Polynucleotide HPTTM-Based Hydrogels Exhibit Scavenging Activity Against Reactive Oxygen Species

Abstract

This study investigates the scavenger activity of Polynucleotide High Purification Technology (PN HPT^TM), alone or in combination with hyaluronic acid (PN HPT^TM + HA) against oxidative stress induced by hydrogen peroxide (H₂O₂). Since oxidative stress is implicated in numerous pathological conditions, identifying effective antioxidants is crucial for therapeutic development. We employed a cell-free fluorometric assay based on Calcein-AM, a fluorescence probe whose signal increases proportionally to the generation of reactive oxygen species (ROS), to evaluate the ability to neutralize ROS under varying oxidative stress conditions and determine the dose- and time-dependent effects of these compounds. PN HPT^TM, HA, and PN HPT^TM + HA were tested at various concentrations over multiple time points. Our results demonstrated that all tested treatments significantly lowered ROS levels compared to the untreated control. Notably, the PN HPT^TM -based compounds exhibited robust scavenging activity, with PN HPT^TM + HA displaying the strongest and most consistent ROS-neutralizing effect across all concentrations and time points. This enhanced performance suggests a synergistic interaction between PN HPT^TM and HA, potentially due to complementary mechanisms of free radical scavenging and structural stabilization. These findings highlight the potential of PN HPT^TM and PN HPT^TM + HA as effective antioxidative agents, offering potential for therapeutic applications where oxidative stress is central, including wound healing and tissue regeneration.

Keywords: antioxidant therapy; hyaluronic acid; oxidative stress; polynucleotides; scavenger activity.

Figure A1

Oxygen-radical absorbance capacity (ORAC) kinetics of Polynucleotide High Purification Technology (PN HPT™), hyaluronic acid (HA) and their combination (PN HPT™ + HA) compared with the radical control. In a cell-free phosphate buffer (75 mM, pH 7.4, 37 °C) fluorescein (80 nM) was challenged with peroxyl radicals generated by AAPH (153 mM) and fluorescence was recorded at 485/535 nm every minute for 30 min. Antioxidants were supplied at the maximal test concentration used throughout the study immediately before radical generation. Curves represent the mean ± SD of three independent experiments; higher fluorescence indicates more effective scavenging of peroxyl radicals. The PN HPT™ + HA group became statistically higher than both the HA and PN HPT™ groups starting at minute 16.

Figure 1

Dose–response relationship between hydrogen peroxide (H₂O₂)-derived reactive-oxygen-species (ROS) and their modulation by Polynucleotide High Purification Technology (PN HPT™), hyaluronic acid (HA), and the combined formulation (PN HPT™ + HA). Increasing H₂O₂ concentrations—0, 150, 300, 600, and 1000 µM—were added to the samples for 60 min, either in the absence (Ctr) or in the presence of each compound supplied at its highest test concentration. ROS accumulation was quantified as fluorescence and expressed in arbitrary units (AU); bars represent mean ± SD of three independent experiments. See Appendix A for statistical analysis.

Figure 2

Scavenger activity of Polynucleotide High Purification Technology (PN HPT™), hyaluronic acid (HA), and their combination (PN HPT™ + HA) against H₂O₂-induced ROS, monitored as calcein fluorescence at 10, 30, 60, 90, and 120 min. Panel (A) shows PN HPT™ at 0, 300, 600, and 900 μg mL⁻¹ and at its maximal commercial concentration (“Max”); panels (B,C) present PN HPT™ + HA and HA, respectively, under the same concentration regimen; in each case 5 mM N-acetyl-cysteine (NAC; green) serves as the positive antioxidant control. Panel (D) compares the three materials at their Max concentrations with a NAC titration (1.25, 2.5, and 5 mM) and the oxidant control (0 μg mL⁻¹). Across doses and time points, both PN HPT™ and PN HPT™ + HA reduced the H₂O₂-driven fluorescence versus the oxidant control; the mixture tended to yield lower signals than PN HPT™ alone at matched doses, although at the highest concentration the two curves overlapped within the SD. Data are mean ± SD (n = 3). See Appendix A for the statistical analysis.

Figure 3

Scavenger activity of Control, HA, PN HPT^TM, and PN HPT^TM + HA products in response to H₂O₂-induced oxidative stress. Fluorescence intensity (A.U.) was measured at 10, 60, and 120 min for the vehicle (control), PN HPT^TM, HA, and PN HPT^TM + HA at maximum concentration. PN HPT^TM and PN HPT^TM + HA significantly reduced fluorescence compared to the vehicle at 60 min (p < 0.001, “a”), and at 120 min (p < 0.001, “b”). Data are expressed as mean ± SD.