Liposome

Preparation of doxorubicin-encapsulated liposomes using Presome™

1. Add 250mM (NH₄)₂SO₄ to Presome ACD-1^※ and disperse it uniformly at 60℃ to form multilamellar liposomes.
2. Pass through a polycarbonate membrane filter of 0.2㎛, 0.1㎛, and 0.08㎛ diameter 5 times each using an extruder.
3. (NH₄)₂SO₄ in the liposome outer water layer is converted to sucrose by ultrafiltration.
4. Add doxorubicin and incubate at 60℃ for 30 minutes to encapsulate doxorubicin in the aqueous liposome layer. Then adjust to ㏗ 6.5 using 0.1 N HCl or 0.1 N NaOH.
※Presome ACD-1: HSPC/Cholesterol/MPEG2000-DSPE=3/1/1 (w/w)

Comparison with the commercial liposomal drug DOXIL®

Lipid and drug concentration

Lipid and doxorubicin concentrations were quantified by HPLC.

Components	Interview Form of DOXIL®	Assay Values	% of Total Lipid
HSPC	9.58 mg/mL	9.74 mg/mL	59.6%
Cholesterol	3.19 mg/mL	3.27 mg/mL	20.0%
MPEG2000-DSPE	3.19 mg/mL	3.33 mg/mL	20.4%
doxorubicin	2.00 mg/mL	2.00 mg/mL	-

Lipid to drug concentration ratio

Interview Form of DOXIL®: 0.125
DOXIL® from Presome ACD-1: 0.122

Drug encapsulation rate

In order to measure the encapsulation rate of doxorubicin, the obtained liposomes were ultracentrifuged and the liposome fraction was collected. The doxorubicin concentration was quantified by HPLC, and the concentration of doxorubicin encapsulated in the liposomes was measured.

The percentage of encapsulated Doxorubicin in DOXIL® from Presome ACD-1: 100%

Particle size distribution and zeta potential

The average particle size, polydispersity index [PDI], and zeta potential of the obtained liposome solution were measured by dynamic scattering light analysis using Malvern Nano-ZS.

Enlarge illustration
Particle size distribution
The average particle size: 100.9± 0.9㎚
PDI: 0.048± 0.004

Zeta potential
-27.1± 0.7㎷ [9% sucrose]

Hydration layer thickness measurement

From the slope of the approximate curve of In ζ-potential vs. Debye-Huckel parameter plot ^1], the thickness of the hydrated layer on the surface of the liposome was measured.

NaCl Conc. [C]	ζ-potential
0 M	-36.05±2.20㎷
0.005 M	-19.36±0.25㎷
0.010 M	-15.06±1.04㎷
0.015 M	-12.26±1.79㎷
0.050 M	-7.22±1.12㎷
0.100 M	-4.75±1.14㎷

The thickness of the fixed aqueous layer of DOXIL® from Presome ACD-1: 2.1㎚

1] K. Shimada, A. Miyagishima, Y. Sadzuka, Y. Nozawa, Y. Mochizuki, H. Ohshima and S. Hirota, Determination of the thickness of the fixed aqueous layer around polyethyleneglycol-coated liposomes. Journal of Drug Targeting, 1995, 3, 283

TEM image

Negative staining: The samples were let to absorbed 400 mesh carbon coated grid and were put into 2% phospho tungstic acid solution [㏗7.0] for a few seconds.

Observation and imaging: The samples were observed by a transmission electron microscope [JEM-1200EX; JEOL Ltd.,] at an acceleration voltage of 80㎸.
Digital images [2048x2048 pixels] were taken with a CCD camera [VELETA, Olympus Soft Imaging Solutions GmbH].

Summary of Comparison with the commercial liposomal drug DOXIL®

As described above, we were able to prepare single membrane liposomes using Presome. In addition, most of the drug was retained in the liposome aqueous layer, and the lipid / drug composition, average particle size, particle distribution, and hydration layer thickness were shown to be equivalent to DOXIL®.
Based on these facts, it is considered that even if Presome is used, a liposomal drug equivalent to DOXIL® can be produced.

Liposome design flow

※2 - 4 will be repeated as necessary.

1. Request from the customer [Conclusion of confidentiality agreement if necessary]
2. Consideration liposome preparation and submit sample
3. Evaluate liposome performance, efficacy, etc. by customer
4. Both companies share results details [Visit, email]
5. Determination of basic lipid composition
Promotion of drug development

Support system

In the Nippon Fine Chemical Lipit Department, we have experienced encapsulating drugs in many liposomes using our abundant high-purity phospholipid series.
The Presome series is also available to provide services tailored to customer needs.

Basic research

Lipid composition design
Pharmacokinetic study
Liposome properties and performance evaluation
Liposome stability assessment
Consideration of commercial production of liposome solution [※1]

Preclinical

Pharmacokinetic study
Drug efficacy test
Product safety testing [GLP]

Clinical trial

Investigational product manufacturing
Conduct clinical trials [GMP]

Approval application / pharmaceutical sales

Final formulation manufacturing [GMP]

Consideration of commercial production of liposome solution [※1]

Requires a large amount of expensive phospholipids.
※66,200 yen for 1g DSPC at reagent manufacturer
Extruder [granulating device] is required for particle size control
If the liposome solution preparation is incomplete, the original effects and results cannot be obtained.
Liposome preparation in flask [Bangham method] is difficult to scale up for commercial production