Fluorescence Imaging Cytometry Research Articles

Synthesis of SulfoCy5‐ and SulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

Abstractα‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer‐antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7‐αGalCer 1 and sulfoCy5‐αGalCer 2. The fluorescent probes were obtained from a linker‐functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5‐αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer‐antigen conjugates as shown here for DC2.4 cells.

Ginger vesicle as a nanocarrier to deliver 10-hydroxycamptothecin

In this study, we developed the ginger vesicles as nanocarrier for the targeted delivery of 10-hydroxy-camptothecin (HCPT), aiming to improve its therapeutic efficacy while minimizing the systemic toxicity. Ginger vesicles exhibit a wide spectrum of biological activities and excellent biocompatibility, rendering them as the promising nanocarriers candidates for anticancer drug delivery. The ginger vesicles with an average diameter of 86.83 nm were successfully prepared by utilizing a gradient centrifugation method. The loading conditions for HCPT into the ginger vesicles were optimized through the addition of an appropriate amount of Ca2+. The loading efficiency, size distribution, stability, and cytotoxicity profile of the ginger vesicles were comprehensively characterized using UV spectroscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and cytotoxicity experiments. Furthermore, in vitro cytotoxicity studies confirmed that ginger vesicles loaded with HCPT exhibited high inhibitory activity against tumor cells as evidenced by fluorescence imaging and flow cytometry analysis. Most importantly, in vivo antitumor assay demonstrated that the ginger vesicles loaded with HCPT displayed remarkable inhibitory effects on tumor growth. In summary, our results demonstrated the potential application of the ginger vesicles as ideal nanocarriers for delivering HCPT.

Design and development of DSPE-PEG2000/DPPC disk-like micelles for targeted delivery of icariin phytochemical in pulmonary fibrosis

Icariin (ICA)-loaded DSPE-PEG2000/DPPC disk-like micelles were synthesized utilizing the thin film hydration method to enhance the solubility and delivery of ICA to the lungs. The micellar formulation significantly improved the water solubility of ICA. This was attributed to the high encapsulation efficiency (95 %) and drug loading capacity (12 %) of the DSPE-PEG2000/DPPC micelles. Comprehensive characterization using Fourier-transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Particle size analysis through dynamic light scattering (DLS) and transmission electron microscopy (TEM) demonstrated the formation of stable micelles with an average particle size around 10 nm.In vitro aerosolization studies using the next-generation impactor (NGI) revealed that the fine particle fraction was 63.5 ± 4 %, which means that over 60 % of the aerosolized ICA/DSPE-PEG2000/DPPC micelles were capable of reaching and targeting the deep lung alveoli, indicating their potential efficacy for pulmonary delivery. Cytotoxicity assessments via the MTT assay showed IC50 values of ICA-loaded DSPE-PEG2000/DPPC micelles were 117 ± 8 μg/mL, 29 ± 3 μg/mL, and 21 ± 1 μg/mL at 24, 48, and 72 h, respectively, highlighting the time-dependent cytotoxic effects of the ICA-loaded micelles on A549 cells. However, the IC50 values of free ICA were > 500 μg/mL, 252 ± 3 μg/mL, and 109 ± 2 μg/mL, respectively at three different time points. That indicated that ICA-loaded nano micelles enhanced the cytotoxicity of ICA. Furthermore, the cellular uptake of the nano micelles by A549 cells was visualized and confirmed using EVOS fluorescence imaging and flow cytometry techniques. In addition, RAW 264.7 M2 polarization studies indicated ICA loaded DSPE-PEG2000/DPPC micelles have potential for treating pulmonary fibrosis.These findings suggest that DSPE-PEG2000/DPPC micelles significantly enhance the solubility and delivery of ICA, presenting a promising nanocarrier system for targeted pulmonary fibrosis therapy.

Targeting Myeloid-Derived Suppressor Cells via Dual-Antibody Fluorescent Nanodiamond Conjugate.

Fluorescent nanodiamonds (FNDs) are carbon-based nanomaterials that emit bright, photostable fluorescence and exhibit a modifiable surface chemistry. Myeloid-derived suppressor cells (MDSCs) are an immunosuppressive cell population known to expand in cancer patients and contribute to worse patient outcomes. To target MDSC, glycidol-coated FND were conjugated with antibodies against the murine MDSC markers, CD11b and GR1 (dual-Ab FND). In vitro, dual-Ab FND uptake by murine MDSC was significantly higher than IgG-coated FND (94.7% vs. 69.0%, p < 0.05). In vivo, intra-tumorally injected dual-Ab FND primarily localized to the tumor 2 and 24 h post-injection, as measured by in vivo fluorescence imaging and flow cytometry analysis of the spleen and tumor. Dual-Ab FND were preferentially taken up by intra-tumoral MDSC, representing 87.1% and 83.0% of FND+ cells in the tumor 2 and 24 h post-injection, respectively. Treatment of mice with anti-PD-L1 immunotherapy prior to intra-tumoral injection of dual-Ab FND did not significantly alter the uptake of FND by MDSC. These results demonstrate the ability of our novel dual-antibody conjugated FND to target MDSC and reveal a potential strategy for targeted delivery to other specific immune cell populations in future cancer research.

Persistence and Culturability of Escherichia coli under Induced Toxin Expression.

Bacteria are well known to enter dormancy under stress conditions. However, the mechanisms of different dormancy-related phenotypes are still under debate and many questions remain unanswered. This study aims to better understand the effects of toxin gene expression on the dormancy of Escherichia coli. The effects of toxin gene expression on growth, persistence, and culturability were characterized. Specifically, we detailed dose- and time-dependent dormancy of E. coli and its susceptibility to ofloxacin via arabinose-induced hipA toxin gene expression under the PBAD promoter. A new plot was developed to better describe the dynamic changes in culturability and persistence. The expression level of hipA was determined using qPCR and cellular activities were monitored using fluorescence imaging and flow cytometry. High-level persister formation and strong tolerance to ofloxacin were observed after high-level hipA induction. The new plot reveals more information than the changes in persistence alone, e.g., reduced culturability of E. coli and thus deeper dormancy under high-level hipA induction. Consistently, controlled hipA induction led to decreased cellular activities at promoter PrrnBP1 and an increase in the non-culturable subpopulation. Overall, this study provides new insights into dormancy induced by toxin gene expression and a more comprehensive view of persistence and culturability. The findings may help develop better control agents against dormant bacterial cells.

Selective Cellular Uptake and Druggability Efficacy through Functionalized Chitosan-Conjugated Polyamidoamine (PAMAM) Dendrimers.

Nanotechnology has ushered in significant advancements in drug design, revolutionizing the prevention, diagnosis, and treatment of various diseases. The strategic utilization of nanotechnology to enhance drug loading, delivery, and release has garnered increasing attention, leveraging the enhanced physical and chemical properties offered by these systems. Polyamidoamine (PAMAM) dendrimers have been pivotal in drug delivery, yet there is room for further enhancement. In this study, we conjugated PAMAM dendrimers with chitosan (CS) to augment cellular internalization in tumor cells. Specifically, doxorubicin (DOX) was initially loaded into PAMAM dendrimers to form DOX-loaded PAMAM (DOX@PAMAM) complexes via intermolecular forces. Subsequently, CS was linked onto the DOX-loaded PAMAM dendrimers to yield CS-conjugated PAMAM loaded with DOX (DOX@CS@PAMAM) through glutaraldehyde crosslinking via the Schiff base reaction. The resultant DOX@CS@PAMAM complexes were comprehensively characterized using Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Notably, while the drug release profile of DOX@CS@PAMAM in acidic environments was inferior to that of DOX@PAMAM, DOX@CS@PAMAM demonstrated effective acid-responsive drug release, with a cumulative release of 70% within 25 h attributed to the imine linkage. Most importantly, DOX@CS@PAMAM exhibited significant selective cellular internalization rates and antitumor efficacy compared to DOX@PAMAM, as validated through cell viability assays, fluorescence imaging, and flow cytometry analysis. In summary, DOX@CS@PAMAM demonstrated superior antitumor effects compared to unconjugated PAMAM dendrimers, thereby broadening the scope of dendrimer-based nanomedicines with enhanced therapeutic efficacy and promising applications in cancer therapy.

Biogenic fabrication and multifunctional therapeutic applications of silver nanoparticles synthesized from rose petal extract

Abstract This study explores a novel, eco-friendly approach to silver nanoparticle (AgNP) synthesis by using rose petal (RP) extracts. Unlike traditional chemical synthesis, this method reduces silver ions (Ag+) to elemental silver (Ag0) with RP, facilitating nanoparticle stabilization. RP-AgNPs were structurally characterized using UV/Vis (ultraviolet/visible) spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, zeta potential, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy which confirmed the synthesis of predominantly spherical to oval RP-AgNPs, with a distinct absorption peak at 410 nm as observed in UV/Vis spectra. The NPs demonstrated exceptional antibacterial efficacy against Escherichia coli and Staphylococcus aureus (inhibitory zones of 12.5 ± 0.20 and 14 ± 0.67 mm at 50 µg) and potent antibiofilm activity. Crucially, they also exhibited selective cytotoxicity toward HeLa cervical cancer cells when assessed by viability assays, fluorescence imaging, and flow cytometry. These findings underscore the potential of green synthesized AgNPs for future biomedical applications in areas such as infection control and cancer treatment.

Tailored design of NHS–SS–NHS cross-linked chitosan nano-hydrogels for enhanced anti-tumor efficacy by GSH-responsive drug release

The traditional chemotherapeutic agents’ disadvantages such as high toxicity, untargeting and poor water solubility lead to disappointing chemotherapy effects, which restricts its clinical application. In this work, novel size-appropriate and glutathione (GSH)-responsive nano-hydrogels were successfully prepared via the active ester method between chitosan (containing –NH2) and cross-linker (containing NHS). Especially, the cross-linker was elaborately designed to possess a disulfide linkage (SS) as well as two terminal NHS groups, namely NHS–SS–NHS. These functionalities endowed chitosan-based cross-linked scaffolds with capabilities for drug loading and delivery, as well as a GSH-responsive mechanism for drug release. The prepared nano-hydrogels demonstrated excellent performance applicable morphology, excellent drug loading efficiency (∼22.5%), suitable size (∼100 nm) and long-term stability. The prepared nano-hydrogels released over 80% doxorubicin (DOX) after incubation in 10 mM GSH while a minimal DOX release less than 25% was tested in normal physiological buffer (pH = 7.4). The unloaded nano-hydrogels did not show any apparent cytotoxicity to A 549 cells. In contrast, DOX-loaded nano-hydrogels exhibited marked anti-tumor activity against A 549 cells, especially in high GSH environment. Finally, through fluorescent imaging and flow cytometry analysis, fluorescein isothiocyanate-labeled nano-hydrogels show obvious specific binding to the GSH high-expressing A549 cells and nonspecific binding to the GSH low-expressing A549 cells. Therefore, with this cross-linking approach, our present finding suggests that cross-linked chitosan nano-hydrogel drug carrier improves the anti-tumor effect of the A 549 cells and may serve as a potential injectable delivery carrier.

Synthesis and comparison of cationic polymers for structural effects of backbones and grafted side chains on their DNA transfection for efficient gene delivery

AbstractMany cationic polymers with varied architectures either in backbones or in side chains have been used for gene delivery. However, these polymers yielding different DNA transfection efficiency cannot answer whether backbones or side chains have more influence on DNA delivery. In this research, polyurethanes (PUs) made from the reaction of l‐lysine diisocyanate and 1,4‐piperazinediethanol were synthesized. Together with poly(l‐lysine) (PLL), these polymers were further modified to carry side chains by connecting to low‐molecular‐weight polyethylenimine (PEI) via imine linkages. The PEI was either linear (423 Da) or branched (600 Da) and grafted to the backbone representing two distinct shapes (i.e. a twig with needle‐leaves or round‐leaves). In addition, one PU derivative was synthesized to have shorter side chains (as PU‐PEI S423) than PU‐PEI423 by aminolysis reaction. These synthesized polymers were compared for their transfection potential as gene vectors into cells. Electrophoretic mobility, dynamic light scattering and zeta potential analyses of polymer/DNA complexes confirmed that, after grafting of PEI (PEI423 or PEI600), PLL‐PEIs and PU‐PEIs were able to self‐assemble with plasmid DNA to form nano‐scaled (ca 200 nm) and positively charged (ca +25 mV) complexes. These cationic polymers hardly showed any cytotoxicity on COS‐7 cells. The outcome of fluorescence imaging and flow cytometry verified that the order of greater transfection efficiency is PLL‐PEI600 = PU‐PEI600 &gt; PLL‐PEI423 = PU‐PEI423 &gt; PEI25K (commercial standard) = PU‐PEI S423 &gt;&gt; PLL &gt; PEI423 = PEI600 (starting materials). These results reveal that the polymers with longer side chains or higher charge density have positive influence on transfection efficiency. Nevertheless, different polymer backbones may reflect their biodegradability and cytotoxicity, but their effect on transfection efficiency is less significant. © 2024 Society of Chemical Industry.

Au/Doc/Quer@PDA/A10-3.2 Nanoparticles for targeted treatment of docetaxel-resistant prostate cancer

Docetaxel (Doc), as a first-line chemotherapy drug for prostate cancer (PC), often loses its therapeutic efficacy due to acquired resistance and lack of targeting specificity. Therefore, there is a need to develop a novel drug that can overcome Doc resistance and enhance its targeting ability to inhibit PC progression. In this study, we prepared Au/Doc/Quer@PDA/A10-3.2 nanoparticles (NPs) composite drug by encapsulating Doc and quercetin (Quer) within polydopamine (PDA)-coated Au NPs and further modifying them with RNA oligonucleotide aptamer A10-3.2. A10-3.2 was used for specific targeting of prostate-specific membrane antigen (PSMA)-positive PC cells (LNCaP). Quer was employed to reverse the resistance of Doc-resistant cell line (LNCaP/R) to Doc. Physical characterization using ultraviolet-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FTIR) confirmed the successful preparation of Au/Doc/Quer@PDA/A10-3.2 NPs. Fluorescence imaging and flow cytometry experiments demonstrated the targeting ability of Au/Doc/Quer@PDA/A10-3.2 NPs towards PSMA-positive LNCaP/R cells. Cell proliferation, apoptosis, invasion, and migration experiments revealed that Quer reversed the resistance of LNCaP/R cells to Doc. Immunoblotting experiments further confirmed the mechanism behind sensitization of chemotherapy by Quer. Finally, we evaluated the therapeutic efficacy of Au/Doc/Quer@PDA/A10-3.2 NPs in a mouse model of PC. In conclusion, this study synthesized and validated a novel nano-composite drug (Au/Doc/Quer@PDA/A10-3.2 NPs) for combating Doc-resistant PC, which could potentially be applied in clinical treatment of PC.

Machine-learning-guided quantitative delineation of cell morphological features and responses to nanomaterials.

Delineation of cell morphological features is essential to decipher cell responses to external stimuli like theranostic nanomaterials. Conventional methods rely on labeled approaches, such as fluorescence imaging and flow cytometry, to assess cell responses. Besides potentially perturbing cell structure and morphology, these approaches are relatively complex, time-consuming, expensive, and may not be compatible with downstream analysis involving live cells. Herein, leveraging label-free phase-contrast or brightfield microscopy imaging and machine learning, the delineation of different cell types, phenotypes, and states for monitoring live cell responses is reported. Notably, pixel classification based on a supervised random forest classifier is used to distinguish between cells and backgrounds from the microscopy images, followed by cell segmentation and morphological feature extraction. Quantitative analysis shows that most of the compared cell groups have distinguishable size and shape features. Principal component analysis and unsupervised k-means clustering of morphological features reveal the possible existence of heterogenous cell subpopulations and treatment responses among the seemingly homogenous cell groups. This shows the merit of the reported approach in complementing conventional techniques for cell analysis. It is anticipated that the demonstrated method will further aid the implementation of machine learning to streamline the analysis of cell morphology and responses for early disease diagnosis and treatment response monitoring.

TfR Aptamer-Functionalized MSNs for Enhancing Targeted Cellular Uptake and Therapy of Cancer Cells.

Mesoporous silica nanoparticles (MSNs), as novel nanocarriers for drug delivery in cancer treatment, have attracted widespread concern because of their rich pore structure, large pore capacity, ease of modification, and biocompatibility. However, the limitation of nontargeting and low uptake efficiency hindered their further application. Considering the overexpression of the transferrin receptor (TfR) on most cancer cell membranes, herein, we propose a strategy to effectively enhance the cellular internalization of MSNs by arming them with the TfR aptamer. Cellular fluorescent imaging and flow cytometry analysis demonstrated that TfR aptamer-functionalized MSNs exhibited superior cellular internalization compared to unmodified or random sequence-modified MSNs toward three different cancer cell lines, including MCF-7, HeLa, and A549. Furthermore, TfR aptamer-functionalized MSNs displayed enhanced drug delivery efficiency compared with MSNs at equivalent doses and incubation times. These results suggested that TfR aptamer-functionalized MSNs have the potential for enhanced delivery of therapeutic agents into TfR-positive cancer cells to improve therapeutic efficacy.

Novel time-resolved reporter mouse reveals spatial and transcriptional heterogeneity during alpha cell differentiation.

Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.

DWH24: a new antibody for fluorescence-based cell death analysis

Antibodies have gained considerable importance in laboratory and clinical settings. Currently, antibodies are extensively employed for the diagnosis and treatment of several human diseases. Herein, using targeted and cell immunisation approaches, we developed and characterised an antibody clone, DWH24. We found that DWH24 is an IgM κ type antibody that enables excellent visualisation and quantification of dead cells using immunofluorescence, fluorescence microscopy, and flow cytometry. This property was proved by the spontaneous cell death of several tumour cell lines and stimulated T cells, as well as after chemo- and photodynamic therapy. Unlike conventional apoptosis and cell death markers, DWH24 binding occurred in a Ca2+- and protein-independent manner and enabled live imaging of cell death progress, as shown using time-lapse microscopy. The binding specificity of DWH24 was analysed using a human proteome microarray, which revealed a complex response profile with very high spot intensities against various proteins, such as tropomyosin variants and FAM131C. Accordingly, DWH24 can be employed as a suitable tool for the cost-effective and universal analysis of cell death using fluorescence imaging and flow cytometry.

95. Hierarchical surface roughness using a 3D-printed interbody implant promotes optimal bone formation and integration on fluorescence image cytometry

95. Hierarchical surface roughness using a 3D-printed interbody implant promotes optimal bone formation and integration on fluorescence image cytometry

Effective and Selective Ru(II)-Arene Complexes Containing 4,4'-Substituted 2,2' Bipyridine Ligands Targeting Human Urinary Bladder Cancer Cells.

Cisplatin-based chemotherapy is a common regimen for bladder cancer, a life-threatening cancer with more than 500,000 new cases worldwide annually. Like many other metallodrugs, cisplatin causes severe side effects for its general toxicity. Organoruthenium is known for its structural stability, good anticancer activity, and possible low general toxicity. Here, we have prepared and characterized a series of water-soluble ruthenium-arene complexes with N,N'-chelating ligands: [Ru(II)-η6-arene-(4,4'-(X)2-2,2'-bipyridine)Cl]Cl (arene = p-cymene, X = C4H9 (1), COOH (2), COOCH3 (3), COOC2H5 (4); arene = benzene, X = C4H9 (5), COOCH3 (6), COOC2H5 (7)). These complexes are carefully characterized using single-crystal X-ray diffraction, UV-vis, IR, 1H NMR, and MALDI-TOF MS spectroscopy. Their DFT-calculated structural and thermodynamic properties are consistent with the experimental observations. Biophysicochemical studies of complex interaction with CTDNA and BSA supported by molecular docking simulations reveal suitable properties of 1-7 as anticancer agents. Cytotoxicities of 1-7 are evaluated on healthy human MCF-10a-breast epithelial and African green monkey Vero cells, and carcinoma human HepG-2-hepatic, T24-bladder, and EAhy-926-endothelial cells. All complexes exhibit much higher cytotoxicity for T24 than cisplatin. Particularly, 1 and 2 are also highly selective toward T24. Fluorescence imaging and flow cytometry demonstrate that 1 and 2 penetrate T24 cell membrane and induce early apoptosis at their respective IC50 concentrations, which ultimately lead to cell death. Statistical analysis suggests that the order of importance for T24 cell antiproliferation is protein binding, Log p, Ru-Cl bond length, while DNA binding is the least important. This study is the first to report the anti-bladder cancer efficacy of Ru-arene-2,2'-bipyridine complexes, and may provide insights for rational design of organoruthenium drugs in the enduring search for new chemotherapeutic agents.

N doped-carbon quantum dots with ultra-high quantum yield photoluminescent property conjugated with folic acid for targeted drug delivery and bioimaging applications

BackgroundImproving targeted therapy, and reducing adverse side effects of antitumor drugs toward normal cells with simultaneous bioimaging properties are being developed in pharmaceutical industries. In this study, N doped-carbon quantum dot (CQD) with ultra-high quantum yield (QY) was synthesized and its surface was modified with folic acid as the targeted anticancer agent. MethodsCQD was synthesized by one-step hydrothermal method. The impact of operating parameters such as pyrolysis temperature, time, and pH on the QY and surface charge was investigated. Then, the CQD surface was modified with folic acid as the targeted anticancer agent followed by doxorubicin (DOX) conjugation on the surface. Cytotoxicity of CQD-FA and CQD-FA-DOX upon 4T1, MCF7 and PC12 cell lines were examined. Furthermore, fluorescent microscopy imaging and flow cytometry analysis for various cell lines were applied to evaluate CQD-FA bioimaging property. In addition, the efficacy of the formulation on the BALB/c mice bearing breast cancer tumors was investigated. ResultsIn CQD preparation, average particle size of 7 nm was confirmed by the characterization results. With optimum synthesis conditions of pH = 11, time = 8 h and temperature = 160 °C, QY was about 90%. XPS analysis confirmed formation of N doped CQD. DOX loading efficiency was 88.6% and about 86% of the loaded DOX was released from CQD-FA-DOX after 72 h at pH = 5.5. Cytotoxicity analysis confirmed a higher toxicity effect of CQD-FA-DOX toward cancerous cell lines (4T1, MCF7) in comparison to free DOX. This nanocarrier could act smartly in identifying folate receptor positive cancerous cells and enter mainly by the receptor-mediated route. In vivo results showed that cellular internalization of CQD-FA-DOX was greater than free DOX, in which the tumor growth in mice treated with CQD-FA-DOX was lower than free DOX. In addition, ex vivo analysis confirmed lower side effects of CQD-FA-DOX on the main organs of mice in comparison to the free drug group. ConclusionThis new synthesized carrier can be a suitable platform for drug delivery applications with lower side effects, targeted cellular uptake and bioimaging properties.

In vitro fertilization with frozen-thawed blue catfish (Ictalurus furcatus) sperm and implications for gene banking

Catfish farming constitutes about 75% of total U.S. finfish aquaculture production, for which the channel catfish female (Ictalurus punctatus) × blue catfish male (I. furcatus) hybrid accounts for nearly 60% of the harvest. Current hatchery technology to produce hybrids is labor intensive and requires the sacrifice of males. This is expensive and time-consuming, as blue catfish males require 4 to 7 years to mature. Sperm, even from mature fish, are often of low quality and quantity and do not necessarily give high fertility and hatch. Therefore, paternal gametes should be used sparingly, using the minimum quantity of sperm for fertilization, while maximizing offspring production. This is particularly important when using cryopreserved cells, as each males' sperm is in limited supply. To optimize the use of available sperm cells, the objectives of this study were to (i) compare sperm swimming kinematics and sperm health metrics before and after cryopreservation; (ii) determine the minimum quantity of frozen-thawed sperm required to maximize hatching success; and (iii) decipher how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. Sperm kinematics were evaluated by computer-assisted sperm analysis, while health metrics (i.e., viability, oxidative stress, DNA fragmentation) were assessed using fluorescent imaging and flow cytometry. Over 2 spawning seasons, eggs from 18 females were collected and fertilized using cryopreserved sperm from 36 males at 10 sperm-to-egg ratios (ranging from 1.0 × 104 to 9.0 × 105 sperm per egg). Embryos sired with fresh and frozen-thawed sperm were then incubated under common environmental conditions and growth performance was documented over 40 days post-hatch. Generally, sperm kinematic traits [curvilinear velocity (VCL), motility, progressive VCL, and progressive motility] and health metrics (cell membrane viability, oxidative stress, and DNA fragmentation) declined after cryopreservation. Despite this decrease in sperm quality, hatching success using cryopreserved sperm was high, but dependent on the sperm-to-egg ratio. For instance, at ratios of 1.0 × 104 to 5.0 × 104 sperm per egg, hatch increased from 18.0% ± 8.5 to 43.9% ± 8.2. Adding >5.0 × 104 sperm per egg had no significant effect on hatching success. Furthermore, there were generally no differences in morphology or deformity rates in offspring sired with cryopreserved sperm, compared to a fresh control. These data improve understanding of frozen-thawed sperm quality for blue catfish to improve reproductive sustainability and reduce production costs.

Chitosan-Imidazolium Core-Shell Nanoparticles of Gd-Mn-Mo Polyoxometalate as Novel Potential MRI Nano-Agent for Breast Cancer Detection.

Polyoxometalates (POMs) are mineral nanoclusters with many advantages in various diagnostic fields, in particular cancer detection. This study aimed to synthesize and evaluate the performance of gadolinium-manganese-molybdenum polyoxometalate (Gd-Mn-Mo; POM) nanoparticles coated with chitosan-imidazolium (POM@CSIm NPs) for detecting 4T1 breast cancer cells by magnetic resonance imaging in vitro and in vivo. The POM@Cs-Im NPs were fabricated and characterized by FTIR, ICP-OES, CHNS, UV-visible, XRD, VSM, DLS, Zeta potential, and SEM. Cytotoxicity, cellular uptake, and MR imaging in vivo and in vitro of L929 and 4T1 cells were also assessed. The efficacy of nanoclusters was demonstrated using MR images of BALB/C mice bearing a 4T1 tumor in vivo. The evaluation of the in vitro cytotoxicity of the designed NPs showed their high biocompatibility. In fluorescence imaging and flow cytometry, NPs had a higher uptake rate by 4T1 than L929 (p < 0.05). Furthermore, NPs significantly increased the signal strength of MR images, and its relaxivity (r1) was calculated as 4.71 mM-1 s-1. MR imaging also confirmed the attachment of nanoclusters to cancer cells and their selective accumulation in the tumor region. Overall, the results showed that fabricated POM@CSIm NPs have considerable potential as an MR imaging nano-agent for early 4T1 cancer detection.

Development of a Golgi-targeted superoxide anion fluorescent probe for elucidating protein GOLPH3 function in myocardial ischemia-reperfusion injury

Superoxide anion (O2•-) is an important reactive oxygen species (ROS) and participates in various physiological and pathological processes in the organism. The O2•- burst induced by ischemia-reperfusion (I/R) is associated with cardiovascular disease and promotes the cell apoptosis. In this work, a turn-on type Golgi-targeting fluorescent probe Gol-Cou-O2•- was rationally designed for sensitive and selective detection of O2•-. The minimum detection limit concentration for O2•- was about 3.9 × 10−7 M in aqueous solution. Gol-Cou-O2•- showed excellent capacity of detecting exogenous and endogenous O2•- in living cells and zebrafish, and was also used to capture the up-regulated O2•- level during the duration of I/R process in cardiomyocytes. Golgi Phosphoprotein 3 (GOLPH3) is a potential Golgi stress marker protein and plays a key role in cells apoptosis during I/R. The fluorescence imaging and flow cytometry assay results indicated that silencing GOLPH3 through siRNA could give rise to the down-regulated O2•- level and alleviation of apoptosis in I/R myocardial cells. Thus, development of Gol-Cou-O2•- provides a diagnostic tool for myocardial oxidative stress injury and distinct insights on roles of GOLPH3 in myocardial I/R injury.