ELECTRICAL METHOD OF DELIVERING HYALURONIC ACID THROUGH THE SKIN

Abstract

An electrical method of delivering hyaluronic acid through the skin that includes 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, for example an aqueous composition, to a biological subject, and transporting different rates of hyaluronic acid across the skin in accordance to the selected current mode.

Claims

2. The electrical method of claim 1, wherein applying an electroporation pulsatile current stimulus to a biological subject includes generating an electroporation pulsatile current stimulus having a voltage below 500 V.

3. The electrical method of claim 1, wherein 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.

4. The electrical method of claim 1, wherein the electroporation pulsatile current stimulus comprises pulses having a pulse duration ranging from 3 milliseconds to 100 milliseconds.

5. The electrical method of claim 1, wherein the electroporation pulsatile current stimulus comprises pulses having a pulse interval ranging from 5 milliseconds to 100 milliseconds.

6. The electrical method of claim 1, wherein the electroporation pulsatile current stimulus comprises a number of pulses ranging from 1 to 200 pulses.

7. The electrical method of claim 1, wherein the electroporation pulsatile current stimulus includes 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.

8. The electrical method of claim 1, further comprising applying to a biological subject an iontophoresis direct current stimulus having an average current density ranging from 0.001 mA/cm.sup.2 to 1 mA/cm.sup.2.

9. The electrical method of claim 1, wherein the iontophoresis direct current stimulus is applied for a duration ranging from 1 minute to 60 minutes.

10. The electrical method of claim 1, further comprising: transdermally delivering a 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 100%.

11. The electrical method of claim 1, wherein the hyaluronic acid has a molecular weight in a range from 1 Da to 4000 kDa.

12. The electrical method according to claim 1, comprising the step of measuring at least one of the temperature of the skin, the impedance of the skin, and a pH of the composition, and wherein the application of current stimulus may be reduced to a safety level when a measured value measured by one of the sensors exceeds a safety range or a safety value.

13. 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. and water present in an amount of at least 0% by weight.

14. An electrical 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 of claim 1.

15. The electrical kit of claim 14, wherein the composition is an aqueous composition.

16. The electrical kit of claim 14, the kit being configured such that hyaluronic acid and water are already mixed in the composition when the composition is applied to the skin.

17. The electrical kit of claim 14, the device comprising at least one of a temperature sensor, an impedance sensor, and a pH sensor, and wherein 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 safdy value.

Description

DESCRIPTION OF THE DRAWINGS

[0118] 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:

[0119] FIG. 1 is a flow diagram of a method in accordance with one embodiment;

[0120] FIG. 2 is a flow diagnm of a method in accordance with one embodiment.

[0121] FIGS. 3a and 3b are graphs showing the impedance change and the relative impedance for the tested treatments;

[0122] FIG. 4 is a graph showing the skin deposition of 120 kDa FL-HA formulation;

[0123] FIG. 5 is a graph showing the comparison of skin deposition of 120 kDa FL-HA formulation after electroporation alone or electroporation plus iontophoresis; and

[0124] FiG. 6 is a view of the skin images obtained.

DETAILED DESCRIPTION

[0125] FIG. 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 concurrently delivering a direct current and an electroporation pulsatile current to a biological subject, the direct current and the pulsed current is of a character and for a duration sufficient to deliver a cosmetic composition to the biological subject.

[0126] In some embodiments, the illustrated steps 604, 606, 608, and 610 are optional. Further, in some embodiments, the sequence of the steps 604, 606, 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 606 for generating current density. In some embodiments, the user makes selections that cause the electrical device to generate the selected current density. 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.

[0127] In some embodiments, the method 600 for concurrently delivering the direct current and 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.

[0128] In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse duration ranging from 5 milliseconds to 100 milliseconds, prefenably from 7 milliseconds to 70 milliseconds and more preferably from 10 milliseconds to 50 milliseconds.

[0129] In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse interval ranging from 5 milliseconds to 100 milliseconds, preferably from 7 milliseconds to 80 milliseconds and more preferably from 10 milliseconds to 60 milliseconds.

[0130] 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.

[0131] In some embodiments, the method 600 for concurrently delivering the direct current and the pulsed current to a biological subject includes generating a direct current stimulus having an average current density ranging from 0.01 mA/cm.sup.2 to 0.5 mA/cm.sup.2, preferably from 0.01 mA/cm.sup.2 to 0.4 mA/cm.sup.2, and more preferably from 0.05 mA/cm.sup.2 to 0.2 mA/cm.sup.2.

[0132] In some embodiments, the method 600 for concurrently delivering the direct current and the pulsed current to a biological subject includes generating a direct current stimulus having an average current density of 0.2 mA/cm.sup.2.

[0133] In some embodiments, the method 600 for concurrently delivering the direct current and the electroporation pulsatile current to a biological subject includes generating a pulsed current stimulus having an average current density ranging from 0.01 mA/cm.sup.2 to 10 mA/cm.sup.2, 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%.

[0134] In some embodiments, the method 600 for concurrently delivering the direct current and the pulsed current to a biological subject includes generating a pulsed alternating current stimulus having an average current density of 0.2 mA/cm.sup.2, a pulse duration of 500 microseconds, and a pulse frequency of 200 Hertz.

[0135] In some embodiments, the method 600 for concurrently delivering the direct current and the pulsed current to a biological subject includes generating a pulsed current having an average current density ranging from 0.01 mA/cm.sup.2 to 10 mA/cm.sup.2, 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%.

[0136] In some embodiments, the method 600 for concurrently delivering the direct current and the pulsed current to a biological subject includes generating a pulsed current stimulus having an average current density ranging from 0.01 mA/cm.sup.2 to 10 mA/cm.sup.2, 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%.

[0137] In some embodiments, the method 600 for concurrently delivering the direct current and the pulsed current to a biological subject includes mnerating a pulsed current having sinusoidal waveforms, non-sinusoidal waveforms, or combinations thereof.

[0138] In some embodiments, the method 600 for concurrently delivering the direct current and 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 wavefornis, or combinations thereof.

[0139] In some embodiments, the method 600 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 seboreg,ulating active agents, or a composition for making up the face or body.

[0140] FIG. 2 illustrates embodiments of a method 700 for delivering an aqueous active agent composition, such as an aqueous hyaluronic acid composition through the skin to a biological subject through the generation of electrical stimuli of certain waveform types.

[0141] In some embodiments, the method 700 includes step 702 for applying a selected current stimulus, either direct current, pulsed current or a combination of both, from any device and/or support comprising at least one electrode to a biological subject, the direct current, the pulsed current or the combination of both is of a character and for a duration sufficient to transdermally deliver an aqueous composition to a biological subject, thus, transporting different rates of hyaluronic acid across the skin in accordance to the selected current mode.

[0142] In some embodiments, the illustrated steps 704, 706, 708, and 710 are optional. Further, in some embodiments, the sequence of the steps 704, 706, 708, and 710 can be in any order and is not confined to the illustration. In some embodiments, the method 700 includes a step 704 for generating waveforms. In some embodiments, the user makes selections that cause the electrical device to generate the selected wavethrm or waveforms that constitute the electrical stimuli. In some embodiments, the method 700 includes a step 706 for generating current density. In some embodiments, the user makes selections that cause the electrical device to generate the selected current density. In some embodiments, the method 700 includes a step 708 for generating pulse duration. In some ernbodiments, the user makes selections that cause the electrical device to generate the selected pulse duration. In some embodiments, the method 700 includes a step 710 for generating pulse frequency. In some embodiments, the user makes selections that cause the electrical device to generate the selected pulse frequency. In step 712, the method 700 includes transdermally delivering an aqueous composition.

[0143] In some embodiments, the method 700 for concurrently delivering the direct current and 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.

[0144] In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse duration ranging from 5 milliseconds to 100 milliseconds, prefenably from 7 milliseconds to 70 milliseconds and more preferably from 10 milliseconds to 50 milliseconds.

[0145] In some embodiments, the electroporation pulsatile current stimulus may comprise pulses having a pulse interval ranging from 5 milliseconds to 100 milliseconds, preferably from 7 milliseconds to 80 milliseconds and more preferably from 10 milliseconds to 60 milliseconds.

[0146] In some embodiments, the method 700 includes generating an 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.

[0147] In some embodiments of the method 700 of delivering an aqueous hyaluronic acid composition through the skin, applying a selected current stimulus to a biological subject includes generating a direct current stimulus having an average current density ranging from 0.01. mA/cm.sup.2 to 0.5 mA/cm.sup.2, preferably from 0.01 mA/cm.sup.2 to 0.4 mA/cm.sup.2, and more preferably from 0.05 mA/cm.sup.2 to 0.3 mA/cm.sup.2.

[0148] In some embodiments of the method 700 of delivering an aqueous hyaluronic acid composition through the skin, applying a selected current stimulus to a biological subject includes generating a. direct current stimulus having an average current density of 0.2 mA/cm.sup.2.

[0149] In some embodiments of the method 700 of delivering an aqueous hyaluronic acid composition through the skin, applying a selected current stimulus to a biological subject includes generating a pulsed current having sinusoidal waveforms, non-sinusoidal waveforms, or combinations thereof.

[0150] In some embodiments of the method 700 of delivering an aqueous hyaluronic acid composition through the skin, applying a selected current stimulus 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 com.binations thereof.

[0151] In some embodiments of the method 700 of delivering an aqueous hyaluronic acid composition through the skin, applying a selected current stimulus to a biological subject includes concurrently delivering the direct current and the pulsed current and generating a pulsed current stimulus having an average current density ranging from 0.05 mA/cm.sup.2 to 0.5 mA/cm.sup.2; a pulse duration ranging from 200 microseconds to 300 microseconds; and a pulse frequency ranging from 100 Hertz to 300 Hertz.

[0152] In some embodiments, the method 700 of delivering an aqueous hyaluronic acid composition through the skin 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 present in amounts ranging from 0.01 to 1.00% by weight, preferably from 0.5 to 60% by weight.

[0153] In some embodiments, the method 700 of delivering an aqueous hyaluronic acid composition through the skin, further comprises transdermally delivering an aqueous composition including hyaluronic acid having a molecular weight in a ramze from 0.8 kDa to 2000 kDa, preferably from. 20 kDa to 1000 kDa and more preferably from 50 kDa to 500 kDa, and in particular from 100 kDa to 200 kDa. In an embodiment, the molecular weight of the hyaluronic acid may be of 120 kDa.

[0154] In some embodiments, the method 700 of delivering an aqueous hyaluronic acid composition through the skin 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 0.1% 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.

[0155] In some embodiments, the method 700 of delivering an aqueous hyaluronic composition through the skin, further comprises transdennally 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 2.0% by weight; and water present in an amount of at least 30% by weight.

[0156] In some embodiments of the method 700 of delivering an aqueous hyaluronic acid composition through the skin, transdennally delivering the aqueous active agent composition includes generating a direct current stimulus having an average current density ranging from 0.01 mA/cm.sup.2 to 0.5 mA/cm.sup.2; and generating a pulsed current stimulus having an average current density ranging from 0.01 mA/cm.sup.2 to 10 mA/cm.sup.2; pulse duration ranging from 10 microseconds to 500 microseconds; and a pulse frequency ranging from 10 Hertz to 500 Hertz; the direct current and the pulsed current of a duration sufficient to transdermally deliver an aqueous active agent composition to a biological subject.

[0157] An electrical composition for use with the electrical methods described above in relation to FIGS. 1 and 2 is disclosed.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] In some embodiments, the electrical composition further comprises a pH ranging from 2 to 7.5.

[0163] 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(dimethyisilylane)], polyvinyl siloxane, cyclohexasiloxane, derivatives thereof, or any combination thereof.

[0164] In some embodiments of the electrical composition, the ionic polymers and nonionic polymers are selected from acrylonitrile/methyl methacrylate/vinylidene chloride copolymer, biosaccharide gum-1, sodium styrene/maleic anhydride copolymer, xanthan gum, ammonium polyacryloyldimethyl taurate, derivatives thereof, their ions, and any combination thereof.

[0165] 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.

[0166] In some embodiments of the electrical composition, the composition includes ionic polymers and nonionic polymers selected from biosaccharide gum-1 (and) sodium levulinate (and) glyceryl caprylate (and) sodium anisate, acrylates/c10-30 alkyl acrylate crosspolym.er, carbomer, sodium styrene/malcie anhydride copolymer, nylon-12, xanthan gum, derivatives thereof, their ions, or any combination thereof.

[0167] In some embodiments, the electrical composition has a pH from 2 to 7.4.

[0168] In some embodiments, the electrical composition has a pH from 2 to 7.

[0169] In some embodiments, the electrical composition has a pH from 5.7 to 6.3.

[0170] In some embodiments, the electrical composition has a pH from 2 to 6.3.

[0171] In some embodiments, the electrical. composition includes one or more vitamins selected from vitamin B5, vitamin A, vitamin B3, and vitamin E.

[0172] In some embodiments, the electrical composition includes one or more fats selected from nut oils, seed oils, and plant oils.

[0173] In some embodiments, the electrical composition includes one or more solvents selected from water, deionized water, and Eau de la Roche-Posay.

[0174] In some embodiments, the electrical composition includes one or more humectants selected from glycerin, caprylyl glycol., and sodium hyaluronate.

[0175] In some embodiments, the electrical composition includes one or more viscosity reducers selected from glycerine.

[0176] In some embodiments, the electrical composition includes one or more preservatives selected from phenoxyethanol, salicylic acid, and sodium methylparaben.

[0177] In some embodiments, the electrical composition includes one or more chelating agents selected from disodium EDTA.

[0178] In some embodiments, the electrical composition includes one or more viscosity controllers selected from disodium EDTA, ammonium polycryldimethyltyltauramide, and nylon-12.

[0179] 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, biosaceharide gum-1, oxothiazolidinecarboxylic acid, ascorbic acid, sodium styrene/maleic anhydride copolymer, salicylic acid, cyclohexasiloxane, hydrogenated polyisobutene, biosaccharide gum-1 and sodium levulinate and glyceryl caprylate and sodium anisate, lemon extract, alcohol and Gentiana lutea root extract, and dimethicone and polyethylene glycol/polypropylene glycol-18/18 dimethicone.

[0180] 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-1 and sodium levulinate and glyceryl caprylate and sodium anisate, and dimethicone and polyethylene. glycol-polypropylene glycol-18118 dimethicone.

[0181] 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, biosaceharide gum-1 and sodium levulinate and glyceryl caprylate and sodium anisate, and dimethicone and polyethylene. glycol/polypropylene glycol-18/18 dimethicone.

[0182] In some embodiments, the electrical. composition includes one or more cleansing agents selected from glyceryl stearate and polyethylene glycol 100 Stearate.

[0183] 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/C10-30 alkylacrylate crosspolymer.

[0184] 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/C10-30 alkylacrylate crosspolymer.

[0185] In some embodiments, the electrical composition includes one or more antioxidants selected from tocopheryl acetate, and ascorbic acid.

[0186] In some embodiments, the electrical composition includes one or more binders selected from xanthan gum.

[0187] In some embodiments, the electrical composition includes one or more skin bleaching agents selected from oxothiazolidinecarboxylic acid.

[0188] In some embodiments, the electrical composition includes one or more pH adjusters selected from triethanolamine, potassium hydroxide, and sodium hydroxide.

[0189] In some embodiments, the electrical composition includes one or more buffering agents selected from potassium hydroxide and hydroxyethylpiperazine ethane sulfonic acid, and sodium hydroxide.

[0190] In some embodiments, the electrical composition includes one or more denaturants selected from sodium hydroxide.

[0191] In some embodiments, the electrical composition includes one or more bulking agents selected from nylon-12.

[0192] In some embodiments, the electrical composition includes one or more opacifying agents selected from nylon-12.

[0193] In some embodiments, a hyaluronic acid composition herein can comprise the specified components, leaving open the possibility of other unspecified components.

[0194] 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

[0195] I-Introduction

[0196] The effect of electroporation parameters on skin integrity and transport of hyaluronic acid (at 1% by weight in water) 120 kDa (HA) labeled with fluorescein into the permeabilzed skin was investigated in vitro using electroporation vs. its passive diffusion.

[0197] A voltage of 50 V and 250 V was used with pulse length between 1-80 ms, pulse interval of 10-50 and number of pulses of 1-80.

[0198] This 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 (x6 compared to passive diffusion).

[0199] The combination of such parameters with iontophoresis at 0.2 mA/cm.sup.2 for 20 min has also been corroborated (x 12 vs. passive diffusion). Micrographs of confocal microscopy also prove such effect and the lack of significant damage to the skin.

[0200] This shows that a combined treatment of electroporation at such conditions plus iontophoresis may decrease the skin impedance and enhance the passage much importantly than electroporation alone.

[0201] II First Experiments

[0202] Two modes have been tested:

[0203] i) transfer mode (50-99 V, longer pulse lengths and high number of pulses) and

[0204] ii) porating mode (100-250 V, shorter pulse lengths and low number of pulses). Franz-diffusion cells were used to evaluate the in vitro delivery.

[0205] The experimental protocol has been as follows:

[0206] 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 min for all samples. Skin was frozen or stored at 4 C. until use.

[0207] 2. Identification of optimal electroporation parameters

[0208] The following procedure was used to evaluate the effect of the electroporation parameters:

[0209] 1. Leave the porcine ear skin for 1 hour at room temperature.

[0210] 2. Set up the program of electroporation condition.

[0211] 3. Rinse the skin well with Milli-Q water.

[0212] 4. Place the skin samples in 3-compartment Franz-type vertical diffusion cells.

[0213] 5. Add PBS buffer to the electrode compartments (i.e. upper compartments) to ensure good electrical contact between the electrodes and the skin.

[0214] 6. Measure the skin resistance before electroporation (5 mm platinum disk electrode).

[0215] 7. Apply electroporation current, as shown in the tables 1 and 2 below.

[0216] 8. Measure the skin resistance after electroporation.

[0217] 9. Take photos to make a visual record of skin condition.

[0218] Transfer mode (conditions 1-9)

TABLE-US-00001 Pulse Pulse Condi- Voltage length interval tion (V) (ms) (ms) Pulses 1 99.9 80 10 80 2 40 10 60 3 20 10 40 4 1 10 20 5 50.0 80 10 80 6 40 10 60 7 20 10 40 8 1 10 20

[0219] Poring Mode (conditions 9-16)

TABLE-US-00002 Pulse Pulse Condi- Voltage length interval tion (V) (ms) (ms) Pulses 9 250 15 50 9 10 10 50 6 11 5 50 3 12 1 50 1 13 150 15 50 9 14 10 50 6 15 5 50 3 16 1 50 1

[0220] The results are shown on FIGS. 3a and 3b.

[0221] On FIG. 3a is shown the impedance change (lmpedance=FinalInitial) and Relative Impedance (Final/Initial) in Transfer Pulse Mode. The absolute change in impedance (lmpedance) is shown by the bars and the relative impedance given by the ratio of the final to the initial impedance is given by the circles. The largest decrease (or lowest relative impedance) is seen for Treatments 1 and 2 which had the most aggressive conditions (the higher voltage, the longer pulse lengths and the greater number of pulses).

[0222] On FIG. 3b is shown the impedance difference ((Impedance=FinalInitial) and Relative Impedance (Final/Initial) for Poring Pulse Mode. The absolute change in impedance (Impedance) is shown by the bars and the relative impedance given by the ratio of the final to the initial impedance is given by the circles. As above, the largest decrease (or lowest relative impedance) is seen for the most aggressive treatmentsin this case, Treatments 9 and 10.

[0223] Conditions 1, 2, 9 and 10 provide the high decrease of skin impedance.

[0224] IISecond Experiments

[0225] In order to test delivery of HA through the skin, conditions 2, 9 and 10 were selected. Application of the formulation was conducted in two different ways, previous (pre-treatmentPT) and simultaneously to the electrical treatment (co-treatmentCT), in order to obtain the optimal conditions for active delivery.

[0226] The experimental protocol has been as follows: 1. Skin source and treatment

[0227] 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.

[0228] 2. Analytical method

[0229] 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.

[0230] 3. Skin transport study

[0231] The aim of the experiments was to quantify penetration of FL-HA (120 kDa) into intact, electroporated porcine ear skin with and without iontophoresis) as a function of duration using the optimal treatment conditions identified in the previous part of the study. Electroporation treatment was performed either as (i) pre-treatment, where electroporation was performed before formulation application, or as (ii) co-treatment, where the elcetroporation treatment was performed simultaneou.sly in the presence of the formulation. All conditions were tested for 10 and 20 min.

[0232] The following specific conditions treatments were tested:

[0233] TRANSFER PULSE MODE (Condition nos, from Preliminary report) 1. Condition 2 (Voltage 99.9 V, Pulse length 40 ms, Pulse interval 10 ms and 60 Pulses), pre-treatment with electroporation

[0234] 2. Condition 2 (Voltage 99.9 V, Pulse length 40 ms, Pulse interval 10 ms and 60 Pulses), simultaneous treatment with electroporation and formulation

[0235] PORING PULSE MODE

[0236] 3. Condition 9 (Voltage 250 V, Pulse length 15 ms, Pulse interval 50 ms and 9 Pulses), pre-treatment with electroporation

[0237] 4. Condition 9 (Voltage 250 V, Pulse length 15 ms, Pulse interval 50 ms and 9 Pulses), simultaneous treatment with electroporation and formulation

[0238] 5. Condition 10 (Voltage 250 V, Pulse length 15 ms, Pulse interval 50 ms and 6 Pulses), pre-treatment with electroporation

[0239] 6. Condition 10 (Voltage 250 V, Pulse length 15 ms, Pulse interval 50 ms and 6 Pulses), simultaneous treatment with electroporation and formulation

[0240] 7. Control: Application of an aqueous solution of FL-HA (120 kDa) formula on (1% w/w) to untreated skin.

[0241] The results of the conditions 2 and 9 with passive diffusion for 10 min or 20 min (vs. single passive diffusion) are shown on FIG. 4. FIG. 4 shows skin deposition of 120 kDa FL-HA formulation following PT (Pre-Treatment; Electroporation BEFORE formulation application), CT (Co-Treatment; Electroporation AFTER formulation application).

[0242] The use of 250 V (pulse length 15; pulse interval 50 ms; 9 pulses) enhanced significantly the amount of fluorescence labeled HA 120 kDa at 1% (w/v) into the skin (x6) compared to the passive diffusion of the active. The use of higher voltages and short pulse length and low number of pulses results more efficient for skin delivery while keeping skin integrity, rather than increased number of pulses at a lower voltage.

[0243] IVThird experiments

[0244] The following specific conditions treatments were tested:

[0245] ELECTROPORATION+IONTOPHORESIS

[0246] Application of an. aqueous solution of each FL-HA formulation (1% w/w) to treated skin following electroporation using previous poring pulse mode (condition 9) and iontophoresis at 0.2 mA/cm.sup.2 for 10 and 20 min. Iontophoresis-combined treatment employed cathodal direct current (DC) at 0.2 mA/cm.sup.2 (Ag/AgCl electrodes, DC generator).

[0247] The results are shown on FIG. 5. FIG. 5 shows the comparison of skin deposition of 120 kDa FL-HA formulation after electroporation alone (CT; Electroporation AFTER formulation application) or electroporation plus iontophoresis (CT+I; Electroporation AFTER formulation application followed by iontophoresis).

[0248] The comparison of the deposition when performing electroporation with and without iontophoresis showed statistical difference with the CT+120 m condition as compared to the rest (p<0.05; ANOVA, Student Newman Keuls test). The similar deposition obtained with 10 min electroporation, indicates that up to 10 min, iontophoresis is not increasing the deposition; nevertheless, after iontophoresis for 20 min, the increase was statistically superior.

[0249] The skin images obtained provide more information about the possible skin damage produced by the electroporation treatment. In the case of condition 9 plus iontophoresis, no damage was observed, as shown on FIG. 6.

[0250] Co-treatment of the formulation leads to optimal conditions for eiectropermeabilization. When the technique is combined with iontophoresis, an important increase is observed compared to the passive diffusion of the active corroborating its efficacy on active transport of hydrophilic and charged molecules,

[0251] It has been demonstrated that electroporation of the skin by electrical shots with short pulse length of 15 ms, pulse interval of 50 ms and low number of pulses (<10) at 250 V is favorable to enhance skin deposition (x6) of HA 120 kDa compared to its passive diffusion, with no visual damage to the membrane. The effectiveness of this technique in combination with iontophoresis is also corroborated, and can be dedicated to durable treatments.

[0252] 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.