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Naturally emulsifying withbiomimetic clinical benefitsn Alain Thibodeau PhD - Innovacos Corp, US

Choosing the right emulsifier remains adecision formulation chemists face at theonset of each project. The natural origin,global regulatory acceptance, functionality,robustness and compatibility with skinstructures are all parameters that need tobe carefully analysed when selecting anemulsifier. In addition, the impact of theemulsifier on the sensorial properties of thefinal formulation remains a key factor. Thisparameter often determines theacceptance of the cosmetic product by endconsumers. Even though oils, emollientsand thickeners may influence the emulsioncharacteristics, emulsifiers remain thecornerstone of a formulations aestheticsand functionality.

Through years of research, Innovacoshas developed an expertise in polyglyceroltechnology . This knowhow led to a newgeneration of polyglycerol-basedemulsifiers known as PolyAquol™. The firstmember of this family is PolyAquol-2W; anall naturally derived O/W emulsifyingtechnology that was assigned the Ecocertand Cosmos certifications. It is made of askilled combination of the componentspolyglyceryl-2 stearate, glyceryl stearateand stearyl alcohol. It is non-ionic and hasself-emulsifying properties meaning it worksindependently of the HLB system and cancreate stable emulsions on its own without

the need of co-emulsifiers or co-factors. Polyglycerol-based emulsifiers are not

new in the field of cosmetics. However,Innovacos succeeded to reinvent thepolyglycerol technology by fine tuning itschemistry. PolyAquol-2W (now referred toas ‘the polyglycerol-based emulsifier’) ismanufactured using highly purified buildingblocks such as diglycerol and stearic acid.The glycerol moiety and the fatty acid arelinked together in a solvent-free systemwhile respecting a very precise molecularstoichiometry. Such a close control of the

glyceryl-OH group density allows for aconsistent polarity of the emulsifier (Fig 1).This becomes important for uniformemulsifying properties between each lotproduced. Furthermore, the reaction iscontrolled to avoid the presence of freediglycerol to maintain optimal sensorialproperties in the final emulsion. Thetechnology of the polyglycerol-basedemulsifier (patent pending) thus emergesfrom a unique knowledge of the polyglycerolmolecularity combined with a meticulouslycontrolled manufacturing process.

Figure 1: Schematic representation of polygylceryl-2 stearate. Stearic acid (C18) forms the lipophilictail interacting with the oil phase and the polyglyceryl-2 moiety creates the hydrophilic head of themolecule interacting with the water phase.

Figure 2: Proposed molecular configuration of the components of PolyAquol-2W to create stable O/W emulsions.

Hydrogen bond interactions between individual

components

Lipophilic environment betweenaliphatic chains trapping

the “oil phase”

Hydrophilic environment created by hydrogen bonds holding

the “water phase”

Hydrophilic head Lypophilic tail

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Once each component of thepolyglycerol-based emulsifier is obtained,they are assembled in a way to favourmolecular interactions. We take advantageof the presence of oxygen molecules ‘O’(present on polyglyceryl-2 and glycerylstearate) and hydroxyl groups ‘OH’ (presenton stearyl alcohol) that can attract eachother through hydrogen electrostaticinteractions. A lamellar-like structure canthus be formed in which molecule poles arespatially oriented. In this manner, thehydrophilic heads can interact with theexternal water phase while the lipophilictails would merge with the oil therebymaximising the O/W emulsion (Fig 2).

This lamellar structure most likelytranslates into a highly organised system in the emulsion particularly at the oil/waterinterface. This is evidenced by theobservation of liquid crystals at theperiphery of oil droplets in a polyglycerol-based emulsifier emulsion (Fig 3). In suchan emulsion, liquid crystals (typicallylyotrophic) self-organise during thehomogenisation and cooling phase and areindicative of a molecular orientational orderas depicted in Figure 2. Liquid crystalstructures possess the dual property of aliquid (fluidity) and that of a solid (structure).It is the latter property that endows liquidcrystals with the birefringence characteristic– the ability to bend polarised light. Theseemingly discrete liquid crystal structuresas seen in Figure 3b are not limited to theimmediate vicinity of the oil droplets butwould also extend – though in a lesserorganised manner – in the continuous waterphase.1 This creates a sort of three-dimensional crystalline meshwork in theemulsion that actively contributes to therheology and the stability of it. In turn, thiswould restrict the movement of oil dropletskeeping them apart to preventcoalescence. In addition to impart stabilityto the emulsion, the fluidity and dynamismof this meshwork contributes to thesensorial properties of the formulation uponapplication on the skin.

Formulation performancesThe molecularity and structuralcharacteristics of the polyglycerol-basedemulsifier make it a highly versatileemulsifier. Indeed, it can be added inconcentrations between 1% and 5% in thewater phase as well as in the oil phase whenformulating O/W systems. Adding it to theoil phase results in a relatively higherviscosity emulsion compared to whenincorporated to the water phase (Fig 4).The density of oil droplets is higher whenthe emulsifier is added in the oil phase. In that case, the average diameter of the oildroplets is 2.0 µm. On the contrary,incorporation of the polyglycerol-basedemulsifier directly into the water phase

produces a relatively lower density of oildroplets with a higher average diameter of3.5 µm. This feature of the polyglycerol-based emulsifier is advantageous to createformulations with different viscosity levelswithout the addition of rheology modifiers.Yet, the polyglycerol-based emulsifier iscompatible with a variety of thickeners orjellifying agents. Regarding the type of thelipid phase, the polyglycerol-basedemulsifier is compatible with oils of differentpolarity and chemistry such as vegetableoils, esters, mineral oils and silicones.Furthermore, increasing the oil phaseconcentration is another way to modify theviscosity of the final emulsion. For instance,increasing the concentration of sweetalmond oil from 5% to 20% brings theemulsion viscosity from 7000 to 18000 cps,

respectively. Those characteristics of thepolyglycerol-based emulsifier allow for thecreation of emulsions with various viscositylevels and textures from sprayable lotions torich creams and butters.

The robustness of the polyglycerol-based emulsifier was observed in differentpH and electrolyte environments.Emulsions made with the emulsifier wereshown to sustain a pH range between 4 to 9after an incubation at 50°C for 30 days.Furthermore, the polyglycerol-basedemulsifier tolerates the presence ofmonovalent or divalent electrolytes in theemulsion. For instance, the viscosity andthe stability of the emulsion were notaffected in the presence of up to 3% NaCl(Table 1) even after 30 days at 50°C.

The polyglycerol-based emulsifier was

Figure 3: PolyAquol-2W emulsifies oil droplets in the continuous water phase (A). Using a set ofdiametrically opposed polarised filters, liquid crystals can be detected due to their birefringenceproperties (B). The average oil droplet diameter is 2.5 μm. Micrographs were taken with a Moticmicroscope BA310E equipped with polarised filters, a LED light source and a Moticam 5 (5.0MegaPixel) camera. Magnification 400X.

Figure 4: Addition of PolyAquol-2W in the water or oil phase. PolyAquol-2W was added at 75°C ineither the water phase or the oil phase during the emulsion and led to viscosity levels of 6 750 cps and10 400 cps, respectively (Brookfield, RVDV-I Prime, Sp 92, 10 rpm). Average oil droplet diameter was3.5 µm (water phase) and 2.0 µm (oil phase).

a b

Viscosity (cPs)

12000

10000

8000

6000

4000

2000

0

PolyAquolTM-2Win water phase

PolyAquolTM-2Win oil phase

Figure 5: Clinical benefits of PolyAquol-2W. A, In the preventive effect protocol, subjects (10) were applied with a formulation made of 5% PolyAquol-2W in95% water (hereafter referred to as a PolyAquol-2W water gel) or a control made of cetearyl alcohol, ceteareth-20 and polyacrylate as a jellifying agent (2mg/cm2) for a period of 10 days. Twenty-four hours after the last application, TEWL was measured (Tewameter 300® - Courage+Khazaka, electronic GmbH)to obtain a baseline value. After measurement, subjects were exposed to a solar simulator (UVA/B). TEWL was again measured 24 hours after UV exposure.B, In the repair action protocol, subjects were first exposed to UVA/B and TEWL was measured 24 hours after to obtain a reference value. Immediately afterthe reference measurement, subjects were applied with the water gel or the control formulation. One single application of each formulation was made.TEWL was re-assessed 30 minutes, 1 hour, 2 hours and 24 hours after formulation applications. Solar simulator exposure was done at 1.5X the minimalerythema dose. All protocols were under dermatologist supervision. *p<0.05; **p<0.01

Variation in TEWL (%) vs baseline 90

80

70

60

50

40

30

20

10

0

Control PolyAquolTM-2W

**

Variation in TEWL (%) vs baseline 5

0

-5

-10

-15

-20

-25

30 mins. 1 hour 2 hours 24 hours

**

** **

*

n Controln PolyAquolTM-2W

** p<0.01 *p<0.05** p<0.01

a b

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shown to outperform other polyglycerol-based emulsifiers such as polyglycerol-3distearate or polyglyceryl-3 methylglucosedistearate. Each emulsifier was separatelyadded at a concentration of 3% or 5% to anoil phase made of 12% or 20% vegetableoil, respectively, to create O/W emulsions.After an incubation at 50°C for 30 days,only the emulsions made with the newpolyglycerol-based emulsifier (in bold)remained stable (Table 2). Those resultsconfirm the self-emulsifying properties ofthe polyglycerol-based emulsifier.

Biomimetic clinical benefitsAfter having characterised its emulsifyingproperties, we wanted to explore how thepolyglycerol-based emulsifier can providebenefits to the skin especially toward theepidermal barrier function. The skin barrierhas two main physiological functions:preventing excessive water loss andimpeding the intrusion of foreign particlessuch as pathogens, pollutants and allergensto viable layers of the skin.2 Moreover, itprotects from physical insults and isinvolved in body thermoregulation. A weak

barrier function would eventually lead toskin dryness and skin hypersensitivity. Anoptimal skin barrier is thus important torestrict outward and inward movementspreserving skin and body homeostasis. The skin barrier function resides in theupper stratum corneum and is a complexhistological and molecular structure thatmainly rely on fully differentiatedkeratinocytes (called corneocytes)embedded in an intercellular lipid matrix.This arrangement is often referred to as thebrick-and-mortar model.3 The lipid matrixconsists of cholesterol, ceramides and fattyacids intermingled together.4 Numerousstudies have been carried out with the goalto unravel the architecture of the lipidmatrix of the skin barrier function. Whilethe formation of a lamellar bilayer lipidmatrix is acknowledged, several resultspoint toward the co-existence of acrystalline gel and a fluid phase.5,6 Thosestructural characteristics are responsible forthe semi-permissive water movement andprotection against mechanical abrasions aswell as the plasticity needed to cope withskin stretching and compression.7,8

The barrier function does not only relyon the intercellular lipid matrix of thestratum corneum but is complemented byother lipids originating from thepilosebaceous unit. Secreted triglyceridesdeposited at the surface of the skin areseparated into glycerol and fatty acids bylipases.9 As mentioned above, fatty acidsare an important component of the inter-corneocyte lipid matrix interacting withceramides and cholesterol. Fatty acidscatabolised from skin surface triglyceridescan diffuse back into the skin and intermixwith the extracellular lipid matrix of thestratum corneum contributing to the skinbarrier function.10 Furthermore,triglyceride-derived glycerol also activelyparticipates in the skin barrier function.Topically added glycerol has been shown torestore skin barrier function11, 12 stabilise theliquid crystalline structure of the barrierextracellular lipid matrix13 and enhance thestratum corneum hydration.14

Interestingly, the glycerol and fatty acidscatalytically released from the triglyceridesproduced by the pilosebaceous unit bringto mind a similarity with the polyglycerol

Table 1% NaCI Viscosity (cPs)* Stability

(1 month at 50°C)

0 (control) 10 400 ✓

0.5 11 200 ✓

1.0 11 700 ✓

2.0 11 200 ✓

3.0 11 000 ✓

Table 1: Electrolyte tolerance of PolyAquol-2W. Theemulsifier (5%) was added in the oil phase that wassubsequently emulsified with a water phase. NaCl wasadded in the emulsion. Viscosity was measured with aBrookfield apparatus, RVDV-I Prime, Sp 92, 10 rpm.

Table 2Emulsifier Vegetable oil Viscosity (cPs)* Stability

(1 month at 50°C)

3%

Polyglyceryl-3- Distearate

12%

3 600 Unstable

Polyglyceryl-3-Methylglucose Distearate 1 550 Unstable

PolyAquol™–2W 6 300 ✓ Stable

5%

Polyglyceryl-3- Distearate

20%

5 350 Unstable

Polyglyceryl-3-Methylglucose Distearate 3 250 Unstable

PolyAquol™–2W 18 000 ✓ Stable

Table 2: Comparison of PolyAquol-2W with other polyglycerol-based emulsifiers. Emulsion stabilitywas assessed by verifying absence of phase separation after 30 days at 50°C. Viscosity was measuredwith a Brookfield apparatus, RVDV-I Prime, Sp 92, 10 rpm.

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derivatives of fatty acids present in thepolyglycerol-based emulsifier. In additionto this molecular resemblance, the ability ofthe polyglycerol-based emulsifier to formliquid crystals extends the analogy with theskin barrier lipid matrix to a more structurallevel. The molecular and structuralbiomimicry features of the polyglycerol-based emulsifier led us to clinically verify itspotential benefits on the skin barrierfunction challenged with UV. UV radiationis a ubiquitous skin stressor that has beenshown to negatively impact the epidermallipid synthesis and the skin barrierintegrity.15,16

The polyglycerol-based emulsifier wasapplied topically as a water gel consistingof 5% polyglycerol-based emulsifier and95% water. The water gel is odourless,stable and has a viscosity in the vicinity of10,000 cps. The clinical trial was twofoldand designed as both ‘preventive effect’and ‘repair action’ protocols. In thepreventive effect protocol the water gel wasapplied daily, for a period of 10 days, priorto UVA/B exposure. In the case of therepair action protocol, the water gel wasapplied, only once, 24 hours after UVA/Bstress. Pre-treating the skin with the watergel significantly reduces the UVA/B-inducedincrease in TEWL compared to a control(Fig 5a). When applied post-UVA/B the

water gel rapidly brings down TEWL (Fig 5b). There is a significant reduction ofthe UV-induced increase in TEWL readily

30 minutes upon water gel application.The repair action of the polyglycerol-basedemulsifier water gel builds up upon time as

Figure 6: Proposed model for the biomimetic action of PolyAquol-2W in maintaining and repairingthe integrity of the skin barrier lipid matrix.

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TEWL progressively decreases up to 24hour upon its application.

The clinical results obtained with thepolyglycerol-based emulsifier could beinterpreted as a biomimetic action of itscomponents improving and also repairingthe skin barrier lipid matrix. The lipophilictails would allow for the integration of themolecules into the lipid matrix and possiblyimprove the structure of it. Thepolyglycerol heads would contribute to thelipid matrix crystalline organisation.Furthermore, due to its hygroscopic nature,the polyglycerol moiety would act as amicro-sponge retaining moisture into theskin. In this way, it would play the role of anextracellular natural moisturising factor. The proposed mechanisms of action areillustrated in Figure 6. UV radiation as wellas other external stresses will causedamage to the skin barrier function.Deterioration of the skin barrier function will lead to an outflux of water (increase inTEWL) and an influx of foreign particlesexacerbating skin sensitivity. Thecomponents of the polyglycerol-basedemulsifier, would integrated into the skinbarrier lipid matrix reinforcing it to improveits resistance (Prevention) to stresses suchas UV. Optimising the skin barrier functionintegrity would help reducing skindehydration and skin hypersensitivity uponexternal aggressions. In case the skinbarrier lipid matrix is already impaired, thepolyglycerol-based emulsifier will mergewith the barrier lipid matrix fixing damage(Repair action).

ConclusionPolyAquol-2W leads a new generation ofpolyglycerol-based emulsifying technology.

Its natural, efficacy, structural and skincompatibility features make it an ideal self-emulsifier for a myriad of applicationsincluding skin care for all ages, pro-moisturising concepts and sensitive skinregimen. PolyAquol-2W can also beformulated with different UV filters andblockers making it highly suitable for O/Wsun care product development.

This sun care application is furthersupported by our clinical results showingprevention and repair of the skin barrierfunction integrity upon UV-induceddamage. Indeed, a unique trait ofPolyAquol-2W resides in its ability toimprove skin barrier function homeostasis.This most likely relies on a skin biomimicryconcept put forward by its molecularcomposition and the formation of tri-dimensional liquid crystal structures.Interestingly, when added in a completelyanhydrous system, PolyAquol-2Wgenerates birefringent – thus highlyorganised – lamellar structures (Fig 7)evocative of the bi-layer organisation of the skin barrier lipid matrix.8

The robustness and clinical efficacy ofPolyAquol-2W ally with the possibility tocreate formulations endowed with uniqueproperties and benefits. The availability ofPolyAquol-2W to all chemists andcosmetologists is likely to open newfrontiers in designing multi-functional andefficacious formulations.

References1. Klein K: Liquid Crystals and Emulsions: A

Wonderful Marriage; in Skin Barrier: Chemistryof Skin Delivery Systems, Cosmetics & Toiletries2008; Chapter 26, p 265-272; ed. Johann W.Wiechers 2008.

PC

2. Boer M, Duchnik E, Maleszka R andMarchlewicz M. Structural and biophysicalcharacteristics of human skiin maintainingproper epidermal barrier function. AdvDermatol Allergol 2016; XXXIII (1): 1–5

3. Nemes Z and Steinert PM. Bricks and mortar ofthe epidermal barrier. Exp. Mol. Med. 1999,31(1): 5-19.

4. Wertz PW, Swartzendruber DC, Kathi CM andDowning DT: Composition and morphology ofepidermal cyst lipids. J Invest Dermatol. 1987;89:419-424.

5. Norlén L. Skin Barrier Formation: TheMembrane Folding Model. J Invest Dermatol.2001; 117: 823-829.

6. Bouwstra J, Gooris G and Ponec M. The LipidOrganisation of the Skin Barrier: Liquid andCrystalline Domains Coexist in Lamellar Phases.Journal of Biological Physics 2002; 28: 211–223.

7. Das C, Noro MG, and Olmsted PD. SimulationStudies of Stratum Corneum Lipid Mixtures.Biophysical Journal 2009; 97: 1941–1951.

8. Iwai I, Han HM, den Hollander L, Svensson S etal. The Human Skin Barrier Is Organized asStacked Bilayers of Fully Extended Ceramideswith Cholesterol Molecules Associated with theCeramide Sphingoid Moiety. Journal ofInvestigative Dermatology 2012; 132: 2215–2225.

9. Pappas A. Epidermal surface lipids. Dermato-Endocrinology 2009; 1(2): 72-76.

10. Ní Raghallaigh S, Bender K, Lacey N, Brennan Land Powell FC. The fatty acid profile of the skinsurface lipid layer in papulopustular rosacea,British Association of Dermatologists 2012;166: 279–287.

11. Fluhr JW, Gloor M, Lehmann L, Lazzerini S,Distante F, Berardesca E. Glycerol acceleratesrecovery of barrier function in vivo. Acta DermVenereol. 1999;79(6):418-21.

12 Breternitz M, Kowatzki D, Langenauer M, ElsnerP and Fluhr JW. Placebo-controlled, double-blind, randomized, prospective study of aglycerol-based emollient on eczematous skin inatopic dermatitis: biophysical and clinicalevaluation. Skin Pharmacol Physiol.2008;21(1):39-45.

13. Froebe CL, Simion FA, Ohlmeyer H et al.Prevention of stratum corneum lipid phasetransition in vitro by glycerol – an alternativemechanism for skin moisturization. J SocCosmet Chem 1990; 41:51–65.

14. Fluhr JW, Mao-Qiang M, Brown BE et al.Glycerol regulates stratum corneum hydrationin sebaceous gland deficient (asebia) mice. J Invest Dermatol. 2003 May;120(5):728-737.

15. Kim EJ, Jin XJ, Kim YK et al. UV decreases thesynthesis of free fatty acids and triglycerides inthe epidermis ofhuman skin in vivo,contributing to development of skinphotoaging. Journal of Dermatological Science2010; 57: 19–26.

16 Biniek K, Levi K, and Reinhold H. Dauskardt RH.Solar UV radiation reduces the barrier functionof human skin. Proc Natl Acad Sci USA 2012;109(42): 17111–17116.

Figure 7: Formation of lamellar organised structures by PolyAquol-2W in an anhydrous system.Micrographs were taken with a Motic microscope BA310E equipped with polarised filters, a LED lightsource and a Moticam 5 (5.0 MegaPixel) camera. Magnification was 400X.