Preparation, characterization, and in-vitro cytotoxicity of nanoliposomes loaded with anti-tubercular drugs and TGF-β1 siRNA for improving spinal tuberculosis therapy

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 by TaKaRa 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 by BD Biosciences (USA).

HRZ-loaded nanoliposomes were prepared by the thin film hydration 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.

Biomics Biotechnologies manufactured the siRNA oligonucleotides targeting TGF-β1 (siTGF-β1), 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 (Ultrapure™ agarose, Life Technologies).

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). A 5:1 mass ratio of cationic nanoliposomes and siRNA was spread over a copper grid and air-dried for 30 min before detection to prepare TEM samples.

As reported previously, INH, RIF, and PZA loading in HRZ/siTGF-β1 nanoliposomes were assessed with slight modifications [34]. 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 16,873 g for 20 min (Centrifuge 5418; Eppendorf AG, Germany). Unencapsulated drugs that remained in the supernatant were quantitated by UV detection at 334.00 nm [35].

Entrapment efficiency (%) was derived as [(weight of drug-loaded initially − the 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 [36]. Then, the PMA media were removed, followed by three PBS rinses, and incubated in a fresh medium for three hours. To evaluate the cytotoxic effect of the developed nanoliposomes on human macrophages, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed directed by the manufacturer. In brief, 5 × 10³ THP-1 cells were added to 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. As described in a previous report, cell viability was quantitated, determining the percentages of viable cells and inhibitory potency (IC50) values [37]. Triplicate assays were carried out.

Flow cytometry (FCM) was conducted 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 to assess 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.

THP1-derived macrophages were administered to 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 [38] was employed to analyze 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 the 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. First of all, the extracted protein is added to the electrophoresis tank for electrophoresis and mold rotation, and after the transfer is completed, the band is cut and blocked according to the molecular weight size of the target protein and the marker, 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.

DLS measured the particle size according to the principle that particles move randomly under Brownian motion. Particle size is significant in determining the cell’s absorption rate. Liposomes of about 200.00 nm could induce membrane fusion with target cells, delivering the encapsulated products into cells with high efficiency [39]. Here, the particle sizes of HRZ/siTGF-β1 nanoliposomes were determined by DLS (Table 1, Fig. 1). The product composed of HRZ nanoliposomes and siTGF-β1 with a weight ratio around 5:1 displayed an average diameter of 168.14 ± 0.54 nm, which was adequate for alveolar epithelium deposition and macrophage internalization [40]. We also found that with increasing weight ratio, the particle size of HRZ/siTGF-β1 nanoliposomes decreased from 237.89 to 91.46 nm.

Zeta potential provides information regarding the electrostatic potential of the particle in solution [40]. The zeta potential of HRZ/siTGF-β1 nanoliposomes with different weight ratios was measured immediately after preparation (Table 2, Fig. 2A). The HRZ nanoliposomes carried a positive charge and absorbed the negatively-charged oligonucleotides by easily mixing the siTGF-β1 to produce the final HRZ/siTGF-β1 nanoliposomes. Zeta potential analysis revealed that the surface charge of HRZ nanoliposomes was 28.13 ± 2.40 mV in an aqueous solution. Upon conjugation of siTGF-β1 (weight ratio of HRZ nanoliposomes/siTGF-β1 = 5:1), the zeta potential was reduced to 4.03 ± 1.32 mV, thereby implying successful conjugation of the components that consumed the surface amino groups. The gel retardation assay was carried out to evaluate the siRNA loading capacity of nanoliposomes (Fig. 2B). HRZ/siTGF-β1 nanoliposomes were generated at various weight ratios of HRZ nanoliposomes to siTGF-β1 between 0:1 and 20:1. Gel electrophoresis showed a small number of bands at weight ratios > 5:1, indicating that most of the siRNA was absorbed by HRZ nanoliposomes. These findings corroborated surface charge data obtained by DLS. By adjusting HRZ nanoliposome-to-siTGF-β1, surface charges varied between 15.33 ± 0.55 mV and 28.13 ± 2.40 mV. In addition, a gradually increasing trend of zeta potential was observed with increasing amounts of HRZ nanoliposomes, in agreement with findings obtained by agarose gel electrophoresis.

Cells internalize nanoparticles with positive charges faster than neutral or negatively charged counterparts [41]. Moreover, excessive surface charge causes cell cytotoxicity [42]. This phenomenon can be prevented by ensuring an efficient loading of siRNA while maintaining a slight positive charge on the surface. Thus, a weight ratio of 5:1 for HRZ nanoliposomes to siTGF-β1 was applied in subsequent assays.

The appearance and morphological properties of the particles were examined by imaging air-dried HRZ/siTGF-β1 nanoliposomes under a transmission electron microscope (TEM) (Fig. 3; scale bar 0.50 µm). TEM images exhibited a spherical shape for the nanoliposomes with a homogenous surface morphology (200.00–300.00 nm), consistent with DLS data.

EE is a valuable index concerning nano-drug delivery. Adequate drug amounts are required in a given polymer for sustained release to the target site [43]. EE (28.13 ± 2.4 mV) is calculated as the percentage amount of drug that is entrapped in the form of nanoliposomes INH, RIF, and PZA loading of HRZ nanoliposomes showed EE values of 90%, 88%, and 37%, respectively (Fig. 4A and B).

The interaction of HRZ/siTGF-β1 nanoliposomes with macrophages was established using a human macrophage model to achieve macrophage targeting and effective concentrations of anti-TB products at the infection site. Appropriate amounts of nanoliposomes were selected for subsequent research of cytotoxicity data based on the MTT assay. This test was carried out to assess increasing concentrations (from 0 to 50.00 mg/ml) of samples compared to untreated nanoliposomes. As shown in Fig. 5, the human macrophage cell line exhibited a gradual proliferation reduction with increasing HRZ/siTGF-β1 nanoliposomes compared with untreated cells, and the IC50 value for nanoliposomes in macrophages was 37.47 mg/ml. Therefore, 35.00 and 40.00 mg/ml nanoliposomes were selected for subsequent experiments. Since nanoliposome components are considered safe or lowly cytotoxic at the levels assessed, the concentration-dependent cytotoxic effects of loaded HRZ/siTGF-β1 nanoliposomes were likely due to significant and heterogeneous particle aggregation decreasing cellular activity and promoting cell death [44]. This phenomenon could be ascribed to the toxicity imparted by the positive surface charge of nanoliposomes [45].

Macrophages were exposed to different nanoliposomes containing HRZ, HRZ/siNC, and HRZ/siTGF-β1 (35.00 and 40.00 mg/ml) for 24 h. The percentages of cells in G1, S, and G2 are shown in Table 3; live, apoptotic (early and late), and necrotic cells were also quantitated (Table 4). The results exhibited that the percentage of G2 cells significantly increased from 21.26% to 38.54% after HRZ/siTGF-β1 (40.00 mg/ml) treatment compared to 17.83% and 17.90% for cells treated with HRZ and HRZ/siNC nanoliposomes, respectively (Table 3, Fig. 6), suggesting that HRZ/siTGF-β1 nanoliposomes (40.00 mg/ml) induced cell accumulation in the G2 phase of the cell cycle in human macrophages. However, no significant differences were detected upon treatment with HRZ/siTGF-β1 nanoliposomes (35.00 mg/ml) in other cell cycle phases.

Moreover, cells treated with HRZ/siTGF-β1 had a slightly higher apoptotic rate than untreated cells (Table 4, Fig. 7). At the same time, no apparent cytotoxicity was observed for HRZ and HRZ/siNC nanoliposomes, which might be due to the high uptake of siTGF-β1. These findings suggested that HRZ/siTGF-β1 nanoliposomes possessed adequate biocompatibility, although TGF-β1 siRNA conjugation slightly increased cytotoxicity.

Total mRNA and protein were obtained 24 h after transfection from human macrophages treated with or without HRZ/siTGF-β1 nanoliposomes to confirm the knockdown efficiency of siTGF-β1. Negative and blank control cells were treated with non-coding siRNA (siNC) and PBS. As shown in Fig. 8, TGF-β1 mRNA and protein amounts were markedly reduced in the HRZ/siTGF-β1 group compared with the negative and blank control groups. Although the resolution of the picture is not high, it does not affect the experimental results. These data indicated that siTGF-β1 successfully repressed TGF-β1 expression at the gene and protein levels.