Ronald X. Xu

Multifunctional microbubbles and nanobubbles for photoacoustic and ultrasound imaging

We develop a novel dual-modal contrast agent-encapsulated-ink poly(lactic-co-glycolic acid) (PLGA) microbubbles and nanobubbles-for photoacoustic and ultrasound imaging. Soft gelatin phantoms with embedded tumor simulators of encapsulated-ink PLGA microbubbles and nanobubbles in various concentrations are clearly shown in both photoacoustic and ultrasound images. In addition, using photoacoustic imaging, we successfully image the samples positioned below 1.8-cm-thick chicken breast tissues. Potentially, simultaneous photoacoustic and ultrasound imaging enhanced by encapsulated-dye PLGA microbubbles or nanobubbles can be a valuable tool for intraoperative assessment of tumor boundaries and therapeutic margins.

Synthesizing and binding dual-mode poly (lactic-co-glycolic acid) (PLGA) nanobubbles for cancer targeting and imaging.

Accurate assessment of tumor boundaries and recognition of occult disease are important oncologic principles in cancer surgeries. However, existing imaging modalities are not optimized for intraoperative cancer imaging applications. We developed a nanobubble (NB) contrast agent for cancer targeting and dual-mode imaging using optical and ultrasound (US) modalities. The contrast agent was fabricated by encapsulating the Texas Red dye in poly (lactic-co-glycolic acid) (PLGA) NBs and conjugating NBs with cancer-targeting ligands. Both one-step and three-step cancer-targeting strategies were tested on the LS174T human colon cancer cell line. For the one-step process, NBs were conjugated with the humanized HuCC49 Delta C(H)2 antibody to target the over-expressed TAG-72 antigen. For the three-step process, cancer cells were targeted by successive application of the biotinylated HuCC49 Delta C(H)2 antibody, streptavidin, and the biotinylated NBs. Both one-step and three-step processes successfully targeted the cancer cells with high binding affinity. NB-assisted dual-mode imaging was demonstrated on a gelatin phantom that embedded multiple tumor simulators at different NB concentrations. Simultaneous fluorescence and US images were acquired for these tumor simulators and linear correlations were observed between the fluorescence/US intensities and the NB concentrations. Our research demonstrated the technical feasibility of using the dual-mode NB contrast agent for cancer targeting and simultaneous fluorescence/US imaging.

Near-infrared fluorescence labeled anti-TAG-72 monoclonal antibodies for tumor imaging in colorectal cancer xenograft mice.

Anti-TAG-72 monoclonal antibodies target the tumor-associated glycoprotein (TAG)-72 in various solid tumors. This study evaluated the use of anti-TAG-72 monoclonal antibodies, both murine CC49 and humanized CC49 (HuCC49deltaCH2), for near-infrared fluorescent (NIR) tumor imaging in colorectal cancer xenograft models. The murine CC49 and HuCC49deltaCH2 were conjugated with Cy7 monofunctional N-hydroxysuccinimide ester (Cy7-NHS). Both in vitro and in vivo anti-TAG-72 antibody binding studies were performed. The in vitro study utilized the human colon adenocarcinoma cell line LS174T that was incubated with Cy7, antibody-Cy7 conjugates, or excessive murine CC49 followed by the antibody-Cy7 conjugates and was imaged by fluorescence microscopy. The in vivo study utilized xenograft mice, bearing LS174T subcutaneous tumor implants, that received tail vein injections of Cy7, murine CC49-Cy7, HuCC49deltaCH2-Cy7, or nonspecific IgG-Cy7 and were imaged by the Xenogen IVIS 100 system from 15 min to 288 h. The biodistribution of the fluorescence labeled antibodies was determined by imaging the dissected tissues. The in vitro study revealed that the antibody-Cy7 conjugates bound to LS174T cells and were blocked by excessive murine CC49. The in vivo study demonstrated that murine CC49 achieved a tumor/blood ratio of 15 at 96 h postinjection. In comparison, HuCC49deltaCH2-Cy7 cleared much faster than murine CC49-Cy7 from the xenograft mice, and HuCC49deltaCH2-Cy7 achieved a tumor/blood ratio of 12 at 18 h postinjection. In contrast, Cy7 and Cy7 labeled nonspecific IgG resulted in no demonstrable tumor accumulation. When mice were injected with excessive unlabeled murine CC49 at 6 h before the injection of murine CC49-Cy7 or HuCC49deltaCH2-Cy7, both the intensity and retention time of the fluorescence from the tumor were reduced. In summary, the Cy7 labeled murine CC49 and HuCC49deltaCH2 demonstrate tumor-targeting capabilities in living colorectal cancer xenograft mice and provide an alternative modality for tumor imaging.

Coaxial electrospray of microparticles and nanoparticles for biomedical applications

Coaxial electrospray is an electrohydrodynamic process that produces multilayer microparticles and nanoparticles by introducing coaxial electrified jets. In comparison with other microencapsulation/nanoencapsulation processes, coaxial electrospray has several potential advantages such as high encapsulation efficiency, effective protection of bioactivity and uniform size distribution. However, process control in coaxial electrospray is challenged by the multiphysical nature of the process and the complex interplay of multiple design, process and material parameters. This paper reviews the previous works and the recent advances in design, modeling and control of a coaxial electrospray process. The review intends to provide general guidance for coaxial electrospray and stimulate further research and development interests in this promising microencapsulation/nanoencapsulation process.

Fabrication of indocyanine green encapsulated biodegradable microbubbles for structural and functional imaging of cancer

We developed a novel dual-modal contrast agent for the structural and functional imaging of cancer. The contrast agent was fabricated by encapsulating indocyanine green (ICG) in poly(lactic-co-glycolic acid) (PLGA) microbubbles using a modified double-emulsion method. More stabilized absorption and fluorescence emission characteristics were observed for aqueous and plasma suspensions of ICG-encapsulated microbubbles. The technical feasibility of concurrent structural and functional imaging was demonstrated through a series of benchtop tests in which the aqueous suspension of ICG-encapsulated microbubbles was injected into a transparent tube embedded in an Intralipid phantom at different flow rates and concentrations. Concurrent fluorescence imaging and B-mode ultrasound imaging successfully captured the changes of microbubble flow rate and concentration with high linearity and accuracy. One potential application of the proposed ICG-encapsulated PLGA microbubbles is for the identification and characterization of peritumoral neovasculature for enhanced coregistration between tumor structural and functional boundaries in ultrasound-guided near-infrared diffuse optical tomography.

Heat-sensitive microbubbles for intraoperative assessment of cancer ablation margins

A heat-sensitive microbubble (HSM) agent, comprising a core of liquid perfluorocarbon (PFC) compound and a shell of biodegradable poly lactic-co-glycolic acid (PLGA), was fabricated using an emulsion evaporation method. Optical microscopic imaging showed that heating the HSM suspension to 55 degrees C activated the HSMs for significant volumetric expansion. After placing a HSM-dispersed agar-agar gel phantom in a 55 degrees C water bath for 10 min, the phantom became ultrasonically hyperechoic due to the HSM expansion. In an ex vivo test, a porcine tissue sample was injected with the HSM suspension and placed in a 55 degrees C water bath for 10 min. US imaging clearly identified the hyperechoic regions resulted from the HSM activation. The hyperechoic regions in the tissue sample kept a strong US contrast for more than 1 h. In a simulated ablation process, a HSM-dispersed agar-agar gel phantom was ablated by a cylindrical heating element. US imaging accurately estimated the ablation margin propagation while thermographic imaging underestimated the ablation margin. Our experiments demonstrated that the HSM agent could be used as a novel contrast agent for intraoperative assessment of ablation margins in cancer thermal ablation therapies.

Diffuse optical imaging and spectroscopy for cancer

Visible light and near infrared light interact with biological tissue by absorption and scattering. Diffuse optical imaging and spectroscopy reconstructs tissue physiologic parameters based on noninvasive measurement of tissue optical properties. This technology can be used to differentiate physiologic and molecular signatures of both malignant and benign tissues, as they relate to the area of cancer research. Major advantages are the use of non-ionizing radiation, real-time continuous data acquisition, low cost, and portability. Limitations include low spatial resolution and limited reproducibility. This paper reviews the currently available state-of-the-art technologies for diffuse optical imaging and spectroscopy and their applications in cancer research.

A prospective pilot clinical trial evaluating the utility of a dynamic near-infrared imaging device for characterizing suspicious breast lesions

IntroductionCharacterizing and differentiating between malignant tumors, benign tumors, and normal breast tissue is increasingly important in the patient presenting with breast problems. Near-infrared diffuse optical imaging and spectroscopy is capable of measuring multiple physiologic parameters of biological tissue systems and may have clinical applications for assessing the development and progression of neoplastic processes, including breast cancer. The currently available application of near-infrared imaging technology for the breast, however, is compromised by low spatial resolution, tissue heterogeneity, and interpatient variation.Materials and methodsWe tested a dynamic near-infrared imaging schema for the characterization of suspicious breast lesions identified on diagnostic clinical ultrasound. A portable handheld near-infrared tissue imaging device (P-Scan; ViOptix Inc., Fremont, CA, USA) was utilized. An external mechanical compression force was applied to breast tissue. The tissue oxygen saturation and hemoglobin concentration were recorded simultaneously by the handheld near-infrared imaging device. Twelve categories of dynamic tissue parameters were derived based on real-time measurements of the tissue hemoglobin concentration and the oxygen saturation.ResultsFifty suspicious breast lesions were evaluated in 48 patients. Statistical analyses were carried out on 36 out of 50 datasets that satisfied our inclusion criteria. Suspicious breast lesions identified on diagnostic clinical ultrasound had lower oxygenation and higher hemoglobin concentration than the surrounding normal breast tissue. Furthermore, histopathologic-proven malignant breast tumors had a lower differential hemoglobin contrast (that is, the difference of hemoglobin concentration variability between the suspicious breast lesion and the normal breast parenchyma located remotely elsewhere within the ipsilateral breast) as compared with histopathologic-proven benign breast lesions.ConclusionThe proposed dynamic near-infrared imaging schema has the potential to differentiate benign processes from those of malignant breast tumors. Further development and refinement of the dynamic imaging device and additional subsequent clinical testing are necessary for optimizing the accuracy of detection.

Ultrasound-Mediated Destruction of LHRHa-Targeted and Paclitaxel-Loaded Lipid Microbubbles for the Treatment of Intraperitoneal Ovarian Cancer Xenografts

Ultrasound-targeted microbubble destruction (UTMD) is a promising technique to facilitate the delivery of chemotherapy in cancer treatment. However, the process typically uses nonspecific microbubbles, leading to low tumor-to-normal tissue uptake ratio and adverse side effects. In this study, we synthesized the LHRH receptor-targeted and paclitaxel (PTX)-loaded lipid microbubbles (TPLMBs) for tumor-specific binding and enhanced therapeutic effect at the tumor site. An ovarian cancer xenograft model was established by injecting A2780/DDP cells intraperitoneally in BALB/c nude mice. Microscopic imaging of tumor sections after intraperitoneal injection of TPLMBs showed effective binding of the microbubbles with cancer cells. Ultrasound mediated destruction of the intraperitoneally injected TPLMBs yielded a superior therapeutic outcome in comparison with other treatment options. Immunohistochemical analyses of the dissected tumor tissue further confirmed the increased tumor apoptosis and reduced angiogenesis. Our experiment suggests that ultrasound-mediated intraperitoneal administration of the targeted drug-loaded microbubbles may be a useful method for the treatment of ovarian cancer.

Development of a handheld near-infrared imager for dynamic characterization of in vivo biological tissue systems

A handheld near-infrared imager was developed for real-time monitoring of tissue physiologic changes in response to dynamic compression stimuli. Both 2D and 3D imaging schemas were developed for reconstruction of tissue heterogeneities based on optical measurements. The handheld imager and the dynamic imaging schema were validated on both benchtop phantoms and in vivo human tissues. The benchtop tests demonstrated that the imager was able to reconstruct absorption properties of the embedded heterogeneity with accuracy and repeatability. The tests on in vivo human tissues demonstrated that the imager was able to generate various dynamic loading profiles with reproducibility and to detect tissue optical, mechanical, and physiologic changes under the dynamic loading condition.