Development of a novel cholesterol tag-based system for trans-membrane transport of protein drugs.

The main technological difficulties of developing an intracellular (transmembrane) transport system for protein drugs lie in two points: i) overcoming the barriers in the cellular membrane, and ii) loading enough protein drugs, and particularly high-dose proteins, into particles. To address these two technological problems, we recently developed a novel cholesterol tag (C-Tag)-based transmembrane transport system. This pilot study found that the C-Tag dramatically improved the cellular uptake of Fab (902-fold, vs. Fab alone) into living cells, indicating that it successfully achieved transmembrane transport. Moreover, C-Tag-mediated membrane transport was verified using micron-scale large unilamellar vesicles (LUVs, approximately 1.5 µm)-based particles. The C-Tagged Fab was able to permeate the liposomal bilayer and it greatly enhanced (a 10.1-fold increase vs. Fab alone) internalization of proteins into the LUV-based particles, indicating that the C-Tag loaded enough proteins into particles for use of high-dose proteins. Accordingly, we established a novel C-Tag-based transport system that has overcome the known technological difficulties of protein transmembrane delivery, and this might be a useful technology for drug development in the future.

Devising novel near-infrared aggregation-induced-emission luminogen labeling for point-of-care diagnosis of Mycobacterium tuberculosis.

Detecting and appropriately diagnosing a Mycobacterium tuberculosis infection remains technologically difficult because the pathogen commonly hides in macrophages in a dormant state. Described here is novel near-infrared aggregation-induced-emission luminogen (AIEgen) labeling developed by the current authors' laboratory for point-of-care (POC) diagnosis of an M. tuberculosis infection. The selectivity of AIEgen labeling, the labeling of intracellular M. tuberculosis by AIEgen, and the labeling of M. tuberculosis in sputum samples by AIEgen, along with its accuracy, sensitivity, and specificity, were preliminarily evaluated. Results indicated that this near-infrared AIEgen labeling had satisfactory selectivity and it labeled intracellular M. tuberculosis and M. tuberculosis in sputum samples. It had a satisfactory accuracy (95.7%), sensitivity (95.5%), and specificity (100%) for diagnosis of an M. tuberculosis infection in sputum samples. The current results indicated that near-infrared AIEgen labeling might be a promising novel diagnostic tool for POC diagnosis of M. tuberculosis infection, though further rigorous verification of these findings is required.

The Preparation of Gen-NH2-MCM-41@SA Nanoparticles and Their Anti-Rotavirus Effects.

Genistein (Gen), a kind of natural isoflavone drug monomer with poor water solubility and low oral absorption, was incorporated into oral nanoparticles with a new mesoporous carrier material, NH2-MCM-41, which was synthesized by copolycondensation. When the ratio of Gen to NH2-MCM-41 was 1:0.5, the maximum adsorption capacity of Gen was 13.15%, the maximum drug loading was 12.65%, and the particle size of the whole core-shell structure was in the range of 370 nm-390 nm. The particles were characterized by a Malvern particle size scanning machine, XRD, Fourier transform infrared spectroscopy, scanning electron microscopy, and nitrogen adsorption and desorption. Finally, Gen-NH2-MCM-41 was encapsulated by sodium alginate (SA), and the chimerism of this material, denoted as GEN-NH2-MCM-41@SA, was investigated. In vitro release experiments showed that, after 5 h in artificial colon fluid (pH = 8.0), the cumulative release reached 99.56%. In addition, its anti-rotavirus (RV) effect showed that the maximum inhibition rate was 62.24% at a concentration of 30 µM in RV-infected Caco-2 cells, and it significantly reduced the diarrhea rate and diarrhea index in an RV-infected-neonatal mice model at a dose of 0.3 mg/g, which was better than the results of Gen. Ultimately, Gen-NH2-MCM-41@SA was successfully prepared, which solves the problems of low solubility and poor absorption and provides an experimental basis for the application of Gen in the clinical treatment of RV infection.

Establishment of a rotavirus-infected zebrafish model and its application in drug screening.

Rotavirus (RV) is one of the main pathogens that induce infantile diarrhea and by now no effective drugs are available for RV-induced infantile diarrhea. Thus the development of novel models is of vital importance for the pathological research of RV-induced infantile diarrhea, as well as the progress of the associated treatment strategy. Here we introduced for the first time that RV-Wa strain and RV-SA-11 strain could infect 5 dpf(day post fertilization) and 28 dpf larvae, to induce infantile diarrhea model that was highly consistent with the clinical infection of infants. RV infection significantly changed the signs, survival rate and inflammation of larvae. Some important indicators, including the levels of RV antigen VP4 and VP6, the in vivo RV tracking, and the RV particles were also analyzed, which collectively demonstrated that the model was successfully established. More importantly, we also determined the potentials of the proposed RV-infected zebrafish model for anti-viral drug assessment. In conclusion, we established a RV-infected zebrafish model with formulated relevant indicators both larvae and adult fish, which might be served as a high throughput platform for antiviral drug screening.

Host miR-4301 promotes rotavirus replication via PPP1R3D in Caco-2 cells.

To investigate the role of miR-4301 in rotavirus (RV)-infected Caco-2 cells. In this experiment, RNAs of RV-infected Caco-2 cells were extracted, and the high-throughput second-generation sequencing was performed to detect the expression profiles of host microRNAs (miRNAs). Synthetic miRNA mimics and inhibitors were examined (quantitative polymerase chain reaction [qPCR], crystalline violet, immunofluorescence and electron microscopy) to evaluate the effect on RV replication. Target genes of miR-4301 were predicted by software analysis. The expression of target genes was evaluated by qPCR and Western blot after transfected with miRNA inhibitor/mimic, and crystalline violet and qPCR were used to detect the downregulation effects of target genes on RV replication. By transfecting miRNA inhibitors/mimics and detecting downstream target genes, the mechanism of miRNA affecting RV replication was analyzed. There were 78 known miRNAs with significant differential expression, including 39 upregulated miRNAs and 39 downregulated miRNAs. The results showed that miR-4301 exerted a key role in enhancing RV replication. PPP1R3D protein which can inhibit RV replication was predicted as the target gene of miR-4301 by software analysis. While upregulating miR-4301 by RV, the expression of PPP1R3D and glycogen synthase (GS) is suppressed. For the first time, the effect of miR-4301 on RV infection, and its influence on GS was investigated. Specifically, RV inhibits host cell glycogen synthesis to utilize the host intracellular glucose for promoting its own replication.