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International Journal of Nanomedicine 2024In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive...
PURPOSE
In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity.
METHODS
The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical "post-formulation" DNA addition vs "pre" addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake.
RESULTS
Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 µg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 µg/mL of pDNA with the "post" method, while maintaining their characteristics up to 100 µg/mL of pDNA with the "pre" addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 µg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes.
CONCLUSION
In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.
Topics: Plasmids; Humans; Cations; DNA; Genetic Therapy; Microfluidics; Particle Size; Nanomedicine; COVID-19; Liposomes; Transfection; Nanoparticles; SARS-CoV-2; COVID-19 Vaccines; Quaternary Ammonium Compounds; Fatty Acids, Monounsaturated
PubMed: 38766661
DOI: 10.2147/IJN.S457302 -
Environmental Research Aug 2024Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are...
Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and bla, irrespective of season. The less explored bla showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and bla varied seasonally. int1 was significantly correlated with bla in autumn and spring, whereas it showed no correlation with bla, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE.
Topics: Wastewater; Japan; Bacteria; Real-Time Polymerase Chain Reaction; Drug Resistance, Microbial; Drug Resistance, Bacterial; Genes, Bacterial; RNA, Ribosomal, 16S; Viruses; Microfluidics
PubMed: 38759773
DOI: 10.1016/j.envres.2024.119156 -
Nature Communications May 2024Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial...
Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. In this work, we develop a multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells during transitions between separate environmental conditions. With this platform, we implement a dynamic single-cell screening for pheno-tuning compounds, which induce a phenotypic change and decrease cell-to-cell variation, aiming to undermine the entire bacterial population and make it more vulnerable to other drugs. We apply this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our lead compound impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that harnessing phenotypic variation represents a successful approach to tackle pathogens that are increasingly difficult to treat.
Topics: Mycobacterium tuberculosis; Antitubercular Agents; Single-Cell Analysis; Tuberculosis; Humans; Microbial Sensitivity Tests; Microfluidics; Phenotype; Drug Discovery; Drug Synergism
PubMed: 38755132
DOI: 10.1038/s41467-024-48269-2 -
Nature Communications May 2024Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches,...
Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. First, we characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles are required.
Topics: Humans; Nanoparticles; Lipid Bilayers; Holography; Extracellular Vesicles; Microfluidics; Female; Microfluidic Analytical Techniques; Cell Line, Tumor; Biosensing Techniques; Biomarkers
PubMed: 38750038
DOI: 10.1038/s41467-024-48132-4 -
Sensors (Basel, Switzerland) May 2024Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as... (Review)
Review
Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as promising diagnostic platforms based on wide-ranging cancer biomarkers owing to the versatility, high sensitivity, and flexibility to perform the multiplexing of beads. This comprehensive review highlights recent trends and innovations in the development of bead-based biosensors for cancer-biomarker detection. We introduce various types of bead-based biosensors such as optical, electrochemical, and magnetic biosensors, along with their respective advantages and limitations. Moreover, the review summarizes the latest advancements, including fabrication techniques, signal-amplification strategies, and integration with microfluidics and nanotechnology. Additionally, the challenges and future perspectives in the field of bead-based biosensors for cancer-biomarker detection are discussed. Understanding these innovations in bead-based biosensors can greatly contribute to improvements in cancer diagnostics, thereby facilitating early detection and personalized treatments.
Topics: Biosensing Techniques; Humans; Neoplasms; Biomarkers, Tumor; Electrochemical Techniques; Nanotechnology; Microfluidics
PubMed: 38733011
DOI: 10.3390/s24092904 -
Polymers Apr 2024Bio-fillers are intensively studied for advanced polymer composite circular design and production. In this context, the algal biomass may be considered an important and...
Bio-fillers are intensively studied for advanced polymer composite circular design and production. In this context, the algal biomass may be considered an important and relatively low-cost resource, when harvested as a by-product from wastewater treatment plants. The biomass of the algal species is frequently used in this type of environmental process, and its macro constituents' composition ranges from around 15-25% carbohydrates, 10-20% lipids, and 50-60% proteins. Poly (styrene-butadiene-styrene) (SBS) copolymers have a matrix composed of glassy polystyrene domains connected by flexible polybutadiene segments. Although the physical-mechanical properties of SBS copolymers recommend them for many industrial applications, they have the drawback of low biodegradability. This study aimed to assess the aerobic biodegradability of polymer composites by integrating biomass from at varying mass percentages of 5, 10, and 20% into SBS copolymer composites. Biodegradation tests were conducted under industrial composting conditions (58 °C and 50% relative humidity) for 180 days. The biodegradability of materials was evaluated by measuring the CO produced in each vessel during the study period. Potential correlations between the amount of carbon dioxide released and the percentage of biomass added to the polymer matrix were examined. Structural and morphological changes were assessed using Fourier Transform infrared spectroscopy (FTIR), thermal analysis (DSC), and scanning electron microscopy (SEM). Physical and chemical testing revealed a decrease in sample density after the industrial composting test, along with noticeable changes in melt flow index (MFI). The observed physical and chemical changes, coupled with FTIR, SEM, and DSC data, indicate increased cross-linking and higher porosity in biodegraded polymer structures with higher biomass content. This behavior is likely due to the formation of cross-linked connections between polymer chains and polypeptide chains resulting from protein degradation, enhancing connections between polystyrene units facilitated by peptide bonds with the benzene units of the styrene blocks within the polymer matrix.
PubMed: 38732710
DOI: 10.3390/polym16091241 -
Polymers Apr 2024The preparation of polymer composites that incorporate material of a biogenic nature in the polymer matrices may lead to a reduction in fossil polymer consumption and a...
The preparation of polymer composites that incorporate material of a biogenic nature in the polymer matrices may lead to a reduction in fossil polymer consumption and a potentially higher biodegradability. Furthermore, microalgae biomass as biogenic filler has the advantage of fast growth and high tolerance to different types of culture media with higher production yields than those provided by the biomass of terrestrial crops. On the other hand, algal biomass can be a secondary product in wastewater treatment processes. For the present study, an SBS polymer composite (SBSC) containing 25% (/) copolymer SBS1 (linear copolymer: 30% styrene and 70% butadiene), 50% (/) copolymer SBS2 (linear copolymer: 40% styrene and 60% butadiene), and 25% (/) paraffin oil was prepared. Arthrospira platensis biomass (moisture content 6.0 ± 0.5%) was incorporated into the SBSC in 5, 10, 20, and 30% (/) ratios to obtain polymer composites with spirulina biomass. For the biodegradation studies, the ISO 14855-1:2012(E) standard was applied, with slight changes, as per the specificity of our experiments. The degradation of the studied materials was followed by quantitatively monitoring the CO resulting from the degradation process and captured by absorption in NaOH solution 0.5 mol/L. The structural and morphological changes induced by the industrial composting test on the materials were followed by physical-mechanical, FTIR, SEM, and DSC analysis. The obtained results were compared to create a picture of the material transformation during the composting period. Thus, the collected data indicate two biodegradation processes, of the polymer and the biomass, which take place at the same time at different rates, which influence each other. On the other hand, it is found that the material becomes less ordered, with a sponge-like morphology; the increase in the percentage of biomass leads to an advanced degree of degradation of the material. The FTIR analysis data suggest the possibility of the formation of peptide bonds between the aromatic nuclei in the styrene block and the molecular residues resulting from biomass biodegradation. It seems that in industrial composting conditions, the area of the polystyrene blocks from the SBS-based composite is preferentially transformed in the process.
PubMed: 38732687
DOI: 10.3390/polym16091218 -
Molecules (Basel, Switzerland) May 2024This study aims to investigate the vegetative buds from (spruce), naturally found in a central region of Romania, through a comprehensive analysis of the chemical...
This study aims to investigate the vegetative buds from (spruce), naturally found in a central region of Romania, through a comprehensive analysis of the chemical composition to identify bioactive compounds responsible for pharmacological properties. Using HPLC/derivatization technique of GC-MS and quantitative spectrophotometric assays, the phenolic profile, and main components of an ethanolic extract from the buds were investigated. The essential oil was characterized by GC-MS. Moreover, the antioxidant activity with the DPPH method, and the antimicrobial activity were tested. Heavy metal detection was performed by graphite furnace atomic absorption spectrometry. The main components of the alcoholic extract were astragalin, quercetin, kaempferol, shikimic acid, and quinic acid. A total content of 25.32 ± 2.65 mg gallic acid equivalent per gram of dry plant (mg GAE/g DW) and of 10.54 ± 0.083 mg rutin equivalents/g of dry plant (mg RE/g DW) were found. The essential oil had D-limonene, α-cadinol, δ-cadinene, 13-epimanool, and δ-3-carene as predominant components. The spruce vegetative buds exhibited significant antioxidant activity (IC50 of 53 μg/mL) and antimicrobial effects against . Furthermore, concentrations of heavy metals Pb and Cd were below detection limits, suggesting that the material was free from potentially harmful contaminants. The results confirmed the potential of this indigenous species to be used as a source of compounds with pharmacological utilities.
Topics: Picea; Antioxidants; Phytochemicals; Anti-Infective Agents; Plant Extracts; Oils, Volatile; Microbial Sensitivity Tests; Gas Chromatography-Mass Spectrometry; Romania; Phenols
PubMed: 38731619
DOI: 10.3390/molecules29092128 -
Nature Communications May 2024Ultra-fast single-photon detectors with high current density and operating temperature can benefit space and ground applications, including quantum optical communication...
Ultra-fast single-photon detectors with high current density and operating temperature can benefit space and ground applications, including quantum optical communication systems, lightweight cryogenics for space crafts, and medical use. Here we demonstrate magnesium diboride (MgB) thin-film superconducting microwires capable of single-photon detection at 1.55 μm optical wavelength. We used helium ions to alter the properties of MgB, resulting in microwire-based detectors exhibiting single-photon sensitivity across a broad temperature range of up to 20 K, and detection efficiency saturation for 1 μm wide microwires at 3.7 K. Linearity of detection rate vs incident power was preserved up to at least 100 Mcps. Despite the large active area of up to 400 × 400 μm, the reset time was found to be as low as ~ 1 ns. Our research provides possibilities for breaking the operating temperature limit and maximum single-pixel count rate, expanding the detector area, and raises inquiries about the fundamental mechanisms of single-photon detection in high-critical-temperature superconductors.
PubMed: 38729944
DOI: 10.1038/s41467-024-47353-x -
Biosensors & Bioelectronics Aug 2024Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive...
Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.
Topics: Humans; Biosensing Techniques; Cell Adhesion; Glycosylation; Skin Neoplasms; Melanoma; Keratinocytes; Skin; Lectins; Cell Line, Tumor; Melanocytes; Microfluidics; Microfluidic Analytical Techniques
PubMed: 38703495
DOI: 10.1016/j.bios.2024.116337