CN101078697B - Method for determining phase change temperature of liposome - Google Patents
Method for determining phase change temperature of liposome Download PDFInfo
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- CN101078697B CN101078697B CN2007100248276A CN200710024827A CN101078697B CN 101078697 B CN101078697 B CN 101078697B CN 2007100248276 A CN2007100248276 A CN 2007100248276A CN 200710024827 A CN200710024827 A CN 200710024827A CN 101078697 B CN101078697 B CN 101078697B
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Abstract
The invention relates to a method of measuring transformation temperature of liposome, belonging to the analysis measuring field. The method is that while transformation temperature of some liposome is measured by DSC the relation curve of drug loading efficiency and incubation temperature; according to the incubation temperature corresponding to the highest peak of drug loading efficiency, actual transformation peak, namely accurate transformation temperature of the liposome can be determined in many heat absorption peaks in DSC map. The method can determine transformation temperature of liposome more accurately. If transformation point of some liposome is adjusted furthermore, only DSC is used to track the change of transformation peak and interference from other mixed peaks is avoided to measure the phase transformation of liposome quickly, conveniently and accurately.
Description
Technical field
The invention belongs to the analytical test field, specifically relate to a kind of method of measuring phase change temperature of liposome.
Background technology
After medicine combined with carrier and forms medicament carrier system, the absorption of medicine and distributing no longer determined by medicine itself, but was subjected to the influence of the physicochemical property of carrier.Select suitable drug carrier material according to clinical requirement, not only can conduct drugs to target organ, and also play useful effect for the physicochemical property and the pharmacologically active of medicine.Liposome has constituted a kind of new medicine controlled releasing system as the carrier of drug delivery and controlled release.This carrier not only can improve the dissolution rate of medicine, helps drug absorption, can also make medicine arrive target site fast, especially is fit to anti-tumor medicinal preparation.
After liposome will wrap the medicine that carries and be transported to the lesions position of human body, spot heating made drug usually, to reach the purpose of treatment.Liposome membrane is made of phosphatide.Phospholipid molecule can generate closed phospholipid bilayer automatically in water, can undergo phase transition with the different liposome film of temperature.When phase transition temperature was following, membrane structure was in the glue crystalline state, and structure is tight relatively; When phase transition temperature was above, liposome was in fluid attitude and liquid crystal state, and the flowability and the permeability of film increase sharply, and the entrained chemicals of liposome can discharge rapidly.Therefore the phase transition temperature method of accurately measuring liposome is the important foundation of technology such as transformation temperature modulation, drug control.
The lipidosome drug carrier of having reported both at home and abroad exists and is difficult to realize that temperature control discharges the problem of medicine at present.The most classical liposome phase transformation assay method is differential scanning calorimetry (DSC).The DSC method is a kind of method of mensuration phase transformation of comparative maturity, but because the heat of transformation of liposome is very little, cause the detection sensitivity of phase transition temperature poor, simultaneously because problem such as the overlapping or assorted peak of exothermic peak, endothermic peak is more in the testing process, the phase transition temperature that causes measuring makes a mistake.
Other assay method commonly used comprises FD method, nephelometry, radio-labeled method, nuclear magnetic resonance method and self-quenching fluorescence method etc.But,, can not be used to the mensuration of liposome transformation temperature because there are shortcomings such as detection sensitivity is low, experimental implementation complexity in said method.In addition, the relation between utilization incubation temperature and the liposome encapsulation, though also can roughly infer the phase transition temperature of liposome, this experimental technique cycle is long, and the experimentation complexity, is not suitable for determining of phase change temperature of liposome.This patent causes outside the variation of enthalpy according to the phase transformation decapacitation of liposome, also can cause the ultimate principle of other physicochemical property significant change, certain relation that liposome encapsulation of finding under study for action in conjunction with us and phase change temperature of liposome exist (when incubation temperature more near the phase transition temperature of liposome, the envelop rate of liposome is just high more), a kind of method of accurate mensuration phase change temperature of liposome is proposed.
Summary of the invention
The method sensitivity that has the mensuration phase change temperature of liposome now is low in order to solve, the deficiency of complicated operation, the invention provides a kind of method of definite phase transition temperature, has overcome the deficiencies in the prior art effectively.
The technical scheme that technical solution problem of the present invention is adopted is:
A kind of method of measuring phase change temperature of liposome, it is characterized in that, when measuring the phase transition temperature of certain liposome with the DSC method, measure the relation curve of entrapment efficiency and incubation temperature, according to the pairing incubation temperature in envelop rate top in the curve, the actual transformation peak of determining this liposome in the numerous endothermic peaks in the DSC collection of illustrative plates i.e. phase transition temperature accurately.
The technique scheme concrete operations are:
1) the DSC method is measured the phase transition temperature of liposome
With the liposome turbid liquor that makes on hydro-extractor with 12000rmin
-1Centrifugal 30min removes supernatant liquor, keeps lower floor's liposome; Take by weighing and place differential scanning calorimeter about 10.0mg with 2 ℃ of min
-1Programming rate detects;
2) measure the relation of incubation temperature to liposome encapsulation
Result according to DSC mensuration, choose a series of incubation temperatures, the preparation liposome turbid liquor, precision is measured the 1mL suspension in the bag filter that has washed, exchanged extracellular fluid dialysis 25mL in per 3 hours for, extracellular fluid dialysis is free drug solution, and the solution of collecting four dialysis after 12 hours mixes, is settled to 100mL.Under certain wavelength, measure above-mentioned solution absorbency then,, calculate the envelop rate of liposome according to following formula by the concentration of typical curve conversion extracellular fluid dialysis Chinese traditional medicine with the uv-spectrophotometric instrument;
Envelop rate=(1-C
Free/ C
Always) * 100%
In the formula: C
FreeFor not wrapping into the amount of liposome free drug; C
AlwaysBe the medicine total amount
With the incubation temperature is horizontal ordinate, and envelop rate is that ordinate is figure;
3) according to 2) in the temperature of curve envelop rate peak correspondence determine 1) in phase transformation peak in the DSC collection of illustrative plates, the pairing temperature in this peak is the phase transition temperature of liposome.
The method of the said mensuration phase change temperature of liposome of the present invention can be determined the phase transition temperature of liposome more exactly.After obtaining accurately phase transition temperature by this method, if this kind liposome is carried out further transformation temperature modulation, the change in location that only needs utilization DSC to follow the tracks of this phase transformation peak is avoided the interference at other assorted peak, and then the convenient and swift phase transformation of measuring liposome exactly.
Description of drawings
Fig. 1: liposome (100% lecithin) DSC collection of illustrative plates
Fig. 2: typical curve
Fig. 3: the relation of liposome (100% lecithin) incubation temperature and envelop rate
Fig. 4: liposome (lecithin is 3 to 1 with the cholesterol mass ratio) DSC collection of illustrative plates
Fig. 5: the relation of liposome (lecithin is 3 to 1 with the cholesterol mass ratio) incubation temperature and envelop rate
Fig. 6: liposome (lecithin is 5 to 1 with the mass ratio of cholesterol) DSC collection of illustrative plates
Fig. 7: the relation of liposome (lecithin is 5 to 1 with the cholesterol mass ratio) incubation temperature and envelop rate
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
Embodiment 1: preparation berberine hydrochloride liposome (100% lecithin) suspension, with the liposome turbid liquor that makes on hydro-extractor with 12000rmin
-1Centrifugal 30min removes supernatant liquor, keeps lower floor's liposome.Take by weighing and place differential scanning calorimeter about 10.0mg with 2 ℃ of min
-1Programming rate detects.According to DSC collection of illustrative plates (see figure 1), a bigger endothermic peak appears in the time of 60.6 ℃.
Choose 25 ℃, 37 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, under 6 kinds of incubation temperatures, prepare berberine hydrochloride liposome, after dialysing 12 hours with bag filter, the constant volume extracellular fluid dialysis is to 100mL, use the determined by ultraviolet spectrophotometry solution absorbency under 345nm, by the concentration of typical curve (see figure 2) conversion solution, the entrapment efficiency of liposome and the relation of incubation temperature are seen shown in Figure 3.Envelop rate was the highest when the result showed 60 ℃.The result that this and DSC measure matches, and the phase transition temperature that can determine liposome is 60.6 ℃.
Embodiment 2: preparation berberine hydrochloride liposome (lecithin is 3 to 1 with the cholesterol mass ratio) suspension, with the liposome turbid liquor that makes on hydro-extractor with 12000rmin
-1Centrifugal 30min removes supernatant liquor, keeps lower floor's liposome.Take by weighing and place differential scanning calorimeter about 10.0mg with 2 ℃ of min
-1Programming rate detects.According to DSC collection of illustrative plates (see figure 4), a bigger endothermic peak appears in the time of 41.8 ℃.
Choose 39 ℃, 41 ℃, 43 ℃, 45 ℃, under 4 kinds of incubation temperatures, prepare berberine hydrochloride liposome, after dialysing 12 hours with bag filter, the constant volume extracellular fluid dialysis is to 100mL, measure solution absorbency with the uv-spectrophotometric instrument under 345nm, by the concentration of typical curve (see figure 2) conversion solution, the entrapment efficiency of liposome and the relation of incubation temperature are seen shown in Figure 5.Envelop rate was the highest when the result showed 43 ℃.The result that this and DSC measure matches, and the phase transition temperature that can determine liposome is 41.8 ℃.
Embodiment 3: preparation berberine hydrochloride liposome (lecithin is 5 to 1 with the cholesterol mass ratio) suspension, with the liposome turbid liquor that makes on hydro-extractor with 12000rmin
-1Centrifugal 30min removes supernatant liquor, keeps lower floor's liposome.Take by weighing and place differential scanning calorimeter about 10.0mg with 2 ℃ of min
-1Programming rate detects.According to DSC collection of illustrative plates (see figure 6), a bigger endothermic peak appears in the time of 55.8 ℃.
Choose 47 ℃, 50 ℃, 53 ℃, 56 ℃, 59 ℃, under 5 kinds of incubation temperatures, prepare berberine hydrochloride liposome, after 12 hours, the constant volume extracellular fluid dialysis is measured solution absorbency with the uv-spectrophotometric instrument to 100mL under 345nm with the bag filter dialysis, by the concentration of typical curve (see figure 2) conversion solution, the entrapment efficiency of liposome and the relation of incubation temperature are seen shown in Figure 7.Envelop rate was the highest near the result showed 53 ℃.The result that this and DSC measure is comparatively identical, and the phase transition temperature that can determine liposome is 41.8 ℃.
Claims (1)
1. method of measuring phase change temperature of liposome, it is characterized in that, when measuring the phase transition temperature of certain liposome first with the DSC method, measure the relation curve of entrapment efficiency and incubation temperature, according to the pairing incubation temperature in envelop rate top in the curve, the actual transformation peak of determining liposome in the numerous endothermic peaks in the DSC collection of illustrative plates i.e. phase transition temperature accurately; Concrete operation is:
1) the DSC method is measured the phase transition temperature of liposome
With the liposome turbid liquor that makes on hydro-extractor with 12000rmin
-1Centrifugal 30min removes supernatant liquor, keeps lower floor's liposome; Take by weighing and place differential scanning calorimeter about 10.0mg with 2 ℃ of min
-1Programming rate detects;
2) measure the relation of incubation temperature to liposome encapsulation
Result according to DSC mensuration, choose a series of incubation temperatures, under every kind of described incubation temperature, prepare liposome turbid liquor, precision is measured the 1mL suspension in the bag filter that has washed, exchanged extracellular fluid dialysis 25mL in per 3 hours for, extracellular fluid dialysis is free drug solution, and the solution of collecting four dialysis after 12 hours mixes, is settled to 100mL; Under certain wavelength, measure above-mentioned solution absorbency then,, calculate the envelop rate of liposome according to following formula by the concentration of typical curve conversion extracellular fluid dialysis Chinese traditional medicine with the uv-spectrophotometric instrument:
Envelop rate=(1-C
Free/ C
Always) * 100%
In the formula: C
FreeFor not wrapping into the amount of liposome free drug; C
AlwaysBe the medicine total amount
With the incubation temperature is horizontal ordinate, and envelop rate is the ordinate mapping;
3) according to 2) in the temperature of curve envelop rate peak correspondence determine 1) in phase transformation peak in the DSC collection of illustrative plates, this pairing temperature in phase transformation peak is the phase transition temperature of liposome.
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| CN102023135B (en) * | 2010-11-19 | 2012-05-16 | 中国航空工业集团公司北京航空材料研究院 | Method for analyzing slanting peak locating spectrum |
| CN102735709B (en) * | 2012-07-02 | 2015-01-07 | 辽宁省食品药品检验所 | Method for rapidly detecting sildenafil citrate illegally added in health food products and traditional Chinese medicine preparations |
| DE102013011730B3 (en) * | 2013-07-12 | 2014-08-14 | Netzsch-Gerätebau GmbH | Method for evaluating a measurement result of a thermal analysis, and use of the method, computer device, computer program product and system for carrying out the method |
| CN104352491B (en) * | 2014-11-13 | 2021-05-04 | 重庆泰通动物药业有限公司 | Compound amoxicillin sodium liposome medicine and preparation method thereof |
| CN105954315A (en) * | 2016-05-03 | 2016-09-21 | 西安电子科技大学 | Method for determining phase transition temperature of two-component liposome |
| CN111638188A (en) * | 2020-05-27 | 2020-09-08 | 东北农业大学 | Method for measuring entrapment rate of lipid liposome |
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Non-Patent Citations (2)
| Title |
|---|
| Qingguo Xu,etc.Encapsulation and release of a hydrophobic drug from hydroxyapatite coated liposomes.《Biomaterials》.2007,(第28期),2688-2694. * |
| 张芬,安学勤.盐酸小檗碱脂质体的制备工艺研究.《南京师大学报(自然科学版)》.2006,第29卷(第1期),56-58. * |
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