A G-Quadruplex Hydrogel via Multicomponent Self-Assembly

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A G-Quadruplex Hydrogel via Multi-Component SelfAssembly: Formation and Zero-Order Controlled Release Yuanfeng Li, Yong Liu, Rujiang Ma, Yanling Xu, Yunliang Zhang, Baoxin Li, Yingli An, and Linqi Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00957 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on April 1, 2017

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ACS Applied Materials & Interfaces

A G-Quadruplex Hydrogel via Multi-Component Self-Assembly: Formation and Zero-Order Controlled Release Yuanfeng Li,1,† Yong Liu,1,† Rujiang Ma,1,2,* Yanling Xu,3 Yunliang Zhang,4 Baoxin Li,4 Yingli An,1 and Linqi Shi,1,2,* 1

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials of

Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, China. 2

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin

300071, China. 3

Department of Biological Pharmacy, College of Basic Science, Tianjin Agricultural University, Tianjin

300384, China. 4

Endocrinology Department, Baoding First Central Hospital, Baoding, Hebei 071000, China.

ABSTRACT:

Stimuli-sensitive hydrogels

are ideal

candidates

for biomedical

and

bioengineering purposes, while 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 of 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 multi-component self-assembly of endogenous guanosine (G), 2formylboronic acid (2-FPBA), and tris(2-aminoethyl)amine (TAEA) in the presence of KCl in an easy and convenient way. The G-quadruplex hydrogel has the natures of (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

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drug release was successfully achieved. This kind of supramolecular G-quadruplex hydrogels may find applications in biological fields. KEYWORDS: supramolecular hydrogels, G-quadruplex, dynamic covalent bonds, zero-order, controlled release 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 cargoes, protect the cargoes from environmental variations and/or control cargo release by designing the gel structure in response to micro-environment 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 hydrogel-based controlled drug release systems exhibit two-step release profiles (fast-then-slow behavior), while an ideal candidate should release cargo at a constant rate.13, 14 The range of application of hydrogels in biological field would be greatly expanded if such issues could be overcome.15 Since Davies and coworkers reported the hydrogel formation by guanosine (G) deviations in 1962,16 endogenous motifs were 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 each other to form a G-quadruplex which could be extended to fibrous network to form supramolecular hydrogels.15, 24, 25 Owing to the dynamic combinatorial chemistry and dynamic covalent bonds involved in the formation of 2/33 ACS Paragon Plus Environment

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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. While, recently, the interest in 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 fivemembered cyclic boronate esters with boronic acids.34, 35 Formylphenylboronic acids (FPBAs) are versatile building blocks for multi-component 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 imino bonds, which can endow a self-assembled system with glucose and acid-triggered drug release respectively.36,41 Therefore, a hydrogel stabilized and cross-linked by iminoboronate bonds will 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 mainly because that diffusion is the main driving force for the drug release.

43,44

Diffusion always occurred because of the random molecular

motion of drugs and related to concentration gradients. Especially for cargos loaded in polymeric systems, with the swelling of carriers and osmotic pressure, burst release may happen. Fast-then-



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slow and burst release resulted in unpredictable release dose and uncontrollable duration. It is desirable to fabricate controlled drug release system with a steady and even a zero-order release profile which is very important for maintaining higher levels and longer effective concentration of drug. In zero-order release system, diffusion of 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 a functional trilayer nanofibers through triaxial electrospinning, which provided linear release of ketoprofen based on loading incremental contents of ketoprofen from exterior layer of fibers to inward layer.46 Solaiappan et al, introduced 3D 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 drug molecules.47 Zhang et al, designed dynamic layer-by-layer films made of two polymers with narrow molecular weight distribution, provided a platform with intelligent and zero-order release profiles because of its dissociation of the interpolymer complex.13 Though different kinds of zero-order drug release systems have been studied, hydrogels with stimuli-responsive and zeroorder release profiles have been rarely reported. More importantly, most of zero-order drug release systems usually involved in complicated synthesis and process, 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 commercial available materials, in order to solve the problem in uncontrolled drug release. The supramolecular hydrogels are constructed by using dynamic


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multi-component self-assembly of guanosine (cis-diol containing motif), 2-formylboronic acid (2-FPBA, boronic acid and aldehyde groups containing motif), tris(2-aminoethyl)amine (TAEA, amino groups containing motif) and KCl (cations) in aqueous solution (Scheme 1). Hydrogelation is driven by (1) the Hoogsteen-type hydrogen-bonding between the guanine groups which 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-quartet each other. 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 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 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.



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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.

2. MATERIALS AND METHODS 2.1. Materials. Guanosine (G, 98%), methylene blue (99%), 2-formylphenylboronic acid (2FPBA, 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. 2,2’,2’’-trianminotriethylamine (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


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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. 2.2. 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. 2.3. 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.



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2.4. 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 PpIXcontaining 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 Gquadruplex structure detection using ThT, the procedure was essentially the same as the PpIXquadruplex method with the using of ThT (30 µL, 0.5 mM) 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. 2.5. 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-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.


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2.6. 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. 2.7. 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. 2.8. 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 9/33 ACS Paragon Plus Environment


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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. 2.9. 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 of drugs. Aliquots (2 mL) of the upper solution were collected every 1 h up to 12 h and the 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 (1) (1)

Mt = CtV +ΣCt – 1Vs

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 10/33 ACS Paragon Plus Environment

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volume (2 mL) taken out every time. All experiments were repeats triply with separately prepared hydrogels and all data were presented as average value. 3. RESULTS AND DISCUSSION 3.1. Formation of G-Quadruplex Hydrogel via Multi-Component 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 under room temperature, it is found to form stable hydrogel with TAEA and 2-FPBA in the presence of K+ at ultrapure water by heating the solution mixture to boiling and then cooling 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 templated by potassium cations, which resulted in the formation of a G-quartet structure and had been reported by Davies et al.24 The second one was the spontaneous and synergistic formation of the iminoboronate bonds among guanosine, 2-FPBA, and TAEA. It was because that the cis-diol on the ribose of guanosine was inclined to combine with phenylboronic acid group on 2-FPBA through a cyclic boronate structure and the primary amino groups on TAEA was also easily combine with the formyl on 2-FPBA through an imino bond. These two kinds of combination were collectively called iminoboronate bonds as they occurred synergistically and reinforced each other.48 The iminoboronate bonds could connect adjacent G-quartets because of the tri-functionality of TAEA. The third important interaction was the supramolecular stacking of the G-quartet each other, leading to the formation of fibrous G-quadruplex structure. As a joint effect of the three kinds of important interactions, G-quadruplex hydrogels were successfully obtained. In this study, Gquadruplex hydrogels were prepared by mixing, heating and cooling of mixture composed of 11/33 ACS Paragon Plus Environment


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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 Gquadruplex hydrogels. The formed hydrogels were visually thermo-reversible as they transformed between solution and gel phase upon alternating heating and cooling cycles. Time autocorrelation function of light scattering was measured by DWS through adding probe particles (polystyrene 222 nm) into the hydrogel solution. In Figure 1A, with temperature decreased from 70°C to 33°C, the time scale for the decay of autocorrelation 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 the temperature decreased. 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 of hydrogel concentration. The gelation temperatures were 35°C, 38°C, and 45°C for hydrogels with concentrations of 25, 30, and 35 mM respectively (Figure 1A and Supporting Information, Figure. S1).49 It should be noted that 25 mM or 0.7 wt% was the lowest critical gelation concentration for guanosine, which was lower than ever reported values 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 anyone or replacement of them by analogues and results were collected in Figure 1B and Table 1. Absence of anyone of KCl, TAEA, and 2-FPBA could lead to the failure of gelation. When K+ was replaced by Na+ and NH4+, precipitation always occurred, indicating K+ was necessary for the hydrogel. As far as molecules with primary amino groups concerned, functionality larger than 2 was necessary and higher functionality could


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facilitate the formation and stability of the hydrogel (i.e. polyethyleneimine > TAEA >> ethylenediamine). TAEA was purposely used in this work for the feather of low-molecular weight hydrogel. If 2-FPBA was replaced by 3-FPBA or 4-FPBA, hydrogel could also be obtained with the only disadvantage of lack of the synergy between the cycloboronate and the imine bond.

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).



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Table 1. Screening of hydrogel formation conditions. Entry

G

KCl

TAEA

2-FPBA

Phenomena a

1

√

√

√

√

white hydrogel

2

√

-

√

√

clear solution

3

√

√

-

√

suspension liquid

4

√

√

√

-

suspension liquid

5

√

NaCl

√

√

clear solution

6

√

NH4Cl

√

√

clear solution

a. For detailed phenomena see Figure 1B.

3.2. Determination of G-Quadruplex Structures in Hydrogels. The stacking of the hydrogen-bonded square planar G-quartet formed by potassium cation-templated assembly of guanosine was one of the main driving forces for the gelation of our systems by forming a Gquadruplex.24,25 To confirm the existence of the G-quadruplex structure, fluorescent spectrometry was used with protoporphyrin IX (PpIX) and thioflavin T (ThT) as the fluorescence probes respectively. In aqueous solution, PpIX gives low fluorescence intensity owning to its poor water-solubility and aggregation. Once PpIX bound to G-rich DNA which contained Gquadruplex 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, while it was greatly enhanced when PpIX was added to G-containing hydrogels. This might be interpreted as the guanosines bonded each other through Hoogsteen-type hydrogen-bonding, which allowed PpIX to stack with via π-π interaction. Though 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


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hydrogels. Unlike PpIX, ThT is a hydrophilic fluorogenic dye and has been applied to identify telomeric G-quadruplex and G-quartets assembled by guanosines in vitro for its strong enhancement in fluorescence after binding to 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). While the fluorescence emission of ThT increased significantly after mingling with G-containing hydrogels and the enhancement of fluorescence (I/I0) at 519 nm was found to be as large as 7 folds. 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 results were consistent with previous studies, indicating the existence of the G-quadruplex structure in the hydrogel and demonstrating the key role of K+ for the stabilization and formation of Gquadruplex structures.



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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.

In order to further validate the formation of G-quadruplex, X-ray powder diffraction (XPRD) experiment was performed to get more evidences and certain parameters about secondary structure of G-quadruplex. Figure 2C showed the XRPD pattern of the lyophilized powder of 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 16/33 ACS Paragon Plus Environment

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= 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 G-quadruplex and gave the microstructural parameters. 3.3. Formation of Iminoboronate Bonds in G-quadruplex Hydrogels. The spontaneous and synergistic formation of iminoboronate bonds among diols (G), 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 analyse the formation of iminoboronate bonds. As the FT-IR spectra shown in Figure 3A and 3B, 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, the 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 (νB-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 Gquadruplex.39



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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 (left), 11B NMR spectra (right).

NMR measurements were also performed to identify iminoboronate bonds 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

11

B 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. While this peak 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, formation of the C=N


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bonds was not detected (Supporting Information, Figure S3), which indicated the imine bonds and the cyclic boronate bonds were formed in a synergetic manner. This may be attributed that the nitrogen atom was electron-rich while the boron atom was electron-deficient, 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 well described in pioneer works.56-58 In order to confirm 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 stronger interaction with the boronic acid than the cis-diol of the G. As showed in Supporting Information, Figure S4A, Gquadruplex hydrogel could not 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 (Supporting Information, Figure S4B).59 The stronger binding between ARS and 2-FPBA prevented the formation of iminoboronate bonds and the G-quadruplex hydrogels as well, which proved the importance of iminoboronate bonds in gelation. 3.4. Morphology and Rheology of the G-Quadruplex Hydrogels. Electron microscopic technologies were applied to investigate the morphology of hydrogels. Scanning electron microscope (SEM) images clearly showed fibrous structure for the hydrogel (Figure 4A, B). Transmission electron microscopy (TEM) image in Figure 4C also showed a dense, entangled fibrils consistent with previous observations.29 The cross-sectional diameters of cylindrical fibres 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 fibres revealed that 19/33 ACS Paragon Plus Environment


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they were composed of several thinner fibres. This fibrous structure also confirmed the formation of G-quadruplex by the supramolecular stacking of G-quartet each other. Additionally, it could be observed from both of SEM and TEM images that the hydrogels were very compact with extraordinarily dense fibres interpenetrated and the pores in hydrogels were usually less than 100 nm in diameters. The rheology of 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.

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


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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.

Figure 5. Rheological tests of G-quadruplex hydrogel (with a guanosine concentration of 35 mM): (A) frequency sweeps at 1% strain, (B) stress sweeps at 10 rad sec-1.

3.5. Stimuli-Responsive Drug Release Behaviours of the G-Quadruplex Hydrogel. As discussed above, 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 acid-responsiveness respectively, as illustrated in Scheme 1. When the G-quadruplex hydrogel was exposed to 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 G-quartets and thus destroyed the cross-linked hydrogel structure. When acid stimulus was used, not only the hydrogen bonds between guanosine were broken, but also the imino bonds between aldehyde group in 2-FPBA and primary amino groups in TAEA were destroyed because of protonation of amino groups. As a result, the quadruplex hydrogel was dissolved. In this study, two kinds of model drugs including methylene blue (Mn = 319.9), and FITClysozyme (Mn = 14000) were encapsulated in the G-quadruplex hydrogels and stimuli-responsive



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drug release was investigated. The G-quadruplex hydrogels are quite different from other hydrogels that cargos are in situ locked during the formation of hydrogel instead of with the help 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 pH 7.4, about 50% of methylene blue was released in 12 h, while hydrogels exposed to a pH 5.0 phosphate buffer released about 80% methylene blue in 12 h incubation, indicating an acid-responsive drug release. Figure 6B showed the glucose-responsive release profiles of methylene blue from the Gquadruplex hydrogels. It was observed that the release rate was significantly increased by the stimuli of glucose and a higher release rate was obtained under higher glucose concentration. When combined 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 stimuli as indicated in Figure 6A (red triangle). Furthermore, the release behavior of FITC-lysozyme with a higher molecular weight from the G-quadruplex hydrogel were also studied. As shown in Figure 6C and 6D, the release profiles of FITC-lysozyme were similar to those of methylene blue while 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 2FPBA via imino bonds. Thus, the FITC-lysozyme may involve in the formation of the Gquadruplex hydrogel to some extent, resulting 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.


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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.

3.6. Proposed Mechanism for Zero-order Drug Release. Through the experiment of stimuliresponsive release, it was found that the release behaviors were different from those general fastthen-slow ones. In order to study the mechanism of drug release, zero-order, first-order, and 23/33 ACS Paragon Plus Environment


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Higuchi model were applied to fit the release profiles. Zero-order release, with a constant release rate for an extended time, is an ultimate goal of most controlled release.60 The equation of zeroorder model is: Mt = M0 + K0t

(2)

The first-order model, which accounts for burst release, is presented as: ln(Mt) = ln(M0) + K1t

(3)

The Higuchi equation, simplifying from Fick’s law which is the best description of diffusioncontrolled delivery system,61 is expresses as: Mt = KHt1/2

(4)

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 Mt against t, In(Mt) against t or Mt against t1/2, for the zero-order, first-order, and Higuchi models respectively (Table 2, 3).62-65 The different rate constants (K) indicated the stimuli-responsiveness of the G-quadruplex hydrogel, larger K meant faster release rate and represented more efficient stimuli-responsiveness. The R2 were used to evaluate the fitness of equations, the closer to 1 the more suitable of equation in fitting the data. The different R2 of three mathematical models indicated the release more likely tend to follow zero-order model with R2 > 0.980 for methylene blue loaded hydrogel, and R2 > 0.930 for FITC- lysozyme loaded hydrogel. The zero-order fit lines were also coherent with data very well as showed in Figure 6. 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.


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Stimuli

Zero-order equation

First-order equation

R2

0.914

15.1

0.780

0.256

0.919

22.9

0.816

0.991

0.264

0.895

32.4

0.918

8.18

0.989

0.245

0.924

29.3

0.823

8.93

0.995

0.243

0.862

31.9

0.859

R

K1

R

pH 7.4 buffer

4.34

0.984

0.238

pH 5.0 buffer

6.43

0.990

6.95

pH 7.4, 10 g L glucose pH 5.0, 10 g L-1 glucose

-1

pH 7.4, 5 g L glucose -1

2

Higuchi equation KH

K0

2

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. Zero-order equation Stimuli

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

-1

pH 7.4, 5 g L glucose

3.11

0.958

0.474

0.824

8.95

0.756

-1

3.88

0.983

0.329

0.819

12.0

0.833

-1

4.24

0.971

0.439

0.824

13.1

0.747

pH 7.4, 10 g L glucose pH 5.0, 10 g L glucose

To understand this zero-order release, the morphologies of hydrogels after exposure to different stimuli were investigated via SEM (Figure 7). As shown in the images, the original dense fibrous network of the hydrogel (Figure 7A) has been eroded to different extent after exposure to 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 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, which pore sizes are much larger than the molecular dimensions of the guest molecules, tended to release cargos in a diffusion-controlled way and unsatisfactory fast-then-



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slow drug release behavior is usually achieved. While for non-porous hydrogels with compact networks, drug diffusion are greatly restricted because of steric hindrance. As far as our Gquadruplex hydrogel is concerned, the compact structure with dense, entangled fibrils may restrict drug diffusion while 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 anti-tumor therapies. The G-quadruplex hydrogels with the 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 anti-tumor therapy. Further studies focusing on the biomedical application of the G-quadruplex hydrogels will be reported in the future.


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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 5.0 for 2 h.

4. CONCLUSIONS In conclusion, we developed supramolecular G-quadruplex hydrogels by multi-component self-assembly of guanosine, TAEA, and 2-FPBA based on the formation of 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 2FPBA, and the stacking of G-quartet each other. The components of the hydrogel were commercially available and the self-assembly was performed in an easy and convenient way. Fluorescent spectrometry and X-ray powder diffraction demonstrated the existence of Gquadruplex structure while FT-IR and NMR analysis confirmed the formation of iminoboronate 27/33 ACS Paragon Plus Environment


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bonds in the hydrogels. SEM and TEM images showed fibrous and compact network for 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 dissolving the hydrogel under the stimuli of acid and glucose. This kind of supramolecular G-quadruplex hydrogel formed by multi-component self-assembly of small molecules with the natures of versatility and commercial availability of building blocks, pH and glucose-responsiveness, and zero-order drug release behavior may be promising candidate for application in biological fields. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Including figures of correlation functions of hydrogels with the concentration of 30 and 35 mM by diffusing wave spectroscopy, synthesis of FITC-labeled lysozyme, calibration curves for cargo release, NMR spectra of source materials and the hydrogel, and the result of ARScompeting experiment. AUTHOR INFORMATION †These two authors contribute equally to this work. Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ACKNOWLEDGMENTS


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This study was financially supported by the National Natural Science Foundation of China (Nos. 21274001, 51390483, 91527306, 21620102005, and 21404082), the Natural Science Foundation of Tianjin, China (No. 15JCYBJC29700), and the PCSIRT (IRT1257). REFERENCES (1) Hoffman, A. S. Hydrogels for Biomedical Applications. Adv. Drug Delivery Rev. 2012, 64, 18–23. (2) Qiu, Y.; Park, K. Environment-Sensitive Hydrogels for Drug Delivery. Adv. Drug Delivery Rev. 2012, 64, 49–60. (3) Vermonden, T.; Censi, R.; Hennink, W. E. Hydrogels for Protein Delivery. Chem. Rev. 2012, 112, 2853– 2888. (4) Yu, J.; Ha, W.; Sun, J. N.; Shi, Y. P. Supramolecular Hybrid Hydrogel Based on Host–Guest Interaction and Its Application in Drug Delivery. ACS Appl. Mater. Interfaces 2014, 6, 19544–19551. (5) Xu, X. D.; Liang, L.; Chen, C. S.; Lu, B.; Wang, N. L.; Jiang, F. G.; Zhang, X. Z.; Zhuo, R. X. Peptide Hydrogel as an Intraocular Drug Delivery System for Inhibition of Postoperative Scarring Formation. ACS Appl. Mater. Interfaces 2010, 2, 2663–2671. (6) Lee, S. C.; Kwon, I. K.; Park, K. Hydrogels for Delivery of Bioactive Agents: A Historical Perspective. Adv. Drug Delivery Rev. 2013, 65, 17–20. (7) Du, X. W.; Zhou, J.; Shi, J. F.; Xu, B. Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem. Rev. 2015, 115, 13165–13307. (8) Seliktar, D. Designing Cell-Compatible Hydrogels for Biomedical Applications. Science 2012, 336, 1124–1128. (9) Ikeda, M.; Tanida, T.; Yoshii, T.; Hamachi, I. Rational Molecular Design of Stimulus-Responsive Supramolecular Hydrogels Based on Dipeptides. Adv. Mater. 2011, 23, 2819–2822. (10) Yesilyurt, V.; Webber, M. J.; Appel, E. A.; Godwin, C.; Langer, R.; Anderson, D. G. Injectable SelfHealing Glucose-Responsive Hydrogels with pH-Regulated Mechanical Properties. Adv. Mater. 2016, 28, 86– 91. (11) Peppas, N., A. ; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Hydrogels in Biology and Medicine: From Molecular Principles to Bionanotechnology. Adv. Mater. 2006, 18, 1345–1360. (12) Li, J.; Mo, L. T.; Lu, C. H.; Fu, T.; Yang, H. H.; Tan, W. H. Functional Nucleic Acid-Based Hydrogels for Bioanalytical and Biomedical Applications. Chem. Soc. Rev. 2016, 45, 1410–1431. (13) Zhao, Y. N.; Yuan, Q. P.; Li, C.; Guan, Y.; Zhang, Y. J. Dynamic Layer-by-Layer films: A Platform for Zero-Order Release. Biomacromolecules 2015, 16, 2032–2039. (14) Zhao, Y. N.; Gu, J. J.; Jia, S. Y.; Guan, Y.; Zhang, Y. J. Zero-Order Release of Polyphenolic Drugs From Dynamic, Hydrogen-Bonded LBL Films. Soft Matter 2016, 12, 1085–1092. (15) Ashley, G. W.; Henise, J.; Reid, R.; Santi, D. V. Hydrogel Drug Delivery System with Predictable and Tunable Drug Release and Degradation Rates. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 2318–2323. (16) Gellert, M.; Lipsett, M. N.; Davies, D. R. Helix Formation by Guanylic Acid. Proc. Natl. Acad. Sci. U. S. A. 1962, 48, 2013–2018. (17) Wang, F.; Liu, X. Q.; Willner, I. DNA Switches: From Principles to Applications. Angew. Chem., Int. Ed. 2015, 54, 1098–1129. (18) Wang, Z. G.; Ding, B. Q. DNA-Based Self-Assembly for Functional Nanomaterials. Adv. Mater. 2013, 25, 3905–3914. (19) Miyata, T.; Asami, N.; Uragami, T. A Reversibly Antigen-Responsive Hydrogel. Nature 1999, 399, 766–769.



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Table of Contents Graphic


A G-Quadruplex Hydrogel via Multicomponent Self-Assembly

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