One-Shot Preparation of Polyacrylamide/Poly(sodium styrenesulfonate) Double-Network Hydrogels for Rapid Optical Tissue Clearing.

In this study, we propose a convenient method for the synthesis of double-network gels by the one-shot radical polymerization for their application to rapid optical tissue clearing. Double-network gels were produced during the radical polymerization of acrylamide (AAm) and sodium styrenesulfonate (SS) in the presence of N,N'-methylenebisacrylamide and sodium divinylbenzenesulfonate as the cross-linkers by simultaneous addition, that is, one-shot polymerization accompanying the delay of polymerization for a second network monomer. We analyzed the polymerization process based on the consumption rates of each monomer during the reactions in the absence of the cross-linkers in order to estimate the repeating unit structure of the resulting polymers. We then fabricated the AAm/SS gels by the polymerization of AAm and SS in the presence of the cross-linkers. We analyzed the swelling, viscoelastic, and mechanical properties of the produced gels to investigate their network structure. Finally, we demonstrated the validity of the double-network gels for the application to rapid optical tissue clearing.

Introduction

Polymer hydrogels with three-dimensional (3D) network structures are used as the key materials for the construction of smart materials and systems, such as water absorbents,[1] contact lenses,[2] membranes for separation,[3] drug delivery systems,[4] shape-memory materials,[5] self-healing materials,[6] actuators,[7] cell scaffolds,[8] tissue engineering,[9] and so forth. Several approaches have been proposed to design high-performance hydrogel materials, for example, interpenetrated polymer networks,[10] bio-responsible gels,[11] slide-ring gels,[12] tetra-PEG gels,[13] nanocomposite gels,[14] anisotropic gels,[15] supramolecular hydrogels,[16] and cyclodextrin-guest interaction.[17] Recently, double-network gels have drawn much attention in various fields because of their high mechanical strength and toughness.[18−20] Typical high-strength double-network gels contain two independent kinds of networks that interact with each other in a space. The double-network gels with a high mechanical strength are usually fabricated by a two-step polymerization process. One component of the networks is highly cross-linked polyelectrolytes (the first network) as the rigid skeleton, and the other component is comprised of neutral polymers with a loose cross-linking structure (the second network).[20] It has been reported that the produced double-network gels possess excellent tensile and compression properties.[21−30]

Optical tissue clearing is an indispensable process for the 3D imaging of tissues combined with a molecular-labeling technique, such as immunofluorescence staining[31] and 4′,6-diamidino-2-phenylindole staining,[32] without preparing thin tissue sections. A smart technique for optical tissue clearing using hydrogels was developed in 2013 by Chung et al., named CLARITY (clear lipid-exchanged acrylamide-hybridized rigid imaging/immuno-staining/in situ-hybridization-compatible tissue-hydrogel),[33] and several modified methods have since been proposed.[34−41] The CLARITY methods include several steps, such as the fixation of proteins in polyacrylamide gels using paraformaldehyde (PFA), the removal of lipids with a detergent-containing buffer, and adjustment of the refractive index using refractive index matching solutions. Ono et al. recently reported the application of polyelectrolyte hydrogels, which were synthesized by the radical copolymerization of acrylamide (AAm) and sodium 4styrenesulfonate (SS) in the presence of N,N′-methylenebisacrylamide (bisAA), to the optical tissue clearing method.[42] The SS unit included in the hydrogel contributed to shortening an optical tissue clearing process because of an electrostatic repulsion and/or an increased osmotic pressure in the hydrogels. The tissues became transparent more rapidly when they were embedded in the SS-containing hydrogels.

When the hydrogels of AAm and SS were synthesized, we noticed the formation of gels with a complex structure because of a different reactivity of these monomers during polymerization; that is, the copolymerization did not simply provide a random copolymer. During a radical copolymerization process using two kinds of monomers with significantly different reactivities, a highly reactive monomer predominantly undergoes polymerization during the initial stage of polymerization, in which only a small amount of another monomer with a lower reactivity is incorporated into the produced polymer. After almost total consumption of the reactive monomer (the first monomer), the polymerization of the less-reactive monomer (the second monomer) gradually occurs. It is also postulated that bisAA as the cross-linker readily reacts with the propagating radical produced from AAm, but it hardly adds to the SS propagating radical with a highly conjugated structure. It was possible that the network of SS as the first monomer was insufficiently grown because of the lower reactivity of bisAA to the SS radical under the polymerization conditions conducted in our previous study.[42] The structure control of the network consisting of SS repeating units as the electrolyte with an anionic charge is important for the tissue clearing. Previously, Aranas et al. reported a simple one-step radical polymerization procedure using potassium 3-sulfo-1-propyl methacrylate and N-vinylpyrrolidone as the conjugating and nonconjugating monomers, respectively, in the presence of the two kind of corresponding cross-linkers for the synthesis of hydrogels.[29] The produced double-network hydrogels were available for rapid cell detachment.

In this study, we carried out the one-shot polymerization of SS and AAm using dual cross-linkers. First, the polymerization of AAm and SS was carried out in the absence of cross-linkers in a buffer or D2O and monitored the consumption of both monomers as a function of the polymerization time using NMR spectroscopy. We analyzed the monomer reactivity and the detailed reaction behaviors during the one-shot polymerization. Next, we synthesized double-network hydrogels by the radical polymerization of AAm and SS via a one-shot process in the presence of dual cross-linkers, bisAA and sodium divinylbenzenesulfonate (DVBS) (Figure ). We also discuss the effect of the combination of the monomers and the cross-linkers with different reactivities on the network structures and the properties of the produced hydrogels. The composition, swelling behavior, viscoelastic, and mechanical properties of the obtained hydrogels were investigated in detail. Finally, the produced double-network hydrogels were applied to the rapid optical clearing of tumor tissues.

Figure 1

Chemical structure of monomers (AAm and SS), cross-linkers (bisAA and DVBS), and radical initiators (VA-044 and APS) used in this study.

Results and Discussion

Polymerization Reactivity of AAm and SS

The polymerizations of AAm or SS were carried in the presence of VA-044 in phosphate-buffered saline (PBS) prepared using D2O (PBS–D2O) at 37 °C to compare the polymerization reactivity of these monomers. As shown in the time–conversion relationship in Figure , the polymerization of AAm rapidly proceeded and reached 95 and 99% conversions after 1 and 3 h, respectively, while the SS polymerized at a lower rate and the conversion still increased even after 3 h.

Figure 2

Time–conversion relationship for independent homopolymerizations of AAm (red circle) and SS (blue square). The polymerizations were carried out in the presence of VA-044 (7.0 mmol/L) in PBS–D2O at 37 °C. [AAm]0 = 0.50 mol/L, [SS]0 = 0.50 mol/L.

For the polymerization of AAm and SS by the simultaneous addition to the reaction system, that is, the one-shot polymerization of AAm and SS, both monomers exhibited a polymerization reactivity that was opposite to the order observed during the independent homopolymerizations. In Figure , the time–conversion relationships and their first-order plots are shown for the one-shot polymerizations of AAm and SS with various monomer ratios in the feed. The conversions of each monomer were simultaneously monitored as a change in the intensities of the characteristic peaks in the 1H NMR spectrum. It clearly showed that SS was more rapidly consumed than AAm during the polymerization (see Figure S1). The polymerization rate of SS was almost independent of the presence or absence of AAm; that is, the conversion was over 90% after a 4 h polymerization, and then no SS was detected in the reaction mixture after 8 h (Figure ). The consumption rate of SS increased according to an increase in the AAm content, but the magnitude of the change was small. In contrast, the polymerization behavior of AAm sensitively changed with or without SS. In the presence of SS, the consumption of AAm was significantly suppressed. The conversion of AAm was less than 20% after 4 h during the polymerization in the presence of an equimolar amount of SS. This was quite different from the quantitative consumption of AAm during homopolymerization under similar conditions in the absence of SS. After the almost consumption of SS, the polymerization of AAm was accelerated. During the one-shot polymerization of AAm and SS, the composition of the remaining monomers continuously changed according to the consumption rates of both monomers. This may lead to the formation of polymers with heterogeneous structures after the complete monomer consumption.

Figure 3

(a) Time–conversion relationships and (b) first-order plots for one-shot polymerization of AAm and SS with various feed compositions in the presence of VA-044 (7.0 mmol/L) in PSB–D2O at 37 °C. [AAm]0/[SS]0 = 1/1 (circle), 3/1 (triangle), 6/1 (square), and 12/1 (rhombus) molar ratio in the feed. The total monomer concentration was 0.50 mol/L.

In general, styrene monomers have a high reactivity as the monomer because of the highly stabilized structure of the corresponding propagating radicals. Under the conditions of the coexistence of a highly conjugated SS monomer and a relatively less-conjugated AAm monomer, the propagation rate of SS was much greater prior to that of AAm. In fact, the monomer reactivity ratios were reported to be r1 = 1.17 and r2 = 0.58 for the copolymerization of styrene (M1) and AAm (M2) in the literature.[43] The SS monomer is one of typical conjugating monomers, for example, r1 = 7.19 and r2 = 0.084 for the copolymerization of SS (M1) and N-vinylpyrrolidone (M2).[44] These literature values also supported the predominant propagation of SS and the lower reactivity of AAm during the copolymerization. Based on the monomer consumption rates in Figure , the comonomer–copolymer composition curve was depicted, and the monomer reactivity ratios were estimated to be r1 = 0.10 and r2 = 6.6 using the Fineman–Ross plots (Figures S2 and S3), where M1 and M2 monomers are AAm and SS, respectively. These parameters indicated that SS possesses a reactivity more than 60 times higher than that of AAm. With the increase in the AAm content in the feed, a time lag between the consumption of both monomers was shortened, and the polymerization behavior became close to the typical copolymerization.

Preparation of the Hydrogels

The polymerization was carried out in the presence of difunctional monomers as the cross-linkers to fabricate the hydrogels of AAm and SS. In this study, we used two types of cross-linkers, bisAA and DVBS, which have a molecular structure similar to AAm and SS, respectively. The other polymerization conditions were the same as those for the one-shot polymerization described in the previous section. Table shows typical results for the preparation of the gels and the evaluation for their swelling property in water. When a small amount of cross-linkers was added to the polymerization system (0.6 mol % to the monomers), all the produced polymer chains were incorporated into the polymer networks, and no soluble polymer was detected by extraction using a large amount of water after polymerization. The 1H NMR spectra indicated the formation of the gels with the composition corresponding to the feed monomer ratio, that is, the AAm/SS ratios incorporated in the hydrogels were 3.16, 5.99, and 8.52 for the feed ratios of 3/1, 6/1, and 9/1, respectively (Figure S4). The heterogeneous polymer structure may be produced during the gel fabrication, based on the results for the one-pot polymerization of AAm and SS without any cross-linker. During the initial stage of the polymerization, the polymers containing SS-rich segments are predominantly produced. In contrast, the consecutive AAm segments are produced as the major components around the final stage of the polymerization. All of the monomers are consumed and incorporated into the polymer networks as the final products.

Table 1

Preparation Conditions and Swelling Ratio for the Hydrogels of AAm and SSa

hydrogel	[AAm]0/[SS]0 (molar ratio)	[bisAA]0 × 103 (mol/L)	[DVBS]0 × 103 (mol/L)	[bisAA]0/[DVBS]0 (molar ratio)	swelling ratio (%)
AAm/SS gel	12/1	3.0	0.2	15/1	5120
	9/1	2.9	0.3	9.7/1	5220
	6/1	2.8	0.5	5.6/1	6180 ± 230
	3/1	2.4	0.8	3/1	6970 ± 300b
	1/1	1.6	1.6	1/1	8440 ± 1130c
	1/2	1.1	2.1	1/1.9	7060
	1/3	0.8	2.4	1/3	9520
	1/6	0.5	2.7	1/5.4	9110
	1/9	0.3	2.9	1/9.7	5240
	1/1	3.2	0	1/0	5610
	1/1	0	3.2	0/1	6910
AAm gel	1/0	3.2	0	1/0	1860 ± 110
SS gel	0/1	0	3.2	0/1	d

Polymerization conditions: The total monomer concentration was 0.55 mol/L. [VA-044] = 7.7 mmol/L in PBS at 37 °C for 4 h (AAm gel) and 24 h (AAm/SS gels) or at 45 °C for 4 h (SS gel). Total concentration of the cross-linkers was 3.2 mmo/L (0.6 mol % to the monomers). The swelling ratios were determined in distilled water at room temperature after 18 h.

The storage modulus (G′) was determined to be 0.70 kPa at 1 Hz and 25 °C with a strain of 0.01 by a viscoelasticity measurement (see also Figure S6).

The G′ value was 0.92 kPa (see Figure S6).

Not determined due to the loose structure of the gel.

The microscopic compositions, sequence distribution, the cross-linking density, and local chain dynamics of the gels determine the performance for the optical tissue clearing, but it is not easy to fully define a polymer network structure. In this study, we examined the effects of the polymerization conditions on the swelling behavior of the obtained hydrogels. The dried samples were gradually swollen during immersion in water at room temperature (Figure S5). The AAm/SS gel exhibited an excellent swelling property, and the infinite swelling ratio was as high as 5120–9520% after 18 h (Table ). Electrostatic repulsion between the negatively charged SS units on the polymer chain resulted in highly swollen and loose hydrogels. Actually, the swelling ratio significantly depended on the composition of the gels; that is, the swelling ratio decreased along with an increase in the AAm/SS ratio. The AAm gel as the nonelectrolyte gel showed a swelling ratio of 1860%, which was much lower than those of the AAm/SS gels. The SS gel was difficult to be used for the swelling ratio measurement because it was too soft and fragile. An increase in the SS content led to an increasing swelling ratio of the gels and finally induced collapse of the gels. Consequently, it reduced the apparent swelling ratio, as shown in the gel prepared with AAm/SS = 1/9 in Table . The much higher reactivity of SS seems to produce a soluble polymer during the copolymerization of AAm and SS using only bisAA as the cross-linker. Actually, however, no soluble polymer was extracted from the gels produced after the complete conversion of both monomers (see also Figure S4), and the swelling property of the gel produced using only bisAA under the AAm/SS = 1/1 condition was similar to those for the other gels (Table ). The swelling ratio was 5610% for the gel produced with bisAA at AAm/SS = 1/1, and this swelling ratio was slightly smaller than those for the gels produced in the presence of only DVBS and both DVBS and bisAA, (6910 and 8440%, respectively). The time–conversion curves indicate a large difference in the consumption rates of SS and AAm, but no induction period was observed in these curves. This means the incorporation of AAm and bisAA even at the early stage of the copolymerization, leading to the production of the gel containing both AAm and SS units as the final product under these copolymerization conditions. The network structures of the gels, such as monomer sequences and the distribution of cross-linking points, possibly depend on the type of the cross-linkers, leading to different performances for optical tissue clearing.

Viscoelastic and Compression Properties

We prepared the harder AAm/SS gels for the polymerization in water in the presence of a larger amount of cross-linkers (2 or 3.3 mol % to the monomers) using ammonium persulfate (APS) as the radical initiator in water at 70 °C. APS was used because it produced no nitrogen gas bubbles in the gels. The obtained gels were used for the rheology and compression mechanical tests. The preparation conditions, the swelling ratios, the elastic modulus, and the fracture strain are summarized in Table .

Table 2

Viscoelasticity and Compression Measurements of the Hydrogels of AAm and SSa

hydrogel	[AAm]0/[SS]0 (molar ratio)	[bisAA]0/[DVBS]0 (molar ratio)	[monomers]0/[cross-linkers]0 (molar ratio)	swelling ratiob (%)	elastic modulusc (kPa)	fracture strainc (%)
AAm/SS gel	6/1	6/1	50/1	6050 ± 900	29.0	64.0
	3/1	3/1	50/1	7050 ± 400	69.2	55.2
	1/1	1/1	50/1	4970 ± 470	50.3	60.3
	1/1	1/1	30/1	3900	93.3	47.9
	2/1d	1/40d	3/4.1d	3900	88.2	38.8
	1/3	1/3	50/1	4810 ± 190	50.3	60.0
	1/6	1/6	50/1	6340 ± 60	11.5	55.0
AAm gel	1/0	1/0	50/1	940 ± 370	28.8	53.3
	1/0	1/0	30/1	450	30.0	45.0
SS gel	0/1	0/1	50/1	>7000	<1

Polymerization conditions: [AAm]0 + [SS]0 = 1.0 mol/L, [bisAA]0 + [DVBS]0 = 20 or 33 mmol/L (2 or 3.3 mol % to the monomers), and [APS] = 10 mmol/L in water at 70 °C for 4 h.

Determined in water at room temperature after 24 h.

Determined by compression test using columnar samples (20 mm diameter and 10 mm height) at a rate of 1 mm/min.

[AAm]0/[bisAA]0/[SS]0/[DVBS]0 = 2/0.1/1/4 in molar ratio.

The equilibrium swelling ratios of the AAm/SS gels were 4810–7050% in water at 25 °C. The G′ values at a small strain (γ < 0.01) were 4–6 kPa for the AAm/SS and AAm gels, while the G′ values for the SS gel was much lower (102 Pa), as shown in the strain dependence of the storage and loss moduli for the gels (Figure S7). These values suggest the formation of the harder gels with a high cross-linking density, compared to the gels prepared under the conditions described in Table . The relationship between the compression strain and stress is shown in Figure . The curves for the AAm/SS gels were different from those for the AAm gel and the SS gel. The AAm/SS gels exhibited the maximum strength with 50–70 kPa at ca. 60% of compression strain, independent of the AAm/SS compositions. The elastic moduli determined from the initial slopes of the compression curves were in the range of 29–69 kPa. The AAm/SS gel prepared under the condition of [AAm]0/[SS]0 = 1/6 was exceptionally different from the other AAm/SS gels (Figure b), that is, the elastic modulus and the strength of that gel were lower. The AAm gel also showed a lower elastic modulus and strength. The SS gel was soft and fragile because of the loose cross-linking even under the preparation conditions shown in Table .

Figure 4

Compression test of (a) AAm/SS gel ([AAm]0/[SS]0 = 1/1), AAm gel, and SS gel and (b) AAm/SS gels with various compositions, (c) AAm/SS gels prepared under the conditions of different monomers and cross-linker ratios, and (d) AAm gels prepared under the conditions of different monomers and cross-linker ratios. The compression rate was 1 mm/min using columnar samples with 20 mm diameter and 10 mm height. The preparation conditions for the gels are shown in Table . The molar ratio of the monomers and the cross-linkers was 50/1 for the preparation of the gels in (a,b).

The mechanical properties of the AAm/SS gels are controlled by the network structures, and they are quite dependent on the ratio of the monomers and cross-linkers.[19−30] In this study, we also prepared the AAm/SS gel and the AAm gel with more dense cross-linking structures by using cross-linkers at a higher concentration. When the ratio of the monomers to the cross-linkers was changed from 50 to 30, harder gels are produced as shown in Table . The swelling ratio decreased from 4970 to 3900% and the elastic modulus increased from 50.3 to 93.3 kPa for the AAm/SS gels. Similar changes were observed for the AAm gels. As shown in the results of the compression test in Figure c, the harder gel exhibited a breaking point at a lower compression strain. In the literature, typical double-network gels have been prepared by the two-step polymerization process,[20] which includes the formation of rigid and brittle polyelectrolyte as the first network and then a soft and ductile neutral polymer as the second network. A large amount of the second network, usually 20–30 times higher, is used. During the fabrication of the first network, the polymerization was carried out in the presence of a high concentration of a cross-linker. We also synthesized an AAm/SS gel at a higher cross-linker concentration to check whether a high-strength hydrogel similar to those described in the literature is produced or not, that is, under the feed condition of [AAm]0/[bisAA]0/[SS]0/[DVBS]0 = 2/0.1/1/4. In this study, however, only a weak gel was produced as shown in Table and Figure c. This was because of the slow propagation rates of the SS and DVBS monomers. The limited solubility of SS and DVBS was also disadvantageous for the formation of a dense and well-developed first network structure. We observed a plateau region for the stress–strain curves during the compression test of the AA/SS gels (Figure ). Similar compression curves with a plateau region were reported for some other double-network hydrogels,[45−47] although the detailed fracture mechanism is unclear for the AAm/SS hydrogels. Because the expansion of the SS first network is insufficient in the present case, the observed S–S curves might be related to that for the second or later cycle during repeating loading/deloading mechanical tests. We are now continuing the investigation of the mechanical properties of the double-network gels prepared by the one-shot radical polymerization. In this study, we focused on the application of the AAm/SS gels with a double-network structure to the rapid optical tissue clearing.

Optical Tissue Clearing

We performed optical clearing of tumor tissues using the passive CLARITY method with the AAm/SS gels prepared in this study. The general CLARITY method consists of the following processes: (i) The tissue is incubated in a cold monomer solution containing AAm, bisAA, a radical initiator, and PFA. The PFA generates formaldehyde as the mediator for cross-linking between the proteins and polymer networks. (ii) The polymerization is initiated by incubation at 37 °C, resulting in the fixation of the protein in the hydrogel. (iii) The lipids are then removed through diffusion in a detergent-containing buffer. (iv) Finally, the refractive index of the media is adjusted by the substitution of the aqueous media to the refractive index matching solutions.[33,42] In the present study, we used the AAm/SS gels ([AAm]0/[SS]0 = 1/1 in molar ratio) prepared in the presence of bisAAm and/or DVBS (0.6 mmol %). The mechanical toughness of the obtained gels was enough for the experiments of optical tissue clearing. The results for the evaluation of the optical tissue clearing are shown in Figure . The opaque tissues became transparent after the lipid removal using sodium dodecyl sulfate (SDS) for 2 days. The tumor tissues treated with the gels prepared in the presence of DVBS became more rapidly transparent. The transparency of the tissues was estimated using ImageJ in this study. The transmittance was determined to be 88 and 74% after the SDS treatment for 2 days, while it was only 23% when bisAA was used. After the removal of the detergent and the immersion in ethylene glycol to adjust the refractive index, each tissue became clearer. The lipid molecules in the tissue-embedded hydrogel were rapidly excluded to reduce the osmotic pressure. The SS networks of the double-network AAm/SS gels play an important role in the efficient removal of the lipid. The use of DVBS as the effective cross-linker for the polymerization of SS led to the expansion of the first networks over the entire gels. In contrast, bisAA is not suitable for the SS network formation during the initial stage of the polymerization for the gel preparation. Probably, the SS-rich domains are discontinuously located in the gels. It was actually disadvantageous for the lipid removal process using SDS, as shown in Figure a. Thus, we have demonstrated the validity of the use of not only SS as the anionic monomer but also DVBS as the cross-linker, which was efficient for the SS network formation.

Figure 5

Optical clearing of tumor tissues in the process of extraction of lipids with SDS for 2 days and the subsequent immersion in ethylene glycol. (a) bisAA, (b) DVBS, and (c) both bisAA and DVBS were used as the cross-linkers for the preparation of the hydrogels. The [AAm]0/[SS]0 ratio was 1/1. The right panels are the images for the evaluation of transmittance, which were produced using ImageJ. T indicates the transmittance.

Conclusions

In the present study, we demonstrated the convenient synthesis of double-network gels by the one-shot radical polymerization of AAm and SS with different reactivities for the propagation in the presence of bifunctional monomers as the cross-linkers. This new method is advantageous because of the one-pot and one-shot process for the polymerization while double-network gels are prepared by a two-step polymerization process in many cases. In addition, it was revealed that the double-network gels containing polyelectrolyte segments with an anionic charge were available for the fixation matrix gels for optical tissue clearing. The new cross-linker DVBS led to the production of the expanded SS network structure, which was valid for shortening the tissue clearing time. The present method for the synthesis of double-network gels by the one-shot polymerization can be applied to many combinations of reactive and less-reactive monomers other than SS and AAm. The mechanical strength of the AAm/SS gels prepared in this study was lower than those for the other double-network gels reported in the literature. It was because of the unexpanded network structure of SS as the first monomer because of the slow propagation rate of SS. We are now continuing our investigation of the synthesis and characterization of the double-network gels using the one-shot polymerization technique.

Experimental Section

Materials

Commercially available AAm (Nacalai Tesque, Kyoto, Japan), SS (Tokyo Chemical Industry Corporation, Ltd., Tokyo, Japan), and bisAA (Tokyo Chemical Industry Corporation, Ltd., Tokyo, Japan) were used without further purification. DVBS was supplied from Tosoh Finechem Corporation, Tokyo, Japan) and used as received. VA-044 and APS (Wako Pure Chemical Industries, Osaka, Japan) as the initiators were used without purification. For the preparation of a PBS solution, commercial sodium chloride, potassium chloride, disodium hydrogen phosphate, and dipotassium hydrogen phosphate (Wako Pure Chemical Industries, Osaka, Japan) were used as received. 1,2-Dimethoxyethane (Tokyo Chemical Industry Corporation, Ltd., Tokyo, Japan), ethylene glycol, sodium hydroxide, PFA, sodium lauryl sulfate, and boric acid (Nacalai Tesque, Kyoto, Japan), and TritonX-100 (Tokyo Chemical Industry Corporation, Ltd., Tokyo, Japan) were used as received.

General Procedures

The NMR spectra were recorded in D2O using JEOL ECS-400 and ESX-400 spectrometers. A compact pH meter LAUAtein (Horiba, Ltd., Kyoto, Japan) was used for the pH measurement. The mechanical properties were characterized using a HAAKE MARS III rheometer (Haake Technik GmbH, Vreden, Germany). The sample hydrogels were placed between parallel plates of 20 mm diameter at 25 °C. A compression test was performed at room temperature using Autograph AGS-X 1 kN (Shimadzu Corporation, Ltd., Kyoto, Japan) and a columnar sample with a 20 mm diameter and 10 mm height at a compression rate of 1 mm/min. Freeze-drying was carried out using FDU-1200 (EYELA Corporation, Ltd., Tokyo, Japan). Tissue clearing was performed using bio-chamber BCP 120-F (Taitec Corporation, Osaka Japan) and a rotary shaker NR-2 (Taitec Corporation, Osaka Japan).

Homopolymerization

To the mixture of AAm (0.200 g) or SS (0.577 g) in 5 mL of the phosphate buffered saline prepared using D2O (PBS–D2O) and 0.5 mL of 1,2-dimethoxyethane as the internal standard, nitrogen gas was bubbled through the solution at 0 °C for 5 min. After the addition of VA-044 (0.0125 g), the solution was transferred to an NMR tube. Nitrogen was charged again, and the polymerization was performed at 37 °C. The monomer and initiator concentrations were 0.55 and 7.7 mmol/L, respectively. After polymerization for a specific time, an NMR spectrum was recorded. The monomer conversions were determined based on a change in the intensity of the characteristic peaks because of the AAm and SS monomers observed at 5.75–5.90 and 5.30–5.45 ppm, respectively. The pH values of the solutions were determined before and after the polymerization. The pH was constant of 6.87 during the polymerization.

One-Shot Polymerization of AAm and SS

The one-shot polymerization of AAm and SS was carried out according to a procedure similar to the homopolymerization. The total monomer concentration was 0.55 mol/L. The ratio of AAm to SS was changed in the range of 12/1 to 12/1 molar ratio in the feed. The conversion of each monomer was determined after polymerization by NMR spectroscopy. The 1H NMR spectra of the reaction mixture of AAm and SS ([AAm]0/[SS]0 = 1/1 in molar ratio) in PBS–D2O before and after the polymerization for 3 h are shown in Figure S1. The conversion of the monomers was determined based on the change in the peak intensity because of the vinyl hydrogens (the peaks a and d for AAm and SS, respectively, in Figure S1).

Preparation of Hydrogels

AAm (0.2 g) and SS (0.58 g) as the monomers and bisAA (4.9 mg) and DVBS (7.4 mg) as the cross-linkers were dissolved in 5 mL of PBS. Nitrogen gas was then bubbled through the solution at 0 °C for 5 min. After the addition of VA-044 (0.0125 g), the solutions were incubated at 37 °C for 4 or 8 h for the AAm/SS gels, at 37 °C for 4 h for the AAm gel, and at 45 °C for 4 h for the SS gels. The obtained hydrogels were immersed in distilled water for 48 h to remove the water-soluble compounds. The weight of the dried gels was measured after freeze-drying. Some portions of the gels were provided for 1H NMR measurement in PBS–D2O. The hydrogels for determination of the mechanical properties were also prepared using APS as the radical initiator at 70 °C for 4 h. Hydrogels were swollen in distilled water, and the swelling ratio was calculated using the following equationwhere Wdry and Wwet are the weights of the dried gel and obtained hydrogel, respectively. The number of samples was 1–4.

Optical Tissue Clearing Method

Tumor tissues obtained from tumor-bearing mice (MDA-MB-231 cell-implanted nude mice) were stored in a formalin solution.[48] The tissues were manually sliced into pieces of 0.7 mm thickness. The size of the test pieces was 6–9 mm × 2–5 mm. The optical tissue clearing was carried out according to previous reports.[42,49] The small tissues were immersed in 10 mL of PBS (pH 7.4) containing AAm and/or SS (0.56 mol/L), bisAA and/or DVBS (3.2 × 10–2 mmol/L), VA-044 (7.73 mmol/L), and PFA (1.33 mol/L) at 4 °C for 24 h. After incubating at 37 °C for 24 h, a hydrogel was formed. The gel outside the tissue was then removed, and the tissues were immersed in 30 mL of 0.8 mol/L borate buffer (pH 8.5) containing 4% SDS at 37 °C and shaken for 2 days. Next, the tissues were placed in 30 mL of a 0.1% (v/v) Triton X-100 solution. After 2 days of shaking at 37 °C, the tissues were immersed in 20 mL of ethylene glycol for 1 h. Images of the tissues were obtained before and after the SDS treatment and after the final step. An image analysis was conducted with ImageJ software version 1.52a (23 April 2018, http://rsb.info.nih.gov/ij/). The transmittance of a black bar (width 0.7 mm) under the tissues in the images was determined.