Professor Metha Rutnakornpituk

Professor Laurent Fontaine

Ph.D.) ………………………………………..Committee ( Sagrario Pascual

Professor Pranee Phinyocheep

Professor Panya Sunintaboon

Professor Rattana Buosonte

Dr Metharutnakornpituk

AZLACTONE FUNCTIONALIZATION OF MAGNETIC NANOPARTICLES USING CRP TECHNIQUES AND THEIR BIOCONJUGATION YINGRAK PRAY-IN

Keywords: Magnetite, nanoparticle, ATRP, RAFT polymerization, PNA, folic acid, antibody Magnetite, nanoparticle, ATRP, RAFT polymerization, PNA, folic acid, antibody. nL

We herein report the surface modification of magnetite nanoparticle (MNP) with copolymers containing active azlactone rings via a grafting 'from' and grafting 'onto' controlled radical polymerization (CRP) for use as a nano-solid support for immobilization with biomolecules. Three different approaches were presented as following.

First, synthesis of poly(poly(ethylene glycol) methyl ether methacrylate-stat-2vinyl-4,4-dimethylazlactone) (PEGMA-stat-VDM)-grafted MNP viaa grafting 'from' atom transfer radical polymerization (ATRP) and its application as a platform for conjugating thymine peptide nucleic acid (PNA) monomer were presented. ATRP of PEGMA and VDM was firstly carried out in a solution system to optimize the reaction condition. It was found that 1/0.2/0.2 molar ratio of [ethyl-2-bromoisobutyrate (EBiB)] 0 / [copper(I) bromide (CuBr)] 0 / [tris-[2-(dimethylamino)ethyl]amine (Me 6 Tren)] 0, respectively, led to a good control of the copolymerization; the resulting polymers have molecular weight ( n M )

close to the theoretical ones and relatively narrow polydispersity indices (PDIs = 1.11-1.43). The optimal condition was then applied in the surface-initiated ATRP of MNP.

The presence of polymeric shell on MNP core was indicated by fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM) techniques.

After immobilization of thymine PNA monomer, thermogravimetric analysis (TGA) results showed that there were 4 wt% of the PNA monomer in the complex (1.2 mol/g complex). The presence of the PNA monomer was also confirmed via FTIR and VSM.

In the second work, the synthesis of MNP grafted with PEGMA and VDM via ATRP for conjugation with folic acid (FA) was studied. A grafting 'from' was applied for the copolymerization of PEGMA and VDM from the particle surface with various molar compositions of PEGMA to VDM (0/100, 30/70, 50/50 and 70/30, respectively). The existence of PEGMA and VDM in the structure was characterized by FTIR, TGA and VSM. It was found that the degree of PEGMA and VDM could be finely tuned by varying the molar ratio of PEGMA and VDM in the copolymerization.

After the FA conjugation, TEM result indicated that the FA-conjugated MNP having high VDM content exhibited good dispersibility in water.

Third, the synthesis of MNP grafted with poly(ethylene oxide)-block-poly(2vinyl-4,4-dimethylazlactone) (PEO-b-PVDM)diblock copolymer via a grafting 'onto'

strategy and its application as recyclable magnetic nano-support for adsorption with antibody were studied. PEO-b-PVDM diblock copolymers were first synthesized using a reversible addition-fragmentation chain transfer (RAFT) polymerization and then grafted onto amino-functionalized MNP by coupling with some azlactone rings of the PVDM block. TEM was used to investigate the particle nanoclustering and its distribution. It was found that increasing the PVDM chain length increased the size of MNP nanoclusters from 20 to 150 nm; approximately 10, 50 and 100 particles/clusterfor PEO-b-PVDM 21 , PEO-b-PVDM 41 and PEO-b-PVDM 84 diblock copolymer, respectively. Moreover, TEM images and photo correlation spectroscopy (PCS) technique indicated an improvement in the particle dispersibility in water after coating with the copolymers. The nanoclusters with PEO-b-PVDM 84 diblock copolymer coating were used as recyclable magnetic nano-supports for adsorption with antibody. The results show that there was 96-99% adsorption ability during eight adsorption-separation-desorption cycles.

Conclusions……………………………………………………

References…………………………………………………………

II

Azlactone functionalization of magnetic nanoparticles using ATRP and their bioconjugation…………………………………….. diagnosis [3][4][5][6][7]. Surface-modification of the MNP core with a suitable and functional organic polymer shell is a method of choice to obtain biocompatible nanoparticle and to enhance dispersibility of the particles in aqueous and organic solvents. In addition, the organic layer on its surface can provide a platform for incorporating biological functional molecules, such as amino acid, protein and enzyme [8][9][10]. Therefore, surface modification and functionalization with a shell of organic polymers have been widely studied.

The objective of this research is to modify the MNP surface with the polymeric layer containing azlactone functional groups (2-vinyl-4,4-dimethylazlactone, VDM) via a controlled radical polymerization (CRP), such as atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization, using either grafting 'from' or grafting 'onto' techniques. In addition, applications of the azlactone-functionalized MNP for bioconjugation have also been demonstrated. In the first approach (Chapter II), surface-initiated ATRP of the copolymer between poly(ethylene glycol) methacrylate (PEGMA) and VDM (poly(PEGMA-stat-VDM)) from MNP was studied. The application of the surfacemodified MNP as a scavenger for thymine peptide nucleic acid (PNA) monomer was also demonstrated. In the second approach (Chapter III), the MNPs coated with poly(PEGMA-stat-VDM) having various azlactone functionalities were used as a scavenger for folic acid (FA), a cancer cell targeting agent. In the third approach (Chapter IV), the copolymer between poly(ethylene oxide) (PEO) and VDM (PEO-co-PVDM) was synthesized via RAFT polymerization in solutions and then grafted 'onto' the MNP surface. The particles were used as magnetic nanosolid support for adsorption of antibody.

Therefore, in this chapter, surface modifications of MNP via CRP reactions, such as ATRP and RAFT polymerizations, are described in details. The studies in the applications of the MNP for bioconjugations, such as PNA, FA and antibody, are also discussed. In addition, the background of azlactone compounds was then reviewed.

Surface modification of MNP

Because MNP is in nano-scale that has a high surface area-to-volume ratio, it is thus not stable, leading to aggregation in dispersions due to various attractive forces among the particles such as Van der Waals force, gravitational force and magnetic force [11][12]. Large clusters of aggregates take place during or after the synthesis of the MNP because of these attractive forces in order to minimize the total surface energy. Therefore, stabilization of MNPs by surface modification is necessary to protect them from aggregation. Electrostatic and steric stabilizations are typically used for stabilization of the MNPs. In the case of electrostatic stabilization, an electrical double layer of charges is created around the MNPs, resulting in repulsive forces between the particles (Figure 1.1 (a)) [13]. While in the steric stabilization strategy, the MNPs are coated by surfactant such as oleic acid, citric acid and sodium oleate [13]. The particles can be coated with surfactants during and after the synthesis to reduce the surface tension [START_REF] Nalwa | Encyclopedia of Nanoscience and Nanotechnology[END_REF]. Polymers are also used as a surfactant by grafting to the particle surface (Figure 1.1 (b)). These polymer shells can increase the steric repulsive force that will prevent the particle agglomeration [13]. In addition, stabilization and surface modification of the MNP with small organic molecules, or polymers, not only prevents uncontrollable aggregation and protects them from the aggregation, but also allows them to be dispersible in various organic solvents or water. The attachment of polymer chains to the MNP surface can be performed via the interaction between the hydroxyl groups on the bare MNP surface and suitable functional groups of polymer chains or active compounds. Two alternative strategies:

grafting 'onto' and 'from' are used to prepare polymer-coated MNP. In the case of grafting 'from' strategy, the polymer chain is grown from the initiator-functionalized particle surface, while in the grafting 'onto' strategy, a pre-formed polymer with an appropriate functional group is grafted onto the particle surface [START_REF] Li | [END_REF]. The combination of these two strategies with CRP technique has widely used for preparation of polymer-coated particles. Figure 1.2 shows the use of grafting 'onto' and 'from' strategies to prepare polymer-coated MNP. ATRP is one of several kinds of CRP, which does not require stringent experimental condition. A wide range of functional monomers such as styrene [16],

methacrylate [17], acrylates [18] and N-isopropylacrylamide [19] can be used to prepare the polymer with a controllable fashion and narrowly dispersed molecular weights. Figure 1.3 presents the general mechanism of ATRP. In the polymerization, initiating radical species (R • ) and an oxidized metal compound (M n+1 X n+1 L m ) will be activated from the carbon-halogen bond in an organic halide (R-X) via one-electron oxidation of the metal center (M n X n L m ). After that R • is reacted with the halogen on the oxidized metal to regenerate R-X or added to the monomer to generate a radical

species [R-CH 2 -C(R 1 )(R 2 ) • ]. The adduct [R-CH 2 -C(R 1 )(R 2 )
-X] of R-X and monomer will be then generated via an abstraction of a halogen atom from M n+1 X n+1 L m. . The carbon-halogen bond of the adduct is subsequently activated by the metal complex, similarly to R-X, to result in a similar carbon-halogen bond at the polymer terminal via a repetitive set of the reactions. The important factors of this reaction are the low concentration of the radical intermediates at a given time and the fast but reversible transformation into the dormant species rather than the addition to monomers [20]. 1.7) [24]. In the first step, MNP was coated with a thin layer of silica of (3-aminopropyl)triethoxysilane to prevent the oxidation and dissolution of the particle. The ATRP initiator (3-chloropropionic acid) was then immobilized onto the silica surface via amidation reaction. PEGMA macromonomer was grafted from the immobilized initiators to give the macroinitiators for the ATRP of the MMA second block. It was found that the biocompatibility of the particles was considerably improved by introducing a monolayer of PEGMA to its surface.

Moreover, the capability of the modified particles in resistance to the proteins nonspecific adsorption was higher than the uncoated particles. 1.9) [26]. The PHEMA-grafted particle was subsequently functionalized with nitrilotriacetate-Ni 2+ to give magnetic beads for selective capture of polyhistidine-tagged (His-tagged) protein from cell extracts (Figue 1.10). First, the MNP was coated with silica and then immobilized with ATRP initiators via silanization. The immobilized initiators were used to polymerize HEMA monomer to give PHEMAgrafted MNP. After that, the polymer brushes were further reacted with succinic anhydride (SA) and N α, N α -bis (carboxymethyl)-L-lysine hydrate (aminobutyl NTA). It was found that His-tagged protein can be rapidly isolated from a cell extract with high purity by using this particle. The 50 nm thickness of polymer brushes can reduce the diffusion limitation and allow protein capture in a short period of time (5 min).

Furthermore, the polymer brush-modified particles provide high protein recoveries and can be recycled. PMDETA: N,N,N',N",N"-pentamethylenetriamine) [27] Furthermore, Rutnakornpituk et al. have presented the surface modification of MNP with poly(t-butyl acrylate) (P(t-BA)) via ATRP, followed by a hydrolysis of t-BA groups to obtain poly(acrylic acid) (PAA) and finally immobilization of folic acid (FA) on its surface (Figure 1.12) [28]. FA was successfully activated with ethylene diamine (EDA) to obtain primary amine-terminated folic acid derivative. This logical strategy enhanced the reactivity of FA to efficiently react with carboxylic acid overexpressed on the surface of PAA-coated MNPs through amidization reaction.

Moreover, PAA on MNP surface can also provide stabilization mechanisms through both steric and electrostatic repulsion.

Surface modification of magnetite nanoparticle (MNP) via grafting 'from' reversible addition-fragmentation chain transfer (RAFT) polymerization

The RAFT process utilizes classic free radical initiators and monomers but also includes the presence of chain transfer agent (CTA) in the reaction. This technique, similar to other CRP techniques, produces polymers with controllable molecular weight with narrow PDIs. Figure 1 -A) and N,N 'dimethylaminoethylacrylate (DMAEA) were polymerized in the presence of an αphosphonic acid RAFT agent. Two types of polymers: α-phosphonic acid-P(OEG-A) and α-phosphonic acid-P(DMAEA), were obtained (Figure 115). It was found that the n M values of the polymers were nearly with those of the targeted n M values. The narrow PDIs were also observed (PDIs below 1.3). Moreover, the first-order kinetics of the monomer consumption confirmed the controlled characteristics of CRP. Each polymer was conjugated to the MNP surface to yield P(OEG-A)-coated MNP and P(DMAEMA)-coated MNP. It was found that the MNP coated with these two polymers were well dispersible in aqueous solutions. In addition, it was also tested that these surface-modified MNPs were non-toxic to human neuroblastoma cells and capable of complexing siRNA.

PAA-PEO

PAA-PAAMPEO PNIPAAm was first synthesized by RAFT polymerization using S,S'-bis(α,α'dimethyl-α"-acetic acid)trithiocarbonate and 4,4'-azobis(4-cyanovaleric acid) (ACVA) as a CTA and initiator, respectively (Figure 1.18 (a)) [33]. Biotin was then conjugated at one end of the polymer chain to yield biotin-PNIPAAm through the coupling reaction between PNIPAAm and a PEO maleimide-activated biotin. The mixture of PNIPAAm and biotin-PNIPAAm was mixed with the particle in an attempt to yield biotin-PNIPAAm-coated MNP via the interaction between carboxylate groups of the polymers and the particle surface (Figure 1.18 (b)). The binding of biotin-coated particle to streptavidin was observed by fluorescence and surface plasmon resonance.

CH 3 O(CH 2 CH 2 O) 46 CH 2 CH 2 OH [MPEO-OH] + C CH 3 CH 3 C O HO S C S S C 12 H 25 DCC C CH 3 CH 3 C O O S C S S C 12 H 25 H 2 C H 2 C O H 2 C H 2 C H 3 CO 46 [PEO-RAFT] [PEO-RAFT] AA/DMF C CH 3 CH 3 C O O PEO PAA S C S S C 12 H 25 [PEO-PAA] H 2 C CH C O OH n CTA, AIBN DMF, 80 o C C O HO C CH 3 CH 3 CH 2 H C C O OH CH 2 H C C S C S S C
It was found that the biotin-coated particles have a high affinity to bind with streptavidin in solution or when the streptavidin was immobilized on the surface. Furthermore, stabilization and bioconjugation of MNP with hetero-telechelic polymers synthesized by RAFT polymerization were reported [Boyer et al. [34]].

Water soluble telechelic polymers of poly(oligoethyleneglycol acrylate) P(OEG-A)

and PNIPAAm were first synthesized using trithiocarbonate compounds containing a dimethyl group as a CTA in the presence of AIBN initiator (Figure 1.19 (a)). After modification of one terminal of the polymers to obtain phosphonic acid end-groups and another terminal to obtain ethylpyridyl disulfide end-groups, the phosphonic acid can conjugate to the particle surface, resulting in stable particles dispersible in water.

It was found that the particles grafted with P(OEG-A) having n M larger than 10,000 g/mol were stable in phosphate buffers for several days. Additionally, the particles grafted with P(OEG-A) having

Bioconjugation on surface of magnetic nanoparticle

Besides the creation of hydrophilic polymers on magnetic nanoparticle surface and making them stable against agglomeration in water, surface modification with polymeric surfactant can also provide a platform for functionalization with bioentities.

Various chemical approaches have been used for conjugation of biomolecules on particle surfaces such as targeting agent, therapeutic and imaging reporter molecules.

These can be achieved by covalent linkage approaches (direct nanoparticle conjugation, click chemistry, covalent linker chemistry) and physical interactions (electrostatic, hydrophilic/hydrophobic interactions). Table 1.1 shows some examples of strategies that can be used for surface modification. Hydrophobic molecules (Ref. [49])

Various biological molecules such as antibodies, proteins, targeting ligands, PNA, DNA and FA, can be chemically bound to nanoparticle surfaces through those linkages. Because the current work involved the immobilization of PNA, FA or antibodies on MNP surfaces, introduction to PNA, FA and antibodies-immobilized nanoparticles have been reviewed.

Surface functionalization of MNP with peptide nucleic acid (PNA)

Peptide nucleic acid (PNA) is a synthetic deoxyribonucleic acid (DNA) mimic in which the phosphate backbone is replaced by repeating units of a flexible pseudopeptide polymer such as N-(2-aminoethyl)-glycine units or d-prolyl-2aminocyclopentanecarboxylic acid units that linked by peptide bonds, so called aegPNA [50] and acpcPNA [51], respectively ( with FA for targeting human cervices carcinogen HeLa cell [45]. In the first step, 2carboxyethyl phosphonic acid (CEPA, ( 14)) was immobilized on the particle surface to form carboxyl group-terminated nanoparticles [START_REF] Li | [END_REF]. In the second step, γ -{N-{2-[2-

(2-aminoethoxy) ethoxy]ethyl} folic acid (FA-EDBE, ( 18)) was then immobilized through amidiation between amine (-NH 2 ) groups of FA-EDBE and carboxyl groups on the particle surface (Figure 1.26). They found that the FA-conjugated particles (19) had a hydrodynamic size of 58 nm with PDI less than 0.2. Change of pH (4.5-8.5) of the medium did not significantly change the hydrodynamic diameter of the samples.

Moreover, the nanoparticles had the ability to target human cervices carcinoma HeLa cell and low cytotoxicity even at relatively high concentration (0.2 mg/mL). and FAMAM were internalized into breast cancer cell (KB cells). The amount of uptake of FAMAM was much higher than that of MAM. This result suggests that the modification of MNP with both MA and FA could be used to facilitate the nanoparticle uptake to specific KB cells for molecular imaging. This strategy prevented to loss of the α-carboxyl group of FA, which is essential for high-affinity binding to folate receptors on human tumors.

+ O Fe 3 O 4 FA "Click" Reaction CuI N N N N N N N N N O O O (A) (B) (C) (1)

Surface functionalization of MNP with antibody

Antibodies or immunoglobulins (Ig), biological products that are parts of the specific immune system, usually possess pathogenic or toxin neutralizer properties.

Also, they show the recruitment of immune elements such as complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells. In addition, they could carry several elements such as toxins, drugs, fluorochroms, or even nanoparticles, etc., and be used in several diagnostic procedures and therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a hybrid product that merges the properties of both. For instance, they combine the special thermal, imaging, drug carrier, or magnetic characteristics of nanoparticles with the specific and selective recognition abilities of antibodies [57].

Xu et al. have demonstrated the separation of circulating cancer cells (CTCs)

from fresh whole blood using an antibody-conjugated MNP (Ab-MNP). First, MNP was modified with amphiphilic polymer ( or Lane 3 (the whole protein extract). It was also found that carboxylated MNP without anti-HER2 conjugated cannot separate HER2 (Lane 4). These results confirm that the capture of the HER2 was only obtained when MNP was conjugated with the specific antibody. After that, the particles were bind with a complementary antibody labeled with fluorescein isothiocyanate (FITC) (anti-goat IgG). According to fluorescence microscopy results, the covalent attachment of the antibody to MNP was efficient with very low non-specific interactions. On the other hand, the physical adsorption of the antibody was not successful with the presence of non-specific interactions, indicated by observation of the secondary antibody labeled with FITC.

Moon et al. have reported the preparation of polymer-coated MNP with various carboxyl vinyl monomers including N-acryloylaminohexanate, N-

acryloylglycine and N-acrloyl-L-glutamic acid by radical polymerization for application as an immunoassay magnetic marker [60]. First, a hydrophilic macromer having a vinyl group at the terminal poly(N-vinyl-2-pyrrolidone) was adsorbed on the particle surface. Then, a carboxyl vinyl monomer and a crosslinking agent (tris(2acryloy-loxyethyl)ammonium chloride) were added to initiate a copolymerization reaction (Figure 1.31). Finally, the particle was conjugated with antibody via a reaction between the carboxyl groups on the particle surface and amino groups of antibody. It was found that the particles were dispersed stably in aqueous media. In addition, the particles exhibited high antibody binding ability and high magnetic sensitivity properties which are suitable for use as magnetic markers. Puertas et al. have recently reported a synthesis of carboxylated MNP for ionic adsorption with antibodies through their richest positive charged regions [48]. The adsorption of antibody was applicable at the pH lower than its isoelectric point and the antibodies could be easily eluted by increasing the ionic strength and/or changing the net charge of the antibodies by changing the solution pH.

The reaction of biomolecules with MNP containing reactive functional groups may require the presence of a catalyst to achieve high yields and it can be accompanied by the formation of a side-product, making purification of the resulting polymer tedious. In the context of biological applications, such reactions should be avoided whenever possible, since the catalyst and/or by-product can be toxic.

Therefore, utilization of MNP coated with reactive polymers containing functional groups that do not require a catalyst to react with biomolecules is very interesting.

Therefore, the background of azlactone compounds is then reviewed. for the synthesis of a series of amine functionalized-polymers [65]. These materials can be used as cationic polymers for DNA delivery. Recently, Fontaine et al. have synthesized PVDM brushes from a polymeric resin using a surface-initiated ATRP [66]. Wang resin, a styrene-based resin, was first immobilized with an ATRP initiator, followed by VDM polymerization from the resin surface. The VDM-functionalized resin was efficiently used as a nucleophilic scavenger to remove benzylamine from solutions.

Chemistry of azlactone compounds

Conclusions

In this chapter, we reviewed some important works regarding the utilization of solution system to optimize the reaction condition and the optimal condition was then applied in the surface-initiated ATRP of MNP. Fourier transform infrared (FTIR) spectroscopy indicated the presence of the copolymer in the MNP complexes. After immobilization of thymine PNA monomer, thermogravimetric analysis (TGA) results indicated that there were 4 wt% of the PNA monomer in the complex (1.2 mmol/g complex). The existence of the PNA monomer in the complex was also confirmed via FTIR and vibrating sample magnetometry (VSM). The MNP complex with active surface might be efficiently used as magnetically guidable nanosolid support for PNA oligomers and other molecules containing affinity functional groups.

CRP

Experimental

Materials

Unless otherwise stated, all reagents were used without further purification: iron(III) acetylacetonate (Fe(acac)

Results and discussion

Synthesis of poly(PEGMA-stat-VDM) via ATRP in solution

In the present work, a statistical copolymer of PEGMA and VDM was first synthesized in toluene using CuBr/Me 6 Tren catalytic complex in the presence of EBiB as an initiator. CuBr and Me 6 Tren were selected as a catalytic complex in the present work. Fontaine et al. have reported that using Me 6 Tren ligand led to a good control in polymerization of VDM: the experimental molecular weights were comparable to the theoretical values and narrow polydispersities were obtained [4]. Molar ratio of CuBr/Me 6 Tren/EBiB was studied in order to determine the best experimental conditions to obtain a good control in molecular weight and molecular weight distribution. The selected reaction conditions would then be used for surface-initiated ATRP of PEGMA and VDM from magnetite nanoparticles, which would be then used for immobilizing thymine PNA monomer on their surface.

A EBiB/CuBr/Me 6 Tren molar ratio of 1/1/1 has successfully been used in our group to synthesize well-defined (co) polymers based on VDM in solution [4].

Therefore, in this work, poly(PEGMA-stat-VDM) was synthesized via ATRP of PEGMA and VDM monomers using the same catalytic/initiator system and similar catalyst/ligand ratios.

M ,th = ([PEGMA] 0 /[EBiB] 0 × conv. PEGMA × M PEGMA ) + ([VDM] 0 /[EBiB] 0 × conv. VDM × M VDM ).
c Measured via SEC (calibrated with polystyrene standard).

A preliminary experiment was performed in toluene at 50 o C using [EBiB] 0 /

[CuBr] 0 /[Me 6 Tren] 0 ratio of 1/1/1 (entry 1, Table 2.1). Then, to prepare statistical copolymers based on PEGMA and VDM grafted onto MNP surface, the optimal condition established previously for ATRP in solution were used. Because the MNP-supported copolymers could not be studied by NMR technique, the free initiator EBiB (also called "sacrificial" initiator) was added to the reaction to easily monitor the reaction progress. Such a strategy was previously applied for ATRP of polystyrene (PS) on Wang resin [5]. It was found that the macromolecular characteristics (molar masses and PDIs) of PS formed in solution and on Wang resin were similar. Therefore, monomer conversions and copolymer compositions, discussed in the latter section, were investigated from the free copolymers via NMR spectroscopy. The reaction was carried out in toluene at 30 o C using

[PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[BTPAm-coated MNP] 0 /[CuBr] 0 /[Me 6
Tren] 0 in a 100/100/1/1/0.4/0.4 molar ratio. This molar ratio was adjusted in accordance with the fact that there were both ATRP initiating sites (BTPAm) grafted on particle surface and a free initiator (EBiB) in this system. After the ATRP reactions, the particles in the dispersion were magnetically separated from the mixture. The aggregate was used in FTIR and VSM characterizations, and the supernatant containing the free copolymers was used in SEC and NMR analyses. 24% and 29%, respectively, after 24 h of reaction, while those in the solution system with the same reaction conditions proceeded in a much shorter time (25% and 31%, respectively, in 30 min reaction) (see Table A3 in the appendix A). This phenomenon has been previously observed in the surface-initiated ATRP of VDM from the Wang resin solid support [5].

n M gradually increased when the reaction conversions increased, indicating the growth of the copolymer chains. In all cases, the n M ,SEC values were higher than the theoretical ones and their distributions were narrow (PDIs = 1.09-1.21) throughout the reaction (Table 2.2).

Immobilization of thymine PNA monomer on poly(PEGMA-stat-VDM)coated MNP

The as-synthesized poly(PEGMA-stat-VDM)-coated MNP containing active azlactone functional groups were then used as a magnetically guidable-nanoscale amine support. In this study, thymine PNA monomer was selected as a model compound for grafting onto the particle surface. It is anticipated that these novel copolymer-coated MNPs can be used as a nanosolid support for PNA oligomer immobilization. Because precedents have reported the utilization of PNA oligomer as a probe for detection of DNA sequences [6][7][8][9], the attachment of PNA oligomers on these copolymer-coated MNP is warrant for further studies.

The immobilization process of thymine PNA monomer on the particle surface was performed in anhydrous DMF at room temperature for 6 h (Scheme 2.2). Figure 2.8C shows the disappearance of cyclic carbonyl of azlactone rings at 1816 cm -1 [5] and the appearance of a signal at 1159 cm -1 corresponding to a C-H bending of thymine [10], indicating the presence of thymine PNA monomer in the complex. The presence of hydrophobic thymine PNA monomer units on surface of the complexes might promote the particle aggregation in DMF. Although there was some in various solvents, such as THF, DMF and toluene, probably due to the presence of the polymeric thin film on their surface. A TEM image showing the presence of polymeric thin films on the particle surface was illustrated in the appendix A.

Conclusions

This work presented the surface modification of MNP with the statistical copolymer between PEGMA and VDM via ATRP to obtain the particles containing active functional groups on its surface. Hydrophilic PEGMA provided good dispersibility to the particle in polar solvents and azlactone rings served as active functional groups for further chemical attachment with nucleophiles of interest.

Surface-initiated ATRP of the copolymer via a grafting 'from' strategy from the particle produced the active polymer layer with a predicable and controllable fashion.

The nanosolid supports were successfully used for immobilization of thymine PNA monomers on its surface. The results signified the feasibility to functionalize the surface of these novel azlactone-based nanoparticles with a broad range of other nucleophilic scavengers such as hydroxyl-and thiol-containing compounds.

HYDROPHILIC AZLACTONE-FUNCTIONALIZED MAGNETITE NANOPARTICLE FOR CONJUGATION WITH FOLIC ACID* Abstract

Herein we report the synthesis of magnetite nanoparticles (MNPs) grafted with poly(poly(ethylene glycol) methyl ether methacrylate-stat-2-vinyl-4,4dimethylazlactone) copolymers (poly(PEGMA-stat-VDM)) prepared via a surfaceinitiated atom transfer radical polymerization (ATRP) and used for the immobilization of folic acid (FA). The MNPs were synthesized using a thermal decomposition method and surface functionalized to obtain ATRP initiating sites. Molar ratio of PEGMA to VDM was systematically varied (0/100, 30/70, 50/50 and 70/30, respectively) in the copolymerization to obtain water dispersible MNP with various amounts of azlactone rings, an electrophilic moiety, on its surface. Grafting density of VDM on the particle surface increased with increased VDM loading in the copolymerization reaction.

These copolymer-coated MNPs were well dispersible in water with some nano-scale aggregation after FA functionalization due to hydrophobic character of FA. Since FA is a cancer-cell targeting agent, it is anticipated that these novel FA-functionalized MNPs could be used as magnetically guidable vehicle for drug delivery, particularly for cancer treatment. The results of this study warrant a future investigation of this promising system.

Experimental

Materials

Unless otherwise stated, all reagents were used without further purification: copper(I) bromide (CuBr, 98%, Acros), triethylamine (TEA, 97%, Carlo Erba), dicyclohexylcarbodiimide (DCC, 99%, Acros), sodium hydroxide (≥ 98%, Aldrich), [2] and N-(2-aminoethyl) folic acid [3] were prepared according to previously reported procedures. N,N-dimethylformamide (DMF, Acros), toluene (Acros) and dichloromethane (CH 2 Cl 2 , ≥ 99.5%, Sigma-Aldrich) were used as received. Acryloyl chloride was synthesized via the reaction between acrylic acid and benzoyl chloride at 75 o C to give a colorless liquid; 60% yield.

Characterization

Fourier transform infrared (FTIR) spectroscopy was performed on Perkin-Elmer Model 1600 Series FTIR Spectrophotometer. The solid samples were mixed with KBr to form pellets. Nuclear magnetic resonance (NMR) spectroscopy was performed on a 400 MHz Bruker NMR spectrometer using CDCl [4]. 2-Methylalanine (5.6966 g, 5.52 x 10 -2 mol, 1 eq.) was added into an aqueous solution of sodium hydroxide (4.4160 g, 11.04 x 10 -2 mol, 2 eq. in 15 mL water) in the presence of BHT (0.0552 g, 2.5051 x 10 -4 mol) as a polymerization inhibitor at 0-10 o C, followed by an addition of acryloyl chloride (5 mL, 5.52 x 10 -2 mol, 1 eq.). After 12 h stirring, conc.

Synthesis of 2-vinyl-4,4-dimethylazlactone (VDM)

Synthesis of N-acryloyl-2-methylalanine

HCl (6.81 mL, 6.90 x 10 -2 mol, 1.25 eq.) was added into the solution, which was kept at 10 o C. The mixture was continuously stirred for another 30 min to form a white solid, which was filtered, washed with water and dried. Yield: 69-72%. C=O). FTIR: 1822 cm -1 (C=O stretching), 1668 cm -1 (C=N stretching), 1204 cm -1 (C-O-C stretching). This procedure was modified from the previously reported article using ethyl chloroformate as a coupling agent and giving rise to about 60% yield [4]. 1 H NMR, 13 C NMR and FTIR spectra are shown in the appendix B. outermost layer of polymeric surfactant coated MNPs, which might limit the grafting efficiency to the particle surface [7][8][9]. A promising strategy in promoting the grafting efficiency of FA to MNP surface is to have reactive functional groups along the polymer coating layers. As compared to other polymeric coatings [3,10], the advantage of using azlactone-containing copolymers is that the azlactone rings react readily with nucleophiles at room temperature without using a catalyst or a coupling agent and leaving no by-products. Therefore, the significance of the current work is that we here report an efficient strategy to enhance the grafting efficiency of FA on water dispersible MNPs coated with well-defined copolymers. It is envisioned that the loading of FA on the poly(PEGMA-stat-VDM) copolymer-coated MNPs should be facilitated due to the increased amount of azlactone rings.

BTPAm, the initiator for ATRP, was first immobilized onto the surface of the particles through the combination of a ligand exchange reaction and condensation of triethoxysilane to obtain ATRP initiating sites on their surface, followed by surfaceinitiated ATRP of PEGMA and VDM. FA was then immobilized on the particle surface via a ring-opening reaction. The TEM samples were prepared from MNP-DMF dispersion.

Percent weight loss of poly(PEGMA-stat-VDM)-coated MNPs having different molar ratios of PEGMA to VDM was investigated to determine the relative amount of the copolymer that can be grafted on its surface. It should be noted that the particles were separated from the ungrafted species with an assistance of an external magnet. Using the hypothesis that percent char yield was the weight of magnetite core remaining at 600 o C, the weight loss of the samples was thus attributed to the decomposition of organic components including BTPAm and the copolymers that partitioned to the particle surface. According to the TGA results, BTPAm content in BTPAm-coated MNP was about 7.6% and those of the copolymers were in the range of 10.5-15.3% (Figure 3.3). This result corroborates the results from FTIR suggesting that the copolymers existed on the particle surface. After the surface-initiated ATRP from the MNPs, it was envisioned that the particles having azlactone ring-enriched surface were obtained. The azlactone rings are reactive with nucleophilic functional groups such as amines, hydroxyls and thiols.

In the current work, FA was covalently immobilized on poly(PEGMA-stat-VDM)coated MNPs through a ring-opening reaction of the VDM moiety. Utilization of FA increasingly attracted much attention for tumor selective drug delivery applications because it provides specific sites that differentiate tumor cells from normal cells. FA also plays a role in receptor-mediated endocytosis and the process involves the access of a folate-receptor complex into the cells as a group, where it is fused with a lysosome [11][12]. In addition, FA is considered appropriate for targeted delivery because it is non-immunogenic, high binding affinity for its receptor, highly stable and inexpensive and easy to be conjugated with imaging agents [13].

In this work, FA needed to be functionalized with ethylene diamine (EDA) to contain a primary aliphatic amine group (N-(2-aminoethyl) folic acid or EDA-FA).

This strategy enabled FA to efficiently couple with azlactone rings present on the particles. Immobilization of EDA-FA on the particle surface was carried out in anh.

DMF at room temperature for 12 h. The D h and zeta potential of FA-functionalized and non-functionalized MNP are given in Table 3.2. The D h of the particles significantly increased after the FA immobilization. This was attributed to the grafting of hydrophobic FA on the particle surface and thus promoting particle agglomeration. This is in good agreement with the decrease in degree of negative charge of the FAfunctionalized MNPs as compared to those before the FA functionalization, which was attributed to the presence of the pterin rings of FA after coupling reactions. It should be mentioned that the first requirement of MNPs for applicability in the biomedical field is that they should be stable in aqueous medium. In order to have the best possible transmembrane permeability, a second requirement is that the final size of the MNPs should be as small as possible. Grafting FA to MNP surface should increase the permeability into cancer cells but, due to FA hydrophobic behavior, it seemed to decrease the overall solubility of the complex. Therefore, optimization between the degree of nano-clustering, reflecting to the final size of the MNP, and FA grafting efficiency needs to be investigated. In addition, transmembrane permeability testing of the FA-grafted MNPs is warranted for future studies.

After FA immobilization, the zeta potential values of the particles decreased from -14.12 to -4.90 mV with decreasing azlactone composition in the copolymer.

These values seemed to be quite low for nanoparticle stabilization via electrostatic repulsion, which might cause an increase in nanoparticle agglomeration tendency.

However, these particles were rather well dispersible in water because of the presence of hydrophilic polymeric coating on their surface providing steric repulsion stabilization mechanism, and this might consequently enhance cell membrane penetration ability. was attributed to the presence of hydrophobic FA coated on the particle surface (Figure 3.5A'-D'). More interestingly, according to the visual observation, FA-grafted MNP having high VDM content exhibited good particle dispersibility and stability in water. For instance, FA-poly(VDM)-coatedMNP (without PEGMA) was well dispersible in water for more than 24 h without noticeable aggregation as opposed to other samples. This result could be explained by the formation of carboxylate ions from a ring-opening reaction of the remaining VDM in water, leading to additional electrostatic repulsion stabilization and thus preventing particle agglomeration.

Therefore, FA-poly(VDM)-coated MNPs were used for study of the percentage of FA in the complex discussed in the later section. The compositions of poly(VDM) and FA on the particles were determined via TGA by analyzing the samples both before and after FA conjugation (Figure 3.6A). It was found that there were 12.3 wt% of poly(VDM) and 10.6 wt% FA grafted to the particles. This number corresponds to about 1.30 FA molecules/nm 2 of the particle surface area (260 FA molecules/particle). An example of the calculation is illustrated in the appendix B.

Conclusions

This work reports the surface modification of MNPs with poly(PEGMA-stat-VDM) via ATRP using a grafting 'from' strategy. The degree of the electrophilic azlactone rings could be adjusted by varying the molar ratio of PEGMA to VDM in the copolymerization. These copolymer-coated MNPs were successfully used for immobilization of FA on their surface. It was found that the particles with good dispersibility and stability in water, and with reasonable FA immobilization capability were obtained. These novel FA-functionalized MNPs might be used as efficient drug delivery vehicles, particularly for cancer treatment. Studies investigating the efficiency of treating cancer using these particles will be conducted in the future.

RECYCLABLE MAGNETIC

Formation of PEO-b-PVDM copolymers-MNP nanocluster

APS-grafted MNP (100 mg) was dispersed in 10 mL 1,4-dioxane with sonication. Then, a PEO-b-PVDM diblock copolymer solution (100 mg of the copolymer in 10 mL 1,4-dioxane) was added to the APS-grafted MNP dispersion with stirring at room temperature for 12 h. The copolymer-coated MNP was magnetically separated from the dispersion and extensively washed with 1,4-dioxane. This process was repeatedly performed to remove ungrafted copolymer from the MNP nanocluster.

The copolymer-MNP nanocluster was then dried in vacuo at room temperature for 12 h.

Adsorption efficiency study of the copolymer-MNP nanoclusters with antibody

The copolymer-MNP nanocluster was first dispersed in water for 12 h to form carboxylate-enriched nanocluster due to the ring-opening reaction of the remaining azlactone rings in the copolymer on particle surface. After drying process, 10 mg of the copolymer-MNP nanoclusters were incubated in 1 mL of 10 mM MES pH 6 solution containing anti-rabbit IgG antibody for 2 h at room temperature. The Bradford assay [4] was used to determine antibody adsorption efficiency. The protein concentration of all samples was determined using a calibration curve of BGG as a protein standard. The copolymer-MNP nanoclusters after adsorption with antibody were separated from the supernatant using an external magnet. The absorption at 595 nm of the antibody solution before and after adsorption process was measured using the Bradford assay. The amount of the antibody adsorbed on the nanoclusters (in the unit of mg antibody/mg MNP) can be directly calculated from the decrease in antibody concentration in the supernatant using the following equation:

Adsorption efficiency = [(A-B)/A] x100……………………………………………..(1)
where A is the loaded concentration of antibody and B is the concentration of adsorbed antibody.

Antigen recognition capacity study of antibody-adsorbed magnetic nanoclusters

After antibody adsorption procedure, the remaining carboxyl groups of magnetic nanoclusters were blocked with 1 mL of 1% BSA in 10 mM MES pH 6 at 25 o C for 16 h. MNP nanoclusters were then washed with 10 mM MES pH 6 to remove an excess BSA. To detect the antigen recognition capacity of antibody-adsorbed MNP nanoclusters, an indirect detection method was used. First, 1 mL of 400 ppm of primary antigen (2.00 times molar excess) in 10 mM MES pH 6 was added into antirabbit IgG antibody-adsorbed MNP nanoclusters and incubated for 30 min. After washing the particles with 10 mM MES pH 6, they were incubated with 1 mL of 400 ppm of anti-rabbit IgG-HRP antibody for another 30 additional min. The presence of antigen on the particle surface was visualized by adding 1 mL of ABTS-H 2 O 2 solution into 10 µl of the MNP nanoclusters dispersion. 4.1. Theoretical number-average molecular weights ( n M ,th ) calculated from the monomer conversion increased as increasing the PVDM block length and this result is in good agreement with SEC results (Table 4.1). Figure 4.1 shows that SEC traces of PEO-b-PVDM diblock copolymers shift to earlier retention times with respect to the SEC trace of PEO-CTA maintaining low polydispersity indices (PDIs ≤ 1.10). This result shows that the copolymerization is well controlled leading to well-defined diblock copolymer structures. In good agreement with TEM results, hydrodynamic diameter (D h ) of APSgrafted MNP was significantly larger than those of the MNPs coated with PEO-b-PVDM copolymers, indicating the improvement in water dispersibility of the particles after polymer coating (Table 4.2). Increasing PVDM block lengths in the copolymers led to the increase in D h (97-166 nm), which corresponded to the TEM results. In addition, hydrodynamic size distributions of the copolymer-MNP nanoclusters were narrow as compared to that of APS-grafted MNP as a result of polymer coating and thus improving their water dispersibility. Zeta potential values of the nanoclusters were significantly increased after copolymer coating (-27 to -34 mV) due to the ring opening of PVDM units in the copolymers, resulting in the formation of the nanoclusters with carboxylate-enriched surface. The negative charge of the carboxylate-coated nanoclusters was essential for magnetic separation of antibody through ionic adsorption mechanism in the next step. well dispersible in the media but not completely separated from the media using an external magnet. Trace of MNP in supernatant after magnetic separation and/or ultracentrifugation could interfere with Bradford assay [4]. The presence of single nanoparticles or magnetic clusters with small size possesses low magnetic sensitivity, resulting in difficulty in removal from the dispersion [5]. A compromise between good water dispersibility and magnetic sensitivity of MNP is crucial for use as a nano-solid support. Therefore, the nanoclusters grafted with these two copolymers were unsuitable to be used as the supports for antibody adsorption. Interestingly, nanoclusters grafted with PEO 44 -b-PVDM 84 copolymer with D h of 166 nm exhibited good dispersibility in the media and more importantly facilitated manipulation by an external magnet without trace of MNP left in the dispersion and interference with Bradford assay.

Results and discussion

PEO

The amount of adsorbed antibody was quantified by the different concentrations of antibody in loading and in supernatant after adsorption process using the Bradford protein concentration assay [4]. The adsorption efficiency can be calculated using the Equation (1). experiments confirm that the antigen recognition of the antibody-adsorbed MNP nanoclusters was preserved. These MNP nanoclusters could be used as a recyclable magnetic marker to immobilize with other specific antibody-antigen conjugates.

A) B) C) D) E) F) G)

Conclusions

Surface modification of MNP with well-defined PEO-b-PVDM diblock copolymer using grafting 'onto' strategy to form magnetic nanoclusters was described.

The nanocluster size could be tuned by adjusting the amount of reactive azlactone rings in the PVDM block to obtain the size below 150 nm with a controllable fashion.

The obtained nanoclusters were well dispersible in water, have good magnetic sensitivity and negatively charged surface. These properties are necessary for use these particles in magnetic separation applications. was then applied in the surface-initiated ATRP of MNP. After the surface modification, thymine PNA monomer was then immobilized on poly(PEGMA-stat-VDM)-coated MNP via ring opening reactions with azlactone rings on the particle surface. TGA results indicated that there were 4 wt% of the PNA monomer in the complex (1.2 µmol/g complex).

In the second work, surface modification of MNP with various degrees of PEGMA and VDM using surface initiated ATRP and its conjugation with FA were studied. It was found that the degree of PEGMA and VDM could be finely tuned by varying the molar ratio of PEGMA and VDM in the loading. TEM indicated that after the FA conjugation, FA-grafted MNP having high VDM content exhibited good particle dispersibility and stability in water. This result could be explained by the formation of carboxylate ions from a ring-opening reaction of the remaining VDM in water, leading to additional electrostatic repulsion stabilization and thus preventing the particle agglomeration.

In addition, modification of MNP surface using grafting 'onto' technique with PEO-b-PVDM diblock copolymer synthesized by RAFT polymerization was prepared.

Its applications for adsorption with antibody were also studied. Three different azlactone block lengths with narrow PDI were first prepared by RAFT polymerization and then grafted onto amino-functionalized MNP to form magnetic nanoclusters with tunable cluster size. The nanocluster size could be tuned from 20 to 150 nm in diameter with 10 to 100 particles/cluster by adjusting the amounts of reactive azlactone rings in the PVDM block. After the copolymer coating, the remaining azlactone rings on the nanocluster surface were hydrolyzed with water to yield the MNP with negatively charged surface. Zeta potential values of the nanoclusters were significantly increased (-27 to -34 mV) as compared with its precursor due to the ringopening of PVDM units in the copolymers. Then, the nanoclusters were used as recyclable nano-supports for adsorption with antibody. The result indicated that there was 96-99% adsorption ability of the antibody during eight adsorption-separationdesorption cycles.

The presence of polymeric layer not only prevents aggregation to the magnetite particles, but the active functional groups can be used for further chemical attachment with biomolecules of interest. Therefore, these novel polymer-coated MNPs can be used as an intermediate for covalent bonding with a variety of molecules containing affinity functional groups. It is anticipated that these azlactone-functionalized MNPs might be used as magnetically guidable vehicles for further attachment with active biomolecules and particularly used in biomedical applications, such as drug delivery vehicle, anticancer treatment and magnetic separation. To a stirred solution of (3-aminopropyl)triethoxysilane (APS) (0.18 mL, 0.8 mmol) and triethylamine (TEA) (0.12 mL, 0.8 mmol) in dried toluene (10 mL), 2bromoisobutyryl bromide (BIBB) (0.1 mL, 0.8 mmol) in dried toluene (10 mL) was added dropwise at 0°C for 2 h under nitrogen. The reaction mixture was warmed to room temperature and stirred for 24 h. The mixture was passed through a filter paper to remove salts and the filtrate was evaporated to remove the unreacted TEA under reduced pressure. The resulting product, BTPAm, was yellowish thick liquid (78% yield).

APPENDIX

Figure A3 Reaction between APS and BIBB to obtain BTPAm

BTPAm was prepared through an amidization reaction between APS and BIBB. Comparing with the FTIR spectrum of APS and BIBB, Figure A4C showed FTIR characteristic absorption peaks of BTPAm (1658 cm -1 of -NH-CO-carbonyl stretching, 1112-1026 cm -1 of Si-O stretching, 1532 cm -1 of N-H bending and 3345 cm -1 of N-H-stretching).

stat-VDM)-coated MNP after dispersing in water

The amounts of carboxyl groups presenting on the particle surface were quantitatively determined by a conductometric titration. Figure B9 shows the conductometric titration curve of the reaction between NaOH and HCl having a Vshape (Blank). During the titration, the reaction that takes place in the titration vessel is following:

In the region I, before the end point, OH -is removed from the solution by reaction with H + , and Cl -is added to the solution. The conductance of the solution decreases prior to the end point. After the end point (region II), no OH -is available to react, and the conductance of the solution increases as a result of the additional of H + and Cl -.

In the case of the titration of HCl with -COOH groups on the particle surface, the conductrometric titration curve exhibits three regions (Figure B9B). Before the titration of -COOH groups on the MNP surface, the -COOH grafted on the MNP surface was dispersed in an excess of NaOH solution. Thus, the reaction that takes place in the vessel is following:

In the region I of the titration, because basicity of excess OH -in the solution is stronger than that of -COO -, the OH -in the solution was first neutralized when HCl was titrated.

In the region II, when the OH -in the solution was completely neutralized, the H + ions reacted with the COO -groups on the MNP surface. After the COO -groups on the MNP surface were completely reacted with H + ions, the solution conductivity sharply increase due to the excess of OH -and Na + (region III). The measurement of

Abstract

We herein report the surface modification of magnetite nanoparticle (MNP) with copolymers containing active azlactone rings via a grafting 'from' and grafting 'onto' controlled radical polymerization (CRP) for use as a nano-solid support for immobilization with biomolecules. Three different approaches were presented as following.

First, synthesis of poly(poly(ethylene glycol) methyl ether methacrylate-stat-2-vinyl-4,4-dimethylazlactone) (PEGMA-stat-VDM)-grafted MNP viaa grafting 'from' atom transfer radical polymerization (ATRP) and its application as a platform for conjugating thymine peptide nucleic acid (PNA) monomer were presented. ATRP of PEGMA and VDM was firstly carried out in a solution system to optimize the reaction condition. It was found that 1/0.2/0.2 molar ratio of [ethyl-2-bromoisobutyrate (EBiB)]0/ [copper(I) bromide (CuBr)]0/[tris- [2-(dimethylamino)ethyl]amine (Me6Tren)]0, respectively, led to a good control of the copolymerization; the resulting polymers have molecular weight ( n M ) close to the theoretical ones and relatively narrow polydispersity indices (PDIs = 1.11-1.43). The optimal condition was then applied in the surface-initiated ATRP of MNP.

The presence of polymeric shell on MNP core was indicated by fourier transform infrared spectroscopy (FTIR) and vibrating sample magnetometry (VSM) techniques.

After immobilization of thymine PNA monomer, thermogravimetric analysis (TGA) results showed that there were 4 wt% of the PNA monomer in the complex (1.2 mol/g complex). The presence of the PNA monomer was also confirmed via FTIR and VSM.

In the second work, the synthesis of MNP grafted with PEGMA and VDM via ATRP for conjugation with folic acid (FA) was studied. A grafting 'from' was applied for the copolymerization of PEGMA and VDM from the particle surface with various molar compositions of PEGMA to VDM (0/100, 30/70, 50/50 and 70/30, respectively). The existence of PEGMA and VDM in the structure was characterized by FTIR, TGA and VSM. It was found that the degree of PEGMA and VDM could be finely tuned by varying the molar ratio of PEGMA and VDM in the copolymerization. After the FA conjugation, TEM result indicated that the FA-conjugated MNP having high VDM content exhibited good dispersibility in water.

Third, the synthesis of MNP grafted with poly(ethylene oxide)block-poly(2-vinyl-4,4-dimethylazlactone) (PEO-b-PVDM)diblock copolymer via a grafting 'onto' strategy and its application as recyclable magnetic nanosupport for adsorption with antibody were studied. PEO-b-PVDM diblock copolymers were first synthesized using a reversible addition-fragmentation chain transfer (RAFT) polymerization and then grafted onto aminofunctionalized MNP by coupling with some azlactone rings of the PVDM block. TEM was used to investigate the particle nanoclustering and its distribution. It was found that increasing the PVDM chain length increased the size of MNP nanoclusters from 20 to 150 nm; approximately 10, 50 and 100 particles/clusterfor PEO-b-PVDM21, PEO-b-PVDM41 and PEO-b-PVDM84 diblock copolymer, respectively. Moreover, TEM images and photo correlation spectroscopy (PCS) technique indicated an improvement in the particle dispersibility in water after coating with the copolymers. The nanoclusters with PEO-b-PVDM84 diblock copolymer coating were used as recyclable magnetic nano-supports for adsorption with antibody. The results show that there was 96-99% adsorption ability during eight adsorption-separation-desorption cycles.

I Introduction and literature review…………………………………. 1 . 1

 11 Surface modification of MNP………………………………… 1.1.1 Surface modification of magnetite nanoparticle (MNP) via atom transfer radical polymerization (ATRP)……………………. 1.1.2 Surface modification of magnetite nanoparticle (MNP) via grafting 'from' reversible addition-fragmentation chain transfer (RAFT) polymerization…………………………………………... 1.2 Bioconjugation on surface of magnetic nanoparticle…………. 1.2.1 Surface functionalization of MNP with peptide nucleic acid (PNA)……………………………………………………….......... 1.2.2 Surface functionalization of MNP with folic acid (FA)……. 1.2.3 Surface functionalization of MNP with antibody…………... 1.3 Chemistry of azlactone compounds…………………………...

3 3 . 1 . 4 III 3 . 1 . 5 1

 33143151 Synthesis of 2-vinyl-4,4-dimethylazlactone (VDM)……….. 3.1.3.1 Synthesis of N-acryloyl-2-methylalanine………………… 3.1.3.2 Cyclization of N-acryloyl-2-methylalanine to form VDM cyclic……………………………………………………………… Synthesis of poly(PEGMA-stat-VDM) copolymer coated on the MNP surface via ATRP………………………………………. Immobilization of folic acid (FA) on the surface of poly(PEGMA-stat-VDM)-coated MNP………………………….. Synthesis of PEO-b-PVDM diblock copolymers by RAFT polymerization……………………………………………………. 4.2.2 Synthesis of (3-aminopropyl)triethoxysilane-grafted MNP (APS-grafted MNP)………………………………………………. 4.2.3 Formation of PEO-b-PVDM copolymers-MNP nanocluster……………………………………………………….. 4.2.4 Adsorption efficiency study of the copolymer-MNP nanoclusters with antibody……………………………………….. 4.2.5 Determination of recycling ability of the PEO-b-PVDM copolymer-MNP nanoclusters in adsorption with anti-rabbit IgG antibody…………………………………………………………... 4.2.6 Antigen recognition capacity study of antibody-adsorbed magnetic nanoclusters……………………………………………….

Figure 1 . 1 Figure 1 . 2

 1112 Figure 1.1 (a) Electrostatic stabilization of MNPs, and (b) steric stabilization of MNPs by coating with a surfactant or a polymeric shell…… Figure 1.2 Surface modification of MNP via grafting 'from' and grafting 'onto' strategies……………………………………………………………..

Figure 1 . 3 Figure 1 . 4

 1314 Figure 1.3 Schematic mechanism of atom transfer radical polymerization (ATRP)……………………………………………………………………… Figure 1.4 Methods for immobilizing ATRP initiators on MNP surfaces in the preparation of functional polymer brushes by surface-initiated ATRP…..........................................................................

Figure 1 . 5

 15 Figure 1.5 Schematic representation for the synthesis of PMMA-coated MNPs by a surface-initiated ATRP……………………………………….....

Figure 1 . 6

 16 Figure 1.6 Illustration of the synthesis route of polystyrene-coated MNP with core/shell structure…..............................................................................

Figure 1 . 7

 17 Figure 1.7 Surface-initiated ATRP of MNP with PEGMA-b-MMA block copolymer brushes…………………………………………………………...

Figure 1 . 8 Figure 1 . 9 Figure 1 . 10 Figure 1 . 11

 1819110111 Figure 1.8 Schematic representation for the preparation of P(PEGMA)coated MNP by a surface-initiated ATRP………………………………..… Figure 1.9 Surface modification of MNP with PHEMA brush…………..… Figure 1.10 Schematic diagrams of PHEMA brush-modified beads and the protein binding……………………………………………………….……… Figure 1.11 Synthesis of MNP/PDMAEMA nanoparticles............................

Figure 1 . 12 Figure 1 . 13 Figure 1 . 14

 112113114 Figure 1.12 Synthesis of PAA-coated MNPs via ATRP reaction and immobilization of FA……………………………………………..………… Figure 1.13 Proposed mechanism of the RAFT process……………………Figure 1.14 Synthetic scheme of chain transfer agent-anchored PS and PSgrafted MNP by grafting 'onto' strategy…………………………………….

Figure 1 .

 1 Figure 1.15 RAFT polymerization of N,N'-dimethylaminoethylacrylate (DMAEMA) and oligo(ethylene glycol) methyl ether acrylate (OEG-A) in the presence of phosphonic acid RAFT agent…………………………….....

Figure 1 . 16

 116 Figure 1.16 Strategies for the synthesis of PAA-PEO and PAA-PAMPEO copolymers…………………………………………………………………..

Figure 1 . 17

 117 Figure 1.17 Proposed stabilization of MNP with PAA-PEO and PAA-PAMPEO copolymers…………………………………………………….....

Figure 1 .

 1 Figure 1.18 (a) Homopolymerization of N-isopropylacrylamide (NIPAAm) by RAFT process and (b) exchange of the surfactant with a mixture of PNIPAAm and biotin-PNIPAAm in dioxane on the MNP……………………………....................................................................

Figure 1 . 19 Figure 1 . 20

 119120 Figure 1.19 Synthesis of poly(OEG-A) and PNIPAAmhaving phosphonic acid end-group and ethylpyridyl disulfide end-groups……………………… Figure 1.20 Chemical structures of A) DNA, B) aegPNA, C) acpcPNA and D) hydrogen bonding of nucleotides via Watson-Crick base paring rules……………………………………………………………………….....

Figure 1 . 21

 121 Figure 1.21 Preparation of PNA-functionalized magnetic nanoparticles (MNPs)............................................................................................................

Figure 1 . 22

 122 Figure 1.22 Functionalized gold shell-cobalt ferrite nanoparticles with thio-lated PNA for use as a PNA/DNA biosensor…………………………………..……..

Figure 1 .Figure 1 .

 11 Figure 1.23 (A) Chemical scheme for the synthesis of a trifluoroethyl ester terminal PEG silane, (B) immobilization of FA and PEG on MNP surface……………………………………………………………………………… Figure 1.24 TEM micrographs of MNP modified with (A) TFEE terminal PEG silane, (B) amine-terminated PEG, and (C) FA-t-BOC-terminal PEG silane................................................................................................................

Figure 1 .

 1 Figure 1.25 (A) Synthesis of folate-NHS (10) and (B) surface modification of nanoparticles with FA (11) or (C) PEG-FA conjugate (13)…………………………………………………………………………..

Figure 1 . 26

 126 Figure 1.26 The surface modification of MNPs with FA...............................

Figure 1 . 27

 127 Figure 1.27 Modification of MNP with MA (MAM) and with FA (FAMAM)…………………………………………………………………...

Figure 1 .

 1 Figure 1.28 (A) Synthesis of Azide-Functionalized MNPs, (B) Synthesis of Alkyne-Bound FA, and (C) Synthesis of FA-Functionalized MNPs via Click Reaction……………………………………………………………….

Figure 1 . 29 Figure 1 .

 1291 Figure 1.29 Chemical structure of amphiphilic polymer…………………… Figure 1.30 SDS-PAGE of HER2 from SK-BR3 cell lysate……………...

Figure 1 . 31

 131 Figure 1.31 Schematic diagram of the process for preparing the magnetite fine particles encapsulated with a polymer……………………………….....

Figure 1 .Figure 1 . 33 Figure 2 . 1 Figure 2 . 2 Figure 2 . 3

 1133212223 Figure 1.32 2-Vinyl-4,4-dimethylazlactone (VDM) monomer………… Figure 1.33 Azlactone-ring opening reaction and proposed mechanism…...

Figure 2 . 4

 24 Figure 2.4 Dependence of n M and w M / n M (PDI) with monomer conversion of ATRP of PEGMA and VDM using [PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 /[Me 6 Tren] 0 molar ratio = 50/50/1/0.5/0.5, in toluene at 30 o C…………………………………………..

Figure 2 . 5

 25 Figure 2.5 The ln([M] 0 /[M]) vs time plot and monomers conversion vs time plot of ATRP of PEGMA and VDM using [PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 /[Me 6 Tren] 0 molar ratio = 50/50/1/0.2/0.2, in toluene at 30°C………………………………………......

Figure 2 . 6

 26 Figure 2.6 Dependence of n M and w M / n M (PDI) with monomer conversion of ATRP of PEGMA and VDM using [PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 /[Me 6 Tren] 0 molar ratio = 50/50/1/0.2/0.2, in toluene at 30 o C…………………………………………..

Figure 2 . 7 Figure 2 . 8 Figure 2 . 9

 272829 Figure 2.7 FTIR spectra of (A) bare MNP, (B) BTPAm-coated MNP and (C) poly(PEGMA-stat-VDM)-coated MNP………………………………… Figure 2.8 FTIR spectra of (A) thymine PNA monomer, (B) poly(PEGMA-stat-VDM)-coated MNP and (C) poly(PEGMA-stat-VDM)coated MNP immobilized with thymine PNA monomer……………………Figure 2.9 TEM images of (A) oleic acid-coated MNP (prepared from toluene dispersion), (B) poly(PEGMA-stat-VDM)-coated MNP (prepared from DMF dispersion) and (C) poly(PEGMA-stat-VDM)-coated MNP immobilized with thymine PNA monomer (prepared from DMF dispersion)…………………………………………………………………...

Figure 2 . 55 Figure 2 .Figure 3 . 1 65 Figure 3 . 2 66 Figure 3 . 3 Figure 3 . 4 Figure 3 . 5

 255231653266333435 Figure 2.10 TGA curves of (A) bare MNP, (B) BTPAm-coated MNP, (C) poly(PEGMA-stat-VDM)-coated MNP and (D) poly(PEGMA-stat-VDM)coated MNP immobilized with thymine PNA monomer…………………… 55 Figure 2.11 M-H curves of A) bare MNP, B) BTPAm-coated MNP, C) poly(PEGMA-stat-VDM)-coated MNP and (D) poly(PEGMA-stat-VDM)coated MNP immobilized with thymine PNA monomer…………………… 56

Figure 3 . 6 (Figure 4 . 1 Figure 4 . 2 A) 1 H 41 copolymer……………………………………………………………………Figure 4 . 3 Figure 4 . 4 Figure 4 . 5 Figure 4 . 6

 36414214143444546 Figure 3.6 (A) TGA thermograms of a) poly(VDM)-coated MNP without FA and b) FA-poly(VDM)-coated MNP and (B) UV-visible absorption spectra of a) poly(VDM)-coated MNP without FA b) FA-poly(VDM)coated MNP and c) FA………………………………………………………

Figure 4 . 7

 47 Figure 4.7 Adsorption efficiency of PEO 44 -b-PVDM 84 -MNP nanoclusters for ionic adsorption with anti-rabbit IgG antibody………………………......

Figure 4 . 8

 48 Figure 4.8 Recycling efficiency of PEO 44 -b-PVDM 84 -MNP nanoclusters in the ionic adsorption of anti-rabbit IgG antibody after 8-recycling process….

Figure 4 . 9 Scheme 2 . 1 43 Scheme 2 . 2 Scheme 3 . 1 62 Scheme 3 . 2 63 Chapter IV Scheme 4 . 1 Scheme 4 . 2 Scheme 4 . 3 2 APS

 49214322316232634142432 Figure 4.9 Scheme showing the indirect method used to visualize the antigen recognition capability; A) 10 mM MES pH 6, B) 1% BSA in 10 mM MES pH 6, C) anti-rabbit IgG-HRP, D) anti-rabbit IgG-adsorbed MNP nanoclusters immobilized with IgG and anti-rabbit IgG-HRP, E) anti-rabbit IgG-adsorbed MNP nanoclusters immobilized with IgG, F) anti-rabbit IgG-adsorbed MNP nanoclusters without IgG (blocked with BSA) and G) MNP nanoclusters………… 91

FeFigure 1 . 1

 11 Figure 1.1 (a) Electrostatic stabilization of MNPs, and (b) steric stabilization of MNPs by coating with a surfactant or a polymeric shell

Figure 1 . 2 1 . 1 . 1

 12111 Figure 1.2 Surface modification of MNP via grafting 'from' and grafting 'onto' strategies

Figure 1 . 3 Figure 1 . 4 A

 1314 Figure 1.3 Schematic mechanism of atom transfer radical polymerization (ATRP)[20]

Figure 1 . 4

 14 Figure 1.4 Methods for immobilizing ATRP initiators on MNP surfaces in the preparation of functional polymer brushes by surface-initiated ATRP (BTPAm: 2-bromo-2-methyl-N-(3-(triethoxysilyl) propyl) propanamide; CTS: [4-(chloromethyl) phenyl] trichlorosilane; APS: (3-aminopropyl)triethoxysilane; CPA: 3chloropropionic acid; CTCS: 2-(4-chlorosulfonylphenyl)ethyl trichlorosilane; BMPA:

Figure 1 . 10

 110 Figure 1.10 Schematic diagrams of PHEMA brush-modified beads and the protein binding[26]

Fe 3 Figure 1 . 11

 3111 Figure 1.11 Synthesis of MNP/PDMAEMA nanoparticles (CuBr: copper(I) bromide;

Figure 1 . 12

 112 Figure 1.12 Synthesis of PAA-coated MNPs via ATRP reaction and immobilization of FA (CuBr: copper(I) bromide; PMDETA: N,N,N',N",N"-pentamethylenetriamine; EDC: 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; DMSO:dimethyl sulfoxide)[28]

8 Figure 1 .

 81 Figure 1.15 RAFT polymerization of N,N'-dimethylaminoethylacrylate (DMAEMA)and oligo(ethylene glycol) methyl ether acrylate (OEG-A) in the presence of phosphonic acid RAFT agent[31]

Figure 1 . 16 Figure 1 . 17

 116117 Figure1.16 Strategies for the synthesis of PAA-PEO and PAA-PAMPEO copolymers[32]

Figure 1 .

 1 Figure 1.18 (a) Homopolymerization of N-isopropylacrylamide (NIPAAm) by RAFT process and (b) exchange of the surfactant with a mixture of PNIPAAm and biotin-PNIPAAm in 1,4-dioxane on the MNP[33]

= 62 ,Figure 1 . 19

 62119 Figure1.19 Synthesis of P(OEG-A) and PNIPAAmhaving phosphonic acid end-group and ethylpyridyl disulfide end-groups[34]

Figure 1 .

 1 20). Because the PNA backbone contains no charged phosphate groups, the hybridization between PNA and DNA strands is stronger than between DNA and DNA strands due to the lack of electrostatic repulsion. Hybridization between DNA and PNA abides by the Watson-Click base pairing rule; adinine (A) bonds with thymine (T) and cytosine (C) bonds with glycine (G) (Figure1.20).In addition, PNA is not recognized by nuclease and protease, making it resistant to enzyme degradation. These unique properties of PNA have led to the development of a wide range of biological assays, especially in the field of PNA-based hybridization technology. In the recent years, many research lines have been developed the modification of magnetic nanoparticle with PNA for use as a PNA probe to separate DNA or ribose nucleic acid (RNA). For example, Wang et al. have reported the preparation of PNA-functionalized magnetic nanoparticles (MNPs) for hybridizing with DNA (Figure1.21)[52]. First, 4-pyridyldithiol-derivertized PNA was used to attach to 3-mercapropropyl-oxysilane-coated MNPs via a thiol-disulfide exchange reaction. After that, it was used as a PNA probe to separate perfect-match DNA from non-complementary DNA. The hybridization of PNA probe with target DNA was characterized using surface-enhanced Raman scattering (SERS). The results showed that PNA probe can be able to hybridize with the perfect-match DNA target and did not bind with non-complementary DNA.

Figure 1 . 20 Figure 1 . 21

 120121 Figure 1.20 Chemical structures of A) DNA, B) aegPNA, C) acpcPNA and D) hydrogen bonding of nucleotides via Watson-Crick base paring rules

Figure 1 . 22 1 . 2 . 2

 122122 Figure 1.22 Functionalized gold shell-cobalt ferrite nanoparticles with thio-lated PNA for use as a PNA/DNA biosensor[53]

Figure 1 .Figure 1 Figure 1 .

 111 Figure 1.23 shows the chemical scheme for surface modification of nanoparticles with TFEE silane and subsequently conjugating with ethylenediamine using carboxylic acid

Figure 1 .

 1 Figure 1.24 TEM micrographs of MNP modified with (A) TFEE terminal PEG silane, (B) amine-terminated PEG, and (C) FA-t-BOC-terminal PEG silane[55]

Figure 1 .

 1 Figure 1.25 (A) Synthesis of folate-NHS (10) and (B) surface modification of nanoparticles with FA(11) or (C) PEG-FA conjugate(13) [36]

Figure 1 . 26

 126 Figure 1.26The surface modification of MNPs with FA[45]

Figure 1 . 27

 127 Figure 1.27 Modification of MNP with MA (MAM) and with FA (FAMAM)[56]

Figure 1 .

 1 Figure 1.28 (A) Synthesis of Azide-Functionalized MNPs, (B) Synthesis of Alkyne-Bound FA, and (C) Synthesis of FA-Functionalized MNPs via Click Reaction[44]

Figure 1 .

 1 29) to yield the water dispersible conjugated with Ab against human epithelial growth factor receptor 2 (anti-HER2 or anti-HER2/neu) through amidation between amine groups (-NH 2 ) of the Ab and carboxyl groups (-COOH) of MNP surface to form Ab-MNP. The Ab-MNP was used to isolate HER2 from human breast cancer cell line SK-BR3. The Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results in Figure 1.30 show that Ab-MNP can selectively separate HER2 protein based on the retarded migration of the Ab-MNP on Lane 5, 6 and 7 in comparison with Lane 2 (the original cell lysate)

Figure 1 . 29 Figure 1 .

 1291 Figure1.29 Chemical structure of amphiphilic polymer[58]

Figure 1 . 31

 131 Figure 1.31 Schematic diagram of the process for preparing the magnetite fineparticles encapsulated with a polymer[60]

2 . 1 . 3

 213 5 mm) followed by two columns (Polymer Laboratories, two PL gel 5 µm Mixed -D columns, 2 × 300 × 7.5 mm). Polystyrene standards (580-483 × 10 3 g.mol -1 ) were used to calibrate the SEC. Fourier transform infrared (FTIR) analyses were recorded using a Nicolet Avatar 370 DTGS spectrometer in the attenuated total reflection (ATR) mode. Elemental analysis was performed by the Service Central d'Analyses du Centre National de Recherche Scientifique, Gif-sur-Yvette (France). Transmission electron microscopy (TEM) images were taken using a Philips Tecnai 12 operated at 120 kV equipped with Gatan model 782 CCD camera. The particles were re-suspended in toluene or DMF with sonication before deposition on a TEM grid. Magnetic properties of the particles were measured at room temperature using a Standard 7403 Series, Lakeshore vibrating sample magnetometer (VSM). Magnetic moment of each sample was investigated over a range of ± 10000 G of applied magnetic fields using 30 min sweep time. Thermogravimetric analysis (TGA) was performed on SDTA 851 Mettler-Toledo at the temperature ranging between 25-600 o C at a heating rate of 20 o C/min under oxygen atmosphere. General procedure for the synthesis of poly(PEGMA-stat-VDM) copolymers via ATRP in solution An example of the synthesis of poly(PEGMA-stat-VDM) using EBiB/CuBr/Me 6 Tren/PEGMA/VDM of 1/1/1/50/50 is illustrated. CuBr (0.0287 g, 0.2 mmol) was added to a Schlenk tube equipped with a stir bar. After sealing with a rubber septum, the Schlenk tube was deoxygenated with three vacuum/argon fill cycles. A degassed solution of PEGMA (6 g, 20 mmol), VDM (2.78 g, 20 mmol), toluene (70 % v/v, 6 mL), EBiB (0.039 g, 0.2 mmol) and DMF (5 % v/v, 0.4 mL) (used as an internal standard) was added to the Schlenk tube containing CuBr via a cannula. The resulting solution was further degassed using freeze/pump/thaw cycles in vacuo. Degassed Me 6 Tren (0.046 g, 0.2 mmol) was added (t= 0) to the Schlenk tube and it was then placed in an oil bath preheated to a desired temperature. The samples were withdrawn periodically via a degassed syringe for conversion measurements and SEC analyses.

2. 1 . 4 1 2. 1 . 5 1 )Scheme 2 . 1 2 . 1 . 6 2 )Scheme 2 . 2

 14115121216222 Scheme 2.1 Surface modification of MNP with poly(PEGMA-stat-VDM) copolymer via ATRP reaction

Figure 2 .

 2 1 shows that conversions of PEGMA and VDM reached 83% and 90%, respectively, after 30 min of polymerization time. Moreover, the ln([M] 0 /[M]) vs time plot shows a curvature and reaches a plateau after 30 min of the reaction, indicating the presence of irreversible terminations. This phenomenon is confirmed by the relative broad polydispersity 0 /[CuBr] 0 /[Me 6 Tren] 0 ratio to 1/0.5/0.5 and a decrease of the reaction temperature to 30 o C resulted in a decrease of PEGMA and VDM conversion to 70% and 82%, respectively (after 90 min, entry 2, Table 2.1 and Figure 2.3). However, the polydispersity indices were still high (PDIs = 1.40 -2.19) for a controlled radical polymerization (Figure 2.4). In addition, the ln([M] 0 /[M]) vs time plot again shows a decrease of active species concentration, which is compatible with the presence of irreversible terminations (Figure 2.3).

Figure 2 . 1 Figure 2 . 2 Figure 2 . 3 Figure 2 . 4

 21222324 Figure 2.1 The ln([M] 0 /[M]) vs time plot and monomers conversions vs time plot of ATRP of PEGMA and VDM using [PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 /[Me 6 Tren] 0 molar ratio = 50/50/1/1/1, in toluene at 50 o C

Figure 2 . 5 Figure 2 . 6 2 . 2 . 2

 2526222 Figure 2.5 The ln([M] 0 /[M]) vs time plot and monomers conversion vs time plot of ATRP of PEGMA and VDM using [PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 /[Me 6 Tren] 0 molar ratio = 50/50/1/0.2/0.2, in toluene at 30°C

Figure 2 .

 2 Figure 2.7C shows FTIR spectrum of poly(PEGMA-stat-VDM)-coated MNP compared with those of bare MNP (Figure 2.7A) and BTPAm-coated MNP (Figure 2.7B). Poly(PEGMA-stat-VDM)-coated MNP exhibited a characteristic signal of azlactone rings of VDM units at 1816 cm -1 (-C=O stretching), 1203 cm -1 (C-O-C stretching) and that of PEGMA at 1722 cm -1 (-C=O stretching) (Figure 2.7C), indicating the presence of the copolymer in the complex. A broad and strong band of Fe-O from MNP cores was also observed at 578 cm -1 .

Figure 2 . 7

 27 Figure 2.7 FTIR spectra of (A) bare MNP, (B) BTPAm-coated MNP and (C) poly(PEGMA-stat-VDM)-coated MNP

Figure 2 . 8 Figure 2 . 9 .

 2829 Figure 2.8 FTIR spectra of (A) thymine PNA monomer, (B) poly(PEGMA-stat-VDM)-coated MNP and (C) poly(PEGMA-stat-VDM)-coated MNP immobilized with thymine PNA monomer

Figure 2 . 9

 29 Figure 2.9 TEM images of (A) oleic acid-coated MNP (prepared from toluene dispersion), (B) poly(PEGMA-stat-VDM)-coated MNP (prepared from DMF dispersion) and (C) poly(PEGMA-stat-VDM)-coated MNP immobilized with thymine PNA monomer (prepared from DMF dispersion)

Figure 2 .

 2 Figure 2.10 TGA curves of (A) bare MNP, (B) BTPAm-coated MNP, (C) poly(PEGMA-stat-VDM)-coated MNP and (D) poly(PEGMA-stat-VDM)-coated MNP immobilized with thymine PNA monomer

Figure 2 .

 2 Figure 2.11 M-H curves of A) bare MNP, B) BTPAm-coated MNP, C) poly(PEGMAstat-VDM)-coated MNP and (D) poly(PEGMA-stat-VDM)-coated MNP immobilized with thymine PNA monomer. The expansion in the range of ± 500 G of applied filed is shown in the inset.

  photo correlation spectroscopy (PCS) using NanoZS4700 nanoseries Malvern instrument. The sample dispersions were sonicated for 30 min before the measurement at 25 o C. The measurement was carried out without filtration. The amounts of azlactone groups present on the particle surfaces were quantitatively determined by a conductometric titration using Seveneasy conductometer (Metter Toledo Bmblt 8603 Swchwerzenbach). The presence of FA was investigated using SPECORD S100 ultraviolet-visible (UV-vis) spectrophotometer (Analytikjena AG) coupled with a photo diode array detector at λ max = 371 nm.

3. 1 . 4 1 )

 141 Synthesis of poly(PEGMA-stat-VDM) copolymer coated on the MNP surface via ATRP (Scheme 3.In this work, surface modification of MNPs with four different molar ratios of PEGMA to VDM (0/100, 30/70, 50/50 and 70/30, respectively) was performed. An example of surface modification of MNPs with 50/50 PEGMA/VDM copolymer was described herein. Other copolymer-MNP complexes were prepared in a similar fashion with appropriate amounts of reagents used. In a typical procedure, BTPAm-coated MNP (0.1 g) was sonicated with toluene (3.0 mL) in a Schlenk tube. A solution of PEGMA (3.6 mL, 0.11 mol), VDM (1.7 g, 0.11 mol), CuBr (0.04 g, 0.002 mol) and Me 6 Tren (0.05 g, 0.002 mol) in toluene (3 mL) were then syringed to the Schlenk tube. After degassing the solution by three freeze-pump-thaw cycles, ATRP was set at 30 o C for 24 h.

Fe 30 Scheme 3 . 1

 3031 Scheme 3.1 Synthesis of poly(PEGMA-stat-VDM)-coated MNPs via surface-initiated ATRP reaction

Figure 3 .Figure 3 . 1

 331 Figure 3.1 FTIR spectra of poly(PEGMA-stat-VDM)-coated MNPs having molar ratio of PEGMA to VDM of A) 0/100, B) 30/70, C) 50/50 and D) 70/30, respectively

Figure 3 . 2

 32 Figure 3.2 TEM images of poly(PEGMA-stat-VDM)-coated MNPs having molar ratio of PEGMA to VDM of A) 0/100, B) 30/70, C) 50/50 and D) 70/30, respectively.

Figure 3 . 3

 33 Figure 3.3 TGA curves of A) bare MNPs, B) BTPAm-coated MNPs and poly(PEGMA-stat-VDM)-coated MNPs having C) 0/100, D) 30/70, E) 50/50 and F) 70/30 molar ratios of PEGMA to VDM, respectively

Figure 3 . 4 M

 34 Figure 3.4 M-H curves of A) bare MNP, B) BTPAm-coated MNP and poly(PEGMAstat-VDM)-coated MNPs having C) 0/100, D) 30/70, E) 50/50 and F) 70/30 molar ratios of PEGMA to VDM

  poly(PEGMA-stat-VDM)-coated MNPs 114.0 ± 4.1 -14.12 FA-70/30 poly(PEGMAstat-VDM)-coated MNPs 270.0 ± 7.0 -4.90 TEM images of poly(PEGMA-stat-VDM)-coated MNPs having 0/100, 30/70, 50/50 and 70/30 molar ratios of PEGMA to VDM, respectively, both before and after FA conjugation are shown in Figure 3.5. These TEM samples were prepared from aqueous dispersions. It was found that, before FA conjugation, all polymer-coated MNPs were well dispersible in water without aggregation (Figure 3.5A-D). After FA conjugation, some nano-scale aggregations of multiple MNPs were observed and this

Figure 3 . 5

 35 Figure 3.5 TEM images of the poly(PEGMA-stat-VDM)-coated MNPs, (A-D) before and (A'-D') after the FA conjugation. The MNPs were coated with the copolymers having (A, A') 0/100, (B, B') 30/70, (C, C') 50/50 and (D, D') 70/30 molar ratios of PEGMA to VDM, respectively. All TEM samples were prepared from aqueous dispersions.

Figure 3 .Figure 3 . 6

 336 Figure 3.6 (A) TGA thermograms of a) poly(VDM)-coated MNP without FA and b) FA-poly(VDM)-coated MNP and (B) UV-visible absorption spectra of a) poly(VDM)coated MNP without FA b) FA-poly(VDM)-coated MNP and c) FA

1 1 ) 65 (Scheme 4 . 1 4 . 2 . 2

 116541422 Scheme 4.1 Synthesis of PEO-b-PVDM diblock copolymers by RAFT polymerization

Scheme 4 . 2

 42 Scheme 4.2 Synthesis of PEO-b-PVDM copolymers-MNP nanocluster

  -b-PVDM copolymers-MNP nanoclusters were obtained by the reaction between primary amine groups coated on MNPs surface and azlactone groups within the backbone of PEO-b-PVDM diblock copolymers via a ring-opening reaction (Scheme 4.2). In the first step, PEO-b-PVDM diblock copolymers were synthesized using PEO-CTA as the macromolecular chain transfer agent to mediate the RAFT polymerization of VDM in the presence of ACVA used as initiator in 1,4-dioxane at 70 o C. The characteristics of the copolymers having three different PVDM block lengths are shown in Table

  b VDM conversion determined by 1 H NMR spectroscopy by comparing the integration areas of the vinylic protons of VDM at 5.93 ppm with the integral area value of the CH proton of DMF at 8.01 ppm. c n M ,th = n M , NMR of PEO-CTA + (([VDM] 0 /[PEO-CTA] 0 ) x VDM conv. x M VDM ). d Determined by SEC in THF using polystyrene standard.

Figure 4 . 1 Figure 4 . 2 A) 1 H

 41421 Figure 4.1 Overlay SEC traces of (a) PEO 44 -CTA, (b) PEO 44 -b-PVDM 21 copolymer (Entry 1, Table 4.1), (c) PEO 44 -b-PVDM 41 copolymer (Entry 2, Table 4.1) and (d) PEO 44 -b-PVDM 84 copolymer (Entry 3, Table 4.1)

Figure 4 . 3 Figure 4 . 4

 4344 Figure 4.3 FTIR spectra of A) APS-grafted MNPs, B) PEO 44 -b-PVDM 41 block copolymers and C) PEO 44 -b-PVDM 41 -MNP nanoclusters

  TGA technique was used to calculate the percentage of organic layers bound to MNP. It was assumed that the percent weight loss was attributed to the weight of organic components and the residual weight was due to completely oxidized iron oxide in the form of magnetite. It was found that APS content in the nanoclusters was about 2.2% and the copolymers in the nanoclusters were in the range of 8.4-11.9% (Figure4.5).

Figure 4 . 5

 45 Figure 4.5 TGA thermograms of A) bare MNPs, B) APS-grafted MNPs, C) PEO 44 -b-PVDM 21 -MNP nanoclusters, D) PEO 44 -b-PVDM 41 -MNP nanoclusters and E) PEO 44b-PVDM 84 -MNP nanoclusters

Figure 4 .

 4 6 shows the calibration of BGG standard for quantification of antibody adsorption studies.

Figure 4 .Figure 4 . 6 Figure 4 . 7 1 M

 446471 Figure 4.6 Calibration curve of BGG standard

Figure 4 .Figure 4 . 8

 448 Figure 4.8 shows the antibody adsorption ability of the nanoclusters in each cycle. After adsorption-separation process, the concentration of adsorbed antibody and desorbed antibody from each cycle was determined using Bradford assay. The results showed that the particles retained higher than 96% adsorption ability of the antibody during eight adsorption-separation-desorption cycles, indicating the potential feasibility in using this novel hybrid nanocluster as recyclable support in cell separation applications.

Figure 4 . 9

 49 Figure 4.9 Scheme showing the indirect method used to visualize the antigen recognition capability; A) 10 mM MES pH 6, B) 1% BSA in 10 mM MES pH 6, C) anti-rabbit IgG-HRP, D) anti-rabbit IgG-adsorbed MNP nanoclusters immobilized with IgG and anti-rabbit IgG-HRP, E) anti-rabbit IgG-adsorbed MNP nanoclusters immobilized with IgG, F) anti-rabbit IgG-adsorbed MNP nanoclusters without IgG (blocked with BSA) and G) MNP nanoclusters

Figure A2 1 H

 1 Figure A2 1 H NMR spectra of (A) tris-(2-aminoethyl) amine (Tren) (solvent: CDCl 3 ), and tris-(2-(dimethylamino)ethyl) amine (Me 6 Tren) (solvent: CDCl 3 )

Figure A5C showed a 1 HFigure A4 Figure A5 1 HPart 2 .

 1A412 Figure A5C showed a 1 H NMR spectrum of BTPAm in comparison with APS and BIBB starting reagents. In good agreement with FTIR results, a distinctive shift of the 1 H NMR signal corresponding to methylene protons adjacent to NH group (from 2.50 ppm, signal a, to 3.22 ppm, signal a') indicated the formation of BTPAm. In addition, slight shifts of other signals such as methylene protons of signal b (1.45 ppm)

Figure A6 Part 3 .FigurePart 1 .Figure B2 1 HFigure B3 13 CFigure B4 FigureFigure B6 1 HeFigure B7 13 CFigure B8 FTIR spectrum of VDM Part 2 .

 A631113B4113B82 Figure A6 Monitoring of monomers conversions by 1 H NMR spectroscopy during atom transfer radical polymerization of PEGMA and VDM using DMF as an internal standard

From

  TGA result, FA-poly(VDM)-coated MNP possessed 71.5% Fe 3 O 4 , 5.6% BTPAm, 12.3% poly(VDM) and 10.6% FA Weight of BTPAm-coated on a single particle = (1.41 x 10 -8 g) x (5.6/71.5) = 1.10 x 10 -19 g Moles of BTPAm = (1.10 x 10 -19 g)/(156 g/mol); MW. Of BTPAm = 156 g/mol = (1.10 x 10 -19 g) (6.02 x 10 23 molecule/mol)/(156 g/mol) = 424 molecules/particle = 424 molecules/201 nm 2 = 2.1 molecule/nm 2 Similarly, weight of poly(VDM) coated on a single particle = (1.41 x 10 -18 g) x (12.3/71.5) = 2.42 x 10 -19 g Moles of azlactone ring = (2.42 x 10 -19 g)/(139.15 g/mol); MW. of VDM unit = 139.15 g/mol Number of azlactone ring = ((2.42 x 10 -19 g) x (6.02 x 10 23 molecules/mol))/(139.15 g/mol) = 1046.74 molecules/particle = 1046.74 molecules/201 nm 2 = 5.2 molecules/nm 2 Similarly, weight of FA in a single particle = (1.41 x 10 -18 g) x (10.6/71.5) = (2.09 x 10 -19 g) Moles of FA = (2.09 x 10 -19 g)/(483 g/mol) ; MW. of EDA-FA = 483 g/mol Number of FA = ((2.09 x 10 -19 g) x (6.02 x 10 23 molecules/mol))/(483 g/mol) . (Industrial Chemistry) Naresuan University, Phitsanulok, Thailand Research Output  Prai-in, Y., Tankanya, K., Rutnakornpituk, B., Wichai, U., Montembault, V., Pascual, S., Fontaine, L., Rutnakornpituk, M. (2012). Azlactone functionalization of magnetic nanoparticles using ATRP and their bioconjugation. Polymer, 53, 113-120  Pray-in, Y., Rutnakornpituk, B., Wichai, U., Vilaivan, T., Rutnakornpituk, M. (2014). Hydrophilic azlactone-functionalized magnetite nanoparticle for conjugation with folic acid. Journal of Nanoparticle Research, 16:2357  Pray-in, Y., Rutnakornpituk, B., Wichai, U., Montembault, V., Pascual, S., Fontaine, L., Rutnakornpituk, M. Recycable magnetic nanoclustercrosslinked with poly(ethylene oxide)-block-poly(2-vinyl-4,4-dimethylazlactone copolymer for adsorption with antibody. (draft manuscript) Presentations  Pray-in, Y., Tankanya, K., Rutnakornpituk, B., Wichai, U., Montembault, V., Pascual, S., Fontaine, L., Rutnakornpituk, M. "Hydrophilic azlactone functionalized magnetic nanoparticles and their application as scavengers", Poster presentation at Pure and Applied Chemistry InternationalFonctionnalisation de l'azlactone de nanoparticules magnétiques à l'aide de techniques de crp et de leur bioconjugation Résumé Nous rapportons ici la modification de surface de nanoparticule de magnétite (MNP) avec des copolymères contenant des noyaux d'azlactone actifs par greffage «from» et greffage Polymérisation radicalaire contrôlée «sur» (CRP) à utiliser comme support nano-solide pour immobilisation avec des biomolécules. Trois approches différentes ont été présentées comme suivant. Premièrement, la synthèse du méthacrylate de méthyléther-stat-2-de poly (éthylène glycol) méthylique vinyl-4,4-diméthylazlactone) (PNG-PEGMA-stat-VDM) greffée via un greffage «à partir d'atome» polymérisation radicalaire par transfert (ATRP) et son application comme plateforme de conjugaison. Le monomère d'acide nucléique peptidique (PNA) de thymine a été présenté. ATRP de PEGMA et VDM a d'abord été réalisée en solution pour optimiser les conditions de réaction. Cela a été trouvé le rapport molaire 1 / 0,2 / 0,2 du [bromure de cuivre-éthyl-2-bromoisobutyrate (EBiB)] 0 / [cuivre (I)] (CuBr)] 0 / [tris-[2-(diméthylamino) éthyle] amine (Me6Tren)] 0, respectivement, ont conduit à un bon contrôle de la copolymérisation; les polymères résultants ont un poids moléculaire (n M) proches des valeurs théoriques et des indices de polydispersité relativement étroits (PDI = 1,11-1,43). La condition optimale a ensuite été appliquée dans l'ATRP du MNP initié en surface.La transformation de la coque en polymère sur le noyau de la MNP était indiquée par la transformation de Fourier la spectroscopie infrarouge (FTIR) et les techniques de magnétométrie à échantillon vibrant (VSM). Après immobilisation du monomère thymine-PNA, analyse thermogravimétrique (TGA) Les résultats ont montré que le complexe contenait 4% en poids de monomère de PNA (1,2 mol / g complexe). La présence du monomère PNA a également été confirmée par FTIR et VSM. Dans le deuxième travail, la synthèse de MNP greffés avec PEGMA et VDM via ATRP pour une conjugaison avec l'acide folique (FA) a été étudiée. Un greffage «from» a été appliqué pour la copolymérisation de PEGMA et de VDM à partir de la surface des particules avec diverses compositions molaires de PEGMA à VDM (0/100, 30/70, 50/50 et 70/30, respectivement). L'existence de PEGMA et de VDM dans la structure a été caractérisée par FTIR, TGA et VSM. Il a été trouvé que le degré de PEGMA et de VDM pouvait être ajusté avec précision en faisant varier le rapport molaire PEGMA et VDM dans la copolymérisation. Après la conjugaison de FA, le résultat de TEM indiquait que le MNP conjugué à FA ayant une teneur élevée en VDM présentait une bonne dispersibilité dans l'eau. Troisièmement, la synthèse de MNP greffé avec un copolymère dibloc poly (oxyde d'éthylène) -bloc-poly (2-vinyl-4,4-diméthylazlactone) (PEO-b-PVDM) via une stratégie de greffage "sur" et son application en tant que source magnétique recyclable nano-support pour l'adsorption avec des anticorps ont été étudiés. Les copolymères diblocs PEO-b-PVDM ont d'abord été synthétisés en utilisant une polymérisation réversible par transfert de chaîne par addition (RAFT), puis greffés sur du MNP à fonctionnalité amino par couplage avec des noyaux azlactone du bloc PVDM. La TEM a été utilisée pour étudier le nanocluster de particules et sa distribution. Il a été constaté que l'augmentation de la longueur de la chaîne PVDM augmentait la taille des nanoclusters de MNP de 20 à 150 nm; environ 10, 50 et 100 particules / groupe pour le copolymère dibloc PEO-b-PVDM21, PEO-b-PVDM41 et PEO-b-PVDM84, respectivement. De plus, les images TEM et la technique de spectroscopie par photo-corrélation (PCS) ont indiqué une amélioration de la dispersibilité des particules dans l'eau après revêtement avec les copolymères. Les nanoclusters avec revêtement de copolymère dibloc PEO-b-PVDM84 ont été utilisés en tant que nano-supports magnétiques recyclables pour une adsorption avec un anticorps. Les résultats montrent qu'il y avait une capacité d'adsorption de 96 à 99% au cours de huit cycles d'adsorption-séparation-désorption.Mots clésMagnétite, nanoparticule, ATRP, polymérisation RAFT, PNA, acide folique, anticorps.

Table 1 . 1

 11 Examples of various MNP surface modifications…………….. 19

	Chapter II

Table 2 .

 2 1 ATRP of mixtures of PEGMA and VDM using various molar ratios of EBiB initiator and CuBr/Me 6 Tren catalytic complexes in

	toluene………………………………………………………………….…	45

Table 2 . 2

 22 Summary of monomers conversions,

n M , and PDIs of poly(PEGMA-stat-VDM) copolymers using 100/100/1/0.4/0.4 molar ratio of [PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 / [Me 6 Tren] 0 , respectively, in toluene at 30 o C………………………………………………………… 52

Chapter III

Table 3 .

 3

1 Grafting density of carboxyl groups on poly(PEGMA-stat-VDM)-coated MNP after dispersing in water……………………………. 69

Table 3 . 2

 32 Hydrodynamic diameter (D h ) and zeta potential of the MNPs in water at pH 7.4, before and after FA conjugation…………………….. 71

	Chapter IV

Table 4 . 1

 41 Diblock copolymers of PEO and PVDM synthesized by

	RAFT polymerization at 70 o C in 1,4-dioxane: experimental conditions
	and characterizations……………………………………………………...	82

Table 4 . 2

 42 Hydrodynamic diameter (D h ), size distribution and zeta potential of the nanoclusters dispersed in 10 mM MES pH 6 solutions….

Table 1 . 1

 11 Examples of various MNP surface modifications

		Covalent bonding attachment
	1. Direct nanoparticle conjugation
	Nanoparticle	Reactive ligand	Conjugate	Typical
				ligands
				Activated
				small
				molecules,
				dyes,
				peptides,
				proteins or
				antibodies
				(Ref. [35-
				38], [40])
				Activated
				dyes,
				peptides,
				proteins or
				antibodies
				(Ref. [39])

Table 1 .

 1 1 (cont.)

		Physical interaction attachment
	Starting	Ligand	Functionalized	Typical ligand
	nanoparticle		nanoparticle
				Modified peptides,
				proteins or antibodies,
				DNA or RNA
				(Ref. [47-48])

  4%, ISOCHEM) was distilled in vacuo and stored at -15 o C after purification. Tris-

	[2-(dimethylamino) ethyl]amine (Me 6 Tren) [1] and 2-bromo-2-methyl-N-(3-
	(triethoxysilyl) propyl) propanamide (BTPAm) [2] were prepared according to
	previously reported procedures. Dichloromethane (CH 2 Cl 2 ), ethanol, methanol and
	diethyl ether were dried with P 2 O 5 and distilled prior to use. N,N-dimethylformamide
	(DMF, 99.8%, Aldrich), toluene (99.8%, Sigma-Aldrich) and deuterated chloroform
	(CDCl 3 , 99%, Euriso-Top) were used as received.
	2.1.2 Characterization
	Proton Nuclear Magnetic Resonance (
	3 , 99.9%, Acros), benzyl alcohol (98%, Unilab),
	oleic acid (Fluka), copper(I) bromide (99.999%, Aldrich), triethylamine (TEA, 97%,
	Carlo Erba), ethyl-2-bromoisobutyrate (EBiB, 98%, Aldrich), and thymine PNA
	(Acros). Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Aldrich) with
	average molecular weight

n M = 300 g.mol -1 was purified by passing through basic alumina and stored at -4 o C after purification. 2-Vinyl-4,4-dimethylazlactone (VDM, 99.1 H NMR) spectra were obtained from a Bruker AC 200-MHz spectrometer using CDCl 3 as a solvent. Molar masses and molar mass distributions were determined via size exclusion chromatography (SEC) at 35 o C on a system equipped with a Spectra System AS1000 autosampler with a guard column (Polymer Laboratories, PL gel 5 µm guard, 50 × 7.

Table 2 .

 2 1 shows the summary of the statistical copolymerizations of 50/50/1 molar ratio of PEGMA/VDM/EBiB at various CuBr/Me 6 Tren ratios.

Table 2 .

 2 1 ATRP of mixtures of PEGMA and VDM using various molar ratios of EBiB initiator and CuBr/Me 6 Tren catalytic complexes in toluene

	entry EBiB/ CuBr /Me 6 Tren	Temp ( o C)	time (min)	conv a (%) PEGMA VDM	n M ,th (g/mol) b	M ,SEC n (g/mol)	c	PDI c
	1	1/1/1	50	30	83	90	18705 29400	2.17
	2	1/0.5/0.5	30	90	70	82	16199 18600	1.72
	3	1/0.2/0.2	30	90	52	61	12039 14400	1.35

a Calculated via 1 H NMR spectroscopy. b n

Table 2 . 2

 22 Summary of monomers conversions, n M , and PDIs of poly(PEGMA-stat-

	VDM)	copolymers	using	100/100/1/0.4/0.4	molar	ratio	of
	[PEGMA] 0 /[VDM] 0 /[EBiB] 0 /[CuBr] 0 / [Me 6 Tren] 0 , respectively, in toluene at 30 o C
		time	conv a (%)	n M ,th	b	n M ,SEC	c	PDI c
		(min)	PEGMA VDM	(g/mol)	(g/mol)
		0	0	0	0		0	0
		30	13	16	3062		3800	1.20
		45	14	17	3282		4100	1.21
		60	15	20	3640		5400	1.19
		90	18	22	4229		7700	1.14
		120	18	25	4438		7900	1.13
		150	20	24	4668		8000	1.13
		180	22	28	5246		8100	1.13
		1440 24	29	5616		8300	1.09

a Determined via 1 H NMR spectroscopy (monomer depletion monitored relative to DMF used as an internal standard). b n M ,th = ([PEGMA]0/[EBiB]0 × conv.PEGMA × MPEGMA) + ([VDM]0/[EBiB]0 × conv.VDM × MVDM). c Measured by SEC (calibrated with polystyrene standard).

1.3.2 Cyclization of N-acryloyl-2-methylalanine to form VDM. The

	mixture of N-acryloyl-2-methylalanine (5.00 g, 3.18 x10 -2 mol) and BHT (0.0549 g,
	2.4912 x 10 -4 mol) in CH 2 Cl 2 (30 mL) was stirred at 0 o C under argon atmosphere to
	form a colloidal dispersion. DCC solution (7.21 g, 6.37 x 10 -3 mol in 40 mL CH 2 Cl 2 )
	was then added to the mixture with continuous stirring. After 12 h reaction, the solid
	dicyclohexylurea by-product was filtered off and the filtrate was evaporated to remove
	CH 2 Cl 2 . The VDM product was purified by distillation under reduced pressure to give
	a colorless mobile liquid. Yield: 60%. 1 H NMR (400 MHz, CDCl 3 ) δ H in ppm: 1.47
	(s, 6H, C(CH 3 ) 2 ], 5.93 (dd, J Htrans-Hcis = 2.0 Hz, J Htrans-Hgem = 9.9 Hz, 1H, CH 2 =CH
	[trans]), 6.25 (dd, J Hcis-Htrans = 2.0 Hz, J Hcis-Hgem = 17.6 Hz, 1H CH 2 =CH [cis]), 6.27

1 H NMR (400 MHz, DMSO-d6) δ H in ppm: 1.37 (s, 6H, (C(CH 3 ) 2 ), 5.58 (dd, J Htrans-Hcis = 2.1 Hz, J Htrans-Hgem = 10.1 Hz, 1H, CH 2 =CH [trans]), 6.03 (dd, J Hcis-Htrans = 2.1 Hz, J Hcis-Hgem = 17.1 Hz, 1H CH 2 =CH [cis]), 6.25 (dd, J Hgem-Htrans = 10.1 Hz, J Hgem-Hcis = 17.1 Hz 1H, CH 2 =CH [gem]), 8.26 (s, 1H, NH), 12.16 (s, 1H, COOH). 13 C NMR (400 MHz, DMSO-d6) δ C in ppm: 25.1 (C(CH 3 ) 2 ), 55.0 (-C(CH 3 ) 2 -), 125.3 (CH 2 =CH), 131.8

(CH 2 =CH), 164.0 (COOH), 175.5 (NH-C=O). FTIR (ATR): 3337 cm -1 (N-H stretching), 1707 cm -1 (C=O of carboxylic acid), 1647 cm -1 (C=O stretching of amide), 1599 cm -1 (C=C stretching), 1551 cm -1 (N-H bending).

1

H NMR,

13

C NMR and FTIR spectra are shown in the appendix B.

3.

(dd, J Hgem-Htrans = 9.9 Hz, J Hgem-Hcis = 17.6 Hz, 1H, CH 2 =CH [gem]).

13

C NMR (100 MHz, DMSO-d 6 ) δ c in ppm: 25.5 [C(CH 3 ) 2 ], 66.6 [C(CH 3 ) 2 ], 124.7 (CH 2 =CH), 129.6 (CH 2 =CH), 159.7 (-C=N-) and 181.5 (-

Table 3 .

 3

	1 Grafting density of carboxyl groups on poly(PEGMA-stat-VDM)-coated
	MNPs after dispersing in water
	Molar ratio of PEGMA to VDM	Density of carboxyl group (azlactone)
	in the copolymer coated on the MNPs	(mmol/g of MNPs)	(molecules/nm 2 of MNPs)
	0/100	4.67 ± 0.00	19.72 ± 0.00
	30/70	3.97 ± 0.23	16.83 ± 0.82
	50/50	3.39 ± 0.17	14.86 ± 0.77
	70/30	2.67 ± 0.21	11.36 ± 0.67

Table 3 . 2

 32 Hydrodynamic diameter (D h ) and zeta potential of the MNPs in water at pH 7.4, before and after FA conjugation

Table 4 . 1

 41 Diblock copolymers of PEO and PVDM synthesized by RAFT polymerization at 70 o C in 1,4-dioxane: experimental conditions and characterizations

	Entry Copolymer a	[VDM] 0 : [PEO-CTA] 0 : [ACVA] 0	Reaction time (h)	VDM (%) conv. a	(g/mol) n M ,th b	(g/mol) n M ,SEC c	PDI c
	1	PEO 44 -b-PVDM 21	25:1:0.2	4	84	5277	7400	1.04
	2	PEO 44 -b-PVDM 41	100:1:0.2	4	41	8060	8800	1.09
	3	PEO 44 -b-PVDM 84	100:1:0.2	8	84	14083	14500	1.06
	a The number of monomer units were calculated from the reaction conversion,
	determined by 1 H NMR spectroscopy.

Table 4 . 2

 42 Hydrodynamic diameter (D h ), size distribution and zeta potential of the

	nanoclusters dispersed in 10 mM MES pH 6 solutions
	Entry Samples	D h (nm) Size	Zeta potential
				distribution	(mV)
	1	APS-coated MNP	984.1	0.484	-3.45
	2	PEO 44 -b-PVDM 21 -MNP	96.7	0.175	-27.2
		nanoclusters
	3	PEO 44 -b-PVDM 41 -MNP	117.5	0.108	-28.3
		nanoclusters
	4	PEO 44 -b-PVDM 84 -MNP	166.1	0.277	-33.9
		nanoclusters

Table A1

 A1 Summary of monomer conversion, molecular weight and PDI of statistical

	copolymers poly(PEGMA-stat-VDM) obtained by ATRP using [EBiB] 0 /[CuBr] 0
	[Me 6 Tren] 0 molar ratio = 1/1/1 in toluene (70% v/v) at 50 o C
	time	conv. a (%)		n M ,th	b	n M ,SEC	c	PDI c
		PEGMA	VDM	(g/mol)	(g/mol)
	0	0	0	0		0		0
	30	83	90	18705	29400		2.17
	60	84	92	18994	54200		2.44
	90	84	93	19064	34800		3.00
	120	88	94	19733	52200		3.12
	180	89	94	53122	19900		3.28
	240	90	95	33327	20100		4.09
	300	89	95	41552	20000		3.71
	a Determined by 1 H NMR spectroscopy (monomer depletion monitored relative to DMF, which was
	used as an internal standard). b	n M ,th = ([PEGMA] 0 /[EBiB] 0 × conv. PEGMA × M PEGMA ] +
	[VDM] 0 /[EBiB] 0 × conv.

VDM x M VDM ]). c Measured by SEC (calibrated with polystyrene standards).

report, these novel magnetic nanoclusters were successfully used as efficient and recyclable nano-solid supports for adsorption with anti-rabbit IgG antibody. This magnetic nanocluster might be advantageous for use as a nanosolid support for efficient and facile separation of positively charged molecules.

materials when necessary, particularly Metha Research Group and Uthai Research Group. The staffs at Naresuan are excellent at making everything function smoothly and are irreplacable.

I especially would like to thank the financial support from the Thailand Research Fund (TRF) (DBG5580002), the Franco-Thai Cooperation Program in Higher Education and Research 2009-2010, supported by the Ministry of Foreign Affairs, Ministry of Higher Education and Research of France and the Commission on Higher Education of Thailand and PHC grant (PHC 2009-2010 n o 20609UF). Financial support from the Royal Golden Jubilee for the scholarship (PHD/0207/2551), Office of the Higher Education Commission, Ministry of Education is also gratefully acknowledged. Finally and certainly not least, I would like to thank Dr.Uthai Wichai and Dr.

Biography…………………………………………………………………….

LIST OF TABLES Chapter I

Immobilization of folic acid (FA) on the surface of poly(PEGMA-stat-VDM)-coated MNPs

Immobilization of FA on MNP surface was accomplished following Scheme 3.2. FA was first functionalized with a primary aliphatic amine to form EDA-FA in order to activate its reactivity toward VDM ring-opening reaction. The detailed synthesis of EDA-FA was previously reported [3]. First, poly(PEGMA-stat-VDM)coated MNPs (100 mg) were dispersed in a 10 mL anh. DMF. Then, an EDA-FA solution (100 mg EDA-FA in 10 mL anh. DMF) was added to the MNP dispersion.

The suspension was agitated at room temperature for 12 h. The particles were then recovered, washed with DMF repeatedly and dried in vacuo.

Poly(PEGMA-stat-VDM)-coated MNP immobilized with FA (EDA-FA) Scheme 3.2 Immobilization of FA onto poly(PEGMA-stat-VDM)-coated MNPs

Results and discussion

The aim of this work was to modify MNP surface with poly(PEGMA-stat-VDM) copolymer and immobilize FA on its surface. PEGMA on the MNP surface provides good dispersibility of the particles in aqueous media, while VDM serves as an active functionality for further immobilization of any nucleophilic molecules such as PNA [5] and fluorescent molecules [6]. In this work, FA was used as a model

Experimental

Materials

Unless otherwise stated, all reagents were used without further purification: iron(III) acetylacetonate (Fe(acac) 3 , 99.9%, Acros), benzyl alcohol (98%, Unilab), oleic acid (90%, Fluka), (3-aminopropyl)triethoxysilane (APS, 99%, Sigma-Aldrich), triethylamine (TEA, ≥ 99%, Sigma-Aldrich), 4,4'-azobis(4-cyanovaleric acid) (ACVA, ≥ 98%, Aldrich), anti-rabbit IgG (antibody produced in goat, Sigma-Aldrich), IgG from rabbit serum (Sigma-Aldrich), Anti-IgG-HRP (Anti-rabbit IgG (whole molecule)-Peroxidase (Sigma-Aldrich), 10% BSA diluents (KPL), ABTS ® Peroxidase Substrate (KPL), Bradford reagent (Sigma), bovine gramma globulin (BGG, Thermo Scientific) and 2-(N-morpholino)ethanesulfonic acid (MES, 99%, Acros). 2dodecylsulfanylthiocarbonylsulfanyl-2-methylpropionic acid (DMP) [1],

poly(ethylene oxide) chain transfer agent (PEO-CTA) [2] and 2-vinyl-4,4dimethylazlactone (VDM) [3] were synthesized following the reported procedures.

Pure water was obtained from a Millipore Direct Q system and had a conductivity of Transmission electron microscopy (TEM) was performed on a Philips Tecnai 12 operated at 120 kV equipped with a Gatan model 782 CCD camera.

Determination of recycling ability of the PEO-b-PVDM copolymer-MNP nanoclusters in adsorption with anti-rabbit IgG antibody

Copolymer-MNP nanoclusters adsorbed with anti-rabbit IgG antibody were thoroughly washed with 1 mL of a washing solution to desorb the antibody on the nanocluster surface. The amount of the antibody after desorption was determined using the Bradford assay. Desorption efficiency was determined from the following equation;

where C is the concentration of desorbed antibody and B is the concentration of adsorbed antibody

The adsorption-separation-desorption process was performed repeatedly to determine the recycling efficiency of the nanocluster (Scheme 4.3).