CN112826795B - Tetrandrine-loaded liposome preparation and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the field of pharmaceutical preparations, and discloses a tetrandrine-loaded liposome preparation which comprises the following raw materials: the soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol, tetrandrine and perfluorooctyl bromide, wherein the mass ratio of the sum of the soybean lecithin and the dipalmitoyl phosphatidyl ethanolamine to the cholesterol to the tetrandrine is 4:1: 2.5-3.5. The preparation method of the tetrandrine-loaded liposome preparation comprises the following steps: dissolving soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol and tetrandrine in a solvent, performing rotary evaporation to obtain a uniform thin liquid membrane, hydrating the thin liquid membrane, adding perfluorooctyl bromide, performing ultrasonic oscillation in ice bath, centrifuging, and collecting precipitate to obtain the tetrandrine-loaded liposome preparation. The tetrandrine-loaded liposome preparation is applied to the preparation of the medicament for treating the xerophthalmia, has small influence on the intraocular pressure, and greatly improves the encapsulation rate and the drug loading rate of the tetrandrine.
Description
Technical Field
The invention relates to the field of medicinal preparations, in particular to a tetrandrine-loaded liposome preparation as well as a preparation method and application thereof.
Background
Dry eye is a multifactorial induced disease with increased tear film permeability and ocular surface inflammation. In the current main strategies for treating dry eye, artificial tears can lubricate eyes and relieve tear evaporation, and have small side effects, so the artificial tears are most commonly used in the treatment of dry eye; because topical anti-inflammatory drugs such as glucocorticoids can break the vicious circle of surface damage, greatly ameliorating the symptoms and clinical signs of moderate to severe dry eye, artificial tears are also commonly used in combination with topical anti-inflammatory drugs. However, topical anti-inflammatory drugs have limited their use due to their long-term use, increasing intraocular pressure and inducing complications such as cataract formation. For this reason, it is desirable to find a formulation that has less effect on ocular pressure to treat dry eye.
Tetrandrine (6,6', 7, 12-tetramethoxy-2, 2' -dimethyl berberine, Tet) is a plant alkaloid extracted from the traditional Chinese medicine tetrandra root and is used for treating hypertension, rheumatoid arthritis and pulmonary fibrosis. With the research infiltration, other biological activities of tetrandrine are gradually discovered, including induction of cancer cell apoptosis, reversal of tumor cells, anti-oxidation, anti-inflammation, etc. In ophthalmology, tetrandrine has also been studied for its therapeutic effects on conjunctivitis, ocular hypertension, sub-corneal haze and uveitis. The anti-inflammatory and antioxidant activity of tetrandrine and its effect on conjunctivitis and uveitis suggest that it is a potential anti-inflammatory drug for treating dry eye disease.
The liposome is an artificial spherical vesicle which consists of one or more phospholipid bilayers and is similar to a cell membrane, is always researched as a water-soluble and fat-soluble drug carrier since the 70 th 20 th century, and is widely researched in ophthalmology due to the complete biodegradability and relative non-toxicity of the liposome. Therefore, it is desired to encapsulate tetrandrine in liposome for preparing a preparation for treating dry eye, so as to obtain a dry eye treatment preparation with less influence on eye pressure.
Disclosure of Invention
The invention aims to provide a tetrandrine-loaded liposome and a preparation method and application thereof, and aims to solve the problem that the existing anti-inflammatory medicinal preparation has great influence on eye pressure when used for treating xerophthalmia.
In order to achieve the purpose, the invention provides a tetrandrine-loaded liposome preparation, which comprises the following raw materials: the soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol, tetrandrine and perfluorooctyl bromide, wherein the mass ratio of the sum of the soybean lecithin and the dipalmitoyl phosphatidyl ethanolamine to the cholesterol to the tetrandrine is 4:1: 2.5-3.5.
The invention also provides a preparation method of the tetrandrine-loaded liposome preparation, which comprises the following steps:
s1, dissolving soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol and tetrandrine in a solvent, and performing rotary evaporation to obtain a uniform thin liquid film;
s2, hydrating the thin liquid film obtained in the step S1, adding perfluorooctyl bromide, and performing ultrasonic oscillation in ice bath to obtain a system containing PFOB @ LIP-Tet nanoparticles;
s3, centrifuging the system obtained in the step S2, and collecting sediments to obtain the tetrandrine-loaded liposome preparation.
The principle and the advantages of the scheme are as follows: according to the scheme, a thin film dispersion-hydration-ultrasonic method is adopted, tetrandrine (Tet) is carried by a liposome and wrapped perfluorooctyl bromide (PFOB) is utilized to prepare PFOB @ LIP-Tet nanoparticles, the particle size of the PFOB @ LIP-Tet nanoparticles is 103.36 +/-7.844 nm, and the encapsulation rate and the drug loading rate of Tet in the PFOB @ LIP-Tet nanoparticles can reach (79.9 +/-0.9)% and (48.0 +/-0.6)%. When the tetrandrine-loaded lipidosome preparation in the scheme is applied to preparation of a medicament for treating xerophthalmia, the influence on intraocular pressure is small, and the problem that the existing anti-inflammatory medicinal preparation has a large influence on intraocular pressure when used for treating xerophthalmia is solved.
Preferably, as an improvement, in step S2, the ratio of the amount of perfluorooctyl bromide to the amount of tetrandrine is 150 μ L:6mg to 250 μ L:6 mg.
Has the advantages that: when the addition amount of the perfluorooctyl bromide is limited in the range, the PFOB @ LIP-Tet nanoparticles with high encapsulation efficiency can be obtained.
Preferably, as a modification, in step S1, the solvent is chloroform, dichloromethane or methanol.
Has the advantages that: chloroform, dichloromethane or methanol are used for dissolving soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol and tetrandrine, so that the four are fully mixed, and the subsequent generation of the liposome carrying the tetrandrine is facilitated.
Preferably, as a modification, in step S2, the thin liquid film is hydrated using a phosphate buffer.
Has the advantages that: the thin liquid membrane is hydrated by using the phosphate buffer solution, so that the perfluorooctyl bromide can be more fully contacted with substances contained in the thin liquid membrane, and the pH value in the reaction process is adjusted, thereby being beneficial to the subsequent generation of the tetrandrine-loaded liposome coated with the perfluorooctyl bromide.
Preferably, as an improvement, in step S2, the ultrasonic frequency is 10kHz to 30kHz, the ultrasonic power is 55w to 100w, and the ultrasonic time is 3 min to 5 min.
Has the advantages that: experiments show that PFOB @ LIP-Tet nanoparticles with high encapsulation efficiency can be obtained when the ultrasonic frequency is 10-30 kHz, the ultrasonic power is 55-100 w and the ultrasonic time is 3-5 min in the ultrasonic oscillation process.
Preferably, as an improvement, in step S3, the centrifugation speed is 5000 to 8000rpm, and the centrifugation time is 5 to 10 min.
Has the beneficial effects that: under the centrifugal condition, the liquid in the system can be well separated from the PFOB @ LIP-Tet nanoparticles.
Preferably, as an improvement, in step S1, the rotation speed of the rotary evaporation is 120 to 150rpm, and the temperature of the rotary evaporation is 45 to 55 ℃.
Has the beneficial effects that: when the rotating speed of the rotary evaporation is 120-150 rpm and the temperature of the rotary evaporation is 45-55 ℃, a uniform thin liquid film can be formed, and the unevenness of the formed thin liquid film is avoided.
Drawings
FIG. 1 is a graph showing the intraocular pressure trend of the model rabbits of the third experiment.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1
A tetrandrine-loaded liposome preparation comprises the following raw materials: the composition comprises soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol, tetrandrine and perfluorobromooctane, wherein the mass ratio of the sum of the mass of the soybean lecithin and the dipalmitoyl phosphatidyl ethanolamine to the mass of the cholesterol to the tetrandrine is 4:1: 2.5-3.5, and the ratio of the amount of the perfluorobromooctane to the amount of the tetrandrine is 150 muL: 6 mg-250 muL: 6 mg.
A preparation method of a tetrandrine-loaded liposome preparation comprises the following steps:
s1, dissolving soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol and tetrandrine in a solvent, and performing rotary evaporation to obtain a uniform thin liquid film; in the embodiment, the soybean lecithin, the dipalmitoyl phosphatidylethanolamine, the cholesterol and the tetrandrine are respectively 7mg, 1mg, 2mg and 6mg, the solvent is chloroform, and the dosage of the chloroform is 10 mL; the rotational speed of the rotary evaporation was 120rpm, and the temperature of the rotary evaporation was 50 ℃. In addition, in other embodiments of this embodiment, the solvent can be dichloromethane or methanol, the rotation speed of the rotary evaporation can be selected from 120 to 150rpm, and the temperature of the rotary evaporation can be selected from 45 to 55 ℃.
S2, hydrating the thin liquid membrane obtained in the step S1 by using 2mL of phosphate buffer solution, adding 200 mu L of perfluorooctyl bromide, and carrying out ultrasonic oscillation for 4min in ice bath with ultrasonic power of 55w and ultrasonic frequency of 30kHz to obtain a system containing PFOB @ LIP-Tet nanoparticles. In other embodiments of this embodiment, the ultrasonic frequency may be selected from 10kHz to 30kHz, the ultrasonic power may be selected from 55w to 100w, and the ultrasonic time may be selected from 3 min to 5 min.
S3, centrifuging the system obtained in the step S2, removing the supernatant, and collecting the sediment to obtain the tetrandrine-loaded liposome preparation. In this embodiment, the centrifugation rotation speed is 6000rpm, and in other embodiments of this embodiment, the centrifugation rotation speed can be selected from 5000 to 8000rpm, and the centrifugation time can be selected from 5 to 10 min. And (3) when the sediments are collected, re-suspending the sediments by using double distilled water to obtain a suspension, and freeze-drying the suspension to obtain the tetrandrine-loaded liposome.
Examples 2 to 5 and comparative examples 1 to 6 are substantially the same as example 1 except for the points shown in table 1. In addition, the inventors studied the amounts of soybean lecithin, dipalmitoyl phosphatidylethanolamine and cholesterol in the course of experiments, and found that when the mass ratio of the sum of the masses of soybean lecithin and dipalmitoyl phosphatidylethanolamine to cholesterol is 4:1 and the mass ratio of soybean lecithin to dipalmitoyl phosphatidylethanolamine is 7:1, the resulting liposome is more stable, and the stability of the resulting liposome is poor beyond the above mass ratio.
TABLE 1 parameter settings for the examples and comparative examples
Experiment one
Particle size and surface potential measurements were made on the formulations obtained in example 1 and comparative example 6, and it was found that the particle size of the formulation obtained in example 1 (PFOB @ LIP-Tet nanoparticles) was 103.36 + -7.844 nm and the surface potential was-21.833 + -0.556 mV, and the particle size of the formulation obtained in comparative example 6 (PFOB @ LIP nanoparticles) was 79.397 + -7.718 nm and the surface potential was-6.013 + -1.219 mV. It is not difficult to find that the particle size and the surface potential of the preparation obtained in example 1 are changed compared with those of the preparation obtained in comparative example 6, and then the preparation obtained in example 1 is observed by combining a transmission electron microscope, and it is found that PFOB @ LIP-Tet nanoparticles take liposomes as shells, PFOB is in the shells, and Tet is loaded in the liposome shells. Thus, particle size, surface potential and transmission electron microscope observations all demonstrate the successful loading of Tet on liposomes. Examples 2-5 compared to example 1, only the amount of PFOB added was different, and it was confirmed that PFOB @ LIP-Tet nanoparticles were successfully prepared in example 1, and PFOB @ LIP-Tet nanoparticles were also successfully prepared in examples 2-5.
Experiment two
The test calculation of Tet encapsulation rate and Tet drug loading rate is carried out on the samples of example 1-example 5 and comparative example 1-comparative example 5, wherein the samples of example 1-example 5 are PFOB @ LIP-Tet nanoparticles, and the samples of comparative example 1-comparative example 5 are LIP-Tet nanoparticles. Specifically, the test calculation of the Tet encapsulation rate and the Tet drug loading rate of example 1 is taken as an example: and (3) resuspending the sediment obtained in the example 1 by using 10mL of double distilled water to obtain a suspension, taking 5mL of the suspension, and freeze-drying to obtain powdery PFOB @ LIP-Tet nanoparticles. Then, dissolving the powdery PFOB @ LIP-Tet nanoparticles in 5mL of methanol, demulsifying, centrifuging at the rotating speed of 8000rpm, taking the supernatant, and detecting the Tet concentration C in the supernatant by using high performance liquid chromatography T Thus calculating the Tet encapsulation rate and the Tet drug loading rate. The formula for calculating the Tet encapsulation efficiency is shown in formula (1), and the formula for calculating the Tet drug loading efficiency is shown in formula (2).
In the formulae (1) and (2), C T The unit of (1) is mg/mL; in the formula (1), M T Is the added mass of Tet; in formula (2), M L Is the sum of the addition mass of the soybean lecithin, the dipalmitoyl phosphatidylethanolamine and the cholesterol.
The Tet encapsulation efficiency and Tet drug loading efficiency of the formulations obtained in examples 1 to 5 and comparative examples 1 to 5 are shown in table 2.
Table 2 Tet encapsulation and Tet drug loading (mean ± SD, n ═ 3) for the formulations obtained in the examples and comparative examples
As can be seen from Table 2, the Tet encapsulation efficiency and the Tet drug loading rate of the examples 1 to 5 are respectively and remarkably higher than those of the comparative examples 1 to 5, and the addition of perfluorooctyl bromide (PFOB) can effectively improve the Tet encapsulation efficiency and the Tet drug loading rate. Moreover, by comparing the Tet encapsulation efficiency and the Tet drug loading rate of examples 1 to 5, it is not difficult to find that the addition amount of PFOB affects the Tet encapsulation efficiency and the Tet drug loading rate of the PFOB @ LIP-Tet nanoparticles, and the addition amount of PFOB is controlled in a proper range, so that PFOB @ LIP-Tet nanoparticles with higher Tet encapsulation efficiency and Tet drug loading rate can be obtained, and the Tet encapsulation efficiency and the Tet drug loading rate of example 4 are significantly lower than those of examples 1 to 3 and example 5, so that the addition amount of PFOB is controlled in the following ranges: the ratio of the dose of PFOB to the dose of Tet is 150 muL: 6 mg-250 muL: 6 mg.
Experiment three
The preparation obtained in example 1 was resuspended in artificial tear fluid (ATS) to obtain PFOB @ LIP-Tet-ATS suspension drug having a Tet equivalent concentration of 0.1mg/mL, and this suspension drug was applied to the eyes of a group of model rabbits (dry eye rabbits) (three drops of drug per day, 40. mu.L each), which was named as PFOB @ LIP-Tet-ATS group).
The preparation obtained in comparative example 6 was resuspended in Artificial Tears (ATS) to obtain a suspension preparation of PFOB @ LIP-ATS having an equivalent concentration of LIP of 0.2mg/mL, and the suspension preparation was applied to the eyes of a group of model rabbits (dry eye rabbits) (three drops of the preparation per day, 40. mu.L each), which was designated as PFOB @ LIP-ATS group.
Tetrandrine was suspended using Artificial Tear (ATS) to obtain Tet-ATS suspension drug with tetrandrine concentration of 0.1mg/mL, and eye treatment was performed according to the same method on a group of model rabbits (dry eye rabbits) (three times daily with 40 μ L of drug added), which was called Tet-ATS group.
Eyes of another group of model rabbits (dry eye rabbits) were treated with an equivalent amount of Artificial Tears (ATS) (three drops of the drug each at 40 μ L each time) and this group was referred to as a control group.
During treatment, the severity Of Dry Eye symptoms was scored in model rabbits Of PFOB @ LIP-Tet-ATS group, PFOB @ LIP-ATS group, Tet-ATS group and control group, with a scoring criteria reference (the criteria described in Grading Of Corneal and Conjunctional Staining in the Context Of Other Dry Eye Tests, Antbony J.Bron, FCOpbtb, FMedSci, et al, CORNEA,22(2003)640-649), wherein the scoring range was 0-5 points, with 0 points representing mild or severe Dry Eye symptoms, and 5 points representing severe Dry Eye symptoms, i.e., greater points representing more severe Dry Eye symptoms. Day of dosing was day 1, and day of scoring was scored prior to dosing. The scoring results of model rabbits of PFOB @ LIP-Tet-ATS group, PFOB @ LIP-ATS group, Tet-ATS group and control group are shown in Table 3.
Table 3 score of each group of model rabbits (mean ± SD, n ═ 6)
| Day 1 | Day 2 | Day 4 | Day 6 | Day 8 | |
| PFOB @ LIP-Tet-ATS component/component | 5.0±0.0 | 5.0±0.0 | 3.2±0.4 | 1.5±0.5 | 0.5±0.5 |
| Tet-ATS component/component | 5.0±0.0 | 5.0±0.0 | 3.7±0.5 | 2.7±0.5 | 1.5±0.5 |
| PFOB @ LIP-ATS Components/Components | 5.0±0.0 | 5.0±0.0 | 4.0±0.0 | 3.7±0.5 | 3.0±0.6 |
| Control group/fraction | 5.0±0.0 | 5.0±0.0 | 4.7±0.5 | 4.3±0.5 | 3.8±0.4 |
During the treatment period, the intraocular pressure of the model rabbits of each group was measured using a small animal tonometer, and the measurement was performed before the administration on the day of measuring the intraocular pressure. The results of ocular pressure in model rabbits of PFOB @ LIP-Tet-ATS group, PFOB @ LIP-ATS group, Tet-ATS group and control group (ATS in FIG. 1) are shown in FIG. 1, where "0 d" in FIG. 1 indicates the measurement of ocular pressure after 0 day of treatment, i.e., before the administration on day 1, "7 d" indicates the measurement of ocular pressure after 7 days of treatment, i.e., before the administration on day 8, "14 d" indicates the measurement of ocular pressure after 14 days of treatment, i.e., before the administration on day 15; "+" indicates that there was a significant difference in intraocular pressure in the Tet-ATS group from that in the control group 14 days after treatment.
As can be seen from Table 3, on day 8, the PFOB @ LIP-Tet-ATS group model rabbits had the lowest score, that is, the PFOB @ LIP-Tet-ATS group model rabbits had the lowest severity of disease symptoms, and the PFOB @ LIP-Tet-ATS group also had cured model rabbits, which proved that PFOB @ LIP-Tet nanoparticles had a therapeutic effect on dry eye diseases, and the therapeutic effect was better than that of other groups.
As can be seen from FIG. 1, after 14 days of treatment, the intraocular pressure of the Tet-ATS group model rabbits was significantly different from that of the control group model rabbits (T test, p < 0.05), the former was significantly decreased, and thus Tet had an effect on intraocular pressure, and Tet caused a decrease in intraocular pressure. After 14 days of treatment, compared with the intraocular pressure of the control group model rabbit, the intraocular pressure of the PFOB @ LIP-Tet-ATS group model rabbit has no statistical difference, which indicates that the PFOB @ LIP-Tet nano-particle has little or no influence on the intraocular pressure during the treatment period. Therefore, the invention loads Tet on the liposome, and the liposome wraps PFOB, so that the influence of Tet on intraocular pressure can be reduced, PFOB @ LIP-Tet nanoparticles with little or even no influence on ocular pressure are obtained, and the better treatment effect on dry eye is ensured.
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be defined by the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (7)
1. A preparation method of a tetrandrine-loaded liposome preparation is characterized by comprising the following steps: the method comprises the following raw materials: the composition comprises soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol, tetrandrine and perfluorooctyl bromide, wherein the mass ratio of the sum of the soybean lecithin and the dipalmitoyl phosphatidyl ethanolamine to the cholesterol to the tetrandrine is 4:1: 2.5-3.5; the method comprises the following steps:
s1, dissolving soybean lecithin, dipalmitoyl phosphatidyl ethanolamine, cholesterol and tetrandrine in a solvent, and performing rotary evaporation to obtain a uniform thin liquid film;
s2, hydrating the thin liquid film obtained in the step S1, adding perfluorooctyl bromide, and performing ultrasonic oscillation in an ice bath to obtain a system containing PFOB @ LIP-Tet nanoparticles;
s3, centrifuging the system obtained in the step S2, and collecting sediments to obtain the tetrandrine-loaded liposome preparation;
the ratio of the dosage of the perfluorooctyl bromide to the dosage of the tetrandrine is 150 mu L to 6mg, 250 mu L to 6 mg.
2. The method for preparing the tetrandrine liposome preparation carrying formulation according to claim 1, wherein the method comprises the following steps: in step S1, the solvent is chloroform, dichloromethane or methanol.
3. The method for preparing the tetrandrine-loaded liposome preparation according to claim 2, wherein the preparation method comprises the following steps: in step S2, the thin liquid film is hydrated using a phosphate buffer.
4. The method for preparing the tetrandrine-loaded liposome preparation according to claim 3, wherein the method comprises the following steps: in the step S2, the ultrasonic frequency is 10 kHz-30 kHz, the ultrasonic power is 55-100 w, and the ultrasonic time is 3-5 min.
5. The method for preparing the tetrandrine-loaded liposome preparation according to claim 4, wherein the method comprises the following steps: in step S3, the centrifugation speed is 5000-8000 rpm, and the centrifugation time is 5-10 min.
6. The method for preparing the tetrandrine liposome preparation carrying formulation according to claim 5, wherein the method comprises the following steps: in step S1, the rotation speed of the rotary evaporation is 120-150 rpm, and the temperature of the rotary evaporation is 45-55 ℃.
7. The use of the tetrandrine-loaded liposome preparation of claim 1 in the preparation of a medicament for the treatment of dry eye.
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