Grafting DTPAA and labeling Re-188 on amino-functionalized magnetic nanocomposites
Figure 1 is the scheme of amino-functionalized magnetic nanoparticles undergone DTPAA modified and Re-188 labeled. Through previous research findings, it is known that the surface of amino-functionalized magnetic nanoparticles contains abundant amino compounds. Especially under Ninhydrin Reaction, it can turn the chromogenic agent into purple. This indicates that the surface of magnetic particles contains active amino compounds. After dispersing particles into DMSO and using the Carboxylic Acid on DTPAA with the amino compounds on the magnetic particles, the results are DTPA grafted nanoparticles. This reaction does not require catalyst, and can easily happen formulating DTPA-SiO2@MNPs. The byproduct of peanut size nanoparticles to conduct a FT-IR Spectrum was selected, the results are shown in Fig. 2.
Through Fig. 2 of the FT-IR Spectrum, can be observed that Fe3O4@SiO2-DTPA complex particle has an absorption peak at 2863 cm− 1, 2923 cm− 1 while -CH3 experience a stretching vibration. At the absorption peak of 1443 cm− 1 is the opposing stretching of -COO−. These three absorption peaks are all recorded on the DTPPA FT-IR Spectrum, which were not appeared on the Fe3O4@SiO2 FT-IR Spectrum. This indicates that DTPA has successfully grafted onto the surface of the particle. Additionally, 3400 cm− 1 and 1600 cm− 1 are inherent to the absorption peaks of –OH, indicating that the expose silicon surface contains substantial amino.
Table 1
Data of elemental analysis of the nanoparticles
Nanoparticles/Element | C% (w%) | N% (w%) | H% (w%) |
Fe3O4@SiO2-NH2 | 6.66 | 1.87 | 2.61 |
Fe3O4@SiO2-DTPAA | 10.12 | 2.71 | 2.91 |
An element analysis on the amino and DTPAA modified nanoparticle was also carried out: the elemental content of Fe3O4@SiO2-NH2 is 6.66% carbon, 2.61% hydrogen, 1.87% nitrogen and calculable amino at 1.335×10− 6 mol/mg. DTPPA modified nanoparticle Fe3O4@SiO2-DTPA has a significant increase in carbon, hydrogen, and nitrogen content, respectively 10.12%, 2.71%, and 2.91%. This increase was attributed by components in DTPAA, illustrating that DTPAA has successfully grafted to the surface of the nanoparticles. This result is in a good agreement with the findings obtained by the FT-IR Spectrum analysis. The hydrogen concentration, however, was not proportionally increase like carbon and nitrogen, and this is duly because the hydrogen concentration in nanoparticle is derived from the hydroxyl group on the surface.
DTPAA are generally use clinically as labeling nuclide because its molecules contain numerous non-binding pair of electron (N, O), such as the preparation of 99Tcm-DTPA nuclide derivative, Ga-DTPA MRI contrast agent. DTPA is an effective coordinator for 188Re3+ to form a coordination center of 188Re3+ as shown in Fig. 1. Re-188 labeled nanoparticles remain radioactive even after numerous magnetic separation, as shown in Table 2, this demonstrates that Re-188 has been labeled on the nanoparticle.
Table 2
radioactive intensity of these three kinds of nanoparticles
Nanoparticles | radioactive intensity (mCi/5 mg) |
S-100 | 1.68 |
P-180 | 1.41 |
S-230 | 1.37 |
TEM and laser particle size analysis
TEM image of S-Fe 3 O 4 , P-Fe3O4 and PG-Fe3O4
Figure 3a, Fig. 3c, and Fig. 3e are TEM images of S-Fe3O4, P-Fe3O4 and PG-Fe3O4, respectively. The figures for S-Fe3O4 and P-Fe3O4 particles show good dispersibility. The particle size of S-Fe3O4 is mainly at about 178 nm in diameter, and the TEM image shows good dispersibility. The HRTEM image of Fig. 3b shows that the lattice of S-Fe3O4 has parallel pore structure oriented in the same direction. The electron diffraction illustration (upper right) shows that the interplanar spacing of S-Fe3O4 is 0.299 nm, 0.250 nm, and 0.210 nm, which are consistent with the interplanar spacing of (220), (311) and (400) of Fe3O4, respectively.
The particle size of P-Fe3O4 is mainly in the range of 60–70 nm, and the particle size distribution is relatively uniform. It indicates that the doping of EuOCl leads to the decrease of the particle size of Fe3O4 nanoparticles, and the P-Fe3O4 particles have the tendency of double-sphere coupling. The HRTEM image of Fig. 3d shows that the lattice of P-Fe3O4 exhibits parallel pore structure oriented in three directions. In the elliptical circle region of Fig. 3d staggered pores appear, and the interplanar spacing is 0.496 nm, it is higher than the interplanar spacing of the parallel aligned channels in other regions. The electron diffraction illustration in the upper right corner shows that the P-Fe3O4 interplanar spacing is 0.309 nm and 0.263 nm, which are consistent with the interplanar spacing of (220) and (311) of Fe3O4, respectively. The P-Fe3O4 crystal plane spacing is 0.500 nm, which is consistent with the measured crystal plane spacing. This is a new interplanar spacing, possibly due to the presence of a new phase between the two coupled P-Fe3O4 spherical shells after doping.
The PG-Fe3O4 nanoparticles synthesized by DTAB as a templating agent have a particle size of about 38 nm. The particles have good dispersibility and polygonal morphology. They contain quadrilaterals and pentagons, mainly hexagonal shapes, as shown in Fig. 3e. It can be seen from the HRTEM image of Fig. 3f that PG-Fe3O4 exhibits an ordered parallel arrangement of lattice fringes, indicating that the particles have a single crystal structure with a crystal face spacing of 0.148 nm in the figure: it is consistent with the crystal plane spacing of (110) of Fe3O4.
TEM image and laser particle size analysis of S-230, P-180 and P-180
Comparing the TEM images Fig. 3 and Fig. 4, it can be seen that the size of SiO2 coated nanoparticles changed significantly. The SiO2 coating S-Fe3O4 with a particle size of 178 nm, the particle size of S-230 is increased to 230 nm (as shown in Fig. 4a), the core-shell structure is very obvious, and the dispersion is uniform: its kinetic diameter is mainly concentrated at 250 nm (as shown in Fig. 5a).
As shown in Fig. 4b, two pairs of coupled P-Fe3O4 particles which grain size of 60–70 nm are recombined after SiO2 coating to form peanut-like P-180 nanoparticles with an aspect ratio (about 180 nm long and 95 nm wide). Its kinetic diameter is mainly at 130 nm, but there is also a distribution between 200–400 nm (as shown in Fig. 5b), which may be caused by the special morphology of P-180 with aspect ratio.
The particle size of PG-Fe3O4 nanoparticles is about 38 nm. After coating, the size of S-100 is mainly concentrated at 105 nm. The nanoparticles are uniformly dispersed and the average particle size is 98 nm, as shown in Fig. 4c. The kinetic diameter is mainly at 110 nm (as shown in Fig. 5c).
The Magnetic Properties of three functionalized nanoparticles of S-230, P-180 and S-100
The Magnetic Properties of S-Fe3O4, P-Fe3O4 and PG-Fe3O4
The magnetic hysteresis (M-H) curve of S-Fe3O4, P-Fe3O4 and PG-Fe3O4 materials is shown in Fig. 6. It can be seen from the figure that S-Fe3O4, P-Fe3O4, and PG-Fe3O4 nanoparticles have no hysteresis loop and exhibit superparamagnetic properties. The saturation magnetization Ms of S-Fe3O4 is 75.4 emu/g, the saturation magnetization of P-Fe3O4 is 43.7emu/g, while the polygonal Fe3O4 nanoparticle PG-Fe3O4 shows superparamagnetic characteristics and has ultra-high saturation magnetization. Strength, up to 134.2 emg /g.
The Magnetic Properties of S-230, P-180 and S-100
Analyzing M-H curves from Fig. 3, the M-H diagram of the three particles does not indicate any hysteresis effect. This illustrates that all three particles are superparamagnetic. Additionally, after modifying, the three particles all exhibit greater saturated magnetic intensity; when S-230 reaching an intensity of 56 emu/mg, P-180 reaching 28 emu/mg, and S-100 reaching 32 emu/mg respectively.
Distribution in vivo
Distribution and excretion in normal lab mouse in vivo
By route of the tail vein, the three different Re-188 labeled nanoparticles were injected into three anesthetic mice respectively. The mice were mounted on a flat paper plate, as shown in figure 8. A series of SPECT images were performed every 1 h to monitor the clearance and distribution of nanoparticles in mice, and SPECT images of 1h, 2h, and 3h were shown in figure 9.
Through the SPECT images, it can be seen that the three sets of rat sample all have a strong response mainly in their organ distribution area and bladder area. The weak response near the head of the sample indicates that the majority of the particles are being captured by the organs and excreted by the kidneys through the bladder. Only a small fraction of the nanoparticles can cross the blood-brain barrier. Group C is S-100 nanoparticles, and after 3 hours the response intensity in the organs is significantly weaker than 1 hour. This demonstrates that the smaller S-100 nanoparticles can mostly escape through the organs. Group A and Group B is S-230 and P-180, organs distribution response intensity is not affected by time, and mainly condense in the lung, liver and kidney areas. A more detail distribution data can be obtained from the distribution data of anatomical organ.
Distributes in many tissues
ID/g% (Inject Dose per Gram of Tissue) is a frequently employed for measurement in Nuclear Medicine to measure the absorption of radioactive dosage per gram of tissue. In our research, nanoparticles are consistently labeled with Re-188 nuclide, therefore, Re-188 distribution is equivalent to nanoparticle distribution. ID/g% relative value represents the intensity of nanoparticle in tissue. As the Fig. 11 shows, we monitored the biodistribution of the 3 different kind of MNPs in the mice’ organs and blood (Fig. 11a-c) and the plot of dose change over time(Fig. 11d). The distribution of nanoparticles in tissue illustrates that the three different particles have a different distribution in the organs. Larger particles, S-230 and P-180 are mainly concentrated in the lung, kidney, liver and the spleen. After 2 hours, the ID/g% measurement of S-230 in lung, kidney, liver and Spleen reached 76.16 ± 4.3, 16.45+-1.1, 16.76 ± 1.1, 21.3 ± 2.6. P-180 measurements are: lung 60.4 ± 3.6, kidney 10.3 ± 0.7, liver 38.00 ± 1.8 and spleen 121.04 ± 2.9: a significant greater ID/g% value than other organs. These organs are mainly the Reticuloendothelial System (RES) distribution area. On the other hand, The observation illustrates that S-100 in organs have a relative similar ID/g%: with measurements of lung 19.7 ± 0.9, kidney 10.3 ± 0.9, liver 12.59 ± 1.8, and spleen 10.54 ± 1.9. This indicates that this kind of nanoparticles are evenly distributed among organs; distinctive distribution is not as significant as larger particles and hence that the size of nanoparticles has a significant impact on the distribution inside organism. The main reason is that RES contains numerous phagocytes: the larger particle are more easily labeled and ingested, and the smaller ones have a higher chance of escaping; therefore, it has a low concentration of organ distribution.
In the changes of ID/g% in blood stream (Fig. 11d), illustrates that the three particles are significantly diluted in bloodstream. This indicates that nanoparticles inside an organism will be rapidly intercepted and metabolized.
Magnetic targeted imaging in vivo in tumor-bearing mice
Nuclide labeled magnetic nanoparticle compound material has a better dispersibility in saline and responsiveness under magnetic fields. Take two nuclide labeled magnetic compound materials and inject into VX2 tumor lab rat samples through the caudate vein, all of the tumor lab rat samples have been injected tumor tissue in their left forelimb and hind limb and with the magnetic pulsation focus at the left forelimb (peak value 0.6T). After a given time, a series of SPECT images were performed on magnetically targeted tumor-bearing mice (Fig. 12). A qualitative study of the enriching effect of magnetic fields on the nanomagnetic particles in tumor-bearing mice was investigated.
Figure 12 is the SPECT imaging of tumor mice after 1 hour, 2 hour, and 3 hours. Area A shows a normal tissue in right forelimb, Area B reports the magnetic targeted tumor area of the left forelimb. Area C correspond to the tumor area in the left hind limb. Area D is the inflamed area of the right hind limb. Comparing of the signals obtained from area B, C, D with area A, the result shows that the three different particles can be saturated in tumor cells. Taking P-180 for example, after 1 hour of magnetic targeting, signal in area B is 2.94 times of area A, signal in area C is 2.1 times of area A. It explains that relative to normal tissue, P-180 can saturate near tumor tissue and with magnetic targeting can increase the effect of saturation of P-180 in tumor area. On the other hand, signal in area D is 1.16 times of area A: this illustrates that P-180 will not saturate in inflamed area. This finding demonstrates the potential of using P-180 as a contrast agent has significant value in differentiating tumor area and inflamed area. In interested area, S-100 and S-230 has a similar distribution trend as P-180, this indicates that the three different particles do not react differently to vitro magnetic pulsation. The responsiveness of nanoparticles to magnetic fields is dependent on the intensity of the saturation and magnetize of the particular nanoparticles[22], as described by Ms. The difference of Ms in the three nanoparticles is not significant enough to create an impact of distribution inside an organism for this level of magnetic fields.