Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Stabilization of RNA Encapsulated in Silk Jiuyang He,†,⊥ Burcin Yavuz,† Jonathan A. Kluge,†,‡ Adrian B. Li,‡ Fiorenzo G. Omenetto,† and David L. Kaplan*,†,§ †
Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States Vaxess Technologies, c/o Pagliuca Harvard Life Lab, 127 Western Avenue, Allston, Massachusetts 02134, United States § Department of Chemical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States ‡
S Supporting Information *
ABSTRACT: The use of mRNA and miRNA as diagnostic parameters and therapeutic agents has drawn wide interest both clinically and in research. However, RNA is a labile molecule, which requires strict storage conditions, often including cold temperatures or dry environments, in order to preserve RNA integrity. Achieving this requires huge costs for storage and added difficulty in transport. To address these issues, we introduce a system to encapsulate and store it longterm in dried silk fibroin matrices. At temperatures up to 45 °C, mRNA samples stored in lyophilized silk matrices showed good stability over 1 week, as measured by real-time PCR to assess transcript recovery. While the presence of the silk interfered with the generation of cDNA required for quantitation at roughly 1% w/v (400:1 silk:RNA mass), this phenomenon was resolved by the use of commercial RNA purification kits for silk concentrations up to 4% w/v. A higher concentration of silk correlated with increased thermal protection. As an alternative to lyophilization, RNA was simply air-dried in the presence of aqueous fibroin to create storage matrices. While air-dried matrices composed of low silk content were not protective, higher concentrations were protective and progressively lost additional water over time of storage because of the overall hydrophobic nature of the system. Taken together, these findings provide a new and potentially simpler method for preserving RNA samples for long-term storage and transportation, acting primarily on a water exclusion mechanism. KEYWORDS: silk, stabilization, RNA, biologics solutions can be stored safely at 4 °C for extended periods of time using the RNAlater products (Qiagen).9 As commercial alternatives to refrigerated options, RNA can be dried on paper, such as FTA cards (Whatman) designed to lyse cellular components and immobilize released nucleic acids on the fibers of a cellulose matrix. RNA can also be dehydrated in the presence of additives (e.g., Biomatrica’s RNAgard/RNAstable, GenTegra’s GenTegra-RNA) in order to protect RNA under ambient conditions.10 These drying techniques primarily protect by inactivating RNase through the action of chelators and inhibitors, but are less effective at protecting RNA in complex samples such as blood at elevated temperatures that might be encountered during transport.11 Stainless steel minicapsules have also been used to stabilize RNA via protection from exposure to the atmosphere,5 but the use of laser welding and atmosphere control during the encapsulation process makes this method complex and costly and offers limited field utility. Recently, silica was used to store RNA in microcapsules that protected from reactive oxidative species and RNase,12 but
1. INTRODUCTION Research with ribonucleic acids (RNA) has grown in importance over the last few decades, from the original understanding of RNA as a messenger, carrying genetic information between DNA to protein, to a versatile functional family of nucleic acids involved in gene regulation1 and with enzymatic roles.2 Moreover, quantification of the transcriptome using quantitative real-time polymerase chain reaction (qRTPCR), microarrays, or next-generation sequencing has become well-established in research and in clinical and diagnostic uses.3 However, despite this growing utility for the study and use of RNA, the labile features of the molecule remain problematic. This labile nature is due to rapid oxidation,4 the presence of hydroxyl groups on the ribose ring resulting in susceptibility to hydrolysis,5 and the ubiquity and robust nature of ribonucleases (RNases).6 RNA stability and integrity are important for downstream applications and diagnostic uses, where high sensitivity, specificity, reproducibility, and accuracy are required for proper analysis.7 To preserve their structure, RNA samples must be maintained at low temperatures, which can lead to economic and logistical burdens.8 To address this issue, several methods for RNA storage have been reported. RNA in tissues and © XXXX American Chemical Society
Received: February 21, 2018 Accepted: March 18, 2018
A
DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
2.3.1. Agarose Gel Electrophoresis. Agarose gels (1% w/v) were prepared according to the manufacturer’s recommendations (BioRad). Silk samples containing RNA (25.0 μg/mL, 8 μL) were mixed with 2 μL of 5× Electrophoresis Sample Loading Dye (Bio-Rad) and loaded into wells, which were run alongside a ladder for comparison (1 kb Molecular Ruler, Bio-Rad). The gels were run using a 1× TAE running buffer at 120 V for 30 min, stained for 15 min with 250 ng/ mL ethidium bromide (Bio-Rad), and then visualized and photographed using the Foto/UV 26 system with Foto Analyst/PC Image software (version 10.21, Fotodyne Incorporated, Hartland, WI). 2.3.2. Quantitative Real-Time Polymerase Chain Reaction. Silk samples containing RNA (12.5 μg/mL, 10 μL) were used to synthesize cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) using an MJ Research PTC100 thermal cycler following the manufacturer’s instructions. Run conditions were as follows: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and finally storage at 4 °C until samples were retrieved. cDNA and TaqMan Gene Expression Master Mix were combined with forward and reverse primers for β-actin (Applied Biosystems) following the manufacturer’s instructions. For final analyses, the 25 μL reaction volume in each well contained 1.5 μL of 1:50 diluted cDNA, 25 nM each forward and reverse primers, and 12.5 μL of SYBR Green Master Mix. qRT-PCR was performed using 96-well sealed optical plates in a 7300 Real Time PC System (Applied Biosystems). Run conditions for qRT-PCR were as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The data from each qRT-PCR plate were processed using SDS Software (Applied Biosystems) and a fluorescence threshold (CT) value was generated for each sample and compared with non-silk controls stored at −80 °C. 2.4. Purification of Silk-Laden RNA Samples To Remove Protein. A study was conducted in order to eliminate silk protein interference in the qRT-PCR assay. Silk-laden RNA samples as described above were treated with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and then purified according to the manufacturer’s instructions. In order to confirm protein removal from the use of the kit columns, a bicinchoninic acid (BCA) assay (Thermo Fisher) was run on samples before and after column treatment following the manufacturer’s recommendations. A side study was conducted in order to evaluate the source of interference of residual silk in the quantitation of RNA. This was approached by doping in silk (at 4 and 1% w/v) either into the RNA as per above or into cDNA generated from non-silk-doped RNA samples, followed by typical qRT-PCR quantitation. cDNA doped with silk was also subsequently treated using a DNA cleanup kit (Qiagen) following the manufacturer’s instructions prior to qRT-PCR analysis. In order to confirm adequate purification of RNA in the presence of silk, fragment analysis was performed. RNA was again prepared to a concentration of 25.0 μg/mL with silk at final concentrations of 4, 2, 1, and 0% w/v. In addition to non-silk RNA controls (0% w/v), additional controls containing silk (4% w/v) but without RNA were prepared both with and without RNeasy purification (denoted as “− Silk” and “+Silk”, respectively). Samples were diluted 10× in running buffers prior to analysis on a Fragment Analyzer Automated CE System (Advanced Analytical, Ankeny, IA). PROSize 2.0 analytical software was used to assess the RNA quality number (RQN), based on the RNA distribution of each sample compared to a standard curve derived from a ladder with the following sizes (bp): 15, 200, 500, 1000, 1500, 2000, 3000, 4000, 6000. 2.5. Evaluating RNA Stability in Lyophilized Silk Matrices. Samples containing RNA (50 μg/mL) and silk from 4 to 0% w/v (60 μL) were transferred to a VirTis Genesis 25L Super XL freeze-dryer (SP Scientific, Stone Ridge, NY) in which all of the 96-well plates were in direct contact with a cooling plate via aluminum blocks in order to facilitate accurate and homogeneous thermal regulation. Samples were frozen from ambient temperature (22 °C) to −45 °C (0.1 °C/min ramp) and held for 480 min, followed by a ramp to −20 °C at 0.2 °C/ min. Primary drying was conducted at −20 °C and 100 mT vacuum for 40 h (sufficient to register a Pirani vapor pressure less than the capacitance manometer pressure25). For secondary drying, samples
the outer layer of the silica required 4 days to grow, also limiting use for the processing of time-sensitive samples. Encapsulation of labile compounds in silk fibroin is a stabilization technique that has emerged because of several useful attributes of the protein. Silk is a high-molecular-weight amphiphilic protein composed of a block copolymer structure dominated by large hydrophobic domains interspersed with small hydrophilic regions.13 The predominantly hydrophobic nature (due to the long domains of repeated glycine-alanine) and high glass transition temperature (T g ) result in thermodynamic stability of compounds in silk materials exposed to elevated temperature and moisture.14 Silk also has a low pI (∼4.2), enabling it to maintain conformational stability at neutral pH and in the presence of buffer salts for many months while providing structural rigidity needed for sample transport when in solid formats. These formats include lyophilized foams,14,15 films,16 hydrogels,17 and microspheres.18 These qualities distinguish silk fibroin as a suitable biomaterial to stabilize plasma and serum proteins in complex samples, as well as enzymes and antibiotics.19−21 Several reviews have summarized these findings as well as the current understanding of the mechanism of stabilization.11,22,23 The objective of the present study was to assess the utility of silk for the stabilization of mRNA and to initially optimize silk− mRNA interactions toward this goal. Since the addition of a large amount of silk protein to generate these matrices interfered with RNA quantity and quality measures, we evaluated the effect of a common RNA purification technique on RNA recovery and silk removal. This scheme was applied for all stability studies. We evaluated both lyophilization and air drying to generate silk matrices as high- and low-resource options, respectively.
2. MATERIALS AND METHODS 2.1. Materials. Whole human heart total RNA samples were obtained commercially from Zyagen (San Diego, CA) at a concentration of 1 mg/mL in a solution containing 10 mM Tris and 1 mM EDTA in RNase-free water. Samples were received on dry ice and immediately frozen to −80 °C prior to use. Silk solutions were generated from silkworm cocoons using previously published methods.14 An extraction time of 60 min in Na2CO3 (0.02 M) was used to remove sericin from fibroin, which was then dissolved in LiBr (9.3 M) and desalted over 48 h using dialysis cassettes with a 3.5 kDa cutoff (Pierce, Thermo Fisher).24 RNA was diluted to a working concentration of 25.0 μg/mL in all studies, unless otherwise indicated. Silk solutions and non-silk-loaded controls were diluted with RNasefree water and TE buffer to working concentrations of 10 mM Tris and 1 mM EDTA. 2.2. RNase Characterization and Deactivation in Silk-Laden RNA Samples. A study was performed to evaluate whether RNase contamination was present in the silk solutions and to evaluate strategies for RNase deactivation compatible with silk. Silk solutions at 4 and 1% w/v were prepared for these studies. In order to potentially deactivate RNase, samples were either doped with diethyl pyrocarbonate (DEPC) (0.2%) and subjected to a standard 20 min steam autoclave cycle (121 °C) and sterile-filtered (0.2 μm, Millipore Steriflip PES membrane) or doped with 1 unit/μL RNase inhibitors (SUPERase·In, Ambion). The RNaseAlert test (Ambion) was run according to the manufacturer’s instructions to evaluate RNase in sterile-filtered 4% w/v silk. 2.3. Evaluation of Assay Compatibility of Silk-Laden RNA: Concentration Effects. Silks at concentrations of 4, 2, 1, 0.5, and 0.1% w/v were mixed with whole heart tissue RNA and subjected to agarose gel electrophoresis, qRT-PCR, and fragmentation analysis to evaluate the threshold for silk protein interference. The RNA concentration was varied by experiment, as indicated. B
DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering were heated to 4 °C at 0.2 °C/min and held for 620 min. The samples were then stored at −4 °C at 800 mT pressure until the recovery protocol was initiated (typically 2−3 h). Following lyophilization, an initial set of samples was reconstituted at time zero, and the remaining samples in 96-well plates were entered into a 1 week stability study. Four different storage temperatures were used: −80 °C (control), room temperature (22 °C), 37 °C, and 45 °C (stress conditions). Samples taken off of stability were solubilized in 60 μL of ultrapure water and treated with the RNeasy Mini Kit as per above, followed by qRT-PCR analysis for β-actin transcripts. Samples stored at 37 °C were also analyzed for RNA fragments. 2.6. Evaluation of RNA Stability in Silk Films. For the following film studies, the fibroin was originally boiled for 120 min in order to aid in film solubility.11 Film formulations were diluted from the stock solution to obtain 0−4% w/v silk solutions and then sterile-filtered (0.2 μm, Millipore Steriflip PES membrane). Samples containing RNA (40 μg/mL) and silk (50 μL) were transferred either to sterile 1.5 mL microcentrifuge tubes (0% and 0.25%) or polydimethylsiloxane (PDMS) molds (4%) and left at room temperature overnight for drying. Following film formation, a set of samples were reconstituted on day zero (D0) to evaluate RNA recovery from films, and the remaining samples were entered into a 2 week stability study in which the samples were kept at room temperature (22 °C), 37 °C, or 45 °C. Initial samples on D0 and stability samples on day 14 (D14) were solubilized in 50 μL of ultrapure water and treated with the RNeasy Mini Kit as per above, followed by qRT-PCR analysis for β-actin transcripts. RNA solutions were also incubated under the same conditions as film samples as a control group to determine the effect of silk and film formation on RNA stability. 2.7. Thermogravimetric Analysis (TGA). TGA was performed to evaluate the effects of time and incubation conditions on the water content in silk films. For this purpose, 4% w/v silk solutions were used to cast films. Aliquots of silk solution (50 μL) were transferred to the PDMS molds and kept at room temperature overnight to dry. Films were collected from the molds and immediately analyzed on D0. The rest of the film samples were placed in microcentrifuge tubes and incubated at room temperature (22 °C), 37 °C, or 45 °C for 7 days (D7). TGA was performed on films of known weight (2.04 to 2.43 mg) using a Universal TA 500Q system (TA Instruments, New Castle, DE). Samples were heated to 1000 °C with a step increase of 20 °C/ min under an inert nitrogen atmosphere. 2.8. Statistical Analysis. Data are expressed as mean ± standard deviation (SD). SPSS Statistics 22 software (IBM, Armonk, NY, USA) was used to perform one-way ANOVA (across a range of independent variables) or Student’s t test (for individual group comparisons). Statistical significance was accepted at the p < 0.05 level and is indicated in the figures as *p < 0.05 or **p < 0.01.
Figure 1. Recovery of RNA from silk using different RNase inactivation techniques. Raw CT values correspond to amplification cycle thresholds of β-actin transcripts. The final silk concentration was 4 or 1% w/v. All data represent mean ± SD (N = 3). The solid line represents the baseline CT value (with ±SD shown by the dotted lines) of RNA without any silk treatment. Each * label indicates significance at the p < 0.05 level compared with the baseline CT value.
the detection of RNase with the assay (see Figure S1). No RNase mitigation strategies were therefore explored in any follow-on work. 3.2. Evaluation of Assay Compatibility of Silk-Laden RNA: Concentration Effects. In light of the interferences with the qRT-PCR assay preliminarily observed for high concentration (4% w/v) silk solutions, we sought to identify the threshold levels at which this interference occurred. As shown in Figure 2A, silk titrated as low as 1% w/v eliminated significant interferences with the qRT-PCR assay, with concentrations at or below 0.5% w/v also appearing comparable to the 0% w/v (non-silk) control case. During RNA electrophoresis in which the silk concentration was varied from 0 to 4% w/v concentration, the silk concentration affected the RNA band (Figure 2B). For the 4% silk the RNA band was trapped at the top of the gel, while this interference was attenuated in a dose-dependent manner until roughly 0.1% silk loading. As a check on the source of interference, silk was directly added to RNA as well as the corresponding library of cDNA. When silk solution was added to RNA to a final concentration of 4% w/v, β-actin quantification was reduced; however, when silk was added directly to cDNA, no significant interference was observed (Figure 3A). This result suggested the importance of protein purification as the first step in silk/RNA processing. RNeasy minicolumns were thus used to purify silk/RNA mixtures prior to the reverse transcription step required for RNA quantitation. The total protein content of silk/RNA mixtures was successfully reduced following column purification, independent of the silk concentration (Figure 3B). The quality of RNA following purification was analyzed by a fragment analyzer, and the resulting quality scores are shown in Table 1. The RNA quality number (RQN) is a score between 1 and 10, from most to least degraded; the neat stock solution stored at −80 °C had a score of 4.3, indicating moderate starting quality. As shown in the column of “Non-Treated” sample values, silk interfered with the assay in a dosedependent fashion. However, because of the enrichment of rRNA fragments in all samples, the RNeasy kit purification step universally improved the RQN for all of the silk-laden samples, most significantly so for the 4% w/v samples. Raw data can be found in Figure S3. 3.3. Evaluation of RNA Stability in Lyophilized Silk Matrices. Use of RNeasy purification kits enabled the
3. RESULTS 3.1. RNase Characterization and Deactivation in SilkLaden RNA Samples. The standard silk purification process results in a nonsterile bulk material with unknown starting RNase content; therefore, different sterilization and RNase deactivation methods were applied to silk solutions prior to addition of RNA. The RNase contents of silk solutions were also evaluated. Purified human whole heart RNA was added to silk solutions pretreated with either addition of RNase In (a Life Technologies product), addition of DEPC (a smallmolecule RNase inhibitor), or sterile filtration (0.2 μm filter), and the solutions were assayed by qRT-PCR for the housekeeping mRNA transcript β-actin. While there were no differential effects of any of the sterilization methods on the ability to recover RNA, the concentration determined the ability to fully recover the RNA, as evidenced by the higher CT responses of 4% vs 1% silk (see Figure 1). Silk did not contribute any detectable RNase as measured by the RNase Alert assay, nor did silk at 4% w/v loading completely inhibit C
DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 2. Silk concentration determines RNA recovery. (A) Raw CT values corresponding to amplification cycle thresholds of β-actin transcripts. The final silk concentration was 4 to 0.25% w/v (vs 0% non-silk control). All data represent mean ± SD (N = 3). Each * label indicates significance at the p < 0.05 level compared with the baseline CT value. (B) Agarose gel containing silk-laden RNA. The final silk concentration was 4 to 0.1% w/v (vs 0% non-silk control). The gel was run alongside a 1 kb ladder as indicated, and all lanes were stained with ethidium bromide. The left arrow indicates the location of the sample loading well, and the right arrow indicates the location of greatest putative rRNA band intensity.
Figure 3. Commercial RNA purification strategies can completely resolve silk interferences. (A) Raw CT values corresponding to amplification cycle thresholds of β-actin transcripts. The final silk concentration was 4 or 1% w/v. All data represent mean ± SD (N = 3). The solid line represents the baseline CT value (with ±SD shown by the dotted lines) of RNA without any silk treatment. (B) Total protein contents of silk/RNA mixtures as measured by bicinchoninic acid (BCA) assay. RNA solutions containing silk at concentrations of 8 to 1% w/v were measured neat (Non-Treated) or following purification by RNeasy Mini Kit. All data represent N = 1.
Table 1. Fragment Analysis of Silk-Containing RNAa Non-Treated sample blank 4% w/v 2% w/v 1% w/v 0% w/v
silk silk silk silk
greater susceptibility to elevated temperatures, especially at 45 °C, where the greatest divergence with −80 °C storage was observed (Figure 4B). Moreover, while RNA was also stable in silk-free samples (0%) stored at −80 °C, room-temperature storage compromised these samples by 1 week, and storage at 45 °C rendered RNA nearly undetectable by qRT-PCR. Consistent with PCR-based measures of RNA stability, fragment analysis confirmed the effect of silk addition on the quality of the recovered and stabilized RNA. In keeping with the behavior of silk solutions (Figure S3), lyophilized silks that were solubilized, purified by RNeasy kits, and fragmentanalyzed showed slight decreases in RNA quantity for samples of higher silk concentration, as shown by the more diffuse gel appearance at day 0. However, following 1 week of storage at 37 °C, this trend was reversed, with good preservation of gel RNA landmarks at the highest silk concentration and nearly complete destruction of RNA in the non-silk-laden control samples (Figure 4C). 3.4. Evaluating RNA Stability in Silk Films. Film samples were stored at temperatures up to 45 °C for 2 weeks and subsequently purified using RNeasy kits, and the amounts of RNA in the samples were quantified using qRT-PCR. RNA stability at −80 °C storage was previously established without silk (Figure 4B), so in order to establish film stability, only nonsilk controls (0%) were included under stress conditions (22, 37, and 45 °C). Results for D0 samples showed a small loss in
RNeasy
RQN
28S/18S
RQN
28S/18S
1.00 2.40 3.20 4.10 4.30
0.00 1.40 0.90 0.80 1.00
1.00 4.40 5.80 5.70 4.4
0.00 0.40 0.90 0.90 0.7
a
Liquid samples containing silk and RNA at a range of concentrations were either purified using the RNeasy kit or left untreated. RQN = RNA quality number; 28S/18S = the ratio of the two major rRNA band intensities.
characterization of RNA stability in the context of silk-laden samples of various concentrations. In stability studies, silk solutions were mixed with RNA followed by drying via lyophilization. RNeasy purification showed a significant improvement on RNA recovery at the 4 and 0.25% w/v silk concentration levels, while non-silk controls appeared unaffected by the purification step (Figure 4A). Lyophilized samples were stored at temperatures up to 45 °C for 1 week and subsequently purified using RNeasy kits to evaluate stability. While RNA stored in silk at 4% w/v was relatively insensitive to temperature, RNA in silk-containing samples of lower concentration (0.25 and 0.1% w/v) showed D
DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 4. RNA purification enables stability tracking of silk-laden RNA samples. (A) Raw CT values corresponding to amplification cycle thresholds of β-actin transcripts. Samples were measured neat (Non-Treated) or following purification (RNeasy Kit) at final silk concentrations of 4 to 0.01% w/v (vs 0% non-silk control). All data represent mean ± SD (N = 3). Each * label indicates significance at the p < 0.05 level comparing Non-Treated with RNAeasy Kit. (B) Samples from (A) were stored at the four temperatures shown for 1 week, reconstituted, purified by RNeasy kit, and again analyzed for β-actin transcripts. Each * label indicates significance at the p < 0.05 level comparing thermally stressed samples with the respective −80 °C groups. (C) RNeasy-treated samples from (A) and 37 °C-stored samples from (B) were analyzed by a fragment analyzer, and gels are shown for comparison of relative gel band intensities.
Figure 5. RNA recovery from air-dried silk films. (A) Film recovery immediately after preparation and (B) after 14 days of incubation. Raw CT values correspond to amplification cycle thresholds of β-actin transcripts. All data represent mean ± SD for N = 6 (3 film × 2 analytical replicates). The bar represents a significant difference between solution samples, and * indicates a significant difference between the film and respective solution samples (p < 0.05).
RNA recovery during film preparation and reconstitution (Figure 5A). The results of the 14 day stability study showed that the RNA solution degraded significantly with increased temperature (Figure 5B). Dried RNA samples without silk (0% samples) were found to be more stable than the solution aliquots at lower temperatures but degraded at the same rate as RNA solutions at 45 °C. The data showed that concentrations of silk as low as 0.25% w/v do not effectively stabilize RNA at elevated temperatures. On the other hand, 4% w/v silk films
were able to stabilize RNA significantly compared with solutions and silk-free air-dried RNA (p < 0.05). Moreover, it was observed that RNA was even more stabilized at higher temperatures with a high silk concentration (4% w/v). 3.5. Thermogravimetric Analysis. Film stability data showed that RNA recovery from films on day 0 was slightly worse than that from day 14 samples at room temperature for all silk concentrations. Thus, we decided to evaluate the effects of time and incubation conditions on the water content of the E
DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering films in order to investigate whether the recovery loss of D0 samples was due to the higher water content of the films. TGA data showed that D0 films lost 23.5, 25.1, and 26.6% more total weight compared with D7 samples incubated at room temperature, 37 °C, and 45 °C respectively (Figure 6). D7
RNA extracted from complex samples such as blood or saliva. While silk has inhibitory effects on reverse transcription, there was no direct interaction between silk and the RNA, presumably as a result of charge repulsion. The pI of intact fibroin is 4.2,28 and is therefore negatively charged at neutral pH, similar to RNA. mRNA samples in lyophilized silk are readily dissolvable in water and can be used directly in processes where the silk has no inhibitory effect (e.g., gene delivery) using this format.29−31 Unlike many of the other drying techniques described elsewhere for mRNA preservation, an alternative, simplified air drying approach was also explored for silk using similarly efficacious concentration windows to impart stability. Air-dried silk films at 4% w/v imparted essentially lossless RNA stability for 2 weeks at 45 °C (under stress conditions) compared to −80 °C, which was a 4−8-fold improvement over the stability imparted by air drying without silk protection. By comparison, it was reported that cell-based RNA stored on FTA paper at room temperature or 4 °C for extended time periods was suboptimal compared with samples stored at temperatures less than or equal to −20 °C.32 Since enhancements from silk were realized only at relatively high concentrations, we presume that the silk loading must surpass a threshold necessary for it to act like a cage, shielding the RNA from oxidative stress and thermally induced fast relaxations.27 This simple technique could be useful in limited-resource settings, such as in the field of diagnostics, to preserve reagents or field-collected mRNA in biospecimens.19 The generally low moisture content of these matrices is conferred from fibroin’s generally hydrophobic amino acid structure,33 in stark contrast to stabilizing sugar glasses, which are generally slow to dry under ambient conditions. The silk system described here is also contrasted to air drying on paper (FTA cards), which generally does not act as a shield but instead leaves RNA on the surface of the cellulose fibers, where it is vulnerable to UV and oxidative stresses. The silk system has the added benefit of improving the recovery yield compared with paper, which must be punched and eluted, since the silk samples can be reconstituted in water without dilution (and in theory with the potential for amplification). Taken together, the fundamental and practical benefits of the silk system make it attractive for further exploration as an RNA entrapment, storage, and recovery matrix.
Figure 6. Weight loss of RNA-laden air-dried silk films with increasing temperature. TGA curves of 4% silk films that were incubated at different temperatures are shown. The samples were analyzed right after preparation (D0) and after 7 days of incubation at room temperature (RT), 37 °C, or 45 °C.
samples that were kept at room temperature, 37 °C, and 45 °C showed similar drying profiles and stabilized after 600 °C. These data support the theory that RNA recovery from D0 samples is lower because of the high water content in the films.
4. DISCUSSION In this work, mRNA was stabilized by incorporation into silk fibroin during long-term storage. The technique involves entrapment and immobilization without chemical modifications. A simple and efficient 1 day lyophilization cycle was pursued because of the low collapse temperature (i.e., high Tg, −11 to −13 °C),14 which is in contrast to traditional lyophilized formulations containing a majority composition of sugars (e.g., trehalose).26 The experimental groups that formed a bulk lyophilization cake (∼1% w/v and higher) had better RNA recovery than silk groups with concentrations too low to form cakes, suggesting the importance of the initial drying step on long-term stability performance. It is possible that even greater stability performance could be achieved through additional formulation efforts (e.g., pH control, addition of excipients, etc.) that could act synergistically with silk.27 While many additives that are beneficial to RNA stability can be incorporated with silk and the RNA, additions such as RNase inhibitors and DEPC were not essential because RNase was not detected in the silk samples. We anticipate that the long-term solubility of these lyophilized cakes will not change on the basis of prior characterization of these systems over 1 year of storage at elevated temperatures.14 Initial attempts at RNA recovery from silk solutions showed low yield in qRT-PCR, likely because the silk protein inhibited enzymatic reactions in qRT-PCR during reverse transcription. This binding is presumably due to the ability of silk to bind proteins, including enzymes, in both solution and solid states.12 To quantify the yield of RNA, a purification step was used to remove all of the proteins from silk-laden RNA samples. Purification requires an extra step in preparing stabilized RNA samples for diagnostics; however, it is a common technique for
5. CONCLUSIONS Using silk fibroin to encapsulate RNA through drying results in a storage-stable format that is protective against thermally induced degradation. The intrinsically high collapse temperature of the fibroin protein enables fast and efficient drying through lyophilization. In order to recover mRNA from fibroin, commercial RNA purification kits were used following fibroin solubilization in water. These recovery methods were translated to a relatively simple fibroin evaporative drying technique, resulting in similarly impressive long-term stability profiles due to low residual water content while stored at elevated temperature. Although we have identified concentration regimes of fibroin that are protective through drying and storage, it may be possible to further augment the results with additional formulation efforts. In the future, we anticipate that these methods will be useful in a number of diverse contexts for recovery or delivery of RNA. F
DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00207. Measurement of residual RNase activity in silk fibroin following purification (Figure S1), use of PCR cleanup kits to demonstrate that contamination of the cDNA library with fibroin ultimately disrupts qPCR amplification for detection (Figure S2), and raw RNA integrity data comparing silk/RNA solutions to those purified using the RNAeasy kit over a range of silk concentrations (Figure S3) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: 617-627-3251. Fax: 617-627-3231. E-mail: david.
[email protected]. ORCID
Burcin Yavuz: 0000-0003-2352-9321 Fiorenzo G. Omenetto: 0000-0002-0327-853X David L. Kaplan: 0000-0002-9245-7774 Present Address
⊥ J.H.: Key Laboratory of Protein and Peptide Pharmaceuticals, CAS-University of Tokyo Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
We thank the NIH (P41 EB002520), AFOSR, and DTRA for support of this work. Notes
The authors declare the following competing financial interest(s): Vaxess is a commercial entity involved in stabilization of biologics in silk matrices.
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ACKNOWLEDGMENTS We thank Albert Tai for assistance running the fragment analyzer at the Tufts Clinical Translational Science Institute, which is supported by the National Center for Advancing Translational Sciences, National Institutes of Health (Award UL1TR001064).
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ABBREVIATIONS h, hours; PDMS, polydimethylsiloxane; min, minutes; °C, degrees Celsius; mL, milliliter; μg, microgram; μL, microliter; w/v, weight/volume; mM, millimolar; mg, milligram; g, gram; mT, millitorr; kDa, kilodalton; ng, nanogram; s, seconds
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DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsbiomaterials.8b00207 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
