Poster O17 - Exploring the Neuroprotective Mechanisms of Poly(pro-curcumin) Polymers

Introduction: Curcumin, the principal phenol of the turmeric plant, exhibits diverse biological effects, including antioxidant, anti-inflammatory, antimicrobial, and tissue-protective abilities.1–5 Although curcumin is promising in biomedical applications, it is limited as a therapeutic due to its highly hydrophobic nature, limiting its bioavailability even when administered in high doses.6,7 Researchers have incorporated curcumin into biomaterial carriers to overcome these limitations, although these complexes destabilize quickly due to a lack of covalent bonding.1,8 Previously, we reported the synthesis of a novel poly(pro-curcumin), which has two benefits: 1) the polymer contains a PEG backbone, increasing the overall hydrophilicity to allow for more efficient curcumin delivery, and 2) curcumin is synthesized into the polymer backbone, increasing the curcumin dose.8 In this study, we utilized two poly(curcumin-co-PEG) polymers, P50 and P75, at multiple concentrations. We assessed the ability of the two polymers across all concentrations to provide neuroprotective effects in a simulated neural injury with a free-radical insult. Additionally, due to the chemical differences between the two polymers, we investigated the unique neuroprotective mechanisms of each polymer at all concentrations.

Materials and

Methods: Previously, two different poly(pro-curcumin) polymers were synthesized using a condensation polymerization reaction of curcumin and PEG in varying molar ratios: P50 and P75 (Px), where x is the molar ratio of curcumin in the monomer feed.8 P50 and P75 films, at 6%, 8%, and 10% (w/w Px/CHCl3), were fabricated by drop casting 40 µL of polymer solution onto 15-mm diameter coverslips over heat. Films were protected from light, air-dried overnight, and subsequently sterilized via ethylene oxide. The toxicity of the films, as well as their ability to rescue neurons by quenching reactive oxygen species (ROS), was assessed by incubating primary rat cortical neuronal cultures with films with and without a hydrogen peroxide insult. Briefly, cortical neurons isolated from E18 Sprague-Dawley rats were cultured for 7 days at 37°C. On day 7, all groups received treatments: 1) control (no polymer); 2) 100 µM H2O2 insult; 3) Px 6% film; 4) Px 8% film; 5) Px 10% film; 6) H2O2 insult + Px 6% film; 7) H2O2 insult + Px 8% film; and 8) H2O2 insult + Px 10% film, where x is 50 or 75. Cell viability was quantified via PrestoBlue, and ICC allowed for visualization of neuronal integrity. To visualize dissolution of the polymers across all concentrations, polymer films were incubated in PBS at 37°C and examined using mass loss calculations and SEM at predetermined time points. Furthermore, profilometry was used to determine if film thickness affected dissolution, while degradation of the polymers was assessed via GPC.

Results, Conclusions, and Discussions: The addition of the Px polymers at 6%, 8%, and 10% displayed little to no fluorescent differences compared with the control, indicating that the polymers were nontoxic at these concentrations. Additionally, the 100 µM hydrogen peroxide insult greatly decreased neuronal viability, while the application of Px films across all concentrations with the insult led to a considerable increase in cell viability, indicating neuronal rescue (Fig. 1 & 2). Interestingly, both P50 and P75 6% films exhibited the greatest neuroprotective effect, while 10% exhibited the least neuroprotective effects. Dissolution studies revealed that P50 is highly soluble; P50 6% solubilizes within 30 minutes while P50 10% loses about half of its mass over 25 hours. On the contrary, P75 is insoluble; across all concentrations, little to no mass loss was observed over 25 hours. Further examination via SEM displayed nanoparticle structures within the P50 films. Additionally, they demonstrated that P50 6% and 8% likely undergo bulk dissolution, while P50 10% undergoes a combination of both bulk and surface dissolution (Fig. 3). P75 across all concentrations displayed no differences in the surface across the time course (Fig. 4). The lack of solubilization at higher polymer concentrations is supported via profilometry data; higher polymer concentrations lead to increases in viscosity, resulting in greater layer thickness, which water molecules have a hard time penetrating (Fig. 5A-B). Furthermore, P75 likely does not dissolve due to the high curcumin amount within the polymer, suggesting that hydrophilicity plays a greater role in dissolution than film thickness. Finally, GPC results revealed that there was slight degradation of P50 during the time course, while P75 did not degrade (Fig. 5C-D). Thus, even though no curcumin was released from P75, it neutralized H2O2 as effectively as P50. In summary, these studies demonstrate that our poly(pro-curcumin) polymers are neuroprotective in their polymer film forms through different mechanisms. This work is the first to date to characterize the protective mechanisms and effects of the polymers at multiple concentrations. By investigating P50 and P75 at multiple concentrations, this study will provide insight into the appropriate material selection for future in vivo and clinical applications.

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