КОЛЛОИДНЫЙ ЖУРНАЛ, 2007, том 69, № 4, с. 501-509
УДК 541.18
PHYSICAL INVESTIGATIONS OF COLLOIDAL IRON-INULIN COMPLEX © 2007 Goran S. Nikolic, Milorad D. Cakic
Faculty of Technology, University of Nis, Bulevar oslobodjenja 124, Leskovac 16000, Serbia Поступила в редакцию 31.10.2006 г.
Iron-inulin complex, which could be applied as parenteral hematinic agents, was studied by physical techniques. Electron microscopy showed that complex contained very small particles, approximately spherical and 10-30 nm in diameter. The examination revealed the presence of an electron-transparent sheath, probably linked inulin oligomers Mw ~ 3000 g mol-1, around the electron-dense iron-containing core of the FeOOH particles. FTIR spectroscopy suggested the existence of stronger hydrogen bonds which have been formed in the complexes between FeOOH and oligosaccharides, compared to the hydrogen bond in the initial compounds. A gel-filtration technique on Sephadex-column showed conclusively that a large proportion of combined inulin oligomers Mw ~ 1500 g mol as secondary fractions were present in complexes. These low molar inulin fractions have a significant influence on the complex stability.
1. INTRODUCTION
Aqueous solutions of iron (more correctly FeOOH) complexes with oligosaccharides are important parenteral hematinic agents [1]. Iron-dextran complexes are preferred as the standard in a modern iron therapy [2]. The possibility of different carbohydrate types to form water-soluble complexes with FeOOH in alkaline solutions shows that there is no strict choice of ligands. Besides dex-tran, the potential application of pullulan (polymaltotri-ose) [3-5] and inulin (polyfructose) [6-8] as ligands are known in literature. Few studies on the complex structures by FTIR [9, 10], ESR [11, 12], UV-VIS [13], SEM-TEM [14], GPC [15] and physical properties of the ligands and complexes [16, 17] have been reported, perhaps because of the difficulties involved in examining polydisperse colloidal particles.
A general structure for the hematinic is now suggested mainly from examination of its physical properties, especially a particle size. The first electron microscopy (EM) studies of the iron-dextran complex [18, 19] showed electron-dense, spherocolloidal particles 2-4 nm in diameter, which are presumed to be the colloidal FeOOH particles, and particles ranging from 11 to 21 nm (with an average value of 14 nm) when examined the whole particle including the dextran layer [20]. It is reasonable to assume that colloidal ferric oxyhydroxide is formed with the dextran acting as the stabilizing hydrophilic colloid attached to it in some way. However, a tendency of the individual particles to aggregate in large masses was noted. Other references [21] to a particle size of 3 ^m have raised doubts on the particulate nature of the complexes.
The nature of the linkage between the iron (i.e. FeOOH) and the oligosaccharides, as well as the molecular weight (Mw) of the product is not well known. That is why the structure of the polynuclear iron complexes has
not been explained in details yet, despite of a number of studies. Aqueous gel filtration chromatography (GFC) on Sephadex columns and ultrafiltration techniques seemed likely to throw light on the nature of the iron-dextran preparation [19]. Physical investigations on colloidal iron-dex-tran complexes, valence investigations and molecular weight have been briefly discussed and reported [22]. The low molecular weight dextran [23, 24], pullulan [3, 7] and inulin [25, 26] derivatives, which are the protective colloid in complexes, have been the subject of a recent paper. The adsorption of dextran on FeOOH sol particles has been studied [27] and the results suggest that adsorption occurs through the terminal glucose unit in the dextran chain. Muller [6] has reviewed both the complexing behavior of various forms of FeOOH with a range of carbohydrates and the application of certain physical techniques for characterizing the complexes.
Inulin is a polydisperse mixture of linear fructose polymers ((3-1,2) with a different chain-length and one glucose at each reducing C2-end (Fig. 1). Inulin is a suitable ligand for complexing with FeOOH because of its physicochemical characteristics [28, 29]. The literature data on this complex (synthesis, structure, nature of the linkage with iron, stability, pharmacological activity) are very rare [6, 7]. The GFC based methods have been reported in the literature [15] for the determination of molecular weight of inulin oligomers. This paper reports certain physical investigations carried out on the established products of iron-inulin complex. The object of this study was to characterize the complex as fully as possible by the available GPC, SEM-TEM and FTIR techniques, with an ultimate aim of correlating its properties and structures.
HOCH2 JO
5 ^
Kh HO /
H\_/ C
OH H HOCH2 O
O
KH H^CH2
OH H
O
O
HOCH2
kH HO
HH
O
a
1 2
2
CH2
O
ß 1-
H
5
CH2OH
HOH2C H ^ 1
H OH
OH
4
'C1HH.
.O,
\
o
-X
C2
ohC5 \
OH
C6HH—O6H
/ c3 c4
o1
OH
C1HH
ro
O,
~ C{ Xo
„O2 \ OH /<C6HH-o6H
C3 c4
OH
n ~ 30
OH H
Fig. 1. Constitutional formula of inulin (n ~ 30) with helicoid (1 x 4.5 nm) chain conformation (^ = 66°, y = 154°, ro = -82°, %0 = 54°).
2. EXPERIMENTAL
Inulin Mw 5000 g mol1 was purchased from Merck (KGaA Darmstat, Germany). Reagents were of analytical grade, eluant of HPLC spectroscopic grade and KBr of IR spectroscopic grade. The dialysis tubes were from Reichelt (Heidelberg, Germany) and the ion-exchangers (Am-berlite IR-120 and IRA-410) were obtained from Fluka. The apparatus used for the titrations combined with a pH glass electrode was purchased from Metrohm (Filderstadt, Germany).
Polynuclear FeOOH and iron-inulin complex were synthesized according to literature data [17]. Fig. 2 shows a flow chart of the synthetic procedure. The amorphous ß2-form of FeOOH, as the only applicable form for this synthesis, was prepared by precipitation from ferric chloride solution using sodium carbonate at room temperature and pH 6.5. The precipitate, after removing the electrolytes, was directly used for the synthesis of the complex.
Native inulin was depolymerized by HCl (pH 2.5 at 85°C for 5 min). Inulin oligomers were reduced with NaBH4 at 40°C [17] and the excess of unreacted NaBH4 was destroyed by HCl and by further treatment of the de-polymerization solution at 70°C. The solution was purified and deionized by acidic and alkaline ion-exchangers (Amberlite IR-120 and IRA-410).
The method of complex preparation involves homoge-nization of FeOOH gel with inulin oligomers in alkaline solutions (pH 10). The mixture was refluxed for 60 min at the boiling temperature and pH 10.5. The synthesis was realized at a mass ratio iron/ligand 1 : 3. Most of the inulin
possibly binds to the complex colloid particles in the reflux process. After cooling, methanol was added up to 48% (w/v) to the precipitated complex. After centrifUga-tion, the precipitate was dialyzed against the running water overnight. The complex solution was adjusted to pH 6.5 with a drop of NaOH solution and concentrated by using an evaporator. The iron content was determined com-plexometrically. Chlorides were determined by potentio-metric titration. The powder sample of the complex was prepared by lyophilization.
A gel chromatography was carried out at the Institute of Pharmaceutical Industry Zdravlje-Actavis, Leskovac (Serbia). The chromatography was performed on a gel chromatography system (LKB Bromma), consisted of a Microperpex P-2132 peristaltic pump, LKB-2142 RI detector, and LKB-2210 recorder. The gel chromatography experiments were conducted on a column (1 x 60 cm, Pharm. Uppsala) of Sephadex G-75 gel (Pharmacia Ltd., Mw disunion 1000-50000). Sodium chloride solution (0.1 M) was used as the mobile phase. With the optimized conditions, mobile phase flow rate was set at 0.5 cm3 min-1 and ambient column temperature. The injection volume of the complex or inulin solution was 0.2 cm3. The eluate was monitored continuously for re-fractivity (An) using the differential refractometer. A blue dextran (Pharmacia Ltd.) was applied to mark the void volume. The GFC method is calibrated using reference standards [15] of know molar masses ranging from 1000 to 10000 g mol-1.
The electron microscopy was carried out at the Institute of Medicine, Nis (Serbia). For a direct microscopy,
4
3
n
2
Native inulin
Depolimerization HCl (pH 2.5, 85°C, 5 min)
Reduction (NaBH4)
Anion-exchange resin
I
Inulin oligomers solution
FeCl3 solution HCl (pH 1.6)
Precipitation Na2CO3 (pH 6.5)
Washing (H2O)
I
P2-FeOOH gel
Homogenezation
1 Alkali-treated mixture, NaOH (pH 10) ■
I ;
; Synthesis ;
' Reflux (pH 10.5, 60 min) :
I ;
: Centrifugation (CH3OH, H2O) : Dialysis Concentration by evaporation Lyophilization
.......".....1.......,.......
Complex powder
Fig. 2. Flow chart of synthetic procedure of (2-FeOOH and
iron-inulin complex.
samples were diluted with water, a drop was placed on a glass plate and a carbon-coated grid was floated on top with its carbon film uppermost. The preparation was then allowed to dry. Measurements of inulin were made with minimum beam intensity to avoid the melting of the sample. For a freeze-drying, the microscope grids were placed on a copper block assembly and a carbon film applied as the support for the sample [14]. The whole assembly was then cooled with solid carbon dioxide in ethanol. The complex was sprayed onto the grids with a fine chromato-graphic spray before the assembly was cooled again and set in the vacuum chamber of a coating unit. A copper bar cooled with liquid nitrogen was introduced into the chambers as a cold trap after which the chamber was pumped down to 1.3 x 10-2 Pa (10-4 Torr) or better for the specified period. The grids were examined at 90 kV on TEM electron microscope (Philips C-12) and at 30 kV on SEM electron microscope (Jeol JSM 53
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