Hyaluronic Acid Nanoparticles with Parameters Required for In Vivo Applications: From Synthesis to Parametrization

Abstract

Hyaluronic acid is an excellent biocompatible material for in vivo applications. Its ability to bind CD44, a cell receptor involved in numerous biological processes, predetermines HA-based nanomaterials as unique carrier for therapeutic and theranostic applications. Although numerous methods for the synthesis of hyaluronic acid nanoparticles (HANPs) are available today, their low reproducibility and wide size distribution hinder the precise assessment of the effect on the organism. A robust and reproducible approach for producing HANPs that meet strict criteria for in vivo applications (e.g., to lung parenchyma) remains challenging. We designed and evaluated four protocols for the preparation of HANPs with those required parameters. The HA molecule was cross-linked by novel combinations of carbodiimide, and four different amine-containing compounds resulted in monodisperse HANPs with a low polydispersity index. By a complex postsynthetic characterization, we confirmed that the prepared HANPs meet the criteria for inhaled therapeutic delivery and other in vivo applications.

Figure 1

Size of nanoparticles. Average size distribution of HANPs via DLS. (A) z-average size distribution of AAD-, BAPA-, EDBE-, and EDA-HANPs. Variability of (B) z-average and (C) PDI of HANPs with average values (dashed line) between independent synthesis (n = 10 for Protocol no. 1 and 2, and n = 5 for Protocol no. 3 and 4). NTA average size distribution of HANPs. The red area around the average curve corresponds to the standard deviation between each measurement. (D) Protocol no. 1 (AAD-HANPs), (E) Protocol no. 2 (BAPA-HANPs), (F) Protocol no. 3 (EDBE-HANPs), and (G) Protocol no. 4 (EDA-HANPs).

Figure 2

Characteristics of nanoparticles. (A) FTIR spectra in the range between 1830 and 1000 cm^–1. Comparison of free HA (red spectrum) and AAD-, BAPA-, EDBE-, and EDA-HANPs. Fractograms obtained by A4F of (B) AAD-HANPs, (C) BAPA-HANPs, (D) EDBE-HANPs, and (E) EDA-HANPs. The TEM images of (F) AAD-HANPs, (G) BAPA-HANPs, (H) EDBE-HANPs, and (I) EDA-HANPs stained with uranyl acetate. (J) SPR binding curves of HANPs and free HA on the CD44-modified CMDP SPR chip. (K) SPR binding curves of AAD-HANPs on the CD44-modified CMDP SPR chip demonstrating the effect of 0.05% Tween 20.