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. Author manuscript; available in PMC: 2024 Apr 24.
Published in final edited form as: Methods Mol Biol. 2022;2371:427–448. doi: 10.1007/978-1-0716-1689-5_23

Hydrogel Formation with Enzyme-Responsive Cyclic Peptides

Andrea S Carlini 1,2,3,4,5,6, Mary F Cassidy 1,2,3,4,5,6, Nathan C Gianneschi 1,2,3,4,5,6
PMCID: PMC11042486  NIHMSID: NIHMS1865433  PMID: 34596862

Abstract

Self-assembling peptides (SAPs), which form hydrogels through physical cross-linking of soluble structures, are an intriguing class of materials that have been applied as tissue engineering scaffolds and drug delivery vehicles. For feasible application of these tissue mimetics via minimally invasive delivery, their bulk mechanical properties must be compatible with current delivery strategies. However, injectable SAPs which possess shear-thinning capacity, as well as the ability to reassemble after cessation of shearing can be technically challenging to generate. Many SAPs either clog the high-gauge needle/catheter at high concentration during delivery or are incapable of reassembly following delivery. In this chapter, we provide a detailed protocol for topological control of enzyme-responsive peptide-based hydrogels that enable the user to access both advantages. These materials are formulated as sterically constrained cyclic peptide progelators to temporarily disrupt self-assembly during injection-based delivery, which avoids issues with clogging of needles and catheters as well as nearby vasculature. Proteolytic cleavage by enzymes produced at the target tissue induces progelator linearization and hydrogelation. The scope of this approach is demonstrated by their ability to flow through a catheter without clogging and activated gelation upon exposure to target enzymes.

Keywords: Enzyme-responsive, Hydrogels, Injectable, Macrocycles, Minimally invasive, Proteolytic cleavage, Self-assembling peptides, Steric constraint, Tissue engineering

1. Introduction

Injectable hydrogel-based scaffolds have gained attention as a therapeutic approach for tissue repair. Biomaterials have included extracellular matrix (ECM) [1], alginate [2], and hyaluronic acid hydrogels [3], as well as therapeutic delivery scaffolds such as naturally derived polymeric hydrogels (e.g., collagen [4], fibrin [5], heparin [6], and gelatin [7]), synthetic polymeric hydrogels (e.g., PNIPAAm [8], ureido-pyrimidinone-modified PEG [9], and chitosan [10]) and microparticles (e.g., PLGA [11] and dextran [12]). Despite many successful preclinical studies, widespread translation into clinical trials has been slow. The majority of hydrogels are not candidates for minimally invasive catheter delivery because of excess material viscosity and their tendency to cause catheter clogging [13–15]. One versatile class of hydrogels, self-assembling peptides (SAPs), are attractive as they are mimetic and resemble native ECM [16, 17]. In addition, they are biocompatible, require no additives to initiate self-assembly, are biodegradable, promote endothelial cell adhesion and capillary formation by virtue of their porosity (~5–200 nm diameter), and can allow rapid cellular migration because of their flexibility. If properly designed they can be re-healable [18, 19], and generally do not suffer from batch-to-batch chemical variability. Perhaps most importantly, they are amenable to sequence modification [20, 21]. However, SAPs have not been demonstrated to be amenable to high-gauge needle/catheter delivery.

Most known SAPs rely on short ionic self-complementary sequences to generate soft fibrous and porous networks, complementary to native tissue ECM [22–26]. One such peptide, KLD-12, is comprised of three amphipathic Lys-Leu-Asp-Leu repeats that enable monomers to stack as antiparallel β-sheets and further self-assemble into highly crosslinked fibrous hydrogels via a combination of electrostatic and hydrophobic interactions [22–24]. In this chapter, we discuss various modifications to the KLD-12 peptide chemistry for functionalizing it as a cyclic peptide progelator. It should be noted that this strategy is not unique to the KLD-12 peptide, and in fact can be applied to various other SAP sequences.

Herein, the synthesis and characterization of cyclic enzyme-responsive peptides as a versatile platform that enables minimally invasive delivery of SAPs for tissue engineering purposes, is described [27]. SAP sequences (green) are prepared as water soluble, dispersed progelators through addition of a substrate recognition sequence for MMP-2/9 and elastase (red), an optional rhodamine label (pink ellipse), and disulfide linkage (black) of terminal cysteines to promote macrocyclization. Enzymatic cleavage of these sterically constrained cyclic progelators results in linearization to generate SAPs (Fig. 1) which can self-assemble with their neighbors, forming re-healable viscoelastic hydrogels. In theory, the enzyme recognition sequence can be substituted for a sequence corresponding to any other protease to induce gelation. Furthermore, this system can be modified for other stimuli that exist outside the scope of this method (e.g., UV activation [28], reduction/oxidation [29], and/or pH change.) Characterization of these progelators, their enzyme-responsive assembly, and resulting hydrogel formation can be achieved with a variety of optical instrumentation (DLS, SLS, CD), electron microscopy (TEM), and mechanical analysis (rheology, catheter injection). Brief instructions on preparing peptide solutions for each of these techniques are provided at the end of this chapter.

Fig. 1.

Fig. 1

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Schematic of cyclic, enzyme-responsive progelator peptides for enzyme-responsive hydrogel formation. (a) Cyclic progelator peptides, containing gelling sequence (green), matrix metalloproteinase (MMP)/elastase enzyme cleavage recognition sequence (red), and disulfide bridge (black), resist assembly due to conformational constraint. Labels such as rhodamine (pink) are appended to a small molar percentage (0.1–10%) of total peptide content for visualization during biological experiments. (b) Enzymatic cleavage of progelators results in linearization into self-assembling peptides (SAPs). (c) SAPs assemble into viscoelastic hydrogels composed of β-sheets fibrils through intermolecular electrostatic interactions and hydrophobic forces. (d) Peptide sequence for Rho-KLDLCyclic and enzymatic cleavage sites. Gray arrow (cleavage not observed). (e) Photographs of KLDLCyclic (2 mol% labeled, 10 mM) without (−) and with (+) thermolysin treatment, demonstrating enzyme-responsive hydrogel formation. Reproduced with modifications from Carlini et al. 2019 with permission from Springer Nature [27]

2. Materials

2.1. Materials for Solid-Phase Synthesis of Linear Peptide Precursors

  1. Peptide synthesizer (optional).

  2. Resin: rink amide MBHA resin (100–200 mesh, 0.71 mmol/g).

  3. Solid-phase peptide (SPPS) synthesis vessel: 20-mL Chromatography Columns with 30 μm polyethylene frit.

  4. Orbital Shaker: ran at 300–500 rpm unless otherwise noted.

  5. Solvents: N, N-dimethylformamide (DMF).

  6. Dichloromethane (DCM).

  7. Methanol (MeOH).

  8. Isopropanol (IPA).

  9. Plastic squirt bottles for solvent.

  10. Liquid waste collection assembly: 2 L Büchner flask equipped with a straight glass vacuum adaptor with PTFE stopcock inserted through a perforated rubber stopper, a rubber septum (14/20 joint) with needle were hose-clamped to the exposed adapter socket as an inlet for liquid waste, and rubber tubing connecting a secondary 1 L Büchner flask, a dry ice/IPA cold trap, and house vacuum.

  11. Fmoc-deprotection solution: 20% (v/v) 4-methylpiperidine in DMF.

  12. Fmoc-protected amino acids for KLDLcyclic: Fmoc-Cys(Acm)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH (see Note 1).

  13. N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate (HBTU) general amino acid coupling agent.

  14. Diisopropylethylamine (DIPEA): SPPS coupling base.

  15. Acetic anhydride: N-terminal capping agent.

  16. Kaiser test kit.

  17. Heat gun.

  18. 5 mL glass tubes.

  19. Lithium chloride (LiCl) (optional).

  20. 5(6)-carboxytetramethyl rhodamine: N-terminal dye (optional label).

  21. 1-Hydroxybenzotriazole (HOBt): Dye Coupling agent.

  22. Refrigerator (8 °C) and freezer (−20 °C).

  23. 50 mL conical centrifuge tubes.

  24. Cleavage cocktail: 95:2.5:2.5 (%v/v) trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and H2O, respectively.

  25. Dithiothreitol (DTT).

  26. Nitrogen (N2) gas.

  27. Vortex mixer.

  28. Rotary evaporator.

  29. Anhydrous diethyl ether, chilled to −20 °C.

  30. Centrifuge.

2.2. Materials for Solution-Phase Macrocyclization of Progelators

  1. Acetic acid (AcOH).

  2. Dimethyl sulfoxide (DMSO).

  3. milliQ water (H2O).

  4. Acetonitrile (ACN).

  5. pH paper: test strips, range pH 0–14.

  6. Iodine (I2).

  7. Homogenizer (optional).

  8. Probe sonicator (optional).

  9. Anion exchange resin.

  10. Bath sonicator.

  11. Liquid nitrogen (N2).

  12. Lyophilizer.

  13. Trifluoroacetic acid (TFA).

2.3. Materials for Peptide Purification and Analysis

  1. Liquid chromatography instrument coupled to mass spectrometer (LCMS): C18 high-performance liquid chromatography (HPLC) column (3 μm particle size) equipped with a with a quadrupole ion trap mass analyzer configured with an electrospray ionization (ESI) source.

  2. Analytical HPLC instrument: C18 column (150 × 4.60 mm, 4 μm particle size, 90 Å pore size) equipped with an ultraviolet–visible (UV–Vis) detector.

  3. Prep-HPLC instrument: C18 column (2050 × 25.0 mm, 4 μm particle size, 90 Å pore size).

  4. Glass vials.

  5. Electrospray ionization mass spectrometer (ESI-MS).

  6. Syringes (1 mL, 20 mL) and needles.

  7. 0.45 μm PES syringe filters.

  8. Buffer A (H2O, 0.1% v/v TFA or formic acid).

  9. Buffer B (ACN, 0.1% v/v TFA or formic acid).

  10. Glass collecting tubes and tube rack: borosilicate glass disposable culture tubes 18× 150 mm.

2.4. Materials for Progelator Sterilization and Formulation

  1. Aluminum foil.

  2. 2-L beakers.

  3. Ammonium hydroxide (NH4OH) solution (28% NH3 in H2O).

  4. 1 kDa MWCO dialysis tubing (capacity 3–20 mL).

  5. 0.22 μm polyethersulfone (PES) filters.

  6. 10× Dulbecco’s phosphate-buffered saline (DPBS) (pH 7.4).

2.5. Materials for Enzymatic Cleavage of Cyclic Peptide Progelators

  1. 1× MMP-9 buffer: 50 mM Tris-HCl, 200 mM NaCl, 5 mM CaCl2, 1 mM ZnCl2, pH 7.5.

  2. 1× elastase buffer: 100 mM Tris-HCl, 0.2 mM NaN3, pH 8.0.

  3. 1× thermolysin buffer: 50 mM Tris-HCl, 0.5 mM CaCl2.

  4. Matrix metalloproteinase 9 (MMP-9): Recombinant human catalytic domain (BML-SEro360), 1.0 mg/mL solution at 40 U/μL in 50 mM Tris-HCl, pH 7.5, 1 mM CaCl2, 300 mM NaCl, 5 μM ZnCl2, 0.1% Brij-35, and 15% glycerol.

  5. Porcine pancreatic elastase, lyophilized powder (22 U/mg).

  6. Thermolysin, derived from Geobacillus stearothermophilus, lyophilized powder (25 mg).

  7. Dry bath block heater.

  8. 0.5 M ethylenediaminetetraacetic acid (EDTA) in water, pH 8.0.

3. Methods

3.1. Solid-Phase Synthesis of Linear Peptide Precursors

The protocol below describes the detailed manual synthesis of linear precursors of macrocyclic progelators. This protocol can be used in lieu of an automated peptide synthesizer (see Note 2). All experiments are performed at ambient temperature unless otherwise stated and assumes the user has access to basic organic laboratory equipment. This protocol may be adapted for synthesis of other cyclic peptide progelators, where a progelator can be defined as a linear gelling peptide sequence that is cyclically constrained to introduce steric hindrance along the backbone. This design significantly reduces side chain interactions and subsequent gelation until cleavage. Our progelators have ranged in length from 14 to 20 amino acid residues in length. We suspect there is an upper and lower boundary to ensure that steric constraint prevents excess self-assembly Cys(Acm) on both terminals has been the most convenient approach for cyclization, however it is possible to cyclize through C to N-terminal amide bond formation using the N-terminal amine (without an acetyl group) and C-terminal carboxylate that can be offered through use of resins such as Wang and 2-chlorotrityl chloride. Alternatively, substitution of the conventional C(Acm) groups with amino acids containing orthogonal chemistries that can bond with each other are an option. Canonical amino acid pair examples include Lysine and Glutamate, which could amide bond through their orthogonal side groups. Noncanonical amino acids pairs, such as ones containing an orthogonal azide and alkyne can engage in copper-mediated cycloaddition, to generate the macrocycle. A large number of other click chemistries are possible options, given they do not sterically hinder macrocyclization, chemically alter the rest of the peptide, inhibit target enzyme cleavage, or cause cytotoxicity.

  1. Transfer 1 g of resin into a 20 mL peptide synthesis vessel and add 15 mL of DMF. Cap the vessel and allow resin to swell on an orbital shaker for 1 h at room temperature.

  2. Drain the DMF under vacuum and wash with additional DMF (5 mL). Drain again.

  3. Deprotect the resin Fmoc group by adding Fmoc-deprotection solution (15 mL) and incubate on an orbital shaker for 5 min at room temperature, then drain the solution.

  4. Without rinsing, incubate an additional 15 mL of Fmoc-deprotection solution for 15 min at room temperature on the orbital shaker, then drain the solution.

  5. Remove excess 4-methylpiperidine by washing with DMF (3 × 5 mL), DCM (1 × 5 mL), and DMF (3 × 5 mL) again (see Note 3). Use of a squirt bottle to agitate the solution while under slow vacuum to remove solvent ensures complete rinsing.

  6. Prepare a coupling solution of the first amino acid, Cys(Acm), corresponding to the C-terminal amino acid of the final peptide. Combine the Fmoc-protected amino acid (3 eq), HBTU (98 mol%, 2.94 eq), and DIPEA (6 eq) in DMF (15 mL) for 1–2 min at room temperature, to form the activated ester.

  7. Add the coupling solution to the resin and incubate on the orbital shaker for 1 h at room temperature. Drain and rinse the resin with DMF (3 × 5 mL), DCM (1 × 5 mL), and DMF (3 × 5 mL).

  8. Double couple the first amino acid by repeating steps 6 and 7 (see Note 4).

  9. Cap any remaining amines on the resin that were not conjugated by the first amino acid by incubating acetic anhydride (30 eq) and DIPEA (30 eq) DMF (10 mL) for 15 min at room temperature on the orbital shaker (see Note 5).

  10. Repeat steps 3–7 for each additional amino acid incorporated during SPPS (see Note 6).

  11. Kaiser Test (optional): At any point, a Kaiser Test Kit can be used to determine if Fmoc deprotection is complete. Take a spatula tip-sized amount of peptide-loaded resin, transfer it to a small glass test tube, add a few drops of test solution, and apply brief heating with a heat gun (see Note 7). If the N-terminus is deprotected, ninhydrin from the test kit will form a deep purple-colored Schiff base. If the solution remains colorless, there are no primary amines exposed.

  12. After addition of the last amino acid, Cys(Acm), it is recommended that the resin be split into different reaction vessels for generating unlabeled (majority) and labeled (minority) cyclic progelators (see Note 8).

  13. Unlabeled precursor: The N-terminus should be temporarily protected (e.g., Fmoc) or permanently capped (e.g., acetylated) during peptide macrocyclization to avoid S-to-N acyl transfer reactions with a free primary amine [30]. In general, temporary Fmoc protection is recommended (see Note 9). Fmoc-protected linear precursors are obtained by skipping the final Fmoc-deprotection steps after addition of Cys(Acm). Acetyl-capped linear precursors are obtained by following the protocol in step 9.

  14. Labeled precursor: A rhodamine can be incorporated during the synthesis at the N-terminus of the sequence by deprotecting the Cyc(Acm) Fmoc group steps 3–5, followed by conjugation of 5(6)-carboxytetramethyl rhodamine to the N-terminal amine (see Note 10). Prepare a coupling cocktail of HBTU (1.47 eq) and HOBt (1.47 eq) in DMF at 0.45 M. Separately, incubate DIPEA (6.0 eq) with 5(6)-carboxytetramethyl rhodamine (1.5 eq) in DMF (10 mL) to generate the closed spirolactam configuration. Combine the coupling cocktail with the dye solution for 3 min at room temperature, before adding the mixture to the peptide-bound resin. Cover the reaction with aluminum foil and incubate overnight at room temperature (see Note 11).

  15. Dry the resin(s) by washing with DMF (3 × 5 mL) followed by MeOH (3 × 5 mL) and DCM (3 × 5 mL) washes to remove DMF. Dry the resin while open to air and under vacuum 5–10 min. This dry peptide-loaded resin should move freely without clumps (see Note 12). If not, repeat this step.

  16. Transfer peptide-loaded resin to a 50 mL conical tube, mix 40 mL of the peptide cleavage cocktail with DTT (500 mg) to release the peptide from resin and simultaneously deprotect side chains (except the Cys(Acm) group). To avoid build-up of pressure from production of gases during cleavage and the high vapor pressure of TFA, leave the solution open to air 10–15 s, then close the tube, invert, and vent gas (repeat venting 3×).

  17. Close the tube and place it on an orbital shaker. Incubate for 3 h at room temperature, adding 1 additional hour to the cleavage time for each arginine present (only applicable if Arg is used in the target sequence) to fully remove any 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf)-protecting groups.

  18. Clamp an open peptide synthesis vessel over a clean 50 mL conical centrifuge tube used for collection of filtrate, and remove the drainage cap. The frit will be used to retain the used resin while the filtrate drains.

  19. Remove the cleavage reaction tube from the orbital shaker and carefully vent before opening. Pour the contents into the peptide synthesis vessel and allow it to drain into a clean 50 mL conical centrifuge tube.

  20. Wash the resin with DCM (3× 2 mL) to collect additional filtrate.

  21. Concentrate the cleavage solution by gently blowing an inert gas (e.g., nitrogen) over the surface to obtain a concentrated yellow semisolid oil. Alternatively, the TFA can be removed through addition of DCM to the peptide in TFA solution, followed by careful rotary evaporation of both solvents. In both instances, good ventilation is necessary and should be performed in a fume hood.

  22. Add 40 mL chilled diethyl ether to the concentrate, close the tube, and vortex mix briefly (10–15 s) to precipitate the peptide, followed by sonication (5 min). Centrifuge at 5842 × g-force for 15 min at 8 °C, then pour off the supernatant. Repeat these steps 3× until a fluffy precipitate is formed. (see Note 13).

  23. Pull vacuum on the remaining precipitate (15–60 min at room temperature) to remove any remaining ether (see Note 14).

  24. Purify the resulting crude semi-protected peptide, containing Cys(Acm), by preparatory-phase HPLC, using a gradient of Buffer A (H2O, 0.1% v/v TFA or formic acid) and Buffer B (ACN, 0.1% v/v TFA or formic acid), and confirm identity by ESI-MS prior to macrocyclization (see Note 15). Refer to Subheading 3.3.

3.2. Solution-Phase Macrocyclization of Progelators

Steric constraint of SAP is a facile and modular engineering solution for preventing network assembly in a biological environment (Fig. 2). Nonviscous solutions of unlabeled and labeled progelators are simply achieved by oxidation of terminal cysteine residues of the functionalized SAP, which contains the substrate recognition sequence and either an Fmoc-protecting group or rhodamine dye on the N-terminus, respectively. Temporary Fmoc protection of unlabeled progelators is used to improve peak separation during purification and to monitor cyclization by LCMS or HPLC and ESI-MS.

Fig. 2.

Fig. 2

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Self-assembling peptide (SAP) macrocyclization produces a facile strategy for generating nonviscous progelator solutions. (a) Synthetic scheme for unlabeled (route I) and labeled (route II) KLDLCyclic progelators. SAPs are cyclized under dilute conditions (500 μM) with 5 eq I2 in a mixture of AcOH/MeOH/H2O to create soluble progelators. Route I uses temporary N-terminal Fmoc-protected SAPs to improve material separation during chromatographic purification. Route II depicts the synthesis of labeled progelators through N-terminal modification with rhodamine prior to cyclization. (b, c) High-performance liquid chromatography (HPLC) (b) monitored at 214 nm and corresponding electrospray ionization (ESI) mass spectra (c) of Fmoc-KLDLLinear (red), Fmoc-KLDLCyclic (blue), and KLDLCyclic (green) (835.79 m/z, 787.76 m/z, and 1070.26 m/z, respectively) verify mass identity and purity. (d) Photographs of KLDLLinear precursor as a hydrogel (left) and the resulting KLDLCyclic progelator as a soluble solution (right). Reproduced with modifications from Carlini et al. 2019 with permission from Springer Nature [27]

  1. Dissolve HPLC-purified semi-protected peptides in a mixture of AcOH/MeOH/H2O (1:16:4) at approximately 10–20 mg/mL peptide (see Note 16). The dissolved peptide is diluted with a 4:1 (v/v) solution of MeOH/H2O to a final concentration of 500 μM peptide. The target bulk solution pH is ~4–5 and can be confirmed with pH paper.

  2. Slowly add iodine (I2) dissolved in MeOH (0.1 M) dropwise to the solution until the yellow color persists (~4–5 eq) (see Note 17). Vigorously stir the reaction for ~2 h at room temperature, or until macrocyclization is complete. Minimize reaction time to avoid unwanted side reactions.

  3. Monitor cyclization kinetics by removing aliquots (300 μL) from the reaction solution at certain time points, adding a spatula tip quantity of anion exchange resin, and sonicating the solution (10 min) until it is clear (see Note 18). Decant the liquid and concentrate it 10× with rotary evaporation for analysis and purification by LCMS (see Subheading 3.3).

  4. After peptide macrocyclization is complete, add anion exchange resin to the bulk reaction solution (~1/3 volume) and stir vigorously for 1 h at room temperature (see Note 19).

  5. Filter the suspension of cyclic peptide progelators and wash the resin with small amounts of water and methanol. Remove excess acetic acid and methanol by rotary evaporation of the combined filtrate.

  6. Dilute the remaining solution with H2O, freeze with liquid nitrogen, and lyophilize it to a white powder (see Note 20).

  7. If applicable, N-terminal Fmoc groups can be removed at this stage by treating dry peptide with 1:1 (v/v) H2O/piperidine (200 μL per 2 μmol peptide) for 3 min at room temperature, followed by cooling the solution on ice. Slowly add an equal volume of TFA dropwise to quench the reaction, and then precipitate the peptide with chilled diethyl ether.

  8. Purify cyclic peptide progelators by prep-HPLC (see Subheading 3.3).

3.3. Peptide Purification and Analysis

Both semi-protected linear precursors as well as cyclic progelators are purified through reverse-phase high-performance liquid chromatography (RP-HPLC). Prior to injection, each peptide is first dissolved at concentrated conditions in a small amount (2–10%) of organic solvent such as AcOH, DMSO, or MeOH in which the peptide has high solubility (see Note 21). Then the solution is diluted with running buffer for injection onto the LC column. Three forms of liquid chromatography are useful for the analysis and purification of these peptides: (1) LCMS, (2) analytical HPLC, and (3) prep-HPLC. LCMS is primarily used for rapid assessment of peptide purity and macrocyclization kinetics (see Note 22). Analytical HPLC is used for determining the necessary elution gradient for prep-HPLC, as it requires small quantities of material and utilizes the identical solvent conditions. Prep-HPLC is used for purifying large quantities of peptide (~200 mg) per run.

A typical elution profile on HPLC or LCMS involves four phases: (1) an isocratic equilibration phase, (2) the gradient phase, (3) the wash phase, and (4) the re-equilibration phase. Dissolved peptide is injected into a column and a solvent gradient, containing Buffer A (H2O, 0.1% v/v TFA or formic acid) and Buffer B (ACN, 0.1% v/v TFA or formic acid), is applied by initially running mostly Buffer A with increasing concentrations of Buffer B over time. Liquid chromatography conditions for the purification of a representative peptide, Fmoc-KLDLLinear, are shown in Table 1. In general, the % Buffer B used at the beginning of the gradient phase is the same value used for dissolution of the peptide and the equilibration steps (see Note 23). The general protocol for KLDLCyclic bulk purification by prep-HPLC with a 20-mL injection loop.

Table 1.

Representative liquid chromatography conditions for analysis and/or purification of Fmoc-KLDLLinear. by liquid chromatography mass spectrometry (LCMS), analytical high-pressure liquid chromatography (HPLC), and preparatory-phase HPLC (prep-HPLC). Gradients ranges represent continuous change over the given time of Buffer B (vol %) in the elution solvent

Buffer A Buffer B Equilibration phase Gradient phase Wash phase Re-equilibration phase Volinjection
LCMS H2O (0.1% v/v formic acid) ACN (0.1% v/v formic acid) 15% 0.5 min 15–35% over 7 min 35–95% over 1 min; 95% 1 min 95–15% over 0.5 min 2–4 μL
Analytical HPLC H2O (0.1% v/v TFA) (0.1% v/v TFA) 30% 3 min 30–50% over 30 min 50–90% over 3 min; 90% over 3 min 90–30% over 1 min; 30% over 3 min 50–80 μL
Prep-HPLC H2O (0.1% v/v TFA) (0.1% v/v TFA) 30% 7 min 30–50% over 43 min 50–90% over 10 min; 90% 5 min 90–30% over 5 min; 30% 5 min 20 mL
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  1. Dissolve the peptide (~200 mg) in AcOH (2 mL) in a glass vial with sonication and heating (see Note 24).

  2. Once dissolved, dilute the solution with Buffers A and B to a max volume of 20 mL until the solution contains 25% (v/v) Buffer B. The final injection mixture is 10:65:25% AcOH/Buffer A/Buffer B, respectively.

  3. Filter the sample through a 0.45 μm pore size PES filter (up to 5×) to remove insoluble aggregates and shear apart assemblies. A well dispersed sample should have low viscosity and allow most material to pass through the filter.

  4. Keep the sample under constant sonication and use heating until loop injection onto the column to prevent self-assembly (see Note 25).

  5. Collect fractions into glass collecting tubes and tube rack. Test each fraction by ESI-MS or LCMS for correct mass and purity. Combine fractions corresponding to the correct product.

  6. Lyophilize the combined fractions to a powder for later use. Store the powder in a dry, sealed 50 mL conical centrifuge tube at —20 °C until further use.

These conditions can be applied to other peptides sequences, with primary modifications being made to the elution gradient percentages used, in order to have full peak separation of the expected product from other impurities as well as a column retention time that occurs during gradient phase (see Note 26).

Table 2 shows selected HPLC elution settings for each peptide discussed in this chapter (see Note 27).

Table 2.

Analytical HPLC conditions for each adduct generated during the synthesis of unlabeled and labeled cyclic KLDL. Rhodamine-labeled peptides elute as two isomeric peaks containing either 5- or 6-carboxytetramethyl rhodamine on the N-terminus

Analytical HPLC gradient Rt (min) UV detection λ (nm)
Fmoc-KLDLLinear 30–50% over 30 min 20.5 min 214, 290 nm
Fmoc-KLDLCyclic 30–50% over 30 min 20.0 min 214, 290 nm
KLDLCyclic 25–45% over 30 min 15.5 min 214 nm
Rho-KLDL Linear 27–37% over 30 min 16.0/20.2 min 214, 565 nm
Rho-KLDLCyclic 27–37% over 30 min 16.6/22.2 min 214, 565 nm
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3.4. Progelator Sterilization and Formulation (See Note 28)

  1. Dissolve lyophilized HPLC-purified peptides into H2O using half volume of the dialysis tubing capacity. Use of additives such as ACN, MeOH, DMSO, NH4OH, and/or denaturing salts is acceptable as they will be removed during dialysis. Pipette the solution into 1 kDa dialysis tubing submerged in 2 L H2O to remove additives TFA from HPLC (see Note 29). Cover all dialysis solutions with aluminum foil.

  2. Equilibrate dialysis tubing 12–24 h, then replace the 2 L bath with fresh H2O (repeat 3×).

  3. Remove the peptide solutions and sterilize them through a 0.22 μm PES filter. Lyophilize the filtrate to a powder for long-term storage.

  4. Reconstitute peptides prior to injection with sterile pH 9 H2O to form a clear solution (11.11 mM), then dilute further with sterile 10× DPBS to a final concentration of 10 mM peptide in 1× DPBS (pH 7.4) or appropriate enzyme cleavage buffer. (see Note 30).

3.5. Enzymatic Cleavage of Cyclic Peptide Progelators (See Note 31)

  1. Test enzyme responsiveness of progelator by performing cleavage experiments at a final peptide concentration of 500 μM in 1× enzyme buffers (see Note 32).

  2. To ensure longevity of enzyme stocks, keep frozen at the recommended temperature (−20 or −80 °C) in separated aliquot vials. Avoid thawing and refreezing enzyme stock solutions. Prior to use in a cleavage reaction, thaw aliquots of enzyme solution in an ice bath (see Note 33).

  3. Dissolve cyclic progelator peptides in the appropriate 1× enzyme buffer at concentration determined in step 1. Dilution from a concentrated formulation in H2O into 1× enzyme buffer is generally preferred to dilution from 1× DPBS due to possible solvent incompatibility or risk of peptide flocculation during a rapid solvent switch.

  4. Incubate the peptide in 1× enzyme buffer solution at the 37 °C (for MMP-9 and elastase) or 65 °C (for thermolysin) for several min in a dry bath block heater to equilibrate the solution temperature.

  5. MMP-9 cleavage: incubate MMP-9 at 1:1000 enzyme/substrate molar ratio in the peptide solution containing 1× MMP-9 buffer for 5 h at 37 °C. Denature MMP-9 to halt the reaction by incubating at 65 °C for 10 min.

  6. Elastase cleavage: incubate elastase at 1:250 enzyme/substrate molar ratio in the peptide solution containing 1× elastase buffer for 5 h at 37 °C. Denature elastase to halt the reaction by incubating at 65 °C for 10 min.

  7. Thermolysin cleavage: incubate thermolysin at 1:4500 enzyme/substrate molar ratio in the peptide solution containing 1× thermolysin buffer for 15 min at 65 °C (see Note 34). Inactivate thermolysin by incubation with 10% (v/v) EDTA solution.

  8. Analyze cleavage products by HPLC coupled with ESI and/or LCMS (see Notes 35 and 36, Subheading 3.3).

  9. Incubation with MMP-9 catalytic domain and porcine elastase cleaves at a single location and produces a single product (see Fig. 1). Thermolysin has several possible potential cut sites along the recognition sequence, producing multiple peaks by LCMS.

3.6. Sample Preparation for Instrumental Analysis

Below are instructions on sample preparation for various instrumental analysis techniques that can be used for confirming studying cyclic peptide progelators and their self-assembly following enzyme activation. Materials required for these techniques are not listed above in the Materials (Subheading 2), as operation of these instruments are outside of the scope of this chapter. Detailed information is provided in Carlini et al. [27]

  1. Transmission electron microscopy (TEM): This technique enables dilute-scale visualization of assemblies at nanoscale resolution. Dilute peptide sample (100 μM) 5× from enzyme cleavage reaction from Subheading 3.5. Use 4–5 μL per TEM grid.

  2. Static and dynamic light scattering (SLS and DLS): Prepare peptide at 1 mg/mL in buffer of interest (e.g., 1× DPBS at pH 7.4 and 37 °C). Perform thermolysin cleavage by incubation at 1:4500 enzyme/substrate molar ratio over a course of several min to hours. Blank samples require solvent with enzyme only. Sample volumes are typically ~50–2000 μL, depending on cuvette geometry.

  3. Circular dichroism (CD): Prepare peptides at ~125–500 μM in 10 mM Tris buffer (pH 7.4, 37 °C). Salts or other buffers such as DPBS cause signal interference during analysis of secondary peptide structures. Concentration or cuvette pathlength can be varied to ensure that sample voltage does not exceed 800 mV within the wavelength range of interest.

  4. Oscillatory rheology: Prepare peptides at 10 mM in 1× DPBS (pH 7.4, 37 °C). A sufficient volume of ~350 μL is necessary for analysis of each sample with a 20 mm diameter parallel plate geometry and gap height of 1000 μm. To initiate thermolysin cleavage, mix the sample with thermolysin (1:4500 enzyme/substrate molar ratio) for 5–15 s via pipette followed by gentle flash centrifugation with a tabletop spinner for 2 s to remove air bubbles. Apply the peptide solution to the rheometer stage with gap height set and deposit mineral oil around the sample to prevent dehydration during rheological measurements.

  5. Catheter injection: Prepare peptides (0.6–0.8 mL) at 10 mM in 1× DPBS (pH 7.4). Load the solution into a 1 mL Leur Lock syringe attached to a syringe pump set to a flow rate of 0.6 mL/min. Inject peptide solution through the 27G inner nitinol tubing of a MyoStar catheter that is immersed in a 37 °C water bath.

Acknowledgments

The authors are grateful for the support of an NHLBI (R01HL139001), an AFORS MURI (FA9550-16-1-0150), and an ARO MURI (W911NF-15-1-0568).

4 Notes

1.

Any number of Fmoc-protected amino acids can be incorporated into the growing chain. Reactive side groups (e.g., amines, hydroxyl, thiols, carboxylates, carboxamide) should be protected during solid-phase chain growth. In the case of sequences that require enhanced proteolytic protection at certain locations, simple substitution with D-amino acid enantiomer adducts or unnatural amino acids can be utilized. However, proceed with care upon lengthening the peptide sequence significantly, such that steric constraint from macrocyclization ensures that progelators do not self-assemble.

2.

Linear peptide precursors can also be synthesized automatically with a peptide synthesizer, which uses nitrogen gas to direct flow of solvents and reagents. It should be noted that use of reagents with high emission coefficients and low solubility on the synthesizer are avoided due to risks ofclogging and staining of instrument tubing. For these steps, as well as cleavage and solution-phase peptide synthesis, manual synthesis is preferred.

3.

Use of DCM is optional and may risk dehydrating the resin if left to dry open to air too long. Take care to keep resin swollen for best yield.

4.

Double coupling is a useful strategy for incorporation of the first amino acid or unusual amino acids that have low coupling efficiencies. Increasing coupling times from 1 to 2 h may also be convenient for more expensive amino acids.

5.

Capping with acetic anhydride is used to improve peptide purity by introducing an inert acetyl group on the N-terminal amine of resin or growing peptide. This prevents further growth of peptides that did not incorporate the previous amino acid, leading to incorrect sequences. Capping can be performed to react with free amines following any coupling step and prior to Fmoc deprotection.

6.

As the peptide continues to grow, aggregation of the resin-bound peptides can limit access of incoming amino acids to the N-terminus. This is especially prevalent with SAPs. Incubation of coupling solutions with chaotropic salts like LiCl (0.4 M) can enhance peptide solubility on resin, and thus swelling [31].

7.

Excess heating of the peptide in ninhydrin solution can cause unwanted Fmoc deprotection and result in a false-positive color change. Additionally, the Kaiser test is not reliable for detecting secondary amines (e.g., proline).

8.

As labels are not intended for participation in self-assembly, they may lower bulk solubility or resulting hydrogel strength depending on the tag used. Thus, it is advised to use only a small molar percentage (0.1–10%) of tagged materials in comparison to unlabeled materials when preparing bulk progelator solutions.

9.

Temporarily leaving an Fmoc on the N-terminus of unlabeled linear precursors and the resulting cyclic progelator can significantly improve peak separation by LCMS and HPLC while monitoring cyclization efficiency and peptide purification, respectively.

10.

5(6)-carboxytetramethyl rhodamine was synthesized as previously described [32]. Dye-labeled cyclic progelators are used for visualization in in vitro and in vivo experiments. This moiety can be substituted with any number of small molecule tags such as contrast agents (e.g., NIR, MRI, PET) or functional handles (e.g., azide, biotin tag), given an available carboxylic acid for amide bond formation at the N-terminal amine of the cyclic peptide progelators. LiCl solution (0.4 M) can be used to help unfold SAPs when adding bulky tags.

11.

If necessary, perform double coupling or increasing the number of equivalents of dye to improve yield.

12.

Dry peptide-loaded resin, containing an N-terminal Fmoc and side chain protecting groups, can be stored in a moisture-free container at −20 °C for several years without adverse effects on purity.

13.

In some instances, the peptide will not precipitate completely and retain a gel-like or sticky consistency despite efforts to precipitate. This can be broken up with addition of 1–2 mL of chilled methanol to the diethyl ether solution, followed by similar vortexing, sonication, and centrifugation steps. Getting a dispersed precipitate is a very important initial step for purification.

14.

This can simply be achieved by applying a large rubber septum upside down to seal the 50-mL conical tube, piercing it with a low gauge needle, and pulling vacuum on the sample. In the initial few seconds, take care to pull the vacuum slowly as the precipitate will dry rapidly and trapped ether may cause the dry sample to bump. If the sample of semi-protected peptide does not appear as a fluffy powder, there may be residual solvents or oily cleavage products remaining. Repeat steps 23 and 24 with ether washes. Solubility, purification, and macrocyclization efficiency of this semi-protected peptide is directly impacted by how well the crude material is cleaned, washed, and dried in this step.

15.

The cyclic peptide isomer elutes at similar retention times (Rt) to that of the linear precursor. Any initial impurities from the linear adduct may be difficult to remove later.

16.

Complete dissolution is extremely important during this process as it minimizes the possibility of dimerization. Dissolution in AcOH/MeOH/H2O (1:16:4) may be easiest if starting in pure AcOH (1–2 mL for every ~200–400 mg peptide) with sonication and mild heating (~1–2 h). Subsequent addition of MeOH and H2O with continued sonication and mild heating has been effective. Use of a homogenizer or probe sonicator may also be useful in breaking up aggregates. In some cases, shearing forces through a high-gauge needle can also be applied. Importantly, if dissolution is still not possible, either the total volume of this mixture or ratio of components may be adjusted.

17.

To synthesize cyclic progelators, iodine is used to simultaneously deprotect Cys(Acm) and initiate disulfide bond formation under dilute conditions to favor intramolecular macrocyclization [33].

18.

Amberlite IRA-400 chloride exchange resin halts the macrocyclization reaction by quenching unreacted I2, sequestering iodide byproducts.

19.

Although efficient, chloride exchange resin may produce peptides with chloride counter ions that limit overall solubility. If so, the chloride exchange resin can be converted to an acetate exchange resin by simply stirring resin in 1.6 N AcOH (10 mL for every 245 mg resin) for 1 h, then washing 3× with 0.16 N AcOH (10 mL for every 245 mg resin). This acetate exchange resin can be immediately used to quench macrocyclization reactions.

20.

If the solution thaws rapidly on the lyophilizer, excess methanol or acetic acid may be present. Try removing these volatile solvents with additional rotary evaporation and/or dilution with more H2O prior to lyophilization.

21.

For determining the best elution gradient and solvation conditions, test a variety of solvent conditions on small quantities of crude peptide material (<1 mg).

22.

The running solvent for LCMS utilizes formic acid to improve mass detection but sacrifices peptide peak separation. Thus, this elution gradients used for LCMS may not be optimal for prep-phase HPLC.

23.

Keep the percentage of Buffer B to a minimum, or else the peptides will elute from the column too soon without separation.

24.

Use shearing forces via aspiration through a needle to break up larger aggregates.

25.

This is necessary to avoid unwanted peptide precipitation and subsequent clogging on the column.

26.

Within increasing hydrophobicity, higher % Buffer B values are necessary. With difficult to separate peaks (e.g., isomers), narrowing the gradient percentages from Δ20% to Δ10–15% may be useful. Furthermore, increasing flow rates (higher pressure) as well as adding a secondary gradient phase may prevent peptide spreading, and thus lower peak resolution, on the column. High-pressure shearing forces during HPLC purification minimizes self-assembly of peptides into dimers, trimers, or other multimers that can appear as low intensity overlapping peaks during purification.

27.

UV detection of all peptides are monitored at 214 nm for total peptide content. Additional wavelengths of use are 290 nm for the Fmoc-protecting group, and 565 nm for the rhodamine label. In some instances, 265 nm is useful for peptides containing aromatic residues such as phenylalanine. Finally, ESI mass spectra of collected fractions from analytical and preparatory-phase HPLC are used to determine sample identity. Due to the large number of cis/trans configurational isomers that cyclic progelators could adopt, and the amphiphilic self-assembling nature of these peptides, peak resolution via HPLC may be low in some instances. Therefore, various other mass spectrometry techniques can be used to definitively identify and verify purity of isolated materials, such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), high resolution mass spectrometry (HRMS), and tandem mass spectrometry (tandem-MS).

28.

It is recommended that stock solutions for biological applications be prepared fresh from sterile and desalted powder. However, formulated stock solutions in 1× DPBS are stable up to 3 days at 8 °C under dilute (100 μM) and concentrated (10 mM) conditions.

29.

Excess TFA salts from HPLC purification may be difficult to remove and cause the peptide solution to remain very acidic despite several rounds of dialysis. Addition of NH4OH to neutralize or lightly basify (pH 9.0) the peptide solution prior to dialysis can be very helpful.

30.

Use of nontoxic quantities of excipients such as DMSO, sucrose, or propylene glycol, can also improve peptide solubility at these concentrations.

31.

The engineered cyclic peptide progelators flow freely in solution and resist assembly into hydrogels until acted upon by enzymes such as MMP-9, elastase, or thermolysin. Enzymatic cleavage by any of these three enzymes induces peptide linearization. Various other proteases can be targeted through simple modification of the substrate recognition sequence during peptide synthesis in Subheading 3.1.

32.

Scale and peptide concentrations can be increased accordingly. Note that with higher concentrations of peptide, diffusion of proteases through solution may be slowed by increasing solidlike behavior of the activated hydrogel. Increasing enzyme concentration, activity doses, or incubation time may aid with in vitro experiments.

33.

Some enzymes have the capacity to digest themselves through autolysis, so keeping stock solutions under reduced temperatures (0 °C) immediately prior to use can maintain activity levels.

34.

Thermolysin is a notoriously robust enzyme that optimally operates with high activity at elevated heat conditions and for prolonged periods. However, this enzyme can operate even at room temperature and in a variety of solvents, such as 1× DPBS (pH 7.4).

35.

In the case of excess proteolysis, there might be smaller peptides that are cleaved. Depending on the resulting sequence and their hydrophilic to hydrophobic content, their solubility could be high or low. Altering the HPLC or LCMS running conditions (e.g., peptide concentration, buffer ratio, pressure, run time, gradient, and choice of column) can enable complete separation of each species. In general, however, once the enzyme substrate sequence has been initially cleaved to produce the linear gelator, self-assembly is rapid. Formation of secondary structures, which entangle to form porous hydrogel networks, incurs resistance to further proteolysis. In our studies, this holds true, with limited additional proteolysis only in the presence of robust enzymes (e.g., thermolysin) at sites of amino acids that do not engage in self-assembly (e.g., remaining recognition sequence). Aliquots analyzed at 0, 1, 5, 10, 30, 60, 120, 210, and 300 min are helpful time points for following peptide cleavage kinetics as well as identifying different product populations from proteases that may recognize multiple locations along the peptide backbone. Some enzymes have the capacity to digest themselves through autolysis, so keeping stock solutions under reduced temperatures (0 °C) immediately prior to use can maintain activity levels.

36.

In selecting specific protease targets or considering biological applications, concerns may arise that the SAP sequence intended for responsive hydrogel formation may be rapidly degraded as well. With many SAP sequences, intermolecular electrostatic interactions, hydrophobic forces, and/or aromatic э-э stacking, significantly lower their proteolytic susceptibility in comparison to soluble monomeric peptide sequences. Conversely, an appended recognition sequence should be more accessible for proteolysis and enable progelator linearization and resulting self-assembly. As a result, the proteolytically susceptible cyclic progelators should exhibit enhanced proteolytic resistance following enzyme-responsive activation.

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