Investigating the Neuroprotective Potential of Poly(pro-curcumin) Polymers

Introduction: Curcumin, a naturally occurring chemical compound found in the roots of turmeric plants, has been extensively utilized in traditional medicines throughout East Asia. Recently, many studies have demonstrated that curcumin exhibits diverse biological effects, including antioxidant, anti-inflammatory, antimicrobial, and tissue-protective abilities^1–5. However, curcumin is extremely hydrophobic and one major limitation preventing its use as a therapeutic is its limited bioavailability, even when administered at high doses⁶. Cortical electrodes are microelectrodes that are implanted into the brain for a variety of purposes, including neuroprosthetic technologies or deep-brain stimulation. However, neuronal tissue is sheared and degraded upon implantation, leading to a secondary injury cascade preventing neuronal regeneration.



Thus, we sought to create novel curcumin polymers capable of enhancing curcumin's bioavailability and assess its neuroprotective effects in vitro. In this study, we synthesized two poly(curcumin-co-PEG) polymers, each with unique amounts of curcumin and PEG. Additionally, we analyzed the ability of these polymers to enable neuroprotection following free-radical insult to cortical neuron cultures, where several concentrations of each polymer were tested. We hypothesized that our polymers would effectively quench free radicals at concentrations lower than unbound curcumin, and that the more soluble polymer would have greater neuroprotective effects. Overall, this study seeks to increase the bioavailability of curcumin by creating polymers that have the ability to be fabricated into a multitude of biomaterials for injury applications.

Materials and Methods:: Two different poly(curcumin-co-PEG) polyesters were synthesized using a condensation polymerization reaction of curcumin and PEG in the presence of sebacoyl chloride and pyridine in varying molar ratios (75 and 50% mol% curcumin)7. These materials are referred to as Px, where x is the molar percentage of curcumin in the monomer feed. To confirm the correct composition of the polymer, NMR experiments were performed. Curcumin release from the polymers was assessed using UV-Vis. Additionally, degradation was analyzed following incubation in PBS over 8 weeks utilizing GPC. P50 and P75 films were fabricated by drop casting either 40 µL of P50/chloroform solution (4% w/w) or 40 µL of P75/chloroform solution (6% w/w) onto 15-mm diameter coverslips. Films were protected from light, dried under vacuum 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) curcumin films (P50: average 2.3 mg per coverslip or P75: average 3.35 mg per coverslip); 3) 100 µM H2O2 insult; 4) 100 µM H2O2 insult + Px film; 5) bolus curcumin (corresponding to P50/P75 amounts); 6) 100 µM H2O2 insult + bolus curcumin. PrestoBlue was added 30 minutes after treatment exposure and quantified 24 hours post-treatment (Fig.1).

Results, Conclusions, and Discussions:: 1H NMR spectroscopy confirmed the desired polymers were synthesized. Release data gathered over 8 weeks using UV-Vis showed that soluble curcumin release of P50 from films was about 0.56 mg, while P75 release from films was about 0.02 mg. GPC degradation data confirmed the release findings, where P50 was mostly degraded and P75 was mostly intact. P50, which is more hydrophilic, is able to undergo more hydrolytic degradation, allowing for more curcumin to be released from the construct. On the contrary, P75 is more hydrophobic than P50 and undergoes little to no hydrolytic degradation. Cortical neuronal culture cell viability was quantified using PrestoBlue fluorescence data. Following incubation with P50 and P75 films, there was no change in cell viability between cultures treated with films compared to control cultures, indicating that these polymers are not toxic to the cortical neuron cultures. The hydrogen peroxide insult decreased neuronal viability, while application of the Px films was able to rescue neurons from H2O2 insult, where viability of these cultures was comparable to that of the control and Px only groups (Fig.2). These results suggest that distinct curcumin films display unique neuroprotective characteristics. P50, which is partially hydrolyzable, contributes to radical scavenging via degradation and neutralization of the H2O2 at the polymer surface. Although P75 releases very little curcumin via degradation, it exhibits neuroprotective benefits primarily through surface interactions. Thus, even with very low amounts of curcumin being released from P75, the P75 polymer also effectively neutralized H2O2. In sum, these studies demonstrate that unique polymers containing curcumin and PEG can be created, each displays unique characteristics, and both are able to rescue neurons from free radical insult. While these polymers are developed to enable neuroprotection following electrode implantation, such polymers could be synthesized into different structures (fibers/micelles) to enable regeneration following injury in other tissue systems.

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