Research ArticleOrgan Transplantation

Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys

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Science Translational Medicine  29 Nov 2017:
Vol. 9, Issue 418, eaam6764
DOI: 10.1126/scitranslmed.aam6764
  • Fig. 1. CD31 is present throughout the human renal vasculature.

    (A) Representative fluorescent immunohistochemistry staining image of a cryosection taken from a wedge biopsy of a human kidney undergoing normothermic machine perfusion (NMP). Section was stained for Ulex to depict vascular endothelium (green). White arrows label different vascular beds and the kidney surface. Multiple 20× images were tiled to reconstruct the entire cryosection. (B) Representative images for CD31 (green) and nuclear 4′,6-diamidino-2-phenylindole (DAPI; blue) staining of glomerular capillaries (left) and interstitial microvessels (right). Representative staining from three different donor kidneys.

  • Fig. 2. CD31 provides robust NP targeting in vitro but slow kinetics.

    (A) Representative transmission electron microscopy image of poly(lactic acid)–poly(ethylene) glycol NPs. Scale bar, 200 nm. (B) Quantification of NP size distribution and polydispersity index as measured by dynamic light scattering for both 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)– and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO)–loaded nanoparticles (NPs; n = 3; a t test was used for statistical analysis). N.S., not statistically significant. (C) Representative 20× images of CD31-NPs versus Control-NPs after a 2-hour incubation with human umbilical vein endothelial cells (HUVECs) in static culture. DiO-loaded NPs are shown in green (right column) overlaid with brightfield images (left column). Scale bars, 150 μm. n = 3. (D) Representative fluorescence-activated cell sorting (FACS) histograms for HUVECs treated identically to (C). (E) Quantification of triplicate FACS experiments as shown in (D). n = 3. MFI, mean fluorescence intensity; a.u., arbitrary units. (F) Representative images of accumulation of DiI NPs as a function of time on HUVECs in a microfluidic cell culture during perfusion with CD31-NPs (100 μg/ml; top) or Control-NPs (100 μg/ml; bottom) at 2 dyne/cm2. Each image is 300 μm wide, representing the full width of the microfluidic channel. NPs are pseudocolored green for better visualization. (G) Quantification of mean area of positive fluorescence for flow experiments as performed in (F) at a range of concentrations (n = 3 independent imaging regions for each dose and time point). Lines through data represent fit to a rate equation. (H) Plot of rates as a function of NP concentration as extracted from quantification in (G). Line through data represents linear fit. Rates are normalized to a concentration of 10 μg/ml (dashed line, 1). All error bars represent 1 SD.

  • Fig. 3. CD31 targeting enhances NP accumulation in perfused human kidney.

    (A) Representative 20× fluorescent images of glomeruli (top) and interstitial microvessels (bottom) for kidneys perfused with either CD31-NPs (left) or Control-NPs (right). Overlay images show vascular stain (Ulex; green), DAPI nuclear stain (blue), and NP accumulation (red). Scale bars, 100 μm. Images are cropped to provide finer details. (B) Quantification of individual image MFI after background subtraction. Each image was normalized to the area of Ulex vascular stain, and every dot represents an individual normalized image mean intensity. The red dots correspond to the images shown in (A). *P < 0.0001 using a Mann-Whitney t test. For the single CD31-NP–treated kidney, n = 35 for images of glomeruli and n = 59 for images of microvasculature; for the single Control-NP–treated kidney, n = 31 for images of glomeruli and n = 56 for images of microvasculature. (C) Time-dependent normalized mean intensity values for CD31-NP versus Control-NP in glomeruli with corresponding fits to a linear equation (CD31-NP, black line) or saturating kinetic equation (Control-NP, dashed gray line). Error bars refer to 95% confidence window of the mean. n for each condition is provided in Table 3.

  • Fig. 4. Specific versus nonspecific NP accumulation.

    Representative thresholded 20× fluorescent images of (A) glomeruli from the NP kidney (10 μg/ml), (B) interstitial microvessels from the NP kidney (50 μg/ml), and (C) glomeruli and interstitial microvessels from the NP kidney (100 μg/ml) with white boxed regions highlighting areas of either specific or nonspecific accumulation within the same field of view or same section (DAPI, blue; CD31-NPs, red; Control-NPs, green; NP overlay, yellow; Ulex, white). The boxed regions are shown at a larger scale in the insets for both (A) and (B). The white dashed lines across the insets in (A) refer to the line scale analysis shown in (D) for nonspecific (region 1) and specific (region 2) accumulation. Scale bars, 50 μm. (D) Line scan plot of highlighted regions of NP glomerular accumulation in (A) showing similar amounts of accumulation in nonspecific regions with many more CD31-NPs relative to Control-NPs in more specific regions. (E) Region quantifications depicting the total relative NPs signal (CD31-NPs/Control-NPs) for the individual highlighted regions in (A) to (C); each bar represents the quantification of the single image shown for the respective panel.

  • Fig. 5. Global analysis of CD31-NP targeting benefit in the renal cortex.

    (A) Quantification of relative NP signal (CD31-NPs/Control-NPs) at 4 hours for kidneys 3 to 5 (10, 50, and 100 μg/ml, respectively). Each point represents an individual image of either glomeruli (light gray) or interstitial microvessels (dark gray) with mean value shown as the black line; nonspecific (1:1) accumulation is represented by dashed gray line. **P < 0.001 for all values compared to 1 (nonspecific) using Wilcoxon signed-rank test. *P < 0.004 using Mann-Whitney test. (B) Relative CD31-NP/Control-NP intensity for images taken from wedge biopsies collected at the given time point for kidneys 3 to 5. Values are shown as mean with error bars signifying a 95% confidence interval of the mean. n for each condition is provided in Table 3.

  • Fig. 6. Effects of CD31-NP targeting in perfused human kidneys.

    Quantification of relative NP signal (CD31-NPs/Control-NPs) at 4 hours (h) for kidneys 6 to 8. Each point represents an individual image of either interstitial microvessels (A) or glomeruli (B); nonspecific (1:1) accumulation is represented by dashed gray line. **P < 0.0001 and *P = 0.0024 according to a Wilcoxon signed-rank test. Colored data points correspond to the quantification of the higher (red) or lower (green) specificity images shown in (C). (C) Representative images of either higher or lower specificity regions of NP accumulation in interstitial microvessels of kidney 6 (left) or glomeruli of kidney 7 (middle) and lower specificity regions in glomeruli of kidney 8 (right; DAPI, blue; CD31-NPs, red; Control-NPs, green; NP overlay, yellow; Ulex, white). Scale bars, 50 μm. n for each condition is provided in Table 3.

  • Fig. 7. NPs accumulate at sites of vascular obstruction.

    (A) Three-dimensional projection of a z-stack of confocal images of a vessel containing a high density of nonspecific NP accumulation in its lumen (DAPI, blue; CD31-NPs, red; Control-NPs, green; NP overlay, yellow; Ulex, white). Dashed white line represents the point of apparent obstruction, and arrows highlight apparent red blood cells (RBCs). Scale bar, 10 μm. (B) Representative image of nonspecific colocalization of NPs with sites of high-density RBC (glycophorin A, white; nonspecific NP, yellow) presumably representing vascular obstruction. Scale bar, 10 μm. (C) Quantification of relative NP signal (CD31-NP/Control-NP) for image in (B). (D) Representative hematoxylin and eosin–stained images of kidney 5 (100 μg/ml) before NP introduction (pre-NP) or after 4 hours of NP perfusion. RBC occlusions are stained red. Scale bars, 100 μm.

  • Table 1. Declined human kidney characteristics and experimental conditions.

    Donor demographics, donor type; donation after brain death (DBD), donation after circulatory death (DCD), reason for decline, warm ischemic time, cold ischemic time, duration of ex vivo normothermic perfusion (NMP), mean renal blood flow (RBF), total urine output, macroscopic appearance, NPs, and time to administration of NPs.

    Case 1Case 2Case 3Case 4Case 5Case 6Case 7Case 8
    Donor age
    (years)
    5725446845646557
    Donor type
    (DBD/DCD)
    DCDDBDDCDDBDDBDDCDDCDDCD
    Left/right kidneyLeftRightRightRightLeftLeftLeftLeft
    Reason for declinePoor in situ
    perfusion
    MalignancySuspected
    malignancy
    Suspected
    malignancy
    Poor in situ
    perfusion + prolonged
    agonal phase
    Suspected
    malignancy
    Histological
    changes
    consistent with
    diabetes
    Damage to
    ureter and
    artery
    Warm ischemic
    time (min)
    1313121411
    Cold ischemic time
    (hour:min)
    13:1317:1734:3716:0322:3827:0518:2025:58
    NMP duration
    (hour:min)
    4:306:505:455:005:008:308:308:30
    Mean RBF
    (ml/min per 100 g)
    88.0 ± 19.6123.7 ± 35.291.6 ± 23.967.9 ± 31.783.5 ± 35.575.0 ± 22.177.3 ± 25.480.7 ± 37.7
    Total urine
    output (ml)
    1777301071045373598451257
    Macroscopic
    appearance
    ModerateModerateModerateModerateModerateExcellentExcellentModerate
    NP typeCD31-NPControl-NPCD31-NP +
    Control-NP
    CD31-NP +
    Control-NP
    CD31-NP +
    Control-NP
    CD31-NP +
    Control-NP
    CD31-NP +
    Control-NP
    CD31-NP +
    Control-NP
    NP circulating
    concentration
    4 μg/ml4 μg/ml10 μg/ml50 μg/ml100 μg/ml50 μg/ml50 μg/ml50 μg/ml
    NP administration
    time (from start of
    perfusion)
    30 min50 min45 min75 min60 min30 min30 min30 min
  • Table 2. Functional characterization and histological analysis of kidneys 6 to 8.

    The mean RBF, urine output, level of oxygen consumption, and perfusate pH levels at baseline (after 30 min of perfusion but before the administration of NPs), 2 hours, 4 hours, 6 hours, and 8 hours of perfusion. ATN, acute tubular necrosis.

    Case 6Case 7Case 8
    RBF (ml/min per 100 g)
      Baseline524125
      2 hours608278
      4 hours769098
      6 hours96102119
      8 hours120107135
    Urine output (ml)
      Baseline5598
      2 hours1115455
      4 hours135126101
      6 hours15412781
      8 hours14313512
      Total598451257
    Oxygen consumption
    (ml/min per g)
      Baseline38.546.057.9
      2 hours43.961.056.6
      4 hours52.267.470.9
      6 hours70.275.852.1
      8 hours84.781.099.4
    pH
      Baseline7.217.237.34
      2 hours7.277.417.35
      4 hours7.347. 427.35
      6 hours7.377.387.28
      8 hours7.377.407.28
    Histology
      BaselineNo ATNNo ATNNo ATN
      2 hoursNo ATNNo ATNNo ATN
      4 hoursNo ATNNo ATNNo ATN
      6 hoursNo ATNFocal mild
    ATN
    No ATN
      8 hoursFocal mild
    ATN
    No ATNFocal mild
    ATN
  • Table 3. Number of images collected per sample and condition.

    N/A refers to conditions that were not analyzed within the manuscript.

    KidneyTime point
    (hours)
    Glomeruli
    (n)
    Microvasculature
    (n)
    Stage 1 (kidneys
    1 and 2)
    10.527N/A
    129N/A
    326N/A
    43559
    20.529N/A
    129N/A
    325N/A
    43156
    Stage 2 (kidneys
    3 to 5)
    30.53148
    12631
    22338
    34450
    44837
    40.52023
    13330
    22239
    32236
    43735
    50.52220
    13022
    22436
    33332
    43831
    Stage 3 (kidneys
    6 to 8)
    623937
    44033
    64036
    723839
    44035
    64040
    824048
    44037
    64036
  • Table 4. Macroscopic assessment, thresholds of RBF, and urine output.
    AssessmentScore
    NMP assessment score during ex vivo NMP
    Macroscopic assessment
      Grade I: excellent perfusion (global pink appearance)1
      Grade II: moderate perfusion (patchy appearance)2
      Grade III: poor perfusion (global mottled and purple
    and black appearance)
    3
    RBF
      Threshold, ≥50 ml/min per 100g0
      Threshold, <50 ml/min per 100g1
    Total urine output
      Threshold, ≥43 ml0
      Threshold, <43 ml1

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/418/eaam6764/DC1

    Materials and Methods

    Fig. S1. CD31-NP characterization.

    Fig. S2. Matching of two-color NP fluorescence.

    Fig. S3. Tissue autofluoresence characterization.

    Fig. S4. Effect of NP dose on average glomerular accumulation.

    Fig. S5. Two-color NP accumulation.

    Fig. S6. Vascular obstruction histology.

    Data file S1. MATLAB image analysis code.

  • Supplementary Material for:

    Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys

    Gregory T. Tietjen, Sarah A. Hosgood, Jenna DiRito, Jiajia Cui, Deeksha Deep, Eric Song, Jan R. Kraehling, Alexandra S. Piotrowski-Daspit, Nancy C. Kirkiles-Smith, Rafia Al-Lamki, Sathia Thiru, J. Andrew Bradley, Kourosh Saeb-Parsy, John R. Bradley, Michael L. Nicholson, W. Mark Saltzman, Jordan S. Pober*

    *Corresponding author. Email: jordan.pober{at}yale.edu

    Published 29 November 2017, Sci. Transl. Med. 9, eaam6764 (2017)
    DOI: 10.1126/scitranslmed.aam6764

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. CD31-NP characterization.
    • Fig. S2. Matching of two-color NP fluorescence.
    • Fig. S3. Tissue autofluoresence characterization.
    • Fig. S4. Effect of NP dose on average glomerular accumulation.
    • Fig. S5. Two-color NP accumulation.
    • Fig. S6. Vascular obstruction histology.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Word format). MATLAB image analysis code.

    [Download Data file S1]

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