RNA Oncological Therapeutics: Intracellular Hairpin RNA Assembly Enables MicroRNA-Triggered Anticancer Functionality.

RNA therapeutics are of global interest because of their versatility in targeting a variety of intracellular and extracellular biomolecules. In that context, long double-stranded RNA (dsRNA) has been studied as an antitumor agent that activates the immune response. However, its performance is constrained by poor cancer selectivity and cell-penetration ability. Here, we designed and synthesized an oncolytic RNA hairpin pair (oHP) that was selectively cytotoxic toward cancer cells expressing abundant oncogenic microRNA-21 (miR-21). Although the structure of each hairpin RNA was thermodynamically metastable, catalytic miR-21 input triggered it to open to generate a long nicked dsRNA. We demonstrated that oHP functioned as a cytotoxic amplifier of information in the presence of miR-21 in various cancer cells and tumor-bearing mice. This work represents the first example of the use of short RNA molecules as build-up-type anticancer agents that are triggered by an oncogenic miRNA.

Hydrophobicity Tuning of Cationic Polyaspartamide Derivatives for Enhanced Antisense Oligonucleotide Delivery.

Various cationic polymers are used to deliver polyplex-mediated antisense oligonucleotides (ASOs). However, few studies have investigated the structural determinants of polyplex functionalities in polymers. This study focused on the polymer hydrophobicity. A series of amphiphilic polyaspartamide derivatives possessing various hydrophobic (R) moieties together with cationic diethylenetriamine (DET) moieties in the side chain (PAsp(DET/R)s) were synthesized to optimize the R moieties (or hydrophobicity) for locked nucleic acid (LNA) gapmer ASO delivery. The gene knockdown efficiencies of PAsp(DET/R) polyplexes were plotted against a hydrophobicity parameter, logD7.3, of PAsp(DET/R), revealing that the gene knockdown efficiency was substantially improved by PAsp(DET/R) with logD7.3 higher than -2.4. This was explained by the increased polyplex stability and improved cellular uptake of ASO payloads. After intratracheal administration, the polyplex samples with a higher logD7.3 than -2.4 induced a significantly higher gene knockdown in the lung tissue compared with counterparts with lower hydrophobicity and naked ASO. These results demonstrate that the hydrophobicity of PAsp(DET/R) is crucial for efficient ASO delivery in vitro and in vivo.

Size-tunable PEG-grafted copolymers as a polymeric nanoruler for passive targeting muscle tissues.

Muscle-targeted drug delivery is a major challenge in nanomedicine. The extravasation of nanomedicines (or nanoparticles) from the bloodstream into muscle tissues is hindered by the continuous endothelium, the so-called blood-muscle barrier. This study aimed to evaluate the optimal size of macromolecular drugs for extravasation (or passive targeting) into muscle tissues. We constructed a size-tunable polymeric delivery platform as a polymeric nanoruler by grafting poly(ethylene glycol)s (PEGs) onto the poly(aspartic acid) (PAsp) backbone. A series of PEG-grafted copolymers (gPEGs) with a narrow size distribution between 11 and 32 nm in hydrodynamic diameter (DH) were prepared by changing the molecular weight of the PEGs. Biodistribution analyses revealed that accumulation amounts of gPEGs in the muscle tissues of normal mice tended to decrease above their size of ~15 nm (or ~11 nm for the heart). The gPEGs accumulated in the skeletal muscles of Duchenne muscular dystrophy model mice (mdx mice) at a 2-3-fold higher level than in the skeletal muscles of normal mice. At the same time, there was a reduced accumulation of gPEGs in the spleen and liver. Intravital confocal laser scanning microscopy and immunohistochemical analysis showed extravasation and locally enhanced accumulation of gPEGs in the skeletal muscle of mdx mice. This study outlined the pivotal role of macromolecular drug size in muscle-targeted drug delivery and demonstrated the enhanced permeability of 11-32 nm-sized macromolecular drugs in mdx mice.

Fine-tuning of polyaspartamide derivatives with alicyclic moieties for systemic mRNA delivery.

Development of efficient delivery vehicles for in vitro transcribed mRNA (IVT mRNA) is currently a major challenge in nanomedicines. For systemic mRNA delivery, we developed a series of cationic amphiphilic polyaspartamide derivatives (PAsp(DET/R)s) carrying various alicyclic (R) moieties with diethylenetriamine (DET) in the side chains to form mRNA-loaded polyplexes bearing stability under physiological conditions and possessing endosomal escape functionality. While the size and ζ-potential of polyplexes were comparable among various PAsp(DET/R)s, the transfection efficiencies of polyplexes were considerably varied due to difference in the R moieties of PAsp(DET/R)s and were described by an octanol-water (or buffer at pH 7.3) distribution coefficient (logD7.3). The critical logD7.3 for the efficient in vitro transfection of mRNA was indicated at -2.7 to -1.8. The polyplexes with logD7.3 > -1.8 elicited the much higher in vitro transfection efficiencies. After systemic administration, the polyplexes with logD7.3 from -1.8 to -1.3 elicited the significant mRNA expression specifically in the lungs. The highest mRNA expression in the lungs was achieved by a polyaspartamide derivative having a cyclohexylethyl group (PAsp(DET/CHE)), which induced more than 10-fold increase in mRNA transfection efficiency compared to commercially available lipid nanoparticles. The higher mRNA expression by polyplexes in the lungs was explained well by the preferential lung accumulation of intact mRNA, as determined by quantitative real-time PCR. Our results demonstrate that PAsp(DET/R)s are a promising synthetic material for the enhanced systemic IVT mRNA delivery.

Installation of a Thermoswitchable Hydrophobic Domain into a Unimer Polyion Complex for Enhanced Cellular Uptake of siRNA.

Whereas small siRNA nanocarriers with a size of 10-20 nm exert high tissue-permeability, they encounter the challenge of inefficient adsorption on the cell surface, resulting in poor cellular uptake of siRNA. To solve this dilemma, this study aims to control the hydrophobicity of a small siRNA nanocarrier, unimer polyion complex (uPIC), with a size of ∼10 nm. The uPICs are fabricated to consist of a single pair between siRNA and a smart triblock copolymer comprising hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx), thermoswitchable poly(2-n-propyl-2-oxazoline) (PnPrOx), and cationic poly(l-lysine) (PLL). The PnPrOx segment is dehydrated at 37 °C (>lower critical solution temperature) to enhance the hydrophobicity of uPICs. The uPICs with a hydrophobic domain facilitates cellular uptake of the siRNA payload through stronger binding to the cell surface, compared with control uPICs without a PnPrOx segment, leading to a significantly enhanced gene silencing effect in cultured cancer cells.