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Conventional drug delivery methods to treat testicular disorders face various challenges, which could be circumvented by using targeted drug delivery. Testicular cell targeting ligands, such as Leydig cell homing peptides, would be an excellent choice to achieve the targeted delivery of drugs to the testis. In this study, Leydig cell homing peptides (LCHPs), LCHP1 and LCHP2, were identified via in vitro , followed by in vivo biopanning of a phage display peptide library and next-generation sequencing. Both of the LCHPs were validated in vitro for their specific Leydig cell and in vivo testis targeting potential. Furthermore, molecular targets of the LCHP1 and LCHP2 were identified using affinity purification mass spectrometry (APMS). The LCHP1 and LCHP2 are able to specifically target Leydig cells of the testis and undergo cell internalization as well as target the testis at the in vivo level, hence providing an opportunity to be utilized as a potential ligand for drug delivery to the testis., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

The testicle, an organ privileged with immunity because of Blood-Testis Barrier (BTB), poses a major impediment to developing and delivering drugs to the testes. These problems can be prevented by targeting testicular cells using specific ligands, such as homing peptides. This is the first study to demonstrate the successful selection of Sertoli cell homing peptides using a phage display peptide library. The identification of peptides is performed with Sanger sequencing and high-throughput NGS. The Sertoli cell and testis targeting potential of the SCHP1 and SCHP2 was confirmed using confocal microscopy and flow cytometry of the FITC-labelled peptides and in vivo bio-distribution of the corresponding Cy5.5-tagged peptides. Secondary structures were predicted in the setting of different polarity by circular dichroism. The results suggest that SCHP1 and SCHP2 can effectively target Sertoli cells. In vivo bio-distribution in mouse models indicated significantly higher uptake of SCHP1 and SCHP2 by testes compared with the heart, brain, and spleen. SCHP1 and SCHP2 can be adopted as molecular steering for targeted male contraceptive delivery, treatment of testicular cancer, and male infertility. Further development of the peptides into peptidomimetics may increase their stability, and information on the molecular targets of these peptides may reveal their therapeutic potential.