CN106546706B - In-vitro release test method for liposome drug prepared by pH gradient active drug loading method - Google Patents
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Abstract
The invention relates to a test method of liposome medicine, in particular to in-vitro release of liposome medicine prepared by a pH gradient active medicine carrying method. The invention controls the release rate of the release promoting agent to release the compound and the dissolution balance of the compound in solution and air by the vertical displacement motion mode of the container, indirectly controls the release rate of the medicine and simulates the dynamic action of blood circulation in vivo on the medicine. The release rate can be accurately adjusted by controlling the parameters such as the addition amount, the temperature or the rotating speed of the release promoting agent. The method has the advantages of high controllability, convenient operation and good repeatability. Ion exchange resin can be added to adsorb released free medicine, so as to achieve the effect of simulating the state of the leakage groove in vivo.
Description
Technical Field
The invention relates to a test method of liposome drugs, in particular to a test method for simulating the drug release process of liposome drugs after entering the body in vitro.
Background
Since the discovery of phospholipid bilayer membranes in the 90's of the 20 th century, the study of liposomes as drug delivery vehicles has been under-developed. Liposomes refer to the microvesicles formed by encapsulating a drug within a lipid bilayer. The phospholipid molecules are inserted into water from the hydrophilic head part of the molecule in the water, and the hydrophobic tail part extends to the air to form the spherical liposome with a lipid bilayer structure, wherein the particle size is usually 25-1000 nm.
The double-layer membrane structure of the liposome is similar to a biological membrane, and has good biocompatibility; the liposome-encapsulated drug has the advantages of targeting property, long-acting property, low toxicity and good encapsulation protection. Thus, liposomes are used for the delivery of a variety of active drugs to improve the blood circulation time after the drug enters the body, increasing accumulation at the target site. The function of the liposome can be changed by changing the particle size, the lipid formulation composition and the surface characteristics of the lipid membrane. Different lipid materials can be used for preparing various functional liposomes such as long-circulating liposome, thermosensitive liposome, pH sensitive liposome, immunoliposome and the like, thereby achieving the purpose of special treatment.
Therefore, in order to rationally design a liposome drug delivery system, detailed studies on the in vivo and in vitro release behaviors of drugs are required, and the drug release process after the liposome drugs enter the body is tested by in vitro simulation.
However, the only test methods available today have serious shortcomings. Taking doxorubicin liposome as an example, the in vitro release investigation method in the FDA guideline only gives two experimental parameters of medium pH and temperature, and simulates the in vivo physiological environment by a large volume of buffer or a buffer containing human plasma. However, because doxorubicin exists in liposome in a precipitated form of doxorubicin sulfate, the solubility is very low, so that the release rate is too slow to meet the requirement of rapid evaluation in a quality control system.
The Low Frequency Ultrasound (LFUS) method adopted by the Avi Schroeder and Yechezkel Barenholz can release 80% of drugs within a short time (3 minutes), but the low frequency ultrasound can cause transient pore-like defects of a lipid membrane, so that the damage to the liposome is severe, and the released liposome has no preparation concept and cannot simulate the physiological environment in vivo. Atsuko Hioki and Yoshie Maitani use Bovine Serum Albumin (BSA) added under high temperature (50 ℃) conditions to accelerate drug release. Although the method can complete the release in a short time, the release curve is quite unstable and has poor repeatability, and the method cannot be used as a standard for quality evaluation.
JingXia Cui et al added ammonium chloride to liposomes and incubated at 52 ℃ to examine doxorubicin release behavior, the principle of which Is that ammonium chloride solution Is decomposed during heating to generate ammonia molecules, and at 52 ℃ close to the phospholipid phase transition, the liposome membrane fluidity Is increased, so that the ammonia molecules can penetrate through the lipid membrane to enter the liposome interior, and then undergo displacement reaction with doxorubicin sulfate doxorubicin, and the displaced free doxorubicin molecules penetrate through the lipid membrane to diffuse to the liposome exterior, thereby achieving doxorubicin release, In which process the pH inside the original liposome Is low, and the ammonia molecules enter the liposome interior, thereby breaking the pH balance, thereby releasing the drug, for a detailed discussion of this method, see documents ① J ControlRelease,2007 r2, 118(2), 204-15, Direct comparison of two-packed liposome oxidation reagents for AUC ②, for biological reactions, 2011 3-333, and 333, 3-3, and 333.
The method is based on the idea that a release-promoting agent is added in a solid preparation dissolution release experiment to promote drug release, and is a recognized method for investigating doxorubicin liposome. However, in this method, the major driving force for doxorubicin release is the amount of ammonium chloride added, which is added in large amounts if the desired doxorubicin release is to be achieved. But simultaneously, the chemical action of the ammonium chloride is very quick, 60 percent of release can be achieved within 0.5h in a short time, and the plateau period can be achieved within 3 h. Therefore, the detail characteristics of the release curve obtained by the method are not obvious, the main release period of the adriamycin cannot be prolonged, so that the distinguishability between different preparation formulas is increased, and the in-vivo and in-vitro release relationship of the medicament cannot be simulated.
Disclosure of Invention
The invention aims to provide a method for testing in-vitro release of liposome medicine, which has high controllability of medicine release rate and good repeatability.
An in vitro release test method of liposome medicine prepared by a pH gradient active drug loading method, wherein the liposome medicine is in a solution form and is dissolved with liposome particles encapsulating the medicine, comprises the following steps:
(1) filling the mixed solution of the liposome medicine and the release-promoting agent into a container at a preset temperature, and closing the container, wherein no more than 30% of the volume of the container is filled with air;
(2) -subjecting the container to a movement with a back and forth displacement in a vertical direction;
(3) samples were taken at desired time points and either the undelivered drug concentration or the released drug concentration of the liposomal drug was determined and the drug release rate was calculated.
Further, the temperature preset in the step (1) is the phospholipid phase transition temperature of the liposome particles +/-20 ℃.
Further, the drug is an anthracycline drug, vincristine or amphotericin B, preferably adriamycin.
Furthermore, the concentration of the release-promoting agent in the mixed solution is 5-100 mM. The pH value of the mixed solution is 5.5-6.5.
Further, the movement in the step (2) is rotation around a non-vertical rotation axis, and the container is strip-shaped and is installed in a manner of being radially perpendicular to the rotation axis.
Further, the rotation motion of the step (2) is completed by using a molecular hybridization instrument, the rotating shaft is horizontal, the container is a centrifugal tube, and the rotating speed is 15-50 rpm.
Further, in the step (1), a predetermined amount of ion exchange resin for adsorbing the released free drug is added to the container, the predetermined amount being not less than an amount required for adsorbing all of the drug encapsulated in the liposome drug; the concentration of undelivered drug in the liposomal drug is determined in step (3).
Still further, the ion exchange resin is a cation exchange resin.
Compared with the prior art, the method has the following advantages:
(1) the dynamic action of in vivo blood circulation on the medicine is simulated through the motion of the container, the release behavior of the medicine after entering the body can be better simulated, and the release process is closer to the real situation of in vivo release.
(2) All operating parameters, including concentration, temperature, speed of rotation of the release-promoting agent, etc., are controllable, thereby conveniently adjusting the release rate, whether short-time rapid release is required or the main release period time is required to be lengthened to enhance the distinctiveness among a plurality of release profiles.
(3) The experimental repeatability is far higher than that of various methods in the prior art, and the reliability is high.
(4) If the ion exchange resin is added into the container, the released free medicine can be immediately adsorbed by the resin, the effect of simulating the in-vivo leakage groove state is achieved, the concentration of the residual unreleased medicine can be rapidly measured, and the experimental efficiency is greatly improved.
In conclusion, the invention is the only test method which can meet the requirements of a drug production quality evaluation system at present.
Drawings
FIG. 1: a release behavior comparison chart under three different motion modes of standing, stirring and rotating;
FIG. 2: a graph comparing the release rate curves of 6 samples at each time point in example 2;
FIG. 3: the temperature and the pH of the PBS buffer affect the release rate;
FIG. 4: a graph comparing the release rates of doxorubicin at different ammonium chloride concentrations in example 12;
FIG. 5: a graph comparing the release rates of the different formulations of liposomes in example 14;
FIG. 6: a graph comparing the release rates of the samples from each set at 3ml loading in example 14;
FIG. 7: comparative release rate for each set of samples at 4ml loading in example 15.
Detailed Description
The test method disclosed by the patent is suitable for all the liposomes prepared by using a pH gradient active drug loading method, such as adriamycin liposome, vincristine liposome and the like.
In this patent, the term release-promoting agent is defined to broadly include all substances that promote the release of liposomal drugs, including surfactants, or compounds that release ammonia molecules in solution, such as ammonium sulfate, ammonium chloride, ammonium acetate, ethanolamine, diethylamine, ethylenediamine, and the like. Any compound which can be decomposed in an aqueous solution and can enter the liposome and break the pH gradient which keeps the drug stably existing in the liposome can be used.
The amount of release-enhancing agent added is one of the important control parameters. In the case of ammonium chloride, the lower the concentration of ammonium chloride in the solution, the less ammonia molecules are released, and the less ammonia molecules the liposome drug in the mixed solution can contact, the lower the release rate. Meanwhile, the addition amount of the release-promoting agent is also related to the type of the release-promoting agent. For example, ammonium acetate is a weak electrolyte relative to ammonium chloride, and thus, if an effective amount of ammonia molecules is to be ionized, a much greater amount of ammonium acetate is added than ammonium chloride.
The concentration of the liposomal drug must be set according to experimental needs, and is an irregulable parameter, and in pharmaceutical experiments it is common to select a concentration that approximates the distribution of the drug in the human body. There is no strict limit to the ratio of liposomal drug to release-enhancing agent in solution. The mass molar ratio range can reach 1 (0.1-5), even wider, because besides the release promoter, the parameters such as temperature, rotating speed and the like can also play a role in regulating the release rate of the medicine, and the effects of the parameters can be mutually offset to a certain extent. Moreover, the experiment fails because too little or excessive release-promoting agent is added, and only the release curve with ideal rate cannot be obtained, but sometimes we need to examine the release condition under extreme conditions, so that there is no absolute 'allowable interval' in the addition amount of the release-promoting agent.
The test temperature is typically selected to be near the phospholipid phase transition temperature of the liposome particles. If the phospholipid phase transition temperature of the adriamycin liposome is 52 degrees, the test is generally carried out at 50 degrees or above. Because the container in the method needs to rotate, and a water bath can not be adopted like a standing method, the temperature control condition is not the same as that of the standing method, the temperature usually fluctuates by 0.5-1 ℃, and the small-amplitude temperature change of the degree has little influence on the experimental result.
In applying motion to the solution, rotation about a horizontal axis using a molecular hybridization apparatus is most convenient. The centrifuge tubes are preferably arranged vertically in a radial centripetal manner around the axis of rotation. The rotation mode can enable a small amount of air in the centrifugal tube to form bubbles to be dispersed in the solution, so that the blood circulation in vivo can be simulated, the release of the medicament can be promoted, and the physical collision between the bubbles and the liposome can damage a lipid membrane to cause the release of the medicament.
However, if too much air is in the centrifugal tube, although the drug release can be accelerated, the process of air dispersion with rotation is a chaotic system and is difficult to control. Under the condition, even if all parameters such as temperature, rotating speed, air volume and the like are kept consistent, the repeatability of the drug release curve obtained by repeated tests is not as good as that of the drug release curve obtained by only retaining a small amount of air, and the experimental result obtained by carrying out chemical release by mainly utilizing the release-promoting agent is mainly used. The air in the container cannot be completely removed either because the ammonia molecules decomposed from ammonium chloride must have an equilibrium of entering the air from the solution and returning to the solution from the air. On the other hand, the released free drug can be adsorbed by the ion exchange resin, and the concentration of the residual drug can be conveniently and directly measured by adsorbing the released free drug by the ion exchange resin.
The cation resin can adsorb anthracycline (daunorubicin, adriamycin, aclarubicin, epirubicin, idarubicin, valrubicin, mitoxantrone), vincristine, amphotericin B, etc., wherein the strong acid type cation exchange resin has strong acidic reactive group such as sulfonic group (-SO)3H) Can widely adsorb all cations; the weak acid type cation exchange resin has a weak reactive group such as carboxyl (-COOH group), and only cations such as Ca in the weak base can be exchanged2 +、Mg2 +. The anion exchange resin is mainly strong base type anion exchange resin, and can exchange and adsorb all anions.
If the ion exchange resin is added before the experiment begins, the effect of simulating the state of the leakage groove in the body can be achieved; if ion exchange resin is added after the experiment is finished and the sample is taken, only the function of adsorbing free drugs is achieved, and the operation of the sample before detection is simplified.
The following examples further illustrate specific embodiments of the present invention.
(1) Investigating the release behavior under three different motion modes of standing, stirring and rotating
Comparative example 1: standing-52-10% of resin-33 mM ammonium chloride
A. Preparing adriamycin liposome solution:
placing Phosphate Buffered Saline (PBS) with pH of 6.0 in an ultrasonic cleaning machine, and ultrasonically cleaning for 20min to remove gas; the initial drug concentration was calculated by pipetting 2.5ml of doxorubicin liposome into a 100ml volumetric flask using a pipette and diluting 40-fold of the volume with the above-mentioned PBS (pH 6.0).
B. Pretreatment with cation exchange resin
Adding cation exchange resin into the chromatographic column, draining water naturally, and blowing and beating with ear washing ball until no continuous liquid flows out.
C. In vitro Release assay
a. Adding 3ml of adriamycin liposome diluent into a 5ml EPPENDOF centrifugal tube;
b. adding ammonium chloride into the solution to form a mixed solution, so that the concentration of the ammonium chloride is 33 mM;
c. sealing the centrifugal tube, carrying out constant-temperature water bath at 52 ℃, then respectively sampling after 3h, 6h and 8h, and taking 3 samples at each time point to average;
d. the tubes were removed and immediately placed in an ice-water bath, after cooling, 0.3g (w/v ═ 10%) of each cation exchange resin was added, vortexed for 30 seconds to adsorb the free drug, and 1ml of the supernatant was subjected to HPLC at 4 ℃ to detect the residual drug concentration in the sample supernatant.
e. Drug release rate ═ (1-residual drug concentration/initial drug concentration) × 100%.
Comparative example 2: stirring-52 deg.C-10% resin-33 mM ammonium chloride
Steps A and B are the same as in comparative example 1.
In step C, 50ml of doxorubicin liposomes were placed in a 250ml eggplant-shaped bottle, ammonium chloride was added at the same final concentration of 33mM, and resin was added at the same amount of 10% (5 g). Then the eggplant-shaped bottle was put into a constant temperature water bath to be subjected to water bath at 52 ℃ while stirring at 210rpm, and samples were taken after 3 hours, 6 hours, and 8 hours, respectively.
During sampling, stirring is stopped firstly, 1ml of supernatant is removed by using a liquid-removing gun after the solution is kept stand, the supernatant is immediately placed into an ice water bath, and the concentration of the residual medicine in the supernatant of the sample is detected by HPLC at 4 ℃.
Drug release rate ═ (1-residual drug concentration/initial drug concentration) × 100%.
Example 1: spin-52 deg.C-10% resin-33 mM ammonium chloride
Steps A and B are the same as in comparative examples 1 and 2.
C. In vitro Release assay
a. Adding 5ml of adriamycin liposome diluent into a 5ml of EPPENDOF centrifugal tube;
b. adding ammonium chloride into the solution to form a mixed solution, so that the concentration of the ammonium chloride is 33 mM;
c. adding 0.5g (w/v ═ 10%) of cation exchange resin to the mixed solution;
d. setting the temperature of the molecular hybridization instrument to be 52 ℃ and the rotating speed to be 0rpm, preheating and simultaneously adding samples; .
e. After the centrifugal tube is closed, the centrifugal tube is fixed on a hybridization tube frame of the molecular hybridization instrument in a radial centripetal manner and perpendicular to a rotating shaft;
f. the temperature of the molecular hybridization apparatus was set at 52 ℃ and the rotation speed was set at 25rpm, and the rotation was started and timed.
g. Sampling after 3h, 6h and 8h respectively, and taking 3 samples at each time point to obtain an average value;
h. immediately after removal of the tube, the tube was placed in an ice-water bath, and the concentration of the residual drug in the sample supernatant was measured by HPLC at 4 ℃.
i. Drug release rate ═ (1-residual drug concentration/initial drug concentration) × 100%.
Adriamycin Release Rate comparison of Table 1, comparative example 2, example 1
The accompanying figure 1, which corresponds to the experimental data in table 1, compares the release behavior of doxorubicin liposomes for three different modes of motion.
From the analysis of mechanism, the common point of the three movement modes is that the main power for promoting the release of the adriamycin is ammonia molecules generated by the decomposition of ammonium chloride, and the main influencing factor is the concentration of the ammonium chloride. The difference is that the ammonia molecules generated by the decomposition of ammonium chloride move differently under different movement modes. Ammonia gas is very soluble in water and is lighter in density than air, and is redissolved in solution immediately after being diffused to the upper layer of the liquid surface in a stirring or rotating motion mode.
Under the standing condition of the comparative example 1, ammonia gas released by ammonium chloride-ammonia gas enters the inside of the liposome through a membrane-doxorubicin is released in a free manner, and the whole process depends on chemical equilibrium and is not interfered by external acting force. The release of ammonia and the transmembrane process are very rapid processes, so that the release of adriamycin also reaches a peak in a short time, and then gradually slows down.
Under the motion conditions of comparative example 2 and example 1, the ammonia gas dissolved and released in the air and the solution reach a dynamic equilibrium. Because the air exists in the centrifuge tube/eggplant-shaped bottle, ammonia molecules are decomposed from the ammonium chloride, and are partially diffused into the air along with the stirring and rotating processes, and then are returned to the solution again in the stirring and rotating processes. This dynamic process allows the ammonia concentration in the system to be kept in equilibrium.
The difference between the comparative example 2 and the example 1 is that when the stirring mode is adopted, the eggplant-shaped bottle is an open system, the test temperature is higher, and part of ammonia gas volatilizes from the bottle mouth. This resulted in the same initial release concentration for both systems, but the late release ratio for comparative example 2 was lower than for the closed system, at the same ammonium chloride concentration.
Under the three systems, the release rate is most uniform by adopting a closed container rotating mode, and the finally achieved release proportion is the highest.
Under the condition of standing, if the initial release rate is wanted to be reduced so as to make a more detailed observation on the release behavior in this stage, the ammonium chloride concentration can be reduced. However, this also results in the final release rate being difficult to achieve, for example, about 55% in fig. 2, but if the ammonium chloride concentration is reduced, it may reach only 40% to enter the plateau phase, and it is not possible to pull the whole release rising phase away under the condition of reducing the ammonium chloride addition (concentration) and simultaneously achieve the total effective release amount.
The method disclosed by the patent has the advantages that the release rate is uniform in the front period and the back period, the final release ratio is high, and the release behavior in a human body can be simulated to the maximum extent by adding the cation adsorption resin.
(2) Investigating the repeatability of the method
Example 2: 52-10% resin-48 mM ammonium chloride
Step A, B, C is the same as in example 1. Except that the final concentration of ammonium chloride in the mixed solution was 48 mM.
Samples were taken at time points of 1h, 2h, 4h, 6h, 8h, and 6 samples were taken at each time point, and the Standard Deviation (SD) was determined. The test results are shown in table 2.
Table 2, release rates of 6 samples per time point in example 2
FIG. 2 is a plot of the data from Table 2. As can be seen from the figure, in the group of 6 simultaneous experiments, the doxorubicin release rates were very close and the method was very reproducible.
(3) Examine the influence of temperature and pH of PBS buffer on the release rate
Example 3: 37-10% resin-33 mM ammonium chloride-pH 5.5
Example 4: 37 ℃ -10% resin-33 mM ammonium chloride-pH 6.0
Example 5: 37-10% resin-33 mM ammonium chloride-pH 6.5
The experimental conditions for examples 3, 4 and 5 are as indicated under the heading, otherwise identical to example 1 except that the temperature was set at 37 ℃; adjusting the pH value of the PBS buffer solution to 5.5, 6.0 and 6.5 respectively; sampling time points were 4h, 8h, and 1 sample was taken at each time point.
Example 6: 47 ℃ -10% resin-33 mM ammonium chloride-pH 5.5
Example 7: 47 ℃ -10% resin-33 mM ammonium chloride-pH 6.0
Example 8: 47 ℃ -10% resin-33 mM ammonium chloride-pH 6.5
The experimental conditions for examples 6, 7 and 8 are as indicated under the headings, otherwise the same as for example 1, except that the temperature was set at 47 ℃; adjusting the pH value of the PBS buffer solution to 5.5, 6.0 and 6.5 respectively; sampling time points were 4h, 8h, and 1 sample was taken at each time point.
Example 9: 52 ℃ -10% resin-33 mM ammonium chloride-pH 5.5
Example 10: 52 ℃ -10% resin-33 mM ammonium chloride-pH 6.0
Example 11: 52 ℃ -10% resin-33 mM ammonium chloride-pH 6.5
The experimental conditions for examples 9, 10 and 11 are as indicated under the headings, otherwise the same as for example 1, except that the temperature was set at 52 ℃; adjusting the pH value of the PBS buffer solution to 5.5, 6.0 and 6.5 respectively; sampling time points were 4h, 8h, and 1 sample was taken at each time point.
The experimental data for examples 3-11 are shown in Table 3 below, while FIG. 4 is a graph of Table 3.
TABLE 3 comparison of doxorubicin release rates for examples 3-11
As can be seen from FIG. 3, the release rate of doxorubicin is greatly increased with the increase of temperature under the same pH condition. Meanwhile, under the same temperature condition, the release of the adriamycin is accelerated by increasing the pH value of the PBS buffer solution, mainly because the decomposition of ammonia molecules in the ammonium chloride is more facilitated by the increase of the pH value.
(4) Examination of the Effect of ammonium chloride concentration on Release Rate
Example 12: 52-10% of resin
The experimental conditions of example 12 were as indicated under the headings, and the same as in example 1 except that the concentrations of ammonium chloride in the mixed solution were adjusted to 0mM, 8mM, 17mM, 33mM, 48mM, 64mM and 77mM, respectively, and the experiments were carried out one by one. Sampling time points were 6h, and 1 sample was taken at each time point.
The data for example 12 is shown in table 4 below, and figure 4 is a plot of the data from table 4.
TABLE 4 comparison of Adriamycin Release Rate for different ammonium chloride concentrations in example 12
As can be seen from fig. 4, the doxorubicin release rate increased significantly with increasing ammonium chloride ratio. The release rate of the adriamycin is mainly determined by the addition amount of ammonium chloride, so that the release rate of the adriamycin can be controlled by controlling the concentration of the ammonium chloride.
(5) Examine the distinctiveness of the test method on doxorubicin liposomes of different formulations
Example 13: 52-10% resin-33 mM ammonium chloride
The experimental conditions for example 13 are as indicated under the title, all the same as example 1. The doxorubicin liposomes tested were distinguished in 3 different formulations of product:
prescription 1: HSPC (lecithin)/Chol (cholesterol)/PEG- (55: 37: 5);
prescription 2: HSPC/Chol/PEG- (46: 46: 5);
prescription 3: HSPC/Chol/PEG- (55: 37: 1).
The characteristic parameters and experimental data of the three formulations are shown in table 5 below, and fig. 6 is a graph of the data of table 5.
Table 5, comparison of Properties and Release Rate for liposomes of different formulations in example 13
In liposome formulations, cholesterol acts as a lipid membrane fluidity modifier. It can not only reduce membrane permeability and reduce drug leakage, but also maintain lipid membrane with certain flexibility and enhance the ability of liposome vesicle to resist external condition change.
As can be seen from Table 5, the change rule of the drug release curves of different prescriptions obtained by the test method conforms to the theoretical inference: the release rate of formula 2 was higher than formula 1, with no significant difference for formula 3. The results show that the test method has good distinguishability for different prescriptions.
Example 14: 49-10% resin-ammonium sulfate
Step A, B, C is the same as in example 1. Except that the temperature was set at 49 ℃ and the release-promoting agent was changed to ammonium sulfate. Samples were taken at time points 1h, 2h, 4h, 6h, 8 h.
Prescription 4: HSPC/Chol/PEG- (56: 37: 5), encapsulation efficiency 95.35%, particle size 101.23, ammonium sulfate concentration 250 mM;
prescription 5: HSPC/Chol/PEG- (62: 37: 5), encapsulation efficiency 96.10%, particle size 97.63, ammonium sulfate concentration 250 mM;
prescription 6: HSPC/Chol/PEG- (56: 37: 5), encapsulation efficiency 95.84%, particle size 97.29, ammonium sulfate concentration 200 mM.
The test results are shown in table 6.
Table 6, characterization of liposomes of different formulations and comparison of release rates in example 14
Fig. 5 is a drawing of table 6. As can be seen from the figure, even if the ratio of phospholipid and cholesterol in the formulation was changed only slightly, a significant difference in the drug release behavior was observed in this experiment.
This is because a change in the ammonium sulphate concentration changes the precipitation morphology of doxorubicin sulphate in the aqueous phase within the liposomes, thereby affecting its release behaviour. From the results, the concentration of ammonium sulfate is reduced, and the precipitation crystalline state of the doxorubicin sulfate is converted into a colloidal state, so that the release of the doxorubicin is facilitated. Thus, the method is well distinguishable for liposomes with minor differences in prescription.
(7) Investigating the influence of the air ratio on the Release Rate
Example 15: 5ml tube-52 ℃ -10% resin-48 mM ammonium chloride-3 ml liposome
Step A, B, C is the same as in example 2. Except that the amount of the liposome drug added was 3 ml. Samples were taken at time points of 2h, 4h, 6h, and 6 samples were taken at each time point and tested, and the SD value was determined. The test results are shown in Table 7.
Table 7, 6 sample release rates per time point in example 15
FIG. 6 is a plot of the data from Table 7. As can be seen from the figure, when the amount of the added liquid is reduced to 3ml, the difference of 6 samples operated in the same group is large, and the SD value is also large. The reproducibility was significantly reduced compared to example 2, which was carried out under the same conditions.
Example 16: 5ml tube-52 ℃ -10% resin-48 mM ammonium chloride-4 ml liposome
Step A, B, C is the same as in example 15. Except that the loading of the liposome drug was 4 ml. The test results are shown in Table 8.
Table 8, 6 sample release rates per time point in example 16
FIG. 7 is a plot of the data from Table 8. As can be seen from the figure, when the amount of addition was increased to 4ml, the reproducibility was significantly improved as compared with example 15, but there was a gap from the results obtained in example 2.
In summary, the comparison of examples 2, 15, 16 verifies the conclusion that an increase in air volume within the container leads to a decrease in experimental reproducibility. Therefore, it is desirable that the volume of gas in operation not exceed 1/3 of the volume of the container. Considering the convenience of experimental operation, under the condition that a centrifugal tube and a molecular hybridization instrument are used as experimental equipment, the medicine can be charged according to the maximum volume defined by the operating specification of the centrifugal tube directly without paying attention to the absolute volume of residual air. Under the condition, the repeatability of the drug release curve obtained by a plurality of tests is enough to meet the requirement of a quality evaluation system.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that these examples are merely illustrative of the desired test results that can be obtained by adjusting various parameters, and are not intended to guide the specific setting of parameters. With the understanding of the nature and amount of release-enhancing agents, and the principle of temperature effect on drug release rates, the best test protocol can be obtained with a limited number of experiments for any given formulation of liposomes and experimental purposes, and is not limited to the specific embodiments described above, nor to the use in testing drug release from doxorubicin or other anthracycline-like liposomes. Various changes or modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (9)
1. An in vitro release test method of liposome medicine prepared by a pH gradient active drug loading method, wherein the liposome medicine is in a solution form and is dissolved with liposome particles encapsulating the medicine, and is characterized by comprising the following steps:
(1) filling the mixed solution of the liposome medicine and the release-promoting agent into a container at a preset temperature, and closing the container, wherein no more than 30% of the volume of the container is filled with air;
(2) -subjecting the container to a movement with a back and forth displacement in a vertical direction;
(3) sampling at a desired time point and determining the concentration of the undiseased drug or the concentration of the released drug of the liposome drug, and calculating the release rate of the drug;
the movement in the step (2) is rotation around a non-vertical rotation axis, and the container is strip-shaped and is installed in a manner of being radially perpendicular to the rotation axis.
2. The in vitro release assay of claim 1, wherein the temperature preset in step (1) is the phospholipid phase transition temperature of the liposome particles ± 2 ℃.
3. The in vitro release test method according to claim 1, wherein the mass molar ratio of the liposome drug to the release promoter in the mixed solution in the step (1) is in the range of 1 (0.1-5).
4. The in vitro release assay of claim 1, wherein the concentration of the release-enhancing agent in the mixed solution in step (1) is 5 to 100 mM.
5. The in vitro release assay of claim 1, wherein the movement is accomplished using a molecular hybridization apparatus, the axis of rotation is horizontal, and the container is a centrifuge tube.
6. The in vitro release test method according to claim 1, wherein in the step (1), a predetermined amount of ion exchange resin for adsorbing the released free drug is added to the container, the predetermined amount being not less than an amount required for adsorbing all of the drug encapsulated in the liposome drug.
7. The in vitro release test method according to claim 6, characterized in that the ion exchange resin is a cation exchange resin.
8. In vitro release assay method according to claim 1, characterized in that said drug is an anthracycline, vincristine or amphotericin B.
9. The in vitro release test method according to claim 8, wherein said anthracycline is doxorubicin.
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