RESUMO
OBJECTIVE: A role for thrombin in the pathogenesis of atherosclerosis has been suggested through clinical and experimental studies revealing a critical link between the coagulation system and inflammation. Although approved drugs for inhibition of thrombin and thrombin-related signaling have demonstrated efficacy, their clinical application to this end may be limited because of significant potential for bleeding side effects. Thus, we sought to implement a plaque-localizing nanoparticle-based approach to interdict thrombin-induced inflammation and hypercoagulability in atherosclerosis. APPROACH AND RESULTS: We deployed a novel magnetic resonance spectroscopic method to quantify the severity of endothelial damage for correlation with traditional metrics of vessel procoagulant activity after dye-laser injury in fat-fed apolipoprotein E-null mice. We demonstrate that a 1-month course of treatment with antithrombin nanoparticles carrying the potent thrombin inhibitor PPACK (d-phenylalanyl-l-prolyl-l-arginyl chloromethylketone) nanoparticle (1) reduces the expression and secretion of proinflammatory and procoagulant molecules, (2) diminishes plaque procoagulant activity without the need for systemic anticoagulation, (3) rapidly restores disrupted vascular endothelial barriers, and (4) retards plaque progression in lesion-prone areas. CONCLUSIONS: These observations illustrate the role of thrombin as a pleiotropic atherogenic molecule under conditions of hypercholesterolemia and suggest the utility of its inhibition with locally acting antithrombin nanoparticle therapeutics as a rapid-acting anti-inflammatory strategy in atherosclerosis to reduce thrombotic risk.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Antitrombinas/farmacologia , Aterosclerose/tratamento farmacológico , Permeabilidade Capilar/efeitos dos fármacos , Lesões das Artérias Carótidas/tratamento farmacológico , Endotélio Vascular/efeitos dos fármacos , Nanopartículas , Trombina/antagonistas & inibidores , Trombose/prevenção & controle , Clorometilcetonas de Aminoácidos/farmacocinética , Animais , Antitrombinas/farmacocinética , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Coagulação Sanguínea/efeitos dos fármacos , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Mediadores da Inflamação/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos Knockout , Placa Aterosclerótica , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Trombose/genética , Trombose/metabolismo , Trombose/patologia , Fatores de TempoRESUMO
OBJECTIVE: Despite significant advances in intravascular stent technology, safe prevention of stent thrombosis over prolonged periods after initial deployment persists as a medical need to decrease device failure. The objective of this project was to assess the potential of perfluorocarbon nanoparticles (NP) conjugated with the direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK-NP) to inhibit stent thrombosis. METHODS: In a static model of stent thrombosis, 3 × 3-mm pieces of stainless steel coronary stents were cut and adsorbed with thrombin to create a procoagulant surface that would facilitate thrombus development. After treatment with PPACK-NP or control NP, stents were exposed to platelet-poor plasma (PPP) or platelet-rich plasma (PRP) for set time points up to 60 minutes. Measurements of final clot weight in grams were used for assessing the effect of NP treatment on limiting thrombosis. Additionally, groups of stents were exposed to flowing plasma containing various treatments (saline, free PPACK, control NP, and PPACK-NP) and generated thrombi were stained and imaged to investigate the treatment effects of PPACK-NP under flow conditions. RESULTS: The static model of stent thrombosis used in this study indicated a significant reduction in thrombus deposition with PPACK-NP treatment (0.00067 ± 0.00026 g; n = 3) compared with control NP (0.0098 ± 0.0015 g; n = 3; P = .026) in PPP. Exposure to PRP demonstrated similar effects with PPACK-NP treatment (0.00033 ± 0.00012 g; n = 3) vs control NP treatment (0.0045 ± 0.00012 g; n = 3; P = .000017). In additional studies, stents were exposed to both PRP pretreated with vorapaxar and PPACK-NP, which illustrated adjunctive benefit to oral platelet inhibitors for prevention of stent thrombosis. Additionally, an in vitro model of stent thrombosis under flow conditions established that PPACK-NP treatment inhibited thrombus deposition on stents significantly. CONCLUSIONS: This study demonstrates that antithrombin perfluorocarbon NPs exert marked focal antithrombin activity to prevent intravascular stent thrombosis and occlusion.
Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Antitrombinas/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Portadores de Fármacos , Fluorocarbonos/química , Nanopartículas , Intervenção Coronária Percutânea/instrumentação , Stents , Trombose/prevenção & controle , Clorometilcetonas de Aminoácidos/química , Antitrombinas/química , Velocidade do Fluxo Sanguíneo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Intervenção Coronária Percutânea/efeitos adversos , Desenho de Prótese , Aço Inoxidável , Propriedades de Superfície , Trombose/sangue , Trombose/etiologia , Trombose/fisiopatologia , Fatores de TempoRESUMO
Currently, there are no generally applicable noninvasive methods for defining the relationship between atherosclerotic vascular damage and risk of focal thrombosis. Herein, we demonstrate methods to delineate the progression and regression of vascular damage in response to an atherogenic diet by quantifying the in vivo accumulation of semipermeable 200-300 nm perfluorocarbon core nanoparticles (PFC-NP) in ApoE null mouse plaques with [(19)F] magnetic resonance spectroscopy (MRS). Permeability to PFC-NP remained minimal until 12 weeks on diet, then increased rapidly following 12 weeks, but regressed to baseline within 8 weeks after diet normalization. Markedly accelerated clotting (53.3% decrease in clotting time) was observed in carotid artery preparations of fat-fed mice subjected to photochemical injury as defined by the time to flow cessation. For all mice on and off diet, an inverse linear relationship was observed between the permeability to PFC-NP and accelerated thrombosis (P = 0.02). Translational feasibility for quantifying plaque permeability and vascular damage in vivo was demonstrated with clinical 3 T MRI of PFC-NP accumulating in plaques of atherosclerotic rabbits. These observations suggest that excessive permeability to PFC-NP may indicate prothrombotic risk in damaged atherosclerotic vasculature, which resolves within weeks after dietary therapy.
Assuntos
Aterosclerose/complicações , Trombose/etiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/dietoterapia , Aterosclerose/etiologia , Permeabilidade Capilar , Colesterol/química , Colesterol/metabolismo , Cristalização , Dieta Aterogênica/efeitos adversos , Dieta Ocidental/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fluorocarbonos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Nanopartículas , Placa Aterosclerótica/complicações , Placa Aterosclerótica/diagnóstico , Coelhos , Fatores de RiscoRESUMO
In the extension phase of acute kidney injury, microvascular thrombosis, inflammation, vasoconstriction, and vascular endothelial cell dysfunction promote progressive damage to renal parenchyma after reperfusion. In this study, we hypothesized that direct targeting and pharmaceutical knockdown of activated thrombin at the sites of injury with a selective nanoparticle (NP)-based thrombin inhibitor, PPACK (phenylalanine-proline-arginine-chloromethylketone), would improve kidney reperfusion and protect renal function after transient warm ischemia in rodent models. Saline- or plain NP-treated animals were employed as controls. In vivo 19F magnetic resonance imaging revealed that kidney nonreperfusion was evident within 3 h after global kidney reperfusion at 34 ± 13% area in the saline group and 43 ± 12% area in the plain NP group and substantially reduced to 17 ± 4% (â¼50% decrease, P < 0.05) in the PPACK NP pretreatment group. PPACK NP pretreatment prevented an increase in serum creatinine concentration within 24 h after ischemia-reperfusion, reflecting preserved renal function. Histologic analysis illustrated substantially reduced intrarenal thrombin accumulation within 24 h after reperfusion for PPACK NP-treated kidneys (0.11% ± 0.06%) compared with saline-treated kidneys (0.58 ± 0.37%). These results suggest a direct role for thrombin in the pathophysiology of AKI and a nanomedicine-based preventative strategy for improving kidney reperfusion after transient warm ischemia.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Inibidores de Cisteína Proteinase/farmacologia , Células Endoteliais/efeitos dos fármacos , Nanopartículas de Magnetita/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Trombina/antagonistas & inibidores , Injúria Renal Aguda/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Células Cultivadas , Creatinina/urina , Modelos Animais de Doenças , Células Endoteliais/patologia , Inflamação/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/patologiaRESUMO
Melittin is a cytolytic peptide derived from bee venom that inserts into lipid membranes and oligomerizes to form membrane pores. Although this peptide is an attractive candidate for treatment of cancers and infectious processes, its nonspecific cytotoxicity and hemolytic activity have limited its therapeutic applications. Several groups have reported the development of cytolytic peptide prodrugs that only exhibit cytotoxicity following activation by site-specific proteases. However, systemic administration of these constructs has proven difficult because of their poor pharmacokinetic properties. Here, we present a platform for the design of protease-activated melittin derivatives that may be used in conjunction with a perfluorocarbon nanoparticle delivery system. Although native melittin was substantially hemolytic (HD50: 1.9 µM) and cytotoxic (IC50: 2.4 µM), the prodrug exhibited 2 orders of magnitude less hemolytic activity (HD50: > 100 µM) and cytotoxicity (IC50: > 100 µM). Incubation with matrix metalloproteinase-9 (MMP-9) led to cleavage of the prodrug at the expected site and restoration of hemolytic activity (HD50: 3.4 µM) and cytotoxicity (IC50: 8.1 µM). Incubation of the prodrug with perfluorocarbon nanoparticles led to stable loading of 10,250 peptides per nanoparticle. Nanoparticle-bound prodrug was also cleaved and activated by MMP-9, albeit at a fourfold slower rate. Intravenous administration of prodrug-loaded nanoparticles in a mouse model of melanoma significantly decreased tumor growth rate (p = 0.01). Because MMPs and other proteases play a key role in cancer invasion and metastasis, this platform holds promise for the development of personalized cancer therapies directed toward a patient's individual protease expression profile.
Assuntos
Sistemas de Liberação de Medicamentos , Fluorocarbonos/química , Metaloproteinase 9 da Matriz/metabolismo , Meliteno/farmacologia , Nanopartículas/administração & dosagem , Fragmentos de Peptídeos/química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Animais , Hemólise/efeitos dos fármacos , Humanos , Espectrometria de Massas , Melanoma Experimental , Meliteno/química , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , CoelhosRESUMO
The goal of the present work was to design and test an acute-use nanoparticle-based antithrombotic agent that exhibits sustained local inhibition of thrombin without requiring a systemic anticoagulant effect to function against acute arterial thrombosis. To demonstrate proof of concept, we functionalized the surface of liposomes with multiple copies of the direct thrombin inhibitor, d-phenylalanyl-l-prolyl-l-arginyl-chloromethyl ketone (PPACK), which exhibits high affinity for thrombin as a free agent but manifests too rapid clearance in vivo to be effective alone. The PPACK-liposomes were formulated as single unilamellar vesicles, with a diameter of 170.78 ± 10.59 nm and a near neutral charge. In vitro models confirmed the inhibitory activity of PPACK-liposomes, demonstrating a KI' of 172.6 nM. In experimental clots in vitro, treatment of formed clots completely abrogated any further clotting upon exposure to human plasma. The liposomes were evaluated in vivo in a model of photochemical-induced carotid artery injury, resulting in significantly prolonged arterial occlusion time over that of controls (69.06 ± 5.65 min for saline treatment, N = 6, 71.33 ± 9.46 min for free PPACK treated; N = 4, 85.75 ± 18.24 min for precursor liposomes; N = 4, 139.75 ± 20.46 min for PPACK-liposomes; P = 0.0049, N = 6). Systemic anticoagulant profiles revealed a rapid return to control levels within 50 min, while still maintaining antithrombin activity at the injury site. The establishment of a potent and long-acting anticoagulant surface over a newly forming clot with the use of thrombin targeted nanoparticles that do not require systemic anticoagulation to be effective offers an alternative site-targeted approach to the management of acute thrombosis.
Assuntos
Anticoagulantes/química , Anticoagulantes/uso terapêutico , Lipossomos/química , Trombina/química , Trombina/metabolismo , Trombose/tratamento farmacológico , Clorometilcetonas de Aminoácidos/química , Animais , Humanos , Camundongos , Lipossomas Unilamelares/químicaRESUMO
BACKGROUND: A direct and independent role of inflammation in atherothrombosis was recently highlighted by the Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial, showing the benefit of inhibiting signaling molecules, eg, interleukins. Accordingly, we sought to devise a flexible platform for preventing the inflammatory drivers at their source to preserve plaque endothelium and mitigate procoagulant risk. METHODS: p5RHH-siRNA nanoparticles were formulated through self-assembly processes. The therapeutic efficacy of p5RHH-JNK2 siRNA nanoparticles was evaluated both in vitro and in vivo. RESULTS: Because JNK2 is critical to macrophage uptake of oxidized lipids through scavenger receptors that engender expression of myriad inflammatory molecules, we designed an RNA-silencing approach based on peptide-siRNA nanoparticles (p5RHH-siRNA) that localize to atherosclerotic plaques exhibiting disrupted endothelial barriers to achieve control of JNK2 expression by macrophages. After seven doses of p5RHH-JNK2 siRNA nanoparticles over 3.5 weeks in ApoE-/- mice on a Western diet, both JNK2 mRNA and protein levels were significantly decreased by 26% (P=0.044) and 42% (P=0.042), respectively. Plaque-macrophage populations were markedly depleted and NFκB and STAT3-signaling pathways inhibited by 47% (P<0.001) and 46% (P=0.004), respectively. Endothelial barrier integrity was restored (2.6-fold reduced permeability to circulating 200 nm nanoparticles in vivo, P=0.003) and thrombotic risk attenuated (200% increased clotting times to carotid artery injury, P=0.02), despite blood-cholesterol levels persistently exceeding 1,000 mg/dL. No adaptive or innate immunoresponses toward the nanoparticles were observed, and blood tests after the completion of treatment confirmed the largely nontoxic nature of this approach. CONCLUSION: The ability to formulate these nanostructures rapidly and easily interchange or multiplex their oligonucleotide content represents a promising approach for controlling deleterious signaling events locally in advanced atherosclerosis.
Assuntos
Aterosclerose/complicações , Endotélio/patologia , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Nanoestruturas/química , Peptídeos/metabolismo , Placa Aterosclerótica/complicações , RNA Interferente Pequeno/metabolismo , Trombose/complicações , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Aterosclerose/patologia , Aterosclerose/terapia , Modelos Animais de Doenças , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Nanopartículas/química , Placa Aterosclerótica/patologia , Placa Aterosclerótica/terapia , Células RAW 264.7 , Interferência de RNA , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Trombose/patologia , Trombose/terapiaRESUMO
High-density lipoprotein (HDL) plays an important role in the transport and metabolism of cholesterol. Mimics of HDL are being explored as potentially powerful therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I and with the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] have been demonstrated to be robust acceptors of cellular cholesterol. However, detailed structural information about this functionalized HDL AuNP is still lacking. In this study, we have used X-ray photoelectron spectroscopy and lecithin/cholesterol acyltransferase activation experiments together with coarse-grained and all-atom molecular dynamics simulations to model the structure and cholesterol uptake properties of the HDL AuNP construct. By simulating different apolipoprotein-loaded AuNPs, we find that lipids are oriented differently in regions with and without apoA-I. We also show that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL.
Assuntos
Nanopartículas Metálicas , Colesterol , Ouro , Lipoproteínas HDL , Simulação de Dinâmica MolecularRESUMO
As atherosclerosis remains one of the most prevalent causes of patient mortality, the ability to diagnose early signs of plaque rupture and thrombosis represents a significant clinical need. With recent advances in nanotechnology, it is now possible to image specific molecular processes noninvasively with MRI, using various types of nanoparticles as contrast agents. In the context of cardiovascular disease, it is possible to specifically deliver contrast agents to an epitope of interest for detecting vascular inflammatory processes, which serve as predecessors to atherosclerotic plaque development. Herein, we review various applications of nanotechnology in detecting atherosclerosis using MRI, with an emphasis on perfluorocarbon nanoparticles and fluorine imaging, along with theranostic prospects of nanotechnology in cardiovascular disease.
Assuntos
Aterosclerose/diagnóstico por imagem , Meios de Contraste/uso terapêutico , Nanopartículas/uso terapêutico , Placa Aterosclerótica/diagnóstico por imagem , Aterosclerose/patologia , Meios de Contraste/química , Humanos , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Placa Aterosclerótica/patologia , RadiografiaRESUMO
Natural products serve as an important source of novel compounds for drug development. Recently, peptides have emerged as a new class of therapeutic agents due to their versatility and specificity for biological targets. Yet, their effective application often requires use of a nanoparticle delivery system. In this chapter, we review the role of natural peptides in the design and creation of nanomedicines, with a particular focus on cell-penetrating peptides, antimicrobial peptides, and peptide toxins. The use of natural peptides in conjunction with nanoparticle delivery systems holds great promise for the development of new therapeutic formulations as well as novel platforms for the delivery of various cargoes.