JP2012522730A - Polysaccharide liposomes, their preparation and use - Google Patents

Abstract

  Polysaccharide liposomes, more particularly lentinan liposomes, methods for their preparation, pharmaceutical compositions comprising liposomes, and the use of liposomes for the production of anti-tumor or immunopreparations are disclosed. Liposomes are prepared from raw materials including polysaccharides, phospholipids, cholesterol, surfactants and polyethylene glycols.

Description

The present invention relates to polysaccharide liposomes, methods for preparing polysaccharide liposomes and uses of polysaccharide liposomes, and more particularly to lentinan liposomes.

BACKGROUND OF THE INVENTION Polysaccharides are widely present in nature. Polysaccharides are an important part of living organisms and are associated with many physiological functions necessary to maintain life. Polysaccharides have special roles in situations such as antitumor effects, antiinfective effects, vascular heart damage, sugar metabolism, and anti-aging effects, and can also improve the body's immunity.

  However, in the actual clinical field, polysaccharides can hardly achieve the desired pharmacological activity intended. This is mainly because the polysaccharide drug is metabolized before reaching the affected lesion in vivo and does not target the lesion. Thus, serious problems that require technically urgent solutions lie in how to minimize the metabolism of polysaccharide drugs before reaching the lesion and how to target the lesion to obtain the desired clinical effect.

  Liposome delivery systems draw many benefits due to the good effects shown in the clinical field. However, the preparation method, particularly the preparation method of polysaccharide liposomes having a high encapsulation rate, has a common problem. It is very difficult to obtain polysaccharide liposomes having a high encapsulation rate and a small particle size.

Summary of the Invention

  As a result of repeated studies, the present inventor obtained a liposome encapsulating a polysaccharide drug and a preparation method thereof. Furthermore, the present inventors have surprisingly found that the polysaccharide liposomes of the present invention have a high encapsulation rate and a small particle size.

  Thus, the present invention provides liposomes prepared from starting materials comprising polysaccharides, phospholipids, cholesterol, surfactants and polyethylene glycol (PEG).

  The present invention also provides a. Dissolving the starting material comprising phospholipid, cholesterol, surfactant and polyethylene glycol in an organic solvent to obtain an oil phase mixture; b. Dissolving a starting material comprising a polysaccharide and a surfactant in water or an aqueous solution to obtain an aqueous phase mixture; c. Adding the aqueous phase mixture to the oil phase mixture to emulsify; d. Removing the solvent by evaporation under a protective gas decompressed at a constant temperature; e. And a step of adding water or an aqueous solution and then leaving the product in a water bath (incubating).

  Furthermore, this invention provides the composition containing the liposome mentioned above.

  Furthermore, the present invention provides the use of a composition comprising a liposome or a liposome for the manufacture of an anti-tumor drug or a drug for limiting the immune function of a human or animal.

  The polysaccharide liposome of the present invention can be controlled to a uniform size distribution with a particle size of 1 μm or less. The polysaccharide liposome of the present invention has an encapsulation rate of 65% or more. The polysaccharide liposome of the present invention exhibits a greatly improved pharmacological activity in vivo after administration as compared to a polysaccharide not encapsulated in the liposome.

1 is a graph showing the particle size distribution of the liposome of the present invention in Example 1. FIG. FIG. 2 is a transmission electron microscope image of the liposome of the present invention in Example 1.

DETAILED DESCRIPTION OF THE INVENTION The compounds, compositions, methods, and reagents disclosed in this application are not limited to those specifically listed herein and may vary. The specific embodiments herein are provided for a better understanding of the present invention and are not intended to limit the scope of the present invention.

  The present invention will be described in detail with reference to specific embodiments.

  As used herein, the term “liposome” means an enclosed vesicle having a lipid bilayer structure encapsulating a pharmacologically active material. The liposome may be a single-layer liposome or a multi-layer liposome. The liposome of the present invention is a liposome having a modified surface.

  The polysaccharide of the present invention comprises lentinan, astragalan, Tremella polysaccharide, Tremella fuciformis, panaxan, ganoderan, umbellatus polysaccharide. It may be selected from a group. Preferably, the polysaccharide is lentinan or astragalin. Most preferably, the polysaccharide is lentinan.

  The term “phospholipid” as used herein refers to soybean phosphatide, lecithin, hydrogenated soybean phosphatide, hydrogenated lecithin, phosphatidylethanolamine (phosphatidyl). It may be selected from the group consisting of ethanolamine), distearoyl phosphatidylcholine, PEGylated phospholipid, and mixtures thereof.

  The polyethylene glycol of the present invention may be selected from the group consisting of PEG 800-5000, phospholipids-modified polyethylene glycol derivatives and mixtures thereof. Preferably, the polyethylene glycol is PEG 1000-2400. More preferably, the polyethylene glycol is PEG2000.

  The surfactant of the present invention may be selected from the group consisting of deoxycholates, poloxamers, polysorbate 80 (Tween-80) and mixtures thereof. Preferably, the surfactant is a deoxycholate such as sodium deoxycholate and potassium deoxycholate.

  Starting materials for preparing the liposomes of the present invention may further include those suitable for preparing liposomes. In one embodiment of the invention, for example, the starting material further comprises an antioxidant to improve the storage stability of the liposomes. In another embodiment of the liposomes of the present invention, the starting material further comprises a protective agent for lyophilization so that the liposome can be formulated into a lyophilized powder injection.

  The antioxidant of the present invention may be any of pharmaceutically acceptable antioxidants such as vitamin E, vitamin C, cysteine, methionine, sodium bisulfite and mixtures thereof.

  Protecting agents for lyophilization according to the present invention include pharmaceutically acceptable lyophilizing agents such as mannitol, trehalose, sucrose, glycine and mixtures thereof. Any of these may be sufficient.

  In one embodiment of the invention, the starting material includes the following ratios of components. 1 part lentinan, 30-100 parts phospholipid and 2-20 parts cholesterol (1 part lentinan, 30-100 parts phospholipid, 2-20 parts cholesterol); preferably 1 part lentinan, 40 ~ 90 parts phospholipid and 5-15 parts cholesterol; more preferably 1 part lentinan, 50-70 parts phospholipid, 5-15 parts cholesterol.

  Unless otherwise specified, “parts” as used herein are parts by weight.

  In one embodiment of the invention, the starting material comprises 3 to 12 parts surfactant. In another embodiment of the invention, the starting material comprises 1-8 parts polyethylene glycol.

  In yet another embodiment of the invention, the starting material comprises an appropriate amount of an antioxidant that can be readily determined according to standard knowledge of those skilled in the art. For example, 1 to 8 parts of antioxidant can be used.

  The liposomes of the present invention can be present in any formulation suitable for administration to a subject. For example, the liposomes of the present invention can be formulated into injections such as injection solutions or powder injections, or oral agents such as tablets, capsules or granules. Preferably, the liposome of the present invention is in the form of an injection solution or a powder injection.

  The liposome of the present invention is obtained by a method comprising the steps a to e.

  When the liposome of the present invention is formulated into a powder injection, the method for producing the liposome of the present invention further comprises a step f in which the solvent obtained from step e is separated and freeze-dried.

  In step a, the solvent used may be any organic solvent suitable for dissolving the starting material such as chloroform, chloroform-methanol or diethyl ether. The organic solvent is used in such an amount that the starting material can be dissolved. The amount depends on the raw material and is generally easily determined according to standard knowledge by those skilled in the art.

  In step b, the aqueous solution used is a buffer such as phosphate buffer, citrate buffer, acetate buffer, sodium bicarbonate buffer or tartrate buffer, or saline. It may be a sodium chloride solution. The water or aqueous solution used in step b is used in such an amount that the starting material can be dissolved. The amount depends on the raw material and is generally easily determined according to standard knowledge by those skilled in the art.

  In step c, emulsification of the preparation method of the invention can be achieved by sonication or operation at high pressure. The emulsification is generally carried out at a low temperature such as 10 ° C. or less. The emulsification time depends on the particle size, but can be appropriately determined by those skilled in the art using known methods. For example, the emulsification time may be 3 to 10 minutes. Preferably, the ultrasonic treatment of the present invention is preferably ultrasonic batch treatment, and the emulsification at high pressure is preferably cyclic emulsification at high pressure.

  In step d of the preparation method of the present invention, the protective gas may be an inert gas containing nitrogen.

  In step d of the preparation method of the present invention, the rotary evaporator may be carried out at a suitable temperature, and is preferably carried out at a constant temperature in the range of 35-45 ° C.

  In step e of the preparation method of the invention, the standing is preferably carried out in a water bath at a temperature in the range of 35-50 ° C, more preferably 40 ° C.

  In step e of the preparation method of the present invention, the standing is preferably performed for 1 to 2 hours.

  In step e of the preparation method of the present invention, the aqueous solution is a sodium chloride solution or a phosphate buffer, a citrate buffer, an acetate buffer, a sodium bicarbonate buffer, a tartrate buffer, or the like. A simple buffer may be used.

  Preferably, the pH value of the reaction environment in step b is different from step e. More preferably, the acidity or basicity of the reaction environment in step b is opposite to step e. The opposite acidity or basicity means that the reaction environment in steps b and e is acidic and the other is basic. Particularly preferably, the reaction environment in step b is basic and the reaction environment in step e is acidic.

  In one embodiment of the liposome of the present invention, the starting material in step a contains an antioxidant.

  In another embodiment of the liposome of the present invention, the aqueous solution in step e contains a protective agent for lyophilization. Protective agents for lyophilization are readily determined according to standard knowledge by those skilled in the art.

  The polysaccharide liposomes of the present invention are specifically targeted to specific tissues or cells such as liver, bone, spinal cord, tumor and inflammatory tissue, and prolong the circulation.

  The polysaccharide liposome of the present invention is for treating tumors such as liver, ovary, intestine, lung, breast, prostate, pancreas, kidney, stomach, endometrium, esophagus, head, neck, malignant pleural effusion, ascites tumor and the like. It can be used as an adjunct therapy or as an adjunct therapy for tumors. The polysaccharide liposome of the present invention also has an immunomodulatory effect. For example, it can be used to improve immunity in patients suffering from pneumonia, rheumatoid arthritis or AIDS. Compared to the case where the polysaccharide is administered as it is, the polysaccharide exhibits a greatly improved efficacy when administered in a form encapsulated in liposomes.

  The polysaccharide liposome of the present invention has a high encapsulation rate. The encapsulation rate of the polysaccharide liposome of the present invention is 65% or more. The encapsulation rate is preferably 70% or more, and more preferably 80% or more. In addition to high encapsulation rates, the polysaccharide liposomes of the present invention have a small particle size. The particle diameter of the liposome is about 1 μm or less, preferably 100 to 500 nm or less, and more preferably 140 to 300 nm.

  The encapsulation rate of the liposome here means the ratio of the amount of polysaccharide encapsulated in the liposome to the total amount of polysaccharide in the starting material for producing the liposome, and the ratio is expressed in weight percent.

A pharmaceutical composition is prepared from the liposomes of the present invention and combined with the following pharmaceutically active ingredients.
Cytotoxic agents such as bleomycin, doxorubicin, mitomycin, carmustin, carboplatin, cisplatin, iproplatin, cyclophosphamide, gemcitabine, gemcitabine (Gemcitabine), Docetaxel (Docetaxel), Etoposide (Etoposide), Fluorouracil (Melphalan), Methotrexate (Methotrexate), Vinorelbine (Vinorelbine), Vincristine (Pacliel), etc. Drugs and aminoglutethinmide, anastrozole, bicalutamide, irinotecan, rituximab, tamoxifen, trastuzumab What kind of hormones or anti-hormone,
Anti-inflammatory agents, such as Ganciclovir, Ribavirin, Vidarabine, Famciclovir, Lamivudine, Abacavir, Nevirapine, Indinavir, etc. Anti-viral agents such as Aspirin, Ibuprofen, Indometacin, Piroxicam, Nimesulide, Glucosamine, and the like, and
An immunomodulatory agent, such as an interferon agent or an interleukin agent.

  The liposome composition of the present invention comprises a filler, an adsorbent, a humectant, an adhesive, a lubricant, a colorant, a fragrance, a stabilizer, a preservative, a buffer, a disintegrant, a coating material, a diffusion agent, and a suspending agent. It can also be used in combination with a pharmaceutically acceptable carrier, such as pharmaceutical additives and adjuvants such as

  The polysaccharide liposomes or compositions of the present invention can be delivered to a target area of interest by various routes. For example, liposomes or compositions have been targeted and improved in situ to be processed in a suitable form via oral administration or injection, such as intravenous injection, intramuscular injection, intraperitoneal injection and subcutaneous injection. An immunomodulatory effect or an antitumor effect is obtained.

  The liposomes of the invention can also be administered in combination with one or more other pharmaceutically active agents, eg, as described above, to obtain a therapeutic effect.

  Embodiments of the present invention are further described with reference to the following examples. The compounds and agents used in this example are either commercially available or can be prepared by those skilled in the art using standard methods known in the art, and the devices used in this experiment are also commercially available. Is.

Example 1
Formulation of starting material for liposome 1

Preparation of liposome 1:
8.0 g soybean phospholipid, 1.0 g cholesterol, 0.2 g PEG2000, and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.9 g of sodium deoxycholate were added to 300 ml of water for injection and an appropriate amount of 0.1 mol / L NaOH solution was added until the lentinan was dissolved. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an oil phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice-water bath, followed by batch emulsification ultrasound for 5 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. 200 ml of 0.9% NaCl solution was added and then the resulting mixture was left at 40 ° C. for 1 hour to obtain a solution of liposome 1. The liposome solution was sterilized through a Millipore filter before being divided into injections of 100 aliquots of lentinan liposomes.

Evaluation of liposome 1:
(1) Encapsulation rate 0.2 ml of liposome 1 was added to a 10 ml tapered centrifuge tube, and 0.2 ml of protein (10 mg / ml) was added. They were shaken uniformly and left for 3 minutes, after which 0.05 mol / L NaOH solution was added so that the total volume reached 5 ml. The resulting mixture was centrifuged at 3000 r / min for 30 minutes at room temperature. 2 ml of supernatant was removed and 4 ml of 0.2% anthrone sulfate solution (prepared from 0.2 g anthrone and 100 ml concentrated sulfuric acid) was added. The resulting mixture was shaken uniformly, heated in a boiling water bath for 6 minutes, cooled in an ice water bath for 2 minutes, allowed to stand at room temperature for 10 minutes, and then the absorbance was measured. In addition, as a target group, an appropriate amount of lentinan dried with P ₂ O ₅ so as to have a constant weight is accurately weighed and dissolved in a small amount of 0.5 mol / L NaOH solution by grinding, Dilution with water made a solution with a concentration of 20 μg / ml. Absorbance was determined as described above. The amount of lentinan that was not encapsulated in the liposomes was calculated. The encapsulation rate was calculated by the following formula.
Encapsulation rate (%) = (1−amount of lentinan not encapsulated / total amount of lentinan) × 100%
The lentinan encapsulation rate of the sample was determined to be 82.8%.

(2) Particle size and microscopic morphology Liposomes 1 have a particle size in the range of 200-400 nm and 316 nm by using a DELSA 440S laser particle size analyzer (Beckman, USA) (FIG. 1). It was determined to have an average diameter of Liposome 1 was observed to have a spherical appearance and a clearly visible contour under a Tecnai G212 transmission electron microscope, FEI, Holland (FIG. 2).

Example 2
Liposome 2 starting material formulation:

Preparation:
8.0 g soybean phospholipid, 1.0 g cholesterol, 0.2 g PEG2000 and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.9 g of sodium deoxycholate were added to 300 ml of water for injection and an appropriate amount of 0.1 mol / L NaOH solution was added until the lentinan was dissolved. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an oil phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice-water bath, followed by batch emulsification ultrasound for 5 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. The pH is 6.0-6.5 and 2% mannitol in 200 ml phospholipid buffer solution was added, after which the resulting mixture was left at 40 ° C. for 1 hour to obtain a solution of liposome 2 .
The liposome solution was sterilized through a Millipore filter and pre-frozen at -50 ° C for 2 hours before being divided into 100 aliquots. The temperature was gradually increased by −6 ° C. over 8 hours and then gradually increased to 30 ° C. over 12 hours to obtain a light yellow mass, that is, a powder injection of liposome 2.

  After reconstituting the produced powder injection, according to the method of Example 1, it was determined that liposome 2 had an encapsulation rate of 84.3% and an average particle size of 318 nm. Liposome 2 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 3
Liposome 3 starting material formulation:

Preparation:
6.1 g soybean phospholipid, 0.8 g cholesterol, 0.2 g PEG1000 and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.5 g of sodium deoxycholate were added to 300 ml of water for injection and an appropriate amount of 0.1 mol / L NaOH solution was added until the lentinan was dissolved. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an oil phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in an ice water bath, followed by batch emulsification sonication for 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. The pH was 6.0-6.5 and 200 ml of phospholipid buffer solution was added, after which the resulting mixture was left at 40 ° C. for 1.5 hours to obtain a solution of liposome 3.
According to the method of Example 1, it was measured that the encapsulation rate of lentinan was 82.2% and the average particle size was 302 nm. Liposome 3 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 4
Liposome 4 starting material formulation:

Preparation:
5.2 g soy phospholipid, 0.7 g cholesterol, 0.2 g PEG2000 and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.7 g of sodium deoxycholate were added to 300 ml of water for injection and an appropriate amount of 0.1 mol / L NaOH solution was added until the lentinan was dissolved. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an oil phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in an ice water bath followed by batch emulsification ultrasound for 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. 200 ml of 2% mannitol was added and then the resulting mixture was left at 40 ° C. for 1.5 hours and sterilized through a Millipore filter to obtain a solution of liposome 4. A powder injection of liposome 4 was prepared according to the method of Example 2. After reconstitution, according to the method of Example 1, it was determined that the encapsulation rate of lentinan was 75.1% and the average particle size was 297 nm. Liposome 4 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 5
Formulation of starting material for liposome 5:

Preparation:
10.0 g of soybean phospholipid, 1.5 g of cholesterol and 0.3 g of PEG2000 were dissolved in 900 ml of chloroform with stirring to obtain an oil phase mixture. 0.2 g of astragalin and 1.0 g of sodium deoxycholate were dissolved in 300 ml of water for injection with stirring to obtain an aqueous phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice-water bath, followed by batch emulsification ultrasound for 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under nitrogen protection, yielding a pale yellow thick milky material. 200 ml of water for injection was added, after which the resulting mixture was left at 40 ° C. for 1.5 hours, sterilized through a Millipore filter and divided into 100 aliquots of astragalin injection. According to the method of Example 1, it was determined that the encapsulation rate of astragalin was 75.2% and the average particle size was 293 nm. Liposome 5 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 6
Liposome 6 starting material formulation:

Preparation:
10.0 g soybean phospholipid, 1.5 g cholesterol and 0.7 g PEG2000 were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.2 g of astragalin and 0.9 g of sodium deoxycholate were dissolved in 300 ml of water for injection with stirring to obtain an aqueous phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice-water bath, followed by batch emulsification ultrasound for 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. At pH 6.5, 2% mannitol in 200 ml phospholipid buffer solution was added and then the resulting mixture was left at 40 ° C. for 1.5 hours to obtain a solution of liposome 6.
A powder injection of liposome 6 was prepared according to the method of Example 2. After liposome reconstitution, according to the method of Example 1, it was determined that the encapsulation rate of astragalin was 84.5% and the average particle size was 308 nm. Liposome 6 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 7
Liposome 7 starting material formulation:

Preparation:
5.0 g soybean phospholipid, 0.6 g cholesterol, 0.2 g PEG2000 and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.4 g of polysorbate 80 were added to 300 ml of water for injection and an appropriate amount of 0.1 mol / L NaOH solution was added until the lentinan was dissolved. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an oil phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice-water bath, followed by batch emulsification ultrasound for 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. At pH 6.0-6.5, 200 ml of phospholipid buffer solution was added and then the resulting mixture was left at 40 ° C. for 1.5 hours to obtain a solution of liposome 7. According to the method of Example 1, it was measured that the encapsulation rate of lentinan was 80.2% and the average particle size was 293 nm. Liposome 7 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 8
Liposome 8 starting material formulation:

Preparation:
6.1 g soybean phospholipid, 1.5 g cholesterol, 0.2 g PEG2000 and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.4 g of polysorbate 80 were added to 300 ml of water for injection and added to an appropriate amount of 0.1 mol / L NaOH solution until the lentinan was dissolved. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an oil phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice-water bath, followed by batch emulsification ultrasound for 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed on a rotary evaporator at 40 ° C. under reduced pressure under protection of nitrogen, yielding a pale yellow thick milky substance. At pH 6.0-6.5, 2% mannitol in 200 ml phospholipid buffer solution is added, then the resulting mixture is left at 40 ° C. for 1.5 hours to obtain a solution of liposome 8 It was. A powder injection of liposome 8 was prepared according to the method of Example 2. After liposome reconstitution, according to the method of Example 1, it was determined that the encapsulation rate of lentinan was 82.1% and the average particle size was 305 nm. Liposome 8 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 9
Liposome 9 starting material formulation:

Preparation:
7.0 g soybean phospholipid, 0.8 g cholesterol, 0.6 g PEG1000 and 0.2 g vitamin E were dissolved in 900 ml chloroform with stirring to obtain an oil phase mixture. 0.1 g of injectable grade lentinan and 0.5 g of deoxysodium cholate are dissolved in 300 ml of water for injection with stirring and the appropriate amount of 0.1 mol / L NaOH solution until lentinan is dissolved. Was added. The solution was adjusted to pH 7.0-8.0 with 0.1 mol / L HCl solution to obtain an aqueous phase mixture. The aqueous phase mixture was slowly added to the oil phase mixture in the ice water bath, followed by batch emulsification ultrasound for about 6 minutes to obtain a homogeneous milky white W / O emulsion. The solvent was removed by rotary evaporation at 35 ° C. under reduced pressure under nitrogen protection, resulting in a pale yellow thick milky substance. At a pH of 6.0-6.5, 200 ml of phospholipid buffer solution was added, then the resulting mixture was left at 40 ° C. for 1.5 hours to obtain a solution of liposome 9. According to the method of Example 1, it was measured that the encapsulation rate of lentinan was 75.0% and the average particle size was 312 nm. Liposome 9 was observed to have a spherical outline and a clearly visible outline under a transmission electron microscope.

Example 10
The in vivo antitumor and immunomodulatory effects of the polysaccharide liposomes of the present invention were tested using lentinan liposomes (liposomes 9) prepared as in the above examples. The therapeutic effect was measured by evaluating the activity of microphages, natural killer cells (NK cells) and lymphocytes, and the tumor inhibition rate was also measured.

Mouse tumor model:
Healthy and mature male Kunming mice weighing 20-24 g (provided by the Animal Research Center of Tongji Medical University of Huazhong University of Technology) were used. Mice (Wuhan was kindly provided by teaching and research unit of the Medical School of the University) H ₂₂ hepatoma cells were inoculated, being at least 3 passages. Ascites was removed from the peritoneal cavity of the passaged mice and diluted with RPMI 1640 culture medium (Gibco) to a cell concentration of 2 × 10 ⁷ / ml. The dilution was injected subcutaneously under the right limb of the mouse at 0.2 ml / mouse once to inoculate the tumor. In model mice, tumors ranged from 1.13 g to 3.1 g with an average weight of 0.4 g.

Grouping and administration:
Mice were grouped into tumor model groups, cyclophosphamide groups, lentinan + cyclophosphamide groups, and lentinan liposomal cyclophosphamide groups depending on the drug being tested and its amount. The grouping information is specifically shown in Table 1 below.

In terms of hepatic macrophage phagocytic activity, splenic lymphocyte transformation activity and NK cell activity, animals were randomly grouped into groups 4 above 2 days after inoculation with H ₂₂ hepatoma cells and the drug was administered for 15 days. It was administered intraperitoneally. In the analysis of the tumor inhibition rate, animals four days after inoculation of H ₂₂ hepatoma cells, are grouped randomly into 4 groups described above chemicals were administered intraperitoneally 15 days.

(1) Analysis of macrophage activity Mice were sacrificed 24 hours after the last dose. Hepatocyte arrest was prepared as follows. Mice were soaked in 75% ethanol for a while, the liver was sterilized and removed, washed with D-Hank's solution for 3 minutes, and weighed. Livers from the same group of animals were placed in RPMI 1640 medium (Gibco) and homogenized. The final concentration of cells was adjusted to 2 × 10 ⁵ / ml.

The obtained hepatocyte suspension was placed in a 96-well microplate at 100 μl / well. The plate was cultured in a CO ₂ incubator for 24 hours, and then washed three times with RPMI 1640 medium (Gibco) to obtain a short molecular layer of adherent macrophages. Thereafter, 0.072% neutral red (Solarbio) in normal saline solution was added in an amount of 10 μ / well. The cells were cultured for 4 hours and the supernatant was discarded. Acetic acid ethanol solution (1: 1) was added in an amount of 150 μm / well. In order to completely dissolve the crystals, a 96-well microplate was shaken with a stirrer for 10 minutes. Thereafter, the absorbance was measured at each well at 492 nm using a plate reader (ZS-3, Xinfeng Mechanical-Electronic Technology Inc., Beijing, China), and the phagocytic activity (OD value) of macrophages was calculated. The results were analyzed by using SPSS statistical software.
Comparison between groups was performed by t-test. The results are shown in Table 2 below.

Note: ^** p <0.01 compared to tumor model group; ^## p <0.01 compared to lentinan + cyclophosphamide group

The results are shown as follows.
The difference in macrophage activity between the cyclophosphamide group and the tumor model group was small, suggesting that cyclophosphamide had a small effect on the phagocytic activity of liver macrophages in tumor-bearing mice.
The difference in macrophage activity between the lentinan + cyclophosphamide group and the cyclophosphamide group was small, suggesting that lentinan also has a small effect on the phagocytic activity of macrophages in tumor-bearing mice.
Macrophage phagocytic activity is significantly improved in lentinans in lentinan liposome + cyclophosphamide group compared to those with tumor model group or cyclophosphamide group (p <0.01), This suggests that lentinan liposomes greatly improve the phagocytic activity of macrophages in tumor-bearing mice.
Macrophage phagocytic activity of tumor-bearing mice in the lentinan liposome + cyclophosphamide group was also significantly improved compared to the same dose (p <0.01) lentinan + cyclophosphamide group.
Thus, lentinan in the form of liposomes has been shown to significantly improve liver macrophage activity.

(2) Natural killer cell analysis Mice were sacrificed 24 hours after the last dose. Hepatocyte arrest was prepared as follows. Mice were soaked in 75% ethanol for a while, the liver was sterilized and removed, washed with D-Hank's solution for 3 minutes, and weighed. Livers from the same group of animals were placed in RPMI 1640 medium (Gibco) and homogenized at a 1: 1 specific weight. The final concentration of cells was adjusted to 2 × 10 ⁶ / ml.

100 ml of effector cells (spleen cells prepared by the method described above) and 100 μl of RPMI 1640 culture solution (Gibco) are placed in Ewell of a 96-well microplate, and 100 μm of target cells (K562 cells, Tongji Medical University of Huazhong Medical University) (Provided by the research center) and 100 μm RPMI 1640 culture solution (Gibco) were placed in Twell, and 100 ml of effector cells and 100 ml of target cells were placed in E + Twell, three each. The plate was incubated for 24 hours at 37 ° C. in a CO ₂ incubator. 4 mg before completion of the culture, 5 mg / ml MTT (Sigma) was added in an amount of 20 μl / well, and the cells were further cultured for 4 hours. The supernatant was discarded and DMSO was added in an amount of 150 μl / well. In order to completely dissolve the crystals, a 96-well microplate was shaken with a stirrer for 10 minutes. Thereafter, DMSO was used as a blank solvent, and the absorbance of each cell was measured at 492 nm. The result was calculated as follows. NK cell kill rate = [1− (D _{E + T} −D _T )] × 100%. The results were analyzed by using SPSS statistical software.
Comparison between groups was performed by t-test. The results are shown in Table 3 below.

Note: ^* p <0.05 compared to cyclophosphamide group; ^# p <0.05 compared to lentinan + cyclophosphamide group

The results are shown as follows.
The killing rate of NK cells in the cyclophosphamide group is very small compared to the tumor model group (P <0.05), which indicates that cyclophosphamide is the killing rate of NK cells in tumor-bearing mice. It was suggested to have an inhibitory effect.
The NK cell killing rate of the lentinan + cyclophosphamide group is very small compared to that of the tumor model group and slightly increased statistically compared to that of the cyclophosphamide group, Lentinan in combination with cyclophosphamide was suggested to partially kill the toxicity and side effects of cyclophosphamide on NK cells of tumor-bearing mice.
The killing rate of NK cells in the lentinan liposome + cyclophosphamide group is significantly improved compared to either the tumor model group or the cyclophosphamide group (P <0.05), Lentinan combined with cyclophosphamide not only partially kills the toxicity and side effects of cyclophosphamide on NK cells in tumor-bearing mice, but also significantly increases the killing rate of NK cells in tumor-bearing mice It was suggested to do.
The killing rate of NK cells in the lentinan liposome + cyclophosphamide group is also significantly improved compared to the lentinan + cyclophosphamide group at the same dose (P <0.05), which is compared to lentinan. It was suggested that lentinan liposomes significantly increase the killing rate of NK cells in tumor-bearing mice.

(3) Lymphocyte proliferation analysis A suspension of spleen cells was prepared as described above. The suspension was placed in a 96-well microplate at 100 μl / well. Control wells and test wells were set for each drug tested. 100 μl (12.5 μg / ml) PHA-P (Sigma) was placed in each test well and 100 μl RPMI 1640 (Gibco) was placed in each control well. Plates were incubated for 24 hours at 37 ° C. in 5% CO ₂ . The next step is described in (2) above. The result was calculated as follows. Decomposition rate = (OD _{test well} −OD _{control well} ) / OD _{control well} × 100%. The results were analyzed by using SPSS statistical software.
Comparison between groups was performed by t-test. The results are shown in Table 4 below.

Note: ^* p <0.05 compared to cyclophosphamide group; ^# p <0.05 compared to lentinan + cyclophosphamide group

The results are shown as follows.
The growth rate of spleen lymphocytes in the cyclophosphamide group is very small compared to that of the tumor model group (P <0.05), which indicates that cyclophosphamide is a spleen lymphocyte in tumor-bearing mice. It was suggested that it has an inhibitory effect on the growth rate.
The growth rate of the lentinan + cyclophosphamide group has changed slightly from that of the tumor model group, but increased compared to that of the cyclophosphamide group, which is why it was combined with cyclophosphamide. Lentinan was suggested to reduce the toxicity and side effects of cyclophosphamide on lymphocytes from tumor-bearing mice.
The proliferation rate of lymphocytes in the lentinan liposome + cyclophosphamide group is significantly increased compared to either the tumor model group or the cyclophosphamide group (P <0.05). Lentinan in combination with cyclophosphamide not only partially kills the toxicity and side effects of cyclophosphamide on lymphocytes in tumor-bearing mice, but also significantly increases the proliferation rate of lymphocytes in tumor-bearing mice It was suggested to do.
The proliferation rate of lymphocytes in the lentinan liposome + cyclophosphamide group was also significantly increased compared to the lentinan + cyclophosphamide group at the same dose (P <0.05). These results suggest that lentinan liposomes significantly increase the proliferation rate of splenic lymphocytes in tumor-bearing mice.

(4) Analysis of tumor suppression rate Mice were sacrificed 24 hours after the last dose. Tumors were separated from the mice and weighed. And the tumor suppression rate was computed. The results were analyzed by using SPSS statistical software.
Comparison between groups was performed by t-test. The results are shown in Table 5 below.

Note: ^* p <0.05 compared to cyclophosphamide group

  According to the criteria for evaluation of therapeutic effects in the pharmacology guidelines for anti-tumor drugs, the ineffective standard is a tumor suppression rate of 40% or less, and the effective standard is a tumor suppression rate of 40% or more. P <0.05 (compared to the tumor model group).

The results are shown as follows.
The tumor suppression rate of the lentinan + cyclophosphamide group is increased compared to that of the cyclophosphamide group, and this indicates that lentinan improves the tumor suppressive effect of cyclophosphamide. It was suggested that is not statistically large.
The tumor inhibition rate of the lentinan liposome + cyclophosphamide group is significantly increased compared to the cyclophosphamide group (p <0.05), which indicates that lentinan liposomes are It was suggested that the tumor suppression effect is greatly improved.
Thus, lentinan liposomes have been shown to have greater anti-tumor activity than lentinan.

Claims (40)

  Liposomes, characterized in that the starting materials for the production of the liposomes include polysaccharides, phospholipids, cholesterol, surfactants and polyethylene glycol.   The liposome according to claim 1, wherein the polysaccharide is lentinan, astragalin, tremela polysaccharide, tremella fushihomis polysaccharide, panaxan, litchi, choreimaitake polysaccharide or a mixture thereof.   The liposome according to claim 1, wherein the polysaccharide is lentinan or astragalin.   The phospholipid is soybean phospholipid, lecithin, hydrogenated soybean phospholipid, hydrogenated lecithin, phosphatidylethanolamine, distearoylphosphatidylcholine, PEG-modified phospholipid, or a mixture thereof, The liposome according to any one of claims 1 to 3.   The liposome according to any one of claims 1 to 3, wherein the surfactant is deoxycholate, poloxamer, polysorbate 80, or a mixture thereof.   The liposome according to claim 5, wherein the surfactant is sodium deoxycholate or potassium deoxycholate.   The liposome according to any one of claims 1 to 3, wherein the polyethylene glycol is selected from the group consisting of PEG 800-5000, a phospholipid-modified polyethylene glycol derivative and a mixture thereof.   The liposome according to claim 7, wherein the polyethylene glycol is PEG1000-2400.   The liposome according to claim 8, wherein the polyethylene glycol is PEG2000.   The liposome according to any one of claims 1 to 3, wherein the starting material further comprises an antioxidant and / or a protective agent for lyophilization.   The starting material comprises 1 part polysaccharide, 30-100 parts phospholipid, 2-20 parts cholesterol, 3-12 parts surfactant and 1-8 parts polyethylene glycol, Item 4. The liposome according to any one of Items 1 to 3.   The liposome according to claim 11, wherein the starting material comprises 1 part by weight of lentinan, 40 to 90 parts by weight of phospholipid and 5 to 15 parts by weight of cholesterol.   12. The liposome according to claim 11, wherein the starting material comprises 1 part lentinan, 50-70 parts phospholipid, and 5-15 parts cholesterol.   The liposome according to claim 12, further comprising 1 to 8 parts of an antioxidant.   The liposome according to any one of claims 1 to 3, wherein the encapsulation rate of the liposome is 65% or more.   The liposome according to any one of claims 1 to 3, wherein the encapsulation rate of the liposome is 70% or more.   The liposome according to any one of claims 1 to 3, wherein the encapsulation rate of the liposome is 80% or more.   The liposome according to any one of claims 1 to 3, wherein the liposome has a particle diameter of 1 µm or less.   The liposome according to any one of claims 1 to 3, wherein the liposome has a particle diameter of 100 to 500 nm.   The liposome according to any one of claims 1 to 3, wherein the liposome has a particle diameter of 140 to 300 nm.   The liposome according to any one of claims 1 to 3, wherein the liposome is in the form of an injection solution or a powder injection.   A composition comprising the liposome according to any one of claims 1 to 21.   24. The composition of claim 22, wherein the composition further comprises a pharmaceutically acceptable carrier.   24. Composition according to claim 22 or 23, characterized in that it further comprises one or more anti-tumor or immunomodulatory active ingredients.   24. Composition according to claim 22 or 23, characterized in that the composition is in the form of an injection or a powder infusion. A method for preparing the liposome according to any one of claims 1 to 21, comprising:
a. Dissolving the starting material comprising phospholipid, cholesterol, surfactant and polyethylene glycol in an organic solvent to obtain an oil phase mixture;
b. Dissolving the starting material comprising a polysaccharide and a surfactant in water or an aqueous solution to obtain an aqueous phase mixture;
c. Adding the aqueous phase mixture to the oil phase mixture and emulsifying;
d. Removing the solvent by evaporation under a protective gas decompressed at a constant temperature;
e. Adding water or an aqueous solution and then leaving the product in a water bath.   f. 27. The method of claim 26, further comprising separating and lyophilizing the solution obtained from step e.   28. The method according to claim 26 or 27, wherein the organic solvent in step a is chloroform or chloroform-methanol.   28. A method according to claim 26 or 27, characterized in that the emulsification in step c is an ultrasonic batch treatment or circulating emulsification at high pressure.   28. Method according to claim 26 or 27, characterized in that the standing in the water bath in step e is carried out at a temperature in the range of 35-50 [deg.] C.   The method according to claim 30, characterized in that the standing in the water bath in step e is carried out at 40 ° C.   28. A method according to claim 26 or 27, characterized in that the pH value of the reaction environment in step b is different from the pH value of the reaction environment in step e.   28. Method according to claim 26 or 27, characterized in that the acidity or basicity of the reaction environment in step b is opposite to the acidity or basicity of the reaction environment in step e.   34. A method according to claim 33, characterized in that said leaving in step e is carried out for 1-2 hours.   28. A method according to claim 26 or 27, wherein the starting material in step a further comprises an antioxidant.   28. A method according to claim 26 or 27, wherein the aqueous solution in step e further comprises a protective agent to be lyophilized.   Use of the liposome according to any one of claims 1 to 21 for the manufacture of an anti-tumor or immunomodulator.   26. Use of a liposome according to any one of claims 22 to 25 for the manufacture of an antitumor or immunomodulatory agent.   A method for preparing a tumor treatment or immunity comprising the step of administering to the subject a liposome according to any one of claims 1 to 21.   26. A method for preparing a tumor treatment or immunity comprising the step of administering to a subject a liposome according to any one of claims 22-25.