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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.

We developed a heat-sensitive microbubble (HSM) agent for intraoperative assessment of thermal ablation margins in cancer ablation therapies. The HSM agent, comprising a core of liquid perfluorocarbon (PFC) compound and a shell of biodegradable poly lactic-coglycolic acid (PLGA), was fabricated using an emulsion evaporation method. In our previous study, significant increase of ultrasound contrast was observed after heat activation of HSMs. In this study, intraoperative ultrasonic assessment of thermal ablation margins by HSMs was demonstrated in vivo in a pig model. HSMs were delivered to the pig liver by portal vein injection. Liver ablation was done using a RF ablation probe. Intraoperative ultrasound imaging with HSMs clearly delineated the ablation margin. Fluorescence images of liver tissue samples confirmed the existence and activation of HSMs. This result demonstrated that the HSM agent can be potentially utilized as a multimodal contrast agent for intraoperative ultrasonic and fluorescence assessment of thermal ablation margins in cancer ablation therapies.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

We synthesize multifunctional microbubbles (MBs) for targeted delivery of antivascular endothelial growth factor (antiVEGF) therapy with multimodal imaging guidance. Poly-lactic-co-glycolic acid (PLGA) MBs encapsulating Texas Red dye are fabricated by a modified double-emulsion process. Simultaneous ultrasound and fluorescence imaging are achieved using Texas Red encapsulated MBs. The MBs are conjugated with Avastin, an antiVEGF antibody for treating neovascular age-related macular degeneration (AMD). The conjugation efficiency is characterized by enzyme-linked immunosorbent assay (ELISA). The efficiency for targeted binding of Avastin-conjugated MBs is characterized by microscopic imaging. Our work demonstrates the technical potential of using multifunctional MBs for targeted delivery of antiVEGF therapy in the treatment of exudative AMD.

Background: Clinical ultrasound (US) uses ultrasonic scattering contrast to characterize subcutaneous anatomic structures. Photoacoustic (PA) imaging detects the functional properties of thick biological tissue with high optical contrast. In the case of image-guided cancer ablation therapy, simultaneous US and PA imaging can be useful for intraoperative assessment of tumor boundaries and ablation margins. In this regard, accurate co-registration between imaging modalities and high sensitivity to cancer cells are important. Methods: We synthesized poly-lactic-co-glycolic acid (PLGA) microbubbles (MBs) and nanobubbles (NBs) encapsulating India ink or indocyanine green (ICG). Multiple tumor simulators were fabricated by entrapping ink MBs or NBs at various concentrations in gelatin phantoms for simultaneous US and PA imaging. MBs and NBs were also conjugated with CC49 antibody to target TAG-72, a human glycoprotein complex expressed in many epithelial-derived cancers. Results: Accurate co-registration and intensity correlation were observed in US and PA images of MB and NB tumor simulators. MBs and NBs conjugating with CC49 effectively bound with over-expressed TAG-72 in LS174T colon cancer cell cultures. ICG was also encapsulated in MBs and NBs for the potential to integrate US, PA, and fluorescence imaging. Conclusions: Multifunctional MBs and NBs can be potentially used as a general contrast agent for multimodal intraoperative imaging of tumor boundaries and therapeutic margins.

Bevacizumab (Avastin) has been used as one of the anti-VEGF therapies to manage neovascular age-related macular degeneration (AMD). The drug delivery system for bevacizumab needs to be improved in order to decrease the frequency of injection and reduce the adverse effects. In our study, bevacizumab was conjugated with poly (lactic-co-glycolic acid) (PLGA) microbubbles by activating carboxyl functional groups. The averaged size of microbubbles was estimated 1.055±0.258μm, allowing for ultrasound guided drug delivery. The binding efficiency between bevacizumab and microbubbles was evaluated in an enzyme-linked immunosorbent assay plate. The test results demonstrated the potential of using PLGA microbubbles to deliver bevacizumab with imaging guidance.

Background: Accurate assessment of tumor boundaries and intraoperative detection of therapeutic margins are important oncologic principles for minimal recurrence rates and improved long-term outcomes. However, many existing cancer imaging tools are based on preoperative image acquisition and do not provide real-time intraoperative information that supports critical decision-making in the operating room. Method: Poly lactic-co-glycolic acid (PLGA) microbubbles (MBs) and nanobubbles (NBs) were synthesized by a modified double emulsion method. The MB and NB surfaces were conjugated with CC49 antibody to target TAG-72 antigen, a human glycoprotein complex expressed in many epithelial-derived cancers. Multiple imaging agents were encapsulated in MBs and NBs for multimodal imaging. Both one-step and multi-step cancer targeting strategies were explored. Active MBs/NBs were also fabricated for therapeutic margin assessment in cancer ablation therapies. Results: The multimodal contrast agents and the cancer-targeting strategies were tested on tissue simulating phantoms, LS174 colon cancer cell cultures, and cancer xenograft nude mice. Concurrent multimodal imaging was demonstrated using fluorescence and ultrasound imaging modalities. Technical feasibility of using active MBs and portable imaging tools such as ultrasound for intraoperative therapeutic margin assessment was demonstrated in a biological tissue model. Conclusion: The cancer-specific multimodal contrast agents described in this paper have the potential for intraoperative detection of tumor boundaries and therapeutic margins.

Accurate assessment of wound oxygenation and perfusion is important for evaluating wound healing/regression and guiding following therapeutic processes. However, many existing techniques and clinical practices are subjective and qualitative due to background bias, tissue heterogeneity, and inter-patient variation. To overcome these limitations, we developed a dual-modal imaging system for in vivo, non-invasive, real-time quantitative assessment of wound tissue oxygenation and perfusion. The imaging system integrated a broadband light source, a high-resolution CCD camera, a highly sensitive thermal camera, and a liquid crystal tunable filter. A user-friendly interface was developed to control all the components systematically. Advanced algorithms were explored for reliable reconstruction of tissue oxygenation and appropriate co-registration between thermal images and multispectral images. Dual-mode oxygenation and perfusion imaging was demonstrated on both benchtop models and human subjects, and compared with measurements using other methods, such as Laser Doppler and tissue oximeter. The test results suggested that the dual-modal imaging system has the potential for non-contact real-time imaging of wound tissue oxygenation and perfusion.

Many advantages of biomedical optical imaging modalities include low cost, portability, no radiation hazard, molecular sensitivity, and real-time non-invasive measurements of multiple tissue parameters. However, clinical acceptance of optical imaging is hampered by the lack of calibration standards and validation techniques. In this context, developing phantoms that simulate tissue structural, functional, and molecular properties is important for reliable performance and successful translation of biomedical optical imaging techniques to clinical applications. Over the years, we have developed various tissue simulating phantoms to validate imaging algorithms, to optimize instrument performance, to test contrast agents, and to calibrate acquisition systems. We also developed phantoms with multimodal contrasts for co-registration between different imaging modalities. In order to study tissue dynamic changes during medical intervention, we develop gel wax phantoms to simulate tissue optical and mechanical dynamics in response to compression load. We also dispersed heat sensitive microbubbles in agar agar gel phantoms to simulate heatinduced tissue coagulative necrosis in a cancer ablation procedure. The phantom systems developed in our lab have the potential to provide standardized traceable tools for multimodal imaging and image-guided intervention.

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.

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.

We synthesize drug-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres for image-guided combinatory epigenetic therapy in MCF-10A human mammary epithelial cells. LY294002 and Nile Red are encapsulated in microspheres for sustained drug release and fluorescence microscopic imaging. Drug-loaded microspheres target MCF-10A cells through a three-step binding process involving biotinylated antibody, streptavidin, and biotinylated microspheres. LY294002 loaded microspheres and 5-Aza-2-deoxycytidine are applied to MCF-10A cells for combinatory PI3K/AKT inhibition and deoxyribonucleic acid (DNA) demethylation. Our study implies the technical potential of disease targeting and image-guided combinatory epigenetic therapy using drug-loaded multifunctional biodegradable PLGA microspheres.

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.