RESUMEN
New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Péptidos/química , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/química , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanotecnología , Neoplasias/patología , Tamaño de la Partícula , Péptidos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report herein thermally responsive elastin-like polypeptides (ELPs) in a linear AB diblock architecture with an N-terminal peptide ligand that self-assemble into spherical micelles when heated slightly above body temperature. A series of 10 ELP block copolymers (ELP(BC)'s ) with different molecular weights and hydrophilic-to-hydrophobic block ratios were genetically synthesized by recursive directional ligation. The self-assembly of these polymers from unimers into micelles was investigated by light scattering, fluorescence spectroscopy, and cryo-TEM. These ELP(BC)'s undergo two phase transitions as a function of solution temperature: a unimer-to-spherical micelle transition at an intermediate temperature and a micelle-to-bulk aggregate transition at a higher temperature when the hydrophilic-to-hydrophobic block ratio is between 1:2 and 2:1. The critical micelle temperature is controlled by the length of the hydrophobic block, and the size of the micelle is controlled by both the total ELP(BC) length and hydrophilic-to-hydrophobic block ratio. These polypeptide micelles display a critical micelle concentration in the range 4-8 microM demonstrating the high stability of these structures. These studies have also identified a subset of ELP(BC)'s bearing terminal peptide ligands that are capable of forming multivalent spherical micelles that present multiple copies of the ligand on their corona in the clinically relevant temperature range 37-42 degrees C and target cancer cells. These ELP(BC)'s may be useful for drug targeting by thermally triggered multivalency. More broadly, the design rules uncovered by this study should be applicable to the design of other thermally reversible nanoparticles for diverse applications in medicine and biology.
Asunto(s)
Elastina/química , Calor , Micelas , Péptidos/química , Microscopía por Crioelectrón , Luz , Dispersión de Radiación , Espectrometría de Fluorescencia , TemperaturaRESUMEN
Peptides are emerging as a new class of biomaterials due to their unique chemical, physical, and biological properties. The development of peptide-based biomaterials is driven by the convergence of protein engineering and macromolecular self-assembly. This review covers the basic principles, applications, and prospects of peptide-based biomaterials. We focus on both chemically synthesized and genetically encoded peptides, including poly-amino acids, elastin-like polypeptides, silk-like polymers and other biopolymers based on repetitive peptide motifs. Applications of these engineered biomolecules in protein purification, controlled drug delivery, tissue engineering, and biosurface engineering are discussed.
RESUMEN
Angle-resolved low coherence interferometry (a/LCI) enables depth-resolved measurements of scattered light that can be used to recover subsurface structural information, such as the size of cell nuclei. Measurements of nuclear morphology, however, can be complicated by coherent scattering between adjacent cell nuclei. Previous studies have eliminated this component by applying a window filter to Fourier transformed angular data, based on the justification that the coherent scattering must necessarily occur over length scales greater than the cell size. To fully study this effect, results of experiments designed to test the validity of this approach are now presented. The a/LCI technique is used to examine light scattered by regular cell arrays, created using stamped adhesive micropatterned substrates. By varying the array spacing, it is demonstrated that cell-to-cell correlations have a predictable effect on light scattering distributions. These results are compared to image analysis of fluorescence micrographs of the cell array samples. The a/LCI results show that the impact of coherent scattering on nuclear morphology measurements can be eliminated through data filtering.
Asunto(s)
Algoritmos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Interferometría/métodos , Tomografía de Coherencia Óptica/métodos , Animales , Almacenamiento y Recuperación de la Información/métodos , Luz , Ratones , Células 3T3 NIH , Reproducibilidad de los Resultados , Dispersión de Radiación , Sensibilidad y Especificidad , Estadística como AsuntoRESUMEN
Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream.
Asunto(s)
Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Animales , Línea Celular Tumoral , Calor , Humanos , Liposomas , Ratones , Microscopía Confocal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antivascular targeting is a promising strategy for tumor therapy. This strategy has the potential to overcome many of the transport barriers associated with targeting tumor cells in solid tumors, because the tumor vasculature is directly accessible to targeting vehicles in systemic circulation. We report a novel nanoscale delivery system consisting of multivalent polymer micelles to target receptors that are preferentially upregulated in the tumor vasculature and perivascular cells, specifically CD13. To this end we utilized amphiphilic block copolymers, composed of a genetically engineered elastin-like polypeptide (ELP) that self-assemble into monodisperse spherical micelles. These polymer micelles were functionalized by incorporating the NGR tripeptide ligand, which targets the CD13 receptor, on their corona. We examined the self-assembly and in vivo tumor targeting by these NGR-functionalized nanoparticles and show that multivalent presentation of NGR by micelle self-assembly selectively targets the tumor vasculature by targeting CD13. Furthermore, we show greater vascular retention and extravascular accumulation of nanoparticles in tumor tissue compared to normal tissue, although the enhancement is modest. These results suggest that enhanced delivery to solid tumors can be achieved by targeting upregulated receptors in the tumor vasculature with multivalent ligand-presenting nanoparticles, but additional work is required to optimize such systems for multivalent targeting.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Antígenos CD13/metabolismo , Portadores de Fármacos/química , Elastina/química , Neovascularización Patológica/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Péptidos/química , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Luz , Ratones , Ratones Desnudos , Micelas , Microscopía Confocal , Microscopía Fluorescente , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Péptidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Dispersión de Radiación , Temperatura , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in nontarget tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy--dynamic affinity modulation (DAM)--using elastin-like polypeptide diblock copolymers (ELP(BC)s) that self-assemble from a low-affinity to high-avidity state by a tunable thermal "switch", thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELP(BC)s on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELP(BC) self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micrometer-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM and provide rational design parameters for future applications of this technology for targeted drug delivery.
Asunto(s)
Elastina/química , Nanopartículas/química , Péptidos/genética , Péptidos/metabolismo , Temperatura , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Integrina alfaVbeta3/metabolismo , Células K562 , Ligandos , Micelas , Péptidos/químicaRESUMEN
This study evaluated a biodegradable drug delivery system for local cancer radiotherapy consisting of a thermally sensitive elastin-like polypeptide (ELP) conjugated to a therapeutic radionuclide. Two ELPs (49 kDa) were synthesized using genetic engineering to test the hypothesis that injectable biopolymeric depots can retain radionuclides locally and reduce the growth of tumors. A thermally sensitive polypeptide, ELP(1), was designed to spontaneously undergo a soluble-insoluble phase transition (forming viscous microparticles) between room temperature and body temperature upon intratumoral injection, while ELP(2) was designed to remain soluble upon injection and to serve as a negative control for the effect of aggregate assembly. After intratumoral administration of radionuclide conjugates of ELPs into implanted tumor xenografts in nude mice, their retention within the tumor, spatio-temporal distribution, and therapeutic effect were quantified. The residence time of the radionuclide-ELP(1) in the tumor was significantly longer than the thermally insensitive ELP(2) conjugate. In addition, the thermal transition of ELP(1) significantly protected the conjugated radionuclide from dehalogenation, whereas the conjugated radionuclide on ELP(2) was quickly eliminated from the tumor and cleaved from the biopolymer. These attributes of the thermally sensitive ELP(1) depot improved the antitumor efficacy of iodine-131 compared to the soluble ELP(2) control. This novel injectable and biodegradable depot has the potential to control advanced-stage cancers by reducing the bulk of inoperable tumors, enabling surgical removal of de-bulked tumors, and preserving healthy tissues.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Péptidos/genética , Animales , Antígenos , Biopolímeros , Formas de Dosificación , Elastina/genética , Femenino , Calor , Inyecciones , Ratones , Ratones Desnudos , Neoplasias , Procesos Neoplásicos , RadioisótoposRESUMEN
In this article, the authors describe new approaches to synthesize and pattern surfaces with poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA) polymer brushes synthesized by surface-initiated atom transfer radical polymerization. These patterned coatings confer "nonfouling" properties protein and cell resistance-to the surface in a biological milieu. The versatile routes for the synthesis of POEGMA demonstrated here offer clear advantages over other techniques previously used in terms of their simplicity, reliability, and ability to pattern large-area substrates. They also demonstrate that POEGMA polymer brushes can be patterned directly by photolithography, plasma ashing, and reactive ion etching to create patterns at the micro- and nanoscale over large areas with high throughput and repeatability, while preserving the protein and cell resistance of the POEGMA brush.