Nanoparticle migration and delivery of Paclitaxel to regional lymph nodes in a large animal model.

BACKGROUND: The aim of this study was to demonstrate feasibility of migration and in situ chemotherapy delivery to regional lymph nodes (LN) in a large animal model using an expansile polymer nanoparticle (eNP) delivery system. STUDY DESIGN: Dual-labeled 50-nm and 100-nm eNP were prepared by encapsulating an IR-813 near-infrared (NIR) fluorescent dye within coumarin-conjugated expansile polymer nanoparticles (NIR-C-eNP). NIR imaging and fluorescent microscopy were used to identify intralymphatic migration of NIR-nanoparticles to draining inguinal or mesenteric LN after injection in swine hind legs or intestine. Nanoparticle-mediated intranodal delivery of chemotherapy was subsequently assessed with Oregon Green paclitaxel-loaded NIR-eNP (NIR-OGpax-eNP). RESULTS: NIR imaging demonstrated direct lymphatic migration of 50-nm, but not 100-nm, NIR-C-eNP and NIR-OGpax-eNP to the draining regional LNs after intradermal injection in the hind leg or subserosal injection in intestine. Fluorescent microscopy demonstrated that IR-813 used for NIR real-time trafficking colocalized with both the coumarin-labeled polymer and paclitaxel chemotherapy and was identified within the subcapsular spaces of the draining LNs. These studies verify nodal migration of both nanoparticle and encapsulated payload, and confirm the feasibility of focusing chemotherapy delivery directly to regional nodes. CONCLUSIONS: Regionally-targeted intranodal chemotherapy can be delivered to draining LNs for both skin and solid organs using 50-nm paclitaxel-loaded eNP.

Protoporphyrin IX fluorescence contrast in invasive glioblastomas is linearly correlated with Gd enhanced magnetic resonance image contrast but has higher diagnostic accuracy.

The sensitivity and specificity of in vivo magnetic resonance (MR) imaging is compared with production of protoporphyrin IX (PpIX), determined ex vivo, in a diffusely infiltrating glioma. A human glioma transfected with green fluorescent protein, displaying diffuse, infiltrative growth, was implanted intracranially in athymic nude mice. Image contrast from corresponding regions of interest (ROIs) in in vivo MR and ex vivo fluorescence images was quantified. It was found that all tumor groups had statistically significant PpIX fluorescence contrast and that PpIX contrast demonstrated the best predictive power for tumor presence. Contrast from gadolinium enhanced T1-weighted (T1W+Gd) and absolute T2 images positively predicted the presence of a tumor, confirmed by the GFP positive (GFP+) and hematoxylin and eosin positive (H&E+) ROIs. However, only the absolute T2 images had predictive power from controls in ROIs that were GFP+ but H&E negative. Additionally, PpIX fluorescence and T1W+Gd image contrast were linearly correlated in both the GFP+ (r = 0.79, p<1×10(-8)) and H&E+ (r = 0.74, p<0.003) ROIs. The trace diffusion images did not have predictive power or significance from controls. This study indicates that gadolinium contrast enhanced MR images can predict the presence of diffuse tumors, but PpIX fluorescence is a better predictor regardless of tumor vascularity.

Toward optimization of imaging system and lymphatic tracer for near-infrared fluorescent sentinel lymph node mapping in breast cancer.

BACKGROUND: Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in breast cancer requires optimized imaging systems and lymphatic tracers. MATERIALS AND METHODS: A small, portable version of the FLARE imaging system, termed Mini-FLARE, was developed for capturing color video and two semi-independent channels of NIR fluorescence (700 and 800 nm) in real time. Initial optimization of lymphatic tracer dose was performed using 35-kg Yorkshire pigs and a 6-patient pilot clinical trial. More refined optimization was performed in 24 consecutive breast cancer patients. All patients received the standard of care using (99m)Technetium-nanocolloid and patent blue. In addition, 1.6 ml of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected directly after patent blue at the same location. Patients were allocated to 1 of 8 escalating ICG:HSA concentration groups from 50 to 1000 µM. RESULTS: The Mini-FLARE system was positioned easily in the operating room and could be used up to 13 in. from the patient. Mini-FLARE enabled visualization of lymphatic channels and SLNs in all patients. A total of 35 SLNs (mean = 1.45, range 1-3) were detected: 35 radioactive (100%), 30 blue (86%), and 35 NIR fluorescent (100%). Contrast agent quenching at the injection site and dilution within lymphatic channels were major contributors to signal strength of the SLN. Optimal injection dose of ICG:HSA ranged between 400 and 800 µM. No adverse reactions were observed. CONCLUSIONS: We describe the clinical translation of a new NIR fluorescence imaging system and define the optimal ICG:HSA dose range for SLN mapping in breast cancer.

Nerve-highlighting fluorescent contrast agents for image-guided surgery.

Nerve damage is the major morbidity of many surgeries, resulting in chronic pain, loss of function, or both. The sparing of nerves during surgical procedures is a vexing problem because surrounding tissue often obscures them. To date, systemically administered nerve-highlighting contrast agents that can be used for nerve-sparing image-guided surgery have not been reported. In the current study, physicochemical and optical properties of 4,4'-[(2-methoxy-1,4-phenylene)di-(1E)-2,1-ethenediyl]bis-benzenamine (BMB) and a newly synthesized, red-shifted derivative 4-[(1E)-2-[4-[(1E)-2-[4-aminophenyl]ethenyl]-3-methoxyphenyl]ethenyl]-benzonitrile (GE3082) were characterized in vitro and in vivo. Both agents crossed the blood-nerve barrier and blood-brain barrier and rendered myelinated nerves fluorescent after a single systemic injection. Although both BMB and GE3082 also exhibited significant uptake in white adipose tissue, GE3082 underwent a hypsochromic shift in adipose tissue that provided a means to eliminate the unwanted signal using hyperspectral deconvolution. Dose and kinetic studies were performed in mice to determine the optimal dose and drug-imaging interval. The results were confirmed in rat and pig, with the latter used to demonstrate, for the first time, simultaneous fluorescence imaging of blood vessels and nerves during surgery using the FLARE™ (Fluorescence-Assisted Resection and Exploration) imaging system. These results lay the foundation for the development of ideal nerve-highlighting fluorophores for image-guided surgery.

Comparing implementations of magnetic-resonance-guided fluorescence molecular tomography for diagnostic classification of brain tumors.

Fluorescence molecular tomography (FMT) systems coupled to conventional imaging modalities such as magnetic resonance imaging (MRI) and computed tomography provide unique opportunities to combine data sets and improve image quality and content. Yet, the ideal approach to combine these complementary data is still not obvious. This preclinical study compares several methods for incorporating MRI spatial prior information into FMT imaging algorithms in the context of in vivo tissue diagnosis. Populations of mice inoculated with brain tumors that expressed either high or low levels of epidermal growth factor receptor (EGFR) were imaged using an EGF-bound near-infrared dye and a spectrometer-based MRI-FMT scanner. All data were spectrally unmixed to extract the dye fluorescence from the tissue autofluorescence. Methods to combine the two data sets were compared using student's t-tests and receiver operating characteristic analysis. Bulk fluorescence measurements that made up the optical imaging data set were also considered in the comparison. While most techniques were able to distinguish EGFR(+) tumors from EGFR(-) tumors and control animals, with area-under-the-curve values=1, only a handful were able to distinguish EGFR(-) tumors from controls. Bulk fluorescence spectroscopy techniques performed as well as most imaging techniques, suggesting that complex imaging algorithms may be unnecessary to diagnose EGFR status in these tissue volumes.

Fluorescent molecular imaging and dosimetry tools in photodynamic therapy.

Measurement of fluorescence and phosphorescence in vivo is readily used to quantify the concentration of specific species that are relevant to photodynamic therapy. However, the tools to make the data quantitatively accurate vary considerably between different applications. Sampling of the signal can be done with point samples, such as specialized fiber probes or from bulk regions with either imaging or sampling, and then in broad region image-guided manner. Each of these methods is described below, the application to imaging photosensitizer uptake is discussed, and developing methods to image molecular responses to therapy are outlined.

Molecular imaging agents specific for the annulus fibrosus of the intervertebral disk.

Low back pain is a prevalent medical condition that is difficult to diagnose and treat. Current imaging methods are unable to correlate pain reliably with spinal structures, and surgical removal of painful damaged or degenerating disks is technically challenging. A contrast agent specific for the intervertebral disk could assist in the detection, diagnosis, and surgical treatment of low back pain. The styryl pyridinium (FM) fluorophores were characterized and structure-activity relationships between chemical structure and in vivo uptake were established. Two novel FM fluorophores with improved optical properties for imaging the intervertebral disks were synthesized and evaluated in mice, rats, and pigs. After a single systemic injection, eight of eight FM fluorophores provided high-contrast imaging of the trigeminal ganglia, whereas six of eight provided high-contrast imaging of the dorsal root ganglia. Unexpectedly, three of eight FM fluorophores provided high-contrast imaging of annulus fibrosus tissue of the intervertebral disks, confirmed histologically. We present the first known contrast agent specific for the intervertebral disks and identify the chemical structural motif that mediates uptake. FM fluorophores could be used for image-guided surgery to assist in the removal of intervertebral disk and lay the foundation for derivatives for magnetic resonance imaging and positron emission tomography.

MRI-coupled fluorescence tomography quantifies EGFR activity in brain tumors.

RATIONALE AND OBJECTIVES: This report demonstrates the diagnostic potential of magnetic resonance imaging (MRI)-coupled fluorescence molecular tomography (FMT) to determine epidermal growth factor receptor (EGFR) status in brain cancer. MATERIALS AND METHODS: Two orthotopic glioma xenograft models were used in this study: one represented high EGFR expression and the other low expression. Nude mice were inoculated with cells from either one of the tumor lines or were used in a sham surgery control group. Animals were imaged using a unique MRI-FMT scanner 48 hours after intravenous injection of a near-infrared fluorophore bound to epidermal growth factor (EGF) ligand. Coronal images of fluorescence activity of the injected dye in the mouse brain were recovered using the MRI images as anatomical templates. RESULTS: In vivo images of fluorescence activity showed significant differences between animal populations, an observation confirmed by receiver operating characteristic analysis that revealed 100% sensitivity and specificity between animal groups implanted with EGFR((+)) and EGFR((-)) tumor lines. Similar performance was observed between EGFR((+)) and sham surgery control animals. CONCLUSIONS: This preclinical study suggests that MRI-FMT with fluorescent EGF provides excellent discrimination between tumors based on EGFR status. Reliable quantification of receptor status using minimally invasive techniques would be an important innovation for investigating new and existing cancer treatments that target these cellular mechanisms in research animals, and may be applied to identify receptor amplification in human brain cancer patients. This study represents the first systematic multianimal validation of receptor-specific imaging using MRI-guided fluorescence tomography.

Detecting epidermal growth factor receptor tumor activity in vivo during cetuximab therapy of murine gliomas.

RATIONALE AND OBJECTIVES: Noninvasive molecular imaging of glioma tumor receptor activity was assessed with diagnostic in vivo fluorescence monitoring during targeted therapy. The study goals were to assess the range of use for treatment monitoring and stratification of tumor types using epidermal growth factor (EGF) receptor (EGFR) status with administration of fluorescently labeled EGF and determine its utility for tumor detection compared to magnetic resonance imaging (MRI). MATERIALS AND METHODS: EGFR+ and EGFR- glioma tumor lines (human glioma [U251-GFP] and rat gliosarcoma [9L-GFP], respectively) were used to assess these goals, having a 20-fold difference between their EGF uptakes. RESULTS: Treatment with cetuximab in the EGFR+ tumor-bearing animals led to decreased EGF tumor uptake, whereas for the EGFR- tumors, no change in fluorescence signal followed treatment. This diagnostic difference in EGFR expression could be used to stratify the tumor-bearing animals into groups of potential responders and nonresponders, and receiver-operating characteristic curve analysis revealed an area under the curve (AUC) of 0.92 in separating these tumors. The nonlocalized growth pattern of U251-GFP tumors resulted in detection difficulty on standard MRI, but high EGFR expression made them detectable by fluorescence imaging (AUC = 1.0). The EGFR+ U251-GFP tumor-bearing animals could be noninvasively stratified into treated and untreated groups on the basis of fluorescence intensity difference (P = .035, AUC = 0.90). CONCLUSIONS: EGFR expression was tracked in vivo with fluorescence and determined to be of use for the stratification of EGFR+ and EGFR- tumors, the detection of EGFR+ tumors, and monitoring of molecular therapy.

Intraoperative localization of insulinoma and normal pancreas using invisible near-infrared fluorescent light.

BACKGROUND: Neuroendocrine tumors of the pancreas, such as insulinoma, are difficult to localize, and complete resection is essential for cure. Our hypothesis is that a near-infrared (NIR) fluorophore exhibiting uptake in insulinoma could provide high-sensitivity detection intraoperatively. MATERIALS AND METHODS: The optical properties of methylene blue (MB) were measured in vitro in 100% serum at 37 degrees C and in vivo after tissue uptake. MB was injected as a rapid intravenous bolus at doses ranging from 0.25 to 2 mg/kg into wildtype rats and pigs, and into insulinoma-bearing transgenic mice. The FLARE imaging system was used to acquire color video and NIR fluorescence images simultaneously and in real-time. The signal-to-background ratios (SBR) of tissues and tumors were quantified using FLARE software. RESULTS: When appropriately diluted, MB exhibits moderate NIR fluorescence emission peaking at 688 nm. At doses > or =1 mg/kg, certain normal tissues, such as pancreas, accumulate MB and remain NIR fluorescent for up to 1 h with an SBR > or = 1.6. MB spectral properties are maintained after uptake into tissue. Interestingly, insulinoma exhibits even higher MB signal than normal pancreas, resulting in insulinoma-to-pancreas ratios of 3.7 and insulinoma-to-muscle ratios of 16.2. MB permitted high-sensitivity, real-time localization of primary, multicentric, and metastatic insulinoma and permitted differentiation among tumor, normal pancreas, and other abdominal structures. CONCLUSION: A single intravenous injection of a clinically available, commonly used NIR fluorophore provides prolonged intraoperative localization of normal pancreas and insulinoma using invisible NIR fluorescent light.

First-in-human clinical trials of imaging devices: an example from optical imaging.

Clinical translation of scientific discoveries is often the long-term goal of academic medical research. However, this goal is not always realized due to the complicated path between bench research and clinical use. In this review, we outline the fundamental steps required for first-in-human testing of a new imaging device, and use the FLARE() (Fluorescence-Assisted Resection and Exploration) near-infrared fluorescence optical imaging platform as an example.

In vivo myocardial distribution of multipotent progenitor cells following intracoronary delivery in a swine model of myocardial infarction.

AIMS: There are few data comparing the fate of multipotent progenitor cells (MPCs) used in cardiac cell therapy after myocardial infarction (MI). To document in vivo distribution of MPCs delivered by intracoronary (IC) injection. METHODS AND RESULTS: Using an anterior MI swine model, near-infrared (NIR) fluorescence was used for in vivo tracking of labelled MPCs [mesenchymal stromal (MSCs), bone marrow mononuclear (BMMNCs), and peripheral blood mononuclear (PBMNCs)] cells early after IC injection. Signal intensity ratios (SIRs) of injected over non-injected (reference) zones were used to report NIR fluorescence emission. Following IC injection, significant differences in mean SIR were documented when MSCs were compared with BMMNCs [1.28 +/- 0.10 vs. 0.77 +/- 0.11, P < 0.001; 95% CI (0.219, 0.805), respectively] or PBMNCs [1.28 +/- 0.10 vs. 0.80 +/- 0.14, P = 0.005; 95% CI (0.148, 0.813), respectively]. Differences were maintained during the 60 min tracking period, with only the MSC-injected groups continuously emitting NIR fluorescence (SIR>1). This is correlated with greater cell retention for MSCs relative to mononuclear cells. However, there was evidence of MSC-related vessel plugging in some swine. CONCLUSION: Our in vivo NIR fluorescence findings suggest that MPC distribution and retention immediately after intracoronary delivery vary depending on cell population and could potentially impact the clinical efficacy of cardiac cell therapy.

Imaging of glioma tumor with endogenous fluorescence tomography.

Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX.

The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping.

BACKGROUND: Invisible NIR fluorescent light can provide high sensitivity, high-resolution, and real-time image-guidance during oncologic surgery, but imaging systems that are presently available do not display this invisible light in the context of surgical anatomy. The FLARE imaging system overcomes this major obstacle. METHODS: Color video was acquired simultaneously, and in real-time, along with two independent channels of NIR fluorescence. Grayscale NIR fluorescence images were converted to visible "pseudo-colors" and overlaid onto the color video image. Yorkshire pigs weighing 35 kg (n = 5) were used for final preclinical validation of the imaging system. A six-patient pilot study was conducted in women undergoing sentinel lymph node (SLN) mapping for breast cancer. Subjects received (99m)Tc-sulfur colloid lymphoscintigraphy. In addition, 12.5 microg of indocyanine green (ICG) diluted in human serum albumin (HSA) was used as an NIR fluorescent lymphatic tracer. RESULTS: The FLARE system permitted facile positioning in the operating room. NIR light did not change the look of the surgical field. Simultaneous pan-lymphatic and SLN mapping was demonstrated in swine using clinically available NIR fluorophores and the dual NIR capabilities of the system. In the pilot clinical trial, a total of nine SLNs were identified by (99m)Tc- lymphoscintigraphy and nine SLNs were identified by NIR fluorescence, although results differed in two patients. No adverse events were encountered. CONCLUSIONS: We describe the successful clinical translation of a new NIR fluorescence imaging system for image-guided oncologic surgery.

Diagnostic detection of diffuse glioma tumors in vive with molecular fluorescent probe-based transmission spectroscopy.

The diffuse spread of glioma tumors leads to the inability to image and properly treat this disease. The optical spectral signature of targeted fluorescent probes provides molecular signals from the diffuse morphologies of glioma tumors, which can be a more effective diagnostic probe than standard morphology-based magnetic resonance imaging (MRI) sequences. Three orthotopic xenograft glioma models were used to examine the potential for transmitted optical fluorescence signal detection in vivo, using endogenously produced protoporphyrin IX (PpIX) and exogenously administered fluorescently labeled epidermal growth factor (EGF). Accurate quantification of the fluorescent signals required spectral filtering and signal normalization, and when optimized, it was possible to improve detection of sparse diffuse glioma tumor morphologies. The signal of endogenously produced PpIX provided similar sensitivity and specificity to MRI, while detection with fluorescently labeled EGF provided maximal specificity for tumors with high EGF receptor activity. Optical transmitted fluorescent signal may add significant benefit for clinical cases of diffuse infiltrative growth pattern glioma tumors given sufficient optimization of the signal acquisition for each patient.

High dose rate radiation treatment of experimental intramuscular prostate carcinoma.

PURPOSE: The Dunning R3327-MLL is a well established transplanted tumour line, and as such it makes a desirable model for evaluative studies of therapy. In the current study, the interstitial growth characteristics as well as the response of this tumour to a single fraction of high dose rate radiation is investigated. MATERIALS AND METHODS: The in vitro response to radiation of the Dunning R3327-MLL was studied via a colony forming assay using a Cs-137 irradiator. In vitro radiosensitivity was determined on tumours implanted intramuscularly in the left gastrocnemius muscle of the rat and irradiated using an Ir-192 afterloader. RESULTS: The results demonstrate a faster growth rate when compared to the reported subcutaneous growth rates. The Dunning R3327-MLL's radiosensitivity is comparable to that of late response tissues. The dose required to achieve a specific radiobiological response (the alpha:beta ratio) of the in vitro cell line is 2.4 Gy, whereas the ratio for the intramuscularly growing tumour was 0.99 Gy. CONCLUSIONS: These findings signify the intramuscularly implanted Dunning R3327-MLL tumour model as a desirable model for the study of single fraction high dose rate radiation treatments.

Noninvasive measurement of aminolevulinic acid-induced protoporphyrin IX fluorescence allowing detection of murine glioma in vivo.

Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium. Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals. However, 2 h after ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0.016, and U251 group p-value=0.004, relative to the control group). The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situ at ex vivo analysis. Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm) is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups. The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups.

Magnetic resonance-coupled fluorescence tomography scanner for molecular imaging of tissue.

A multichannel spectrally resolved optical tomography system to image molecular targets in small animals from within a clinical MRI is described. Long source/detector fibers operate in contact mode and couple light from the tissue surface in the magnet bore to 16 spectrometers, each containing two optical gratings optimized for the near infrared wavelength range. High sensitivity, cooled charge coupled devices connected to each spectrograph provide detection of the spectrally resolved signal, with exposure times that are automated for acquisition at each fiber. The design allows spectral fitting of the remission light, thereby separating the fluorescence signal from the nonspecific background, which improves the accuracy and sensitivity when imaging low fluorophore concentrations. Images of fluorescence yield are recovered using a nonlinear reconstruction approach based on the diffusion approximation of photon propagation in tissue. The tissue morphology derived from the MR images serves as an imaging template to guide the optical reconstruction algorithm. Sensitivity studies show that recovered values of indocyanine green fluorescence yield are linear to concentrations of 1 nM in a 70 mm diameter homogeneous phantom, and detection is feasible to near 10 pM. Phantom data also demonstrate imaging capabilities of imperfect fluorophore uptake in tissue volumes of clinically relevant sizes. A unique rodent MR coil provides optical fiber access for simultaneous optical and MR data acquisition of small animals. A pilot murine study using an orthotopic glioma tumor model demonstrates optical-MRI imaging of an epidermal growth factor receptor targeted fluorescent probe in vivo.