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Label-free characterization of cell subpopulations is a very promising biomedical approach. Nowadays, there are several label-free methods based on different physical properties such as size, density, stiffness, etc. allowing the characterization of biological objects. However, fluorescence properties are the most suitable feature for the label-free study of tissue and cells. Understanding the autofluorescence level peculiarities of normal and pathological / live and dead cells can become a helpful tool for cells' metabolic activity, viability evaluation, and diagnostics of a number of diseases. In this study, we applied a series of mouse cell lines (RAW 264.7 - macrophages, L929 - fibroblasts, C2C12 - myoblasts, and B16-F10 - melanoma) to compare cell autofluorescence of live and dead cells under 488 nm laser excitation and found the difference between their autofluorescence depending on a cell state and type.
Assuntos
Técnicas Citológicas , Fluorescência , Animais , Linhagem Celular , Sobrevivência Celular , CamundongosRESUMO
Flow cytometry nowadays is among the main working instruments in modern biology paving the way for clinics to provide early, quick, and reliable diagnostics of many blood-related diseases. The major problem for clinical applications is the detection of rare pathogenic objects in patient blood. These objects can be circulating tumor cells, very rare during the early stages of cancer development, various microorganisms and parasites in the blood during acute blood infections. All of these rare diagnostic objects can be detected and identified very rapidly to save a patient's life. This review outlines the main techniques of visualization of rare objects in the blood flow, methods for extraction of such objects from the blood flow for further investigations and new approaches to identify the objects automatically with the modern deep learning methods.
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Separação Celular/métodos , Aprendizado Profundo , Diagnóstico por Imagem/métodos , Citometria de Fluxo/métodos , Automação , Circulação Sanguínea , Separação Celular/instrumentação , Rastreamento de Células , Diagnóstico por Imagem/instrumentação , Testes Diagnósticos de Rotina , Citometria de Fluxo/instrumentação , Corantes Fluorescentes , Doenças Hematológicas/diagnóstico , Humanos , Magnetismo , Células Neoplásicas Circulantes/patologia , Doenças Raras/diagnóstico , Coloração e Rotulagem/métodosRESUMO
Layer-by-layer assembled polymeric multilayer capsules (PMC) of micrometer sizes are permeable for molecules below 1 KDa; therefore, the efficacy of such capsules in the delivery of low molecular weight water soluble bioactive compounds and drugs is frequently challenged. Thermally induced contraction of hollow PMC is explored here to enhance their loading efficacy with model compound, fluorescent rhodamine B (RhB). Four bilayered capsules obtained of poly(diallyldimethylammonium chloride)/polystyrene sulfonate ([PDADMAC/PSS]4 ) or poly-l-arginine/dextran sulfate ([PARG/DS]4 ) on sacrificial CaCO3 spherical microparticles are postloaded with RhB at ambient or elevated temperatures. The influence of heat on capsule loading is determined quantitatively by varying the amounts of capsules in the batch and keeping the concentration of RhB constant. The applied heat improves the loading efficacy of [PDADMAC/PSS]4 capsules at concentrations up to 2.25 × 109 capsules mL-1 , but has a reversed effect on [PARG/DS]4 capsules at all studied concentrations ((0-3.5) × 109 capsules mL-1 ).
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Cápsulas/química , Temperatura Alta , Polímeros/química , Rodaminas/química , Sulfato de Dextrana/química , Peptídeos/química , Polietilenos/química , Poliestirenos/química , Compostos de Amônio Quaternário/químicaRESUMO
The paradigm of drug delivery via particulate formulations is one of the leading ideas that enable overcoming limitations of traditional chemotherapeutic agents. The trend toward more complex multifunctional drug carriers is well-traced in the literature. Nowadays, the prospectiveness of stimuli-responsive systems capable of controlled cargo release in the lesion nidus is widely accepted. Both endogenous and exogenous stimuli are employed for this purpose; however, endogenous pH is the most common trigger. Unfortunately, scientists encounter multiple challenges on the way to the implementation of this idea related to the vehicles' accumulation in off-target tissues, their immunogenicity, the complexity of drug delivery to intracellular targets, and finally, the difficulties in the fabrication of carriers matching all imposed requirements. Here, we discuss fundamental strategies for pH-responsive drug delivery, as well as limitations related to such carriers' application, and reveal the main problems, weaknesses, and reasons for poor clinical results. Moreover, we attempted to formulate the profiles of an "ideal" drug carrier in the frame of different strategies drawing on the example of metal-comprising materials and considered recently published studies through the lens of these profiles. We believe that this approach will facilitate the formulation of the main challenges facing researchers and the identification of the most promising trends in technology development.
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The development of advanced methods for the synthesis of nano- and microparticles in the field of biomedicine is of high interest due to a range of reasons. The current synthesis methods may have limitations in terms of efficiency, scalability, and uniformity of the particles. Here, we investigate the synthesis of submicron calcium carbonate using a microfluidic chip with a T-shaped oil supply for droplet-based synthesis to facilitate control over the formation of submicron calcium carbonate particles. The design of the chip allowed for the precise manipulation of reaction parameters, resulting in improved porosity while maintaining an efficient synthesis rate. The pore size distribution within calcium carbonate particles was estimated via small-angle X-ray scattering. This study showed that the high porosity and reduced size of the particles facilitated the higher loading of a model peptide: 16 vs. 9 mass.% for the particles synthesized in a microfluidic device and in bulk, correspondingly. The biosafety of the developed particles in the concentration range of 0.08-0.8 mg per plate was established by the results of the cytotoxicity study using mouse fibroblasts. This innovative approach of microfluidically assisted synthesis provides a promising avenue for future research in the field of particle synthesis and drug delivery systems.
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Naturally inspired biomaterials such as calcium carbonate, produced in biological systems under specific conditions, exhibit superior properties that are difficult to reproduce in a laboratory. The emergence of microfluidic technologies provides an effective approach for the synthesis of such materials, which increases the interest of researchers in the creation and investigation of crystallization processes. Besides accurate tuning of the synthesis parameters, microfluidic technologies also enable an analysis of the process in situ with a range of methods. Understanding the mechanisms behind the microfluidic biomineralization processes could open a venue for new strategies in the development of advanced materials. In this review, we summarize recent advances in microfluidic synthesis and analysis of CaCO3-based bioinspired nano- and microparticles as well as core-shell structures on its basis. Particular attention is given to the application of calcium carbonate particles for drug delivery.
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Superficial fungal infections are of serious concern worldwide due to their morbidity and increasing distribution across the globe in this era of growing antimicrobial resistance. The delivery of antifungals to the target regions of the skin and sustaining the effective drug concentration are essential for successful treatment of such mycoses. Topical formulations get extra benefits here if they penetrate into the hair follicles since fungal hyphae can proliferate and produce spores in such reservoirs. We designed a novel particulate system for the encapsulation and intrafollicular delivery of griseofulvin (Gf) antifungal drug, which is water-insoluble and currently commercially available in oral dosage forms. Micron-sized calcium carbonate (vaterite) carriers containing 25 ± 3% (w/w) of Gf were prepared via the wet chemical method. The successful in vivo transportation of the carriers into the hair follicles of rats was demonstrated using scanning electron and confocal laser scanning microscopy. In addition, we introduced an approach toward Gf release prolongation for the proposed system. The stabilizing coatings were formed on the surface of the obtained particles via the layer-by-layer technique. The formulations displayed sufficient biocompatibility and good cellular uptake in contact with fibroblast cells in vitro. Four different coatings were tested for their preserving ability in the course of continued carrier incubation in the model media. The best release prolonging formulation liberated 38% of the loaded Gf during 5 days, while the uncoated carriers demonstrated more than 50% drug release within the first 24 h in water. To assess the in vivo release properties, free Gf drug and Gf-loaded carriers (uncovered and covered with the stabilizing shell) were administered topically in rats and the drug excretion profiles were further studied. By comparing the daily Gf levels in urine, we verified the sustained effect (longer than a week) of the stabilizing shell formed on the carrier surface. Conversely, the application of the free drug did not provide reliable Gf detection for this period. These findings open new prospects for the efficiency enhancement of topical therapeutics. Importantly, the elaborated system could be adapted for the dermal delivery of various water-insoluble drugs beyond the scope of antifungal therapy.
Assuntos
Antifúngicos , Folículo Piloso , Animais , Antifúngicos/farmacologia , Carbonato de Cálcio , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Excipientes , Ratos , Absorção Cutânea , ÁguaRESUMO
The microvascular changes caused by disorders of host immune response to oral microorganisms resulting in long-lasting inflammation of gums play a critical role in the periodontal lesion in the pathogenesis of chronic periodontitis. Current strategies of non-surgical periodontal therapy are aimed at the attainment of anti-inflammatory effects. We hypothesized that the usage of the microencapsulated form of anti-inflammatory substances with vasoactive effects could enhance the efficiency of the therapy by the prolonged release of active components. The prepared suspension of silver-alginate microcapsules loaded with tannic acid in the hydrogel was applied in vivo to the experimental model of periodontitis in rats induced by a ligature. The effect of this formulation was assessed by monitoring changes in local microcirculation performed by the Laser Doppler Flowmetry (1 and 24 h after application of hydrogel on intact gums and 21-days after the start of periodontitis' modeling). Application of the hydrogel containing multicomponent microcapsules to the affected area of gums allows correction of inflammatory microcirculatory disorders in model periodontitis. Immobilization of tannic acid into microcapsules allows increasing the correction of the following parameters: perfusion disorders, neurogenic tone of arterioles, myogenic tone of precapillary sphincters, as well as a venous outflow in the microvasculature of the gums. The hydrogel containing multicomponent microcapsules reduces the vascular inflammatory response in the model of periodontitis. Loading of silver-alginate microcapsules with tannic acid enhances the efficiency of microvascular disorders' correction in the model of periodontitis that suggests the prospects for application of this drug delivery system for non-surgical treatment of periodontitis.
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Alginatos , Periodontite , Animais , Cápsulas , Microcirculação , Periodontite/tratamento farmacológico , Ratos , Prata , Taninos/farmacologiaRESUMO
Detection and extraction of circulating tumor cells and other rare objects in the bloodstream are of great interest for modern diagnostics, but devices that can solve this problem for the whole blood volume of laboratory animals are still rare. Here we have developed SPIM-based lightsheet flow cytometer for the detection of fluorescently-labeled objects in whole blood. The bypass channel between two blood vessels connected with the external flow cell was used to visualize, detect, and magnetically separate fluorescently-labeled objects without hydrodynamic focusing. Carriers for targeted drug delivery were used as model objects to test the device performance. They were injected into the bloodstream of the rat, detected fluorescently, and then captured from the bloodstream by a magnetic separator prior to filtration in organs. Carriers extracted from the whole blood were studied by a number of in vitro methods.
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Microencapsulation and targeted delivery of cytotoxic and antibacterial agents of photodynamic therapy (PDT) improve the treatment outcomes for infectious diseases and cancer. In many cases, the loss of activity, poor encapsulation efficiency, and inadequate drug dosing hamper the success of this strategy. Therefore, the development of novel and reliable microencapsulated drug formulations granting high efficacy is of paramount importance. Here we report the in vitro delivery of a water-soluble cationic PDT drug, zinc phthalocyanine choline derivative (Cholosens), by biodegradable microcapsules assembled from dextran sulfate (DS) and poly-l-arginine (PArg). A photosensitizer was loaded in pre-formed [DS/PArg]4 hollow microcapsules with or without exposure to heat. Loading efficacy and drug release were quantitatively studied depending on the capsule concentration to emphasize the interactions between the DS/PArg multilayer network and Cholosens. The loading data were used to determine the dosage for heated and intact capsules to measure their PDT activity in vitro. The capsules were tested using human cervical adenocarcinoma (HeLa) and normal human dermal fibroblast (NHDF) cell lines, and two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Our results provide compelling evidence that encapsulated forms of Cholosens are efficient as PDT drugs for both eukaryotic cells and bacteria at specified capsule-to-cell ratios.
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Non-destructive, controllable, remote light-induced release inside cells enables studying of time- and space-specific surface-mediated delivery of bioactive compounds, which is an important approach in a wide range of biomedical tasks, especially those related to the control of cell growth, regenerative medicine, and self-disinfecting structures such as catheters. In this regard, the elaboration of encapsulation and controlled release of oxidative species is in high demand due to its versatile applications. One of the obvious candidates for such species is hydrogen peroxide. However, the delivery of hydrogen peroxide to the site of interest with high temporal and spatial precision remains challenging due to the active and unstable nature of the substance. We hereby present an approach to encapsulate and store a hydrogen peroxide-containing solid compound (sodium percarbonate) in the free-standing arrays of biopolymer-based microchambers. In this regard, we use solid-state encapsulation enabling high payload ability, followed by isolated storage in order to prevent contact of the cargo with water. Monitoring of the release profiles reveals the encapsulation of sodium percarbonate with little leakage for up to 24 hours. Microchambers are fabricated with predetermined size and spatial distribution, which allows the release of extremely small amounts of cargo (10-30 pg) with high spatial accuracy. Microchambers are made of polylactic acid and functionalized by carbon nanodots, which provide biocompatibility and biodegradability of the whole system together with responsiveness towards NIR light. These chambers facilitate both ultrasound-assisted burst release and laser-driven carbon nanoparticle-assisted precise release of extremely small, controlled amounts of a few picograms of hydrogen peroxide in submerged conditions. Microchambers loaded with sodium percarbonate provided adhesion and high viability of mouse fibroblasts over 24 h of exposure. The developed system opens an exciting avenue for prospective delivery routes in a number of areas such as wound healing by time and site-specific release.