Phospholipid for Liposome
Formulation
Empty Liposomes <COATSOME® EL Series>
In general , preparation of liposomal drugs requires adjustments of the liposomal composition and also control of the particle size and encapsulation efficiency, which make researchers usually reluctant to formulate liposomal drugs. NOF has successfully overcome these problems by developing unique experimental kits for the investigation of liposomal drug delivery systems (DDS).
Freeze-dried ready-to-use liposome powder, called Empty Liposomes, filled in vials is composed of several kinds of phospholipids and electrolytes. When a drug solution is poured into a vial and gently shaken, the drug is easily encapsulated in the liposomes. Empty Liposomes have a special advantage in that even drugs such as the anthracyclines and aminoglycosides can be efficiently encapsulated without the need for special technologies such as extrusion.
Freeze-dried ready-to-use liposome powder, called Empty Liposomes, filled in vials is composed of several kinds of phospholipids and electrolytes. When a drug solution is poured into a vial and gently shaken, the drug is easily encapsulated in the liposomes. Empty Liposomes have a special advantage in that even drugs such as the anthracyclines and aminoglycosides can be efficiently encapsulated without the need for special technologies such as extrusion.
How to use
(1) COATSOME® EL from a cool storage temperature and allow it to come to room temperature.
(2) Add an aqueous solution of a drug in the temperature range of 16℃ to 40℃.
(3) Shake the vial gently three to five times by hand.
(4) The liposomal drug is now ready for use.
(2) Add an aqueous solution of a drug in the temperature range of 16℃ to 40℃.
(3) Shake the vial gently three to five times by hand.
(4) The liposomal drug is now ready for use.
EL Series Product Characteristics
The product compositions and characteristics are shown below. Cationic charged liposomes are prepared by incorporating stearylamine as a charged lipid into the formulation. Nonionic and anionic charged liposomes are prepared by altering the molarity of DPPG as the charged lipid. While preparing an aqueous solution of the drug in water (Distilled Water for Injection), it is necessary to decide the concentrations according to the molarity of the DPPG as the charged lipid. For further details, please refer to Suggestions for Use in the next section.
COATSOME® EL series
COATSOME® EL-P series
COATSOME® EL-01-D
We have also provided new cationic liposomes (COATSOME EL-01-D) applicable to gene delivery systems. This novel cationic liposome contains not only O, O’-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, but also DOPE and cholesterol, and confers the characteristics of efficient transfection activity and expression ability in both in- vitro serum-containing media and in- vivo assay systems.
We have also provided new cationic liposomes (COATSOME EL-01-D) applicable to gene delivery systems. This novel cationic liposome contains not only O, O’-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, but also DOPE and cholesterol, and confers the characteristics of efficient transfection activity and expression ability in both in- vitro serum-containing media and in- vivo assay systems.
Suggestions for Use
COATSOME® EL
How to use
| a) | When 2 mL of distilled water is added, the osmotic pressure ratio of the product becomes 0.8 to 1.1. The osmotic pressure should be adjusted according to the need in the intended experiment. |
| b) | For enhancement of the encapsulation efficiency of the drug by electrostatic interaction, the molar ratio of the charged lipids (either stearylamine or DPPG) against the drug solution plays an important role; the molar ratio should be at least more than 2, and preferably not less than 3. |
| c) | Adjust the temperature range for storage of the aqueous drug solution between 16℃ and 40℃. |
| d) | Use the liposomal drugs immediately after the adjustment. (This is not for use in humans.) |
Determination of the Encapsulation Efficiency
Method for removal of the unencapsulated drugs
| a) | Dialysis Place the prepared drug liposome solution in a dialyzing tube and dialyze it against an isotonic solution: the unencapsulated drug leaks into the isotonic solution outside the tube. |
| b) | Gel Filtration Pass the prepared liposome solution through a column filled with a packing material for gel filtration. The liposomes and the unencapsulated drug are eluted from the column in that order. Select the packing materials according to the molecular weight of the drug to be removed (e.g., Sephadex G-50 for low molecular weight, Sepharose 4B for high molecular weight). |
| c) | Centrifugation When centrifugation is performed at high speeds after the addition of physiological saline (150mM NaCl), the liposomal drug is precipitated. After removal of the supernatant, add saline once again. This procedure usually needs to be repeated two or three times at 100,000rpm. Regardless of the procedure employed, it is absolutely essential to maintain both the inside and outside layers of the liposomes isotonic, and the temperature below the liposome phase -transition temperature throughout the procedure. During centrifugation, the temperature should be below 40℃. |
Quantitative determination of the encapsulated drug
After removal of the non-encapsulated drug according to the methods described above, perform quantitative determination of the drug encapsulated in the liposomes after destroying the liposomes. Two methods are available for the destruction of liposomes; addition of surfactants and separation of the aqueous layer from the solvent layer by the addition of an appropriate solvent (chloroform). After destroying the liposomes, conduct the quantitative assay of the released drug as directed in Ordinary Procedures.
After removal of the non-encapsulated drug according to the methods described above, perform quantitative determination of the drug encapsulated in the liposomes after destroying the liposomes. Two methods are available for the destruction of liposomes; addition of surfactants and separation of the aqueous layer from the solvent layer by the addition of an appropriate solvent (chloroform). After destroying the liposomes, conduct the quantitative assay of the released drug as directed in Ordinary Procedures.
Application Data for the COATSOME® EL Series
Cationic Liposomes, COATSOME® EL-01-C
Nonionic Liposomes, COATSOME® EL-01-N
For enhancement of the encapsulation efficiency, the concentration of the cationic drug needs to be controlled at below 1mM. Thus, to achieve higher encapsulation efficiency, the molar ratio of DPPG (3mM) against the drug should be not less than 3.
For enhancement of the encapsulation efficiency, the concentration of the cationic drug needs to be controlled at below 1mM. Thus, to achieve higher encapsulation efficiency, the molar ratio of DPPG (3mM) against the drug should be not less than 3.
Anionic Liposomes, COATSOME® EL-01-A
Improvement of the encapsulation efficiency requires maintenance of the cationic drug concentration below 5mM. If higher encapsulation efficiency is desired, the molar ratio of DPPG (15mM) against the drug should be kept at not less than 3.
Improvement of the encapsulation efficiency requires maintenance of the cationic drug concentration below 5mM. If higher encapsulation efficiency is desired, the molar ratio of DPPG (15mM) against the drug should be kept at not less than 3.
Comparison of activity in vivo*
*K.Yachi et al, Biopharm. & Drug Dispos., 17, 589-605(1996):Composition:DPPC:DPPG:Chol=27:53:20 (Anionic Liposomes)
*K.Yachi et al, Biopharm. & Drug Dispos., 17, 589-605(1996):Composition:DPPC:DPPG:Chol=27:53:20 (Anionic Liposomes)
Cationic Liposomes for gene delivery (COATSOME® EL-01-D)
Transfection Activities in vitro*1 by Luciferase Assay*5 (Light units/mg protein・sec)
Transfection Activities in vitro*1 by Luciferase Assay*5 (Light units/mg protein・sec)
Transfection Activities in vivo*2 in intraperitoneal disseminated tumors*5
Relationship Between Transfection Efficiencies and Cell Mitotic Activities*3,*5
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| Transfection performed in the absence or presence of 10% fetal bovine serum (FBS). Data shown represent mean of three experiments. |
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| mEIIL cells growing in peritoneal cavities of nude mice were transfected with liposome/CAG-lacZ (20 μ g) and the percentage of lacZ-positive cells was determined. Data shown represent mean: ± SD. |
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| Percentages of LacZ-positive cells and labeling indexes were determined. Transfection was performed in the presence of 10% FBS. Data shown represent mean ±SD. n=3 |
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| ND = Not done |
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| Reference: A. Kikuchi et al., HUMAN GENE THERAPY, 10: 947-955 (1999). |
   
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