Figure 1. Chemical structures of VG (1,1’-bis(4-aminophenyl)-[4,4’-bipyridine]-1,1’-diium), ALG (alginic acid), and the cucurbit[8]uril macrocycle (CB8).
The role of CB8 in triggering cellular internalization to white blood cancer cells is investigated. While it is not uncommon to see such systems at work in small organic molecules that change their cellular uptake under the influence of CB macrocycles,[23]using such systems for extended networked structures such as the present CB8-based alginate hydrogels (VGCB8ALG) is purely academic and should unfold more information.
METHODS
Samples.
All starting reagents cucurbit[8]uril (CB8), 1,1′-bis(4-aminophenyl)-[4,4′-bipyridine]-1,1′-diium dichloride (VG), alginic acid (ALG), 4-dimethylamino pyridine (DMAP), and N,N′-dicyclohexylcarbodiimide (DCC), as well as methanol and DMSO solvents, including the deuterated ones, were obtained from Sigma-Aldrich (St. Louis, MO) and used as received. All chemicals had purities exceeding 95%, except for CB8, which was assumed to contain 20% (w/w) water in the supplied bottles.
Preparation of VGALG and VGCB8ALG Conjugate .
ALG (0.41 g), DCC (∼0.08 g, 0.38 mmol), and DMAP (∼0.051 g, 0.42 mmol) were dissolved in dimethyl sulfoxide (DMSO), purged under nitrogen for one hour at 295 K (50 mL), and the solution was stirred for sixteen hours. A 1:1 mixture (0.032 mmol) of VG and CB8 was dissolved in DMSO/ water (20 mL, 1:1 v/v) in a separate flask with six hours of stirring under nitrogen at 298 K. The two solutions were mixed and stirred for two hours under nitrogen at 328 K and then purified by dialysis against water (pH 7) for several days to separate the solid conjugates from unreacted starting materials. The final suspension was centrifuged for eight hours, and the solid product was washed with water and freeze-dried for ten hours.
Fourier Transform Infrared (FTIR) Spectroscopy.
The FTIR spectra were recorded on a Perkin Elmer spectrometer, and the data were processed with Spectrum IR software. The solid samples were mixed with dry KBr (FT-IR grade Sigma-Aldrich, St.Louis, MO) in a ratio of 1:100 and compressed into pellets using a hydraulic press. The resulting pellet’s transmittance was recorded within a range of 4000–450 cm–1 with thirty-two scans.
Proton Nuclear Magnetic Resonance (NMR) Spectroscopy.
Proton NMR spectra were recorded on a 400 MHz Varian spectrometer (Varian, Inc., Palo Alto, CA), all the measurements were done in DMSO-d6 solvent, and all the peaks were referenced to residual protonated solvent with chemical shift (δ ) =2.49 ppm.
Thermogravimetric Analysis (TGA).
Thermogravimetric analysis was conducted on a Mettler Toledo thermal analyzer (TGA 2, Switzerland). All solid samples were measured in the 25–600° C temperature range at a heating rate of 10 °C/min under the gaseous nitrogen flux, and the data was processed with STAR software.
Differential Scanning Calorimetry (DSC).
The differential scanning calorimetric measurements were recorded on a Shimadzu DSc-60 Plus (DSC instrument, Japan). The software workstation LabSolutions TA controlled thermal analysis. The sample was precisely weighed on a microbalance taken on an aluminum crimp pan with a top lid. All samples were measured in the 25–400 °C temperature range at a heating rate of 10 °C/min under a constant nitrogen flow of 100 mL/min.
Size Distribution and Zeta Potential Analysis.
The particle size (z-average d.nm) and ζ potential (mV) were recorded on a Malvern zetasizer Nano ZS instrument (Malvern Instruments UK). All solid samples dispersed in methanol (Honeywell HPLC grade) ≈ 0.1 mg/mL concentration, vortexed and analyzed by using a disposable sizing cuvette for DLS measurement and electrophoretic measurement were performed on a zetasizer cell at 25 °C after 120 seconds temperature equilibration at a backscattering angle of 173°.
Diffuse Reflectance Spectroscopy (DRS).
The absorption spectra of the solid samples were obtained by using the Kubelka–Munk conversion (K–M = (1 − R)2/2R) of the recorded diffusive-reflectance spectra at room temperature for the solid samples on an FS5 spectrometer (Edinburgh, UK) equipped with an SC-30 (integrating sphere) as the sample holder. The specular reflection of the sample surface light was removed from the signal by directing the incident light at the sample at an angle of 0o; only the diffusive reflected light was measured. Polytetrafluoroethylene (PTFE) polymer was used as the reference. The bandgap energy (Eg) values of the solid samples from the DRS spectra were calculated using Eg = 1240 eV nm l−1, where l is the absorption edge (in nm).
Photoluminescence (PL) and Photoluminescence-Excitation (PLE) Measurements .
Fluorescence spectra were recorded on a spectrofluorometer FS5 instrument (Edinburg, United Kingdom) using slit widths of 2.0 nm for both the excitation and the emission monochromators in all experiments. The estimated experimental error was 2% for a lifetime value of less than one ns and 20% for a lifetime of around five ns.
Excited-State PL Lifetime Measurements and Time-Resolved Photoluminescence (TRPL) Measurements.
The time-resolved photoluminescence measurements were performed on a LifeSpec-II spectrometer (Edinburgh Inc., Edinburgh, United Kingdom) based on the Time-Correlated Single Photon Counting (TCSPC) method. The excitation diode laser source was set at 475 nm and used on an Edinburgh instrument (Edinburgh Inc., Edinburgh, United Kingdom) with a repetition rate of 20 MHz, a time resolution of 30 ps, and a red-sensitive high-speed photomultiplier tube detector (Hamamatsu, H5773-04). The data were analyzed by the iterative reconvolution method using the instrument’s software that utilizes the Levenberg–Marquardt algorithm to minimize χ2. The fluorescence decay was analyzed in terms of the multi-exponential model to calculate the average lifetime value, given by:
\(\overset{\overline{}}{\tau}=\sum_{i}{f_{i}\tau_{i}}\ \) (1)
And the contribution of each component to the steady-state intensity is given by:
\(f_{i}=\frac{\alpha_{i}\tau_{i}}{\sum_{j}{\alpha_{j}\tau_{j}}}\ \)(2)
Where τi are the lifetimes with amplitudes αi and ∑i = 1.0. And the sum in the denominator is for all the decay times and amplitudes.
Medicinal Research Materials.
Human T-cell leukemia cell line 1301 (European collection of authenticated cell cultures, Sigma Aldrich, Merck KGaA, Germany) and Peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 7) were used as the material for the study. The ethics committee of RIFCI, Russia, approved the design of the study and recruitment of donors. Donors gave written informed consent.
PBMCs Isolation.
PBMCs were isolated from the heparinized venous blood of healthy donors using the ficoll-urografin (1.077 g/mL) density gradient centrifugation method. The fresh heparinized venous blood (5 ml) was layered into a test tube for 3 mL ficoll-urografin (1.077 g/mL). The tubes were centrifuged for 20 minutes at 2700 rpm. After centrifugation, mononuclear rings were collected into separate tubes, followed by double washing in 10 mL of phosphate-buffered saline (PBS) end ethylenedinitrilotetraacetic acid disodium salt dihydrateNa2EDTA 1%. The washed cells were placed in 1 mL of RPMI-1640 (Gibco, Invitrogen, Carlsbad, CA, USA). Then they were counted using the Goryaev camera.
Cultivation.
Cells at a concentration of 1 million/mL (PBMCs) or 150,000 cells/ml (1301 cell line) were cultured for 1, 24, or 72 hours in 96-well plates (in a volume of 100 µL) in RPMI-1640 medium (Gibco, Cat. No. 27016021) containing 0.3% L-glutamine, 50 µg/mL gentamicin, 25 µg/mL of thienam and 10% inactivated fetal calf serum (FCS) (Hyclone, Chicago, IL, USA) in the presence of various concentrations of VGALG and VGCB8ALG (1.8 mg /mL; 0.18 mg/mL; 0.018 mg/ mL; 0.0018 mg/mL; 0 mg/mL).
Cytotoxicity of Hydrogels.
The cytotoxicity of hydrogels was evaluated using WST-1 assay kit (Takara Bio, Kusatsu, Japan) and LDH-Cytox™ Assay Kit (BioLegend, San Diego, CA, USA). Ten μl WST-1 was added to the cell culture media and incubated for four hours at 37°C in the incubator. As a positive control, 10% DMSO was used. The cytotoxicity of VGALG and VGCB8ALG was analyzed by the amount of formazan dye produced by measuring the absorbance at 450 nm. To perform the lactate dehydrogenase (LDH) assay for the cellular cytotoxicity of the compounds, the reaction mixture was added to the wells of the cell culture medium. After 30 min of incubation with lysis buffer and 30 min of incubation with assay buffer with protection from light, the reaction was stopped by adding a stop solution, and the absorbance was measured using a microplate reader Infinite F50 (Tecan, Grödig, Austria) at 490 nm. The average absorbance of each triplicate set of wells was calculated, and the background control value was subtracted. The percent cytotoxicity was calculated with the following equation:
\(\mathrm{Cytotoxicity(\%)=}\frac{A-C}{B-C}\times 100\) (3)
where A: Test Substance, B: High Control, C: Low Control.
Internalization.
After cultivation, the cells were collected, and PBMCs were labeled with surface markers using anti-CD3-FITC and anti-CD14-APC antibodies (all Biolegend, San Diego, CA, USA). Internalization was determined by the modified viologen in the composition of hydrogels, whose fluorescence spectrum was shifted to longer wavelengths and was detected by flow cytometry (Ex 488 nm, Em 695 nm). Internalization evaluation was performed cytometrically using flow cytometry (BD FACSCanto II cytometer) and FACSDiva software (Becton Dickinson, Franklin Lakes, NJ, USA). Further, to establish the uptake route of hydrogels, 1301 cells were exposed to various pharmacological inhibitors, including methyl-beta-cyclodextrin (MβCD), chlorpromazine, nystatin, N-ethylisopropylamiloride (EIPA), dynasore, cytochalasin or wortmannin, in the 12-well plates at the concentration of 150,000 cells/mL for one hour. Later, the hydrogels (0.18 mg/mL) were added and co-incubated for an additional hour. Here was another group with a low temperature. 1301 cells were precooled at 4℃ for 30 minutes, and then, the treatment was the same as the previous four inhibitor groups but at 4℃ all the time. As for the flow cytometry analysis, the cells were harvested using centrifugation, then washed twice and made into a single-cell suspension for flow cytometry analysis. 10000 events were collected for each sample.
Fluorescence Microscopy.
The cellular uptake of hydrogels was investigated by fluorescence microscopy. Cells were incubated with VGCB8ALG or VGALG (0.18 mg/mL) for 24 hours. Then, cells were washed twice with 2 mM EDTA solution in PBS and fixed with a mixture of ethanol: glacial acetic acid (3:1, v/v). Cells were resuspended thoroughly to avoid clumps after the fixation step and placed on slides. Then, the slides were analyzed using phase-contrast microscopy for cell cytoplasm and nuclei visualization in transmitted light. After that, slides were mounted with a Pro Long Gold antifade containing 6-diamidino-2-phenylindole (DAPI) (Invitrogen MP, Waltham, MA, USA) to prevent dye photo-bleaching and identify cell nuclei further. Phase-contrast and fluorescent microscopy was performed with the Axioscope 40 fluorescence microscope (Zeiss, Germany). DAPI-stained nuclei images and hydrogel signals were captured separately with the software package ZEN-2012 (Zeiss, Germany) on the magnitude X1000. Exposure time was adjusted automatically.
Statistical data.
Analysis was performed using GraphPad Prism 9.3.1 using Friedman’s test using Dunn’s test for multiple comparisons. A p value <0.05 was considered the minimum criterion for statistical significance.
RESULTS AND DISCUSSION
Material Characterizations of Hydrogels (Functionalization, Encapsulation, Stoichiometry, Composition, Thermal Stability, Size Distribution, and Charges)
The FTIR spectra of the modified hydrogels in Figure 2 show distinct, entirely different stretching and bending bands compared to those peaks of ALG and VG starting materials (Table S1 in the Supporting Information) or to the previously reported FTIR spectrum of CB8[24] confirming the formation of VGALG and VGCB8ALG.