Biomimetic approaches toward the control of bacterial infections
Li, Yuanfeng
DOI:
10.33612/diss.171588622
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Publication date: 2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Li, Y. (2021). Biomimetic approaches toward the control of bacterial infections. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.171588622
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CHAPTER 3
A G‑quadruplex Hydrogel via Multicomponent
Self‑Assembly: Formation and Zero‑Order
Controlled Release
Y. Li, Y. Liu, R, Ma, Y. Xu, Y. Zhang, B. Li, Y. An, L. Shi.
ACS Applied Materials & Interfaces. 2017, 9, 13056-13067.
Reproduced with permission from ACS publications.
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ABSTRACT
Stimuli-sensitive hydrogels are ideal candidates for biomedical and bioengineering purposes, although applications of hydrogels may be limited, due in part to the limited choice of suitable materials for constructing hydrogels, the complexity in the synthesis of the source materials, and the undesired fast-then-slow drug-release behaviors of usual hydrogels. Herein, we describe the fabrication of a new supramolecular guanosine (G)-quadruplex hydrogel by multicomponent self-assembly of endogenous guanosine (G), 2-formylphenylboronic acid (2-FPBA), and tris(2-aminoethyl)amine (TAEA) in the presence of KCl in an easy and convenient way. The features of the G-quadruplex hydrogel include (1) versatility and commercial availability of building blocks with different functions, (2) dynamic iminoboronate bonds with pH and glucose responsiveness, and (3) zero-order drug-release behavior because of the superficial peel-off of the hydrogel in response to stimuli. The structure, morphology, and properties of the G-quadruplex hydrogel were well-characterized, and satisfactory zero-order drug release was successfully achieved. This kind of supramolecular G-quadruplex hydrogels may find applications in biological fields.
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1. INTRODUCTION
In recent years, hydrogels have been applied extensively in the development of platforms for biomedical and bioengineering purposes.1−5 With a three-dimensional network, hydrogels
can encapsulate a large amount of cargos, protect the cargos from environmental variations, and/or control cargo release by designing the gel structure in response to microenvironment variations.6,7 Stimuli-sensitive hydrogels are ideal candidates for developing self-regulated
drug-delivery and -release systems.2,8−10 However, hydrogels are generally constructed from
limited natural polymers or synthetic polymers via complex procedures, which restricts the application of hydrogels in the biomedical and bioengineering fields to some extent.11,12 To
make things worse, most of the hydrogel-based controlled drug-release systems exhibit two-step release profiles (fast-then-slow behavior), whereas an ideal candidate should release the cargo at a constant rate.13,14 The range of applications of hydrogels in the biological field
would be greatly expanded if such issues could be overcome.15
Since Davies and co-workers reported the hydrogel formation by guanosine (G) deviations in 1962,16 endogenous motifs have been incorporated into hydrogels, including DNA,17,18
antibodies,19 polypeptides,20 and enzymes.21−23 The G-quartet, a hydrogen-bonded square
planar structure formed by cation-templated assembly of guanosine, can stack together to form a G-quadruplex, which could be extended to a fibrous network to form supramolecular hydrogels.15,24,25 Owing to the dynamic combinatorial chemistry and dynamic covalent
bonds involved in the formation of guanosine-based hydrogels, researchers can reversibly modify,26,27 exchange, or rearrange the building blocks in hydrogels, which can
greatly enlarge the range of material source for the preparation of hydrogels. Lehn and Sreenivasachary demonstrated that a hydrazide gelator deviated from guanosine readily forms G-quartets with carbonyl compounds through acylhydrazone bonds.27,28 More
recently, Davis and Peters described a stable G-quadruplex hydrogel made from guanosine and 0.5 equiv of KB(OH)4.29,30 To our knowledge, most of the previous works mainly
focused on the formation and stability of the G-quadruplex gels. However, recently, the interest in the G-quadruplex structures has focused on their potential biological applications (i.e., aptamers, drug-release systems).31−33
It is well known that the cis-diols on the furanose ring of guanosines are inclined to form five-membered cyclic boronate esters with boronic acids.34,35 Formylphenylboronic acids
(FPBAs) are versatile building blocks for multicomponent self-assembly via iminoboronate bonds, which allow for multiple applications, including protein modification,36 bacteria
targeting,37 and organized architectures.38−40 More importantly, the iminoboronate bonds are
essentially composed of two kinds of typical dynamic covalent bonds, the cyclic boronates and the imino bonds, which can endow a self assembled system with glucose- and acid-triggered drug release, respectively.36,41 Therefore, a hydrogel stabilized and crosslinked by
iminoboronate bonds has promising applications in the biological fields.42
In the past decades, lots of drug-delivery devices were designed. However, most of them showed fast-then-slow release profiles, which is mainly because diffusion is the main driving force for the drug release.43,44 Diffusion always occurred because of the random
molecular motion of the drugs and due to concentration gradients. Especially for cargos loaded in polymeric systems, with the swelling of carriers and osmotic pressure, burst release may happen. Fast-then-slow and burst release resulted in unpredictable release doses and uncontrollable durations. It is desirable to fabricate a controlled drugrelease system
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with a steady and even a zero-order release profile, which is very important for maintaining higher levels and longer effective concentrations of the drug. In the zero-order release system, diffusion of the drug may be limited as far as possible and surface erosion of the carrier from the exterior toward the interior may be necessary.45 Many efforts have been
made to design and fabricate zero-order release systems. Williams et al. created functional trilayer nanofibers through triaxial electrospinning, which provided a linear release of ketoprofen based on loading incremental contents of ketoprofen from the exterior layer of fibers to the inward layer.46 Solaiappan et al. introduced three-dimensional aluminosiloxane
aerogel microspheres to uptake the drug and release with a zero-order kinetics following the Korsmeyer−Peppas model because of the multidimensional hierarchically porous structure, which controlled the diffusion of the drug molecules.47 Zhang et al. designed dynamic
layer-by-layer films made of two polymers with a narrow molecular weight distribution, which provided a platform with intelligent and zero-order release profiles because of its dissociation of the interpolymer complex.13 Although different kinds of zero-order
drug-release systems have been studied, hydrogels with stimuli-responsive and zero-order drug-release profiles have been rarely reported. More importantly, the synthesis and processes involved in most of the zero-order drug-release systems are usually complicated, which essentially restricted their extensive and in-depth application in biological fields. It is still a big challenge to fabricate hydrogels with stimuli-responsive and zero-order release profiles by using commercially available materials in an easy and convenient way.
In this study, G-quadruplex structures were used to form stimuli-sensitive hydrogels with zero-order release from commercially available materials, to solve the problem of uncontrolled drug release. The supramolecular hydrogels are constructed by the dynamic multicomponent self-assembly of guanosine (cis-diol-containing motif), 2-formylphenylboronic acid (2-FPBA, boronic acid and aldehyde group-containing motif), tris(2-aminoethyl)amine (TAEA, amino group-containing motif), and KCl (cations) in an aqueous solution (Scheme 1). Hydrogelation is driven by (1) Hoogsteen-type hydrogen
bonding between the guanine groups that are templated by potassium cations; (2) the iminoboronate bonds spontaneously and synergistically formed among guanosine (diols), TAEA (primary amino groups), and 2-FPBA (aldehyde and boronic acid groups), and (3) the stacking of the formed G-quartets. This kind of supramolecular hydrogel has supreme features including (1) versatility and commercial availability of building blocks with different functions, (2) dynamic iminoboronate bonds with pH and glucose-responsiveness, and (3) zero-order drug-release behavior because of the superficial peel-off planar structure of the component units and dense fibrils of the hydrogel in response to stimuli. The crucial factors of hydrogelation are studied and spectrometry methods are applied to confirm the formation of the G-quadruplex structures and iminoboronate bonds. Morphologies of hydrogels were compared before and after exposure to stimuli via microscopic technologies. The G-quadruplex hydrogels are subsequently loaded with model drugs, and their zero-order drug release behavior is evaluated.
2. RESULTS AND DISCUSSION
2.1 Formation of the G‑quadruplex hydrogel via multicomponent self‑assembly. Guanosine is a purine nucleoside comprising guanine attached to a ribose (ribofuranose) ring via a β-N9-glycosidic bond (Wikipedia). Although guanosine is not water-soluble
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the presence of K+ in ultrapure water by heating the solution mixture to boiling and then
cooling it down. Gelation of the solution mixture was mainly attributed to three kinds of important interactions among the components, as illustrated in Scheme 1. The first one
was a supramolecular interaction of the Hoogsteen-type hydrogen bonding between the guanine groups that were templated by potassium cations, which resulted in the formation of a G-quartet structure and has been reported by Davies et al.16 The second one was the
spontaneous and synergistic formation of the iminoboronate bonds among guanosine, 2-FPBA, and TAEA. The cis-diol on the ribose of guanosine is inclined to combine with the phenylboronic acid group on 2-FPBA through a cyclic boronate structure, and the primary amino groups on TAEA also easily combines with the formyl on 2-FPBA through an imino bond. These two kinds of combinations are collectively called iminoboronate bonds as they occurr synergistically and reinforce each other.48 The iminoboronate bonds can
connect adjacent G-quartets because of the trifunctionality of TAEA. The third important interaction was the supramolecular stacking of the G-quartets, leading to the formation of a fibrous G-quadruplex structure. As a joint effect of the three kinds of important interactions, the G-quadruplex hydrogels were successfully obtained. In this study, the G-quadruplex hydrogels were prepared by mixing, heating, and cooling a mixture composed of guanosine, 2-FPBA, TAEA, and KCl at a molar ratio of 1:1:1/3:1/4 in ultrapure water (Scheme 2).
It was found that temperature and concentration played key roles in the formation of the G-quadruplex hydrogels. The formed hydrogels were visually thermoreversible as they
Scheme 1. Schematic presentation of the multi-component self-assembly of guanosine, 2-FPBA,
TAEA and KCl for the formation of G-quartet, G-quadruplex and hydrogels and the dissociation of the G-quadruplex hydrogels in response to the stimuli of glucose and acid.
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transformed between solution and gel phases upon alternating heating and cooling cycles. The time−autocorrelation function of light scattering was measured using DWS by adding probe particles (polystyrene 222 nm) into the hydrogel solution. In Figure 1A, with the
temperature decreased from 70 to 33 °C, the time scale for the decay of the autocorrelation
Scheme 2. Synthesis of the G-quadruplex hydrogel.
Figure 1. (A) Autocorrelation curves of hydrogel (25 mM, 0.5‰ 222 nm polystyrene particles were
loaded) recorded by diffusing wave spectroscopy under different temperatures 70 °C to 33 °C. (B) Pictures of formed hydrogels (1) and the mixtures in the absence of KCl (2), TAEA (3), 2-FPBA (4), and after replacement of KCl with NaCl (5) or NH4Cl (6). All experiments were kept with the same
guanosine concentrations (25 mM).
Table 1. Screening of hydrogel formation conditions.
a. For detailed phenomena see Figure 1B.
Entry G KCl TAEA 2‑FPBA Phenomena a
1 √ √ √ √ white hydrogel
2 √ - √ √ clear solution
3 √ √ - √ suspension liquid
4 √ √ √ - suspension liquid
5 √ NaCl √ √ clear solution
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curve greatly increased from 10−3 to 10−1, which indicated that the freely diffusing state
of the probe particles was restricted because of gelation with decreased temperature. Gelation point was the temperature at which decays of autocorrelation function showed an obviously longer time scale. For hydrogels with different concentrations, gelation occurred in a narrow temperature range, and the gelation temperature increased with the increasing in the hydrogel concentration. The gelation temperatures were 35, 38, and 45 °C for hydrogels with concentrations of 25, 30, and 35 mM, respectively (Figures 1A and Figure S1, Supporting Information).49 It should be noted that 25 mM or 0.7 wt % was
the lowest critical gelation concentration for guanosine, which is the lowest reported value for hydrogels made of guanosine analogues.29 These hydrogels were stable at ambient
conditions for more than one month without any visible variations. For demonstration of the roles of KCl, TAEA, and 2-FPBA played in the gelation, tests were performed with the absence of either one or replacement of them by analogues, and the results are given in
Figure 1B and Table 1. The absence of either one of KCl, TAEA, or 2-FPBA could lead
to the failure of gelation. When K+ was replaced by Na+ and NH
4+, precipitation always
occurred, indicating that K+ was necessary for the hydrogel formation. As far as molecules
with primary amino groups are concerned, a functionality larger than 2 was necessary, and a higher functionality could facilitate the formation and stability of the hydrogel (i.e., polyethyleneimine > TAEA >> ethylenediamine). TAEA was purposely used in this work for the formation of low-molecular-weight hydrogels. If 2-FPBA was replaced by 3-FPBA or 4-FPBA, a hydrogel could also be obtained with the only disadvantage of a lack of synergy between the cycloboronate and the imine bond.
2.2 Determination of G‑quadruplex structures in hydrogels.
The stacking of the hydrogen-bonded square planar G-quartet formed by the potassium cation-templated assembly of guanosine was one of the main driving forces for the gelation of our systems by forming a G-quadruplex.24,25 To confirm the existence of the G-quadruplex
structure, fluorescent spectrometry was used with PpIX and ThT as the fluorescence probes. In aqueous solution, PpIX gives a low fluorescence intensity owing to its poor water-solubility and aggregation. Once PpIX binds to the G-rich DNA, which contains the G-quadruplex self-assembled by G, the fluorescence intensity of PpIX can be remarkably enhanced via π−π stacking interactions.50,51 As shown in Figure 2A, the fluorescence
emission of PpIX itself in aqueous solution was relatively low, whereas it was greatly enhanced when PpIX was added to the G-containing hydrogels. This might be interpreted as being caused by the bonding among guanosines through Hoogsteen-type hydrogen bonding, which allowed PpIX to stack on them via π−π interactions. Although the addition of PpIX into a mixture without K+ could also enhance the fluorescence emission of PpIX, it
was much lower than the emission in the K+-templated stable hydrogels. Unlike PpIX, ThT
is a hydrophilic fluorogenic dye and has been applied to identify telomeric G-quadruplexes and G-quartets assembled by guanosines in vitro for its strong enhancement in fluorescence after binding to the G-quartet motifs.52−54 It was found that ThT (7.5 μM) alone showed a
very weak fluorescence emission profile centered at 527 nm (Figure 2B). However, the
fluorescence emission of ThT increased significantly after mingling with the G-containing hydrogels, and the enhancement of fluorescence (I/I0) at 519 nm was found to be as large
as 7 fold. If K+ was absent in the solution, the fluorescence emission spectrum of ThT was
centered at 490 nm, which was different from that of K+-containing counterparts. These
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structure in the hydrogel and demonstrating the key role of K+ in the stabilization and
formation of G-quadruplex structures.
To further validate the formation of the G-quadruplex, an XPRD experiment was performed to get more evidences and study certain parameters about the secondary structure of the G-quadruplex. Figure 2C shows the XRPD pattern of the lyophilized powder of the
formed hydrogels. The Bragg diffraction peak at 2θ = 3.40° corresponded to a distance d = 25.9 Å, compatible with the width of a single G-quartet. The sample also displayed a broad Bragg diffraction peak at 2θ = 20.9° (d = 4.25 Å), just consistent with the data reported.55
The peak at 2θ = 26.9° (d = 3.31 Å) corresponded to the π−π stacking distance between two G-quartets. The additional peaks at 2θ = 28.3° (d = 3.15 Å) and 40.4° (d = 2.23 Å) corresponded to crystalline KCl. The XPRD test proved the existence of a G-quadruplex and gave the microstructural parameters.
2.3 Formation of iminoboronate bonds in G‑quadruplex hydrogels.
The spontaneous and synergistic formation of iminoboronate bonds among diols (G),
Figure 2. G-quadruplex structure detection. (A) Fluorescence emission spectra of PpIX in phosphate
buffer (10 mM, pH 7.4, blue dotted line), mixtures in the absence of K+ (black dotted line) and
hydrogel (red solid line). (B) Fluorescence emission spectra of ThT in phosphate buffer (10 mM, pH 7.4, blue dotted line), mixtures in the absence of K+ (black dotted line) and hydrogel (red solid line).
(C) X-ray powder diffraction spectrum of G-quadruplex hydrogel with an inset G-quartet structure to present crystal data.
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primary amino groups (TAEA), and 2-FPBA was also one of the main driving forces for the gelation of our systems. To get insight into the G-quadruplex hydrogel, FT-IR and NMR spectra were used to analyze the formation of iminoboronate bonds. As shown in the FT-IR spectra in Figure 3A and B, the free νC=O = 1674 cm–1 band disappeared, and νC=N = 1694
cm–1 in the G-quadruplex hydrogel appeared, indicating the reaction of the aldehyde groups
on 2-FPBA with the primary amino groups on TAEA. At the same time, νO‒H = 3340, 3070
cm-1 in the 2-FPBA disappeared after gelation, and the G-quadruplex hydrogel showed an
absorption band at around 1014 cm-1 (ν
B-OC) instead of the vibration at 1296 cm-1 (νB-OH),
which supported the formation of the cyclic boronate ester. These results demonstrated the formation of iminoboronate bonds in the G-quadruplex.39
NMR measurements were also performed to identify iminoboronate bond formation. As shown in Figure 3C, the 1H NMR spectra of 2-FPBA showed an obvious peak at δ
9.89 ppm (–CHO), and this peak was replaced by a δ 8.36 ppm peak (–CH=N–), which indicated the formation of imine bonds. In the 11B NMR spectra of 2-FPBA, there was only
a single sharp peak at δ 30.26 ppm, which was attributed to the free boronic acid. This peak,
Figure 3. Iminoboronate bonds detection and hydrogel characterizations. (A) FT-IR spectra
(wavenumbers from 600 to 4000 cm-1) of guanosine (red line), 2-FPBA (blue line), and G-quadruplex
hydrogel (black line), (B) details of FT-IR spectra, wavenumbers from 500 to 1800 cm-1, (C) nuclear
magnetic resonance spectra of formed G-quadruplex hydrogel and 2-FPBA in D2O, 1H NMR spectra
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however, shifted to δ 8.69 ppm and became a broad absorption band after hydrogelation, which suggested the chemical change of boron atoms because of the formation of boronate bonds.13 However, when 2-FPBA and TAEA were mixed together in the absence of
guanosine, the formation of the C=N bonds was not detected (Figure S3, Supporting Information), which indicates that the imine bonds and the cyclic boronate bonds were
formed in a synergetic manner. This may be attributed to the fact that the nitrogen atom was electron-rich, whereas the boron atom was electron-deficient, and their vicinal position facilitated the N → B coordination. Therefore, the cyclic boronate bonds and the imino bonds, as well as the N → B coordination, were collectively called iminoboronate bonds as they occurred synergistically and reinforced each other, as described well in pioneer works.56−58
To confirm whether the iminoboronate bonds were crucial for hydrogelation, a control experiment was carried out with the addition of Alizarin red S (ARS) into the solution mixture for hydrogelation. ARS carries a catechol structure, which has a stronger interaction with the boronic acid than the cis-diol of G. As showed in the Figure S4A, Supporting Information, the G-quadruplex hydrogel could not be formed in the presence
of stoichiometric equivalent ARS in the system; only a viscous dark brown suspension was obtained. The dramatically increased fluorescence emission intensity of ARS indicated the binding of ARS with boronic acids (Figure S4B, Supporting Information).59 The stronger
binding between ARS and 2-FPBA prevented the formation of iminoboronate bonds as well as the G-quadruplex hydrogels, which proved the importance of iminoboronate bonds in gelation.
Figure 4. Morphology characterizations of G-quadruplex hydrogel (with a guanosine concentration
of 35 mM): (A, B) SEM images with different magnifications, (C) TEM image to present the fibrous network structures of G-quadruplex hydrogels and (D) statistical data of the width of fibres in panel A quantified via Image J software and data were expressed in percentage.
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2.4 Morphology and rheology of the G‑quadruplex hydrogels.
Electron microscopic technologies were applied to investigate the morphology of hydrogels. SEM images clearly show a fibrous structure for the hydrogel (Figure 4A and B).
The TEM image in Figure 4C also shows dense, entangled fibrils consistent with previous
observations.29 The cross-sectional diameters of cylindrical fibers were in the range from
about 20 to 85 nm but more commonly from 25 to 45 nm (Figure 4D) according to the
SEM image in Figure 4A. Further observation of the thicker fibers revealed that they were
composed of several thinner fibers. This fibrous structure also confirmed the formation of a G-quadruplex by the supramolecular stacking of G-quartets. In addition, it could be observed from both SEM and TEM images that the hydrogels were very compact with extraordinarily dense fibers interpenetrated, and the pores in hydrogels were usually less than 100 nm in diameter. The rheology of the G-quadruplex hydrogel was measured on a rheometer. When examined as a function of frequency, the storage modulus (G′) remained larger than its loss modulus (G″), indicating a viscoelasticity for the hydrogel (Figure 5A).
G′ and G″ exhibited weak frequency and strain dependence (Figure 5B), indicating that
the fibrous network did not relax even over long time scales. These fine morphology and rheology properties provided the hydrogel with potentials for biomedical applications. 2.5 Stimuli‑responsive drug‑release behaviors of the G‑quadruplex hydrogel.
As discussed above, the formation of the iminoboronate bonds was one of the main driving forces for gelation, and the iminoboronate bonds were composed of cyclic boronates and imino bonds, which endowed the G-quadruplex hydrogel with glucose and acidresponsiveness, respectively, as illustrated in Scheme 1. When the G-quadruplex
hydrogel was exposed to an aqueous solution with glucose, the cis-diols in glucose could react with the formed cyclic boronate ester by substituting guanosine, which broke the connections between the G-quartets and thus destroyed the cross-linked hydrogel structure. When an acid stimulus was used, not only were the hydrogen bonds between guanosines broken, but also the imino bonds between the aldehyde group in 2-FPBA and the primary amino groups in TAEA were destroyed because of protonation of the amino groups. As a result, the quadruplex hydrogel was dissolved.
Figure 5. Rheological tests of G-quadruplex hydrogel (with a guanosine concentration of 35 mM): (A)
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In this study, two kinds of model drugs including methylene blue (Mn = 319.9) and FITC−lysozyme (Mn = 14000) were encapsulated in the G-quadruplex hydrogels and stimuliresponsive drug release was investigated. The G-quadruplex hydrogels are quite different from other hydrogels in which the cargos are locked in situ during the formation of the hydrogel instead of the use of other specific interactions. In fact, any cargo can be easily loaded in the G-quadruplex hydrogel provided it is evenly dispersed in the solution mixture before gelation. As shown in Figure 6A, when the hydrogels were exposed to
phosphate buffer of pH 7.4, about 50% of methylene blue was released in 12 h, whereas hydrogels exposed to a pH 5.0 phosphate buffer released about 80% methylene blue in 12 h of incubation, indicating an acid-responsive drug release. Figure 6B showed the
glucose-responsive release profiles of methylene blue from the G-quadruplex hydrogels. It was observed that the release rate was significantly increased by the glucose stimulus, and a
Figure 6. Stimuli-triggered release of methylene blue and FITC-lysozyme from G-quadruplex
hydrogel in vitro. (A) Cumulative methylene blue release as a function of time after exposure to buffer control (10 mM phosphate buffer pH 7.4, black square), acidic stimulus (10 mM acetate buffer pH 5.0, green cycle), and glucose in acidic aqueous solution (10 g L-1 glucose in pH 5.0 acetate buffer,
red triangle), (B) Cumulative methylene blue release as a function of time after exposure to buffer control (10 mM phosphate buffer pH 7.4, black square), glucose solution (5 g L-1 glucose in pH 7.4
phosphate buffer, yellow cycle), and glucose solution (10 g L-1 glucose in pH 7.4 phosphate buffer,
blue triangle). (C) and (D) Cumulative FITC-lysozyme release as a function of time after exposure to the same stimuli (A) and (B) respectively. All data were fitted using zero-order model, and the fit lines were shown in each figure.
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higher release rate was obtained under higher glucose concentrations. On combining the two stimuli of acid and glucose together, the G-quadruplex hydrogels had the fastest release rate at pH 5.0 with a 10 g L−1 glucose stimulus, as indicated in Figure 6A (red triangle).
Furthermore, the release behavior of FITC−lysozyme with a higher molecular weight from the G-quadruplex hydrogel was also studied. As shown in Figure 6C and D, the release
profiles of FITC−lysozyme were similar to those of methylene blue, whereas the release rates were relatively slower, and a short induction period was observed. The slower release rates may be attributed to the lysozyme carrying multiple primary amino groups, which could react with 2-FPBA via imino bonds. Thus, the FITC−lysozyme may be involved in the formation of the G-quadruplex hydrogel to some extent, resulting in a close loading of the drug and a denser structure of the hydrogel as indicated by a visually lower transparency and a physically higher stiffness. The dissolution of the hydrogel and the release of FITC− lysozyme were slower.10 Actually, all hydrogels were completely dissolved in response to
different stimuli at last, and the cargos were 100% released. 2.6 Proposed mechanism for zero‑order drug release.
Through the experiment of stimuli-responsive release, it was found that the release
Table 2. Kinetic parameters of three equations (zero-order equation, first-order equation and Higuchi
equation) in fitting the release data of methylene blue from G-quadruplex hydrogel. K represented the rate of release, R2 evaluated the fitness of equation in fitting data.
Table 3. Kinetic parameters of three equations (zero-order equation, first-order equation and Higuchi
equation) in fitting the release data of FITC-lysozyme from G-quadruplex hydrogel. K represented the rate of release, R2 evaluated the fitness of equation in fitting data.
Stimuli Zero‑order equation First‑order equation Higuchi equation
K0 R2 K1 R2 KH R2 pH 7.4 buffer 4.34 0.984 0.238 0.914 15.1 0.780 pH 5.0 buffer 6.43 0.990 0.256 0.919 22.9 0.816 pH 7.4, 5 g L-1 glucose 6.95 0.991 0.264 0.895 32.4 0.918 pH 7.4, 10 g L-1 glucose 8.18 0.989 0.245 0.924 29.3 0.823 pH 5.0, 5 g L-1 glucose 8.93 0.995 0.243 0.862 31.9 0.859
Stimuli Zero‑order equation First‑order equation Higuchi equation
K0 R2 K1 R2 KH R2 pH 7.4 buffer 2.05 0.941 0.537 0.885 5.76 0.681 pH 5.0 buffer 2.65 0.940 0.505 0.883 7.56 0.656 pH 7.4, 5 g L-1 glucose 3.11 0.958 0.474 0.824 8.95 0.756 pH 7.4, 10 g L-1 glucose 3.88 0.983 0.329 0.819 12.0 0.833 pH 5.0, 5 g L-1 glucose 4.24 0.971 0.439 0.824 13.1 0.747
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behaviors were different from those in the general fast-then-slow release profiles. To study the mechanism of drug release, zero-order, first-order, and Higuchi models were applied to fit the release profiles. Zero-order release, with a constant release rate for an extended time, is the ultimate goal of most controlled-release formulations.60 The equation for the
zero-order model is
Mt = M0 + K0t (1)
The first-order model, which accounts for burst release, is presented as: ln(Mt) = ln(M0) + K1t (2)
The Higuchi equation, simplifying from Fick’s law which is the best description of diffusion-controlled delivery system,61 is expresses as:
Mt = KHt1/2 (3)
where Mt is the amount of drug released in time t, M0 is the initial amount of drug in the
solution, K0 is the zero-order release rate constant, K1 is the first-order release rate constant, KH is the Higuchi model rate constant, and t is the release time. The rate constants (K) and R2 were calculated from the plots of M
t against t, In(Mt) against t, or Mt against t1/2, for the
zero-order, first-order, and Higuchi models, respectively (Tables 2 and 3).62−65 The different
rate constants (K) indicated the stimuli-responsiveness of the G-quadruplex hydrogel; larger
K values indicate faster release rates and represent more efficient stimuli-responsiveness.
The R2 values were used to evaluate the fitness of equations; the closer the value is to 1,
the more suitable is the equation in fitting the data. The different R2 values of the three
mathematical models indicates that the release more likely tends to follow the zero-order
Figure 7. SEM images of G-quadruplex hydrogels’ surface (A) 25 mM hydrogel after drying in air.
The surfaces of hydrogels after exposure to (B) 10 mM acetate buffer pH 5.0, (C) 10 g L-1 glucose
solution in 10 mM phosphate buffer pH 7.4, (D) 10 g L-1 glucose solution in 10 mM acetate buffer pH
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model with R2 > 0.980 for the methylene blue-loaded hydrogel and R2 > 0.930 for the
FITC−lysozyme-loaded hydrogel. The zero-order fit lines were also coherent with the data, as showed in Figure 6.
To understand this zero-order release, the morphologies of hydrogels after exposure to different stimuli were investigated by SEM (Figure 7). As shown in the images, the original
dense fibrous network of the hydrogel (Figure 7A) has been eroded to different extents after
exposure to the phosphate buffer at pH 5.0 (Figure 7B), 10 g L−1 glucose solution at pH
7.4 (Figure 7C), and 10 g L−1 glucose solution at pH 5.0 (Figure 7D). Larger pores were
not shown after exposure to stimuli, indicating that the hydrogels were gradually peeled off instead of swelled. This was consistent with the observation that the hydrogel was dissolved at the surface in contact with the stimuli without apparent swelling, and finally a clear solution was obtained. It is well known that porous hydrogels, the pore sizes of which are much larger than the molecular dimensions of the guest molecules, tended to release cargos in a diffusion controlled manner, and an unsatisfactory fast-then-slow drug release behavior is usually achieved. However, for nonporous hydrogels with compact networks, drug diffusion is greatly restricted because of steric hindrance. As far as our G-quadruplex hydrogel is concerned, the compact structure with dense, entangled fibrils may restrict drug diffusion, whereas the dissolution of the hydrogel because of superficial peeling-off in response to stimuli can enable the drug release. As comprehensive results, zero-order drug-release behaviors are achieved. It is well known that decrease of systemic toxicity by inhibiting burst drug release and increase of therapeutic efficacy by maintaining prolonged serum levels are always desired for anti-infection and antitumor therapies. The G-quadruplex hydrogels with properties including zero-order drug release, weak acid-responsiveness, and degradation products as small molecules that can be easily cleaned may be promising candidates for delivery of ibuprofen and aspirin in anti-infection therapy and for delivery of paclitaxel in antitumor therapy. Further studies focusing on the biomedical application of the G-quadruplex hydrogels will be reported in the future.
3. CONCLUSIONS
In conclusion, we developed supramolecular G-quadruplex hydrogels by multicomponent self-assembly of guanosine, TAEA, and 2-FPBA on the basis of the formation of a G-quartet via Hoogsteen-type hydrogen bonding among four guanosines mediated by potassium cations, the iminoboronate bonds spontaneously and synergistically formed among guanosine, TAEA, and 2-FPBA, and the stacking of G-quartets. The components of the hydrogel were commercially available, and the self-assembly was performed in an easy and convenient manner. Fluorescent spectrometry and XPRD demonstrated the existence of a G-quadruplex structure, whereas FT-IR and NMR analyses confirmed the formation of iminoboronate bonds in the hydrogels. SEM and TEM images showed a fibrous and compact network of the hydrogel. The iminoboronate bonds not only facilitated the formation of the hydrogel but also endowed it with glucose and acid-responsiveness. Zero-order releases of methylene blue and FITC−lysozyme were achieved because of the superficial peeling-off and dissolution of the hydrogel under the stimuli of acid and glucose. This kind of supramolecular G-quadruplex hydrogel formed by multicomponent self-assembly of small molecules with features of versatility and commercial availability of building blocks, pH and glucose-responsiveness, and zero-order drug-release behavior may be a promising candidate for application in biological fields.
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MATERIALS AND METHODS
Materials. Guanosine (G, 98%), methylene blue (99%), 2-formylphenylboronic acid
(2-FPBA, 97%), and fluorescein isothiocyanate (FITC, 90%) were purchased from Heowns Biochem Technologies. Potassium chloride (KCl, 99.5%), glucose (99%), alizarin red S (ARS), were purchased from J&K Chemical. tris(2-aminoethyl)amine (TAEA, 97%) were purchased from Strem Chemical). Protoporphyrin IX (PpIX, 95%), thioflavin T (ThT, 95%), chicken egg white lysozyme (lysozyme, 90%) were purchased from Sigma-Aldrich. All the reagents were used as received. Buffers we used are phosphate buffer (PB) solution (10 mM, pH 7.4), acetate buffer (10 mM, pH 5.0) and borate buffer (100 mM, pH 9.0). All aqueous solutions were prepared with ultrapure water (> 18 MΩ) from a Millipore Milli-Q system.
Preparation of G‑quadruplex hydrogel. For the preparation of G-quadruplex hydrogel,
G (7.06 mg, 25 mmol), 2-FPBA (3.75 mg, 1 equiv. to G), TAEA (1.22 mg, 0.33 equiv. to G), and KCl (0.47 mg, 0.25 equiv. to G) were added to a clean test tube, followed by addition of ultrapure water (pH 6.5, 1.0 mL). The resulting mixtures were heated to boiling, and kept for 2 min until a clear, fine solution was obtained. The G-quadruplex hydrogel formed when the solution gradually cooled to room temperature with a final guanosine concentration of 25 mM. Unless otherwise noted, hydrogels were prepared at a molar ratio of 1:1:1/3:1/4 corresponding to G:2-FPBA:TAEA:MCl (M = Na, K, NH4). For the preparation of drug
loaded hydrogels, the solution mixture without drug was first heated to boiling and cooled to temperature slightly higher than the gelation point. Then drug was added and evenly dispersed in the gel solution under slight vortex for 5 min. Finally, drug loaded hydrogel was obtained after cooling the mixture to room temperature.
Gelation point test via diffusing wave spectroscopy (DWS). To understand the gelation
point, hydrogels with different concentrations (25 mM, 30 mM, and 35 mM) were tested via diffusing wave spectroscopy. The hydrogels were prepared through the general procedure with 0.5‰ (weight) polystyrene nanoparticles (diameter = 222 nm) were added into the hot solution as tracking probes. Time autocorrelation functions of light scattering were recorded on a RheoLab Ⅲ diffusing wave spectroscopy (LS Instruments AG, Switzerland). The beam laser of wavelength λ = 685 nm, 40 mW, was focused to the sample cell (10.0 mm length). Temperature was set varying from 70 °C to 30 °C, and stayed for 15 min at each time point before testing.
Characterization of G‑quadruplex structure via spectrometry methods. To determine
the G-quadruplex structures in the formed hydrogels, PpIX-containing G-quadruplex hydrogel was also prepared according to the general procedure with extra freshly prepared PpIX solution aliquot (50 μL, 2.3 mM) added after heating. Upon cooling to about 45 °C, the PpIX-containing G-quadruplex solution was then diluted with Milli-Q water to a total volume of 2 mL and kept at room temperature in the dark for 0.5 h to ensure the complete interactions between PpIX and G-quadruplex structures. For fluorescence spectroscopic analysis, spectra were recorded on a Hitachi F-4600 fluorescence spectrophotometer (Japan) with a 410 nm light to excite PpIX and fluorescence emission spectra were collected from 550 to 750 nm. For controls, PpIX solution (without hydrogel) and PpIX-guanosine solution mixture in the absence of potassium ion (K+) were also examined according to the
above-mentioned methods. For G-quadruplex structure detection using ThT, the procedure was essentially the same as the PpIX-quadruplex method with the using of ThT (30 μL, 0.5 mM)
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and the excitation wavelength of 450 nm for collecting the fluorescence emission spectra from 470 to 600 nm. The ThT solution (without hydrogel) and ThT-guanosine solution mixture were also used as controls.
X‑ray powder diffraction (XPRD) and Fourier transform infrared spectroscopy (FT‑IR) measurements. For XPRD and FT-IR measurements, G-quadruplex hydrogel
with the concentration of 35 mM was lyophilized. X-ray powder diffraction (XPRD) were recorded on a D/Max-2500 X-ray diffractometer using Cu-Kα radiation at 20 °C while FT-IR spectra were collected on a Bio-Rad FTS 6000 FT-FT-IR instrument using 128 scans at an 8 cm−1 resolution. For comparisons, the FT-IR spectra of small molecules of 2-FPBA and
guanosine were also collected after drying in vacuum at room temperature overnight.
Nuclear magnetic resonance (NMR) spectroscopy. G-quadruplex hydrogel sample for
NMR measurement was prepared as described above, with deuterated water (D2O) used for
replacing ultrapure water. After heating the sample to boiling, the clear solution (0.5 mL) was transferred into an NMR tube, allowed to cool to room temperature gradually and set for 24 h. 1H NMR and 11B NMR spectra were recorded on a Bruker AVANCE III 400 MHz
spectrometer at 25 °C. For comparisons, NMR spectra of small molecules like 2-FPBA, TAEA and guanosine in D2O were also collected.
Scanning electron microscope (SEM) and transmission electron microscopy (TEM) measurements. In order to get the morphology information, G-quadruplex hydrogel with
the concentration of 35 mM was lyophilized for SEM measurements with a JEOL JSM-7500F field emission microscope. For the characterization of the morphology changes after exposure to different stimuli, freshly prepared G-quadruplex hydrogel (35 mM) was prepared, and different stimuli were added on the surface of the hydrogel for 2 h followed by air-drying and analyzing on SEM. Stimuli including 10 mM phosphate buffer solutions of pH 7.4 with 10 g L–1 glucose and 10 mM acetate buffer solutions of pH 5.0 with and/or
without 10 g L–1 glucose respectively. For TEM test, G-quadruplex hydrogel solution with
the concentration of 25 mM was prepared, 10 μL hot solution mixture was dropped onto a carbon-coated copper grid and blotted with filter paper to remove excess liquid before hydrogelation. The sample was vacuum-dried and TEM measurements were performed on a Philips T20ST electron microscope at an acceleration voltage of 100 kV.
Evaluation of rheological behavior. Dynamic rheological analysis of freshly prepared
G-quadruplex hydrogel (25 mM) was accessed on an AR2000ex rheometer (TA Instrument, America). Parallel plate geometry with a diameter of 40 mm was used. The temperature was set at 20 °C. The gel sample was allowed to well-formed on the plate for 10 min. Frequency sweeps were performed at 1% strain. Stress sweeps were performed at 10 rad sec–1 by
ramping the stress from 0.5 Pa to 1000 Pa.
Stimulated cargo release from hydrogels in vitro. To study the release of cargos from
the G-quadruplex hydrogels, two kinds of model drug molecules including methylene blue, and FITC−lysozyme were encapsulated respectively into the hydrogel (35 mM) during gelation with their final concentrations were 21.4 mg L–1 and 22.4 mg L–1 respectively. 1 mL
drug-containing hydrogel in test tube was exposed to 8 mL stimuli solutions (including 10 mM phosphate buffer at pH 7.4, 10 mM acetate buffer at pH 5.0, 10 mM phosphate buffer at pH 7.4 with 5 g L–1 glucose, 10 mM phosphate buffer at pH 7.4 with 10 g L–1 glucose,
and 10 mM acetate buffer pH 5.0 with 10 g L–1 glucose respectively) at 37 °C for the release
3
UV-Vis (methylene blue) or fluorescence (FITC−lysozyme) absorbance of the solutions was recorded on an UV-Vis spectrophotometer (UV-2550, Shimadzu, Japan) or a fluorescence spectrophotometer (F-4600, Hitachi, Japan). The volume of the stimuli solution was kept constant by adding 2 mL of fresh buffer solution after each aliquot was taken. Details for the synthesis of FITC-labeled lysozyme and the monitoring of the drug release from the hydrogel by UV-Vis and fluorescence spectrophotometer could be found in the Supporting Information. The cumulative mass (Mt) of cargo released from hydrogels at certain time
point t was calculated according to following equation (4)
Mt = CtV +ΣCt– 1Vs (4)
where Ct is the concentration of cargo molecule measured at time point t, while V refers
to the total volume of hydrogel and the stimuli solution in the test tube (9 mL), Vs is the
aliquots volume (2 mL) taken out every time. All experiments were repeats triply with separately prepared hydrogels and all data were presented as average value.
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Figure S1. Autocorrelation curves of hydrogels with different concentrations (0.5‰ polystyrene
particles of 222 nm were loaded), (A) 30 mM with the temperature recorded from 70 to 36 °C and (B) 35 mM with the temperature was recorded from 70 to 44 °C.
Figure S2. The calibration curves of two guest molecules and the equations between concentration
and UV-VIS Abs/fluorescence intensity. (A) standard curve of methylene blue (MB), (B) standard curve of FITC-lysozyme.
SUPPORTING INFORMATION
Autocorrelation curves of hydrogels from diffusing wave spectroscopy.
Synthesis of FITC‑labeled lysozyme.For preparation of FITC-lysozyme, 20 mg
lysozyme was dissolved in 3 mL pH 9.0 borate buffer (100 mM) followed by a dropwise addition of 1 mg FITC in 1 mL DMF. After remove the unbounded FITC and DMF upon dialysis against phosphate buffer (pH 7.4, 10 mM) for 3 days, the FITC-lysozyme solution was used to prepare lysozyme-containing hydrogels.1
Calibration curves for cargo release. Two calibration curves were used to measure the
amount of cargos from the G-quadruplex hydrogels. Figure S2A is the calibration curve of
methylene blue (MB) that UV absorbance correlated to the concentration, ranging from 0.52 to 16.7 µg mL–1. The UV absorbance of methylene blue was measured by UV spectrometry
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Figure S3. The 1H NMR spectra of G, TAEA, 2-FPBA, TAEA + 2-FPBA and G-quadruplex hydrogel.
FITC-lysozyme concentrations range from 0.136 to 4.3 µg mL–1. The fluorescence intensity
of FITC-lysozyme was measured at 517 nm upon excitation at 494 nm on fluorescence spectrophotometer.
3
Determination of iminoboronate bonds in G‑quadruplex hydrogels.
Figure S4. ARS-competing experiments. (A) Photographs of G-quadruplex-ARS complexes (upper),
G-quadruplex hydrogel (below), (B) Fluorescence emission spectra of ARS and diluted G-quadruplex-ARS complexes.
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