Ganoderma sinense is a traditional and protein-rich edible fungus with outstanding immunoregulatory activity. However, the mechanism through which G. sinense protein hydrolysates and peptides exert their immunomodulatory effects is unclear. The objective of this study was to prepare and isolate immunologically active peptides from G. sinense protein hydrolysates (GSP) and to investigate their impact on the activation of macrophages. G. sinense peptides with low molecular weights (< 1 kDa, 87.76%) markedly promoted RAW264.7 cell proliferation; increased their phagocytic capacity, nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and IL-6 secretion and reactive oxygen species (ROS) production; and upregulated Tlr2 mRNA expression. In addition, GSP promoted nuclear factor kappa-B (NF-κB) activation and translocation through the upregulation of p65 and p-p65 protein expression. Virtual molecular screening technology revealed that compared with other peptides, the SFAGNIPVNR, YGDAFIR and TVSYLPAPQR peptides derived from GSP showed stronger binding affinities. Interaction site map analysis indicated that the SFAGNIPVNR and YGDAFIR peptides formed stable hydrogen bonds with toll-like receptor 2 (TLR2). Together, these results suggested that G. sinense peptides can serve as important ingredients in nutraceuticals or functional foods.

Intraocular injection of anti-vascular endothelial growth factor (VEGF) antibodies is the first-line treatment for ocular neovascular diseases. However, the invasive nature of this administration method often reduces patient compliance and negatively affects treatment outcomes. Noninvasive formulations of anti-VEGF antibody are urgently needed, but their development remains challenging due to the complex ocular barriers. This study identified an anti-VEGF single-domain antibody (sdVE01) that is three times smaller than the commercially available ranibizumab, yet retains a comparable anti-angiogenic effect to the heavy-chain region of ranibizumab (VHHL). Additionally, four dithiolane molecules (DM) were designed to construct DM-based antibody nanoformulations, which effectively penetrate both the anterior and posterior segments of the eye. Upon eyedrop administration, DM-based antibody nanoformulations significantly inhibited the VEGF pathway and reduced neovascularization in a corneal alkali-burn rat model. Notably, the therapeutic effects of the antibody eyedrops were comparable to those of ranibizumab administered via subconjunctival injection. Overall, the dithiolane-based antibody eyedrops represent a promising noninvasive strategy for treating ocular neovascularization diseases.