pH-Dependent Cellular Internalization of Paramagnetic Nanoparticle

A hallmark of the tumor microenvironment in malignant tumor is extracellular acidosis, which can be exploited for targeted delivery of drugs and imaging agents. A pH sensitive paramagnetic nanoaparticle (NP) is developed by incorporating GdDOTA-4AmP MRI contrast agent and pHLIP (pH Low Insertion Peptide) into the surface of a G5–PAMAM dendrimer. pHLIP showed pH-selective insertion and folding into cell membranes, but only in acidic conditions. We demonstrated that pHLIP-conjugated Gd₄₄-G5 paramagnetic nanoparticle binds and fuses with cellular membrane at low pH, but not at normal physiological pH, and that it promotes cellular uptake. Intracellular trafficking of NPs showed endosomal/lysosomal path ways

Effect of FePro labeling on THP-1 cell chemotaxis in response to MCP-1 and Rantes.

<p>THP-1 cells incubated in the presence of MCP-1 (A) and Rantes (B). Cell migration determined by fluorescence based assay, values expressed as units of fluorescence. Experiment included non-labeled and FePro labeled THP-1 cells incubated without the treatment (white bars), incubated in the presence of 10% FBS (grey bars) and 300 ng/ml of MCP-1 or 50 ng/ml of Rantes (black bars). Bars, means±SD. * p<0.05 compared to non-labeled control cells.</p

Effect of FePro labeling on THP-1 cell ability to respond to LPS stimulation.

<p>Effect of LPS on the levels of CD83 and CD54 cell surface expression (A), TNF-α production (B) and activation of NFκB signaling pathway (C) in non-labeled and Fe-labeled THP-1 cells. (A) After incubation with LPS, cells were stained with PE conjugated anti-CD54 and anti-CD83 antibodies. Flow cytometric histograms of the non treated (control) cells (solid green lines), TPA treated (solid blue lines) and TPA and LPS treated (solid yellow lines) from one representative experiment are shown (n = 3). At least 10,000 live-gated cells were analyzed for PE expression. Isotype control shown as solid blue histograms. (B) TNF-α protein levels in THP-1 cells supernatants after 4 h of stimulation with 100 ng/ml of LPS, determined by ELISA. Data expressed as means±SD. * p<0.05 compared to non-labeled, TPA differentiated and LPS stimulated control cells whose maximum response was set to be 100%. (C) Images of Western blots of nuclear protein fraction probed with anti- p65NFκB antibody (top panel) and cytoplasmic protein fraction probed with anti-IκBα antibody (lower panel). The data depicts the levels of protein in non-labeled and FePro labeled THP-1 cells after 24 h incubation with 100 nM of TPA and following 30 minutes with 100 ng/ml of LPS. Single representative experiment is shown (n = 3). The same patterns of relative NFκB and IκBα expression were observed in all three experiments.</p

Effect of FePro labeling on THP-1 cell ability to modulate T cells.

<p>The data depicts the levels of CD markers expression after 24 h of THP-1 cell-T cell co-culture. T cells were stained with FITC conjugated anti-CD3, PE/Cy5 conjugated CD4 and PE conjugated anti-CD8. Cells were gated based on the forward and side scatter characteristics (R1) and on the expression of CD3 marker (R2) (A). Flow cytometric histograms of T cells cultured without the presence of THP-1 cells (red line), T cells co-cultured with non-labeled THP-1 cells (blue line) and T cells co-cultured with FePro labeled THP-1 cells (green line) from representative experiment are shown. THP-1 cells induced modulation of T cell expression levels of CD4 and CD8 (B). Note the complete overlap of blue and green lines. At least 10,000 live-gated cells were analyzed for CD4 and CD8 expression.</p

Effect of FePro labeling on THP-1 cell differentiation.

<p>THP-1 cells were labeled with FePro complexes and then differentiated in the presence of 100 nM of TPA for 24 h. Phase contrast photomicrograhs of non-labeled THP-1 cells before (A) and 24 h after incubation with 100 nM of TPA (B). FePro labeled THP-1 cell before (C) and 24 h after incubation with 100 nM of TPA (D).</p

Effect of FePro labeling on THP-1 ability to induce T cell proliferation.

<p>Non-labeled and FePro labeled, gamma irradiated, THP-1 cells co-cultured with T cells for 7 days. MTT proliferation assay performed at day 3 (gray bars) and day 7 (black bars). Data are presented as a mean±SD of values of optical density measured at 570 nm wavelength. Statistical analysis was performed by comparing proliferation at day 7 with day 3 within the group (labeled or non-labeled THP-1 co-culture and labeled or non-labeled THP-1 cells only or T cells only) and by comparing proliferation at day 7 between the groups (labeled vs. non-labeled THP-1 co-culture). * = p<0.05 day 7 compared to day 3. No significant difference was observed between labeled and non-labeled conditions in co-culture and in THP-1 cells only samples.</p

Effect of long term <i>in vitro</i> expansion on CB AC133+ cell chemotaxis in response to SDF1-α and Rantes.

<p>At days 15 and 30 of primary culture, CB AC133+ cells were incubated for 4 h in the presence of 50 ng/ml of either Rantes or SDF-1α. Cell migration in response to these to chemokines was significantly higher (p<0.05) than that observed in control conditions (w/o chemoatractant; gray bars), with no difference between the cells cultured for 15 and the cells cultured for 30 days. Cells incubated in the presence of 10% FBS (black bars) were used as a positive control. Bars, means ± SD. * p<0.05.</p

Morphological changes in CB AC133+ cells differentiated for 2 weeks.

<p>The similar pattern of morphological changes was observed in cells differentiated at day 15 (<b>A, B</b>) and at day 30 (<b>C, D</b>) of primary culture. Differentiated cells exhibited “cobble stone”-like organizational pattern. Spindle shaped adherent cells sprouted from cell clusters in an attempt to organize into linear, tube-like structures. Phase contrast photomicrographs from 2 representative experiments. Magnifications used: 10x (A and C) and 25x (B and D). Scale bar  = 100 µm.</p

Expression of cell surface markers in CB AC133+ progenitor cells differentiated for two weeks.

<p>CB AC133+ progenitor cells at day 31 of primary culture were differentiated for two weeks. Flow cytometric analysis showed that after 2 weeks of differentiation cells were positive for CD31, CD54, CD184, KDR, CD62E, CD29, CD150, CD195, CD105 and VE Cadherin. Flow cytometric histograms from one representative experiment are shown (n = 3). At least 10,000 live gated cells were analyzed for FITC, PE or PE-Cy5 expression. Isotype controls are shown as solid blue histograms.</p

Functional analysis of long term expanded CB AC133+ cells to form tubes in Matrigel.

<p>Tube like structures after 24 h of CB AC133+ cells and HDMVECs co-culture (<b>A-D</b>). Complete tubes in matrigel formed by HDMVECs incubated in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h (<b>E</b>). When plated alone, CB AC133+ did not form tube like structures (<b>F</b>). Note in panels A-D, HDMVECs labeled with Calcein (<b>C</b>, green fluorescence) and CB AC133+ cells labeled with DiI (<b>D</b>, red fluorescence) co-localized (yellow; panel <b>A</b>) to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered between the tubes. Overlays of bright light microscopy and fluorescent microscopy images (<b>A, C, D</b>). Bright light microscopy only, images shown in panels B, E and F. Magnification 10x.</p