CN117180195A - Efficient entrapment and stable liposome gel of protein polypeptide drugs and preparation method thereof - Google Patents
Efficient entrapment and stable liposome gel of protein polypeptide drugs and preparation method thereof Download PDFInfo
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Abstract
The invention provides a liposome gel for efficiently entrapping and stabilizing protein polypeptide drugs and a preparation method thereof, belonging to the technical field of pharmaceutical preparations. The preparation method of the invention comprises the following steps: (1) preparing phospholipid into liposome suspension; (2) After regulating the pH of the liposome suspension, obtaining a stable liposome solution after freezing and thawing; (3) And mixing the liposome solution with twelve boron cluster ions and protein polypeptide drugs to obtain the liposome gel. The liposome gel disclosed by the invention can solve the problems that the drug loading rate is low, the drug is easy to inactivate and the loaded drug is easy to leak in the existing liposome drug loading technology. The invention does not need to prepare the drug-loaded liposome first, does not need to use a polymer gel material, has simple formula and process, and is easier to realize amplified production.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a liposome gel for efficiently entrapping and stabilizing protein polypeptide drugs and a preparation method thereof.
Background
Biological drugs include polypeptides, proteins, antibodies, glycans, nucleic acid drugs, and the like. The biological macromolecular medicament has unique advantages in treating serious clinical diseases such as heart and brain, refractory wound surfaces and the like, and has the advantages of strong pharmacological action, low treatment dosage and small side effect. In recent years, the global biopharmaceutical market has developed rapidly, and has assumed a high growth in accordance with the F & S report, and the global biopharmaceutical market is expected to increase from $2402 billion in 2017 to $4040 billion in 2022 with a composite annual growth rate of 11.0%. With the continuous development of new technologies and novel antibodies, clinical applications have also expanded from tumor and immune diseases to the fields of cardiovascular, gastrointestinal, respiratory and infectious diseases, and the like, and patient populations also show a diversified trend. In recent years, among TOP 10-free drugs worldwide, biopharmaceutical formulations occupy as many as eight. However, most of the biological agents obtained in clinic are administered by injection due to the fact that the biological agents are easily decomposed by metabolism through gastric acid, enzymes and liver and intestine circulation. However, the biological drugs have the problems of large molecular weight, poor permeability and rapid aggregation and degradation, and the problems that most biological drugs are difficult to penetrate natural physiological barriers of human bodies, such as skin, gastrointestinal epithelium and the like, and the biological drugs are degraded in the gastrointestinal tract by oral administration routes are all the time an insurmountable barrier for macromolecules in non-invasive administration routes. The key to the efficient treatment of biopharmaceuticals is the design and construction of novel delivery vehicles.
The liposome is a vesicle formed by closing phospholipid bilayer, and has an inner water phase which can be used for wrapping macromolecular drugs such as protein polypeptides and the like. Liposome-encapsulated biomacromolecules currently mainly utilize reverse evaporation. The method has low drug loading, high loss of biological molecule activity, residual toxic organic solvent in the preparation process, and complex preparation process. To overcome these problems, liposome in situ drug delivery for macromolecular drugs has been developed using membrane porogens, iontophoresis techniques, counter-ion membrane permeation techniques, and the like. Among these in situ carrier technologies, charged amphiphilic molecules mediate membrane transport of cargo in wide use. The principle is that the hydrophobic tail of the charged amphoteric molecule is used as an anchor group to be inserted into a lipid membrane, and the ionic head group is used for loading cargoes through electrostatic attraction to form an electrically neutral compound, triggering the transport in the lipid membrane and realizing the drug loading. The method has the defects that charged amphoteric molecules are inserted into liposome bilayer membranes in advance, the transport goods need to have opposite charges with high density, and certain toxicity exists in the amphoteric molecules. And the liposome is unstable, the particle size is easy to change in the storage process, and the leakage of the encapsulated medicine is easy to occur. The conversion of liposomes to semi-solid gels is an effective strategy to improve liposome stability. Due to the nature of the phospholipid molecules spontaneously assembling in water to form vesicles, simple liposome solutions are difficult to aggregate to form hydrogels without additives. Thus, the method employed is generally to prepare a liposome gel by dispersing drug-loaded liposomes in a high molecular polymer gel matrix. For example, chinese patent (publication No. 109464298A, publication No. 2019.03.15) discloses that PVA/HA/EGCG liposome gel is prepared by loading EGCG entrapped liposome into PVA/HA gel matrix material. Based on the problems that the liposome has low drug loading on water-soluble drugs, particularly protein polypeptide drugs, and the liposome is difficult to aggregate into gel, it is highly desirable to provide a liposome gel capable of realizing efficient entrapment and stability of the water-soluble drugs and a preparation method thereof.
Disclosure of Invention
The invention aims to provide a liposome gel for efficiently entrapping and stabilizing protein polypeptide drugs and a preparation method thereof. After the blank liposome is prepared, the in-situ drug loading of twelve boron cluster ions is combined, so that the liposome gel with high-efficiency entrapment and stability of the protein polypeptide drug can be prepared, and the problems that the drug loading rate is low, the drug is easy to inactivate and the loaded drug is easy to leak in the existing liposome drug loading technology can be solved. The invention does not need to prepare the drug-loaded liposome first, does not need to use a polymer gel material, has simple formula and process, and is easier to realize amplified production.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of liposome gel for efficiently entrapping and stabilizing protein polypeptide drugs, which comprises the following steps:
(1) Preparing phospholipid into liposome suspension;
(2) After regulating the pH of the liposome suspension, obtaining a stable liposome solution after freezing and thawing;
(3) And mixing the liposome solution with twelve boron cluster ions and protein polypeptide drugs to obtain the liposome gel.
Preferably, when preparing the liposome suspension in the step (1), oleic acid is also added according to the mass ratio of phospholipid to oleic acid of 90 (10-30).
Preferably, the method for preparing the liposome suspension is a film hydration method, a lyophilization method or an injection method.
Preferably, the pH of the liposome suspension after adjustment is 6-9.
Preferably, the freezing time is 1-3 h, and the freezing temperature is-30 to-10 ℃; the temperature during melting is 50-85 ℃.
Preferably, the temperature of the mixing is 3-5 ℃, and the mixing time is 12-18 hours.
Preferably, the mass concentration of the phospholipid in the liposome gel is 1% -30%; the mass concentration of the oleic acid is 0-3%; the mass concentration of the twelve boron cluster ions is 0.05% -4%; the mass concentration of the protein polypeptide medicine is 0.001% -5%; the balance being water.
Preferably, the molar ratio of the phospholipid to the twelve boron cluster ions is 10: (0.01-5); the molar ratio of the twelve boron cluster ions to the protein polypeptide drug is 1: (0.1-20).
Preferably, the phospholipid comprises any one or more of hydrogenated soybean phospholipid, distearoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine; the twelve boron cluster ions comprise 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- Any one or more of the following.
The invention also provides a liposome gel which is obtained by the preparation method and is used for efficiently encapsulating and stabilizing the protein polypeptide drugs.
The invention provides a liposome gel for efficiently entrapping and stabilizing protein polypeptide drugs and a preparation method thereof. The invention prepares blank liposome by taking phospholipid or a mixture of phospholipid and oleic acid as a material, combines twelve boron cluster ions, prepares ion cluster membrane chimeric liposome gel, and realizes the high-efficiency loading and stabilization of protein polypeptide medicines such as epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, superoxide dismutase, catalase and the like. Compared with the prior art, the method has the following advantages: (1) The liposome gel disclosed by the invention does not contain a polymer gel matrix material, only contains phospholipid with high biocompatibility and a small amount of oleic acid, and is simple in formula; (2) The liposome gel is a three-dimensional network skeleton formed by liposome aggregation, twelve boron cluster ions are embedded in a lipid bilayer membrane or an inner water phase, and in-situ drug loading can be realized after drugs are added, so that the drugs do not need to be wrapped in advance; (3) The twelve boron cluster ions are combined with the protein polypeptide drugs in a hydrophobic way through a chaotropic effect, so that the efficient loading and stabilization of the drugs are realized; (4) The liposome in the liposome gel has good stability of the form and the particle size, and is not easy to leak and burst in vivo; (5) The blank liposome is directly combined with twelve boron cluster ions, the medicine is loaded in situ in an aqueous environment based on a chaotropic effect, an organic solvent is not needed, the stability of the protein polypeptide medicine is improved, the preparation method is simple, and the large-scale production is easy to realize.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The invention provides a liposome gel for efficiently encapsulating and stabilizing protein polypeptide drugs. According to the formula and preparation process parameters shown in Table 1, liposome gel is prepared by the following specific preparation process:
film dispersion method (groups 1 to 6, groups 8 to 16, pairs 1 to 3, pairs 5 to 7): according to the formulation of Table 1, phospholipid and oleic acid (group 16 only phospholipid) were weighed and placed in a round bottom flask, 5mL of methylene chloride was added to dissolve to form a clear solution, and the clear solution was placed in a rotary evaporator to completely remove the organic solvent, thereby preparing a uniform film; 2mL of distilled water was added to completely hydrate the membrane to obtain a liposome suspension. Adjusting the liposome suspension to the corresponding pH shown in Table 1 by using a dilute NaOH solution (10 mM), pouring into a penicillin bottle, and freezing in a refrigerator at-20 ℃ for 2 hours to completely freeze the sample; subsequently, the samples were thawed in a water bath at the melting temperature shown in table 1 to convert the liposomal suspension to a clear liquid; finally, the clarified liposome suspension is cooled at room temperature (25 ℃), boron cluster ions and medicines with the formula amount shown in table 1 are added, and the mixture is stirred uniformly and is placed in a refrigerator at 5 ℃ for overnight storage, so that liposome gel is prepared.
Lyophilization (group 7): according to the formulation of Table 1, phospholipid and oleic acid were weighed and placed in a round bottom flask, 10mL of t-butanol was added to dissolve to form a clear solution, the clear solution was freeze-dried to prepare a loose proliposome powder, and 2mL of distilled water was added to completely re-dissolve and disperse to prepare a liposome suspension. Adjusting the liposome suspension to the corresponding pH shown in Table 1 by using a dilute NaOH solution, pouring into a penicillin bottle, and freezing for 2 hours at the temperature of minus 20 ℃ in a refrigerator to completely freeze the sample; subsequently, the samples were thawed in a water bath at the melting temperature shown in table 1 to convert the liposomal suspension to a clear liquid; finally, after the clarified liposome suspension is cooled at room temperature, boron cluster ions and medicines with the formula amounts shown in the table 1 are added, and the mixture is stirred uniformly and is placed in a refrigerator at 5 ℃ for overnight storage, so that liposome gel is prepared.
Injection method (groups 17 to 24, pairs 8 to 11): according to the formulation of Table 1, phospholipid and oleic acid were weighed and placed in a round bottom flask, 5mL of diethyl ether was added to dissolve to form a clear solution, and 2mL of distilled water was slowly injected to completely hydrate the membrane to prepare a liposome suspension. Adjusting the liposome suspension to the corresponding pH shown in Table 1 by using a dilute NaOH solution, pouring into a penicillin bottle, and freezing for 2 hours at the temperature of minus 20 ℃ in a refrigerator to completely freeze the sample; subsequently, the samples were thawed in a water bath at the melting temperature shown in table 1 to convert the liposomal suspension to a clear liquid; finally, after the clarified liposome suspension is cooled at room temperature, boron cluster ions and medicines with the formula amounts shown in the table 1 are added, and the mixture is stirred uniformly and is placed in a refrigerator at 5 ℃ for overnight storage, so that liposome gel is prepared.
Wherein, in the table 1, "pair 4" firstly adopts a reverse evaporation method to prepare liposome for encapsulating the drug, and then adopts a freezing multiple sol-gel method to prepare liposome gel, and the specific preparation process is as follows:
according to the formulation of table 1, the formulated drug was dissolved in 0.1mL of phosphate buffer (ph=7.4) as an aqueous phase; weighing phospholipid and oleic acid, placing in a round-bottom flask, adding 5mL of dichloromethane, and dissolving to form a clear solution serving as an organic phase; slowly adding the water phase into the organic phase under the ice water bath condition, and vigorously stirring to prepare a W/O emulsion; completely removing the organic solvent by a rotary evaporator to prepare a uniform film; adding 2mL of distilled water to completely hydrate the film, and preparing liposome suspension; regulating the liposome suspension to 6.5 by using a dilute NaOH solution, pouring into a penicillin bottle, and freezing for 2 hours at the temperature of minus 20 ℃ in a refrigerator to completely freeze the sample; subsequently, the sample was thawed under 70 ℃ water bath conditions to convert the liposomal suspension to a clear liquid; finally, the clarified liposome suspension is cooled at room temperature and then placed in a refrigerator at 5 ℃ for overnight storage, so as to prepare liposome gel.
Table 1 liposome gel formulation and preparation process parameters
Note that: film: preparing liposome by a film hydration method; and (3) injection: preparing liposome by injection method; and (3) freeze-drying: preparing liposome by lyophilization; sodium salts of twelve boron cluster ions include BBr: b (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- The method comprises the steps of carrying out a first treatment on the surface of the -means free of the substance; EGF: an epidermal growth factor; VEGF: vascular endothelial growth factor; KGF: a keratinocyte growth factor; SOD: superoxide dismutase; CAT: a catalase; KPV is anti-inflammatory tripeptide.
Test example 1
The gel properties of the liposome suspensions prepared in each group of example 1, and the drug entrapment parameters (drug loading, encapsulation efficiency, drug activity) were measured, and the measurement results are shown in table 2. The specific detection method comprises the following steps:
drug loading and encapsulation efficiency determination: 200 μg of each group of liposome gel was weighed, 5mL of phosphate buffer (pH=7.4) was added to disperse into suspension, centrifugation was performed at 12000rpm for 10min, the liposome was precipitated, the supernatant was taken, the free protein concentration was measured by BCA method, and the drug loading and encapsulation efficiency were calculated by the following formula:
drug loading (%) = (total weight of drug added to formulation-free drug weight) ×100/(weight of phospholipid + weight of oleic acid);
encapsulation efficiency (%) = (total weight of drug added to formulation-free drug weight) ×100/(total weight of drug added to formulation).
Determination of pharmaceutical Activity: weighing 200 mug of each group of liposome gel, adding 5mL of phosphate buffer solution (pH=7.4) to disperse into suspension, adding 1% of triton-100 to mix and destroy lipid double-fraction, releasing entrapped medicine, centrifuging at 12000rpm for 10min to precipitate lipid components, and measuring the activity of supernatant medicine by ELISA method; meanwhile, weighing the medicines with equal weight according to the formula of the table 1, and adding 2mL of distilled water to prepare a medicine solution; precisely weighing 200 μl of the drug solution, adding 5mL of phosphate buffer (ph=7.4) to prepare an equal concentration drug solution, and measuring the activity of the equal concentration drug solution by ELISA method, wherein the drug activity in liposome gel is calculated by the following formula:
pharmaceutical activity (%) =liposomal gel pharmaceutical activity×100/isoconcentration solution pharmaceutical activity.
Table 2 liposomal suspension gelation and drug entrapment parameters prepared in the examples
Note that: y represents that the liposome suspension can gel; n indicates that the liposome suspension is not gellable.
Compared with the group 4, the group 1-11 can be seen that compared with the liposome gel prepared by the reverse evaporation method drug loading in the boron cluster ion in-situ drug loading mode, the drug activity is obviously improved, and the method has obvious advantages.
Compared with groups 1-3 and groups 5-8 and 1, it can be seen that the gelation properties, drug encapsulation efficiency and drug activity of liposomes prepared with HSPC and BBr are affected by the molar ratio of phospholipid to boron cluster ions in the formulation. When the mole ratio of the phospholipid to the dodecaboron cluster ions is 10 (0.01-5), the encapsulation rate is more than 80%, and the pharmaceutical activity is higher than 85%; when the mole ratio of phospholipid to dodecaboron cluster ion is higher than 10:5, flocculent precipitation occurs, liposome is not gelled, the medicine encapsulation rate is lower than 60%, and the medicine activity is lower than 40%.
Compared with groups 1-2 and groups 2-3, the activity and the encapsulation efficiency of the medicine are influenced by the ratio of boron cluster ions to the medicine under the condition that the mol ratio of the fixed HSPC to the BBr is 10:1. Optimal encapsulation efficiency and highest drug activity are obtained when the molar ratio of the twelve boron cluster ions to the drug is 1:2.
Compared with groups 1 to 11 and 5 to 6, it can be seen that the mass concentration of phospholipids in the liposome gel has an important effect on the gelling properties of the liposome suspension; when the mass concentration of the phospholipid is 1-30%, the liposome suspension can form semisolid gel; at concentrations below 1% or above 30%, liposomal suspensions do not form a homogeneous gel, but rather form a thinner dispersion or a thicker suspension.
Compared with groups 12-16 and 7, it can be seen that the mass concentration of oleic acid in the liposome gel has a significant effect on liposome gelation, drug encapsulation efficiency and drug activity, and when the mass concentration of oleic acid is higher than 3%, the liposome suspension cannot form semi-solid gel; however, oleic acid is not an essential component in the formulation, and when the formulation does not contain oleic acid, the liposome suspension can still form liposome gel, and the drug encapsulation efficiency is higher than 80%, and the drug activity is also higher than 85%.
Compared with groups 17 to 21 and 8 to 9, it can be seen that in the preparation method of the liposome gel, the pH of the liposome suspension has a remarkable influence on the gelation property, the drug encapsulation efficiency and the drug activity of the liposome suspension, the pH of the liposome suspension is in the range of 6 to 9, the liposome suspension can form semi-solid gel, and the drug encapsulation efficiency and the drug activity are both higher than 80%. The pH of the liposome suspension is lower than 6 or higher than 9, the liposome suspension can not form semisolid gel, and the medicine encapsulation rate and the activity are both lower than 30%.
Compared with the comparative groups 10 to 11, the liposome gel preparation method has the advantages that the rapid re-melting temperature after the liposome suspension is frozen has obvious influence on the gelation property, the drug encapsulation efficiency and the drug activity of the liposome suspension, the re-melting temperature of the liposome suspension is 50-85 ℃, the liposome suspension can form semi-solid gel, and the drug encapsulation efficiency and the drug activity are both higher than 80%. The liposome suspension has a melting temperature lower than 50 ℃ or higher than 85 ℃, and the liposome suspension can not form semisolid gel, and the drug encapsulation rate and the activity are both lower than 15%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the liposome gel for efficiently encapsulating and stabilizing the protein polypeptide drugs is characterized by comprising the following steps:
(1) Preparing phospholipid into liposome suspension;
(2) After regulating the pH of the liposome suspension, obtaining a stable liposome solution after freezing and thawing;
(3) And mixing the liposome solution with twelve boron cluster ions and protein polypeptide drugs to obtain the liposome gel.
2. The method of claim 1, wherein oleic acid is added in the mass ratio of phospholipid to oleic acid of 90 (10 to 30) in the preparation of the liposome suspension in step (1).
3. The method of claim 1 or 2, wherein the method of preparing the liposome suspension is a thin film hydration method, a lyophilization method, or an infusion method.
4. A method of preparation as claimed in claim 3 wherein the liposome suspension has a pH of 6 to 9 after adjustment.
5. The method according to claim 4, wherein the freezing time is 1 to 3 hours, and the freezing temperature is-30 to-10 ℃; the temperature during melting is 50-85 ℃.
6. The method according to claim 5, wherein the temperature of the mixing is 3 to 5℃and the time of the mixing is 12 to 18 hours.
7. The method according to claim 6, wherein the mass concentration of the phospholipid in the liposome gel is 1% to 30%; the mass concentration of the oleic acid is 0-3%; the mass concentration of the twelve boron cluster ions is 0.05% -4%; the mass concentration of the protein polypeptide medicine is 0.001% -5%; the balance being water.
8. The method of claim 7, wherein the molar ratio of the phospholipid to the twelve boron cluster ion is 10: (0.01-5); the molar ratio of the twelve boron cluster ions to the protein polypeptide drug is 1: (0.1-20).
9. The method of claim 8, wherein the phospholipid comprises any one or more of hydrogenated soybean phospholipid, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine; the twelve boron cluster ions comprise 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- Any one or more of the following.
10. A liposome gel of high efficiency entrapping and stabilizing protein polypeptide drugs obtained by the preparation method of any one of claims 1-9.
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