Integration of Zn2+, ATP, and bFGF to Nanodressing with Core-Shell Structure Fabricated by Emulsion Electrospinning for Wound Healing.

Basic fibroblast growth factor (bFGF) plays an important role in active wound repair. However, the existing dosage forms in clinical applications are mainly sprays and freeze-dried powders, which are prone to inactivation and cannot achieve a controlled release. In this study, a bioactive wound dressing named bFGF-ATP-Zn/polycaprolactone (PCL) nanodressing with a "core-shell" structure was fabricated by emulsion electrospinning, enabling the sustained release of bFGF. Based on the coordination and electrostatic interactions among bFGF, ATP, and Zn2+, as well as their synergistic effect on promoting wound healing, a bFGF-ATP-Zn ternary combination system was prepared with higher cell proliferation activity and used as the water phase for emulsion electrospinning. The bFGF-ATP-Zn/PCL nanodressing demonstrated improved mechanical properties, sustained release of bFGF, cytocompatibility, and hemocompatibility. It increased the proliferation activity of human dermal fibroblasts (HDFs) and enhanced collagen secretion by 1.39 and 3.45 times, respectively, while reducing the hemolysis rate to 3.13%. The application of the bFGF-ATP-Zn/PCL nanodressing in mouse full-thickness skin defect repair showed its ability to accelerate wound healing and reduce wound scarring within 14 days. These results provide a research basis for the development and application of this bioactive wound dressing product.

On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Positional assembly of multi-enzyme cascade reaction in polyelectrolyte doped microcapsule through electrospray and layer-by-layer assembly.

Polyelectrolyte-doped microcapsules (PDM) was fabricated by coaxial electrospray of a mixture of glycerol and water containing 10 mg/mL cationic polyelectrolyte poly(allylamine hydrochloride) (PAH) fed as the core phase solution, and a N,N-dimethylacetylamide solution of 10 wt% polyurethane fed as the shell phase solution. Multi-enzyme system involving Candida Antarctica lipase B (CALB), glucose oxidase (GOD), and horseradish peroxidase (HRP) for cascade reaction was assembled in the PDM at three different places, namely, surface, shell, and lumen. Placing of enzyme inside aqueous lumen of the PDM was realized by in situ encapsulation through adding the enzyme in the core-phase solution for coaxial electrospray. By ion-pairing of enzyme with cationic surfactant CTAB, an organic soluble enzyme-CTAB complex was prepared, so that in situ embedding of enzyme in the shell of the PDM was realized by adding it into the shell phase solution. Surface attachment of enzymes was achieved by layer-by-layer (LbL) technology, which is based on the ion-exchange interactions between oppositely charged enzymes and PAH that was doped in PDM. The enzyme-decorated microcapsule was then studied as a micro-bioreactor, in which 1-Oxododecyla-α-glucopyranoside was converted by CALB to glucose, which was oxidised by GOD to gluconolactone in a second step. The hydrogen peroxide produced was then used by HRP to oxidize ABTS to form coloured radical cation ABTS• + for activity analysis. The successful fabrication of the PDM and precise localization of enzymes in the PDM by different strategies were fully characterized. By varying the immobilization strategy, totally six PDM bioreactors with three enzymes precisely positional assembled in different strategies were constructed and their activities for the cascade reaction were investigated and compared. The PDM micro-bioreactor prepared by novel electrospray technologies provide a smart platform for positional assembly of multi-enzyme cascade reaction in a precise and well-controlled manner.

Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.