Nanoformulations of Cosmetic Interest for the Cutaneous Uptake of Nickel

Abstract

Cosmetic products contain numerous metals used as pigments, UV filters, preservatives, antiperspirants and antimicrobial agents, which are responsible for allergic skin reactions, with the most common being nickel. To reduce skin penetration of Ni, innovative pharmaceutical formulations such as lipogels with chelating action against the metal ions themselves can be used. Chelation therapy allows a chelating agent to combine with metal ions to form a stable ring structure called a chelate. The chelate structure is more soluble in water than the toxic metal, which facilitates removal of the toxic metal from the tissue and its excretion by the kidneys. The aim of the following work was to evaluate the chelating properties against nickel ions of different types of lipogels containing flavonoids such as resveratrol and epigallocatechin gallate with chelating activities largely dependent on the number and position of their hydroxyl groups. The results obtained showed that lipogels based on epigallocatechin gallate show high chelating action against nickel, especially at low concentrations. In addition, rheological studies showed an ideal profile to ensure viscoelasticity and swelling of the lipogel within 48 h, confirming reports of 75% epigallocatechin release from the lipogel after 48 h. Tests have shown that lipogels based on epigallocatechin gallate have high chelating action against nickel, especially at low concentrations.

1. Introduction

The use of cosmetics can lead to various adverse effects on the body due to the presence of toxic metals [1]. Researchers have confirmed that cosmetic products contain heavy metals such as lead, cadmium, chromium, arsenic, mercury, and nickel, which can exist as either ingredients or impurities in these products [2]. Nickel can be present in cosmetics due to the manufacturing process of the cosmetic itself, through the industrial machinery used for production, which is often steel turbo-mills [3]. The use of cosmetics has increased over the past few years and so has the possibility of being in contact with this metal, which is present as a processing residue in cosmetic products. There are three main ways in which metals can enter cosmetics [4]: the addition of metal compounds such as UV filters and pigments, the use of raw materials and water containing metal elements as impurities, and the use of equipment with metal coatings. Nickel has not been added as an ingredient by the cosmetics industry [5]. There is no regulation requiring manufacturers to test or declare whether their products contain nickel residues. As a result, very few cosmetics companies carry out specific tests to ensure that their products are free of contamination and therefore offer consumers a greater sense of security [6]. This is because there is a limit of uncertainty in the detection method which makes it impossible to claim that nickel is totally absent from any cosmetic. However, heavy metals in cosmetics are well regulated in Europe in Annex II of R.1223/2009 containing the list of prohibited substances in cosmetic products. The Annex mentions that the presence of heavy metals such as arsenic (As), cobalt (Co), cadmium (Cd), antimony (Sb), chromium (Cr), lead (Pb), and nickel (Ni) is prohibited [7]. These metals pose potential health risks even in small quantities. For example, in allergic subjects, dermatitis can occur following skin exposure to nickel, to the point of producing eczema-like skin lesions with redness and itching [8]. Recognizing an allergy to nickel can sometimes be tricky, as the symptoms are similar to other skin conditions. Some common signs include itching, redness, swelling, blistering, and a burning sensation on the skin [9]. The symptoms can range from mild to severe, depending on an individual’s degree of sensitivity and the amount of nickel they are exposed to [10]. Nickel ions are transferred to the skin from contact with nickel-containing materials, household products, and cosmetics [11]. According to a study, it has been documented that less than 1% of nickel can cross the skin layer due to its slow transport speed; however, several factors influence this speed, such as the anatomical site, duration of exposure, dosing time, oxidizing capacity of sweat, and counter-ions (i.e., sulfate, acetate, nitrate, chloride) [12]. In recent decades, the development of adsorbent gels for heavy metal ions has increased considerably, especially in the cosmetic field, where the development of porous structures in polymer gels can potentially increase the number of effective adsorption sites. Among the numerous methods for the synthesis of porous gels, the formation of emulsions is of considerable importance [13]. Emulsions are considered biphasic colloidal dispersions that differ in the qualitative/quantitative composition of the phases, in the emulsifying systems used, or, simply, in the functional substances incorporated in them [14,15]. In general, when the inner phase is watery and the outer phase is oily, one speaks of water-in-oil (A/O) emulsions; otherwise, one speaks of oil-in-water (O/A) emulsions [15,16].

Recently, colloidal dispersions known as lipogels, in which the dispersed phase is no longer free to move but forms a three-dimensional lattice that retains the dispersant phase within it, have shown high potential for the effective removal of heavy metals [17]. This nano-system absorbs heavy metals in a three-dimensional interstitial structure, ensuring multiple sites per unit volume. Unlike other adsorbents, the gels absorb heavy metals in a highly porous three-dimensional network, which leads to high adsorption efficiency mainly due to the surface chemistry and the presence of hydrophilic functional groups (-COOH, -NH₂, -OH, -SO, -H, etc.) that act as complexing agents for heavy metal removal [18]. Recently, the use of natural compounds, such as flavonoids, with numerous biological activities, including antioxidant properties, which depend largely on the number and position of hydroxyl groups, has attracted considerable attention with regard to their ability to chelate metal ions [19,20].

In this work, particular attention has been paid to resveratrol and epigallocatechin gallate; these are flavonoids with chelating properties towards metal ions related to three possible metal-chelating sites, including (i) the 3-hydroxy-4-ketone groups in the C-ring, (ii) the 5-hydroxy group in the A-ring and 4-carbonyl group in the C–ring; and (iii) 3′,4′-dihydroxy groups located on the B-ring [21]. The aim of the following work was to assess the chelating properties towards nickel ions of different types of lipogels based on resveratrol and epigallocatechin gallate (Figure 1).

The aim was to identify, via ICP-MS analysis, which of the above molecules has the best chelating capacity against nickel.

2. Results and Discussion

2.1. Analysis via ICP-MS

All of the samples were analyzed via ICP-MS. Data were collected three times and the mean was taken. Gels containing oleic acid proved unsuitable for chelation. The graph in Figure 2 shows the interaction between other synthetized gels and the nickel solution after 6 h of contact. A preliminary evaluation of results obtained during the other two time periods (i.e., 1 and 3 h) showed that contact time of 6 h was the best one to obtain a major uptake of nickel ion.

What emerges from the graph is that the most effective gels for the chelation of nickel were the lipogels with epigallocatechin gallate (numbers 1 and 3 in Figure 2). Interestingly, a comparison between lipogel with resveratrol and catechin (EGCG), number 1, and lipogel containing only free resveratrol, number 2, that is unsuitable for chelation, shows that the highest chelating activity is still only given by EGCG. This can be easily explained considering the structures of the molecules involved. In fact, nickel ions can bind to phenolic groups in EGCG, which have higher chelating activity compared with the phenolic groups present in resveratrol. This introduces the chelating ability into polyphenolic compounds without the requirement for an external chelator. The protonated phenolic group, once deprotonated, could generate an oxygen center with a high charge density, or the so-called hard ligand. In addition to having their own functional groups, the molecules considered for this study are biocompatible and reducible. The hydroxyl group in positions 3 or 5 and the adjacent carbonyl (C=O) group are the main groups involved in the formation of complexes with metal ions [22]. Also, the hydroxyl groups eventually present in the B-ring could form chelate complexes. Secondary, in the case of flavonoid glycosides, the hydroxyl groups belonging to the sugar moiety can also participate in metal binding [23]. After the encouraging results obtained from the analysis of lipophilic gels containing free EGCG, another set composed of lipophilic gels containing 0.020 g, 0.030 g, 0.060 g, and 0.090 g of the active ingredients was tested anew. The goal was to detect the amount of EGCG that worked better in terms of the chelation of nickel. Results of the interaction between these gels and the nickel solution after 6 h of contact are listed in

Table 1. Interaction between EGCG gels and the nickel solution after 6 h of contact.

Time (h)	EGCG 20 mg	EGCG 30 mg	EGCG 60 mg	EGCG 90 mg
0	880 ± 1 µg/L Ni	880 ± 1 µg/L Ni	880 ± 1 µg/L Ni	880 ± 1 µg/L Ni
6	742 ± 1 µg/L Ni	740 ± 2 µg/L Ni	738 ± 2 µg/L Ni	734 ± 3 µg/L Ni

.

After the interaction with 6 h kinetics, the data show that the nickel percentage gradually decreases as the concentration of EGCG increases, as assumed at the beginning of this experimental study. This trend confirms the chelating activity of EGCG towards nickel ions also at a lower concentration of the active ingredient.

2.2. Rheological Test

Figure 3 below reports the amplitude sweeps of the used materials in a stress range between 0.008 Pa and 1407 Pa.

For each lipogel, the breaking point is reached for stress values higher than 45 Pa. Linear-viscoelastic (LVE) regions, assumed as the region in which the moduli trend is flat, can be defined according to graphs and are reported in

Table 2. LVE trends for selected lipogels.

Material	LVE Region
	from	to
EGCG 0.03	0.21 [Pa]	9.91 [Pa]
EGCG 0.06	0.21 [Pa]	9.93 [Pa]
EGCG 0.09	0.09 [Pa]	9.95 [Pa]
Catechin 0.02	0.21 [Pa]	9.94 [Pa]

below:

As shown in these sweeps, the storage modulus G’ increases, passing from 0.03 to 0.06 and 0.09 for EGCG lipogels. Moreover, frequency sweeps were performed to define the behavior of the materials used in the tests in a range of frequency between 0.1 and 10 Hz under the same test conditions of amplitude sweeps. Stress was fixed for all materials at 2 Pa, ensuring it was inside the viscoelastic region. Each frequency sweep was repeated two times. Figure 4 shows the average values of storage modulus (blue) and loss modulus (red) with standard deviation of the sweeps executed for each material.

Each lipogel exhibits predominant elastic behavior as reported by the higher storage modulus compared to the loss modulus in the range of frequency investigated. No crossover between moduli was observed in the frequency sweeps. Lipogel 0.09 reported the high value of moduli for the EGCG series.

2.3. Swelling Studies

To evaluate the stability of the gels and their affinity towards the physiological environment, swelling studies were performed at pH 7.4. The data (Figure 5) show that the degree of swelling increases as the amount of ECGC decreases, confirming the greater capacity of the gel containing the lower amount of ECGC (20 mg) to retain nickel. Data are expressed as mean of n = 3 replicates in experiments.

2.4. In Vitro Release Studies

The release of EGCG from lipogel was observed at pH 7.4, at different time intervals (i.e., 1, 2, 4, 8, 16, 24, and 48 h), using a cellulose acetate membrane of 12 kDa cut-off to eliminate the risk of possible skin component interferences. EGCG release profile was determined by UV-Vis spectrometry at 274 nm and expressed as a percentage of the substance released in respect to the total loaded amount in a function of time. Obtained data showed that EGCG was released within 48 h from the lipogel in quantities of 75.75% (Figure 6). Data are expressed as mean of n = 3 replications of experiments.

3. Materials and Methods

3.1. Materials and Instruments

The substances used were resveratrol, epigallocatechin gallate (EGCG), olive oil, and silica purchased from Sigma Aldrich, St. Louis, MI, USA, VWR Chemical Prolabo and Alfa Aesar, Haverhill, MA, USA. The release of nickel was measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) analysis. The determinations were carried out utilizing an Elan DRC-e ICP-MS instrument (Perkin-Elmer SCIEX, Concord, ON, Canada) and the sample delivery system consisted of a Perkin-Elmer autosampler model AS-93 Plus with peristaltic pump and a cross-flow nebulizer with a Scott type spray chamber. The ICP torch was a standard torch (Fassel type torch) with platinum injector. A solution containing Rh, Mg, Pb, Ba, and Ce (10 μg/L, Merck, Milan, Italy) was used to optimize the instrument in terms of sensitivity, resolution, and mass calibration. Quantitative data were obtained using an external calibration curve built by analyzing standard solutions of Ni at increasing concentrations prepared from a multi-element solution containing nickel at 10 mg/L (Multi-element Calibration Standard 3, Perkin-Elmer).

3.2. Lipophilic Gel Preparation

The lipophilic gels were prepared as follows: The active ingredient was placed in the beaker containing olive oil and stirred until completely dissolved. The mixture was then poured into the beaker containing silica by stirring with a glass rod, without lifting it up so as not to incorporate air, until the gel formed. To prepare 50 g of empty lipogel, the following were used: 2.5 g silica and 47.5 g olive oil. The lipogels containing the active substances were prepared in the same way as the quantities shown in

Table 3. Amounts of substances used.

Typologies	Resveratrol (g)	Catechin (g)	Resveratrol + Catechin (g)
Resveratrol-based lipogel	0.025	-	-
Catechin (EGCG)-based lipogel with three different concentrations	-	0.020	-
		0.030
		0.060
		0.090
Resveratrol + Catechin (EGCG)-based lipogel	0.025	-	0.025

.

3.3. Nickel Solution and Gel Sample Preparation for ICP-MS Analysis

As reported in the literature [4], 1 g of gel was put into contact with 20 mL of NiCl₂ 1.234 × 10⁻⁵ M. The samples were placed on a tilting shaker and divided according to their contact time (i.e., 1 h, 3 h, and 6 h). At the end, the resulting suspension was collected after the respective time passed and 150 µL of 65% HNO₃ was added for the following instrumental analysis. Lipophilic gels containing free epigallocatechin gallate, 0.020 g, 0.030 g, 0.060 g, and 0.090 g, were tested again with a method that involved a rotating agitation system. At the end, the resulting suspension was collected in a sterile conic tube and 150 µL of 65% HNO₃ was added.

3.4. Rheological Test

The materials used in this work were lipogel EGCG with different ratios (% p/p): 0.04%, 0.06%, 0.12%, and 0.18%. The rheological characterization of the materials was performed to define their structure and viscoelastic behavior. All measurements were performed on a parallel plate rheometer MCR 702e (Anton Paar, Ankerstraße, Austria). First, amplitude sweeps were executed to outline the linear viscoelastic region for each material using the following conditions: plate temperature of 25 °C degrees, gap between plates of 1.2 mm, frequency of 1 Hz, and plate diameters of 25 mm. Waiting time after loading of the samples was 200 s.

3.5. Swelling Studies

The affinity of gels towards the aqueous environment was determined by studying their swelling degree (WR%). The sample (100 mg) was placed in glass filters (porosity G2/3), previously wetted, centrifuged (2000 rpm for 5 min), and then weighed. Subsequently, the filters were put in contact with solutions at pH 7.4 and at 37 °C. At predetermined times (i.e., 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h), the excess of water was removed from the filters by percolation at atmospheric pressure. Subsequently, the filters were centrifuged (3500 rpm for 15 min) and then weighed. The weights recorded at the times indicated above were averaged and used to calculate the swelling degree, using the following equation:

WR% = (Ws − Wd) Wd∙100

where Ws and Wd are the weights of swollen and dried samples, respectively.

To evaluate the stability of the gels and their affinity towards the physiological environment, swelling studies were performed at pH 7.4 [23,24].

3.6. In Vitro Release Studies

In vitro release studies were performed using Franz diffusion cell apparatus with cellulose acetate membranes for 48 h. The apparatus was maintained at 36.5 °C to mimic physiological conditions. Receptor chambers (6.0 mL) were filled with phosphate buffer (pH 7.4) solution and stirred continuously to maintain sink conditions. Empty lipogel was used as control. At specific time intervals (1, 2, 4, 8, 16, 24, and 48 h), an aliquot (1 mL) of each sample was withdrawn from receptor chambers and replaced with fresh release medium. Samples were analyzed through UV-Vis spectrophotometry and EGCG release profile was expressed as a percentage of substance released with respect to the total loaded amount in a function of time [25].

4. Conclusions

One of the main challenges facing the cosmetics industry is the presence of heavy metals both as intentional ingredients and as contaminants of raw materials, which can have negative effects on consumers’ health. Consequently, there is an urgent need to regulate the levels of these metals, especially nickel, which is considered the most commonly present compound in cosmetics. For this reason, the aim of the present work was to make flavonoid-based lipogels as nickel chelating systems. Tests showed that, in particular, lipogels based on epigallocatechin gallate show higher chelating action against nickel and, above all, ideal rheologic and swelling properties to ensure lipogel viscoelasticity and swelling within 48 h. Furthermore, release tests showed a 75% release of flavonoid from the lipogel after 48 h. These results would suggest the possibility of using such lipogels as reuptake systems for nickel in cosmetic products, ensuring greater safety of these products, which have now become commonplace.