Exploring the use of injectable and erodible poly(urethane)-based supramolecular hydrogels as carriers for localized gene therapy delivery

Genetic disorders are conditions caused by DNA alterations that affect cell structure and functionality. Gene therapy represents a potential innovative approach aimed at directly correcting the underlying genetic mutations. However, one of the main challenges of gene therapy is ensuring the safe and effective delivery of therapeutic genes to target cells. The purpose of this project was to assess the potential of bioerodible, injectable, and supramolecular (SM) hydrogels as carriers for the localized release of siRNA-containing polyplexes (PXs). SM hydrogels were designed by mixing aqueous solutions of a custom-synthesized amphiphilic Poloxamer®407-based poly(ether urethane) (PEU) and commercial &#945;-cyclodextrins (&#945;-CDs). In the final formulations, PEU and &#945;-CDs concentration varied in the range 1.43%-2.57% w/v and 8.56%-10% w/v, respectively. In parallel, PXs were prepared in 10 mM HEPES buffer and in 10 mM HEPES buffer containing &#945;-CDs (from 12 to 14% w/v) using a custom-synthesized poly(&#946;-amino ester). PX formulation was tuned by varying siRNA concentration (50-300 pmol/100 &#956;l) and the N/P ratio (N/P within 1 and 15). Before loading into the hydrogel, PX size, polydispersity index, zeta potential and concentration were assessed by Dynamic light scattering (DLS), Laser Doppler Anemometry (LDA), and Nanoparticle tracking Analysis (NTA), while their encapsulation efficiency was assessed though the SYBR Gold assay. Compared to PXs prepared in pure HEPES buffer, the presence of &#945;-CDs made the PXs larger (100 nm vs. 250-400 nm), more polydisperse (<0.2 vs. >0.4), with more positive zeta potential values (e.g., -20 mV vs.+30mV at N/P ratio of 5; +20 mV vs. +30 mV at N/P ratio of 10), and with a significantly or slightly lower encapsulation efficiency depending on the N/P ratio (e.g., 80% vs. 55% at N/P of 5; 100% vs. 95% at N/P of 10). Moreover, PXs concentration increased up to 50 billion nanoparticles/ml, as siRNA content increased. PX loading in the SM hydrogels slowed down their gelation kinetics, as assessed through the tube inverting test (6-8 hours vs. overnight-36 hours for unloaded and PXs loaded hydrogels, respectively). Rheological tests indicated that higher PXs concentrations negatively impacted thixotropic and self-healing properties and induced the formation of gels with lower storage modulus (G’) values (985 Pa at 50 pmol/100 &#956;l vs. 260 Pa at 200 pmol/100 &#956;l). Afterward, PX release from the hydrogels was tested through DLS, NTA and SYBR Gold assay, confirming the presence of PXs within the release medium (siRNA release between 40% and 100% at 48 h) and highlighting that hydrogel network properties affected the release kinetics (higher release as gel G' decreased). Lastly, the developed formulations were validated through in vitro experiments with H1299/eGFP and HeLa/eGFP cell lines. Regarding the silencing ability of released PXs, flow cytometry data indicated the lack of reduction in the mean fluorescent intensity, probably due to an increased cellular metabolic activity caused by the uptake of gel erosion products, without yielding the expected gene silencing effects. Overall, this study provided a basis for the design of a new SM hydrogel platform as potential carriers for localized siRNA delivery, showcasing the benefits, issues and still open questions related to their use. The lack of effective gene silencing stressed the importance of further improving the hydrogel composition, release kinetics and targeted gene delivery, while maintaining proper biocompatibility.