Direct in Vivo Functionalizing Silkworm Fibroin via Molecular

Article pubs.acs.org/journal/abseba

Direct in Vivo Functionalizing Silkworm Fibroin via Molecular Recognition Kun Li,† Junli Zhao,† Jianjun Zhang,† Jinyan Ji,† Yu Ma,*,†,‡ Xiangyang Liu,*,†,§,∥ and Hongyao Xu†,‡ †

College of Material Science and Engineering and ‡State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, P. R. China § Research Institute for Biomimetics and Soft Matter, College of Materials, Xiamen University, Xiamen 361005, P. R. China ∥ Department of Physics and Department of Chemistry, Faculty of Science, National University of Singapore, Singapore 117542 ABSTRACT: Recent works by Tansil et al. suggested a promising way to introduce functionalities into silk fibroin by simply feeding, which was expected to allow silk-based functional biomaterials to have scalable production and direct application. In this research, we aimed to obtain deeper understanding on such a smart strategy of selectively absorbing additives via molecular recognition and the impact of additives on the secondary structures of fibroin. We suggested that the partition ratio of the additive in fibroin and sericin was a critical parameter to evaluate the ability of the additive to enter fibroin, which showed a strong correlation with the isoelectric point (pI) of the additive. On the basis of the classical micelle model, we suggested that silk in vivo recognized additives with low pI and amphiphile chemical structure from other chemical similar additives and assembled them into fibroin. Fibroin-additive assemblies were rather stable that hindered the formation of β-sheet and also crystallites, as indicated by FTIR and WAXD. KEYWORDS: functionalization, silk fibroin, isoelectric point lack of strong interactions between protein and fluorescent dyes.14 Compared with the previous two approaches, feeding silkworms on functionally modified diets is nontoxic, greener, more convenient, and economic on massive production of silk fibroin-based functional biomaterials. Unfortunately, after tentatively feeding silkworms on hundreds of additives,15,16 people sadly find that most of additives are nonabsorbable, and the others are still rejected by fibroin, dominantly located in sericin. It is partially attributed to the hydrophobic nature and highly crystalline secondary structures of fibroin. On the other hand, additives randomly chosen are usually lack of specific interaction with fibroin protein on the molecular scale. As what has been revealed, silk fibroin consists of long hydrophobic GAGAGS motifs and intersecting short hydrophilic segments.17,18 Such multiblock molecules start to assemble into micelles even at the posterior division of gland. So fibroin micelles have sufficient time to clear out those thermodynamically unfavorable additives before spinning. Therefore, a negative viewpoint in functionalizing fibroin by direct feeding is dominant for a long time. Recently, Tansil et al. reported a successful example of introducing fluorescent dye Rhodamine B (RhB) and its derivatives into fibroin, which opened the door to search more candidate substance to modify fibroin in vivo.11,16 Meanwhile, remarkable

1. INTRODUCTION Silkworm silk fibroin, as one of the most important natural biomaterials, currently still attracts growing research interests, even though they have been used by humans for thousands of years. Its extensively adjustable hierarchical structures combined with good biological compatibility promise its wide potential applications in tissue engineering, drug delivery, and other emerging fields.1−6 Till now, most modified fibroin-based materials are produced by reassembling those yet solved fibroin in nonsolvent environments and functionalized by blending with various additives. For example, luminescent regenerated fibroin was produced by introducing various fluorescent dyes including small organic molecules, quantum dots, and metal nanoclusters.7−9 Those luminescent silk fibroin indeed show their applications in textile, visual scaffold, and solar concentrator fields.10−12 However, those indirect modifying approaches are generally hard to control, resource wasting, and carry the risk of getting polluted. For such reasons, direct obtainment of nascent silkworm silk with functionalities becomes a fascinating and more realistic way to produce fibroin-based materials, especially in a large quantity. Moreover, outstanding mechanical properties of silk fiber can be inherited. To achieve this goal, currently three major routes are developed in fabrication of silk fiber with luminesce, e.g., genetic modification, post treatment, and modified diets.13 However, genetic modification is rather restricted in functionalizing, the post treatment is usually conducted under harsh conditions, and the modification is always unstable due to the © 2015 American Chemical Society

Received: December 2, 2014 Accepted: May 27, 2015 Published: May 27, 2015 494

DOI: 10.1021/ab5001468 ACS Biomater. Sci. Eng. 2015, 1, 494−503

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ACS Biomaterials Science & Engineering

Figure 1. Molecular structures of fluorescent dyes: (a) fluorescent sodium (hydrophobic parameter = −0.79), (b) RhB (hydrophobic parameter = 2.43), (c) Rh101 (hydrophobic parameter = 2.19), (d) Rh110 (hydrophobic parameter = 1.17), and (e) acridine orange (hydrophobic parameter = 1.80). Hydrophobic parameters were cited from ref 11.

2. MATERIALS AND METHODS

absorption ratio difference in tested additives was observed despite only a slight variation in chemical structures. The origin was rather ambiguous and contradictory according to their discussion based on hydrophobicity. For example, the distribution of additives in fibroin and sericin exhibited a sharp reversion between rhodamine B (RhB) and rhodamine 110 (Rh110), although their hydrophobicity parameters were very close. Actually, similar selective phenomena of fibroin were also observed during mixing various chemically similar organic molecules with two-photon fluorescence properties into fibroin.19−21 Recognition between fibroin and organic additives via hydrogen bonding was demonstrated to be essential, in order to assemble functionalities into fibroin matrices successfully. Till now, however, how the silkworm recognizes precisely a particular substance from others in vivo, such as recognizing and selective absorbing RhB rather than Rh110, is still an open question. In fact, silk fibroin solution undergoes an extensive environment variation from the posterior division to the anterior division and the spinneret, such as the concentration of fibroin increased from 10 to about 30% and the pH drops from about 6.9 to 4.8, surface charges of micelles change from strong negative to almost neutral (PI of fibroin is 4.53), etc.26,27 Therefore, the role of additives in the fibroin assembling process, and the recognition between additives and micelles need to be further clarified by examining detail relationship between absorption ratios and certain characteristic properties of molecules. Meanwhile, investigations on the secondary structures of modified fibroin protein also provide information on interaction between fibroin and additives. In this research, we were aiming to find a more general principle of fibroin selectively assembled particular additives in vivo. We found a combinational effect of amphiphile molecular structure and negative charges that assisted additives to be successfully recognized and uptaken by fibroin micelles. We also elucidated the correlated effect of additives on altering secondary structures of fibroin. Meanwhile, we discussed some side effects on fibroin assembly, such as the longitudinal nonuniform of secondary structures and lowered mechanical properties.

2.1. Production of Fluorescent Diet Modified Silk. Production of fluorescent dye modified silk was conducted following the description by Tansil et al.11 Briefly, a 1:2 mixture of mulberry leaf powder (100g, Shandong sericultural research institute, China) and distilled water (200 mL) was heated in the microwave oven for about 5 min. And then individual fluorescent additives were added into the prepared feeds. Domesticated silkworms (Bombyx mori larvae, Liang Guang II, China) were fed on fluorescent additive containing diets since the beginning of the fifth instar. Normal feeds without special additives were used before the fifth instar. Fluorescent sodium, rhodamine B (RhB), rhodamine 101 (Rh101), rhodamin 110 (Rh110), and acridine orange were selected as fluorescent additives to introduce luminescent properties into silk. The luminescent cocoons were reeled into silk fibers. All chosen fluorescent additives had exactly similar chemical structures with conjugated fluorescent xanthene or acridine backbone, except for differences in the substituted groups at 3,6,9 positions, as shown in Figure 1. Fluorescent sodium contained two highly

water-solvable phenolate sodium end groups, whereas substituted groups of Rh110 were replaced by two hydrophilic amino groups. For RhB and Rh101, the two substituted end groups were hydrophobic diethylamino and nitrogen heterocycle, respectively. Acridine orange had the same substituted groups as RhB, but without the hydrophilic benzoic acid end. We noted that the amount of fluorescent sodium uptaken by both silk fibroin and sericin was below the detectable limit according to our studies, so we did not present data related to fluorescent sodium in the following characterizations. 2.2. Degumming of Silk. Silk was boiled in an aqueous solution of Na2CO3 (0.5 wt %) for 45 min and then the degummed silk was rinsed with distilled water for three times. Silk fibroin fibers were obtained after drying in a room condition. 2.3. Fluorescence Spectroscopy Analysis. The fluorescence spectra measurements were performed on a JASCO FP-6600 type fluorescence spectrometer. Sericin was collected during degumming, and fibroin was dissolved in 9 M LiBr aqueous solution. Both sericin and fibroin solution were diluted to constant volume for concentration analysis. A similar solution with specified concentration of fluorescent dyes was manually prepared in a low concentration range (∼1 × 10−9 M) and then their fluorescence intensity was measured to obtain a concentration-intensity working curve. The corresponding concentration of fluorescence dye in sericin solution and fibroin solution were 495


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ACS Biomaterials Science & Engineering therefore assigned according to the working curve. In the test, the response time was 0.2 s and the scanning speed was 500 nm/min. Both Ex and Em were 10 nm. For RhB, Rh101, Rh110, and acridine orange, the exciting wavelengths were 540, 560, 490, and 490 nm, respectively. Correspondingly, the detected wavelengths were 580, 600, 520, and 520 nm, respectively. 2.4. Zeta Potential Analysis. The zeta potential measurements were performed on Malvern Nano ZS particle size and zeta potential analyzer. The concentration of each fluorescent dye in an aqueous solution was 5.45 × 10−5 M. 0.1 M NaOH and 0.1 M HCl aqueous solutions were gradually added, in order to obtain a series of samples with different pH. The obtained zeta potential points under different pH were used to find the isoelectric points (pI). 2.5. Fourier Transform Infrared Microspectroscopy Analysis. Secondary structures of fibroin fibers were analyzed by ThermoFisher iN10 Fourier transform infrared microspectroscopy (micro-FTIR) using transmission model with a liquid nitrogen cooled MCT detector. The aperture was 7 μm × 30 μm because of the diameter of single fiber was about 8 μm. The final curve was averaged based on 128 repetitive scans. The resolution was 4 cm−1 and the scanning wavenumber range was from 675 to 4000 cm−1. The decomposition of Amide III (1200 cm−1-1300 cm‑1) curves was operated on a OMNIC 9 software. First, the intensity of Amide III was corrected by subtracting a straight baseline at bottom of the peak. Then the peak was fitted with Gaussian function as suggested by ref 22. The peaks at 1239 cm−1 were assigned as random coil (silk I) and the peaks at 1266 cm−1 were assigned as β-sheet (silk II) according to literature.22−24 For a better fitting, the centers of peaks were allowed to shift around 1239 cm−1 for silk I and around 1266 cm−1 for silk II. 2.6. Wide-Angle X-ray Diffraction (WAXD) Analysis. WAXD measurements were performed on the Beamline 16 (BL16B1) in Shanghai Synchrotron Radiation Facility (SSRF) with X-ray wavelength of 0.124 nm. The distance between the CCD X-ray detector (MAR CCD 165) and samples was 110 mm and the scanning time was 100 s. The samples were a bunch of ∼200 parallel degummed silk fibroin fibers. All the WAXS data was processed with the Fit2D software. 2.7. Mechanical Analysis. Fibroin single fibers of 100 mm long were tested 30 times on the XQ-1A Monofilament Strength Tester (Shanghai Lipu Institute of Applied Science and technology) with a strain rate of 10 mm/min. The humidity was controlled around 60%, and temperature was maintained at 20 °C.

Figure 2. Pictures of silkworms, cocoons, and fibroin fibers under natural light and fluorescent light. Pictures were obtained after feeding silkworms on modified diets containing various florescent dyes. The silkworms feeding on the fluorescent sodium modified diet was not shown in this figure because there was no detectable difference from those feeding on a normal diet (control).

molecules with inner environment of silkworms plus their affinity to fibroin protein. Here, we introduced a parameter named partition ratio, defined as the ratio of the additive between fibroin and sericin. So the partition ratio was a direct parameter to describe the recognition and selection of fibroin to a certain additive. Apparently, the higher partition ratio was preferred in selecting proper additives to functionalize fibroin. Maximum weight concentrations in fibroin and sericin and partition ratios were summarized in Table 1. Among the four fluorescent additives, the concentration of RhB in fibroin (14.29 × 10−3 wt %) was the highest, which was more than two magnitudes higher than that of Rh110 (0.04 × 10−3 wt %), although they only had a slight difference in substituted groups. Meanwhile, RhB and Rh110 showed a distinct reverse tendency in their concentration between fibroin and sericin. RhB (partition ratio = 4.37) was more likely to enter fibroin, and Rh110 (partition ratio = 0.125) preferred to stay outside in sericin. One possible reason as revealed previously was that the RhB (hydrophobic parameter = 2.43) was a bit more hydrophobic than Rh110 (hydrophobic parameter = 1.17), resulting in higher affinity to hydrophobic fibroin.11 However, such theory failed in explaining the highest partition ratio of Rh101 (partition ratio=10.80, hydrophobic parameter = 2.19), which was more hydrophilic compared with RhB. Acridine orange showed the lowest concentration in silk, which might be attributed to the lack of hydrophilic end groups on its molecular structure.

3. RESULTS AND DISCUSSION 3.1. Amounts of Fluorescent Additives in Fibroin and Sericin. As shown in Figure 2, all tested fluorescent additives except fluorescent sodium were successfully uptaken by silkworms and displayed in cocoons and fibroin fibers. Different from most of other additives, fibroin obtained after degumming of silk still contained a certain amount of dyes, as evidenced by their strong fluorescence emission. Similar results were also presented by Tansil et al.11 Taking advantage of fluorescence emission of dyes, it was rather convenient to precisely determine the amounts of fluorescent dyes within fibroin (Wfibroin) and sericin (Wsericin), by measuring intensity of fluorescence emission. We noted that Wfibroin was corresponding to the remaining fluorescent dyes in fibroin after degumming, which was exactly correlated with the absorption amount in fibroin. The amounts of fluorescent dyes in silk were acquired according to the relationship: Wsilk = 75%Wfibroin+25% Wsericin. Figure 3 illustrated a linear relationship between concentrations of fluorescent dyes in diets and in fibroin, until reaching saturated at high dye concentration. The linear dependence might indicate that silkworms had no special preference to absorb or reject certain tested additives, so the efficiency in uptake was mainly decided by the compatibility of 496


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Figure 3. Amounts of RhB, Rh101, Rh110, and acridine orange in silk, sericin, and fibroin with different concentrations in modified diets. The error bars reflected the scatter of individual silk obtained on the basis of three samples.

Table 1. Concentrations of Fluorescent Dyes in Silk, Fibroin, and Sericin and Partition Ratiosa

a

The presented concentrations of Rh110, RhB, Rh101, and acridine orange were the highest values in the experiment, where amounts of rhodamine dyes in feeds were 0.1, 0.1, 0.2, and 0.3 wt %, respectively.

energy configuration. Upon reaching equilibrium, the defined partition ratio indicated thermodynamically the most favorable configuration of the additive was decided by their chemical natures. Therefore, we suggested the partition of the additive between fibroin and sericin (partition ratio) could be a more reliable parameter to evaluate the possibility of the additive to be uptaken by fibroin, i.e., the higher partition ratio, the more likely the candidate additive to enter fibroin. Thus, our next goal was to build the relationship between partition ratios and certain characteristic properties of additives. 3.2. Isoelectric Point (pI) Analysis. The amphiphile nature of molecules undoubtedly played a key role in assisting additives in entering glands of silkworms. But it was somehow

We needed to emphasize that the total amounts of additives existing in fibroin might be correlated with many factors, such as the willingness of silkworms to eat modified diets, absorption and evacuation process, etc. Therefore, although it was frequently discussed in Tansil’s work, actually the hydrophobic parameter was not an ideal parameter to correlate with the most important questions that why some additives could successfully enter fibroin and how fibroin recognized the particular molecules. In fact, before solidification, fibroin protein in the gland of silkworms had already automatically assembled into the micelle structure with a hydrophobic fibroin core.25 Those additives were freely moving into and out of micelles in such a liquid environment to seek its lowest free 497


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Figure 4. Zeta potentials of RhB, Rh101, Rh110, and acridine orange in different pH aqueous solution. Their pI values were 5.63, 4.94, 6.66, and 11.06, respectively. The decreasing pI values of Rh110, RhB, and Rh101 followed exactly same sequence as increasing partition ratios.

Cys-c20 and Cys-172 of light chain.28−31 Due to the highest pH in posterior division, fibroin chains in this division were extended and carried negative charges. Ca2+ and/or perhaps other ions with positive charges surrounded the fibroin micelles as counterions in the aqueous environment.27 When the fibroin micelles were transported into the middle division, three different kinds of sericin were secreted and dissolved in water.29,32 Through the removal of water, the increase of the Ca2+ concentration made contributions to promoting proper folding of fibroin chains. Comparing the pI values of various additives (Figure 4) with the pH values in middle division, we could easily find that Rh110 and arcidine orange dominantly carried positive charges while RhB and Rh101 carried negative charges. Thus, negatively charged fibroin micelles were likely to distinguish particular molecules existing in the gland through the charges of them. On basis of the classical electric double layer theory, molecules with positive charges, such as Rh110 and arcidine orange, played the role of counterions around the micelles. On the other hand, the negative charged molecules, such as RhB and Rh101, were easier and more likely to assemble into the negatively charged micelles and increase density of surface charges and stability of micelles. Arguments based on pI analysis showed good accordance with the measurements of partition ratios. Moreover, increasing partition ratios of Rh110, RhB and Rh101 followed exactly similar sequence to their decreasing pI. Particularly for Rh101, which had the lowest pI, it carried the negative charges throughout the whole transportation of micelles in the gland and finally exhibited the concentration was over ten times higher in fibroin than that in sericin. Therefore, we suggested that the pI values or the charges of additives in the gland were critical for the fibroin to recognize and selectively absorb a certain additive. The lower pI value of the additive, the more likely it entered fibroin, and vice versa. A

insufficient to predict the partition ratio solely based on the amphiphile property. For example, RhB and Rh110 possessed both hydrophilic end and hydrophobic end in a single molecule, but their partition ratios were quite opposite. Hydrogen bonding between fibroin and additives was suggested as another origin of molecular recognition.19 However, in our study, all fluorescent additives could form hydrogen bonding with fibroin on their amino groups. Moreover, hydrogen bonding was a rather short-range and local interaction compared with other interactions like Coulombic and hydrophobic interaction, which were more significant in assembled system. Therefore, hydrogen bonding was not the origin of big difference of partition ratios among different fluorescent additives in the current case. As was already known, fibroin protein consisted of long hydrophobic GAGAGS repetitive units that were intersected by short hydrophilic residue segments. This amphiphile nature allowed fibroin molecules to assemble into the micelle structure with a hydrophobic core even at the posterior division of the gland as the concentration was higher than critical micelle concentration (CMC). Micelles were stabilized by the exterior hydrophilic segments and negative charges carried by fibroin protein. The isoelectric point (pI) of fibroin protein had been demonstrated to be 4.53,26 which was much lower than the pH in most parts of the silk gland. Generally, from the anterior to the spinneret, the silk gland could be divided into five divisions, i.e. posterior division (pH 6.9), posterior part of middle division (pH 5.6), middle part of middle division (pH 5.2), anterior part of middle division (pH 5.0) and anterior division (pH 4.8) where the pH was almost equal to the isoelectric point of fibroin protein.27 The posterior division was the region to secrete the heavy chain (350k Da) and light chain (25k Da) fibroin that linked by a single disulfide bond between the twentieth residue from the carboxyl terminus of heavy chain 498


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Figure 5. FTIR spectra of fluorescent additives modified silk fibroin fibers. (A) FTIR absorbance of fibroin obtained by feeding on the normal diet (control) and modified diets with 0.1 wt % RhB, 0.2 wt % Rh101, 0.1 wt % Rh110, and 0.3 wt % acridine orange, respectively. (B) Wavenumber of silk I vibration of corresponding fibroin. Error bars reflected the scatter of structures that was obtained based on over 16 positions of fibroin fiber.

Figure 6. (A−E) Decomposition of amide III bands of silk fibroin fibers obtained by feeding on diets containing the normal diet (control), 0.1 wt % RhB, 0.2 wt % Rh101, 0.1 wt % Rh110, and 0.3 wt % acridine orange, respectively. (F) Comparison of according fibers’ secondary structures. The hollow circles represented experimental data, the red solid lines were fitted spectra based on decomposition into silk I (blue dash lines) and silk II (magenta dash lines). Addition of fluorescent dyes resulted in a drop of silk II content.

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Figure 7. (A−E) Decomposition of amide III bands of silk fibroin fibers obtained by feeding on diets containing 0, 0.025, 0.050, 0.075, and 0.100 wt % RhB, respectively. (F) Comparison of according fibroin’s secondary structures. The hollow circles represented experimental data, the red solid lines were fitted spectra based on decomposition into silk I (blue dash lines) and silk II (magenta dash lines). Silk II decreased gradually with the increase of RhB in feeds.

the pI values could be the dominant factor for those additives with amphiphile structure and moderate solubility (hydrophobic parameter). Third, other interactions, such as hydrogen bonding, might stabilize the attachment of additives to fibroin protein. Short range interaction could also induce formation of local premature structures and show significant effects on secondary structures of fibroin as indicated by following structural analysis. 3.3. Effects of Fluorescent Dyes on Secondary Structures. Effects of fluorescent dyes on the secondary structures of fibroin in modified silk could provide more insights into the roles of additives on formation of hierarchical structures of fibroin. Analysis of secondary structures of silk fibroin by FTIR had been demonstrated to be a simple way to analyze the conformation of protein. Generally, Amide I, II, III bands could be decomposed to distinguish the contents of silk I (dominantly random coil and α-helix) and silk II (dominantly β-sheet).24,33−36 Figure 5A showed the FTIR spectra of Amide III band of controlled and modified silk, where 1239 cm−1 peak

slight variation on the substituted groups of additives resulted in a distinct difference in isoelectric points and their partition ratios. Several effects related to charged additives in the gland should be mentioned. First, most of additives actually were screened out of the gland so that the concentration of additive ions in the gland was rather low (less than 12 × 10−3%), especially compared with the native ions like Ca2+ (59.7 × 10−3% in anterior division),27 so the slight change of ion density would not severely alter the natural process of silkworm spinning and the environment inside the gland. Second, although the pI value showed its exact relationship with the partition ratio, it might not be the only origin to determine the ratio of partition. For example, the fluorescent sodium had a very low pI value, but its high solubility made them easily clear out of the gland. On the other hand, arcidine orange had the highest pI value, however, due to its strong hydrophobic tendency, it still showed a definite concentration in fibroin, whose partition ratio was even higher than Rh110. Therefore, 500


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Figure 8. 2D-WAXD pattern of silk fibroin prepared by feeding on different RhB concentrations. The concentrations were (a) 0 wt %, (b) 0.025 wt %, (c) 0.050 wt %, (d) 0.075 wt % and (e) 0.100 wt %, respectively. Crystalline planes (020), (210), (030), (021), and (002) were denoted.

Figure 9. (A) Tensile strength and (B) modulus of modified fibroin (concentrations in diets, 0, 0.025, 0.050, 0.075, and 0.100 wt %) decreased. The error bars reflected the scatter in results obtained from 30 individual measurements.

was assigned to silk I and the 1266 cm−1 peak was assigned to silk II.22−24 Silk I peaks shifted to low wavenumber for all three fluorescent dyes that had relatively higher partition ratios (RhB, Rh101 and aricidine orange). According to Figure 6, contents of silk I exhibited the similar tendency that if dyes were concentrated in fibroin (RhB and Rh101), silk I was more than that of parallel controlled silk. More silk I, or fewer crystalline silk II, might be related with the premature and stabilized ordered domains surrounding additive ions in the gland before spinning. Those local ordered structures binding to additive ions with hydrogen bonding were unlikely to transit into more ordered silk II (β-sheet conformation) during spinning and remained in random coil or α-helix conformation. Aricidine orange also showed a strong effect on suppressing formation of silk II, although it had a rather low concentration in fibroin. Another effect on fibroin’s secondary structures was observed by modifying fibroin via feeding. Due to the low concentration of additives and high viscosity in the gland, and discontinuous food intake habits of silkworms, there was apparent structural inhomogeneous along a single silk stem, as evidenced by statistically measuring more than 16 individual positions by micro-FTIR (shown in Figure 5B). Such structural fluctuation might deteriorate the mechanical properties of a single fiber and restrict its applications in particular fields that required uniform fibers. We took RhB-modified fibroin as an example to further demonstrate the effects of additives on secondary structures. As shown in Figure 7, content of silk I continued increasing with increasing amounts of RhB in diets, while fewer silk II structure content was reduced. It was in accordance with the X-ray

diffraction measurement as shown in Figure 8. Additives had negligible effects on orientation of β-sheet structure. In contrast, because of the existence of increasing RhB in fibroin micelles, the (020), (210), (021), and (002) peaks became weaker,37−41 and (030) peaks and other high order diffraction peaks disappeared, which indicated smaller crystal size and more defects within crystals. On the other hand, based on our previous study, modification of fiber by post treatment, i.e., immersing fibroin into RhB aqueous solution at a high temperature, did not bring significant change to crystalline structures, even though the concentration of RhB could reach one magnitude higher.14 The RhB molecules introduced via post treatment were dominantly diffused into amorphous regions, and had low interaction with crystals so that it could be easily removed by washing. In the current study, RhB molecules took part in the whole process of crystallization of fibroin during spinning, as it had already existed in fibroin micelles in the gland. It confined the size of crystals and introduced defects into crystals; meanwhile, it was tightly bonded to crystalline domains, and hard to remove by simply washing. 3.4. Effects of Fluorescent Dyes on Mechanical Properties. Figure 9 illustrated the changes of mechanical properties of modified silk fibroin fibers compared with natural silk fibroin fiber. Both tensile strength and modulus decreased due to additives in diets, especially severe for that containing acridine orange and Rh101, which had the lowest silk II content (Figure 6F) and the largest structural fluctuation (Figure 5B). Nevertheless, they still possessed excellent mechanical properties as compared with unmodified silk. 501


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functionalities to be integrated into fibroins required in many bioengineering fields.

3.5. Mechanism of Fluorescent Dyes Uptaken into Silk Fibroin Fiber. On the basis of the above analysis, additives were involved into the whole process of hierarchical structure formation in the gland (micelle) until out of spinneret (crystal). To promote the recognition of fibroin for a certain additive, optimal additives should possess both an amphiphile molecular structure and pI lower than 5. Mechanism of additives uptaken into fibroin was illustrated in Figure 10. The hydrophilic end

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

Y.M. supervised all the research and the publication. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This work was financially supported by the National Natural Science Foundation of China (Grant 21204011), the Major Project of Chinese National Programs for Fundamental Research and Development (973 Program) (2011CB606100), National Higher-education Institution General Research and Development Project and State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (SKLFPM). Notes

The authors declare no competing financial interest.

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Figure 10. Mechanism of charged additives involved into silk fibroin micelles. Additional cations dominantly acted as counterions around the micelles, whereas additional anions were likely to be involved into fibroin micelles.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grant 21204011), the Major Project of Chinese National Programs for Fundamental Research and Development (973 Program) (2011CB606100), Fundamental Research Funds for the Central Universities. The WAXS experiment was performed at Shanghai Synchrotron Radiation Facility (SSRF), China.

group guaranteed that the additives could be uptaken into the humor and the hydrophobic end group could interact with hydrophobic blocks by forming hydrogen bonding. The pI values of additives extensively decided the partition ratios of additives in fibroin to those in sericin. Additives with higher pI carried positive charges and acted as counterions in acid environment in the gland; on the other hand, additives with lower pI and hydrophobic groups had greater chance to get involved into fibroin micelles with negative surface charges, especially during the lower pH and the increase in the micelle concentration in the gland. Incorporated additives might bond with fibroin by hydrogen bonding or other interactions, and promote the formation of preordered structures nearby, which were even stable upon shear or elongation in spinning. As a result, the final fibroin exhibited lower β-sheet content, poor crystal regularity, and thus slightly reduced mechanical properties.

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REFERENCES

(1) Kundu, B.; Rajkhowa, R.; Kundu, S. C.; Wang, X. Silk fibroin biomaterials for tissue regenerations. Adv. Drug Delivery Rev. 2013, 65, 457−470. (2) Nakazawa, Y.; Asano, A.; Nakazawa, C. T.; Tsukatani, T.; Asakura, T. Structural characterization of silk-polyurethane composite material for biomaterials using solid-state NMR. Polym. J. 2012, 44, 802−807. (3) Wang, X.; Wenk, E.; Matsumoto, A.; Meinel, L.; Li, C.; Kaplan, D. L. Silk microspheres for encapsulation and controlled release. J. Controlled Release 2007, 117, 360−370. (4) Wang, X.; Hu, X.; Daley, A.; Rabotyagova, O.; Cebe, P.; Kaplan, D. L. Nanolayer biomaterial coatings of silk fibroin for controlled release. J. Controlled Release 2007, 121, 190−199. (5) Kim, Y. J.; Abe, Y.; Yanagiura, T.; Park, K. C.; Shimizu, M.; Iwazaki, T.; Nakagawa, S.; Endo, M.; Dresselhaus, M. S. Easy preparation of nitrogen-enriched carbon materials from peptides of silk fibroins and their use to produce a high volumetric energy density in supercapacitors. Carbon 2007, 45, 2116−2125. (6) Yun, Y. S.; Cho, S. Y.; Shim, J.; Kim, B. H.; Chang, S. J.; Baek, S. J.; Huh, Y. S.; Tak, Y.; Park, Y. W.; Park, S.; Jin, H. J. Microporous carbon nanoplates from regenerated silk proteins for supercapacitors. Adv. Mater. 2013, 25, 1993−1998. (7) Somashekarappa, H.; Annadurai, V.; Subramanya, G.; Somashekar, R. Structure-property relation in varieties of acid dye processed silk fibers. Mater. Lett. 2002, 53, 415−420. (8) Chang, S.; Dai, Y.; Kang, B.; Han, W.; Chen, D. Fabrication of silk fibroin coated ZnSe: Mn2+ quantum dots under γ-radiation and their magnetic properties. Solid State Commun. 2009, 149, 1180−1183. (9) Wang, X.; Gao, W.; Xu, S.; Xu, W. Luminescent fibers: In situ synthesis of silver nanoclusters on silk via ultraviolet light-induced reduction and their antibacterial activity. Chem. Eng. J. 2012, 210, 585−589.

4. CONCLUSIONS Feeding silkworms on functional additives is an effective and environment-friendly approach to obtain silk fiber with various functions in a large quantity. A general principle of selecting or designing proper additives is proposed, according to the assembly mechanism of fibroin before spinning. Those additives should have certain solubility or dispersion in water, low toxicity, preferably small size, and amphiphile structure. Moreover, the partition ratio of the additive is strongly correlated with its pI, which indicates that fibroin recognizes and assembles a certain additive according to its charges in the gland. Therefore, it is crucial to choose additives with low pI in order to introduce fibroin with functionalities in vivo. Additives may have potential effects on controlling formation of fibroin’s secondary structures, as they suppress the formation of β-sheet and crystallites. The proposed additive selection principle may apply to both organic and inorganic molecules and even nanoclusters or their mixtures, which allows combinational 502


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Direct in Vivo Functionalizing Silkworm Fibroin via Molecular

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