Effects of the microbubble shell physicochemical properties on ultrasound-mediated drug delivery to the brain
Graphical abstract
Introduction
Microbubbles, gas-filled microspheres (1–10 μm) initially used merely as contrast agents for ultrasound imaging, have recently been shown critical in ultrasound-mediated therapeutic applications such as sonothrombolysis [1], [2], molecular delivery to the cell via sonoporation [3], [4] and/or endocytosis [5], [6], and to the brain parenchyma via blood–brain barrier (BBB) opening [7], [8] paracellularly or transcellularly. For molecular delivery purposes, although the biological mechanisms may vary, sonoporation and/or endocytosis and BBB opening share the same physical mechanism that cavitation increases the permeability of the cell membranes. In all the cases, the microbubble properties play important roles in determining the delivery efficiency. For example, larger microbubbles (4–5 μm in diameter) induce larger BBB opening and delivery efficiency than smaller microbubbles (1–2 μm in diameter) [9], [10]; soft-shelled (lipid or protein) microbubbles gave higher cell viability and transfection rate of gene delivery than hard-shelled (polymer) microbubbles [11].
Overall, the main goal of drug delivery is to achieve high efficiency without causing cell damage, and with the use of lipid-coated microbubbles it is achievable. In fact, with lipid-coated microbubbles the overall drug delivery efficiency could be influenced by changing the lipid hydrophobic chain length that modulates the overall physicochemical properties of the monolayer shell. Borden et al. have shown that increasing the lipid hydrophobic chain length increased the gas permeation resistance to the environment [12], decreased the acoustic dissolution rate while enhancing the lipid-shedding phenomenon during insonification [13]. Kwan et al. have reported that bubbles with longer lipid hydrophobic chains required longer re-stabilization following shell rupture, and longer to dissolve after the onset of collapse due to stronger attractive intermolecular forces [14], [15]. Longer acyl chains can also increase the lipid monolayer thickness [16] and microbubble mechanical properties such as in-plane rigidity [17], thereby modulating cavitation response and the shear stress applied on the cell membrane [18], [19]. Those results suggest that the physicochemical properties of the lipid-shelled microbubbles may play a role in affecting the drug delivery efficiency, but the exact effects remain to be discovered.
This study aimed at investigating the shell effect of lipid-coated microbubbles on the drug delivery efficiency in vivo. We hypothesize that increasing the lipid hydrophobic chain length would enhance the drug delivery efficiency after focused ultrasound (FUS)-induced BBB opening. The microbubbles used were coated with phosphatidylcholine (PC) lipids of various acyl chains (C16, C18, C24), and the phospholipid:lipopolymer ratio was fixed in order to isolate the effect of the PC acyl chain length. In addition, the diameter of the microbubble samples was kept constant at 4–5 μm in all experiments in order to exclude the influence of the microbubble size. Both molecular size (3 kDa and 40 kDa dextran been delivered), acoustic pressure (225–600 kPa), and pulse length (100 cycles and 1000 cycles) dependences were investigated in order to fully assess the microbubble shell effects on the drug delivery efficiency.
The different shelled microbubble dynamics in vivo were also captured during insonification using passive acoustic cavitation detection (PCD) in order to potentially uncover the physical mechanisms affecting the delivery efficiency such as micro-streaming and micro-jetting. The signal recorded by PCD is the acoustic emission from the cavitating bubbles, which represents the cavitation intensity with the signature of stable and/or inertial cavitation. We assume that the detected harmonics and ultraharmonics relate to stable cavitation (low and high amplitude bubble pulsation, or decaying oscillation) resulting in micro-streaming in a short or long period, and the detected broadband emission to inertial cavitation (violent bubble oscillation, bubble breakup or rebound) causing micro-jetting or shock wave emission, based on the bubble activities categorized by Leighton [20]. Both types of cavitation are thought to contribute to ultrasound-mediated drug delivery.
Section snippets
Microbubble generation
All the lipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC or C16), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC or C18), 1,2-dilignoceroyl-sn-glycero-3-phosphocholine (DLiPC or C24) and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (DSPE-PEG2000). The perfluorobutane gas (PFB, 99 wt.% purity) used for microbubble generation was obtained from FluoroMed (Round Rock, TX,
Microbubble generation and size-isolation
For each microbubble shell type, the probe sonication and differential centrifugation method produced an opaque milky microbubble suspension that was stable during the experimental timeframe. The mean, median and mode diameters of all microbubble samples, regardless of their shell lipid components, all fell within the range of 4 to 5 μm (Table 2). The representative number-weighted and volume-weighted size distributions of the microbubble samples as measured by the Coulter Multisizer III are
Discussion
In this study, microbubbles with different phospholipid shell components were utilized to facilitate targeted drug delivery in the brain after FUS-induced BBB opening in mice. Based on the fluorescence analysis, our results clearly showed that increasing the hydrophobic chain length of the phospholipid had a substantial effect on the larger 40-kDa dextran delivery (Fig. 4) while such effects were minimal for smaller molecules (3 kDa) (Fig. 3). Increasing the acoustic pulse length did not alter
Conclusion
The effects of the microbubble shell physicochemical properties on drug delivery efficiency using ultrasound have been characterized using microbubbles of three phospholipids with increasing hydrophobic chain lengths (C16, C18, C24) for drug delivery to the brain through BBB opening. The entire process was monitored using passive acoustic cavitation detection (PCD) in order to shed light on the physical mechanisms behind the shell effects. The dependence on both the molecular size and acoustic
Acknowledgments
This research was supported in part by the National Institutes of Health grants R01 EB009041 and R01 AG038961. The authors would like to thank Wenlan Zheng and Hong Chen at Columbia University for assisting with the manuscript.
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