In vivo photoacoustic potassium imaging of the tumor microenvironment.

The accumulation of potassium (K+) in the tumor microenvironment (TME) has been recently shown to inhibit immune cell efficacy, and thus immunotherapy. Despite the abundance of K+ in the body, few ways exist to measure it in vivo. To address this technology gap, we combine an optical K+ nanosensor with photoacoustic (PA) imaging. Using multi-wavelength deconvolution, we are able to quantitatively evaluate the TME K+ concentration in vivo, and its distribution. Significantly elevated K+ levels were found in the TME, with an average concentration of approximately 29 mM, compared to 19 mM found in muscle. These PA measurements were confirmed by extraction of the tumor interstitial fluid and subsequent measurement via inductively coupled plasma mass spectrometry.

In Vivo Photoacoustic Lifetime Based Oxygen Imaging with Tumor Targeted G2 Polyacrylamide Nanosonophores.

Lifetime imaging methods using phosphorescence quenching by oxygen for molecular oxygen concentration measurement have been developed and used for noninvasive oxygen monitoring. This study reports photoacoustic (PA) oxygen imaging powered by polyacrylamide (PAAm) hydrogel nanoparticles (NP) which offer advantages including improved biocompatibility, reduced toxicity, and active tumor targeting. A known oxygen indicator, oxyphor G2, was conjugated with the matrix of the NPs, giving G2-PAA NPs, followed by PEGylation for biocompatibility and F3 surface modification for tumor targeting. Using two lasers providing pump and probe pulses, respectively, PA imaging was performed so as to quantitatively map the oxygen concentration in biological tissues in vivo, including cancer tumors and normal thigh muscles. Furthermore, via the imaging at the pump wavelength and two additional wavelengths, the accumulation of the G2-PAA NPs in the tumors were also determined. The successful imaging experiment accomplished on animal models renders a method for in vivo noninvasive imaging and assessment of hypoxic tumor microenvironments, which is critical for assessing cancer progression, metastasis, and treatment.

Photoacoustic imaging of clofazimine hydrochloride nanoparticle accumulation in cancerous vs normal prostates.

Prostate cancer was the most common form and had the second highest death rate of male cancer in the United States in 2015. Current diagnosis techniques, such as prostate-specific antigen tests, transrectal ultrasound scans, and biopsies, are often inconclusive, and in the latter case, invasive. Here, we explore the use of clofazimine hydrochloride nanoparticles (CFZ-HCl NPs), a repurposed formulation from an FDA-approved antimycobacterial agent, as a photoacoustic contrast agent for the evaluation of prostate cancer due to its macrophage-targeting capabilities and high optical absorbance at 495 nm. Using a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model, our results indicate a preferential accumulation of intravenously injected CFZ-HCl NPs in cancerous prostates over normal prostates. Differences in accumulation of CFZ-HCl NPs between cancerous and normal prostates were determined using a two-wavelength unmixing technique via ex vivo photoacoustic imaging. Thus, intravenous injection of CFZ-HCl NPs leads to differences in the interactions of the particles with cancerous vs normal prostates, while allowing for photoacoustic detection and analysis of prostate cancer. These findings could lead to the development of a new noninvasive technique for the detection and monitoring of prostate cancer progression in an animal model that can potentially be translated to human patients.

Chemical Imaging in Vivo: Photoacoustic-Based 4-Dimensional Chemical Analysis.

We describe how 4-dimensional in vivo biochemical analysis can be performed using photoacoustic contrast nanoagents that have been designed to probe both structural and chemical information in vivo, enabling noninvasive, real time, spatially resolved chemical imaging. Early chemical imaging of a patient's tumor can inform the decision of effective treatment, regarding choices of chemotherapy, radiation, or immunotherapy.

Synthesis and Characterization of a Biomimetic Formulation of Clofazimine Hydrochloride Microcrystals for Parenteral Administration.

Clofazimine (CFZ) is a broad spectrum antimycobacterial agent recommended by the World Health Organization as a first line treatment for leprosy and second line treatment for multidrug resistant tuberculosis. Oral administration of CFZ leads to a red skin pigmentation side effect. Since CFZ is a weakly basic, red phenazine dye, the skin pigmentation side effect results from lipophilic partitioning of the circulating, free base (neutral) form of CFZ into the skin. Here, we developed a stable and biocompatible formulation of CFZ-HCl microcrystals that mimics the predominant form of the drug that bioaccumulates in macrophages, following long term oral CFZ administration. In mice, intravenous injection of these biomimetic CFZ-HCl microcrystals led to visible drug accumulation in macrophages of the reticuloendothelial system with minimal skin accumulation or pigmentation. In fact, no skin pigmentation was observed when the total amount of CFZ-HCl administered was equivalent to the total oral dose leading to maximal skin pigmentation. Thus, parenteral (injected or inhaled) biomimetic formulations of CFZ-HCl could be instrumental to avoid the pigmentation side effect of oral CFZ therapy.

Transient Triplet Differential (TTD) Method for Background Free Photoacoustic Imaging.

With the capability of presenting endogenous tissue contrast or exogenous contrast agents in deep biological samples at high spatial resolution, photoacoustic (PA) imaging has shown significant potential for many preclinical and clinical applications. However, due to strong background signals from various intrinsic chromophores in biological tissue, such as hemoglobin, achieving highly sensitive PA imaging of targeting probes labeled by contrast agents has remained a challenge. In this study, we introduce a novel technique called transient triplet differential (TTD) imaging which allows for substantial reduction of tissue background signals. TTD imaging detects directly the triplet state absorption, which is a special characteristic of phosphorescence capable dyes not normally present among intrinsic chromophores of biological tissue. Thus, these triplet state absorption PA images can facilitate "true" background free molecular imaging. We prepared a known phosphorescent dye probe, methylene blue conjugated polyacrylamide nanoparticles, with peak absorption at 660 nm and peak lowest triplet state absorption at 840 nm. We find, through studies on phantoms and on an in vivo tumor model, that TTD imaging can generate a superior contrast-to-noise ratio, compared to other image enhancement techniques, through the removal of noise generated by strongly absorbing intrinsic chromophores, regardless of their identity.

Ion-Selective Nanosensor for Photoacoustic and Fluorescence Imaging of Potassium.

Ion-selective optodes (ISOs), the optical analog of ion-selective electrodes, have played an increasingly important role in chemical and biochemical analysis. Here we extend this technique to ion-selective photoacoustic optodes (ISPAOs) that serve at the same time as fluorescence-based ISOs, and apply it specifically to potassium (K+). Notably, the potassium ion is one of the most abundant cations in biological systems, involved in numerous physiological and pathological processes. Furthermore, it has been recently reported that the presence of abnormal extracellular potassium concentrations in tumors suppresses the immune responses and thus suppresses immunotherapy. However, unfortunately, sensors capable of providing potassium images in vivo are still a future proposition. Here, we prepared an ion-selective potassium nanosensor (NS) aimed at in vivo photoacoustic (PA) chemical imaging of the extracellular environment, while being also capable of fluorescence based intracellular ion-selective imaging. This potassium nanosensor (K+ NS) modulates its optical properties (absorbance and fluorescence) according to the potassium concentration. The K+ NS is capable of measuring potassium, in the range of 1 mM to 100 mM, with high sensitivity and selectivity, by ISPAO based measurements. Also, a near infrared dye surface modified K+ NS allows fluorescence-based potassium sensing in the range of 20 mM to 1 M. The K+ NS serves thus as both PA and fluorescence based nanosensor, with response across the biologically relevant K+ concentrations, from the extracellular 5 mM typical values (through PA imaging) to the intracellular 150 mM typical values (through fluorescence imaging).

Photoacoustic and ultrasound imaging of cancellous bone tissue.

We used ultrasound (US) and photoacoustic (PA) imaging modalities to characterize cattle trabecular bones. The PA signals were generated with an 805-nm continuous wave laser used for optimally deep optical penetration depth. The detector for both modalities was a 2.25-MHz US transducer with a lateral resolution of ~1 mm at its focal point. Using a lateral pixel size much larger than the size of the trabeculae, raster scanning generated PA images related to the averaged values of the optical and thermoelastic properties, as well as density measurements in the focal volume. US backscatter yielded images related to mechanical properties and density in the focal volume. The depth of interest was selected by time-gating the signals for both modalities. The raster scanned PA and US images were compared with microcomputed tomography (µCT) images averaged over the same volume to generate similar spatial resolution as US and PA. The comparison revealed correlations between PA and US modalities with the mineral volume fraction of the bone tissue. Various features and properties of these modalities such as detectable depth, resolution, and sensitivity are discussed.