CN115634200A - Water-insoluble drug liposome composition and preparation method and application thereof - Google Patents
Water-insoluble drug liposome composition and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a water-insoluble drug liposome composition and a preparation method and application thereof. A water-insoluble drug liposome composition is characterized in that the drug liposome is composed of water-insoluble drugs, phospholipid, cholesterol and sulfobutylbetacyclodextrin; the weight ratio of the water-insoluble drug to the phospholipid to the cholesterol to the sulfobutyl-beta-cyclodextrin is 1.0-10.0. The invention has the main advantages that the passive targeting effect due to uniform particle size is more obvious and more beneficial to the concentration on tumor cells, thereby improving the anti-tumor treatment effect, and meanwhile, the modified phospholipid has better biocompatibility due to the limitation of safety.
Description
Technical Field
The invention relates to a water-insoluble drug liposome composition, a preparation method and application thereof.
Background
The liposome is a vesicle with lipid bilayers, can be used for directional drug carriers, and belongs to a new dosage form of a targeted drug delivery system. The medicine powder or solution can be embedded in micro-particles with the diameter of micron or nanometer, and the micro-particles are similar to bilayer micro-vesicles with a biological membrane structure and have good biocompatibility.
Particles and foreign substances in human blood are gradually removed from the circulatory system by vascular filtering organs of the human body, such as the pancreas, lungs and liver, and generally, the particles in blood include red blood cells (about 8 μm in diameter), white blood cells (about 6-8 μm in diameter) and platelets (about 1-3 μm in diameter). The microcirculation of most organs and tissues allows these blood cells to pass freely. When small blood (clots) of size greater than 10-15 μm are present in the circulation, there is a risk of capillary occlusion or obstruction leading to ischemia or hypoxia and possibly tissue death. Therefore, injection of particles with a diameter greater than 10-15 μm into the circulation must be avoided. However, suspensions of particles with diameters less than 7-8 μm are relatively safe and can be used to deliver pharmacologically active substances in the form of liposomes and emulsions, nutritional agents and contrast agents.
The size of the particles in the body and their mode of delivery determine their biological behavior. The behavior of particles ranging in size from a few nanometers (nm) to 100nm in the human body: after gap injection, the mixture enters lymphatic capillaries and phagocytosis can be generated in lymph nodes; particles with a diameter of less than 2 μm after intravenous/intraarterial injection: it is rapidly cleared from the blood by the reticuloendothelial system (RES), also known as the Mononuclear Phagocyte System (MPS); particles with a diameter greater than 7 μm: after intravenous injection, these particles are trapped by the pulmonary capillaries and after intra-arterial injection, the first capillary bed reached and the inhaled particles are captured by alveolar macrophages.
Similarly, the liposome can be a targeted drug delivery system, on one hand, after the liposome without modified surface enters into human body, the liposome is usually easy to be phagocytized by reticuloendothelial system to activate the autoimmune function of the body, and the in vivo distribution of the encapsulated drug is changed, so that the drug is mainly accumulated in tissues and organs such as liver, spleen and the like, and the liposome with the particle size in nanometer range can be effectively accumulated in tumor sites due to the high permeability and retention effect (namely EPR effect) of the tumor, and the property can be called as passive targeting of the liposome; on the other hand, the surface of the liposome can be modified by special covalent or non-covalent special ligands, including antibodies, polypeptides, aptamers, glycosyl groups, small molecules and the like, and the efficient uptake of the liposome by specific target cells is mediated through ligand-receptor interaction, which is called active targeting and is often likened to 'biological missile'. The targeted drug delivery capability of the liposome can improve the therapeutic index of the drug, reduce the therapeutic dose of the drug and reduce the toxicity of the drug. The drug-loading mode of the liposome can be divided into two modes, namely passive drug-loading and active drug-loading, generally speaking, a passive targeting technology is adopted for hydrophobic drugs, and an active targeting technology is mostly adopted for water-soluble drugs.
The advantages of liposome such as targeting, low toxicity and high efficiency are increasingly recognized, and the release of liposome drugs is influenced by various factors including particle size, lipid membrane composition, internal water phase, drug loading mode and the like, and for general liposome carrying drugs passively, the influence on drug release is mainly influenced by particle size and encapsulation efficiency.
The liposome has the advantages that the problems of stability, low encapsulation efficiency, leakage and the like of the liposome caused by the materials (easy oxidation and hydrolysis) or the process (simultaneously, toxic organic solvents are selected in the liposome preparation process and incomplete removal of the process) or the composition (combination of different materials) of the liposome easily cause aggregation, fusion and drug permeation during storage, injection toxicity of the solvents, dosage dumping (dose doubling), poor targeting effect and other medical accidents, so in order to solve the problems, the prior art mainly focuses on improving the preparation methods such as air freeze drying, spray drying and the like, and simultaneously, toughness is increased by modifying film-forming materials, or certain excipient protective agents are added to improve the stability and the encapsulation efficiency and control the particle size.
CN200610028725.7A discloses a stable liposome composition, which is prepared from saturated phospholipid and cholesterol as membrane-forming lipid, vitamin E as antioxidant, sucrose as lyophilized excipient, and paclitaxel by thin film evaporation-freeze drying method.
CN201310376983.4 discloses docetaxel nanoparticles for injection and a preparation method thereof, and the docetaxel nanoparticles are prepared by adopting a freeze-drying method to reduce the residue of an organic solvent.
Adding thermosensitive adjuvants into liposome to obtain thermosensitive liposome, adding pH sensitive substance to obtain pH sensitive liposome, adding cation or anion to obtain cation liposome or anion liposome, or adding surfactant.
However, the above method still has problems that the freeze-drying period is long, the productivity is affected, the required investment cost is high, the requirements of the preparation process by the added stabilizer are high, the toxic organic solvent is affected, because it is difficult to precisely control the temperature of the product, the quality of the product is not uniform from batch to batch and from batch to batch, the appearance color, shape, encapsulation rate and the like of the product are affected, the rejection rate in the production process is high, and the liposome of a special material has problems in material safety and manufacturing process.
Meanwhile, due to the difference of physicochemical properties of different types of drugs, such as structure, solubility, stability and the like, the drugs have corresponding preparation technical requirements, and meanwhile, links such as technology, membrane materials, pilot scale amplification and the like need to be continuously improved, so that liposome materials and processes adopted by different drugs have great difference, and a method suitable for most drugs is difficult to form. Therefore, the liposome with different preparation processes and different principles needs to be evaluated by establishing corresponding quality standards, and currently, the quality evaluation of the liposome mainly comprises 8 indexes, the shape and the particle size (including dispersity), the entrapment rate, the drug-loading rate, the burst release and leakage rate, the in vitro release degree, the phospholipid oxidation degree, the organic solvent residue and the in vitro and in vivo function evaluation. However, in general, the existing liposome still has the defects of low entrapment efficiency, poor stability, complex preparation process and the like, the targeting property of which needs to be further improved.
Therefore, the research of a liposome which has the advantages of high efficiency, safety, stability, strong targeting property, good uniformity, uniform particle size distribution, stable and reliable quality and simple and convenient preparation process is always the focus and direction of the research of the liposome.
The pharmaceutical adjuvant cyclodextrin has a hollow hydrophobic three-dimensional chiral inner cavity, the structural characteristics of 'inner hydrophobicity and outer hydrophilicity' of the cyclodextrin can encapsulate various organic small molecules (substrates) with proper space sizes to form a non-covalent host-guest complex (inclusion compound), the most remarkable pharmaceutical effect of the cyclodextrin is to increase the water solubility of insoluble drugs and increase the stability of the drugs, the cyclodextrin and derivatives thereof are mainly used for paclitaxel and busulfan at present, CN100486645C, the composition is prepared by adding paclitaxel to the cyclodextrin and the pharmaceutical adjuvant, but the formed composition is a new cyclodextrin derivative with longer stabilization time under clinical use concentration, the safety is not clinically verified, and potential safety hazards exist. US2018318249A1/CN201880044721.9 and CN201810888451.1 adopt cyclodextrin and derivatives thereof to solubilize busulfan to obtain a certain effect, but the drug effect and pharmacokinetics are not changed, the solubility is basically equivalent to that of busulfan injection in clinic, the tolerance is improved, but a large amount of acetone and a large amount of cyclodextrin are used, so that the process is complex, and the repeatability is poor.
Paclitaxel and busulfan are both water-insoluble drugs and can be solved by adopting a passive targeting liposome, but the related researches of the current commercially available paclitaxel liposome and busulfan liposome show that the problems still exist, so that the improvement space is provided.
The taxol is a natural secondary metabolite separated and purified from the bark of a gymnosperm yew, and has good anti-tumor effect through clinical verification, and particularly has special effects on ovarian cancer, uterine cancer, breast cancer and the like with high incidence rate of cancer. Paclitaxel is the most popular anticancer drug in the international market in recent years, and is considered to be one of the most effective anticancer drugs in human for the next 20 years. However, paclitaxel is difficult to dissolve in water (the solubility in water is only 0.006 mg/mL), but can be dissolved by using organic solvents, such as ethanol, methanol, chloroform, DMA, petroleum ether and the like, the earliest paclitaxel adopts polyoxyethylene castor oil and absolute ethanol for solubilization, and a commercial paclitaxel injection is approved by the U.S. FDA in 1992 and is on the market, because the polyoxyethylene castor oil can promote the release of histamine in vivo, a human body generates anaphylaxis, the incidence rate of moderate anaphylaxis is as high as 50%, even the life is threatened, and the toxic and side effects of the paclitaxel, such as peripheral neurotoxicity, liver toxicity, influence on the antitumor effect and the like can be aggravated, so that the clinical application of the traditional paclitaxel injection is limited.
The paclitaxel liposome for injection is marketed in China in 2003, lecithin, cholesterol, threonine and glucose are used as auxiliary materials, a special oscillator is used for shaking for 5min for clinical use, the most common problem in the clinical use process is that only 5% of glucose injection can be used for dissolving and diluting, physiological saline or other solutions cannot be used for dissolving and diluting, and meanwhile, in order to avoid liposome aggregation, the oscillator is used for oscillating to carry out solution recombination, the clinical use is extremely inconvenient, and medical data waste is caused.
Busulfan, a dual-functional alkylating agent of the bis-methylsulphonate type, was first discovered in 1953. Busulfan is mainly suitable for proliferative diseases in blood and bone marrow, and has good therapeutic effect on chronic myelocytic leukemia, essential thrombocythemia, polycythemia vera, essential myelofibrosis, etc. However, the solubility of the busulfan is poor, particularly the water solubility is the worst, the busulfan is difficult to absorb after being orally taken, the dosage required by oral administration is high, and the individual difference of drug absorption is large, so that the dosage is difficult to accurately control, therefore, the busulfan product sold on the market is still mainly injection, and the busulfan injection on the market at present
The use of DMA/PEG solubilization has limited the widespread use of DMA due to its neurotoxicity, hepatotoxicity, reproductive toxicity.
EP 1089727B 1 adopts liposome technology to embed busulfan, and US7351427B2 liposome is added with Glutathione (GSH) or a compound for synthesizing a GSH precursor for reducing the hematologic toxicity of the busulfan liposome, but cosolvent such as chloroform, dichloromethane and the like is still adopted, so that the residue is high, the hematologic toxicity is caused, and the particle size distribution range of the formed liposome is wide between 50nm and 300nm, so that the in-vivo distribution is influenced.
At present, liposome technology and cyclodextrin solubilization technology are combined for research of drugs, related research is less, US20090196918A1 uses liposome research of hydrophobic lactone drugs in the presence of metal ions, CN102596178B uses liposome research of sulfobutyl ether cyclodextrin as an internal water phase, and the liposome research belongs to the type of active targeting liposomes. Although the products formed by the researches are still liposomes, fundamental changes are generated on certain indexes, such as more stable liposome quality, improved entrapment rate, improved stability in batches, smaller particle size distribution range, improved in vivo targeting effect and the like.
The simultaneous use of liposome technology and cyclodextrin technology for the study of paclitaxel and busulfan has not been found.
Therefore, aiming at the characteristics and the defects of the existing liposome and cyclodextrin technologies, the inventor combines the passive targeting liposome technology and the cyclodextrin wrapping technology for use and uses the liposome and the cyclodextrin wrapping technology for water-insoluble drugs of paclitaxel and busulfan, and the prepared unilocular liposome with uniform liposome particle size has the average particle size of 10-100nm, obviously improves the activity of tumor cells on the cell level, and achieves the effects of attenuation and synergy.
Disclosure of Invention
The invention provides a medicinal composition of an antitumor medicinal liposome with uniform particle size, small particle size and good medicinal effect and a preparation method thereof.
The purpose of the invention can be realized by the following technical scheme:
the invention specifically provides a medicinal composition of water-insoluble medicament liposome, which comprises a combination of water-insoluble medicament, phospholipid, cholesterol and sulfobutyl betacyclodextrin, wherein the water-insoluble medicament: 1.0-10.0 weight ratio of phospholipid, water insoluble drug: the weight ratio of cholesterol is 1: the weight ratio of the sulfobutyl beta cyclodextrin is 1.
As a preferable aspect of the present invention, the weight ratio of the water-insoluble drug, phospholipid, cholesterol, and sulfobutyl-betacyclodextrin is 1.0-10.0: 1.0 to 5.0, and more preferably: 1.0-3.0, 0.1-0.5, wherein the water-insoluble drug is paclitaxel or busulfan.
The invention also provides a preparation method of the water-insoluble drug liposome, which is characterized in that the water-insoluble drug liposome with the prescription amount, phospholipid, cholesterol, SBECD, water and organic solvent are placed in a high-pressure micro-jet device, circulated for 5-30min at 4-40 ℃ and 1-200MPa to obtain a uniform solution, the organic solvent for removal is removed at 25-40 ℃, filtered, added with water for injection to the prescription amount, subpackaged in penicillin bottles, placed in a freeze drier, rapidly cooled to-50 ℃ within 0.5-2 h and kept for 4-6 h for freezing, the primary drying temperature is-30 ℃, 24-48 h, the vacuum degree is 5-15 pa, the resolution drying temperature is 30 ℃, 6-8 h and the vacuum degree is 15pa, and nitrogen is introduced or not introduced to obtain the water-insoluble drug liposome, wherein the organic solvent is ethanol or acetone, and the water-insoluble drug is paclitaxel or busulfan.
The invention also provides a preparation method of the water-insoluble drug liposome, which can also be characterized in that the water-insoluble drug, phospholipid, cholesterol, SBECD, water and organic solvent with the prescription dose are placed in a high-pressure micro-jet device, circulated for 5-30min at 4-40 ℃ and 1-200MPa to obtain a uniform solution, filtered, subpackaged in penicillin bottles, placed in a freeze drier, rapidly cooled to-50 ℃ within 0.5-2 h and kept for 4-6 h for freezing, the primary drying temperature is-30 ℃, 24-48 h, the vacuum degree is 5-15 pa, the analytic drying temperature is 30 ℃, 6-8 h and the vacuum degree is 15pa, nitrogen is introduced or not introduced to obtain the water-insoluble drug liposome, wherein the organic solvent is ethanol or acetone, and the water-insoluble drug is paclitaxel or busulfan.
The liposome prepared by the invention has the entrapment rate of more than or equal to 95 percent and the average particle size of 20-100nm
The liposome is recombined by water for injection, 5% glucose injection and 0.9% sodium chloride injection to be used at clinical use concentration, and the stability time is longer.
The liposome can be used for treating breast cancer, lung cancer, blood tumor and other diseases.
Has the beneficial effects that:
the invention adopts common phospholipid with good biocompatibility and cholesterol to form liposome, SBECD and drug molecules are arranged in the center of the liposome, and the formed liposome has the characteristics of high entrapment rate, small particle size, uniform distribution and good stability. Compared with the traditional liposome, the liposome has the main advantages that the passive targeting effect due to uniform particle size is more obvious and more beneficial to the concentration on tumor cells, so that the anti-tumor treatment effect is improved, and meanwhile, the liposome has better biocompatibility due to the limitation of safety problems different from modified phospholipid.
Specifically, the liposome is characterized in that liposome materials can be favorably formed into the liposome, the dispersion and solubilization of SBECD on certain drug molecules are favorably realized, the using amount of an organic solvent is reduced, the organic solvent residue of the liposome is reduced, and the drug safety is improved.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are intended to better illustrate the present invention, but should not be construed as limiting the scope thereof.
Example 1: preparation of paclitaxel liposomes
Preparing paclitaxel liposome according to the following formula, placing paclitaxel, phospholipid, cholesterol, SBECD, water and an organic solvent of the formula amount in a high-pressure micro-jet device, circulating for 5-30min at 4-40 ℃ and 1-100MPa to obtain a uniform solution, removing the organic solvent at 25-40 ℃, filtering, adding water for injection to the formula amount, subpackaging the solution in a penicillin bottle, placing the solution in a freeze dryer, rapidly cooling to-50 ℃ within 0.5-2 h, keeping the temperature for 4-6 h for freezing, drying at-30 ℃ for one time, 24-48 h, vacuum degree of 5-15 pa, analyzing the drying temperature of 30 ℃, 6-8 h and vacuum degree of 15pa, and introducing nitrogen or not to obtain the paclitaxel liposome.
TABLE 1 paclitaxel liposome formulation
The encapsulation efficiency and the particle size distribution of the paclitaxel liposome are measured, and the results are as follows:
TABLE 2 mean particle size and encapsulation efficiency results for paclitaxel liposomes
| Prescription | Average particle diameter (nm) | Encapsulation efficiency (%) |
| Prescription 1 | 33.9 | 98.5 |
| Prescription 2 | 34.4 | 98.2 |
| Prescription 3 | 38.4 | 98.0 |
| Prescription 4 | 42.0 | 95.2 |
| Prescription 5 | 20.2 | 99.6 |
| Prescription 6 | 40.1 | 98.4 |
The recipe 1 and the recipe 2 were placed at 25 ℃. + -. 2 ℃ and 60% RH. + -. 10% RH, and measured for 6 months, and the contents of the main measurement indices, impurities, and particle diameters were measured, and the results thereof were as follows,
the method for measuring the encapsulation rate of the paclitaxel liposome comprises the following steps:
chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, methanol and water (3:1) are used as mobile phases, and the detection wavelength is 227nm. The number of theoretical plates is not less than 1000 calculated according to the peak of paclitaxel.
Preparation of a test solution: taking 1 bottle of the product, adding 7.5mL of 5% glucose injection, and mixing uniformly for 10 minutes under swirling flow to obtain an emulsion containing about 4mg of paclitaxel per 1 mL.
The determination method comprises the following steps: adding Sephadex G-50 (100-300 μ) fully swollen with water into a chromatographic column with inner diameter of 0.7cm to make gel layer about 21cm high, adding 100mL of water into the column in several times to balance the column, sucking 0.2mL of sample solution precisely, adding the sample solution onto the gel layer along the tube wall, washing the column wall with a small amount of water, 1mL each time, twice, allowing the sample to enter the gel column, and gently stirring the sample layer (about 1cm high) with a fine glass rod. Eluting with water, collecting eluate 15mL when the eluate is slightly turbid, shaking, precisely measuring 2mL, placing in 10mL measuring flask, adding methanol to dilute to scale, and shaking to obtain the final product E The solution of (1). Eluting the gel column with gradient ethanol of different concentrations (33% ethanol, 3mL,66% ethanol, and anhydrous ethanol added slowly along the tube wall), discarding 10mL of eluate, collecting the free paclitaxel fraction immediately, collecting 5mL of eluate as assay M F The solution of (1). According to the chromatographic method under the content measurement item, taking M E And M F The solutions were each 20. Mu.L, and each solution was injected into a liquid chromatograph, and the chromatogram was recorded. The encapsulation efficiency was calculated as the peak area according to the following formula:
in the formula: m E Peak area for paclitaxel encapsulated in liposomes
M F Peak area of unencapsulated free paclitaxel.
The content and impurity determination method comprises the following steps: the same as paclitaxel injection method in Chinese pharmacopoeia
Particle size and particle size distribution measurement method: taking a proper amount of finished liposome, diluting with water for injection to obtain a liposome containing paclitaxel: 0.5mg/mL, placed in a hundred T-90 nanometer particle size, and the particle size and particle size distribution are recorded.
TABLE 3 stability results
Example 2
Preparation of Busulfan liposome
The busulfan liposome is prepared according to the following formula, busulfan, phospholipid, cholesterol, SBECD, water and acetone in the formula amount are placed in a high-pressure micro-jet device, circulation is carried out for 5-30min at 4-40 ℃ and 1-100MPa to obtain a uniform solution, organic solvent for removal is removed at the temperature of 30 ℃, filtration is carried out, water for injection is added to the formula amount, the solution is filled into penicillin bottles, the penicillin bottles are placed in a freeze drier, the temperature is rapidly reduced to minus 50 ℃ within 0.5-2 h and kept for 4-6 h for freezing, the primary drying temperature is minus 30 ℃, 24-48 h, the vacuum degree is 5-15 pa, the analytic drying temperature is 30 ℃, 6-8 h and the vacuum degree is 15pa, and nitrogen gas is introduced or blocked, so that the busulfan liposome is obtained.
TABLE 4 Busulfan liposome formulation
The encapsulation efficiency and the particle size distribution of the busulfan liposome are measured, and the results are as follows:
TABLE 5 mean particle size and encapsulation efficiency results for busulfan liposomes
Determination of encapsulation rate of Busulfan liposome:
chromatographic conditions are as follows: chromatographic column Zorbax SB C 8 250X 4.6mm,5 μm, in acetonitrile: trifluoroacetic acid: water (65.
Preparation of a test solution: taking 1 bottle of the product, adding 7.5mL of 5% glucose injection, and mixing for 10 minutes in a vortex manner to obtain an emulsion containing about 4mg of busulfan in each 1 mL.
The determination method comprises the following steps: adding SephadexG-50 (100-300 μ) fully swollen with water into a chromatographic column with an inner diameter of 0.7cm to make the gel layer about 21cm high, adding 100mL of water into the column in several steps to balance the column, precisely sucking 0.2mL of a sample solution, adding the sample solution onto the gel layer along the tube wall, washing the chromatographic column wall with a small amount of water, 1mL each time twice, allowing the sample to enter the gel column, and gently stirring the sample layer (about 1cm high) with a fine glass rod. Eluting with water, collecting eluate 15mL when slight turbidity appears, shaking, precisely measuring 2mL, placing in 10mL measuring flask, adding acetone alcohol to dilute to scale, and shaking to obtain determination of encapsulated busulfan content M 1 The solution of (1). Eluting with acetone of different concentrations (33% acetone 3mL,66% acetone 3mL and acetone slowly and sequentially along the tube wall), discarding 10mL of eluate, collecting free busulfan part immediately, and collecting 5mL of free busulfan part as M 2 A solution of (A) M 1 And M 2 The solution is respectively added into a proper amount of sodium ethyldithiocarbamate for derivatization. According to the chromatographic method under the content measurement item, the derivative is takenM of (A) 1 And M 2 The solutions were each 50. Mu.L, and each solution was injected into a liquid chromatograph, and the chromatogram was recorded. The encapsulation efficiency was calculated as the peak area according to the following formula:
in the formula: m is a group of 1 Peak area for busulfan encapsulated in liposomes
M 2 Peak area of unencapsulated free busulfan.
Particle size and particle size distribution measurement method: taking a proper amount of finished liposome, diluting with water for injection to obtain a liposome containing busulfan: 0.5mg/mL, placed in the hundred T-90 nanometer particle size, and the particle size and particle size distribution are recorded.
Example 3
Stability testing after reconstitution of formula 2 and formula 7 samples, examples formula 2 and formula 7 samples were reconstituted diluted to 0.5mg/mL with 5% dextrose injection and the particle size of the samples was determined as shown in table 6, and the selected solvent was also 0.9% sodium chloride injection or water for injection.
TABLE 6
The results show that: the liposome using SBECD has longer in-vitro stability time, is in a uniform suspension state within 24h, and has no precipitation or aggregation phenomenon.
Example 4
The formula 2 and the in vitro pharmacodynamics in vitro antitumor activity are studied.
Paclitaxel liposome prepared by MTT method and paclitaxel injection
For human breast cancer cells (MCF-7, MDA-MB-435 s), human lung adenocarcinoma cells (A549), human small cell lung cancer (NCI-H446) and human non-small cell lung cancer (NCI-H460), human ovarian cancer cellsInhibitory Effect of cells (Ho-8910, bcap-37).
And (3) experimental design:
the paclitaxel liposome is dissolved by PBS and diluted to the following concentration of 200 mug/mL, 20 mug/mL, 2 mug/mL, 0.2 mug/mL, 0.02 mug/mL, 0.002 mug/mL and 0.0002 mug/mL, and is prepared for use; positive control paclitaxel injection
Diluted to the following concentration, 200. Mu.g/mL, 20. Mu.g/mL, 2. Mu.g/mL, 0.2. Mu.g/mLl, 0.02. Mu.g/mL, 0.002. Mu.g/mL, 0.0002. Mu.g/mL, and prepared for use.
Taking each cell in logarithmic phase, digesting with conventional pancreatin to obtain single cell suspension, and diluting with DMEM medium containing 10% newborn calf serum to 5 × 10 4 cells/mL, 100. Mu.L per well inoculated in a 96-well plate, 5% CO 2 And culturing at 37 deg.c for 24 hr. The serum-containing DMEM and 90 mu L of serum-free DMEM and 10 mu L of paclitaxel liposome solution with corresponding concentration are discarded, the same amount of PBS is added into a blank control group, and each group is provided with 6 multiple wells.
Detection method and frequency: continued to 5% CO after administration 2 Incubation was carried out in an incubator at 37 ℃ for 72 hours, 10. Mu.L of MTT (5 mg/mL) was added to each well, incubation was carried out for 4 hours, the medium was discarded, 100. Mu.L of DMSO was added to each well, shaking was carried out for 10min, the absorbance value of each well was measured at a wavelength of 570nm on a microplate reader, and the tumor cell inhibition was calculated according to the following formula:
cell inhibition (%) = (control OD value-drug OD value)/control OD value × 100%
The experiment was repeated three times, the mean was calculated, and the SPSS software calculated the IC50.
Reference substance
The IC50 was calculated in the same way and the results are as follows:
TABLE 7 IC50 values of paclitaxel liposomes on individual tumor cell lines
The IC50 values of paclitaxel liposomes on 8 cell lines calculated by SPSS are shown in Table 6. The paclitaxel liposome and Taxol of 8 cell strains are both less than 10 mug/mL, and both have the effect of inhibiting the proliferation of tumor cells, compared with Taxol liposome, the inhibition proliferation effect of MCF-7, A549 and Bcap-37 tumor strains is better than that of paclitaxel injection, which shows that the activity of paclitaxel on some tumor cells is improved after the liposome is improved, and the possible reason is that the concentration of paclitaxel in some tumor tissues is increased due to the targeting effect of the liposome, so that the drug effect is improved.
Claims (7)
1. A water-insoluble medicinal liposome composition is characterized by mainly comprising water-insoluble medicaments, phospholipid, cholesterol and sulfobutylbetacyclodextrin.
2. The water-insoluble pharmaceutical liposome composition of claim 1, wherein the ratio of water-insoluble drug: 1.0-10.0 weight ratio of phospholipid, water insoluble drug: the weight ratio of cholesterol is 1: the weight ratio of the sulfobutyl beta cyclodextrin is 1; preferably, the weight ratio of the water-insoluble drug, the phospholipid, the cholesterol and the sulfobutyl-beta-cyclodextrin is 1:1.0 to 5.0, more preferably: 1.0-3.0.
3. The water-insoluble pharmaceutical liposome composition of claim 1, wherein the water-insoluble drug is paclitaxel or busulfan.
4. The water-insoluble pharmaceutical liposome composition of claim 1, wherein the liposome has a particle size of 20-100nm, preferably 20-50nm.
5. The water-insoluble pharmaceutical liposome composition of any one of claims 1 to 4, wherein the composition is prepared by the following method: placing the water-insoluble drug, phospholipid, cholesterol, SBECD, water and organic solvent in the formula amount in a high-pressure micro-jet device, circulating for 5-30min at 4-40 ℃ and 1-200MPa to obtain a uniform solution, filtering, subpackaging in a penicillin bottle, placing in a freeze dryer, rapidly cooling to-50 ℃ within 0.5-2 h, keeping for 4-6 h for freezing, performing primary drying at-30 ℃, 24-48 h, vacuum degree of 5-15 pa, resolution drying at 30 ℃, 6-8 h and vacuum degree of 15pa, and introducing nitrogen or stopping to obtain the water-insoluble drug liposome, wherein the organic solvent is ethanol or acetone, and the water-insoluble drug is taxol or busulfan.
6. The preparation method according to claim 5, characterized in that the prescribed amount of water-insoluble drugs, phospholipids, cholesterol, SBECD, water and organic solvents are placed in a high-pressure micro-jet device, the mixture is circulated for 5-30min at 4-40 ℃ and 1-200MPa to obtain a uniform solution, the organic solvents for removal are removed at 25-40 ℃, the uniform solution is filtered, the water for injection is added to the prescribed amount, the mixture is placed in a vial, the vial is placed in a freeze drier, the temperature is rapidly reduced to-50 ℃ within 0.5-2 h and kept for 4-6 h for freezing, the primary drying temperature is-30 ℃, 24-48 h, the vacuum degree is 5-15 pa, the resolution drying temperature is 30 ℃, 6-8 h and the vacuum degree is 15pa, nitrogen is introduced or not communicated, and the water-insoluble drug liposome is obtained by pressing, wherein the organic solvents are ethanol or acetone, and the water-insoluble drugs are paclitaxel or busulfan.
7. Use of the water-insoluble pharmaceutical liposome composition of any one of claims 1-4 for the preparation of a medicament for the treatment of breast cancer, lung cancer, hematological neoplasms.
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