Near Infrared Fluorescent Nanoplatform for Targeted Intraoperative Resection and Chemotherapeutic Treatment of Glioblastoma.

Despite significant efforts to improve glioblastoma multiforme (GBM) treatment, GBM remains one of the most lethal cancers. Effective GBM treatments require sensitive intraoperative tumor visualization and effective postoperative chemotherapeutic delivery. Unfortunately, the diffusive and infiltrating nature of GBM limits the detection of GBM tumors, and current intraoperative visualization methods limit complete tumor resection. In addition, although chemotherapy is often used to eliminate any cancerous tissue remaining after surgery, most chemotherapeutic drugs do not effectively cross the brain-blood barrier (BBB) or enter GBM tumors. As a result, GBM has limited treatment options with high recurrence rates, and methods that improve its complete visualization during surgery and treatment are needed. Herein, we report a fluorescent nanoparticle platform for the near-infrared fluorescence (NIRF)-based tumor boundary visualization and image-guided drug delivery into GBM tumors. Our nanoplatform is based on ferumoxytol (FMX), an FDA-approved magnetic resonance imaging-sensitive superparamagnetic iron oxide nanoparticle, which is conjugated with hepthamethine cyanine (HMC), a NIRF ligand that specifically targets the organic anion transporter polypeptides that are overexpressed in GBM. We have shown that HMC-FMX nanoparticles cross the BBB and selectively accumulate in the tumor using orthotopic GBM mouse models, enabling NIRF-based visualization of infiltrating tumor tissue. In addition, HMC-FMX can encapsulate chemotherapeutic drugs, such as paclitaxel or cisplatin, and deliver these agents into GBM tumors, reducing tumor size and increasing survival. Taken together, these observations indicate that HMC-FMX is a promising nanoprobe for GBM surgical visualization and drug delivery.

Tumor-Activatable Clinical Nanoprobe for Cancer Imaging.

Purpose: A successful cancer surgery requires the complete removal of cancerous tissue, while also sparing as much healthy, non-cancerous tissue as possible. To achieve this, an accurate identification of tumor boundaries during surgery is critical, but intra-operative tumor visualization remains challenging. Fluorescence imaging is a promising method to improve tumor detection and delineate tumor boundaries during surgery, but the lack of stable, long-circulating, clinically-translatable fluorescent probes that can identify tumors with high signal-to-noise ratios and low background fluorescence signals have prevented its widespread application. Methods: We screened the optical properties of several fluorescent dyes before and after nanoprobe encapsulation, and then identified nanoprobes with quenched fluorescence that were re-activated upon dye release. The physical and biological properties of these nanoprobes leading to fluorescence activation were investigated in vitro. Further, the cancer imaging properties of both free dyes and nanoprobe-encapsulated dyes were compared in vivo. Results: A novel fluorescent nanoprobe was prepared by combining two FDA-approved agents commonly used in the clinic: Feraheme (FH) and indocyanine green (ICG). The resulting FH-entrapped ICG nanoprobe [FH(ICG)] displayed quenched fluorescence compared to other nanoprobes, and this quenched fluorescence was re-activated in acidic tumor microenvironment conditions (pH 6.8) and upon uptake into cancer cells. Finally, in vivo studies in a prostate cancer mouse model demonstrated that FH(ICG) treatments enhance long-term fluorescence signals in tumors compared to ICG treatments, allowing for fluorescence-guided tumor identification using clinically relevant fluorescence cameras. Conclusions: FH(ICG) nanoprobes were identified as fluorescent nanoprobes with beneficial fluorescence activation properties compared to other FH-entrapped dyes. The activatable nature of this nanoprobe allows for a low background fluorescence signal and high signal-to-noise ratio within a long-circulating nanoagent, which allows for long-term fluorescence signals from tumors that enabled their fluorescence-guided detection. This activatable nanoprobe offers tremendous potential as a clinically translatable image-guided cancer therapy modality that can be prepared in a clinical setting.

Phase 1 Safety, Pharmacokinetics, and Fluorescence Imaging Study of Tozuleristide (BLZ-100) in Adults With Newly Diagnosed or Recurrent Gliomas.

BACKGROUND: Fluorescence-guided surgery (FGS) can improve extent of resection in gliomas. Tozuleristide (BLZ-100), a near-infrared imaging agent composed of the peptide chlorotoxin and a near-infrared fluorophore indocyanine green, is a candidate molecule for FGS of glioma and other tumor types. OBJECTIVE: To perform a phase 1 dose-escalation study to characterize the safety, pharmacokinetics, and fluorescence imaging of tozuleristide in adults with suspected glioma. METHODS: Patients received a single intravenous dose of tozuleristide 3 to 29 h before surgery. Fluorescence images of tumor and cavity in Situ before and after resection and of excised tissue ex Vivo were acquired, along with safety and pharmacokinetic measures. RESULTS: A total of 17 subjects received doses between 3 and 30 mg. No dose-limiting toxicity was observed, and no reported adverse events were considered related to tozuleristide. At doses of 9 mg and above, the terminal serum half-life for tozuleristide was approximately 30 min. Fluorescence signal was detected in both high- and low-grade glial tumors, with high-grade tumors generally showing greater fluorescence intensity compared to lower grade tumors. In high-grade tumors, signal intensity increased with increased dose levels of tozuleristide, regardless of the time of dosing relative to surgery. CONCLUSION: These results support the safety of tozuleristide at doses up to 30 mg and suggest that tozuleristide imaging may be useful for FGS of gliomas.

Polymalic acid chlorotoxin nanoconjugate for near-infrared fluorescence guided resection of glioblastoma multiforme.

Maximal surgical resection of glioma remains the single most effective treatment. Tools to guide the resection while avoiding removal of normal brain tissues can aid surgeons in achieving optimal results. One strategy to achieve this goal is to rely upon interoperative fluorescence staining of tumor cells in vivo, that can be visualized by the surgeon during resection. Towards this goal we have designed a biodegradable fluorescent mini nano imaging agent (NIA) with high specificity for U87MG glioma cells and previously unmet high light emission. The NIA is the conjugate of polymalic acid (PMLA) with chlorotoxin for tumor targeting, indocyanine green (ICG) for NIR fluorescence and the tri-leucin peptide as fluorescence enhancer. PMLA as a multivalent platform carries several molecules of ICG and the other ligands. The NIA recognizes multiple sites on glioma cell surface, demonstrated by the effects of single and combined competitors. Systemic IV injection into xenogeneic mouse model carrying human U87MG glioblastoma indicated vivid tumor cell binding and internalization of NIA resulting in intensive and long-lasting tumor fluorescence. The NIA is shown to greatly improve tumor removal supporting its utility in clinical applications.

Real time optical Biopsy: Time-resolved Fluorescence Spectroscopy instrumentation and validation.

The Time-resolved fluorescence spectroscopy (TR-FS) has the potential to differentiate tumor and normal tissue in real time during surgical excision. In this manuscript, we describe the design of a novel TR-FS device, along with preliminary data on detection accuracy for fluorophores in a mixture. The instrument is capable of near real-time fluorescence lifetime acquisition in multiple spectral bands and analysis. It is also able to recover fluorescence lifetime with sub-20ps accuracy as validated with individual organic fluorescence dyes and dye mixtures yielding lifetime values for standard fluorescence dyes that closely match with published data. We also show that TR-FS is able to quantify the relative concentration of fluorescence dyes in a mixture by the unmixing of lifetime decays. We show that the TR-FS prototype is able to identify in near-real time the concentrations of dyes in a complex mixture based on previously trained data. As a result, we demonstrate that in complex mixtures of fluorophores, the relative concentration information is encoded in the fluorescence lifetime across multiple spectral bands. We show for the first time the temporal and spectral measurements of a mixture of fluorochromes and the ability to differentiate relative concentrations of each fluorochrome mixture in real time.

Transcranial Near-Infrared Laser Transmission (NILT) Profiles (800 nm): Systematic Comparison in Four Common Research Species.

BACKGROUND AND PURPOSE: Transcranial near-infrared laser therapy (TLT) is a promising and novel method to promote neuroprotection and clinical improvement in both acute and chronic neurodegenerative diseases such as acute ischemic stroke (AIS), traumatic brain injury (TBI), and Alzheimer's disease (AD) patients based upon efficacy in translational animal models. However, there is limited information in the peer-reviewed literature pertaining to transcranial near-infrared laser transmission (NILT) profiles in various species. Thus, in the present study we systematically evaluated NILT characteristics through the skull of 4 different species: mouse, rat, rabbit and human. RESULTS: Using dehydrated skulls from 3 animal species, using a wavelength of 800nm and a surface power density of 700 mW/cm2, NILT decreased from 40.10% (mouse) to 21.24% (rat) to 11.36% (rabbit) as skull thickness measured at bregma increased from 0.44 mm in mouse to 0.83 mm in rat and then 2.11 mm in rabbit. NILT also significantly increased (p<0.05) when animal skulls were hydrated (i.e. compared to dehydrated); but there was no measurable change in thickness due to hydration. In human calvaria, where mean thickness ranged from 7.19 mm at bregma to 5.91 mm in the parietal skull, only 4.18% and 4.24% of applied near-infrared light was transmitted through the skull. There was a slight (9.2-13.4%), but insignificant effect of hydration state on NILT transmission of human skulls, but there was a significant positive correlation between NILT and thickness at bregma and parietal skull, in both hydrated and dehydrated states. CONCLUSION: This is the first systematic study to demonstrate differential NILT through the skulls of 4 different species; with an inverse relationship between NILT and skull thickness. With animal skulls, transmission profiles are dependent upon the hydration state of the skull, with significantly greater penetration through hydrated skulls compared to dehydrated skulls. Using human skulls, we demonstrate a significant correlation between thickness and penetration, but there was no correlation with skull density. The results suggest that TLT should be optimized in animals using novel approaches incorporating human skull characteristics, because of significant variance of NILT profiles directly related to skull thickness.

Advances in Imaging: Brain Tumors to Alzheimer's Disease.

Professor Black and colleagues have been working to improve the quality and sensitivity of imaging in the early detection of conditions from brain tumors to Alzheimer's disease to enhance treatment protocols and patient management. Professor Black et al introduced nanoparticles to improve MRI imaging. These nanoparticles consist of poly (b-L- malic acid (PMLA)) conjugates with monoclonal antibodies ((mAbs)) and Gd-DOTA. These are known as MRI nano-imaging agents (NIA). Most importantly, they can penetrate the endothelial blood-brain barrier (BBB) to reach brain tumors (primary or metastasis). This is effective in cases of brain tumors or breast cancer or other cancers such as lung cancer and gastric cancer having HER2 and/or EGFR positive crossing BBB. By the covalent conjugation of MR contrast (NIA), the MRI virtual biopsy can differentiate brain tumors from infections or other brain pathological conditions. The brain's intrinsic natural fluorescence such as NADH, FAD, lipopigments and porphyrin in the brain tissue can be identified by using time resolved fluorescence spectroscopy (TRFS) which is operated through the use of ultra-short laser. TRFS produces various color bands to differentiate the tumor from normal brain tissue in real time and registers the data on a 3D map. This is significant, as this will provide a greatly improved assessment methodology of tissue type. Consequently, this will potentially result in shorter operation times as well as more satisfactory tumor removal. In the detection of Alzheimer disease, amyloid plaque is deposited in retina tissue (including the RGC, RNFL and inner plexiform layer) which can produce a fluorescence effect by using curcumin as a contrast. This is then shown by human retina amyloid imaging device. Immunotherapies with glatiramer acetate (GA) have been shown to reduce amyloid deposits in brain and retinal AB deposits in mice. The study of advanced imaging technology and techniques including NIA, TRFS and the detection of amyloid plaque in Alzheimer disease are very important approaches to create a new era for diagnostic and therapeutic management of brain tumors and other cancers (HER2 and/or EGFR positive). This pioneering work by Professor Black, and colleagues, gives rise to a new hope for cancer patients for targeted therapy and for immunotherapies in Alzheimer's disease.

Near-infrared imaging of brain tumors using the Tumor Paint BLZ-100 to achieve near-complete resection of brain tumors.

OBJECT: The intraoperative clear delineation between brain tumor and normal tissue in real time is required to ensure near-complete resection without damaging the nearby eloquent brain. Tumor Paint BLZ-100, a tumor ligand chlorotoxin (CTX) conjugated to indocyanine green (ICG), has shown potential to be a targeted contrast agent. There are many infrared imaging systems in use, but they are not optimized to the low concentration and amount of ICG. The authors present a novel proof-of-concept near-infrared (NIR) imaging system using a standard charge-coupled device (CCD) camera for visualizing low levels of ICG attached to the tumors. This system is small, inexpensive, and sensitive. The imaging system uses a narrow-band laser at 785 nm and a notch filter in front of the sensor at the band. The camera is a 2-CCD camera, which uses identical CCDs for both visible and NIR light. METHODS: The NIR system is tested with serial dilution of BLZ-100 from 1 µM to 50 pM in 5% Intralipid solution while the excitation energy is varied from 5 to 40 mW/cm(2). The analog gain of the CCD was changed from 0, 6, and 12 dB to determine the signal-to-noise ratio. In addition to the Intralipid solution, BLZ-100 was injected 48 hours before euthanizing the mice that were implanted with the human glioma cell line. The brain was removed and imaged using the NIR imaging system. RESULTS: The authors' results show that the NIR imaging system using a standard CCD is able to visualize the ICG down to 50 nM of concentration with a high signal-to-noise ratio. The preliminary experiment on human glioma implanted in mouse brains demonstrated that BLZ-100 has a high affinity for glioma compared with normal brain tissue. Additionally, the results show that NIR excitation is able to penetrate deeply and has a potential to visualize metastatic lesions that are separate from the main tumor. CONCLUSIONS: The authors have seen that BLZ-100 has a very high affinity toward human gliomas. They also describe a small, cost-effective, and sensitive NIR system for visualizing brain tumors tagged using BLZ-100. The authors hope that the use of BLZ-100 along with NIR imaging will be useful to delineate the brain tumors in real time and assist surgeons in near-complete tumor removal to increase survival and reduce neurological deficits.

Development of transcranial sonothrombolysis as an alternative stroke therapy: incremental scientific advances toward overcoming substantial barriers.

Transcranial ultrasound and high intensity focused ultrasound technologies have been developed as a method of thrombolysis to be applied to the treatment of acute ischemic stroke. The originating idea to apply ultrasound to treat disease states dates back from the 1930s to 1940s when seminal research findings suggested that ultrasound could have an effect on biological systems and the brain, but the mechanism(s) involved in the effects were unknown. This exciting field of research has flourished since the potential exists to effectively utilize ultrasound to induce thrombolysis noninvasively or perhaps in combination with a thrombolytic agent, such as tissue plasminogen activator or secondary pharmaceutical such as microbubbles to promote cerebral reperfusion and clinical improvement. While there is great enthusiasm in this field of stroke treatment, specific parameters required for optimal sonothrombolysis such as output power, duty cycle, pulse width, and exposure time, as well as the impact of skull bone characteristics and flow mechanics, remain to be defined. This article analyzes relevant ultrasound studies to provide a synthesis of insight in the field of sonothrombolysis to attempt to provide direction for possible future use in stroke patients.

Fluorescence lifetime spectroscopy for guided therapy of brain tumors.

This study evaluates the potential of time-resolved laser induced fluorescence spectroscopy (TR-LIFS) as intra-operative tool for the delineation of brain tumor from normal brain. Forty two patients undergoing glioma (WHO grade I-IV) surgery were enrolled in this study. A TR-LIFS prototype apparatus (gated detection, fast digitizer) was used to induce in-vivo fluorescence using a pulsed N2 laser (337 nm excitation, 0.7 ns pulse width) and to record the time-resolved spectrum (360-550 nm range, 10 nm interval). The sites of TR-LIFS measurement were validated by conventional histopathology (H&E staining). Parameters derived from the TR-LIFS data including intensity values and time-resolved intensity decay features (average fluorescence lifetime and Laguerre coefficients values) were used for tissue characterization and classification. 71 areas of tumor and normal brain were analyzed. Several parameters allowed for the differentiation of distinct tissue types. For example, normal cortex (N=35) and normal white matter (N=12) exhibit a longer-lasting fluorescence emission at 390 nm (τ390=2.12±0.10 ns) when compared with 460 nm (τ460=1.16±0.08 ns). High grade glioma (grades III and IV) samples (N=17) demonstrate emission peaks at 460 nm, with large variation at 390 nm while low grade glioma (I and II) samples (N=7) demonstrated a peak fluorescence emission at 460 nm. A linear discriminant algorithm allowed for the classification of low-grade gliomas with 100% sensitivity and 98% specificity. High-grade glioma demonstrated a high degree of heterogeneity thus reducing the discrimination accuracy of these tumors to 47% sensitivity and 94% specificity. Current findings demonstrate that TR-LIFS holds the potential to diagnose brain tumors intra-operatively and to provide a valuable tool for aiding the neurosurgeon-neuropathologist team in to rapidly distinguish between tumor and normal brain during surgery.

Intraoperative delineation of primary brain tumors using time-resolved fluorescence spectroscopy.

The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8+/-0.3 ns) and 460 nm (lifetime=0.8+/-0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7+/-0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.

Distinction of brain tissue, low grade and high grade glioma with time-resolved fluorescence spectroscopy.

Neuropathology frozen section diagnoses are difficult in part because of the small tissue samples and the paucity of adjunctive rapid intraoperative stains. This study aims to explore the use of time-resolved laser-induced fluorescence spectroscopy as a rapid adjunctive tool for the diagnosis of glioma specimens and for distinction of glioma from normal tissues intraoperatively. Ten low grade gliomas, 15 high grade gliomas without necrosis, 6 high grade gliomas with necrosis and/or radiation effect, and 14 histologically uninvolved "normal" brain specimens are spectroscopicaly analyzed and contrasted. Tissue autofluorescence was induced with a pulsed Nitrogen laser (337 nm, 1.2 ns) and the transient intensity decay profiles were recorded in the 370-500 nm spectral range with a fast digitized (0.2 ns time resolution). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site were used for tissue characterization. A linear discriminant analysis diagnostic algorithm was used for tissue classification. Both low and high grade gliomas can be distinguished from histologically uninvolved cerebral cortex and white matter with high accuracy (above 90%). In addition, the presence or absence of treatment effect and/or necrosis can be identified in high grade gliomas. Taking advantage of tissue autofluorescence, this technique facilitates a direct and rapid investigation of surgically obtained tissue.

Diagnosis of meningioma by time-resolved fluorescence spectroscopy.

We investigate the use of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for the intraoperative rapid evaluation of tumor specimens and delineation of tumor from surrounding normal tissue. Tissue autofluorescence is induced with a pulsed nitrogen laser (337 nm, 1.2 ns) and the intensity decay profiles are recorded in the 370 to 500 nm spectral range with a fast digitizer (0.2 ns resolution). Experiments are conducted on excised specimens (meningioma, dura mater, cerebral cortex) from 26 patients (97 sites). Spectral intensities and time-dependent parameters derived from the time-resolved spectra of each site are used for tissue characterization. A linear discriminant analysis algorithm is used for tissue classification. Our results reveal that meningioma is characterized by unique fluorescence characteristics that enable discrimination of tumor from normal tissue with high sensitivity (>89%) and specificity (100%). The accuracy of classification is found to increase (92.8% cases in the training set and 91.8% in the cross-validated set correctly classified) when parameters from both the spectral and the time domain are used for discrimination. Our findings establish the feasibility of using TR-LIFS as a tool for the identification of meningiomas and enables further development of real-time diagnostic tools for analyzing surgical tissue specimens of meningioma or other brain tumors.

Fluorescence lifetime spectroscopy of glioblastoma multiforme.

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360-550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectral- and the time domain can enhance the ability of fluorescence-based techniques to diagnose and detect brain tumor margins intraoperatively.