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\begin{document}

\title{Alginate Supramolecular Hydrogels Based on Viologen and
Cucurbit{[}8{]}uril: Host-Induced Caveolae-Mediated Endocytosis to White
Blood Cancer Cells}



\author[1]{Falguni  Chandra}%
\author[2]{Marya  Bykova}%
\author[1]{Vijo  Polous}%
\author[1]{Paltan  Laha}%
\author[2]{	Alina  Aktanova}%
\author[2]{	Olga  Boeva}%
\author[2]{Margarita  Barkovskaya}%
\author[2]{Ekaterina  Pashkina}%
\author[3]{Na'il  Saleh}%
\affil[1]{United Arab Emirates University}%
\affil[2]{Novosibirskij gosudarstvennyj medicinskij universitet Ministerstva zdravoohranenia Rossijskoj Federacii}%
\affil[3]{UAE University}%


\vspace{-1em}



  
  \date{June 15, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
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 (VG) fluorescent tag was engulfed by
cucurbit{[}8{]}uril (CB8) and covalently linked to alginate
polysaccharides (ALG) as the modified drug vehicle. When adsorbed on the
ALG surface, the encapsulation of VG was first confirmed utilizing FTIR
and NMR spectroscopic methods. Solid optical measurements (DRS, PL, and
TRPL) revealed emissive materials at around 650 nm and that CB8 enhanced
the rigidity of the modified hydrogel. The molar composition of 2 to 1
for the complexation of VG to CB8 on the alginate surface and the
thermal stabilities were also confirmed using TGA and DSC 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 to CB8 host on top of
the sugar platform dramatically enhanced the internalization to 1301
cells (viz. from 1 to 99\%) at a concentration of 1.8 mg/mL via
caveolae-mediated endocytosis (CvME) 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%
\end{abstract}%



\sloppy


Alginate Supramolecular Hydrogels Based on Viologen and
Cucurbit{[}8{]}uril: Host-Induced Caveolae-Mediated Endocytosis to White
Blood Cancer Cells

Falguni Chandra,\textsuperscript{+§}\selectlanguage{ngerman} Maria
Bykova,\selectlanguage{english}\textsuperscript{++}Vijo Polous,\selectlanguage{english}\textsuperscript{+} Paltan Laha,
\selectlanguage{english}\textsuperscript{+SS} Alina Aktanova,\selectlanguage{english}\textsuperscript{++[?]} Olga
Boeva,\selectlanguage{english}\textsuperscript{++} Margarita Barkovskaya, \selectlanguage{english}\textsuperscript{++}
Ekaterina Pashkina,*\selectlanguage{english}\textsuperscript{,++[?]} and Na'il
Saleh*\selectlanguage{english}\textsuperscript{+SS}

\selectlanguage{english}\emph{\textsuperscript{+}Department of Chemistry, College of Science,
United Arab Emirates University, P.O. Box 15551, Al Ain, United Arab
Emirates}

\emph{\textsuperscript{SS}Zayed Bin Sultan Center for Health Sciences,
United Arab Emirates University, PO. Box 15551 Al Ain, United Arab
Emirates}

\selectlanguage{english}\emph{\textsuperscript{++}Laboratory of Clinical Immunopathology,
Research Institute of Fundamental and Clinical Immunology, 14
Yadrintsevskaya, 6300099 Novosibirsk, Russia}

\selectlanguage{english}\textsuperscript{[?]}\emph{Novosibirsk State Medical University, 52,
Krasny Prospect, 630091 Novosibirsk, Russia}

\emph{*Corresponding and submitting author: n.saleh@uaeu.ac.ae (N.S.).
Tel.: +971-(0)3-713-6138,eapashkina@niikim.ru (E.P.)
Tel.:+7923-190-4706}

\textbf{ABSTRACT.} 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 (VG) fluorescent tag was engulfed by
cucurbit{[}8{]}uril (CB8) and covalently linked to alginate
polysaccharides (ALG) as the modified drug vehicle. When adsorbed on the
ALG surface, the encapsulation of VG was first confirmed utilizing FTIR
and NMR spectroscopic methods. Solid optical measurements (DRS, PL, and
TRPL) revealed emissive materials at around 650 nm and that CB8 enhanced
the rigidity of the modified hydrogel. The molar composition of 2 to 1
for the complexation of VG to CB8 on the alginate surface and the
thermal stabilities were also confirmed using TGA and DSC 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 to CB8 host on top of
the sugar platform dramatically enhanced the internalization to 1301
cells (viz. from 1 to 99\%) at a concentration of 1.8 mg/mL via
caveolae-mediated endocytosis (CvME) 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.

\textbf{KEYWORDS:} Cucurbiturils, alginates, viologen, time-resolved
photoluminescence, cellular uptake, blood cancer cells.

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.\textsuperscript{{[}1{]}}

Using a dynamic host-guest system, the supramolecular scientists set
their goals by providing specific, tunable, and thermodynamically
reversible bonds. This concept also led them to develop water-soluble
nanocontainer-based drug delivery systems such as cucurbituril
(CB)-based drug-delivery systems.\textsuperscript{{[}2{]}} Nature is the
primary inspiration for studying supramolecular chemistry in
biorecognition, which includes multimolecular host-guest complexes
formed by noncovalent interaction between suitable complementary guest
molecules and nanocavity-based macrocyclic molecules known as molecular
recognition.\textsuperscript{{[}3{]}}

The reversible associations are highly selective and have been employed
over several decades in generating supramolecular hydrogels such as
polysaccharides-based hydrogels.\textsuperscript{{[}4,5{]}} Generally,
supramolecular hydrogels' excellent elastic and mobility
nature\textsuperscript{{[}6{]}} offers superior performance in
biomedical applications over chemically crosslinked. UV light-induced
crosslinking or in situ crosslinking can irreversibly generate the
latter. Yet, the resulting covalently crosslinked hydrogels are rigid
and brittle and cannot be tuned. Due to several dynamic physical bonding
and a shorter time for gelation formation, the supramolecular hydrogels
gained unique properties such as excellent
shear-thinning\textsuperscript{{[}7{]}}elasticity,\textsuperscript{{[}8{]}}
mouldability, sol-gel switching,\textsuperscript{{[}9{]}} capability for
self-healing,\textsuperscript{{[}10{]}} and control release of growth
factors. The supramolecular hydrogels are known to have a well-defined
stoichiometry, contiguous network, and high biocompatibility due to
their high selectivity and supercilious binding
strength.\textsuperscript{{[}11{]}} The external environmental
biochemical and physical variation can regulate the reversible
association and dissociation of the host-guest supramolecular
system.\textsuperscript{{[}12{]}} Also, supramolecular hydrogels can be
tailored for application \emph{in vivo} due to their significant water
content and biocompatibility.\textsuperscript{{[}13{]}} Specifically,
employing supramolecular hydrogels based on polysaccharides for
biomedical applications utilizing CB macromolecules is not
original.\textsuperscript{{[}5{]}} It has also been described for
various biological and clinical applications. For instance, biomedical
researchers reported different designs using CB for crosslinking the
supramolecular hydrogel 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 WHO
estimation, cancer death will increase globally by 80\% by
2030.\textsuperscript{{[}14{]}} On a global level, seven out of ten
cancer deaths happened in Asia, Africa, Central and South
America.\textsuperscript{{[}15{]}} 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.

The nanocavity cucubit{[}\emph{n} {]}urils (CB\emph{n} ) are more
biocompatible than other water-soluble macrocyclic hosts because
CB\emph{n} is dexterity to
functionalization.\textsuperscript{{[}16{]}}The water-soluble
macrocyclic molecules offer their hydrophobic cavity for encapsulating
polar and non-polar guest molecules with a reasonable binding
constant.\textsuperscript{{[}17{]}} CB\emph{n} is made from
acid-catalyzed polymerization of glycoluril and formaldehyde to form
macrocycles with different numbers of monomers unit.

The eight-monomer unit containing cucurbit{[}8{]}uril (CB8) forms a
complex with viologen guests
non-covalently.\textsuperscript{{[}18{]}}The viologens are functional
organic materials first discovered by Michaelis in
1932.\textsuperscript{{[}19{]}} The viologens are the classic example of
redox and electron-deficient compounds. Several viologen-based
crystalline and amorphous photochromic materials have been synthesized
for the last ten years, enabling photochemists to develop substantial
multifunctional viologen-based photochromic
materials.\textsuperscript{{[}20{]}} To 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 is not
easy. 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 viologen diamines salts
(VG)\textsuperscript{{[}21{]}} are covalently linked to the surface of
alginate polysaccharides (ALG)\textsuperscript{{[}22{]}} (Figure 1).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{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,\textsuperscript{{[}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.

\textbf{METHODS}

\emph{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.

\emph{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.

\emph{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\textsuperscript{--1} with thirty-two scans.

\emph{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-d\textsubscript{6} solvent, and all the peaks were referenced to
residual protonated solvent with chemical shift (\selectlanguage{greek}\emph{δ} \selectlanguage{english}) =2.49 ppm.

\emph{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\selectlanguage{ngerman}° C temperature range at a heating rate of 10 °C/min under the
gaseous nitrogen flux, and the data was processed with STAR software.

\emph{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.

\emph{Size Distribution and Zeta Potential Analysis.}

The particle size (z-average d.nm) and \selectlanguage{greek}ζ \selectlanguage{english}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 \selectlanguage{ngerman}°C after 120 seconds temperature
equilibration at a backscattering angle of 173°.

\emph{Diffuse Reflectance Spectroscopy (DRS).}

The absorption spectra of the solid samples were obtained by using the
Kubelka--Munk conversion (K--M = (1 \selectlanguage{english}- R)\textsuperscript{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 0\textsuperscript{o}; 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
E\textsubscript{g} = 1240 eV nm l\selectlanguage{english}\textsuperscript{-1}, where l is the
absorption edge (in nm).

\emph{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.

\emph{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 \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}. 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 \selectlanguage{greek}τ\selectlanguage{english}\textsubscript{i} are the lifetimes with amplitudes
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{i} and [?]\textsubscript{i} = 1.0. And the sum in the
denominator is for all the decay times and amplitudes.

\emph{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.

\emph{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
dihydrateNa\textsubscript{2}EDTA 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.

\emph{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 \selectlanguage{greek}µ\selectlanguage{english}L) in RPMI-1640 medium (Gibco, Cat. No. 27016021)
containing 0.3\% L-glutamine, 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL gentamicin, 25 \selectlanguage{greek}µ\selectlanguage{english}g/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).

\emph{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 \selectlanguage{greek}μ\selectlanguage{english}l WST-1 was added to the cell culture media and
incubated for four hours at 37\selectlanguage{ngerman}°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.

\emph{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 (M\selectlanguage{greek}β\selectlanguage{english}CD), 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.

\emph{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.

\emph{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 \textless{}0.05
was considered the minimum criterion for statistical significance.

RESULTS AND DISCUSSION

\emph{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\textsuperscript{{[}24{]}} confirming the formation of VGALG and
VGCB8ALG.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. FTIR spectra of VG, ALG, VGALG, and VGCB8ALG hydrogels in KBr
solids.

Notably, the peak for (NH\textsubscript{2}) primary amine
stretching\textsuperscript{{[}21{]}} at 3202 cm\textsuperscript{--1} and
bending at 1634 and 1596 cm\textsuperscript{--1} (in VG) has evolved
into the amide (CONH) peak at 3329 cm\textsuperscript{--1} and 1627 and
1574 cm\textsuperscript{--1}, respectively (in the modified ALG).
Moreover, a significant enhancement in the intensity of the CH
stretching (in ALG)\textsuperscript{{[}22{]}} at 2927 and 2850
cm\textsuperscript{--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.\textsuperscript{{[}24{]}} Only peaks in the range from 1500 to
1400 cm\textsuperscript{--1} due to the stretching vibration of the
benzene rings of VG were slightly weakened.\textsuperscript{{[}24{]}}

In the proton NMR spectra (aromatic region), ALG shows a single broad
resonance peak at \selectlanguage{greek}δ \selectlanguage{english}= 12.30 ppm associated with the -OH group in ALG
(Figure S1 in the Supporting Information). The NMR spectrum for VG shows
five peaks, including one singlet \selectlanguage{greek}δ \selectlanguage{english}= 5.30 ppm (for
-NH\textsubscript{2}) and four doublets with resonances at \selectlanguage{greek}δ \selectlanguage{english}= 6.0,
6.75, 8.2, and 8.75 ppm.\textsuperscript{{[}25--27{]}} A new peak at \selectlanguage{greek}δ \selectlanguage{english}=
12.25 ppm appears when VG is linked to ALG due to forming 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, the peaks b-e
of the new hydrogels were further broadened (almost disappeared) upon
engulfing VG in CB8.\textsuperscript{{[}26{]}} 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 Supporting Information) 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 with CB8), 275, and
475 nm (490 with CB8).

Figure 3 shows the TGA of VG, CB8, ALG, VGALG, and VGCB8ALG in the
temperature range from 35\selectlanguage{ngerman}°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.\textsuperscript{{[}28{]}} More importantly, the results confirm
the host-guest interaction between VG and CB8 on the ALG platform.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Thermo-gravimetric analysis showing the weight losses
of VG (green), ALG (red), CB8 (dark red), VGALG (blue), and VGCB8ALG
(dark cyan).

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 in the
Supporting Information). We divided the weight \% by the molecular
weight, giving us several moles. We then concluded the percentage by
moles of VG (\textasciitilde{}20 \%) in both alginate polymers. We also
compared the number of moles of VG with CB8, which allowed us to
conclude to 2:1 VG: CB8 interaction on the alginate surface, which
agrees with other supramolecular architectures that contain
CB8-complexed viologen dyes.\textsuperscript{{[}18{]}} Table 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.

DSC data characterize the thermal behavior of individual ALG, VG, and
CB8 in terms of their simple structure, hydrophilic properties, and
association rate.\textsuperscript{{[}29{]}} The 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 in the
Supporting Information shows an endothermic peak at 94.8\selectlanguage{ngerman}°C followed by a
strong exothermic peak 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 in the Supporting
Information 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 (\selectlanguage{greek}ζ \selectlanguage{english}potential (mV)) of VG, ALG, VGALG, and VGCB8ALG
were studied by dynamic light scattering (DLS) and electrophoretic light
scattering (ELS) as shown in Figure 4.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure 4. Particle size distribution of modified hydrogels and starting
materials (A) and surface charge of different hydrogels and their
components (B).

The samples were dissolved in methanol, and the size distribution and \selectlanguage{greek}ζ
\selectlanguage{english}potential were recorded. The measurement for ALG shows particles with a
large size of 1540 nm. The \selectlanguage{greek}ζ \selectlanguage{english}potential measured consistently higher
value of -13.8 mV. The particle size measured for VG is 280 nm and has a
relatively high \selectlanguage{greek}ζ \selectlanguage{english}potential value of +34.9. As Figure 4 shows, the size
distribution for VGALG revealed a particle size of 485 nm, and a \selectlanguage{greek}ζ
\selectlanguage{english}potential of -19.8 mV, indicating the robust binding of VG to ALG. The
polydispersity index (PDI), 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 \selectlanguage{greek}ζ \selectlanguage{english}potential value shifted to +14.4 mV, indicating the
guest-host interaction in VGCB8ALG.\textsuperscript{{[}12{]}} The
manipulation in the size and charge of hydrogels upon the addition of
CB8 explains the results of cellular internalization below.

\emph{Optical Properties (Functionalization, Encapsulation,
Implementation for Cellular Uptake, and Rigidity Enhancement)}

The solid UV--Visible spectra (Figure S4 in the Supporting Information)
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.\textsuperscript{{[}30{]}}

The 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 5. PL and 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.

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 PL and PLE spectra for VG or
VGCB7\textsuperscript{{[}21,25{]}} 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 Methods section, Figure S5, and
Table S4 in the Supporting Information) increased from 0.28 to 0.85 ns
with the addition of CB8 because of the confinement
effect.\textsuperscript{{[}21,25--27{]}} A more rigid biopolymer helps
interpret the results of cellular internalization below.

\emph{Immunomodulatory Properties (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 peripheral blood mononuclear cells
(PBMCs) from healthy donors using the WST-1 test (Figure S6 in the
Supporting Information) 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 in the Supporting Information). 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. That
allows us to conclude that hydrogels under study have high
biocompatibility characteristics and low toxic effects on immune cells.
Following the literature, we expected a high safety profile from VGALG
and VGCB8ALG since each component of the hydrogels,
CB8,\textsuperscript{{[}31{]}} alginate,
\textsuperscript{{[}32,33{]}}and viologen, \textsuperscript{{[}34{]}} is
safe, biocompatible and has already been used separately to create
similar hydrogels. The hydrogels (0.18 mg/mL) were then used for
investigating the drug internalization to the PBMCs
(CD3\textsuperscript{-}and CD3\textsuperscript{+}T-lymphocytes) by flow
cytometry (Figure S8 in the Supporting Information). PBMCs uptake of
VGALG has no significant differences from the control (non-treated
cells). In the case of VGCB8ALG, internalization into
CD3\textsuperscript{-} cells was low and was measured at about 6\%,
while the median uptake by T cells was 62\%, which is ten times higher.
Noticeably, CB8 is a critical factor for internalization into the T
cells. In the case of CD14\textsuperscript{+}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 in
the Supporting Information). 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 hours of treatment (Figures S10
and S11 in the Supporting Information). Specifically, after 24 hours of
treatment with 1301 hydrogels (Figure S10 in the Supporting
Information), 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
hours of treatment with 1301 hydrogels, the percentage of viable 1301
cells differed when treated with different concentrations of VGALG and
VGCB8ALG (Figure S11 in the Supporting Information). 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 mg/ml 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 hours. In summary, VGALG can
enhance the proliferative activity of cells at average concentrations of
0.18 mg/ml and 0.018 mg/ml with long-term treatment for 72 h.
Contrarily, VGCB8ALG increased the proliferative activity of these cells
only at high concentrations (1.8 mg/ml) in culture for 24 and 72 hours.
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 in the
Supporting Information). The VGCB8ALG hydrogel was shown to be rapidly
internalized by cells; after 1 hour, more than 80 percent of 1301 cells
had absorbed the gel. After 24 and 72 hours, 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 to 15\% at
the lowest concentration. VGALG was poorly taken up by cells at all
concentrations used.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 6. Evaluation of internalization of 1301 cells 24 hours of
treatment by different concentrations of hydrogels (n=4).

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 four \selectlanguage{ngerman}°C resulted in potent inhibition of
endocytosis. The results showed a significant difference in uptake when
inhibited with nystatin, thus suggesting caveolae-mediated endocytosis
was preferred for VGCB8ALG.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 7. Effect of endocytosis inhibitors on the uptake of VGCB8ALG
(0.18 mg/mL) by 1301 cells. (n=3)

To confirm our results, 1301 cells were treated with hydrogels for 24
hours, 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 in it.
VGCB8ALG is localized in the cytoplasm of cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Figure 8. Upper cases (A, B) represent merged fluorescent images on the
magnitude X1000. The red spots represent labeled hydrogel, and the
nucleus is stained with DAPI (blue). (A) and (B) represent cellular
uptake of hydrogel alone and in complex with CB8 correspondingly. Lower
cases (a, b) represent the correspondent phase-contrast images on the
magnitude X400 where arrows indicate the cytoplasm. The concentration of
each hydrogel was 0.18 mg/mL.

\emph{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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Figure 9.} Schematic representation of blood cellular uptake of
VGALG and VGCB8ALG hydrogels. Chemical structures of VG, ALG, and CB8
are also shown. The 2:1 binary complex of CB8 with the viologen dimers
is not shown for simplicity.

As described in the introduction, employing supramolecular hydrogels
based on polysaccharides for biomedical applications and utilizing
cucurbituril macromolecules is not original and has received significant
attention. It has also been described for various biological and
clinical applications. Hydrogels are themselves a widely researched
field of (bio)materials. They have been enormously reported, and the
field is not novel.\textsuperscript{{[}12{]}} For example, hydrogel drug
delivery has already implemented the host-guest association between
cucurbit{[}7{]}uril (CB7) and
cisplatin.\textsuperscript{{[}35{]}}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.\textsuperscript{{[}36{]}} The
research concluded the significant role of size. Three different
size-dependent mechanisms for cellular uptake were suggested. Oleoyl ALG
ester nanoparticles whose sizes are 50, 120, or 730 nm enter the colon
cancer cells via Clathrin-Mediated Endocytosis (CME), CvME, or
micropinocytosis, respectively.\textsuperscript{{[}36{]}} From 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\textsuperscript{{[}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.\textsuperscript{{[}38{]}} From 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 \emph{N} ,\emph{N} -diethyl acrylamide, and
2-hydroxyethyl methacrylate cross-linked with~\emph{N} ,\emph{N}
'-methylene-bis-acrylamide were better internalized to RAW 264.7 murine
macrophage cells compared to soft
nanoparticles.\textsuperscript{{[}39{]}} From Figure S5 in the
Supporting Information, 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 involves a macromolecular system or other
drug vehicles. Although, nowadays, the field of application, material
type, and drug loading need to be precisely selected and extensively
studied, we envisage that the successful modulation of cellular
internalization upon the addition of CB8 on VGALG must have a wide
impact in the broader research field and benefits similar cellular
studies performed on other macrocycle-based biomaterials for other
biomedical applications. The technology is patentable, which would then
be of interest to international pharmaceutical companies for commercial
development.

\textbf{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.

\par\null

\textbf{Supplementary Information} The online version contains
supplementary material available at \textbf{XXXXXX} .

Author Contributions

F.C.; optical data (UV, PL, TRPL) acquisition and analysis and writing
the initial draft, V. P.; material characterization by FTIR, DLS, TGA,
DSC, and NMR, P.L.; data analysis, M.B., A.A., O.B., M.B., and E.P.;
clinical studies and contribute to manuscript writing, N.S. created the
idea, conceptualization, supervision, and final manuscript writing. All
authors have approved the final version of the manuscript.

Funding Sources

This study was supported by Grant 12R113 from UAE University.

\textbf{Data availability:} All relevant data are within the paper.

\textbf{Declarations}

\textbf{Ethical approval} The study was conducted according to the
guidelines of the Declaration of Helsinki and approved by the
Institutional Local Ethical Committee of the Research Institute of
Fundamental and Clinical Immunology (protocol No. 141; 20 April 2023).

\textbf{Consent to participate} The participants consented.

\textbf{Consent for publication} The authors consented.

\textbf{Conflict of interest} The authors declare no competing
interests.

AUTHOR INFORMATION

Corresponding Authors

n.saleh@uaeu.ac.ae; pashkina.e.a@yandex.ru.

ACKNOWLEDGMENTS

We thank the research program at United Arab Emirates University for
financial grant number Grant 12R113 and the Russian Science Foundation
for financial grant number 19-15-00192.

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\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\textbf{Graphical Abstract.}

CB8 turns over the charge and decreases the size of VGALG hydrogel,
enabling it to enter the blood cancer cells.

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