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Lateral-flow immunoassays (LFAs) can be used to diagnose urinary tract infections caused by Escherichia coli (E. coli) at the point of care. Unfortunately, urine samples containing dilute concentrations of E. coli can yield false negative results on LFAs. Our laboratory was first to implement aqueous two-phase systems (ATPSs) to preconcentrate samples into smaller volumes prior to their application on LFAs. This is achieved by manipulating the ratio of the volume of the top phase to that of the bottom phase (volume ratio; VR) and concentrating biomarkers in the bottom phase which, when applied to LFAs in fixed volumes, leads to corresponding improvements in sensitivity. This work is the first demonstration that the same LOD can be achieved irrespective of the VR when the entire bottom phase is added to LFAs. A custom 3D-printed device was also developed to decrease liquid handling steps. Across different VRs expected from patient urine variability, this diagnostic workflow successfully detected E. coli concentrations down to 2 × 105 colony-forming units (cfu) mL-1 in synthetic urine, demonstrating consistent 10-fold improvements in sensitivity compared to trials conducted without ATPS preconcentration. This method successfully addresses the variability of patient samples while remaining easy to use at the point of care.
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In this work, we present a novel point-of-care hydrogel-based diagnostic device for the rapid detection of elevated bicarbonate levels in serum for the diagnosis of mild to severe cases of metabolic alkalosis. Our system consists of hydrogel beads composed of calcium alginate and the nonionic polymer dextran. This assay utilizes the reaction of sodium bicarbonate and citric acid to produce citrate, a metal chelator capable of competitively binding to calcium cations in the gel matrix to trigger hydrogel degradation. This results in successful detection of elevated bicarbonate concentrations in less than one hour. Specifically, critically high bicarbonate concentrations of 50, 45, and 40 mmol L-1 in human serum were detected in as little as 10, 15, and 20 min, respectively. To demonstrate the assay's feasibility for use in resource-limited settings, we developed a simple electronic device that achieved similar results and could be used by untrained individuals with no lab equipment and minimal power. To our knowledge, this is the first demonstration of the use of nonionic polymers to synthesize and improve the morphology of calcium alginate hydrogel beads using a simple processing method that involves minimal labor and equipment. The simplified bead synthesis protocol combined with the user-friendly device allows for the rapid detection of metabolic alkalosis at the point of care.
Assuntos
Alcalose , Bicarbonatos , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Hidrogéis , Ácido Cítrico , AlginatosRESUMO
We are the first to combine the lateral-flow immunoassay (LFA) with gold nanorod (GNR) etching to achieve a multicolor readout where the color produced was correlated with digoxin concentrations in human serum in the relevant range for therapeutic drug monitoring of 0.5-3.0 ng mL-1.
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Ouro , Nanotubos , Digoxina , Humanos , Imunoensaio , Limite de DetecçãoRESUMO
Malaria is an infectious disease that can cause severe sickness and death if not diagnosed and treated in a timely manner. The current gold standard technique for malaria diagnosis is microscopy, which requires a dedicated laboratory setting and trained personnel and can have a long time to result. These requirements can be alleviated using paper-based diagnostic devices that enable rapid and inexpensive diagnosis at the point of care, which can allow patients to receive treatment before their symptoms progress when used for early detection of diseases. The lateral-flow immunoassay (LFA) is one such device, but currently available LFAs are susceptible to false negative results caused by low parasite density. To improve sensitivity and detection, we utilized the aqueous two-phase system (ATPS) to concentrate and purify the sample, and nanozyme signal enhancement to increase the intensity of the visible signal on the test strip. We were able to achieve a limit of detection (LOD) of 0.01 ng/mL for the malaria biomarker Plasmodium lactate dehydrogenase (pLDH) in human serum using a multi-step assay combining the LFA format with the ATPS and nanozyme signal enhancement.
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Malária , Plasmodium , Humanos , L-Lactato Desidrogenase , Imunoensaio/métodos , Limite de Detecção , Malária/diagnósticoRESUMO
We developed an innovative 3D printed casing that incorporates a lateral-flow immunoassay, dehydrated signal enhancement reagents, and a sealed buffer chamber. With only the push of a button for signal enhancement, our device detected the SARS-CoV-2 N-protein in 40 min at concentrations as low as 0.1 ng mL-1 in undiluted serum.
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COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Humanos , Imunoensaio , Sensibilidade e EspecificidadeRESUMO
Infectious diseases remain one of the leading causes of deaths in developing countries because of a lack of basic sanitation, healthcare clinics, and centralized laboratories. Paper-based rapid diagnostic tests, such as the lateral-flow immunoassay (LFA), provide a promising alternative to the traditional laboratory-based tests; however, they typically suffer from having a poor sensitivity. Biomarker preconcentration and signal enhancement are two common methods to improve the sensitivity of paper-based assays. While effective, these methods often require multiple liquid handling steps which are not ideal for use by untrained personnel in a point-of-care setting. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of concentration and detection of biomarkers on the LFA. In this work, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS. We applied this technology to automate biomarker concentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step.
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Automação , Escherichia coli O157/isolamento & purificação , Ouro/química , Imunoensaio , Papel , Platina/química , Biomarcadores/análise , Células Cultivadas , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Infectious diseases remain one of the major causes of death worldwide in developing countries. While screening via conventional polymerase chain reaction (PCR) is the gold standard in laboratory testing, its limited applications at the point-of-care have prompted the development of more portable nucleic acid detection systems. These include isothermal DNA amplification techniques, which are less equipment-intensive than PCR. Unfortunately, these techniques still require extensive sample preparation, limiting user accessibility. In this study, we introduce a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar aqueous two-phase system (ATPS) to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to amplify and detect a target gene from whole-cell samples containing as low as 102 cfu/mL, and is the first known application of ATPSs to isothermal DNA amplification. This system's ease-of-use and sensitivity underlie its potential as a point-of-care diagnostic platform to detect for infectious diseases. Graphical abstract á .
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DNA Bacteriano/genética , Escherichia coli O157/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , DNA Helicases/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Limite de Detecção , Micelas , Octoxinol/química , Transição de Fase , Sistemas Automatizados de Assistência Junto ao Leito , Temperatura , Água/químicaRESUMO
The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.
Assuntos
Toxinas Bacterianas/genética , Modelos Teóricos , Neoplasias/tratamento farmacológico , Transferrina/genética , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Toxina Diftérica , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mutagênese Sítio-Dirigida , Mutação , Transferrina/análogos & derivados , Transferrina/uso terapêuticoRESUMO
The development of point-of-need (PON) diagnostics for viruses has the potential to prevent pandemics and protects against biological warfare threats. Here we discuss the approach of using aqueous two-phase systems (ATPSs) to concentrate biomolecules prior to the lateral-flow immunoassay (LFA) for improved viral detection. In this paper, we developed a rapid PON detection assay as an extension to our previous proof-of-concept studies which used a micellar ATPS. We present our investigation of a more rapid polymer-salt ATPS that can drastically improve the assay time, and show that the phase containing the concentrated biomolecule can be extracted prior to macroscopic phase separation equilibrium without affecting the measured biomolecule concentration in that phase. We could therefore significantly decrease the time of the diagnostic assay with an early extraction time of just 30 min. Using this rapid ATPS, the model virus bacteriophage M13 was concentrated between approximately 2 and 10-fold by altering the volume ratio between the two phases. As the extracted virus-rich phase contained a high salt concentration which destabilized the colloidal gold indicator used in LFA, we decorated the gold nanoprobes with polyethylene glycol (PEG) to provide steric stabilization, and used these nanoprobes to demonstrate a 10-fold improvement in the LFA detection limit. Lastly, a MATLAB script was used to quantify the LFA results with and without the pre-concentration step. This approach of combining a rapid ATPS with LFA has great potential for PON applications, especially as greater concentration-fold improvements can be achieved by further varying the volume ratio. Biotechnol. Bioeng. 2014;111: 2499-2507. © 2014 Wiley Periodicals, Inc.
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Imunoensaio/métodos , Polietilenoglicóis/química , Cloreto de Sódio/química , Vírus/isolamento & purificação , Bacteriófago M13 , Coloides , Ouro , Limite de Detecção , Técnicas de Sonda Molecular , Nanoestruturas , Cultura de Vírus , Vírus/químicaRESUMO
Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Transporte Biológico Ativo , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Citoesqueleto/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Biológicos , Modelos Moleculares , Pinocitose , Lipossomas Unilamelares/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. SCOPE OF REVIEW: We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). MAJOR CONCLUSIONS: Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. GENERAL SIGNIFICANCE: Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.
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Ferro/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Endocitose , Endossomos/metabolismo , Humanos , Transporte de Íons , Receptores da Transferrina/químicaRESUMO
Block polypeptides are an emerging class of materials that have the potential to be used in many biomedical applications, including the field of drug delivery. We have previously developed a negatively charged block copolypeptide, poly(L-glutamate)60-b-poly(L-leucine)20 (E60L20), which forms spherical vesicles in aqueous solution. Since these vesicles are negatively charged, they are minimally toxic toward cells. However, the negative charge also inhibits these vesicles from effectively being internalized by cells, which can be problematic as many therapeutics have intracellular targets. To overcome this limitation of the E60L20 vesicles, transferrin (Tf) was conjugated onto the vesicle surface, since the receptor for Tf is overexpressed on cancer cells. The enhanced uptake of the Tf-conjugated vesicle was verified through confocal microscopy. Furthermore, endocytosis and immunostaining experiments confirmed that the Tf conjugated on the vesicle surface plays a critical role in the internalization and subsequent intracellular trafficking behavior of the vesicles.
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Portadores de Fármacos/síntese química , Endocitose , Peptídeos/química , Ácido Poliglutâmico/análogos & derivados , Transferrina/química , Transporte Biológico , Linhagem Celular Tumoral , Portadores de Fármacos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Microscopia Confocal , Peptídeos/metabolismo , Ácido Poliglutâmico/metabolismo , Receptores da Transferrina/metabolismo , Eletricidade Estática , Transferrina/metabolismo , ÁguaRESUMO
The development of nanoscale drug delivery vehicles is an exciting field due to the ability of these vehicles to improve the pharmacokinetic and pharmacodynamic properties of existing therapeutics. These vehicles can improve drug effectiveness and safety by providing benefits such as increased blood circulation, targeted delivery, and controlled release. With regard to the building blocks, amphiphilic polypeptide and polypeptide hybrid (i.e., a macromolecule comprised of a polypeptide and another type of polymer) systems have been recently investigated for their abilities to self-assemble into vesicles. Advances in synthesis methodologies have allowed the development and characterization of many different amphiphilic polypeptide and polypeptide hybrid systems. In this review, we will discuss these vesicle-forming materials in terms of their synthesis, processing, and characterization. In addition, current efforts to use them for drug delivery purposes will be discussed.
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Materiais Biocompatíveis/química , Preparações de Ação Retardada/química , Portadores de Fármacos/química , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Tensoativos/metabolismo , Animais , Materiais Biocompatíveis/metabolismo , Preparações de Ação Retardada/metabolismo , Portadores de Fármacos/metabolismo , Composição de Medicamentos , Humanos , Camundongos , Microscopia , Peptídeos/química , Polimerização , Técnicas de Síntese em Fase Sólida , Tensoativos/químicaRESUMO
We demonstrate that recent observed increases in cell permeation activity of cyclic peptides via transporter sequences is due to the underlying phase behavior of peptide-lipid complexes and its relation to the topological requirement of membrane permeation. We also show how these effects can be amplified by incorporating hydrophobicity in these sequences.
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The lateral-flow (immuno)assay (LFA) has been widely investigated for the detection of molecular, macromolecular, and particle targets at the point-of-need due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements. However, for some analytes, such as certain proteins, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay, and needs to be improved. One solution for improving the detection limit of LFA is to concentrate the target protein in a solution prior to the detection step. In this study, a novel approach was used in the context of an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 to concentrate a model protein, namely transferrin, prior to LFA. Proteins have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin were used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by tenfold from 0.5 to 0.05 µg/mL in a predictive manner. In addition to enhancing the sensitivity of LFA, this universal concentration method could also be used to improve other detection assays.
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Fracionamento Químico/métodos , Imunoensaio/métodos , Transferrina/análise , Transferrina/isolamento & purificação , Imunoensaio/instrumentação , Limite de Detecção , MicelasRESUMO
The block copolypeptide poly(l-homoarginine)(60)-b-poly(l-leucine)(20) (R(60)L(20)) was previously found to self-assemble into versatile vesicles with controllable size and encapsulate hydrophilic cargo. These R(60)L(20) vesicles also demonstrated the ability to cross the cell membrane and transport encapsulated cargo into different cell lines. To assess the potential for using the R(60)L(20) vesicles as drug delivery vehicles further, we have investigated their endocytosis and intracellular trafficking behavior. Using drugs that inhibit different endocytosis pathways, we identified macropinocytosis to be a major process by which the R(60)L(20) vesicles enter HeLa cells. Subsequent immunostaining experiments demonstrated that the vesicles entered the early endosomes but not the lysosomes, suggesting that they recycle back to the cell surface. Overall, our studies indicate that the R(60)L(20) vesicles are able to enter cells intact with their cargos, and although some manage to escape from early endosomes, most are trapped within these intracellular compartments.
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Sistemas de Liberação de Medicamentos/métodos , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Homoarginina , Lisossomos/metabolismo , Peptídeos/farmacocinética , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Foodborne illness is a major public health issue that results in millions of global infections annually. The burden of such illness sits mostly with developing countries, as access to advanced laboratory equipment and skilled lab technicians, as well as consistent power sources, is limited and expensive. Current gold standards in foodborne pathogen screening involve labor-intensive sample enrichment steps, pathogen isolation and purification, and costly readout machinery. Overall, time to detection can take multiple days, excluding the time it takes to ship samples to off-site laboratories. Efforts have been made to simplify the workflow of such tests by integrating multiple steps of foodborne pathogen screening procedures into a singular device, as well as implementing more point-of-need readout methods. In this review, we explore recent advancements in developing point-of-need devices for foodborne pathogen screening. We discuss the detection of surface markers, nucleic acids, and metabolic products using both paper-based and microfluidic devices, focusing primarily on developments that have been made between 2015 and mid-2020.
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Doenças Transmitidas por Alimentos , Ácidos Nucleicos , Doenças Transmitidas por Alimentos/diagnóstico , Humanos , Dispositivos Lab-On-A-Chip , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
We generated stable amphiphilic copolymer-based polymeric micelles (PMs) with temperature-responsive properties utilizing Pluronic® L35 and a variety of ionic liquids (ILs) to generate different aqueous two-phase micellar systems (ATPMSs). The partitioning of the hydrophobic model compound curcumin (CCM) into the PM-rich phase and the drug delivery capabilities of the PMs were investigated. ATPMSs formed using more hydrophobic ILs (i.e., [Ch][Hex] ≈ [Ch][But] > [Ch][Pro] > [Ch][Ac] ≈ [Ch]Cl) were the most effective in partitioning (KCCM) and recovering (RECRich) CCM into the PM-rich phase (15.2 < KCCM < 22.0 and 90% < RECRich < 95%, respectively). Moreover, using 1.2 M [Ch][But] and 0.2 M [Ch][Hex] ILs yielded higher encapsulation efficiency (EE) (94.1 and 96.0%, respectively) and drug loading (DL) capacity (14.8 and 16.2%, respectively), together with an increase in the average hydrodynamic diameter of the PMs (DH) (42.5 and 45.6 nm, respectively). The CCM-PM formulations were stable at 4.0, 25.0, and 37.0 °C and the release of CCM was faster with the less hydrophobic ILs (i.e., [Ch]Cl and [Ch][Ac]). Furthermore, due to the lower critical solution temperature properties of Pluronic® L35, the PMs exhibit temperature responsiveness at 37.0 °C. In vitro cytotoxicity assays were also performed to determine the potency of CCM-PM formulations, and a 1.8-fold decrease in IC50 values was observed between the CCM-PMs/[Ch][Hex] and CCM-PMs/[Ch]Cl formulations for PC3 cells. The lower IC50 value for the [Ch][Hex] version corresponded to a greater potency compared to the [Ch]Cl version, since a lower concentration of CCM was required to achieve the same therapeutic effect. The ATPMSs investigated in this study serve as a novel platform for Pluronic® L35/PBS buffer (pH 7.4) + IL-based ATPMS development. The unique properties reported here may be useful in applications such as controlled-release drug delivery systems (DDS), encapsulation, and bioseparations.
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Líquidos Iônicos , Micelas , Portadores de Fármacos , Interações Hidrofóbicas e Hidrofílicas , PolímerosRESUMO
Development of cancer therapeutics requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Three-dimensional (3-D) in vitro models have therefore been investigated for drug screening. In this study, we have developed a novel in vitro model in which multicellular aggregates, or spheroids, were incorporated into 3-D porous scaffolds. Drug resistance assays showed that spheroid-seeded scaffolds have much higher drug resistance than monolayer cultures, spheroids on flat substrates, or scaffolds seeded with dispersed cells. Furthermore, spheroid-seeded scaffolds demonstrated higher lactate production leading to acidosis, and higher expression of angiogenic factors. These data suggest that the spheroid-seeded 3-D scaffolds might serve as a useful in vitro system for screening cancer therapeutics.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Esferoides Celulares/efeitos dos fármacos , Alicerces Teciduais , Células Tumorais Cultivadas/efeitos dos fármacos , Humanos , Esferoides Celulares/citologia , Células Tumorais Cultivadas/citologiaRESUMO
Availability of a rapid, accurate, and reliable point-of-care (POC) device for detection of infectious agents and pandemic pathogens, such as swine-origin influenza A (H1N1) virus, is crucial for effective patient management and outbreak prevention. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the lateral-flow (immuno)assay (LFA) has gained much attention in recent years as a possible solution. However, since the sensitivity of LFA has been shown to be inferior to that of the gold standards of pathogen detection, namely cell culture and real-time PCR, LFA remains an ineffective POC assay for preventing pandemic outbreaks. A practical solution for increasing the sensitivity of LFA is to concentrate the target agent in a solution prior to the detection step. In this study, an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 was investigated for concentrating a model virus, namely bacteriophage M13 (M13), prior to LFA. The volume ratio of the two coexisting micellar phases was manipulated to concentrate M13 in the top, micelle-poor phase. The concentration step effectively improved the M13 detection limit of the assay by tenfold from 5 × 10(8) plaque forming units (pfu)/mL to 5 × 10(7) pfu/mL. In the future, the volume ratio can be further manipulated to yield a greater concentration of a target virus and further decrease the detection limits of the LFA.