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Polynucleotides HPTTM -based compounds exhibit Scavenging Activity Against Reactive Oxygen Species

A peer-reviewed version of this preprint was published in:
Antioxidants 2025, 14(9), 1089. https://doi.org/10.3390/antiox14091089

Submitted:

27 May 2025

Posted:

28 May 2025

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Abstract
This study investigates the scavenger activity of Polynucleotides High Purification Technology (PN-HPTTM) and hyaluronic acid (HA) products and their combination (PN-HPTTM +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 using Calcein-AM to evaluate the dose- and time-dependent effects of these compounds in neutralizing reactive oxygen species (ROS). PN-HPTTM, HA, and PN-HPTTM +HA products 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-HPTTM -based compound exhibited robust scavenging activity, while the PN-HPTTM +HA based compound displayed the strongest and most consistent ROS-neutralizing effect across all concentrations and time points. This enhanced performance suggests a synergistic interaction between PN-HPTTM and HA, potentially due to complementary mechanisms of free radical scavenging and structural stabilization. These findings highlight the potential of PN-HPTTM and PN-HPTTM +HA based compounds as effective antioxidative agents, offering potential for therapeutic applications where oxidative stress is central, including wound healing and tissue regeneration.
Keywords: 
polynucleotides
;  
hyaluronic acid
;  
oxidative stress
;  
scavenger activity
;  
antioxidant therapy
Subject: 
Biology and Life Sciences  -   Other

1. Introduction

Reactive oxygen species (ROS) are a diverse family of oxidants derived from molecular oxygen during cell processes such as respiration and include both radical species like superoxide (O₂·⁻) and hydroxyl radicals (·OH), as well as non-radical molecules such as hydrogen peroxide (H₂O₂) [1]. Under normal physiological conditions, ROS are necessary components to various cellular processes, such as signaling and metabolic pathways, and the maintenance of redox homeostasis [1,2]. These physiological levels of ROS contribute to what is known as oxidative eustress, which is essential for regular cell function by activating transcription factors like nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) cascades [3]. However, if the production of ROS exceeds the capacity of cellular antioxidant defenses, the resulting imbalance may lead to oxidative stress which causes cell and tissue damage [4].
One significant target of ROS during oxidative stress is the extracellular matrix (ECM). The ECM is a complex network of structural proteins such as collagens and elastin, glycoproteins like fibronectin, and proteoglycans, including glycosaminoglycans like hyaluronic acid (HA) [5]. Beyond providing structural support to tissues, the ECM regulates crucial processes, including cell adhesion, migration, proliferation, and differentiation [6,7]. Chemical modifications to ECM components caused by ROS can severely impact tissue integrity and repair [8,9]. For example, oxidative damage to collagen fibers compromises their structural stability, making them more susceptible to enzymatic degradation [10]. Similarly, alterations to HA can result in increased fragmentation and in the loss of its viscoelastic and hydrating properties [11]. These changes disrupt the ECM’s ability to mediate cell signaling and bind growth factors, further impairing tissue regeneration and repair [12].
ECM degradation is particularly pronounced in chronic inflammatory conditions such as periodontitis, aging, and impaired wound healing [13]. Excessive ROS production destabilizes the delicate balance between ECM synthesis and turnover, initiating destructive processes [14]. For instance, in the gingival tissues affected by periodontitis, ROS-induced modifications to proteoglycans alter their core proteins and glycosaminoglycan chains, impairing their ability to regulate tissue homeostasis [15].
Addressing ROS-induced ECM damage is crucial, given the ECM’s pivotal role in maintaining tissue function and facilitating repair. Numerous research efforts are actively focused on this challenge [16,17,18,19]. However, effective solutions must go beyond neutralizing ROS; they must also provide specific biochemical and structural cues to restore tissue integrity. To this effect, interventions capable of reestablishing a regenerative microenvironment under oxidative stress conditions are needed [20,21,22].
Biomaterial scaffolds are particularly well-suited for this purpose, as they are a commonly used strategy to promote tissue repair and regeneration [23,24]. In particular, hydrogels have attracted attention as promising tools in tissue engineering [25,26,27,28]. Hydrogels are versatile, three-dimensional networks capable of mimicking the natural structure and function of the ECM [29]. They are highly biocompatible and possess unique physicochemical properties, including high water content and porosity, making them ideal scaffolds for supporting cell adhesion and viability [30,31,32,33,34]. By providing a biomimetic microenvironment, hydrogels facilitate cellular processes that are essential for tissue repair and regeneration [35].
Polynucleotides HPTTM and hyaluronic acid (HA) have shown significant potential as components of hydrogel scaffolds due to their combined biochemical and structural contributions. PN-HPTTM, derived from DNA fragments, support cell vitality by providing a stable and physiological microenvironment [36,37]. HA, a major glycosaminoglycan in the ECM, is known for its viscoelasticity, hydrating properties, and useful scaffold characteristics to modulate tissue repair [38,39,40,41]. More recently, products with PN-HPTTM and with PN-HPTTM combined with HA have been proposed for improved tissue conditions and have demonstrated considerable clinical potential [42,43,44,45,46,47,48,49,50,51,52].
Despite these advancements, the potential of PN-HPTTM and PN-HPTTM +HA hydrogels for mitigating oxidative stress has not been explored. We hypothesize that these hydrogels can effectively reduce ROS levels and thereby help preserve ECM integrity under oxidative stress conditions. Therefore, in this brief report, to investigate this hypothesis, we utilized a cell free in vitro model of H₂O₂-induced oxidative stress to assess the direct ROS-scavenging capabilities of these hydrogels.

2. Results and Discussion

The purpose of our investigation was to assess whether Polynucleotides High Purification Technology (PN- HPTTM) could hamper ROS under challenging conditions that mimic the oxidative stress associated with harmful tissue and organ conditions [53]. To this purpose, we relied on a cell-free model we previously developed and chacterized to evaluate H₂O₂-induced ROS [54]. This approach allowed us to focus on direct ROS-scavenging activity, minimizing confounding variables such as cellular metabolism or signaling.
Figure 1 illustrates fluorescence levels measured using the Calcein-AM probe for PN-HPTTM (Figure 1A), HA (Figure 1B), and PN- HPTTM +HA (Figure 1C) products at different concentrations of these compounds (300, 600, and 900 μg/mL, including the maximum concentration) over time (10, 30, 60, 90, and 120 minutes after H₂O₂ addition).
As visible in Figure 1, the rising intensity of Calcein-AM fluorescence in the control group (0 μg/mL, red line) indicated that ROS levels progressively increased over time after addition of H₂O₂. PN-HPTTM products exhibited a strong, dose-dependent scavenging activity, as evidenced by significantly reduced fluorescence at concentrations ≥900 μg/mL (Figure 1A). At the highest concentration, PN-HPTTM products significantly lowered fluorescence as early as 30 minutes after H₂O₂ exposure. Lower concentrations produced statistically significant reductions later, around 60–120 minutes. These observations underscore the robust antioxidant properties of PN-HPTTM compounds and suggest that sufficient PN-HPTTM concentration can rapidly and effectively neutralize free radicals. This aligns with reports suggesting that nucleotides or nucleotide-containing fragments can act as scavengers for multiple ROS species, possibly by directly donating electrons or forming stable complexes that inhibit radical propagation [55].
Hyaluronic acid (HA) alone based-products also reduced fluorescence intensity compared to the control group, though their effects were less pronounced than those of PN-HPTTM or PN-HPTTM +HA (Figure 1B). HA’s scavenging activity was not strongly concentration-dependent, with statistically significant reductions observed starting at 90 minutes and only for the concentration of 600 μg/mL. Albeit limitedly, HA reduced ROS levels; however, its scavenging activity remained lower than that of PN-HPTTM products at any concentration.
The PN-HPTTM +HA combination demonstrated the most pronounced reduction in fluorescence intensity across all time points (Figure 1C). At 60-120 minutes, PN- HPTTM +HA significantly reduced ROS levels compared to the control, even at 600 μg/mL. This enhanced activity may result from complementary mechanisms of PN- HPTTM and HA; we hypothesize that while specific chemical groups in Polynucleotides HPTTM (e.g., nitrogenous bases) can directly interact with and neutralize free radicals, HA’s large, charged glycosaminoglycan structure might provide multiple additional reactive sites for binding or quenching ROS. However, the peculiar behavior of PN- HPTTM +HA compounds could be also centered on HA’s high water-binding capacity, which could create a hydrophilic microenvironment that dilutes ROS or slows their diffusion, thus reducing their local concentration. Collectively, these properties suggest a synergistic interaction in which HA’s structural and hydrating functions support and amplify PN- HPTTM’s direct radical-scavenging potential.
Figure 2 illustrates fluorescence levels at the highest compound concentrations at 10, 60, and 120 minutes post-H₂O₂ exposure, highlighting the superior scavenging efficacy of PN-HPTTM and PN- HPTTM + HA products over HA alone based products.
The significant reductions in fluorescence intensity observed for PN- HPTTM and PN- HPTTM +HA products reflect their capacity to neutralize reactive oxygen species. The superior performance of the PN- HPTTM +HA combination across different concentrations suggests a synergistic effect, potentially arising from the complementary mechanisms of PN- HPTTM and HA. Taken together, these combined properties of PN-HPTTM and HA produce a more potent antioxidant effect than either component alone.
The implications of these findings are significant, given the central role of oxidative stress in the pathogenesis of numerous conditions, including aging [4,56,57,58] , periodontitis [59,60], and osteoarthrosis [61,62,63,64,65]. This study demonstrates antioxidant activity of PN-HPTTM and PN-HPTTM + HA products, positioning them as promising candidates for therapeutic applications aimed at locally reducing oxidative damage. Their robust activity against ROS could also partially explain the favourable clinical outcomes observed with PN-HPTTM -containing products across diverse clinical contexts, including dermatology, wound healing, gynecology, osteoarthrosis, dentistry and aesthetic medicine [45,46,47,48,49,50,51,52,66,67,68,69].
Nevertheless, our approach has limitations. While the cell-free system effectively isolates scavenging activity, it does not recapitulate the complexity of living tissues, where factors like enzymatic activity, local pH, and cellular signaling may influence ROS levels. Further in vitro and in vivo experiments are needed to clarify how PN- HPTTM and PN- HPTTM +HA functions within the physiological milieu, how quickly it is metabolized or replaced, and whether its protective effects extend to diverse cell types and tissues. Investigating the molecular details of how PN- HPTTM and HA interact—both with each other and with endogenous antioxidant systems—will yield deeper insights into optimizing their use against oxidative stress.

3. Conclusions

This study demonstrates the significant antioxidant properties of Polynucleotides High Purification Technology (PN- HPTTM), Hyaluronic Acid (HA), and their combination (PN- HPTTM +HA) in a cell-free model of oxidative stress induced by hydrogen peroxide. The findings indicate that PN- HPTTM and PN- HPTTM +HA effectively reduce ROS activity in a dose- and time-dependent manner, with PN- HPTTM +HA showing the most pronounced scavenger effect.
The synergistic activity observed in the PN- HPTTM +HA combination underscores its potential as a powerful therapeutic tool for mitigating oxidative damage. These results suggest promising applications in medical and aesthetic fields, particularly in contexts such as tissue repair, wound healing, osteoarthrosis and anti-aging therapies.
Further research is needed to validate these findings in more complex biological systems and to explore the mechanisms underlying their antioxidant effects. The insights provided by this study form a solid foundation for advancing the development of PN- HPTTM and PN- HPTTM +HA as innovative solutions to oxidative stress-related challenges.

4. Materials and Methods

4.1. Polynucleotides High Purification Technology (PN- HPTTM)

Polynucleotides High Purification Technology (PN- HPTTM) used in this study were obtained from Mastelli S.r.l. (Sanremo, Italy). PN- HPTTM is a compound containing DNA fragments of varying chain lengths, extracted from the gonads of salmon trout (Oncorhynchus mykiss) through an original high-purification technology (HPT™). This technology provides high-quality DNA while minimizing immunological side effects [34]. The products employed in the present study are commercially available Class III medical devices: PN- HPTTM (20 mg/mL), hyaluronic acid (HA) (20 mg/mL), and a combination of PN- HPTTM and HA (PN- HPTTM +HA) containing 10 mg/mL PN- HPTTM and 10 mg/mL HA.

4.2. Oxidative Stress Assay

The scavenger activity of PN-HPTTM, HA, and PN-HPTTM +HA products was evaluated using a cell-free fluorimetric assay with Calcein-AM fluorescence as a marker for oxidative stress. Stock solutions of PN-HPT™, HA, and PN-HPT™ + HA were tested at the commercially available concentration (undiluted, indicated as “Max” in the figures) and at 900, 600, and 300 μg/ml, obtained by dilution in PBS (Sigma-Aldrich, St. Louis, MO, USA).The samples were then aliquoted into a 96-well black microplate (Thermo-Fisher), with 100 μl added to each well. PBS alone was used as control.
To initiate the assay, 2 μM Calcein-AM (Thermo-Fisher, Waltham, MA, USA) was added to each well, and the plate was incubated at room temperature for 10 minutes to allow for fluorescence development. Following this, 600 μM H₂O₂ was introduced to induce oxidative stress. Fluorescence signals were measured at an emission wavelength of 530 nm using a microplate reader (Infinite F200 TECAN, Switzerland) at time intervals of 10, 30, 60, 90, and 120 minutes. This experimental setup allowed for the assessment of the scavenger effects of the treatments over time.

4.3. Statistical Analysis

Fluorescence data were reported as mean ± standard deviation. Differences between groups were assessed using two-way ANOVA followed by Bonferroni’s post-hoc test to compare treated samples against the control, using Prism (GraphPad, La Jolla, CA, USA). Statistical significance was set at p < 0.05. Each experiment was conducted in triplicate, and the entire procedure was repeated three times to ensure reproducibility.

Author Contributions

Conceptualization, C.G. and S.G.; methodology, S.B. and M.T.C.; formal analysis, S.B. and M.T.C.; resources, S.G.; writing—original draft preparation, C.G.; writing—review and editing, S.G.; visualization, C.G. and M.T.C.; supervision, S.G. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Mastelli s.r.l., Sanremo Italy.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data available on request.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Scavenging activity of Polynucleotides High Purification Technology (PN- HPTTM), Hyaluronic acid (HA), or Polynucleotides HPTTM + Hyaluronic Acid (PN- HPTTM +HA) compounds in response to H₂O₂-induced oxidative stress. Results of the Bonferroni post-test for the experiment in Figure 1 are presented below.
PN- HPTTM:
30’: a p=0.002 Control vs Max concentration
60’: b p=0.0004 Control vs Max concentration, p=0.01 Control vs 900 μg/ml
90’: c p=0.0002 Control vs Max concentration, p=0.005 Control vs 900 μg/ml
120’: d p<0.001 Control vs Max concentration, p=0.004 Control vs 900 μg/ml.
HA
90’: a p=0.05 Control vs 600 μg/ml.
120’: b p=0.04 Control vs 600 μg/ml.
PN- HPTTM +HA
30’: a p=0.01 Control vs Max concentration, p=0.02 Control vs 900 μg/ml.
60’: b p=0.004 Control vs Max concentration, p=0.006 Control vs 900 μg/ml, p= 0.02 Control vs 600 μg/ml.
90’: c p=0.004 Control vs Max concentration, p=0.002 Control vs 900 μg/ml and Control vs 600 μg/ml.
120’: d p=0.004 Control vs Max concentration, p=0.001 Control vs 900 μg/ml, p=0.01 Control vs 600 μg/ml.

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Figure 1. Scavenger activity of Polynucleotides High Purification Technology (PN- HPTTM), Hyaluronic acid (HA), or Polynucleotides HPTTM + Hyaluronic Acid (PN- HPTTM +HA) compounds in response to H₂O₂-induced oxidative stress. Fluorescence intensity (A.U.) produced by Calcein-AM oxidation was measured at 10, 30, 60, 90, and 120 minutes for PN- HPTTM (1A), HA (1B) and PN- HPTTM +HA (1C) at different concentrations (0, 300, 600, 900 μg/ml, and maximum concentration). Both PN- HPTTM and PN- HPTTM +HA reduced fluorescence in a dose- and time-dependent manner compared to the control (0 μg/ml, red line). PN- HPTTM +HA exhibited greater scavenger activity across all time points and concentrations, with significant reductions observed as early as 30 minutes for the highest concentration. Results are presented as mean ± SD. See Appendix A for the results of the statistical analysis.
Figure 1. Scavenger activity of Polynucleotides High Purification Technology (PN- HPTTM), Hyaluronic acid (HA), or Polynucleotides HPTTM + Hyaluronic Acid (PN- HPTTM +HA) compounds in response to H₂O₂-induced oxidative stress. Fluorescence intensity (A.U.) produced by Calcein-AM oxidation was measured at 10, 30, 60, 90, and 120 minutes for PN- HPTTM (1A), HA (1B) and PN- HPTTM +HA (1C) at different concentrations (0, 300, 600, 900 μg/ml, and maximum concentration). Both PN- HPTTM and PN- HPTTM +HA reduced fluorescence in a dose- and time-dependent manner compared to the control (0 μg/ml, red line). PN- HPTTM +HA exhibited greater scavenger activity across all time points and concentrations, with significant reductions observed as early as 30 minutes for the highest concentration. Results are presented as mean ± SD. See Appendix A for the results of the statistical analysis.
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Figure 2. Scavenger activity of Control, HA, PN- HPTTM, and PN- HPTTM +HA products in response to H₂O₂-induced oxidative stress. Fluorescence intensity (A.U.) was measured at 10, 60, and 120 minutes for the vehicle (control), PN- HPTTM, HA, and PN- HPTTM + HA at maximum concentration. PN- HPTTM and PN- HPTTM +HA significantly reduced fluorescence compared to the vehicle at 60 minutes (p < 0.001, “a”), and at 120 minutes (p < 0.001, “b”). Data are expressed as mean ± SD.
Figure 2. Scavenger activity of Control, HA, PN- HPTTM, and PN- HPTTM +HA products in response to H₂O₂-induced oxidative stress. Fluorescence intensity (A.U.) was measured at 10, 60, and 120 minutes for the vehicle (control), PN- HPTTM, HA, and PN- HPTTM + HA at maximum concentration. PN- HPTTM and PN- HPTTM +HA significantly reduced fluorescence compared to the vehicle at 60 minutes (p < 0.001, “a”), and at 120 minutes (p < 0.001, “b”). Data are expressed as mean ± SD.
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