CN109646401A - A kind of preparation method for mixing liposome containing targeting ester polypeptide - Google Patents
A kind of preparation method for mixing liposome containing targeting ester polypeptide Download PDFInfo
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- CN109646401A CN109646401A CN201811549582.3A CN201811549582A CN109646401A CN 109646401 A CN109646401 A CN 109646401A CN 201811549582 A CN201811549582 A CN 201811549582A CN 109646401 A CN109646401 A CN 109646401A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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Abstract
本发明涉及一种含有靶向性酯多肽的混杂脂质体的制备方法，属于制药领域，所述制备方法包括以下步骤：首先称取卵磷脂、胆固醇和酯多肽，置于玻璃容器中，向玻璃容器中加入无水乙醇溶解，得混合液；其次，向混合液中加入姜黄素无水乙醇溶液，混匀，在旋转蒸发仪上减压蒸发除去有机相至瓶壁上形成均匀透明的薄膜；随即趁热加入磷酸缓冲盐溶液将薄膜洗脱分散，水浴常压旋转水合，超声溶解，得混杂脂质体溶液。所述制备方法采用一种含RGD多肽和C12序列的两亲性多肽作为靶向材料，姜黄素为模型药物，制备出的脂质体具有包封率高、粒径分布均匀、稳定性好、安全低毒的优点。
The invention relates to a preparation method of a hybrid liposome containing a targeting ester polypeptide, belonging to the field of pharmacy. The preparation method comprises the following steps: firstly, weighing lecithin, cholesterol and ester polypeptide, placing them in a glass container, and placing them in a glass container. Add anhydrous ethanol to the glass container to dissolve to obtain a mixed solution; secondly, add curcumin anhydrous ethanol solution to the mixed solution, mix well, evaporate the organic phase under reduced pressure on a rotary evaporator to form a uniform and transparent film on the bottle wall ; Immediately add phosphate buffered saline solution while hot to elute and disperse the film, rotate in a water bath at normal pressure for hydration, and dissolve by ultrasonic to obtain a mixed liposome solution. The preparation method adopts an amphiphilic polypeptide containing an RGD polypeptide and a C12 sequence as a targeting material, and curcumin is a model drug, and the prepared liposome has high encapsulation efficiency, uniform particle size distribution, good stability, The advantages of safety and low toxicity.
Description
Technical field
The invention belongs to pharmaceutical fields, and in particular, to a kind of preparation for mixing liposome containing targeting ester polypeptide
Method.
Background technique
Liposome refers to drug encapsulation in the miniature vesicular body formed in lipoids bilayer.Because it has sustained release
The features such as type and hypotoxicity, plays an important role in drug delivery system.Liposome is the property or right by itself
The purpose for modifying available different target administration or slow controlled release on its surface.Curcumin (CUR) is from zingiberaceous plant turmeric
A kind of phenolic compound of middle extraction is the main active of Turmeric.Studies have shown that CUR be it is a kind of safely and effectively
Drug with a variety of effects such as anticancer, anti-inflammatory, anti-oxidant, wherein curcumin is for preventing and treating tumour, swelling to a variety of
Generation, proliferation, the transfer of oncocyte all have inhibiting effect, and therefore, CUR is often used as anti-tumor drug.But CUR is not
It is dissolved in water, and aqueous solution is unstable under neutrality to alkaline pH, is stored three days at 4 DEG C of aqueous solution, Anticancer Activity in vitro
It is decreased obviously.CUR is encapsulated in the direction for preparing that liposome formation medicine-carried system is research in lipoids bilayer, is related to
And the stability of liposome, drug contain rate, how to extend the retention time after liposome enters body, improve biology benefit
The problems such as quantization with rate and in the industrial production.
Ester polypeptide is as a kind of functional polypeptide material, since it is with hydrophobic saturated carbon chains and hydrophilic polypeptide chain
Section, this amphipathic structure and the structure of lipoids molecule are extremely similar, excellent self assembly performance and targeting in water
Liposome can be improved drug to the targeting of destination organization and with the affinity and conjugation of destination organization.It is peptide modified
The liposome for containing drug can increase the selectivity of drug in vivo, reduce the toxic side effect of drug, improve drug therapy
Index.
Peptide molecule is a kind of important bioactive substance in body, special with ligand-receptor as fairlead
Property combine mode be applied to targeted drug delivery system, have good researching value and application prospect.By designing amino
Acid sequence can obtain a series of self assembly polypeptide of 8~16 amino acid residues.Self-assembling polypeptide mainly passes through a system
The drivings such as the non-covalent bond effect power of column such as hydrogen bond, electrostatic interaction, hydrophobic effect, Van der Waals force, fragrant accumulation, aquation
It realizes.Amphiphilic peptide has amphipathic, good biocompatibility and biodegradability, and is easy to carry out molecule and sets
The features such as meter and structural modification, thus self assembly polypeptide has huge potentiality in terms of as hydrophobic drug carrier.For
How different hydrophobic drugs effectively improves encapsulation rate and reduces its leakage, improves bioavilability, then need root
Most suitable preparation process is found out according to formulation requirements.
Summary of the invention
For above situation, the purpose of the present invention is to provide a kind of systems for mixing liposome containing targeting ester polypeptide
Preparation Method, the preparation method is using a kind of RGD containing peptides and the Amphiphilic peptide of C12 sequence as targeting material, curcumin
Prepare liposome for model drug, solve hydrophobic drug because in water solubility it is low due to be administered that difficult, bioavilability is low
Problem, while the liposome prepared has the advantages that encapsulation rate is high, particle diameter distribution is uniform, stability is good, safety and low toxicity.
To achieve the goals above, the present invention use the specific scheme is that
A kind of preparation method for mixing liposome containing targeting ester polypeptide, it is characterised in that: the following steps are included:
Step 1: weighing lecithin, cholesterol and ester polypeptide, it is placed in glass container, dehydrated alcohol is added into glass container
Dissolution, obtains mixed liquor；The chemical structural formula of the ester polypeptide are as follows:
Step 2: curcumin ethanol solution is added into mixed liquor, mix, being evaporated under reduced pressure to remove on a rotary evaporator has
Machine is mutually to the film of formation homogeneous transparent in bottle wall；Phosphate buffered saline solution is added while hot immediately and elutes film and disperses, 42-48
DEG C water-bath normal pressure rotation hydration 20-40min, ultrasonic 10-20min make sufficiently to dissolve, and must mix liposome solutions.
It is advanced optimized as to above scheme, the mass ratio of the lecithin and cholesterol is 1:1~3:1；Turmeric
Cellulose content is 0.1~0.25mg；The additional amount of phosphate buffered saline solution is 30-50mL, concentration 0.01mol/L；Ester polypeptide
Content is 0.001g.
As the further optimization to above scheme, the mass ratio of the lecithin and cholesterol is 2:1；Curcumin contains
Amount is 0.15mg；The additional amount of phosphate buffered saline solution is 50mL.
It is advanced optimized as to above scheme, the frequency of the ultrasound is 30KHZ, and temperature is 30 DEG C.
It is advanced optimized as to above scheme, the absolute pressure of reduction vaporization described in step 2 is 0.
The utility model has the advantages that
1, the peptide molecule that the present invention uses is C12KKGRGDS sequence, RGD sequence therein are by arginine, glycine
It is formed with aspartic acid, can be specifically bound with 11 kinds of integrins, adherency of the cell to biomaterial can be effectively facilitated, mentioned
Adherency of the high lesion tissue to drug target.Preparation method of the present invention utilizes a kind of RGD containing peptides and C12 aliphatic chain
For Amphiphilic peptide as targeting material, the liposome that curcumin is model drug preparation, targeting is strong, solves because hydrophobicity exists
Solubility is low in water and the problem that causes administration difficult, and liposome has that encapsulation rate is high, particle diameter distribution is uniform, percolation ratio is low, steady
The advantages that qualitative good, safety and low toxicity, enables drug high efficiency to reach lesions position and plays drug action, improves biological utilisation
Rate.
2, the prescription that the method for the present invention uses is simple, and stable process conditions are good, is easy to industrialization, has larger popularization
Value.Curcumin liposome encapsulation rate made from this method is high.The in vitro toxicity test proof of liposome prepares the material of liposome
Lecithin and cholesterol and the targeting material C 12 of addition for cell, toxicity be it is very small, can ignore substantially, accord with
The requirement of composite medicine carrier low toxicity, can be used as pharmaceutical carrier.In addition, the ester polypeptide mixes liposome there is targeting positioning to make
With, the accumulation by load drug in lesion tissue can be significantly improved, and drug effect is effectively improved, and the poison for reducing drug is secondary
Effect, is expected to be developed as novel target medicine carrier.
Detailed description of the invention
Fig. 1 is liposomal particle size distribution map；
Fig. 2 is cytotoxicity comparison diagram of the conventional liposome of various concentration with C12 liposome to C6 cell.
Specific embodiment
Below in conjunction with the embodiment of the present invention and attached drawing, technical solution in the embodiment of the present invention carries out clear, complete
Ground description.
A kind of preparation method for mixing liposome containing targeting ester polypeptide:
1, experimental procedure
Liposome is prepared using film dispersion method.Precision weighs lecithin, cholesterol and C12KKGRGDS polypeptide in
In 500mL round-bottomed flask, appropriate dehydrated alcohol is added, ultrasound is allowed to be completely dissolved；Add the anhydrous second of the curcumin prepared
Alcoholic solution is evaporated under reduced pressure on removing organic phase (control temperature is at 45 DEG C) to bottle wall on a rotary evaporator and forms homogeneous transparent
Film；Phosphate buffered saline solution is added while hot immediately and elutes film and disperses, 42-48 DEG C of water-bath normal pressure rotation hydration 20-
40min, ultrasonic 10-20min make sufficiently to dissolve, and must mix liposome solutions.
The C12KKGRGDS polypeptide is purchased from Shanghai Qiang Yao Bioisystech Co., Ltd, purity > 95%；
In the process, about the mass ratio of lecithin and cholesterol, the additional amount of curcumin, the additional amount and use of ester polypeptide
In the additional amount of the phosphate buffer of dissolution be vital to the performance indicator of the liposome of preparation.
The frequency of above-mentioned ultrasound is 30KHZ, and temperature is 30 DEG C；The absolute pressure of reduction vaporization be 0, relative pressure be-
0.01Mpa；The mass ratio of the lecithin and cholesterol is 1:1~3:1；Turmeric cellulose content is 0.1~0.25mg；Phosphoric acid buffer
The additional amount of salting liquid is 30-50mL, concentration 0.01mol/L.
2, the optimum material proportion of liposome preparation is studied
Optimum proportioning is found using orthogonal test, it is final to determine most using the encapsulation rate of liposome as the index investigated
Excellent prescription.The test method of reasonable arrangement and analysis many factors, has finally chosen three principal elements and is analyzed, is i.e. ovum
Phosphatide and cholesterol ratio A, curcumin dosage are B, PBS dosage C, and each factor determines three levels, each number of levels of A factor
It is followed successively by 1:1,2:1,8:1；Each number of levels of B factor is followed successively by 0.1,0.15,0.25；Each number of levels of C factor is followed successively by
30,40,50, L9 (33) orthogonal array is selected, as shown in table 1 below.
Table 1: Orthogonal Experiment and Design optimizes liposome prescription and the experimental design and result of preparation process
Measurement result: with the increase of lecithin and cholesterol ratio, the encapsulation rate first increases and then decreases of liposome；With
The encapsulation rate of the increase of curcumin dosage, liposome is consequently increased；With the increase of PBS dosage, the encapsulation rate of liposome
Increase therewith.So optimal level group is combined into A2B2C3, i.e. the ratio of lecithin and cholesterol is 2:1, wherein lecithin
0.02g, cholesterol 0.01g；The quality of curcumin is 0.15mg；PBS is 50mL.
3, formulation optimization
Under the optimal conditions optimized above, different amounts of targeting material C is added12KKGRGDS polypeptide, is prepared respectively
Target liposomes.Using encapsulation rate as inspection target, C is finally determined12The dosage of KKGRGDS polypeptide is 0.001g.
4, the particle diameter distribution of target liposomes
Particle diameter distribution is detected using dynamic light scattering (DLS).Resulting liposome turbid liquor is prepared by 0.2mL is fresh
With the dilution of 2.5mL secondary distilled water.Measurement is repeated 3 times every time.
Its average grain diameter is 115nm to measurement result as shown in Figure 1: (coefficient of dispersion PDI is 0.131).By Fig. 1 analysis Lai
It sees, the liposomal particle size prepared is smaller, is evenly distributed.
5, stability test
It contains the liposome of RGD peptide and blank liposome sample liquid stands at 4 ± 2 DEG C and is kept in dark place 5 days, and the 1st,
3,5 days measurement percolation ratios.The results are shown in Table 2.
Table 2: the percolation ratio of target liposomes containing peptide and blank liposome
| Time | 1 day | 3 days | 5 days | Percolation ratio |
| Containing peptidoliposome | 82.78 | 75.08 | 73.34 | 11.4% |
| Blank liposome | 92.83 | 87.69 | 80.58 | 13.2% |
Measurement result: the percolation ratio measured in five days is smaller than the percolation ratio of Placebo liposomes containing peptidoliposome.
6, the in vitro toxicity test of liposome
Non- drug-loaded liposome selects C6 cell to carry out in vitro toxicity test by mtt assay.C6 cell is by 6000/hole
Concentration kind is in 96 orifice plates.After DMEM (200 hole the μ L/) culture for 24 hours containing 10%FBS, 200 μ L of change contain specific dense
The fresh culture of degree liposome continues to cultivate 48h.Portalled after middle solution with 200 μ L fresh culture mediums, is added to every hole
20 μ L MTT solution (5mg/mL) cultivate 4h.Culture medium is removed, and 150 μ L DMSO are added in each hole.At 570nm,
Absorbance value (OD) is measured through microplate reader (Bio-Rad, Model 550, USA).Versus cell activity is calculated by following formula:
Cell activity (%)=[OD570 (sample)/OD570 (control)] × 100.Wherein, OD570 (control) indicates to be free of liposome
Measured value, and OD570 (sample) indicate the measured value containing liposome.
Measurement result is as shown in Fig. 2, when the concentration of liposome reaches 200 μ g/mL, and the relative survival rate of C6 cell is all
80% or more, show the targeting material C of the materials lecithin for preparing liposome and cholesterol and addition12KKGRGDS is to cell
For, toxicity be it is very small, can ignore substantially, meet the requirement of pharmaceutical carrier low toxicity, can be used as pharmaceutical carrier.
Claims (5)
1. a kind of preparation method for mixing liposome containing targeting ester polypeptide, it is characterised in that: the following steps are included:
Step 1: weighing lecithin, cholesterol and ester polypeptide, it is placed in glass container, dehydrated alcohol is added into glass container
Dissolution, obtains mixed liquor；The chemical structural formula of the ester polypeptide are as follows:
Step 2: curcumin ethanol solution is added into mixed liquor, mix, being evaporated under reduced pressure to remove on a rotary evaporator has
Machine is mutually to the film of formation homogeneous transparent in bottle wall；Phosphate buffered saline solution is added while hot immediately and elutes film and disperses, 42-48
DEG C water-bath normal pressure rotation hydration 20-40min, ultrasonic 10-20min make sufficiently to dissolve, and must mix liposome solutions.
2. a kind of preparation method for mixing liposome containing targeting ester polypeptide as described in claim 1, it is characterised in that:
The mass ratio of the lecithin and cholesterol is 1:1 ~ 3:1；Turmeric cellulose content is 0.1 ~ 0.25mg；Phosphate buffered saline solution adds
Entering amount is 30-50mL, concentration 0.01mol/L；The content of ester polypeptide is 0.001g.
3. a kind of preparation method for mixing liposome containing targeting ester polypeptide as claimed in claim 2, it is characterised in that:
The mass ratio of the lecithin and cholesterol is 2:1；Turmeric cellulose content is 0.15mg；The additional amount of phosphate buffered saline solution is
50mL。
4. a kind of preparation method for mixing liposome containing targeting ester polypeptide as described in claim 1, it is characterised in that:
The frequency of the ultrasound is 30KHZ, and temperature is 30 DEG C.
5. a kind of preparation method for mixing liposome containing targeting ester polypeptide as described in claim 1, it is characterised in that:
The absolute pressure of reduction vaporization described in step 2 is 0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110201147A (en) * | 2019-05-23 | 2019-09-06 | 中山市粤美医疗生物科技有限公司 | A kind of composition and preparation method thereof comprising peptide |
| CN114532535A (en) * | 2022-01-05 | 2022-05-27 | 齐鲁工业大学 | Preparation method of curcumin nano-liposome |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06219967A (en) * | 1993-01-22 | 1994-08-09 | D D S Kenkyusho:Kk | Peptide-lipid derivative and liposome |
| CN101538312A (en) * | 2009-05-08 | 2009-09-23 | 首都医科大学 | Preparation and applications of RGD-fatty amine series compound as tumor targeting vector material |
-
2018
- 2018-12-18 CN CN201811549582.3A patent/CN109646401A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06219967A (en) * | 1993-01-22 | 1994-08-09 | D D S Kenkyusho:Kk | Peptide-lipid derivative and liposome |
| CN101538312A (en) * | 2009-05-08 | 2009-09-23 | 首都医科大学 | Preparation and applications of RGD-fatty amine series compound as tumor targeting vector material |
Non-Patent Citations (2)
| Title |
|---|
| JU L ET AL: "Enhanced solubility and targeted delivery of curcumin by lipopeptide micelles", 《JOURNAL OF BIOMATERIALS SCIENCE》 * |
| 许汉林等: "姜黄素脂质体制备工艺的研究", 《湖北中医学院学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110201147A (en) * | 2019-05-23 | 2019-09-06 | 中山市粤美医疗生物科技有限公司 | A kind of composition and preparation method thereof comprising peptide |
| CN114532535A (en) * | 2022-01-05 | 2022-05-27 | 齐鲁工业大学 | Preparation method of curcumin nano-liposome |
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