rHSA-P53i and rHSA-PMI bind to MDM2 and MDMX.

<p>To detect the interaction between MDM2/MDMX and rHSA fusion proteins, 4 µg each of biotinylated rHSA (lane 1), rHSA-P53i (lane 2), or rHSA-PMI (lane 3) were added to 200 µg of SJSA-1 whole cell lysate. MDM2 or MDMX antibody was added to the lysate followed by pulling down MDM2/MDMX and rHSA complexes using Protein A/G (1:1) resins. Samples were then analyzed by SDS-PAGE and Western blotting using MDM2, MDMX, and Streptavidin-HRP (Strep-HRP) antibodies. </p

Schematic diagram of rHSA-mediated co-delivery technology.

<p>Recombinant HSA-delivery complexes were conceived as a co-delivery technology in that 1) therapeutic peptides can be fused to the C-terminal of HSA for both extracellular and intracellular targeting and 2) FA-Drugs can form stable complexes with rHSA fusion proteins to promote synergistic therapeutic efficacy. </p

rHSA-p53 and rHSA-PMI are efficiently taken up into SJSA-1 cells.

<p>FITC-labeled rHSA (5 μM), FITC-rHSA-P53i (5 μM), and FITC-rHSA-PMI (5 μM) were added to SJSA-1 cells as described in <i>Methods</i>. Visualization at 60X magnification revealed efficient uptake of FITC-rHSA (A), FITC-rHSA-P53i (B) and FITC-rHSA-PMI (C) occurred following 24-hour incubation. FITC staining of vesicular cargo suggests significantly greater uptake of rHSA-P53i and rHSA-PMI, compared to rHSA.</p

rHSA-P53i and rHSA-PMI induce p53 accumulation, but not MDM2.

<p>SJSA-1 cells were plated and allowed to attach overnight. On day 2, culture media with 10 µM rHSA (lane 1), 10 µM nutlin (lane 2), 10 µM rHSA-P53i (lane 3), or 10 µM rHSA-PMI (lane 4) were added to respective wells and allowed to incubate for 24 hrs. Cells were then washed, lysed and immunoblotted for p53 and MDM2. Western blot analysis to detect p53 protein (middle panel) reveals treatment with rHSA-P53i or rHSA-PMI resulted in modest accumulation of p53. Densitometry analysis reveals p53 accumulation following rHSA-P53i and rHSA-PMI treatment is on average, 1.5 and 2.9 orders of magnitude above control wells, respectively. As expected, nutlin-treatment promotes robust p53 accumulation (11.5-fold average increase). However, unlike nutlin, which promotes a 5-fold increase in MDM2 expression, MDM2 protein remained at basal levels following treatment with rHSA-P53i or rHSA-PMI (1.1- and 1.0-fold change, respectively). </p

rHSA-P53i and rHSA-PMI promote cytotoxicity in SJSA-1 cells via caspase activation.

<p>rHSA fusion proteins, as well as nutlin (to serve as a p53-MDM2 antagonist control) were added at the indicated concentrations and allowed to incubate for 24 hrs. A). Cytotoxicities were measured by CyQuant Assay and normalized according to 10 μM rHSA-treated cells. B). Caspase activation was quantitated using the Homogeneous Caspase Assay as described in <i>Methods</i> and normalized according to untreated cells. Results are displayed as percent cell death (A) or fold change (B) relative to 10 μM rHSA-treated wells. Data are representative of 3 independent experiments performed in triplicate. Error bars indicate ± SD.</p

rHSA fusion proteins are able to form stable complexes with FA-FITC.

<p><b>A</b>). rHSA-PMI (lane 1-3), rHSA-P53i (lane 4-6) and rHSA (lane 7-9) were incubated at the indicated molar ratios (rHSA:FA-FITC) with FA-FITC (lane 1, 4, and 7 (1:1); lane 2, 5, and 8 (1:2); lane 3, 6, and 9 (1:4); lane 10, FA-FITC only). The upper band in the gel corresponds to the HSA/FA-FITC complex, while the lower band indicates unbound FA-FITC. Incorporation of FA-FITC into rHSA was achieved up to a 1:4 rHSA:FA-FITC molar ratio. <b>B</b>). rHSA/FA-FITC complexes were pre-formed at a 1:4 molar ratio (HSA:FA-FITC; 30 pmol:120 pmol) as described in <i>Methods</i>. Unlabeled FA was then added at the indicated concentrations to mimic the competition of free FA present under physiological conditions. The minimal dissociation of FA-FITC from pre-formed rHSA/FA-FITC complexes at the 8 times excess concentration of unlabeled FA (lane 8) indicates FA-FITC and rHSA complex was highly stable in the presence of free FA. <b>C</b>). Pre-formed biotin-rHSA and FA-FITC complexes (biotin-rHSA:FA-FITC; 1:2) were incubated with PBS (Lane 1 and 3) and 10% serum (lane 2 and 4) for 1 and 24 hours. Lanes 1 and 2 represent total FA-FITC incorporation into rHSA without and with 10% FBS, respectively, prior to the addition of streptavidin resins. Lane 3 (with PBS only) and lane 4 (with 10% FBS) correspond to the supernatants of samples after incubation with streptavidin resins and pulling down rHSA/FA-FITC complexes. The absence of rHSA/FA-FITC complexes in lane 3 indicates that all biotin-rHSA/FA-FITC complexes (in PBS) were efficiently pulled down. Any FA-FITC present in lane 4 would imply the displacement of rHSA-bound FA-FITC by serum components. The presence of only a weak band in lane 4 indicates the majority of FA-FITC remained bound to rHSA (pulled down by streptavidin resins). Quantitation of the amount of FA-FITC in lane 4 (Image J, <i>NIH</i>) revealed approximately 15% and 17% of FA-FITC was removed from biotin-rHSA in the presence of serum following 1 and 24 hour incubations, respectively. </p

IGF-1C domain-modified hydrogel enhances therapeutic potential of mesenchymal stem cells for hindlimb ischemia

Abstract Background Poor cell engraftment and survival after transplantation limited the application of stem cell therapy. Synthetic biomaterials could provide an artificial microenvironment for stem cells, thereby improve cell survival and enhance the therapeutic efficiency of stem cells. Methods We synthesized a hydrogel by conjugating C domain peptide of insulin-like growth factor-1 (IGF-1C) onto chitosan (CS-IGF-1C hydrogel). Human placenta-derived mesenchymal stem cells (hP-MSCs), which constitutively express a red fluorescent protein (RFP) and renilla luciferase (Rluc), were co-transplanted with CS-IGF-1C hydrogel into a murine hindlimb ischemia model. Transgenic mice expressing firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (VEGFR2-Luc) were used. Dual bioluminescence imaging (BLI) was applied for tracking the survival of hP-MSCs by Rluc imaging and the VEGFR2 signal pathway activation by Fluc imaging. To investigate the therapeutic mechanism of CS-IGF-1C hydrogel, angiographic, real-time PCR, and histological analysis were carried out. Results CS-IGF-1C hydrogel could improve hP-MSCs survival as well as promote angiogenesis as confirmed by dual BLI. These results were consistent with accelerated skeletal muscle structural and functional recovery. Histology analysis confirmed that CS-IGF-1C hydrogel robustly prevented fibrosis as shown by reduced collagen deposition, along with increased angiogenesis. In addition, the protective effects of CS-IGF-1C hydrogel, such as inhibiting H2O2-induced apoptosis and reducing inflammatory responses, were proved by in vitro experiments. Conclusions Taken together, IGF-1Cs provides a conducive niche for hP-MSCs to exert pro-mitogenic, anti-apoptotic, and pro-angiogenic effects, as well as to inhibit fibrosis. Thus, the incorporation of functional peptide into bioscaffolds represents a safe and feasible approach to augment the therapeutic efficacy of stem cells