Chris Jun Hui Ho

In vivo covalent cross-linking of photon-converted rare-earth nanostructures for tumour localization and theranostics

The development of precision nanomedicines to direct nanostructure-based reagents into tumour-targeted areas remains a critical challenge in clinics. Chemical reaction-mediated localization in response to tumour environmental perturbations offers promising opportunities for rational design of effective nano-theranostics. Here, we present a unique microenvironment-sensitive strategy for localization of peptide-premodified upconversion nanocrystals (UCNs) within tumour areas. Upon tumour-specific cathepsin protease reactions, the cleavage of peptides induces covalent cross-linking between the exposed cysteine and 2-cyanobenzothiazole on neighbouring particles, thus triggering the accumulation of UCNs into tumour site. Such enzyme-triggered cross-linking of UCNs leads to enhanced upconversion emission upon 808 nm laser irradiation, and in turn amplifies the singlet oxygen generation from the photosensitizers attached on UCNs. Importantly, this design enables remarkable tumour inhibition through either intratumoral UCNs injection or intravenous injection of nanoparticles modified with the targeting ligand. Our strategy may provide a multimodality solution for effective molecular sensing and site-specific tumour treatment.

Multifunctional photosensitizer-based contrast agents for photoacoustic imaging.

Photoacoustic imaging is a novel hybrid imaging modality combining the high spatial resolution of optical imaging with the high penetration depth of ultrasound imaging. Here, for the first time, we evaluate the efficacy of various photosensitizers that are widely used as photodynamic therapeutic (PDT) agents as photoacoustic contrast agents. Photoacoustic imaging of photosensitizers exhibits advantages over fluorescence imaging, which is prone to photobleaching and autofluorescence interference. In this work, we examined the photoacoustic activity of 5 photosensitizers: zinc phthalocyanine, protoporphyrin IX, 2,4-bis [4-(N,N-dibenzylamino)-2,6-dihydroxyphenyl] squaraine, chlorin e6 and methylene blue in phantoms, among which zinc phthalocyanine showed the highest photoacoustic activity. Subsequently, we evaluated its tumor localization efficiency and biodistribution at multiple time points in a murine model using photoacoustic imaging. We observed that the probe localized at the tumor within 10 minutes post injection, reaching peak accumulation around 1 hour and was cleared within 24 hours, thus, demonstrating the potential of photosensitizers as photoacoustic imaging contrast agents in vivo. This means that the known advantages of photosensitizers such as preferential tumor uptake and PDT efficacy can be combined with photoacoustic imaging capabilities to achieve longitudinal monitoring of cancer progression and therapy in vivo.

Sensitive SERS glucose sensing in biological media using alkyne functionalized boronic acid on planar substrates

In this work, we propose a novel glucose binding mechanism on a highly sensitive SERS substrate, in order to overcome challenges in specific glucose detection in bio-fluids. We make use of phenylboronic acid as a receptor for saccharide capture onto the substrate and the ability of the captured glucose molecule to undergo secondary binding with an alkyne-functionalized boronic acid to form a glucose-alkyne-boronic acid complex. The formation of this complex shows high selectivity for glucose, over other saccharides. In addition, the alkyne group of the alkyne-functionalized boronic acid exhibits a distinct Raman peak at 1996 cm(-1) in a biological silent region (1800-2800 cm(-1)) where most endogenous molecules, including glucose, show no Raman scattering, thus offering a high sensitivity over other SERS glucose sensing. The substrate offers long-term stability, as well as high SERS enhancement to the glucose-alkyne boronic acid complex on substrate. In addition, the reversibility of SERS signals at various incubation stages also shows reusability capabilities, whereas positive results in clinical urine samples demonstrate clinical feasibility. All these strongly suggest that this newly developed SERS-based assay offers great potential in glucose sensing.

Molecular photoacoustic imaging of breast cancer using an actively targeted conjugated polymer

Conjugated polymers (CPs) are upcoming optical contrast agents in view of their unique optical properties and versatile synthetic chemistry. Biofunctionalization of these polymer-based nanoparticles enables molecular imaging of biological processes. In this work, we propose the concept of using a biofunctionalized CP for noninvasive photoacoustic (PA) molecular imaging of breast cancer. In particular, after verifying the PA activity of a CP nanoparticle (CP dots) in phantoms and the targeting efficacy of a folate-functionalized version of the same (folate-CP dots) in vitro, we systemically administered the probe into a folate receptor-positive (FR+ve) MCF-7 breast cancer xenograft model to demonstrate the possible application of folate-CP dots for imaging FR+ve breast cancers in comparison to CP dots with no folate moieties. We observed a strong PA signal at the tumor site of folate-CP dots-administered mice as early as 1 hour after administration as a result of the active targeting of the folate-CP dots to the FR+ve tumor cells but a weak PA signal at the tumor site of CP-dots-administered mice as a result of the passive accumulation of the probe by enhanced permeability and retention effect. We also observed that folate-CP dots produced ~4-fold enhancement in the PA signal in the tumor, when compared to CP dots. These observations demonstrate the great potential of this active-targeting CP to be used as a contrast agent for molecular PA diagnostic imaging in various biomedical applications.

Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma.

Multi-modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non-invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation. In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800-2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.

Noninvasive real-time characterization of non-melanoma skin cancers with handheld optoacoustic probes

Currently, imaging technologies that enable dermsurgeons to visualize non-melanoma skin cancers (NMSC) in vivo preoperatively are lacking, resulting in excessive or incomplete removal. Multispectral optoacoustic tomography (MSOT) is a volumetric imaging tool to differentiate tissue chromophores and exogenous contrast agents, based on differences in their spectral signatures and used for high-resolution imaging of functional and molecular contrast at centimeter scale depth. We performed MSOT imaging with two- and three-dimensional handheld scanners on 21 Asian patients with NMSC. The tumors and their oxygenation parameters could be distinguished from normal skin endogenously. The lesion dimensions and depths were extracted from the spectral melanin component with three-dimensional spatial resolution up to 80 μm. The intraclass correlation coefficient correlating tumor dimension measurements between MSOT and ex vivo histology of excised tumors, showed good correlation. Real-time 3D imaging was found to provide information on lesion morphology and its underlying neovasculature, indicators of the tumor’s aggressiveness.

Structural and functional 3D mapping of skin tumours with non-invasive multispectral optoacoustic tomography

Recent advances in technology have enabled the development of various non‐invasive skin imaging tools to aid real‐time diagnosis of both benign and malignant skin tumours, minimizing the need for invasive skin biopsy. Multispectral optoacoustic tomography (MSOT) is a recently developed non‐invasive imaging tool, which offers the unique capacity for high resolution three dimensional (3D) optical mapping of tissue by further delivering highly specific optical contrast from a depth of several millimetres to centimetres in living tissues. MSOT enables volumetric, spectroscopic differentiation of tissue, both in vivo and in real time, with and without the application of biomarker‐specific probes, and is further able of providing spatial maps of skin chromophores, as well as underlying blood vasculature.

Multispectral photoacoustic imaging artifact removal and denoising using time series model-based spectral noise estimation.

The aim of this study is to solve a problem of denoising and artifact removal from in vivo multispectral photoacoustic imaging when the level of noise is not known a priori. The study analyzes Wiener filtering in Fourier domain when a family of anisotropic shape filters is considered. The unknown noise and signal power spectral densities are estimated using spectral information of images and the autoregressive of the power 1 ( AR(1)) model. Edge preservation is achieved by detecting image edges in the original and the denoised image and superimposing a weighted contribution of the two edge images to the resulting denoised image. The method is tested on multispectral photoacoustic images from simulations, a tissue-mimicking phantom, as well as in vivo imaging of the mouse, with its performance compared against that of the standard Wiener filtering in Fourier domain. The results reveal better denoising and fine details preservation capabilities of the proposed method when compared to that of the standard Wiener filtering in Fourier domain, suggesting that this could be a useful denoising technique for other multispectral photoacoustic studies.

Noninvasive Anatomical and Functional Imaging of Orthotopic Glioblastoma Development and Therapy using Multispectral Optoacoustic Tomography

PURPOSE: Here we demonstrate the potential of multispectral optoacoustic tomography (MSOT), a new non-invasive structural and functional imaging modality, to track the growth and changes in blood oxygen saturation (sO2) in orthotopic glioblastoma (GBMs) and the surrounding brain tissues upon administration of a vascular disruptive agent (VDA). METHODS: Nude mice injected with U87MG tumor cells were longitudinally monitored for the development of orthotopic GBMs up to 15 days and observed for changes in sO2 upon administration of combretastatin A4 phosphate (CA4P, 30 mg/kg), an FDA approved VDA for treating solid tumors. We employed a newly-developed non-negative constrained approach for combined MSOT image reconstruction and unmixing in order to quantitatively map sO2 in whole mouse brains. RESULTS: Upon longitudinal monitoring, tumors could be detected in mouse brains using single-wavelength data as early as 6 days post tumor cell inoculation. Fifteen days post-inoculation, tumors had higher sO2 of 63 ± 11% (n = 5, P < .05) against 48 ± 7% in the corresponding contralateral brain, indicating their hyperoxic status. In a different set of animals, 42 days post-inoculation, tumors had lower sO2 of 42 ± 5% against 49 ± 4% (n = 3, P < .05) in the contralateral side, indicating their hypoxic status. Upon CA4P administration, sO2 in 15 days post-inoculation tumors dropped from 61 ± 9% to 36 ± 1% (n = 4, P < .01) within one hour, then reverted to pre CA4P treatment values (63 ± 6%) and remained constant until the last observation time point of 6 hours. CONCLUSION: With the help of advanced post processing algorithms, MSOT was capable of monitoring the tumor growth and assessing hemodynamic changes upon administration of VDAs in orthotopic GBMs.

Advances in Optoacoustic Imaging: From Benchside to Clinic

Optoacoustic imaging has been widely used for in vivo disease diagnosis and therapy monitoring. Acquisition hardware, analysis, and contrast agents have been subject to much innovation, creating access to an ever-growing range of biomedical applications. In this review, a broad overview of optoacoustic theory, instrumentation and data processing is provided, together with the various categories of contrast agents that have been developed. In addition, the application of these techniques and contrast agents in preclinical and clinical imaging applications will be discussed in detail, ranging from imaging of cancer and various organs like skin, brain and breast to sentinel lymph node mapping. Finally under conclusions, we highlighted future perspectives in this field, in the context of instrumentation and software development, as well as advances in clinical translation.