Core-based lipid nanoparticles as a nanoplatform for delivery of near-infrared fluorescent imaging agents.

Pyropheophorbide a (Pyro) is a near-infrared (NIR) fluorescent dye and photosensitizer with high quantum yield that makes the dye suitable for tumor treatment both as an imaging and therapy agent. We have designed and synthesized a series of a Pyro-based NIR probes, based on the conjugation of Pyro with lipids. The nature of our probes requires the use of a lipophilic carrier to deliver the probes to cancer cell membranes. To address this, we have utilized lipid-based nanoparticles (LNPs) consisting of PEGylated lipids, which form the nanoparticle shell, and a lipid core. To endow the LNPs with targeting properties, nitrilotriacetic acid (NTA) lipids were included in the composition that enables the non-covalent attachment of His-tag targeting proteins preserving their functional activity. We found that the nature of the core molecules influence the nanoparticle size, shelf-life and stability at physiological temperature. Two different Pyro-lipid conjugates were loaded either into the core or shell of the LNPs. The conjugates revealed differential ability to be accumulated in the cell membrane of the target cells with time. Thus, the modular organization of the core-shell LNPs allows facile adjustment of their composition with goal to fine tuning the nanoparticle properties for in vivo application

Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors

Choline kinase alpha (ChoKα) overexpression is associated with an aggressive tumor phenotype. ChoKα inhibitors induce apoptosis in tumors, however validation of their specificity is difficult in vivo. We report the use of optical imaging to assess ChoKα status in cells and in vivo using JAS239, a carbocyanine-based ChoKα inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoKα independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoKα from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic

415 Intraoperative Molecular Imaging of Gliomas using Indocyanine-Conjugated Choline Kinase Alpha Inhibitor

OBJECTIVES/GOALS: Distinguishing tumor tissue from normal brain parenchyma remains a major challenge during the resection of gliomas, leading to the persistence of tumor cells. This study aims to assess the choline kinase alpha-targeting fluorophore JAS239 as a novel fluorescent agent to intraoperatively visualize gliomas in an orthotopic murine model. METHODS/STUDY POPULATION: The human glioblastoma-derived U87 MG-Luc2 cell line will be intracranially implanted in nude mice and tumor growth will be assessed using bioluminescence imaging. After 14 days, the mice will be treated with either antiangiogenic therapy (10 mg/kg bevacizumab, twice/week) or saline (control). Tumor growth will be monitored until 21-28 days after initial implantation, at which point JAS239 (4.0 mg/kg, 90 min before sacrifice) and Evans Blue (4 ml/kg, 60 min before sacrifice) will be administered. The mice will be sacrificed, and their brains will be harvested and sectioned for near-infrared imaging. The brain sections will be processed for histopathologic analysis, allowing for the correlation of observed fluorescence with the distribution of tumor and comparison of signal-to-background ratios. RESULTS/ANTICIPATED RESULTS: JAS239 is an indocyanine-based choline mimetic (excitation 745 nm, emission 775 nm) that has been shown to cross the blood-tumor barrier (BTB) in rodent glioblastoma studies. PET imaging with choline-based radiotracers like 18F-choline has also been shown to delineate both contrast-enhancing tumor (CET) and non-contrast-enhancing tumor (NCET) regions, supporting the hypothesis that JAS239 will be able to visualize heterogeneous glioma tissue in our mouse model. Evans Blue is a passive dye in the visible light spectrum (excitation 620 nm, emission 680 nm) expected to only fluoresce in CET regions due to the disruption of the BTB. JAS239 is expected to fluoresce in both CET and NCET regions, which will be assessed by the fluorescence in mice treated with bevacizumab (expected to renormalize the BTB and model NCETs). DISCUSSION/SIGNIFICANCE: JAS239 may allow for real-time visualization of heterogeneous glioma tissue, which is important because there are no current intraoperative imaging agents for NCETs. Future research and clinical translation of this class of agents may allow surgeons to maximize the safe resection of gliomas, improving progression-free and overall survival rates

Identification of a Unique Inhibitor-Binding Site on Choline Kinase α

Choline kinase α (ChoKα) is an enzyme that is upregulated in many types of cancer and has been shown to be tumorigenic. As such, it makes a promising target for inhibiting tumor growth. Though there have been several inhibitors synthesized for ChoKα, not all of them demonstrate the same efficacy <i>in vivo</i>, though the reasons behind this difference in potency are not clear. One particular inhibitor, designated TCD-717, has recently completed phase I clinical trials. Cell culture and <i>in vitro</i> studies support the powerful inhibitory effect TCD-717 has on ChoKα, but an examination of the inhibitor’s interaction with the ChoKα enzyme has been missing prior to this work. Here we detail the 2.35 Å structure of ChoKα in complex with TCD-717. Examination of this structure in conjunction with kinetic assays reveals that TCD-717 does not bind directly in the choline pocket as do previously characterized ChoKα inhibitors, but rather in a proximal but novel location near the surface of the enzyme. The unique binding site identified for TCD-717 lends insight for the future design of more potent <i>in vivo</i> inhibitors for ChoKα

Implementation of two-dimensional L-COSY at 7 Tesla: an investigation of reproducibility in human brain.

PurposeTo evaluate the utility of two-dimensional (2D) Localized Correlated Spectroscopy (L-COSY) in metabolic profiling of the human brain at 7 Tesla (T).Materials and methodsThe 2D L-COSY sequence was implemented at 7 T and its reliability was assessed by test-retest studies of a metabolite phantom and a healthy volunteer. L-COSY data were acquired from the occipital lobe of healthy subjects (n = 6; all male; age, 30-72 years) to assess intersubject variability. Additionally, two subjects underwent scans from the parieto-occipital region, basal ganglia, frontal lobe or dorsolateral prefrontal cortex to test the versatility of L-COSY in studying differing anatomy. Integral/volume measurements of L-COSY spectra were used to estimate normalized metabolite-to-creatine concentrations.ResultsPhantom test-retest studies revealed coefficients of variation (CVs) of 3-20% for most metabolites. Human 2D L-COSY spectra permitted detection of several metabolite resonances from multiple locations and inter-subject variation studies demonstrated CVs of 4-26%. Cross-peaks from gamma-aminobutyric acid (GABA), isoleucine (Ile), lysine (Lys) and Ethanolamine (Eth) were quantified, which are not readily resolvable with conventional one-dimensional (1D) MR spectroscopy.Conclusion2D L-COSY at 7 T demonstrated improved sensitivity in detecting additional metabolites with reliability comparable to established techniques at lower fields, which may aid in the metabolic assessment of diseased states

Implementation of two-dimensional L-COSY at 7 Tesla: an investigation of reproducibility in human brain.

PurposeTo evaluate the utility of two-dimensional (2D) Localized Correlated Spectroscopy (L-COSY) in metabolic profiling of the human brain at 7 Tesla (T).Materials and methodsThe 2D L-COSY sequence was implemented at 7 T and its reliability was assessed by test-retest studies of a metabolite phantom and a healthy volunteer. L-COSY data were acquired from the occipital lobe of healthy subjects (n = 6; all male; age, 30-72 years) to assess intersubject variability. Additionally, two subjects underwent scans from the parieto-occipital region, basal ganglia, frontal lobe or dorsolateral prefrontal cortex to test the versatility of L-COSY in studying differing anatomy. Integral/volume measurements of L-COSY spectra were used to estimate normalized metabolite-to-creatine concentrations.ResultsPhantom test-retest studies revealed coefficients of variation (CVs) of 3-20% for most metabolites. Human 2D L-COSY spectra permitted detection of several metabolite resonances from multiple locations and inter-subject variation studies demonstrated CVs of 4-26%. Cross-peaks from gamma-aminobutyric acid (GABA), isoleucine (Ile), lysine (Lys) and Ethanolamine (Eth) were quantified, which are not readily resolvable with conventional one-dimensional (1D) MR spectroscopy.Conclusion2D L-COSY at 7 T demonstrated improved sensitivity in detecting additional metabolites with reliability comparable to established techniques at lower fields, which may aid in the metabolic assessment of diseased states

Synthesis and Evaluation of Cytosolic Phospholipase A₂ Activatable Fluorophores for Cancer Imaging

Activatable fluorophores selective to cytosolic phospholipase A₂ (cPLA₂) were synthesized and evaluated for their ability to image triple negative breast cancer cells. The activatable constructs were synthesized by esterification of a small molecule fluorophore with a fatty acid resulting in ablated fluorescence. Selectivity for cPLA₂ was generated through the choice of fluorophore and fatty acid. Esterification with arachidonic acid was sufficient to impart specificity to cPLA₂ when compared to esterification with palmitic acid. <i>In vitro</i> analysis of probes incorporated into phosphatidylcholine liposomes demonstrated that a nonselective phospholipase (sPLA₂ group IB) was able to hydrolyze both arachidonate and palmitate coupled fluorophores resulting in the generation of fluorescence. Of the four fluorophores tested, DDAO (7-hydroxy-9<i>H</i>-(1,3-dichloro-9,9-dimethylacridin-2-one)) was observed to perform optimally <i>in vitro</i> and was analyzed further in 4175-Luc+ cells, a metastatic triple negative human breast cancer cell line expressing high levels of cPLA₂. In contrast to the <i>in vitro</i> analysis, DDAO arachidonate was shown to activate selectively in 4175-Luc+ cells compared to the control DDAO palmitate as measured by fluorescence microscopy and quantitated with fluorescence spectroscopy. The addition of two agents known to activate cPLA₂ enhanced DDAO arachidonate fluorescence without inducing any change to DDAO palmitate. Inhibition of cPLA₂ resulted in reduced fluorescence of DDAO arachidonate but not DDAO palmitate. Together, we report the synthesis of a cPLA₂ selective activatable fluorophore capable of detecting cPLA₂ in triple negative breast cancer cells