RESUMO
The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1(+) cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1-coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[(18)F]fluorobenzoic acid ([(18)F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [(18)F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [(18)F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.
Assuntos
Apolipoproteínas E , Aterosclerose/metabolismo , Compostos Férricos/farmacocinética , Nanopartículas , Peptídeos Cíclicos/farmacocinética , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/patologia , Sistemas de Liberação de Medicamentos/métodos , Feminino , Compostos Férricos/farmacologia , Camundongos , Camundongos Mutantes , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologiaRESUMO
The rapid development and translation of targeted molecular imaging agents from bench to bedside is currently a slow process, with a clear bottleneck between the discovery of new compounds and the development of an appropriate molecular imaging agent. The ability to identify promising new molecular imaging agents, as well as failures, much earlier in the development process using high-throughput screening techniques could save significant time and money. This work combines the advantages of combinatorial chemistry, site-specific solid-phase radiolabeling, and in vivo imaging for the rapid screening of molecular imaging agents. A one-bead-one-compound library was prepared and evaluated in vitro, leading to the identification of 42 promising lead peptides. Over 11 consecutive days, these peptides, along with a control peptide, were successfully radiolabeled with 4-[(18)F]fluorobenzoic acid and evaluated in vivo using microPET. Four peptides were radiolabeled per day, followed by simultaneous injection of each individual peptide into 2 animals. As a result, 4 promising new molecular imaging agents were identified that otherwise would not have been selected based solely on in vitro data. This study is the first example of the practical application of a high-throughput screening approach using microPET imaging of [(18)F]-labeled peptides for the rapid in vivo identification of potential new molecular imaging agents.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Compostos Radiofarmacêuticos/química , Animais , Técnicas de Química Combinatória , Radioisótopos de Flúor , Camundongos , Neoplasias Experimentais/diagnóstico por imagem , Oligopeptídeos/química , Biblioteca de Peptídeos , Tomografia por Emissão de PósitronsRESUMO
Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies.
Assuntos
Microbolhas , Imagem Molecular/métodos , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Peptídeos/farmacocinética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ultrassonografia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , NucleolinaRESUMO
The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (â¼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.
Assuntos
Aterosclerose/terapia , Células Endoteliais/metabolismo , MicroRNAs/administração & dosagem , Nanopartículas/administração & dosagem , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Técnicas de Transferência de Genes , Humanos , Camundongos , MicroRNAs/genética , Terapia de Alvo Molecular , Nanopartículas/químicaRESUMO
Our goal is to provide a physiological perspective on the use of imaging to optimize and monitor the accumulation of nanotherapeutics within target tissues, with an emphasis on evaluating the pharmacokinetics of organic particles. Positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound technologies, as well as methods to label nanotherapeutic constructs, have created tremendous opportunities for preclinical optimization of therapeutics and for personalized treatments in challenging disease states. Within the methodology summarized here, the accumulation of the construct is estimated directly from the image intensity. Particle extravasation is then estimated based on classical physiological measures. Specifically, the transport of nanotherapeutics is described using the concept of apparent permeability, which is defined as the net flux of solute across a blood vessel wall per unit surface area of the blood vessel and per unit solute concentration difference across the blood vessel wall. The apparent permeability to small molecule MRI constructs is accurately shown to be far larger than that estimated for proteins such as albumin or nanoconstructs such as liposomes. Further, the quantitative measurements of vascular permeability are shown to facilitate detection of the transition from a pre-malignant to a malignant cancer and to quantify the delivery enhancement resulting from interventions such as ultrasound. While PET-based estimates facilitate quantitative comparisons of many constructs, high field MRI proves useful in the visualization of model drugs within small lesions and in the evaluation of the release and intracellular trafficking of nanoparticles and cargo.
Assuntos
Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Tomografia por Emissão de Pósitrons , Ultrassonografia , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Humanos , Tomografia por Emissão de Pósitrons/métodos , Tomografia por Emissão de Pósitrons/tendências , Ultrassonografia/métodos , Ultrassonografia/tendênciasRESUMO
The identification of novel, synthetic targeting ligands to endothelial receptors has led to the rapid development of targeted nanoparticles for drug, gene and imaging probe delivery. Central to development and optimization are effective models for assessing particle binding in vitro. Here, we developed a simple and cost effective method to quantitatively assess nanoparticle accumulation under physiologically-relevant laminar flow. We designed reversibly vacuum-sealed PDMS microfluidic chambers compatible with 35 mm petri dishes, which deliver uniform or gradient shear stress. These chambers have sufficient surface area for facile cell collection for particle accumulation quantitation through FACS. We tested this model by synthesizing and flowing liposomes coated with APN (K (D) ~ 300 µM) and VCAM-1-targeting (K (D) ~ 30 µM) peptides over HUVEC. Particle binding significantly increased with ligand concentration (up to 6 mol%) and decreased with excess PEG. While the accumulation of particles with the lower affinity ligand decreased with shear, accumulation of those with the higher affinity ligand was highest in a low shear environment (2.4 dyne/cm(2)), as compared with greater shear or the absence of shear. We describe here a robust flow chamber model that is applied to optimize the properties of 100 nm liposomes targeted to inflamed endothelium.
Assuntos
Microfluídica , Nanopartículas , Anticorpos Monoclonais/farmacologia , Antígenos CD13/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Lipossomos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Significant upregulation of the integrin alpha(v)beta(6) has been described as a prognostic indicator in several cancers, making it an attractive target for tumor imaging. This study compares variants of a PEGylated alpha(v)beta(6)-targeting peptide, bearing either an [(18)F]fluorobenzoyl prosthetic group ([(18)F]FBA-PEG-A20FMDV2) or different [(64)Cu]copper chelators (DOTA-PEG-A20FMDV2, CB-TE2A-PEG-A20FMDV2). The compounds were evaluated in vitro by enzyme-linked immunosorbent assay (against the integrin alpha(v)beta(6) and the closely related integrin alpha(v)beta(3)) and by cell labeling (alpha(v)beta(6)-positive DX3purobeta6/alpha(v)beta(6)-negative DX3puro) and in vivo using micro-positron emission tomography in a mouse model bearing paired DX3purobeta6/Dx3puro xenografts. In vitro, all three compounds showed excellent alpha(v)beta(6)-specific binding (50% inhibitory concentration [IC(50)](alpha(v)beta(6)) = 3 to 6 nmol/L; IC(50)(alpha(v)beta(3)) > 10 micromol/L). In vivo, they displayed comparable, preferential uptake for the alpha(v)beta(6)-expressing xenograft over the alpha(v)beta(6)-negative control (> 4:1 ratio at 4 hours postinjection). Whereas [(64)Cu]Cu-DOTA-PEG-A20FMDV2 resulted in increased levels of radioactivity in the liver, [(64)Cu]Cu-CB-TE2A-PEG-A20FMDV2 did not. Significantly, both (64)Cu-labeled tracers showed unexpectedly high and persistent levels of radioactivity in the kidneys (> 40% injected dose/g at 4 and 12 hours postinjection). The findings underscore the potential influence of the prosthetic group on targeted in vivo imaging of clinically relevant markers such as alpha(v)beta(6). Despite identical targeting peptide moiety and largely equal in vitro behavior, both (64)Cu-labeled tracers displayed inferior pharmacokinetics, making them in their present form less suitable candidates than the (18)F-labeled tracer for in vivo imaging of alpha(v)beta(6).
Assuntos
Antígenos de Neoplasias/metabolismo , Compostos Heterocíclicos com 1 Anel , Integrinas/metabolismo , Neoplasias Experimentais/diagnóstico por imagem , Compostos Organometálicos , Tomografia por Emissão de Pósitrons/métodos , Animais , Linhagem Celular , Radioisótopos de Cobre , Interpretação Estatística de Dados , Ensaio de Imunoadsorção Enzimática , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/metabolismo , Compostos Heterocíclicos com 1 Anel/farmacocinética , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacocinética , Peptídeos/metabolismo , Ligação Proteica , Distribuição TecidualRESUMO
Numerous radiolabeled peptides have been utilized for in vivo imaging of a variety of cell surface receptors. For applications in PET using [(18)F]fluorine, peptides are radiolabeled via a prosthetic group approach. We previously developed solution-phase (18)F-"click" radiolabeling and solid-phase radiolabeling using 4-[(18)F]fluorobenzoic and 2-[(18)F]fluoropropionic acids. Here we compare the three different radiolabeling approaches and report the effects on PET imaging and pharmacokinetics. The prosthetic groups did have an effect; metabolites with significantly different polarities were observed.
Assuntos
Antígenos de Neoplasias/efeitos dos fármacos , Benzoatos/química , Integrinas/efeitos dos fármacos , Neoplasias/diagnóstico , Peptídeos , Tomografia por Emissão de Pósitrons/métodos , Propionatos/química , Compostos Radiofarmacêuticos , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Radioisótopos de Flúor , Humanos , Integrinas/química , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Sensibilidade e Especificidade , Estereoisomerismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 2-[(18)F]fluoropropionic (2-[(18)F]FPA) acid is used as a prosthetic group for radiolabeling proteins and peptides for targeted imaging using positron emission tomography (PET). Radiolabeling of compounds with more than one acylable functional group can lead to complex mixtures of products; however, peptides can be labeled regioselectively on the solid phase. We investigated the use of a solid-phase approach for the preparation of 2-[(18)F]fluoropropionyl peptides. [(18)F]FPA was prepared and conjugated to the peptides attached to the solid phase support. The (18)F-labeled peptides were obtained in 175 min with decay corrected yields of 10% (related to [(18)F]fluoride) and with a purity of 76-99% prior HPLC purification. The suitability of various coupling reagents and solid supports were tested for radiolabeling of several peptides of various lengths.