Materials. Pluronic surfactant F108, cyclohexane, tetraethyl orthosilicate (TEOS), diethoxydimethylsilane (DEDMS), dimethyl sulfoxide (DMSO), carbon tetrachloride (CCl4), corn oil, rosiglitazone were purchased from Sigma-Aldrich. Near-infrared fluorescent dye Cy5.5-NHS ester was purchased from Lumiprobe Corporation. Rat HSC cell line (HSC-T6) was purchased from the Procell Life Sciences&Technology Co, Ltd. CTCE9908 peptide (sequence: Lys-Gly-Val-Ser-Leu-Ser-Tyr-Arg-Cys-Arg-Tyr-Ser-Leu-Ser-Val-Gly-Lys) were purchased from Nanjing Peptide Industry Biotechnology Co., Ltd. Male ICR mice (20 ± 2 g) were obtained from the Animal Centre of Yangzhou University.
Synthesis of SCLMs, PSCLMs, Hp-PSCLMs. Briefly, Pluronic copolymer F108 (0.25 g) was dissolved in aqueous HCl solution (7.5 mL, 2.0 M) under vigorous stirring at room temperature. Then, cyclohexane (240 µL) was added and dispersed by sonication, and the emulsion was stirred for another 30 min. Afterwards, TEOS (268 µL) was added and stirred for 4 h at room temperature before the addition of DEDMS (120 µL). 24 h later, the product was dialyzed in deionized water for 5 times (20 000 Da molecular weight membrane) to purify the product. Cyclohexane was removed at by rotary evaporation at 50°C under 150 mbar for 15 min. To remove the potential precipitates, the product was centrifuged at 5000 rpm for 15 min and the supernatant with SCLMs was filtered by a 0.22 µm membrane before the characterization and applications.
Mal-F108-Mal was synthesized as described by Frayne et al57 (Fig. S16). Furan (1.05 g) and maleimide (1 g) were dissolved in diethyl ether (15 mL) in a sealed tube, and heated for 12 h at 100°C. After cooling down to room temperature, the precipitiation was filtered, washed with diethyl ether and dried to afford compound 1 (Fig. S17). 1H NMR (300 MHz, DMSO) δ 11.15 (s, 1H), 6.53 (s, 2H), 5.11 (s, 2H), 2.85 (s, 2H). F108 (7.3 g, 0.5 mmol) and p-toluenesulfonyl chloride (0.955 g, 5 mmol) was dissolved in tetrahydrofuran (100 mL). The aqueous solution of sodium hydroxide (0.8 g, 10 mmol) was slowly added to the solution under stirring. The resulting mixture was warmed to 50°C and stirred overnight. After the reaction, the mixture was concentrated under reduced pressure and the solid was dissolved in dichloromethane. The organic layers were washed with water and saturated saline solution, then dried by anhydrous sodium sulfate. The solvent was evaporated to afford the crude product Ts-F108-Ts (Fig. S18). 1H NMR (300 MHz, D2O) δ 7.79 (d, J = 7.7 Hz, 4H), 7.46 (d, J = 7.8 Hz, 4H), 3.63 (s, 1471H), 3.49 (dt, J = 13.4, 6.7 Hz, 457H), 2.40 (s, 6H), 1.20–0.99 (m, 637H). Compound 1 (0.33 g, 2 mmol) and Ts-F108-Ts (2.92 g, 0.2 mmol) were dissolved in acetone, potassium carbonate (0.278 g, 2 mmol) was then added, the mixture was refluxed for 60 hours under an inert N2 atmosphere. After completion, the solids were removed by filtration and the filtrate was concentrated under reduced pressure to afford compound 2 (Fig. S19). 1H NMR (300 MHz, D2O) δ 6.64 (s, 4H), 5.32 (s, 4H), 3.72 (s, 1416H), 3.63–3.43 (m, 131H), 3.14 (s, 5H), 1.18 (d, J = 5.7 Hz, 179H). Compound 2 (3 g, 0.2 mmol) was dissolved in toluene (20 mL) and refluxed under an inert N2 atmosphere overnight. The solvent was removed and the residue was dissolved in 20 mL dichloromethane, the mixture was added dropwise to stirred diethyl ether. The precipitation was collected and washed with diethyl ether repeatedly, then dried under high vacuum to afford Mal-F108-Mal (Fig. S20). 1H NMR (300 MHz, D2O) δ 6.93 (s, 2H), 3.75 (s, 1190H), 3.61 (d, J = 4.9 Hz, 102H), 1.21 (d, J = 5.9 Hz, 150H)
In the synthesis of Mal-SCLMs, Mal-F108-Mal was doped in F108 in a weight ratio of 7% (0.0175 g Mal-F108-Mal and 0.2325 g F108), and solubilized in HCl (7.5 mL, 2.0 M). The next steps are the same as the above synthetic method of SCLMs to obtain Mal-SCLMs. PSCLMs was obtained through the click reaction between the sulfhydryl group of CTCE9908 peptide and maleimide group. CTCE9908 (1.8 mg) was then added to Mal-SCLMs and stirred at room temperature for 2 h. The solution was collected and dialysis in deionized water for 5 times (20 000 Da molecular weight membrane) to remove the excessive reactants and impurities. Then the solution was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered using a 0.22 µm filter to obtain PSCLMs. The concentration of SCLMs and CT-SCLMs was determined by lyophilization.
The sulfhydryl group was modified to FEN1 by Traut’s reagent to obtain FEN1-SH. HpDNA-SH was synthesized by the modification of the 5′-end of the strand of hpDNA with the sulfhydryl group. FEN1-SH (400 pmol) and hpDNA-SH (3600 pmol) were added to SCLMs (0.78 mg) and reacted at room temperature for 2 h. The product was denoted as Hp-SCLMs with a ratio of FEN1 to hpDNA of 1:9. Hp-PSCLMs was synthesized in the same method using the PSCLMs as substrate.
Characterizations. Transmission electron microscopy (TEM) images were captured on a JEOL JEM-1400 transmission electron microscope with operation voltage of 100 kV. Dynamic light scattering (DLS) and zeta potential experiments were performed at 25°C using an Anton Paar Litesizer 500 nm particle size analyzer. Absorption spectra were obtained by a Philes G9-9S Uv-visible spectrophotometer. Confocal images of cells and tissues were recorded on an Olympus FV3000 Confocal Laser Scanning Microscope (CLSM).
In vitro RNA cleavage assay. A 10 µL mixture consisting of single-strand RNA (ssRNA) substrate (10 pmol) (see Table S1 for the sequence), 3-(4-Morpholino) propanesulphonic acid (10 mM), 0.05% Tween-20, 0.01% nonidet P-40, MgCl2 (7.5 mM), and Hp-PSCLMs (1 µg) with designed ratio of hpDNA and FEN1 was incubated at 37°C for 2h. The 5’-end of the target ssRNA was modified with fluorescent FAM. The products were analyzed by denatured-polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. The loading buffer contained 90% formamide, 0.5% EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue. Before loading, 10 µL loading buffer was added to the 10 µL reaction mixture. The sample was then loaded onto a 20% PAGE gel at room temperature for the electrophoresis in a buffer containing urea (8.7 M) and triborate (89 mM) with a speed of 9.6 V·cm-1 for 2 h. The gel was imaged by an Amersham Imager 600 (GE Healthcare). The protocol of the cleavage using the Hp-SCLMs was the same as the above method using free HpSGN.
Loading of rosiglitazone. Rosiglitazone was dissolved in DMSO to form a solution with a concentration of 100 mg·mL-1. Then, the 10 µL DMSO solution containing rosiglitazone were added to a solution with the drug carriers (20 mg·mL-1) under vigorous sonication to load the hydrophobic molecules into the PPO core of SCLMs. The rosiglitazone-loaded drug carriers were denoted as R@SCLMs, R@PSCLMs and R@Hp-PSCLMs, respectively. The loading ratio of rosiglitazone was determined by UV-vis as 5%.
In vitro release of rosiglitazone. The release rate of rosiglitazone was monitored under sink conditions in a PBS solution (pH 7.4) with 1% Tween 80. The drug carrier (5 mL, 4 mg·mL-1) containing rosiglitazone (0.2 mg·mL-1) was sealed in a dialysis membrane with MWCO of 8000–14000. The dialysis bag was then soaked in PBS (45 mL) at 37°C with constant stirring (120 rpm). At designed time, 1 mL of the release medium was withdrawn and replaced with the same amount of fresh PBS with 1% Tween 80. The released amount of the drug was determined by UV-absorption using a PHILES G-9S ultraviolet spectrophotometer.
Cell culture. HSC-T6 rat hepatic stellate cells were cultured in DMEM supplemented with 10% FBS. All cells were incubated at 37°C in a humidified 5% CO2 atmosphere.
Toxicity Studies. The in vitro cytotoxicity of the nanocarriers were investigated by MTT assay. HSC-T6 cells were seeded to a 96-well plate (1 × 104 cells per well) and cultured overnight at 37°C in 200 µL medium containing 10% FBS. Then the DMEM medium in the plate was replaced by 200 µL FBS-free medium containing different concentrations of the drug carrier (0, 3.125,6.25,12.5,25, 50, 100, 200 and 400 µg·mL-1), and the cells were incubated for another 24 h. Afterwards, 20 µL MTT solution (5 mg·mL-1) was added to each well in the 96-well plate and the plates were further incubated at 37°C for 4 h in the dark. After that, the medium was removed and 150 µL dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The plate was shaken gently on the shaker at room temperature for 15 min. Cell viability was estimated based on the absorbance at 570 nm in each well. Cell viability was calculated according to the following formula:
$$\text{C}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{{Abs}^{treatment}}{{Abs}^{control}}\times 100\%$$
The biosafety of PSCLMs and Hp-PSCLMs were further tested in vivo. The mice were intravenously injected with PSCLMs or Hp-PSCLMs (4 mg·mL-1, 200 µL) or saline for 24 h then the mice were sacrificed and the main organs (heart, liver, spleen, lung, and kidney) were harvested for H&E staining. The blood were collected for the ALT, AST, CRE, BUN test using commercial test kits (Jiancheng BioTech, Nanjing, China).
Hemolytic activity study. Hemolytic activity was evaluated using the blood from mice. Different concentrations of SCLMs, PSCLMs and Hp-PSCLMs (0.05, 0.1 and 0.5 mg·mL-1) were incubated with 2% red blood cell suspension at 37°C for 3 h. Blood (200 µL) mixed with saline and distilled water served as negative and positive controls, respectively. Following, all samples were centrifuged at 680×g for 5 min and the supernatant was added to a 96-well plate to measure the absorbance at 540 nm. The calculation of hemolysis rate is as follows:
$$\text{H}\text{e}\text{m}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s} \text{r}\text{a}\text{t}\text{e}\left(\text{%}\right)=\frac{Abs-{Abs}^{negative control}}{{Abs}^{positive control}-{Abs}^{negative control}}\times 100\text{\%}$$
In vitro inhibitory effect of rosiglitazone on cell proliferation. HSC-T6 cells were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured for 12 h. Then the medium was removed and cells were incubated for 24 h with fresh medium containing 10 ng·mL-1 TGF-β to activate HSCs. The fresh medium was replaced with the medium containing different concentrations of R@SCLMs or R@PSCLMs (with rosiglitazone of 1.56, 3.13, 6.25, 12.5, 25.0 and 50.0 µg·mL-1) for another 24 h before the MTT assay. The group with no treatment was used as a control. The calculation formula of the inhibitory rate of cell proliferation is as follow:
$$\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{o}\text{r}\text{y} \text{r}\text{a}\text{t}\text{e} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l} \text{p}\text{r}\text{o}\text{l}\text{i}\text{f}\text{e}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\left(\text{%}\right)=\frac{{Abs}^{Control}-Abs}{{Abs}^{Control}}\times 100\%$$
In vitro cellular uptake analysis of PSCLMs. HSCs were seeded in confocal dishes at a density of 5 × 103 cells per dish and incubated in 1 mL of DMEM supplemented with 10% FBS for 12 h. Then the cells were incubated for 24 h with fresh medium containing 10 ng·mL-1 TGF-β to activate HSCs. Activated HSCs were treated with SCLMs or PSCLMs loaded with FITC (100 µg·mL-1). For the free ligand competitive inhibition assay, the CXCR4 on the activated HSCs were saturated by free CTCE9908 (50 µg·mL-1) for 30 min before incubation with FITC-labeled PSCLMs. 4 h after the addition of the drug carriers, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Afterwards, the cells were washed three times with PBS and stained with DAPI for 10 min. Finally, after washed with PBS for three times, the fluorescence of FITC and nuclei was observed by confocal laser scanning microscopy.
Animal models of liver fibrosis. Six-week-old male ICR mice (20 ± 2 g) were obtained from the Animal Centre of Yangzhou University (Yangzhou, China). All animal experiments were conducted under protocols approved by the Ministry of Health of the People's Republic of China and following Guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University (2022-04-001). All mice were housed at 25°C at 40–60% humidity. Liver fibrosis was induced by CCl4 in ICR mice. CCl4 and corn oil mixture (1:9 in volume ratio) was given by intraperitoneal injection twice a week for 4 weeks (at the 1st, 4th, 8th, 11th, 15th, 18th, 22nd, 25th day) at a dose of 10 mL·kg-1.
Biodistribution and intrahepatic distribution of nanoparticles. For the biodistribution study, the fibrotic mice were administered with a single intravenous injection of the nanoparticles prepared with Cy5.5-labeled Hp-SCLMs or Hp-PSCLMs (4 mg·mL-1, 200 µL). The fluorescence signals of the main organs (heart, liver, spleen, lung, and kidney) were monitored and analyzed at 2, 12, 24, 48 and 96 h after injection using a Vilber FUSION FX7 imaging system (λex = 680 nm, λem = 750 ± 10 nm).
For the intrahepatic distribution, the mice were sacrificed 24 h after the injection of the drug carriers (4 mg·mL-1, 200 µL) and the liver tissues were sectioned for immunofluorescence staining. α-SMA was stained with an anti-α-SMA antibody and a 488-conjugated secondary antibody to label activated HSCs. Nuclei were stained with DAPI. Immunofluorescence images were captured using a CLSM.
Therapeutic efficacy. The liver fibrosis mice were established by the aforementioned method. During the 4-week administration of CCl4, the mice were intravenously injected with 200 µL of 4 mg·mL-1 NTHp-PSCLMs, R@PSCLMs, Hp-PSCLMs and R@Hp-PSCLMs (at the 16th, 19th, 23nd, 26th day). As negative control, the free rosiglitazone (2 mg·kg-1, 200 µL) and HpSGN (4 nmol per mice) were also injected intravenously to another two groups of mice at the same time. The concentrations of rosiglitazone and HpSGN in the drug carriers were the same as the negative control groups. The body weights of the mice were recorded. Afterwards, all the mice were sacrificed, the liver and blood were harvested for further analysis.
The levels of ALT, AST and T-BIL in blood and hydroxyproline (Hyp) in the liver were determined using analysis kits according to the manufacturers’ instructions (Nanjing JianCheng Bioengineering Institute, China). H&E and Masson were stained according to the standard histological procedures. Protein expression of collagen I, α-SMA, TIMP-1, PPARγ and MMP1 in liver after different treatments were measured by western blot and the bands were analyzed by ImageJ. The expression of TIMP1 and α-SMA were determined by immunofluorescence and the fluorescence were quantified by ImageJ. The expression of collagen I was determined by immunocytochemistry and their positive areas were analyzed using ImageJ. The level of TIMP1 was analyzed by qPCR and the sequences of the primers were shown in Table S2.
$$\text{C}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y} \left(\text{\%}\right)= \frac{\text{m}\text{e}\text{a}\text{n} \text{A}\text{b}\text{s} \text{v}\text{a}\text{l}\text{u}\text{e} \text{o}\text{f} \text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t} \text{g}\text{r}\text{o}\text{u}\text{p}}{\text{m}\text{e}\text{a}\text{n} \text{A}\text{b}\text{s} \text{v}\text{a}\text{l}\text{u}\text{e} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{g}\text{r}\text{o}\text{u}\text{p}} \times 100\%$$
Statistical analyses. All data are presented as the mean ± standard deviation obtained from at least three independent experiments. The two-tailed Student’s t-test was used to compare the data in two groups. P < 0.05 was considered significant.