Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires inv... more Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires invasive surgical procedures for its removal. Nanomedicine offers a minimally invasive approach to alleviate plaque burden by targeted therapeutic delivery. However, nanocarriers are limited without the ability to sense and respond to the diseased microenvironment. In this study, targeted self-assembled peptide amphiphile (PA) nanofibers are developed that cleave in response to biochemical cues expressed in atherosclerotic lesions-reactive oxygen species (ROS) and intracellular glutathione-to deliver a liver X receptor agonist (LXR) to enhance macrophage cholesterol efflux. The PAs release LXR in response to physiological levels of ROS and reducing agents and can be co-assembled with plaque-targeting PAs to form nanofibers. The resulting LXR PA nanofibers promoted cholesterol efflux from macrophages in vitro as well as LXR alone and with lower cytotoxicity. Further, the ApoA1-LXR PA nanofibers target plaque within an atherosclerotic mouse model in vivo and activate ATP-binding cassette A1 (ABCA1) expression as well as LXR alone with reduced liver toxicity. Taken together, these results demonstrate the potential of self-assembled PA nanofibers for controlled therapeutic delivery to the atherosclerotic niche.
This special issue presents the latest advances in nanomaterials for managing infectious disease.... more This special issue presents the latest advances in nanomaterials for managing infectious disease. The contributions are from a diverse, global panel of experts leading efforts to enhance vaccine and antibiotic delivery as well as disrupt fungal and biofilm adhesions found in hospital-acquired infections.
Atherosclerosis, Thrombosis, and Vascular Biology, 2020
Objective: Cardiovascular disease is the leading cause of death in the world, with atherosclerosi... more Objective: Cardiovascular disease is the leading cause of death in the world, with atherosclerosis a major contributor. Atherosclerotic plaque buildup is caused by an impaired lipid metabolism in macrophage foam cells. Liver X Receptor agonists (LXR) have the potential to restore macrophage cholesterol efflux by increasing the expression of ABCA1 cholesterol transporters. However, translation of LXR is limited by systemic toxicity. We hypothesized attaching LXR to peptide amphiphiles (PAs) via niche-cleavable linkages, and subsequent co-assembly with plaque-targeting PAs, would allow targeted, controlled delivery that reduces LXR toxicity while retaining its therapeutic effects.
Methods: We investigated two linkages for tethering LXR, reactive oxygen species (ROS)-cleavable prolines and glutathione-cleavable succinimidyl 3-(2-pyridyldithio)propionate (SPDP). We attached the LXR to PAs using solid phase peptide synthesis and characterized the PAs for LXR release. To allow targeted delivery to atherosclerotic lesions, we co-assembled the niche-responsive LXR PAs with PAs containing apolipoprotein A1-derived peptide 4F (ApoA1-LXR PAs), and determined processing parameters that enabled nanofiber formation. Lastly, we evaluated the effect of ApoA1-LXR PAs on macrophage viability and cholesterol efflux in vitro.
Results: We found 46% cleavage of ROS-LXR PAs after 24 hours of treatment with 250 μM H2O2 + CuSO4, and 100% cleavage by 4 days. In comparison, the SPDP-LXR PAs showed 100% cleavage in response to 24 hours of treatment with 10 mM glutathione. At 10 mol%, the ROS-LXR PAs formed nanofibers upon co-assembly with 40 mol% ApoA1 PAs and 50 mol% diluent PA after 24 hours of aging at 4°C. Similarly, the SPDP-LXR PAs formed the best nanofibers at 10 mol% co-assembly, yet required annealing at 80°C for 30 minutes. Both the ApoA1-ROS-LXR and ApoA1-SPDP-LXR PAs were able to promote macrophage cholesterol efflux (>70%) to a similar extent as free LXR at concentrations >32 μM LXR (p>0.53) while improving cell viability by at least 3-fold.
Conclusion: Our results demonstrate tethering LXR to bioresponsive PAs has the potential to increase the safety of LXR to alleviate plaque burden, and supports the use of PA nanofibers for atherosclerosis nanomedicine.
Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patenc... more Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patency due to limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia. Multifunctional elastomers that coat the ePTFE graft surface to promote EC adhesion while simultaneously inhibiting intimal hyperplasia are highly desirable. Poly(diol-co-citrate) (PDC), a thermoset elastomer, is biodegradable, biocompatible, and mimics vascular mechanical properties. Engineering antioxidant components into PDC polymeric structures improves biocompatibility by attenuating oxidative stress yet is limited by bioavailability. Herein, we develop a new ascorbate protection and deprotection strategy (APDS) for loading bioactive ascorbic acid into the structure of PDC elastomers to improve poly(1,8-octanediol-co-citrate-co-ascorbate) (POCA) prepolymer ascorbate activity. Elastomers cured from APDS POCA prepolymers provide twice the active ascorbate sites on the elastomer surface (35.19 ± 1.64 ng mg–1 cm–2) versus unprotected POCA (Un.POCA, 18.31 ± 0.97 ng mg–1 cm–2). APDS POCA elastomers displayed suitable mechanical properties for vascular graft coating [Young’s modulus (2.15–2.61 MPa), elongation (189.5–214.6%) and ultimate tensile strength (2.73–3.61 MPa)], and superior surface antioxidant performance through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging and lipid peroxidation inhibition as compared to poly(1,8-octanediol-co-citrate) (POC) and Un.POCA. Hydrolytic degradation of APDS POCA occurred within 12 weeks under physiological conditions with a mass loss of 25.8 ± 3.4% and the degradation product retaining ascorbate activity. APDS POCA elastomer surfaces supported human aortic endothelial cell proliferation while inhibiting human aortic smooth muscle cell proliferation in vitro. APDS POCA elastomer surfaces displayed superior decomposition of S-nitrosothiols compared to POC and Un.POCA. Taken together, these findings indicate the potential of APDS POCA elastomers to serve as bioactive, therapeutic coatings that enhance the long-term patency of small diameter ePTFE grafts.
Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths.... more 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.
Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. Howeve... more 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.
Coassembled peptide amphiphile nanofibers designed to target atherosclerotic plaque and enhance c... more Coassembled peptide amphiphile nanofibers designed to target atherosclerotic plaque and enhance cholesterol efflux are shown to encapsulate and deliver a liver X receptor agonist to increase efflux from murine macrophages in vitro. Fluorescence microscopy reveals that the nanofibers, which display an apolipoprotein-mimetic peptide, localize at plaque sites in low density lipoprotein receptor knockout (LDLR KO) mice with or without the encapsulated molecule, while nanofibers displaying a scrambled, nontargeting peptide sequence do not demonstrate comparable binding. These results show that nanofibers functionalized with apolipoprotein-mimetic peptides may be effective vehicles for intravascular targeted drug delivery to treat atherosclerosis.
The recently developed synthetic oligonucleotides referred to as “Click” Nucleic Acids (CNAs) are... more The recently developed synthetic oligonucleotides referred to as “Click” Nucleic Acids (CNAs) are promising due to their relatively simple synthesis based on thiol-X reactions with numerous potential applications in biotechnology, biodetection, gene silencing, and drug delivery. Here, the cytocompatibility and cellular uptake of rhodamine tagged, PEGylated CNA copolymers (PEG-CNA-RHO) were evaluated. NIH 3T3 fibroblast cells treated for 1 hour with 1, 10 or 100 μg/mL PEG-CNA-RHO maintained an average cell viability of 86%, which was not significantly different from the untreated control. Cellular uptake of PEG-CNA-RHO was detected within 30 seconds and the amount internalized increased over the course of 1 hour. Moreover, these copolymers were internalized within cells to a higher degree than controls consisting of either rhodamine tagged PEG or the rhodamine alone. Uptake was not affected by temperature (i.e., 4°C or 37°C), suggesting a passive uptake mechanism. Subcellular colocalization analysis failed to indicate significant correlations between the internalized PEG-CNA-RHO and the organelles examined (mitochondria, endoplasmic reticulum, endosomes and lysosomes). These results indicate that CNA copolymers are cytocompatible and are readily internalized by cells, supporting the idea that CNAs are a promising alternative to DNA in antisense therapy applications.
Self‐assembled microvasculature from cocultures of endothelial cells and stromal cells have signi... more Self‐assembled microvasculature from cocultures of endothelial cells and stromal cells have significantly advanced efforts to vascularize engineered tissues by enhancing perfusion rates in vivo and producing investigative platforms for microvascular morphogenesis in vitro. However, in order to clinically translate prevascularized constructs, the issue of endothelial cell source must be resolved. Endothelial progenitor cells (EPCs) can be noninvasively supplied from the recipient through adult peripheral and umbilical cord blood, as well as derived from induced pluripotent stem cells, alleviating antigenicity issues. EPCs can also differentiate into all tissue endothelium, and have demonstrated potential for therapeutic vascularization. Yet, EPCs are not the standard endothelial cell choice to vascularize tissue constructs in vitro. Possible reasons include unresolved issues with EPC identity and characterization, as well as uncertainty in the selection of coculture, scaffold, and culture media combinations that promote EPC microvessel formation. This review addresses these issues through a summary of EPC vascular biology and the effects of tissue engineering design parameters upon EPC microvessel formation. Also included are perspectives to integrate EPCs with emerging technologies to produce functional, organotypic vascularized tissues.
In this study, we tested the hypotheses that endothelial cells (ECs) derived from human umbilical... more 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.
The development of stable, functional microvessels remains an important obstacle to overcome for ... more 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 polyethylene glycol 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-β 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 represents a promising cell source for pro-angiogenic therapies. The present... more 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-β, 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.
Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell so... more Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for an-giogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular en-dothelial growth factor (VEGF)-A or changes in TGF-b1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45 + cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements , however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression , as evidenced by a lack of significant differences in total segment length (p = 0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45 + cells and improves upon the addition of soluble growth factors.
Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent ... more Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood–derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24 h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.
Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires inv... more Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires invasive surgical procedures for its removal. Nanomedicine offers a minimally invasive approach to alleviate plaque burden by targeted therapeutic delivery. However, nanocarriers are limited without the ability to sense and respond to the diseased microenvironment. In this study, targeted self-assembled peptide amphiphile (PA) nanofibers are developed that cleave in response to biochemical cues expressed in atherosclerotic lesions-reactive oxygen species (ROS) and intracellular glutathione-to deliver a liver X receptor agonist (LXR) to enhance macrophage cholesterol efflux. The PAs release LXR in response to physiological levels of ROS and reducing agents and can be co-assembled with plaque-targeting PAs to form nanofibers. The resulting LXR PA nanofibers promoted cholesterol efflux from macrophages in vitro as well as LXR alone and with lower cytotoxicity. Further, the ApoA1-LXR PA nanofibers target plaque within an atherosclerotic mouse model in vivo and activate ATP-binding cassette A1 (ABCA1) expression as well as LXR alone with reduced liver toxicity. Taken together, these results demonstrate the potential of self-assembled PA nanofibers for controlled therapeutic delivery to the atherosclerotic niche.
This special issue presents the latest advances in nanomaterials for managing infectious disease.... more This special issue presents the latest advances in nanomaterials for managing infectious disease. The contributions are from a diverse, global panel of experts leading efforts to enhance vaccine and antibiotic delivery as well as disrupt fungal and biofilm adhesions found in hospital-acquired infections.
Atherosclerosis, Thrombosis, and Vascular Biology, 2020
Objective: Cardiovascular disease is the leading cause of death in the world, with atherosclerosi... more Objective: Cardiovascular disease is the leading cause of death in the world, with atherosclerosis a major contributor. Atherosclerotic plaque buildup is caused by an impaired lipid metabolism in macrophage foam cells. Liver X Receptor agonists (LXR) have the potential to restore macrophage cholesterol efflux by increasing the expression of ABCA1 cholesterol transporters. However, translation of LXR is limited by systemic toxicity. We hypothesized attaching LXR to peptide amphiphiles (PAs) via niche-cleavable linkages, and subsequent co-assembly with plaque-targeting PAs, would allow targeted, controlled delivery that reduces LXR toxicity while retaining its therapeutic effects.
Methods: We investigated two linkages for tethering LXR, reactive oxygen species (ROS)-cleavable prolines and glutathione-cleavable succinimidyl 3-(2-pyridyldithio)propionate (SPDP). We attached the LXR to PAs using solid phase peptide synthesis and characterized the PAs for LXR release. To allow targeted delivery to atherosclerotic lesions, we co-assembled the niche-responsive LXR PAs with PAs containing apolipoprotein A1-derived peptide 4F (ApoA1-LXR PAs), and determined processing parameters that enabled nanofiber formation. Lastly, we evaluated the effect of ApoA1-LXR PAs on macrophage viability and cholesterol efflux in vitro.
Results: We found 46% cleavage of ROS-LXR PAs after 24 hours of treatment with 250 μM H2O2 + CuSO4, and 100% cleavage by 4 days. In comparison, the SPDP-LXR PAs showed 100% cleavage in response to 24 hours of treatment with 10 mM glutathione. At 10 mol%, the ROS-LXR PAs formed nanofibers upon co-assembly with 40 mol% ApoA1 PAs and 50 mol% diluent PA after 24 hours of aging at 4°C. Similarly, the SPDP-LXR PAs formed the best nanofibers at 10 mol% co-assembly, yet required annealing at 80°C for 30 minutes. Both the ApoA1-ROS-LXR and ApoA1-SPDP-LXR PAs were able to promote macrophage cholesterol efflux (>70%) to a similar extent as free LXR at concentrations >32 μM LXR (p>0.53) while improving cell viability by at least 3-fold.
Conclusion: Our results demonstrate tethering LXR to bioresponsive PAs has the potential to increase the safety of LXR to alleviate plaque burden, and supports the use of PA nanofibers for atherosclerosis nanomedicine.
Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patenc... more Small-diameter expanded polytetrafluoroethylene (ePTFE) graft surfaces have poor long-term patency due to limited endothelial cell (EC) coverage and anastomotic intimal hyperplasia. Multifunctional elastomers that coat the ePTFE graft surface to promote EC adhesion while simultaneously inhibiting intimal hyperplasia are highly desirable. Poly(diol-co-citrate) (PDC), a thermoset elastomer, is biodegradable, biocompatible, and mimics vascular mechanical properties. Engineering antioxidant components into PDC polymeric structures improves biocompatibility by attenuating oxidative stress yet is limited by bioavailability. Herein, we develop a new ascorbate protection and deprotection strategy (APDS) for loading bioactive ascorbic acid into the structure of PDC elastomers to improve poly(1,8-octanediol-co-citrate-co-ascorbate) (POCA) prepolymer ascorbate activity. Elastomers cured from APDS POCA prepolymers provide twice the active ascorbate sites on the elastomer surface (35.19 ± 1.64 ng mg–1 cm–2) versus unprotected POCA (Un.POCA, 18.31 ± 0.97 ng mg–1 cm–2). APDS POCA elastomers displayed suitable mechanical properties for vascular graft coating [Young’s modulus (2.15–2.61 MPa), elongation (189.5–214.6%) and ultimate tensile strength (2.73–3.61 MPa)], and superior surface antioxidant performance through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging and lipid peroxidation inhibition as compared to poly(1,8-octanediol-co-citrate) (POC) and Un.POCA. Hydrolytic degradation of APDS POCA occurred within 12 weeks under physiological conditions with a mass loss of 25.8 ± 3.4% and the degradation product retaining ascorbate activity. APDS POCA elastomer surfaces supported human aortic endothelial cell proliferation while inhibiting human aortic smooth muscle cell proliferation in vitro. APDS POCA elastomer surfaces displayed superior decomposition of S-nitrosothiols compared to POC and Un.POCA. Taken together, these findings indicate the potential of APDS POCA elastomers to serve as bioactive, therapeutic coatings that enhance the long-term patency of small diameter ePTFE grafts.
Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths.... more 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.
Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. Howeve... more 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.
Coassembled peptide amphiphile nanofibers designed to target atherosclerotic plaque and enhance c... more Coassembled peptide amphiphile nanofibers designed to target atherosclerotic plaque and enhance cholesterol efflux are shown to encapsulate and deliver a liver X receptor agonist to increase efflux from murine macrophages in vitro. Fluorescence microscopy reveals that the nanofibers, which display an apolipoprotein-mimetic peptide, localize at plaque sites in low density lipoprotein receptor knockout (LDLR KO) mice with or without the encapsulated molecule, while nanofibers displaying a scrambled, nontargeting peptide sequence do not demonstrate comparable binding. These results show that nanofibers functionalized with apolipoprotein-mimetic peptides may be effective vehicles for intravascular targeted drug delivery to treat atherosclerosis.
The recently developed synthetic oligonucleotides referred to as “Click” Nucleic Acids (CNAs) are... more The recently developed synthetic oligonucleotides referred to as “Click” Nucleic Acids (CNAs) are promising due to their relatively simple synthesis based on thiol-X reactions with numerous potential applications in biotechnology, biodetection, gene silencing, and drug delivery. Here, the cytocompatibility and cellular uptake of rhodamine tagged, PEGylated CNA copolymers (PEG-CNA-RHO) were evaluated. NIH 3T3 fibroblast cells treated for 1 hour with 1, 10 or 100 μg/mL PEG-CNA-RHO maintained an average cell viability of 86%, which was not significantly different from the untreated control. Cellular uptake of PEG-CNA-RHO was detected within 30 seconds and the amount internalized increased over the course of 1 hour. Moreover, these copolymers were internalized within cells to a higher degree than controls consisting of either rhodamine tagged PEG or the rhodamine alone. Uptake was not affected by temperature (i.e., 4°C or 37°C), suggesting a passive uptake mechanism. Subcellular colocalization analysis failed to indicate significant correlations between the internalized PEG-CNA-RHO and the organelles examined (mitochondria, endoplasmic reticulum, endosomes and lysosomes). These results indicate that CNA copolymers are cytocompatible and are readily internalized by cells, supporting the idea that CNAs are a promising alternative to DNA in antisense therapy applications.
Self‐assembled microvasculature from cocultures of endothelial cells and stromal cells have signi... more Self‐assembled microvasculature from cocultures of endothelial cells and stromal cells have significantly advanced efforts to vascularize engineered tissues by enhancing perfusion rates in vivo and producing investigative platforms for microvascular morphogenesis in vitro. However, in order to clinically translate prevascularized constructs, the issue of endothelial cell source must be resolved. Endothelial progenitor cells (EPCs) can be noninvasively supplied from the recipient through adult peripheral and umbilical cord blood, as well as derived from induced pluripotent stem cells, alleviating antigenicity issues. EPCs can also differentiate into all tissue endothelium, and have demonstrated potential for therapeutic vascularization. Yet, EPCs are not the standard endothelial cell choice to vascularize tissue constructs in vitro. Possible reasons include unresolved issues with EPC identity and characterization, as well as uncertainty in the selection of coculture, scaffold, and culture media combinations that promote EPC microvessel formation. This review addresses these issues through a summary of EPC vascular biology and the effects of tissue engineering design parameters upon EPC microvessel formation. Also included are perspectives to integrate EPCs with emerging technologies to produce functional, organotypic vascularized tissues.
In this study, we tested the hypotheses that endothelial cells (ECs) derived from human umbilical... more 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.
The development of stable, functional microvessels remains an important obstacle to overcome for ... more 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 polyethylene glycol 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-β 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 represents a promising cell source for pro-angiogenic therapies. The present... more 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-β, 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.
Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell so... more Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for an-giogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular en-dothelial growth factor (VEGF)-A or changes in TGF-b1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45 + cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements , however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression , as evidenced by a lack of significant differences in total segment length (p = 0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45 + cells and improves upon the addition of soluble growth factors.
Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent ... more Coculture of endothelial cells (ECs) and smooth muscle cells (SMCs) in vitro can yield confluent monolayers or EC networks. Factors influencing this transition are not known. In this study, we examined whether the spatial arrangement of EC-SMC cocultures affected EC migration, network morphology, and angiogenic protein secretion. Human umbilical cord blood–derived ECs (hCB-ECs) were grown in coculture with human aortic SMCs in either a mixed or lamellar spatial geometry and analyzed over a culture period of 12 days. The hCB-ECs cultured on SMCs in a mixed system had higher cell speeds, shorter persistence times, and lower random motility coefficients than ECs in a lamellar system. By day 12 of coculture, mixed systems demonstrated greater anastomoses and capillary loop formation than lamellar systems as evidenced by a higher number of branch points, angle of curvature between branch points, and percentage of imaged area covered by networks. The network morphology was more uniform in the mixed systems than the lamellar systems with fewer EC clusters present after several days in culture. Proliferation of hCB-ECs was higher for mixed cocultures during the first 24 h of coculture, and then declined dramatically suggesting that proliferation only contributed to network formation during the early stages of coculture. Proteome assay results show reduced solution levels, but no change in intracellular levels of angiogenic proteins in lamellar systems compared to mixed systems. These data suggest that mixing ECs and SMCs together favors the formation of EC networks to a greater extent than a lamellar arrangement in which ECs form a cell layer above a confluent, quiescent layer of SMCs.
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Papers by Erica Peters
Methods: We investigated two linkages for tethering LXR, reactive oxygen species (ROS)-cleavable prolines and glutathione-cleavable succinimidyl 3-(2-pyridyldithio)propionate (SPDP). We attached the LXR to PAs using solid phase peptide synthesis and characterized the PAs for LXR release. To allow targeted delivery to atherosclerotic lesions, we co-assembled the niche-responsive LXR PAs with PAs containing apolipoprotein A1-derived peptide 4F (ApoA1-LXR PAs), and determined processing parameters that enabled nanofiber formation. Lastly, we evaluated the effect of ApoA1-LXR PAs on macrophage viability and cholesterol efflux in vitro.
Results: We found 46% cleavage of ROS-LXR PAs after 24 hours of treatment with 250 μM H2O2 + CuSO4, and 100% cleavage by 4 days. In comparison, the SPDP-LXR PAs showed 100% cleavage in response to 24 hours of treatment with 10 mM glutathione. At 10 mol%, the ROS-LXR PAs formed nanofibers upon co-assembly with 40 mol% ApoA1 PAs and 50 mol% diluent PA after 24 hours of aging at 4°C. Similarly, the SPDP-LXR PAs formed the best nanofibers at 10 mol% co-assembly, yet required annealing at 80°C for 30 minutes. Both the ApoA1-ROS-LXR and ApoA1-SPDP-LXR PAs were able to promote macrophage cholesterol efflux (>70%) to a similar extent as free LXR at concentrations >32 μM LXR (p>0.53) while improving cell viability by at least 3-fold.
Conclusion: Our results demonstrate tethering LXR to bioresponsive PAs has the potential to increase the safety of LXR to alleviate plaque burden, and supports the use of PA nanofibers for atherosclerosis nanomedicine.
Methods: We investigated two linkages for tethering LXR, reactive oxygen species (ROS)-cleavable prolines and glutathione-cleavable succinimidyl 3-(2-pyridyldithio)propionate (SPDP). We attached the LXR to PAs using solid phase peptide synthesis and characterized the PAs for LXR release. To allow targeted delivery to atherosclerotic lesions, we co-assembled the niche-responsive LXR PAs with PAs containing apolipoprotein A1-derived peptide 4F (ApoA1-LXR PAs), and determined processing parameters that enabled nanofiber formation. Lastly, we evaluated the effect of ApoA1-LXR PAs on macrophage viability and cholesterol efflux in vitro.
Results: We found 46% cleavage of ROS-LXR PAs after 24 hours of treatment with 250 μM H2O2 + CuSO4, and 100% cleavage by 4 days. In comparison, the SPDP-LXR PAs showed 100% cleavage in response to 24 hours of treatment with 10 mM glutathione. At 10 mol%, the ROS-LXR PAs formed nanofibers upon co-assembly with 40 mol% ApoA1 PAs and 50 mol% diluent PA after 24 hours of aging at 4°C. Similarly, the SPDP-LXR PAs formed the best nanofibers at 10 mol% co-assembly, yet required annealing at 80°C for 30 minutes. Both the ApoA1-ROS-LXR and ApoA1-SPDP-LXR PAs were able to promote macrophage cholesterol efflux (>70%) to a similar extent as free LXR at concentrations >32 μM LXR (p>0.53) while improving cell viability by at least 3-fold.
Conclusion: Our results demonstrate tethering LXR to bioresponsive PAs has the potential to increase the safety of LXR to alleviate plaque burden, and supports the use of PA nanofibers for atherosclerosis nanomedicine.