Acoustic destructibility studies

The studies were directed towards investigation of the acoustic destructibility of MBs depending on the US frequency and intensity. Unloaded model phospholipid-shelled MBs were prepared according to the thin-film hydration method. Briefly, 80 mol% DPPC and 20 mol% DPPG at a total concentration of 5 mM were blended in HPLC-grade chloroform. The organic solvent was eliminated under vacuum at 65 °C vor 60 min using a Büchi Rotavapor R-114 (Büchi Labortechnik GmbH, Essen, Germany). The phospholipid film was hydratized with highly-purified water (Purelab Plus®_{, USF Elga Ionpure GmbH, Germany).}

The resulting liposome formulation was aliquoted in amounts of 400 µl into 2 ml round bottom safe-lock tubes (Eppendorf AG, Hamburg, Germany) and covered with octafluoropropane gas. The liposomal dispersion was mechanically agitated for 20 s using a CapMixTM _{(3M Deutschland GmbH, Neuss, Germany).}

MBs were mounted into 1.2 µm filtered 5% w/w bovine serum albumin solution in Dulbecco’s phosphate-buffered saline, imitating blood plasma in viscosity, ionic strength, and pH.

Prior to each experiment the flow-cell of the PCATL model was filled with medium and conditioned to 37 °C. The background particle concentration in the medium was measured. The freshly prepared MB suspensions were introduced into the running PCATL model by a calibrated 1 ml syringe through the septum gap. After the distribution of MBs in the entire setup volume, the pressure was adjusted to 110 mmHg and a sample of 100 μl was drawn through the septum, indicating the starting MB concentration. The drawn volume was replaced with medium in order to prevent pressure loss. US was applied and the above procedure was repeated. The collected sample was diluted in 40 ml particle free deionized water (Purelab Plus®, USF Elga Ionpure GmbH, Germany) and MBs size distribution and concentration were measured with light blockage using PAMAS SVSS-C (PAMAS GmbH, Rutesheim, Germany). The procedures of sampling and measuring were repeated after each sonication frame.

MBs were exposed to US with a lower frequency of 1 MHz, and a higher frequency of 3 MHz at a constant US intensity of 4 W/cm2_{. This was in order to ascertain whether there is a}

dependency of MB destructibility from the MB size as known from the theory of Apfel et al. (1991) [129] and Holland et al. (1989) [130].

In the second study, MBs were exposed to US with a frequency of 3 MHz and output intensity, increasing from 1 W/cm2 _{to 4 W/cm}2 _{in order to characterize the correlation of MB}

destructibility and US intensity. In both experiments, MBs were exposed to six sonication frames of 10 s at a duty cycle of 50% using a 5 mm sonication probe.

In contrast to the in-vitro setup of Lazewatsky et al. (1999) [124] (Table 7), the implementation of whole blood into the PCATL model was impracticable due to the presence of formed blood elements which cannot be distinguished by the particle counting method used.

For both studies, collected data from five repeated measurements was averaged. The standard deviation was determined and the significance of the results was assessed by single factor variance analysis (ANOVA). Mathematical curve data fitting was performed using SigmaPlot®_{, software version 9.0 (Systat Software, GmbH, Erkrath, Germany). The}

coefficient of determination R2 for each sonication decay curve was calculated. The experimental data was fitted using a single exponent decay two parameter function (Equation 9), where a represents the initial population coefficient, b – the decay coefficient, and t – the sonication time. The MB half-life t1/2 during US exposure was calculated using Equation 10.

t

b

e

a

y

₌

_.

−

.

( )

b

t

=

ln

2

4. Results and Discussion

4.1. Establishing the in-vitro model

The optimal flow rate through the membrane cell of the PCATL model was determined to be 4-5 ml/min (data not shown). Higher flow rates led to insufficient interaction of MBs with the US beam and therefore too slow destruction paces. A slower circulation caused MBs to buoy up and form a foam layer in the tubing, causing reduced reproducibility.

At optimum flow rate the minimum residence time for a MB in the membrane cell was calculated to be 1.5 s at a flow rate of 5 ml/min. Hence, a MB flowing through the membrane cell will be exposed to at least two US pulses when using a duty-cycle of 50%.

The standard deviation of the sampling syringe was considered acceptable (σ = 1.7%) in a series of twenty repeats. The standard deviation of the PCATL model was derived from the measurement data. It was found to be 10.3% ± 3.2%.

The focal distance between the flow-cell and the sonication probe was measured to be approximately 15 mm. At this distance the measured peak-negative acoustic pressure in the flow-cell both for 1 MHz and 3 MHz and 100% duty cycle was 0.037 ± 0.008 MPa at 1 W/cm2 _{and 0.131 ± 0.027 MPa at 4 W/cm}2 _{output intensity.}

Compared to related circulation in-vitro setups [124, 131], the PCATL model possessed a membrane flow-cell with a specific egg-shape which provided optimized flow conditions (Figure 17, black arrows) and minimized compartments with low flow velocity (dead volume compartments). Dead volume compartments could accumulate MBs and substantially distort experimental results. Within the flow-cell MBs were pumped in an upright direction, approached into the broader bottom part of the cell and were slowed down. During their habitation in the bottom cell part MBs could interact with the US beam.

The upper flow-cell part was the most likely place for dead volume locations due to the affinity of MBs to float (buoyancy). Therefore, the outflow part was shaped so as to allow MBs to accelerate before leaving the cell.

4.2. Acoustic destructibility studies

According to the in-vitro studies, there was an apparent dependence of MB destructibility on the US frequency and intensity as well as on the MB diameter.

When US with a lower frequency of 1 MHz was applied, acoustic destructibility of MBs did not correlate with their diameter, resulting to half-lives of less than 50 s for MBs of any size (Figure 18, dashed line).

In contrast to that at a higher US frequency of 3 MHz MBs of sizes from 1.0 µm to 1.4 µm were burst 5.04 ± 3.42-fold faster than MBs of sizes from 4.0 µm to 6.0 µm (Figure 18, solid line).

Figure 18: Half-life (t1/2) of unloaded phospholipid-monolayer shelled MBs during exposure to US with an

intensity of 4 W/cm2 _{and frequencies of 1 MHz (dashed line) and 3 MHz (solid line).}

Specific size-dependent destruction patterns for MBs at different US frequencies have been explained by different thresholds of transient cavitation which is required for MB destruction. This suggestion links the observed phenomenon to the analytical model established by Apfel et al. (1991) [129] and Holland et al. (1989) [130] (Figure 19) and recently related to SonoVue® by Greis et al. (2004) [132]. The authors proposed an approximate mathematical model, allowing for the calculation of acoustic pressure thresholds for transient cavitation over a variety of frequencies and MB diameters.

Figure 19: Computed plot of the cavitation threshold in water as a function of initial bubble radius for three frequencies of sonication: 1, 5, and 10 MHz. From: Apfel et al. (1991) [129].

In the context of the current study, MBs with larger diameters, exposed to ultrasound with a higher frequency (in this case 3 MHz), reach their bursting threshold at much higher peak- negative acoustic pressures than smaller MBs.

However, when exposed to US with a lower frequency (in this case 1 MHz) and the same output intensity, the influence of the MB size on the bursting threshold minimum decreases. As a consequence, at a frequency of 1 MHz both smaller and larger MBs are fragmented with a similar decay pace.

Figure 20: MB half-life during US exposure at a frequency of 3 MHz and an increasing intensity.

The dependence of the acoustic destructibility of unloaded MBs during US exposure at a frequency of 3 MHz and an intensity increasing from 1 W/cm2 to 4 W/cm2 is represented on Figure 20. Thereby, the MB life-time during US exposure decreases with 49.45% ± 2.73%, following a polynomial inverse second order equation (R2 = 0.9934).

5. Summary

In recent years the knowledge about microbubbles (MBs) as targeted drug and gene-carreers grew rapidly. Since the relevance of this promising approach steadily increases and new drug- loaded formulations emerge on a pre-clinical stage, an accurate and fast screening method has

been necessary to characterize their acoustic destructibility. Expectably, any new drug-loaded MB formulation possesses different physico-chemical characteristics which affect MB acoustic characteristics such as echogenicity and destructibility.

In-vivo techniques are generally not suitable for high-throughput formulation screening. Therefore, near to physiologic conditions have to be imitated in-vitro, if clinically relevant data is to be obtained.

In this chapter, an improved in-vitro model was developed and tested for pre-clinical assessment of the acoustic destructibility of newly developed MB formulations, based on previous literature work. This tool combines many physical aspects of the in-vivo setup, such as flow conditions, pressurizing, temperature conditioning, and mounting of MBs in artificial blood plasma.

The near to physiological conditions acoustic transmission line (PCATL) in-vitro model proved as an useful tool for reproducible and reliable characterization of the MB acoustic destruction kinetics. Thus, it is possible to predict which drug-loaded MB formulations are appropriate candidates for further in-vivo evaluation, with regard to their sufficient acoustic destructibility.

In the above studies, MB acoustic destructibility of model unloaded phospholipid MBs was correlated to their diameter and an empirical evidence to the theory of Apfel and Holland [129, 130] was provided. According to it, at higher ultrasound frequencies MBs of smaller size are better destructible than larger ones. At lower frequencies no size-dependent difference could be observed.

Furthermore, MB destructibility was studied at various ultrasound intensities with no regard to the MB size. Thereby, MB destruction pace increased non-linearly with ultrasound intensity, following a polynomial inverse second order kinetics.

Further in this thesis, the PCATL in-vitro model will be implemented for characterization of doxorubicin-loaded MBs and acoustically active lipospheres, comprising an additional oil layer. The in-vitro data will be correlated with in-vivo studies and a correlation model will be established. The formulation with better acoustic properties – echogenicity and acoustic destructibility, will be selected for further in-vivo studies.

IV. CHAPTER: Ultrasound targeted tumor therapy