the invention, the application of the self-splitting multifunctional liposome in the preparation of tumor targeted therapy medicaments.
Ferroferric oxide nano particles as contrast agents have been successfully applied to magnetic resonance imaging and have important application in some disease diagnosis. Meanwhile, the magnetic nano-particles can orient the target part under the guidance of an external magnetic field, and play a role in physical targeting. Therefore, the magnetic nano-particles and the phospholipid are connected in a covalent bond mode to prepare the nano-liposome carrier with magnetic targeting property, so that the magnetic targeting effect can be kept, and the defects of blood toxicity and the like caused by the nano-particles can be reduced.
Nitric Oxide (NO), an important signaling molecule or effector, is involved in a variety of signal pathways essential for the regulation of tumor cells, and plays an important role in various biological systems. NO donors are compounds that are capable of generating NO. Compared with NO gas, the NO donor is easier to obtain and convenient to process, and can effectively release NO molecules to play the role of NO as a signal molecule and also can play the effect of being cooperated with some anti-tumor drugs.
Based on the method, the applicant carries out chemical modification on the surface of the liposome and combines the superparamagnetic Fe through covalent bonds3O4The nano particles are introduced to the tail end of the phospholipid to realize the magnetic targeting and magnetic resonance imaging functions; on the basis, the furazan nitrogen oxide NO donor is further introduced into the molecular structure, on one hand, after entering tumor cells, the furazan nitrogen oxide NO donor releases NO gas molecules under the promotion of a promoting substance (such as high-concentration glutathione), the bilayer structure of the liposome can be damaged, the release of the encapsulated medicine is facilitated, and on the other hand, the released NO molecules can be used as signal molecules and play a role in cooperation with anti-tumor medicines, so that the anti-tumor activity is improved. In addition, NO can inhibit ATP binding transport protein over-expressed in multi-drug resistant tumor cells by nitrifying key tyrosine residues, thereby preventing the tumor cells from discharging drugs and overcoming drug resistance.
Therefore, in the application, the nitrogen monoxide donor, the polyethylene glycol and the superparamagnetic ferroferric oxide nano particles are sequentially introduced into the amino terminal of the distearoyl phosphatidyl ethanolamine, so that the magnetic targeting phospholipid of the NO donor bridge chain can be obtained.
The NO donor bridge chain type magnetic targeting phospholipid disclosed by the invention can be used as a main component and is matched with distearoyl phosphatidylcholine (DSPC), cholesterol and other auxiliary materials to prepare the nano-liposome. The research result shows that the liposome can wrap antitumor drugs such as adriamycin, the obtained drug delivery system has good stability under physiological conditions, and meanwhile, the liposome can be enriched in a magnetic field area under the condition of an external magnetic field to realize magnetic targeting. In addition, under the condition of being rich in glutathione, the NO donor in the drug delivery system is promoted to release a large amount of NO gas molecules, so that the antitumor activity is promoted, the tumor drug resistance is overcome, the nanoliposome can be disintegrated to release the loaded drug, the drug release efficiency of the drug delivery system is improved, and the tumor treatment effect is improved.
The application of the self-cracking multifunctional nano liposome drug delivery system is that the nano drug delivery system can wrap hydrophobic anti-tumor drugs, and in vitro research shows that the drug delivery system has good stability under physiological conditions, and simultaneously can be enriched in a magnetic field area under the condition of an external magnetic field to realize magnetic targeting. In addition, under the condition of being rich in glutathione, the NO donor in the drug delivery system is promoted to release a large amount of NO gas molecules, so that the antitumor activity is promoted, the tumor drug resistance is overcome, the nanoliposome can be disintegrated to release the loaded drug, and the drug release efficiency of the drug delivery system is improved, so that the nano-liposome can be applied to tumor targeted therapy.
Compared with the prior art, the invention has the following beneficial effects because the technology is adopted:
the invention discloses a self-cracking multifunctional liposome drug delivery system and application thereof, wherein the self-cracking multifunctional nano liposome drug delivery system can be enriched in a magnetic field area under the condition of an external magnetic field so as to realize magnetic targeting. The nano drug delivery system can wrap hydrophobic anti-tumor drugs, has good stability under physiological conditions, but under the condition of being rich in glutathione, NO donor in the drug delivery system can be promoted to release a large amount of NO gas molecules, so that the nano liposome is self-cracked to release loaded drugs; meanwhile, the released NO is beneficial to improving the anti-tumor activity, overcoming the tumor drug resistance and improving the tumor treatment effect.
Drawings
FIG. 1 is a plot of isothermal magnetization of magnetically targeted phospholipids;
FIG. 2 is a particle size distribution and Transmission Electron Microscope (TEM) image of blank liposomes (A, B) and drug-loaded liposomes (C, D);
FIG. 3 shows the release behavior of DOX-loaded liposomes under different conditions;
FIG. 4 shows the results of the in vitro anti-tumor activity of the drug delivery system under the condition of an applied magnetic field, wherein the shapes and positions of the applied magnetic field are marked by black dashed lines;
FIG. 5 is an intracellular NO release assay;
Detailed Description
The present invention will be further illustrated with reference to the following specific embodiments.
Example 1
A preparation method of self-cracking multifunctional liposome comprises the following steps:
(1) preparation of intermediate 1
Figure GDA0003080327840000051
P-toluenesulfonyl phenol (6.21g,0.05mmol) and sodium hydroxide (2.00g,0.05mmol) were dissolved in 40mL of ethanol, 50mL of an aqueous solution prepared from bromoacetic acid (6.95g,0.05mmol) and sodium carbonate (2.88g,0.03mmol) were added, and the mixture was stirred at room temperature for 4 hours and then refluxed for 1 hour. Cooling to room temperature, rotary evaporating to remove ethanol, adjusting pH to 2 with concentrated hydrochloric acid, precipitating a large amount of white solid, vacuum filtering, and drying to obtain 8.65g white solid with yield of 95.0%.
Intermediate 1: ESI-MS: m/z [ M-COOH]-＝137.04.
(2) Preparation of intermediate 2
Figure GDA0003080327840000052
Intermediate 1(3.00g,16.48mmol) was dissolved in 50mL of glacial acetic acid, magnetically stirred, and 6mL of 30% H was slowly added dropwise at 45 ℃2O2(diluted with 10mL of glacial acetic acid), heated to 70 ℃ for 4h, and then heated to 90 ℃ for 11 h. After the reaction was stopped, the reaction mixture was diluted with about 130mL of water, extracted with ethyl acetate (80 mL. times.3), and concentrated. The product was isolated and purified by column chromatography with DCM: MeOH 60:1 to give 3.00g of a white solid with 85.1% yield.
Intermediate 2: ESI-MS: m/z [ M + Na ]]+＝237.01.
1H NMR(300MHz,CDCl3):δ7.84(d,J＝8.0Hz,2H),7.39(d,J＝8.0Hz,2H),4.13(s,2H),2.47(s,3H).
(3) Preparation of intermediate 3
Figure GDA0003080327840000053
Intermediate 2(5.00g,23.36mmol) was dissolved in 100mL MeCN, magnetically stirred, N-bromosuccinimide (5.00g,28.04mmol) and benzoyl peroxide (0.85g,3.5mmol) were added, and the reaction was carried out at 45 ℃ for 12h under UV irradiation. After the reaction was stopped, acetonitrile was removed by rotary evaporation and purified by column chromatography with DCM: MeOH 100:1 to give 6.50g of a white solid in 87.6% yield.
Intermediate 3: ESI-MS: m/z [ M-COOH]-＝248.94.
1H NMR(300MHz,CDCl3):δ8.34(s,1H),7.97(d,J＝8.2Hz,2H),7.63(d,J＝8.2Hz,2H),4.53(s,2H),4.18(s,2H).
(4) Preparation of intermediate 4
Figure GDA0003080327840000061
6.50g of intermediate 3 are dissolved in 100mL of glacial acetic acid, 20mL of fuming nitric acid are slowly added dropwise with stirring and ice-bath, and the temperature is raised to 120 ℃ after the dropwise addition procedure and the reflux is carried out for 8 h. A part of glacial acetic acid is removed by rotary evaporation, 100ml of water is added into the reaction solution, and the reaction solution is placed into a refrigerator for freezing for 12 h. White solid is separated out, filtered and dried to obtain white solid.
Intermediate 4:1H NMR(300MHz,CDCl3):δ8.13(t,J＝8.1Hz,4H),7.67(t,J＝7.7Hz,4H),4.52(d,J＝8.2Hz,4H).
IR(KBr,cm-1):3436.94,1700.46,1622.06,1381.04,1311.91,1165.86,851.33,769.40,614.89.
(5) preparation of intermediate 5
Figure GDA0003080327840000062
Intermediate 4(0.30g,0.54mmol) was dissolved in 150mL tetrahydrofuran and 1.5 equivalents of N were added3-PEG2000(1.63g,0.81mmol), then, placing the reaction device in an ice-water bath, controlling the temperature at 0-10 ℃, slowly dropwise adding 2mL of 25% sodium hydroxide solution, changing the solution from colorless to yellow, and reacting at room temperature for 6 h. After the reaction was completed, the tetrahydrofuran solvent was removed by rotary evaporation, and dissolved in 20mL of dichloromethane, extracted with 20mL of water, separated to obtain an organic layer, dried, filtered, concentrated, and subjected to column chromatography (PE: DCM: MeOH: 15:1) to obtain 0.45g of a product with a yield of 43.2%.
IR(KBr,cm-1):3431.81,3099.81,2547.79,1943.37,1701.18,1621.55,1376.89,1310.25,1232.78,1166.65,769.77,611.73.
(6) Preparation of intermediate 6
Figure GDA0003080327840000063
50mg of distearoyl phosphatidyl ethanolamine DSPE (0.07mmol) was weighed out and dissolved in 20mL of a solution of LDMF, and 150mg of intermediate 5(0.07mmol) was added thereto, followed by reflux reaction overnight. After the reaction is finished by TCL method, the DMF solvent is removed by rotary evaporation, and the mixture is washed 3 times by dichloromethane and water and subjected to column chromatography (PE: DCM: CH)3OH 20:20:1) to yield 83mg of product in 41.5% yield.
1H NMR(300MHz,CDCl3):δ0.87(-CH3 of DSPE),δ1.27(-CH2-of DSPE),δ3.57(-CH2CH2O-of PEG),δ7.34andδ7.78(Ph-H).
IR(KBr,cm-1):3426.76,2920.09,2852.89,1714.21,1622.89,1376,92,1306.86,1230.26,1169.05,1114.87,1083.78,852.28,696.74,575.89.
(7) Preparation of magnetically targeted phospholipids for NO donor bridge chain
Figure GDA0003080327840000071
Adding 1.0g of ferroferric oxide nanoparticles with modified amino groups on the surface into 30mL of methanol solution, performing ultrasonic-assisted dispersion, adding 10mL of 3-bromo-1-propyne, stirring for 10min, adding 2mL of triethylamine, stirring at room temperature for reaction for 24h, and performing magnetic settling to obtain an intermediate 7. Dissolving the intermediate 6(60mg) in a mixed solution of ethanol/water (1:1), adding the intermediate 7(20mg), performing ultrasonic dispersion, adding sodium ascorbate, and stirring under nitrogen protection at room temperature for 10 min. The reaction solution was poured into a copper sulfate pentahydrate solution (5% equivalent) by a syringe and reacted overnight under dark conditions. Magnetic sedimentation was used to give a black solid product (magnetically targeted phospholipid of NO donor bridge).
IR(KBr,cm-1):3744.36,3445.66,2920.06,2312.12,1629.82,1388.73,1109.64,782.67,591.18.
XPS(1Scan,1mins 8.8secs,CAE 150.0 1.00eV Step):Fe2p(710.62eV),Fe3p(55.63eV),N1s(399.57eV),C1s(284.88eV),Si2p(102.01eV),P2s(191.62eV).
Example 2
Preparation of blank liposome and drug-loaded liposome
Blank liposomes: weighing membrane materials such as 30mg of DSPC, 18mg of cholesterol and 2mg of magnetic phospholipid, dissolving in 10mL of chloroform solution, performing ultrasonic promotion of dissolution, removing the organic solvent by using a vacuum rotary evaporator after complete dissolution to form a layer of film, performing freeze drying for 12h to completely remove the organic solvent, adding 9mL of deionized water, performing rotary hydration in a water bath at 45 ℃ to form liposome, performing ultrasonic treatment for 8min by using a probe, and filtering to obtain a blank liposome solution.
Carrying a medicine liposome: weighing membrane materials such as 30mg of DSPC, 18mg of cholesterol and 2mg of magnetic phospholipid, dissolving in 10mL of chloroform solution, ultrasonically promoting dissolution to obtain a membrane material solution, weighing 4.5mg of adriamycin, dissolving in methanol solution, adding into the membrane material solution, ultrasonically promoting dissolution, removing the organic solvent by using a vacuum rotary evaporator after complete dissolution to form a layer of membrane, freeze-drying for 12 hours to completely remove the organic solvent, adding 9mL of deionized water, carrying out rotary hydration in a water bath at 45 ℃ to form liposome, carrying out ultrasonic treatment for 8 minutes by using a probe, filtering, and dialyzing for 24 hours to obtain a drug-loaded liposome solution.
Example 3
Paramagnetic assay
The saturation magnetization and coercive force of the magnetic targeting phospholipid of the NO donor bridge chain are measured by a Vibrating Sample Magnetometer (VSM), and the experimental scheme is as follows: sample quality: 5 mg; a sample stage: a cylindrical sample stage; tape patch position: sticking to the bottom surface of a cylindrical sample table; temperature: keeping the temperature constant at room temperature; measuring the magnetic field range: -20000G to 20000G; maximum magnetic moment: <700 memu; and (3) testing time: 2 h; experimental data: magnetization curve (ordinate: emu/g), saturation magnetization (Ms), mass (m), coercive force (Hc), remanence (Br); setting a point: a total of 51 points (as shown in Table one) were taken and the sample magnetic moment-field relationship was measured point by point.
TABLE 1 VSM test Point taking settings
Figure GDA0003080327840000081
As shown in FIG. 1, when the external magnetic field goes from negative maximum to 0 to positive maximum and then to 0 to negative maximum, the remanence of the material is almost 0, indicating that the obtained magnetic phospholipid has superparamagnetism.
Example 4
Liposome particle size distribution, Zeta potential and morphological observation
Particle size distribution, Zeta potential and morphology of the hollow white liposomes and drug-loaded liposomes of example 2 were observed.
Particle size and Zeta potential of liposomes: liposome solutions with concentrations of 0.5-1.0mg/mL were prepared, the particle size distribution and Zeta potential of the liposomes were measured by a particle size potentiostat at 25 deg.C, and each sample was tested in triplicate and the average value was recorded.
The shape of the liposome is as follows: preparing a liposome solution with the concentration of 0.5-1.0mg/mL, dropwise adding the liposome solution to the carbon film surface of a copper mesh, standing for half a minute, sucking off redundant liquid from the side surface, and dropwise adding a small amount of phosphotungstic acid solution (1% w/w) to the surface of the copper mesh for dyeing after the solution is dried. After standing for half a minute, the excess liquid was aspirated from the side, evaporated to dryness, and the size and morphology of the liposomes were observed using a Transmission Electron Microscope (TEM).
As a result, as shown in FIG. 2, the obtained liposome was in the form of a spherical vesicle having a relatively uniform particle size distribution as measured by DLS, a particle size of 103. + -. 2.1nm and a Zeta potential of-11.5. + -. 0.2mV, and after loading with DOX, the morphology did not change and the hydrated particle size slightly increased (112. + -. 1.3nm), and the potential was measured to be-12.2. + -. 0.3 mV.
Example 5
Drug-loaded liposome encapsulation efficiency and drug-loaded rate test
Selection of measurement wavelength: 0.05mg/mL of Doxorubicin (DOX) solution, DSPC solution, cholesterol solution and magnetic phospholipid solution are prepared respectively, and scanning is carried out by an ultraviolet-visible spectrophotometer (the wavelength is 290nm-600nm), so that doxorubicin has the largest absorption peak at the wavelength of 480nm, and the absorbance of a phospholipid material at the wavelength is nearly 0, so that the wavelength of 480nm can be selected as the detection wavelength.
And (3) drawing an adriamycin absorbance-concentration standard curve: accurately weighing 5.0mg of adriamycin, dissolving the adriamycin in methanol, placing the solution in a 50mL volumetric flask, fixing the volume to a scale mark, uniformly mixing, preparing a series of adriamycin methanol solutions with concentration gradients on the basis of the adriamycin methanol solutions, carrying out ultraviolet test, recording the absorbance A of the adriamycin solutions with different concentrations c at the wavelength of 480nm, drawing a standard curve between the absorbance A and the concentration c of DOX at the position of 480nm in the methanol solution, and solving a linear equation relational expression. Each concentration was tested in triplicate. In this way, the standard curves of DOX between absorbance a and concentration c at 480nm in phosphate buffer at pH 7.4 and pH 5.5, respectively, were tested and the linear equation was found, respectively.
The test method of liposome encapsulation efficiency and drug loading capacity comprises the following steps: measuring 0.5mL of drug-loaded liposome, adding 1mL of the drug-loaded liposome, demulsifying, diluting to 10mL by using methanol, measuring the absorbance A of the drug-loaded liposome at the wavelength of 480nm by using an ultraviolet-visible spectrophotometer, and calculating the concentration c of DOX by using the standard curve of the DOX in the methanol obtained in the previous step. Each test is carried out 3 times in parallel, and the entrapment rate and the drug-loading capacity of the drug-loaded liposome are respectively calculated by the following two equations:
encapsulation ratio (%) - (mass of DOX in liposome/total mass of DOX) × 100%
The drug loading (%) × 100% (mass of DOX in liposome/total mass of phospholipid)
The encapsulation rate of the prepared DOX-loaded liposome is 82.7% and the drug-loading rate is 9.1% through measurement and calculation.
Example 6:
drug Release test
The liposome drug release was performed by dialysis and an ultraviolet-visible spectrophotometer was used to test the in vitro drug release capacity of the drug-loaded liposomes under different conditions. Measuring 5mL of DOX-loaded liposome solution, transferring the solution into a dialysis bag (MWCO 3500), placing the solution into 100mL of phosphate buffer solution with pH 7.4 and without 1mM glutathione, keeping the temperature at 37 ℃, measuring 5mL of dialysis external solution at intervals, supplementing fresh phosphate buffer solution with equal amount of pH, measuring the content of DOX in the taken phosphate buffer by an ultraviolet-visible spectrophotometer, detecting the wavelength lambda of 480nm, and calculating the release amount of DOX by using a standard curve of DOX in different media. Each time point was measured 3 times.
The results show (figure 3) that the drug-loaded liposome delivery system is very stable at pH 7.4 with less than 15% drug released within 48h, suggesting that the system is not prone to leakage in normal body fluid environment (pH 7.4); under the condition that a trigger factor (such as high-level glutathione contained in tumor cells) exists, the sulfydryl on the glutathione can promote furazan nitric oxide to open rings to release NO gas molecules, so that the liposome is cracked, the drug release is obviously increased, and the drug delivery system is prompted to have the self-cracking characteristic of environmental response.
Example 7:
antitumor Activity effect of drug-loaded liposomes
An MTT method is adopted to detect the inhibiting effect of a conventional DOX drug-loaded liposome drug delivery system (namely, a liposome prepared by DSPE replacing the magnetic targeting phospholipid disclosed by the invention) and the magnetic targeting phospholipid drug-loaded liposome on tumor cells, and adriamycin is selected as a positive control group. The results are shown in Table 2.
TABLE 2 IC of drug-loaded liposomes on tumor cell lines50Value of
Figure GDA0003080327840000101
The drug-loaded liposome drug delivery system disclosed by the invention is found to have effects on various tumor cell lines, wherein the best effect is the SGC7901 cell line and IC thereof50The value was 0.94. + -. 0.22. mu.g/mL. The magnetic targeting phospholipid drug-loaded liposome group has slightly lower activity than that of positive control adriamycin, which is probably because a drug release process exists when a drug delivery system exerts drug effect, and the loaded drug cannot be completely released under the test condition, so the activity is lower than that of free adriamycin. Compared with the conventional liposome group, the activity of the prepared magnetic target drug-loaded liposome is obviously improved, which may be derived from that the phospholipid disclosed by the invention contains NO donor fragments, and the NO donor fragments can release NO to play a synergistic anti-tumor role through promotion, and meanwhile, the liposome is promoted to crack to release adriamycin. For the adriamycin drug-resistant cell strain, the activities of free adriamycin and a conventional liposome group are obviously reduced, while the activity of the magnetic targeting drug-loaded liposome group is only slightly reduced, so that the NO release function of the adriamycin drug-resistant cell strain is favorable for overcoming tumor drug resistance.
Example 8
In-vitro anti-tumor activity investigation of drug delivery system under external magnetic field condition
SGC7901 is cultured in a cell culture dish, a ring magnet is additionally arranged at the bottom of the cell culture dish to serve as an external magnetic field, when the cell proliferation amount reaches 70%, 0.5 mu g/mL of free adriamycin, a common adriamycin-loaded liposome drug and a magnetic target adriamycin-loaded liposome are respectively added, after 24 hours of culture, crystal violet is used for dyeing treatment, living cells can be dyed by the crystal violet, dead cells cannot be dyed, and an unstained area is a tumor cell apoptosis area. The result (fig. 4) shows that in the annular magnetic field area of the external magnetic field, the magnetic targeting doxorubicin-loaded liposome group has an obvious annular blank area and is consistent with the position of the external magnetic field, which indicates that the drug can be enriched at the high magnetic field under the condition of the external magnetic field, so that the tumor cells at the position can be effectively killed; the effect is not seen in the free adriamycin and common liposome groups, which shows that the liposome obtained by the invention has magnetic targeting effect.
Example 9
Target liposome in vitro NO release assay
DAF-FM DA (Beyotime) was used as a fluorescent indicator of intracellular NO. When 80% of the cells grown in the small dish were reached, they were washed with PBS. After incubation at 37 ℃ for 20min with 5. mu.M DAF-FM DA, cells were rinsed 3 times with PBS and incubated with test compounds for 24h, and then fluorescence photographs were taken using a confocal microscope at excitation and emission wavelengths of 495 and 515nm, and NO production was measured separately using a flow cytometer.
The results (fig. 5) show that when NO is released in cells, the DAF-FM-DA probe fluoresces, A, B two groups are blank control groups, the fluorescence shown in the a group is weak because cells can generate trace NO, the B group is a confocal bright field picture thereof, the C group is a target liposome group, the fluorescence is obviously enhanced, a large amount of NO is released under experimental conditions, and the D group is a single NO donor administration group and also has strong fluorescence.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.

Claims (3)

1. A self-splitting multifunctional liposome is characterized by having the following structural formula:
Figure 24532DEST_PATH_IMAGE001
；
the preparation method comprises the following steps:
(1) synthesis of NO donor:
step 1: p-methyl thiophenol reacts with bromoacetic acid under alkaline condition to obtain a thioether intermediate 1;
step 2: oxidizing the intermediate 1 with hydrogen peroxide to obtain an intermediate 2;
and step 3: carrying out NBS bromination reaction on the intermediate 2 to obtain an intermediate 3;
and 4, step 4: the intermediate 3 reacts with fuming nitric acid to cyclize to obtain a furazan nitrogen oxide NO donor 4;
Figure 613777DEST_PATH_IMAGE002
；
(2) synthesis of magnetically targeted phospholipids for NO donor bridge chain:
step 1: NO Donor 4 with azido substituted PEG2000Reacting to obtain an intermediate 5;
step 2: the intermediate 5 reacts with distearoyl phosphatidyl ethanolamine (DSPE) to obtain an intermediate 6;
and step 3: reacting the nano ferroferric oxide nano particles with the surfaces modified with amino with propargyl bromide to prepare an intermediate 7; carrying out click reaction on the intermediate 7 and the intermediate 6 to obtain the magnetic targeting phospholipid of the NO donor bridge chain;
Figure 190252DEST_PATH_IMAGE004
。
2. the self-lysing multifunctional liposome of claim 1, which is prepared by the following steps: and (3) sequentially introducing a nitric oxide donor, polyethylene glycol and superparamagnetic ferroferric oxide nanoparticles into the amino terminal of the distearoyl phosphatidyl ethanolamine to obtain the magnetic targeting phospholipid of the NO donor bridge chain.
3. Use of the self-lysing multifunctional liposome according to any of claims 1-2 for the preparation of a medicament for tumor targeted therapy.
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