Hydrophilic azlactone-functionalized magnetite nanoparticle for conjugation with folic acid

RESEARCH PAPER

Hydrophilic azlactone-functionalized magnetitenanoparticle for conjugation with folic acid

Yingrak Pray-in • Boonjira Rutnakornpituk •

Uthai Wichai • Tirayut Vilaivan •

Metha Rutnakornpituk

Received: 16 December 2013 / Accepted: 6 March 2014 / Published online: 22 March 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Herein, we report the synthesis of magne-

tite nanoparticles (MNPs) grafted with poly(poly(eth-

ylene glycol) methyl ether methacrylate-stat-2-vinyl-

4,4-dimethylazlactone) copolymers (poly(PEGMA-

stat-VDM)) prepared via a surface-initiated 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. The particle size was in the range of 5–10 nm

with the average of 8.0 ± 1.2 nm in diameter. 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 electro-

philic 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.

Keywords Magnetite � Nanoparticle �Azlactone � Folic acid � Atom transfer radical

polymerization

Introduction

Currently, a significant amount of interest is focused

on the synthesis of magnetite nanoparticles (MNPs)

coated with water soluble polymers targeted for

biomedical applications such as magnetic resonance

imaging (MRI) (Yallapu et al. 2012), drug delivery

systems (Wang et al. 2013; Yu et al. 2013; Mosivand

et al. 2013), enzyme and protein immobilization

(Johnson et al. 2011; Kang et al. 2009), ribonucleic

acid (RNA) and deoxyribonucleic acid (DNA) puri-

fication (Kang et al. 2009; Sarkar and Irudayaraj

Electronic supplementary material The online version ofthis article (doi:10.1007/s11051-014-2357-7) contains supple-mentary material, which is available to authorized users.

Y. Pray-in � B. Rutnakornpituk � U. Wichai �M. Rutnakornpituk

Department of Chemistry and Center of Excellence for

Innovation in Chemistry, Faculty of Science, Naresuan

University, Phitsanulok 65000, Thailand

B. Rutnakornpituk � U. Wichai � M. Rutnakornpituk (&)

Center of Excellence in Biomaterials, Faculty of Science,

Naresuan University, Phitsanulok 65000, Thailand

e-mail: [email protected]

T. Vilaivan

Organic Synthesis Research Unit, Department of

Chemistry, Faculty of Science, Chulalongkorn University,

Phayathai Road, Patumwan, Bangkok 10330, Thailand

123

J Nanopart Res (2014) 16:2357

DOI 10.1007/s11051-014-2357-7

http://dx.doi.org/10.1007/s11051-014-2357-7

2008), and gene therapy (Jenkins et al. 2011). A thin

layer of polymeric coating on the particle surface is

necessary to prevent the particle from agglomeration

and also to provide active functionality on its surface

for further coupling with biomolecules such as amino

acids (Ebrahiminezhad et al. 2013; Qu et al. 2012),

proteins (Wang and Lee 2003; Fu et al. 2009),

streptavidin (Narain et al. 2007), and peptide nucleic

acid (PNA) (Wang et al. 2006). Therefore, surface

modification of MNPs is an important step for

controlling chemical composition and functionality

on their surface.

Different strategies have been developed to coat

active organic compounds (Culita et al. 2013) and

polymers on particle surface such as physical adsorp-

tion (Ahmad et al. 2008), emulsion polymerization

(Horak and Chekina 2006), and the so-called grafting

to (Aqil et al. 2008; Pothayee et al. 2011) and grafting

from methods (Sun et al. 2007; Wang et al. 2012;

Khodadust et al. 2013). The ‘‘grafting to’’ method

involves the reactions between reactive functional

groups in the polymer and complementary active

groups on the particle surface. However, loading

density of the polymers in this approach is rather

limited due to steric hindrance from the pre-grafted

polymers. Conversely, the ‘‘grafting from’’ approach

involves the polymerization initiated from active

species previously grafted on the particle surface.

Dense polymer structure on the particle surface is

usually obtained with this approach (Dong et al. 2011).

Atom transfer radical polymerization (ATRP), one

of the well-known controlled radical polymerization

(CRP) reactions, has been widely reported as an

efficient method for the ‘‘grafting from’’ approach.

This reaction allows for homopolymerization, ran-

dom, and block copolymerizations of various mono-

mers such as styrene (Mendonca et al. 2011; Sun et al.

2007), methylmethacrylate (Mendonca et al. 2011),

acrylate (Lacerda et al. 2013), and N-isopropylacryl-

amide (Lu et al. 2007; Mauricio et al. 2011). In

addition, it can produce polymers with narrow

molecular weight distribution and a good control in

functionality and composition distribution (Lacerda

et al. 2013; Lu et al. 2007; Mauricio et al. 2011;

Mendonca et al. 2011; Sun et al. 2007).

In our previous work, we have reported the

immobilization of thymine PNA monomer onto the

MNPs grafted with 50/50 molar ratio of poly(PEG-

MA) to poly(azlactone). It was found that 4 wt% of

thymine PNA monomer conjugated on the MNP

surface through the azlactone ring opening reaction

(Prai-in et al. 2012). This result signified the potential

of this MNP for conjugation with a wide range of other

bioentities having nucleophilic sites, such as amine,

hydroxyl, or thiol. Folic acid (FA) is of used as a model

compound in this work because it can specifically

interact with folate receptors overexpressed on human

tumor cells (Zhang et al. 2002). Precedents have

reported the immobilization of FA on MNP surface for

use as a cancer cell targeting agent by complexing FA

on the outermost polymeric layer of the MNP surface

(Lin et al. 2009; Mohapatra et al. 2007; Sahu et al.

2012; Viota et al. 2013). Relatively small number of

publications has reported surface modifications of

MNPs to obtain reactive functional groups along the

polymeric coating layers such as poly(acrylic acid)

(Rutnakornpituk et al. 2011) and poly(succinimide)

(Yang et al. 2013). In the current report, hydrophilic

poly(azlactone)-containing copolymers were used to

modify MNP surface such that FA can be readily

grafted not only on the periphery but also inside the

polymeric layer of the MNPs via the azlactone ring-

opening reaction.

In the present work, poly(ethylene glycol) methyl

ether methacrylate (PEGMA)-co-2-vinyl-4,4-dime-

thylazlactone (VDM) copolymers (poly(PEGMA-

stat-VDM)) was synthesized via a surface-initiated

ATRP from MNP. The hydrophilic poly(PEGMA)

coating allowed the particle to disperse well in

aqueous media, which is a critical requirement for

biomedical uses, and the azlactone ring of the VDM

served as an electrophilic site for covalent attachment

of FA. Precedents have reported the stabilization of

nanoparticles with hydrophilic (co)polymers contain-

ing the reactive functionalities toward amines such as

N-hydroxysuccinimide ester (Mohapatra et al. 2007;

Sun et al. 2006; Chen et al. 2010). The main drawback

of using this functional group is the formation of small

molecule products after the reaction with amine.

Therefore, in the current work, we take the advantages

of using 2-vinyl-4,4-dimethylazlactone (VDM) to

stabilize MNPs and use it as an electrophilic site for

covalent bonding with FA. The azlactone reacts

readily with nucleophiles and leaves no by-products

after the ring-opening reaction (Levere et al. 2011).

The novelty of this work stems from the synthesis of

the MNPs bearing various amounts of electrophilic

moieties on their surface. Varying the molar ratios of

2357 Page 2 of 12 J Nanopart Res (2014) 16:2357

123

PEGMA and VDM monomers in the polymerization

step controlled this parameter. This is the first report,

to our knowledge, of fine-tuning the properties of

poly(PEGMA-stat-VDM)-coated MNPs with an

attempt to obtain the particle with good dispersibility

and stability in water, and with suitable FA immobi-

lization capability.

Particle size and its distribution for the MNPs

were investigated using transmission electron

microscopy (TEM). Photocorrelation spectroscopy

(PCS) was used to study the hydrodynamic size and

zeta potential of the MNPs dispersed in water both

before and after grafting with FA. Thermogavimetric

analysis (TGA) was used to determine the compo-

sitions of the polymer-MNP complexes and vibrat-

ing sample magnetometry (VSM) was used to study

their magnetic properties. It is anticipated that this

multifunctional FA-grafted MNPs should efficiently

bind to cancer cell membranes and improve MNP-

binding capability. The efficiency of treating cancer

cells with the polymer-grafted MNP is warranted for

future studies.

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), copper(I) bromide (CuBr, 98 %,

Acros), triethylamine (TEA, 97 %, CarloErba), dicyclo-

hexylcarbodiimide (DCC, 99 %, Acros), sodium

hydroxide (C98 %, Aldrich), 2,6-di-tert-butyl-p-cresol

(C99 %, Aldrich) (BHT), and concentrated hydrochloric

acid (37 %, Sigma-Aldrich). Poly(ethylene glycol)

methyl ether methacrylate (PEGMA, Aldrich) with

average molecular weight Mn ¼ 300 g mol-1 was

purified by passing through basic alumina column

and stored at -4 �C after purification. Tris-[2-

(dimethylamino)ethyl]amine (Me6Tren) (Ciampolini

and Nardi 1966), 2-bromo-2-methyl-N-(3-(triethoxy-

silyl)propyl) propanamide (BTPAm) (Sun et al. 2007)

and N-(2-aminoethyl) folic acid (Rutnakornpituk et al.

2011) were prepared according to previously reported

procedures. N,N-dimethylformamide (DMF, Acros),

toluene (Acros), and methylene chloride (CH2Cl2,

C99.5 %, Sigma-Aldrich) were used as received.

Acryloyl chloride was synthesized via the coupling

reaction between acrylic acid and benzoyl chloride at

75 �C to give a colorless liquid; 60 % yield.

Characterization

Fourier transform infrared spectrophotometry (FTIR)

was performed on Perkin-Elmer Model 1600 Series

FTIR Spectrophotometer. The solid samples were

mixed with KBr to form pellets. Nuclear magnetic

resonance spectroscopy (NMR) was performed on a

400 MHz Bruker NMR spectrometer using CDCl3 and

DMSOd6 as solvents. TEM was performed on a Philips

Tecnai 12 operated at 120 kV equipped with a Gatan

model 782 CCD camera. TGA was performed on

SDTA 851 Mettler-Toledo at the temperature ranging

between 25 and 600 �C at a heating rate of 20 �C/min

under oxygen atmosphere. VSM was performed at

room temperature using a Standard 7403 Series,

Lakeshore vibrating sample magnetometer. Hydrody-

namic and zeta potential of the particles were

measured via PCS using NanoZS4700 nanoseries

Malvern instrument. The sample dispersions were

sonicated for 30 min before the measurement at

25 �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 con-

ductometer (Metter Toledo Bmblt 8603 Swchwerzen-

bach). The presence of FA was investigated using

SPECORD S100 UV–Visible spectrophotometer

(Analytikjena AG) coupled with a photo diode array

detector at kmax = 371 nm.

Synthesis of MNPs coated with ATRP initiators

(BTPAm-coated MNP)

The MNPs grafted with an ATRP initiator were

prepared via a three-step reaction; (1) synthesis of

MNP core, (2) coating the MNPs with oleic acid, and

(3) coating the particle with BTPAm (an ATRP

initiator). First, a thermal decomposition reaction

between Fe(acac)3 (5 g, 14.05 mmol) and benzyl

alcohol (90 mL) under a nitrogen blanket was carried

out at 180 �C for 48 h to form the MNPs. The particles

were then separated from the solvent with the help of

an external magnet and washed with ethanol and

CH2Cl2 repeatedly to remove benzyl alcohol and then

J Nanopart Res (2014) 16:2357 Page 3 of 12 2357

123

dried under reduced pressure. To prepare oleic acid-

coated MNPs, oleic acid (4 mL) was dropped into a

MNP-toluene dispersion (0.8 g of dried MNPs in

30 mL of toluene) with sonication. To coat BTPAm

onto the MNPs, BTPAm (0.6 g) was then added to the

oleic acid-coated MNP dispersed in toluene (0.8 g of

the MNP in 30 mL of toluene) containing 2 M TEA

(5 mL) as a catalyst. After stirring for 24 h, the

particles were precipitated in methanol and washed

with toluene to remove oleic acid and ungrafted

BTPAm from the dispersion.

Synthesis of 2-vinyl-4,4-dimethylazlactone

(VDM)

Synthesis of N-acryloyl-2-methylalanine (Taylor et al.

1982)

2-Methylalanine (5.6966 g, 5.52 9 10-2 mol, 1 eq.)

was added into an aqueous solution of sodium

hydroxide (4.4160 g, 11.04 9 10-2 mol, 2 eq. in

15 mL water) in the presence of BHT (0.0552 g,

2.5051 9 10-4 mol) as a polymerization inhibitor at

0–10 �C, followed by an addition of acryloyl chloride

(5 mL, 5.52 9 10-2 mol, 1 eq.). After 12 h stirring,

conc. HCl (6.81 mL, 6.90 9 10-2 mol, 1.25 eq.) was

added into the solution, which was kept at 10 �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 %. 1H NMR, 13C

NMR, and FTIR spectra are shown in the supporting

information.

Cyclization of N-acryloyl-2-methylalanine to form

VDM

The mixture of N-acryloyl-2-methylalanine (5.00 g,

3.18 9 10-2 mol) and BHT (0.0549 g, 2.4912 9

10-4 mol) in CH2Cl2 (30 mL) was stirred at 0 �C

under argon atmosphere to form a colloidal dispersion.

DCC solution (7.21 g, 6.37 9 10-3 mol in 40 mL

CH2Cl2) was then added to the mixture with contin-

uous stirring. After 12 h reaction, the solid dicyclo-

hexylurea by-product was filtered off, and the filtrate

was evaporated to remove CH2Cl2. The VDM product

was purified by distillation under reduced pressure to

give a colorless mobile liquid. Yield: 60 %. 1H NMR

(400 MHz, CDCl3) dH in ppm: 1.47 (s, 6H, C(CH3)2],

5.93 (dd, JHtrans-Hcis = 2.0 Hz, JHtrans-Hgem =

9.9 Hz, 1H, CH2 = CH [trans]), 6.25 (dd,

JHcis-Htrans = 2.0 Hz, JHcis-Hgem = 17.6 Hz, 1H

CH2=CH [cis]), 6.27 (dd, JHgem-Htrans = 9.9 Hz,

JHgem-Hcis = 17.6 Hz, 1H, CH2=CH [gem]). 13C

NMR (100 MHz, DMSO-d6) dc in ppm: 25.5

[C(CH3)2], 66.6 [C(CH3)2], 124.7 (CH2=CH), 129.6

(CH2=CH), 159.7 (–C=N–) and 181.5 (–C=O). FTIR:

1,822 cm-1 (C=O stretching), 1,668 cm-1 (C=N

stretching), 1,204 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 (Taylor et al. 1982).

Synthesis of poly(PEGMA-stat-VDM) copolymer

coated on the MNP surface via ATRP (Scheme 1)

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 pre-

pared 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 Me6Tren (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 �C for 24 h.

Immobilization of folic acid (FA) on the surface

of poly(PEGMA-stat-VDM)-coated MNPs

Immobilization of FA on MNP surface was accom-

plished using following Scheme 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 (Rutnakornpituk

et al. 2011). 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 disper-

sion. The suspension was agitated at room temperature

for 12 h. The particles were then recovered, washed

with DMF repeatedly and dried in vacuo.


123

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 mole-

cules such as PNA (Prai-in et al. 2012) and fluorescent

molecules (Ho et al. 2010). In this work, FA was used

as a model molecule for covalently grafting on the

MNP surface through the ring-opening reaction of the

VDM. Molar ratios of PEGMA to VDM on the MNPs

were systematically varied (0/100, 30/70, 50/50, and

70/30, respectively) in an attempt to obtain water

dispersible MNPs with various amounts of the active

azlactone rings on their surface. Previous works have

mostly reported the immobilization of FA on the

outermost layer of polymeric surfactant-coated MNPs,

which might limit the grafting efficiency to the particle

Fe(acac)3

1) Benzyl alcohol,180oC, 48 h2) Oleic acid/Toluene

3) BTPAm4) PEGMA,VDM/CuBr,Me6Tren

O SiNH

O

OO

O

SiO

O

Si

OSi

OSi O

O

OO

O

O

stat

O N

O

H3C

Poly(PEGMA-stat-VDM)-coated MNP

nm

om : n = 0 : 100= 30 :70= 50 : 50= 70 : 30

Scheme 1 Synthesis of poly(PEGMA-stat-VDM)-coated MNPs via surface-initiated ATRP reaction

O SiNH

O

OO

O

SiO

O

Si

OSi

OSi O

O

O

+

O

O

O

stat

O N

O

H3C

Poly(PEGMA-stat-VDM)-coated MNP

O SiNH

O

OO

O

SiO

O

Si

OSi

OSi O

O

OO

O

O

stat

H3C

O NH

O

nm

o

m n

o

NH

OHOOCHN

ONH

NN

N

NHO NH2

H2N

N-(2-aminoethyl) folic acid

NH

OHOOCHN

ONH

NN

N

NHO NH2

HN

Poly(PEGMA-stat -VDM)-coated MNP immobilized with FA

(EDA-FA)

Scheme 2 Immobilization of FA onto poly(PEGMA-stat-VDM)-coated MNPs


123

surface (Lin et al. 2009; Mohapatra et al. 2007; Sahu

et al. 2012). 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

(Rutnakornpituk et al. 2011; Yang et al. 2013), the

advantage of using poly(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 copoly-

mers. 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 immobi-

lized 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 surface-

initiated ATRP of PEGMA and VDM. FA was then

immobilized on the particle surface via a ring-opening

reaction. Figure 1 shows FTIR spectra of poly(PEG-

MA-stat-VDM)-coated MNPs having the copolymer

molar ratio of 0/100, 30/70, 50/50, and 70/30,

respectively (Fig. 1A–D). The success of the grafting

reaction was signified by the presence of the charac-

teristic signals of PEGMA at 1,719–1,722 cm-1

[O(C=O) stretching] and those of VDM at

1,815–1,821 cm-1 (C=O stretching). The decrease

of the intensity of ester bonds [–O(C=O) stretching,

1,719–1,722 cm-1] of PEGMA relative to those of the

Fe–O bonds from MNP core (578 cm-1) corresponds

to the decrease of the amount of PEGMA units in the

copolymer. Additionally, the increase of C=O signals

of VDM (1,815–1,821 cm-1) was also observed as the

percentage of VDM in the copolymer was increased. It

should be pointed out that the signals of the Fe–O

bonds (578 cm-1) of the MNP core did not signifi-

cantly change in their intensity in all samples.

TEM images of poly(PEGMA-stat-VDM)-coated

MNPs (0/100, 30/70, 50/50, and 70/30, respectively)

are shown in Fig. 2. The particle size is in the range of

5–10 nm with the average of 8.0 ± 1.2 nm in diam-

eter. Dispersibility of the particles in DMF improved

wavenumber (cm-1)

4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400

A

B

C

D

1821 cm-1

1816 cm-1

1815 cm-1

1816 cm-1

1722 cm-1

1721 cm-1

1719 cm-1

% T

Fig. 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


123

with increasing PEGMA-to-VDM ratio in the copoly-

mer due to the presence of higher number of polar

PEGMA units on their surface. However, some nano-

scale clustering of these particles was observed in

TEM images, and this was attributed to the existence

of relatively non-polar poly(VDM) moieties on the

particle surface. It should be noted that these particles

were well dispersible in DMF without visually

noticeable aggregation despite the observation of

nanoclusters in TEM images.

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 assis-

tance of an external magnet. Using the hypothesis that

percent char yield was the weight of magnetite core

remaining at 600 �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

D

20 nm

C

20 nm

B

20 nm

A

20 nm

Fig. 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. The TEM

samples were prepared from

MNP-DMF dispersion

60

70

80

90

100

0 100 200 300 400 500 600 700

Res

idua

l Wei

ght (

%)

Temperatue (oC)

A

B

F

C, DE

Fig. 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


123

MNP was about 7.6 % and those of the copolymers

were in the range of 10.5–15.3 % (Fig. 3). This result

corroborates the results from FTIR suggesting that the

copolymers existed on the particle surface.

M–H curves of bare MNP, BTPAm-coated MNPs,

and poly(PEGMA-stat-VDM)-coated MNPs are

shown in Fig. 4. The particles exhibit superparamag-

netic behavior at room temperature as indicated by the

absence of remanence and coercivity. Bare MNPs and

BTPAm-coated MNPs showed relatively high satura-

tion magnetization (Ms) (48.9–56.3 emu/g) due to a

lack of or only trace of organic component in the

samples (Fig. 4A, B). A slight decrease of Ms values of

the copolymer-coated MNPs (34.4–42.8 emu/g) as

opposed to their precursors was attributed to the

presence of the copolymers on the particle surface,

giving rise to the decrease of magnetite content in the

complex (Fig. 4C–F).

Grafting density of VDM on the particle surface

was quantitatively determined by a conductometric

titration. First, the copolymer-coated MNPs were

dispersed in water, leading to a ring-opening reaction

of the azlactone rings grafted on the MNP surface and

giving rise to the formation of carboxyl groups. After

drying to obtain the exact weight of the samples, they

were then re-dispersed in a 0.005 M NaOH solution,

and the amounts of carboxyl groups were quantified

from back titrations with a 0.005 M HCl solution. An

example of the calculation of the grafting density of

VDM on the MNP surface is shown in the supporting

information. It was found that the VDM loading

ranged between 2.67 and 4.67 mmol/mg of MNPs,

and its grafting density consistently increased from

11.36 to 19.72 molecules/nm2 as VDM in the copoly-

mer increased from 30 to 100 %, respectively

(Table 1). This implies that the grafting density of

VDM on the MNP surface can be fine tuned by simply

modulating the molar ratio of VDM to PEGMA

monomers in the copolymerization reaction. It should

be noted that the density of carboxyl groups on the

MNP was reproducible as indicated by the low values

of the standard deviations.

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(PEG-

MA-stat-VDM)-coated MNPs through a ring-opening

reaction of the VDM moiety. Utilization of FA

increasingly attracted much attention for tumor selec-

tive 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 (Sauzedde et al.

1999a, b). In addition, FA is considered appropriate

for targeted delivery because it is non-immunogenic,

high binding affinity for its receptor (Kd = 10-10 M),

highly stable and low cost and easy to be conjugated

with imaging agents (Low et al. 2008).

-60

-40

-20

0

20

40

60

-12000 -8000 -4000 0 4000 8000 12000

Mag

net

izat

ion

(em

u/g

)

Applied field (G)

A)B)

E)

F)

C)D)

Fig. 4 M–H curves of (A) bare MNP, (B) BTPAm-coated MNP

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

Table 1 Grafting density of carboxyl groups on poly(PEG-

MA-stat-VDM)-coated MNP after dispersing in water

Molar ratio of PEGMA

to VDM in the copolymer

coated on the MNP

Density of carboxyl

group (azlactone)

(mmol/mg

of MNP)

(Molecules/nm2

of MNP)

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


123

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. Immo-

bilization of EDA-FA on the particle surface was

carried out in anh. DMF at room temperature for 12 h.

The hydrodynamic (HD) size and zeta potential of FA-

functionalized and non-functionalized MNP are given

in Table 2. The results showed reproducible HD size

and zeta potential of the polymer-coated MNPs as

indicated by acceptable values of the standard devi-

ations. The HD size 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 agglom-

eration. This is in good agreement with the decrease in

degree of negative charge of the FA-functionalized

MNPs as compared to those before the FA function-

alization, 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

as 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 pro-

viding steric repulsion stabilization mechanism, and

this might consequently enhance cell membrane

penetration ability.

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 Fig. 5. These

TEM samples were prepared from aqueous disper-

sions. It was found that, before FA conjugation, all

polymer-coated MNPs were well dispersible in water

without aggregation (Fig. 5A–D). After FA conjuga-

tion, some nano-scale aggregations of multiple MNPs

were observed, and this was attributed to the presence

of hydrophobic FA coated on the particle surface

(Fig. 5A0–D0). 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 aggrega-

tion 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 stabiliza-

tion and thus preventing particle agglomeration.

Therefore, FA-poly(VDM)-coated MNPs were be

Table 2 Hydrodynamic (HD) size and zeta potential of the MNPs in water at pH 7.4, before and after FA conjugation

MNP (before FA conjugation) HD size

(nm)

Zeta

potential

(mV)

MNP (after FA conjugation) HD size

(nm)

Zeta

potential

(mV)

Poly(VDM)-coated MNP 96.3 ± 1.1 -17.39 FA-poly(VDM)-coated MNP 107.7 ± 1.4 -14.12

30/70 poly(PEGMA-stat-VDM)-

coated MNP

131.0 ± 5.0 -15.92 FA-30/70 poly(PEGMA-stat-VDM)-

coated MNP

210.3 ± 4.5 -9.80


coated MNP


coated MNP

168.0 ± 1.2 -6.38


coated MNP


coated MNP

270.0 ± 7.0 -4.90


123

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

(Fig. 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/

nm2 of the particle surface area [260 FA molecules/

particle). An example of the calculation is illustrated

in the supporting information. Figure 6B exhibits the

UV–Visible spectra of poly(VDM)-coated MNP both

before and after FA conjugations. FA shows a kmax

value at 371 nm (Fig. 6B-c)], and suspension of

poly(VDM)-coated MNPs after FA conjugation exhi-

bit a weak absorbance signal at the same wavelength

(Fig. 6B-b). Poly(VDM)-coated MNPs before FA

conjugation do not show any absorbance signal at

A

20 nm

C

20 nm

B

20 nm

D

20 nm

C’

20 nm

A’

20 nm

B’

20 nm

D’

20 nm

Fig. 5 TEM images of the poly(PEGMA-stat-VDM)-coated

MNPs, (A–D) before and (A0–D0) after the FA conjugation. The

MNPs were coated with the copolymers having (A, A0) 0/100,

(A, B0) 30/70, (C, C0) 50/50, and (D, D0) 70/30 molar ratios of

PEGMA to VDM, respectively. All TEM samples were

prepared from aqueous dispersions

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

340 360 380 400 420 440

Abs

orba

nce

Wavelength (nm)

c)

b)

a)40

50

60

70

80

90

100

0 100 200 300 400 500 600 700

Temperature (oC)

a)

b)

A B

Res

idua

l Wei

ght (

%)

Fig. 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


123

the same wavelength (Fig. 6B-a). This result signifies

that FA was, to some extent, covalently conjugated to

the MNP surface.

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 electro-

philic azlactone rings could be adjusted by varying the

molar ratio of PEGMA to VDM in the copolymeriza-

tion. These copolymer-coated MNPs were success-

fully used for immobilization of FA on their surface.

To obtain well-defined polymeric coating on the

MNPs with high FA grafting efficiency, many con-

trollable strategies have been employed, such as a

thermal decomposition method to obtain MNP with

well define size and ATRP ‘‘grafting from’’ strategy

for a good control in degree of PVDM and/or PEGMA

(co)polymerization from MNP surface. It was found

that the MNPs with good dispersibility and stability in

water, and with reasonable FA immobilization capa-

bility were obtained. These azlactone-grafted MNPs

might be useful for further conjugation with a broad

range of molecules containing nucleophilic groups

such as amino, hydroxyl, or thiol. These novel FA-

functionalized MNPs might be used as efficient drug

delivery vehicles, particularly for cancer treatment.

The studies in the efficiency of treating cancer using

these particles will be conducted in the future.

Acknowledgments The authors thank the Thailand Research

Fund (TRF) (DBG5580002) for financial support. We also thank

the Franco-Thai Cooperation Program in Higher Education and

Research 2009–2010, provided 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 No. 20609UF). YP specially

acknowledges the Royal Golden Jubilee Ph.D. Program for

the scholarship (PHD/0207/2551). The Center of Excellence for

Innovation in Chemistry (PERCH-CIC), Commission on Higher

Education, Ministry of Education is also gratefully

acknowledged for financial support.

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Hydrophilic azlactone-functionalized magnetite nanoparticle for conjugation with folic acid