Supramolecular Protein Cage Composite MR Contrast Agents with Extremely Efficient Relaxivity Properties

A DTPA-Gd containing polymer was grown in the interior of a heat shock protein cage resulting in T1 particle relaxivities of 4200 mM−1 sec−1 for the 12 nm particle. Relaxivity parameters were determined, and this analysis suggests that the rotational correlation time has been optimized while the water exchange lifetime is longer than optimal. This synthetic approach holds much promise for the development of next generation contrast agents and this report will aid in their design

FePro labeled THP-1 and U-251 cells.

<p>Microphotography of cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h and stained using PB method to visualize the intracellular iron incorporation in THP-1 cells (A, bar = 50 µm) and U-251 cells (B, bar = 50 µm). Electron microscopy images of U251 cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (C, D, bar = 1 µm) and control unlabeled U251 cells (E, bar = 1 µm). Note; extracellular FePro granules attached to the cell membranes and on their way to be engulfed and endocytosed (arrows) (C) and endocytosed intracellular iron within the endosomal compartment (arrows in circles) (D). Very little to no extracellular particles of FePro complexes seen compared to our previous method of labeling cells (F, G). Prussian blue staining of magnetically labeled hMSCs using our previous method showing extracellular complexes (F, bar = 10 µm) which are also confirmed by electron microscopic images (G bar = 2 µm). Extracellular complexes pointed by arrows in F–G.</p

Global gene expression and multidimensional scaling analysis of FePro labeled BMSCs.

<p>BMSC samples from 3 donors (FePro-labeled, gold nanoparticle-labeled and unlabeled control) and control cells (3 samples from human embryonic stem cells and 3 samples of adult cells) were analyzed by an oligonucleotide microarray. The multidimensional scaling plot similarly grouped the hES cells together, the adult cells other than BMSCs together in another group, and all the BMSC samples into a third group. The BMSCs did not cluster according to the type of labeling method. hES- human embryonic stem cell; adult indicated the adult cells: Fb-fibroblasts, EC endothelial cells, SMC-smooth muscle cells; BMSC-FePro: bone marrow stromal cellslabeled with FePro; BMSC-Gold: bone marrow stromal cells labeled with gold nanoparticle; BMSC-control: unlabeled BMSC control; D1: donor 1; D2-donor 2; D3 donor 3.</p

FePro labeled T and AC133+ hHSCs cells.

<p>Microphotography DAB-enhanced PB staining of non-labeled T-cells (A, bar = 50 µm) and T cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (B, bar = 50 µm). Note the intracellular localization of FePro complexes that appear as the dark brown granules on electron microscopic images (arrows). Electron microscopy images of T-cells (C–D, bar = 1 µm) and AC133+ HSCs (F–G, bar = 1 µm) cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h. Prussian blue staining of AC133+ HSCs cells (E, bar = 20 µm).</p

Immunohistochemical staining of ossicles derived from FePro or GFP labeled and unlabeled BMSCs.

<p>A representative ossicle derived from unlabeled (A) and FePro labeled (B) BMSCs at 8 weeks, stained with H & E showing comparable abundant bone formation and abundant hematopoiesis. Immunohistochemistry staining for GFP of a representative ossicle derived from BMSCs labeled with both FePro and lentivirus carrying GFP (C) and control unlabeled BMSCs (D).</p

CD 146 expression in FePro-labeled BMSCs.

<p>(A) Representative flow cytometry histogram with overlay of the two groups showing no difference in CD146 expression after SPION labeling of BMSCs. (B) Bar graph showing mean CD146 expression in FePro labeled and unlabeled BMSC. Note the lack of statistically significant difference in CD146 expression after SPION labeling (solid colored bars), Student t test, p>0.5. Data shown as mean +/− S.D. of CD 146 expression in 5 donors.</p

Colony forming efficiency in FePro-labeled BMSCs.

<p>Secondary colony forming efficiency of BMSCs plated at clonal density (A) or high density (B) from 5 donors. Data are represented as mean +/− S.D. of colony forming units for each donor done in triplicates. Note the lack of a statistically different change in number of colonies in SPION-labeled BMSCs (solid colored bars), Student t test, p>0.5. A similar lack of a statistically different change in the number of colonies were found when the secondary colony forming efficiency experiments from 5 donors were repeated independently by two other scientists.</p

Prussian blue staining of ossicles derived from FePro or unlabeled BMSCs.

<p>Prussian blue (PB) staining of a representative ossicle derived from BMSCs labeled with FePro (A) and control unlabeled BMSCs (B). PB staining of a representative ossicle derived from BMSCs labeled with FePro showing PB⁺ adipocytes (C). PB staining of a representative ossicle derived from labeled BMSCs showing PB⁺ pericytes (D).</p

Dilution of FePro in cultured FePro-labeled and unlabeled BMSCs.

<p>The microphotographs show PB staining of BMSCs cultured <i>in vitro</i> at passages 3, 4, 5 and 6 after BMSCs at passage 2 were labeled with FePro.</p

Unlabeled and FePro labeled hMSC (A–C) and mNSC (D–F) cells.

<p>Microphotography of PB staining of non-labeled (A) and PB (B) and DAB-enhanced PB (C) staining of hMSC cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h (B–C, show images taken from two different fields in the same slide). PB staining of non-labeled (D) and PB (E) and DAB-enhanced PB (F) staining of mNSC cells labeled with 100 µg/ml Fe and 3 µg/ml Pro for 4 h. Note the intracellular localization of FePro complexes that appear as blue (PB stain) and the dark brown granules (DAB enhanced PB stain). (A–F, bar = 100 µm).</p