After RNAi was first discovered over 20 years ago, siRNA-based therapeutics are finally becoming reality. However, the delivery of siRNA has remained a challenge. In our previous research, we found that spermine-based poly(β-amino ester)s are very promising for siRNA delivery. However, the role of hydrophobic modification in siRNA delivery of spermine-based poly(β-amino ester)s is not fully understood yet. In the current work, we synthesized spermine-based poly(β-amino ester)s with different percentages of oleylamine side chains, named P(SpOABAE). The chemical structures of the polymers were characterized by ¹H NMR. The polymers showed efficient siRNA encapsulation determined by SYBR Gold assays. The hydrodynamic diameters of the P(SpOABAE) polyplexes from charge ratio N/P 1 to 20 were 30-100 nm except for aggregation phenomena observed at N/P 3. Morphology of the polyplexes was visualized by atomic force microscopy, and cellular uptake was determined by flow cytometry in H1299 cells, where all the polyplexes showed significantly higher cellular uptake than hyperbranched polyethylenimine (25 kDa). The most hydrophobic P(SpOABAE) polyplexes were able to achieve more than 90% GFP knockdown in H1299/eGFP cells. The fact that gene silencing efficacy increased with hydrophobicity but cellular uptake was affected by both charge and hydrophobic interactions highlights the importance of endosomal escape. For pulmonary administration and improved storage stability, the polyplexes were spray-dried. Results confirmed the maintained siRNA activity after storage for 3 months at room temperature, indicating potential for dry powder inhalation.

RNA interference (RNAi) is an efficient strategy to post‐transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N‐acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine‐based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine‐copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid‐based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease‐activated receptor 2 (PAR2), a G protein‐coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Glioblastoma multiforme is a devastating disease that has attracted enormous attention due to poor prognosis and high recurrence. Small interfering RNA (siRNA) in principle offers a promising therapeutic approach by the downregulation of disease-related genes via RNA interference. For efficient siRNA delivery to target sites, cationic polymers are often used in preclinical studies for the protection of siRNA and complex formation based on electrostatic interactions. In an effort to develop biocompatible and efficient nanocarriers with a translational outlook for optimal gene silencing at reduced toxicity, we synthesized two sets of nylon-3 copolymers with variable cationic content (DM or NM monomer) and hydrophobic subunits (CP monomer) and evaluated their suitability for in vitro siRNA delivery into glioblastoma cells. DM_0.4/CP_0.6 and NM_0.4/CP_0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits. Additionally, we utilized NM_0.2/CP_0.8 polymers to evaluate the impact of the different hydrophobic content in the polymer chain. The siRNA condensation ability and polymer–siRNA complex stability was evaluated by unmodified and modified SYBR gold assays, respectively. Further physicochemical characteristics, e.g., particle size and surface charge, were evaluated by dynamic light scattering and laser Doppler anemometry, whereas a relatively new method for polyplex size distribution analysis—tunable resistive pulse sensing—was additionally developed and compared to DLS measurements. Transfection efficiencies, the route of cell internalization, and protein knockdown abilities in glioblastoma cells were investigated by flow cytometry. Furthermore, cellular tolerability was evaluated by MTT and LDH assays. All the polymers efficiently condensed siRNA at N/P ratios of three, whereas polymers with NM cationic subunits demonstrated smaller particle size and lower polyplex stability. Furthermore, NM_0.2/CP_0.8 polyplexes with the highest hydrophobic content displayed significantly higher cellular internalization in comparison to more cationic formulations and successful knockdown capabilities. Detailed investigations of the cellular uptake route demonstrated that these polyplexes mainly follow clathrin-mediated endocytotic uptake mechanisms, implying high interaction capacity with cellular membranes. Taken together with conducive toxicity profiles, highly hydrophobic nylon-3 polymers provide an appropriate siRNA delivery agent for the potential treatment of glioblastoma.

Ocular gene therapy targets eye diseases at the genetic level. Systemic transport of nucleic acids to ocular tissues poses a significant challenge, as the effectiveness of crossing the blood‐retina barrier limits nucleic acid penetration. Therefore, local administration, such as topical, periocular, or intraocular, can improve the outcome of in vivo gene therapy by bypassing the first‐pass effect and minimizing the systemic toxic effects. The eye is an immune‐privileged organ with limited local immune response, making it an ideal candidate for local gene therapy. Ocular gene therapy offers a promising solution for the treatment of a wide range of retinal diseases including age‐related macular degeneration, diabetic retinopathy, retinitis pigmentosa, and Leber congenital amaurosis. Gene therapy enables replacement of mutated genes essential for visual function, delivery of genes expressing neurotrophic factors and anti‐apoptosis factors for retinal degeneration, and delivery of genes expressing anti‐angiogenic proteins for ocular neovascularization. This perspective discusses the potential of nanoparticles for nucleic acid delivery to the retina, explores challenges, and evaluates different delivery methods, including non‐viral agents such as liposomes and polymers. These nonviral agents present advantages over traditional viral vectors, showing promise in overcoming limitations and offering a viable option for retinal gene therapy.

Pyridine-based heteroaromatic yttrium-bis(phenolate) complexes were investigated in ring-opening polymerization of racemic β-butyrolactone (rac-BBL) and (−)-menthide. In addition, AB and BAB copolymers from poly((−)-menthide) (PM, block A) and poly(3-hydroxybutyrate) (PHB, block B) were synthesized via sequential addition. BAB copolymers were accessible through introduction of a bifunctional pyrazine initiator enabling chain growth in two directions. Block copolymers were analyzed using thermal analysis, X-ray diffraction, small-angle X-ray scattering, NMR analysis and stress–strain experiments. These measurements confirmed a microphase separation within the semicrystalline BAB materials. Regarding the mechanical behavior, two hot-moldable PHB-based (co)polymers were investigated. The syndiotactic PHB is less brittle than natural PHB, resulting in a lower Young's modulus. Incorporating PM in the structure, the elastic modulus is further lowered with increasing elongation at break.