Colloids and Surfaces A: Physicochem. Eng.

Aspects 293 (2007) 255–261

Structure behaviors of hemoglobin in PEG 6000/Tween

80/Span 80/H2O niosome system
Tianqing Liu, Rong Guo ∗ , Wei Hua, Jing Qiu
School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, Jinagsu, 25002, PR China
Received 30 March 2006; received in revised form 18 July 2006; accepted 24 July 2006
Available online 12 August 2006

Abstract
The structure behaviors of Hemoglobin (Hb) are in detail studied by the methods of UV–vis and fluorescence spectra, circular dichroism, negative
staining-TEM, FF-TEM and electrochemistry techniques in PEG 6000/Tween 80/Span 80/H2 O niosome system. The obtained results show that
Hb can be adsorbed and outspread on the surface of the niosome membrane. The intensities of the UV–vis and fluorescence spectra of Hb in
Hb/niosome system are greater than those in Hb/H2 O system. The structure behaviors of Hb are partially stabilized and protected in Hb/niosome
system. With the increase of PEG 6000 content in the niosome system, the content of ␣-helix structure decreases but the contents of ␤-sheet and
random increase for Hb. The content of ␤-turn structure is almost independent of PEG 6000 content. The negative zeta potential of Hb and the
conductivity of the system all decrease. The zeta potential of the niosome firstly decreases and then increases.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Hemoglobin; Niosome; Structure behavior; Vesicle

1. Introduction mixture of polymerizable matrix lipid and receptor were formed

by a modified probe sonication method [26]. There are reports
As one form of amphiphilic molecular organized assemblies, about the fabrication and characterization of human serum albu-
vesicle is spherical or ellipsoidal monocellular or multicellular min (HSA) and l-r-dimyristoylphosphatidic acid microcapsules
[1–4]. Vesicles can be formed spontaneously in life body, and based on template technique, and microcapsule assembly of
also prepared from phospholipids, surfactants, mixed cationic HSA at the liquid/liquid interface [27,28]. However, little reports
and anionic surfactant [5–15]. The vesicles formed from phos- are about the interactions of vesicles with proteins and the effects
pholipids and nonionic surfactants are known as lipsome and of vesicles on the protein structure behaviors in life systems. It
noisome, respectively. They are widely used not only as the is very important to study the structure behaviors of protein and
modeling cell membranes, but also as micro-reactors (for exam- the interaction of Hb with niosome in niosome system in order to
ple, providing various special reaction environments, actualizing deeply understand and validate the vesicles functions. It is ben-
and controlling some chemical reactions, the release of drugs, efit to efficiently support the dependability of vesicles used as
genic carriers and biological mineralization) [16–21] and as the biology modeling membranes and to develop the functions
targeted drug carriers to decrease the drug toxicity. So, the of vesicles.
properties and applications of the niosome are one of the most Tween 80 and Span 80 are nonionic surfactants. Tween 80,
noticeable researches in the amphiphilic molecular organized Span 80 and PEG 6000 are provided with low cost and toxicity
assemblies. [29–33]. They can be assembled to form highly steady niosome,
More surfactants can interact with some proteins and then which have hydrotrope-solubilization action on both hydrophilic
affect the protein properties and structures [22–25]. Vesicles of a and hydrophobic drugs [34]. So, the noisome may be applied
in some medicine. When the medicine contained the noisome
interacts and kills virus, the noisome also interacts with the pro-
∗ Corresponding author. Tel.: +86 514 7975590 9517; fax: +86 514 7975244.
teins in human body. In present paper, the structure behaviors of
E-mail addresses: [email protected] (T. Liu), [email protected] hemoglobin (Hb) and the interactions of the niosome with Hb
(R. Guo). are in detail studied by circular dichroism spectrum, negative

0927-7757/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2006.07.053
256 T. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 293 (2007) 255–261

staining-TEM, freeze fracture replication-electron microscopy 2.5. Viscosity measurement

and electrochemistry methods in PEG 6000/Tween 80/Span
80/H2 O niosome system. The obtained results show that Hb can The viscosity measurement of the solution was performed
be adsorbed and outspread on the surface of the niosome mem- on a low-shear rheoanalyzer (Haake Rheostress 600, Thermo
brane. The behaviors of Hb are partially stabilized and protected Electron Co., German) with Haake rheopwin data manager. All
in Hb/niosome system. The results will be helpful to understand samples were equilibrated for 48 h before the measurement.
and apply deeply the structure and functions of biological mem- Each result was the average of six runs.
brane in life system, and the relation of protein properties with
vesicles. 2.6. Negative staining-TEM (NS-TEM) determination

2. Experiment The size and morphology of niosome is also determined by

negative staining-transmission electron microscopy [35]. After
2.1. Materials niosome was negatively stained, the size and morphology of
the niosome were clearly observed by TECNAIR transmission
Tween 80 was bought from Shanghai Biological Engineering electron microscopy (Philip Apparatus Co., USA). The size dis-
Ltd. (99%), Span 80 from Shanghai Commonage Pharmacy Co. tribution and the stability of niosome were also observed and
(99%), PEG 6000 from Shanghai Pudong Goulian Chemical proofread.
Plant. Doubly distilled and deionized water was used for the
preparation of the solutions. 2.7. Dynamic light scattering technique (DLS)
determination
2.2. Niosome preparation
Dynamic light scattering measurements were performed with
a spectrometer (ALV-5000/E/WIN multiple digital correlator)
Tween 80, PEG 6000 and water were vortex mixed at a
and a Spectra-Physics 2017 200-mW Ar laser, and a computer-
given mass ratio. The niosome was prepared by sonicating the
controlled and stepping-motor-driven variable angle detection
mixed solution for 6–25 min (CQX25-06 Sonicator, Shanghai
system. The data acquisition was carried out for 10 min and
Bilieng Sonicator Co. Ltd.). Span 80 was added to the Tween
each experiment was repeated three times. The data were ana-
80/PEG 6000/water niosome. And then the sample was soni-
lyzed by cumulant method using the software provided by the
cated for 5–25 min. The stability of niosome was studied by
manufacturer.
measuring the particle size and the mean size distribution as
a function of time using a computerized micrographs image
2.8. Zeta potentials of protein and niosome determination
analyzing meter (model SK-882, Sanko Co., Japan) (imag-
ing microgram). Its size is about 122 ± 2 nm. The particle
The protein electrophoresis can be taken on under an elec-
size distribution of the niosome varied little after 48 h of the
tric field. Each sample was centrifuged at 15,000 rpm for 15 min
preparation.
for the Hb/niosome system. The supernatant (the niosome) and
the precipitate (the solution with Hb) were separated out. The
2.3. Circular dichroism measurement zeta potentials of Hb and niosome were measured from the
electrophoresis by Js-94F microelectrophoresis meter (Shang-
Circular dichroism (CD) measurement was carried out hai Zhongcheng Instrument Co. China) and high performance
with Jasco-810 Spectropolarimeter (Japan). The scanning rate capillary electrophoresis (Beckman Co. USA), respectively. The
was set at 100 nm/min. The contents of the ␣-helicity, ␤- principle of the zeta potential measurement is similar to the ref-
sheet, ␤-turn and random structures in protein were calcu- erence [36]. The measured results were gotten by the average
lated using the software provided by the manufacturer (Jasco- replicates of five times. The measured difference was less than
810 Analytical Manager System) corresponding to standard ±0.1 mV.
substances.
2.9. Conductivity determination
2.4. Freeze fracture replication-electron microscopy
(FF-TEM) of niosome The system conductivity was determined by DDS-11A con-
ductormeter (Shanghai No. 2 Analytical Instrument Co., China).
The samples for transmission electron microscopy were The calibration of the conductormeter had been made by
prepared by freeze fracture replication according to standard 0.01 mol L−1 KCl solution.
techniques. Fracturing and replication were carried out in a All of above measurements were carried out at 25 ± 0.1 ◦ C.
high vacuum freeze-etching system (Balzers BAF-400D). Repli- All the solutions with protein must be placed at least
cas were examined in a transmission electron microscope 30 min for the interaction equilibrium. Hb concentration is
(model TECNAIR, Philip Apparatus Co.). The polydispersity 5 × 10−6 mol L−1 . The pH of the system is 7.0. The mass ratio
was less than 0.03, which indicates that the distribution is of Tween 80/PEG 6000/Span 80/H2 O = 0.180/0.015/0.100/1.00
homogeneous. in the niosome system.
 T. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 293 (2007) 255–261 257

3. Results and discussion membrane. The peptide chains may be gradually spread in the
conformation.
3.1. Effects of niosome on CD spectrum and secondary
structure parameters of Hb 3.2. Effects of niosome on Hb conformation

PEG 6000/Tween 80/Span 80/H2 O noisome is highly stable Comparing the UV–vis spectrum and the fluorescence spec-
[34]. Its size is about 122 ± 2 nm from dynamic light scatter- trum of Hb in same Hb concentration, the spectra intensities in
ing, FF-TEM and NS-TEM. When Hb is added in the nio- the Hb/niosome system are greater than those in Hb/H2 O system
some, the secondary structure of Hb is changed. The content (Fig. 2). The hydrophilic and hydrophobic interactions and the
of ␣-helix decreases, but the contents of ␤-sheet and random hydrogen bonding of Hb with niosome lead to the absorption of
increase (Fig. 1). The content of ␤-turn does not obviously Hb and the spread of the peptide chains on the surface of the
change. niosome membrane [26]. So, the amino acids in Hb are exposed
When the peptide chains of protein tend to be bended into a gradually. The spectra intensities all increase.
dense global, the “walk” direction of the peptide chains needs The photographs of the niosome (Figs. 3 and 4) in the nio-
to change. Here, the structure pattern is known as ␤-turn. When some/Hb system further approve the existence of the interaction
Hb is added into the noisome, due to the hydrophilic interac- of Hb with the niosome. The size of Hb is about 4–5 nm. From
tion, hydrophobic interaction and hydrogen bonding between the Figures, There are the effects of the niosome on the structure
the niosome and Hb, Hb can be adsorbed on the surface of the behaviors of Hb. Hb can be existed in the niosome membrane,
niosome membrane. This brings the peptide chains of Hb to be that is, Hb is obviously absorbed and located on the membrane
adsorbed and outspread on the surface of the membrane gradu- surface. With the increase of PEG 6000 content, the imaging
ally, which makes the ␣-helix draw and extend into ␤-sheet. But of the protein on the membrane surface is much more dark
the “walk” direction of the peptide chains does not obviously and distinct. This result indicates that the hydrophilic action
change. with long chain of PEG 6000 links and enhances the interaction
With the increase of PEG 6000 content, for Hb, the con- of Hb with the noisome. PEG 6000 makes the peptide chains
tent of ␣-helix structure decrease but the contents of ␤- in Hb be spread gradually and the secondary structure of Hb
sheet and random increase. The content of ␤-turn structure changed.
is almost independent of PEG 6000 content (Fig. 1). PEG The ␣-helix in Hb is unwound. The content of ␣-helix struc-
6000 is provided with strong hydrophilic ability, so that it ture decreases (Fig. 1). The niosomes are also aggregated each
is existed in between water phase of the niosome membrane other through the hydrophilic actions and hydrogen bonding.
and the medium. PEG 6000 can also be inserted in the nio-
some membrane [34]. The protein is also provided with many 3.3. Electric properties of Hb and niosome
hydrophilic groups. This leads the hydrophilic ability and the
viscosity of the membrane to increase (from 1.06 × 10−3 to The isoelectric point of Hb is 6.4 so that the Hb charge dis-
1.13 × 10−3 kg m−1 s−1 ), and does the hydrophilic interaction plays negative in neutral solution. The niosome self is neutral
and hydrogen bonding of Hb with the niosome to enhance. and does not display any charge in the niosome system. In the
The number of Hb on the niosome surface increases simultane- Hb/niosome system, the zeta potential of Hb increases and the
ity, which brings the sheet structure of the protein molecular net negative charge decrease (Fig. 5a). And the niosome is pro-
to be relaxed and the some groups inside the sheet structure vided with the zeta potential and charge. The charges of Hb and
to be exposed. Hb is spread on the surface of the niosome the niosome all are negative. These phenomena and the results

Fig. 1. Effects of PEG 6000 on CD spectrum and secondary structure parameters of Hb in the niosome system. () ␣-helix, (䊉) ␤-sheet, () ␤-turn, () random.
cHb (mol L−1 ): 5 × 10−6 ; wPEG 6000 (%): 0-0, 1-1, 2-2, 3-3, 4-4, 5-5, 6-7, 7-10 (wPEG 6000 = 0 for Hb/H2 O system).
258 T. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 293 (2007) 255–261

Fig. 2. UV–vis spectrum and fluorescence spectrum of Hb. cHb (mol L−1 ): 5 × 10−6 , (1) UV–vis spectrum of Hb, (2) fluorescence spectrum of Hb. (a) Hb/H2 O
system, (b) Hb/niosome system.

in the Section 3.2 all indicate that Hb is in deed adsorbed on the The increase of PEG 6000 content makes the niosome volume
niosome surface or the adsorption of Hb on the surface makes expand.
the niosome be electrified and charged. The moving rate of the niosome goes down in a given elec-
From Fig. 5, with the increase of PEG 6000 content, the neg- tric field. Its zeta potential also does. However, when PEG
ative zeta potential of Hb and the system conductivity decrease. 6000 content is great, PEG 6000 brings the niosome mem-
The zeta potential of the niosome firstly decreases and then brane to be solubilized and disrupted. The stability and the
increases. The changing rate of the zeta potential of Hb is greater volume of the niosome decrease [34]. The size and mov-
than that of the niosome. The reasons may be: (1) the increase ing rate of the niosome also do. Therefore, the zeta potential
of PEG 6000 content causes the system viscosity to increase. increases. (3) The hydrophilic interaction and hydrogen bond-
The interaction of Hb with the niosome goes up through the ing between Hb and the niosome make the peptide chains of Hb
hydrophilic interaction and hydrogen bonding. Therefore, the be partially twisted and spread. The second structure of Hb is
moving rates of the charged particles go down. The conduc- changed.
tivity decreases. The apparent net negative-charge and the zeta When PEG 6000 content is more than 3%, single free Hb is
potential of Hb also do. (2) PEG 6000 is existed in the water difficultly found from the NS-TEM. Hb may be almost interacted
continuous phase and the bilayer of the niosome membrane. with PEG 6000 and adsorbed on the niosome surface. So, the

Fig. 3. Negative staining-TEM photograph of niosome/Hb system. cHb (mol L−1 ): 5 × 10−6 ; wPEG 6000 (%): (1) 0, (2) 1, (3) 2, (4) 4, (5) 5, (6) 7.
 T. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 293 (2007) 255–261 259

Fig. 4. FF-TEM photograph of niosome/Hb system. cHb (mol L−1 ): 5 × 10−6 ; wPEG 6000 (%): (1) 0, (2) 1, (3) 2, (4) 4, (5) 5, (6) 7.

zeta potential of Hb ends when PEG 6000 content is above 3% be adsorbed on the surface of the niosome membrane (Fig. 3) and
in Fig. 5a. the viscosity of the niosome system increases (from 1.13 × 10−3
to 1.21 × 10−3 kg m −1 s−1 ), the contacting area between Hb and
3.4. Stabilities of Hb and niosome in niosome system oxygen decreases. These make Hb be not easily oxidized. The
structure behaviors of Hb are partially stabilized and protected.
PEG 6000/Tween 80/Span 80/H2 O niosome is provided with The stabilizing time of Hb in the Hb/niosome system is longer
highly stability [34]. If Hb is added to the noisome and its than that in Hb/H2 O system.
concentration is reached at 6.00 × 10−6 mol L−1 , the stability From Fig. 6, it also is saw that the stabilizing time of Hb
and the average size of the noisome change from 360 days and increases first and then decreases with the increase of PEG 6000
122 nm to 381 days and 115 nm. There are not obviously effects content. This may be related with the stability of the niosome.
of Hb on the stability and size of the niosome from the exper- The long hydrophilic chains in PEG 6000 are mostly coiled and
iments of negative staining-TEM, FF-TEM, DLS and imaging adsorbed on the niosome and act as a stabilizing agent, some
micrograph. inserts partially in the film phase of the niosome [34]. This brings
Hb is easily oxidized and its oxidized stability is low in the stability of the niosome membrane and Hb to be enhanced.
Hb/H2 O system, about 56 h. However, the oxidized stability Alternately, more hydrophilic PEG 6000 leads hydrophilicity
increases in the Hb/niosome system (Fig. 6) from the obtained of the niosome to increase and thickness and the intensity of
and evaluated results using UV–vis and fluorescence spectra, cir- the membrane to decrease until the structure with organized-
cular dichroism, electrochemistry techniques. Because Hb can assemble is disrupted. When PEG 6000 content is high, its

Fig. 5. Effects of PEG 6000 on zeta potentials of Hb and niosome and system conductivity in Hb/niosome system (wPEG 6000 = 0 corresponds to Hb/H2 O system).
() Zeta potentials of Hb, (䊉) zeta potentials of noisome, () system conductivity.
260 T. Liu et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 293 (2007) 255–261

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