Solvent-Free Strategy To Encapsulate Degradable, Implantable Metals in Silk Fibroin

Abstract

Implantable electronics hold enormous clinical potential for diagnosis and treatment of neurodegenerative and cardiac diseases and abnormalities. Transient devices are attractive alternatives to conventional silicon electrodes, as they can provide short-term electrical stimulation/recording followed by complete device degradation, mitigating the need for removal surgeries. Packaging transient metals is inherently challenging as they degrade upon contact with aqueous conditions. Development of new transient metal packaging strategies is a critical step toward transient device development. In this fundamental work, a solvent-free compression molding approach to encapsulate magnesium, a resorbable metal, in silk fibroin protein is reported. Silk fibroin was selected because of its processing versatility, desirable mechanical properties, compatibility with biological environments, and controllable degradation behavior in aqueous environments. The silk/magnesium composites were fabricated via compression molding, followed by water annealing to modify the secondary structure of the silk protein matrix to tune physical properties. Transient composite properties as a function of water annealing time are presented, which elucidate synergies between silk physical properties and degradation kinetics of the encapsulated magnesium, information useful in the design of multifunctional, transient metal-based constructs.

Graphical Abstract

INTRODUCTION

A critical need in the field of bioelectronics lies in developing implantable devices that can integrate with biological tissue, especially those with spatially controlled signaling capabilities for diagnosis and treatment of neurodegenerative and cardiac conditions and diseases.^1–3 Transient probe-type electronic devices are attractive alternatives to conventional silicon devices,^4,5 if they can fully degrade in physiological environments in a prescribed time frame, thus mitigating the need for high-risk removal surgeries. Precise control of device lifetime is an essential aspect of transient implant technology, where lifetime needs may range by multiple orders of magnitude (hours to years). For example, electrodes implanted in the subthalamic nucleus for deep brain stimulation will have a different degradation time frame requirement than a temporary pacemaker, where functional clinical use is considered to be >5 years and 1–30 days, respectively.^6–9 A wide range of transient devices have been fabricated, where applications include electrophysiological skin sensors,¹⁰ drug delivery vehicles,¹¹ neural sensors,¹² thermal therapies,¹³ and batteries.¹⁴ Regardless of application, both chemical identity and material format of the conductive agent and the packaging are crucial to device transience and function.

Conductive agents for implantable transient electronics include degradable semiconductor forms, such as silicon nanowires or porous microneedles, ^10–13 and resorbable metals such as magnesium (Mg)/Mg-based alloys, zinc, iron, tungsten, and molybdenum (some in conjunction with silicon components).^13–20 Conductive agent passivation is essential to control corrosion and ultimate degradation.^21,22 Furthermore, it is desirable to combine conductive agents with one or more soft material packaging strategies to customize the biological environment/device interface in terms of both chemical and mechanical properties.^23,24 “Multifunctional” materials that offer both conductivity and tunable moduli capable of matching favorably with soft tissue environments include intrinsically conducting polymer systems (e.g., those based on poly-aniline, poly-pyrrole, or poly-thiophene derivatives).^25–28 Even by modifying conductive polymer systems to “enhance biocompatibility”, achieving wide-range compatibility between this class of polymers and biological systems remains challenging.²⁷ Because semiconductors and metals as discussed above typically have higher conductivity and well-established degradation pathways in comparison with most inherently conductive polymers, their overall utility in transient devices is usually superior to that of conductive polymers.

Selecting an appropriate transient device packaging strategy is inherently challenging. For an implantable device, the packaging should be degradable and compatible with the surrounding biological environment. Multiple natural^16,29,30 and synthetic^12,31–33 polymer packaging strategies have been explored in the context of transient electronic devices, often requiring complex lithography. Such lithography-based fabrication methods often utilize a solid polymer substrate. This approach offers limited utility for fabricating nonplanar devices. It is often difficult to employ traditional methods of polymer encapsulation (dip coating, drop-casting) because semiconductor/metal components degrade rapidly when subjected to the aqueous processing conditions required for other methods to couple the transient metal component. Thus, one or more pretreatment steps are often required to passivate the semiconductor/metal surface before encapsulation. Very few methods utilize one-step, nonaqueous-based processing (a recent report from Kim et al. is a notable exception).³⁴ Developing simpler transient metal packaging strategies to both control device lifetime and tune interactions at the device/tissue interface represents an area in need of innovation.

In this work, we report a compression molding fabrication strategy to passivate resorbable metals. We utilize this strategy to develop proof-of-concept composites containing silk fibroin protein and Mg wires for which we present comprehensive materials characterization and performance analyses. Mg was selected as a proof-of-principle transient material because it has been integrated into numerous electronic and nonelectronic implantable devices,^35–38 owing to favorable electrical properties (electrical conductivity = 2.3 × 10⁷ S/m, resistivity = 4.4 × 10⁻⁸ mΩ), general compatibility, and natural presence in the human body.³⁹ Furthermore, Mg can be alloyed with varying metals to enhance stability.³⁷ For example, Mg and Mg-based alloys subcutaneously implanted in mice exhibited varying stability in vivo, yet demonstrated wide-range biocompatibility, where the heart, kidney, lung, skin, and liver were not abnormally affected during the corrosion period of two months.³⁸ Mg wires were encapsulated using silk fibroin, which was chosen as a natural polymeric material owing to its well-established processability, cytocompatibility, nonimmunogenic nature, tunable degradation, bioresorbability, and controllable mechanical properties.^40–45 Although silk has been employed for bioelectronics development in various formats and functions,⁴⁶ its role in this work is distinctive from previous reports as it serves three unique purposes: (1) is a platform for solvent-free encapsulation of metal components with 3D geometries; (2) acts as a barrier with tunable physical properties to control transient metal degradation; (3) softens rapidly upon introduction in an aqueous environment to provide favorable mechanical matching at the device/biological environment interface. Using these silk/Mg composites, we validate a route to tune transient metal corrosion through altering silk crystallinity. Notably, this fabrication strategy represents a versatile platform that could be extended to encapsulate devices with a wide range of geometries and compositions and could be used to fabricate future devices as the encapsulation process occurs in a single solvent-free step.

METHODS

Silk Processing.

Regenerated silk fibroin (hereafter referred to as silk) solutions were prepared on the basis of previously published protocols with minor adjustments.⁴³ Briefly, 5.0 g of Bombyx mori cocoons (Tajima Shoji, Yokohama, Japan) were cut into 1 cm² pieces and degummed by boiling in 0.02 M sodium carbonate (Na₂CO₃, Sigma-Aldrich) for 30 min to remove sericin. During the degumming process, fibers were gently pulled apart at frequent time intervals to ensure uniform processing. Fibers were removed from boiling Na₂CO₃ immersed into room temperature deionized (DI) water (resistivity ≥18 MΩ). After rinsing for several minutes under running DI water, fibers were stretched and placed on aluminum foil in the fume hood at ambient temperature to dry overnight. Dry silk fibers were packed tightly into a clean Pyrex beaker, to which a 20% w/v solution of 9.3 M lithium bromide (Sigma-Aldrich) was added. The beaker was tightly covered with aluminum foil and placed in a 60 °C oven for 3–4 h or until the white silk fibers were no longer visible and the solution was transparent. The solution was cooled slightly before transferring to cellulose dialysis tubing (molecular wight cut-off = 3500 Da, Spectra/Por), and filled tubing was placed in DI water. A stir bar was added to slowly agitate the dialysis water to promote salt extraction without applying shear stress to the silk. Water was refreshed six times over 3 days. Following dialysis, the silk solution was transferred into 50 mL Falcon tubes and centrifuged three times (20 min, ~12 700g, 4 °C) to remove impurities. Regenerated (dialyzed and centrifuged) silk concentration was determined by dispensing a known volume in a weigh boat and measuring mass after drying at 60 °C. Standard concentrations ranged from 6% to 8% w/v. Silk solutions were stored in tightly capped 50 mL Falcon tubes at 4 °C for up to three months.

Materials Fabrication.

To prepare lyophilized scaffolds, a 2% w/v silk solution was prepared by diluting a regenerated silk solution with DI water and gently inverting to homogenize. Multiple lyophilized silk substrates were prepared simultaneously by pipetting the dilute silk solution into a plastic cube-shaped mold and bringing the base of the mold in contact with an aluminum plate cooled by liquid nitrogen for ~10 min to induce unidirectional freezing.⁴⁷ This fabrication technique creates a freezing front beginning at the interface between the metal plate and the mold, resulting in ice crystal formation perpendicular to the metal plate. Ice crystals are surrounded by sheets of silk fibroin. Constructs were then placed in a lyophilizer. This process yielded cube-shaped silk scaffolds with an aligned morphology (~1.3 cm × 1.3 cm × 1.3 cm), where dimensions were selected for convenience in subsequent processing steps. Lyophilized silk scaffolds were stored in a vacuum desiccator at room temperature. To fabricate composite Mg/silk materials, an as-received Mg wire segment of ~0.3 cm in length (Goodfellow USA, 99.99% as-drawn, d = 125 μm) was inserted into a lyophilized silk scaffold parallel to the direction of alignment. Lyophilized silk was inserted in a 13 mm die assembly (PerkinElmer) perpendicular to silk alignment and compression molded by applying a 700 MPa load for 5 min using a pellet press (Specac). This was the maximum pressure obtainable by our pellet press machinery, resulting in dense packing of the silk to produce a mechanically robust construct that could be easily handled. The composite relaxed throughout the 5 min molding process, necessitating frequent reloading of the sample to the targeted pressure. This high-throughput, solution-free process yielded robust, semitransparent silk filmsi (d = 1.3 cm) between 250 and 350 μm thick with masses between 40 and 50 mg. Blank silk films (i.e., those without Mg wires inserted) were also prepared as controls. A schematic outlining the silk processing and compression molding methodology is presented in Figure 1a.

Figure 1.

(a) Schematic overview of silk/Mg composite fabrication, starting from B. mori silk cocoons and ending with compression molded composites. SEM images of the lyophilized silk construct before (b) and after (c) compression molding demonstrate differences in morphology between the lyophilized anisotropic silk and resulting silk films.

Water annealing was utilized to modify silk construct physical properties, as previously reported.^43,44,48 Briefly, pressed silk materials were placed in a vacuum chamber (Isotemp Vacuum Oven, Model 281A) along with a shallow dish containing 250 mL of room temperature tap water. The silk materials did not directly contact the dish of water. Vacuum chamber pressure was decreased to 25 in. H₂O (~0.06 atm) and maintained for 1, 6, or 15 h, which created a humid environment at ambient temperature. Notably, water annealing is not expected to induce Mg wire degradation as the Mg wires are protected by a native oxide layer prior to compression molding. Furthermore, water annealing does not provide anionic conditions necessary for Mg hydrolysis, which has been previously published as the major mechanism underlying Mg corrosion.^36,49–51 After water annealing, films were dried at room temperature in the fume hood and were stored at room temperature until further use. Nonwater annealed controls were also utilized (non-WA, water annealing time = 0 h).

Characterization.

Morphology of lyophilized silk scaffolds, pressed silk films, and Mg wires was assessed using scanning electron microscopy (SEM, Zeiss EVO MA10). Most silk-containing specimens were coated with a thin layer of gold to mitigate charging (Emitech SC7620). Cross sections of silk films (with and without Mg wires embedded) were exposed by slicing through the entire construct using surgical scissors (Henry Schien), creating a clean edge to image via SEM. Film thickness was measured using on-board software (Zeiss SmartSEM). Top-down views of Mg wires embedded in pressed silk films were visualized using the bright-field setting on a Macro Zoom System Microscope (Olympus, MVX10) and captured using the on-board software (Olympus cellSens Dimension version 1.8.1).

X-ray photoelectron spectroscopy was utilized to evaluate surface chemistry of silk/Mg composites and as-received Mg wires (Thermo Scientific K-Alpha XPS system). X-rays were generated using a monochromatic aluminum Kα source (energy = 1.4866 keV, line width = 0.85 eV). An X-ray spot size of 50 μm was utilized to collect all survey spectra (pass energy = 200 eV, step size = 1 eV, 5 scans/sample). For silk/Mg composites, survey spectra were collected in the form of a line scan, with 5 equidistant points over the top of the wire. The first and last points were off of the wire, which were treated as silk-only controls. Depth profiling was performed on Mg wire samples using an argon sputtering gun in 30 s etch time increments (energy = 3000 eV). Onboard Thermo Advantage software was employed for all data analysis.

Silk material secondary structure was determined using Fouriertransform infrared spectroscopy with an attenuated total reflection accessory (FTIR-ATR, Jasco 6200 spectrometer coupled with a PIKE MIRacle accessory containing a germanium crystal). FTIR-ATR data were acquired by coadding 32 scans over the wavenumber range of 600–4000 cm⁻¹ with 2 cm⁻¹ resolution. The amide I region (1720–1580 cm⁻¹) was deconvoluted using the second derivative method and known literature values,^52–54 revealing contributions from four secondary structure classes: β-sheets, α-helices, β-turns, and random coils.

To evaluate swelling characteristics of compression molded silk films, samples were weighed to determine dry mass $\left.(m_{\text{dry}\text{initial}}\right)$ and then immersed in 37 °C PBS for various amounts of time (30 min, 3 h, 24 h). After immersion, surface moisture was removed from the sample using a Kimwipe (Kimberly-Clark, USA), and the wet mass of the sample ${(m}_{\text{wet}})$ was measured. Swelling ratios were calculated using eq 1.

$$\text{swelling}\text{ratio}=\frac{\left(m_{\text{wet}}-m_{\text{dry}\text{initial}}\right)}{m_{\text{dry}\text{initial}}}$$ (1)

After immersion in PBS, films were removed and dried in a 60 °C oven overnight. Mass loss was quantified via percentage change using eq 2.

$$\text{massloss}=\frac{\left(m_{\text{dry}\text{initial}}-m_{\text{dry}\text{final}}\right)}{m_{\text{dry}\text{initial}}}$$ (2)

Magnesium degradation was assessed by immersing samples in 150 mM sodium chloride in scintillation vials for 3 weeks. Three separate replicates of each type of sample were used. Aliquots were collected as a function of immersion time after gentle mixing via pipet and used in a colorimetric coupled enzyme absorbance assay following manufacturer recommendations (Sigma-Aldrich). Briefly, magnesium chloride standards and aliquots from immersion tests were dispensed into individual wells of a clear-bottomed 96-well plate. The total sample size for each type of sample was 9 (N = 9, three technical replicates on three of each type of sample at each time point). The well plate was incubated at 37 °C for the duration of the assay in a temperature-controlled plate reader (Molecular Devices, SpectraMax M2). After 10 min of static incubation, absorbance data at 450 nm were collected every 5 min for 40 min with 3 s of shaking before each read. A standard curve was constructed using magnesium chloride standard absorbance values, and sample concentrations were determined by converting absorbance at 450 nm to concentration using a linear regression of the standard curve.

Atomic force microscopy (AFM) was utilized to measure stiffness of compression molded silk films in the as-prepared and hydrated state. The nanoindentation method used for these experiments has been described in detail in previous publications.^55,56 In brief, a Veeco Dimension 3100 AFM fit with Novascan borosilicate glass particle probes (bead diameter = 10 μm, rated spring constant = 0.6 N/m) was used to collect stiffness data. Indentation response between the AFM probe and the sample can be described using the Hertz model, such that Hertzian theory can be used to calculate Young’s moduli values. The Young’s modulus was calculated for each indentation curve over an entire 2D force volume using a MATLAB code. In these calculations, a Poisson ratio of 0.5 was assumed because protein networks such as silk fibroin obey rubber elasticity. Young’s moduli values presented in this work represent an average of 256 measurements per sample type.

Statistical Analyses.

Quantitative data are presented as the mean ± one standard deviation. A minimum of three technical replicates were analyzed on a minimum of three separate samples (N = 9) unless otherwise noted. One-way analysis of variance tests (ANOVA, significance level = 0.05) with Tukey post-hoc tests were run to compare silk film thickness, silk secondary structure, and swelling/mass loss data. Linear regression analysis was performed on magnesium release data to predict release at extended time points.

RESULTS

Lyophilized anisotropic silk was utilized as a solvent-free material system to fabricate compression molded constructs with tunable mechanical properties. SEM images of lyophilized silk displayed long-range anisotropy (Figure 1b). The fibrillar morphology present in the lyophilized silk precursor was no longer visible upon compression molding (Figure 1c). A representative top-down image of an Mg wire encapsulated in silk via compression molding indicates the wire maintained its macroscale structure (i.e., the wire remained straight and did not bend upon insertion into the lyophilized silk), which was presumably facilitated by the inherent directional porous structure of the silk construct (Figure 2a). Cross-sectional images revealed the wire was fully encapsulated, although slight wire deformation occurred in the direction perpendicular to compression (Figure 2b). This can be clearly visualized by comparing the encapsulated wire and as-received wire profiles, where the former is elliptical and the latter is circular (Figure 2b, inset). Wire encapsulation conformality was assessed via XPS survey data (Figure 2c), where spectra collected directly over the wire displayed identical chemical environments to those collected elsewhere on the silk film (carbon, oxygen, nitrogen with trace silicon). Signal attributed to Mg (expected around 1300 eV) was absent from all spectra, indicating conformal silk film coverage over the wire length.

Figure 2.

Optical characterization and surface chemical properties of silk-encapsulated Mg wires. (a) Representative top-down view of an encapsulated wire after pressing, demonstrating that the wire remains straight upon insertion and compression. (b) Representative cross-sectional SEM image of an encapsulated wire after encapsulation, demonstrating that the wire becomes slightly compressed during processing (blue dashed lines indicate the silk film border, and dashed orange lines indicates the outline of the wire). Compression of the embedded wire is more apparent when compared with the circular cross section of as-received wires (b, inset). (c) XPS survey spectra collected from a representative line scan over the embedded Mg wire, where the cartoon depicts data collection locations relative to the wire position.

After compression molding, constructs were water annealed with the goal of tuning physical properties in an aqueous environment. Comprehensive characterization of compression molded constructs as a function of water annealing time presented in Figure 3, Table 1, and Figure 4 reflect data collected on blank, silk-only constructs (e.g., without a Mg wire inserted). This decision was made to evaluate silk properties separately than that of the composite, as well as to conserve resources. Amide I regions from FTIR absorbance spectra provide qualitative and quantitative assessment of silk secondary structure, which profoundly influences degradation under aqueous conditions (Figure 3a,b).⁴⁴ As observed in Figure 3a, the amide I region shifts from a single broad peak centered at ~1640 cm⁻¹ (for non-WA WA films) to a peak centered at ~1620 cm⁻¹ with a shoulder around ~1655 cm⁻¹ (for 15 h WA films). Amide I region deconvolution reveals differences in secondary structure, most notably an enhancement in β-sheet crystalline structures as a function of water annealing time (Figure 3b). It is important to note that relative error associated with deconvolution decreases as a function of water annealing time. This error likely arises from challenges in performing deconvolution on spectra comprising one broad peak (e.g., for non-WA WA films), when compared to spectra with well-defined peak structures (e.g., for 15 h WA films). In addition to changes in secondary structure, a notable shift in film morphology was observed as a function of water annealing time (Figure 3c). Specifically, non-WA films display a layered structure akin to that of the native lyophilized silk construct (Figure 1a), which shifts to a distinctive morphology comprising round pores upon longer exposure to the water annealing environment (Figure 3c). Mixed morphologies comprising both layered structures and round pores are observed for intermediate water annealing times. Difference in morphology was accompanied by statistically significant differences in silk film thickness as determined by one-way ANOVA (p < 0.0001). A Tukey post-hoc test demonstrated that, although thickness of non-WA films and those water annealed for 1 h were not significantly different (p = 0.325), films WA for 6 and 15 h were significantly thicker than non-WA (p = 0.004 and <0.0001, respectively).

Figure 3.

Water annealing was utilized as a method to tune compression molded film secondary structure, morphology, and thickness. FTIR spectra highlighting the amide I region (a) with corresponding secondary structures from spectral deconvolution (b) are presented, as well as SEM cross-sectional film images (c) with corresponding film thickness measurements (d). All data are presented as a function of water annealing time. In (b) and (d), mean values with standard deviation-based error bars are shown (N ≥ 9), and significant differences are indicated with asterisks (*, determined by one-way ANOVA with Tukey post-hoc test, p ≤ 0.05).

Table 1.

Young’s Modulus of Compression Molded Silk Films as a Function of Water Annealing Time^a

water annealing time (h)	Young’s modulus, as- prepared state (GPa)	Young’s modulus, hydrated state (GPa)
0b	0.87 ± 0.49
1	1.08 ± 0.08	0.74 ± 0.86
6	1.81 ± 0.23	0.56 ± 0.23
15	2.79 ± 0.16	1.52 ± 1.37

^aData are presented for films as-prepared, as well as those hydrated in water.

^bNon-WA WA film completely dissolved upon immersion in water, not possible to determine hydrated state modulus.

Figure 4.

Silk films were immersed in PBS and incubated at 37 °C to evaluate swelling and degradation, quantified by swelling ratios (a) and mass loss (b). Data are presented as a function of water annealing time. Average values with standard deviation-based error bars are shown (N = 9). Significant differences within groups in (a) are indicated with asterisks and pound symbols (* and #, determined by one-way ANOVA with Tukey post-hoc test, p ≤ 0.05).

Adding to the comprehensive characterization of compression molded silk films, mechanical properties were evaluated as a function of water annealing time (Table 1). For as-prepared silk films, AFM nanoindentation data reveal an increase in Young’s modulus as a function of water annealing time, with moduli ranging from ~0.8 to 2.8 GPa. Films in the hydrated state demonstrate a similar trend, with moduli ranging from ~0.7 to 1.5 GPa. To our knowledge, this is the first report where nanoindentation has been used to determine the modulus of compression molded silk films. Thus, values reported in Table 1 are not necessarily expected to match with previously reported data collected on silk films (as-prepared or hydrated) fabricated using other methods.⁵⁷ Interestingly, non-WA films completely degraded when immersed in water such that mechanical properties could not be measured using this analytical method. The hydrated state data have high variability, likely arising from technical challenges when collecting these data. For each water annealing condition, as-prepared films have a higher average modulus than their hydrated state counterparts.

As stated previously, one crucial function of the silk encapsulation layer is to provide a physical barrier to embedded transient electronic components. The silk and magnesium components are expected to degrade independently under physiological conditions. Thus, evaluating how each constituent degrades in aqueous environments, as well as synergies present in degradation behavior of the composite silk/Mg constructs, is essential. Silk film swelling ratios and mass loss were utilized as two complementary metrics to evaluate silk behavior in aqueous environments (eqs 1 and 2, Figure 4). Notably, non-WA films were challenging to handle with tweezers and became gelatinous in texture after only 30 min of immersion, which was also observed for 1 h water annealed films after 3 h of immersion. Thus, contributions from opposing swelling and dissolution processes are likely present for 0 and 1 h water annealed films, which contributes to the relatively large error associated with swelling ratios for these films regardless of immersion time. After 30 min of immersion, all films had a positive swelling ratio, indicating the wet mass was greater than the dry mass. Additionally, swelling ratios at 30 min displayed a decreasing trend as a function of water annealing time. Subsequent immersion caused a statistically significant decrease in swelling ratios for 1 h water annealed films, which is likely more reflective of partial dissolution rather than a decrease in swelling. A similar trend was observed for non-WA films, although a large error associated with swelling ratios likely impeded statistical significance. Dissolution is corroborated via mass loss, wherein non-WA films and those water annealed for 1 h display nonzero mass loss regardless of immersion time. Films water annealed for 6 and 15 h exhibited small but nonsignificant gains in swelling ratio as a function of immersion time, suggesting relative film stability of up to 24 h. Mass loss data support swelling ratio observations, indicating no change in mass as a function of immersion time for films water annealed for 6 and 15 h.

Corrosion of Mg wires embedded in silk films was quantified by immersion in aqueous salt solution and measuring resulting Mg²⁺ concentration in the bulk solution via colorimetric assay (magnesium corrosion occurs via oxidation and hydrolysis in chloride ion-containing environments, which is well-documented in the literature^36,49–51). Magnesium ion concentrations as a function of immersion time are displayed in Figure 5a. The nonencapsulated wire control displayed an expected burst release after 1 day of immersion, with Mg²⁺ concentration plateauing to ~0.12 nmol/μL after 4 days. Silk encapsulation resulted in a lower Mg²⁺ concentration than that observed in wire-only control regardless of water annealing time, for up to 4 days of immersion. After 8 days of immersion, 0 and 1 h water annealed constructs demonstrated a statistically similar Mg²⁺ concentration to the wire-only control. Constructs water annealed for 6 and 15 h, however, displayed statistically lower Mg²⁺ concentrations than the wire-only control after immersion for 21 days (3 weeks). Linear regression analysis was performed on 15 h water annealed materials to predict the time to reach an identical Mg²⁺ concentration as that observed for the wire-only control. Extrapolation of linear regression results indicated the 15 h water annealed samples would reach the same Mg²⁺ concentration as the wire-only control only after ~35 days of immersion. Notably, the silk-only control demonstrates negligible absorbance over the immersion time frame used for this study. Altogether, these data illustrate the ability to customize transient metal corrosion/degradation kinetics using a compression molded silk encapsulation strategy.

Figure 5.

Silk/magnesium construct response to immersion in 150 mM NaCl. (a) Magnesium ion concentration, determined via colorimetric coupled enzyme absorbance assay, as a function of immersion time. Concentration data are shown for silk-encapsulated magnesium samples for four unique water annealing times, as well as magnesium-only and silk-only controls. Linear regression performed on the 15 h WA data set is omitted for clarity; regression line: y = 0.004x + 0.009, R² = 0.976. (b) Representative cross-sectional SEM images of silk/magnesium constructs at 0 day, 4 day, 1 week, and 3 week immersion time points (scale bar = 100 μm). (c) Percentage of magnesium wire remaining in the composite as a function of immersion time, corresponding with data presented in panel a. Percentage of wire remaining was estimated from Mg wire control absorbance data.

One alternative interpretation of the magnesium wire degradation data presented in Figure 5a is that Mg wires could undergo complete corrosion and degradation on a shorter time scale than suggested by data in Figure 5a, whereby corrosion products including Mg²⁺ could become entrapped in the silk matrix instead of diffusing into the bulk media. Thus, measured Mg²⁺ concentrations in the bulk media would not directly correlate to the extent of wire degradation. To investigate this possibility, silk/Mg wire samples were removed from solution at different stages of immersion, rinsed with DI water, cut to reveal cross-sectional segments, and dried under ambient conditions.

After imaging samples, it is clear that Mg wires encapsulated in non-WA silk films remain visible for up to 1 week of immersion. The wire is no longer visible after extended immersion, and a void is left behind in the space the wire had previously occupied. The void can be visualized in the bottom left cross-sectional image of Figure 5b, where the silk film appears to have folded over itself. Wires encapsulated in films water annealed for 15 h, however, remain visible for at least 3 weeks of immersion (bottom right cross-sectional image of Figure 5b).

Composite cross-section imaging supported our original hypothesis that magnesium wire degradation can be tuned by altering physical properties of the silk matrix. This finding can be visualized in Figure 5c, where the percent of magnesium wire remaining in the composite is estimated by comparing corresponding absorbance data in Figure 5a with absorbance values for magnesium-only controls. Non-WA constructs exhibit near complete magnesium degradation in 14 days (2 weeks). After immersing 6 and 15 h WA constructs for the same time frame, magnesium wires are not completely degraded. Rather, they have exhibited less than 50% corrosion via oxidation. Wires in these constructs exhibited ~35% corrosion after immersion for 3 weeks, the maximum immersion time utilized in this study. Collectively, immersion study data demonstrate the ability to customize magnesium degradation time via alteration silk matrix physical properties over time frames ranging from 2 weeks to over 1 month.

DISCUSSION

Here, we utilized a compression molding strategy to encapsulate transient metals in silk. Protein-based compression molding-based fabrication holds promise for creating tunable, transient implants for bioelectronics applications. This work extends the utility of silk in transient devices by presenting a one-step solvent-free process to prepare silk/magnesium composites, effectively avoiding aqueous and organic solution-based processing previously reported.⁴⁶ Postprocessing constructs using a completely “green” water vapor annealing process provided a simple route to control composite degradation. Previous reports on water annealing studies with silk present a proposed mechanism to explain enhanced β-sheet crystallinity as a function of water annealing time, whereby this process effectively decreases the protein glass transition temperature, resulting in enhanced protein chain mobility.^43,44,48 Enhanced chain mobility explains the enhancement in β-sheet crystallinity observed for compression molded silk films as well (Figure 3b).

Differences in cross-sectional network morphology and thickness upon water annealing (Figure 3c,d) are less likely attributed to chain mobility and more likely attributed to water uptake of the silk network. The anisotropic, porous silk starting material was prepared via unidirectional freezing and lyophilization, whereby the silk protein has a primarily amorphous structure with a relatively low β-sheet content. The humid water annealing environment likely facilitates diffusion of water into the amorphous silk network, effectively swelling the material. Because the crystallinity (β-sheet content) of the silk network increases concomitantly with water annealing time, this swollen structure becomes more crystalline/rigid and is thus able to be maintained after drying.

It is likely that silk secondary structure, mechanical properties, film morphology/pore structure, and film degradation synergistically control degradation behavior of the encapsulated magnesium wire (Figure 5). Magnesium corrosion occurs via oxidation followed by anion-mediated hydrolysis, which is well-documented by previous publications.^36,49–51 The aforementioned properties of the exterior silk film, therefore, control diffusion of the anion-containing aqueous solution into the silk network, thereby controlling magnesium degradation. The non-WA and 1 h water annealed composites exhibit the fastest magnesium degradation rates (Figure 5a), as well as relatively low crystallinity (Figure 3) and relatively fast silk film degradation (Figure 4). Thus, lower crystallinity allows these silk films to degrade relatively quickly in an aqueous environment and no longer adequately protect the underlying magnesium. Conversely, films that were water annealed for 6 and 15 h exhibit the slowest magnesium degradation rates coupled with relatively high crystallinity and relatively slow silk film degradation. The higher crystallinity present in these 15 h WA films renders them less water-soluble. Thus, these films can offer better long-term protection to the encapsulated wire. Using this approach, the encapsulated wire could be designed to fully corrode from anywhere from 2 weeks to over 1 month. Results presented in Figure 5a are somewhat surprising when interpreted in the context of silk film morphology and pore size. Specifically, pore size increases as a function of water annealing time (Figure 3c), where larger pore size would be hypothesized to provide increased diffusion and corrosion of the Mg wire. This hypothesis is not supported by experimental results, suggesting degradation of the silk film (secondary structure and mechanical properties) plays a more prominent role in controlling degradation of the underlying Mg wire than does film morphology.

Shorter complete magnesium corrosion times (<2 weeks) could be attained by fabricating composites with a thinner silk encapsulation layer, as this layer would provide a smaller barrier to diffusion compared to that in the present study. Conversely, longer complete magnesium corrosion times (>1 month) could be attained by fabricating thicker constructs. Increasing device size, however, will limit application of compression molded constructs as implantable electronics. Alternate strategies to extend the magnesium corrosion time frame could include the use of additional hydrophobic polymers to act as a diffusion barrier, as well as chemical modification of the magnesium wire itself to further passivate its surface (e.g., adding oxide/hydroxide conversion coatings). Although further passivation could offer corrosion resistance, it will simultaneously enhance the resistivity of the magnesium wire. The trade-off between magnesium wire conductivity and protecting magnesium from corrosion is important to consider for future applications.

Developing an understanding of mechanical properties of these composite material systems in the dry and hydrated state is imperative for translation into applications. Notably, all silk materials presented in this work have Young’s moduli that are orders of magnitude lower than conventional Si shanks, which holds promise in terms of reducing the mechanical mismatch between implanted electronics and soft tissues. An additional aspect that is promising for the translation of materials from this fundamental study into a multifunctional electronic implant is presented in Figure 6. Although this work utilized anisoptropic constructs, which facilitated transient metal insertion parallel to the direction of silk fiber alignment, more recent technology developed in our laboratories utilizes silk powder for compression molding, allowing us to create thinner, more flexible constructs (Figure 6a), to insert wires in multiple directions (such as those shown crossing in Figure 6b), or to laminate silk with 2D metal materials such as magnesium foils (Figure 6c). These preliminary extensions demonstrate promise toward developing devices with a wide range of geometries, thus providing the opportunity to customize devices to meet a given clinical need. We anticipate that using free-flowing powder could be used to encapsulate existing functional transient metal devices and plan to evaluate the efficacy of interfacing currently utilized devices (discussed in the Introduction) with silk using these methods in future studies. Although fabricating a (multi)functional device was not the focus of the fundamental work reported here, silk is effectively a blank canvas for further modification, including chemical modification^58–61 and therapeutic loading,^62–67 both of which can be exploited to enhance the functionality of compression molded silk constructs.

Figure 6.

Demonstration of how the compression molding composite preparation strategy could be used to fabricate materials in additional formats, including (a) flexible constructs in the dry state, (b) constructs with transient metals in multiple directions, and (c) laminates with transient metal foils.

CONCLUSION

A unique solvent-free approach for fabricating silk-based transient constructs was developed. We employed this approach to reproducibly encapsulate magnesium wires as proof-of-concept for transient conductive agents. The physical properties of silk encapsulation layers were successfully tuned via room temperature water annealing, where significant differences in secondary structural motifs were observed as a function of water annealing time. Specifically, films displayed enhanced β-sheet crystallinity after prolonged water annealing, resulting in decreased silk film degradation and enhanced protection of the underlying magnesium. Through proof-of-principle experiments presented here, we demonstrate the capacity to control degradation of both the silk coating and the encapsulated resorbable metal, showcasing the tunability of this compression molded-based method. We anticipate that the versatility of this method will be especially applicable to implantable transient electronic probes, as all parameters of the system (geometry, mechanical properties, surface chemistry, device lifetime) are customizable to fit a given stimulation/recording need. Overall, this fabrication platform holds promise for multifunctional transient device development.