Design and characterization of injectable supramolecular formulations for the localized delivery of nucleic acids

Gene technology is facing years of crucial development with the promise of bringing new opportunities for the treatment of several diseases, targeting the causes instead of the symptoms. However, finding a proper delivery vehicle able to overcome the countless barriers present in the physiological environment is still the main challenge, that needs to be addressed to improve the clinical translation. Local nucleic acid delivery allows to reduce the doses, obstacles, and side effects. The present work represents an exploratory study with the main goal of finding out whether a well-established injectable and bioerodible supramolecular (SM) hydrogel based on a customized poly(ether urethane) (PEU) and α-cyclodextrins (CDs) could be used as a platform for the localized delivery of polyethylenimine/small interfering RNA (PEI/siRNA) polyplexes. First, a Poloxamer 407-based amphiphilic PEU was successfully synthesized, as proved by infrared spectroscopy and size exclusion chromatography. SM hydrogels were then prepared by simply mixing PEU and CD aqueous solutions to achieve final 1 and 10% w/V concentrations, respectively. To embed the polyplexes in the SM hydrogel, it is necessary to mix the polyplexes with the CD solution first, and then use the resulting dispersion to assemble the hydrogel. Hence, PEI/siRNA polyplexes were prepared in a 14% w/V CD solution and their physico-chemical properties were studied via dynamic light scattering (DLS) and transmission electron microscopy (TEM), showing particles with a size around 200 nm and spherical shape. Moreover, we observed that CDs increase the already high encapsulation efficiency of PEI by SYBR Gold assay. Polyplexes inside the CD solution were then used to prepare the SM hydrogel, and this system was characterized in comparison to the unloaded hydrogel, to determine if polyplex presence could influence the hydrogel's properties. Rheological studies demonstrated that the hydrogel does not lose its thixotropic and self-healing properties when loaded with the therapeutic cargo; however, a slight weakening in terms of mechanical features was observed. When stability tests were carried out in watery medium at 37°C, results showed that the hydrogel progressively underwent erosion over five days, with very little difference if polyplexes are present. Finally, it was of critical importance to understand if the polyplexes can be released from the hydrogel intact and the release kinetics during the process. For this purpose, DLS and TEM were used to demonstrate that intact polyplexes were present in the collected release medium, approx. 200 nm in size. Then, to investigate the release kinetics, PEI and siRNA were labeled with two different dyes, allowing to quantify the cumulative release using fluorescence. A dye quenching effect affecting labeled siRNA molecules was noticed, allowing to hypothesize that the siRNA is still located inside the particles upon release. These results demonstrate the potentiality of using this SM hydrogel for gene delivery applications, with injectability, bioerodibility, and good release profile being its more interesting features. However, there is still space for improvements: it would be interesting to develop a release protocol with reliable in vitro-in vivo correlation, to have a better evaluation of the release kinetics of this system. Moreover, the formulation's efficacy in cell culture experiments should be addressed, maybe with the use of a more functional gene vector than PEI.