As already mentioned earlier, for an application of the nanoparticles in aqueous environment, coating with a water-soluble ligand is necessary to obtain stable particle dispersions. To avoid particle aggregation, the coating was performed using the poly(ethylene glycol) biphosphonate ligand, which was already used in chapter 8.1.2. to render the “polyol” route synthesized particles water dispersable. In figure 14 TEM images of the non coated and coated particles are displayed.
Figure 14: TEM images of uncoated and PEG-bisphosphonate coated Gd2O3 nanoparticles
The uncoated particles are, as expected, strongly aggregated due to the disadvantageous preparation method of the TEM specimen. In contrast, the coated particles are discrete, round shaped nanocrystals with visible, less contrasted, ligand on the surface, indicated by the particle roughness. Again, as already mentioned, the problem of the correlation between TEM and PCS analysis is given, suggesting once more the inherent problem of the performed PCS analysis. The particles shown on the TEM image (~ 80 nm) give a size of about 170 nm for their hydrodynamic diameter as shown in figure x, second bar. Nevertheless, it was possible to synthesize coated nanoparticles of different sizes by varying the reaction parameters as exemplary shown in figure 15. The lowest measurable hydrodynamic diameter was about 50 nm in size indicating that the measured particles are even smaller.
0 50 100 150
Size /nm
0,0 0,2 0,4 0,6 0,8 1,0
1 eq urea
Polydispersitiy index
0.2 eq urea
PEG-bisphosphonate coated Gd2O3
Figure 15: PCS analysis of PEG-bisphosphonate coated Gd2O3 nanoparticles synthesized in two different sizes.
These as coated particles of different diameters were applied in an in vitro CT experiment.
For this experiment particles of different sizes coated with PEG-bisphosphonate and dispersed in water, were filled into small vessels of 2 mL volume and put into the computer tomograph.
The as-derived HU amounts for the samples were correlated to the gadolinium content of the samples in table 2.
Table 2: CT contrast, corresponding gadolinium content and HU per mg gadolinium of in vitro CT experiment.
Composition of sample Hydrodynamic diameter /nm
CT contrast of sample /HU
Concentration /mg/mL
HU per mg Gd
GdCl3 in H2O - 209 3.28 64
H2O - 71 0 -
Air - 0 0 -
Gd2O3@PEG-bisphosphonate ~ 170 244 4.14 59
~ 80 234 3.72 63
~ 50 255 4.24 60
All concentrations of the gadolinium samples were checked with ICP-MS and put into relation to their CT contrast. Furthermore, the attenuation of water and air, which must be
zero, was measured. The coated nanoparticles give good in vitro CT contrast, with about 60 HU per mg gadolinium. These, about 60 HU correspond to the HU values of gadolinium chloride in aqueous solution, and marks the inherent attenuation of gadolinium. Therefore, the measured CT contrast of the particulate samples is in very good accordance to the expected values, and no alteration with particle formation and particle size is visible. In figure 16, a cross section of the investigated samples with the respective HU values is shown.
(1) 255 HU
(5) 0 HU
(3) 244 HU
(4) 209 HU
(2) 234 HU
(6) 71 HU (1) 255 HU
(5) 0 HU
(3) 244 HU
(4) 209 HU
(2) 234 HU
(6) 71 HU
Figure16: CT image of nanopariclulate G2O3@PEG-bisphosphonate in 3 different sizes and the corresponding control vials (1) Gd2O3 50 nm; (2) Gd2O3 80 nm; (3) Gd2O3 170 nm; (4) GdCl3 in H2O; (5) air; (6) water.
The brightness of the samples marks their attenuation, and therefore a good visibility of the nanoparticle dilutions in vivo is expected. Moreover, the particles, functionalized with targeting functions, will give remarkably higher contrast at the site of interest and longer circulation times due to the targeting function. A last thing to investigate is the cytotoxicity of these nanoparticles.
8.2.3. Cytotoxicity tests of Gadolinium Oxide Nanoparticles with different coatings The cytotoxicity of the coated gadolinium oxide nanoparticles and free gadolinium ions was tested according to the MTT cytotoxicity test described in chapter 7.5.7. For the assay freshly prepared dispersions of coated nanoparticles in medium, using two different serial dilutions were applied. The chosen concentrations of the nanoparticulate samples and the gadolinium ion concentrations are based on the known toxic gadolinium amount [13]. The amount of particles used was only related to the overall gadolinium concentration in the sample detected
by ICP-OES or ICP-MS, due to the imprecise PCS data. According to this data, no accurate determination of the particle diameter is possible, and therefore, no calculation of the particle concentration. However, all investigated particles had hydrodynamic diameters of about 200 nm, making the experiment comparable. Moreover, the investigated range of gadolinium ion concentration well covers the theoretical amounts of the element in all of the nanoparticulate samples (0.5 – 0.01 µM). The variations of the used nanoparticle amounts, respecting gadolinium concentration, result from the preparation procedure of the particles.
Table 3: Concentrations of nanopartilulate and gadolinium ion samples and their respective cytotoxicity
Concentration Gd3+ Cytotocicity /%
Composition of sample
/µmol/L /µg/mL L929 CHO
0.539 85.0 1.2 15.7
Gd2O3@PA
0.054 8.5 -6.8 12.0
0.119 18.8 6.2 2.8
Gd2O3@CA
0.012 1.9 2.3 -5.4
0.445 70.0 5.7 4.5
Gd2O3@ES
0.045 7.0 0.1 -10.3
0.774 121.8 11.7 6.6
Gd2O3@PEG-bisphosphonate
0.077 12.2 -8.0 -10.2
6.5 1.03·103 66.3 32.4
0.65 103.0 2.2 -13.8
0.07 10.3 10.7 -2.6
Gd3+
0.01 1.0 9.8 -7.3
All investigated nanoparticulate concentrations show, as expected, only very low to no observable toxicity toward the chosen cell lines. In contrast, the highest gadolinium concentration shows a considerable effect on the viability of the cells. In table 3 the gadolinium ion concentrations of the nanoparticles and the control with their respective cytotoxicity on two different cell lines are shown. Figure 17 displays the viability of both cell lines under nanoparticulate and control treatment.
0
0.539 0.054 0.119 0.012 0.445 0.045 0.774 0.077 Gd3+/µM
0.539 0.054 0.119 0.012 0.445 0.045 0.774 0.077 Gd3+/µM
Figure 17: Viability of L929 and CHO cells under treatment with differently coated Gd2O3 nanoparticles and Gd3+, * significantly different p < 0.01.
For the different gadolinium ion solutions, the faster proliferating and more robust CHO cells always showed a slightly lower response in comparison to the L929 cell line. In contrast, for the particulate sample no general trend was observed. This indicates that the observed variations are due to minor variations of the cell numbers or growth activity, but not founded
in toxic or proliferative effects. Therefore, the gadolinium oxide nanoparticles can be said to be not toxic in the tested concentration ranges.
9. References
[1] F. Caruso, Nanoengineering of Particle Surfaces, Adv. Mater. 13(1) (2001) 11--22.
[2] B.M. Silverman, K.A. Wieghaus, J. Schwartz, Comparative Properties of Siloxane vs. Phosphonate Monolayers on A Key Titanium Alloy, Langmuir 21(1) (2005) 225--228.
[3] S. Biggs, P.J. Scales, Y.-K. leong, T.W. Healy, Effects of citrate adsorption on the interactions between zirconia surfaces, J. Chem. Soc. Farad T 91(17) (1995) 2921--2928.
[4] A. Sehgal, Y. Lalatonne, J.-F. Berret, M. Morvan, Precipitation-Redispersion of Cerium Oxide Nanoparticles with Poly(acrylic acid): Toward Stable Dispersions, Langmuir 21(20) (2005) 9359--9364.
[5] M. Aguilar, N. Miralles, A.M. Sastre, Metal complexes with phosphonic and phosphinic acids, Rev. Inorg. Chem. 10(1-3) (1989) 93--119.
[6] P.H. Mutin, G. Guerrero, A. Vioux, Organic-inorganic hybrid materials based on organophosphorus coupling molecules: from metal phosphonates to surface modification of oxides. Cr Acad. Sci II C 6(8-10) (2003) 1153--1164.
[7] L. Qi, A. Sehgal, J.-C. Castaing, J.-P. Chapel, J. Fresnais, J.-F. Berret, F. Cousin, Redispersible Hybrid Nanopowders: Cerium Oxide Nanoparticle Complexes with Phosphonated-PEG Oligomers, ACS Nano 2(5) (2008) 879--888.
[8] A.A. Datea, V.B. Patravaleb, Current strategies for engineering drug nanoparticles, Cur Opin Colloid Interface Sci 9(3-4) (2004) 222--235.
[9] A.M. Pires, M.F. Santos, M.R. Davolos, E.B. Stucchi, The effect of Eu3+ ion doping concentration in Gd2O3 fine spherical particles, J Alloy Compounds 344(1-2) (200344(1-2) 276--279.
[10] U. Rambabu, A. Mathur, S. Buddhudu, Fluorescence spectra of Eu3+ and Tb3+ -doped lanthanide oxychloride powder phosphors, Mater. Chem. Phys. 61(2) (1999) 156--162.
[11] U. Rambabu, K. Annapurna, T. Balaji, S. Buddhudu, Fluorescence spectra of Er3+ : REOCl (RE = La, Gd, Y) powder phosphors. Mater. Lett. 23(1,2,3) (1995) 143--146.
[12] E. Antic-Fidancev, M. Lemaitre-Blaise, P. Porcher, J. Hölsä, Ovservation and Simulation of the Energy Levels of Trivalent Rare Earth Ions in RE Oxyhalide Matrices, Phys. Stat. Sol. A 130 (1992) K142--K153.
[13] Y. Kubota, S.I. Takahashi, G. Patrick, Different cytotoxic response to gadolinium between mouse and rat alveolar macrophages. Toxicol. in Vitro 14(4) (2000) 309--319.