Introduction: Photodynamic therapy (PDT) is a non-invasive therapeutic approach that generates reactive oxygen species (ROS) through the light activation of a photosensitizer, leading to a localized response of oxidative stress and subsequent cell death. Zinc phthalocyanine (ZnPc) is a promising photosensitizer that is advantageous due to its intense light absorption and high level of ROS generation upon light activation¹, but its poor aqueous solubility and low tumor selectivity has limited its clinical use. These shortcomings could be improved through conjugation with a hydrophilic targeting molecule such as the integrin α_vβ₆-selective peptide ABM-5G². The integrin α_vβ₆ is an epithelial specific cell surface receptor that is overexpressed on numerous cancer types^{3, 4}, making it an attractive target for PDT. Notably, ABM-5G has shown α_vβ₆-selective tumor targeting in preclinical studies and is currently being evaluated in Phase I clinical trials (NCT04665947, NCT06228482 and NCT06389123). Here we report the in vitro and in vivo evaluation of ZnPc-ABM-5G (1) as a potential photodynamic therapy agent. 

Methods: ZnPc-ABM-5G (1, Fig. 1A) was synthesized by coupling ZnPc-COOH onto the ABM-5G peptide on solid phase. The singlet oxygen quantum yield (Φ_Δ)  of 1 was determined in DMSO and aqueous solubility was evaluated by the optical absorbance of 1 (2 μM) in comparison to unmodified ZnPc in fetal bovine serum (FBS). The phototoxic effects of 1 were evaluated in vitro by a WST-1 cell viability assay using DX3puroβ6 (α_vβ₆+) and DX3puro (α_vβ₆-) cells with or without near infrared (NIR) laser irradiation (671 nm, 40 mW/cm², 6 min). Pharmacokinetics and biodistribution were studied in mice bearing paired DX3puroβ6/DX3puro xenograft tumors at 1, 2, 4 and 24 h, and 24h post-intravenous (IV) injection of 1 (5 nmol), respectively, using an In Vivo Imaging System (IVIS) with Ex/Em filters of 675/720 nm. For PDT, mice bearing DX3puroβ6 tumors were randomly assigned to the PDT (1 + NIR) or control group (saline + NIR; n = 5/group); forty-eight hours after IV injection of either 1 (20 nmol) or saline, mice were exposed to NIR laser irradiation (671 nm, 100 mW/cm², 6 min/tumor) and imaged (IVIS) pre- and post-NIR exposure. Tumor volume and body weight were measured twice weekly.  

Results: Peptide 1 was successfully synthesized in 32% yield and the singlet oxygen quantum yield was measured as Φ_Δ = 0.67. In FBS, 1 exhibited a sharp absorption peak in contrast to the broad peak of unmodified ZnPc, indicating improved aqueous solubility of the ABM-5G conjugate 1 vs the unmodified ZnPc (Fig. 1B). Integrin α_vβ₆-selective phototoxicity was observed following NIR laser irradiation of DX3puroβ6 (α_vβ₆+) and DX3puro (α_vβ₆-) cells with 1, with an EC₅₀ of 1.5 nM and 66.6 nM, respectively (Fig. 1C). The pharmacokinetics study revealed good tumor uptake and low fluorescent signal in the surrounding tissue (Fig 1D), and the biodistribution study yielded a high tumor-to-skin ratio of 9.3±1.3 at 24 h post-injection. Optical imaging in the PDT study confirmed uptake of 1 in DX3puroβ6 tumors and significantly reduced fluorescence post-NIR laser irradiation, indicating photosensitizer quenching (Fig. 1E, F). At 21 days post-irradiation, significantly smaller tumors were observed in the PDT group compared to the control group (p = 0.0009). 

Conclusion: The α_vβ₆-targeted photosensitizer 1 was successfully synthesized and demonstrated high singlet oxygen quantum yield and good aqueous solubility. In vitro, α_vβ₆-selective phototoxicity was demonstrated. Current observations indicate tumor growth suppression in the PDT group (1 + NIR) in comparison to rapid tumor growth in the control group (saline + NIR) and suggest 1 as a potential PDT agent for α_vβ₆+ tumors.

Acknowledgements: Staff at Center of Molecular and Genomic Imaging of UC Davis. SU2C-AACR-PCC-06-18 and U01CA217665.

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Figure 1. (A) Structure of the α_vβ₆-targeted photosensitizer 1. (B) UV-Vis absorption spectra of 1 and unmodified ZnPc in FBS (1 μM, light path length = 10 mm). (C) Cell viability determined by WST-1 assay. (D) Representative in vivo fluorescence-bright field overlay images at 1, 2, 4, and 24 h after IV injection of 1 (5 nmol) in a mouse bearing DX3puroβ6 (α_vβ₆+, left) and DX3puro (α_vβ₆-, right) tumors on the upper left and right flank, respectively (color scale settings: 0.2 x 10⁸ – 8.0 x 10⁸ p/s/sr/μW). (E, F) Representative in vivo fluorescence-bright field overlay images during the PDT study, at 48 h after IV injection of 1 (20 nmol) before (E) and after (F) NIR irradiation of a mouse bearing an α_vβ₆+ DX3puroβ6 tumor on the lower left flank (arrow).

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