The evolution of peptidomimetic hybrid molecules for preoperative imaging and guided surgery targeting the prostate-specific membrane antigen (PSMA) significantly progressed over the past few years, and some approaches are currently being evaluated for further clinical translation. However, accumulation in nonmalignant tissue such as kidney, bladder, spleen, or liver might limit tumor-to-background contrast for precise lesion delineation, particularly in a surgical setting. To overcome these limitations, a rational linker design aims at the development of a second generation of PSMA-11–based hybrid molecules with an enhanced pharmacokinetic profile and improved imaging contrast. Methods: A selection of rationally designed linkers was introduced to the PSMA-targeting hybrid molecule Glu-urea-Lys-HBED-CC-IRDye800CW, resulting in a second-generation peptidomimetic hybrid molecule library. The biologic properties were investigated in cell-based assays. In a preclinical proof-of-concept study with the radionuclide ⁶⁸Ga, the impact of the modifications was evaluated by determination of specific tumor uptake, pharmacokinetics, and fluorescence imaging in tumor-bearing mice. Results: The modified hybrid molecules carrying various selected linkers revealed high PSMA-specific binding affinity and effective internalization. The highest tumor-to-background contrast of all modifications investigated was identified for the introduction of a histidine- (H) and glutamic acid (E)–containing linker ((HE)₃-linker) between the PSMA-binding motif and the chelator. In comparison to the parental core structure, uptake in nonmalignant tissue was significantly reduced to a minimum, as exemplified by an 11-fold reduced spleen uptake from 38.12 ± 14.62 percentage injected dose (%ID)/g to 3.47 ± 1.39 %ID/g (1 h after injection). The specific tumor uptake of this compound (7.59 ± 0.95 %ID/g, 1 h after injection) was detected to be significantly higher than that of the parental tracer PSMA-11. These findings confirmed by PET and fluorescence imaging are accompanied by an enhanced pharmacokinetic profile with accelerated background clearance at early time points after injection. Conclusion: The novel generation of PSMA-targeting hybrid molecules reveals fast elimination, reduced background organ enrichment, and high PSMA-specific tumor uptake meeting the key demands for potent tracers in nuclear medicine and fluorescence-guided surgery. The approach’s efficacy in improving the pharmacokinetic profile highlights the strengths of rational linker design as a powerful tool in strategic hybrid-molecule development.

用于针对前列腺特异性膜抗原（PSMA）的术前成像和引导手术的拟肽混合分子的进化在过去几年中取得了显着进展，目前正在评估一些方法以进行进一步的临床转化。然而，肾脏、膀胱、脾脏或肝脏等非恶性组织中的积累可能会限制肿瘤与背景的对比度，从而无法精确描绘病变，特别是在手术环境中。为了克服这些限制，合理的接头设计旨在开发第二代基于 PSMA-11 的杂合分子，该分子具有增强的药代动力学特征和改善的成像对比度。方法：将合理设计的连接子引入到PSMA靶向杂合分子Glu-urea-Lys-HBED-CC-IRDye800CW中，形成第二代拟肽杂合分子库。在基于细胞的测定中研究了生物学特性。在一项放射性核素⁶⁸ Ga 的临床前概念验证研究中，通过确定荷瘤小鼠的特定肿瘤摄取、药代动力学和荧光成像来评估修饰的影响。结果：携带各种选定接头的修饰杂合分子显示出高 PSMA 特异性结合亲和力和有效的内化。所有研究的修饰中肿瘤与背景对比度最高的原因是在 PSMA 结合基序和螯合剂之间引入了含有组氨酸 (H) 和谷氨酸 (E) 的接头（(HE) ₃ -接头） 。 与亲代核心结构相比，非恶性组织的摄取显着降至最低，例如脾脏摄取量从 38.12 ± 14.62 百分比注射剂量 (%ID)/g 减少到 3.47 ± 1.39 %ID/g，减少了 11 倍。 （注射后1小时）。检测到该化合物的特异性肿瘤摄取（7.59 ± 0.95 %ID/g，注射后 1 小时）显着高于亲本示踪剂 PSMA-11。 PET 和荧光成像证实的这些发现伴随着注射后早期时间点的药代动力学特征的增强和背景清除的加速。结论：新一代 PSMA 靶向混合分子具有快速消除、降低背景器官富集和高 PSMA 特异性肿瘤摄取的特点，满足核医学和荧光引导手术中对有效示踪剂的关键需求。该方法在改善药代动力学特征方面的功效凸显了合理连接子设计作为战略混合分子开发的强大工具的优势。