Systemic peptide amphiphile nanofiber delivery following subcutaneous injection.

Peptide amphiphile (PA) nanofibers have been shown to target and deliver drugs when administered via an intravenous (IV) injection. Subcutaneous administration can broaden the applicability of PA nanofibers in the medical field. The ability of PA nanofibers to be absorbed into systemic circulation after subcutaneous administration was investigated. Four PA molecules with different amino acid sequences were designed to understand the effect of nanofiber cohesion and charge on uptake. Solution small-angle X-ray scattering confirmed nanostructure morphology and provided characteristic lengths for co-assemblies. Circular dichroism and solution wide-angle X-ray scattering confirmed PA secondary structure and molecular order. PAs were co-assembled in a 95 %:5 % molar ratio of unlabeled PA to fluorescently labeled PA. Male and female Sprague Dawley rats were injected in the nape of the neck with PA co-assemblies. In vivo normalized abdominal fluorescence was measured 1-72 h after injection. PA nanofibers with a negative charge and low internal order showed the highest amount of systemic absorption at 1, 6, and 24 h. At 24 h after injection, white blood cell count decreased and glucose was elevated. Glucose began to decrease at 48 h. These data indicate that PA nanofibers can be absorbed into the systemic circulation after subcutaneous injection.

Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Self-Assembled Peptide Amphiphile Nanofibers for Controlled Therapeutic Delivery to the Atherosclerotic Niche.

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 were 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 released LXR in response to physiological levels of ROS and reducing agents and could 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 targeted plaque within an atherosclerotic mouse model in vivo and activated 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.

Coating small-diameter ePTFE vascular grafts with tunable poly(diol-co-citrate-co-ascorbate) elastomers to reduce neointimal hyperplasia.

Lack of long-term patency has hindered the clinical use of small-diameter prosthetic vascular grafts with the majority of these failures due to the development of neointimal hyperplasia. Previous studies by our laboratory revealed that small-diameter expanded polytetrafluoroethylene (ePTFE) grafts coated with antioxidant elastomers are a promising localized therapy to inhibit neointimal hyperplasia. This work is focused on the development of poly(diol-co-citrate-co-ascorbate) (POCA) elastomers with tunable properties for coating ePTFE vascular grafts. A bioactive POCA elastomer (@20 : 20 : 8, [citrate] : [diol] : [ascorbate]) coating was applied on a 1.5 mm diameter ePTFE vascular graft as the most promising therapeutic candidate for reducing neointimal hyperplasia. Surface ascorbate density on the POCA elastomer was increased to 67.5 ± 7.3 ng mg-1 cm-2. The mechanical, antioxidant, biodegradable, and biocompatible properties of POCA demonstrated desirable performance for in vivo use, inhibiting human aortic smooth muscle cell proliferation, while supporting human aortic endothelial cells. POCA elastomer coating number was adjusted by a modified spin-coating method to prepare small-diameter ePTFE vascular grafts similar to natural vessels. A significant reduction in neointimal hyperplasia was observed after implanting POCA-coated ePTFE vascular grafts in a guinea pig aortic interposition bypass graft model. POCA elastomer thus offers a new avenue that shows promise for use in vascular engineering to improve long-term patency rates by coating small-diameter ePTFE vascular grafts.

Intravenous Delivery of Lung-Targeted Nanofibers for Pulmonary Hypertension in Mice.

Pulmonary hypertension is a highly morbid disease with no cure. Available treatments are limited by systemic adverse effects due to non-specific biodistribution. Self-assembled peptide amphiphile (PA) nanofibers are biocompatible nanomaterials that can be modified to recognize specific biological markers to provide targeted drug delivery and reduce off-target toxicity. Here, PA nanofibers that target the angiotensin I-converting enzyme and the receptor for advanced glycation end-products (RAGE) are developed, as both proteins are overexpressed in the lung with pulmonary hypertension. It is demonstrated that intravenous delivery of RAGE-targeted nanofibers containing the targeting epitope LVFFAED (LVFF) significantly accumulated within the lung in a chronic hypoxia-induced pulmonary hypertension mouse model. Using 3D light sheet fluorescence microscopy, it is shown that LVFF nanofiber localization is specific to the diseased pulmonary tissue with immunofluorescence analysis demonstrating colocalization of the targeted nanofiber to RAGE in the hypoxic lung. Furthermore, biodistribution studies show that significantly more LVFF nanofibers localized to the lung compared to major off-target organs. Targeted nanofibers are retained within the pulmonary tissue for 24 h after injection. Collectively, these data demonstrate the potential of a RAGE-targeted nanomaterial as a drug delivery platform to treat pulmonary hypertension.

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.

A comparative study of a preclinical survival model of smoke inhalation injury in mice and rats.

Smoke inhalation injury increases morbidity and mortality. Clinically relevant animal models are necessary for the continued investigation of the pathophysiology of inhalation injury and the development of therapeutics. The goal of our research was threefold: 1) to develop a reproducible survival model of smoke inhalation injury in rats that closely resembled our previous mouse model, 2) to validate the rat smoke inhalation injury model using a variety of laboratory techniques, and 3) to compare and contrast our rat model with both the well-established mouse model and previously published rat models to highlight our improvements on smoke delivery and lung injury. Mice and rats were anesthetized, intubated, and placed in custom-built smoke chambers to passively inhale woodchip-generated smoke. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for confirmatory tests. Lung sections were hematoxylin and eosin stained, lung edema was assessed with wet-to-dry (W/D) ratio, and inflammatory cell infiltration and cytokine elevation were evaluated using flow cytometry, immunohistochemistry, and ELISA. We confirmed that our mouse and rat models of smoke inhalation injury mimic the injury seen after human burn inhalation injury with evidence of pulmonary edema, neutrophil infiltration, and inflammatory cytokine elevation. Interestingly, rats mounted a more severe immunological response compared with mice. In summary, we successfully validated a reliable and clinically translatable survival model of lung injury and immune response in rats and mice and characterized the extent of this injury. These animal models allow for the continued study of smoke inhalation pathophysiology to ultimately develop a better therapeutic.

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.

Development of Fractalkine-Targeted Nanofibers that Localize to Sites of Arterial Injury.

Atherosclerosis is the leading cause of death and disability around the world, with current treatments limited by neointimal hyperplasia. Our goal was to synthesize, characterize, and evaluate an injectable, targeted nanomaterial that will specifically bind to the site of arterial injury. Our target protein is fractalkine, a chemokine involved in both neointimal hyperplasia and atherosclerosis. We showed increased fractalkine staining in rat carotid arteries 24 h following arterial injury and in the aorta of low-density lipoprotein receptor knockout (LDLR-/-) mice fed a high-fat diet for 16 weeks. Three peptide amphiphiles (PAs) were synthesized: fractalkine-targeted, scrambled, and a backbone PA. PAs were ≥90% pure on liquid chromatography/mass spectrometry (LCMS) and showed nanofiber formation on transmission electron microscopy (TEM). Rats systemically injected with fractalkine-targeted nanofibers 24 h after carotid artery balloon injury exhibited a 4.2-fold increase in fluorescence in the injured artery compared to the scrambled nanofiber (p < 0.001). No localization was observed in the non-injured artery or with the backbone nanofiber. Fluorescence of the fractalkine-targeted nanofiber increased in a dose dependent manner and was observed for up to 48 h. These data demonstrate the presence of fractalkine after arterial injury and the localization of our fractalkine-targeted nanofiber to the site of injury and serve as the foundation to develop this technology further.

The Occurrence and Characterization of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolated from Clinical Diagnostic Specimens of Equine Origin.

Escherichia coli isolates were recovered from clinical specimens of equine patients admitted to the Texas A&M Veterinary Medical Teaching Hospital over a five-year period. Ceftiofur resistance was used as a marker for potential extended-spectrum beta-lactamase (ESBL)-activity, and of the 48 ceftiofur-resistant E. coli isolates, 27.08% (n = 13) were phenotypically ESBL-positive. Conventional PCR analysis followed by the large-scalebla Finder multiplex PCR detected the ESBL genes, CTX-M-1 and SHV, in seven out of the 13 isolates. Moreover, beta-lactamase genes of TEM-1-type, BER-type (AmpC), and OXA-type were also identified. Sequencing of these genes resulted in identification of a novel TEM-1-type gene, called blaTEM-233, and a study is currently underway to determine if this gene confers the ESBL phenotype. Furthermore, this report is the first to have found E. coli ST1308 in horses. This subtype, which has been reported in other herbivores, harbored the SHV-type ESBL gene. Finally, one out of 13 E. coli isolates was PCR-positive for the carbapenemase gene, blaIMP-1 despite the lack of phenotypically proven resistance to imipenem. With the identification of novel ESBL gene variant and the demonstrated expansion of E. coli sequence types in equine patients, this study underscores the need for more investigation of equines as reservoirs for ESBL-producing pathogens.

Phenotypic and genotypic characteristics of Trueperella pyogenes isolated from ruminants.

Trueperella pyogenes is an opportunistic pathogen that causes suppurative infections in animals including humans. Data on phenotypic and genotypic properties of T. pyogenes isolated from ruminants, particularly goats and sheep, are lacking. We characterized, by phenotypic and genotypic means, T. pyogenes of caprine and ovine origin, and established their phylogenetic relationship with isolates from other ruminants. T. pyogenes isolates ( n = 50) from diagnostic specimens of bovine ( n = 25), caprine ( n = 19), and ovine ( n = 6) origin were analyzed. Overall, variable biochemical activities were observed among the T. pyogenes isolates. The fimbriae-encoding gene, fimE, and neuraminidase-encoding gene, nanH, were, respectively, more frequently detected in the large ( p = 0.0006) and small ( p = 0.0001) ruminant isolates. Moreover, genotype V ( plo/ nanH/ nanP/ fimA/ fimC) was only detected in the caprine and ovine isolates, whereas genotype IX ( plo/ nanP/ fimA/ fimC/ fimE) was solely present in the isolates of bovine origin ( p = 0.0223). The 16S rRNA gene sequences of all T. pyogenes isolates were clustered with the reference T. pyogenes strain ATCC 19411 and displayed a high degree of identity to each other. Our results highlight phenotypic and genotypic diversity among ruminant isolates of T. pyogenes and reinforce the importance of characterization of more clinical isolates to better understand the pathogenesis of this bacterium in different animal species.

Diversity of Borrelia spirochetes and other zoonotic agents in ticks from Kyiv, Ukraine.

Lyme borreliosis (LB) is caused by tick-borne spirochetes of the Borrelia burgdorferi sensu lato complex. LB is the most prevalent vector-borne illness in Ukraine, but current data on the prevalence of LB pathogens in their tick vector, Ixodes ricinus, are lacking. I. ricinus ticks may also carry Borrelia miyamotoi, an emerging relapsing fever group spirochete that has been implicated in human illness. Despite its zoonotic potential, the prevalence of B. miyamotoi in ticks has not been examined in Ukraine. Similarly, data on the prevalence of other important tick-borne pathogens, Anaplasma phagocytophilum, Babesia spp., Bartonella spp., Francisella tularensis, and Rickettsia spp., in ixodid ticks are scarce or even absent. Thus, the overall objective of this study was to investigate the prevalence of these tick-borne pathogens in questing I. ricinus and Dermacentor reticulatus ticks collected in recreational parks of Kyiv, the most densely populated city of Ukraine. A total of 182 adult I. ricinus, 98 nymphal I. ricinus, and 98 adult D. reticulatus ticks were molecularly analyzed for the presence of these pathogens. As a result, the study shows a greater diversity of Borrelia genospecies in questing I. ricinus ticks than previously reported. The most prevalent genospecies in adult I. ricinus ticks were B. afzelii (7.7%), followed by B. burgdorferi sensu stricto (s.s.) (2.2%) and B. garinii (0.5%). In contrast, B. burgdorferi s.s. was most dominant in unfed I. ricinus nymphs (67.3%). Moreover, B. afzelii was detected in 11.2% of nymphs, but only 1.0% of nymphal ticks were positive for B. garinii and B. valaisiana. Importantly, this study provides the first record of B. miyamotoi detected in I. ricinus ticks from Ukraine (1.1%). Furthermore, the report is also the first to document other vector-borne pathogens, Bartonella henselae, Rickettsia conorii, and Rickettsia mendelii, in ixodid ticks from Ukraine. In summary, this work offers the latest data on the diversity and prevalence of the important zoonotic tick-borne agents in questing ticks from Kyiv, Ukraine. The data will help to better gauge the risk associated with vector-borne infections to which residents and guests of Ukraine's capital may be exposed.

Antibody Response to Lyme Disease Spirochetes in the Context of VlsE-Mediated Immune Evasion.

Lyme disease (LD), the most prevalent tick-borne illness in North America, is caused by Borrelia burgdorferi The long-term survival of B. burgdorferi spirochetes in the mammalian host is achieved though VlsE-mediated antigenic variation. It is mathematically predicted that a highly variable surface antigen prolongs bacterial infection sufficiently to exhaust the immune response directed toward invariant surface antigens. If the prediction is correct, it is expected that the antibody response to B. burgdorferi invariant antigens will become nonprotective as B. burgdorferi infection progresses. To test this assumption, changes in the protective efficacy of the immune response to B. burgdorferi surface antigens were monitored via a superinfection model over the course of 70 days. B. burgdorferi-infected mice were subjected to secondary challenge by heterologous B. burgdorferi at different time points postinfection (p.i.). When the infected mice were superinfected with a VlsE-deficient clone (ΔVlsE) at day 28 p.i., the active anti-B. burgdorferi immune response did not prevent ΔVlsE-induced spirochetemia. In contrast, most mice blocked culture-detectable spirochetemia induced by wild-type B. burgdorferi (WT), indicating that VlsE was likely the primary target of the antibody response. As the B. burgdorferi infection further progressed, however, reversed outcomes were observed. At day 70 p.i. the host immune response to non-VlsE antigens became sufficiently potent to clear spirochetemia induced by ΔVlsE and yet failed to prevent WT-induced spirochetemia. To test if any significant changes in the anti-B. burgdorferi antibody repertoire accounted for the observed outcomes, global profiles of antibody specificities were determined. However, comparison of mimotopes revealed no major difference between day 28 and day 70 antibody repertoires.

Nebulin binding impedes mutant desmin filament assembly.

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160-164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.