Time-Evolution Contrast of Target MRI Using High-Stability Antibody Functionalized Magnetic Nanoparticles : An Animal Model

1 Department of Surgery & Hepatitis Research Center, National Taiwan University Hospital, Taipei 100, Taiwan 2Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan 3MagQu Co., Ltd., New Taipei City 231, Taiwan 4 Institute of Electro-Optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan 5Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei 100, Taiwan 6Department of Primary Care Medicine, National Taiwan University, Taipei 100, Taiwan 7Department of Material Engineering, Kun Shan University, Tainan City 710, Taiwan 8Center for Molecular Imaging and Translational Medicine, Xiamen University, Xiamen 361, China 9Graduate Institute of Electronics Engineering, National Taiwan University, Taipei 106, Taiwan 10Department of Electro-Optical Engineering, Kun Shan University, Tainan City 710, Taiwan


Introduction
Due to its nontoxicity, nanosized, high-stability, iron oxide magnetic nanoparticles have been applied to in vitro and in vivo medical applications.For in vitro medical applications, several groups have published papers about the immunoaasay using biofunctionalized magnetic nanoparticles as labeling markers [1][2][3][4][5][6].These papers reveal that the magnetically labeled immunoassay show high sensitivity, low interference, and versatility in clinics [7,8].The in vivo applications using magnetic nanoparticles may include the magnetic labeling on cells [9,10], drug delivery [11,12], hyperthermia [13,14], and so forth.Another important application of magnetic nanoparticles in in vivo medicine is the contrast medium for magnetic resonance (MR) imaging [15][16][17][18].
From the metabolism point of view, the injected magnetic nanoparticles injected into beings are eventually digested with liver.It has been further demonstrated that only normal liver cells can digest the injected magnetic nanoparticles, while liver tumor cells can not absorb the injected magnetic nanoparticles.With the magnetic nanoparticles, the relaxation time of MR like T 2 for normal liver cells is reduced.Thus, for T 2 -weighted MR imaging, the brightness of normal cells is reduced.However, the brightness of T 2 -weighted MR image for liver tumor cells remains unchanged.Therefore, by injecting magnetic nanoparticles into beings, the bright spots on liver MR image are regarded as tumors [19].Nowadays, iron oxide magnetic nanoparticles are used as contrast medium in clinics for imaging liver tumors.
For other kinds of tumors, target MR imaging using biofunctionalized Fe 3 O 4 magnetic nanoparticles is utilized [20][21][22].For a given kind of tumor, specific antigens are expressed by the tumor.Magnetic nanoparticles immobilized with antibodies against the antigens are able to bind with the tumor and attribute to the reduction in the relaxation times of MR.Thus, the MR image of the tumor becomes darker when compared with that of surrounding normal cells.Practically, as the antibody functionalized magnetic nanoparticles are injected to beings and are circulated to the tumor, both the tumor and surrounding normal cells are rich in magnetic nanoparticles.Thus, MR images of the tumor and surrounding normal cells become dark.It is impossible to differentiate the tumor from the surrounding normal cells.After a period of time, the tumor keeps the antibody functionalized magnetic nanoparticles, while the magnetic nanoparticles around the normal cells are washed away.During this period of time, the MR image of the tumor is darker than that of the surrounding normal cells.It is easy to find the tumor according to the MR image.But, the binding of the antibody functionalized magnetic nanoparticles with the tumor is not permanent.The bound magnetic nanoparticles with the tumor eventually are digested or washed away after certain period of time.The above descriptions imply that there exists a suitable time window for imaging the tumor using MR.However, the nanoparticles of the imaging time window of MR for target tumors are very rare.
In this work, the time-evolution of MR images of mice bearing with colorectal tumors is recorded after the injection of antibody functionalized Fe 3 O 4 magnetic nanoparticles.Besides, the synthesis of antibody functionalized Fe 3 O 4 magnetic nanoparticles is introduced.Since colorectal tumors express carcinoembryonic antigen (CEA), antibody against CEA is conjugated onto Fe 3 O 4 magnetic nanoparticles.The characterizations, such as particle size, MR relaxivity, stability and magnetization, of these magnetic nanoparticles are examined.

Experimental Details
The protocol of synthesizing magnetic Fe To make antibodies against carcinoembryonic antigen (CEA) for colorectal cancer, that is, anti-CEA (10C-CR2014M5, Fitzgerald; AT-CEA, MagQu), bound to the dextran on the outmost shell of magnetic nanoparticles, NaIO 4 solution was added into the magnetic solution to oxide dextran, which was then used to create aldehyde groups (-CHO).Then, dextran can react with anti-CEA via the linking of -CH=N-.Thus, anti-CEA is bound covalently to dextran.Through magnetic separation, unbound anti-CEA was separated from the solution.
The size distribution of Fe 3 O 4 magnetic nanoparticles biofunctionalized without/with anti-CEA was analyzed by using dynamic laser scattering (Nanotrac 150, Microtrac).The morphology of anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles is analyzed by using a scanning electronic microscope (JSM-6700F, Jeol).The relaxivity of MR for the reagent is measured by 3-T MR imaging instrument (Biospec System, Bruker).
To check the immunoreactivity of anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles, the technology so-called immunomagnetic reduction is used [24][25][26].The working principle of immunomagnetic reduction (IMR) is illustrated in Figure 2. In IMR, antibody functionalized magnetic nanoparticles are under the actions of multiple ac magnetic fields.Thus, magnetic nanoparticles exhibit an ac magnetic susceptibility  ac .As the antibody functionalized magnetic nanoparticles are mixed with a solution with target biomolecules.Via the antibodies on magnetic nanoparticles, nanoparticles associate with target biomolecules.Before the association, magnetic nanoparticles exhibit an ac magnetic susceptibility, denoted by  ac,o in Figure 2(a).After the association, some magnetic nanoparticles become larger, which results in the reduction in the ac magnetic susceptibility.The ac magnetic susceptibility of magnetic nanoparticles after biomoleculeparticle association is denoted by  ac, in Figure 2(b). ac, is smaller than  ac,o .The reduction percentage in  ac of magnetic nanoparticles due to the biomolecule-particle association is defined as IMR signal as follows: The ac magnetosusuceptometer (XacPro-E, MagQu) is used to monitor the time dependent  ac of the anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles after mixing with CEA solution.A significant reduction in the ac magnetic susceptibility of magnetic nanoparticles can be found once CEA molecules bind with active anti-CEA on magnetic nanoparticles.Furthermore, more reduction in ac magnetic susceptibility is obtained for more CEA molecules.That is, the sample with higher CEA concentrations shows higher IMR signals.On the other hand, the activity of anti-CEA on magnetic nanoparticles fails; no significant IMR signal can be observed.
For implanting the colorectal tumors, the injections of the CT-26 cell line were processed through the skin on the backs of 8-week-old mice.Three weeks later, 0.06 emu/g and 100 L of anti-CEA magnetic reagent were injected into the tail veins of five mice.Two groups of mice are used for examinations of MR imaging and Prussian blue (PB) staining, respectively, as tabulated in Table 1.Group 1 formed with two mice, numbered as mouse 1 and mouse 2, is examined using MR imaging instrument.The MR examination schedule was at the 0th, 18th, 30th, 48th, 72nd, and 96th hours for mouse 1 and at the 0th, 12th, 24th, and 46th hours for mouse 2. Here, 0th hour represents the time just before injection.Proving that the anti-CEA Fe 3 O 4 magnetic nanoparticles were bound to the tumor tissue required determining the Fe using Prussian blue (PB) staining to examine the tumor tissue of Group 2, which is formed with mouse 3, mouse 4, and mouse 5, which were euthanized at the 0th, 24th, and 98th hours, respectively.The tissue staining was processed (Laboratory Animal Center, National Taiwan University, Taipei, Taiwan), and the ×400 magnification of the optical images was observed using a light microscope.The 3-T MR imaging (Biospec System, Bruker) and a volume coil were used for T 2 -weighted images.In parallel with the arrangement of the anesthetized mouse, a long tube filled with deionized (DI) water was inserted as the intensity reference to dismiss the instrument drift at various times.Producing the coronal images of each entire mice body at 2 mm intervals required nearly 2 hours.

Results and Discussion
The distribution of particle diameter of dextran-coated Fe 3 O 4 magnetic nanoparticles is analyzed using dynamic laser scattering.The results are shown in the upper part of Figure 3(a) with the schematically illustration of dextran-coated Fe 3 O 4 magnetic nanoparticle.The mean diameter of dextran-coated Fe 3 O 4 magnetic nanoparticles was found to be 42.80 nm.After immobilizing anti-CEA on the dextran-coated Fe 3 O 4 magnetic nanoparticles, the distribution of particle diameter is analyzed, as shown in the lower part of Figure 3(a).In the inset, the Ys on the dextran-coated Fe 3 O 4 magnetic nanoparticle denote anti-CEA.The mean diameter of anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles was found to be 51.3 nm, which is larger than that of dextran-coated Fe 3 O 4 magnetic nanoparticles by 8.5 nm.Hence, the thickness of anti-CEA on the outmost layer of Fe 3 O 4 magnetic nanoparticles is around 4.25 nm.The shapes of the antibody functionalized magnetic nanoparticles are examined by using scanning electronic microscope and are shown in Figure 3(b).It is obvious that each particle is almost identical.Hence, the homogeneity of particle size is high.
To investigate the stability of the suspension of antibody functionalized Fe 3 O 4 magnetic nanoparticles in PBS solution, the storing-period dependent mean diameter of nanoparticles is explored.In case of low-stability suspension, the magnetic nanoparticles initially suspended in individual in PBS solution agglomerate with each other as time goes by.An increase in the mean diameter of nanoparticles will be observed.On the other hand, if the mean diameter of nanoparticles was observed to be unchanged, the stability of suspension of magnetic nanoparticles in PBS solution was high.The experimental results are shown in Figure 4.The reagent of antibody functionalized Fe 3 O 4 magnetic nanoparticles is stored at 2-8 ∘ C. It is clear in Figure 4 that the mean diameter of antibody functionalized Fe 3 O 4 magnetic nanoparticles in PBS solution remains around 50 nm for 7.7-month storage.The results evidence the high-stability suspension of antibody functionalized Fe 3 O 4 magnetic nanoparticles in PBS solution.
It is important to check the immunoreactivity of anti-CEA as conjugated onto dextran-coated Fe 3 O 4 functionalized magnetic nanoparticles.One of methods for this check is to observe the association between the anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles and CEA molecules in PBS solution.The method used in this experiment for observing the nanoparticle-CEA association is so-called immunomagnetic reduction [24][25][26].The time dependent ac magnetic susceptibility  ac of the reagent mixing with 5 ng/mL CEA solution was recorded and shown with dots in Figure 5.
It was found that  ac of the reagent keeps unchanged just after mixing with the CEA solution, as labeled with  ac,o in Figure 5.After one hour, the  ac of the reagent starts to reduce.The reduction in  ac of the reagent is not terminated until 3.8 hours after the mix of the reagent and the CEA solution.The IMR signal via (1) was found to be 1.68%.The reduction in  ac of the reagent proves the association between the anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles and CEA molecules.As the CEA concentration is increased, say 1000 ng/mL, the reduction in  ac of the reagent is enhanced, as presented with cross symbols in Figure 5.The IMR signal for 1000 ng/mL CEA solution is found to be 3.17% via (1).The observed reduction in the  ac of the reagent in Figure 5 demonstrates that the anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles are capable of binding with CEA molecules.
To examine the MR stability of the Fe 3 O 4 magnetic nanoparticles suspended in PBS solution, the time-evolution relaxivity of MR, for example,  1 , is detected.It is worth noting that the solution of dextran-coated Fe 3 O 4 magnetic nanoparticles is separated into two groups stored at 4 ∘ C and 25 ∘ C, respectively.The measured relaxivity  1 of MR as a function of the storing period of time is shown in Figure 6.It was found that  1 is around 145 mM −1 s −1 for the solution of dextran-coated magnetic nanoparticles.Furthermore, the  1 keeps unchanged for six months no matter the solution of dextran-coated magnetic nanoparticles is stored at 4 ∘ C or 25 ∘ C.This means that the magnetic stability of dextrancoated Fe 3 O 4 magnetic nanoparticles can be six months at least.
The anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles with high-stability magnetism and suspension are injected into mice implanted with colorectal tumor cells, CT-26.The time-evolution MR images of the tumor are taken after the injection into mouse 1, as shown in Figure 7.The tumor is circled and indicated with an arrow.The brightness of the images at a certain instant is normalized to that of water.The normalized brightness is referred to as image contrast.The image contrast of the circled tumor is analyzed at every image-taking instant.Then, the image contrast of the circled tumor at 0 h is used as a reference value.The ratio in the images contrast of the circled tumor at every image-taking instant to that at 0 h is calculated and shown with dots in Figure 8.The cross symbols in Figure 8

Conclusions
High-stability antibody functionalized Fe 3 O 4 magnetic nanoparticles are synthesized for the use of contrast medium of MR imaging.By injecting these nanoparticles into mice, target MR imaging, such as imaging colorectal tumor, is demonstrated.It is further clarified that the suitable time window for MR imaging for the target tumor is from 20 hours to 40 hours after the tail-vein injection.

Figure 2 :
Figure 2: Illustration for the mechanism of immunomagnetic reduction.

Figure 3 :
Figure 3: Analysis of particle diameter for Fe 3 O 4 magnetic nanoparticles (a) without (upper) and with (lower) antibodies suspended in PBS solution using dynamic laser scattering and (b) with antibodies using scanning electronic microscope.

1 Figure 6 :
Figure 6: Storing period of time dependent relaxivity  1 of MR for the solutions of dextran-coated magnetic nanoparticles stored at 4 ∘ C and 25 ∘ C, respectively.

Figure 8 ,Figure 9 :
Figure 8, the brightness of the tumor is reduced by 22% at 20 h as compared to that at 0 h.The brightness of the colorectal tumor recovers after 50 hours after the injection.The reduction in the brightness of MR image for the colorectal tumor is deduced to be due to the association of anti-CEA functionalized Fe 3 O 4 magnetic nanoparticles with the tumor.To prove this deduction, the biopsy of the colorectal tumors in other mice injected with anti-CEA 3 O 4 nanoparticles was proposed by MagQu Co., Ltd.[23].The flow chart for pH-7.4 phosphate buffered saline (PBS) solution.

Table 1 :
Two groups of mice used for the examinations of MR imaging and PB staining.