Abstract
Exceptionally long lived microbubbles containing a fluorocarbon as part of their filling gas have been obtained by using a fluorinated phospholipid instead of a standard phospholipid as shell component. An unexpected, strong synergistic effect between the fluorocarbon gas and the fluorinated phospholipid has been discovered. Such bubbles could be used for in vivo oxygen delivery, ultrasound contrast imaging and drug delivery.
INTRODUCTION
Intravascular administration of oxygen in the form of stabilized micron-size bubbles is, in principle, a simple, elegant and cost-effective means of delivering oxygen to tissues [Citation[1-3]]. Injectable compressible gas microbubbles that scatter ultrasound (US) several orders of magnitude more effectively than any other materials already provide efficacious contrast agents for US imaging [Citation[4-7]]. In addition to enhancing the intensity of backscatter signals, microbubbles interact with sound waves. The response of microbubbles to US is non-linear, generating useful harmonic and sub-harmonic sound waves. Microbubbles can also be destroyed by US pulse. All these phenomena are being exploited in various new imaging modalities, as well as in new drug delivery systems.
One limitation to the intravascular use of oxygen microbubbles is that they dissolve rapidly in the blood under the combined action of Laplace pressure and arterial pressure. Their short half-life limits their efficacy as O2-carriers in most practical applications. Perfluorocarbons (PFCs), when used as part of the bubble filling gas, retard bubble dissolution significantly, due to very low water solubility, allowing the half-life of the bubbles to increase from a few seconds to several minutes. Because of high biological inertness, high oxygen solubility and extremely low solubility in water, PFCs are being investigated for various biomedical uses, including intravascular oxygen transport,diagnosis and drug delivery [Citation[3], Citation[8-11]].
Bubble shell engineering is an interdisciplinary field that involves surfactant, polymer and fluorous materials sciences, and acoustic physics. Besides the PFC contribution to stabilization, microbubbles can also be stabilized against both dissolution and coalescence by an appropriate shell. Diverse hard shells (made of gelatin, alginate, poly(ter-butyloxycarbonylmethyl)glutamate [Citation[12]], the biodegradable block copolymer poly(D,L-lactide-co-glycolide) [Citation[13]], polyelectrolyte multilayers [Citation[14]], etc.) have been investigated. An elastic solid polymeric shell can oppose the effect of surface tension. The shell-forming material can also consist of a molecular surfactant (or combination of surfactants) that promotes stability by decreasing the interfacial tension, hence the Laplace pressure. Synthetic phosphatidylcholines, ethanolamines, and glycerol diesters are being used in commercial products [Citation[3]]. Soft shells that only minimally dampen oscillations are usually preferred for contrast agent applications.
We have investigated the stability of microbubbles stabilized by perfluorohexane (PFH) and coated with a perfluoroalkylated phosphatidylcholine (F-PC). The stability of PFH-stabilized microbubbles coated with dimyristoyl phosphatidylcholine (DMPC) was also studied for comparison.
F-PCs have been shown to form highly stable liposomes with low shell permeability [Citation[15]]. Measurements of ultrasound transmission through a dispersion of microbubbles provides a convenient method for assessing microbubble stability.
MATERIALS AND METHODS
Perfluorohexane (PFH, C6F14, purity > 99%), D,L-α-1,2-dimyristoyl-sn-3-glycero-phosphatidylcholine (DMPC, purity > 99%) and Pluronic F-68 (MW ∼ 8300, poly(propylene glycol) : poly(ethylene glycol) = 1:4, purity > 99%) were purchased from Sigma. The perfluoroalkylated phosphatidylcholine F-PC was synthesized according to Ref [Citation[16]]. Water was purified using a Millipore system (pH = 5.5; surface tension: 72.1 mN m−1 at 20°C, resistivity: 18 MΩ cm).
The microbubble shell components, F-PC or DMPC (24 mM), were first allowed to fully rehydrate at 25°C under agitation during 2–4 hours in a phosphate buffered saline (PBS) solution consisting of 121.5 mM NaCl, 25.2 mM Na2HPO4 and 4.8 mM NaH2PO4 (all components from Fluka). The pH of the PBS solution was 7.4. The PBS solution was complemented by Pluronic F-68 (2.4 mM). The dispersions were then sonicated (20 kHz, 3 mm probe, 600 W) by positioning the tip of the probe on top of the solution, the volume above the dispersion being filled either with PFH-saturated N2 or with N2. The time of sonication was adjusted in order to obtain similarly sized microbubbles (30 s for F-PC and 5 s for DMPC microbubbles both filled with PFH-saturated N2). The average diameter of the microbubbles was measured by static light scattering (Coulter LS 100) immediately after dilution of the resulting foam in PBS (5 µL in 10 mL of PBS, 0.05% v/v) and over time.
Measurements of the intensity of the US transmitted through a microbubble suspension was achieved using an original experimental setup. In this set–up, an emitting transducer is excited with five sinusoids (2.2 MHz, 20 V peak-to-peak) with a repetitive frequency of 100 Hz. The ultrasound signal goes through a glass cell (140 mL) containing the test material and is received by a second transducer that converts it into an electrical signal. The transmitted US intensity is proportional to the number of microbubbles (approximatively 2.10Citation7) present between the PZT transducers. Its measurement upon time directly provides the life-time of the microbubbles under US irradiation. The volume of microbubbles injected in the cell was 50 µL (0.036% v/v).
RESULTS AND DISCUSSION
The microbubbles investigated here contained either pure N2 or PFH-saturated N2 as filling gases. They were obtained by sonication of an aqueous dispersion of F-PC or of DMPC (control), the volume above the dispersion being filled either with PFH-saturated N2 or with N2. The average diameter of the microbubbles, as well as the intensity of the US transmitted through a microbubble-containing cell, were measured upon time.
shows the transmitted US intensity, which reflects the life-time of the microbubbles under US irradiation, and hence their stability, as a function of time for similarly sized (∼ 14 µm) PFH–stabilized microbubbles encapsulated in DMPC or F-PC shells. It is remakable that the half-life of bubbles with F-PC shells is ∼ 70 min, as compared to ∼ 5 min for the reference bubbles with a DMPC shell. This demonstrates that the diffusion rate of the internal gaseous phase is substantially different for F-PC-coated and DMPC-coated microbubbles.
Figure 1 Variation of the transmitted ultrasound intensity, (I0 is the ultrasound intensity in the absence of bubbles) at 25°C, as a function of time, for PFH–containing microbubbles stabilized with a) DMPC alone and b) F-PC. The mean diameter of all the microbubbles investigated is ∼ 14µm. The half-lives are 5 and 70 min for the microbubbles with shells made of DMPC and F-PC, respectively.
Contrary to the regular monotonous curve found with DMPC shells, the curve for F-PC shells presents two regimes, with an initial slow increase of US signal intensity from ∼ 0 to only 20% during the first 60 min, followed by a much faster increase after ths period. The second regime, although comparable to the DMPC profile, occurred at a slower rate.
Particle size measurements over time also showed dramatic differences in behavior between F-PC- and DMPC-coated PFH-stabilized microbubbles (). The mean diameter of the DMPC microbubbles decreased from ∼14 µm to ∼ 4 µm in only about 10 min (inset), whilst it took ∼ 60 min for the mean diameter of the F-PC microbubbles to decrease from ∼ 14 µm to ∼ 3 µm.
Figure 2 Size distributions of microbubbles filled with N2 saturated with PFH and having a shell made of F-PC measured immediately after dilution of the initial foam, after 30 min and after 60 min, or a shell made of DMPC (inset) at 0 min, 5 min and 10 min.
These size measurements can be tied up with the US transmission experiments. For DMPC microbubbles, about 80% of the US signal intensity is recovered within 10 min and corresponds to a decrease in bubble size from 14 to 4 µm. By contrast, the F-PC bubbles remain in this size range (14 to 3 µm) for 60 min. After this time period, still only 25% of the US signal is transmitted, likely due to the fact that long-living small F-PC-bubbles absorb US very effectively. These results establish that the nature of the shell strongly influences the diffusion rate of the internal gas phase.
Remarkably, it was found that microbubbles that have both F-PC in their shell and PFH in their filling gas last about two orders of magnitude longer than those filled with N2 only, independently of the nature of the lipidic shell (DMPC or F-PC). In the latter cases, the half-life of the bubbles was less than 100 s. This establishes the existence of a hitherto unknown and unpredicted dramatic synergistic effect between filling gas and shell component.
The potential of our long-lived microbubbles should be of value for US diagnostic imaging, in particular for the detection of deep buried tumors. They are also expected to be useful for intravascular oxygen and drug delivery, where the use of microbubbles is promising.
The authors gratefully acknowledge the Centre National de la Recherche Scientifique (CNRS) for funding.
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