Volume 12 Issue 6 - January 29, 2010 PDF
Multifunctional pH-Sensitive Magnetic Nanoparticles for Simultaneous Imaging, Sensing and Targeted Intracellular Anticancer Drug Delivery
Shashwat S. Banerjee, Dong-Hwang Chen*
Department of Chemical Engineering, College of Engineering, National Cheng Kung University
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Targeted drug delivery systems which interact with intracellular components or events such as pH change are becoming increasingly important for controlled and triggered release of drugs. In the last decade nanotechnology and nanofabrication have significantly impacted the field of drug delivery. Enabled by their size and supramolecular structures, nanoparticles promise to be particularly capable agents in the detection, diagnosis, and treatment of cancer. In this work, we developed a novel multifunctional nanomedical system for simultaneous cancer-targeted optical imaging, sensing and magnetically guided pH triggered drug delivery. As shown in scheme 1, this nanomedical system was synthesized by coupling doxorubicin (DOX) to adipic dihydrazide-grafted gum arabic modified magnetic nanoparticles (ADH-GAMNP) via hydrolytically degradable pH-sensitive hydrazone bonds to selectively deliver the drugs to tumor cells. In contrast to classic conjugates with enzymatically induced drug release, the advantage of having DOX coupled with hydrazone bond is that the presence of lysosomal enzymes for drug activation is not essential for the synthesized novel system but the drug can be released by mild acid hydrolysis modeling environment in endosomes of tumor cells. Also, the hydrazone bond was found to be relatively stable in buffer at neutral pH modeling conditions of blood circulation. In addition, the gum arabic modified magnetic nanoparticle (GAMNP) was found to possess fluorescence property in the near-infrared (NIR) region due to the two-photon absorption mechanism. The benefit of fluorescence in NIR region is that it enables the use of light in the tissue transparent window (750-1000 nm) allowing deeper light penetration and reduced risk of laser hyperthermia. The fluorescence and magnetic property of the nanoparticles offers new avenue both as optical probes for intravital fluorescence microscopy and contrast agents for magnetic resonance imaging (MRI).
Scheme 1. Fabrication of DOX coupled GAMNP. The hydrazone bond formed between the CO- group of DOX and the hydrazide group of ADH-GAMNP could be effectively cleaved under acidic condition around pH 5.0 which corresponds to that of endosomes/lysosomes in the cells.

GAMNP were prepared by co-precipitation of Fe2+ and Fe3+ ions with ammonia solution and the followed surface modification with GA. For the linking of ADH to GAMNP, 0.5 g of GAMNP was dispersed in 10 mL of buffer A solution (0.003 M phosphate, pH 6, 0.1 M NaCl) by sonicating for 10 min. Then, 2.5 mL of carbodiimide solution (0.025 g/mL in buffer A) and 10 mL of ADH solution (0.5M in buffer A) were added and then the mixture was sonicated for another 60 min at 4-7℃. The reaction mixture was kept in a refrigerator at about 4℃ for 24 h. The resultant ADH-GAMNP was magnetically recovered, then washed repeatedly with water, and finally vacuum dried. For the synthesis of DOX-ADH-GAMNP, 0.25 g of ADH-GAMNP was placed in 10 mL of acetate buffer solution (0.5M, pH 6.0) and the mixture was magnetically stirred at room temperature. After 10 min, 25 mL of DOX solution (0.144 mg/mL in 50:50 methanol:dioxane) was added and further stirred for 24 h at room temperature. A hydrazone bond was formed between the COCH3 group of DOX and the hydrazide group of ADH-GAMNP. The product was magnetically recovered, then washed several times with NaOH solution of pH 7.5, and finally vacuum dried.

The resultant DOX-ADH-GAMNP had a mean diameter of 13.8 nm (Fig. 1). By varying the concentration and volume of ADH solution, the maximum amount of ADH that could be grafted onto GAMNP was determined to be 3.5 mg/g via the thermogravimetric analysis. From the spectrophotometrical analysis, the amount of DOX coupled to the ADH-GAMNP was found to be 6.52 mg/g. In-vitro drug release studies of DOX from the resultant DOX-ADH-GAMNP were performed under the physiological condition (PBS, pH 7.4) and slightly acidic environment (pH 5.0) at 37℃ to simulate the pH of endosomal and lysosomal microenvironments. The release profiles (Fig. 2) revealed that DOX-ADH-GAMNP exhibited pH dependent characteristic significantly. The drug release at pH 7.4 was considerably lower (with an initial burst of about 15%) than that at pH 5.0 (with approximately 74% of drug released after 5 h), confirming the property of pH-triggered drug release. Thus, in practical application, the amount of DOX released from DOX-ADH-GAMNP in the bloodstream (pH≈7.4) during the transport to the target cells will be less, and the majority of active drug could be released from the nanocarrier after entering the target cancer cell due to a pH decrease in the endosomes/lysosomes (pH ≈ 4-6) as shown in scheme 2.
Fig. 2. Release profiles of DOX form DOX-ADH-GAMNP in PBS of pH 5.0(Δ) and pH 7.4(□).
Fig. 1. Typical TEM image of DOX-ADH-GAMNP.

Scheme 2. (a)Schematic representation of DOX-ADH-GAMNP Bi-FRET system. In the step, DOX is coupled with GAMNP which results in quenching of fluorescence through Bi-FRET mechanism. (b) Schematic illustration of specific suptake of DOX-ADH-GAMNP into the targeted cancer cell. The release of fluorescence from both GAMNP and DOX, thereby sensing the intracellular delivery of DOX.

Fig. 3. Nomalized absorption spectra (---) of DOX and florescence spectra(–) of GAMNP at an excitation wavelength of 850nm.
GAMNP possessed fluorescence property with a typical fluorescence peak at ~572 nm when excited at a wavelength of 850 nm (Fig. 3). It can function as a photon up-converting material by converting lower-energy light to higher-energy light through excitation with multiple photons. Also, it is well known that anthracycline class of drugs such as DOX has fluorescence property. When excited at a wavelength of 850 nm, DOX also showed fluorescence emission at ~571 nm, close to that observed for GAMNP. Fig. 3 reveals that the absorption of DOX in PBS media had a spectrum overlap with the fluorescence of GAMNP, which enables the energy transfer between them. This permits DOX molecule to act as the photon acceptor of GAMNP which emits light in the range of 560-620 nm.

Fig. 4. Fluorescenc spectra of DOX-ADH-GAMNP in PBS of pH 5.0(a) and 7.4(b) at an excitation wavelength of 850 nm.
To further understand the phenomenon of energy transfer in the nanosystem, we used fluorescence spectroscopy to monitor the effect of DOX release on fluorescence intensity at an excitation wavelength of 850 nm. As shown in Fig. 4, a sequential increase in the intensity of fluorescence emission was observed when DOX-ADH-GAMNP (25 ppm) was dispersed in PBS of pH 5.0. On the other hand, when the DOX-ADH-GAMNP was dispersed in PBS of pH 7.4, a sequential increase of very low magnitude as compared to that observed in PBS 5.0 was found. This can be explained based on the phenomenon of donor acceptor model fluorescence resonant energy transfer (FRET). DOX coupling with GAMNP might result in quenching of the fluorescence of GAMNP through FRET between the donor GAMNP and acceptor DOX. The sequential increase in fluorescence emission of DOX-ADH-GAMNP when exposed to an acidic media might be due to the decrease in the energy transfer between GAMNP and DOX as a consequence of drifting apart of DOX from GAMNP because of the faster release. The increase in fluorescence emission for DOX-ADH-GAMNP was not pronounced in PBS 7.4 as the energy transfer between GAMNP and DOX was much more feasible due to the presence of higher amount of DOX on GAMNP. It has also been reported that the decrease in fluorescence intensity of DOX was observed when it was associated with macromolecule due to the quenching of fluorescence through FRET. The presence of GA on the surface of MNP might result in the quenching of DOX fluorescence because of the interaction between DOX and GA. Thus, the sequential increase in the fluorescence intensity of DOX-ADH-GAMNP dispersion might be attributed to the release of DOX which enabled it to regain normal fluorescence emission. This suggested that DOX coupling might result in a Bi-FRET system. The change in the fluorescence intensity of DOX-ADH-GAMNP on release of DOX could act as a potential sensor to sense the delivery of the drug as illustrated in scheme 2.

In summary, a novel multifunctional nanomedical system with cumulative effects of pH sensitive hydrazone bond bearing drug nanoparticle conjugate, GA which acts as targeting ligands as well as the combined properties of fluorescence and magnetism has been developed. Such a promising nanocarrier for optical imaging, sensing by change in fluorescence intensity, and delivering anticancer drugs to cancer cells by specific targeting and releasing the drug molecules inside the cells to the cytosols is a significant breakthrough in the development of drug delivery vehicles.
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