Factors affecting the endothelial retention of targeted microbubbles: influence of microbubble shell design and cell surface projection of the endothelial target molecule

Abstract

Background: In biologic systems, the arrest of circulating cells is mediated by adhesion molecules projecting their active binding domain above the cell surface to enhance bond formation and tether strength. Similarly, molecular spacers are used for ligands on particle-based molecular imaging agents. The aim of this study was to evaluate the influence of tether length for targeting ligands on ultrasound molecular imaging agents.

Methods: Microbubbles bearing biotin at the end of variable-length polyethylene glycol spacer arms (MB(2000) and MB(3400)) were prepared. To assess in vivo attachment efficiency to endothelial counterligands that vary in their distance from the endothelial cell surface, contrast-enhanced ultrasound (CEU) molecular imaging of tumor necrosis factor-α-induced P-selectin (long distance) or intercellular adhesion molecule-2 (short distance) was performed with each agent in murine hind limbs. To assess the influence of the glycocalyx on microbubble attachment, CEU molecular imaging of intercellular adhesion molecule-2 was performed after degradation of the glycocalyx.

Results: CEU molecular imaging targeted to P-selectin showed signal enhancement above control agent for MB(2000) and MB(3400), the degree of which was significantly higher for MB(3400) compared with MB(2000). CEU molecular imaging targeted to intercellular adhesion molecule-2 showed low overall signal for all agents and signal enhancement above control for MB(3400) only. Glycocalyx degradation increased signal for MB(3400) and MB(2000).

Conclusions: Microbubble targeting to endothelial ligands is influenced by (1) the tether length of the ligand, (2) the degree to which the endothelial target is projected from the cell surface, and (3) the status of the glycocalyx. These considerations are important for designing targeted imaging probes and understanding potential obstacles to molecular imaging.

Figure 1

(A) Mean ± SD diameter for microbubble preparations containing PEG spacer molecules with a PEG 2000, PEG 3400, or PEG 10000 spacer length. Data are from ≥10 microbubble preparations for each PEG spacer length. (B) Histograms illustrating the size distribution of the three microbubble preparations. ANOVA, Analysis of variance.

Figure 2

CEU data used to evaluated recirculation time for the microbubble agents that varied according to PEG length. (A) Time versus mean ± SEM video intensity plots on CEU used to assess the duration of recirculation for the three microbubble preparations (n = 5 mice with injection of the three microbubble preparations in random order). (B) Examples of CEU images at incremental intervals after bolus injection of the three microbubble preparations. Time intervals after bolus injection are provided at the bottom of each image. (C) Mean ± SEM circulation half-life for each agent. P = NS between the three agents. ANOVA, Analysis of variance.

Figure 3

Molecular imaging data for the three microbubble preparations targeted to P-selectin (n = 16 mice). (A) Mean ± SEM of background-subtracted targeted signal intensity for the three microbubble preparations bearing a P-selectin antibody and microbubbles bearing a control antibody. Examples of background-subtracted color-coded images of targeted signal from one animal are illustrated for MB_PSel with a PEG 2000 spacer arm (B), MB_PSel with a PEG 3400 spacer arm (C), and MB_Ctr (D). Color scales at bottom. *P < .001 versus MB_Ctr; ^†P < .01 versus PEG 2000 and PEG 10000; ^¥P < .01 versus MB_Ctr.

Figure 4

Molecular imaging data for microbubble preparations targeted to ICAM-2 in murine hind limb skeletal muscle. (A) Mean ± SEM of background-subtracted targeted signal intensity for the three microbubble preparations bearing an ICAM-2 antibody and microbubbles bearing a control antibody (n = 16 mice). Examples of background-subtracted color-coded images of targeted signal from one animal are illustrated for MB_ICAM with a PEG 3400 spacer arm (B) and MB_Ctr (C). (D) Mean ± SEM of background-subtracted signal intensity for MB_PEG3400-ICAM, MB_PEG2000-ICAM, and MB_Ctr 60 min after intravenous injection of hyaluronidase (n = 16). Examples of background-subtracted color-coded images of targeted signal from one animal are illustrated for MB_ICAM with a PEG 2000 spacer arm (E) and MB_Ctr (F). *P < .01 versus MB_Ctr; ^†P < .05 versus PEG 2000; ^¥P < .05 versus MB_Ctr.