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ISSN: 2157-7439
Journal of Nanomedicine & Nanotechnology
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Hemocompatibility and Biomedical Potential of Poly(Gallic Acid) Coated Iron Oxide Nanoparticles for Theranostic Use

Márta Szekeres1*, Erzsébet Illés1, Christina Janko2, Katalin Farkas3, Ildikó Y Tóth1, Dániel Nesztor1, István Zupkó4, Imre Földesi3, Christoph Alexiou2 and Etelka Tombácz1*

1Department of Physical Chemistry and Materials Science, University of Szeged, Aradi vt 1, 6720 Szeged, Hungary

2Department of Otorhinolaryngology, Head and Neck Surgery, Section for Experimental Oncology and Nanomedicine (SEON), University Hospital Erlangen, Glückstraße 10a, 91054 Erlangen, Germany

3Institute of Laboratory Medicine, Faculty of Medicine, University of Szeged, Semmelweis u. 6, 6720 Szeged, Hungary

4Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 1, 6720 Szeged, Hungary

*Corresponding Author:
Szekeres M
Department of Physical Chemistry and Materials Science
University of Szeged, Aradi vt 1
6720 Szeged, Hungary
Tel: 36-62544212
E-mail: [email protected]

Tombácz E
Department of Physical Chemistry and Materials Science
University of Szeged, Aradi vt 1
6720 Szeged, Hungary
Tel: 36-62544212;
E-mail: [email protected]

Received Date: October 13, 2014; Accepted Date: December 10, 2014; Published Date: January 01, 2015

Citation: Szekeres M, Illés E, Janko C, Farkas K, Tóth IY, et al. (2015) Hemocompatibility and Biomedical Potential of Poly(Gallic Acid) Coated Iron Oxide Nanoparticles for Theranostic Use. J Nanomed Nanotechnol 5:252. doi:10.4172/2157-7439.1000252

Copyright: © 2015 Szekeres M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Polyacid covered core-shell iron oxide nanoparticles were designed for potential use in biomedicine with special attention to theranostics - magnetic resonance imaging (MRI), magnetic hyperthermia and magnetic drug targeting. The magnetite nanoparticles coated with a gallic acid shell polymerized in situ on the nanoparticle surface ([email protected]) were tested for hemocompatibility in blood, sedimentation rate, blood smear and blood cell viability experiments and for antioxidant capacity in Jurkat cells in the presence of H2O2 as reactive oxygen species. No signs of interaction of the nanoparticles with whole blood cells were found. In addition, the [email protected] reduced significantly the oxidative stress mediated by H2O2 supporting earlier findings of MTT tests, namely, the improvement of cell viability in their presence. The in vitro tests revealed that [email protected] are not only biocompatible but also bioactive. Preliminary experiments revealed that the nanoparticles are especially efficient MRI and magnetic hyperthermia agents. The r2 relaxivity was found to be one of the highest among published values (387 mM-1s-1) and they possess a relatively significant specific absorption rate (SAR) value of 11 W/g magnetite.

Keywords

Iron oxide nanoparticles; HeLa, Jurkat and blood cells; Hemocompatibility; Antioxidant effect; MRI contrast enhancement; Magnetic hyperthermia

Introduction

A wide variety of core-shell magnetic iron oxide (magnetite) nanoparticles (MNPs) has been designed up to date for the aims of magnetic imaging (magnetic resonance imaging, MRI; magnetic particle imaging, MPI), magnetic hyperthermia and targeted drug delivery [1-10]. The combined use of MNPs in both diagnosis and therapy (called theranostics) exploits the superparamagnetic property of nano-sized magnetite (also called SPION, “superparamagnetic iron oxide nanoparticle”) and its specific surface properties that allow easy surface modification by physical or chemical adsorption of different organics. The shell around the core particles ensures colloidal stability and salt tolerance, which are necessary to prevent aggregation in the biological environment. In addition, the coating layer can host biologically active molecules such as drugs and targeting agents.

Using surface-polymerized gallic acid (PGA) as protecting shell of nanoparticles can be highly advantageous in biomedicine, because gallic acid (GA) is not only biocompatible but also bioactive. GA is one of the most important antioxidant components of a healthy diet. The anti-inflammatory, antibiotic, antifungal and anticancer effects of GA are well known as well as its role in preventing cardiovascular diseases and neurodegenerative disorders [11-15]. Many GA-like compounds (phenolic acids) are present in large concentrations in different plants [16]. GA has been used as a model for food polyphenols [17] and the concentration of dietary polyphenols is often expressed in gallic acid equivalents. GA-like compounds are also used in biomedicine as drugs for toxic metal removal due to their capability of chelating heavy metal and transition metal ions [18]. Their application for curing degenerative diseases is promising, because they can cross the blood brain barrier and chelate toxic ions in the brain thus preventing or even inverting the formation of plaques [13,14]. When attached to magnetic nanoparticles, the curing efficiency can be enhanced via magnetic targeting.

We prepared GA-coated magnetite nanoparticles and allowed surface-induced polymerization to proceed during the course of five weeks resulting in PGA-coated MNPs ([email protected]). Our hypothesis was that the autogenic polymerization of GA catalyzed by the surface of MNPs should proceed similarly to the known natural process of humic matter formation on the surface of clay minerals, the precursor molecules of which are structurally similar to GA [19]. This way, the synthesis of [email protected] would proceed in an all-natural soft method and the emergence of toxic by-products would be excluded. The [email protected] have indeed been successfully produced and we have published their physico-chemical and colloidal characterization relevant to possible biomedical applications [20-22]. In the present study, we utilized in vitro experiments for testing the biodistribution, biocompatibility and antioxidant capacity of the polygallate stabilized SPION product. In addition, we studied the MRI contrasting and hyperthermia efficiency of the coated MNPs.

Materials and Methods

FeCl2·4H2O, FeCl3·6H2O (Molar, Hungary), gallic acid (GA, Carlo Erba) and NaCl, NaOH and HCl (Molar, Hungary) to set the ionic strength and pH in the experiments were analytical grade reagents. Ultrapure water (18 MΩ) from a HumanCorp Zeneer water purification system was used in the experiments.

Magnetite (Fe3O4) nanoparticles were synthesized by alkaline hydrolysis of iron(II) and iron(III) salts [22]. Briefly, the concentrated iron salt solutions were mixed at the ratio of 1:2 and precipitated by NaOH solution. We washed and acidified the formed MNPs in order to fully disperse them in the aqueous medium, then dialyzed against 1 mM HCl solution and stored in the dark at 4°C.

The [email protected] for the biomedical tests were prepared on the basis of GA adsorption experiments detailed by us earlier [21,22]. The [email protected] dispersions were prepared at a concentration of 15 g/L in aqueous medium. The pH of magnetite dispersions was adjusted to ∼6.5 and GA was added in concentration of 0.6 mmol GA/g magnetite. The pH was readjusted subsequently for five weeks, during which period GA polymerized at the surface as it is demonstrated in [21,22]. The sample was stored in dark at 4oC. The values of electrokinetic potential (-40 mV), hydrodynamic diameter (∼170 nm, as compared to that of naked MNPs of ∼100 nm) and critical coagulation concentration (500 mM, while that of naked MNPs of is only 1 mM) revealed the presence of a thick coating layer, which in turn, stabilizes the particles even in the presence of salt at high concentrations. TEM pictures of naked and PGA-coated MNPs are given in Seciton 1 of Supplementary material (Figures S1 and S2) demonstrating also the formation of a thick PGA coating. Particles with not fully polymerized GA shell ([email protected]) were also characterized earlier [21,22]. Although they were found to be highly biocompatible (cell viability inhibition < 5%), their low colloidal stability made them inapplicable for biomedical application.

Interaction of [email protected] with HeLa cells

The accumulation of [email protected] at the surface of or inside cancer cells was tested via examining their interaction with HeLa cells in a cell culture within 48 hours using Prussian blue staining. The procedure is described in detail in [22].

Hemocompatibility tests

Hemocompatibility of [email protected], i.e. their interaction with human blood was studied in erythrocyte sedimentation rate (ESR) experiments utilizing a Sedi-15 automated sedimentation rate measuring device (BD Inc., Franklin Lakes, NJ, USA) and Seditainer 1.8 vacutainer tubes (BD Inc., USA) as given in our previous publication [22]. [email protected] samples were mixed at room temperature with citrate-, and EDTA-anticoagulated blood of three healthy donors (Donors #1-#3) to achieve a [email protected] concentration of 0.32 mg/mL.

Peripheral blood smear tests of the whole blood (EDTAanticoagulated) of the donors were carried out by an automated slide preparation system (Sysmex SP4000i) at room temperature using the May-Grünwald Giemsa (MGG, Biolyon, Dardilly, France) staining technique in a CellaVisionTM DM96 automation device (CellaVision AB, Ideon, Science Park, Lund, Sweden) as detailed in [22]. The influence of [email protected] on platelet aggregation was studied at a concentration of 0.32 mg/mL. The acquisition and classification software of the instrument was used to differentiate the normal and abnormal cells (white blood cells (WBC), red blood cells (RBC) and platelets (PLT)).

Blood cell viability measurements

WBC viability factor (WVF), i.e., the fraction of viable WBCs present in the sample was determined by CELL-DYN Sapphire hematology analyser (Abbott Diagnostics, USA). This routine diagnostic method is used to differentiate the necrotic, apoptotic and normal cells in EDTA-anticoagulated blood based on the selective staining of nuclei of damaged or nonviable cells with a cell membrane impermeable fluorescent dye, propidium iodide. After hydrodynamic focusing, WVF was determined by the emitted red fluorescence (at 617 nm) of the dye bound to the nucleic acid of injured or dead cells. The effect of PGA coated nanoparticles on WBC viability was examined at 37°C. [email protected] concentration in blood was 0.4 mg/mL. The WVF values were determined 10, 30, 60, 90, 120, 180 and 240 minutes after mixing the MNPs with the blood of Donors #1-#3 at a final concentration of 0.4 mg [email protected]/mL. The accuracy of the measured WVFs is above 97% as calculated from 5 parallel runs. The cell count of WBC, RBC and PLT comprising all viable, damaged and dead cells was determined by impedance measurements in the same samples. The impedance measurements were also used for determination of the volumes of thrombocytes and red blood cells.

Determination of antioxidant capacity

The non-adherent human T-cell leukemia cell line Jurkat (DSMZ ACC 282) was used for in vitro experiments. The cells were cultured in RPMI medium supplemented with 10% fetal calf serum and 1 % glutamine (all from Thermo Fisher Scientific, Waltham, MA, USA) under standard cell culture conditions in a humidified incubator at 37°C and 5% CO2. For the experiments, the cells were counted and their viability was determined using MUSE Cell Analyzer (Merck Millipore, Billerica, MA, USA). The antioxidative capacity of free GA and [email protected] was determined according to the following procedure. Intracellular oxidative stress was determined using 2′,7′-Dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, Taufkirchen, Germany). The cells were adjusted to a density of 5×105 / mL in PBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and pre-incubated with 20 μM DCFH-DA for 30 minutes at 37°C. Then the cells were washed once with PBS and subsequently incubated in cell culture medium with free GA or [email protected] in the indicated concentrations for 1 h at 37°C without further washing steps. As an oxidizing agent H2O2 (Roth, Karlsruhe, Germany) was added to the cells for further 2 h at 37°C. Then the cells were analyzed using a Gallios flow cytometer (Beckman Coulter, Fullerton, CA, USA). Intracellular DCF fluorescence (excitation at 488 nm and emission at 525 nm) was measured by gating of morphologically viable cells. Data analysis was performed with Kaluza software version 2.0 (Beckman Coulter).

MRI studies

The MRI contrast enhancement efficiency of the [email protected] was studied by determining the T1 (longitudinal) and T2 (transversal) relaxation times and calculating r1 and r2 relaxivities as described in our previously published paper [23] by using a clinical MRI instrument GE Excite HDxt (GE Medical Systems, Milwaukee, WI) at magnetic field strength of 1.5 T. The contrast enhancement of coated particles was measured using the standard “birdcage” head coil. The MNP dispersions were prepared at nominal Fe concentrations of 0.009, 0.018, 0.045, 0.09, 0.135, 0.18 and 0.22 mM and the exact iron content of the samples was measured by inductively coupled plasma (ICP) method using an Optima 7000 DV ICP-OES instrument (Perkin- Elmer, Shelton, CT, USA). 4 mL of samples was placed in a plastic box filled with water and put in the centre of the head coil. The r1 and r2 relaxivities were determined as the slopes of the 1/T1 and 1/T2 vs. Fe concentration plots to quantify the contrast enhancement efficiency of the [email protected] The quality of fitting was estimated by the coefficient of determination (R-squared).

Hyperthermia experiments

The magnetic hyperthermia efficiency of [email protected] was tested in a magneThermTM (nanoTherics Ltd., Keele, Staffordshire, UK) instrument. The volume of the MNP dispersion was 4 mL, and the concentration 15 g/L. The measurements were performed at a resonant frequency of 110.7 kHz in a magnetic field of B=25 mT (H=19.9 kA/m, 17 turn coil and 200 nF capacitor). The measurement time in the hyperthermia setup was 15 minutes and the specific absorption rate (SAR, W/g magnetite) values for the initial rate of temperature change were calculated according to the approximating equation (Eq.1):

SAR=(cp,s mwater /m[email protected]) (ΔT/Δt) (1)

where cp,s is the specific heat capacity of the medium (water), mwater and m[email protected] are the masses of the medium and the nanoparticles, respectively, and ΔT/Δt is the initial temperature rate.

Results and Discussion

Interaction of [email protected] with Hela cells

The Prussian blue staining method allowed detecting the distribution of [email protected] in the matrix of a HeLa cancer cell culture. As it is seen in Figure 1, the particles contacted with the cancer cells intimately either by adsorbing at the outer surface or by penetrating into the cytoplasm. The adsorption was observed as the presence of particle agglomerates linked to the cells. In case of penetration, the nuclei remained unstained. Both types of interaction are advantageous for the medical use of the particles. The attachment to the surface and internalization identify the cancer cells in MRI and localize the heat production in hyperthermia. The [email protected] can also be used for targeting specific anticancer drugs, which extends their potential use in the wider field of theranostics. The tendency of cells to internalize negatively charged core-shell nanoparticles, such as [email protected] at physiological pH [20], has been reported frequently [24].

nanomedicine-nanotechnology-Prussian

Figure 1: Prussian blue staining of HeLa cell culture. (A) Control and (B) [email protected] loading at 7.35 mg/mL concentration.

Hemocompatibility of [email protected]

Three standard in vitro assays [25,26], blood sedimentation (i.e., erythrocyte sedimentation rate, ESR), smear and WBC viability tests were used to assess the hemocompatibility of [email protected] Blood sedimentation experiments were performed with citrate-anticoagulated blood samples to examine whether the coated magnetic nanoparticles induce RBC aggregation or not. Although the citrate anticoagulant seems to induce thrombocyte aggregation [22], it can be safely used for blood sedimentation rate measurements in accordance with the daily laboratory routine, since it does not influence the sedimentation of RBCs. In addition, the thin and long citrate-anticoagulated test tubes are suited well for visual observations similarly to the EDTAanticoagulated tubes, but use significantly smaller sample volume. Representative sedimentation results are shown in Figure 2. The height of the settled RBCs is virtually the same both in the absence and the presence of [email protected], but a brownish colour appeared in the plasma after addition of [email protected] Similar effect was found for other types of polyacid-coated MNPs as well [22,27]. The recorded ESR values coincide in the absence and presence of coated MNPs (11 and 8 mm/h, respectively) within the formal accuracy of the measurements ±3 mm/h. The same RBC sedimentation feature was observed in EDTA-anticoagulated test tubes as well. The presence of [email protected] did not influence the aggregation of RBCs in either of the experiments. The same picture was observed previously when GA-coated MNPs (GA adsorbed but not polymerized) were added to whole blood samples [22], with ESR value of 9 mm/h. This suggests that hemocompatibility is determined by the chemistry of the surface of MNPs but does not depend on the thickness of the coating layer.

nanomedicine-nanotechnology-sedimentation

Figure 2: Blood sedimentation test of native blood sample of Donor #3 (left) and of [email protected] mixture at 0.32 mg/mL [email protected] concentration (right).

Peripheral blood smear tests were performed to study the influence of MNPs on platelet aggregation. EDTA-anticoagulated blood samples were used to exclude thrombocyte aggregation observed previously in citrate-anticoagulated blood [22]. The morphological analysis of human blood cells showed neither deformation nor decomposition of RBCs in the presence of PGA coated nanoparticles. As it is seen in Figure 3, no aggregation of RBCs was detected revealing excellent hemocompatibility of [email protected] comparable to that of other polyacid- [22,27], polyethylehene oxide (PEG)- [23] and dehydroascorbic acid (DHA)- [28] coated MNPs. Similar pictures were taken for all the three donor’s blood.

nanomedicine-nanotechnology-smears

Figure 3: Microscopic pictures of the blood smears prepared from EDTAanticoagulated blood of Donor #3 (A) without and (B) with [email protected]

The effect of [email protected] on viability of white blood cells (WBCs) was tested by measuring the viability factor (WVF) and the results obtained for the blood of Donor #3 are demonstrated in Figure 4. The viable fraction of WBCs of EDTA-coagulated native blood decreased from 0.99 to 0.97 after 240 minutes, which is a spontaneous process in in vitro experiments. The oxygen and nutrients concentration of blood decreases with time initiating a gradual cell death right after the sample collection [26]. The PGA coated nanoparticles were dispersed in an aqueous medium and so the effect of the added water on the WBC viability was studied as well. Addition of ultrapure water induced a decrease in WVF from 0.98 to 0.91 within 240 minutes. The dilution caused a stronger decrease in WVF as compared to the undiluted sample probably due to the decrease in the osmotic pressure and an additional decrease in nutrient content [26]. No further change in the time-dependence of WVF was observed upon addition of [email protected] MNP revealing the excellent hemocompatibility of the PGA coated nanoparticles. The relatively empty right region of scatter plot of the blood containing [email protected] (inset of Figure 4) shows that only negligible amount of necrotic and apoptotic cells are present in the sample. The same picture was observed for the water-diluted blood sample, indicating that WBC viability was influenced by dilution, but not by the presence of PGA coated nanoparticles. Similar results were obtained for the blood samples of Donors #1 and #2.

nanomedicine-nanotechnology-anticoagulated

Figure 4: Time-dependent WBC viability in EDTA-anticoagulated blood with and without [email protected] Inset: Fluorescence scatter plot of blood samples with [email protected] FL3-DNA is the fluorescence intensity of DNA-bound propidium iodide and SIZE is cell size determined from intensity loss. The blue and red dots indicate different types of WBC.

Exploiting the multichannel structure of the CELL-DYN Sapphire hematology analyzer, we investigated the change in the cell counts of RBC, WBC and PLT in parallel as a function of time, dilution and [email protected] addition. The results are shown in Figure 5. The total amount of the three blood components did not change significantly during the 240 minutes of the experiment, indicating the stability of the blood sample for the time of inspection. Dilution with ultrapure water reduced the cell count values of WBC, RBC and PLT and the decrease coincided well with the degree of dilution (16%) for all three components. Dilution with [email protected] dispersion led to the same ∼16% decrease in the cell counts evidencing that the presence of the nanoparticles did not affect the stability of the sample.

nanomedicine-nanotechnology-dispersion

Figure 5: Time-dependent change in the cell count of WBCs (6.5-5×106 /L), RBCs (3.7-3.2×109 /L) and PLTs (2.4-2×104 /L) in the blood sample of Donor #3 without dilution and diluted with water and [email protected] dispersion at 37°C.

The mean platelet volume (MPV=8.47 ± 0.2 10-15 L) did not change with time in the [email protected] mixture, which also supports the results from smear experiments that no thrombocyte aggregation occurred in the presence of PGA coated nanoparticles. However, a slight increase was observed in the mean RB cell volume over time (Δ MCV ∼2%, from 87.9 to 90.0 10-15 L) indicating that swelling took place during the experiments. This can be explained either by the change in the osmotic conditions after dilution or by thermal instability of RBCs as the temperature-dependence of their swelling property was observed in other human studies as well [29]. The hemocompatibility experiments, altogether, did not show any tendency for [email protected] to interfere negatively with any of the blood components considered in our experiments. This evidence of hemocompatibility makes the particles good candidates for use as MRI or drug targeting agents being injected intravenously.

Antioxidant effect of [email protected]

The effect of free GA or [email protected] on the cellular morphology of Jurkat cells was analyzed in flow cytometry by means of forward scatter (FS) and side scatter (SS) (shown in Figure S3A, Section 2 of Supplementary material), providing information about cellular size and granularity. Cells undergoing cell death change their morphology, reflected by scatter alterations [30], thus viable cells can be clearly separated from dying or dead cells [31]. Morphological analysis showed that treatment of Jurkat cells with low concentrations of H2O2 (25 μM and 50 μM) and/or GA solutions only induced minimal amounts of cell death during the observation period of 2 h.

Intracellular oxidative stress was determined using the cell permeable non-fluorescent dye DCFH-DA, which is enzymatically hydrolyzed inside the cells by cellular esterases to non-fluorescent DCFH. In the presence of oxidative stress, DCFH is subsequently oxidized to fluorescent DCF [32]. To be sure that the DCF fluorescence is located inside the cells, only cells of the viable gate were considered for analysis of oxidative stress [33]. After treatment with H2O2, an increase in the DCF fluorescence was measured as a function of H2O2 and GA dosing. Pre-incubation of the cells with free GA significantly reduced the H2O2 mediated DCF fluorescence at all tested GA concentrations (shown in Figure S3B, Section 2 of Supplementary material), showing its antioxidative properties.

Next, the question arises whether [email protected] also exhibit antioxidative capacity or not. Since we speculated that GA might be less potent when immobilized on nanoparticles, we used only the higher nominal GA concentrations 25 μM and 50 μM added in the form of [email protected] The morphological analysis showed high biocompatibility of [email protected] during the observation period of 2 h (Figure 6A). After induction of oxidative stress using H2O2, a dose dependent increase in DCF fluorescence was observed. Adding free GA at 50 μM, as reference, reduced the fluorescence intensity similarly as in the above set of experiments using free GA as antioxidant. Two different [email protected] samples were used for the experiments. The first one contained free GA in the supernatant as a result of the adsorption/ polymerization process, i.e., dissolved GA in equilibrium with that on the surface of MNPs. Free GA, on the other hand, was removed from the second sample by washing. The [email protected] dispersions both with and without free GA reduced the H2O2 mediated oxidative stress as it is seen in Figure 6B. However, their effect was weaker as compared to that of free GA. In addition, [email protected] with free GA in the supernatant showed a stronger antioxidative capacity than in the absence of that.

nanomedicine-nanotechnology-oxidative

Figure 6: Reduction of H2O2 mediated oxidative stress by [email protected] (A) Observed resistance of the cells to morphological changes induced by free GA, [email protected], or H2O2during the observation period of 2 h. (B) The changes in mean fluorescence intensity (MFI) of DCF measured in the population of morphologically viable cells in function of dosing of H2O2, free GA and [email protected]

The observed antioxidative capacity of [email protected] is in harmony with our earlier cell viability experiments performed with [email protected] on healthy and cancer cells MRC-5, HeLa and A431 for several types of polyacid-coated MNPs [21]. As compared to the cell inhibition values between 3 and 25% found for citrate, humate, polyacrylate and poly(acrylate-co-maleate) coated MNPs, for [email protected] we have found negative values around -5% at the highest concentration. Thus, cell viability has even appeared to be enhanced in the presence of [email protected] in the cell culture. This result becomes fully explicable after their antioxidant effect was experimentally proved in the present studies of oxidative stress reduction.

MRI contrasting properties of [email protected]

The contrast enhancement efficiency of [email protected] was tested in MRI measurements. The longitudinal and transverse relaxivity values (r1 and r2, respectively) were calculated by plotting the relaxation rates (R1=1/T1 and R2=1/T2) against the Fe concentration as described in our previous papers [23,34]. According to our expectations, small r1 value (1.8 mM-1s-1) was obtained for [email protected], which is characteristic for superparamagnetic iron oxide nanoparticles (SPION) in general [23]. However, remarkable T2 contrast enhancement was recorded for the PGA coated MNPs, shown in Figure 7 as the concentration dependence of the R2 relaxation rate. The calculated value of transverse relaxivity was extremely high (r2 ∼387 mM-1s-1) exceeding the majority of data published for MNPs (50-306 mM-1s-1) [28,34-38]. Resovist (r2 306 mM- 1s-1) [34], in addition, is generally used as reference superparamagnetic iron oxide MRI contrast agent despite its FDA approval has not yet been completed [22]. The high transverse relaxivity of [email protected] is probably due the strong hydration of the highly negatively charged PGA shell near the magnetite core. The enhanced relaxation of large amount of protons near the MNP surface leads to the decrease in signal intensity and increase in the negative contrast [23]. The r2 relaxivity of [email protected] ∼387 mM-1s-1 is somewhat lower than that of naked MNPs (measured at pH ∼4 as 429.5 ± 9.5 mM-1s-1 [34]). The naked MNPs can be stabilized only at low pH, where they hold positive charges due to the accumulation of protons directly on the MNP surface causing the large r2 relaxivity. In the same time, PGA-coated MNPs are negatively charged (stable in wide range of pH [20], necessary for biocompatibility) and they accumulate water dipoles somewhat farther from the MNP surface, where the effect of the local magnetic field on proton relaxation is weaker.

nanomedicine-nanotechnology-relaxation

Figure 7: Transverse relaxation rate (R2=1/T2) of the [email protected] sample as a function of iron concentration and the resulting r2relaxivity value. Inset: T2- weighted image of samples in phantom water (TR=3000 ms, TE=40 ms, Fe concentration increases from left to right).

Magnetic hyperthermia efficiency of [email protected]

The heating effect of two different PGA coated samples was tested and compared to the result obtained for naked MNPs (Figure 8A). The gallic acid modified magnetite samples [email protected] (1) and [email protected] MNP (2) are two different lots produced by the same protocol detailed in the Materials and methods. The frequency and the field strengths (amplitude) of the alternate magnetic field were selected to be appropriate for medical application. The SAR of the MNP and [email protected] MNP samples were calculated from the slopes of the heating rate curves below the clear kinks in the [email protected] heating rate curves at 70-80 seconds [39] as seen in Figure 8B. The obtained SAR values of all the dispersions were nearly identical, around 11 W/g magnetite. This value is fully consistent with others found in the literature, albeit the choice of the parameters of the magnetic field and the magnetization and particle size of the particles among its other relevant properties have tremendous effect on the SAR values [40-44]. The observed heating efficiency of our samples should be sufficient to rise temperature ~5°C above body temperature necessary to induce apoptosis and necrosis of cells in the case when the same particle concentration can be achieved by accumulating them in specific target locations during administration.

nanomedicine-nanotechnology-temperature

Figure 8: (A) Increase in the temperature of aqueous dispersions of naked and PGA-coated MNPs at particle concentration of 15 mg/mL measured in alternating magnetic field during 15 minutes. (B) The initial part of temperature increase.

The finding that PGA coating did not alter the SAR of the MNPs is in agreement with the fact that the hyperthermic effect of the nanoparticles with ∼8 nm diameter is primarily via Neél relaxation [43], i.e., to the alteration of the direction of a single magnetic dipoles but not the particle as a whole. The coating shell could be expected to affect the heating rate in case of the Brownian type of relaxation or relaxation caused by the obvious magnetic hysteresis of larger magnetic particles.

Conclusions

We have performed in vitro biomedical testing of magnetite nanoparticles coated with gallic acid shell polymerized in situ at the surface of the particles in a soft and reagent-free process, similar to that of formation of humic matter in nature. The high chemical and colloidal stability of the particles ([email protected]) were studied and discussed thoroughly in our previous publications [20-22], and the paper on the mechanism of surface polymerization of GA on MNPs is published in parallel [45]. The ease and low cost of preparation combined with the exceptional durability and stability of these particles make them superior candidates for biomedical applications.

A series of in vitro blood compatibility experiments (ESR, blood smears, viability tests of different particulate components of blood WBC, RBC and PLT) revealed that [email protected] do not interact with whole blood, which make them promising candidates for designing intravenous injection formulations as MRI contrast or drug targeting agents. The significant antioxidant capability of the PGA coating measured in ROS tests may be beneficial in the future in vivo applications.

Measurements of r2 relaxivities for MRI and SAR values for hyperthermia applications proved the reliability of the use of [email protected] MNPs as theranostic agents in biomedicine.

Acknowledgements

This work was supported, in part, by the Hungarian Scientific Research Found OTKA (NK84014) and by the Bavarian State Ministry of the Environment and Consumer Protection.

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Nursing & Health Care Journals

Stephanie Skinner

[email protected]

1-702-714-7001Extn: 9039

Medical Journals

Nimmi Anna

[email protected]

1-702-714-7001Extn: 9038

Neuroscience & Psychology Journals

Nathan T

[email protected]

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

Ann Jose

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1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

[email protected]

1-702-714-7001Extn: 9042

 
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