Tsitsos Fotios N, Batts Alec J, Jimenez Daniella A, Macsemchuk Craig A, Li Chunqi, Ji Robin, Bae Sua, Ciaccio Gillian L, Theodorou Angeliki, Ramirez Rashell K, Gorman Samantha L, Lugg Chloe L, Konofagou Elisa E
Department of Biomedical Engineering, Columbia University, New York, NY, USA.
Department of Biomedical Engineering, Columbia University, New York, NY, USA.
J Control Release. 2025 Jun 27:113986. doi: 10.1016/j.jconrel.2025.113986.
The characterization of microbubble (MB) activity in ultrasound-mediated blood-brain barrier (BBB) opening has proven critical in assessing the method's safety and efficacy in drug delivery. In this study, we build upon our previous work on theranostic ultrasound (ThUS)-mediated BBB opening (ThUS-BBBO) and conduct for the first time a comprehensive characterization of the role of MB cavitation in ThUS-BBBO and gene delivery with adeno-associated viruses (AAV).
A repurposed diagnostic ultrasound imaging phased array was used to generate ultra-short focused transmits at 1.5 MHz center frequency and simultaneously record acoustic emissions. MB activity was first characterized using a separate passive cavitation detector (PCD) in a flow phantom using pulse lengths ranging from 1.5 to 20 cycles and varying MB flow rates. A comprehensive in vivo study in mice was then conducted to characterize and correlate the resulting cavitation with AAV transgene expression. The transcranial MB activity was first detected using a PCD to confirm the findings of the flow phantom study. Next, three mouse studies were conducted to evaluate the relationship between cavitation and AAV delivery; one with different MB size distributions including polydisperse and size-isolated MB, one with variable burst length and burst repetition frequency, and one with different AAV serotypes and injection doses. Electronic beam steering enabled bilateral BBB opening with 1.5 cycle on the left and 10 cycles on the right hemisphere. Cavitation dose, BBBO volume, transgene expression, and histological safety were assessed following each experimental condition.
Frequency domain analysis in the phantoms revealed increasing harmonic cavitation with longer pulses. However, analysis in the time domain showed that longer pulses induce higher MB collapse. In the transcranial in vivo experiments, the PCD detected increased harmonic cavitation for 10-cycle pulses. The MB study showed that 3-5 μm MB resulted in the largest cavitation doses, BBBO volumes and transgene expression compared to the smaller MB groups. However, non-significant differences in BBBO volume and transgene expression were found when the MB dose was normalized by gas volume. The burst sequence study revealed that shorter bursts and faster burst repetition frequencies induce larger BBBO volumes and AAV transduction due to MB replenishment within the focal volume. Transgene expression was also increased with injection dose, and AAV9 showed the highest brain transduction efficiency compared to AAV2 and AAV5.
The phantom and in vivo studies showed that the mechanism of ThUS-BBBO is transient cavitation dominant and MB collapse increases with pulse length. Increased cavitation dose resulted in larger BBBO volumes and transgene expression in vivo. Overall, these results offer insights into the mechanism of AAV delivery across the BBB with ThUS and indicate that despite modulation of ThUS parameters and MB sizes, particular AAV serotypes are better-suited for maximal ThUS-mediated transgene expression in the brain after systemic injection.
事实证明，在评估超声介导的血脑屏障（BBB）开放技术在药物递送中的安全性和有效性时，对微泡（MB）活性的表征至关重要。在本研究中，我们基于之前关于治疗诊断超声（ThUS）介导的血脑屏障开放（ThUS - BBBO）的工作，首次全面表征了MB空化在ThUS - BBBO和腺相关病毒（AAV）基因递送中的作用。
使用重新利用的诊断超声成像相控阵，以1.5MHz中心频率产生超短聚焦发射，并同时记录声发射。首先在流动模型中使用单独的被动空化探测器（PCD），通过1.5至20个周期的脉冲长度和变化的MB流速来表征MB活性。然后在小鼠中进行了一项全面的体内研究，以表征所产生的空化并将其与AAV转基因表达相关联。首先使用PCD检测经颅MB活性，以确认流动模型研究的结果。接下来，进行了三项小鼠研究以评估空化与AAV递送之间的关系；一项研究使用不同的MB大小分布，包括多分散和大小分离的MB，一项研究使用可变的脉冲长度和脉冲重复频率，还有一项研究使用不同的AAV血清型和注射剂量。电子束转向实现了双侧BBB开放，左半球为1.5个周期，右半球为10个周期。在每种实验条件后评估空化剂量、BBBO体积、转基因表达和组织学安全性。
模型中的频域分析显示，脉冲越长，谐波空化增加。然而，时域分析表明，较长的脉冲会诱导更高的MB塌陷。在经颅体内实验中，PCD检测到10个周期脉冲的谐波空化增加。MB研究表明，与较小的MB组相比，3 - 5μm的MB产生的空化剂量、BBBO体积和转基因表达最大。然而，当按气体体积对MB剂量进行归一化时，在BBBO体积和转基因表达方面未发现显著差异。脉冲序列研究表明，由于焦点体积内的MB补充，较短的脉冲和较快的脉冲重复频率会诱导更大的BBBO体积和AAV转导。转基因表达也随注射剂量增加，并且与AAV2和AAV5相比，AAV9显示出最高的脑转导效率。
模型和体内研究表明，ThUS - BBBO的机制以瞬时空化为主，并且MB塌陷随脉冲长度增加。空化剂量增加导致体内更大的BBBO体积和转基因表达。总体而言，这些结果为ThUS介导的AAV跨BBB递送机制提供了见解，并表明尽管对ThUS参数和MB大小进行了调节，但特定的AAV血清型更适合在全身注射后在脑中实现最大的ThUS介导的转基因表达。
