CN103630508A - Method for measuring encapsulation efficiency of doxorubicin hydrochloride liposomal - Google Patents
Method for measuring encapsulation efficiency of doxorubicin hydrochloride liposomal Download PDFInfo
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- CN103630508A CN103630508A CN201310585408.5A CN201310585408A CN103630508A CN 103630508 A CN103630508 A CN 103630508A CN 201310585408 A CN201310585408 A CN 201310585408A CN 103630508 A CN103630508 A CN 103630508A
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- 238000000034 method Methods 0.000 title abstract description 23
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 title abstract description 10
- 229960002918 doxorubicin hydrochloride Drugs 0.000 title abstract description 10
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Abstract
The invention belongs to the field of chemical medicine detection and in particular relates to a method for measuring the encapsulation efficiency of doxorubicin hydrochloride liposomal. The doxorubicin hydrochloride liposomal is a nano small ball formed by wrapping doxorubicin hydrochloride with a phospholipid double-layer film, and the uncovered doxorubicin hydrochloride is free outside the ball. When alkaline liquid is added to a system, the free doxorubicin hydrochloride and alkaline react; the absorption wavelength of the free doxorubicin hydrochloride is long, and the doxorubicin hydrochloride in the film and the alkaline do not reacts due to isolation from the film, and an original absorption wavelength is kept. The encapsulation efficiency of the liposomal is calculated due to the change of the wavelengths through an ultraviolet-visible light photometer. The method has the characteristics of short measurement time, convenience, accuracy, small error and high reproducibility and is suitable for control on the industrial production process.
Description
Technical field
The invention belongs to chemicals detection field, particularly a kind of assay method about Doxil envelop rate.
Background technology
Envelop rate is an important index of liposome and nanoparticle quality control, has reflected the degree that medicine is sealed by carrier, the concentration of medicine and carrier, and the condition in preparation process is the principal element that affects its envelop rate.Envelop rate comprises the mensuration of percentage envelop rate and parcel volume, and general document is mainly investigated percentage envelop rate (Encapsulation percentage, EN%).Its expression formula is EN%=(1-Cf/Ct) * 100%.The amount that in formula, Cf is free drug; Ct is the total amount of nanoparticle or liposome suspension Chinese traditional medicine.For Accurate Determining envelop rate, by nanoparticle or liposome separated with free drug be crucial step.Because liposome or nanoparticle are than large many of wrapped drug particle, that can utilize them varies in size the separated not medicine of parcel of removing, and these methods have gel filtration column chromatography, dialysis etc.; If wrapped medicine is protein or DNA etc., or not wrapped medicine may form larger agglomerated thing, can utilize they from nanoparticle or liposome buoyancy, density is different and adopt the method such as centrifugal to carry out separated.Common envelop rate detection method is as follows:
1. column chromatography
Gel permeation chromatography technology because of its effectively and in laboratory, be used widely fast.It can be used for separation remove not packaging medicine also can be to the liposome in suspension or nanoparticle size packets.It should be noted after liposome or nanoparticle are by wash-out medium and may need to increase concentration step.The conventional glucosan of column chromatography filler is as Sephadex G-50, also there is bibliographical information to use Sephadex G-25 and Sephadex G-200, concrete steps are: the Sephadex G-50 that packs water or physiological saline or phosphate buffer swelling in chromatographic column into, by suspension upper prop, water or physiological saline or phosphate buffer coutroi velocity wash-out.Select the particle size of gel, the speed of the kind of eluent, wash-out, temperature have important impact to separation, in concrete experiment, need to make elution curve to determine best separating effect.
Korea Spro waits people quietly and when measuring the envelop rate of solid lipid nanoparticles containing volatile oil of Lignum dalbergia odoriferae, adopts column chromatography separated.Condition is: Sephadex G-50 post (16cm * 2cm), and eluent is distilled water, flow velocity is 1ml/min, room temperature.Pipette dalbergia wood volatile oil solid nano grain 0.5ml upper prop, with column chromatography condition wash-out, every 3ml collects once.The HPLC condition that entrapped medication amount application content of dispersion is measured is measured.
The people such as He Jun, by the swelling Sephadex G-25 of 12 hours dress post, use 80ml water elution, flow velocity 1ml/min, and after collecting, 40ml eluent is measured (2).The entrapment efficiency determination of taxol long-circulation fat matter nanoparticle is selected Sephadex gel column equally, and water is eluent, accesses wherein and measures with the part of blue-opalescent.
Luan Libiao etc. find gel filtration chromatography method except having accurately when measuring Ketoprofen gelatine microsphere envelop rate, convenient, particulate is survivable, high repeatability and other advantages, all right purifying microballoon, not only can remove not the low molecular weight impurities such as medicine, inorganic salts of parcel, and can isolate Tween-20.
The condition that Gao Xiaoli etc. measure liposome encapsulation to dextrane gel chromatography is screened.To glass column, the particle diameter of gel is not the smaller the better, if gel particle is too thin, larger liposome may be trapped on gel column, will consider molecular size and the character of separated compound while selecting.The gel (particle size 50-150 μ m) of thick level in should selecting for multilamellar liposome, can be with any grade of other gel for small unilamellar vesicle.In column chromatography process, the blade diameter length ratio of chromatographic column used, the number of the flow velocity of eluent and applied sample amount all affects separated free medicine from liposome, should selection can make liposome reach the elution requirement of maximum separation degree, and guarantee is envelop rate accurately.It should also be noted that glucosan surface exists can be combined with liposome membrane and interactional minute sites.Although this effect does not affect the flow characteristics of liposome on gel column, but still can cause the loss of a small amount of lipid, film instability be increased, thereby cause the change of membrane permeability and the seepage of encapsulate substances.This phenomenon be should give special attention in the situation that lipid concentration is lower, generally can be by strengthening liposomal samples upper column quantity or solving pillar is saturated in advance with empty liposome.
2. dialysis
Dialysis is to utilize liposome or nanoparticle particle and drug molecule to vary in size, the method for free drug being removed by the fenestra crown_interception of semipermeable membrane material.Liposome and nanoparticle can not see through semi-permeable diaphragm more greatly, and free drug can pass through, and generally liposome and nanoparticle is placed in semi-permeable diaphragm, and conventional semipermeable membrane material has parchment, animal's bladder and collodion etc.Film is put neat solvent outward, and due to concentration difference factor, the free drug in film, constantly to film exosmosis, is often changed the solvent outside film, and free drug is separated completely.This method is to be the most also the most frequently used not method of entrapped drug (except macromolecular compound) of removing.It does not need complicated technology, also without expensive instrument, and can expanding production.By continuous change dialysis medium, can remove all free drugs.But this method is very time-consuming, under general room temperature condition, the free drug that remove more than 95% at least needs to change dialysis medium three times, and the time is more than 10-24h.In addition, the osmotic strength of dialysis medium should be consistent with the concentration of liposome or nanoparticle suspension, otherwise in dialysis, will change the volume of liposome and nanoparticle suspension, and may cause the seepage of encapsulate substances.
The separation application dialysis of insulin nanoparticles, insulin liposome is mixed with suitable concn with pH7.4PBS, get 1ml and put into bag filter, pH7.4PBS10.0ml is dissolution medium, be placed in triangle tool plug flask, Erlenmeyer flask is placed in oscillator and is vibrated, 37 ℃ of constant temperature, oscillation frequency is 150r/min.After 3h, get dislysate, measure its concentration.
Zhang Guifang etc. measure Buddhist nun not in liquid nano liposomes envelop rate by liposome solutions 1ml in processed bag filter (molecular weight 10000), bag filter is immersed to bag filter (while preparing this liposome used hydration medium) 45ml, be placed on magnetic stirring apparatus and stir, regularly change dislysate.Because liposome particles less (about 30-150nm) in this experiment, centrifuge method separating effect is not fine, and the effect of dialysis is relatively good.
Lv Wenli etc. add every bottle of lipid freeze-dry powder after the hydration of 1ml redistilled water in entrapment efficiency for liposomal formulation of breviscapine is measured, precision pipettes 0.5ml, add in the bag filter of wound packages, put into the conical flask that fills 100ml0.9%NaCl, magnetic agitation in 37 ℃ of water-baths.In preliminary experiment, determine that at 4h, getting outer liquid measures.The people such as Xiong Fei have applied anti-dialysis in the assay method of Breviscapinun nano liposomes envelop rate.Concrete grammar is that liposome solutions is placed in outside bag filter, and hydration medium, in bag filter, stirs the outer liquid of bag filter, dialysis 16h.The accurate interior liquid of bag filter of drawing is measured.
Because the liposome of preparation is less, mean grain size 20.8nm, adopts sephadex chromatography method, exchanges different loading volumes for and is all difficult to separated liposome and free drug with mobile phase, and liposome is larger from loading and complete wash-out extension rate, likely causes liposome seepage.The particle diameter of liposome is less, also cannot separated liposome and free drug with the ultracentrifugation of long period.Conventional dialysis is that liposome is placed in bag filter, dialysis material is placed in the outer liquid of bag filter, free drug can see through bag filter, until the concentration of free drug reaches balance with dialysis in medium in bag filter, but because the volume of the outer liquid of bag filter is generally the long-pending 20-50 of interior liquid doubly, when dialysis is complete, liposome around drug concentration can dilute larger multiple, may be broken due to the mobile equilibrium of liposome and free drug the seepage that causes liposome medicament in dialysis procedure.So hyperfiltration can effectively be avoided seepage.
3. centrifuge method
Under different centrifugal force, centrifuging is the effective ways that free drug in variety classes liposome or nanoparticle is removed in separation.In order to remove free drug completely, usually need to repeat to suspend with repeatedly centrifugal.Make liposome or the nanoparticle needed centrifugal force that sinks depend on the size of liposome and nanoparticle, also depend on to a certain extent the coagulation of suspension.If liposome or nanoparticle are little and narrowly distributing, just need high speed centrifugation and freezing condition.Low speed (2000-4000r/min) is centrifugal can only make large liposome or nanoparticle sedimentation.For a large amount of liposomes and nanoparticle, utilize the freezing centrifugal and consumed energy and more expensive of high speed, so this method is unsuitable for separated small liposome or nanoparticle.
In bibliographical information, nanoparticle is separated with the larger multiplex centrifuge method of liposome.Zhou little Ju etc. are separated with its free drug by Ursolic Acid Phospholipid Nanoparticles by centrifuge method, after low temperature ultracentrifugation (4 ℃, 100000r/min, 60min), get supernatant and measure.Aclarubicin A PLA milimicron particle, eye are all that application of cold temperature high speed centrifugation carrys out separated free medicine with Norfloxacin milimicron particle, Praziquantel-Liposome etc.The people such as Tasana Pitaksuteepong prepare in water-in-oil microemulsion interfacial polymerization the separation condition of using in the nanoparticle of hydrophilic substance and are: 51500 * g, 1h, 25 ℃ (14).For larger liposome, low-speed centrifugal can shorten the time and can rarer liposome turbid liquor be concentrated to required concentration simultaneously.For fear of liposome, destroyed, be must be noted that to guarantee that the osmotic pressure of repetition suspension medium is consistent with the osmotic pressure of liposome suspension.
4. ultrafiltration
Ultrafiltration (being called for short UF), is to take pressure as expulsive force, utilizes ultra filtration membrane different pore size to carry out separated physics screening process to liquid.Its molecule cutting quantity (CWCO) is generally 6000 to 500,000, and aperture is 100nm(nanometer).Originating from is 1748, and Schmidt divides filter solution with cotton glued membrane or fine jade film, and when applying certain pressure, solution (water) sees through film, and the materials such as protein, colloid are retained down, and its filtering accuracy is considerably beyond filter paper, therefore claim ultrafiltration.The separation characteristic of ultrafiltration has: 1. detachment process, without phase change, is therefore saved the energy; 2. separation is to carry out at normal temperatures, is suitable for the concentrated of some heat sensitivity materials (as fruit juice, medicine, biopreparate) and purifies; 3. the ultra filtration membrane of seriation, can carry out molecular-weight gradation by the large molecular solution system of many components; 4. equipment is simple, easy operating, management and maintenance; 5. ultrafiltration is a uniform non-individual body of being made by high molecular polymer, and in use, film itself comes off without any chip or particulate, guarantees to see through water pure.
In entrapment efficiency determination, ultrafilter is also used to separated liposome or nanoparticle and free drug.In the research of camptothecine solid lipid nanoparticle, with SS-5 hollow fiber membrane ultrafiltration device (holding back mean molecular weight 10000), carry out ultrafiltration.The red grade of Wang Zhun is used SS-14A type ultrafilter, ultra filtration membrane molecule interception 30,000 in the entrapment efficiency of Penciclovir liposome is measured.
Summary of the invention
Technical matters to be solved by this invention is: in prior art, measure envelop rate, conventionally need to liposome is separated with not entrapped medicine, and complicated operation, accuracy rate declines.
For solving this technical matters, the technical solution used in the present invention is:
The invention provides a kind of assay method of Doxil envelop rate, be divided into 3 steps:
(1) total drug monitoring: prepare certain density Doxil sample solution, measure the absorbance log of this sample solution,
In Doxorubicin, the concentration of Doxil is controlled as 0.00001mg/ml～2mg/ml, and total drug monitoring wavelength is 249-259nm,
As preferably, can adopt following operation, get Doxil, be diluted to 0.004mg/ml, take methyl alcohol as blank, utilize ultraviolet-visible photometer, at 254nm place, measure absorbance log A
1;
(2) free drug is measured: to step (1) be same batch, in concentration Doxil solution, add excessive alkali reaction, after reaction finishes, mensuration absorbance log.
Wherein, alkali is selected from NaOH, potassium hydroxide, lithium hydroxide, ammoniacal liquor one or more, and the concentration of alkali is 0.001mol/L～3mol/L, and alkalescence is stronger, and the reaction time is shorter, reacts more complete,
Temperature of reaction is 0 ℃～55 ℃, and temperature usury is in reaction, but liposome membrane has phase transition temperature, and under too high condition, film just becomes and is easy to penetratingly, and alkali can enter liposome center like this, thereby affects measurement result.
Envelop rate of the present invention detection time is short is its distinguishing feature, and when alkali adds in liposome, free Doxorubicin reacts at once, makes system become blue, and its reaction time is 0.001 hour～0.5 hour.
In Doxorubicin, the concentration of Doxil is controlled as 0.001mg/ml～2.5mg/ml, and it is 570-610nm that free drug is measured wavelength,
As preferably, can adopt following operation, get the Doxil 1ml of same batch of 2mg/ml, after diluting 500 times, add excessive alkali reaction, after reaction finishes, the Doxil of same concentration of take is blank, utilize ultraviolet-visible photometer, at 600nm place, measure absorbance log A
2;
(3) by correction factor computational envelope rate, envelop rate=(1-A
2* f/A
1) * 100%, the difference of consideration testing tool and brand, correction factor f is 1.488～1.522, in the present invention, measuring f is 1.500.
Because liposome duplicature has buffer action.While adding alkali lye in system, free Doxorubicin and alkali react, and free Doxorubicin absorbing wavelength length is moved, and is wrapped in the Doxorubicin in film, because the isolation of film does not react with alkali, keep original absorbing wavelength, do not have interference.Thereby can directly measure free drug, calculate envelop rate.
The invention has the beneficial effects as follows: the present invention measures a kind of novel method is provided for liposome encapsulation, without employing separation means in the situation that, have measure consuming time short, convenient, accurate, error is little, reappear, and is applicable to the feature of commercial process control.
Accompanying drawing explanation
Fig. 1 is the scintigram of doxorubicin hydrochloride, at 600nm place, does not have absorption.
Fig. 2 is the doxorubicin hydrochloride scintigram adding after alkali, obviously at 600nm place, exists and absorbs as seen.
Fig. 3 is the Doxil scintigram of 70% envelop rate, at 600nm place, does not have absorption.
Fig. 4 is the Doxil of 70% envelop rate, adds the scintigram after alkali, obviously at 600nm place, exists and absorbs as seen.
Embodiment
Embodiment 1
Doxil lot number 0344689 envelop rate approximately 80%
(1) mensuration of total medicine: get the Doxil 1ml of 2mg/ml in the volumetric flask of 50ml, add methyl alcohol to scale and shake up.Get wherein 1ml and, in the volumetric flask of 10ml, add methyl alcohol to scale, shake up, it is A that the methyl alcohol of take is measured absorbance log as blank at 254nm place
1 is equal=0.382.
(2) free drug is measured: get 2mg/ml with step (1) the Doxil 1ml that is same batch in the volumetric flask of 50ml, add water to scale and shake up.Get wherein 1ml and, in the volumetric flask of 10ml, at 2～8 ℃, add the NaOH of 0.1mol/L to scale, shake up, react 5 minutes.
Getting the Doxil of same concentration (same batch), get 1ml in the volumetric flask of 50ml, add water to scale and shake up, then get wherein 1ml and, in the volumetric flask of 10ml, at room temperature, add water to scale, shake up, is blank.
Utilize ultraviolet-visible photometer, at 600nm place, measuring absorbance log is A
2 is equal=0.051.
(3) by correction factor computational envelope rate, envelop rate=(1-A
2* f/A
1) * 100%, correction factor f is 1.5.
Measurement result is analyzed
Above-mentioned supercentrifugal process is measured, and with reference to State Food and Drug Administration's standard (YBH02522012), carries out.
Embodiment 2
Doxil lot number 0344466 envelop rate approximately 90%,
(1) mensuration of total medicine: get the Doxil 2ml of 2mg/ml in the volumetric flask of 50ml, add methyl alcohol to scale and shake up.Get wherein 1ml and, in the volumetric flask of 10ml, add methyl alcohol to scale, shake up, it is A that the methyl alcohol of take is measured absorbance log as blank at 259nm place
1 is equal=0.453.
(2) free drug is measured: get 2mg/ml with step (1) the Doxil 2ml that is same batch in the volumetric flask of 50ml, add water to scale and shake up.Get wherein 1ml and, in the volumetric flask of 10ml, at room temperature, add the potassium hydroxide of 1mol/L to scale, shake up, measure immediately,
Getting the Doxil of same concentration (same batch), get 2ml in the volumetric flask of 50ml, add water to scale and shake up, then get wherein 1ml and, in the volumetric flask of 10ml, at room temperature, add water to scale, shake up, is blank.
Utilize ultraviolet-visible photometer, at 600nm place, measuring absorbance log is A
2 is equal=0.030.
(3) by correction factor computational envelope rate, envelop rate=(1-A
2* f/A
1) * 100%, correction factor f is 1.5.
Measurement result is analyzed
Above-mentioned supercentrifugal process is measured, and with reference to State Food and Drug Administration's standard (YBH02522012), carries out.
Claims (7)
1. an assay method for Doxil envelop rate, is characterized in that: described assay method is,
(1) total drug monitoring: prepare certain density Doxil sample solution, measure the absorbance log of this sample solution;
(2) free drug is measured: to step (1) be same batch, in concentration Doxil solution, add excessive alkali reaction, after reaction finishes, mensuration absorbance log;
(3) by correction factor computational envelope rate.
2. the assay method of Doxil envelop rate as claimed in claim 1, is characterized in that: the alkali described in step (2), is selected from NaOH, potassium hydroxide, lithium hydroxide, ammoniacal liquor one or more.
3. the assay method of Doxil envelop rate as claimed in claim 1, is characterized in that: in step (2), the concentration that adds alkali is 0.001mol/L～3mol/L.
4. the assay method of Doxil envelop rate as claimed in claim 1, is characterized in that: the temperature of reaction described in step (2) is 0 ℃～55 ℃.
5. the assay method of Doxil envelop rate as claimed in claim 1, is characterized in that: in step (1), total drug monitoring wavelength is 249-259nm; In step (2), free drug monitoring wavelength is 570-610nm.
6. the assay method of Doxil envelop rate as claimed in claim 1, is characterized in that: the correction factor described in step (3) is 1.488～1.522.
7. the assay method of Doxil envelop rate as claimed in claim 1, is characterized in that: in step (1) or step (2), in Doxorubicin, the concentration of Doxil is 0.001mg/ml～2.5mg/ml.
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| CN108776114A (en) * | 2018-05-28 | 2018-11-09 | 常州大学 | A kind of hyaluronic acid liposome ultraviolet fingerprint method measurement hyaluronic acid contents |
| CN116003493A (en) * | 2022-12-16 | 2023-04-25 | 常州金远药业制造有限公司 | Method for recovering cupric ion and cytarabine |
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