Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

Nanodevices for magnetic resonance imaging of cancer were self-assembled to core-shell micellar structures by metal complex formation of K(2)PtCl(6) with diethylenetriaminepentaacetic acid gadolinium (III) dihydrogen (Gd-DTPA), a T(1)-contrast agent, and poly(ethylene glycol)-b-poly{N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-PAsp(DET)) copolymer in aqueous solution. Gd-DTPA-loaded polymeric micelles (Gd-DTPA/m) showed a hydrodynamic diameter of 45 nm and a core size of 22 nm. Confining Gd-DTPA inside the core of the micelles increased the relaxivity of Gd-DTPA more than 13 times (48 mM(-1) s(-1)). In physiological conditions Gd-DTPA/m sustainedly released Gd-DTPA, while the Pt(IV) complexes remain bound to the polymer. Gd-DTPA/m extended the circulation time in plasma and augmented the tumor accumulation of Gd-DTPA leading to successful contrast enhancement of solid tumors. μ-Synchrotron radiation-X-ray fluorescence results confirmed that Gd-DTPA was delivered to the tumor site by the micelles. Our study provides a facile strategy for incorporating contrast agents, dyes and bioactive molecules into nanodevices for developing safe and efficient drug carriers for clinical application.

PR domain zinc finger protein 14 (PRDM14) maintains stemness in embryonic stem cells via epigenetic mechanisms. Although PRDM14 is elevated in several cancers, it is unclear if and how PRDM14 confers stem cell-like properties and epigenetic changes to cancer cells. Here, we examined the phenotypic characteristics and epigenetic and gene expression profiles of cancer cells that differentially express PRDM14, and assessed the potential of PRDM14-targeted cancer therapy. PRDM14 expression was markedly increased in many different cancer types and correlated with poor survival of breast cancer patients. PRDM14 conferred stem cell-like phenotypes to cancer cells and regulated the expression of genes involved in cancer stemness, metastasis, and chemoresistance. PRDM14 also reduced the methylation of proto-oncogene and stemness gene promoters and PRDM14-binding regions were primarily occupied by histone H3 Lys-4 trimethylation (H3K4me3), both of which are positively correlated with gene expression. Moreover, strong PRDM14 binding sites coincided with promoters containing both H3K4me3 and H3K27me3 histone marks. Using calcium phosphate hybrid micelles as an RNAi delivery system, silencing of PRDM14 expression by chimera RNAi reduced tumor size and metastasis in vivo without causing adverse effects. Conditional loss of PRDM14 function also improved survival of MMTV-Wnt-1 transgenic mice, a spontaneous model of murine breast cancer. Our findings suggest that PRDM14 inhibition may be an effective and novel therapy for cancer stem cells.

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.

In boron neutron capture therapy (BNCT), boron drugs should accumulate selectively within a tumor and be quickly cleared from blood and normal organs. However, it is usually challenging to achieve the efficient tumor accumulation and the quick clearance simultaneously. Here we report the complex composed of a fructose-modified poly(ethylene glycol)-poly(l-lysine) block copolymer and p-boronophenylalanine, termed PEG-P[Lys/Lys(fructose)]-BPA, as a boron delivery system permitting selective accumulation within the target tumor with quick clearance from normal organs as well as blood. Our PEG-P[Lys/Lys(fructose)]-BPA could be internalized into tumor cells through LAT1 amino acid transporter-mediated endocytosis and retain in the targeted cells, thereby accomplishing more efficient accumulation and retention in a subcutaneous tumor than clinically used fructose-BPA complexes. Importantly, the moderately cationic property of the polymer facilitated renal clearance and PEG-P[Lys/Lys(fructose)]-BPA exhibited high accumulation contrast between the target tumor and the blood/normal organ. Finally, upon thermal neutron irradiation, PEG-P[Lys/Lys(fructose)]-BPA significantly inhibited the tumor growth in mice. PEG-P[Lys/Lys(fructose)]-BPA may be a promising boron delivery system for BNCT.

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.

Nano-sized contrast agents (NCAs) hold potential for highly specific tumor contrast enhancement during magnetic resonance imaging. Given the quantity of contrast agents loaded into a single nano-carrier and the anticipated relaxation effects, the current molecular design approaches its limits. In this study, a novel molecular mechanism to augment the relaxation of NCAs is introduced and demonstrated. NCA formation is driven by the intramolecular self-folding of a single polymer chain that possesses systematically arranged hydrophilic and hydrophobic segments in water. Utilizing this self-folding molecular design, the relaxivity value can be elevated with minimal loading of gadolinium complexes, enabling sharp tumor imaging. Furthermore, the study reveals that this NCA can selectively accumulate into tumor tissues, offering effective anti-tumor results through gadolinium neutron capture therapy. The efficacy and versatility of this self-folding molecular design underscore its promise for cancer diagnosis and treatment.

For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking.

Although automated external defibrillators (AEDs) have contributed to a better survival of out-of-hospital cardiac arrests, there have been reports of their malfunctioning. We investigated the diagnostic accuracy of commercially available AEDs using surface ECGs of ventricular fibrillation (VF), ventricular tachycardia (VT), and supraventricular tachycardia (SVT).

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.

More than 400 syndromes associated with hearing loss and other symptoms have been described, corresponding to 30% of cases of hereditary hearing loss. In this study we aimed to clarify the mutation spectrum of syndromic hearing loss patients in Japan by using next-generation sequencing analysis with a multiple syndromic targeted resequencing panel (36 target genes). We analyzed single nucleotide variants, small insertions, deletions and copy number variations in the target genes. We enrolled 140 patients with any of 14 syndromes (BOR syndrome, Waardenburg syndrome, osteogenesis imperfecta, spondyloepiphyseal dysplasia congenita, Stickler syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Pendred syndrome, Klippel-Feil syndrome, Alport syndrome, Norrie disease, Treacher-Collins syndrome, Perrault syndrome and auditory neuropathy with optic atrophy) and identified the causative variants in 56% of the patients. This analysis could identify the causative variants in syndromic hearing loss patients in a short time with a high diagnostic rate. In addition, it was useful for the analysis of the cases who only partially fulfilled the diagnostic criteria.

Although growth factors have great therapeutic potential because of their regenerative functions, they often have intrinsic drawbacks, such as low thermal stability and high production cost. Oligonucleotides have recently emerged as promising chemical entities for designing synthetic alternatives to growth factors. However, their applications in vivo have been recognized as a challenge because of their susceptibility to nucleases and limited distribution to a target tissue. Here, we present the first example of oligonucleotide-based growth factor mimetics that exerts therapeutic effects at a target tissue after systemic injection. The aptamer was designed to dimerize a growth factor receptor for its activation and mitigated the progression of Fas-induced fulminant hepatitis in a mouse model. This unprecedented functionality of the aptamer can be reasonably explained by its high nuclease stability and migration to the liver parenchyma. These mechanistic analyses provided insights for the successful application of aptamer-based receptor agonists.

In the current clinical boron neutron capture therapy (BNCT), p-boronophenylalanine (BPA) has been the most powerful drug owing to its ability to accumulate selectively within cancers through cancer-related amino acid transporters including LAT1. However, the therapeutic success of BPA has been sometimes compromised by its unfavorable efflux from cytosol due to their antiport mechanism. Here, we report that poly(vinyl alcohol) (PVA) can form complexes with BPA through reversible boronate esters in aqueous solution, and the complex termed PVA-BPA can be internalized into cancer cells through LAT1-mediated endocytosis, thereby enhancing cellular uptake and slowing the untoward efflux. In in vivo study, compared with clinically used fructose-BPA complexes, PVA-BPA exhibited efficient tumor accumulation and prolonged tumor retention with quick clearance from bloodstream and normal organs. Ultimately, PVA-BPA showed critically enhanced antitumor activity in BNCT. The facile technique proposed in this study offers an approach for drug delivery focusing on drug metabolism.

Gemcitabine (GEM) is a powerful anticancer drug for various cancers. However, the anticancer efficacy and the side effects should be addressed for effective therapeutics. To this end, we created a GEM-conjugated polymer (P-GEM) based on cyclic acetal linkage as a delivery carrier of GEM. The obtained P-GEM stably conjugated GEM at physiological pH (i.e., bloodstream), but released GEM in response to acidic environments such as endosome/lysosome. After systemic administration of P-GEM for mice bearing subcutaneous tumors, it achieved prolonged blood circulation and enhanced tumor accumulation relative to free GEM system. In addition, the polymer-drug conjugate structure of P-GEM realized effective distribution in the tumor tissues toward the induction of apoptosis in most areas of the tumor sites. Of note, the molecular design of P-GEM achieved minimal accumulation in normal tissues, resulting in negligible GEM-derived adverse effects (e.g., gastrointestinal toxicity and hematotoxicity). Ultimately, even four times smaller dose of P-GEM on a GEM basis realized comparable/higher tumor growth suppression effect for two distinct pancreatic tumor models, compared to free GEM system. The obtained results suggest the huge potential of the present design of GEM-conjugated polymer for anticancer therapeutics.

Increased glutamine uptake toward the elevated glutaminolysis is one of the hallmarks of tumour cells. This aberrant glutamine metabolism has recently attracted considerable attention as a diagnostic and therapeutic target. Herein, we developed glutamine-functionalized polymer to achieve a selective high affinity to tumour cells overexpressing glutaminolysis-related transporter ASCT2. In in vitro study, our developed polymer exhibited faster and higher cellular uptake in tumour cells than that in normal cells. Uptake inhibition study revealed the dominant contribution of ASCT2 to the polymer-cell interaction. Furthermore, the binding affinity of the polymer to tumour cells was estimated to be comparable to that of the potent ligand molecules reported in the literature. In in vivo study, the polymer showed prolonged retention at tumour site after intratumoral injection. This study offers a novel approach for designing tumour cell-binding synthetic polymers through the recognition of dense transporters related to tumour-associated metabolism.

Cancer stem-like cells (CSCs) treatment is a plausible strategy for enhanced cancer therapy. Here we report a glucose-installed sub-50-nm nanocarrier for the targeted delivery of small interfering RNA (siRNA) to CSCs through selective recognition of the glucose ligand to the glucose transporter 1 (GLUT1) overexpressed on the CSC surface. The siRNA nanocarrier was constructed via a two-step assembling process. First, a glucose-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (Glu-PEG-PLL-LA) was associated with a single siRNA to form a unimer polyion complex (uPIC). Second, a 20 nm gold nanoparticle (AuNP) was decorated with ~65 uPICs through AuS bonding. The glucose-installed targeted nanoparticles (Glu-NPs) exhibited higher cellular uptake of siRNA payloads in a spheroid breast cancer (MBA-MB-231) cell culture compared with glucose-unconjugated control nanoparticles (MeO-NPs). Notably, the Glu-NPs became more efficiently internalized into the CSC fraction, which was defined by aldehyde dehydrogenase (ALDH) activity assay, than the other fractions, probably due to the higher GLUT1 expression level on the CSCs. The Glu-NPs elicited significantly enhanced gene silencing in a CSC-rich orthotopic MDA-MB-231 tumor tissue following systemic administration to tumor-bearing mice. Ultimately, the repeated administrations of polo-like kinase 1 (PLK1) siRNA-loaded Glu-NPs significantly suppressed the growth of orthotopic MDA-MB-231 tumors. These results demonstrate that Glu-NP is a promising nanocarrier design for CSC-targeted cancer treatment.

Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

Boron neutron capture therapy is a promising tumor treatment method, though its wide application has been limited due to the poor tumor selectivity and intracellular delivery of 10B-compounds. Here, block copolymer-boron cluster conjugate based on the clinically used sodium borocaptate (BSH) and poly(ethylene glycol)-b-poly(glutamic acid) copolymer have been developed for effectively penetrating tumor tissues and homogeneously delivering the boron clusters into cancer cells towards safe and efficient boron neutron capture therapy. The PEGylated block copolymer-boron cluster (BSH) conjugate has demonstrated significant higher cellular uptake and tumor accumulation when compared to the non-PEGylated formulations and BSH. Moreover, the enhanced delivery to tumors of the conjugates, as well as their superior intratumoral penetration, which facilitated reaching the intracellular space of most cells in tumors, allowed the effective ablation of tumors after neutron irradiation.

An efficient targeting delivery technology is needed for functional oligonucleotides to exert their potential effect on the target gene without an adverse effect in vivo. Development of enteral delivery systems for nucleic acids is a major challenge because of their large molecular size and instability. Here, we describe a new enteral delivery technique that enables small interfering RNA (siRNA) selectively delivered to the liver to silence its target Apolipoprotein B gene expression. A nuclease-resistant synthetic siRNA was conjugated with α-tochopherol and administered as lipid nanoparticle to the large intestine of the mice in a postprandial state. The selective transport into the liver, effective gene silence, and consequently significant reduction in serum low density lipoprotein-cholesterol level, were demonstrated. The chylomicron-mediated pathway via the lymphatic route was suggested as major mechanism. This unique approach may provide a basis for developing oral and rectal delivery systems for nucleic acids targeting liver.

We set out to examine the activity of γ-glutamyltranspeptidase (GGT) in lung cancer and the validity of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) for intraoperative imaging of primary lung cancer.