Enzyme-Responsive Nanoparticles for the Targeted Delivery of an MMP Inhibitor to Acute Myocardial Infarction.

Herein, we have developed a drug-loaded matrix metalloproteinase (MMP)-responsive micellar nanoparticle (NP) intended for minimally invasive intravenous injection during the acute phase of myocardial infarction (MI) and prolonged retention in the heart for small-molecule drug delivery. Peptide-polymer amphiphiles (PPAs) bearing a small-molecule MMP inhibitor (MMPi), PD166793, were synthesized via ring-opening metathesis polymerization (ROMP) and formulated into spherical micelles by transitioning to aqueous solution. The resulting micellar NPs underwent MMP-induced aggregation, demonstrating enzyme responsiveness. Using a rat MI model, we observed that these NPs were capable of successfully extravasating into the infarcted region of the heart where they were retained due to the active, enzyme-mediated targeting, remaining detectable after 1 week post administration without increasing macrophage recruitment. Furthermore, in vitro studies show that these NPs demonstrated successful drug release following MMP treatment and maintained drug bioactivity as evidenced by comparable MMP inhibition to free MMPi. This work establishes a targeted NP platform for delivering small-molecule therapeutics to the heart after MI, opening possibilities for myocardial infarction treatment.

Inflammation-Responsive Micellar Nanoparticles from Degradable Polyphosphoramidates for Targeted Delivery to Myocardial Infarction.

Nanoparticles that undergo a localized morphology change to target areas of inflammation have been previously developed but are limited by their lack of biodegradability. In this paper, we describe a low-ring-strain cyclic olefin monomer, 1,3-dimethyl-2-phenoxy-1,3,4,7-tetrahydro-1,3,2-diazaphosphepine 2-oxide (MePTDO), that rapidly polymerizes via ring-opening metathesis polymerization at room temperature to generate well-defined degradable polyphosphoramidates with high monomer conversion (>84%). Efficient MePTDO copolymerizations with norbornene-based monomers are demonstrated, including a norbornenyl monomer functionalized with a peptide substrate for inflammation-associated matrix metalloproteinases (MMPs). The resulting amphiphilic peptide brush copolymers self-assembled in aqueous solution to generate micellar nanoparticles (30 nm in diameter) which exhibit excellent cyto- and hemocompatibility and undergo MMP-induced assembly into micron-scale aggregates. As MMPs are upregulated in the heart postmyocardial infarction (MI), the MMP-responsive micelles were applied to target and accumulate in the infarcted heart following intravenous administration in a rat model of MI. These particles displayed a distinct biodistribution and clearance pattern in comparison to nondegradable analogues. Specifically, accumulation at the site of MI competed with elimination predominantly through the kidney rather than the liver. Together, these results suggest this as a promising new biodegradable platform for inflammation targeted delivery.

Intravascularly infused extracellular matrix as a biomaterial for targeting and treating inflamed tissues.

Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Myocardial matrix hydrogel acts as a reactive oxygen species scavenger and supports a proliferative microenvironment for cardiomyocytes.

As the native regenerative potential of adult cardiac tissue is limited post-injury, stimulating endogenous repair mechanisms in the mammalian myocardium is a potential goal of regenerative medicine therapeutics. Injection of myocardial matrix hydrogels into the heart post-myocardial infarction (MI) has demonstrated increased cardiac muscle and promotion of pathways associated with cardiac development, suggesting potential promotion of cardiomyocyte turnover. In this study, the myocardial matrix hydrogel was shown to have native capability as an effective reactive oxygen species scavenger and protect against oxidative stress induced cell cycle inhibition in vitro. Encapsulation of cardiomyocytes demonstrated an enhanced turnover in in vitro studies, and in vivo assessments of myocardial matrix hydrogel treatment post-MI showed increased thymidine analog uptake in cardiomyocyte nuclei compared to saline controls. Overall, this study provides evidence that properties of the myocardial matrix material provide a microenvironment mitigating oxidative damage and supportive of cardiomyocytes undergoing DNA synthesis, toward possible DNA repair or cell cycle activation. STATEMENT OF SIGNIFICANCE: Loss of adult mammalian cardiomyocyte turnover is influenced by shifts in oxidative damage, which represents a potential mechanism for improving restoration of cardiac muscle after myocardial infarction (MI). Injection of a myocardial matrix hydrogel into the heart post-MI previously demonstrated increased cardiac muscle and promotion of pathways associated with cardiac development, suggesting potential in promoting proliferation of cardiomyocytes. In this study, the myocardial matrix hydrogel was shown to protect cells from oxidative stress and increase proliferation in vitro. In a rat MI model, greater presence of tissue free thiol content spared from oxidative damage, lesser mitochondrial superoxide content, and increased thymidine analog uptake in cardiomyocytes was found in matrix injected animals compared to saline controls. Overall, this study provides evidence that properties of the myocardial matrix material provide a microenvironment supportive of cardiomyocytes undergoing DNA synthesis, toward possible DNA repair or cell cycle activation.

Injectable Myocardial Matrix Hydrogel Mitigates Negative Left Ventricular Remodeling in a Chronic Myocardial Infarction Model.

A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.

Development of a Translatable Ultrasound Molecular Imaging Agent for Inflammation.

This study details the development, characterization and non-clinical efficacy of an ultrasound molecular imaging agent intended for molecular imaging of P-selectin in humans. A targeting ligand based on a recently discovered human selectin ligand was manufactured as fusion protein, and activity for human and mouse P- and E-selectin was evaluated by functional immunoassay. The targeting ligand was covalently conjugated to a lipophilic anchor inserted into a phospholipid microbubble shell. Three lots of the targeted microbubble drug product, TS-07-009, were produced, and assays for size distribution, zeta potential and morphology were established. The suitability of TS-07-009 as a molecular imaging agent was evaluated in vitro in a flow-based adhesion assay and in vivo using a canine model of transient myocardial ischemia. Selectivity for P-selectin over E-selectin was observed in both the human and murine systems. Contrast agent adhesion increased with P-selectin concentration in a dynamic adhesion assay. Significant contrast enhancement was observed on ultrasound imaging with TS-07-009 in post-ischemic canine myocardium at 30 or 90 min of re-perfusion. Negligible enhancement was observed in resting (no prior ischemia) hearts or with a control microbubble 90 min after ischemia. The microbubble contrast agent described here exhibits physiochemical properties and in vivo behavior suitable for development as a clinical imaging agent.

Enzyme-responsive progelator cyclic peptides for minimally invasive delivery to the heart post-myocardial infarction.

Injectable biopolymer hydrogels have gained attention for use as scaffolds to promote cardiac function and prevent negative left ventricular (LV) remodeling post-myocardial infarction (MI). However, most hydrogels tested in preclinical studies are not candidates for minimally invasive catheter delivery due to excess material viscosity, rapid gelation times, and/or concerns regarding hemocompatibility and potential for embolism. We describe a platform technology for progelator materials formulated as sterically constrained cyclic peptides which flow freely for low resistance injection, and rapidly assemble into hydrogels when linearized by disease-associated enzymes. Their utility in vivo is demonstrated by their ability to flow through a syringe and gel at the site of MI in rat models. Additionally, synthetic functionalization enables these materials to flow through a cardiac injection catheter without clogging, without compromising hemocompatibility or cytotoxicity. These studies set the stage for the development of structurally dynamic biomaterials for therapeutic hydrogel delivery to the MI.

Evidence for Mechanisms Underlying the Functional Benefits of a Myocardial Matrix Hydrogel for Post-MI Treatment.

BACKGROUND: There is increasing need for better therapies to prevent the development of heart failure after myocardial infarction (MI). An injectable hydrogel derived from decellularized porcine ventricular myocardium has been shown to halt the post-infarction progression of negative left ventricular remodeling and decline in cardiac function in both small and large animal models. OBJECTIVES: This study sought to elucidate the tissue-level mechanisms underlying the therapeutic benefits of myocardial matrix injection. METHODS: Myocardial matrix or saline was injected into infarcted myocardium 1 week after ischemia-reperfusion in Sprague-Dawley rats. Cardiac function was evaluated by magnetic resonance imaging and hemodynamic measurements at 5 weeks after injection. Whole transcriptome microarrays were performed on RNA isolated from the infarct at 3 days and 1 week after injection. Quantitative polymerase chain reaction and histologic quantification confirmed expression of key genes and their activation in altered pathways. RESULTS: Principal component analysis of the transcriptomes showed that samples collected from myocardial matrix-injected infarcts are distinct and cluster separately from saline-injected control subjects. Pathway analysis indicated that these differences are due to changes in several tissue processes that may contribute to improved cardiac healing after MI. Matrix-injected infarcted myocardium exhibits an altered inflammatory response, reduced cardiomyocyte apoptosis, enhanced infarct neovascularization, diminished cardiac hypertrophy and fibrosis, altered metabolic enzyme expression, increased cardiac transcription factor expression, and progenitor cell recruitment, along with improvements in global cardiac function and hemodynamics. CONCLUSIONS: These results indicate that the myocardial matrix alters several key pathways after MI creating a pro-regenerative environment, further demonstrating its promise as a potential post-MI therapy.

Degradable acetalated dextran microparticles for tunable release of an engineered hepatocyte growth factor fragment.

Injectable biomaterials are promising as new therapies to treat myocardial infarction (MI). One useful property of biomaterials is the ability to protect and sustain release of therapeutic payloads. In order to create a platform for optimizing the release rate of cardioprotective molecules we utilized the tunable degradation of acetalated dextran (AcDex). We created microparticles with three distinct degradation profiles and showed that the consequent protein release profiles could be modulated within the infarcted heart. This enabled us to determine how delivery rate impacted the efficacy of a model therapeutic, an engineered hepatocyte growth factor fragment (HGF-f). Our results showed that the cardioprotective efficacy of HGF-f was optimal when delivered over three days post-intramyocardial injection, yielding the largest arterioles, fewest apoptotic cardiomyocytes bordering the infarct and the smallest infarcts compared to empty particle treatment four weeks after injection. This work demonstrates the potential of using AcDex particles as a delivery platform to optimize the time frame for delivering therapeutic proteins to the heart.

Enzyme-Responsive Nanoparticles for Targeted Accumulation and Prolonged Retention in Heart Tissue after Myocardial Infarction.

A method for targeting to and retaining intravenously injected nanoparticles at the site of acute myocardial infarction in a rat model is described. Enzyme-responsive peptide-polymer amphiphiles are assembled as spherical micellar nanoparticles, and undergo a morphological transition from spherical-shaped, discrete materials to network-like assemblies when acted upon by matrix metalloproteinases (MMP-2 and MMP-9), which are up-regulated in heart tissue post-myocardial infarction.

Coevolution within a transcriptional network by compensatory trans and cis mutations.

Transcriptional networks have been shown to evolve very rapidly, prompting questions as to how such changes arise and are tolerated. Recent comparisons of transcriptional networks across species have implicated variations in the cis-acting DNA sequences near genes as the main cause of divergence. What is less clear is how these changes interact with trans-acting changes occurring elsewhere in the genetic circuit. Here, we report the discovery of a system of compensatory trans and cis mutations in the yeast AP-1 transcriptional network that allows for conserved transcriptional regulation despite continued genetic change. We pinpoint a single species, the fungal pathogen Candida glabrata, in which a trans mutation has occurred very recently in a single AP-1 family member, distinguishing it from its Saccharomyces ortholog. Comparison of chromatin immunoprecipitation profiles between Candida and Saccharomyces shows that, despite their different DNA-binding domains, the AP-1 orthologs regulate a conserved block of genes. This conservation is enabled by concomitant changes in the cis-regulatory motifs upstream of each gene. Thus, both trans and cis mutations have perturbed the yeast AP-1 regulatory system in such a way as to compensate for one another. This demonstrates an example of "coevolution" between a DNA-binding transcription factor and its cis-regulatory site, reminiscent of the coevolution of protein binding partners.

A systems approach to delineate functions of paralogous transcription factors: role of the Yap family in the DNA damage response.

Duplication of genes encoding transcription factors plays an essential role in driving phenotypic variation. Because regulation can occur at multiple levels, it is often difficult to discern how each duplicated factor achieves its regulatory specificity. In these cases, a "systems approach" may distinguish the role of each factor by integrating complementary large-scale measurements of the regulatory network. To explore such an approach, we integrate growth phenotypes, promoter binding profiles, and gene expression patterns to model the DNA damage response network controlled by the Yeast-specific AP-1 (YAP) family of transcription factors. This analysis reveals that YAP regulatory specificity is achieved by at least three mechanisms: (i) divergence of DNA-binding sequences into two subfamilies; (ii) condition-specific combinatorial regulation by multiple Yap factors; and (iii) interactions of Yap 1, 4, and 6 with chromatin remodeling proteins. Additional microarray experiments establish that Yap 4 and 6 regulate gene expression through interactions with the histone deacetylase, Hda1. The data further highlight differences among Yap paralogs in terms of their regulatory mode of action (activation vs. repression). This study suggests how other large TF families might be disentangled in the future.

TNF-alpha downregulates the leukotriene C4 synthase gene in mononuclear phagocytes.

We studied the effect of tumor necrosis factor (TNF)-alpha exposure on cysteinyl leukotriene (LT) synthesis by cells of monocyte/macrophage lineage. TNF-alpha conditioning of monocytic THP-1 cells and primary human monocytes resulted in a decreased capacity for LTC(4) release. TNF-alpha exposure (for 16-24 h) decreased LTC(4) synthase mRNA in THP-1 cells, primary mouse bone marrow-derived macrophages, and eosinophilic AML14.3D10 cells. TNF-alpha downregulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with downregulation observed as early as 4 h. The effect of TNF-alpha on LTC(4) synthase mRNA expression was mediated via the MEK/ERK pathway, but not via cyclooxygenase or nitric oxide synthase pathways. Conditioning of actinomycin D-treated cells with TNF-alpha did not accelerate degradation of LTC(4) synthase mRNA. TNF-alpha produced sustained activation of p50 and p65, which were previously reported by our group to decrease LTC(4) synthase promoter activity. In transiently transfected THP-1 cells, TNF-alpha decreased promoter activity via an element located within the first 620 bp of the promoter. We conclude that TNF-alpha exposure downregulates the synthetic capacity for cysteinyl LT release and LTC(4) synthase gene expression in monocytes/macrophages via a transcriptional mechanism.