Multifunctional Nanocarriers for Cell Imaging, Drug Delivery, and Near

Jan 22, 2010 - ... of Oncology, Affiliated Drum Tower Hospital, Medical College, and .... cancer cell line LoVo (Shanghai Institute of Cell Biology, C...
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Multifunctional Nanocarriers for Cell Imaging, Drug Delivery, and Near-IR Photothermal Therapy Rui Guo,†, Leyang Zhang,† Hanqing Qian,† Rutian Li,‡ Xiqun Jiang,*,†,§ and Baorui Liu‡ Laboratory of Mesoscopic Chemistry and Department of Polymer Science & Engineering, College of Chemistry & Chemical Engineering, ‡Department of Oncology, Affiliated Drum Tower Hospital, Medical College, and § Jiangsu Provincial Laboratory for Nanotechnology, Nanjing University, Nanjing 210093, PR China, and College of Chemistry, Chemical Engineering & Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China )

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Received October 13, 2009. Revised Manuscript Received December 18, 2009 Multifunctional nanocarriers based on chitosan/gold nanorod (CS-AuNR) hybrid nanospheres have been successfully fabricated by a simple nonsolvent-aided counterion complexation method. Anticancer drug cisplatin was subsequently loaded into the obtained hybrid nanospheres, utilizing the loading space provided by the chitosan spherical matrix. In vitro cell experiments demonstrated that the CS-AuNR hybrid nanospheres can not only be utilized as contrast agents for real-time cell imaging but also serve as a near-infrared (NIR) thermotherapy nanodevice to achieve irradiation-induced cancer cell death owing to the unique optical properties endowed by the encapsulated gold nanorods. In addition, an effective attack on the cancer cells by the loaded anticancer drug cisplatin has also been observed, rendering the obtained nanocarriers an all-in-one system possessing drug delivery, cell imaging, and photothermal therapy functionalities.

Introduction Nanotechnology has been intensively explored in the constant battle against cancer in the past few decades.1 Recently, the concept of multifunctional nanocarriers that can simultaneously perform multiple functions, such as anticancer drug delivery, optical imaging, and controllable cancerous cell death, has emerged and attracted increasing attention.2-17 The advantages of using a multifunctional nanocarrier system could be manifold. First, certain beneficial synergies can be achieved with much less administration of the adjuvant carriers. Second, multifunctional systems can endow researchers with more control over the *To whom correspondence should be addressed. Fax: 86 25 83317761. E-mail: [email protected].

(1) Ferrari, M. Nat. Rev. Cancer 2005, 5, 161–171. (2) Peer, D.; Karp, J. M.; Hong, S.; Farkhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotechnol. 2007, 2, 751–760. (3) Nie, S.; Xing, Y.; Kim, G. I.; Simons, J. W. Annu. Rev. Biomed. Eng. 2007, 9, I2.1–2.32. (4) Sanvicens, N. M.; Marco, P. Trends Biotechnol. 2008, 26, 425–433. (5) Gao, Y.; Cui, Y.; Levenson, R. M.; L. Chung, W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969–976. (6) Kim, J.; Lee, J. E.; Lee, S. H.; Hu, J. H.; Lee, J. H.; Park, T. G.; Hyeon, T. Adv. Mater. 2008, 20, 478–483. (7) Yang, J.; Lee, C.; Ko, H.; Suh, J.; Yoon, H.; Lee, K.; Huh, Y.; Haam, S. Angew. Chem., Int. Ed. 2007, 46, 8836–8839. (8) Liong, M.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. ACS Nano 2008, 2, 889–896. (9) Guo, Y.; Shi, D.; Cho, H.; Dong, Z.; Kulkarni, A.; Pauletti, G. M.; Wang, W.; Lian, J.; Liu, W.; Ren, L.; et al. Adv. Funct. Mater. 2008, 18, 2489–2497. (10) Kumar, S.; Harrison, N.; Richards-Kortum, R.; Sokoloy, K. Nano Lett. 2007, 7, 1338–1343. (11) Huang, X.; El-sayed, I. H.; Qian, W.; El-sayed, M. A. J. Am. Chem. Soc. 2006, 128, 2116–2120. (12) Norman, R. S.; Stone, J. W.; Gole, A.; Murphy, C. J.; Sabo-Attwood, T. L. Nano Lett. 2008, 8, 302–306. (13) Huang, Y.; Sefah, K.; Bamrungsap, S.; Chang, H.; Tan, W. Langmuir 2008, 24, 11860–11865. (14) Li, J. L.; Day, D.; Gu, M. Adv. Mater. 2008, 20, 3866–3871. (15) Dickerson, E. B.; Dreaden, E. C.; Huang, X.; El-sayed, I. H.; Chu, H.; Pushpanketh, S.; McDonald, J. F.; El-Sayed, M. A. Cancer Lett. 2008, 269, 57–66. (16) Angelatos, A. S.; Radt, B.; Caruso, F. J. Phys. Chem. 2005, 109, 3071–3076. (17) Skirtach, A. G.; Javier, A. M.; Kreft, O.; Kohler, K.; Alberola, A. P.; Mohwald, H.; Parak, W. J.; Sukhorukov, G. B. Angew. Chem., Int. Ed. 2006, 45, 4612–4617.

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nanocarriers and provide more in-depth information about the fate of those carriers. By combining functional inorganic nanomaterials, including quantum dots, gold nanoparticles, magnetic particles, and carbon nanotubes, with a protective organic matrix that embeds other active agents, people have been able to fabricate various kinds of the aforementioned multifunctional nanocarriers.5-20 However, most of the multifunctional nanocarriers reported so far require complicated synthesis processes and the synthesis is usually very system-specific, meaning that one preparation protocol can hardly be adapted to another system. Furthermore, the poor biocompatibility and potential toxicity of some systems, which arise from either the inorganic nanomaterials or the use of surfactants, may present a serious problem in real clinical applications. More importantly, instead of constructing real multifunctional nanocarriers, many of the reported systems simply change the surface properties of inorganic functional nanomaterials by coating them with a thin polymer layer mainly for stability or biocompatibility reasons10-15 and actually cannot provide enough space for the loading of other active agents, which makes it hard to extend their application into areas such as drug delivery. Therefore, it is still very challenging and highly desirable to develop a nanocarrier system that combines facile and versatile synthesis, good extendability, good biocompatibility as well as multifunctional character. Herein, we report an easy-to-fabricate multifunctional nanocarrier system based on drug-loaded chitosan/gold nanorod (CSAuNR) hybrid nanospheres as schematically shown in Figure 1. Low-molecular-weight water-soluble chitosan (CS) was chosen as the material for the polymer matrix considering its good biocompatibility.21,22 Gold nanorods (AuNR) were employed as the (18) Takahashi, H.; Niidome, Y.; Niidome, T.; Kaneko, K.; Kawasaki, H.; Yamada, S. Langmuir 2006, 22, 2–5. (19) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stlzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331–338. (20) Sumner, J. P.; Kopelman, R. Analyst 2005, 130, 528–533. (21) Richardson, S.; Kolbe, H.; Duncan, R. Int. J. Pharm. 1999, 178, 231–243. (22) Chae, S.; Jang, M.; Nah, J. J. Controlled Release 2005, 102, 383–394.

Published on Web 01/22/2010

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Article clear solution turned opalescent, which indicated the formation of colloidal particles. Finally, 30 μL of GA solution (25%) was introduced to cross link the obtained hybrid nanospheres at room temperature for 4 h. The cross-linked nanospheres were purified by dialyzing against distilled water for 48 h to remove the EDTA and ethanol.

Characterizations of CS-AuNR Hybrid Nanospheres.

Figure 1. Schematic illustration of multifunctional CS-AuNR hybrid nanospheres with encapsulated gold nanorods, the loaded anticancer drug, and the fluorescent probe attached to the surface.

functional inorganic material because of their unique optical properties and potential applications in cell imaging and photothermal therapy.10-15 Meanwhile, fluorescein isothiocyanate (FITC) was covalently attached to the nanospheres as a fluorescent probe. Finally, anticancer drug cisplatin was successfully loaded into the resultant CS-AuNR hybrid nanospheres, taking advantage of the ample loading space provided by the chitosan polymeric matrix. In vitro cell experiments demonstrated that the obtained hybrid nanospheres were very promising biocompatible carriers for the loading and delivery of anticancer drugs and could be utilized as contrast agents for real-time cell imaging by dark-field microscopy and fluorescence microscopy. In addition, the encapsulated gold nanorods enabled the hybrid nanospheres to be used as a near-infrared (NIR) thermotherapy nanodevice to achieve external-irradiation-induced cancer cell death, rendering the obtained nanocarriers an all-in-one system possessing drug delivery, cell imaging, and photothermal therapy functionalities.

Experimental Section Materials. Chitosan (CS) with an average molecular weight (Mn) of 5000 was purchased from Yuhuan Biomedical Company (Zhejiang, China) and used without further purification. Ethylene diamine tetraacetic acid (EDTA), cetyltrimethylammonium bromide (CTAB), glutaraldehyde (GA), sodium borohydride (NaBH4), and HAuCl4 were used as received. All of the other ingredients were of analytical grade unless otherwise stated. Synthesis of Gold Nanorods. The nanorods were synthesized according to the seed-mediated growth method with some modifications.23-25 Briefly, a seed solution was prepared by reducing 0.8 mM HAuCl4 in a 0.2 M CTAB solution with cold NaBH4 (0.01 M) at room temperature. In the meantime, 0.4 mL of HAuCl4 (8 mM) and 30 μL of AgNO3 (0.01 M) were added to 20 mL of CTAB (0.1 M), and the obtained mixture was reduced by 30 μL of ascorbic acid (0.1 M) to yield a growth solution. The seed solution (40 μL) was then introduced into 20 mL of the growth solution, and nanorods were obtained after several hours of growth in the darkness. Preparation of CS-AuNR Hybrid Nanospheres. The CSAuNR hybrid nanospheres were synthesized by a nonsolventaided counterion complexation method. First, 50 mg of CS was dissolved in 10 mL of water. Then a predetermined amount of EDTA was added to the CS aqueous solution, and the mixture was stirred until the EDTA fully dissolved. The gold nanorods prepared earlier were centrifuged and redispersed twice to remove the redundant CTAB and then added subsequently to the CSEDTA mixture solution. The resultant mixture was sonicated for 2 min. After that, a certain amount of ethanol (nonsolvent) was added dropwise to the system under vigorous stirring until the (23) Murphy, C. J.; Jana, N. R. Adv. Mater. 2002, 14, 80–82. (24) Gole, A.; Murphy, C. J. Chem. Mater. 2004, 16, 3633–3640. (25) Busbee, B. D.; Obare, S. O.; Murphy, C. J. Adv. Mater. 2003, 15, 414–416.

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Morphological studies of the hybrid nanospheres were carried out using TEM (JEM-100S, JEOL, Japan). One drop of a properly diluted sample was placed on a copper grid covered with a nitrocellulose membrane and air dried before examination. The mean diameter, size distribution, and zeta potentials of the prepared nanospheres were determined by a DLS method using a Brookhaven BI-9000AT instrument (Brookhaven Instruments Corporation). Each sample was diluted and sonicated before measurement. Each result was an average of triplicate measurements. The UV-vis absorption spectra were recorded on a Shimadzu UV3100 spectrophotometer (Shimadzu, Japan).

In Vitro Cytotoxicity of CS-AuNR Hybrid Nanospheres. The in vitro cytotoxicity of CS-AuNR hybrid nanosphere was determined by standard MTT assays, and human colorectal cancer cell line LoVo (Shanghai Institute of Cell Biology, China) was used here. Cells were seeded into a 96-well plate at a density of 5000 cells per well and incubated at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was a 1640 medium supplemented with 10% calf blood serum and changed every other day until 80% confluence had been reached. The medium was then replaced with 200 μL of a medium with gold nanorods, CS nanospheres, and CS-AuNR hybrid nanosphere solutions of different concentrations. One row of 96-well plates was used as a control with 200 μL of a culture medium only. After incubation, 20 μL of a 10 mg mL-1 MTT solution was added to each well, and the plate was incubated for 4 h, allowing the viable cells to reduce the yellow MTT to dark-blue formazan crystals, which were dissolved in 200 μL of dimethyl sulphoxide (DMSO). The absorbance of individual wells was measured at 570 nm by an ELISA reader (Huadong, DG-5031, Nanjing). The cell viability was determined by eq 1: cell viability ð%Þ ¼

Abstest cells  100% Absreference cells

ð1Þ

Dark-Field and Fluorescence Microscopy Measurements. A predetermined amount of fluorescein isothiocyanate (FITC) dissolved in dimethylsulfoxide (DMSO) was added dropwise to a CS-AuNR solution, and the system was allowed to react for 4 h, after which the FITC-labeled CS-AuNR hybrid nanospheres were purified by dialyzing against pure water for 24 h. Human colorectal cancer cell line LoVo was cultured in a 1640 medium supplemented with 10% calf blood serum at 37 °C in a humidified atmosphere with 5% CO2. The cells were detached by trypsinization and replated onto 20 mm glass coverslips in a sixwell plate and allowed to grow for 2 days. Then the medium was replaced with 2 mL of a medium containing FITC-labeled CSAuNR hybrid nanospheres at different concentrations. After incubation for 2 h, the cell monolayer on the coverslip was taken out of the medium from the incubator, repeatedly rinsed with PBS, and then sealed with a microscope glass slide without fixation. Bright-field, dark-field, and fluorescence images were collected with a BX51 Olympus System microscope (Olympus, Tokyo) with a dark-field condenser (U-DCW).

Preparation of Cisplatin-Loaded Hybrid Nanospheres. The drug-loaded nanospheres were prepared by mixing cisplatin aqueous solution (2 mg mL-1) with a CS-AuNR hybrid nanosphere solution overnight at room temperature in the dark to allow cisplatin to equilibrate between the aqueous phase and the nanosphere matrix. Then the mixture was centrifuged and redispersed in pure water to remove the free drug. The drug-loading content was determined by centrifugation of the samples at 15 000 rpm for 30 min. The free cisplatin in the DOI: 10.1021/la903893n

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supernatant was quantified by a modified colorimetric o-phenylenediamine method at 704 nm.26 The encapsulation efficiency and drug-loading content were calculated from eqs 2 and 3: encapsulation efficiency ð%Þ ¼

M total drug - M drug in supernatant M total drug  100%

drug loading content ð%Þ ¼

M drug in nanospheres  100% M nanospheres

ð2Þ ð3Þ

In Vitro Release and Cytotoxicity of Cisplatin-Loaded Hybrid Nanospheres. The in vitro release of cisplatin from the nanospheres was evaluated using a dialysis bag diffusion technique right after the preparation of drug-loaded nanospheres. A cisplatin-loaded hybrid nanosphere solution (0.5 mL, 10 mg/mL) was placed into a preswelled dialysis bag with a 12 kDa molecular weight cutoff and immersed in 10 mL of a 0.1 mol L-1 PBS solution at pH 7.4 and 37 °C with gentle agitation. Periodically, 5 mL of the release medium was withdrawn and then 5 mL of fresh PBS was added to the system. The cisplatin concentration in the sampled medium was measured by the colorimetric method with appropriate dilution. BGC823 cell lines were used to assess the in vitro cytotoxicities of empty CS nanospheres, hybrid nanospheres, free cisplatin, and cisplatin-loaded empty and hybrid nanospheres by standard MTT assays as described previously. Cell inhibition was determined by eq 4: cell inhibition ð%Þ ¼

Absreference cells -Abstest cells  100% ð4Þ Absreference cells

NIR Photothermal Therapy. LoVo cells were seeded in a 24well plate and incubated at 37 °C in a humidified atmosphere with 5% CO2 for 2 days. Then the medium was replaced with 2 mL of a medium containing empty CS nanospheres or CS-AuNR hybrid nanosphere solutions at the same concentration. Nanospheres with no drug loading were used here to exclude the potential influence of the cisplatin on cell viability. After incubation for 2 h, the cell monolayer in the wells was repeatedly rinsed with PBS buffer to remove the nonspecifically adsorbed nanospheres and nanospheres remaining in the medium and then exposed to the red laser at 808 nm (LD808, Coherent Inc.) with power densities of 1, 2, and 3 W/cm2 separately for 2 min. The laser was focused on an area of 2 cm2, which can just overlay the area of a single well (1.9 cm2). Finally, cells were treated with 0.4% trypan blue for 10 min to evaluate their viability. Living cells can get rid of trypan blue and keep themselves colorless, but dead ones will collect the blue dye. Therefore, the cell viability can be qualitatively determined from the color of the cell monolayer. The images of stained cells were collected with an Olympus microscope equipped with a digital camera.

Results and Discussions Construction of CS-AuNR Hybrid Nanospheres. To synthesize multifunctional CS-AuNR hybrid nanospheres, gold nanorods with proper optical properties were first prepared. Gold nanorods are known to have two principal plasmon absorption bands. One is the transverse plasmon band at about 520 nm, and the other is the longitudinal plasmon band changing from the visible to the NIR region with the increase in the aspect ratios of gold nanorods.27 Because of the minimum extinction of human tissues and much higher penetration depth in the NIR region,28 (26) Yan, X.; Gemeinhart, R. A. J. Controlled Release 2005, 106, 198–208. (27) Jain, P.; Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J. Phys. Chem. B 2006, 110, 7238–7248. (28) Weissleder, R. Nat. Biotechnol. 2001, 19, 316–317.

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Figure 2. Schematic illustration of the preparation of CS-AuNR hybrid nanospheres: brown chains, chitosan molecule; blue dots, EDTA; magenta rods, gold nanorods.

gold nanorods with strong absorption and scattering in the NIR region have the potential to be used as contrast agents for darkfield optical imaging and photothermal absorbing agents for photothermal therapy.10,11 Here, gold nanorods with a longitudinal absorption band at around 800 nm and an aspect ratio of 3.6 (Supporting Information Figure S1) were prepared using cationic cetyltrimethylammonium bromide (CTAB) as the stabilizer23 and used for the construction of CS-AuNR hybrid nanospheres after purification because their absorption band is in the NIR region and overlaps well with the laser wavelength that we used in the following photothermal experiment. CS-AuNR hybrid nanospheres were synthesized by a nonsolvent-aided counterion complexation method as schematically shown in Figure 2 using gold nanorods, ethylene diamine tetraacetic acid (EDTA), and low-molecular-weight chitosan as the starting materials. The gold nanorods purified by repeated ultrasonication/centrifugation/redispersion cycles are first dispersed in an aqueous solution containing chitosan and EDTA. EDTA will attach to the gold nanorods through electrostatic interaction with the residual cationic CTAB at the surface of AuNR. Subsequently, ethanol, a nonsolvent for both chitosan and EDTA, is added to the mixture to facilitate counterion interactions between the positively charged chitosan chains and the anionic EDTA. Then the gold nanorods can act as the nucleation center for the precipitation of the ionic complexes formed between chitosan and EDTA and can be effectively encapsulated within the chitosanEDTA complex, which leads to the formation of colloidal chitosan-EDTA-AuNR hybrid spheres. After the chitosan moiety is cross linked by glutaraldehyde, EDTA is easily removed by dialysis against water thanks to its small-molecule nature, but gold nanorods are left inside the cross-linked chitosan matrix, yielding CS-AuNR hybrid nanospheres. Empty chitosan nanospheres have also been prepared as the control without adding gold nanorods. The hybrid nanospheres are stabilized solely by the hydrophilically cationic chitosan spherical matrix, which is nice for biomedical applications. It is also worth mentioning that the nonsolvent-aided counterion complexation method used here is very versatile and can be directly adapted to the preparation of hybrid nanospheres entrapping other functional nanomaterials (e.g., Fe3O4 magnetic nanoparticles as demonstrated in the Supporting Information), which shows the good extendability of this method. According to our previous study,29 the electrostatic interaction between two oppositely charged substances plays a key role in the (29) Guo, R.; Zhang, L.; Zhu, Z.; Jiang, X. Langmuir 2008, 24, 3459–3464.

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Figure 4. TEM micrograph of (a) empty chitosan nanospheres and (b) CS-AuNR hybrid nanospheres.

formation of nanospheres by the nonsolvent-aided counterion complexation method. Therefore, to optimize our synthesis parameters, various ratios of EDTA, CS, and gold nanorods were utilized to prepare CS-AuNR hybrid nanospheres. First, with a constant weight ratio of AuNR to CS (1:100), hybrid nanospheres were synthesized by changing the weight ratio of CS to EDTA from 10:1 to 3:1 and monitored by dynamic light scattering (DLS). As can be seen in Figure 3a, the particle size of CS-AuNR hybrid nanospheres increased from 122 to 227 nm corresponding to the decrease in the ratio of CS to EDTA, indicating that this system is flexible in terms of size control. It is worth pointing out that the gold nanorod feeding ratio also plays a role in determining the particle size. As mentioned earlier, the gold nanorods can act as the nucleation centers for counterion complexation between chitosan and EDTA. Normally, the more the nucleation centers, the smaller the particle size. As shown in Figure 3b, a higher AuNR to CS ratio resulted in smaller hybrid nanospheres. Figure 3c shows the UV-vis spectra of gold nanorods, CS nanospheres, and CS-AuNR hybrid nanospheres prepared with different gold nanorod feeding ratios from 1:100 to 1:7.5. We can see that after nanorods are encapsulated into chitosan nanospheres the longitudinal absorption of gold nanorods red shifts only very slightly to 810 nm because of the higher dielectric constant of the surrounding chitosan matrix.30 Also, the absorption intensity increases significantly with increasing gold nanorod feeding ratio because of the increased effective concentration of AuNR encapsulated in the hybrid nanospheres. It is apparent that the CS-AuNR hybrid nanospheres well preserve the optical properties of the encapsulated gold nanorods and hence the hybrid nanospheres should also possess the functionalities originating from the gold nanorods’ unique optical characteristics. Although a higher loading level of gold nanorods could endow

CS-AuNR hybrid nanospheres with more prominent absorption and scattering properties, it also compromises the dispersion stability of hybrid nanospheres. We found that the irreversible agglomeration of the CS-AuNR hybrid nanospheres occurred after 3 days of storage when the feeding ratio of AuNR to CS was higher than 1:5 whereas nanospheres prepared with lower feeding ratios were quite stable in both water and cell culture medium. Therefore, in subsequent experiments, to achieve a good balance between the nanosphere stability and the longitudinal absorption intensity, the CS-AuNR hybrid nanospheres prepared at 1:7.5 w/w AuNR/CS and 5:1 w/w CS/EDTA were used. It is worth mentioning, however, that because the high density of the gold nanorods’ gradual sedimentation could still take place after 1 week of storage but gentle agitation by hand can fully redisperse the sedimented nanospheres and DLS analysis indicated that the particle size did not change after redispersion. Figure 4a,b shows the TEM micrographs of empty CS nanospheres and the CS-AuNR hybrid nanospheres. It can be seen that both the empty CS nanospheres and the CS-AuNR hybrid nanospheres are well dispersed with spherical outline and uniform size. The particle size observed under TEM is slightly smaller than the DLS result because of shrinking in the dry state. It is apparent from Figure 4b that all of the gold nanorods are located in the interior of the hybrid nanospheres, highlighting the effectiveness of encapsulation by the nonsolvent-aided counterion complexation method. It is well known that the strong cytotoxicity originating from the use of CTAB in the preparation and subsequent stabilization of gold nanorods may greatly limit their practical use in biomedical areas.18,31 Thus, it is expected that by encapsulating the purified gold nanorods into a biocompatible polymer matrix the biocompatibility problem can be circumvented without sacrificing their dispersing stability. This speculation has been confirmed by our in vitro cell viability tests as shown in Figure 5. It can be seen that the cell survival rates in the presence of the as-prepared gold nanorods are below 25%, indicating significant cytotoxicity. In contrast, no obvious inhibition of cell proliferation has been observed in the cases of empty CS nanospheres and CS-AuNR hybrid nanospheres. These facts imply that the prepared hybrid nanospheres have good biocompatibility, which is an important prerequisite for nanocarriers for biomedical applications. Cell Imaging with CS-AuNR Hybrid Nanospheres. By taking advantage of the inherited optical characteristics from the encapsulated gold nanorod, the CS-AuNR hybrid nanospheres were evaluated as contrast agents in real-time cell imaging by dark-field optical microscopy. Fluorescent FITC was also covalently attached to the nanospheres as a secondary probe to utilize the abundant amino groups on chitosan chains. This makes the

(30) Liz-Marzan, L. M.; Giersig, M.; Mulvaney, P. Langmuir 1996, 12, 4329– 4335.

(31) Mirska, D.; Schirmer, K.; Funari, S. S.; Langer, A.; Dobner, B.; Brezesinski, G. Colloids Surf., B 2005, 40, 51–59.

Figure 3. (a) Lognormal size distributions of CS-AuNR hybrid nanospheres obtained using different weight ratios of CS to EDTA (9: CS:EDTA = 10:1; 2: CS:EDTA = 5:1; (: CS:EDTA = 3:1) with the same weight ratio of AuNR to CS (1:100). (b) Mean hydrodynamic diameters of CS-AuNR hybrid nanospheres prepared with different weight ratios of AuNR to CS over the range of 1:100 to 1:5. The weight ratio of CS to EDTA was fixed at 5:1. (c) UV-vis spectra of gold nanorods (AuNR), empty chitosan nanospheres (CS NS), and CS-AuNR hybrid nanospheres prepared with various weight ratios of AuNR to CS from 1:100 to 1:7.5.

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Figure 5. Viability of cells (LoVo) incubated with as-prepared gold nanorods (AuNR), CS-AuNR hybrid nanospheres, and empty chitosan nanospheres (CS NS) at different concentrations (chitosan-weight-based). The bare AuNR concentration was set at the same level as the embedded gold nanorods in the CS-AuNR group according to the gold-loading content of the hybrid nanospheres.

Figure 6. (a) Bright-field, (b) dark-field, and (c) fluorescence microscopy images of LoVo cells incubated with CS-AuNR nanospheres at 40.

resultant CS-AuNR nanospheres observable via fluorescence microscopy as well, and hence we can obtain complementary information on the location of the CS-AuNR hybrid nanospheres. Figure 6a-c shows the bright-field, dark-field, and fluorescence-field microscopy images of LoVo cells after 2 h of incubation in a medium containing CS-AuNR hybrid nanospheres. From the microscopy images of LoVo cells, one can see that the cells incubated with CS-AuNR hybrid nanospheres are still well attached to the glass slide and maintain their normal morphology, which corroborates our conclusion about the good biocompatibility of CS-AuNR hybrid nanospheres. In the darkfield microscopy image, the hybrid nanospheres as the bright pinkish dots can be easily distinguished by virtue of the strong longitudinal surface plasmon oscillation of gold nanorods in the NIR region, which makes individual cells more identifiable. Meanwhile, in the fluorescence images, the strong fluorescent signals correlate very well with the signals observed in dark-field microscopy, implying that the fluorescence of FITC in CS-AuNR nanospheres is not affected much by the fluorescence quenching effect occurring in gold nanoparticles coated with polyelectrolytes.32 This may be due to the relatively larger size of gold nanorods and the large distance between gold nanorods and FITC in this study. These two sets of results provide reinforced evidence of the intracellular distribution of the hybrid nanospheres. Although we cannot fully exclude the existence of the nonspecific adsorption of hybrid nanospheres on the cell surface despite the fact that the cells were washed extensively with PBS buffer prior to optical observations, a further careful examination of Figure 6b,c revealed that those signals are not evenly or randomly distributed in cells as would be in the case of nonspecific adsorption but obviously enrich the cytoplasm of the cells. (32) Schneider, G.; Decher, G.; Nerambourg, N.; Praho, R.; Werts, M. H. V.; Blanchard-Desce, M. Nano Lett. 2006, 6, 530–536.

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Thus, it is reasonable to speculate that a substantial fraction of the CS-AuNR hybrid nanospheres were endocytosed upon incubation with cancer cells by virtue of the nanometer size of the hybrid nanospheres and the bioadhesion effect of many polysaccharides,33 which increased the contrast of cells under optical observations. Therefore, the CS-AuNR hybrid nanospheres may be employed as a bimodal contrast agent for real-time optical cell imaging, which could be very desirable for cancer diagnosis and treatment. Moreover, these results clearly indicate that the CSAuNR hybrid nanospheres may serve as a promising anticancer drug delivery system because of their potential to help the loaded drugs enter tumor cells. Drug Delivery with CS-AuNR Hybrid Nanospheres. Compared with other commonly reported multifunctional nanodevices, which only have a very thin layer of surfactants or polymer brushes,11-15 the CS-AuNR hybrid nanospheres reported here have an obvious advantage in that they have a relatively larger polymer matrix that may be used as a reservoir for the loading of various active agents, thus making the resultant nanospheres multifunctional and practically useful for drugdelivery purposes. Accordingly, cisplatin, a widely used anticancer drug for the clinic treatment of many malignancies,34 was loaded into the CS-AuNR hybrid nanospheres with a drug loading content of 3% (w/w) by physical interactions that probably involve ligand exchange.35 Figure 7a shows the release profiles of cisplatin from both CS nanospheres and CS-AuNR hybrid nanospheres at 37 °C in PBS (pH 7.4). The two release curves look quite similar, indicating that the drug loading and release characteristic is mainly dependent on the chitosan matrix and the encapsulation of AuNR has negligible effect on it. In both cases, an initial burst release of about 35% of the total loaded drug in the first hour was observed, and about 80% of the loaded drug was released in 150 h. Compared with the release profile of free cisplatin solution, which is a total release of more than 93% in the first 2 h (Supporting Information Figure S2),36 this system showed a sustained release profile achieved with the help of the nanospheres, which is also a desirable property for anticancer drug delivery systems. In vitro cytotoxicity tests against human gastric cancer cell line BGC823 were conducted subsequently to investigate the pharmaceutical activity of the released cisplatin. Cell inhibition rates of empty CS and CSAuNR hybrid nanospheres, drug-loaded CS and hybrid nanospheres, and free cisplatin at different concentrations are shown in Figure 7b. The results show that cisplatin-loaded hybrid nanospheres have a comparable cytotoxic activity to the free cisplatin after 48 h of exposure, which also makes the hybrid nanospheres a weapon to kill cancer cells in addition to a contrast agent. Hybrid nanospheres with no drug loading were once again found to have negligible cytotoxicity. Photothermal Therapy with CS-AuNR Hybrid Nanospheres. One important reason for using gold nanorods in this study is that they can not only absorb and scatter NIR irradiation but also convert the absorbed energy into heat and generate localized hyperthermia, which can be exploited to destroy malignant cells by controlling the position of incident radiation. Here, we provide an in vitro demonstration of the photothermal therapy application of CS-AuNR hybrid nanospheres. LoVo cells were (33) Henriksen, I.; Green, K. L.; Smart, J. D.; Smistad, G.; Karlsen, J. Int. J. Pharm. 1996, 145, 231–240. (34) Jamieson, E. R.; Lippard, S. J. Chem. Rev. 1999, 99, 2467–2498. (35) Najajreh, Y.; Peleg-Shulman, T.; Moshel, O.; Farrell, N.; Gibson, D. J. Biol. Inorg. Chem. 2003, 8, 167–175. (36) Avgoustakis, K.; Beletsi, A.; Panagi, Z.; Klepetsanis, P.; Karydas, A. G.; Ithakissios, D. S. J. Controlled Release 2002, 79, 123–135.

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Figure 7. (a) Release profiles of cisplatin from drug-loaded CS and CS-AuNR nanospheres at 37 °C and pH 7.4. (b) In vitro cytotoxicity of cisplatin-loaded nanospheres in comparison with free cisplatin and empty nanospheres. The concentration of empty nanospheres was set to the same value as the nanosphere concentration used in the drug-loaded ones.

that CS-AuNR hybrid nanospheres with no drug loading were used in the photothermal experiments instead of drugloaded nanospheres so as to fully eliminate the potential influence of the released cisplatin on cell viability and to better demonstrate the photothermal property of our multifunctional nanocarriers. After exposure to laser power at 1 W/cm2, both the cells incubated with CS-AuNR hybrid nanospheres and those incubated with CS nanospheres were still living well. However, when the laser power density was increased to 2 W/cm2, several blue dots appeared as a sign of cell death in the group with CS-AuNR nanospheres. When the laser power density reached 3 W/cm2, almost all of the cells incubated with CS-AuNR hybrid nanospheres were stained with trypan blue. In comparison, no destruction of cells was observed in the group incubated with empty CS nanospheres even at the highest laser power density of 3 W/cm2. Therefore, it can be deduced that the cells incubated with CSAuNR nanospheres died as a consequence of the local heating generated from the hybrid nanospheres under NIR irradiation. It is worth noting that the radiation dose needed to achieve substantial cancer cell death in our work was only 3 W/cm2 for 2 min, a value 2 or 3 times lower than reported in other studies.11,13-15 We suspect that this is because the CS-AuNR hybrid nanospheres can be effectively endocytosed by cells upon incubation as demonstrated in the above text, which will likely increase the thermotherapy efficacy. It is noteworthy that the clinical therapeutic efficacy can be improved significantly by the combination of localized hyperthermia treatment and traditional chemotherapy (including cisplatin) as a result of beneficial synergy.37,38 Thus, the concept of multifunctional nanocarriers that combine stimuliresponsive localized hyperthermia and conventional anticancer drug delivery may be of great scientific significance in the cancer research area, and the CS-AuNR hybrid system reported here may be used as a potential prototype system for the realization of such a concept. Figure 8. Microscopic images of LoVo cells incubated with CS nanospheres (left) and CS-AuNR nanospheres (right) after being exposed to laser at different power densities and being stained with trypan blue. (a, b) 1 W/cm2, 10; (c, d) 2 W/cm2, 10; (e, f) 3 W/cm2, 10; and (g, h) 3 W/cm2, 40.

incubated with CS-AuNR hybrid nanospheres or empty CS nanospheres for 2 h and then exposed to laser irradiation with a wavelength of 808 nm at different power densities for 2 min each. Finally, the cells were treated with 0.4% trypan blue for 10 min. Because living cells can eliminate trypan blue and keep themselves colorless but dead ones will collect the dye and become blue, the cell viability of different groups could be evaluated directly from optical observations (Figure 8). It is worth mentioning Langmuir 2010, 26(8), 5428–5434

Conclusions CS-AuNR hybrid nanospheres have been effectively prepared using a facile nonsolvent-aided counterion complexation method. The desired particle size and gold nanorod loading level can be easily synthesized by varying the ratio among EDTA, CS, and AuNR. The obtained CS-AuNR hybrid nanospheres were found to have good biocompatibility and stability. Anticancer drug cisplatin was successfully incorporated into the obtained hybrid (37) Ohtsubo, T.; Saito, H.; Tanaka, N.; Matsumooto, H.; Sugimoto, C.; Saito, T.; Hayashi, S.; Kano, E. Cancer Lett. 1997, 119, 47–52. (38) Paiva, M. B.; Bublik, M.; Castro, D. J.; Udwitz, M.; Wang, M. B.; Kowalski, L. P.; Sercarz, J. Photomed. Laser Surg. 2005, 23, 531–535.

DOI: 10.1021/la903893n

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nanospheres by utilizing the loading space provided by the chitosan matrix. In vitro cell experiments have demonstrated the feasibility of using such hybrid nanospheres simultaneously to image cancer cells optically, attack cancer cells with a loaded anticancer drug, and use NIR irradiation to induce cancer cell death. Furthermore, this system holds advantages in terms of facile, versatile synthesis, the abundant functional groups on the hybrid nanospheres for further modification, and the high loading capacity provided by the polymeric matrix for the embedding of other active agents. Thus, the CS-AuNR hybrid nanospheres may serve as a good starting point for the construction of multifunctional nanodevices for cancer research and development and may also be extended to other advanced biomedical applications.

5434 DOI: 10.1021/la903893n

Guo et al.

Acknowledgment. This work is supported by the National Natural Science Foundation of China (nos. 50625311 and 20874042) and by the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (no. 707028). We thank Professor H. T. Wang for his help with laser irradiation experiments. Supporting Information Available: TEM micrograph and UV-vis spectra of gold nanorods used in this study. Release profile of free cisplatin solution in PBS at 37 °C and pH 7.4. TEM micrographs of CS-Fe3O4 hybrid nanospheres synthesized by the nonsolvent-aided counterion complexation method. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(8), 5428–5434