Indocyanine green-encapsulating calcium phosphosilicate nanoparticles: Bifunctional theranostic vectors for near infrared diagnostic imaging and photodynamic therapy

The synthesis, laundering, and properties of calcium phosphosilicate nanoparticles (CPSNPs) that encapsulate the NIR fluorophore indocyanine green (ICG) related to multifunctional fluorescent photosensitization is presented. Imaging with transmission electron microscopy (TEM) revealed the well dispersed state of the nanoparticles, the spherical morphology, and the log normal mean particle diameter of 16 nm. Electron energy loss spectroscopy (EELS) mapping identified a Ca:P:Si ratio of 1:1.72:0.41 and a homogeneous composition without evidence of an element rich or deficient architecture. Zeta potential of the as-synthesized, citrate-functionalized CPSNPs was -29 +/-3 mV. A theoretical solids loading of 1.9 x 1013 CPSNP/mL was calculated for a standard suspension. The mean ICG content per suspension is 2 x 10 -6 M, which equates to approximately 63 fluorophore molecules encapsulated per CPSNP.;For imaging and diagnostic considerations, the doped CPSNPs exhibited significantly greater intensity at the maximum emission wavelength relative to the free constituent fluorophore. The quantum efficiency of the fluorescent agent is 200% greater at 0.053+/-0.003 over the free fluorophore in PBS. Also, photostability based on fluorescence half-life of encapsulated ICG in PBS is 500% longer under typical clinical imaging conditions relative to the free dye. These performance enhancements are attributed to the matrix shielding effect of the NP around the internalized fluorophore molecules.;The in vivo emission signal stability from ICG-CPSNPs was compared to the free fluorophore by whole animal NIR imaging. The duration of fluorescent signal from the ICG-CPSPNPs was extended to up to four days post-injection, highlighting the potential for long-term imaging and sensitive tracking applications using ICG when encapsulated within the protective matrix of CPSNPs. The surfaces of the ICG-CPSNPs were covalently bound with polyethylene glycol (PEG). The pharmacokinetic behavior of the PEGylated ICG-CPSNPs revealed that ICG-CPSNP-PEG passively localize within solid tumor xenografts within 24 hours of systemic administration via the enhanced permeation and retention (EPR) effect.;To impart tissue specificity, the ICG-CPSNP-PEGs were bioconjugated with gastrin-10 with the intention of targeting BxPC-3 pancreatic cancer cells by specifically binding the over expressed receptors for this hormone. In vitro assessment acknowledged the faculty of this functionalization to preferentially target the cells of interest; fluorescence microscopy visually revealed this targeting capacity, while flow cell cytometry explicitly characterized the preferential cellular uptake of the ICG-CPSNP-PEG-Gastrin-10 by BxPC-3 cancer cells. An NIR whole animal imaging study further verified that gastrin functionalization provides a direct means for targeting orthotopic pancreatic tumors in vivo, with emission signal intensities from excised tumors measuring higher relative to the controls. This result highlights the ability of targeted ICG-CPSNPs to provide the high in vivo selectivity needed for the most effective diagnostics imaging.;Initial in vitro toxicity trials were conducted in four distinct cell lines to identify an ICG-CPSNP-PEG dosing limit. It was revealed that acute toxicity is subject to the particle number concentration (LD 50 of 2 x108 CPSNP/cell) and not the dose of encapsulated ICG. Next, cell viability was examined as a function of photodynamic therapy (PDT) dose. An unmistakable drop in cell viability in vitro relative to the control was observed for all cell lines. The significance of these results rests in the drastically low applied fluence (1 J/cm 2), which suggests a plausibly greater efficacy in cell lethality at significantly higher, more customary laser powers.;This enhancement in photodynamic response was supplemented by the exceptional in vivo PDT effect on tumor growth. ICG-CPSNP-PEGs arrested human breast adenocarcinoma tumor growth over 36 days after only a single, low dose systemic administration (44 nM) and laser activation (12.5 J/cm2). Such heightened photodynamic cell lethality with ICG-CPSNPs emphasizes the tremendous potential this composite nanovector has for low dose PDT applications, particularly considering the non-optimized nature of the preliminary experimentation. (Abstract shortened by UMI.)...