hAAT Immunohistochemistry.

<p>Non-transfected pig liver hAAT staining (a; 40x) versus transfection employing endovascular closed catheterization (b; 40x). hAAT protein staining in liver sections of transfected mice is shown (c; 40x); human liver hAAT immunostaining can be observed (d; 40x). Black arrows indicate representative specific immunohistochemical reaction against hAAT protein in pig samples. Tissue was counterstained with hematoxylin.</p

hAAT gene translation index in liver tissue.

<p>Fig 3 represents the gene translation index per cell, calculated per ELISA, after both the endovascular and surgery closed perfusion procedures. Translation in each liver lobe is represented (mean ± SD). First letter: R = right, L = left; Second letter: M = medial, L = lateral; Number: 1 = upper, 2 = lower. Statistical analysis: linear mixed model with pig as random factor, hAAT protein as response variable, and group, liver lobe and their interaction as explicative variables.</p

Pig hAAT gene decoding by catheterization and surgical closed procedure versus standards.

<p>The average copy contents (mean ± SD) of hAAT DNA, RNA and protein (tissue and plasma) in pigs after closed liver hydrofection (A) are compared with those obtained in standards (human liver and hydrodynamically transfected mouse liver, B). Tissue indexes are shown as copy number per cell in each case. Protein expression in plasma is expressed as concentration (μg/ml). Statistical analysis: Linear mixed model with expression level as response variable and sample type (DNA, RNA, protein or plasma), species and their interaction as explicative variables; statistically significant interaction between pig and protein in serum (p < 0.001). †: p<0.05 (p = 0.027); ††: p<0.01 (p = 0.007).</p

Open and closed endovascular catheterization procedure.

<p>The endovascular closed procedure is represented in Fig 1A. A schematic representation of the procedure is provided. An 8Fr balloon introducer and a 10Fr balloon catheter were placed in the vena cava, limiting entrance to the liver. Another transhepatic 10Fr balloon catheter was placed in the portal vein to block outflow. Fig 1B provides a schematic representation of the surgery closed model. After midline laparotomy, the liver is exposed and its vasculature (veins and artery), except the infrahepatic inferior vena cava, is referenced and ligated, thus completely sealing the organ. Retrograde gene transfer is performed through an 11 Fr catheter.</p

hAAT protein expression in liver tissue.

<p>hAAT protein expression in liver tissue is represented. The part at left corresponds to controls. Positive controls: pure hAAT protein (1 and 5 ng). Negative control: homogenate from eGFP transfected pig liver tissue (50 μg of total protein). The part at right in the figure represents hAAT protein presence in liver tissue after both the endovascular and surgery closed perfusion procedures. Translation in each liver lobe is represented. Equivalent amounts of total protein from distal and proximal tissue samples of each liver lobe were mixed. First letter: R = right, L = left; Second letter: M = medial, L = lateral; The id of pigs follows the internal nomenclature employed in our laboratory.</p

hAAT protein concentration in plasma on day 14.

<p>hAAT protein concentration in plasma on day 14.</p

Histological and cellular scheme of hydrofection and nanoparticles distribution within liver tissue.

<p>The schematic area (left) provides a representation of how hydrodynamic retrograde injection, under the described conditions, mediates greater separation between endothelial cells and their fenestrations, as well as enlargement of the Disse space and the formation of massive endocytic vesicles. These changes result in DNA molecule entry into the liver cell nucleus, where DNA transcription to RNA occurs. Then, mRNA is released into the cell cytoplasm, where it is translated to protein. Finally, after the necessary modifications, the mature protein, if to be secreted, is released out of the tissue. The image at right provides a transmission electron microscopy view of the nuclear compartment of hepatocytes after transferring 4 nm and 15 nm gold nanoparticles by the surgery closed procedure (lower panel) and open procedure (upper panel). Nanoparticles only reach the nucleus with more energetic conditions of the open procedure. N: nucleus; C: cytoplasm; *: nuclear envelope. White arrows: nanoparticles 4–8 nm in diameter.</p