Superparamagnetic iron oxide nanoparticles functionalized with peptides by electrostatic interactions

Superparamagnetic iron oxide nanoparticles coated with dextran were functionalized with negatively charged functionalities in order to connect specific peptide labels by electrostatic interactions. Peptide binding on the nanoparticles was indicated by HR-TEM, an electrospray scanning mobility particle sizer, and fluorescence measurements.


Introduction
Contrast agents provide an established tool in clinical magnetic resonance imaging (MRI).All contrast agents induce a decrease in the T 1 and T 2 relaxation times of surrounding water protons and thereby manipulate the signal intensity of the imaged tissue. 1 While traditional contrast agents distribute rather non-specifically, molecular imaging probes have been developed that specifically label body tissue or cells. 2 Furthermore, visualizing a single property of a pathological entity with one agent is generally insufficient to characterize it accurately in clinical MRI.Therefore, we aim at developing an imaging probe that permits study of several aspects of a pathological entity with only one intravenous injection.With respect to our interest in atherosclerosis, an angiogram would show the arteries.A specific ligand would bind the probe acute arterial thrombus which carries a high risk for stroke and heart attack.A second ligand would mediate transendothelial transport and indicate macrophage accumulation in inflammatory atherosclerotic wall plaque.Although preliminary studies have shown the potential of the appropriate MRI techniques to readily identify and differentiate these three characteristics, previous superparamagnetic iron-oxide nanoparticles (SPIO's) as diagnostic agents had insufficient unspecific binding for acute thrombus and some limited uptake in macrophage cells of inflammatory atherosclerotic plaque. 3,4PIO's are small superparamagnetic iron-oxide nanoparticles with a crystalline magnetite structure coated with dextran or dextran derivatives. 5They are a promising group of imaging probes since MR signal intensity is manipulated, they have a favorable toxicity, and genuine potential for cellular uptake.Furthermore, SPIO's are incorporated by several phagocytic cells, in particular macrophages in liver and spleen even with a regular dextran coat. 1 Modification of the dextran coating by carboxylation leads to a shorter clearance half-life in blood. 6,7Ferumoxytol, a carboxyalkylated polysaccharide coating, 8 is already described as a good first-pass contrast agent, 9 but uptake by macrophages is unspecific and too fast to enhance the uptake in macrophage-rich plaques.In order to improve the cell uptake in general, Weissleder et al. combined the tat peptide from the HIV tat protein with particles (CLIO, cross-linked iron oxide) covalently linked by a thioether. 10This leads to an increase of particle incorporation into spleen and liver.However, they are enriched close to the nucleus, indicating a different uptake mechanism caused by the peptide.Therefore, it is our intention to use specific peptide labels to address the particles towards relevant targets in blood vessels. 11For example, the peptide with the sequence IELLQAR is known for selective binding to the E-selectin receptor on injured artery walls. 12Cell uptake and specific direction of particles might be obtained by small particles labeled with the respective peptide sequence.
The attachment of peptide oligomers to the dextran-coated iron oxide particles was addressed by electrostatic interactions as a general non-specific and efficient method for recognition.This way of labeling is independent of the specific recognition sequence.Similarly, electrostatic interactions are used already for the deposition of polysaccharide multilayers on damaged arteries by an alternating aggregation of polycationic chitosan and polyanionic hyaluronan. 13Also, dextran sulfate can be used to form layer-by-layer assemblies with proteins based on charge-charge interactions. 14SPIO's modified by electrostatic interactions with transition agents such as poly-lysine are used for magnetic labeling of mammalian cells. 15In this case, the negative charges on the particle surface resulting from its preparation seem sufficient to bind the large polycation.
Within this study, small peptides were bound to SPIO´s using electrostatic interactions.Peptides containing a specific recognition sequence and amino acids that are positively charged under physiological conditions were bound to the negatively charged surface of a modified dextran.It needed to be proved that iron oxide particles are still stabilized by polysaccharide coating even the dextran derivative contains negative charges.Furthermore, the influence of functionalization by charge-charge interactions on the particle stability and the amount of charges required for attachment of the peptide label was investigated.Superparamagnetic particles were provided that, in principle, can be combined with any peptide carrying a recognition sequence shortly before injection.In particular, SPIO's labeled with a set of different peptides with individual targeting might be of interest in the future.The binding was estimated by size distribution and fluorescence measurements.In comparison with TEM measurements, the electrospray scanning mobility particle sizer (ES-SMPS) was introduced for the first time to analyze charged particles.

Results and Discussion
As the first example, the peptide sequence IELLQAR was chosen as the recognition motif known for inhibition of sialyl Lewis X binding to the E-selectin receptor. 12For electrostatic labeling of the nanoparticles three or six lysine amino acids, respectively, were added to the Cterminal end.At the N-terminus a dansyl group was connected as the fluorescent dye allowing for later detection of the particle-bound peptides.The dansyl group is stable under peptidecleavage conditions and the UV absorption of the particle (λ abs.max = 270 nm) does not affect its excitation (λ exc = 325 nm) or fluorescence (λ fl.max = 525 nm).Synthesis of the peptides was provided by standard automated Fmoc solid phase peptide synthesis protocols on a Wang resin. 16he dansyl group was introduced by addition of dansyl chloride and triethylamine in methylene chloride to the resin-bound oligomer. 17After cleavage from the solid support the two oligomers, dansyl-IELLQARKKK-OH (1) and dansyl-IELLQARKKKKKK-OH (2), were obtained, 18 differing in the amount of lysine residues for electrostatic interaction with the modified dextran coating of the particles.
In order to generate iron oxide nanoparticles with negatively charged surfaces we decided on a dextran-methylcarboxylate coating 3 as indicated in Scheme 1. Dextran (M w =40000 g mol -1 ) was modified with monochloracetic acid according to a known protocol. 18The carboxyl groups were introduced without any regiocontrol providing 4.6 mmol COO -g -1 as determined by titration with 0.1 M HCl.Whereas formation of dextran-methylcarboxylate iron oxide nanoparticles (4) by modification of the dextran-coated SPIO's was not successful, the respective particles have been obtained by iron oxide coating with the carboxylated dextran.In analogy to the dextran coated SPIO's, dextran-methylcarboxylate (3) was transformed with FeCl 2 , FeCl 3 and ammonium hydroxide at 80 °C for 2 h. 19The amount of carboxyl groups was proved to be unchanged after particle formation.The size of the dextran-methylcarboxylate particles 4 was investigated by HR-TEM (high resolution transmission electron microscopy) resulting in a size distribution around 20 nm (Figures 1 and 2).The accuracy of the TEM measurements was estimated to be higher than 95%.As a second method for size-determination of the nanoparticles, electrospray (ES) was used in combination with a scanning mobility particle sizer (SMPS). 20The solution containing nanoparticles was sprayed in the ES with subsequent solvent evaporation.The size-distribution of the generated aerosol was determined with the SMPS, resulting in a distribution of particles 4 around 8.4 nm assuming singly charged particles (Figure 3).Both methods clearly indicate the successful formation of particles.Nevertheless, the differing results obtained for the diameters of the particles are probably due to a higher population of doubly-charged particles in the ES-SMPS measurements; this would imply an increase of the real particle size by a factor of two.In addition, the particle size in the TEM measurements is usually overestimated with respect to ES, since higher concentrations are required for TEM solutions resulting in correspondingly larger amounts of impurities from the synthesis which might cover the nanoparticles after drying.Overall, the results obtained by ES-SMPS and TEM seem to agree reasonably well, considering the two points mentioned.
For the attachment of the respective peptides 1 or 2 to particle 4 an aqueous solution of the components was kept overnight at room temperature to guarantee an electrostatic equilibrium.HR-TEM and ES-SMPS experiments were performed for the functionalization of the particles with peptide 1. 20 The particle diameter increased significantly by about 15% from 20 nm to 23 nm as indicated by HR-TEM measurements (Figure 2).Also by ES-SMPS an increase of particle size from 8.4 nm to 9.1 nm was detected (assuming singly charged particles) (Figure 3).Both methods indicate an enlargement of the particle size, probably owing to loading of the negatively charged surface of the dextran-methylcarboxylate coated particles with peptides.The attachment of peptides by electrostatic interactions seems to be quite stable under aqueous conditions at neutral pH.
Further proof for particle functionalization was obtained by fluorescence measurements' detecting the dansyl labeling of the particle-bound peptides.In addition, this method allows estimation of the influence of the amount of positive charged amino acids on the stability of complex formation.Particle-peptide combinations 4 + 1 and 4 + 2 were independently separated from all unbound peptides using an ultrafiltration device with a molecular-weight cut of 5000 D in combination with centrifugation.After thoroughly washing with water, the fluorescence was determined (Table 1).a Fluorescence experiments were performed in twice-distilled water, λ exc = 325 nm, λ em = 400-600 nm.Calibration curves with known concentrations were measured for peptide 1 and 2, respectively.b Measurements were performed after washing with water using an ultrafiltration device.c Measurements were performed after washing with 0.1 M NaCl using an ultrafiltration device.
In both of the cases above, the peptide seems to be well bound to the particle; peptide 1 with three lysines required 2.7 carboxylic acid groups for binding whereas peptide 2 with six lysine residues was already bound by 4.8 carboxylic acid residues.In order to estimate the stability of the electrostatic interaction, both samples were washed with a 0.1 M NaCl solution using the ultrafiltration device.Peptide 1 was completely cleaved from the particle, since no fluorescence was detectable any more, whereas about half of the peptide loading was still present for the case of peptide 2. Therefore, six lysine residues provide significantly more stability than three lysines.Further improvement of the binding stability might be obtained by linear or dendrimer-like extension of the lysine sequence.
In order to estimate the need for the dextran coat functionalization with negative charges for binding peptides, a particle covered with regular dextran was synthesized. 19It was incubated with peptide 1 and separated from all unbound peptides using the ultrafiltration device in combination with centrifugation.After thoroughly washing with water no fluorescence was determined, indicating that negative charges resulting from the particle preparation are not sufficient to bind peptides with three positive charges by electrostatic interaction, even though this effect was reported for poly-lysine. 15 tag with the length of about six positively charged amino acids seemed to be sufficient to address dextran-coated iron oxide nanoparticles.Therefore, we introduced a general method of selectively functionalizing these kinds of particles with specific-recognition-as well as celltransport units.Before targeting selective binding sites in vitro and in vivo, further stability tests under protein-containing conditions are planned to ensure that the peptide stays associated in human blood.

Experimental Section
General Procedures. 1 H-NMR spectra were obtained on a Varian U 300 at 300 MHz, with chemical shifts being reported as ppm from trimethylsilane as internal standard.IR spectra were recorded on a Perkin Elmer FTIR 1600.Analytical and preparative HPLC were performed on an Amersham Pharmacia Biotech P 900.The mass spectra were measured on a Finnigan LQC spectrometer.For the concentration and cleaning of particle solutions, a Vivaspin 6 ml concentrator (MWCO 5000, PET membrane) was used as ultrafiltration device.Millipore H 2 O was employed for the preparation of all aqueous solutions.High resolution transmission electron microscopy (HR-TEM) measurements were carried out using a CM 200 FEG-UT.To prepare the samples, a drop of a solution containing the desired dextran-methylcarboxylate particle (4) (c = ca.0.3 nmol mL -1 , assuming that the average molecular weight of particle 4 is 60,000 g mol -1 ) was dried on a carbon-layered copper grid (Formvar/carbon film, 400 mesh, d = 3.05 mm).For ES-SMPS (electrospray-scanning mobility particle sizer) measurements, particle solutions with different concentrations were sprayed in the ES (TSI 3480) at 2.0 kV and examined by the SMPS equipped with a differential mobility analyzer for small particles (TSI 3936N25).The particle concentration of the sample was decreased until the maximum diameter measured was constant.The best results were examined at a dextran-methylcarboxylate particle concentration of ca.75 pmol mL -1 (assuming that the average molecular weight of particle 4 is 60,000 g mol -1 ).Fluorescence experiments were recorded on a Jasco FP-6200 spectrometer.The samples were dissolved in H 2 O.With excitation at λ exc = 325 nm fluorescence was measured at λ em = 400 -600 nm.The peptide concentration of the sample was linked to the peak-area by a linear relationship.For each sample the concentration was adjusted to an UV absorbance of 0.1 (λ abs.= 325 nm).Afterwards, the concentration was continuously enhanced.

Figure 1 .particle 4 and peptide 1 Figure 2 .
Figure 1.HR-TEM (CM 200 FEG-UT) of particle 4. One drop of a 0.3 nmol/mL solution of 4 was added on a copper grid with a lacey-carbon film and air-dried.The size is indicated by the bar).

Figure 3 .
Figure 3. Size distribution of nanoparticle 4 and nanoparticle 4 + peptide 1 measured by ES-SMPS assuming singly charged particles.For doubly charged particles sizes of 16.8 nm and 18.2 nm, respectively, would result.

Table 1 .
Ratio of peptide/amount of carboxylic acid groups calculated according to fluorescence experiments a

Issue in Honor of Prof. Lutz F. Tietze ARKIVOC 2007 (v) 79-90 ISSN 1424-6376 Page 87
For fluorescence experiments, calibration curves for the peptides 1 and 2 were needed.Therefore, peptide 1 (0.90 mg, 0.62 µmol) and peptide 2 (1.2 mg, 0.66 µmol), respectively, were dissolved in H 2 O (1 mL).Known amounts of these solutions were added to a cuvette containing H 2 O (1.2 mL), fluorescence spectra were measured, and the peak areas λ em = 400 -600 nm determined.A linear fit of the calibration curves yielded the following relationship between peak © ARKAT

Electrostatic binding of peptide 1 to particle 4. Peptide
1 (0.33 mg, 0.23 µmol, 1 eq.) and particle 4 (1.0 mg, 4.6 µmol COO -, 20 eq.) were dissolved in H 2 O (500 µL) and the solution was shaken for 12 h at RT.To purify the product an ultrafiltration device was used for centrifugation and after concentration the sample was washed with H 2 O (3 x 2 mL).Size-distribution experiments were carried out as described above.For fluorescence measurements the complex (0.39 mg) was dissolved in H 2 O (200 µL) (solution L).Known amounts of this solution were placed in a cuvette containing H 2 O (500 µL) (Table2).

Table 2
Afterwards the sample was transferred in an ultrafiltration device again, and washed with 0.1 M NaCl (2 mL) and H 2 O (2 x 2 mL).For fluorescence measurements the complex (0.16 mg) was dissolved in H 2 O (200 µL).Known amounts of this solution were placed in a cuvette already containing H 2 O (1.2 mL).No fluorescence was measured at any concentration.