Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting.

PURPOSE: A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection. METHODS: Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60 min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR). RESULTS: Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30 min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05). CONCLUSION: CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

Calcium Silicate-Activated Gelatin Methacrylate Hydrogel for Accelerating Human Dermal Fibroblast Proliferation and Differentiation.

Wound healing is a complex process that requires specific interactions between multiple cells such as fibroblasts, mesenchymal, endothelial, and neural stem cells. Recent studies have shown that calcium silicate (CS)-based biomaterials can enhance the secretion of growth factors from fibroblasts, which further increased wound healing and skin regeneration. In addition, gelatin methacrylate (GelMa) is a compatible biomaterial that is commonly used in tissue engineering. However, it has low mechanical properties, thus restricting its fullest potential for clinical applications. In this study, we infused Si ions into GelMa hydrogel and assessed for its feasibility for skin regeneration applications by observing for its influences on human dermal fibroblasts (hDF). Initial studies showed that Si could be successfully incorporated into GelMa, and printability was not affected. The degradability of Si-GelMa was approximately 20% slower than GelMa hydrogels, thus allowing for better wound healing and regeneration. Furthermore, Si-GelMa enhanced cellular adhesion and proliferation, therefore leading to the increased secretion of collagen I other important extracellular matrix (ECM) remodeling-related proteins including Ki67, MMP9, and decorin. This study showed that the Si-GelMa hydrogels were able to enhance the activity of hDF due to the gradual release of Si ions, thus making it a potential candidate for future skin regeneration clinical applications.

Establishment of a trimodality analytical platform for tracing, imaging and quantification of gold nanoparticles in animals by radiotracer techniques.

This study aims to establish a (198)Au-radiotracer technique for in vivo tracing, rapid quantification, and ex vivo visualization of PEGylated gold nanoparticles (GNPs) in animals, organs and tissue dissections. The advantages of GNPs lie in its superior optical property, biocompatibility and versatile conjugation chemistry, which are promising to develop diagnostic probes and drug delivery systems. (198)Au is used as a radiotracer because it simultaneously emits beta and gamma radiations with proper energy and half-life; therefore, (198)Au can be used for bioanalytical purposes. The (198)Au-tagged radioactive gold nanoparticles ((198)Au-GNPs) were prepared simply by irradiating the GNPs in a nuclear reactor through the (197)Au(n,γ)(198)Au reaction and subsequently the (198)Au-GNPs were subjected to surface modification with polyethylene glycol to form PEGylated (198)Au-GNPs. The (198)Au-GNPs retained physicochemical properties that were the same as those of GNP before neutron irradiation. Pharmacokinetic and biodisposition studies were performed by intravenously injecting three types of (198)Au-GNPs with or without PEGylation into mice; the γ radiation in blood specimens and dissected organs was then measured. The (198)Au-radiotracer technique enables rapid quantification freed from tedious sample preparation and shows more than 95% recovery of injected GNPs. Clinical gamma scintigraphy was proved feasible to explore spatial- and temporal-resolved biodisposition of (198)Au-GNPs in living animals. Moreover, autoradiography, which recorded beta particles from (198)Au, enabled visualizing the heterogeneous biodisposition of (198)Au-GNPs in different microenvironments and tissues. In this study, the (198)Au-radiotracer technique facilitated creating a trimodality analytical platform for tracing, quantifying and imaging GNPs in animals.

In-situ formation and assembly of gold nanoparticles by gum arabic as efficient photothermal agent for killing cancer cells.

Gold nanoparticles (AuNPs) have been established to sufficiently eradicate tumors by means of heat production for photothermal therapy. However, the translation of the AuNPs from bench to the clinic still remains to be solved until realizing high bioclearance after treatment. Herein, we developed a simple strategy for simultaneous formation and assembly of small-size gold nanoparticles (Au-SNPs) to form a novel nanocomposite in the presence of gum arabic (GA) by synchrotron X-ray irradiation in an aqueous solution within 5 min. GA, a porous polysaccharide, can not only provide a confined space in which to produce uniform Au-SNPs (1.6 ± 0.7 nm in diameter), but can also facilitate the formation of Au-SNPs@GA (diameter ≈ 40 nm) after irradiating synchrotron X-rays. Specifically, the Au-SNPs@GA possesses high thermal stability and a strong photothermal effect for killing cancer cells. Importantly, a bioclearance study demonstrated that the Au-SNPs@GA can be gradually excreted by the renal and hepatobiliary system, which might be due to the breakdown and oxidation of GA under irradiating synchrotron X-rays. Thus, the novel gold nanocomposite can be promising photothermal agents for cancer treatment at the therapeutic level, minimizing toxicity concerns regarding long-term accumulation in vivo.

A single-monomer derived linear-like PEI-co-PEG for siRNA delivery and silencing.

Polyethylenimines (PEIs) are commonly used as a vehicle to deliver and protect siRNA, but the strong interaction still remains to be modulated for efficient siRNA release and silencing. Herein, a single-monomer derived linear-like PEI-co-PEG (LPEI-co-PEG, P(2)) was synthesized to substantially enhance the siRNA release, but not affect the efficiency of protection. The linear-like copolymer (P(2)) was only synthesized from a single-monomer by intensive synchrotron X-ray irradiation within 5 min, randomly producing both PEI and PEG segments. The counterpart vehicle, LPEI (P(1)), was also synthesized for comparison. We found that the P(1) and P(2) were able to prevent siRNA against enzymatic degradation. Most importantly, efficient siRNA release (52%) was only observed in the siRNA/P(2) complexes and not in the siRNA/P(1) complexes (<5%), suggesting that the PEG segment may modulate the interaction between siRNA and P(2) segment. Specifically, P(2) as well as P(1) can emit photoluminescence; cancer cells exhibited a detectable photoluminescence after treatment with P(1) and P(2), indicative of their excellent transfection efficiency. Subsequently, the siGFP/P(2) complexes knocked down GFP with excellent efficiency (75%) above the siGFP/P(1) complexes (19%) and siGFP/Lipofectamine complexes (20%). Importantly, the siRNA with anti-VEGF function being associated with P(2) have been demonstrated an excellent efficiency in the suppression of tumor growth.

One-pot synthesis of linear-like and photoluminescent polyethylenimines for intracellular imaging and siRNA delivery.

Linear-like and photoluminescent polyethylenimines (LPEIs) were synthesized through a one-pot reaction within 5 min using synchrotron radiation for intracellular imaging and siRNA delivery.

Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometry.

Fluorescence-based tagging in proteomics is useful in tracking and quantifying target proteins during sample preparation or chromatographic processes. In this study, we report a novel cysteinyl tagging method using a popular fluorophore, fluorescein derivative. Such visible dyes were shown to have multiple unique characteristics, including a unique reporter ion containing the dye moiety caused by collision-induced dissociation (CID) and high affinity toward multicarboxylate functional groups, which could be useful for enhanced selectivity in MS-based proteomics. We used sulfhydryl-reactive 5-iodoacetamidofluorescein to target cysteinyl residues on the intact protein of ovalbumin and bovine serum albumin as well as proteins in MCF-7 cells. After trypsin digestion, the digests were analyzed by nanoLC-ESI-Q-TOF or MALDI-TOF. The resulting MS spectra of tryptic fragments were similar to those of unlabeled or iodoacetamide-derivatized proteins, and the MS/MS fragmentation of all fluorescein-tagged peptides was readily interpretable with intact label. Thus, fluorescein-derivatized proteins can be identified by automatic mass mapping or peptide sequencing with high confidence. It is notable that, in MS/MS mode, a strong reporter ion (m/z 422) containing the fluorescein moiety was readily detected and was believed to derive from the immonium fragment of fluorescein-labeled cysteine residues, f C (m/z 463), under CID conditions. Using a precursor scan of the reporter ion, a cysteinyl protein, ovomucoid, was identified to be present in the ovalbumin sample as an impurity. The fluorescein derivatives were further shown to have high affinities toward metal-chelating materials that have iminodiacetic acid functional groups either with or without the presence of bound metal ions. When coupling with stable isotope dimethyl labeling, fluorescein-tagged peptides could be selectively enriched, identified, and quantified. In view of its popularity, visible tracking, and unique characteristics for developing selective methods, fluorescein tagging holds great promises for targeting proteomics.