CN112773766A

CN112773766A - Liposome delivery system for tumor treatment and preparation method and application thereof

Info

Publication number: CN112773766A
Application number: CN202011598830.0A
Authority: CN
Inventors: 王维; 蔡洁旋; 洪美静; 关柏鸥
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-05-11
Anticipated expiration: 2040-12-29
Also published as: CN112773766B

Abstract

The invention discloses a liposome delivery system for tumor treatment and a preparation method and application thereof, belonging to the field of preparation of medicine raw materials. Dissolving an alkylamine compound in a solvent, adding 3,4,5, 6-tetrahydrophthalic anhydride, 2, 3-dimethylmaleic anhydride or 2,2,3, 3-tetramethylsuccinic acid, reacting to obtain a negatively charged lipid PHL, and mixing with neutral phospholipid molecules and positively charged phospholipids to prepare the liposome delivery system with the characteristic of pH sensitivity. The liposome delivery system can load drugs and is hydrolyzed in a weak acid tumor microenvironment at a specific part, so that the effect of releasing the drugs at the tumor part is achieved, and the drugs can stably exist in normal tissues. The liposome delivery system has high biocompatibility and tumor targeting property, is easy to activate in a tumor environment, improves the controllability and accuracy of in-vivo anti-tumor treatment, and can be used as an effective tool for treating cancers.

Description

Liposome delivery system for tumor treatment and preparation method and application thereof

Technical Field

The invention belongs to the field of preparation of medicine raw materials, and particularly relates to a liposome delivery system for tumor treatment and a preparation method and application thereof.

Background

Recent studies have shown great advantage in drug delivery to deep cancer tissues using nanomaterials. The traditional tumor treatment has the limitations of nonspecific and inflammatory reaction caused by injury after treatment due to the lack of tumor targeting and the full study of pharmacokinetics. Wherein the lack of tumor targeting can damage the integrity of adjacent tissues, accelerate the proliferation of residual tumor cells and promote the migration and infiltration of surviving tumor cells. In recent years, through scientific research, nanomaterials such as particles, micelles, liposomes, polymeric microcapsules and the like, which have a large number of drug loading and release and tumor targeting capabilities, have been developed.

Liposomes are recognized as the first therapeutic class of Nanoscale Drug Delivery Systems (NDDSs) due to their superior biocompatibility and adjustable pharmacokinetics^[1]Can be used as a carrier of drugs, polypeptides, proteins, DNA, antisense oligonucleotides or ribozymes. When used as a drug carrier, liposomes can reduce toxic side effects on sensitive organs (e.g., heart and kidney) and enhance specific tissue effects (e.g., tumors). Since tumors have a range of pathophysiological features both in tumor cells and in tumor microenvironment, e.g., tumor acidosis is an important hallmark of solid tumor microenvironment, extracellular tumor acidosis has been shown to promote tumor invasion^[2]And delayed immune monitoring^[3]Therefore, the liposome can provide conditions for targeting of the liposome.

The liposome with targeting property is applied for the first time in 1980^[4]Later on, there was a great progress in liposome research, where liposomes of different materials have different functionalities after synthesis, e.g. ability to respond to the environment to improve drug delivery. Chinese patent application CN108721643A Cholesterol modified with a succinic group and blended with dioleoyl phospholipid ethanolamine (DOPE) and soya lecithin (SPC) was selected to prepare pH sensitive liposomes for use in immunochemistry. Chinese patent application CN 108186572A discloses a pH-sensitive konjac glucomannan-liposome composite nano-drug carrier for injection, and preparation and application thereof. In the invention, the preparation process is complex, the selected pH response mechanism material needs hydrophilic and hydrophobic modification, and the pH response group is embedded in the liposome phospholipid bilayer rather than exposing the surface of the liposome, so that the response speed is limited. In addition, the liposome has a wide pH response range, so that sensitive response (the pH of a tumor microenvironment is 5.6-6.8) can not be made in a small pH change, and the practical application effect of the liposome is further limited. Therefore, there is a need to develop a novel liposome to solve the existing problems.

Reference documents:

[1]Torchilin V P.Multifunctional,stimuli-sensitive nanoparticulate systems for drug delivery[J].Nature reviews Drug discovery,2014,13(11):813-827.

[2]a)Yan Y,Jiang K,Liu P,et al.Bafilomycin A1 induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways[J].Scientific reports,2016,6(1):1-13；b)Mekhail K,Gunaratnam L,Bonicalzi M E,et al.HIF activation by pH-dependent nucleolar sequestration of VHL[J].Nature cell biology,2004,6(7):642-647.

[3]a)Chang C H,Qiu J,O’Sullivan D,et al.Metabolic competition in the tumor microenvironment is a driver of cancer progression[J].Cell,2015,162(6):1229-1241；b)Hulikova A,Aveyard N,Harris A L,et al.Intracellular carbonic anhydrase activity sensitizes cancer cell pH signaling to dynamic changes in CO₂ partial pressure[J].Journal of Biological Chemistry,2014,289(37):25418-25430.

[4]Heidel J D,Yu Z,Liu J Y C,et al.Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA[J].Proceedings of the National Academy of Sciences,2007,104(14):5715-5721.

disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the present invention is primarily directed to a method for preparing a liposome delivery system for tumor therapy.

The invention also aims to provide a liposome delivery system for tumor treatment prepared by the preparation method.

It is a further object of the present invention to provide the use of the above liposome delivery system for tumor therapy.

The invention provides a pH-sensitive liposome which can be encapsulated with a functional drug in a self-assembly or physical coating mode, wherein the liposome comprises neutral phospholipid molecules, positively charged phospholipid molecules and negatively charged lipid molecules.

The present invention provides liposome delivery systems capable of drug release at the tumor site activated by acidosis characteristics in the tumor microenvironment. In normal tissues, the liposome has certain stability, and the liposome is stably destroyed only in a weak acid environment (low pH value, pH 5.6-6.8) of the tumor, so that the liposome is dissociated, and the drug is released into target cancer tissues and cells. The liposome system is activated only in the environment of cancerous tissue and does not work in non-cancerous tissue, controlled by pH (6.5-6.8). The inventive liposome improves controllability and accuracy of in vivo antitumor therapy, and can be used as an effective tool for cancer therapy.

The purpose of the invention is realized by the following technical scheme:

a method of preparing a liposome delivery system for tumor therapy, comprising the steps of:

(1) preparation of negatively charged lipid PHL: dissolving an alkylamine compound in a solvent, adding 3,4,5, 6-tetrahydrophthalic anhydride, 2, 3-dimethylmaleic anhydride or 2,2,3, 3-tetramethylsuccinic acid, and reacting; purifying the reaction solution to obtain the lipid PHL with negative charge;

(2) preparation of liposome delivery system: mixing neutral phospholipid molecules, phospholipid with positive charges and the lipid PHL with negative charges obtained in the step (1), dissolving the mixture in a solvent, evaporating, concentrating and drying to obtain a lipid membrane; hydrating the obtained lipid membrane to obtain a mixed lipid membrane suspension; and (3) carrying out ultrasonic treatment and freeze-thaw cycling on the obtained suspension, and extruding in a micro extruder to obtain the liposome delivery system for tumor treatment.

Preferably, the formula of the alkylamine compound in the step (1) is C_nNH₂Wherein n is an integer of 6-18, represents a saturated or unsaturated alkane carbon chain, and has a length of C6-C18.

Preferably, the solvent in the step (1) is one of chloroform, dichloroethane, 1, 4-dioxane, toluene, acetonitrile, ethyl acetate, N-methylpyrrolidone, dimethyl sulfoxide and N, N-dimethylformamide; more preferably, the solvent is dichloromethane, DCM.

Preferably, the amount of the 3,4,5, 6-tetrahydrophthalic anhydride, 2, 3-dimethylmaleic anhydride or 2,2,3, 3-tetramethylsuccinic acid used in step (1) is 1-1.5: 1-1.5, and preferably, the molar ratio of 1: 1 meter.

Preferably, the reaction condition in the step (1) is stirring at room temperature for 1-2 h.

Preferably, the purification in step (1) is carried out by removing the solvent by a rotary evaporator or distillation under reduced pressure, and purifying the residue by silica gel column chromatography.

Preferably, the solvent in step (2) is a chloroform/methanol mixture, wherein the volume ratio of chloroform to methanol is 2: 1.

Preferably, the evaporative concentration described in step (2) is carried out by vacuum rotary evaporator at room temperature.

Preferably, the reagent used in the hydration treatment in step (2) is a phosphate buffer, wherein the concentration of the phosphate buffer is 0.1mol/L, and the pH is 7.3 to 7.5.

Preferably, the time of the ultrasonic treatment in the step (2) is 5-15 min.

Preferably, the number of times of freezing and thawing in the step (2) is 4-6.

Preferably, the pore diameter of the polycarbonate filtering membrane of the micro-extruder in the step (2) is 0.1 μm, and the number of times of extrusion is 40-50.

Preferably, the ratio of the positively charged phospholipid to the negatively charged lipid PHL in the neutral phospholipid molecules in step (2) is preferably 35-45: 35-45: 17-23; more preferably, the molar ratio is 40: 40: 20.

the neutral phospholipid molecules described in the above step (2) include, but are not limited to, 1, 2-Dioleoylphosphatidylethanolamine (DOPE), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1, 2-pentacosanyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-heptacosanoyl-sn-glycero-3-phosphoethanolamine (DHPE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-docosyl-sn-glycero-3-phosphocholine (DEPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-Dioleoylphosphatidylcholine (DOPC), 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-palmitoyl-2-myristoyl lecithin (PMPC), 1-palmitoyl-2-stearoyl lecithin (PSPC), 1-stearoyl-2-palmitoyl lecithin (SPPC), hydrogenated soybean lecithin (HSPC), 1-myristoyl-2-palmitoyl lecithin (MPPC), soybean lecithin (SPC), egg yolk lecithin (EPC), and the like.

The positively charged phospholipid described in the above step (2) includes, but is not limited to, 1, 2-dioleoyl propyl trimethylammonium chloride (DOTMA), (2, 3-dioleoyl-propyl) -trimethylammonium-hydrochloride (DOTAP), 1, 2-dioleyl-3-dimethylamino-propane (DODMA), 1, 2-dilauroyl-sn-glycerol-3-ethylphosphonic acid choline, and the like.

A liposome delivery system for tumor therapy is prepared by the method.

The liposome delivery system for tumor treatment or the preparation method thereof is applied to the preparation of tumor treatment preparations.

In the application, the required medicine is loaded in the liposome delivery system for treating the tumor; the specific operation is as shown in the above preparation method of liposome delivery system for tumor therapy, except that in step (2), neutral phospholipid molecules, positively charged phospholipids, the negatively charged lipids PHL obtained in step (1) and the desired drug are mixed and dissolved in a solvent, or neutral phospholipid molecules, positively charged phospholipids and the negatively charged lipids PHL obtained in step (1) are mixed and dissolved in a solvent, evaporated, concentrated and dried to obtain a lipid membrane; then, the obtained lipid membrane is hydrated by a hydration reagent containing the required medicine to obtain a mixed lipid membrane suspension, and then the subsequent operation is carried out.

The drug includes, but is not limited to, at least one of a drug for treating cancer, a photo-thermal agent, an imaging agent.

The drugs for treating cancer include but are not limited to

(doxorubicin, a compound of formula (I)),

(paclitaxel) and (C) a pharmaceutically acceptable carrier,

(the daunorubicin) is prepared,

(vincristine) which is a derivative of the general formula (I),

(Azithromycin) is added into the raw materials,

(Triptorelin)，

(naltrexone) in a liquid medium,

(minocycline) is added to the reaction mixture,

(risperidone) is added to the mixture,

the LAR depot (octreotide),

(Somatropin)，

(

(cytarabine) in a pharmaceutically acceptable carrier,

(morphine),

(Exenatide), Somatuline LA (lanreotide).

The photothermal agent includes, but is not limited to, cyanine-based organic dyes such as indocyanine green ICG, Cy-5, Cy-5.5, Cy-7, Cy-7.5; porphyrins, BODIPY, phthalocyanines, theobromine, and the like; nano metal photothermal agents such as nano gold, nano silver, nano platinum, nano palladium, metal sulfides such as copper sulfide, silver sulfide, nickel sulfide; carbon-based nanomaterials, such as nanographene, carbon nanotubes; two-dimensional nanomaterials such as black scale nanoplatelets, boron nitride, graphitic carbon nitride, MXenes, and nano-semiconducting polymer materials, among others.

When the photo-thermal agent is indocyanine green ICG, the dosage of the photo-thermal agent is preferably as follows according to the total phospholipid mass: ICG mass ratio 1: 0.1-0.3 meter; more preferably, the ratio by total phospholipid mass: ICG mass ratio 1: and (4) 0.2.

A preparation for treating tumor comprises the liposome delivery system for treating tumor and required drugs.

The tumor treating preparation consists of liposome and carried medicine, the liposome is pH sensitive, and the carried medicine may be tumor treating medicine, imaging agent, photo-thermal agent, etc. The pH sensitive liposome comprises neutral lipid molecules, positively charged lipid molecules and artificially synthesized lipid molecules PHL with negative charges. In normal tissues, non-covalent bonding (hydrophobicity, charge interactions, hydrogen bonding) between lipid molecules helps to maintain liposome stability. However, in acidic tumor tissue, the β -carboxylic acid amide of the negatively charged lipid molecule PHL can be hydrolyzed in acidic tumor tissue, thereby changing the head group charge from negative to positive. This disrupts the structure and charge balance within the liposome, resulting in dissociation of the liposome shell and release of the loaded drug into the target cancer tissue and cells, thereby achieving the effect of treating or imaging the tumor, etc. The liposome delivery system formed by liposome encapsulated drugs can be activated by the key cancer characteristics of acidosis in a tumor microenvironment to release the drugs. Compared with the prior art, the method has the following characteristics: firstly, the negatively charged lipid PHL prepared by the invention enables the surface of the liposome to be negatively charged, enhances the surface hydrophilicity, can coordinate with the interaction between solid-phase phospholipids, maintains the stability of the liposome in plasma, prolongs the half-life period of the liposome in blood, and simultaneously reduces the non-specificity caused by the cell uptake of liver and spleen. Secondly, the prepared liposome has the characteristic of sensitivity to pH, can be hydrolyzed in a weak acid tumor microenvironment at a specific part to achieve the effect of releasing the drug at the tumor part, and can stably exist at the normal tissue pH of 7.4. The liposome delivery system has high biocompatibility, tumor targeting property and easy activation in tumor environment. In particular, the liposome delivery system has a significant therapeutic effect in a mouse breast cancer model, with minimized inflammatory response and collateral damage. More importantly, tumor cells and tumor-associated microvessels are rapidly ablated, reducing the likelihood of tumor recurrence. The innovative liposome-enhanced therapeutic approach can serve as an effective tool for both cancer diagnosis and treatment.

The invention aims to solve the problems of tumor targeting property and safety caused by lack of a delivery carrier in the common antitumor treatment medicines, not only reduces the economic cost, but also can improve the early tumor targeted treatment effect. Provides a new theoretical support for developing anticancer medicaments and relevant clinical detection and treatment, and has important scientific significance, practical value and economic value.

Drawings

Fig. 1 is a schematic diagram of the operation of the liposome delivery system for tumor therapy of the present invention.

FIG. 2 is a graph showing the average particle size of Lipo-PTA liposome prepared in example 1.

FIG. 3 is a transmission electron micrograph of Lipo-PTA liposome prepared in example 1.

FIG. 4 is a TEM picture of Lipo-PTA liposome prepared in example 1 before and after acid treatment; wherein, the pH in the graph (a) is 7.4, the pH in the graph (b) is 6.8, the pH in the graph (c) is 6.5, and the pH in the graph (d) is 6.0.

FIG. 5 is a graph showing the results of the cytotoxicity test of red blood cells by Lipo-PTA prepared in example 1.

FIG. 6 is a graph showing the results of a hemolysis experiment of red blood cells with Lipo-PTA prepared in example 1.

FIG. 7 is a graph showing the blood circulation after intravenous injection in Lipo-PTA mice prepared in example 1.

FIG. 8 is a graph showing the results of measurement of the accumulation of Lipo-PTA prepared in example 1 in mouse tumor tissues.

FIG. 9 is a photograph of tumor site taken by thermal imaging during the treatment of tumor mice and 24 hours after the treatment; in this figure, (a) is an image photograph and (b) is a photograph of a tumor site.

FIG. 10 is a graph showing the results of examination of the inhibitory effect of Lipo-DOX prepared in example 5 on tumor tissues.

Detailed Description

The technical solution of the present invention is further explained by the following detailed description and the accompanying drawings. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical manufacturers.

Example 1: preparation of liposome delivery system for tumor photothermal therapy

(1) Preparation of photothermal agent loaded liposome (Lipo-PTA):

a. 2.69g of octadecylamine was dissolved in 60mL of dry DCM. 1.52g of 3,4,5, 6-tetrahydrophthalic anhydride were added. The resulting mixture was stirred at room temperature for 1h, the solvent was removed by rotary evaporator, and the residue was purified by silica gel column chromatography to give PHL as a white product. Yield: 96 percent.

b. Three different phospholipid molecules: 3.74mg DOTAP, 4.22mg PHL, 6.98mg DOPE were mixed in a molar ratio (PHL: DOTAP: DOPE: 40: 20) while incorporating 2.98mg ICG (total phospholipid mass: ICG mass: 1: 0.2) in a volume ratio of 3mL to 2:1 chloroform/methanol, then concentrated under vacuum at room temperature using a rotary evaporator, after evaporation of the solvent, the mixture formed a thin film on the wall of the flask, and the mixture was dried under vacuum overnight. The lipid membrane was hydrated with 2.5mL of PBS buffer (0.1mol/L, pH 7.4) while the total concentration of lipids was brought to 10 mM. The suspension was sonicated at room temperature under argon for 10min, then subjected to 5 freeze-thaw cycles (15 min in liquid nitrogen for each cycle, >15 min in a room temperature water bath). The liposome suspension (10mM, 1mL) was extruded 45 times through a single 0.1 μm polycarbonate filter in an Avanti Mini Extruder apparatus at room temperature. The resulting liposomes (large unilamellar vesicles, LUVs) were stored in glass vials at 4 ℃.

(2) Characterization of liposomes

The liposomes were morphologically observed by transmission electron microscopy. The liposomes had a uniform particle size distribution and the particle size analysis indicated an average diameter of about 85nm (FIG. 2). Under a transmission electron microscope, the liposome is uniform, hollow and spherical, and the shell thickness is about 15nm (figure 3).

(3) Dispersibility and stability of liposome

As shown in FIG. 4, liposomes were able to retain their size and monodispersity for at least two months in PBS buffer (pH 7.4) at 4 ℃ (FIG. 4 (a)). In contrast, soaking in acidified PBS (pH 6.0) for 24h released 79% of the liposome content. Physiological conditions in blood (PBS buffer, pH 7.4) were simulated with a simple water system and it was found that Lipo-PTA could remain intact for more than 2 months, but gradually degraded within 24 hours in weakly acidic simulated intratumoral conditions (PBS buffer, pH 6.0). After dissociation of the liposomes in an acidic tumor microenvironment, the compounds can effectively leave the tumor stroma and enter the tumor cells adjacent to the tumor stroma.

(4) Measurement of Lipo-PTA Encapsulated Rate and Encapsulated amount

Subjecting 5mL ICG-loaded liposome to Sephadex ultrafiltration column chromatography (see below)

G-50, Sigma), non-encapsulated probes in liposomes were isolated and the drug loading rate and drug loading of the liposomes were determined. The concentration of ICG in the sephadex eluent is determined by adopting an ultraviolet-visible spectrophotometry, the drug loading rate of ICG in liposome is determined, and the drug loading rate is 16.2%. The result of the simultaneous absorption spectrophotometry shows that the absorption spectra and the maximum absorption peak positions of the ICG-coated liposome (Lipo-PTA) and the ICG are similar, and the optical properties of the ICG are not changed by the liposome coating.

Example 2: study of Lipo-PTA Liposome delivery System on Normal cytotoxicity and hemolysis

The cytotoxicity of Lipo-PTA on 4T-1 cells is detected by MTT method. All cells were at 37 ℃ with 5% CO₂Culturing in medium. 4T-1 cells (ATCC) were cultured with Lipo-PTA diluted in a concentration gradient for 24 hours, and then the cell viability was calculated. As shown in FIG. 5, even at a liposome concentration of 1000. mu.g/mL, cell viability was still higher than 50% after 24 hours of co-culture with Lipo-PTA, with no significant cytotoxicity in 4T-1 cells.

About 1mL of whole blood drawn from a healthy mouse was taken, added to heparin sodium diluted with 10mL of physiological saline, centrifuged, washed with physiological saline, and red blood cells were collected. Adding 20 μ L mouse erythrocyte suspension into liposome suspension (1mL) with different concentrations, incubating at 37 deg.C for 60min, wherein the negative control group contains normal saline and blood, and no other substances, and the positive control group uses distilled water instead of normal saline of the negative control group. The reaction mixture was then centrifuged at 1000rpm for 5min and the absorbance of the supernatant was measured at 545nm using a spectrophotometer. As shown in FIG. 6, only pure water samples were hemolyzed, and no significant hemolysis was induced by different concentrations (20-500. mu.g/mL) of Lipo-PTA, demonstrating that the liposome has good blood compatibility.

Example 3: research on distribution and pharmacokinetics of Lipo-PTA liposome delivery system in organisms

The Lipo-PTA preparation is injected intravenously after being purified by a sephadex column. Six weeks old BALB/c mice were selected. 200-300. mu.L of 800. mu.g/mL Lipo-PTA (absorbance 825nm) salt solution was injected intravenously (i.v.) into the tail vein of each mouse. Before injecting the Lipo-PTA solution, the absorption spectrum was recorded and used to calculate the Lipo-PTA concentration according to the concentration calibration curve. At various time points (6h, 12h, 24h, 48h) after injection (pi), approximately 100 μ L of blood was collected from the tail vein (using a different vein from the injected vein) and dissolved in 200 μ L of lysis buffer (1% SDS, 1% Triton X-100, 40mM Tris acetate, 10mM EDTA, 10mM DTT) for detection of Lipo-PTO in the blood by absorption measurements. The absorption peak area at 825nm was measured to calculate the Lipo-PTO concentration in blood, and the percentage injected dose per gram of blood (% ID/g) was calculated. At each time point p.i, three to four mice were used per group to obtain mean and standard deviation of blood circulation and biodistribution measurements. The experimental result shows that the blood circulation curve of Lipo-PTA follows a two-chamber model, the half-lives of the first stage and the second stage are respectively 0.18h and 16h, and the result is shown in figure 7, which proves that the Lipo-PTA is suitable for long-term circulation and retention in vivo before being used as a therapeutic agent, and is beneficial to targeted enrichment at a tumor site. In addition, Lipo-PTA accumulates efficiently in highly vascularized tissues (liver, spleen and tumor) early. After 48h, the Lipo-PTA concentration in each organ is far lower than the maximum value, which indicates that Lipo-PTA can be gradually eliminated from the body, and the bionic liposome property is helpful for reducing tissue toxicity and improving biodegradability. The pharmacokinetics of the tumor was monitored in vivo and the accumulation of the photothermal agent in the tumor was found 24h after injectionUp to 10.23% ID g^–1The results are shown in figure 8, which is gradually metabolized in the tumor over time.

Overall, these results convincingly demonstrate the stability and biocompatibility of Lipo-PTA prior to activation, enabling its long-term retention in vivo and accumulation at the tumor site.

Example 4: research on curative effect of Lipo-PTA liposome delivery system in organism

BALB/c mice (4-6 weeks old, 16-20g) were treated with 4T1 cells (1X 10 cells) to establish a mammary xenograft tumor model⁶) Suspended in 50. mu.L PBS and injected subcutaneously into the left forelimb axillary region of mice. The tumor volume reaches 60-80 mm³The 4T-1 tumor-bearing mice were randomly divided into 4 groups (4 groups), namely, a normal saline group (control group), a laser irradiation group (laser irradiation only), a Lipo-PTA administration group (Lipo-PTA only), a Lipo-PTA administration group and a laser irradiation group (Lipo-PTA + laser irradiation group). Lipo-PTA administration groups liposomes (800. mu.g/mL in 5% glucose solution) were injected intravenously at a dose of 120mg/kg body weight per mouse, approximately 300. mu.L per tail injection. The injection time of the Lipo-PTA administration and laser irradiation group is 1.0 w.cm after 24h^-2825nm laser is continuously irradiated, the ambient temperature is kept at 25 ℃ to avoid temperature interference, and the total irradiation time is 5 min. The body temperature of the mice was monitored radiatively with an infrared thermography. The group receiving Lipo-PTA treatment reaches about 64 ℃ within 3min of irradiation; while the temperature change of the non-tumor subcutaneous tissue was small, see fig. 9 (a). In addition, no strong thermal signal was observed at the tumor site in the other control groups. These results demonstrate that Lipo-PTA can efficiently release PTA at the tumor growth site, producing photothermal effects, rather than normal subcutaneous tissue.

After laser irradiation, the mice tumor ablated successfully, forming visible burn scars at the tumor site, whereas the laser only group and the Lipo-PTA only group did not, see fig. 9 (b). After 22 days, the burn wound is completely healed, and no tumor relapse occurs after long-term observation. All mice that were irradiated with Lipo-PTA + laser survived and treatment had no effect on mouse body weight, confirming the biocompatibility of photothermal treatment of PTT with Lipo-PTA liposomes. The tumor growth of the liposome group and the laser group is continuous and obvious, and the tumor volume has no significant difference. The PTT of Lipo-PTA is an effective experimental method for eradicating primary tumors.

Example 5: preparation of liposome delivery system for tumor drug therapy

(1) Preparation of photothermal agent-loaded liposome (Lipo-DOX):

three different phospholipid molecules: 3.74mg of DOTAP, 4.22mg of PHL, and 6.98mg of DOPE were mixed in a molar ratio (PHL: DOTAP: DOPE: 40: 20). Then, the mixture is mixed in a volume ratio of 3mL to 2:1 chloroform/methanol, then concentrated under vacuum at room temperature using a rotary evaporator, after evaporation of the solvent, the mixture formed a thin film on the wall of the flask, and the mixture was dried under vacuum overnight. The lipid membrane was hydrated with 1.49mL of DOX-containing PBS buffer (0.1mol/L, pH 7.4, DOX concentration 5mg/mL) while the total lipid concentration was 10 mg/mL. The suspension was sonicated at room temperature under argon for 10min, then subjected to 5 freeze-thaw cycles (15 min in liquid nitrogen for each cycle, >15 min in a room temperature water bath). The liposome suspension (10mg/mL, 1.0mL) was extruded 45 times through a single 0.1 μm polycarbonate filter in an Avanti Mini Extruder apparatus at room temperature. The resulting liposomes (large unilamellar vesicles, LUVs) were stored in glass vials at 4 ℃.

(2) Measurement of Lipo-DOX Encapsulated Rate and Encapsulated amount

Subjecting 1.5mL DOX-loaded liposomes to Sephadex ultrafiltration column chromatography (see below)

G-50, Sigma), non-encapsulated probes in liposomes were isolated and the drug loading rate and drug loading of the liposomes were determined. And (3) measuring the concentration of DOX in the sephadex eluent by adopting an ultraviolet-visible spectrophotometry, and measuring the drug loading rate of DOX in the liposome, wherein the drug loading rate is 36.9%.

Example 6: research on curative effect of Lipo-DOX liposome delivery system in organisms

BALB/c mice (4-6 weeks old, 18-22g) were treated with 4T1 cells (1X 10) to create a mammary xenograft tumor model⁶) Suspended in 50. mu.L PBS and injected subcutaneously into the left anterior of miceThe axilla area of the limb. The tumor volume reaches 60-80 mm³The 4T-1 tumor-bearing mice were randomly divided into 4 groups (4 groups), namely a control group (normal saline group), a blank liposome group and a lipo-DOX administration group. The Lipo-DOX administration group was composed of three dose groups (10, 20, 50mg/kg, 5% glucose solution as a diluent solvent), and liposomes were administered by tail vein injection, about 200 μ L each by tail injection, once every other day, for a total of 9 times. The mice are fed normally, the survival condition of the mice is observed every day, the weight of the mice is recorded every two days, the length and the short diameter of the tumor of the mice are measured by a vernier caliper, the tumor volume is calculated, and a time-mouse tumor volume change curve is drawn.

After 25 days, mice injected with Lipo-DOX survived and treatment had no effect on mouse body weight, confirming the biocompatibility of tumor treatment with Lipo-DOX liposomes. Due to the tumor inhibition effect of DOX, the tumor inhibition effect of the group with higher administration concentration (50mg/kg) in the treatment group is obvious, as shown in figure 10, the tumor growth of the blank liposome group is continuous and obvious, and the tumor volume has no significant difference.

The Lipo-DOX is a high-efficiency tumor delivery drug carrier, and achieves the anti-tumor effect by carrying and releasing the anti-tumor drug on the tumor part.

The embodiments of the present invention are not limited to the above-described embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit of the present invention are intended to be equivalent replacements within the scope of the present invention.

Claims

1. A method for preparing a liposome delivery system for tumor therapy, characterized in that: the method comprises the following steps:

2. The method for preparing a liposome delivery system for tumor therapy according to claim 1, characterized in that:

the neutral phospholipid molecule in the step (2) has a molar ratio of the positively charged phospholipid to the negatively charged lipid PHL of 35-45: 35-45: 17-23.

3. The method for preparing a liposome delivery system for tumor therapy according to claim 1, characterized in that:

the structural formula of the alkylamine compound in the step (1) is C_nNH₂Wherein n is an integer of 6-18, represents a saturated or unsaturated alkane carbon chain, and has a length of C6-C18;

the neutral phospholipid molecule in the step (2) is 1, 2-dioleoyl phosphatidyl ethanolamine, 1, 2-dihexanoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-pentacosanyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-heptacosanoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-docosyl-sn-glycerol-3-phosphocholine, 1, 2-dilauroyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoylphosphatidylcholine, 1, 2-dihexoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, 1, 2-distearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl lecithin, 1-palmitoyl-2-stearoyl lecithin, 1-stearoyl-2-palmitoyl lecithin (SPPC), hydrogenated soybean lecithin, 1-myristoyl-2-palmitoyl lecithin, hydrogenated soybean lecithin, and mixtures thereof, One or more of soybean phospholipid and egg yolk lecithin;

the phospholipid with positive charge in the step (2) is any one or more of 1, 2-dioleoyl propyl trimethyl ammonium chloride, (2, 3-dioleoyl propyl) -trimethyl ammonium-hydrochloride, 1, 2-dioleoyl-3-dimethylamino-propane and 1, 2-dilauroyl-sn-glycerol-3-ethyl phosphorylcholine.

4. The method for preparing a liposome delivery system for tumor therapy according to claim 1, characterized in that:

the solvent in the step (1) is one of chloroform, dichloroethane, 1, 4-dioxane, toluene, acetonitrile, ethyl acetate, N-methylpyrrolidone, dimethyl sulfoxide and N, N-dimethylformamide;

the amount of the 3,4,5, 6-tetrahydrophthalic anhydride, 2, 3-dimethylmaleic anhydride or 2,2,3, 3-tetramethylsuccinic acid used in the step (1) is calculated according to the mass ratio of the 3,4,5, 6-tetrahydrophthalic anhydride to the alkylamine compound of 2-3: 1-2;

the reaction condition in the step (1) is stirring for 1-2 h at room temperature;

the specific operation of the purification in the step (1) is that the solvent is removed by a rotary evaporator or a reduced pressure distillation method, and then the residue is purified by silica gel column chromatography.

5. The method for preparing a liposome delivery system for tumor therapy according to claim 1, characterized in that:

the solvent in the step (2) is a chloroform/methanol mixture, wherein the volume ratio of chloroform to methanol is 2: 1;

the evaporation concentration in the step (2) is carried out at room temperature by a vacuum rotary evaporator;

the reagent used in the hydration treatment in the step (2) is phosphate buffer solution, wherein the concentration of the phosphate buffer solution is 0.1mol/L, and the pH value is 7.3-7.5;

the ultrasonic treatment time in the step (2) is 5-15 min;

the number of times of freezing and thawing in the step (2) is 4-6;

the aperture of the polycarbonate filtering membrane of the micro-extruder in the step (2) is 0.1 mu m, and the extruding times are 40-50 times.

6. A liposome delivery system for tumor therapy, which is prepared by the method for preparing the liposome delivery system for tumor therapy according to any one of claims 1 to 5.

7. A method of preparing a liposome delivery system for the treatment of tumours as claimed in any one of claims 1 to 5 or the use of a liposome delivery system for the treatment of tumours as claimed in claim 5 in the preparation of a formulation for the treatment of tumours.

8. Use according to claim 7, characterized in that:

in the above-mentioned application, a desired drug is entrapped in the liposome delivery system for tumor therapy of claim 6; specifically, the method for preparing the liposome delivery system for tumor therapy according to any one of claims 1 to 5 is characterized in that, in the step (2), neutral phospholipid molecules, positively charged phospholipids, the negatively charged lipids PHL obtained in the step (1) and the desired drug are mixed and dissolved in a solvent, or, neutral phospholipid molecules, positively charged phospholipids and the negatively charged lipids PHL obtained in the step (1) are mixed and dissolved in a solvent, evaporated, concentrated and dried to obtain a lipid membrane; then, the obtained lipid membrane is hydrated by a hydration reagent containing the required medicine to obtain a mixed lipid membrane suspension, and then the subsequent operation is carried out.

9. Use according to claim 7, characterized in that:

the medicine is at least one of medicine, photo-thermal agent and imaging agent for treating cancer.

10. Use according to claim 9, characterized in that:

the drug for treating the cancer is any one or combination of more of adriamycin, paclitaxel, daunorubicin, vincristine, azithromycin, Triptorelin, naltrexone, minocycline, risperidone, octreotide, Somatropin, cytarabine, morphine, exenatide and lanreotide;

the photo-thermal agent is any one or combination of more of cyanine organic dye, nano metal photo-thermal agent, metal sulfide, two-dimensional nano material and nano semiconductor polymer material.