Background: Prostate‐specific membrane antigen (PSMA), overexpressed on prostate cancer (PCa), is a well‐characterized cell surface protein to selectively diagnose PCa. PSMA's unique characteristics and its 1000‐fold higher expression in PCa compared with other tissues renders it as a suitable biomarker for detection of PCa in its early stage. In this report, we critically analyze and recommend the requirements needed for the development of variety of PSMA‐targeted molecular imaging agents based on antibodies, small molecule ligands, peptides, and aptamers. The targeting moieties are either conjugated to radionuclear isotopes or near‐infrared agents for efficient diagnosis of PCa.Recent findings: From the analysis, it was found that several small molecule–derived PCa imaging agents are approved for clinical trials in Europe and the United States, and few are already in the clinical use for diagnosis of PCa. Even though ¹¹¹In‐labeled capromab pendetide was approved by the Food and Drug Administration (FDA) and other engineered antibodies are available for detection of PCa, but high production cost, low shelf life (less than 1 month at 4°C), possibility of human immuno reactions, and low blood clearance rate necessitated a need for developing new imaging agents, which are serum stable, cost‐effective, and possesses longer shelf life (6 months), have fast clearance rate from nontargeted tissues during the diagnosis process. It is found that small molecule ligand‐derived imaging agents possesses most of the desired properties expected for an ideal diagnostic agent when compared with other targeting moieties.Conclusion: This report discusses in detail the homing moieties used in the development of targeted diagnostic tools for detection of PCa. The merits and demerits of monoclonal antibodies, small molecule ligands, peptides, and aptamers for imaging of PCa and intraoperative guided surgery are extensively analyzed. Among all, urea‐based ligands were found to be most successful in preclinical and clinical trials and show a major promise for future commercialization.

The current challenge in fluorescence guided surgery (FGS) for prostate cancer (PCa) is in the design of imaging probes with high selectivity, clear visualization of tumour margins, and minimal toxicity. This report aims to design and develop a novel NIR-nanoprobe, and evaluate its potential in the penetration of PCa tumour tissues. The PSMA receptor-targeted quantum dot (PSMA-QD655) is a NIR, deep-tissue imaging agent, which has the potential for intraoperative navigation during surgery and improved detection specificity for PCa. The probe was designed and synthesized by conjugating functionalized amino-PEG quantum dots (QDs) through a heterobifunctional linker to a DUPA targeted polypeptide construct. The nanoprobe was evaluated in vitro in PSMA⁺ PCa cell lines for specificity and its binding affinity was determined by flow cytometric analysis. The penetration efficacy was tested further on large PCa 3D tumour spheroids (dia ∼1200 μm, thickness ∼450 μm) by deep tissue multiphoton imaging. PSMA-QD655 was found to be an efficient deep tissue intra-operative guided surgical tool with a high affinity (K_D = 15.3 nM) and penetrative capacity. The results have been demonstrated in vitro in 2D and 3D tissue models, mimicking cancer lesions in vivo. In summary, we have developed a deep-tissue imaging NIR nanoprobe targeting prostatic lesions that (i) binds to PSMA⁺ tumour with sub-nanomolar affinity and high specificity, (ii) shows an excellent safety profile in primary cell lines in vitro and (iii) shows high penetrative capacity in a 3D prostate tumour model (∼450 μm tissue depth).

In this article, we have successfully designed and demonstrated a novel continuous process for assembling targeting ligands, peptidic spacers, fluorescent tags and a chelating core for the attachment of cytotoxic molecules, radiotracers, nanomaterials in a standard Fmoc solid-phase peptide synthesis in high yield and purity. The differentially protected Fmoc-Lys-(Tfa)-OH plays a vital role in attaching fluorescent tags while growing the peptide chain in an uninterrupted manner. The methodology is versatile for solid-phase resins that are sensitive to mild and strong acidic conditions when acid-sensitive side chain amino protecting groups such as Trt (chlorotrityl), Mtt (4-methyltrityl), Mmt (4-methoxytrityl) are employed to synthesise the ligand targeted fluorescent tagged bioconjugates. Using this methodology, DUPA rhodamine B conjugate (DUPA = 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid), targeting prostate specific membrane antigen (PSMA) expressed on prostate, breast, bladder and brain cancers and pteroate rhodamine B, targeting folate receptor positive cancers such as ovarian, lung, endometrium as well as inflammatory diseases have been synthesized. In vitro studies using LNCaP (PSMA +ve), PC-3 (PSMA −ve, FR −ve) and CHO-β (FR +ve) cell lines and their respective competition experiments demonstrate the specificity of the newly synthesized bioconstructs for future application in fluorescent guided intra-operative imaging.