The blood-brain barrier (BBB) limits transport of nanoparticles from the circulation to the brain parenchyma. Angiopep-2, a peptide which functions as a brain transport vector, can be coupled to nanoparticles in order to facilitate binding and internalization by brain endothelial cells (ECs), and subsequent BBB penetration. This multi-step process may be affected by blood flow over brain ECs, as flow influences endothelial cell phenotype as well as interactions of nanoparticles with ECs. In the present study a microfluidic BBB model was constructed to evaluate binding and internalization by brain ECs, as well as BBB penetration of Angiopep-2 coupled liposomes (Ang2-Liposomes) in static and flow conditions. Ang2 conjugation to liposomes markedly improved binding relative to unconjugated liposomes. Ang2-Liposomes bound and were internalized efficiently by brain endothelial cells after static incubation or with 1 dyne/cm2 of fluid shear stress (FSS), while binding was reduced at a FSS of 6 dyne/cm2. Penetration of the model microfluidic BBB by Ang2-Liposomes was higher at a FSS of 1 dyne/cm2 and 6 dyne/cm2 than with static incubation. Analysis of barrier function and control experiments for receptor-mediated penetration provided insight into the magnitude of transcellular versus paracellular transport at each tested FSS. Overall, the results demonstrate that flow impacted the binding and BBB penetration of Ang2-functionalized nanoparticles. This highlights the relevance of the local flow environment for in vitro modeling of the performance of nanoparticles functionalized with BBB penetrating ligands.

Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolar-mediated pathways, allows uptake of nano- and micro-carriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and micro-carriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size-restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems.

Modification of drug delivery nanomaterials with affinity molecules that facilitate targeting, has rendered a new class of ligands for cell receptors, which often possess valency and dimensions different from natural counterparts. Designing strategies to target multiple receptors or, never explored, multiple epitopes on the same receptor may modulate the biodistribution properties of these nanomaterials. We examined this using antibody-directed targeting of polymer nanocarriers to transferrin receptor (TfR) and intercellular adhesion molecule 1 (ICAM-1). Regarding epitopes on one receptor, nanocarriers addressed with anti-TfR-R17 maintained brain and lung targeting in mice, compared with “free” antibody, while anti-TfR-8D3 nanocarriers lost specificity. Coating nanocarriers with both antibodies decreased targeting in brain and liver, not lungs, modulating biodistribution. Regarding different receptors, nanocarriers coated with both anti-ICAM and anti-TfR displayed intermediate specific accumulation in lungs and higher in liver, compared to single-targeted nanocarriers, while brain targeting was comparable to TfR- and lower than ICAM-1-targeted nanocarriers. Tracing a model therapeutic cargo, acid sphingomyelinase (enzyme replacement for Niemann-Pick Disease A-B), showed that combined-targeted anti-ICAM/TfR nanocarriers enhanced enzyme delivery versus “free” enzyme, with biodistribution patterns different from single-targeted nanocarriers. Hence, targeting nanocarriers to multiple epitopes or receptors holds promise to control distribution of drug delivery nanomaterials in the body.

Brain drug delivery is a major challenge for therapy of central nervous system (CNS) diseases. Biochemical modifications of drugs or drug nanocarriers, methods of local delivery, and blood–brain barrier (BBB) disruption with focused ultrasound and microbubbles are promising approaches which enhance transport or bypass the BBB. These approaches are discussed in the context of brain cancer as an example in CNS drug development. Targeting to receptors enabling transport across the BBB offers noninvasive delivery of small molecule and biological cancer therapeutics. Local delivery methods enable high dose delivery while avoiding systemic exposure. BBB disruption with focused ultrasound and microbubbles offers local and noninvasive treatment. Clinical trials show the prospects of these technologies and point to challenges for the future.