The Liposome‘s Diameter Measured by the Computerized Program of Image Pro Express 4.5
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
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.
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.
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.
The steps of measuring the diameter of
liposome using Image Pro Express 4.56:
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008
191
Karangan Asli
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
192
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
7
using
“Nicomp”
particle-seizer .
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
7
the liposome diameter tend to be broad. .
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
day of 7 in other lining position.
The pixel was 76 (400X).
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008
Ernie H. Purwaningsih
The Liposome‘s Diameter Measured...
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.
Figure 5. The diameter of small liposome at the
day of 7. The pixel was 35 (400X).
REFERENCES
1.
Lasic DD (ed). Liposomes as a drug
delivery system Liposomes from Physics
to Application. Elsevier Science Publisher
BV 1993, p.265-324.
2.
Papahadjopoulos D, Allen TM, Gabizon
A, et al. Sterically stabilized liposomes:
Improvements in pharmacokinetics and
antitumor therapeutic efficacy. Proc Natl
Acad Sci 1991; 88: 11460-64.
3.
Huang SK, Mayhew E, Gilani S, Lasic
DD, Martin FJ, Papahadjopoulos D.
Pharmacokinetics and therapeutics of
sterically stabilized liposomes in mice
bearing C-26 colon carcinoma. Cancer
Research 1992; 52: 6774-81.
4.
Gabizon AA. Selective tumor localization
and improved therapeutic index of
anthracyclines encapsulated in longcirculating liposomes. Cancer Research 1992;
52: 891-6.
5.
Freisleben HJ, Bormann J, Litzinger DC,
Lehr F, Rudolph P, Schatton W, Huang
L. Toxicity and biodistribution of
liposomes of the main phospholipid from
the
Archaebacterium
Thermoplasma
acidophilum in mice. J Liposome Research
1995; 5 (1):215-23.
6.
Sugai A, Sakuma R, Fukuda I, Kurosawa
N, Itoh YH, Kon K, Ando S, Itoh T. The
structure of the core polyol of the ether
lipids from Sulfolobus acidocaldarius.
Lipids 1995;30 (4):339-44.
7.
Image Pro Express
Cybernetics 2002.
8.
Purwaningsih EH, Freisleben HJ, Sadikin
M. Peningkatan inkorporasi metalprednisolon palmitat pada liposom yang
mengandung
tetraeter
lipid
dari
Sulfolobus
acidocaldarius
membran
membentuk sediaan baru liposomal
metilprednisolon palmitat. Jurnal Farmasi
Indonesia 2002; 1(1):24-30.
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
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008
4.5
in
Media
193
the Computerized Program of Image Pro Express 4.5
Ernie H. Purwaningsih
Department of Medical Pharmacy, Faculty of Medicine, Indonesia University, Jakarta
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.
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.
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.
The steps of measuring the diameter of
liposome using Image Pro Express 4.56:
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008
191
Karangan Asli
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
192
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
7
using
“Nicomp”
particle-seizer .
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
7
the liposome diameter tend to be broad. .
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
day of 7 in other lining position.
The pixel was 76 (400X).
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008
Ernie H. Purwaningsih
The Liposome‘s Diameter Measured...
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.
Figure 5. The diameter of small liposome at the
day of 7. The pixel was 35 (400X).
REFERENCES
1.
Lasic DD (ed). Liposomes as a drug
delivery system Liposomes from Physics
to Application. Elsevier Science Publisher
BV 1993, p.265-324.
2.
Papahadjopoulos D, Allen TM, Gabizon
A, et al. Sterically stabilized liposomes:
Improvements in pharmacokinetics and
antitumor therapeutic efficacy. Proc Natl
Acad Sci 1991; 88: 11460-64.
3.
Huang SK, Mayhew E, Gilani S, Lasic
DD, Martin FJ, Papahadjopoulos D.
Pharmacokinetics and therapeutics of
sterically stabilized liposomes in mice
bearing C-26 colon carcinoma. Cancer
Research 1992; 52: 6774-81.
4.
Gabizon AA. Selective tumor localization
and improved therapeutic index of
anthracyclines encapsulated in longcirculating liposomes. Cancer Research 1992;
52: 891-6.
5.
Freisleben HJ, Bormann J, Litzinger DC,
Lehr F, Rudolph P, Schatton W, Huang
L. Toxicity and biodistribution of
liposomes of the main phospholipid from
the
Archaebacterium
Thermoplasma
acidophilum in mice. J Liposome Research
1995; 5 (1):215-23.
6.
Sugai A, Sakuma R, Fukuda I, Kurosawa
N, Itoh YH, Kon K, Ando S, Itoh T. The
structure of the core polyol of the ether
lipids from Sulfolobus acidocaldarius.
Lipids 1995;30 (4):339-44.
7.
Image Pro Express
Cybernetics 2002.
8.
Purwaningsih EH, Freisleben HJ, Sadikin
M. Peningkatan inkorporasi metalprednisolon palmitat pada liposom yang
mengandung
tetraeter
lipid
dari
Sulfolobus
acidocaldarius
membran
membentuk sediaan baru liposomal
metilprednisolon palmitat. Jurnal Farmasi
Indonesia 2002; 1(1):24-30.
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
Majalah Kedokteran Nusantara Volume 41 y No. 3 y September 2008
4.5
in
Media
193