J. Biol. Macromol. 11(1), 15-22 (2011)

Regular Paper
Concentric Nano-sized Vesicle Formation by Immunosuppressant FTY720 Revealedwith Small-angle X-ray Scattering using Synchrotron Radiation Source
Jun’ichi Katakawa1,*, Hideki Minami2, Tetsuro Fujita1, and Yoh Sano1
1

Faculty of Pharmaceutical Sciences, Setsunan University, 45-1, Nagaotoge-cho, Hirakata,
Osaka 573-0101, Japan and 2Hiroshima International University, 5-1-1, Hirokoshingai, Kure,
Hiroshima 737-0112, Japan
Received January 15, 2011; Accepted March 31, 2011
Tertiary structure of nano-sized vesicle in aqueous solution of immunosuppressant
FTY720 was studied. The molecular weight of FTY720 self-assembling in solution wasdetermined by a laser light scattering method and was 20,800. Angular dependency of
the intensities of the scattered light in small-angle X-ray scattering showed a typical
pattern of the concentric spherical vesicle. The determined values through thetheoretical curve fitting were 0.8 nm and 3.6 nm for the inner and our radius of theconcentric vesicle, respectively. This means that the hydrophilic site of FTY720 situates
at inner core and the hydrophobic group of FTY720 forms the dimer by end-to-end
aggregation at the outer core.
Keywords: Tertiary structure in solution; immunosuppressant; FTY720; Small-angle X-ray
scattering; synchrotron radiation source; concentric spherical vesicle shell; Kratky plot
Introduction
Immunosuppressants are clinicallyimportant for organ transplantations and thetreatment of autoimmune diseases. Since
cyclosporin A (CsA) [1] was introduced, thesuccess rate in organ transplantations has
increased remarkably. Immunosuppressantisolated from Streptomyces FK-506 [2] wasfound to be 10-100-fold more potent thanCsA as an immunosuppressant.
*Author to whom correspondence should be
addressed, Phone: +81-72-866-3150,
Fax: +81-72-866-3150,
E-mail: katakawa@pharm.setsunan.ac.jp.

These compounds have very similar
mechanisms of action, inhibiting the
production of interleukin-2, a signal
molecule that induces cytotoxic T cell, buthigher doses of both compounds induce
renal dysfunction and other side effects [3].
Therefore, less toxic drugs for theprevention of graft rejection are needed.
Several immunosuppressants, ISP-I,
were isolated from culture broths of Isaria
sinclairii, which is an imperfect stage ofCordyceps sinclairii and Mycelia sterilia,
respectively [4, 5]. The immunosuppressive
activity of ISP-I was 10-100 times more

16

Tertiary Structure of FTY720 in Solution

potent than that of CsA in terms ofsuppressing both lymphocyte proliferation inmouse allogeneic mixed lymphocyte

fact FTY720 in aqueous solution showed
higher molecular weight by gel filtration
method than monomer whose molecular

reaction in vitro and generation of
allo-reactive cytotoxic T lymphocytes inmice in vivo [4, 5].
FTY720, 2-amino-2-[2-(4-octylphenyl)
ethyl]propane-1,3-diol hydrochloride, wassynthesized with chemical modification
technique as one of ISP-I derivatives.
FTY720 prolonged rat skin allograft
survival more effectively than CsA andshowed activity in other administration

weight was 344 according to mass analysisof this sample. However, the detailed
structure of FTY720 in aqueous solution
remains exclusive.
In the present paper, we investigated thedetailed molecular structure of vesicle of
FTY720 in aqueous solution with Smallangle X-ray Scattering (SAXS) using
Synchrotron Radiation Source.

routes [6]. FTY720 sequesters circulating
mature lymphocytes into peripheral patchesby acceleration of lymphocyte homing andthereby decreases
thenumberoflymphocytes in peripheral blood [7].
FTY720 also had no inhibitory effect onserinepalmitoyltransferaseataconcentration of 1000 nmol/L or less,suggestingthat
FTY720possess
considerable activity and is expected to be

2. Materials and Methods
2. 1. Materials
FTY720 was prepared according to themethod by Fujita et al. [6]. The sample wasconverted to hydrochloride salt by treatment
with ethanol and 1N HCl solution in diethyl
ether and was recrystallized as white solidfrom ethanol and ethyl acetate mixture. The
melting point of purified sample was107-108℃ (decompose). The mass analysis

useful as an immunosuppressive drug fororgan transplantation [6].
FTY720 consists of a hydrophilicgroup(2-amino-2-ethylpropane-1,3-diol)and a hydrophobic group (hydrocarbon
chain and phenyl ring) in a molecule as isshown in Fig. 1.

(EI: Electron Ionization method) gave also
as m/z 344 (M+) and element analysis
showed C19H33NO2･HCl.
2. 2. Determination of Molecular Weight
The apparent weight-average molecularweight of FTY720 in aqueous solution wasmeasured by the intensity of light scatteredat 90°angle and at a wavelength of 436 nmwith the instrument of a modified
ellipsometer, an automatic light scattering
analyzer AEP-100 (Shimadzu Co. Ltd.) [8].
The apparatus constant was obtained byusing a solution of bovine serum albumin
whose molecular weight was taken as
65,800. The sample FTY720 solution andthe solvent of distilled water for light

Fig. 1 Monomeric structure of FTY720
The characteristics of an amphiphile of
FTY720 may induce nano-sized micelle-likevesicle formation in aqueous solutions. In

2

Katakawa, Minami, Fujita, and Sano17

scattering measurements were opticallyclarified through a Gelman (Germany) filter(pore size 0.45 micro meter). The refractive
index increment of FTY720 was measured
as 0.140 ml/g.
2. 3. Small-angle X-ray Scattering (SAXS)SAXS experiments were performed
with the optics and detector system of
SAXES (Small-angle X-ray Scattering
Equipment for Solutions) installed at the 2.5
GeV storage ring in the Photon Factory of
the National Laboratory for High Energy
Physics, Tsukuba [9-12]. The X-ray beam ismonochromatized by using a double-crystal
monochromator and focused with atoroidal-focusing mirror. A wavelength, λ,of 0.149 nm was used. The Small-angle
scattering from Q (Q = 4πsin θ/λ, where2θ is the scattering angle) ＝0.11 nm-1 to
3.8 nm-1 was registered at 512 different
angles by using the one-dimensional
position-sensitive proportional counter with
an effective length of 200 mm (Rigaku
Denshi Co. Ltd). The scattered intensities
were corrected with regard to the variationin the incident X-ray flux by monitoring thebeam with an ionization chamber placed in
front of the temperature controlled specimen
chamber. The counting time was 1800 sec
for each measurement. The net scattering
intensities were calculated by subtracting thescattering intensities of a blank buffer
solution from those of the sample after thecorrection of the effect of the X-ray beamtransmissions.
3. Results
3. 1. Molecular weight of FTY720
aggregate in solution

Fig. 2 Concentration dependence of inverse
scattering intensity for FTY720 in aqueous
solution.
Figure 2 showed the light scattering
pattern of FTY720 in aqueous solution,
where Kc/R90 was plotted against FTY720
concentration c (mg/ml). Here K is the
optical constant and R90 is the reduced
scattering intensity observed at the
scattering angle 90°. Considering no effectof the internal interference factor because ofsmall molecular size of FTY720 thefollowing equation is derived as [13],Kc/R90 = 1/M + 2A2c
(1)
Where M is the molecular weight and A2 thesecond virial coefficient.
As was shown in Fig. 2, the inverse
scattering intensity plot was almost linear.
Therefore, the vesicle size is almost thesame between 3 to 30 mg/mL concentration.
The molecular weight of FTY720 in solution
was obtained from the intercept of thisstraight line and was 20,800 whichsuggested that FTY720 in aqueous solution
was situated in an aggregate state andformed a micelle-like vesicle structure,
because monomeric molecular weight of
FTY720 was 344.

18

Tertiary Structure of FTY720 in Solution

reflects theaggregates.

3. 2. Size and shape of FTY720 aggregatesparticle

size

of

FTY7203. 3. Kratky plot
150

I(Q)*Q*Q

120
90
60
30
0.0

Fig. 3 Angular dependency of the scattering
intensity profile of SAXS in 20 mg/mL
FTY720 solution in distilled water. Open

1.0

2.0

3.0

4.0

-1

Q (nm )

Fig. 4 Kratky plot of FTY720 solution of
SAXS pattern shown in Fig. 1.

circles indicate the experimental values. The
theoretical curve as the two concentric
spherical vesicle shell model is shown with

In synthetic polymer chemistry the
Kratky plot is usually employed to evaluate
the shape and the order of extension of alinear or branched chain polymer. Even ifpolymers have the same radius of gyration
value in solution, Kratky plot reveals thedifference according to their shape and chain
extension, e.g., a spherical shape yields a
sharp peak, a Gaussian chain a horizontal

the solid line taking account of the sphericalfactor, P(Q).
SAXS experiments were performed
with the solution of FTY720 from 15 to 20
mg/ml in distilled water. The scattering
curve showed the well-defined maximum atabout Q=l.5 nm-1, as was shown in Fig. 3.
The shape and size of the aggregates were
determined by comparing the experimental
angular scattering intensity profiles with thecalculated theoretical values for suitablemodels. The theoretical equations of the
shape factor, P(Q), are given as a function of
scattering angle Q for various models [13,14]. As was shown in Fig. 3, the scattering
pattern indicated typical two concentric
vesicle shell molecules. Therefore, FTY720

line and a rigid rod a straight line with aslope one [16]. As was shown in Fig. 4, theKratky plot showed one sharp peak ofscattering curve, indicating that FTY720
aggregate is approximated as vesicle shape.
The peak in Kratky plot also indicatedsymmetrically and therefore the size
distribution of the vesicle formed may be
mono distribution.
4. Discussion
Total scattering intensity I(Q) is
generally proportional to P(Q). For the twoconcentric vesicle shell model, P(Q) is given

aggregatesinsolution
couldbeapproximated as two concentric vesicle
shells model with different scattering length
as in the case of dodecyl sulfate micelles(SDS) or glucose lipid micelles [15]. In thelarger Q region the scattering curve mostly
12
P(Q) =  3
 R 23 F(Q R 2)  R1 3 F(Q R1)3 2
 (R 2  R1 ) 
3  (sin Q R i - Q R i cos Q R i)
F(Q R i) =(QRi)3

4

(2)

(3)

Katakawa, Minami, Fujita, and Sano19

as [14] and Ri is the inner (R1) or outer (R2)
radius, respectively. Each spherical portionof the two concentric vesicle shell model ishydrophobic region is calculated as 193 nm3from the values of the two concentric vesicle
shell model. If FTY720 molecule is assumed

expressed with the normal spherical particle
factor.
The best fitted values of thetheoretical curve shown with solid line in
Fig. 3 as the two concentric spherical vesicle
shell model were 0.8 nm for the inner radius
R1 and 3.6 nm for outer R2. As is shown in
Fig. 3, the theoretical curve is controlledwith a position of the sharp valley and themoderate peak. Therefore, the radii values

as a conic cylinder whose height is 1.7 nm
and the diameters are 0.38 nm as the uppersurface and 7.0 nm as the bottom surface
according to the atomic molecular model
because the bond angle between thehydrophilic and the hydrophobic parts isabout 140 degree, the volume of one
molecule of FTY720 is calculated as 2.9
nm3. Therefore, about 67 molecules of
FTY720are
included
withintheare determined uniquely and the errors ofthe determined values may be appreciably
zero.
According to the atomic molecularmodel calculated with the computer using
the energy minimum method, FTY720
monomer is approximated as two short rods
bent at a phenyl ring as is shown in Fig. 1.
The one part consists of a hydrophilic group(2-amino-propane-1,3-diol and phenyl ring)hydrophobic region whose value isconsistent well with the result of molecularweight shown in Fig. 2.
The tip length of the hydrophilic
region of FTY720 shown in Fig. 1 iscalculated as 0.24 nm according to theatomic molecular model and the radius ofchloride ion is assumed as 0.17 nm. The
inner core of the hydrophilic region of thetwo concentric vesicle shell models is

whose length is 0.89 nm. The other partconsists of a hydrophobic group of thehydrocarbon chain whose length is 1.0 nm.
The stretched end-to-end distance of
FTY720 monomer is 1.8 nm. The region
between the inner and the outer shell has theradius of 2.4 nm as is shown with theschematic model in Fig. 5 whose lengthagrees almost with the twice value of thestretched end-to-end distance of FTY720
monomer. This means that the hydrophiliccompatible well with the experimental value.
This indicated that the tip hydrophilic regionof FTY720 molecule in the core regions
situates as the mixtures of dissociative andundissociative states.
In physiologically, it is recognizedthat the effect of FTY720 was continuous[17,18] as was already shown inIntroduction. Clinically, results of ashort-term trial indicated that FTY720 incombination with steroids and CsA wasparts of FTY720 situates at inner core andthe hydrophobic group of FTY720
molecules forms dimer by end-to-end
aggregation through hydrophobic interaction
and situates at the outer shell core.
On the other hand, the volume ofefficient in preventing acute rejection [17].
As is shown in Fig. 5, FTY720 has aself-assembling property in physiologicalsolution and can form the two concentric
vesicle shell molecule which consists of thelarge external hydrophobic core and the20

Tertiary Structure of FTY720 in Solution

small internal hydrophilic core.
HOH2C

7.2 nm

1.6 nm

HOH2C
H2N
HOH2C

CH2OH
NH2
CH2OH

CH2OH
HOH2C
NH2
CH2OH H2N
HOH2C

HOH2C
H2N
HOH2C

concentric vesicle shell. The best fitted
values of the theoretical curves as the twoconcentric spherical vesicle shell model was

NH2
CH2OH

HOH2C
H2N
HOH2C

0.8 nm for the inner radius and 3.6 nm forouter radius, indicating that the hydrophilic
sites of FTY720 situated at inner core andthe hydrophobic group of FTY720 molecule
forms dimer by end-to-end aggregation
through the hydrophobic interaction andsituated at the outer shell core. These
dimensions were consistent with the values
calculated from the molecular structure of
FTY720. The two concentric vesicle shell

CH2OH
NH2
CH2OH

CH2OH
NH2
CH2OH
HOH2C

CH OH
NH2 2

Fig. 5 Schematic two concentric vesicle
shell model of FTY720 aggregates instructure of FTY720 aggregates may relatewith the continuous physiological effect of
FTY720.

aqueous solution. Closed circles indicatehydrophilic parts (blank part at center) andzigzag line and benzene signals showhydrocarbon chains and phenyl rings ofhydrophobic parts (gray zone) of FTY720
molecule shown in Fig. 1.

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Under the physiological condition atneutral pH, the monomeric FTY720 is fairly
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Communicated by Tate Shin-ichi

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