2.1 Chemicals and reagents
Pluronic F127 was purchased from Sigma, China. SAHA was purchased from Nanjing Duolun Chemical Co., Ltd., China. Propidium Iodide (PI) was purchased from Santa Cruz Biotechnology. Acetonitrile, Dimethyl Sulfoxide (DMSO), Ammonium Persulfate, Sodium Chloride, Dodecyl Sodium Sulfate, Tween 20, Methanol, Ethanol, Isopropanol and Chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd., China. Phosphate Buffered Saline, Dulbecco's Modified Eagle Medium (DMEM), 1640 Medium, Trypsin were purchased from Solarbio. Fetal Bovine Serum was purchased from Corning. MTT, Glycine, Tris(hydroxymethyl)aminomethane, Acrylamide were purchased from Aladdin, China.
2.2 Preparation of SAHA-Pluronic F127 Nanoparticles
200 mg of Pluronic F127 and 3 mg of SAHA were dissolved into 10 ml of acetonitrile. Subsequently, the solvent was removed by rotary evaporation at 55℃ with decompression. The solid copolymer matrix obtained was then preheated at 65℃ for 1 h and eventually hydrated with phosphate buffer solution (PBS, 10mM or 150mM NaCl) or H2O. The nanomicellar solution was filtered with a 0.22 μm filter to remove any free drug. PI encapsulation was similarly achieved. 10 mg PI and 200 mg were dissolved in 10 ml of acetonitrile, and followed the same process explained above. The dispersion, size and zeta potential of nanomicelles were measured by dynamic light scattering (Particle size analyzer, Malvern, UK).
2.3 Atomic Force Microscope (AFM) characterization
10 μL of nanomicelles aliquots were spotted on mica substrates at a concentration of 100 μg/mL (Agar Scientific, UK) and dried at room temperature. Sample topography was obtained in air using a Bruker BioScope Catalyst (Bruker Instruments, Santa Barbara, California, USA) AFM. Bruker ScanAsyst-Air cantilevers were used, with a nominal spring constant of 0.4 N/m and a nominal resonant frequency of 70 kHz. All imaging was conducted using Peak Force Tapping (PFT) in ScanAsyst Mode. Images were processed with first-order flattening and planefit using Bruker Nanoscope Analysis 1.5. Gwyddion in-built grain analysis was used to identify nanomicelles and to calculate their size.
2.4 Nanomicelle stability
Nanoparticles were resuspended in either H2O, PBS (10mM NaCl) or PBS (150 mM NaCl) and stored at 4℃. In order to assess the stability of each formulation over time, size and poly-dispersion (PDI) measurements were taken at 0, 5, 10, 15, 20, 25 and 30 days.
2.5 Drug release assessment
A high performance liquid chromatography (HPLC) system (Waters 2535, Milford, MA, US) equipped with a photodiode array detector was used for the analysis of the drug release potential of the Pluronic formulations. A C18 HPLC column (GraceSmart RP C18, 4.6 mm × 250 mm, 5 μm) was used for quantitative analysis of SAHA. Mobile phase A contained HPLC grade H2O, and mobile phase B contained HPLC grade acetonitrile. SAHA was eluted with 50% mobile phase A and mobile phase B at a flow rate of 1 ml/min, with a retention time of 3.6 min and UV detection at 265 nm. Standard curves of concentration peaks and areas were drawn. Five-point calibration curves for SAHA in the range of 31.25–500 μM were considered reliable (r2≥ 0.999).
2.6 Determination of drug loading and entrapment efficiency.
200 μl of nanomicelle solution were added with 800 μl acetonitrile and centrifuged for 5 min at 10,225 x g. The supernatant was used to determine the concentration of drug by HPLC.
The entrapment efficiency (EE) and drug loading efficiency (DL) were calculated as follows:
2.7 In vitro drug release
In order to measure the release of SAHA from nanomicelles, a 20 ml solution containing SAHA loaded nanomicelles was loaded into a dialysis bag (MWCO: 8000~14,000 Da, Spectrum®, Rancho Dominquez, CA, USA), which was immersed in 500 ml of 10mM PBS (pH 7.4). Temperature was maintained at 37°C. At predetermined time intervals, 1 mL of release medium (PBS) was withdrawn and replaced with the same volume of fresh PBS into the system. The concentration of SAHA inside the solution was determined by HPLC.
2.8 Cell lines
HeLa (human epithelial cervical cancer) and MCF-7 (human breast adenocarcinoma) cell lines were kindly donated by Suzhou Institute of Nano-Tech and Nano-Bionics (SINANO), Chinese Academy of Sciences. HeLa cells were grown in DMEM and MCF-7 cells in RMPI. All media was supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) and 10% FBS at 37℃ in a humidified 5% CO2 and 95% air atmosphere.
2.9 Cell proliferation assay
The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x104cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China).
2.10 Protein blot
2.5x105 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).
2.11 Quantitative RT-PCR (qRT-PCR)
Hela and MCF-7 cells were treated with the SAHA and SAHA loaded nanomicelles for 24 h or 48 h. Total RNA was isolated using the RNAiso Plus kit (Takara, Japan). 10ug of total RNA was converted into complementary DNA (cDNA) with PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). SYBR Premix Ex TaqTMⅡ (Takara, Japan) solution was used according to manufacturer’s protocol to measure for mRNA expression of p53, p21, E-cadherin and N-cadherin with by qPCR. GAPDH was used as a control to determine relative mRNA expression. The table below shows the primer sequences used.
2.12 Cellular uptake of nanomicelles
1.5x104 HeLa and MCF-7 cells/well were seeded in 8-well chambered coverslips (Ibidi). After 24 h from seeding, cells were treated with 1 µM of PI loaded Pluronic F127 nanoparticles and incubated at 37oC in a humidified atmosphere. PBS was added as the untreated control. After 4 h, 24 h and 48 h cells were washed with 1X PBS and nuclei were counterstained with Hoechst 33342 (Life Technologies). 0.1% Triton X-100 was used as a positive control and added to cells for 10 min to permeabilize them, followed by 1 µM PI treatment for 10 min. Cells were imaged live on a Zeiss LSM710 fluorescent confocal microscope (Carl Zeiss Microscopy, Jena) at a 40X magnification using the 543 nm and 405 nm laser lines.
2.13 Cell migration assay
MCF7 and HeLa cells were seeded at a 5x105 cells/well concentration in 6-well plates. When cells reached 90% confluence scratches were performed with a 200µl sterile pipette tip and detached cells were washed in 1X PBS. Subsequently, empty nanomicelles, SAHA-nanomicelles and free SAHA were added to each well and the final volume was brought to 2 ml with FBS-free media. Images were acquired at 0 h, 24 h and 48 h using a Zeiss inverted microscope at a 4X magnification.
Reference marks on the bottom of each well along the scratches were made to align the same fields in each image acquisition, at each time point. Image analysis of the scratches was performed using the Wound Healing Size Tool, an ImageJ/Fiji® plugin that allows for the quantification of the wounded area (Suarez-Arnedo et al. 2020). The scratch area was calculated for each field and time point, and the percentage of wound closure was calculated according to the following formula:
Where At=0 is the area of a specific field at time 0h and At=Δt is the area of the same field after n hours of the initial scratch. The data was graphed and analyzed using GraphPad Prism. For the statistical analysis it was used one-way ANOVA with Dunnett’s multiple comparison test.
2.14 Statistical analysis
Data were expressed as mean ± standard deviation and analyzed using SPSS software. According to the distribution type of the data, the samples were processed by T-test and one-way ANOVA analysis with Dunnett’s multiple comparison test. A pvalue P<0.05 was considered statistically significant.