CN104983684A - Oleanolic acid polycystic lipidosome and preparation method and application thereof - Google Patents
Oleanolic acid polycystic lipidosome and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides oleanolic acid polycystic lipidosome and a preparation method and application thereof. The method comprises the following steps: 1 weighing granulesten, cholesterol, olein, oleanolic acid and stearic acid by mass, and dissolving the ingredients into organic solvent, wherein oil phase is formed through the dissolution; 2 taking and adding a specified amount of oil phase into internal water phase, and using an emulsifying machine for conducting shearing and dispersing for forming primary emulsion; 3 taking and adding a specified amount of primary emulsion into external water phase, and conducting vortex oscillation for forming compound emulsion; 4 transferring the compound emulsion rapidly into a round-bottom flask, conducting rotary evaporation for removing the organic solvent, and obtaining the oleanolic acid polycystic lipidosome. According to the oleanolic acid polycystic lipidosome and the preparation method and application thereof, the oleanolic acid is loaded into a lipoid layer of polycystic lipidosome for preparing the oleanolic acid polycystic lipidosome (OA-MVLs), and it is expected to improve the bioavailability and achieve the purposes of releasing medicine slowly, reducing administration times and improving the adaptability of a patient.
Description
Technical field
The present invention relates to medical art, particularly relate to a kind of oleanolic acid multivesicular liposome, preparation method and application thereof.
Background technology
Oleanolic acid (Oleanolic acid, be called for short OA), have another name called oleanolic acid, another name (3BETA)-3-Hydroxyolean-12-en-28-oic acid, chemistry (3 β)-3-Hy-droxyolean-12-en-28-oic-acid by name is white tufted crystalline powder, odorless, tasteless, water insoluble, dissolve in methanol, ethanol, chloroform and acetone.OA belongs to pentacyclic triterpenoid, is mainly present in the plants such as Spica Prunellae, Fructus Forsythiae, Fructus oleae europaeae, Fructus Ligustri Lucidi and Fructus Chaenomelis with free or saponin form.1908, OA was separated in the leaf of oleaceae plant Oleaeuro-peae L by FB Powers first, and determined its structure by Ruzicka in nineteen forty-six.Hu'nan Inst. of Plarmaceutical Industry of China is in the chemistry to the anti-hepatitis effective ingredient of Herba Swertiae Mileensis in 1975 and pharmacological research, and be separated and identify OA monomer, after this researcheres have just carried out research extensively and profoundly to the pharmacological action of OA.OA protects the liver except having, antiinflammatory, antiviral, hypoglycemic, two-way immunomodulating, except antihypertensive biological thing activity, also there is the multiple pharmacological effect such as heart tonifying, arrhythmia, anticoagulant and anti-peroxidation, OA preparation is mainly used in treatment acute icteric and chronic poisoning type hepatitis clinically, and as anticancer ancillary drug.In addition, the features such as OA also relies on its high-efficiency low-toxicity, material is easily got become the focus for the treatment of and prevention of tumour research aspect, and its mechanism of anticancer action has almost run through each stage of tumor development.The related preparations mainly oleanolic acid tablet of current oleanolic acid clinically, but oleanolic acid is fat-soluble medicine, there is the shortcomings such as low at gastrointestinal tract dissolution, absorbability is poor, liver first-pass effect is obvious, its bioavailability is reduced greatly, therefore, the novel form researching and developing oleanolic acid is very necessary.
Summary of the invention
The object of the invention is to the defect solving the existence of above-mentioned prior art, the oleanolic acid multivesicular liposome providing a kind of bioavailability high, preparation method and application thereof.
A preparation method for oleanolic acid multivesicular liposome, comprises the following steps:
1) take soybean phospholipid, according to quantity, cholesterol, glycerol trioleate, oleanolic acid, stearic acid be dissolved in organic solvent, dissolve and form oil phase;
2), add in interior aqueous phase by the oil phase measuring ormal weight, shear dispersion with mulser and form colostrum;
3), join in outer aqueous phase by the colostrum measuring ormal weight, vortex vibration forms emulsion;
4), by emulsion be transferred to rapidly in round-bottomed flask, rotary evaporation removes organic solvent, obtains oleanolic acid multivesicular liposome.
Further, the preparation method of oleanolic acid multivesicular liposome as above, the mass ratio of described soybean phospholipid, cholesterol, glycerol trioleate, oleanolic acid is: 1: 0.57: 0.38: 0.2; The interpolation concentration of stearic acid in oil phase is: 1mg/ml.
Further, the preparation method of oleanolic acid multivesicular liposome as above, the volume ratio of described oil phase and interior aqueous phase is 1-1.5: 1, and the volume ratio of colostrum and outer aqueous phase is 0.3-0.5: 1.
Further, the preparation method of oleanolic acid multivesicular liposome as above, described interior aqueous phase by mass percentage, comprising:
Glucose 5%
Surplus: water;
Described outer aqueous phase by mass percentage, comprising:
Glucose 4%
Tween 80 2%
Polyvinyl alcohol 3%
Surplus: water.
Further, the preparation method of oleanolic acid multivesicular liposome as above, to be chloroform and ether be described organic solvent by volume mixes at 1: 1.
Further, the preparation method of oleanolic acid multivesicular liposome as above, in step 2, the shear rate of colostrum is 13500rpm, shear time is 150s.
Further, the preparation method of oleanolic acid multivesicular liposome as above, in step 3, the time of vortex vibration is 25s.
A kind of basis is the oleanolic acid multivesicular liposome for preparing of arbitrary preparation method as above.
The application of oleanolic acid multivesicular liposome as above in preparation treatment cancer therapy drug.
Application as above, described medicine is the medicine being used for the treatment of hepatocarcinoma.
Oleanolic acid is loaded in the class lipid layer of multivesicular liposome and prepares oleanolic acid multivesicular liposome (OA-MVLs) by the present invention, to improving bioavailability, reaches slow releasing medicine, reduces administration number of times, improves the object of patient's compliance.
Accompanying drawing explanation
Fig. 1 is OA-MVLs form (× 400) under an optical microscope;
Fig. 2 is the particle size distribution figure of OA-MVLs;
Fig. 3 is the preparation curve chart of OA-MVLs and free OA in release medium;
Fig. 4 A is that OA-MVLs acts on HepG2 model cell, after hatching 4h, and the nucleus microscope figure of DAPI blue-fluorescence labelling;
Fig. 4 B is that OA-MVLs acts on HepG2 model cell, after hatching 4h, and the OA-MVLs microscope figure of DHPE green fluorescent label;
Fig. 5 is each factor reciprocal action to the response surface design of envelop rate and average grain diameter influence and contour map; Fig. 5 A is X
1(mass ratio of C/PL) and X
2(medicine fat ratio) impact on envelop rate; Fig. 5 B is X
1(mass ratio of C/PL) and X
3(mass ratio of glycerol trioleate and phospholipid) impact on envelop rate; Fig. 5 C is X
2(medicine fat ratio) and X
3(mass ratio of glycerol trioleate and phospholipid) impact on envelop rate; Fig. 5 D is X
1(mass ratio of C/PL) and X
2(medicine fat ratio) impact on particle diameter; Fig. 5 E is X
2(medicine fat ratio) and X
3(mass ratio of glycerol trioleate and phospholipid) impact on particle diameter; Fig. 5 F is X
1(mass ratio of C/PL) and X
3(mass ratio of glycerol trioleate and phospholipid) impact on particle diameter;
Fig. 6 is the Cell Image Analyzer change microscope figure after variable concentrations OA-MVLs acts on HepG2 cell.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearly, below the technical scheme in the present invention is clearly and completely described.Obviously, described embodiment is the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1:
A preparation method for oleanolic acid multivesicular liposome, comprises the following steps:
1) take soybean phospholipid, according to quantity, cholesterol, glycerol trioleate, oleanolic acid, stearic acid be dissolved in organic solvent, dissolve and form oil phase; Wherein, the mass ratio of described soybean phospholipid, cholesterol, glycerol trioleate, oleanolic acid is: 1: 0.57: 0.38: 0.2; The interpolation concentration of stearic acid in oil phase is: 1mg/ml;
2) oil phase, measuring 1ml adds in 1ml in aqueous phase, with mulser shearing dispersion formation colostrum; Shear rate is 13500rpm, shear time is 150s;
3), the colostrum that measures 0.3ml joins in the outer aqueous phase of 1ml, and vortex vibration forms emulsion; The time of vortex vibration is 25s;
4), by emulsion be transferred to rapidly in round-bottomed flask, rotary evaporation removes organic solvent, obtains oleanolic acid multivesicular liposome.
Embodiment 2:
1) take soybean phospholipid, according to quantity, cholesterol, glycerol trioleate, oleanolic acid, stearic acid be dissolved in organic solvent, dissolve and form oil phase; Wherein, the mass ratio of described soybean phospholipid, cholesterol, glycerol trioleate, oleanolic acid is: 1: 0.57: 0.38: 0.2; The interpolation concentration of stearic acid in oil phase is: 1mg/ml;
2) oil phase, measuring 1.5ml adds in 1ml in aqueous phase, with mulser shearing dispersion formation colostrum; Shear rate is 13500rpm, shear time is 150s;
3), the colostrum that measures 0.5ml joins in the outer aqueous phase of 1ml, and vortex vibration forms emulsion; The time of vortex vibration is 25s;
4), by emulsion be transferred to rapidly in round-bottomed flask, rotary evaporation removes organic solvent, obtains oleanolic acid multivesicular liposome.
In above-described embodiment, described interior aqueous phase by mass percentage, comprising: 5% glucose, surplus: water.Described outer aqueous phase by mass percentage, comprising: 4% glucose, 2% tween 80,3% polyvinyl alcohol, surplus: water.To be chloroform and ether be described organic solvent by volume mixes at 1: 1.
Oleanolic acid (OA) belongs to pentacyclic triterpenoid, is mainly used in treatment acute icteric and chronic poisoning type hepatitis clinically.Current its related preparations mainly oleanolic acid tablet clinically, but oleanolic acid is fat-soluble medicine, there is the shortcomings such as low at gastrointestinal tract dissolution, absorbability is poor, liver first-pass effect is obvious, its bioavailability is reduced greatly.Therefore, oleanolic acid is loaded in the class lipid layer of multivesicular liposome and prepares oleanolic acid multivesicular liposome (OA-MVLs) by the present invention, to improving bioavailability, reaches slow releasing medicine, reduces administration number of times, improves the object of patient's compliance.
Below experimental technique of the present invention is sketched:
1, the present invention adopts multi-emulsion method to prepare OA-MVLs, and optimizes its prescription and preparation technology with center combination design.
2, the form of OA-MVLs, envelop rate, particle diameter and the drug release behavior in the PBS buffer of pH7.4 thereof is investigated.
3, adopt mtt assay and DAPI staining research OA-MVLs to the suppression of HepG2 cell and apoptosis-promoting effect, and adopt laser co-focusing to measure OA-MVLs enter born of the same parents' situation, measure the change of HepG2 intraor extracellular oleanolic acid concentration after administration.
4, SD rat is selected to be animal model, with oleanolic acid raw material medicine solution for contrast, by subcutaneous administrations, the slow release effect of research OA-MVLs in rat body.
Result:
1, the OA-MVLs form rule that this experiment is obtained, particle diameter meets normal distribution, and envelop rate is 82.3 ± 0.61%.
2, OA-MVLs is significantly not prominent in the PBS buffer of pH7.4 releases phenomenon, 120h cumulative release 80.56 ± 1.27%, shows good slow release and long-acting.Matching is carried out to the drug release profiles of OA-MVLs, meets Ritger-Peppas equation most, show that the release of OA-MVLs may be diffused as master with Fick.
3, OA-MVLs acts on HepG2 cell, energy antiproliferative effect, and can impel apoptosis; OA-MVLs is distributed on nucleus and cell membrane after entering born of the same parents.Along with OA-MVLs is to the increase of HepG2 cytosis time, in cell, OA concentration increases gradually, and EC change is little; Along with the increase of OA-MVLs concentration, the OA concentration of intraor extracellular also increases gradually.
4, compared with OA solution, the half-life of OA-MVLs obviously extends, t
1/2for (4.38 ± 4.34) VS (48.14 ± 27.67) h; AUC significantly increases, AUC
(0-t)for (5007.51 ± 1346.55) VS (24530.94 ± 1293.42) ng/mLh; Mean residence time significant prolongation, MRT is (6.30 ± 3.63) VS (56.84 ± 28.99) h, after injection 96h, still medicine can be detected in blood plasma.
Major experimental reagent
Soybean phospholipid (PC, Shanghai Taiwei Pharmaceutical Co., Ltd.)
Glycerol trioleate (TO, Aladdin chemistry Co.Ltd)
Cholesterol (Chol, Chengdu Ke Long chemical reagent factory)
Oleanolic acid (AO, source bio tech ltd, east, Mianyang)
Tween 80 (Chengdu Ke Long chemical reagent factory)
Stearic acid (Chemical Reagent Co., Ltd., Sinopharm Group)
Polyvinyl alcohol (PVA, Chengdu Ke Long chemical reagent factory)
All the other reagent are domestic analytical reagent.
The ratio of 1.1 phospholipid and cholesterol
Because cholesterol (Cholesterel, Chol) has the dual regulation to immobilized artificial membrane mobility, the percolation ratio of medicine can be reduced and become the important component of liposomal preparation.Test under the prerequisite keeping preparation technology and other components unchanged, its impact on OA-MVLs envelop rate is investigated by regulating the mass ratio (80: 20,80: 30,80: 40) of phospholipid and cholesterol, result shows, PC: Chol=80: 30 time, the envelop rate of medicine is the highest.
The ratio of 1.2 phospholipid and glycerol trioleate
Glycerol trioleate, as neutral lipid, there is no film forming ability, but is filled in the hydrophobic chamber in lipid bilayer, stablizes interconnection place, forms discontinuous drug solution vesicle.This experiment prepares OA-MVLs by adjusting the addition of glycerol trioleate, and measures envelop rate, and result shows that envelop rate is the highest when the mass values of phospholipid and glycerol trioleate is 80: 33.
1.3 medicine fat ratios
In the preparation process of OA-MVLs, when other condition is constant, change the medicine fat ratio addition of oleanolic acid (namely in oil phase), and measure envelop rate, result shows the increase along with medicine proportion, and the envelop rate of MVLs obviously raises, but increasing along with medicine addition, its dissolubility reduces, best when medicine fat ratio is 1: 10.
1.4 outer aqueous phases
Outer aqueous phase has larger impact to the outward appearance of OA-MVLs and stability, experiment is using polyvinyl alcohol (PVA), glucose (Glu) and tween 80 (Tween-80) as outer aqueous phase, different outer aqueous phases is prepared by the concentration ratio changing these three kinds of compositions, investigate its envelop rate, to obtaining outer watr-proportion preferably.Result shows, when polyvinyl alcohol: glucose: tween 80=3: the MVLs envelop rate obtained when 4: 2 is the highest.
1.5 stearic acid
According to reported in literature, preparation MVL adds bear electricity phospholipid often as film protective agent, to improve the electrostatic repulsion between phospholipid electric double layer, stops merging and the destruction of immobilized artificial membrane.But the phospholipid of bear electricity is expensive and not easily obtain.Studies have found that, add a small amount of oleic acid or stearic acid in oil phase, also can significantly improve the mouldability of MVL, the MVL size formed is even, significantly can reduce the generation of phospholipid fragment and eliminate the phenomenon of aggregate and precipitate.
Result shows, after adding stearic acid, the envelop rate of MVL raises to some extent, but stearic acid adds the excessive seepage of medicine that will cause increases, and therefore, stearic addition is that 1mg/ml is more suitable.
1.6 oil phases are compared with interior aqueous phase
In the preparation process of colostrum, the stability investigating the colostrum of formation by regulating the volume ratio of oil phase and interior aqueous phase and the envelop rate of OA-MVLs prepared.Result shows because OA is fat-soluble medicine, the addition increasing oil phase is conducive to obtaining the higher liposome of drug loading, and the preparation of these three kinds of volume ratios does not all occur lamination in preparation process, therefore preferred higher one group of envelop rate, namely volume ratio is 0.75: 0.5.
2.1 shear rate and the times of preparing colostrum
Through shear history at a high speed, energy need be provided for forming Emulsion when preparing colostrum.Shear rate and time has a certain impact to the particle diameter of colostrum and stability, also the remote-effects quality of emulsion.Experiment, under identical formulation and technology condition, respectively with 12000rpm, 13500rpm and 15000rpm high speed shear 120s, compares particle diameter and the envelop rate of gained liposome.Result shows, the liposome encapsulation obtained when 13500rpm is higher, and the particle diameter examined under a microscope is also less.
The envelop rate of shear time to medicine also has a certain impact.When result shows that shear time is too short, colostrum cannot reach high degree of dispersion state, and Emulsion is unstable, and the OA-MVL envelop rate of gained is lower, and during shearing 150s, envelop rate is higher, and experiment selects 13500rpm to shear 150s.
2.2 emulsion vortex times
Under identical formulation and technology condition, the intensity of stationary vortex mixed instrument and change incorporation time to prepare emulsion, with envelop rate and particle diameter for index, single factor exploration vortex incorporation time is on the impact of OA-MVLs envelop rate, and experimental result shows, the vortex time of emulsion is little in the change of the envelop rate of 10s, 15s, 25s, but when examining under a microscope, find the increase along with vortex time, the particle diameter of MVL reduces to some extent, and historical facts or anecdotes is tested and selected vortex oscillation 25s to prepare emulsion.
Central combination design
According to experiment of single factor result in early stage, in conjunction with related documents, select to affect comparatively significant 3 factors as investigation object, i.e. cholesterol and soybean phospholipid mass ratio (X to particle diameter and envelop rate
1), medicine fat ratio (i.e. oleanolic acid: phospholipid) (X
2), glycerol trioleate and soybean phospholipid mass ratio (X
3), each factor level of central combination design is in table 1.
The actual value of table 1 the effects variable and experimental level coding
Test number (TN) required in center combination design is 2
f+ 2f+1 (f is the quantity of studied factor), this test is the center combination design of 3 factors, and required test number (TN) is 15 times.In addition, in order to investigate the error size of test, the test of central point is repeated 5 times.The experimental program that other experiment conditions adopt experiment of single factor to determine.Using multivesicular liposome mean diameter and envelop rate as index screening best prescription condition, experimental design and experimental result are in table 2.
Table 2CCD experimental design table and inspection target experimental result
Wherein, X
1: the mass ratio X of C/PL
2: the mass ratio of glycerol trioleate and phospholipid, X
3: medicine fat ratio; Y
1: envelop rate, Y
2: particle diameter.
Date processing and equation model
Using mean diameter, envelop rate as evaluation index (dependent variable), statistics software DesignExpert 7.0 is adopted to carry out multiple linear regression and fitting of a polynomial to each factor (independent variable) respectively, and check with F, the statistics index such as Lack of fit and R-Squared investigates the accuracy of gained regression equation, the results are shown in Table 3, table 4:
By checking each fit equation F, P value, the integrated survey of Lack of fit item, delete some and reach the object simplified polynomial equation, fit equation is as follows:
Y
1＝67.07+69.36*X
1+1.20*X
2+22.82*X
3-1.10*X
1*X
2+24.89*X
1*X
3+2.10*X
2*X
3-60.84*X
1^2-0.053*X
2^2-92.44*X
3^2(P＜0.0001，R
2＝0.93)
Y2＝+0.58-7.50*X
1+1.28*X
2+63.98*X
3-81.86*X
1*X
3-5.06*X
2*X
3+22.12*X
1^2+78.17*X
3^2(P＜0.0003，R
2＝0.85)；
As can be seen from each equation degree of fitting and statistical test result, all available polynomial equation of 2 indexs investigated carries out good matching.
Table 3Y
1fit equation the results of analysis of variance
Table 4Y2 fit equation the results of analysis of variance
Owing to affecting the many factors of multivesicular liposome particle diameter and envelop rate, therefore the present invention first adopts experiment of single factor primary dcreening operation on the most significant factor of its impact, then optimizes its prescription and process conditions by center combination design method.
Response surface design is optimized and checking
According to the fit equation of each index, fix the level of a factor, adopt statistics software Statistica10.0, draw out the three-dimensional response surface design graph of a relation of each inspection target and two other empirical factor respectively, as Fig. 5.
According to fit equation and response surface design graph of a relation, determine that the optimum prescription preparing OA-MVLs is: Chol:PC (X
1)=0.57, medicine fat ratio (X
2)=16, TO:PC (X
3)=0.39.Sample is prepared by the side of clicking here, and measures its mean diameter and envelop rate, and each index prediction value and measured value the results are shown in Table 5.
The mean diameter of table 5 optimization formulation and the predictive value of envelop rate and measured value comparing result (n=3)
As can be seen from Table 5, each index measured value after optimization, all close to Optimization Prediction value, illustrates that fit equation that the present invention obtains can describe the relation of factor and index preferably.
Response surface design figure can represent each factor intuitively on the interaction relation between the impact of index and factor, and curvature is higher, shows that between factor, interactive impact is more remarkable; Isocontour shape also can reflect the power of interaction, and ellipse shows factor significant interaction, otherwise circle then, and namely the central point of the minimum ellipse in equal pitch contour is the peak of response surface (envelop rate peak).
From Fig. 5-A to Fig. 5-C, when Chol content, TO content, medicine fat is conducive to improving the envelop rate of MVLs than investigating value near in the middle of scope in factor, and medicine fat is than the most remarkable with the reciprocal effect of TO content, and cholesterol level takes second place than reciprocal effect with medicine fat.
From Fig. 5-D to Fig. 5-F, to OA-MVLs grain diameter influence comparatively greatly, medicine fat ratio and the effect of TO content reciprocal effect are comparatively strong, and cholesterol level is more general than reciprocal effect with medicine fat for medicine fat ratio and TO content.
Comprehensive above optimum results, the preparation method of OA-MVLs of the present invention is:
Step 1: precision takes soybean phospholipid 0.0800g, cholesterol 0.0456g, glycerol trioleate 0.0300g, oleanolic acid 0.0160g, stearic acid 0.0010g is in 1ml chloroform: in the organic solvent of ether (v/v=1: 1), dissolves and forms oil phase.
Step 2: precision measures 0.75ml oil phase, adds in 0.5ml in aqueous phase (5% glucose), shears 150s form colostrum with mulser with 13500rpm.
Step 3: precision measures 0.4ml colostrum, join in the outer aqueous phase of 1ml (4% glucose, 2% tween 80,3% polyvinyl alcohol) fast, vortex vibration 25s forms emulsion.
Step 4: be transferred to rapidly in round-bottomed flask by emulsion, rotary evaporation removes organic solvent, obtains OA-MVLs.
In the process of driving away the organic solvent in emulsion, if organic solvent volatilization is too fast, the destruction of emulsion can be caused, and slow emulsion stability of volatilizing is inadequate, can not obtain the good MVLs of form.This experiment adopts vacuum rotary steam to drive away organic solvent, need carry out under the condition of ice bath simultaneously.
The outward appearance of oleanolic acid multivesicular liposome and structure
Experimental technique
OA-MVLs morphologic observation
Get appropriate OA-MVLs suspension, drip on microscope slide, be placed in its form of optical microphotograph Microscopic observation.
OA-MVLs particle size distribution
The common method measuring liposomal particle size has: laser scattering method, optical microscopy, centrifugal settling method, electron microscope method and microporous filter membrane-optical densitometric method etc.This experiment adopts microscope photographing high definition photo, utilizes Image-pro plus 6.0 professional image software to record mean diameter.
From Fig. 1, Fig. 2, OA-MVLs outward appearance rounding, evenly, to be made up of multiple non-concentric vesicle.Particle size distribution meets normal distribution.
The mensuration of OA-MVLs envelop rate
The envelop rate (Encapsulation Efficiency) of liposome refers to that the dose be encapsulated in liposome accounts for the percentage ratio of total dose in system, be one of important indicator of liposome quality evaluation, the assay method of conventional envelop rate has sephadex chromatography method, mini-column centrifugation, ultrafiltration membrance filter method and dialysis etc.Because multivesicular liposome particle diameter is comparatively large, just can be separated liposome and free drug preferably with low-speed centrifugation, and can unilamelar liposome and micelle etc. be removed, improve the quality of the pharmaceutical preparations.This experiment adopts low-speed centrifugation to measure envelop rate.
Precision measures 500 μ l OA-MVLs in 5ml volumetric flask, adds methanol constant volume, with 0.22 μm of filtering with microporous membrane after ultrasonic emulsion breaking, measures to obtain total dose (W is total) by regulation chromatographic condition sample introduction.Separately get same volume OA-MVLs, centrifugal after adding 1ml normal saline dilution, get supernatant methanol constant volume to 5ml, through 0.22 μm of filtering with microporous membrane, measure to obtain free dose (W trip) by rated condition chromatographic condition sample introduction.
The envelop rate computing formula of medicine carrying multivesicular liposome is:
EE (%)=(total-W trip of W)/W always × 100%
Wherein, described rated condition chromatographic condition is:
This experiment adopts high effective liquid chromatography for measuring OA content.
Chromatographic column: China's spectrum Unitary C18 post (4.6*250mm, 5 μm)
Mobile phase: methanol: water: acetic acid: triethylamine=90: 10: 0.04: 0.02
Determined wavelength: 210nm
Flow velocity: 0.8ml/min
Column temperature: 30 DEG C
Sample size: 20 μ l
Centrifugation time:
The present invention adopts low-speed centrifugation to measure the envelop rate of OA-MVLs, and centrifugal rotating speed and time also can affect separating effect and the envelop rate of OA-MVL.On the basis of preliminary experiment determination in early stage rotating speed (2500rpm), then investigate separating effect and the envelop rate of MVL by changing centrifugation time.As shown in Figure 3, when centrifugation time is 10 minutes, the envelop rate of MVL is lower for result, is because centrifugation time is shorter, and MVL is separated not exclusively with free drug, and the visible MVL of naked eyes is cotton-shaped swimming in centrifuge tube.Along with the increase of centrifugation time, the envelop rate of MVL has a larger increase, but the long meeting of centrifugation time causes the redispersibility of MVL poor, therefore select 12 minutes comparatively suitable as centrifugation time.
Finally recording OA-MVLs envelop rate is 82.3 ± 0.61%.
The extracorporeal releasing experiment of OA-MVLs
The OA-MVLs that 3 crowdes adopt the inventive method to prepare is placed in bag filter respectively, two ends are put in 500ml dissolution medium (containing 0.3%Tween-80 after tightening, the PBS of pH=7.4), constant temperature (37 DEG C) constant speed (50r/min) stirs, respectively at 0.5,1,2,4,6,8,12,24,36,48,72,96,120h quantitative sampling 1ml, add the dissolution medium of isothermal same volume after sampling immediately.Separately establish parallel group of the crude drug (i.e. OA) that 3 are equal with above-mentioned OA-MVLs dose, in 0.5,1,2,4,6,8,12h quantitative sampling 1ml, add the dissolution medium of isothermal same volume after sampling immediately.Sampling sample, after 0.22 μm of filtering with microporous membrane, measures by described fixing chromatographic condition sample introduction, calculates release amount, draws the accumulative release rate of medicine.The results are shown in Table 6.
The accumulative release rate of table 6 medicine
More than for each time point dissociates the preparation of OA and OA-MVLs.
Just reach more than 80%, OA-MVLs at the preparation of 12h reach 80% according to table 6 OA that dissociates after 120h, describe OA-MVLs and there is good slowly releasing effect.
In order to understand the tablets in vitro rule of OA-MVLs in release medium further, experimental data is carried out matching with zero level equation model, First-order equation model, Higuchi equation model, Ritger-Peppas equation model and Korsmeyer-Peppas equation model respectively, and fitting result is in table 7.
Table 7 release in vitro models fitting result
From above fitting result, the release behavior Ritger-Peppas equation model of OA-MVLs in the PBS of pH7.4 is comparatively suitable, its fitting correlation coefficient R
2be 0.9526, describe the release rule of medicine preferably.And zero order kinetics degree of fitting is poor.According to Ritger-Peppas model theory, when k≤0.45, the releasing mechanism of medicine is Fick diffusion (Fickian Diffusion).
Oleanolic acid multivesicular liposome affects HepG2 cell proliferation and the experiment of its apoptotic effect of induction
Experimental technique
The cultivation of human hepatoma HepG2 cell
The recovery of cell
Prepare superclean bench, first use ultra violet lamp half an hour with front, the used time is with alcohol swab wiping table top.HepG2 cell is taken out from liquid nitrogen, be placed in 37 DEG C of thermostat water bath joltings, make its fast melt to liquid condition, after thawing, the cell suspending liquid in cryopreservation tube to be transferred in 10ml centrifuge tube with 1200rpm centrifugal 5 minutes, abandon supernatant, add 2ml 1640 complete medium (containing 10% hyclone, 100U/ml streptomycin, 100U/ml penicillin) re-suspended cell, add appropriate culture medium again after resuspended cell is transferred to culture bottle, 37 DEG C are positioned over, 5%CO gently after piping and druming mixing
2cell culture incubator in cultivate.
Going down to posterity of cell
Observation of cell under inverted microscope, can go down to posterity when the stand density of cell reaches the 80-90% of culture bottle floor space.Cell is put into superclean bench, discard original culture medium in bottle, softly cell is washed 2-3 time with PBS, add the trypsin digestion cell of appropriate 0.25%, the addition of pancreatin is advisable just to cover cell, add well and keep flat jog culture bottle, to ensure that trypsin touches all cells.Put into incubator and cultivate 3-5 minute, observation of cell under inverted microscope, when cell dissociation to form shrinkage and become single circular time, add culture medium and stop digestion.Abundant piping and druming cell, cell suspending liquid is transferred in 10ml centrifuge tube, put into centrifuge with 1200rpm centrifugal 5 minutes, abandon supernatant, add 2ml culture medium re-suspended cell, in ready two culture bottles, add 1ml cell suspending liquid respectively, then add appropriate culture medium, 37 DEG C are positioned over, 5%CO gently after piping and druming mixing
2cell culture incubator in cultivate.
The conservation of cell
Cell is put into superclean bench, discards original culture medium in bottle, softly wash cell 2-3 time with PBS, add the trypsin digestion cell of appropriate 0.25%, when becoming bowlder at Microscopic observation to cellular contraction, adding culture medium and stopping digestion.Cell suspending liquid is transferred in 10ml centrifuge tube, put into centrifuge with 1200rpm centrifugal 5 minutes, abandon supernatant, add cells frozen storing liquid (culture medium: serum: DMSO=7: 2: 1) re-suspended cell, divide to be filled in cryopreservation tube and put into-20 DEG C of refrigerator 2h, put into-80 DEG C of refrigerator overnight again, be transferred in liquid nitrogen.
The counting of cell
Cell is put into superclean bench, discards original culture medium in bottle, softly wash cell 2-3 time with PBS, add the trypsin digestion cell of appropriate 0.25%, after stopping digestion, cell is transferred in 10ml centrifuge tube.Take out 100 μ l cell suspending liquids after piping and druming mixing in 2ml EP pipe, add 900 μ l culture medium mixings, drop on counting chamber, count under inverted microscope after building coverslip.
N: the total cellular score of 4 medium squares on counting chamber; D: extension rate; V: Cell suspension volumes
Mtt assay detects OA-MVLs and suppresses HepG2 cell proliferation
To take the logarithm the HepG2 cell of trophophase, after the trypsin digestion cell of 0.25%, with 1*10
4the density in individual/hole is inoculated in 96 orifice plates, for getting rid of edge effect, outmost for a 96 orifice plates circle only being added PBS and does not add cell.
Observe the growth conditions of cell in 96 orifice plates, when growing to logarithmic growth after date, give the OA-MVLs solution (OA-MVLs being dissolved in 1640 cell culture complete medium to obtain) that final concentration is 10,20,40,80,120,160,200,220,240,250,260,270,280,290,300,310,320,330 and 350 μm of ol/L respectively, each concentration group establishes 3 multiple holes, and a matched group (only having cell not administration) is established in experiment.Continue cultivation after administration 24 hours, every hole lucifuge adds the MTT 20 μ l that concentration is 5mg/ml, continues to put to stop cultivating after cell culture incubator cultivates 4 hours.The supernatant in hole is abandoned in careful suction, and every hole adds DMSO 150 μ l, is then placed on vibration in microplate reader and crystallization is dissolved completely in 10 minutes, survey each hole absorbance, replication three times, calculating suppression ratio of averaging at 490nm place.
Suppression ratio=1-[(processed group-blank group)/(matched group-blank group)] * 100%.
DAPI staining is observed OA-MVLs and is induced HepG2 apoptosis
To take the logarithm the HepG2 cell of trophophase, after the trypsin digestion cell of 0.25%, with 2*10
6the density in individual/hole is inoculated in 12 orifice plates, when growing to the administration of logarithmic growth after date, give the OA-MVLs that final concentration is 20,40,80,120,160,180,200,240,270,290 μm of ol/L respectively, experiment is established one blank group (cellar culture adds DAPI dye liquor).The supernatant abandoned in hole is carefully inhaled in administration after 24 hours, cell is softly washed twice with PBS, the paraformaldehyde adding 1ml 4% fixes 5 minutes, cell is softly washed twice with PBS again after sucking-off paraformaldehyde, then lucifuge adds DAPI dye liquor effect 3-5 minute, sop up dye liquor, add PBS and wash cell twice, then put the form of observation of cell core to fluorescence microscope.
Result:
Table 8 variable concentrations OA-MVLs is on the impact (n=3) of HepG2 cell proliferation
* p < 0.05, * * p < 0.01, compares with matched group
MTT testing result shows: the OA-MVLs of same time point variable concentrations is different on the impact of HepG2 cell proliferation.With the rising of OA-MVLs concentration, the suppression ratio of HepG2 cell is also increased gradually, when OA-MVLs concentration is within the scope of 160-200 μm of ol/L, increase the fastest to the suppression ratio of HepG2 cell, along with raising gradually again of concentration, the suppression of cell is tended towards stability, suppression degree is dose dependent, IC
50=169.4 μm of ol/L.Compare with matched group, all there is statistical significance (p < 0.01).
Fluorescence microscope karyomorphism
Visible at fluorescence microscopy Microscopic observation, cellular control unit core is complete, the even fluorescence in disperse, and has cell to carry out mitosis.Experimental group cell is along with the increase of OA-MVLs concentration, and nucleus starts to become circle, pyknosis, occurs apoptosis phenomenon, and then chromatin starts to occur fine and close dense right bulk or graininess fluorescence, and visible apoptotic body.Wherein, Fig. 6 is the morphocytology change Electronic Speculum figure after variable concentrations OA-MVLs acts on HepG2 cell.(A) matched group (the normal cultured cells of administration), (B) OA-MVLs concentration is 20 μm of ol/L, (C) OA-MVLs concentration is 80 μm of ol/L, (D) OA-MVLs concentration is 160 μm of ol/L, (E) OA-MVLs concentration is 240 μm of ol/L, and (F) OA-MVLs concentration is 290 μm of ol/L.
Confocal laser scanning microscope HepG2 cellular uptake oleanolic acid multivesicular liposome is tested
DHPE (the immobilized artificial membrane dyestuff by the marked by fluorescein isothiocyanate) concentration with 10% is added in oil phase, prepares OA-MVLs by the inventive method.
Taking the logarithm the HepG2 cell of trophophase, after the trypsin digestion cell of 0.25%, being inoculated in the density in 2*106/hole in 24 orifice plates that placed the special creep plate of cell in advance, when growing to the administration of logarithmic growth after date.The supernatant abandoned in hole is carefully inhaled after administration 4h, cell is softly washed twice with PBS, the paraformaldehyde adding 1ml4% fixes 5 minutes, softly washes cell twice again after sucking-off paraformaldehyde with PBS, and then lucifuge adds DAPI dye liquor effect 5 minutes, sop up dye liquor, add PBS and wash cell five times, press from both sides out creep plate, covered on microscope slide, be positioned over the picked-up situation of observation of cell under laser confocal microscope immediately, the results are shown in Figure 4A and Fig. 4 B.
From Fig. 4 A and Fig. 4 B, mainly be distributed in nucleus by the OA-MVLs of DHPE green fluorescent label, secondly on cell membrane, also have distribution, this prompting medicine can enter in cell, and assemble in a large number on nucleus, its anti-tumor activity may synthesize with suppression DNA and divide relevant.
Pharmacokinetics in Rat is tested
Laboratory animal
Female sd inbred rats, about 200g.
Experimental technique
The determination of dosage
The dosage of this experiment converts according to the dose,equivalent in pharmacological testing between animals and human beings body, with the dosage 1.00-1.33mg/kg of the ordinary people of standard body weight, becomes rat dosage according to following formula scales:
Db＝Da*Sa*Rab*Sb；
Db: rat dosage (mg/kg);
Da: ordinary people's dosage (mg/kg);
Rab: conversion coefficient (table look-up and know that by the conversion coefficient Rab of ordinary people a to rat b be 6.17);
Sa, Sb: conversion factor (when animal is standard body weight, Sa, Sb=1, when animal is Unstandard weight, can obtain Sa, Sb value by tabling look-up);
The dosage finally conversing 0.2kg standard body weight rat according to above formula is: 6.17-16.40mg/kg.The dosage setting this experimental rat is 10mg/kg.
Experimental design
10 SD rats are divided into two groups at random, often organize each 5, one group is experimental group, and one group is matched group, all adopts the mode administration of dorsal sc injection, and before administration, water 12h is can't help in fasting.Experimental group injection OA-MVLs, the OA solution (add in 20mlPEG400 by 40mg OA, magnetic agitation adds 20ml water for injection after dissolving again) of matched group injection same dose.After administration experimental group respectively at 0.5,1,2,4,8,12,24,36,48,60,72,96,120h time point eyeground vein clump blood sampling 0.5-1ml, matched group respectively at 0.25,0.5,1,1.5,2,3,5,7,9,11h time point eyeground vein clump blood sampling 0.5-1ml.After blood sampling rapid with 4000rpm/min centrifugal 10 minutes, be separated the supernatant obtained and be blood plasma.Blood plasma temporally with after sequence number label is put into-20 DEG C of Refrigerator stores, for subsequent use.
Result
In the body of rat respectively after subcutaneous injection OA-MVLs and OA solution, the change of blood drug level is in table 9 and table 10.
The mean blood plasma concentration (n=5) of each time point after table 9 rat skin lower injection OA-MVLs
The mean blood plasma concentration (n=5) of each time point after table 10 rat skin lower injection OA solution
From experimental result, after subcutaneous injection OA raw material medicine solution and OA-MVLs, the two blood concentration-time curve difference significantly.After OA raw material medicine solution subcutaneous injection, reach peak concentration rapidly, decline immediately.And after subcutaneous injection OA-MVLs, because liposome exists part free drug, just reach the dense point of the highest medicine at 0.5h, but peak concentration comparatively OA solution group significantly reduces, blood drug level reduces gradually subsequently, and the elimination time significant prolongation of medicine in rat body, until 96h drug release is substantially complete.This illustrates that OA-MVLs has good slow release effect, serves the effect of drug-reservoir.
By the data PKSolver analyzing and processing of table 9 and table 10, determine best compartment model, ask calculation pharmacokinetic parameters.Between group, data acquisition t inspection compares: during P > 0.05, there was no significant difference; During P < 0.05, difference tool significance; P < 0.01, difference has highly significant meaning.Result shows, and the pharmacokinetic characteristic after OA and OA-MVLs subcutaneous injection in rat body all meets two-compartment model, and main pharmacokinetic parameters is in table 11.
Table 11 rat distinguishes the pharmacokinetic parameter (n=5) after subcutaneous injection 10mg/kg OA-MVLs suspension and OA solution
*: p < 0.01, * *: p < 0.05, compared with OA solution
As seen from the results in Table 6, compared with OA solution, the AUC (O-t) of OA-MVLs enlarges markedly, half-life significant prolongation, mean residence time MRT also significant prolongation, peak concentration reduces, and illustrates and OA is made multivesicular liposome, not only there is good slowly releasing effect, the bioavailability of medicine can also be increased.
The present invention have studied preparation method and the release behaviour in vitro thereof of OA-MVLs, the picked-up degree of hepatoma Hep G 2 cells to OA-MVLs is have studied by cell experiment, and have studied the pharmacokinetics process of OA-MVLs in SD rat body, for the application clinically of this medicine provides certain theoretical basis.Now result is summarized as follows:
1, first, we have adopted experiment of single factor to screen affects particle diameter and the most significant three factors of envelop rate, then utilizes center combination design to be optimized these three factors, obtains optimum prescription, successfully prepared OA-MVLs; And establish the high performance liquid chromatography of external test OA.
2, we are studied OA-MVLs physicochemical property further.Perusal OA-MVLs is the suspension of white translucent shape, and optical microphotograph Microscopic observation OA-MVLs outward appearance scrotiform rounding, each particle is made up of a lot of nonconcentric(al) vesicles.The mean diameter utilizing Image-pro plus 6.0 professional image software to record OA-MVLs meets normal distribution.In order to study OA-MVLs release characteristics in vitro, we adopt dialysis to have studied the release of OA-MVLs in the PBS of pH7.4, and matching has been carried out to releasing theory, result display OA-MVLs has good sustained release performance, the cumulative release percentage rate of slow release about 120h, 120h 80.56 ± 1.27% can be reached in the PBS of pH7.4.
3, afterwards we in order to investigate the picked-up degree of human liver cancer cell HepG2 to OA-MVLs, have employed mtt assay and DAPI staining research OA-MVLs to the suppression of HepG2 cell and apoptosis-promoting effect, and have employed laser co-focusing measure OA-MVLs enter born of the same parents' situation.Result display OA-MVLs has obvious inhibitory action to HepG2 cell, and can impel apoptosis, and OA-MVLs is mainly distributed in nucleus after entering born of the same parents, and next is distributed on cell membrane.We also establish the HPLC method that intraor extracellular measures OA content simultaneously, and the blank response rate of the method, precision, accuracy all meets analysis requirement.Testing result shows, and along with OA-MVLs is to the increase of HepG2 cytosis time, in cell, OA concentration increases gradually, and EC change is little; Along with the increase of OA-MVLs concentration, the OA concentration of intraor extracellular also increases gradually.
4, in order to investigate the release feature of OA-MVLs in rat body, we are studied its pharmacokinetic property in rat body.First establish the HPLC method of in vivoassay OA content, result shows the method linear dependence good (r=0.993) in 100-900ng/mL concentration range, and precision, the response rate, stability all meet Determination of Biological Samples requirement.Respectively at SD rat skin lower injection OA raw material medicine solution and OA-MVLs suspension (dosage is 10mg/kg), after result display OA-MVLs subcutaneous injection, reduce compared to OA solution maximum plasma concentration in vivo, Increased Plasma Half-life, reaches slow releasing function.
To sum up, the present invention is loaded into multivesicular liposome by OA, and the OA-MVLs inside and outside slow release prepared is good, achieves object of the present invention, provides new approaches for fat-soluble medicine loading multivesicular liposome reaches slow controlled release.
Conclusion:
1, the present invention has successfully prepared OA-MVLs, and obtains optimum prescription and preparation technology with center combination design optimization;
2, OA-MVLs has obvious slowly releasing effect in vitro;
3, OA-MVLs is more easily by HepG2 cellular uptake, and has obvious inhibitory action to it;
4, OA-MVLs also has slow releasing function in vivo.
Last it is noted that above embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to previous embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (10)
1. a preparation method for oleanolic acid multivesicular liposome, is characterized in that, comprises the following steps:
1) take soybean phospholipid, according to quantity, cholesterol, glycerol trioleate, oleanolic acid, stearic acid be dissolved in organic solvent, dissolve and form oil phase;
2), add in interior aqueous phase by the oil phase measuring ormal weight, shear dispersion with mulser and form colostrum;
3), join in outer aqueous phase by the colostrum measuring ormal weight, vortex vibration forms emulsion;
4), by emulsion be transferred to rapidly in round-bottomed flask, rotary evaporation removes organic solvent, obtains oleanolic acid multivesicular liposome.
2. the preparation method of oleanolic acid multivesicular liposome according to claim 1, is characterized in that, the mass ratio of described soybean phospholipid, cholesterol, glycerol trioleate, oleanolic acid is 1: 0.57: 0.38: 0.2; The interpolation concentration of stearic acid in oil phase is: 1mg/ml.
3. the preparation method of oleanolic acid multivesicular liposome according to claim 1, is characterized in that, the volume ratio of described oil phase and interior aqueous phase is 1-1.5: 1, and the volume ratio of colostrum and outer aqueous phase is 0.3-0.5: 1.
4. the preparation method of oleanolic acid multivesicular liposome according to claim 1, is characterized in that, described interior aqueous phase by mass percentage, comprising:
Glucose 5%
Surplus: water;
Described outer aqueous phase by mass percentage, comprising:
Glucose 4%
Tween 80 2%
Polyvinyl alcohol 3%
Surplus: water.
5. the preparation method of oleanolic acid multivesicular liposome according to claim 1, is characterized in that, to be chloroform and ether be described organic solvent by volume mixes at 1: 1.
6. the preparation method of oleanolic acid multivesicular liposome according to claim 1, is characterized in that, in step 2, the shear rate of colostrum is 13500rpm, shear time is 150s.
7. the preparation method of oleanolic acid multivesicular liposome according to claim 1, is characterized in that, in step 3, the time of vortex vibration is 25s.
8. according to the oleanolic acid multivesicular liposome that arbitrary claim 1-7 preparation method prepares.
9. the application of oleanolic acid multivesicular liposome according to claim 8 in preparation treatment cancer therapy drug.
10. application according to claim 9, described medicine is the medicine being used for the treatment of hepatocarcinoma.
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| CN106361702A (en) * | 2016-10-28 | 2017-02-01 | 西南民族大学 | Berberine sulfate or hydrochloride multi-vesicular liposome and preparation method thereof |
| CN110582275A (en) * | 2017-03-02 | 2019-12-17 | 丹迪生物科技有限公司 | multi-domain vesicles comprising an immunostimulatory substance, methods of making the same, and immunomodulatory compositions comprising the same |
| CN115067460A (en) * | 2022-06-16 | 2022-09-20 | 南京林业大学 | Red date pigment nano-particles and preparation method thereof |
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| WO2007028154A2 (en) * | 2005-09-02 | 2007-03-08 | Northwestern University | Encapsulated arsenic drugs |
| WO2008058156A2 (en) * | 2006-11-06 | 2008-05-15 | Jina Pharmaceuticals Inc. | Guggulphospholipid methods and compositions |
| CN103381143A (en) * | 2012-05-02 | 2013-11-06 | 江西中医学院 | Polymer-coated oleanolic acid liposome and preparation method thereof |
| CN104013576A (en) * | 2014-06-06 | 2014-09-03 | 重庆医科大学 | Uricase multivesicular liposome and preparation method thereof |
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| WO2007028154A2 (en) * | 2005-09-02 | 2007-03-08 | Northwestern University | Encapsulated arsenic drugs |
| WO2008058156A2 (en) * | 2006-11-06 | 2008-05-15 | Jina Pharmaceuticals Inc. | Guggulphospholipid methods and compositions |
| CN103381143A (en) * | 2012-05-02 | 2013-11-06 | 江西中医学院 | Polymer-coated oleanolic acid liposome and preparation method thereof |
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| CN106361702A (en) * | 2016-10-28 | 2017-02-01 | 西南民族大学 | Berberine sulfate or hydrochloride multi-vesicular liposome and preparation method thereof |
| CN106361702B (en) * | 2016-10-28 | 2019-12-13 | 西南民族大学 | Sulfuric acid or berberine hydrochloride multivesicular liposome and preparation method thereof |
| CN110582275A (en) * | 2017-03-02 | 2019-12-17 | 丹迪生物科技有限公司 | multi-domain vesicles comprising an immunostimulatory substance, methods of making the same, and immunomodulatory compositions comprising the same |
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