The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.

miR-145 micelles mitigate atherosclerosis by modulating vascular smooth muscle cell phenotype.

In atherosclerosis, resident vascular smooth muscle cells (VSMCs) in the blood vessels become highly plastic and undergo phenotypic switching from the quiescent, contractile phenotype to the migratory and proliferative, synthetic phenotype. Additionally, recent VSMC lineage-tracing mouse models of atherosclerosis have found that VSMCs transdifferentiate into macrophage-like and osteochondrogenic cells and make up to 70% of cells found in atherosclerotic plaques. Given VSMC phenotypic switching is regulated by microRNA-145 (miR-145), we hypothesized that nanoparticle-mediated delivery of miR-145 to VSMCs has the potential to mitigate atherosclerosis development by inhibiting plaque-propagating cell types derived from VSMCs. To test our hypothesis, we synthesized miR-145 micelles targeting the C-C chemokine receptor-2 (CCR2), which is highly expressed on synthetic VSMCs. When miR-145 micelles were incubated with human aortic VSMCs in vitro, >90% miR-145 micelles escaped the lysosomal pathway in 4 hours and released the miR cargo under cytosolic levels of glutathione, an endogenous reducing agent. As such, miR-145 micelles rescued atheroprotective contractile markers, myocardin, α-SMA, and calponin, in synthetic VSMCs in vitro. In early-stage atherosclerotic ApoE-/- mice, one dose of miR-145 micelles prevented lesion growth by 49% and sustained an increased level of miR-145 expression after 2 weeks post-treatment. Additionally, miR-145 micelles inhibited 35% and 43% plaque growth compared to free miR-145 and PBS, respectively, in mid-stage atherosclerotic ApoE-/- mice. Collectively, we present a novel therapeutic strategy and cell target for atherosclerosis, and present miR-145 micelles as a viable nanotherapeutic that can intervene atherosclerosis progression at both early and later stages of disease.

CCR2-targeted micelles for anti-cancer peptide delivery and immune stimulation.

Signaling between the CC chemokine receptor 2 (CCR2) with its ligand, monocyte chemoattractant protein-1 (MCP-1) promotes cancer progression by directly stimulating tumor cell proliferation and downregulating the expression of apoptotic proteins. Additionally, the MCP-1/CCR2 signaling axis drives the migration of circulating monocytes into the tumor microenvironment, where they mature into tumor-associated macrophages (TAMs) that promote disease progression through induction of angiogenesis, tissue remodeling, and suppression of the cytotoxic T lymphocyte (CTL) response. In order to simultaneously disrupt MCP-1/CCR2 signaling and target CCR2-expressing cancer cells for drug delivery, KLAK-MCP-1 micelles consisting of a CCR2-targeting peptide sequence (MCP-1 peptide) and the apoptotic KLAKLAK peptide were synthesized. In vitro, KLAK-MCP-1 micelles were observed to bind and induce cytotoxicity to cancer cells through interaction with CCR2. In vivo, KLAK-MCP-1 micelles inhibited tumor growth (34 ± 11%) in a subcutaneous B16F10 murine melanoma model despite minimal tumor accumulation upon intravenous injection. Tumors treated with KLAK-MCP1 demonstrated reduced intratumor CCR2 expression and altered infiltration of TAMs and CTLs as evidenced by immunohistochemical and flow cytometric analysis. These studies highlight the potential application of CCR2-targeted nanotherapeutic micelles in cancer treatment.

Collagenase-Cleavable Peptide Amphiphile Micelles as a Novel Theranostic Strategy in Atherosclerosis.

Atherosclerosis is an inflammatory disease characterized by plaques that can cause sudden myocardial infarction upon rupture. Such rupture-prone plaques have thin fibrous caps due to collagenase degradation, and a noninvasive diagnostic tool and targeted therapy that can identify and treat vulnerable plaques and may inhibit the onset of acute cardiac events. Toward this goal, monocyte-binding, collagenase-inhibiting, and gadolinium-modified peptide amphiphile micelles (MCG PAMs) are developed. Monocyte chemoattractant protein-1 (MCP-1) binds to C-C chemokine receptor-2 expressed on pathological cell types present within plaques. Through the peptide binding motif of MCP-1, MCG PAMs bind to monocytes and vascular smooth muscle cells in vitro. Moreover, using magnetic resonance imaging, MCG PAMs show enhanced targeting and successful detection of plaques in diseased mice in vivo and act as contrast agents for molecular imaging. Through the collagenase-cleaving peptide sequence of collagen [VPMS-MRGG], MCG PAMs can compete for collagenases that degrade the fibrous cap of plaques, providing therapy. MCG PAM-treated mice show increased fibrous cap thickness by 61% and 113% histologically compared to nontargeting micelle- or PBS-treated mice (p = 0.0075 and 0.001, respectively). Overall, this novel multimodal nanoparticle offers new theranostic opportunities for noninvasive diagnosis and treatment of atherosclerotic plaques.

Hybrid, metal oxide-peptide amphiphile micelles for molecular magnetic resonance imaging of atherosclerosis.

BACKGROUND: Atherosclerosis, a major source of cardiovascular disease, is asymptomatic for decades until the activation of thrombosis and the rupture of enlarged plaques, resulting in acute coronary syndromes and sudden cardiac arrest. Magnetic resonance imaging (MRI) is a noninvasive nuclear imaging technique to assess the degree of atherosclerotic plaque with high spatial resolution and excellent soft tissue contrast. However, MRI lacks sensitivity for preventive medicine, which limits the ability to observe the onset of vulnerable plaques. In this study, we engineered hybrid metal oxide-peptide amphiphile micelles (HMO-Ms) that combine an inorganic, magnetic iron oxide or manganese oxide inner core with organic, fibrin-targeting peptide amphiphiles, consisting of the sequence CREKA, for potential MRI imaging of thrombosis on atherosclerotic plaques. RESULTS: Hybrid metal oxide-peptide amphiphile micelles, consisting of an iron oxide (Fe-Ms) or manganese oxide (Mn-Ms) core with CREKA peptides, were self-assembled into 20-30 nm spherical nanoparticles, as confirmed by dynamic light scattering and transmission electron microscopy. These hybrid nanoparticles were found to be biocompatible with human aortic endothelial cells in vitro, and HMO-Ms bound to human clots three to five times more efficiently than its non-targeted counterparts. Relaxivity studies showed ultra-high r2 value of 457 mM-1 s-1 and r1 value of 0.48 mM-1 s-1 for Fe-Ms and Mn-Ms, respectively. In vitro, MR imaging studies demonstrated the targeting capability of CREKA-functionalized hybrid nanoparticles with twofold enhancement of MR signals. CONCLUSION: This novel hybrid class of MR agents has potential as a non-invasive imaging method that specifically detects thrombosis during the pathogenesis of atherosclerosis.

Shape Effects of Peptide Amphiphile Micelles for Targeting Monocytes.

Peptide amphiphile micelles (PAMs) are a nanoparticle platform that have gained popularity for their targeting versatility in a wide range of disease models. An important aspect of micelle design is considering the type of hydrophobic moiety used to synthesize the PAM, which can act as a contributing factor regarding their morphology and targeting capabilities. To delineate and compare the characteristics of spherical and cylindrical micelles, we incorporated the monocyte-targeting chemokine, monocyte chemoattractant protein-1 (MCP-1), into our micelles (MCP-1 PAMs). We report that both shapes of nanoparticles were biocompatible with monocytes and enhanced the secondary structure of the MCP-1 peptide, thereby improving the ability of the micelles to mimic the native MCP-1 protein structure. As a result, both shapes of MCP-1 PAMs effectively targeted monocytes in an in vitro binding assay with murine monocytes. Interestingly, cylindrical PAMs showed a greater ability to attract monocytes compared to spherical PAMs in a chemotaxis assay. However, the surface area, the multivalent display of peptides, and the zeta potential of PAMs may also influence their biomimetic properties. Herein, we introduce variations in the methods of PAM synthesis and discuss the differences in PAM characteristics that can impact the recruitment of monocytes, a process associated with disease and cancer progression.