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[Nanami Fujisawa](https://orcid.org/0000-0002-8894-1790), [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350)

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[Layer-by-layer assembled film can serve as an enhanced reaction environment for Diels–Alder reaction](https://mdr.nims.go.jp/datasets/7a1edf31-9184-4729-980a-30f3e1a4222c)

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Layer-by-layer assembled filmcan serve as an enhanced reactionenvironment for Diels–AlderreactionNanami Fujisawa1,2 and Mitsuhiro Ebara1,2,3*1Research Center for Macromolecules and Biomaterials, National Institute for Materials Science (NIMS),Tsukuba, Japan, 2Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan,3Department of Materials Science and Technology, Tokyo University of Science, Tokyo, JapanAlthough the Diels–Alder reaction (DA) has garnered significant attention due toits numerous advantages, its long reaction time is a drawback. Herein, weinvestigated the effects of polarity difference on DA using Layer-by-Layer(LbL) films comprising polycationic polyallylamine hydrochloride andpolyanionic poly (styrenesulfonic acid-co-furfuryl methacrylate) [poly (SS-co-FMA)] as the reaction environment. First, furan composition in poly (SS-co-FMA)was adjusted to be 19mol% to achieve good water solubility and layer deposition.The successful formation of LbL films with 8 and 40 layers was confirmed byquartz crystal microbalance. The polarity within films and, consequently, the DAefficiency between furfuryl methacrylate and the maleimide in MAL-PEG2-NHSincreased with an increasing number of layers up to 40 layers without requiringchemical modification on the reaction site of DA or any catalysts. Furthermore,we employed the LbL coating on the surface of magnetic nanoparticles (MNPs).The retro DA reaction (rDA) was successfully triggered by heating theMNPs by ACmagnetic field. We believe that the proposed technology can serve as anenhanced DA reaction environment as well as temporal/spatial control of rDAin various applications.KEYWORDSDiels-Alder reaction, layer-by-layer, quartz crystal microbalance, magneticnanoparticles, AC magnetic field1 IntroductionDynamic covalent bonds are chemical bonds that reversibly form under equilibrium-controlled conditions. These bonds first reach transition states upon being subjected toexternal stimuli such as heating or catalysis, following which they achieve stable states thatare not their initial states. The Diels–Alder (DA) reaction, which is highly direct and doesnot require the use of a catalyst, (Rowan et al., 2002; Maeda et al., 2009; Gregoritza andBrandl, 2015), is based on the chemically selective [4 + 2] cyclization between the electron-withdrawing group of a diene and the electron-donating group of a dienophile; it isparticularly important in the formation of ring structures and is applied for the productionof compounds such as small-molecule pharmaceuticals (Nicolaou et al., 2002). However,the reaction time for DA reactions can be relatively long, which can hinder practicalapplication; in our previous study, a DA reaction required 120 h to reach completion(Fujisawa et al., 2021). The reaction time can be controlled by changing the energy state ofthe reaction site via chemical modification (Froidevaux et al., 2015). In addition, reactionOPEN ACCESSEDITED BYUrara Hasegawa,The Pennsylvania State University (PSU),United StatesREVIEWED BYAndre Van Der Vlies,The Pennsylvania State University (PSU),United StatesShota Fujii,University of Massachusetts Amherst,United States*CORRESPONDENCEMitsuhiro Ebara,Ebara.Mitsuhiro@nims.go.jpRECEIVED 07 November 2024ACCEPTED 25 November 2024PUBLISHED 12 December 2024CITATIONFujisawa N and Ebara M (2024) Layer-by-layerassembled film can serve as an enhancedreaction environment for Diels–Alder reaction.Front. Chem. 12:1524096.doi: 10.3389/fchem.2024.1524096COPYRIGHT© 2024 Fujisawa and Ebara. This is an open-access article distributed under the terms of theCreative Commons Attribution License (CC BY).The use, distribution or reproduction in otherforums is permitted, provided the originalauthor(s) and the copyright owner(s) arecredited and that the original publication in thisjournal is cited, in accordance with acceptedacademic practice. No use, distribution orreproduction is permitted which does notcomply with these terms.Frontiers in Chemistry frontiersin.org01TYPE Original ResearchPUBLISHED 12 December 2024DOI 10.3389/fchem.2024.1524096https://www.frontiersin.org/articles/10.3389/fchem.2024.1524096/fullhttps://www.frontiersin.org/articles/10.3389/fchem.2024.1524096/fullhttps://www.frontiersin.org/articles/10.3389/fchem.2024.1524096/fullhttps://www.frontiersin.org/articles/10.3389/fchem.2024.1524096/fullhttps://crossmark.crossref.org/dialog/?doi=10.3389/fchem.2024.1524096&domain=pdf&date_stamp=2024-12-12mailto:Ebara.Mitsuhiro@nims.go.jpmailto:Ebara.Mitsuhiro@nims.go.jphttps://doi.org/10.3389/fchem.2024.1524096https://creativecommons.org/licenses/by/4.0/https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.org/journals/chemistry#editorial-boardhttps://www.frontiersin.org/journals/chemistry#editorial-boardhttps://doi.org/10.3389/fchem.2024.1524096times are highly dependent on the external environment, (Kiselevand Konovalov, 2009), including the nature of the reaction solvent(e.g., solvent polarity, hydrogen bond formation with reactants, andthe use of ionic liquids), the presence of catalysts, the formation ofreaction environments by molecular aggregates (Itami et al., 2002),and the reaction temperature. From this background, Baker et al.,has recently proposed to use an enzyme as a reaction filed which cancatalyze the DA reaction (Siegel et al., 2010). They described the denovo computational design and experimental characterization ofenzymes catalyzing a bimolecular DA reaction with highstereoselectivity and substrate specificity.In general, the progression of an endothermic reactions wasaccelerated with an increase in temperature; however, in the case ofa DA reaction, when the energetic threshold is exceeded, the reverereaction, so called retro DA reaction (rDA), proceeds backward to formthe original structure (Gregoritza and Brandl, 2015). Also of particularnote in the DA reaction is the fact that this chemo selective reaction canproceed in both organic solvents and water requiring no catalyst.Therefore, many applications of DA/rDA reaction in the biomedicalfields have been reported by taking advantages of the thermalreversibility and biocompatibility (e,.g., controlled drug releasetechnologies, etc.). On the other hand, the major limitation is itsrelatively high working temperature. For example, we havepreviously shown that the rDA reaction occurred at 90°C, which istoo high for living organisms (Fujisawa et al., 2021). From theseperspectives, we consider that discovering and establishing anefficient DA reaction environment are significantly important toovercome current limitations.In this study, we aimed to investigate the effects of the polarity ofthe reaction environment on DA reactions through a novel approach.As mentioned previously, the reaction rate of a DA reaction can becontrolled without changing the chemical structure of the reaction sitebut its polarity. For instance, Jung et al. measured the reaction rates ofDA reactions in various solvents with different polarities and foundthem to be accelerated in highly polar solvents such as DMSO (Jungand Gervay, 1989). Notably, we used the Layer-by-Layer (LbL) self-assembly technology instead of solvents to alter the externalenvironment (Figure 1A). LbL has been widely used to fabricatenanometer-scale multilayered membranes and has been adapted tosequentially adsorb oppositely charged polyelectrolytes (PEs) ontosubstrates to fabricate membranes comprising a variety of polymerspecies (Caruso et al., 2001; Ariga et al., 2006; Ariga et al., 2019).Interestingly, Tedeschi et al. reported that LbL layers are highly polar;(Tedeschi et al., 2001); they prepared LbL layers of various polyanionsto form the polycation polyallylamine hydrochloride (PAH) andshowed that the difference in internal polarity can be measured asan indicator of the difference in pyrene fluorescence (Bains et al.,2011). Though LbL has been used for various applications, there havebeen no reports on techniques that utilize the internal polarity of thelayers. Herein, we show that the use of LbL layers can replace chemicalmodification to promote DA reactions and potentially increase theirreaction rates. We evaluated the polarities of the LbL membranesusing pyrene and, correspondingly, the amounts of MAL-PEG2-NHSbound in the DA reaction. In addition, we applied the LbL coating onthe surface of magnetic nanoparticles (MNPs). Because theMNPs cangenerate heat by irradiating AC magnetic field, the progression ofreverse reaction (retro DA reaction) was also evaluated.2 Results and discussion2.1 Synthesis of poly (SS-co-FMA)First, the free-radical polymerization of copolymers of furfurylmethacrylate (FMA) and styrene sulfonic acid sodium salt (SS) wasperformed at their respective proportions to provide reaction sitesFIGURE 1(A) Layer-by-Layer assembled reaction environment comprising PAH and poly (SS-co-FMA). (B) Diels–Alder reaction between poly (SS-co-FMA)and MAL-PEG2-NHS. Styrene sulfonic acid sodium salt; SS, furfuryl methacrylate; FMA, polyallylamine hydrochloride; PAH.Frontiers in Chemistry frontiersin.org02Fujisawa and Ebara 10.3389/fchem.2024.1524096https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://doi.org/10.3389/fchem.2024.1524096for the DA reaction. The reason for copolymerization with SS isthat FMA must be dispersed in water owing to its hydrophobicity,but it does not have a charge for the electrostatic interactions inLbLs; therefore, it must be copolymerized with SS, an anion. Theamount of FMA to be introduced was calculated using 1H nuclearmagnetic resonance (1H NMR) spectroscopy and was found to be19 mol% (Figure 2B). Gel permeation chromatography (GPC)revealed that the synthesized polymer, poly (styrenesulfonic acid-co-furfuryl methacrylate (poly (SS-co-FMA)), had a molecularweight of approximately 50k and a polydispersity index (PDI)of 2.9. To determine the composition of poly (SS-co-FMA), UV-visspectra were recorded for a poly (SS-co-FMA) solution(Supplementary Figure S1). Absorption peaks were observed at262 and 300–350 nm, corresponding to the phenyl and furangroups (Yılmaztürk et al., 2009; Liu et al., 2013), respectively(Supplementary Figure S1A); the phenyl absorption tended todecrease with increasing furan content.2.2 Layer-by-layer thin film formationPoly (SS-co-FMA) with 19 mol% of furan group, which wassoluble in water, was used to evaluate the LbL process. Poly (SS-co-FMA) is entirely anionic; therefore, it was stacked with thecationic PAH via electrostatic interactions. Alternately stackedpolymer layers were achieved by repeatedly immersing quartzsubstrates in alternating polymer solutions followed by washingand drying. The adsorbed polymers were evaluated using aquartz crystal microbalance (QCM) (Figure 3); the resultsshowed that alternating stacking can be achieved even withfuran-containing polymer. Up to 40 layers were evaluated; itwas found that alternating stacking continued stably even forthis high number of layers. We therefore concluded that DAreaction sites were successfully introduced into theLbL membrane.2.3 Polarity in layer-by-layer filmTo investigate the relative polarity changes within the LbLlayers, the LbL layers stacked on the quartz slide surface wereimmersed in a 1.0 × 10−4 g L–1 pyrene solution; pyrene is acommonly used polarity-sensitive probe. The emission spectrumof the pyrene monomer showed five prominent fluorescence peaksbetween 370 and 400 nm. The first band (I) increased in intensitycompared to the third band (III) when exposed to the polarity ofthe solvent owing to coupling between the electronic andFIGURE 2(A) Synthesis scheme of poly (SS-co-FMA) and (B) 1H NMR spectrum of poly (SS-co-FMA) in D2O by using 400 MHz NMR. The feed ratio of SS andFMA was 60 mol% and 40 mol%, respectively.FIGURE 3Adsorption of [PAH/PolySS] (gray) and [PAH/Poly (SS-co-FMA)](blue). PAH (open), polySS and poly (SS-co-FMA) (closed).Frontiers in Chemistry frontiersin.org03Fujisawa and Ebara 10.3389/fchem.2024.1524096https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://doi.org/10.3389/fchem.2024.1524096vibrational states (Supplementary Figure S2). The first and thirdvibronic peaks ratio, I/III, is generally referred to as the Py value(Tedeschi et al., 2001), and indicates the polarity of the solvent.Here, an increase in Py value was observed for the 40 layers filmcompared to the 8 layers film, indicating an increase in polarity asthe number of layers is increased (Figure 4). Generally, polaritywithin polymer layers is known to be influenced by theirenvironments, such as dielectric constant. Because of the densestacking layer structure of PAH/Poly (SS-co-FMA), the dielectricconstant increases with an increase of layer numbers. The Py valueof the dry film and the film immersed in MiliQ water arecompared, and both show a similar trend of increased Py value,indicating that it is not the fluorescence of pyrene seeping from thefilm, but the fluorescence of pyrene loaded on the film.2.4 Diels–Alder reaction in poly (SS-co-FMA) solutionThe DA reaction is widely used in small-molecule synthesis to formcyclic structures in molecules. Because the reaction proceeds in water ororganic solvents and does not require a catalyst, it is applicable tobiomaterials. Furan efficiently undergoes the DA reaction withmaleimide because furan is an electron-donating group andmaleimide is an electron-withdrawing group, and the pericyclicreaction proceeds efficiently because of the large energy gap of thehighest occupied molecular orbital/lowest unoccupied molecularorbital. (Froidevaux et al., 2015). The progression of the DA reactionwas confirmed by 1H NMR spectroscopy (Figure 5) and was evaluatedaccording to the K peaks of the newly formed protons after cyclization,which were found to be stereoisomeric K-endo (3.60 ppm) and K-exo(3.49 ppm). The conversion rate of the DA reaction between furan andMAL-PEG2-NHS in the polymer increased with time, with saturation atapproximately 50%–60% after 144 h. This rate depends strongly on thereaction environment, including the reaction solvent and temperature.2.5 Diels–Alder reaction in poly (SS-co-FMA)layer-by-layer filmFinally, MAL-PEG2-NHS was permeated into the membraneswith 8 and 40 layers, and the DA reaction was investigated. Asshown in Figure 6A, 8 and 40 layers were stacked on quartz slides,immersed in a 1.0 mg mL–1 of MAL-PEG2-NHS solution, and thefrequencies of the quartz substrates in the dry state were measured.The frequencies decreased as the weight of the material bonded tothe crystals increased. The Sauerbrey’s equation was then used tocalculate the actual amounts of the adsorbed substance bycalculating the total weight of the bound material; the totalweight of the introduced MAL-PEG2-NHS was plotted againstthe frequencies of the 8 and 40 layers samples. Figure 6B showsthe weight change of the membranes stacked with 8 layers of the[PAH/Poly (SS-co-FMA)] film and 8 layers of a [PAH/PolySS] film.PolySS is a styrene sulfonic acid sodium salt homopolymer that doesnot contain furans. However, even with the [PAH/PolySS] film, aweight gain was observed. This may be attributed to the nonspecificinteraction of a MAL-PEG2-NHS between the layers. Therefore, weevaluated 40 layers of [PAH/PolySS] and [PAH/Poly (SS-co-FMA)]to observe the differences in adsorption with and without furan(Figure 6C). Even with 40 layers of [PAH/PolySS], MAL-PEG2-NHSadsorption was observed after a reaction time of 12 h. However, thetotal weight of the 40 layers of [PAH/PolySS] decreased after 24 h,whereas that of the 40 layers of [PAH/Poly (SS-co-FMA)] continuedto increase with reaction time. This is thought to be because in the40 layers of [PAH/PolySS], the MAL-PEG2-NHS were notchemically bonded but only dispersed, and thus were releasedout of the layer by diffusion. In contrast, in the 40 layers of[PAH/Poly (SS-co-FMA)], the MAL-PEG2-NHS was chemicallybound to FMA via the DA reaction, which is thought to haveinhibited MAL-PEG2-NHS release. However, of the total weight of[PAH/Poly (SS-co-FMA)] after 48 h, 14,904 ng of the MAL-PEG2-NHS was adsorbed, which was 137 times more than the amount offuran groups in the 40 layers of [PAH/Poly (SS-co-FMA)]. Thus, itshould be noted that this weight includes the MAL-PEG2-NHSdispersed between the layers. However, there is a difference inadsorption with respect to stacking.2.6 MAL-PEG2-NHS release in response toAC magnetic field from layer-by-layer filmFinally, a MAL-PEG2-NHS was introduced to the polymer-coated MNPs via the Diels–Alder reaction. MNPs have beenexploited as heating agents by taking advantage of heatgeneration by the irradiation of AC magnetic field. This enableslocal heating aroundMNPs. Because DA-based covalent bonds havebeen known to turn to its original form upon heating (called retroDA reaction; rDA), AC magnetic field irradiation can trigger therelease of MAL-PEG2-NHS from MNPs via rDA reaction. Themagnetic nanoparticles decorated with 8 layers of [PAH/Poly(SS-co-FMA)] were measured by dynamic light scattering (DLS)with a particle size of 271 nm and a PDI of 0.16. Figure 7 comparesthe released amount of MAL-PEG2-NHS from MNPs with 8 layersof [PAH/PolySS)] and [PAH/Poly (SS-co-FMA)] with/without ACmagnetic field application for 15 min. As expected, the acceleratedFIGURE 4Fluorescence intensity ratio (I/III, Py-value) of pyrene in 8 and40 layers of [PAH/Poly (SS-co-FMA)]. Dry state (open) and immersed inMiliQ water (closed).Frontiers in Chemistry frontiersin.org04Fujisawa and Ebara 10.3389/fchem.2024.1524096https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://doi.org/10.3389/fchem.2024.1524096release of MAL-PEG2-NHS has been observed upon AC magneticfield application, while no or little release was observed without ACmagnetic field. According to our previous study, heat generationfrom MNPs was calculated to be approximately 6.0 mJ mg−1 for15 min and this energy is much larger than that required for the rDA(52.0 mJ mg−1). (Fujisawa et al., 2024). Therefore, rDA-based MAL-FIGURE 5Scheme of the Diels–Alder reaction (A) and 1H NMR spectral changes after 144 h of Diels–Alder reaction (B).FIGURE 6(A) DA reaction of LbL layers in MAL-PEG2-NHS solution. Adsorption of MAL-PEG2-NHS over time in (B) 8 layers and (C) 40 layers of [PAH/PolySS)](open) and [PAH/Poly (SS-co-FMA)] (closed).Frontiers in Chemistry frontiersin.org05Fujisawa and Ebara 10.3389/fchem.2024.1524096https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://doi.org/10.3389/fchem.2024.1524096PEG2-NHS release was successfully achieved using LbL-coatedMNPs. On the other hand, MAL-PEG2-NHS release was alsoobserved even in [PAH/PolySS)], which does not contain furangroups. This is due to the release of the MAL-PEG2-NHS entrappedwithin the layers noncovalently. Although optimization of theproposed system is still required to achieve perfect ON-OFFMAL-PEG2-NHS release control, these results indicate atremendous potential for facile regulation of rDA reaction inbiomedical fields.3 Experimental3.1 Materialsp-Styrenesulfonic acid sodium salt (SS), dimethyl sulfoxide(DMSO), 2,2′-azobis (isobutyronitrile) (AIBN), 1 mol L-1 sodiumchloride (NaCl) solution, pyrene, deuterium oxide (99.8%, forNMR), lithium chloride (LiCl), acetonitrile (ACN; high-performance liquid chromatography grade), sulfuric acid(H2SO4), and hydrogen peroxide (H2O2, 30 wt%) were purchasedfrom FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).Maleimide-PEG2-NHS (MAL-PEG2-NHS, >98.0%) was purchasedfrom Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ultrapuredistilled Milli-Q water was used in this study (Merck, Darmstadt,Germany). Furfuryl methacrylate (FMA, 97%) containing 200 ppmmonomethyl ether hydroquinone as an inhibitor) was removed bypassing through an alumina oxide column before use.Polyallylamine hydrochloride (PAH; average molecular weight(Mw) of 50k) was purchased from Sigma–Aldrich (MO, USA).An iron oxide nanoparticle water suspension and EMG 607(cationic surfactant) were purchased from Ferrotec MaterialTechnologies Corporation (CA, USA).3.2 Synthesis of poly (SS-co-FMA)Poly (SS-co-FMA) was polymerized via free-radicalpolymerization. Briefly, SS (4.0 g), FMA, and AIBN (0.065 mol%of the total monomer concentration) were dissolved in 11.0 mL ofDMSO; the total amount of monomer was 19.4 mmol. All thereactants were placed in a 20 mL glass vial, mixed well, and sealedwith a rubber cap. The reaction solution was purged with nitrogengas for 20 min. The polymerization was conducted overnight at60 °C, following which the polymer solution was loaded into Spectra/Por® dialysis tubing with a Mw cutoff of 3.5 kDa (Repligen, MA,USA) against distilled water for 3 days. The dialyzed solutions werefreeze-dried for 2 days, and the polymer was obtained as a whitepowder. The chemical structure of poly (SS-co-FMA) was confirmedby 1HNMR spectroscopy at 400MHz (JEOL Ltd., Tokyo, Japan). Allthe NMR samples were prepared at a concentration of 10.0 mg mL-1in D2O. The average molecular weight of the polymers wasdetermined via gel permeation chromatography (GPC; GPC101,Shodex Corporation, Tokyo, Japan). The mobile phase had a flowrate of 0.8 mL min-1 at 40 °C and consisted of 0.5 wt% LiCl in ACN:H2O (4:6). A polystyrene standard was used to calculate the Mw,Mn and PDI.3.3 Characterization of I/III ratio in poly (SS-co-FMA)/PAH layersAn aqueous solution of pyrene was prepared to a concentration of1.0 × 10−7 g mL-1. Because pyrene is difficult to dissolve in water at highconcentrations, 49.0 mg of pyrene was first weighed and mixed up with100 mL of acetone using a measuring flask (4.9 × 10−4 g mL-1). Then,204 µL was taken from the prepared pyrene solution and was meteredup with 100 mL of MiliQ water using a volumetric flask (1.0 ×10−7 g mL-1). Layers of 8 and 40 films were prepared on the quartzslides as described above, and then immersed in a pyrene solution for3 days to incorporate pyrene into the polymer layers. The crystal slideswere removed from the pyrene solution, washed three times withMiliQwater, and placed in a cuvette filled with and without MiliQ water byfluorescence intensity measurement.3.4 Characterization of PAH/poly (SS-co-FMA) assembly by QCMPoly (SS-co-FMA) and PAH solutions were prepared in advanceat concentrations of 5.0 mg mL-1 in Milli-Q water and 150 mMNaCl. Before the nanolayer formation on quartz crystal substrate,piranha solutions were prepared in H2SO4:H2O2 (30 wt% in water) =3:1 v/v% with ice cooling and then dropped with a Pasteur pipetteonto the quartz crystal slides for 1 min. Subsequently, the electrodeswere rinsed with Milli-Q water and dried with nitrogen gas to cleanthe electrode surface via blasting. Prior to the formation of [PAH/Poly (SS-co-FMA)] assembly on the gold-coated quartz crystalsubstrate (QA-A9M-AU(SEP), SEIKO, E.G.,&G Co., Ltd., Tokyo,Japan), the QCM leads were protected with silicone rubber gel (KE-1830, Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) to preventdegradation during immersion in the polymer solutions. Piranhasolutions were dropped with a Pasteur pipette onto the QCMelectrodes for 1 min. Subsequently, the electrodes were rinsedwith Milli-Q water and dried with nitrogen gas to clean theelectrode surface via blasting. The cleaned QCM electrodes wereimmersed in a 5.0 mgmL-1 PAH aqueous solution for 5 min,FIGURE 7Release of MAL-PEG2-NHS from MNPs coated with 8 layers of[PAH/PolySS)] and [PAH/Poly (SS-co-FMA)] with (red) and without(blue) AC magnetic field irradiation for 15 min.Frontiers in Chemistry frontiersin.org06Fujisawa and Ebara 10.3389/fchem.2024.1524096https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://doi.org/10.3389/fchem.2024.1524096removed, washed thoroughly with Milli-Q water, and dried withnitrogen gas. The frequencies of the QCM electrodes were thenmeasured using a QCA917 device (SEIKO, E.G.,&G Co., Ltd.,Tokyo, Japan). Next, the QCM electrodes were immersed againin a 5.0 mg mL-1 of poly (SS-co-FMA) aqueous solution for 5 min,removed, washed, and dried, and their frequencies were measuredagain. This process was repeated eight times, and the reduction inthe frequency of each polymer layer was plotted. Error barsrepresent standard deviations for n > 6 of QCM measurements.3.5 Characterization of Diels–Alder reactionin poly (SS-co-FMA) solutionMAL-PEG2-NHS (161.0 mg) was dissolved in 11.5 mL of D2O.Poly (SS-co-FMA19) (100.0 mg) was placed in the glass vial. AnMAL-PEG2-NHS solution (1.0 mL) was added to the polymer-containing in the glass vial and allowed to react with stirring. Thereaction solution (0.7 mL) was collected at various time points(0 and 144 h) and directly analyzed via 1H NMR spectroscopy. Theprogress of the DA reaction corresponded to K-endo (3.60 ppm) andK-exo (3.49 ppm), which appeared after the reaction.3.6 Characterization of Diels–Alder reactionin PAH/poly (SS-co-FMA) nanolayersby QCMMAL-PEG2-NHS solution was prepared in Mili-Q water to1.0 mg mL-1. Quartz slides with stacked polymer layers wereimmersed and the frequency of the quartz crystal was measuredat each time (3–48 h). Here, the mass of MAL-PEG2-NHS adsorbed(Δm) were calculated from the decrease in frequency usingSauerbrey’s equation (Vogt et al., 2004; Uto et al., 2008). Thedensity of the quartz crystal (ρq) was 2.648 g cm-3, the modulusof elasticity of the crystal (μq) was 2.947 × 10−13 g m-1 s-2, and thestandard resonance frequency of the crystal (f0) was calculated fromthe data of QA-A9M-AU (SEP) as 8.95 MHz and the electrodediameter of the gold substrate of the quartz crystal (A) as 5 mm φ.−Δf � 2f02A����ρqμq√ × Δm3.7 MAL-PEG2-NHS release in response toAC magnetic field from layer-by-layer filmon magnetic nanoparticlesThe MAL-PEG2-NHS release from LbL film on magneticnanoparticles (Fe3O4) was conducted following the article (Fujisawaet al., 2024). A solution of poly (SS-co-FMA) (4,900 µL)—a polyanionadjusted to a concentration of 5.0 mg mL-1 in Milli-Q water containing150 mM NaCl—was added to a 10 mL sample tube. Subsequently,100 µL of an MNP dispersion was added to the poly (SS-co-FMA)solution and stirred for 15 min. A sample tube containing the MNP/poly (SS-co-FMA) solution was placed on a neodymium magnet for10min, and the supernatant (1.0mL) was placed in a 1.5mL Eppendorftube. To wash the polymer-coated MNPs, the aliquoted MNPdispersion was separated in a centrifuge pre-cooled to 4°C at15,000 rpm for 15 min, and the supernatant was removed. Fresh1.0 mL Milli-Q water was added to the polymer-coated MNPs andpipetted. The particles were separated via centrifugation and cooled to4 °C at 15,000 rpm for 15 min. These washing process was repeatedtwice to obtainMNPswith a single layer of poly (SS-co-FMA). The PAHsolution (a polycation) was then prepared in Milli-Q water at aconcentration of 5 mg mL-1 with 150 mM NaCl. PAH solution(1.0 mL) was added to the MNPs coated with poly (SS-co-FMA)and stirred for 15 min. The sample tube containing the MNP/poly(SS-co-FMA) solution was placed on a neodymiummagnet for 10 min,and the 1.0 mL of supernatant was placed in a 1.5 mL Eppendorf tube.The aliquotedMNP dispersion was separated in a centrifuge pre-cooledto 4 °C at 15,000 rpm for 15 min. To wash the polymer-coated MNPs,the supernatant was removed, and fresh 1.0 mL Milli-Q water wasadded to the polymer-coated MNPs and pipetted. The particles wereseparated via centrifugation and cooled to 4 °C at 15,000 rpm for 15min.The washing process with Milli-Q water was repeated twice to obtainMNPs with PAHs stacked on the poly (SS-co-FMA) layer. By repeatingthese processes an arbitrary number of times, MNPs with up to eightpolymer layers were obtained. Hydrodynamic diameter measurementswere performed using an ELSZ-2000 instrument (Otsuka ElectronicsCo., Ltd., Osaka, Japan). A high-power semiconductor laser was used asthe incident beam. After the concentration of each polymer-coatedMNPwas adjusted to a level in themeasurable rangewithMilli-Qwater,at each layer number, filtered by a 0.45 μm pore size, 13 mm-diameterpolytetrafluoroethylene syringe filters (Membrane Solutions, LLC, WA,USA) then added the solution in disposable cuvettes were used for theparticle size measurements at 25°C.To evaluate the MAL-PEG2-NHS release from MNPs, the ACmagnetic field irradiation time was fixed, and the amount of MAL-PEG2-NHS released was evaluated. MAL-PEG2-NHS was dissolved in1.0 mL at a concentration of 5.0 mgmL-1 of anMNPs solution with upto eight layers of polymer and bound via the DA reaction overnight.To remove the unbound MAL-PEG2-NHS, the MNPs solution wascentrifuged and washed with 4.0 mL of Milli-Q water, excluding thesupernatant. The washing process was repeated three times.Subsequently, 1.0 mL of the MNPs solution was placed in a1.5 mL microtube such that the sample was at the center of thecoil of the AC magnetic field apparatus and was subjected to an ACmagnetic field using HOSHOT2 at 283 kHz and 184 A for 15 min.The samples were then cooled in a coil using water. The supernatantcontaining the released MAL-PEG2-NHS was collected. To calculatethe concentration of releasedMAL-PEG2-NHS, the ultraviolet–visible(UV–vis) absorption of the MAL-PEG2-NHS in 10 µL of the collectedsupernatant was measured using a NanoDrop OneC (Thermo FisherScientific K.K., MA, USA). The concentration of the released MAL-PEG2-NHS was calculated from the calibration curve of MAL-PEG2-NHS dissolved in Milli-Q water at various concentrations. Aminimum of three measurements were performed for each sample.4 ConclusionIn summary, we successfully formed an LbL-based reactionenvironment for the DA reaction using FMA copolymers. ThePy-value, which indicates the degree of polarity, increased whenFrontiers in Chemistry frontiersin.org07Fujisawa and Ebara 10.3389/fchem.2024.1524096https://www.frontiersin.org/journals/chemistryhttps://www.frontiersin.orghttps://doi.org/10.3389/fchem.2024.1524096the number of polymer layers was 40 compared to 8, indicating thatthe polarity of the reaction environment could be controlled whileusing the same polymer by simply increasing the number of layers.Notably, we succeeded in promoting the binding of DAMAL-PEG2-NHS in the layers more effectively than the binding achieved in thetypical solvent system. To the best of our knowledge, this is the firstreport on the construction of an enhanced reaction environment forthe DA reaction on the surface of a substrate. This technology canpave the way for the industrialization of dynamic covalentchemistry, as the reaction rate can be accelerated withoutchanging the chemical structures of the substrates. Furthermore,revere reaction (rDA) was successfully triggered by AC magneticfield irradiation by employing the LbL-coating on MNPs.Data availability statementThe raw data supporting the conclusions of this article will bemade available by the authors, without undue reservation.Author contributionsNF: Conceptualization, Data curation, Investigation,Methodology, Writing–original draft. ME: Conceptualization,Funding acquisition, Supervision, Writing–original draft,Writing–review and editing.FundingThe author(s) declare that financial support was received for theresearch, authorship, and/or publication of this article. This studywas supported by the JSPS KAKENHI Grant-in-Aid for ScientificResearch (B) (JP19H04476) and Grant-in-Aid for TransformativeResearch Areas (A) (JP20H05877).Conflict of interestThe authors declare that the research was conducted in theabsence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.Generative AI statementThe author(s) declare that no Generative AI was used in thecreation of this manuscript.Publisher’s noteAll claims expressed in this article are solely those of the authorsand do not necessarily represent those of their affiliatedorganizations, or those of the publisher, the editors and thereviewers. 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