Reactive Oxygen Species‐Responsive Nanoparticles Toward Extracellular Matrix Normalization for Pancreatic Fibrosis Regression

Abstract Pancreatic fibrosis (PF) is primarily characterized by aberrant production and degradation modes of extracellular matrix (ECM) components, resulting from the activation of pancreatic stellate cells (PSCs) and the pathological cross‐linking of ECM mediated by lysyl oxidase (LOX) family members. The excessively deposited ECM increases matrix stiffness, and the over‐accumulated reactive oxygen species (ROS) induces oxidative stress, which further stimulates the continuous activation of PSCs and advancing PF; challenging the strategy toward normalizing ECM homeostasis for the regression of PF. Herein, ROS‐responsive and Vitamin A (VA) decorated micelles (named LR‐SSVA) to reverse the imbalanced ECM homeostasis for ameliorating PF are designed and synthesized. Specifically, LR‐SSVA selectively targets PSCs via VA, thereby effectively delivering siLOXL1 and resveratrol (RES) into the pancreas. The ROS‐responsive released RES inhibits the overproduction of ECM by eliminating ROS and inactivating PSCs, meanwhile, the decreased expression of LOXL1 ameliorates the cross‐linked collagen for easier degradation by collagenase which jointly normalizes ECM homeostasis and alleviates PF. This research shows that LR‐SSVA is a safe and efficient ROS‐response and PSC‐targeted drug‐delivery system for ECM normalization, which will propose an innovative and ideal platform for the reversal of PF.


Preparation of LR-SSVA
LR-SSVA was synthesized by self-assembly and charge adsorption methods.The nanocarrier SSVA and RES were dissolved in 50 μL DMSO; the mixture was added into 2 mL of normal saline drop by drop, and stirred overnight at RT to obtain slightly milky micelle solution.Subsequently, siLoxl1 was dissolved in DEPC water, with the volume ratio of DEPC water and micelle solution 1:1, and then LR-SSVA was obtained after vortex for 30 s. C6-SSVA and siC6-SSVA used hereinafter were prepared in the same way.

Characterizations of LR-SSVA
The size distribution, polymer dispersity index (PDI) and zeta potential were measured by Zetasizer Nano (ZS90, Malvern, UK) for 10 runs.Transmission electron microscope (TEM) system (H-7650, Hitachi, Tokyo, Japan) was applied for the micromorphology of various micelles.
ROS was detected by DCFH-DA method.mPSCs were cultured in a 6-well plate with a density of 8×10 4 cells per well for 24 h.After washed by PBS for 3 times, PBS, LR-SVA (non-ROS response) and LR-SSVA were added into the plate for another 24 h.Subsequently, the culture medium was removed and 1 mL of DCFH-DA was added to each well and incubated at 37℃ for 30 min to monitor the green fluorescence intensity at 488 nm.

Assay of Loading Ability
The content of RES was monitored by detecting the absorbance at 468 nm with Multiskan GO (Thermo Fisher Scientific, USA).The encapsulation efficiency (EE) and the drug loading efficiency (DLE) of the LR-SSVA were calculated referring to Equation ( 1) and (2), respectively.

Stability of Nanoparticles
The stability of LR-SSVA was determined by measuring the change of particle size with time (6, 12, 24, 36, 48, 60 and 72 h) in various buffers.Briefly, LR-SSVA was suspended in saline, phosphate buffered solution (PBS) and 1 mL DME/F12 with 10% fetal bovine serum (FBS), respectively, and the particle size of them were measured at the specified time point.

Assay of Drug Response Release
2 ml of LR-SSVA solution was put into the pre-treated dialysis bag (3500D), accordingly, 20 mL of PBS with 1% Tween 80 (v/v) was added into the Erlenmeyer flask, and shaken at 37°C with the rate of 120 rpm.One group was added with hydrogen peroxide solution (20 mM), and the other with equal quality of PBS for negative control.Samples were taken at 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h, respectively, for the detection of RES concentration.

Gene Binding Capacity and Degradation Resistance of LR-SSVA
The siRNA binding capacity of SSVA was verified by agarose gel electrophoresis, and the electrophoresis conditions were 100 V, 15 min and 0.5×Tris/Borate/EDTA buffer solution.Specifically, 0.5 g of agarose powder was put into a conical flask, and was heat to dissolve evenly in a microwave oven.The agarose solution was poured into the glue mold after cooling to 50-60℃, and then a comb is inserted in the appropriate position.After the gel was solidified, it was transferred to an electrophoresis tank, and samples with different mass ratios (SSVA/siRNA, 0.5~20) were carefully added to the sample tank.When the band moved to about 2 cm from the front of the gel, electrophoresis was stopped for Observation of images under the gel imaging system.
Gel electrophoresis was applied to evaluate the protective ability of SSVA to siRNA, and the electrophoresis conditions were 100 V, 25 min and 0.5×Tris/Borate/EDTA buffer solution.Specifically, the sample with mass ratio of 10: 1 was prepared (siRNA, 0.2 μg), and the RNase A/Mg2+ and/or heparin sodium (5%) were added to incubate at RT for 2 h according to different grouping.After electrophoresis, the images were observed under the gel imaging system.

Cytotoxicity Assay
Firstly, a density of 1×10 4 cells per well of 266-6 cells and PSCs were seeded in 96-well plates to incubate for 24h, after which the growth medium was replaced by 100 μL of two vehicles with/without RES loaded at different concentrations.Then, 20 μL of MTT (5mg mL -1 ) was added in each well after further incubation for 24 h, which should be replaced by DMSO after incubating at 37 °C for 4 h.Finally, microplate reader (Thermo Fisher Scientific, USA) was applied to detect the absorbance of each well at 570 nm.Experiments were all conducted with three secondary wells.

Cellular Uptake and Lysosome Escape
Coumarin 6 (C6) was regarded as fluorescent probe for the investigation of In vitro uptake of micelles.C6-loaded micelles were synthesized as previous description.
1×10 5 cells per dish of mPSCs were seeded in glass-bottomed petri dish to incubate overnight.Thereafter, serum-free medium containing C6, C6-S and C6-SSVA was added and the cells continued to incubate for 2 h to 4 h.DAPI was used to stain cell nucleus after washed by PBS, and the images were obtained by Confocal laser scanning microscopy (CLSM) (Olympus, Tokyo, Japan).Quantitative analysis of cellular uptake was conducted with cell suspension after digestion, by detecting fluorescence intensity of C6 with flow cytometry.
In order to explore the uptake mechanism of LR-SSVA, the uptake inhibitors genistein (5 μg/mL), chlorpromazine (10 μg/mL) and amiloride (50 μM) were incubated with cells for 1 h in advance, respectively.The cells were washed with PBS and digested with trypsin to harvest the cell suspension.After centrifuged (2000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 400 μL PBS for detection of C6 fluorescence intensity by flow cytometry.
The cells were treated with siFAMR-SSVA for 3 h to 6 h, and lysosome was stained by Lyso Tracker Red DND.CLSM was used to obtain the images and to analyze co-localization.

Immunofluorescence Staining
mPSCs were washed by PBS and fixed by 4% paraformaldehyde after treating with different preparations.Hank's balanced salt solution (HBSS) was used to wash cells and 5% donkey serum was applied for blocking.Subsequently, rabbit anti-human αalpha smooth muscle actin (α-SMA) primary antibody (Abcam, Cambridge, UK) was added and incubated 4°C.After which, cells were incubated with AF657-conjugated goat anti-rabbit secondary antibody (Beyotime Biotechnology, Shanghai, China), and DAPI dye was used to counterstain the nuclei.At last, CLSM was used to assess cell fluorescence and the images were quantified by Image J software.

Assay of Cell Invasion and Migration
Transwell device was coated with rat tail collagen I (0.3 mg mL-1, 50 μL) in advance; 500 μL of mPSCs in FBS-free medium were seeded onto the collagen layer, and equal growth medium was simultaneously added into the 24-well plates.After co-incubating with various preparations for 24 h, the cells in the upper layer were gently scraped, while those outside the chamber were fixed with 4% paraformaldehyde for further staining by 0.1% crystal violet.Inverted fluorescence microscope was applied for imaging, and Image J software was used for semi-quantification.
mPSCs were seeded onto the upper chamber and cultured for 24 h, after which the cells were treated with various preparations to incubate another one day.The transepithelial electrical resistance (TEER) was determined by recording the resultant resistance after inserting the TEER probe into the apical and basal compartment of the transwell.TEER was recorded before and after treatments to assess the migration ability.

Acute and Short-term Toxicity Studies
Six healthy mice were divided into 2 groups.One group was given LR-SSVA at three times of the therapeutic dose for three days, and the other group was given PBS as negative control.Blood samples and main organs (heart, liver, spleen, lung and kidney) were collected for H&E staining and liver/kidney function assessment to evaluate the acute toxicity of micelles.
Additionally, blood and organs (liver, lung, heart, spleen and kidney) were harvested after the final injection of pharmacodynamic studies, for H&E staining and liver/kidney function detecting to evaluate the short-term toxicity of micelles.

Pharmacodynamics and In vivo Imaging Studies
For the pharmacodynamic studies, 81 of the fibrotic mice were assigned into 9 groups from the fourth week, and injected through tail vein with free RES, R-SS, L-SS, R-SSVA, L-SSVA, LR-SS and LR-SSVA in PBS or PBS alone twice a week.
After the final injection, mice were sacrificed to collect blood and organs (pancreas, liver, lung, heart, spleen and kidney) for further experimentation.
For the living imaging studies, 21 of fibrotic mice were divided into 3 groups, and DiR was chosen as fluorescent probes.Each group of mice were injected with free DiR, DiR-SS and DiR-SSVA (DiR dose, 2 mg kg -1 body weight), respectively; afterwards the fluorescence at various time points (3 h, 6 h, 12 h, 24 h and 48 h) was imaged with excitation and emission wavelengths of 745 nm and 800 nm, by In vivo imaging system (FX-Pro; Bruker).
For the localization analysis, 21 of fibrotic mice were included for 3 groups.Free DiI, DiI-SS and DiI-SSVA (DiI dose, 2 mg kg -1 body weight) were injected via tail vein for three consecutive days.After the last injection, mice were sacrificed to harvest pancreas for frozen sections.Rabbit anti-mouse antibody of α-SMA and DAPI dye were used for section staining, and the co-localization of α-SMA and DiI was visualized by CLSM.

Scanning Electron Microscope (SEM)
The pancreas samples of different groups were cut into small pieces and decellularized for 4-12h, in PBS with 0.1% penicillin/streptomycin (Sigma-Aldrich) and 1% sodium dodecyl sulfate (Sigma-Aldrich).Later on, the tissues were washed with distilled water, lyophilized and sputter-coated with gold for observation under a JSM-IT200LA (JEOL, Tokyo, Japan).

Western Blotting
Protein from cells and pancreas tissues was extracted according to the standard procedures, and was transferred to polyvinylidene difluoride (PVDF) membranes after isolation by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE).After blocking in 5% skim milk for 2 h at RT, membranes were incubated with primary antibodies (collagen I, a-SMA, fibronectin, LOXL1) and β-actin rabbit anti-mouse antibodies) at 4°C overnight, and followed with secondary antibodies at RT for 2 h.
Protein bands were visualized by CCD image system (Tanon 4200, Shanghai, China), and Image J software was used for semi-quantitative analysis.
aminotransferase (ALT), aspartate aminotransferase (AST) and serum amylase (AMY) activity were determined by automatic biochemical analyzer in Servicebio Biotechnology Co., Ltd.(Wuhan, China).Hydroxyproline levels in pancreas, serum SOD, MDA and GSH-PX were tested by standard assay kit following the manufacturer's instructions.The serum and pancreatic concentrations of transforming growth factor-β (TGF-β), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and LOXL1 were detected by enzyme-linked immunosorbent assay (ELISA) kit against their specifications.Histological AnalysisPancreas tissues were embedded by paraffin, and sectioned for staining.H&E staining Kit, Masson's trichrome staining Kit, and and 0.1% (w/v) Sirius Red were applied in accordance with the specifications.Immunohistochemistry was carried out referred to the standard protocols, during which the rabbit anti-mouse collagen I, a-SMA, fibronectin and LOXL1 primary antibody and goat anti-rabbit secondary antibody were used for stanning.Primary antibodies of collagen I, a-SMA and fibronectin were purchased from Abcam (Cambridge, UK), LOXL1 was from Novus (USA), and secondary antibody was obtained from Servicebio Biotechnology Co., Ltd.Dihydroethidium (DHE) can freely get into living cells and be oxidized by intracellular ROS to form ethidium oxide, which can be mixed into chromosome DNA to produce red fluorescence and reflect ROS content.

Fig. S10
Fig. S10 Detection of cellular ROS by DCFH-DA method.Green fluorescence represents ROS accumulation.

Fig. S11
Fig. S11The images of the general changes of pancreas in the control group, the

Fig. S15
Fig. S15 Acute toxicity In vivo of LR-SSVA.(a-d) Serum activities of ALT, AST, BUN and Cr, as measured by biochemical assays.(e) Representative of H&E staining

Fig. S16
Fig. S16 Short-term toxicity In vivo of LR-SSVA over the experimental period.(a-d) Serum activities of ALT, AST, BUN and Cr, as measured by biochemical assays.(e) Visual observation of hemolysis caused by siRES/SSVA in PBS at pH 7.4.(f) Representative of H&E staining of heart, liver, spleen, lung and kidney tissue sections from mice after different treatments.Scale bar: 100 μm.200×.Values are expressed as means ± SD (*P < 0.05, **P < 0.01, NS, no significant difference; n = 3).

Fig. S17
Fig. S17 Semi-quantitative results of DHE staining of ROS in healthy and PF mice All of the animal experiments were conducted under the protocols approved by the Ministry of Health of the People's Republic of China and followed the Guidelines for the Care and Use of mice of six-week-old were purchased from Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).