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The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.
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Lentivirus , Estomatite Vesicular , Animais , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Células HEK293 , Peptídeos/metabolismo , Vesiculovirus/genética , Vetores GenéticosRESUMO
Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.
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The study of plant electrophysiology offers promising techniques to track plant health and stress in vivo for both agricultural and environmental monitoring applications. Use of superficial electrodes on the plant body to record surface potentials may provide new phenotyping insights. Bacterial nanocellulose (BNC) is a flexible, optically translucent, and water-vapor-permeable material with low manufacturing costs, making it an ideal substrate for non-invasive and non-destructive plant electrodes. This work presents BNC electrodes with screen-printed carbon (graphite) ink-based conductive traces and pads. It investigates the potential of these electrodes for plant surface electrophysiology measurements in comparison to commercially available standard wet gel and needle electrodes. The electrochemically active surface area and impedance of the BNC electrodes varied based on the annealing temperature and time over the ranges of 50 °C to 90 °C and 5 to 60 min, respectively. The water vapor transfer rate and optical transmittance of the BNC substrate were measured to estimate the level of occlusion caused by these surface electrodes on the plant tissue. The total reduction in chlorophyll content under the electrodes was measured after the electrodes were placed on maize leaves for up to 300 h, showing that the BNC caused only a 16% reduction. Maize leaf transpiration was reduced by only 20% under the BNC electrodes after 72 h compared to a 60% reduction under wet gel electrodes in 48 h. On three different model plants, BNC-carbon ink surface electrodes and standard invasive needle electrodes were shown to have a comparable signal quality, with a correlation coefficient of >0.9, when measuring surface biopotentials induced by acute environmental stressors. These are strong indications of the superior performance of the BNC substrate with screen-printed graphite ink as an electrode material for plant surface biopotential recordings.
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Grafite , Agricultura , Transporte Biológico , Carbono , Clorofila , VaporRESUMO
The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene-peptide (CAP) hybrid ligands for the rapid and discrete photo-responsive capture and release of blood coagulation Factor VIII (FVIII). A predictive method - based on amino acid sequence and molecular architecture of CAPs - was developed to correlate the conformation of cis/trans CAP photo-isomers to FVIII binding and release. The combined in silico and in vitro analysis of FVIII:peptide interactions guided the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G-cycloAZOB[Lys-YYKHLYN-Lys]-G on translucent chromatographic beads, featured high binding capacity (> 6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics (τ < 30s) when exposed to 420-450 nm light at the intensity of 0.1 W·cm-2. The adsorbent purified FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life-saving biotherapeutics.
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Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands-typically camelid antibodies-that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH = 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (KD ~ 10-5 -10- 6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC10% > 1013 vp/mL of resin) and product yields (~50%-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50%-80%), 80- to 400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.
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Dependovirus , Peptídeos , Humanos , Dependovirus/genética , Células HEK293 , Ligantes , Peptídeos/genética , Peptídeos/metabolismo , Sequência de Aminoácidos , Vetores GenéticosRESUMO
Neural interfaces provide a window into the workings of the nervous system-enabling both biosignal recording and modulation. Traditionally, neural interfaces have been restricted to implanted electrodes to record or modulate electrical activity of the nervous system. Although these electrode systems are both mechanically and operationally robust, they have limited utility due to the resultant macroscale damage from invasive implantation. For this reason, novel nanomaterials are being investigated to enable new strategies to chronically interact with the nervous system at both the cellular and network level. In this feature article, the use of nanomaterials to improve current electrophysiological interfaces, as well as enable new nano-interfaces to modulate neural activity via alternative mechanisms, such as remote transduction of electromagnetic fields are explored. Specifically, this article will review the current use of nanoparticle coatings to enhance electrode function, then an analysis of the cutting-edge, targeted nanoparticle technologies being utilized to interface with both the electrophysiological and biochemical behavior of the nervous system will be provided. Furthermore, an emerging, specialized-use case for neural interfaces will be presented: the modulation of the blood-brain barrier.
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Viral vectors are an emerging, exciting class of biologics whose application in vaccines, oncology, and gene therapy has grown exponentially in recent years. Following first regulatory approval, this class of therapeutics has been vigorously pursued to treat monogenic disorders including orphan diseases, entering hundreds of new products into pipelines. Viral vector manufacturing supporting clinical efforts has spurred the introduction of a broad swath of analytical techniques dedicated to assessing the diverse and evolving panel of Critical Quality Attributes (CQAs) of these products. Herein, we provide an overview of the current state of analytics enabling measurement of CQAs such as capsid and vector identities, product titer, transduction efficiency, impurity clearance etc. We highlight orthogonal methods and discuss the advantages and limitations of these techniques while evaluating their adaptation as process analytical technologies. Finally, we identify gaps and propose opportunities in enabling existing technologies for real-time monitoring from hardware, software, and data analysis viewpoints for technology development within viral vector biomanufacturing.
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Vetores Genéticos , Humanos , Animais , Terapia Genética , Vírus/genética , Vírus/isolamento & purificação , Controle de QualidadeRESUMO
Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈1014 vp mL-1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%).
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Capsídeo , Dependovirus , Humanos , Dependovirus/genética , Capsídeo/química , Células HEK293 , Transdução Genética , Peptídeos/metabolismo , Ligantes , Cromatografia de Afinidade , Vetores Genéticos/genéticaRESUMO
Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 1014 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy.
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BACKGROUND AND AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). METHODS: hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.
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Inflamação , Interleucinas , Humanos , Interleucinas/metabolismo , Inflamação/metabolismo , Células-Tronco/metabolismo , Hipóxia , Oxigênio/metabolismoRESUMO
Background & Aims: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces 'inflammatory hypoxia', a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. Methods: hISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1α response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. Results: The novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1α at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins. Conclusions: Hypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.
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The isolation of a single type of protein from a complex mixture is vital for the characterization of the function, structure, and interactions of the protein of interest and is typically the most laborious aspect of the protein purification process. In this work, a model system is utilized to show the efficacy of synthesizing a "baited" nanoparticle to capture and recycle enzymes (proteins that catalyze chemical reactions) from crude cell lysate. Enzyme trapping and recycling is illustrated with the carbazole 1,9a-dioxygenase (CARDO) system, an enzyme important in bioremediation and natural product synthesis. The enzymes are baited with azide-modified carbazolyl moieties attached to poly(propargyl acrylate) nanoparticles through a click transformation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates the single-step procedure to immobilize the enzymes on the particles is capable of significantly concentrating the protein from raw lysate and sequestering all required components of the protein to maintain bioactivity. These results establish a universal model applicable to concentrating and extracting known substrate-protein pairs, but it can be an invaluable tool in recognizing unknown protein-ligand affinities.
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Enzimas/isolamento & purificação , Enzimas/metabolismo , Nanopartículas/química , Química Click/métodos , Enzimas/química , Nanotecnologia/métodosRESUMO
Carbon nanotubes (CNTs) are known for their excellent conductive properties. Here, we present two novel methods, "sandwich" (sCNT) and dual deposition (DD CNT), for incorporating CNTs into electrospun polycaprolactone (PCL) and gelatin scaffolds to increase their conductance. Based on CNT percentage, the DD CNT scaffolds contain significantly higher quantities of CNTs than the sCNT scaffolds. The inclusion of CNTs increased the electrical conductance of scaffolds from 0.0 ± 0.00 kS (non-CNT) to 0.54 ± 0.10 kS (sCNT) and 5.22 ± 0.49 kS (DD CNT) when measured parallel to CNT arrays and to 0.25 ± 0.003 kS (sCNT) and 2.85 ± 1.12 (DD CNT) when measured orthogonally to CNT arrays. The inclusion of CNTs increased fiber diameter and pore size, promoting cellular migration into the scaffolds. CNT inclusion also decreased the degradation rate and increased hydrophobicity of scaffolds. Additionally, CNT inclusion increased Young's modulus and failure load of scaffolds, increasing their mechanical robustness. Murine fibroblasts were maintained on the scaffolds for 30 days, demonstrating high cytocompatibility. The increased conductivity and high cytocompatibility of the CNT-incorporated scaffolds make them appropriate candidates for future use in cardiac and neural tissue engineering.
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Wearable and wireless monitoring of biomarkers such as lactate in sweat can provide a deeper understanding of a subject's metabolic stressors, cardiovascular health, and physiological response to exercise. However, the state-of-the-art wearable and wireless electrochemical systems rely on active sweat released either via high-exertion exercise, electrical stimulation (such as iontophoresis requiring electrical power), or chemical stimulation (such as by delivering pilocarpine or carbachol inside skin), to extract sweat under low-perspiring conditions such as at rest. Here, we present a continuous sweat lactate monitoring platform combining a hydrogel for osmotic sweat extraction, with a paper microfluidic channel for facilitating sweat transport and management, a screen-printed electrochemical lactate sensor, and a custom-built wireless wearable potentiostat system. Osmosis enables zero-electrical power sweat extraction at rest, while continuous evaporation at the end of a paper channel allows long-term sensing from fresh sweat. The positioning of the lactate sensors provides near-instantaneous sensing at low sweat volume, and the custom-designed potentiostat supports continuous monitoring with ultra-low power consumption. For a proof of concept, the prototype system was evaluated for continuous measurement of sweat lactate across a range of physiological activities with changing lactate concentrations and sweat rates: for 2 h at the resting state, 1 h during medium-intensity exercise, and 30 min during high-intensity exercise. Overall, this wearable system holds the potential of providing comprehensive and long-term continuous analysis of sweat lactate trends in the human body during rest and under exercising conditions.
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Suor , Dispositivos Eletrônicos Vestíveis , Humanos , Ácido Láctico/análise , Monitorização Fisiológica , Osmose , Suor/químicaRESUMO
Advances in materials, bio-recognition elements, transducers, and microfabrication techniques, as well as progress in electronics, signal processing, and wireless communication have generated a new class of skin-interfaced wearable health monitoring systems for applications in personalized medicine and digital health. In comparison to conventional medical devices, these wearable systems are at the cusp of initiating a new era of longitudinal and noninvasive sensing for the prevention, detection, diagnosis, and treatment of diseases at the molecular level. Herein, we provide a review of recent developments in wearable biochemical and biophysical systems. We survey the sweat sampling and collection methods for biochemical systems, followed by an assessment of biochemical and biophysical sensors deployed in current wearable systems with an emphasis on their hardware specifications. Specifically, we address how sweat collection and sample handling platforms may be a rate limiting technology to realizing the clinical translation of wearable health monitoring systems; moreover, we highlight the importance of achieving both longitudinal sensing and assessment of intrapersonal variation in sweat-blood correlations to have the greatest clinical impact. Lastly, we assess a snapshot of integrated wireless wearable systems with multimodal sensing capabilities, and we conclude with our perspective on the state-of-the-art and the required developments to achieve the next-generation of integrated wearable health and performance monitoring systems.
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Técnicas Biossensoriais , Dispositivos Eletrônicos Vestíveis , Eletrônica , Monitorização Fisiológica , SuorRESUMO
Operating at low sweat rates, such as those experienced by humans at rest, is still an unmet need for state-of-the-art wearable sweat harvesting and testing devices for lactate. Here, we report the on-skin performance of a non-invasive wearable sweat sampling patch that can harvest sweat at rest, during exercise, and post-exercise. The patch simultaneously uses osmosis and evaporation for long-term (several hours) sampling of sweat. Osmotic sweat withdrawal is achieved by skin-interfacing a hydrogel containing a concentrated solute. The gel interfaces with a paper strip that transports the fluid via wicking and evaporation. Proof of concept results show that the patch was able to sample sweat during resting and post-exercise conditions, where the lactate concentration was successfully quantified. The patch detected the increase in sweat lactate levels during medium level exercise. Blood lactate remained invariant with exercise as expected. We also developed a continuous sensing version of the patch by including enzymatic electrochemical sensors. Such a battery-free, passive, wearable sweat sampling patch can potentially provide useful information about the human metabolic activity.
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Suor , Dispositivos Eletrônicos Vestíveis , Humanos , Hidrogéis , Ácido Láctico , SudoreseRESUMO
Ultrasound-powered implants (UPIs) represent cutting edge power sources for implantable medical devices (IMDs), as their powering strategy allows for extended functional lifetime, decreased size, increased implant depth, and improved biocompatibility. IMDs are limited by their reliance on batteries. While batteries proved a stable power supply, batteries feature relatively large sizes, limited life spans, and toxic material compositions. Accordingly, energy harvesting and wireless power transfer (WPT) strategies are attracting increasing attention by researchers as alternative reliable power sources. Piezoelectric energy scavenging has shown promise for low power applications. However, energy scavenging devices need be located near sources of movement, and the power stream may suffer from occasional interruptions. WPT overcomes such challenges by more stable, on-demand power to IMDs. Among the various forms of WPT, ultrasound powering offers distinct advantages such as low tissue-mediated attenuation, a higher approved safe dose (720 mW cm-2 ), and improved efficiency at smaller device sizes. This study presents and discusses the state-of-the-art in UPIs by reviewing piezoelectric materials and harvesting devices including lead-based inorganic, lead-free inorganic, and organic polymers. A comparative discussion is also presented of the functional material properties, architecture, and performance metrics, together with an overview of the applications where UPIs are being deployed.
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Fontes de Energia Elétrica , Próteses e Implantes , Movimento , UltrassonografiaRESUMO
Chronic wounds can occur when the healing process is disrupted and the wound remains in a prolonged inflammatory stage that leads to severe tissue damage and poor healing outcomes. Clinically used treatments, such as high density, FDA-approved fibrin sealants, do not provide an optimal environment for native cell proliferation and subsequent tissue regeneration. Therefore, new treatments outside the confines of these conventional fibrin bulk gel therapies are required. We have previously developed flowable, low-density fibrin nanoparticles that, when coupled to keratinocyte growth factor, promote cell migration and epithelial wound closure in vivo. Here, we report a new high throughput method for generating the fibrin nanoparticles using probe sonication, which is less time intensive than the previously reported microfluidic method, and investigate the ability of the sonicated fibrin nanoparticles (SFBN) to promote clot formation and cell migration in vitro. The SFBNs can form a fibrin gel when combined with fibrinogen in the absence of exogenous thrombin, and the polymerization rate and fiber density in these fibrin clots is tunable based on SFBN concentration. Furthermore, fibrin gels made with SFBNs support cell migration in an in vitro angiogenic sprouting assay, which is relevant for wound healing. In this report, we show that SFBNs may be a promising wound healing therapy that can be easily produced and delivered in a flowable formulation.
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Fibrina , Nanopartículas , Adesivo Tecidual de Fibrina , Polimerização , CicatrizaçãoRESUMO
The development of wearable multiplexed biosensors has been focused on systems to measure sweat l-lactate and other metabolites, where the employment of the direct electron transfer (DET) principle is expected. In this paper, a fusion enzyme between an engineered l-lactate oxidase derived from Aerococcus viridans, AvLOx A96L/N212K mutant, which is minimized its oxidase activity and b-type cytochrome protein was constructed to realize multiplexed DET-type lactate and glucose sensors. The sensor with a fusion enzyme showed DET to a gold electrode, with a limited operational range less than 0.5 mM. A mutation was introduced into the fusion enzyme to increase Km value and eliminate its substrate inhibition to construct "b2LOxS". Together with the employment of an outer membrane, the detection range of the sensor with b2LOxS was expanded up to 10 mM. A simultaneous lactate and glucose monitoring system was constructed using a flexible thin-film multiplexed electrodes with b2LOxS and a DET-type glucose dehydrogenase, and evaluated their performance in the artificial sweat. The sensors achieved simultaneous detection of lactate and glucose without cross-talking error, with the detected linear ranges of 0.5-20 mM for lactate and 0.1-5 mM for glucose, sensitivities of 4.1 nA/mMâmm2 for lactate and 56 nA/mMâmm2 for glucose, and limit of detections of 0.41 mM for lactate and 0.057 mM for glucose. The impact of the presence of electrochemical interferants (ascorbic acid, acetaminophen and uric acid), was revealed to be negligible. This is the first report of the DET-type enzyme based lactate and glucose dual sensing systems.
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Técnicas Biossensoriais , Glicemia , Automonitorização da Glicemia , Eletrodos , Elétrons , Glucose , Glucose Oxidase , L-Lactato DesidrogenaseRESUMO
The ability to measure microtissue contraction in vitro can provide important information when modeling cardiac, cardiovascular, respiratory, digestive, dermal, and skeletal tissues. However, measuring tissue contraction in vitro often requires the use of high number of cells per tissue construct along with time-consuming microscopy and image analysis. Here, we present an inexpensive, versatile, high-throughput platform to measure microtissue contraction in a 96-well plate configuration using one-step batch imaging. More specifically, optical fiber microprobes are embedded in microtissues, and contraction is measured as a function of the deflection of optical signals emitted from the end of the fibers. Signals can be measured from all the filled wells on the plate simultaneously using a digital camera. An algorithm uses pixel-based image analysis and computer vision techniques for the accurate multiwell quantification of positional changes in the optical microprobes caused by the contraction of the microtissues. Microtissue constructs containing 20,000-100,000 human ventricular cardiac fibroblasts (NHCF-V) in 6 mg/mL collagen type I showed contractile displacements ranging from 20-200 µm. This highly sensitive and versatile platform can be used for the high-throughput screening of microtissues in disease modeling, drug screening for therapeutics, physiology research, and safety pharmacology.