RESUMO
Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using ß-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.
Assuntos
Antineoplásicos/farmacologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Imunotoxinas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Polietilenoglicóis/química , Proteínas Recombinantes/farmacologia , Animais , Antineoplásicos/química , Apoptose , Proliferação de Células , Feminino , Meia-Vida , Humanos , Imunotoxinas/química , Mesotelina , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Recombinantes/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Amphiphilic graft copolymers having polysulfone (PSf) backbones and poly(ethylene glycol) (PEG) side chains were synthesized via reaction of an alkoxide formed from PEG and a base (sodium hydride) with chloromethylated polysulfone. The resulting polysulfone-graft-poly(ethylene glycol), PSf-g-PEG, materials were hydrophilic but water insoluble, rendering them potentially useful as biomaterial coatings. PSf-g-PEG films exhibited high resistance to protein adsorption and cell attachment. When used as an additive in PSf membranes prepared by immersion precipitation, the graft copolymer preferentially segregates to the membrane surface, delivering enhanced wettability, porosity and protein resistance compared to unmodified PSf membranes. The surface properties of PSf-g-PEG modified membranes render them desirable candidates for hemodialysis.
Assuntos
Polietilenoglicóis/química , Polímeros/química , Sulfonas/química , Animais , Linhagem Celular , Cloro/química , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Microscopia Eletrônica de Varredura , Estrutura Molecular , Polietilenoglicóis/síntese químicaRESUMO
The development of antidrug antibodies (ADAs) is a common cause for the failure of biotherapeutic treatments and adverse hypersensitivity reactions. Here we demonstrate that poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying rapamycin, but not free rapamycin, are capable of inducing durable immunological tolerance to co-administered proteins that is characterized by the induction of tolerogenic dendritic cells, an increase in regulatory T cells, a reduction in B cell activation and germinal centre formation, and the inhibition of antigen-specific hypersensitivity reactions. Intravenous co-administration of tolerogenic nanoparticles with pegylated uricase inhibited the formation of ADAs in mice and non-human primates and normalized serum uric acid levels in uricase-deficient mice. Similarly, the subcutaneous co-administration of nanoparticles with adalimumab resulted in the durable inhibition of ADAs, leading to normalized pharmacokinetics of the anti-TNFα antibody and protection against arthritis in TNFα transgenic mice. Adjunct therapy with tolerogenic nanoparticles represents a novel and broadly applicable approach to prevent the formation of ADAs against biologic therapies.
Assuntos
Tolerância Imunológica/efeitos dos fármacos , Nanopartículas/administração & dosagem , Sirolimo/administração & dosagem , Vacinas Sintéticas/imunologia , Adalimumab/administração & dosagem , Adalimumab/imunologia , Anafilaxia , Animais , Artrite Experimental/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Feminino , Hiperuricemia/tratamento farmacológico , Ácido Láctico , Macaca fascicularis , Camundongos Transgênicos , Nanopartículas/efeitos adversos , Nanopartículas/química , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos Sprague-Dawley , Sirolimo/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Vacinas Sintéticas/administração & dosagemRESUMO
We report on spatial control of nanoporosity in polyelectrolyte multilayer (PEM) films using photopatterning and its effects on film optical and adsorption properties. Multilayers assembled from poly(acrylic acid-ran-vinylbenzyl acrylate) (PAArVBA), a photo-cross-linking polymer, and poly(allylamine hydrochloric acid) (PAH) were patterned using ultraviolet light followed by immersion in low pH and then neutral pH solutions to induce nanoporosity in unexposed regions. Model charged small molecules rhodamine B, fluorescein, and propidium iodide and the model protein albumin exhibit increased adsorption to nanoporous regions of patterned PEM films as shown by fluorescence microscopy and radiolabeling experiments. Films assembled with alternating stacks of PAH/poly(sodium-4-styrene sulfonate) (SPS), which do not become nanoporous, and stacks of PAH/PAArVBA were patterned to create nanoporous capillary channels. Interdigitated channels demonstrated simultaneous, separate wicking of dimethyl sulfoxide-solvated fluorescein and rhodamine B. In addition, these heterostack structures exhibited patternable Bragg reflectivity of greater than 25% due to refractive index differences between the nanoporous and nonporous stacks. Finally, the PEM assembly process coupled with photo-cross-linking was used to create films with two separate stacked reflective patterns with a doubling in reflectivity where patterns overlapped. The combined adsorptive and reflective properties of these films hold promise for applications in diagnostic arrays and therapeutics delivery.
RESUMO
The effects of tether length on cell adhesion to poly(methyl methacrylate)-graft-poly(ethylene oxide), PMMA-g-PEO, comb copolymer films functionalized with the adhesion peptide RGD were investigated. Copolymers having PEO tether lengths of 10 and 22 EO segments were synthesized and coupled with a synthetic peptide that contained both RGD and the synergy sequence PHSRN. Cell spreading assays revealed that the longer polymer tethers increased the rate of spreading and reduced the time required for fibroblasts to form focal adhesions. Fluorescence resonance energy transfer (FRET) measurements indicated a mean separation between integrin-bound peptides of 15.6 +/- 1.4 nm for combs with long (22-mer) tethers, compared with 17.5 +/- 1.3 nm for short (10-mer) tethers, on films of comparable peptide density (approximately 2500 peptides/microm2). The results suggest that the added mobility afforded by the more extensible tethers encouraged the formation of focal adhesions by allowing cells to reorganize tethered peptides on the nanometer length scale. In addition, adhesion peptides were selectively coupled to 10-mer or 22-mer PEO tethers within a bimodal brush to investigate stratification effects on cell adhesion. Peptides bound by short tethers in a bed of long unsubstituted chains resulted in surfaces that resisted, rather than promoted, cell adhesion. By contrast, when long peptide tethers were employed with short unsubstituted chains, cell attachment and spreading were comparable to that found on a monomodal brush of long chains at equivalent peptide density.
Assuntos
Materiais Biocompatíveis/química , Oligopeptídeos/química , Polietilenoglicóis/química , Polímeros/química , Polimetil Metacrilato/química , Actinas/química , Adesão Celular , Transferência Ressonante de Energia de Fluorescência , Ligantes , Modelos Químicos , Peso Molecular , Peptídeos/química , Engenharia de Proteínas/métodos , Propriedades de Superfície , Fatores de TempoRESUMO
Comb copolymers comprising a poly(methyl methacrylate) (PMMA) backbone and short, polyethylene oxide (PEO) side chains, PMMA-g-PEO, have been proposed to self-organize at the polymer/water interface, resulting in the quasi-2D confinement of the backbone for chains at the immediate surface of PMMA-g-PEO films (D.J. Irvine et al., Biomacromolecules2001, 2, 85-94). To directly probe such 2D conformations, combs modified with maleimide groups on the PEO chain ends were blended at 0.5-10 wt% into unmodified PMMA-g-PEO (M(n) 142 kg/mol, PDI 3.2, 32 wt% PEO) and cast into films â¼35 nm thick. Films were immersed in aqueous solution to induce orientation of surface molecules, and maleimide-functionalized chains at the film/water interface were labeled with 1.4 nm dia. Au nanoparticles. Transmission electron microscopy (TEM) was then used to trace the 2D trajectories of nanoparticle-decorated chains. The distribution of observed chain lengths was in good agreement with that from gel permeation chromatography. The 2D radius of gyration (R(g)) calculated from the observed conformations scaled with number of backbone segments (N) as R(g)â¼N(0.69±0.02). Monte Carlo simulations of a 2D melt of comparable chain length distribution yielded a scaling exponent ν=0.67±0.03, suggesting that the deviation from 2D melt behavior arose from polydispersity.