Nanodiamonds (NDs) are an innovative material in biomedical applications based on their excellent biocompatibility, nanoscale dimensions, and high surface area. In this study, we evaluated the potential of ND-in-oil emulsion to induce potent antibody responses in animals immunized with cobra venom. NDs demonstrated the capacity to bind complex venom proteins as stable conjugates, well dispersed in aqueous solution. Immunization of mice with cobra venom incorporated with ND-in-oil emulsion adjuvant (ND/venom) elicited strong venom-specific antibody responses with titers comparable to those induced by venom formulation with conventional Freund’s adjuvants (FA/venom). IgG subclass analysis revealed that ND- and FA-based formulations induced a Th2-biased immune response in mice. Moreover, antibodies elicited by ND/venom or FA/venom immunization specifically recognized the epitopes of the lethal component of short-chain neurotoxin and conferred full protection against lethal cobra venom challenge (3LD₅₀). Further, ND/venom hyperimmunization was capable of inducing high levels of neutralizing antibodies in larger animals, rabbits, highlighting the potential for antivenom manufacturing. Notably, there were no obvious lesions at the injection sites of animals that received ND/venom, in contrast to those that received FA/venom. These findings indicated NDs as an effective and safe additive in venom formulation for antivenom production.

Introduction: This study investigates the transdermal permeation efficiency and traceability of fluorescent nanodiamond-conjugated small extracellular vesicles (FND-sEVs) derived from umbilical cord mesenchymal stem cells. Methods: First, the sEVs were isolated and characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis, confirming their typical cup-shaped morphology and size distribution (62.5–141.1 nm) consistent with canonical sEV properties. Subsequently, FNDs were conjugated to sEVs, forming FND-sEVs, whose composite structure – featuring a high-electron-density FND core surrounded by a vesicular sEV membrane – was verified by TEM. Conjugation efficiency was then validated via confocal microscopy, showing complete colocalization between FND fluorescence and Alexa Fluor 488-labeled wheat germ agglutinin-tagged sEVs. Transdermal assessment was conducted using a skin tissue model, with magnetically modulated fluorescence (MMF) spectroscopy applied for background-free quantification. Results: Leveraging the unique nitrogen-vacancy centers in FNDs (fluorescence lifetime ∼20 ns), MMF eliminated interference from skin autofluorescence (∼3 ns) and enabled direct quantification in tissue digests without pre-separation. FND-sEVs were discovered to remain predominantly in the supernatant, with a measurable fraction permeating skin tissue layers, while their presence in the filtrate was negligible, thereby indicating limited transdermal passage. Conclusion: Collectively, these findings establish FND-sEVs as a traceable platform for transdermal studies and highlight MMF as a robust tool for quantifying nanoscale vesicle distributions in complex biological matrices.