Melanin is a kind of dark insoluble pigment that can cause pigmentation and free-radical clearance, inducing melasma, freckles, and chloasma, affecting the quality of life of patients. Due to poor water solubility and low safety, the absorption of poorly water-soluble drugs is limited by the hinderance of a skin barrier. Therefore, it is necessary to develop new, safe, and highly efficient drugs to improve their transdermal absorption efficiency and thus to inhibit the production of melanin. To address these issues, we developed a new nicotinamide (NIC)-stabilized phloretin nanocrystals (PHL-NCs). First, NC technology significantly increased the solubility of PHL. The in vitro release results indicated that at 6 h, the dissolution of the PHL-NIC-NCs was 101.39% ± 2.40% and of the PHL-NCs was 84.92% ± 4.30%, while that of the physical mixture of the two drugs was only 64.43% ± 0.02%. Second, NIC acted not only as a stabilizer to enlarge the storage time of PHL-NIC-NCs (improved to 10-day in vitro stability) but also as a melanin transfer inhibitor to inhibit melanin production. Finally, we verified the melanin inhibition effect of PHL-NIC-NCs evaluated by the zebrafish model. It showed that 0.38 mM/L PHL-NIC-NCs have a lower tyrosinase activity at 62.97% ± 0.52% and have less melanin at 36.57% ± 0.44%. The inhibition effect of PHL-NCs and PHL-NIC-NCs was stronger compared to the positive control arbutin. In conclusion, the combination of NIC and PHL achieves better inhibition of tyrosinase and inhibition of melanin production through synergism. This will provide a direction to the subsequent development of melanin-inhibiting drugs and the combined use of pharmaceutical agents.

Antigen cross-presentation by dendritic cells (DCs) is essential for inducing cytotoxic immune responses, which are crucial for eliminating infected or malignant cells. Although microbial signal processing reveals that the temporal relationship between DC activation and antigen uptake dictates cross-presentation efficiency, these two fundamental events are poorly coordinated in current vaccine design. Here, we report an oligosaccharide (OG) adjuvant that induces rapid DC activation but deferred antigen uptake, by sequentially activating two separate pathogen-recognition receptors on the same cells. Specifically, we synthesize a series of glucomannan hexasaccharides with varying degrees of acetylation (acOG6) and identify a low-acetylated candidate (0.6; acOG6-L) that could rapidly activate DCs into a pro-inflammatory state through toll-like receptor 2 (TLR2) signaling while, after 6 h, triggering cluster of differentiation 14 (CD14)-mediated antigen internalization. Molecular dynamics (MD) simulations provide structural insight into how acetylation affects the assembly of acOG6-L and its interaction with TLR2 pockets. This temporal deferment enables the trafficking of ovalbumin (OVA), which is readily conjugated with acOG6-L, into endosomal compartments, where the recruited nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) contributes to the alkalization of the antigen-containing phagosome - a critical step for promoting cross-presentation. Comprehensive in vitro and in vivo tests validate that OVA-acOG6 elicits enhanced antigen-specific CD8⁺ T cell -mediated cellular immunity against tumor cells in a subcutaneous B16 ovalbumin-expressing (B16-OVA) melanoma model. This work demonstrates that temporally coordinating cellular activation and antigen uptake by an acetyl hexasaccharide effectively enhances antigen cross-presentation.