Castillo Jair I, Navarro-Becerra J Angel, Angelini Ilaria, Kokoshinskiy Maxim, Borden Mark A
Biomedical Engineering Program, University of Colorado, Boulder, Colorado 80309, United States.
Mechanical Engineering Department, University of Colorado, Boulder, Colorado 80309, United States.
ACS Appl Bio Mater. 2025 Mar 17;8(3):2128-2140. doi: 10.1021/acsabm.4c01699. Epub 2025 Feb 12.
Ultrasound molecular imaging (USMI) utilizing targeted microbubbles (tMBs) and primary acoustic radiation force () pulses has demonstrated enhanced sensitivity in recent studies. However, current USMI techniques are limited to a single ligand-receptor pair per imaging scan. With the advent of the buried-ligand architecture (BLA), "cloaked" ligand-receptor binding and tMB adhesion can be activated by pulses, enabling multicolor USMI. This approach permits the selective activation of two or more tMB species, each binding to its cognate receptors based on distinct resonance frequencies () tuned by pulses. The goal of this study was to demonstrate frequency-selective tMB adhesion to receptor-bearing microvessel tubes . Size-isolated BLA tMBs of 1 and 5 μm diameter were synthesized with equal to 7 and 4 MHz, respectively (within the frequency limits of our ultrasound probe). The 1 μm tMBs were conjugated with IELLQAR peptide for P-selectin targeting, while the 5 μm tMBs were conjugated with cyclo-RGD peptide for αβ integrin targeting. The MB gas volume fraction (φ) was used to unify size and concentration into a single parameter. Frequency-selective tMB binding was quantified using fluorescence microscopy. Specific targeting was evaluated by comparing RGD- or IELLQAR-MB attachment to control RAD- or nonligand-bearing MBs, respectively. The results confirmed specific frequency-selective targeting of the two tMB species to their cognate receptors when activated by pulses at their respective , both individually and in a cocktail. In the cocktail population, φ of RGD-MB targeting increased 18-fold at 4 MHz compared to 7 MHz, while IELLQAR-MB targeting φ increased 5-fold at 7 MHz compared to 4 MHz. In conclusion, this study presents the first demonstration of frequency-selective targeting of two different receptor species by two different tMB species, representing a significant step toward multicolor USMI and the potential for simultaneous imaging of multiple biomarkers within a single scan.
利用靶向微泡（tMBs）和初级声辐射力（）脉冲的超声分子成像（USMI）在最近的研究中已显示出更高的灵敏度。然而，当前的USMI技术在每次成像扫描中仅限于单个配体-受体对。随着埋藏配体结构（BLA）的出现，“隐形”配体-受体结合和tMB粘附可通过脉冲激活，从而实现多色USMI。这种方法允许选择性激活两种或更多种tMB，每种tMB基于由脉冲调谐的不同共振频率（）与其同源受体结合。本研究的目的是证明频率选择性tMB对带有受体的微血管管的粘附。合成了直径为1和5μm的尺寸分离的BLA tMB，其分别等于7和4MHz（在我们超声探头的频率范围内）。1μm的tMB与用于靶向P-选择素的IELLQAR肽偶联，而5μm的tMB与用于靶向αβ整合素的环RGD肽偶联。微泡气体体积分数（φ）用于将尺寸和浓度统一为单个参数。使用荧光显微镜对频率选择性tMB结合进行定量。通过分别比较RGD或IELLQAR微泡与对照RAD或无配体微泡的附着来评估特异性靶向。结果证实，当在各自的频率下通过脉冲单独或在混合液中激活时，两种tMB对其同源受体具有特异性频率选择性靶向。在混合群体中，与7MHz相比，4MHz时RGD微泡靶向的φ增加了18倍，而与4MHz相比，7MHz时IELLQAR微泡靶向的φ增加了5倍。总之，本研究首次证明了两种不同的tMB对两种不同受体的频率选择性靶向，这代表了朝着多色USMI以及在单次扫描中同时成像多种生物标志物的潜力迈出的重要一步。
