RESUMEN
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
Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5'-triphosphate (5'-ppp) RNA and mediate IFN production. Cytosolic 5'-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-ß independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5'-ppp dsRNA and induces IFN-ß through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-ß induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-ß production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.
Asunto(s)
Infecciones por Alphavirus/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN Bicatenario/biosíntesis , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Virus de los Bosques Semliki/fisiología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Infecciones por Alphavirus/genética , Animales , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Humanos , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/metabolismo , Ratones , ARN Bicatenario/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Receptores Inmunológicos , Proteínas Virales/genéticaRESUMEN
The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 1. The HTML has now been updated to include a corrected version of the Supplementary Information.
RESUMEN
Bacterial resistance to antibiotics has made it necessary to resort to antibiotics that have considerable toxicities. Here, we show that the cyclic 9-amino acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of CARG-conjugated vancomycin-loaded nanoparticles in S. aureus-infected tissues and reduces the needed overall systemic dose, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing the peptide in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissue may help combat difficult-to-treat infections.
RESUMEN
The aim of this survey was to investigate human immunodeficiency virus type 1 (HIV-1) coreceptor, chemokine receptor 5 (CCR5), polymorphism among Estonian HIV-1-infected individuals. Homozygous CCR5Delta32 genotypes have been associated with resistance to HIV-1 infection; however, inconsistent evidence exists as to whether a single copy of a mutant allele among heterozygotes confers protection from HIV-1 infection. In an Estonian population the frequency of the CCR5Delta32 allele has been found to be among the greatest observed to date. Ironically, Estonia is concomitantly characterized by a very high HIV-1 prevalence. We compared the allele frequencies in a healthy control population to the HIV-positive group. The frequency of heterozygous individuals did not differ significantly between the HIV-positive group and the control population. Allele frequencies were analyzed among different risk groups as well as groups with different HIV genetic backgrounds. We did not find a difference between CCR5Delta32 allele frequencies among intravenous drug users (IDUs) and sexually infected persons. Likewise, the distribution of CCR5Delta32 allele frequencies among patients infected with different subtypes did not differ while data from "pure" subtypes A, B, and CRF06_cpx were pooled and evaluated against unique recombinant forms.
Asunto(s)
Predisposición Genética a la Enfermedad/epidemiología , Infecciones por VIH/genética , VIH-1/inmunología , Polimorfismo de Nucleótido Simple , Receptores CCR5/genética , Abuso de Sustancias por Vía Intravenosa/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Estonia/epidemiología , Femenino , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas , Infecciones por VIH/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Factores de RiesgoRESUMEN
Anti-angiogenic and vascular disrupting therapies rely on the dependence of tumors on new blood vessels to sustain tumor growth. We previously reported a potent vascular disrupting agent, a theranostic nanosystem consisting of a tumor vasculature-homing peptide (CGKRK) fused to a pro-apoptotic peptide [D(KLAKLAK)2] coated on iron oxide nanoparticles. This nanosystem showed promising therapeutic efficacy in glioblastoma (GBM) and breast cancer models. However, complete control of the tumors was not achieved, and some tumors became non-responsive to the treatment. Here we examined the non-responder phenomenon in an aggressive MCF10-CA1a breast tumor model. In the treatment-resistant tumors we noted the emergence of CD31-negative patent neovessels and a concomitant loss of tumor homing of the nanosystem. In vivo phage library screening in mice bearing non-responder tumors showed that compared to untreated and treatment-sensitive tumors, treatment sensitive tumors yield a distinct landscape of vascular homing peptides characterized by over-representation of peptides that target αv integrins. Our approach may be generally applicable to the development of targeted therapies for tumors that have failed treatment.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Nanopartículas/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Péptidos/uso terapéutico , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/patología , Biblioteca de PéptidosRESUMEN
Cerebrovascular changes occur in Alzheimer's disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson's disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.
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Enfermedad de Alzheimer/metabolismo , Péptidos Cíclicos/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Hipocampo/irrigación sanguínea , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Biblioteca de Péptidos , Péptidos Cíclicos/química , Unión ProteicaRESUMEN
Tumor penetrating peptides contain a cryptic (R/K)XX(R/K) CendR element that must be C-terminally exposed to trigger neuropilin-1 (NRP-1) binding, cellular internalization and malignant tissue penetration. The specific proteases that are involved in processing of tumor penetrating peptides identified using phage display are not known. Here we design de novo a tumor-penetrating peptide based on consensus cleavage motif of urokinase-type plasminogen activator (uPA). We expressed the peptide, uCendR (RPARSGR↓SAGGSVA, ↓ shows cleavage site), on phage or coated it onto silver nanoparticles and showed that it is cleaved by uPA, and that the cleavage triggers binding to recombinant NRP-1 and to NPR-1-expressing cells. Upon systemic administration to mice bearing uPA-overexpressing breast tumors, FAM-labeled uCendR peptide and uCendR-coated nanoparticles preferentially accumulated in tumor tissue. We also show that uCendR phage internalization into cultured cancer cells and its penetration in explants of murine tumors and clinical tumor explants can be potentiated by combining the uCendR peptide with tumor-homing module, CRGDC. Our work demonstrates the feasibility of designing tumor-penetrating peptides that are activated by a specific tumor protease. As upregulation of protease expression is one of the hallmarks of cancer, and numerous tumor proteases have substrate specificities compatible with proteolytic unmasking of cryptic CendR motifs, the strategy described here may provide a generic approach for designing proteolytically-actuated peptides for tumor-penetrative payload delivery.
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Portadores de Fármacos/administración & dosificación , Neoplasias Mamarias Animales/metabolismo , Péptidos/administración & dosificación , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Bacteriófago T7 , Línea Celular Tumoral , Portadores de Fármacos/farmacocinética , Humanos , Nanopartículas del Metal/administración & dosificación , Ratones Endogámicos BALB C , Péptidos/farmacocinética , Plata/administración & dosificación , Plata/farmacocinéticaRESUMEN
Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 ± 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 ± 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.
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Técnicas Citológicas , Nanopartículas del Metal , Plata , Línea Celular Tumoral , Humanos , Isótopos , Masculino , Péptidos , FenotipoRESUMEN
Traumatic brain injury (TBI) is a major health and socio-economic problem, but no pharmacological agent is currently approved for the treatment of acute TBI. Thus, there is a great need for advances in this field. Here, we describe a short peptide (sequence CAQK) identified by in vivo phage display screening in mice with acute brain injury. The CAQK peptide selectively binds to injured mouse and human brain, and systemically injected CAQK specifically homes to sites of brain injury in mouse models. The CAQK target is a proteoglycan complex upregulated in brain injuries. Coupling to CAQK increased injury site accumulation of systemically administered molecules ranging from a drug-sized molecule to nanoparticles. CAQK-coated nanoparticles containing silencing oligonucleotides provided the first evidence of gene silencing in injured brain parenchyma by systemically administered siRNA. These findings present an effective targeting strategy for the delivery of therapeutics in clinical management of acute brain injuries.
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Lesiones Traumáticas del Encéfalo/terapia , Sistemas de Liberación de Medicamentos , Péptidos , Anciano , Animales , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Matriz Extracelular/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Broad CTL response against HIV-1 is one factor that helps to control the viral replication. We have constructed a DNA vaccine that encodes a large artificial fusion protein (MultiHIV) and shown it to be immunogenic in mice, swine and macaques. Inbred mice revealed CTL response only against two epitopes due to limited MHC class I variability. To assess the quality of the CTL response we addressed this question in domestic swine. Number of presented epitopes varied between 7 and 14 among the five selected animals. Epitopes detected in swine are localised in the same antigenic regions recognised in humans. This can be explained by the fact that swine MHC-I (SLA-I) complex is remarkably similar to human HLA-I. These results also indicate that immunogenicity profile of vaccines in domestic swine may predict the outcome of human immunisation.