Biomaterials 31 (2010) 5588e5597
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Superparamagnetic iron oxide e Loaded poly (lactic acid)–D-a-tocopherol polyethylene glycol 1000 succinate copolymer nanoparticles as MRI contrast agent Chandrasekharan Prashant a, Maity Dipak b, Chang-Tong Yang c, Kai-Hsiang Chuang c, Ding Jun b, Si-Shen Feng a, d, e, * a
Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576 Department of Materials Science and Engineering, Faculty of Engineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117574 Laboratory of Molecular Imaging, Singapore Bioimaging Consortium, Agency for Science, Technology and Research Singapore, 11 Biopolis Way, #02-02 Helios, Singapore 138667 d Division of Bioengineering, Faculty of Engineering, National University of Singapore, Block E3A #04-15, 7 Engineering Drive 1, Singapore 117574 e NUS Nanoscience & Nanotechnology Initiative (NUSNNI), National University of Singapore, Blk E3-05-29, 2 Engineering Drive 3, Singapore 117581 b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2010 Accepted 26 March 2010 Available online 29 April 2010
We developed a strategy to formulate supraparamagnetic iron oxides (SPIOs) in nanoparticles (NPs) of biodegradable copolymer made up of poly(lactic acid) (PLA) and D-a-tocopherol polyethylene glycol 1000 succinate (TPGS) for medical imaging by magnetic resonance imaging (MRI) of high contrast and low side effects. The IOs-loaded PLAeTPGS NPs (IOs-PNPs) were prepared by the single emulsion method and the nanoprecipitation method. Effects of the process parameters such as the emulsifier concentration, IOs loading in the nanoparticles, and the solvent to non-solvent ratio on the IOs distribution within the polymeric matrix were investigated and the formulation was then optimized. The transmission electron microscopy (TEM) showed direct visual evidence for the well dispersed distribution of the IOs within the NPs. We further investigated the biocompatibility and cellular uptake of the IOs-PNPs in vitro with MCF-7 breast cancer cells and NIH-3T3 mouse fibroblast in close comparison with the commercial IOs imaging agent ResovistÒ. MRI imaging was further carried out to investigate the biodistribution of the IOs formulated in the IOs-PNPs, especially in the liver to understand the liver clearance process, which was also made in close comparison with ResovistÒ. We found that the PLAeTPGS NPs formulation at the clinically approved dose of 0.8 mg Fe/kg could be cleared within 24 h in comparison with several weeks for ResovistÒ. Xenograft tumor model MRI confirmed the advantages of the IOs-PNPs formulation versus ResovistÒ through the enhanced permeation and retention (EPR) effect of the tumor vasculature. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Biodegradable polymers Biocompatibility Cellular and molecular imaging Microencapsulation Nanoprecipitation Nanocomposite
1. Introduction Versatility in the use of supraparamagnetic iron oxide nanocrystals (SPIOs or IOs) has gained tremendous importance in the field of biomedical application. In cancer research, for example, IOs have been used for molecular imaging, tumor imaging, cancer hyperthermia therapy, and other techniques [1e4]. IOs have been extensively investigated as a contrast agent for MRI, which are easily assimilated by the human body [5e7]. IOs are primarily used
* Corresponding author. Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576. Tel.: þ65 6516 3835; fax: þ65 6779 1936. E-mail address:
[email protected] (S.-S. Feng). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.03.070
in MRI as liver signal erasers since they create a reduction in signal intensity in the reticuloendothelial tissues, in which they are greatly concentrated and thus result in greater T2 shortening, to produce more conspicuous area of liver not containing the reticuloendothelial tissues. However, long term retention of IOs in liver is a concern for the side effects of IOs imaging agent [8]. Several other side effects are also associated with administering of the IOs, which may create clinical complications [9]. Such problems could be addressed by two strategies. One is by coating of the IOs core and another is by formulating of the IOs in nanoparticles (NPs) of biodegradable polymers. Various coating techniques have been suggested to provide IOs with functionality of desired surface properties. For instance, thermally sensitive coatings provide the IOs with dual functionality for treatment of cancer by hyperthermia and through a release of the entrapped
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anti-cancer agent [10]. In another study, IOs with their surface coated with PEG and further linked to biotin or neutravidin along with a peptide enabled detection of specific cancer type that produces a proteolytic enzyme which enabled the peptide blocked biotin and neutravidin to self assemble and thus influences the T2 relaxivity in MRI. Such a coating strategy thus enables detection of the tumor as well as specification of the tumor type [11]. Moreover, coating IOs by hydrophilic or amphiphilic biodegradable polymer provides them with other functionality such as biocompatibility and long circulation in plasma. Formulation of the IOs in nanoparticles (NPs) can be realized by using various FDA-approved biodegradable polymers such as poly (lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) and various novel biodegradable copolymers such as poly(lactic acid)-poly (ethylene glycol) (PLEA) copolymer and poly(lactic acid)-D-atocopherol polyethylene glycol 1000 succinate (PLAeTPGS) copolymer [12,13,28]. Encapsulation by using preformed polymer can be done by several methods such as the micro/nanoemulsion method [14,15], the nanoprecipitation method [16,17] and the micelle formation [18e20]. Emulsion polymerization makes use of monomers that polymerize over the encapsulant, which is stabilized by using a surfactant [21e23], which is of great concern due to the presence of certain chemical precursors that were not removed and could have some cytotoxic effect [24]. The solvent displacement technique was used in the preparation of nanocapsules [24]. The nanocapsules were formed when the non-solvent replaces the solvent in the presence of a surfactant followed by deposition of the preformed polymer at the interface. The encapsulant was remained in an oily phase within the polymer layer. This process has been extended to the formation of nanospheres without the surfactant [16] and it has been gaining increasing importance over time for preparing nanoparticles. Various parameter optimization of this method has been done. For instance, it was suggested that the diffusion of the solvent into the non-solvent results in the phase transformation of the polymer and thus in the formation of the nanoparticles [25]. It was also shown how the nanoparticles formation could be affected by changing the concentration of the polymer in the organic phase or the concentration of the surfactant in the aqueous phase, which may influence the size of the formed nanoparticles [26]. Other research in the nanoprecipitation method included the choice of the solvent [27]. It was suggested that the solubility parameter of the solvent and the non-solvent system strongly influence the particle formation, in which the particle size was usually used as the primary criteria for optimization in the literature. In the present research, we developed a strategy to formulate IOs in the NPs of PLAeTPGS copolymer, for enhanced MRI of higher image contrast and less system side effects. The amphiphilic nature and enhanced biocompatibility of this copolymer make it promising for nanoparticle delivery of diagnostic and therapeutic agents [28,29]. We have exploited the different treatment between drug and the IOs in the nanoparticle encapsulation process. A crystalline drug after encapsulation could become in an amorphous state, resulting in approved solubility [30e32], whereas the IOs do not undergo a phase transition after encapsulation process. Rather these nanoparticles get entrapped within the polymeric matrix and retain their crystalline state. Thus diffusion of the solvent into the polymeric matrix plays a key role during the preparation of the nanoparticle encapsulation. Two commonly used methods for polymeric nanoparticle preparation, namely the single emulsion method and the nanoprecipitation method, are employed in this research for the nanoparticle preparation in close comparison with optimization of the various synthesis parameters such as the emulsifier concentration in the aqueous phase, solvent/non-solvent ratio, loading of IOs/ drug and more. The effect of synthesis parameters on the size and
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size distribution of the polymeric nanoparticles and the distribution of IOs within the polymeric matrix were studied by laser light scattering and transmission electron microscopy (TEM). The optimally synthesized nanoparticles were further investigated for in vitro cytotoxicity and cellular uptake of the IOs-loaded nanoparticles in close comparison with the commercial ResovistÒ. Due to their desired particles size and their long half-life in the plasma, the IOs-loaded PLAeTPGS nanoparticles should have the property to accumulate in regions of leaky and high permeable vasculature and lymphatic vessels such as those in tumor, which is also referred to as the enhanced permeability and retention (EPR). The nanoparticle formulation of IOs can thus be exploited for passively targeted delivery of the imaging agent for clinical MRI with better contrast and less side effects [33]. 2. Materials and methods 2.1. Materials Lactide (3,6-dimethyl-1,4-dioxane-2,5-dione, C6H8O4) was purchased from Aldrich, which was recrystallized twice from ethyl acetate before use. Vitamin E TPGS (D-a-tocopherol polyethylene glycol 1000 succinate, C33O5H54(CH2CH2O)23) was from Eastman chemical company (USA), which was freeze dried for two days before use. Stannous octoate (Sn(OOCC7H15)2) was purchased from Sigma and was used as 1% distilled toluene solution. Millipore water was prepared by a Milli-Q Plus System (Millipore Corporation, Breford, USA). Oleylamine (OM, 70%), Oleic acid (OA, 90%), Iron(III) acetylacetonate (Fe(acac)3, 97%) and Nitric acid (69%) were purchased from SigmaeAldrich. All chemicals including absolute ethanol, dichloromethane (DCM), tetrahydrofuran (THF) and ethyl acetate were HPLC grade. They were used without further purification. 2.2. Synthesis of PLAeTPGS polymer The PLAeTPGS copolymer of 90:10 PLA:TPGS w/w ratio was synthesized by a process discussed in our earlier publication [28]. In brief, weighted amounts of lactide, TPGS and 0.5 wt% stannous octoate (in distilled toluene) were added in an ampoule. The ampoule was evacuated in liquid nitrogen for 45 min. After that the ampoule was sealed by butane burner and reacted in silicone oil bath at 145 C. After 12 h, the reaction product was dissolved in DCM and then precipitated in excess cold methanol to remove unreacted lactide monomers and TPGS. The final product was collected by filtration and vacuum dried at 45 C for two days. The product formed were analyzed and confirmed by FTIR and 1H NMR at 500 Hz (Bruker AMX500). The weight-average molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC, Waters GPC analysis system with RI-G1362A refractive index detector). 2.3. Synthesis of IOs Iron oxide nanoparticles (IOs) were prepared by thermal decomposition method of Fe(acac)3 as done earlier [34]. In brief, 2 mmol of Fe(acac)3 was dissolved in a 20 ml surfactant mixture of oleic acid (OA) and oleylamine (OM) and magnetically stirred under a flow of argon. The solution was dehydrated at 120 C for 1 h, and then quickly heated to 300 C temperature and kept at this temperature for 1 h. The black solution was cooled to room temperature by removing the heat source. A total of 20 ml of ethanol was added into the solution and the precipitated IOs were collected by centrifugation at 10,000 rpm, followed by washing with ethanol three times. The as-prepared IOs were dispersed in THF. The iron content and coating was confirmed by using inductively coupled plasma-mass spectrometry (ICP-MS). 2.4. Preparation of IOs-encapsulated polymeric nanoparticles IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs) were prepared by two methods, namely the single emulsion solvent extraction/evaporation method and the nanoprecipitation method. In the single emulsion method, 100 mg of the polymer along with known amount of IOs and 8 ml of DCM were added and the solution was vortexed till the polymer is fully dissolved in the organic solvent and a clear solution is obtained. The organic phase was then added into 120 ml of aqueous phase containing the emulsifier at a given concentration (TPGS of 15, 5 and 1% w/v) and a probe sonicator was used to disperse the emulsion for 90 s. The solution was allowed to stir overnight to remove the solvent. In the nanoprecipitation method, 100 mg of the polymer along with known amount of IOs and 8 ml of THF were added and vortexed till the polymer was fully dissolved in the organic solvent and a clear solution is obtained. 30 ml of aqueous phase containing a known concentration of the emulsifier (TPGS of 15, 5 and 1% w/v) was well dispersed using a probe sonicator for 30 s. The organic phase was then added into the aqueous phase. The solution was vigorously stirred till uniformity and
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diluted with large amount of water. The solution was then allowed to be stirred overnight to evaporate the organic solvent. The nanoparticles were obtained after centrifugation at 10,000 rpm for 15 min. The duration of centrifugation was increased for samples with higher concentration of the emulsifier. Several washes were done to remove the emulsifier. The nanoparticles were then characterized for distribution of IOs within the polymer matrix by using TEM (JEM 2010F, JEOL, Japan) and for surface morphology by using FESEM (JEOL, JSM-6700F, Japan) as well as the size and size distribution by using Zetasizer (Nano ZS, Malvern Instruments Ltd, UK). 2.5. X-ray photoelectron spectroscopy (XPS) XPS was carried out for qualitative determination of the iron content and the carbon content on the surface of the IOs-PNPs. A small amount of the dry nanoparticle powder was analyzed using XPS (AXIS His-165 Ultra, Kratos Analytical, Shimadzu, Japan). Data were analyzed with the software provided by the manufacturer. 2.6. ICP-MS analysis A known amount of IOs-PNPs were put in a glass test tube. 2 ml of concentrated nitric acid was added. The tube was then heated to 110 C for 45 min and the samples were analyzed using ICP-MS (Agilent ICP-MS 7500 Series) after sufficient dilution with milli-Q water. The analysis of sample was done in comparison with the ICP-MS standard (Sigma). 2.7. Magnetic property of IOs-PNPs The superparamagnetic properties of the nanoparticle formulation of IOs were investigated by the vibrating sample magnetometer (VSM, Lakeshore 7300 Series, USA).
2.10. MRI Transverse relaxivity measurement and in vivo MRI were conducted on a Varian 9.4T MRI system (Palo Alto, CA, USA). 2.10.1. Calculation of relaxivity The transverse relaxation time T2 of the IOs-PNPs were measured in 1% Agarose phantoms with concentration from 0.4 to 0.0066 mM [Fe], using multiple spin echo sequence at TR ¼ 10,000 ms and TE ¼ 5.69 ms. Transverse relaxivity (r2 in mM1 s1) were obtained from linear least-squares determination of the slopes of 1/T2 versus the concentration of [Fe] plots. The r2* measured by multiple gradient echo sequence with same concentrations with those measured by T2. The longitudinal relaxation time (T1) of the IOs-PNPs were measured by using inversion recovery spin echo sequence. 2.10.2. In vivo MRI study In vivo study was conducted on Wistar rats (male, weight 320e340 g) under 2% isoflurane anesthesia using 72 mm volume coil. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC), National University of Singapore (#802/05(A10)09). The polymeric contrast agents were injected at a dosage of 0.8 mg [Fe]/kg body weight through tail vein. T2-weighted images were acquired every 48 s for 32 min with T2-weighted fast spin echo sequence (TR/ TE ¼ 1000/28.24 ms, flip angle ¼ 90 , field of view ¼ 8 cm, thickness ¼ 2 mm, intersection gap ¼ 0.3 mm, matrix ¼ 256 192, under fat saturation and external trigger. Respiration rate ¼ 60/min). The coronal and axial sections of the whole body of the rat were imaged at different time periods. The SNR (signal to noise ratio) from the liver were measured and plotted against time. SNR ¼
ROI mean signal Background standard deviation
2.8. In vitro cytotoxicity MCF-7 breast cancer cells and NIH-3T3 mice fibroblast cells were seeded in 96well plates (Costar, IL, USA) at the density of 3 105 viable cells/well using Dulbecco’s modified eagle medium (DMEM), containing 5% fetal bovine serum and 1% antibiotics, and incubated for 24 h with MCF-7 or 48 h with NIH-3T3 cells to allow cell attachment. The media was removed and the replaced with fresh media. To each well, 10 ml of the IOs-PNPs suspension or ResovistÒ at concentrations ranging from 2.5 to 10 mmol [Fe]/L was added and incubated for 24 h. Untreated wells were used as control. Five hours prior the time point, 10 ml of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) in PBS was added into the 96-well plates. The cells were incubated till the designated time point. After incubation, 100 ml of a stop mix solution composing of 20% sodium dodecyl sulfate (SDS) in 50% dimethylformamide (DMF) was added into each well. The plate was incubated for an hour to dissolve the formazan crystals that are formed and the absorbance in each well was measured by microplate reader (GENios, Tecan, Switzerland) at 550 nm and 630 nm as reference wavelength. 2.9. In vitro cellular uptake of NPs 2.9.1. Quantitative evaluation MCF-7 breast cancer cells and NIH-3T3 mouse fibroblast cells were seeded in 24 well plates (Costar, IL, USA) at a density 1 105 viable cells/ml/well. The cells were allowed to grow for 24 h. After 24 h, the medium in the wells were replaced with fresh medium and 100 ml of the IOs-PNPs suspension or the ResovistÒ at the various designated [Fe] concentration were added to the wells. Wells with neither the nanoparticles suspension nor the ResovistÒ added were used as control. After incubation for 24 h with MCF-7 or 48 h with NIH-3T3 cells, the culture medium was removed. The plates were washed 4 times with PBS, each time with fresh exchange of PBS. The cells were then trypsinated and then centrifuged at 2000 rpm for 5 min. To the cell pellet, 2 ml of concentrated nitric acid was added and was heated to 120 C. This dissolved and converted the IOs-PNPs internalized by the cells into its corresponding ions. The solution was diluted using Milli-Q water, and the samples were analyzed by inductively coupled plasma-mass spectrometer (ICP-MS, Agilent ICP-MS 7500). 2.9.2. Qualitative analysis To assess the cellular uptake of the IOs-PNPs qualitatively, the MCF-7 cancer cells were grown in 175 cm2 culture flask at a cell density of 1 106 cells/ml. After 24 h, the medium was replaced by fresh medium containing the IOs-PNPs at 2 mmol of [Fe]/L equivalent concentration. After 4 h of incubation, the medium was removed; the cells were washed 4 times with PBS, and fixed with 2.5% glutaraldehyde at 37 C. After 2 h, the glutaraldehyde solution was removed and the cells were washed with PBS. The cells were then scrapped and pelleted at 3500 rpm for 10 min. The pellet was post-fixed using 1% Osmium tetroxide at PH 7.4 for 1 h, followed by washing with PBS two times. The cell pellet was then fixed in 6% gelatine, followed by a series of dehydration process using ethanol and acetone, and finally embedded in araldite resin. The sections were stained using lead citrate prior to viewing. The samples were viewed using Transmission Electron Microscope (TEM, (JEOL, JEM-1220)).
% Contrast change ¼
SNR at any time t 100 SNR at time zero before contrast
Xenograft model was developed using severe combined immune deficiency (SCID) mice (female, 20 g). A 5 106 number of MCF-7 cancer cells were injected into the subcutaneous part of the mice near the right flank. The tumor was then developed and allowed to grow until a volume of 150e200 mm3. The SCID mice were imaged using a Bruker 7T Clinscan MRI system. The protocol was approved by the A*STAR Institutional Animal Care and Use Committee (IACUC) (#060188). Contrast agent was injected (dosage: 5 mg of [Fe]/kg body weight) through tail veins of the mice under 1% isoflurane anesthesia. T2-weighted images were acquired at various time points using T2-weighted turbo spin echo sequence (TR/TE ¼ 1500/36 ms, resolution ¼ 100 mm, thickness ¼ 1 mm). Signals from region of interest (ROI) of the tumor was taken and normalized with the signal of the phantom filled with saline.
3. Results and discussion 3.1. Synthesis of PLAeTPGS copolymer PLAeTPGS (with a ratio 90:10 of PLA:TPGS) was successfully synthesized by ring opening polymerization. The polymer was characterized using FTIR and 1H NMR as shown in Fig. 1. The carbonyl band shift from 1730 cm1 for TPGS to 1755 cm1 for PLAeTPGS polymer and also the CH stretching at 2945 cm1 for PLA to at 2880 cm1 for that for TPGS can be observed from the FTIR spectra (Fig. 1A). The successful synthesis of PLAeTPGS copolymer was further verified by 1H NMR for the presence of CH proton peak at 5.2 ppm for PLA-TPGS compared with 5.07 ppm for PLA. The signals at 5.2 and 1.69 ppm were assigned to the PLA segment and that at 3.65 ppm was assigned to the TPGS segment (Fig. 1B). The number-averaged molecular weight of the PLAeTPGS copolymer was determined to be Mn ¼ 17,027 by NMR and Mn ¼ 9856 by GPC respectively. The weight-averaged molecular weight was found by GPC to be Mw ¼ 13559 with polydispersity of 1.36. Our results are quite close to those given in our earlier work in the literature for PLAeTPGS of 88:12 PLA:TPGS component ratio [28]. 3.2. Synthesis of hydrophobic IOs We have successfully synthesized hydrophobic IOs which coated with oleic acid (OA) and oleylamine (OM). Fig. 2A shows a TEM image of the hydrophobic IOs, from which it can be seen that
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Fig. 1. (A) Fourier Transformation Infrared Spectroscopy (FTIR) of amphiphilic D-a-tocopherol polyethylene glycol 1000 succinate (TPGS) and PLAeTPGS copolymer, (B) 1H NMR spectrum of PLAeTPGS copolymer.
the IOs are highly monodispersed and non-agglomerated. The particle size is found to be 10 nm which is in the superparamagnetic size range. The OA and OM coating was confirmed by FTIR as shown in Fig. 2B. The peaks at 2926 and 2854 cm1 are associated with the CeH stretching vibration, which arises from the surface adsorbed OA and OM coatings respectively. The peaks at 1562 and 1440 cm1 are due to the COO stretching vibration and the peak at 1411 cm1 (which is merged with the 1440 cm1 peak) is due to the CeN bending vibration, which arises from the surface adsorbed OA and OM coatings, respectively. The broad band between 3600 and 3000 cm1 centered at about 3400 cm1 is due to the OeH stretching vibration arising from surface adsorbed water. In addition, the strong absorption band at 584 cm1 is due to the FeeO stretching vibration of Fe3O4 IOs. 3.3. Synthesis of IOs-encapsulated polymeric nanoparticles (IOs-PNPs) The IOs-PNPs were synthesized in this research by the single emulsion method and the nanoprecipitation method, respectively. Table 1 summarizes the synthesis parameters and the results obtained from the IOs-PNPs prepared by the single emulsion method. Fig. 3AeC show the TEM image of the IOs-PNPs prepared
by the emulsion method at emulsifier concentration of 1%, 5% and 15% respectively. It can be seen that for the IOs-PNPs prepared by the single emulsion method, the distribution of the IOs within the polymer matrix is not significantly affected by the increase in the emulsifier concentration. Moreover, there seems a trend that the IOs are distributed more towards the periphery. This kind of IOs distribution in the polymeric matrix was seen previously in others work as well [35,36]. However the size of the IOs-PNPs was found reduced with increase of the surfactant concentration at the same IOs loading. This is understandable since more surfactant molecules could cover larger surfaces of the nanoparticles of smaller size as described in the literature [27]. There was also considerable reduction in the IOs encapsulation efficiency (EE) of the IOs-PNPs with increase in the emulsifier concentration. This can be due to the stabilization of the hydrophobic IOs by the micelles of the surfactant which might be formed and then washed away in the preparation process. It should be noticed that the surfactant concentration used in the IOs-PNPs preparation process fell in the micelle forming region of TPGS which is between the critical micelle concentration (CMC) 0.03e20%. The characters of the IOs-PNPs prepared by the nanoprecipitation method are summarized in Table 2. The TEM images of such IOs-PNPs prepared with the emulsifier concentration of 0%, 1%, 5% and 15% are
Fig. 2. (A) TEM image of the as-synthesized hydrophobic IOs (scale ¼ 10 nm), (B) FTIR confirmation of the oleic acid and oleylamine coating on the as-synthesized hydrophobic IOs.
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Table 1 Characters of the IOs-loaded PLAeTPGS nanoparticIes (IOs-PNPs) prepared by the single emulsion method. Polymer
% Emulsifier (w/v) in aqueous phase
IO Loading
EE%a
% Fe per IOs-PNPsb
Size
PDI
PLAeTPGS PLAeTPGS PLAeTPGS
1 5 15
2% 2% 2%
36.67 3.51 24.02 2.33 16.03 3.31
0.815 0.078 0.600 0.0.58 0.464 0.096
352.03 18.28 307.44 16.46 282.22 14.70
0.379 0.058 0.236 0.044 0.153 0.043
Amount of iron oxide present in the IOsePNP obtained Amount of iron oxide used in the preparation of IOsePNP
a
EE% ¼
b
% Iron content ¼
100.
Weight of iron in X grams of IOsePNP ðin gramsÞ X ðin gramsÞ
100.
Fig. 3. TEM images of IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs) prepared by the single emulsion method at the emulsifier concentration of (A) 1%, (B) 5% and (C) 15% and by the nanoprecipitation method at the emulsifier concentration of (D) 0%, (E) 1%, (F) 5% and (G) 15% respectively (scale bar ¼ 100 nm).
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Table 2 Characters of the IOs-loaded PLAeTPGS nanoparticIes (IOs-PNPs) prepared by the nanoprecipitation method. Polymer
% Emulsifier
IO loading
EE%a
PLAeTPGS PLAeTPGS PLAeTPGS PLAeTPGS
0 1 5 15
2% 2% 2% 2%
16.58 54.33 58.17 64.17
Amount of iron oxide present in the IOsePNP obtained Amount of iron oxide used in the preparation of IOsePNP
a
EE% ¼
b
Hydrodynamic diameter measurement, n ¼ 3.
c
% Iron content ¼
% Iron contentc
1.90 3.33 3.60 2.32
0.368 0.987 1.292 1.283
0.042 0.061 0.080 0.046
Sizeb 356.77 302.53 272.80 249.43
PDI
07.34 15.83 02.53 19.89
0.321 0.283 0.128 0.093
0.063 0.022 0.016 0.008
100.
Weight of iron in X grams of IOsePNP ðin gramsÞ X ðin gramsÞ
100.
shown in Fig. 3DeG, respectively. From these figures, the effect of emulsifier concentration on the IOs distribution within the polymeric matrix can be observed, which shows that with increase of the surfactant concentration in the aqueous phase, the IOs were distributed more and more to the interior of the polymer matrix, leading to almost homogenous distribution at high emulsifier concentration. Moreover, both of the size of the IOs-PNPs and the polydispersity index (PDI) were greatly decreased with increase of the emulsifier concentration at the same IOs loading. The EE increased as well. Fig. 4AeC show the FESEM images of the IOs-PNPs synthesized by the nanoprecipitation method at emulsifier concentration of 1%, 5% and 15%, from which the size of the IOs-PNPs was reconfirmed. The ratio of the solvent to non-solvent and IOs loading level could be important factors that may affect the size of the IOs-PNPs and the IOs distribution within the polymeric NPs. In one set of the experiment, IOs-PNPs were prepared with different loading level of 1%, 2% and 3% IOs (w/w based on polymer) were prepared by the nanoprecipitation method at 15% emulsifier concentration. It was observed that the distribution of IOs in the polymeric matrix was not affected (TEM not shown). However, the size did increase with the IOs loading in the order of 225.60 15.037 nm (PDI: 0.073 0.055) for 1% IOs loading, 249.43 19.89 nm (PDI: 0.093 0.008) for 2% IOs loading, and 448.6 20.03 nm (PDI: 0.497 0.044) for 3% IOs loading respectively. This finding agrees with that observed from the nanoparticle formulation of hydrophobic drugs, in which the nanoparticle size was also found to increase with the drug loading [17]. The ratio of the solvent to non-solvent and IOs loading level could jointly affect the size of the IOs-PNPs and the IOs distribution within the polymeric NPs. It seems that increase in the former parameter and/or decease in the later parameter would lead to smaller particle size. We found that the solvent/non-solvent ratio of 1:15, 4:15 and 3:5 yielded nanoparticles of iron content of 1.1%, 1.2% and 0.8% and size of 283 5.80, 249 19.9 and 203 8.93 nm respectively, and the IOs-PNPs prepared at the 1:15 solvent/non-
solvent ratio were polydispersed while IOs distribution in the NPs prepared at the 4:15 solvent/non-solvent ratio looked more uniform and had a higher iron content, thus was considered optimal (the TEM images not shown). We can thus conclude that the nanoprecipitation preparation method seems preferable to prepare the IOs-PNPs of more desired particle size and IOs distribution, for which the surfactant concentration, the solvent to non-solvent ratio and the IOs loading level are three important parameters that may determine the particle size and size distribution as well as the IOs distribution within the polymeric NPs. Taking into consideration the size of the IOs-PNPs and the iron content, the IOs-PNPs prepared at the 4:15 solvent/nonsolvent ratio and 2% IOs loading level was found to be optimal. 3.4. XPS analysis XPS can qualitatively and quantitatively determine the chemical composition up to a depth of 5e10 nm. In the XPS spectrum, a peak characterizes a corresponding chemical bond and the area-underthe curve at that peak gives information about the relative % of that chemical bond. 5e10 nm is the depth to which the X-ray can penetrate under the surface and thus the XPS peak obtained is for the chemical structure of the surface up to a 5e10 nm depth. The XPS spectra of the IOs-PNPs prepared by the nanoprecipitation method at the various emulsifier concentrations are shown in Fig. 5. It can be found from this figure that with increase in the emulsifier concentration in the NPs preparation process, the iron content in the IOs-PNPs, which is denoted by the peaks at binding energy 708 eV (2p3/2) and 722 eV (2p1/2), diminished. This means that all loaded IOs have been located within the polymeric matrix. The iron content on the surface becomes almost nil for the IOs-PNPs synthesized at 15% emulsifier concentration. The IOs-PNPs prepared with no emulsifier showed the highest content of iron on the surface. On the other hand, the area-under-the curve at the CeOeC peak 286.1 eV, which denotes the PEG element of the TPGS
Fig. 4. Field emission scanning electron microscopy (FESEM) images of IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs) prepared by the nanoprecipitation method at the emulsifier concentration of (left) 1%, (middle) 5% and (right) 15%, respectively.
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A
B 2P1/2
2P3/2 5% TPGS C-O-C O-C=O
C-C/C-H
C-O-C=O
1% TPGS 292
290 288 286 284 Binding Energy (eV)
282
No Emulsifier
C 15% TPGS C-C/C-H
740
730
720 710 Binding Energy (eV)
700
C-O-C O-C=O
292
C-O-C=O
290 288 286 284 Binding Energy (eV)
282
Fig. 5. (A) X-ray photoelectron spectroscopy (XPS) spectrum of iron bond on the surface of the IOs-loaded PLAeTPGS nanoparticles prepared by the nanoprecipitation method at the concentration of emulsifier of 0% (i.e. no emulsifier), 1%, 5% and 15%, respectively, (B) XPS spectrum of carbon bond on the surface of the IOs-loaded PLAeTPGS nanoparticles prepared by the nanoprecipitation method without emulsifier, (C) XPS spectrum of carbon bond on the surface of the IOs-loaded PLAeTPGS nanoparticles prepared by the nanoprecipitation method at 15% emulsifier concentration.
macromolecule, increases from 22.56% for the IOs-PNPs synthesized without emulsifier (Fig. 5B) to 33.73% for those prepared with 15% TPGS emulsifier concentration (Fig. 5C). This shows that the emulsifier macromolecules do remain on the surface of the polymeric NPs to stabilize the NPs formulation. Such observation was not as significant for the IOs-PNPs prepared with emulsifier concentration lower than 15%. This can be understandable since the carbon peak now could be contributed more from the oleic acid and oleylamine coating of the IOs near the surface than from the emulsifier macromolecules.
3.5. Hysteresis Fig. 6 shows the hysteretic MeH curves of the IOs-PNPs prepared by the nanoprecipitation method at the various emulsifier concentrations. The normalized saturation magnetization (ss) of the hydrophobic IO crystals is 84.5 emu/g while that of the IOsPNPs were 10.26, 44.46, 70.11, and 79.23 emu/g when prepared at the 0%, 1%, 5% and 15% emulsifier concentration, respectively. The ss values of the IOs-PNPs are found to be always lower than that of hydrophobic IO crystals due to the encapsulation within the nonmagnetic polymer matrix [37]. Moreover, the ss values of the IOsPNPs are found to increase with increasing the emulsifier concentration in the NPs preparation process. This could be due to the increment of the iron content within the IOs-PNPs with increasing the emulsifier concentration. Conversely, the ss values are found to decrease with the increase of the non-magnetic polymer content over the IOs in the NPs (i.e. decrease in IOs encapsulation in the IOs-PNPs) with decreasing the emulsifier concentration. The zero coercivity and remenance of the MeH curves indicate that all of the encapsulated IOs are superparamagnetic in nature [37]. 3.6. Cytotoxicity
Fig. 6. Hysteresis curve of IOs and IOs-loaded PLAeTPGS nanoparticles (IOs-PNPs) prepared by the nanoprecipitation method at the emulsifier concentration of 0%, 1%, 5% and 15%, respectively.
Cytotoxicity of the IOs-PNPs was measured by the MTT assay 24 h after incubation with MCF-7 breast cancer cells and NIH-3T3 cells in close comparison with the clinical IOs agent ResovistÒ at the equivalent 2.5, 5, 10 mmol [Fe]/L, respectively. The results are shown in Fig. 7, from which it can be seen that both formulation of supraparamagnetic IOs, the IOs-PNPs and ResovistÒ, have little toxicity on MCF-7 cancer cells at least at the designated equivalent IO concentration ranged (Fig. 7A). However, ResovistÒ has much
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Fig. 7. Cytotoxicity of the IOs-PNPs was measured by the MTT assay 24 h after incubation with (A) MCF-7 breast cancer cells and (B) NIH-3T3 cells in close comparison with the clinical IOs agent ResovistÒ at the equivalent 2.5, 5, 10 mmol [Fe]/L, respectively (n ¼ 8).
higher cytotoxicity for the NIH-3T3 mouse fibroblast cells than the IOs-PNPs, which demonstrates the advantages of the latter over the former (Fig. 7B). It was found that the IOs-PNPs were approximately 15, 17 and 10 fold less toxic than ResovistÒ at the same iron concentration of 10, 5 and 2.5 mmol Fe/L, respectively.
3.8.2. In vivo liver clearance As mentioned before, the mostly used formulation of IOs used in current MRI is ResovistÒ, which has raised concerns for its low imagining effect and toxicity due to their accumulation in the visceral organs. As a preliminary study, we investigated the
3.7. Cellular uptake of IOs-PNPs 3.7.1. Quantitative investigation Cellular uptake of the IOs formulated in the PLAeTPGS NPs (the IOs-PNPs) was measured by ICP-MS, which was conducted in close comparison with the IOs formulated in the ResovistÒ. For MCF-7 breast cancer cells, the cellular uptake of IOs formulated in the IOsPNPs were found 1.2, 16.4, 20.4 fold higher than that formulated in ResovistÒ at the equivalent iron concentrations of 2.5, 1.25 and 0.75 mmol Fe/L, respectively. For the NIH-3T3 mouse fibroblast cells, the cellular uptake of IOs formulated in the PLAeTPGS NPs was found 16.23 and 6.5 fold higher than the ResovistÒ formulation at the equivalent iron concentrations of 2.5 and 0.75 mmol Fe/L, respectively although ResovistsÒ showed higher cellular uptake than the IOs-PNPs formulation at the equivalent iron concentration of 1.25 mmol Fe/L due to the irregular cellular behaviour. 3.7.2. Qualitative investigation Qualitatively investigation of cellular uptake of the IOs-PNPs in MCF-7 breast cancer cells was visualized by TEM. Fig. 8 shows TEM images of the MCF-7 cells 4 h after incubation with the IOs-PNPs at equivalent 2 mmol Fe/L NP concentration. It can be seen from Fig. 8A that the IOs-PNPs are confined within a meso vesicle in the cytoplasm of the cell (scale bar ¼ 200 nm). Fig. 8B shows the meso vesicle in Fig. 8A at higher magnification (scale ¼ 100 nm). In Fig. 8 A and B, the arrow marks the meso vesicle. It seems that the IOsPNPs were internalized by a mechanism called endocytosis (penocytosis), in which the NP sacrifice its interfacial energy to bend a small piece of the lipid bilayer membrane to form a small meso vesicle in a scale of 200e300 nm that will bring the IOs-PNPs into the cells. Moreover, the surface of the nanoparticle seem to have been disturbed, indicating the process of metabolism by the cell. 3.8. In vivo MRI 3.8.1. Relaxivity studies The IOs-PNPs had a relaxivity of r2 ¼ 164.09, r2* ¼ 280.73 and r1 ¼ 0.3372 (L mmol Fe1 s1). This indicated that the IOs-PNPs can be used for T2-weighted MRI imaging. All in vivo experiments were carried out under T2-weighted sequence.
Fig. 8. Shows the TEM image of MCF-7 cancer cell line incubated with IOs-PNPs; A. image shows that the IOs-PNPs are confined to a vesicle in the cytoplasm of the cell (Scale ¼ 200 nm); B. image shows a magnified view of the vesicle marked with the middle arrow in image A. The vesicle carries the IOs-PNPs into the cell. The walls of the particle seem to be disturbed, indicating the process of metabolism by the cell (scale ¼ 100 nm).
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intensity of the liver is lost within 24 h as shown by the % contrast change curve versus time in Fig. 9. This indicates the advantages of the IOs-PNPs formulation over ResovistÒ for earlier clearance of the IOs from the animal that can thus greatly reduce the IOs toxicity to the organs.
Fig. 9. The signal trend in the liver obtained after i.v. injection of the IOs-PNPs suspension at an equivalent iron dose of 0.8 mg Fe/kg of body weight of Wistar rats, which was imaged by a 9.4 T MRI Varian scanner. The insert shows the MRI images (the axial section) of the liver taken at various designated time. The arrow indicates the liver of the rat.
3.8.3. In vivo tumor MRI We further investigated the potential of the IOs-PNPs for MRI imaging on a xenograft model, which is shown in Fig. 10. The signal trend shown in this figure was from the tumor obtained before and 0.33, 2, 5 and 12 h after i.v. injection of the IOs-PNPs suspension at an equivalent iron dose of 5 mg Fe/kg of body weight of SCID mice, in which a tumor has been induced. The MRI was conducted by a 7 T MRI Bruker scanner. The insets show the MRI images (the axial section) of the tumors which were taken off from the animal before and 5 h after the injection of the contrast, respectively. The arrow indicates the tumor. It can be seen from the inserts that the IOsPNPs have been internalized in the tumor developed in SCID mice as shown by the heterogeneity developed in the tumor after 5 h compared to the tumor scanned before administration of the IOsPNPs at time zero. This indicates that the IOs-PNPs can be used to passively target tumor by EPR. 4. Conclusion
biodistribution of the IOs-PNPs in the various organs 1 h after intravenous (i.v.) injection of the IOs-PNPs suspension at the equivalent iron dose of 0.8 mg Fe/kg through the tail of rats. Such a dose was determined from a report in the literature, in which the average concentration of IOs used on human MRI was around 0.54 mg Fe/Kg [38]. Fig. 9 shows the signal trend in the liver obtained before 0.6, 24, 48, and 120 h after i.v. injection of the IOsPNPs suspension at an equivalent iron dose of 0.8 mg Fe/kg of body weight of Wistar rats, which was imaged by a 9.4 T MRI Varian scanner. The insert shows the MRI images (the axial section) of the liver taken at various designated time. The arrow indicates the liver of the rat. It can be seen from the Fig. 9 inset that the IOs-PNPs are accumulated to large extent in the liver. Kidney and other organs had a reduced uptake (images not shown). However, the signal
The IOs-loaded PLAeTPGS NPs (IOs-PNPs) prepared by the single emulsion method and the nanoprecipitation method at the various process parameters, especially the IOs loading level in the NPs and the solvent/non-solvent ratio used in the process, were characterized for their physicochemical and supraparamagnetic properties for an optional formulation using nanoprecipitation method. We found that the IOs-PNPs prepared by the nanoprecipitation method at the 4:15 solvent/non-solvent ratio and 2% IOs loading level would have desired properties for MRI. The cellular uptake of the IOs-PNPs by the MCF-7 breast cancer cells and NIH-3T3 mice fibroblast cells was measured by ICP-MS and visualized by TEM. The in vitro cytotoxicity of the IOs-PNPs was also investigated by MTT assay in close comparison with ResovistÒ. In vivo MRI investigation found that the IOs-PNPs have high relaxivity and suitable for T2-weighted MRI imaging. Moreover, the IOs-PNPs formulation has advantages over ResovistÒ for earlier clearance of the IOs from the liver that can thus greatly reduce the IOs toxicity to the organs. The xenograft tumor model MRI indicated the great potential of the IOs-PNPs formulation for clinical tumor MRI as well as for other medical imaging. Acknowledgement The authors are thankful for the financial support from the NUS FOE Grant R-279-000-226-112 and NanoCore Grant R-279-000284-646 as well as A Singapore A*STAR SBIC (Singapore Bioimaging Consortium, Agency of Science Technology and Research). Appendix Figures with essential color discrimination. Figs. 5, 6 and 7 in this article are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/ j.biomaterials.2010.03.070.
Fig. 10. Xenograft tumor model of the signal trend in the liver obtained after i.v. injection of the IOs-PNPs suspension at an equivalent iron dose of 5 mg Fe/kg of body weight of SCIF mice in which a tumor has been induced, which was imaged by a 7 T MRI Bruker scanner. The insets show the MRI images (the axial section) of the tumors which were taken off from the animal before and 5 h after the injection of the contrast, respectively. The arrow indicates the tumor.
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