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Development of Stable, Water-Dispersible, and Biofunctionalizable Superparamagnetic Iron Oxide Nanoparticles N. Miguel-Sancho,†,‡ O. Bomatí-Miguel,*,†,|| Gloria Colom,§,† J.-Pablo Salvador,†,§ M.-Pilar Marco,§,† and J. Santamaría*,*,†,‡ †
CIBER de Bioingeniería, Biomateriales y Nanomedicina and ‡Aragon Institute of Nanoscience, University of Zaragoza, Campus Río Ebro-Edificio IþD, C/Poeta Mariano Esquillor S/N, 50018-Zaragoza, Spain § Institute of Advanced Chemistry of Catalonia, Spanish Council for Scientific Research (IQAC-CSIC), C/Jordi Girona, 18-26, 08034-Barcelona, Spain ABSTRACT: Water-dispersible superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by thermal decomposition of iron(III) acetylacetonate in the presence of triethylene glycol (TREG). The resulting TREG-coated SPIONs were not stable, undergoing agglomeration and loss of the TREG coating under prolonged storage at 37 °C or in the presence of increased saline concentrations. To avoid these problems, stable colloidal TREG-coated SPIONs were obtained by two different procedures: (i) dimercaptosuccinic acid (DMSA) ligand-exchange reactions to obtain DMSA-coated SPIONs and (ii) chemical modification of the TREG coating. Both procedures, but especially the DMSA exchange, increased the stability of the SPION suspension. Finally, the functionality of both types of particles for biological applications was demonstrated by conjugating a model antibody to the end carboxyl groups of the SPIONs and testing the immunoreactivity of the final antibodyparticle conjugates by an enzyme-linked immunosorbent assay (ELISA). KEYWORDS: superparamagnetic iron oxide nanoparticles, polyol-mediated synthesis, hydrophilic coating, colloidal stability, surface derivatization, antibody conjugation
’ INTRODUCTION Functionalized magnetic nanoparticles have been intensely investigated in recent years, mainly on account of their potential biomedical applications.15 These entities usually have a magnetic nanoparticle as the core, surrounded by a hydrophilic and biocompatible coating, that is often functionalized to achieve a certain function (e.g., targeting, membrane crossing, endosomal escape). Particularly, colloidal dispersions made by mixtures of superparamagnetic iron oxide nanoparticles (SPIONs), such as magnetite (Fe3O4) and maghemite (γ-Fe3O4), have attracted considerable attention because of their ability to induce a dephasing on the transverse relaxation rates (T2 decay) of the magnetic moments of water protons placed around these nanoparticles, which leads to a negative contrast in T2-weighted magnetic resonance images (MRI) of organic samples containing SPIONs.6,7 For instance, commercial SPION preparations, such as Feridex (AMI-25, Advanced Magnetics, Cambridge, MA) and Ferrixan (SHU555A, Schering AG, Berlin), are currently used as passive contrast agents for MR anatomic imaging of malignant diseases in organs associated with the reticuloendothelial system (e.g., liver, spleen)811 and the lymph nodes.1214 Besides their imaging capability, SPIONs have other therapeutic applications, including magnetic guidance and magnetic hyperthermia, that is, the ability to heat when placed in an oscillating magnetic field, which has been used for thermal ablation5,15 and triggered drug delivery.16 Furthermore, SPIONs can be attached to a wide range of biologically active molecules by well-established surface conjugation chemistry.1719 Finally, SPIONs are useful platforms to integrate in vivo therapeutic and diagnostic uses (theranostics).2022 r 2011 American Chemical Society
However, the in vivo medical use of SPIONs still faces formidable challenges. Among the main ones is their biofouling in blood plasma and rapid sequestration by the phagocytes of the reticuloendothelial system (RES), which results in an efficient clearing from the bloodstream a few minutes after their intravenous injection.7 This problem is often addressed by masking surface charges and by coating the particles with hydrophilic polymers. A second challenge relates to the lack of surface functional groups in SPIONs, which are highly desirable for conjugation with molecules that can be used to increase targeting selectivity.19 A third key aspect addresses the need to obtain sufficient hydrodynamic stability of the particles under biologically relevant conditions (temperature, ionic concentration). Ideally, these three problems could be addressed simultaneously (e.g., by developing coatings that are hydrophilic, deliver surface groups for conjugation of targeting moieties, and provide sufficient hydrodynamic stability), but there is a surprising scarcity of research works that confront these problems. One of the most popular routes to prepare high-quality waterstable SPIONs is polyol-mediated synthesis. In this method, the use of a high-boiling solvent such as ethylene glycol,23 triethlylene glycol (TREG),23,24 tetraethylene glycol (TEG),22,23 poly(ethylene glycol) (PEG),24 and poly(vinyl alcohol) (PVA)25 or 2-pyrrolidone26 allows reaching the high temperatures needed for decomposition of the chemical precursor (e.g., iron Received: December 22, 2010 Revised: April 20, 2011 Published: May 20, 2011 2795
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Chemistry of Materials acetylacetonate, [Fe(acac)3]). Because of the reaction environment used, this method produces SPIONs with a hydrophilic coating (a desirable characteristic to retard detection and removal by the RES), acceptable size uniformity, and good crystallinity, enabling their use in many applications. For instance, recent in vitro studies have demostrated the ability of TREG-coated SPIONs to act as superparamagnetic contrast agents in MRI27 or as agents for magnetic hyperthermia.28 However, the TREG coatings on the SPIONs produced by this synthesis route are not covanlently linked on nanoparticle surfaces. The relatively weak interaction between the coating and the nanoparticle may favor the desorption of TREG, compromising the long-term stability of these materials.29 In addition, TREG molecules show a low concentration of reactive functional end groups to facilitate covalent anchoring of biomolecules, which also limits the versatility and applicability of these nanoparticles. In this work we have used polyol-mediated synthesis to obtain TREG-coated SPIONs and then studied the stability of the resulting dispersions under different environmental conditions (temperature and salinity). We have also modified the TREGcoated SPIONs to obtain (a) dimercaptosuccinic acid (DMSA) coatings via ligand-exchange reactions on the SPION surfaces and (b) TREG coatings with carboxyl end groups via an aminationcarboxylation process. The hydrodynamic stability of these two types of particles is compared to that of TREG-coated SPIONs. Finally, a specific-recognition antibody was immobilized on the more stable coatings as a test to evaluate the suitability of these functionalized SPIONs for conjugation with biologically active molecules. The biofunctionality of the SPIONs-antibody conjugates was determined by an enzyme-linked immunosorbent assay (ELISA) test.
’ EXPERIMENTAL SECTION Chemicals. Nanoparticle synthesis and functionalization experiments were carried out by use of commercially available analytical-grade reagents without further purification. Succinic acid anhydride and epichlorohydrin were purchased from Aldrich Chemical Co. (Milwaukee, WI). Absolute ethanol, triethylene glycol (TREG), ethyl acetate, iron(III) acetylacetonate [Fe(acac)3], meso-2,3-dimercaptosuccinic acid (DMSA), N-hydroxysuccinimide acid (NHS), Kaiser test kit (ninhydrin, phenol, and potassium cyanide solution), hydrogen peroxide (30%), N-ethylN0 -[3-dimethylamino)propyl]carbodiimide hydrochloride (EDC), bis(2methoxyethyl) ether (diglyme), N,N-dimethylformamide (DMF), polyoxyethylenesorbitan monolaurate (Tween 20), and 3,30 ,5,50 -tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis, MO). The composition of the buffers used in the TREG-coated-SPIONs colloidal dispersion and functionalization experiments were the following: PBS is 0.01 M phosphate buffer in a 0.8% saline solution (137 mM NaCl, 2.7 mM KCl), and the pH is 7.5. PBST is PBS with 0.05% Tween 20. Borax buffer is 50 mL of 0.2 M boric acid and 22.5 mL of 0.05 M sodium borate diluted in 200 mL of Milli-Q water, pH 8.6. Coating buffer is 0.05 M carbonatebicarbonate buffer, pH 9.6. Citrate buffer is a 0.04 M solution of sodium citrate, pH 5.5. Substrate solution contains 0.01% TMB (3,30 ,5,50 -tetramethylbenzidine) and 0.004% H2O2 in citrate buffer. Sodium hydroxide, sulfuric acid (98%), and other salts were purchased from Merck (Darmstadt, Germany), and ammonia (30%) was purchased from Panreac (Rodano, Italy). Immunochemicals. The specific antibody As143 was produced by immunization of a methylboldenone-haptenized conjugate coupled to HCH (antibody production will be published elsewhere). Methylboldenone-haptenized protein conjugate 15-BSA was prepared by the active ester method.30 The avidity of the antiserum versus 15-BSA was assessed
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Figure 1. Schematic illustration of the derivatization process of waterdispersible TREG-coated magnetite nanoparticles. (a) Preparation of DMSA-coated SPIONs. (b) Preparation of COOH-TREG-coated SPIONs. by measuring the binding of serial dilutions of the antiserum in PBST to microtiter plates coated with 15-BSA (1D ELISA). As a nonspecific antiserum against 15-BSA, we used As167, antiserum produced for the detection of sulfonamides.31 Both antibodies were purified by precipitation through ammonium sulfate method at 35%. Anti-rabbit IgG (whole molecule)/horseradish peroxidase conjugate (anti-IgGHRP) and bovine serum albumin (BSA), were purchased from Sigma (St. Louis, MO). Nanoparticle Synthesis. Water-soluble magnetic iron oxide nanoparticles were synthesized by a polyol-mediated method according to the synthesis procedure described by Wei and Wan.23 In a typical synthesis, 0.2 g of [Fe(acac)3] were vigorously mixed with 30 mL of TREG in a three-neck round-bottom flask equipped with a mechanical stirrer and degassed with Ar. The resulting mixture was heated at 180 °C and held at this temperature for 30 min in order to achieve the decomposition of the [Fe(acac)3] precursor, which takes place at temperatures around 180190 °C. After that, the mixture was heated under reflux at 280 °C and kept at this temperature for 30 min. The resulting dark solution was allowed to cool at room temperature. Under ambient conditions, ethyl acetate and ethanol were added to the mixture and a black magnetic material was precipitated and separated via magnetic separation with a magnetic field strength of 0.3 T. The precipitated material was redispersed in polar solvents such as water or PBS. Finally, the resulting dispersion was refined and made sterile by filtration through a 0.2 μm pore size filter. This procedure yielded SPIONs with a TREG coating adsorbed on their surface. Modification of Nanoparticle Coatings. The TREG-coated SPIONs were modified to increase particle stability and ease of functionalization. In particular, carboxylic functional groups were introduced by either (i) replacement of the TREG coating by DMSA (termed DMSA-SPIONs in the remainder of this work) or (ii) in situ functionalization of the TREG coating (termed COOH-TREG-SPIONs). DMSA-SPIONs were obtained via a ligand-exchange reaction process described previously in the literature32 with some modifications (Figure 1a). Briefly, a DMSA aqueous solution (30 mg/mL) was vigorously mixed with a TREG-coated SPIONs aqueous solution (10 mg/mL) at room temperature. The resulting mixture was kept under continuous stirring overnight to achieve the total displacement of TREG by DMSA on the nanoparticle surfaces. After that, the DMSA-SPIONs were separated via magnetic separation and repeatedly washed with distilled water. The solid was dispersed in distilled water and immediately 2796
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Chemistry of Materials dialyzed in 5 L for 24 h by using a 12 00014 000 nominal molecular weight cutoff membrane (CelluSep F3, Membrane Filtration Products Inc.) in order to eliminate the excess DMSA. The resulting functionalized SPIONs were dispersed in a NaOH aqueous solution, subsequently redispersed in PBS, and finally made sterile by filtration through a 0.2 μm pore size filter. COOH-TREG-SPIONs were obtained through a more complex process that was expected to functionalize the TREG coating and, to a certain extent, induce some cross-linking of the coating to increase its stability. To this end, the derivatization strategy described by Gr€uttner et al.33 was followed, with some modifications (Figure 1b). In a typical functionalization process, epichlorohydrin (47 μL, 600 mM) were added to TREG-coated SPIONs (1 mL, previously centrifuged) resuspended with a mixture of sodium hydroxide (500 μL, 400 mM) and dyglime (500 μl) to obtain 1 mL of TREG-coated-SPIONs with an epoxy terminal group, as schematized in Figure 1b. The reaction was carried out for 4 h with continuous circular stirring, and then the mixture was centrifuged at 14 000 rpm and 25 °C for 1 h and the supernatant was removed. The nanoparticles were magnetically separated and washed once with distilled water and twice with ethanol. The TREG-coated-SPIONs were redispersed in distilled water, 1 mL of 30 wt % ammonia solution was added, and the mixture was allowed to
Figure 2. Schematic illustration of the conjugation of antibodies on the surface of functionalized magnetite nanoparticles.
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react overnight under stirring to conduct the amination of the hydroxyl TREG end groups. The magnetic nanoparticles were removed via magnetic separation and washed once with distilled water and twice with PBS. The particles were then redispersed in PBS and a standard Kaiser’s test was carried out on this dispersion to confirm the functionalization with amino groups. The final step was carboxylation through reaction of the amino groups. To this end, 500 μL of the NH2-TREGcoated-SPIONs in PBS were mixed into a borax buffer containing 50 μL of a 200 mM solution of succinic anhydride in DMF. The mixture was allowed to react overnight at room temperature, and then the functionalized COOH-TREG-SPIONs were magnetically separated, washed three times with distilled water, and redispersed in PBS. A Kaiser’s test was again conducted to confirm the absence of amino groups on the particle surface and verify their carboxylic conversion. Finally, these materials were sterilized by filtration through a 0.2 μm pore size. Antibody Conjugation. As a proof of concept of the viability of the functionalized SPIONs, the DMSA-coated SPIONs and the COOH-TREG-coated SPIONs were linked to a specific rabbit antiserum, As143, employed for the determination of methylboldenone (Figure 2). In this procedure, 25 μL of 400 mM EDC and 25 μL of 400 mM NHS were added to 200 μL of a solution containing the functionalized SPIONs. Then 50 μL of specific purified rabbit antiserum As143 (1 mg/mL, in PBS) were added dropwise to the SPIONactivated solution. The final mixture was circularly shaken overnight at room temperature to achieve nanoparticle functionalization. The same procedure was carried out with a nonspecific purified antiserum, As167, which was used as reference to compare the ability of both bioconjugated SPIONs to recognize specifically the same coating antigen in the immunoassay analysis. The yield of the antibody conjugation process was determined by measuring the antibody concentration remaining in the supernatant via Bradford’s test. The result obtained was a 90% ( 6% efficiency yield (N = 6).
Figure 3. Structural characterization of as-prepared TREG-coated SPIONs: (a) XRD pattern (reflections of Fe3O4 are included for comparison). (b) TEM micrograph of TREG-coated magnetite nanoparticles. (c) Particle size distribution calculated from statistical analysis of TEM images: (—) log-normal curve fitting; histograms, size distribution obtained by TEM. (d) HRTEM image of Fe3O4 nanoparticles showing discernible lattice plane fringes and Fourier transform of the indicated zone. (e) Magnetization curve for TREG-coated SPIONs at 300 K. 2797
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Figure 4. Stability of dispersions of TREG-coated-SPIONs. (a, b, d) Hydrodynamic size distribution determined by DLS of TREG-coated SPIONS (a) as prepared, (b) after incubation for 1 week at 37 °C, and (d) after exposure to a high saline concentration. (c) Variation of ζ-potential with pH for naked and TREG-coated SPIONs.
Characterization Methods. A battery of characterization techniques was used to characterize the SPIONs synthesized. Phase identification was performed by X-ray diffraction analysis (XRD, Rygaku/Max System RU 300). The shape, microstructure, size, and particle size distribution of the iron oxide nanoparticles were determined by use of a T200 Philips Tecnai transmission electron microscope (TEM) operated at 200 kV. The hydrodynamic diameter distribution of the SPIONs dispersed in different media and their surface charge were measured by dynamic light scattering spectroscopy (DLS, 90-Plus Brookhaven Instruments Corporation). Both the hydrodynamic size and the ζ-potential were used as means to characterize the stability of the different suspensions of coated SPIONs during long-term storage or against changes in the environmental conditions: increases in temperature (to 37 °C) or ionic concentration (to an electric conductivity of up to 16 S 3 m1). Each measurement was repeated three times. The iron concentration in the SPION colloidal dispersions was measured via inductively coupled plasma mass spectrometry (ICP-MS) on a Perkin Elmer, Elan 6000 spectrometer. The magnetic properties of the nanoparticles were studied with a superconducting quantum interference device (SQUID MPMS-5S, Quantum Design). The biofunctionalty of SPIONantibody bioconjugates was evaluated in one-dimensional indirect enzyme-linked immunosorbent assays (1D-ELISA). In this analysis, a polystyrene microtiter plate (Nunc Maxisorp, Roskilde, Denmark) was coated with 15-BSA (1 μg/mL in coating buffer, 100 μL/well) during 4 h at room temperature. The plate was washed four times with PBST by using a SLY96 PW microplate washer (SLT Labinstruments GmbH, Salzburg, Austria). A colloidal dispersion containing SPIONs conjugated with specific and preimmune antibodies was half-diluted in PBS and added respectively in the first well of two different columns from the plate (250 μL). After addition of 125 μL of PBS in the other wells of both columns, SPION dispersions were done starting from the first to the second-to-last well; the last one represented the blank. The same was done with a colloidal dispersion containing antibodies (As143 and As167) that were not conjugated to
nanoparticles, starting with 250 μL of 0.1 μg/mL antibody solution. The plate was washed again according the method described above. After that, a solution of anti-IgG-HRP (1/6000 in PBS) was added (100 μL/ well), incubated for 30 min at room temperature, and subsequently washed. Finally, the substrate solution was added (100 μL/well) and the enzymatic reaction was stopped after 30 min at room temperature with 4 N H2SO4 (50 μL/well). The absorbance of the SPIONantibody conjugates was read at 450 nm by using a SpectramaxPlus (Molecular Devices, Sunnyvale, CA).
’ RESULTS AND DISCUSSION TREG-Coated SPIONs. As described in the Experimental Section, after reaction, TREG-coated SPIONS were obtained, which were magnetically separated from the reaction byproducts and the polyol solvent to obtain a colloidal dispersion in water. The typical X-ray diffraction pattern of TREG-coated SPIONs is shown in Figure 3a. The XRD pattern of this sample was assigned to the phase of bulk magnetite [Joint Committee on Powder Diffraction Standards (JCPDS) card number 89-0691]. The lattice parameter (a) of this material calculated from the position of the (311) peak was approximately 8.36 Å, which could be associated with magnetite (8.39 Å), maghemite (8.338.39 Å), or a solid solution between the two phases. The average crystallite size for the iron oxide nanoparticles was calculated from the XRD peak broadening by use of DebyeScherrer’s equation, and a mean crystallite size of 7 nm was obtained. Figure 3b presents a representative TEM micrograph of the asobtained TREG-coated magnetite nanoparticles dispersed in water. The magnetite nanoparticles were of roughly spherical morphology, rather uniform in size, and apparently well dispersed. Figure 3c displays the particle size distribution determined by statistical analysis of the dimensions of at least 100 particles, 2798
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Figure 5. Stability of dispersions of (a, b) COOH-TREG-SPIONs and (c, d) DMSA-SPIONs: comparison of hydrodynamic size distributions for the particles (a, c) as prepared and (b, d) after incubation for 1 week at 37 °C.
measured on the TEM micrographs. Fitting to a log-normal distribution yielded an average particle size of 4.8 nm, with 2 nm standard deviation. HRTEM image of the as-synthesized iron oxide nanoparticles is displayed in Figure 3d. Selected area electron diffraction pattern (inset in Figure 3d) of these nanoparticles showed the characteristic diffraction spots of iron oxide nanoparticles. The spacings of the lattice fringes were approximately 4.84, 2.96, and 2.53 Å, which are close to the expected lattice fringes for the (111), (220), and (311) planes of an inverse iron spinel structure of magnetite, thus confirming the results obtained by XRD. Finally, the magnetic behavior of the as-prepared nanoparticles at 300 K is shown in Figure 3e. The nanoparticles displayed superparamagnetic behavior, with a coercivity of 10 Oe and a saturation magnetization of 64 emu/g. Above 150 K the coercivity values were comparable to those of the remanent field of the coils in the magnetometer, that is, the coercivity of the samples is negligible, in agreement with their SPION characteristics. The TREG-coated SPIONs were dispersed in water or PBS to obtain an iron concentration (estimated by ICP-MS) of 1 mg/mL. Figure 4a displays the hydrodynamic particle size distribution in PBS obtained by DLS. The distribution was fitted to a log-normal distribution function with a mean size of 15.7 nm and a standard deviation of 2 nm. This hydrodynamic diameter is about 3 times larger than that obtained by TEM analysis, indicating that the TREG-coated SPIONS form colloidal magnetic aggregates of ca. 10 particles when dispersed in PBS. This is a rather moderate degree of aggregation that shows the effectiveness of the TREG coating to prevent particle agglomeration. However, the TREG molecules are physisorbed on the nanoparticle surfaces, and only a weak interaction is expected, which means that the coating is likely to be unstable. To test this hypothesis, the TREG-coated SPIONS were stored at 37 °C for 1 week, and the particle size distribution was measured
again (Figure 4b). It can be seen that a strong aggregation of the particles takes place: the initial monomodal size distribution at 15.7 nm becomes a bimodal distribution, with an average particle size of 143 nm, and clusters with sizes close to 200 nm can be found. This increase in average particle size is accompanied by a significant increase in polydispersity (standard deviation increased from 2 to 60 nm). At the same time, the ζ-potential measured at pH = 7 changed from 7.7 to 20 mV over the same 1-week period at 37 °C. In Figure 4c, the ζ-potential is plotted as a function of pH for both naked and TREG-coated SPIONs. As expected, the TREG coating shields electrostatic charges on the particles, and as a consequence, the absolute values of the ζ-potential of the TREG-coated-SPION dispersions at low and high pH values are considerably smaller than the values obtained from naked-SPION dispersions. At the same time, the isoelectric point for TREGcoated particles is approximately 6.2, compared to 5.5 for naked SPIONs. This change in the surface-charge potential has often been reported in the literature for hydrophilic polymer-coated SPIONs and has been explained as a consequence of the progressive ionization of the hydroxyl end groups of TREG as pH increases.34 In any case, the shift in ζ-potential at neutral pH from 7.7 to 20 mV (which is close to that corresponding to naked SPIONs) strongly suggests the detachment of part of the TREG coating upon storage at 37 °C, a detachment that would also explain the strong agglomeration observed in Figure 4b. The instability of TREG-coated SPIONs became even more apparent when the particles were subjected to an environment of a higher ionic strength. Figure 4d shows the particle size distribution measured after dispersion of the TREG-coated SPIONs in a 0.18 M solution of NaCl. The aggregation is intense, with an average particle size that is now around 409 nm, and the presence of some clusters approaching a size of 800 nm can be detected. 2799
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Figure 6. ζ-Potential (at pH = 7) of TREG-coate SPIONs, COOHTREG-SPIONs, and DMSA-SPIONs before and after incubation for 1 week at 37 °C.
Most potential biomedical applications of SPION dispersions require their storage at least for moderate periods of time before their clinical application. In addition, they will often be required to be stable under exposure to higher temperatures (e.g., 37 °C) and increased saline concentrations. It is clear that, in spite of their good initial hydrodynamic size distribution, TREG-coatedSPIONs lack stability, due to the weak interaction of the physisorbed TREG molecules with the particles. COOH-TREG-SPIONs and DMSA-SPIONs. The colloidal stability of PBS dispersions of SPIONs with modified coatings (i.e., after replacement of the TREG coating by DMSA, in DMSASPIONs, or after chemical modification of the TREG coating in COOH-TREG-SPIONs) regarding 1 week of storage at 37 °C was measured and the results are presented in Figure 5. From Figure 5, it can be seen that in both cases the stability improved with respect to that of TREG-coated SPIONs shown in Figure 4. In the case of COOH-TREG-SPIONs (Figure 5a), chemical modification of the coating is carried out at the expense of a significant change in hydrodynamic size: the monomodal size distribution of the initial TREG-coated SPIONs (Figure 4a) becomes a bimodal distribution, with the main body of particles centered at around 55 nm and a small fraction at 200 nm. The average size of the as-prepared COOH-TREG-SPIONs is 104 nm. After 1 week at 37 °C, this dual particle size distribution is preserved, and the contribution of the larger size fraction is still minimal, but there is a clear shift in the mean particle sizes, which are roughly doubled (Figure 4b), with an increase in the mean particle size to 120 nm. Therefore, while it can be said that the COOH-TREGSPIONs behave more stably than the TREG-coated SPIONs, (where a significant proportion of the particle populations formed large agglomerates), the increase in stability is not sufficient to avoid some degree of agglomeration upon prolonged exposure at 37 °C. The best results in terms of hydrodynamic stability were obtained with the DMSA-SPIONs. The particle size distribution is unchanged after replacement of the TREG coating with DMSA (Figure 5c) and it stays almost unchanged after 1 week at 37 °C (Figure 5d). This indicates a considerably higher stability of the DMSA coating, even when compared against the chemically modified TREG coating on the SPIONs. The same trend is confirmed when the changes in ζ-potential are compared for the three types of SPIONs after incubation for 1 week at 37 °C (Figure 6). As already discussed, the ζ-potential of the TREG-coated
Figure 7. Hydrodynamic size distribution of DMSA-SPIONs (a) after exposure to high saline concentration and (b) after 10 months of exposure to ambient conditions (light, temperature) in the laboratory.
SPIONs becomes significantly more negative (approaching that of the naked SPIONs), which strongly suggests the loss of part of the TREG coating. In contrast, both DMSA-SPIONs and COOH-TREG-SPIONs present similar values of their ζ-potential before and after incubation, indicating a higher stability of the nanoparticle coating. Especially the DMSA coating is able to form a stable coating through its carboxylic chelate (COOH) bonding, and further stabilization of the ligand shells is attained through intermolecular disulfide cross-linkages between the thiol groups (SH) of the ligands under ambient conditions.32 In both cases, the coating presented reactive carboxyl (COOH) groups that could be used for the later attachment of specific targeting ligands. The ionization of these carboxyl groups increase the negative ζ-potential of carboxilated particles35 and explains the higher negative charge of DMSA-SPIONs and COOH-TREGSPIONs with respect to TREG-coated-SPIONs. Furthermore, the difference in ζ-potential between COOH-TREG-SPIONs and DMSA-SPIONs can probably be attributed to the presence of residual SH groups of the DMSA ligands on the surface.36 The particles with the best stability (DMSA-SPIONs) were also tested at a higher saline concentration. The results are shown in Figure 7a, where it can be seen that the excellent colloidal stability of the functionalized SPIONs is maintained in the presence of an environment with higher ionic strength. A 2800
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Figure 8. Comparison of antigen recognition between antibodies conjugated to nanoparticles and a model anti-rabbit IgGHRP in a one-dimensional indirect ELISA assay. Plates are coated with a rabbit antiserum 15-BSA to IgGHRP-related antibody, a PBS dilution of IgGHRP antibody was added, and a PBS dilution containing rabbit antiserum to IgGHRP (specific As143 and nonspecific As167) attached (right) and not attached (left) to nanoparticles was used as indicator reagent.
with dimensional indirect ELISA conducted after 6 months did not reveal any loss in the biofunctionality of these materials.
’ CONCLUSIONS The TREG-coated SPIONs resulting from a conventional polyol-mediated synthesis present particle size homogeneity (4.8 ( 2 nm) and a good initial particle size distribution, with a mean hydrodynamic size of 15.7 ( 2 nm. However, the TREG layer adsorbed on the magnetite nanoparticles showed weak adherence and was progressively lost when the particles were subjected to temperatures of 37 °C or to environments of increased saline concentration, as indicated by hydrodynamic size measurements and by the changes in the ζ-potential of the nanoparticles. The modification of the TREG coating by a multistep process (involving amination of hydroxyl end groups and the introduction of carboxylic groups) yielded only a moderate increase in stability of the SPION against prolonged exposure to 37 °C (exposure against increased saline concentrations was not tested) but produced a coating with terminal carboxylic groups capable of anchoring a biofunctional antibody. The best results were obtained with DMSA-SPIONs, which yielded a well-dispersed suspension of particles and showed excellent long-term stability, even when subjected to variations in the temperature and saline concentration. Also, in the same way as the COOH-TREG-SPIONs, the carboxylic group was able to immobilize a model antibody with high specific targeting functionality. Finally, the methods described in this work to modify the coating on the TREG-coated nanoparticles are relatively easy to implement and use well-established chemistry and procedures. Because of this, they have the potential to be applied in a wide variety of nanoparticulate systems for biomedical applications. ’ AUTHOR INFORMATION Corresponding Author
*(J.S.) E-mail
[email protected]; tel þ34 976 761153; fax þ34 976 762142. (O.B.-M.) E-mail
[email protected]; tel þ34-91-4978608; fax þ34-91-4973969. Present Addresses
)
secondary population appears at around 55 nm, but the bulk of the nanoparticles remain close to their initial average size, and the mean size of the particle population is virtually the same (13.5 nm versus 17.2 nm). The long-term stability of DMSA-SPIONs is also excellent: Figure 7b shows the particle size distribution of a suspension of DMSA-SPIONs, measured after 10 months under ambient conditions on the laboratory bench (i.e., in the presence of natural light and temperatures varying from 18 to 29 °C), is very similar to that of the as-prepared DMSA-SPIONs. Since DMSA-SPIONs and COOH-TREG-SPIONs presented better stability than TREG-coated SPIONs and also showed reactive COOH end groups, we attempted covalent bonding of a model antibody in order to evaluate the potential application of these materials in biomedical applications. We have selected a purified As143 due to its specific binding properties against anabolic androgenic steroids, such as a methylboldenonehaptenized protein (15-BSA), whose chemistry conjugation and detection procedures have previously been established in our laboratories and therefore form a good basis to judge the functionality of the antibody-SPION conjugates. The conjugation of the functionalized SPIONs with As143 was made through wellknown carbodiimide chemistry, leading to the formation of an amide bound between the carboxylic groups of the functionalizated SPIONs and the amino groups of antibody lysines. The same procedure was conducted for a nonspecific rabbit antibody (As167) that was used as reference. The effectiveness of the antibody conjugation process was analyzed by Bradford’s test. In both cases, the yield of the conjugation reaction was calculated to be around 90%, which indicated that almost all the loaded antibody reacted with the functionalized SPIONs. The specific recognition biofunctionality of the antibody SPION bioconjugates was evaluated in one-dimensional ELISA experiments. In these analyses 15-BSA was coated on the plate, and subsequently serial dilutions of the two different functionalized SPIONs (DMSA-SPIONs and COOH-TREG-SPIONs) coupled with the antibodies (specific As143 and nonspecific As167) were added to the plate. As can be seen in Figure 8, a nonsignal result was observed in the place of As167SPION conjugates, whereas, as expected, a positive signal result was detected in the place of both As143SPION conjugates. These results demonstrate the efficiency of the coupling of the antibody As143 to the functionalized SPION surfaces, as well as the minimal nonspecific adhesion of the conjugates, giving excellent selectivity. In addition, a comparison
Departamento de Física Aplicada, Facultad de Ciencias, Modulo 12, Universidad Autonoma de Madrid, Avda. Francisco Tomas y Valiente 7, 28049, Madrid.
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’ ACKNOWLEDGMENT This work has been supported by the CIBER-BBN (IMAFEN, MICROPLEX, and NANOMAG intramural projects), by the European Commission (contract number ENIAC-120215), and from MICINN, Spain. G.C., J.-P.S., and M.-P.M. acknowledge support as a consolidated Grup de Recerca from the Departament d’Universitats, Recerca i Societat de la Informacio de la Generalitat de Catalunya (expedient 2009SGR1343). G.C. thanks AGAUR of Generalitat de Catalunya for her FI predoctoral grant. ’ REFERENCES (1) Barnejee, R.; Katsenovich, Y.; Lagos, L.; Clintosh, M.; Zhang, X.; Lu., C.-Z. Curr. Med. Chem. 2010, 17 (27), 3120. (2) Figuerola, A.; Di Corato, R.; Manna, L.; Pellegrino, T. Pharmacol. Res. 2010, 62, 126. (3) Lacroix, L.-M.; Hon, D.; Shouheng, S. Curr. Top. Med. Chem. 2010, 10 (12), 1184. (4) Giouroudi, I.; Kosel, J. Rec. Pat. Nanotechnol. 2010, 4 (2), 111. (5) Pankhurst, Q. A.; Thanh, N. K. T.; Jones, S. K.; Dobson, J. J Phys. D: Appl. Phys. 2009, No. 42, 224001. (6) Gould, P. Nano Today 2006, 1 (4), 34. (7) Corot, C.; Robert, P.; Idee, J.-M.; Port, M. Adv. Drug Delivery Rev. 2006, 58, 1471. (8) Namkung, S.; Zech, C. J.; Heimberger, T.; Reiser, M. F.; Schoenberg., S. O. J. Magn. Reson. Imaging 2007, 25 (4), 755. (9) Moran, G.; Salviato, E.; Alessandro, A. Cancer Imaging 2007, 7, S24. (10) Savranoglu, P.; Obuz, F.; Karasu, S.; Coker, A.; Secil, M.; Sagol, O.; Igci, E.; Dicle, O.; Astarcioglu, I. Clin. Imaging 2006, 30 (6), 377. (11) Stark, D.; Weissleder, R.; Elisondo, G. Radiology 1988, 168, 297. (12) Oghabian, M. A.; Gharehaghaji, N.; Amirmohseni, S.; Khoe, S.; Guiti, M. Nanomed.-Nanotechnol. Biol. Med. 2010, 6 (3), 496. (13) Pultrum, B. B.; Van der Jagt, E. J.; Van Westreenen, H. L.; Van Dullemen, H. M.; Kappet, P.; Groen, H.; Sietsma, J.; Oudkerk, M.; Plukker, J.; Th., M.; Van Dam, G. M. Cancer Imaging 2009, 9, 19. (14) Hudgins, P. A.; Anzai, Y.; Morris, M. R.; Lucas, M. A. Am. Neuroradiol. 2002, 23, 649. (15) Gazeau, F.; Levy, M.; Wilhelm, C. Nanomedicine 2008, 3 (6), 831. (16) Hoare, T.; Santamaria, J.; Goya, G.; Irusta, S.; Lau, S.; Lin, D.; Padera, R.; Langer, R.; Kohane, D. Nano Lett. 2009, 9, 3651. (17) Corchero, J. L.; Villaverde, A. Trends Biotechnol. 2009, 27 (8), 468. (18) Barakat, N. S. Nanomedicine 2009, 4 (7), 799. (19) Berry, C. J. Phys. D: Appl. Phys. 2009, 42, No. 224003. (20) Pan, D.; Lanza, G. M.; Wickline, S. A.; Caruthers, S. D. Eur. J. Radiol. 2009, 70, 274. (21) Mornet, S.; Vasseur, S.; Grasset, F.; Durguet, E. J. Mater. Chem. 2004, 14, 2161. (22) Liu, T.-Y.; Hu, S.-H.; Liu, D.-M.; Chen, S.-Y.; Chen, I.-W. Nano Today 2009, 4, 52. (23) Wei, C.; Wan, J. J. Colloid Interface Sci. 2007, 305, 366. (24) Mondini, S.; Cenedese., S.; Marioni, G.; Molteni, G.; Santo, N.; Bianchi, C. L.; Ponti, A. J. Colloid Interface Sic. 2008, 322, 173. (25) Theppaleak, T.; Tumcharern, G.; Wichai, U.; Rutnakornpituk, M. Polym. Bull. 2009, 63, 79. (26) Li, Z.; Chen, H.; Bao, H.; Gao, M. Chem. Mater. 2004, 16 (8), 1391. (27) Wan, J.; Cai, W.; Meng, X.; Liu, E. Chem. Commun. 2007, 47, 5004. (28) Maity, D.; Kale, S. N.; Kaul-Ghanekar, R.; Xue, J.-M.; Ding, J. J. Magn. Magn. Mater. 2009, 321, 3093. (29) Yu, W. W.; Chang, E.; Falkner, J. C.; Zhang, J. Y.; Al-Somali, A. M.; Sayes, C. M.; Johns, J.; Drezek, R.; Colvin, V. L. J. Am. Chem. Soc. 2007, 129, 2871.
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