(T0930-07-42) Tumor-Targeting Polymeric Micelles for Nose-to-Brain Delivery of Near-Infrared Imaging Agents for Optical Image-Guided Brain Surgery

Purpose: The purpose of this study was to design, develop, and optimize a tumor-targeting nanosized polymeric micelle system for intranasal (nose-to-brain) delivery of the near-infrared (NIR) imaging agent DiR. This innovative approach aims to bypass the blood-brain barrier, enabling localized and sustained retention of the imaging agent at the tumor site, minimizing systemic exposure and phototoxicity, and providing a non-invasive, tumor-specific fluorescence "on/off" activation for safer and more precise optical image-guided brain tumor surgery.

Methods: Polymeric micelles were prepared by conjugating the near-infrared fluorescent dye DiR with NHS-activated PEG-PLA copolymers across a range of DiR-to-polymer w/w ratios (1:10 to 1:400) to optimize dye loading. The mixture was stirred at 400 RPM, and a pH 8.5 buffer was added dropwise to trigger micelle self-assembly. The organic solvent was then evaporated under controlled conditions at 40°C. After micelle formation, the tumor-targeting peptide Cyclo(RGDfK) was conjugated to the micelles to enhance specificity toward tumor tissues expressing αvβ3 integrins. The resulting micelles were characterized for particle size, polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS). DiR loading content was quantified using UV-Visible spectroscopy at 775 nm, while peptide conjugation efficiency was developed by reverse-phase high-performance liquid chromatography (HPLC). The fluorescence "on/off" behavior of the micelles was evaluated using in vitro imaging systems such as IVIS and Fluobeam 800, to confirm that DiR fluorescence remained quenched until micelle disassembly in the tumor microenvironment. For in-vivo studies, micelles were administered intranasally to mice bearing brain tumors. Following imaging to visualize tumor-associated signals, the tumor-bearing mice were re-anesthetized, and the original surgical incision was reopened to perform the surgery followed by post-surgical imaging to assess the effectiveness of tumor removal.

Results: The micelles demonstrated uniform particle sizes ranging from 49 to 160 nm, with low polydispersity indices (PDI < 0.3), indicating a narrow size distribution, and exhibited stable zeta potentials between –15 and –19 mV, suggesting good colloidal stability. Through optimization of temperature and reaction parameters, the conjugation efficiency of the tumor-targeting peptide Cyclo(RGDfK) reached approximately 80%. The fluorescence behavior of the DiR-loaded micelles was highly dependent on dye packing density; at high DiR loading, fluorescence remained quenched ("off" state), but was rapidly activated ("on" state) upon micelle disassembly or tumor-specific binding. Maximum fluorescence activation was observed at a DiR-to-polymer molar ratio of 1:200, indicating optimal dye incorporation without self-quenching. In-vivo studies confirmed that intranasal administration of the micelles resulted in strong and sustained accumulation in brain tumors, with detectable fluorescence retained for up to 72 hours, followed by gradual clearance. During optical image-guided surgery, these micelles enabled precise visualization of tumor margins, facilitating accurate resection with minimal background fluorescence from surrounding healthy tissues.

Conclusion: In summary, we developed a tumor-targeted, fluorescence-switchable polymeric micelle system for intranasal, nose-to-brain delivery of NIR imaging agents. This strategy bypasses the blood–brain barrier without systemic injection, enabling prolonged tumor retention, minimal systemic toxicity, and precise image-guided brain tumor resection. The micelles showed uniform size (49–160 nm), low PDI ( < 0.3), stable zeta potential (–15 to –19 mV), and ~70% peptide conjugation efficiency. Fluorescence remained quenched at high DiR loading and switched "on" upon micelle disassembly or tumor binding. In vivo, micelles localized effectively to brain tumors for up to 48 hours, and fluorescence-guided surgery achieved about 94% signal reduction post-resection, indicating successful tumor removal. This non-invasive system offers rapid tumor targeting and high clinical potential. Future work will focus on detailed pharmacokinetics, and clinical translation.

References: 1. McKenzie M, Betts D, Suh A, Bui K, Tang R, Liang K, et al. Proof-of-Concept of Polymeric Sol-Gels in Multi-Drug Delivery and Intraoperative Image-Guided Surgery for Peritoneal Ovarian Cancer. Pharm Res. 2016;33:2298–306.
2. Cho H, Indig GL, Weichert J, Shin H-C, Kwon GS. In vivo cancer imaging by poly (ethylene glycol)-b-poly (ɛ-caprolactone) micelles containing a near-infrared probe. Nanomedicine: Nanotechnology, Biology and Medicine. 2012;8:228–36.
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Acknowledgements:

Funding: Research reported in this work was sponsored by the NIGMS (National Institute of General Medical Sciences), NIH (National Institutes of Health) under award number R16GM154661.


Figure-1 Characterization of Polymeric Micelles loaded with DiR (1:10) by a) Cryo TEM, b) Particle Size c) Zetapotential and its Fluorescence signals d) Fluobeam & e) IVIS for polymeric micelles carrying DiR at various occupancies


Figure 2. In vivo imaging of a) fluorescence (FLU) and bioluminescent (BLU) signals of mice Regions of interest (ROIs) are highlighted in red boxes, indicating strong localized signal at the tumor site. b) Before and after surgical intervention signals in two mice bearing tumors with significant reduction in signal is observed post-surgery, suggesting successful removal of tumor tissue


Figure-3. Overall Graphical Abstract