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. 2018 Sep 9;8(18):4912-4924.
doi: 10.7150/thno.27608. eCollection 2018.

Blood Exosomes Regulate the Tissue Distribution of Grapefruit-Derived Nanovector via CD36 and IGFR1 Pathways

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Free PMC article

Blood Exosomes Regulate the Tissue Distribution of Grapefruit-Derived Nanovector via CD36 and IGFR1 Pathways

Qi-Long Wang et al. Theranostics. .
Free PMC article

Abstract

Tumor-specific delivery of therapeutics is challenging. One of the major hurdles for successfully delivering targeted agents by nanovectors is the filtering role of the liver in rapidly sequestering nanovectors from the circulation. Exosomes, a type of endogenous nanoparticle, circulate continuously in the peripheral blood and play a role in intercellular communication. The aim of this study was to determine whether the level of endogenous exosomes has an effect on nanovector delivery efficiency of targeted agents. Methods: Exosomes were isolated from peripheral blood and intravenously (I.V.) injected into tumor-bearing mice. Subsequently, 1,1-dioctadecyl-3,3,3'3'-tetramethylindotricarbocyanine-iodide (DiR) fluorescent dye-labeled nanoparticles, including grapefruit nanovectors (GNV) and standard liposomes, were I.V. injected in the mice. The efficiency of redirecting GNVs from liver to other organs of injected mice was further analyzed with in vivo imaging. The concentration of chemo drugs delivered by GNV was measured by HPLC and the anti-lung metastasis therapeutic effects of chemo drugs delivered by GNVs in mouse breast cancer and melanoma cancer models were evaluated. Results: We show that tail vein-injected exosomes isolated from mouse peripheral blood were predominately taken up by liver Kupffer cells. Injection of peripheral blood-derived exosomes before I.V. injection of grapefruit-derived nanovector (GNV) decreased the deposition of GNV in the liver and redirected the GNV to the lung and to the tumor in breast and melanoma tumor-bearing mouse models. Enhanced therapeutic efficiency of doxorubicin (Dox) or paclitaxel (PTX) carried by GNVs for lung metastases was demonstrated when there was an I.V. injection of exosomes before therapeutic treatment. Furthermore, we found that CD36 and IGFR1 receptor-mediated pathways played a critical role in the exosome-mediated inhibitory effect of GNV entry into liver macrophages. Conclusions: Collectively, our findings provide a foundation for using autologous exosomes to enhance therapeutic vector targeted delivery to the lung.

Keywords: Blood exosomes; CD36 and IGFR1; liver Kupffer cells; lung metastasis; nanovector uptake; outer nuclear membrane cluster.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
The majority of circulating exosomes are taken up by liver F4/80 macrophages and pre-injection of exosomes leads to redirection of subsequently injected nanovectors from the liver to the lungs. Exosomes from normal mouse plasma were isolated using the PureExo® Exosomes Isolation kit. (A) Representative samples of Western blotting for exosome CD63, CD81, CD9 are shown. (B) The morphology of exosomes was examined and imaged using transmission electron microscopy (TEM). The size distribution (C) and surface Zeta potential (D) of exosomes were measured using a ZetaSizer. (E) Distribution of DiR-labeled exosomes in normal mice. Mice were I.V. injected with 200 μg of exosomes and DiR signals in the liver, lung, spleen, kidney, heart, thymus, brain and stomach were analyzed by scanning using a Kodak Image System. (F) Livers from mice were removed over a 24-72 h period after I.V. injection and liver tissue sections were stained with anti-mouse F4/80 antibody. Representative images of DiR-labeled exosomes from mice and F4/80-stained liver section. (G) Exosomes (200 μg) were isolated from plasma of normal mice and injected I.V. into mice. DiR dye-labeled nanovectors including grapefruit lipid-derived GNVs (#1), lymphocyte membrane-coated GNVs-IGNVs (#2), DOTAP:DOPE liposomes (#3) and liposomes from Avanti (#4) were I.V. injected into mice 30 min after an injection of exosomes. Accumulation of nanovectors in mouse liver was examined in living mice (left panel) and ex vivo livers (right panel). Representative images of DiR-labeled nanovectors from mice (left panel) and livers (right panel), followed by bar charts of the mean net intensity (sum intensity/area, n=5). For statistics, see method section “Statistics”. (H) PKH67-labeled GNVs (200 nmol) were I.V. injected into mice that were previously I.V. injected with exosomes (200 μg/mouse). Liver, lung and spleen were removed and PKH67-GNVs in tissues sections stained with anti-F4/80 antibody were imaged using confocal microscopy. Representative images are shown. (I) Inhibition of liver accumulation of nanovectors. Different doses of C57BL/6 mice serum-derived exosomes (25, 50, 100 and 200 μg) were I.V. injected into C57BL/6 mice. 30 min after injection of exosomes, mice were injected with 200 nmol DiR dye-labeled GNVs. DiR dye signals in living mice (top panel), liver (middle panel) and lung (bottom panels) were quantitatively analyzed using a Kodak Image System. The data are presented as the mean net intensity (sum intensity/area, n=5). Data are presented as mean ± SD, **p < 0.01, ***p < 0.001. Error bars represent SD. The data (A-F, H) shown are representative of at least 3 independent experiments (n=5).
Figure 2
Figure 2
Exosomes redirect GNVs from the liver to the lungs and tumor. (A) DiR dye-labeled GNVs were I.V. injected into 6-week-old female BALB/c mice pretreated with exosomes or clodrosomes to deplete Kupffer cells, or PBS as a control (Normal). Peripheral blood was collected into anticoagulant tubes 30, 60, and 180 min after injection. The DiR dye signals in blood were quantified by scanning using a Kodak Image System. The data are presented as the mean net intensity (sum intensity/area, n=5). (B) DiR dye-labeled GNVs were I.V. injected into 4T1 tumor-bearing mice pretreated with exosomes or PBS as a control. 4T1 tumor-bearing mice without any treatment were used as a negative control (NC). Representative images collected at 1 h, 3 h, 6 h and 20 h after injection. The data are presented as the mean net intensity (sum intensity/area, n=5). (C) Mice were sacrificed, tumors and organs (liver, lung, spleen, heart, thymus, kidney, and lymph node) were removed, scanned using a Kodak Image System and quantitatively analyzed. The data are presented as the mean net intensity (sum intensity/area, n=5). (D) Distribution of paclitaxel in B16F10 tumor-bearing mice. B16F10 tumor-bearing mice were I.V. injected with paclitaxel-loaded GNV (GNV-PTX) 3 times. The concentration of paclitaxel in mouse tumor, liver, lung and spleen was analyzed using HPLC. (n=3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SD.
Figure 3
Figure 3
Pre-injection of blood-derived exosomes enhances anti-tumor metastasis of therapeutic agents delivered by GNVs. 1×105 4T1 cells were injected into a mammary fat pad of female BALB/c mice. On day 5 after the injection, mice were tail vein injected every 3 days for a total of 10 times with PBS, Dox, GNV-Dox, Exo/GNV-Dox. (A) Mice were sacrificed and lungs were imaged and the pulmonary metastatic nodules were quantified. (B) Lung tissue sections were stained with H&E. (C) Representative images of lung and sectioned lung tissue (n=5), and survival rates of mice were recorded. B16F10 cells (5×104) were I.V. injected into C57BL/6 mice. 5 days later, mice were tail vein injected every 3 days for a total of 10 times with PBS, PTX, GNV-PTX or Exo/GNV-PTX. Lungs were removed, imaged and the metastatic nodules in lungs were quantitative analyzed (D), tissue sections were stained with H&E (E), and survival rates of mice were recorded (F). Data are presented as mean ± SD, *p<0.05, p**<0.01 and p***<0.001. Error bars represent SD. The data (A-D) shown are representative of at least 3 independent experiments (n=5).
Figure 4
Figure 4
Pre-injection of exosomes prevents co-localization of CD36 and GNVs and knockout of CD36 leads to canceling of exosomes-mediated inhibition of liver uptake of GNVs. Six-week-old B6 mice were I.V. injected with DiR-GNVs (2 nmol/100 μL). 30 min after the injection, mice were sacrificed and liver sections were immuno-stained with CD36 and F4/80. (A) Representative images of sectioned liver tissue (n=5). Six-week old B6 mice were I.V. injected with exosomes (200 μg/mouse) isolated from peripheral blood or PBS as a control. 30 min after the injection, DiR-GNVs (200 nmol) was administered intravenously. The mice were sacrificed and F4/80-positive cells were FACS-sorted and stained with CD36 antibody. (B) Representative confocal images of F4/80 positive cells isolated from liver (n=5). Wild-type B6 mice and age/sex-matched CD36 KO mice were I.V. injected with C57BJ/6 plasma exosomes (200 μg/mouse, n=5) followed by DiR-labeled GNVs (200 nmol) at 30 min intervals. (C) The DiR signals in living mice (top panel) and liver tissue (bottom panel) were imaged 30 min after the GNV injection.
Figure 5
Figure 5
siRNA knockdown of IGFR1 reversed exosomes-mediated inhibition of GNV uptake by human monocytes. (A) U937 human monocytes were incubated for 30 min with exosomes (2 μg/100 culture media) isolated from healthy subjects. Treated cells were then incubated with PKH26-labeled GNVs (2.0 nmol) for additional 0-120 min and the cells were subsequently FACS analyzed. Representative FACS images of GNV-positive cells (n=5). (B) 48 h siRNA-transfected U937 human monocytes were incubated with/without exosomes for 30 min. Then, PKH26-labeled GNVs were added to the treated cells and incubated for an additional 60 min before cells were harvested for FACS analysis of PKH26-positive cells. Representative FACS images of GNV-positive cells (left panel) and the percentages of GNVs+ U937 cells as a result of siRNA knockout of IGFR1 are presented as mean ± SD (right panel), *p < 0.05. Error bars represent SD.

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