Separation and purification method for liposome containing encapsulated material

Abstract

The invention discloses a method for separating and purifying liposome containing encapsulated material through gel electrophoresis and belongs to the field of biomedicine. The method comprises the following steps: firstly preparing liposome containing encapsulated drug, then preparing polymer gel, and separating the liposome from non-encapsulated drugs by gel electrophoresis. Compared with the prior art, the invention has the following characteristics: (1), based on the difference in migration of different components under the action of an electric field, the method achieves the effective separation of the liposome from the non-encapsulated drugs; (2), with the polymer gel as the medium, the method can separate the liposomes of different sizes and different types from the non-encapsulated drugs by changing the pore size of the gel; (3), the method has a wide application range and is applied to the separation of small molecule drugs as well as DNAs, siRNAs and proteins; and (4), the method simplifies the separation operation and achieves the high-efficiency recovery of the liposome and the non-encapsulated drugs after separation.

Description

technical field

[0001] The invention relates to the separation and purification of liposomes containing drugs, belonging to the field of biomedicine. Background technique

[0002] Liposome drugs have received more and more attention in the field of modern medicine, and are also playing an increasingly important role. Liposome-encapsulated drugs can not only effectively improve the biological activity of drugs, but also improve and regulate the absorption and release of drugs through targeted methods, prevent the destruction of sensitive drugs, reduce side effects, and so on. As a drug carrier, liposomes can encapsulate hydrophilic and hydrophobic drugs, proteins and nucleic acids, and have been widely used in many biomedical fields such as cancer treatment, antibacterial, vaccine and gene therapy.

[0003] The common method of encapsulating drugs in liposomes is to mix drugs during liposome preparation, or encapsulate drugs by fusion after liposome formation. According to ...

Examples

Embodiment 1

[0019] Embodiment 1. Taking the encapsulated substance as a negatively charged small molecule (such as the commonly used model fluorescent molecule calcein, and the commonly used analgesic drug ibuprofen) as an example

[0020] 1. Preparation of electrophoresis buffer

[0021] Fully dissolve Tris (10.78g), boric acid (5.50g), and EDTA (0.74g) in 100mL of water, and filter through a filter membrane with a pore size of 0.45um to obtain a stock solution of the electrophoresis buffer. Diluted 20 times for use.

[0022] 2. Preparation of drug model solution

[0023] Here, calcein is selected as a model drug molecule to test the separation effect of this method. The molecular structure of calcein is It is not the actual drug used and has no medicinal effect. But because of the fluorescence, it is convenient for characterization. Calcein (311 mg, 0.5 mmol) was added to 5 mL of electrophoresis buffer, stirred evenly until the calcein was completely dissolved, and then 4 mol / L Na...

Embodiment 2

[0034] Embodiment 2, taking the encapsulated substance as a positively charged small molecule (such as the commonly used anticancer drug doxorubicin) as an example:

[0035] The molecular structural formula of doxorubicin is:

[0036] 1. Preparation of liposomes encapsulated with doxorubicin

[0037] The chloroform solution of distearoylphosphatidylcholine and cholesterol was taken, and the chloroform solution was removed by rotary evaporation to obtain a phospholipid film. The liposomes were prepared by freezing and thawing or extrusion by hydration with a citrate buffer solution with a pH of 4.0. The final concentration of phospholipids was 30 mM, and then the liposomes were eluted with a gel column saturated with hydroxyethylpiperazineethanesulfonic acid buffer solution to form a pH gradient inside and outside the liposomes. Dissolve doxorubicin in normal saline at a concentration of 2 mg / mL. The liposome and doxorubicin solution were mixed at a volume ratio of 5:1, an...

Embodiment 3

[0045] Embodiment 3, taking encapsulated substances as charged macromolecules (such as DNA, siRNA, etc.) as an example:

[0046] 1, the preparation method of the liposome that has encapsulated DNA

[0047] Firstly, liposomes composed of egg yolk phosphatidylcholine were prepared at a concentration of 80 mM. Take 250 μL of liposome solution and blend with 100 μL of protist DNA at a concentration of 1 mg / mL. Add ethanol and CaCl to the above mixed solution 2 Aqueous solution, vortexed for 30s and then dialyzed for 24 hours.

[0048] 2. Gel electrophoresis separation of liposomes encapsulating DNA

[0049] Prepare an agarose gel with a concentration of 0.5%, and you can choose spot staining or bubble staining to determine the separation of DNA. Acrylamide gel can also be used as the separation medium, the voltage is 9V / cm, electrophoresis is carried out for 2 hours, and the unencapsulated DNA can be separated from the liposome. Liposomes and free DNA molecules can be recover...

technical field

[0001] The invention relates to the separation and purification of liposomes containing drugs, belonging to the field of biomedicine. Background technique

[0002] Liposome drugs have received more and more attention in the field of modern medicine, and are also playing an increasingly important role. Liposome-encapsulated drugs can not only effectively improve the biological activity of drugs, but also improve and regulate the absorption and release of drugs through targeted methods, prevent the destruction of sensitive drugs, reduce side effects, and so on. As a drug carrier, liposomes can encapsulate hydrophilic and hydrophobic drugs, proteins and nucleic acids, and have been widely used in many biomedical fields such as cancer treatment, antibacterial, vaccine and gene therapy.

[0003] The common method of encapsulating drugs in liposomes is to mix drugs during liposome preparation, or encapsulate drugs by fusion after liposome formation. According to ...

Examples

Embodiment 1

[0019] Embodiment 1. Taking the encapsulated substance as a negatively charged small molecule (such as the commonly used model fluorescent molecule calcein, and the commonly used analgesic drug ibuprofen) as an example

[0020] 1. Preparation of electrophoresis buffer

[0021] Fully dissolve Tris (10.78g), boric acid (5.50g), and EDTA (0.74g) in 100mL of water, and filter through a filter membrane with a pore size of 0.45um to obtain a stock solution of the electrophoresis buffer. Diluted 20 times for use.

[0022] 2. Preparation of drug model solution

[0023] Here, calcein is selected as a model drug molecule to test the separation effect of this method. The molecular structure of calcein is It is not the actual drug used and has no medicinal effect. But because of the fluorescence, it is convenient for characterization. Calcein (311 mg, 0.5 mmol) was added to 5 mL of electrophoresis buffer, stirred evenly until the calcein was completely dissolved, and then 4 mol / L Na...

Embodiment 2

[0034] Embodiment 2, taking the encapsulated substance as a positively charged small molecule (such as the commonly used anticancer drug doxorubicin) as an example:

[0035] The molecular structural formula of doxorubicin is:

[0036] 1. Preparation of liposomes encapsulated with doxorubicin

[0037] The chloroform solution of distearoylphosphatidylcholine and cholesterol was taken, and the chloroform solution was removed by rotary evaporation to obtain a phospholipid film. The liposomes were prepared by freezing and thawing or extrusion by hydration with a citrate buffer solution with a pH of 4.0. The final concentration of phospholipids was 30 mM, and then the liposomes were eluted with a gel column saturated with hydroxyethylpiperazineethanesulfonic acid buffer solution to form a pH gradient inside and outside the liposomes. Dissolve doxorubicin in normal saline at a concentration of 2 mg / mL. The liposome and doxorubicin solution were mixed at a volume ratio of 5:1, an...

Embodiment 3

[0045] Embodiment 3, taking encapsulated substances as charged macromolecules (such as DNA, siRNA, etc.) as an example:

[0046] 1, the preparation method of the liposome that has encapsulated DNA

[0047] Firstly, liposomes composed of egg yolk phosphatidylcholine were prepared at a concentration of 80 mM. Take 250 μL of liposome solution and blend with 100 μL of protist DNA at a concentration of 1 mg / mL. Add ethanol and CaCl to the above mixed solution 2 Aqueous solution, vortexed for 30s and then dialyzed for 24 hours.

[0048] 2. Gel electrophoresis separation of liposomes encapsulating DNA

[0049] Prepare an agarose gel with a concentration of 0.5%, and you can choose spot staining or bubble staining to determine the separation of DNA. Acrylamide gel can also be used as the separation medium, the voltage is 9V / cm, electrophoresis is carried out for 2 hours, and the unencapsulated DNA can be separated from the liposome. Liposomes and free DNA molecules can be recover...

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