Enhanced Stabilization in Dried Silk Fibroin Matrices - ACS Publications

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Enhanced Stabilization in Dried Silk Fibroin Matrices Adrian B. Li, Jonathan A. Kluge, Miaochan Zhi, Marcus T. Cicerone, Fiorenzo G Omenetto, and David L Kaplan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00857 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

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Enhanced Stabilization in Dried Silk Fibroin Matrices Adrian B. Li1, Jonathan A. Kluge2, Miaochan Zhi3, Marcus T. Cicerone3, Fiorenzo G. Omenetto1, David L. Kaplan1,2*

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Department of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, MA 02155.

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Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155.

Materials Measurement Lab, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899

ACS Paragon Plus Environment

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Abstract

Preliminary studies have shown that silk fibroin can protect biomacromolecules from thermal degradation, but a deeper understanding of underlying mechanisms needed to fully leverage the stabilizing potential of this matrix has not been realized. In this study, we investigate stabilization of plasma C-reactive protein, a diagnostic indicator of infection or inflammation, to gain insight into stabilizing mechanisms of silk. We observed that the addition of antiplasticizing excipients that suppress β-relaxation amplitudes in silk matrices resulted in enhanced stability of plasma CRP. These observations are consistent with those made in sugar-glass based protein stabilizing matrices and suggest fundamental insight into mechanisms, as well as practical strategies to employ with silk protein matrices for enhanced stabilization utility.

Keywords: silk, beta relaxation, stabilization, C-reactive protein Introduction Silk fibroin, a biologically derived protein harvested from Bombyx mori silkworm cocoons, has demonstrated ability to stabilize a wide range of biologics1 including labile enzymes,2,

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antibodies,4 and blood proteins.5 Silk fibroin (hereafter referred to as silk), is a high molecular weight amphiphilic protein (up to 390kDa)6, 7 that is generated in an aqueous state and at neutral pH and is thus readily miscible with biologic solutions.8 Silk’s unique protein structure (amphiphilic, block copolymer, self-assembly features) allows for formation of mechanically robust constructs including films,9 cakes,10 and gels,11 through a simple dehydration mechanism.


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Although silk has also demonstrated a stabilizing effect on numerous labile analytes, some have remained unstable, even after solid-state encapsulation in silk.12 A clear understanding of stabilization mechanisms in silk would illuminate the path to improved stability of these difficult analytes, but such an understanding is currently lacking. Two hypotheses, which were developed to explain why sugar-glasses could stabilize proteins, could explain stability of proteins encapsulated in silks. One theory, known as the water replacement theory, hypothesizes that sugars displace water at the surface of the protein thus leading to a thermodynamically favored folding state by providing sites for hydrogen-bonding.13 The other theory, known as the vitrification theory, hypothesizes that through dehydration, degradation processes are impeded by slowing the dynamics of the matrix.14 The effects of the three characteristic dynamic processes exhibited by amorphous encapsulating solids on protein degradation pathways has been evaluated.15 Reduction of matrix α-relaxation, which represents global or structural scale relaxation and is oft compared to the glass transition temperature (Tg), has not consistently resulted in slower degradation rates of encapsulated protein.16 On the other hand, of particular interest is work demonstrating a robust correlation between smaller scale β-relaxations, βJG and βfast, and protein stability in sugar-glass matrices,16-19 which is attributed to a relationship between β-relaxation and transport. It seems that β-relaxation dynamics are also coupled to protein conformational motion20,

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and to small-molecule mass transport.22 Thus, strongly

suppressed β-relaxation of a matrix improves stability along degradation pathways involving partial unfolding of the protein, or mass-transport limited degradation reactions such as oxidation or deamidation. In the present work, we explore the hypothesis that protein stability in silk formulations will correlate with matrix β-relaxation, as it does in sugar-based dry formulations. To do so, we dry


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C-reactive protein (CRP) in several silk formulations having a range of β-relaxation values, then evaluate stability in those formulations. CRP, a plasma protein that plays a role in inflammatory diseases including cardiovascular,23 diabetes,24 and obesity,25 has become a prominent focus in the clinical chemistry space, as evidenced by the increased development of CRP diagnostic assays,26 due to the need for more robust analytical methods. One strategy to enhance assay sensitivity and selectivity is to minimize the effect of stresses imposed on specimens prior to analysis. CRP has poor stability at 37°C within one week of drying,27 and was thus chosen as a candidate for assessing improvements in stabilization. We present physical analyses of silk-based matrices after the addition of plasticizers and antiplasticizers, which modulate β-relaxation in the matrix. We then provide evidence that we can control the stabilizing performance of silk using these excipients. Specifically, we show that plasticizers facilitate β-relaxation and accelerate degradation rates while antiplasticizers suppress β-relaxation and decelerate degradation rates. Ultimately, we obtain an apparently robust correlation between plasma CRP stability trends and β-relaxation properties of the glass, suggesting that similar degradation mechanisms are important in dried silk formulations as are found to be important in dried sugar formulations, and thus provide a foundation for formulating silks to stabilize biospecimens and other biologicals. Materials and Methods Materials Bombyx mori silkworm cocoons were purchased from Tajima Shoji Co., LTD1 (Sumiyashicho, Naka-Ku, Yokohama, Japan). All chemicals used in the production of silk and subsequent

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Certain commercial equipment, instruments, or materials are identified in this paper to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.


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studies were reagent grade and purchased from Sigma-Aldrich (St. Louis, MO). All aqueous solutions were prepared using deionized ultrapure water (UPW) produced by a Millipore purification system (Billerica, MA). Fresh donor plasma was received in Na-citrate Vacutainer tubes (Research Blood Components, Brighton, MA) and was delivered within 1 hour of venipuncture and kept on ice packs. Aliquots were immediately frozen at -80°C to serve as positive controls. Silk fibroin purification Aqueous silk fibroin stock solutions were prepared as previously described.28 Briefly, silk fibroin was purified from cladding sericin by boiling silk cocoon pieces in 0.02M sodium carbonate solution for 60min. The silk fibroin fibers were rinsed for 20min, three times, with UPW, and air dried at ambient temperature for a minimum of 12h. After drying, the fibroin was solubilized at 20% (mass/vol) in 9.3M lithium bromide at 60°C for 120min. The resulting solution was then dialyzed against UPW using 3500Da molecular weight cut-off dialysis tubing (Fisher Scientific, Pittsburgh, PA). The concentration of the dialyzed solution was determined by comparing the mass of 0.5mL of aqueous silk and silk dried at 60°C for a minimum of 12h. Silk solutions were stored at 4°C until fabrication. Silk encapsulated plasma films and cakes Plasma laden silk films were cast from mixtures of biospecimen and purified silk at a 1:7 volume

ratio

(350µL

silk:50µL

serum,

final

silk

concentration,

4%)

on

28mm

polydimethylsiloxane molds. The silk formulations were buffered with 20mM Tris-HCl, 1% bovine serum albumin (BSA) and titrated to pH 8.0 with 1N sodium hydroxide prior to mixing with biospecimen. Resulting 28mm films were punched to 20mm and reconstituted with UPW at a volume commensurate with the fraction of mass remaining (punch mass/film mass). Silk and


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plasma solutions were also lyophilized in 2mL serum vials using a VirTis 25L Genesis SQ SuperXL-70 Freeze Dryer (SP Scientific, Warminster, PA). Samples were frozen at -45°C for 180min. Primary drying occurred at -25°C for 600min, and secondary drying occurred at 4°C for 120 min. This temperature was below the glass transition temperature of the frozen silk (Tg’) and therefore sufficient to prevent cake collapse. Samples were backfilled with nitrogen and stoppered at 500Torr. Films stored under nitrogen were punched and placed in 0.6mL Eppendorf tubes. The tubes were then placed in 20mL serum vials and stoppered at the same condition as the cakes. Serum vials, stoppers, and seals were supplied by Fisher Scientific (Waltham, MA). Both films and cakes were reconstituted with 0.1% polysorbate-20 in UPW. For stability experiments, CRP was tracked for three months at 22°C, 37°C, and 45°C, in solution, DPS, and silk film formats. These temperatures were chosen to simulate shipping scenarios in which the cold chain may not be available. CRP was assayed via DuoSet ELISA (DY1707, R&D Systems, Minneapolis, MN) following manufacturer protocols and utilizing manufacturer reagents. Final dilution level for all samples was 3200x. A BioTek Synergy HT plate reader (Winooski, VT) was used to detect the analytes colorimetrically. Additives for plasticization and antiplasticization of silk matrices Silk stock was mixed with glycerol or sucrose (both provided by Sigma-Aldrich, St. Louis, MO) to achieve a final solution of 4% solids. The dry mass contained increasing amounts of glycerol (10, 20% mass/mass) and sucrose (5, 10, 20, 50% mass/mass). For plasma stability studies, the silk and glycerol/sucrose mixtures described above were used to dilute biospecimens at a 1:7 volume ratio to reach a final solids ratio of 4%. Temperature modulated differential scanning calorimetry (TMDSC)


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TMDSC was used to determine the Tg of non-plasma loaded freeze-dried cakes. Samples of approximately 10mg were hermetically sealed in aluminum pants (TA Instruments, New Castle, DE) underwent TMDSC in a Q100 DSC (TA Instruments) with a dry nitrogen gas flow of 50mL/min. The samples were equilibrated to 20°C for 5min and heated to 230°C at a rate of 5°C/min with ±0.8°C modulation amplitude every 60sec. The half-height method was used to define the Tg. Dynamic mechanical thermal analysis (DMTA) Air dried non-plasma loaded films were cut to approximately 28mm length x 5mm width and loaded on to stainless steel grips 10mm apart on a RSA3 Dynamic Mechanical Analyzer (TA Instruments). Samples were frozen with boiloff from a liquid nitrogen dewar to -120°C at 10°C/min, and held for 5min. The films were then heated under purge with dried compressed air to 200°C at 3°C/min at an oscillation frequency of δ=1Hz and a strain of 0.1%. The program was set to auto adjust strain, with a max strain of 10%, as the sample was heated to maintain contact with the film and avoid artifacts resulting from film deformation. The storage modulus, loss modulus, and loss tangent were recorded during the dynamic temperature sweep, and tan(δ) (the ratio of loss modulus to storage modulus) was reported. Neutron scattering analysis Neutron backscattering from silk samples was measured using the high flux backscattering (HFBS) spectrometer at the NIST Center for Neutron Research on the NG2 beam line. A neutron wavelength of 6.271 Å and a kinetic energy of 2.08MeV were used, and scattering over the momentum transfer (Q) range (0.71 to 1.75)Å-1 was used in the analysis. In these studies, the spectrometer operated in the fixed-window scanning mode where the elastic scattering intensity (I) was recorded as a function of Q while the sample was heated at 1K/min from 40K to about


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330K. The elastic scattering intensity was normalized to its intensity at 40K, and the temperature-dependent Debye–Waller factor () was extracted, assuming a harmonic oscillator model for hydrogen motion as done previously.17 Obtained in this way, represents the mean-square displacement of hydrogen atoms on timescales of ≤ 5ns, and length scales of ≤ 9Å.

Results and Discussion: Stability of plasma C-reactive protein in dried silk matrices In order to assess the stabilizing effect of silk, a storage study was initiated at elevated temperatures to assess stability of plasma C-reactive protein in air-dried silk and plasma films, and lyophilized silk and plasma cakes stored for 3 months at 22°C, 37°C, and 45°C. Prior to fabrication, all plasma samples were aliquoted and frozen at -80°C to create a consistent positive control. Absolute readings of the freshly drawn plasma as received, and the frozen plasma aliquot assayed at each time-point can be found in Figure S1. It is important to note that the single freeze-thaw did not negatively affect the assayed CRP level in relation to the levels in the plasma as received. Nonetheless, to account for plate-to-plate variability, samples were normalized to a time-matched -80°C stored plasma aliquot.


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Figure 1: Stability of plasma CRP in air-dried silk films and lyophilized silk cakes. Plasma encapsulated in silk films (left) and lyophilized cakes (right) were subject to temperatures of 22°C, 37°C, and 45°C storage over 84 days. An additional group was stored under 500Torr nitrogen at 22°C. Data are average ± SD of n=4 replicates. Data were normalized to timematched thawed frozen plasma aliquot control. The results of encapsulation, as seen in Figure 1, demonstrates the comparability between the air-dried films and lyophilized cakes. In silk films, the formulation resulted in complete recovery after fabrication. After 3 months of storage at 22°C, 37°C, and 45°C, ~90%, ~55%, and ~40% of CRP was retained, respectively. When comparing films stored under nitrogen, a small improvement was observed in the nitrogen-stored films, although not statistically different at day 84. The cakes, despite using controlled lyophilization, showed ~10% loss of recovery after fabrication, and exhibited similar but more erratic stability profiles when compared to the films. This result could be attributed to additional freeze-thaw cycle undergone by the plasma sample. Taken together, this preliminary study indicates that additional optimization to obtain complete recovery after storage at elevated temperatures is needed. Physical analysis of relaxation properties in plasticized and antiplasticized silk materials


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Over the past decade or so, it has become increasingly clear that reduced β-relaxation amplitude corresponds with improved stability of proteins in dry sugar formulations.29 We hypothesized that suppression of β-relaxation through addition of excipients to the silk matrix would be effective in improving stability profiles of plasma CRP. To achieve this goal, it was first necessary to quantify physical changes imparted by additives to the silk-based matrix. Once additive concentration boundaries that notably imparted changes in dynamics were identified using TMDSC and DMTA, it was then necessary to investigate β-relaxation in silk matrices to allow a more in-depth investigation of dynamics. To do so, we first performed TMDSC on lyophilized silk systems. The effect of two additives on relaxation behavior of silk formulations in cakes was investigated. Glycerol has demonstrated the ability to facilitate β-relaxations in starch-polymer matrices at concentrations above 10%,17, 30 and was used as a plasticizer. Sucrose is commonly used as freeze-dried excipient due to its ability to form glasses in the solid state,14 and appears to function as an antiplasticizer to the silks. Thermograms in Figure 2A indicated that at low ratios, glycerol drastically facilitated αrelaxation of silk as indicated by a reduction of the Tg. At ratios >10%, Tg was not attainable due to transition broadening and increased sensitivity requirements to detect silk’s transition from a glassy to rubbery state. The result was unsurprising as plasticizers have been used to improve ductility, or yield at break, in many polymer-additive compositions.31 Thermograms in Figure 2B indicated that addition of sucrose also facilitated α-relaxation of silk. The plasticization effect of sucrose on silk was not as significant as glycerol, as a much higher mass loading (50%) was required to reach a similar Tg to that of silk with 10% glycerol. Given these results, we were confident that we had established the concentration ranges of silk and glycerol that would affect silk dynamics.


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To monitor sub-Tg relaxation behavior, we characterized the silk and glycerol films using DMTA, as shown in Figure 3C. Typically, DMTA is not used to examine β-relaxation properties of stabilizing matrices, due to the inability of materials that are typically used for stabilization to form free-standing constructs that are compatible with DMTA loading vices. However, because silk has robust mechanical properties in film format, we could characterize the β-relaxation behavior using mechanical analysis. We chose to study the glycerol formulations instead of the sucrose formulations because the sucrose films were more brittle and susceptible to shattering, whereas glycerol induced more flexibility in the silk films. In each formulation, two transitions were observed. The rapid increase in tan(δ) at higher temperature represents the point of mechanical failure observed in the films, which occurs at the Tg of the material. We recall that the Tg observed via DSC corresponds to the Tg seen here. Similar to results seen via DSC, increasing concentrations of glycerol reduced Tg rapidly. In the case of the film with 20% glycerol, the onset of the 1 Hz α-relaxation peak occurs at approximately 0°C. The peak at the lower temperatures represents the slow β-relaxation process, the Johari-Goldstein relaxation (βJG), which occurs at Tβ. The dotted line represents the Tβ of the silk film without glycerol. In contrast to the facilitation of α-relaxation dynamics evidenced by the reduction of Tg, inclusion of glycerol slows β-relaxation, as indicated by increasing Tβ. This behavior is characteristic of antiplasticization, and has been observed before in several polymer-polyol systems.30, 32 In this instance, the amplitude and temperature of the βJG was monitored in addition to the αrelaxation and Tg. In recent studies, both the fast and the slow β processes have correlated strongly with protein or drug stability.16 Because the timescale of the βJG is linked to the amplitude of βfast, and small molecule transport appears to be coupled to both relaxation processes,22 we consider whether DMTA presents as a potential formulation screening tool for


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protein stability in silk. In such considerations, it is important to recognize that DMTA monitors dynamic response at a fixed, relatively low frequency (1 Hz here). This means that it will be impossible to monitor the fast β process, and samples must be taken to very low temperature to sense the βJG process. This presents a bit of complexity. Because the antiplasticization phenomenon is strongly temperature dependent, naive interpretation of DMTA measurements made at a temperature other than the intended storage temperature could give misleading results. For example, the DMTA results show that 20% glycerol formulation has an elevated Tβ relative to formulations with less glycerol. This indicates that glycerol at 20% antiplasticizes silk, and could be interpreted to suggest that 20% glycerol would be a better stabilizer than other formulations. However, due to frequency bandwidth limitations of DMTA, we can detect plasticization or antiplasticization for this system only at temperatures near -50°C. At the intended storage temperatures, however, this level of glycerol acts as a strong plasticizer rather than an antiplasticizer. On the other hand, an informed interpretation of DMTA data may yield results relevant to the intended storage temperature. Psurek et al.32 have shown that changes to β-relaxation with antiplasticization occurs in highly regular ways, suggesting that, if DMTA experiments could be performed in even a narrow band of frequencies, data over a small range of low temperatures could be extrapolation from an experimentally accessible temperature regime to the temperature regime of interest for stabilization. With this proviso, DMTA has potential to be a useful predictive tool. Once it was determined that the formulations screened with TMDSC and DMTA would produce significant changes in dynamics, we sought to gain more insight into the high-frequency βfast relaxation processes. Lyophilized silk samples doped with sucrose were analyzed using


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neutron scattering, as shown in Figure 3D. Lyophilized samples for this analysis were chosen in order to remove the confound of varying residual moisture, a factor the measurement is highly sensitive to. Sucrose was chosen for this analysis in order to compare results to the literature.33 This method measures mean squared displacement of hydrogen atoms () in a glass matrix, which is correlated to the amplitude of the fast β-relaxation process, which is related the timescale of the slow β-relaxation process.17 Results demonstrated that indeed inclusion of sucrose to silk matrices suppressed β-relaxation at temperatures above 75 K. One caveat to note, however, is that residual moisture was not explicitly quantified. Because water acts as a plasticizer to polymers34, we suggest residual moisture analysis to become a standard part of the workflow in future studies.

Figure 2: Physical analysis of dried silk systems. All data are single measurements on each respective analytical method. A) Temperature modulated differential scanning calorimetry of lyophilized silk and glycerol (A) or sucrose (B) cakes. All composition references are to mass fraction of additive in dried cake. Tg=glass transition temperature, Tβ=temperature of β-


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relaxation. C) DMTA of air-dried silk and glycerol films. Dotted line indicates temperature at which the silk alone film undergoes β-relaxation. D) Neutron scattering analysis of silk and sucrose cakes. Inset: mean squared displacement of hydrogen atoms () as function of sucrose mass fraction. Correlation of matrix and stability of plasma CRP Knowing that both glycerol and sucrose had effects on silk relaxation properties, we decided to incorporate the excipients into lyophilized silk and plasma stabilization matrices. Lyophilization was chosen over film drying to control residual water, which can be a confounding factor that obfuscates trends of interest. We chose to exclude pure sucrose groups from these studies in order to expedite sample fabrication time. Because Tg’ of sucrose, estimated to be -32°C35 is dramatically higher than that of silk’s, -12°C, the predominantly silk samples were able to be freeze-dried in less than one day. The results shown in Figure 3A, as hypothesized, demonstrated that inclusion of glycerol at high levels plasticized the matrix and lead to more degradation of CRP. Results also demonstrated that inclusion of sucrose antiplasticized the matrix and improved stability of plasma CRP, producing levels that were significantly higher in the 20% and 50% sucrose formulations. Taken together, this experiment demonstrates our ability to modulate stability of plasma CRP in silk matrices using additives. To determine whether β-relaxation processes in silk matrices correlates with protein stability, we extracted values at 45°C (Figure 3B) from neutron scattering data in Figure 2D. These values were then plotted against degradation rates of plasma CRP in silk and glycerol glasses taken over 1 month (days 7, 14, 28), as seen in Fig 3A. Degradation rates were extracted from stability data by regressing CRP recovery to an exponential decay function, and can be found in


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Figure S2. Results demonstrated that indeed degradation rate correlated with β relaxation processes of the encapsulating matrix.

Figure 3: Effect of additives and matrix on stability of plasma CRP. A) G=glycerol. S=sucrose. % indicates mass ratio of additive in dried cake. Data are after storage at 45°C for 28 days and normalized to time-matched frozen control. Asterisks indicate significant differences from the silk control formulation at p < 0.05 level. Data are average ± SD of n=4 replicates. B) Plasma CRP degradation rates (k) in silk and glycerol or sucrose cakes plotted against of the encapsulating matrix. Dotted line indicates linear regression of the points. Y-axis error bars indicate sum of squares error from linear regression of stability data used to calculate k (Fig S2). X-axis error bars indicate SD of n=2 replicates. Conclusions These studies represent the first attempt to uncover the mechanism(s) controlling stability of proteins in silk matrices. In sugar-based dry formulations it has been established that matrix βrelaxation correlates with protein stability because the former controls transport and protein conformational flexibility (at the length scale of a few residues). We have tested and confirmed the hypothesis that β-relaxation will also correlate with protein stability in silk formulations. We


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surmise that, as with sugar formulations, protein stability in silk formulations is profoundly influenced by local protein mobility and transport of reactive species. Furthermore, two screening techniques were utilized to outline excipient concentration ranges that affect silk matrix dynamics. Taken together, the approach herein provides a platform for analyzing silk stabilizing materials, and demonstrates that that rational design of silk formulations based on underlying fundamental mechanisms of stabilization can be used as an approach to optimize stability of encapsulated macromolecules.

ASSOCIATED CONTENT

Supporting Information CRP levels in frozen plasma aliquots analyzed at each stability study time-point for use as normalization basis for Figure 1, as well as degradation rate regression for Figure 3B. Corresponding Author *E-mail: [email protected]. Tel.: +1-617-627-3251 Fax: +1-617-627-3231 ORCID David L. Kaplan: 0000-0002-9245-7774 Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT We thank the NIH (P41EB00252), DTRA and AFOSR (FA9550-14-1-0015) for support of this research. ABBREVIATIONS CRP, C-reactive protein; TMDSC, temperature modulated differential scanning calorimetry; DMTA, dynamic mechanical thermal analysis, βJG, Johari-Goldstein β-relaxation REFERENCES 1.

Li, A. B.; Kluge, J. A.; Guziewicz, N. A.; Omenetto, F. G.; Kaplan, D. L., Silk-based

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TABLE OF CONTENTS GRAPHIC

Encapsulated CRP degradation rate

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Enhanced Stabilization in Dried Silk Fibroin Matrices - ACS Publications

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