Inhibition of post-trabeculectomy fibrosis via topically instilled antisense oligonucleotide complexes co-loaded with fluorouracil.

Trabeculectomy is the mainstay of surgical glaucoma treatment, while the success rate was unsatisfying due to postoperative scarring of the filtering blebs. Clinical countermeasures for scar prevention are intraoperative intervention or repeated subconjunctival injections. Herein, we designed a co-delivery system capable of transporting fluorouracil and anti-TGF-ß2 oligonucleotide to synergistically inhibit fibroblast proliferation via topical instillation. This co-delivery system was built based on a cationic dendrimer core (PAMAM), which encapsulated fluorouracil within hydrophobic cavity and condensed oligonucleotide with surface amino groups, and was further modified with hyaluronic acid and cell-penetrating peptide penetratin. The co-delivery system was self-assembled into nanoscale complexes with increased cellular uptake and enabled efficient inhibition on proliferation of fibroblast cells. In vivo studies on rabbit trabeculectomy models further confirmed the anti-fibrosis efficiency of the complexes, which prolonged survival time of filtering blebs and maintained their height and extent during wound healing process, exhibiting an equivalent effect on scar prevention compared to intraoperative infiltration with fluorouracil. Qualitative observation by immunohistochemistry staining and quantitative analysis by Western blotting both suggested that TGF-ß2 expression was inhibited by the co-delivery complexes. Our study provided a potential approach promising to guarantee success rate of trabeculectomy and prolong survival time of filtering blebs.

Hollow carbon dots labeled with FITC or TRITC for use in fluorescent cellular imaging.

Hollow carbon dots (HCDs) were prepared by a solvothermal method and conjugated to either tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein-5-isothiocyanate (FITC). This resulted in HCDs with bright red or green fluorescence, with excitation/emission peaks at 550/580 and 491/520 nm, respectively. The nanocomposites are well water-soluble, remarkably photostable and biocompatible. In addition, the fluorescence of the composites is more stable in a reactive oxygen environment than the free dyes. Confocal images indicate that the nanoparticles quickly enter A549 cells and mainly accumulate in the cytoplasm. The wavelength of functionalized HCDs can be regulating via coupling the HCDs to different dyes. These results demonstrate that these composite materials can be very promising reagents for biological labeling and imaging. Graphical abstract Schematic of the preparation of hollow carbon dots conjugated to tetramethyl rhodamine isothiocyanate (RHCDs) by solvothermal method. The material is water-soluble, remarkably photostable and biocompatible. It was applied to cellular labeling and imaging.

Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

Development and evaluation of folate functionalized albumin nanoparticles for targeted delivery of gemcitabine.

Gemcitabine is one of the most potent anticancer agents acting on a wide range of solid tumors, however, its use is limited by short half life and high dose leading to serious side effects. The present investigation describes the development and characterization of folate functionalized gemcitabine loaded bovine serum albumin nanoparticles (Fa-Gem-BSANPs). The nanoparticles were prepared by desolvation cross-linking technique and characterized for various parameters including morphology, particle size, zeta potential, drug loading and release profile. The particle size of Gem-BSANPs and Fa-Gem-BSANPs was found to be 159.1±5.29 and 208.7±1.80 nm, respectively. DSC and XRD analysis indicated amorphous nature of the drug within the particles. The encapsulated gemcitabine exhibited less hemolytic properties as compared to native drug. The anticancer activity of Fa-Gem-BSANPs was evaluated in folate receptor over expressing cell lines (Ovcar-5 and MCF-7) and folate receptor deficient cell line (MIAPaCa-2). The Fa-Gem-BSANPs showed superior anticancer activity as compared to Gem-BSANPs in Ovcar-5 and MCF-7 cells while no significant difference in cytotoxicity was found with MIAPaCa-2 cells. Confocal microscopy indicated facilitated intracellular uptake of Fa-Gem-BSANPs in MCF-7, which in turn result in a higher potential for apoptosis. Further, Fa-Gem-BSANPs exhibited improved anti-tumor activity in Ehrlich solid tumor model in mice. In conclusion, our study indicates that folate functionalized nanoparticles confer enhance cellular uptake and cytotoxicity for gemcitabine.

The immunomodulatory and anti-apoptotic effect of dexamethasone in imminent preterm labor: an experimental study.

The study was designed to investigate the effect of dexamethasone (DEX) on the latency period to delivery in a murine model of preterm labor. To this purpose, pregnant mice were randomly assigned in groups: the control group received water for injection (n=20), the preterm labor group was injected with lipopolysaccharide (LPS) (n=22), while the glucocorticoids group was administered DEX either 1h before (n=17) or after (n=7) lipopolysaccharide. In a first set of experiments animals were monitored to record perinatal outcomes. In another set of experiments, the remaining animals were sacrificed eight h after interventions. Fetuses were homogenized to measure tumor necrosis alpha in supernatants. Maternal splenocytes were isolated and stimulated for cytokine production. Serum of mice was incubated with donor cells from healthy pregnant and non-pregnant animals to induce apoptosis. LPS induced preterm labor but treatment or pretreatment with DEX delayed parturition exerting a favorable impact on survival of delivered fetuses. DEX inverted the increase of fetoplacental tumor necrosis alpha levels. Serum of LPS-stimulated mice induced apoptosis of splenocytes of either pregnant or non-pregnant healthy mice; this was reversed after incubation of splenocytes with serum coming from DEX pre-treated mice. The presented findings suggest that DEX administered either as pre-treatment or treatment prolonged gestation and promoted neonatal survival in a sterile murine model of preterm labor. These favorable outcomes were closely linked to alterations in both immune and apoptotic responses of animals.

Development of a rapid and high-performance chemiluminescence immunoassay based on magnetic particles for protein S100B in human serum.

Protein S100B is a clinically useful non-invasive biomarker for brain cell damage. A rapid chemiluminescence immunoassay (CLIA) for S100B in human serum has been developed. Fluorescein isothiocyanate (FITC) and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) are used to label two different monoclonal antibodies of anti-S100B. Protein S100B in serum combines with labeled antibodies and can form a sandwiched immunoreaction. A simplified separation procedure based on the use of magnetic particles (MPs) that were coated with anti-FITC antibody is performed to remove the unwanted materials. After adding the substrate solution, the relative light unit (RLU) of ABEI is measured and is found to be directly proportional to the concentration of S100B in serum. The relevant variables involved in the CLIA signals are optimized and the parameters of the proposed method are evaluated. The results demonstrate that the method is linear to 25 ng/mL S100B with a detection limit of 0.02 ng/mL. The coefficient of variation (CV) is < 5% and < 6% for intra- and interassay precision, respectively. The average recoveries are between 97 and 107%. The linearity-dilution effect produces a linear correlation coefficient of 0.9988. Compared with the commercial kit, the proposed method shows a correlation of 0.9897. The proposed method displays acceptable performance for quantification of S100B and is appropriate for use in clinical diagnosis.