Potential of Plant-derived Exosome-like Nanoparticles from Physalis peruviana Fruit for Human Dermal Fibroblast Regeneration and Remodeling.

AIM: This research aimed to study the potential of PDEN from P. peruviana fruits (PENC) for regenerating and remodeling HDF. BACKGROUND: Large wounds are dangerous and require prompt and effective healing. Various efforts have been undertaken, but have been somewhat ineffective. Plant-derived exosome-like nanoparticles (PDEN) are easily sampled, relatively cost-effective, exhibit high yields, and are nonimmunogenic. OBJECTIVE: The objective of the study was to isolate and characterize PDEN from Physalis peruviana (PENC), and determine PENC's internalization and toxicity on HDF cells, PENC's ability to regenerate HDF (proliferation and migration), and PENC ability's to remodel HDF (collagen I and MMP- 1 production). METHOD: PENC was isolated using gradual filtration and centrifugation, followed by sedimentation using PEG6000. Characterization was done using a particle size analyzer, zeta potential analyzer, TEM, and BCA assay. Internalization was done using PKH67 staining. Toxicity and proliferation assays were conducted using MTT assay; meanwhile, migration assay was carried out by employing the scratch assay. Collagen I production was performed using immunocytochemistry and MMP-1 production was conducted using ELISA. RESULT: MTT assay showed a PENC concentration of 2.5 until 500 µg/mL and being non-toxic to cells. PENC has been found to induce cell proliferation in 1, 3, 5, and 7 days. PENC at a concentration of 2.5, 5, and 7.5 µg/mL, also accelerated HDF migration using the scratch assay in two days. In remodeling, PENC upregulated collagen-1 expression from day 7 to 14 compared to control. MMP-1 declined from day 2 to 7 in every PENC concentration and increased on day 14. Meanwhile, ROS declined from day 2 to 7 and 14, with PENC inducing a rise in the ROS level compared to control. Overall, PENC at concentrations of 2.5, 5, and 7.5 µg/mL induced HDF proliferation and migration, upregulated collagen I production, and decreased MMP-1 levels. CONCLUSION: Isolated PENC was 190-220 nm in size, circular, covered with membrane, and its zeta potential was -6.7 mV; it could also be stored at 4°C for up to 2 weeks in aqua bidest. Protein concentration ranged between 170-1,395 µg/mL. Using PKH67, PENC could enter HDF within 6 hours. PENC was non-toxic up to a concentration of 500 µg/mL. Using MTT and scratch assay, PENC was found to elevate HDF proliferation and migration, and reorganize actin. Using immunocytochemistry, collagen I was upregulated by PENC, whereas MMP-1 concentration was reduced.

Development and Characterization of a Gel Formulation Containing Golden Cherry Exosomes (Physalis minima) as a Potential Anti-Photoaging.

AIM: The present study aims to produce a novel therapeutic approach for the treatment of photoaging. BACKGROUND: Plant-derived exosome-like nanoparticles (PDENs) are nano-sized vesicles containing biomolecules released by multivesicular bodies. Recently, studies have shown the efficacy of exosomes in treating photoaging through increasing collagen synthesis and decreasing collagen degradation. In addition, some PDENs were also proven to contain bioactive metabolites, which also have potential antioxidant activity to mitigate the risk of photoaging. OBJECTIVE: Formulating and developing a gel and incorporating it with exosomes derived from golden cherry (Physalis minima). METHOD: The formulation was developed by first preparing various base formulations with different compositions and selecting the best through evaluation tests. The results showed that only polymer base natrosol with a concentration of 0.25% was suitable for incorporating exosomes. The selected base was then incorporated with various concentrations of golden cherry exosomes and was evaluated regarding its physical and stability profile. RESULT: The result demonstrates that the incorporated gel displayed pleasant organoleptic properties and a pH compatible with the skin, with pseudoplastic flow and a suitable viscosity for topical application. The stability study also only revealed minor changes in viscosity and pH without affecting the general stability of the formulation. Formulation incorporating 0.25% golden cherry exosomes had shown the best stability profile compared to other concentrations. On characterization, although the incorporated exosomes showed heterogeneous particle size distribution (PI > 0.3), they still maintained their structural integrity. In addition, the incorporated exosomes showed antioxidant activity with IC 50 of 372.435 µg/mL, which can help mitigate the risk of photoaging. CONCLUSION: Golden cherry exosomes have been successfully incorporated into gel and, thus, can be potentially utilized as a novel therapeutic approach for the treatment of photoaging.

Novel approach to enhance aggregate migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer-immobilized surface.

The dynamic migratory behavior of human mesenchymal stem cells (hMSCs) has a significant impact on the epigenetic profiles that determine fate choice and lineage commitment during differentiation. Here we report a novel approach to enhance repeated migration-driven epigenetic memory which induces cardiomyogenic differentiation on a dendrimer surface with fifth generation (G5). Cells exhibited the formation of cell aggregates on the G5 surface through active migration with morphological changes, and these aggregates showed strong expression of the cardiac-specific marker cardiac troponin T (cTnT) at 10 days. When cell aggregates were passaged onto a fresh G5 surface over three passages of 40 days, the expression levels of the multiple cardiac-specific markers including GATA4, NKX2.5, MYH7, and TNNT2 were higher compared to those passaged as single cells. To investigate whether cardiomyogenic differentiation of hMSCs was enhanced by repeated aggregate migration-driven epigenetic memory, cells on the G5 surface were reseeded onto a fresh G5 surface during three passages using aggregate-based and single cell-based passage methods. Analyses of global changes in H3 histone modifications exhibited pattern of increased H3K9ac and H3K27me3, and decreased H3K9me3 in aggregate-based passage cultures during three passages. However, the pattern of their histone modification on the PS surface was repeated after the initialization and reformation during three passages in single cell-based passage cultures. Thus, repetitive aggregate migratory behavior during aggregate-based passage led to a greater degree of histone modification, as well as gene expression changes suggestive of cardiomyogenic differentiation.

Muscle lineage switching by migratory behaviour-driven epigenetic modifications of human mesenchymal stem cells on a dendrimer-immobilized surface.

Understanding of the fundamental mechanisms of epigenetic modification in the migration of human mesenchymal stem cells (hMSCs) provides surface design strategies for controlling self-renewal and lineage commitment. We investigated the mechanism underlying muscle lineage switching of hMSCs by cellular and nuclear deformation during cell migration on polyamidoamine dendrimer surfaces. With an increase in the dendrimer generation number, cells exhibited increased nuclear deformation and decreased lamin A/C and lamin B1 expression. Analysis of two repressive modifications (H3K9me3 and H3K27me3) and one activating modification (H3K9ac) revealed that H3K9me3 was suppressed, and H3K9ac and H3K27me3 were upregulated in the cultures on a higher-generation dendrimer surface. This induced significant hMSC lineage switching to smooth, skeletal, and cardiac muscle lineages. Thus, reorganizations of the nuclear lamina and cytoskeleton related to migration changes on dendrimer surfaces are responsible for the integrated regulation of histone modifications in hMSCs, thereby shifting the cells from the multipotent state to muscle lineages. These findings improve our understanding of the role of epigenetic modification in cell migration and provide new insights into how designed surfaces can be applied as cell-instructive materials in the field of biomaterial-guided differentiation of hMSCs to different cell types. STATEMENT OF SIGNIFICANCE: Stem cell engineering strategies currently applied the mechanical cues that emerge from cellular microenvironment to regulate stem cell behaviour. This study significantly improved our understanding of the mechanotransduction mechanism involving cell-ECM and cytoskeleton-nucleoskeleton interactions, and of nuclear genome regulation based on cellular responses to biomaterial modifications. The new insights into how the physical environment on a culture surface influences cell behaviour improve our understanding of mechanical control mechanisms of the interactions of cells with the extracellular environment. Our findings are also expected to contribute to and play an essential role in the development of future material strategies for creating artificial cell-instructive niches.