Indocyanine Green-Encapsulating Calcium Phosphosilicate Nanoparticles: Bifunctional Theranostic Vectors for Near Infrared Diagnostic Imaging and Photodynamic Therapy

Open Access
Altinoglu, Erhan I
Graduate Program:
Materials Science and Engineering
Doctor of Philosophy
Document Type:
Date of Defense:
June 14, 2010
Committee Members:
  • James Hansell Adair, Dissertation Advisor
  • James Hansell Adair, Committee Chair
  • Mark Kester, Committee Member
  • Carlo G Pantano, Committee Member
  • Gary Lynn Messing, Committee Member
  • photodynamic therapy
  • diagnostic imaging
  • near infrared
  • encapsulation
  • nanoparticles
  • indocyanine green
  • calcium phosphate
The American Cancer Society projected 1.5 million new cancer cases to be diagnosed in 2009. Of these diagnoses, a mere 20% were expected to be detected in their early, localized stage. With such advantageous early detection comes various low risk and highly effective treatment options, resulting in 5-year relative survival rates of over 90%. Unfortunately, the remaining diagnoses were expected in a later stage of the progressive disease, where treatment requires infusions of toxic pharmaceuticals and blasts of brutal radiation, and the prognosis is as miserable as the experience itself. Thus, the advantages associated with timely detection and treatment has generated interest in a simultaneous approach to early diagnosis and therapy, a combined modality termed theranostics. Recently, the concept of multifunctional nanocarriers that can concurrently perform these distinct tasks has emerged and is attracting increased attention. The full potential of a theranostic strategy lies in the ability to engineer such a bifunctional vector with both of the optical and therapeutic properties necessary to complete the separate functions. Specifically, much interest has been initiated on fluorescent photosensitive agents that respond in the near infrared (NIR) by concurrently emitting intense and sustained fluorescence signals for deep tissue imaging, as well as generating a photodynamic response to trigger localized cell death for minimally invasive, molecular-scale 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. Likewise, the therapeutic capacity of the ICG-CPSNP nanocomposites is greater when comparing the ex situ generation of singlet oxygen (1O2) to that from the unencapsulated sensitizer. The photoactivation of singlet oxygen in a hypoxic aqueous environment was found to be 60% higher from the ICG molecules upon encapsulation within the CPSNP matrix. 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 (LD50 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/cm2), 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. Coupled with the demonstrated improvements in bioimaging performance, the efficient in vivo therapeutic execution of ICG-CPSNPs serves as a convincing validation of the apposite bifunctionality of these vectors. Together with the established biocompatibility and favorable pharmacokinetics of calcium phosphates, these composite nanoparticles possess the potential to serve as a model theranostic agent in the early stage diagnosis and treatment of disease.