Cell-penetrating peptide enhanced intracellular Raman imaging and photodynamic therapy

Abstract

We present the application of a theranostic system combining Raman imaging and the photodynamic therapy (PDT) effect. The theranostic nanoplatform was created by loading the photosensitizer, protoporphyrin IX, onto a Raman-labeled gold nanostar. A cell-penetrating peptide, TAT, enhanced intracellular accumulation of the nanoparticles in order to improve their delivery and efficacy. The plasmonic gold nanostar platform was designed to increase the Raman signal via the surface-enhanced resonance Raman scattering (SERRS) effect. Theranostic SERS imaging and photodynamic therapy using this construct were demonstrated on BT-549 breast cancer cells. The TAT peptide allowed for effective Raman imaging and photosensitization with the nanoparticle construct after a 1 h incubation period. In the absence of the TAT peptide, nanoparticle accumulation in the cells was not sufficient to be observed by Raman imaging or to produce any photosensitization effect after this short incubation period. There was no cytotoxic effect observed after nanoparticle incubation, prior to light activation of the photosensitizer. This report shows the first application of combined SERS imaging and photosensitization from a theranostic nanoparticle construct.

Figure 1

SERRS spectra of AuNS-DTDC solution (solid, top), AuNS-DTDC@SiO2-PpIX-TAT solution (dotted, middle), and a point collection from a cell that had been incubated with AuNS-DTDC@SiO2-PpIX-TAT (dashed, bottom). All spectra were acquired at 633 nm excitation (8 mW) with a 10 second integration time. The solution spectra were recorded using a 10× objective with the particles suspended in water, while the intracellular Raman spectrum was recorded with a 40× objective. Spectra are baseline-subtracted and offset for clarity.

Figure 2

(A) Absorption spectra of free PpIX (solid) and DTDC (dashed) in ethanol. (B) Absorption spectra of the AuNS-DTDC before (solid, left axis) and after (dotted, left axis) silica coating (particles dispersed in water) and fluorescence emission from the AuNS-DTDC@SiO2-PpIX-TAT (dispersed in ethanol) under 415 nm excitation (dashed, right axis).

Figure 3

TEM micrograph of the silica coated AuNS. Scale bar is 100 nm.

Figure 4

Cell viability after a 1-hour incubation with various particle samples at a concentration of 0.1 nM. The error bars are ± one standard deviation of 8 measurements.

Figure 5

Three representative Raman images of cells incubated with AuNS-DTDC@SiO₂-PpIX-TAT, collected with 633 nm excitation (8 mW). The top row shows the brightfield image and selected area while the bottom row displays the calculated Raman image. The calculated intensity values came from the integrated peak intensity of the baseline-subtracted DTDC peak between 1120 and 1150 cm⁻¹. The intensity scale is shown beneath the images and was kept constant across all acquisitions.

Figure 6

Viability staining of cells incubated with AuNS-DTDC@SiO₂-PpIX-TAT (left) and AuNS-DTDC@SiO₂-TAT (right) after 30 seconds of light irradiation. Live cells are stained green and dead cells are stained red. Scale bars are 250 µm.

Scheme 1

Schematic depiction of the nanocomposite synthesis.