Accepted Manuscript Title: Synthesis and Surface Functionalization of Fe3 O4 -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 PII: DOI: Reference: S0927-7757(16)30316-8 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.05.008 COLSUA 20632 To appear in: Colloids and Surfaces A: Physicochem Eng Aspects Received date: Revised date: Accepted date: 18-1-2016 27-4-2016 3-5-2016 Please cite this article as: 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, Synthesis and Surface Functionalization of Fe3O4-SiO2 Core-Shell Nanoparticles with 3-glycidoxypropyltrimethoxysilane and 1,1 -carbonyldiimidazole For Bioapplications, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2016.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Synthesis and Surface Functionalization of Fe3O4-SiO2 Core-Shell Nanoparticles with 3glycidoxypropyltrimethoxysilane and 1,1’-carbonyldiimidazole For Bio-applications Thi Kieu Hanh Ta,1 Minh-Thuong Trinh,2 Nguyen Viet Long,6,* Thi Thanh My Nguyen,1 Thi 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,* Faculty of Materials Science, University of Science, Vietnam National University, Ho Chi Minh, Faculty of Biology and Biotechnology, University of Science, Vietnam National University, Ho Vietnam Chi Minh, Vietnam Faculty of Resources and Environment, Thu Dau Mot University, Binh Duong, Vietnam Laboratory of Advanced Materials, University of Science, Vietnam National University, Ho Chi Minh, Vietnam School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Graphical abstract Highlights The core-shell Fe3 O4 -SiO2 nanoparticles functionalized with GPS and CDI The Fe3O4-SiO2-GPS-CDI core-shell nanoparticles could bind BSA, pA/G and anti-T cell IgG antibodies 23 mg pA/G could be coupled on g Fe3O4-SiO2 -GPS-CDI nanoparticles The g Fe3O4 -SiO2-GPS-CDI-pA/G nanoparticles could bind mg anti-T cell IgG antibodies The Fe3 O4-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 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 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-tovolume 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-SiO24 GPS-IDA-Zn2+ 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 [175 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 3Glycidoxypropyltrimethoxysilane (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 coreshell NPs The evidence of TEM observation of the 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-Zn2+ [7] Additionally, the prepared Fe3O4-SiO2-GPSCDI-pA/G NPs can bind about mg T IgG antibodies per 1g NPs Notably, the prepared Fe3O4SiO2 -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 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 Fe3O4 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 at 50 °C in Ar atmosphere for homogeneity 40 mL NaOH (1M) was added dropwise into A slowly over 90 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