Dual Functioned Hexapeptide‐Coated Lipid‐Core Nanomicelles Suppress Toll‐Like Receptor‐Mediated Inflammatory Responses through Endotoxin Scavenging and Endosomal pH Modulation

Abstract Excessive activation of Toll‐like receptor (TLR) signaling pathways and the circulating endotoxin are key players in the pathogenesis of many acute and chronic inflammatory diseases. Regulation of TLR‐mediated inflammatory responses by bioactive nanodevices represents a promising strategy for treating these diseases. In searching for novel, clinically applicable nanodevices with potent TLR inhibitory activities, three types of hexapeptide‐modified nano‐hybrids with different cores of phospholipid nanomicelles, liposomes, and poly(lactic‐co‐glycolic acid) nanoparticles are constructed. Interestingly, only the peptide‐modified lipid–core nanomicelles (M‐P12) display potent TLR inhibitory activities. Further mechanistic studies disclose that lipid–core nanomicelles have a generic property to bind to and scavenge lipophilic TLR ligands including lipopolysaccharide to block the ligand–receptor interaction and down‐regulate the TLR signaling extracellularly. In addition, the peptide modification enables M‐P12 a unique capability to modulate endosomal acidification upon being endocytosed into macrophages, which subsequently regulates the endosomal TLR signal transduction. In an acute lung injury mouse model, intratracheal administration of M‐P12 can effectively target lung macrophages and reduce lung inflammation and injuries. This work defines a dual mechanism of action of the peptide‐modified lipid–core nanomicelles in regulating TLR signaling, and provides new strategies for the development of therapeutic nanodevices for treating inflammatory diseases.


Figure S18
The stability of M-P12 and M DSPE-mPEG over time.

Figure S19
The gating strategy of flow cytometry analysis to identify different immune cells in the lung.

Figure S20
The protective effects of M-P12 on the dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model.

Materials
Mouse IL-6 and TNF- ELISA kits were purchased from InvitroGen (Carlsbad, CA, USA), and mouse KC/CXCL1 ELISA kit was from R&D Systems (Minneapolis, MN, USA).

Post-treatment of M-P12 on TLR3-mediated activation of NF-B/AP-1 and IRF
For post-treatment cell model, reporter cell-derived macrophages were first stimulated with Poly I:C (50 g/mL) for 2 h. Cells were washed three times with PBS to remove Poly I:C, follwed by M-P12 treatment for 24 h. The culture medium was then collected and incubated with QUANTI-Blue solution until the solution color turned into dark blue. The absorption at 655 nm was measured by a microplate reader (TECAN, Mannedorf, Zurich, Switzerland).

Cellular uptake of M-P12 at different temperatures
To study whether the uptake of M-P12 was energy dependent, THP-1 cells-derived macrophages were treated with DiD-labelled M-P12 for 3.5 h at 4℃ or 37℃; the untreated cells were used as a negative control. Cells were resuspended in PBS for flow cytometry analysis of DiD fluorescence in macrophages on a flow cytometer (BD FACSVerse, BD, San Diego, CA, USA), and the data was processed using FlowJo software (TreeStar, Ashland, OR, USA).

DSS-induced ulcerative colitis mouse model
Female C57BL/6 mice (8-10 weeks) were randomly divided into WaterPBS group, DSSPBS group and DSSM-P12 group. DSS (3%) was prepared in sterilized drinking water and given to the mice for 7 days continuously; mice were then fed with sterilized drinking water for another 2 days. PBS or M-P12 (phospholipids: 7.2 g/kg) were injected intraperitoneally one day before DSS treatment and every other day during the DSS feeding period (a total of 5 doses).
The colitis severity was scored by evaluating the disease activity through daily observations of the following parameters: weight loss (0 point:1%, 1 point:1-3%, 2 points: 3-6%, 3 points:6-9%, and 4 points: ≥9%), stool consistency (0 point: normal, 1 point: loose, 6 blood, 1 point: observed on the blood test strip, 2 points: visulized trace of blood, 3 points: obvious blood in the stool, 4 points: blood around the stool and the anus). The disease activity index (DAI) was calculated based on the above three parameters to obtain an average value from 0-4. The mice were sacrificed at Day 9, and the colon length was measured; the colon tissues were processed for further analysis.
For histological analyses, half of the distal colon from each mouse was fixed in 4% paraformaldehyde for 24 h, followed by dehydration and paraffin embedding. They were cut into 5-m thick sections, which were furher processed for hematoxylin and eosin (H&E) staining. The disease severity was evaulated on the basis of a histopathological score that includes 7 parameters: extent of inflammation, inflammatory cell infiltration, extent of crypt damage, crypt abscesses, sub-mucosal edema, loss of goblet cells and reactive epithelial hyperplasia.
A fixed mass of each colon tissue was homogenized in modified RIPA lysis buffer containing phosphotase and protease inhibitors (Beyotime, Shanghai, China) to obtained tissue lysates for cytokine measurements of IL-6, TNF- and KC/CXCL1 by ELISA kits.