Biomedical

Communication

Biosci. Biotech. Res. Comm. 10(3): 445-454 (2017)

Fe3+-EDTA-zinc oxide nano-diagnostics: Synthesis and

in vitro cellular evaluation

Kimia Roshani

, Mazyar Etemadzade

, Ramin Farhoudi

, Seyed Esmaeil Sadat Ebrahimi

Morteza Pirali Hamedani

, Artin Assadi

and Mehdi Sha ee Ardestani

Faculty of Pharmacy, International Campus, Tehran University of Medical Sciences, Tehran, Iran

Natioanl Institute for Genetic Engineering and Biotechnology (NIGEB)

Department of Laboratory Animal Science, Pasteur Institute of Iran, Karaj, Iran

Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

ABSTRACT

Resulting from many efforts in opportune recognizing and correct treating brie y molecular imaging and therapy,

some methods or molecules have been developed by now to overcome any unwanted defaults in imaging and

therapy, speci cally nanoparticles. Generally, synthesis new compounds, for example Zinc Oxide-Iron nano-complex

consisting non-toxic paramagnetic ion [Fe3+] and its cellular uptake vehicle [zinc oxide] as a lowering risk of toxicity

and increase in cellular uptake liability, could be useful and noticeable in molecular imaging purposes. The isolated

nano-contrast was structurally analyzed by variety of techniques such as EDAX, AFM, Zeta and size measuring, SEM,

FTIR and UV spectrums. Finally for monitoring the nano-complex toxicity and cellular uptake on human embryonic

kidney cells named as HEK 293 was assessed respectively. The analytical result showed a very good promising size

at nanoscale and zeta potential as well as iron content for the suggested ferric containing nano-complex as well as

paramagnetic properties. Also outcome of cell study were resulted in no signi cant cellular toxicity comparing to

62% toxicity of control drug Magnevist and higher cellular uptake of 56% comparing to 9% for that of Magnevist

as well. In summary, it seems that the proposed ZnO-Fe nano-complex may be useful as a novel low risk contrast

agent to increase resolution in molecular Imaging like MRI and improve the current situation with the minimum cost.

KEY WORDS: SYNTHESIS, CONTRAST AGENT, MOLECULAR IMAGING, ZNO-FE3+ NANOCOMPLEX, HUMAN EMBRYONIC KIDNEY CELL

445

ARTICLE INFORMATION:

*Corresponding Author: sha eeardestani@gmail.com

Received 10

July, 2017

Accepted after revision 28

Sep, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.3/18

446 CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Kimia Roshani et al.

INTRODUCTION

As per numerous endeavors in perfect perceiving and

right treating [early conclusion and treatment], expan-

sive quantities of creating strategies have been utilized

as of recently. One of the imperative one in this  eld

is sub-atomic imaging, an advanced innovation which

gives legitimate component to the early recognition and

portrayal of the ailments, checking of natural process in

body, assessment of treatment and observing reaction,

almost assessing drug pharmacokinetics. Atomic imag-

ing as shows is the molecularly focused on, constant,

and noninvasive imaging of wonders and procedures

at cell and subcellular levels, (Meade et al. 2009). For

upgraded determination execution imaging specialist as

little particles, built protein nanoparticles has been per-

formed, (James etal. 2012 Rameshwar etal. 2015 Langer

etal. 2015).

At present there are a few imaging modalities usually

utilized for atomic imaging like Magnetic Resonance

which bene ts balance operators with paramagnetic

owing to properties and the others incorporate positron

emission tomography (PET), single-photon emission

computed tomography (SPECT), computerized tomogra-

phy (CT), optical imaging [ uorescence and biolumines-

cence], photo acoustic imaging. Contrasted with other

imaging modalities, the principle points of interest of

attractive reverberation imaging is its great determina-

tion which can enhance by upgrading contrast special-

ist. The  rst kind of clinically applied radio-opaque usu-

ally known as contrast agents were salts of chemically

designed complexes by paramagnetic diagnostics, such

as Ferric (Fe III), gadolinium (III) and manganese (II)

which their mechanisms show that such complexes of

mentionedparamagnetic radio-metalsdecrease the longi-

tudinal (T1) and transverse (T2) relaxation parameters of

surrounded water molecules, (Lauffer etal.1987), (Toth

etal. 2002) (Schwert etal. 2002).

Indeed it is notifying about paramagnetic compounds

as chemically designed agents ready to go about as dif-

ferentiation operators in the locales where they disperse

in body and improve differentiate amid imaging. Among

the difference operator the greater part of them are low

sub-atomic weight metal edi ces. Also the utilization of

paramagnetic metal for this propose, nanomedicine for-

mulation by employing different types of nanoparticles

such as polymeric or metal based such as zinc oxide

nanoparticles have been vastly used for imaging appli-

cations beside for therapeutic goals. Little size of nano-

particles encourage the auspicious identi cation of little

different changes furthermore give a high surface range

to stacking different atoms. Attractive nanoparticles

have additionally been utilized to convey medications

to a sick range, (Barakat, 2009).

It is another test utilizing nanoparticle as a part

of both indicative and restorative objectives. In such

framework the name as theranostic has been more rec-

ognizable in scientist’s mentality, nanoparticles assume

an essential part as growing high  ag force and limit

with regards to various application, (Jain et al. 2008).

The fundamental advantages of utilizing nanoparticle

as a part of symptomatic and imaging are low lethal-

ity, site-particular collection and hours of course time

next to their wellbeing and biocompatibility, (Jesse etal.

2011). In spite of broadly utilization of difference oper-

ator up to now, many looks into are progressing due

symptoms and lethality of complexity specialist espe-

cially in crucial organs, for example, kidney and here

and there low determination that are the primary issues

as dependably uncommonly in the individuals who uti-

lize this strategy to catch up the treatment procedure

forever. Even so, synthesis new compounds with all the

ideal index have still been restricted and have no com-

plete in vitro or in vivo data. Herein, we synthesis a

new Compound including Zink Oxide nanoparticle in

chelation with Fe

-EDTA in optimal condition with the

aim of creating low risk contrast agent and improving

the current situation along with minimum cost. Brie y

in current experimental observations, stable complex of

iron with a chelator was formed and then such complex

loaded into zinc oxide nanoparticle for increase in cel-

lular uptake liability of tumors.

MATERIAL AND METHODS

Zincsulfate hepta hydrate [ZnSO

.7H

O, Merck], Sodium

Hydroxide [Merck], Deionized Water, EthyleneDiamin

Tetra Acetic acid [99.5 %, Merck], Ferric-Cholorid

hexa hydrate [FeCl

.6H

O, Merck] were used for the

synthesis of the materials. In the following, scanning

electron microscope [SEM] with EDAX analysis was

provided by Phenom-Prox model made in Holland

Atomic-force microscopy[AFM] image was obtained

by CP-RESEARCH[CP-R] model, VEECO Manufacturer

Company made in America. Zeta potential and size on

nanoparticle in water as a solvent and ultimate nano-

complex in DMSO as a solvent were performed by Mal-

vern Nano-Zetasizer from Pasteur Institute in Tehran.

Fourier Transform Infrared spectroscopy [FTIR] result

was measured by Perkin Elmer, Spectrum Two FT-IR and

at the end ultraviolet-visible spectrum was measured on

VARIAN, CARY 100 Bio UV-VIS. Cells and related medi-

ums were provided from Pasteur Institute of Iran.

SYNTHESIS OF ZINC OXIDE NANOPARTICLE

In order to synthesize ZnO nanoparticle water based

solution [0.2 M] of zinc nitrate as well as a prepared

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON 447

Kimia Roshani et al.

[0.4 M] of NaOH was prepared by using deionized water,

respectively. The accessible NaOH solution was dropped

into zinc nitrate medium at room temperature[rt] with

accurate stirring, which advanced in the accumulation

of abeyance with a white color. Resulting white  nished

compound was elaborately centrifuged at 5000 rpm for

at least 20 min and then washed three times with eluting

solution of distilled water as well as washing with abso-

lute alcohol  nally. The acquired artefact was  nally

calci ed at 500 °C in air atmosphere for 3 hrs to achieve

zinc oxide NP respectively.

SYNTHESIS FE

-EDTA COMPLEX

1 mol EDTA equivalent 0.25g [M=292.2 g/mol] was

mixed with 20ml distilled water and after 5 minutes of

stirring, 1 mol Ferric Choloride hexa hydrate equivalent

0.27g [M=270 g/mol] was added to the container. The

mixture was heated to 90

C for 10 minutes with stirring.

Then after wrapping the foil placed 7-10 days without

moving in laboratory condition to form the intended

complex. Eventually puri ed by sephadex G25 column

or Dialysis bags [cut off 1000Da] as well.

SYNTHESIS ZNO-FE-EDTA NANOCOMPLEX

To synthesis the ultimate complex, half of the amount of

Fe-EDTA complex was added to the double amount of

Zinc Oxide nanoparticle with 5-7ml distilled water and

were stirred for 20 minutes to obtaine light brick color

transparent solution and placed 7-10 days without mov-

ing in 25

C, covering with aluminum foil. Henceforth

puri cation by sephadex G25 was performed as previ-

ous step.

In order to con rm the  nal structure, nanocomplex

was structurally analyzed by variety of techniques. To

characterize size, morphology, elements Scanning Elec-

tron Microscopy[SEM] withEDAX analysis was used,

andcalculation of the roughness parameters meas-

ured by means of the Atomic Force Microscopy[AFM],

Zeta and size measuring ,also Fourier transform infra-

red spectroscopy[FTIR] is an established tool for the

structural characterization of the new complex and in

additionUltraviolet[UV] spectrum was used to identify

the active site in nanocomplex, measuring zeta potential

and size of nanoparticle was done eventually.

IN VITRO CELLULAR TOXICITY [XTT]

In order to  nd and assesse the possible toxic param-

eters of synthesized zinc oxide nanoparticles contain-

ing trivalent iron-EDTA, XTT method was employed

as described in detail in the literature. Furthermore, as

stated in literature one of the possible route of discretion

of nanoparticle from the body as well as major affected

body part of human by the contrast agents is kidney,

HEK-293 was elected for XTT experimental analysis and

observations based on the effects of different dosages of

zinc oxide nanoparticles containing trivalent iron-EDTA,

-EDTA and Magnevist [as a FDA approved MR con-

trast agent] as controls. XTT data were performed for 72

h period of time.

IN VITRO CELLULAR UPTAKE

To evaluate the cancerous cellular permeability of zinc

oxide nanoparticles containing trivalent iron-EDTA,

MCF-7 was chosen and different dosages of zinc oxide

nanoparticles containing trivalent iron-EDTA, Fe

-EDTA

and Magnevist [as a FDA approved MR contrast agent]

as controls were assessed at an obvious time of 6 hours

after exposure. After a subjected times cells treated by

the agents were eluted by cell culture medium and the

cells were centrifuged and lysed by acidic medium. The

resultant solution was transferred to detect amunts of

and Fe

by ICP-Mass spectroscopy as well. Inter-

cellular concentration of metals were considered as per-

centage of cellular uptake.

RESULTS AND DISCUSSION

In Figure 1 SEM with EDAX analysis for ZnO nanopar-

ticle is presented, in addition the image of the sample in

various dimension, the result of SEM also be displayed as

spectrums. The EDAX pattern exhibited different peaks

that each of them shows a speci c element percentage.

The higher peak represents greater concentration. In

this way, each element has a special atomic structure

containing series of peaks. We have followed the same

procedure after preparation of Fe-EDTA complex and

ultimate nanocomplex and prominent additional peaks

for new elements are obvious whereas those peaks are

completely absent in the case of EDAX spectra of the

ZnO nanoparticle.

In Figure 2 AFM topography of the as prepared ZnO

nanoparticle and the ultimate nanocomplex are pre-

sented. The diagram and two-dimensional images of the

initial nanoparticle and conclusive nanocomplex are

shown in Figure 2a-b which shows the approximate the

height of the sample. Three-dimensional images of ZnO

nanoparticle and  nal complex are displayed in Figure

2c-d that shows peak-valley more concretely. The Fe-

EDTA complex is inside ZnO nanoparticle pores ran-

domly resulting in the formation and size of nanocom-

plex. As the load increases, the values of area roughness,

line roughness, and also peak−valley height increases

obviously. Thus surface morphology veri cation of

nanoparticle and terminal nanocomplex was deter-

mined by AFM technique. The changes are explicated by

448 CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Kimia Roshani et al.

FIGURE 1. SEM with EDAX analysis, [A] EDAX of ZnO nanoparticle, [B] EDAX of Fe-EDTA

complex, [C] EDAX of ZnO nanoparticle with Fe-EDTA as a nanocomplex.

comparing the graphs. Another physicochemical param-

eter which has been investigated by Zetasizer is particle

size and zeta potential. The average diameter, particle

size distribution index, intercept of ZnO nanoparticle

and  nal nanocomplex is shown in Figure 3 and in

addition the nanoparticles charge surface before and

after reactionsare-7.31mv and -5.63mv respectively.

The FTIR spectrum support the result obtained from

SEM, AFM, Zetasizer. After each reaction new peaks

appear that proverecent atoms and links as an example

a FTIR absorption band is observed at 3436 cm

-1

which

represents NH link related an amino group of EDTA or

the peak in  ngerprint zone, about 440 cm

-1

reveals

presence of halogen of FeCl

in  nal nanocomplex and

background spectrum of ZnO nanoparticle including a

peak at 2926 cm

-1

con rms the presence of zinc oxide

nanoparticle.Figure.4 give more explanation about spec-

trums.

At the end UV-VIS spectra absorption is recorded

between 200-800nm wavelengths. In Figure 5 there is

a clear indication of Fe-EDTA complex in the absorp-

tion spectrum after loading. The absorption spectrum

of the Fe-EDTA complex shows two peaks with absorp-

tion between 1.5-2.5 which the  rst one is related to

Carboxylic Acid group of EDTA and the second one

demonstrates Ferric Choloride that because of its orbital

properties is tended to long wavelengths or visible light.

Also the  nal nanocomplex dissolved in DMSO shows

a sharp and narrow peak between 0.2-1nm comparably

the previous one.

Moreover, as stated in Figure. 6 zinc oxide nanoparti-

cles containing trivalent iron-EDTA, Fe

-EDTA at all the

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON 449

Kimia Roshani et al.

FIGURE 2. [a] 2D image and diagram of ZnO nanoparticle, [b] 2D image and diagram of

ultimate nanocomplex, [c] 3D image of ZnO nanoparticle at the previous zone, [d] 3D

image of ultimate nanocomplex at the previous zone.

used dosages did not rise any signi cant toxic effects on

human kidney cells otherwise a signi cant toxic effects

was shown by Magnevist 100 μM [as a FDA approved

MR contrast agent] as control caused 62% cellular mor-

tality respectively. Additionally a very good promising

cancer cell uptake was recorded for zinc oxide nanopar-

ticles containing trivalent iron-EDTA 56% comparing to

9% for that of Magnevist and 5% for Fe-EDTA as well

[more details Figure 7].

As it is clear utilizing contrast specialist as a part of

sub-atomic imaging is a discussable subject in atomic

prescription, so pro ted a low dangerous balance opera-

tor with ideal impact has dependably been a worry. Too

Many diverse diagnosis of some targeted human body

sections in molecular imaging methodology including

Magnetic Resonance Imaging MRI showed noticeable

drawbacks that could be considered as dif culties to

remain them also without other con rmation techniques

that mostly would be invasive or even de ned as impos-

sible, (Rameshwar etal. 2015).

Nephrotoxicity and unfavorably susceptible response

of many difference operator are the other test because

Kimia Roshani et al.

450 CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 3. [a] size of ZnO nanparticle is shown at the left side, and the graph on the right

related to  nal nanocomplex. [b] ZP of ZnO nanoparticle is on the left and the right one is ZP

of ultimate nanocomplex.

of their structure and phisico-concoction portrayal. In

such cases FDA caution is not immaterial showing the

improving shot of Nephrogenic Systemic Fibosis [NSF]

and or Nephrogenic Fibrosing Dermopathy [NFD] for

the determined patients confounded to have renal dis-

appointment maladies. [Enzo et al. 2010] What’s more

moderate discharge and high lethality of numerous mac-

romolecular complex, for example, Gd III, are because of

the digestion system which can discharge Gd III parti-

cle in body, (Zheng-Rong etal. 2003). Magnetic nano-

bio-materials like fe-EDTA-prepared zinc oxide NP

nanocomplex using in MRI have been well offered for

the next subsequent imaging such viable tumor places

(Langer etal. 2015) then again different techniques which

require keeps imaging amid treatment. However nano-

drugs like nano-complex can defeat natural obstructions

which can impact on conclusion and treatment because

of passing particles. Then again this nano scale give a

fast dissemination amid systemic conveyance further-

more can be cleared up easily (Frank etal. 2008). These

qualities gather least danger and time to get the most

extreme impact and tissue aggregation. Numerous mol-

ecule in view of nanotechnology have been considered

to conform the pharmacokinetic properties furthermore

upgrade the relaxivity which metal-based nanoparticle

can be noted, (Enzo etal. 2010, Na etal. 2008).

In this work we have used FeCl

chelating with EDTA,

a six dentate ligand, and react the resulting complex to

ZnO nanoparticle to be placed in its pores and structure

properly. Ferric as main recognized paramagnetic iona-

ble to provide adequate magnetic effects on surrounding

water atoms and causes to reduce in T

and or T

relaxa-

tion time quantities. Subsequently this decline makes a

brighter picture in light of T1 relaxation time lessening

and darker picture consequently diminishing T2 relaxa-

tion time, (Brown etal. 2003) (Na etal. 2008). Contrast-

ing with present difference operator, Gd buildings are

delegated the specialists that impact on T1 relaxation

time and can’t wide water proton resonance and also

T2 specialist, for example, paramagnetic and superpara-

magnetic specialist containing iron. [(Luca etal. 2012))

(Na etal. 2008)]. So creating new compound by having

both T1 and T2 diminishment can be so useful. There-

for in this paper we synthesized ZnO-Fe nanocomplex

besides EDTA as a safe FDA approved and low cost

effective chelator in optimum condition.

The result of physico-chemical study showed a good

and acceptable data and also low toxicity, at least cost

and availability of the material needed are the other pos-

itive aspects. The small size of nanocomplex and exist-

ing Fe

as a paramagnetic agent in ultimate complex

displayed a stable and appropriate complex in different

Kimia Roshani et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON 451

FIGURE 4. FTIR spectrums regarding prepared nanoparticle containing Fe-EDTA [A],

Zinc Oxide NP [B], Fe-EDTA [C], EDTA [D] as well as FeCl3 [E].

features such as size, surface charge, topography, func-

tional groups and fundamental elements. Ease of entry

and low risk of cytotoxicity make this nanocomplex as

a novel carrier in loading some drugs and having thera-

peutic effects in addition to diagnostic effects especially

in tumor cells with prominent uptake.Consequently,

based on these points and outcomes synthesized ZnO-Fe

nanocomplex can freely play an important and effective

role in the replacement of present contrast agent in MR

molecular imaging erelong.

A novel nanocomplex of ZnO-Fe along with EDTA

has been prepared by a simple chemical approach as a

low toxicMR contrast agent. Thus after chelating iron by

EDTA, composed complex reacts with ZnO nanoparti-

Kimia Roshani et al.

452 CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 5. U.V spectroscopy data regarding prepared nanoparticle containing Fe-EDTA [A], Fe-EDTA [B], EDTA [C] as

well as FeCl3 [D].

FIGURE 4. (Continued)

Kimia Roshani et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON 453

FIGURE 7. Depiction of competitive MCF-7 cellular uptake regarding Magnevist, Fe-EDTA

and Fe-EDTA-Zinc Oxide NP respectively. Obtained data were recorded from a period of 6hr

at the same concentration of 50 μM.

FIGURE 6. Illustrations of HEK-293 Cellular Toxicity resulting from 72 hrs.

Exposure to different concentration of Magnevist, Fe-EDTA and Fe-EDTA-Zinc

Oxide NP respectively. Potential signi cant toxicity regarding to Magnevist was

observed otherwise no toxic effects were recorded for proposed NP as well.

454 CELLULAR EVALUATION OF ZINC OXIDE NANOPARTICLE CONTAINING IRON BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Kimia Roshani et al.

cle and a new stable nanocomplex is formed. Small size

of ZnO in nano scale helped to uptake more by cells.

The presence of Ferric Chlorideas a valuable magnetic

agent makes opportune recognizing easier in molecular

imaging like Magnetic Resonance Imaging. The phys-

icochemical characteristics were monitored by different

analysis resulting in desired achievements that con rm

the  nal nanocomplex without any structural damage.

In conclusion, along the development of nanoparticles

for targeted diagnosis and therapy for biomedical appli-

cation, proposed ZnO-Fe nanocomplex may be useful

as a novel low risk contrast agent to increase resolution

in molecular Imaging like MRI and improve the current

situation with the minimum cost. Considering lower

normal human cellular toxicity as well as higher and

promising cancerous human cellular uptake comparing

to Magnevist it could be concluded that such iron con-

taining nanoparticle marked as new target for future in

vivo studies as well as clinical practice.

ACKNOWLEDGMENTS

The authors would to thank Tehran University of Medical

Sciences as well as all laboratories and technicians who

provided supported during within the course of the study.

CONFLICT OF INTEREST

The authors declared no con ict of interest.

REFERENCES

Barakat, N. S. (2009). Magnetically modulated nano systems: a

unique drug-delivery platform. Nanomedicine, 4 [7], 799–812.

Brown, M. A.; Semelka, R. C. (2003).MRI: Basic Principles and

Applications; Wiley-Liss: New York.

Enzo T, Daniela D C, Alessandra V, Silvio A. (2010).Challenges

for Molecular Magnetic Resonance Imaging. Chem. Rev, 110,

3019–3042.

Frank ,Eric P,Linda K. M,Omid C. (

2008).Factors Affecting the

Clearance and Biodistribution of Polymeric Nanoparticles.Mol.

Pharmaceutics,5[4], pp 505–515

Jain, P. K.; Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. (2008)

.Noble metals on the nanoscale: optical and photothermal

properties and some applications in imaging, sensing, biology,

and medicine. Acc. Chem. Res, 41 [12], 1578–1586.

James, M. L., and Gambhir, S. S. (2012) . A molecular imaging

primer: modalities, imaging agents, and applications. Physiol.

Rev. 92, 897−965

Jesse V. J, Sanjiv S. G. (

2011).Molecular Imaging with Ther-

anostic Nanoparticles.

Acc. Chem.Res., 44 [10], pp 1050–

1060

Langer, R. and Weissleder, R. (2015). Nanotechnology. JAMA.

313, 135-136

Lauffer, R. B. [1987]. Paramagnetic metal complexes as water

proton relaxation agents for NMR imaging: theory and design.

Chem. Rev. 87, 901−927.

Luca F, Junqing Z, Changning W, Chunying W, Robert H.

M, Yanming W.

(2012) Myelin Imaging Compound [MIC]

Enhanced Magnetic Resonance Imaging of Myelination. J.

Med. Chem.,55[1], pp 94–105

Meade, T. J., and Aime, S. (2009). Chemistry of molecular

imaging. Acc. Chem. Res. 42, 821−821.

Na, H. B.; Hyeon, T. (2009). Nanostructured T1 MRI contrast

agents. J. Mater. Chem., 19, 6267.

Na, H. B.; Song, I. C.; Hyeon, T. (2009). Inorganic Nanoparticles

for MRI Contrast Agents. Adv. Mater., 21, 2133–2148.

Rameshwar P, Alexander V. L, Pallavi R. G, Hui D, Jose

P.A,Shawn W,Satoshi I,Bindu K,Arthur R,Alexandra C,Janet

L. M,Vladimir A. L,Debiao L,Ravi S. P,Keith L. B,Eggehard

H, andJulia Y. L.

(2015). MRI Virtual Biopsy and Treatment

of Brain Metastatic Tumors with Targeted Nanobioconjugates:

Nanoclinic in the Brain.ACS Nano,9[5], pp 5594–5608

Schwert, D., Davies, J., and Richardson, N. (2002) NonGadolin-

ium-Based MRI Contrast Agents [Krause, W., Ed.] pp 165−199,

Springer, Berlin

Toth, E., Helm, L., and Merbach, A. (2002) Relaxivity of

MRI Contrast Agents [Krause, W., Ed.] pp 61−101, Springer,

Berlin.

Zheng-Rong L, Xinghe W, Dennis L. P, K. Craig G, Henry R. B.

(2003). Poly [L-glutamic acid] Gd[III]-DOTA Conjugate with a

Degradable Spacer for Magnetic Resonance Imaging. Biocon-

jugate Chem, 14, 715−719