Photoacoustic lifetime oxygen imaging of radiotherapy-induced tumor reoxygenation In Vivo.

Purpose: Early detection and diagnosis of cancer is critical for achieving positive therapeutic outcomes. Biomarkers that can provide clinicians with clues to the outcome of a given therapeutic course are highly desired. Oxygen is a small molecule that is nearly universally present in biological tissues and plays a critical role in the effectiveness of radiotherapies by reacting with DNA radicals and subsequently impairing cellular repair of double strand breaks.Techniques for measuring oxygen in biological tissues often use blood oxygen saturation to approximate the oxygen partial pressure in surrounding tissues despite the complex, nonlinear, and dynamic relationship between these two separate oxygen populations. Methods and materials: We combined a directly oxygen-sensitive, tumor-targeted, chemical contrast nanoelement with the photoacoustic lifetime-based (PALT) oxygen imaging technique to obtain image maps of oxygen in breast cancer tumors in vivo. The oxygen levels of patient-derived xenografts in a mouse model were characterized before and after a course of radiotherapy. Results: We show that, independent of tumor size, radiotherapy induced an increase in the overall oxygenation levels of the tumor. Further, this increase in the oxygenation of the tumor significantly correlated with a positive response to radiotherapy, as demonstrated by a reduction in tumor volume over the twenty-day monitoring period following therapy and histological staining. Conclusion: Our PALT imaging presented here is simple, fast, and non-invasive. Facilized by the PALT approach, imaging of tumor reoxygenation may be utilized as a simple, early indicator for evaluating cancer response to radiotherapy. Further characterization of the reoxygenation degree, temporal onset, and possible theragnostic implications are warranted.

Photoacoustic Chemical Imaging Sodium Nano-Sensor Utilizing a Solvatochromic Dye Transducer for In Vivo Application.

Sodium has many vital and diverse roles in the human body, including maintaining the cellular pH, generating action potential, and regulating osmotic pressure. In cancer, sodium dysregulation has been correlated with tumor growth, metastasis, and immune cell inhibition. However, most in vivo sodium measurements are performed via Na23 NMR, which is handicapped by slow acquisition times, a low spatial resolution (in mm), and low signal-to-noise ratios. We present here a plasticizer-free, ionophore-based sodium-sensing nanoparticle that utilizes a solvatochromic dye transducer to circumvent the pH cross-sensitivity of most previously reported sodium nano-sensors. We demonstrate that this nano-sensor is non-toxic, boasts a 200 µM detection limit, and is over 1000 times more selective for sodium than potassium. Further, the in vitro photoacoustic calibration curve presented demonstrates the potential of this nano-sensor for performing the in vivo chemical imaging of sodium over the entire physiologically relevant concentration range.

Photoacoustic Spectral Analysis for Evaluating the Aggressiveness of Prostate Cancer Labeled by Methylene Blue Polyacrylamide Nanoparticles.

Evaluating the aggressiveness of prostate cancer (PCa) is crucial for PCa diagnosis and prognosis. Previously, studies have shown that photoacoustic spectral analysis (PASA) can assess prostate tissue microarchitecture for evaluating the aggressiveness of PCa. In this study, in a transgenic mouse (TRAMP) model of PCa, we utilized methylene blue polyacrylamide nanoparticles (MB PAA NPs) to label the cancer cells in prostate in vivo. MB PAA NPs can specifically target proliferating cancer cells as a contrast agent, allowing photoacoustic (PA) imaging to better detect PCa tumors, and also assessing prostate glandular architecture. With the PA signals from the prostates measured simultaneously by a needle hydrophone and a PA and ultrasound (US) dual-imaging system, we conducted PASA and correlated the quantified spectral parameter slopes with the cancer grading from histopathology. The PASA results from 18 mice showed significant differences between normal and cancer, and also between low-score cancer and high-score cancer. This study in the clinically relevant TRAMP model of PCa demonstrated that PA imaging and PASA, powered by MB PAA NPs that can label the PCa microarchitectures in vivo after systemic administration, can detect PCa and, more importantly, evaluate cancer aggressiveness.

Personalized Oncology by In Vivo Chemical Imaging: Photoacoustic Mapping of Tumor Oxygen Predicts Radiotherapy Efficacy.

We hereby apply the approach of photoacoustic chemical imaging, performing an in vivo chemical analysis that is spatially resolved (200 µm) and in real time, to predict a given tumor's response to therapy. Using triple negative breast cancer as a model, we took photoacoustic images of tumors' oxygen distributions in patient-derived xenografts (PDXs) in mice using biocompatible, oxygen-sensitive tumor-targeted chemical contrast nanoelements (nanosonophores), which function as contrast agents for photoacoustic imaging. Following radiation therapy, we established a quantitatively significant correlation between the spatial distribution of the initial oxygen levels in the tumor and its spatial distribution of the therapy's efficacy: the lower the local oxygen, the lower the local radiation therapy efficacy. We thus provide a simple, noninvasive, and inexpensive method to both predict the efficacy of radiation therapy for a given tumor and identify treatment-resistant regions within the tumor's microenvironment.

Tumor Biochemical Heterogeneity and Cancer Radiochemotherapy: Network Breakdown Zone-Model.

Breakdowns of two-zone random networks of the Erdos-Rényi type are investigated. They are used as mathematical models for understanding the incompleteness of the tumor network breakdown under radiochemotherapy, an incompleteness that may result from a tumor's physical and/or chemical heterogeneity. Mathematically, having a reduced node removal probability in the network's inner zone hampers the network's breakdown. The latter is described quantitatively as a function of reduction in the inner zone's removal probability, where the network breakdown is described in terms of the largest remaining clusters and their size distributions. The effects on the efficacy of radiochemotherapy due to the tumor micro-environment (TME)'s chemical make-up, and its heterogeneity, are discussed, with the goal of using such TME chemical heterogeneity imaging to inform precision oncology.

Tumor Hypoxia Heterogeneity Affects Radiotherapy: Inverse-Percolation Shell-Model Monte Carlo Simulations.

Tumor hypoxia was discovered a century ago, and the interference of hypoxia with all radiotherapies is well known. Here, we demonstrate the potentially extreme effects of hypoxia heterogeneity on radiotherapy and combination radiochemotherapy. We observe that there is a decrease in hypoxia from tumor periphery to tumor center, due to oxygen diffusion, resulting in a gradient of radiative cell-kill probability, mathematically expressed as a probability gradient of occupied space removal. The radiotherapy-induced break-up of the tumor/TME network is modeled by the physics model of inverse percolation in a shell-like medium, using Monte Carlo simulations. The different shells now have different probabilities of space removal, spanning from higher probability in the periphery to lower probability in the center of the tumor. Mathematical results regarding the variability of the critical percolation concentration show an increase in the critical threshold with the applied increase in the probability of space removal. Such an observation will have an important medical implication: a much larger than expected radiation dose is needed for a tumor breakup enabling successful follow-up chemotherapy. Information on the TME's hypoxia heterogeneity, as shown here with the numerical percolation model, may enable personalized precision radiation oncology therapy.

Cell-morphodynamic phenotype classification with application to cancer metastasis using cell magnetorotation and machine-learning.

We define cell morphodynamics as the cell's time dependent morphology. It could be called the cell's shape shifting ability. To measure it we use a biomarker free, dynamic histology method, which is based on multiplexed Cell Magneto-Rotation and Machine Learning. We note that standard studies looking at cells immobilized on microscope slides cannot reveal their shape shifting, no more than pinned butterfly collections can reveal their flight patterns. Using cell magnetorotation, with the aid of cell embedded magnetic nanoparticles, our method allows each cell to move freely in 3 dimensions, with a rapid following of cell deformations in all 3-dimensions, so as to identify and classify a cell by its dynamic morphology. Using object recognition and machine learning algorithms, we continuously measure the real-time shape dynamics of each cell, where from we successfully resolve the inherent broad heterogeneity of the morphological phenotypes found in a given cancer cell population. In three illustrative experiments we have achieved clustering, differentiation, and identification of cells from (A) two distinct cell lines, (B) cells having gone through the epithelial-to-mesenchymal transition, and (C) cells differing only by their motility. This microfluidic method may enable a fast screening and identification of invasive cells, e.g., metastatic cancer cells, even in the absence of biomarkers, thus providing a rapid diagnostics and assessment protocol for effective personalized cancer therapy.

Combining Active Carbonic Anhydrase with Nanogels: Enzyme Protection and Zinc Sensing.

BACKGROUND: Due to its excellent biocompatibility, the polyacrylamide (PAAm) hydrogel has shown great potential for the immobilization of enzymes used in biomedical applications. The major challenge involved is to preserve, during the immobilization process, both the biological activity and the structural integrity of the enzymes. Here we report, for the first time, a proof-of-concept study for embedding active carbonic anhydrase (CA) into polyacrylamide (PAAm) nanogels. By immobilizing CA in these nanogels, we hope to provide important advantages, such as matrix protection of the CA as well as its targeted delivery, and also for potentially using these nanogels as zinc nano-biosensors, both in-vitro and in-vivo. METHODS AND RESULTS: Two methods are reported here for CA immobilization: encapsulation and surface conjugation. In the encapsulation method, the common process was improved, so as to best preserve the CA, by 1) using a novel biofriendly nonionic surfactant system (Span 80/Tween 80/Brij 30) and 2) using an Al2O3 adsorptive filtration purification procedure. In the surface conjugation method, blank PAAm nanogels were activated by N-hydroxysuccinimide and the CA was cross-linked to the nanogels. The amount of active CA immobilized in the nanoparticles was quantified for both methods. Per 1 g nanogels, the CA encapsulated nanogels contain 11.3 mg active CA, while the CA conjugated nanogels contain 22.5 mg active CA. Also, the CA conjugated nanoparticles successfully measured free Zn2+ levels in solution, with the Zn2+ dissociation constant determined to be 9 pM. CONCLUSION: This work demonstrates universal methods for immobilizing highly fragile bio-macromolecules inside nanoparticle carriers, while preserving their structural integrity and biological activity. The advantages and limitations are discussed, as well as the potential biomedical applications.

Intracellular detection of singlet oxygen using fluorescent nanosensors.

Detection of singlet oxygen is of great importance for a range of therapeutic applications, particularly photodynamic therapy, plasma therapy and also during photo-endosomolytic activity. Here we present a novel method of intracellular detection of singlet oxygen using biocompatible polymeric nanosensors, encapsulating the organic fluorescent dye, Singlet Oxygen Sensor Green (SOSG) within its hydrophobic core. The singlet oxygen detection efficiency of the nanosensors was quantified experimentally by treating them with a plasma source and these results were further validated by using Monte Carlo simulations. The change in fluorescence intensity of the nanosensors serves as a metric to detect singlet oxygen in the local micro-environment inside mammalian cancer cells. We used these nanosensors for monitoring singlet oxygen inside endosomes and lysosomes of cancer cells, during cold plasma therapy, using a room-temperature Helium plasma jet.

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.

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.

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.

Ultracompact Nanotheranostic PEG Platform for Cancer Applications.

We present a new targetable nanoconstruct (NC) capable of simultaneously serving as a therapeutic platform for photodynamic therapy (PDT) as well as a magnetic resonance (MR) molecular imaging agent, free of heavy metal atoms. PDT has seen much interest with the introduction of NC-assisted cell-specific targeting of the photosensitizer (PS). The previously reported ultrasmall 8-arm polyethylene glycol amine (8PEGA) NC, with an attached chlorin e6 (Ce6) PS, yielded promising results for PDT of heart arrhythmia, in vivo and ex vivo, on live rat and sheep hearts, respectively, when using targeting peptides for cell-specific ablation of cardio-myocytes. Here we explore the extension of this NC-based PDT to cancer. For this purpose, we switched the targeting peptide from CTP-cys to F3-cys. Notably, the 8PEGA-Ce6 NCs have a superior reactive oxygen species (ROS) production compared to traditional Ce6 encapsulated polyacrylamide (PAAm) NCs, which should be advantageous for PDT. This NC is also cyto-compatible and offers chemical flexibility for the attachment of a choice of targeting peptides. Finally, this label-free 8PEGA NC can be directly and selectively imaged by MRI, using standard spin-echo imaging sequences with large diffusion magnetic field gradients to suppress the water signal. Notably, due to its ultrasmall size this NC is also expected to have improved in vivo penetration and bioelimination, as was already shown in previous biodistribution studies.

In vivo quantitative imaging of tumor pH by nanosonophore assisted multispectral photoacoustic imaging.

Changes of physiological pH are correlated with several pathologies, therefore the development of more effective medical pH imaging methods is of paramount importance. Here, we report on an in vivo pH mapping nanotechnology. This subsurface chemical imaging is based on tumor-targeted, pH sensing nanoprobes and multi-wavelength photoacoustic imaging (PAI). The nanotechnology consists of an optical pH indicator, SNARF-5F, 5-(and-6)-Carboxylic Acid, encapsulated into polyacrylamide nanoparticles with surface modification for tumor targeting. Facilitated by multi-wavelength PAI plus a spectral unmixing technique, the accuracy of pH measurement inside the biological environment is not susceptible to the background optical absorption of biomolecules, i.e., hemoglobins. As a result, both the pH levels and the hemodynamic properties across the entire tumor can be quantitatively evaluated with high sensitivity and high spatial resolution in in vivo cancer models. The imaging technology reported here holds the potential for both research on and clinical management of a variety of cancers.Background optical absorption of several biomolecules impedes an effective in vivo pH imaging in tumors. Here, the authors developed a visible light-based in vivo pH mapping method by coupling photoacoustic imaging and pH-responsive modified nanoparticles that selectively target tumor cells.

Matrix Density Engineering of Hydrogel Nanoparticles with Simulation-Guided Synthesis for Tuning Drug Release and Cellular Uptake.

The use of a nanoparticle (NP)-based antitumor drug carrier has been an emerging strategy for selectively delivering the drugs to the tumor area and, thus, reducing the side effects that are associated with a high systemic dose of antitumor drugs. Precise control of drug loading and release is critical so as to maximize the therapeutic index of the NPs. Here, we propose a simple method of synthesizing NPs with tunable drug release while maintaining their loading ability, by varying the polymer matrix density of amine- or carboxyl-functionalized hydrogel NPs. We find that the NPs with a loose matrix released more cisplatin, with up to a 33 times faster rate. Also, carboxyl-functionalized NPs loaded more cisplatin and released it at a faster rate than amine-functionalized NPs. We performed detailed Monte Carlo computer simulations that elucidate the relation between the matrix density and drug release kinetics. We found good agreement between the simulation model and the experimental results for drug release as a function of time. Also, we compared the cellular uptake between amine-functionalized NPs and carboxyl-functionalized NPs, as a higher cellular uptake of NPs leads to improved cisplatin delivery. The amine-functionalized NPs can deliver 3.5 times more cisplatin into cells than the carboxyl-functionalized NPs. The cytotoxic efficacy of both the amine-functionalized NPs and the carboxyl-functionalized NPs showed a strong correlation with the cisplatin release profile, and the latter showed a strong correlation with the NP matrix density.

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).

Scattering based hyperspectral imaging of plasmonic nanoplate clusters towards biomedical applications.

A new optical scattering contrast-agent based on polymer-nanoparticle encapsulated silver nanoplates (PESNs) is presented. Silver nanoplates were chosen due to the flexibility of tuning their plasmon frequencies. The polymer coating preserves their physical and optical properties and confers other advantages such as controlled contrast agent delivery. Finite difference time domain (FDTD) simulations model the interaction of light with the nanoplates in different orientations in the cluster. Hyperspectral dark field microscopy (HYDFM) observes the scattering spectra of the PESNs. An unsupervised sequential maximum angle convex cone (SMACC) image analysis resolves spectral endmembers corresponding to different stacking orientations of the nanoplates. The orientation-dependent endmembers qualitatively agree with the FDTD results. For contrast enhancement, the uptake and spatial distribution of PESNs are demonstrated by an HYDFM study of single melanoma cells to result in an enhanced contrast of up to 400%. A supervised spatial mapping of the endmembers obtained by the unsupervised SMACC algorithm reveals spatial distributions of PESNs with various clustering orientations of encapsulated nanoplates. Our study demonstrates tunability in plasmonics properties in clustered metal nanoparticles and its utility for the development of scatter-based imaging contrast agents for a broad range of applications, including studies of single cells and other biomedical systems.

Intracellular Photodynamic Activity of Chlorin e6 Containing Nanoparticles.

Nanoparticles (NPs) containing the photo-therapeutic dye Chlorin e6 (Ce6) have been explored in multiple studies for photo-dynamic therapy (PDT). However, little work has been carried out regarding their PDT efficacy, relative to other dye containing NPs. Here polyacrylamide nanoparticles (PAAm NPs) containing Ce6 were prepared and their PDT efficacy compared to previously reported methylene blue (MB) containing PAAmNPs. It was found that, for identical NP dosages and photon doses, the Ce6 NPs are an order of magnitude more potent in killing cancer cells.