@Article{biocell.2025.065199,
AUTHOR = {Seo Yeon Shin, Nu Ri Song, Sa Rang Choi, Ki Min Kim, Jae Hee Byun, Su Jung Kim, Dai Hyun Jung, Seong Sim Kim, Seong Ju Park, So Jeong Chu, Kyung Mok Park},
TITLE = {Human-Derived Low-Molecular-Weight Protamine (hLMWP) Conjugates Enhance Skin Cell Penetration and Physiological Activity},
JOURNAL = {BIOCELL},
VOLUME = {49},
YEAR = {2025},
NUMBER = {8},
PAGES = {1435--1448},
URL = {http://www.techscience.com/biocell/v49n8/63612},
ISSN = {1667-5746},
ABSTRACT = {Background: The efficient transdermal delivery of biologically active molecules remains a major challenge because of the structural barrier of the stratum corneum, which limits the penetration of large or hydrophilic molecules. Low-molecular-weight protamine (LMWP) has a structure similar to that of the HIV TAT protein-derived peptide and is a representative cell-penetrating peptide (CPP) used to increase cell permeability. However, protamine has been reported to have many toxicities and side effects. Objectives: We developed human-derived low-molecular-weight protamine (hLMWP), which is based on fish-derived LMWP but designed using human protein sequences to improve safety and functionality. As it is derived from human proteins, it may reduce side effects and immune rejection during long-term or repeated administration. Additionally, we confirmed in our preliminary study that hLMWP enhances permeability compared to LMWP. In this study, we evaluated physiological activities and skin cell penetration of hLMWP conjugates to assess the potential applications of hLMWP. Methods: cDNA sequences for hLMWP-EGF (His) and hLMWP-SOD-1 (His) were synthesized by connecting hLMWP (RSRRRRRRSCQTRRR) to the N-terminus of Epidermal growth factor (EGF) and Superoxide dismutase 1 (SOD-1), respectively, with a 6 His-tag added to the C-terminus. The constructs were cloned into a pET-41b(+) expression vector and expressed in <i>E. coli</i> BL21-CodonPlus (DE3)-RIL cells. Expressed proteins were purified using a nickel column and eluted with imidazole buffer. Protein purity was confirmed by SDS-PAGE, and concentrations were quantified using a BCA assay. To evaluate the functional properties of these hLMWP-protein conjugates, a series of <i>in vitro</i> assays were conducted using keratinocyte and macrophage cell lines. These included assessments of permeability, proliferation, wound healing, and anti-inflammatory activity. Results: The results demonstrated that hLMWP-EGF and hLMWP-SOD-1 exhibited superior biological activities, including increased cell proliferation, wound healing, and anti-inflammatory effects, compared to EGF and SOD-1. Moreover, hLMWP-EGF and hLMWP-SOD-1 significantly enhanced the skin permeability of both EGF and SOD-1, as shown by Franz diffusion cell assay and immunofluorescence analysis. Conclusion: Our findings demonstrate that hLMWP significantly enhances skin permeability and biological activity of functional proteins such as EGF and SOD-1 while maintaining safety. This suggests its potential for application in transdermal drug delivery, regenerative medicine, and cosmeceutical formulations.},
DOI = {10.32604/biocell.2025.065199}
}