Michal
Kačenka
a,
Ondřej
Kaman
*b,
Jan
Kotek
a,
Lukáš
Falteisek
c,
Jan
Černý
c,
Daniel
Jirák
d,
Vít
Herynek
d,
Klára
Zacharovová
d,
Zuzana
Berková
d,
Pavla
Jendelová
e,
Jaroslav
Kupčík
f,
Emil
Pollert
b,
Pavel
Veverka
b and
Ivan
Lukeš
a
aDepartment of Inorganic Chemistry, Faculty of Science, Charles University, Albertov 6, 128 43, Praha 2, Czech Republic
bInstitute of Physics AS CR, Cukrovarnická 10, 162 53, Praha 6, Czech Republic. E-mail: kamano@seznam.cz; Tel: +420 220 318 418
cDepartment of Cell Biology, Faculty of Science, Charles University, Albertov 6, 128 43, Praha 2, Czech Republic
dInstitute of Clinical and Experimental Medicine, Vídeňská 1958, 140 21, Praha 4, Czech Republic
eInstitute of Experimental Medicine AS CR, Vídeňská 1083, 142 40, Praha 4, Czech Republic
fInstitute of Inorganic Chemistry AS CR, Řež u Prahy, 250 68, Czech Republic
First published on 20th October 2010
The present study reveals the potential of magnetic nanoparticles based on the La0.75Sr0.25MnO3 perovskite manganite for magnetic resonance imaging (MRI). Moreover, it describes the development of the dual imaging probe where the magnetic cores are combined with a fluorescent moiety while the improved colloidal stability is achieved by a two-ply silica shell. At first, the magnetic cores of La0.75Sr0.25MnO3 are coated with a hybrid silica layer, comprising a covalently attached fluorescein moiety that is subsequently covered by a pure silica layer providing the enhanced stability. The detailed characterization of the intermediate and the final product reveals the importance of the complex two-ply shell. Viability tests show that the complete particles are suitable for biological studies. Internalization of the particles and their presence in intracellular vesicles are observed by fluorescence microscopy in different cell types. Further experiments prove no fatal interference with the vitality and insulin releasing ability of labeled pancreatic islets. Relaxometric measurements confirm high spin–spin relaxivities at magnetic fields of B0 = 0.5–3 T, while visualisation of in vitro labeled pancreatic islets by MRI is successfully tested.
Although MRI itself is very efficient, the utilization of a contrast agent (CA) can further improve the resolution and informative content of the obtained images. From the physical point of view, there are two major families of CAs classified according to the relaxation process they predominantly accelerate.2 Whereas T1-CAs induce a positive contrast, i.e. a 1H NMR signal of the affected tissue increases, compounds affecting the T2 relaxation cause lowering of a local proton signal and, thus, they show a “negative enhancement” pattern.3 From the chemical point of view, T1-CAs are complexes of paramagnetic metal ions, such as Gd(III) or Mn(II), with suitable organic ligands. On the other hand, T2-CAs, developed slightly later, are based on magnetic nanoparticles, at present predominantly iron oxide nanoparticles. Basically, they consist of magnetite (Fe3O4) or maghemite cores (γ-Fe2O3) of various sizes ranging from several nanometres to several tens of nanometres covered by e.g.dextrans or polysiloxanes. Hence, their overall size reaches several tens to several hundreds of nanometres. In clinical practice, the iron oxides nanoparticles are used as blood-pool and more recently as organ-specific CAs.4,5
Even though the clinical use of T2-CAs is not so wide-spread as the use of T1-CAs, research in this field has continued because of their exceptional efficiency for high field MRI scanners.4 This is the reason why other nanomaterials exhibiting tunable magnetic properties and extraordinary relaxometric properties have been introduced.6 Among these, the most important are the complex oxide nanoparticles (ferrites,7 manganites8,9) and the metallic nanoparticles (Fe,10FePt,11FeCo12) but the utilization of the latter is limited due to their high chemical reactivity.
As mentioned above, MRI shows excellent spatial resolution; on the other hand, the sensitivities of other techniques, such as optical methods, single-photon emission computed tomography (SPECT) or positron emission tomography (PET), are much higher. Thus, the design and synthesis of so called dual or multimodal probes is an important field. The combination of both respective approaches utilizing only one dual probe, e.g. magnetic nanoparticles tagged with fluorescent moieties, establishes a very useful method for bioimaging. Such an application enables efficient cellular labelingin vitro followed by in vivo tracking of the cells implanted into the organism.13
Moreover, fluorescent magnetic nanoparticles are promising materials for other medical applications, where the same tool might be used either for diagnostics or for therapy, like for magnetic hyperthermia and optically driven surgery. The positioning of the magnetic cores with the external magnetic field could be used in cell micromanipulation.14,15
While bare magnetic nanoparticles exhibit neither sufficient colloidal stability in water nor fluorescent properties, it is necessary to introduce some surface coating able to tag a fluorescent agent and to provide solubilization and biocompatibility of the whole particles. Bifunctional low-molecular compounds possessing strong affinity to the surface of particles and simultaneously a highly hydrophilic moiety (e.g.dimercaptosuccinic acid, citric acid) seems to be the simplest way to meet these requirements. However, surface modification by biopolymers (e.g.dextran, chitosan), synthetic polymers (e.g. polyethyleneglycol, polyvinylalcohol) or a silica layer represents a genuine coating provided that the surface layer is stable and tightly attached. Consequently, such a shell protects the magnetic core from the surrounding biological environment and vice versa.6,16 The fluorescent agents employed in the composite nanoparticle architecture involve mainly organic fluorescent dyes (e.g.fluorescein, rhodamine),17 lanthanide and transition metal complexes covalently attached to the coating material,18,26 or nanoparticle devices like CdSe quantum dots,19carbon nanotubes,20etc. In spite of the mentioned potential of dual probes, their development is still in the early stages. Only very few publications have been dealing with the preparation of fluorescent magnetic particles, that were tested either in vitro or in vivo, thus proving they are suitable for biological and biomedical utilization, and simultaneously such particles that are based on other magnetic cores than iron oxides.14,18
The present study reports on the preparation and properties of new fluorescent magnetic nanoparticles based on the perovskite manganite La1−xSrxMnO3 (LSMO) where x = 0.25 that are coated by a double silica layer. The magnetic properties (i.e. magnetization and Curie temperature) of LSMO nanoparticles may be efficiently tuned by varying both the compositional parameter x (describing the level of hole doping of the mother phase LaMnO3) and the particle size.21,22 The LSMO nanoparticles are promising mediators for magnetically induced hyperthermia due to the self-controlled heating efficiency ruling out the risk of local overheating.22 However, bare LSMO nanoparticles are neither colloidally stable in water nor acceptable for biological applications and thus a surface coating has to be employed. Encapsulation into silica was successfully used to prepare colloidally stable hybrid nanoparticles possessing high magnetization, heating efficiency and low toxicity.9,23 Bhayani et al. reported also low toxicity for LSMO (x = 0.3) encapsulated in dextran or coated by bovine serum albumin.24 More recently, a detailed study of relaxometric properties of silica coated LSMO nanoparticles performed by our group has shown substantial r2 relaxivity far exceeding the relaxivities of iron oxides.9
In order to extend the possible applications of these particles, the present report describes the preparation of LSMO (x = 0.25) nanoparticles coated by a double silica layer containing fluorescein moieties covalently attached to the inner layer. The size and morphology of the prepared particles are investigated by transmission electron microscopy (TEM) and photon correlation spectroscopy (PCS). The silica layer is characterized by means of ζ-potential measurements, infrared (IR) and luminescence spectroscopies. The properties important for contrast agents are investigated using relaxometric techniques accompanied by SQUID magnetometry, while the fluorescence microscopy used in biological experiments evaluates the suitability of the nanoparticles for intended applications.
Scheme 1 Preparation scheme of fluorescent nanoparticles. |
The other method (preparation of LSMO@siF@si-w) employed gentle separation of LSMO@siF by centrifugation (4000 rpm, 70 min, r = 10.4 cm), followed by a washing cycle in ethanol. The solid residue was redispersed in the new reaction mixture (400 mL of ethanol and 64 mL of ammonia) and TEOS (167 μL, 745 μmol) was added. The ultrasound irradiation was terminated after 5 min and then reaction mixture was stirred mechanically for 12 h in the dark at ≈40 °C. Both products were collected viacentrifugation (6000 rpm, 40 min, r = 8.5 cm) and washed three times with ethanol and three times with water. In both cases, the heavy fractions were removed by gentle centrifugation (3000 rpm, 15 min, r = 8.5 cm) of their diluted water suspensions, while final products were isolated from the corresponding supernatants and redispersed in water (50 mL). In order to remove residual ethanol, that could negatively affect biological studies, the suspensions were treated in a vacuum drier at 35 °C for 1 h. Thereafter the products were drained from the beakers without the material deposited on the walls. The overall yield of the final fraction was typically 10–15%.
Rat pancreatic islets (PIs) were incubated for 24 h in CMRL-1066 medium containing the product at a concentration corresponding to 0.11 mmol(Mn) L−1. The vitality of labeled PIs was determined by counting dead cells under the fluorescence microscope in distinct islets after counterstaining with acridine orange and propidium iodide. The ability to produce insulin was tested using a static incubation technique. The uptake of nanoparticles into PIs was investigated by means of immunofluorescence detection employing fluorescent dye tagged antibodies for distinguishing different cell types within the islet. Gel samples of labeled PIs were prepared for visualization in the 4.7 T MRI device. For a more detailed description see ESI.†
Fig. 1 The pH dependence of ζ-potential for the intermediate and both the final products. |
The preparation of FITC derivatized silica and silica coated nanoparticles is widely reported in the literature, but only a few studies discuss the colloidal stability in detail. Generally, different ratios of fluorescein, APS and TEOS are used, but mostly relatively small amounts of the dye that hardly influences the stability of the suspension. Nevertheless, a similar lack of colloidal stability in the case of the surface layer containing fluorescein was also described.27
Therefore the LSMO@siF nanoparticles were subsequently coated in the additional step by a thin secondary silica layer prepared from TEOS. Two different approaches, the first being the formation in situ after the first coating step, and the second one involving the separation and purification of LSMO@siF followed by encapsulation in the new reaction mixture, were tested. The single-vessel preparation of the product LSMO@siF@si-u (the intermediate LSMO@siF was not isolated) is facile in comparison to more elaborate work leading to the latter one LSMO@siF@si-w (the intermediate LSMO@siF was washed prior to the next reaction step), but the latter definitely exhibits much higher stability. Simple observation of water suspensions showed that LSMO@siF@si-u is stable in water for approximately one week, after which sedimentation occurs, in contrast to LSMO@siF@si-w providing suspensions which are stable for much longer periods. Furthermore, the fluorescein is partially released to the supernatant of the LSMO@siF@si-u suspension after approximately one week whereas no fluorescein leaching was observed for LSMO@siF@si-w during several weeks of storage. As can be seen in Fig. 1, the ζ-potential dependences on pH differ for these samples, as well. The isoelectric point IEP of LSMO@siF@si-w is ≈4.5, whereas the IEP of LSMO@siF@si-u, being ≈5.5, is the same as for the intermediate product LSMO@siF. A comparison of the product LSMO@siF@si-u and LSMO@siF indicates similar acidobasic surface properties given by the free amino groups. The IEP of LSMO@siF@si-w is still not equal to the value reported for pure silica nanoparticles (IEP = 2.9 found for particles prepared by the Stöber process at 40 °C)28 or pure silica coated LSMO particles (IEP = 3.5),23 but the colloidal behaviour of its suspension is sufficient for the intended studies. The PCS measurement of a fresh LSMO@siF@si-u sample and the LSMO@siF@si-w product evidenced their colloidal stabilities in water and showed reasonable distributions of the hydrodynamic sizes (see Fig. S3 in ESI†) with the mean values dhydro = 147 nm and 153 nm, respectively. The differences between the size distributions of the samples are not significant taking into account the uncertainties and repeatability of determination and sample preparation, but the nanoparticles seem to be larger and exhibit a broader distribution than the silica coated LSMO nanoparticles of the same dXRD covered by an approximately 20 nm thick silica shell, for which dhydro,50 = 135 nm was reported in the previous study.23 Nevertheless, the particles coated with pure silica were measured by Photon Cross Correlation Spectroscopy (PCCS) eliminating the multiple scattering and thus providing lower values than PCS. On the other hand, the more complex preparation of fluorescent magnetic particles LSMO@siF@si could lead to a broader size distribution. Besides the hydrodynamic size, the mean size of manganite crystallites in LSMO@siF@si-u and LSMO@siF@si-w was determined to be almost the same, 26 and 25 nm respectively, which is larger by 5–6 nm than the mean value found for the starting LSMO nanoparticles. Its increase is an expected consequence of the size fractionation that occurred mainly during separation of LSMO particles from the PVP solution, when the exhaustive centrifugation was not carried out thus the lightest fraction of the nanoparticles (the smallest fragments from mechanical processing) was removed with the supernatant.
The morphology of all three products was checked by TEM and HRTEM studies (see Fig. 2). The image analysis of the TEM data employing the spherical approximation provided the silica shell thickness of ≈11 nm for LSMO@siF and revealed its increase by ≈5 nm during the secondary encapsulation. The overall diameter of LSMO@siF@si-u was estimated to be dTEM = 89 (19) nm, while the diameter of the manganite core was found to be 57 (17) nm, i.e. it is significantly larger than the dXRD value (see also Fig. 2). It implies that besides the single manganite cores the particles consist of connate crystallites not broken during the mechanical treatment, eventually of physically aggregated crystallites (see Fig. S4 in ESI†). Almost the same results were obtained for LSMO@siF@si-w. The employed spherical approximation remains controversial due to the broad distribution of core shapes and thus the calculated parameters need not describe the actual dimensions precisely. However, for the coated products with the same type of core these results enable a relative comparison of the shell thicknesses and the overall sizes, providing also their rough estimations.
Fig. 2 TEM images of a) the intermediate product LSMO@siF and both the final products: b) LSMO@siF@si-u and c,d) LSMO@siF@si-w. |
The IR spectra (see Fig. S5 in ESI†) were recorded for both the final and the intermediate products and in addition for the nanoparticles obtained by the encapsulation employing TEOS and APS (LSMO@siA)9 in order to unambiguously resolve the origin of the bands. The spectra of all these samples show four absorptions bands typical for hydrated silica with silanol groups. The maxima at around 1100, 945, 800 and 470 cm−1 are given by the asymmetric stretching vibration of Si–O–Si, the bending of silanol groups, the symmetric stretching vibration of Si–O–Si and its bending vibration, respectively.29 The band of the asymmetric stretching vibration νas(Si–O–Si) is more or less divided into two components as a consequence of transverse optical–longitudinal optical (TO–LO) splitting observed for both the crystalline and vitreous SiO2.30 The transverse optical vibrational mode occurs at lower energies while the longitudinal optical mode manifests at higher wavenumbers as a shoulder or even a separate maximum. The intermediate product LSMO@siF exhibits other band, not observable in the IR spectra of the final products due to the shielding effect of the whole shell or its top silica layer. Namely the band found at ≈615 cm−1 corresponds to MnO6 octahedra31 and its wavenumber is characteristic for LSMO nanoparticles.23 The weak bands at 1480 and 1380 cm−1 are connected with the organic moiety of APS, as is obvious from the comparison to the LSMO@siA spectrum. Probably, the first band corresponds to the symmetric deformation mode32 of NH3+ and the latter to the scissoring of CH2.33
Fig. 3 The r2 relaxivity dependence on temperature for LSMO@siF@si-w that is related to the decrease of its specific magnetization M750kA m−1 with increasing temperature depicted in the inset. |
The fluorescent properties of the final products were investigated and the corresponding spectra are depicted in Fig. S6 in ESI† together with the spectra of the pure silica coated LSMO nanoparticles LSMO@si that were prepared for comparison.23 Similarly, the reference spectra of the starting FITC, the reaction mixture of FITC-APS in anhydrous ethanol and the mixture after its hydrolysis in water were recorded (see Fig. S7†). The excitation spectrum of LSMO@siF@si-u shows more complex structure while its broad emission maximum is found at around 514 nm. Namely the main excitation maximum at 492 nm and the mentioned emission maximum correspond well to the maxima reported for fluorescein itself.35 The reference excitation spectra of pure FITC and its subsequent products exhibit also few maxima that explain together with the scattering of the particles the complex nature of the LSMO@siF@si-u spectrum. The actual scattering contribution should be comparable to the spectrum of pure LSMO@si (Fig. S6†). The excitation spectrum of LSMO@siF@si-w measured at λem = 514 nm is similar to the corresponding excitation scan of LSMO@siF@si-u, although the maximum at 492 nm is relatively weaker taking into account the concentration of the nanoparticles that determines the actual amount of scattered light. In the case of LSMO@siF@si-u, the amount of scattered light, forming the background, and consequently the concentration of particles were lower in comparison to those of LSMO@siF@si-w. Furthermore, the differences between the emission spectra (λex = 492 nm) of the two products markedly demonstrate the lower fluorescein content in LSMO@siF@si-w since its band at 514 nm, though well visible in the spectrum of LSMO@siF@si-u, is superimposed by the significant scattering contribution (see also the emission spectrum of LSMO@si). On the other hand, the comparison of the emission spectrum of LSMO@siF@si-w obtained with λex = 465 nm and the one of LSMO@si better indicates the presence of the fluorescein band in the former spectrum.
Fig. 4 Viability tests of HeLa cells and fibroblasts exposed to various concentrations of LSMO@siF@si-u and LSMO@siF@si-w particles. |
The mean relative fluorescence intensities of the cells, measured by flow cytometry in the FITC channel, are plotted against the concentration of appropriate nanoparticles involved in the incubation in Fig. S8 in ESI.† For HeLa cells the fluorescence intensities are lower for LSMO@siF@si-w in accordance with lower content of fluorescein, but the values could be affected by the different uptake of the distinct products of LSMO@siF@si-w and LSMO@siF@si-u by the cells as well. The fluorescence microscopy confirmed that tested nanoparticles are localized inside the cells and revealed that the particles are present in numerous intracellular vesicles with patterns similar to late endosomes and lysosomes and becoming apparently expanded at higher concentrations. They are visible as perinuclear patches (see Fig. 5) whilst the bulk area of the cell remained clear. At higher concentration of the labeling agents (0.22 and 0.33 mmol(Mn) L−1), the fibroblasts were obviously affected by the nanoparticles and certain morphological changes occurred in contrast to the incubation at lower concentrations (0.011, 0.055 and 0.11 mmol(Mn) L−1) where the cells remained unchanged and the fluorescence intensity was still very high. For all the studied concentrations, the specific fluorescence of the nanoparticles in the cells was much higher that the autofluorescence of the lysosomes.
Fig. 5 Fluorescence microscopy images of the cells incubated with nanoparticles at the given concentration and subsequently washed with the PBS buffer: a) HeLa cells – LSMO@siF@si-u, 0.011 mmol(Mn) L−1, overlay image of fluorescence and bright field; b) HeLa cells – LSMO@siF@si-w, 0.33 mmol(Mn) L−1; c) fibroblasts – LSMO@siF@si-w, 0.055 mmol(Mn) L−1 and d) rMSCs – LSMO@siF@si-w, 0.13 mmol(Mn) L−1; blue spots – DAPI stained nuclei; green spots – LSMO@siF@si-w. |
Considering the large potential of stem cell transplantation for organ repair and consequent efforts in contemporary biomedicinal research, an efficient dual contrast agent for labeling and tracking these cells would be very useful. As a model for testing the suitability of the studied nanoparticles for cell labeling, rat mesenchymal stem cells (rMSCs) were chosen as an example of adult stem cells, since they are easily derived from bone marrow, they can differentiate into a variety of specialized cell populations, and efficiently proliferate in vitro. In addition, they can migrate to tumors and therefore serve in gene therapy as gene carriers. Tests carried out in the similar concentration range of 0.026–0.51 mmol(Mn) L−1 for 48 h showed viability higher than 85% in all the experiments with LSMO@siF@si-w and LSMO@siF@si-u. These observations indicated that the particles can be used for rMSCs labeling in spite of the generally higher sensitivity of stem cells. Similar green vesicles to those in HeLa cells and fibroblasts were observed inside the rMSCs using fluorescence microscope (see Fig. 5d) whereas the nuclei remained completely dark due to the absence of the fluorescent particles.
Results obtained with rMSCs are generally comparable with results obtained from labeling experiments with fibroblasts, also adherent primary cell culture. The presence of the nanoparticles obviously affected cell proliferation and adherent properties—after incubation approximately 25% of cells were floating. It is not clear if this is due to changes in surface properties of the culture plastic well (caused by sedimentation of silica particles), or if the cells have partially lost their ability to adhere in the presence of nanoparticles. The cells remained alive, however a type of apoptosis called anoikis can be initiated once the cell adhesion to the matrix is disrupted.36 No difference in viability in rMSCs labeled with LSMO@siF@si-u or LSMO@siF@si-w was found. However, the percentage of floating cells was lower (10%) in cells labeled with LSMO@siF@si-w and thus the preparation of the dual probe involving the isolation of the intermediate product proved to be more convenient for subsequent stem cell labeling.
Type 1 diabetes mellitus characterized by autoimmune pancreatic islet (PIs) destruction can be treated by transplantation of the PIs into the liver. The success of such transplantation can be efficiently monitored by MRI, but bioimaging of PIs themselves still constitutes an important topic of current biomedical research. The in vitrolabeling of PIs with LSMO@siF@si-u and LSMO@siF@si-w, carried out at the concentration of 0.11 mmol(Mn) L−1 for 24 h, provided promising results. The vitality of PIs exceeded 75% in all experiments, namely vitalities of 75 and 83% were found for LSMO@siF@si-u and vitality of 87% was observed two times for LSMO@siF@si-w. The study of the insulin releasing ability of PIs treated with LSMO@siF@si-u provided stimulation indexes of 2.3 and 4.9 in two experiments, thus showing that their activity is not impaired completely even after incubation with the mentioned product.
Microscopic investigation after immunofluorescent staining of the labeled PIs revealed that the nanoparticles were present inside the peripheral islet cells. They were internalized in various cell types, including insulin producing beta-cells, without any observable differences in the corresponding fluorescence intensity of the particular types (see Fig. 6). Also the contrast agent was not preferentially taken by the rare islet macrophages. The material internalized in the islet cells is, similarly to the above mentioned labeling experiments, present in vesicles of lysosomal/endosomal pattern.
Fig. 6 a) Immunofluorescent staining of the labeled PIs: red spots – immunostained c-peptide indicating beta-cells, green spots – LSMO@siF@si-w, blue spots – DAPI stained cell nuclei; b) T2w MR image of the labeled PIs mounted in agar (4.7 T, well diameter 35 mm). |
The possibility of visualization of labeled PIs by means of MRI was proved by the T2w MR image measurement (see Fig. 6) of the sample in a gel at B0 = 4.7 T. The corresponding MR signal of the labeled PIs was so strong that they were observable in MR images acquired even with one acquisition, while usually many acquisitions are necessary. Let us mention that until this time only iron oxide based particles have been used for labeling of PIs by T2 contrast agents.
TEM studies showed uniformly coated magnetic cores that do not form larger aggregates in the final product and further it confirmed together with IR spectroscopy the increase of the shell thickness during the two-step procedure. Enhanced colloidal stability was proved by the ζ-potential dependence on pH, while the luminescence spectroscopy demonstrated the presence of fluorescein moieties in the nanoparticles. The relaxometric study at magnetic fields of B0 = 0.5, 1.5 and 3 T at 20 °C revealed extraordinarily high spin–spin relaxivities r2 = 580, 540 and 520 s−1mmol(Mn)−1 L, respectively, sharply exceeding the relaxivities of iron oxides. The relaxometric results were also discussed in relation to magnetic characterizations in static field, namely supporting the temperature dependence of r2.
In vitro experiments performed on HeLa cells, fibroblasts and rMSCs indicated high cell viability, generally exceeding 85%. Simultaneously significant uptake of the nanoparticles and their localization in the intracellular vesicles of late endosomal or lysosomal pattern were observed directly by fluorescence microscopy. Islets of Langerhans exhibited good vitalities during in vitrolabeling whereas their insulin releasing capability was retained. On the other hand the labeled islets provided very strong MR signals due to the uptake of the nanoparticles.
Footnote |
† Electronic supplementary information (ESI) available: Detailed experimental section (description of characterization methods settings, preparation of starting materials, biological experiments), additional figures mentioned within the text. See DOI: 10.1039/c0jm01258k |
This journal is © The Royal Society of Chemistry 2011 |