Development of novel nanofibers targeted to smoke-injured lungs.

Smoke inhalation injury is associated with significant mortality and current therapies remain supportive. The purpose of our study was to identify proteins upregulated in the lung after smoke inhalation injury and develop peptide amphiphile nanofibers that target these proteins. We hypothesize that nanofibers targeted to angiotensin-converting enzyme or receptor for advanced glycation end products will localize to smoke-injured lungs. METHODS: Five targeting sequences were incorporated into peptide amphiphile monomers methodically to optimize nanofiber formation. Nanofiber formation was assessed by conventional transmission electron microscopy. Rats received 8 min of wood smoke. Levels of angiotensin-converting enzyme and receptor for advanced glycation end products were evaluated by immunofluorescence. Rats received the targeted nanofiber 23 h after injury via tail vein injection. Nanofiber localization was determined by fluorescence quantification. RESULTS: Peptide amphiphile purity (>95%) and nanofiber formation were confirmed. Target proteins were increased in smoke inhalation versus sham (p < 0.001). After smoke inhalation and injection of targeted nanofibers, we found a 10-fold increase in angiotensin-converting enzyme-targeted nanofiber localization to lung (p < 0.001) versus sham with minimal localization of non-targeted nanofiber (p < 0.001). CONCLUSIONS: We synthesized, characterized, and evaluated systemically delivered targeted nanofibers that localized to the site of smoke inhalation injury in vivo. Angiotensin-converting enzyme-targeted nanofibers serve as the foundation for developing a novel nanotherapeutic that treats smoke inhalation lung injury.

Development of Optimized Tissue-Factor-Targeted Peptide Amphiphile Nanofibers to Slow Noncompressible Torso Hemorrhage.

Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths. We report here on the development of injectable, targeted supramolecular nanotherapeutics based on peptide amphiphile (PA) molecules that are designed to target tissue factor (TF) and, therefore, selectively localize to sites of injury to slow hemorrhage. Eight TF-targeting sequences were identified, synthesized into PA molecules, coassembled with nontargeted backbone PA at various weight percentages, and characterized via circular dichroism spectroscopy, transmission electron microscopy, and X-ray scattering. Following intravenous injection in a rat liver hemorrhage model, two of these PA nanofiber coassemblies exhibited the most specific localization to the site of injury compared to controls (p < 0.05), as quantified using immunofluorescence imaging of injured liver and uninjured organs. To determine if the nanofibers were targeting TF in vivo, a mouse saphenous vein laser injury model was performed and showed that TF-targeted nanofibers colocalized with fibrin, demonstrating increased levels of nanofiber at TF-rich sites. Thromboelastograms obtained using samples of heparinized rat whole blood containing TF demonstrated that no clots were formed in the absence of TF-targeted nanofibers. Lastly, both PA nanofiber coassemblies decreased blood loss in comparison to sham and backbone nanofiber controls by 35-59% (p < 0.05). These data demonstrate an optimal TF-targeted nanofiber that localizes selectively to sites of injury and TF exposure, and, interestingly, reduces blood loss. This research represents a promising initial phase in the development of a TF-targeted injectable therapeutic to reduce preventable deaths from hemorrhage.

Peptide Amphiphile Supramolecular Nanostructures as a Targeted Therapy for Atherosclerosis.

The rising prevalence of cardiovascular disease worldwide necessitates novel therapeutic approaches to manage atherosclerosis. Intravenously administered nanostructures are a promising noninvasive approach to deliver therapeutics that reduce plaque burden. The drug liver X receptor agonist GW3965 (LXR) can reduce atherosclerosis by promoting cholesterol efflux from plaque but causes liver toxicity when administered systemically at effective doses, thus preventing its clinical use. The ability of peptide amphiphile nanofibers containing apolipoprotein A1-derived targeting peptide 4F to serve as nanocarriers for LXR delivery (ApoA1-LXR PA) in vivo is investigated here. These nanostructures are found to successfully target atherosclerotic lesions in a mouse model within 24 h of injection. After 8 weeks of intravenous administration, the nanostructures significantly reduce plaque burden in both male and female mice to a similar extent as LXR alone in comparison to saline-treated controls. Furthermore, they do not cause increased liver toxicity in comparison to LXR treatments, which may be related to more controlled release by the nanostructure. These findings demonstrate the potential of supramolecular nanostructures as safe, effective drug nanocarriers to manage atherosclerosis.

Atheroma Niche-Responsive Nanocarriers for Immunotherapeutic Delivery.

Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. However, drug delivery is limited without the ability of nanocarriers to sense and respond to the diseased microenvironment. In this study, nanomaterials are developed from peptide amphiphiles (PAs) that respond to the increased levels of matrix metalloproteinases 2 and 9 (MMP2/9) or reactive oxygen species (ROS) found within the atherosclerotic niche. A pro-resolving therapeutic, Ac2-26, derived from annexin-A1 protein, is tethered to PAs using peptide linkages that cleave in response to MMP2/9 or ROS. By adjusting the molar ratios and processing conditions, the Ac2-26 PA can be co-assembled with a PA containing an apolipoprotein A1-mimetic peptide to create a targeted, therapeutic nanofiber (ApoA1-Ac226 PA). The ApoA1-Ac2-26 PAs demonstrate release of Ac2-26 within 24 h after treatment with MMP2 or ROS. The niche-responsive ApoA1-Ac2-26 PAs are cytocompatible and reduce macrophage activation from interferon gamma and lipopolysaccharide treatment, evidenced by decreased nitric oxide production. Interestingly, the linkage chemistry of ApoA1-Ac2-26 PAs significantly affects macrophage uptake and retention. Taken together, these findings demonstrate the potential of PAs to serve as an atheroma niche-responsive nanocarrier system to modulate the inflammatory microenvironment, with implications for atherosclerosis treatment.

Poly(ethylene glycol) Hydrogel Scaffolds Containing Cell-Adhesive and Protease-Sensitive Peptides Support Microvessel Formation by Endothelial Progenitor Cells.

The development of stable, functional microvessels remains an important obstacle to overcome for tissue engineered organs and treatment of ischemia. Endothelial progenitor cells (EPCs) are a promising cell source for vascular tissue engineering as they are readily obtainable and carry the potential to differentiate towards all endothelial phenotypes. The aim of this study was to investigate the ability of human umbilical cord blood-derived EPCs to form vessel-like structures within a tissue engineering scaffold material, a cell-adhesive and proteolytically degradable poly(ethylene glycol) (PEG) hydrogel. EPCs in co-culture with angiogenic mural cells were encapsulated in hydrogel scaffolds by mixing with polymeric precursors and using a mild photocrosslinking process to form hydrogels with homogeneously dispersed cells. EPCs formed 3D microvessels networks that were stable for at least 30 days in culture, without the need for supplemental angiogenic growth factors. These 3D EPC microvessels displayed aspects of physiological microvasculature with lumen formation, expression of endothelial cell proteins (connexin 32, VE-cadherin, eNOS), basement membrane formation with collagen IV and laminin, perivascular investment of PDGFR-ß and α-SMA positive cells, and EPC quiescence (<1% proliferating cells) by 2 weeks of co-culture. Our findings demonstrate the development of a novel, reductionist system that is well-defined and reproducible for studying progenitor cell-driven microvessel formation.

Umbilical Cord Blood-Derived Mononuclear Cells Exhibit Pericyte-Like Phenotype and Support Network Formation of Endothelial Progenitor Cells In Vitro.

Umbilical cord blood represents a promising cell source for pro-angiogenic therapies. The present study examined the potential of mononuclear cells (MNCs) from umbilical cord blood to support endothelial progenitor cell (EPC) microvessel formation. MNCs were isolated from the cord blood of 20 separate donors and selected for further characterization based upon their proliferation potential and morphological resemblance to human vascular pericytes (HVPs). MNCs were screened for their ability to support EPC network formation using an in vitro assay (Matrigel™) as well as a reductionist, coculture system consisting of no additional angiogenic cytokines beyond those present in serum. In less than 15% of the isolations, we identified a population of highly proliferative MNCs that phenotypically resembled HVPs as assessed by expression of PDGFR-ß, NG2, α-SMA, and ephrin-B2. Within a Matrigel™ system, MNCs demonstrated pericyte-like function through colocalization to EPC networks and similar effects as HVPs upon total EPC tubule length (p = 0.95) and number of branch points (p = 0.93). In a reductionist coculture system, MNCs served as pro-angiogenic mural cells by supporting EPC network formation to a significantly greater extent than HVP cocultures, by day 14 of coculture, as evidenced through EPC total tubule length (p < 0.0001) and number of branch points (p < 0.0001). Our findings are significant as we demonstrate mural cell progenitors can be isolated from umbilical cord blood and develop culture conditions to support their use in microvascular tissue engineering applications.

Effect of cellular senescence on the albumin permeability of blood-derived endothelial cells.

In this study, we tested the hypotheses that endothelial cells (ECs) derived from human umbilical cord blood (hCB-ECs) exhibit low permeability, which increases as hCB-ECs age and undergo senescence, and that the change in the permeability of hCB-ECs is due to changes in tight junction protein localization and the activity of exchange protein activated by cAMP (Epac)1. Albumin permeability across low-passage hCB-EC monolayers on Transwell membranes was 10 times lower than for human aortic ECs (HAECs) (P < 0.01) but similar to in vivo values in arteries. Expression of the tight junction protein occludin and tyrosine phosphorylation of occludin were less in hCB-ECs than in HAECs (P < 0.05). More hCB-ECs than HAECs underwent mitosis (P < 0.01). hCB-ECs that underwent >44 population doublings since isolation had a significantly higher permeability than hCB-ECs that underwent <31 population doublings (P < 0.05). This age-related increase in hCB-EC permeability was associated with an increase in tyrosine phosphorylation of occludin (P < 0.01); permeability and occludin phosphorylation were reduced by treatment with 2 µM resveratrol. Tyrosine phosphorylation of occludin and cell age influence the permeability of hCB-ECs, whereas levels of EC proliferation and expression of tight junction proteins did not explain the differences between hCB-EC and HAEC permeability. The elevated permeability in late passage hCB-ECs was reduced by 25-40% by elevation of membrane-associated cAMP and activation of the Epac1 pathway. Given the similarity to in vivo permeability to albumin and the high proliferation potential, hCB-ECs may be a suitable in vitro model to study transport-related pathologies and cell aging.