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.

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.

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.

In situ-formable, dynamic crosslinked poly(ethylene glycol) carrier for localized adeno-associated virus infection and reduced off-target effects.

The adeno-associated virus (AAV) is a potent vector for in vivo gene transduction and local therapeutic applications of AAVs, such as for skin ulcers, are expected. Localization of gene expression is important for the safety and efficiency of genetic therapies. We hypothesized that gene expression could be localized by designing biomaterials using poly(ethylene glycol) (PEG) as a carrier. Here we show one of the designed PEG carriers effectively localized gene expression on the ulcer surface and reduced off-target effects in the deep skin layer and the liver, as a representative organ to assess distant off-target effects, using a mouse skin ulcer model. The dissolution dynamics resulted in localization of the AAV gene transduction. The designed PEG carrier may be useful for in vivo gene therapies using AAVs, especially for localized expression.

Molecular Weight-Dependent Diffusion, Biodistribution, and Clearance Behavior of Tetra-Armed Poly(ethylene glycol) Subcutaneously Injected into the Back of Mice.

Four-armed poly(ethylene glycol) (PEG)s are essential hydrophilic polymers extensively utilized to prepare PEG hydrogels, which are valuable tissue scaffolds. When hydrogels are used in vivo, they eventually dissociate due to cleavage of the backbone structure. When the cleavage occurs at the cross-linking point, the hydrogel elutes as an original polymer unit, i.e., four-armed PEG. Although four-armed PEGs have been utilized as subcutaneously implanted biomaterials, the diffusion, biodistribution, and clearance behavior of four-armed PEG from the skin are not fully understood. This paper investigates time-wise diffusion from the skin, biodistribution to distant organs, and clearance of fluorescence-labeled four-armed PEGs with molecular weight (Mw) ranging from 5-40 kg/mol subcutaneously injected into the back of mice. Changes over time indicated that the fate of subcutaneously injected PEGs is Mw-dependent. Four-armed PEGs with Mw ≤ 10 kg/mol gradually diffused to deep adipose tissue beneath the injection site and distributed dominantly to distant organs, such as the kidney. PEGs with Mw ≥ 20 kg/mol stagnated in the skin and deep adipose tissue and were mainly delivered to the heart, lung, and liver. The fundamental understanding of the Mw-dependent behavior of four-armed PEGs is beneficial for preparing biomaterials using PEGs, providing a reference in the field of tissue engineering.

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.

Effects of Oxygen Gas Injection on the Subsequent Development of Chick Embryos in a Shell-Less Culture System.

The effects of oxygen gas injection starting on day 17 of incubation (D17) in a chick shell-less culture system (cSLC) on the subsequent embryo development were examined on day 19 of incubation (D19). On D19 of cSLC, the plasma phosphorus and total cholesterol concentrations of the embryos were significantly higher (P<0.05), while the plasma calcium concentrations were significantly lower (P<0.05) than those in the intact control (IC) group. However, no significant differences in embryo viability and other major blood component levels were observed among the experimental groups (P>0.05). The percutaneous oxygen saturation was lower in D17-cSLC embryos before oxygen gas supplementation than in the IC (P<0.05) embryos. Severe renal tubular degeneration of the metanephros was observed in D19-cSLC embryos despite oxygen gas injection starting from D17. These results indicate that D19-cSLC embryos are hypoxia even after injecting oxygen gas starting on D17.

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.

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.

Bridging mRNA and Polycation Using RNA Oligonucleotide Derivatives Improves the Robustness of Polyplex Micelles for Efficient mRNA Delivery.

Polyplex for messenger RNA (mRNA) delivery requires strong yet reversible association between mRNA and polycation for extracellular robustness and selective intracellular disintegration. Herein, RNA oligonucleotide (OligoRNA) derivatives that bridge mRNA and polycation are developed to stabilize polyplex micelles (PMs). A set of the OligoRNAs introduced with a polyol moiety in their 5' end is designed to hybridize to fixed positions along mRNA strand. After PM preparation from the hybridized mRNA and poly(ethylene glycol)-polycation block copolymer derived with phenylboronic acid (PBA) moieties in its cationic segment, PBA moieties form reversible phenylboronate ester linkages with a polyol moiety at 5' end of OligoRNAs and a diol moiety at their 3' end ribose, in the PM core. The OligoRNAs work as a node to bridge ionically complexed mRNA and polycation, thereby improving PM stability against polyion exchange reaction and ribonuclease attack in extracellular environment. After cellular uptake, intracellular high concentration of adenosine triphosphate triggers the cleavage of phenylboronate ester linkages, resulting in mRNA release from PM. Ultimately, the PM provides efficient mRNA introduction in cultured cells and mouse lungs after intratracheal administration, demonstrating the potential of the bridging strategy in polyplex-based mRNA delivery.

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.

Cancer-Specific Targeting of Taurine-Upregulated Gene 1 Enhances the Effects of Chemotherapy in Pancreatic Cancer.

Overcoming drug resistance is one of the biggest challenges in cancer chemotherapy. In this study, we examine whether targeting the long noncoding RNA taurine upregulated gene 1 (TUG1) could be an effective therapeutic approach to overcome drug resistance in pancreatic ductal adenocarcinoma (PDAC). TUG1 was expressed at significantly higher levels across 197 PDAC tissues compared with normal pancreatic tissues. Overall survival of patients with PDAC who had undergone 5-FU-based chemotherapy was shorter in high TUG1 group than in low TUG1 group. Mechanistically, TUG1 antagonized miR-376b-3p and upregulated dihydropyrimidine dehydrogenase (DPD). TUG1 depletion induced susceptibility to 5-FU in BxPC-3 and PK-9 pancreatic cell lines. Consistently, the cellular concentration of 5-FU was significantly higher under TUG1-depleted conditions. In PDAC xenograft models, intravenous treatment with a cancer-specific drug delivery system (TUG1-DDS) and 5-FU significantly suppressed PDAC tumor growth compared with 5-FU treatment alone. This novel approach using TUG1-DDS in combination with 5-FU may serve as an effective therapeutic option to attenuate DPD activity and meet appropriate 5-FU dosage requirements in targeted PDAC cells, which can reduce the systemic adverse effects of chemotherapy. SIGNIFICANCE: Targeting TUG1 coupled with a cancer-specific drug delivery system effectively modulates 5-FU catabolism in TUG1-overexpressing PDAC cells, thus contributing to a new combinatorial strategy for cancer treatment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1654/F1.large.jpg.

Structural tuning of oligonucleotides for enhanced blood circulation properties of unit polyion complexes prepared from two-branched poly(ethylene glycol)-block-poly(l-lysine).

Downsizing nanocarriers is a promising strategy for systemically targeting fibrotic cancers, such as pancreatic cancer, owing to enhanced tissue permeability. We recently developed a small oligonucleotide nanocarrier called a unit polyion complex (uPIC) using a single oligonucleotide molecule and one or two molecule(s) of two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The uPIC is a dynamic polyion-pair equilibrated with free bPEG-PLys, and thus, is highly stabilized in the presence of excess amounts of free bPEG-PLys in the bloodstream. However, the dynamic polyion-pairing behavior of uPICs needs to be further investigated for longevity in the bloodstream, especially under lower amounts of free bPEG-PLys. Herein, the polyion-pairing behavior of uPICs was investigated by highlighting oligonucleotide stability and negative charge number. To this end, small interfering RNA (siRNA) and antisense oligonucleotides (ASO) were chemically modified to acquire nuclease resistance, and the ASO was hybridized with complementary RNA (cRNA) to form a hetero-duplex oligonucleotide (HDO) with twice the negative charges. While all oligonucleotides similarly formed sub-20 nm-sized uPICs from a single oligonucleotide molecule, the association number of bPEG-PLys (ANbPEG-PLys) in uPICs varied based on the negative charge number of oligonucleotides (N-), that is, ANbPEG-PLys = ~2 at N- = ~40 (i.e., siRNA and HDO) and ANbPEG-PLys = ~1 at N- = 20 (i.e., ASO), presumably because of the balanced charge neutralization between the oligonucleotide and bPEG-PLys with a positive charge number (N+) of ~20. Ultimately, the uPICs prepared from the chemically modified oligonucleotide with higher negative charges showed considerably longer blood retention than those from the control oligonucleotides without chemical modifications or with lower negative charges. The difference in the blood circulation properties of uPICs was more pronounced under lower amounts of free bPEG-PLys. These results demonstrate that the chemical modification and higher negative charge in oligonucleotides facilitated the polyion-pairing between the oligonucleotide and bPEG-PLys under harsh biological conditions, facilitating enhanced blood circulation of uPICs.

Bioinspired Silicification of mRNA-Loaded Polyion Complexes for Macrophage-Targeted mRNA Delivery.

In vitro transcribed messenger RNA (mRNA) delivery to macrophages is a promising therapeutic modality for inflammatory diseases because it can modulate the immunological activity of macrophages. However, efficient macrophage-targeted mRNA delivery remains challenging. Herein, we fabricated silica-coated polyion complexes (PICs), termed SilPICs, via bioinspired silicification for stable encapsulation of mRNA and scavenger receptor (SR)-mediated macrophage targeting. Silica coating was readily performed by simply mixing mRNA-loaded PICs with tetramethyl orthosilicate in aqueous media at 25 °C. The silica shell formation was verified by a slight increase in size (∼18 nm), a conversion of ζ-potential from positive (+22 mV) to negative (-23 mV), the peak appearance derived from silanol groups and siloxane bonds in the IR spectra, and elemental analyses by scanning transmission electron microscopy-energy-dispersive X-ray spectrometry (STEM-EDS). The silica shell efficiently protected the mRNA payload from enzymatic degradation in a fetal bovine serum-containing medium. Meanwhile, the reversibility of the silica shell allowed mRNA release from SilPICs after silica dissolution into silicic acids under diluted conditions. Furthermore, SilPICs elicited 20-fold higher mRNA transfection efficiency in the macrophage cell line RAW264.7 compared to noncoated PICs, presumably due to the facilitated cellular internalization by the silica shell. These enhancements were compromised in the RAW264.7 cells incubated with dextran sulfate and poly(inosinic acid) as inhibitors of SR type A1 and were not observed in cultured CT26 colon cancer cells, which are SR-negative cells. Collectively, SilPIC is a promising mRNA delivery vehicle with both mRNA protectability and macrophage targetability.

mRNA loading into ATP-responsive polyplex micelles with optimal density of phenylboronate ester crosslinking to balance robustness in the biological milieu and intracellular translational efficiency.

Carriers for messenger RNA (mRNA) delivery require propensities to protect the mRNA from enzymatic degradation and to selectively release mRNA in the cytosol for smooth mRNA translation. To meet these requirements, we designed mRNA-loaded polyplex micelles (PMs) with ATP-responsive crosslinking in the inner core by complexing mRNA with poly(ethylene glycol)-polycation block copolymers derivatized with phenylboronic acid and polyol groups, which form crosslinking structures via spontaneous phenylboronate ester formation. PMs thus prepared are tolerable against enzymatic attack and, in turn, disintegrate in the cytosol to release mRNA when triggered by the cleavage of phenylboronate ester linkages in response to elevated ATP concentration. Two structural factors of the PM, including (i) the introduction ratios of phenylboronate ester crosslinkers and (ii) the structure and protonation degree of amino groups in the polycation segment, are critical for maximizing protein expression in cultured cells due to the optimized balance between the robustness in the biological milieu and the ATP-responsive mRNA release in the cytosol. The optimal PM formulation was further stabilized by installing cholesterol moieties into both the mRNA and ω-end of the block copolymer to elicit longevity in blood circulation after intravenous injection.

Noncovalent Stabilization of Vesicular Polyion Complexes with Chemically Modified/Single-Stranded Oligonucleotides and PEG-b-guanidinylated Polypeptides for Intracavity Encapsulation of Effector Enzymes Aimed at Cooperative Gene Knockdown.

For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate a multimolecular (or secondary) association of unit PICs for noncovalent (or chemical cross-linking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be ∼2500 using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at ∼10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of noncovalently stabilized nanovesicles for the codelivery of two varying bio-macromolecule payloads for ensuring their cooperative biological activity.

Photo-reactive oligodeoxynucleotide-embedded nanovesicles (PROsomes) with switchable stability for efficient cellular uptake and gene knockdown.

A photo-responsive nanovesicle is fabricated by polyion complex (PIC) formation between poly(ethylene glycol) (PEG)-block-polypeptides and photo-reactive oligodeoxynucleotides (PROs)/anti-sense oligonucleotides (ASOs). The ultraviolet (UV) light triggers reversible crosslinking between PROs and ASOs in the vesicular membrane, providing the nanovesicle with switchable stability under physiological conditions. The resulting nanovesicle allows efficient cellular internalization, leading to significant UV-triggered gene knockdown in cultured cells.

Proliferation-associated long noncoding RNA, TMPO-AS1, is a potential therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer compared with luminal or epidermal growth factor receptor 2 subtypes, thus effective therapeutic options for TNBC are yet to be developed. Nowadays, oncogenic long noncoding RNAs (lncRNAs) are applied to cancer management as a new class of therapeutic targets. We previously showed that thymopoietin antisense transcript 1 (TMPO-AS1) is a proliferation-associated lncRNA that contributes to hormone-dependent breast cancer progression by stabilizing estrogen receptor-α mRNA. We here showed that TMPO-AS1 is abundantly expressed in basal-like breast cancer subtype based on the transcriptomic data in The Cancer Genome Atlas as well as in TNBC cell lines and patient-derived cells. Small interfering RNA-based loss-of-function analyses showed that TMPO-AS1 knockdown substantially represses the proliferation and migration of TNBC cells. Expression microarray analysis showed that TMPO-AS1 alters gene signatures related to transforming growth factor-ß signaling in addition to proliferative E2F signaling pathways. TMPO-AS1-targeted siRNA treatment through engineered drug delivery systems using cancer-targeted polyion complex micelle or nanoball technology significantly impaired the in vivo growth of primary and metastatic TNBC xenograft tumors. Our findings suggest that TMPO-AS1 plays a key role in TNBC pathophysiology and could be a potential therapeutic target for TNBC.

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.