An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells.

Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell-culturing time using two defined culturing media containing TGF-ß, and Wnt/ß-catenin pathway inhibitors, and bFGF growth factor over 25 days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-ß and Wnt/ß-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

The Application of the in-Situ Hyperthermia Emission from Acoustic Nanodroplets for Theranostic Dual-Imaging and Antitumor Modalities.

In this study, we have developed a theranostic nanocarrier that can emit heat upon the exposure to ultrasound (US) irradiation as well as the generation of a contrast signal that can be detected with ultrasonography. The prepared acoustic nanodroplets (NDs) made with liquid perfluporopentane (PFPn) had an average size of 197.7 ± 3.6 nm in diameter and were stable in vitro for 60 min. US irradiation at 2 W.cm-2 induced phase change of NDs into bubbles in vitro. On the other hand, the intra-tumor injection of NDs in combination with US irradiation induced thermal emission in situ in B16BL6 melanoma tumor implanted into mice and the emission areas have mostly covered the tumor site. Also, the combination between NDs and US irradiation has inhibited the tumor growth. Under this condition, the heat shock protein (HSP70) in tumor was significantly upregulated after 6 h of the treatment of NDs with US. Thus, we have developed a therapeutic system with multiple theranostic modalities composed of acoustic NDs and US irradiation applicable to the tumor treatment on the external surface of the body.

Evaluation of the Theranostic Potential of Perfluorohexane-Based Acoustic Nanodroplets.

In this study, we have prepared perfluorohexane (PFH)-based acoustic nanodroplets (PFH-NDs) and evaluated their theranostic characteristics. Nile Red (NR) was incorporated into PFH-NDs as a model of hydrophobic drugs (NR-PFH-NDs). The mean particle diameters of PFH-NDs and NR-PFH-NDs were 205 ± 1.8 nm and 346.3 ± 6 nm, respectively. There was no significant PFH leakage from PFH-NDs during 90 min incubation at 37°C in the presence of 10% rat serum. The in vitro ultrasonography showed that the phase transition of PFH-NDs from liquid droplets to gassed bubbles could be induced by therapeutic low-intensity ultrasound with a frequency of 1 MHz and an intensity of 5 W/cm2. Irradiation of ultrasound in combination with NR-PFH-NDs enhanced uptake of NR in murine adenocarcinoma cells (C26). After intravenous injection of PFH-NDs to mice, PFH gradually disappeared from blood circulation with an elimination half-life of 43.3 min. Intravenous injection of PFH-NDs also resulted in significant contrast enhancement in the mouse carotid artery upon therapeutic low-intensity ultrasound irradiation. These results suggest the potential of PFH-NDs as a novel contrast agent for further theranostic applications.

The development of mechanically formed stable nanobubbles intended for sonoporation-mediated gene transfection.

In this study, stable nano-sized bubbles (nanobubbles [NBs]) were produced using the mechanical agitation method in the presence of perfluorocarbon gases. NBs made with perfluoropropane had a smaller size (around 400 nm) compared to that of those made with perfluorobutane or nitrogen gas. The lipid concentration in NBs affected both their initial size and post-formulation stability. NBs formed with a final lipid concentration of 0.5 mg/ml tended to be more stable, having a uniform size distribution for 24 h at room temperature and 50 h at 4 °C. In vitro gene expression revealed that NBs/pDNA in combination with ultrasound (US) irradiation had significantly higher transfection efficacy in colon C26 cells. Moreover, for in vivo gene transfection in mice left limb muscles, there was notable local transfection activity by NBs/pDNA when combined with US irradiation. In addition, the aged NBs kept at room temperature or 4 °C were still functional at enhancing gene transfection in mice. We succeeded in preparing stable NBs for efficient in vivo gene transfection, using the mechanical agitation method.

Evaluation of the potential of doxorubicin loaded microbubbles as a theranostic modality using a murine tumor model.

In this study, a novel phospholipid-based microbubble formulation containing doxorubicin and perfluoropropane gas (DLMB) was developed. The DLMBs were prepared by mechanical agitation of a phospholipid dispersion in the presence of perfluoropropane (PFP) gas. An anionic phospholipid, distearoyl phosphatidylglycerol (DSPG) was selected to load doxorubicin in the microbubbles by means of electrostatic interaction. The particle size, zeta potential, echogenicity and stability of the DLMBs were measured. Drug loading was ⩾ 92%. The potential of the DLMBs for use as a theranostic modality was evaluated in tumor bearing mice. Gas chromatography analysis of PFP showed significant enhancement of PFP retention when doxorubicin was used at concentrations of 10-82% equivalent to DSPG. The inhibitory effects on the proliferation of B16BL6 melanoma murine cells in vitro were enhanced using a combination of ultrasound (US) irradiation and DLMBs. Moreover, in vivo DLMBs in combination with (US) irradiation significantly inhibited the growth of B16BL6 melanoma tumor in mice. Additionally, US echo imaging showed high contrast enhancement of the DLMBs in the tumor vasculature. These results suggest that DLMBs could serve as US triggered carriers of doxorubicin as well as tumor imaging agents in cancer therapy.