Hyaluronic Acid Nanogel as a Protein Delivery System
Purpose
Hyaluronic acid (HA) nanogel has hyaluronic acid in the main chain, a hydrophilic, biocompatible material with low toxicity, and a hydrophobic group in the side chain. A nanoordered gel is spontaneously formed, due to its hydrophilic-hydrophobic interactions, in which it is possible to load small molecules, peptides, and proteins. (1,2) Unique characteristics are predicted, such as improved targeting ability, reduced toxicity and solubility of the active pharmaceutical ingredients (APIs).
HA nanogel can load various proteins; however, the correlation between the physicochemical property of proteins and the loading capacity has not been investigated. In order to explore the function of HA nanogel-protein complex preparations, we investigated stability of protein activity, and inhibition of aggregation and denaturation with Cholesteryl-modified Hyaluronic Acid (CHHA) as our HA nanogel.
Objectives
Aiming to understand the interaction between the protein and HA nanogel, the relationship between TSA analysis and the loading capacities, and the influence on the aggregation and activity of the proteins when the nanogels were encapsulated were examined. This study contributes to the development of formulations with high protein stability.
Methods
For this study, we used CHHA whose degree of substitution by the cholesteryl group is 12 % in HA units, and whose molecular weight is 80 kDa.
Sustained release of hGH from HA nanogel/hGH complex in VIVO
CHHA solution and hGH solution in 10wt% sucrose was mixed in various concentration and it was incubated at 37C for 24 hours. The CHHA/hGH formulations were injected subcutaneously into the back of seven-week-old male Sprague-Dawley with a 25-gauge needle at a dose in the table of figure 1. Blood was collected from the jugular vein and rhGH levels in plasma were determined using an hGH enzyme-linked immunosorbent assay (ELISA) kit.
TSA (Thermal Shift Assay) Analysis
SYPRO® Orange protein gel stain (50 times diluted, 1 μL) was added to the protein solution (0.151 mg/mL, 19μL). The fluorescence intensity was measured by the real-time polymerase chain reaction (RT-PCR), with a scan mode of FRET.
Loading Proteins in HA Nanogels
Protein was incubated with CHHA at 37 oC for 24 hours ([protein] = [CHHA] = 1 mg/mL), and then the solution (200 μL) was put into ×5 PBS buffer (50 μL) to precipitate the complex of CHHA and protein. After centrifuging at 9000g for 10 min, the protein in the supernatant was subjected to GPC measurement to determine the amount of encapsulated protein. Loading capacity of protein = [amount of encapsulated protein] / [amount of CHHA].
Antibody Stability in HA nanogels
Antibody (anti-OVA antibody, IgG from rabbit) was incubated with or without CHHA, at 4 or 80 oC for 24 hours ([antibody] = 4 μg/mL, [HA nanogel] = 6 mg/mL), and O.D. was measured using a plate reader, to evaluate the thermal stability of antibodies in HA nanogel.
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Article information: Toru Katsumata, Keisuke Fukumoto, Kohei Yabuuchi, Yoshiyuki Nakagawa, Yoshiro Tahara, Yurika Tanaka, Tsuyoshi Shimoboji. Provided by Asahi Kasei