Polyester with Pendent Acetylcholine-Mimicking Functionalities Promotes Neurite Growth

Successful regeneration of nerves can benefit from biomaterials that provide a supportive biochemical and mechanical environment while also degrading with controlled inflammation and minimal scar formation. Herein, we report a neuroactive polymer functionalized by covalent attachment of the neurotransmitter acetylcholine (Ach). The polymer was readily synthesized in two steps from poly(sebacoyl diglyceride) (PSeD), which previously demonstrated biocompatibility and biodegradation in vivo. Distinct from prior acetylcholine-biomimetic polymers, PSeD-Ach contains both quaternary ammonium and free acetyl moieties, closely resembling native acetylcholine structure. The polymer structure was confirmed via (1)H nuclear magnetic resonance and Fourier-transform infrared spectroscopy. Hydrophilicity, charge, and thermal properties of PSeD-Ach were determined by tensiometer, zetasizer, differential scanning calorimetry, and thermal gravimetric analysis, respectively. PC12 cells exhibited the greatest proliferation and neurite outgrowth on PSeD-Ach and laminin substrates, with no significant difference between these groups. PSeD-Ach yielded much longer neurite outgrowth than the control polymer containing ammonium but no the acetyl group, confirming the importance of the entire acetylcholine-like moiety. Furthermore, PSeD-Ach supports adhesion of primary rat dorsal root ganglions and subsequent neurite sprouting and extension. The sprouting rate is comparable to the best conditions from previous report. Our findings are significant in that they were obtained with acetylcholine-like functionalities in 100% repeating units, a condition shown to yield significant toxicity in prior publications. Moreover, PSeD-Ach exhibited favorable mechanical and degradation properties for nerve tissue engineering application. Humidified PSeD-Ach had an elastic modulus of 76.9 kPa, close to native neural tissue, and could well recover from cyclic dynamic compression. PSeD-Ach showed a gradual in vitro degradation under physiologic conditions with a mass loss of 60% within 4 weeks. Overall, this simple and versatile synthesis provides a useful tool to produce biomaterials for creating the appropriate stimulatory environment for nerve regeneration.