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Accepted ManuscriptTitle: Synthesis and Surface Functionalization of Fe3O4-SiO2 Core-Shell Nanoparticles with 3-glycidoxypropyltrimethoxysilane and 1,1-carbonyldiimidazole For Bio-applic

Accepted Manuscript

Title: Synthesis and Surface Functionalization of Fe3O4-SiO2

Core-Shell Nanoparticles with

3-glycidoxypropyltrimethoxysilane and

1,1-carbonyldiimidazole For Bio-applications

Author: Thi Kieu Hanh Ta Minh-Thuong Trinh Long Viet

Nguyen Thi Thanh My Nguyen Thi Lien Thuong Nguyen

Tran Linh Thuoc Bach Thang Phan Derrick Mott Shinya

Maenosono Hieu Tran-Van Van Hieu Le

Bio-applications, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2016.05.008

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Synthesis and Surface Functionalization of Fe 3 O 4 -SiO 2 Core-Shell Nanoparticles with glycidoxypropyltrimethoxysilane and 1,1 ’ -carbonyldiimidazole For Bio-applications

3-Thi Kieu Hanh Ta,1 Minh-Thuong Trinh,2 Nguyen Viet Long,6,* Thi Thanh My Nguyen,1Thi Lien Thuong Nguyen,3 Tran Linh Thuoc,2 Bach Thang Phan,1,4 Derrick Mott,5

Shinya Maenosono,5 Hieu Tran-Van 2,*, Van Hieu Le1,*

1 Faculty of Materials Science, University of Science, Vietnam National University, Ho Chi Minh,

Vietnam

2 Faculty of Biology and Biotechnology, University of Science, Vietnam National University, Ho

Chi Minh, Vietnam

3 Faculty of Resources and Environment, Thu Dau Mot University, Binh Duong, Vietnam

4 Laboratory of Advanced Materials, University of Science, Vietnam National University, Ho Chi

Minh, Vietnam

5 School of Materials Science, Japan Advanced Institute of Science and Technology,

1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan

6 Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Graphical abstract

Highlights

1 The core-shell Fe3O4-SiO2 nanoparticles functionalized with GPS and CDI

2 The Fe3O4-SiO2-GPS-CDI core-shell nanoparticles could bind BSA, pA/G and anti-T cell IgG antibodies

3 23 mg pA/G could be coupled on 1 g Fe3O4-SiO2-GPS-CDI nanoparticles

4 The 1 g Fe3O4-SiO2-GPS-CDI-pA/G nanoparticles could bind 9 mg anti-T cell IgG antibodies

5 The Fe3O4-SiO2-GPS-CDI-pA/G NPs can be applied in bone marrow transplantation

Abstract

In our research, we have presented the controlled synthesis of Fe3O4 nanoparticles (NPs) with a size

of about 10 nm coated with SiO2 shells for bio-applications On this basis, the controlled synthesis

of Fe3O4-SiO2 core-shell nanoparticles and their surface functionalization with

3-glycidoxypropyltrimethoxysilane (GPS) and 1,1’-carbonyldiimidazole (CDI) has been presented with a facile synthetic process The as-prepared Fe3O4-SiO2-GPS-CDI core-multishell NPs can bind

proteins Therefore, recombinant protein A/G (pA/G) was efficiently coupled onto the surface of

NPs via CDI groups, creating a complete coverage Antibodies (T IgG) were also conjugated on

Fe3O4-SiO2-GPS-CDI-pA/G, i.e 9 mg T antibodies per 1g NPs After the surface

functionalization of the magnetic nanoparticles, their superparamagnetism was reduced by a factor

of about threefold in Fe3O4-SiO2, and fivefold in Fe3O4-SiO2-GPS-CDI-pA/G in comparison with

that of the naked Fe3O4 NPs The NPs conjugated with T IgG could bind and remove 1x105 cells

per 0.25 mg NPs in vitro Finally, the new models of surface functionalization of magnetic

nanoparticles have been proposed for promising bioconjugations in our further research

Keywords: Core-shell Fe3O4-SiO2 nanoparticles, 3-Glycidoxypropyltrimethoxysilane, 1,1 ’Carbonyldiimidazole, Protein BSA, Fe3O4-SiO2-GPS-CDI-pA/G-T

-*Corresponding author:

nguyenviet_long@yahoo.com, lvhieu@hcmus.edu.vn, tvhieu@hcmus.edu.vn

1 Introduction

So far, magnetite (Fe3O4) nanoparticles (NPs) have received very large notable interest in

the field of biotechnology and nanomedicine for probing, manipulating biological systems and

delivering targeted drugs [1-5] However, Fe3O4 NPs are not very stable under ambient conditions

due to their high oxidation and instability in various acidic media Fe3O4 NPs show large

surface-to-volume ratio in the particle size range of 20 nm Therefore they possess high surface energies,

leading to aggregation, which minimizes the surface energy Additionally, the naked Fe3O4 NPs

have high chemical activity on their surfaces but are highly prone to oxidization in air, which can

lead to significant loss of their beneficial magnetism and dispersibility [1-5] In order to avoid these

challenges, the Fe3O4 NPs need to be protected by thin or thick non-magnetic materials to maintain

individual particle stability and durability It is required that the protecting materials not only

stabilize the magnetic iron oxide NPs, but can also be used for the specific processes of further

targeting functionalization Inorganic materials such as silica (SiO2) can satisfy the above key

requirements since SiO2 is known to be very stable under acidic conditions, inert to redox reactions

and abundant in surface hydroxyl groups, which offers ease of successful functionalization of Fe3O4

NPs for binding various biological ligands [1-8]

Most importantly, scientists have proven that SiO2 can provide better protection against high

toxicity [1-9] In addition, doping an organic dye into the SiO2 shell can extend the applications of

such core–shell structures to fascinating biomedical imaging applications through its luminescent

properties To further facilitate the subsequent biomolecule conjugation, surface modification has

often been explored to enable specific functional groups on the Fe3O4-SiO2 core-shell structures

The parameters affecting Fe3O4-SiO2 NPs and biomolecule interaction consist of physiochemical

properties, including surface chemistries, particle size, shape, charge, surface area, surface defects,

the functional groups of Fe3O4-SiO2 NPs and the composition of the biological fluid Ma etal

showed that Fe3O4-SiO2-GPS coated with iminodiacetic acid (IDA) and Zn2+ to form

Fe3O4-SiO2-GPS-IDA-Zn core-multishell NPs can only bind bovine haemoglobin (BHb protein) rather than

bovine serum albumin (BSA) protein [7]

Another interesting study revealed that 3-glycidoxypropyltrimethoxysilane (GPS) can be a

potential agent in bio-applications [7,8] In our approach, GPS was used as a coupling agent to react

with Fe3O4-SiO2 to prepare Fe3O4-SiO2-GPS, whose surface possesses epoxy groups The high

chemical activity of epoxy groups enables the easy attachment mechanisms of the specific ligands

[7,8, 10-12] The long-term goal of this study is to use the Fe3O4-SiO2 NPs for bio-application, such

as in bone marrow transplant Recently, bone marrow transplantation is a therapy for blood-related

diseases including acute myeloid leukemia Allogeneic transplants, using stem cells from a donor,

have steadily increased for the past several years [13,14]

The transplants have several advantages, but also come with severe complications, including

Graft-Versus-Host-Disease – GVHD, caused by the reaction of donor’s T cells to the recipient’s

cells [14] In the murine model of GVHD, T cell depletion by radiation increases the survival rate

after transplantation up to 100 % [15] In humans, a body of clinical trials shows T cell removal

prior to transplant reduces GVHD from 53-60 to 10-13 % [16] These results indicate the need of T

cell removal or depletion before bone marrow transplantation There are several approaches for T

cell removal, in which using anti-T cell (T) magnetic beads is one of the most effective methods

because the procedure is fast and leaves stem cells untouched [13-16]

We think that incorporating surface functionalized Fe3O4-SiO2 NPs into marrow allows for

the remote manipulation of T cells using an external magnetic field Hence, specific antibodies (T

IgG) should be coupled onto the Fe3O4-SiO2 NPs The antibody has two regions, an antigen-binding

region (Fab) and a function-mediated region (Fc) Only the former region specifically binds to the

target, hence keeping Fab outwardly-oriented is preferential There is a class of protein, namely

protein A, protein G, protein L or recombinantly expressed protein A/G (pA/G), can specifically

bind to the Fc region of an antibody with species-specific affinity, thus keeping Fab outward

[17-19] Currently, recombinant pA/G is commonly used due to its wide range of species-specific

binding

To deal with the critical issues, a new method of preparation of the Fe3O4-SiO2 core-shell

NPs has been carried out under the sequential functionalization of their surfaces with

3-Glycidoxypropyltrimethoxysilane (GPS) and 1,1’-Carbonyldiimidazole (CDI) Then, the BSA

protein, pA/G and antibody-binding capability of the prepared Fe3O4-SiO2-GPS-CDI core-shell NPs

is discussed

Since amino or carboxyl groups are the most common functional groups in biomolecules,

either of these two functionalities should be present on the Fe3O4 NPs’ surface to allow further

derivatization with biological ligands In this study, we used 1,1’-carbonyldiimidazole (CDI), a

highly reactive carboxylating agent that contains two acyl imidazole leaving groups, to form

reactive carbonyl groups on the hydroxyl particles, and thereby couples the amino group on the

biomolecules to the hydroxyl group through an amide linkage We expect to use Fe3O4 NPs in cell

separation Since cell size is larger than protein size, the functionalized Fe3O4-SiO2 NPs should

have long binding arms, which allows immobilized ligands to circumrotate freely to make it easy

for the biomacromolecules to have access to specific binding sites on the Fe3O4-SiO2 NPs surface

In our study, therefore, GPS acts as the long binding arm coupling agent and CDI is the

immobilized ligands

In this research, our results show that XRD and FTIR indicate Fe3O4 phase, Si-O-Si

vibrations, C-H groups, and carbonyl groups (C=O) of the prepared Fe3O4-SiO2-GPS-CDI

core-shell NPs The evidence of TEM observation of the core-core-shell structures are confirmed after the

surface functionalization Despite the presence of thick SiO2 shells on the Fe3O4 cores, the obtained

values of saturation magnetization of Fe3O4-SiO2-GPS-CDI NPs are enough for bio-applications

The amount of BSA and pA/G coupled per gram NPs was around 27 mg and 23 mg, respectively

This binding efficiency of BSA protein with our prepared Fe3O4-SiO2-GPS-CDI NPs was proven to

be better than that of Fe3O4-SiO2-GPS-IDA-Zn [7] Additionally, the prepared

SiO2-GPS-CDI-pA/G NPs can bind about 9 mg T IgG antibodies per 1g NPs Notably, the prepared

Fe3O4-SiO2-GPS-CDI-pA/G-T NPs can be used for targeting T cells which should be ex vivo eliminated

from bone marrow prior to transplantation

2 Materials and Methods

2.1 Chemical

Chemical reagents for controlled synthesis of Fe3O4-SiO2-GPS-CDI NPs were purchased

from Merck and used as received This includes iron(II) chloride tetrahydrate (FeCl2·4H2O, purity ≥

98 %), Iron(III) chloride hexahydrate (FeCl3·6H2O, purity ≥ 98 %), tetraethyl orthosilicate (TEOS,

purity ≥ 99 %), sodium hydroxide (NaOH, purity ≥ 99 %), sodium chloride (NaCl, purity ≥ 99 %),

ammonia solution (NH3, 25%), sulfuric acid (H2SO4, ≥ 95 %), hydrogen peroxide (H2O2), ethanol,

toluene, acetonitrile, and bovine serum albumin (BSA) 3-Glycidyloxypropyltrimethoxysilane

(GPS, purity ≥ 99 %) and 1,1’-carbonyldiimidazole (CDI, purity ≥ 90 %) were purchased from

Sigma-Aldrich and used as received

2.2 Synthesis of superparamagnetic Fe 3 O 4 NPs

In our typical process, Fe3O4 NPs were synthesized by the coprecipitation method [1-5]

Briefly, a mixture of FeCl2·4H2O (4.73 mmol) and FeCl3·6H2O (9.46 mmol) was dissolved in

distilled water (40 mL), which was called Solution A Then, A was mixed under ultrasonication for

15 min at 50 °C in Ar atmosphere for homogeneity 40 mL NaOH (1M) was added dropwise into A

slowly over 90 min to form Fe3O4 nuclei during slow coprecipitation process In order to grow

Fe3O4 particles with a sphere shape and control the particle size, we have kept the pH value of the

reaction solution in a range of about 10<11<12 by Duotest pH 1-12 After the completion of the

addition of NaOH into A, the mixed reaction solution was continuously ultrasonicated for 90 min at

50 °C in Ar atmosphere for Fe3O4 NP growth The reaction occurred at 50 °C and Ar gas was used

to prevent oxidation of the formed Fe3O4 NPs The black as-prepared products were separated by a

small magnet bar (SmCo), washed several times with distilled water/ethanol mixture, and then dried

at 50 °C for 9 h in vacuum Finally, the black dried product of Fe3O4 NPs was obtained with a size

range of about 10 nm

2.3 Synthesis of Fe 3 O 4 -SiO 2 core-shell NPs

To synthesize the Fe3O4-SiO2 core-shell structure for bioconjugation, 40 mg of Fe3O4 NPs

was redispersed in a mixture of 16.8 mL of distilled water, 64.0 mL of ethanol and 4.0 mL of NH3

The pH value of the dispersion was kept at ca 11 The mixture was sonicated for 30 min at room

temperature The Fe3O4 NPs were coated with SiO2 through hydrolysis and condensation of 0.4 mL

TEOS added into the mixture, which was continuously stirred for 4 h at room temperature in air

Finally, the obtained Fe3O4-SiO2 NPs were thoroughly purified repeatedly by centrifugation,

washed several times with distilled water and ethanol to remove blank silica NPs, then dried at 80

°C for 7 h in vacuum The size of obtained Fe3O4-SiO2 NPs is in the range of 100 nm

2.4 Surface functionalization of Fe 3 O 4 -SiO 2 core-shell NPs with GPS and CDI

In our tests of surface functionalization, 100 mg of Fe3O4-SiO2 NPs was hydroxylated in

100 mL of piranha solution (H2SO4-H2O2) followed by sonication for 10 min at room temperature

to create hydroxyl groups on the NPs surface for coupling to GPS The surface modified

Fe3O4-SiO2 NPs were purified repeatedly by centrifugation, washed several times with distilled water and

ethanol, then dried at 80 °C for 7 h in vacuum 70 mg of surface modified Fe3O4-SiO2 NPs was

redispersed in an aqueous solution of 210 mL GPS and toluene in order to bind the silane groups of

GPS to hydroxyl groups on the surface of the modified Fe3O4-SiO2 NPs The mixed dispersion was

sonicated overnight at 70 °C, completeing the silanization process The obtained product was

abbreviated as Fe3O4-SiO2-GPS NPs, and collected repeatedly by centrifugation, washed several

times with distilled water and ethanol, and dried at 80 °C for 7 h in vacuum The obtained

Fe3O4-SiO2-GPS NPs have an epoxy group on their own surface

Before surface functionalization of the Fe3O4-SiO2-GPS NPs with CDI, the epoxy groups of

the NPs were converted into hydroxyl groups through dispersion of 100 mg of Fe3O4-SiO2-GPS

NPs into 100 mL of NaCl solution (0.1 M) Then the dispersion was sonicated for 30 min at 70 °C

with stirring The hydroxylated Fe3O4-SiO2-GPS NPs was purified repeatedly by centrifugation,

washed several times with distilled water and ethanol, then dried at 80 °C for 7 h in vacuum for the

removal of water and solvent Finally, 50 mg of the hydroxylated Fe3O4-SiO2-GPS NPs were

redispersed into 100 mL of acetonitrile containing 250 mg of CDI and was sonicated for 1 h at room

temperature During the sonication, acyl imidazole leaving groups of CDI bind to hydroxyl groups

to form reactive carbonyl imidazolegroups Since amino or carboxyl groups are the most common

functional groups in biomolecules, either of these two functionalities should be present on the

Fe3O4-SiO2-GPS-CDI NPs surface to allow further derivatization with biological ligands

Therefore, the Fe3O4-SiO2-GPS-CDI NPs with available carbonyl imidazolegroups can couple the

amino group on the biomolecules The obtained Fe3O4-SiO2-GPS-CDI core-shell NPs were purified

repeatedly by centrifugation, washed several times with acetone, then stored in acetone for later use

The synthetic scheme of Fe3O4-SiO2-GPS-CDI core-shell NPs with detailed steps is illustrated in

Figure 1

2.5 Structure of Fe 3 O 4 NPs

The crystalline structure of the as-prepared Fe3O4 NPs was characterized in -2 mode

using a Bruker D8 Advance X-ray diffractometer (XRD) with Cu-Kα radiation (λ = 0.154 nm)

Fourier transform infrared spectroscopy (FTIR) was conducted on both the unfunctionalized and

functionalized magnetic NPs to confirm functionalization of SiO2, GPS and CDI on the Fe3O4 NPs

The size and morphology of the magnetic NPs was studied using a JEOL JEM-2100F transmission

electron microscope (TEM) operated at 100 kV Magnetic properties of magnetic NPs were

investigated using a vibrating sample magnetometer (VSM, MicroSense EasyVSM)

2.6 Magnetic nanoparticles concentration for protein-coupling reaction

Due to a limited amount of recombinant pA/G, BSA was used for optimization of the

protein coupling reaction BSA solution was prepared in carbonate buffer (pH = 9.0) with a final

concentration of 30 µg/mL After that, different amounts of Fe3O4-SiO2-GPS-CDI NPs (0.125, 0.25,

0.5, and 1.0 mg) prepared in different Eppendorf tubes were mixed with 1 mL of BSA solution, and

then incubated in an end-over-end rotator with a speed of 15 rpm at 4 °C for 24 h The Eppendorf

tubes containing the reacting mixtures were put close to a permanent magnet bar for 5 min to

separate NPs and supernatants. The supernatants were withdrawn away from the NPs to new

Eppendorf tubes Excess protein concentration in the supernatant was quantified using Bradford

assay and the NPs were washed twice with carbonate buffer The concentration of protein in the

wash fraction was also determined by Bradford assay In addition, for checking the interaction

between NPs and BSA, the NPs after coupling reaction were analyzed by SDS-polyacrylamide gel

electrophoresis (SDS-PAGE) The overall amount of coupled BSA per milligram of NPs was

calculated using the following equation:

M V C V C V C

XBSA ( B B A A W W / (1)

where XBSA (µg) denotes the overall amount of coupled BSA per 1 mg of NPs; CB (µg/mL) and VB

(mL) are the concentration and the volume of BSA solution before coupling reaction, respectively;

CA (µg/mL) and VA (mL) are the BSA concentration and the volume of the supernatant after

coupling reaction, respectively; CW (µg/mL) and VW (mL) are the BSA concentration and the

volume of wash fraction, respectively; M (mg) is the initial weight of NPs

2.7 Coupling reaction of recombinant protein A/G onto NPs

The coupling reaction between Fe3O4-SiO2-GPS-CDI NPs and recombinant protein (pA/G)

(purchased from Bio Basic Inc.) was conducted under the same conditions as coupling BSA with

the optimum concentration of reacting NPs After the coupling reaction, the NPs were then analyzed

by SDS-PAGE The total coupled pA/G amount on the NPs was calculated using the following

equation:

M V C V C V C

W

* W

* A

* A

* B

* B

C and V are the pA/G concentration and the volume of wash fraction, respectively; M is the W*

initial weight of the NPs

2.8 Conjugation of antibodies to pA/G-coupled NPs

The polyclonal antibodies were diluted in tris-buffered saline with Tween20 (TBST) to a

final concentration at 50 µg/mL and were then mixed with 0.2 mg of pA/G-coupled

Fe3O4-SiO2-GPS-CDI NPs (pA/G NPs) or 0.2 mg of non-pA/G-coupled NPs (non-pA/G NPs) These

dispersions were mixed using an end-over-end rotator with a speed of 15 rpm at 4°C for 1 h

Dispersed antibody-conjugated NPs were magnetically collected by pouring of the solvent The

collected NPs were washed with TBST twice and then eluted by using glycine solution (pH = 2.5)

for 15 min at room temperature The elution fraction was neutralized with Tris buffer (pH = 9.0) in

a ratio of 9:1, and the total protein concentration was quantified by Bradford protein quantitation

assay Finally, the amount of antibodies conjugated onto pA/G NPs was determined based on the

equation:

M V C

XG G G/

(3)

where XG denotes the amount of antibodies conjugated to 1 mg of pA/G NPs, CG and VG represent

the antibody concentration and volume of elution fraction, respectively Here, M is the initial weight

of pA/G NPs

2.9 Evaluation of T-cell binding ability of T-pA/G NPs

The NPs coupling with specific T antibodies (T-pA/G NPs) were prepared in phosphate

buffer saline (PBS) containing 1 % BSA (w/v) and 2 nM EDTA with final concentration of 0.25

mg/mL After that, 1 mL of NP solution was added into 5.103 Jurkat T cells, a common T cell line The mixture was incubated in an end-over-end rotator with a speed of 15 rpm at 4 °C for 40 min,

and then was put close to a permanent magnet bar for 5 min to separate cells into NPs fraction and

supernatant The cell number in the NP fraction and supernatant were determined by

Hemacytometer T-pA/G NPs were also incubated with TF-1 cells, a hematopoietic cell line, at the

same condition as a control In addition, antibody pA/G NPs were used for determining

non-specific binding of NPs to cells

3 Results and discussion

3.1 Characterization of Fe 3 O 4 , Fe 3 O 4 -SiO 2 , and functionalized Fe 3 O 4 -SiO 2 NPs

3.1.1 XRD

An XRD pattern of the Fe3O4 NPs is shown in Figure 2 Representative diffraction peaks at

2 = 30.31o, 35.71o, 43.36o, 57.34o, and 62.89o can be clearly identified Those diffraction peaks are characteristic of (220), (311), (400), (511) and (440) planes, respectively The XRD pattern shows a

single spinel phase of magnetite Fe3O4 (JCPDS card No.79-0417) [4,5,9,11] The average size of

Fe3O4 NPs can be calculated by the width of the reflection according to the Debye-Scherrer

equation: D = 0.9λ/(β cos θ), where β, θ, and λ are the full width at half maximum (FWHM) of the

peak, the angle of diffraction, and the wavelength of the X-ray radiation.The average size of the

sample, calculated from the Scherer equation in the use of the most intense peak (311) is about 12

nm as an estimation

3.1.2 FTIR

Figure 3 shows the FTIR spectra of both the unfunctionalized (curve a) and functionalized

magnetic NPs (curve b and c) The characteristic absorption of Fe-O vibration was observed at 585

cm−1 and O–H deformed vibration at 1635 cm-1 were observed for three NPs [curve a, b and c] The strong absorption bands at around 585 cm−1 (Fe-O) confirmed the Fe3O4 phase, which is consistent

to XRD analysis [20-22]

In curve b and c, the bands at 467 cm-1, 800 cm-1, 945 cm-1, and 1100 cm-1 were assigned to the asymmetric vibration of the Si–O–Si bond, the symmetric stretching of Si–O–Si, and the

symmetric stretching of the Si–OH bond, respectively Those bands are indicative of the existence

of SiO2 in the Fe3O4 NPs The most typical band at 3420 cm-1 was assigned to the stretching vibration of OH [20-22]

3-glycidoxypropyltrimethoxysilane (GPS) is an organic compound with the molecular

formula C9H20O5Si In curve c, the appearance of bands around 2927 cm-1 was attributed to stretching in C-H groups of GPS [20-24] In the case of CDI functionalization, since 1,1'-

Carbonyldiimidazole is an organic compound with the molecular formula (C₃H₃N₂)₂CO, it is

expected that acyl imidazole leaving groups of CDI bind to hydroxyl groups to form reactive

carbonyl groups (C=O), which record in the region 1690 – 1800 cm-1 [21, 22, 24, 25] Therefore, the band at 1720 cm-1 was assigned to the vibration absorption of carbonyl groups (C=O) of CDI In general, the -NH stretching vibration occurs in the region 3500 – 3000 cm-1 [24, 26] However, there is a wide superposition due to co-operation of stretching vibration of -OH group and -NH in

the region 3500 – 3000 cm-1, leading to no observation of the vibration band of –NH

The appearance of C=O and C-H bands provided the evidence that the GPS and CDI were

successfully attached to the surface of the designed SiO2 NPs Therefore, the prepared

Fe3O4-SiO2-GPS-CDI NPs with available carbonyl groups can be used to couple the amino group on the

biomolecules

In brief summary, both the XRD and FTIR results support the presence of Fe3O4 phase, as

well as Si-O-Si vibrations, C-H groups of GPS, and carbonylgroups (C=O) of CDI in the prepared

Fe3O4-SiO2-GPS-CDI NPs

3.1.3 Size and shape: TEM

Figure 4 shows TEM images of Fe3O4, Fe3O4-SiO2 and Fe3O4-SiO2-GPS-CDI NPs As seen

in Figure 4a, the Fe3O4 NPs are mostly spherical/spheroidal in shape and the mean diameter is

estimated to be 10 ± 3 nm, in agreement with the size calculated from XRD data As can be seen in

Figure 4b, Fe3O4 NP cores were successfully coated with SiO2 shell (thickness of 35 ± 5 nm)

resulting in the Fe3O4-SiO2 shell structure (thickness of 80 ± 5 nm) A single Fe3O4-SiO2

core-shell NP actually contains multiparticle Fe3O4 cores as seen in Figure 4b It is noted that the

Fe3O4-SiO2-GPS-CDI NPs retained the core-shell structure after the surface functionalization as shown in

Figure 4c

3.1.4 Magnetism

The magnetic measurements were performed on dried samples as shown in Figure 5 For all

the samples, the magnetization curves measured at room temperature do not show any hysteresis

loop, indicating that these NPs are superparamagnetic Moreover, this fact indicates that the Fe3O4

NPs are still well separated Saturation magnetization values, MS, were found to be 61.2, 18.4 and

11.6 emu/g for naked Fe3O4, Fe3O4-SiO2 and Fe3O4-SiO2-GPS-CDI NPs, respectively The MS

values of Fe3O4-SiO2 and Fe3O4-SiO2-GPS-CDI NPs are somewhat lower than that of naked Fe3O4

NPs because of the surface coating Despite the presence of a thick SiO2 shell, the obtained values

of saturation magnetization MS of Fe3O4-SiO2-GPS-CDI nanoparticles are considered to be enough

for potential practical bio-application [7]

3.2 Magnetic nanoparticle concentration for the protein-coupling reaction

The coupling efficiency (XBSA) was calculated for different NP concentrations (CNPs) as

shown in Figure 6 XBSA decreased with increasing CNPs At lower CNPs, i.e CNPs = 0.125 and 0.25

mg/mL, the XBSA values were 35 and 27 mg/g NPs, respectively On the other hand, at higher CNPs,

i.e 0.5 and 1.0 mg/mL, the XBSA value was quite small or could not be determined by Bradford assay

The prepared Fe3O4-SiO2-GPS-CDI NPs were hydrophobic, which leads to their interaction

and aggregation at higher CNPs level Because the aggregation lowered the available coupling sites

on the NP surfaces, XBSA might decrease dramatically Therefore, the lower CNPs lead to higher

XBSA However, there is no significant difference between the cases of CNPs = 0.125 and 0.25 mg/mL (unpaired t-test, not significant) Thus, the NP concentration of 0.25 mg/mL was used for

protein coupling reaction Besides that, the interaction between NPs and BSA was determined

through SDS-PAGE analysis, which had a limit of detection around 10 ng of protein when using the

colloidal coomassie method of Weiss et al and other scholars [27,28] As shown in Figure 7, there

are no bands apparent either in the wells of NPs coupled with BSA (Figure 7, 2–5) or in the wells of

their wash fractions (Figure 7, 6–9) regardless of CNPs This result clearly demonstrates that BSA

was successfully coupled onto Fe3O4-SiO2-GPS-CDI NPs via covalent interaction Hence, CNPs =

0.25 mg/mL was used as the optimized concentration for coupling protein A/G onto NPs

3.3 Coupling of pA/G on the surfaces of NPs

To enhance the antibody-binding capability of NPs and orient the antigen-binding site of

antibodies, pA/G was coupled onto the surface of NPs via CDI groups instead of BSA Average

XpA/G (4 experiments) was calculated to be 22.4 ± 2.4 g Although XpA/G was slightly less than XBSA (26.7 ± 3.2 µg), the moles of pA/G bound onto NPs (0.44 nmol) was nearly equal to that of BSA

(0.41 nmol) The interaction between pA/G and NPs was checked by SDS-PAGE method (Figure

8) The result showed that there was no band at 50.5 kDa, which is the molecular weight of pA/G,

either in the well of pA/G NPs (Figure 8, 4) or the wash fraction (Figure 8, 5) This demonstrated

that pA/G was bound onto NPs via a stable and strong linkage which might be the covalent

interaction between the CDI group on NPs and the amine group on pA/G Therefore, it can be

concluded that pA/G NPs were successfully formed Although the MS value of

Fe3O4-SiO2-GPS-CDI NPs was relatively low, there was a high possiblity to magnetically collect the pA/G NPs

dispersed in solution Figure 9 shows TEM images of pA/G NPs The core-shell structures still

remained after the coupling

In short summary, 1 gram of our Fe3O4-SiO2-GPS-CDI NPs can couple of 27 mg BSA and

23 mg pA/G, respectively In comparison, Ma et al., reported that their Fe3O4-SiO2-GPS-IDA-Zn2+core-shell NPs bound the BSA protein with low coupling efficiency (< 10 mg/g NPs) [7]

Therefore, our prepared Fe3O4-SiO2-GPS-CDI NPs have higher coupling efficiency in binding BSA

protein (27 mg/g NPs) compared with Ma’s NPs [7]

3.4 Antibodies conjugation to the surfaces of pA/G NPs (T -pA/G NPs)

The antibody-binding capacity of pA/G NPs (XG) is illustrated in Figure 10 While the

prepared Fe3O4-SiO2-GPS-CDI-pA/G NPs could bind about 8.7 ± 1.0 µg T antibodies per 1 mg

NPs, the Fe3O4-SiO2-GPS-CDI NPs without pA/G only bound about 3.40 ± 0.98 µg T antibodies

per 1 mg NPs

The result reveals that pA/G NPs are capable of binding more antibodies than the NPs

without pA/G on their surface This result indicates that the pA/G enhanced the

antibody-conjugating capacity of NPs because recombinant pA/G contains 6 antibody-binding sites which

can bind to the Fc domain of antibodies from a variety of species Theoretically, the pA/G NPs can

bind an antibody amount six times more than non-pA/G NPs but in this experiment, the

antibody-conjugating capacity of pA/G NPs is just about two times more than non-pA/G NPs (8.7 µg vs 3.4

µg) This might arise from the limitation of dimensional space for interaction between pA/G on the

NPs’ surface and the antibodies The dimensional limitations included the coverage or deactivation

of some antibody-binding sites happening when pA/G interacts with the CDI group on the NPs’

surface, and the near distance between pA/G and other pA/G on the NPs’ surface leading to a lack

of space for antibody bound onto pA/G’s surface site Other reasons for reduction of antibody

binding amount onto the pA/G NPs includes the stringent conditions of the coupling reaction

between pA/G and the NPs This leads to partially denatured pA/G on NPs, causing a decrease in

antibody-binding efficiency

3.5 Evaluation of T-cell binding ability of T-pA/G NPs

The T cell separation efficiency of T-pA/G NPs is summarized in Figure 11 The result

shows that the Jurkat T and TF-1 cell number did not change after incubating with pA/G NPs (data

not shown), thereby the NPs did not bind non-specifically onto Jurkat T cells or TF-1 cells

In the Jurkat T cell separation (Figure 11, left columns), the cell number in the supernatant

decreased by 37.7 % compared with the total sample, which was not incubated with NPs (1.733x105

to 2.733x105 cells) In addition, the cell number calculated from the NP fraction after separating was about 0.9x105 cells which almost agrees with the cell number reduction in the supernatant (0.9x105compared with 1x105 cells) Thus, the data indicates that T-pA/G NPs could bind and separate Jurkat T cells In contrast, the NPs did not show a non-specific binding on TF-1 cells (Figure 11,

right columns) since the cell number in the supernatant is similar to the total sample as well as TF-1

cells found in the NP fraction were few (0.133x105 cell) In summary, T antibodies maintained their ability to specifically recognize and bind to Jurkat T cell after coupling onto pA/G NPs

4 Conclusion

In this paper we reported on the synthesis of core-shell Fe3O4-SiO2 nanoparticles and its

surface functionalization with 3-glycidoxypropyltrimethoxysilane (GPS) and 1,1’carbonyldiimidazole (CDI) Here, XRD and FTIR results indicated the existence of Fe3O4 phase, Si-

-O-Si vibrations, C-H bands and C=O groups of the prepared Fe3O4-SiO2-GPS-CDI core-shell

nanoparticles TEM observation confirmed that the core-shell structure remains after the surface