Hyaluronic acid (MW 130 kDa) was purchased from LifeCore Biomedical (Chaska, USA). 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), DL-Dithiothreitol (DTT), Cysteamine hydrochloride, 2,2′-Dithiodipyridine, 4-Nitrophenyl chloroformate were purchased from Sigma-Aldrich. Dialysis membranes used for purification were purchased from Spectra Por-6 (MWCO 3500). All solvents were of analytical quality. Nuclear Magnetic Resonance (NMR) spectroscopy experiments were performed with a Varian Mercury 300 MHz NMR Spectrometer (Palo Alto, USA). All spectrophotometric analysis was carried out on Shimadzu UV-3600 plus UV-VIS-NIR spectrophotometer.
2D Cell culture
Both Ishikawa (ECACC 99040201 Public Health England, UK) a type I endometrial cancer (TIEC) model and Hec-50B (Cat-No 1145, JCRB Cell Bank, Japan) a type II endometrial cancer (TIIEC) model , were maintained in DMEM:F-12 (1:1) + GlutaMax™ full media (Cat-No 31331-028, Gibco, ThermoFisher Scientific, UK) supplemented with 10% foetal bovine serum (FBS), sodium bicarbonate 1 mM, sodium pyruvate 1 mM and 1% antibiotic-antimycotic solution in plastic culture flasks at 37 °C and 5% CO2 incubator (Nuaire). Cells were supplemented with full serum media every 2 days and passaged when confluent.
3D cell culture
Spheroids were prepared for viability experiments using an ultra-low attachment 96 well plate . 4 × 103 cells per well were added to a 96-well Ultra-Low Attachment surface (ULA) microplate (Corning™ 4520) supplemented with stripped serum media. After 48 h treatment incubation, CellTiter-Glo® 3D Cell Viability Assay reagents (Promega) were added following manufacturer’s instructions and luminescence recorded using a microplate reader (FLUOstar Omega) at RT. Spheroids for protein expression were prepared using the liquid overlay technique in clear 96 well plates . Briefly, 96 well plates were conditioned for spheroid seeding by coating the bottom of each well with 2% agarose. The agarose was left to set for 1 h in the flow hood under ultraviolet (UV) light to ensure sterility. 6 × 104 cells/mL were used to form the spheroids for western blot experiments. Spheroid morphology following treatments was assessed using Image J (FIJI). Images were taken using a Zeiss microscope at 40X objective with scale bar exported from the proprietary Zen software (Carl Zeiss Ltd, UK). Scale bar consistent exported images were analyzed in Image J (FijiLOCI, University of Wisconsin, USA), setting measurements to include area, perimeter and shape descriptors using the particle analyzer function.
Synthesis of thiolated HA (HA-SH)
HA-SH conjugates were synthesized by coupling dithiobis (propanoic hydrazide) (DTPH) using carbodiimide chemistry to the carboxylic groups of HA and subsequently reducing the disulphide bond using dithiothreitol (DTT). Briefly, 1 equivalent HA (400 mg, 1 mmol with respect to the disaccharide repeat units) was dissolved in 120 ml of deionized water followed by the addition of 1 equivalent 3,3’-dithiobis (propanoic hydrazide) (DTPH 238.3 mg, 1 mmol) and 1 equivalent HOBt (153 mg, 1 mmol) and stirred at room temperature until the reaction becomes homogeneous. Thereafter, the pH of the reaction mixture was adjusted to 4.7 by careful addition of 1 M NaOH and 1 M HCl. Finally, 0.25 mmol EDC·HCl (48 mg, 0.25 equivalent) was added and allowed to stir overnight. Then the reaction mixture was loaded into a dialysis bag (Spectra Por-6, MWCO 3500 g/ mol) and dialyzed against dilute HCl (pH = 3.5) containing 100 mM NaCl (4 × 2L, 48 h) and then dialyzed against deionized water (2 × 2L, 24 h). The solution was lyophilized to obtained as white fluffy material. The degree of hydrazide modification was estimated to be 10.5% (with respect to the disaccharide repeat units) as determined by trinitrobenzene sulfonic acid (TNBS) assay by measuring absorbance at 500 nm, using UV spectroscopy .
In the second step, the lyophilized HA-DTPH derivative was dissolved in 100 ml deionized water and pH of the solution was adjusted to 9 with 1 M NaOH. Subsequently, dl-Dithiothreitol (DTT, 124 mg, 0.8 mmol) was added dropwise to the solution. The mixture was stirred overnight, after which the solution was transferred to a dialysis bag (Spectra Por-6, MWCO 3500 g/ mol) and dialyzed against deionized water (3 × 2L, 24 h). The dialyzed solution was lyophilized to give white fluffy thiol-modified HA (HA-SH). The concentration of thiol groups was estimated to be 7% (with respect to the disaccharide unit) by Ellman's assay using the extinction coefficient of 14 150 M− 1cm− 1 at 412 nm using UV spectroscopy .
Synthesis of thiol-terminated Pluronic® F-127 (NP-S-S-Py)
Activation of Pluronic F127
The synthesis of pluronic F127 functionalized with terminal disulphide pyridyl was performed following the reported procedure . Briefly, the terminal PEG diols of pluronic F127 was activated using 4-nitrochloroformate. The degree of activation was found to be 78%, determined spectrophotometrically by measuring the amounts of 4-nitrophenolate ions released in the alkaline solution by measuring the absorbance at 402 nm using a molar extinction coefficient of 18400 cm− 1M− 1.
Synthesis of pyridyl disulphide ligand (2-(2-pyridyldithio) ethylamine)
Cysteamine hydrochloride (2.288 gm, 20.1408 mmol) was dissolved in methanol (17.5 ml) followed by the addition of glacial acetic acid (1.6 ml). Above solution was then dropwise added to a stirred solution of 2,2-dithiopyridine (8.815 gm, 40.2817 mmol) in methanol (41.6 ml). Reaction mixture was stirred for 48 h at room temperature and product was precipitated from stirred diethyl ether (200 ml). The product was dissolved in a small volume of methanol and was again precipitated by diethyl ether to afford white solid compound. The 1H NMR of the pyridyl disulphide product was consistent with the reported data .
Synthesis of F127 pyridyl disulphide derivative (NP-S-S-Py)
Activated pluronic F127 (1 gm, 0.069 mmol) dissolved in 10 ml DCM was reacted with extracted disulphide ligand (154 mg, 0.69 mmol) dissolved in 1 ml DCM. The reaction mixture was refluxed overnight. It was then concentrated and diluted with methanol:water (1:1, v/v, 10 ml) and dialyzed (membrane MWCO 3500 Da) against 2 L deionized water for two days. The product was obtained as white fluffy material after lyophilization. The modification was determined using UV (343 nm, molar extinction coefficient of 8060 cm− 1M− 1). The UV absorbance of product (1 mg/ml in PBS buffer at pH 9) was measured before as well as 10 min after the addition of 0.1 ml of DTT (15 mg/1000 ml in PBS at pH 9). The percentage modification was found to be 78%.
Pluronic micelle fabrication
Pluronic® F-127 (NP) and thiol-terminated Pluronic® F-127 (NP-S-S) micelles were fabricated using thin-film hydration method described by Caldwell et al. The polymer constituent and 10% SAHA (Cayman Chemical Company, 10009929) / propidium iodide (Sigma-Aldrich, P4170-10MG) were dissolved in acetonitrile (ACN). The acetonitrile was removed by rotary evaporation at 65 °C and under 226 mbar for 1 h, and the flask placed into a desiccator under vacuum overnight, to ensure the film was void of all moisture and residual copolymer matrix acetonitrile removed. Prior to hydration, samples were heated to 65 °C in a water bath under mild rotation for 1 h until the sample resembled a viscous thin film coating the flask. NPs were hydrated in deionized water and returned to heated rotation for 30 min to ensure maximum micelle formation and drug loading. HA-thiol was added in excess (2:1) to NP-S-S and mixed using a magnetic stirrer at 700 rpm for 1 h to form NP-HA; prior to use HA-thiol was sterilized under UV for 1 h. An 0.22 µm filter (Millipore) was applied on all samples – except NP-HA samples – to remove non-encapsulated drugs agglomeration.
Size and Zeta potential analysis with dynamic light scattering
Polymeric micelle size and zeta potential were determined using Zetasizer Nano ZS™ (Malvern, UK), at a concentration of 100 µg/ml. A series of data indicating size, zeta- average, polydispersity index was generated. All data is presented as average and standard deviation, from a minimum of three independent biological repeats.
Atomic Force Microscope (AFM)
10 µL nanoparticle aliquots were spotted on mica substrates at a concentration of 100 µg/mL (AGG250-1, Agar Scientific, UK) and dried at RT. 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, the height of the particles was calculated using freeware AFM software WsXM 5.0 .
An Agilent detector equipped with autosampler and a C18 reversed-phase column (Xbridge C18, 130 Å, 3.5 µm, 2.1 mm x150 mm) was ran isocratically at room temperature (RT). All samples were diluted using acetonitrile and loaded into the 0.3 mL polypropylene, 9 mm thread, screw thread vials (Sigma, 29377-U); mobile phase throughout analysis was 0.1% formic acid:acetonitrile 78:22. A flow rate of 0.25 µL/min and sample injection volume of 2 µL gave a 7 min analyte retention time. Following elution from the column, the sample was run through the UV/vis detector recording at 241 nm. Nanoparticle samples were diluted 1:10, 1:100 and 1:200 to ensure measurements were occurring within the range of the standard curve. Encapsulation efficiency was determined using the equation below (1).
Drug release profile
Cellulose dialysis sacks with a molecular weight cut-off pore size of 12 kDa (Sigma Aldrich, D6191-25EA) were used to monitor drug release profiles over 80 h. Dialysis sacks were soaked in PBS buffer overnight, 1.5 mL of sample was loaded into the cellulose tubing and placed in a beaker containing 20 mL of PBS and a small magnetic fly. The buffer was mixed continually throughout the experiment by stirring at 300 rpm. For the first 2 h of dialysis, 1 mL samples were taken at 30 min intervals, for the remainder of the first 48 h samples were taken every 60 min. Following each sampling, 1 mL of fresh PBS was reintroduced to maintain a total volume of 20 mL. Samples were diluted in ACN to dissociate polymer micelles and frozen until analysis.
2D / 3D Cell viability assays
Cell viability assays were performed in 2D using Realtime-Glo™ MT Cell Viability RT-Glo (Promega UK, G9711) and in 3D using Celltiter-Glo 3D (Promega UK, G9682). Both methods were performed according to the manufacturer’s instructions [94, 95]. Readings were taken using a FLUOstar Omega microplate reader at 0-, 24-, 48- and 72 h time points. For 3D experiments, only 48 h and 72 h timepoint was investigated. The spectrophotometer was heated to 37 °C prior to readings. All SAHA-NP preparations were compared to free drug SAHA and all data was normalized against the control cell line without any treatment.
Protein blotting and antibodies
Total protein lysates (20 µg) were resolved on a precast 4–20% polyacrylamide gels (mini-PROTEAN® TGX stain-free™ gels, 456–8094), transferred and immobilized onto polyvinylidene fluoride (PVDF) membranes (mini format Trans-Blot® Turbo™ transfer pack, 170–4156), incubated for 60 min at room temperature in blocking solution (TRIS-buffered saline containing 5% BSA and 0.1% Tween 20), followed by an overnight incubation in primary antibodies at 4 °C at 1:1000 dilution. The following antibodies were used: Cell Signalling (Danvers, MA): p21 (#2947s; 1:1000), p53 (#9282s; 1:1000), Santa Cruz: GAPDH (# sc-47724; 1:1000). Abcam (Cambridge, MA): E-Cadherin (#1416; 1:1000), N-Cadherin (#18203; 1:1000), GE Healthcare Lifescience: Anti-Rabbit (#10794347, 1:2000), Anti-Mouse (#10094724, 1:2000). Membranes were then washed three times and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h at 1:2000 dilution.
Data were normalized against the respective cell line without any treatment. Statistical analyses used to in this study to validate results, unless stated otherwise, were pairwise student’s T-Tests and ANOVA where treatments were compared against the free drug only. Significance is indicated on graphs or tables, when appropriate in the following fashion: p ≤ 0.05 denoted by *, p ≤ 0.01 by **, p ≤ 0.001 by *** and p ≤ 0.0001 by ****.