Injectable tissue integrating networks from recombinant polypeptides with tunable order

Abstract

Emergent properties of natural biomaterials result from the collective effects of nanoscale interactions among ordered and disordered domains. Here, using recombinant sequence design, we have created a set of partially ordered polypeptides to study emergent hierarchical structures by precisely encoding nanoscale order-disorder interactions. These materials, which combine the stimuli-responsiveness of disordered elastin-like polypeptides and the structural stability of polyalanine helices, are thermally responsive with tunable thermal hysteresis and the ability to reversibly form porous, viscoelastic networks above threshold temperatures. Through coarse-grain simulations, we show that hysteresis arises from physical crosslinking due to mesoscale phase separation of ordered and disordered domains. On injection of partially ordered polypeptides designed to transition at body temperature, they form stable, porous scaffolds that rapidly integrate into surrounding tissue with minimal inflammation and a high degree of vascularization. Sequence-level modulation of structural order and disorder is an untapped principle for the design of functional protein-based biomaterials.

Figure 1:. Partially ordered polymer library and structural characterization

(a) Recombinant POPs were constructed with 3 ELP components and 4 polyalanine helices at amino acid percentages up to 50%. UV-CD reveals definitive helical peaks at 222 and 208 nm, with peak amplitudes minimally altered by (b) polyalanine domain and (c) ELP but highly dependent on (d) total alanine content (dynode voltage >500 at <200nm; data not used for analysis). (e) This structural signature is consistent with helix-coil predictions (Agadir). (f) ¹⁵N-HSQC and (g) H(N)CO (residue labels are the associated C’ of the previous residue) 2D solution NMR spectra for E1-H2–25% were used to more precisely quantify total structural content. Each polyalanine domain was determined to have an average helicity of 90% (Supplementary Methods).

Figure 2:. Phase behavior and tunable hysteresis

(a) OD measurements as a function of temperature show sharp, reversible phase behavior and hysteresis (ΔT_t). Hysteresis scales as a function of total helical content. (b-d) For a given E(X), the composition of the alanine domain modulates the T_t-heating and T_t-cooling with greater hydrophilicity leading to increased temperatures. Hysteresis is also dependent on the composition (charge distribution) of the polyalanine domains with an increase in charge producing a decrease in hysteresis. The T_t-cooling is concentration independent and solely determined by the polyalanine domains. (e) Therefore, for a given H(X), the T_t-heating can be independently controlled with ELP composition, providing a method to orthogonally control T_t-heating and T_t-cooling. (f) Polymers can be cyclically heated and cooled with no change in thermal behavior. Optical density measurements were taken at 350nm in PBS at 50μM unless otherwise indicated. Heating and cooling rates were kept at 1°C/min. OD amplitudes are non-interpretable due to difference in aggregate formation and settling.

Figure 3:. Proposed mechanism for hysteresis

(a) Simulations of the hysteretic cycle were performed using a coarse-grained bead-spring model for the POPs. Heating and cooling were achieved by modulating the interaction strengths between ELP domains. The interactions become favorable as temperature increases and the converse is true upon cooling. Snapshots extracted from phenomenological simulations of POPs are shown in the middle, surrounded by cartoon representations of the four states observed for POP during heating and cooling. Rod-like objects represent polyalanine domains and string-like tethers represent ELPs. The colors indicate their initial cluster with shading indicating different proteins in the same initial cluster. The one-sided arrows provide a pictorial summary of the expected rates for transitions between different states (fast for 2–3 and slow for 4–1). Within entangled aggregates we observe two types of morphologies viz., entangled spheres or entangled cylinders. There is a reversible spheres to cylinders transition at even higher temperatures. (b) A sketch of the experimental observable as a function of heating / cooling viz., the optical density is annotated by the species populating each regime. (c-d) Enlarged snapshots from the cooling arm of panel (a) demonstrate that the highlighted POP is not able to isolate itself into a single cluster and that the decrease in aggregate density is limited by the presence of domain swapped proteins.

Figure 4:. Arrested phase separation into fractal networks

(a) E1-H5–25% (2 mM, PBS) aggregation during a heating and cooling cycle shows a reversible transition from an optically translucent liquid to an opaque solid-like structure (passes inversion test) with syneresis observed at higher temperatures. (b) At the microscale, E1 and E1-H5–25% (400 μM, PBS) form liquid-like coacervates and fractal networks, respectively; scale bar 50 μm and 10μM for insert. (c) The intricacy of the network is more clearly seen with a 20 μm thick 3D reconstruction of E1-H5–25% (200 μM, PBS); scale bar 50 μM and 10 μM for the insert. (d) Network architecture at the meso scale is that of interconnected beads, as revealed by SIM; scale bars 10 μm (left) and 1 μm (right).

Figure 5:. Network stability and void volume

(a) As determined by the limited fluorescence recovery 25 min after bleaching, 12.5% and 25% networks have a high kinetic stability and limited liquid-like properties; Inset pictures are shown for E1-H5–25% at 400 μM. (b-c) Void volumes can be tuned from 60–90% by altering polymer concentration. Data represent mean ± SEM (n=4 independent samples). Scale bars are 50 μm.

Figure 6:. In vivo stability and tissue incorporation of POPs

(a) E1-H5–25% POP s.c. injections were significantly more stable than their E1 counterparts with just 5% of the injected dose degraded at 120hrs; 200μl 250μM injections; p<0.05 for all data points after 0hr, determined by two-tailed t-tests (n=6 mice); data represent mean ± SEM. (b) Whereas ELPs diffuse into the s.c. space, POP depots were externally apparent, retaining the shape and volume of the initial injection up to dissection and ex vivo analysis. (c) Representative CT-SPECT images of the depots confirm increased diffusivity of ELPs and increased stability of POPs. (d) POPs were injected into BL/6 mice and explanted for analysis over 21 days. Representative images are shown with arrows pointing at externally evident vascularization. Scale bars 5mm. (e) POPs rapidly integrated into the subcutaneous environment with sufficient strength to endure moderate extension less than 24 hours after injection. (f) There is a high initial cell incorporation with some change over the observed time periods; for *, p<0.05 determined by ANOVA with Tukey post-hoc (D1 n=3, D3–21 n=4); data presented as 10–90% box plots. (g) Flow cytometry for cells involved in the innate immune reveals subsequent spikes in neutrophils, inflammatory monocytes, and macrophages, with a loss in all hematopoietic cells (CD45+) by day 21; for *, p<0.05 determined by ANOVA with Tukey post-hoc (D1 n=3, D3–21 n=4); data represent mean ± SEM. (i) The loss in inflammation corresponds with an increase in vascularization, quantified by number of visible capillaries in histological sections; for *, p<0.05 as determined by ANOVA with Tukey post-hoc (n=3); data represent mean ± SEM. (j) An example tissue slice 10 days post injection shows an area of particularly high vascularization density (scale bar 100μm). Additional images given in the supplementary information.