Supplies
DSPE-PEG2000, DSPE-PEG2000-FA, Ldl cholesterol, and DOTAP had been bought from Ruixibio Co., Ltd. (Xi’an, China). Iron oxide nanoparticles with oleic acid (OA-Fe3O4) (Product Quantity: SOR10-02, 25 mg/mL, 5 nm) had been bought from Shanghai Sunr Biotech Co., Ltd (Shanghai, China), the preparation of Iron oxide nanoparticles by thermal decomposition technique. Iron Oxide Nanoparticles in Chloroform have been coated with oleic acid for particle stability, and the oleic acid molecules’ hydrophobic tail teams (hydrocarbon chains) line as much as type a shell across the nanoparticle. PFP was bought from Chongqing Zihai (Chongqing, China). Reagents corresponding to trichloromethane (CHCl3) and others of analytical grade had been purchased from Chongqing Chuandong Chemical substances (Chongqing, China). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s Medium (DMEM), and Opti MEM had been obtained from GIBCO BRL (Grand Island, USA). Agarose, Cell Counting Equipment 8, 4′, 6-Diamidino-2-phenylindole (DAPI), trypsin, Annexin V-FITC/PI apoptosis detection equipment, phosphate-buffered saline (PBS), and serum-free cell freezing medium had been obtained from Biosharp (Anhui, China). Lipofectamine RNAiMAX Reagent and carboxyfluorescein succinimidyl ester (CFSE) had been bought from Thermo Fisher Scientific (Thermo Fisher, MA, USA). Recombinant Interleukin-4(IL-4) was bought from PeproTech (USA). Trizol, primescript RT equipment (036A), and SYBR Inexperienced PCR Grasp Combine had been bought from Takara (Shiga, Japan). DNase I, collagenase IV, hyaluronidase, and RBC lysis buffer had been bought from Solrabio (Beijing, China). The three double-stranded SIRPα siRNA and Cy5-NC siRNA used to knock down SIRPα gene expression within the examine had been obtained from Genepharma (Shanghai, China). The sequence of SIRPα siRNA is proven in Desk S1. The circulate cytometry antibodies used on this examine are listed in Desk S2, and the antibodies concerned within the Western Blotting assay are proven in Desk S3. Primers had been obtained from ZerKcorp Biotech (Xi’an, China), and their particular sequences may be present in Desk S4.
Screening of siRNA focused SIRPα
To generate M2-like macrophages, RAW 264.7 cells had been incubated in DMEM containing 20 ng/mL of IL-4 in a single day. Particular siRNAs had been designed to focus on SIRPα for silencing. Western blotting was carried out to detect the impact of SIRPα gene silencing. Focused siRNAs had been transfected into M2-like macrophages utilizing Lipofectamine RNAiMAX transfection reagent. M2-like macrophages had been divided into 4 teams: the experimental group was transfected with siRNA focusing on SIRPα (SIRPα-1, SIRPα-2, and SIRPα-3), and the management group was not transfected with siRNAs.
Synthesis of FA-PFNB-SIRPα siRNA
FA-PFNB was synthesized utilizing a typical skinny movie hydration technique [32]. Initially, DOTAP, ldl cholesterol, and DSPE-PEG2000-FA had been dissolved in 6 mL of trichloromethane in a round-bottom flask with a molar ratio of three:1:1. Concurrently, OA-Fe3O4 nanoparticles had been dissolved in CHCl3 in a transparent glass bottle and mixed with the lipid resolution at 25% of the lipid weight. The combined resolution was evaporated in a round-bottom flask utilizing rotary evaporation to create a skinny lipid movie. Subsequently, 4 mL of PBS was launched, and the answer was sonicated for 1 h at 60 °C. After including 200 µL of PFP, the suspension was emulsified utilizing a sonicator (Sonics and Supplies Inc., USA) at 125 W energy in an ice-water tub. Sonication was carried out for 8-min intervals of 5 s on and off. To take away extra reactants and free lipids, the emulsion was centrifuged at 8,000 rpm for 10 min at 4 °C, with this step repeated thrice for purification. The ensuing vesicles had been extruded by a 200-nm membrane and purified by way of dialysis.
To finalize the binding of siRNA to FA-PFNB (FA@PFP@Fe3O4@LNBs), the positively charged FA-PFNB was merged with the siRNA resolution in enzyme-free water and incubated at room temperature for 10 min. The ensuing ready samples had been denoted as FA-PFNB-siRNA. Inside this designation, FA-PFNB-SIRPα siRNA recognized mixtures with SIRPα siRNA, whereas FA-PFNB-NC siRNA, serving because the management group, represented bindings with NC siRNA.
Characterization
Dynamic gentle scattering (DLS) (Nano-ZS, Malvern, UK) was used to detect Particle measurement and floor potential, together with PFP@LNBs (PNB), PFP@Fe3O4 @LNBs-NC siRNA (PFNB-NC siRNA), PFP@LNBs-SIRP α siRNA (PNB-SIRPα siRNA), FA@ PFP@Fe3O4@LNBs (FA-PFNB), and FA@ PFP@Fe3O4 @LNBs-SIRPα siRNA (FA-PFNB-SIRPα siRNA). To guage the steadiness of FA-PFNB-SIRPα siRNA nanocarriers, the hydromechanical particle measurement of the nanocomposites was measured at 1, 3, 5, 7, and 10 days, respectively. Droplets of the diluted nanoparticle resolution had been added to the copper mesh and silicon wafer and dried at room temperature. The interior construction and morphology of FA-PFNB-SIRPα siRNA had been obtained by Transmission electron microscope (TEM) (H-7600; Hitachi Ltd., Tokyo, Japan). The Fe focus was analyzed with an atomic absorption spectrometer (Hitachi mannequin Z-5000, Hitachi Ltd., Tokyo, Japan). The encapsulation effectivity and loading capability of Fe3O4 had been decided utilizing the next equation:
$${textual content{Encapsulation effectivity}}left(%proper)={textual content{mass of }}frac{{{textual content{Fe}}_{3}{textual content{O}}_{4}}}{{textual content{complete}}}{textual content{Fe}}_{3}{textual content{O}}_{4}instances 100%;$$
$${textual content{Loading content material }}left(%proper)={textual content {mass of} {textual content{Fe}}}_{3}{textual content{O}}_{4}/textual content{complete liposomes}instances 100%.$$
Gel retardation assay
Agarose gel electrophoresis was used to check the SIRPα siRNA-carrying capability of FA-PFNB nanoparticles [33]. First, SIRPα siRNA was incubated with FA-PFNB at mass ratios of 1:0, 1:1, 1:2, 1:4, 1:8, 1:16, 1:20, and 1:25 for 10 min at 25°C temperature. After that, add 6 × loading buffer to the incubated combination. Utilizing 3% agarose gel at a voltage of 100 V, the pattern was subjected to electrophoresis for 30 min. After electrophoresis, the gel was positioned within the gel imaging system to watch the band adjustments of SIRPα siRNA.
In vitro CEUS imaging
To discover the section transition dynamics of PNB-SIRPα siRNA and the operate of PFP inside this course of, PNB-SIRPα siRNA nano/microbubbles and nano/microparticles loaded with SIRPα siRNA underwent LIFU publicity at different acoustic intensities (starting from 0 to 1.5 W/cm2, 50% responsibility cycle). The irradiation lasted completely different durations from 1 to 4 min. B-mode and contrast-enhanced ultrasound (CEUS) modes had been utilized for picture seize, and pre-irradiation photographs had been in contrast with them as controls. Picture evaluation and analysis had been carried out utilizing ultrasound imaging software program (FDY-II; Institute of Ultrasound Imaging, Chongqing, China). Optical microscopy (DMIL LED Fluo, Leica, Germany) underneath the optimum ultrasound parameters allowed for the visualization and detection of the section transition of PNB-SIRPα siRNA.
Cells and animals
The RAW264.7 cell line (representing M0-like macrophages) and Lewis’s lung most cancers (LLC) cell line used on this examine had been obtained from Procell Life Science & Know-how Co., Ltd. (Wuhan, China). LLC cells had been cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. The cells had been incubated in a humidified ambiance at 37 °C with 5% CO2. C57BL/6 feminine mice aged 5–6 weeks had been procured from Lanzhou Veterinary Analysis Institute, Chinese language Academy of Agricultural Sciences Co. Ltd. (Lanzhou, China). All mouse experiments had been carried out per the accepted tips by the Ethics Committee of Lanzhou College Second Hospital.
Cell viability assays
The Cell Counting Equipment-8 (CCK-8) assay was used to judge completely different concentrations and teams of NBs biocompatibility with RAW264.7 cells and assess the cytotoxicity of various ultrasound parameters on RAW264.7 and LLC cells [34]. Various concentrations (25–400 µg/mL) and different teams of NBs had been launched into the tradition medium of RAW264.7 cells, and the cells had been additional incubated for an extra 24 h. RAW264.7 cell viability assays had been then carried out. RAW264.7 and LLC cells had been subjected to a variety of ultrasound energy (0–1.5 W/cm2) for 1 to 4 min. Following a 24-h incubation, cell viability was assessed utilizing the CCK-8 equipment. Subsequently, the PNB-SIRPα siRNA and FA-PFNB-SIRPα siRNA teams had been uncovered to various ultrasound parameters on the specified concentrations, adopted by the analysis of cell viability.
Analysis of transfection effectivity of NBs loading PNB-SIRPα siRNA
To find out the optimum gene transfection parameters utilizing LIFU, varied components, corresponding to ultrasound depth and period of irradiation had been investigated. The PNB-SIRPα siRNA was incubated with cells for 10 min in a cell incubator earlier than publicity to LIFU at completely different ultrasound parameters (0–1.5 W/cm2, responsibility cycle of fifty%). The period of ultrasound publicity different from 1 to 4 min. Earlier than ultrasound remedy, the M2-like macrophages had been cultured for an extra 6 h, after which the medium was changed earlier than persevering with the tradition for an additional 24 h. Transfection effectivity was evaluated within the cells by circulate cytometry (CytoFLEX, Beckman Coulter, USA) evaluation utilizing Cy5-NC/SIRPα siRNA.
Mobile uptake/transfection
M2-like macrophages (2 × 106 cells/pores) had been plated onto confocal dishes and incubated at 37 °C. Varied media (PBS, free Cy5-SIRPα siRNA [120 nM], Lipo-Cy5-SIRPα siRNA, PNB-Cy5-SIRPα siRNA, PFNB-Cy5-NC siRNA, FA-PFNB-Cy5-SIRPα siRNA, and FA-PFNB-Cy5-SIRPα siRNA + US [200 µg/mL, Cy5-SIRPα siRNA: 120 nM]) had been added to the dishes and cells had been cultured for twenty-four h. The ultrasonic remedy course of remained unchanged as described earlier for FA-PFNB-Cy5-SIRPα siRNA + US. Following incubation, cell nuclei had been stained with DAPI, and pictures had been captured utilizing a laser confocal scanner [35].
M2-like macrophages (2 × 106 cells/pores) had been ready. Totally different medium circumstances had been used (PBS, free Cy5-SIRPα siRNA [120 nM], Lipo-Cy5-SIRPα siRNA [120 nM], PNB -Cy5-SIRPα siRNA, PFNB-Cy5-SIRPα siRNA, FA-PFNB-Cy5-SIRPα siRNA and FA-PFNB-Cy5-SIRPα siRNA + US [200 µg/mL, Cy5-SIRPα siRNA: 120 nM]), the ultrasonic remedy technique is similar as above. After 24 h of tradition, the cells had been examined for nanomaterial uptake by circulate cytometry, and the outcomes had been analyzed utilizing FlowJo software program.
Downregulation of SIRPα gene expression after remedy
M2-like macrophages (2 × 106 cells/properly) had been plated and cultured in a single day. Varied media formulations had been then added to the cells: PBS, Lipo-SIRPα siRNA, PNB-SIRPα siRNA, FA-PNB-SIRPα siRNA, FA-PFNB-SIRPα siRNA, and FA-PFNB-SIRPα siRNA + US (200 µg/mL, SIRPα siRNA: 120 nM). Lipofectamine RNAiMAX Reagent was used as a constructive management for SIRPα siRNA supply. The ultrasonic remedy process remained constant, as described earlier. After an extra 48-h incubation, a Western blot evaluation was carried out to judge the degrees of SIRPα protein expression.
Analysis of the repolarization of M2-like macrophages
M2-like macrophages had been cultured with various concentrations of PFNB-NC siRNA for twenty-four h. Subsequently, the macrophages had been washed thrice. The M2-like macrophage samples had been instantly collected and stained by fluorescence-labeled antibodies, i.e., PE-CD206 and FITC-CD86. The samples had been run on a circulate cytometer [36].
M2-like macrophages (2 × 106 cells/pores) had been inoculated into 6-well plates and acquired completely different remedies (PBS, PNB-SIRPα siRNA, PFNB-SIRPα siRNA, FA-PFNB -SIRPα siRNA and FA-PFNB-SIRPα siRNA + US, (200 µg/mL, different teams with the equal Fe3O4 or SIRPα siRNA (120 nM) content material) primarily based on the LIFU (1W/cm2, 2min). After 24 h, the cells had been stained with fluorescence-labeled antibodies, i.e., PE-CD206 and FITC-CD86 for fluorescence commentary. Equally, the quantized fluorescence depth was additionally detected by circulate cytometry.
Macrophage-associated cytokine secretion evaluation
M2-like macrophages (2 × 106 cells/properly) had been handled with completely different formulations within the tradition medium, together with PBS, PNB-SIRPα siRNA, PFNB-NC siRNA, PFNB-SIRPα siRNA, FA-PFNB-SIRPα siRNA, and FA-PFNB-SIRPα siRNA + US. They had been cultured at 37 °C for twenty-four h to acquire handled macrophages. RT-PCR evaluation was carried out to evaluate Interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α) and Interleukin-10(IL-10) expression ranges [37].
Phagocytosis assay
M2-like macrophages (2 × 106 cells/properly) had been pretreated with varied substances: PBS, PNB-SIRPα siRNA, PFNB-NC siRNA, PFNB-SIRPα siRNA, FA-PFNB-SIRPα siRNA, and FA-PFNB-SIRPα siRNA + US (200 µg/mL). Moreover, all teams had been uncovered to equal Fe3O4 or SIRPα siRNA (120 nM) for 48 h. Subsequently, CFSE-labeled LLC cells (5 × 105) had been added to the macrophage-containing plate at a 4:1 cell ratio. Following a 6-h incubation, the macrophages had been stained with APC-F4/80, and the ratio of double-positive cells (CFSE/APC) inside the complete macrophage inhabitants (APC) was decided utilizing circulate cytometry [38].
Apoptosis assays
M2-like macrophages had been handled with a special medium situation for 48 h (PBS, PNB-SIRPα siRNA, PFNB-NC siRNA, PFNB-SIRPα siRNA, FA-PFNB -SIRPα siRNA and FA-PFNB-SIRPα siRNA + US). 5 × 105 LCC cells had been suspended in 500 µL of M2 conditioned medium in numerous circumstances with out FBS. Cell apoptosis was evaluated utilizing the Annexin V-FITC Apoptosis Detection Equipment I [39].
Migration and invasion experiment
Cell Migration and invasion exams had been carried out utilizing a 24-well Transwell (8 µm aperture) plate precoated with matrix glue [40]. 5 × 105 LLC cells had been suspended in M2 conditioned medium (PBS, PNB-SIRPα siRNA, PFNB-NC siRNA, PFNB-SIRPα siRNA, FA-PFNB -SIRPα siRNA and FA-PFNB-SIRPα siRNA + US) with completely different circumstances with out FBS and added to the higher chamber, and 600 µL RPMI 1640 containing 20% FBS was added to the decrease chamber. After a 24-h tradition, the matrix glue and residual cells within the higher chamber had been eliminated utilizing a cotton swab. Matrix glue was used within the higher layer of the invasion assay, and no matrix glue was used for migration experiments. The cells beneath the membrane had been mounted with a 4% paraformaldehyde fixative for 30 min, adopted by staining with 0.5% crystal violet for 10 min and subsequent washing thrice. Lastly, the cells passing by the membrane are noticed with an optical microscope, counted, and photographed.
Cell proliferation inhibition experiment
LLC cells had been inoculated into 6-well plates at a density of three × 103 cells per properly and cultured in a single day at 37 °C. Totally different circumstances of M2-like macrophages conditioned medium (PBS, PNB-SIRPα siRNA, PFNB-NC siRNA, PFNB-SIRPα siRNA, FA-PFNB -SIRPα siRNA, and FA-PFNB-SIRPα siRNA + US) had been used, and the medium was modified each two days. After 7 days of remedy, the cells had been mounted with 4% paraformaldehyde and stained with 0.5% crystal violet. The mounted and stained cells had been then evaluated and documented utilizing organic microscopy to find out the proliferation standing of the completely different cell teams [41].
In vivo fluorescence imaging
To determine the NSCLC tumor-bearing mouse mannequin, 5 × 107 LLC cells in 200 µL of PBS had been injected subcutaneously into the correct thigh of feminine C57BL/6 mice (6–8 weeks outdated). When the tumor quantity of the mice grew to roughly 800 mm3, they had been intravenously administrated with PFNB-Cy5-SIRPα siRNA, FA-PFNB-Cy5-SIRPα siRNA by way of tail vein (injection dose of NBs: 200 μL, 3 mg/mL). At 1, 2, 4, 8, and 24 h, the fluorescence biodistribution of Cy5 within the tumor was analyzed. Within the FA-PFNB-Cy5-SIRPα siRNA group, the best fluorescence aggregation time was given to the LIFU stimulation (1 W/cm2, 2 min, 50% responsibility cycle) to watch the in vivo imaging impact. On the peak of fluorescence depth, the mice had been euthanized, and organs, together with the guts, liver, spleen, lung, kidney, and tumors, had been harvested. These tissues underwent ex vivo imaging for Cy5 fluorescence utilizing the IVIS Spectrum Imaging System (VISQUE Invivo Good-LF) [42].
In vitro and in vivo PA imaging
To find out the optimum excitation wavelength, we used FA-PFNB-SIRPα siRNA (200 µg/ml, 10 µL) for steady photoacoustic imaging (Vevo LAZR Photoacoustic Imaging System) (VisualSonics Inc., Toronto, Canada) of the nanomaterials throughout the excitation wavelength spectrum of 680–970 nm) [43]. When the tumor quantity of the mice grew to roughly 800 mm3, the mannequin rats had been randomly divided into three teams for PA imaging: PFNB-SIRPα siRNA, FA-PFNB-SIRPα siRNA, and FA-PFNB-SIRPα siRNA + US. The medicine had been administered to the teams by way of tail vein injections (injection dose of NBs: 200 μL, 3 mg/mL). The FA-PFNB-SIRPα siRNA + US group underwent LIFU remedy for two min with 1 W/cm2 in 2 h after administering the drug. PA photographs had been acquired after injection at 1 h, 2 h, 4 h, 8 h and 24 h.
In vivo US imaging
The impact of PFNB-Cy5-SIRPα siRNA, FA-PFNB-Cy5-SIRPα siRNA nanocomplexes on in vivo CEUS imaging was examined utilizing an LLC subcutaneous tumor mannequin. When the tumor quantity of mice grew to roughly 800 mm3, CEUS imaging was carried out by intravenous injection of nanocomplex resolution [44]. The drug was injected by the tail vein and acquired completely different remedies (injection dose of NBs: 200 μL, 3 mg/mL). LIFU stimulation was given 2 h after the tail vein injection, and additional contrast-enhanced ultrasound was carried out. The B-mode and CEUS photographs had been captured earlier than and after LIFU irradiation, and the depth of the Area of Curiosity (ROI) was subsequently in contrast.
In vivo mixture most cancers remedy
The NSCLC tumor-bearing C57BL/6 mice mannequin (roughly 200 mm3) was randomly divided into seven teams and handled with varied interventions (injection quantity: 200 µL, PBS: 10 mM; PNB -SIRPα siRNA, PFNB–NC siRNA, PFNB -SIRPα siRNA, FA-PFNB-SIRPα siRNA, FA-PFNB-SIRPα siRNA + US, and (PNB-SIRPα siRNA + PFNB-NC siRNA) (injection dose of NBs: 200 μL, 3 mg/mL). Tumor quantity was monitored utilizing a vernier caliper. Survival evaluation was carried out on the mice, and steady commentary was carried out till the thirty fifth day. Along with the spontaneous demise of the mice, when the tumor quantity reached 2000 mm3, or the size of the tumor was > 20 mm, it is also thought-about the demise endpoint.
Intertumoral lymphocyte evaluation
On the 14th day after the remedy, the mice in every group had been euthanized, and tumor tissues and draining lymph nodes had been extracted to organize a single-cell suspension for analyzing the tumor immune microenvironment [45]. To evaluate SIRPα expression on the macrophage floor, macrophages had been labeled with anti-F4/80-APC and anti-SIRPα-FITC antibodies for circulate cytometric evaluation. To investigate cytotoxic T lymphocytes (CTLs, CD3CD4, CD3CD8), T lymphocytes had been stained with anti-CD3-PE, anti-CD4-FITC, and anti-CD8-FITC antibodies. For assessing T cell activation and proliferation frequency, lymphocytes had been stained with anti-CD4-FITC, anti-CD8-FITC, anti-CD69-PE, anti-IFN-γ-PE, and anti-ki67-PE antibodies. The frequency of regulatory T cells (Tregs) (CD4 Foxp3) was analyzed by staining lymphocytes with anti-CD4-FITC and anti-Foxp3-PE antibodies. M2-like macrophages (F4/80CD206) and M1 macrophages (F4/80CD86) in tumor tissue cell suspensions had been recognized utilizing anti-F4/80-APC, anti-CD86-FITC, and anti-CD206-PE antibodies by circulate cytometric evaluation. TNF-α and IFN-γ ranges in tumor tissues had been decided utilizing enzyme-linked immunosorbent assay (ELISA). Subsequently, the frequency of M1-like macrophages (F4/80CD86), CTLs (CD3CD4, CD3CD8), and T cell activation and proliferation (CD4 IFN-γ, CD8 IFN-γ, CD8Ki67) within the tumor-draining lymph nodes (TDLNs) was examined utilizing circulate cytometric evaluation [36, 46].
Detection of apoptosis in tumor tissues
TUNEL staining was carried out to detect post-treatment apoptosis in tumor tissue. The stained sections had been examined underneath a fluorescence microscope (Olympus, Japan) to determine apoptosis. The apoptotic index was decided and analyzed in 5 randomly chosen samples. Moreover, tumor tissues had been stained with hematoxylin and eosin (H&E) to evaluate the pathological alterations [47].
Transcriptomic evaluation
The NSCLC tumor-bearing mice had been divided into PBS and FA-PFNB-SIRPα siRNA + US teams. After remedy completion, tumor tissues had been collected for transcriptome sequencing evaluation [48].
In vitro assays for immune cell stimulation
Main murine spleen-derived lymphocytes (PBMC) had been remoted based on beforehand described protocols [49]. The collected major lymphocytes had been added to the three teams with completely different remedies: PBMC + LLC + M0 group, PBMC + LLC + M2(PBS) group, PBMC + LLC + M2(FA-PFNB-SIRPα siRNA + US). The cells had been co-cultured for twenty-four h, and lymphocytes had been stained with anti-CD8-FITC, anti-CD69-PE, anti-CD4-FITC, and anti-CD69-PE, in addition to anti-CD8-FITC and anti-Ki67-PE to evaluate lymphocyte activation and proliferation by way of circulate cytometry. Moreover, supernatants from stimulated lymphocytes had been collected and used to tradition LLC cells for twenty-four h. Following incubation, LLC cell viability and apoptotic price had been assessed utilizing the CCK-8 assay and an apoptosis equipment.
Toxicity evaluation
A histopathological analysis of significant organs was carried out to evaluate potential toxicity from varied remedies. Coronary heart, liver, spleen, lung, and kidney specimens had been harvested from every group of mice, mounted in paraffin, and sectioned for hematoxylin and eosin (H&E) staining. Observations and evaluations of the stained sections had been carried out utilizing a light-weight microscope. Blood samples had been collected from the mice for routine exams. Following in a single day refrigeration at 4 °C, serum samples had been obtained by centrifugation and analyzed for key serological biochemical indicators utilizing an automatic analyzer (Hitachi Excessive-Applied sciences Corp., Japan).
Statistical evaluation
Statistical evaluation was carried out utilizing GraphPad Prism 7 (GraphPad, San Diego, USA). Scholar’s t-test and one-way evaluation of variance (ANOVA) had been employed for knowledge evaluation. Survival analyses had been carried out utilizing the Kaplan–Meier technique with a log-rank take a look at. All statistical outcomes are offered as imply ± normal error of measurement. Statistical significance was outlined as a P-value < 0.05 (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
