2.1. Materials
paclitaxel (PTX), tetrahydrofuran (THF), Oxalyl chloride, 4-(N,N-dimethylamino)pyridine (DMAP), 1-Hydroxybenzotriazole (HOBT), 1-Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), and caffeic acid (CA) were bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). 4-(1,2,2-triphenylethenyl)phenol (TPE-OH) was purchased from Saen Chemical Technology Co., Ltd. (Shanghai, China). Fucoidan (FUC) was purchased from Mingyue Seaweed Group Co., Ltd. (Qingdao, China). D-α-Tocopherol was purchased from Jingming Biotechnology Co., Ltd. (Beijing, China). 2,2'-[propane-2,2-diylbis(thio)]diacetic acid (TK) was purchased from Chuangyan Chemical Technology Co., Ltd. (Shanghai, China).
2.2. Synthesis of CFTT
The synthetic routes of CFTT were revealed in Fig. 1A.
First, 0.187 mM of TK was dissolved in THF, then oxaloyl chloride (0.187 mM) was slowly added at 0 ℃[34]. After stirring at room temperature for 3 h, TK with one end of carboxyl group activated was obtained, and the system was described as solution 1. An appropriate amount of triethylamine was dropped into the THF solution of TPE-OH (0.187 mM) to obtain solution 2. Under the ice bath condition, solution 2 was slowly added into solution 1 and stirred at 55 ℃ for 6 h without light. At the end of the reaction, THF and unreacted oxaloyl chloride and triethylamine were removed using a rotary evaporator. Single-substituted TK-TPE purified products were obtained by column chromatography.
The purified TK-TPE (0.187 mM) was dissolved in formamide, then 1.5 eq of EDC and 1.5 eq of DMAP were added to activate the carboxyl group of TK-TPE at 35 ℃ for 2 h. After activation, the formamide solution of FUC was added and stirred at 35 ℃ for 48 h without light. After 48 h, the reaction solution was dialysed in deionized water for 12 h, and the deionized water was changed every 2 h. FUC-TK-TPE (FTT) was obtained by lyophilization after dialysis.
0.187 mM of CA was dissolved in formamide, then 1.2 eq of EDC and HOBT were added to activate the carboxyl group for 3 h. After the activation, the formamide solution of FTT was added into the activated CA reaction solution, and the reaction was stirred at room temperature without light for 48 h. After the reaction, CA-FUC-TK-TPE (CFTT) was obtained by dialysis and freeze-drying. 1H-NMR was used to verify the structure of CFTT.
2.3. Synthesis of FTVE
The synthetic route of FTVE was shown in Fig. 1B.
After one end of the carboxyl group of TK was activated by the acyl chloride method, THF solution containing 80.54 mg of D-α-tocopherol (VE) was slowly added under ice bath condition, and a small amount of triethylamine was injected at the same time. After stirring at 55 ℃ for 6 h, the reaction solution was rotated to remove volatile components. Pure VE-TK was obtained by column chromatography. After activation at room temperature for 2 h, the formamide solution of FUC was added and reacted at 35 ℃ for 36 h. Finally, FUC-TK-VE (FTVE) was obtained through dialysis and lyophilization. 1H-NMR was used to verify the structure of FTVE.
2.4. Preparation of CT/PTX nanoparticles
8 mg of CFTT and 2 mg of FTVE were dissolved in 4 mL of formamide, and then PTX (1 mg/mL) was slowly and successively added into the carrier material solution. After the solution was mixed evenly by ultrasound, the solution was dialysed in the 2000 Da dialysis bag for 10 h, and appropriate FeCl3·6H2O was added for further dialysis. After dialysis, CT/PTX was obtained through 0.22µm membrane filtration.
2.5. Characterizations
The particle size and polydispersity index (PDI) of CT/PTX were determined by Delsa Nano C Particle Analyzer[35]. TEM was used to observe the morphology of CT/PTX nanoparticles. The content of PTX was determined by HPLC at 227 nm[36]. The DL% were calculated as follows:
DL% = weight of drugs in nanoparticles / weight of nanoparticles × 100%
2.6. In vitro ROS- responsive assay
1 mL of concentrated CT/PTX nanoparticles solution was packed into dialysis bags of 2000 Da respectively. These dialysis bags were put into centrifuge tubes containing 47 mL PBS (pH 7.4, containing 0.5% Tween 80) with different concentrations of H2O2 (0, 0.1, 1, and 10 mM). The samples were incubated on a thermostatic water bath shaker at 37°C. At different time points specified, 1.0 mL of the release medium was collected, and 1.0 mL of fresh release medium was subsequently added to keep the medium volume constant. The concentration of PTX in the collected release medium was determined by HPLC.
In order to prove that CT/PTX nanoparticles could induce Fenton reaction to produce ROS, methylene blue (MB) was used to study Fenton catalytic activity of CT/PTX nanoparticles in the presence of H2O2[11]. •OH can degrade MB, and the absorbance value of MB at 664 nm decreases, which indirectly reflects the formation of •OH. Compared with MB alone and MB containing H2O2, the changes of absorbance values at 664 nm were recorded after 3 h co-incubation with MB, H2O2, and CT/PTX nanoparticles.
2.7. In vitro cytotoxicity assays
FT/PTX and CT/PTX nanoparticles were prepared by the dialysis method. Different from the preparation of CT/PTX, the carrier material of FT/PTX is a mixture of FTT and FTVE, and the mass ratio of FTT to FTVE is 4:1. The cytotoxicity of CT/PTX and FT/PTX nanoparticles against A549 was determined by MTT assay[37, 38]. A549 cells were respectively plated in 96-well plates. After incubation for 24 h, the old culture medium was discarded, and a fresh complete culture medium containing different preparations (Free PTX, FT/PTX, CT/PTX) was added. After incubation for 24 h or 48 h, the cells were incubated with MTT solution for 4 h. Then, DMSO was added to dissolve the violet formazan crystals. Finally, the absorbance was measured at 490 nm using a microplate reader.
2.8. Cellular uptake and distribution
A549 cells and B16F10 cells were incubated in a 24-well plate for 24 h. When the cells adhered to the wall, CT/PTX at a dose of 10 µg mL− 1 (PTX) were added and incubated for 1 h. After washing and fixing, cell images were obtained with an inverted fluorescent microscope to observe the cellular uptake.
2.9. ROS detection assay
The ROS generation capacities of Free TPE-OH, FT/PTX and CT/PTX were evaluated by DCFDA, which is non-fluorescent however transformed into DCF with green fluorescence in the presence of ROS[39, 40]. The A549 cells were incubated in a 6-well plate and treated with FT/PTX and CT/PTX (Fe3+concentration: 8 µg/mL) for 5 h. After incubation, an appropriate amount of DCFDA working solution was used to detect the ROS generation.
2.10. In vivo distribution and imaging
0.2 mL of Free DiR, FT/DiR and CT/DiR were delivered into nude mice bearing A549 tumors by caudal vein injection when the tumor had grown to an appropriate size. At different time points (2, 4, 8, 12, 24 h), and in vivo fluorescence imaging system was used to monitor the distribution of fluorescence in the body of the tumor-bearing nude mice.
2.11. In vivo antitumor efficiency and histological analysis
The nude mice bearing A549 tumors were randomly divided into four groups, including saline, Free PTX, FT/PTX, and CT/PTX. The A549 tumor-bearing nude mice were intravenously injected with different PTX preparations (PTX dosage: 10 mg/kg) every three days. The tumor volume and bodyweight of the nude mice were measured and recorded before each administration. After the nude mice were humanely sacrificed, major organs and tumors were collected and preserved in 4% paraformaldehyde solution quickly. Hematoxylin and eosin (H&E) staining assay was conducted to study the anti-tumor effect of different PTX preparations. The expression of bcl-2 and MMP-9 in tumor tissues was investigated by immunohistochemistry assay to explore the mechanism of tumor cell apoptosis.