Abstract
A niosome-based delivery system incorporating hyaluronic acid-modified Poloxamer 407 (P407-LHA) was developed to enhance the topical delivery of resveratrol (RSV). It was proposed that the system targets CD44 receptors on human fibroblast cells to improve skin penetration, drug accumulation, and anti-aging efficacy. In this study, the niosome formulations composed of non-ionic P407, P407-LHA, and their blends (P407/P407-LHA(2/1) and P407/P407-LHA(1/2)) were investigated. The grafting ratio of LHA on P407-LHA was 32.93 ± 0.73 %. RSV-loaded niosomes (RSV@niosomes) exhibited particle sizes ranging from 200 to 300 nm and a negative zeta potential, and the LHA-modified niosomes enhanced the RSV payload. In vitro skin penetration demonstrated that RSV@P407/P407-LHA(1/2) niosomes achieved the highest RSV accumulation in both the epidermis and dermis compared to the other formulations. Additionally, RSV@P407/P407-LHA(1/2) niosomes induced the highest collagen production in H2O2-stimulated L929 cells (142.64 ± 7.44 %) compared to untreated cells (66.94 ± 0.38 %). In aged Wistar rat skin, drug deposition in the epidermis and dermis followed the order: RSV@P407/P407-LHA(1/2) niosomes > RSV@P407 niosomes > RSV solution. The RSV@P407/P407-LHA(1/2) niosomes not only efficiently inhibited ROS generation (41.27 ± 5.25 % relative to the control) but also promoted collagen production, increasing from 65.93 ± 2.88 % to 172.95 ± 2.79 %. These results elucidate that the combination of resveratrol and LHA-based niosomes effectively enhances anti-aging efficacy through improved skin penetration and drug accumulation.
Introduction
Niosomes are bilayer vesicles formed from non-ionic surfactants and stabilized by cholesterol. It can be encapsulated both hydrophilic and hydrophobic payloads where the hydrophilic drugs will be loaded in the core and the hydrophobic drugs will be encapsulated in the bilayer [1]. The niosome delivery system has been utilized in the treatment of skin infections, cancer therapy, neurodegenerative brain diseases, and in the field of cosmetics [[2], [3], [4], [5]]. Kulkarni et al. encapsulated the hydrophilic drug D-cycloserine and the lipophilic drug ethionamide in niosomes for the treatment of drug-resistant tuberculosis. This combination demonstrated enhanced bacterial inhibition compared to the free drugs [6]. They also employed niosomes for the nasal delivery of dual drugs to the brain, offering a nose-to-brain targeting advantage [7]. The hydrophilic-lipophilic balance (HLB) and critical packing parameter (CPP) are two critical factors which affect noisome performance [8,9]. It compares favorably to liposomes, with advantages of high stability and low cost [10]. The bilayer conformation, in contrast to the monolayer micelle, ensures encapsulation of payloads with varying hydrophilic/hydrophobic properties [11]. Niosomes are generally reported to transport through the skin via the intercellular pathway. The non-ionic surfactant softens the stratum corneum, and the elastic bilayer vesicle further enhances percutaneous drug absorption [[12], [13], [14]]. Niosomes with active targeting properties have been developed by conjugating specific ligands to their surface, enabling them to bind to and internalize receptor-overexpressing cells [15]. The polyethylene glycolated niosomes, modified with the penetrating peptide tLyP-1 (CGNKRTR), showed a reduced IC50 for the peptide-conjugated niosomes compared to the peptide-free niosomes [16].
Resveratrol (trans-3,4′,5-trihydroxystilbene, RSV) is a polyphenolic compound derived from plants, with antioxidant, anti-inflammatory, neuroprotective, antitumor, antibacterial, and anti-aging activities [[17], [18], [19], [20]]. RSV was served as an antioxidant where the hydroxyl group as an electron donor could terminate the free radical propagation. It was found that RSV can inhibit nuclear factor-κ B (NF-κ B) as well as MMP expression in oxidant-stimulated mice resulting in reduction of collagen degradation [21]. RSV is rapidly degraded to glucuronides and sulfonates by uridine diphosphate glycosyltransferase (UGT) and sulfotransferase (SULT) in liver with very short half-life after oral administration [22,23]. In addition, its low water solubility and instability in light limit its application in medications [24]. Therefore, the proper delivery system is highly needed for RSV.
The fibroblast cells in the skin express two important receptors, CD44 and PHAMM, which are involved in cell proliferation, lymphocyte homing, and recirculation. CD44 is a glycoprotein receptor overexpressed in several tumors, and hyaluronic acid (HA), a linear polysaccharide, has a high affinity for CD44 receptors and is widely used in drug and gene delivery [25]. HA is biocompatible, biodegradable, and hygroscopic, with wound healing activity. HA-based nanocarriers can control drug release and target therapeutic agent delivery, improving efficacy and reducing side effects [[26], [27], [28]]. HA can be an effective nanocarrier for both topical and transdermal delivery. Several properties, including specific receptor binding, skin hydration, bioadhesion, and viscoelasticity, contribute to its potential role in enhancing skin penetration and cellular uptake [29,30]. It has been reported that HA can bind to the CD44 receptor and activate mitogen-activated protein kinase (MAPK) to enhance collagen generation [31,32]. Meanwhile, HA binds to CD44 in human normal polymorphonuclear neutrophils to inhibit p38-MAPK phosphorylation, matrix metalloproteinases (MMP) expression, and collagen degradation [33]. The molecular weights of HA-based assemblies play a critical role in adhesion to CD44-overexpressing tumor cells, driven by the HA-CD44 interaction [34].
Niosomes have been employed as a nano-delivery system, offering advantages such as excellent transdermal absorption, high stability, and cost-effectiveness. Niosomes primarily rely on passive transport mechanisms. However, the absence of active targeting capabilities may limit their therapeutic potential. In this study, a niosome-based delivery system incorporating hyaluronic acid-modified P407 (P407-LHA) was designed to facilitate the topical delivery of resveratrol (RSV) and target the CD44 receptor on human fibroblast cells. LHA-modified P407 niosomes are expected to enhance skin penetration, selectively bind to the CD44 receptor, and accumulate in the skin, thus enhancing its anti-aging effects. The skin penetration, targeting efficiency, antioxidant activity, and collagen production of RSV-loaded niosomes (RSV@niosomes) were evaluated through in vitro cell models and in vivo animal studies.
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Materials
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), phorbol 12-myristate 13-acetate (TPA, 99 + %), and sodium cyanoborohydride (NaCNBH3, 95 %) were from Alfa Aesar (Heysham, England). Kolliphor® P407 was from BASF Co., Ltd. (Washington, NJ, USA). N-hydroxysuccinimide (NHS, 98 %) was from Acros Organics Co., Inc. (New Jersey, USA). Resveratrol (trans-3,4′,5-trihydroxystilbene, RSV) was from Tokyo Chemical Industry CO., Ltd. (Tokyo, Japan). Sodium hyaluronate (LHA, MW 29,000 Da) was from Long Chen Shing Trading Co., Ltd. (Kaohsiung, Taiwan). Cholesterol, d-chloroform, and 2′,7′-dichlorofluorescin diacetate (DCFDA, >97 %) were from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium powder (high glucose) was from Gibco by life technologies Co. (Grand Island, NY, USA). Fetal bovine serum (FBS), penicillin-streptomycin-amphotericin B solution, and trypsin EDTA solution (0.5 % trypsin, 0.2 % EDTA Na4) were from Biological Industries Israel Beit-Haemek Ltd. (Kibbutz Beit-Haemek, Israel). Hydrogen peroxide (30 % aqueous solution) was from SHOWA Chemical Co., Ltd. (Tokyo, Japan). L929 mouse fibroblast cell line (ATCC®CCL-1™) was from Bioresource Collection and Research Center (Hsinchu, Taiwan). Sicrol soluble collagen assay kit was from Biocolor Ltd. (Antrim, UK).
Wei Tsung Chen, Wen Jen Lin, Advanced hyaluronic acid-modified niosomes for percutaneous delivery of resveratrol in the treatment of aged skin, International Journal of Biological Macromolecules, Volume 325, 2025, 147250, ISSN 0141-8130, https://doi.org/10.1016/j.ijbiomac.2025.147250.
