CN110613686A - Photostimulation-response liposome, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention relates to a photostimulation-response liposome, a pharmaceutical composition and application thereof. The photostimulation-responsive liposome comprises at least one photosensitizer and at least one lipid, and is sensitive to light and capable of releasing the entrapped contents under laser irradiation. The photostimulation-responsive liposomes further comprise at least one material having long circulation properties and at least one lipid-soluble active agent and/or water-soluble active agent. The invention also relates to a pharmaceutical composition comprising the photostimulation-responsive liposome. The invention also relates to the application of the photostimulation-response liposome and the pharmaceutical composition in preparing the drugs for treating tumors.

Description

Photostimulus-responsive liposomes, pharmaceutical compositions and uses thereof

technical field

The present invention relates to the field of liposomes, in particular to a light stimulation-response liposome, a pharmaceutical composition containing the light stimulation-response liposome, and the light stimulation-response liposome and medicine The use of the composition for preparing antitumor drugs.

Background technique

The treatment of malignant tumors has always been a worldwide problem. With the in-depth understanding of tumors and the continuous improvement of diagnosis and treatment techniques, human beings have made great progress in the diagnosis and treatment of tumors. For now, chemotherapy is still the main means of tumor treatment, but chemotherapy has problems such as poor physical and chemical stability, short half-life in vivo, and large toxic and side effects on healthy tissues.

Studies have found that the application of various nano-drug delivery systems, such as liposomes, polymer micelles, polymer nanoparticles, drug-polymer conjugates, etc., can change the kinetic properties of drugs in vivo, Increase the accumulation concentration of drugs in the tumor site and prolong their residence time, thereby improving drug efficacy and reducing toxic and side effects.

Liposome is a tiny membranous structure composed of ordered lipid bilayers (lipid membrane), with a size ranging from tens to hundreds of nanometers. Drugs can be encapsulated in phospholipid membranes or packed in vesicles. Although the drug delivery system of liposome-loaded anti-tumor drugs can make up for the deficiency of chemotherapy to a certain extent, the main problem faced by traditional liposome treatment of tumors is that liposomes have poor targeting of tumors in vivo. The release control is not precise enough.

In response to this phenomenon, researchers use the specific environment of the tumor site (such as pH, enzymes, ions, etc.), or use physical signals applied to the tumor site (such as light, ultrasound, heating, etc.) to design targeted drug delivery to the tumor site. system. For the controlled release method that depends on the stimulation response of internal factors, the way of triggering the delivery system to release drugs based on the specific environment of the tumor site is largely dependent on internal factors, and these factors have large individual differences, which leads to the reliability of triggering. not tall. For the controlled release method that relies on in vitro stimulus response, the physical signal triggering method is less dependent on internal environmental factors, and can also monitor the intensity and location of the physical stimulus signal, which has higher controllability and higher clinical practicability .

Among the many physical signals, laser is an ideal in vitro triggering method, which can be very precisely positioned in the target area, easy to use, rapid excitation and less interference. In recent years, photodynamic therapy (PDT) based on laser irradiation and photosensitizers has been widely used clinically (the first case in the United States in 1976, and the first case in China in 1981). Its principle is to activate a photosensitizer (such as chlorin e6, etc.) by a specific wavelength of laser light, start a photochemical reaction, and generate singlet oxygen ( ¹ O ₂ ). ¹ O ₂ is a powerful oxidant, but its lifetime is very short (less than 0.1 μs), and its diffusion range is extremely short. Only the substances very close to the photosensitizer can be affected by ¹ O ₂ . When the photosensitizer gathers at the tumor site, the photosensitizer is excited to generate ¹ O ₂ by illuminating the tumor site, and the ¹ O ₂ destroys the integrity of the tumor cell membrane structure, thereby killing the tumor cells. A large number of PDT clinical practices have shown that photosensitizers such as chlorin e6 have minimal phototoxicity to non-irradiated areas and can be used safely in humans. Clinically, in addition to treating superficial lesions with PDT, doctors can also introduce optical fibers through imaging and interventional equipment such as puncture needles, endoscopes, ultrasound or CT, and perform laser irradiation on deep lesions in patients. The increasing maturity of PDT technology provides a basis for the research of light-triggered nano-drug delivery system.

Reference 1 discloses a light-responsive liposome and its preparation method and application, which uses an azobenzene derivative containing a quaternary ammonium salt group that can respond to ultraviolet light to realize the light-responsive liposome. Controlled release of drugs. However, the preparation process of the azobenzene derivatives containing quaternary ammonium salt groups in the preparation method in Reference 1 is complicated and the production cost is relatively high. In addition, in reference 1, the azobenzene derivatives containing quaternary ammonium salt groups undergo trans-cis isomerization under light irradiation, which disturbs the phospholipid membrane and forms drug-releasing channels in the phospholipid bilayer , although the controllable drug release has achieved good results, but there is still room for improvement in its release.

Based on the above background, the present invention proposes a design idea of constructing light-stimulus-responsive liposomes based on the principle of photodynamics, using photosensitizers as photodynamic trigger groups. In the present invention, by applying light stimulation to the tumor site, the photodynamic control group in the light stimulation-responsive liposome - the photosensitizer - undergoes a photodynamic reaction to generate ¹ O ₂ , which in turn leads to the disintegration of the lipid membrane of the liposome , the drug encapsulated in the liposome is quickly released at the tumor site, which increases the drug concentration at the tumor site, thereby enhancing the anti-tumor effect of the drug.

Reference 1: CN 106822895A

Contents of the invention

The problem to be solved by the invention

The main purpose of the present invention is to improve the targeting and effectiveness of drugs and reduce the clinical needs of drug side effects, to provide a light-stimuli-responsive liposome that is easy to prepare and easy to industrialize, and contains the A light stimulation-responsive liposome pharmaceutical composition, and the use of the light stimulation-response liposome and the pharmaceutical composition in the preparation of antitumor drugs.

solutions to problems

The inventors have found in long-term research that when a certain amount of photosensitizer is added to the lipid film of liposomes, and then after light stimulation under certain conditions, the photosensitizer in the lipid film will undergo photodynamic reactions. ¹ O ₂ is generated, leading to the disintegration of the lipid membrane, and then the drug encapsulated in the liposome is released rapidly. The present invention has been accomplished based on the above findings.

One aspect of the present invention relates to a photostimuli-responsive liposome comprising at least one lipid and at least one photosensitizer, characterized in that it is sensitive to light waves and can disintegrate under the action of light waves to release the The contents of the package. Specifically, the photosensitizer can undergo a photodynamic reaction to generate ¹ O ₂ under the trigger of a light wave such as a laser, thereby causing the photo-stimulus-responsive liposome to disintegrate and release the contained content.

According to the light stimulation-response liposome described above, the photosensitizer is selected from the group consisting of methylene blue and analogs, riboflavin and derivatives thereof, chlorophyll a and b, retinoids, porphyrins, hematoporphyrins Compounds, gallium porphyrin compounds and various purine compounds, or their mixtures; preferably, the photosensitizer is methylene blue and the like.

According to the light stimulation-response liposome described above, the methylene blue compound is a compound of the following formula:

Wherein, R ₁ is H, or a C ₁ -C ₆ saturated or unsaturated hydrocarbon group, preferably, R ₁ is H, CH ₃ , CH ₂ -CH ₃ or CH=CH ₂ ;

R ₂ is H, or a C ₁ -C ₆ saturated or unsaturated hydrocarbon group, preferably, R ₂ is H, CH ₃ , CH ₂ -CH ₃ or CH=CH ₂ ;

R ₃ is H, or C ₁ -C ₆ alkyl, preferably, R ₃ is H, CH ₃ , CH ₂ -CH ₃ ;

R ₄ is H, or C ₁ -C ₆ alkyl, preferably, R ₄ is H, CH ₃ , CH ₂ -CH ₃ ;

R ₅ is H, or C ₁ -C ₆ alkyl, preferably, R ₅ is H, CH ₃ , CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH ₃ ;

R ₆ is H, or C ₁ -C ₆ alkyl, preferably, R ₆ is H, CH ₃ , CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH ₃ ;

Preferably, the methylene blue compound is 7-(ethyl(methyl)amino)-3-(ethylamino)-2-toluidine blue, as shown in the following formula:

According to the light stimulation-responsive liposome described above, the lipid is selected from phospholipids, glycolipids, fatty acids, dialkyldimethylammonium amphiphilic molecules, polyglycerol alkyl ethers, polyoxyethylene alkyl ethers, or combination; preferably, the lipid is selected from dipalmitoylphosphatidylcholine, diacetylglycerophosphatidylcholine, egg yolk lecithin, cholesterol or a combination thereof.

According to the above-mentioned photostimulus-responsive liposome, the light wave is selected from various light waves that can cause the photosensitizer to generate singlet oxygen ¹ O ₂ , preferably 600-1100nm light wave.

According to the light stimulation-response liposome described above, the light stimulation-response liposome further comprises at least one material with long circulation properties, preferably pegylated phospholipids, gangliosides, non-ionic Surfactants or polymers, more preferably different molecular weight polyethylene glycol modified phosphatidylethanolamine phospholipids, gangliosides, polyvinyl alcohol, polyvinylpyrrolidone, Tween, poloxamer, particularly preferably distearoyl Phosphatidylethanolamine-Polyethylene Glycol 2000.

According to the photostimuli-responsive liposome described above, the photostimuli-responsive liposome further comprises at least one fat-soluble active agent and/or water-soluble active agent, preferably a fat-soluble and/or water-soluble anticancer agent , more preferably cisplatin, carboplatin, nedaplatin, doxorubicin or paclitaxel.

According to the above-mentioned photostimulus-response liposome, the weight ratio of the lipid, the photosensitizer, and the material with long-cycle properties is 50-90:0.5-12:3-18.

According to the above-mentioned photostimulus-response liposome, the weight ratio of the total weight of the lipid, photosensitizer and material with long circulation properties to the fat-soluble active agent and/or water-soluble active agent is 10-135 :1-15.

According to the light stimulation-response liposome described above, the light stimulation-response liposome is granular, and its particle diameter is 60-300nm; preferably, its particle diameter is 60-250nm; preferably, its particle The particle size is 80-200nm; preferably, the particle size is 100-200nm; preferably, the particle size is 100-150nm.

In addition, the present invention also provides a pharmaceutical composition, which comprises the above-mentioned photostimuli-responsive liposome and a pharmaceutically acceptable carrier.

Further, the present invention also provides the use of the above-mentioned photostimuli-responsive liposome or the above-mentioned pharmaceutical composition in the preparation of antitumor drugs.

The effect of the invention

The photostimulation-response liposome of the present invention can improve the targeting and effectiveness of drugs, reduce the clinical demand of toxic and side effects of drugs, and provide a photo-stimulation-response liposome with simple preparation method and easy industrial production.

The photostimulation-responsive liposome in the present invention can improve the encapsulation rate of drugs, can provide appropriate drug concentration to tumor sites, and promote the killing effect on tumor cells.

Compared with the prior art, when a certain amount of photosensitizer is added to the lipid membrane of the liposome, and then stimulated by light under certain conditions, the photosensitizer in the lipid membrane will undergo a photodynamic reaction to generate ¹ O ₂ , lead to the disintegration of the lipid membrane, and then release the drug encapsulated in the liposome quickly at the tumor site, increase the drug concentration at the tumor site, and then enhance the anti-tumor effect of the drug, making the targeting and effectiveness of the anti-tumor drug be greatly improved.

Detailed ways

The main purpose of the present invention is to improve the targeting and effectiveness of drugs and reduce the clinical needs of drug side effects, to provide a light-stimuli-responsive liposome that is easy to prepare and easy to industrialize, and contains the A light stimulation-responsive liposome pharmaceutical composition, and the use of the light stimulation-response liposome and the pharmaceutical composition in the preparation of antitumor drugs.

The inventors have found in long-term research that when a certain amount of photosensitizer is added to the lipid film of liposomes, and then after light stimulation under certain conditions, the photosensitizer in the lipid film will undergo photodynamic reactions. ¹ O ₂ is generated, leading to the disintegration of the lipid membrane, and then the drug encapsulated in the liposome is released rapidly. The present invention has been accomplished based on the above findings.

One aspect of the present invention relates to a light stimulus-responsive liposome, characterized in that it comprises at least one lipid and at least one photosensitizer, is characterized in that it is sensitive to light waves, and can disintegrate under the action of light waves and release the contained contents. Specifically, the lipid and photosensitizer form a lipid film that can carry the drug. The photosensitizer can undergo a photodynamic reaction to generate ¹ O ₂ under the trigger of light waves such as laser light, and then lead to the disintegration of the light stimulus-responsive liposome and release the contained content.

The photostimuli-responsive liposomes further comprise at least one material having long-circulation properties.

The photostimuli-responsive liposome further comprises at least one fat-soluble active agent and/or water-soluble active agent. Specifically, the fat-soluble active agent and/or water-soluble active agent is encapsulated in a lipid membrane, or encapsulated in a vesicle formed by a lipid membrane. The fat-soluble active agent and/or water-soluble active agent is released when the lipid membrane is disintegrated.

Wherein said lipid, photosensitizer, the weight ratio of the material with long circulation characteristic is 50-90:0.5-12:3-18, preferably 60-90:1-10:5-15, more preferably 70 -90:5-10:8-15, more preferably 80-90:8-10:10-15.

The weight ratio of the total weight of the lipid, the photosensitizer and the material with long cycle characteristics to the fat-soluble active agent and/or water-soluble active agent is 10-135:1-15, preferably 20-120:5- 15, more preferably 50-100:8-15, even more preferably 80-100:10-15.

In the present invention, the lipid is not particularly limited, and it can be any known lipid, such as phospholipids, glycolipids, fatty acids, dialkyldimethylammonium amphiphilic molecules, polyglycerol alkyl ethers or Polyoxyethylene alkyl ether, etc.

Examples of the above-mentioned phospholipids include natural or synthetic phospholipids such as lecithin (soybean lecithin, egg yolk lecithin, dilauroyl lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, or distearoyl lecithin, etc.) , Phosphatidylethanolamine (dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine or distearoylphosphatidylethanolamine, etc.), phosphatidylserine (dilauroylphosphatidylethanolamine, dimeat Myristoylphosphatidylserine, dipalmitoylphosphatidylserine or distearoylphosphatidylserine, etc.), phosphatidic acid, phosphatidylglycerol (dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol glycerol or distearoylphosphatidylglycerol, etc.), phosphatidylinositol (dilauroylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, or distearoylphosphatidylinositol etc.), lysolecithin, sphingomyelin, hydrogenated phospholipid, etc.

Examples of the aforementioned glycolipids include glyceroglycolipids, glycosphingolipids, sterols and the like.

Examples of the aforementioned glyceroglycolipids include digalactosyl diglycerides (digalactosyl dilauroyl diglycerides, digalactosyl dimyristoyl glycerides, digalactosyl dipalmitoyl glycerides, or digalactosyl distearoyl Glycerides, etc.) or galactose diglycerides (galactose dilauroylglycerides, galactose dimyristoylglycerides, galactose dipalmitoylglycerides, galactose distearoylglycerides, etc.) and the like.

Examples of the aforementioned glycosphingolipids include galactocerebroside, lactocerebroside, or ganglioside and the like.

Examples of the aforementioned sterols include cholesterol, carbamoyl cholesterol, cholesterol hemisuccinate, ergosterol or lanosterol, and the like.

In the present invention, the lipids may be used alone or in combination of two or more of them.

In particular, the lipid is selected from dipalmitoylphosphatidylcholine, diethynylglycerophosphatidylcholine, egg yolk lecithin, cholesterol.

In the present invention, the material with long-term circulation characteristics refers to the material that can endow liposomes with the ability to evade the clearance of the reticuloendothelial system in vivo, and mainly refers to materials with relatively large molecular weight and hydrophilic or hydrophilic groups. Material. This type of material can be staggered on the surface of the carrier material to form a dense conformational cloud, forming a thicker steric hindrance layer, hindering the function of the reticuloendothelial system, or enhancing the solvation of the carrier due to its hydrophilicity, effectively preventing The opsonization of its surface, thereby reducing the affinity with macrophages of the mononuclear phagocyte system, allows liposomes to stabilize in the circulation and prolong their half-life. The material with long-term cycle characteristics can be, for example, PEGylated phospholipids, gangliosides, non-ionic surfactants or polymers, and the like.

In the present invention, the materials with long cycle characteristics include but are not limited to polyethylene glycol-hydrogenated soybean phosphatidylethanolamine (HSPE-PEG2000) with an average molecular weight of 2000, polyethylene glycol-hydrogenated soybean with an average molecular weight of 5000 Phosphatidylethanolamine (HSPE-PEG5000), polyethylene glycol-distearoylphosphatidylethanolamine (DSPE-PEG2000) with an average molecular weight of 2000, polyethylene glycol-dipalmitoylphosphatidylethanolamine (DPPE) with an average molecular weight of 2000 -PEG2000) and other different molecular weight polyethylene glycol modified phosphatidylethanolamine phospholipids, gangliosides, polyvinyl alcohol, polyvinylpyrrolidone, Tween, poloxamer, etc.

In particular, the material with long cycle characteristics is selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000.

In the present invention, the term "photosensitizer" refers to substances that can produce singlet oxygen and other substances that can cause changes in lipid structure under the stimulation of light waves. The photosensitizer is selected from the group consisting of methylene blue and analogs, riboflavin and its derivatives, chlorophyll a and b, retinoids, porphyrin compounds, hematoporphyrin compounds, gallium porphyrin compounds and various purine compounds , or a mixture of them. The photosensitizer mentioned above is preferably selected from methylene blue compounds.

Described methylene blue compound is the compound of following formula:

Wherein, R ₁ is H, or a C ₁ -C ₆ saturated or unsaturated hydrocarbon group, preferably, R ₁ is H, CH ₃ , CH ₂ -CH ₃ or CH=CH ₂ ;

R ₂ is H, or a C ₁ -C ₆ saturated or unsaturated hydrocarbon group, preferably, R ₂ is H, CH ₃ , CH ₂ -CH ₃ or CH=CH ₂ ;

R ₃ is H, or C ₁ -C ₆ alkyl, preferably, R ₃ is H, CH ₃ , CH ₂ -CH ₃ ;

R ₄ is H, or C ₁ -C ₆ alkyl, preferably, R ₄ is H, CH ₃ , CH ₂ -CH ₃ ;

R ₅ is H, or C ₁ -C ₆ alkyl, preferably, R ₅ is H, CH ₃ , CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH ₃ ;

R ₆ is H, or C ₁ -C ₆ alkyl, preferably, R ₆ is H, CH ₃ , CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH ₃ ;

Particularly, the methylene blue compound is 7-(ethyl(methyl)amino)-3-(ethylamino)-2-toluidine blue, as shown in the following formula:

The light waves used in the present invention can be various light waves that can cause the photosensitizer to generate singlet oxygen ( ¹ O ₂ ), preferably near-infrared light.

The wavelength range of the above-mentioned near-infrared light is 600-1100nm, the irradiation power density is generally 100-300mW/cm ² , the energy density is 100-600J/cm ² , and the action time is 0.25-90 minutes. The sources of infrared light are usually divided into three types: thermal radiation infrared light source, gas discharge infrared light source and laser infrared light source. Specifically, the source of the infrared light can be a black body; an electrified silicon carbide rod; a xenon lamp; a semiconductor laser; a carbon dioxide gas laser;

In an embodiment of the present invention, said fat-soluble active agent and/or water-soluble active agent is an anticancer agent. Other fat-soluble and/or water-soluble active agents include, but are not limited to, antibiotics, antifungals, immunosuppressants, antivirals, and the like.

In the present invention, the anticancer agent is selected from alkylating agents, antimetabolites, spindle toxin plant alkaloids, cytotoxic antitumor antibiotics, topoisomerase inhibitors, monoclonal antibodies or fragments thereof, kinase inhibitors , anti-tumor enzymes and enzyme inhibitors, apoptosis inducers, antihormones, retinoids, platinum-containing compounds, proteins, polypeptides and nucleic acid drugs.

In particular, the fat-soluble active agent and/or water-soluble active agent is selected from cisplatin, carboplatin, nedaplatin, doxorubicin or paclitaxel.

In the present invention, the particle size of the photostimulus-responsive liposome is 60-300nm; further, its particle size is 60-250nm; further, its particle size is 70-250nm; further, its particle size is 80-250nm. 200nm; further, the particle diameter is 100-200nm; further, the particle diameter is 100-150nm.

Another aspect of the present invention relates to a pharmaceutical composition comprising the photostimuli-responsive liposome according to the present invention and a pharmaceutically acceptable carrier. The carrier is selected from liposomes prepared from dipalmitoylphosphatidylcholine, diethynylglycerophosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and the like.

Another aspect of the present invention relates to the use of the photostimuli-responsive liposome or pharmaceutical composition according to the present invention in the preparation of antitumor drugs.

The photostimuli-responsive liposomes of the present invention may be administered to a subject using any suitable route, for example, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intraarticular Administration, intrathecal administration, intracerebroventricular administration, nasal spray, pulmonary inhalation, oral administration, and other suitable routes of administration known to those skilled in the art. Tissues that may be treated using the methods of the present invention include, but are not limited to, nose, lung, liver, kidney, soft tissue, muscle, adrenal tissue, and breast. Any tissue or body fluid that can be reached by light waves can be treated with the photostimuli-responsive liposomes of the present invention.

Dosages of active agents to be administered to a subject using the photostimuli-responsive liposomes of the invention are readily determined by those skilled in the art. The dosage of the active agent may be adjusted according to the active agent contained in the carrier, as known in the art.

During and/or after administration of the photostimuli-responsive liposomes of the present invention, the subject's target site may be irradiated with light. In one embodiment of the invention, the photostimuli-responsive liposomes of the invention are delivered to a subject, and the target site is irradiated with light as soon as possible after administration.

The beneficial effect of the present invention is that the present invention is based on the photodynamic principle, adding a photosensitizer as a photodynamic trigger group in the existing liposome formulation, so that the liposome has good light-stimulus response characteristics. Triggered by light radiation, the drug encapsulated in the liposome can be quickly released at the target site, which increases the drug concentration at the target site, thereby enhancing the therapeutic effect of the drug, reducing the systemic toxicity of the drug, and has a good application prospect.

Example

The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

The present invention provides general and/or specific descriptions of the materials and test methods used in the tests. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention has been described here in as much detail as possible.

The abbreviations used in the following examples are as follows:

DPPC: Dipalmitoylphosphatidylcholine

DC _8,9 PC: Diethynylglycerophosphatidylcholine

DSPE-PEG2000: Distearoylphosphatidylethanolamine-Polyethylene Glycol 2000

EEMI: 7-(ethyl(methyl)amino)-3-(ethylamino)-2-toluidine blue

HEPES: Hydroxyethylpiperazine Ethiosulfate Buffer

EPC: egg yolk lecithin

Chol: Cholesterol

Example 1: Preparation of doxorubicin photostimulation-responsive liposomes

(1) Materials and dosage: (the dosage of 100ml liposome, the following table 1)

Table 1

(2) Preparation method

Weigh various lipid materials, materials with long cycle characteristics and photosensitizer according to the amount in Table 1, dissolve in 200ml of chloroform:methanol (4:1) solution, remove the organic solvent by rotary evaporation at 50-60°C, Obtain a film that is dry and evenly adhered to the bottom of the glass bottle. The obtained film was hydrated at 50-60° C. for 20 minutes with 250 ml of citric acid solution with pH=4.0 to form a uniform liposome solution. Then, a polycarbonate membrane with a pore size of 100 nm was extruded, and the extrusion was repeated 5 times. The obtained liposomes were adjusted with an appropriate amount of sodium carbonate aqueous solution (500 mM) to the pH value of the external aqueous phase to 7.0-7.4. The obtained liposomes were co-cultured with 270ml of an aqueous solution of doxorubicin (Shenzhen Wanle Pharmaceutical Co., Ltd., batch number 20160905) (1 mg/ml of doxorubicin) at 37°C for 1 hour to obtain photostimuli-responsive lipids loaded with doxorubicin body. Afterwards, store at 4-7°C for later use.

Embodiment 2: Doxorubicin photostimulation-response liposome encapsulation efficiency assay

Take 1 ml of the doxorubicin light stimulation-responsive liposome prepared in Example 1 in a 1.5 ml EP tube, centrifuge at 10000 r/min for 10 min. Take 0.2ml of the supernatant in a 10ml volumetric flask, use methanol solution to permeate the membrane and make up the volume, and inject into high performance liquid chromatography (HPLC) for analysis (C18 column, Agilent SB-C18, 4.6×150mm, 1.8μm), methanol-acetonitrile -Phosphate buffer solution (25mM NH ₄ H ₂ PO ₄ -30mM H ₃ PO ₄ , pH 5.0) (5:2:3) as mobile phase, flow rate 1.0ml·min ^-1 , detection wavelength 233nm), to load lipid The doxorubicin drug content (C1) in the body is 0.202 mg; take another 0.2 ml of non-centrifuged doxorubicin photostimulation-response liposome sample in a 10 ml volumetric flask, dilute the membrane with methanol solution and dilute to the mark. High-performance liquid chromatography (HPLC) sample injection analysis, the total drug content (C2) of doxorubicin in the doxorubicin light stimulation-responsive liposome suspension was 0.208 mg. Encapsulation efficiency (E)=C1/C2×100%. The results showed that the encapsulation efficiency of doxorubicin light stimulation-responsive liposome was 97.1%.

Embodiment 3: In vitro photostimulation-response test of doxorubicin photostimulation-response liposome

Take 0.5ml of the doxorubicin photostimulation-response liposomes prepared in Example 1 and put them into a cellulose acetate dialysis bag (cellulose acetate dialysis bag, MWCO 8-14kDa) (BioVision, Inc., batch number C413103001), and soak in 20ml of HEPES solution was stirred magnetically at 60r/min. Irradiated by laser (660nm, 200mW/cm ² ), after irradiating for 10 minutes, take out 0.5ml release solution and pass through 0.22μm filter membrane to measure the drug content in the release solution by HPLC (C18 column, Agilent SB-C18, 4.6×150mm, 1.8μm), methanol-acetonitrile-phosphate buffer (25mM NH ₄ H ₂ PO ₄ -30mM H ₃ PO ₄ , pH 5.0) (5:2:3) as mobile phase, flow rate 1.0ml·min ^{- 1} , detection wavelength 233nm). A sample not irradiated with light was used as a control, and the drug content was determined by HPLC in the same way. Release amount%=total amount of released drug/total amount of entrapped drug×100%.

After the doxorubicin light-stimulated-responsive liposome was treated with light, the drug release amount (81.2±9.4%) was significantly higher than that of the control group without light (6.4±1.6%). Therefore, the doxorubicin photostimulation-response liposome of the present invention has obvious photo-stimulation-response drug release characteristics in vitro.

Embodiment 4: In vivo photostimulation-response test of doxorubicin photostimulation-response liposome

Fifteen Kunming mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., KM) were randomly divided into three groups (5 mice in each group).

The first group was injected with (0.5 ml, 1 mg) doxorubicin injection (Italpha Macia, 10 mg/bottle, batch number 132550013) through the tail vein (injection group). The second group was injected with (0.5ml, 0.5mg) doxorubicin common liposome (Shipharm Ouyi Pharmaceutical Co., Ltd., 10ml:20mg, lot number 691160402) through tail vein (common liposome group). The third group was injected into the tail vein (0.5ml, 0.5mg) of the doxorubicin photostimulation-responsive liposome of Example 1 of the present invention (photostimulation-responsive liposome group).

Immediately after administration, the left foreleg of each mouse was irradiated with laser (660nm, 200mW/cm ² ), and the right foreleg without light was used as its own control. Doxorubicin content. The measurement method is as follows.

C18 column (Agilent SB-C18, 4.6×150mm, 1.8μm), the mobile phase is methanol-acetonitrile-0.01mol L ^-1 ammonium dihydrogen phosphate-glacial acetic acid (50:22:28:0.6), the flow rate is 1.0ml min ^-1 , the excitation wavelength is 479 nm, the emission wavelength is 587 nm, and the column temperature is 30°C. With daunomycin as the internal standard, take 0.5 g of muscle tissue, add 1 mL of phosphate buffer solution (pH 7.4), homogenate, add internal standard solution, add chloroform-methanol (4:1) mixture to extract, and the residue is extracted with methanol Dissolve, filter, inject.

Content = drug amount in tissue/tissue weight.

The results are as follows: the injection group (2.6 ± 0.3 μg/g), the common liposome group (2.1 ± 0.4 μg/g) and the photostimuli-response liposome group (2.2 ± 0.4 μg/g) in the non-irradiated part of the A There was no significant difference in the content of mymycin; the drug content of the injection group (2.6 ± 0.4 μg/g) and the ordinary liposome group (2.2 ± 0.4 μg/g) compared with the non-irradiated site did not have a significant difference. However, in the doxorubicin photostimulus-responsive liposome group, the drug content in the light site (4.9±0.7μg/g) was nearly double that of the non-light site (2.2±0.4μg/g), which significantly increased drug content. Therefore, the doxorubicin photostimulation-response liposome of the present invention has obvious photo-stimulation-response drug release characteristics in vivo.

Example 5: Pharmacodynamic observation of doxorubicin light-stimulated-responsive liposomes

Take MCF-7 breast cancer cells in the logarithmic growth phase (Shanghai Meixuan Biotechnology Co., Ltd.), and inoculate mice with 0.2ml of MCF-7 breast cancer cell suspension (medium solution diluted to 2×10 ⁶ /mL) (60, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., BALB/c nude mice) subcutaneous on the back of the right axillary side.

On the 10th day after tumor cell inoculation, 50 mice with relatively uniform tumor size were selected (sorted by tumor volume and randomly divided into each group), and divided into five groups (10 mice in each group): control group, normal injection Liquid group, common liposome group, photostimulus-responsive liposome group (lighted) and photostimulus-responsive liposome group (unlit). Except for the tail vein injection of normal saline in the control group, the other four groups were injected with common injection equivalent to 10 mg/kg doxorubicin, common liposomes and the doxorubicin light-stimuli-responsive liposomes of Example 1 respectively.

Immediately after the injection, the above-mentioned laser (660nm, 200mW/cm ² , 10min) was used to irradiate, and the mice in other groups were not treated. A total of three administrations were given, once every two days. The tumors of the mice in each group were weighed, and the tumor inhibition rate was calculated according to the formula. The results are shown in Table 2.

Tumor inhibition rate=(1-Ws/Wx)×100%

Among them, Ws is the tumor mass of the treatment group; Wx is the tumor mass of the normal saline group. (x±s means mean±standard deviation; tumor volume=length×width×width/2)

Table 2 pharmacodynamic test results

The above data show that the combination of doxorubicin photostimulation-responsive liposomes with light significantly enhances the tumor-inhibitory effect. Therefore, it can be used to prepare antitumor drugs.

Industrial availability

The photostimulation-response liposome of the present invention can improve the targeting and effectiveness of drugs, and provides a photo-stimulation-response liposome with a simple preparation method and easy industrial production. The photostimuli-responsive liposomes of the present invention can be implemented in industrial production.

Claims (11)

1. A photostimulation-responsive liposome comprising at least one photosensitizer and at least one lipid, characterized in that it is sensitive to light waves and is capable of dissociating and releasing the entrapped contents under the action of light waves,

the photosensitizer is selected from methylene blue and analogues, riboflavin and derivatives thereof, chlorophyll a and b, retinol, porphyrin compounds, hematoporphyrin compounds, gallium porphyrin compounds and various purine compounds, or a mixture thereof; preferably, the photosensitizer is methylene blue and the like.

2. The photostimulation-responsive liposome of claim 1, wherein said methylene blue-like compound is of the formula:

wherein R is₁Is H or C₁～C₆Saturated or unsaturated hydrocarbon radical, preferably, R₁Is H, CH₃、CH₂-CH₃Or CH ═ CH₂；

R₂Is H or C₁～C₆Saturated or unsaturated hydrocarbon radical, preferably, R₂Is H, CH₃、CH₂-CH₃Or CH ═ CH₂；

R₃Is H or C₁～C₆Alkyl, preferably, R₃Is H, CH₃、CH₂-CH₃；

R₄Is H or C₁～C₆Alkyl, preferably, R₄Is H, CH₃、CH₂-CH₃；

R₅Is H or C₁～C₆Alkyl, preferably, R₅Is H, CH₃、CH₂-CH₃Or CH₂-CH₂-CH₃；

R₆Is H or C₁～C₆Alkyl, preferably, R₆Is H, CH₃、CH₂-CH₃Or CH₂-CH₂-CH₃；

Preferably, the methylene blue compound is 7- (ethyl (methyl) amino) -3- (ethylamino) -2-toluidine blue, which is represented by the following formula:

3. the photostimulation-responsive liposome according to claim 1 or 2, wherein said lipid is selected from phospholipids, glycolipids, fatty acids, dialkyldimethylammonium amphothers, polyglycerol alkyl ethers, polyoxyethylene alkyl ethers, or combinations thereof; preferably, the lipid is selected from dipalmitoylphosphatidylcholine, diacetylene glycerol phosphatidylcholine, egg yolk lecithin, cholesterol or combinations thereof.

4. The photostimulation-responsive liposome of any of claims 1 to 3, wherein said light waves are selected from the group consisting of those which cause the photosensitizer to produce singlet oxygen¹O₂Preferably 600-1100 nm.

5. Photostimulation-responsive liposome according to any of claims 1 to 4, characterized in that it further comprises at least one material with long circulation properties, preferably a pegylated phospholipid, ganglioside, non-ionic surfactant or polymer, more preferably a different molecular weight polyethylene glycol modified phosphatidylethanolamine phospholipid, ganglioside, polyvinyl alcohol, polyvinylpyrrolidone, tween, poloxamer, particularly preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000.

6. The photostimulation-responsive liposome of claim 5, wherein the weight ratio of said lipid, photosensitizer, material with long circulation properties is 50-90:0.5-12: 3-18.

7. Photostimulation-responsive liposome according to any of claims 1 to 6, characterized in that it further comprises at least one liposoluble active agent and/or water-soluble active agent, preferably a liposoluble and/or water-soluble anticancer agent, more preferably cisplatin, carboplatin, nedaplatin, doxorubicin or paclitaxel.

8. The photostimulation-responsive liposome of claim 7, wherein the weight ratio of the total weight of said lipid, photosensitizer and material with long circulation properties to the weight of fat-soluble active agent and/or water-soluble active agent is 10-135: 1-15.

9. The photostimulation-responsive liposome according to any one of claims 1 to 8, wherein the photostimulation-responsive liposome is in the form of particles with a particle size of 60 to 300 nm; preferably, the particle size is 60-250 nm; preferably, the particle size is 80-200 nm; preferably, the particle size is 100-200 nm; preferably, the particle size is 100 to 150 nm.

10. A pharmaceutical composition comprising the photostimulation-responsive liposome of any of claims 1-9 and a pharmaceutically acceptable carrier.

11. Use of a photostimulation-responsive liposome according to any of claims 1-9 or a pharmaceutical composition according to claim 10 for the preparation of an antitumor medicament.