Supramolecular Environmental Sensitive Micelles for Nucleic Acid Delivery

Ectrodactyly-ectodermal dysplasia-cleft (EEC) syndrome is a rare genetic disease caused by heterozygous missense mutations in the TP63 gene. This gene encodes the p63 transcription factor, highly expressed in the stem cells of epithelial basal layers, where its altered form impedes the correct epithelial renewal. In the corneal epithelium, the TP63 mutations in limbal epithelial stem cells (LESCs) induce a progressive limbal stem cell deficiency, leading to total blindness. Currently, no therapeutic options are available for treating these ocular morbidities. A promising published approach consists of the local silencing of the TP63 mutated allele by siRNAs, allowing a phenotypic correction of LESCs. Advantages of this treatment include higher specificity towards the targets than conventional small molecules, but the exploitation of oligonucleotides as drugs suffers from their poor biopharmaceutical profile. mPEG5kDa-Cholane is a well-established amphiphilic polymer able to assemble in stable biocompatible micelles. We hypothesized to exploit the physical PEGylation with mPEG5kDa-Cholane for the local siRNA treatment of EEC syndrome ocular morbidities. An amphiphilic siRNA, conjugated with a cholesterol molecule on the 3’-end of the sense strand (“siRNA-Chol”), was exploited to allow the complexation in the micelles through hydrophobic interactions, obtaining nanostructures named “micelloplexes”. mPEG5kDa-Cholane proved to effectively encapsulate siRNA-Chol into neutrally charged and small micelloplexes (which should be suitable to permeate into anterior eye tissues following topical administration), providing siRNA-Chol protection from the RNases. Micelloplexes showed high in vitro biocompatibility, induced efficient siRNA-Chol cell uptake, and good in vitro gene silencing. Further studies are needed to find safe strategies for enhancing the endosomal escape of micelloplexes in the presence of FBS. Nevertheless, the preliminary results suggest that the siRNA physical PEGylation can be a promising strategy for the local treatment of EEC syndrome ocular morbidities. The second chapter of this work focused on the design, development, and characterization of a shell-crosslinked version of the mPEG5kDa-Cholane micelles. Despite their good performances as drug delivery systems, simple micelles are often limited by low stability, especially after systemic administration in the bloodstream, where high dilution can lead to disassembly. The crosslinking of the micellar core or shell is an established procedure to overcome these limitations. A new amphiphilic polymer was successfully synthesized by introducing a cysteine dimer within the PEG chain of mPEG-Cholane. The thiol groups of the cysteine molecules, protected with a pyridine-2-thiol, were kept available as pendant groups to mediate a shell crosslinking in the micelles. This type of crosslinking was designed to ensure better encapsulation and protection of amphiphilic drugs, such as hydrophobic peptides and oligonucleotide conjugates. A dithiol linker displaced the cysteine thiol group protections through a thiol-disulfide exchange reaction, creating new disulfide bonds between the polymer chains and the crosslinking agent. The crosslinked micelles exhibited good stability, low PDI, and their final size could be fine-tuned by adjusting the amount of crosslinking agent added to polymer solutions. The obtained results suggest that the developed nanocarrier represents a promising drug delivery system for diverse applications. Moreover, the presence of disulfide bonds in the crosslinking network may confer responsiveness to reducing environments, such as tumoral tissues or the intracellular space, allowing selective drug release at the target site.