1. Volume 138, Issue 3, Pages 843-853.e2 (March 2010)
Drug-Loaded Nanoparticles Targeted to the Colon With Polysaccharide Hydrogel Reduce Colitis in a Mouse Model 
Hamed Laroui, Guillaume Dalmasso, Hang Thi Thu Nguyen, Yutao Yan, Shanthi V. Sitaraman, Didier Merlin 
Gastroenterology 
Volume 138, Issue 3, Pages e2 (March 2010)
DOI: /j.gastro
Copyright © 2010 AGA Institute Terms and Conditions

2. Figure 1
(A) Schematic process of NP synthesis. A hydrophilic drug is encapsulated by double emulsion water in oil in water (W/O/W). (B) Schematic representation of PLA NPs loaded with KPV, a hydrophilic drug, and coated with PVA.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
Schematic representation of biomaterial encapsulation of KPV-loaded NPs. (A) An alginate and chitosan hydrogel was formed by double linking of Ca2+ and SO42- ions. Suspension of NPs in the polysaccharide solution loads the final formed hydrogel with NPs. (B) Optical microscopy image of KPV-loaded NPs encapsulated into a hydrogel bead of alginate-chitosan linked via Ca2+ and SO42- ions.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
Double-gavage procedure of encapsulated KPV-loaded NPs. (A) The first gavage delivered 100 μL of the polymer mix solution (alginate, 7 g/L; chitosan, 3 g/L) containing a homogenous suspension of NPs (2 mg/mL). Red Ponceau has been added for visualization. (B) The second gavage delivered 50 μL of a solution containing 70 mmol/L calcium chloride and 30 mmol/L sodium sulfate. (C) Visualization of the mixed hydrogel formed by chelation of the polymers in the stomach. (D) The hydrogel after extraction from a mouse stomach 5 minutes after the double-gavage method.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Physicochemical characterization and biocompatibility of KPV-loaded NPs. (A) Nanoparticles size average (nm) and polydispersity index of KPV-loaded NPs and latex NPs (size calibration standard) by photon correlation spectroscopy method (n = 5). (B) Scanning electron microscopy picture of KPV-loaded NP suspension (5 μg/mL). (C) Cumulative drug release kinetic (percentage of total encapsulated drug). NPs at 5 mg/mL containing BSA–FITC and KPV were incubated in PBS containing 5 μg/mL pepsin and 30 μg/mL trypsin at pH 6 (n = 5). (D) WST-1 cell viability test on Caco2–BBE (percentage over control) after 72 hours of exposure to a 1-mg/mL suspension of 3 different NP samples. (Control, culture medium; NanoKPV, KPV-loaded NPs; NanoFITC, dextran–FITC–loaded NPs; NanoBSA, BSA-loaded NPs) (n = 8). (E) Electrical impedance sensing method on Caco2–BBE cells to determine cell viability (percentage over control) after a long exposure to a 1-mg/mL suspension of 2 different NP samples. (culture medium without NP as control; latex particles [culture medium without latex NP as control] and KPV-NP) (n = 3).
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
Cell interactions of KPV-loaded NPs and messenger RNA (mRNA) expression of proinflammatory cytokines (IL-8 and COX-2) on Caco2–BBE monolayers treated or not with KPV-loaded NPs. (A) Transmission electron microcopy picture of KPV–NPs (500 μg/mL) on monolayer of Caco2–BBE cells after a LPS stimulation (10 μg/mL, 1 h). Bar, 1 μm. KPV-loaded NP in the extracellular domain (1). KPV-loaded NP interacting with the cell membrane starting an endocytose process (2). KPV-loaded NP in intracellular domain after endocytosis (3). (B) Caco2–BBE cells were stimulated with LPS (10 μg/mL, 1 h), alone or after pretreatment with different concentrations of KPV-loaded NPs (50, 200, or 500 μg/mL) or the equivalent concentration of free KPV in solution (free KPV 41 μg/L corresponds to 500 μg/mL KPV-loaded NPs) (41 μg/L) for 72 hours (n = 3). (**P < .001, *P < .01 for IL-8; and ##P < .001, #P < .01 for COX-2).
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

7. Figure 6
Localization of encapsulated dextran–FITC NPs (μg dextran–FITC/μg protein of tissue) on the digestive tract after 3 days of gavage. Localization of dextran–FITC NPs (μg dextran–FITC/μg protein of tissue) on the digestive tract after 3 days of gavage. Digestive tract was divided into 6 sections as follows: stomach, proximal (1/3) small intestine, medial (2/3) small intestine, and distal (3/3) small intestine, cecum, and colon (n = 12).
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

8. Figure 7
Histology of DSS-induced colitis in 7 different biomaterial treatments on mice.
During in vivo experiments, all mice received a daily gavage of 150 μL of biomaterial. Control group received the hydrogel with empty NPs (alginate, 7 g/L; chitosan, 3 g/L) and regular water as drinking solution. DSS treated group received the hydrogel with empty NPs (alginate, 7 g/L; chitosan, 3 g/L) and 3% DSS solution as drinking solution. KPV–NP–treated group received the hydrogel (alginate, 7 g/L; chitosan, 3 g/L) loaded with encapsulated KPV-loaded NPs and 3% DSS solution as drinking solution. Histologic section of colon after 7 days of a daily gavage in groups 1, 2, and 3.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

9. Figure 8
In vivo parameters of DSS-induced colitis in 7 different biomaterial treatments on mice. During in vivo experiments, mice received (or not for group 7) a daily gavage of 150 μL of biomaterial with or without NPs. All groups had DSS as drinkable water excepted group 1 (water). Groups numbers were defined as followed: number = biomaterial + daily drink; hydrogel, alginate 7 g/L and chitosan 3 g/L; DSS, 30 g/L; Ø-NP, empty NP; 1, hydrogel + water; 2, hydrogel + DSS; 3, hydrogel + KPV–NP (encapsulated KPV-loaded NPs) + DSS; 4, hydrogel + Ø-NP + DSS; 5, hydrogel with 41 μg/L free KPV (free KPV in hydrogel) + DSS; 6, hydrogel + dextran–FITC NP (encapsulated FITC-loaded NPs); 7, DSS only. ##P < (A) Percentage of initial body weight (%) after 7 days of a daily gavage in groups 1–7. (B) Percentage increase over control of MPO activity after 7 days in groups 1, 2, 3 4, 5, and 7. (C) mRNA expression over control of TNF-α after 7 days in groups 1, 2, 3, 4, and 5. (D) mRNA expression over control of IL-β after 7 days in groups 1, 2, 3, 4, and 5.
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

10. Supplementary figure 1
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

11. Supplementary figure 2A
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

12. Supplementary figure 2B
Gastroenterology  , e2DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions