Novel approach to safe and highly efficient cancer imaging

Improved early disease detection is one of the major promises of nanotechnology. For many diseases, including cancer, early detection is the single most important determinant of successful treatment. Near-IR imaging has been used as a safe and noninvasive method of bioimaging for at least the past 25 years, most notably for monitoring cerebral functions. Combining near-IR imaging with enhanced fluorescence from nanoencapsulated contrast agents could greatly increase our ability to detect breast and other cancers earlier and possibly with greater safety and less expense than using x-ray or magnetic-resonance imaging.¹ From a broader perspective, encapsulating near-IR fluorophores provides the enhanced properties of brighter fluorescence intensity and much improved photostability for a wide range of near-IR applications.

Figure 1. (Top) Schematic of calcium-phosphate nanoparticles (CPNPs), (bottom) transmission-electron micrograph of ~16nm (mean diameter) indocyanine-green (ICG) CPNPs.

Indocyanine green (ICG) is the only near-IR organic dye approved by the Food and Drug Administration for use in the human body. Due to its low toxicity, ICG is used clinically as a contrast agent for optical imaging in angiography and guiding biopsies as well as for evaluating blood flow and hepatic function. Like many organic dyes, ICG is prone to various instabilities in environments such as those found in the human body. Free-ICG molecules in the blood often bind with proteins, causing agglomeration leading to elimination from the body within minutes, while those retained in circulation undergo rapid photobleaching, thereby limiting long-term imaging. However, for a fluorescent-imaging probe to be effective, it should retain strong and prolonged image intensity, avoid binding with proteins, and have the ability to accumulate and be retained in a targeted area over an extended time period.

We have developed a nanoparticulate-encapsulation system that meets these needs using a biocompatible material—calcium phosphate—that is already present in the blood system in significant amounts.^1–3 Here, we report the results of in vivo testing of calcium-phosphate nanoparticles (CPNPs) containing ICG using human breast tumors injected into a live nude mouse model.

Spherical CPNPs of the critical size for efficient cellular uptake (~16nm mean diameter) doped with ICG were synthesized using aqueous coprecipitation of calcium chloride and disodium hydrogen phosphate in the presence of disodium silicate within water-in-oil microemulsions. ICG doping was accomplished through the addition of the fluorphore into the microemulsion during precipitation. Particle stability was fostered through electrosteric repulsion by means of citrate-surface functionalization. Particle suspensions were then laundered via a van der Waals high-performance liquid-chromatography protocol to remove residual synthetic components and concentrate the particles in an ethanol-water mixture. Figure 1 shows a transmission-electron-microscope image of the ICG-doped CPNPs.

One of the most common methods for in vivo particle encapsulation uses polymer-based carriers because of their high biocompatibility. However, these polymer networks give the encapsulated molecules little protection from dimerization or photoisomerization. Unlike polymeric encapsulation, the rigid calcium-phosphate matrix protects the encapsulated fluorophore from deleterious structural or chemical alterations that adversely affect photon absorption and fluorescence-photon emission under near-IR illumination. Figure 2 shows the similar shapes of the absorption and emission curves of the dye-doped CPNPs and the free-ICG dye of matching concentration, which indicates the absence of chemical or structural alterations due to encapsulation. In addition, the fluorescence-emission intensity of one dye-doped CPNP is approximately 10³ times that of one ICG dye molecule (see Figure 2). This enhanced brightness is at least partially due to the nanoparticle architecture, with multiple dye molecules being encapsulated in a single particle.

Figure 2. Absorption (a) and fluorescence spectra (b) of free ICG (red) and ICG-CPNPs (black) in aqueous solution. The CPNP suspension (10¹³ particles/ml) had an apparent fluorophore content of roughly 10^-5M based on absorption standards. A matching concentration of free dye was used for the comparison. The shape and position of both the absorption and emission spectra are similar, indicating the absence of either chemical modification or dimerization upon encapsulating the dye in the calcium-phosphate matrix. Emission from the ICG-CPNPs (b) is significantly brighter than the matching free-dye solution, which suffers from quenching interactions due, in part, to free-dye self-aggregation.

For sensitive, deep-tissue, real-time imaging of human breast cancers, both high quantum efficiency and photostability are imperative. The fluorescence effect must be both bright and long lasting. Brightness is enhanced of order 1000 times in CPNPs. Photostability is also greatly improved by the calcium-phosphate matrix (see Figure 3). Photobleaching is typically credited to a reaction between the fluorophore molecule and dissolved oxygen. The CPNP has a matrix-shielding effect that protects it from environmental oxygen and permits prolonged periods of excitation without significant degradation in emission intensity. The data in Figure 3 shows that CPNP encapsulation provides significant improvements in emission half-life across a range of laser powers. In particular, an approximate 400% increase in emission half-life is granted by encapsulation at laser powers typically used in clinical near-IR imaging applications (indicated by the yellow band).

Figure 3. Half-life of ICG emission intensity at 850nm as a function of power density for the excitation wavelength (780nm). Two environments are reported, a 70:30 volume percent ethanol-water mixture and PBS (10mM phosphate buffer, 0.14M NaCl, 0.01M KCl solution). For both environments the encapsulated fluorophore, ICG-CPNPs, has a significantly longer half-life than the free dye. In particular, there is an approximate 400% increase in emission half-life for ICG-CPNPs in physiological environments (PBS) under laser powers typical for clinical-imaging applications (yellow band).

Five sets of live nude mice implanted with subcutaneous human breast tumors were injected via the tail with either a free-ICG dye, a CPNP without dye, or a surface-functionalized CPNP with polyethylene glycol (PEG). The latter has been shown to provide physiological dispersion and inhibit protein absorption. Our preliminary in vivo imaging study (see Figure 4) shows the benefits of long retention and an extended luminescence half-life as nanoparticles accumulate in the breast tumors over time through a passive mechanism referred to as the enhanced permeability and retention effect. This initial data provides strong evidence that CPNP encapsulation of ICG is sufficient for in vivo shielding to provide prolonged fluorescence emission over four days following systemic injection.

Figure 4. Near-IR transillumination images (excitation: 755nm, emission: 830nm) taken at various times track the fluorescence signals and pharmacokinetic distributions for the ICG-CPNPs and controls delivered systemically via tail-vein injections in nude mice implanted with subcutaneous human-breast adenocarcinoma tumors. Hash marks next to each mouse indicate the position of the 5mm tumors. Two control samples, (i) carboxylate-terminated CPNPs without ICG encapsulant and (ii) free ICG, match the particle concentration and fluorophore content (10¹³particles/ml and 10⁵M, respectively) of a (iii) PEGylated ICG-CPNP sample. (b ii) No fluorescence signal is detected from the free ICG at 24 hours post injection while the PEG-ICG-CPNP sample (c iii) retains significant signal even after 96 hours. (b iii) The fluorescence signal is unmistakably localized to the tumors 24 hours after administration with PEGylated ICG-CPNPs.

The encapsulation of a near-IR emitting fluorophore into a bioabsorbable PEGylated CPNP provides enhanced emission and photostability as well as long term in vivo retention times for imaging applications. With a critical size for cellular uptake (<50nm) and the colloidal properties of robust dispersion and prolonged circulation lifetimes under physiological conditions, ICG-CPNPs fulfill the requirements needed for effective long-term bioimaging applications. Current and future work aims toward the encapsulation of tumor-inhibiting drugs with ICG for simultaneous treatment and tracking of cancer tumors in the breast and elsewhere.