Summary - Core/Single-Crystal-Shell Nanospheres for Controlled Drug Release

Chapter 5 Core/Single-Crystal-Shell Nanospheres for Controlled Drug Release

5.8 Summary

In conclusion, we have demonstrated a novel core-shell nanosphere with PVP-modified silica core following a functional deposition of a single-crystal iron oxide shell. Such a core-shell nanosphere offers a surprisingly outstanding controlled release and non-release behavior for the molecules encapsulated inside the silica core.

The dense, single crystalline shell is efficiently preventing the fluorescence dye from un-desired release, giving that an undesirable leakage of the molecule during the course of delivery is completely inhibited. More than that, the molecules encapsulated in the core can be released with a highly controllable manner, through the use of a magnetic stimulus. It is envisioned that the drug of interest can be released in précised dosage in a remotely controlled manner or burst as the shell being ruptured when reached the diseased sites. We also envision from this study that these core-shell nanospheres are expected to play a significant role in the development of new generation of site-specific controlled-release drug delivery nano-device.

Chapter 6 A Multifunctional Nanodevice capable of Imaging, Magnetically Controlling, and In-situ Monitoring Drug Release

6.1 Introduction

Surface modification with functional attachments is a widely used technical strategy to enhance biological, optical and chemical functionality of materials in a wide variety of biomedical applications, such as imaging, diagnosis, drug delivery, implants, and so on. Recently, the development of multifunctional, nanomedical platforms, through skillful combination of different nanostructured materials, has been proposed. Owing to the specific advantages of these nanomaterials, many studies have been done on multimodal imaging and simultaneous therapy. For example, magnetic nanoparticles were infused with fluorescent dye to construct multimodal imaging probes. Among the many nanoparticulate systems, semiconductor nanocrystals known as quantum dots (QDs) confer advantages over traditional fluorescent molecules, such as organic dyes. This is because of their unique optical properties, including narrow photoluminescence spectra, low photo-leaching, and high resistance to chemical degradation. QDs have also been reported to carry therapeutic agents for healing applications. The combination of both imaging and therapeutic functions in nanoparticles introduces an attractive advance in the field of biomedicine, diagnosis, and pharmaceutics.

However, there has been little investigation into fast-response, controlled drug release from nano-platforms under magnetic stimulation, or monitoring of the release of therapeutic molecules in a quantitative manner. Therefore, an integration of these functions will be technically significant in developing a multifunctional nanodevice capable of simultaneous imaging, controlled drug delivery and in-situ monitoring of

drug release with molecular resolution.

In our earlier research, we developed a new type of magnetic core-shell nanocapsule which allows active molecules to be expelled in the presence of a remotely controlled magnetic field. The active molecules released from the nanocapsules can be finely tuned according to exposure time in the magnetic field.

However, it would be desirable to further engineer the nanocapsules to monitor the release of active molecules at nanometric or cellular resolution. We also believe that a nanocapsule should be able to provide easily monitored, high-resolution optical information, viewable via simple spectroscopic methods, rather than conventional MRI imaging. On this basis, here, we designed a new drug delivery nanodevice by deposition of quantum dots (QDs), i.e., Zn-Cu-In-S (ZCIS) nancrystals, onto the surface of a core-shell drug delivery nanocapsules recently developed, to form a nanometric multifunctional platform. The core-shell nanocapsule consists of a polymer core covered with a thin layer of single-crystal iron oxide shell. This unique core-shell structure offers great therapeutic potential for controllable drug release, with a dosage controlled via an external magnetic field. The quantum dots deposited on the surface of the magnetic nanoparticles are used for directly monitoring the release of the drugs. This is achieved by observing variations in the fluorescence intensity of quantum dots. The fluorescence intensity of quantum dots varies with absorption of thermal energy matching the excitation binding energy of QDs. Free carriers generated by exciton dissociation can tunnel between nearby QDs, causing a decrease in the fluorescence intensity. While the QDs are attached to the surface of the magnetic nanoparticles, the heat energy induced by the external HFMF can transfer from the magnetic shell to QDs via magnetic nanoparticles. Since this design is conceptually and technically achievable, it is further believed that the attachment of the ZCIS QDs on the surface of the nanocapsules may act not only as a strong

fluorescence-emitting agent, but also as a nanometric sensor to monitor drug release, in real-time, within a magnetic field.

6.2 Experimental section of multifuntional nanodevices

Preparation of the nanodevices. Synthesis of the PVP-Fe₃O₄ core/shell nanospheres was reported in the previous section. To encapsulate the model drug, fluorescence molecules, in the polymer matrix, 4 Wt. % Polyvinylpyrrolidone (PVP;

Mw~10,000; Sigma) was dissolved in D.I. water and preheated to 80 ^°C. Then fluorescence molecules were added into the solution and mixed for 6 hours. In suitable concentrations, the PVP will assembly themselves as nanospheres in the solution, forming drug-loaded PVP nanoparticles. Under nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O with a FeCl2/FeCl3 molar ratio of 2:1 were dissolved into water and mixed with the drug-loaded PVP nanoparticles under vigorous stirring at 80^°C. After 4 hr, the iron salts were deposited on the surface of the drug-loaded PVP nanoparticles. This was achieved via slow addition of 2 ml of ammonium water (NH4OH, 33%), causing precipitation so that iron oxide shells were immediately formed on the surface of PVP nanoparticles. At this stage, a drug-loaded PVP core-iron oxide shell nanosphere exists in the solution. Precipitated powders were collected by centrifugation at 6000 rpm, removed from the solution, and washed in D.I. water four times. The PVP-Fe3O4 core/shell nanospheres were separated by the centrifugation. The average diameter of the core-shell nanospheres was about 12-20 nm.

To grow the ZCIS quantum dots on the surface of the PVP-Fe₃O₄ core/shell nanospheres, they were re-dispersed in trioctylphosphine (TOP, 90%, technical grade) with diethyldithiocarbamic acid zinc salt, [(C₂H₅)₂NCSS]₂Zn. The solution was diluted with octadecene (ODE, 90%, technical grade) to form Solution 1. Then, CuCl

and InCl₃ were dissolved in oleylamine at 50 ^°C to form Solution 2. The two solutions were mixed and heated to 140^°C in a nitrogen atmosphere for several minutes.

Deposition of quantum dots (QDs) on the surface of the PVP/Fe₃O₄ core-shell nanospheres caused the solution to turn yellow.

Characterization. X-ray photoelectron spectroscopy (XPS) was performed in an ESCALAB 250 (Thermo VG Scientific, West Sussex, UK), equipped with Mg Kα peaks were standardized with respect to C 1s peak at 284.6 eV.

Cell Culture and In-Situ Monitoring of Drug Release. HeLa, human cervical cancer cells, were maintained in Dulbecco‘s modified Eagle‘s medium (DMEM) with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37 ^°C in a humidified atmosphere of 5% CO2 in air. The nanodevices were incubated with the cells for 12 hours. The cells were then subjected to a high frequency magnetic field (HFMF) for 0, 90 and 180 sec. Results were observed by PL microscopy (Nikon TE-2000U, Japan). Digital analysis software (Nikon, Japan) was used to analyze the fluorescence intensities of the model drug and the nanodevices.

The conditions of exposure were the same for each color channel. Analysis was done by Nikon C1 software, which defined the fluorescence intensity from 1 to 255. The range of the fluorescence intensities were: Blue channel (60-255), Green channel (40-255), and Red channel (30-255).

6.3 Structures of multifunctional nanodevices

The procedure for synthesis of magnetic nanocarriers is schematically illustrated in Figure 6.1a. Poly-(N-vinyl-2-pyrrolidone) (PVP) nanoparticles containing a test molecule, such as fluorescent dye, were prepared through a self-assembly process in benzyl ether. After self-assembly, the PVP molecules are aggregated into a micelle

Figure 6.1 (a) Schematic of the stimuli-response nanodevice delivery system where quantum dots were deposited on the shell of the PVP/Fe3O4 core-shell nanospheres. Attachment of the ZCIS QDs on the surface of the nanospheres acts not only as a strong fluorescence-emitting agent, but also as a sensor to monitor the drug release in a real-time basis under magnetic induction. (b) TEM image and (c) HRTEM image of the PVP-Fe3O4

core-shell nanospheres. (d) HRTEM image of the ZCIS-doped nanospheres. After incorporation of the ZCIS QDs on the core-shell nanospheres, the suspension displayed a fluorescence character under the UV light (inset picture).

structure. Drug molecules are embedded within the amphiphilic nature of the PVP.

Following micelle formation, a thin layer of iron oxide, is deposited on the core surface in single-crystal form. The resulting PVP-Fe₃O₄ core-shell nanocapsules, shown in Figure 6.1b, display a spherical geometry ranging from 10 nm to 15 nm in diameter. High-resolution transmission electron microscopy (HR-TEM), shown in

Figure 6.1c and in the supporting information of Figure 6.2,

confirms that the structure of the nanospheres is an amorphous core and a thin single-crystal shell which suggests self-organization of the iron oxide precursor upon nucleation and

Iron salts Q-dots

Single-Crystal Fe3O4 Shell

ZCIS Q-dots

(a)

Model Drug

Polymer Matrix

Nanocapsule Nanodevice

(b)

10 nm

(c)

2 nm

(d)

2 nm

Figure 6.2 Local Fourier transfer patterns of single-crystal iron oxide shell indicate that the shell exhibits uniform and homogenous crystalline orderliness along the surface of the core phase.

Figure 6.3 (a) The TEM image and local Fourier transfer patterns of ZCIS QD-Single crystal Fe3O4 shell nanoplatform. The local Fourier transfer pattern also demonstrated a high crystallinity of the ZCIS QD. (b) The TEM image of CIS QD-Single crystal Fe3O4 shell nanoplatform. The EDS investigated the Fe3O4 shell and ZCIS QD, corresponding to the regions 1 and 2 in the HRTEM image, respectively. HRTEM image shows the solid nanoparticle attaching the ring-like shell region being ZCIS QDs in the heterodimer. The energy dispersive X-ray spectrometer (EDS) analysis confirms that the ring-like region mainly consists of Fe and the small solid particle consists of Cu and Se.

(a) (b)

(c) (d)

Region 1

Region 2

Region 1 Region 2

-10000 -5000 0 5000 10000

-60 -40 -20 0 20 40 60

Applied Field (Oe)

Magnetization (emu/g)

Nanodevices without QDs Nanodevices

Figure 6.4 Field-dependent magnetization curve of nanodevices with and without quantum dots.

growth in the presence of the PVP. Although limited to metallic crystals, this agrees with an earlier study of a similar synthesis scenario. In the current core-shell system, iron ions can be efficiently anchored onto the pyrrolidone ring of PVP. This provides epitaxial-like growth of the oxide to form single-crystal structure. After the core-shell nanocapsules were synthesized, ZCIS quantum dots were prepared and grown on the shell surface. A procedure described by Nakamura et al. is used to form Fe₃O₄shell -ZCIS heterodimers.^[147] An HRTEM image, shown in Figure 6.1d, shows the solid particle residing on the ring-like shell, corresponding to a ZCIS heterodimer. Energy dispersive X-ray spectrometer (EDS) analysis confirms that the ring-like region mainly consists of Fe and the small solid particle consists of Cu and S shown in the

Figure 6.3. The local Fourier transfer pattern also demonstrates a high crystallinity of

the ZCIS QD. After incorporation of the ZCIS QDs onto the shells, the resulting suspension shows a strong yellow appearance under UV exposure. This can be seen in the inset picture of Figure 1d and suggests that these ZCIS-modified nanocapsules (hereafter called nanodevices) can be used not only as drug nanocarriers,

Figure 6.5 Schematic drawing showing multi-functionalities of each compartment from as-designed nanodevices for nanoimaging, controlled drug release, and in-situ motoring of drug release.

Figure 6.6 Cellular uptake of the nanodevice was evaluated using HeLa cells, by incubating the cell line with both mercaptoundecanoic acid-modified nanodevice (MUA-NDs) and folic

• Changeable colors with drug release and deformation of Fe₃O₄ shell

acid-modified nanodevice (FA-NDs). Both the MUA-NDs and FA-NDs were uptaken by the cells in 4 hours of period, probably through endocytosis, with different degrees of efficiency.

In comparison, the majority of the FA-NDs can be clearly observed in the cytoplasm region of the cell, but only a few MUA-NDs was uptaken by HeLa cells, indicating that the folic acid-modified version promotes a stronger cell-specific intake by the HeLa cell line than that of the mercaptoundecanoic acid-modified version. The folic-acid-modified nanodevices showed excellent cell-specific uptake efficiency, through possibly endocytosis.

but also as nano-probes for imaging. (Figure 6.5) The magnetic properties of the nanodevices and nanocapsules were measured by SQUID at 298 K, with the magnetic field sweeping from -10000 to +10000 G. The correlation of magnetization with magnetic field for both the nanodevices and the nanocapsules, shown in Figure 6.4, demonstrates a similar shape with negligible hysteresis, showing superparamagnetic behavior. The saturation magnetization (Ms) of the nanodevices is smaller than that of the nanocapsules, due to dilution effect.

6.4 Drug release and in-situ monitoring abilities of the nanodevices

The nanodevices were surface-modified and conjugated with a targeting ligand, folic acid (FA), to cause hydrophilic behavior. The process is illustrated in Figure 6.6 After modification of the nanodevices, a stable aqueous suspension was prepared. The fluorescence spectrum of the ZCIS remained identical to the initial preparation, indicating that folic acid imparts no adverse effect on the optical properties of the ZCIS QDs. The suspension was subjected to a high frequency magnetic field (HFMF) for investigation of the drug release mechanism. Prior to magnetic stimulation, the green-fluorescence loaded nanocapsules showed no sign of release in 24 hours of storage in an ambient environment. This was confirmed via PL spectroscopic monitoring and suggests that the dye molecules are encapsulated in the core phase for a long period of time without any unwanted leakage. However, subjection to the

HFMF for varying lengths of time, as shown in Figure 6.7a, reveals an interesting phenomenon. The intensity of the dye emission peak at 517 nm increased with the duration of the magnetic field. In contrast, the intensity of the emission peak from the ZCIS QDs showed an opposite relationship, a decrease in emission intensity. The increase in peak intensity with time upon magnetic stimulation indicates a magnetically-induced release of dye molecules from the nanodevices. In a recent

Figure 6.7 (a) Emission spectra of dye-loaded nanodevices (30 mg per 10 mL water) under HFMF treatment over a time period from 0 s to 100 s. Before HFMF exposure, the dye-loaded nanodevices displayed no sign of dye release, which causes green fluorescence at an emission wavelength of 517 nm, as determined by fluorescence spectrophotometer.

However, a strong emission signal from the QDs after exposure, red fluorescence at an emission wavelength of 581-614 nm, show that dye was released from the nanodevices. A degenerative green florescence appeared concurrently. (b) Model drug intensity versus quantum dot intensity curves originate from both the dye and ZCIS emitting spectra and show a near symmetrical profile under different magnetic field strengths.

500 550 600 650 700

study, such a release can be easily and precisely controlled with manipulation of the surrounding magnetic field; from a burst-like profile upon stimulation to a zero release profile immediately after removal of the magnetic field. The emission peak from the ZCIS quantum dots showed strong PL intensity at the beginning of the stimulus. However, as time elapsed, a gradual reduction in peak intensity together with a red shift of the PL spectra for the QDs was clearly detected over an operation time of 100 seconds. The peaks for the dye and QDs reacted oppositely under the same magnetic stimulus, implying a potential correlation of both spectra.

Bias may arise when determining whether the correlation is simply coincidence or a rule in this nanosystem. To avoid this, magnetic fields of varying intensities were applied. This aimed at determining whether the correlation of the spectra between drug release and quantum dot emission is sustainable over a range of operating intensities, from 0.8 kA/m to 2.5 kA/m in the current magnetic system. The resulting correlation in the magnetically-induced changes of spectra are given in Figure 6.7b.

There exists a linear relationship between the spectral intensity of dye molecules and the QD emission. The correlation coefficient over a range of the data reaches 0.97-0.99 and can be obtained for different magnetic intensities. The quantity of drug released relied mainly on the strength of magnetic field under short-term induction.

This implies that a weaker magnetic field results in the slower release of drugs. A linear relationship exists over the entire range of operating conditions. Although the emission spectra were measured from two independent sources, this finding strongly suggests that the drug release from the nanodevice can be quantitatively correlated with in-vitro, doped ZCIS QDs. to high precision. Dye release can be precisely monitored along with the spectral variation of the ZCIS QDs from the nanodevices.

This finding is in agreement with the hypothesis that two independent mechanisms can be triggered simultaneously from a given magnetic field, and that the mechanisms

will also be quantitatively correlated. We believe that this highly correlated relationship may also be adopted for in-situ monitoring of cellular systems. This will be explained further.

Figure 6.8 Temperature curve of nanodevices during application of HFMFs of different strengths.

6.5 Spectrum variation of the ZCIS QD under magnetic induction

The mechanism causing the spectral variation of the ZCIS QDs upon magnetic induction is of great interest. To further explore the possible causes, a separate photoluminescence test for the ZCIS QDs was performed in the same magnetic field;

no detectable sign of change in the emitted spectrum was observed. This indicates an invariance in the spectral properties of the ZCIS QDs with respect to magnetic field.

Since the variation of emitted spectrum for quantum dots has been proven to be associated with a change in the band energy structure or surface composition of the QDs, it is highly plausible that a number of factors cause changes in the resulting spectrum of the ZCIS QDs. These changes may include magnetically-induced heating, surface adsorption of the dye molecules, and/or surface corrosion due to the presence of water. However, according to experimental observation, the above factors have to

be rapidly responsive and probably interdependent since the decrease in the emission intensity and a red shift of the spectra occurred relatively quickly, in less than 20 seconds, in the presence of the magnetic stimulation at 2.5 kA/m. More interestingly, it is evident that the spectral variation has experimentally proven to be irreversible.

These observations suggest that a permanent change in either chemical or physical structure has been induced rapidly on the ZCIS QDs while the nanodevices were subjected to magnetic stimulus.

To further explain the reasons behind the spectral degeneration of the QDs under short-term induction, nanodevices not carrying the dye molecules were prepared and exposed to the same magnetic field. The resulting emission spectra were identical to the case of the nanodevices with the dye. This indicates that dye molecules exerted little or no influence on resulting PL emission spectra of the ZCIS QDs. The PL spectrum of the dye molecules remained identical in shape and emission peak position to that given in Fig. 6.2, suggesting that the dye molecules, after release into an aqueous environment, remained chemically and physically stable. There is a magnetically-induced temperature rise, or hot spot, on the shell of the nanodevice due to magnetic energy dissipation from single-domain particles, such as a Brown and/or Neel relaxation, where this has been verified in a number of recent studies.^[137] From experimental observation, the temperature of the solution that contained the nano-devices gradually rises. As shown in Figure 6.8, while applying a high strength magnetic field of 2.5 kA/m, the temperature of the solution increased to about 40^oC in 4 minutes. Since the temperature rise of the solution is solely resulting from the energy dissipated from the magnetic nanodevices, the temperature of the nanodevice itself should be much higher and can be calculated by the thermodynamic relationship,

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