DNA Polyplexes as Combinatory Drug Carriers of Doxorubicin and Cisplatin: An in Vitro Study

Double helix nucleic acids were used as a combination drug carrier for doxorubicin (DOX), which physically intercalates with DNA double helices, and cisplatin (CDDP), which binds to DNA without an alkylation reaction. DNA interacting with DOX, CDDP, or both was complexed with positively charged, endosomolytic polymers. Compared with the free drug, the polyplexes (100–170 nm in size) delivered more drug into the cytosol and the nucleus and demonstrated similar or superior (up to a 7-fold increase) in vitro cell-killing activity. Additionally, the gene expression activities of most of the chemical drug-loaded plasmid DNA (pDNA) polyplexes were not impaired by the physical interactions between the nucleic acid and DOX/CDDP. When a model reporter pDNA (luciferase) was employed, it expressed luciferase protein at 0.7- to 1.4-fold the amount expressed by the polyplex with no bound drugs (a control), which indicated the fast translocation of the intercalated or bound drugs from the “carrier DNA” to the “nuclear DNA” of target cells. The proposed concept may offer the possibility of versatile combination therapies of genetic materials and small molecule drugs that bind to nucleic acids to treat various diseases

Oral Nanoparticles Exhibit Specific High-Efficiency Intestinal Uptake and Lymphatic Transport

Herein, we describe a simple and promising nanoparticle oral delivery phenomenon and propose pathways for oral nanoparticle absorption from the gastrointestinal tract (GIT), combining apical sodium-dependent bile acid transporter-mediated cellular uptake and chylomicron transport pathways. This strategy is proven to employ bile-acid-conjugated, solid fluorescent probe nanoparticles (100 nm diameter) to exclude any potential artifacts and instability issues in observing transport pathways and measuring oral bioavailability. The results of the <i>in vitro</i> studies showed that there is no interference from bile acid and no simultaneous uptake of nanoparticles and dextran. The probe nanoparticle exhibited a significantly enhanced average oral bioavailability (47%) with sustained absorption in rats. Particle-size- and dose-dependent oral bioavailability was observed for oral nanoparticle dosing up to 20 mg/kg. The probe nanoparticles appear to be transported to systemic circulation <i>via</i> the gut lymphatic system. Thus, we propose a pathway for oral nanoparticle absorption from the GIT, combining apical bile acid transporter-mediated cellular uptake and chylomicron transport pathways

Oral Nanoparticles Exhibit Specific High-Efficiency Intestinal Uptake and Lymphatic Transport

Herein, we describe a simple and promising nanoparticle oral delivery phenomenon and propose pathways for oral nanoparticle absorption from the gastrointestinal tract (GIT), combining apical sodium-dependent bile acid transporter-mediated cellular uptake and chylomicron transport pathways. This strategy is proven to employ bile-acid-conjugated, solid fluorescent probe nanoparticles (100 nm diameter) to exclude any potential artifacts and instability issues in observing transport pathways and measuring oral bioavailability. The results of the <i>in vitro</i> studies showed that there is no interference from bile acid and no simultaneous uptake of nanoparticles and dextran. The probe nanoparticle exhibited a significantly enhanced average oral bioavailability (47%) with sustained absorption in rats. Particle-size- and dose-dependent oral bioavailability was observed for oral nanoparticle dosing up to 20 mg/kg. The probe nanoparticles appear to be transported to systemic circulation <i>via</i> the gut lymphatic system. Thus, we propose a pathway for oral nanoparticle absorption from the GIT, combining apical bile acid transporter-mediated cellular uptake and chylomicron transport pathways

Tempo-spatial Activation of Sequential Quadruple Stimuli for High Gene Expression of Polymeric Gene Nanocomplexes

The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications

Time-Resolved X‑ray Spectroscopy in the Water Window: Elucidating Transient Valence Charge Distributions in an Aqueous Fe(II) Complex

Time-resolved nitrogen-1s spectroscopy in the X-ray water window is presented as a novel probe of metal–ligand interactions and transient states in nitrogen-containing organic compounds. New information on iron­(II) polypyridyl complexes via nitrogen core-level transitions yields insight into the charge density of the photoinduced high-spin state by comparing experimental results with time-dependent density functional theory. In the transient high-spin state, the 3d electrons of the metal center are more delocalized over the nearest-neighbor nitrogen atoms despite increased bond lengths. Our findings point to a strong coupling of electronic states with charge-transfer character, facilitating the ultrafast intersystem crossing cascade in these systems. The study also highlights the importance of local charge density measures to complement chemical interaction concepts of charge donation and back-bonding with molecular orbital descriptions of states

Electronic and Molecular Structure of the Transient Radical Photocatalyst Mn(CO)₅ and Its Parent Compound Mn₂(CO)₁₀

We present a time-resolved X-ray spectroscopic study of the structural and electronic rearrangements of the photocatalyst Mn₂(CO)₁₀ upon photocleavage of the metal–metal bond. Our study of the manganese K-edge fine structure reveals details of both the molecular structure and valence charge distribution of the photodissociated radical product. Transient X-ray absorption spectra of the formation of the Mn­(CO)₅ radical demonstrate surprisingly small structural modifications between the parent molecule and the resulting two identical manganese monomers. Small modifications of the local valence charge distribution are decisive for the catalytic activity of the radical product. The spectral changes reflect altered hybridization of metal-3d, metal-4p, and ligand-2p orbitals, particularly loss of interligand interaction, accompanied by the necessary spin transition due to radical formation. The spectral changes in the manganese pre- and main-edge region are well-reproduced by time-dependent density functional theory and <i>ab initio</i> multiple scattering calculations

Probing the Electronic Structure of a Photoexcited Solar Cell Dye with Transient X-ray Absorption Spectroscopy

This study uses transient X-ray absorption (XA) spectroscopy and time-dependent density functional theory (TD-DFT) to directly visualize the charge density around the metal atom and the surrounding ligands following an ultrafast metal-to-ligand charge-transfer (MLCT) process in the widely used Ru^II solar cell dye, Ru­(dcbpy)₂(NCS)₂ (termed N3). We measure the Ru L-edge XA spectra of the singlet ground (¹A₁) and the transient triplet (³MLCT) excited state of N3^4– and perform TD-DFT calculations of 2p core-level excitations, which identify a unique spectral signature of the electron density on the NCS ligands. We find that the Ru 2p, Ru e_g, and NCS π* orbitals are stabilized by 2.0, 1.0, and 0.6 eV, respectively, in the transient ³MLCT state of the dye. These results highlight the role of the NCS ligands in governing the oxidation state of the Ru center

Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., <i>N</i>²-ethyl­(Et)­G, <i>O</i>⁶-methyl­(Me)­G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, <i>N</i>²-EtG, <i>O</i>⁶-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the <i>N</i>-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg²⁺ (but not with Mn²⁺), coinciding with its steady-state kinetic data showing a ∼10-fold increase in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates (only with Mg²⁺). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates either with Mg²⁺ or Mn²⁺, except for that opposite <i>N</i>²-EtG with Mn²⁺ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg²⁺), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn²⁺ than with Mg²⁺. The results indicate that the R96G variation severely impairs most of the Mg²⁺- and Mn²⁺-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg²⁺-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens

The reduction in Kir2.1 activity impaired myoblast differentiation in Cdo-depleted cells.

<p>(A) Western blot analysis of C2C12 cell lysates from various differentiation time courses for indicated proteins. (B) C2C12 cells were stably transfected with the control (pcDNA3.1) or Kir2.1 expression vectors, cultured in DM for 3 days and subjected to immunostaining with anti-MHC antibody (red). Cell nuclei were visualized by DAPI staining (blue). Scale bar indicates 200μm. (C) Quantification of the MHC-positive cells shown in B. Values represent means ± SEM in three random fields. *<i>P</i> <0.05. (D) Western blot analysis for expression of Cdo or β-tubulin in lysates of C2C12/pSuper and C2C12/Cdo shRNA cells, which were differentiated for 3 days. β-tubulin was used as a loading control. (E) C2C12 cells were stably transfected with pSuper or Cdo shRNA expression vectors. Cells were then induced to differentiate for 3 days and analyzed by immunostaining with anti-MHC antibody (red). Cell nuclei were visualized by DAPI staining (blue). Scale bar = 200μm. (F) Western blot analysis for expression of Kir2.1, Myogenin and MHC in C2C12/pSuper or C2C12/Cdo shRNA cells, which were induced to differentiate for indicated time courses. β-tubulin was used as a loading control. (G) Current-voltage relationships of inwardly rectifying K⁺ (IRK) current of C2C12 cells expressing either pSuper (left) or Cdo shRNA (middle). IRK current was obtained by subtraction of steady state currents at the end of test pulse, in the absence or presence of 0.5 mM Ba²⁺. Voltage-steps were from -140 mV to +60 mV from a holding potential at -70 mV. The graph on the right is the summary of IRK density. The current density is the whole-cell current amplitude divided by the membrane capacity (which is directly correlated with the surface area of the cell), thus allowing comparison between cells. IRK amplitude was measured during a step to –140 mV from a holding potential of –70 mV. (H) Western blot analysis for expression of CFP, Cdo and MHC in C2C12/Control or C2C12/Kir2.1-CFP cells, which were induced to differentiate for indicated time. Cadherin was used as a loading control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158707#pone.0158707.ref008" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158707#pone.0158707.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158707#pone.0158707.ref034" target="_blank">34</a>]. (I) Western blot analysis for expression of Kir2.1, Cdo and MHC in C2C12/Control or C2C12/Kir2.1 shRNA cells, which were induced to differentiate for indicated time. Cadherin was used as a loading control.</p

p38MAPK signaling was required for the translocation of Kir2.1 to the plasma membrane.

<p>(A) Current-voltage relationships of inwardly rectifying K⁺ (IRK) current of C2C12 cells in the absence (left) or the presence (middle) of SB203580 (2.5 μM). The current density of IRK at -140 mV before or after SB203580 is shown on the right. (B) DMSO (0.25%)- or SB203580-treated C2C12 cells were induced to differentiate for indicated time, and subjected to biotinylation assay. The biotinylated (surface) and non-biotinylated (lysate) protein fractions were blotted with an anti-Kir2.1 antibody. Cadherin was used as a loading control. (C) Confocal immunofluorescence detection of Kir2.1 (green) in DMSO-, or SB203580-treated C2C12 cells after differentiation induction for 6 hours. Cell membrane and nuclei were visualized by pan-Cadherin (red) and DAPI (blue), respectively. The regions of plasma membrane, where cell-cell contact occurs, are indicated with rectangles. The scale bar denotes 10μm. (D) Changes in resting membrane potential (RMP) in Cdo-depleted C2C12 cells with or without MKK6(EE).</p