RESUMEN
The term "molecular ZIP (or area) codes" refers to an originally hypothetical system of cell adhesion molecules that would control cell trafficking in the body. Subsequent discovery of the integrins, cadherins, and other cell adhesion molecules confirmed this hypothesis. The recognition system encompassing integrins and their ligands came particularly close to fulfilling the original ZIP code hypothesis, as multiple integrins with closely related specificities mediate cell adhesion by binding to an RGD or related sequence in various extracellular matrix proteins. Diseased tissues have their own molecular addresses that, although not necessarily involved in cell trafficking, can be made use of in targeted drug delivery. This article discusses the molecular basis of ZIP codes and the extensive effort under way to harness them for drug delivery purposes.
Asunto(s)
Moléculas de Adhesión Celular , Sistemas de Liberación de Medicamentos , Integrinas , Animales , Cadherinas/química , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Humanos , Integrinas/química , Integrinas/genética , Integrinas/metabolismo , Ligandos , Oligopéptidos/química , Oligopéptidos/metabolismoRESUMEN
Nucleotide-based drugs, such as antisense oligonucleotides (ASOs), have unique advantages in treating human diseases as they provide virtually unlimited ability to target any gene. However, their clinical translation faces many challenges, one of which is poor delivery to the target tissue in vivo. This problem is particularly evident in solid tumors. Here, we functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment. The iRGD-liposomes exhibited a high loading efficiency of AR-ASO, and an efficient knockdown of AR gene products was achieved in vitro, including AR splice variants. In vivo, iRGD-liposomes significantly increased AR-ASO accumulation in the tumor tissue and decreased AR expression relative to free ASOs in prostate tumors established as subcutaneous xenografts. Similar results were obtained with intra-tibial xenografts modeling metastasis to bones, the predominant site of metastasis for prostate cancer. In treatment studies, iRGD-liposomes markedly improved the AR-ASO efficacy in suppressing the growth of both subcutaneous xenografts and intra-tibial xenografts. The inhibitory effect on tumor growth was also significantly prolonged by the delivery of the AR-ASO in the iRGD-liposomes. Meanwhile, iRGD-liposomes did not increase ASO accumulation or toxicity in healthy organs. Overall, we provide here a delivery system that can significantly increase ASO accumulation and efficacy in solid tumors. These benefits are achieved without significant side effects, providing a way to increase the antitumor efficacy of ASOs.
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Antiangiogenic and vascular disrupting compounds have shown promise in cancer therapy, but tend to be only partially effective. We previously reported a potent theranostic nanosystem that was highly effective in glioblastoma and breast cancer mouse models, retarding tumor growth and producing some cures [ Agemy , L. et al. Proc. Natl. Acad. Sci. U.S.A. 2011 , 108 , 17450 - 17455 . Agemy , L. et al. Mol. Ther. 2013 , 21 , 2195 - 2204 .]. The nanosystem consists of iron oxide NPs ("nanoworms") coated with a composite peptide with tumor-homing and pro-apoptotic domains. The homing component targets tumor vessels by binding to p32/gC1qR at the surface or tumor endothelial cells. We sought to further improve the efficacy nanosystem by searching for an optimally effective homing peptide that would also incorporate a tumor-penetrating function. To this effect, we tested a panel of candidate p32 binding peptides with a sequence motif that conveys tumor-penetrating activity (CendR motif). We identified a peptide designated as Linear TT1 (Lin TT1) (sequence: AKRGARSTA) as most effective in causing tumor homing and penetration of the nanosystem. This peptide had the lowest affinity for p32 among the peptides tested. The low affinity may have moderated the avidity effect from the multivalent presentation on nanoparticles (NPs), such that the NPs avoid getting trapped by the so-called "binding-site barrier", which can hinder tissue penetration of compounds with a high affinity for their receptors. Treatment of breast cancer mice with the LinTT1 nanosystem showed greatly improved efficacy compared to the original system. These results identify a promising treatment modality and underscore the value of tumor penetration effect in improving the efficacy tumor treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Nanopartículas/uso terapéutico , Péptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Ratones , Nanomedicina , Nanopartículas/química , Nanopartículas/metabolismo , Péptidos/química , Péptidos/metabolismoRESUMEN
This Viewpoint discusses the rapid advances in molecular cell biological approaches over the past 50 years and the many avenues for future advances that have been opened, including direct applications for therapeutic and regenerative medicine.
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Distinciones y Premios , Biología Celular , Integrinas , Investigación Biomédica , Biología Celular/historia , Biología Celular/tendencias , Historia del Siglo XXI , Integrinas/fisiología , Estados UnidosRESUMEN
The rapid development of fluorescence imaging technologies requires concurrent improvements in the performance of fluorescent probes. Quantum dots have been extensively used as an imaging probe in various research areas because of their inherent advantages based on unique optical and electronic properties. However, their clinical translation has been limited by the potential toxicity especially from cadmium. Here, a versatile bioimaging probe is developed by using highly luminescent cadmium-free CuInSe2/ZnS core/shell quantum dots conjugated with CGKRK (Cys-Gly-Lys-Arg-Lys) tumor-targeting peptides. This probe exhibits excellent photostability, reasonably long circulation time, minimal toxicity, and strong tumor-specific homing property. The most important feature of this probe is that it shows distinctive versatility in tumor-targeted multimodal imaging including near-infrared, time-gated, and two-photon imaging in different tumor models. In a glioblastoma mouse model, the targeted probe clearly denotes tumor boundaries and positively labels a population of diffusely infiltrating tumor cells, suggesting its utility in precise tumor detection during surgery. This work lays a foundation for potential clinical translation of the probe.
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Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.
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Aminopiridinas/química , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Etilenodiaminas/química , Proteínas Mitocondriales/administración & dosificación , Péptidos Cíclicos/administración & dosificación , Aminopiridinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Proteínas Portadoras , Línea Celular Tumoral , Etilenodiaminas/farmacología , Femenino , Humanos , Ligandos , Ratones , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Nanopartículas/químicaRESUMEN
Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.
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ADN/química , Lípidos/química , Liposomas/química , Péptidos Cíclicos/síntesis química , Polietilenglicoles/química , Cationes/química , Humanos , Liposomas/síntesis química , Estructura Molecular , Nanopartículas/química , Péptidos Cíclicos/químicaRESUMEN
Vascular endothelium offers a variety of therapeutic targets for the treatment of cancer, cardiovascular diseases, inflammation, and oxidative stress. Significant research has been focused on developing agents to target the endothelium in diseased tissues. This includes identification of antibodies against adhesion molecules and neovascular expression markers or peptides discovered using phage display. Such targeting molecules also have been used to deliver nanoparticles to the endothelium of the diseased tissue. Here we report, based on in vitro and in vivo studies, that the specificity of endothelial targeting can be enhanced further by engineering the shape of ligand-displaying nanoparticles. In vitro studies performed using microfluidic systems that mimic the vasculature (synthetic microvascular networks) showed that rod-shaped nanoparticles exhibit higher specific and lower nonspecific accumulation under flow at the target compared with their spherical counterparts. Mathematical modeling of particle-surface interactions suggests that the higher avidity and specificity of nanorods originate from the balance of polyvalent interactions that favor adhesion and entropic losses as well as shear-induced detachment that reduce binding. In vivo experiments in mice confirmed that shape-induced enhancement of vascular targeting is also observed under physiological conditions in lungs and brain for nanoparticles displaying anti-intracellular adhesion molecule 1 and anti-transferrin receptor antibodies.
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Anticuerpos Monoclonales/administración & dosificación , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Endotelio Vascular/inmunología , Pulmón/irrigación sanguínea , Pulmón/inmunología , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , Adhesividad , Animales , Anticuerpos Monoclonales/farmacocinética , Línea Celular , Sistemas de Liberación de Medicamentos/métodos , Endotelio Vascular/ultraestructura , Femenino , Ratones , Ratones Endogámicos BALB C , Microfluídica , Microscopía Electrónica de Rastreo , Nanosferas/administración & dosificación , Nanosferas/ultraestructura , Nanotecnología , Nanotubos/ultraestructura , Tamaño de la Partícula , RatasRESUMEN
Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αß complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.
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Anticuerpos Antineoplásicos/inmunología , Biomarcadores de Tumor/inmunología , Inmunoterapia/métodos , Proteínas Musculares/metabolismo , Neoplasias de la Próstata/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Anticuerpos Antineoplásicos/metabolismo , Especificidad de Anticuerpos , Western Blotting , Técnicas de Visualización de Superficie Celular , Cromatografía de Afinidad , Cromatografía Liquida , Sistemas de Liberación de Medicamentos/métodos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Inmunoprecipitación , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/terapia , Estadísticas no Paramétricas , Espectrometría de Masas en TándemRESUMEN
There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.
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Células/metabolismo , Nanopartículas del Metal , Imagen Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Plata/química , Plata/metabolismo , Animales , Avidina/química , Transporte Biológico , Línea Celular Tumoral , Femenino , Humanos , Ratones , Sondas Moleculares/análisis , Sondas Moleculares/toxicidad , Polietilenglicoles/química , Plata/toxicidadRESUMEN
A major limitation in the pharmacological treatment of pulmonary arterial hypertension (PAH) is the lack of pulmonary vascular selectivity. Recent studies have identified a tissue-penetrating homing peptide, CARSKNKDC (CAR), which specifically homes to hypertensive pulmonary arteries but not to normal pulmonary vessels or other tissues. Some tissue-penetrating vascular homing peptides have a unique ability to facilitate transport of co-administered drugs into the targeted cells/tissues without requiring physical conjugation of the drug to the peptide (bystander effect). We tested the hypothesis that co-administered CAR would selectively enhance the pulmonary vascular effects of i.v. vasodilators in Sugen5416/hypoxia/normoxia-exposed PAH rats. Systemically administered CAR was predominantly detected in cells of remodeled pulmonary arteries. Intravenously co-administered CAR enhanced pulmonary, but not systemic, effects of the vasodilators, fasudil and imatinib, in PAH rats. CAR increased lung tissue imatinib concentration in isolated PAH lungs without increasing pulmonary vascular permeability. Sublingual CAR was also effective in selectively enhancing the pulmonary vasodilation by imatinib and sildenafil. Our results suggest a new paradigm in the treatment of PAH, using an i.v./sublingual tissue-penetrating homing peptide to selectively augment pulmonary vascular effects of nonselective drugs without the potentially problematic conjugation process. CAR may be particularly useful as an add-on therapy to selectively enhance the pulmonary vascular efficacy of any ongoing drug treatment in patients with PAH.
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Sistemas de Liberación de Medicamentos/métodos , Hipertensión Pulmonar/tratamiento farmacológico , Péptidos/química , Vasodilatadores/uso terapéutico , Administración Sublingual , Secuencia de Aminoácidos , Animales , Arteriopatías Oclusivas/tratamiento farmacológico , Arteriopatías Oclusivas/patología , Benzamidas/farmacología , Benzamidas/uso terapéutico , Hipertensión Pulmonar Primaria Familiar , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Mesilato de Imatinib , Infusiones Intravenosas , Inyecciones Intravenosas , Masculino , Datos de Secuencia Molecular , Piperazinas/farmacología , Piperazinas/uso terapéutico , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Resultado del Tratamiento , Vasodilatadores/administración & dosificación , Vasodilatadores/farmacologíaRESUMEN
The ability to detect and quantify macrophage accumulation can provide important diagnostic and prognostic information for atherosclerotic plaque. We have previously shown that LyP-1, a cyclic 9-amino acid peptide, binds to p32 proteins on activated macrophages, facilitating the visualization of atherosclerotic plaque with PET. Yet, the in vivo plaque accumulation of monomeric [(18)F]FBA-LyP-1 was low (0.31 ± 0.05%ID/g). To increase the avidity of LyP-1 constructs to p32, we synthesized a dendritic form of LyP-1 on solid phase using lysine as the core structural element. Imaging probes (FAM or 6-BAT) were conjugated to a lysine or cysteine on the dendrimer for optical and PET studies. The N-terminus of the dendrimer was further modified with an aminooxy group in order to conjugate LyP-1 and ARAL peptides bearing a ketone. Oxime ligation of peptides to both dendrimers resulted in (LyP-1)4- and (ARAL)4-dendrimers with optical (FAM) and PET probes (6-BAT). For PET-CT studies, (LyP-1)4- and (ARAL)4-dendrimer-6-BAT were labeled with (64)Cu (t1/2 = 12.7 h) and intravenously injected into the atherosclerotic (ApoE(-/-)) mice. After two hours of circulation, PET-CT coregistered images demonstrated greater uptake of the (LyP-1)4-dendrimer-(64)Cu than the (ARAL)4-dendrimer-(64)Cu in the aortic root and descending aorta. Ex vivo images and the biodistribution acquired at three hours after injection also demonstrated a significantly higher uptake of the (LyP-1)4-dendrimer-(64)Cu (1.1 ± 0.26%ID/g) than the (ARAL)4-dendrimer-(64)Cu (0.22 ± 0.05%ID/g) in the aorta. Similarly, subcutaneous injection of the LyP-1-dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. In the same model system, ex vivo fluorescence images within aortic plaque depict an increased accumulation and penetration of the (LyP-1)4-dendrimer-FAM as compared to the (ARAL)4-dendrimer-FAM. Taken together, the results suggest that the (LyP-1)4-dendrimer can be applied for in vivo PET imaging of plaque and that LyP-1 could be further exploited for the delivery of therapeutics with multivalent carriers or nanoparticles.
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Aterosclerosis/diagnóstico por imagen , Radioisótopos de Cobre/química , Dendrímeros/química , Imagen Multimodal , Péptidos Cíclicos/química , Tomografía de Emisión de Positrones/métodos , Tomografía Computarizada por Rayos X/métodos , Secuencia de Aminoácidos , Animales , Apolipoproteínas E/genética , Dendrímeros/farmacocinética , Ratones , Ratones Noqueados , Péptidos Cíclicos/farmacocinética , Distribución TisularRESUMEN
Antiangiogenic therapy is a promising new treatment modality for cancer, but it generally produces only transient tumor regression. We have previously devised a tumor-targeted nanosystem, in which a pentapeptide, CGKRK, delivers a proapoptotic peptide into the mitochondria of tumor blood vessel endothelial cells and tumor cells. The treatment was highly effective in glioblastoma mouse models completely refractory to other antiangiogenic treatments. Here, we identify p32/gC1qR/HABP, a mitochondrial protein that is also expressed at the cell surface of activated (angiogenic) endothelial cells and tumor cells, as a receptor for the CGKRK peptide. The results demonstrate the ability of p32 to cause internalization of a payload bound to p32 into the cytoplasm. We also show that nardilysin, a protease capable of cleaving CGKRK, plays a role in the internalization of a p32-bound payload. As p32 is overexpressed and surface displayed in breast cancers, we studied the efficacy of the nanosystem in this cancer. We show highly significant treatment results in an orthotopic model of breast cancer. The specificity of cell surface p32 for tumor-associated cells, its ability to carry payloads to mitochondria, and the efficacy of the system in important types of cancer make the nanosystem a promising candidate for further development.
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Inhibidores de la Angiogénesis/farmacología , Neoplasias de la Mama/terapia , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Nanopartículas/química , Péptidos/farmacología , Receptores de Complemento/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias Mamarias Experimentales , Glicoproteínas de Membrana/genética , Metaloendopeptidasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Terapia Molecular Dirigida , Especificidad de Órganos , Péptidos/administración & dosificación , Péptidos/uso terapéutico , Receptores de Complemento/genéticaRESUMEN
Autoimmune diseases, such as rheumatoid arthritis, frequently target one major tissue/organ despite the systemic nature of the immune response. This is particularly perplexing in the case of ubiquitously distributed antigens invoked in arthritis induction. We reasoned that selective targeting of the synovial joints in autoimmune arthritis might be due in part to the unique attributes of the joint vasculature. We examined this proposition using the adjuvant-induced arthritis model of human rheumatoid arthritis, and profiled the synovial vasculature using ex vivo and in vivo screening of a defined phage peptide-display library. We identified phage that preferentially homed to the inflamed joints. The corresponding synthetic peptides showed binding to the joint-derived endothelial cells, as well as specificity in inhibiting binding of the respective phage to the synovial vasculature. Intriguingly, the treatment of arthritic rats with one such peptide resulted in efficient inhibition of the progression of arthritis. The suppression of arthritis was attributable in part to the peptide-induced reduction of T-cell trafficking into the joints and the inhibition of angiogenesis. This peptide differed in sequence, in receptor binding specificity, and in angiogenesis/inflammation-related cell signaling from the previously characterized arginine-glycine-aspartic acid-containing peptide. Thus, our study reveals joint-homing peptides that can be further exploited for the selective delivery of antiarthritic agents into the inflamed joints to enhance their efficacy while reducing systemic toxicity, and also for examining intricacies of the pathogenesis of arthritis. This approach can be customized for application to other organ-specific autoimmune diseases as well.
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Artritis Experimental/inmunología , Péptidos/inmunología , Membrana Sinovial/inmunología , Vasculitis/inmunología , Secuencia de Aminoácidos , Animales , Artritis Experimental/patología , Artritis Experimental/prevención & control , Western Blotting , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Humanos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Biblioteca de Péptidos , Péptidos/genética , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Endogámicas Lew , Membrana Sinovial/irrigación sanguínea , Membrana Sinovial/patología , Vasculitis/prevención & controlRESUMEN
Antiangiogenic therapy can produce transient tumor regression in glioblastoma (GBM), but no prolongation in patient survival has been achieved. We have constructed a nanosystem targeted to tumor vasculature that incorporates three elements: (i) a tumor-homing peptide that specifically delivers its payload to the mitochondria of tumor endothelial cells and tumor cells, (ii) conjugation of this homing peptide with a proapoptotic peptide that acts on mitochondria, and (iii) multivalent presentation on iron oxide nanoparticles, which enhances the proapoptotic activity. The iron oxide component of the nanoparticles enabled imaging of GBM tumors in mice. Systemic treatment of GBM-bearing mice with the nanoparticles eradicated most tumors in one GBM mouse model and significantly delayed tumor development in another. Coinjecting the nanoparticles with a tumor-penetrating peptide further enhanced the therapeutic effect. Both models used have proven completely resistant to other therapies, suggesting clinical potential of our nanosystem.
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Inhibidores de la Angiogénesis/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Nanopartículas de Magnetita/administración & dosificación , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oligopéptidos/químicaRESUMEN
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.
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Apolipoproteínas E , Aterosclerosis/metabolismo , Compuestos Férricos/farmacocinética , Nanopartículas , Péptidos Cíclicos/farmacocinética , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/patología , Sistemas de Liberación de Medicamentos/métodos , Femenino , Compuestos Férricos/farmacología , Ratones , Ratones Mutantes , Oligopéptidos/farmacocinética , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacologíaRESUMEN
Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.
Asunto(s)
Bacteriófago T7/genética , Técnicas Analíticas Microfluídicas/métodos , Neuropilina-1/antagonistas & inhibidores , Biblioteca de Péptidos , Línea Celular Tumoral , Humanos , Neuropilina-1/genética , Neuropilina-1/metabolismoRESUMEN
This article recounts my journey as a scientist in the early days of extracellular matrix research through the discovery of fibronectin, the RGD sequence as a key recognition motif in fibronectin and other adhesion proteins, and isolation and cloning of integrins. I also discuss more recent work on identification of molecular "zip codes" by in vivo screening of peptide libraries expressed on phage, which led us right back to RGD and integrins. Many disease-specific zip codes have turned out to be based on altered expression of extracellular matrix molecules and integrins. Homing peptides and antibodies recognizing zip code molecules are being used in drug delivery applications, some of which have advanced into clinical trials.
RESUMEN
We present a chemical strategy to engineer analogs of the tumor-homing peptide CREKA (Cys-Arg-Glu-Lys-Ala), which binds to fibrin and fibrin-associated clotted plasma proteins in tumor vessels (Simberg et al. in Proc Natl Acad Sci USA 104:932-936, 2007) with improved ability to inhibit tumor growth. Computer modeling using a combination of simulated annealing and molecular dynamics were carried out to design targeted replacements aimed at enhancing the stability of the bioactive conformation of CREKA. Because this conformation presents a pocket-like shape with the charged groups of Arg, Glu and Lys pointing outward, non-proteinogenic amino acids α-methyl and N-methyl derivatives of Arg, Glu and Lys were selected, rationally designed and incorporated into CREKA analogs. The stabilization of the bioactive conformation predicted by the modeling for the different CREKA analogs matched the tumor fluorescence results, with tumor accumulation increasing with stabilization. Here we report the modeling, synthetic procedures, and new biological assays used to test the efficacy and utility of the analogs. Combined, our results show how studies based on multi-disciplinary collaboration can converge and lead to useful biomedical advances.
Asunto(s)
Antineoplásicos/química , Diseño de Fármacos , Oligopéptidos/química , Aminoácidos/síntesis química , Animales , Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Ratones , Simulación de Dinámica Molecular , Nanoestructuras/química , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Péptidos/química , Conformación ProteicaRESUMEN
Tumour blood vessels express markers that are not present in resting blood vessels of normal tissues, but that can be shared by angiogenic vessels in non-malignant conditions. Many of these proteins are functionally important in the angiogenic process. Some tumours also contain lymphatic vessels, as well as channels that consist of cancer cells and their extracellular matrix. These special features of tumour vessels are good targets for cancer therapies.