DSpace at VNU: Design of iron oxide nanoparticles decorated oleic acid and bovine serum albumin for drug delivery

Accepted Manuscript
Title: Design of iron oxide nanoparticles decorated oleic acid
and bovine serum albumin for drug delivery
Author: Thao Truong-Dinh Tran Toi Van Vo Phuong Ha-Lien
Tran
PII:
DOI:
Reference:

S0263-8762(15)00011-8
/>CHERD 1765

To appear in:
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Revised date:
Accepted date:

7-7-2014
23-10-2014
19-12-2014

Please cite this article as: Tran, T.T.-D., Van Vo, T., Tran, P.H.-L.,Design
of iron oxide nanoparticles decorated oleic acid and bovine serum albumin
for drug delivery, Chemical Engineering Research and Design (2015),
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Design of iron oxide nanoparticles decorated oleic acid and bovine serum

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albumin for drug delivery

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Thao Truong-Dinh Tran*, Toi Van Vo and Phuong Ha-Lien Tran*

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Pharmaceutical Engineering Laboratory, Biomedical Engineering Department, International

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University, Vietnam National University – Ho Chi Minh City, Vietnam

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*Correspondence to:

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Phone: (84-8) - 37244270 Ext. 3337

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Fax: (84-8) - 37244271

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E-mail address: (Thao Truong-Dinh Tran)

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(Phuong Ha-Lien Tran)

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Abstract
This study aimed to originally develop a new nanoparticulate drug delivery system of

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iron oxide nanoparticles (Fe3O4) for biomedical applications. Oleic acid and bovine serum

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albumin were decorated on the surface of iron oxide nanoparticles in new pattern by

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conjugation. The decoration was kicked off by the functionalization of arginine on the surface

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of the iron oxide nanoparticles. It was then followed by the conjugation of oleic acid and

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bovine serum albumin through the amide bond. Scanning electron microscopy, transmission

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electron microscopy, powder X-ray diffraction and Fourier transform infrared spectroscopy

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were used to characterize and determine mechanism of the decorated nanoparticles.

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Paclitaxel was chosen as the model drug in the study. The nanoparticles demonstrated a

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potential utility in delivery of anticancer drugs.

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Keywords: iron oxide nanoparticles, drug delivery, anticancer drug, oleic acid, bovine serum

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albumin.

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1. Introduction
One of the most important applications of nanotechnology is nanomedicine, which

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applies the technique to the prevention, diagnosis and treatment of diseases [1-3]. Fabrication

35

of nanoparticles has drawn much interest in developing a new generation of more effective

36

cancer therapies since nanoparticles show highly promising in the improvement of drug

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efficacy, especially drugs with a narrow therapeutic window or low bioavailability such as

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anticancer drugs [4]. Moreover, nanoparticles are under particular researches since they can

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selectively access to tumor due to their small size and versatile modified physicochemical

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properties [5]. Solid tumors facilitate preferential accumulation of nanosized drug delivery

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systems due to their specific structure where the vasculature is different in both functional

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and morphological aspects, from the one in normal tissues [6, 7]. Generally, tumor blood

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vessels are larger in size, more heterogeneous in distribution and more permeable [8]. The

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increased vascular permeability and the impaired lymphatic drainage in rapidly growing

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tumors allow an accumulation of nanoparticles in the tumor [9]. When the absorption occurs,

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the drug is released. The technique overcomes disadvantages of the conventional solution

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including rapid clearance from the blood circulation due to low molecular weight, and low

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accumulation at the tumor site for treatment. In addition, anticancer drugs tend to present

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with a large volume of distribution leading to toxicity towards healthy tissues due to their

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small size and/or their high hydrophobicity in the conventional treatment.

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A tool for observation of tumor response during cancer therapy is very important and

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indispensable in treatment of this disease. Magnetic resonance imaging (MRI) is a common

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approach widely used for diagnosis in biomedical application. Development of contrast

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agents for further improving tissue resolution on the image hence, have drawn much interest.
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Novel nanomedicine based drug delivery systems as directions to deliver anticancer drugs to

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tumor for effective therapy and diagnostics have been incessantly investigated [10]. Those

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systems not only reduce side effects of anticancer drugs but also utilize the nano structure as

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an MRI contrast agent. Iron oxide nanoparticles (IONPs) have emerged as feasible materials

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for tumor imaging and targeted anticancer drug delivery [11-15]. Various IONPs have been

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clinically used as contrast agents due to their high contrast effects and biocompatibility.

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However, the use of these products as a drug carrier system has been still under investigation.

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It has been reported that under physiological pH conditions the IONPs are not charge and

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precipitated because the isoelectric point of IONPs is 7 [16]. Consequently, agglomerated

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particles are rapidly cleared by macrophages in the reticuloendothelial system (RES) before

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they can reach to target cells [17-20]. One of the feasible approaches is coating the

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nanoparticles by a biocompatible material [21] which can act to shield the IONPs from

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surrounding environment and can also be functionalized then. Type of surface coating, its

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concentration and a wide variety of experimental factors such as stirring rate, stirring time,

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pH, etc. determine the overall size of the colloids which may also play a significant role in

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biodistribution [22-24]. Besides, the drug loading capacity of the hydrophobic part depends

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on compatibility between hydrophobic functional groups and poorly water-soluble drugs

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encapsulated [25]. A design of surface-modified IONPs hence, would determine drug loading

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capacity and probably encapsulation efficiency also [25]. Oleic acid (OA) is a biocompatible

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fatty acid and also an agent that induces the stability of many nanoparticle systems. It can

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play a role of a capping agent for the particles to form a protective monolayer through a

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strong bond. Nanoparticles with a hydrophobic coating through the attachment of the polar

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end groups to the surface hence are obtained with monodisperse and highly uniform [26, 27].

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The system with oleic acid coating only is not suitable for biomedical applications because

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they possess hydrophobic surfaces with a large surface area to volume ratio which cause

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agglomeration and formation of large clusters, resulting in the increased particle size [28].

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However, OA is the essential part of the IONPs coating for anticancer hydrophobic drug to be

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loaded [29]. Therefore, for biomedical applications in aqueous environments, in addition to

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OA part, the presence of a hydrophilic coating is favorable. Albumin nanoparticles have

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recently withdrawn attraction due to the preparation under mild conditions and the capability

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of various kinds of molecules incorporation [30]. Bovine serum albumin (BSA) is a

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preferable carrier in drug delivery systems to facilitate sophisticated biological nanostructures

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easily adaptable to human body [31]. The surface of the IONPs hence, was further conjugated

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with BSA. Paclitaxel, a hydrophobic anticancer agent, was chosen in the study as the model

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drug. The decorated multifunctional nanoparticles in this research is expected to offer

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advantages over conventional formulations in further studies including combination of

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effective tumor treatment and tumor observation during the therapy, and reducing the side

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effects of chemotherapy.

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2. Materials and Methods

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2.1 Materials

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L-arginine,

iron

oxide
(NHS),

nanoparticles

(Fe3O4

-

LOT#MKBG0737V),

dicyclohexylcarbodiimide

(DCC),

N-

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hydroxysuccinimide

2-(N-

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morpholino)ethanesulfonic acid (MES) were purchased from Sigma-Aldrich (St Louis, MO,

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USA). N,N-Dimethylformamide, oleic acid, triethylamine, sodium hydroxide, potassium
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dihydrogen phosphate were purchased from Xilong Group (China). Bovine serum albumin

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(BSA – LOT#0000079719) powder was purchased from Himedia Laboratories Pvt. Ltd.

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(India). Sodium phosphate was purchase from Guangdong Guanhua Sci-Tech Co., Ltd.

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(China). 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) was

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purchased from Merck Schuchardt (Germany). The solvents used were high-performance

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liquid chromatography (HPLC) grade. All other chemicals were of analytical grade and were

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used without further purification.

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2.2 Methods

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2.2.1. Preparation of IONPs decorated by OA and BSA

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2.2.1.1 Amine functionalization of IONPs

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500 mg IONPs was firstly dispersed in 50 ml pH 6 (KH2PO4 0.1M, adjusted by NaOH

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1M) by the tip sonicator in 5 min with 25 W of power supply (Qsonica, Model No. Q700) at

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room temperature. L-arginine was dissolved in pH 6 buffer (prepared as mentioned above) to

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yield a solution of 1.25 mg/ml. Then, 50 ml L-arginine solution was added to dispersed-

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IONPs. This mixture was continuously sonicated in 30 min with 10 W of power supply at

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room temperature. The amine-functionalized IONPs (A-IONPs) were separated by an

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external magnet (5 cm in length) and the solution was discarded. 100 ml of distilled water

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was then added to the nanoparticles for washing. The washing process was repeated thrice.

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The sample was then dried in oven at 40 ˚C.

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2.2.1.2 Conjugation of OA to A-IONPs

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OA was conjugated to the free amine on IONPs through an amide bond linkages

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between carboxylates and amines [32]. Firstly, 100 mg OA was activated by DCC and NHS

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(1:1:1) in 20 ml dimethylformamide containing 1% triethylamine (DMF-TEA) for 30 min. A

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dispersion of 500 mg A-IONPs in 50 ml DMF-TEA was then added to the above mixture so

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that the activated OA could react with the free amine on IONPs. This mixture was

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magnetically stirred in 120 min for the reaction (stirring rate 700 rpm with 5cm in length of

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magnetic bar). The final product (OA-IONPs) was separated by an external magnet and

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washed with dimethylformamide (similar to washing process in 2.2.1.1). The sample was

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then dried in oven at 40 ˚C.

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2.2.1.3 Conjugation of BSA to OA-IONPs

BSA was also conjugated to the residual free amine on OA-IONPs through an amide

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bond linkages between carboxylates and amines [32]. First, 150 mg of BSA was activated by

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408 mg of EDC and 302 mg of NHS in 250 ml MES buffer (pH 6). This mixture was kept

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magnetically stirring for 30 min at room temperature (stirring rate 700 rpm with 5cm in

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length of magnetic bar). The activated BSA was then reacted with the residual amine group

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by adding 250 ml of pH 7.5 (16% of NaH2PO4 0.1M and 84% of Na2HPO4 0.1M, adjusted by

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NaOH 1M) containing 300 mg of OA-IONPs to the above mixture. The BSA-conjugated

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OA-IONPs (BOA-IONPs) were obtained after 120 min and introduced to the separation by

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an external magnet and then, washed with distilled water (similar to washing process in

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2.2.1.1). The sample was finally dried in oven at 40 ˚C.

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2.2.2. Paclitaxel loading in BOA-IONPs
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Firstly, BOA-IONPs were dispersed in distilled water at the concentration of 2.5

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mg/ml. Then, solution of 100 µl of paclitaxel in ethanol (50 mg/ml) was added to 20 ml of

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BOA-IONPs suspension. The nanoparticle suspension was left for 5h under stirring.

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Paclitaxel-loaded BOA-IONPs were separated by a magnet and washed several times with

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distilled water. The total amount of paclitaxel in the supernatant and washing solution was

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measured to determine the percentage of drug loading and loading efficiency. All

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measurements were performed in triplicate. The Paclitaxel loading efficiency and % drug

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loading of the process were calculated as follows:

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− =- Where:

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%

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 x: initial amount of paclitaxel for loading
 y: amount of free paclitaxel in supernatant and washing solution
 z: total amount of blank nanoparticles and loaded paclitaxel

2.2.3. Paclitaxel release studies

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Paclitaxel-loaded BOA-IONPs (5 mg) were placed in a test tube containing 10 ml of

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phosphate buffer (pH 7.4) and then, incubated in a shaking water bath at 37 °C (The shaking

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frequency is 120 rpm). At pre-determined time intervals, paclitaxel-loaded BOA-IONPs were

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separated using an external magnet. While these nanoparticles were re-suspended in 10 ml of

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pH 7.4 fresh buffer for the continuous release study, the amount of paclitaxel released was

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determined from the aliquot using HPLC. All experiments were performed in triplicate.

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Percent cumulative release rate of paclitaxel were calculated as follows:

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xn: % drug release at n hour

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xbefore n: % drug release at the time point just before n hour

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2.2.4. HPLC analysis

Paclitaxel concentration was determined by HPLC system (Dionex UltiMate 3000,

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Thermoscientific Inc., USA) with Luna 5µ C18 analytical column (150x4.6 mm).

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Acetonitrile and distilled water at the ratio 67:33 was used as mobile phase with the flow rate

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at 1 ml/min. The UV detector was set to a wavelength of 227 nm. The running time was 5

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min. The standard solutions were constructed in the range of 0.5–20 ppm for

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calibration with good linearity (R2= 0.9997). Acetonitrile was used as diluted solution and

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blank solution. Twenty microliters of samples were injected into HPLC system for analysis.

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2.2.5. Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM)

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SEM (JSM-6480LV, Jeol, USA) was used to characterize surface morphology of

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nanoparticles. Fried sample was deposited on a carbon tape and coated with a thin layer of

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Platin. All samples were examined under accelerating voltage of 10 kV.

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Also, TEM (JEM-1400 plus, Jeol, USA) was used to examine nanoparticle

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morphology. A drop of nanoparticles dispersion was placed on a copper grid. Acceleration

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voltage was kept constant at 100kV.

2.2.6. Powder X-ray diffraction (PXRD)

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A D8 Advance diffractometer (Bruker, Germany) using Ni-filtered, CuKα (λ =

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1.54060 Å) radiation, was used to investigate the crystallinity of the samples. Samples were

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held on quartz frame. Diffraction pattern was obtained at a voltage of 40kV and at a current

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of 40 mA. The samples were scanned in increments of 0.02o from 5o to 80o (diffraction angle

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2θ) at 1 sec/step, using a zero background sample holder.

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2.2.7. Fourier transform infrared spectroscopy (FTIR)

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A FTIR spectrophotometer (Bruker Vertex 70, Germany) was used to investigate the

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spectra of functionalized iron oxide nanoparticles. The wavelength was scanned from 500 to

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4000 cm-1 with a resolution of 2 cm-1. KBr pellets were prepared by gently mixing 1 mg of

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the sample with 200 mg KBr.
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3. Results and discussion

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3.1. Characterization of BOA-IONPs
In this study, surface of IONPs was decorated with OA and BSA for biomedical

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applications of theranostics (Figure 1). Arginine was firstly coated on the surface of IONPs.

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The presence of the free amine group provided an opportunity for the conjugation of OA and

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BSA through the formation of amide bond. BSA offered the biocompatibility enhancement of

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nanoparticles; whereas, OA molecule was used to carry the poorly water-soluble drug.

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NH2

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NH
2

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C

NH
NH

O

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O

C

O

NH

NH

C

R1

O

NH

N

H

222

R1

C

221

O

O

C

C

C

O

R2

NH

220

C
H

N

219

R2

O

218

2
NH

R1

R2

217

NH2

NH2

216

HN

NH
2

215

2
NH

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R2

R2

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Figure 1. Illustration of synthesis iron oxide nanoparticles decorated oleic acid and bovine

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serum albumin (R1 - conjugated oleic acid, R2 – conjugated bovine serum albumin).

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The morphology of BOA-IONPs was observed by SEM (Figure 2A) and TEM

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(Figure 2C, 2D). Generally, these nanoparticles had an average diameter of 28.33 ± 5.77 nm.

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They are a fairly uniform size distribution, spherical shape with a smooth surface as showed

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in the SEM image. Figure 2C and 2D show that BOA-IONPs were covered by the outer layer

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as compared to IONPs (Figure 2B) and had the diameter below 50 nm. These results

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indicated that OA and BSA were potentially functionalized on the surface of IONPs.

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(B)

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(C)

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(D)

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Figure 2. SEM of BOA-IONPs (A) and TEM images of IONPs (B), BOA-IONPs (C, D)

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To investigate whether the crystalline structure of IONPs in BOA-IONPs was

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changed or not, PXRD was used to analyze the samples of IONPs and BOA-IONPs. The

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PXRD diffractogram of IONPs indicated a highly crystalline structure with characteristic

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peaks at 30.2º, 35.7º, 43.3º, 57.2º and 62.9º (Figure 3). The functionalization of amine groups

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and conjugation of OA and BSA on the surface on IONPs did not affect those peaks. In other

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words, the characteristic peaks of IONPs still maintained under the conditions. Therefore, the

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PXRD results confirmed the the nanocrystalline structure of Fe3O4 [33] in BOA-IONPs.

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The formation mechanism of nanoparticles was elucidated through FTIR analysis.

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Figure 4 shows FTIR spectra of OA, A-IONPs and OA-IONPs. Regarding the FTIR

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spectrum of A-IONPs, the peak at 580 cm-1 can be attributed to the vibration of Fe-O [34].

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The peaks of arginine at 1610 and 1419 cm−1 are those of COO− asymmetric and symmetric

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stretch, respectively [35] which were shifted to 1648 and 1400 cm−1 in the coated particles.

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These results suggested the binding of the carboxylic group of arginine to the IONPs. The

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presence of amine groups from arginine provides a means to conjugate OA and BSA.

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Regarding the FTIR spectrum of OA, the carbonyl group of OA was showed at 1710 cm-1. It

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has been reported that the strong C=O absorption will be shifted to lower wavenumbers if the

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molecule is conjugated [36, 37]. In this case, the C=O peak was shifted to right at 1640 cm-1

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which was attributed to the peak of amide bond formation [38-40]. This result confirmed that

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OA was successfully conjugated to the amine groups. Moreover, BSA was also conjugated to

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the surface of nanoparticles to provide hydrophilicity and increase the motility in biological

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fluids. As showed in Figure 5, the peak of amide bond in OA-IONPs was at 1640 cm-1 as

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explained above. However, this peak was stronger in the spectrum of BOA-IONPS which

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was attributed to the formation of another amide bond because of the conjugation of BSA and

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amine group [39]. Besides, the peak at 580 cm-1 which can be attributed to the vibration of

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Fe-O also appeared in BOA-IONPs. Therefore, these results confirmed the conjugation of

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BSA and OA-IONPs.

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1400

an

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1648

cr

1710

A - IONPs

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OA - IONPs

2000

1500

1000

500

-1

Wavelength (cm )

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2500

M

3000

1640

Figure 4. FT-IR spectra of oleic acid (OA), arginine-functionalization iron oxide

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nannoparticles (A-IONPs) and iron oxide nanoparticles decorated oleic acid (OA-IONPs).

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1540

BOA - IONPs

1560

1580

an

1640

1540

1580

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1560

M

BSA

te

OA - IONPs

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1640

2000
274

1800

1600

1400

1200

1000

800

600

Wavelength (cm-1)

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Figure 5. FT-IR spectra of OA-IONPs, bovine serum albumin (BSA) and iron oxide

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nanoparticles decorated oleic acid and bovine serum albumin (BOA-IONPs).

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3.3. Paclitaxel loading and encapsulation efficiency
In this study, paclitaxel was chosen as anticancer drug. Paclitaxel was loaded in BOA-

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IONPs by adsorption on oleic moiety under stirring. Percentage of drug loading and

281

encapsulation efficiency were measured by indirect method. The amount of paclitaxel loading

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was calculated from unencapsulated drug in the supernatant by HPLC analysis. Paclitaxel

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was successfully loaded in BOA-IONPs with high encapsulation efficiency. Specifically,

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percentage of encapsulation efficiency was measured to be 93% ± 2.8%. Percentage of

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paclitaxel loading was 8.5% ± 0.23%.

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3.4. In vitro release study

Figure 6 shows that the percent cumulative release rate of paclitaxel from BOA-IONPs

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at pH 7.4 in 30 days. The release profile demonstrates a burst release observed at the initial

290

stage, followed by a slower and continuous release. Particularly, paclitaxel was released 14%,

291

24% and 34 % after 1, 2, 4 days, respectively. A gradual decrease in release rate was

292

observed after 4 days. The initial burst release offers an opportunity to obtain high

293

concentrations of paclitaxel in the target tissue while the potential of prolonged release offers

294

the ability to prevent persistent excessive vascular smooth muscle cell proliferation [41]. The

295

rapid release at the initial time may be due to the adsorption of drug on the exterior surface

296

[42] and hydrophilic regions. The prolonged release may be attributed to the drug attached to

297

OA [43].

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demonstrated the successful conjugation of OA and BSA on the surface of iron oxide

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nanoparticles. More importantly, it was found that paclitaxel was loaded in BOA-IONPs with

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a high encapsulated efficiency. The in vitro release paclitaxel suggested that BOA-IONPs

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could be a promising dug carrier for cancer therapy. Moreover, the current nanoparticles with

311

magnetic core are potential for theranostics. Further studies are required to evaluate the

312

application of these nanoparticles in diagnostic.

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Acknowledgments:

This research is funded by Vietnam National University – Ho Chi Minh City under

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grant number C2014-28-09. We would like to thank to International University for their

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continued, generous and invaluable support to our studies as well as greatly boost the

318

efficiency of our research activities. We also thank to Mr. Khanh Nghia Tran for his research

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assistance in the preparation of nanoparticles.

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 Oleic acid and bovine serum albumin were decorated on the surface of iron oxide
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 Potential utility in delivery of anticancer drugs

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