Time-Resolved Fluorescence Spectroscopy Reveals Fine Structure and Dynamics of Poly(l‑lysine) and Polyethylenimine Based DNA Polyplexes

Structural dynamics of the polyethylenimine–DNA and poly­(l-lysine)–DNA complexes (polyplexes) was studied by steady-state and time-resolved fluorescence spectroscopy using the fluorescence resonance energy transfer (FRET) technique. During the formation of the DNA polyplexes, the negative phosphate groups (P) of DNA are bound by the positive amine groups (N) of the polymer. At N/P ratio 2, nearly all of the DNA’s P groups are bound by the polymer N groups: these complexes form the core of the polyplexes. The excess polymer, added to this system to increase the N/P ratio to the values giving efficient gene delivery, forms a positively charged shell around the core polyplex. We investigated whether the exchange between the core and shell regions of PEI and PLL polyplexes takes place. Our results demonstrated a clear difference between the two studied polymers. Shell PEI can replace PEIs previously attached to DNA in the polyplex core, while PLL cannot. Such a dynamic structure of PEI polyplexes compared to a more static one found for PLL polyplexes partially explains the observed difference in the DNA transfection efficiency of these polyplexes. Moreover, the time-resolved fluorescence spectroscopy revealed additional details on the structure of PLL polyplexes: in between the core and shell, there is an intermediate layer where both core and shell PLLs or their parts overlap

The L-type Ca²⁺ Channels Blocker Nifedipine Represses Mesodermal Fate Determination in Murine Embryonic Stem Cells

<div><p>Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca²⁺ channels of different cell types. Moreover, influx of Ca²⁺ through L-type Ca²⁺ channels (LTCCs) activates Ca²⁺ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage <em>in vitro</em>. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca²⁺ transient and reduction of LTCCs current density (<em>I</em>_CaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca²⁺ channel α_1C (Ca_V1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca²⁺ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.</p> </div

Nifedipine does not affect the generation of specific cardiac subtypes as well as the β-adrenergic and muscarinic regulation pathways.

<p>(<b><i>A</i></b>) Representative AP recordings from spontaneously beating ES cell-derived CMs revealed differentiation of nifedipine-treated cell into the different cardiac subtypes: nodal-, embryonic atrial- and embryonic ventricular-like CMs, note the presence of unspecified CMs (left panel). Statistic analysis (right panel) of different cardiac subtypes generated under control and nifedipine-treated cultures. (<b><i>B–C</i></b>) Representative AP of untreated (<i>B</i>) and nifedipine-treated (<i>C</i>) ES cells-derived CMs showed a prominent positive chronotropic effect of Iso (1 µM) (middle left) and negative chronotropic response to CCh (1 µM) (middle right) application. These effects could be partially reversed by washout. The dotted lines indicate the zero current level.</p

Functional characteristics of Na⁺ channel and depolarization-activated outward K⁺ currents.

<p>(<b><i>A–B</i></b>) Representative traces (A) and currents densities (B) of Na⁺ current recorded from a control and nifedipine-treated cells at days 4 (non-beating cell) and 12 (beating CM) of differentiation. (<b><i>C–D</i></b>) Representative traces and current-voltage relationships (<i>I/V</i>) of the peak depolarization-activated outward K⁺ current (<i>I</i>_peak). (<b><i>E–F</i></b>) Effect of acute application of Na⁺ channel (<i>E</i>) blocker TTX (1 µM) and non-specific K⁺ channel (<i>F</i>) blocker TEA (10 mM). The dotted lines indicate the zero current level.</p

Nifedipine repressed the expression level of most specific cardiac and mesoderm genes.

<p>(<b><i>A</i></b>) RT-PCR for L-type Ca²⁺ channel α-subunit isoforms in undifferentiated ES cells and CMs at day 12 of differentiation, demonstrating (i) presence of L-type Ca²⁺ channel mRNA even in undifferentiated ES cells and (ii) an increase in mRNA encoding the CaV1.2 subunit of the channel. (<b><i>B</i></b>) RT-PCR analyses of cardiac-specific and transcription factors <i>α-MHC/Myh6, ANF/Nppa, MLC2v/Myl2, NKx2.5</i> and <i>GATA4</i> in beating cluster at day 12 of both ES and iPS differentiation. (<b><i>C</i></b>) RT-PCR analyses of representative markers, PECAM-1 (mesoderm, endothelial cell), AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) and α-SMA (mesoderm) in ES and iPS beating cluster at day 12 of differentiation. (<b><i>D</i></b>) Quantitative real-time PCR analyses of mesoderm and cardiac markers and specific transcript (<i>Brachyury T, Mesp1, NKx2.5, Tbx5, Mest, Myocd, Myh6/α-MHC, Myl2/MLC2v, and Nppa/ANF</i>), as well as cardiac ionic channels (<i>CACNA1c, KCNH2</i> and <i>SCN5A</i>) expression at different stage of ES cell differentiation. GAPDH was used as housekeeping gene and served to normalize the result. Results are reported as the means±SEM (n = 3). *<i>P</i><0.05 vs. control CMs.</p

The presence of nifedipine in culture medium inhibits differentiation of ES cell-derived CMs.

<p>(<b><i>A</i></b>) Protocol used to determine the effect of nifedipine application during cell differentiation. Note that for beating EBs and single cell electrophysiology and Ca2+ imaging experiments, analysis was performed at least 24 hours after washout to reflect long-term changes instead of acute drugs effects. (<b><i>B–C</i></b>) Representative experiments showing EBs with GFP expressing areas cultured under control conditions (<i>B</i>) and after nifedipine-treatment (10 µM) (<i>C</i>) of ES cells. (<b><i>D–F</i></b>) Representative EBs showing EGFP+ CMs differentiated of ES cells under control conditions (<b><i>D</i></b>), after nifedipine- (<b><i>E</i></b>) and BayK8644- (<b><i>F</i></b>) treatment. (<b>G</b>) Representative FACS analysis of ES cell-derived CMs generated under control, nifedipine- and BayK8644-treated conditions. (Scale bars 50 µm (B, C) and 20 µm (D–F).</p

Nifedipine reduced spontaneous Ca²⁺ transient activity in ES cell-derived CMs.

<p>(<b><i>A</i></b>) Representative tracings of spontaneous Ca²⁺ transients recorded in spontaneously beating CMs from control and nifedipine-treated cultures. Fluorescence images (labeled with Fura 2 AM) of the recorded cells are shown on the left panel. (<b><i>B</i></b>) Left, spontaneous Ca²⁺ transients recorded from CMs derived under untreated (top) and nifedipine-treated (bottom) condition before and after caffeine application. Right, caffeine-induced peak amplitude (top, right) of the Ca²⁺ signals and Fractional release (bottom, right) calculated as the ratio of peak Ca²⁺ concentration under control condition to peak Ca²⁺ concentration induced by caffeine. (<b><i>C–F</i></b>) Comparison of the frequency (<i>C</i>), amplitude (<i>D</i>), maximum upstroke (<i>E</i>) and decay (<i>F</i>) velocity of spontaneous Ca²⁺ transients on day 12 in controls (<i>n</i> = 10) and nifedipine-treated <i>(n</i> = 8) cultures.</p

The presence of nifedipine in differentiation medium altered the cardiac <i>I</i>_CaL density of ES cell-derived CMs.

<p>(<b><i>A–C</i></b>), Representative L-type Ca²⁺ current traces recorded as indicated in protocol (<i>A</i>) from CM cultured in control (<i>B</i>) and nifedipine-treated (<i>C</i>) conditions. (<b><i>D</i></b>) Current densities recorded at the maximal peak of the current (10 mV) of cells generated in control and under nifedipine-treatment at days 8 and 12 of differentiation. (<b><i>E–F</i></b>) Current-voltage relationships (<i>E</i>) and activation curves (<i>F</i>) of <i>I</i>_CaL recorded in control (open symbols) and nifedipine-treated (filled symbols) CMs. (<b><i>G</i></b>), Steady-state inactivation curves of control (open symbols) and nifedipine-treated (filled symbols) CMs. (<b><i>H–I</i></b>) shows the time constants of fast τ_f (<i>H</i>) and slow τ_s (<i>I</i>) phases of <i>I</i>_CaL inactivation recorded in ES cell-derived CMs from control and nifedipine-treated cultures. (<b><i>J</i></b>) Immunolocalization of L-type calcium Ca_V1.2 subunit (α_1C) in undifferentiated ES cells suggests enrichment in the cell periphery and cellular membrane. (<b><i>K–L</i></b>) Immunocytochemistry of L-type calcium Ca_V1.2 subunit (α_1C) in CMs generated under both control (<i>K</i>) and nifedipine-treated (<i>L</i>) conditions. Insert are positive control experiments from the same bath of cells performed with mouse anti-α actinin. Hoechst 33342 was used to stain nuclei (blue). (Scale bars: 20 µm). * denote significant differences to control and <b>^†</b> significant differences to day 12 of differentiation.</p

AP parameters of CMs obtained from EBs generated under control and nifedipine-treated conditions.

<p><b>Abbreviations:</b> n, indicates the cell number; APD50/APD90, AP duration measured at 50% or 90% of repolarization; dV/dtmax, maximum rate of rise of AP; and MDP, maximum diastolic potential.</p>*<p>P<0.05 compared with control condition.</p

Biodistribution of pre-targeted and non-targeted liposomes in mice bearing i.p. SKOV3.ip1 xenografts.

<p>Either neutravidin-cetuximab, at a dose of 20 µg of antibody, or PBS was injected i.p. to the mice. ^99mTc-labeled biotin-liposomes were injected 24 h later i.v. (A) or i.p. (B). Radioactivity of the indicated tissues was determined 24 h from liposome injections. The results are expressed as %ID/g tissue ± SD for pre-targeted liposomes and for non-targeted liposomes (<i>n</i> = 3–4).</p