Journal of Polymer Science, Part A: Polymer Chemistry
Synthesis and Characterization of PDEAEMA-based Magneto-Nanogels: Preliminary Results on the Biocompatibility with Cells of Human Peripheral Blood
Aintzane Pikabea, 1 Jose Ramos, 1 Nikos Papachristos, 2 Dimosthenis Stamopoulos,* , 2,3 and
Jacqueline Forcada* ,1
1 POLYMAT, Bionanoparticles Group, Department of Applied Chemistry, UFI 11/56, Faculty of
Chemistry, University of the Basque Country UPV/EHU, Apdo. 1072, Donostia-San Sebastián, 20080,
Spain.
2 Institute of Nanoscience and Nanotechnology, NCSR ‘Demokritos’, Patriarchou Grigoriou &
Neapoleos Str., 153 10, Athens, Greece.
3 Department of Solid State Physics, National and Kapodistrian University of Athens, Zografou
Panepistimioupolis, 157 84, Zografou, Greece.
*Correspondence to: Dimosthenis Stamopoulos (E-mail: d.stamopoulos@inn.demokritos.gr
), Jacqueline
Forcada (E-mail: jacqueline.forcada@ehu.es
)
S1. Energy-Dispersive X-ray Spectroscopy of the synthesized MNPs
Figure S1 : EDS analysis of MNPs.
S2.
Effect of the lyophilization process on nanogels swelling behavior
Figure S2
: Hydrodynamic diameters of nanogel PM0 (before lyophilization) (□), after lyophilization (+) and MNG PM0m (■) dispersed at pH 6.4 and as a function of temperature.
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S3.
Preparation of single-layered films of MNGs and of MNGs-incubated blood cells
To perform high-throughput microscopy experiments and obtain reliable information, the MNGs and MNGsincubated blood cells should be prepared in single-layered film form with appropriate characteristics. The employed procedure was originally presented for the case of pristine blood cells in [D. Stamopoulos, N. Bakirtzi, E. Manios and E. Grapsa, Int. J. Nanomedicine, 2013, 8, 3887–3894].
Films of MNGs: After redispersing the MNGs in a pH 6 buffer at a solid content of 0.05 wt%, the dispersion was sonicated for 15 min and filtrated by using homemade filters based on standardized cotton-columns of known retention response to remove aggregates above a threshold size. Single-layered films of MNGs were prepared on standard microscope glass slides (Corning, NY, USA) thoroughly cleaned with ethanol, as described in [D.
Stamopoulos, D. Benaki, P. Bouziotis and P. N. Zirogiannis, Nanotechnology, 2007, 18, 495102-495115]. Briefly, a small drop of the MNGs dispersion (50 μL) is deposited onto the glass slide, near one of its corners, that is placed onto the centrifugal spinner, an electronically controlled platform (Headway Research INC MOTOR CONTROL-
101) that can rotate at prefixed conditions (angular speed and duration). The centrifugal force exerted onto the dispersion drop produces a ‘dry’ film in the sense that the particles are left in air environment after their deposition, as the buffer liquid medium evaporates during the process. Most important, by careful adjustment of the angular speed (practically denoted by the rotation frequency in rounds per min, rpm) and of the rotation duration we can produce single layered films of uniformly distributed, however well-separated particles. For the specific concentration of MNGs used here 0.05 wt% and the distance of the dispersion drop from the rotation axis (2.5 cm), the rotation frequency was set to 2.000 rpm and the rotation duration was set to 10 sec. Using the parameters described above, a film of uniformly distributed and well-separated MNG particles was produced onto the glass slide.
Films of MNGs-incubated blood cells: After the incubation experiments, films of the MNGs-incubated blood cells were prepared for the imaging investigations and elemental analysis by microscopy techniques. For the preparation of single-layered films of the MNGs-incubated blood cells the same instrument was used with the same nominal parameters as described in the above paragraph ‘ Films of MNGs ’. Briefly, a drop of 50 μL was placed onto the glass slide, near one of its corners. During rotation the sample was dispersed uniformly on the specific area of the glass slide and was left in the ‘dry’ form of closely packed cells as the plasma or saline evaporated (see section ‘ In vitro experiments’; Sample Series I and III for plasma and Sample Series II for saline).
S4. Microscopy analyses
All MNPs, MNGs, and the MNGs-incubated blood cells were analyzed by microscopy to attain information on the morphological and geometrical characteristics and the elemental composition, as described below.
S4a. Optical microscopy (OM)
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A conventional optical microscope (OM) was employed to observe the MNGs-incubated blood cells.
OM images were recorded in either transmission mode with a LEICA DMRXP (Leica, Wertzlar, Germany).
S4b. Atomic force microscopy (AFM)
Atomic force microscopy (AFM) was employed to acquire data on the morphological and geometrical characteristics of the MNG particles and the MNGs-incubated blood cells by means of a scanning probe microscope
(Solver PRO; NT-MDT Co, Moscow, Russia) with a 100x100x5 μm 3 XYZ scanner hosted on an active vibration isolation table (MOD-1M plus; Halcyonics GmbH, Goettingen, Germany). Measurements were performed in the semi-contact scanning mode with NCH (Non-Contact/tapping mode-High resonance frequency) cantilevers that end with silicon nitride tips (Nano and More GmbH, Wetzlar, Germany) and have the nominal parameters, spring constant = 42 N/m and resonance frequency = 320 kHz. The optimum imaging results were obtained with the following scanning parameters: line frequency 1.5-4 Hz, area = 0.5x0.5 - 30x30 μm 2 , and lines per image = 256-512.
In our AFM measurements we recorded the ‘phase’ signal during each single scan. It is well known that the phase signal provides information referring to the viscoelasticity/adhesion/friction [B. Bhushan and J. Qi, Nanotechnology,
2003, 14, 886-895] of the imaged sample. In addition, referring to the study of MNGs with AFM, it is well known that the semi-contact mode is the optimum mode of operation for soft biomaterials since any problems that can be caused by tip deformation are minimized in this mode [D. Losic, K. Short, J.J. Gooding and J.G. Shapter, J. Serb.
Chem. Soc., 2004, 69 , 93–106].
S4c. Scanning electron microscopy (SEM)
For scanning electron microscopy (SEM) measurements the MNGs-incubated blood cells were deposited (using the technique discussed above) on ultra-smooth silicon substrates that were precoated with a relatively thick (100 nm) underlayer of Niobium (Nb) by means of a sputtering Edwards 306A unit (Edwards, Sanborn, NY, USA). The SEM data were acquired by using an Inspect microscope (FEI, Hillsboro, OR, USA) working with W (tungsten) filament.
Regarding the sample preparation stage, the Nb-precoated silicon substrate hosting the single-layered film of MNGs or blood cells was glued with silver paste onto conventional pin stubs with extra care to short-circuit the holder (pin stub) with the surface of the film at, at least, two different corners of the silicon substrate. Then a thin overlayer (5-
20 nm) of gold (Au) was deposited under medium vacuum (10 -1 Torr) by means of a typical SEM sputtering unit
(E5100; Quorum Technologies Ltd, East Sussex, UK). The double role of the Au overlayer is: to make the sample conductive and to adequately protect it from possible cumulative heating and ultimate evaporation originating from the exposition to the electronic beam. During SEM imaging, an acceleration voltage of 20-30 kV, a working distance of 10 mm, and spot size of 3 were employed.
S4d. Energy-Dispersive X-ray Spectroscopy
Elemental analysis was performed by means of Energy Dispersive X-ray Spectroscopy (EDS). The built-in energydispersive X-ray ability of the SEM was used.
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S4e. Transmission electron microscopy (TEM)
MNPs samples were prepared re-dispersing the MNPs in ethanol at a concentration of 0.33 mg/mL, after grinding the sample using a mortar and pestle. Then, they were sonicated at low temperature in order to avoid overheating.
MNGs samples were prepared re-dispersing the MNGs in a buffer of pH 6 at a concentration of 0.05 mg/mL and sonicated for 1-1.5 h at low temperature, so as to avoid overheating. All the samples were placed onto a Formvarcoated copper grid, which has been previously hydrophilized by a glow discharge process and dried at room temperature. TEM images were obtained by using a high-resolution transmission electron microscope (TECNAI G2
20 TWIN, 200 kV, filament laB6).
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