Structure–Chemical Modification Relationships with Silk Materials

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Structure−Chemical Modification Relationships with Silk Materials Yingjie Hang,† Jie Ma,‡ Siyuan Li,† Xiaoyi Zhang,§ Bing Liu,† Zhaozhao Ding,§ Qiang Lu,*,†,§ Hong Chen,*,† and David L Kaplan∥ †

College of Chemistry, Chemical Engineering and Materials Science & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, People’s Republic of China ‡ Department of Burns, Gansu Provincial Hospital, Lanzhou 730000, People’s Republic of China § National Engineering Laboratory for Modern Silk, Soochow University, Suzhou 215123, People’s Republic of China ∥ Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by WESTERN UNIV on 05/09/19. For personal use only.

S Supporting Information *

ABSTRACT: Chemical modifications used with silk materials can be challenging due to heterogeneous reactions, in part due to the assembly state of the protein chains. Here, we assess factors that determine the efficiency of chemical modifications with silk materials. Unlike other natural macromolecules, silk presents changeable self-assembled or aggregation states in aqueous solution, which affect the chemical reactions based on reactive group distribution or accessibility. To confirm this hypothesis, silk nanofibers in various conformation and aggregation states in solution were exposed to the same reaction conditions. Amorphous silk nanofibers provided improved control for consistent chemical modification outcomes, while silk nanofibers with control of structure could be utilized to generate bifunctional materials through multiple chemical modifications. The results of the chemical modifications demonstrated that control of the conformational transitions of silk nanofibers provided a feasible strategy for developing multifunctional silk materials with improved chemical outcomes. KEYWORDS: silk, chemical modification, group distribution, multifunction, biomedical applications

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INTRODUCTION Silk fibroins are useful biomaterials due to their sophisticated conformations,1 hierarchical structures,2,3 tunable assembly,3 ease of fabrication,4 biocompatibility,5 robust mechanical propertie,s6,7 and biodegradability.8 The use of silk fibroins has developed from traditional textile applications9,10 to now include biomaterials,11,12 drug carriers,13−15 bioelectrical16,17 and optical devices,18,19 and energy materials.20 The expanding utility of silk fibroin is due to the versatility of the protein and the improved understanding of structure−function relationships.8,21 Versatile silk-based materials with various microstructures, shapes, and performance have been fabricated to match multiple requirements,7,22,23 while further improvements are still wanted to optimize the utility of silk fibroin in some applications. The different conformations of silk fibroin have stimulated the development of physical modification methods to form various material formats with tunable microstructures, mechanical properties, and bioactivity.3,23 Silk fibroin contains many reactive groups via the amino acid composition;24 thus, chemical modifications are a natural choice to expand silk materials with enhanced physicochemical properties and added functionalities.25,26 Different chemical functionalizations have been pursued, including grafting on threonine,27 tyrosine,28,29 glutamic acid,30 and serine31,32 to immobilize peptides, enzymes, drugs, and polymers.24,26,33−35 These chemically modified silk materials exhibit altered cell attachment and © XXXX American Chemical Society

proliferation, control of cell differentiation, responsiveness to specific analytes or conditions, and improved applicability in tissue engineering.25,36 Therefore, chemical modifications provide for the design of smart silk-based materials with designed physiochemical and biological properties. At the same time, few studies have focused on chemical modifications in the context of the physical state of the starting materials and undergoing the modifications. In some cases, alternative biosynthesis routes are utilized to gain more consistent control of outcomes, through combining silklike peptides37 and other functional peptides such as elastin to generate new materials.38−40 Through this approach, several biomaterials with preferred mechanical and responsive properties were obtained with potential utility in tissue regeneration and drug delivery.41,42 However, the complex biosynthesis process involved in generating these recombinant DNA systems, including costs and low yields, may be hindrances to broader applications in the short term. For the chemical modification of silk biomaterials, the amount of active groups is usually calculated from the theoretical amino acid composition of fibroin molecules.25,43 It is often assumed that all of these active groups can be chemically modified; Received: March 15, 2019 Accepted: May 2, 2019 Published: May 2, 2019 A

DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 1. Nanostructures and conformations of silk in various solutions: (a) AFM images showing short nanofiber (200−500 nm), particle (10−100 nm), and long nanofiber (1−2 μm) morphologies of ASS, NS, and BLS solutions, respectively. Scale bar: 500 nm. (b, c) FTIR spectra and CD curves illustrating the conformational comparisons of ASS, NS, and BLS solutions. (d) Fourier self-deconvolution of the amide I region showing conformation contents.

Figure 2. Chemical modification distinctions between silk solutions with different aggregates and conformations: (a) reaction time curves of sulfonic acid groups via diazonium coupling chemistry based on phenol groups of silk solutions; (b) estimated grafting density based on the azo bond absorbance peak at 328 nm using Beer’s Law (data are presented as the mean ± SD (n = 6), ***p < 0.001); (c) digital photos of sulfonic acid groups modified in silk solutions with degree of grafting via color changes; (d) digital images of silk solutions after HRP/H2O2 reaction showing different states (solution or hydrogel); (e) turbidity curves presenting silk hydrogel formation monitored by optical density changes at 550 nm.

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however, the degree of active group participation in reactions is usually significantly less than 100%, and this can be due to the state of protein assembly or aggregation before and during the reactions, along with the time scale of the reactions due to the dynamic nature of silk protein assembly.

RESULTS AND DISCUSSION

Recent studies found that silk fibroin in solution changed with time in terms of assembly state, with continuous conformational transitions rather than stable single-molecule chains.23,44−46 We deduced that the change in aggregation and conformations B

DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Table 1. ζ Potential of ASS, NS, BLS, and BSS Solutions ζ potential (mV)

ASS

NS

BLS

BSS

−3.9 ± 0.55

−15.7 ± 2.50

−59.3 ± 1.71

−32.4 ± 1.42

in these solutions due to changeable colors from the newly formed azo bond. A darker color means a higher content of azo bonds, suggesting that the reacted ASS solution with the darkest color had the highest degree of grafting. Since the conformations of silk molecules in BLS solution were different from those in ASS and NS solutions, the results suggested that the conformations of silk molecules could affect chemical modification. Although similar conformations existed in the silk molecules in NS and ASS solutions, different reactions also occurred, suggesting an effect of the nanoaggregates where the nanoparicles in NS, the further aggregates of the nanofibers,54 faced higher steric hindrance in chemical modifications. HRP cross-linking showed similar variations, which facilitated an understanding of the role of conformation and aggregates in chemical modifications. Silk hydrogels can be formed from solution after HRP cross-linking.51 An inverted test tube method was performed to show different gelation behaviors of the three silk solutions (Figure 2d). Within 10 min, the ASS solution became a hydrogel, whereas BLS and NS solutions remained liquid, indicting limited reactivity in the cross-linking. The gelation time for the NS solution was longer than that reported previously due to lower silk concentration in the present systems.51 After 85 min, the NS solution also transformed into a hydrogel, while the BLS remained a solution. Even after 24 h, no hydrogel formed in the BLS samples. Turbidity curves (Figure 2e) illustrated initial hydrogel formation at about 10 min and faster reaction rates within 17 min for ASS samples, while the initial hydrogel formation time and reaction time increased to 15 and 65 min, respectively, for the NS samples. Turbidity results confirmed that BLS solutions remained inert to HRP crosslinking, without hydrogel formation. Quantitative analysis of dityrosine fluorescence formed during the HRP cross-linking further revealed a significantly higher degree of reaction in the ASS solution (Figure S2).55 Since both reactions, diazonium coupling and HRP cross-linking, targeted phenol groups but showed different reaction behaviors in the different silk solutions, it is reasonable to infer that the various conformations resulted in different distributions of the tyrosine groups inside and outside of the silk aggregates, determining the level of reactivity. The ζ potential (Table 1) of the silk solutions further indicated changes in reactive group distribution and an opposite tendency to the graft density results. Theoretically, the reactive groups outside of the aggregates (nanofibers or particles) are more reactive, while those within the aggregates face steric hindrance for reactions. Therefore, the difference in reactions between HRP cross-linking and diazonium coupling in BLS solutions makes sense, as HRP (40000 Da, molecular radius ∼30 Å) is a much larger molecule than the diazonium salt of sulfanilic acid (184 Da, molecular radius ∼3 Å) and thus faces greater steric hindrance and higher diffusion time, to limit cross-linking. Unlike the NS solutions that gradually change aggregate size over time,46 the ASS solutions remain as stable nanofibers, avoiding changes in aggregate sizes in aqueous solutions. Therefore, ASS is a better candidate to achieve more controlled chemical modifications of silk materials, when this is the target. After thermal treatment, ASS can be transformed from an amorphous into a β-sheet structure (Figure S3), accompanied by a significant increase of ζ potential from −10 mV to >−30 mV

could influence chemical modifications, leading to variable outcomes in such reactions. To address this variability, we conducted various chemical modifications with several silk solutions with typical aggregates and conformations to confirm their influence on the reaction outcomes.23,24 Normal silk solution prepared via traditional methods,47 amorphous silk nanofiber solutions,23 and β-sheet-rich silk nanofiber solutions48 were termed NS, ASS, and BLS (Table S1), respectively. The microstructures of silk in solutions were characterized by AFM images, where the ASS solution was composed of silk nanofibers with length of 200−500 nm, the NS solution consisted of particles with different sizes in the range of 10−100 nm, and the BLS solution showed nanofibers with lengths of about 1−2 μm (Figure 1a). FTIR spectra and CD curves indicated conformational differences in these silk solutions (Figure 1b−d). Infrared peaks at 1648−1654 cm−1 are characteristic of amorphous structures, whereas the absorptions at 1610−1630 cm−1 are indicative of β-sheet structures. Both ASS and NS showed amorphous structures but with slight differences in the infrared spectral region from 1648 to 1654 cm−1. In comparison to NS, ASS showed a small blue shift at the 1648−1654 cm−1 peak, suggesting more amorphous content. Unlike ASS and NS solutions, BLS solution displayed high β-sheet content with a strong infrared peak at 1620 cm−1. CD curves further confirmed the conformational differences among the solutions. BLS showed positive ellipticity at 190−200 nm and a negative ellipticity at 210−220 nm, suggestive of β-sheet conformations, while both ASS and NS solutions illustrated negative ellipticity at 190−200 nm, a feature of an amorphous structure. The various conformations (Figure 1d) obtained with Fourier selfdeconvolution of the amide I region suggested that ASS and NS solutions have similar secondary conformations while BLS had higher β-sheet content (∼43.1%). The phenol groups from the tyrosines (∼5%) of silk chains were chosen for chemical modifications through diazonium coupling43,49 and oxidative coupling strategies50,51 in ASS, NS, and BLS solutions (Schemes S1 and S2), respectively. The grafting processes were carried out under same conditions (silk and reagent concentrations, temperature, and time). For the diazonium coupling reaction, the FTIR results (Figure S1) indicated successful grafting of sulfonic acid groups in ASS, NS, and BLS solutions, yet the graft rate and degree were significantly different. The azo bond formed had an absorbance at 328 nm, which reflected the reaction rate. The grafting density for each condition was calculated using Beer’s law (Table S2) with an extinction coefficient of 22000 M−1 cm−1.52,53 The ASS solution achieved the highest reaction rate and the highest degree of grafting (Figure 2a), in which the grafting density was close to the theoretical limit of tyrosine residues in silk molecules (∼40%).25 In comparison to ASS solution, the reaction in NS solution showed a slower reaction rate and less degree of reaction or substitution. The reaction was further restrained in the BLS solution, resulting in the lowest reaction conversion. Grafting density analysis (Figure 2b) confirmed a higher grafting reaction in the ASS and NS solutions in comparison to the BLS system; the reaction increased by 13.7% and 6.2%, respectively, in terms of degree of substitution in ASS and NS solutions. The pictures in Figure 2c confirmed various degrees of modification C

DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 3. Influence of silk conformations on chemical modifications: (a) schematic of conformational transition from ASS to BSS solution; (b, c) reaction time curves and relative absorbance intensity of sulfonic acid groups via diazonium coupling chemistry based on phenol groups of ASS and BSS solutions (the insert is a digital photo of sulfonic acid groups modified in silk solutions with degree of grafting via color changes; for (c), the absorbance value of ASS-sul at 328 nm was defined as 1); (d, e) fluorescence spectra and fluorescence intensity (523 nm) of silk solutions excited at 442 nm after modification with a fluorescent molecule via NHS/EDC reaction based on carboxylic acid groups of silk solutions (the insert is a magnification of fluorescence intensity curves of ASS and BSS solutions; for (e), the fluorescence intensity of BSS-flu was defined as 1; data are presented as mean ± SD (n = 6), ***p < 0.001).

Figure 4. Dual modification of silk based on conformational transitions: (a) schematic depicting the procedure for preparation of ASS-sul-b-flu solution; (b) comparison of fluorescence intensity of silk solution grafting; (c) absorbance of azo bond at different steps (the fluorescence intensity and absorbance of ASS-sul-b-flu was defined as 1; data are presented as the mean ± SD (n = 6), ***p < 0.001).

(Table 1). The results indicated that conformational conversion changed the reactive group distribution without adjustment of aggregate morphology (Figure 3a). The β-sheet-rich silk solutions were termed as BSS (Table S1). The two silk nanofiber solutions (ASS and BSS) were then processed by diazonium coupling under the same reaction conditions, and the

modified solutions were termed as ASS-sul and BSS-sul (Table S1), respectively. Figure S4 shows successful grafting of sulfonic acid groups. As expected, a higher grafting rate occurred for the ASS solution in comparison to the BSS solution (Figure 3b). Further relative absorbance value analysis (Figure 3c) shows that the relative absorbance at 328 nm of ASS-sul and BSS-sul D

DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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modification, ASS-sul-b-flu showed higher fluorescence intensity, implying a successful second reaction (Figure 4b). Although the fluorescence intensity of ASS-sul-b-flu was lower than that of BSS-flu due to its lower ratio of β-sheet structure (Table S3), significantly higher fluorescence was achieved in comparison to that of ASS-sul-flu (Figure S8) due to the redistribution of the carboxylic groups on the surface of the nanofibers. Since ASSsul-b-flu also showed higher grafting density with sulfonic acid groups (Figure 4c), it is feasible to achieve better use of phenol and carboxylic groups in dual-modification processes through the above strategy. More interestingly, silk fibroin maintained a nanofiber structure during the dual-modification process (Figure S9). Therefore, the present work also provides a strategy of designing multifunctional silk biomaterials where control of multiple chemical modifications can be enacted on the same starting material without the need for blocking and deblocking of functional groups. Considering that the same structure−chemical modification relationships might exist for the non-mulberry groups of silk materials, it is interesting to further evaluate other non-mulberry silk systems to reveal the universality of the mechanism, which we will perform in our future studies.

solutions increased 90% and 70%, respectively, in comparison to the untreated silk solutions (ASS and BSS), confirming ∼10% higher graft level for the ASS solutions (Figure S1). The results indicated that conformal transitions can be utilized to change the distribution or accessibility of reactive groups to then control the chemical modifications. Similar to the case for BLS solutions, the BSS solutions remained inert to HRP cross-linking (Figure S5), which confirmed steric hindrance together with the reactive group distribution as inaccessible. The ζ potential and chemical modifications based on the phenol group accessibility suggested that phenol groups shifted inward into the nanofibers while the carboxylic groups migrated to the outer regions of the fibers following β-sheet structure formation. EDC/NHS chemistry based on carboxylic groups56−59 was therefore used to modify the two solutions (Scheme S3). The modified solutions were termed BSS-flu and ASS-flu (Table S1), respectively, and their FT-IR spectra were assessed (Figure S6). As expected, the fluorescence emission spectrum (Figure 3d) and fluorescence intensity analysis (Figure 3e) indicated a higher modification in the BSS-flu solutions, confirming a higher accessible carboxylic group content toward the outside of the nanofibers in BSS solutions. All of these results suggest that the conformational transitions resulted in the redistribution of reactive groups, which then influenced the extent of potential chemical modification. Since the ASS solutions remain unchanged as nanostructures during conformational transitions, the ASS avoided differences in reactivity and provided more consistent reaction outcomes. The chemical modification behavior of silk materials was similar to that of enzymes, where hierarchical structures of enzymes can determine catalysis efficiency.60−62 Different strategies were developed to reveal and control the structures of enzymes to optimize catalysis capacity.63−65 For better control of chemical modifications of silk materials, it is necessary to reveal suitable indicators of group distributions in silk aggregates used as reactants. The ζ potential was considered a suitable indicator to judge silk status in the ASS solutions, which would then increase the predicted reliability of the chemical modifications. Therefore, choosing suitable silk solutions with definite nanostructures and conformations is a key factor to achieving reproducible chemical modifications. The selectivity of different chemical modifications on silk nanofibers with various conformations implies the interesting possibility of actively designing multifunctional silk materials by choosing different chemical modification systems with suitable steric hindrances. As shown in Figure 4a, sulfonic acid groups were efficiently grafted onto the ASS nanofibers via diazonium coupling. The grafted nanofibers, ASS-sul, transformed into βsheet-rich nanofibers, ASS-sul-b (Table S1), resulting in the exposure of the carboxylic groups for further chemical modification. The grafted nanofibers, ASS-sul, transformed into β-sheet-rich nanofibers, ASS-sul-b, resulting in the exposure of the carboxylic groups for further chemical modification. EDC/NHS reactions, the second modification process, was then performed in ASS-sul-b for reaction with a fluorescent molecule. Two different functional molecules were finally cross-linked on silk nanofibers more effectively via this strategy, and the treated solution was termed as ASS-sul-b-flu (Table S1). FTIR (Figure S7) shows successful conformational transformation and chemical modification. As illustrated previously, ASS-sul showed the highest sulfonic acid group degree of grafting among the silk solutions due to the phenol groups enriched on the surface of the silk nanofibers. After β-sheet transformation and the second

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CONCLUSIONS Silk materials remain in various aggregate states in aqueous solutions, which resulted in different distributions of reactive groups sequestered in silk material systems. The distribution of reactive groups was a critical determinant of chemical modification efficiency for silk materials. Amorphous silk nanofibers that avoid the uncontrollable influence of the aggregate states could improve the control of chemical modification, providing a better choice for developing functional biomaterials. Silk nanofibers together with controllable transformations further provided a promising platform for designing bifunctional materials through various chemical modifications. Therefore, our study opens up novel strategies of developing silk biomaterials through chemical modifications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.9b00369. General experimental procedures and characterization details for silk materials (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Q.L.: tel, (+86)-512-67061649; e-mail, [email protected]. cn. *H.C.: tel, (+86)-512-65880827; e-mail, [email protected]. ORCID

Qiang Lu: 0000-0003-4889-5299 Hong Chen: 0000-0001-7799-4961 David L Kaplan: 0000-0002-9245-7774 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Key R&D Program of China (2016YFE0204400), the National Natural Science Foundation E

DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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of China (21674074, 21704072), and the NIH (R01NS094218, R01AR070975). We also thank the Social Development Program of Jiangsu Province (BE2018626) for support of this work.

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DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.9b00369 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX