Microbubble-assisted pLIFU enhances riboflavin delivery and corneal cross-linking in an in vivo rabbit keratoconus-like model.

Epithelium-off corneal collagen cross-linking (CXL) slows keratoconus progression but disrupts the epithelial barrier, whereas epithelium-on CXL is constrained by limited stromal riboflavin loading and insufficient evidence that physical delivery enhancement improves CXL outcomes in vivo keratoconus-like ectasia. Here, we developed microbubble-assisted pulsed low-intensity focused ultrasound (pLIFU) to enhance riboflavin transport through cavitation and evaluated its therapeutic effect in a collagenase-induced rabbit keratoconus-like model. Passive cavitation detection and temperature monitoring identified a treatment condition of 1 Hz pulse repetition frequency, 20 % duty cycle, 1.43 MPa focal-zone peak negative pressure, and 200 μl SonoVue solution, with maximum temperature rise limited to 1.5 °C. Hydrophone measurements showed low posterior-segment acoustic action, with a retinal-position mechanical index of approximately 0.076. Confocal fluorescence imaging showed that pLIFU with microbubbles increased peak stromal riboflavin fluorescence by 9.27 ± 2.16-fold (p < 0.0001) and effective penetration depth by 4.09-fold (p < 0.001) relative to control. In vivo, pLIFU-microbubble-enhanced CXL produced greater corneal flattening and a larger shear-wave-speed response than the US group. One month after treatment, central corneal curvature decreased by 45.85 ± 6.32 % and shear-wave speed (cs) increased by 52.50 ± 12.89 % relative to pretreatment in the USMB group. Short-term histology and inflammatory cytokine assays showed no sustained inflammatory elevation or overt retinal laminar disruption within the observation window. These findings support the early feasibility of pLIFU cavitation-enhanced riboflavin delivery as a physical augmentation strategy for epithelium-on CXL in keratoconus-like ectasia, with favourable short-term ocular tissue tolerance for the assessed endpoints; they do not establish non-inferiority to epithelium-off CXL or long-term safety and durability.