Proceedings of the 2010 3rd IEEE RAS & EMBS

International Conference on Biomedical Robotics and Biomechatronics,

The University of Tokyo, Tokyo, Japan, September 26-29, 2010

Experimental Study and Model of the Physical Chemical Properties

of Liposome under Ultrasonic Irradiation
Roberto Furukawa, Member, IEEE

Abstract— In the current method of drug administration for

optimal dose and to the affected area (Drug Delivery System:
DDS), a research of the study on permeability effect of liposome
under external stimulation is conduced. Different conditions of
ultrasonic irradiation are considered for experimental research
such as time of irradiation, frequency of irradiation, position of
the probe, water level, different concentration of liposomes and
light. Due to a better understand of each stage of the process, a
physical chemistry model is developed.
Fig. 1. Process diagram of the experiment. The experiment starts with
I. INTRODUCTION the ingredients, transforming it in liposome by preparation. Then the
liposomes are irradiated with ultrasound and other effects. Samples of

M any studies in medical treatment are developed;

conventional drug administration is administered
throughout the body in a diffusion process, causing side
liposomes are analyzed by Spectrofluorometer and TEM observation.

effects. Controlled release drug (Drug Delivery System:

DDS) is studied for optimal dose to the affected area
(targeting) using liposomes. The current applications are in
medicine and pharmaceutics, as carriers of drugs. [1]
There are studies with temperature sensitive liposomes,
changing the temperature will destabilize the liposome
membrane. Other studies use pH sensitive liposome, by
changing the pH affect the membrane.
Physical chemical properties of liposomes are
biocompatible and biodegradable. The size is small enough
compared with blood cells ( 10m ), bacteria ( 3m ) and
virus ( 100nm ) but not small as DNA ( 2nm ), and
fullerenes ( 1nm ).
The first part of this study is experimental from the
liposome preparation, ultrasound irradiation,
Spectrofluorometer analysis and TEM observation. The
second part of this study, due to a better understand of the
external stimulation a development of a Physical Chemistry
model on permeability effect of liposome under external
stimulation research on Liposome is conduced. This research
consider variables such as temperature, light, position of the
liposome’s sample, preparation’s method of liposome, size of
liposome, concentration of liposomes, and frequency of the
ultrasound radiation.
Manuscript received April 16, 2010. This work was supported in part by
the Yamaguchi Prefecture Government for the KENPI Scholarship.
R. F. was with Yamaguchi University, Yamaguchi, Japan. He is now with
the Mechanical Engineer Department, Universidad Nacional de Ingenieria,
Lima, Peru (e-mail: royoshii@yahoo.com).
Fig. 2. Liposome preparation process diagram. In this process,
ingredients are transformed to liposome with different concentrations
and properties.
II. LIPOSOME PREPARATION PROCESS
A. Liposomes
Liposomes are biological material with a hydrophilic (water
like) and hydrophobic (water dislike) part. They are lipids
such as body’s cell membrane and other biological
component [2]. The size of liposomes is from few tens of
nanometers to micrometers. Liposomes are composed of
lipids known as artificial cell membrane.
B. Structure and organization of lipid
The physical description of lipids is cylinder, cone and
inverted cone.
1) Phosphatidylcholine (PC) belongs to the cylinder. The
electric polarity of the head is neutral, pH will not be affected.
2) Phosphatidylethanolamine (PE) belongs to the cone. It is
difficult to close the endoplasmic reticulum.
3) Mi-lysolecithin, inverted cone, it alone doesn’t form
membranes.
C. Leakage model
Two theories on permeable membrane mechanism is
considered. [3]
1) Solubility-Diffusion model: This model of the membrane
can become unstable under some kind of stimulus. The
contents can spread out by the permeability-phase.
2) Transient-Defects model: This model of the membrane
can form temporally pores by adding external stimulus. The
contents can spread out also.

978-1-4244-7709-8/10/$26.00 ©2010 IEEE 843

 In this study we are considered the two models of leak. Ultrasonic devices are driven by a sinusoidal AC voltage at
Temperature, pH, ultrasound, magnetic, light and other the resonance frequency transmitting energy [4]. In medical
factors affect the body’s structure. diagnostic, frequencies of ultrasound irradiation are between
250 to 2000 kHz. Ultrasound irradiation can disintegrate
D. The form of liposomes
biological cells by producing cavitation [5].
A. Transmission of ultrasound
It 1
I   (1)
I i 1  0.25 ( Z 2 / Z 1  Z 1 / Z 2 ) 2 sin 2 (2l /  2 )
I : Intensity of ultrasound
Z : Acoustic impedance
Considering the following figure
TABLE II
MATERIAL PROPERTIES
Acoustic
Fig. 3. Form of liposome by making method. Different method of Sonic velocity Density  impedance
preparation can produce Multi Lamellar Vesicle (MLV), Small
Material
c (m/s) (kg/m3) Z=c･ 
Unilamellar Vesicle (SUV), Large Unilamellar Vesicle (LUV). (N･s/m3)
Water(23-27 1500 1000 1.5×106
E. Components in liposome preparation ℃)
1) 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocoline Glass (Flint) 5100 2500 12.8×106
(POPC): Principal component and phospholipids In the experiment with ultrasound, the medium which ultrasound
passes through is water and glass. In the experiment, the density of glass
2) 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine
was 2230 (kg/m3) and sonic velocity was 5100 (m/s); the density of
(DPPE-2000): Susceptible to the effects of ultrasound. water was 1000 (kg/m3) and sonic velocity was 1483 (m/s).
3) Cholesterol: Stabilize the film.

TABLE I
TYPES OF LIPOSOME AND THEM CONCENTRATION

liposomes POPC Cholesterol DPPE-2000

CREV(451) 40 50 10
CREV(851) 57 36 7
SREV(45) 44 56 ―
Using these components we prepare different type of liposome by
different concentration on molar ratio.
Another component for this experiment is calcein, which is Fig. 5. Heterogeneity of the transmission and reflection. We have three
a fluorescent dye. We use this property for measure the medium I, II and III. I and III are the water and II is the glass with a
permeability. The only inconvenient, as a fluorescence, is that width of l. Part of the intensity goes through and another one reflects or
energy difference between the absorbed and emitted photons absorbs.
ends up as molecular rotations, vibrations or heat. TABLE III
TRANSPARENCY AT DIFFERENT FREQUENCIES
F. Liposome preparation method
Transparency Value
For the liposome preparation, we use the reverser phase
evaporation method between the thin-film and acid removal  28 kHz  0.9762 High
methods. For the adjustment of the grain size, we use a  45 kHz  0.9409 High
polycarbonate membrane extruder of 100nm of hole’s  100 kHz  0.7644 Medium
diameter. For calcein separation, we use gel filtration Using (1), transparency was calculated. In 28 kHz is almost
chromatography. Finishing this process we obtain our transparent. Near cero the transparency is low, and is when the
liposome with different concentrations and properties. frequency is 100 kHz.

III. ULTRASOUND IRRADIATION PROCESS

Fig. 6. Spectrofluorometer analysis process diagram. We can measure

the leakage.
Fig. 4. Ultrasound irradiation process diagram. When the liposome is
irradiated with ultrasound, properties of liposomes changes.

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 IV. SPECTROFLUOROMETER ANALYSIS PROCESS dI
  k1 ' db (12)
A. Fluorescent leakage rate I
The fluorescent intensity is measured by (1) [6]. I : Light intensity of absorption
I (t )  I (0) I
R(t )  x100 (2)  log( )  k1b (13)
I ()  I (0) I0
I (0) : Initial fluorescent intensity of liposome
I () : Maximum fluorescent intensity of liposome
B. Calcein permeable membrane leakage
The transmission speed of leakage from the interior [7].
dC
 K (Ci  C ) (3)
dt
t : Transmission time
K : Transmission coefficient Fig. 7. Absorption of light. The probe has a width b and concentration
C : Concentration of fluorescent C. The light intensity of absorption changes from a initial value.
C i : Concentration of fluorescent in the interior
D. Fluorescence quantitative method
At t=0, C= C 0
Absorption of light by Lambert-Beer law [9]
Integrating (3) at initial conditions.
I  I 0e  abC (14)
ln(C  C )  ln(C  C0 )  Kt (4)
a : Constant
Using fluorescent intensity as a parameter of concentration.
b : Length of medium
ln( I   I )  ln( I   I 0 )  Kt (5)
C : Concentration (mol/l)
Using mass balance
F  K ' ( I 0  I )  KI 0 (1  e  abC ) (15)
 dC 
 VL  K (Cin  C )Vs (6) K : Irradiation area
 dt  F : Fluorescence intensity
 : Ratio of surface area of liposome  : Fluorescence yield (ratio between the total amount of
V L : Volume of liposome light absorbed by the excited amount of fluorescent)
V S : Volume of liposome water phase For abC < 0.05
Overall balance of material F  KI 0abC (16)
C (VL  VS )  CinVS  CVL (7) For 0.05  abC  0.25
The initial conditions at t=0, C= C0 abC
C V L  C VS  CV L F  KI 0abC(1  ) (17)
C in  (8) 2
VS
From (5) and (7) organizing V. TEM ANALYSIS PROCESS
dC V
 K (C   C )( S  1) (9)
dt VL
Vs <<VL, organizing and integrate
C t
1 1 Fig. 8. TEM Analysis process diagram. We can see by microscope

C C C dC  K 0 dt (10) liposomes before and after ultrasound irradiation.

01  By TEM observation, we observe the exposure of liposome

C before and after under ultrasound irradiation. Liposome’ sizes
In terms of intensity vary from 20nm to 200nm.
I I
ln(1  )  ln(1  0 )  Kt (11) VI. EXPERIMENTAL RESULTS
I I
Experimental conditions
C. Absorption spectrometry Concentration of lipid: 50 (μM)
Volume: 3 (ml)
By using the Lambert law [8].
Power of ultrasound: 100 (W)
Frequency: 28 (kHz), 45(kHz) and 100(kHz)

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Time of exposure: 20s until complete 4 minutes.
Liposome: CREV4:5:1
A=26(mm), C=1(mm), H=55(mm)
Fig. 9. Schematic diagram of the experiment. H is the level of water, A
is the level of liposome in the test tube and C is the distance between
the ultrasound device and test tube.
The practicable meaning of the water level is the medium
where the ultrasound device is applied. In the body, there is
different tissue and size of tissues. With a combination of H
and frequency, it is possible to see cavitation.
Fig. 10. Conditions: Frequency 45 (kHz), each 20s for 4 minutes. The
result shows that the leakage increase with the time of ultrasound
irradiation.
Fig. 11. Conditions: Frequency 45[kHz], 20s:40s:60s of ultrasound
irradiation. The graph shows that intensity, from 20s to 40s, increases
210 Int; and from 40s to 60s, increases 70 Int
Fig. 12. Conditions: Frequency 45[kHz], 20s of ultrasound irradiation
and different samples. The result show range of the intensity is 40
(230-190)
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Fig. 13. Conditions: Wrapped and unwrapped probe with foil for 1
hour. The result show that light causes a variation on the intensity
around 0.19. Wrapped is the up line and unwrapped on is the down line.
Fig. 14. Conditions: Only buffer. The result shows that buffer has an
intensity of almost 0. We use buffer to reach the concentration of the
probe.
At this moment our results show a variation on the leakage
of liposome with different time of exposure at a specific
frequency, different type of leakage with same time of
ultrasound radiation; and variation of leakage causing by the
light. In consequence our parameters are leakage measured
with the intensity (I), time of ultrasound irradiation (t),
frequency of ultrasound (f), and light (L).
In order to understand more about other parameters, I
search on past researches on the laboratory. And the studies
are:
1) From a research by Mr Shibata in 2007, where his
parameters are position of the probe (C), water level (H),
frequency of ultrasound (f), different liposomes (CREV451,
CREV 851 and SREV 45), and different time of exposure (t).
2) From a research by Yoshida in 2007, where his
parameters are different liposomes (CREV451, CREV 851
and SREV 45), and different frequencies (f) with different
time of exposure (t).
3) From a research by Yoshida from 2006 to 2008, where
his parameters are water level (H), different time of exposure
(t), different frequencies (f), position of the probe (C), and
TEM observations.
With that, we have correlations between intensity (I), time
of ultrasound irradiation (t), frequency of ultrasound (f), light
(L), position of the probe (C), water level (H), different
liposomes (CREV451, CREV851, SREV45), and TEM
observations.
VII. MODEL OF PHYSICAL CHEMICAL PROPERTIES OF
LIPOSOME UNDER ULTRASOUND RADIATION
 C. CREV451
TABLE VI
SREV851 EXPERIMENTAL DATA

%I t (s) f (kHz) L(h) C(mm) H(mm)

17 20 28 1 1 27
28 40 28 1.5 1 27
36 60 28 2 1 27
55 20 28 1 1 53
80 40 28 1.5 1 53
92 60 28 2 1 53
12 20 45 1 1 33
41 40 45 1.5 1 33
Fig. 15. Procedure to validate the model.
62 60 45 2 1 33
Experimentally, we have these relations:
１） At 28(kHz), the leakage is proportional to the time and at
Studies of prediction on the contribution of diffusion and 53(mm), is more than 27(mm).
disintegration were conduced with Cisplatin, Doxorubicin ２） At 45(kHz), the leakage is proportional to the time and at
and MPS [10]. 33(mm), is similar with 28(kHz) at 27(mm).

A. SREV51
VIII. CONCLUSIONS
TABLE IV Experimentally the permeability of liposome under
SREV51 EXPERIMENTAL DATA ultrasound irradiation is predictable measuring the leakage of
%I t (s) f (kHz) L(h) C(mm) H(mm)
calcein, considering the with parameters as frequency (f),
time of exposure (t), type of liposome (CREV451, CREV851
8 20 28 1 1 27 and SREV 51), position of the probe (C), water level (H), and
18 40 28 1.5 1 27
22 60 28 2 1 27
time of light’s exposure (L).
22 20 28 1 1 53 The importance of this work is related to the ultrasound
30 40 28 1.5 1 53 device that can produce permeability on the liposome with
37 60 28 2 1 53 parameters of time of exposure, frequency of ultrasound
3 20 45 1 1 33
13 40 45 1.5 1 33
irradiation, position of the probe, water level as the size of the
22 60 45 2 1 33 medium, and light that affect the permeability almost
Experimentally, we have these relations: insignificant.
１） At 28(kHz), the leakage is proportional to the time and at
53(mm) is more than 27(mm). ACKNOWLEDGMENT
２） At 45(kHz), the leakage is proportional to the time and at
33(mm), is similar with 28(kHz) at 27(mm). The author thanks Mr. Makoto Yoshida for assistance in
A possible equation using concepts of energy: experimental studies. RF is grateful to Professor Zhongwei
b Jiang from Micromechatronics Laboratory at Yamaguchi
 fH  University and Professor Alberto Coronado from Multiscale
% I  at  (18)
Model and Simulation Group at Universidad Nacional de
 C 
Ingenieria for their support.
B. CREV851
TABLE V REFERENCES
SREV851 EXPERIMENTAL DATA [1] A. Schroeder, J. Kost, Y. Barenholz. Ultrasound, liposomes, and drug
delivery: principles for using ultrasound to control the release of drugs
%I t (s) f (kHz) L(h) C(mm) H(mm) from liposomes, Chemistry and Physics of Lipids, 2009, 162, pp. 1-16.
[2] A. Berk, L. Zipursky, P. Matsudaira, D. Baltimore, J. Darnell, H.
38 20 28 1 1 27
Lodish, Molecular Cell Biology, W H Freeman & Co, 1999, pp. 29-50.
58 40 28 1.5 1 27
65 60 28 2 1 27 [3] C．Dordas，P.H. Brown, Permeability of Basic Acid Across Lipid
68 20 28 1 1 53 Bilayers and Factor Affecting It, J. Membrane Biol., 2000, 175, pp.
92 40 28 1.5 1 53 95-105.
95 60 28 2 1 53 [4] K. Uchino, J. Giniewicz, Micromechatronics, Marcel Dekker Inc, 2003,
38 20 45 1 1 33 pp. 403-460.
66 40 45 1.5 1 33 [5] U. Neis, K. Nickel and A. Tiehm. Enhancement of anaerobic sludge
75 60 45 2 1 33 digestion by ultrasonic disintegration. Water Science & Technology,
2000, pp. 9-73.
Experimentally, we have these relations:
[6] V. P . Torchilin and V. Weissing, Liposomes, Second Edition, Oxford
１） At 28(kHz), the leakage is proportional to the time and at University Press 2003.
53(mm) is more than 27(mm). [7] H. Komatsu，S. Okada，Increased permeability of phase-separated
２） At 45(kHz), the leakage is proportional to the time and at liposomal membranes with mixtures of ethanol-included interdigitated
33(mm), is similar with 28(kHz) at 27(mm). and non-interdigitated structures，Biochimica et Biophysica, 1995,
1237，pp. 169-175.

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[8] P. Atkins and J. De Paula, Atkins’s Physical Chemistry, Oxford
University Press, 2006, pp. 430-508.
[9] C. Egusa, Study on release characteristic of medicine encapsulated
liposomes by ultrasonic irradiation, MSc. dissertation, Dept. Mech.
Eng., Yamaguchi Univ., Japan, 2007.
[10] G. Enden, A. Schroeder. A Mathematical Model of Drug Release from
liposomes by low frequency Ultrasound, Annals of Biomedical
Engineering. 37(12) pp. 2640-2645. 2009.

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