Synthesis and cytotoxicity of quercetin/hyaluronic acid containing ether block segment

Abstract

This study is about preparation of natural quercetin type surfactants. The preparation involves polycondensing quercetin (QT) and propylene glycol in the presence of an acid catalyst so as to make quercetin more water soluble; performing ring opening polymerization between polyoxyethylene chains (MW: 2000, 4000, 6000, 8000, 10,000) changed by polyethylene glycol and succinic anhydride so as to obtain an ether block surfactant; and then introducing hydrolyzed hyaluronic acid (HA) by means of esterification while preparing a series of natural QT/HA-type surfactants through condensation. The synthetized products were analyzed using Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (¹H-NMR,¹³C-NMR) for their structures, and assayed for their surface active properties, including their surface tension, fluorescence, contact angles, and emulsification properties. The experimental results show that the surface activity of the QT/HA-type surfactants decreased with the ethylene oxide chain length. Among all the surfactants, the QP2H surfactant exhibited the most efficient emulsification ability, lowest surface tension, and good emulsification stability. The synthetized products were assessed for safety through in vitro cytotoxicity and MTT assays with HaCaT cell lines. According to the results, QP2H, that was similar to oil in terms of HLB value, showed outstanding emulsion stability, and its zeta potential suggested low risk of colloidal agglutination due to repulsion between its emulsion drops. However, as proven in tests with HaCaT cell lines, QT-HA containing ether block surfactants when applied in cosmetics are safe. No significant cytotoxicity was observed in any case with a concentration less than 0.01 wt% (10 mg/mL). With CMC lower than 0.01 wt%, they would not hurt skin when used in cosmetics.

Introduction

Surfactants are extensively seen in products for daily use and can be used in various processes [1]. They are mainly used as cleaning agents, wetting agents, emulsifiers, and leveling agents, and used for increased dye solubility and stability in printing and dyeing processes. However, synthetic surfactants made from petroleum are too stable to be decomposed by microorganisms naturally, and can reduce microorganisms, roe, and larvae of fish and shellfish at just 0.5 ppm concentration [2], making them a huge threat to ecological balance. Not only lethal to aquarium creatures, they are an irritant to human skin, and can permeate into human organs such as the eyes, brain, heart and liver. Some synthetic surfactants can even disrupt hormones and mimic the hormone estrogen, causing lasting damage to the body. Also, there are usually ethylene oxide, nitrosamines, and 1,4 dioxane in their compositions [3]; these substances can contaminate the environment and increase cancer risk. Following the food chain, these substances will eventually enter the human body and accumulate in body fat to disrupt hormones and lead to irremediable damage. They also cause lasting pollution and harm to the biophysical environment and organisms.

In addition, free radicals are generated every day in the human body due to biosynthesis and metabolism, plus external free radical inducing factors, such as air pollution, water pollution, cigarettes, medicine, pesticide, preservatives, and stress. When grabbing electrons from outside, free radicals attack normal cells and tissues nonselectively, and produce more new free radicals, leading to chain peroxidation. Free radicals are responsible for oxidation and in turn oxidative stress, which can cause cell aging and many chronic diseases. When there are too many oxygen-derived free radicals oxidizing low-density lipoprotein, lipid peroxidation will happen and endanger integrity of cell membranes. This can cause cardiovascular atherosclerosis, and even change the metabolic program, causing gene mutation and, in turn, tumors and cancers [4].

In order to discover new and safe surfactants with regard to the environment, new environmentally friendly nonionic surface active agents were synthesized by the reaction of tannic acid (as a natural product presents in several plants) and polyethylene glycol fatty acids containing different numbers of ethylene glycol units [5]. The antiradical efficiency of gallic acid (GA) and hydroxytyrosol (HT) demonstrated that scavenging capacity was maintained. The DPPH ED50 values of the GA derivatives showed a higher antiradical activity compared with the HT derivatives. The content of the cutaneously absorbed compound is higher for the antioxidant surfactants (ester derivatives). This particular behavior could be due to the higher hydrophobicity of these compounds and the presence of surface activity in the antioxidant surfactants [6].

Quercetin (QT) is also known as sophoretin, or pentahydroxy flavone, and is one of the most widely distributed flavone compounds [7]. It is a reducing compound having phenolic hydroxyl groups, and being an antioxidant itself can be easily oxidized. Most research has focused on the antioxidant properties of quercetin, its effects on several enzyme systems, and effects on biological pathways involved in carcinogenesis, inflammation and cardiovascular diseases [[8], [9], [10]]. An antioxidant works by having its phenolic hydroxyl groups reacting with detrimental free radicals to form relatively stable semiquinone radicals, thereby stopping the chain reaction that would be otherwise caused by detrimental free radicals [[11], [12], [13]]. QT manifested a remarkable ability to increase the proliferation of the glioma cells within 24 h of treatment at a concentration ≤100 μM. The expected cytotoxic effect was instead observed toward the gliosarcoma cells, the most polar QT derivatives did not show any toxic effects [14]. No cytotoxic effects were observed on the fibroblast cells for quercetin in the tested concentrations (0.2–25.0 μg/mL) [15].

This study uses hyaluronic acid, which has a disaccharide structure as the hydrophilic group, as a biodegradable material for synthesis of surfactants. Hyaluronic acid (HA) is also known as aldonic acid, and it is a high molecular weight polymer which has a linear disaccharide structure composed of repeating units of d- glucuronic acid and N- acetylglucosamine, alternately connected by β-1,3, β-1,4 glycosidic bonds [16]. The hydroxyl and carboxylic functional groups on the HA molecule backbone provide active sites for conjugating drugs directly or indirectly. In general, direct conjugation is inefficient owing to huge steric hindrance of the backbone and poor reactivity of the carboxy groups. In the case of indirect conjugation, HA may be extensively derived via a chemical linker, mostly adipic acid dihydrazide. This method provides HA molecule with functional groups (NH₂-NH₂) which allow the attachment of terminal groups in the drug backbone [17]. Hyaluronic acid is known to have outstanding biocompatibility and great humectancy, and has been extensively used in cosmetics. However, due to its poor surface activity, HA fails to stabilize emulsions so cannot be used as an emulsifier.

Combining antioxidative polyphenols and biodegradable carbohydrates to prepare surfactants is an important development of this century, which is both sustainable and economic. The combination of hyaluronic acid (HA) and quercetin (QU) exhibited much higher cytotoxicity in vitro and antitumor efficacy in vivo, with less toxicity compared to Taxotere® [18]. Surfactants of the next generation shall be environmentally friendly, biodegradable, hypoirritant, hypo-allergenic, and resistant to free radicals. For meeting environmental laws and regulations, reducing manufacturers’ costs, and providing consumers with special chemicals that are antioxidant and environmentally friendly, this study proposes biodegradable natural QT-HA surfactants that are hypoirritant, hypotoxic, and no significant cytotoxicity.