Prevention of Obesity Related Diseases through Laminarin-induced targeted delivery of Bindarit

Rationale: The developement of oral targeted therapeutics for obesity and obesity-related diseases is challenging, as these diseases involve multiple lesions distributed throughout the whole body. Herein, we report a successful stragety for targeted oral delivery of bindarit to multiple obesity-related lesions including inflamed adipose tissue, fatty liver and atherosclerotic plaques. Methods: The computer simulation from atomstic to mesoscale was first applied for designing bindarit-loaded nanoparticles (pBIN) and laminarin-modified bindarit-loaded nanoparticles (LApBIN). Then pBIN were suceesfully prepared using a dialysis procedure, and LApBIN were prepared though the interaction bewtween laminarin and pBIN. The physiochemical properties, in vitro and in vivo pharmacokinetics, oral targeting capability and in vivo efficacy of LApBIN in various obesity-related diseases were examined. Results: LApBIN were sucessfully designed and prepared. Following oral administration of LApBIN, the nanoparticles could be sucessully orally adsorbed and translocated to monocytes. Contributed by the recruitment of monocytes to multiple obesity-related lesions, LApBIN successfully delivered bindarit to these lesions, and effectively suppressed inflammation there, which exerted successful preventive effects on high-fat-diet-induced obesity, insulin resistance, fatty liver and atherosclerosis. Conclusions:This strategy could represent a promising approach to develop effective oral treatments for obesity and other metabolic diseases.

other reagents are commercially available and used as received.

MD Simulations
All the components were described by the COMPASS force field. The BIN/PEI or LAM/BIN/PEI complex obtained from Adsorption module was employed to run molecular dynamic simulation. To eliminate unfavorable contacts, the initial configurations were subjected to 10000 steps of energy minimization with an energy convergence threshold of 10 −4 Kcal/mol and a force convergence of 0.005 kcal mol −1 Å −1 . The van der Waals interactions were calculated with a cutoff of 12.5 Å, a spline width of 1 Å, and a buffer width of 0.5 Å, while the electrostatic interactions were estimated by the Ewald summation with an accuracy of 0.001 Kcal/mol. After minimization, the lowest energy configuration were chosen for simulation. 200 ns MD simulation using isothermal and isochoric (NVT) was carried out at 298 K using the Nose thermostat and 1 bar using the Andersen barostat for pure components.

DPD Simulation
Firstly, polymer blocks (PEI), BIN and water molecules were coarse-grained to corresponding beads ( Figure S30). Flory−Huggins interaction parameters between these beads (Table S4) were calculated by Blends module in MS. Then a DPD simulation was activated at an ultra-fine level to investigate the self-assembly of BIN/PEI in a box of 100×100×100 rc 3 at the BIN/PEI/Water ratio of 1/1/8. According to the suggestion of Groot and Warren [1], the maximum repulsion between coarse grain i and j are obtained from Equation S1: The repulsions αij at 298K used in this work were given in Table S5. Consequently, the size and number of the simulated aggregates were direct and important criteria to value the effects of simulation box size and integration time step. DPD simulations were performed in 100×100×100 rc 3 with 20 ns. The DPD length unit or the cut-off radius rc = 6.46 Å in our work. Thus, the box size in our work was characterized by effective dimensions of 1938Å×1938Å×1938 Å. All the simulations were performed using Mesocite program incorporated in Materials Studio 2017 at 298 K, and total simulation time is 20 ns with integration time step of 150 fs.

Preparation of Cy5.5 labeled nanoparticles
To synthesize Cy5.5-conjugated PEI (Cy5.5-PEI), 15 mg Cy5.5 NHS ester in 5 mL of DMSO was added into 5 mL of DMSO containing 160 mg PEI, the obtained solution was stirred at 40 °C for 12 h in dark. After complete reaction, Cy5.5-PEI were obtained by dialysis of the solution against deionized water for 24 h. Then 5 mg Cy5.5-PEI and 5 mg BIN were firstly dissolved in 1 mL DMSO to dialyze against deionized water at 25 °C. The deionized water was exchanged every 2 h. After 12 h of dialysis, Cy5.5-pBIN were obtained by collecting the solution in the dialysis bag without further treatment. For preparing Cy5.5 labeled LApBIN, 0.1 mL LAM solution (10 mg/mL) was added into 1 mL Cy5.5-pBIN suspension (1 mg/mL), and then incubated for 1 min under ultrasonic vibration. The unabsorbed LAM was removed by twice water washing after high speed centrifugation at 12000 rpm.

Characterization of various nanoparticles
Dynamic light scattering (DLS) and ξ-potential measurements were carried out on a

Migration of macrophages after LApBIN internalization
Raw 264.7 cells were seeded into 6-well plates at 3 × 10 5 cells per well in 2 ml of growth medium. LApBIN were added in each well at 2.5 μg/mL, and incubated for 4 h.
The cells were washed with PBS, detached by trypsin digestion, centrifuged, and resuspended in the medium containing 0.5% FBS. Then, these Raw 264.7 cells were seeded into the upper chambers of Transwell (6.5 mm Transwell® with 5.0 μm pore polycarbonate membrane, Corning) and cultured with 100 μL of medium. In the lower chambers, 600 μL of medium supplemented with 3 × 10 5 cells of foam cells or endothelial cells was added. After incubation for 6 h at 37 °C, the cells were fixed, and stained with crystal violet. The upside membrane surface was scraped to remove remaining cells, and the cells migrated to the downside surface were counted in four high-power fields per membrane under a microscope.