Poster O1 - Antioxidant Activity of Chemically Modified Naturally Derived Polymers

Introduction: Hyaluronic acid (HA) and tanfloc, a condensed tannin (CT) representative, exhibit remarkable biological properties and processability. Combining them as polyelectrolyte multilayers (PEMs) creates novel, biodegradable biomaterials. Their antibacterial, antioxidant, and anti-inflammatory activities make such PEMs suitable for biomedical applications and metallic implant coatings.
The antioxidant properties of biomaterials play a critical role in scavenging reactive oxygen species (ROS) generated by activated inflammatory and immune cells in response to injury. Excessive ROS production leads to oxidative stress, resulting in damage to biomolecules and biomaterial itself, and can cause necrosis, protein, and DNA damage.
In this study, we introduce modified hyaluronic acid and tanfloc to enhanced antioxidant activity, validated through three antioxidant assays.
Three tanfloc derivatives are obtained and tested on their antioxidant activity: 3-O-gallate tanfloc, O-methylated tanfloc, and quaternary ammonium tanfloc (Figure 1). The highest antioxidant activity is expected for 3-O-gallate tanfloc due to the presence of catechol and pyrogallol moieties. And the lowest antioxidant activity is expected for O-methylated tanfloc due to the absence of hydroxyl groups. Additionally, an antioxidant study on HA-dopamine complex is performed to assess the effect of catechol group addition on antioxidant activity.
PEMs incorporating both modified and unmodified tanfloc and HA hold the potential to provide biodegradable, multifunctional biomaterials derived from sustainable sources, that can obviate or reduce the need for therapeutic drug delivery. The result of this study is a library of modified biopolymers with a range of functional groups, from which we can test hypotheses about how chemistry modifies the antioxidant activity.

Materials and

Methods: 3-O-gallate tanfloc is synthesized by adding galloyl group to the A ring. To do so phenyl hydroxyl groups on the aromatic B and C rings are protected by O-benzyl groups. O-methylated tanfloc is obtained by complete methylation of all hydroxyl groups. To prevent methylation of amine group, it is protected by N-phthaloyl group. Quaternary ammonium tanfloc is obtained by introduction of quaternary ammonium moiety. HA is modified to contain catechol groups by addition of dopamine to the carboxylic acid through chemistries similar to those already reported by others.
Three solutions are prepared to quantify the mechanisms of antioxidant activity in in the modified biopolymer library. For hydrogen atom transfers 2,2-diphenyl-1-picrylhydrazyl (DPPH·), for single electron transfers 2,29-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and for metal chelation an iron (Fe3+) solution. Each solution has a species that has a peak absorbance that changes upon reduction, the methods for these assays have been well established in literature.
The DPPH· solution is generated by dissolving DPPH· in methanol. The ABTS radical is synthesized using ABTS and potassium persulfate. The Fe3+ solution is created using acetate buffer, 2,4,6-tripyridyl-s-triazine (TPTZ) with HCl, and FeCl3 * 6H2O.
The polymer solutions are incubated with the solutions containing DPPH·, ABTS·+, or Fe3+ in a 1:1 volumetric ratio, 100 μL will be used for all solutions. The peak absorbance is measured at 30 minutes, 1 hour, and 2 hours. Antioxidant activity is compared to ascorbic acid, glutathione, and a vitamin E analog to express the radical scavenging activity as equivalents to common antioxidants.

Results, Conclusions, and Discussions: Quaternary ammonium and N-phthaloylated tanfloc were synthesized. Figure 2 shows the FTIR spectra of both tanfloc and modified tanfloc. These spectra indicate the presence of C=C stretching vibrations from the aromatic ring, -C-OH stretching and deformation vibrations from catechol groups, which are intrinsic to CT. The FTIR spectra of N-phthaloylated tanfloc also reveal the presence of carboxyl groups, supporting the introduction of the phthaloyl group. To further confirm the successful modification, NMR spectra and additional FTIR spectra will be reported.
Figure 1 demonstrates tanfloc derivatives and identifies moieties that are expected to improve antioxidant activity. The 3-O-gallate tanfloc is expected to have the highest antioxidant activity, followed by the quaternary ammonium tanfloc, then the unmodified tanfloc, and finally the methylated tanfloc. In tanfloc the expected antioxidant activity comes from the combination of hydroxyl groups with the aromatic ring. By adding gallic acid to ring A, additional pyrogallol moieties on tanfloc can aid in the reduction of ROS. At the same time methyl groups will provide absence of hydroxyl groups essential for antioxidant activity
A hyaluronic acid - dopamine complex has been synthesized, confirmed by the proton NMR in Figure 3. The NMR shows peaks from dopamine and hyaluronic acid. Peaks highlighted in red and blue in Figure 3 have been used to calculate the degree of substitution for dopamine. Using current methods, the degree of substitution has been calculated to be between 0.15 to 0.20.
In this study synthesis of tanfloc modifications and of HA-dopamine complex is reported. Overall, the findings of this study are meant to highlight the potential of HA, polydopamine and tanfloc-based PEMs to offer biodegradable and multifunctional properties that are highly beneficial for various biomedical applications. These materials can be particularly effective in medical device coatings and metallic implants, where the reduction of oxidative stress and prevention of infection are crucial. HA, polydopamine and tanfloc-based PEMs represent a new solution reduce or eliminate the need for either systemic or local delivery of drugs to control undesirable biological outcomes.

Acknowledgements (Optional): This work was funded by the National Science Foundation, award number 2306983.