• In Situ Self-Crosslinkable and Long-Term Stable Hyaluronic Acid Hydrogel Dermal Filler with Vitamin E Release on Fibroblast Cells for Tissue Augmentation and Skin Wrinkle Correction
  • Nikoo Ansar,1,*
    1. n.ansar@merc.ac.ir


  • Introduction: Dermal fillers, particularly hyaluronic acid (HA)-based ones, are widely used in aesthetic medicine to correct wrinkles and restore subcutaneous tissue volume. HA, a natural carbohydrate, supports skin hydration, tissue repair, and collagen/elastin synthesis. Despite their popularity due to biocompatibility, ease of injection, and low immunogenicity, HA fillers have a short half-life and rapid absorption in vivo. They are classified as monophasic or biphasic, with biphasic fillers offering better stability and volumizing effects. Challenges include enhancing mechanical properties and longevity while minimizing side effects like pain or toxicity from chemical crosslinkers. This study investigates in situ enzymatic crosslinking of HA hydrogels with peroxidase, long-term stability, and vitamin E release for skin rejuvenation, evaluating crosslinking degree, water retention, and rheological properties.
  • Methods: Hyaluronic acid (HA) was functionalized with dopamine hydrochloride in 0.5 M MES buffer (pH 5.5). HA (0.5% w/v) was mixed with EDC, NHS, and dopamine, dialyzed (3,500 Da), and lyophilized. Aldehyde-functionalized HA (AHA) was synthesized using sodium periodate, adjusted to pH 5.5, dialyzed, and lyophilized. AHA was reacted with dopamine to form DAHA via Schiff base reaction. Hydrogels were formed by mixing 2% DAHA with HRP (0.6 U/mL) and 0.6 M H₂O₂ at 37°C. Vitamin E (1.5 mM) was loaded into hydrogels in ethanol. Swelling was tested in PBS for 1200 hours. Enzymatic degradation used hyaluronidase (0.5–50 U/mL). Vitamin E release was monitored at 530–540 nm. Cytotoxicity was assessed via MTT assay on NIH/3T3 cells.
  • Results: 3-1 Mechanism of In Situ Hydrogel Formation Hyaluronic acid (HA) was functionalized with dopamine via carbodiimide chemistry, forming amide bonds. EDC activates HA carboxyl groups, reacting with dopamine’s amine groups, with NHS enhancing the reaction. ^1H NMR confirmed functionalization. Alternatively, HA was oxidized with NaIO₄ to form aldehyde-functionalized HA (OHA), enabling Schiff base reactions with amines. OHA was purified by dialysis and lyophilized, with oxidation degree verified by hydroxylamine hydrochloride titration. 3-2 Self-Crosslinking and Hydrogel Formation HA modified with gallol formed hydrogels via enzymatic auto-oxidation. ^1H NMR confirmed gallol conjugation (peaks at 2.8, 6.5 ppm). Hydrogels formed in PBS (pH 7.4, 2% w/v) with HRP (0.06 U/mL), achieving gelation in 5 minutes. H₂O₂ from gallol oxidation accelerates crosslinking, enhancing biocompatibility and mechanical properties. 3-3 Swelling Test Swelling ratios of 2% (w/v) HA hydrogels (with/without vitamin E) were measured in PBS (pH 7.4). Vitamin E-loaded hydrogels showed lower swelling (peak: 16 vs. 20 at 100 hours), reaching zero at 840 hours (vs. 1200 hours for unloaded). High initial swelling caused burst drug release. 3-4 Rheological Test Rheological tests (0.1–14 Hz) showed gel-like behavior (G′ > G′′). Vitamin E-loaded hydrogels had a 0.33% lower storage modulus, increased loss modulus, and 24% reduced viscosity, indicating minor softening but retained stability. 3-5 Elastic Modulus Crosslinking reduced hydrogel fluidity. Vitamin E-loaded hydrogels showed a negligible 0.33% decrease in elastic modulus. 3-6 Loss Modulus Increased crosslinking raised loss modulus. Vitamin E-loaded hydrogels dissipated more energy, aiding tissue integration. 3-7 Complex Viscosity Vitamin E-loaded hydrogels exhibited a 24% viscosity reduction, consistent with hydrophobic drug-loaded HA hydrogels. 3-8 In Vitro Drug Release Vitamin E release from 1.5 mM-loaded hydrogels was nearly complete in 12 hours, while 0.75 mM-loaded hydrogels released gradually over 84 hours, due to vitamin E’s hydrophobicity.
  • Conclusion: The results indicated that the hydrogel prepared via the enzymatic method exhibited a three-dimensional porous structure, in which all pores were filled with water upon swelling. Furthermore, the drug release profile was directly influenced by the swelling behavior, crosslinking density, and mechanical strength of the hydrogel. The initial burst release and subsequent drug liberation suggested the accumulation of the drug on the surface or outer layers of the system. By increasing the concentration of hydrogen peroxide, the crosslinking density of the hydrogel was enhanced, while the average pore size was reduced. This, in turn, led to improved mechanical strength, lower swelling capacity, and consequently, a reduced drug release rate. Moreover, an increase in the concentration of both the polymer and the enzyme resulted in a shorter gelation time. Collectively, these findings demonstrated that the in situ enzymatically formed hydrogel system possesses favorable properties, including suitable gelation time, controllable swelling and stability, adequate mechanical integrity, injectability, and desirable in vitro drug release behavior. Considering the abundance and accessibility of hyaluronic acid in Iran, this system can be regarded as a promising and cost-effective platform for controlled drug delivery and further biomedical applications.
  • Keywords: Hyaluronic acid hydrogel, in situ crosslinking, vitamin E release, fibroblasts, dermal filler, tissu