WO2019185168A1

WO2019185168A1 - Electroporation of hyaluronic acid and heating

Info

Publication number: WO2019185168A1
Application number: PCT/EP2018/058345
Authority: WO
Inventors: Patrycja NZOUNZA
Original assignee: L'oreal
Priority date: 2018-03-30
Filing date: 2018-03-30
Publication date: 2019-10-03

Abstract

The invention relates to an electrical method of delivering hyaluronic acid through the skin, the electrical method comprising: applying a selected electroporation pulsatile current stimulus, from any device and/or support comprising at least one electrode, to a biological subject, the electrical method comprising heating the skin at a temperature between 39 °C and 42 °C.

Description

ELECTROPORATION OF HYALURONIC ACID AND HEATING

The present invention relates to the field of skincare and/or the care of skin appendages. The term“skin and/or its appendages” is intended to mean in particular the skin, the mucous membranes, the lips, the scalp, the eyelashes, the eyebrows and the hair.

A subject of the invention is a cosmetic treatment method for the skin and/or appendages thereof, comprising at least one step consisting in applying at least one composition comprising at least one hyaluronic acid or a derivative thereof to the skin and at least one step consisting in heating said skin and/or appendages thereof. The method of the invention is other than therapeutic.

The present invention also relates to a topical cosmetic, in particular a care cosmetic composition comprising, in a physiologically acceptable medium, at least one hyaluronic acid or a derivative thereof.

Hyaluronic acid and its various forms (e.g., hyaluronic acid salts, sodium hyaluronate, oxidized forms hyaluronic acid, anions of hyaluronic acid, and the like) may have one or more beneficial uses. For example, hyaluronic acid may act as an antiaging in humans. Hyaluronic acid is most abundant in the skin, accounting for 50% of the total hyaluronic acid of the human body and has the unique capacity to attach and retain large amounts of moisture. This highly contributes to the healthy and young appearance of the skin.

One problem, however, is that conventional hyaluronic acids use in topically applied anti-aging preparations, have molecules with high molecular weight (usually superior to 1,000 kDa) and diameter. Substances with molecular weight superiror to 500 kDa cannot be absorbed into the skin. Conventional hyaluronic acids may be around 3 pm in diameter, whereas the intercellular space is only 0.015 to 0.050 pm and just 0.006 to 0.010 pm at the hyaline membrane. This makes it impossible for conventional hyaluronic acids to penetrate passively into deep layers of the skin (Jegasothy et al, 2014; Desai et ah, 2010).

Electroporation is a method used to transiently create aqueous pores in cell membranes, using electric pulses of high voltage and short duration. It has been used to enhance transdermal delivery of molecules with different lipophilicities and sizes including high molecular weight actives (proteins, peptides, and oligonucleotides). As used herein, the term“electroporation” means the temporary creation of holes or pores by an applied electrical potential and through which high molecular cosmetic agents may pass. Electroporation is now widely used in biology, particularly for transfection studies, where plasmids, DNA fragments and other genetic material are introduced into living cells. During electroporation pulsing, molecules which are not normally skin permeant are able to pass through the skin during the period of induced reversible membrane permeabilization. The permeabilized state is caused by the generation of an electrical field in the cell suspension or tissue of sufficient field strength to perturb the cell surface membrane's proteolipid structure. During this short period of permeabilization, external agents can rapidly transfer across the surface membrane via these pores. With appropriate electrical parameters for the poration (field strength, pulse width, number of pulses etc), resealing of the membrane begins almost immediately after the pulsing. The surface membrane can reorganize with a full restoration of its former structural integrity, receptor status and other functional properties. Electrical fields for poration are commonly generated by capacitor discharge power units using pulses of very short (micro to millisecond) time course. Square wave and radio frequency pulses have also been used for cell electroporation.

Electroporation (also called electropermeabilization) may transitory change the structural perturbation of the lipid bilayer of the skin due to the application of relatively high voltage pulses, allowing a reduction in skin impedance and significant increase in skin deposition. It is a highly efficient strategy for the introduction of foreign high molecular weight substances into many cell types.

The main advantage of electroporation is its applicability for transient and stable transfection of all cell types. Furthermore, because electroporation is easy and rapid, it is able to transfect a large number of cells in a short time once optimum electroporation conditions are determined.

An electrical pulse at an optimized voltage and only lasting a few microseconds to a millisecond is discharged through the cell suspension. This disturbs the phospholipid bilayer of the membrane and results in the formation of temporary pores. The electric potential across the cell membrane simultaneously rises to allow charged molecules like DNA to be driven across the membrane through the pores in a manner similar to electrophoresis. It is known from WO 03/013615 an electroporative method for delivering a polynucleotide into cells of an organ.

It is also known from WO 2005/035755 a method of introducing nucleic acid into cells by electroporation, comprising the step of causing an electrode surface to support a nucleic acid and to effect adhesion of cells on the obtained nucleic acid supporting electrode surface.

WO 98/52613 discloses a method of administration of hyaluronic acid with an electrical assisted delivery method for treating a disease. The electrical current which is used is not specified.

The publication“Temperature Influences the Postelectroporation, Permeability State of the Skin’’, S. NARASIMHA MURTHY, ARINDAM SEN, YA-LI ZHAO, SEK WEN HUI, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 4, p.908 APRIL 2004 discloses the influence of temperature on the electrical conductance and transport of macro molecules across porcine epidermis during and after electroporation were studied.

The publication“Thermal Assisted In Vivo Gene Electrotransfer”, Amy Donate and ah, Curr Gene Ther. 2016 ; 16(2): 83-89 discloses that increased temperature during electrotransfer increased expression or maintained expression of gene with a reduction in applied voltage.

The publication“Controllable Moderate Heating Enhances the Therapeutic Efficacy of Irreversible Electroporation for Pancreatic Cancer”, Chelsea M. Edelblute and al, Scientific reports | 7: 11767 | DOI:l0. l038/s4l598-0l7-l2227-4 , published online: September 18, 2017 discloses that moderate heating (MH) at 43 °C for 1-2 minutes significantly enhanced ex vivo IRE tumor ablation of Pan02 cells by 5.67-fold at 750 V/cm and by 1.67-fold at 1500 V/cm.

W02014151850 discloses systems and methods that enable tissue cooling applications and delivery of electrical energy to adipose tissue for alteration and reduction of body fat are described herein. Aspects of the disclosure are directed to, for example, temperature-controlled electroporation of subcutaneous lipid-rich cells. Additional aspects of the disclosure are directed to treatment methods for treating a target region of a human subject's body to achieve an alteration of subcutaneous adipose tissue. European patent application n° 17306221.7 filed September 20 2017 by the Applicant describes a cosmetic treatment by electropermeabilization to improve delivery of moderate-high size compounds.

Electroporation is an effective technique for increasing the penetration of high molecular weight molecules (polymers such as hyaluronic acid). However, the quantity delivered is limited and this procedure requires the application of high voltages causing side effects such as pain and muscle contraction. The goal is to improve penetration with moderate voltage to avoid side effects.

There is a need for improving the delivering of moderate-high size compounds, such as hyaluronic acid, through the skin, without altering the skin.

Electrical method

In some embodiments, an electrical cosmetic method of delivering hyaluronic acid, for example an aqueous hyaluronic acid composition, through the skin, comprises applying a selected electroporation pulsatile current stimulus from any device and/or support, comprising at least one electrode, the electroporation pulsatile current stimulus being of a character and for a duration sufficient to transdermally deliver hyaluronic acid, notably an aqueous composition, to a biological subject, and transporting different rates of hyaluronic acid across the skin in accordance to the selected current mode, the electrical method comprises heating the skin at a temperature between 39 °C and 42 °C.

The method according to the invention delivers hyaluronic acid through electroporation and heats the skin. This idea of applying both heat and electroporation is at the basis of the present invention.

According to the invention, thermal energy is applied to treat, pre-treat, post treat the Stratum Comeum layer. This energy lowers the threshold energy required to permeabilize this layer. Synergy between thermal energy and electrical energy leads to greater penetration of molecules by simultaneous supply of both energies.

An increased penetration of hyaluronic acid polymers allows to obtain: a better filling of the wrinkles or lines, an improvement of the aspect of the texture of the skin (smoothing), a remanent hydration, a correction of volume on the face or the body leading to an improvement of the general appearance of the skin (anti-aging). This method may allow significant changes in skin impedance and thus an increase permeability of the hyaluronic acid through keratinous material, and in particular through the skin.

The method of the invention is cosmetic and non-therapeutic.

In the invention, the selected current stimulus is applied to the keratinous material of the biological subject, in particular the skin.

In some embodiments, the administration of hyaluronic acid is conducted on live, human beings. In some embodiments, the administration of hyaluronic acid is conducted on any biological subject. In some embodiments, biological subjects include, but are not limited to, mammals, including human beings.

In some embodiments of the electrical method, applying an electroporation pulsatile current stimulus to a biological subject may include generating an electroporation pulsatile current stimulus having a voltage below 500 V, preferably below 400 V and more preferably below 300 V.

The given value of the voltage corresponds to the peak value of the voltage.

In some embodiments of the electrical method, applying an electroporation pulsatile current stimulus to a biological subject may include generating an electroporation pulsatile current stimulus having a voltage higher than 20 V, preferably higher than 50 V, and more preferably higher than 80 V and even higher thanlOO V.

In an embodiment, the voltage may be of 250 V. In another embodiment, the voltage may be of 99.9 V.

Such electroporation pulsatile current stimulus may enable to reduce the impedance of keratinous material, and in particular of the skin. The reduction of the impedance of the skin may enable to allow the passage of moderate-high size compounds, such as hyaluronic acid.

In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse duration ranging from 0.5 milliseconds to 100 milliseconds, preferably from 5 milliseconds to 70 milliseconds and more preferably from 7 milliseconds to 50 milliseconds.

In an embodiment, the pulse duration may be of 5 ms. In another embodiment, the pulse duration may be of 40 ms. In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse interval ranging from 400 to 1000 ms, preferably from 500 milliseconds to 800 milliseconds, preferably from 600 milliseconds to 700 milliseconds.

In some embodiments, the electroporation pulsatile current stimulus may comprise a number of pulses ranging from 1 to 200 pulses, preferably from 3 to 150 pulses, and more preferably from 5 to 100 pulses.

In an embodiment, the number of pulses may be of 60 pulses. In another embodiment, the number of pulses may be of 6 or 9 pulses.

In an embodiment, the electroporation pulsatile current stimulus may comprise generating a non-constant current, which may comprise a very short pulsatile current.

In some embodiments, the electroporation pulsatile current stimulus may comprise generating a pulsed current having periodic square waveforms, rectangular waveforms, saw tooth waveforms, spiked waveforms, trapezoidal waveforms, triangle waveforms, or combinations thereof, and/or sinusoidal waveforms, non-sinusoidal waveforms, or combinations thereof. In an embodiment, the electroporation pulsatile current stimulus may have square waveforms or rectangular waveforms.

APPLICATION OF HEAT

The process according to the invention comprises a step of heating (ii).

Advantageously, the process according to the invention comprises a step of heating the skin.

Preferably, the skin is heated to a temperature between 39°C and 42°C, more preferably between 40° C and 4l°C.

Whatever the embodiments considered, the duration of step of heating (ii) may vary from 1 second to 60 minutes, preferably 1 minute to 30 minutes, preferably 5 minutes to 20 minutes.

Whatever the embodiments considered, the step (ii) may be repeated, for example between 1 and 10 times, preferably between 1 and 3 times. In this case, short times of treatments can be selected, with a rest time between each treatment.

The temperature difference between the heated skin surface and the non-heated skin may be less than or equal to 5°C, or less than or equal to 3°C, or indeed less than 2°C. More preferably, the process according to the invention comprises a step of heating the composition at a temperature between 39°C and 42°C, more preferably between 40° C and 4l°C before applying it on the skin.

The temperature difference between the heated composition and the non-heated composition may be less than or equal to 5°C, or less than or equal to 3°C, or indeed less than 2°C.

Even more preferably, the process according to the invention comprises a step of heating the electrode at a temperature between 39°C and 42°C, more preferably between 40° C and 4l°C, before it contacts the skin.

The temperature difference between the heated electrode and the non-heated electrode may be less than or equal to 5°C, or less than or equal to 3°C, or indeed less than 2°C.

The heater may be a system that heats by emitting infrared radiation, from a halogen or an incandescent lamp, or by blowing hot air.

In some embodiments, the composition and the electrode may be heated by being exposed to RF radiation, e.g., microwave radiation. In still other embodiments, they may be heated by ultrasound vibration.

The heating may also be performed through exposure to infrared (IR) radiation, and/or by convection, and/or by vibration, and/or by radio frequency (RF) radiation, or any other desired method for providing heat. Preferably, the heat is performed through exposure to infrared (IR).

By way of example, the temperature of the skin may be measured at the surface using a device including a microcontroller or a thermometer.

The skin is preferably heated at a rate of 2° C per minute, even better 1° C per minute.

The composition is preferably heated at a rate of 2° C per minute, even better 1° C per minute.

The electrode is preferably heated at a rate of 2° C per minute, even better 1 ° C per minute.

According to the invention, the step of heating (ii) is prior or simultaneous or post to the step of applying a selected electroporation pulsatile current stimulus (i). HYALURONIC ACID

In the context of the present invention, the term“hyaluronic acid or a derivative thereof’ covers in particular the basic unit of hyaluronic acid of formula:

It is the smallest fraction of hyaluronic acid comprising a disaccharide dimer, namely D glucuronic acid and N acetylglucosamine.

The term“hyaluronic acid or a derivative thereof’ also comprises, in the context of the present invention, the linear polymer comprising the polymeric unit described above, linked together in the chain via alternating b(1 ,4) and b(1 ,3) glycosidic linkages, having a molecular weight (MW) that can range between 380 and 13 000 000 daltons. This molecular weight depends in large part on the source from which the hyaluronic acid is obtained and/or on the preparation methods.

The term“hyaluronic acid or a derivative thereof’ also comprises, in the context of the present invention, the hyaluronic acid salts, and in particular the alkali metals salts such as the sodium salt and the potassium salt.

In the natural state, hyaluronic acid is present in pericellular gels, in the base substance of the connective tissues of vertebrate organs such as the dermis and epithelial tissues, and in particular in the epidermis, in the synovial fluid of the joints, in the vitreous humor, in the human umbilical cord and in the crista galli apophysis.

Hyaluronic acid is a predominant glucosaminoglycan found in the skin. Thus, the fibroblasts synthesize predominantly collagens, matrix glycoproteins other than collagens (fibronectin, laminin), proteoglycans and elastin. The keratinocytes, for their part, synthesize predominantly sulfated glycosaminoglycans and hyaluronic acid. Hyaluronic acid is also called hyaluronan (HA).

Hyaluronic acid is present in the free state in the epidermis and in the dermis and is responsible for turgescence of the skin. This polysaccharide can in fact retain a large volume of water, corresponding to up to 1000 times its weight. In this sense, hyaluronic acid plays an important role in increasing the amounts of water bound in the tissue, and also in the mechanical properties of the skin and in wrinkle formation.

Thus, the term“hyaluronic acid or a derivative thereof’ comprises all the fractions or subunits of hyaluronic acid having a molecular weight in particular within the molecular weight range recalled above.

In the context of the present invention, hyaluronic acid fractions which do not have an inflammatory activity are preferably used.

By way of illustration of the various hyaluronic acid fractions, reference may be made to the document“Hyaluronan fragments: an information-rich system”, R. Stem et al, European Journal of Cell Biology 58 (2006) 699 715, which reviews the listed biological activities of hyaluronic acid according to its molecular weight.

According to an embodiment of the invention, the hyaluronic acid has a molecular weight in a range from 1 Da to 4000 kDa, preferably from 1 kDa to 2000 kDa and more preferably from 5 kDa to 200 kDa.

According to a preferred embodiment of the invention, the hyaluronic acid fractions suitable for the use covered by the present invention have a molecular weight of between 800 Da and 2 000 000 Da, preferably between 20 000 and 1 000 000 Da, in particular between 50 000 and 500 000, especially between 100 000 and 200 000 Da.

The hyaluronic acid may have a molecular weight in a range from 800 Da to 2000 kDa, preferably from 20 kDa to 1000 kDa and more preferably from 50 kDa to 500 kDa, even better from 100 kDa to 200 kDa. In an embodiment, the molecular weight of the hyaluronic acid is of 120 000 Da. In this case, the term used is intermediate-molecular- weight hyaluronic acid.

Finally, the term“hyaluronic acid or a derivative thereof’ also comprises hyaluronic acid esters in particular those in which all or some of the carboxylic groups of the acid functions are esterified with oxyethylenated alkyls or alcohols, containing from 1 to 20 carbon atoms, in particular with a degree of substitution at the level of the D glucuronic acid of the hyaluronic acid ranging from 0.5 to 50%.

Mention may in particular be made of methyl, ethyl, n propyl, n pentyl, benzyl and dodecyl esters of hyaluronic acid. Such esters have in particular been described in D. Campoccia et al. “Semisynthetic resorbable materials from hyaluronan esterification”, Biomaterials 19 (1998) 2101 2127.

The molecular weights indicated above are also valid for the hyaluronic acid esters.

Hyaluronic acid may in particular be hyaluronic acid supplied by the company Hyactive under the trade name CPN (MW: 10 to 150 kDa), by the company Soliance under the trade name Cristalhyal (MW: 1.1 x 106), by the company Bio land under the name Nutra HA (MW: 820 000 Da), by the company Bio land under the name Nutra AF (MW: 69 000 Da, by the company Bio land under the name Oligo HA (MW: 6100 Da) or else by the company Vam Farmacos Metica under the name D Factor (MW: 380 Da).

In one embodiment, the hyaluronic acid is present in the form of spheres. In particular, such spheres are sold by the company BASF under the name Sphere d’Acide Hyaluronique [hyaluronic acid sphere]. It is a mixture of hyaluronic acid of various molecular weights, i.e. of MW 1.5 x 106, 400 000 and 600 000 Da.

In some embodiments of the electrical method, the method further comprises transdermally delivering a composition including one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives.

The one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives may be present in amounts ranging from 0.01 % to 100 % by weight, preferably from 0.5 % to 60 % by weight.

The amounts by weight are given relative to the total weight of the composition.

The hyaluronic acid or a derivative thereof may be present in the composition according to the present invention at a content of between 0.001% and 20%, preferably between 0.01% and 10%, and more particularly between 0.01% and 5% by weight, relative to the total weight of the composition.

In an embodiment, the composition comprises 1% by weight of hyaluronic acid.

In an embodiment, the composition comprises 1% by weight of hyaluronic acid, and water.

In an embodiment, the hyaluronic acid may be sodium hyaluronate.

In an embodiment, the molecular weight of the hyaluronic acid may be of 120 kDa. In some embodiments of the electrical method, the method further comprises transdermally delivering a composition, for example an aqueous composition, including, one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.1 % to 20 % by weight; one or more silicon surface-active agents present in amounts ranging from 0.01 % to 30 % by weight; one or more ionic polymers present in amounts ranging from 0.01 % to 10% by weight; and water present in an amount of at least 30 % by weight.

In some embodiments of the electrical method, the method further comprises transdermally delivering an aqueous composition including,

one or more silicon surface-active agents present in amounts ranging from 0.01 % to 30 % by weight;

one or more ionic polymers present in amounts ranging from 0.01 % to 10 % by weight; and

water present in an amount of at least 30 % by weight.

In some embodiments of the electrical method, the method further comprises transdermally delivering an aqueous composition including, one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.01 % to 30 % by weight; one or more silicon surface- active agents present in amounts ranging from 0.01 % to 30 % by weight; one or more non ionic polymers present in amounts ranging from 0.01 % to 20 % by weight; and water present in an amount of at least 30 % by weight.

In some embodiments of the electrical method, the method comprises a step of measuring at least one of the temperature of the skin, the impedance of the skin, and a pH of the composition.

In some embodiments of the electrical method, the application of current stimulus is reduced to a safety level when a measured value measured by one of the sensors exceeds a safety range or a safety value.

In some embodiments of the electrical method, the method comprises a step of measuring the pH of the composition. When the measured pH exceeds a pH safety range, the application of current stimulus is switched to a safety level, for example a safety level less than IV, such as 0,5V. The pH safety range may be pH 4 to 7. In some embodiment, when the measured pH exceeds a pH safety range, for example the range from 4 to 7, the device switches the polarity during a short time to enable to reequilibrate the pH.

In some embodiments of the electrical method, the method comprises a step of measuring the impedance of the skin. When the measured impedance exceeds an impedance safety range, the application of current stimulus is reduced to a safety level to avoid adverse event. The safety level may be less than IV, such as 0,5V. The impedance safety range may be 50 W to 1 MW.

In some embodiments of the electrical method, the method comprises a step of measuring the temperature of the skin. When the measured temperature exceeds a temperature safety value, the application of current stimulus is switched to a safety level, for example less than IV, such as 0,5V. The temperature safety value may be chosen less than 42°C.

In a preferred embodiment of the electrical method, the method comprises the steps of: measuring the temperature of the skin, and

measuring the impedance with the composition, and

measuring the pH of the composition.

Composition

The present invention also relates to a topical cosmetic, in particular a care cosmetic, composition comprising, in a physiologically acceptable medium, at least one hyaluronic acid or a derivative thereof.

In some embodiments, the invention also relates to an electrical composition comprising one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.005 % to 30 % by weight, preferably in an amounts ranging from 0.01 % to 20 % by weight, 0.05 % to 10 % by weight.

The composition may be deprived of water.

In some embodiments, the invention also relates to an electrical composition comprising one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.01 % to 30 % by weight, preferably in an amounts ranging from 0.1 % to 20 % by weight, 0.1% to 5 % by weight, and

water present in an amount of at least 0 % by weight, preferably in an amount of at least 10 % by weight, more preferably in an amount of at least 60 % by weight, more preferably in an amount of at least 90 % by weight.

In some embodiments, the invention also relates to an electrical composition comprising

one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.005 % to 30 % by weight, preferably in an amounts ranging from 0.01 % to 20 % by weight, 0.05 % to 10 % by weight, and

water present in an amount of at least 0 % by weight, preferably in an amount of at least 10 % by weight, more preferably in an amount of at least 60 % by weight.

In particular, the composition of the invention may be for improving the barrier function of the skin. It can thus be used in hydrating the skin, in improving the suppleness of the skin, in improving and/or decreasing the microrelief of the skin, and also in combating the signs of aging of the skin.

In some embodiments of the electrical composition, the composition comprises one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.1 % to 30 % by weight; one or more silicon materials present in amounts ranging from 0.1 % to 30 % by weight; and water present in an amount of at least 20 % by weight; the electrical composition having an aqueous phase that is at least 30% by weight relative to the total weight of the electrical composition.

In some embodiments of the electrical composition, the composition further comprises one or more ionic polymers present in amounts ranging from 0.01 % to 10% by weight; wherein the one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives are present in amounts ranging from 0.1 % to 30 % by weight. In some embodiments of the electrical composition, the composition further comprises one or more non- ionic polymers present in amounts ranging from 0.01 % to 20% by weight; wherein the one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives are present in amounts ranging from 0.01 % to 30 % by weight.

In some embodiments of the electrical composition, the composition further comprises a pH ranging from 2 to 7.5.

In some embodiment, when the measured pH exceeds a pH safety range, for example the range from 4 to 7, the device switches the polarity during a short time to enable to reequilibrate the pH.

Electrical kit

In some embodiments, the invention also provides an electrical cosmetic kit comprising:

- an electrical composition including one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives, and

- an electrical device for carrying the electrical method as described above.

The composition may be an aqueous composition.

The electrical kit may be configured such that hyaluronic acid and water are already mixed in the composition when the composition is applied to the skin.

In some embodiments, the electrical device may comprise at least one of a temperature sensor, an impedance sensor, and a pH sensor. The electrical device may comprise at least two of a temperature sensor, an impedance sensor, and a pH sensor. In an embodiment, the electrical device may comprise a temperature sensor, an impedance sensor, and a pH sensor.

The device may be configured such that the application of current stimulus is reduced to a safety level when a measured value measured by one of the sensors exceeds a safety range or a safety value.

In a preferred embodiment, the method, the composition and the kit above enable to treat wrinkles and ageing signs, to improve smoothness, quality of skin and appearance of the skin. It can thus be used in hydrating the skin, in improving the suppleness of the skin, in improving and/or decreasing the microrelief of the skin, and also in combating the signs of aging of the skin.

In another embodiment, the method is used to minimize skin anti-aging, and/or pigmentation, and/or volume, and/ or sagging wrinkle, and/or event tone and/or spots, and/or to improve firmness, and/or radiance, and/or smoothness, and/or softness of the skin. The method of the invention may be associated with the application of active agents associated to milli current (mcurrent) and/or micro current (pcurrent).

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of the disclosed subject matter will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 is a flow diagram of a method in accordance with one embodiment;

FIGURE 2 is a graph showing the comparison of skin deposition of l20kDa FL- HA formulation after electroporation alone or electroporation plus heating.

DETAILED DESCRIPTION

FIGURE 1 illustrates embodiments of a method 600 for delivering a cosmetic composition through the generation of electrical stimuli. In some embodiments, the method includes a step 602 for delivering an electroporation pulsatile current to a biological subject, the pulsed current is of a character and for a duration sufficient to deliver a cosmetic composition to the biological subject.

In some embodiments, the illustrated steps 604, 608, and 610 are optional. Further, in some embodiments, the sequence of the steps 604, 608, and 610 can be in any order and is not confined to the illustration. In some embodiments, the method 600 includes a step 604 for generating waveforms. In some embodiments, the user makes selections that cause the electrical device to generate the selected waveform or waveforms that constitute the electrical stimuli. In some embodiments, the method 600 includes a step 608 for generating pulse duration. In some embodiments, the user makes selections that cause the electrical device to generate the selected pulse duration. In some embodiments, the method 600 includes a step 610 for generating pulse frequency. In some embodiments, the user makes selections that cause the electrical device to generate the selected pulse frequency.

In some embodiments, the method 600 for delivering the pulsed current to a biological subject includes generating an electroporation pulsatile current stimulus having a voltage below 500 V, preferably below 250 V and more preferably below 200 V. The voltage may be higher than 20 V, preferably higher than 50 V, and more preferably higher than 100 V.

In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse duration ranging from 5 milliseconds to 100 milliseconds, preferably from 7 milliseconds to 70 milliseconds and more preferably from 10 milliseconds to 50 milliseconds.

In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse interval ranging from 500 milliseconds to 800 milliseconds, preferably from 600 milliseconds to 700 milliseconds.

In some embodiments, the method 600 includes generating an the electroporation pulsatile current stimulus may comprise generating a pulsed current having periodic square waveforms, rectangular waveforms, saw tooth waveforms, spiked waveforms, trapezoidal waveforms, triangle waveforms, or combinations thereof, and/or sinusoidal waveforms, non-sinusoidal waveforms, or combinations thereof. In an embodiment, the electroporation pulsatile current stimulus may have square waveforms or rectangular waveforms.

In some embodiments, the method 600 for delivering the electroporation pulsatile current to a biological subject includes generating a pulsed current stimulus having an average current density ranging from 0.01 mA/cm² to 10 mA/cm², a pulse duration ranging from 50 microseconds to 1 milliseconds, and a pulse frequency ranging from 10 Hertz to 500 Hertz, and a duty cycle of pulses ranging from 1% to 90%.

In some embodiments, the method 600 for delivering the pulsed current to a biological subject includes generating a pulsed alternating current stimulus having an average current density of 0.2 mA/cm², a pulse duration of 500 microseconds, and a pulse frequency of 200 Hertz.

In some embodiments, the method for delivering the pulsed current to a biological subject includes generating a pulsed current having an average current density ranging from 0.01 mA/cm² to 10 mA/cm², a pulse width ranging from 50 microseconds to 1 milliseconds, at least one wave packet (or wave train) ranging from 2 to 20 pulses, a frequency of wave packets ranging from 10 Hertz to 500 Hertz, and a duty of pulses ranging from 1% to 90%.

In some embodiments, the method 600 for delivering the pulsed current to a biological subject includes generating a pulsed current stimulus having an average current density ranging from 0.01 mA/cm² to 10 mA/cm², a pulse width ranging from 50 microseconds to 1 milliseconds, at least one wave packet (wave train) having from 2 to 20 pulses with alternating polarity, a frequency of wave packets ranging from 10 Hertz to 500 Hertz, and a duty cycle of pulses ranging from 1% to 90%.

In some embodiments, the method 600 for delivering the pulsed current to a biological subject includes generating a pulsed current having sinusoidal waveforms, non- sinusoidal waveforms, or combinations thereof.

In some embodiments, the method 600 for delivering the pulsed current to a biological subject includes generating a pulsed current having periodic square waveforms, rectangular waveforms, saw tooth waveforms, spiked waveforms, trapezoidal waveforms, triangle waveforms, or combinations thereof.

In some embodiments, the method comprises delivering a cosmetic composition chosen from a face care or body care composition, comprising in particular, an active agent chosen from humectant or moisturizing active agents, anti-ageing active agents, for example depigmenting active agents, active agents that act on cutaneous microcirculation, or seboregulating active agents, or a composition for making up the face or body.

An electrical composition for use with the electrical methods described above in relation to FIGURES 1 and 2 is disclosed.

In some embodiments, the electrical composition comprises one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.1 % to 30 % by weight, preferably in an amounts ranging from 0.5 % to 20 % by weight, 0.7 % to 10 % by weight, and water present in an amount of at least 20 % by weight, preferably in an amount of at least 40 % by weight, more preferably in an amount of at least 60 % by weight.

In some embodiments, the electrical composition includes one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives present in amounts ranging from 0.1 % to 30 % by weight; one or more silicon materials present in amounts ranging from 0.1 % to 30 % by weight; and water present in an amount of at least 20 % by weight; the electrical composition having an aqueous phase that is at least 30% by weight relative to the total weight of the electrical composition.

In some embodiments, the electrical composition further comprises one or more ionic polymers present in amounts ranging from 0.01 % to 10% by weight; wherein the one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives are present in amounts ranging from 0.1 % to 30 % by weight.

In some embodiments, the electrical composition further comprises one or more non- ionic polymers present in amounts ranging from 0.01 % to 20% by weight; wherein the one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives are present in amounts ranging from 0.01 % to 30 % by weight.

In some embodiments, the electrical composition further comprises a pH ranging from 2 to 7.5.

In some embodiments of the electrical composition, the one or more silicon materials include one or more silicon surface-active agents. In some embodiments of an electrical composition, the silicon-containing surface active agents are selected from polydimethylsiloxane, poly[oxy(dimethylsilylane)], polyvinyl siloxane, cyclohexasiloxane, derivatives thereof, or any combination thereof.

In some embodiments of the electrical composition, the ionic polymers and nonionic polymers are selected from acrylonitrile/methyl methacrylate/vinylidene chloride copolymer, biosaccharide gum-l, sodium styrene/maleic anhydride copolymer, xanthan gum, ammonium polyacryloyldimethyl taurate, derivatives thereof, their ions, and any combination thereof.

In some embodiments of the electrical composition, the composition further comprises a vitamin, a fat, a solvent, a humectant, a viscosity reducer, a preservative, a chelating agent, a viscosity controller, a skin conditioner, an emollient, an emulsifier, a cleansing agent, an emulsion stabilizer, a viscosity increaser, an antioxidant, a binder, a skin bleaching agent, a pH adjuster, a buffering agent, a denaturant, a bulking agent, an opacifying agent.

In some embodiments of the electrical composition, the composition includes ionic polymers and nonionic polymers selected from biosaccharide gum-l (and) sodium levulinate (and) glyceryl caprylate (and) sodium anisate, acrylates/c 10-30 alkyl acrylate crosspolymer, carbomer, sodium styrene/maleic anhydride copolymer, nylon- 12, xanthan gum, derivatives thereof, their ions, or any combination thereof.

In some embodiments, the electrical composition has a pH from 2 to 7.4.

In some embodiments, the electrical composition has a pH from 2 to 7.

In some embodiments, the electrical composition has a pH from 5.7 to 6.3.

In some embodiments, the electrical composition has a pH from 2 to 6.3.

In some embodiments, the electrical composition includes one or more vitamins selected from vitamin B5, vitamin A, vitamin B3, and vitamin E.

In some embodiments, the electrical composition includes one or more fats selected from nut oils, seed oils, and plant oils.

In some embodiments, the electrical composition includes one or more solvents selected from water, deionized water, and Eau de la Roche-Posay™.

In some embodiments, the electrical composition includes one or more humectants selected from glycerin, caprylyl glycol, and sodium hyaluronate.

In some embodiments, the electrical composition includes one or more viscosity reducers selected from glycerine.

In some embodiments, the electrical composition includes one or more preservatives selected from phenoxy ethanol, salicylic acid, and sodium methylparaben.

In some embodiments, the electrical composition includes one or more chelating agents selected from disodium EDTA.

In some embodiments, the electrical composition includes one or more viscosity controllers selected from disodium EDTA, ammonium polyacryldimethyltauramide, and nylon- 12.

In some embodiments, the electrical composition includes one or more skin conditioners selected from C12-15 alkyl benzoate, caprylyl glycol, glyceryl stearate and polyethylene glycol 100 Stearate, tocopheryl acetate, sodium hyaluronate, ethylhexyl palmitate, dimethicone and dimethiconol, dimethicone, dimethicone and dimethicone/vinyl dimethicone crosspolymer, biosaccharide gum-l, oxothiazolidinecarboxylic acid, ascorbic acid, sodium styrene/maleic anhydride copolymer, salicylic acid, cyclohexasiloxane, hydrogenated polyisobutene, biosaccharide gum-l and sodium levulinate and glyceryl caprylate and sodium anisate, lemon extract, alcohol and Gentiana lutea root extract, and dimethicone and polyethylene glycol/polypropylene glycol-l8/l8 dimethicone.

In some embodiments, the electrical composition includes one or more emollients selected from C12-15 alkyl benzoate, caprylyl glycol, glyceryl stearate and polyethylene glycol 100 Stearate, ethylhexyl palmitate, dimethicone and dimethiconol, dimethicone, dimethicone and dimethicone/vinyl dimethicone crosspolymer, cyclohexasiloxane, hydrogenated polyisobutene, biosaccharide gum-l and sodium levulinate and glyceryl caprylate and sodium anisate, and dimethicone and polyethylene glycol/polypropylene glycol- 18/ 18 dimethicone.

In some embodiments, the electrical composition includes one or more emulsifiers selected from glyceryl stearate and polyethylene glycol 100 Stearate, cetyl alcohol, xanthan gum, triethanolamine, biosaccharide gum-l and sodium levulinate and glyceryl caprylate and sodium anisate, and dimethicone and polyethylene glycol/polypropylene glycol- 18/ 18 dimethicone.

In some embodiments, the electrical composition includes one or more cleansing agents selected from glyceryl stearate and polyethylene glycol 100 Stearate.

In some embodiments, the electrical composition includes one or more stabilizers selected from cetyl alcohol, xanthan gum, ammonium polyacryldimethyltauramide, sodium styrene/maleic anhydride copolymer, carbomer, and acrylates/C 10-30 alkylacrylate crosspolymer.

In some embodiments, the electrical composition includes one or more viscosity increasers selected from cetyl alcohol, xanthan gum, dimethicone and dimethicone/vinyl dimethicone crosspolymer, carbomer, and acrylates/C 10-30 alkylacrylate crosspolymer.

In some embodiments, the electrical composition includes one or more antioxidants selected from tocopheryl acetate, and ascorbic acid.

In some embodiments, the electrical composition includes one or more binders selected from xanthan gum.

In some embodiments, the electrical composition includes one or more skin bleaching agents selected from oxothiazolidinecarboxylic acid.

In some embodiments, the electrical composition includes one or more pH adjusters selected from triethanolamine, potassium hydroxide, and sodium hydroxide. In some embodiments, the electrical composition includes one or more buffering agents selected from potassium hydroxide and hydroxyethylpiperazine ethane sulfonic acid, and sodium hydroxide.

In some embodiments, the electrical composition includes one or more denaturants selected from sodium hydroxide.

In some embodiments, the electrical composition includes one or more bulking agents selected from nylon- 12.

In some embodiments, the electrical composition includes one or more opacifying agents selected from nylon- 12.

In some embodiments, a hyaluronic acid composition herein can "comprise" the specified components, leaving open the possibility of other unspecified components.

In some embodiments, a hyaluronic acid composition herein can "consist" of the specific components, meaning the composition only includes the specified components. Stated another way, the specified components constitute 100% by weight of the hyaluronic acid composition.

EXAMPLES

I - Introduction

The effect of electroporation parameters and temperature on skin integrity and transport of hyaluronic acid (at 1 % by weight in water) 120 kDa (HA) labeled with fluorescein into the permeabilized skin was investigated in vitro using electroporation vs. its passive diffusion.

Passives diffusions at 32° C and at 40° C were implemented on samples and labelled respectively condition 3 and condition 4.

Temperature conducts to significant changes in skin impedances and thus, an increase permeability of HA 120 kDa. It has been demonstrated a significant increase in the skin deposited into the skin due to the temperature.

II - Experiments

One mode has been tested: the porating mode (150-300V, short pulse lengths and low number of pulses). The heater is a IR lamp.

For conditions 4 and 6:

The composition and the electrodes are pre heated to a temperature of 40° C during 10 minutes.

Before the application of the heated composition, the skin is pre heated at 40° C during 5 minutes.

The heated composition is applied on the skin.

Then, during 20 minutes, the skin, the composition and the electrodes are heated together at 40°C during 20 minutes.

Ill - Measurements

In order to test delivery of HA through the skin, conditions 3, 4, 5, 6 and 7 were selected.

The experimental protocol has been as follows:

1. Skin source and treatment

Porcine ear skin was used for the in vitro studies. It is accepted as a good model for the human skin barrier. Hairs were excised from the skin surface using clippers. The skin was“sliced” so as to obtain samples with an average thickness of 1.5 mm for all samples. Skin was frozen (-20°C) or stored at 4°C until use.

2. Analytical method

The FL-HA was quantified using by fluorimetry. The measurements were performed using a microplate reader. The excitation and emission wavelengths were 494 and 514 nm. The gain was adjusted to 80 in order to obtain better sensitivity of the samples. All samples were put in polystyrene 96-well plates and protected from the light until analysis. Furthermore, a calibration curve was included in every plate.

3. Skin transport study

The aim of the experiments was to quantify penetration of FL-HA (120 kDa) into intact, electroporated porcine ear skin as a function of the temperature. Electroporation treatment was performed as pre-treatment, where electroporation was performed before formulation application. All conditions were tested for 20 min.

The following specific conditions treatments were tested:

PORING PULSE MODE AND HEATING Condition 5 (Voltage 150 V, Pulse length 40 ms, Pulse interval 1000 ms and 9 Pulses), 32°C, pre-treatment with electroporation followed by formulation application.

Condition 6 (Voltage 150 V, Pulse length 40 ms, Pulse interval 1000 ms and 9 Pulses), 40°C, pre-treatment with electroporation followed by formulation application

Condition 7 (Voltage 300 V, Pulse length 10 ms, Pulse interval 1000 ms and 10

Pulses), 32°C, pre-treatment with electroporation followed by formulation application

Control: Application of an aqueous solution of FL-HA (120 kDa) formulation (1% w/w) to“untreated” skin.

The results of the conditions 3 and 4 with passive diffusion for 20 min are shown on figure 2 as well as the results of the conditions 5, 6 and 7 with poring pulse mode and heating for condition 6.

The use of 150 V and of a temperature of 40° C enhanced significantly the amount of fluorescence labeled HA 120 kDa at 1% (w/v) into the skin compared to the passive diffusion of the active at 40° C (x2,5) and at 32°C (x5).

The use of 150 V and of a temperature of 40° C enhanced significantly the amount of fluorescence labeled HA 120 kDa at 1% (w/v) into the skin compared to the electroporation 300 V at 32° C(xl,2).

The use of 150 V and of a temperature of 40° C enhanced significantly the amount of fluorescence labeled HA 120 kDa at 1% (w/v) into the skin compared to the electroporation 150 V at 32° C (x2,2).

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the claimed subject matter.

Claims

1. An electrical cosmetic method of delivering hyaluronic acid through the skin, the electrical method comprising: applying a selected electroporation pulsatile current stimulus, from any device and/or support comprising at least one electrode, the electroporation pulsatile current stimulus being of a character and for a duration sufficient to transdermally deliver hyaluronic acid, notably an aqueous composition, to a biological subject, and transporting different rates of hyaluronic acid across the skin in accordance to the selected current mode, wherein the electrical method comprises heating the skin at a temperature between 39 °C and 42 °C.

2. The electrical method according to claim 1, wherein the duration of heating varies from 1 second to 60 minutes, preferably 1 minute to 30 minutes, preferably 5 minutes to 20 minutes.

3. The electrical method of any preceding claim, wherein the composition is heated at a temperature between 39°C and 42°C, more preferably between 40° C and 4l°C before being applied on the skin.

4. The electrical method of any preceding claim, wherein the heating is prior or simultaneous or post to the applying of a selected electroporation pulsatile current stimulus.

5. The electrical method of any preceding claim, wherein the electrode is heated at a temperature between 39°C and 42°C, more preferably between 40° C and 4l°C, before it contacts the skin.

6. The electrical method of any preceding claim, wherein the electroporation pulsatile current stimulus comprises pulses having a pulse duration ranging from 0,5 milliseconds to 100 milliseconds, preferably from 5 milliseconds to 70 milliseconds and more preferably from 7 milliseconds to 50 milliseconds.

7. The electrical method of any preceding claim, wherein the electroporation pulsatile current stimulus comprises pulses having a pulse interval ranging from 400 to 1000 ms, preferably from 500 milliseconds to 800 milliseconds, preferably from 600 milliseconds to 700 milliseconds.

8. An electrical cosmetic kit comprising: - An electrical composition including one or more of hyaluronic acid, hyaluronic acid derivatives, ions of hyaluronic acid, and ions of hyaluronic acid derivatives, and

- An electrical device and a heating device for carrying the electrical method of anyone of the preceding claims.