CN104922063A - Nanoemulsion for transdermal drug delivery and method for producing same - Google Patents

Abstract

The present invention relates to a nanoemulsion for transdermal drug delivery. The present invention provides chemicals for preparing nanoemulsions and methods of preparing the nanoemulsions. The nanoemulsion has a desired particle size for effective transdermal administration, and does not involve any organic solvent that is harmful to human skin. The corresponding manufacturing method is simple.

Description

Nanoemulsion for transdermal administration and method for its manufacture

Cross References to Related Applications

Pursuant to 35 U.S.C. §119(e), this case is a non-provisional patent application claiming the rights to U.S. Provisional Patent Application Serial No. 61/967,454, filed March 19, 2014, the disclosure of which is incorporated by reference in its entirety way incorporated into this article.

technical field

The invention relates to a nanoemulsion drug delivery system for transdermal drug delivery and a manufacturing method thereof.

Background technique

The skin, the largest organ of the body, has long been considered a promising route for the administration of therapeutic agents. Because the skin is an excellent natural barrier against foreign substances, it has a low permeability to therapeutic agents. To increase skin permeability, scientists have tried many different penetration-enhancing techniques, including the addition of chemical enhancers, sonophoresis, iontophoresis, and microneedling. Compared with the aforementioned techniques, nanovesicle drug delivery systems have gained a lot of attention due to various advantages such as minimized drug degradation and drug loss, increased drug bioavailability and drug accumulation in the target area, harmful Toxic effects are prevented, broadness and flexibility in drug handling are achieved and patient compliance is improved.

On the basis of the main formulation components, nanovesicle drug delivery systems can be broadly classified into lipid-based carriers and polymer-based colloidal carriers. These two families share common advantages such as controlled particle size, enhanced skin penetration and controlled release. The main difference between lipid-based carriers and polymer-based carriers is that the former are mainly composed of physiological lipids and are therefore more biocompatible and can be degraded into non-toxic residues without polymeric materials causing security issues. Various lipid nanocarriers have been disclosed for skin care, such as liposomes, ethosomes, microemulsions, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and oil-in-oil carriers. Solid nanodispersion. The three types of lipid nanovesicles (including microemulsions, NLCs, and solid-in-oil nanodispersions) are proposed in the present invention as examples for promoting skin penetration of hydrophobic or hydrophilic active ingredients . These examples are only for better understanding of the present invention and are not intended to limit the scope of the present invention in any way.

Microemulsions or nanoemulsions are thermodynamically stable isotropic dispersions that are clear, low in viscosity, and consist of oil and water stabilized by an interfacial film of surfactant molecules, typically in combination with cosurfactants . The skin penetration enhancing efficacy of nanoemulsions can be attributed to the combined effect of both the lipophilic and hydrophilic regions of the microemulsions. Lipid domains can partition directly into the lipids of the stratum corneum, or the lipid vesicles themselves can intervene between the lipid chains of the stratum corneum, thereby destabilizing the bilayer structure of the stratum corneum, creating barriers for drug penetration. path of. On the other hand, the hydrophilic region of the nanoemulsion can hydrate the stratum corneum to a greater extent, resulting in increased passive transdermal drug flux. Because some lipid chains are covalently attached to keratinocytes, hydration of these proteins will also lead to disorder of the lipid bilayer, which further facilitates drug delivery. Many studies have demonstrated the improved transdermal and dermal delivery properties of microemulsion formulations for different drugs such as estradiol, meloxicam, methotrexate and clindamycin phosphate. In order to achieve better skin penetration of active ingredients, nanoemulsions incorporating transdermal penetration enhancers have been developed. V. Prasad Shastri's group demonstrated a novel microemulsion system containing oleyl alcohol and N-methylpyrrolidone (NMP) as penetration enhancers for the delivery of two hydrophilic drugs (diltiazem HCl and lidocaine hydrochloride (lidocaine HCl)) and two hydrophobic drugs (estradiol and lidocaine free base). A comparison between oil-in-water (o/w) nanoemulsions and water-in-oil (w/o) nanoemulsions shows that the former provides higher promotion for both hydrophilic and hydrophobic drugs. When compared to aqueous solutions, o/w microemulsion systems facilitate drug penetration 17-fold for lidocaine free base, 30-fold for lidocaine HCl, and 58-fold for estradiol , and 520 times for diltiazem hydrochloride.

Nanostructured lipid carriers (NLCs) are derived from emulsions by replacing only some of the liquid lipids (oils) with solid lipids (ie solid at body temperature). There are five main advantages of NLC over other conventional carriers: (1) low toxicity and excellent tolerance due to the presence of biodegradable physiological lipids; Closer contact and increased drug skin penetration; (3) allow controlled release of many substances due to the solid matrix; (4) increased skin hydration due to the obstructive properties of the lipid nanoparticles; and (5) improved resistance to Chemical stability of compounds sensitive to light, oxidation and hydrolysis. Because of the hydrophobic matrix, solid lipid particles have been widely used to deliver poorly water-soluble drugs in order to facilitate their skin penetration and stability.

Solid-in-oil nanodispersions are another emulsion-based drug delivery system. Typically, it is prepared by creating a water-in-oil emulsion followed by removal of solvent and water. The resulting nanoparticles are dispersed in oil to form a solid-in-oil nanodispersion comprising active ingredient, surfactant and oil. Solid-in-oil nanodispersions have long been used in the transdermal delivery of active ingredients, especially hydrophilic proteins. Masahiro Goto's group has demonstrated that skin penetration of 6kDa insulin, 27kDa enhanced green fluorescent protein (EGFP) and 40kDa horseradish peroxidase (HRP) can be enhanced in solid-in-oil nanodispersions compared to controls . The team also demonstrated a 2- to 3-fold increase in the penetration efficiency of an antirheumatic drug called methotrexate through the skin in the solid-in-oil nanodispersion compared to a control aqueous solution. The addition of urea as an accelerant to the aforementioned dispersion resulted in an approximately 8.8-fold increase over the control aqueous solution after 24 hours.

However, the above-mentioned conventional techniques, especially lipid vesicles, have disadvantages that their processing steps are complicated and that they involve organic solvents that are harmful to human skin.

Contents of the invention

Accordingly, a first aspect of the present invention is to provide a chemical formulation for the preparation of nanoemulsions.

According to one embodiment of the present invention, the chemical formulation for preparing the nanoemulsion comprises: oil, surfactant, ethanol, water, and active ingredients; wherein the water is mixed with the ethanol+the oil+the surfactant The weight ratio is in the range of 3:7 to 2:8.

The ratio of the water to the ethanol + the oil + the surfactant is a key technical feature as the ratio enables the size reduction of the nanoemulsions below 100nm and this size range provides more efficient skin penetration . Additionally, this ratio can maximize the concentration of the encapsulating phase while maintaining the carrier phase to provide better transdermal delivery.

Preferably, the ratio of ethanol to oil to surfactant is about 1:1:2. Similarly, this ratio can also reduce the size of the nanoemulsion.

Preferably, the chemical formulation additionally comprises a penetration enhancer.

Preferably, the active ingredient is schisandrin or epidermal growth factor.

According to another embodiment of the present invention, the chemical formulation for preparing the nanoemulsion comprises: oil, surfactant, penetration enhancer, water, and active ingredients; wherein the weight ratio of the oil to the surfactant is between 1 :9 to 2:8 range.

The weight ratio of the oil to the surfactant is a key technical feature as it enables the size reduction of the nanoemulsion below 100 nm, thus providing better skin penetration. Additionally, this ratio can maximize the concentration of the encapsulating phase while maintaining the carrier phase to provide better transdermal delivery.

Preferably, the active ingredient is palmitoyl pentapeptide-3.

A second aspect of the present invention is to provide a method for preparing nanoemulsions.

According to one embodiment of the present invention, the method for preparing a nanoemulsion comprises: mixing said oil, said surfactant, said ethanol and said active ingredient to form a first mixture; adding water dropwise to said first mixture; mixture to form a second mixture; and agitating or homogenizing the second mixture to form the nanoemulsion.

Preferably, the active ingredient is Schisandrin.

According to another embodiment of the present invention, a method of preparing a nanoemulsion comprises: mixing said oil, said surfactant, said ethanol and said penetration enhancer to form a first mixture; mixing said active ingredient with said water to form a second mixture; adding the second mixture dropwise to the first mixture to form a third mixture; stirring the third mixture to form the nanoemulsion.

Preferably, the active ingredient is epidermal growth factor.

According to another embodiment of the present invention, the method of preparing a nanoemulsion comprises: mixing the active ingredient, the oil, the surfactant and the penetration enhancer to form a first mixture; adding water dropwise to the said first mixture to form a second mixture; and agitating or homogenizing said second mixture to form said nanoemulsion.

Preferably, the active ingredient is palmitoyl pentapeptide-3.

The chemical formulations and methods of the present invention provide nanoemulsions with the desired particle size (below 100 nm) for effective transdermal delivery. Thus, the nanoemulsions of the present invention are smaller than conventional lipid vesicles with a size of approximately 500 nm. Furthermore, the chemical formulation of the present invention does not involve any organic solvents that are harmful to human skin, and the corresponding manufacturing method is simple.

Description of drawings

Embodiments of the invention will be described in more detail hereinafter with reference to the accompanying drawings, in which:

Fig. 1 is a schematic diagram of the nano-preparation of Sch B according to an embodiment of the present invention;

Figure 2 is a graph showing the effect of homogenization speed on particle size at a homogenization time of 20 minutes in a Sch B (1.5%) nanoemulsion formulation according to one embodiment of the present invention;

Figure 3 is a graph showing the effect of homogenization time on particle size at a homogenization speed of 20000 rpm in Sch B (1.5%) nanoemulsion formulations according to one embodiment of the present invention;

Figure 4 is a graph showing the effect of Sch B concentration on particle size in nanoemulsion formulations, homogenized at a homogenization speed of 20,000 rpm for 20 minutes, according to one embodiment of the present invention;

Figure 5 is a graph showing a comparison of different methods for forming particles in a 1.5% Sch B-nanoemulsion according to one embodiment of the present invention;

Figure 6 is a graph showing the effect of incubation time of Sch B-nanoemulsion formulations on skin penetration enhancement in in vitro studies according to one embodiment of the claimed invention;

Figure 7 is a graph showing the effect of the concentration of Sch B in nanoscale Sch B formulations on skin penetration enhancement, according to one embodiment of the present invention;

Figure 8 is a graph showing the percentage of Sch B permeated into the skin in nanonized Sch B formulations and a control group after a 6 hour diffusion cell experiment according to one embodiment of the present invention;

Figure 9 is a schematic diagram of the nano-preparation of Pal-KTTKS according to one embodiment of the present invention;

Figure 10 is a graph showing the effect of homogenization speed on particle size at a homogenization time of 2 minutes in a Pal-KTTKS (0.2%) nanoemulsion formulation according to one embodiment of the present invention;

Figure 11 is a chart showing the effect of homogenization time on particle size at a homogenization speed of 12000 rpm in a Pal-KTTKS (0.2%) nanoemulsion formulation according to one embodiment of the present invention;

Figure 12 is a graph showing the effect of stirring speed on particle size at a stirring time of 1 minute in a Pal-KTTKS (0.2%) nanoemulsion formulation according to one embodiment of the present invention;

Figure 13 is a chart showing the effect of stirring time on particle size at a stirring speed of 1500 rpm in a Pal-KTTKS (0.2%) nanoemulsion formulation according to one embodiment of the present invention;

Figure 14 is a graph showing the effect of Pal-KTTKS concentration on particle size in nanoemulsion formulations at a stirring speed of 1500 rpm for 1 minute according to an embodiment of the present invention;

Figure 15 is a graph showing an in vitro study of the effect of 3 different chemical enhancers in Pal-KTTKS-nanoemulsions on skin penetration enhancement compared to a control group, according to an embodiment of the claimed invention;

FIG. 16 is a graph showing the effect of phospholipid concentration in nanosized Pal-KTTKS formulations on skin penetration enhancement, according to one embodiment of the present invention;

Figure 17 is a graph showing the effect of incubation time of Pal-KTTKS-nanoemulsion formulations on skin penetration enhancement in in vitro studies according to one embodiment of the present invention;

Figure 18 is a graph showing the effect of the concentration of Pal-KTTKS in a nanosized Pal-KTTKS formulation on skin penetration enhancement, according to one embodiment of the present invention;

Figure 19 is a graph showing the percentage of Pal-KTTKS penetrated into the skin in nanonized formulations and control formulations after 6 hours of diffusion cell experiments according to one embodiment of the present invention;

Figure 20 is a schematic diagram of the nano-preparation of EGF according to one embodiment of the present invention;

Figure 21 is a graph showing the effect of geraniol concentration in nanosized EGF formulations on skin penetration enhancement, according to one embodiment of the present invention;

Fig. 22 is a graph, it shows according to one embodiment of the present invention, the influence of the incubation time of the EGF-nanoemulsion formulation in the in vitro study on skin penetration promotion;

Figure 23 is a graph showing the effect of the concentration of EGF in nanosized EGF formulations on skin penetration enhancement, according to one embodiment of the present invention; and

Figure 24 is a graph showing the percentage of nanosized EGF formulations and controls in skin after a 6 hour diffusion cell experiment according to one embodiment of the present invention.

Detailed ways

In the following description, chemical formulations for preparing nanoemulsions, and methods for preparing said nanoemulsions for effective transdermal administration are illustrated as preferred examples. It will be obvious to those skilled in the art that changes, including additions and/or substitutions, can be made without departing from the scope and spirit of the present invention. Specific details may be omitted without obscuring the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation.

Example

Example 1

A. Nanoization of Schizandrin B (Sch B)

A.1 Preparation of Sch B-nanoemulsion

First mix together the prescribed amounts of Sch B, oil and surfactant with ethanol. The mixture was then sonicated for 10 minutes in a sonication bath to obtain a clear homogeneous solution. The required amount of water was then added dropwise under magnetic stirring (1000-2000 rpm) or homogenization (5000-20000 rpm) to form a nanoemulsion. The resulting nanoemulsion was further stirred for a specified time (2-20 minutes). Figure 1 illustrates the preparation strategy of the nanoemulsion. The nanoemulsions were stored at room temperature until later use.

The nanoemulsion system consists of water, ethanol, oil and surfactant. The ratio of water to ethanol + oil + surfactant is a critical ratio of 27:73. The ratio of ethanol to oil to surfactant is about 1:1:2. Oils that can be used in nanoemulsions include and are preferably selected from the group formed by (1) esters of polyols and fatty acids, such as isopropyl myristate, caprylocaproyl polyoxyglycerides or ethyl oleate; (2) animal fats and oils and vegetable fats and oils containing saturated alkyl chains of about 10 to 12 carbons in length attached to polar head groups, such as oleic acid, lauric acid or linoleic acid; (3) natural or synthetic essential oils such as limonene or menthol. The amount of oil is preferably in the range of 15% to 16% by weight relative to the total weight of the nanoemulsion. The surfactant is preferably selected from nonionic surfactants such as span 80, Capryol 90, tween 20 or tween80. The amount of surfactant used is preferably in the range of 37% to 39% by weight. The content of ethanol and water in the nanoemulsion is preferably in the range of 17% to 18% by weight and 26% to 27% by weight, respectively. The active ingredients to be loaded in the nanoemulsion system can be biphenyl cyclooctadiene lignans and their derivatives, such as Schisandrin A, Schisandrin B, Schisandrin C, gomisin (gomisin) A or gomisin J. The preferred percentage range of said active ingredient is 0.15% to 3.6%. The percentage ranges and processing parameters for each component in the nanoemulsion are shown in Table 1.

Table 1. Percentage ranges of components of Sch B-nanoemulsions

An example of the components used to prepare Sch B-nanoemulsions is shown in Table 2.

Table 2. An example of components in Sch B-nanoemulsions

A.2 Optimization of Sch B-nanoemulsion

To facilitate skin penetration of nanoemulsions, the homogenization speed, homogenization time and effect of homogenization or agitation method were optimized and selected based on particle size. The components of the Sch B-nanoemulsions in these studies are listed in Table 2.

Sch B-nanoemulsions were prepared with different homogenization speeds (5000, 10000, 15000 and 20000 rpm) and the homogenization time was kept at 20 minutes. The Sch B concentration in the nanoemulsion was 1.5%. The results are shown in Figure 2. The results show a particle size of less than 15 nm at all homogenization speeds. The smallest particle size was found to be 9.8 nm at a homogenization speed of 20000 rpm. So choose 20000rpm.

The effect of homogenization time (2, 5, 10 and 20 min) on particle size is shown in Figure 3. The test was performed at a homogenization speed of 20000 rpm. The Sch B concentration in the nanoemulsion was 1.5%. The particle size at all different times was less than 20 nm. The smallest particle size was found to be 9.8 nm at 20 minutes of homogenization. The homogenization time was chosen to be 20 minutes.

In addition, the effect of Sch B concentration (0.15%, 0.3%, 0.6% and 1.5%) on the nanoemulsion particle size was studied at a homogenization speed set at 20000 rpm and a homogenization time set at 20 minutes. Referring to Figure 4, it was found that the particle size decreased when the weight % of Sch B was increased. 0.15% to 1.5% Sch B can produce similar small particles, which are smaller than 80nm. Therefore, this study demonstrates that Sch B concentrations ranging from 0.15% to 1.5% can be applied for nanoemulsion formulations.

A comparison of the particle size results produced by homogenization or stirring is shown in Fig. 5. The particles (9.8 nm) prepared by homogenization at 20000 rpm were comparable to the particles (14.2 nm) prepared by stirring at 1000 rpm. 20 minutes of mixing was used under both conditions. The Sch B concentration in the nanoemulsion was 1.5%. Both homogenization and stirring can achieve particle sizes below 20 nm. However, it is more preferred to use agitation for the production of active-encapsulating nanovesicles. Considering the industrial scale production and equipment cost, it is more appropriate to choose to prepare Sch B-nanoemulsion by stirring method.

A.3 Preparation of nano-sized Sch B preparation cream and control samples

The nanoscaled SchB formulation cream was prepared by mixing the SchB-nanoemulsion with the cream base in the specimen container. Then Sch B dissolved in an ethanol/water mixture (EtOH:water weight ratio = 3:2) was mixed with a cream base in another specimen container as a control. Both mixtures were swirled for 2 minutes to obtain a homogeneous cream. The volume ratio of nano-Sch B or non-nano-Sch B to cream base is 3:7.

A.4 In vitro skin penetration studies

A vertical Franz-type diffusion cell was used in the in vitro skin permeation assay. The study was performed in triplicate using porcine ear skin samples with a total area of 6.25 ^cm2 . Before the experiment, the skin samples were cleaned with double deionized water and then with 0.9% sodium chloride solution. Cleaned porcine skin was mounted on the diffusion cell with the cuticle facing the sample donor chamber. The nanoemulsion formulation containing the active ingredient or the control (0.5 mL) was applied on the stratum corneum of the skin by means of a syringe protruding into the donor chamber. The donor chamber is then covered with parafilm to prevent evaporation. The dermal side of the skin was contacted with the receptor solution (0.9% sodium chloride), which was stirred continuously at 350 rpm. The experiment was performed at a temperature of 32.5°C. After the defined period, 2 mL of the receptor solution was withdrawn through the sampling port of the receptor compartment. The remaining formulation on the skin was wiped off with a cotton pad, and a skin sample was taken for extraction of the active ingredient. The amount of Sch B in skin and recipient solutions was determined by high performance liquid chromatography (HPLC).

A.5 Effect of incubation time on skin penetration in in vitro studies

In the in vitro study of the percutaneous absorption of the Sch B nanoemulsion formulation cream, 4 different incubation time points (1.5 hours, 3 hours, 6 hours and 24 hours) were studied. Figure 6 shows the effect of different incubation time points on the facilitation of skin penetration. The greatest enhancement of skin penetration was obtained when the formulation was incubated on the skin for 6 hours, which showed a 3.6-fold increase in skin penetration compared to the control group.

A.6 The impact of the concentration of Sch B in the nano-sized Sch B preparation on skin penetration

The in vitro percutaneous absorption of three different Sch B concentrations (0.045%, 0.45%, 1.08%) in the nano-Sch B preparation cream was tested. These creams were prepared from nanoemulsions containing 0.15%, 1.5% and 3.6% of Sch B, respectively. Figure 7 shows the effect of Sch B concentration in Sch B nanoemulsion formulations on skin penetration enhancement. Skin penetration increased with increasing Sch B concentration, and a maximum facilitation of 354% was found at a concentration of 0.45%. Further increasing the concentration of Sch B in the formulation from 0.45% to 1.08% attenuated the skin penetration enhancing effect. Therefore, the optimum SchB concentration in the formulation cream is 0.45%.

Optimized Sch B nanoemulsion formulation (working example)

A.7 Skin penetration of nano-sized Sch B preparation cream compared with control group

This is a skin penetration study of the optimized Sch B nanoemulsions presented in Table 2. Figure 8 shows the percentage of the nanosized Sch B formulation cream and control group that penetrated into the skin. The tested nanonized Sch B formulation cream and the control contained 0.45% Sch B. The nanonized Sch B formulation cream was prepared from a 1.5% Sch B nanoemulsion. When tested for 6 hours, the amount of Sch B that the nano-sized Sch B formulation penetrated into the skin was 3 times that of the control group. This result indicates that the skin penetration ability of Sch B can be greatly improved by nanonized transdermal drug delivery. The average particle size of the tested Sch B-nanoemulsions in this formulation was less than 10 nm.

Example 2

B. Nanoscale of palmitoyl pentapeptide 3 (Pal-KTTKS)

B.1 Preparation of Pal-KTTKS-nanoemulsion

Weigh the required amount of Pal-KTTKS, surfactant, oil and accelerator in predetermined weight ratios into 20ml vials. The mixture was then sonicated for 30 seconds. Double deionized water was then added dropwise to the mixture under magnetic stirring (500-1500 rpm) or homogenization (8000-20000 rpm). The resulting nanoemulsion was kept stirring for a specified time (1-10 minutes) to reach an equilibrium state (Figure 9). The nanoemulsions were stored at room temperature until later use.

Pal-KTTKS-nanoemulsion system consists of water, oil phase, surfactant and penetration enhancer, wherein the water content is up to 90% by weight or equal to 70% by weight, and the weight ratio of oil:surfactant is 1:9 critical ratio. The oil phase that can be used in the nanoemulsion includes the following and is preferably selected from the group formed by: (1) esters of polyhydric alcohols and fatty acids, such as isopropyl myristate, caprylocaproyl polyoxyglycerol esters or ethyl oleate; (2) animal fats and oils and vegetable fats and oils containing saturated alkyl chains of about 10 to 12 carbons in length attached to polar head groups, such as oleic acid, lauric acid, (3) natural or synthetic essential oils, such as limonene or menthol; (4) low carbon C1-C8 glycols, such as Capryol 90 or polyethylene glycol. The amount of oil is preferably in the range of 1% to 3% by weight relative to the total weight of the nanoemulsion. The surfactant is preferably selected from nonionic surfactants such as span 80, Labrasol, tween 20 or tween 80. The amount of surfactant used is preferably in the range of 9% to 27% by weight. Additionally, penetration enhancers that may be used in the nanoemulsion include and are preferably selected from the group formed by: (1) terpenes, such as carvone, geraniol or menthol; (2) phospholipids , such as phosphatidylcholine; (3) urea. The amount of penetration enhancer used is preferably in the range of 0.5% to 4% by weight. The active ingredient to be loaded in the nanoemulsion system can be palmitoyl peptide, such as palmitoyl dipeptide 6, palmitoyl tripeptide 5, palmitoyl tetrapeptide 3, palmitoyl pentapeptide 3 or palmitoyl hexapeptide. The preferred percentage range of said active ingredient is 0.05% to 6.7%. The percentage ranges and processing parameters for each component in the nanoemulsion are shown in Table 3.

Table 3. Percentage range of components of Pal-KTTKS-nanoemulsion

An example of the components used to prepare the Pal-KTTKS-nanoemulsion is shown in Table 4.

Table 4. An example of the components in the Pal-KTTKS-nanoemulsion

B.2 Optimization of Pal-KTTKS-nanoemulsion

To facilitate skin penetration of nanoemulsions, the effects of homogenization speed and homogenization time were optimized and selected based on particle size. In addition, the feasibility of the stirring method for the fabrication of nano-sized particles was investigated. The components of the Pal-KTTKS-nanoemulsions in these studies are listed in Table 4.

The effect of homogenization speed on the particle size of Pal-KTTKS was studied at 8000, 12000, 16000 and 20000 rpm, and the homogenization time was kept at 2 minutes. The concentration of Pal-KTTKS in the nanoemulsion was 0.2%. Figure 10 shows that the particle size is less than 10 nm at different homogenization speeds. The smallest particle size was found to be 5.2 nm at a homogenization speed of 12000 rpm. Therefore, a homogenization speed of 12000 rpm was chosen.

The effect of homogenization time on particle size was studied at 2, 5, 10 and 20 minutes with the homogenization speed kept at 12000 rpm. The concentration of Pal-KTTKS in the nanoemulsion was 0.2%. The results are shown in Figure 11. All particles were found to be smaller than 10 nm at different homogenization times. The smallest particle size was found to be 1.8 nm under 20 minutes of homogenization. This suggests that homogenization conditions of 20 minutes should be chosen.

In addition to homogenization, stirring can also be used to produce Pal-KTTKS-nanoemulsions. The effect of the stirring speed for the preparation of the Pal-KTTKS-nanoemulsion was investigated at 200, 1000 and 1500 rpm with the stirring time set at 1 min. The concentration of Pal-KTTKS in the nanoemulsion was 0.2%. The results (Figure 12) show particle sizes of less than 10 nm at all stirring speeds. The smallest particle size was found to be 1.8 nm at a stirring speed of 1500 rpm. Therefore choose to stir under 1500rpm.

The effect of stirring time on particle size was studied at 1, 2, 5 and 10 minutes with the stirring speed kept at 1500 rpm. The results are shown in Figure 13. All particles at different stirring times were smaller than 10 nm. After stirring for 1 minute, the smallest particle size was 1.8 nm. This indicates that a stirring time condition of 1 minute should be chosen. From the point of view of industrial scale production and equipment cost, stirring method is better than homogenization. Therefore, stirring at 1500 rpm for 1 min was chosen to prepare the Pal-KTTKS-nanoemulsion.

In addition, the effect of Pal-KTTKS concentration (0.05%, 0.1% and 0.2%) on the nanoemulsion particle size was investigated. The components and processing parameters of the Pal-KTTKS-nanoemulsion in this study are listed in Table 4. Figure 14 shows the results of the effect of Pal-KTTKS concentration on particle size. The results show that the particle size is less than 10 nm at different concentrations of Pal-KTTKS. Therefore, this study demonstrates that Pal-KTTKS concentrations ranging from 0.05% to 0.2% can be applied for nanoemulsion preparation.

B.3 Preparation of Pal-KTTKS-nanoemulsion formulation cream and control samples

The Pal-KTTKS-nanoemulsion formulation cream was prepared by mixing the Pal-KTTKS-nanoemulsion with the cream base in a specimen container. Pal-KTTKS dissolved in water was then mixed with a cream base in another specimen container as a control. Both mixtures were swirled for 2 minutes to obtain a homogeneous cream. The volume ratio of nano-sized Pal-KTTKS or non-nano-sized Pal-KTTKS to cream base is 3:7.

B.4 In vitro skin penetration studies

The procedure is the same as described in 'In Vitro Skin Penetration Studies' of Sch B. The amount of Pal-KTTKS in skin and receptor solutions was analyzed by LC/MS/MS.

B.5 Effect of Chemical Accelerators in Pal-KTTKS-Nanoemulsions on Skin Penetration

The effect of chemical accelerators on the skin penetration of Pal-KTTKS-nanoemulsions was investigated using three different accelerators (limonene, phospholipids and oleic acid). Figure 15 shows that the strongest enhancement of skin penetration can be obtained by adding phospholipids to the formulation, which shows a 174% increase in skin penetration. Therefore, phospholipids were chosen as chemical accelerators in Pal-KTTKS nanoemulsions.

The in vitro transdermal absorption of Pal-KTTKS-nanoemulsions with 3 different phospholipid concentrations (0.25%, 0.5% and 1%) was tested. Figure 16 shows the effect of phospholipid concentration in Pal-KTTKS nanoemulsion on skin penetration enhancement. When 0.5% of phospholipids were added to the formulation, the highest increase in skin penetration of 147% was achieved compared to the control. The results indicated that 0.5% phospholipid should be used as a chemical accelerator in the formulation.

B.6 Effect of incubation time on skin penetration in in vitro studies

In the in vitro study of transdermal absorption of Pal-KTTKS nanoemulsion formulations, 4 different incubation time points (1.5 hours, 3 hours, 6 hours and 24 hours) were studied. Figure 17 shows the effect of different incubation time points on skin penetration enhancement. It was found that a significant increase in skin penetration could be achieved at incubation times of 6 h and 24 h, which showed increases of 179% and 182%, respectively, compared to the control group. Since the enhancement of skin penetration at 6 hours and 24 hours is comparable, the shorter time will be considered as the optimal incubation time.

B.7 The effect of the Pal-KTTKS concentration in the nano-sized Pal-KTTKS preparation cream on skin penetration Influence

The in vitro percutaneous absorption of three different Pal-KTTKS concentrations (0.02%, 0.2%, 2%) in the nano-sized Pal-KTTKS preparation cream was tested. These creams were prepared from nanoemulsions containing 0.067%, 0.67% and 6.7% of Pal-KTTKS, respectively. Figure 18 shows the effect of Pal-KTTKS concentration in Pal-KTTKS nanoemulsion formulations on skin penetration enhancement. The highest skin penetration enhancement was obtained at a concentration of 2% Pal-KTTKS. Cosmetics marketed as Pal-KTTKS range below 1%, so 0.2% was chosen as an appropriate concentration.

Optimized Pal-KTTKS nanoemulsion formulation (working example)

B.8 The skin penetration of nano-sized Pal-KTTKS preparation cream compared with the control group

This is a skin penetration study of the optimized Pal-KTTKS nanoemulsions presented in Table 4. Figure 19 shows the percent skin penetration of Pal-KTTKS in nanosized formulations and control formulations. Both the tested nanosized Pal-KTTKS formulation cream and the control contained 0.2% Pal-KTTKS. Nanoized Pal-KTTKS formulation cream was prepared from 0.67% Pal-KTTKS nanoemulsion. When tested for 6 hours, the skin penetration of the Pal-KTTKS-nanoformulation cream increased by 1.6 times compared to the control group. The results indicate that Pal-KTTKS can be effectively penetrated into the skin by this formulation. The average particle size of the tested Pal-KTTKS-nanoemulsions in this formulation was less than 10 nm.

Example 3

C. Nanoization of Epidermal Growth Factor (EGF)

C.1 Preparation of EGF-nanoemulsion

First mix the specified amount of oil and surfactant with ethanol. Terpenes are added as skin penetration enhancers. The mixture was then sonicated for 10 minutes in a sonication bath to obtain a clear homogeneous solution. The required amount of water containing EGF was added dropwise under magnetic stirring (1000-2000 rpm) to form a nanoemulsion. The resulting nanoemulsion was further stirred for a specified time (1-20 minutes). Figure 20 illustrates the preparation strategy of nanoemulsions. Nanoemulsions were stored at room temperature for subsequent use.

The EGF nanoemulsion system is composed of water, ethanol, oil, surfactant and penetration enhancer. The ratio of water to ethanol + oil + surfactant is a critical ratio of 22:78. The ratio of ethanol to oil to surfactant is about 1:1:2. Oils that can be used in nanoemulsions include and are preferably selected from the group formed by (1) esters of polyols and fatty acids, such as isopropyl myristate, caprylocaproyl polyoxyglycerides or ethyl oleate; (2) animal fats and oils and vegetable fats and oils containing saturated alkyl chains of about 10 to 12 carbons in length attached to polar head groups, such as oleic acid, lauric acid or linoleic acid; (3) natural or synthetic essential oils such as limonene or menthol. The amount of oil phase is preferably in the range of 16% to 17% by weight relative to the total weight of the nanoemulsion. The surfactant is preferably selected from nonionic surfactants such as span 80, Capryol 90, tween 20 or tween 80. The amount of surfactant used is preferably in the range of 39% to 42% by weight. The content of ethanol and water in the nanoemulsion is preferably in the range of 18 to 19% by weight and 19 to 21% by weight, respectively. Additionally, penetration enhancers that may be used in the nanoemulsion include and are preferably selected from the group formed by: (1) terpenes, such as carvone, geraniol or menthol; (2) phospholipids , such as phosphatidylcholine; (3) urea. The amount of penetration enhancer used is preferably in the range of 0.5% to 4% by weight. The active ingredients to be loaded in the nanoemulsion system can be water-soluble growth factors such as epidermal growth factor, transforming growth factor beta, vascular endothelial growth factor, keratinocyte growth factor, interleukin or insulin-like growth factor 1. The preferred percentage range of the active ingredient is 0.0067-0.1333%. The percentage ranges and processing parameters for each component in the nanoemulsion are shown in Table 5.

Table 5. Percentage ranges of components of EGF-nanoemulsions

An example of the composition and ingredients used to prepare EGF nanoemulsions is shown in Table 6.

Table 6. An example of components in EGF-nanoemulsions

C.2 Preparation of nano-sized EGF preparations and control samples

EGF-formulations were prepared by mixing EGF-nanoemulsions with a cream base in specimen containers. EGF dissolved in water was then mixed with a cream base in another specimen container as a control. Both mixtures were swirled for 2 minutes to obtain a homogeneous cream. The volume ratio of nano-sized EGF or non-nano-sized EGF to cream base is 3:7.

C.3 In vitro skin penetration studies

The procedure is the same as described in 'In Vitro Skin Penetration Studies' of Sch B. The amount of EGF in skin and receptor solutions was determined by ELISA.

C.4 The influence of the terpene concentration in the nano-sized EGF preparation on skin penetration

The components of the EGF-nanoemulsions in the following studies are listed in Table 6. Since the stratum corneum provides the greatest barrier against transdermal absorption, chemical accelerators such as terpenes are added to achieve better skin penetration. In this study, the promotion effect of different geraniol concentrations (0.5%, 1%, 2% and 4%) on the in vitro percutaneous absorption of nanosized EGF preparations was investigated. Figure 21 shows the effect of geraniol concentration in the nanosized EGF formulation on skin penetration enhancement. When 0.5% geraniol was used, skin penetration increased by 20% compared to the control group. When the geraniol concentration was further increased to 1%, a 113% increase in skin penetration was achieved. However, increasing the concentration of geraniol above 1% resulted in a significant decrease in skin penetration enhancement. Therefore, it was suggested that 1% geraniol should be used as a chemical accelerator in the formulation.

C.5 Effect of incubation time on skin penetration in in vitro studies

In the in vitro study of the transdermal absorption of nanosized EGF formulations, 5 different incubation time points (1.5 hours, 3 hours, 6 hours, 9 hours and 24 hours) were studied. Figure 22 shows the effect of different incubation time points on skin penetration enhancement. All time points showed no skin penetration enhancement except for the 6 hour incubation time which showed a 113% increase in skin penetration compared to the control.

C.6 The influence of the EGF concentration in the nanometerization EGF preparation emulsifiable cream on skin penetration

The in vitro transdermal absorption of three different EGF concentrations (0.002%, 0.02%, 0.04%) in the nano-sized EGF preparation cream was tested. These creams were prepared from nanoemulsions containing 0.0067%, 0.067% and 0.67% EGF, respectively. Figure 23 shows the effect of the EGF concentration in the nano-sized EGF formulation on the promotion of skin penetration. EGF concentrations lower or higher than 0.02% did not show skin penetration enhancement. Therefore, a concentration of 0.02% EGF should be chosen in the formulation.

The skin penetration of C.7 nano-sized EGF preparation cream compared with the control group

This is a skin penetration study of the optimized EGF nanoemulsions set forth in Table 6. Figure 24 shows the percentage of nano EGF preparation cream and control group that penetrated into the skin. Both the tested nanosized EGF formulation cream and the control contained 0.02% EGF. Nanosized EGF formulations were prepared from 0.067% EGF nanoemulsions. When tested for 6 hours, the skin penetration of the nanosized EGF formulation was 1.2 times higher than that of the control group. The results indicate that transdermal administration of this formulation can enhance the skin penetration of EGF. The average particle size of the tested EGF-nanoemulsions in this formulation was less than 15 nm.

The foregoing description of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention in accordance with the various embodiments and with various modifications as are suited to the particular use contemplated. invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Claims (19)

1., for the preparation of a chemicals for nanoemulsions, described chemicals comprises:

Oil;

Surfactant;

Ethanol;

Water; With

Active component;

The weight ratio of wherein said water and described ethanol+described oil+described surfactant is in the scope of 3:7 to 2:8.

2. chemicals according to claim 1, wherein said active component is schizandrin, and described chemicals comprises the described schizandrin of the described oil of 15-16wt%, the described surfactant of 37-39wt%, the described ethanol of 17-18wt%, the described water of 26-27wt% and 0.15-3.6wt%.

3. chemicals according to claim 2, wherein said oil is isopropyl myristate, and described surfactant is Tween 80, and described schizandrin is wuweizisu B.

4. chemicals according to claim 3, wherein said chemicals comprises the described wuweizisu B of the described isopropyl myristate of 15.8wt%, the described Tween 80 of 38.4wt%, the described ethanol of 17.7wt%, the described water of 26.6wt% and 1.5wt%.

5. prepare a method for the nanoemulsions of the chemicals containing claim 1, described method comprises:

Mix described oil, described surfactant, described ethanol and described active component to form the first mixture;

Described water is dropwise joined in described first mixture to form the second mixture; With

Stir described second mixture or described second mixture that homogenizes to form described nanoemulsions.

6. method according to claim 5, the step wherein stirring described second mixture is included in 1000-2000rpm speed lower magnetic force and stirs 2-20 minute, or the step of described second mixture that homogenizes be included in the speed that homogenizes of 5000-20000rpm under to homogenize 2-20 minute.

7. chemicals according to claim 1, it comprises penetration enhancer in addition.

8. chemicals according to claim 7, wherein said active component is epidermal growth factor, and described chemicals comprises the described penetration enhancer of the described oil of 16-17wt%, the described surfactant of 39-42wt%, the described ethanol of 18-19wt%, the described water of 19-21wt%, the described epidermal growth factor of 0.0067-0.1333wt% and 0.5-4wt%.

9. chemicals according to claim 7, wherein said active component is epidermal growth factor, and described oil is isopropyl myristate, and described surfactant is Tween 80, and described penetration enhancer is terpenes.

10. chemicals according to claim 9, wherein said chemicals comprises the described terpenes of the described isopropyl myristate of 17wt%, the described Tween 80 of 42wt%, the described ethanol of 19wt%, the described water of 21wt%, the described epidermal growth factor of 0.067wt% and 1wt%.

11. 1 kinds of methods preparing the nanoemulsions of the chemicals containing claim 7, described method comprises:

Mix described oil, described surfactant, described ethanol and described penetration enhancer to form the first mixture;

Mix described active component and described water to form the second mixture;

Described second mixture is dropwise joined in described first mixture to form the 3rd mixture;

Stir described 3rd mixture to form described nanoemulsions.

12. methods according to claim 11, the step wherein stirring described second mixture is included in 1000-2000rpm speed lower magnetic force and stirs 1-20 minute.

13. 1 kinds of chemicals for the preparation of nanoemulsions, described chemicals comprises:

Oil;

Surfactant;

Penetration enhancer;

Water; With

Active component;

The weight ratio of wherein said oil and described surfactant is in the scope of 1:9 to 2:8.

14. chemicals according to claim 13, wherein said active component is Matrixyl-3, and described chemicals comprises the described Matrixyl-3 of the described oil of 1-3wt%, the described surfactant of 9-27wt%, the described water of 70-90wt%, the described penetration enhancer of 0.5-4wt% and 0.05-6.7wt%.

15. chemicals according to claim 13, wherein said oil is Capryol 90, and described surfactant is Tween 20, and described penetration enhancer is phospholipid, and described active component is Matrixyl-3.

16. chemicals according to claim 15, wherein said chemicals comprises the described phospholipid of the described Capryol 90 of 1wt%, the described Tween 20 of 9wt%, the water of 89wt%, the described Matrixyl-3 of 0.67wt% and 0.5wt%.

17. 1 kinds of methods preparing the nanoemulsions of the chemicals containing claim 13, described method comprises:

Mix described active component, described oil, described surfactant and described penetration enhancer to form the first mixture;

Described water is dropwise joined in described first mixture to form the second mixture; With

Stir described second mixture or described second mixture that homogenizes to form described nanoemulsions.

18. methods according to claim 17, the step wherein stirring described second mixture is included in 500-1500rpm speed lower magnetic force and stirs 1-10 minute, or the step of described second mixture that homogenizes is included in 8000-20000rpm and homogenizes the 1-10 minute that to homogenize under speed.

19. 1 kinds of chemicals for the preparation of nanoemulsions, described chemicals comprises:

The isopropyl myristate of 15.8wt%;

The Tween 80 of 38.4wt%;

The ethanol of 17.7wt%;

The water of 26.6wt%; With

The wuweizisu B of 1.5wt%;

The weight ratio of wherein said water and described ethanol+described oil+described surfactant is 27:73.