(214) Vesicle-Based Male Contraceptive Advances for Reversible Efficiency (VESI-CARE)

Sandeep Urandur¹; Lisa C. Rohan^1,2,3; William Walker^2,3; Kyle Orwig^2,3

1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, USA
2. Magee-Womens Research Institute, Pittsburgh, USA
3. Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, USA

Abstract Text:

Despite significant unmet need, male contraceptive options remain limited to condoms, withdrawal, and vasectomy. While hormonal contraceptive approaches under development act on the hypothalamic-pituitary-testicular axis to reduce intratesticular testosterone, they face significant limitations including delayed onset (3-4 months), bone loss, metabolic alterations, decreased libido, and weight gain on long-term use. In contrast, non-hormonal approaches offer potential advantages through direct targeting of spermatogenesis or sperm function, enabling faster onset and reduced systemic effects. Adjudin, a promising non-hormonal drug, disrupts spermatogenesis by targeting Sertoli-germ cell adhesion junctions, offering reversible contraception. However, adjudin displays poor bioavailability, with less than 1% reaching the testis following oral administration, largely due to its poor water solubility and the presence of efflux drug transporters (P-glycoprotein, multidrug resistance-associated protein 1) expressed by Sertoli cells, germ cells, peritubular myoid cells, and endothelial cells. These transporters actively pump exogenous compounds out of the testis, limiting drug bioavailability. Extracellular vesicles (EVs) naturally serve as intercellular communicators that can be internalized by specific testicular cells through membrane fusion and transcytosis without disrupting normal testicular function, making them ideal carriers for drug delivery. We aimed to develop testicular EVs (T-EVs) as biological nanocarriers to enhance adjudin delivery to its site of action. T-EVs were isolated from mouse testes (n=8) using enzymatic digestion with Accumax (20 mg/ml, 1 hour, 37°C), followed by differential centrifugation and membrane affinity chromatography. Vesicles were characterized for morphology (scanning electron microscopy), size (dynamic light scattering), concentration (nanoparticle tracking analysis), and molecular markers (Western blot). Adjudin loading was optimized by comparing passive incubation (37°C, 60 min) versus electroporation (400V, 125 μF) with varying drug-to-vesicle ratios. Loading efficiency was determined by high-performance liquid chromatography. Our methodology yielded T-EVs with uniform spherical morphology and optimal size distribution (mean 179.5 ± 1.1 nm), expressing characteristic markers CD81 and CD63, with protein content of 0.156 ± 0.005 mg/mL across three batches. Passive incubation achieved superior drug loading, with Adjudin concentration reaching 18.5 ± 5.2 nM (12-fold higher than the target concentration, i.e. 1.5 nM). We have successfully developed an in vitro Sertoli-germ cell co-culture model that will enable future assessment of drug-loaded vesicles on cellular junctions. We conclude that testicular extracellular vesicles can efficiently encapsulate Adjudin while maintaining vesicle integrity, potentially enabling enhanced local drug delivery through their natural cellular internalization mechanisms. Our approach represents a promising strategy for overcoming delivery challenges in non-hormonal male contraception development, addressing significant gaps in reproductive medicine, and potentially reaching millions of men seeking shared contraceptive responsibility. Our upcoming investigations include the development of advanced 3D testicular organoids to further validate this platform, alongside planned in vivo biodistribution and fertility impact studies that will advance this technology toward clinical application. The broader impact of this research extends beyond contraception—our T-EV platform represents a novel approach to enhancing testicular drug delivery for various therapeutic applications including treatment of testicular cancer, preservation of fertility during chemotherapy, and treatment of inflammatory conditions affecting reproductive tissues. Financial support for this research is provided by the Magee-Womens Research Institute Pilot Grant Program.