Cytotoxicity Evaluation of Photosensitizer-Conjugated Hexagonal Upconverting Nanoparticles

Abstract

In this report, we synthesized hexagonal NaYF₄:Yb,Er upconverting nanoparticles (UCNPs) of 171 nm in size with a narrow particle size distribution. To address their colloidal stabi-lity in aqueous media and to incorporate a photosensitizer that can produce reactive singlet oxygen (¹O₂) to kill tumor cells, UCNPs were conjugated with 6-bromohexanoic acid-functionalized Rose Bengal (RB) and coated with PEG-alendronate (PEG-Ale). The particles were thoroughly characterized by transmission electron microscopy, dynamic light scattering, ATR FTIR, X-ray photoelectron spectroscopy, thermogravimetric analysis, and spectrofluorometry, and ¹O₂ formation was detected using a 9,10-diphenylanthracene spectrophotometric probe. Cytotoxicity determination on rat mesenchymal stem cells by using the MTT assay showed that neutralization of the large positive surface charge of neat UCNPs with PEG-Ale and the bound RB sensitizer significantly reduced the concentration-dependent cytotoxicity. The presented strategy shows great potential for the use of these particles as a novel agent for the photodynamic therapy of tumors.

Keywords: cytotoxicity; nanoparticles; photosensitizer; rose bengal; upcoverting.

Figure 1

(a) Preparation of 2-[(5-carboxypentyloxy)carbonyl]- rose bengal (RB-CPC) and (b) modification of hexagonal UCNP with RB-CPC and PEG-Ale.

Figure 2

TEM micrographs of hexagonal (a) UCNP@OA, (b) UCNPs, (c) UCNP@Ale, (d) UCNP@Ale-RB-CPC, and (e) UCNP@Ale-RB-CPC/Ale-PEG nanoparticles. The inset shows a thin layer of Ale on the particles.

Figure 3

(a) Luminescence intensity of UCNP@OA particles excited at 980 nm and (b) their TEM micrographs at different reaction times.

Figure 3

(a) Luminescence intensity of UCNP@OA particles excited at 980 nm and (b) their TEM micrographs at different reaction times.

Figure 4

Comparison of the high-resolution XPS spectra of the initial hexagonal UCNP@OA (black), UCNP@Ale (magenta), UCNP@Ale-RB-CPC (green), and UCNP@Ale-RB-CPC/Ale-PEG (navy) in the region of (a) P 2p, Y 3d, Er 4d, Yb 4d, P 2s, Cl 2p, and (b) C 1s (red, fitted data; blue, individual contributions of functional groups on the UNCP surface).

Figure 5

(a) FTIR spectra, (b) thermogravimetric analysis, and (c) luminescence spectra of surface-modified hexagonal UCNPs excited at 980 nm.

Figure 6

Time-dependent decrease in DPA absorbance in the presence of hexagonal UCNP@Ale-RB-CPC/Ale-PEG particles excited at 980 nm and measured by UV-Vis spectroscopy. The arrow points to the generation of ¹O₂. The black curve represents the beginning of the experiment; each subsequent curve was measured after 10 min.

Figure 7

Cytotoxicity of neat hexagonal UCNPs (orange) and UCNP@Ale-RB-CPC/PEG-Ale particles (dark red) incubated with rMSCs for 24 h and measured using the MTT cell viability assay. Error bars represent the standard error of the mean (S.E.M.) calculated from at least three different experiments performed in triplicate; *^,† p < 0.05, ^†† p < 0.01, ***^,††† p < 0.001, and ^†††† p < 0.0001; one-way ANOVA with Dunnett’s post hoc test (ꝉ) to compare the particular treatment relative to the control and the two-tailed unpaired Student’s t-test (*) to compare differences between the UCNPs and UCNP@Ale-RB-CPC/PEG-Ale.