Biodegradable thermoresponsive polymeric magnetic nanoparticles: a ...

J Nanopart Res (2011) 13:1677–1688 DOI 10.1007/s11051-010-9921-6

RESEARCH PAPER

Biodegradable thermoresponsive polymeric magnetic nanoparticles: a new drug delivery platform for doxorubicin Nidhi Andhariya • Bhupendra Chudasama R. V. Mehta • R. V. Upadhyay

•

Received: 29 October 2009 / Accepted: 2 April 2010 / Published online: 20 April 2010 Ó Springer Science+Business Media B.V. 2010

Abstract The use of nanoparticles as drug delivery systems for anticancer therapeutics has great potential to revolutionize the future of cancer therapy. The aim of this study is to construct a novel drug delivery platform comprising a magnetic core and biodegradable thermoresponsive shell of tri-block-copolymer. Oleic acid-coated Fe3O4 nanoparticles and hydrophilic anticancer drug ‘‘doxorubicin’’ are encapsulated with PEO–PLGA–PEO (polyethylene oxide–poly D, L lactide-co-glycolide–polyethylene oxide) tri-blockcopolymer. Structural, magnetic, and physical properties of Fe3O4 core are determined by X-ray diffraction, vibrating sample magnetometer, and transmission electron microscopy techniques, respectively. The hydrodynamic size of composite nanoparticles is determined by dynamic light scattering and is found to be *36.4 nm at 25 °C. The functionalization of magnetic core with various polymeric chain molecules N. Andhariya R. V. Mehta Department of Physics, Bhavnagar University, Bhavnagar 364022, India B. Chudasama (&) School of Physics & Materials Science, Thapar University, Patiala 147004, India e-mail: [email protected]; [email protected] R. V. Upadhyay P.D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Education Campus, Changa 388421, India

and their weight proportions are determined by Fourier transform infrared spectroscopy and thermogravimetric analysis, respectively. Encapsulation of doxorubicin into the polymeric magnetic nanoparticles, its loading efficiency, and kinetics of drug release are investigated by UV–vis spectroscopy. The loading efficiency of drug is 89% with a rapid release for the initial 7 h followed by the sustained release over a period of 36 h. The release of drug is envisaged to occur in response to the physiological temperature by deswelling of thermoresponsive PEO–PLGA–PEO block-copolymer. This study demonstrates that temperature can be exploited successfully as an external parameter to control the release of drug. Keywords Biocompatible materials Encapsulation Drug delivery IR spectroscopy Thermoresponsive polymer Cancer therapy Nanomedicine

Introduction Precise targeting of drug to diseased cells or locations within organs defines the ‘‘magic bullet’’ in medical therapy but has not been achieved by current drug delivery methods (Mann 1999). Targeting occurs when the drug or drug vehicle can identify a specific molecular feature in the diseased area (Vasir and Labhasetwar 2005). It has long been recognized that there is great difficulty in constructing a chemical

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sensor that can discriminate the target cells from all other cells in the body. One way to circumvent the problem or reduce the importance of chemical specificity while improving targeting efficiency is to physically manipulate the position of the drug (Lu¨bbe et al. 2001; Vasir and Labhasetwar 2005). Accurate drug delivery can avoid high concentration intake of drug and thus decreases the possibilities of side effects (Alexiou et al. 2000; Lu¨bbe et al. 1996). The introduction of superparamagnetic nanoparticles in the 1970s produced a novel transport vehicle for the drug delivery, which can be manipulated with external magnetic field. A number of studies have focused on systemic delivery of nanoparticles carrying drugs and radioisotopes to specific sites in the body (Gu et al. 2005; Jurgons et al. 2006; Koneracka et al. 2008; Mohapatra et al. 2007; Saiyed et al. 2003; Sun et al. 2006; Zhang et al. 2007). For systematic delivery of drugs, it is required that the nanoparticles circulate for longer time in blood without occluding arteries, veins, or capillaries, and the physical components must be non-toxic, biocompatible, biodegradable, and acceptable for human medication in all other aspects (Arshady et al. 1999). In designing drug delivery systems for cancer therapy, it is preferred to opt for composite nanoparticles that are characterized by a functionalized magnetic core and a biodegradable polymer shell because the uncovered anticancer drugs can lead to possible side effects during the delivery of the drugs (Gupta and Curtis 2004; Gupta and Wells 2004; Kohaler et al. 2004; Yu et al. 2006). However, most of the studies found in the literature (Gu et al. 2005; Jurgons et al. 2006; Koneracka et al. 2008; Mohapatra et al. 2007; Saiyed et al. 2003; Sun et al. 2006; Zhang et al. 2007) failed to fulfill these criteria. The objective of the work presented here is to design and synthesize a drug delivery platform, which fulfills all above-outlined standards. Superparamagnetic Fe3O4 nanoparticles coated with a monolayer of oleic acid constitute the core of the composite nanoparticles. The molecules in the blood that are responsible for enhancing the uptake of particles by the reticuloendothelial system (RES) prefer to associate with hydrophobic surfaces. Accordingly, we have tackled this challenge by modifying the particles with a hydrophilic coating of poloxamer (pluronic F-127), which has a proven ability to mask recognition by the RES (Jain et al. 2005). To avoid the uncontrolled release of drug from the system while they are transported to the desired site (Zhang and

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Misra 2007), we have encapsulated the functionalized core along with the model drug doxorubicin into the rapidly biodegradable, thermoresponsive, blockcopolymer (PEO–PLGA–PEO). Composite nanoparticles are characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), UV–vis spectroscopy, and vibrating sample magnetometer (VSM). Drug loading efficiency and kinetics of drug release has also been investigated in the presence and absence of static magnetic field at elevated temperatures. The unique and novel carrier combines the stimuli-responsive and highly biodegradable properties of the smart block-copolymer with the advantages of magnetic nanoparticles, having potential applications in a magnetic drug delivery system for controlled release of anticancer drugs.

Materials and methods Materials FeCl3 (97%), FeCl24H2O (99%), pluronic F-127, Tris(hydroxymethyl) aminomethane, and PLGA (Poly D, L-lactide-co-glycolide) whose copolymer ratio of D, L-lactide to glycolide is 85:15 were obtained from Sigma-Aldrich. Doxorubicin hydrochloride (DOX–HCl) was purchased from Dabur Pharma Ltd. Ammonium hydroxide solution (25% NH3), HPLC grade water and acetone were procured from Merck. Analytical grade oleic acid was purchased from S.d. fine chem. Ltd. All chemicals were used as received without purification. Synthesis of Fe3O4 nanoparticles Fe3O4 nanoparticles were synthesized by chemical coprecipitation technique (Mehta et al. 1997; Sastry et al. 1995). Stoichiometric aqueous solutions of ferric (40 mM) and ferrous (20 mM) salts were prepared from their chlorides and homogenized. In this mixture, 0.2 M ammonium hydroxide solution was added drop wise under continuous stirring. Black precipitates form immediately. The pH of the solution was maintained at 10.5. After continuously stirring for 20 min at room temperature, the black precipitates of Fe3O4 were magnetically decanted and washed several times with

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warm distilled water. Finally water-wet slurry of nanoparticles was equally divided into two parts. One part was kept as control (Fe3O4), which was dried at room temperature after several acetone washes. Chemical synthesis of oleic acid-coated magnetic nanoparticles Magnetic nanoparticles have large surface to volume ratio. They have a tendency to agglomerate in order to minimize their surface energy. This leads to large size clusters with deteriorated magnetic properties. These agglomerated nanoparticles cannot be used in magnetic drug targeting as clustering destroys the superparamagnetic properties. Therefore, the magnetic properties of these agglomerated nanoparticles cannot be switched ON and OFF with the help of external magnetic field. Steric repulsion is usually used to avoid the agglomeration (Jain et al. 2005). We have used monolayer coating of short chain length fatty acid (oleic acid) to produce sterically stabilized Fe3O4 nanoparticles. In short, 0.72 mM oleic acid was added to 20 mL ammoniated water to form ammonium oleate. This was added to water-wet particles’ slurry and stirred at room temperature for 1 h followed by heating at 93°C. The mixture was cooled to room temperature and centrifuged at 12,000 rpm for 10 min. Excess oleic acid was removed by discarding the supernatants. Oleic acidcoated Fe3O4 (OA–Fe3O4) particles were extracted by adding 30 mL HPLC grade water. The water-wet particles’ slurry was equally divided into two parts. One part was kept for further processing, while the other was flocculated with dilute HCl followed by acetone washes. These OA–Fe3O4 particles were dried at room temperature. Surface modification of OA–Fe3O4 nanoparticles with pluronic F-127 Oleic acid prevents the agglomeration of nanoparticles, but made them hydrophobic. For use in biomedical applications, nanoparticles should be hydrophilic in nature, as it can prevent the absorption of plasma proteins in blood (Gupta and Gupta 2005; Jain et al. 2005). Hydrophobic OA–Fe3O4 nanoparticles were converted into the hydrophilic nanoparticles by using thermoreversible poloxamer, pluronic F-127. Under

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continuous stirring, pluronic F-127 was added to water-wet slurry of OA–Fe3O4 in the ratio of 1:1 w/w. The mixture was stirred at room temperature for 3 h. Pluronic stabilized OA–Fe3O4 (PLU–OA–Fe3O4) fluid was preserved at 4 °C for further process. Encapsulation of doxorubicin and PLU–OA– Fe3O4 nanoparticles into smart polymer Magnetic nanoparticles are generally coated with hydrophilic polymers such as starch or dextran, and the therapeutic agent is either chemically conjugated or ionically bound to the outer layer of polymer (Alexiou et al. 2000; Bergemann et al. 1999; Koneracka et al. 1999, 2002; Mehta et al. 1997). This approach is complex, involves multiple steps and usually results in limited drug loading capacity (Jain et al. 2005). In the present investigation, we have developed emulsion–precipitation process to encapsulate anticancer drug doxorubicin into biocompatible and biodegradable block-copolymer PEO–PLGA–PEO. Hydrophilic–hydrophilic interaction between doxorubicin and PEO blocks of pluronic F-127 acts as a driving force for grafting of doxorubicin into the nanocavities formed by the block-copolymer. 4 mg DOX– HCl was dissolved in Tris(hydroxymethyl) aminomethane buffer (pH *7) by keeping the drug concentration as 1 mg/mL. The aqueous phase was prepared by adding 1.8 mL of PLU–OA–Fe3O4 fluid into the drug solution. 40 mg PLGA was dissolved in 30 mL acetone to prepare the organic phase. Under continuous stirring, aqueous phase was added drop wise into the organic phase. The emulsion was covered with a perforated aluminum foil and stirred at room temperature until the organic phase evaporated completely. Drug-loaded nanoparticles were separated from the unentrapped drug using a permanent magnet. After that, they were washed with HPLC grade water till the absorption at 480 nm was detected in the absorption spectrum of the supernatant. The supernatant was collected and preserved at 4 °C to determine the drug content and loading efficiency. Drug-loaded PLGA nanoparticles (PLGA–DOX–PLU–OA–Fe3O4) were also preserved at 4 °C for subsequent analysis. Following the same procedure PLGA–PLU–OA–Fe3O4 nanoparticles were prepared without incorporating the drug.

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Investigation of nanoparticles Structural characterization of Fe3O4 nanoparticles was carried out by recording their powder XRD pattern. The pattern was recorded on Bruker’s D8 Advance X-ray diffractometer at room temperature using monochromatic radiation of CuKa (k = 0.15406 nm) and analyzed by Rietveld refinement technique. TEM images were obtained on Philips CM200 transmission electron microscope operated at 200 kV. For this purpose, a fine drop of Fe3O4 nanoparticles dispersed in ethanol was placed on a carbon-coated copper grid and ethanol was allowed to evaporate slowly at room temperature. Hydrodynamic particle size and its distribution of core and composite nanoparticles were determined on Beckman Coulter’s particle size analyzer (model Delsa Nano C). The functionalization of nanoparticles by oleic acid, pluronic F-127, and PLGA was confirmed by FTIR. The FTIR spectra of samples Fe3O4, OA–Fe3O4, PLGA–PLU–OA–Fe3O4, and pluronic F-127 were recorded on Spectrum GX (Perkin Elmer) single beam spectrophotometer. The measurements were carried out in 4,000–400 cm-1 spectrum range using KBr pellet method. TGA of Fe3O4, OA– Fe3O4, and PLGA–PLU–OA–Fe3O4 were performed on Mettler Toledo TGA/SDTA 851e system in the temperature range of 30–800 °C at a heating rate of 10 °C/min in nitrogen environment. Magnetic properties of bare and functionalized nanoparticles were investigated on indigenously built VSM. The measurements were carried out at room temperature in the field range of -318 to ?318 kA m-1. UV–vis spectra of aqueous solution of doxorubicin and PLGA–DOX– PLU–OA–Fe3O4 nanoparticles were recorded at room temperature on Elico’s biospectrophotometer (model no. BL198).

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Drug content ¼

Weight of drug in nanoparticles Total weight of nanoparticles 100; ð1Þ

Drug loading efficiency ¼ ½ðW1 W2Þ=W1 100; ð2Þ where W1 is the total weight of drug and W2 is the weight of free drug, which did not incorporated into the nanoparticles. Kinetics of drug release A method similar to drug loading capacity was established to study kinetics and response of drug release from the drug-conjugated nanoparticles. The in vitro release of drug from the nanoparticles was studied under the sink condition, using double diffusion chambers separated by a membrane (Durapore, Millipore) with a porosity of 10 nm. A suspension of drugloaded nanoparticles in PBS buffer (pH *7.4) was placed in the donor chamber and pure PBS in the receiver chamber. The chambers were placed in an orbital shaker (NOVA). The shaker was maintained at elevated temperatures (30, 37, 45, and 50 °C) with a shaking speed of 100 rpm. At different time intervals, the entire volume of the receiver chamber was removed and replaced with fresh PBS. The amount of drug release was determined by measuring the optical density of sample extracted from the receiver chamber. Identical experiments were performed in the presence of static magnetic field (477 kA m-1). A permanent slab magnet was used to produce static magnetic field.

Results and discussion Drug content and drug loading efficiency Structure of PLGA–DOX–PLU–OA–Fe3O4 Optical density of collected supernatant was measured at 480 nm using UV–vis spectrophotometer. The concentration of doxorubicin that remained unincorporated into the polymeric nanoparticles was determined by Beer–Lambert law, A = ecl, where A is the optical density at sample concentration c, l the path length of sample cell, and e is molar absorptivity. The drug content and loading efficiency were determined using the following equations:

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Schematic representation of the proposed structure of PLGA–DOX–PLU–OA–Fe3O4 nanoparticles is shown in Fig. 1. The structure was deduced from the analysis of physical and chemical characterization of nanostructures. Fe3O4 nanoparticles constitute the core of composite nanostructures, which are coated with monolayer of oleic acid. Coating of oleic acid renders them hydrophobic. Polypropylene oxide (PPO) chains of

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Oleic acid Pluronic F-127 Fe3O4 Doxorubicin PLGA

Fig. 1 Schematic representation of structure of PLGA–DOX– PLU–OA–Fe3O4 magnetic polymeric nanoparticles (311)

400

Yobs Ycal Yobs-Ycal

Intensity (cps)

300

(440)

(220)

200 (511)

(400) (422)

100 0 -100 20

30

40

50

60

70

2θ Fig. 2 Refined XRD spectrum of as-synthesized Fe3O4 nanoparticles. All the reflections are indexed with inverse spinel structure

pluronic F-127 encroached at the tail of OA and hydrophilic part (PEO) remained unattached, which keeps them dispersible in water. The PEO block of pluronic F-127 makes a covalent bond with the PLGA and forms a tri-block-copolymer PEO–PLGA–PEO. Hydrophilic interaction between doxorubicin and PEO blocks of pluronic provides the driving force for its encapsulation in the pores of tri-block-copolymers. Since the composite nanostructures of PLGA–DOX– PLU–OA–Fe3O4 are hydrophilic (dispersible in water), we believe that water loving chains (PEO) of pluronic F127 are only partially entrapped into the block-copolymer matrix. The unentrapped part of PEOs keeps the composite nanoparticles dispersible in water. Investigation of nanoparticles X-ray diffraction spectrum of as-synthesized Fe3O4 nanoparticles is shown in Fig. 2. The spectrum is refined

using Rietveld refinement technique. All the reflections present in the spectrum are in well agreement with the inverse spinel structure. The value of refined lattice ˚ . The line broadening of XRD peaks parameter is 8.24 A is primarily due to the small size of particles. The average crystallite size obtained by classical Scherrer equation (Cullity 1978) with a geometric factor of 0.9 over (311) reflection is 10.7 nm. A transmission electron micrograph of Fe3O4 nanoparticles and the corresponding SAED (specific area electron diffraction) pattern are presented in Fig. 3. The micrograph suggests that the Fe3O4 nanoparticles are characterized by near spherical morphology. The average particle size is found to be 11.1 ± 2.7 nm. In the SAED pattern, the diffraction rings are relatively broad because of the smaller size of the particle. Using dynamic light scattering, hydrodynamic size and size distribution of Fe3O4, OA–Fe3O4, PLU–OA– Fe3O4, and PLGA–DOX–PLU–OA–Fe3O4 are determined at room temperature (25 °C). The size distribution histograms are shown in Fig. 4. The average hydrodynamic size and polydispersity index of each system are presented in Table 1. Hydrodynamic size of OA–Fe3O4 nanoparticles is 12.8 nm and that of bare Fe3O4 is 10.7 nm. Therefore, the increase in the hydrodynamic size due to the oleic acid coating is 2.1 nm, which corresponds to monolayer coating of oleic acid (Wooding et al. 1992). Further, functionalization of Fe3O4 nanoparticles with oleic acid and pluronic has no significant effect on the polydispersity index, which suggests that individual Fe3O4 nanoparticles are coated with a primary monolayer of oleic acid and a secondary monolayer of pluronic F127. The hydrodynamic size of composite nanostructures, i.e., PLGA–DOX–PLU–OA–Fe3O4 is found to be 36.3 nm (identical to PLU–OA–Fe3O4 nanoparticles), which further support our claim that PEO chains are only partially entrapped into the tri-blockcopolymer (Fig. 1). In order to evaluate the effect of temperature on thermoresponse of drug-loaded tri-block-copolymers, DLS experiments were performed at elevated temperatures (from 20 to 55 °C). Results are presented in Fig. 5 and summarized in Table 2. Thermoresponse of PLGA–DOX–PLU–OA–Fe3O4 nanoparticles is clearly evidenced by swelling and deswelling of nanostructures below and above a lower critical solution temperature (LCST). For PLGA–DOX–PLU–OA–Fe3O4 nanostructures, this transition temperature is found to be

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Fig. 3 Transmission electron micrograph of assynthesized Fe3O4 nanoparticles. Inset shows the specific area electron diffraction (SAED) pattern of nanoparticles

45–50 °C. The observed behavior of nanostructures is due to the thermoresponse of the tri-block-copolymer PEO–PLGA–PEO. Temperature induced sol to gel transition was also observed by Kwon et al. (2002) in these tri-block-copolymer. The volume of nanostructures was contracted by 25% at 45 °C and the contraction reaches to 44% at 55 °C. This property of nanostructures will be utilized to release the drug from the drug delivery system. Fourier transform infrared spectroscopy is an appropriate technique to understand chemical adsorption or functionalization of nanoparticles with polymers. The FTIR absorption spectra of Fe3O4, OA– Fe3O4, PLGA–PLU–OA–Fe3O4, and pluronic F-127 are presented in Fig. 6, and analyzed in Table 3. Only the salient features of each spectrum are described here. The characteristic absorption bands at 440, 580, and 620 cm-1 in the FTIR spectra of Fe3O4 (Fig. 6a) are attributed to Fe–O. Bands at 580 and 620 cm-1 are due to the splitting of the absorption band observed at 570 cm-1 in the spectrum of bulk Fe3O4. Similarly, the band at 440 cm-1 originates from another absorption band of Fe–O of bulk Fe3O4 located at 375 cm-1. The results are consistent with those observed by Zhang et al. (2007, 2008). In the case of OA–Fe3O4 nanoparticles (Fig. 6b), a careful comparison and analysis of FTIR spectra of as-synthesized Fe3O4 and OA–Fe3O4 nanoparticles

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suggest that oleic acid is coated on the surface of Fe3O4. The presence of absorption bands centered at 1,428 and 1,521 cm-1 are due to the symmetric and asymmetric stretching vibrations of –CH3. The absence of absorption band at 1,705 cm-1, which is attributed to the C=O stretch, suggest that carboxylic group of OA is bound with Fe3O4. Further, peaks centered at 2,854 and 2,924 cm-1 are attributed to the symmetric and asymmetric stretch of C–H, respectively. A strong and broad peak at 3,439 cm-1 suggests chemisorption of OA on Fe3O4 nanoparticles. The suppression of OH vibrational mode in the 3,000–3,700 cm-1 region has been related to evidence of host–guest interaction as consequence of water release upon chemisorption of OA. The FTIR spectra of PLGA–PLU–OA–Fe3O4 and pluronic F-127 are shown in Fig. 6c and d, respectively. The presence of absorption band at 961 and 1109 cm-1 in Fig. 6c is due to the CH2 rocking and C– O–C stretching vibrations of pluronic F-127. The sharp absorption band observed at 1,757 cm-1 attributed to the C=O stretch of PLGA, confirms the formation of tri-block-copolymer. Further, the concentration of pluronic used in the present formulation is below its critical micelle concentration (CMC), therefore, it is possible that pluronic could have been encroached at the interface of OA-coated nanoparticles in the form of a layered deposit rather than as micelles.

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1683

40

a

6

d 30

3

20

0 6

b

3

0 40

20

30

40

Differential volume (%)

10

50

c

30

Frequency (% )

20 10 0 40

20

30

40

c

3 0 6

d

3 0 6

e

3 0 9 6 3 0 12 9 6 3 0

50

b 30 20 10 0 40

0 6

f

g

20

10

12

14

16

40

30

60

80 100 120 140 160 180 200

Particle Size (nm)

a h

20 10 0 8

10

12

14

Size (nm) Fig. 4 Size distribution histogram of a as-synthesized Fe3O4, b oleic acid-coated Fe3O4 nanoparticles (OA–Fe3O4), c pluronic functionalized oleic acid coated Fe3O4 nanoparticles (PLU–OA–Fe3O4), and d doxorubicin-loaded nanoparticles (PLGA–DOX–PLU–OA–Fe3O4)

Table 1 Mean hydrodynamic size and polydispersity index of bare and functionalized Fe3O4 nanoparticles System

Mean hydrodynamic size (nm)

Polydispersity index

Fe3O4

10.7

0.31

OA–Fe3O4

12.8

0.27

PLU–OA–Fe3O4

36.1

0.25

PLGA–DOX–PLU– OA–Fe3O4

36.39

0.25

T < LCST

T > LCST

Fig. 5 Size distribution histogram of PLGA–DOX–PLU–OA– Fe3O4 nanoparticles obtained from DLS at a 20 °C, b 25 °C, c 30 °C, d 37 °C, e 45 °C, f 50 °C, g 55 °C, and h schematic representation of structure of PLGA–DOX–PLU–OA–Fe3O4 below and above the LCST (45 °C)

Thermogravimetric analysis is an appropriate technique to determine the weight proportion of individual components in composite polymeric nanoparticles. TGA thermogram of as-synthesized Fe3O4 (Fig. 7a) shows only 1.8% weight loss in the temperature range of 30–190 °C is due to the loss of physical water from the surface of nanoparticles. Additional 3.2% weight loss observed in the second step (190–360 °C) can be attributed to desorption of

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Table 2 Mean hydrodynamic size of PLGA–DOX–PLU–OA– Fe3O4 nanoparticles at elevated temperatures

Table 3 Assignment of FTIR spectra of different samples presented in Fig. 6

Temperature (°C)

Size (nm)

System

20

34.5

25

36.4

Fe3O4

IR bands (cm-1)

Description

440

Absorption band of Fe–O

41.1

580


37

44.5

620


45

33

50

26.5

55

24.3

30

3,402 OA–Fe3O4

d

PLGA–PLU– OA–Fe3O4


569


623


1,428

ms (–CH3)

1,521

mas (–CH3)

2,854

ms (–CH)

2,924 3,439

mas (–CH) Absorption band due to chemisorbtion of OA onto Fe3O4

437


590


627


961

CH2 rocking vibration of Pluronic F-127

c

%T

1,109

b

Pluronic F127

a

4000

3000

2000

Wavenumber

1000

0

(cm-1)

Fig. 6 FTIR spectrum of a as-synthesized Fe3O4, b OA– Fe3O4, c PLU–OA–Fe3O4, and d PLGA–PLU–OA–Fe3O4

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–OH vibrations

437

C–O-C stretching vibration of Pluronic F-127

1,188

CH2 twist

1,278

CH2 wag

1,347

CH2 wag

1,458

ms (–CH3)

1,757

C=O stretch band of PLGA

2,893

ms (–CH)

2,926

mas (–CH)

3,437

Absorption band due to chemisorbtion of OA onto Fe3O4 CH2 rocking vibration

961 1,111

C–O–C stretching vibration

1,243

CH2 twist

1,283

CH2 twist

1,346

CH2 wag

2,887

ms (–CH)

2,972

mas (–CH)

chemically absorbed water. A gradual and steady negligible weight loss of 0.9% from 360 to 800 °C is due to the release of trapped impurities. Thermogravimetric analysis thermogram of OA– Fe3O4 nanoparticles is shown in Fig. 7b. 1.4% weight loss observed in the first step (30–190 °C) is due to

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a

Weight (%)

80

b

60

40

c

20 150

300

450

600

750

T (°C) Fig. 7 Typical thermogram of a as-synthesized Fe3O4, b OA– Fe3O4, and c PLGA–PLU–OA–Fe3O4

the loss of surface hydroxyl groups. A sharp weight loss of 17.7% from 190 to 400 °C confirms the formation of oleic acid monolayer on Fe3O4 nanoparticles. The observed weight loss is in good agreement with those reported by Jain et al. (2005) and Fu et al. (2001) for the monolayer coating of oleic acid on Fe3O4 nanoparticles. Out of 24% oleic acid used as a starting composition, 6.3% remain unattached as excess oleic acid and removed during the washing cycles. The mass loss spectrum of PLGA–PLU–OA– Fe3O4 is presented in Fig. 7c. The entire thermogram is divided into three distinct steps. The first step commences from 30 °C and last up to 200 °C. The 1.65% weight loss in this step is attributed to the loss of surface water. In the second step (200–332 °C), the observed weight loss of 49.3% is due to the simultaneous decomposition of oleic acid and

pluronic. However, from the TGA of OA–Fe3O4, it is known that 17.7% weight loss is due to the monolayer coating of oleic acid. Hence, the remaining 31.6% weight loss in the second step is due to the encroachment of pluronic. In the third and final step (332–800 °C), 24.7% weight loss is observed. This loss corresponds to the decomposition of PLGA. At 800 °C, the residual weight of the sample is 24.6%, which corresponds to the core mass of Fe3O4 in PLGA–PLU–OA–Fe3O4. The results are summarized in Table 4 and mass losses in the thermogram are in good agreement with the starting compositions. The encapsulation of doxorubicin in tri-blockcopolymer is confirmed by UV–vis spectroscopy. Figure 8 shows the absorption spectra of aqueous solution of doxorubicin and PLGA–DOX–PLU–OA–

0.14 0.12 0.10

OD

100

a

0.08 0.06

b

0.04 0.02 0.00

350

400

450

500

550

600

650

Wavelength (nm) Fig. 8 UV–visible absorption spectrum of aqueous solution of a doxorubicin and b PLGA–DOX–PLU–OA–Fe3O4 nanoparticles. The spectrum was recorded at 25 °C with HPLC grade water as reference

Table 4 Thermogravimetric analysis of bare and functionalized Fe3O4 nanoparticles System

Step

Temperature range (°C)

% Weight loss Observed

Fe3O4

OA–Fe3O4 PLGA–PLU–OA–Fe3O4

Calculated

I

30–190

1.80

–

II

190–360

3.20

–

III

360–800

0.90

–

I

30–190

1.40

–

II

190–400

I

30–200

II

200–332

49.3

51.0

III

332–800

24.6

21.8

17.7 1.65

24.0 –

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a b

Fe 3O4 Fe 3O4OA

40

M (emu/g)

Drug content and drug loading efficiency of PLGA– PLU–OA–Fe3O4 nanoparticles are determined using Eqs. 1 and 2, respectively. For the present system, the calculated value of drug content is 9.1% and the experimental value is 8.1%. The drug loading efficiency is found to be 89%. The observed value of drug loading efficiency is much higher than that obtained with conventional polymers like, PEG (50%) and dextran (51%) (Zhang et al. 2008; Xu et al. 2005).

Fe 3O4OAPLUPLGA

20

c

0 -20 -40 -60 -400 -300 -200 -100

0

100

200

300

400

H (kA/m) Fig. 9 Magnetization curves of a as-synthesized Fe3O4 nanoparticles, b OA–Fe3O4 nanoparticles, and c PLGA– PLU–OA–Fe3O4

Fe3O4 nanoparticles. The peak observed at 470 nm in the absorption spectrum of PLGA–DOX–PLU–OA– Fe3O4 is the characteristic peak of doxorubicin. The same peak was observed at 480 nm in the free solution of doxorubicin (Bertin et al. 2005). The blue shift in the absorption maximum confirms the encapsulation of doxorubicin in tri-block-copolymer. Magnetic properties of as-synthesized Fe3O4, OA– Fe3O4, and PLGA–PLU–OA–Fe3O4 nanoparticles are shown in Fig. 9. The saturation magnetization (Ms) of Fe3O4, OA–Fe3O4, and PLGA–PLU–OA–Fe3O4 are found to be 64, 54, and 16.4 emu g-1, respectively. The Ms value of as-synthesized Fe3O4 is 70% of its bulk value (92 emu g-1). This decrease in the Ms value might be due to the decrease in particle size accompanied by an increase in surface area, and is consistent with the results observed by Mohapatra et al. (2007). The Ms value of OA–Fe3O4 is 84% of as-synthesized Fe3O4. The 16% decrease in the Ms is due to the diamagnetic contribution of organic coating. The result is consistent with the 17.7% weight loss in TGA. The Ms value of PLGA–PLU– OA–Fe3O4 is 26% of the as-synthesized Fe3O4. Again, this is in good agreement with the 74% weight loss observed in the thermogram for the same system. All the three systems are superparamagnetic in nature as no remnant magnetic field and coersivity have been found. The M versus H curve of PLGA– DOX–PLU–OA–Fe3O4 is analogous to PLGA–PLU– OA–Fe3O4 hence, omitted from Fig. 9 for the sake of clarity.

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Drug content and drug loading efficiency

Kinetics of drug release The drug release profiles of doxorubicin from PLGA– DOX–PLU–OA–Fe3O4 nanoparticles at four temperatures (30, 37, 45, and 50 °C) are shown in Fig. 10. Measurements were carried out in the presence and absence of static magnetic field. The drug is continuously released from the system over a period of 36 h at all studied temperatures. No significant drug release has been observed in the presence or absence of static magnetic field, when the nanostructures are subject to temperatures (30 and 37 °C) that are below

H=477 kA/m

80 60

Cumulative drug release (%)

60

30°C 37°C

40

45°C 20

50°C

0

H=0 kA/m

80 60 40 20 0 0

5

10

15

20

25

30

35

40

Time (h) Fig. 10 Drug release profile of PLGA–DOX–PLU–OA–Fe3O4 nanoparticles in the absence and presence of static magnetic field (477 kA/m) at T = 30, 37, 45, and 50 °C

J Nanopart Res (2011) 13:1677–1688

the LCST of tri-block-copolymer PEO–PLGA–PEO. At these temperatures, the diffusion process (Klose et al. 2006) solely governs the kinetics of drug release. Burst release of doxorubicin is observed in both the absence and presence of magnetic field for the initial 7 h. After 7 h, 39% of the accumulated drug is released at 45 °C in both the absence and presence of external magnetic field. Similar, behavior of burst release is observed at 50 °C. In this case, slightly higher value (45%) of drug is released again in both the presence and absence of static magnetic field. The rapid release of doxorubicin from the polymeric magnetic nanoparticles at 45 and 50 °C can be attributed to deswelling of tri-block-copolymer PEO– PLGA–PEO at LCST (Shameem et al. 1999). At this temperature (as observed from the temperature dependent DLS measurements), volume of the polymeric nanostructures were contracted by 44%. This sharp thermal response of polymer leads to the rapid release of doxorubicin during the initial 7 h. A slow and controlled release of doxorubicin is further observed in the second stage of release profile. In this stage, release mechanism is partially governed by thermal response of tri-block-copolymer and diffusion of doxorubicin in the medium. At 45 °C, the amount of cumulative drug release is 72.5% in both the presence and absence of magnetic field after 36 h. The cumulative release in the absence of magnetic field increases to 77.7% and that in the presence of magnetic field is 74.6% when the temperature is raised to 50 °C. No significant effect of static magnetic field on the release behavior of doxorubicin has been observed at any temperature. Hence drug release is mainly governed by the thermal response of polymeric magnetic particles while, static magnetic field will be useful for targeting this composite nanostructures to the sight of action.

Conclusions In this study, we have designed and developed doxorubicin-loaded magnetic polymeric nanoparticles that can release the drug at a specified rate to take advantage of unique pharmacodynamic properties of doxorubicin. The drug loading efficiency is 89%, which is much higher than that obtained with conventional biocompatible polymers like PEG and

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dextran. The drug release is high and rapid for the initial 7 h followed by the sustained release over a period of 36 h. Better control on the release profile has been achieved with the help of temperature. The same carrier system can be loaded with multiple anticancer drugs to further improve its efficiency. Acknowledgments The work was carried out under Ramanna Fellowship Project awarded to Prof. R.V. Mehta by the Department of Science and Technology (DST), New Delhi. Authors are thankful to K. Padmanabhan, Beckman Coulter India Pvt Ltd. for his help in temperature dependent DLS measurements.

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