Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain.

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Microphysiological Engineering of Self-Assembled and Perfusable Microvascular Beds for the Production of Vascularized Three-Dimensional Human Microtissues.

The vasculature is an essential component of the circulatory system that plays a vital role in the development, homeostasis, and disease of various organs in the human body. The ability to emulate the architecture and transport function of blood vessels in the integrated context of their associated organs represents an important requirement for studying a wide range of physiological processes. Traditional in vitro models of the vasculature, however, largely fail to offer such capabilities. Here we combine microfluidic three-dimensional (3D) cell culture with the principle of vasculogenic self-assembly to engineer perfusable 3D microvascular beds in vitro. Our system is created in a micropatterned hydrogel construct housed in an elastomeric microdevice that enables coculture of primary human vascular endothelial cells and fibroblasts to achieve de novo formation, anastomosis, and controlled perfusion of 3D vascular networks. An open-top chamber design adopted in this hybrid platform also makes it possible to integrate the microengineered 3D vasculature with other cell types to recapitulate organ-specific cellular heterogeneity and structural organization of vascularized human tissues. Using these capabilities, we developed stem cell-derived microphysiological models of vascularized human adipose tissue and the blood-retinal barrier. Our approach was also leveraged to construct a 3D organotypic model of vascularized human lung adenocarcinoma as a high-content drug screening platform to simulate intravascular delivery, tumor-killing effects, and vascular toxicity of a clinical chemotherapeutic agent. Furthermore, we demonstrated the potential of our platform for applications in nanomedicine by creating microengineered models of vascular inflammation to evaluate a nanoengineered drug delivery system based on active targeting liposomal nanocarriers. These results represent a significant improvement in our ability to model the complexity of native human tissues and may provide a basis for developing predictive preclinical models for biopharmaceutical applications.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain.

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1.

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

Carbon Nanomaterials for Drug Delivery and Cancer Therapy.

Nanotechnology is one of the most exciting disciplines and it incorporates physics, chemistry, materials science, and biology. It can be applied to design cancer medicines with improved therapeutic indices. At the basic level, carbon nanotubes (CNTs) and graphene are sp2 carbon nanomaterials. Their unique physical and chemical properties make them interesting candidates of research in a wide range of areas including biological systems and different diseases. Recent research has been focused on exploring the potential of the CNTs as a carrier or vehicle for intracellular transport of drugs, proteins, and targeted genes in vitro and in vivo. Several research groups are actively involved to find out a functional CNT carrier capable of transporting targeted drug molecules in animal models with least toxicity. Current investigations are also focused on graphene, an allotrope of carbon, which appears to be a promising agent for successful delivery of biomolecules in various animal models. But potential clinical implementations of CNTs are still hampered by distinctive barriers such as poor bioavailability and intrinsic toxicity, which pose difficulties in tumor targeting and penetration as well as in improving therapeutic outcome. This article presents recent progresses in the design and evaluation of closely related CNTs for experimental cancer therapy and explores their implications in bringing nanomedicines into the clinics.