RESUMEN
Among inorganic materials, divalent cations modulate thousands of physiological processes that support life. Their roles in protein assembly and aggregation are less known, although they are progressively being brought to light. We review the structural roles of divalent cations here, as well as the novel protein materials that are under development, in which they are used as glue-like agents. More specifically, we discuss how mechanically stable nanoparticles, fibers, matrices, and hydrogels are generated through their coordination with histidine-rich proteins. We also describe how the rational use of divalent cations combined with simple protein engineering offers unexpected and very simple biochemical approaches to biomaterial design that might address unmet clinical needs in precision medicine.
Asunto(s)
Cationes Bivalentes/química , Proteínas/química , Humanos , Medicina de Precisión , Ingeniería de ProteínasRESUMEN
One-third of diffuse large B-cell lymphoma patients are refractory to initial treatment or relapse after rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone chemotherapy. In these patients, CXCR4 overexpression (CXCR4+) associates with lower overall and disease-free survival. Nanomedicine pursues active targeting to selectively deliver antitumor agents to cancer cells; a novel approach that promises to revolutionize therapy by dramatically increasing drug concentration in target tumor cells. In this study, we intravenously administered a liganded protein nanocarrier (T22-GFP-H6) targeting CXCR4+ lymphoma cells in mouse models to assess its selectivity as a nanocarrier by measuring its tissue biodistribution in cancer and normal cells. No previous protein-based nanocarrier has been described as specifically targeting lymphoma cells. T22-GFP-H6 achieved a highly selective tumor uptake in a CXCR4+ lymphoma subcutaneous model, as detected by fluorescent emission. We demonstrated that tumor uptake was CXCR4-dependent because pretreatment with AMD3100, a CXCR4 antagonist, significantly reduced tumor uptake. Moreover, in contrast to CXCR4+ subcutaneous models, CXCR4- tumors did not accumulate the nanocarrier. Most importantly, after intravenous injection in a disseminated model, the nanocarrier accumulated and internalized in all clinically relevant organs affected by lymphoma cells with negligible distribution to unaffected tissues. Finally, we obtained antitumor effect without toxicity in a CXCR4+ lymphoma model by administration of T22-DITOX-H6, a nanoparticle incorporating a toxin with the same structure as the nanocarrier. Hence, the use of the T22-GFP-H6 nanocarrier could be a good strategy to load and deliver drugs or toxins to treat specifically CXCR4-mediated refractory or relapsed diffuse large B-cell lymphoma without systemic toxicity.
Asunto(s)
Antineoplásicos , Linfoma de Células B Grandes Difuso , Animales , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Ciclofosfamida/uso terapéutico , Doxorrubicina/uso terapéutico , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Ratones , Recurrencia Local de Neoplasia/tratamiento farmacológico , Prednisona/uso terapéutico , Receptores CXCR4/genética , Rituximab/uso terapéutico , Transducción de Señal , Distribución Tisular , Vincristina/uso terapéuticoRESUMEN
Quatsomes are outstanding new lipid-based nanovesicles that are highly homogeneous and stable in different media for years, but the composition must be carefully chosen to avoid any potentially toxic side effects in in vivo applications. To this end, we have developed and studied a novel type of Quatsomes composed of cholesterol and myristalkonium chloride (MKC), the latter being extensively used as antimicrobial preservative in many ophthalmic and parenteral formulations on the EU and USA market. We have synthesized these novel MKC-Quatsomes in different media that are suitable for parenteral administration, and confirmed their stability in these media for 18 months, as well as the stability in human serum for 24 hours. Biodistribution assays were performed after intravenous injection of fluorescently labeled MKC-Quatsomes in live mice bearing xenografted colorectal tumors, showing nanovesicle accumulation in tumors, liver, spleen, and kidneys. No histological alteration or toxicity was observed in any of these organs.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Animales , Colesterol/química , Cromatografía Líquida de Alta Presión , Humanos , Ratones , Modelos Teóricos , Nanomedicina/métodosRESUMEN
Constitutive activation of the chemokine receptor CXCR4 has been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma, its biological relevance in this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue levels of CXCL12 correlated with high microvessel density and bone marrow involvement at diagnosis, supporting a role for the CXCL12-CXCR4 axis in disease progression. We then identified the tetra-amine IQS-01.01RS as a potent inverse agonist of the receptor, preventing CXCL12-mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than those of the standard agent. IQS-01.01RS activity was associated with downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allowing a synergistic interaction with the bromodomain and extra-terminal domain inhibitor, CPI203. In a xenotransplant model of diffuse large B-cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased tumor burden through MYC and p-AKT downregulation, and enhanced the induction of apoptosis. Thus, our results point out an emerging role of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lymphoma and support the simultaneous targeting of CXCR4 and bromodomain proteins as a promising, rationale-based strategy for the treatment of this disease.
Asunto(s)
Acetamidas/farmacología , Azepinas/farmacología , Linfoma de Células B Grandes Difuso , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores CXCR4/metabolismo , Animales , Biopsia , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Femenino , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The membrane pore-forming activities of the antimicrobial peptide GWH1 have been evaluated in combination with the CXCR4-binding properties of the peptide T22, in self-assembling protein nanoparticles with high clinical potential. The resulting materials, of 25 nm in size and with regular morphologies, show a dramatically improved cell penetrability into CXCR4+ cells (more than 10-fold) and enhanced endosomal escape (the lysosomal degradation dropping from 90% to 50%), when compared with equivalent protein nanoparticles lacking GWH1. These data reveal that GWH1 retains its potent membrane activity in form of nanostructured protein complexes. On the other hand, the specificity of T22 in the CXCR4 receptor binding is subsequently minimized but, unexpectedly, not abolished by the presence of the antimicrobial peptide. The functional combination T22-GWH1 results in 30% of the nanoparticles entering cells via CXCR4 while also exploiting pore-based uptake. Such functional materials are capable to selectively deliver highly potent cytotoxic drugs upon chemical conjugation, promoting CXCR4-dependent cell death. These data support the further development of GWH1-empowered cell-targeted proteins as nanoscale drug carriers for precision medicines. This is a very promising approach to overcome lysosomal degradation of protein nanostructured materials with therapeutic value.
Asunto(s)
Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Portadores de Fármacos/química , Nanopartículas/química , Péptidos/química , Receptores CXCR4/antagonistas & inhibidores , Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Endocitosis , Endosomas/metabolismo , Humanos , Nanopartículas/ultraestructura , Péptidos/metabolismo , Receptores CXCR4/metabolismoRESUMEN
Under the unmet need of efficient tumor-targeting drugs for oncology, a recombinant version of the plant toxin ricin (the modular protein T22-mRTA-H6) is engineered to self-assemble as protein-only, CXCR4-targeted nanoparticles. The soluble version of the construct self-organizes as regular 11 nm planar entities that are highly cytotoxic in cultured CXCR4+ cancer cells upon short time exposure, with a determined IC50 in the nanomolar order of magnitude. The chemical inhibition of CXCR4 binding sites in exposed cells results in a dramatic reduction of the cytotoxic potency, proving the receptor-dependent mechanism of cytotoxicity. The insoluble version of T22-mRTA-H6 is, contrarily, moderately active, indicating that free, nanostructured protein is the optimal drug form. In animal models of acute myeloid leukemia, T22-mRTA-H6 nanoparticles show an impressive and highly selective therapeutic effect, dramatically reducing the leukemia cells affectation of clinically relevant organs. Functionalized T22-mRTA-H6 nanoparticles are then promising prototypes of chemically homogeneous, highly potent antitumor nanostructured toxins for precise oncotherapies based on self-mediated intracellular drug delivery.
Asunto(s)
Antineoplásicos/farmacología , Nanoestructuras/química , Neoplasias/patología , Receptores CXCR4/metabolismo , Proteínas Recombinantes/farmacología , Ricina/farmacología , Secuencia de Aminoácidos , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células HeLa , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Proteínas Recombinantes/química , Ricina/químicaAsunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/deficiencia , Colágeno Tipo I/biosíntesis , Metilación de ADN , Enfermedad de Hodgkin/enzimología , Proteínas de Neoplasias/metabolismo , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN Neoplásico/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Conjuntos de Datos como Asunto , Decitabina/farmacología , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Linfocitos/metabolismo , Metilación/efectos de los fármacos , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Alineación de Secuencia , ARNt Metiltransferasas/metabolismoRESUMEN
Protein materials are rapidly gaining interest in materials sciences and nanomedicine because of their intrinsic biocompatibility and full biodegradability. The controlled construction of supramolecular entities relies on the controlled oligomerization of individual polypeptides, achievable through different strategies. Because of the potential toxicity of amyloids, those based on alternative molecular organizations are particularly appealing, but the structural bases on nonamylogenic oligomerization remain poorly studied. We have applied spectrofluorimetry and spectropolarimetry to identify the conformational conversion during the oligomerization of His-tagged cationic stretches into regular nanoparticles ranging around 11 nm, useful for tumor-targeted drug delivery. We demonstrate that the novel conformation acquired by the proteins, as building blocks of these supramolecular assemblies, shows different extents of compactness and results in a beta structure enrichment that enhances their structural stability. The conformational profiling presented here offers clear clues for understanding and tailoring the process of nanoparticle formation through the use of cationic and histidine rich stretches in the context of protein materials usable in advanced nanomedical strategies.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Nanopartículas/química , Multimerización de Proteína , Péptidos Catiónicos Antimicrobianos/genética , Antineoplásicos/administración & dosificación , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Conformación Proteica en Lámina beta , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown. We have here explored the cell penetrability of differently charged polyarginines in two alternative presentations, namely as unassembled fusion proteins or assembled in multimeric protein nanoparticles. By this, we have observed that arginine-rich peptides switch between receptor-mediated and receptor-independent mechanisms of cell penetration. The relative weight of these activities is determined by the electrostatic charge of the construct and the oligomerization status of the nanoscale material, both regulatable by conventional protein engineering approaches.
Asunto(s)
Arginina/química , Membrana Celular/metabolismo , Péptidos de Penetración Celular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Nanopartículas/química , Receptores CXCR4/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Ligandos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Nanopartículas/química , Péptidos/farmacología , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/síntesis química , Bencilaminas , Ciclamas , Expresión Génica , Células HeLa , Compuestos Heterocíclicos/farmacología , Humanos , Imidazoles/química , Nanopartículas/ultraestructura , Nanotecnología/métodos , Tamaño de la Partícula , Péptidos/síntesis química , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Cloruro de Sodio/químicaRESUMEN
Bacterial inclusion bodies are non-toxic, mechanically stable and functional protein amyloids within the nanoscale size range that are able to naturally penetrate into mammalian cells, where they deliver the embedded protein in a functional form. The potential use of inclusion bodies in protein delivery or protein replacement therapies is strongly impaired by the absence of specificity in cell binding and penetration, thus preventing targeting. To address this issue, we have here explored whether the genetic fusion of two tumor-homing peptides, the CXCR4 ligands R9 and T22, to an inclusion body-forming green fluorescent protein (GFP), would keep the interaction potential and the functionality of the fused peptides and then confer CXCR4 specificity in cell binding and further uptake of the materials. The fusion proteins have been well produced in Escherichia coli in their full-length form, keeping the potential for fluorescence emission of the partner GFP. By using specific inhibitors of CXCR4 binding, we have demonstrated that the engineered protein particles are able to penetrate CXCR4+ cells, in a receptor-mediated way, without toxicity or visible cytopathic effects, proving the availability of the peptide ligands on the surface of inclusion bodies. Since no further modification is required upon their purification, the biological production of genetically targeted inclusion bodies opens a plethora of cost-effective possibilities in the tissue-specific intracellular transfer of functional proteins through the use of structurally and functionally tailored soft materials.
Asunto(s)
Amiloide/administración & dosificación , Amiloide/química , Cuerpos de Inclusión/química , Nanoestructuras/administración & dosificación , Nanoestructuras/química , Neoplasias/tratamiento farmacológico , Amiloide/metabolismo , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Cuerpos de Inclusión/metabolismo , Péptidos/administración & dosificación , Péptidos/química , Péptidos/metabolismo , Receptores CXCR4/administración & dosificación , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Although all KRas (protein that in humans is encoded by the KRas gene) point mutants are considered to have a similar prognostic capacity, their transformation and tumorigenic capacities vary widely. We compared the metastatic efficiency of KRas G12V (Kirsten rat sarcoma viral oncogene homolog with valine mutation at codon 12) and KRas G13D (Kirsten rat sarcoma viral oncogene homolog with aspartic mutation at codon 13) oncogenes in an orthotopic colorectal cancer (CRC) model. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild-type (Kras WT)] expressing the KRas G12V or KRas G13D allele were microinjected in the mouse cecum. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Microscopic, macroscopic, and visible lymphatic foci were 1.5- to 3.0-fold larger in KRas G12V than in KRas G13D mice (P < 0.05). In the lung, only microfoci were developed in both groups. KRas G12V primary tumors had lower apoptosis (7.0 ± 1.2 vs. 7.4 ± 1.0 per field, P = 0.02), higher tumor budding at the invasion front (1.2 ± 0.2 vs. 0.6 ± 0.1, P = 0.04), and a higher percentage of C-X-C chemokine receptor type 4 (CXCR4)-overexpressing intravasated tumor emboli (49.8 ± 9.4% vs. 12.8 ± 4.4%, P < 0.001) than KRas G13D tumors. KRas G12V primary tumors showed Akt activation, and ß5 integrin, vascular endothelial growth factor A (VEGFA), and Serpine-1 overexpression, whereas KRas G13D tumors showed integrin ß1 and angiopoietin 2 (Angpt2) overexpression. The increased cell survival, invasion, intravasation, and specific molecular regulation observed in KRas G12V tumors is consistent with the higher aggressiveness observed in patients with CRC expressing this oncogene.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Angiopoyetina 2/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Femenino , Humanos , Integrina beta1/metabolismo , Metástasis Linfática , Ratones , Ratones Desnudos , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Unión al ARN/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Diabetes, growth or clotting disorders are among the spectrum of human diseases related to protein absence or malfunction. Since these pathologies cannot be yet regularly treated by gene therapy, the administration of functional proteins produced ex vivo is required. As both protein extraction from natural producers and chemical synthesis undergo inherent constraints that limit regular large-scale production, recombinant DNA technologies have rapidly become a choice for therapeutic protein production. The spectrum of organisms exploited as recombinant cell factories has expanded from the early predominating Escherichia coli to alternative bacteria, yeasts, insect cells and especially mammalian cells, which benefit from metabolic and protein processing pathways similar to those in human cells. Up to date, around 650 protein drugs have been worldwide approved, among which about 400 are obtained by recombinant technologies. Other 1300 recombinant pharmaceuticals are under development, with a clear tendency towards engineered versions with improved performance and new functionalities regarding the conventional, plain protein species. This trend is exemplified by the examination of the contemporary protein-based drugs developed for cancer treatment.
Asunto(s)
Bacterias/citología , Preparaciones Farmacéuticas/metabolismo , Proteínas Recombinantes/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Bacterias/metabolismo , Aprobación de Drogas , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
BACKGROUND: Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials. RESULTS: We have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions. DISCUSSION: The observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.
Asunto(s)
Escherichia coli/metabolismo , Nanopartículas , Multimerización de Proteína , Proteínas/química , Proteínas/metabolismo , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Células HeLa , Humanos , Ratones , Ratones Desnudos , Nanopartículas/química , Nanopartículas/metabolismo , Tamaño de la Partícula , Polimerizacion , Ingeniería de Proteínas/métodos , Proteínas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Relación Estructura-ActividadRESUMEN
The chemokine receptor CXCR4 has been implicated in the migration and trafficking of malignant B cells in several haematological malignancies. Over-expression of CXCR4 has been identified in haematological tumours, but data concerning the role of this receptor in diffuse large B cell lymphoma (DLBCL) are lacking. CXCR4 is a marker of poor prognosis in various neoplasms, correlating with metastatic disease and decreased survival of patients. We studied CXCR4 involvement in cell migration in vitro and dissemination in vivo. We also evaluated the prognostic significance of CXCR4 in 94 biopsies of DLBCL patients. We observed that the level of expression of CXCR4 in DLBCL cell lines correlated positively with in vitro migration. Expression of the receptor was also associated with increased engraftment and dissemination, and decreased survival time in NOD/SCID mice. Furthermore, administration of a specific CXCR4 antagonist, AMD3100, decreased dissemination of DLBCL cells in a xenograft mouse model. In addition, we found that CXCR4 expression is an independent prognostic factor for shorter overall survival and progression-free survival in DLBCL patients. These results show that CXCR4 mediates dissemination of DLBCL cells and define for the first time its value as an independent prognostic marker in DLBCL patients.
Asunto(s)
Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Linfoma de Células B Grandes Difuso/mortalidad , Linfoma de Células B Grandes Difuso/fisiopatología , Receptores CXCR4/fisiología , Animales , Bencilaminas , Línea Celular Tumoral , Ciclamas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Técnicas In Vitro , Linfoma de Células B Grandes Difuso/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica/fisiopatología , Pronóstico , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles.
Asunto(s)
Barrera Hematoencefálica , Sistemas de Liberación de Medicamentos , Nanopartículas , Péptidos , Humanos , Receptores de LDL , Distribución TisularRESUMEN
Unliganded drug-nanoconjugates accumulate passively in the tumor whereas liganded nanoconjugates promote drug internalization in tumor cells via endocytosis and increase antitumor efficacy. Whether or not tumor cell internalization associates with enhanced tumor uptake is still under debate. We here compared tumor uptake of T22-GFP-H6, a liganded protein carrier targeting the CXCR4 receptor, and the unliganded GFP-H6 carrier in subcutaneous and metastatic colorectal cancer models. T22-GFP-H6 had a higher tumor uptake in primary tumor and metastatic foci than GFP-H6, with no biodistribution or toxicity on normal tissues. T22-GFP-H6 was detected in target CXCR4+ tumor cell cytosol whereas GFP-H6 was detected in tumor stroma. SDF1-α co-administration switched T22-GFP-H6 internalization from CXCR4+ tumor epithelial cells to the stroma. Therefore, the incorporation of a targeting ligand promotes selective accumulation of the nanocarrier inside target tumor cells while increasing whole tumor uptake in a CXCR4-dependent manner, validating T22-GFP-H6 as a CXCR4-targeted drug carrier.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Receptores CXCR4 , Portadores de Fármacos , Endocitosis , Humanos , Ligandos , Nanotecnología , Péptidos , Transducción de Señal , Distribución TisularRESUMEN
AIMS: Focal adhesions have been associated with poor prognosis in multiple cancer types, but their prognostic value in diffuse large B-cell lymphoma (DLBCL) has not been evaluated. The aim of this study was to investigate the expression patterns and the prognostic value of the focal adhesion proteins FAK, Pyk2, p130Cas and HEF1 in DLBCL. METHODS AND RESULTS: Focal adhesion protein expression was examined using immunohistochemistry in normal lymphoid tissues and in 60 DLBCL patient samples. Kaplan-Meier survival and Cox regression analysis were performed to evaluate the correlation of focal adhesion protein expression with patient prognosis. FAK, Pyk2, p130Cas and HEF1 expression was mostly found in the germinal centres of normal human lymphoid tissues. When assessed in DLBCL samples, FAK, Pyk2, p130Cas and HEF1 were highly expressed in 45%, 34%, 42% and 45% of the samples, respectively. Multivariate Cox analysis revealed that decreased FAK expression was a significant independent predictor of poorer disease outcome. CONCLUSIONS: FAK expression is an independent prognostic factor in DLBCL. Our results suggest that the addition of FAK immunostaining to the current immunohistochemical algorithms may facilitate risk stratification of DLBCL patients.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Biomarcadores de Tumor/análisis , Proteína Sustrato Asociada a CrK/biosíntesis , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Linfoma de Células B Grandes Difuso/patología , Fosfoproteínas/biosíntesis , Anciano , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/mortalidad , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Análisis de Matrices TisularesRESUMEN
By recruiting functional domains supporting DNA condensation, cell binding, internalization, endosomal escape and nuclear transport, modular single-chain polypeptides can be tailored to associate with cargo DNA for cell-targeted gene therapy. Recently, an emerging architectonic principle at the nanoscale has permitted tagging protein monomers for self-organization as protein-only nanoparticles. We have studied here the accommodation of plasmid DNA into protein nanoparticles assembled with the synergistic assistance of end terminal poly-arginines (R9) and poly-histidines (H6). Data indicate a virus-like organization of the complexes, in which a DNA core is surrounded by a solvent-exposed protein layer. This finding validates end-terminal cationic peptides as pleiotropic tags in protein building blocks for the mimicry of viral architecture in artificial viruses, representing a promising alternative to the conventional use of viruses and virus-like particles for nanomedicine and gene therapy. FROM THE CLINICAL EDITOR: Finding efficient gene delivery methods still represents a challenge and is one of the bottlenecks to the more widespread application of gene therapy. The findings presented in this paper validate the application of end-terminal cationic peptides as pleiotropic tags in protein building blocks for "viral architecture mimicking" in artificial viruses, representing a promising alternative to the use of viruses and virus-like particles for gene delivery.
Asunto(s)
ADN/administración & dosificación , Técnicas de Transferencia de Gen , Nanopartículas/química , Proteínas/química , Secuencia de Aminoácidos , ADN/genética , Terapia Genética , Células HeLa , Histidina/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
Surface-exposed calreticulin (CRT) serves as a crucial cell damage-associated molecular pattern for immunogenic apoptosis, by generating an "eat me" signal to macrophages. Aiming at precision immunotherapies we intended to artificially label tumoral cells in vivo with a recombinant CRT, in a targeted way. For that, we have constructed a CRT fusion protein intended to surface attach CXCR4+ cancer cells, to stimulate their immunological destruction. As a targeting ligand of the CRT construct and to drive its specific cell adhesion, we used the peptide V1, a derivative of the vMIP-II cytokine and an antagonist of CXCR4. The modular protein tends to self-assemble as regular 16 nm nanoparticles, assisted by ionic Zn. Through both in vivo and in vitro experiments, we have determined that CRT itself confers cell targeting capabilities to the construct overcoming those of V1, that are only moderate. In particular, CRT binds HeLa cells in absence of further internalization, by a route fully independent of CXCR4. Furthermore, by cytometry in THP-1 cells, we observed that the binding of the protein is preferential for dead cells over live cells, a fact that cannot be associated to a mere artefactual adsorption. These data are discussed in the context of the oligomerizing properties of CRT and the potential clinical applicability of proteins and protein materials functionalized with this novel cell surface ligand.