Optimizing Molecular Weight of Lyophilized Silk As a Shelf-Stable

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Optimizing molecular weight of lyophilized silk as a shelf-stable source material Jonathan A. Kluge, Brooke Kahn, Joseph Brown, Fiorenzo G Omenetto, and David L Kaplan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00556 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 8, 2016

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ACS Biomaterials Science & Engineering

Optimizing molecular weight of lyophilized silk as a shelf-stable source material Jonathan A. Kluge12, Brooke Kahn1, Joseph Brown1, Fiorenzo G. Omenetto1, David L. Kaplan1,3

1

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

2

Vaxess Technologies, c/o Lab Central, 700 Main Street, Cambridge MA, 02139, USA

3

Department of Chemical Engineering, Tufts University, 4 Colby Street, Medford, MA 02155, USA

KEYWORDS: silk, stabilization, recovery, biologics

ACS Paragon Plus Environment

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ABSTRACT

Storage of silk proteins in liquid form can lead to excessive waste from premature gelation, thus an alternative storage strategy is proposed using lyophilization to generate soluble and shelfstable powder formats for on-demand use. Initial solution stability studies highlighted instabilities of higher molecular weight silks that could not be resolved by solution modifications such as autoclaving, pH increases, dilution or combinations thereof. Conversely, shelf-stable lyophilized stock powders of silk fibroin of moderate to low molecular weights, were developed which could be fully constituted even after one year of storage at elevated temperatures. Increasing dried silk powder loading in aqueous solution facilitated increased silk solution concentrations – here up to 80 mg/mL solubility was demonstrated across a range of formulations. Powders generated from silk solutions with higher molecular weight distributions were less soluble than moderate or lower molecular weight versions, despite no differences in their solution glass transition temperatures. Instead, the aggregation and β-sheet content of lyophilized higher molecular weight stock solutions were identified as the cause of the reduced powder solubility by circular dichroism and dynamic light scattering analyses. The solubility and molecular weight profiles of all formulations investigated were preserved after storing the lyophilized materials over 1 year, even at 37°C. No long-term powder stability behaviors were influenced by the addition of a secondary drying step in the lyophilization procedure, suggesting that this protocol could be scaled without the burden of lengthy process times. Taken together, these findings provide a very flexible and potentially cost-saving approach to producing shelfstable silk fibroin stock materials based on the use of moderate to lower molecular weight lyophilized preparations. This utility is demonstrated with the formation of silk material formats from the stored powders, including films, gels, and salt-leached porous scaffolds. In turn, a more


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efficient system allowing full re-solubilization will enable stockpiling powder for on-demand usage and for deployment of dried silks for application demands in field settings.


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1 Introduction

Silkworm silks have been a mainstay of the global textile industry, with approximately 60,000 tons1 of raw silk produced annually around the world supporting a $5B industry.2 Silk-based sutures remain a dominant medical device, with 1,000s of tons produced and used annually.3 Recently, silk has emerged from these longstanding native fiber-based applications to new biomaterial systems building upon this feedstock of renewable raw protein material. The expanding list of applications for silk-based materials reflects the versatility of the protein polymer in meeting a diverse range of technical specifications. Silk-based solutions can be used to generate fibers, films, gels, sponges, foams, blocks, and particles. These formats range from biomedical utility in the form of implantable biomaterials4 and pharmaceuticals,5 to components in more technical formats like devices and sensors.6

Processing native silkworm cocoons into usable materials first requires separation of the core fibroin protein from the cladding sericin proteins, typically involving immersion in boiling sodium carbonate solution in a process called de-gumming,7 followed by disruption of the crystalline structure to generate solutions of the proteins. The native protein that makes up the core of the fibers is silk fibroin, which is formed as a dimer, composed of a heavy ~390kDa chain and a light ~25kDa chain.8 Depending on the time of exposure to the heat during the degumming step, the final silk fibroin protein polydispersity per molecular weight distribution can be tuned.9 Further, control over the polymorphic structure of silk in solution and solid state impacts solution stability and water solubility,10 a process influenced by physiochemical parameters including shear, pH, concentration, and ionic charges, as well as time. These time-


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dependent changes on silk polymorphism have a direct consequence on the material properties generated from these solutions.

In order to exploit new material uses and technological applications for reconstituted silk fibroin, a major limitation is the challenge in stabilizing aqueous fibroin in solution. When stored at 4°C, aqueous fibroin remains in a liquid state for up to 3-4 months, prior to the onset of a sol-gel transition characterized by rapid increases in beta sheet structure.11 This transition occurs much faster at ambient temperatures.12 Several solution features such as high pH and lower silk fibroin solution concentrations delay this sol-gel transition time frame.13 Therefore, new approaches are needed to circumvent silk fibroin liquid storage requirements in order to provide a more readilyavailable and stable feedstock of processed silk fibroin proteins.

Here we report new shelf-stable lyophilized silk materials generated from low and moderate molecular weight silk protein compositions, which overcome the long-standing solution stability limitation with silk solution preparations. As a dried and room temperature-stable format, these new options are not only more reliable by avoiding the eventual gel formation process, but also reduce storage space requirements and improve cost-effectiveness by removing cold storage expenses. The reconstituted materials are assessed for long term storage, reconstitution yields, and functional materials formation to determine the potential impact of this new approach to the field of silk supply chains.


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2 Materials and Methods 2.1 Silk solution preparation Silk fibroin solution was prepared as reported previously.14 Briefly, pure silk fibroin was extracted from Bombyx mori cocoons by boiling the cocoons in a sodium carbonate solution (0.02 M) (Sigma-Aldrich, St. Louis, MO) for 10, 20, 30 or 60 minutes to remove sericin. The purified silk fibroin was solubilized in aqueous lithium bromide (9.3 M) (Sigma-Aldrich, St.Louis, MO) for 4 hours at 60ºC. The 10 minute extracted (10 min) silk fibers were dissolved in lithium bromide solution at 15% wt/v, while 20, 30 and 60 minute (20 min, 30 min, and 60 min, respectively) extracted fibers were dissolved at 20% wt/v. The silk solutions were dialyzed using 3.5 kDa Mw cutoff Slide-A-Lyzer™ Cassettes (Life Technologies) against deionized water for 48 hours. The concentration of the silk solution was determined by drying a known volume of the solution and assessing the mass of the remaining solids. This protocol resulted in a 6-8% wt/v silk solutions. These silk solutions were stored at 4°C prior to use, which was within 1 week for each of the following studies unless otherwise specified. Following purification, some silk solutions were also sterilized using a standard 20 minute liquid autoclave cycle at 121°C (15 psi pressure) using 30 mL solution in a 100 ml glass vials covered with aluminum foil, as previously reported.14

2.2 Silk solution aging study Silk solutions of 10 min, 20 min, and 60 min were prepared from ~8 w/v% stock, diluted to 1% or 4% wt/v in either Milli-Q water or 1X PBS (using 10X PBS buffer pH 7.4, Life Technologies Cat# AM9624), which contains 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, and 2.0 mM KH2PO4. Half of the groups were autoclaved and half were not (i.e. left as-processed). All 24


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formulations were stored in UV-transparent 96-well microplates (Corning, Catalog #3635) at N=3 aliquots for 1 year at 4°C, 22°C and 37°C. At twice-weekly intervals, the solutions were measured for absorbance (optical density, OD) at 550nm using a plate reader (BioTek Synergy HT, Winooski, VT) in order to define the onset of gelation, following prior methodology.12 The OD values at 550nm for each formulation were different at baseline and at equilibrium as a function of concentration and processing (e.g., autoclaving, PBS), so all high-concentration autoclaved groups were normalized to average week 1 absorbance values for data presentation purposes.

Table 1: Freeze-Drying Conditions Step

Temp [°C]

Ramp [°Cmin-1]

Freeze

-45

0.8

480

N/A

Primary Dry

-20

0.2

2400

100

4

0.2

620

100

Secondary Dry

Hold Vacuum [min] [mTorr]

2.3 Preparation of lyophilized silks Silk solutions of 10 min, 20 min, 30 min, and 60 min were prepared starting from ~8% wt/v stock solutions, diluted to 4% wt/v in Milli-Q water. Then 1.5mL samples were aliquot into 2 mL Eppendorf tubes and transferred to a VirTis Genesis 25L Super XL Freeze Dryer (SP Scientific, Stone Ridge, NY), utilizing a protocol previously described.15 Table 1 summarizes the freeze drying conditions. Briefly, samples were frozen from ambient 22°C temperature to 45°C (0.1°C·min-1 ramp) and held for 480 min, followed by a ramp to -20°C at 0.2°C·min-1. Primary drying was conducted at -20°C and 100 mT vacuum for 40hrs (sufficient to register pirani pressure < capacitance manometer pressure).16 If secondary drying was employed, then


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samples were heated to 4°C at 0.2°C·min-1 and held for 620 minutes. Following either primary (P) or primary and secondary drying (P+S), the samples were then stored at -40°C at 800mT pressure until the recovery protocol was initiated, typically 2-3 hrs. Samples were capped prior to use or storage. Temperature-modulated differential scanning calorimetry (TM-DSC) was used to evaluate the sub-zero glass transition temperature of the four solutions (10-60 min extracted) in order to validate the lyophilization cycle conditions shown in Table 1. Briefly, a hermetic pan was filled with 10 µL of 4 w/v% solution, sealed, and placed on a Q100 TA Instruments DSC opposite a water reference of equivalent weight. Samples were equilibrated at 4 °C, then frozen to -45 °C at a rate of -0.8 °C·min-1, followed by a heating ramp of 2.0 °C·min-1 to 20 °C modulated at +/0.32 °C per minute. Reversing heat flow [W/g] thermographs were analyzed by TA Instruments Universal Analysis Software. Tg’ was defined by the midpoint of the temperature range bounded

Table 2: Study Designs Study Name

Processing Variables

Extract Time Loading Concentration Autoclaving Extract Time Stability Lyo Condition Autoclaving

Mass [mg]

Vol. H2O [µL]

Max Load [w/v %]

20

980

2.0

40

960

4.0

80

920

8.0

15

985

1.5

by the tangents to relatively flat regions of the reversing heat flow curves. 2.4 Loading Study: Solubility Characterization of Lyophilized Silks Lyophilized silk was pulverized using a Benchtop Analytical Mill (Cole Parmer, Vernon Hills, IL) for 15 seconds, and collected powder was weighed using an analytical balance (Mettler Toledo, Billerica, MA) according to the “Loading Study” described in Table 2. All samples were


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added to Milli-Q water sufficient to bring the final mixture volume to 1mL, assuming the silk powder would displace 1mL per 1g mass (e.g. 960 µL water was added to 40 mg powder to generate 4% w/v solutions). The mixtures were vortexed for 10 seconds, and centrifuged for 10 minutes at 10,000 x g in fresh 2 mL Eppendorf tubes. In order to quantify solubility of the silk powder in water, 250 µL of the resultant supernatant was cast onto polydimethylsiloxane (PDMS, General Electric RTV615, Momentive) surfaces, allowed to air dry, and the residual dried mass was measured using an analytical balance for silk w/v% measurements, as had been done for initial solution preparations.

2.5 Fabrication of Porous Salt-Leached Scaffolds from Reconstituted Solutions In order to generate homogenous 500-600µm pore-sized silk scaffolds an aqueous solution approach was used,17 requiring the combination of defined 500–600 µm NaCl particles (Sigma) and 6 wt/v% reconstituted silk solutions from lyophilized powders in a 2:1 ratio (i.e. 4g salt particles for 2mL of reconstituted silk solutions). Sifted NaCl salt particles were combined with silk solutions in polyethylene cylindrical molds (Ø = 1.8 cm, height = 2.0 cm). In order to achieve a concentration of silk ≥ 6 wt/v% for the 10 min, 20 min, and 30 min solution cases, different silk powder:water ratio loadings were used (i.e. 120 mg: 880 µL, 100 mg: 900 µL, 80 mg: 920 µL, respectively). For this study lyophilization and reconstitution steps followed the mass loading vs. solubility approach described above. The cylindrical molds containing silk solutions and salt were covered and left at room temperature for 48 h. After 48 h, the cylindrical molds were immersed in distilled water for 2 days to dissolve away the NaCl, leaving behind the porous silk scaffolds.


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2.6 SDS-PAGE Gel electrophoresis was used to determine the molecular weight distribution of silk fibroin solutions and the supernatant of reconstituted solutions after solubilization of silk powder under the different temperature storage conditions and extraction times. The electrophoretic mobility of the fibroin molecules was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For each condition, 0.15% (1.5 mg/mL) silk fibroin solution was loaded into a 3-8% Tris Acetate gel (NuPAGE, Life Technologies, Grand Island, NY). The gels were run with one of two high molecular weight ladders as a reference (HiMark and Mark12 Protein Standards, Life Technologies) and stained with a Colloidal Blue staining kit (Life Technologies). Analysis of gels using ImageJ software is shown in Supplemental Figure 1. The use of gel permeation chromatography as an orthogonal approach to confirm the distribution of molecular weight is shown in Supplemental Figure 3. 2.7 Reconstituted Solution Structure by DLS and CD Additional samples were prepared in order to study liquid state structure changes and aggregation of silk solutions following reconstitution for all as-processed and autoclaved formulations, using a 2 w/v% final loading (i.e. 33, 25.5, 25, and 20mg powder loadings into a 1mL final volume for the 10, 20, 30, and 60 min groups, respectively). Loading ratios followed results of the previous “Loading Study”, and as such mixtures were also vortexed for 10 seconds, centrifuged for 10 minutes at 10,000 x g in 2 mL Eppendorf tubes, and supernatant removed immediately for analysis. Original stock solutions were prepared at 2 w/v% for comparison, labeled as (S). These stock solutions were then centrifuged as above (10,000 x g) and then measured again, labeled as (C). Dynamic Light Scattering (DLS) experiments were performed on a ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY) and


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data analyzed using Particle Solutions Version 2.5 software. A laser with wavelength of 659 nm and 90° scattering angle detection was used. An effective diameter [nm] was determined from the average of three consecutive measurements. Samples were diluted 200x (to 0.01 w/v%) and circular dichroism (CD) spectra were collected using an AVIV Biomedical Model 410 CD spectrometer (Lakewood, NJ), as previous described.18 Stock solutions or lyophilized and reconstituted (lyo) samples were used from both as-processed (AP) or autoclaved (AC) solutions. Following dilution, solutions were immediately loaded in a 1.0 mm quartz cell (Hellma Analytics, Plainview, NY) within a temperaturecontrolled cell holder. CD wavelength scans were conducted 3 times at 25°C between 210 and 260 nm using 0.5nm steps. Signal intensity data is presented after a 7-point smoothing factor and normalization on a 0 to 1 scale.

As-Purified Solutions 10 + 20 + 30 + 60 min

Lyo

Primary Dry Only

Autoclaved Solutions 60 min

Primary + Secondary Dry

4C, 22C, 37C Storage

Prep Analysis

Grind powder, weigh, solubilize

SDS-PAGE, dry film weight

Figure 1: The process flow diagram for the stability study of silk extracted for 10, 20, 30, or 60 minutes. Mass loadings are shown in Table 1 and the freeze-drying conditions are shown in Table 2.

2.8 Long-Term Stability Study Lyophilized samples stored at 3 different temperatures (4°C, 25°C, 37°C) were removed at designated time points (day 0, 6 months, 1 year). As described above, two different


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lyophilization protocols were used to generate dried silk for stability studies: one using just freezing followed by primary drying and a second using both primary and secondary drying steps. While as-processed 10-60 min formulations were used, the only autoclaved formulation used for the stability study was the 60 min group since it was the lowest reported molecular weight distribution studied here. For all groups, a 1.5 w/v% reconstituted silk solution was generated at each time point requiring 15 mg of dried powder. Following storage, samples for the “Stability Study” were ground and solubilized as per the “Loading Study” and analyzed by SDS-PAGE or dried down to films for solubility measurements (Figure 1). At the 1-year time point, the resultant gels run on SDS-PAGE were further analyzed as described in the supplemental methods.

2.9 Statistical Analyses Data are expressed as mean ± standard deviation (SD). Statistically significant differences were determined by one- or two-way analysis of variance (ANOVA) and the Tukey post-test. Statistical significance was accepted at the p < 0.05 level and indicated in the figures as *p< 0.05 or **p< 0.01.


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3 Results 3.1 Silk solution aging study Silk solutions of varying i) boil time, ii) concentration, iii) autoclave cycle (vs. as-processed), and iv) content of 1x PBS were stored for 1 year at either 4°C, 22°C, or 37°C in order to evaluate the cooperative effects of known solution modifiers12-13 on long-term rates of gelation. Samples gelled faster at high concentrations or without PBS (or the combination thereof), consistent with previous findings (Figure 2).11-12, 19 At 4°C, higher molecular weight resulted in faster gelation rates at each respective formulation (concentration, PBS). During the 365 day study, the only groups that did not gel at 4 °C were the as-processed 60 min 1% PBS group and the autoclaved 20 min 1% PBS, 60 min 4% and 1% PBS groups. By 1.5 years, the only group that had not yet gelled at 4°C was the autoclaved 60 min 1% PBS group (data not shown). All silk solutions stored at higher temperatures (22°C and 37°C) universally gelled faster than their 4°C stored counterparts, on the order of days in the case of high Mw groups to months for low Mw (low concentration) groups. At 37°C, all groups gelled within 80 days except the 60 min 1% PBS group, which required 120 days. In summary, the ability to decrease the concentration or molecular weight of the solutions (via control of the extraction time or autoclave treatment) and/or to raise the pH (from ~6.8 to 7.4) can significantly extend the shelf life of silk solutions, although none of these strategies are effective at delaying gelation beyond 3-4 months when stored at ambient or higher temperatures.


13


10 min 20 min 60 min

A 3

1% PBS

4% PBS

1%

4%

4°C Storage As-Processed

Absorbance (OD)

2.5 2 1.5 1 0.5 0 0

50

100

150

200 250 Days

B 10 min

20 min

450

400

400

350

350

300 250 200 150

350

400

60 min

300 250 200 150

100

100

50

50

0

0 1%

1% PBS

4%

4% PBS

1%


100

120

Days to Gel

140

80 60 40

1% PBS

4%

4% PBS

22°C Storage Autoclaved

120

Days to Gel

300


450

Days to Gel

Days to Gel


20

100 80 60 40 20

0

0 1%

1% PBS

4%

4% PBS

1%

37°C Storage As-Processed 70

140

60

120

50

100

40 30 20 10

1% PBS

4%

4% PBS


Days to Gel

Days to Gel

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 20

0

0 1%

1% PBS

4%

4% PBS

1%

1% PBS

4%

4% PBS

Figure 2: (A) Optical density changes (at 550 nm) of the as-processed fibroin solutions at 200 µL in a 96 well plate at 4°C. Fibroin was extracted for 10 min. (green), 20 min. (red), or 60 min. (blue). Two concentrations, 1 w/v% (squares) or 4w/v% (triangles), were used with 1xPBS (open symbols) or without (closed symbols). Each set of data is truncated to the last two points for improved visualization. N=3 samples per data point. (B) Raw data from (A) was reduced to the average time to gel for each formulation. Three samples were taken for each condition and averages and standard deviation displayed. Groups that required >450 days to gel are indicated by an arrow.


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3.2 Silk Solution Molecular Weight and Glass Transition Temperatures The molecular weight distributions of silks extracted for 10-60 minutes (as-processed) and after autoclave cycle are shown in Figure 3. As shown previously, 9 silks extracted for longer time

Figure 3: A) SDS-PAGE of supernatants dissolved from the 20mg loading group (7.5 mg of total protein) was run under reducing conditions on a 3-8% Tris-Acetate Gel (Invitrogen). The 9 ladder positions correspond to Mw markers 500, 290, 240, 160, 116, 97, 66.3, 55, and 40 kDa, from top to bottom (HiMark™, Life Technologies).(B) Reversible heat flow thermographs resulting from temperature-modulated differential scanning calorimetry (TMDSC) measurements of as-processed silk solutions (4 w/v%, 10µL) inside hermetic pans.


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periods showed progressively lower molecular weight distributions, and this was even more advanced by 20-minute autoclaving treatment. The range of molecular weights estimated by comparing gel mobility to known molecular weight ladders was similar to the range of molecular weights estimated by comparing the elution of silk from a size exclusion column to monodisperse protein standards, although SEC estimated a larger sub-population of lower molecular weight fragments compared to SDS-PAGE analysis (Supplemental Figure S3). Despite extraction time decreasing molecular weight, there was a negligible effect on the solution glass transition temperature (Figure 3) as measured by liquid DSC in the sub-zero regime.

3.3 Solubility of Lyophilized Silks of Different Boil Times and Concentrations Having observed the practical bounds of storing silk solutions at high concentrations, we investigated the ability to prepare silk dry and reconstitute to high concentration as an alternative solution preparation method. Samples of 4 w/v% silk solutions from the four extraction times were lyophilized, with or without first autoclaving the solutions, then reconstituted to varying target concentrations (2%, 4%, 8% w/v). Lyophilized samples were pulverized into a powder using an analytical mill, distributed to known loading weights (20, 40, and 80 mg, respectively) and vortexed in the presence of water in order to dissolve. As a summary, Table 2 lists the silk masses and water volumes used to generate reconstituted solutions of various concentrations for this “Loading Study”. Figure 3 shows silk powder dissolving behavior in terms of final solution concentration, which was measured by pipetting out the dissolved supernatant and allowing the sample to air dry. This concentration was normalized to the expected concentration (e.g., 20, 40, and 80 mg/mL, respectively) and was represented as a percentage in order to define solubility.


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Figure 4 A) Solubility of silk powders of different mass loadings (20 mg, 40 mg, 80 mg) generated from lyophilized silk solutions of various extraction times (10-60 min). Four samples were taken for each condition and averages and standard deviations are displayed. % solubility is defined as the measured supernatant w/v concentration divided by theoretical loading (namely 20, 40, and 80 mg/mL, respectively) x100. (B) Photographs of salt-leached porous silk scaffolds formed from as-processed stock solutions or reconstituted powders for three (10-30 min) extraction conditions.


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Extraction time significantly affected the solubility of the powders, independent of loading mass with longer boil times increasing solubility in a dose-dependent manner. There was no statistically significant solubility difference between powders generated from autoclaved solutions and as-processed solutions, although the autoclaved groups were on average more soluble and allowed near 100% solubility even at the highest 80mg loading condition explored in this study. The ability to use the reconstituted solutions in order to generate solid silk-based materials requiring higher concentration feedstock was also studied. Following prior downstream methodology,17 porous scaffolds were generated containing gross morphologies comparable to prior studies comparing the different effects of boil time effects on scaffold fabrication.9 Images of these scaffolds are shown in Figure 4. As before, 60 min solutions failed to form robust scaffolds, instead generating incomplete and weakened structures, while 20 min and 30 min reconstituted solutions generated ideal self-standing scaffolds comparable to prior results.17

3.4 Structure of Reconstituted Solutions Films generated for solubility (i.e. dry weight) measurements as above were evaluated by FTIR spectroscopy to confirm that film secondary structure was not influenced by the mass loading or extraction time used to prepare the lyophilized materials. The Amide I and II regions showed characteristic peaks centered around 1650 cm-1 and 1525 cm-1 for the as-cast films, suggesting a largely amorphous “Silk I” conformation (i.e. non-physically cross-linked or crystallized) (Supplemental Figure S4). However, following treatment of these films with 90% v/v methanol for 1 hour, the Amide I region was shifted right and included a small shoulder at 1700 cm-1, while the Amide II shifted to a broader shoulder at 1535 cm-1. These methanol-induced changes


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are due to the formation of β-sheet structures characteristic of a “Silk II” confirmation (physically crosslinked and crystallized) and demonstrate the ability to further process reconstituted silks formed using this new method. Solutions generated from reconstituting the silk powder at 2 w/v% were analyzed by circular dichroism (CD) following 200x dilution in order to evaluate changes in secondary structure from the lyophilization procedure and subsequent reconstitution protocol. As shown in Figure 5, all as-processed (AP) and autoclaved (AC) stock solutions had near perfectly overlaid CD spectra for each extraction condition. With decreased extraction times, especially apparent for the 10 min group, there was a general decrease in the local minima observed at 199 nm and increase in the local minima observed around 217nm, suggestive of an increase in β-sheet content.12 This peak was nearly non-existent in the 60 min groups. However, when lyophilized powders were reconstituted via vortexing, 10 min and 20 min (i.e. high Mw) groups developed small but consistent enhancements of β-sheet formation, while these were noticeably absent for 30 min and 60 min (i.e. low Mw) formulations.


19


Stock Solutions 1

0.8 0.6 0.4

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0.8 0.6 0.4


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Figure 5: Circular dichroism spectra of stock silk solutions before lyophilization (A-D) and solutions of reconstituted silk after lyophilization (E-H). Each graph shows the spectra of both silk solution “as processed” (AP, solid line) and after autoclaving (AC, dotted line). Minutes refer to the length of time for silk extraction. Typical peak assignment for β-sheet structure is at 199 nm and 217nm (see reference 12).

The aggregation behavior of the various formulations were analyzed by dynamic light scattering


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(DLS), and results are shown in Figure 6. At baseline, the as-processed stock (S) solutions contained large effective diameters with shorter extraction times (10 and 20 min), consistent with the more prevalent formation of aggregates in these solutions and general cloudier appearance compared to the higher extracted counterparts. Autoclaved stocks, however, contained aggregates around 600nm independent of formulation. Centrifuging solutions resulted in pelleted aggregates in the sample tubes and universally correlated with significantly decreased effective diameters. Compared to centrifuging stock solutions, the effect of lyophilizing, reconstituting powders, and then centrifuging the water/silk mixtures resulted in supernatants with generally low effective diameters (i.e. ranging from 100-500nm).

Figure 5: Effective diameter of silk fiboin stock solutions (Stock), centrifuged stock solutions (Centrifuge), and reconstituted silk after lyophilization (Reconstitute) as measured by Dynamic Light Scattering. 10-60 min extracted solutions taken “as-processed” and “autoclaved” were analyzed. Three samples were taken for each condition and averages and standard deviations are displayed.


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3.5 Long-Term Stability Studies Having shown that lyophilized silk powder could be used to prepare reconstituted silk solutions with similarities to the original feedstock solutions, we then set out to evaluate the long-term shelf stability of these various formulations over the course of 1 year. Table 2 shows the ratios of silk to water used for study and Figure 1 shows the sample preparation methodology for this “Stability Study”. Data for the 6 month time point can be found in the Supplemental Figures. Figures 7 and 8 shows the solubility and molecular weight distribution, respectively, of silk fibroin solutions obtained from lyophilized silk stored at 4°C, 22°C, and 37°C for 1 year.

Figure 7: Solubility of silk powders at day zero and stored at 4°C, 22°C, and 37°C for 1 year based on 15 mg/mL powder loading (i.e 1.5 w/v % theoretical concentration). Powders were generated from lyophilized silk solutions of various extraction times (10-60 min). Primary drying (left) and primary and secondary drying (right) lyophilization protocols were evaluated. Three samples were taken for each condition and averages and standard deviations are displayed.

Consistent with the loading study at concentrations of 20-80 mg/mL, the 15 mg/mL loading utilized in the stability study resulted in less soluble powder generated from higher molecular weight feedstocks in a dose-dependent manner. Also consistent with the findings at 6 months (see Supplemental Figures S5 and S6), samples stored at 1 year did not generally change in solubility behavior (except at 4°C for 20 min, 30 min primary drying and 60 min autoclaved


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secondary drying where the solubility increased by ~0.5%). Likewise, the molecular weight distribution remained unchanged over time (Figure 8). For all dissolved silk powders examined by SDS-PAGE densitometry at 1 year, the percentage of silk fibroin having molecular weight of 200 kDa or higher decreased as the degumming time increased (Figure S2), as previously described.9 Meanwhile, the percentage of silk fibroin having molecular weight of no more than 120kDa increased as the degumming time increased. Silk extracted for 60 minutes (e.g., the lowest molecular weight group in this study) was comprised of no more than 15% of total weight exceeding 200 kDa (confirmed by SEC analysis, Figure S3), and at least 50% of the total weight between 3.5 kDa and about 120 kDa. This behavior did not change with storage temperature or lyophilization condition.


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Figure 8: Supernatants from dissolved powders stored for 1 year corresponding to Fig. 7 were analyzed by SDSPAGE as in the “solubility study”. Both set of lanes 1-5 and 6-10 correspond to the five formulations put on stability (10, 20, 30, 60, 60A). The 7 ladder positions correspond to Mw markers 200, 116.3, 97.4, 66.3, 55.4, 36.5, and 31 kDa, from top to bottom (Mark 12, Life Technologies).


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4 Discussion

The goal of this study was to evaluate different storage methods of silk fibroin in order to determine methods of feedstock preparation for on-demand utility. Purified silk fibroin is customarily stored as a solution in a refrigerator at 4°C; however, liquid storage is a dynamic, time-limited process, whereby subtle changes in turbidity increases and viscosity decreases occur over the course of weeks to months, until ultimately a gelation occurs.11 Moreover, the shear sensitivity of silk in solution and its amphiphilic nature (e.g., bubble-forming nature) limit the distribution of the raw material in this liquid format. These changes are concentration-dependent and reflect the gradual formation of intra- and/or intermolecular hydrogen bonds.20 While, for many applications, this limited shelf life does not prohibit use on the batch scale, eventual gelation results in waste and can compromise efforts to scale up the process. An alternative strategy for long-term recovery not explored here would be the use of chaotropes20 to stabilize or dissolve gelled materials, although the added processing time needed to de-salt these formulations and the potential to interact with other embedded biologics makes this approach impractical for many applications.

We observed that by either reducing the molecular weight or decreasing the concentration of the silk solution we could extend the solution shelf life of the silk fibroin from weeks/months to nearly 1 year, which provides a starting point for optimization. Indeed, we also demonstrated that pH and sterilization control provide another option for optimization for certain formulation needs. One limitation of this study was the limited range of concentrations and molecular weights employed which can be extended well beyond the current parameter space (both higher and lower) and may be used as a suitable feedstock supply for a subset of applications.


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As opposed to options of air/vacuum drying or spray drying the silk solutions, we chose to sublimate water via lyophilization in order to maximize solubility of the stored material by minimizing the exposure to the air-liquid interface during elimination of residual water. Air- or vacuum-assisted drying of silk can result in the formation of partially insoluble mixtures of Silk I (predominantly random coil) and II (β-sheet) structures.21 Sublimation is a more appropriate water removal method for silk-based storage formats because it results in insoluble Silk I structures22 due to a lack of freezing-induced crystallization when performed below the solution glass transition temperature (Tg’).23 In turn, Silk I can be easily converted into silk II structure on-demand by temperature changes,12 solvent effects,24 and other physiochemical stresses18, 25 in order to generate useful insoluble material formats. Despite limited theoretical structure changes during lyophilization, we still observed some insoluble material after aggressively dispersing and dissolving the powders across all formulations. The ratios of insoluble-to-soluble fractions did not appear to change as powder loading increased. This suggests that, instead of reaching a saturation point in the loading ranges studied here, for all the material in solution there is a fixed fraction that participates in the self-assembly/ aggregation process during lyophilization or reconstitution. We estimate this fraction to be about 40-50% for the 10 min extracted groups ranging to ~5-10% for the 60 min extracted groups used in this study.

We observed significantly different solubility behaviors of lyophilized powders across silk extraction times (10 min – 60 min), independent of powder loading amount. Historically, increased extraction time (i.e. lower molecular weight, Mw) has correlated with faster drug diffusion in silk films, supporting the idea that it is easier to wet the lower Mw systems when dried down.26 Here we used a generally well-accepted freezing rate (0.8°C/·min-1), freezing


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temperature (-45°C shelf), and primary drying temperature (-20°C shelf) during lyophilization in order to accelerate drying while preventing product collapse. Higher Mw (i.e. more native-like) silk solutions may be more susceptible to self-assembly when forced to concentrate during freezing as a result of ice crystal expansion. As a route to offset these extraction-dependent solubility behaviors one can consider varying the lyophilization parameters to modify residual water or minimize the effects of freezing-induced amorphous-to-crystallization transitions in the silk via changes to the rates or set points of freezing or primary drying.27

Designing the loading study relied on only a subset of available reconstitution techniques that could have been explored in order to modify solubility. As a starting point, we chose to pulverize the lyophilized dry materials using a 15-second milling process that could have been further modified for either longer treatments or different grinding techniques in order to overcome the inherent solubility issues of powders.28 Vortexing was used to disperse the powder in solution due to convenience and ease of use, although this process is known to induce insolubility in silk.18 Indeed, by comparing the CD spectra of solutions prepared by vortexing reconstituted powders to the original stock solutions, there was an increase in β-sheet formation independent of extraction conditions for the vortexed samples. These changes are small, however, compared to the changes that typically accompany a more significant shear-induced phase changes via sonication or extended vortexing.18,

25

This was confirmed by studying films cast from

reconstituted supernatants of all powder loading ratios, which also revealed no β-sheet formation (as contrasted by the same films crosslinked by methanol treatment). We can also see that the effective diameter of particles in reconstituted solutions is significantly higher than centrifuged stock solutions in the case of in 30 and 60 min extracted groups, suggesting a higher prevalence


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of aggregates formed from vortexing. Thus, future studies will investigate alternative mixing strategies via orbital shaking or brief heating in order to improve solubility while minimizing aggregates which, in turn, could increase post-reconstitution shelf life.

The results of the loading study highlighted additional drawbacks of long-term storage of the high molecular weight silks in that the solubility of powders was quite poor (~50%) even at low 1.5-2% loadings. Interestingly, while we hypothesized that the insoluble fraction of the low molecular powders was predominantly high molecular weight in content, it appeared that the soluble supernatant was instead composed of the full distribution of molecular weights of the original feedstock. Although not shown, the pellets taken from the centrifuged samples were also fully distributed in molecular weights. We therefore speculate that it is the cooperative effects of both high- and low-molecular weight fractions of the lyophilized materials in generating insoluble secondary structures. CD data suggests that the as-processed silks of high molecular weight (10 min) contain some β-sheet signatures prior to lyophilization that are not found in lower molecular weight systems (20-60 min). Likewise, we confirmed via DLS that there are large aggregates already formed in the lower molecular weight groups prior to lyophilization, that likely come out of solution during the reconstitution process. Therefore, the inherent insolubility of high molecular weight silks may be a persistent challenge without chemical modification. With appropriate consideration for end usage compatibility, alternative strategies may involve addition of surfactants, buffers, or molecules of known charge to lyophilized solutions in order to inhibit intramolecular interactions favoring insolubility.


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The lyophilized silk powder materials stored for 6- and 12-months proved nearly indistinguishable from the samples analyzed at day zero, suggesting that this method could be used to allow room temperature storage for at least 2-3 years, or perhaps indefinitely. A comparable system is gelatin, which is another high molecular weight structural protein manufactured via lyophilization as a powder form and likewise widely used in biomedical contexts (e.g. tissue engineering substrate, stabilizer, implantable materials, etc.).29 Dry gelatin stored in airtight containers at room temperature remains unchanged for many years, according to the manufacturers (Sigma, CAS RN 9000-70-8). Also similar to our findings, higher Mw gelatin powders are more difficult to dissolve, and this was attributed to the higher aggregateforming potential.29 Indeed, years of experience utilizing gelatin dried powders may provide insights into routes to improve the lyophilized silk systems.

This new system of drying silk fibroin via lyophilization provides a new option to long-term storage of field-ready silk materials for a wide variety of storage and shipment needs based on its lightweight nature (e.g. ~25x weight reduction for a 4 wt/v% solution) and ease of reconstitution, especially in lower molecular weight forms. We also expect that the dried formats will allow silk to be deployed in field settings without the need for refrigeration based on the stability of the dried products at elevated temperatures. Although not investigated here, one can consider other additives or active biologics for the lyophilized formulations that can enhance solubility of the silk component or the utility of the approach for controllable deployment.15, 30-31 Based on the approaches described here, we expect that this technique will be further refined in order to offer a more robust and high yield method to secure both high- and low-molecular weight silks to fill a range of future needs.


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5 Conclusions

Low and moderate molecular weight silk fibroin-based materials, generated by control of extraction/purification conditions, provide unique stability and solubilization properties suitable for storage and recovery applications. These features are distinct from higher molecular weight feedstocks, which are more prone to premature β-sheet formation in solution state, in turn limiting solution shelf life and promoting aggregation during drying. Moreover, as large quantities of high-concentration silk fibroin are required for certain downstream processes, the scalable nature of the approach was demonstrated. Storing dried lyophilized materials at elevated temperatures for upwards of one year did not inhibit the baseline solubility profiles and resulted in retention of molecular weight distributions. Future efforts to improve the system in terms of solubility could utilize alternative lyophilization conditions and/or reconstitution protocols. Taken together, these findings offer a new strategy to form silk feedstocks free of refrigeration for a variety of downstream applications.

Supporting information

Detailed methods and images describing the densitometry analysis of SDS-PAGE results for the 1-year stability study (Figure S1). Results of densiometry analysis of SDS-PAGE results for the 1-year stability study (Figure S2). An orthogonal size exclusion chromatography technique was used to measure molecular weight distribution of silk extracted for 60 minutes as a case study (Figure S3). Methods and results for FTIR structural analysis of silk films generated from different loadings (per the “loading study”) (Figure S4). Result of solubility testing of powders stored for 6 months and resultant SDS-PAGE (Figure S5 and S6, respectively).


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Corresponding Author *Corresponding author. Phone: 617-627-3251; Fax: 617-627-3231; Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources We thank the NIH P41 EB002520, AFOSR and DTRA for support of this work. ACKNOWLEDGMENT Thanks to Dr. Qiaobing Xu and Dr. Ming Wang for their assistance with DLS measurements. Thanks to Melissa Adler for her help with biophysical analyses. Thanks to Nishant Jain, Kathryn Kosuda and Carter Palmer at Vaxess Technologies for their help with size exclusion chromatography. ABBREVIATIONS PBS, Phosphate Buffered Saline; FTIR, Fourier Transform Infrared Spectroscopy; SDS-PAGE, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; PDMS, polydimethylsiloxane; optical density, OD; NaCl, sodium chloride; KCl, potassium chloride; Na2HPO4, sodium phosphate dibasic; KH2PO4, potassium phosphate monobasic; cm, centimeters; min, minutes; ºC, degrees Centigrade; psi, pounds per square inch; mL, milliliter; µL, microliter; wt/v, weight / volume; mM, millimolar; UV, ultraviolet; mg, milligram; g, grams; mT, millitorr; kDa, kilodalton; SEC, size exclusion chromatography.


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30. Guziewicz, N. A.; Massetti, A. J.; Perez-Ramirez, B. J.; Kaplan, D. L. Mechanisms of monoclonal antibody stabilization and release from silk biomaterials. Biomaterials 2013, 34, 7766-75, DOI: 10.1016/j.biomaterials.2013.06.039. 31. Pritchard, E. M.; Dennis, P. B.; Omenetto, F.; Naik, R. R.; Kaplan, D. L. Review physical and chemical aspects of stabilization of compounds in silk. Biopolymers 2012, 97, 479-98, DOI: 10.1002/bip.22026.


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For Table of Contents Use Only

Optimizing molecular weight of lyophilized silk as a shelf-stable source material Jonathan A. Kluge, Brooke Kahn, Joseph Brown, Fiorenzo G. Omenetto, David L. Kaplan


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Optimizing Molecular Weight of Lyophilized Silk As a Shelf-Stable

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