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Background: Position of gadolinium atom(s) plays a key role in contrast enhancement of gadolinium-based contrast agents. To gain a better understanding of effects of distance of gadolinium in relation to the nanoconjugate platform, we designed and synthesized single- and multi-arm ("star") gadolinium conjugates equipped with antibody and peptides for targeting. The contrast agents were studied for their tumor imaging performance in a glioma mouse model., Materials and Methods: Antibody- and peptide-targeted nano contrast agents (NCAs) were synthesized using polymalic acid platforms of different sizes. Gadolinium-DOTA and intermediates were attached as amides and targeting agents such as antibodies and peptides as thioethers. For in vivo experiments, we used human U87MG xenografts as glioma models. Magnetic resonance imaging (MRI) was performed on a Bruker BioSpec 94/20USR 9.4 T small-animal scanner. Delivery of contrast agents across the blood-brain barrier was studied by fluorescent microscopy., Results: All contrast agents accumulated into tumor and showed composition-dependent imaging performance. Peptide-targeted mini-NCAs had hydrodynamic diameters in the range 5.2-9.4 nm and antibody-targeted NCAs had diameters in the range 15.8-20.5 nm. Zeta potentials were in the range of -5.4--8.2 mV and -4.6--8.8 mV, respectively. NCAs showed superior relaxivities compared to MultiHance at 9.4 T. The signal enhancement indicated maximum accumulation in tumor 30-60 minutes after intravenous injection of the mouse tail vein. Only targeted NCAs were retained in tumor for up to 3 hours and displayed contrast enhancement., Conclusion: The novel targeted NCAs with star-PEG features displayed improved relaxivity and greater contrast compared with commercial MultiHance contrast agent. The enhancement by mini-NCAs showed clearance of tumor contrast after 3 hours providing a suitable time window for tumor diagnosis in clinics. The technology provides a great tool with the promise of differential MRI diagnosis of brain tumors., Competing Interests: KLB, JYL and EH are shareholders of Arrogene, Inc. Keith L Black reports grants from NIH, during the conduct of the study. The authors report no other conflicts of interest in this work., (© 2020 Patil et al.)

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., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4β1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma. Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth., (©2019 American Association for Cancer Research.)