(C-84) Injectable, Antioxidant, Iron Chelating Hydrogel for Spinal Cord Injury Neural Progenitor Cell Delivery

This study proposes a new method for spinal cord repair using an injectable, thermoresponsive hydrogel made of poly(poly(ethylene glycol) citrate-co-N-isopropylacrylamide) (PPCN), which can protect and deliver neural progenitor cells (NPCs). Based solely on the material properties, PPCN incorporates antioxidants to counteract oxidative stress and iron chelators to regulate iron levels, creating an optimal environment for spinal cord regeneration. Simultaneously, we incorporate NPCs into PPCN to promote spinal cord regeneration. NPCs encapsulated in PPCN benefit from the hydrogel’s mechanical properties, which can be controlled to resemble the spinal cord matrix, and can, when encapsulated, be precisely delivered to the injury site due to the injectability of PPCN. This approach has the potential to advance spinal cord regeneration and treat neurodegenerative disorders.

The PPCN hydrogel was synthesized by reacting Poly(ethylene glycol), Glycerol 1,3-diglycerolate, and citric acid, followed by polymerization with N-isopropylacrylamide. To determine the free radical oxygen species (ROS) scavenging potential of the material, a Fenton reaction generated free radicals to reduce Safranin O. If ROS scavenging was occurring, a red color was preserved and read using UV/Vis Spectroscopy (λ = 492 nm). A ferrozine indicator confirmed iron chelation, which could be read using UV/Vis Spectroscopy (λ = 562 nm). PPCN structure was confirmed using Scanning Electron Microscopy (SEM). Finally, rheological properties with a focus on Storage Modulus (G’), Loss Modulus (G’’) and viscosity were calculated using MCR302e Rheometer to determine optimal concentration of PPCN to mimic the mechanical properties of the spinal matrix.

Results:

The ROS scavenging assay confirmed that the hydrogel, PPCN, exhibited antioxidant properties with a percent scavenging capacity of 21 ± 14%. The iron chelation assay demonstrated that PPCN possessed excellent iron chelating capabilities, sequestering 99.2 ± 0.4% of the iron ions. Rheological analysis (Figure 1A) demonstrated that the storage modulus of PPCN increased with concentration. At a concentration of about 65 mg/mL, PPCN achieved close to the optimal storage modulus of 180 Pa, which is ideal for promoting neuronal growth and proliferation (Banerjee et al., 2009). The thermoresponsive properties of PPCN were also confirmed by rheology in Figure 1C, as the storage modulus became greater than the loss modulus around 28ºC, indicating a change from viscoelastic liquid to solid. SEM imaging confirmed that the hydrogel forms a net-like matrix that should be capable of encapsulating NPCs, as shown in Figure 1B.

Conclusions

In summary, the results of this study indicate that PPCN has significant antioxidant and iron-chelating properties. It effectively scavenges reactive oxygen species and chelates iron ions, which are crucial for reducing oxidative stress and preventing further neuronal damage. Additionally, the rheological analysis demonstrates that PPCN's mechanical properties can be optimized to support neuronal growth and proliferation. PPCN’s thermoresponsive properties confirm it can be injected as a liquid at room temperature and become more solid-like at body temperature.

Discussions:

By reducing oxidative stress and maintaining iron homeostasis, PPCN can create a favorable microenvironment for the survival, proliferation, and differentiation of neural progenitor cells within the spinal cord as a treatment to enable spinal cord regeneration. The achieved storage modulus at 65 mg/mL further indicates its suitability for mimicking the mechanical properties of the natural spinal cord extracellular matrix.

Future studies will focus on investigating the functional integration of neural progenitor cells encapsulated within PPCN. In vivo experiments and animal models will be necessary to evaluate the efficacy of PPCN in promoting spinal cord regeneration and functional recovery. Additionally, exploring the controlled release of growth factors or other bioactive molecules from PPCN could enhance the therapeutic outcomes by providing additional cues for neuronal survival and differentiation.

Banerjee, A.; Arha, M.; Choudhary, S.; Ashton, R. S.; Bhatia, S. R.; Schaffer, D. V.; Kane, R. S. The Influence of Hydrogel Modulus on the Proliferation and Differentiation of Encapsulated Neural Stem Cells. Biomaterials 2009, 30 (27), 4695–4699. DOI:10.1016/j.biomaterials.2009.05.050.