Synthesis and Characterization of Conjugated Hyaluronic Acids. Application to Stability Studies of Chitosan-Hyaluronic Acid Nanogels Based on Fluorescence Resonance Energy Transfer

Abstract

Hyaluronic acid (HA) was functionalized with a series of amino synthons (octylamine, polyethylene glycol amine, trifluoropropyl amine, rhodamine). Sodium hyaluronate (HAs) was first converted into its protonated form (HAp) and the reaction was conducted in DMSO by varying the initial ratio (-NH₂ (synthon)/COOH (HAp)). HA derivatives were characterized by a combination of techniques (FTIR, ¹H NMR, 1D diffusion-filtered ¹⁹F NMR, DOSY experiments), and degrees of substitution (DS_HA) varying from 0.3% to 47% were determined, according to the grafted synthon. Nanohydrogels were then obtained by ionic gelation between functionalized hyaluronic acids and chitosan (CS) and tripolyphosphate (TPP) as a cross-linker. Nanohydrogels for which HA and CS were respectively labeled by rhodamine and fluorescein which are a fluorescent donor-acceptor pair were subjected to FRET experiments to evaluate the stability of these nano-assemblies.

Keywords: fluorinated and fluorescent HA conjugates–hyaluronic acid degree of substitution–diffusion ordered spectroscopy (DOSY)–1D diffusion-filtered 19F NMR–atomic force microscopy–FRET experiments–hyaluronidase–nanohydrogel stability; nanohydrogels–hyaluronic acid–HA-mPEG2000.

Scheme 1

Syntheses of functionalized HA described in the paper.

Figure 1

FTIR spectra of octylamine functionalized HA samples: (a) carbonyl vibrations region (1500–1800 cm⁻¹) and (b) C–H stretching vibrations region (2700–3000 cm⁻¹).

Figure 2

¹H NMR spectra of octylamine functionalized HA: Hap, HA-C8a, HA-C8b, HA-C8c, and HA-C8d) (500 MHz, 318 K, DMSO d₆, and DMSO peak is omitted for clarity).

Figure 3

(a) ¹⁹F NMR spectra of trifluoropropyl functionalized HA: HA-TFPa, HA-TFPb, HA-TFPc and HA-TFPd for initial molar ratios (NH₂/COOH) = 10 (bottom), 20, 50, and 100 (top) %, respectively; TFP-NH₂ ¹⁹F spectrum is given as a reference (470.64 MHz, 318 K, DMSO-d₆). (b) One-dimensional (1D) diffusion-filtered ¹⁹F NMR spectra of HA-TFPd and TFP-NH₂ with a gradient g of 2% and 95%.

Figure 4

Diffusion curves and diffusion coefficients extracted from DOSY spectra (ref δ (¹HRhod) = 1.1 ppm) for (a)—HA-Rhod a, (b)—HA-Rhod b, (c)—HA-Rhod c, and (d)—HA-Rhod d and (e) Rhod-NH alone and (f) HA alone as controls. See Equation (1) for I_UG and I_G definitions (UG corresponds to ungrafted Rhod synthon, while G corresponds to grafted Rhod synthon).

Figure 5

(a) Emission spectrum of CS-Fluo-TPP/HA-Rhod nanogels in PBS (◆), by comparison of CS-Fluo-TPP/HA (△) and CS-TPP/HA-Rhod (◯) in CS-Fluo-TPP/HA-Rhod nanogels in PBS (λ_ex = ${\lambda }_{ex}^D$ = 470 nm, CS-Fluo being the donor dye and HA-Rhod being the acceptor dye). (b) Fluorescence measurements of CS-Fluo-TPP/HA-Rhod nanogels ([A]/[D] ratio = 0.5) without (◆) and in the presence of hyaluronidase (×).