J. Biol. Macromol., 6 (2) , 30-35 (2006)
ArticlePrepar ation and proper ties of protein par ticles from human hairToshihiro Fujii,* Kaoru Nakai, Hideto Kikuta, Shunichi Kobayashi,
and Toshihiro Hirai
Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567,
Japan

Received June 8, 2006; Accepted July 25, 2006
Protein microspheres were conveniently prepared from human hair without chemical
modification. The par ticle size was dependent on the mechanical stimuli and the aver age diameter
was approximately 0.5 m when the wet protein films were homogenized by mechanical stimuli usinga Polytron and sonicator. The protein par ticles mainly consisted of -ker atin types I and II.
Confocal microscopic obser vations showed that the par ticles were irregular in shape and eachpar ticle consisted of smaller par ticle aggregates. The aver age size of the protein par ticles slightly
increased when the ionic strength of the solution was high, whereas the disper sion of the par ticles
was har dly affected by divalent cations, pH, and temper ature.
Keywor ds: human hair, protein particle, keratin, individual productIntroduction
Collagen, a representative component of
extracellular matrix proteins, and its denatured
substance, gelatin, appear to be good sources forprotein particles. Chowdhury and Mitra reported
that low- (deoxyuridine) and high- (FITC-dextran)
molecular-weight materials were entrapped in thecollagen and collagen/gelatin microspheres using
glutaraldehyde [1]. The binding of retinol to calfcollagen microspheres caused the effective
transport in mouse skin [2]. Balthasan et al.
showed that gelatin nanoparticles attached to theanti-CD3 antibodies using the avidin-biotin system
exhibited a slightly toxic effect at low
concentrations [3]. Rising concern of bovine
spongiform encephalopathy (BSE) resulted in the
restricted use of a number of proteins including
collagen, gelatin, casein, and albumin that havebeen used in the food, medical, and cosmetics
fields. In order to avoid BSE, proteins andprotein products from less hazardous animals andplants are beginning to be used in these fields.
We believe that some problems will be solved
by using biomaterials gathered from the same
species as the application object, and not from the*Corresponding author (Fax: +81-268-21-5511,
E-mail: fujiit1@shinshu-u.ac.jp)

other origins. From this viewpoint, human hair isa valuable source for the use of biomaterials
containing particles, film, and fibers, because hairis collected in large quantities from specificindividuals and the protein content is high(approximately 80%). In human hair, two majorgroups of proteins are known. One is the hard-keratins forming microfibrous intermediatefilaments and the other is matrix proteins forminga nonfilamentous matrix as intermediatefilaments-associated proteins [4].
The hard-keratins are further resolved into two
subfamilies consisting of at least 4-9 distinct type I
acidic (40-50 kDa) and 4-6 type II neutral/basic(55-65 kDa) members.
Matrix proteins areclassified into high-sulfur proteins (10-20 kDa)
and high-tyrosine proteins (6-9 kDa). Little is
known about the protein microspheres from hairproteins and -keratins. Recently, we reported anovel method of producing protein particles fromhuman hair protein film and used the solution as a
blood analog fluid [5].
In this paper, we compared hair protein
particles from three kinds of films based on thesize, morphology, and protein components. The
biochemical properties of the protein particles
were also examined.

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Human hair protein particle

Mater ials and Methods
Preparation of human hair protein particles
Human hair proteins were extracted fromethanol-treated human hair using ‘Shindai
solution’, which is 20 mM Tris-HCl buffer solution(pH 8.5) containing 2.6 M thiourea, 5 M urea, and
5% 2-mercaptoethanol, at 50°C for 3-4 days [6].
After filtration and centrifugation of the extracted
solution, the obtained ca. 5% (w/v) solution of hairprotein was used to form the films. We prepared
three kinds of protein films using the Pre-cast
method, Post-cast method, and soft Post-cast
method [7, 8]. The hair proteins in the Shindai
solution were mixed with guanidine-HCl at a final
concentration of 5% and then poured onto tissue
cultured dishes containing distilled water (Pre-cast
method). On the other hand, as for Post-cast andsoft Post-cast methods, the protein solution was
directly exposed in tissue cultured dishes
containing 100 mM acetate buffer solution (pH 4)
and 40 mM MgCl2 solution, respectively, andprotein aggregates were formed. After washingwith tap water for 24 h or more and distilled waterfor 6 h, these protein aggregates, that are protein
films, were crushed to pieces by mechanicalprocessing and collected in a bottle. The protein
aggregate solution was homogenized using a
Polytron homogenizer to provide an apparently
uniform protein particle solution. Furthermore,
the protein particle solution was dispersed byultrasonic agitation.
The hair protein particles were recovered aspellets by centrifugation at 15,000 g for 20 min.
The obtained pellets were mixed with the Shindai
solution and incubated for 1 day at 50°C for
perfect dissolution. The protein recovery as theparticles from the protein solution was calculatedon the basis of the protein concentrations. The
protein concentrations were determined according
to Bradford using bovine serum albumin as astandard [9].
The protein components wereanalyzedbysodiumdodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE)[10].
Particle size analysis
The diameter and diameter distribution of theprotein particle suspension were measured using alaser diffraction particle size analyzer (Shimadzu
SALD-200V). The mechanical stimuli were done
at 8 mg/ml in distilled water, and the protein
particle solution was diluted to 0.2 mg/ml indistilled water or 25 mM Tris-HCl (pH 7.6)
containing 75 mM NaCl. The average particle
size was expressed as both the number average
diameter and the volume average one.
Morphological observation
The protein particles after dying with 0.05
mg/ml rhodamine 6G were observed at the protein
concentration of 0.4 mg/ml in the solution stateusing a confocal microscope (Bio-Rad, Radiance2000).
Results and Discussion

Table 1. Average diameter of protein particles
Human hair protein films prepared by the Pre-cast, Post-cast, and soft Post-cast methods were disintegrated
in distilled water using a Polytron homogenizer (intensity 9, for 5 min × 5 times) and a sonicator (intensity
5 and for 10 min). Samples were diluted to 0.2 mg/ml in distilled water, and the particle diameters were
measured by a laser diffraction particle size analyzer. The number average diameters and the volume
average ones indicated in the columns denoted with ‘N’ and ‘V’, respectively.

Conditions

Pre-cast

Average particle diameter ( m)Post-castSoft Post-cast

N

V

N

V

N

V

Polytron

3.4

21

2.3

22

4.6

21

Polytron+sonication

0.6

2.5

0.6

2.8

0.5

1.7

T. Fujii et al.

Par ticle amount (% )Preparation of human hair protein particles
During the course of our continuing research on
the application of human hair and nail proteins, wehave developed a rapid and convenient procedure
for protein extraction that was called the "Shindai
Method" [6]. Using the extracted solution, we
have developed novel preparation procedures forhuman hair protein films and called them thePre-cast, Post-cast, and soft Post-cast methods [7].
More recently, we reported the preparation ofprotein particles with the average diameter of 9 m
from the wet films by soft Post-cast method andused them as a model of red blood cells [5, 8].
SEM observations revealed that the films consistedof particles with a diameter of 0.5-2 m, filaments
and their associated architecture. In addition, thefine structure was dependent on the preparation
method to some extent.
From the wet hair protein films prepared byusing the three methods, we prepared a protein
particle suspension by mechanical stimuli(Polytron treatment). The size distribution ofeach protein suspension was measured using alaser diffraction particle size analyzer and thenumber average diameter and the volume average
one were 2.3-4.6 m and 21-22 m, respectively(Table 1). Further stimulation of the protein
particle suspension by ultrasonic treatment reduced
the average diameters in protein suspension to be
0.5-0.6 m in number average value and 1.7-2.8
m in volume average one. The average sizes ofthe protein particles were slightly different among
the films formed by the three methods. The soft
Post-cast method was the most effective to produce
fine particles after sonication among these methods.
Representative size distribution data obtained fromthe Post-cast film by the double mechanicaltreatments are shown in Fig. 1. The particle sizes
at the peaks were 0.6 m in number average and
1.0 and 5.3 m in volume average.
The original human hair protein solution
mainly consisted of -keratins and matrix proteins.
In the protein particle suspension, the -keratinswere definitely certainly contained, while thecontent of the matrix proteins was low comparedwith that in the hair protein solution (Fig. 2). No
significant protein degradation was found after themechanical stimulation.
The recoveries of net proteins in the protein
solution from the human hair and that in protein

32
22
20
18
16
14
12
10
8
6
4
2
0

0.1

1
Particle diamenter ( m)10

Fig. 1. Particle diameter distribution. Hair
protein particles were prepared from the films
formed by the Post-cast method. After treatmentof the protein suspension at 8 mg/ml by the
Polytron plus sonication, the particle diameter at
0.2 mg/ml was measured using a laser diffraction
particle size analyzer. The plots for number
average diameters and volume average ones are
exhibited with closed and open circles,
respectively.

( kDa ) C
94
67
43

1

2

3
Keratintype Ⅱ
type Ⅰ30
20

Matrixproteins

14

Fig. 2. SDS-PAGE analysis of the protein
particles.
Hair protein particles wereprepared from the films formed by the Pre-cast(lane 1), Post-cast (lane 2), and soft Post-cast(lane 3) methods. After centrifugation at
15,000 g for 20 min, the pellets were incubatedin the Shindai solution for 1 day at 50°C, andthe extracts were analyzed by SDS-PAGE. C,
original hair proteins.

33

Human hair protein particle

Fig. 3. Confocal microscopic observations of human hair protein particles. The protein particles
fabricated from the films prepared by the Pre-cast (a and d), Post-cast (b and e), and soft Post-cast (c and f)
methods were incubated with rhodamine for 30 min at 25°C. After washing, the samples were dispersed by
the Polytron (a-c) and ultrasonic (d-f) treatments and then observed by confocal microscopy.

films from the hair protein solution were 50-60%
and 60-85%, respectively [6-8]. Thus, it ispossible to obtain 300-500 mg of a protein particle
suspension from 1 g of human hair that can usually
be bio-synthesized within 4-7 days on the head.

slightly affected by the preparation methods.
Therefore, in order to reveal the particle-particle
interactions, the following experiments were
undertaken using the protein particles from the
Post-cast film (100 mM acetate buffer, pH 4).

Morphology of the hair protein particles
When the protein particles were prepared fromthe films formed by the Pre-cast, Post-cast, andsoft Post-cast methods, a fine structure in thesolution was observed using a confocalmicroscope(Fig. 3). The protein particles were irregular inshape and had a rough surface with cracks. Someof small particles with the diameter of 0.5-2 m
seemed to be aggregated together. These images
agreed with the scanning electron microscopic
observations (data not shown).
Although thefine structure was different in the hair protein films
prepared by the three methods, the size andmorphology of the protein particles were only

Effects of ionic strength, divalent cations, pH,
and temperature on hair protein particles
The effects of the ionic strength, divalentcations, pH, and temperature on the size andprotein composition of the hair protein particles
were examined (Table 2). After letting theparticle suspension (4 mg/ml) stand under various
conditions for 1-3 days, the samples werevigorously shaken by the hand, and the size andthe protein components were examined. The
average diameters, especially the volume average
diameters, increased with the increasing ionicstrengths during the period, suggesting thehydrophobic interaction promotes the aggregation

T. Fujii et al.

34

Table 2. Effects of various factors on average diameter of hair protein particles
Samples (8 mg/ml) under various conditions were stored at 25°C and diluted to 0.2 mg/ml in the solution
as mentioned above. The particle diameters were measured as shown in Table 1.

0 day

Average particle diameter ( m)
1 day

3 days

N

V

N

V

N

V

Control

0.7

1.8

0.8

2.5

0.8

2.1

100mM NaCl

0.7

1.7

0.8

2.6

0.8

2.3

500mM NaCl
100mM KCl

0.7
0.7

1.8
1.8

1.1
0.8

5.7
2.5

1.2
0.8

4.2
2.3

500mM KCl

0.7

1.9

1.1

4.8

1.1

4.0

2mM EDTA
5mM MgCl2
2mM CaCl2
pH 6.0pH 9.0
4℃
37℃
50℃

0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7

1.8
1.8
1.7
1.9
2.0
2.0
2.0
2.0

0.8
0.8
0.8
0.7
0.8
0.7
0.7
0.8

2.1
2.4
2.2
1.9
2.2
1.9
2.2
2.2

0.8
0.8
0.8
0.8
0.8
0.7
0.7
0.8

2.2
2.1
2.1
2.1
2.7
2.2
2.6
2.7

Conditionsof particles to some extent under high-saltconditions. Whereas, the diameters were notsignificantly changed by the addition of EDTA,
MgCl2, and CaCl2. Furthermore, the average
diameters were hardly affected over wide pH (pH6-9) and temperature ranges (4-50°C). The data
listed in Table 2 suggests that the conditions of lessthan 100 mM salt and nearly neutral pH at 4°C are
suitable for the purpose of longtime storage ofprotein particles. After the suspension was stored
under such conditions for 3 days, no detectable
degradation was observed by SDS-PAGE (data notshown). We have reported that the keratin of theprotein particle was only slightly degraded evenafter storage for 8 months at 4°C [5]. These datasuggested that human hair protein particles were
extremely stable not only at the molecular level,
but also at the microspherical level.
In this report, we presented a procedure for the
preparation of stable protein particles with adiameter of 0.6-4.6 m from human hair and someproperties of the protein particles. The particle

samples with fine size and high recovery as aprotein were obtained from the films prepared bysoft Post-cast method and Post-cast method,respectively. We have succeeded in trapping
alkaline phosphatase with a molecular mass of 95
kDa in the hair protein film using the Post-cast
method [7]. The protein particles were prepared
from
the
film,
indicatingthat
high-molecular-weight components such asalkaline phosphatase will be incorporated into theparticles without chemical modification. The
application of protein particles is being sought inthe cosmetic and medical fields [1-3,11,12].
Human hair protein particles will be promising as
safe and biocompatible materials, because the haircan be individually collected to be self-originatingproducts.
Acknowledgments
This study was supported by Grants-in-aid forthe 21st Century COE Program (10CE2003), the
CLUSTER, and Science Research (B) (16350123)

35

Human hair protein particle

from The Ministry of Education, Culture, Sports,
Science and Technology of Japan and theCosmetology Research Foundation. We are alsoindebted to the Division of Gene Research Center,Research Center for Human and Environmental
Science, Shinshu University, for allowing the useof their facilities.
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Communicated by Hisayuki Morii