CN101017164B - Determination method for entrapment efficiency of liposome - Google Patents
Determination method for entrapment efficiency of liposome Download PDFInfo
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Abstract
This invention discloses one test method with drug fat package rate, which comprises the following steps: adding the liquid not reacting with resin double molecule layer into resin mixture hanging liquid to achieve balance through dialysis water concentration to get outer phase for fat test; this invention adds double molecule layer without reaction into sample to be tested; filtering to get property liquid to test its medicine and liquid concentration to compute discrete bus to pack rate.
Description
Technical field
The present invention relates to the assay method of pastille entrapment efficiency of liposome.
Background technology
Liposome (Liposomes) is the sealing cystidium of being made for the framework film material by phosphatide and (or not with) additives with bilayer structure.Two long hydrophobic hydrocarbon chain and hydrophilic radicals are arranged in the common phospholipid molecule structure, an amount of phosphatide is added in water or the buffer solution, and phospholipid molecule aligns, and its hydrophilic radical is towards the water of both sides, hydrophobic hydrocarbon chain is formed toward each other and is bilayer, constitutes liposome.The phosphatide that is used to prepare liposome has natural phospholipid, as Fabaceous Lecithin, yolk phospholipid etc.; Synthetic phospholipid, as dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine etc.Additives commonly used are cholesterol.Cholesterol also is an amphiphilic species, mixes use with phosphatide, can make stabilized liposomes, and its effect is the flowability of regulating bilayer, lowers the permeability of liposome membrane.Other additives have octadecylamine, phosphatidic acid etc., and these two kinds of additives can change the charge property of surface of liposome, thereby change envelop rate, other parameters of inside and outside of liposome.
Liposome can be divided three classes: little single chamber (layer) liposome, and particle diameter is 20-50nm, through the liposome of ultrasonic Treatment, the overwhelming majority is a small unilamellar vesicle; Multicell (layer) liposome, particle diameter is about 400-3500nm, and microscopically can be observed especially the sandwich construction as onion section or staff fingerprint; Large unilamellar vesicle, particle diameter is about 200-1000nm, and the liposome for preparing with the ether injection method mostly is this class.
The method for making of liposome has multiple, according to the character of medicine maybe needs select.(1) film dispersion method: this is a kind of preparation method of classics, and it can form multilamelar liposome, obtains small unilamellar vesicle after sonicated.This method advantage is easy and simple to handle, the liposome structure typical case, but envelop rate is lower.(2) injection method: ether injection method and alcohol injection etc. are arranged." ether injection method " is that membrane materials such as phosphatide are dissolved in the ether, under agitation slowly drips in 55-65 ℃ of pastille or not in the aqueous medium of pastille, boils off ether, continues to stir 1-2h, can form liposome.(3) reverse phase evaporation: be that liposoluble constituents such as phosphatide are dissolved in organic solvent, in chloroform, mix with the damping fluid of pastille by a certain percentage again, emulsification that the pressure reducing and steaming organic solvent can form liposome then.This method is suitable for water soluble drug, big molecular activity material, and the liposome preparation as insulin etc. can improve envelop rate.(4) freeze-drying: be suitable for the preparation of labile drug liposome in water.(5) fusion method: adopt the multiphasic liposomes of this method preparation, its physical stability is good, but heat sterilization.
When preparation pastille liposome, the mechanism difference according to medicine loads can be divided into " initiatively medicine carrying " and " passive medicine carrying " two big classes.So-called " initiatively medicine carrying " promptly carries out medicine carrying by the different ions or the compound gradient of inside and outside water, mainly contains K
+-Na
+Gradient and H
+Gradient (being the pH gradient) etc.Traditionally, people to adopt maximum methods be " passive medicine carrying " method.So-called " passive medicine carrying ", promptly at first medicine is soluble in the aqueous phase or organic phase (fat-soluble medicine) in, prepare the pastille liposome by selected method for preparing lipidosome then, its common feature is: the inside and outside water of liposome or the drug concentration basically identical on the bimolecular tunic in loading process, the factor that determines its envelop rate be medicine with interior water volume, liposome number and the medicine fat of the composition of the acting force of immobilized artificial membrane, film material, liposome than (medicine and immobilized artificial membrane material than) etc.For fat-soluble, with the high medicine of immobilized artificial membrane affinity, " passive medicine carrying " method is comparatively suitable.And for amphipathic medicine, its profit partition factor is subjected to the influence of the pH value of medium and ionic strength bigger, and the less variation of sealing condition just might make envelop rate that bigger variation is arranged.
The index of estimating the liposome quality has particle diameter, size distribution and envelop rate etc.Wherein the envelop rate of liposome (that is, wrapping into the interior medication amount of liposome and the percentage by weight of inventory) is an important indicator weighing the liposome inherent quality.Yet often there is bigger problem in entrapment efficiency determination method commonly used at present.As accuracy, the poor repeatability of method, the measurement result of different measuring methods is difficult to unanimity especially, and same liposomal samples adopts diverse ways to measure and often obtains different results.Generally speaking; the entrapment efficiency determination method of liposome is divided into balancing method and imbalance method; the data that the former records are more reliable; the data that the latter records through regular meeting owing to the mensuration process in the leakage of entrapped drug reduce; but; if medicine anelasticity in liposome is better, can not leak because outer water drug concentration reduces, the result that balancing method and imbalance method record can be more consistent.People's such as Teshima research has also proved this point (Teshima M, Kawahami S, Nishida K, et al.Prednisone retention in integrated liposomes by chemical approachand pharmaceutical approach[J] .J Control Release, 2004,97 (2): 211-218.), they use ultrafiltration, gel chromatography is measured the envelop rate of prednisolone liposome, the measurement result of preceding method is more than 90%, then the measurement result of method is all less than 4.5%, after prednisolone being prepared into palmitate, make liposome again, carry out entrapment efficiency determination with above-mentioned two kinds of methods, the result is more than 85%.Balancing method comprises supercentrifugation, super (height) speed is centrifugal and the coupling of centrifugal ultrafiltration method, these two kinds of methods need expensive hydro-extractor or ultrafiltration pipe, while length consuming time, therefore cost (comprising time cost) is very high, more importantly, these two kinds of methods can not be separated with outer water exactly to liposome, and the outer water that the former separates has small unilamellar vesicle to exist probably, cause the outer aqueous-phase concentration that records higher, this will influence the accuracy of measurement result.
People such as Xiong Fei have reported the envelop rate (Xiong Fei that measures the Breviscapinun liposome with anti-dialysis, Zhu Jiabi, Wang Wei, Deng. Breviscapinun nano liposomes entrapment efficiency determination method research [J]. Acta Pharmaceutica Sinica, 2004,39 (9): 755-757.), this method is because only add the volume of relative system dislysate seldom own to the liposome turbid liquor system, diluting effect to system in the mensuration process can be ignored, so this method can be regarded as a kind of approximate equilibrium method.Therefore can be used to measure the dilution back medicine envelop rate of the liposome of leakage easily.They are calculated as follows the envelop rate of liposome,
In the formula (1): EE is an envelop rate, C
TotalBe the total concentration of liposome Chinese traditional medicine to be measured, C
FreeBe outer aqueous phase drug concentrations, V
0Be the sample volume of liposomal samples to be measured, V
1Be V
0With anti-dialysate volumes sum.Because anti-dialysate volumes is very little, so V
1With V
0Very approaching.Be not difficult to find out C in this formula
Free* V
1The total amount of expression free drug, the author uses V
1Representing outer water cumulative volume, obviously is improper, because liposome itself also will account for certain volume in the liposome turbid liquor system, unless the volume of liposome is medium and small to ignoring in whole system.Therefore will record envelop rate accurately, must accurately measure outer water volume, a kind of conventional method is centrifugal with the liposome deposition with super (height) speed, realizes separating with outer water, claims to decide weight then.It is high that but this method requires instrument, length consuming time, and also the big shortcoming of resultant error is mentioned in the above.
Summary of the invention
At the above-mentioned deficiency of prior art, technical matters to be solved by this invention is to propose a kind of method that can accurately measure the pastille liposome encapsulation.This assay method has designed a kind of brand-new mensuration method of water volume outward, and then can accurately measure the pastille liposome encapsulation.
In order to solve the problems of the technologies described above, technical conceive of the present invention is to utilize a kind of water miscible material, this material and lipid bilayer do not interact, survey in the process of liposome encapsulation in anti-dialysis, a certain amount of this material is added in the liposome turbid liquor, reach the volume that to try to achieve outer water behind the dialysis equilibrium by the concentration of measuring this material in the bag filter, with the V in the outer water volume place of equation (1)
1Can try to achieve liposome encapsulation accurately.
Similarly, the present invention also combines the envelop rate of measuring liposome with the method for the outer surely water volume of this auxiliary substance with the centrifugal ultrafiltration method, promptly, before centrifugal ultrafiltration, a certain amount of this water miscible material is joined in the testing sample, the concentration that ultrafiltration obtains measuring respectively after an amount of filter liquor its Chinese traditional medicine and this water miscible material can calculate the free drug total amount, and then tries to achieve envelop rate.
The inventor has prepared a kind of easily by the blank liposome of centrifugation, 5 FU 5 fluorouracil (5-Fluorouracil with certain volume, 5-Fu) solution is added in the above-mentioned liposome turbid liquor, try to achieve the 5-Fu concentration of outer water behind the mixing with dialysis, then record the volume of the outer water of liposome with centrifugal, try to achieve the total amount of outer aqueous phase 5-Fu according to outer water volume and 5-Fu concentration, this value can be tried to achieve the recovery of 5-Fu in the liposome turbid liquor divided by the 5-Fu total amount that adds, the result who measures for three times is respectively 94.8%, 93.2%, 95.2%, the recovery and precision result thereof are good, have proved that 5-Fu can be in order to the outer water volume of accurate mensuration liposome turbid liquor.
Utilize identical method measure phenol red in the blank liposome suspension recovery, three times measurement result is respectively 95.5%, 96.1%, 96.6%, the recovery and precision result thereof are good, have proved the phenol red outer water volume that also can accurately measure liposome turbid liquor thus.
Thus, the inventor finds that above-mentioned water-soluble substances satisfies and interactional condition does not take place lipid bilayer, and then proposes the method for following mensuration pastille liposome encapsulation.
One of concrete technical solution that the present invention proposes: a kind of liposome encapsulation assay method, get liposome turbid liquor sample m to be measured
s, be V with volume
5-FuConcentration is C
5-FuThe solution that interactional water-soluble substances does not take place with lipid bilayer join in the liposome turbid liquor, mix; With bag filter input that hydration medium is housed wherein, reach dialysis equilibrium after, the concentration of measuring the dislysate Chinese traditional medicine is C
AE, this water-soluble substances concentration be C
FE, the content of measuring the liposome turbid liquor Chinese traditional medicine is C
T, be calculated as follows the entrapment efficiency EE of liposome.
As the preferred version of one of technical solution, interactional water-soluble substances does not take place and is preferably 5 FU 5 fluorouracil or phenol red in above-mentioned and lipid bilayer.
Two of the concrete technical solution that the present invention proposes: a kind of liposome encapsulation assay method, get liposome turbid liquor sample W to be measured
S, be V with volume
5-FuConcentration is C
5-FuThe solution that interactional water-soluble substances does not take place with lipid bilayer join in the liposome turbid liquor, mix, centrifugal, get clear liquid after the layering and place ultra filtration unit to carry out centrifugal ultrafiltration, obtain filter liquor, the concentration of measuring the filter liquor Chinese traditional medicine is C
AF, this water-soluble substances concentration be C
FF, measuring liposome turbid liquor Chinese traditional medicine content is C
T, be calculated as follows the entrapment efficiency EE of liposome.
As two preferred version of technical solution, above-mentionedly with lipid bilayer interactional water-soluble substances does not take place and be preferably 5 FU 5 fluorouracil or phenol red.
With respect to prior art, the anti-dialysis and the centrifugal-ultrafiltration of the outer water volume of water-soluble substances mensuration of work are not two kinds of methods of measuring liposome encapsulations under the approximate equilibrium condition not take place mutually with lipid bilayer in the present invention, therefore all advantages that possess balance determination, while two kinds of method precision height of the present invention, accuracy is good, weak point consuming time, experimental cost is low.
With respect to prior art, the sample size that centrifugal-ultrafiltration that the present invention proposes needs is very little, can carry out the mensuration of multiple sample easily with experiment once, be the Perfected process of measuring the envelop rate of liposome, concentrate little consumption sample amount, accurately, advantage such as efficient, large sample handling capacity.
With respect to prior art, two kinds of assay methods of the present invention (balancing method that comprises other) are very valuable for the interaction of verifying liposome and encapsulated medicine, for a liposome turbid liquor, can understand the retention characteristics of medicine in lipid bilayer as long as measure the variation of dilution suitable multiple front and back entrapment efficiency, if dilution back entrapment efficiency does not descend or descends not obvious, just illustrate that medicine is detained in lipid bilayer, if dilution back entrapment efficiency obviously descends, just illustrate that medicine is detained bad at lipid bilayer.
Description of drawings
Fig. 1 is the concentration change synoptic diagram of integerrimine in the different time bag filter.
Fig. 2 is the concentration change synoptic diagram of 5-Fu in the different time bag filter.
Embodiment
Below in conjunction with drawings and Examples the present invention is described in further detail.
Embodiment 1 (measuring the envelop rate of the anti-dialysis mensuration integerrimine liposome of outer water volume with auxiliary substance).
1.1 the preparation of integerrimine liposomal samples
Adopt the dry film dispersion method to prepare integerrimine (A alkali) liposome.Get A alkali bulk drug 0.2g, injection stage soybean lecithin 2g, cholesterol 0.5g, vitamin E 0.1g places the 500ml eggplant-shape bottle, add chloroform 50ml and make dissolving, 60 ℃, 50r/min rotation pressure reducing and steaming chloroform makes and forms the lipid dry film, room temperature vacuum drying 12h is to remove remaining chloroform, and with 50ml CBS 4.0 aquation dry films, the shaking table jolting makes molten the loosing of film on bottle wall behind the inflated with nitrogen, leave standstill 12h at 4 ℃ of refrigerators again, disperse to make whole system even with the high shear separating apparatus at last, promptly get the liposome finished product, refrigeration is standby behind the inflated with nitrogen.
1.2 the assay method of A alkali concn in outer water and the liposome
Adopt the HPLC method to measure the concentration of A alkali in outer water and the liposome, destroy liposome with an amount of 10% triton x-100 ethanolic solution when measuring liposomal samples.
1.3 the method for measurement of concentration of outer aqueous phase 5-Fu
Adopt the HPLC method to measure the concentration of outer aqueous phase 5-Fu.
1.4 the investigation of anti-dialysis equilibrium time
Get the 10ml liposome turbid liquor and place beaker, add 1.626mg/ml 5-Fu solution 0.2ml, mix, 0.5ml CBS 4.0 hydrating fluids of packing in the bag filter simultaneously, bag filter is put into liposome turbid liquor, dialyse under the magnetic agitation, draw at regular intervals dislysate 25 μ l in the bag, after the dilution suitable multiple respectively sample introduction 20 μ l HPLC under different condition measure the wherein concentration of A alkali and 5-Fu.Two kinds of drug concentrations of different time in the bag filter were mapped to the time, as shown in Figure 1, 2.A alkali reaches dialysis equilibrium substantially in 4 h as can be seen from Figure, and 5-Fu reaches dialysis equilibrium in 2h, all fully reaches dialysis equilibrium for guaranteeing two medicines, so the anti-dialysis equilibrium time is defined as 6h.
1.5 the investigation of the anti-recovery of dialysing
Get four parts of A alkali bulk drugs of different amounts, respectively with CBS 4.0 preparations 0.8,2,3, the solution of 4mg/ml, respectively get 10ml and place beaker, the 5-Fu solution 500 that in the A of variable concentrations aqueous slkali, adds 1.626mg/ml successively, 300,400,500 μ l, mix the every part of solution in back and get two parts on the sample of 25 μ l respectively, be respectively applied for the concentration of measuring before the dialysis A alkali concn and 5-Fu in the solution, the bag filter that 400 μ lCBS, 4.0 hydrating fluids will be housed is subsequently put into different mixed solution (bag filter that removes first portion of mixed liquor is equipped with 500 μ l hydrating fluids) and is carried out retrodialysis, dialysis finishes behind the 6h, from each bag filter, get two parts on the sample of 25 μ l respectively, be respectively applied for the concentration of measuring A alkali and 5-Fu in the bag filter, the concentration determination of all samples presses 2.2,2.3 in condition carry out.The computing formula of the dialysis recovery is as follows:
V
0′＝V
0+V
F-V
S1 (4)
R in formula (2), (3), (4)
A, R
FBe respectively the recovery of A alkali and 5-Fu, C
A0, C
F0Be respectively before the dialysis concentration of A alkali and 5-Fu in the mixed solution, C
A1, C
F1Be respectively dialysis concentration of A alkali and 5-Fu in the bag filter when finishing, V
0Be the amount of getting of variable concentrations A aqueous slkali, V
FBe the amount of getting of 5-Fu solution, V
S1For being used for measuring the cumulative volume of two duplicate samples of mixed liquor two drug concentrations before the dialysis, V
0' be the cumulative volume of the preceding two medicine mixed solutions of dialysis.Sample solution concentration determination result and recovery result of calculation see Table 1,2, by the table in as can be seen, the recovery of A alkali and auxiliary substance 5-Fu is all good in the anti-dialysis procedure, and repeatability is fine, illustrate anti-dialysis can be accurately, critically measure outer aqueous phase drug concentration.
The recovery of integerrimine in the anti-dialysis of table 1
Annotate: *: C
INTThe sign concentration of integerrimine.
The recovery of 5-Fu in the anti-dialysis of table 2
Annotate: *: V
5-FuFor adding to the volume of the 5-Fu solution (1.626mg/ml) in the mixed solution.
1.6 whether contain the inspection of liposome in the anti-dislysate
Adopt whether microscopic examination method and A450 turbidity inspection technique are checked liposome in the dislysate existence.
1.6.1 microscopic examination method
The negate dislysate prepares a plurality of samples, observes at microscopically (under 10 * 40,10 * 100 two enlargement factors), and each sample is all observed the visual field more than 10, does not all find the existence of liposome.
1.6.2 A450 turbidity inspection technique
Get 3ml CBS 4.0 hydrating fluids and place in the bag filter, bag is outer to be A alkali liposome turbid liquor sample 10ml, and behind the dialysis 6h, liquid is surveyed turbidity at 450nm in the bag taking, and is consistent with distilled water.Illustrate that no liposome exists in the dislysate.
1.7 improved anti-dialysis is measured liposome encapsulation
Liposome turbid liquor to be measured is shaken up, and microscopically observes in the liposome turbid liquor whether drug crystallization is arranged, if drug crystallization is arranged, then abandons this law; If there is not drug crystallization, begin to measure.Get liposome turbid liquor sample m to be measured
s(g), the accurate title, decide, place beaker, add 1.626mg/ml 5-Fu solution 200 μ l, the bag filter input of 200 μ l hydration mediums will be housed after mixing, dialyse, take out bag filter behind the 6h, from bag, take out two parts on the sample of 25 μ l, dilute the concentration (C that is used for measuring dislysate A alkali after the suitable multiple respectively
AE) with the concentration (C of 5-Fu
FE).Simultaneously, measure A alkali content C in the liposome turbid liquor
T(mg/g suspension).Be calculated as follows the entrapment efficiency (EE) of liposome.
Table 3 liposomal samples entrapment efficiency determination result
| Sequence number | m s(g) | C T(mg?INT/g?Gel?) | C AE(mg/ml) | C FE(mg/ml) | EE(％) |
| 1 | 10.1425 | 3.62 | 2.940 | 0.0426 | 38.87 |
| 2 | 10.5523 | 3.62 | 3.020 | 0.0411 | 37.38 |
| 3 | 9.9914 | 3.62 | 2.975 | 0.0423 | 36.78 |
Liposomal samples to 1.1 preparations is carried out entrapment efficiency determination, parallel 3 parts, the results are shown in Table 3, by the repeatability of sample determination is better as can be seen in the table.This sample is measured with a centrifugal ultrafiltration, measures twice, and the result is respectively 39.23%, the measurement result basically identical of 35.61%, two kind of method.
Measurement result to the liposomal samples of another part pH gradient medicine carrying sees Table 4, and the entrapment efficiency determination value before this liposome pH gradient medicine carrying is seen with table.
Table 4 liposomal samples entrapment efficiency determination result
| The sample name | m s(g) | C T(mg?INT/g) | C AE(mg/ml) | C FE(mg/ml) | EE(％) |
| Before the PH gradient medicine carrying | 6.4522 | 19.02 | 8.639 | 0.0542 | 57.75 |
| Behind the PH gradient medicine carrying | 9.2123 | 19.13 | 19.369 | 0.0409 | 12.53 |
Embodiment 2 (measuring the envelop rate of the centrifugal ultrafiltration method mensuration liposome of outer water volume with auxiliary substance)
2.1 ultrafiltration is to the influence of free drug solution concentration
Get each three parts of the A aqueous alkalis of three variable concentrations (be respectively 1.22,0.50,0.10mg/ml), every part 200 μ L, (ultra filtration unit adopts Microcon YM 100K, Millipore), measures before filter liquor and the ultrafiltration the corresponding peak area of A alkali in the solution respectively with HPLC to carry out ultrafiltration.The results are shown in Table 5, as can be seen, ultra filtration membrane has a small amount of absorption to medicine, when drug concentration is enough big, can think that ultrafiltration do not have influence to drug solution concentration.
Table 5 ultrafiltration is to the influence of free integerrimine concentration
Get each three parts of the 5-Fu aqueous solution of three variable concentrations (be respectively 0.01626,0.008130,0.003252mg/ml), every part 200 μ L, (ultra filtration unit adopts Microcon YM100K to carry out ultrafiltration, Millipore), measure before filter liquor and the ultrafiltration the corresponding peak area of 5-Fu in the solution respectively with HPLC.The results are shown in Table 6, as can be seen, ultra filtration membrane has a small amount of absorption to medicine, when drug concentration is enough big, can think that ultrafiltration do not have influence to drug solution concentration.
Table 6 ultrafiltration is to the influence of free 5-Fu concentration
2.2 improved centrifugal-ultrafiltration measures liposome encapsulation
Get liposomal samples to be measured, begin to measure after no drug crystallization exists in the confirmatory sample, otherwise, this law abandoned.Get the about 1g of testing sample, weight (W decided in accurate title
S), place 1.5ml ependoff pipe, add the 5-Fu solution 20 μ l of 1.626mg/ml, mixing, 5000r/min, 4 ℃ are centrifugal down.After sample has certain layering, get an amount of clear liquid and place ultra filtration unit (Microcon YM 100K, Millipore) carry out centrifugal ultrafiltration in, obtain stopping after an amount of filter liquor centrifugal, from filter liquor, take out two parts on the sample of 25 μ l, dilute the concentration (C that is used for measuring filter liquor A alkali after the suitable multiple respectively
AF) with the concentration (C of 5-Fu
FF), simultaneously, measure A alkali content C in the liposome turbid liquor
T(mg/g suspension).Be calculated as follows the entrapment efficiency (EE) of liposome.
Table 7 liposomal samples entrapment efficiency determination result
| Sequence number | W S(g) | C T(mg?INT/g?Gel) | C AF(mg/ml) | C FF(mg/ml) | EE(％) |
| 1 | 0.9881 | 5.62 | 3.700 | 0.0403 | 46.22 |
| 2 | 0.9949 | 5.62 | 3.674 | 0.0401 | 46.68 |
| 3 | 1.0040 | 5.62 | 3.848 | 0.0424 | 47.65 |
| 4 | 1.0092 | 5.62 | 3.773 | 0.0416 | 48.05 |
Use this law a A alkali liposomal samples is carried out entrapment efficiency determination, parallel 4 parts, the results are shown in Table 7, by the repeatability of sample determination better (RSD=1.8%) as can be seen in the table.
Check that through microscopic examination method and A450 nephelometry the result shows: no liposome exists in the ultrafiltration filter liquor.
Embodiment 3 (measuring the envelop rate of the anti-dialysis mensuration integerrimine liposome of outer water volume with auxiliary substance).
Test procedure and test condition are with embodiment 1, and with phenol red replacement 5-Fu, test findings is identical with embodiment 1.
Embodiment 4 (measuring the envelop rate of the centrifugal ultrafiltration method mensuration liposome of outer water volume with auxiliary substance)
Test procedure and test condition are with embodiment 2, and with phenol red replacement 5-Fu, test findings is identical with embodiment 2.
Claims (6)
1. a determination method for entrapment efficiency of liposome is characterized in that, getting weight is m
sLiposome turbid liquor sample to be measured, be V with volume
5-FuConcentration is C
5-FuThe solution that interactional water-soluble substances does not take place with lipid bilayer join in the liposome turbid liquor, mix; With bag filter input that hydration medium is housed wherein, reach dialysis equilibrium after, the concentration of measuring the dislysate Chinese traditional medicine is C
AE, this water-soluble substances concentration be C
FE, the content of measuring the liposome turbid liquor Chinese traditional medicine is C
T, be calculated as follows the entrapment efficiency EE of liposome.
2. determination method for entrapment efficiency of liposome as claimed in claim 1 is characterized in that, interactional water-soluble substances does not take place described and lipid bilayer is 5 FU 5 fluorouracil.
3. determination method for entrapment efficiency of liposome as claimed in claim 1 is characterized in that, interactional water-soluble substances does not take place described and lipid bilayer is phenol red.
4. a determination method for entrapment efficiency of liposome is characterized in that, getting weight is W
SLiposome turbid liquor sample to be measured, be V with volume
5-FuConcentration is C
5-FuThe solution that interactional water-soluble substances does not take place with lipid bilayer join in the liposome turbid liquor, mix, centrifugal, get clear liquid after the layering and place ultra filtration unit to carry out centrifugal ultrafiltration, obtain filter liquor, the concentration of measuring the filter liquor Chinese traditional medicine is C
AF, this water-soluble substances concentration be C
FF, measuring liposome turbid liquor Chinese traditional medicine content is C
T, be calculated as follows the entrapment efficiency EE of liposome.
5. determination method for entrapment efficiency of liposome as claimed in claim 4 is characterized in that, interactional water-soluble substances does not take place described and lipid bilayer is 5 FU 5 fluorouracil.
6. determination method for entrapment efficiency of liposome as claimed in claim 4 is characterized in that, interactional water-soluble substances does not take place described and lipid bilayer is phenol red.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102980963A (en) * | 2011-09-07 | 2013-03-20 | 北京泰德制药股份有限公司 | Method for determining drug encapsulation efficiency in liposomes |
| CN107884529A (en) * | 2016-09-29 | 2018-04-06 | 辽宁远大诺康生物制药有限公司 | A kind of method and its application for determining fat emulsion formulation entrapment efficiency |
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| EP2882780A1 (en) * | 2012-09-24 | 2015-06-17 | Rohm and Haas Company | Method for measuring encapsulation efficiency for hydrophobic actives |
| CN103575868B (en) * | 2013-11-13 | 2017-02-08 | 北京泰德制药股份有限公司 | Method for measuring distribution coefficient of main medicine phase in lipid microsphere injection |
| CN108414393A (en) * | 2018-06-13 | 2018-08-17 | 安徽中医药大学 | A method of measuring entrapment efficiency of liposome |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102980963A (en) * | 2011-09-07 | 2013-03-20 | 北京泰德制药股份有限公司 | Method for determining drug encapsulation efficiency in liposomes |
| CN102980963B (en) * | 2011-09-07 | 2014-08-13 | 北京泰德制药股份有限公司 | Method for determining drug encapsulation efficiency in liposomes |
| CN107884529A (en) * | 2016-09-29 | 2018-04-06 | 辽宁远大诺康生物制药有限公司 | A kind of method and its application for determining fat emulsion formulation entrapment efficiency |
| CN107884529B (en) * | 2016-09-29 | 2020-12-08 | 辽宁远大诺康生物制药有限公司 | Method for measuring drug encapsulation efficiency of fat emulsion preparation and application thereof |
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| CN101017164A (en) | 2007-08-15 |
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