44 Methylselenocysteine-Hyaluronic Acid Conjugates to Prevent Therapy Resistance in Head and Neck Cancer

Faculty Mentor: Abigail Pulsipher (Surgery, University of Utah)

Background

Head and neck squamous cell carcinoma (HNSCC), a malignancy of head and neck cancers, remains an extensive issue, accounting for over 890,000 new cases and 450,000 deaths worldwide each year.^[1] While early-stage HNSCC is often curable, advanced-stage disease, which accounts for two-thirds of cases, has a poor long-term survival rate due to treatment failure, recurrence, and therapy resistance.^{[2][3] [4] [5]}This is a result of tumors’ ability to adapt to oxidative stress and evade cell death through redox-regulating and pro-survival mechanisms, leading to treatment resistance.^[6][7]

Methylselenocysteine (MSC), an organic selenium compound, has shown significant potential in reinstating sensitivity to chemotherapy by generating reactive oxygen species (ROS) and impairing the antioxidant mechanisms of cancer cells.^[8][9] However, clinical translation has been limited by the high doses required to achieve therapeutically effective concentrations, resulting in systemic toxicity.^[10][11]

To overcome this barrier, we developed a selenium functionalized hyaluronic acid (sHASe 011), by covalently conjugating MSC to hyaluronic acid (HA), a natural glycosaminoglycan found in the body with strong affinity for CD44, a highly prevalent surface receptor on HNSCC tumor cells.^[12][13] This HA-MSC conjugate is designed to directly target the tumor microenvironment only to enhance intracellular delivery of MSC, maximize therapeutic efficacy, and minimize systemic side effects.

Our approach offers a solution to the persistent problem of therapy resistance in HNSCC by combining targeted delivery with redox-based mechanisms. This strategy holds strong translational potential and the ability to significantly improve outcomes for patients with aggressive, treatment-refractory cancers.

Methods

sHASe 011 was synthesized by covalently conjugating MSC to low molecular weight HA using carbodiimide-mediated coupling with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (Figure 1a). The reaction proceeded at room temperature, stirring for 4 hours. The compound-containing solution was purified by 48-hour dialysis against deionized water using 2 kDa molecular weight cut-off membranes with frequent buffer changes, followed by lyophilization.^[14] Successful conjugation and structural integrity were confirmed by proton nuclear magnetic resonance (¹H NMR), selenium (Se) content was quantified by inductively coupled plasma mass spectrometry (ICP-MS), and molecular weight was characterized using gel permeation chromatography (GPC). Cytocompatibility of sHASe 011 was assessed in human umbilical vein endothelial cells (HUVECs) and FaDu pharyngeal squamous carcinoma cells using the Cell Counting Kit-8 (CCK- 8) assay after 48 hours of incubation. FaDu cells were exposed to sHASe 011 at concentrations ranging from 0 to 7 μM, while HUVECs experienced concentrations ranging from 0 to 16 μM. To evaluate ROS sensitization, FaDu cells were incubated with sHASe 011 for 48 hours, followed by a 6-hour ROS induction with tert-butyl hydroperoxide (TBHP), and cell viability was evaluated via CCK-8 assay. All experiments were performed in triplicate, and the data were analyzed using one-way analysis of variance (ANOVA) with Dunnett’s post hoc test to determine statistical significance.

Results, Discussion, and Conclusion

¹H NMR confirmed successful conjugation of MSC to HA, as evidenced by indicated MSC peaks at 2.83 and 2.69 ppm (Figure 1b). Percent functionalization was quantified by integrating the methylene peak of MSC at 2.83 ppm relative to the N-acetyl peak of HA at 2.00 ppm, yielding a conjugation efficiency of 28.5%. This was calculated using the formula: %Functionalization = ((I2.83/2)(I2.0/3))×100; where I2.83 and I2.0 are the integrals of the peaks at 2.83 and 2.0, respectively. GPC analysis further validated the formation of the sHASe 011 conjugate, showing an increase in molecular weight compared to HA. The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) of sHASe 011 were determined to be 5.14 ± 0.27 kDa, 7.25 ± 0.18 kDa, and 1.41 ± 0.32, respectively, compared to native HA (Mn: 3.28 ± 0.26 kDa; Mw: 4.20 ± 0.20 kDa; PDI: 1.28 ± 0.33) (Figure 1c). ICP-MS confirmed selenium conjugation, with a measured Se content of 5.941 µg/g (Figure 1d).

Cell viability assays demonstrated that sHASe 011 exhibited excellent cytocompatibility with HUVECs for all concentrations tested (0–16 µM), indicating cytocompatibility in healthy cells (Figure 2a). FaDu cells treated with sHASe 011 alone (0–7 µM) showed no significant reduction in viability, suggesting limited cytotoxicity (Figure 2b). However, under oxidative stress conditions, FaDu cells exhibited a statistically significant reduction in viability at 0.28 µM (p<0.0045) and 1.4 µM (p<0.0004) of sHASe 011, indicating ROS-sensitizing effects (Figure 2c). In conclusion, sHASe 011 was successfully synthesized and characterized, with positive evidence of MSC conjugation, appropriate molecular weight distribution, and selenium content. The compound demonstrated selective ROS-sensitization in tumor cells without affecting healthy cells, indicating its potential as a redox-modulating, tumor-targeted therapeutic. These findings support the continued development of sHASe 011 as a novel design to overcome oxidative stress resistance in head and neck cancers.