Chapter 5 Core/Single-Crystal-Shell Nanospheres for Controlled Drug Release
5.7 in vitro controlled release
Since the core-shell nanospheres are able to provide precise control of release and non-release characters for the molecules, which provides greatest advantages for drug delivery uses, however, it is important to investigate the endocytosis following a magnetically-triggered drug release behavior of the nano-device within cells. To further elucidate such a behavior, HeLa (human cervical cancer) cells were incubated overnight with the magnetic nanospheres loaded with green fluorescence dye to allow the endocytosis of nanospheres to be optically examined. Figure 5.6a shows the optical and fluorescence images of the cell line before HFMF treatment where a relatively weak green fluorescence was observed. However, after 30 seconds of HFMF stimulus, green fluorescence was clearly observed within the cell bodies of these HeLa cells upon excitation at 494 nm (Figure 5.6b) which strongly indicates that the dye-loaded nanospheres were efficiently uptaken by the cells and were release rapidly under a well-controllable manner within the cells. The dye molecules were also illustrated a well-controlled non-release behavior during the time period of the test. Therefore, we envision from the appearance of healthy intact and the visibility of fully grown cells suggested that the nanospheres are biocompatible in vitro under experimental conditions and precisely released desirable molecule for therapeutic purposes.
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Figure 5.6 Fluorescence micrographs of HeLa cells after 10 hours incubation with fluorescence-loaded PVP-modified Silica/Fe3O4 core-shell nanospheres. (a) without HFMF treatment; (b) after 30 seconds of HFMF stimulus, green fluorescence was clearly observed within the cell bodies of these HeLa cells upon excitation at 494 nm.
5.8 Specific power absorption rate (SAR) of nanocapsules
Functional magnetic nano-capsules (f-MNCs) can provide dual effect to therapy cancer; one is to magnetically control drug release in specific site, and the other is hyperthermia treatment of cancer. When the magnetic nanoparticles were subjected to a high-frequency magnetic field (HFMF), the resulting eddy current, hysteresis, and Brownian rotational losses can produced a source of heat capable of imparting cell death. The ability to combine MRI contrast enhancement and therapy in the form of hyperthermia and controlled drug release as well as the possibility of functionalizing particles specific to biomarkers may further advance the use of magnetic nanoparticles in biomedical application. To further understand the ability of hyperthermia of f-MNCs, the different strength of high-frequency magnetic field
Before HFMF Stimulus
(a)
After HFMF Stimulus
(b)
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Figure 5.7 (a)Temperature curve of nanocapsules during application of HFMFs. (b) Drug release behavoirs of nanocapsules under HFMFs, and their release rates (k).
was used. The heating field is always generated by AC typically in the radio-frequency (RF) range, 104~105 Hz. Because an AC field can produce an eddy current, induction heating is always feasible for conductor, and it exhibits more efficient for a magnetic material in which magnetic hysteresis causes additional energy dissipation. To enhance the sum of eddy current heating and magnetic heating, the relative large magnetic coercivity (mainly due to the resistance to domain wall movement) is preferred. However, functional magnetic nano-capsules (f-MNCs) are superparamagnetic because of the single-crystalline and the particle size, it is a
(a)
(b)
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Table 1 The specific power absorption rates (SAR) of functional magnetic nanocapsules (f-MNCs) under the specific strength of magnetic field. H is the strength of magnetic field with an alternating frequency of 50 kHz. dT/dt is the initial heating rate, and C is the specific capacity of f-MNCs.
single-domain ferromagnet free to switch following a quasi-state field without apparent coercivity. There is only little coercivity contribution, so the energy dissipation must come from some sort of internal or boundary ―friction‖ which drags the magnetic moment letting it lag the AC field. In linear-response medium, the Debye theory describes this lag in terms of a relaxation time. Under magnetic heating, the temperature of the f-MNCs solution gradually rises reaching a steady of several to several tens of degrees of centigrade higher. At this temperature, the heat of f-MNCs absorption equals the heat loss at the external boundary (container, solution). The initial heating rate of magnetic dissipation is informative, the order of 0.1oC/s to 0.5oC/s for f-MNCs, which is depending on the strength of magnetic field. As shown in Figure 5.7a, while applying the magnetic-field strength of 2.5 kA/m, the temperature of the solution increased about 25 oC in 10 minutes. In order to compare the energy absorption and drug release behavior of f-MNCs induced by the high-frequency magnetic field, the specific power absorption rate (SAR) was calculated. The SAR values of magnetic fluids strongly depend on the alternating magnetic field and the magnetic fluids properties, such as particle size, size distribution, anisotropy constant, saturation magnetization. In our study, a high frequency magnetic field with different strength at 50 kHz was applied to the functional magnetic nano-capsules (f-MNCs). The SAR of a magnetic fluid is
H (kA/m) dT/dt C (J/gK) SAR (W/g) k R2
0.8 1.25 1.04 1.30 0.0011 0.986
1.4 2.00 1.04 2.07 0.0031 0.998
2.0 2.9 1.03 3.00 0.0061 0.998
2.5 3.75 1.04 3.89 0.011 0.998
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determined by the initial linear temperature rise of a fluid measured after subjecting to the magnetic field:
where C is the heat capacity of the sample and (△T/△t) is the initial slope of the temperature data. The SAR values were summarized in Table 5.1 to further compare with the drug release behaviors. To investigate the release mechanism of the drug
where Mt is the mass of drug released at time t, M is the mass released at time infinity, and Mt/M is the fractional mass of released drug; k is a rate constant, and n is a measured by PL spectroscopy. After 3-min period operation of HFMF, the cumulative release of f-MNCs can reach to 87 % under strength of 2.5 kA/m. With decreasing power of magnetic field to trigger drug release, the cumulative release of f-MNCs showed a relative lower amount, suggesting the release rate is high corresponding to the magnetic field power. More interestingly, the release profile of the f-MNCs
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1.0 1.5 2.0 2.5 3.0 3.5 4.0
0.000 0.002 0.004 0.006 0.008 0.010 0.012
R=0.98
SAR
k
Figure 5.8 A near-linear relationship between the rate constant k and SAR values, with correlation coefficient as high as 0.98 can be obtained.
displayed a zero-order release pattern under the stimulus. In other words, the drug molecules released from the nanocarriers, albeit in a burst-like profile under HFMF stimulus, can still be well regulated with a controllable dosage. By taking Eq. (3), a kinetic analysis of drug release from the f-MNCs can be obtained, as shown in Figure
5.7b, where release kinetics under different strength of magnetic field treatment. Both
exponent constant, n and rate constant k, are estimated. For the release kinetics, the exponent constant is in a range of 0.83-1.05, indicating the release mode under the high-frequency magnetic field (HFMF) treatment similar. However, the n values under the HFMF can not be explained by typical diffusion mode because an external energy powerfully controlled the release behaviors. The rate constant, k, for f-MNCs under strong magnetic field (2.5 kA/m) is apparently higher than others, indicating that the strength highly affects the burst release property. With the decreasing the strength of magnetic field, the rate constants relatively decreased. In order to compare the relation between strength of magnetic field and release rate, SAR values were defined as the energy that the external magnetic field donated to nanoparticles because SAR values can express the energy absorption of nanoparticles. The resulting80
correlations of the magnetically-induced drug release rate are given in Figure 5.8, where a near-linear relationship between the rate constant k and SAR values, with correlation coefficient as high as 0.98 can be obtained. The finding strongly suggests, albeit the heating production and the drug release behavior were seemingly independent, that drug release rate from the f-MNCs can be correlated over a relatively high precision with SAR value. To this end, we can use the SAR values calculated by the energy absorption of f-MNCs to estimate the drug release rate in specific strength range of magnetic-field based on this newly-designed drug nano-carriers may also adopted for cellular or animal systems in the future.
Applying magnetic field to trigger drug release from nanocapsules is a convenient way because it is a non-contact energy for a specific-site therapy. However, the intensity of magnetic field is not easy to form uniformly in the magnetic coil, especially for large volume of magnetic field. For this reason, a 9-cm diameter of magnetic field was estimated to understand the different regions of magnetic strength and the relative drug release behaviors of nanocapsules in the cell. First, as shown in
Figure 5.9, the magnetic field was divided into 5 regions from center defined by
specific power absorption rates (SAR). The highest SAR is 3 W/g in the red region of center, and the magnetic strength gradually decreased form red region to the blue region of 1.4 W/g. The f-MNCs was uptaken through the endocytosis in the the A549 cell lines by incubating of 12 hours.The possible mechanism of magnetically-triggered drug release form functional magnetic nanocapsules (f-MNCs) has been conjectured in our previous report. The high frequency magnetic field (HFMF) is able to induce vibration of the magnetic shell, resulting that an irreversible change of the core-shell nanostructure if the physical deformation is large enough to cause the permanent damage under long-term MF treatment. To further investigate the behaviors of the deformation or even rupture of the f-MNCs, the HFMF treatment and directly-heating the nano-capsules were
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Figure 5.9 SAR effects on the different manetic field regions. Drug release behaviors in cells are highly relative with the strength of SAR.
carried out individually in this study to interpret the effect of heat and magnetically-induced force to the nanostructure. To estimate the temperature of the f-MNCs after the HFMF treatment, the initial heating rate of magnetic dissipation is informative, the order of 0.1oC/s to 0.5oC/s for f-MNCs, which is depending on the strength of magnetic field. Since the energy input of the solution is entirely from the energy input of the magnetic nanoparticles, the temperature of the f-MNC should be calculated by the thermodynamic relationship, ΔH=mwCwΔTw= mmCmΔTm. Here Cw and Cm are the specific heat capacity of water and the magnetic material, respectively, and m is the weight. If without any heat transport to the surrounding water, the temperature of the f-MNCs after subjecting the HFMF for 1 min can rise to 200 oC under the strength of 2.5 kA/m. However, the high specific surface area of the nanocarriers would rapidly decrease theirs temperature through the heat transfer to the surrounding solution so the temperature of the particles should be much lower than the theory calculation. After subjecting the f-MNCs to the HFMF for 15 minutes, the crystallographic phase was examined by the X-ray diffractometer, at a scanning rate of 6o per minute. The X-ray diffraction patterns exhibited the similar crystalline
-4 -3 -2 -1 0 1 2 3 4
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Figure 5.10 The TEM electron beam is focus on the nanocapsule for different time durations.
While increasing the heating time to 40 minutes, the obvious deformation of melting was observed.
phases of f-MNCs with different time of HFMF treatment, which are Fe3O4 (magnetite, according to JCPDS [85-1436]). The finding demonstrated that the temperature of the f-MNCs under the HFMF is lower than the temperature of the iron oxide phase transition, indicating the f-MNCs still maintain low temperature even under the long-term of HFMF treatment. In order to understand the behaviors of the f-MNCs with heating, the high-resolution transmission electron microscopy (HR-TEM) was applied to continue focusing on the nanoparticles for 40 minutes, and the images were monitored as shown in Figure 5.10. In such high magnification of TEM images, the electron beam can rapidly heat the nanoparticles more than 200 oC within 10 minutes. With continue focusing for 10 minutes, the morphology of the nanocapsule was still similar to the original one. However, in Figure 3c, increasing to 15 minutes of electron beam heating, the surface of nanocapsules starts to slight melt.
While increasing the heating time to 40 minutes, the obvious deformation of melting was observed. These results demonstrated that the heat can only melt the nanocapsules, but not to crack the nanocapsules. The phenomena of heat melting and cracking inducing by HFMF are significantly different, suggesting that HFMF probably provided energy to induce some mechanical force in the inner structure of magnetic nanocapsules, and then to rupture the structures. Some sort of internal or
10 min
0 min 15 min 40 min
Time under the electron beam
(a) (b) (c) (d)
Surface melting
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boundary ―friction‖ were believed to drags the magnetic moment with the Néel relaxation which implied the magnetic moment rotates within the crystal.[27] This inner energy can cause the unstable boundaries of nanocapsules to crack or even rupture, especially in the ulta-thin magnetic shells. Such a nanostructural evolution under HFMF stimulus surely enhances the burst release behavior from the magnetic nanocapsules.
Figure 5.11 (a) Field-dependent magnetization curve and (b) XPS analysis of nanocapsules for different time of HFMF treatment.
The magnetic property of functional magnetic nanocapsules (f-MNCs) was estimated by SQUID at 298 K with the magnetic field sweeping from -10000 to +10000 G. Figure 5.11a shows the correlation of the magnetization with magnetic
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field for functional magnetic nanocapsules (f-MNCs) with different duration time of HFMF treatments, where the curves show similar shape with negligible hysteresis.
The result shows there is no obviously difference between magnetic properties with the HFMF treatment, indicating that the nanocapsules didn‘t change the crystal phase or chemical states. This finding is also corresponding to the XRD results as previous section discussion. In this duration of HFMF treatment, the nanocapsules can receive enough energy to rupture or crack but not to cause phase transfer, demonstrating the inner force produced by Néel relaxation leading a strong physical destruction. X-ray photoelectron spectroscopy (XPS) was employed to clarify surface chemistry of the f-MNCs after magnetic induction. A change in the binding energy spectrum of O 1s,
Figure 5.12b, of the f-MNCs from 529 eV (before the stimulus) to 533 eV (after the
stimulus) is clearly detected. The peak of iron oxide was at 529 eV, which is reasonably consistent with literature report, i.e., 528-531 eV. After applying HFMF for 15 minutes, some of the Fe-O-Fe bonds were broken, and different binding of oxide appeared, which represented the oxide binding to the hydroxide. The finding suggested that new interfaces between iron oxide shells and water produced after long-term HFMF treatment, in the other words, the nanocapsules cracked and then to cause these new interfaces.In conclusion, we have demonstrated a novel functional magnetic nanocapsule (f-MNC) with polymer core following a functional deposition of a single-crystal iron oxide shell. Through an external high-frequency magnetic field, these f-MNCs offer an excellent controlled release behavior for molecules encapsulated inside the polymer core by a non-contact force. By the specific power absorption rate (SAR) values investigate previously, the precise drug release rate can be predicted in the body. It is envisioned that the drug of interest can be released in precise dosage in a remotely controlled manner or burst as the shell being ruptured when reached the diseased sites. On the other hand, the mechanisms of f-MNCs rupture were examined
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through heating directly and applying magnetic field. The results demonstrated that the heat induced by HFMF only can melt the iron oxide but not to crack the nanostructures, indicating the HFMF provided an inner mechanical force to rupture the nanocapsules. From this study, we envision that these f-MNCs are expected to play a significant role in the development of new generation of site-specific controlled-release drug delivery nano-device.