Self-assembled Trehalose Amphiphiles as Molecular Gels: A Unique Formulation to Wax-free Cosmetics

Abstract

Trehalose has been used as an emollient and antioxidant in cosmetics. However, we aimed to explore trehalose amphiphiles as oil structuring agents for the preparation of gel-based lip balms as part of wax-free cosmetics. This article describes the synthesis of trehalose fatty acyl amphiphiles and their corresponding oleogel-based lip balms. Trehalose dialkanoates were synthesized by esterifying the two primary hydroxyls of trehalose with fatty acids (C4–C12) using a facile, regioselective lipase catalysis. The gelation potential of as-synthesized amphiphiles was evaluated in organic solvents and vegetable oils. Stable oleogels were subjected to X-ray diffraction (XRD), thermal (DSC), and rheological studies and further used for the preparation of lip balms. Trehalose dioctanoate (Tr8), trehalose didecanoate (Tr10) were found to be super gelators as their minimum gelation concentration is ≤ 0.2 wt%. XRD studies revealed their hexagonal columnar molecular packing while forming the fibrillar networks. Rheometry proved that the fatty acyl chain length of amphiphiles can influence the strength and flow properties of oleogels. Further rheometry (at 25 °C, 37 °C, and 50 °C) and DSC studies have validated that Tr8- and Tr10-based oleogels are stable for commercial applications. Tr8- and Tr10-based olive oil oleogels were used for the preparation of lip balms. The preliminary results suggested that the cumulative effect of trehalose’s emolliency and vegetable oil gelling nature can be achieved with trehalose amphiphiles, specifically, Tr8 and Tr10. This study has also demonstrated that Tr8- and Tr10-based lip balms can be used as an alternative to beeswax and plant wax lip balms, indicating their huge potential to succeed as a new paradigm to formulate wax-free cosmetics.

Introduction

The concept of “sustainability” has been embraced by the personal care industry, especially cosmetics, in terms of products and their manufacturing practices. Over the decades, public demand for green and sustainable products has been growing in this sector. However, while choosing, the consumer expects the quality and efficacy of a non-sustainable product from a sustainable product. This demand has become a major challenge for cosmetic industries. To meet this challenge, plant and animal waxes were prioritized against petroleum-based microcrystalline wax, parabens, and mineral oil in lipsticks, lip balms, and facial creams (Chuberre et al. 2019). While formulating these products, waxes have been used as base materials, structuring agents, and thickeners. Waxes are primarily composed of complex mixtures of hydrocarbons and long-chain fatty acid esters (Behr and Seidensticker 2020). These chemicals endow cosmetics, especially lip balms and lipsticks with hardness, brittleness, and glossy appearance. To these waxes, vegetable oils such as olive oil, coconut oil, and jojoba oil are added as plasticizers so that additives such as colorants, flavoring agents (taste and smell), and stabilizers can be dissolved or dispersed easily (Behr and Seidensticker 2020; Shimizu, Nomura, and Bui 2021). In addition to plasticity, vegetable oils mainly provide moisturization and emollience to the lips. The success of waxes as a base material can be attributed to the long-chain fatty acid esters. They facilitate strength (hardness), stability (melting point) and vegetable oil entrapment in cosmetics (Chuberre et al. 2019). Inspired from waxes, we are aimed to synthesize trehalose-based fatty acyl esters as alternative to commercial waxes.

Candelilla wax, carnauba wax, and beeswax are popularly used in the cosmetic industry (Fernandes et al. 2013; Heldermann 2016). Though these waxes are natural, sustainable, and promising, their availability is limited. The dwindling supply of natural waxes and the corresponding rise in cost can be attributed to their increased demand (in cosmetics and foods) and sensitive climatic conditions in tropical countries (the majority of waxes are produced by Brazil (carnauba wax) and Mexico (candelilla wax)) (Heldermann 2016). In addition to sustainability, waxes pose functional and operating issues. Once the wax in lip balms is exposed to air, it gradually crystallizes and affects the viscosity and rigidity of the formulation (Shimizu, Nomura, and Bui 2021). Given these pitfalls, it seems inevitable to substitute waxes with new ingredients to produce next-generation cosmetics. As of today, the overall cost of waxes seems to be more advantageous than that of trehalose sugar. However, one needs to take into consideration a continuous rise in the pricing of waxes observed since 2003. To keep up with the availability issue and rising cost, beeswax users have reported several instances of deterioration of beeswax quality which comes from the adulteration of beeswax with cheaper materials such as animal tallow, plant oils, and most importantly paraffin (G.L. Yadeta 2014). Using trehalose-based amphiphiles in a formulation would allow avoiding important quality and adulteration issues.

Sugar fatty acid esters (SFAEs) have been extensively used as surfactants, emulsifiers, stabilizers, and emollients in a variety of cosmetic products (Pérez, Anankanbil, and Guo 2017). At the industrial level, SFAEs synthesis is carried out by chemical methods, involving severe conditions such as high temperature, use of hazardous chemicals, and generation of undesirable byproducts (Buzatu et al. 2020). However, chemical methods are not safe as they could leave traces of organic solvents. Henceforth, we have adopted a facile, one-step enzymatic method to synthesize trehalose fatty acyl esters (TFAEs). Trehalose is a non-reducing disaccharide with two α−1,1 linked glucose molecules. Trehalose is having excellent moisture retention properties and hydration potential even under harsh freezing and dehydrating environments (Yang et al. 2010; Mori et al. 2010). These properties have enabled trehalose to be used as an antioxidant, moisturizer, and skin protectant in certain cosmetics. Due to its protective function, trehalose is considered an ideal ingredient for sensitive skin formulations. Since lips are one of the most sensitive external organs of our body, the use of trehalose in lip balms could provide extra protection and emollience to lips. As mentioned before, waxes are the preferred choice as structuring base material in lip care products. In this regard, to emulate the role of waxes, value-added trehalose fatty acid esters are designed. After the synthesis, we are poised to check the structuring ability of trehalose esters in vegetable oils and to use thus formed molecular oleogels as the base material for preparing lip balms.

Sugar-based molecular oleogels represent a relatively new direction in the field of supramolecular gels. Under high entropy (provided by agitation and/or temperature), sugar derivatives aggregate as self-assembled fibrillary networks (SAFiNs) and structure vegetable oils to form molecular oleogels. Over the past few years, our research group has been exploiting sugar alcohols such as sorbitol, mannitol, xylitol, and raspberry ketone glucoside as raw materials for the synthesis of SFAEs to create molecular oleogels (Jadhav et al. 2013; Sai Sateesh Sagiri, Samateh, and John 2017b; Samateh et al. 2020; Silverman and John 2015). Further, SFAEs potential in food applications was also well documented (Sai Sateesh Sagiri, Samateh, and John 2017a; Samateh, Sagiri, and John 2018). However, the use of sugar fatty acid esters-based oleogels in cosmetics is not explored. The use of skin-protective trehalose to create base material could add high value to lip balms. In this manuscript, TFAEs will be synthesized by following a facile enzymatic reaction. The self-assembling and structuring ability of pure trehalose diesters was studied in various organic solvents and commercial vegetable oils. Physico-chemical and thermal properties of molecular gels were also studied prior to their use as base material in lip balms.

Materials and Methods

Materials

Trehalose anhydrous, and respective vinyl esters were purchased from TCI America (Portland, OR). Lipase from Candida antarctica was provided by Novozymes North America as Novozyme 435 (Franklinton, NC). Hexanes and acetone were purchased from Fisher Scientific Company (Suwannee, GA). Unscented Lip Balm by Badger (W.S. Badger Co., Inc, Gilsum, NH) and Natural Vegan Hemp Balm by The Merry Hempsters (Eugene, OK) were purchased from a local Whole Foods store.

Trehalose Fatty Acid Ester Synthesis

Trehalose-based amphiphiles were synthesized using a previously reported reaction of enzymatic transesterification of sugar alcohol with a respective vinyl ester in a dry organic solvent (Jadhav et al. 2013; Silverman and John 2015). Solid Novozyme 435 lipase (0.146 g) was added to a mixture of trehalose (1.46 mmol, 0.5 g), vinyl ester (7.3 mmol) and 14.6 mL dried acetone in a 500 mL screw-cap Erlenmeyer flask. Acetone has been used as an industrially preferred solvent for conducting green chemistry in synthetic reactions (G. Jessop et al. 2012; Babij et al. 2016). The respective reaction was set up in an orbital shaker at 250 RPM and 50 °C. The reaction progress was monitored by thin-layer chromatography (TLC) using two solvent systems: 1:9 methanol/dichloromethane and 1:1 hexanes/ethyl acetate. The results of the TLC were visualized using KMnO₄ solution and gentle heating. The product spot varied depending on the vinyl ester used with the observed range of Rf = 0.35–0.57. Once the product formation was confirmed after 48 hours, the enzymes were filtered out, rinsed with acetone, air-dried and stored for reuse. After the evaporation of acetone from the filtrate, another TLC test was performed to reveal the presence of trehalose-diester product, unreacted vinyl ester (Rf =1) and free fatty acid (Rf = 0.82). The solid mixture was washed with hexanes three times to remove impurities. Another TLC test was performed on the purified product to confirm the absence of impurities. The solid product was allowed to air dry. Purity of the products and absence of any trace amount of solvent was confirmed by ¹H NMR, ¹³C NMR and FTIR studies.

Preparation of Molecular Gels

The gelation ability of trehalose amphiphiles in various solvents was tested by weighing 10 mg of the respective gelator and dissolving it in 200 μL of a solvent. The mixture was heated, then the obtained homogenous solution was allowed to cool at room temperature. A vial inversion test was performed to confirm the gel formation (S. S. Sagiri et al. 2015). No flow of constituents under gravity was regarded as gel formation. The efficiency of the trehalose amphiphiles was assessed by finding the minimum gelation concentration (MGC) in a particular solvent.

The thermal stability of the oleogels was assessed by measuring gel-to-sol transition temperature (T_g). The T_g for the trehalose-based gels was determined by immersing 2 mL inverted vials containing respective gels into an oil bath and slowly heated. The temperature at which the gel turned into a sol was reported as T_g of the respective gel. In case of organogels, the solvent was found to be escaping from the gel matrix when kept at room temperature. In this regard, the temperature at which the gel destabilizes was noted as gel decomposition temperature, T_d, instead of gel-to-sol transition temperature, T_g.

Characterization of Molecular Gels and Lip Balms

Optical Microscopy

Leica DM 2000 LED (Leica Microsystems, Germany) microscope was used to observe the microstructure of trehalose-based gels. Molten oleogels were applied on a glass slide and placed under the microscope to study the self-assembled structures.

Field Emission-Scanning Electron Microscopy (FE-SEM)

Helios-FIB-FEI 600 was used to investigate the nature of the gelators after self-assembly. After the formation of gels, the solvent (ethyl acetate) was evaporated by placing them under a vacuum on a silicon 111 wafer for two days (Holey et al. 2022). Thus formed xerogels were further used in the experiment. Gold was then sputtered onto the sample surface and FE-SEM images were acquired in immersion mode with an operating voltage of 3 kV and operating current of 25 pA, and dwell time of 300 ns.

X-ray Powder Diffraction (XRD)

PANalytical X’Pert Pro powder X-ray Diffractometer (Philips Panlytical, USA) was used to investigate the molecular assembly of the gelators. Two types of samples were investigated: pure gelators as a powder and their respective xerogels. Pure gelators were ground into a fine powder and placed onto a glass cover slide which was then placed onto the holder. Similarly, xerogels were prepared by following the protocol as mentioned in FE-SEM studies. The glass slide with the xerogel was then placed onto a holder. The experiments were performed with the X-ray beam source of Cu K_α at 40 kV and 40 mA. All the figures representing Powder the X-ray diffraction (XRD) data are provided in the supporting information (Fig S6–S9, SI). XRD data at smaller (2° – 7°) and wider (5° - 50°) were performed using the 1/16 and ¼ anti-scatter slits.

Ultraviolet-visible (UV-Vis) spectroscopy

Evolution 300 (Thermo Scientific, USA) UV-Vis spectrometer was used to investigate the intermolecular interactions of the trehalose derivatives in the solid state as compared to that of the xerogel state and compare the results to those obtained from the XRD studies. Pure gelators were placed onto a glass cover slide which was then inserted into the spectrometer. Similarly, the glass cover with the respective xerogel was then inserted into the spectrometer for measurements. For both solid samples and xerogels, the scan was performed in the range of 200 nm-800 nm.

Rheological Measurements

All rheological measurements were performed on molecular oleogels using 302 modular compact rheometer (Anton Paar, GmbH, Germany). For measurements, a parallel plate of diameter 10 mm (geometry gap: 1 mm) was used for (a) amplitude sweeps: oscillatory strain, 0.001% - 100%; frequency, 1 Hz at 25 °C (b) frequency sweeps: oscillatory strain, 0.0251%; angular frequency, 0.1–10 rad/s at 25 °C. The samples were prepared by dissolving 5 mg of the respective gelator in 200 μL of canola oil by heating in a vial. Upon the formation of a homogenous liquid, the vial was emptied into a mold that was previously placed onto the rheometer’s stage. The gel was allowed to cool to room temperature for 5 minutes, after which a rheological experiment was performed.

For rheological measurements of trehalose-based lip balms, a parallel plate of diameter 10 mm (geometry gap: 1 mm) was used for (a) amplitude sweeps: oscillatory strain, 0.001% −100%; frequency 1 Hz, at 25 °C, 37 °C, and 50 °C; b) frequency sweeps: oscillatory strain, 0.01%; angular frequency 0.1–10 rad/s, at 25 °C, 37 °C and 50 °C, except beeswax-containing commercial lip balms for which oscillatory strain of 0.005% at 25 °C, 0.004% at 37 °C; and 0.001% at 50 °C

The rheological properties of trehalose-based samples were compared to the commercially available lip balms: beeswax-based unscented lip balm by Badger and candelilla-based natural vegan hemp balm by The Merry Hempsters. A thin uniform slice of the respective lip balms was cut out from the main lip balm stick and placed directly onto the rheometer’s stage for rheological experiments.

Preparation of Trehalose-Based Lip Balms

Three types of trehalose-based lip balms were prepared: trehalose dioctanoate (Tr8)-based lip balm, trehalose didecanoate (Tr10)-based lip balm, and 50% trehalose dioctanoate (Tr8) - 50% trehalose didecanoate (Tr10) based lip balm. For the formulations containing 100% of the respective gelator, 200 mg of the gelator (Tr8 or Tr10) was added to a vial containing 350 mg of shea butter, 100 mg of coconut oil, and 350 mg of olive oil. The mixture was then heated until a homogenous solution was formed. Then the mixture was poured into a lip balm container and allowed to cool down at room temperature. For the lip balm formulations containing 50% Tr8–50% Tr10, 100 mg of Tr8 and 100 mg of Tr10 were added to the vial and the same procedure was followed.

Differential Scanning Calorimetric (DSC) Analysis

DSC 822e (Mettler Toledo, USA) was used to understand and correlate the thermal properties of both commercial and trehalose-based lip balms. The samples (5 to 15 mg) were taken in pure aluminum crucibles and were hermetically sealed with a pierced aluminum lid. An empty pure aluminum crucible with pierced lid was used as the reference. The crucibles were kept in the furnace and the experiment was conducted under inert nitrogen atmosphere. Initially, samples were kept at 25 °C for two minutes and then heated to 170 °C at 2°/min. Then, the samples were kept at the maximum temperature for two minutes and cooled down to 25 °C at 2°/min.

Results and Discussion

The lipase-mediated enzymatic reaction was found to be working efficiently with trehalose and fatty acids of different chain lengths. A schematic representation of the enzymatic reaction is given in Scheme 1. Of all the tested fatty acids, five of them produced better yields when reacted with trehalose. Derivatives of the corresponding fatty acids are trehalose dibutyrate (Tr4), trehalose dihexanoate (Tr6), trehalose dioctanoate (Tr8), trehalose didecanoate (Tr10), trehalose didodecanoate (Tr12). Since lipase acts on the primary hydroxyl group(s), the two primary hydroxyls on C6 of the two glucose units were esterified to yield trehalose-based diesters. After the purification steps, all the diesters were obtained with higher yields as white powders or flakes with a difference in crystallinity (explained later in XRD studies). The successful synthesis of TFAEs was confirmed by ¹H, ¹³C NMR, and FTIR spectrophotometric studies. (Data is given in supplementary file).

Scheme 1.

General reaction scheme of trehalose dialkanoates synthesis

Gelation Efficiency

The gelation efficiency of TFAEs was evaluated using a variety of organic solvents and vegetable oils with different polarity and unsaturation, respectively. The order of relative polarity of the solvents in this study is toluene (0.09) < ethyl acetate (0.228) < Hexanol (0.559) < DMSO (0.644) < ethanol (0.654). TFAEs have formed organogels with solvents having a less polar nature, as compared to the solvents with more polarity (Table 1). A reverse trend was observed in the case of ethyl cellulose gelators (Davidovich-Pinhas, Barbut, and Marangoni 2015). These polymeric gelators formed stable oleogels when the polarity of the solvent was raised and vice versa. Formation of additional hydrogen bonds between the unsubstituted hydroxyl groups of ethyl cellulose and polar moieties of the solvent were responsible for the better gelation in polar solvents. Unlike ethyl cellulose polymeric gelators, the gelation behavior of TFAEs is driven by self-assembly therefore, a different gelation mechanism takes place. In the present scenario, the unsubstituted hydroxyl groups of trehalose would have been involved in gelator-gelator interactions. On the other hand, gelator-solvent interactions were mediated by apolar tail groups (Gaudino et al. 2019). This kind of interaction can only be possible with the inverse micellar assembly of gelators (Sai S. Sagiri and Rao 2020). The molecular association of gelators as inverse micelles (primary structures) and their propagation as bilayers (secondary structures) lead to the formation of the fibrous network (tertiary structures). The bilayer arrangement and fibrous network of gelators were confirmed by XRD and microscopic studies.

Table 1.

Gelation ability of trehalose amphiphiles in various solvents

Solvent	Tr4	Tr6	Tr8	Tr10	Tr12
Toluene	In	G (1.0)	G (1.0)	G (2.0)	G (2.0)
Ethyl acetate	G (0.5)	G (1.0)	G (1.0)	G (2.0)	G (2.0)
Hexanol	G (0.5)	G (1.0)	Pt	In	In
DMSO	In	In	In	In	In
Ethanol	In	In	In	In	In
Olive oil	G (0.8)	G (0.4)	G (0.2)	G (0.06)	G (0.4)
Canola oil	G (0.8)	G (0.4)	G (0.2)	G (0.06)	G (0.4)
Grapeseed oil	G (0.8)	G (0.4)	G (0.2)	G (0.06)	G (0.4)

G: gel formed, In: Insoluble, Pt: Precipitate

To explain this behavior, HLB of gelators is empirically used, as the difference in the length of the fatty acyl chain has directly influenced the HLB of gelators. Hydrophilic-lipophilic balance (HLB) of the amphiphilic gelators was calculated using Griffin’s equation (Royer et al. 2018).

$$\mathit{\text{HLB}}=20*M_h/M$$ (1)

Where, M_h is the molecular mass of the hydrophilic moiety of the molecule and M is the whole mass of the molecule.

The calculated HLB values of TFAEs are given in Table S1. The difference in HLB of the gelators seems to have affected their self-assembly, which in turn affected their gelation efficiency in organic liquids (Sekhar et al. 2020). The higher HLB value indicates the predominant hydrophilic nature which leads to Tr4 precipitating in highly non-polar toluene. Although Tr4 did not form any gel, others have formed stable gels in toluene. On the contrary, Tr4 and Tr6 have formed stable gels in hexanol but not others. When the solvent polarity is further increased by means of DMSO and ethanol, no gelator has formed stable gels. This suggests that optimal solvent polarity is needed for the self-assembly of TFAEs. Supramolecular self-assembly involves a very sensitive mechanism, in which the gelator molecules should not be either completely dissolved or precipitated in the solvent (Dassanayake et al. 2009; Marangoni and Garti 2011). In other words, the formation and stability of a metastable gel phase are based on the equilibrium between the solvophobic (gelator-gelator) and solvophilic (gelator-solvent) interactions. The finely dispersed or dissolved gelators should phase out of the solvent and self-assemble to form network structures without precipitation. If the polarity of the solvent does not comprehend the HLB of the gelator, the molecules tend to aggregate and precipitate out from the solvent. Henceforth, a difference in gelation behavior was observed concerning the change in polarity of solvents and trehalose-based gelators.

In the case of vegetable oils, all gelators have formed stable gels (Table 1). In this study, vegetable oils were chosen in such a way that their unsaturated fatty acid content is distinct from each other. The order of % of PUFAs (Polyunsaturated Fatty Acids) is olive oil (10.5) < canola oil (28.1) < grapeseed oil (74.7) (Asadi, Shahriari, and Chahardah-Cheric 2010). Although % of PUFAs are different from one another, the total unsaturation is above 80% in all the tested vegetable oils. Henceforth, % of unsaturation did not show any significant effect on the gelation behavior of TFAEs as they have formed stable gels. Of all the tested oils, olive oil was chosen for the lip balm studies because of its physiological health benefits and high commercial value (García-González, Aparicio-Ruiz, and Aparicio 2008; Al-Waili 2005). Although gelators have formed stable gels in organic solvents and all the tested vegetable oils, their efficiency has been significantly different from each other. MGC was found to have decreased with the increase in fatty acyl chain length from Tr4 to Tr10 and then increased for Tr12 (Fig. 1). This suggests that Tr10 is the most efficient trehalose-based gelator. This could be due to the optimal balance in polarity between the hydrophilic trehalose moiety and lipophilic decanoate(s) moiety (Bascuas et al. 2020). Based on the MGC values, trehalose amphiphiles, namely, Tr8 and Tr10, can be regarded as super gelators. In general, if the MGC is ≤ 0.2, then the gelator is regarded as a super gelator (Farahani et al. 2019; Ou et al. 2013). MGC of Tr8 and Tr10 was found to be 0.2 % and 0.06% (w/v), respectively. There are numerous synthetic or chemical-derived super hydrogelators and super organogelators (K. Das and K. Gavel 2020; Podder et al. 2019), but bioderived gelators as super gelators are very rare. Trehalose-based gelators could be the first of its kind in the family of sugar fatty acid esters to be regarded as super gelators. Though the super gelation is predominantly due to the optimal balance between trehalose and fatty acyl groups, this behavior is also aided by a high % of unsaturated fatty acids in vegetable oils. The greater degree of conformational freedom associated with the unsaturated fatty acids leads to a higher molar volume of solvent (J. Martins et al. 2018), which in turn could facilitate the creation of more intergelator junction zones. These junction zones enhance the 3D gelator network propagation, thereby, gelation is possible at very low concentrations. Oleogels stability also seems to be governed by these favorable gelator-solvent (oil) interactions. The high stability of all trehalose-based oleogels can be attributed to the balance in between the solvophobic and solvophilic critical forces. On the contrary, in the case of organogels, the equilibrium is relatively more shifted towards the solvophobic interactions leading to the time-dependent rupture of the metastable phase. Our results unambiguously characterized the selective preference of trehalose-based gelators toward vegetable oil, rather than organic solvents.

Figure 1.

MGC (left), gel-to-sol transition temperature (T_g) and decomposition temperature (T_d) T_g and T_d (right) of trehalose-based gels

Thermal stability

Thermal strength or stability was evaluated by determining the gel-to-sol transition temperature, T_g, gel decomposition temperature, T_d, of oleogels and organogels, respectively. All oleogels have exhibited high thermal stability as their T_g values were found to be above 100 °C (Fig. 2). The order of thermal stability of oleogels in terms of TFAEs is expressed as Tr8 (151 °C) > Tr10 (144 °C) > Tr6 (142 °C) > Tr12 (140 °C) > Tr4 (129 °C). The order of thermal strength is in correlation with the MGC of the gelators. In the case of MGC studies too, Tr10 and Tr8 have shown better performance, followed by Tr6, Tr12, and Tr4. Since uniform gelator concentration (1 % (w/v)) was maintained in all the samples, gelator network density and gelator-solvent interactions played a crucial role in the strength of oleogels (Doan et al. 2017). At 1 % (w/v), higher network density can be expected for Tr8 and Tr10 as their MGC was too low. Henceforth, more amount of heat was needed to disperse and dissolve these gelators, compared to others.

Figure 2.

Optical micrographs of trehalose-based oleogels

In the case of organogels, the solvent was found to be escaping from the gel matrix when kept at room temperature. In this regard, the temperature at which the gel destabilizes was noted as gel decomposition temperature, T_d, instead of the gel-to-sol transition temperature, T_g. The T_d values were found to be decreasing with the increase in fatty acyl chain length of gelators, except for Tr6 (Fig. 1). Order of T_d is almost consistent with the MGC of gelators in ethyl acetate. This suggests that ethyl acetate has strong interactions with gelators having more polarity.

Morphological Studies: Optical Microscopy and FE-SEM

The microstructure of gelator network in oleogels was studied using a bright-field optical microscope and field emission scanning electron microscope (FE-SEM). Optical micrographs have revealed the fine fibrous network of gelators in all oleogels. This was confirmed by examining them under FE-SEM. Supramolecular self-assembly of amphiphilic gelators has resulted in translucent and transparent oleogels (shown as inserts in Fig. 2). Higher transparency was observed in Tr10 oleogels and least in Tr4 oleogels. Opacity has been influenced by the length, diameter, and density of fibrous networks in oleogels. Optical micrographs showed long and dense fibrous architecture in Tr4 oleogels. On the other hand, short and fine fibers in Tr10 oleogels. Higher magnification was needed to observe the fibers in Tr10 and Tr8 oleogels. This suggests that the fibrous network is less conspicuous to visible light and can lead to transparency in oleogels. Hence, Tr8 and Tr10 have formed highly transparent oleogels compared to others. It has also been noticed that the diameter of fibers increased with the increase in the aliphatic chain length of gelators. The diameter of Tr6 fibers was found to be within the range of 40–64 nm, while Tr8 was in the range of 82–107 nm (Fig. 3). In addition to diameter, length of fibers also seems to be affected by the length of the fatty acyl chain. In case of Tr12 oleogels, random aggregates of fibrous network were seen (Fig. 2). Higher hydrophobicity of gelators might have influenced the formation of a network in Tr12 oleogels. In general, intermolecular hydrogen bonds between the hydroxyl groups of trehalose and Van der Waals forces between the alkyl chains, guide their self-assembly mechanism (Chen et al. 2021). Any alteration in these interactions directly influences the length, diameter, and growth of fibers in the network (Datta and Bhattacharya 2015). Higher hydrophobicity between Tr12 molecules (evident from HLB values) might have enhanced their random aggregation before the defined growth of fibers into needle-like structures (as observed with other gelators).

Figure 3.

FE-SEM images of trehalose-based organogels

XRD and UV-Vis spectroscopic studies

The ability of gelator molecules to self-assemble is at the core of their ability to produce molecular gels. Any variation in molecular interactions would cause significant changes in the physical properties of a supramolecular system (Doan et al. 2015). The influence of the difference in the self-assembly of trehalose amphiphiles on physical properties was studied for both oleo- and organogels. Currently, we do not have a proper rationalization for the difference in physical properties of both oleo- and organogels. However, physical properties could be significantly related to the molecular self-assembly or how the molecules are arranged within the matrix (Fayaz et al. 2017). Therefore, the self-assembly of organogels was investigated by using powder X-ray diffraction (vide Supplemental Information for details).

Based on PXRD studies, molecular assembly patterns of pure gelators were identified. Both Tr4 and Tr10 exhibit biphasic tetragonal columnar (Col_t) and hexagonal columnar (Col_h) molecular assembly, (Table 2). Gelators with the intermediate fatty acyl chain length such as Tr6 and Tr8 exhibit monophasic assembly with rectangular columnar (Col_r) and Col_h packing patterns, respectively. Interestingly, the biphasic molecular assembly of Tr4 changes to monophasic with the relative increase in chain length (Tr6 and Tr8). Further increase in fatty acyl chain length to Tr10 brings the molecular assembly back to biphasic with the retention of the monophasic features again in Tr12. As expected, with the lattice constant being directly proportional to the molecular length, d, they both increase with the aliphatic chain length of the gelators, where the most significant increase was observed from Tr4 to Tr6.

Table 2.

XRD results and respective packing patterns of trehalose-based gelators

Molecule	d values, Å	Reciprocal ratios	Packing pattern	‘a’ and ‘b’ values, Å
Tr4	16.5:11.56	1:1/$\sqrt{2}$	Colt,	a=33, b=12.3
	14.28:8.23	1: 1/$\sqrt{3}$	Colh	a=16.5
Tr6	30.05:21.09:18.26	1:1/$\sqrt{2}$: 1/$\sqrt{3}$.	Colr	a=61, b=22.5
Tr8	19.08:12.18:10.05	1: 1/$\sqrt{3}$: 1/$\sqrt{4}$	Colh	a=22
Tr10	30.87:22.34	1:1/$\sqrt{2}$	Colt	a=61.7, b=23.9
	25.35:14.91	1: 1/$\sqrt{3}$	Colh	a=29.3
Tr12	27.39:14.40	1:1/$\sqrt{3}$	Colh	a=31.6

In contrast to the solid gelators, all the xerogels (Tr4 to Tr12) exhibited monophasic Col_h molecular assembly, giving rise to high aspect ratio fibers (Table 3). The ‘d’ values correspond to a reciprocal spacing ratio of 1:1/√3:1/√4 which suggests a Col_h packing as evidenced from the previous studies (Satapathy, Prabakaran, and Prasad 2018; Kim et al. 2005; Percec et al. 1996; Kato et al. 2009). The schematic representation of Col_h molecular packing is presented in Scheme 2.

Table 3.

XRD results and packing patterns for trehalose-based xerogels.

Gelator	‘d’ values, Å	Reciprocal ratios	Packing pattern	‘a’ value, Å
Tr4	16.57:10.56:8.37:7.00	1: 1/$\sqrt{3}$: 1/$\sqrt{4}$	Colh	19.13
Tr6	29.28:16.73:14.05:12.06	1: 1/$\sqrt{3}$: 1/$\sqrt{4}$	Colh	33.81
Tr8	18.87:12.22:10.18:7.30	1: 1/$\sqrt{3}$: 1/$\sqrt{4}$	Colh	21.79
Tr10	21.99:12.18:10.46:8.30	1: 1/$\sqrt{3}$: 1/$\sqrt{4}$.	Colh	25.39
Tr12	24.01:13.33:12.06		Colh	27.72

Scheme 2.

Schematic representation of hexagonal columnar (Col_h) unit cell (left) and its corresponding dimer (right) which leads to Col_h self-assembly in Tr8 gels.

The geometry of the monomers is optimized via B3LYP/6–31g(d). Two units of monomers were further optimized to estimate a lattice constant of a = 21.8 Å value of the unit cell which agrees well with the experimental value (21.79 Å). The increase in molecular length of pure gelators Tr10 and Tr12 relative to the respective xerogels was confirmed by the XRD results (vide Supplemental Information, Fig. S7–S9), where a red shift was observed for both powders. A decrease in the thermal stability of Tr12 when compared to other trehalose derivatives is an indication of relatively lesser intermolecular interactions in its fiber network. This observation was confirmed by a decrease in crystallinity for Tr12 xerogel relative to other xerogels where the order of crystallinity increased with the increase in the chain length. From PXRD analysis, the drastic increase in the columnar length a, for Tr6 can be related to its bilayer assembly owing to the interdigitation of hydrophobic tails.

The broadening of UV-Vis features (vide Supplemental Information, Fig. S10), greater Mie scattering (tails extending above the baseline) and red shift of λ_max signifies significant aggregation existing behavior in case of solids. On the other hand, as deduced from the UV-Vis results, the aggregation behavior is significantly reduced in case of all the xerogels. stronger intermolecular interactions (π-π and Van der Waal forces) of all the trehalose amphiphiles in solid aggregated state as compared to that in the xerogel state. This validates well with the XRD results (Fig, S7–S9, SI) which have also identified the hexagonal columnar packing (Table 3) in xerogels due to the increased intermolecular interactions (π-π and Van der Waal forces).

Rheological studies

Rheological analysis was conducted on oleogels but not organogels, as the organic solvent evaporated from the latter during the experiment. Flow behavior of oleogels was characterized by subjecting them to amplitude and frequency sweep tests. The LVR (Linear Viscoelastic Region) obtained from amplitude sweep curves was interpreted to explain the strength of oleogels by observing three factors, namely, maximum G′ in LVR (G′_LVR), the difference between G′ and G″ during LVR, and length of the linear region in the curve (Patel et al. 2014). All the curves have clearly shown higher G′ over G″, indicating the semi-solid gel formation by all trehalose amphiphiles (Fig. 4). But they differ from each other in their gel strength as Tr8 had the highest G′_LVR (95 kPa) and Tr4 had lowest (110 Pa). However, Tr6 and Tr10 oleogels also exhibited a strong gel property as their G′_LVR was found to be around 10 kPa and 15 kPa, respectively (Fig. 4). Similar to Tr4 oleogels, Tr12 oleogels also had poor strength with G′_LVR around 2500 Pa. Lower strength of Tr4 and Tr12 oleogels is in accordance with their poor gelation efficiency (evident from MGC values) and lower thermal stability. Despite the poor strength, Tr4 and Tr12 oleogels are considered as true gels since their G′ was higher than G″ (characteristic of a gel). Poor gelation efficiency, in other words, weak microarchitecture of Tr4 and Tr12 gelators has led to the poor strength of corresponding oleogels.

Figure 4.

Amplitude sweep curves (G′ is in black and G″ is in red color) and G′_LVR of oleogels

The shear strength of oleogels can also be determined by identifying the stress at which gel-to-sol transformation occurs during the shear. The concept of gel-to-sol transformation was also used earlier to determine the thermal strength, T_g of oleogels. In rheology studies, the gel-to-sol transition is determined as the cross-over point at which G″ takes over G′ (tan delta (tan ẟ) = 1; G′ = G″) (Mezger 2020). The cross-over point is a function of applied stress and strength of the material, and upon reaching it, the material turns to a liquid predominant state (G″ > G′) with further application of stress. Tr8 and Tr10 oleogels have revealed higher yield stress (Y_s) as compared to others (Fig. 5), suggesting that these oleogels are more stable and possess a strong gelator network. A lower cross-over point or Y_s was clearly seen with Tr4 oleogels, indicating their weak microstructure and instability. It is also interesting to note that there was a small difference in Y_s of Tr6 and Tr12 oleogels, although they differ high in their G′_LVR. Higher elastic modulus (G′) and Y_s of Tr8 oleogels indicate that these oleogels have rigid, elastic, and brittle nature. On the contrary, Tr4, Tr6, and Tr12 oleogels are soft and viscous, based on their poor G′_LVR and Y_s values. On the other hand, Tr10 oleogels possess better G′_LVR (15.6 kPa) and high Y_s values, indicating that these are having better elastic (rigid) and viscous (flexible or soft) properties (Doan et al. 2015). The viscoelastic nature of oleogels is further explained by evaluating the critical stress (C_s) and yield zone (Y_z) from amplitude sweep curves.

Figure 5.

Rheological properties of trehalose-based oleogels

The critical stress (C_s) value of oleogels was determined in the LVR, where the curve of G′ deviates by 10% (Fayaz et al. 2017). C_s provides the preferable stress range in which the oleogel can have linear viscoelastic behavior during processing. In other words, below the C_s value, the viscoelastic or rheological behavior of oleogels is independent of the applied stress. Beyond C_s, oleogels start undergoing permanent deformation (yielding). The partial breakup of gelator microarchitecture starts at C_s, continues during the Y_z and complete breakup occurs at Y_s. Values of C_s also followed the same order as that of Y_s, with the exception that Tr6 > Tr12. The corresponding critical strain values of C_s provide clarity regarding the strength of SAFiNs of gelators. Tr8 oleogels have undergone the least deformation (strain, 0.03 %) at the applied critical stress (29.4 Pa). This indicates that a strong gelator network is associated with Tr8 oleogels. On the other hand, Tr4 and Tr6 oleogels have undergone the same deformation (0.1%) although their corresponding C_s values (Tr4: 0.1 Pa, Tr6: 8.8 Pa) are distinctly different from each other. A similar kind of observation was made with Tr10 and Tr12 oleogels. The difference in the obtained critical strain values (Tr10: 0.16 %, Tr6: 0.13%) was found to be insignificant although the corresponding C_s values (Tr10: 23.1 Pa, Tr12: 3.1 Pa) vary from each other. These results support that Tr6- and Tr10-based gels possess a strong gelator network over Tr4- and Tr12-based gels, respectively. All the gels have started yielding after C_s. The highest yielding (via Y_z) was noticed with Tr4 and Tr12 gels, followed by Tr10, and lowest with Tr8 and Tr6 oleogels (Fig. 5). Higher yielding confirms that Tr12, Tr10, and Tr4 gels are soft and viscous and, Tr6 and Tr8 oleogels are rigid and elastic. These results corroborate with the G′_LVR and Y_s values. The soft (lower G′_LVR, C_s, and Y_s) and viscous (higher Y_z and critical strain) nature of Tr4, Tr6, and Tr12 oleogels was confirmed by these results. Interestingly, Tr10 oleogels possess both rigid (higher G′_LVR, C_s, and Y_s) and viscous (higher Y_z and critical strain) properties. Of all, Tr8 gels possess higher rigidity and elastic properties and are brittle in nature.

To ensure the gel and viscoelastic nature of oleogels, a stress value within the LVR was chosen to perform frequency sweep studies. Time-dependent deformation behavior of oleogels was determined in the angular frequency range of 0.1–10 rad/s. In the applied frequency range, G′ and G″ curves were mostly linear with G′ > G″, indicating the gel nature of samples (Fig. 6). Parallel/linear G′ and G″ curves with respect to frequency axis also suggest that the rate of deformation and bonds forming the microarchitecture of gelator network at the given amplitude are almost in equilibrium within the time frame of the test. Thus, the viscoelasticity and stability of gels could be maintained while processing them for food and cosmetics. Although oleogels seem stable, all of them showed a marginal dependence on frequency, as substantiated by the slightly positive slope of G′ and G″ curves. This type of rheological behavior is typical of weak colloidal gels in which viscous nature predominates at low frequency and elastic nature at high frequency (Trappe and Weitz 2000; Hesarinejad, Koocheki, and Razavi 2014).

Figure 6.

Frequency sweep curves (G′ is in black and G″ is in red color) of trehalose oleogels

The amplitude and frequency sweep studies inferred that small changes in hydrophobicity (fatty acyl chain length) have significantly affected the flow behavior of oleogels. The use of trehalose as a structuring agent instead of an additive in cosmetics can be possible by conjoining it with either caprylate or decanoate molecules. Tr8 and Tr10 gelators possess the optimal molecular properties (evident from HLB values) to generate stable oleogels with the requisite flow properties while preparing wax-free lip balms. These results suggest that Tr8 and Tr10 oleogels are the best-suited gels for cosmetics applications.

Trehalose-Based Lip Balm Formulations

Based on the microscopic and rheological data of molecular gels, Tr8 and Tr10 gelators were chosen as the model gelators to prepare lip balms. These gelators were used to completely replace the wax in the lip balm formulation. Apart from the pristine Tr8 and Tr10-based lip balms, a third lip balm was prepared using an equal (1:1) mixture of the two gelators. All the formulations were found to be in off-white color and stable at room temperature; more importantly, shape was retained after pouring into the lip balm mold (stick) (vide supplementary file, Fig. S11). Performance of these lip balms was compared against two commercial lip balms made using bees wax and vegetable wax-based formulations by conducting rheological and thermal (DSC) studies.

Rheological Assessment of Lip Balm Formulations

Amplitude sweep studies showed that the G′_LVR of Tr8-based lip balm is in the magnitude of commercial lip balms (Fig. 7). On the other hand, G′_LVR of Tr10 and Tr8/Tr10-lip balms were found to be lower by an order of 10 against the commercial lip balms. A comparative account of C_s, Y_s, Y_z, and critical strain and of lip balms is graphically illustrated in Fig. 8. Tr10-based lip balm was found to be less rigid and has shown higher Y_s along with large Y_z. This indicates that Tr10-based lip balms are having soft (due to lower G′_LVR), highly viscoelastic (due to longer LVR), less brittle (large Y_z), and higher shear strength (due to high Y_s) properties. Comparatively, Tr8-lip balms are strong (high G′_LVR and Y_s) in nature like commercial lip balms. However, Tr8-lip balms also possess high Y_z as that Tr10-lip balms. This indicates that trehalose-based oleogels possess the viscous property that facilitates malleability during processing. Rheological properties of Tr8/Tr10-lip balm was found to be in between Tr8- and Tr10-based lip balm properties. This suggests that the rheological flow properties of the final product (lip balm) can be controlled by adjusting the % composition of two gelators. In other words, trehalose derivatives facilitate fine-tuning of the lip balm’s flow properties.

Figure 7.

Amplitude sweep curves (G′ is in black and G″ is in red) and G′_LVR of lip balms

Figure 8.

Rheological properties of lip balms at 25 °C. BW: Commercial lip balm with bees wax; PW: Commercial lip balm with plant wax; Tr8: Lip balm with Tr8 oleogel; Tr10: Lip balm with Tr10 oleogel; Tr8/Tr10: Lip balm with 50% Tr8 and 50% Tr10 oleogels

The retention of viscoelasticity in lip balms during shear was confirmed further by frequency sweep tests. The obtained G′ and G″ curves of all the samples were almost parallel to each other along with a positive slope with respect to frequency sweep (vide supplementary file, Fig. S12). Frequency-independent elastic and viscous moduli and slightly increased elasticity during shear are necessary to use these formulations for commercial applications. It is worth mentioning that the values of viscoelastic moduli of all the samples are alike from both amplitude and frequency sweeps which confirms the accuracy of the obtained results. The rheological properties of trehalose derivative-based lip balms are consistent with their corresponding molecular gels. This indicates the influence of molecular gelators on the flow properties of formulations, although their concentration is less significant.

The influence of temperature on the rheological properties of lip balms was evaluated by conducting amplitude and frequency sweep tests at elevated temperatures, 37 °C, and 50 °C. The order of G′_LVR from amplitude sweep (vide supplementary file, Fig. S13, and S15) of lip balms at 37 °C is the same as that observed at 25 °C, that is Tr8>BW>PW>Tr8/Tr10>Tr10. When the testing temperature was raised to 50 °C, the order changed slightly with the lip balm containing Tr8/T10 gelators outperforming the BW-based commercial lip balm. Even at 50 °C, no significant change in G′_LVR values of Tr8- and Tr10-based lip balms was observed. But wax-based lip balms have lost their strength (in terms of G′_LVR) by an order of 10. Since the melting point of trehalose derivatives is around 150 °C (evident from DSC studies, given in the following section), the structuring ability of gelators was not affected at the testing temperature (50 °C). On the other hand, waxes exhibited melting phenomenon at about 50 °C (evident from DSC studies), which in turn affected the rheological/flow properties of corresponding lip balms. In addition to G′_LVR, Y_s and Y_z also got affected at higher temperature. Y_s and Y_z of all the samples were found to be increased when the test was conducted at 50 °C. However, Tr8-based formulation showed narrowest yield zone, suggesting the enhanced brittleness of the sample at higher temperature.

The temperature has also affected the rheological behavior of the formulations during frequency sweeps (vide supplementary file, Fig. S14, and S16). There was no discrepancy or variation in their behavior when the frequency sweep was conducted at 37 °C, against 25 °C. The viscoelastic moduli (G′ and G″) were independent of the frequency with a slight positive slope. However, moduli were found to be dependent on angular frequency when the sweep was carried out at 50 °C. This suggests that the samples are losing their viscoelasticity, henceforth not suitable to be used for commercial applications at this elevated temperature.

Thermal Stability of Lip Balm Formulations

Differential scanning calorimetry was conducted to evaluate the melting profile of gelators and waxes and in turn to assess the thermal stability of lip balms (Fig. 9). Commercial lip balms exhibited lower melting point as expected and were found to be at 57 °C for the formulation with bees wax and 54 °C for the one with plant wax. On the other hand, lip balms with trehalose derivatives showed higher thermal stability by exhibiting melting points at 150 °C for Tr10 and 156 °C for Tr8. Similar to the rheological profiles, lip balm with both the gelators exhibited a melting point in between those two temperature limits, at 152 °C. The melting points of gelators were found to be matching with the gel-to-sol transition temperature of oleogels, evaluated from oil-bath heating studies. This indicates that the effect of gelators is very significant on the properties of formulations, irrespective of their concentration and the type of characterization being conducted. While cooling, crystallization peaks of gelators and waxes were formed below their corresponding melting temperatures. This indicates that the formulations are thermoreversible in nature (Wijarnprecha et al. 2019).

Figure 9.

DSC thermograms of lip balms

Conclusion

Regiospecific transesterification reaction was successfully conducted using lipase-mediated biocatalysis on trehalose (a disaccharide) and fatty acids of different chain lengths. Successful synthesis and purity of trehalose-fatty acyl amphiphiles were confirmed by ¹H and ¹³C NMR and FT-IR characterization techniques. All the amphiphiles have shown excellent gelation properties in both organic solvents and vegetable oils. Amongst all, Tr8 and Tr10 are identified as super gelators as their MGC was found to be ≤ 0.2 wt%. Optical microscopy and FE-SEM studies confirmed the formation of the 3D fibrous network by gelators. XRD studies indicated the hexagonal columnar packing of the self-assembled amphiphiles during gelation. The viscoelastic nature and flow properties of oleogels were evaluated in detail by performing rheological studies. These studies have inferred that Tr8 and Tr10 gelators attribute antagonist properties to oleogels like rigidity and softness, brittleness and flexibility respectively. Interestingly, such antagonistic features suits for lip balms formulation and thus Tr8 and Tr10 were chosen to prepare wax-free lip balms. The designed new formulations are not only comparable to the currently available commercial beeswax and plant wax-based lip balms, but they exhibited increased rheological and thermal properties. It was also found that the functional properties of lip balms can be finely tuned by using Tr8 and Tr10 gelator mixture. Based on this study, we are confident that trehalose-fatty acyl amphiphiles have huge potential in the field of cosmetics as structuring agents in modulating the rheological and thermal properties of formulations, especially lip balms.