of magnetic fields on the drug diffusion behavior. We have prepared PVA networks loaded with randomly and vertically, parallelly distributed iron oxide nanoparticles,
modulated by random and uniform magnetic field (vertical and parallel direction), as illustrated in Fig. 6.11. Fig. 6.11-(a) represents that the arrangement of iron oxide nanopartices of ferrogel is parallel to the direction of magnetic field; whereas that is
“vertical” to the drug diffusion direction, and Fig. 6.11-(b) illustrates that the arrangement of iron oxide nanopartices of ferrogel is vertical to the direction of magnetic field; whereas that is “parallel” to the drug diffusion direction. In addiiton, the drug diffusion behavior was described in Fig. 6.12. The result shows that the drug diffusion rate would change with different the arrangement of iron oxide nanoparticles, implying the parallel direction exhibits the highest rate of drug diffusion whereas the vertical direction is lowest. It demonstrated that the drug might be restricred and thus difficult to pass through in the vertical ferrogel, but it is freely flowing in the parallel ferrogel. Therefore, it was anticipated to control the arrangement of iron oxide nanoparticles in the ferrogel could modulate the drug diffusion rate. It would be useful for the application in the dialysis membrane by magnetic field controlling.
Fig. 6.11 Schematic representation of the experimental set-up for ferrogel preparation under uniform magnetic field: (a) & (a1) drug diffusion direction is vertical to the arrangement of iron oxide nanopaticle; (b) &
(b1) drug diffusion direction is parallel to the the arrangement of iron oxide nanopaticle [Varga, 2006]
Diffusion
Diffusion
(a) (b)
(a1) (b1)
(Vertical)
(Parallel)
0.00 0.01 0.02 0.03 0.04 0.05
0 50 100 150 200
Time (min)
P re m ea bilit y ( A BS )
RandomVertical Parallel
Fig. 6.12 Drug diffusion behavior in the various ferrogel; random, vertical and parallel means the arrangement of iron oxide nanoparticle is random, vertical and parallel to the drug diffucion direction, respectively
Chapter 7
Thermo-sensitive Ferrofluids
7.1. Introduction
Recently, ferrofluids are widely employed in the fields of biology and biomedicine such as enzyme and protein immobilization, genes, radiopharmaceuticals, magnetic resonance imaging (MRI), diagnostics, immunoassays, RNA and DNA purification, separation, and targeting drug delivery devices [Mak, 2005; Kim, 2002]. In order to develop functional nano-sized magnetite particles, a stable dispersion of the magnetic nanoparticles in organic or aqueous media are critically required an effective surface modification with organic compounds or polymers as dispersing agents. A delicate balance of hydrophilicity/ hydrophobicity in the polymer structure is responsible for exhibiting a lower critical solution temperature (LCST) phenomenon or critical micellization temperature (CMT). A series of tri-block copolymers composed of poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) (Pluronics) are a kind of temperature-sensitive polymers, and they also demonstrate reversible solution transition behaviors in aqueous solution [Bromberg, 2003 & 2004]. Consequently, the novel thermo-sensitive ferrofluids that combine Pluronics-based-polymers (Pluronics F127) with magnetite nanoparticles could lead to a temperature responsive drug carrier system, because the magnetite would provide a source of heat in the alternating magnetic field by hyperthermia [Park, 2005].
7.2 Fabrication of thermo-sensitive ferrofluids
The stable thermo-sensitive ferrofluids was synthesized using the method of in-situ co-precipitation of Fe(II) and Fe(III) salts in the presence of Pluronic F127 (F127, Sigma). In this process, 0.05g of F127, 1.35g of FeCl3·6H2O (5mmol, Riedel-deHaën), and 0.498g of FeCl2·4H2O (2.5mmole, Fluka) were dissolved in 50 ml of water with vigorous stirring at 60oC. Then, the ammonia solution (33%, Riedel-deHaën) was quickly added to the reactor and stirred until pH arrives 10, followed by hydrothermal treatment at 80oC for 30 min. After washed five times, filtrated, and freezing-dried, core (magnet)-shell (F127) nano-magnetic-particles (NMPs) were successfully prepared. Finally, the 1g of core-shell NMPs were
dispersed in the 10 ml of 40% (w/v) F127 aqueous solution (pure F127-fluids, dissolved at 4oC beforehand) by sonication at 4oC for 6 hr. The well-dispersed thermo-sensitive ferrofluids were fabricated. Besides, Fe3O4 NMPs (diameter ca. 5-10 nm) fabricated by in-situ co-precipitation process would be used for reference [Mak, 2005].
7.3 Characterization of thermo-sensitive ferrofluids
TEM photos (JEOL-2000FX) in Figure 7.1-(a) show the core-shell structure of magnet-F127 NMPs. According to the TEM photos, the average thickness of F127 layers was found to be 1-2 nm, and the magnet diameter was about 5-7 nm.
Fig. 7.1 (a) TEM photo of core-shell NMPs; (b)XRD pattern of core-shell NMPs.;
(c) Magnetization curve of core-shell NMPs using VSM
Moreover, it was found in the XRD (Rigaku) pattern [Fig. 7.1-(b)] that six
diffraction peaks at 2θ= 30.1o, 35.6o, 43.3o, 53.5o, 57.2o, 62.9o are the characteristic peak of standard Fe3O4 crystal. Hence, the resulting magnet NMPs can be defined as Fe3O4 NMPs. An additional diffraction peak at ca. 2θ= 32.3o was detected in the NMPs, which is a characteristic peak of standard Fe2O3 crystal, indicating a small amount of Fe2O3 was associated with the core-shell NMPs but the superparamagnetic behavior of the core-shell NMPs was not affected. Furthermore, the pure F127 showed two characteristic peaks at 2θ= 18.8o, and 23.1o, indicating that the F127 displays high degree of crystallization. However, the core-shell NMPs exhibits a broad diffraction pattern peaked at 21o, which is believed due to a too thin layer of F127 to be more difficult to crystalize or subjecting to a much slower crystallization of the F127 lattices. In addition, Figure 7.1-(c) shows that Fe3O4 NMPs display a magnetization (Ms) (60.9 emu/g), which is higher than core-shell NMPs i.e., Ms (28.4 emu/g), measured using the vibrating sample magnetometer (VSM, Toei VSM-5). An incorporation of the F127 may cause damages in some domains of Fe3O4 resulting in a decreased Ms. From these results, it can be confirmed that F127 is truly existed around Fe3O4 nanoparticles.
Fig. 7.2 DSC heating and cooling scans of pure F127-fluids and F127-ferrofluids
Figure 7.2 shows the DSC (Perkin Elmer, Diamond DSC) spectra with heating and cooling thermograms (5oC/min) for both pure F127-fluids and F127-ferrofluids with a process of the gellation and liquefaction, respectively. An endothermic peak of pure F127-fluids and F127-ferrofluids in the heating run was detected which is due to
Liquid state (Ferrofluid) Low Temp. (4oC)
0 min 5 min
gel state (Ferrogel)
High Temp. (30oC)
(a) (b) (c)
(d)
the aggregation transition (gel formation) of these two fluids in aqueous suspension.
Such aggregation is caused by the temperature sensitivity of the PPO segments of the Pluronic that being anchored to the gel network. Much like the un-cross-linked Pluronic, polymers can form intra- and intermolecular micelle-like aggregates due to the hydrophobic interaction [Bromberg, Langmuir, 2004]. The gellation started at 21.8oC and maximized at 32.5oC for pure F127-fluids, and it commenced at 22.8oC and maximized at 35.6oC for F127-ferrofluids. It is more difficult to identify where CMT point is with so broad peak. In this case, the exothermic peak in the cooling run, i.e., melting thermospectrum, can be used to identify the CMT, due to its sharper peak.
The liquefaction started at 24.9oC and maximized at 21.4oC for pure F127-fluids, but commenced at 28.5oC and maximized at 23.5oC for F127-ferrofluids. Therefore, CMT of the pure F127-fluids can be defined in the range of 21-25oC, which is similar to literature data [Bromberg, 2003]. However, it is lower than the CMT of F127-ferrofluids in the range of 23-28oC. The reason that CMT point shift was that core-shell NMPs might play a role of cross-linked point, and hence increased CMT point. Similar results can be found in PNIPAAm /clay systems [Haraguchi, 2002].
The thermo respose behavior of ferrofluid by temperature incrase was illustrated in Fig. 7.3. It was found that the ferrofluid (liquid state, Fig. 7.3-(a)&(b)) transferred rapidly to ferrogel (gel state, Fig. 7.3-(c)&(d)) while the temperature from 4 to 30oC for 5min. It demonstrated that the thermo sensitive ferrofluids could transfer to ferrogel very rapidly.
Fig. 7.3 Series image of thermo response behavior of ferrofluids: (a)&(b) liquid state (ferrofuids); (c)&(d) gel state (ferrogel)
In addition, a model drug can be dispersed homogenously in F127-ferrofluids below CMT, and then encapsulated into F127-ferrogel above CMT, and higher drug uptake was obtained. Moreover, it could be anticipated that the temperature increased of F127-ferrofluids modulated by hyperthermia of magnetic nanoparticles in external alternating magnetic fields [Park, 2005] can transform to F127-ferrogels. The further investigation is now in progress in our group
Chapter 8
Thermal and Magnetic Nano Ferrospheres
8.1 Introduction
Drug carriers that are responsive to external stimuli, such as temperature, pH, light, mechanical signal, electric field, and magnetic fields have received great attention in recent years [Kim, 2002; Zhang, 2004; Chiu, 2005; Lu, 2005; Qiu, 2001;
Etrych, 2001; Chen, 2004; Murdan, 2003; Mamada, 1990]. A wide variety of materials, such as carbon nanotubes, dendrimers, biodegradable nanoparticles (PLGA or PLA), lipid vesicles (micelles), and gold or magnetic nanoparticles have been employed as matrix materials to deliver drugs. Among them, magnetic nanoparticles (MNPs) provide more interesting opportunities since they can be effectively activated in a controllable manner through a non-contact stimulus [Kohler, 2006], as compared with other materials. Although gold nanoparticles also displayed a similar inductive heating ability by infrared light stimulus, the manipulation and heating ability of magnetic nanoparticles were superior to those of the gold nanoparticles. The ability of magnets to convert magnetic energy into heat by the hysteresis effect has been known for a long period of time [Kim, 2005]; for instance, the ability has been used as antenna material for inductive heating in anti-tumor therapy, but the efficiency has not been as good as desired without combining inductive heating with drug delivery. In order to enhance the tumor-inhibition ability, the combination of the heat transfer and drug release in nanoparticles will be more interesting. In our previous work, we developed magnetic hydrogels (ferrogels) [Liu, 2006 & 2008; Hu, 2007] and magnetically sensitive nanospheres for controlled drug release by a high-frequency magnetic field (HFMF) [Hu, 2007 & 2008]. However, the silica seems to be a thermally insensitive material, and it was believed that the nanostructure did not change with temperature increases. In order to accelerate the rate of drug release, a thermo-sensitive polymer was used in this system because it exhibited a huge volume change when the temperature was increased [Choi, 2006].
A number of polymeric materials are known to exhibit a discontinuous change of properties when subjected to a temperature change [Choi, 2006; Schmidt, 2007;
Coughlan, 2004; Bhattacharya, 2007; Li, 2006; Bae, 2007; Desai, 2001]. Some of these systems, e.g. thermo-reversible gels, have been based on the existence of a lower critical solution temperature (LCST), or critical micellization temperature (CMT), and have usually been applied as intelligent drug carriers [17-23].
Poly(N-isopropylacrylamide) (PNIPAAm), poly(ethylene glycol)-poly(lactic acid)-poly(ethylene glycol) triblocks (PEG-PLA-PEG), and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblocks have been among the most important reverse thermal gelation-displaying polymers. These triblocks, especially Poloxamer 407, also known by the trade name Pluronic® F127 (PEO100-PPO65-PEO100), have been investigated for wound covering and controlled drug delivery. Furthermore, Pluronic® series copolymer has excellent biocompatibility and is one of the very few synthetic polymers approved by the US Food and Drug Administration (FDA) for use as a food additive and pharmaceutical ingredient [Bae, 2007; Desai, 2001]. Pluronic® series copolymer is also a potential candidate for biomedical applications.
In the present work, a dual-functional nanoparticle was developed by integrating a nanomagnetic core with a shell layer of thermo-sensitive hydrogel to encapsulate an anti-cancer agent. Inductive heat was generated in the presence of HFMF. When heated, the thermo-sensitive polymer collapsed around the iron oxide nanoparticles, inducing an accelerative drug release. In recent years, some researchers [Zhang, 2007; Chen, 2007; Deng, 2005; Detlef, 2006] have reported that dual-functional drug carriers included magnetic/thermal sensitivity, but few reports emphasized the controlled drug release using HFMF. In addition, raising the temperature of the drug carriers from the surrounding heat source has been insufficient; a better alternative would be to supply the heat source by the drug carrier, e.g., the inductive heating effect induced by HFMF to a magnet core. This type of drug carrier can have great potential for disease treatment.
Moreover, the use of high-frequency magnetic stimulus can also achieve an
“instantaneous” burst release of drug by the rapid heating to induce an instant shrinkage of the F127 hydrogel. Choi et al. [Choi, 2006] reported that Pluronic/Heparin nanocapsules exhibit a 1000-fold thermally reversible volume transition when the
temperature changes from 25 to 37oC. The violent volume-shrinkage would induce a rapid drug release. Therefore, the combination of magnetic and thermal properties would be a very interesting alternative for tumor therapy. Here, we developed a novel Pluronic®/iron oxide magnetic nanoparticle formulated by an in-situ co-precipitation process and evaluated it as a drug carrier. The proposed synthesis process of F127-MNPs is depicted in Fig.8.1-(a) and is characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), spectrofluorophotometry (PL), and dynamic light scattering (DLS). Thereby, we demonstrated an approach to form water-dispersible and magnetic/thermo-responsive nanoparticles formulated with F127 and iron oxide nanoparticles, which can be applied to the biomedical devices, such as drug carriers.
Fig. 8.1 Diagram showing the proposed synthesis process of F127-MNPs: (a1) F127/iron salts nanosphere formation by sonication; (a2) F127-MNPs formation after adding ammonia solution to pH=10; Proposed mechanism for drug encapsulation and delivery process of F127-MNPs by HFMF: (b1) Drug diffuses into the nanospheres at lower temperature (4oC, swelling state); (b2) Slight volume shrinkage and drug encapsulated in the nanospheres after slight heating to 15oC; (b3) Sharp volume shrinkage with accelerative drug release under HFMF treatment
F127/Iron Salts Nanospheres by Sonication
Fe(II) & Fe(III) salts and F127 nanospheres
F127-MNPs
F127 Iron Oxide Nanoparticles
10 nm
pH 10
External HFMF
DOX into F127 layer Swelling state at 4oC
DOX squeezing-out Volume Compressing
Heat
Encapsulated at 15oC Collapsed state at 35oC
(Hydrophilicity) (Hydrophobicity)
Slightly Heating
5 nm
(a1) (a2)
(b1) (b2) (b3)
8.2 Fabrication of Pluronic® F127 ferrosphere
Nanospheres of Pluronic® F127 (F127) and magnetic nanoparticles (F127-MNPs) were synthesized using an in-situ co-precipitation of Fe(II) and Fe(III) salts in the presence of Pluronic® F127 (F127, Sigma). In this process, the optimum ratio of iron oxide precursor and F127 hydrogel was described. 0.05 g of F127, 5 mmol of FeCl3.6H2O (Riedel-deHaën), and 2.5 mmole of FeCl2.4H2O (Fluka) were dissolved in 50 ml of water while being vigorously stirred at 60o C. The dissolved solution was sonicated by a horn-type ultrasonic homogenizer (VC-500 watts, Cole-Parmer Instruments, USA) operating at a constant frequency of 20 kHz for 10 min. Then, Pluronic® F127/iron oxide salt nanospheres were prepared, as shown in Fig. 8.1-(a1) (where pure F127 nanospheres (appropriate size: 10-30 nm) were made in a similar process with sonication). Later, the pH was adjusted to 10 by adding ammonia solution (33%) to the reactor while stirring. This was followed by heating to 60°C, where it remained for 30 min. After washing five times with phosphate buffered saline (PBS, pH 7.4), subsequent filtering by Acrodisc® syringe filters (Aldrich, diameter 25 mm, pore size 0.2 µm), and vacuum-drying, F127-MNPs were then successfully prepared, as shown in Fig. 8.1-(a2).
Doxorubicin (DOX, MW: 580, Sigma, USA), a well-known anti-cancer drug, was used as a model molecule. DOX was employed because of its amphoteric characteristics; it can dissolve in an aqueous environment (ca. 10 mg/ml) or in an organic solvent (high solubility in methanol and chloroform). For incorporation in the F127-MNPs, 2 ml of an aqueous solution of DOX (2 mg/ml) was added drop by drop to 3 ml of an aqueous dispersion of F127-MNPs (100 mg of particles in the 3 ml PBS solution) while stirring. Stirring was continued overnight at 4oC to allow the drug to disperse into the F127-MNPs (see Fig. 8.1-(b1)). Because the F127-MNPs exhibit swelling at lower temperatures (4oC), DOX easily diffused into the ferrospheres. Then, DOX was well encapsulated under 15oC (the collapsing state was at a higher
temperature, which formed the protecting-like layer (skin layer) and prevented drug escape), as shown in Fig. 8.1-(b2). Then, drug-loaded ferrospheres were separated from the un-entrapped drug using a magnet. F127-MNPs were washed three times by being re-suspended in distilled water and separated, as described above, using the magnetic field. The resultant samples were dialyzed by a dialysis membrane with an MW cutoff at 14,000 and stored at 4oC before the nanospheres were used.
8.3 Morphology and Characterization of F127-MNPs
Morphology of F127-shell MNPs was examined by TEM (JEM-2010, JEOL, Japan). Figure 8.2-(a) shows the TEM images of pure F127 nanospheres, displaying a ring-like structure, which was a phenomenon due to the block-copolymer (Pluronic®
F127) self-assembly. After the precursors of Fe(II) and Fe(III) salts and the ammonia solution were added, the iron oxide nanoparticles started to form by co-precipitation.
Figure 8.2-(b) shows that well-distributed F127-MNPs have a spherical geometry of 10-20 nm in diameter. Moreover, under high resolution TEM [Fig. 8.2-(b1)], the iron oxide nanoparticles were encapsulated in the interior region of F127 nanospheres, and it was a crystalline structure [in the insert, Fig. 8.2-(b2)]. In addition, the selected area diffraction pattern of the F127-shell MNPs showed five planes, namely, [220], [311], [400], [511], and [440], indicating the presence of iron oxide nanoparticles, as well as an amorphous phase, e.g., cloudy ring (see Fig. 8.2-(b3)). This again suggests that the iron oxide nanoparticles were encapsulated by F127 hydrogel.
10 nm
(a)
Fig. 8.2 TEM image of the morphology of (a) Pure F127 nanospheres; (b) Distribution of F127-MNPs; (b1) TEM image of F127-MNPs nanospheres (iron oxide nanoparticles were encapsulated in the F127 nanospheres);
(b2) High resolution TEM image showing crystalline condition of iron oxide in the F127 nanosphere; (c) Diffraction pattern of F127-MNPs
Figure 8.3 shows the results of the XRD (M18XHF, Mac Science, Japan) of the F127 nanospheres and F127-MNPs. In Fig. 8.3-(a), six diffraction peaks at 2θ = 30.1°, 35.6°, 43.3°, 53.5°, 57.2°, and 62.9° were the characteristic peaks of the crystal plane, which was the same as JCPDS 85-1436 [Long, 2004], s suggesting the nanoparticles were Fe3O4. Furthermore, the XRD spectrum of pure F127 nanospheres showed two characteristic peaks at 2θ= 18.8° and 23.1°, representing a high degree of ring-like crystallization of the F127, which was in good agreement with the results of the TEM image [Fig. 8.2-(a)]. In addition, F127-MNPs not only exhibited six characteristic peaks of Fe3O4 but also showed a broad diffraction pattern, which peaked at 21°. The diffraction pattern was assumed to be due to the presence of iron oxide that deferred the ring-like crystallographic structure of the F127 phase. These results were similar to those of the diffraction pattern of the TEM (cloudy ring), indicating that both phases were intimately contacted.
Raman spectra are potentially more useful than diffraction techniques to track (511)
(311) (220) (400) (440) (b1) (b2) (b3)
10 nm (b)
subtle structural differences between the vibration frequencies of F127 and Fe3O4
MNPs. Figure 8.3-(b) shows that, although F127 hydrogel were surrounded with iron oxide nanoparticles, a significant difference between these phases can be recognized from the Raman spectra. Both 665 and 704 cm-1 bands, assigned to the characteristic band of Fe3O4 [Long, 2004] were attributed to the vibrational modes. These consisted of the stretching mode of oxygen atoms along Fe-O bonds (metal oxide bond), and four characteristic peaks at 492, 537, 603, and 709 cm-1 were assigned to F127 nanospheres Furthermore, from the sample of F127-MNPs, six peaks, including the peaks from both F127 and Fe3O4 MNPs, were observed where the two peaks of 492 and 537 cm-1 were almost combined to form a broad peak. This suggests that there was some interaction between metal oxide and the -OH group in the F127 when the iron oxide nanoparticles were formed inside the F127 hydrogel.
In addition, the change in chemical-binding energy in F127-MNPs was investigated in the XPS (ESCALAB 250, Thermo VG Scientific, West Sussex, UK), equipped with Mg Kα at 1253.6 eV at the anode. As illustrated in Fig. 8.3-(c), the binding energy of O1s was detected from 526.0 to 538.0 eV, and the peak of iron oxide was at 530.0 eV. This peak represented the metal oxide, which was reasonably consistent with peak reported in the literature, i.e., 528-531 eV. Also, the -OH group peak of pure F127 nanospheres was found in the 532.8 eV, which was similar to the report of Anderson et al. [Anderson, 2001], showing a highly ordered arrangement of ring-like crystallization of the F127 nanospheres. However, after incorporation into the iron oxide nanoparticles (F127-MNPs), the peak at 532.8 eV shifted to a lower energy, 532.4 eV, indicating that the ring-like crystallization of the F127 nanospheres had been deteriorated to an amorphous-like structure due to the presence of iron oxide nanoparticles. In the meantime, the peak of the metal oxide in the F127-MNPs shifted to a higher energy, i.e., from 530.0 to 530.3 eV, suggesting that some interactions occurred between the metal oxide and the –OH group in the F127 hydrogel when the iron oxide was formed inside the F127 hydrogel. Although the actual mechanism for the shift of the binding energy of metal oxide was unclear at present, it is believed that iron ions can be encapsulated by the F127 hydrogel. This hydrogel promoted a self-assembly of the iron oxide salt to form crystal structure while increasing solution
pH upon synthesis, and it thus accompanies some ion-ion interaction to change the binding energy of metal oxide [Hu, 2007].
Fig. 8.3 Characterization analysis of F127 and F127-MNP nanospheres: (a) X-ray diffraction, (b) Raman spectra, and (c) XPS spectra
10 15 20 25 30 35 40 45 50 55 60 65
526 528 530 532 534 536 538
Binding Energy /eV
Binding Energy /eV