(E-180) CT Imaging of Gold-Loaded Polymersomes to Detect Glioblastoma

Glioblastoma multiforme (GBM) is one of the most common and deadly malignant brain and central nervous system (CNS) tumors, with a survival rate of less than 5% after five years1. Imaging plays an important role in diagnosis, monitoring, and potential theranostic treatments for GBM. While magnetic resonance imaging (MRI) is the most prominent imaging technique used to detect GBM, its limitations in image resolution hinder the early detection of small tumors, often leading to delayed diagnosis at advanced stages of the disease. To address this challenge, we utilize computed tomography (CT), a cheaper and more widely available imaging technique compared to MRI. CT imaging uses X-rays to produce detailed images with enhanced spatial resolution for detecting tumor margins. However, CT provides limited contrast resolution compared to MRI and requires long-term exposure to radioactive contrast agents to produce high-quality images, and high doses for clinical usage are not approved by the FDA2. To overcome these limitations, we developed a biocompatible alternative to standard contrast agents by encapsulating gold nanoparticles (AuNPs) in polymersomes. Polymersomes are self-assembled polymeric nanovesicles capable of transporting various cargo and permeating the blood-brain barrier (BBB) while protecting the cargo from premature elimination before reaching the target site. Due to their high x-ray attenuation, simple surface chemistry, and biocompatibility, these vesicles are ideal for CT use. Our study aims to develop AuNP-loaded polymersomes (AuPs) and determine their ability to detect GBM through clinical CT imaging. 

Polymersomes were synthesized using a 50:50 ratio of polyethylene glycol-b-polylactic acid (PEG-PLA) and maleimide-functionalized PEG-PLA (MAL-PEG-PLA). Additionally, TAT peptide was incorporated to facilitate the cellular uptake of AuPs. Polymersomes were characterized using dynamic light scattering (DLS) and freeze-dried before sterile AuNPs were loaded into polymersomes using a dry heat bath method. Loading efficiency for various concentrations of AuNPs was determined and transmission electron microscopy (TEM) images were taken of both loaded and unloaded polymersomes. To assess the imaging capabilities, clinical CT images were acquired using various concentrations of AuNPs and AuPs. Medical imaging software was used to quantify x-ray attenuation values for each treatment. For in vivo assessment, an orthotopic GBM model was developed using U87-MG-Luciferase cells intracranially injected into mus musculus NCr-Foxn1nu female mice to form GBM tumor models, and mice were imaged 2 weeks post-injection using IVIS imaging to confirm tumor establishment. 

Polymersomes synthesized using a 50:50 ratio of PEG-PLA:MAL-PEG-PLA and labeled with TAT were positively charged, confirming successful TAT binding, with an average diameter and polydispersity index of 114.77 ± 0.94 nm and 0.075 ± 0.013, respectively (Fig 1A). TEM images confirmed the spherical shape of polymersomes, as well as successful encapsulation of AuNPs in polymersomes. AuNPs were loaded into polymersomes at an encapsulation efficiency of 99.7 ± 0.6%. CT images of AuNPs revealed increasing intensity directly relating to increasing concentration of AuNPs, which is promising towards the use of AuNPs to obtain higher intensity images of GBM using CT (Fig 1B). Medical imaging software confirmed increasing concentrations of AuNPs increases x-ray attenuation values, and AuPs demonstrated an increase in x-ray attenuation at certain concentrations. MTS assays showed greater than 80% viability of U87-MG cells across all concentrations of AuPs, indicating the polymersomes are cytocompatible. Fluorescent images indicated successful internalization of AuPs in U87-MG cells (Fig 1C). Lastly, mice that were intracranially injected with U87-MG-Luciferase cells were positive for luciferase one-week post-injection, verified via IVIS imaging, indicating successful tumor establishment (Fig 1D) for future in vivo evaluation of the AuPs. 

These results demonstrate that TAT-modified PEG-PLA:MAL-PEG-PLA polymersomes are capable of encapsulating AuNPs with desired size and surface chemistry for cellular uptake. Increasing concentrations of AuNPs correlated with increased x-ray attenuation values, and therefore resulted in enhanced CT imaging. Moreover, AuPs are biocompatible and readily internalized into GBM cells. Altogether, these results demonstrate the promising potential of AuNP-loaded polymersomes for enhanced CT imaging and warrant further evaluation of their in vivo performance for tumor detection and contrast enhancement of GBM tumors. 

This work was supported in part by the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. This work was supported in part by Clemson’s Creative Inquiry Program and Clemson University’s Core Incentivized Access (CU-CIA) initiative.

1. Ostrom et. all, Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012

2.  Hess, Exploring the Brain: How are Brain Images made with CT?