Exploring the biphasic dose-response effects of photobiomodulation on the viability, migration, and extracellular vesicle secretion of human adipose mesenchymal stem cells.

Photobiomodulation (PBM) is a well-established medical technology that employs diverse light sources like lasers or light-emitting diodes to generate diverse photochemical and photophysical reactions in cells, thereby producing beneficial clinical outcomes. In this study, we introduced an 830 nm near-infrared (NIR) laser irradiation system combined with a microscope objective to precisely and controllably investigate the impact of PBM on the migration and viability of human adipose mesenchymal stem cells (hADSCs). We observed a biphasic dose-response in hADSCs' viability and migration after PBM exposure (0-10 J/cm2), with the 5 J/cm2 group showing significantly higher cell viability and migration ability than other groups. Additionally, at the optimal dose of 5 J/cm2, we used nanoparticle tracking analysis (NTA) and found a 6.25-fold increase in the concentration of extracellular vesicles (EVs) derived from hADSCs (PBM/ADSC-EVs) compared to untreated cells (ADSC-EVs). Both PBM/ADSC-EVs and ADSC-EVs remained the same size, with an average diameter of 56 nm measured by the ExoView R200 system, which falls within the typical size range for exosomes. These findings demonstrate that PBM not only improves the viability and migration of hADSCs but also significantly increases the EV yield.

Functional Peptide-Loaded Gelatin Nanoparticles as Eyedrops for Cornea Neovascularization Treatment.

Background: Corneal neovascularization (NV) is a process of abnormal vessel growth into the transparent cornea from the limbus and can disturb the light passing through the cornea, resulting in vision loss or even blindness. The use of nanomedicine as an effective therapeutic formulation in ophthalmology has led to higher drug bioavailability and a slow drug release rate. In this research, we designed and explored the feasibility of a new nanomedicine, gp91 ds-tat (gp91) peptide-encapsulated gelatin nanoparticles (GNP-gp91), for inhibiting corneal angiogenesis. Methods: GNP-gp91 were prepared by a two-step desolvation method. The characterization and cytocompatibility of GNP-gp91 were analyzed. The inhibition effect of GNP-gp91 on HUVEC cell migration and tube formation was observed by an inverted microscope. The drug retention test in mouse cornea was observed by in vivo imaging system, fluorescence microscope, and DAPI/TAMRA staining. Finally, the therapeutic efficacy and evaluation of neovascularization-related factors were conducted through the in vivo corneal NV mice model via topical delivery. Results: The prepared GNP-gp91 had a nano-scale diameter (550.6 nm) with positive charge (21.7 mV) slow-release behavior (25%, 240hr). In vitro test revealed that GNP-gp91 enhanced the inhibition of cell migration and tube formation capacity via higher internalization of HUVEC. Topical administration (eyedrops) of the GNP-gp91 significantly prolongs the retention time (46%, 20 min) in the mouse cornea. In chemically burned corneal neovascularization models, corneal vessel area with a significant reduction in GNP-gp91 group (7.89%) was revealed when compared with PBS (33.99%) and gp91 (19.67%) treated groups via every two days dosing. Moreover, GNP-gp91 significantly reduced the concentration of Nox2, VEGF and MMP9 in NV's cornea. Conclusion: The nanomedicine, GNP-gp91, was successfully synthesized for ophthalmological application. These data suggest that GNP-gp91 contained eyedrops that not only have a longer retention time on the cornea but also can treat mice corneal NV effectively delivered in a low dosing frequency, GNP-gp91 eyedrops provides an alternative strategy for clinical ocular disease treatment in the culture.

Topical Application of Hyaluronic Acid-RGD Peptide-Coated Gelatin/Epigallocatechin-3 Gallate (EGCG) Nanoparticles Inhibits Corneal Neovascularization Via Inhibition of VEGF Production.

Neovascularization (NV) of the cornea disrupts vision which leads to blindness. Investigation of antiangiogenic, slow-release and biocompatible approaches for treating corneal NV is of great importance. We designed an eye drop formulation containing gelatin/epigallocatechin-3-gallate (EGCG) nanoparticles (NPs) for targeted therapy in corneal NV. Gelatin-EGCG self-assembled NPs with hyaluronic acid (HA) coating on its surface (named GEH) and hyaluronic acid conjugated with arginine-glycine-aspartic acid (RGD) (GEH-RGD) were synthesized. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the antiangiogenic effect of GEH-RGD NPs in vitro. Moreover, a mouse model of chemical corneal cauterization was employed to evaluate the antiangiogenic effects of GEH-RGD NPs in vivo. GEH-RGD NP treatment significantly reduced endothelial cell tube formation and inhibited metalloproteinase (MMP)-2 and MMP-9 activity in HUVECs in vitro. Topical application of GEH-RGD NPs (once daily for a week) significantly attenuated the formation of pathological vessels in the mouse cornea after chemical cauterization. Reduction in both vascular endothelial growth factor (VEGF) and MMP-9 protein in the GEH-RGD NP-treated cauterized corneas was observed. These results confirm the molecular mechanism of the antiangiogenic effect of GEH-RGD NPs in suppressing pathological corneal NV.

Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvß3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvß3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 µg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.