Electric Field-Responsive Gold Nanoantennas for the Induction of a Locoregional Tumor pH Change Using Electrolytic Ablation Therapy.

Electrolytic ablation (EA) is a burgeoning treatment for solid tumors, in which electrical energy catalyzes a chemical reaction to generate reactive species that can eradicate cancer cells. However, the application of this technique has been constrained owing to the limited spatial effectiveness and complexity of the electrode designs. Therefore, the incorporation of nanotechnology into EA is anticipated to be a significant improvement. Herein, we present a therapeutic approach based on difructose dianhydride IV-conjugated polyethylenimine-polyethylene glycol-modified gold nanorods as electric nanoantennas and nanoelectrocatalysts for EA. We demonstrate that square-wave direct current (DC) fields trigger a reaction between water molecules and chloride ions on the gold nanorod surface, generating electrolytic products including hydrogen, oxygen, and chlorine gases near the electrodes, changing the pH, and inducing cell death. These electric nanoantennas showed significant efficacy in treating colorectal cancer both in vitro and in vivo after DC treatment. These findings clearly indicate that gold nanoantennas enhance the effectiveness of EA by creating a localized electric field and catalyzing electrolytic reactions for the induction of locoregional pH changes within the tumor. By overcoming the limitations of traditional EA and offering an enhanced level of tumor specificity and control, this nanotechnology-integrated approach advances further innovations in cancer therapies.

Triphenylphosphonium-Functionalized Gold Nanorod/Zinc Oxide Core-Shell Nanocomposites for Mitochondrial-Targeted Phototherapy.

Phototherapies, such as photothermal therapy (PTT) and photodynamic therapy (PDT), combined with novel all-in-one light-responsive nanocomposites have recently emerged as new therapeutic modalities for the treatment of cancer. Herein, we developed novel all-in-one triphenylphosphonium-functionalized gold nanorod/zinc oxide core-shell nanocomposites (CTPP-GNR@ZnO) for mitochondrial-targeted PTT/PDT owing to their good biocompatibility, tunable and high optical absorption, photothermal conversion efficiency, highest reactive oxygen species (ROS) generation, and high mitochondrial-targeting capability. Under laser irradiation of 780 nm, the CTPP-GNR@ZnO core-shell nanocomposites effectively produced heat in addition to generating ROS to induce cell death, implying a synergistic effect of mild PTT and PDT in combating cancer. Notably, the in vitro PTT/PDT effect of CTPP-GNR@ZnO core-shell nanocomposites exhibited effective cell ablation (95%) and induced significant intracellular ROS after the 780 nm laser irradiation for 50 min, indicating that CTPP in CTPP-GNR@ZnO core-shell nanocomposites can specifically target the mitochondria of CT-26 cells, as well as generate heat and ROS to completely kill cancer cells. Overall, this light-responsive nanocomposite-based phototherapy provides a new approach for cancer synergistic therapy.

Mesoporous Silica-Layered Gold Nanorod Core@Silver Shell Nanostructures for Intracellular SERS Imaging and Phototherapy.

Precision diagnosis-guided efficient treatment is crucial to extending the lives of cancer patients. The integration of surface-enhanced Raman scattering (SERS) imaging and phototherapy into a single nanoplatform has been considered a more accurate diagnosis and treatment strategy for cancer nanotheranostics. Herein, we constructed a new type of mesoporous silica-layered gold nanorod core@silver shell nanostructures loaded with methylene blue (GNR@Ag@mSiO2-MB) as a multifunctional nanotheranostic agent for intracellular SERS imaging and phototherapy. The synthesized GNR@Ag@mSiO2-MB nanostructures possessed a uniform core-shell structure, strong near-infrared (NIR) absorbance, photothermal conversion efficiency (65%), dye loading ability, SERS signal, and Raman stability under phototherapy conditions. Under single 785 nm NIR laser irradiation, the intracellular GNR@Ag@mSiO2-MB nanostructures were dramatically decreased to <9%, which showed excellent photothermal and photodynamic effects toward cancer cell killing, indicating that the combination of photothermal therapy (PTT) and photodynamic therapy (PDT) of the GNR@Ag@mSiO2-MB nanostructures could greatly enhance the therapeutic efficacy of cancer cell death. GNR@Ag@mSiO2-MB nanostructures demonstrated a strong Raman signal at 450 and 502 cm-1, corresponding to the δ(C-N-C) mode, suggesting that the Raman bands of GNR@Ag@mSiO2-MB nanostructures were more efficient to detect CT-26 cell SERS imaging with high specificity. Our results indicate that GNR@Ag@mSiO2-MB nanostructures offer an excellent multifunctional nanotheranostic platform for SERS imaging and synergistic anticancer phototherapy in the future.

Generation of a CAG-EGFP tagged cell line (KSCBi017-A-2) from human induced pluripotent stem cells using CRISPR/Cas9.

Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.

Biocompatible Calcium Ion-Doped Magnesium Ferrite Nanoparticles as a New Family of Photothermal Therapeutic Materials for Cancer Treatment.

Novel biocompatible and efficient photothermal (PT) therapeutic materials for cancer treatment have recently garnered significant attention, owing to their effective ablation of cancer cells, minimal invasiveness, quick recovery, and minimal damage to healthy cells. In this study, we designed and developed calcium ion-doped magnesium ferrite nanoparticles (Ca2+-doped MgFe2O4 NPs) as novel and effective PT therapeutic materials for cancer treatment, owing to their good biocompatibility, biosafety, high near-infrared (NIR) absorption, easy localization, short treatment period, remote controllability, high efficiency, and high specificity. The studied Ca2+-doped MgFe2O4 NPs exhibited a uniform spherical morphology with particle sizes of 14.24 ± 1.32 nm and a strong PT conversion efficiency (30.12%), making them promising for cancer photothermal therapy (PTT). In vitro experiments showed that Ca2+-doped MgFe2O4 NPs had no significant cytotoxic effects on non-laser-irradiated MDA-MB-231 cells, confirming that Ca2+-doped MgFe2O4 NPs exhibited high biocompatibility. More interestingly, Ca2+-doped MgFe2O4 NPs exhibited superior cytotoxicity to laser-irradiated MDA-MB-231 cells, inducing significant cell death. Our study proposes novel, safe, high-efficiency, and biocompatible PT therapeutics for treating cancers, opening new vistas for the future development of cancer PTT.

Antibody-conjugated and streptomycin-chitosan oligosaccharide-modified gold nanoshells for synergistic chemo-photothermal therapy of drug-resistant bacterial infection.

Despite the many advanced strategies that are available, rapid gene mutation in multidrug-resistant bacterial infections remains a major challenge. Combining new therapeutic strategies such as chemo-photothermal therapy (PTT) with high antibacterial efficiency against drug-resistant Listeria monocytogenes (LM) is urgently needed. Here, we report synergistic chemo-PTT against drug-resistant LM based on antibody-conjugated and streptomycin-chitosan oligosaccharide-modified gold nanoshells (anti-STR-CO-GNSs) as all-in-one nanotheranostic agents for the first time, which was used for accurate antibacterial applications. The anti-STR-CO-GNSs showed excellent photothermal conversion efficiency (31.97 %) and were responsive to near-infrared (NIR) and pH dual stimuli-triggered antibiotic release, resulting in outstanding chemo-photothermal effects against LM. In vitro chemo-photothermal effect of anti-STR-CO-GNSs with laser irradiation caused a greater antibacterial effect (1.37 %), resulting in more rapid killing of LM and prevention of LM regrowth. Most importantly, the mice receiving the anti-STR-CO-GNSs with laser irradiation specifically at the sites of LM infections healed almost completely, leaving only scars on the surface of the skin and resulting in superior inhibitory effects from combined chemo-PTT. Overall, our findings suggest that chemo-PTT using smart biocompatible anti-STR-CO-GNSs is a favorable potential alternative to combat the increasing threat of drug-resistant LM, which opens a new door for clinical anti-infection therapy in the future.

Methylene Blue-Loaded Mesoporous Silica-Coated Gold Nanorods on Graphene Oxide for Synergistic Photothermal and Photodynamic Therapy.

Photo-nanotheranostics integrates near-infrared (NIR) light-triggered diagnostics and therapeutics, which are combined into a novel all-in-one phototheranostic nanomaterial that holds great promise for the early detection and precise treatment of cancer. In this study, we developed methylene blue-loaded mesoporous silica-coated gold nanorods on graphene oxide (MB-GNR@mSiO2-GO) as an all-in-one photo-nanotheranostic agent for intracellular surface-enhanced Raman scattering (SERS) imaging-guided photothermal therapy (PTT)/photodynamic therapy (PDT) for cancer. Amine functionalization of the MB-GNR@mSiO2 surfaces was performed using 3-aminopropyltriethoxysilane (APTES), which was well anchored on the carboxyl groups of graphene oxide (GO) nanosheets uniformly, and showed a remarkably higher photothermal conversion efficiency (48.93%), resulting in outstanding PTT/PDT for cancer. The in vitro photothermal/photodynamic effect of MB-GNR@mSiO2-GO with laser irradiation showed significantly reduced cell viability (6.32%), indicating that MB-GNR@mSiO2-GO with laser irradiation induced significantly more cell deaths. Under laser irradiation, MB-GNR@mSiO2-GO showed a strong SERS effect, which permits accurate cancer cell detection by SERS imaging. Subsequently, the same Raman laser can focus on highly detected MDA-MB-23l cells for a prolonged time to perform PTT/PDT. Therefore, MB-GNR@mSiO2-GO has great potential for precise SERS imaging-guided synergistic PTT/PDT for cancer.

Recent advances in multifunctional nanomaterials for photothermal-enhanced Fenton-based chemodynamic tumor therapy.

Photothermal (PT)-enhanced Fenton-based chemodynamic therapy (CDT) has attracted a significant amount of research attention over the last five years as a highly effective, safe, and tumor-specific nanomedicine-based therapy. CDT is a new emerging nanocatalyst-based therapeutic strategy for the in situ treatment of tumors via the Fenton reaction or Fenton-like reaction, which has got fast progress in recent years because of its high specificity and activation by endogenous substances. A variety of multifunctional nanomaterials such as metal-, metal oxide-, and metal-sulfide-based nanocatalysts have been designed and constructed to trigger the in situ Fenton or Fenton-like reaction within the tumor microenvironment (TME) to generate highly cytotoxic hydroxyl radicals (•OH), which is highly efficient for the killing of tumor cells. However, research is still required to enhance the curative outcomes and minimize its side effects. Specifically, the therapeutic efficiency of certain CDTs is still hindered by the TME, including low levels of endogenous hydrogen peroxide (H2O2), overexpression of reduced glutathione (GSH), and low catalytic efficacy of Fenton or Fenton-like reactions (pH 5.6-6.8), which makes it difficult to completely cure cancer using monotherapy. For this reason, photothermal therapy (PTT) has been utilized in combination with CDT to enhance therapeutic efficacy. More interestingly, tumor heating during PTT not only causes damage to the tumor cells but can also accelerate the generation of •OH via the Fenton and Fenton-like reactions, thus enhancing the CDT efficacy, providing more effective cancer treatment when compared with monotherapy. Currently, synergistic PT-enhanced CDT using multifunctional nanomaterials with both PT and chemodynamic properties has made enormous progress in cancer theranostics. However, there has been no comprehensive review on this subject published to date. In this review, we first summarize the recent progress in PT-enhanced Fenton-based CDT for cancer treatment. We then discuss the potential and challenges in the future development of PT-enhanced Fenton-based nanocatalytic tumor therapy for clinical application.

Single-Cell RNA Sequencing of Human Pluripotent Stem Cell-Derived Macrophages for Quality Control of The Cell Therapy Product.

Macrophages exhibit high plasticity to achieve their roles in maintaining tissue homeostasis, innate immunity, tissue repair and regeneration. Therefore, macrophages are being evaluated for cell-based therapeutics against inflammatory disorders and cancer. To overcome the limitation related to expansion of primary macrophages and cell numbers, human pluripotent stem cell (hPSC)-derived macrophages are considered as an alternative source of primary macrophages for clinical application. However, the quality of hPSC-derived macrophages with respect to the biological homogeneity remains still unclear. We previously reported a technique to produce hPSC-derived macrophages referred to as iMACs, which is amenable for scale-up. In this study, we have evaluated the biological homogeneity of the iMACs using a transcriptome dataset of 6,230 iMACs obtained by single-cell RNA sequencing. The dataset provides a valuable genomic profile for understanding the molecular characteristics of hPSC-derived macrophage cells and provide a measurement of transcriptomic homogeneity. Our study highlights the usefulness of single cell RNA-seq data in quality control of the cell-based therapy products.

Modelling Toxoplasma gondii infection in human cerebral organoids.

Pluripotent stem cell-derived cerebral organoids have the potential to recapitulate the pathophysiology of in vivo human brain tissue, constituting a valuable resource for modelling brain disorders, including infectious diseases. Toxoplasma gondii, an intracellular protozoan parasite, infects most warm-blooded animals, including humans, causing toxoplasmosis. In immunodeficient patients and pregnant women, infection often results in severe central nervous system disease and fetal miscarriage. However, understanding the molecular pathophysiology of the disease has been challenging due to limited in vitro model systems. Here, we developed a new in vitro model system of T. gondii infection using human brain organoids. We observed that tachyzoites can infect human cerebral organoids and are transformed to bradyzoites and replicate in parasitophorous vacuoles to form cysts, indicating that the T. gondii asexual life cycle is efficiently simulated in the brain organoids. Transcriptomic analysis of T. gondii-infected organoids revealed the activation of the type I interferon immune response against infection. In addition, in brain organoids, T. gondii exhibited a changed transcriptome related to protozoan invasion and replication. This study shows cerebral organoids as physiologically relevant in vitro model systems useful for advancing the understanding of T. gondii infections and host interactions.

Generation of a TLR7 heterozygous knockout line (WAe009-A-18) from human embryonic stem cells using CRISPR/Cas9.

Toll-like receptor 7 (TLR7) is a member of the toll-like receptor (TLR) family that is essential in the innate immune system. In this study, we established a heterozygous TLR7 knockout H9 cell line using CRISPR/Cas9. TLR7 knockout H9 cells maintained their pluripotency and exhibited the ability to differentiate into the three germ layers without any karyotype abnormalities.

Development of genetic quality tests for good manufacturing practice-compliant induced pluripotent stem cells and their derivatives.

Although human induced pluripotent stem cell (hiPSC) lines are karyotypically normal, they retain the potential for mutation in the genome. Accordingly, intensive and relevant quality controls for clinical-grade hiPSCs remain imperative. As a conceptual approach, we performed RNA-seq-based broad-range genetic quality tests on GMP-compliant human leucocyte antigen (HLA)-homozygous hiPSCs and their derivatives under postdistribution conditions to investigate whether sequencing data could provide a basis for future quality control. We found differences in the degree of single-nucleotide polymorphism (SNP) occurring in cells cultured at three collaborating institutes. However, the cells cultured at each centre showed similar trends, in which more SNPs occurred in late-passage hiPSCs than in early-passage hiPSCs after differentiation. In eSNP karyotyping analysis, none of the predicted copy number variations (CNVs) were identified, which confirmed the results of SNP chip-based CNV analysis. HLA genotyping analysis revealed that each cell line was homozygous for HLA-A, HLA-B, and DRB1 and heterozygous for HLA-DPB type. Gene expression profiling showed a similar differentiation ability of early- and late-passage hiPSCs into cardiomyocyte-like, hepatic-like, and neuronal cell types. However, time-course analysis identified five clusters showing different patterns of gene expression, which were mainly related to the immune response. In conclusion, RNA-seq analysis appears to offer an informative genetic quality testing approach for such cell types and allows the early screening of candidate hiPSC seed stocks for clinical use by facilitating safety and potential risk evaluation.

BMP4 and perivascular cells promote hematopoietic differentiation of human pluripotent stem cells in a differentiation stage-specific manner.

The efficient and reproducible derivation and maturation of multipotent hematopoietic progenitors from human pluripotent stem cells (hPSCs) requires the recapitulation of appropriate developmental stages and the microenvironment. Here, using serum-, xeno-, and feeder-free stepwise hematopoietic induction protocols, we showed that short-term and high-concentration treatment of hPSCs with bone morphogenetic protein 4 (BMP4) strongly promoted early mesoderm induction followed by increased hematopoietic commitment. This method reduced variations in hematopoietic differentiation among hPSC lines maintained under chemically defined Essential 8 medium compared to those maintained under less-defined mTeSR medium. We also found that perivascular niche cells (PVCs) significantly augmented the production of hematopoietic cells via paracrine signaling mechanisms only when they were present during the hematopoietic commitment phase. A protein array revealed 86 differentially expressed (>1.5-fold) secretion factors in PVC-conditioned medium compared with serum-free control medium, of which the transforming growth factor-ß inducible gene H3 significantly increased the number of hematopoietic colony-forming colonies. Our data suggest that BMP4 and PVCs promote the hematopoietic differentiation of hPSCs in a differentiation stage-specific manner. This will increase our understanding of hematopoietic development and expedite the development of hPSC-derived blood products for therapeutic use.

Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages.

A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains.

Generation of a KSCBi005-A-5(TLR8KO-A10) homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.

The Toll like Receptor (TLR) family plays an essential role in pathogen recognition and innate immunity activation. TLR8, an endosomal receptor, can recognize single-stranded RNA viruses, such as influenza virus, Sendai virus, Coxsackie B virus, HIV, and HCV. TLR8 binding to the viral RNA recruits MyD88 and leads to activation of the transcription factor NF-kB and antiviral response. We generated biallelic mutants of the TLR8 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). The TLR8 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.

Generation of a TLR7 homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.

Toll Like Receptor (TLR) family plays an important role in the activation of innate immunity against pathogens. TLR7 mediates the recognition of single-stranded RNA viruses, such as human immunodeficiency virus, hepatitis C virus, and influenza virus in endosomes. Here, we generated a TLR7 homozygous knockout human induced pluripotent cell (hiPSC) line, hiPSC-TLR7KO-A59, using the CRISPR/Cas9 genome editing method. The hiPSC-TLR7KO-A59 line maintains normal morphology, pluripotency, and differentiation capacity into three germ layers.

Generation of a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9.

Nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2) is a well-known transcription factor that regulates the expression of a large number of anti-oxidant genes in mammalian cells (J.H. Kim et al., 2014). Here, we generated a homozygous Nrf2 knockout human embryonic stem cell (hESC) line, H9Nrf2KO-A13, using the CRISPR/Cas9 genome editing method. The Nrf2 homozygous knockout H9 cell line maintains pluripotency, differentiation potential into three germ layers, and a normal karyotype.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential.

NIR-light-induced surface-enhanced Raman scattering for detection and photothermal/photodynamic therapy of cancer cells using methylene blue-embedded gold nanorod@SiO2 nanocomposites.

Methylene blue-loaded gold nanorod@SiO2 (MB-GNR@SiO2) core@shell nanoparticles are synthesized for use in cancer imaging and photothermal/photodynamic dual therapy. For the preparation of GNR@SiO2 nanoparticles, we found that the silica coating rate of hexadecylcetyltrimethylammonium bromide (CTAB)-capped GNRs is much slower than that of PEGylated GNRs due to the densely coated CTAB bilayer. Encapsulated MB molecules have both monomer and dimer forms that result in an increase in the photosensitizing effect through different photochemical pathways. As a consequence of the excellent plasmonic properties of GNRs at near-infrared (NIR) light, the embedded MB molecules showed NIR light-induced SERS performance with a Raman enhancement factor of 3.0 × 10(10), which is enough for the detection of a single cancer cell. Moreover, the MB-GNR@SiO2 nanoparticles exhibit a synergistic effect of photodynamic and photothermal therapies of cancer under single-wavelength NIR laser irradiation.

Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells.

BACKGROUND: Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs. RESULTS: From hESCs, we derived NESs, the in-vitro version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of in-vivo-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their in-vivo counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a gamma-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons. CONCLUSION: Our results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an in-vitro model for in-vivo neurogenesis.