Patent Application
20230077199
This application claims priority to Taiwan Patent Application No. 110131131, filed on Aug. 23, 2021. The entire content of the application is hereby incorporated by reference.
The present invention relates to a nanocomposite material and the use thereof. More specifically, the present invention relates to a nanocomposite material comprising a palmitoyl tetrapeptide-20 conjugated nanosilver particle, which exhibits excellent free radical scavenging ability and cutaneous penetration ability, as well as the use thereof in promoting the synthesis of natural hair pigment.
According to analysis of Statista, the global hair care market size reached USD 92.52 billion in 2020 and is growing gradually, which is expected to reach USD 105.14 billion by 2025. It shows that there is a great need for hair care, such as problems regarding excessive sebum secretion, dandruff, dry and broken hair, hair graying and susceptible to hair loss, etc. Hair or scalp damages may be caused by numerous factors. For example, external factors such as ultraviolet lights, air pollution, excessive combing, hair dyeing or perming, diet, or external injuries and so on, or internal factors such as genetics, etc.
Hair follicle is the basic unit for producing hairs. The hair bulge is located in the upper half part of the hair follicle and in the outer root sheath, and in which contains many stem cells including melanocyte stem cells. Hair bulb, lying at the bottom of the hair root, is a wide spherical structure. The bottom of the hair bulb concaves inward, forming a pear-shape tissue called dermal papilla. Dermal papilla is made of connective tissue and contains blood capillaries, responsible for uptake required nutrients and oxygen. A group of stem cells exists around the dermal papilla called hair matrix, which is formed by epithelial cells and melanocytes. Melanocytes can produce melanin and then the melanin is transferred to the surrounding keratinocytes.
Generally, hair graying may be caused by the following factors: (1) oxidative stress (such as ultraviolet lights, chemicals, etc.) leads to depletion or dysfunction of melanocytes that surrounds the dermal papilla, resulting in reduced melanin production or failing to transfer melanosomes, etc.; (2) genotoxic stress (such as ultraviolet lights, IR, etc.) causes defective melanocyte stem cells in the hair bulge; (3) oxidative or genotoxic stress associated with active hair growth. Research has shown that the diameter of white hair is thicker than black hair as well as the growth rate of white hair is 3 times faster than black hair; (4) lack of growth factors for promoting the formation of black hair, such as α-melanocyte stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), β-endorphin, stem cell factor (SCF), hepatocyte growth factor (HGF), and nerve growth factor (NGF), etc. may cause hair graying.
Hair graying is one of the noticeable signs of aging, and many people are eager to recover their original hair color. However, the solution for covering gray hair provided on the current market is merely dyeing the hair artificially, which has many disadvantages such as poor sustainability, long operating time, repeated re-dyeing, unclear color, and scalp irritation caused by chemicals, etc.
In view of these technical problems, the objective of the present invention is to provide a nanocomposite material that is safe and can promote the production of natural hair pigment, thereby restoring the original hair color.
The objective of the present invention is to provide a nanocomposite material, which is formed by conjugation of palmitoyl tetrapeptide-20 with nanosilver particle, thereby improving the problems of palmitoyl tetrapeptide-20 being difficult to be absorb by the skin. The nanocomposite material of the present invention is expected to achieve good cutaneous penetration ability, stimulate production of natural hair pigment, and reduce free radical damages. The present invention provides a viable solution for premature or mid-aged hair graying, and thus restores original hair color.
In order to achieve the abovementioned objective, the present invention provides the following technical means.
In an aspect, the present invention provides a nanocomposite material, comprising a palmitoyl tetrapeptide-20 conjugated nanosilver particle formed by conjugation of palmitoyl tetrapeptide-20 and nanosilver particle.
In some embodiments, the palmitoyl tetrapeptide-20 conjugated nanosilver particle is analyzed in the form of nanosilver aqueous solution by an ultraviolet/visible spectrophotometer; the absorption spectrum may have a specific absorption spectrum peak within 390 nm to 430 nm, preferably 395 nm to 425 nm, more preferably 400 nm to 420 nm, even more preferably within 405 nm to 415 nm.
In some embodiments, the particle size of the palmitoyl tetrapeptide-20 conjugated nanosilver particle may be within 10 nm to 200 nm, preferably within 10 nm to 150 nm, more preferably within 10 nm to 100 nm, more preferably within 10 nm to 90 nm, even more preferably within 10 nm to 80 nm. In certain embodiments, the mean particle size of the palmitoyl tetrapeptide-20 conjugated nanosilver particle may be within 80 nm to 110 nm, preferably within 85 nm to 105 nm, more preferably within 90 nm to 100 nm.
In some embodiments, the Zeta potential of the palmitoyl tetrapeptide-20 conjugated nanosilver particle may be greater than +15 mV, preferably greater than +20 mV, more preferably greater than +30 mV, even more preferably between +30 mV and +38 mV.
In some embodiments, the palmitoyl tetrapeptide-20 conjugated nanosilver particle has the effects of improving cutaneous penetration ability, reducing free radical damages, and promoting the production of natural hair pigments. In some embodiments, the palmitoyl tetrapeptide-20 conjugated nanosilver particle significantly increases the melanin content in newly born hair. In some embodiments, the palmitoyl tetrapeptide-20 conjugated nanosilver particle increases the melanin synthesis in melanocytes in hair follicle. In some embodiments, the palmitoyl tetrapeptide-20 conjugated nanosilver particle promotes the activity of tyrosinase. In some embodiments, the palmitoyl tetrapeptide-20 conjugated nanosilver particle promotes the expression of melanogenic proteins. In some embodiments, the melanogenic protein is selected from TRP-1, TRP-2, or the combination thereof.
In some embodiments, compared to palmitoyl tetrapeptide-20, the palmitoyl tetrapeptide-20 conjugated nanosilver particle enhances percutaneous absorption efficiency preferably by more than 20 times, more preferably by 20 times to 80 times.
In another aspect, the present invention provides a hair care composition, comprising any nanocomposite material described herein, and the hair care composition ameliorates the gray hair in a subject in need through allowing the newly born hair to recover to its original color.
In another aspect, the present invention provides a method of ameliorating gray hair in a subject in need, comprising the step of contacting the scalp of the subject with any nanocomposite material described herein, thereby increasing the melanin content in newly born hair.
In another aspect, the present invention provides a use of the nanocomposite material as described herein for maintaining natural hair color or ameliorating gray hair appearance in a subject in need.
The nanocomposite material comprising palmitoyl tetrapeptide-20 conjugated nanosilver particle described herein has good stability, which can be stored for long term and does not easily deteriorate, and has no cytotoxicity. Applying the nanocomposite material described herein exhibits the effects of excellent cutaneous penetration ability, reduced free radical damages, and promoting melanin synthesis in hair. Established by human subject research, the nanocomposite material described herein prevents the growth of graying hair and restores the subject's hair to their original hair color, and thus provides safe and effective hair care products. Further, the palmitoyl tetrapeptide-20 conjugated nanosilver particle of the present invention improves the poor efficiency of skin absorption of palmitoyl tetrapeptide-20, which increases the percutaneous absorption efficiency to more than 20 times. The penetration of the nanocomposite material described herein can reach to the hair bulb, located at the bottom of hair follicle, and thus can more effectively stimulates the melanocytes to produce melanin, thereby improving the pigment concentration of newly hair born from the hair follicle.
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
The present invention will be further exemplified by the following examples, which are not to be seen as limiting. The embodiments and description are used for illustrating the details and effect of the present invention.
Method of preparing palmitoyl tetrapeptide-20 conjugated nanosilver particle
The tetrapeptide-20 of the palmitoyl tetrapeptide-20 used herein is a peptide sequence derived from α-melanocyte stimulating hormone (α-MSH), enabling the selected sequence to interact with melanocortin 1 receptor (MC1R). The tetrapeptide-20 is a synthetic peptide consisting of arginine, histidine, phenylalanine, and tryptophan.
In a 100 mL beaker, 20 mL of AgNO3 solution (2 mM in water) is mixed with 2 mL of sodium citrate solution (2.5 mM in water) and 23.2 mL of palmitoyl tetrapeptide-20 solution (in concentration of 300 ppm). Subsequently, 1.2 mL freshly prepared NaBH4 solution (10 mM in ice-cold water) is added to the AgNO3 solution. The color turns yellow upon addition of NaBH4, indicating the formation of palmitoyl tetrapeptide-20 conjugated nanosilver particle. The appearance of the sample is shown in
Analysis of Particles Size Distribution and Zeta Potential
Take 0.1 μL of palmitoyl tetrapeptide-20 conjugated nanosilver particle, add 1980 μL deionized water filtered once with 0.22 μm pore-sized filter, and use Vortex to evenly disperse the palmitoyl tetrapeptide-20 conjugated nanosilver particle. After putting the analysis solution into the sample bottle, use Malvern ZEN 3600 Zetasizer to analyze the particle size distribution and Zeta potential. The analysis is repeated for three independent experiments.
The data of particle size distribution is shown in
After the particle is in equilibrium, it is found that the greater the electric potential difference between dispersant and dispersed particles, the easier it is to maintain a stable dispersion state. When the absolute value of electric potential difference is greater than 30 mV, it is in a stable dispersion state; when the absolute value of electric potential difference is less than 30 mV, the system is unstable and susceptible to aggregate; when the electric potential difference is 0, it represents isoelectric point. As shown in
Observation of Transmission Electron Microscope (TEM) View
As shown in
Determination of Wavelength and Absorption Spectrum
The wavelength and absorption spectrum of the palmitoyl tetrapeptide-20 conjugated nanosilver particle in this invention is analyzed by an ultraviolet-visible spectrophotometer to determine whether the wavelength corresponding to the maximum absorption of the palmitoyl tetrapeptide-20 conjugated nanosilver particle is within the normal range of 395 nm to 415 nm. Take 0.1 mL of palmitoyl tetrapeptide-20 conjugated nanosilver particle, put it into a 96-well plate, and scan the absorption spectrum at a wavelength within 300 nm to 600 nm with Epoch™ 2 Microplate Spectrophotometer. The analysis is repeated for three independent experiments.
As shown in
Long-Term Assessment of Stability
Two batches of palmitoyl tetrapeptide-20 conjugated nanosilver particle are produced at different time points. After being stored for 6 months and 8 months respectively, their respective absorption spectrum is analyzed by an ultraviolet-visible spectrophotometer. As shown in
The following examples are provided to better illustrate the nanocomposite material of the present invention, and the advantageous effects thereof through using the nanocomposite material of the present invention. It should be noted that these examples are not to be interpreted as limiting the scope of the claimed invention.
The concentrations of the palmitoyl tetrapeptide-20 conjugated nanosilver particle used in the following cellular assay are 0.03%, 0.1%, 0.3%, 1% and 3%, wherein the peptide concentration ranges from 10−8 M to 10−6 M.
Cell Culture
Human Hair Follicle Dermal Papilla Cells (HFDPC)
HFDPC are cultured in a complete cell culture medium in a 10-cm dish, which is pre-coated with 3 mL collagen coating solution, and then maintain the HFDPC cells in a cell culture incubator at 37° C. with 5% CO2 atmosphere.
When the cells have reach 80% to 90% confluency, remove the used culture medium from the dish and wash the cells with HBSS. Add 5 mL of Trypsin-EDTA solution to cover the dish evenly, immediately remove 4.5 mL of Trypsin-EDTA solution from the dish, and incubated at room temperature for 3 minutes. Subsequently, add 5 mL trypsin neutralizing solution, and the cells will then detach from the dish. Collect the cells to a centrifuge tube for centrifugation. Then, remove the supernatant, add appropriate amount of culture medium, mix evenly, and seed the cells to a new culture dish for maintaining.
Murine Melanoma Cells (B16F10)
B16F10 cells are cultured in DMEM medium (containing 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin-G, and 100 μg/mL streptomycin sulfate) and maintained in a cell culture incubator at 37° C. with 5% CO2 atmosphere.
The cells are passaged at 80% to 90% confluency. Remove used cell medium in the culture dish and wash the cells with PBS twice. The cells are treated with Trypsin-EDTA solution at 37° C. for 3 minutes, and then the cells will detach from the culture dish. Collect the cells to a centrifuge tube for centrifugation. Then, remove the supernatant, add appropriate amount of culture medium, mix evenly, and seed the cells to a new culture dish for maintaining.
HFDPC Cell Line
HFDPC are seeded in 96-well plates at the density of 2×104 cells/well with 100 μL of cell culture medium per well and incubated for 24 hours for cell attachment. After removing the used medium, the cells are treated with 200 μL of fresh culture medium (control group) or culture medium containing various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle, and incubated for 24 hours. At the end of treatment, cells are incubated with 5 mg/mL MTT solution at 37° C. for 1.5 hours. Subsequently, the solution is removed and replaced with 100 μL of DMSO to dissolve the purple formazan crystals at the bottom of the wells. Absorbance is determined at 570 nm (A570 nm) using a microplate reader to calculate cell viability. The equation for calculating cell viability is described as following: Cell viability (%)=(A570 nm treatment group/A570 nm control group)×100%.
B16F10 Cell Line
B16F10 are seeded in 96-well plates at the density of 3×104 cells/well and treated with various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle for 24 hours. At the end of treatment, cells are incubated with 5 mg/mL MTT solution at 37° C. for 40 minutes. Subsequently, the solution is removed and replaced with 100 μL of DMSO to fully dissolve the crystals at the bottom of the wells. Absorbance is determined at 570 nm using a microplate reader. Cell viability is calculated through the equation described above.
The experimental results are shown in
B16F10 are seeded in 24-well plates at the density of 8×104 cells/well and incubated for 24 hours at 37° C. with 5% CO2 atmosphere. After removing the used cell medium, the cells are treated with fresh cell culture medium (control group), 200 nM α-MSH (α-MSH group), combination of 200 nM α-MSH and various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle (α-MSH+sample group), and various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle (sample group), for 24 hours. After treatment, the treated solution in the well is removed with further incubation with fresh cell culture medium for 24 hours.
The cells are washed twice with PBS. The cells are transferred into a 1.5 mL microtube and centrifuged at 10,000 rpm for 5 minutes to obtain cell pellets. Add 100 μL of solution (1 N NaOH containing 10% DMSO) to the pellet for 60 minutes at 80° C. to dissolve intracellular melanin. Extract 80 μL of dissolved melanin and place in 96-well plates, and melanin contents of control group and treatment group are determined by measuring the absorbance at 405 nm. The assay is repeated for three independent experiments. The equation for calculating melanin content is described as following: Total melanin content (%)=(A405 nm treatment group/A405 nm control group)×100%.
This assay compares the effects of co-treatment with α-MSH and palmitoyl tetrapeptide-20 conjugated nanosilver particle, and single treatment with α-MSH or palmitoyl tetrapeptide-20 conjugated nanosilver particle, on the melanogenesis in B16F10 cells. As shown in
Tyrosinase is a critical enzyme in melanogenic pathway, which catalyzes the first two steps of melanin synthesis, respectively: (1) catalyzing the hydroxylation of L-tyrosine to form L-DOPA; (2) subsequently catalyzing the oxidation of L-DOPA to form L-DOPAquinone. L-DOPAquinone is then converted to DOPAchrome through auto-oxidation, and then tyrosinase related proteins (TRP-1, TRP-2) catalyze a series of following reactions to form melanin.
Intracellular tyrosinase activity in B16F10 cells is examined by the following assay. B16F10 are seeded in 24-well plates at the density of 8×104 cells/well and incubated for 24 hours at 37° C. with 5% CO2 atmosphere. After removing the used cell medium, the cells are treated with fresh cell culture medium (control group), 200 nM α-MSH (α-MSH group), combination of 200 nM α-MSH and various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle (α-MSH+sample group), or various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle (sample group), for 24 hours. After treatment, the treated solution in the well is removed with additional incubation with fresh cell culture medium for 24 hours.
The cells are washed twice with cold PBS. The cells are transferred into a 1.5 mL microtube and centrifuged at 10,000 rpm for 5 minutes. The cell pellets are then lysed with 100 μL of PBS containing 1% Triton X-100 (pH 6.8) at −80° C. for 30 minutes, followed by thawing at room temperature for 30 minutes. After repeating freeze-thaw cycles 3 times, the lysate is centrifuged at 12,000 rpm for 10 minutes. 90 μL of supernatant and 20 μL of reaction substrate of 10 mM L-DOPA are added in the well of a 96-well plate. After the wells are incubated at 37° C. for 30 minutes in the dark, absorbance is measured at 475 nm by a microplate reader to determine the production of dopachrome. The experimental data is expressed as a percentage compared to the control group. This assay is repeated for three independent experiments. The equation for calculating cellular tyrosinase activity is described as following: Intracellular tyrosinase activity (%)=(A475 nm treatment group/A475 nm control group)×100%.
As the results shown in
In addition to the involvement of tyrosinase, TRP-1 and TRP-2 mentioned above in melanogenesis, microphthalmia-associated transcription factor (MITF) also plays a key role in melanin synthesis, as well as the occurrence and transport of melanosomes. MITF regulates the expression of enzymes related to melanogenesis such as tyrosinase, TRP-1 and TRP-2.
Binding of α-melanocyte stimulating hormone (α-MSH) to melanocortin 1 receptor (MC1R) on the cell membrane activates adenylate cyclase to convert ATP into cAMP, leading to an increase of cAMP in a cell serving as second messenger to activate protein kinase A. Subsequently, protein kinase A translocates to the nucleus where it phosphorylates CREB, leading to increase the expression of MITF. MITF binds to the promoter of genes responsible for pigment synthesis (tyrosinase, TRP-1 and TRP-2), which upregulates the expression of such genes, ultimately promoting melanin synthesis.
Therefore, the expression level of such melanogenic proteins will be examined by the following assay. B16F10 are seeded in 6-cm dishes at the density of 2×105 cells and treated with cell culture medium (control group), 200 nM α-MSH (α-MSH group), combination of 200 nM α-MSH and various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle (α-MSH+sample group), or various concentrations of palmitoyl tetrapeptide-20 conjugated nanosilver particle (sample group). After treatment for 24 hours, the proteins in the cells are extracted by RIPA lysis buffer and then quantified. The expression of melanogenic proteins in B16F10 is analyzed by western blotting using TRP-1 and TRP-2 specific antibodies with the GAPDH as internal control.
As shown in
This result shows that palmitoyl tetrapeptide-20 conjugated nanosilver particle of the present invention significantly improves the expression level of melanogenic proteins (TRP-1, TRP-2). In addition, it shows synergistic effects for α-melanocyte stimulating hormone when administrating palmitoyl tetrapeptide-20 conjugated nanosilver particle, which can further stimulate the melanogenesis.
Preparation of Hair Care Composition
Formulation of hair care composition is shown in Table 1. Each ingredient in Phase A is evenly mixed with each other followed by stirring for 5 to 10 minutes to complete hydration. Subsequently, ingredients in Phase B are added in order and mixed evenly. pH value is adjusted to 5.5 with citric acid. The product is then stored in a bottle and placed in dark at low temperature.
Efficacy Evaluation
The subjects, 20 in total, aged 20 to 65 containing males and females with hair graying issues are recruited. No hair dyeing is allowed within three weeks prior to testing. The hair care composition containing palmitoyl tetrapeptide-20 conjugated nanosilver particle is applied to the subjects every day after washing hair. The hair care composition is sprayed evenly on top of the scalp (8 times in total, approximately 2 mL) and massage scalp for 3 minutes to absorb. There is no need to wash afterwards. The treatment is 84 days in total, and the differences on the melanin synthesis in hair and hair lightness between before and after using hair care composition are evaluated. Measurement is performed before and after the treatment, the subjects stayed in a room with constant humidity of 50±10% and constant temperature at 20±2° C. for 15 minutes. Then, a Color reader (Konica Minolta's, CR-10) is used to determine the hair on the following locations: top, front hairline, sideburns at right and left sides. Meanwhile, conditions of the subjects' scalps and hairs are filmed and recorded with camera. After the treatment, ANOVA test and paired student's T test are performed to analyze the experiment results. When p-value<0.05, it indicates there is significant difference before and after the treatment.
Calibration Curve of Different Hair Color Levels Over Lightness L Values
As shown in
Changes in Hair Color at Different Locations of Head After Treatment of Hair Care Composition
Left Side of the Head
As shown in
Right Side of the Head
As shown in
Top of the Head
As shown in
Hairline
As shown in
After using hair care composition of the present invention for 12 weeks by 20 subjects, the changes in hair color are shown in Table. 6. This result indicates that palmitoyl tetrapeptide-20 conjugated nanosilver particle of the present invention promotes melanin production in hair. The present invention darkens hair color and reverses the process of hair graying, thereby providing safe and effective hair care products.
Scalp absorption efficiency of palmitoyl tetrapeptide-20 conjugated nanosilver particle of the present invention is evaluated using Franz cells. The skin of pig ear is washed with ddH2O, and the epithelium of the pig ear is obtained using a scalpel and then cut into a penetrating membrane with 1.5×1.5 cm2 in area and 650 μm in thickness, followed by washing with PBS. The penetrating membrane is fixed to the Franz-type diffusion cell with diffusion area of 0.636 cm2. 1.0 mL of tested sample (blank, palmitoyl tetrapeptide-20 conjugated nanosilver particle, palmitoyl tetrapeptide-20, nanosilver particle, unconjugated mixture of palmitoyl tetrapeptide-20 and nanosilver particle) is placed in donor compartment. 5.3 mL of PBS is filled in acceptor compartment. The experiment is conducted in a thermostatic bath at 32±1° C. with a stir bar to balance the concentration of the tested sample in donor compartment.
The result shows that palmitoyl tetrapeptide-20 cannot penetrate the skin, whereas high content of palmitoyl tetrapeptide-20 conjugated nanosilver particle penetrates more internal layers such as epidermis and dermis. Compared to the unconjugated mixture of palmitoyl tetrapeptide-20 and nanosilver particle, the palmitoyl tetrapeptide-20 conjugated nanosilver particle of the present invention increases cutaneous penetration efficiency by 20 to 80 times or more.
Therefore, the palmitoyl tetrapeptide-20 conjugated nanosilver particle of the present invention overcomes the poor skin absorption of palmitoyl tetrapeptide-20 by 20 to 80 times or more. The penetration of the nanocomposite material of the present invention can reach to the hair bulb, located at the bottom of hair follicle, and thus can more effectively stimulates the melanocytes to produce melanin, thereby improving the pigment concentration of new hair born from the hair follicle.
| Number | Date | Country | Kind |
|---|---|---|---|
| 110131131 | Aug 2021 | TW | national |
