Chapter 6 A Multifunctional Nanodevice capable of Imaging, Magnetically
6.6 Operation mechanism of the nanodevice
Based on the mechanism of degeneration of the emission spectrum of the ZCIS QDs and the deformation mechanism of the shell, outlined in a previous investigation , we propose an operation mechanism for the nano-device in a magnetic field. Figure
6.11a shows a schematic drawing of the mechanism of drug release and shell
deformation associated with the structural degeneration of the attached QDs.Accordingly, after 60 seconds of exposure to the stimulus, the single crystal nanoshell structure was subjected to lattice deformation, forming nano-sized polycrystals of varying orientations, shown in Figure 6.11b. Nanometric scale boundaries between the nano-polycrystalline domains formed and provided numerous nano-conduits that allow leaking of the dye molecules to the environment. In other words, for short-term induction, extremely small cracks or crevices were formed along the boundary regions of the thin iron oxide shell and are kept open by the presence of the magnetic field.
Extended magnetic stimulus enlarged the nano-crevices until permanent rupturing
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Figure 6.11 (a) Schematic illustration of the nanodevice with a proposed mechanism for controlled release of the dye molecules, as well as the degeneration of fluorescence intensity of the ZCIS QDs (b) Shell vibration causing enlargement of the dimensions of nano-crevices along the deformed single-crystal shell, rendering dye release upon short-term magnetic stimulation. (c) After long-term exposure, the deformed shell has received a sufficient amount of the energy to cause a final, permanent, mechanical rupture. Meanwhile, a rapid surface oxidation altered the surface structure of the QDs, leading to substantial degeneration of the fluorescence intensity.
of the shell occurred. At this time, the dye molecules were released from the nanodevice easily and completely, as shown in Figure 6.11c. Meanwhile, the energy structure of the ZCIS QDs altered considerably as a result of thermally-induced photo-leaching along with surface oxidation. This resulted in degeneration of the fluorescence intensity. While the shell was undergoing permanent rupture, the bandgap structure of the QDs was irreversibly altered, turning a dark red after about 100 sec at a magnetic field strength of 2.5 kA/m.
6.7 In-situ-monitoring of drug release in cancerous cells (b)
2 nm
QD
(c)
2 nm
QD (b)
Single-crystal Shell (Yellow)
Poly-crystal Shell (Red)
Rupture (Dark)
Oxidation (C)
(a)
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12 h 18 h 24 h 48 h
0 20 40 60 80 100 120
Cell Viability (%)
Time
0.1 L/cc NDs 0.5 L/cc NDs 1 L/cc NDs
To understand the optical behavior of the nanodevices within cells, they were modified using mercaptoundecanoic acid (MUA) followed by cross linking with lysine to form a hydrophilic layer exhibiting both carboxyl and amine groups on its
Figure 6.12 Cell viability of HeLa cells after 12 to 48 hours of incubation with increasing amounts of folic acid modified nanodevices (FA-NDs). Cell viability was measured using an MTT assay.
surface.[148] Folic acid molecules were attached to the functional layer on the surface using carbodiimide chemistry to form bioconjugates. Folate receptors (FR) act as cancer-cell targeting ligands due to overexpression in many human cancerous cells, including mammary gland, lung, kidney, colon, prostate and throat cells.[149] However, they are only minimally distributed in normal cells. Folic acid exhibits high affinity to FR and is expected to allow the nanodevice to efficiently attach on or enter into cancerous cells via receptor mediated endocytosis. Incubation of the HeLa cell line with the MUA modified nanodevices (MUA-NDs) and folic acid modified NDs (FA-NDs) were completed separately. Both the MUA-NDs and FA-NDs were incubated with the cells for a period of 4 hours. The majority of the FA-NDs can be clearly observed in the cytoplasm region of the cell, but only a few of the MUA-NDs were attached or taken by HeLa cells, indicating that the folic acid-modified version
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promotes cellular uptake, as shown in the Supporting Information, Figure S5. It is expected that higher attachment or cellular uptake due to folic acid modification imparts enhanced therapeutic potential for the nanodevices. Figure 6.12 shows the results of the MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl Tetrazolium Bromide) assay, as a measure of metabolic competence of the cell with FA-NDs of different concentrations. The difference in the cytotoxicity over an incubation period of 12 to 48 hours is negligibly small. At the highest concentration of 1 L/cc FA-NDs, the cell viability remained at approximately 96 %. The results suggest that the nanodevices show minimal toxicity for the HeLa cells.
After cellular uptake, controlled drug release and real-time, in-situ monitoring of the ZCIS QDs were performed in a magnetic field with a strength of 2.5 kA/m. As illustrated in Figure 6.13 and 6.15, an increase in the duration of the field from 0 to 180 sec caused the dye molecules (Green channel) to be released rapidly within the cells, while the corresponding fluorescence intensity of the ZCIS QDs (Red channel) decreased. Digital software (Nikon, Japan) was used to analyze the fluorescence intensities of both the dye molecules and ZCIS Qds. Bsum, Gsum and Rsum represent the total intensity of the blue channel, the green channel and the red channel in the images, respectively. The blue fluorescence was due to the DAPI dyed nuclei and was expected to be relatively similar for each cell. Therefore, the intensity of the blue channel was assumed to be standard for each image. Gsum and Rsum are from drug release and quantum dots respectively. Gsum/Bsum, the ratio of the green channel intensity to the blue channel intensity, is indicative of the relative concentration of the dye molecules in each cell. Similarly, Rsum/Bsum is the value of the relative intensities of the nanodevices in each cell. After measurement, statistical analysis of fluorescence intensities exhibited a strong correlation between dye release and spectral variation of the ZCIS within the cells, shown in Figure 6.14. The ratios of Gsum/Bsum and
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Figure 6.13 Fluorescent combination of the HeLa cells with the dye-loaded nanodevices after 12 hours incubation. With increasing duration of HFMF treatment, both the controlled release of the dye molecules (green channel intensity increased) and the associated real-time, in-situ monitoring capability of the doped ZCIS QDs (red channel intensity decreased) can be manipulated simultaneously to single-cell resolution. This implies that dye release can be precisely monitored by the variation of the ZCIS QDs from the nanodevices.
Gsum/Bsum represents the ratio of the green channel intensity to blue channel intensity, and is indicative of the relative concentration of the model drug in each cell. Rsum/Bsum is then defined as the relative intensities of the nanodevices in each cell.
Rsum/Bsum versus duration of the magnetic field in the cells gives rise to two separate curves. These curves show that the relative drug concentration, represented by Gsum/Bsum, in the cells increases with the duration of stimulus. The fluorescence intensity of the nanodevice, originating from the ZCIS QDs and represented by Rsum/Bsum, decreased in proportion at the same time. There are two purposes of the cell culture test. The first purpose is a test of the short-term cyto-compatible nature of the nanodevice, where the HeLa cells appeared to be viable over the duration of the culturing step. The second and most critical purpose is the test of the in vitro
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0 90 180
0 1 2 3
Relative Intensity (a.u.)
MF stimulus time (sec) Gsum/Bsum Rsum/B
sum
monitoring capability of the nanodevices, where drug dosage can be quantitatively traced within a single cell.
Figure 6.14 The ratio of Gsum/Bsum and Rsum/Bsum versus the duration of magnetic stimulus in the cells gives rise to two curves. The results demonstrate the relative drug concentration, represented by Gsum/Bsum, in the cells with increasing duration of stimulus. The fluorescence intensity originating from the ZCIS QDS, represented by Rsum/Bsum, decreased in proportion at the same time.
6.8 Summary
The multifunctional drug delivery nanodevice makes use of quantum dots to successfully image, target, and deliver drugs via remote control. The devices also have the capability to monitor the in-situ drug release within a model cancerous cell line, HeLa cells, to cellular resolution. These nanodevices offer outstanding control of release and retention for the molecules encapsulated inside their polymer core. The dense, single-crystalline shell prevents the fluorescence dye from escaping prior to the desired release. Furthermore, the nanodevices are able to monitor real-time drug dosage through corresponding variation in emission spectrum of the quantum dots within the HeLa cells. The nanodevices have great potential advantages as a cell-specific drug delivery system for nanotherapeutic applications. With the in-situ monitoring capability of the nanodevice, we believe that both target-oriented therapy
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and diagnosis can be integrated and managed within a single cell. Multifunctional nano-platforms are expected to open a new avenue in the development of multifunctional therapeutic nanosystems. An in-vivo analysis is currently underway to treat brain tumors in mice.
Figure 6.15 Fluorescent morphologies of HeLa cells after 12 hours incubation with drug loaded nanodevices. With increasing the time duration of HFMF treatment, the control of drug release associated with a real-time self monitoring of the ZCIS QDs from the nanodevices were manipulated simultaneously. The digital analysis software (Nikon, Japan) was use to analyze the fluorescence intensities of model drug and nanodevices. The conditions of the exposure are the same for each color channels. The analyzed areas were determined by the software which defined the fluorescence intensity from 1 to 255. The ranges of the fluorescence intensities: Blue channel (60-255), Green channel (40-255), and Red channel (30-255).
Overlay Blue channel Nuclei (DAPI)
Red channel NanoDevice
Green channel Model drug MF stimulus
time 0 sec
90 sec 180 sec
90 sec 180 sec
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Table 6.1. The statistic data of fluorescence intensity was calculated by digital analysis software (Nikon, Japan). We employed the blue channel as a standard value because the fluorescence intensities of blue channel (DAPI) were assumed identical for each cell.
Gsum/Bsum represents the ratio of the green channel intensity to blue channel intensity, and is indicative of the relative concentration of the model drug in each cell. In such a way, Rsum/Bsum is then defining as the value of the relative intensities of the nanodevices in each cell.
MF stimulus time
Blue channel Nuclei (DAPI)
Green channel Model drug
Red channel
NanoDevice
G
sum/B
sumR
sum/B
sumMean Sum Mean Sum Mean Sum
0 sec 75 1.24×106 63 3.52×105 53 4.21×106 0.28 3.40
90 sec 78 1.39×106 68 1.82×106 44 89320 1.31 0.64
180 sec 89 1.94×106 74 6.10×106 33 25570 3.14 0.013
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Chapter 7
PVA-Iron Oxide /Silica Core-Shell Nanocarriers for Magnetically Controlled Drug Release and Cancer Cell Uptake Efficiency
7.1 Introduction
Control release of therapeutic agents from nanometric carriers has been received increasingly interest because it provides numerous advantages, such as high delivery efficiency and site-specific therapy, compared to traditional dosing techniques. Owing to these advantages, many researches proposed to integrate active drug molecules with host materials, aimed at manipulating drug release profile. It is desirable that drug release behavior can be optimized with either a slow, zero-order release pattern or a burst fashion mimicking natural release of biological molecules, such as hormones like insulin or thyroxine formed in endocrine glands, in the body.Real-time release upon a short-time stimulus is also hard to achieve for traditional stimuli-responsive polymeric materials, which is especially critical for a certain clinical complications. Therefore, a practical development of a desired drug carrier should possess real-time responsive to the stimuli when an urgent need is required for disease control and/or slow, sustained release to meet different clinical complications.
The use of a magnetic field to modulate drug release through magnetic nanoparticles from drug carriers was previously developed. Recently, the core/single-crystal iron oxide shell nanospheres for magnetically triggered release were also developed by our team. However, nano-carriers with controllable drug release property is highly desirable because such small carriers can be designed to deliver drug to a specific site of disease, and then, drop the therapeutic molecules in a right position at a right time with a therapeutically effective dose. It is far from achievable under current drug delivery systems, especially those of particulate drug delivery nano-devices, and it seems to have a need to bring a successful marriage among the field of materials technology, biology, pharmaceutics, and stimuli
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environment.
It is more clinically desirable if a ―zero or near-zero release profile‖ can be tailored as practically needed before the drug-containing nano-carriers reached the targeted sites because the nature of diffusion of drug molecules from the nano-carriers to the environment is virtually thermodynamically unavoidable in the presence of a concentration gradient of drug. Once such a mechanism was triggered right after administration, a risk of undesired clinical complications may result, included a reduction of therapeutic efficacy. Here, a novel nanodevice was designed and constructed by preparing a self-assembly iron oxide (SAIO) nanoparticles with drug molecules embedded in an ultra-thin silica nanoshell. A structurally dense silica shell has designed as a physical barrier to eliminate undesirable drug release before reaching the target sites appears to be practically desired. A mixture of magnetic nanoparticles and amphiphilic PVA was employed as a functional phase in the resulting nanocarriers which allow the response of the resulting drug carriers to be activated more dynamically and efficiently, aimed to achieve a real-time response to an immediate environmental change, i.e., magnetic field. In the meantime, the PVA phase provides not only a glue to assemble the nanoparticles, but also a matrix to immobilize therapeutical active agents of either hydrophilic or hydrophobic nature within. It is more interesting to know that high-frequency magnetic field, in the range of tens to hundreds of KHz, allows a pulsatile release of drug to be easily achieved on a real-time responsive base without undesirable delay in dosing accuracy under administration and more desirably, restores to zero or near-zero release immediately after removal of the high-frequency magnetic field. In addition, the cell uptake efficiency of these novel SAIO@SiO2 nanocarriers was examined.
7.2 Experimental section
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Materials: The synthesis was carried out under airless processes and using commercially available reagents. Absolute ethanol (99.5%), benzyl ether (99%), 1,2-hexadecanediol (97%), oleic acid (90%), oleylamine (>70%) and Iron(III) acetylacetonate were purchased from Aldrich Chemical Co. Polyvinyl alcohol (PVA, Mw: 72k) were purchased from Fluka Chemical Co. Tetraethylorthosilicate (TEOS) and 3-aminopropyltrimethoxy silane (APTMS) were purchased from Merck.
Fluorescein isothiocyanate (FITC, Sigma) were used to label, which help nano-carriers visualization under fluorescence microscope. Ibuprofen (IBU, Fluka) was used as the model drug.
Synthesis of Magnetic nanoparticles: Monodisperse iron oxide nanoparticles were synthesized by a method developed by Sun et al. Briefly, 5 nm of iron oxide nanoparticles were synthesized by mixing 2 mmol Fe(acac)3, 10 mmol 1,2-dodecanediol, 6 mmol oleic acid, 6 mmol oleylamine and 20 mL benzyl ether under a flow of nitrogen. The mixture was stirred magnetically and pre-heated to reflux (200 oC) for 30 minutes, and then, heated to 300 oC for another 1 hour under nitrogen atmosphere. The black-brown mixture was allowed to cool to room temperature and added 50 mL ethanol to participate. The products were collected by centrifugation at 6000 rpm for 10 minutes, and then washed with excess pure ethanol for 4 times. The product, iron oxide nanoparticles, was centrifuged to remove solvent, and redispersed into hexane.
Synthesis of self-assemble iron oxide nanoparticles/silica core-shell (SAIO@SiO2) nanocarriers: To prepare the self-assembly iron oxide nanoparticles (SAIO), 10 mg of iron oxide nanoparticles were centrifuged at 6000 rpm for 10 minutes, and then redispersed in 4 mL chloroform to form an uniform organic phase. 200 mg of polyvinyl alcohol (PVA) as a polymer binder was dissolved in 10 mL D.I. water at 70
oC. After PVA totally dissolving in the solution, the clear solution was cooled to room
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temperature. Then, organic phase was added into the PVA solution. The mixture was emulsified for 5 minutes with an ultrasonicator at 100 W. Under the ultrasonicating, the mixture was heated and evaporated the organic solvent. The mixture was stirred magnetically and heated again to 50 oC on hot plate to ensure the organic phase removing. After evaporation of organic solvent, the products were washed by D. I.
water for 3 times, and centrifuged at 6000 rpm to collect the products. The precipitates were redispersed in water, and the diameter of the redispersed particles (hereinafter termed as SAIO) is about 77 nm. Once the solvent was removed, a mixture of PVA and iron oxide nanoparticles was formed where the iron oxide nanoparticles appeared to assemble in a somewhat uniform and regular configuration in the resulting SAIO phase. A silica ultra-thin shell was then coated on the SAIO nanoparticles by a modifying Stöber method. In brief, 5 mg of SAIO were dispersed in the 4 mL of 99.5 % ethanol and 0.1 mL of 33 % NaOH4
Tetraethylorthosilicate (TEOS) was slowly added to the mixture and stirred for 12 hours. After hydrolysis and condensation, a silica nanoshell was coated on the SAIO nanoparticle to form a resulting PVA/iron oxide/silica core-shell (termed as SAIO@SiO2) nanocarriers.
Drug Loading and Release: In this investigation, ibuprofen (IBU) was used as a model drug to estimate the drug release behaviors of the SAIO@SiO2 nanocarriers, loading in nanocarriers through an in-situ process. First, the 4% IBU was dissolved in the chloroform with iron oxide nanoparticles to form the organic phase. This organic phase was used to prepare the self-assembly iron oxide nanoparticles (SAIO).
Ibuprofen can be encapsulated in the SAIO nanoparticles through the amphiphilic polyvinyl alcohol (PVA). Then, the process of constructing SAIO and silica shells was also applied to prepare drug-loading SAIO@SiO2 nanocarriers. Before drug release test, the nanocarriers were washed by the phosphate buffered saline (pH 7.4),
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following by washing with D.I. water. Ibuprofen-containing nanocarriers were used in PBS buffer solution for all the drug-release experiments. Quantitative estimate of the IBU loading was obtained by UV-vis spectrophotometer. The drug release behavior from the nanocarriers was measured in a 20 ml phosphate buffered saline per sponge cube (pH 7.4). To measure the concentration of drug release, 1.5 ml PBS medium with the dispersed nanospheres was taken out, and separated by centrifuge with 4000 rpm. The clear solution without nanospheres was used to estimate the concentration of drug release UV-Visible spectroscopy (Agilent, 8453 1UV-Visible spectrophotometer) was used for characterization of absorption peak at 264 nm (Imax of free IBU). The nanocarriers were absent and did not affect the measurements.
Cell Culture: HeLa (human cervical cancer) cells were maintained in DMEM (Dulbecco‘s modified Eagle‘s medium) containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured with complete medium at 37 °C in a humidified atmosphere of 5% CO2 in air. For all of the experiments, cells were harvested from subconfluent cultures by use of trypsin and were resuspended in fresh complete medium before plating. A comparison of in vitro cytotoxicity of SAIO@SiO2 nanocarriers with different concentrations and times was performed on HeLa cells with an in vitro proliferation method using MTT. Briefly, 1×104 cells were plated in 96-well plates to allow the cells to attach, and then, exposed to the serial concentrations of SAIO@SiO2 nanocarriers at 37 oC. At the end of the incubation, 20 μL of MTT solution was added and incubated for another 4 hours. Then, the medium was replaced with 200 μL of DMSO and the absorbance was monitored using a Sunrise absorbance microplate reader at dual wavelengths of 570 and 650 nm.
In order to estimate the cellular uptake of the nanocarriers, green emitting fluorescein dye was attached SAIO@SiO2 nanocarriers (FITC-SAIO@SiO2) for the
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study. First, fluorescein isothiocyanate (FITC) was mixed with ethanolic 3-aminopropyltrimethoxysilane (APTMS) solution for 24 h at room temperature to form N-1-(3-trimethoxysilylpropyl)- N-fluoresceyl thiourea (FITC-APTMS).
Separately, 5 mg of SAIO were dispersed in 4 mL of 99.5 % ethanol and 0.1 mL of 33
FITC-APTMS was slowly added to the mixture, and then, stirred for 12 hours. After hydrolysis and condensation, the FITC-silica was coated on the SAIO to form FITC-SAIO@SiO2 nanocarriers. The un-reacted chemicals were removed by rinsed
FITC-APTMS was slowly added to the mixture, and then, stirred for 12 hours. After hydrolysis and condensation, the FITC-silica was coated on the SAIO to form FITC-SAIO@SiO2 nanocarriers. The un-reacted chemicals were removed by rinsed