Interaction of hyaluronan binding peptides with glycosaminoglycans in poly(ethylene glycol) hydrogels

Abstract

This study investigates the incorporation of hyaluronan (HA) binding peptides into poly(ethylene glycol) (PEG) hydrogels as a mechanism to bind and retain hyaluronan for applications in tissue engineering. The specificity of the peptide sequence (native RYPISRPRKRC vs non-native RPSRPRIRYKC), the role of basic amino acids, and specificity to hyaluronan over other GAGs in contributing to the peptide-hyaluronan interaction were probed through experiments and simulations. Hydrogels containing the native or non-native peptide retained hyaluronan in a dose-dependent manner. Ionic interactions were the dominating mechanism. In diH2O the peptides interacted strongly with HA and chondroitin sulfate, but in phosphate buffered saline the peptides interacted more strongly with HA. For cartilage tissue engineering, chondrocyte-laden PEG hydrogels containing increasing amounts of HA binding peptide and exogenous HA had increased retention and decreased loss of cell-secreted proteoglycans in and from the hydrogel at 28 days. This new matrix-interactive hydrogel platform holds promise for tissue regeneration.

Figure 1

(a) Macromolecular monomers used for the fabrication of PEG-based gels. The precursors included PEGTNB (n ∼ 30), the dithiol linker PEG (n ∼ 80), and cysteine-terminated peptide. (b) Depiction of an idealized cross-linked network formed these macromolecular monomers. (c) Reaction scheme between the thiol containing monomer with the norbornene containing monomer. R denotes PEG and R¹ denotes PEG or peptide.

Figure 2

Hyaluronan (HA) loading and retention capabilities of a HA binding peptide (RYPISRPRKRC), non-native HA binding peptide (RPSRPRIRYKC), and charge control peptide (GYPISGPGGGC). (a) Schematic of the experimental setup (f-HA loading for 48 h, f-HA release in diH₂O for 48 h). (b) Representative images of hydrogels (containing the native HA binding peptide or charge control peptide) in solution corresponding to each 48 h step (i.e., the latter two images of the experimental setup). The fluorescein can be visualized by the yellow color showing retention of the f-HA in hydrogels with the native HA binding peptide in diH₂O and with the charge control peptide showing continued release of f-HA in diH₂O. (c) Total amount of f-HA loading into hydrogels after 48 h of immersion in a solution of 100 μg of f-HA in diH₂O. (d) Release of loaded f-HA after 48 h of immersion in diH₂O. (e) Percent-retained of loaded f-HA after immersion in diH₂O. Data represent mean(SD) with a sample size of 5; *indicates samples were compared to no peptide control (0 mM).

Figure 3

Chondroitin Sulfate (ChS) loading and retention capabilities of a HA binding peptide (RYPISRPRKRC), non-native HA binding peptide (RPSRPRIRYKC), and charge control peptide (GYPISGPGGGC). (a) Total amount of ChS loading into hydrogels after 48 h of immersion in a solution of 100 μg of ChS in diH₂O. (b) Release of loaded ChS after 48 h of immersion in diH₂O. (c) Percent-retained of loaded ChS after immersion in diH₂O. Data represent mean(SD) with a sample size of 5; *indicates samples were compared to no peptide control (0 mM).

Figure 4

Loading of (a) f-HA and (b) ChS over 144 h (6 days) for the native peptide (square, solid line), non-native peptide (×, dashed line), control peptide (circle, solid line), and no peptide (diamond, dashed line).

Figure 5

Amount of GAG retained within constructs having 5 mM native peptide after GAG-loaded constructs were immersed for 48 h in diH₂O, similar to Figures 2c and 3c, is given to the left of the dashed line. To the right of the dashed line, the % of the total loaded GAG that was released from hydrogels containing 5 mM native peptide in varying concentrations of NaCl ranging from the physiological range (0.15 M) to supraphysiological (0.5–2 M NaCl). This was evaluated with both (a) f-HA and (b) ChS.

Figure 6

(a) Representative simulation snapshots of peptide–GAG complexes for the positively charged native HA binding peptide (RYPISRPRKRC) and the electrically neutral charge control peptide (GYPISGPGGGC). The simulation snapshots for non-native peptide–GAG systems are similar visually to that of the native peptides, so it is not shown for brevity. Glycosaminoglycans are shown with a “ball-and-stick” representation, while peptides are shown with a “licorice” representation. Atoms are colored in the following manner: carbon, cyan; hydrogen, white; oxygen, red; nitrogen, blue; sulfur, yellow. Hydrogen bonds are indicated with pink dashed lines. Panels (b) and (c) quantify the energetics of the interactions of the native HA binding peptide (RYPISRPRKRC), non-native HA binding peptide (RPSRPRIRYKC), or charge control peptide (GYPISGPGGGC) with either hyaluronan (HA) or chondroitin-4-sulfate (ChS). (b) Electrostatic and total interaction energy (sum of van der Waals and electrostatic energy) between peptide and GAG. The interaction is primarily electrostatic, and the interaction of the GAG with the charged peptides (native and non-native HA binding peptides) is therefore much stronger than with the electrically neutral charge control peptide. Interactions with ChS are much stronger than those with HA because of the higher charge density of ChS. (c) Total peptide–GAG interaction energy of each residue along the peptides. All of the residues in the charge control peptide generally show weak interactions with the GAG. The charged residues in the positively charged peptides are the primary source of the peptide-GAG interaction energy. Data represent mean(SD) of three independent simulation trials.

Figure 7

(a) Total amount of f-HA loading into hydrogels after 48 h of immersion in a solution of 100 μg of f-HA in PBS. (b) Percent release of loaded f-HA after 48 h of immersion in PBS. (c) Total amount of ChS loading into hydrogels after 48 h of immersion in a solution of 100 μg of ChS in PBS. (d) Percent release of loaded ChS after 48 h of immersion in PBS. Data represent mean(SD) with a sample size of 5; *indicates samples were compared to no peptide control (0 mM).

Figure 8

(a) Schematic of hydrogel formation from a solution of PEG macromers, peptide, HA and chondrocytes. Effect of peptide concentration and peptide chemistry on tissue synthesis showing total amount accumulated in the hydrogel after 28 days (b) and cumulative amount that was released to the culture medium between 14 and 28 days (c). Data represent mean(SD) with a sample size of 3–4; *indicates that samples were compared to nonpeptide control (0 mM).