Alginate supramolecular hydrogels based on viologen and cucurbit[8]uril: Host‐induced caveolae‐mediated endocytosis to white blood cancer cells

The cellular uptake of drug carriers to the cytosol of a specific cell remains challenging, and a non‐classical supramolecular strategy is motivated. Here, we select a model host–guest complex in which a diamino‐viologen (1,1′‐bis(4‐aminophenyl)‐[4,4′‐bipyridine]−1,1′‐diium dichloride [VG]) fluorescent tag was engulfed by cucurbit[8]uril (CB8) and covalently linked to alginate polysaccharides (alginic acid [ALG]) as the modified drug vehicle. When adsorbed on the ALG surface, the encapsulation of VG was first confirmed utilizing Fourier transform infrared and nuclear magnetic resonance spectroscopic methods. Solid optical measurements (diffuse reflectance spectroscopy, photoluminescence, and time‐resolved photoluminescence) revealed emissive materials at around 650 nm and that CB8 enhanced the rigidity of the modified hydrogel. The molar composition of 2:1 for the complexation of VG to CB8 on the alginate surface and the thermal stabilities were also confirmed using thermogravimetric analysis and differential scanning calorimetry techniques. CB8 induced a dramatic decrease in the average size of the VGALG polysaccharides from 485 to 165 nm and a turnover in their charge from –19.8 to +14.4 mV. Flow cytometry with inhibitors of various endocytosis pathways was employed to track the cellular uptake across different blood cell types: human T‐cell leukemia 1301 and peripheral blood mononuclear cells. Noticeably, complexation of VG with the CB8 host on top of the sugar platform dramatically enhanced the internalization into 1301 cells (viz. from 1% to 99%) at a concentration of 1.8 mg/mL via caveolae‐mediated endocytosis because of the size reduction, turnover in the charge from negative to positive, and rigidity induction. These observations reveal a more profound understanding of the macrocyclic effects on drug delivery.


INTRODUCTION
Optimizing drug-delivery methods to improve immunotherapeutic efficiency is often aided by different strategies that modulate cell or tissue targeting, controlled release, and the response of the drug to suitable triggers. 1sing a dynamic host-guest system, the supramolecular scientists set their goals by providing specific, tunable, and thermodynamically reversible bonds.][4] The reversible associations are highly selective and have been employed over several decades in generating supramolecular hydrogels such as polysaccharidebased hydrogels. 5,6Generally, supramolecular hydrogels' excellent elastic and mobility nature 7 offers superior performance in biomedical applications over chemically crosslinked hydrogels.The external environmental biochemical and physical variation can regulate the reversible association and dissociation of the host-guest supramolecular system. 8Also, supramolecular hydrogels can be tailored for application in vivo due to their significant water content and biocompatibility. 9Specifically, employing supramolecular hydrogels based on polysaccharides for biomedical applications utilizing CB macromolecules is not original. 6It has also been described for various biological and clinical applications.For instance, biomedical researchers reported different designs using CB for crosslinking the supramolecular hydrogels based on various functional tags.Yet, the exact mechanism of cellular uptake by macrocycle-based hydrogels is to be explored.
Nowadays, treating and curing cancer is the world's most pressing health-challenging task for clinical research.According to the World Health Organization estimation, cancer death will increase globally by 80% by 2030. 10 Leukemia is the most challenging treatment among all types of cancer.Clinical scientists are continuously working hard to prevent this grim projection.With care that aims to balance the effectiveness of treatment and the importance of quality of life, more patients than ever are living longer.
CB is more biocompatible than other watersoluble macrocyclic hosts because CB is dexterity to functionalization. 11Water-soluble macrocyclic molecules offer their hydrophobic cavities for encapsulating polar and non-polar guest molecules with a reasonable binding constants. 12The eight-monomer unit containing cucurbit [8]uril (CB8 in Figure 1) forms a complex with viologen guests non-covalently.Viologens are functional organic materials first discovered by Michaelis and Hill. 13Viologens are classic examples of redox and electron-deficient compounds.Several viologen-based crystalline and amorphous photochromic materials have been synthesized for the last 10 years. 14In particular, viologens containing disubstituted-4,4′-bipyridinium salts (1,1′-bis(4-aminophenyl)-[4,4′-bipyridine]−1,1′diium dichloride [VG] 15 in Figure 1) are unique organic molecules with hydrophilic and hydrophobic properties.Derivatives similar to VG were used by several researchers to form inclusion complexes with CB8, leading to various supramolecular architectures, 16 such as supramolecular organic frameworks 17,18 and hydrogels. 19,20To be effective, precise structural control of such materials is required.This, of course, puts a premium on the ability to control structural parameters during their tedious synthesis.However, the precise structural control of hydrogels such as alginate polysaccharides (alginic acid [ALG] in Figure 1) 21 is difficult.It requires the simultaneous adjustment of several parameters and/or possibly different processing techniques to achieve any size, charge, or rigidity.Here, a supramolecular hydrogel as a drug delivery system is designed in which CB8-encapsulated VG salts are covalently linked to the surface of ALG.
The role of CB8 in triggering cellular internalization to white blood cancer cells was 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, 22 using such systems for extended networked structures such as the present CB8based alginate hydrogels (VGCB8ALG) is purely academic and should unfold more information.

Samples
All starting reagents CB8, VG, ALG, 4-dimethylamino pyridine (DMAP), and N,N′-dicyclohexylcarbodiimide (DCC), as well as methanol and dimethyl sulfoxide (DMSO) solvents, including the deuterated ones, were obtained from Sigma-Aldrich 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 DMSO, purged under nitrogen for 1 h at 295 K (50 mL), and the solution was stirred for 16 h.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 6 h 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 8 h, and the solid product was washed with water and freeze-dried for 10 h.

Fourier transform infrared spectroscopy
Fourier transform infrared (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 (FTIR grade, Sigma-Aldrich) at 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 32 scans.

Proton nuclear magnetic resonance spectroscopy
Proton nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz Varian spectrometer (Varian, Inc.), all the measurements were done in DMSO-d 6 solvent, and all the peaks were referenced to residual protonated solvent with chemical shift (δ) = 2.49 ppm.

Thermogravimetric analysis
Thermogravimetric analysis was conducted on a Mettler Toledo thermal analyzer (TGA 2).All solid samples were measured in the 25 • C-600 • C temperature range at a heating rate of 10 • C/min under the gaseous nitrogen flux, and the data were processed with STAR software.

Differential scanning calorimetry
The differential scanning calorimetric measurements were recorded on a Shimadzu DSc-60 Plus (DSC Instrument).
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 • C-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 diameter [nm]) and ζ potential (mV) were recorded on a Malvern Zetasizer Nano ZS instrument (Malvern Instruments).All solid samples were dispersed in methanol (Honeywell HPLC grade) at a concentration of ≈0.1 mg/mL, vortexed and analyzed by using a disposable sizing cuvette for dynamic light scattering (DLS) measurements and electrophoretic measurements were performed on a Zetasizer cell at 25 • C after 120 s temperature equilibration at a backscattering angle of 173 • .

Diffuse reflectance spectroscopy
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) 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 0 • ; only the diffusely reflected light was measured.Polytetrafluoroethylene polymer was used as the reference.The bandgap energy (E g ) values of the solid samples from the diffuse reflectance spectroscopy spectra were calculated using E g = 1240 eV nm l −1 , where l is the absorption edge (in nm).

Photoluminescence and photoluminescence-excitation measurements
Fluorescence spectra were recorded on a spectrofluorometer FS5 instrument (Edinburg) 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 1 ns and 20% for a lifetime of around 5 ns.

Excited-state photoluminescence lifetime measurements and time-resolved photoluminescence measurements
The time-resolved photoluminescence measurements were performed on a LifeSpec-II spectrometer (Edinburgh Inc.) based on the time-correlated single photon counting method.The excitation diode laser source was set at 475 nm and used on an Edinburgh instrument (Edinburgh Inc.) 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 χ 2 .The fluorescence decay was analyzed in terms of the multi-exponential model to calculate the average lifetime value, given by: And the contribution of each component to the steadystate intensity is given by: where τ i are the lifetimes with amplitudes α i and ∑ i = 1.0.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) and peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 7) were used as the materials for the study.The ethics committee of RIFCI, Russia, approved the design of the study and recruitment of donors.Donors gave written informed consent.

PBMC isolation
PBMCs were isolated from the heparinized venous blood of healthy donors using the Ficoll-Urografin (1.077 g/mL) density gradient centrifugation method.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 min at 2700 rpm.After centrifugation, mononuclear rings were collected into separate tubes, followed by double washing in 10 mL of phosphate-buffered saline (PBS) and ethylenedinitrilotetraacetic acid disodium salt dihydrate (Na 2 EDTA 1%).The washed cells were placed in 1 mL of RPMI-1640 (Gibco, Invitrogen).Then, they were counted using the Goryaev camera.

Cytotoxicity of hydrogels
The cytotoxicity of hydrogels was evaluated using a WST-1 assay kit (Takara Bio) and LDH-Cytox Assay Kit (BioLegend).Ten microliters of WST-1 was added to the cell culture media and incubated for 4 h 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) 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: where A is the test substance, B is the high control, and C is the 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).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 (E x 488 nm, E m 695 nm).
Internalization evaluation was performed cytometrically using flow cytometry (BD FACSCanto II cytometer) and FACSDiva software (Becton Dickinson).Furthermore, to establish the uptake route of hydrogels, 1301 cells were exposed to various pharmacological inhibitors, including methyl-beta-cyclodextrin, chlorpromazine, nystatin, Nethylisopropylamiloride, dynasore, cytochalasin, or wortmannin, in 12-well plates at a concentration of 150,000 cells/mL for 1 h.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 • C for 30 min, and then, the treatment was the same as the previous four inhibitor groups but at 4 • C all the time.As for the flow cytometry analysis, the cells were harvested using centrifugation, washed twice, and made into a single-cell suspension for flow cytometry analysis.A total of 10,000 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 h.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) to prevent dye photo-bleaching and identify cell nuclei further.Phase-contrast and fluorescent

Statistical data
Analysis was performed using GraphPad Prism 9.3.1 using Friedman's test and Dunn's test for multiple comparisons.
A p-value <.05 was considered the minimum criterion for statistical significance.

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) or to the previously reported FTIR spectrum of CB8 23 confirming the formation of VGALG and VGCB8ALG.
Notably, the peak for (NH 2 ) primary amine stretching 15 at 3202 cm -1 and bending at 1634 and 1596 cm -1 (in VG) has evolved into the amide (CONH) peak at 3329 cm -1 and 1627 and 1574 cm -1 , respectively (in the modified ALG).Moreover, a significant enhancement in the intensity of the CH stretching (in ALG) 21 at 2927 and 2850 cm -1 was observed upon its covalent modification by VG or VGCB8.The two FTIR spectra measured for VGALG and VGCB8ALG hydrogels were similar because encapsulation by CB8 does not usually affect the vibration of the guest molecule's bonds. 23Only peaks in the range from 1500 to 1400 cm -1 due to the stretching vibration of the benzene rings of VG were slightly weakened. 23n the proton NMR spectra (aromatic region), ALG shows a single broad resonance peak at δ = 12.30 ppm associated with the -OH group in ALG (Figure S1).5][26] A new peak at δ = 12.25 ppm appears when VG is linked to ALG due to formation of an amide linkage in the VGALG complex with a concomitant shift to lower ppm and broadening of peaks b-e.As expected in similar reports, 25,27 the peaks b-e of the new hydrogels were further broadened (almost disappeared) upon engulfing VG in CB8.
As additional evidence for the non-covalent entrapment of VG by CB8 in DMSO solution, we measured the UV-visible absorption spectra of all alginate samples.We noticed several isosbestic points at 250, 300, and 550 nm (Figure S2) in the spectra of the VG-modified ALG in the absence and presence of CB8.The addition of CB8 also shifts the absorption peaks from 245 and 475 nm to 250 and 490 nm, respectively.Additionally, the data served us to confirm the covalent conjugation of VG to ALG by the emergence of new peaks at 245 (250 nm with CB8), 275, and 475 nm (490 nm with CB8).
Figure 3 shows the thermogravimetric analysis (TGA) of VG, CB8, ALG, VGALG, and VGCB8ALG in the temperature range from 35 • C to 600 • C at a heating rate of 10 • C. The traces provide information about the composition and thermal decomposition of the new hydrogels VGALG and VGCB8ALG compared to individual viologen and unmodified alginate samples. 28More importantly, the results confirm the host-guest interaction between VG and CB8 on the ALG platform.
The weight loss percentage values at different temperatures were calculated from the TGA curves for all the samples to estimate the weight % of VG and CB8 in the modified alginates (Tables S2).We divided the weight % by the molecular weight, giving us several moles.We then concluded the percentage by moles of VG (∼20%) in both alginate polymers.We also compared the number of moles of VG with CB8, which allowed us to conclude a 2:1 VG:CB8 interaction on the alginate surface, which agrees with other supramolecular architectures that contain CB8-complexed viologen dyes. 18Table S2 also includes the percentage of humidity, decomposition, complete organic degradation mass losses, and residues for ALG, VG, VGALG, CB8, and VGCB8ALG.It was observed that VGCB8ALG samples released a higher percentage of absorbed water than the ALG sample.This result indicates a more robust immobilization of water molecules because of the linking of ALG with VGCB8.The thermal stability of materials is manifested by a significant weight loss in the degradation stage when complete thermal degradation starts.
Differential scanning calorimetry (DSC) data characterize the thermal behavior of individual ALG, VG, and CB8 in terms of their simple structure, hydrophilic properties, and association rate. 29The results confirm the linking of VG to ALG and its host-guest complexation to CB8 while linked to ALG.For the unmodified ALG, the DSC curve in Figure S3 shows an endothermic peak at 94.8 • C followed by a strong exothermic peak at around 288 • C. Endothermic peaks were correlated with the loss of water associated with hydrophilic groups of the biopolymer.In contrast, exothermic peaks resulted from the decomposition of ALG, and subsequently, the decomposition of carbonaceous material occurred above 400 • C. The DSC thermogram of VG unfolded an endothermic peak at 117 • C attributed to the water release and an exothermic peak at 354 • C due to the decomposition of viologen.Upon linking VG to ALG, the two exothermic peaks for ALG and VG were shifted to 232 • C and 360 • C, respectively.Table S3 presents the exothermic and endothermic peaks and the heat flow changes associated with each peak.The thermogram of CB8 shows an endothermic peak at 96 • C, which is associated with the water release, and an exothermic peak at 387 • C because of the decomposition of CB8.In contrast, the DSC of VGCB8ALG indicates the guest-host association as evidenced by the evolution of an endothermic peak at 387 • C instead of an exothermic one in VGALG.
The particle size distribution (z-average hydrodynamic diameter [nm]) and surface charge (ζ potential [mV]) of VG, ALG, VGALG, and VGCB8ALG were studied by DLS and electrophoretic light scattering, as shown in Figure 4.
The samples were dissolved in methanol, and the size distribution and ζ potential were recorded.The measurement for ALG shows particles with a large size of 1540 nm.The ζ potential measured consistently higher value of −13.8 mV.The particle size measured for VG is 280 nm and has a relatively high ζ potential value of +34.9.As Figure 4 shows, the size distribution for VGALG revealed a particle size of 485 nm and a ζ potential of −19.8 mV, indicating the robust binding of VG to ALG.The polydispersity index, a parameter for non-uniform particle distribution, shows a slightly higher result of 0.580, which also supports the non-uniform distribution of VGALG particles in the dissolution media.The measured size distribution for VGCB8ALG was 165 nm, and the ζ potential value shifted to +14.4 mV, indicating the guest-host interaction in VGCB8ALG. 8The manipulation of the size and charge of hydrogels upon the addition of CB8 explains the results of cellular internalization below.

Optical properties (functionalization, encapsulation, implementation for cellular uptake, and rigidity enhancement)
The solid UV-visible spectra (Figure S4) showed a slight difference in the band gap (0.06 eV) between the two hydrogels with the addition of CB8.Yet, both indicated distinct optical properties with onsets at approximately 700 nm compared to the published data for unmodified ALG with 212 and 271 nm peaks. 30he excitation and emission spectra were recorded for the new hydrogels VGALG and VGCB8ALG in Figure 5 and found to be consistent with the solid UV-visible data.
The maxima shift from 475 to 540 nm and 653 to 675 nm in the excitation and emissions spectra, respectively, with the complexation of VG to CB8.The literature reveals entirely different photoluminescence (PL) and photoluminescence-excitation (PLE) spectra for VG 15 or F I G U R E 5 Photoluminescence (PL) and photoluminescence-excitation (PLE) spectra in the solid state of the two modified hydrogels: VGALG (green), measured with excitation wavelength at 475 nm and emission wavelength at 653 nm; and VGCB8ALG (red), measured with excitation wavelength at 454 nm and emission wavelength at 675 nm.
VGCB7 24 in the solid state, and ALG is known to be non-emissive.The results confirm that the chemical engineering of ALG conceived novel solid PL/PLE properties of the new hydrogels, enabling us to monitor their cellular uptake utilizing the flow cytometry technique.Moreover, the excited-state average lifetime values of the hydrogels in the solid state (see Section 2, Figure S5, and Table S4) increased from 0.28 to 0.85 ns with the addition of CB8 because of the confinement effect. 25A more rigid biopolymer helps interpret the results of cellular internalization below.

Cellular internalization
The present study explored the potential effects of newly designed and prepared hydrogels on immunocompetent cells.Thus, the effects of VGALG and VGCB8ALG on the viability of PBMCs from healthy donors using the WST-1 test (Figure S6) were first evaluated.It was shown that the percentage of viable cells decreased only when cells were treated with VGALG and not VGCB8ALG, at the maximum 1.8 mg/mL concentration used.We also evaluated the concentration of LDH in the culture medium after treatment with hydrogels to determine a possible cytotoxic effect (Figure S7).LDH is released into the cell culture supernatant when the plasma membrane is damaged.Treatment of cells with both hydrogels resulted in cell damage only at high concentrations of 1.8 mg/mL.Therefore, hydrogels at concentrations of 0.18 mg/mL and below do not have a cytotoxic effect on PBMCs.This allows us to conclude that hydrogels under study have high biocompatibility characteristics and low toxic effects on immune cells, as verified by estimating the half-maximal inhibitory concentration (IC 50 in Table S5).Following the literature, we expected a safety profile from VGALG and VGCB8ALG since the main components of the hydrogels, CB8 31 and alginate, 32,33 are safe and biocompatible.The hydrogels (0.18 mg/mL) were then used for investigating the drug internalization by the PBMCs (CD3 − and CD3 + T-lymphocytes) by flow cytometry (Figure S8).PBMC uptake of VGALG has no significant differences from the control (non-treated cells).
In the case of VGCB8ALG, internalization into CD3 − cells was low and was measured at about 6%, while the median uptake by T cells was 62%, which is 10 times higher.Noticeably, CB8 is a critical factor for internalization into the T cells.In the case of CD14 + monocytes, internalization was carried out efficiently by both VGALG and VGCB8ALG (0.18 mg/mL), and no significant differences were observed between hydrogels (Figure S9).The observed differences in internalization are associated with the mechanism of particle capture by various subpopulations of PBMCs; monocytes are capable of active phagocytosis of particles from the surrounding space, while T-lymphocytes, like most other body cells, do not have such a function.Motivated by our results on the high level of internalization of the VGCB8ALG hydrogel into T-lymphocytes of healthy donors, we decided to evaluate the effect of hydrogels on human T-cell lymphoma 1301 cells.It was found that at all concentrations used, both hydrogels did not reduce the viability of 1301 cells at 24 and 72 h of treatment (Figures S10 and S11).Specifically, after 24 h of treatment with 1301 hydrogels (Figure S10), cell viability increased under the influence of VGCB8ALG compared to VGALG at a concentration of 1.8 mg/mL.Thus, VGCB8ALG at a high concentration can increase the number of viable cells in the culture, increasing the proliferation of the cells.Moreover, after 72 h of treatment with 1301 hydrogels, the percentage of viable 1301 cells differed when treated with different concentrations of VGALG and VGCB8ALG (Figure S11).For example, at a concentration of 1.8 mg/mL, there was an increase in viability in the VGCB8ALG group compared to VGALG.In contrast, treatment with VGALG increased the number of viable cells compared to treatment with VGCB8ALG at concentrations of 0.18 and 0.018 mg/mL.Nonetheless, the two hydrogels do not have a toxic effect on 1301 human T-cell lymphoma cells with such long-term treatment for 72 h.In summary, VGALG can enhance the proliferative activity of cells at average concentrations of 0.18 and 0.018 mg/mL with long-term treatment for 72 h.In contrast, VGCB8ALG increased the proliferative activity of these cells only at high concentrations (1.8 mg/mL) in culture for 24 and 72 h.Following the viability results, we decided to evaluate the internalization of hydrogels at a concentration of 1.8 mg/mL by T-cell lymphoma cells at different treatment times (Figure S12).The VGCB8ALG hydrogel was shown to be rapidly internalized by cells; after 1 h, more than 80% of 1301 cells had absorbed the gel.After 24 and 72 h, the percentage of internalized VGCB8ALG cells increased to more than 90%.Interestingly, VGALG was practically not absorbed by the cells at all times of treatment.To better explain this result, we evaluated the effect of hydrogel concentration on the uptake of T-cell lymphoma by the cell line (Figure 6).VGCB8ALG was efficiently taken up by cells at all concentrations used.The relative number of cells that took up VGCB8ALG varied with concentration, ranging from 99% at the highest concentration to 15% at the lowest concentration.VGALG was poorly taken up by cells at all concentrations used.
To identify a possible pathway for hydrogel endocytosis into T-lymphocytes, 1301 cells were treated with 0.18 mg/mL VGCB8ALG together with inhibitors of various endocytosis pathways (Figure 7).As expected, exposure to hydrogels at 4 • C resulted in potent inhibition of endocytosis.The results showed a significant difference in uptake when inhibited with nystatin, thus suggesting that caveolae-mediated endocytosis was preferred for VGCB8ALG.
To confirm our results, 1301 cells were treated with hydrogels for 24 h, after which we performed fluorescence microscopy (Figure 8).Fluorescent microscopy images confirmed that VGCB8ALG, compared to VGALG, is strongly internalized into the cell and evenly distributed.VGCB8ALG is localized in the cytoplasm of cells.A report by Studzian et al. 34 has shown that CCRF-1301 cells take up glycodendrimer macromolecules via the endocytosis process, which is slow, cholesterol dependent, clathrin dependent, and caveolin independent.Only surface binding was observed after 2 h of incubation, as in our study in the case of the VGALG.On the other hand, similar T-cell lines have shown the ability to rapid uptake. 35 I G U R E 9 Schematic representation of blood cellular uptake of VGALG and VGCB8ALG hydrogels.The chemical structures of 1,1′-bis(4-aminophenyl)-[4,4′-bipyridine]−1,1′-diium (VG), alginic acid (ALG), and cucurbit [8]uril (CB8) are also shown.Modifying the hydrogels involves first encapsulating the parts of VG with CB8 and then covalently attaching the complex to the sugar ALG via the NH 2 groups.The 2:1 binary complex of CB8 with the viologen dimers is not shown for simplicity.

Supramolecular effects on cellular internalization (role of size, charge, and rigidity)
Overall, these data show that the modified ALG (drug carriers), by covalently anchoring VG tags into the polysaccharides that are readily non-covalently complexed to CB8 macromolecules, have a distinct ability to cross leukemia cancer cells (Figure 9), giving a more explicit description of the cellular uptake mechanisms of supramolecular hydrogels in cancer therapy.
As described in the introduction, employing supramolecular hydrogels based on polysaccharides for biomedical applications and utilizing CB macromolecules is not original and has received significant attention.It has also been described for various biological and clinical applications.Nonetheless, elucidating the effects of macromolecules on the mechanism of cellular uptake is still motivated.The biocompatibility and low toxicity have been confirmed for the modified ALG in the present work.The translation of the unmodified ALG has already been explored in literature. 36The research concluded the significant role of size.Three different size-dependent mechanisms for cellular uptake were suggested.Oleoyl ALG ester nanoparticles with sizes of 50, 120, or 730 nm enter the colon cancer cells via clathrin-mediated endocytosis, caveolae-mediated endocytosis (CvME), or micropinocytosis, respectively. 36rom Figure 4, the CB8-modified ALG has a size that is 165 nm compared to 1540 nm for the undecorated ALG.The uptake mechanism was confirmed to be CvME, which agrees with the literature.In another account, the authors highlighted the effect of charges on the uptake of nanoparticles.Because the lung cancer cell's surface has a negative charge, cationic cerium oxide nanoparticles 37 were better internalized than neutrally or negatively charged ones, such as the cellular internalization of polyplexes, lipoplexes, and lipopolyplexes into a myoblast cell line. 38From Figure 4, the modified VGCB8ALG has a positive charge (viz.+14.4 mV) compared to the negatively charged VGALG (−19.8 mV) and thus was better internalized into leukemia cancer in agreement with those reports.The rigidity factor was demonstrated to modulate the type of cellular uptake, in which rigid nanoparticles of N,N-diethyl acrylamide, and 2-hydroxyethyl methacrylate cross-linked with N,N′-methylene-bis-acrylamide were better internalized by RAW 264.7 murine macrophage cells than soft nanoparticles. 39From Figure S5, adding CB8 renders the modified hydrogels more rigid than VGALG.Thus, the increase in endocytosis is unsurprising.The exciting point remains the straightforward elucidation of how CB8 has modulated the cellular uptake, which can be further utilized for other clinical applications in the future.
Collectively, these data unfold the factors that biomedical researchers need to consider (among others) to get more efficient drug delivery systems in cancer therapy that involve a macromolecular system or other drug vehicles.Specifically, the present work offers a new supramolecular architecture that utilizes the unique properties of the 4,4′-bipyridine unit, which follows the most recent current literature 40,41 in the context of their prospect implications on developing stimuli-responsive hydrogels in biomedicine, exploring and promoting more applications of smart materials in practical production.

CONCLUSION
The present work offers a supramolecular strategy to modulate the cellular uptake and intracellular processing of drug delivery systems, paving the way for a more remarkable improvement in the development of drug therapeutics of alginates in cancer therapy.We showed that supramolecular encapsulation is the key required for the efficient delivery of a model drug system via CvME, as manifested in the substantial decrease in the size of the alginate hydrogels.The employment of a supramolecular approach to modify drug carriers is not new.Still, the results here confirm a distinct, unique modulation of the intracellular processing of alginate carriers, unfolding a thorough understanding of the macrocyclic effects on drug delivery compared to previous reports.

F I G U R E 4
Particle size distribution of modified hydrogels and starting materials (A) and surface charge of different hydrogels and their components (B).

F I G U R E 6
Evaluation of internalization of 1301 cells after 24 h of treatment with different concentrations of hydrogels (n = 4).

F I G U R E 7
Effect of endocytosis inhibitors on the uptake of VGCB8ALG (0.18 mg/mL) by 1301 cells (n = 3).EIPA, N-ethylisopropylamiloride; MβCD, methyl-beta-cyclodextrin.F I G U R E 8 Upper cases (A and B) represent merged fluorescent images on the magnitude ×1000.The red spots represent labeled hydrogel, and the nucleus is stained with 6-diamidino-2-phenylindole (DAPI) (blue).(A and B) Cellular uptake of hydrogel alone and in complex with cucurbit[8]uril (CB8) correspondingly.Lower cases (a and b) represent the correspondent phase-contrast images on the magnitude ×400, where arrows indicate the cytoplasm.The concentration of each hydrogel was 0.18 mg/mL.