Optimization of a nanomedicine-based silicon phthalocyanine 4 photodynamic therapy (Pc 4-PDT) strategy for targeted treatment of EGFR-overexpressing cancers

Abstract

The current clinical mainstays for cancer treatment, namely, surgical resection, chemotherapy, and radiotherapy, can cause significant trauma, systemic toxicity, and functional/cosmetic debilitation of tissue, especially if repetitive treatment becomes necessary due to tumor recurrence. Hence there is significant clinical interest in alternate treatment strategies like photodynamic therapy (PDT) which can effectively and selectively eradicate tumors and can be safely repeated if needed. We have previously demonstrated that the second-generation photosensitizer Pc 4 (silicon phthalocyanine 4) can be formulated within polymeric micelles, and these micelles can be specifically targeted to EGFR-overexpressing cancer cells using GE11 peptide ligands, to enhance cell-specific Pc 4 delivery and internalization. In the current study, we report on the in vitro optimization of the EGFR-targeting, Pc 4 loading of the micellar nanoformulation, along with optimization of the corresponding photoirradiation conditions to maximize Pc 4 delivery, internalization, and subsequent PDT-induced cytotoxicity in EGFR-overexpressing cells in vitro. In our studies, absorption and fluorescence spectroscopy were used to monitor the cell-specific uptake of the GE11-decorated Pc 4-loaded micelles and the cytotoxic singlet oxygen production from the micelle-encapsulated Pc 4, to determine the optimum ligand density and Pc 4 loading. It was found that the micelle formulations bearing 10 mol % of GE11-modified polymer component resulted in the highest cellular uptake in EGFR-overexpressing A431 cells within the shortest incubation periods. Also, the loading of ∼ 50 μg of Pc 4 per mg of polymer in these micellar formulations resulted in the highest levels of singlet oxygen production. When formulations bearing these optimized parameters were tested in vitro on A431 cells for PDT effect, a formulation dose containing 400 nM Pc 4 and photoirradiation duration of 400 s at a fluence of 200 mJ/cm(2) yielded close to 100% cell death.

Figure 1

Optimization strategies to enhance the efficacy of nanomedicine-based cell-targeted PDT strategy.

Figure 2

Representative fluorescence images (A-L) comparing the Pc 4 uptake in A431 cells at various incubation time periods when delivered via EGFR-targeted micelles bearing different mole percentages of GE11 peptide incorporation; (M) shows quantitative data for the Pc 4 fluorescence in these cells for the various targeted nanoformulations at the different incubation periods; all levels of significance are for p < 0.05 for the different formulations at each timepoint.

Figure 3

(A) Representative fluorescence emission spectra of SOSG in solution with either free Pc 4 or Pc 4-nanoformulations of different loading extents, with or without photoirradiation; only three representative loading values are shown for convenience; the loading capacity of around 50 μg of Pc 4 per mg of polymer showed maximum SOSG fluorescence upon photoirradiation. (B) Plot of peak SOSG fluorescence intensity versus loading capacity (μg of Pc 4 per mg of polymer) showing maximum intensity (hence maximum singlet oxygen production) for formulations having about 50 μg Pc 4 per mg of polymer.

Figure 4

Representative images of A431 cells when incubated with free Pc 4 (D1-D2) versus Pc 4 nanoformulation (E1-E2) formulations with (light) or without (dark) photoirradiation; appropriate controls consisted of cells without any Pc 4 or SOSG (A1-A2), cells with SOSG only (B1-B2) and cells with free Pc 4 only (C1-C2); significantly enhanced singlet oxygen production was observed for cells incubated with Pc 4-NP and SOSG; (M) shows quantitative data of SOSG fluorescence intensity in the cells for the various test and control samples.

Figure 5

A431 cell response to PDT with various doses of Pc 4 delivered via micelles bearing 10 mole% EGFR-targeted ligands (the optimum targeting ligand parameter) and 50 μg Pc 4 per mg of polymer loading (the optimum loading parameter), with various irradiation times using 200 mJ/cm² fluence.