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Biomaterials

Biomaterials - Poster Session E

Poster N10 - Determining the Role of Peptide Chirality in a Hydrogel-Forming Vaccine Delivery System

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Hydrogels are polymer networks composed of up to 99% water with numerous potential biomedical applications. Physically crosslinked hydrogels, which are held together by intermolecular forces, offer several advantages including shear-thinning and self-healing behavior, allowing for minimally invasive administration. Peptide-based supramolecular hydrogels are physical hydrogels formed by self-assembling amino acid chains. They are biocompatible and biodegradable with tunable mechanical and biological properties.

Multidomain peptides (MDPs) are peptide-based supramolecular hydrogels, consisting of alternating polar and nonpolar residues flanked by charged amino acids that create β-sheet-rich fibrils in neutral pH environments. K2(SL)6K2, termed K2, is a well-studied MDP and proven effective vaccine adjuvant with sustained antigen release comparable to clinical adjuvants like alum. K2’s positive charge has been shown to aid in recruiting immune cells and provoke inflammatory responses in vivo.

In contrast to traditional levorotatory (L-) peptides prone to enzymatic degradation, enantiomeric dextrorotatory (D-) peptides show altered interactions with biological molecules, making them resistant to protease breakdown. This resistance to proteolytic degradation and subsequent sustained activity underscores D-peptides' potential as vaccine adjuvants.

This study investigates D-K2, the enantiomer of K2, to explore how chirality affects hydrogel formation, degradation, antigen release from hydrogels, and adjuvanticity. While expecting similar in vitro properties to L-K2, we anticipated divergent in vivo behavior due to biological systems' reduced interactions with D-amino acids. Using ovalbumin (OVA) as a model antigen, our findings demonstrate that D-K2 delays hydrogel degradation without significantly altering material characteristics, antigen release rate, or the humoral immune response to co-delivered antigens.

Materials and

Methods:  K2 and D-K2 peptides were manually synthesized using solid-phase synthesis, purified using high-performance liquid chromatography (HPLC), and confirmed for peptide identity and purity using autoflex matrix-assisted laser desorption/ionization mass spectrometer (MALDI MS) and ultra-performance liquid chromatography (UPLC). 1% (w/v) hydrogels of K2 and D-K2 were characterized and evaluated in vitro using circular dichroism (CD), oscillatory rheology, and cryo-electron microscopy to confirm secondary structure. Fluorescence recovery after photobleaching (FRAP) studies were used to investigate transport and measure OVA diffusivity through K2 and D-K2 hydrogels in vitro. In vivo imaging experiments in SKH-1 mice were conducted to evaluate hydrogel clearance using fluorescently labeled K2 and D-K2 peptides as well as antigen release from the hydrogels using fluorescently labeled OVA. At 13 weeks, the skin surrounding the injection site of the hydrogels was harvested for histology and analyzed using Masson’s Trichrome and Hematoxylin and Eosin staining. Submandibular bleeds were performed one day prior to injection as well as at 2, 4, 6, 8, 10, and 12 weeks after administration. OVA-specific IgG antibody titers were assessed and quantified by ELISA to understand the effect on peptide chirality on humoral immunity.

Results, Conclusions, and Discussions: In vitro, K2 and D-K2 hydrogels had inverse CD, confirming the chirality of D-K2. CD measures the absorption difference of left and right circularly polarized light, meaning enantiomeric structures would appear inverted, as K2 and D-K2 did (Figure 1a). Both hydrogels had similar tertiary structure and rheological properties. FRAP experiments showed no statistically significant difference in the recovery rate of both hydrogels, indicating similar in vitro diffusion rates of OVA.

Over the course of 86 days hydrogel degradation in vivo was tracked via imaging and D-K2 degraded at a significantly slower rate (p < 0.0001) than K2 (Figure 1b,c). Tissue sectioning revealed that only D-K2 hydrogels remained present and showed both prevalent cellular infiltration throughout the area of the gel and a collagenous fibrous capsule around the hydrogel (Figure 1d). In a previous study of K2, no formation of a fibrous capsule was found at 6 weeks, indicating that this response may be specific to D-K2.

In vivo release rates of OVA from K2 and D-K2 hydrogels were not significantly different, reaching 100% release over 11 days for both hydrogels. These similar antigen release rates in vivo suggest that peptide chirality and clearance do not meaningfully influence OVA release. Both peptides’ release rates were comparable to that of alum and much slower than non-adjuvanted OVA. Both hydrogels had similar IgG titers, suggesting that humoral immunity is largely unaffected by MDP chirality (Figure 1e). However, the titer levels of both peptides are significantly higher than the 2.3 log2 titers achieved by mice receiving non-adjuvanted OVA administered via the same dose and route, showing that both peptides act as potent adjuvants.

In this research, we have shown that D-K2, the chiral counterpart of K2, forms a supramolecular hydrogel with rheological properties, antigen release rates, and adjuvancy comparable to K2. However, D-K2 has a markedly slower degradation rate in vivo, likely attributable to its nonrecognition by endogenous proteases. Taken together, these data suggest that self-assembling peptides with D-amino acids could be particularly advantageous for biomedical applications requiring extended hydrogel persistence in vivo while maintaining the desirable injectability of the shear-thinning peptides.

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