Radiodynamic Therapy Using TAT Peptide-Targeted Verteporfin-Encapsulated PLGA Nanoparticles

Abstract

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.

Keywords: PLGA; RDT; ROS; TAT peptide; X-PDT; nanoparticles; nuclear targeting; photosensitizer; radiation; radiation therapy; radiodynamic therapy; radiosensitization; reactive oxygen species; singlet oxygen; verteporfin.

Figure 1

Characterization of PLGA–VP–TAT and PGLA–VP nanoparticles in solution (a) SEM image, (b) Zeta potential in water, (c) florescence spectra of VP (425 nm ex/690 nm em) from the NPs in water, (d) absorbance spectra in water, showing the VP and TAT peaks, (e) PLGA–VP–TAT NP stability in PBS with and without FBS, as measured by the nanoparticle peak size, (f) % release of VP from PLGA–VP–TAT NPs in PBS at 37 °C.

Figure 2

Representative confocal images showing the uptake of PLGA–VP and PLGA–VP–TAT nanoparticles in PANC-1 cells after 4 h incubation: blue-Hoechst nuclear stain, red-VP fluorescence. Magnified single-cell images are shown on the right. Scale bar: 50 µm.

Figure 3

Cell viability and singlet oxygen generation following RDT. (a) representative confocal fluorescence image of live (green) and dead (red) cells following RDT with PLGA–VP–TAT NP and 4 Gy, compared to no-treatment controls, (b) corresponding cell viability, (c) representative confocal fluorescence image of SOSG fluorescence (green) and nuclear staining (blue) following 4 Gy RDT with PLGA–VP–TAT NPs compared to untreated controls, (d) SOSG fluorescence in treated and control cells, * p < 0.05 and ** p < 0.5. Scale bar: 50 µm.

Figure 4

RDT effects at the cellular level. (a) representative confocal microscopy images showing DNA double strand breaks indicated by γ-H2AX staining (red: γ-H2AX, blue: Hoechst nuclear stain), (b) quantification of γ-H2AX fluorescence, (c) lipid peroxidation in the treatment and control groups. Scale bar: 50 µm.

Figure 5

Clonogenic assay. (a) example of stained colonies at 14 d following treatment, (b) survival fraction for RDT in various treatment groups.

Figure 6

Schematic illustration of PLGA–VP–TAT nanoparticle formulation.