Cell-penetrating peptides (CPPs) have been widely used as vehicles for delivering therapeutic molecules to the site of action. Apart from their delivering potential, the biological effects of CPPs have not been explored in detail. JTS-1 is a CPP that has been reported to have gene delivery functions, although its biological role is yet to be determined. Hence, in this study, we revealed the biological mechanism such as its uptake mechanism and immunogenic potential and function using primary human tenon fibroblast (TF) cells collected from patients undergoing glaucoma trabeculectomy surgery. Our results showed that the JTS-1 peptide has an α-helical structure and is nontoxic up to 1 μM concentration. It was found to be colocalized with early endosome (Rab5), recycling endosome (Rab7), and Rab11 and interacted with major histocompatibility complex (MHC) class I and II. The peptide also affected actin polymerization, which is regulated by cofilin phosphorylation and ROCK1 localization. It also inhibited TF cell proliferation. Therefore, the JTS-1 peptide could be used as a possible therapeutic agent for modifying the fibrosis process, where TF proliferation is a key cause of surgery failure.

The signalling response is determined by the cell's reaction to different biochemical and biophysical inputs such as stiffness, topological, and structural alignment. The surface patterns at the nano-scale can be an influential factor in cell signalling behaviour. It is important to understand the cellular response to the biophysical cues for biomedical applications. Biomaterials have an important role in regenerative tissue engineering. In this study, we have fabricated electrospun polycaprolactone (PCL) and PCL- Aloe vera (PCL-AV) nanofibrous matrix and studied its effect on the human tenon fibroblast (HTF) cellular and morphological changes. The electrospun fibers were characterized using Scanning Electron Microscope (SEM), Fourier Transform Infrared spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Brunaur, Emette and Teller (BET) analysis for their morphology, composition, topography, surface area and porosity. The results revealed fiber size, roughness and porosity has been altered by addition of AV. The HTF cell viability, proliferation and expression of focal adhesion proteins, such as FAK, Ezrin, Vasp and Cofilin on the PCL-AV fiber matrix were examined. The results showed a change in cellular morphology and a significant change in the cofilin phosphorylation on PCL-AV nanofiber. The influence of Aloe vera composition on the nano-dimension of the PCL has made a significant impact on the cellular morphology at both gene and protein levels. This observation suggests that AV composition in the nanofiber can significantly influence the HTF cellular adhesions.

Fibrosis is the primary factor influencing the prognosis of glaucoma post-trabeculectomy surgery, an eye condition characterized by increased intraocular pressure (IOP). Despite advancements in surgical procedures and aftercare, it continues to be a serious impediment. During the clinical intervention of scarring, fibrosis is managed by using topical application of combined antifibrotic drugs (mitomycin C). But still, scarring remains a key problem due to minimal drug penetration and nonbioavailability. In this study, we synthesized a cell-specific peptide for modulating scarring in human tenon fibroblasts undergoing epithelial− mesenchymal transition (EMT). The peptide was also conjugated with mitomycin C in order to investigate the effect of the drug conjugation on human tenon fibroblasts from the nanofiber composite system and to evaluate the fibrosis process. Peptide VRF2019 was identified using a subtractive proteomics approach, including solubility, cell penetration, and amphipathic properties. The peptide structure was determined using circular dichroism spectroscopy. The peptide and drug was conjugated using N-ethyl-N′-(3-(dimethylamino)propyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry, and the conjugation efficiency was evaluated using highpressure liquid chromatography. The conjugated product and polycaprolactone (PCL) were electrospun to form a composite nanofiber, which was tested for cytotoxicity and drug release on human tenon fibroblast cells. The modeled VRF2019 peptide structure formed an α-helical structure with all residues spanning the allowed regions of the Ramachandran plot. Subsequent molecular dynamics simulations also demonstrated its membrane penetration potential. The peptide uptake was also studied in human tenon fibroblast cells. High-pressure liquid chromatography (HPLC) and mass spectrometry measurements confirmed peptide−drug conjugation and stability. Furthermore, scanning electron microscopy (SEM) investigation revealed the structure and size of the PCL composite nanofiber. We infer from early research that the PCL composite nanofiber matrix can greatly increase drug delivery and bioavailability.