Nanofilamentous Virus-Based Dynamic Hydrogels with Tunable

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Nanofilamentous Virus Based Dynamic Hydrogels with Tunable Internal Structures, Injectability, Selfhealing, and Sugar Responsiveness at Physiological pH Xueli Zhi, Chunxiong Zheng, Jie Xiong, Jianyao Li, Chenxi Zhao, Linqi Shi, and Zhenkun Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02526 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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Nanofilamentous Virus Based Dynamic Hydrogels with Tunable Internal Structures, Injectability, Self-healing, and Sugar Responsiveness at Physiological pH Xueli Zhi+,1, Chunxiong Zheng+,1, Jie Xiong1, Jianyao Li2, Chenxi Zhao1, Linqi Shi1, Zhenkun Zhang1,* 1) Key Laboratory of Functional Polymer Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China; 2) School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Email: [email protected] + Equal contribution

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Abstract. With expanding applications of hydrogels in diverse fields ranging from biomaterials to sensors, actuators and soft robotics, there are urgent needs to endow one single gel with multiple physicochemical properties such as stimuli-responsiveness, injectability, self-healing and tunable internal structures. However, it is challenging to simultaneously incorporate these highly sought-after properties into one single gel. Herein, a conceptual hydrogel system with all of these properties is presented via combining bioconjugate chemistry, filamentous viruses and dynamic covalent bonds. Nanofilamentous bioconjugates with diol affinity were prepared by coupling a tailor-synthesized low-pKa phenylboronic acid derivative to a well-defined green nanofiber-the M13 virus with a high aspect ratio (PBA-M13). Dynamic hydrogels with tunable mechanical strength were prepared by using multiple diol containing agents such as poly(vinyl alcohol) (PVA) to crosslink such PBA-M13 via the classic boronicdiol dynamic bonds. The as-prepared hydrogels exhibit excellent injectability and self-healing behaviors as well as easy chemical accessibility of the PBA moieties on the virus backbone inside the gel matrix. Ordered internal structures were imparted into virus-based hydrogels by simple shear-induced alignment of the virus nanofibers. Furthermore, unique hydrogels with chiral internal structures were fabricated through in situ gelation induced by diffusion of diol-containing molecules to fix the chiral liquid crystal phase of the PBA-M13 virus. Sugar responsiveness of this gel leads to a glucose regulated release behaviors of payloads such as insulin. All of these properties have been implemented at physiological pH, which will facilitate future applications of these hydrogels as biomaterials.

Keywords: Hydrogel, Dynamic bond, Virus, Phenylboronic acid, Structured hydrogel, Nanofiber


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Introduction With explosively expanding applications of hydrogels in diverse fields ranging from tissue engineering, scaffoldings for cell culture, platforms for drug delivery, and biological glues to sensors, actuators and soft robotics, there are urgent needs to impart multiple physicochemical properties into one single gel.1-8 Among the desired properties, stimuli-responsiveness, injectability, self-healing have been intensely explored with most of hydrogels. Stimuli-responsive hydrogels can undergo either sol-gel transition, volume or shape change as response to external stimuli such as pH, temperature, light or chemical signals.9-13 This property is critical to some applications such as controlled delivery of functional payloads, actuators or soft robotics.11-14 Hydrogels with injectability can be delivered via injection with minimum efforts to target sites or some miniature cavities and the gel can instantly recover its original material properties right after injection.4-6, 15 Another enthusiastically sought-after property of hydrogels is self-healing, with which a gel can self-repair damages or recover from large strain induced disruption without detrimental loss of its original properties.1, 16-22 Reversible physical crosslinking, supramolecular interactions, dynamic bonds are some strategies that have been explored to realize self-healing.17-18, 22-23 The internal structure of hydrogels has recently emerged as another key material property, as inspired by many gel-like biological systems with intricate hierarchal internal structures that have profound functions. Imparting regular internal structure into otherwise random hydrogels is expected to produce novel multiple-stimuli responsiveness such as compression/extension induced structure color change, tunable photonic bandgap and unidirectional (de)swelling, as well as enhanced mechanical strength. These properties have further extended the application of the hydrogel into many other fields, including but not limited to directional growth of


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stem cell for tissue engineering, mechanochromic sensors, deformation-based display devices, soft actuators, artificial muscles, color-changed naked eye sensors and auxiliary independent soft robotics, etc.2-5,

24-27

Hydrogels made from superamolecuar nanofibers or doped with nanofibers such as

cellulose nanocrystals have the advantage to construct hydrogels with partially regular internal structure due to shear-induced orientation of the fibrous constitutes and their tendency to form liquid crystal like structure.5, 25-26, 28-34 Embedding preformed structured templates into hydrogels has also been harnessed to construct anisotropic hydrogels with a more complex internal structure.3, 27, 35-37 Hydrogels with one or two of these above properties have been reported.4, 8, 15, 23, 34, 37-40 However, simultaneously incorporating stimuli-responsiveness, injectability, self-healing and tunable internal structures into one single gel are difficult to achieve and there exit few examples. Herein, we shall report a conceptual hydrogel system that has all of the above properties (Scheme 1). The key component of our hydrogel is a filamentous bacteriophage-the M13 virus with a diameter of 6.6 nm and a length of ca. 900 nm. This biocompatible green nanofiber from nature has chemically and genetically addressable surface and their monodisperse rodlike shape with a high aspect ratio of ca. 150 endows these viruses with rich liquid crystal phases.41-44 Recent works have also demonstrated that such rodlike viruses are valuable candidates as the backbone for multiple responsive hydrogels.45-46 In the current work, a phenylboronic acid (PBA) derivative (1 in Scheme 1) with a pKa that is close to the physiological pH was tailored-synthesized and coupled to the nanofibrous M13 virus, leading to fibrous bioconjugates of the PBA-M13 virus that can form dynamic covalent bonds with 1,2 or 1,3 diol-containing agents at physiological pH.47 Dynamic hydrogels are instantly formed upon mixing poly(vinyl alcohol) (PVA) and PBA-M13 at the physiological pH, due to binding of the


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multiple diols of the linear PVA chain with several viruses that crosslinks the virus backbone into a three dimensional network.22,

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The mechanical strength can be tuned by the amount of each

component. Excellent injectability and self-healing properties were demonstrated with the current gels. Furthermore, thanks to the shear-induced alignment of rodlike virus backbone, hydogels with an internal structure can be prepared. Especially, diffusion of diol-containing agents induced in-situ gelation of the chiral liquid crystal phase of the virus backbone leads to unique hydrogels with an internal chiral feature. Glucose responsiveness regulated insulin release behaviors were also demonstrated.

Scheme 1. Preparation of viral biocunjugates of M13 viruses with low-pKa phenylboronic acid derivative (PBA-M13, A) and fabrication of dynamic hydrogels via binding of PBA-M13 with mulple diol containing polymers (B~D).

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Materials. Unless otherwise noted, most of the chemicals were obtained from J&K Scientific (Beijing, China) and used without further purification. Anhydrous solvents were supplied by local suppliers and further dried following the standard drying procedure. Ultrapure water was always produced by a Milli-Q UltraPure system (18.2 mΩ·cm) (Millipore). Following the standard biochemical protocol, the M13 virus was prepared at the scale of grams using the ER2738 strain of E. coli as the host bacteria. All the biological agents for growing virus were supplyed by Dianguo Biotechnology (Tianjin, China). Preparation of bioconjugates of the M13 virus with PBA (PBA-M13). The PBA derivative, 4-((3-((2,5-dioxopyrrolidinyl)oxy)-3-oxopropyl)carbamoyl) phenylboronic acid (DOCPBA, 1 in Scheme 1) was prepared following the procedure as detailed in SI. DOCPBA was coupled to the surface of the M13 virus via reacting the NHS group with the surface amino groups of the N-terminal and the lysine residue of the major P8 coat protein. For this, the M13 virus was suspended in phosphate buffer (100 mM, pH = 8.0) to form suspensions with a concentration of 2 mg mL-1. DOCPBA was dissolved in anhydrous DMSO and 4 mL of the resulted solution was added dropwise to 50 mL of the M13 virus suspension that was pre-cooled in an ice-water bath under stirring. The molar ratio of DOCPBA to the surface amino groups was kept at 20:1 in order to achieve maximum labeling efficacy. After the addition of DOCPBA in anhydrous DMSO, the mixture was kept stirring in an ice-water bath with a temperature of 4℃ for another 2 hours. The mixture was placed into a dialysis bag with a MWCO of 20000 Dalton against first 100 mM NaHCO3 buffer and then phosphate buffer (5 mM, pH=7.4). The dialyzed PBA-M13 in PB buffer was further purified by three rounds of ultracentrifugation and redispersing in PB buffer. The PBA-M13 was always refrigerated and bacteria


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were removed by centrifuge at 8,000 rpm before any further use. pH dependent liquid crystal behavior. The nematic liquid crystal phase of PBA-M13 was investigated at three pH values. For pH < pKa of the surface PBA moieties, a phosphate buffer with pH = 6.0 (0.1M) was used. For pH  pKa of the surface PBA moieties, a phosphate buffer with pH = 7.4 and a carbonate buffer with pH = 9.0 were used. PBA-M13 was first transferred into each buffer by dialysis and then concentrated by ultracentrifuge into concentrated virus suspensions with a virus concentration of more than 30 mg mL-1. Each suspension (20 ~ 30 L) was placed into a glass capillary with an inner diameter of 1.5 mm and both ends of the glass capillary were then sealed with wax to prevent solvent evaporation. To exclude influences of any contaminants on the liquid crystal phase, the internal surface of the glass capillaries were cleaned with the potassium dichromate-sulphuric acid cleaning solution (Caution: extremely toxic and highly corrosive.) and thoroughly rinsed with ultrapure water. The suspension inside the capillary was observed with an Olympus BX 53 polarized optical microcopy (POM) and images were recorded with a HTC 2000 CCD camera (Weitu, China). Affinity of the surface PBA moieties of PBA-M13 for diol-containing compounds and binding with glucose. The classic diol-containing, fluorescent dye-alizarin red S (ARS) based fluorogenetic method was used to assess the dynamic covalent bond formation of PBA moieties on the virus surface with diols.47 PBA-M13 was suspended in phosphate buffer (pH 7.4, 100 mM) to form 2 mg mL-1 suspension. To 600 L of the suspension was added 1 L 0.6 mg mL-1 ARS in a stepwise fashion. After each addition, the mixture was fully mixed. The fluorescent spectra were recorded by excitation at 469 nm and scanning the emission in the range of 500 - 700 nm.


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To investigate dynamic bonding between diol-containing compounds and the surface PBA moieties, ARS-glucose exchange experiments were performed. To a suspension of 2 mg mL-1 PBA-M13 in phosphate buffer (pH 7.4, 100 mM) was added 10 μL 0.6 mg mL-1 ARS solution. The resulted mixture had very strong fluorescent emission when excited at 469 nm due to most of the surface PBA moieties on the surface of PBA-M13 was bound with ARS. To the mixture, the solution of glucose in the same buffer (0.72 mg mL-1) was added stepwise with each step of 1 L. After each addition of the glucose solution, the mixture was fully mixed. The fluorescent spectra were recorded by excitation at 469 nm and scanning the emission in the range of 500 -700 nm. Fabrication and self-healing behavior of dynamic hydrogels of PBA-M13 with poly(vinyl alcohol) (PVA). Poly(vinyl alcohol) (100% hydrolyzed) with an average molecular weights (Mn) 86000 was used as the multiple diol containing agent. Suspensions of PBA-M13 and solutions of PVA in a phosphate buffer (5 mM) with pH 7.4 were prepared separately. Certain volumes of each solution were mixed and gentle vortex was applied to facilitate the mixing of the two solutions. The initial gelation behavior was qualitatively assessed by inverting the glass vial. The three kinds of hydrogels were listed in Table S1 of SI, in which some rheological results were also listed. The No.1 gel in Table S1 was used to demonstrate the self-healing behavior. Two hydrogels were prepared separately and loaded with brilliant blue and rhodamine for visual observation. The two gels were made adjacent to each other to promote healing. After one hour, the healed gel was picked up by a forceps. In addition, a gel was cut into two pieces which were then made adjacent to each other (Figure S7 in SI). The joint interface was monitored until the interface completely disappeared. Injectability and tuning the internal structures of the dynamic hydrogels of PBA-M13 with


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poly(vinyl alcohol) (PVA) hdyrogel. Hydrogels were loaded with rhodamine to endow the gel with color for visual observation. The gel was loaded into a syringe with a 21 gauge needle. The gel was injected out and settled on a glass slides. Writing characters was achieved by moving the syringe when injecting out the hydrogel. To induce alignment of the rodlike virus inside the hydrogel, the cylindrical hydrogels that come out of the needle were let rest on glass slides. The cylindrical gel rods with a diameter of 1~2 mm were then observed with POM. Diffusion of PVA induced in situ gelation to prepare hydrogels with chiral internal structures. Twenty L suspensions of PBA-M13 with a concentration of 30 mg mL-1 in a phosphate buffer (5 mM, pH 7.4) was placed into the middle part of a clean glass capillary with a diameter of 2 mm (Figure 6A). The suspension inside the capillary exists as a cylindrical shape with two open interfaces and a length of 1 cm. The capillary was stored at 4 oC until uniform fingerprints appeared in the virus suspension, indicating the formation of the chiral nematic liquid crystal phase. After this step, 10 L PVA solution (1 wt%) in the same PB buffer was carefully placed at one of the interface of the virus suspensions. The interface was slightly disturbed due to the initial mixing of the two liquids. The texture of the virus suspension after adding of the PVA solution was monitored until no change occurred to the fingerprints. To remove the hydrogel out of the glass capillary, the glass capillary was broken by carefully applying external pressing force.

In-gel diffusion of ARS inside the hydrogels. In order to assess the accessibility of the PBA moieties on the surface of viruses in the gelled state, ARS was used as the external probes. A hydrogel with the same components as the No.1 gel in Table S1 of SI was first formed in a quartz cuvette with a light path of 1 mm. The ARS solution (7 μL, 0.1 mg mL-1) in the same buffer as the hydrogel was injected


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into the hydrogel by a microsyringe at a point away from the point where the excitation light entered the quartz cuvette. Take the ending of the injection of ARS solution as the starting points, the fluorescent spectra were recorded by excitation at 469 nm and scanning in the range of 500 - 700 nm. It is expected that starting from the injection point, ARS will diffuse around the whole gel and binding to PBA moieties. When ARS arrived to the point where the excitation light entered the quartz cuvette, binding of ARS to PBA moieties was turned on the fluorescent emission of ARS. To visualize the in-gel diffusion of ARS, the same gel was created in a glass vial and the ARS solution was injected by a microsyringe. Glucose regulated insulin release behavior. Hydrogels with the same components as the No.1 gel in Table S1 of SI was used to demonstrate glucose regulated insulin release behavior under physiological conditions (pH = 7.4, T = 37℃). Before forming hydrogels, 35 L PBA-M13 was mixed with 15 L FITC-insulin. The mixture was mixed with 10 L PVA solution to form hydrogels. The final concentration of FITC-insulin was 50 M. Two identical gels were prepared and incubated in a water bath at 37℃. To each of the hydrogel was added 1 mL PBS buffer (pH 7.4) and PBS buffer (pH 7.4) containing 6 mg mL-1 glucose, respectively. At preset intervals, 600 L of the solution above the gel was taken away to determine the concentration of released insulin and 600 L PBS buffer was added to the sample. The fluorescent spectra were recorded by excitation at 495 nm and scanning in the range of 500 -700 nm. Rheological measurement. All of the rheological measurements were performed on an AR2000ex rheometer (TA Instruments) with a 20 mm plate geometry at 25 oC. The gap was fixed at 0.5 mm. For time sweep, the two components of the hydrogel were quickly mixed in a vial by vortex and then


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loaded onto the plate. Solvent evaporation was alleviated by covering the top plate with a layer of silicon oil. Time dependent oscillatory measurements were performed at constant strain of 1% and frequency of 1 Hz. For frequency sweep, all of the gels were tested from 0.01 to 100 Hz at constant strain of 1%. The crossover frequency (c) of each hydrogel was recorded when storage modulus G is equal to loss modulus G. To confirm the self-healing properties, step-strain tests at a frequency of 1Hz was carried out by repeating large strain (300%, 300s) for disrupting the hydrogels and small strain (1%, 300s) for mechanical recovery. Instrumental. 1H NMR and

11B

NMR spectra were recorded on an AVANCE III 400MHz

spectrometer (Bruker). Centrifugation and ultracentrifugation were performed on an Allegra X15R benchtop centrifuge and Optima L-90K ultracentrifuge (Beckman Coulter, USA), respectively. The atomic force microscopy (AFM) images were obtained with an etched silicon tip (RTESP, Bruker; spring constant k = 40 N/m; frequency f0 = 300 kHz) by the tapping mode on a NanoScopeR IIIaAFM system (Veeco). Image processing was performed with NanoScope 5.31r1 software. The liquid crystal behavior of the virus suspension was investigated with an Olympus BX53P polarized optical microscopy (POM). Liquid chromatography–mass spectrometry (LC-MS) was performed with an Agilent 6520 Q-TOF LC/MS instrument (Agilent). Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALTI-TOF MS) was performed on the AutoflexIII LRF200-CID (Bruker Daltonics). The concentration of the virus was determined with a UV-Vis 2550 UV-Vis spectrometer (Shimadzu). Fluorescent spectra were recorded on an F-4600 spectrometer (Hitachi).

Results and Discussion Preparation of phenylboronic acid-M13 virus bioconjugates.


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The phenylboronic acid (PBA) derivative (1 in Scheme 1) was synthesized and thoroughly characterized by 1H NMR, 11B NMR and mass spectroscopy (as detailed in SI). The succinimidyl ester (NHS) group can facilitate conveniently labeling of this PBA derivative to proteins or other biomacromolecules to construct bioconjugates via reacting with the amino group that is normally abundant in biomacromolecules.51-52 The M13 virus consists of 2700 major P8 coat proteins that self-assemble in a helical way into a rodlike capsid and the amino groups on the virus surface offer enormous opportunities for chemical modifications.42, 53 Successful labeling of the PBA derivative (1) onto the virus surface (PBA-M13) was confirmed by MOLT-TOF mass spectrometry and X-Ray Photoelectron Spectroscopy (XPS) (Figure 1A, S2 and S4 in SI). The peak at Mw = 5238 corresponding to the intact P8 coat protein becomes very weak, while there appear new peaks that are attributed to the coat proteins labeled with one or two PBA moieties (Figure 1A and S2 in SI). In addition, since there is no B element in the native M13 virus. The clear XPS peak of B1s (192 eV) of PBA-M13 must be due to the coupled PBA moieties (Figure S4). Each virus was labeled with more than one thousand of PBA. The PBA-M13 virus still has the rodlike shape, as revealed by atomic force microscopy (AFM) (Inset of Figure 1A). The pKa of PBA derivatives is vital for many properties of boronic acid−based materials since exchange between boronic acid/diol and boronate esters is most effective at pH near the pKa.47 The pKa of the PBA

moieties on the virus surface was determined by taking advantage of the pH dependent intrinsic fluorescence property of PBA (Figure 1B and S3 in SI).54 A value of 7.7 was obtained, which is nearby the physiological pH and is much lower than many aryl boronic acids with a pKa of more than 9.23 This decreasing pKa of the current PBA derivative (1) is due to the electronic withdrawing amide


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carbonyl on the phenylboronic acid moiety.55

Figure 1. Characterization of the PBA-M13 virus (A), pKa determination of the PBA moieties on the virus surface (B). The inset in (A) is AFM analysis of the PBA-M13 virus. Each peak in (A) is


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assigned to the major P8 coat protein that modified with one or two PBA moieties (Schematic illustrations in the inset of (A)).

In the next, the dynamic covalent bond formation of the PBA moieties on the virus surface with diols was assessed by the classic diol-containing, fluorescent dye alizarin red S (ARS) based fluorogenetic method (Figure 2A).47 At a fixed virus concentration, increasing the amount of ARS leaded to pronounced increasing of the fluorescent intensity. This is due to binding of ARS to PBA moieties, which turns on the fluorescent emission of ARS (Figure 2B, C). In contrast, the fluorescent intensity of the ARS/PBA-M13 mixture gradually decreased upon titration with glucose (Figure 2D, E). Such phenomena stems from binding of glucose to the PBA moieties on the virus surface by replacing ARS (Figure 2A). It is noted here that the diol affinity of the PBA-M13 were demonstrated at pH = 7.4, thanks to the much lower pKa of the tailor-synthesized PBA derivative (1).


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Figure 2. Diol affinity of the PBA moieties on the virus surface. (A) Schematic illustration of binding of ARS to the PBA moieties on the virus surface and subsequent replacement by glucose. (B) and (C) are fluorescent emission spectra and the maximum emission intensity versus the ARS concentration during adding ARS to the PBA-M13 virus suspension, respectively. (D) and (E) are fluorescent emission spectra and the maximum emission intensity versus the glucose concentration during titration of glucose to a fluorescent mixture of ARS and the PBA-M13 virus, respectively. The virus suspension consists of 2 mg mL-1 PBA-M13 in phosphate buffer (pH = 7.4, 100 mM).

pH dependent liquid crystal behaviors of rodlike bioconjugates of PBA-M13. Compared to many superamolecular nanofibers for hydrogels,5, 24, 34, 56 the current virus nanofiber has one unique capability to form the chiral nematic liquid crystal (CLC) phase, in which the rodlike


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viruses arrange relative to each other in a three-dimensional chiral way. Our previous works have indicated that the CLC phase of the M13 virus is sensitive to the virus surface property.56-57 For the current PBA-M13, at pH < pKa of PBA where the PBA moieties on the virus surface are in the uncharged hydrophobic state, the concentrated suspension of the PBA-M13 virus only forms the normal nematic LC phase, in which the long axis of the rodlike virus point in one direction but without any positional ordering (Figure 3A, S5 in SI). However, above pKa where the PBA moieties on the virus surface turn into the hydrophilic and negatively charged boronates, the CLC phase forms, as indicted by the typical fingerprints (Figure 3B, S6 in SI). The nature of the nematic LC phase at several pH indicates that the nematic to chiral LC phase transition occurs at pH ~ 7.4 which is comparable to the pKa of the PBA moieties on the virus surface as determined by the pH dependent intrinsic fluoresce (Figure 1B).


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Figure 3. Influence of pH on the chiral nematic liquid crystal (LC) phase bahavior of the M13-PBA viruses. (A) and (B) are the photos of the nematic and chiral nematic LC phase under polarized optical microscope, respectively. Insets in (A) and (B) are the schematic illustration of the arrangement of the rodlike M13-PBA viruses in each LC phase. The right panels are the charge state of the PBA moieties on the virus surface.

Self-healing dynamic hydrogels of PBA coupled viruses with polymers containing multiple diols. The PBA-M13, a semi-flexible thin nanofiber with thousands of PBA moieties ready for forming dynamic covalent boronate bonds, is expected to be crosslinked by agents containing multiple diols into hydrogels. We chose a biocompatible linear polymer-PVA with a low molecular weight as the crosslinking ingredient to build up hydrogels with a rodlike component and a flexible polymer.22 As expected, mixing the PBA-M13 suspension and PVA solution at physiological pH resulted in a transparent hydrogel that can self-support its own weight (Figure 4A). cryo-SEM revealed the entangled virus-nanofiber inside the hydrated hydrogels formed by gently vortexing the two components (Figure 4B). Time dependent oscillatory rheology characterization revealed that the hydogel forms instantly right after mixing, as indicated by the facts that both the elastic modulus (G) and loss modulus (G) becomes constant and G is much higher than G (Figure 4C). With increasing concentration of either the virus or polymer, the G increases from 100 Pa to above 400 Pa, suggesting tunable mechanical strength (Table S1 in SI). Dynamic frequency sweep measurements revealed frequency-dependent viscoelastic behaviors (Figure 4D and S8 in SI), typical characteristics of dynamic gel networks.23, 51 There exists a crossover frequency (c), at which G is equal to G and


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above which G > G. With the current gel, c typically has values of less than 0.5 rad s−1 (Figure S8), which is significantly lower than most of hydrogels based on similar mechanism.23, 58 Therefore, the current gel has much stable mechanical properties in a wide range of angular frequency that is advantageous in terms of injectability and self-healing.


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Figure 4. Dynamic hydrogels formed upon mixing the PBA-M13 virus with PVA at pH 7.4. (A) Hydrogel in an inverted vial and (B) corresponding internal structure as revealed by cryo-SEM. (C) and (D) are the time dependent oscillatory and frequency sweep measurements, respectively. (E) Step-strain measurements with the strain changing from 1% to 300% and vice versa. Inset of (E): demonstration of the self-healing behavior. The hydrogel consists of 2.5 wt% PBA-M13 and 0.10 wt% PVA.

In order to investigate the self-healing characteristics, two pieces of identical hydrogels prepared

separately or cut from a preformed hydrogel were placed adjacent to each other. The interface became smeared during 15 mins, and then disappears during one hour (Inset of Figure 4E, S7 in SI). The self-healing was further quantitatively confirmed by step-strain sweep following a recently benchmarked protocol (Figure 4E).21 At high strain (300%), both G and G decreased sharply and G


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became lower than G, suggesting the hydrogel was destroyed or transformed into the fluid state. When reversing to lower strain (1%), the G and G recovered instantly to the normal values of the original gels and G became much higher than G, confirming the instant gel reformation at a very short time scale. This procedure can be repeated many cycles (Figure 4E). Such excellent self-healing property of our current gels stems from the fact that all cross-linking points within the current hydrogel are dynamic covalent bonds with their rapid exchange due to boronate ester formation/hydrolysis.22

Compared to previous works based on similar mechanisms in which gelation only occurred at pH  9,23, 58-59 both instant gelation upon mixing the two gel precursors and the self-healing property of the resulted hydrogel occur at physiological pH, thanks to the much lower pKa of the tailor-made PBA moieties.

Injectability and tunable internal structure. To investigate injectability of the current gels, the hydrogel was loaded into a syringe and the gel could be injected out effortlessly through a 21-gauge needle (Figure 5A). As soon as the gel came out, it remained the gel shape and can be molded into any shape as a gel ink (Figure 5A and S7 in SI). The tendency of rodlike particles to align with the flow field is expected to impart the shear-thinning property into the current gel.5,

28

In addition, the fast dissociation-reformation of the boronic-diol

dynamic bonds endows the current hydrogel with self-healing capability. These two synergetic factors contribute to the injectability. Shearing-induced alignment of the nanofibers inside hydrogels has often been explored to form anisotropic hydrogels with a regular internal structure.2, 4-5 Similarly, after injecting the current system out of the long needle to form cylindrical hydrogels, birefringence was observed under polarized


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optical microscopy (POM) (Figure 5B), indicating the ordering arrangement of the virus nanofibers inside the hydrogels.26 The intensity of birefringence, i.e., the degree of the alignment was enhanced by increasing the virus concentrations (Figure 5C). Corresponding cryo-SEM analysis of the internal structure of the birefringent hydrogels in the hydrated state reveals increasing ordering of the virus backbone inside the hydrogel, as indicated by the layer-like structure that contains fiberous bundles consisting of several viruses aligning with their long axis (Figure 5D, E).5 In the dried gels under SEM, there are fiberlike structures pointing at one direction (Figure S9A in SI). This is in contrast to the gel prepared by gently shaking the two components, in which virus nanofibers randomly ordered (Figure 4B).

Figure 5. Injectability and tunable internal structure of hydrogels. (A) Gel was injectable via a syringe with a 21-gauge needle. (B) ~ (E) Optical images of the hydrogels by POM and corresponding cryo-SEM of the internal structures. Cylindrical hydrogels in (B, D) and (C, E) were obtained by injecting out the hydrogel from a 21 gauge needle. The hydrogels in (B) ~ (C) consists of 1 and 2.5 wt % PBA-M13, respectively, while PVA was kept at 0.1 wt%. Insets: Schematic illustration of the ordering of the PBA-M13 nanofibers inside the hydrogels.


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The common strategy so far to construct anisotropic hydrogels is to induce alignment of the rod components inside the gel matrix by external fields such as shearing, electronic, magnetic fields.2, 5, 35 However, the resulted internal structure is often limited to simple parallel alignment of the nanofiberous components inside hydrogels and it is very challenging to achieve other arrangements. As shown in Figure 3, the PBA-M13 bioconjugate can spontaneously form the CLC phase at pH  7.4, one unique property that other supramolecular nanofibers are short of.24, 26 In addition, gelation by mixing two components offers a versatile way to fine tune the internal structure. We first formed a well-defined CLC phase of the PBA-M13 virus in a glass capillary and then placed the PVA solution adjacent to one of the interface (Figure 6A). With gradual diffusion of PVA into the preformed CLC phase of the PBA-M13 virus, gelation started at the interface and eventually the whole system turned into a hydrogel at the time scale of 70 hours (Figure 6). During this procedure, the fingerprints of the CLC phase were distorted, clearly due to the fact that inter-virus crosslinking by PVA influences the chiral ordering of the viruses relative to each other. After taking out of the hydrogels from the glass capillary, the CLC structure was partially remained as indicated by the clear but less regular fingerprint textures (Figure 6E). Corresponding cryo-SEM analysis of this chiral hydrogel in the hydrated state revealed an internal structure consisting of densely packed layers (Figure 6F). In the cross section of the dried chiral hydrogel, there exists a lamellar structure typical of the materials originated from the CLC phase (Figure S9B in SI).60 Therefore, together with gentle vortexing and unidirectional shearing, preparation conditions offer a versatile way to fine-tune the internal structure of the dynamic hydrogel.


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Figure 6. In situ gelation induced by diffusion of PVA into the chiral nematic liquid crystal (CLC) phase of the PBA-M13 virus. (A) Schematic illustration of the diffusion procedure of PVA into the preformed CLC phase of the PBA-M13 virus in a glass capillary. (B) CLC phase of the PBA-M13 virus before adding the PVA solution. (C) ~ (E) Different stages during diffusion of PVA into the CLC phase of the PBA-M13 virus. The red arrows indicate the diffusion direction of the PVA solution. (F) Corresponding cryo-SEM analysis of the internal structure of the final hydrogel with an irregular chiral internal structure. The virus suspension consists of 30 mg mL-1 PBA-M13 suspended in phosphate buffer with a pH 7.4 (5 mM). The PVA solution is 1 wt% PVA in the same buffer.

Chemical Accessibility of the BPA Moieties of the virus backbone inside the hyrogel and glucose responsiveness regulated insulin release behaviors. Previous works have functionalized the nanofibers surface inside hydrogels with functional groups


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such as the cell adhesion peptide Arg-Gly-Asp (RGD), signaling moieties or bioactive epitopes.61 However, the chemical accessibility of these surface bearing functional groups in the gel state has not been fully addressed. To test the chemical accessibility of the PBA moieties on the virus nanofiberous backbones inside the current dynamic hydrogels, ARS was used as an external probe and injected into the hydrogel at one point. With diffusion of ARS inside the whole hdyrogels, the gels became a combination of red/orange during one hour (Figure 7A), due to the binding of ANS with the PBA moieties.47 The fluorescent emission due to the complexation of the PBA with ARS was monitored at a

point far away from the injection point. After an induction time during which ARS diffused to the monitoring point, significant fluorescent emission was recorded and leveled off during 15 min (Figure 7B). This is an elegant demonstration that the PBA moieties on the virus surface inside the gel can be easily accessed.


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Figure 7. Chemical accessibility of the PBA moieties on the virus backbone inside the hydrogel (A, B). (A) Visualization of ARS diffusion inside the hydrogel after injecting the ARS solution into the hydrogel at one point by a microsyringe. (B) Fluorescent emission spectra versus time due to binding of ARS to PBA moieties. The ARS solution was injected into the hydrogel in a quartz cuvette at one point by a microsyringe and the fluorescent emission spectra were monitored at a point far away from the injection point. (C) Glucose responsiveness regulated insulin release behaviors. FITC-insulin was loaded into the hydrogel and then placed into PBS buffer with or without glucose. The released FITC-insulin was monitored by fluorescent measurements. The asterisks represent the statistical significance which is calculated by multiple t tests-one per row: * p < 0.05, ** p < 0.01, *** p < 0.001. The hydrogels consist of 1 wt% PBA-M13 and 0.15 wt% PVA.

Payloads such as fluorescein isothiocyanate (FITC)-insulin can be conveniently loaded into the current hydrogel by mixing with either the M13-PBA virus suspension or the PVA solution. Glucose regulated release behavior of the gels was monitored. As shown in Figure 7C, an initial burst release behavior of insulin in less than 15 hours was observed and there is no difference either in the presence or absence of glucose. This behavior can be explained by the large mesh size of the nanofiber based hydrogels, which is poorly selective to such small proteins as insulin.58 Influence of glucose on the release behaviors was seen at the later release stage, where the hydrogel in the presence of glucose released most of the insulin with a speed faster than that without glucose. Glucose diffuses into the hydrogel and partially replaces diols of PVA or forms charged complex with the dangling free PBA moieties on the virus backbone. This might lead to the swelling of the gel matrix, which facilitates the


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release of insulin.58

Conclusions In summary, we tailor-synthesized a PBA derivative that has a NHS group for the general construction of bioconjugates of PBA-biomacomolecules and a PBA moiety with a lower pKa that facilities gelation at physiological pH via the classic boronic-diol dynamic covalent bonds. Bioconjugates of the M13 virus with PBA (PBA-M13) were then prepared by chemically coupling the PBA derivative onto the surface of the rodlike M13 virus. The low-pKa PBA decorated virus nanofibers have an affinity for cis 1,2- or 1,3- diol containing compounds. Mixing the PBA-M13 virus bioconjugate with multiple diol-bearing agents such as poly(vinyl alcohol) (PVA) leads to instant gelation and the as-prepared hydrogel exhibits excellent injectability and self-healing behavior. Sugar responsiveness of this gel leads to glucose regulated release behaviors of payloads such as insulin. Ordered internal structures were imparted into the virus-based hydrogel by shear-induced orientation of the rodlike virus nanofibers. Furthermore, in situ gelation by diffusion of diol-containing molecules can be used to further fix the chiral liquid crystal phase of the PBA-M13 virus into the hydrogel, leading to unique hydrogels with an internal chiral structure. All of these properties have been implemented at physiological pH, while pervious hydrogels relying on similar mechanisms have only one or two of the above properties that have to be realized at pH of more than 8.

Acknowledgements This work was supported by the National Natural Science Foundation of China (nos. 21774064, 21274067, 21620102005, 91527306 and 51390483), the Fundamental Research Funds for the Central


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Universities, Natural Science Foundation of Tianjin, China (no. 17JCYBJC16900) and PCSIRT (IRT1257). We would like to thank Prof. Zhimou Yang for his supporting with the rheological measurements.

ASSOCIATED CONTENT Supporting Information. Experimental details of synthesis of the PBA derivative, MALDI-TOF MS analysis of the P8 coat protein of the M13 virus and M13-PBA, pKa determination of the PBA moieties on the surface of the PBA-M13 virus, cryo-SEM characterization and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Email: [email protected] + These authors contribute equally to this work


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Table of Contents (TOC)


Nanofilamentous Virus-Based Dynamic Hydrogels with Tunable

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