Methods, compositions and kits for imaging cells and tissues using nanoparticles and spatial frequency heterodyne imaging

#Systems, compositions, methods and kits are provided using nanoparticle compositions and spatial frequency heterodyne imaging for visualizing and detecting cells or a tissue such as a tumor, for identifying a potential therapeutic agent for treating a disease condition, and for preparing the nanoparticle compositions.

#Hepatocellular carcinoma (HCC) is the most common form of liver cancer in adults, accounting for approximately three of every four cancers in the liver (El-Sarag et al. 1999, New England Journal of Medicine 340: 745-750). The American Cancer Society estimates that more than 24,000 new cases of primary liver cancer develop each year in the United States, of which approximately 19,000 result in death. HCC is common in developing countries, particularly in sub-Saharan Africa and Southeast Asia (Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and O'Brien 2004 Cancer Journal 10: 67-73).

#More than 500,000 people are diagnosed with HCC each year worldwide (Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and Bruix et al. 2006 Oncogene 25: 3848-3856). HCC is difficult to diagnose in its earliest stages because there are currently no screening tests available, and HCC generally becomes symptomatic when the tumor is approximately 4.5 centimeters to eight centimeters in diameter (Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and Colombo 1992 Hepatol 15: 225-236).

#FIG. 1 is a drawing of methods for layer-by-layer polyelectrolyte coating of gold nanoparticles using an anionic poly(acrylic acid) (PAA), and a cationic poly(allylamine hydrochloride) (PAH). FIG. 1 Gold (Au) nanoparticles were contacted with PAA to produce PAA-encapsulated nanoparticles (AU-PAA) having carboxylic acid functional groups (—COOH) and de-protonated carboxylic acid functional groups (—COO−) extending from the nanoparticles. The PAA-encapsulated nanoparticles were contacted with PAH to form PAA-PAH encapsulated nanoparticles (Au-PAA-PAH). The PAH layer lies outside the PAA layer of PAA-PAH encapsulated nanoparticle resulting in amine functional groups (—NH2) and protonated amino functional groups (—NH3+) extending from the nanoparticle.

#FIG. 2 is a drawing of the X-ray imaging system used in examples herein. An X-ray source directs electromagnetic radiation to an absorption grid that is a periodic structure positioned in the radiation propagation direction and positioned proximal to a sample and a charged-coupled device (CCD). The grid scatters the directed radiation to the sample positioned in a container or vial. The X-ray radiation passes through the grid and the sample respectively, and is then detected using the CCD.

#FIG. 3 is a set of visible light images and X-ray scatter images after Fourier transformation of scattered X-radiation of a sample obtained by the imaging system shown in FIG. 2. A beam of X-radiation was directed through an absorption grid and sample in a vial, and was detected using an CCD. The original image (F) is shown in the lower left of FIG. 3. A Fourier transformation was performed resulting in an image in the spatial frequency domain. Different peaks in the spatial frequency image contained different information regarding scattering of incident x-radiation by the sample. Selecting an area around a specific peak in the convolution and Fourier back-transforming this area returns the logarithm of the scattered intensities to real space and gives a processed image that contains anisotropic information regarding scattering of the incident X-rays by the sample. The area surrounding the central 0th-order peak (F−1; S0: 0th order; FIG. 3 bottom right) corresponds to the original X-ray absorption image without scatter and is used for normalization. The image from the central 0th-order peak is subtracted from the higher order images to remove all absorption features. The area around the 1st-order peak (F−1; S1: 1st order left; FIG. 3 center bottom) corresponds to scattering in the x-direction, and therefore gives a processed left 1st-order scatter image upon Fourier back-transformation and normalization. Similarly, the area around the 1st-order peak immediately above the 0th-order peak (FIG. 3 top right box) corresponds to scattering in the y-direction, and therefore gives a processed upper 1st-order scatter image upon Fourier back-transformation and normalization. Each X-ray image yields at least two processed images; one image results from X-radiation scattered horizontally (F−1; S1: 1st order left; FIG. 3 center bottom), and the other image results from X-radiation scattered vertically (FIG. 3 top right box). Both 1st order images measure identical scatter signals because of the isotropic scattering of the spherical nanoparticles.

#Nanoparticles are structures/particles that have an approximate size of one nm to 100 nm and are used in applications, including addition to surfaces or fluids for catalytic reactions, self-cleaning and antibacterial products, glass dyeing, sunscreen lotions and manufacturing of optical components (e.g., optical fibers). See Rajala et al. U.S. Pat. No. 8,231,369 issued Jul. 31, 2012. Nanoparticles are composed of a variety of materials including metals, metal oxides, MRI agents, semiconductors, and polymers, and possess unique characteristics because of their small size.

#Laboratory-scale and industrial-scale techniques are used to manufacture nanoparticles having for example a specific size distribution (mono-dispersivity), anti-agglomeration, and homogeneity (see Davis et al. U.S. Pat. No. 8,263,035 issued Sep. 11, 2012; Magdassi et al. U.S. Pat. No. 8,227,022 issued Jul. 24, 2012; and Fiannaca et al. U.S. patent application number US 2008/0050592 A1 published Feb. 28, 2008). Nanoparticles are produced for example by wet chemical processes and by vapor phase processes (see Murphy et al. U.S. Pat. No. 8,241,922 issued Aug. 14, 2012; and Brooks et al. U.S. Pat. No. 7,985,398 issued Jul. 26, 2011). Vapor phase processes (also known as aerosol reactor processes) use a number of different devices and techniques including: flame reactors, hot-wall reactors, plasma reactors, gas condensation methods, laser ablation and spray pyrolysis (see Jun et al. U.S. Pat. No. 7,988,761 issued Aug. 2, 2011; and Yang, Y. et al. 2006 Catalysis Communications 7:281-284) to synthesize nanoparticles of controlled size and composition.

#Nanoparticles composed of metals are used as imaging agents that are imaged using a number of different techniques including MRI, ultrasonography, and X-ray computer tomography (see Grinstaff et al. U.S. Pat. No. 5,505,932 issued Apr. 9, 1996). Imaging of nanoparticles containing for example gadolinium, dysoprium, manganese, iron, and platinum involves specialized excitation and data manipulation of detected radiofrequency signals from non-zero spin nuclei which have a non-equilibrium nuclear spin state distribution. (Weiler et al. U.S. Pat. No. 6,370,415 issued Apr. 9, 2002; and Weiler et al. U.S. Pat. No. 6,595,211 issued Jul. 22, 2003). In conventional MRI the nuclei responsible for the detected signals are protons (e.g., protons of water), and the non-equilibrium spin state distribution is achieved by placing the subject in a strong magnetic field (to enhance the population difference between the proton spin states at equilibrium), and then exposing the subject to pulses of radiofrequency radiation at the proton Larmor frequency, which excites spin state transitions and creates a non-equilibrium spin state distribution (see Seri et al. U.S. Pat. No. 5,811,077 issued Sep. 22, 1998).