Poster 160 - Verteporfin-Loaded Fucoidan-Coated Nanoparticles for targeted Glioblastoma therapy

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Lucia Wang (she/her/hers)

High School Student Intern of University of Maryland
Montgomery Blair High School
Potomac, Maryland, United States

Co-Author(s)

James G. Shamul, MS

Postdoctoral Fellow
University of Maryland, College Park
Lanham, Maryland, United States

Primary Investigator(s)

Xiaoming He, PhD (he/him/his)

Professor
University of Maryland, College Park
College Park, Maryland, United States

Introduction: Glioblastoma (GBM) represents one of the most aggressive and deadly forms of brain cancer. Despite surgery, radiation, and temozolomide chemotherapy, only about 5% of patients survive five years post-diagnosis (1). GBM tumors are highly heterogeneous and resistant to standard therapies, contributing to the dismal survival (2). Therefore, there is an urgent need for novel therapeutic approaches that can more effectively target and eradicate GBM cells.

Nanotechnology offers significant advantages in sustaining small molecules within the bloodstream and improving biodistribution to target tissues (3). Biodegradable materials can be used to synthesize nanoparticles loaded with hydrophobic drugs that have low solubility in aqueous, physiological fluids, for treating cancer and other diseases.

Verteporfin (VP), a drug clinically used in photodynamic therapy for age-related macular degeneration, has shown chemotherapeutic potential for GBM. It is able to suppress the activity of Yes-associated protein (YAP), a transcriptional coactivator in the Hippo pathway with oncogenic properties (4). Fucoidan, a sulfated polysaccharide, naturally targets P-selectin overexpressed in cancer cells, including GBM cells, and activated blood vessels in tumors (5). Fucoidan-coated nanoparticles have shown enhanced tumor targeting and cancer cell uptake (6). Therefore, we propose to encapsulate VP in nanoparticles coated with fucoidan for targeting both GBM cells and activated blood vessels, as shown in Figure 1a, to investigate its improved performance and potential as a therapy for GBM compared to passive-targeting, VP-loaded nanoparticles. This work represents the first dual-targeting, VP-loaded nanoparticle for GBM therapy.

Materials and

Methods: To synthesize nanoparticles, 20 mg PLGA, 10 mg DPPC, and 3 mg VP were dissolved in 2 mL dichloromethane (DCM) and emulsified with 20 mg fucoidan or 5 mg dextran in 8 mL 2% polyvinyl alcohol (PVA) using a sonifier at 18% amplitude for 1 min. The nanoparticles were washed twice in deionized water (DI) and lyophilized for 12-24 h.

Nanoparticle size and zeta potential were measured using a Malvern Zetasizer Nano ZS with nanoparticles resuspended in DI water. Loading efficiency and capacity were determined by converting the fluorescence intensity of VP in the nanoparticle sample dissolved in dimethyl sulfoxide (DMSO) into a concentration using a free VP standard curve.

Brain microvascular endothelial cells (BMECs) were seeded on collagen-coated coverslips and treated with 50 ng/mL tumor necrosis factor-alpha (TNF-α) for 24 h. The activated cells were treated with nanoparticles for 1 h and imaged with confocal microscopy to measure binding. ImageJ was used to quantify VP signal intensity.

Patient-derived GBM1A cells were originally derived and characterized as described by Galli et al (7). The cells were cultured in a serum-free, defined medium as performed previously (8). Cell viability was measured using cell counting kit-8 (CCK-8) after treating seeded GBM1A cells in a Matrigel-coated plate with VP-loaded nanoparticles, blank nanoparticles, and free VP for 24 h.

Results, Conclusions, and Discussions: As shown in Figure 1b, the VP-loaded nanoparticles without any further surface modification (VP@NP), the VP-loaded, fucoidan-coated nanoparticles (VP@NP-F) and VP-loaded, dextran-coated nanoparticles (VP@NP-D) have similar and narrow size distribution, although the VP@NP-F and VP@NP-D are slightly larger than the VP@NP on average. The quantitative characterization data of all the nanoparticle formulations is shown in Figure 1c. VP@NP-F and VP@NP-D are 157.6 ± 2.4 nm and 148.4 ± 6.3 nm, respectively. The zeta potentials of VP@NP-F and VP@NP-D are -38.6 ± 0.4 mV and -38.3 ± 1.4 mV, respectively. Since dextran is similarly-charged as fucoidan, with a different binding affinity to P-selectin, VP@NP-D is compared to VP@NP-F for in vitro studies. The uncoated VP-loaded nanoparticles (VP@NP) are smaller in size than the coated nanoparticles at 129.5 ± 6.7 nm and more positive in surface charge at -28.1 ± 0.6 mV.

As visualized in the images in Figure 1d,e for 1 h treatment, the affinity of the nanoparticle fucoidan-coating for P-selectin is demonstrated, in which the nanoparticles bind to BMECs activated with TNF-α after 2 h of cooling to minimize metabolic activity. The fluorescence intensity quantification of the bound VP is shown in Figure 1f,g for 1 h and 3 h treatment incubation times, respectively. The VP@NP-D treatment is ~7-fold less in VP intensity after activation for the 1 h binding study compared to VP@NP-F. After 3 h binding, VP@NP-D treatment has a greater VP signal post-activation than VP@NP-F.

Finally, after treatment with various VP formulations on GBM1A cells for 24 h, the VP@NP-F and VP@NP-D treatments have similar and the greatest killing effects of all the treatments, as shown in Figure 1h. However, VP@NP is much less effective in GBM1A cell killing than VP@NP-F and VP@NP-D. This may suggest that the GBM cell, P-selectin targeting with fucoidan and dextran is critical for effective GBM cell killing compared to treatments without any targeting effect. This data highlights the potential of P-selectin targeting in a nanoformulation encapsulated with VP and demonstrates the promise of fucoidan and surprisingly dextran for targeting activated BMECs and GBM cells in GBM therapy.

Acknowledgements (Optional): This work was supported by the National Institutes of Health grant R01CA243023.

References (Optional): 1. Seker-Polat, F., Pinarbasi Degirmenci, N., Solaroglu, I. & Bagci-Onder, T. Tumor Cell Infiltration into the Brain in Glioblastoma: From Mechanisms to Clinical Perspectives. Cancers (Basel) 14 (2022).
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6. DuRoss, A. N., Landry, M. R., Thomas, C. R., Jr., Neufeld, M. J. & Sun, C. Fucoidan-coated nanoparticles target radiation-induced P-selectin to enhance chemoradiotherapy in murine colorectal cancer. Cancer Lett 500, 208-219 (2021).
7. Galli, R. et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64, 7011-7021 (2004).
8. Shamul, J. G. et al. Verteporfin-Loaded Anisotropic Poly(Beta-Amino Ester)-Based Micelles Demonstrate Brain Cancer-Selective Cytotoxicity and Enhanced Pharmacokinetics. Int J Nanomedicine 14, 10047-10060 (2019).