Temperature-Responsive Gene Silencing by a Smart Polymer

Intracellular siRNA release is a crucial step in efficient gene silencing mediated by cationic polymers. Here, we show an example of temperature change-induced intracellular siRNA release and silencing using a temperature-responsive polymer consisting of dendrimer, poly­(<i>N</i>-isopropylacrylamide) and phenylboronic acid. The smart polymer can trigger the release of loaded siRNA in a controlled manner upon cooling the surrounding solution below its lower critical solution temperature. Gene silencing efficacy of the polymer was significantly increased by cool treatment after its cellular uptake. The polymer and the cool treatment cause minimal toxicity to the transfected cells. The results provide a facile and promising strategy to design stimuli-responsive polymers for efficient gene silencing

Janus Liposomes: Gel-Assisted Formation and Bioaffinity-Directed Clustering

This article reports a high-yield procedure for preparing microsized (giant) Janus liposomes via gel-assisted lipid swelling and clustering behavior of these liposomes directed by biotin-avidin affinity binding. Confocal fluorescence microscopy reveals in detail that these new lipid colloidal particles display broken symmetry and heterogeneous surface chemistry similar to other types of Janus particles. An optimized formation procedure is presented, which reproducibly yields large liposome populations dominated by a single-domain configuration. This work further demonstrates that biotin-conjugated 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine preferentially partitions into the liquid-disordered phase of the lipid matrix, rendering these Janus liposomes asymmetrical binding capacity toward avidin. This affinity binding drives irreversible and domain-specific cluster formation among Janus liposomes, whose structure and size are found to depend on the domain configuration of individual liposomes and incubation time

Phospholipid/Aromatic Thiol Hybrid Bilayers

Gold-supported hybrid bilayers comprising phospholipids and alkanethiols have been found to be highly useful in biomembrane mimicking as well as biosensing ever since their introduction by Plant in 1993 (Plant, A. L. <i>Langmuir</i> <b>1993</b>, <i>9</i>, 2764–2767). Generalizing the mechanism (i.e., hydrophobic/hydrophobic interaction) that primarily drives bilayer formation, we report here that such a bilayer structure can also be successfully obtained when aromatic thiols are employed in place of alkanethiols. Four aromatic thiols were studied here (thiophenol, 2-naphthalene thiol, biphenyl-4-thiol, and diphenylenevinylene methanethiol), all affording reliable bilayer formation when 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine liposomes were incubated with self-assembled monolayers of these thiols. Characterization of the resultant structures, using cyclic voltammetry, impedance analysis, and atomic force microscopy, confirms the bilayer formation. Significant differences in electrochemical blocking and mechanical characteristics of these new bilayers were identified in comparison to their alkanethiol counterparts. Taking advantage of these new features, we present a new scheme for the straightforward biorecognition of a lipolytic enzyme (phospholipase A₂) using these phospholipid/aromatic thiol bilayers

Mimicking Photosynthesis with Supercomplexed Lipid Nanoassemblies: Design, Performance, and Enhancement Role of Cholesterol

We report here a new approach to mimicking photosynthesis that relies on supercomplexed lipid nanoassemblies to organize small organic species for coordinated light harvesting, energy/electron transfer, and photo-to-electrochemical energy conversion. Specifically, we demonstrate efficient photoinduced electron transfer (PeT) between rhodamine and fullerene assembled together via electrostatically bound liposome and lipid bilayer hosts. The remarkable impact of the lipid matrix on the photoconversion efficiency is further revealed by cholesterol, whose addition is found to modify the distribution and organization of the coassembled rhodamine dyes and thus their photodynamics. This significantly expedites the energy transfer (ET) among rhodamine dyes, as well as the PeT between rhodamines and fullerenes. A respectable 14% photon-to-electron conversion efficiency was achieved for this supercomplexed system containing 5% rhodamines, 5% fullerenes, and 30% cholesterol. The morphology, photodynamics, and photoelectrochemical behavior of these lipid supercomplexes were thoroughly characterized using atomic force microscopy (AFM), fluorescence microscopy, steady-state and time-resolved fluorescence spectroscopy, and transient absorption (TA) and photoaction spectroscopy. A detailed discussion on enhancement mechanisms of cholesterol in this lipid-complexed photosynthesis-mimicking system is provided at the end

Structure characteristics of the bovine <i>PDHB</i> gene.

<p>a. Here we show the genomic, mRNA and protein components in detail. 5’- UTR:5’- untranslated region, 3’- UTR:3’- untranslated region, ORF: open reading frame, Transketpyr: transketolase, pyrimidine binding domain. b. 5’-RACE. Lane 1 and 2 are products of the first and second PCR, respectively. Lane M represents the marker of DL2000. c. 5’-regulatory region sequence of bovine <i>PDHB</i> gene. Arrows mark the transcription initiation sites. The cytosine residue is designated as +1. The transcription factor binding sites are boxed. The primers are underlined with the respective names below the line. The CpG island is indicated with red color.</p

Promoter activity analysis of the bovine <i>PDHB</i> gene.

<p>a. We transferred six serial deletion constructs in pGL3-basic into C2C12 cells. After 5 h we replaced the transfection mixture with DMEM with 5% FBS (myoblasts) or 2% HS (myotubes). b. We transferred the same constructs into 3T3-L1 cells. We normalized relative luciferase activities to Renilla luciferase activity. The transcription factor binding sites of MYOG and C/EBPß are indicated with closed circles and ellipses, respectively. *, P<0.05. Error bars represent the SD (n = 3).</p

ChIP assay of MYOG and C/EBPß binding to PDHB promoter in vivo.

<p>We analyzed immunoprecipitated products for MYOG (a) and C/EBPß (b) antibodies via RT-PCR. We analyzed immunoprecipitated products for MYOG (c) and C/EBPß (d) antibodies via ChIP-QPCR. We used total chromatin from muscle (a and c) and fat (b and d) as the input. We used normal mouse IgG as the negative control antibodies. **, P<0.01. Error bars represent the SD (n = 3).</p

Functional analysis of the mutated MYOG and C/EBPß sites.

<p>We transferred the mutated sites MYOG and C/EBPß into C2C12 myotubes (a) and 3T3-L1 cells (b). **, P<0.01. Error bars represent the SD (n = 3).</p

Spatial expression analysis of bovine <i>PDHB</i> mRNA.

<p>We normalized the mRNA expression levels of <i>PDHB</i> to those of <i>GAPDH</i>. Error bars represent the standard deviation (SD) (n = 3).</p

Primers utilized in this study.

<p>Primers utilized in this study.</p