Interaction between novel porphyrin-dextran nanoparticles and human serum albumin

Article abstract of DOI:10.1142/S1088424610001969

A novel porphyrin-dextran coated Fe₃O₄ nanoparticle (5) was designed and synthesized. The structure of 5 was confirmed by IR, UV-vis and inductively coupled plasma atomic emission spectrometry, and dynamic laser scattering (DLS); magnetic property was measured by a vibrating sample magnetometer (VSM). The interaction between compound 5 and human serum albumin (HSA) was investigated through UV-vis absorbance spectra and fluorospectrophotometer, compared with the 5-(4-aminophenyl)-10,15,20-tris-(4- sulfonatophenyl)porphyrin, trisodium salt (3). The results showed compound 5 containing porphyrin moiety and 3 could interact with HSA. The quenching constant (K_sv) was 4.739 × 10⁵ M^-1 for 3, and 2.846 × 10⁵ M^-1 for 5; the apparent affinity binding constant (K_A) was 8.562 × 10³ M^-1 for 3, and 4.978 × 10⁴ M^-1 for 5.

Full text of DOI:10.1142/S1088424610001969

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2010; 14: 264–270
DOI: 10.1142/S1088424610001969
Published at http://www.worldscinet.com/jpp/
Interaction between novel porphyrin-dextran nanoparticles
and human serum albumin
Zhi Zhanga, Qian-ni Guoa, Yun-guo Lua, Tao Jiab, Kun Yan*a and Zao-ying Li*a
a College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P.R. China
b College of Pharmacy, Wuhan University, Wuhan 430072, P.R. China
Received 18 February 2009
Accepted 16 June 2009
ABSTRACT: A novel porphyrin-dextran coated Fe3O4 nanoparticle (5) was designed and synthesized.
The structure of 5 was confirmed by IR, UV-vis and inductively coupled plasma atomic emission
spectrometry, and dynamic laser scattering (DLS); magnetic property was measured by a vibrating
sample magnetometer (VSM). The interaction between compound 5 and human serum albumin (HSA)
was investigated through UV-vis absorbance spectra and fluorospectrophotometer, compared with the
5-(4-aminophenyl)-10,15,20-tris-(4-sulfonatophenyl)porphyrin, trisodium salt (3). The results showed
compound 5 containing porphyrin moiety and 3 could interact with HSA. The quenching constant (Ksv)
was 4.739 × 105 M-1 for 3, and 2.846 × 105 M-1 for 5; the apparent affinity binding constant (KA) was
8.562 × 103 M-1 for 3, and 4.978 × 104 M-1 for 5.
KEYWORDS: dextran, nanoparticles, human serum albumin, interaction.
intracranial metallic material), the use of magnetic micro-
INTRODUCTION
and nanoparticles has attracted increasing attention in the
Porphyrin compounds tend to accumulate in neo-
areas of biomedicine and biology, with a view to reduc-
plastic tissue to higher concentrations than in surround-
ing the systemic distribution of cytotoxic compounds and
ing normal tissue, and they might be photo-triggered to
enhancing uptake at the target site with effective treat-
produce singlet-state oxygen and destroy the cancer cells
ment at lower dose [6–8]. The coating of polymer onto
when irradiated by light. Therefore, they have recently
magnetic nanoparticles was applied to reduce aggrega-
attracted significant attention in cancer diagnosis and
tion and enhance the biocompatibility of the nanopar-
treatment using photodynamic therapy (PDT) [1, 2].
ticles in vivo [9, 10]. Dextran is one of the candidates.
As a potential valuable anti-tumor and antibiotic drug,
Dextran is a water-soluble polysaccharide and its clinical
porphyrin derivatives depend strongly on their physico-
use over the past 50 years has provided impressive safety
chemical properties to enhance biological effects [3], and
and quality [11]. Recently, it has been investigated as a
it is a challenge to formulate the porphyrin into chemi-
potential macromolecular carrier for delivery of drugs
cally stable, effective, and safe dosage forms [4, 5].
and proteins, primarily to increase the longevity of thera-
Since it is now accepted that magnetic fields are not
peutic agents [12].
especially contraindicated for humans, except for patients
Human serum albumin (HSA) is the major soluble pro-
whose body contains magnetizable material (medical
tein constituent in the human circulatory system [13, 14],
devices with batteries or computer chips, vascular or
and plays an important role in metabolism, the transmis-
sion and distribution of fatty acids, amino acids, drugs and
metal ions, and other functions. Therefore, it is important
to study the interaction between drugs and HSA [15]. In the
*Correspondence to: Kun Yan, email: kun_yan18@hotmail.
present work, we designed and synthesized the porphyrin-
dextran Fe3O4 nanoparticles for the first time (Scheme 1),
and investigated the interaction between 5 and HSA.
com, tel: +86 27-87219084, fax: +86 27-68754067 and Zao-
ying Li, email: zyliwuc@whu.edu.cn, tel: +86 27-87219084,
fax: +86 27-68754067
Copyright © 2010 World Scientific Publishing Company

INTERACTION BETWEEN NOVEL PORPHYRIN-DEXTRAN NANOPARTICLES AND HSA
265
Scheme 1. Synthetic route of compound 5
steps: 3 was conjugated with 4 via Schiff base, then the
RESULTS AND DISCUSSION
corresponding C=N bond was reduced by NaBH4. In order
to describe the characteristic absorption band in FT-IR, we
prepared the pure oxidized dextran specially. Figure 1 lines
(a) and (b) shows the FT-IR spectra of oxidized dextran
and 4, respectively. Both exhibited an obvious peak at 1637
cm-1 in Fig. 1a,b due to the stretch vibration of -HC=O, but
only in Fig. 1b could the peak at 646 cm-1 be observed, cor-
responding to the characteristics of Fe3O4 peak [17].
Porphyrin 3 shows max absorbed (Soret band) at 423
nm in UV-vis spectra [18], while the dextran shows no
absorbance near the Soret band [19]. Therefore, the con-
centration of porphyrin moiety of 5 in solution was cal-
culated with corresponding value at Soret band in UV-vis
from the standard working curve of pure compound 3.
The porphyrin moiety in 5 was 35% of the entire molecu-
lar weight. The concentration of Fe in 5 was determined
With the addition of aqueous ammonia into the mixture
solution of FeCl2 and FeCl3 in the presence of dextran,
Fe3O4 nanoparticles coated with dextran were synthesized.
Then the dextran-coated Fe3O4 was oxidized by NaIO4 to
produce the aldehyde (4). The size of 4 was smallest when
the ratio of FeCl3 to FeCl2 was close to 2:1. In addition, the
concentration of dextran, aqueous ammonia, as well as the
stirring speed will all affect the size of 4 [16]. From porphy-
rin 3 and 4, compound 5 was prepared by two consecutive
Fig. 1. The FT-IR spectra of (a) oxidized dextran and
(b) compound 4
Fig. 2. Diameter distribution of compound 5 by DLS method
Copyright © 2010 World Scientific Publishing Company
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266
Z. ZHANG ET AL.
Fig. 3. The changes of UV-vis absorbance spectra of (a) 3 or (b) 5 with increasing concentration of HSA in PBS (pH = 7.4)
Table 1. Compared date of the interaction of 3 and 5 with HSA
emission of HSA presented a remarkable
decrease as shown in Fig. 4a,b, respectively;
the values are summarized in Table 1.
The quenching of HSA could be caused
by either static quenching or dynamic
quenching. The former could change the
configuration and the bioactivities of HSA,
Compound
UV-vis on the Soret band
Fluorescence emission
Decrease of intensity, %
35.94
Hypochromicity, % Red shift, nm
3
5
12.26
20.46
2
3
33.75
while the latter was only a process of energy and electron
transfer. Since the quenching accords with Stern-Volmer
equation [22]:
by ICP-AES to be 4.87 mg per gram. The diameter of 5
measured by DLS was about 204.9 nm (in Fig. 2).
The UV-vis absorbance changes to the porphyrin Soret
band when HSA was titrated into compound 3 or 5 are
shown in Fig. 3. As can be seen, the Soret band of both 3
and 5 red-shifted and showed hypochromism in different
degrees [20, 21]. This indicates both compounds 3 and
5 are able to bind HSA. As summarized in Table 1, the
Soret band of 5 exhibited 20.46% hypochromism when
excess HSA was added. Only 12.26% hypochromism
was observed for 3 under the same conditions, probably
because the biocompatible dextran moiety made the bind-
ing between 5 and HSA easier and more efficient.
F0/F = 1 + Ksv(C) = 1 + Kqt0(C)
(1)
(t0 = 10-8 s, F, (C) were for the quenching intensity and
concentration of 3 and 5, respectively).
Figure 5a,b shows the average quenching constants of
HSA in presence of 3 and 5; the quenching constant (Ksv)
and quenching velocity (Kq) are listed in Table 2.
From the value of Kq, it could be confirmed that static
quenching happened between the HSA and 3 and 5, for the
maximum value of dynamic quenching velocity (Kq) was
only 2.0 × 105 L.s.mol-1, which was way smaller [23].
Based on the static quenching theory [24, 25], we
obtained the apparent affinity binding constant (KA) and
Fluorescence emission spectra for HSA with an in-
creased concentration of compound 3 or 5 were recorded.
With the titration of 3 or 5, intensity of fluorescence
Fig. 4. Fluorescence emission spectra of HSA (2 µM) in PBS (pH = 7.4) with increasing concentration of (a) 3 or (b) 5 from
0 to 2 µM
Copyright © 2010 World Scientific Publishing Company
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INTERACTION BETWEEN NOVEL PORPHYRIN-DEXTRAN NANOPARTICLES AND HSA
267
Fig. 5. The average quenching constants of HSA in presence of (a) 3 and (b) 5
Table 2. The Stern-Volmer constants of fluorescence quenching of
porphyrin-HSA
based on the intrinsic fluorescence of trypto-
phan residue. Site II contains a hydrophobic,
branched, T-shaped cavity. According to pub-
lished literature, if there were no binding sites
other than Site I, the KA would be smaller than
Ksv [26]. As shown in Tables 2 and 3, the value
of binding point (n) of both 3 and 5 was almost
Compound
Stern-Volmer equation
Ksv, L.mol
Kq, L.s.mol-1
2.846 × 1013
4.739 × 1013
3
5
F0/F = 1.009 + 2.846 × 105 C 2.846 × 105
F0/F = 1.034 + 4.739 × 105 C 4.739 × 105
the same and the KA was smaller than Ksv, which indicated
that probably no other binding sites existed except for the
one binding site near tryptophan [26]. In other words, it is
probably the porphyrin moiety of 5 that was the only moi-
ety located at the binding site in HSA. However, while the
Ksv of 5 (4.739 × 105 M-1) was bigger than that of 3 (2.846 ×
105 M-1), the KA of 5 (8.562 × 103 M-1) was smaller than
that of 3 (4.978 × 104 M-1). The Ksv is in response to the
degree of fluorescence quenching. With the existence of
dextran moiety in 5, the porphyrin moiety should come
into collision with HSA more easily and as a result
quenched HSA more efficiently, and the deduction could
explain the appearance in UV-vis absorbance spectra. The
KA represented the intensity of drug binding with HSA.
The interaction between HSA and the porphyrin was
a course of π-π stacking [27], and the steric resistance
could be a greater factor. The size of 5 was much bigger
than 3, therefore the KA value of 5 was smaller.
Table 3. The apparent affinity binding constant (KA) and the
value of binding point (n) of porphyrin-HSA
Compound
Equation
KA, M-1
n
3
lg((F0-F)/F) = 4.697 +
4.978 × 104 0.874
0.874 lg((D))
5
lg((F0-F)/F) = 3.932 +
8.562 × 103 0.714
0.714 lg((D))
average binding point (n) of compounds 3 and 5 accord-
ing to Equation 2. The data is tabulated in Table 3 and
shown graphically in Fig. 6a,b.
lg((F0 − F)/F) = KA + nlg(C)
(2)
HSA has multiple binding sites for drug molecules.
The primary binding sites of HSA are Site I and Site II.
Site I involves the lone tryptophan residue of the pro-
tein (Trp-214), and fluorescence quenching is performed
Fig. 6. The apparent affinity binding constant and binding point of porphyrin ((a) 3 or (b) 5)-HSA
Copyright © 2010 World Scientific Publishing Company
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268
Z. ZHANG ET AL.
mg, 0.81 mmol) was dissolved in chloroform (150 mL).
The solution was stirred, red fuming nitric acid (6.8 g,
108 mmol) was added at 0 °C over 2 h, and the solution
was stirred for another 1 h. The reaction mixture was next
diluted with ice water (100 mL), neutralized with satura-
tion sodium hydroxide, then extracted with chloroform.
The organic phase was dried over magnesium sulfate.
The resulting solution was concentrated under reduced
pressure. The residue solution was chromatographed on
silica gel with chloroform as eluent to afford violet crys-
tals (250 mg, 46% yield). IR (KBr): ν, cm-1 1597 (NO2).
1H NMR (CDCl3): δH, ppm 8.87 (d, 2 H, J = 5.2 Hz,
β-pyrrole), 8.86 (s, 6 H, β-pyrrole), 8.54 (d, 2 H, J = 8.9
Hz, nitrophenyl), 8.30 (d, 2 H, J = 8.9 Hz, nitrophenyl),
8.19–8.21 (m, 6 H, ortho phenyl), 7.98–8.01 (m, 9 H,
meta/para phenyls), -2.84 (s, 2 H, pyrrole NH). UV-vis
(CHCl3): λmax, nm 418, 516, 552, 595, 645.
Fig. 7. Magnetization loop of compound 5
Magnetization reflects the magnetic response capa-
bility. The greater the susceptibility of the samples, the
higher the corresponding magnetic capacity. Figure 7
shows the magnetization loop of 5 measured at room
temperature. As shown in Fig. 7, although with the inten-
sification of the magnetic field, the value of magnetiza-
tion increased, the relationship is nonlinear. When the
magnetic field increased to a certain value, magnetiza-
tion was unchanged, and at this moment magnetic satu-
ration was reached. The saturation magnetization (Ms)
of fine Fe3O4 particles was 415 emu.g-1. The magnetiza-
tion loop is a single curve over the starting point, and
that means when the magnetic field was 0, there was no
residual magnetism. In other words, Fig. 7 indicated that
the Fe3O4 particles were superparamagnetic.
Preparation of 5-(4-aminophenyl)-10,15,20-tri-
phenyl porphyrin (2). The synthesis of 2 was modified
from the literature methods [28]. To compound 1 (200
mg, 0.3 mmol) in hydrochloric acid (50 mL, 6 M), tin(II)
chloride dihydrate (3.0 g, 13.3 mmol) was added under
nitrogen. The mixture solution was stirred at 60 °C for 1 h.
The reaction mixture was neutralized with saturation
sodium hydroxide to pH = 8 and extracted with chloro-
form. The organic phase was washed with water, followed
by drying over magnesium sulfate. The solvent was con-
centrated under reduced pressure and the residue solution
was chromatographed on silica gel with chloroform as elu-
ent to give violet crystals (132 mg, 70% yield). 1H NMR
(CDCl3): δH, ppm 8.95 (d, 2 H, J = 5.2 Hz, β-pyrrole), 8.84
(s, 6 H, β-pyrrole), 8.24–8.26 (m, 6 H, ortho phenyl), 7.96
(d, 2 H, J = 8.2 Hz, 4-aminophenyl), 7.70–7.72 (m, 9 H,
meta/para phenyls), 7.06 (d, 2 H, J = 8.2 Hz, 4-aminophe-
nyl), 4.02 (s, 2 H, amino), -2.74 (s, 2 H, pyrrole NH).
Preparation of 5-(4-aminophenyl)-10,15,20-tris-
(4-sulfonatophenyl)porphyrin, trisodium salt (3). The
synthesis of 3 was modified from literature methods [28].
The solution of compound 2 (100 mg, 0.16 mmol) in
30 mL sulfuric acid was heated to 70 °C for 48 h. The
dark green solution was stirred under nitrogen for 72 h
at room temperature. The reaction mixture was poured
into 200 mL ice water and neutralized with saturated
sodium hydroxide to pH = 8. The solution was concen-
trated under reduced pressure and methanol was added.
After filtration, the precipitate was washed several times
with methanol. The filtrate was combined and the sol-
vent was evaporated to dryness. The residue was taken up
in methanol and precipitated by addition of ethyl ether,
then filtered off and dried under vacuum to afford violet
EXPERIMENTAL
General
All reagents and solvents were purchased from com-
mercial sources and used without further purification.
Chromatographic separations were performed using sil-
ica gel G (200–300 mesh). All UV-visible spectra were
obtained on a Shimadzu 1601 spectrophotometer. Fluo-
rescence spectra were recorded on PerkinElmer LS-55
fluorospectrophotometer. Proton NMR spectra were
measured using a Varian Mercury VX 300 spectrometer.
In all measurements of interaction between compound
3 or 5 and HSA, individual components were dissolved
respectively in phosphate buffer (PBS) with pre-adjusted
pH, then pH was adjusted again in each solution before
mixing, and the final pH value was verified thereafter.
Buffer solution was made with bidistilled water.
1
crystals (97 mg, 65% yield). H NMR (DMSO-d6): δH,
ppm 8.95 (s, 2 H, J = 5.1 Hz, β-pyrrole), 8.94 (s, 6 H,
β-pyrrole), 8.17 (d, 6 H, J = 8.1 Hz, 4-sulfonatophenyl),
8.03 (d, 6 H, J = 8.2 Hz, 4-sulfonatophenyl), 7.86 (d, 2 H,
J = 8.3 Hz, 4-aminophenyl), 7.01 (d, 2 H, J = 8.2 Hz,
4-aminophenyl), 5.56 (s, 2 H, amino), -2.84 (s, 2 H, pyr-
role NH).
Synthesis of compounds 1–5
Preparation of 5-(4-nitrophenyl)-10,15,20-tri-
phenyl porphyrin (1). The synthesis of 1 was modified
from literature methods [28]. Tetraphenylporphyrin (500
Copyright © 2010 World Scientific Publishing Company
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INTERACTION BETWEEN NOVEL PORPHYRIN-DEXTRAN NANOPARTICLES AND HSA
269
Preparationofoxidizeddextran-coatedFe3O4 nano-
solution added was less than 100 µL. All results were
averaged over at least five independent experiments.
particles (4) [29]. Dextran T-40 (795 mg, 0.02 mmol),
ferric chloride hexahydrate (48 mg, 24.1 mmol) and fer-
rous chloride tetrahydrate (129.3 mg, 45.8 mmol) were
dissolved in H2O (10 mL). Aqueous ammonia (7.5%, v/v)
was added slowly into the above stirred solution (pH =
10–11) at 60 °C for 30 min. The aggregates were removed
from the suspension by 3 cycles of centrifugation at 3000
R/min for 10 min. The solution of sodium periodate
(5 mL, 5 mM) was added into the concentrated filtrate
(5 mL). The mixture was stirred at room temperature for
1 h. The mixture was dialyzed overnight in sodium borate
buffer (20 mM, pH = 8.5) at 4 °C. The residue was sepa-
rated by gel filtration chromatography on Sephacryl 300,
eluting with mixture solution of sodium acetate (0.1 M)
and NaCl (0.15 M) at pH 6.5. The filtrate was freezed-
dried and yellowy powder was obtained (215 mg, 22%
yield). IR (KBr): ν, cm-1 1637 (C=O), 646 (Fe-O).
HSA fluorescence quenching by 3 and 5
Compounds 3 and 5 were titrated into HSA at room
temperature using excitation at 280 nm and emission at
340 nm. The excitation and emission slits were set at 15
and 20 nm, respectively. The concentration of porphyrin
moiety of compound 5 was calculated from UV-vis absor-
bance spectra standard working curve of compound 3.
All results were averaged over at least five independent
experiments.
Magnetic property measured
Magnetization loop was measured by a vibrating sam-
ple magnetometer (VSM) (Model 4AF-VSM, ADE Cor-
poration, USA) at room temperature.
Preparation of porphyrin-dextran-coated Fe3O4
nanoparticles (5). To compound 4 (300 mg) in H2O
(15 mL), compound 3 (100 mg, 0.11 mmol) was added.
The mixture solution was stirred under nitrogen at room
temperature for 8 h, followed by addition of sodium
borohydride solution (10 mL, 5 mM). The mixture solu-
tion was stirred under nitrogen for 40 min. The reaction
solution was dialyzed overnight in sodium borate buffer
(20 mM, pH = 8.5) at room temperature. The residue was
separated by gel filtration chromatography on Sephacryl
300, eluting with mixture solution of sodium acetate
(0.1 M) and NaCl (0.15 M) at pH 6.5. The final resi-
due was freezed-dried to afford violet powder (100 mg,
25% yield). IR (KBr): ν, cm-1 3312 (pyrrole N-H), 1663
(C=O). UV-vis (H2O): λmax, nm 423 (Soret band), 520,
560, 596, 645 nm.
CONCLUSION
Novel porphyrin-dextran coated Fe3O4 nanoparticles
(5) were designed and synthesized which could interact
with HSA effectively due to the introduction of biocom-
patible dextran moiety. Compound 5 containing magnetic
nanopartices moiety was superparamagnetic. The in vivo
cellular uptake properties of 5 in the magnetic field are
currently under investigation.
Acknowledgements
The present research has been financed by the
National Natural Science Foundation of China (Grant
CN 20672082).
Measurement of iron concentration by ICP-AES
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