Multimodal Golden DNA Superstructures (GDSs) for Highly Efficient Photothermal Immunotherapy.

DNA-templated metallization has emerged as an efficient strategy for creating nanoscale-metal DNA hybrid structures with a desirable conformation and function. Despite the potential of DNA-metal hybrids, their use as combinatory therapeutic agents has rarely been examined. Herein, we present a simple approach for fabricating a multipurpose DNA superstructure that serves as an efficient photoimmunotherapy agent. Specifically, we adsorb and locally concentrate Au ions onto DNA superstructures through induced local reduction, resulting in the formation of Au nanoclusters. The mechanical and optical properties of these metallic nanoclusters can be rationally controlled by their conformations and metal ions. The resulting golden DNA superstructures (GDSs) exhibit significant photothermal effects that induce cancer cell apoptosis. When sequence-specific immunostimulatory effects of DNA are combined, GDSs provide a synergistic effect to eradicate cancer and inhibit metastasis, demonstrating potential as a combinatory therapeutic agent for tumor treatment. Altogether, the DNA superstructure-templated metal casting system offers promising materials for future biomedical applications.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Construction of a two-dimensional DNA-RNA hybridized membrane for collecting tumor-derived exosomes.

Macroscopic nucleic acid-based structures have attracted much attention in biomedical fields. Here, we introduce a novel DNA-RNA hybridized membrane structure via enzymatic dual polymerization. The membrane exhibited enhanced rigidity and functionality. Encoded with an aptamer, the membrane showed great potential as a collecting platform of tumor-derived exosomes without additional labeling.

Focused ultrasound-triggered chemo-gene therapy with multifunctional nanocomplex for enhancing therapeutic efficacy.

Nanotechnology-based combination therapies, especially chemo-gene therapy, have been spotlighted as promising alternatives for cancer therapy. However, only a small amount of systemically administered nanomedicines reach the tumor site by the enhanced permeability and retention (EPR) effect, resulting in the limited therapeutic efficacy. Furthermore, the design of ideal drug delivery system for chemo-gene therapy has been impeded by the chemical and physical differences between nucleic acids and chemotherapeutics. Herein, we report a precisely designed nanocomplex which exhibits a focused ultrasound (FU)-responsive release and enhanced accumulation of released therapeutics to tumor site. After the nanocomplex composed of siRNA nanoparticles (siRNA-NP) and chemotherapeutics-loaded microbubbles was systemically injected, the nanocomplex was collapsed around the tumor tissue by FU exposure, and both siRNA-NP and chemotherapeutics were penetrated the dense extracellular matrix (ECM) of tumor site, leading to the enhanced chemo-gene therapeutic efficacy. The two-in-one nanocomplex is expected as a promising platform for combination therapy that can enhance the therapeutic efficiency of combination drugs at the cell and/or tissue levels with high drug loading ratio.

Engineered extracellular vesicles and their mimetics for clinical translation.

Cells secrete extracellular vesicles (EVs) to external environments to achieve cellular homeostasis and cell-to-cell communication. Their therapeutic potential has been constantly spotlighted since they mirror both cytoplasmic and membranous components of parental cells. Meanwhile, growing evidence suggests that EV engineering could further promote EVs with a maximized capacity. In this review, a range of engineering techniques as well as upscaling approaches to exploit EVs and their mimetics are introduced. By laying out the pros and cons of each technique from different perspectives, we sought to provide an overview potentially helpful for understanding the current state of the art EV engineering and a guideline for choosing a suitable technique for engineering EVs. Furthermore, we envision that the advances in each technique will give rise to the combinatorial engineering of EVs, taking us a step closer to a clinical translation of EV-based therapeutics.

BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo.

USE1 has been demonstrated to play crucial roles in the development and progression of human lung cancer. However, the antitumor efficacy of RNA interference (RNAi) targeting of USE1 has not yet been evaluated as a possible clinical application. We here synthesized USE1 targeting bubbled RNA-based cargo (BRC) composed of densely packed multimeric pre-siRNAs with specific Dicer cleavage sites to enable efficient siRNA release upon entry to target cells. The physical entanglement and continuous networking of RNAs via hybridization during enzymatic replication serve as a driving force for the self-assembly of BRCs. These molecules effectively suppressed the transcription of their target genes, leading to tumor growth suppression in vitro and in vivo. Moreover, their repeated intravenous administration efficiently inhibited the growth of A549 tumor xenografts. Based on these findings of a reduced cancer cell viability following a USE1 knockdown, we further explored cell cycle arrest and apoptosis pathways. The observed tumor cell growth suppression was found to be controlled by cell cycle arrest and apoptosis signals induced by the USE1 reduction. These results suggest that USE1 BRCs may have future clinical applications as an RNAi-based cancer therapy.

Immunostimulatory Effects Triggered by Self-Assembled Microspheres with Tandem Repeats of Polymerized RNA Strands.

Self-assembled RNA particles have been exploited widely to maximize the therapeutic potential of RNA. However, the immune response via RNA particles is not fully understood. In addition, the investigation of the immunogenicity from RNA-based particles is required owing to inherent immunostimulatory effects of RNA for clinical translation. To examine the immune stimulating potency, rationally designed microsized RNA particles, called RNA microspheres (RMSs), are generated with single or double strands via rolling circle transcription. The RMSs show an exceptional stability in the presence of serum, while they are selectively degraded under endolysosomal conditions. With precisely controlled size, both RMSs are successfully taken up by macrophages. Unlike the nature of RNA fragments, RMSs induce only basal-level expression of inflammatory cytokines as well as type I interferon from macrophages, suggesting that RMSs are immunocompatible in the therapeutic dose range. Taken together, this study could help accelerate clinical translation and broaden the applicability of the self-assembled RNA-based particles without being limited by their potential immunotoxicity, while a systematic controllability study observing the release of RNA fragments from RMSs would provide self-assembled RNA-based structures with a great potential for immunomodulation.

A Novel Technique for Cervical Laminoplasty Fusion: Simultaneously Enhancing Stabilization and Decompression in Various Cervical Myelopathies: A Technical Note and Outcomes.

BACKGROUND: Cervical laminectomy has 2 major disadvantages: postlaminectomy adhesion of dural membrane and lack of a fusion bed. The objective of this study was to determine whether simultaneous cervical laminoplasty with fusion (CLPF) might overcome these unwanted outcomes. METHODS: Patients who underwent CLPF for treating cervical myelopathy with instability who were followed up for at least 12 months were enrolled. Preoperative and postoperative Neck Disability Index (NDI) and Japanese Orthopedic Association (JOA) scores before and after surgery, recovery rates (RRs), C2-C7 lordosis, and fusion success rates were evaluated. RESULTS: The study cohort comprised 50 patients (35 males and 15 females; mean age, 60.5 ± 14.0 years) who underwent CLPF. The average duration of clinical follow-up was 24.6 ± 16.1 months. Mean preoperative and postoperative NDI scores were 27.0 ± 10.6 and 17.6 ± 7.2, respectively (P = 0.004). Mean preoperative and postoperative JOA scores were 10.4 ± 4.2 and 13.6 ± 3.0, respectively (P = 0.001). The mean JOA RR was 49.8 ± 42.2%. No significant changes in C2-7 lordosis were noted after surgery (preoperative, 7.0 ± 8.0°; postoperative, 7.3 ± 6.3°; P = 0.789). The fusion success rate was 96% (48 of 50 patients). Fusion mass areas at C5 level were significantly different between the opening side and the hinge side (opening side, 15.8 ± 13.1 mm2; hinge side, 50.8 ± 27.2 mm2; P < 0.001). There was no postoperative restenosis or epidural fibrosis. CONCLUSIONS: CLPF might be useful for canal decompression and a good fusion bed while avoiding postoperative epidural fibrosis.

Therapeutic effects of a novel siRNA-based anti-VEGF (siVEGF) nanoball for the treatment of choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries and is characterized by the development of choroidal neovascularization (CNV). Therapies for AMD have focused on suppressing angiogenic factors, such as vascular endothelial growth factor (VEGF), mainly via conventional anti-VEGF antibody agents. However, additional efforts have been made to develop effective small-interfering RNA (siRNA)-based intracellular therapeutic agents. In this study, we have manufactured a novel siRNA-based anti-VEGF nanoball (siVEGF NB). The siVEGF NB was composed of a siRNA hydrogel with a core of anti-VEGF sequence siRNA coated with branched PEI (bPEI) and hyaluronic acid (HA) in order by applying an electrical force. The novel siVEGF NBs, which were employed in a laser-induced CNV mouse model, were optimized as a retinal and choroidal delivery system through the vitreous humor to the sub-retinal space via CD44 receptor endocytosis on the inner limiting membrane, and showed therapeutic effects via pathways bypassing the TLR3-induced siRNA-class effect. The therapeutic effects of siVEGF NBs lasted for 2 weeks after intravitreal injection showing high targeting efficiency to the sub-retinal space. Thus, the newly developed siVEGF NB may have great potential for the delivery of RNAi-based therapeutics for ocular diseases, including AMD.

Synthesis of a multi-functional DNA nanosphere barcode system for direct cell detection.

Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling circle amplification (RCA) with several functionalized nucleotides. DNANSs with a barcode system were designed to exhibit fluorescence for coding enhanced signals and contain biotin for more functionalities, including targeting through the biotin-streptavidin (biotin-STA) interaction. Functionalized deoxynucleotide triphosphates (dNTPs) were mixed in the RCA process and functional moieties can be expressed on the DNANSs. The anti-epidermal growth factor receptor antibodies (anti-EGFR Abs) can be conjugated on DNANSs for targeting cancer cells specifically. As a proof of concept, the potential of the multi-functional DNANS barcode was demonstrated by direct cell detection as a simple detection method. The DNANS barcode provides a new route for the simple and rapid selective recognition of cancer cells.

Bubbled RNA-Based Cargo for Boosting RNA Interference.

As ribonucleic acid (RNA) nanotechnology has advanced, it has been applied widely in RNA-based therapeutics. Among the range of approaches, enzymatically synthesized RNA structures for inducing RNA interference in cancer cells have potential for silencing genes in a target-specific manner. On the other hand, the efficiency of gene silencing needs to be improved to utilize the RNA-based system for RNAi therapeutics. This paper introduces a new approach for efficient generation of siRNA from bubbled RNA-based cargo (BRC). The presence of bubbles in between to avoid nonfunctional short dsRNAs allows the RNA-based cargoes to contain multiple Dicer-cleavage sites to release the functional siRNAs when introduced to cells. BRCs can be synthesized easily in a one-pot process and be purified by simple centrifugation. Furthermore, efficient target gene silencing by the bubbled structure is confirmed both in vitro and in vivo. Therefore, this bubbled RNA cargo system can be utilized for target-specific RNAi therapeutics with high efficiency in the generation of functional siRNAs in the target cells.

Poly-sgRNA/siRNA ribonucleoprotein nanoparticles for targeted gene disruption.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) can be used for the specific disruption of a target gene to permanently suppress the expression of the protein encoded by the target gene. Efficient delivery of the system to an intracellular target site should be achieved to utilize the tremendous potential of the genome-editing tool in biomedical applications such as the knock-out of disease-related genes and the correction of defect genes. Here, we devise polymeric CRISPR/Cas9 system based on poly-ribonucleoprotein (RNP) nanoparticles consisting of polymeric sgRNA, siRNA, and Cas9 endonuclease in order to improve the delivery efficiency. When delivered by cationic lipids, the RNP nanoparticles built with chimeric poly-sgRNA/siRNA sequences generate multiple sgRNA-Cas9 RNP complexes upon the Dicer-mediated digestion of the siRNA parts, leading to more efficient disruption of the target gene in cells and animal models, compared with the monomeric sgRNA-Cas9 RNP complex.

Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for Efficient Gene Expression.

Although mRNA has several advantages over plasmid DNA when delivered into cells for gene expression, mRNA transfection is a very rare occurrence in gene delivery. This is mainly because of the labile nature of RNA, resulting in a low expression level of the desired protein. In this study, self-assembled mRNA nanoparticles (mRNA-NPs) packed with multiple repeats of mRNA were synthesized to achieve efficient gene expression. This approach required only a one-step process to synthesize particles with a minimal amount of plasmid DNA to produce the RNA transcripts via rolling circle transcription. Moreover, there are no concerns for cytotoxicity which can be caused by chemical condensates because mRNA-NPs are made entirely of mRNA. An examination of the cells transfected with the mRNA-NPs encoding the green fluorescence protein (GFP) confirmed that the mRNA-NPs can be used as a novel platform for effective gene delivery.

Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics.

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

Controlled release of an anti-cancer drug from DNA structured nano-films.

We demonstrate the generation of systemically releasable anti-cancer drugs from multilayer nanofilms. Nanofilms designed to drug release profiles in programmable fashion are promising new and alternative way for drug delivery. For the nanofilm structure, we synthesized various unique 3-dimensional anti cancer drug incorporated DNA origami structures (hairpin, Y, and X shaped) and assembled with peptide via layer-by-layer (LbL) deposition method. The key to the successful application of these nanofilms requires a novel approach of the influence of DNA architecture for the drug release from functional nano-sized surface. Herein, we have taken first steps in building and controlling the drug incorporated DNA origami based multilayered nanostructure. Our finding highlights the novel and unique drug release character of LbL systems in serum condition taken full advantages of DNA origami structure. This multilayer thin film dramatically affects not only the release profiles but also the structure stability in protein rich serum condition.

Graphene multilayers as gates for multi-week sequential release of proteins from surfaces.

The ability to control the timing and order of release of different therapeutic drugs will play a pivotal role in improving patient care and simplifying treatment regimes in the clinic. The controlled sequential release of a broad range of small and macromolecules from thin film coatings offers a simple way to provide complex localized dosing in vivo. Here we show that it is possible to take advantage of the structure of certain nanomaterials to control release regimes from a scale of hours to months. Graphene oxide (GO) is a two-dimensional charged nanomaterial that can be used to create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. Protein-loaded polyelectrolyte multilayer films were fabricated using layer-by-layer assembly incorporating a hydrolytically degradable cationic poly(ß-amino ester) (Poly1) with a model protein antigen, ovalbumin (ova), in a bilayer architecture along with positively and negatively functionalized GO capping layers for the degradable protein films. Ova release without the GO layers takes place in less than 1 h but can be tuned to release from 30 to 90 days by varying the number of bilayers of functionalized GO in the multilayer architecture. We demonstrate that proteins can be released in sequence with multi-day gaps between the release of each species by incorporating GO layers between protein loaded layers. In vitro toxicity assays of the individual materials on proliferating hematopoietic stem cells (HSCs) indicated limited cytotoxic effects with HSCs able to survive for the full 10 days of normal culture in the presence of Poly1 and the GO sheets. This approach provides a new route for storage of therapeutics in a solid-state thin film for subsequent delivery in a time-controlled and sequential fashion.

Controlling in vivo stability and biodistribution in electrostatically assembled nanoparticles for systemic delivery.

This paper demonstrates the generation of systemically deliverable layer-by-layer (LbL) nanoparticles for cancer applications. LbL-based nanoparticles designed to navigate the body and deliver therapeutics in a programmable fashion are promising new and alternative systems for drug delivery, but there have been very few demonstrations of their systemic delivery in vivo due to a lack of knowledge in building LbL nanofilms that mimic traditional nanoparticle design to optimize delivery. The key to the successful application of these nanocarriers in vivo requires a systematic analysis of the influence of film architecture and adsorbed polyelectrolyte outer layer on their pharmacokinetics, which has thus far not been examined for this new approach to nanoparticle delivery. Herein, we have taken the first steps in stabilizing and controlling the systemic distribution of multilayer nanoparticles. Our findings highlight the unique character of LbL systems; the electrostatically assembled nanoparticles gain increased stability in vivo with larger numbers of deposited layers, and the final layer adsorbed generates a critical surface cascade, which dictates the surface chemistry and biological properties of the nanoparticle. This outer polyelectrolyte layer dramatically affects not only the degree of nonspecific particle uptake, but also the nanoparticle biodistribution. For hyaluronic acid (HA) outer layers, a long blood elimination half-life (∼9 h) and low accumulation (∼10-15% recovered fluorescence/g) in the liver were observed, illustrating that these systems can be designed to be highly appropriate for clinical translation.