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Bioprinting of hydrogel-based bioinks can allow for the fabrication of elaborate, cell-laden 3D structures. In addition to providing an adequate extracellular matrix mimetic environment and high cell viability, the hydrogels must offer facile extrusion through the printing nozzle and retain the shape of the printed structure. We demonstrate a strategy to incorporate cellulose oxalate nanofibrils in hyaluronan-based hydrogels to generate shear thinning bioinks that allowed for printing of free-standing multilayer structures, covalently cross-linked after bioprinting, yielding long-term stability. The storage modulus of the hydrogels was tunable between 0.5 and 1.5 kPa. The nanocellulose containing hydrogels showed good biocompatibility, with viability of primary human dermal fibroblasts above 80% at day 7 after seeding. The cells were also shown to tolerate the printing process well, with viability above 80% 24 h after printing. We anticipate that this hydrogel system can find broad use as a bioink to produce complex geometries that can support cell growth.
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Bioimpressão , Ácido Hialurônico , Humanos , Impressão Tridimensional , Reologia , Hidrogéis/química , Sobrevivência Celular , Alicerces Teciduais/química , Engenharia TecidualRESUMO
Hydrogels of cellulose nanofibrils (CNFs) are promising wound dressing candidates due to their biocompatibility, high water absorption, and transparency. Herein, two different commercially available wood species, softwood and hardwood, were subjected to TEMPO-mediated oxidation to proceed with delignification and oxidation in a one-pot process, and thereafter, nanofibrils were isolated using a high-pressure microfluidizer. Furthermore, transparent nanofibril hydrogel networks were prepared by vacuum filtration. Nanofibril properties and network performance correlated with oxidation were investigated and compared with commercially available TEMPO-oxidized pulp nanofibrils and their networks. Softwood nanofibril hydrogel networks exhibited the best mechanical properties, and in vitro toxicological risk assessment showed no detrimental effect for any of the studied hydrogels on human fibroblast or keratinocyte cells. This study demonstrates a straightforward processing route for direct oxidation of different wood species to obtain nanofibril hydrogels for potential use as wound dressings, with softwood having the most potential.
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Celulose , Hidrogéis , Humanos , Bandagens , Oxirredução , FibroblastosRESUMO
The self-assembly of nanocellulose in the form of cellulose nanofibers (CNFs) can be accomplished via hydrogen-bonding assistance into completely bio-based hydrogels. This study aimed to use the intrinsic properties of CNFs, such as their ability to form strong networks and high absorption capacity and exploit them in the sustainable development of effective wound dressing materials. First, TEMPO-oxidized CNFs were separated directly from wood (W-CNFs) and compared with CNFs separated from wood pulp (P-CNFs). Second, two approaches were evaluated for hydrogel self-assembly from W-CNFs, where water was removed from the suspensions via evaporation through suspension casting (SC) or vacuum-assisted filtration (VF). Third, the W-CNF-VF hydrogel was compared to commercial bacterial cellulose (BC). The study demonstrates that the self-assembly via VF of nanocellulose hydrogels from wood was the most promising material as wound dressing and displayed comparable properties to that of BC and strength to that of soft tissue.
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Celulose Oxidada , Nanofibras , Celulose , Hidrogéis , Bactérias , BandagensRESUMO
Astrocytes play an important role in the central nervous system, contributing to the development of and maintenance of synapses, recycling of neurotransmitters, and the integrity and function of the blood-brain barrier. Astrocytes are also linked to the pathophysiology of various neurodegenerative diseases. Astrocyte function and organization are tightly regulated by interactions mediated by the extracellular matrix (ECM). Engineered hydrogels can mimic key aspects of the ECM and can allow for systematic studies of ECM-related factors that govern astrocyte behaviour. In this study, we explore the interactions between neuroblastoma (SH-SY5Y) and glioblastoma (U87) cell lines and human fetal primary astrocytes (FPA) with a modular hyaluronan-based hydrogel system. Morphological analysis reveals that FPA have a higher degree of interactions with the hyaluronan-based gels compared to the cell lines. This interaction is enhanced by conjugation of cell-adhesion peptides (cRGD and IKVAV) to the hyaluronan backbone. These effects are retained and pronounced in 3D bioprinted structures. Bioprinted FPA using cRGD functionalized hyaluronan show extensive and defined protrusions and multiple connections between neighboring cells. Possibilities to tailor and optimize astrocyte-compatible ECM-mimicking hydrogels that can be processed by means of additive biofabrication can facilitate the development of advanced tissue and disease models of the central nervous system.
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Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A-mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein A-functionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 µg/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 µg/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.
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Antineoplásicos Imunológicos , Técnicas Biossensoriais , Imunoglobulina G/química , Anticorpos Monoclonais/química , Limite de Detecção , Técnicas Biossensoriais/métodos , Agregados ProteicosRESUMO
Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.
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Bicamadas Lipídicas , Peptídeos , Colesterol/química , Sistemas de Liberação de Medicamentos , Bicamadas Lipídicas/química , Lipossomos/química , Peptídeos/químicaRESUMO
Solid-state nanopores provide a highly versatile platform for rapid electrical detection and analysis of single molecules. Lipid bilayer coating of the nanopores can reduce nonspecific analyte adsorption to the nanopore sidewalls and increase the sensing selectivity by providing possibilities for tethering specific ligands in a cell-membrane mimicking environment. However, the mechanism and kinetics of lipid bilayer formation from vesicles remain unclear in the presence of nanopores. In this work, we used a silicon-based, truncated pyramidal nanopore array as the support for lipid bilayer formation. Lipid bilayer formation in the nanopores was monitored in real time by the change in ionic current through the nanopores. Statistical analysis revealed that a lipid bilayer is formed from the instantaneous rupture of individual vesicle upon adsorption in the nanopores, differing from the generally agreed mechanism that lipid bilayer forms at a high vesicle surface coverage on a planar support. The dependence of the lipid bilayer formation process on the applied bias, vesicle size, and concentration was systematically studied. In addition, the nonfouling properties of the lipid bilayer coated nanopores were demonstrated during long single-stranded DNA translocation through the nanopore array. The findings indicate that the lipid bilayer formation process can be modulated by introducing nanocavities intentionally on the planar surface to create active sites or changing the vesicle size and concentration.
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Localized surface plasmon resonance (LSPR) constitutes a versatile technique for biodetection, exploiting the sensitivity of plasmonic nanostructures to small changes in refractive index. The optical shift in the LSPR band caused by molecular interactions in the vicinity of the nanostructures are typically <5 nm and can readily be detected by a spectrophotometer. Widespread use of LSPR-based sensors require cost-effective devices and would benefit from sensing schemes that enables use of very simple spectrophotometers or even naked-eye detection. This paper describes a new strategy facilitating visualization of minute optical responses in nanoplasmonic bioassays by taking into account the physiology of human color vision. We demonstrate, using a set of nine different plasmonic nanoparticles, that the cyan to green transition zone at â¼500 nm is optimal for naked-eye detection of color changes. In this wavelength range, it is possible to detect a color change corresponding to a wavelength shift of â¼2-3 nm induced by refractive index changes in the medium or by molecular binding to the surface of the nanoparticles. This strategy also can be utilized to improve the performance of aggregation-based nanoplasmonic colorimetric assays, which enables semiquantitative naked-eye detection of matrix metalloproteinase 7 (MMP7) activity at concentrations that are at least 5 times lower than previously reported assays using spherical gold nanoparticles. We foresee significant potential of this strategy in medical diagnostic and environmental monitoring, especially in situations where basic laboratory infrastructure is sparse or even nonexistent. Finally, we demonstrate that the developed concept can be used in combination with cell phone technology and red-green-blue (RGB) analysis for sensitive and quantitative detection of MMP7.
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Visão de Cores , Colorimetria , Metaloproteinase 7 da Matriz/análise , Nanopartículas/química , Ressonância de Plasmônio de Superfície , Telefone Celular , Humanos , Metaloproteinase 7 da Matriz/metabolismo , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Membrane-active peptides that enable the triggered release of liposomal cargo are of great interest for the development of liposome-based drug delivery systems but require peptide-lipid membrane interactions that are highly defined and tunable. To this end, we have explored the possibility to use the competing interactions between membrane partitioning and heterodimerization and the folding of a set of four different de novo designed coiled coil peptides. Covalent conjugation of the cationic peptides triggered rapid destabilization of membrane integrity and the release of encapsulated species. The release was inhibited when introducing complementary peptides as a result of heterodimerization and folding into coiled coils. The degree of inhibition was shown to be dictated by the coiled coil peptide heterodimer dissociation constants, and liposomal release could be reactivated by a heterodimer exchange to render the membrane bound peptide free and thus membrane-active. The possibility to tune the permeability of lipid membranes using highly specific peptide-folding-dependent interactions delineates a new possible approach for the further development of responsive liposome-based drug delivery systems.
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There is a need for biochemical contrast mediators with high signal-to-noise ratios enabling noninvasive biomedical sensing, for example, for neural sensing and protein-protein interactions, in addition to cancer diagnostics. The translational challenge is to develop a biocompatible approach ensuring high biochemical contrast while avoiding a raise of the background signal. We here present a concept where gold nanoparticles (AuNPs) can be utilized as a stimuli responsive contrast medium by chemically triggering their ability to exhibit multiphoton-induced luminescence (MIL) when performing multiphoton laser scanning microscopy (MPM). Proof-of-principle is demonstrated using peptide-functionalized AuNPs sensitive to zinc ions (Zn2+). Dispersed particles are invisible in the MPM until addition of millimolar concentrations of Zn2+ upon which MIL is enabled through particle aggregation caused by specific peptide interactions and folding. The process can be reversed by removal of the Zn2+ using a chelator, thereby resuspending the AuNPs. In addition, the concept was demonstrated by exposing the particles to matrix metalloproteinase-7 (MMP-7) causing peptide digestion resulting in AuNP aggregation, significantly elevating the MIL signal from the background. The approach is based on the principle that aggregation shifts the plasmon resonance, elevating the absorption cross section in the near-infrared wavelength region enabling onset of MIL. This Letter demonstrates how biochemical sensing can be obtained in far-field MPM and should be further exploited as a future tool for noninvasive optical biosensing.
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Meios de Contraste/química , Ouro/química , Nanopartículas Metálicas/química , Microscopia Confocal/métodos , Peptídeos/química , Sequência de Aminoácidos , Cátions Bivalentes/análise , Dimerização , Humanos , Luminescência , Metaloproteinase 7 da Matriz/análise , Nanopartículas Metálicas/ultraestrutura , Fótons , Zinco/análiseRESUMO
OBJECTIVE: Wound dressings that use biosynthetic cellulose may be a good alternative to dressings currently used to treat chronic and acute ulcers because their nanostructure is similar to collagen. The objective of this study was to evaluate a wound dressing created with a new material that is composed of a fibrillary network of biosynthetic cellulose. METHODS: A case series of 8 patients in primary healthcare centers in Östergötland county council, Sweden, with chronic and acute lower limb wounds were treated with a wound dressing based on eiratex (S2Medical AB, Linköping, Sweden). The dressing was applied to traumatic (n = 5) and venous ulcers (n = 3). All ulcers were considered healed at the end of the treatment. MAIN OUTCOME MEASURE: The wounds were examined at regular intervals by a physician to determine healing time, number of dressing changes, and number of visits. MAIN RESULTS: Mean healing time was 43 ± 6 days after the first application of the dressing. The mean number of visits was 5.7 ± 0.6, and the mean number of dressings used per patient was 1.7 ± 0.2. CONCLUSIONS: These results demonstrate the efficacy of a wound dressing made of eiratex to heal chronic and acute ulcers. The data show that the number of dressings used and dressing changes needed to heal the ulcers are lower than what have been reported in the literature for other dressing materials.
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Curativos Hidrocoloides , Celulose/administração & dosagem , Úlcera do Pé/terapia , Úlcera Varicosa/terapia , Cicatrização/fisiologia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Extremidade Inferior , Masculino , Pessoa de Meia-Idade , Absorção Cutânea/fisiologia , SuéciaRESUMO
A novel approach for rapid and sensitive detection of matrilysin (MMP-7, a biomarker involved in the degradation of various macromolecules) based on a polypeptide (JR2EC) functionalized reduced graphene oxide (rGO) field effect transistor (FET) is reported. MMP-7 specifically digests negatively charged JR2EC immobilized on rGO, thereby modulating the conductance of rGO-FET. The proposed assay enabled detection of MMP-7 at clinically relevant concentrations with a limit of detection (LOD) of 10 ng/mL (400 pM), attributed to the significant reduction of the net charge of JR2EC upon digestion by MMP-7. Quantitative detection of MMP-7 in human plasma was further demonstrated with a LOD of 40 ng/mL, illustrating the potential for the proposed methodology for tumor detection and carcinoma diagnostic (e.g., lung cancer and salivary gland cancer). Additionally, excellent specificity of the proposed assay was demonstrated using matrix metallopeptidase 1 (MMP-1), a protease of the same family. With appropriate selection and modification of polypeptides, the proposed assay could be extended for detection of other enzymes with polypeptide digestion capability.
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Grafite/química , Metaloproteinase 7 da Matriz/metabolismo , Peptídeos/química , Limite de Detecção , Microscopia Eletrônica de Varredura , Óxidos/químicaRESUMO
Shape-controlled synthesis of gold nanoparticles generally involves the use of surfactants, typically cetyltrimethylammonium (CTAX, X = Cl(-) , Br(-)), to regulate the nucleation growth process and to obtain colloidally stable nanoparticles. The surfactants adsorb on the nanoparticle surface making further functionalization difficult and therefore limit their use in many applications. Herein, the influence of CTAX on nanoparticle sensitivity to local dielectric environment changes is reported. It is shown, both experimentally and theoretically, that the CTAX bilayer significantly reduces the refractive index (RI) sensitivity of anisotropic gold nanoparticles such as nanocubes and concave nanocubes, nanorods, and nanoprisms. The RI sensitivity can be increased by up to 40% by removing the surfactant layer from nanoparticles immobilized on a solid substrate using oxygen plasma treatment. This increase compensates for the otherwise problematic decrease in RI sensitivity caused by the substrate effect. Moreover, the removal of the surfactants both facilitates nanoparticle biofunctionalization and significantly improves their catalytic properties. The strategy presented herein is a simple yet effective universal method for enhancing the RI sensitivity of CTAX-stabilized gold nanoparticles and increasing their potential as transducers in nanoplasmonic sensors, as well as in catalytic and biomedical applications.
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BACKGROUND: The complications in healthcare systems associated with antibiotic-resistant microorganisms have resulted in an intense search for new effective antimicrobials. Attractive substances from which novel antibiotics may be developed are the bacteriocins. These naturally occurring peptides are generally considered to be safe and efficient at eliminating pathogenic bacteria. Among specific keystone pathogens in periodontitis, Porphyromonas gingivalis is considered to be the most important pathogen in the development and progression of chronic inflammatory disease. The aim of the present study was to investigate the antimicrobial effects of different Lactobacillus species and the two-peptide bacteriocin PLNC8 αß on P. gingivalis. RESULTS: Growth inhibition of P. gingivalis was obtained by viable Lactobacillus and culture media from L. plantarum NC8 and 44048, but not L. brevis 30670. The two-peptide bacteriocin from L. plantarum NC8 (PLNC8 αß) was found to be efficient against P. gingivalis through binding followed by permeabilization of the membranes, using Surface plasmon resonance analysis and DNA staining with Sytox Green. Liposomal systems were acquired to verify membrane permeabilization by PLNC8 αß. The antimicrobial activity of PLNC8 αß was found to be rapid (1 min) and visualized by TEM to cause cellular distortion through detachment of the outer membrane and bacterial lysis. CONCLUSION: Soluble or immobilized PLNC8 αß bacteriocins may be used to prevent P. gingivalis colonization and subsequent pathogenicity, and thus supplement the host immune system against invading pathogens associated with periodontitis.
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Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Lactobacillus/fisiologia , Periodontite/prevenção & controle , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/fisiologia , Bacteriocinas/química , Parede Celular/efeitos dos fármacos , Dicroísmo Circular/métodos , Técnicas de Cocultura , Difusão Dinâmica da Luz/métodos , Concentração de Íons de Hidrogênio , Lipossomos/farmacologia , Testes de Sensibilidade Microbiana , Periodontite/microbiologia , Estrutura Secundária de ProteínaRESUMO
Physical hydrogels are extensively used in a wide range of biomedical applications. However, different applications require hydrogels with different mechanical and structural properties. Tailoring these properties demands exquisite control over the supramolecular interactions involved. Here we show that it is possible to control the mechanical properties of hydrogels using de novo designed coiled coil peptides with different affinities for dimerization. Four different nonorthogonal peptides, designed to fold into four different coiled coil heterodimers with dissociation constants spanning from µM to pM, were conjugated to star-shaped 4-arm poly(ethylene glycol) (PEG). The different PEG-coiled coil conjugates self-assemble as a result of peptide heterodimerization. Different combinations of PEG-peptide conjugates assemble into PEG-peptide networks and hydrogels with distinctly different thermal stabilities, supramolecular, and rheological properties, reflecting the peptide dimer affinities. We also demonstrate that it is possible to rationally modulate the self-assembly process by means of thermodynamic self-sorting by sequential additions of nonpegylated peptides. The specific interactions involved in peptide dimerization thus provides means for programmable and reversible self-assembly of hydrogels with precise control over rheological properties, which can significantly facilitate optimization of their overall performance and adaption to different processing requirements and applications.
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Hidrogéis/química , Fragmentos de Peptídeos/química , Polietilenoglicóis/química , Polímeros/química , Dimerização , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , Reologia , TermodinâmicaRESUMO
Botulinum neurotoxin is considered as one of the most toxic food-borne substances and is a potential bioweapon accessible to terrorists. The development of an accurate, convenient, and rapid assay for botulinum neurotoxins is therefore highly desirable for addressing biosafety concerns. Herein, novel biotinylated peptide substrates designed to mimic synaptosomal-associated protein 25 (SNAP-25) are utilized in gold nanoparticle-based assays for colorimetric detection of botulinum neurotoxin serotype A light chain (BoLcA). In these proteolytic assays, biotinylated peptides serve as triggers for the aggregation of gold nanoparticles, while the cleavage of these peptides by BoLcA prevents nanoparticle aggregation. Two different assay strategies are described, demonstrating limits of detection ranging from 5 to 0.1 nM of BoLcA with an overall assay time of 4 h. These hybrid enzyme-responsive nanomaterials provide rapid and sensitive detection for one of the most toxic substances known to man.
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Toxinas Botulínicas Tipo A/química , Colorimetria/métodos , Ouro/química , Nanopartículas Metálicas , Sequência de Aminoácidos , Espectrometria de Massas , Dados de Sequência Molecular , Proteólise , Proteína 25 Associada a Sinaptossoma/químicaRESUMO
Possibilities for controlling the release of pharmaceuticals from liposomal drug delivery systems can enhance their efficacy and reduce their side effects. Membrane-active peptides (MAPs) can be tailored to promote liposomal release when conjugated to lipid head groups using thiol-maleimide chemistry. However, the rapid oxidation of thiols hampers the optimization of such conjugation-dependent release strategies. Here, we demonstrate a de novo designed MAP modified with an enzyme-labile Cys-protection group (phenylacetamidomethyl (Phacm)) that prevents oxidation and facilitates in situ peptide lipidation. Before deprotection, the peptide lacks a defined secondary structure and does not interact with maleimide-functionalized vesicles. After deprotection of Cys using penicillin G acylase (PGA), the peptide adopts an α-helical conformation and triggers rapid release of vesicle content. Both the peptide and PGA concentrations significantly influence the conjugation process and, consequently, the release kinetics. At a PGA concentration of 5 µM the conjugation and release kinetics closely mirror those of fully reduced, unprotected peptides. We anticipate that these findings will enable further refinement of MAP conjugation and release processes, facilitating the development of sophisticated bioresponsive MAP-based liposomal drug delivery systems.
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In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.
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Bioimpressão , Neoplasias da Mama , Humanos , Feminino , Microambiente Tumoral , Azidas , Peptídeos/química , Metaloproteinases da Matriz/metabolismo , Hidrogéis/químicaRESUMO
Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.
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Anticorpos Monoclonais , Cromatografia de Afinidade , Imunoglobulina G , Proteína Estafilocócica A , Ressonância de Plasmônio de Superfície , Imunoglobulina G/isolamento & purificação , Cromatografia de Afinidade/métodos , Cromatografia de Afinidade/instrumentação , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Proteína Estafilocócica A/química , Agregados Proteicos , Concentração de Íons de HidrogênioRESUMO
High mammographic density, associated with increased tissue stiffness, is a strong risk factor for breast cancer per se. In postmenopausal women there is no differences in the occurrence of ductal carcinoma in situ (DCIS) depending on breast density. Preliminary data suggest that dense breast tissue is associated with a pro-inflammatory microenvironment including infiltrating monocytes. However, the underlying mechanism(s) remains largely unknown. A major roadblock to understanding this risk factor is the lack of relevant in vitro models. A biologically relevant 3D model with tunable stiffness was developed by cross-linking hyaluronic acid. Breast cancer cells were cultured with and without freshly isolated human monocytes. In a unique clinical setting, extracellular proteins were sampled using microdialysis in situ from women with various breast densities. We show that tissue stiffness resembling high mammographic density increases the attachment of monocytes to the cancer cells, increase the expression of adhesion molecules and epithelia-mesenchymal-transition proteins in estrogen receptor (ER) positive breast cancer. Increased tissue stiffness results in increased secretion of similar pro-tumorigenic proteins as those found in human dense breast tissue including inflammatory cytokines, proteases, and growth factors. ER negative breast cancer cells were mostly unaffected suggesting that diverse cancer cell phenotypes may respond differently to tissue stiffness. We introduce a biological relevant model with tunable stiffness that resembles the densities found in normal breast tissue in women. The model will be key for further mechanistic studies. Additionally, our data revealed several pro-tumorigenic pathways that may be exploited for prevention and therapy against breast cancer. STATEMENT OF SIGNIFICANCE: Women with mammographic high-density breasts have a 4-6-fold higher risk of breast cancer than low-density breasts. Biological mechanisms behind this increase are not fully understood and no preventive therapeutics are available. One major reason being a lack of suitable experimental models. Having such models available would greatly enhance the discovery of relevant targets for breast cancer prevention. We present a biologically relevant 3D-model for studies of human dense breasts, providing a platform for investigating both biophysical and biochemical properties that may affect cancer progression. This model will have a major scientific impact on studies for identification of novel targets for breast cancer prevention.