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Hydrophilic multi-walled carbon nanotubes decorated with magnetite nanoparticles as lymphatic targeted drug delivery vehiclesw Dong Yang,a Feng Yang,b Jianhua Hu,a Jiang Long,b Changchun Wang,*a Deliang Fu*b and Quanxing Nib Received (in Cambridge, UK) 22nd April 2009, Accepted 20th May 2009 First published as an Advance Article on the web 12th June 2009 DOI: 10.1039/b908012k Hydrophilic multi-walled carbon nanotubes decorated with magnetite nanoparticles were readily taken up into lymph vessels and delivered gemcitabine to lymph nodes with high efficiency under the guidance of a magnetic field. Lymphatic metastasis occurs extensively in cancers, and has a decisive influence on the prognosis of malignant tumors.1 To eradicate metastatic cancer cells from the regional lymphatic system, it is of fundamental importance to develop new and efficient lymphatic targeted drug delivery systems. They must deliver and retain high doses of drug at specific sites of lymphatic systems for maximum treatment efficiency, while minimizing side effects to normal organs.2 Carbon nanotubes (CNTs) are considered ideal candidates for drug delivery, due to their ultra-high surface area, high mechanical strength but ultra-light weight, excellent chemical and thermal stability, and rich electronic polyaromatic structure.3 CNTs have been proved to be a versatile carrier for a wide variety of biomolecules, including drugs,4 proteins,5 and peptides.6 For biological and biomedical applications, some key factors, e.g. the dispersibility of CNTs in aqueous media, and the toxicity and metabolic pathway of CNTs in vivo, have caused wide concern over recent years. To disperse the CNTs in aqueous media, many strategies, including covalent7 or noncovalent8 modification methods, have been proposed. At the same time, various groups have investigated the toxicity of CNTs, and found that functionalized CNTs display very low toxicity in vivo in contrast to pristine CNTs, even at high concentrations.9 The metabolic pathway of CNTs in animals is critical to potential applications in vivo, Wang et al.10 and Singh et al.11 have reported that after being intravenously injected into animals, functionalized CNTs were mainly excreted through the urine with little uptake by the liver or other organs. These results provide a basis for further exploration of CNTs for therapeutic applications.
Herein, we prepared hydrophilic magnetite nanoparticledecorated multi-walled carbon nanotubes (MN-MWNTs). The detailed preparation procedure is illustrated in Fig. 1. The prepared MN-MWNTs possess excellent dispersibility in water and high magnetic susceptibility. Gemcitabine, a commonly used anti-cancer drug, could be easily loaded on the MN-MWNTs. The in vivo lymphatic targeted drug delivery properties of MN-MWNTs were investigated through subcutaneous administration in Sprague–Dawley (SD) rats. The results showed that MN-MWNTs can be readily taken up into lymph vessels, and deliver cancer chemotherapy drug (gemcitabine) into the targeted lymph nodes with high efficiency. As shown in Fig. 1, PAA-g-MWNTs with a poly(acrylic acid) grafting ratio of 15 wt% were prepared according to the method from our previous work.12 MN-MWNTs were prepared by chemical co-precipitation of Fe2+ and Fe3+ onto the outer surface of PAA-g-MWNTs. Typically, 25 mg of PAA-g-MWNTs were dispersed in 50 mL of deionized water, and then 10.7 mg of FeCl24H2O and 29.1 mg of FeCl36H2O were added. After sonication for 5 min, the solution was purged with N2 for 0.5 h. Then, 2 mL of ammonia was added, and the reaction system was kept at 90 1C for 2 h. The feed molar ratio of Fe2+ : Fe3+ is 1 : 2, and the theoretical reaction yield of Fe3O4 is 12.5 mg. The MN-MWNTs produced were
a
Key Laboratory of Molecular Engineering of Polymers (Ministry of Education), Department of Macromolecular Science, Advanced Materials Laboratory, Fudan University, Shanghai 200433, China. E-mail:
[email protected]; Fax: 86 21 65640293; Tel: 86 21 65642385 b Pancreatic Disease Institute, Department of Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China. E-mail:
[email protected]; Fax: +86 21 52888277; Tel: +86 21 52888277 w Electronic Supplementary Information (ESI) available: Experimental details and additional characterization data (TEM, XRD, and microscopy). See DOI: 10.1039/b908012k
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Fig. 1 An illustration of the preparation of hydrophilic multi-walled carbon nanotubes decorated with magnetite nanoparticles (MN-MWNTs). (A) Grafting poly(acrylate acid) (PAA) on the surface of MWNTs (PAA-g-MWNTs) by in-situ free radical polymerization; (B) depositing Fe3O4 nanoparticles on the surface of PAA-g-MWNTs by a chemical co-precipitation method; (C) loading gemcitabine onto MN-MWNTs by physical adsorption.
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Fig. 2 (a) TEM image of MN-MWNTs, and (b) magnetization curves of dried MN-MWNTs (insert: optical photograph of MN-MWNTs dispersed in water with or without magnet).
Fig. 3 (a) Microscopic image (400) and (b) TEM image of the left popliteal lymph node after H&E staining.
collected by applying an external magnetic field, and then washed several times with deionized water. Fig. 2a shows a transmission electron microscopy (TEM) image of MN-MWNTs. Fe3O4 nanoparticles are clearly observed on the outer surface of the nanotube. The size of the attached Fe3O4 nanoparticles is relatively uniform, and the amount of attached nanoparticles can be easily manipulated by altering the feeding amounts of the raw materials. On the contrary, when using pristine MWNTs instead of PAA-g-MWNTs, most of the Fe3O4 nanoparticles are aggregated together to produce large clusters (Fig. S1, ESIw). This indicates that the pre-functionalization of MWNTs with PAA is critical in this process, since the grafted PAA chains contain a large number of carboxyl groups, which can interact with Fe3O4 nanoparticles and so prevent them from aggregation. Actually, even after sonicatation for 10 min (bath sonicator, 60 kHz), no obvious detachment or aggregation of Fe3O4 nanoparticles was detected. In order to characterize the magnetic properties of the MN-MWNTs, a vibrating sample magnetometer was used to record hysteresis loops of the prepared samples. Fig. 2b shows the magnetization curves of MN-MWNT powder. Due to the existence of nano-sized magnetite particles, the magnetization curves exhibit superparamagnetic behavior, i.e. no remanence remains when the applied magnetic field is removed. The MN-MWNTs can be easily dispersed in water to form a homogeneous black solution, as shown in Fig. 2b insert. When an external magnetic field was applied, the MN-MWNTs could be separated from the solution within ten seconds. After removing the magnet and shaking the bottle, the MN-MWNTs were redispersed again. The process of magnetism-driven aggregation and redispersion could be repeated many times. Overall, this shows that MN-MWNTs can be aggregated at a special site by application of an external magnetic field. It is well known that nano-sized activated carbon (NSAC) is an excellent lymphatic tracer and drug carrier. After injection into organs, NSAC is preferentially absorbed by lymph vessels through the enhanced permeability and retention (EPR) effect, and transferred into lymph nodes.13 To examine whether MN-MWNTs possess a similar property to NSAC, MN-MWNTs in physiological saline solution was subcutaneously injected into the left footpad of SD rats. During the whole experiment, all rats showed no abnormal behavior. No evidence of local toxicity, such as drug allergy, infection, ulceration, erosion or necrosis of skin, was detected. Serial macroscopic examinations revealed that the left
popliteal lymph nodes were dyed black by MN-MWNTs as early as 3 h after subcutaneous administration. Identification of MN-MWNTs in the left popliteal lymph nodes was apparent by microscopy (400) and TEM examination (Fig. 3). In contrast, MN-MWNTs were not identified in the major internal organs such as the liver, spleen, kidney, heart and lung, throughout the whole course of the experiment (Fig. S3, ESIw). These findings suggest that after subcutaneous injection, MN-MWNTs were preferentially absorbed by lymph vessels, and transferred into lymph nodes. Finally, the MN-MWNTs were used to deliver gemcitabine (GEM), a chemotherapy drug that is given as a treatment for some types of cancer, into the lymphatic system of the SD rats. To evaluate the drug delivery performance of MN-MWNTs, five groups of comparative experiments were conducted: GEM without drug carrier and external magnetic field (GEM-Control); GEM using MN-MWNTs as drug carrier with (MN-MWNTsGEM-Magnet) and without (MN-MWNTs-GEM) applying an external magnetic field; and GEM using MN-ACs (nano-sized activated carbon decorated with magnetic nanoparticles) as drug carrier, with (MN-ACs-GEM-Magnet) and without (MN-ACs-GEM) applying an external magnetic field. In the two groups where external magnetic fields were applied, a permanent magnet of 1800 Gs was sutured onto the projection surface of the left popliteal lymph nodes, as shown in the Fig. 4 insert. In each group, the GEM concentration in the left popliteal lymph nodes and blood was measured at different times after subcutaneous administration. As seen from Fig. 4a, the GEM concentration in the left popliteal lymph nodes is low in the GEM-Control group during the whole course of the experiment, which indicates that GEM is not preferentially absorbed by the lymphatic system without the help of drug carrier. Compared with other groups, the MN-MWNTs-GEM-Magnet group shows a highest GEM concentration throughout the experiment, and shows a significant difference (P o 0.01) at 6, 12, 24 and 192 h. The concentration of GEM reached a maximum after 24 h in all of the four groups with drug carriers, and in the MN-MWNTs-GEM-Magnet group, the concentration of GEM was 9.12 0.80 mg g 1, much higher than that in the MN-MWNTs-GEM group (6.28 0.73 mg g 1), MN-ACs-GEM-Magnet group (3.50 0.29 mg g 1), and MN-ACs-GEM group (3.36 0.67 mg g 1). MN-MWNTs show a higher efficiency than MN-ACs in delivering GEM into the lymphatic system, especially while applying an external magnetic field, which aggregates the MN-MWNTs together at a specific
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standards required by law and also complied with the guidelines for the use of experimental animals in China. This work was supported by China Postdoctoral Science Foundation Funded Project (20080440569), National Science Foundation of China (20674009, 50873029, 30701007), National Science Foundation for Distinguished Young Scholars of China (50525310), Shanghai Scientific and Technological Project (08431902300, 08XD14010), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (No.707023), Shanghai Educational Development Foundation (2008CG05) and Shanghai Medical College Research Fellow Award, Shanghai Leading Academic Discipline Project (B113).
Notes and references
Fig. 4 The curves of gemcitabine concentrations at different times after subcutaneous administration: (a) in the left popliteal lymph nodes, and (b) in blood plasma.
location. At the same time, the concentration of GEM in blood plasma distinctly decreased while using MN-MWNTs as drug carrier (Fig. 4b). This indicates that MN-MWNTs drug delivery is a very promising nanoplatform for future lymphatic cancer therapeutics. In conclusion, MN-MWNTs were successfully prepared by chemical co-precipitation of Fe3O4 nanoparticles on the surface of PAA-g-MWNTs in aqueous media. The prefunctionalization of MWNTs with PAA is essential to prepare MN-MWNTs with uniform attachment of magnetic nanoparticles. The MN-MWNTs can be readily dispersed in aqueous media, and show a high magnetic responsivity to external magnetic fields. MN-MWNTs can be preferentially taken up into lymph vessels by the enhanced permeability and retention (EPR) effect, then transferred into specific lymph nodes under the further guidance by magnetic field (positive guidance), and show a high efficiency in delivering GEM into the lymphatic node of SD rats. These results demonstrate that carbon nanotubes may have a prospective potential application in lymphatic targeted drug delivery. All animal procedures were approved by the institutional animal care committee. All guidelines met the ethical
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