Volume 22, Issue 3, Pages 522-534 (March 2014) Targeted Drug Delivery to Intestinal Macrophages by Bioactive Nanovesicles Released from Grapefruit  Baomei Wang, Xiaoying Zhuang, Zhong-Bin Deng, Hong Jiang, Jingyao Mu, Qilong Wang, Xiaoyu Xiang, Haixun Guo, Lifeng Zhang, Gerald Dryden, Jun Yan, Donald Miller, Huang-Ge Zhang  Molecular Therapy  Volume 22, Issue 3, Pages 522-534 (March 2014) DOI: 10.1038/mt.2013.190 Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Characterization of grapefruit-derived nanoparticles (GDNs). (a) Sucrose-gradient band 2 indicated by the arrow (left) was collected for electron microscopy (EM) examination and an (EM) imaging of GDNs. The scale bar indicates 200 nm. (b) Particle size and surface charge were measured using a Zetasizer. (c) Pie chart of the lipid profile of GDNs, reported as percentage of total lipids in GDNs. PS, Phosphatidylserine; PI, Phosphatidylinositol; PE, Phosphatidylethanolamines; PC, Phosphatidylcholines; PG, Phosphatidylglycerol. (d) Representative high-performance liquid chromatography chromatograms of the standards naringin (NAR), naringenin (NE) and GDN extract and the quantification of the content of NAR and NE in GDNs. GDNs were incubated in either distilled water, a 0.5N HCl or a 0.5N NaOH solution at 37 °C for 30 minutes, then the change of particle size (e) and surface charge (f) were measured using a Zetasizer (n = 6). Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Grapefruit-derived nanoparticles (GDN) pretreatment ameliorates DSS-induced colitis in mice. C57/B6 mice were treated with either PBS/DSS or GDN/DSS. (a) Body weight (n = 15). (b) Colon length, values are represented as percentage of untreated control mice (n = 15). On day 7 of DSS treatment, colons were harvested. (c) Histological analysis (n = 15). (d) Histological scoring was evaluated by the combined score of epithelial damage and extension of leukocyte infiltration (n = 15). (e) Immunofluorescent staining for E-cadherin of representative inflamed areas of colon (n = 6). Dotted line indicates basement membrane. (f) Distal colons were harvested for qPCR analysis of inflammatory cytokines and chemokines. Values are shown relative to the mRNA levels of naive mice (n = 15). Alternatively, distal colons isolated from the indicated mice were cultured overnight and then IL-6, TNF-α and IL-1β in the supernatants were measured by ELISA (n = 15). On day 7 of DSS treatment, colons were harvested and digested. Doublets were excluded from colonic digests on the basis of FSC-Aand FSC-H, hematopoietic cells were gated on (g) CD45.2 and myeloid-derived cells were selected as CD11b+. The resulting cells were then analyzed for (h) Ly6C or (i) Ly6G (n = 15). Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The majority of GDNs are taken up by intestinal and systemic macrophages. Sections of (a) small intestine and colon, (b) Peyer's patches and mesenteric lymph node and spleen and (c) liver revealed uptake of PKH-26 (red) labeled GDNs specifically by F4/80+ (green) macrophages. Nuclei were labeled with DAPI. Dotted line indicates basement membrane. Original magnification was ×40 (left panel) with enlargement of the indicated area shown in the right panels. Percentage of macrophages that phagocytized GDNs is shown (n = 15). Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 GDNs utilize both micropinocytosis and clathrin-dependent uptake mechanism for entry into macrophages. (a) The uptake of GDNs by Raw264.7 macrophages. (b) Raw 264.7 macrophages were pretreated with 50 µmol/l amiloride (Amil.), 12.5 µmol/l chlorpromazine (Chlor.) or 100 µmol/l of indomethacin (Indo.) for 30 minutes and then incubated with 2 µg/ml PKH26-labeled GDNs for 3 hours. To exclude membrane contamination, we stained the cell surface with F4/80 antibody. (c) The percentage of GDN uptake relative to control (n = 6). Nuclei were labeled with DAPI; original magnification was ×40. Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Five GDNs enhance the anti-inflammatory capacity of resident intestinal macrophages. Intestinal resident macrophages were isolated from colons of B6 mice treated with PBS or GDNs for 7 days. (a) The expression of HO-1, IL-10, and COX-2 were analyzed by real-time RT-PCR. Values are shown relative to the mRNA levels of PBS group. (b) The upregulation of HO-1 expression by GDNs was confirmed by western blot. (c) Isolated macrophages were stimulated with heat-killed E. coli. (MOI = 50) for 24 hours with 2 µg/ml of GDNs in the GDN-treated group. The amounts of IL-10 and TNF-α in the culture supernatants were measured by ELISA. Data show means ± SEM of five independent experiments from pooled macrophages of 15 mice per group. (d) Mice were orally given 2% DSS for 5 days, fasted overnight, and then gavaged twice with 30 mg/kg of PKH26-labeled GDNs 2 and 4 hours before harvesting the colon. Confocal images showed the uptake of PKH26 (red)-labeled GDNs by F4/80+ (green) macrophages. Nuclei were labeled with DAPI. Original magnification was ×40 (left panel) with enlargement of the indicated area shown in the right panels. Dotted line indicates basement membrane. (e) Percentage of macrophages that phagocytized GDNs is shown, n = 6. (f) Mice were pretreated with GDNs for 7 days and then given 2% DSS for 5 days with continued GDN administration. Colonic macrophages were isolated and incubated with GDNs for 36 hours. The amounts of TNF-α, IL-1β, IL-6, and IL-10 in the culture supernatants were measured by ELISA. Data show means ± SEM of five independent experiments from pooled macrophages of five mice per group. Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Preparation and characterization of GDN–MTX conjugates (GMTX). (a) UV spectra of standard-free MTX (mg/ml) and GMTX (n = 15). (b) The size and surface charge of GMTX were measured using a Zetasizer. (c) Comparative antiproliferative effect of GMTX versus free MTX on mouse macrophage cell line. The bold numbers within each histogram represent the percentage of cells containing CFSE (n = 6). (d) Confocal images show the uptake of PKH26 (red)-labeled GMTX by F4/80+ (green) macrophages. Nuclei were labeled with DAPI. Original magnification was ×40 (left panel) with enlargement of the indicated area shown in the right panel. Dotted line indicates basement membrane. (e) Percentage of macrophages that phagocytized GMTXs is shown, n = 6. Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 The therapeutic effect of GMTX on active colitis. The therapeutic effects of GMTX were evaluated by (a) body weight, (b) colon length, (c) pathology changes, (d) colitis score, (e) the epithelial integrity and (f) cytokine expression and secretion by inflamed colon. Nuclei were labeled with DAPI. Dotted line indicates basement membrane. Data show means ± SEM of five independent experiments with five mice per group. Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Immunomodulation but not cytotoxic effect of GMTX on activated intestinal macrophages. Colitis was induced in mice with 2% dextran sulfate sodium and then mice were given orally either PBS, MTX, or GMTX on day 3, 5, and 6 of dextran sulfate sodium treatment. (a) Colons were collected on day 7 and stained with F4/80 (red) and TUNEL (green). Nuclei were labeled with DAPI, n = 9. Dotted line indicates basement membrane. (b) Percentage of TUNEL+ macrophages is shown. (c) Colonic macrophages were isolated and cultured in 96-well plate for 36 hours. The amounts of TNF-α, IL-1β, IL-6 and IL-10 in the culture supernatants were measured by ELISA. Data show means ± SEM of five independent experiments from pooled macrophages of five mice per group. Molecular Therapy 2014 22, 522-534DOI: (10.1038/mt.2013.190) Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions