Phosphorus dendron nanomicelles as a platform for combination anti-inflammatory and antioxidative therapy of acute lung injury

Rationale: Development of novel nanomedicines to inhibit pro-inflammatory cytokine expression and reactive oxygen species (ROS) generation for anti-inflammatory therapy of acute lung injury (ALI) remains challenging. Here, we present a new nanomedicine platform based on tyramine-bearing two dimethylphosphonate sodium salt (TBP)-modified amphiphilic phosphorus dendron (C11G3) nanomicelles encapsulated with antioxidant drug curcumin (Cur). Methods: C11G3-TBP dendrons were synthesized via divergent synthesis and self-assembled to generate nanomicelles in a water environment to load hydrophobic drug Cur. The created C11G3-TBP@Cur nanomicelles were well characterized and systematically examined in their cytotoxicity, cellular uptake, intracellular ROS elimination, pro-inflammatory cytokine inhibition and alveolar macrophages M2 type repolarization in vitro, and evaluated to assay their anti-inflammatory and antioxidative therapy effects of ALI mice model through pro-inflammatory cytokine expression level in bronchoalveolar lavage fluid and lung tissue, histological analysis and micro-CT imaging detection of lung tissue injury in vivo. Results: The nanomicelles with rigid phosphorous dendron structure enable high-capacity and stable Cur loading. Very strikingly, the drug-free C11G3-TBP micelles exhibit excellent cytocompatibility and intrinsic anti-inflammatory activity through inhibition of nuclear transcription factor-kappa B, thus causing repolarization of alveolar macrophages from M1 type to anti-inflammatory M2 type. Taken together with the strong ROS scavenging property of the encapsulated Cur, the developed nanomicelles enable effective therapy of inflammatory alveolar macrophages in vitro and an ALI mouse model in vivo after atomization administration. Conclusion: The created phosphorus dendron nanomicelles can be developed as a general nanomedicine platform for combination anti-inflammatory and antioxidative therapy of inflammatory diseases.

S-3 spectrometer (Fä llanden, Switzerland). All 31 P NMR spectra were generally recorded by avoiding the disturbance of { 1 H}. The structures of all synthesized compounds are shown in Figure S1.
Compound 1: This compound was prepared and characterized according to the literature [1]. The solution of hexachlorocyclotriphosphazene (17.25 mmol, 50 mL tetrahydrofuran (THF)) was added into the mixture of 4-hydroxybenzaldehyde (86.25 mmol) and anhydrous potassium carbonate (172.5 mmol) in 50 mL THF under an ice bath for 20 min. The reaction mixture was stirred for 12 h at room temperature. Salts were then removed by centrifugation and the clear solution was concentrated under reduced pressure. The residue was then purified by silica column chromatography (hexane/ethyl acetate, 8/2 to 6/4, v/v) to afford compound 1 as a colorless oil in 76% yield. 1  Compound 3: According to the literature [2], the solution of thiophosphoryl chloride (30.7 mmol, 100 mL chloroform (CHCl3)) was dropwise added into the solution of methylhydrazine (61.4 mmol, 10 mL CHCl3) at -61 ℃ under an acetone/liquid nitrogen slurry bath. The reaction mixture was stirred overnight at room temperature and then reactants were filtered to obtain the methylhydrazine-modified thiophosphoryl (compound 3) in 91% yield.

Compound 4:
The solution of compound 2 (0.495 mmol, 10 mL THF) was added into the mixture of cesium carbonate (0.99 mmol) and compound 1 (0.33 mmol) in THF (10 mL) at 0 ℃. The reaction S-4 mixture was stirred for 12 h at room temperature and then centrifuged (8000 rpm, 10min Compound 11: Compound 10 (0.05 mmol) and cesium carbonate (0.5 mmol) were dissolved in 10 mL DCM at 0 ℃. Compound 3 (1 mmol, 5 mL DCM) was dropwise added and the reaction mixture was stirred for 6 h at room temperature. The resulting precipitate was filtered off and then crude product was concentrated under reduced pressure. Crude product was dissolved in 10 mL anhydrous THF, and 100 mL pentane was dropwise added. The mixture was stirred for 0.5 h at room temperature and then vacuum dried to obtain compound 11 as a white powder in 92.4% yield. 1

Transmission electron microscopy (TEM) and atomic force microscope (AFM) imaging.
C11G3-TBP and C11G3-TBP@Cur were respectively dissolved into water to reach a final concentration of 2.5 mg/mL, sonicated for 30 min, and then kept at least 2 h at room temperature.
After the micelle formation, each sample was diluted to a working concentration of 0.25 mg/mL. Then, a TEM sample was prepared by dropping the aqueous solution of each sample onto a carbon-coated copper grid and air dried before measurements. TEM imaging was executed using a JEOL 2010 analytical electron microscope (JEOL, Tokyo, Japan) at an operating voltage of 200 kV. images were randomly selected and analyzed. Besides, an AFM sample was prepared by dropping an aqueous particle suspension onto a silicon wafer and nitrogen-dried before measurements. AFM imaging was carried out using a Molecular Force Probe 3D analytical electron microscope (Asylum Research, Santa Barbara, CA) to observe the size and morphology of micelles.
Hydrodynamic size and zeta-potential measurements. C11G3-TBP and C11G3-TBP@Cur were respectively dispersed in water (1 mg/mL), sonicated for 5 min, and then kept at least 2 h at room temperature. The nanomicelles were diluted to have a concentration of 0.1 mg/mL, and dynamic light scattering and zeta-potential measurements were carried out using a Malvern Zetasizer Nano-ZS Nanoseries 3 equipped with a standard 633-nm laser (Worcestershire, UK). All measurements were performed at room temperature, and three parallel measurements were performed for each sample. property of C11G3-TBP@Cur, the antioxidation-related mRNAs (HO-1, SOD-2 and NOX-2) were examined using RT-PCR under the protocols mentioned above. The upstream and downstream primer sequences of each gene are shown in Table S1. were sacrificed to obtain lung tissues and BALF. Lung tissues were collected and weighed to obtain the "wet" weight, dried at 80 °C for 72 h to obtain the "dry" weight, and the wet/dry weight ratio was calculated to evaluate the anti-inflammatory effect of lung tissue. BALF was centrifuged (1000 rpm for 5 min) to collect the supernatant for quantification of pro-inflammatory and anti-inflammatory factors (TNF-α, IL-1β, IL-6, Arg-1, CD206 and IL-10) and neutrophils infiltration marker MPO using commercial ELISA kits. In addition, the parallel lung tissues were homogenized to collect total RNA via a Trizol reagent for RT-PCR determination of the mRNA expression levels of pro-inflammatory and anti-inflammatory factors (TNF-α, IL-1β, IL-6, Arg-1, CD206 and IL-10). The assay was carried out according to the aforementioned protocols and GAPDH was employed as a reference gene. Further, the homogenized lung tissues were treated with Nuclear and Cytoplasmic Protein Extraction Kit to determine the nuclear and cytoplasmic NF-κB protein content through Western blotting [3]. Statistical analysis. A one-way analysis of variance statistical method was adopted to evaluate the significance of the data for comparison of in-between groups using IBM SPSS Statistic 26 software (IBM, Armonk, NY). A value of 0.05 was considered as the level of significance, and the associated data were indicated as (*) for p < 0.05, (**) for p < 0.01, (***) for p < 0.001, respectively.  TNF-α  5′-AGTGGAGGAGCAGCTGGAGT -3  5′-TCCCAGCATCTTGTGTTTCTG-3′   IL-1β  5′-CAACCAACAAGTGATATTCTCCATG-3′  5′-ATCCACACTCTCCAGCTGCA-3′   IL-6  5′-TTCTTGGGACTGATGCTG-3′  5′-CTGGCTTTGTCTTTCTTGTT-3′   iNOS  5'-TCCTGGAGGAAGTGGGCCGAAG-3'  5' -CCTCCACGGGCCCGGTACTC-3'