Self-Assembled Thermoresponsive Nanogel from Grafted Hyaluronic Acid as a Biocompatible Delivery Platform for Curcumin with Enhanced Drug Loading and Biological Activities

Abstract

A hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-pNIPAM) was synthesized as a polymeric nanogel platform for encapsulation and delivery of hydrophobic bioactive compounds using curcumin as a model drug. As demonstrated by transmission electron microscopy and dynamic light scattering techniques, the HA-pNIPAM was simply assembled into spherical nano-sized particles with the thermoresponsive behavior. The success of curcumin aqueous solubilization was confirmed by fluorescent spectroscopy. The resulting nanogel formulation enhanced the aqueous solubility and uptake into NIH-3T3 cells of curcumin. This nanogel formulation also demonstrates cytocompatibility against NIH-3T3 cells, which deems it safe as a delivery vehicle. Moreover, the formulation has a slight skin-protection effect using an artificial skin equivalence model. The curcumin-loaded HA-pNIPAM nanogel showed an anti-proliferative activity against MDA-MB-231, Caco-2, HepG2, HT-29, and TNF-α-induced hyperproliferation of keratinocyte (HaCaT) cells. The thermoresponsive HA-pNIPAM nanogel reported here could be further optimized as a platform for controlled-release systems to encapsulate pharmaceuticals for therapeutic applications.

Keywords: HA-pNIPAM; curcumin; hyaluronic acid; thermoresponsive nanogel.

Figure 1

Schematic representation of (A) the preparation of pNIPAM-grafted hyaluronic acid (HA-pNIPAM) and (B) the assembly of HA-g- pN nanogel and curcumin release.

Figure 2

The characterization of HA-pNIPAM nanogel. (A) the thermal degradation profile of pNIPAM-grafted HA, in which the green line shows the total weight reduction; the blue line shows the derived weight reduction profile representing the grafted-polymer that decomposed at two different temperatures. (B) Particle size measured by DLS shows the thermoresponsive behavior of the HA-pNIPAM 05 nanogel. (C) The corresponding TEM micrograph represents the spherical morphology of the nanogel at 25 °C (negatively stained) with an inset image from the higher magnification TEM (positively stained). The average size of the particle strikingly changed at the LCST.

Figure 3

Size, morphology, and loading capacity of CUR-HA-pNIPAM 05 formulation. (A) TEM micrograph of CUR-HA-pNIPAM 05 formulation shows that the particles were sized around 100–300 nm. (B) A magnified TEM image for a single CUR-HA-pNIPAM 05 nanogel particle; (C) DLS results demonstrate the curcumin nanogels in the size range correlated with TEM at PDI = 0.2. (D) Loading capacity of the formulation compared to the curcumin in aqueous solution. Scale bars indicate 0.5 µm for (A) and for 0.1 µm for (B). The values expressed in (D) are means ± SD, n = 3.

Figure 4

Confocal microscopy shows the cellular drug uptake in NIH-3T3 cells. DAPI (nuclear staining) and rhodamine-phalloidin (actin staining) were used as counter-stains. The images were obtained using Olympus IX81 with DSU confocal mode under a 20× oil lens (NA = 0.85). Exposure times were kept constant for all samples (500 ms for DAPI and rhodamine-phalloidin, 1000 ms for curcumin).

Figure 5

Histological analysis for acute skin irritation test. Hematoxylin and Eosin (H&E) stained EpiSkin^®. The scale bars for each left and right column indicate 20 µm and 100 µm, respectively.

Figure 6

(A) The cell viability of four different cancer cell lines per increasing doses of curcumin in HA-pNIPAM 05 nanogel formulation. (B) IC₅₀ of curcumin encapsulated in the nanogel formulation against four different cancer cell lines; (C) the anti-proliferation assay of curcumin and CUR-HA-pNIPAM 05 (1 and 5 µg/mL) against TNF-α-induced HaCaT cell proliferation. Data presented are mean ± SD values of the four replications. ^a p < 0.05 indicates significant differences from the control group; ^b p < 0.05 indicates significant differences from the TNF-α group; ^c p < 0.05 indicates significant differences from the TNF-α + curcumin 5 µg/mL group.