A liposome gel for efficiently loading and stabilizing protein polypeptide drugs and a preparation method thereof

Abstract

The application provides a protein polypeptide drug efficient loading and stable liposome gel and a preparation method thereof, and belongs to the technical field of pharmaceutical preparations.The preparation method comprises the following steps: (1) preparing phospholipid into a liposome suspension; (2) adjusting the pH of the liposome suspension, and then freezing, melting and obtaining a stable liposome solution; and (3) mixing the liposome solution with dodecaborate cluster ions and a protein polypeptide drug to obtain the liposome gel.The liposome gel can solve the problems of low drug loading capacity, easy drug inactivation and easy leakage of the loaded drug in the current liposome drug loading technology.The liposome gel does not need to prepare drug-loaded liposomes in advance, and does not need to use high molecular gel materials, so that the formula and process are simple, and the scale-up production is easier to realize.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparation, in particular to a protein polypeptide drug efficient loading and stable liposome gel and a preparation method thereof. BACKGROUND

[0002] Biological drugs include polypeptides, proteins, antibodies, polysaccharides and nucleic acid drugs. Biological macromolecular drugs show unique advantages in the treatment of major clinical diseases such as heart and brain, refractory wounds, etc., and have the advantages of strong pharmacological effect, low treatment dose and small side effects. In recent years, the global biological pharmaceutical market has developed rapidly and shown a high growth trend. With the continuous development of new technologies and new antibodies, clinical applications have also expanded from tumors and immune diseases to cardiovascular, gastrointestinal, respiratory and infectious diseases, and the patient population has also shown a diversification trend. In recent years, among the top 10 best-selling drugs in the world, biological drug preparations account for eight seats. However, biological drugs are easily metabolized and decomposed by gastric acid, enzymes and hepatic intestinal circulation, and most of the clinically approved biological preparations adopt injection administration. However, due to the large molecular weight and poor permeability of biological drugs, most of them are prone to aggregation and rapid degradation, and it is difficult to penetrate the natural physiological barriers of the human body, such as skin, gastrointestinal epithelium, etc., and the problems of biological drugs in oral administration, such as degradation in the gastrointestinal tract, have been an insurmountable obstacle for macromolecules in non-invasive drug delivery. The key to the efficient treatment of biological drugs lies in the design and construction of new delivery carriers.

[0003] Liposomes are vesicles formed by the closure of phospholipid bilayers, which have an inner water phase and can be used to encapsulate large molecule drugs such as proteins and polypeptides. Currently, the main method for encapsulating biological macromolecules in liposomes is reverse evaporation. This method has low drug loading capacity, high loss of biological molecule activity, residual toxic organic solvents in the preparation process, and complex preparation process. In order to overcome these problems, in-situ drug loading of liposomes for macromolecular drugs is used by using membrane pore-forming agents, ion introduction technology, and reverse ion membrane penetration technology. Among these in-situ carrier technologies, the membrane transport of charged amphiphilic molecules is widely used. The principle is that the hydrophobic tail of the charged amphiphilic molecule serves as an anchor group inserted into the lipid membrane, and the ionic head group loads the cargo by electrostatic attraction to form an electrically neutral complex, triggering the transport in the lipid membrane, and achieving drug loading. This method requires the pre-insertion of charged amphiphilic molecules into the bilayer membrane of the liposome, and the transport of the cargo requires a high density of opposite charges, and the amphiphilic molecules have certain toxicity and other defects. Moreover, the liposome is unstable, and during storage, it is prone to changes in particle size, leakage of encapsulated drugs, and other problems. The transformation of liposomes into semi-solid gels is an effective strategy to improve the stability of liposomes. Due to the characteristic of phospholipid molecules spontaneously assembling into vesicles in water, pure liposome solution is difficult to aggregate to form a hydrogel without additives. Therefore, the method commonly used is to disperse drug-loaded liposomes in a high-molecular-weight polymer gel matrix to prepare a liposome gel. For example, Chinese invention patent (publication number: 109464298A, publication date: 2019.03.15) loaded EGCG-loaded liposomes into a PVA / HA gel matrix material to prepare a PVA / HA / EGCG liposome gel. Based on the above problems of low drug loading of liposomes for water-soluble drugs, especially protein and polypeptide drugs, and the difficulty of liposomes to aggregate into gels, it is urgent to provide a kind of liposome gel capable of achieving high-efficiency encapsulation of water-soluble drugs and stable and a preparation method thereof. SUMMARY

[0004] The purpose of the present application is to provide a kind of liposome gel capable of achieving high-efficiency encapsulation of protein and polypeptide drugs and stable and a preparation method thereof. After preparing blank liposomes, in-situ drug loading of dodecaboron cluster ions can be used to prepare a kind of liposome gel capable of achieving high-efficiency encapsulation of protein and polypeptide drugs and stable, which can solve the problems of low drug loading capacity, drug inactivation and drug leakage in current liposome drug loading technology. Moreover, the present application does not need to prepare drug-loaded liposomes, and does not need to use high-molecular-weight gel materials, so the formula and process are simple, and it is easier to realize scale-up production.

[0005] In order to achieve the above purpose, the present application provides the following technical solutions:

[0006] The present application provides a kind of liposome gel capable of achieving high-efficiency encapsulation of protein and polypeptide drugs and stable and a preparation method thereof, comprising the following steps:

[0007] (1) prepare phospholipid into liposome suspension;

[0008] (2) After adjusting the pH of the liposome suspension, a stable liposome solution is obtained after freezing and thawing;

[0009] (3) The liposome solution is mixed with dodecanoboride ion and protein polypeptide drugs to obtain the liposome gel.

[0010] Preferably, in step (1), when preparing the liposome suspension, oleic acid is also added according to a mass ratio of phospholipid to oleic acid of 90: (10-30).

[0011] Preferably, the method for preparing the liposome suspension is a thin film hydration method, a freeze-drying method, or an injection method.

[0012] Preferably, the adjusted pH of the liposome suspension is 6-9.

[0013] Preferably, the freezing time is 1-3 h, and the freezing temperature is -30 to -10℃; the thawing temperature is 50℃-85℃.

[0014] Preferably, the mixing temperature is 3-5℃, and the mixing time is 12-18 hours.

[0015] Preferably, in the liposome gel, the mass concentration of the phospholipid is 1%-30%; the mass concentration of the oleic acid is 0-3%; the mass concentration of the dodecanoboride ion is 0.05%-4%; the mass concentration of the protein polypeptide drug is 0.001%-5%; and the balance is water.

[0016] Preferably, the molar ratio of the phospholipid to the dodecanoboride ion is 10: (0.01-5); and the molar ratio of the dodecanoboride ion to the protein polypeptide drug is 1: (0.1-20).

[0017] Preferably, the phospholipid includes any one or several of hydrogenated soybean phospholipid, distearoyl phosphatidylcholine, and dipalmitoyl phosphatidylcholine; and the dodecanoboride ion includes any one or several of B 12 H 12 2- , B 12 Cl 12 2- , B 12 Br 12 2- , B 12 I 12 2- , and B 12 H 11 SH 2- .

[0018] The application also provides the protein polypeptide drug loaded and stabilized liposome gel prepared by the preparation method.

[0019] The application provides a protein polypeptide drug loaded and stabilized liposome gel and a preparation method thereof. The protein polypeptide drug loaded and stabilized liposome gel is prepared by using phospholipid or a mixture of phospholipid and oleic acid as a material to prepare blank liposomes, combine dodecaboron cluster ions, and prepare ion cluster membrane chimeric liposome gel, so as to realize high-efficiency loading and stabilization of protein polypeptide drugs such as epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, superoxide dismutase and catalase. Compared with the prior art, the liposome gel has the following advantages: (1) the liposome gel does not contain a polymer gel matrix material, but only contains phospholipid with high biocompatibility and a small amount of oleic acid, and the formula is simple; (2) the liposome gel is a three-dimensional network skeleton aggregated by liposomes, and dodecaboron cluster ions are inlaid in the lipid bilayer membrane or the inner water phase, so that the liposome gel can realize in-situ drug loading after the drug is added, and the drug does not need to be pre-wrapped; (3) dodecaboron cluster ions and protein polypeptide drugs are combined by hydrophobic effect, so that high-efficiency loading and stabilization of the drug are realized; (4) the liposome in the liposome gel has good stability in shape and particle size, and drug leakage and in-vivo drug burst are not prone to occur; (5) the blank liposomes are directly combined with dodecaboron cluster ions to realize in-situ drug loading in an aqueous environment based on the salting-out effect, and organic solvents are not needed, so that the stability of the protein polypeptide drug is improved, the preparation method is simple, and large-scale production is easy to realize. DETAILED DESCRIPTION

[0020] The technical solutions provided by the application are described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0021] Example 1

[0022] The application provides a protein polypeptide drug loaded and stabilized liposome gel. The liposome gel is prepared according to the formula and preparation process parameters shown in Table 1, and the specific preparation process is as follows:

[0023] Thin film dispersion method (Group 1-6, Group 8-16, Pair 1-3, Pair 5-7): according to the formula in Table 1, phospholipid and oleic acid (Group 16 only phospholipid) are weighed into a round-bottom flask, 5 mL of dichloromethane is added to dissolve to form a clear solution, and the organic solvent is completely removed on a rotary evaporator to prepare a uniform thin film; 2 mL of distilled water is added to completely hydrate the thin film to obtain a liposome suspension. The liposome suspension is adjusted to the corresponding pH shown in Table 1 using a dilute NaOH solution (10 mM), poured into a vial, and frozen in a -20°C refrigerator for 2 h to completely freeze the sample; then, the sample is melted in a water bath at the melting temperature shown in Table 1 to convert the liposome suspension into a clear liquid; finally, the clear liposome suspension is cooled to room temperature (25°C), and the boron cluster ions and drugs in the amount shown in Table 1 are added, stirred uniformly, and stored in a 5°C refrigerator overnight to prepare a liposome gel.

[0024] Freeze-drying method (Group 7): according to the formula in Table 1, phospholipid and oleic acid are weighed into a round-bottom flask, 10 mL of tert-butyl alcohol is added to dissolve to form a clear solution, and the solution is freeze-dried to prepare loose precursor liposome powder, which is completely redissolved and dispersed in 2 mL of distilled water to prepare a liposome suspension. The liposome suspension is adjusted to the corresponding pH shown in Table 1 using a dilute NaOH solution, poured into a vial, and frozen in a -20°C refrigerator for 2 h to completely freeze the sample; then, the sample is melted in a water bath at the melting temperature shown in Table 1 to convert the liposome suspension into a clear liquid; finally, the clear liposome suspension is cooled to room temperature, and the boron cluster ions and drugs in the amount shown in Table 1 are added, stirred uniformly, and stored in a 5°C refrigerator overnight to prepare a liposome gel.

[0025] Injection method (Group 17-24, Pair 8-11): according to the formula in Table 1, phospholipid and oleic acid are weighed into a round-bottom flask, 5 mL of diethyl ether is added to dissolve to form a clear solution, and 2 mL of distilled water is slowly injected to completely hydrate the thin film to prepare a liposome suspension. The liposome suspension is adjusted to the corresponding pH shown in Table 1 using a dilute NaOH solution, poured into a vial, and frozen in a -20°C refrigerator for 2 h to completely freeze the sample; then, the sample is melted in a water bath at the melting temperature shown in Table 1 to convert the liposome suspension into a clear liquid; finally, the clear liposome suspension is cooled to room temperature, and the boron cluster ions and drugs in the amount shown in Table 1 are added, stirred uniformly, and stored in a 5°C refrigerator overnight to prepare a liposome gel.

[0026] Among them, "Pair 4" in Table 1 first uses the reverse evaporation method to prepare drug-loaded liposomes, and then uses the freeze-redissolving gelation method to prepare a liposome gel, and the specific preparation process is as follows:

[0027] The drug was dissolved in 0.1 mL of phosphate buffer (pH = 7.4) according to the formulation in Table 1 as the aqueous phase; the phospholipid and oleic acid were weighed into a round-bottom flask and dissolved in 5 mL of dichloromethane to form a clear solution as the organic phase; the aqueous phase was slowly added to the organic phase under ice water bath conditions, and the W / O emulsion was prepared by vigorous stirring; the organic solvent was completely removed by rotary evaporation to prepare a uniform film; 2 mL of distilled water was added to completely hydrate the film to prepare a liposome suspension; the liposome suspension was adjusted to 6.5 using a dilute NaOH solution, poured into a vial, and frozen in a -20 °C refrigerator for 2 h to completely freeze the sample; then, the sample was melted in a 70 °C water bath to convert the liposome suspension into a clear liquid; finally, the clear liposome suspension was cooled to room temperature and stored in a 5 °C refrigerator overnight to prepare a liposome gel.

[0028] Table 1 Formulation and preparation process parameters of liposome gel

[0029]

[0030] Note: Film: liposomes prepared by film hydration method; Injection: liposomes prepared by injection method; Freeze-drying: liposomes prepared by freeze-drying method; Sodium salt of dodecaborate cluster includes BBr: B 12 Br 12 2- ; BI: B 12 I 12 2- ; BH: B 12 H 12 2- ; BSH: B 12 H 11 SH 2- ; BCl: B 12 Cl 12 2- ; - indicates that the substance is not contained; EGF: epidermal growth factor; VEGF: vascular endothelial growth factor; KGF: keratinocyte growth factor; SOD: superoxide dismutase; CAT: catalase; KPV is an anti-inflammatory tripeptide.

[0031] Test Example 1

[0032] The gelling properties of the liposome suspensions prepared in each group of Example 1, as well as the drug loading parameters (drug loading, encapsulation efficiency, and drug activity), were detected, and the detection results are shown in Table 2. The specific detection methods are as follows:

[0033] Drug loading and encapsulation efficiency determination: 200 μg of each group of liposome gel was weighed, added to 5 mL phosphate buffer (pH = 7.4) to disperse into suspension, centrifuged at 12000 rpm for 10 min to precipitate the liposome, take the supernatant, determine the free protein concentration by BCA method, and calculate the drug loading and encapsulation efficiency by the following formula:

[0034] Drug loading (%) = (total weight of drug added in the formula - weight of free drug) x 100 / (phospholipid weight + oleic acid weight);

[0035] Encapsulation efficiency (%) = (total weight of drug added in the formula - weight of free drug) x 100 / (total weight of drug added in the formula).

[0036] Drug activity determination: 200 μg of each group of liposome gel was weighed, added to 5 mL phosphate buffer (pH = 7.4) to disperse into suspension, 1% triton-100 was added to destroy the lipid bilayer and release the encapsulated drug, centrifuged at 12000 rpm for 10 min to precipitate the liposome component, and the drug activity of the supernatant was determined by ELISA method; at the same time, according to the formula in table 1, an equal weight of drug was weighed, added to 2 mL distilled water to prepare a drug solution; 200 μL of drug solution was accurately measured, added to 5 mL phosphate buffer (pH = 7.4) to prepare an equal concentration drug solution, and the activity of the equal concentration drug solution was determined by ELISA method, and the drug activity in the liposome gel was calculated by the following formula:

[0037] Drug activity (%) = liposome gel drug activity x 100 / equal concentration solution drug activity.

[0038] Table 2 Liposome suspension gelation and drug encapsulation parameters prepared in examples

[0039]

[0040] Note: Y indicates that the liposome suspension can be gelled; N indicates that the liposome suspension cannot be gelled.

[0041] Compared with group 4, groups 1-11 can be seen that the in-situ drug loading method of boron cluster ions has obvious advantages in significantly improving the drug activity of liposome gel prepared by reverse evaporation method.

[0042] Compared with Pair 1~3, Pair 5~8 and Pair 1, it can be seen that the liposome gelation performance, drug encapsulation rate and drug activity prepared by HSPC and BBr are affected by the molar ratio of phospholipid to boron cluster ion in the formula. When the molar ratio of phospholipid to dodecaboron cluster ion is 10: (0.01~5), the encapsulation rate is greater than 80%, and the drug activity is higher than 85%; when the molar ratio of phospholipid to dodecaboron cluster ion is higher than 10:5, flocculent precipitate appears, the liposome does not gel, the drug encapsulation rate is lower than 60%, and the drug activity is lower than 40%.

[0043] Compared with Pair 1~2 and Pair 2~3, it can be seen that under the condition of fixed molar ratio of HSPC and BBr as 10:1, the drug activity and encapsulation rate are affected by the molar ratio of boron cluster ion to drug. The optimal encapsulation rate and the highest drug activity are obtained when the molar ratio of dodecaboron cluster ion to drug is 1:2.

[0044] Compared with Pair 1~11 and Pair 5~6, it can be seen that the mass concentration of phospholipid in the liposome gel has an important influence on the gelation of the liposome suspension; when the mass concentration of phospholipid is 1~30%, the liposome suspension can form a semi-solid gel; when the concentration is lower than 1% or higher than 30%, the liposome suspension cannot form a uniform gel, but forms a relatively dilute dispersion or a relatively viscous suspension.

[0045] Compared with Pair 12~16 and Pair 7, it can be seen that the mass concentration of oleic acid in the liposome gel has a significant influence on the liposome gelation, drug encapsulation rate and drug activity; when the mass concentration of oleic acid is higher than 3%, the liposome suspension cannot form a semi-solid gel; however, oleic acid is not a necessary component in the formula, and when the formula does not contain oleic acid, the liposome suspension can still form a liposome gel, and the drug encapsulation rate is higher than 80% and the drug activity is higher than 85%.

[0046] Compared with Pair 17~21 and Pair 8~9, it can be seen that in the preparation method of the liposome gel, the pH of the liposome suspension has a significant influence on the gelation of the liposome suspension, the drug encapsulation rate and the drug activity; when the pH of the liposome suspension is in the range of 6~9, the liposome suspension can form a semi-solid gel, and the drug encapsulation rate and the drug activity are both higher than 80%. When the pH of the liposome suspension is lower than 6 or higher than 9, the liposome suspension cannot form a semi-solid gel, and the drug encapsulation rate and the activity are both lower than 30%.

[0047] Compared with the control groups 10-11, it can be seen that the temperature of the quick thawing after the freezing of the liposome suspension has a significant influence on the gelation of the liposome suspension, the drug encapsulation rate and the drug activity in the liposome gel preparation method. When the temperature of the quick thawing of the liposome suspension is in the range of 50-85℃, the liposome suspension can form a semi-solid gel, and the drug encapsulation rate and the drug activity are both higher than 80%. When the temperature of the quick thawing of the liposome suspension is lower than 50℃ or higher than 85℃, the liposome suspension cannot form a semi-solid gel, and the drug encapsulation rate and the drug activity are both lower than 15%.

[0048] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a liposome gel for efficiently loading and stabilizing a protein polypeptide drug, characterized by, The method comprises the following steps: (1) preparing a liposome suspension from phospholipids; optionally, in the step (1), the liposome suspension is prepared by adding oleic acid in a mass ratio of 90: (10-30) to the phospholipids; the mass concentration of the oleic acid is 0-3%; (2) adjusting the pH of the liposome suspension to 6-9, freezing, and then thawing at 50-85°C to obtain a stable liposome solution; (3) mixing the liposome solution with dodecanoboride ions and a protein polypeptide drug to obtain the liposome gel; the mass concentration of the phospholipids in the liposome gel is 1%-30%; the molar ratio of the phospholipids to the dodecanoboride ions is 10: (0.01-5); the molar ratio of the dodecanoboride ions to the protein polypeptide drug is 1: (0.1-20).

2. The production method according to claim 1, wherein The method for preparing the liposome suspension is a thin film hydration method, a freeze-drying method, or an injection method.

3. The production method according to claim 1, wherein The freezing time is 1-3 hours, and the freezing temperature is -30--10°C.

4. The production method according to claim 3, wherein The mixing temperature is 3-5°C, and the mixing time is 12-18 hours.

5. The production method according to claim 4, wherein The mass concentration of the dodecanoboride ions is 0.05%-4%, and the mass concentration of the protein polypeptide drug is 0.001%-5%; the rest is water.

6. The production method according to claim 5, wherein The phospholipid includes any one or more of hydrogenated soybean phospholipid, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine; the dodecaboron cluster ion includes B 12 H 12 2- , B 12 Cl 12 2- , B 12 Br 12 2- , B 12 I 12 2- and B 12 H 11 SH 2- .

7. A protein polypeptide drug high-efficiency encapsulated and stable liposome gel prepared by the method of any one of claims 1-6.

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