2.1 Materials
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[iRGD-(polyethylene glycol)-2000] (DSPE-PEG-iRGD) were obtained from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an, China). 3-mercaptopropionic acid, acetone, anhydrous N,N-dimethylformamide (DMF), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and ursolic acid (UA) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazoliumbromide (MTT), RPMI 1640 medium, and DAPI were purchased from Beyotime Biotechnology (Shanghai, China).
Cell and animals
Human gastric cancer cell lines SGC 7901 and NIH-3T3 cells were obtained from KeyGEN BioTECH (Jiangsu, China) and cultured using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator containing 5% CO2 at 37°C.
Male BALB/c nude mice were supplied by the Bengbu Medical University Experimental Animal Center (Bengbu, China). All in vivo procedures were performed in adherence to the guidelines of the Institutional Animal Care and Use and the study was approved by the Ethics Committee of the Bengbu Medical University.
2.2 TK fabrication
TK was synthesized as previously reported.[32, 33] Briefly, acetone and 3-mercaptopionic acid were mixed at a 9: 5 molar ratio and stirred at room temperature under a dry hydrogen chloride atmosphere for 6 h. After the reaction, the mixture was crystallized under an ice-salt mixture environment. Then, the mixture was filtered to obtain crystals, which were purified by washing with hexane and cold water many times, and then dried under vacuum to obtain TK (yield: 41.2%). TK was confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR AVANCE Ⅲ, BRUKER, Switzerland) and mass spectrometry (MS, AB SCIEX 6500 Qtrap, Applied Biology, Inc., USA).
2.3 TK-UA2 fabrication
In brief, EDC (4.58 g, 0.024 mol) and NHS (2.76 g, 0.024 mmol) were added to the solution of TK (2.51 g, 0.01 mol) in 20.0 mL of anhydrous DMF. The mixture was stirred under an N2 environment at 20°C for 2 h. Subsequently, UA (10.05 g, 0.022 mol) solution in 20 mL of anhydrous DMF was added to the above mixture and reacted for a further 24 h at 20°C in N2. Then, the mixture was concentrated in vacuo to remove the solution. The obtained crude product was purified by silica column chromatography using ethyl acetate/hexane = 4: 1 as the mobile phase. Finally, TK-UA2 was obtained with a yield of 67.3%.
As a control, the ROS-insensitive dimeric prodrug, CC-UA2, was also prepared by conjugating UA to adipic acid using the same method. The yield of CC-UA2 was 71.3%. TK-UA2 and CC-UA2 were characterized by 1H NMR and MS.
2.4 Preparation of drug-loading nanoparticles
The NPs formed by TK-UA2, DSPE-PEG-iRGD, and DSPE-PEG was denoted as iRGD-TK-NPs; NPs formed by TK-UA2 and DSPE-PEG were named as TK-NPs, and NPs formed by CC-UA2 DSPE-PEG-iRGD, and DSPE-PEG were dubbed iRGD-CC-NPs. The ethanol injection method was used to prepare these NPs. Typically, 8.0 mg TK-UA2, 1.0 mg DSPE-PEG-iRGD, and 2.0 mg DSPE-PEG were dissolved in 4.0 mL of ethanol. Subsequently, the mixture was added dropwise to 10.0 mL distilled water under vigorous stirring. After stirring for 2 h, ethanol was removed by evaporating at 25°C under vacuum. Then, the colloidal solution was diluted to 10.0 mL with distilled water to obtain the iRGD-TK-NPs. The zeta potential and hydrodynamic diameter of NPs were determined by dynamic light scattering (DLS) on a Zetasizer (Zs90, Malvern, UK). Additionally, coumarin-6 loaded NPs were also prepared by the same operating procedures.
2.5 Stability assay
NPs were cultured at 37°C in PBS containing 10% fetal bovine serum (FBS). At prescriptive intervals time points, the size of NPs was measured by DLS.
2.6 Evaluation of ROS-responsive ability
Changes in size and in vitro drug release assay were performed to investigate the ROS-responsive ability of NPs. For size changes assay, iRGD-TK-NPs and iRGD-CC-NPs were incubated in PBS containing 10 mM H2O2 at 37°C. After incubation for 2, 4, 8, 12, and 24 h, the size of NPs was measured by DLS.
For drug release analysis, PBS (pH 7.4) containing 20% ethanol (v/v) and various concentrations of H2O2 (1.0, 0.1, and 0 mM) was used as the release medium. A quantitative amount of iRGD-TK-NPs was added to the release medium and incubated at 37°C with gentle stirring. At prescriptive intervals, the amount of UA released was measured by high-performance liquid chromatography (HPLC) method (mobile phase: methanol/water/trifluoroacetate (80: 20: 0.05, v/v), 1 mL/min, UV-vis detector set at 210 nm according to full wavelength scan using a Shimadzu HPLC system (LC20A, Japan).
2.7 Cellular uptake investigation
Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) assay were performed to investigate the cell internalization. For the CLSM study, ten thousand SGC 7901 cells were seeded into a 35-mm glass-bottom culture dish and cultured in medium overnight at 37°C. After incubation, coumarin-6 loaded iRG-TK-NPs and TK-NPs solution diluted with FBS-free DMEM at a final concentration of 500 ng/mL (equal to coumarin-6) was added, and incubated for a further 1 h or 3 h. After treatment, cells were washed with PBS, stained with DAPI, fixed in 4% paraformaldehyde, and then observed by confocal laser scanning microscopy (CLSM, LSM 780, Zeiss, Germany).
For FCM assay, SGC 7901 and NIH-3T3 cells were seeded into a six-well plate at a density of ten thousand cells per well, respectively, and cultured overnight at 37°C. After incubation, coumarin-6 loaded NPs solution diluted with FBS-free DMEM at a final concentration of 500 ng/mL (equal to coumarin-6) were added and incubated for 1, 2, 3, or 4 h. After treatment, cells were washed with PBS and detected by FCM (BD FACSCalibur, Aria Ⅲ, USA).
2.8 In vitro cytotoxicity
SGC 7901 and NIH-3T3 cells were seeded in 96-well plates at a density of 3,000 cells per well and incubated for 24 h. Subsequently, cells were treated with UA, iRGD-TK-NPs, TK-NPs, or iRGD-CC-NPs at various concentrations for 48 h at 37°C. After treatment, 20 μL of MTT solution was added into each well and cultured for a further 4 h. At the end of the incubation period, the medium was removed and 200 μL of DMSO was added; then, the absorbance was determined using a microplate reader (Bio-Rad, USA). Cell viability was calculated according to the following equation: cell viability = (ODtreatment/ODcontrol) × 100%.
2.9 Hemolysis assay
Sprague-Dawley (SD) rat blood was obtained and washed with saline. Then, RBCs were collected and mixed with NPs solution at various concentrations. The mixture was maintained at 37°C with slight stirring for 2 h. Subsequently, the absorbance was detected using a microplate reader at 577 nm. Saline and Triton X-100 were employed as negative and positive controls. The following equation was used to calculate hemolysis: Hemolysis (%) = (ODsample - ODnegative)/(ODpositive – ODnegative) × 100%.
2.10 Pharmacokinetics and biodistribution
The SD rat was used as the animal model to investigate the in vivo pharmacokinetics of all drug formulations. UA, iRGD-TK-NPs, TK-NPs, and iRGD-CC-NPs were injected into SD rats via the tail vein at a UA-equivalent concentration of 11.0 mg/kg, respectively. At pre-set time points, blood samples were obtained from the orbital plexus and centrifuged at 3000 g for 10 min. The supernatant was collected and mixed with methanol/chloroform (1:2, v/v) to extract the UA prodrug and free UA. After centrifugation, the supernatant was collected, dried under vacuum, redissolved with methanol, and measured by HPLC as described above.
The SGC 7901 xenograft tumor mouse was used as the animal model to study the biodistribution of all drug formations. SGC 7901 tumor-bearing mice were prepared by subcutaneous inoculation of 6.0 × 106 cells into the hind flank of each mouse. Two weeks after injected of cells, mice were intravenously injected with UA, iRGD-TK-NPs, TK-NPs, and iRGD-CC-NPs at the UA-equivalent concentration of 11.0 mg/kg, respectively. After treatment for 6 h and 12 h, six mice were euthanized by cervical dislocation; then, the major organs (kidney, spleen, liver, heart, and lung) and tumor tissues were collected, washed with PBS, weighed, and detected by HPLC.
2.11 In vivo antitumor effect
SGC 7901 xenograft tumor mice were randomly divided into four groups when the tumor volume reached about 80 mm3 and then treated with saline, UA, iRGD-TK-UA, TK-NPs, and iRGD-CC-UA, respectively, at UA-equivalent 11.0 mg/kg five times every three days. After the first administration, the tumor’s length/width and mouse body weight were measured every three days. The following equation was used to calculate the tumor volume: Volume = 1/2 × length × (width)2. After treatment for 21 days, mice were sacrificed and tumor tissues were collected. The tumors were imaged, weighed, and tumor growth suppression (TGS) was calculated as follows: TGS (%) = (weightsaline – weighttreatment) / weightsaline × 100%.