Multi-dye theranostic nanoparticle platform for bioimaging and cancer therapy

Abstract

Background

Theranostic nanomaterials composed of fluorescent and photothermal agents can both image and provide a method of disease treatment in clinical oncology. For in vivo use, the near-infrared (NIR) window has been the focus of the majority of studies, because of greater light penetration due to lower absorption and scatter of biological components. Therefore, having both fluorescent and photothermal agents with optical properties in the NIR provides the best chance of improved theranostic capabilities utilizing nanotechnology.

Methods

We developed nonplasmonic multi-dye theranostic silica nanoparticles (MDT-NPs), combining NIR fluorescence visualization and photothermal therapy within a single nanoconstruct comprised of molecular components. A modified NIR fluorescent heptamethine cyanine dye was covalently incorporated into a mesoporous silica matrix and a hydrophobic metallo-naphthalocyanine dye with large molar absorptivity was loaded into the pores of these fluorescent particles. The imaging and therapeutic capabilities of these nanoparticles were demonstrated in vivo using a direct tumor injection model.

Results

The fluorescent nanoparticles are bright probes (300-fold enhancement in quantum yield versus free dye) that have a large Stokes shift (>110 nm). Incorporation of the naphthalocyanine dye and exposure to NIR laser excitation results in a temperature increase of the surrounding environment of the MDT-NPs. Tumors injected with these NPs are easily visible with NIR imaging and produce significantly elevated levels of tumor necrosis (95%) upon photothermal ablation compared with controls, as evaluated by bioluminescence and histological analysis.

Conclusion

MDT-NPs are novel, multifunctional nanomaterials that have optical properties dependent upon the unique incorporation of NIR fluorescent and NIR photothermal dyes within a mesoporous silica platform.

Keywords:

• bioluminescence
• in vivo imaging
• mesoporous silica nanoparticles
• NIR fluorescence
• photothermal ablation
• theranostic

Acknowledgments

The authors acknowledge the financial support of the Particle Engineering Research Center at the University of Florida. This work was supported in part by the Department of Defense Breast Cancer Research Program (grant number 00085128), the National Science Foundation (grant number 00082755), and the Florida Department of Health Bankhead- Coley Cancer Research Program. The transmission electron microscopy imaging was performed by Kerry Siebein at the Major Analytical Instrumentation Center, College of Engineering, University of Florida, which also houses the scanning electron microscope and X-ray diffractometer.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary information

Modification of IR780 dye

Figure S1. Reaction scheme for the synthesis of modified NIR fluorescent IR780 dye. Synthetic procedures are described in Materials and methods.

Abbreviations: APTES, 3-aminopropyltriethoxysilane; IR780, IR780 iodide; NIR, near-infrared.

In vitro cytotoxicity

Figure S2. Effect of MDT-NPs on MDA-MDB-231 cells.

Notes: The MDT-NPs in water were incubated with MDA-MDB-231 breast epithelial cancer cells in culture and were demonstrated to be nontoxic by the lactate dehydrogenase membrane permeability assay following 24 hours exposure to NP concentrations up to 1 mg mL⁻¹. These results support the complete removal of the C₁₆TAB surfactant from the mesoporous NP pores.

Abbreviations: C₁₆TAB, cetyltrimethylammonium bromide; MDT-NPs, multi-dye theranostic silica nanoparticles; NP, nanoparticle.

Comparison with ICG

Figure S3. Quantum yield (QY) of IR780 dye increases upon its encapsulation in a mesoporous matrix.

Notes: A maximum enhancement of 283-fold was observed when compared to free IR780 dye in water. The maximum QY of encapsulated IR780 also was compared with the QY of ICG in free state and encapsulated state (QY values for ICG were obtained from the literature).

Abbreviations: ICG, indocyanine green; IR780, IR780 iodide; qy, quantum yield.

Pore size measurement

Figure S4. X-ray diffractometer pattern of near-infrared fluorescence mesoporous silica nanoparticles (x-axis: angle 2θ (degree), y-axis: intensity [CPS]).

Note: The peak represents a pore center-to-center distance of 3.6 nm, calculated using Bragg’s equation.

Abbreviation: CPS, counts per second.

Table S1 Effect of laser irradiation on different metallo-(na)phthalocyanine dyes in chloroform. All dyes were exposed to continuous laser excitation (785 nm, 637 mW cm⁻²) for 2 minutes

Absorbance of Si-naphthalocyanine dye

Figure S5. UV-vis absorbance spectrum of silicon 2,3-naphthalocyanine dihydroxide in dimethylformamide.

In vivo experiments: histological analysis

Figure S6A shows a viable tumor nodule for the saline/+ablation experiment, while Figure S6B demonstrates areas of necrosis, hemorrhage, and edema caused by localized heat generation of the MDT-NPs/+ablation set. In addition to in vivo imaging, MDT-NPs have the ability to be imaged in the visible region using confocal microscopy. With their broad and well- separated absorption and emission spectra, additional in vitro fluorescence assays are possible, such as flow cytometry for particle uptake studies and ex vivo histopathological analyses.

Figure S6. Histological analysis of tumor sections: (A) saline/+ablation and (B) multi-dye theranostic silica nanoparticles (MDT-NPs)/+ablation stained with hematoxylin and eosin demonstrate areas of viability and necrosis, respectively. (C) Histogram shows significantly more necrosis in tumors irradiated with laser in presence of MDT-NPs, as determined by a board-certified pathologist (JAK). (D) Confocal microscope image (10× magnification) of a representative tissue section (stained using CellMaskTM Orange) containing MDT-NPs (shown by arrow).