Claudin-5 binder enhances focused ultrasound-mediated opening in an in vitro blood-brain barrier model

Abstract

Rationale: The blood-brain barrier (BBB) while functioning as a gatekeeper of the brain, impedes cerebral drug delivery. An emerging technology to overcome this limitation is focused ultrasound (FUS). When FUS interacts with intravenously injected microbubbles (FUS +MB ), the BBB opens, transiently allowing the access of therapeutic agents into the brain. However, the ultrasound parameters need to be tightly tuned: when the acoustic pressure is too low there is no opening, and when it is too high, tissue damage can occur. We therefore asked whether barrier permeability can be increased by combining FUS +MB with a second modality such that in a clinical setting lower acoustic pressures could be used. Methods: Given that FUS +MB achieves BBB opening in part by disruption of tight junction (TJ) proteins such as claudin-5 of brain endothelial cells, we generated a stable MDCK (Madin-Darby Canine Kidney) II cell line (eGFP-hCldn5-MDCK II) that expresses fluorescently tagged human claudin-5. Two claudin-5 binders, the peptide mC5C2 and cCPEm (truncated form of an enterotoxin), reported previously to weaken the barrier, were synthesized and assessed for their abilities to enhance the permeability of cellular monolayers. We then performed a comparative analysis of single and combination treatments, measuring transendothelial electrical resistance (TEER) and cargo leakage, combined with confocal image analysis. Results: We successfully generated a novel cell line that formed functional monolayers as validated by an increased TEER reading and a low (< 0.2%) permeability to sodium fluorescein (376 Da). We found that the binders exerted a time- and concentration-dependent effect on barrier opening when incubated over an extended period, whereas FUS +MB caused a rapid opening followed by recovery after 12 hours within the tested pressure range. Importantly, preincubation with cCPEm prior to FUS +MB treatment resulted in greater barrier opening compared to either FUS +MB or cCPEm alone as measured by reduced TEER values and an increased permeability to fluorescently labelled 40 kDa dextran (FD40). Conclusion: The data suggest that pre incubation with clinically suitable binders to TJ proteins may be a general strategy to facilitate safer and more effective ultrasound-mediated BBB opening in cellular and animal systems and potentially also for the treatment of human diseases of the brain.

A sporadic Alzheimer's blood-brain barrier model for developing ultrasound-mediated delivery of Aducanumab and anti-Tau antibodies

“…Therefore, we performed bulk RNA-sequencing (RNA-seq) on FUS +MB -treated APOE3 and E4 iBECs. To achieve this, APOE3 and E4 iBECs were treated with FUS +MB and collected at 1 h and 24 h post sonication, analogous to the previously reported timeline of BBB opening and spontaneous closing post FUS +MB in in vitro models 18 , 45 . Corresponding untreated (UT) controls at the respective timepoints were included.…”

“…In addition, the physical dynamics of the free-floating MBs in our system may differ from those observed within the constrained brain capillaries, with BBB opening in vitro being primarily achieved due to the formation of standing waves generated by the wave reflection from the media-air interphase (as reported in 84 ). Although FUS +MB has been previously shown to achieve BBB opening in such a configuration in vitro 18 , 45 , alternative model settings that create a more biologically and physically accurate environment for FUS +MB exposure, might have higher translational capability. Finally, our model is currently limited to a fluorescence-based approach.…”

Design of an L-Valine-Modified Nanomicelle-Based Drug Delivery System for Overcoming Ocular Surface Barriers

“…ZO-1 is the marker protein of tight junctions in the corneal epithelium, and claudin-1 is the main protein expressed in claudins [43][44][45]. The effects of ophthalmic preparations on tight junction proteins can be determined by PCR, WB or immunofluorescence staining [45][46][47]. In this study, the expression changes of ZO-1 and claudin-1 were detected with WB, proving that the cellular uptake mechanism of nanomicelles inhibits the overexpression of ZO-1 and claudin-1 induced by drug stimulation, which is a novel and noteworthy discovery.…”

Low-Intensity Focused Ultrasound Technique in Glioblastoma Multiforme Treatment

“…Moreover, these parameters also determine whether the therapeutics uptake will be early/fast or slow/late ( 8 ). Other factors such as the introduction of TJ protein binders (claudin-5 binder) can greatly facilitate the BBB/BTB disruption which can be more effective and safer ( 76 ). It is important to carefully investigate and pick the appropriate set of parameters as the correct parameters can yield in BBB/BTB disruption that can be reversible.…”