Polyacrylic Acid-Derivatized Superparamagnetic Iron Oxide Nanoparticles for Delivery of Tissue Plasminogen Activator

Jyh-Ping Chen,* Pei-Chin Yang, and Hwei-Fang Hwang PAA oligomer, in which case PAA are chemically bound to MNP. For this method (designated as SMP), 500 mg (SMP500) synthesized by two different approaches and studied for or 333 mg (SMP333) PAA oligomer, Fe+2 and Fe+3 were chemicophysical properties. PAA-MNP prepared by physical reacted in 40 ml DI water with vigorous stirring at 60 C under adsorption of PAA on pre-formed MNP showed better properties N , followed by adding 8 M NH OH and stirring for another 30 2 4 for covalent binding with a thrombolytic drug, recombinant tissue min. PAA-MNP was purified by centrifugation, washing, and plasminogen activator (rtPA). The PAA-MNP-rtPA conjugate re-dispersion cycles and dialyzed against DI water. showed full retention of thrombolytic activity and is useful as a Particle size and morphology was examined by transmission magnetically targeted drug carrier to improve clinical electron microscopy (TEM). X-ray diffraction (XRD) thrombolytic therapy. measurements were taken to investigate particle crystal structure. Zeta potential and particle size distribution were I. INTRODUCTION Lysis of fibrin clot by recombinant tissue plasminogen determined by dynamic light scattering (DLS). The activator (rtPA) is currently the only approved therapy for magnetization of PAA-MNP was measured by a treatment of acute ischemic stroke [1]. It will be highly superconducting quantum interference device (SQUID) desirable to deliver magnetic nanoparticle (MNP)-bound rtPA magnetometer. Surface concentration of -COOH group was under magnetic guidance for targeted thrombolysis, which will determined by Toluidine Blue O. rtPA (Actilyse from also reduce its hemorrhagic side effects [2]. When used as a Boehringer Ingelheim) was immobilized to PAA-MNP by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide drug carrier, MNP is usually composed of a magnetite core using hydrochloride as a cross-linking agent. For fibrinolysis assay, with superparamagnetic characteristics, and a polymer-coated 125 I-labelled fibrin clot was incubated with plasminogen (4 surface layer that provides functional groups for drug binding and increasing particle stability. rtPA bound to MNP will Pg/ml) and rtPA (free or bound) at 37 oC for 30 min. Maximal ensure the amount of drug delivered to a specific area in fibrinolysis was defined by the fibrinolytic activity of trypsin during 30-min incubation. Aliquots of supernatant were circulation, and be useful for targeting fibrin clots [3]. Polyacrylic acid (PAA) has been used to stabilize MNP by counted with a gamma counter to determine fibrinolytic providing electrostatic repulsion to against particle aggregation. activity, which is calculated as % maximal fibrinolysis. Both in vivo and in vitro biocompatibility of PAA have been III. RESULTS AND DISCUSSION demonstrated [4]. A facile synthesis of PAA-MNP is highly desirable for its application as a magnetic drug carrier. In this From TEM micrographs, PAA-MNP showed uniform study, we compare two routes for preparing PAA-MNP and particle morphology with an average diameter from 5.5 to 8.7 examine the thrombolysis efficacy of PAA-MNP-rtPA. nm (Table 1). For SMP since PAA oligomer acts as a template for Fe3O4 nucleation, the growth of Fe3O4 MNP was hindered II. MATERIALS AND METHODS by PAA and the size of Fe3O4 MNP (visible under TEM) of Two methods for preparing PAA-MNP were carried out in SNP is smaller than that of MP. DLS measurements show the the experiments. Method A is the physically adsorption of same trend of change in particle diameter observed from TEM, PAA oligomer (MW = 2000) on pre-formed Fe3O4 MNP. For but the hydrodynamic diameter difference between MP and this method (designated as MP), Fe3O4 MNP was first obtained SMP shows more drastic change from 49 to 344 nm (Table 1). by co-precipitation of Fe+2 and Fe+3 in de-ionized (DI) water at For MP, agglomerate formation is possible with a single PAA 60 C under N2, followed by adding 8 M NH4OH to the oligomer chain bound to multiple Fe3O4 MNP and leads to solution. PAA-MNP was prepared by reacting 1 g of Fe3O4 increased hydrodynamic diameter. Particle size also decreases MNP with 750 mg (MP750) or 500 mg (MP500) PAA in 100 with increasing PAA content used in the synthesis for both MP and SMP. Electrophoretic mobility measurements give highly ml DI water at 60C for 20 min. Method B is co-precipitation of Fe+2 and Fe+3 in the presence negative zeta potentials from -25.7 to -49.1 mV (Table 1), which correlates well with the increase of surface density of COOH group. SMP has substantial less COOH functional All authors are at the Department of Chemical and Materials Engineering, Chang groups on particle surface compared with MP. Abundance of Gung University, Kwei-San, Taoyuan 333, Taiwan, ROC. COOH functional groups on MP surface will facilitate the * Contacting author (e-mail: jpchen@mail.cgu.edu.tw). Abstract-Polyacrylic acid (PAA)-derivatized Fe3O4 magnetic nanoparticle (MNP) intended as a drug carrier were

978-1-4244-3544-9/10/$25.00 2010 IEEE

formation of covalent amide bonds between the COOH groups of PAA-MNP and NH2 groups of rtPA. The particles crystalline structure is confirmed to be consistent with magnetite from XRD measurement (Fig. 1). All PAA-MNP show five diffraction peaks at 2T = 30.1, 35.4, 43.1, 56.9, and 62.5, representing corresponding indices (220), (311), (400), (511), and (440), respectively, which confirms that the magnetic particles are pure Fe3O4 with spinel structure. The intensity of the highest peak at 35.4 is substantially higher for MP than SMP. Unsymmetrical peaks at 35.4 are also observed for SMP, indicating defects in the crystalline structure formed. Also, the broader diffraction peak of SMP indicates the particle size of magnetite of SMP is smaller, according to the Debye-Scherrer equation from the full width at half maximum. The average crystalline size calculated from XRD shows that the presence of PAA during co-precipitation of Fe3O4 reduces the size of MNP dramatically due to that COOH groups of PAA inhibit growth of the Fe3O4 particles (Table 1) . The results of SQUID analysis is shown in Fig. 2 as function of applied magnetic field. The saturation magnetization of MP decreased with increasing amount of PAA used during synthesis; with MP750 giving 61.0 emu/g and MP500 giving 30.5 emu/g. SMP shows considerable lower saturation magnetizations than MP, which are 19.2 and 20.9 for SMP500 and SMP333, respectively. Although decrease in saturation magnetization may be due to coverage by PAA molecules, it may mainly result from difference in particle size found from XRD calculation and the fact that saturation magnetization will be reduced significantly when the particle size is smaller than 10 nm. From the magnetization curve, the remanence and coercive force are found to be zero and there is no magnetic hysteresis loop observed, indicating typical superparamagnetic behavior for PAA-MNP. After binding with rtPA, PAA-MNP still retains its saturation magnetization as is observed for MP500. With its superior properties, MP750 is deemed as the best PAA-MNP preparation for rtPA delivery. The fibrinolytic activities of PAA-MNP-rtPA based on MP750 is no different from those of free rtPA measured by 125 I-fibrinolysis assays at two rtPA concentrations (p < 0.05), with negligible background fibrinolysis from MP750 as the control (Fig. 3). This suggests that covalent binding of rtPA to PAA-MNP will preserve most of its fibrinolytic activity, which is critical for effective thrombolysis in vivo. REFERENCES
[1] [2] H.B. van der Worp, and J. van Gijn, Acute ischemic stroke, New Eng. J. Med., vol. 357, pp. 572-5299, 2007. M.D. Kaminski, Y. Xie, C.J. Mertz, M.R. Finck, H. Chen, and A.J. Rosengart, Encapsulation and release of plasminogen activator from biodegradable magnetic microcarriers, Eur. J. Pharm. Sci., vol. 35, pp. 96-103, 2008. Y.H. Ma, S.Y. Wu, T. Wu, Y.J. Chang, M.Y. Hua, and J.P. Chen, Magnetically targeted thrombolysis with recombinant tissue plasminogen activator bound to polyacrylic acid-coated nanoparticles, Biomaterials, vol. 30, pp. 3343-3351, 2009. R. Ghavamzadeh, V. Haddadi-Asl, and H. Mirzadeh, Bioadhesion and biocompatibility evaluations of gelatin and polyacrylic acid as a crosslinked hydrogel in vitro, J. Biomater. Sci. Polym. Ed., vol. 15, pp. 1019-1031, 2004.

35.4

30.1

43.1

56.9 Fe3O4

62.5

Intensuty

MP750

MP500 SMP500

SMP333

20

30

40

50

60

70

2T angle

Fig. 1. X-ray diffraction patterns of PAA-MNP.

80 60 MP750 MP500 SMP500 SMP333 MP500 with bound rtPA

Magnetization, M (emu/g)

40 20 0 -20 -40 -60 -80 -15000

-10000

-5000

5000

10000

15000

Applied Magnetic Field,H (Oe)

Fig. 2. SQUID magnetization curves of PAA-MNP.

60

Maximum fibrinolysis (%)

50 40 30 20 10 0 -10

Control Bound rtPA Free rtPA

0.0003

0.003

rtPA concentration (mg/ml)

Fig. 3. Fibrinolytic activities of free rtPA and rtPA bound to MP750

TABLE I PROPERTIES OF MAGNETIC NANOPARTICLES Sample MP750 MP500 SMP500 SMP333 Particle size (nm) TEM 8.7 6.7 6.0 5.5 DLS 344 210 86.5 49.3 XRD 7.5 9.4 3.9 4.9 Zeta potential (mV) -49.1 -46.5 -37.7 -25.7 -COOH (mmol/g) 36.2 32.7 15.8 12.8

[3]

[4]