UV-responsive cyclic peptide progelator bioinks

Abstract

We describe cyclic peptide progelators which cleave in response to UV light to generate linearized peptides which then self-assemble into gel networks. Cyclic peptide progelators were synthesized, where the peptides were sterically constrained, but upon UV irradiation, predictable cleavage products were generated. Amino acid sequences and formulation conditions were altered to tune the mechanical properties of the resulting gels. Characterization of the resulting morphologies and chemistry was achieved through liquid phase and standard TEM methods, combined with matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).

Fig. 1

Schematic of UV-responsive ink activation and characterization. (a and b) Sterically constrained cyclic peptide linearize in response to UV activation, and self-assemble through amphiphilic interactions. (b) Bulk scale activation from a solution of cyclic peptide ink provides a means for 4D printing of an ECM-like gel comprised of (c) entangled nanofiber networks. (d) Characterization of silicon nitride (SiN_x) chip surfaces to explore morphology by transmission electron microscopy (TEM) and chemical signatures by matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).

Fig. 2

Design of the self-assembling peptide gelator. (a) Schematic of the self-assembling peptide gelator, with amino acid sequence Ac-GFFFLGSGS, showing that the peptide and salt concentration are directly correlated to unimer packing and shear forces induce disassembly. (b) Fibrous morphology of the gelator (100 μM) by dry state TEM. (c–e) Bulk scale rheological characterization of the gelator (15 mg mL⁻¹). Frequency sweeps showing storage (G′) and loss (G″) moduli of gelator prepared in (c) H₂O (pH 6.4) and (d) 1× PBS (pH 7.4). (e) Corresponding complex viscosity as a function of shear rate.

Fig. 3

Photoactivation of a linear ink analogue produces reliable cleavage products. (a) Linear ink analogue with photocleavable ANP residue. (b) Proposed photocleavage mechanism showing conversion of the (1) starting material to a (2) nitroso compound, (3) dehydrated product, and (4) methanol adduct. (c) HPLC spectra vial pictures of linear ink analogue during cleavage at 0, 1, and 3 h. Peaks labeled according to (b). (d) Corresponding observed and expected mass spectra of peaks.

Fig. 4

Peptide macrocyclization to generate photocleavable inks. (a) Synthetic scheme for the generation of macrocycle, cyc(ANP) from the resin-bound peptide. Synthesis involved (1) cleavage from resin and Lys(Mtt) deprotection, (2) amide bond formation between the Lys R-group amine and C-terminal carboxylate under dilute conditions (500 μM in DMF), and (3) serine R-group deprotection. (b) LCMS of fully deprotected peptides at 0 h and 24 h of cyclization. (c) Corresponding ESI mass spectra. (d) Table of expected and observed masses with corresponding mass identities.

Fig. 5

UV cleavage of cyclic peptide ink. (a) Chemical structures of cyclic peptide (1) proposed cleavage products (2, 3, and 4). (b) HPLC spectra of UV-treated ink after 0, 1, 2, 3, and 4 h of UV (365 nm) treatment, with relevant peaks labeled. (c) Corresponding ESI mass spectra of collected peaks from (b).

Fig. 6

Morphological analysis of cyc(ANP) ink with and without UV treatment. (a and b) Formulation 1 conditions: dissolution in H₂O/ACN (66 : 33% v/v) with heating (60 °C), and heating (80 °C) to remove acetonitrile. (a) Picture of vial flips from (left) untreated and (right) treated peptide sample (10 mg mL⁻¹). (b) Corresponding dry state TEM of (left) untreated and (right) treated samples (500 μM). (c and d) Formulation 2 conditions: dissolution in H₂O/ACN/AcOH (85 : 10 : 5% v/v) with heating (60 °C). Neutralization to pH6.4 with NH₄OH. (c) Picture of vial flips from (left) untreated and (right) treated peptide sample (10 mg mL⁻¹). (d) Corresponding dry state TEM of (left) untreated and (right) treated samples (500 μM).

Fig. 7

Dry-state TEM imaging and MALDI-IMS mapping of linGelator-II. (a) Conventional dry-state, uranyl acetate stained TEM image of linGelator-II fibers. (b) Dry-state, unstained image of linGelator-II fibers on a SiN_x chip. (c) MALDI-IMS map of the surface of the chip shown in (b) with mass filter for linGelator-II (1089 Da ± 5 Da) applied. (d) Optical picture of linGelator-II spot on ITO slide with MALDI-IMS measurement region outlined (top) and corresponding MALDI-IMS map with mass filter for linGelator-II applied (bottom). (e) Overall MALDI mass spectra for linGelator-II spot (bottom) and chip surface (top).

Fig. 8

Activating peptide assembly with the electron beam of a TEM. (a and b) LCTEM snapshots of a region of a liquid cell containing cyc(ANP), where t = 0 is when the region imaged was first irradiated with the electron beam. Images at (a) 30 s and (b) 2 min 33 s are shown. Arrows point out one structure change over time. Electron flux = 0.1 e⁻/Ų s. (c and d) Snapshots of another region of the same liquid cell at (c) 10 s and (d) 1 min 24 s. (e and f) MALDI-IMS 2D mapping of the two chip surfaces from the liquid cell experiment shown in (a–d). Applied mass filter for (e) cyc(ANP) (1263 ± 5%) and (f) a new previously unobserved mass, (880 ± 5%). (g) Zoom-in of a region of interest on the chip surface shown in (f, left). (h) Zoom-in of a region of interest on the chip surface shown in (f, right). (i) Optical picture and MALDI-IMS 2D map overlay of a dried spot of UV peptide. (j) 2D map overlay only of the dried spot of cyc(ANP) shown in (i). (k) MALDI spectra for cyc(ANP) (purple), the entire surface of chip 1 (red), the entire surface of chip 2 (green), the window of chip 1 (teal), the window of chip 2 (blue).