Journal of Cosmetics, Dermatological Sciences and Applications, 2013, 3, 129-134
http://dx.doi.org/10.4236/jcdsa.2013.31020 Published Online March 2013 (http://www.scirp.org/journal/jcdsa)
129
Development of a Novel Penetration-Enhancing Agent
for Hair Products
Teruaki Nagasawa1, Hiroyuki Suzuki1, Masako Koyama1, Toshiko Sato1, Kimio Kawamura1,
Yoko Yamaguchi1,2
1NANOEGG® Research Laboratories, Inc., Kawasaki, Japan; 2Institute of Medical Science, St. Marianna University School of Medi-
cine, Kawasaki, Japan.
Received November 15th, 2012; revised December 20th, 2012; accepted December 30th, 2012
ABSTRACT
HAIRCARECUBETM (HCC) is a new additive for hair products that allows the active ingredient to penetrate into the
hair. During the course of percutaneous absorption studies, we discovered that lyotropic liquid crystal promotes the
skin’s absorptiveness to medicines. We conducted a study to determine whether or not lyotropic liquid crystal could be
applied as a penetration-enhancing agent in hair, and as a result we have succeeded in developing HCC. In place of hair
dye, we used fluorescein-HCC to evaluate hair permeability, and strong fluorescence was observed as deep as the core
of the hair. Moreover, the strength of the fluorescence was dependent on HCC concentration. This result did not change
under acidic or basic conditions. In addition, the same trends were observed when using an oxidative hair dye. Further-
more, when HCC was used together with hydrolyzed keratin, which repairs hair damage, a stronger restorative effect
was observed. These results confirmed that HCC has the effect of promoting the permeation of pigments and other ac-
tive ingredients into hair. HCC is expected to be very useful as an additive for developing functional cosmetic hair
products.
Keywords: Permeation; Hair Care; Hair Dye; Hair Repair; HAIRCARECUBETM (HCC)
1. Introduction
Hair care needs have diversified over the years. In recent
times, treatments to combat the graying and thinning of
hair with age have been of particular interest, and the
availability of hair care products and hair dyes has in-
creased accordingly. In addition, various commercial pro-
ducts have been developed with the help of new tech-
nologies to beautify the hair in numerous ways, such as
polishing, coloring, or perming the hair [1,2]. Thus, the
number of hair care products has been increasing dra-
matically, and consumers are on the lookout for simple
but highly effective products. Efficacy is dependent on
the successful delivery of active ingredients to the de-
sired location in the hair, namely through the cuticle and
into the cortex.
Just as a drug delivery system (DDS) is essential for
drug development in the pharmaceutical industry, DDS
technology is also applicable to the development of func-
tional hair care products. In the pharmaceutical industry,
DDS technology is essential for ensuring the successful
arrival of the drug at the desired location in the body. For
example, liposomes [3], a typical example of a DDS, are
already in clinical use, and polymetric micelles, currently
in the clinical trial stage, are following close behind [4,5].
Recently, our group developed a novel transdermal de-
livery system (TDS) with a special liquid crystal struc-
ture that allows drugs to penetrate the skin through the
intercellular lipids in the stratum corneum [6-10].
In order to meet consumer demand, it is necessary to
introduce this DDS technology to the hair care industry
and enhance the permeability of the hair to desirable ac-
tive ingredients. Up until now, there has been a strong
focus on macroscopic properties, and hair penetration-
enhancing products have been created using organic sol-
vents, oil-based mediums, and surfactants—but there are
concerns that these methods may actually disrupt the
structure of the hair and cause serious damage [11]. By
contrast, we focused on the nanostructure of the hair and
targeted the cell-membrane complex (CMC) [12], a layer
separating the cuticle surrounding the hair from the cor-
tex at the core of the hair. With the results of our TDS re-
search in mind, we succeeded in creating HAIRCARE-
CUBETM (HCC), a novel penetration aid for hair care
products.
In this paper, we introduce the properties of HCC and
investigate the impact of HCC on the permeability of hair

Development of a Novel Penetration-Enhancing Agent for Hair Products 130
to active ingredients such as pigments and damage-repair
agents. In order to directly test the degree of penetration,
we used fluorescein as a model pigment, and we evalu-
ated the impact of HCC on the level of fluorescence in-
side the hair. These experiments were carried out using
damaged hair rather than healthy hair since it is believed
that the damaged state has become more common. In
addition to the damage incurred every day as a result of
UV ray exposure, the friction from hair brushes, and the
heat from hair dryers, the level of damage has skyrock-
eted since the advent of bleaching, dying, and perming
the hair in the 1990s. We investigated whether or not
HCC could improve the penetration of hair repair agents
as well as dyes. As keratin is known to increase the
strength of hair fibers, we conducted experiments using
hydrolyzed low molecular weight keratin, which alone
has little effect on hair fiber strength [13-15]. We deter-
mined the level of penetration indirectly by comparing
the strengths of the hair fibers after different treatments.
2. Materials and Methods
2.1. HAIRCARECUBETM (HCC)
HCC is novel penetration aid with a unique mechanism
that allows active ingredients to permeate the CMC. The
lyotropic liquid crystal structure of HCC is created by
mixing the proper amounts of four ingredients together in
series: glycerin, water, oil, and a surfactant. When dis-
solved in water, HCC dissolves to transparency due to its
specific nano-scale structure; however, when the ingre-
dients that compose HCC are dissolved in water together
at equal concentrations, the mixture is cloudy and pre-
cipitates form [16].
Because pure HCC is a thick, semi-transparent gel re-
sembling Vaseline™, HCC should be diluted before use
in hair products, and it is usually present at only a few
percent. Due to its solubility and stability in water, it is
thought that HCC can be easily blended into various hair
products without threatening the stability of the mer-
chandise.
2.2. Fluorescein Study
In order to effectively assess the capacity of HCC to in-
crease the permeability of the hair to pigment, the easily
visualizable and quantifiable fluorescein was used as a
model pigment. Bleached hair was prepared by immers-
ing commercially available human hair in a 1:1 mixture
of aqueous 10% hydrogen peroxide and 5% ammonia
(Wako Pure Chemical Industries, Ltd., Japan) and pro-
cessing for 30 min at 37˚C. Fluorescein (Sigma-Aldrich®
Co., Ltd., Japan) was dissolved to a final concentration of
0.1% in aqueous solutions with fixed concentrations of
HCC (0%, 0.1%, 0.5%, 1%, 2%, and 5%). For the pur-
poses of comparison, fluorescein was also added to two
control solutions: 1) a simple mixture of the four ingre-
dients composing HCC, each present at a 2% concentra-
tion; and 2) a commercially available product. After
processing again for 30 min at 37˚C, the hair was rinsed
to remove any residual pigment and dried. The dried hair
was embedded in Tissue-Tek O.C.T. Compound (Sakura
Finetek Japan Co., Ltd., Japan), frozen, sectioned at 5 μm,
and mounted on glass slides. The sections were observed
under a fluorescent microscope (Biozero Fluorescence
microscope BZ-8100, KEYENCE, Japan) and analyzed
using the included software (BZ analyzer, KEYENCE,
Japan).
To assess the efficacy of HCC under acidic and alka-
line conditions, fluorescein was dissolved in aqueous so-
lutions of 0.5% HCC with varying pH. Hair was pro-
cessed as above, and these cross-sections were also ob-
served under the fluorescent microscope and analyzed as
above.
2.3. Oxidizing Dye Study
An experiment using an oxidizing dye was conducted in
a similar fashion to that of the experiment described
above using the model pigment fluorescein. Bleached
hair was soaked in solutions containing oxidizing dye
and HCC at various concentrations (0%, 2%, and 5%),
allowed to process for 30 min at 37˚C, rinsed, and dried.
The hair was then embedded in the same O.C.T. com-
pound as above, frozen, sectioned at 15 μm, and mounted
on glass slides. The slides were observed under an opti-
cal microscope (Biozero Fluorescence microscope BZ-
8100, KEYENCE, Japan), and image analysis was per-
formed using image processing software (Image J Win-
dows).
2.4. Keratin Study
The level of keratin penetration was determined indi-
rectly by measuring the strength of hair fibers after vari-
ous treatments. Hair processed with bleach was soaked in
aqueous solutions containing 5% hydrolyzed keratin (mo-
lecular weight of approximately 400 g/mol) and various
concentrations of HCC (0%, 2%, 5%, and 10%). For the
purposes of comparison, two control solutions were also
prepared as before: 1) a simple mixture of the four ingre-
dients composing HCC, each present at a 2% concentra-
tion; and 2) a commercially available product. After al-
lowing the hair to process for 30 min at 37˚C, the hair
was rinsed and dried. These hair samples, along with
samples of untreated healthy hair and bleached hair, were
tested for resistance to breaking under the strain of
weight as an indicator of hair fiber strength and therefore
an indicator of the efficacy of the keratin treatment. The
data was normalized such that a unit of 1 corresponds to

Development of a Novel Penetration-Enhancing Agent for Hair Products 131
the amount of weight required to break a single fiber of
healthy hair.
3. Results and Discussion
3.1. Relative Efficacy of HCC
The fluorescent photomicrographs of hair cross-sections
shown in Figure 1 (top) revealed that HCC increased the
capacity of the fluorescein model pigment to penetrate
the hair fibers. When compared to the 0% HCC group, it
was clear that the 2% HCC group displayed not only
stronger fluorescence around the circumference of the
hair but also greater overall fluorescence. Moreover, the
5% HCC group showed strong fluorescence throughout
the entire cross-section, indicating that the fluorescein
pigment permeated throughout the entire hair fiber.
As noted earlier, a simple mixture of the ingredients
that compose HCC should not have the same effect as
properly formulated HCC. As expected, the mixture of
the four HCC components, each present at a concentra-
tion of 2%, showed little effect when compared to 2%
HCC or 5% HCC. In fact, the pattern of fluorescence in
this group was very similar to that of the commercial
product group. It is suspected that this small effect was
due to the presence of a surfactant among the HCC com-
ponents.
The graph in Figure 1 (bottom) shows a graphical
representation of the fluorescence shown in the photomi-
crographs; the intensity along the diameter of each
cross-section is displayed beginning from the cuticle on
one side, passing through the cortex into the medulla, and
continuing across to the cuticle on the other side. As
compared to the commercial product, remarkably high
intensity fluorescence was observed in the HCC groups
throughout the cuticle and cortex layers, which was con-
sistent with the results shown in the photomicrographs.
Figure 2 shows a numerical representation of the fluo-
rescence intensity of fluorescein in the hair fibers after
various treatments. It was determined that the increases
in fluorescence in all HCC groups (0.1% HCC and higher)
were statistically significant when compared to the sim-
ple aqueous fluorescein treatment group (0% HCC). In
fact, the level of fluorescein permeation in the hair in-
creased in an HCC concentration-dependent manner. Treat-
ments containing 2% or more HCC displayed fluores-
cence significantly greater than that of the simple mix-
ture of the HCC components or the commercial pro-
duct.
Figure 1. Penetration of fluorescein model pigment into hair fibers. Representative photomicrographs (top) of hair cross-
sections visualized under a fluorescent microscope and a plot (bottom) displaying the quantified levels of fluorescence along
the diameters of the circular cross-sections.

Development of a Novel Penetration-Enhancing Agent for Hair Products 132
Figure 2. Relative amount of penetration of fluorescein model pigment into hair fibers with respect to HCC concentration.
Asterisks denote significant differences from the 0% HCC control: *p < 0.01; **p < 0.05.
3.2. pH-Insensitive Stability of HCC
Neither acidity nor alkalinity adversely affected the per-
formance of HCC. The representative photomicrographs
in Figure 3 illustrate that even under acidic (pH 2.5) or
alkaline (pH 10.9) conditions, the fluorescence inside the
hair fibers appeared similar to that of the fibers treated
with HCC under neutral (pH 7.0) conditions, indicating
that the effectiveness of HCC was unchanged by the va-
riation in pH.
This pH-insensitive stability is thought to be due to the
special composition of HCC. Firstly, HCC is composed
of four ingredients, each of which is very stable even un-
der acidic or alkaline conditions: water, oil, glycerin, and
a surfactant. Secondly, HCC exists in the form of a liquid
crystal, which is an especially stable type of structure in
aqueous environments. As the composition of commer-
cial hair products is widely varied, this stability is con-
siderably valuable in the hair care industry.
3.3. Efficacy of HCC with Oxidative Dyes
The degree of penetration of the oxidative dye increased
with the addition of HCC in a concentration-dependent
manner, exhibiting the same trend as was the case with
the fluorescein model pigment. The graph in Figure 4
shows that increasing the concentration of HCC from 0%
to merely 2% yielded a statistically significant increase
in the amount of dye that penetrated the hair fibers. This
study served to confirm that HCC can be added to oxida-
tive dyes to enhance penetration, thereby enhancing the
Figure 3. Photomicrographs showing level of HCC-enhanc-
ed penetration of fluorescein model pigment with respect to
pH of treatment.
effectiveness of the hair dye. This remarkable increase in
the amount and depth of penetration implies that the ad-
dition of HCC to hair dyes would markedly improve co-
lor retention.
3.4. Efficacy of HCC with Hydrolyzed Keratin
The effectiveness of hydrolyzed keratin in repairing
damage and restoring strength to hair was significantly

Development of a Novel Penetration-Enhancing Agent for Hair Products 133
Figure 4. Relative amount of penetration of oxidative dye
into hair fibers with respect to HCC concentration. Aste-
risks denote significant differences from the 0% HCC con-
trol: *p < 0.01.
amplified by the addition of HCC. The graph in Figure 5
shows the relative strengths of the tested hair fibers,
where strength was equated to the amount of weight re-
quired to break a strand of hair. The data was normalized
such that a value of 1 represents the weight required to
break a single strand of healthy hair, and therefore a
value of 1 represents the strength of a healthy strand of
hair. At a concentration of 2% or greater, the addition of
HCC enhanced the reparative effect of hydrolyzed kera-
tin beyond that exhibited by the commercial product. At
a concentration of 5% or greater, the addition of HCC
yielded a significant difference in strength compared to
the hydrolyzed keratin alone (0% HCC) treatment group.
As was the case with fluorescein, the addition of a simple
combination of the ingredients that compose HCC did
not have any effect, and the result was similar to that of
the hydrolyzed keratin alone (0% HCC) treatment group.
This study demonstrated that HCC can be effective in
hair repair products as well as in hair dyes.
4. Conclusions
The demand for hair dyes and hair repair products is on
the rise, and one should not underestimate the signifi-
cance of a simple additive capable of increasing the ef-
fectiveness of the dyes and reparative agents.
We adapted the research we conducted on transdermal
drug delivery systems, and we created HAIRCARE-
CUBETM (HCC). In this study, we established that HCC
possesses the capacity to increase the penetration of pig-
ments and other active ingredients into the core of the
hair fibers. Even at low concentrations, HCC is able to
significantly increase the effectiveness of hair products,
and HCC is stable even under acidic or basic conditions.
In addition, the liquid crystal structure of HCC has
nano-scale water and oil components, and therefore it is
expected that HCC would also be an effective additive in
Figure 5. Relative strength of hair as determined by the amount of weight necessary to break a single strand of hair.

Development of a Novel Penetration-Enhancing Agent for Hair Products 134
products that modify the surface of the hair by coating it
with a mixture of oil and water, which improves the feel
to the touch of the hair.
To meet the needs of all the consumers, it is indeed
important to develop various effective active ingredients;
however, it is equally important to ensure that those in-
gredients can be delivered past the cuticle to the core of
the hair. It is expected that demand for effective penetra-
tion-enhancing agents will continue to rise, and that there
will be a wide variety of potential applications for HCC.
5. Acknowledgements
We would like to thank Miss Ariella Coler-Reilly who
wrote the English translation of this paper.
REFERENCES
[1] Japan Hair Color Industry Association (JHCIA), “The Kinds
of Hair Coloring Products,” 2011. http://www.jhcia.org
[2] T. Ikeda, K. Yamada and M. Ono, “Functional Charac-
teristics of the Raw Materials in Hair Care,” Fragrance
Journal, Vol. 35, No. 6, 2007, pp. 55-60.
[3] S. Nojima, J. Sunamoto and K. Inoue, “Liposome,” Nan-
kodo Co., Ltd., Tokyo, 1988.
[4] Y. Yamaguchi, “Treatment Utilizing DDS,” In: H. Kuwano,
Ed., Fundamentals and applications of MEMS/NEMS Engi-
neering, Technosystem Co., Ltd., Tokyo, 2009, pp. 228-233.
[5] University of Tokyo Center for NanoBio Integration, “Na-
noBio Future Seen from Interdisciplinary Research in
Pharmaceutical Science and Technology,” X-Knowledge
Co., Ltd., Tokyo, 2010.
[6] Y. Yamaguchi and R. Igarashi, “DDS to Support Skin Re-
generation and Percutaneous Absorption: Development of
Biomimetic NANOCUBE SYSTEM,” Biotechnology Jour-
nal, Vol. 6, No. 5, 2006, pp. 585-589.
[7] M. Musashi and Y. Yamaguchi, “Development of Cos-
metic Formulations ‘NANOCUBE’ Encourages Skin Regen-
eration by the Self-Healing Ability,” COSMETICSTAGE,
Vol. 1, No. 7, 2007, pp. 56-60.
[8] Y. Yamaguchi, “Development of Topical Base for Pro-
moting Transdermal Delivery. Effective Use of the Inter-
cellular Lipid Present in the Outermost Layer of the
Skin,” Drug Delivery Using Functional DDS Carriers,
CMC Publishing Co., Ltd., Tokyo, 2006, pp. 306-312.
[9] Y. Yamaguchi, “Research Trends of Trandermal Delivery
in the Field of Cosmetics,” Percutaneous Absorption in
Aromatherapy, Aromatopia, Vol. 17, No. 6, 2008, pp. 1-
5.
[10] Y. Yamaguchi, Y. Suzuki, T. Nagasawa, Y. Kubota and
M. Isogai, “Pigmentation and Improve Wrinkles by Multi-
functional Soft Matter. New Concept for Trandermal De-
livery System,” Fragrance Journal, Vol. 37, No. 5, 2009,
pp. 97-100.
[11] M. Hosokawa, “Damage by the Hair Perm, Coloring, Sun,
Heat and Mechanical Force,” In: Hair Scientists Associa-
tion, Ed., Latest Hair Science, Fragrance Journal Ltd.,
Toky o, 2003, pp. 220-259.
[12] A. K. Allen, J. Ellis and D. E. Rivett, “The Presence of
Glycoproteins in the Cell Membrane Complex of a Vari-
ety of Keratin Fibres,” Biochimica et Biophysica Acta,
Vol. 1074, No. 2, 1991, pp. 331-333.
doi:10.1016/0304-4165(91)90172-D
[13] A. Kanaya, R. Maruyama, K. Fujii and K. Kanayama,
“Hair Repair Agent,” Japan Patent No. 267844, 2003.
[14] M. Yoshioka, H. Araya, K. Adachi and S. Uchida, “Hair
Cosmetic,” Japan Patent No. 247692, 2005.
[15] Y. Tuda, “Effectiveness as Hair Care Ingredient Derived
from the Hydrolysis of Waterfowl Feather Keratin,” Fra-
grance Journal, Vol. 38, No. 7, 2010, pp. 74-77.
[16] Y. Yamashita, T. Sato, K. Sakamoto and Y. Yamaguchi
“Shear-Induced Release Model of Solubilized Oil in Di-
luted Liquid Crystal Solution,” Proceedings of Interna-
tional Association of Colloid and Interface Scientists
Conference, Sendai, 13-18 May 2012.