CN110613686A - Photostimulation-response liposome, pharmaceutical composition and application thereof - Google Patents
Photostimulation-response liposome, pharmaceutical composition and application thereof Download PDFInfo
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- CN110613686A CN110613686A CN201810638657.9A CN201810638657A CN110613686A CN 110613686 A CN110613686 A CN 110613686A CN 201810638657 A CN201810638657 A CN 201810638657A CN 110613686 A CN110613686 A CN 110613686A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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
Technical Field
The invention relates to the field of liposomes, in particular to a photostimulation-response liposome, a pharmaceutical composition containing the photostimulation-response liposome, and application of the photostimulation-response liposome and the pharmaceutical composition in preparation of antitumor drugs.
Background
The treatment of malignant tumor is a worldwide problem, and with the deep understanding of tumor and the continuous improvement of diagnosis and treatment technology, human beings make great progress in tumor diagnosis and treatment. At present, chemotherapy is still the main means of tumor treatment, but the chemotherapy has the problems of poor physicochemical stability, short in vivo half-life, great toxic and side effects on healthy tissues and the like.
The research finds that the application of various nano-drug delivery systems, such as liposome, polymer micelle, polymer nanoparticle, drug-polymer conjugate and the like for encapsulating drugs can change the dynamic characteristics of the drugs in vivo, improve the aggregation concentration of the drugs at tumor parts, prolong the detention time of the drugs, thereby improving the curative effect of the drugs and reducing the toxic and side effects.
Liposomes are minute membranous structures consisting of ordered arrangements of lipid bilayers (lipid membranes), which can range in size from tens to hundreds of nanometers. The medicine can be wrapped in the phospholipid membrane or filled in the formed vesicle. Although the drug delivery system of the liposome-encapsulated anti-tumor drug can make up the deficiency of chemotherapy to a certain extent, the main problems of the traditional liposome for treating tumors at present are that the targeting of the liposome to the tumors in vivo is poor, and the drug release control is not accurate enough.
In response to this phenomenon, researchers have utilized specific environments (e.g., pH, enzymes, ions, etc.) at the tumor site, or physical signals (e.g., light, ultrasound, heat, etc.) applied to the tumor site to design a drug delivery system targeted to the tumor site. For the controlled release mode depending on the stimulus response of the in vivo factors, the mode of triggering the release system to release the drug based on the specific environment of the tumor site largely depends on the in vivo factors, and the individual differences of the factors are large, so that the triggering reliability is not high. For the controlled release mode depending on the in vitro stimulation response, the physical signal triggering mode has less dependence on in vivo environmental factors, and can monitor the strength and the position of the physical stimulation signal, so that the controllability is higher, and the clinical practicability is higher.
Among the numerous physical signals, laser is an ideal in vitro triggering means that can be very precisely located at the target area, is convenient to use, is rapid to excite, and is less disturbed. In recent years, photodynamic therapy (PDT) techniques based on laser irradiation and photosensitizers have become widely used clinically (first us in 1976 and first national in 1981). The principle is that a photosensitizer (such as chlorin e6 and the like) is activated by laser with specific wavelength to start a photochemical reaction to generate singlet oxygen (A)1O2)。1O2Is a powerful oxidant, but has a very short lifetime (less than 0.1 mus), a very short diffusion range, and only species very close to the photosensitizer are able to be exposed to1O2The function of (1). After the photosensitizer is gathered at the tumor part, the photosensitizer is excited to generate by illuminating the tumor part1O2，1O2The integrity of the tumor cell membrane structure is damaged, thereby generating the killing effect on the tumor cells. A large number of clinical PDT practices show that photosensitizer such as chlorin e6 has extremely low phototoxicity to non-illumination areas and can be safely used in human bodies. In clinic, PDT can treat superficial lesions, and doctors can introduce optical fibers through images and interventional devices such as puncture needles, endoscopes, ultrasound or CT to irradiate the lesions deep in the body of a patient with laser. The growing development of PDT technology provides the basis for the development of light-triggered nano-drug delivery systems.
Reference 1 discloses a photoresponsive liposome, and a preparation method and application thereof, wherein a quaternary ammonium salt group-containing azobenzene derivative capable of responding to ultraviolet light is used for realizing controllable release of a drug in the photoresponsive liposome. However, in the preparation method of reference 1, the preparation process of the azobenzene derivative containing a quaternary ammonium salt group is complicated, and the production cost is high. In addition, in reference 1, since the azobenzene derivative containing a quaternary ammonium salt group undergoes trans-cis isomerization upon irradiation with light, and disturbs the phospholipid membrane, and forms a drug-releasable channel in the phospholipid bilayer, although a good effect is obtained on the controllability of drug release, there is still room for improvement in the amount of release.
Based on the background, the invention provides a design idea of constructing the photostimulation-response liposome by taking a photosensitizer as a photodynamic trigger group based on a photodynamic principle. The invention triggers photodynamic reaction of photodynamic control group-photosensitizer in the photostimulation-response liposome by applying photostimulation to the tumor part to generate1O2So as to lead the lipid membrane of the liposome to be disintegrated, and the medicine wrapped in the liposome is quickly released at the tumor part, thereby improving the medicine concentration at the tumor part and further enhancing the anti-tumor effect of the medicine.
Reference 1: CN 106822895A
Disclosure of Invention
Problems to be solved by the invention
The invention mainly aims to provide a photostimulation-response liposome which has simple preparation method and is easy for industrial production, a pharmaceutical composition containing the photostimulation-response liposome, and application of the photostimulation-response liposome and the pharmaceutical composition in preparing antitumor drugs aiming at the clinical requirements of improving the targeting property and the effectiveness of drugs and reducing the toxic and side effects of the drugs.
Means for solving the problems
The inventor finds that in long-term research, when a certain amount of photosensitizer is added into a lipid membrane of liposome and then is subjected to light stimulation under certain conditions, the photosensitizer in the lipid membrane can generate photodynamic reaction to generate1O2Leading to the disintegration of the lipid membraneAnd the drug encapsulated in the liposome is rapidly released. The present invention has been completed based on the above findings.
One aspect of the present invention relates to a photostimulation-responsive liposome comprising at least one lipid and at least one photosensitizer, characterized in that it is sensitive to light waves, capable of disintegrating and releasing the entrapped contents under the action of light waves. In particular, the photosensitizer is capable of photodynamic reaction generation triggered by light waves such as laser light1O2Which in turn causes the photostimulation-responsive liposomes to disintegrate, releasing the entrapped contents.
The photostimulation-responsive liposome according to the above, said photosensitizer being selected from methylene blue and its analogues, riboflavin and its derivatives, chlorophylls a and b, retinoids, porphyrins, hematoporphyrins, gallium porphyrins and various purine compounds, or mixtures thereof; preferably, the photosensitizer is methylene blue and the like.
The photostimulation-responsive liposome according to the above, wherein the methylene blue compound is a compound of the following 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:
the photostimulation-responsive liposome as described above, said lipid being selected from the group consisting of phospholipids, glycolipids, fatty acids, dialkyldimethylammonium ampholytes, 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.
The photostimulation-responsive liposome according to the above, wherein said light wave is selected from a variety of light waves capable of causing a photosensitizer to produce singlet oxygen1O2Preferably 600-1100 nm.
The photostimulation-responsive liposome according to the above, further comprising at least one material having 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, poloxamers, particularly preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000.
The photostimulation-responsive liposome according to the above, further comprising at least one liposoluble active agent and/or water-soluble active agent, preferably liposoluble and/or water-soluble anticancer agent, more preferably cisplatin, carboplatin, nedaplatin, doxorubicin or paclitaxel.
According to the photostimulation-response liposome, the weight ratio of the lipid, the photosensitizer and the material with long circulation property is 50-90:0.5-12: 3-18.
According to the photostimulation-response liposome, the weight ratio of the total weight of the lipid, the photosensitizer and the material with long circulation property to the fat-soluble active agent and/or the water-soluble active agent is 10-135: 1-15.
The photostimulation-response liposome is granular, and the particle size of the photostimulation-response liposome is 60-300 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.
Furthermore, the present invention provides a pharmaceutical composition comprising a photostimulation-responsive liposome according to the above and a pharmaceutically acceptable carrier.
Further, the invention also provides the application of the photostimulation-response liposome or the pharmaceutical composition in preparing the antitumor drugs.
ADVANTAGEOUS EFFECTS OF INVENTION
The photostimulation-response liposome can improve the targeting property and effectiveness of the medicament, reduce the clinical requirement of toxic and side effects of the medicament, and provide the photostimulation-response liposome which has simple and convenient preparation method and is easy for industrial production.
The photostimulation-response liposome can improve the entrapment rate of the medicament, can provide proper medicament concentration for a tumor part, and promotes the killing effect on tumor cells.
Compared with the prior art, when a certain amount of photosensitizer is added into the lipid membrane of the liposome and is stimulated by light under certain conditions, the photosensitizer in the lipid membrane can generate photodynamic reaction to generate1O2Leading to the disintegration of lipid membrane, and further releasing the drug wrapped in the liposome at the tumor part quickly, improving the tumorThe drug concentration of the tumor part further enhances the anti-tumor effect of the drug, so that the targeting property and the effectiveness of the anti-tumor drug are greatly improved.
Detailed Description
The invention mainly aims to provide a photostimulation-response liposome which has simple preparation method and is easy for industrial production, a pharmaceutical composition containing the photostimulation-response liposome, and application of the photostimulation-response liposome and the pharmaceutical composition in preparing antitumor drugs aiming at the clinical requirements of improving the targeting property and the effectiveness of drugs and reducing the toxic and side effects of the drugs.
The inventor finds that in long-term research, when a certain amount of photosensitizer is added into a lipid membrane of liposome and then is subjected to light stimulation under certain conditions, the photosensitizer in the lipid membrane can generate photodynamic reaction to generate1O2Resulting in disintegration of the lipid film and thus rapid release of the drug encapsulated in the liposomes. The present invention has been completed based on the above findings.
One aspect of the present invention relates to a photostimulation-responsive liposome, characterized in that it comprises at least one lipid and at least one photosensitizer, and in that it is sensitive to light waves and is capable of disintegrating and releasing the entrapped contents under the action of light waves. In particular, the lipid and photosensitizer form a lipid membrane that can carry a drug. The photosensitizer can be generated by photodynamic reaction triggered by light waves such as laser1O2Which in turn causes the photostimulation-responsive liposomes to disintegrate, releasing the entrapped contents.
The photostimulation-responsive liposome further comprises at least one material having long circulation properties.
The photostimulation-responsive liposome further comprises at least one lipid-soluble active agent and/or water-soluble active agent. Specifically, the fat-soluble active agent and/or the water-soluble active agent are encapsulated in a lipid membrane or encapsulated in a vesicle formed by the lipid membrane. When the lipid membrane disintegrates, the fat-soluble active agent and/or the water-soluble active agent are released.
Wherein the weight ratio of the lipid, the photosensitizer and 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, and further preferably 80-90:8-10: 10-15.
The weight ratio of the total weight of the lipid, the photosensitizer and the material with long circulation characteristic to the fat-soluble active agent and/or the water-soluble active agent is 10-135:1-15, preferably 20-120:5-15, more preferably 50-100:8-15, and further preferably 80-100: 10-15.
In the present invention, the lipid is not particularly limited, and it may be any known lipid such as phospholipid, glycolipid, fatty acid, dialkyldimethylammonium amphoteric molecule, polyglycerol alkyl ether 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 (dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine or distearoyl phosphatidylethanolamine, etc.), phosphatidylserine (dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine or distearoyl phosphatidylserine, etc.), phosphatidic acid, phosphatidylglycerol (dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol or distearoyl phosphatidylglycerol, etc.), phosphatidylinositol (dilauroyl phosphatidylinositol, dimyristoyl phosphatidylinositol, distearoyl phosphatidylinositol, etc.), phosphatidylinositol, Dipalmitoylphosphatidyinositol or distearoylphosphatidylinositol), lysolecithin, sphingomyelin, hydrogenated phospholipid, and the like.
Examples of the above glycolipids include glyceroglycolipids, glycosphingolipids, sterols and the like.
Examples of the above-mentioned glyceroglycolipids include digalactosyldiglyceride (digalactosyldiglyceride, digalactosyldimyristoyl glyceride, digalactosyldipalmitoyl glyceride, digalactosyldistearoyl glyceride, etc.) or galactodiglyceride (digalactosyl glyceride, digalacitoyl glyceride, digalactosyl glyceride, etc.), etc.
Examples of the aforementioned glycosphingolipids include galactosylcerebroside, lactocerebroside, ganglioside and the like.
Examples of such sterols include cholesterol, carbamoyl cholesterol, cholesterol hemisuccinate, ergosterol, lanosterol, and the like.
In the present invention, the lipids may be used alone or in combination by combining two or more thereof.
In particular, the lipid is selected from dipalmitoylphosphatidylcholine, diacetyl glycerol phosphatidylcholine, egg yolk lecithin, cholesterol.
In the present invention, the material having a long circulation characteristic is a material capable of providing a characteristic that the liposome escapes from the reticuloendothelial system in vivo, and mainly refers to a material having a relatively large molecular weight and having a hydrophilic group or a hydrophilic group. The material can be coated on the surface of a carrier material in a staggered manner to form a dense conformation cloud to form a thicker steric barrier layer, so that the action of a reticuloendothelial system is blocked, or the solvation action of the carrier is enhanced due to the hydrophilicity of the material, so that the opsonization of the surface of the carrier is effectively prevented, the affinity of the material with macrophages of a mononuclear phagocyte system is reduced, and the liposome can stably exist in a circulatory system and has a prolonged half-life period. The material with long circulation properties may be, for example, pegylated phospholipids, gangliosides, non-ionic surfactants or polymers, etc.
In the present invention, the material having a long circulation property includes, but is not limited to, polyethylene glycol-hydrogenated soybean phosphatidylethanolamine having an average molecular weight of 2000 (HSPE-PEG2000), polyethylene glycol-hydrogenated soybean phosphatidylethanolamine having an average molecular weight of 5000 (HSPE-PEG5000), polyethylene glycol-distearoylphosphatidylethanolamine having an average molecular weight of 2000 (DSPE-PEG2000), polyethylene glycol-dipalmitoylphosphatidylethanolamine having an average molecular weight of 2000 (DPPE-PEG2000), and various molecular weight polyethylene glycol-modified phosphatidylethanolamine phospholipids, gangliosides, polyvinyl alcohol, polyvinylpyrrolidone, tween, poloxamer, and the like.
In particular, the material with long circulation properties is selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000.
In the present invention, the term "photosensitizer" refers to a substance capable of generating singlet oxygen under the stimulation of light waves, which can cause the lipid structure to change. 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 of the above compounds. The above-mentioned photosensitizer is preferably selected from methylene blue compounds.
The methylene blue compound is a compound of the following 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；
In particular, the methylene blue compound is 7- (ethyl (methyl) amino) -3- (ethylamino) -2-toluidine blue, which is shown as the following formula:
the light wave used in the present invention can be any of various light waves that can cause the photosensitizer to generate singlet oxygen1O2) Preferably near infrared light.
The wavelength range of the near-infrared light wave is 600-2The energy density is 100 to 600J/cm2The action time is 0.25-90 minutes. The generation source of the infrared light is generally divided into 3 types of thermal radiation infrared light source, gas discharge infrared light source and laser infrared light source. Specifically, the generation source of the infrared light may be a black body; electrifying the silicon carbide rod; a xenon lamp; a semiconductor laser; a carbon dioxide gas laser; and so on.
In an embodiment of the invention, the fat-soluble active agent and/or the water-soluble active agent is an anti-cancer agent. Other fat-soluble and/or water-soluble active agents include, but are not limited to, antibiotics, antifungal agents, immunosuppressive agents, antiviral agents, and the like.
In the present invention, the anti-cancer agent is selected from the group consisting of alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic antitumor antibiotics, topoisomerase inhibitors, monoclonal antibodies or fragments thereof, kinase inhibitors, antitumor enzymes and enzyme inhibitors, apoptosis-inducing agents, anti-hormones, retinoids, platinum-containing compounds, proteins, polypeptides and nucleic acid drugs.
In particular, the fat-soluble active agent and/or the water-soluble active agent is selected from cisplatin, carboplatin, nedaplatin, doxorubicin or paclitaxel.
In the invention, the particle size of the photostimulation-response liposome is 60-300 nm; further, the particle size is 60-250 nm; further, the particle size is 70-250 nm; further, the particle size is 80-200 nm; further, the particle size is 100-200 nm; further, the particle size is 100 to 150 nm.
Another aspect of the invention relates to a pharmaceutical composition comprising a photostimulation-responsive liposome according to the invention and a pharmaceutically acceptable carrier. The carrier is selected from liposome prepared from dipalmitoyl phosphatidylcholine, diacetylene glycerol phosphatidylcholine, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and the like.
A further aspect of the invention relates to the use of a photostimulation-responsive liposome or a pharmaceutical composition according to the invention for the preparation of an anti-tumor medicament.
Photostimulation-responsive liposomes of the present invention can be administered to a subject using any suitable route, for example, intravenous administration, intraarterial administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, 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 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 photostimulation-responsive liposomes of the invention.
The dosage of an active agent administered to a subject using the photostimulation-responsive liposomes of the invention can be readily determined by one skilled in the art. As is known in the art, the dosage of the active agent can be adjusted depending on the active agent contained in the carrier.
During and/or after administration of the photostimulation-responsive liposomes of the invention, light irradiation can be performed on the target site of the subject. In one embodiment of the invention, the photostimulation-responsive liposomes of the invention are delivered to a subject and the target site is illuminated with light as soon as possible after administration.
The invention has the beneficial effects that the invention is based on the photodynamic principle, and the photosensitizer is added in the existing liposome prescription as the photodynamic trigger group, so that the liposome has good photostimulation response characteristic. The medicine wrapped in the liposome can be quickly released at a target position by optical radiation triggering, so that the medicine concentration at the target position is improved, the treatment effect of the medicine is further enhanced, the systemic toxicity of the medicine is reduced, and the application prospect is good.
Examples
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
Abbreviations used in the following examples are as follows:
DPPC: dipalmitoyl phosphatidylcholine
DC8,9PC: diacetylene glycerol phosphatidylcholine
DSPE-PEG 2000: distearoylphosphatidylethanolamine-polyethylene glycol 2000
EEMI: 7- (Ethyl (methyl) amino) -3- (ethylamino) -2-toluidine blue
HEPES (high efficiency particulate air): hydroxyethyl piperazine ethanethiosulfonic acid buffer
EPC: egg yolk lecithin
Chol: cholesterol
Example 1: preparation of adriamycin photostimulation-response liposome
(1) Materials and dosage: (amount of 100ml liposomes, Table 1 below)
TABLE 1
(2) Preparation method
Weighing various lipid materials, materials with long-cycle characteristics and photosensitizer according to the dosage in the table 1, dissolving the lipid materials, the materials and the photosensitizer in 200ml of chloroform-methanol (4:1) solution, and rotationally evaporating at 50-60 ℃ to remove the organic solvent to obtain a dry film uniformly attached to the bottom of a glass bottle. The obtained film is hydrated for 20min at 50-60 ℃ by using 250ml of citric acid solution with the pH value of 4.0 to form a uniform liposome solution. Then, the mixture was extruded through a polycarbonate membrane with a pore diameter of 100nm, and the extrusion was repeated 5 times. The obtained liposome adopts sodium carbonate water solution (500mM) to adjust pH value of external water phase to 7.0-7.4. The obtained liposome and 270ml (adriamycin 1mg/ml) of adriamycin (Shenzhen Wanle drug industry, batch No. 20160905) aqueous solution are co-cultured for 1h at 37 ℃, and the photostimulation-response liposome carrying the adriamycin is obtained. And then storing for later use at 4-7 ℃.
Example 2: encapsulation efficiency assay for doxorubicin photostimulation-responsive liposomes
1ml of the doxorubicin photostimulation-responsive liposome prepared in example 1 was taken in a 1.5ml EP tube at 10000r/min and centrifuged for 10 min. Taking 0.2ml of supernatant, placing in a 10ml volumetric flask, breaking membrane with methanol solution to constant volume, injecting sample by High Performance Liquid Chromatography (HPLC) for analysis (C18 column, Agilent SB-C18, 4.6 × 150mm, 1.8 μm), methanol-acetonitrile-phosphoric acid buffer solution (25mM NH)4H2PO4-30mM H3PO4pH 5.0) (5:2:3) as mobile phase at a flow rate of 1.0 ml/min-1Detecting the wavelength of 233nm) to obtain the content (C1) of the adriamycin medicine loaded in the liposome of 0.202 mg; and taking 0.2ml of the non-centrifuged adriamycin light stimulation-response liposome sample into a 10ml volumetric flask, breaking a membrane by using a methanol solution, diluting the solution to a constant volume to a scale mark, and carrying out sample injection analysis by High Performance Liquid Chromatography (HPLC) to obtain 0.208mg of the total drug content (C2) of the adriamycin in the adriamycin light stimulation-response liposome suspension. The encapsulation efficiency (E) is C1/C2 × 100%. The results showed that the entrapment rate of doxorubicin photostimulation-responsive liposomes was 97.1%.
Example 3: in vitro photostimulation-response assay for doxorubicin photostimulation-response liposomes
0.5ml of each of the doxorubicin photostimulation-responsive liposomes prepared in example 1 was placed in a cellulose acetate dialysis bag (MWCO 8-14kDa) (BioVision, Inc., Lot. C413103001) and immersed in 20ml of HEPES solution under magnetic stirring at 60 r/min. By means of a laser (660nm,200mW/cm2) After irradiation for 10 minutes, 0.5ml of the release medium was removed and passed through a 0.22 μm filter and the drug content of the release medium was determined by HPLC (C18 column, Agilent SB-C18, 4.6X 150mM, 1.8 μm), methanol-acetonitrile-phosphate buffer (25mM NH-N-N), and the4H2PO4-30mM H3PO4pH 5.0) (5:2:3) as mobile phase at a flow rate of 1.0 ml/min-1Detection wavelength 233 nm). The samples not irradiated with light were used as controls and drug content was determined by HPLC in the same manner. Release% (% released) is total amount of released drug/total amount of entrapped drug × 100%.
After the adriamycin light stimulation-response liposome is subjected to light treatment, the drug release amount (81.2 +/-9.4%) is obviously higher than that of a control group without light (6.4 +/-1.6%). Therefore, the adriamycin light stimulation-response liposome has obvious light stimulation-response drug release characteristics in vitro.
Example 4: in vivo photostimulation-response assay for doxorubicin photostimulation-response liposomes
15 Kunming mice (Km laboratory animal technology Co., Ltd., Beijing Wittiulihua) were randomly divided into three groups (5 mice each).
Tail vein injection (0.5ml, 1mg) of doxorubicin injection (sefma, 10 mg/vial, lot 132550013) was performed on the first group (injection group). The second group was administered tail vein (0.5ml, 0.5mg) doxorubicin common liposomes (Shiyao Europe, 10ml:20mg, lot 691160402) (common liposomes group). The tail vein of the third group was injected (0.5ml, 0.5mg) with the adriamycin photostimulation-responsive liposome of example 1 of the present invention (photostimulation-responsive liposome group).
Immediately after administration, laser light (660nm, 200 mW/cm) was applied to the left foreleg of each mouse2) And (3) irradiating, wherein the right front leg is not irradiated as a self control, and the content of the adriamycin in the leg muscle of the mouse which is irradiated and not irradiated is respectively measured after the mouse is irradiated for 10 minutes. The measurement method is as follows.
C18 column (Agilent SB-C18, 4.6X 150mm, 1.8 μm), mobile phase methanol-acetonitrile-0.01 mol. L-1Ammonium dihydrogen phosphate-glacial acetic acid (50:22:28:0.6) at a flow rate of 1.0 ml/min-1Excitation wavelength 479 nm, emission wavelength 587nm, columnThe temperature was 30 ℃. Taking the orthopyristin as an internal standard substance, taking 0.5g of muscle tissue, adding 1mL of phosphate buffer solution (pH 7.4), homogenizing, adding the internal standard solution, adding chloroform-methanol (4:1) mixed solution for extraction, dissolving residues with methanol, filtering, and injecting samples.
The content is the amount of drug in the tissue/weight of the tissue.
The results are as follows: the content of adriamycin in the non-illuminated part of the injection group (2.6 +/-0.3 mu g/g), the common liposome group (2.1 +/-0.4 mu g/g) and the photostimulation-response liposome group (2.2 +/-0.4 mu g/g) is not obviously different; compared with the non-illuminated part, the drug content of the illuminated part of the injection group (2.6 +/-0.4 mu g/g) and the common liposome group (2.2 +/-0.4 mu g/g) has no significant difference, while the drug content of the illuminated part (4.9 +/-0.7 mu g/g) of the adriamycin light stimulation-response liposome group is increased by more than 1 time than that of the non-illuminated part (2.2 +/-0.4 mu g/g), thereby significantly improving the drug content of the illuminated part. Therefore, the adriamycin light stimulation-response liposome has obvious light stimulation-response medicine release characteristics in vivo.
Example 5: pharmacodynamic observation of doxorubicin photostimulation-response liposomes
Collecting MCF-7 breast cancer cells in logarithmic growth phase (Shanghai Meixuan Biotech Co., Ltd.), and diluting to 2 × 10 with 0.2ml MCF-7 breast cancer cell suspension per each cell (culture medium liquid)6mL) was inoculated to mice (60, BALB/c nude mice, beijing vindolizhihua laboratory animal technology ltd) with dorsal subcutaneous right axillary.
On the 10 th day after tumor cell inoculation, 50 mice with relatively uniform tumor sizes (randomly and uniformly divided into groups after being sorted according to tumor volume size) were selected and divided into five groups (10 in each group): control group, general injection group, general liposome group, photostimulation-response liposome group (light), and photostimulation-response liposome group (no light). Four groups were separately administered tail vein injection of a normal injection solution equivalent to 10mg/kg of doxorubicin, normal liposomes, and the doxorubicin photostimulation-responsive liposomes of example 1, except for the control group which was administered tail vein injection of physiological saline.
Immediately after injection, the laser (660nm, 200 mW/cm)210min) irradiation, mice of other groups were left untreated. Co-feedingThe medicine is applied three times every two days. Tumors of each group of mice were weighed, and tumor inhibition rates were calculated according to the formula, and the results are shown in table 2.
Tumor inhibition rate ═ 1-Ws/Wx). times.100%
Wherein Ws is the tumor mass of the treatment group; wx is the tumor mass of the normal saline group. (x. + -.s represents mean. + -. standard deviation; tumor volume. length. times. width/2)
TABLE 2 pharmacodynamic test results
The data show that the tumor inhibition effect is obviously enhanced after the adriamycin light stimulation-response liposome is combined with illumination. Therefore, the compound can be used for preparing anti-tumor drugs.
Industrial applicability
The photostimulation-response liposome can improve the targeting property and the effectiveness of the medicament, and provides the photostimulation-response liposome which is simple and convenient in preparation method and easy for industrial production. The photostimulation-responsive liposomes of the present invention can be practiced 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.
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| CN111494338A (en) * | 2020-04-13 | 2020-08-07 | 大连理工大学 | Ovalbumin nanocapsule with synergistic effect of chemotherapy and photodynamic therapy and preparation method and application thereof |
| CN111803446A (en) * | 2020-07-14 | 2020-10-23 | 南方医科大学 | Photosensitive liposome and application thereof |
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