Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008 191
The Liposome‘s Diameter Measured by
the Computerized Program of Image Pro Express 4.5
Ernie H. Purwaningsih
Department of Medical Pharmacy, Faculty of Medicine, Indonesia University, Jakarta
4-5.
The stability of liposome was determined by several parameters, such as the amount and the diameter of liposome. It has to prove before clinically used. Their stability is influenced by physical or chemical condition, and biological activity.
Measuring the liposome’s diameter in our laboratory has to become crucial because of several conditions. For example, the equipment for measuring the diameter of liposome, particle seizer, is too expensive, and the Olympus scale measurements could not seen in a focus of liposome’s picture at the microscope, simultaneously. That’s why it was not only has time consuming because of the measurement was done one by one manually, but also the diameter data were un exactly obtained.
To solve the problems, this study has done to create the computer’s program of Image Pro Express 4.5 for measuring and the Olympus scale as a standard measurement is still using.
MATERIAL AND METHOD
This study has already conducted at the Department of Medical Pharmacy and Medical Physics, Faculty of Medicine, Indonesia University during 3 months, from June to August 2007.
Liposome of EPC (egg yolk Phosphatidylcholine) and TEL (Tetra Ether Lipid) which have named of liposome EPC-TEL 2.5, and the Olympus scale of 0.01 mm in the light microscope which has magnification of 400 have photographed and recorded in Adobe Photo-Shop 0.7 and “jpeg” system. Quinacrine solution of 0.05% was used in this study as liposome’s marker.
The control group of liposome EPC-TEL 2.5 before sonication or extrusion, the sonication and the extrusion liposome have studied previously on physical and chemical stability, at different temperatures (4º C, 37º C, and room temperature) and different times (at the day of 1; 7, 28; 56; 84). For this paper, part of the sonication data of liposome stability will be studied and reported as the liposome’s diameter using the computer program of Image Pro Express 4.5.
Karangan Asli
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008 192
1. Part (1/10) of the Olympus scale of 0.01 mm that contains 100 stripes were photographed in magnification of 400 and recorded in “jpeg” system. The area between two stripes was 100 nm.
2. That Olympus scale was entered into the Image Pro Express 4.5 and click measure for calibrating and measuring each area. On menu of “line profile” will show their pixels that identical with the length of the pixels. Calibrate all areas.
3. The picture of liposome EPC-TEL 2.5 was measured as same as the scale and noted the pixels of liposome. The measuring data were obtained from two to three samples each group.
4. Divide the liposome’s pixels with the pixels of scale and then, multiply by 100 nm. The result was the diameter’s liposome.
RESULTS AND DISCUSSION
Each area of the scale (x2-x1) have pixels of 48; 49; 48; 46; 50; 47; 47; 50; 48; 47 respectively, those the mean pixel of each area was 47. This pixel correlated with 100 nm (Figure 1).
Figure 1. The scale of “Olympus”. Each area between two stripes was 100 nm, identical with the pixels of 47 (400 X)
The pixel of each liposome from the control group, after sonication, was 27, 30, 28, respectively. By means of the steps above, the diameter of liposome as a control was (27/47X100 nm; 30/47X100 nm; 28/47X100 nm) 57.4 nm; 63.8 nm; and 59.8 nm, respectively (Figure 2). The large diameter of liposome at the day of 7 was shown at Figure 3 and 4, and the small diameter at Figure 5 and 6. The pixel of the large diameter was 75 (Figure 3) or 76 (Figure 4). Using the same formula, the exact diameter that has measured was 159.6 nm or 161.7 nm. The pixel of the small diameter
was 35; 30; 36, that was identical with 74.5 nm 63.8 nm; 76.6 nm (Figure 5, 6)
These results rather similar to the diameter of the same composition of liposome
using “Nicomp” particle-seizer7
. The difference result between the Image Pro Express 4.5 and the particle-seizer was highly influenced by the Brownian movement of liposome. That’s why, the distribution of
the liposome diameter tend to be broad. 7
. Unfortunately, this measuring model has had consumed long enough time, so we should try to innovate it to cut the steps.
Figure 2. The liposome’s diameter of the control group. The pixel was 28, see the arrow (400X).
Figure 3. The diameter of large liposome in the day of 7. The pixel was 75 (400X).
Figure 4. The diameter of large liposome at the
Ernie H. Purwaningsih The Liposome‘s Diameter Measured...
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008 193 Figure 5. The diameter of small liposome at the
day of 7. The pixel was 35 (400X).
Figure 6. The small diameter of liposome at the
day of 7 in other lining position. The pixel was 30 and 36 (400X).
Based on these results, the Image Pro Express 4.5 could be come a model for measuring other objects such as cells, component of cells, etc.
CONCLUSION AND SUGGESTION Conclusion
The Image Pro Express 4.5 could be used for measuring the liposome diameter, faster than manually measurement, but slower than the particle-seizer spectrophotometer. This program could also be used to change the categorical data (bellow or upper 100 nm) into the numerical data of liposome’s diameter (60 nm, 76 nm, or 160 nm)
Suggestion
For measuring the diameter of other objects, such as cells, component of cells, etc, the Image Pro Express 4.5 could also be a model because of these exact results. This program should be innovated, not only for
measuring the diameter, but also the amount of the nanometer’s objects, such as liposome.
Acknowledgments
Many thanks to all Staff member of the Department of MedicalPphysics,Indonesia University especially to DR. Hamdani zain (alm), for generously supports to help improving our liposome’s measurement.
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