Post-assembly Fabrication of a Functional Multicomponent

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Post-assembly fabrication of a functional multicomponent supramolecular hydrogel based on a self-sorting double network Wataru Tanaka, Hajime Shigemitsu, Takahiro Fujisaku, Ryou Kubota, Saori Minami, Kenji Urayama, and Itaru Hamachi J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Journal of the American Chemical Society

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Post-assembly fabrication of a functional multicomponent supramolecular hydrogel

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based on a self-sorting double network

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Wataru Tanaka1,§, Hajime Shigemitsu1,†,‡,§, Takahiro Fujisaku1, Ryou Kubota1, Saori

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Minami2, Kenji Urayama2, Itaru Hamachi1,3*

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1

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Engineering, Kyoto University, Katsura, Kyoto 615-8510, JAPAN

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of

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2

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Matsugasaki, Kyoto 606-8585, JAPAN

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3

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Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, JAPAN

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†

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Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, JAPAN

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‡

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of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, JAPAN

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§

Department of Macromolecular Science and Engineering, Kyoto Institute of Technology,

Core Research for Evolutional Science and Technology (CREST), Japan Science and

Present address 1: Department of Applied Chemistry, Graduate School of Engineering,

Present address 2: Frontier Research Base for Global Young Researchers, Graduate School

These authors contributed equally to this work.

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Correspondence: [email protected]

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Abstract

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Living cells exhibit sophisticated functions because they contain numerous endogenous

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stimuli-responsive molecular systems that independently and cooperatively act in response to

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an external circumstance. On the other hand, artificial soft materials containing multiple

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stimuli-responsive molecular systems are still rare. Herein, we demonstrate a unique

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multicomponent hydrogel composed of a self-sorting double network prepared through a

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post-assembly fabrication (PAF) protocol. The PAF protocol allowed the construction of a

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well-ordered hydrogel with a dual-biomolecule-response to two important biomolecules

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(adenosine triphosphate (ATP) and sarcosine). Such a hydrogel could not be prepared

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through a one-step mixing protocol. The resultant multicomponent hydrogel responded to

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ATP and sarcosine through a gel–sol transition behavior programmed in an AND logic gate

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fashion. Finally, we applied the multicomponent hydrogel to a controlled release of an

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antibody.

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Main text

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Stimuli-responsive supramolecular hydrogels have been used as excellent soft materials for

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controlled drug release1, a scaffold for regenerative medicine2, and a self-supported diagnosis

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tool3. Recently, the preparation of multicomponent hydrogels has been a promising strategy

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to obtain hydrogels with intelligent functions4. For instance, multicomponent supramolecular

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hydrogels consisting of an enzyme-loaded liposome enabled precise control of the drug

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release rate by controlling the heating time5. Supramolecular hydrogels including inorganic

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materials (mesoporous silica or layered clay), fluorescent dyes, and an enzyme were used to

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form a unique fluorescent sensor array6. Moreover, biomarker-responsive supramolecular

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hydrogels have been constructed by coupling chemically reactive supramolecular fibers and

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enzymes within a gel matrix7. It has also been reported that the encapsulation of a signal

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amplification system into the biomarker-responsive hydrogels allowed for excellent

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improvement of the sensitivity8. Multicomponent supramolecular hydrogels involving

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orthogonally well-ordered stimuli-response systems would lead to next generation intelligent

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materials equipped with autonomic response to diverse environmental change like living cells,

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supreme natural soft-materials comprising multiple components9.

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A self-sorting double network (SDN) hydrogel is an attractive multicomponent soft

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material10. Adams and co-workers fabricated self-sorting supramolecular nanofibers by

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applying gradual pH changes11 and subsequently developed SDN hydrogels that could be

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spatially resolved by UV light12. Smith and co-workers also reported a phototunable SDN

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hydrogel containing a phototriggered acid generator13. We recently demonstrated hydrogels

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with unique adaptive functions that directly rely on the orthogonality of the SDN hydrogel14.

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An SDN requires a delicate balance of the noncovalent interactions between both the intra3 ACS Paragon Plus Environment


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and interfiber components, which is very difficult to control under aqueous conditions15.

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Therefore, there is a limited variety of SDN hydrogels. An SDN should provide an excellent

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platform to realize well-ordered stimuli-responsive systems.

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Herein, we demonstrate a powerful post-assembly fabrication (PAF) strategy for

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the construction of a multicomponent SDN hydrogel with a dual-stimuli-responsive system

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(Figure 1a). A unique biomolecule-responsive multicomponent hydrogel was successfully

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prepared through the PAF protocol from the original SDN hydrogel. Naked eye observation,

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confocal laser scanning microscopy (CLSM) imaging, and rheological analysis revealed that

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the SDN in the clear hydrogel was well-retained even after the dropwise addition of Ca2+ ions

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and sarcosine oxidase (SOx) (i.e. additional components for acquiring a biomolecule

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response) to the original SDN hydrogel; whereas an inhomogeneous suspension containing

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disordered aggregates was obtained by a one-step mixing protocol of the components. The

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multicomponent hydrogel prepared by PAF undergoes a macroscopic gel–sol transition in

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response to two distinct biomolecules, ATP and sarcosine, in the AND logic gate fashion16,

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which allowed for the controlled release of an antibody, a pharmaceutically important

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protein.


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Figure 1. (a) Schematic representation of the post-assembly fabrication (PAF) strategy for

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the construction of a multicomponent hydrogel based on a self-sorting double network (SDN).

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The additional components (sarcosine oxidase (SOx) and Ca2+ ions) are sequentially added to

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the SDN hydrogel composed of Phos-cycC6 and BPmoc-F3. The multicomponent

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(Phos-cycC6/BPmoc-F3/Ca2+/SOx) hydrogel exhibits designed functions (i.e. gel–sol

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transition in AND logic gate fashion by ATP and sarcosine). (b) Chemical structures of the

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self-sorting hydrogelator pair, Phos-cycC6 and BPmoc-F3.

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The strategy for development of a multicomponent hydrogel by post-assembly

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fabrication (PAF)

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The appropriate combination of additional components can equip an SDN hydrogel with new

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functions without perturbing the orthogonal double fiber networks17. However, the

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components may compromise the orthogonal self-assembly property of the SDN pair in a

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one-step mixing protocol and the resultant material would not exhibit the designed functions

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derived from the well-ordered SDN. Our PAF strategy for the construction of a

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multicomponent hydrogel is illustrated in Figure 1a. In the PAF strategy, additional

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components were sequentially mixed with the SDN hydrogel after the formation of the

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self-sorting nanofibers. Therefore, the kinetically stable well-ordered SDN can be retained

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after the addition of the components without the generation of disordered co-assembled

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structures11,12.

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The sophisticated functionalization of the original properties of the SDN requires

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an elaborate design that can effectively interact the additional components with the fibers.

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We employed Phos-cycC6 and BPmoc-F3 as a gelator pair, which have been previously

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demonstrated to orthogonally self-assemble (Figure 1b)15. Previous structural and

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spectroscopic analyses showed that a few noncovalent interactions (hydrogen-bonding, π/π

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interactions, van der Waals interactions) were distinctly present between the two gelators,

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through which the two different fibers (Phos-cycC6 and BPmoc-F3) were orthogonally

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formed. A coordination bond was not involved in this pair; we have previously reported that

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the strength of the Phos-cycC6 hydrogel was modulated by Ca2+ ions18. It has already been

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revealed that the hybridization of BPmoc-F3 hydrogel and sarcosine oxidase (SOx) affords

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the gel–sol transition response to sarcosine, a biomarker of prostate cancer (Figure S1)7b, 19. 6 ACS Paragon Plus Environment

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Therefore, we decided to use Ca2+ ions and SOx as additional components that may

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functionalize Phos-cycC6 and BPmoc-F3, respectively, in the PAF protocol.

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PAF of ATP-responsive hydrogel based on single Phos-cycC6 fibers

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According to our previous report, a viscous solution containing Phos-cycC6 fibers showed

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Ca2+ ions-induced hydrogelation through a coordination bond with the phosphate head groups

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(Figure 2a)18. The resultant Phos-cycC6/Ca2+ hydrogel was weakened (the gel–sol transition)

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by addition of ethylenediaminetetraacetic acid (EDTA), a Ca2+ ion chelator. These results let

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us expect that ATP (Figure S2) could also be used instead of EDTA as a biologically relevant

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Ca2+ ion chelator to tune the strength of the Phos-cycC6/Ca2+ hydrogel because of its

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sufficient affinity for the Ca2+ ions (the dissociation constant (Kd): 170 µM)20.

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Figure 2. (a) Schematic representation of the preparation of Phos-cycC6/Ca2+ hydrogel by

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PAF. (b) Photo of Phos-cycC6/Ca2+ hydrogels (20 µL) on a flat glass slide 2 h after the

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addition of nucleotide derivatives (ATP, ADP, AMP, and UTP). Conditions: [Phos-cycC6] =

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0.2 wt% (3.2 mM), [Ca2+] = 1.3 mM, 100 mM MES buffer (pH 7.0), 25 °C. (c) Time-course

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CLSM images and (d) fluorescent recovery profiles of Phos-cycC6 fibers stained with

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Alexa546-cycC6 after photobleaching. Conditions: [Phos-cycC6] = 0.1 wt% (1.6 mM),

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[Alexa546-cycC6] = 4.0 µM, [Ca2+] = 4.9 mM, [ATP] = 49 mM, 100 mM MES buffer (pH

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7.0), room temperature (rt). Scale bar: 1 µm. (e) Diffusion coefficients of Phos-cycC6 fibers

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determined by FRAP experiments (n = 5) with or without Ca2+ ions and ATP. The data

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represent the mean ± standard deviation (s.d).

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First, we confirmed the ATP response of the Phos-cycC6/Ca2+ hydrogel. A

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Phos-cycC6/Ca2+ hydrogel was prepared through the PAF protocol (Figure 2a). A 100 mM

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aqueous MES buffer solution (pH 7.0) was added to a powder of Phos-cycC6 and subsequent

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heating yielded a clear solution in which the gelator was completely dissolved (see 8 ACS Paragon Plus Environment

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supporting information for the detailed protocol). After incubation at 25 °C for 1 h to form a

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dispersed solution of Phos-cycC6 fibers, Ca2+ ions were added to afford a clear

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Phos-cycC6/Ca2+ hydrogel (Phos-cycC6: 0.20 wt% (3.2 mM), Ca2+: 1.3 mM) (Figure S3). In

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this manuscript, we define the gel or sol state by the tube inversion test (visual confirmation

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of fluidity of the samples). We also tested a one-step mixing protocol, that is, mixing of two

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components (Phos-cycC6 and Ca2+ ions) followed by heating/cooling. However, we failed to

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prepare a hydrogel but obtained an inhomogeneous suspension containing a large amount of

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precipitate (Figure S4). This result indicated that the PAF process was indispensable for the

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formation of the Phos-cycC6/Ca2+ hydrogel.

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We examined the ATP-response of the Phos-cycC6/Ca2+ hydrogel. The hydrogel

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changed to the sol state 2 h after the addition of ATP, as we expected (Figure 2b, S3). The

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minimal concentration of ATP for the gel–sol transition was 3.2 mM (2.5 equivalents of the

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Ca2+ ions). Adenosine diphosphate (ADP) and uridine triphosphate (UTP) similarly induced

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the gel–sol transition (the minimal concentration of ADP and UTP was 16 mM (12.5 eq.) and

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3.2 mM (2.5 eq.), respectively); whereas adenosine monophosphate (AMP) could not induce

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the gel–sol transition at 16 mM (12.5 eq.) (Figure 2b, S2). It was clear that the order of the

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minimal concentration of these nucleotide derivatives for the transition corresponded well

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with their affinity to the Ca2+ ions (Kd of ATP: 170 µM, ADP: 1.51 mM, AMP: 17.4 mM,

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UTP: 195 µM)20, implying that the gel–sol transition was caused by the removal of the Ca2+

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ions from the Phos-cycC6/Ca2+ network, probably by the oligophosphate groups of these

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nucleotides. In contrast, a Phos-cycC6 hydrogel without Ca2+ ions (8.1 mM, the same as the

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critical gelation concentration (CGC)) retained the gel state even upon ATP addition (24 mM,

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3.0 eq. of Phos-cycC6) (Figure S5). These results clearly revealed that coordination of Ca2+ 9 ACS Paragon Plus Environment


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ions can afford an ATP response in the original Phos-cycC6 network through the PAF

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protocol.

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The impact of Ca2+ ions on the Phos-cycC6 fibers was evaluated by CLSM

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imaging after staining with an appropriate fluorescence probe, Alexa546-cycC6 (Figure

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S6)15b. As shown in Figure S7, the Ca2+ ions did not significantly alter the morphologies of

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the Phos-cycC6 fibers, suggesting that the Ca2+ ions hardly disturbed the fiber network.

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Unfortunately, we could not obtain direct evidence of the interaction of the Ca2+ ions with the

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fibers by CLSM imaging. Therefore, we carefully evaluated the fluidity of the Phos-cycC6

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fibers with/without Ca2+ ions using the fluorescence recovery after photobleaching (FRAP)

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technique21. Time-lapse CLSM images of the stained Phos-cycC6 fibers after photobleaching

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and the fluorescence recovery profiles are shown in Figure 2c and 2d, respectively. The

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diffusion coefficients of the Phos-cycC6 fibers in the absence and presence of Ca2+ ions were

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(3.0 ± 1.8) × 10–2 and (0.16 ± 0.06) × 10–2 µm2/s, respectively (Figure 2e). These results

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indicated that the Ca2+ ions suppressed the fluidity of the Phos-cycC6 fibers owing to

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coordination bonding between the Ca2+ ions and the phosphate head groups of the

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Phos-cycC6 fiber surface. The decreased diffusion coefficient of Phos-cycC6 fibers was

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recovered to (1.5 ± 0.4) × 10–2 µm2/s by the addition of ATP owing to the removal of the

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Ca2+ ions from the Phos-cycC6 fibers (Figure 2e). These FRAP experiments explained the

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macroscopic gel–sol transition of the Phos-cycC6/Ca2+ hydrogel resulting from the addition

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of ATP.

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PAF of a multicomponent (Phos-cycC6/BPmoc-F3/Ca2+/SOx) hydrogel with a

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dual-stimuli-responsive system

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With the two biomolecule-responsive hydrogels (ATP-responsive system: Phos-cycC6/Ca2+,

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sarcosine-responsive system: BPmoc-F3/SOx) in hand, we hybridized the two systems

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through the PAF protocol (Figure 3a). Utilizing the self-sorting feature of the original SDN

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pair, we initially prepared a template SDN using Phos-cycC6 and BPmoc-F3. A mixed

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suspension of Phos-cycC6 and BPmoc-F3 powders in aqueous 100 mM MES buffer (pH 7.0)

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was heated until the powders dissolved. The solution was incubated for 1 h at 25 ºC to give

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an SDN hydrogel (Phos-cycC6: 0.20 wt% (3.2 mM), BPmoc-F3: 0.075 wt% (1.2 mM), the

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CGC of Phos-cycC6 and BPmoc-F3 are 0.5 wt% and 0.05 wt%7b, respectively). Subsequently,

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an aqueous solution of SOx was added and then a Ca2+ ion solution was added dropwise to

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the hydrogel, which afforded a well-ordered Phos-cycC6/BPmoc-F3/Ca2+/SOx hydrogel

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(hereinafter, referred to as “multicomponent hydrogel”) (Figure 3b)22.



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Figure 3. (a) Schematic representation of the preparation of the multicomponent

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(Phos-cycC6/BPmoc-F3/Ca2+/SOx)

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multicomponent hydrogel. (c) CLSM images of Phos-cycC6/BPmoc-F3 (left) and

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multicomponent

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Alexa546-cycC6 (red) and BP-OG (green). The Pearson’s correlation coefficients are shown

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in the upper right of the images. Scale bar: 20 µm. Conditions: ((b) and (c)) [Phos-cycC6] =

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0.2 wt% (3.2 mM), [Alexa546-cycC6] = 4.0 µM, [BPmoc-F3] = 0.075 wt% (1.2 mM),

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[BP-OG] = 4.0 µM, [Ca2+] = 1.3 mM, [SOx] = 0.5 mg/mL (7.7 µM), 100 mM MES buffer

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(pH 7.0), 25 °C (b), 30 °C (c). (d) Rheological properties of the Phos-cycC6/BPmoc-F3/SOx

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hydrogels at an angular frequency of 10 rad/s with or without Ca2+ ions. Conditions:

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[Phos-cycC6] = 0.20 wt% (3.2 mM), [BPmoc-F3] = 0.30 wt% (4.7 mM), [SOx] = 2.0 mg/mL

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(30 µM), [Ca2+] = 3.2 mM, 100 mM MES buffer (pH 7.0), 25 °C. (e) Fluorescent recovery

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profiles of Phos-cycC6 fibers stained with Alexa546-cycC6 after photobleaching in

hydrogel

by

PAF.

(Phos-cycC6/BPmoc-F3/SOx/Ca2+)

(b)

(right)


Optical hydrogels

image

of

stained

the with

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Phos-cycC6/BPmoc-F3/SOx hydrogels with or without Ca2+ ions. (f) Diffusion coefficients

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of Phos-cycC6/BPmoc-F3/SOx hydrogel with or without Ca2+ ions determined by FRAP

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experiments (n = 8). The data represent the mean ± s.d. Conditions: ((e) and (f))

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[Phos-cycC6] = 0.1 wt% (1.6 mM), [Alexa546-cycC6] = 4 µM, [BPmoc-F3] = 0.1 wt% (1.6

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mM), [BP-OG] = 4 µM, 0.5 mg/mL (7.7 µM), [Ca2+] = 4.9 mM, 100 mM MES buffer (pH

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7.0), rt.

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The influence of the additional components (SOx and Ca2+ ions) on the

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orthogonality of the fiber networks was confirmed by in situ CLSM imaging using

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fluorescent probes, Alexa546-cycC6 and BP-OG (Figure S6)15b. As shown in Figure S9, the

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CLSM images of the single-fiber networks revealed that the two probes could selectively

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stain Phos-cycC6 and BPmoc-F3 fibers, respectively, even in the presence of SOx and Ca2+

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ions. The CLSM images of the SDN hydrogel without SOx and Ca2+ ions clearly showed that

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BPmoc-F3 and Phos-cycC6 formed into well-entangled self-sorting nanofiber networks

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(Figure 3c (left), Pearson’s correlation coefficient23, 24: 0.11). The orthogonality of the double

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network remained intact upon sequential addition of SOx and Ca2+ ions (Figure 3c (right),

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Pearson’s correlation coefficient: 0.16), suggesting that both SOx and Ca2+ ions did not have

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crucial impact on the orthogonal fiber networks of BPmoc-F3 and Phos-cycC6 (Figure 3c,

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S10).

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The effect of the Ca2+ ions on the Phos-cycC6/BPmoc-F3/SOx hydrogel was

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evaluated in more detail with dynamic viscoelastic measurement and FRAP analysis. The

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storage modulus (G’) of Phos-cycC6/BPmoc-F3/SOx hydrogel was increased from 5.5 kPa to

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22.9 kPa by the addition of Ca2+ ions, indicating that the Ca2+ ions were bound to the

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Phos-cycC6 fibers and stabilized the hydrogel (Figure 3d, S11, S12). The Ca2+ ions can not

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only bind to Phos-cycC6, but to BPmoc-F3 fibers in this multicomponent hydrogel. However 13 ACS Paragon Plus Environment


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it is conceivable that the Ca2+ ions were mainly bound to Phos-cycC6 fibers, because the

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carboxylic acid groups of BPmoc-F3 have a smaller affinity to Ca2+ ions than the phosphate

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group of Phos-cycC6 (Kd of carboxylic acid: 250 mM, phosphate group: 32 mM)25, 26. The

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FRAP experiments showed that the diffusion coefficient of the Phos-cycC6 fibers in the

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Phos-cycC6/BPmocF3/SOx hydrogel was decreased from (3.8 ± 2.2) × 10–2 µm2/s to (0.72 ±

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0.10) × 10–2 µm2/s by the addition of Ca2+ ions (Figure 3e, 3f, S13). This suggested that a

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sufficient amount of Ca2+ ions bound to the Phos-cycC6 fibers in this multicomponent

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hydrogel.

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As a control experiment, we conducted a one-step mixing protocol for the

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preparation of this multicomponent hydrogel (Figure 4a). The powders of two gelators

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(Phos-cycC6 and BPmoc-F3) and Ca2+ ions were mixed in a buffer solution, followed by

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heating of the mixture and subsequent incubation at 25 °C. This one-step mixing protocol did

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not afford hydrogels but resulted in an inhomogeneous suspension containing precipitates

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(Figure 4a). The CLSM observation of this suspension showed co-assembled spherical

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aggregates together with fibers that were selectively stained with BP-OG (Figure 4b). Given

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the spherical aggregates were stained by both the fluorescent probes, Phos-cycC6 and

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BPmoc-F3 were randomly mixed in the presence of Ca2+ ions during the one-step mixing

18

protocol, resulting in undesired mixtures. It was apparent that hybridization of SOx should be

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performed without heating to avoid thermal denaturation (The enzyme activity of the SOx

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was completely lost at 45 °C for 10 min incubation27). Overall, it was concluded that the PAF

21

protocol was invaluable for preparing a well-ordered multicomponent hydrogel.


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Figure 4. (a) Schematic representation of the one-step mixing protocol and an optical image

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of the obtained inhomogeneous suspension. (b) CLSM images of the inhomogeneous

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suspension prepared by a one-step mixing protocol. Left, middle, and right images show

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Alexa546-cycC6, BP-OG, and the merged channels, respectively. Scale bar: 20 µm.

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Conditions: [Phos-cycC6] = 0.2 wt% (3.2 mM), [Alexa546-cycC6] = 4 µM, [BPmoc-F3] =

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0.075 wt% (1.2 mM), [BP-OG] = 4 µM, [Ca2+] = 1.3 mM, 100 mM MES buffer (pH 7.0),

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25 °C (a), rt (b).

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Controlled protein release from the multicomponent hydrogel in AND logic gate fashion

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by ATP and sarcosine

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Because the two biomolecule-responsive systems were hybridized in a hydrogel through the

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PAF protocol, it was rationally expected that each fiber in the multicomponent hydrogel

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responds to the corresponding stimuli, that is Phos-cycC6/Ca2+ to ATP and BPmoc-F3/SOx

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to sarcosine. Therefore, the multicomponent hydrogel should exhibit a gel–sol transition in

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response to ATP and sarcosine in an AND logic gate fashion (Figure 5a)16. Indeed, when

8

only one stimulus, either ATP or sarcosine, was added to the multicomponent hydrogel, the

9

gel–sol transition did not occur because the single gel fiber network remained intact (Figure

10

5a, S14)28. Conversely, the addition of both ATP and sarcosine induced the gel–sol transition

11

(in an AND logic gate fashion). As shown in Figure S11, S12 and S15, the rheological

12

properties supported the response behavior observed by the naked eye. The initial G’ value of

13

the multicomponent hydrogel was 22.9 kPa, which decreased to 8.6 kPa or 5.4 kPa after the

14

addition of ATP or sarcosine, respectively. Furthermore, the concurrent addition of the two

15

molecules significantly decreased the G’ value to 1.9 kPa (Figure S11, S12, S15)29. These

16

results indicated that the two biomolecule-responsive systems independently work in the

17

multicomponent hydrogel.


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1 2

Figure 5. (a) AND logic gate-type response (gel–sol transition) of the multicomponent

3

hydrogel to ATP and sarcosine. (b) Schematic representation of controlled Fl-IgG release

4

experiments from the multicomponent hydrogel. (c) Photos of the multicomponent hydrogels

5

encapsulating Fl-IgG before and after addition of ATP and/or sarcosine. The photos were

6

taken under UV light in a dark room. (d) Fl-IgG release profiles from the multicomponent

7

hydrogel under various conditions (n = 3). The data represent the mean ± s.d. Conditions: (a)

8

[Phos-cycC6] = 0.2 wt% (3.2 mM), [BPmoc-F3] = 0.075 wt% (1.2 mM), [SOx] = 0.5 mg/mL

9

(7.7 µM), [Ca2+] = 1.3 mM, [ATP] = 9.7 mM, [Sarcosine] = 3.5 mM, 100 mM MES buffer

10

(pH 7.0), 25 °C. ((c) and (d)) [Phos-cycC6] = 0.2 wt% (3.2 mM), [BPmoc-F3] = 0.075 wt%

11

(1.2 mM), [SOx] = 1.0 mg/mL (15 µM), [Ca2+] = 1.3 mM, [Fl-IgG] = 133 nM, [ATP] = 9.7

12

mM

13

V(gel) : V(buffer) = 1: 4.5, 100 mM MES buffer (pH 7.0), rt.

(buffer),

[Sarcosine]

=

14


3.5

mM

(buffer),


1

Finally, the AND-type gel–sol response was applied to the controlled release of an

2

antibody embedded in the multicomponent hydrogel (Figure 5b). The multicomponent

3

hydrogel encapsulating fluorescein-labeled immunoglobulin G (Fl-IgG: 133 nM) was

4

prepared in a quartz cell and then an aqueous buffer solution was poured on the hydrogel.

5

Subsequently, ATP and/or sarcosine were added to the supernatant aqueous solution. The

6

Fl-IgG release rates from the hydrogel to the aqueous solution were evaluated by the

7

fluorescence intensities of the aqueous buffer solution (Figure 5c, 5d, S19). Without any

8

stimuli, the Fl-IgG release was very slow (Release rate: 16 ± 6% for 12 h), indicating the

9

efficient Fl-IgG retention of the multicomponent hydrogel. A similar slow release of 24 ± 4%

10

and 15 ± 1% was observed 12 h after the addition of the single stimulus, ATP or sarcosine,

11

respectively. Although the mesh-size of the multicomponent hydrogel network should be

12

expanded through modulation of the one-fiber network by the corresponding stimulus, the

13

impact on the Fl-IgG release was not so crucial. In contrast, an enhanced Fl-IgG release was

14

observed in the presence of both ATP and sarcosine; 83 ± 5% of Fl-IgG was released from

15

the multicomponent hydrogel after 12 h, as shown in Figure 5c, d. The release rate of Fl-IgG

16

was 3.5 and 5.5-fold larger than those using the single stimulus, ATP and sarcosine,

17

respectively, which clearly implied that the AND logic gate mode of antibody release was

18

accomplished in response to ATP and sarcosine in this multicomponent hydrogel. These

19

results suggested that the release rate of FL-IgG was mainly controlled by the state (gel or

20

sol) of the embedded matrix in this case, rather than the rheological properties. Conversely,

21

the inhomogeneous suspension prepared by the one-step mixing protocol showed an

22

immediate release of Fl-IgG even in the absence of any stimuli (Figure S20), revealing that

23

the PAF protocol was essential for such a sophisticated functionalization. 18 ACS Paragon Plus Environment

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1

Conclusion

2

A unique multicomponent hydrogel composed of a dual-biomolecule-responsive system was

3

prepared through a post-assembly fabrication (PAF) protocol. The programmed hybridization

4

allowed us to produce a hydrogel with a designer AND logic gate mode gel–sol transition in

5

response to two biologically important molecules (ATP and sarcosine). Such a well-ordered

6

multicomponent hydrogel was not obtained with a simple one-step mixing protocol. To the

7

best of our knowledge, this is the first multicomponent hydrogel based on an SDN whose

8

function was positively modified by PAF. This fabrication method relying on the self-sorting

9

and PAF protocol should be more powerful for the development of novel intelligent materials,

10

including more multiple components. We believe that this strategy could open up next

11

generation smart materials for a variety of applications such as therapy, diagnosis, drug

12

delivery systems, and regenerative medicine.

13



1

Associated content

2

Methods

3

The materials, instruments, experimental methods and syntheses of the compounds are

4

described in supporting information. The experimental conditions (concentration, pH and

5

temperature) are described in the figure captions.

6 7

Author information

8

Corresponding author

9

*E-mail: [email protected]

10

ORCID

11

Hajime Shigemitsu: 0000-0002-3104-049X

12

Ryou Kubota: 0000-0001-8112-8169

13

Itaru Hamachi: 0000-0002-3327-3916

14

Acknowledgements

15

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas

16

“Chemistry for Multimolecular Crowding Biosystems” (JSPS KAKENHI Grant 17H06348),

17

the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and

18

Technology (CREST), and JST ERATO Grant Number JPMJER1802 to I.H., and by a

19

Grant-in-Aid for Young Scientists (JSPS KAKENHI Grant 18K14333) to R.K. H.S.

20

acknowledge JSPS Research Fellowships for Young Scientists (16J10716). The authors

21

acknowledge financial support from The Mitsubishi Foundation.

22

Competing financial interests

23

The authors declare no competing financial interests.

24


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19. BPmoc-F3/SOx hydrogel was fabricated by addition of SOx to a BPmoc-F3 hydrogel.

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Because SOx can produce hydrogen peroxide (H2O2) during oxidation of sarcosine, the 24 ACS Paragon Plus Environment

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H2O2-sensitive BPmoc-F3 was decomposed by addition of sarcosine (Figure S1). We

2

confirmed the response of the BPmoc-F3/SOx hydrogel to sarcosine at room

3

temperature. As shown in Figure S1b, the BPmoc-F3/SOx hydrogel changed to the sol

4

state 2 h after addition of sarcosine (3 eq. of BPmoc-F3). The high performance liquid

5

chromatography (HPLC) analysis revealed that 90% of BPmoc-F3 molecules were

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decomposed and the resultant BPmoc-F3 concentration was below the critical gelation

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concentration (0.05 wt%) (Figure S1c).

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18 19

22. The SDN (Phos-cycC6/BPmoc-F3) hydrogel did not show swelling/contraction behaviors by the addition of SOx and Ca2+ ions (Figure S8).

20

23. The Pearson’s correlation coefficient indicates the degree of the linear correlation

21

between the two different channel images. In most case, if gelators are orthogonally

22

self-assembled, the values of Pearson’s correlation coefficients show lower than 0.3.



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24. Dunn, K. W.; Kamocka, M. M.; and McDonald, J. H. A practical guide to evaluating

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C723−C742.

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25. (a) Saha, A.; Saha, N.; Ji, L.; Zhao, J.; Gregáň, F.; Sajadi, S. A. A.; Song, B.; Sigel, H.

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26. The Kd values of acetic acid and methyl phosphate in aqueous solution at an ionic

10 11 12 13 14

strength of 0.1 M and 25 °C were referred. 27. Suzuki, M.; Purification and Some Properties of Sarcosine Oxidase from Corynebacterium sp. U-96. J. Biochem. 1981, 89, 599-607 28. The multicomponent hydrogel did not show swelling/contraction behaviors after the addition of ATP or sarcosine (Figure S14).

15

29. To evaluate the gel-sol transition more quantitatively, we tried to measure the

16

rheological property of the multicomponent hydrogel under the same condition with the

17

tube inversion test (Figure S16-18). The multicomponent hydrogel showed rheological

18

properties of gel state, i.e., frequency-independent G’ (Figure S17a), and G’ >> G’’ (G’

19

= 1082 Pa, G’’ = 174 Pa, Figure S18). On the other hand, the viscous solution obtained

20

after addition of both stimuli (ATP and sarcosine) showed very small G’ value (29 Pa)

21

which may be consistent with the naked eye observation. The tan δ (tan (G’’/G’)) value

22

of the viscous solution was 2.0-fold larger than that of the initial multicomponent

23

hydrogel, indicating the multicomponent hydrogel came close to the sol state by addition 26 ACS Paragon Plus Environment

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1

of the both stimuli, while the G’ value is still larger than G’’. The discrepancy between

2

the rheological data (larger G’ value than G’’) and visual results would be derived from

3

the residual gel fragments in the sol.

4



1

TOC

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Post-assembly Fabrication of a Functional Multicomponent

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