Design of an injectable and bioerodible supramolecular hydrogel for local siRNA delivery

The potential of gene technology to revolutionize disease management by addressing their underlying causes is undeniable. Nevertheless, a substantial challenge lies ahead: the efficient administration of these treatments through the intricate physiological barriers of the human body. The aim of this research was to evaluate the potential of a supramolecular (SM), bioerodible and injectable hydrogel, composed of an ad−hoc engineered mixture of a custom synthetized poly(ether urethane) (PEU) and commercial α-cyclodextrins (CDs), as a conduit for localized gene therapy delivery. Specifically, two types of nanoparticle formulations encapsulating siRNA were examined: poly(lactic-co-glycolic acid) (PLGA) nanoparticles and poly(β-amino ester) (PBAE)-based polyplexes. The initial phase involved the characterization of the amphiphilic PEU, synthesized using Poloxamer®407 as a core component, in order to validate the persistence of its distinctive molecular attributes. After this analysis, SM hydrogels were prepared by mixing aqueous solutions of PEU and CDs, resulting in 3% or 5%, and 10% w/v concentrations, respectively. Incorporating polyplexes within the SM hydrogel matrix mandated a sequential approach, starting with the preparation of PBAE/siRNA polyplexes in α-CD solution. Polyplexes were characterized through Dynamic iLght Scattering (DLS) and Laser Doppler Anemometry (LDA) techniques, including particle sizes hovering around 200 nm and a positively charged zeta potential of approximately +30 mV. Intriguingly, the presence of CDs exerted minimal influence on the encapsulation efficiency of PBAE polyplexes (95%), a fact corroborated by the SYBR Gold assay. In contrast, the formulation of PLGA nanoparticles adhered to a well-established protocol, followed by their suspension within an α-CD aqueous solution, thus obtaining particle sizes of 200 nm, coupled with a negative zeta potential. After performing a RiboGreen assay, the encapsulation efficiency observed was significantly lower in comparison to polyplexes (55%). The subsequent stage involved the incorporation of polyplexes and PLGA NPs into the SM hydrogel. A comparative analysis against the unloaded hydrogel was conducted to ascertain potential alterations in hydrogel properties resulting from nanoparticle integration. Rheological evaluations underscored the hydrogel’s preservation of thixotropic and self-healing characteristics. Nevertheless, delayed gelation kinetics were particularly pronounced in scenarios involving lower PEU concentrations. A crucial aspect of investigation was the polyplex release from the hydrogel. Techniques encompassing DLS, NTA (Nanoparticle Tracking analysis), and TEM converged to affirm the presence of intact polyplexes within the released medium, exhibiting a progressive size expansion over time. Release kinetics found comprehensive elucidation through fluorescence-based assays. Parallel analyses probed the release kinetics of PLGA NPs, revealing a uniform release curve even as differential sizes of PLGA NPs were liberated at distinct time points, presumably entwined within hydrogel degradation debris. Validation of these formulations unfolded through cell culture experiments across diverse cell lines. Strikingly, polyplexes released from the hydrogel manifested pronounced efficacy across specific cell types, inducing a phagocytic response substantiated through flow cytometry and qPCR. In contrast, H1299 cell lines exhibited uptake of PLGA NPs without translating the expected gene silencing outcomes.