Amyloid Fibrils Enhance the Topical Bio-Adhesivity of Liquid Crystalline Mesophase-Based Drug Formulations

Abstract

Despite their distinctive secondary structure based on cross β-strands, amyloid fibrils (AF) are stable fibrous protein aggregates with features similar to collagen, one of the main components of the extracellular matrix, and thus constitute a potential scaffold for enhancing cell adhesion for topical applications. Here, the contribution of AF to skin bio-adhesivity aiming toward topical treatments is investigated. Liquid crystalline mesophase (LCM) based on phytantriol is formulated, with the aqueous phase containing either water or a solution of 4 wt% amyloid fibrils. Then resveratrol is added as a model anti-inflammatory molecule. The developed LCM presents a double gyroid Ia3d mesophase. The incorporation of AF into the LCM increases its bio-adhesive properties. In vitro release and ex vivo permeation and retention confirm the controlled release property of the system, and that resveratrol is retained in epidermis and dermis, but is also permeated through the skin. All formulations are biocompatible with L929 cells. The in vivo assay confirms that systems with AF lead to a higher anti-inflammatory effect of resveratrol. These results confirm the hypothesis that the incorporation of AF in the LCM increases the bio-adhesiveness and efficiency of the system for topical treatment, and consequently, the therapeutical action of the encapsulated drug.

Keywords: amyloid fibrils; bio-adhesiveness; inflammatory diseases; liquid crystalline mesophases; topical treatments.

Figure 1

Study outline. A) Components of the liquid crystalline mesophase (LCM) composed of phytantriol with: water (LCW), water and resveratrol (LCWR), amyloid fibrils (LCAF), and amyloid fibrils and resveratrol (LCAFR). B) Phase diagram of phytantriol. Adapted with permission.^{[ 40 ]} Copyright 2020, American Institute of Physics. C) Cubic Ia3d mesophase loaded with resveratrol, which interacts with the AF. D) Scheme of topical application of LCAFR, and the delivery of resveratrol through the skin. E) Scheme of the enhanced bio‐adhesive property of LCAFR.

Figure 2

A) SAXS curves of LCW, LCWR, LCAF, and LCAFR at 25 and 32 °C. B) Flow rheograms of LCW and LCWR at 25 and 32 °C. (C) Flow rheograms of LCAF and LCAFR at 25 and 32 °C. The closed symbols mean the up‐flow curves, and the open symbols mean the down‐flow curves. D) Frequency sweep profile of the storage modulus G′ (closed symbols) and the loss modulus G′′ (opened symbols) of the LCW and LCWR at 25 and 32 °C. E) Frequency sweep profile of the storage modulus G′ (closed symbols) and the loss modulus G′′ (opened symbols) of the LCAF and LCAFR at 25 and 32 °C. F) AFM image of βlg fibrils in water (pH 2). The scale bar is of 1 µm.

Figure 3

A) Schematic of the bio‐adhesive and permeation and retention assay. B) Bio‐adhesive force of the LCW, LCWR, LCAF, and LCAFR at 32 °C. The results are expressed as an average of five measurements, and the error is reported as standard deviation. C) In vitro release profile (%) of RES from LCWR, LCAFR, and RES suspension (R suspension) through a 0.45‐µm polyethersulfone membrane. D) Ex vivo retention (%) in epidermis + dermis through porcine skin of RES from LCWR, LCAFR, and RES suspension (R suspension). E) Ex vivo permeation (%) of RES from LCWR, LCAFR, and RES suspension (R suspension) through porcine skin.

Figure 4

Cytotoxicity assessment by L929 cell line and anti‐inflammatory activity of the formulations. A) Scheme of the in vitro agar diffusion methodology. (B) Cytotoxicity halos obtained for LCW, LCWR, LCAF, and LCAFR in L929 cell line. Data are shown as mean ± SD of three individual experiments. C) In vivo anti‐inflammatory activity of PBS (negative control), dexamethasone (positive control), free RES, LCW, LCWR, LCAF, and LCAFR. Data are shown as mean ± SD of six individual experiments. D) Scheme of the in vivo ear edema assay methodology.