S.J. Lu, C.Y. Chuang and Jenho Tsao
Dept. of EE. and Inst. of Comm., National Taiwan University, Taipei, Taiwan, ROC Abstract- For drug delivery applications, dosage prediction
before release and estimation after release are required functions. In this study, we attempted to establish a method to evaluate liposome concentrations and liposome shell thickness for dosage prediction. We use the Trilling model with parameter of phospholipids bilayers to simulate the frequency responses under the different acoustic pressure and establish an experimental protocol to evaluate the liposome concentrations and the liposome shell thickness.
Our results illustrate the changes on the signal strength for different concentrations and show that it is relatively stable to estimate the concentrations when the cycles are lower (15cycles). Besides, it is verified that the second harmonic signal is more sensitive in analyzing different concentrations.
On the other hand, it is proved that the liposome shell thickness affect signal strength and thinner thickness will increase the second harmonic response.
Therefore, in accordance with the theoretical and experimental results, we would be able to estimate the concentration and the shell thickness of the liposomes. By numerical analysis methods, dosage prediction would be built.
I. INTRODUCTION
In medical ultrasound applications, microbubbles are closely tied to the diagnostic and therapeutic uses. In diagnostic applications, their sound scattering properties yield improved imaging, when the microbubbles are used as contrast agents [1]. The harmonics, subharmonics, and second harmonic responses from the bubbles assist in distinguishing the acoustic scattering of blood flow from that of the surrounding tissue[2]. The therapeutic use of microbubbles has recently become a subject of much interest, one of which is to serve as modified-release dosage forms.
The modified-release dosage forms start to develop prosperously recently. It is the purpose that we can properly handle modified-release dosage forms for controlling the time of medicine release and determining the perfect timing to release. However, the modified-release dosage forms have obvious effect on diminishing toxicity, raising curative effect and decreasing the times of medication. Hence, we could expect that modified-release dosage forms will still be a prospect of the medical development .
For drug delivery system , the drug must be preserved for This work is supported by the National Science Council, Taiwan, ROC. (NSC95-2221-E002-442).
E-mail: tsaor215@cc.ee.ntu.edu.tw
a period of time in order to utilize bubble characteristics for targeting and control the release at the best time. Dosage prediction before release and estimation after release is considerably significant.
The difference of harmonic responses generated from multi-lamellar vesicles with different numbers of layers and the number of bubbles can be used to estimate the shell thickness related to numbers of layers and predict the concentration of vesicles. However, there is no appropriate mathematical model and experimental protocol to determine the shell thickness of multi-lamellar vesicles and the concentration of vesicles.
In this study, we tried to adopt the Trilling model with parameter of phospholipids bilayers to simulate the frequency responses and establish an experimental protocol to evaluate the effect of shell thickness of liposomes to the estimation of its concentration. And we also attempted to get the optimal condition for measuring the concentration of liposomes. Further, we might use the study to predict the dosage via concentration.
II.MATERIALS AND METHODS
In this section, we will describe our liposomes as drug carrier first and then the simulations and experiments on the liposome concentration. Besides, the liposome shell thickness has a deep connection with signal strength, so we do simulation and experiments to evaluate the effect of shell thickness.
A. Materials
In this study, we have manufactured our own microbubble with lipid-shell (a kind of liposomes) for the ultrasound drug carrier, whose diameter is about 1.4µm (figure.1). The lipid-shelled microbubble is composed of a multi-lamellar lipid shells, a little nitrogen and some normal saline. The shell thickness can be expressed in terms of number of layers also, which is known to range from 1 to 5 layers [3]. The lipid shell comprises DSPC, Cholesterol and DSPE-PEG-Ome. The shell thickness of liposome is controlled by sonicator which controls the numbers of layers. In the size analysis, the optical measurement of N4-PLUS COULTER was used to analyze size distribution of the lipid-shell microbubble. ( as shown in figure 1.)
Proceedings of the 29th Annual International Conference of the IEEE EMBS
Cité Internationale, Lyon, France August 23-26, 2007.
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B. Simulation
Although the behavior of a bubble in an acoustic field has been studied for a long time, few theoretical describe the simulation of a real population of variably sized microbubbles in a finite beam. As the first step in our simulation, we will select the trilling model to simulate our microbubbles (liposomes) and summation all the radiation signals from large number of such bubbles. The bubblesim [4] is the main software to simulate the single bubble response, and we will modify the software to fit this study.
The simulations include both the simulation of the concentration of liposomes and the simulation of the layers of liposome shell.
The concentration simulation
We started the process of simulation by deciding on a sample volume with N microbubbles whose size distribution is Gaussian distribution after the shell parameters of liposomes; Moreover, in order to fit the probe response , we use randomly different focal pressures in the simulation. The cycles of pulse are given 15, 20,and 25 cycles. The selection of concentrations will fit the experimental concentration.
The shell thickness simulation
We started the process of simulation by deciding on a sample volume with N microbubbles whose size distribution is Gaussian distribution after the shell parameters of liposomes; we adopted transmitted narrow-band pulse ( fc =2MHz, cycles = 15 , PRF=1KHz) to simulate 3 different thicknesses of liposomes. We simulated the drug carrier (liposomes) consisting of gas, with mean diameter being 1. 4 micron meter and shell thickness being 1 ,3 ,and 5 layers.(3.5, 10.5, and 17.5 nm).
Using several numerical analysis methods, we can get the frequency response of all bubbles.
The equations and parameters used in our simulations are summarized below.
Equation of motion: Rayleigh-Plesset with radiation damping
Boundary condition: Pressure PL at the bubble wall, using the exponential shell model
0 /
The Trilling model
The Trilling model [4] is derived from the acoustic approximation, the speed of sound is modeled as constant.
It is only meaningful to the first order in the acoustic Mach-number M= a c. The order term of 1 c2are removed from the ODE for the bubble surface, reducing it to
3 2 4
This equation of motion for a gas bubble was published by Trilling in 1952.
With a driving pressurep ti( ), the Trilling equation is
The parameters used in simulations are Density of water = 998 kg/m3, Shear Modulus = 50MPa, Polytropic exponent of gas = 1.4, External pressure Range from 0.5 to 1.5Mpa, hydrostatic pressure = 1.01×105 N/m2, Distance = 2cm, Acoustic speed = 1540 m/s and Shear viscosity of liposome
= 0.8Pa·s.
C. Experiments of concentration estimation
In the experimental setting (as shown in figure2), we use the ultrasound RF signals to measure the acoustic reflection.
In this study, pulse trains (PRF=1KHz, cycles=15, 20,and 25 cycles) are transmitted with the 2.25MHz center frequencies, then backscattered signals are received and digitized for processing. We record the second-harmonic signal strength in several concentrations to findt the relation between concentrations and strength. In addition, we like to find the optimal method for the prediction of concentrations.
Figure1.The size disturbition of original lipsomes in N4PLUS Coulter
D. Experiments for shell thickness effect
In the experimental setting (as shown in figure2), we use the ultrasound RF signals to measure the acoustic reflection.
In this study, pulse trains (PRF=1KHz, BW=5%) are transmitted with the 2MHz center frequencies, then backscattered signals are received and digitized for processing. We attempted to get whether different layers have difference in the frequency domain to develop the forecast of layers of liposomes.
III. RESULTS
A. simulation and experimental evaluation of concentrations
Results for the second harmonic signals of simulation and experiments for several concentration are plotted in the figure 3, 4, and 5. These results illustrate the changes on the signal strength for different concentrations (circle: the experimental data, line: the simulation data). We compare the results of simulation with the results of experiments under three kinds of cycles.( 15, 20,and 25 cycles). When the concentration of liposomes is below around 40% of the concentration ( PS: when 100% of the concentration, DSPC is10−4 mole, cholesterol is 5 10× −5 mole, and DSPE- PEG-OMe is 2.5 10× −6mole in 30 ml.), the ultrasound strength is sensitive for the concentration of liposomes. As shown in [5], the linear scattering assumed in the simulation may no longer be valid, multiple scattering would happen and causes apparent discrepency. In three kinds of conditions, we could observe that in the thiner concentrations there are precise relations between the simulations and the experimental results. In the condition
of 15cycles, the relation is better than others in the all concentrations. And what is more, it is obvious that when the cycles are higher, it excites the nonlinear response of microbubbles more easily, but it is relatively unstable to detect the concentrations. On the contrary, when the cycles are lower, it is relatively stable to detect the concentrations.
It is verified that the second harmonic signal is sensitive in different concentrations; Therefore, it is sufficient to prove the possibility of prediction of concentrations by second harmonic signals.
B. The simulation and experimental evaluation of thickness of shell
In the study, we adopted transmitted narrow-band pulse ( fc =2MHz, cycles = 15 , PRF=1KHz) to simulate 3 different thicknesses of shell. In this study, the thickness of a layer of liposomes is about 3.5 nm under room temperature, the original liposomes generally have 3-5 layer ( the thicknesses of shells are 10.5 - 17.5nm), and the handled liposomes generally have 1 layer (the thickness of shell is 3.5nm), so we simulated three kinds of shells (3.5, 10.5, and 17.5nm by thickness).
As a result of the simulation, we could clearly see that the thicker the shells of liposomes, the lower the strength of non-linear response. Therefore, in the figure 6, it is shown that the difference of strength of second-harmonic response between three and five layers of liposomes was 12dB and the difference of strength of fundamental response between three and five layers of liposomes was about 4dB in our experiments.
In the experiments, we reduce the layers of liposomes with the help of a sonicator. Utilizing microscope, it is verified that the layers of shells of liposomes could be reduced to about 1 layer from 3-5 layers.
According to the experiments in the figure 7, we would discover that the second- harmonic response had larger difference than fundamental response whether the center frequency is 2MHz. The difference of strength of second-harmonic response was about 6dB. Similarly, in the actual experiments, it was obviously observed that the thicker the shells of liposomes, the lower the strength of non-linear response. In Figure 7, we could find stronger nonlinear response.
IV. CONCLUSION
To sum up, it is demonstrated that we could get the concentration of liposomes by second harmonic signals under different focal pressures. In the meanwhile, it is obvious that the shell thickness would affect the signal Figure2. The setup of the ultrasound
strength making the concentration prediction confused. It is useful for us to understand the liposome shell affecting drug packaged.
Therefore, we could realize the drug carrier structure and predict the drug dosage by simple volume calculation and drug binding estimation. In the future work, we would build a model to build a set of dosage prediction system.
REFERENCES
[1] Chin, Chien Ting; Burns, Peter N. “Predicting the acoustic response of a microbubble population for contrast imaging in medical ultrasound” Ultrasound in Medicine and Biology Volume: 26, Issue: 8, October, 2000, pp. 1293-1300
[2] Shih-Jen Lu, Chung-Yuo Wu, and Yi-Hong Chou,
“Bandwidth-Dependent Subharmonic Response of Microbubbles for Pressure Estimation,” 6th International Symposium on Ultrasound Contrast Imaging 2004.
[3] Roger R. C. Liposomes: a practical approach, New York:
Oxford University Press, 1990.
[4] L. Hoff. Acoustic Characterization of Contrast Agents for Medical Ultrasound Imaging, Kluwer Academic Publisher2001.
[5] E. Stride and N. Saffari, “Investigating the Significance of Multiple Scattering in Ultrasound Contrast Agent Particle Populations,” IEEE Trans. Ultrason., Ferroelect., Freq. Contr., vol. 52, pp. 2332- 2345, 2005
Figure3.The results of simulation and experiments in 15cycles pulse. circle: the experimental data, line: the simulation data.
Figure5.The results of simulation and experiments in 25 cycles pulse. circle: the experimental data, line: the simulation data.
Figure6 .The spectrum of simulation of thickness
Figure4.The results of simulation and experiments in 20 cycles pulse. circle: experimental data, line: simulation data.
Figure7 .The spectrum of simulation of thickness