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Rationally Designed Redox-Sensitive Protein Hydrogels with Tunable Mechanical Properties Ming-Liang Zhou, Zhigang Qian, Liang Chen, David L Kaplan, and Xiaoxia Xia Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00973 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016

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Biomacromolecules

Submitted to Biomacromolecules as an Article

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Rationally Designed Redox-Sensitive Protein Hydrogels with Tunable

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Mechanical Properties

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Ming-Liang Zhou,† Zhi-Gang Qian, † Liang Chen, † David L. Kaplan,‡ and Xiao-Xia Xia*,†

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†

State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of

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Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai

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Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China

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‡

Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford,

Massachusetts 02155, United States

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AUTHOR INFORMATION

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Corresponding Author

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*E-mail: [email protected].

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Notes

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The authors declare no competing financial interest.

1 ACS Paragon Plus Environment

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ABSTRACT

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Protein hydrogels are an important class of materials for applications in biotechnology and

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medicine. The fine tuning of their sequence, molecular weight, and stereochemistry offers unique

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opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these

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materials. Here we report a new family of redox-sensitive protein hydrogels with controllable

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mechanical properties, composed of recombinant silk-elastin-like protein polymers (SELPs). The

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SELPs were designed and synthesized SELPs with different ratios of silk-to-elastin blocks that

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incorporated periodic cysteine residues. The cysteine-containing SELPs were thermally

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responsive in solution and rapidly formed hydrogels at body temperature under physiologically

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relevant, mild oxidative conditions. Upon addition of a low concentration of hydrogen peroxide

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at 0.05% (w/v), gelation occurred within minutes for the SELPs with a protein concentration of

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approximately 4% (w/v). The gelation time and mechanical properties of the hydrogels were

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dependent on the ratio of silk to elastin. These polymer designs also significantly affected redox-

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sensitive release of a highly polar model drug from the hydrogels in vitro. Furthermore, oxidative

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gelation was performed at other physiologically relevant temperatures, and this resulted in

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hydrogels with tunable mechanical properties, thus providing a secondary level of control over

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hydrogel stiffness. These newly developed injectable SELP hydrogels with redox-sensitive

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features and tunable mechanical properties may be potentially useful as biomaterials with broad

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applications in controlled drug delivery and tissue engineering.

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Keywords: silk-elastin-like protein polymers, redox-sensitive hydrogel, controllable drug

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delivery, genetic engineering, tissue engineering


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INTRODUCTION

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Protein hydrogels are an important class of biomaterials for potential applications in

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biotechnology and medicine. They are typically fabricated via physical or chemical cross-linking

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of soluble protein polymers that form insoluble three dimensional networks with an ability to

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retain an appreciable amount of water and mimic aspects of the microenvironment of native

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tissue extracellular matrices.1-4 Recent trends in the design of protein hydrogels have shifted

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from static to responsive systems, to address broader biomedical needs such as controllable drug

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release and tissue engineering.5 Two types of dynamic, responsive protein hydrogels are of

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particular interest. In the first scenario, protein polymers are designed to undergo dynamic

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gelation upon triggers of physiological relevance that initiate a sol-gel phase transition.6-8 For

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example, an injectable solution of recombinant elastin-like polypeptide was developed that

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underwent rapid in situ gelation following intramuscular and intratumoral administration in

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mice.9 In the second scenario, responsive protein hydrogels are designed and fabricated that can

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significantly change their volume, shape, pore size, mechanical properties, optical transparency

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and other properties in response to stimuli such as temperature, pH, and certain biological signals.

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10,11

In particular, disulfide cross-linked protein hydrogels have attracted much attention because

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they are redox-sensitive, as disulfides are rapidly reduced to thiols under the reductive

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environment inside cells, thus allowing the quantitative release of the payload incorporated

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within the materials. Moreover, such redox-sensitive protein hydrogels as three-dimensional cell-

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culture scaffolds can be degraded under cytocompatible mild reductive conditions without

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affecting the vitality of the embedded cells.12,13

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Genetically engineered protein polymers offer a unique opportunity for the design of

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responsive hydrogels with tunable mechanical properties, dynamic responses, and utility.14-16


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First, compared to synthetic polymers, protein polymers are generally biocompatible and fully

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degradable in vivo, which is critical for many biomedical needs.15,16 Second, nature has evolved a

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variety of highly functional materials that are composed of unique peptide motifs or domains

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with well-defined structures, such as α-helices, β-sheets, β-turns, and coiled coils.17-19 These

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peptide motifs or domains can be employed either individually or in combinations for the design

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of novel responsive hydrogels.20-22 Indeed, the conserved carboxyl-terminal domain (CTD) of

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spider dragline silk protein, with a structure of five α-helix bundle fold, was recently fabricated

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into protein hydrogels with dual thermosensitive behavior.23 With advances in genetic and

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protein engineering, protein hydrogels can be modularly redesigned at the polypeptide level to

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introduce functions by exploiting the constituent modules of fibrous proteins and even of

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globular, structural proteins. Third, protein polymers are relatively easy to modify at the genetic

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level for functionalization through insertion of diverse amino acids in distinct regions of the

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polymer backbone. 24 Therefore, protein polymers are ideal for designing new stimuli-responsive

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hydrogels and have drawn increased attention. To date, a variety of proteins have been studied for the development of responsive protein

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hydrogels, including elastin,9,25,26 collagen,27 fibrinogen,28 silkworm silk fibroin,29 and spider silk. 23,30

Elastin and recombinant elastin-like polypeptides are particularly attractive since the

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pioneering studies of Urry et al. provided a foundation that linked peptide sequence chemistry,

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the coacervation of elastin-like proteins in solution, and stimuli-responses.31 While the stimuli-

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responsive properties of elastin-like polypeptides were mostly explored when they were in

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solution, quite a few examples have been shown where these proteins can be cross-linked for

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temperature sensitive, soft, elastomeric matrices.6,9,25,26,32-36 However, these hydrogel

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biomaterials are usually weak in mechanical strength9,26,32,34,35 and exhibit sensitivity to


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restrained environmental conditions such as temperature,6,9,25,26,32-36 pH,26 salt,25 and elastolytic

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enzymes,11 which thus limits their uses for drug delivery and tissue engineering. To overcome

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these obstacles, we and others have recently begun to explore the feasibility of designing silk-

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elastin-like polymers (SELPs) and SELP hydrogels with an attempt to combine diverse

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responsive properties of elastin and high tensile strength of silk.37-39, 41-44 This is triggered by the

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assumption that the silk blocks in SELPs might be able to crystallize into β-sheets via hydrogen

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bonding, thus enabling robust materials formation.40 Our initial exploration demonstrated that the

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ratio of silk-to-elastin blocks in a SELP’s repeating unit played an essential role in tuning the

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fundamental self-assembly characteristics of these remarkable polymers in solution.41,42 Going a

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step further, we recently developed a robust high-throughput synthesis and characterization

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method to screen for SELPs that were responsive against diverse environmental stimuli,

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including temperature, pH, ionic strength, redox, phosphorylation, and electric field.43 The new

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family of SELPs with tyrosine residues in the polymer backbone was more recently cross-linked

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with horseradish peroxidase and hydrogen peroxide to form robust hydrogels with temperature

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responsive properties.44 However, SELP hydrogels with responsiveness other than temperature

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remain to be fully explored, to fill the high need for such materials in biomedical-related use.

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Herein, we report the design and fabrication of a new family of SELP hydrogels through

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rapid gelation of the protein polymers under physiologically relevant, mild oxidative conditions.

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First, we synthesized SELPs genetically engineered with cysteine residues in the elastin block

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and varying the ratio of silk-to-elastin blocks in each repeating unit. The proteins were then

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characterized for thermally-induced phase transition behavior in solution. Next, the protein

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polymers were treated with hydrogen peroxide at concentrations as low as 0.05% (w/v) and body

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temperature to rapidly form disulfide crosslinked hydrogels. The utility of these hydrogels for


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dithiothreitol-responsive release of a model polar drug was examined in vitro. To further

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modulate mechanical properties of the hydrogels, oxidative gelation was performed at other

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physiologically relevant temperatures, thus providing a secondary level of control over hydrogel

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stiffness.

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MATERIALS AND METHODS

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Materials.

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Chemically competent cells of E. coli DH5α and BL21(DE3), TIANprep Mini Plasmid Kit and

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TIANgel Midi Purification Kit were purchased from TIANGEN Biotech (Beijing, China).

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Restriction enzymes, alkaline phosphatase and T4 DNA ligase were obtained from New England

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Biolabs (Ipswich, MA). The Pierce™ BCA Protein Assay Kit was purchased from Thermo

125

Fisher Scientific Inc. (Rockford, IL). The membrane dialysis tubing with molecular weight cut

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off (MWCO) at 3.5 kDa was obtained from Spectrumlabs (Phoenix, AZ). Ampicillin, β-

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mercaptoethanol, dithiothreitol (DTT), hydrogen peroxide 30% (w/v) solution, imidazole and

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isopropyl-β-ᴅ-thiogalactopyranoside (IPTG) were purchased from Sangon Biotech (Shanghai,

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China). Ni-NTA agarose (Catalog # 30230) and rhodamine B (Catalog # 83689) were obtained

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from Qiagen (Hilden, Germany) and Sigma (St. Louis, MO), respectively. Coomassie Brilliant

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Blue R-250 (Catalog #161-0400) was obtained from Bio-Rad (Hercules, CA). Tryptone and

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yeast extract were obtained from Oxoid (Basingstoke, Hampshire, UK). All other chemicals were

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of the highest purity available from commercial suppliers.

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Construction of Expression Plasmids.

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The tailor-made vector pET-19b3, was employed to construct plasmids for recombinant

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expression of the SELPs under the IPTG-inducible T7 promoter.41 A DNA sequence encoding the


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silk-elastin-like sequence SE8C [(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)] was purchased as

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a synthetic gene that was cloned into the EcoRV site of plasmid pUC57 from Genewiz (Suzhou,

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China). The pUC57 derivative was digested with the restriction enzyme BanII, and the liberated

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monomer DNA isolated by agarose gel electrophoresis and purified using the TIANgel

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Purification Kit. The purified monomer DNA was then self-ligated by T4 DNA ligase at 16 °C

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for 12 h. The mixture containing the resulting DNA multimers was added with the BanII- and

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alkaline phosphatase-treated pET-19b3, and incubated at 16 °C for an additional 12 h. The

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ligation mixture was then used to transform chemically competent cells of E. coli DH5α. The

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transformants were selected on lysogeny broth (LB) agar plates (per liter: 10 g tryptone, 5 g

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yeast extract, 10 g NaCl, and 15 g agar) supplemented with 50 µg mL-1 of ampicillin. The

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transformants were then grown in the LB liquid medium for extraction of the recombinant

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plasmids using the TIANprep Mini Plasmid Kit. The expression plasmids carrying the repetitive

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SE8C genes of varying lengths were identified by double digest with NcoI and BamHI and

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further confirmed by dideoxy sequencing with primers derived from the T7 promoter and T7

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terminator sequences. Plasmid pSE8C-12 was thus obtained for the recombinant expression of

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12 repeats of SE8C. Plasmids pS2E8C-12 and pS4E8C-11 were constructed as described

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previously,43

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[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)2]

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[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)4], respectively.

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Protein Expression, Purification and Identification.

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The recombinant plasmids were transformed into the common expression host, E. coli BL21

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(DE3), and plated on the LB agar plates with 50 µg mL-1 of ampicillin. A single colony was

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inoculated in a 15 mL tube containing 4 mL of LB medium and cultured overnight at 37 °C and

which

allowed

expression

of and

12 11


repeats repeats

of

S2E8C

of

S4E8C

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220 rpm in a rotary shaker. Unless otherwise indicated, 50 µg mL-1 of ampicillin was routinely

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added into the culture media for the selection of plasmid-carrying recombinant cells.

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Subsequently, 1 mL of the overnight culture was transferred into a 250 mL Erlenmeyer flask

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containing 100 mL of the fresh LB medium. Cell growth was monitored by measuring the

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absorbance at 600 nm (OD600) using an Eppendorf BioPhotometer plus spectrophotometer

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(Hamburg, Germany). After cultivation for 4 h (OD600 ~3-4), the 100 mL seed culture was

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transferred into a 2 L baffled flask containing 800 mL of the Terrific broth (per liter: 12 g

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tryptone, 24 g yeast extract, 5 g glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4). The cultures were

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incubated at 37 °C and 220 rpm for ~6 h, shifted to 16 °C, and induced overnight with IPTG at

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the final concentration of 1 mM. Cells were harvested by centrifugation at 9000g for 15 min at

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10 °C. The cell pellets were resuspended in the 20 mM Tris-HCl buffer (pH 8.0) and then lysed

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using a high pressure homogenizer (AH-1500; ATS Engineering Limited, Vancouver, Canada).

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The homogenate was centrifuged at 9000g for 10 min at 10 °C. The resulting supernatant was

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loaded onto a Ni-NTA agarose column that had been equilibrated with the Tris-HCl buffer

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supplemented with 300 mM NaCl and 5 mM imidazole. The column was washed and eluted with

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the Tris-HCl buffer containing 300 mM NaCl and imidazole at 50 mM and 250 mM, respectively.

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To minimize disulfide bond formation, the eluted proteins of interest were added with 10 mM

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DTT and then dialyzed against deionized water at 4 °C for 2 days, with water changes every 4-8

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h. Following dialysis, the protein solutions were concentrated to ~50 mg mL-1 at 4 °C using

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Amicon Ultra-0.5 mL 10K centrifugal filter devices (Millipore, Billerica, MA). The purity of the

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purified proteins was analyzed by 10% sodium dodecyl sulfate polyacrylamide gel

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electrophoresis (SDS-PAGE) using a gel loading buffer with 1% β-mercaptoethanol (a reducing

182

agent). The gels were stained with Coomassie Brilliant Blue R-250, and the stained gels were


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scanned by the model GS-800 Calibrated Imaging Densitometer (Bio-Rad, Hercules, CA).

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Protein concentrations were quantified using the BCA Protein Assay Kit with bovine serum

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albumin as the standard. To verify molecular weights of the purified proteins, mass spectrometry

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was performed on a SolariX-70FT-MS Bruker spectrometer (Bruker Daltonics Inc., Billerica,

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MA). All the protein samples were freshly prepared and temporarily maintained at 4 °C in a

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refrigerator before use.

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Hydrogel Formation.

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Hydrogen peroxide induced hydrogel formation was performed for the recombinant proteins in

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glass tubes. Briefly, 90 microliters of each protein solution at 4.5% (w/v) were gently mixed with

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10 µL of 0.5% (w/v) H2O2, and promptly incubated at 37 °C in a water bath for 10 min. The

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proteins without H2O2 treatment were taken as controls and also incubated at 37 °C for 10 min.

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Tubes were then taken out and inversed to evaluate the formation of a self-supporting hydrogel.

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Photographs were quickly taken using a Canon EOS 700D camera (Canon, Tokyo, Japan).

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Characterization of Phase Transition.

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The phase transition behavior of the proteins was characterized by monitoring the absorbance of

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protein solutions at 350 nm (OD350) as a function of temperature on a Shimadzu UV-2600 UV-

199

Vis spectrophotometer (Shimadzu Corp., Kyoto, Japan) equipped with a constant-temperature

200

water circulator (Model SDC-6; Scientz, Ningbo, China). Each protein solution at either 1 mg

201

mL-1 or 40 mg mL-1 was loaded in a Suprasil quartz micro cuvette (10 mm lightpath; Hellma,

202

Müllheim, Germany) and tested from 15 °C with a heating rate of 1 °C min-1. The inverse

203

transition temperature (Tt) was defined as the solution temperature corresponding to 50% of the

204

maximum value of OD350.


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Rheological Monitoring of Gelation.

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Rheology monitoring of gelation was performed using a stress-controlled AR-G2 rheometer (TA

207

Instruments, New Castle, DE) with a 40-mm parallel-plate configuration. A 360 µL aliquot of

208

each protein solution (4.5%, w/v) was gently mixed with 40 µL of 0.5% (w/v) H2O2, and the

209

mixture was immediately transferred onto the Peltier plate that had been precooled at 10 °C. The

210

top plate was then lowered to set the gap distance at 300 µm, and hydrogenated silicone oil was

211

added to the outer edge of the samples to minimize water evaporation. Time sweeps were carried

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out at 37 °C with a frequency of 1 Hz and a strain of 1%, which was within the linear

213

viscoelastic range. Frequency sweep tests were performed after 30 min gelation at the indicated

214

temperatures with a constant strain of 1% and logarithmic ramping from 0.1 to 100 rad s-1.

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Dynamic Light Scattering (DLS).

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DLS was carried out on a Zetasizer Nano S system (Malvern Instruments, Worcestershire, UK)

217

equipped with a temperature controller. A mixture of each protein at 1 mg mL-1 and 0.05% (w/v)

218

H2O2 was introduced into quartz cuvettes and the samples stabilized at the desired temperatures

219

(10, 25, 45 or 65 °C) for 10 min prior to measurement. Number distribution data were collected

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from three biological replicates and analyzed using the Zetasizer version 7.04 software (Malvern

221

Instruments).

222

Atomic Force Microscopy (AFM).

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A solution of each protein was mixed with hydrogen peroxide to reach the final concentrations of

224

1 mg mL-1 and 0.05% (w/v), respectively. The mixtures were casted on mica surfaces at

225

indicated temperatures and allowed to dry for ∼2 days. AFM was performed in tapping mode

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using a multimode Nanoscope IIIa atomic force microscope (Bruker, Germany). The silicon tip

227

probe had a spring constant of ~3 N m-1, and the scan rate was 1.97 Hz. The AFM images were


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collected with a scanning window of 10 µm and analyzed using the Nanoscope analysis v5.30

229

software (Bruker).

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Scanning Electron Microscopy (SEM).

231

The recombinant proteins were allowed to form hydrogels at 37 °C upon H2O2 treatment as

232

described above. The hydrogels were then lyophilized with a FreeZone Plus 6 Liter cascade

233

console freeze dry system (Labconco, Kansas City, MO), and the lyophilized hydrogels were

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sputter coated with gold for SEM observation. Images of the microstructure of the hydrogels

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were taken using a Hitachi S-3400N scanning electron microscope (Tokyo, Japan).

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Fourier Transform Infrared Spectroscopy (FTIR)

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FTIR analysis of the hydrogels was carried out in transmission mode using a Nicolet 6700

238

spectrometer (Thermo Fisher Scientific Inc., Madison, WI) equipped with a deuterated triglycine

239

sulfate detector. For each measurement, the wave numbers ranged from 400 to 4000 cm-1 with a

240

resolution of 4 cm-1. The infrared spectra covering the Amide I region (1600-1700 cm-1) were

241

analyzed using the OMNIC software (Thermo Fisher Scientific). The background spectra were

242

taken under the same conditions and subtracted from each sample scan.

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Drug Release Assay.

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The fluorescent dye, rhodamine B, was used as a model of a highly polar drug to examine in

245

vitro drug release from the protein hydrogels. Briefly, 90 µL of each protein at 4.5% (w/v) was

246

first mixed with 5 µL of an aqueous rhodamine B solution (1 mg mL-1) and then with 5 µL of 1%

247

(w/v) H2O2 on a 96-well cell culture plate (Nest Biotechnology Co., Ltd, Wuxi, China). The

248

mixtures were incubated at 37 °C for 30 min to allow the formation of hydrogels. Rhodamine B

249

release was initiated by dropping on the hydrogel surface 200 µL of phosphate buffered saline

250

(PBS) either with or without 10 mM DTT. A 10 µL aliquot of the PBS solution was sampled


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from the wells at the indicated time points, and an equal volume of pre-warmed fresh buffer was

252

added back to each well. Rhodamine B concentrations in the PBS solutions were quantified

253

using a standard calibration curve experimentally obtained. The rhodamine B fluorescence (553

254

nm excitation, 627 nm emission) was measured on a fluorescence microplate reader (SpectraMax

255

M5; Molecular Devices Corp., Sunnyvale, CA). Data are the average of three biological

256

replicates with standard deviation.

257 258

RESULTS AND DISCUSSION

259

Design and Biosynthesis of Cysteine-Containing SELPs.

260

Our earlier study demonstrated that a cysteine-containing SELP with silk-to-elastin block ratio at

261

1:4 displayed redox-sensitive properties in solution.43 Inspired by this observation, we

262

hypothesized that the cysteine residues in the elastin block could be potentially oxidized to form

263

intramolecular and intermolecular disulfide crosslinks leading to a supramolecular network, and

264

the covalent bonds in the resulting hydrogels might also be reduced under a mild reductive

265

condition. To test this hypothesis, a new family of hydrogels was designed based on three SELPs

266

that were genetically engineered with varying ratios of silk-to-elastin blocks at 1:8, 1:4, and 1:2,

267

and

268

(GVGVP)4(GXGVP)(GVGVP)3. Sequence features in this design included the soft elastin

269

domain providing elasticity and stimuli-sensitive properties, and the hard silk domain,

270

GAGAGS, providing tunable mechanical stiffness.

a

cysteine

residue

at

the

X

position

of

the

central

elastin

block

271

We next constructed expression plasmids encoding the desirable cysteine-containing SELPs,

272

which consisted of 12 repeats of SE8C [(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)], 12 repeats

273

of

S2E8C

[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)2],


and

11

repeats

of

S4E8C

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[(GVGVP)4(GCGVP)(GVGVP)3(GAGAGS)4], respectively (Figure 1A). The three SELPs,

275

hereafter termed as SE8C, S2E8C and S4E8C, had a comparable number of the monomer repeats

276

and theoretical molecular weights (Figure 1B). These recombinant proteins were expressed with

277

an N-terminal tag (MGHHHHHHHHHHSSGHIDDDDKHMGAGAGS) in the expression host E.

278

coli BL21(DE3) and purified using immobilized-metal-affinity chromatography. The yield of the

279

purified SELPs was in the range of 80-100 mg L-1 of bacterial culture in shake flasks. All the

280

purified protein polymers had a purity greater than 95% confirmed by SDS-PAGE analysis

281

(Figure 1C). These proteins were further verified by mass spectrometry, displaying identified

282

molecular weights at 47.48 kDa, 52.16 kDa, and 56.89 kDa, respectively, which were all within

283

0.27% difference of the expected theoretical values (Figure S1).

284

285

Figure 1. (A) Constructs of recombinant SELPs that contain cysteine residues and varying ratios

286

of silk-to-elastin blocks in each monomer repeat. (B) The number of monomer repeats, the

287

number of amino acids, theoretical and mass spectrometry-identified molecular weight (Mw) of

288

the three SELPs. (C) Coomassie-stained 10% SDS-PAGE gel analysis of the purified SELPs.


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Thermally Responsive Behavior of the SELPs in Solution.

290

To test whether the cysteine-containing SELPs exhibited thermal transition behavior,

291

changes in optical density were monitored at 350 nm (OD350) of the protein solutions upon

292

heating from 15 to 80 °C. When SE8C, S2E8C and S4E8C were tested at a low concentration of

293

1 mg mL-1, they all exhibited an inverse phase transition typical of elastin, with Tt at

294

approximately 42 °C, 45 °C and 62 °C, respectively (Figure 2A). This indicated a link between

295

the ratio of silk-to-elastin blocks and Tt of the resulting SELPs. A likely explanation was that

296

coacervation of the elastin blocks was hampered in the presence of a higher proportion of the silk

297

blocks in the SELPs. We also examined the thermally triggered phase transition behavior for the

298

SELPs at 40 mg mL-1, a protein concentration relevant for oxidative gelation. In this scenario,

299

the sharpness of the phase transition weakened and the Tt of the three SELPs was also

300

significantly decreased (Figure 2B). SE8C had a Tt of ~30 °C, which is below body temperature

301

(37°C), whereas S2E8C and S4E8C had a Tt of 40 and 45°C, respectively. Collectively, the

302

results indicated that the three SELPs exhibited inverse phase transition and their Tt could be

303

finely modulated by adjusting molecular design and the concentrations of the protein polymers.

304 14 ACS Paragon Plus Environment

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Figure 2. Thermally responsive behavior of the protein solutions at 1 mg mL-1 (A) and 40 mg

306

mL-1 (B) as a function of temperature. Turbidity profiles were obtained by monitoring optical

307

density at 350 nm as the aqueous solutions were heated at a rate of 1 °C min-1.

308 309 310

Hydrogel Formation Under Oxidative Condition.

311

To test whether the cysteine-containing SELPs underwent oxidative gelation via disulfide bond

312

formation, solutions of the recombinant proteins were treated with an externally added oxidant.

313

H2O2 was chosen for gelation studies as it is a common, mild oxidant that is not toxic at

314

reasonable concentrations.9,44 Hydrogel formation of the three SELPs was judged initially at

315

body temperature, 37 °C, with the addition of a low concentration of H2O2 at 0.05% (w/v), which

316

was minimally required according to our preliminary experiments. All the protein polymers

317

formed self-supporting hydrogels at a protein concentration of 4.05% (w/v) within 10 min in the

318

presence of H2O2, while these proteins did not gel without H2O2 treatment (Figure 3A). In

319

addition, SE8C formed an opaque hydrogel, while S2E8C and S4E8C formed a semi-transparent

320

and transparent hydrogels, respectively. SE8C at 4.05% (w/v) should have undergone a complete

321

phase transition into coacervates at 37 °C, while the degree of coacervation was significantly

322

lower for S2E8C and S4E8C under the same conditions (Figure 2B). It appeared that

323

coacervation of the protein polymers contributed to optical transparency of the H2O2-triggered

324

hydrogels. The phenomenon coincided with that observed earlier, in which a transparent elastin-

325

like polypeptide hydrogel that was formed on ice became turbid upon incubation at a temperature

326

above the solution Tt of the polymer.9


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327 328

Figure 3. (A) Formation of self-supporting SELP hydrogels. Vials containing 4.05% (w/v)

329

protein with or without 0.05% (w/v) H2O2 (as controls) were incubated at 37 °C for 10 min, and

330

then inverted for image collection. (B) Oscillatory rheological profiles for the SELP hydrogels. A

331

mixture of 4.05% (w/v) protein solution and 0.05% (w/v) H2O2 was loaded into the rheometer,

332

and time sweeps were carried out at 37 °C with a frequency of 1 Hz and a strain of 1% (linear

333

regime). Elastic modulus (G’) and loss modulus (G”) are shown as a function of time.

334 335

To study gelation kinetics and quantify the hydrogel mechanical behavior, the storage (G′)

336

and loss (G″) moduli were recorded as a function of temperature using oscillatory rheology

337

(Figure 3B). For all the SELP hydrogels, G′ values were larger than their respective G″ values

338

from the very beginning, indicating immediate formation of a network hydrogel structure. For

339

accurate estimation of the time needed to form a robust hydrogel, gelation time was defined as

340

the time when G′ reached a plateau value in the rheological studies. The gelation times for the

341

SE8C, S2E8C and S4E8C were approximately 300 s, 500 s and 900 s, respectively. Notably, the

342

H2O2-triggered SE8C hydrogel at 37 °C exhibited appreciably high elastic modulus, with plateau

343

G′ value at ~870 Pa, which was significantly higher than those of S2E8C (~470 Pa) and S4E8C

344

(~160 Pa), respectively. Taken together, the results indicated that the ratio of silk-to-elastin 16 ACS Paragon Plus Environment

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blocks, one of the key design parameters for the SELPs, was critical in modulating the gelation

346

time and mechanical properties of the resulting hydrogels. Previously, a family of cysteine-

347

containing elastin-like polypeptides (ELPs) with different degrees of hydrophobicity was

348

designed that displayed tunable, thermally responsive behavior, and these ELPs at 2.5 wt% could

349

be rapidly fabricated into disulfide cross-linked hydrogels with storage moduli in the range of

350

~20-200 Pa. Again, this earlier study stressed the important role of molecular design in

351

determining the polymer properties in solution and hydrogel states.

352 353

Redox-Sensitivity of the SELP Hydrogels.

354

To explore the utility of these hydrogels in biomedical needs such as controllable drug delivery,

355

we tested whether the hydrogels permitted the redox-sensitive release of small molecule drugs.

356

Rhodamine B, which is highly hydrophilic, was selected to simulate polar drugs because it

357

exhibits strong fluorescent signals, which can be accurately monitored by fluorescence

358

spectrometry.45 A mild reductive reagent, dithiothreitol (DTT), was used as the trigger to reduce

359

the disulfide bonds in the hydrogels. The kinetics of release from SE8C, S2E8C and S4E8C

360

hydrogels are shown in Figure 4, which tracks the cumulative rhodamine B released (%) as a

361

function of time at 37 °C. For all the hydrogels, the release of rhodamine B was enhanced upon

362

the introduction of 10 mM DTT, implying redox-sensitive behavior. For example, the SE8C

363

hydrogel released 44.4% of the loaded dye at 72 h with DTT in the release buffer, while this

364

hydrogel released only 28.9% in the buffer without DTT. In addition, we observed a significant

365

difference in the release rate among the three hydrogels. The SE8C hydrogel exhibited a

366

significantly lower release rate than those of S2E8C and S4E8C hydrogels, which might be

367

related with their microstructures. To verify this, we performed scanning electron microscopy

368

(SEM) on the hydrogel samples following lyophilization, a procedure generally known to have a 17 ACS Paragon Plus Environment

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Page 18 of 30

369

minimal impact on the structure of hydrogels.23,30 The SE8C hydrogel exhibited a more compact

370

structure with pore sizes at 20-35 μm, which coincided with its higher mechanical strength as

371

observed from the oscillatory rheology analysis (Figure 5). In contrast, S2E8C and S4E8C

372

showed a relatively loose and weak structure with larger pore sizes (40-80 µm).

373 374

Figure 4. Cumulative release of rhodamine B from the hydrogels in an in vitro assay. A mixture

375

of 50 µg mL-1 rhodamine B, 0.05% (w/v) H2O2, and protein at 4.05% (w/v) were allowed to form

376

hydrogels at 37 °C on a 96-well plate, and rhodamine B release was initiated by dropping on the

377

hydrogel surface PBS buffer either with or without 10 mM DTT.

378

379 380

Figure 5. Scanning electron microscopy (SEM) of the lyophilized hydrogels fabricated with 4.05% 18 ACS Paragon Plus Environment

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381

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(w/v) protein and 0.05% (w/v) H2O2 at 37 °C.

382 383

Oxidative Gelation at Diverse Temperatures Resulted in Hydrogels with Tunable

384

Mechanical Properties.

385

Having demonstrated oxidative gelation and redox-sensitivity of the resulting hydrogels at body

386

temperature, we next investigated gelation of the polymers at other physiologically relevant

387

temperatures. This was inspired by the fact that all the three SELPs were thermally responsive in

388

solution, and their coacervation states might affect disulfide crosslinking and thus the mechanical

389

properties of the resulting hydrogels. Therefore, we performed gelation of 4.05% (w/v) SELPs

390

with 0.05% (w/v) H2O2 at 25 °C, 37 °C, and 45 °C on the rheometer. After gelation equilibrium

391

for 30 min to obtain a stable network hydrogel, the rheological data were obtained by linear

392

oscillatory frequency sweep (Figure 6). As expected, the SE8C hydrogels showed superior

393

material stiffness (G′), followed by the S2E8C and S4E8C hydrogels, if the three proteins were

394

gelled at the same temperature. In addition, for each type of protein polymer, an increase in

395

gelation temperature resulted in hydrogels with elevated material stiffness. Notably, when 4.05%

396

(w/v) SE8C was gelled at 37 °C corresponding to full coacervation of the protein in solution, the

397

hydrogel stiffness was enhanced, compared with gelation at 25 °C under which the polymer was

398

partially coacervated. In addition, a further increase in gelation temperature from 37 to 45 °C

399

was beneficial for enhancing hydrogel stiffness, even though 4.05% (w/v) SE8C was fully

400

coacervated at these two temperatures. Taken together, the results demonstrated that gelation

401

temperature played a significant yet complicated role in modulating mechanical properties of the

402

hydrogels fabricated from the thermally responsive SELPs.


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403 404

Figure 6. Storage moduli (G′) as a function of frequency for the SELP hydrogels formed from

405

4.05% (w/v) protein with 0.05% H2O2 (w/v) at 25 °C, 37 °C, and 45 °C, respectively.

406 407

To examine whether the silk blocks in the SELPs were directly involved in hydrogel

408

formation, we performed Fourier transform infrared spectroscopy (FTIR) analysis for the

409

lyophilized hydrogels (Figure 7). It is generally recognized that the region from 1600 to 1640

410

cm-1 of FTIR spectra is related to the intermolecular and intramolecular beta-sheet bands,

411

whereas the region between 1640 and 1660 cm-1 is associated with the presence of random coils

412

and alpha-helices.4,46 Surprisingly, the SE8C and S2E8C hydrogels fabricated at all the three

413

temperatures showed signals in the region between 1600 and 1640 cm-1, which is indicative of

414

the formation of β-sheet structures, whereas S4E8C with higher ratio of silk-blocks did not show

415

obvious β-sheet structures. This result indicated that silk crystallization also contributed to

416

formation of the SE8C and S2E8C hydrogels except disulfide crosslinking, which might explain,

417

at least partially, why the SE8C and S2E8C hydrogels showed higher material stiffness than

418

those of S4E8C (Figure 6).


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419 420

Figure 7. FTIR absorbance spectra of the SELP hydrogels. The hydrogels were fabricated from

421

4.05% (w/v) protein with 0.05% H2O2 (w/v) at 25 °C, 37 °C, or 45 °C for 30 min, and freeze

422

dried before FTIR analysis. The shift toward lower wavenumbers is indicative of the formation

423

of β-sheets.

424 425

Microscopic Structures of the SELPs.


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426 427

Figure 8. Dynamic light scattering (DLS) size distribution profiles. A mixture of each protein at

428

1 mg mL-1 and 0.05% (w/v) H2O2 was incubated at the indicated temperatures before DLS

429

analysis.

430 431

To further explore the molecular and structural events that gave rise to the macroscopic

432

properties described above, we performed dynamic light scattering (DLS) analysis for solutions

433

of the three SELPs with H2O2 oxidation at temperatures ranging from 10 to 65 °C (Figure 8). At

434

low temperatures of 10 and 25 °C (below Tt), SE8C existed mostly as nanostructures with small

435

hydrodynamic diameter sizes of 12.98 ± 0.74 nm and 15.79 ± 1.36 nm, respectively, which was

436

suggestive of the presence of free chains in solution. Upon increasing the temperature to 45 °C

437

(above Tt), the size of SE8C particles was approximately 622 nm, indicating coacervation of this 22 ACS Paragon Plus Environment

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438

protein polymer. Interestingly, self-assembly of S2E8C into large particles with diameter sizes of

439

~150 nm was observed even at 10 and 25 °C, although this polymer mainly existed as free chains

440

at these low temperatures below Tt of the polymer. However, most of S2E8C was self-assembled

441

into larger nanostructures with diameters of 218.87 ± 17.59 nm as the temperature increased to

442

45 °C (around Tt). More interestingly, most of S4E8C existed as self-assembled particles with

443

diameters of 97.38 ± 10.55 nm at 10 °C, and the size distribution of this polymer exhibited as

444

similar profiles at 25 and 45 °C (below Tt). This might be due to the existence of more silk

445

blocks leading to the preassembly of the proteins into micellar-like particles at a low temperature,

446

which partially masked the effect of elevated temperature on coacervation of the elastin blocks in

447

the SELPs.

448 449

Figure 9. Representative AFM images of the cross-linked nanostructures for the SELPs under

450

oxidative condition. A mixture of each protein at 1 mg mL-1 and 0.05% (w/v) H2O2 was

451

deposited on mica surfaces and allowed to dry at the indicated temperatures before analysis.

452 23 ACS Paragon Plus Environment

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453

To verify the microscopic structures, atomic force microscopy (AFM) analysis was

454

performed for the three SELPs under oxidative condition (Figure 9). As expected, the formation

455

of globules was observed, suggestive of micellar-like particles for S2E8C and S4E8C at 25 °C,

456

respectively, whereas SE8C did not form any obvious particles at this temperature. This result

457

stressed that the assembly capability of the cysteine-containing SELPs was dependent on the

458

ratio of silk-to-elastin blocks, which coincided well with that observed for the tyrosine-

459

containing SELPs in our earlier study.41 On the other hand, we also observed the formation of

460

large spherical particles for SE8C and S2E8C at 45 °C, which were most probably formed

461

through cross-linking of the protein coacervates. For S4E8C, no obvious large particles were

462

observed, which might be due to the fact that 45 °C was not high enough to trigger coacervation

463

of this protein.

Page 24 of 30

464

Taken together, oxidative formation of the above SELPs hydrogels at diverse temperatures

465

was complicated and affected by the dual cross-linking mechanisms of disulfide formation and

466

silk crystallization, different from the previously reported SELP hydrogels that were formed

467

from soluble protein polymers at high concentrations via silk physical cross-linking.3,10,37,38 In

468

another interesting study of Fernández-Colino et al., a dual physical gelation mechanism was

469

proposed for gelation of a silk-elastin-like corecombinamer from a 15 wt % aqueous solution of

470

the (EIS)×2 corecombinamer.4 In the first stage, a rapid, thermally driven gelation of the polymer

471

solution occurred upon an increase in temperature due to self-assembly of the elastin blocks as a

472

result of their characteristic inverse transition temperature. In the second stage, folding of the

473

elastin blocks favored the interaction between the silk blocks, which triggered the emergence and

474

maturation of irreversible β-sheet structures with silk annealing at a time scale of days to months.

475

With regard to our newly developed SELP hydrogels, an in-depth investigation of the dual cross-


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476

linking mechanisms at the molecular level may be necessary in future studies to further

477

understand the synergies between these interactions to gain further fine control of the assembly

478

and resulting material properties.

479 480

CONCLUSIONS

481

We have developed a new family of redox-sensitive protein hydrogels via fast oxidative gelation

482

with low concentrations of the mild oxidant, H2O2. This type of material was completely

483

composed of protein polymers that vary in the ratio of silk-to-elastin blocks and contain multiple

484

periodic cysteine residues that provide a means of chemical cross-linking through disulfide bond

485

formation. Such protein designs made it possible for the concerted action of the dynamic, elastin

486

blocks and the silk blocks. By doing so, proteins under physiologically relevant temperatures

487

displayed diverse coacervation states with varying microscopic nanostructures, which can be

488

further crosslinked into hydrogels with tunable mechanical properties and redox-responsive

489

behavior. Tunable control over the material properties is thus possible at polymer design and

490

hydrogel fabrication levels. Such cysteine-containing SELP hydrogels with redox responsiveness

491

and tunable mechanical properties are anticipated to find broader applications in drug delivery,

492

tissue engineering and regenerative medicine.

493 494

ASSOCIATED CONTENT

495

Supporting Information

496

The amino acid sequences of the three SELPs, quantification of free thiol groups (Table S1), and

497

mass spectroscopy analysis of the purified recombinant proteins (Figure S1). This material is

498

available free of charge via the Internet at http://pubs.acs.org.


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499

ACKNOWLEDGEMENTS

500

Financial support was provided by the National Natural Science Foundation of China (31470216,

501

21406138, 21674061), and the Shanghai Pujiang Program (14PJ1405200 to Z.-G.Q). Support

502

from the National Institutes of Health (NIH P41 EB002520) is also greatly appreciated. The

503

authors appreciate Instrumental Analysis Center of Shanghai Jiao Tong University for allowing

504

us to use the AFM and SEM equipments.

505 506

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