Research ArticleNanomedicine

Imaging-assisted nanoimmunotherapy for atherosclerosis in multiple species

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Science Translational Medicine  21 Aug 2019:
Vol. 11, Issue 506, eaaw7736
DOI: 10.1126/scitranslmed.aaw7736
  • Fig. 1 Nanoimmunotherapy production scale-up and evaluation in Apoe−/− mice.

    (A) Schematic of S-HDL production by high-pressure microfluidic homogenization. Transmission electron microscopy (TEM) image of S-HDL. Scale bar, 25 nm. (B) Schematic of radiolabeling of S-HDL with 89Zr by incorporating the phospholipid chelator DSPE-DFO in the formulation. (C) Blood time-activity curve for [89Zr]-S-HDL intravenously injected in Apoe−/− mice (n = 4; 12 weeks on Western diet). ID, injected dose. (D) Representative 3D-rendered PET/CT fusion image of an Apoe−/− mouse 26 hours after injection of [89Zr]-S-HDL. (E) Quantitation of tissue radioactivity distribution 26 hours after injection of [89Zr]-S-HDL in Apoe−/− mice (n = 4; 12 weeks on Western diet). On the right, representative autoradiograph showing radioactivity distribution on the aorta of an Apoe−/− mouse 26 hours after injection of [89Zr]-S-HDL. (F) Gating procedure used in the flow cytometry evaluation of microfluidizer-produced DiO-S-HDL’s cell specificity in Apoe−/− mouse aortas. (G) DiO mean fluorescence intensity (MFI) in different cell types from Apoe−/− mouse aortas. Control, uninjected animal. (H) Gating procedure used in the flow cytometry analysis to evaluate S-HDL treatment efficacy in aortas of Apoe−/− mice. (I) Quantitation of aortic macrophages (MØ) and monocytes (Ly6Chi Mo) after treatment with PBS (placebo) or S-HDL in Apoe−/− mice (n = 10 per group; 12 weeks on Western diet). (J) Representative aortic sections from Apoe−/− mice (n = 2 per group; 12 weeks on Western diet) treated with PBS (placebo) or S-HDL. Scale bars, 200 μm. CD68 is expressed on macrophages. aq., aqueous; Li, liver; Sp, spleen; Ki, kidney; LN, lymph node; Leu, leukocyte; Lin+, lineage positive; Neu, neutrophil. Data are presented as means ± SD from one experiment. *P < 0.05 (Mann-Whitney test).

  • Fig. 2 S-HDL nanoimmunotherapy in vivo evaluation by noninvasive imaging in rabbits and pigs.

    (A) Schematic description of the rabbit and porcine atherosclerosis models used in this study. (B) Schematic of noninvasive biodistribution and plaque targeting using 89Zr-S-HDL with PET/CT and PET/MRI, respectively. (C) Representative 3D-rendered PET/CT fusion images of atherosclerotic rabbits (top) and pigs (bottom) at 1, 24, and 48 hours after administration of [89Zr]-S-HDL. (D) PET imaging–derived time-activity curves in selected tissues in rabbits (n = 2; top) and pigs (n = 2; bottom) injected with [89Zr]-S-HDL. The x represents the value obtained ex vivo by gamma counting. The gray line is the muscle time-activity curve, included for reference. (E) PET/MRI assessment of plaque targeting and quantification of standardized uptake values (SUVs) in one rabbit (top) and one pig (bottom), 48 hours after injection of [89Zr]-S-HDL, in the low and high uptake regions shown in the red- and yellow-bordered squares, respectively. Data shown in (D) and (E) were obtained from one experiment; no statistical analysis was carried out because of the limited number of subjects (n ≤ 2). (F) Regional distribution of S-HDL in atherosclerotic samples from rabbits (top) and pigs (bottom), as determined by autoradiography (AR; [89Zr]-S-HDL) and near-infrared fluorescence (NIRF; DiD-S-HDL) 48 hours after injection.

  • Fig. 3 Imaging-guided S-HDL nanoimmunotherapy in rabbits and pigs: PET-based readouts.

    (A) Schematic representation of S-HDL imaging–guided treatment. Treatment response was evaluated longitudinally by performing a baseline scan before the first S-HDL administration and a terminal scan 48 hours after the last infusion. Animals received four infusions over 2 weeks. (B)18F-FDG-PET imaging–based assessment of vessel wall inflammation in rabbits (n = 5 per group; top) and pigs (n = 4 per group; bottom) treated with PBS (placebo) or S-HDL. Scale bars, 10 mm. (C) 18F-FLT-PET imaging–based assessment of cellular proliferation in the vessel wall in rabbits (n = 4 per group; top) and pigs (n = 4 per group; bottom) treated with PBS (placebo) or S-HDL. Scale bars, 10 mm. In (B) and (C), dots are color-coded for individual animals, and two data points are represented per pig, corresponding to values obtained from analysis of each of the femoral arteries. Line is situated at median. TBRmax, maximal target-to-background ratio. Data were obtained in one experiment and analyzed using a linear mixed model (see the “Statistical analysis” section for details).

  • Fig. 4 Imaging-guided S-HDL nanoimmunotherapy in rabbits and pigs: MRI-based readouts.

    (A) 3D dynamic contrast–enhanced MRI-based vessel wall permeability measurements in rabbits (n = 5 per group; top) and pigs (n = 4 per group; bottom) treated with PBS (placebo) or S-HDL. Scale bars, 10 mm. (B) Ex vivo vessel wall permeability assessed by Evans Blue NIRF imaging in rabbit aortas and porcine femoral arteries. Data are presented as means ± SD from one experiment. (C) T2-weighted MRI-based vessel wall area measurements in rabbits (n = 5 per group; top) and pigs (n = 4 per group; bottom) treated with PBS (placebo) or S-HDL. Scale bars, 10 mm. In (A) and (C), dots are color-coded for individual animals, and two data points are represented per pig, corresponding to values obtained from analysis of each of the femoral arteries. Line is situated at median. (D) Proportion of scans that afforded increased or decreased imaging marker values in rabbits treated with PBS (placebo) or S-HDL. (E) Pooled representation of the variation in the four independent imaging parameters in rabbits treated with PBS (placebo) or S-HDL (line is situated at median). (F) Proportion of scans that afforded increased or decreased imaging marker values in pigs treated with PBS (placebo) or S-HDL. (G) Pooled representation of the variation in the four independent imaging parameters in pigs treated with PBS (placebo) or S-HDL (line is situated at median). Results shown in (A) and (C) were obtained in one experiment and analyzed using a linear mixed model (see the “Statistical analysis” section for details), whereas results shown in (B), (E), and (G) were analyzed using the Mann-Whitney test. *P < 0.05 and **P < 0.01.

  • Table 1 Blood biochemistry results from pigs with atherosclerosis treated with PBS (placebo) or S-HDL.

    Data are presented as median [interquartile range]. Bolded values indicate P < 0.05. ALP, alkaline phosphatase; SGPT, serum glutamic pyruvic transaminase; ALT, alanine aminotransferase; SGOT, serum glutamic-oxaloacetic transaminase; GGT, gamma-glutamyl transferase; AST, aspartate aminotransferase; CPK, creatine phosphokinase; BUN, blood urea nitrogen.

    PlaceboS-HDLP
    ALP (U/liter)127 [87–175]85 [70–132]0.15
    SGPT (ALT)
    (U/liter)
    45 [39–67]51 [38–65]0.94
    SGOT (AST)
    (U/liter)
    69 [43–222]67 [53–160]0.80
    GGT (U/liter)40 [34–78]41 [39–56]0.67
    CPK (U/liter)740 [406–1817]796 [431–1024]0.94
    Total bilirubin
    (mg/dl)
    0.2 [0.1–0.2]0.2 [0.2–0.3]0.28
      Direct bilirubin
    (mg/dl)
    0.0 [0.0–0.1]0.0 [0.0–0.1]>0.99
      Indirect bilirubin
    (mg/dl)
    0.1 [0.1–0.2]0.2 [0.1–0.3]0.37
    Total protein (g/dl)6.6 [5.9–6.7]6.8 [6.5–7.1]0.21
      Albumin (g/dl)3.5 [3.4–4.0]4.0 [3.7–4.5]0.29
      Globulin (g/dl)2.6 [2.5–3.1]2.8 [2.5–2.9]0.81
      Albumin/
    globulin ratio
    1.4 [1.2–1.6]1.4 [1.3–1.8]0.75
    BUN (mg/dl)12 [10–14]10 [8.5–17]0.79
    Creatinine (mg/dl)0.9 [0.8–1.2]1.0 [0.9–1.2]0.69
    BUN/creatinine
    ratio
    13 [9.1–17]11 [9.0–15]0.94
    Glucose (mg/dl)76 [71–80]67 [34–69]0.03
    Cholesterol (mg/dl)573 [450–628]507 [426–747]0.80
    Triglycerides
    (mg/dl)
    62 [26–72]72 [48–186]0.31
    Sodium (mM)140 [140–141]139 [136–141]0.20
    Potassium (mM)4.4 [4.3–5.1]4.6 [4.1–7.5]0.44
    Na/K ratio32 [28–33]30 [21–35]0.44
    Magnesium (mg/dl)2.0 [1.9–2.4]2.1 [1.9–2.8]0.81
    Calcium (mg/dl)10 [9.4–11]10 [9.9–10.2]0.88
    Phosphorus (mg/dl)7.4 [7.2–7.9]6.9 [6.4–8.6]0.53
    Bicarbonate (mM)22 [19–24]19 [17–24]0.49
    Chloride (mM)99 [98–101]102 [97–105]0.49
  • Table 2 Complete blood count results from pigs with atherosclerosis treated with PBS (placebo) or S-HDL.

    Data are presented as median [interquartile range]. Bolded values indicate P < 0.05. MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin.

    PlaceboS-HDLP
    Red blood cell #
    (M/μl)
    8.1 [6.3–9.7]5.0 [3.8–6.5]0.06
    Hemoglobin
    (g/dl)
    13 [10–16]8.9 [7.3–11]0.19
    Hematocrit (%)43 [32–54]31 [25–45]0.29
    MCV (fl)53 [51–56]66 [62–69]0.02
    MCH (pg)16 [16–17]18 [17–19]0.03
    MCH
    concentration
    (g/dl)
    30 [29–31]28 [26–30]0.11
    Reticulocyte #
    (K/μl)
    27 [20–42]65 [38–161]0.06
    Reticulocyte (%)0.28 [0.25–0.60]1.5 [0.7–3.2]0.06
    Platelet # (K/μl)175 [142–193]226 [166–393]0.19
    White blood cell
    # (K/μl)
    6.9 [5.5–9.6]5.5 [5.2–5.9]0.25
    Neutrophil #
    (K/μl)
    3.3 [2.4–5.2]1.6 [1.1–2.1]0.06
    Lymphocyte #
    (K/μl)
    3.4 [2.8–3.8]3.8 [3.1–4.1]0.68
    Monocyte #
    (K/μl)
    0.35 [0.24–0.49]0.24 [0.17–0.36]0.26
    Eosinophil #
    (K/μl)
    0.08 [0.05–0.16]0.03 [0.02–0.12]0.44
    Basophil # (K/μl)0.0 [0.0–0.01]0.01 [0.0–0.01]0.52
    Neutrophil (%)51 [41–56]33 [20–37]0.03
    Lymphocyte (%)41 [39–52]63 [59–72]0.03
    Monocyte (%)4.4 [3.8–5.9]4.3 [3.1–6.3]0.71
    Eosinophil (%)1.0 [0.6–2.1]0.5 [0.3–2.0]0.44
    Basophil (%)0.0 [0.0–0.1]0.2 [0.0–0.2]0.29

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/506/eaaw7736/DC1

    Materials and Methods

    Fig. S1. S-HDL characterization.

    Fig. S2. In vivo S-HDL cell specificity evaluation.

    Fig. S3. Pharmacokinetics and plaque targeting of S-HDL in rabbits and pigs.

    Fig. S4. S-HDL treatment effect in rabbits and pigs.

    Fig. S5. Histological evaluation of S-HDL treatment.

    Fig. S6. S-HDL treatment effect in individual rabbits and pigs.

    Table S1. Composition and size of different S-HDL batches prepared for this study.

    Data file S1. Primary data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. S-HDL characterization.
    • Fig. S2. In vivo S-HDL cell specificity evaluation.
    • Fig. S3. Pharmacokinetics and plaque targeting of S-HDL in rabbits and pigs.
    • Fig. S4. S-HDL treatment effect in rabbits and pigs.
    • Fig. S5. Histological evaluation of S-HDL treatment.
    • Fig. S6. S-HDL treatment effect in individual rabbits and pigs.
    • Table S1. Composition and size of different S-HDL batches prepared for this study.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

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