Long noncoding RNA TUG1 promotes cisplatin resistance in ovarian cancer via upregulation of DNA polymerase eta.

Chemoresistance is a major cause of high mortality and poor survival in patients with ovarian cancer (OVCA). Understanding the mechanisms of chemoresistance is urgently required to develop effective therapeutic approaches to OVCA. Here, we show that expression of the long noncoding RNA, taurine upregulated gene 1 (TUG1), is markedly upregulated in samples from OVCA patients who developed resistance to primary platinum-based therapy. Depletion of TUG1 increased sensitivity to cisplatin in the OVCA cell lines, SKOV3 and KURAMOCHI. Combination therapy of cisplatin with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system effectively relieved the tumor burden in xenograft mouse models. Mechanistically, TUG1 acts as a competing endogenous RNA by downregulating miR-4687-3p and miR-6088, both of which target DNA polymerase eta (POLH), an enzyme required for translesion DNA synthesis. Overexpression of POLH reversed the effect of TUG1 depletion on cisplatin-induced cytotoxicity. Our data suggest that TUG1 upregulation allows OVCA to tolerate DNA damage via upregulation of POLH; this provides a strong rationale for targeting TUG1 to overcome cisplatin resistance in OVCA.

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.

Recent Progress in the Endosomal Escape Mechanism and Chemical Structures of Polycations for Nucleic Acid Delivery.

Nucleic acid-based therapies are seeing a spiralling surge. Stimuli-responsive polymers, especially pH-responsive ones, are gaining widespread attention because of their ability to efficiently deliver nucleic acids. These polymers can be synthesized and modified according to target requirements, such as delivery sites and the nature of nucleic acids. In this regard, the endosomal escape mechanism of polymer-nucleic acid complexes (polyplexes) remains a topic of considerable interest owing to various plausible escape mechanisms. This review describes current progress in the endosomal escape mechanism of polyplexes and state-of-the-art chemical designs for pH-responsive polymers. The importance is also discussed of the acid dissociation constant (i.e., pKa) in designing the new generation of pH-responsive polymers, along with assays to monitor and quantify the endosomal escape behavior. Further, the use of machine learning is addressed in pKa prediction and polymer design to find novel chemical structures for pH responsiveness. This review will facilitate the design of new pH-responsive polymers for advanced and efficient nucleic acid delivery.

TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite for cancer cell proliferation.

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

Terbium-Rose Bengal Coordination Nanocrystals-Induced ROS Production under Low-Dose X-rays in Cultured Cancer Cells for Photodynamic Therapy.

X-ray-triggered scintillators (Sc) and photosensitizers (Ps) have been developed for X-ray-induced photodynamic therapy (X-PDT) to selectively destruct deep tissue tumors with a low X-ray dose. This study designed terbium (Tb)-rose bengal (RB) coordination nanocrystals (T-RBNs) by a solvothermal treatment, aiming to reduce photon energy dissipation between Tb3+ and RB and thus increase the reactive oxygen species (ROS) production efficiency. T-RBNs synthesized at a molar ratio of [RB]/[Tb] = 3 exhibited a size of 6.8 ± 1.2 nm with a crystalline property. Fourier transform infrared analyses of T-RBNs indicated successful coordination between RB and Tb3+. T-RBNs generated singlet oxygen (1O2) and hydroxyl radicals (•OH) under low-dose X-ray irradiation (0.5 Gy) via scintillating and radiosensitizing pathways. T-RBNs produced ∼8-fold higher ROS amounts than bare RB and ∼3.6-fold higher ROS amounts than inorganic nanoparticle-based controls. T-RBNs did not exhibit severe cytotoxicity up to 2 mg/mL concentration in cultured luciferase-expressing murine epithelial breast cancer (4T1-luc) cells. Furthermore, T-RBNs were efficiently internalized into cultured 4T1-luc cells and induced DNA double strand damage, as evidenced by an immunofluorescence staining assay with phosphorylated γ-H2AX. Ultimately, under 0.5 Gy X-ray irradiation, T-RBNs induced >70% 4T1-luc cell death via simultaneous apoptosis/necrosis pathways. Overall, T-RBNs provided a promising Sc/Ps platform under low-dose X-PDT for advanced cancer therapy.

Polyplex designs for improving the stability and safety of RNA therapeutics.

Nanoparticle-based delivery systems have contributed to the recent clinical success of RNA therapeutics, including siRNA and mRNA. RNA delivery using polymers has several distinct properties, such as enabling RNA delivery into extra-hepatic organs, modulation of immune responses to RNA, and regulation of intracellular RNA release. However, delivery systems should overcome safety and stability issues to achieve widespread therapeutic applications. Safety concerns include direct damage to cellular components, innate and adaptive immune responses, complement activation, and interaction with surrounding molecules and cells in the blood circulation. The stability of the delivery systems should balance extracellular RNA protection and controlled intracellular RNA release, which requires optimization for each RNA species. Further, polymer designs for improving safety and stability often conflict with each other. This review covers advances in polymer-based approaches to address these issues over several years, focusing on biological understanding and design concepts for delivery systems rather than material chemistry.

Decoupling between Translational Diffusion and Viscoelasticity in Transient Networks with Controlled Network Connectivity.

The mobility of sustained molecules is influenced by viscoelasticity, which is strongly correlated with the diffusional property in polymeric liquid. However, the study of transient networks formed by a reversible crosslink, which is the viscoelastic liquid, was insufficient due to the absence of a model system. We compare the viscoelastic and diffusional properties of the transient networks, using the model system with controlled network connectivity (Tetra-PEG slime). According to independent measurements of viscoelasticity and diffusion, the root-mean-square distance the polymer diffuses during the viscoelastic relaxation time shows a large deviation from the self-size of the polymer, which is contrary to the conventional understanding. This decoupling between viscoelasticity and diffusion is unique for transient networks, suggesting that the viscoelastic relaxation is not induced by the diffusion of one prepolymer, particularly in the network with low connectivity. These findings will provide a definite basis for discussion to understand the viscoelasticity in transient networks.

Rose Bengal Decorated NaYF4:Tb Nanoparticles for Low Dose X-ray-Induced Photodynamic Therapy in Cancer Cells.

The use of scintillating nanoparticles (ScNPs) in X-ray-induced photodynamic therapy (X-PDT) is a technique for deep tissue-localized tumor therapy with few side effects. ScNPs transfer X-ray-induced energy to photosensitizers, which generate massive amounts of reactive oxygen species (ROS) and kill cancer cells. Here we fabricated rose bengal (RB)-installed, Tb3+-rich NaYF4 nanocrystals (NaYF4:Tb@RB), in which optically inert Y3+ enables highly efficient energy transfer via high amounts of Tb3+ doping. NaYF4:Tb was prepared via solvothermal synthesis to have an average size of 7.6 nm, followed by coating with poly(maleic anhydride-alt-1-octedecene)-poly(ethylene glycol) with a molecular weight of 2000 (C18PMH-PEG2k). Further, RB was covalently conjugated to carboxyl groups generated from PMH on NaYF4:Tb using an ethylenediamine linker. NaYF4:Tb@RB exhibited a hydrodynamic diameter of ∼75 nm with a ζ-potential of -12 mV. NaYF4:Tb@RB efficiently generated ROS in cultured luciferase-expressing murine epithelial breast cancer (4T1-luc) cells under low dose X-ray irradiation (0.5 Gy). The ROS generation amounts of NaYF4:Tb@RB were 1.5-2-fold higher than those of NaGdF4:Tb@RB, in which host nanocrystals were prepared with optically active Gd3+. Flow cytometric and confocal microscopic analyses showed higher intracellular ROS production of NaYF4:Tb@RB, compared to NaYF4:Tb and RB, resulting in higher X-ray-induced DNA damage in cultured 4T1-luc cells. Ultimately, NaYF4:Tb@RB elicited significant cytotoxicity after X-ray irradiation (0.5 Gy), while inducing marginal cytotoxicity without X-ray irradiation. Altogether, this research proposes a promising ScNP design for efficient X-PDT agents that make the better use of incident X-ray energy while causing the fewest side effects.

Systemic single administration of anti-inflammatory microRNA 146a-5p loaded in polymeric nanomedicines with active targetability attenuates neointimal hyperplasia by controlling inflammation in injured arteries in a rat model.

Neointimal hyperplasia (NIH) after revascularization is a key unsolved clinical problem. Various studies have shown that attenuation of the acute inflammatory response on the vascular wall can prevent NIH. MicroRNA146a-5p (miR146a-5p) has been reported to show anti-inflammatory effects by inhibiting the NF-κB pathway, a well-known key player of inflammation of the vascular wall. Here, a nanomedicine, which can reach the vascular injury site, based on polymeric micelles was applied to deliver miR146a-5p in a rat carotid artery balloon injury model. In vitro studies using inflammation-induced vascular smooth muscle cell (VSMC) was performed. Results showed anti-inflammatory response as an inhibitor of the NF-κB pathway and VSMC migration, suppression of reactive oxygen species production, and proinflammatory cytokine gene expression in VSMCs. A single systemic administration of miR146a-5p attenuated NIH and vessel remodeling in a carotid artery balloon injury model in both male and female rats in vivo. MiR146a-5p reduced proinflammatory cytokine gene expression in injured arteries and monocyte/macrophage infiltration into the vascular wall. Therefore, miR146a-5p delivery to the injury site demonstrated therapeutic potential against NIH after revascularization.

Halofuginone micelle nanoparticles eradicate Nrf2-activated lung adenocarcinoma without systemic toxicity.

The Keap1-Nrf2 system is the master regulator of the cellular response against oxidative and xenobiotic stresses. Constitutive activation of Nrf2 is frequently observed in various types of cancers. Nrf2 hyperactivation induces metabolic reprogramming in cancer cells, which supports the increased energy demand required for rapid proliferation and confers high-level resistance against anticancer radio/chemotherapy. Hence, Nrf2 inhibition has emerged as an attractive therapeutic strategy to counter such acquired resistance in Nrf2-activated tumors. We previously identified Halofuginone (HF) as a promising Nrf2 inhibitor. In this study, we pursued preclinical characterization of HF and found that while HF markedly reduced the viability of cancer cells, it also caused severe hematopoietic and immune cell suppression in a dose-dependent manner. Hence, to overcome this toxicity, we decided to employ a nanomedicine approach to HF. We found that encapsulation of HF into a polymeric micelle (HF micelle; HFm) largely relieved the systemic toxicity exhibited by free HF while maintaining the tumor-suppressive properties of HF. LC-MS/MS analysis revealed that the reduction in the magnitude of adverse effects was the result of the ability to release HF from the HFm core in a slow and sustained manner. These results thus support the contention that HFm will potentially counteract Nrf2-activated cancers in the clinical settings.

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.

Experimental Comparison of Bond Lifetime and Viscoelastic Relaxation in Transient Networks with Well-Controlled Structures.

We demonstrate an experimental comparison of the bond lifetime, estimated using surface plasmon resonance (SPR), and the viscoelastic relaxation time of transient networks with well-controlled structures (dynamically cross-linked Tetra-PEG gel). SPR and viscoelastic measurements revealed that the temperature dependences of the two characteristic times are in agreement, while the viscoelastic response is delayed with respect to the lifetime by a factor of 2-3, dependent on the network strand length. Polymers cross-linked by temporary interactions form transient networks, which show fascinating viscoelasticity with a single relaxation mode. However, the molecular understanding of such simple viscoelasticity has remained incomplete because of the difficulty of experimentally evaluating bond lifetimes and heterogeneous structures in conventional transient networks. Our results suggest that bond dissociation and recombination both contribute to the macromechanical response. This report on direct bond-lifetime-viscoelastic-relaxation time comparison provides important information for the molecular design of transient network materials.

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.

Star-Polymer-DNA Gels Showing Highly Predictable and Tunable Mechanical Responses.

Dynamically crosslinked gels are appealing materials for applications that require time-dependent mechanical responses. DNA duplexes are ideal crosslinkers for building such gels because of their excellent sequence addressability and flexible tunability in bond energy. However, the mechanical responses of most DNA gels are complicated and unpredictable. Here, a DNA gel with a highly homogeneous gel network and well predictable mechanical behaviors is demonstrated by using a pair of star-polymer-DNA precursors with presimulated DNA sequences showing the two-state transition. The melting curve analysis of the DNA gels reveals the good correspondence between the thermodynamic potentials of the DNA crosslinkers and the presimulated values by DNA calculators. Stress-relaxation tests and dissociation kinetics measurements show that the macroscopic relaxation time of the DNA gels is approximately equal to the lifetime of the DNA crosslinkers over 4 orders of magnitude from 0.1-2000 s. Furthermore, a series of durability tests find the DNA gels are hysteresis-less and self-healable after the applications of repeated temperature and mechanical stimuli. These results demonstrate the great potential of star-polymer-DNA precursors for building gels with predictable and tunable viscoelastic properties, suitable for applications such as stress-response extracellular matrices, injectable solids, and soft robotics.

Synthetic molecule libraries for nucleic acid delivery: Design parameters in cationic/ionizable lipids and polymers.

Recent progress in the design of cationic lipids and polymers has successfully translated nucleic acid drugs into clinical applications, such as the treatment of liver diseases and the prevention of virus infection. Small or large libraries of delivery molecules have been used to find the key chemical structures to protect nucleic acids from nucleases in the extracellular milieu and to facilitate the endosomal escape after endocytosis. This review introduces three essential design parameters (i.e., acid dissociation constant, hydrophobicity, and biodegradability) to develop synthetic molecules for nucleic acid delivery. The significance and mechanism of each parameter are described based on the results obtained from in vitro and in vivo evaluations. Other design parameters were then discussed to create the next generation of delivery molecules for future nucleic acid therapeutics.

Dynamic Stabilization of Unit Polyion Complexes Incorporating Small Interfering RNA by Fine-Tuning of Cationic Block Length in Two-Branched Poly(ethylene glycol)-b-poly(l-lysine).

To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The blood retention time of uPICs is dramatically increased in the presence of free bPEG-PLys, suggesting dynamic stabilization of uPICs by free bPEG-PLys based on their equilibrium. Herein, we examined how the degree of polymerization of PLys (DPPLys) affected the dynamic stability of uPICs in the bloodstream during prolonged circulation. We prepared a series of bPEG-PLys with DPPLys values of 10, 13, 20, 40, and 80 for the uPIC formation and siRNA with 40 negative charges. These bPEG-PLys were then evaluated in physicochemical characterization and pharmacokinetic analyses. Structural analyses revealed that the uPIC size and association numbers were mainly determined by the molecular weights of PEG and DPPLys, respectively. Under bPEG-PLys-rich conditions, the hydrodynamic diameters of uPICs were 15-20 nm, which were comparable to that of the bPEG block (i.e., ∼18 nm). Importantly, DPPLys significantly affected the association constant of bPEG-PLys to siRNA (Ka) and blood retention of free bPEG-PLys. A smaller DPPLys resulted in a lower Ka and a longer blood retention time of free bPEG-PLys. Thus, DPPLys can control the dynamic stability of uPICs, i.e., the balance between Ka and blood concentration of free bPEG-PLys. Ultimately, the bPEG-PLys with DPPLys values of 14 and 19 prolonged the blood circulation of siRNA-loaded uPICs with relatively small amounts of free bPEG-PLys. This study revealed that the uPIC formation between siRNA and bPEG-PLys can be controlled by their charges, which may be helpful for designing PIC-based delivery systems.

Block catiomer with flexible cationic segment enhances complexation with siRNA and the delivery performance in vitro.

RNA interference (RNAi) by small interfering RNAs (siRNAs) is a promising therapeutic approach. Because siRNA has limited intracellular access and is rapidly cleared in vivo, the success of RNAi depends on efficient delivery technologies. Particularly, polyion complexation between block catiomers and siRNA is a versatile approach for constructing effective carriers, such as unit polyion complexes (uPIC), core-shell polyion complex (PIC) micelles and vesicular siRNAsomes, by engineering the structure of block catiomers. In this regard, the flexibility of block catiomers could be an important parameter in the formation of PIC nanostructures with siRNA, though its effect remains unknown. Here, we studied the influence of block catiomer flexibility on the assembly of PIC structures with siRNA using a complementary polymeric system, i.e. poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and PEG-poly(glycidylbutylamine) (PEG-PGBA), which has a relatively more flexible polycation segment than PEG-PLL. Mixing PEG-PGBA with siRNA at molar ratios of primary amines in polymer to phosphates in the siRNA (N/P ratios) higher than 1.5 promoted the multimolecular association of uPICs, whereas PEG-PLL formed uPIC at all N/P ratios higher than 1. Moreover, uPICs from PEG-PGBA were more stable against counter polyanion exchange than uPICs from PEG-PLL, probably due to a favorable complexation process, as suggested by computational studies of siRNA/block catiomer binding. In in vitro experiments, PEG-PGBA uPICs promoted effective intracellular delivery of siRNA and efficient gene knockdown. Our results indicate the significance of polycation flexibility on assembling PIC structures with siRNA, and its potential for developing innovative delivery systems.

Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.