Reversibly Modulating the Blood-brain Barrier by Laser Stimulation of Endothelial-targeted Nanoparticles

The blood-brain barrier (BBB) excludes or limits over 98% of approved and investigational drugs and, as such, represents a major challenge in developing effective treatment strategies for the myriad of acute and chronic brain diseases. There is increasing recognition that BBB dysfunction is an integral component of many brain diseases, including neurodegenerative diseases and primary malignancies, which contribute to neurocognitive dysfunction. Thus, it is critically important that the strategies used to increase BBB permeability minimize the risks of additional brain injury. Various methods have been developed to modulate the BBB permeability. Currently, there are no molecularly targeted approaches for the non-invasive modulation of BBB permeability. Here, we first demonstrate that short pulse laser stimulation of gold-nanoparticles (AuNPs), functionalized to target an integral protein of the BBB tight-junction complex, JAM-A, causes a graded and reversible increase in BBB permeability in vivo, referred to as OptoBBB. A short pulse laser excitation of JAM-A targeted AuNPs can lead to sufficient loosening of the tight-junction complex to allow passage of blood-circulating molecules (600 Da-70 kDa) through the opposing faces of the tight-junction complex but without permanently compromising its integrity. This approach allows delivery of immunoglobulins, viral gene therapy vectors, and liposomes to specific locations in the brain. It provides high regional specificity and does not lead to significant disruption in the spontaneous vasomotion or the structure of the neurovascular unit. To better understand this technology, we further explored the targeting efficiency and cellular mechanisms involved in OptoBBB using a human cerebral microvascular endothelial/D3 cell line to establish an in vitro transwell BBB model. We demonstrate that targeting glycoprotein on the BBB leads to >20-fold higher targeting efficiency compared with tight junction targeting. Using live calcium (Ca2+), we uncover that OptoBBB is associated with a transient elevation of Ca2+ that propagates among the endothelial cells after laser excitation and extends the region of BBB opening. The Ca2+ response involves both internal Ca2+ depletion and Ca2+ influx. Furthermore, we demonstrate the involvement of actin polymerization and phosphorylation of ERK1/2 (one of the downstream messengers of Ca2+ signaling pathway) after laser treatment, which can contribute to cytoskeletal contraction and BBB opening. In summary, the OptoBBB is a promising strategy to screen and deliver therapeutic agents into the central nervous system in preclinical models noninvasively and for clinical translation using fiberoptics. The findings from the targeting efficiency and cellular mechanism study provide a mechanistic insight into the BBB opening by laser excitation of AuNP and help guide future development of this technology for brain diseases treatment.