2,3-dioleoyl-3-trimethylammonium-propane(DOTAP) and 1, 2-distearoyl-sn-glycero-3-hosphoethanolamine-n-[methoxy (polyethylene glycol) 2000] (DSPE-PEG 2000) were provided by Sigma-Aldrich (USA).Cholesterol, INH, RIF, and PZA (>98% purity) were manufactured by Tokyo Chemical Industry (Japan). RPMI 1640 medium, trypsin, and fetal bovine serum (FBS) were provided by Hyclone (USA). Anti-TGF-1 (Cat No. ab 92486) was from Abcam (UK). SYBR® Premix Ex Taq, PrimeScript™ RT reagent Kit with gDNA Eraser, and RNAiso Plus were manufactured byTaKaRa Biotechnology (Japan). 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide salt (MTT) was provided by Biosharp (China). Annexin V-FITC/PI Apoptosis Detection kit and propidium iodide (PI) staining solution were provided BD Biosciences (USA). Preparation of HRZ/siTGF-β1 nanoliposomes
Preparation of HRZ-loaded nanoliposomes
HRZ-loaded nanoliposomes were prepared by the reverse-phase evaporation method. Briefly, DOTAP (36 mg), DSPE-PEG2000 (50 mg), cholesterol (1 mg), INH (7.2 mg), RFP (10.9 mg), and PZA (1.8 mg) at the molar ratio 20:10:1:21:5.3:5.9 were solubilized in chloroform/methanol (4:1, v/v). After solvent evaporation (rotary evaporator, 37°C), further drying was performed under vacuum for 1 h. The resulting inclusion complex was dissolved in 5 mL of deionized water, and a clear orange-red solution was obtained post-filtration.
The resulting nanoliposome solution was transferred into a 10 kDa ultrafiltration tube, and subjected to ultrafiltration at 5000 ×g for 10 min and repeated 5 times until a colorless filtrate was obtained. The upper layer of the preserved orange-red liquid encompassed cationic liposomes containing the anti-TB drugs. Then, 10% mannitol was added to the liquid and lyophilized to obtain 67 mg of an orange-red oily HRZ-loaded nanoliposome product.
Conjugation of HRZ-loaded nanoliposomes with siTGF-β1
The siRNA oligonucleotides targeting TGF-β1 (siTGF-β1) were manufactured by Biomics Biotechnologies, and their sequences were as follows: siTGF-β1: sense 5’-GGA GUC AGA UCC UCA GCA AGC-3’ and antisense 5’-UUG CUG AGG AUC UGA CUC CUG-3’; non-coding control siRNA (siNC), sense 5’-GAA GGC CCA TAG CCA GTG ACT-3’ and antisense 5’-AGU CAC UGG CUA UGG GCC UUC-3’. Cationic HRZ nanoliposomes were mixed with siTGF-β1 in weight ratios of 2:1, 5:1, 10:1 and 20:1, respectively, and further underwent incubation at ambient for 30 min. The binding efficiency of the HRZ nanoliposomes with siTGF-β1 was determined by the gel retardation assay using 1.5% agarose gel (UltrapureTM agarose, Life Technologies).
Characterization of HRZ/siTGF-β1 nanoliposomes
Particle size and zeta potential
The size and zeta potential of HRZ/siTGF-β1 nanoliposomes were assessed by dynamic light scattering (DLS) on a Malvern ZetasizerNano ZS (Malvern Instruments, UK) in triplicate at ambient, after dilution with double-distilled water.
The surface morphology of HRZ/siTGF-β1 nanoliposomes was assessed by transmission electron microscope (TEM) (TEM Jeol JEM-1400; JEOL, Japan). To prepare TEM samples, cationic nanoliposomes and siRNA at a mass ratio of 5:1 were spread over a copper grid and air-dried for 30 min before detection.
INH, RIF and PZA loading in HRZ/siTGF-β1 nanoliposomes was assessed as reported previously, with slight modifications. Briefly, the mobile phase was formulated to an optimal concentration to detect the EE of HRZ/siTGF-β1 nanoliposomes on a high-performance liquid chromatography (HPLC) system (Agilent Technologies, USA). After the loading procedure, the suspensions were submitted to centrifugation at 16873 g for 20 min (Centrifuge 5418; Eppendorf AG, Germany) and unencapsulated drugs that remained in the supernatant were quantitated by UV detection at 334 nm.
Entrapment efficiency (%) was derived as [(weight of drug loaded initially − weight of unencapsulated drug)/weight of drug loaded initially] × 100%
Human monocytes THP-1 cells provided by American Type Culture Collection (ATCC) underwent culture at 2×105 cells/mL in RPMI 1640 medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 g/mL). The media were replaced twice or thrice weekly, and the cells were sub-cultured until 80–90% confluency.
THP-1 cell differentiation into adherent macrophages was performed with 100 nM phorbol 12-myristate 13-acetate (PMA) for 48 h in RPMI 1640 containing 10% FBS. Then, the PMA media were removed, followed by three PBS rinses and incubation in fresh medium for 3 h.
In-vitro cytotoxicity assays
To assess the developed nanoliposomes for cytotoxic effects on human macrophages, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out as directed by the manufacturer. In brief, 5×103 THP-1 cells were added into each well of a 96-well plate, and allowed to differentiate into macrophages by PMA induction at 100 ng/mL for 48 h. Then, they were incubated with HRZ/siTGF-β1 nanoliposomes at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg/mL at 37°C in 5% CO2 for 24 h. Subsequently, the medium was replaced by MTT containing culture medium. Incubation was carried out for an additional 4 h, and the reaction was stopped with an equivalent volume of DMSO for formazan crystal solubilization. Optical density was obtained at 570 nm. Cell viability was quantitated as described in a previous report, determining the percentages of viable cells and inhibitory potency (IC50) values. Triplicate assays were carried out.
Assessment of cell cycle distribution and apoptosis
Flow cytometry (FCM) was carried out to assess cell cycle distribution and apoptosis upon treatment with nanoliposomes. PMA-induced macrophages were added at 5×103 cells per well of a 96-well plate. Upon overnight incubation, the siNC group was treated with HRZ/siNC nanoliposomes, while the siTGF-β1 groups were administered various amounts of HRZ/siTGF-β1 nanoliposomes (35 and 40 mg/mL); the HRZ group was treated with HRZ nanoliposomes. On the other hand, control cells were administered an identical volume of cell culture medium. Upon treatment, the cells underwent trypsinization, centrifugation (1000 rpm, 5 min), and staining with Annexin V-FITC/PI double-labeling kit (eBioscience, USA) before analysis for cell apoptosis. Next, cell resuspension was performed in PBS with 40 µg/mL PI followed by a 30-min incubation at 37°C away from light for assessing cell cycle distribution. After filtering through 35 µm nylon meshes, FCM on FACSCalibur (BD Biosciences) was performed for analysis. Then, the rates of apoptosis in various cell cycle phases were determined.
Gene knockdown efficiency of TGF-β1 siRNA
Real-time quantitative PCR (RT-qPCR)
THP1-derived macrophages were administered different nanoliposomes containing HRZ, HRZ/siNC, and HRZ/siTGF-β1 (35 and 40 mg/mL) for 6 h. Total RNA from human macrophages was obtained using TRIzol, and reverse transcription was performed with PrimeScript Reverse Transcriptase Kit (TaKaRa), as directed by the manufacturer. RNA quality and amounts were assessed spectrophotometrically on a NanoDrop 1000 (Thermo Fisher Scientific). Then, qRT-PCR was carried out on an ABI PRISM Real-Time PCR system (Applied Biosystems) with the QuantiTect SYBR Green Master Mix kit (Qiagen). PCR was performed at 95°C (10 min), followed by 40 cycles of 95°C (5 s) and 60°C (1 min), with the melting curve obtained at 95°. Fluorescence was collected at 60°C every 0.3°C until 95°C. The primers employed were: TGF-β1, Forward 5’-GTC CTG GTG GAA TGG GTT ATA C-3’ and reverse 5’-GTT GAG TGT TCT TTG GCT TGA C-3’; GAPDH, Forward 5’‑GGT GTG AAC CAT GAG AAG TAT GA-3’ and reverse 5’-GAG TCC TTC CAC GAT ACC AAA G-3’. The 2-ΔΔCq method was employed for the analysis of triplicate assays, normalizing the data to GAPDH expression.
TGF-β1 protein amounts were determined by Western blot assays. After the treatment of THP1-derived macrophages with nanoliposomes containing HRZ, HRZ/siNC, and HRZ/siTGF-β1 (35 and 40 mg/mL), respectively, total protein was obtained with Total Protein Extraction Kit (Bestbio, China) and quantitated by the Bradford assay (Bio-Rad, USA) as described by the manufacturer. Equal amounts of total protein were resolved by 10% SDS-PAGE. Rabbit polyclonal anti-TGF-β1 (Abcam) and anti-GAPDH (Wuhan Boster Biological Technology, China) primary antibodies were reacted overnight at 4°C, followed by incubation with secondary antibodies linked to horseradish peroxidase (HRP) (Wuhan Boster Biological Technology) at ambient for 2 h. Immunoreactive bands were detected with an enhanced chemiluminescence system (Sino-American Biotechnology, China), and quantitated with Image J version 1.441 (National Institutes of Health, USA).
Data are mean±standard deviation (SD). Descriptive statistics and one-way analysis of variance (ANOVA) were performed for analysis. Independent sample Student’s t-test was carried out for group pair compassions. P<0.05 indicated statistical significance.