Loading iron(III)porphyrin as the gas/anion binding site into methylated β-cyclodextrin-incorporated polymer hydrogels

Abstract

Per-O-methylated β-cyclodextrin (CD^Me) was incorporated into an N-acryloylglycinamide (NAGAm) polymer. Hydrogels of the CD^Me polymer were obtained. Fe^IIITPPS was loaded into CD^Me inside the hydrogels, in which binding of N₃⁻/NO to Fe^IIITPPS was confirmed. Functions of the Fe^IIITPPS/CD^Me supramolecular complex as a simple heme protein model were demonstrated in the hydrogels.

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Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucopyranose units linked by α-(1,4) glycosidic bonds. Typically, glucopyranose units of 6 (α-CD), 7 (β-CD), and 8 (γ-CD) are widely used.¹ CDs have a hydrophobic cavity that can trap or encapsulate guest molecules. This unique property leads to a “host–guest” type relationship that can modify and/or improve properties such as physical, chemical and biological characteristics of guest molecules.² Incorporation of CDs into polymer chains has great potential due to the supramolecular interactions exhibited in polymer materials, thereby enabling their application in both industrial and medical fields.^3–5 Harada et al. reported a series of CD-based supramolecular polymers demonstrating macroscopic self-assembly between CD host gels and guest gels.^6–9 Ito et al. successfully established CD-based slide-ring materials (SRMs) that show enhanced toughness and durability.^10,11 In SRMs, dimerized CDs form polyrotaxanes (PRs), in which each CD encircles a polymer axis, and functions as mobile cross-linking points. In the medical field, Tamura and Yui reported a reduction-induced RNA release by CD-based PRs inside cells.¹² Other stimuli-responsive drug releasing properties of CD-based polymers were also reported.^13–15

We have continuously studied supramolecular complexes of per-O-methylated β-cyclodextrin (CD^Me) with 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphinato iron(III) (Fe^IIITPPS) in aqueous media. Encapsulation of Fe^IIITPPS by CD^Me mimics the hydrophobic environment in heme proteins, so that the CD^Me/Fe^IIITPPS system works as a simple heme protein model in water.^16,17 Heme proteins are ubiquitous metalloproteins that play essential roles in various processes such as gas/anion binding, catalysis for chemical reactions, and electron transfer in the biological system.^18,19 Recently, protein–polymer hybrids (PPHs) have attracted broad interest because enzymatic reactions are reproduced/improved or altered in materials that can be applied to biocatalysis, sensing and biomarker detection.^20–22 For example, increases in denaturation temperature in protein–polymer conjugates²⁰ and self-assembling protein materials^23,24 have been reported in heme protein-based PPHs. Herein, we provide a novel approach by constructing a CD^Me/Fe^IIITPPS system within polymer materials. This functional material without proteins is mass-producible and expected to exhibit improved stability against varying temperature and pH conditions compared to current protein-based materials.

Poly(N-acryloylglycinamide) (PNAGAm) was chosen as a base polymer because it forms a hydrogel at lower concentration (>2 wt%)^25,26 and shows biocompatibility and biodegradability.^27–30 Copolymerization of mono-6-acrylamide-6-deoxy-per-O-methyl-β-CD (AACD) with NAGAm successfully yielded P(NAGAm_1−x-co-AACD_x), which can form an inclusion complex with Fe^IIITPPS (Fig. 1). The detailed synthetic method is described in the SI (Scheme S1 and Fig. S1–S6). AACD was synthesized on the gram-scale in 5 steps from β-CD. The incorporation of AACD into the polymer was confirmed by ¹H NMR spectra, where characteristic signals due to H-1 protons of CD^Me were observed around 5 ppm as well as signals of the main chain (1–2 ppm) and amide protons in the side chain (7–9 ppm) of the polymer (Fig. S5). As summarized in Table 1, 0.9–3.4 mol% of AACD (70–90% against the feed composition) was found to be introduced into the polymer chain, and each polymer was named P_x in this text, where “x” represents the incorporation ratio of CD^Me. The inherent viscosity analysis of polymers (P₀–P_3.4) was carried out to determine the viscosity-average molecular weight (M_v).

Fig. 1 (a) Synthesis of P(NAGAm_1−x-co-AACD_x). (b) The complexation between P_x and Fe^IIITPPS in hydrogels.

Table 1 Summary of the synthesis and characterization of P_x prepared in this study

Samples	Feed composition		Incorporated CD (x)^a (mol%)	Mv^b (g mol^−1)
	NAGAm (mol%)	AACD (mol%)
a The molar ratio of the incorporated CD was determined by NMR analysis.b The viscosity-average molecular weight.
P0	100	0	—	5.0 × 10^5
P0.9	99.0	1.0	0.9	3.5 × 10^5
P1.7	98.0	2.0	1.7	1.5 × 10^5
P2.2	97.0	3.0	2.2	1.0 × 10^5
P3.4	95.0	5.0	3.4	7.1 × 10^4

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The inclusion phenomenon between P_x and Fe^IIITPPS was investigated in water at pH 4, where Fe^IIITPPS was coordinated by two H₂O molecules (diaqua form).¹⁶ The UV-vis spectral changes of Fe^IIITPPS upon addition of P_x were observed (Fig. 2a and Fig. S7), whose final spectrum corresponded to that observed for TMe–β-CD/Fe^IIITPPS,¹⁶ suggesting the formation of the CD^Me/Fe^IIITPPS complex in P_x (Fig. S8). Interestingly, the binding isotherm for Fe^IIITPPS was significantly different between P_x and TMe–β-CD. The absorbance changes of Fe^IIITPPS as a function of [CD^Me] were relatively large and sharply saturated in P_x (Fig. 2b and Fig. S9), indicating stronger binding of CD^Me in P_x than TMe–β-CD.

Fig. 2 (a) UV-Vis absorption spectral changes of Fe^IIITPPS (5 µM) upon stepwise addition of P_2.2 (22.8 µg each) in 0.05 M succinate buffer at pH 4.0 and 25 °C. The inset shows the differential spectra. (b) The plots of the absorbance changes of Fe^IIITPPS (5 µM) versus [CD^Me] upon addition of P_2.2 and TMe–β-CD. (c) Calorimetric titrations of the Fe^IIITPPS (0.5 mM) solution (25 aliquots, 10 µL each) into solutions containing 69.7 µg of P_2.2 in 0.05 M succinate buffer at pH 4.0 and 298.15 K. (d) Integrated heat plots of P_2.2 and TMe–β-CD as a function of [Fe^IIITPPS]/[CD^Me] ratios. Solid and dashed lines represent the best fit of the experimental data to the two binding site model in the ORIGIN software. (e) A proposed interaction between NAGAm in P_2.2 and Fe^IIITPPS by hydrogen bonds.

Titration experiments were also conducted at pH 7, where OH⁻ is bound to Fe^IIITPPS (Fig. S10). An anion binding onto Fe^IIITPPS brings a stronger interaction between CD^Me and Fe^IIITPPS,¹⁶ exhibiting clear saturation points (Fig. S11). In such a case, the amount of CD^Me can be determined from the saturation points. As summarized in Table S1, the incorporation ratios determined by UV-vis spectroscopy corresponded to those determined by NMR measurements (shown in Table 1), suggesting that almost all of the CD's cavity is available for complexation with Fe^IIITPPS. Isothermal titration calorimetry (ITC) measurements were also carried out (Fig. 2c). Integrated heat values upon titration of Fe^IIITPPS into P_2.2 or TMe–β-CD were plotted and analysed (Fig. 2d). Thermodynamic parameters summarized in Table 2 indicate that the P_2.2/Fe^IIITPPS complex was more enthalpically stabilized than the TMe–β-CD/Fe^IIITPPS complex. We hypothesized that the side chain of the NAGAm moiety caused further stabilization of the P_2.2/Fe^IIITPPS complex by forming hydrogen bonds between amide groups in NAGAm and sulfonate groups in Fe^IIITPPS.^31–33 To examine the effect of NAGAm, we tested NAGMe (an amide group of NAGAm was replaced with a methyl ester group). P(NAGMe_0.98-co-AACD_0.02) was synthesized using NAGMe and AACD in the same manner as P_2.2. The complexation of P(NAGMe_0.98-co-AACD_0.02)/Fe^IIITPPS was confirmed. As we expected, the complexation was comparable to that of TMe–β-CD/Fe^IIITPPS without a polymer chain (Fig. S12 and Table S2). As a further control, PNAGAm (homopolymer without a CD^Me moiety) exhibited a significant interaction with Fe^IIITPPS compared to PNAGMe (Fig. S13). Therefore, we conclude that the P_x/Fe^IIITPPS complex was stabilized by hydrogen bonds between Fe^IIITPPS and NAGAm (Fig. 2e), resulting in larger binding constants than the TMe–β-CD/Fe^IIITPPS system. The inclusion phenomenon between TPPS (without an Fe atom) and P_x was also confirmed (Fig. S14 and S15).^34,35

Table 2 Thermodynamic parameters determined by ITC measurements for the interaction of Fe^IIITPPS with TM-β-CD and P_2.2 in 0.05 M succinate buffer at pH 4 and 298.15 K

Samples	K1 [M^−1]	ΔH1 [kJ mol^−1]	ΔS1 [J mol^−1 K^−1]	K2 [M^−1]	ΔH2 [kJ mol^−1]	ΔS2 [J mol^−1 K^−1]
TMe–β-CD	(1.2 ± 0.7) × 10^6	−58 ± 3.8	−78 ± 12	(1.2 ± 0.9) × 10^5	−26 ± 1.4	9.1 ± 2.2
P2.2	(2.2 ± 0.4) × 10^6	−69 ± 5.4	−109 ± 20	(4.3 ± 1.4) × 10^6	−64 ± 3.6	−86 ± 1

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Hydrogels of P_x and P_x/Fe^IIITPPS at different polymer concentrations were prepared and characterized. As shown in Fig. 3a, P_2.2 and P_2.2/Fe^IIITPPS formed hydrogels above 2 wt% (hydrogels of P_0.9, P_1.7, and P_3.4 were also obtained above 2 wt% (Fig. S16)). From the rheological analysis, the storage moduli (G′) were greater than the loss moduli (G″) in both P_2.2 and P_2.2/Fe^IIITPPS over the frequency range of 0.1–100 rad s⁻¹ at 25 °C, indicating a stable network structure (Fig. 3b and Fig. S17 for P₀, P_0.9, P_1.7, and P_3.4). The G′ values at a frequency of 6.3 rad s⁻¹ are summarized in Fig. 3c. In P_x, the G′ values gradually decreased as a function of “x” in P_x, probably due to the disruption of the original intermolecular interaction of NAGAm by CD^Me. In P_x/Fe^IIITPPS, P_x forms the inclusion complex with Fe^IIITPPS both inter- and intra-molecularly in the current polymer design. The results showed a great increase in G′ values upon addition of Fe^IIITPPS, in which ratios tend to rise as “x” increased (Fig. 3c). P₀ (without CD^Me) showed little change in the G′ value upon addition of Fe^IIITPPS. The enhancement of the mechanical properties of P_x by Fe^IIITPPS supports the formation of the inclusion complex between CD^Me and Fe^IIITPPS in hydrogels.

Fig. 3 (a) Tube inversion test of P_2.2 and P_2.2/Fe^IIITPPS hydrogels. (b) Frequency dependencies of the storage (G′) and the loss (G″) moduli, and tan δ of P_2.2 (empty shapes) and P_2.2/Fe^IIITPPS (filled shapes) hydrogels (polymer conc.: 3 wt%) from 0.01 to 100 rad s⁻¹ at 25 °C. (c) The G′ values at 6.3 rad s⁻¹ at 25 °C of various hydrogels (polymer conc.: 3 wt%) in the absence and in the presence of Fe^IIITPPS and ratios.

Finally, functions of the P_x/Fe^IIITPPS hydrogel as a simple heme protein model were evaluated. In this study, azide (N₃⁻) and nitric oxide (NO) bindings to the polymer were investigated. These are well-recognized ligands that could coordinate to heme without the axial 5th ligand in heme proteins.^16,37 A piece of the P_2.2/Fe^IIITPPS hydrogel was soaked in a solution containing NaN₃ or NOC7 (1-hydroxy-2-oxo-3-(N-methyl-3-amino-propyl)-3-methyl-1-triazene, a NO-releasing molecule) for one hour; the absorption spectrum of the hydrogel was measured using a demountable cuvette cell. The colour changes of the P_2.2/Fe^IIITPPS hydrogel were noticeable after the treatment with NaN₃ or NOC7 when compared to the P₀ control system (Fig. 4a). In aqueous solution, Fe^IIITPPS/TMe–β-CD forms five-coordinate Fe^IIITPPS–X/TMe–β-CD complexes (X = N₃⁻, Cl⁻, CN⁻, OH⁻, etc.) by binding of anions at the fifth axial position.¹⁶ As shown in Fig. 4b, the obtained spectrum of the NaN₃-treated P_2.2/Fe^IIITPPS hydrogel matched that of the N₃⁻–Fe^IIITPPS/TMe–β-CD complex,¹⁶ suggesting that the azide-capturing phenomenon occurred in the P_2.2/Fe^IIITPPS hydrogel. In addition, the spectral change of P_2.2/Fe^IIITPPS hydrogels was dependent on the amount of NaN₃ (Fig. S18). The plot of the absorbance changes of the hydrogels as a function of [NaN₃] well correlated to that obtained in a P_2.2/Fe^IIITPPS dilute solution, indicating that the azide-binding ability was highly maintained in the hydrogel (Fig. S19). Both the Fe^IIITPPS solution and the P₀/Fe^IIITPPS hydrogel showed no significant spectral changes upon addition of NaN₃ because the binding is significantly prevented by the hydrated azide anion (Fig. S20). The interaction of P_2.2/Fe^IIITPPS with NO was then investigated. The same spectral changes upon addition of NO were observed between TMe–β-CD/Fe^IIITPPS in solution and P_2.2/Fe^IIITPPS in hydrogel (Fig. 4c). The reaction of TMe–β-CD/Fe^IIITPPS with NO results in the formation of a five-coordinate ferrous nitrosyl complex of Fe^IITPPS–NO/TMe–β-CD due to reductive nitrosylation.^36,37 Without CD scaffolds, Fe^IIITPPS also undergoes reductive nitrosylation.^38,39 However, we found that the generated Fe^IITPPS–NO decomposed rapidly in air (t_1/2 < 3 min) (Fig. S21). In the P₀/Fe^IIITPPS hydrogel, the Fe^IITPPS–NO complex was not detected, probably due to the instability of the Fe^IITPPS–NO complex under aerobic conditions (Fig. S22). Therefore, we conclude that functions of TMe–β-CD/Fe^IIITPPS were preserved in the P_2.2/Fe^IIITPPS hydrogels, evidenced by the azide/NO binding phenomena. To the best of our knowledge, this is the first study to observe the heme protein functions of TMe–β-CD/FeTPPS in a concentrated system of hydrogels.

Fig. 4 (a) Photos of hydrogels of P₀/Fe^IIITPPS and P_2.2/Fe^IIITPPS before and after treatment with NaN₃ and NOC7. UV-Vis spectral changes of the TMe–β-CD/Fe^IIITPPS solution and P_2.2/Fe^IIITPPS hydrogel (polymer conc.: 3 wt%) before and after addition of (b) NaN₃ (0.4 mM for TMe–β-CD/Fe^IIITPPS and 3.5 mM for the P_2.2/Fe^IIITPPS hydrogel) in 0.05 M succinate buffer at pH 4 and (c) NO (2 mM) from NOC7 in 0.05 M phosphate buffer at pH 7.

Conclusions

In summary, we successfully synthesized a per-O-methylated β-cyclodextrin-incorporated NAGAm polymer (P_x). The inclusion complex of P_x with Fe^IIITPPS was stabilized more than the TMe–β-CD/FeTPPS system due to hydrogen bonds between sulfonate groups of Fe^IIITPPS and amide groups of NAGAm. P_x/Fe^IIITPPS hydrogels were prepared, in which the enhancement of the mechanical properties was observed. The function of the P_x/Fe^IIITPPS hydrogels as simple heme protein models was studied by monitoring N₃⁻ and NO bindings. As the current P_x/Fe^IIITPPS system lacks the axial 5th ligand like native heme proteins and hemoCD, O₂ binding and activation in the Fe-porphyrin-containing hydrogels should be the next step. We are currently working on a system that could be used as an alternative PPH without proteins.

Author contributions

S. N. and H. K. conceived the study. A. N. and S. N. conducted the experiments. A. N. and H. K. wrote the manuscript. H. K., T. K. and M. K. supervised the study. All co-authors reviewed the manuscript and participated in the revision process.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data have been included in the main manuscript and supplementary information (SI). The supporting data have been provided as part of the SI. Supplementary information: experimental details, NMR and mass spectra of relevant compounds, Scheme S1, Fig. S1–S22, and Tables S1 and S2. See DOI: https://doi.org/10.1039/d6sm00256k.

Acknowledgements

This work was financially supported by JSPS KAKENHI (24K01640, 25K01790, and 25KJ2204), AMED (24ym0126808j), JST (JPMJSF2305), the Grant-in-Aid from the KOSÉ Cosmetology Research Foundation, the Japan Prize Foundation, the Lotte Research Promotion Grant, and the Harris Science Research Institute (Doshisha University).

Notes and references

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