Background The therapeutic efficacy of small molecular agents is often hampered by low retention at tumor sites due to rapid efflux. To address this challenge, we present a novel in situ self-assembly strategy that employs a disulfide-containing phenylalanine (PEP) polypeptide chain, coupled with a targeting group and a radiometal chelator. We have demonstrated that PEP can be specifically targeted to glioma and melanoma using c(RGD)yk and FPN peptides, respectively, evidenced by PET imaging. When PEP is reduced by glutathione (GSH), which is often elevated in the tumor microenvironment, nanomicelles are formed through hydrophobic interactions, thus increasing retention time. Importantly, tumor uptake was enhanced due to in situ self-assembly, significantly improving the efficacy of radioligand therapy.

Methods The probe, designated as either PEP-c(RGD)yk or PEP-FPN, was formed using a solid-phase peptide synthesis. It consists of a GSH-reactive, self-assembling PEP as the skeleton, NOTA or DOTA as the metal chelating group, and either c(RGD)yk or FPN as the targeting group. Characterization was conducted using HPLC, HRMS, and TEM. In vitro cell viability and cellular uptake were performed. PET imaging using either ⁶⁴Cu or ⁶⁸Ga was performed on tumor-bearing mice (n = 3) at various time points following intravenous injection. Afterward, the mice were sacrificed, and their major organs (heart, lung, liver, spleen, and kidney) and tumors were collected for biodistribution analysis. For targeted radioligand therapy, glioma-bearing mice were treated with ¹⁷⁷Lu-PEP-c(RGD)yk, ¹⁷⁷Lu-c(RGD)yk, free ¹⁷⁷Lu, and PBS (n = 5). Tumor growth and body weight were recorded post treatment. In addition, major tissues were sectioned and stained with H&E for histological analysis. Statistical differences between groups were assessed using the t-test, with P < 0.05 denoting statistical significance.

Results Both PEP-c(RGD)yk and PEP-FPN were successfully synthesized and found to undergo self-assembly in the presence of GSH in vitro. The micellar structures were well observed under TEM. Each formulation with purity greater than 97 % was used in radiolabeling experiments. Cell viability was not affected by these formulations, and the nanoprobe was internalized after receptor binding as evidenced by the accumulation of radioactivity in tumor cells. The nanoprobe with PEP exhibited sustained tumor retention by at least 3-fold compared to the control probe without PEP, as evidenced by PET imaging. Histopathological analysis and in vivo cytotoxicity evaluation revealed no adverse effects on the major organ tissues or blood parameters from these nanoprobes. Furthermore, U87MG tumor-bearing mice treated with ¹⁷⁷Lu-PEP-c(RGD)yk showed a significant reduction in tumor volume over time, and the survival rate of the treated group was substantially improved. There was also no significant impact on body weight from the treatment, indicating the excellent biocompatibility of the agent.

Conclusion To prolong tumor retention time, we have synthesized a self-assembled nanoprobe using phenylalanine polypeptides as the primary chain. The cleavage of disulfide bonds in GSH-rich tumor microenvironment initiated the self-assembly process. Furthermore, in vivo PET imaging revealed a considerable uptake of PEP-c(RGD)yk in U87-MG and PEP-FPN in melanoma tumors. Moreover, radioligand therapy using ¹⁷⁷Lu-PEP-c(RGD)yk significantly inhibited tumor growth and improved survival rates without affecting the health of the mice, indicating an effective and safe treatment approach. In conclusion, the self-assembled nanoprobe can prolong tumor retention time and improve cancer detection and therapy. Notably, our versatile phenylalanine-based self-assembly platform readily allows for modifications with a variety of targeting ligands and stimuli-responsive linkers. This flexibility showcases its potential for applications in a wide range of diseases.

Image/Figure:

Click to view full size

Author