In Situ Self-assembly Strategy for Modifying FPN Probes Targeting Melanoma to Prolong Retention Time

Introduction: Malignant melanoma (MM) is a serious form of invasive skin cancer with the highest risk of death. Because of the fast growth rate of MM, early detection and staging sometimes may mean the difference between life and death. In this scenario, both prompt diagnosis and accurate disease staging are paramount to reduce mortality. Melanoma comes from skin cells called melanocytes which can produce melanin. Fluoro-N-(2-[diethylamino]ethyl)picolinamide (FPN) with high melanin affinity and favorable pharmacokinetic properties has shown promising preclinical value for identifying small nodules and distant metastases in MM. However, due to the short retention time of small molecule drugs at the tumor site, rapid efflux may lead to the omission of lesions. Here, we report an in situ self-assembly strategy to enhance the diagnostic value of FPN for MM.

Methods: The probe (named as ⁶⁸Ga-NOTA-PEP-FPN) was made of a glutathione (GSH)-reactive self-assembling polypeptide (PEP) as the skeleton, NOTA as metal chelating group and FPN as targeting group. Its characterization was analyzed by high-performance liquid chromatography (HPLC), high-resolution mass spectrometry (HRMS) and transmission electron microscopy (TEM). Different ⁶⁸Ga-NOTA-PEP-FPN and ⁶⁸Ga-NOTA-FPN PET imaging of MM tumor model mice (n = 3) at different time points (30, 60 and 120 min) post tail vein injection (p.i.) were obtained. Then mice were sacrificed to obtain the major organs (including heart, lung, liver, spleen, and kidney) and tumors to conduct the biological distribution. In addition, these tissues were sectioned and stained with H&E for histological analysis. The t-test were used to compare the differences between groups. P < 0.05 indicates statistical significance.

Results: ⁶⁸Ga-NOTA-PEP-FPN was successfully synthesized and identified to be assembled in glutathione environment in vitro. The micellar structures were well observed under TEM. NOTA-PEP-FPN with purity greater than 97 % were used in radiolabeling experiments. High and uniform tumor distribution were seen in ⁶⁸Ga-NOTA-PEP-FPN group at different time point. Additionally, the probe provided clearer tumor imaging, and longer tumor retention time for 3.80 times than ⁶⁸Ga-NOTA-FPN at 120 min (1.60 ± 0.51 vs. 0.42 ± 0.20, P = 0.04). Ex vivo imaging corroborated these results. In vivo toxicity evaluation showed that ⁶⁸Ga-NOTA-PEP-FPN can not cause significant changes in liver and kidney function nor blood parameters in healthy mice.

Conclusions: For long-term tumor accumulation, we have created a self-assembled FPN-based probe using phenylalanine polypeptides as the primary chain. The insertion of disulfide bonds into phenylalanine polypeptides resulted in persistent self-assembly in a high GSH tumor environment, considerably prolonging the retention effect. Furthermore, in vivo PET imaging revealed a considerable concentration of ⁶⁸Ga-NOTA-PEP-FPN in melanoma. The features of long-term retention and production of nanomaterials are more conducive to therapeutic possibility. In conclusion, our self-assembled probe adds diagnostic value to the diagnosis of melanoma. Additionally, because of the ease with which chemical synthesis features may be included into the phenylalanine self-assembly platform, it has the potential to be widely applied in different disorders.

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