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Biomaterials 30 (2009) 6041–6047

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Biomaterials

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Targeted delivery and controlled release of doxorubicin to cancer cells usingmodified single wall carbon nanotubes

Xiaoke Zhang a, Lingjie Meng a, Qinghua Lu a,b,*, Zhaofu Fei c, Paul J. Dyson c

a Department of Polymer Science and Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, Shanghai 200240, PR Chinab State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, PR Chinac Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland

a r t i c l e i n f o

Article history:Received 4 June 2009Accepted 14 July 2009Available online 29 July 2009

Keywords:Single wall carbon nanotubes (SWCNTs)Targeted drug deliveryChitosanAlginate sodiumNanomedicine

* Corresponding author. Department of Polymer Sciof Chemistry and Chemical Technology, Shanghai JiaDongChuan Road, Shanghai 200240, PR China. Tel.: þ86293 2607.

E-mail address: qhlu@sjtu.edu.cn (Q. Lu).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.07.025

a b s t r a c t

A targeted drug delivery system that is triggered by changes in pH based on single wall carbon nanotubes(SWCNTs), derivatized with carboxylate groups and coated with a polysaccharide material, can be loadedwith the anticancer drug doxorubicin (DOX). The drug binds at physiological pH (pH 7.4) and is onlyreleased at a lower pH, for example, lysosomal pH and the pH characteristic of certain tumor environ-ments. By manipulating the surface potentials of the modified nanotubes through modification of thepolysaccharide coating, both the loading efficiency and release rate of the associated DOX can becontrolled. Folic acid (FA), a targeting agent for many tumors, can be additionally tethered to the SWCNTsto selectively deliver DOX into the lysosomes of HeLa cells with much higher efficiency than free DOX.The DOX released from the modified nanotubes has been shown to damage nuclear DNA and inhibit thecell proliferation.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The ideal drug delivery system combines targeted delivery (i.e.a strong affinity for target cells or target tissue) with controlledrelease (i.e. release triggered by a characteristic feature of thediseased cells) such that the drug is delivered and released ina selective and discriminatory fashion [1,2]. Such a system not onlyimproves the efficacy of the drug, but minimizes the systemictoxicity to improve the quality of the patients’ life.

In recent years a wide range of different nanoscale drug deliveryvectors have been evaluated [3–5]. Notably, single wall carbonnanotubes (SWCNTs) have attracted considerable interest in thisregard, as they offer potential advantages over the more widelystudied metal nanoparticle systems, including their ability to carrya high cargo loading, their intrinsic stability and structural flexibility,which could prolong the circulation time and hence the bioavail-ability of the carried drug molecules [6–9]. Moreover, SWCNTs havebeen shown to enter mammalian cells [10–13] and due to theirpromising properties, SWCNT-based materials have already been

ence and Engineering, Schoolo Tong University, Num.880,6 21 6293 2997; fax: þ86 21

All rights reserved.

investigated as potential delivery vehicles for intracellular transportof nucleic acids [10,14,15], proteins [12,16] and drug molecules[1,13,17,18].

SWCNTs have been functionalized with antibodies and lowmolecular weight targeting agents providing a high efficiency fornanotube internalization into cells [17–25]. Such systems havebeen loaded with drug molecules such as doxorubicin (DOX) viap-p stacking interactions and the release rate of DOX has even beenshown to be controllable by using nanotubes with different dia-meters [18]. Nevertheless, the first generation SWCNTs proved to beunsuitable as drug delivery vectors as they tend to form bundlesthat disperse poorly in aqueous solutions unsuitable for pharma-cological use. To overcome this drawback, synthetic polymers suchas poly(phenylacetylene) [26,27] and natural polymers such aspolysaccharides [22,28–33] have been used to encase SWCNT vianon-covalent interactions improving their compatibility with waterand physiological environments more generally.

Despite excellent progress in using SWCNTs as drug deliveryvehicles, more research is needed to further optimize their abilityto selectively accumulate in diseased tissues and to release theirtoxic payload in a controlled manner. In this paper we describea system which appears to meet all the relevant criteria, which isbased on polysaccharide [sodium alginate (ALG) and chitosan(CHI)] modified SWCNTs for controlled release of DOX (Fig. 1), alsoincluding folic acid (FA, see Fig. 1) as a targeting group. These newmaterials were observed, using transmission electronic microscopy

Fig. 1. Molecular structures of doxorubicin hydrochloride and folic acid.

Fig. 2. Preparation of modified SWCNTs. (a) Modification of SWCNTs (derivatized with–CO2H groups) with ALG, CHI and DOX. (b) UV-Vis absorption spectra of DOX and DOXloaded SWCNTs.

X. Zhang et al. / Biomaterials 30 (2009) 6041–60476042

and fluorescence microscopy, to enter cells via the FA receptor-mediated pathway, and following internalization the drug isselectively released into the acidic environment of the lysosomes.

2. Materials and methods

2.1. Materials and measurements

The SWCNTs (purity, >90%, length, >50 mm, diameter, 1–2 nm, Chengdu OrganicChemistry Co., Ltd), sodium ALG (Acros), CHI (TCI, Tokyo), FA (Shanghai ChemicalReagents Corporation), DOX hydrochloride (Shanghai Yingxuan Chempharm), N,N-(3-dimethylaminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC$HCl, Fluka),porous poly (vinylidene chloride) (PVDC, 0.22 mm pore size, Shanghai ANPEL Instru-ment Co. Ltd), fetal bovine serum (FBS, Hyclone), high glucose Dulbecco’s ModifiedEagle’s medium (DMEM, Hyclone), and WST-1 reagent (Beyondtime Bio-Tech, China)were used as received.

High resolution-transmission electron microscopy (HR-TEM) was conducted ona JEOL TEM-2100 operates at 200 kV. Transmission electron microscopy (TEM) wascarried out on a CM120 (Philips). UV-Visible (UV-Vis) absorption spectra wererecorded on a Lambda 20 spectrometer (Perkin Elmer, Inc.). The fluorescence imageswere obtained using an inverted fluorescence microscope (IX 71, Olympus) anda charge coupled device (CCD, Cascade 650). Zeta potentials were measured ona zeta potential analyzer (Zetasizer Nano ZS90, Malvern).

2.2. Preparation of the SWCNTs

Cutting and purification of the SWCNTs was carried out using a modified liter-ature procedure [34]. The SWCNTs (500 mg) were added to a mixture of 98% H2SO4

and 65% HNO3 (V:V¼ 3:1, 200 mL) and exposed to sonic irradiation at 0 �C for 24 h.The cut SWCNTs were thoroughly washed with ultrapure water (18.2 MU) andfiltered through a micro-porous filtration membrane (F 0.22 mm). They were re-dispersed in HNO3 (2.6 M, 200 mL) and refluxed for 24 h, collected by filtration andwashed with ultrapure water to neutrality. The product was then dried undervacuum at 50 �C for 24 h.

2.3. Preparation of ALG-SWCNTs

Cut SWCNTs (20 mg) were sonicated in sodium ALG solution (40 mg in 0.1 M

aqueous NaCl, 40 mL) for 20 min and then stirred at room temperature for 16 h. Themodified SWCNTs were collected and washed with ultrapure water by ultracentri-fugation to remove unbound ALG, then collected and dried at room temperature toobtain ALG-SWCNTs.

2.4. Preparation of CHI-SWCNTs

CHI-SWCNTs were prepared in the same way as that described above for theALG-SWCNTs by replacing the ALG solution for CHI solution.

2.5. Preparation of CHI/ALG-SWCNTs

The ALG-SWCNTs (10 mg) were sonicated for 20 min and then a CHI solution(20 mg in 0.1 M aqueous NaCl and 0.02 M acetic acid, 20 mL) was added. The mixturewas stirred for 16 h at room temperature to give the product following ultracen-trifugation, washing and drying as described above.

2.6. Preparation of FA-CHI/ALG-SWCNTs

The CHI/ALG-SWCNTs (4 mg) were suspended with FA (6 mg) in a pH 7.4 PBSbuffer solution (8 mL) and then EDC$HCl (5 mg) was added. After stirring thereaction mixture at room temperature for 16 h, the product was washed with

ultrapure water several times by repeated ultracentrifugation to remove unreactedreagents and then dried at room temperature.

2.7. DOX loading onto the nanotubes

DOX hydrochloride (9 mg) was stirred with the modified nanotubes (3 mg)dispersed in a pH 7.4 PBS buffered solution (6 mL) and stirred for 16 h at roomtemperature. The products (denoted as DOX-SWCNTs, DOX-ALG-SWCNTs, DOX-CHI-SWCNTs, DOX-CHI/ALG-SWCNTs and DOX-FA-CHI/ALG-SWCNTs) were collected byrepeated ultracentrifugation with PBS until the supernatant became colour free. Theamount of unbound DOX was determined by measuring the absorbance at 490 nm(the characteristic absorbance of DOX) relative to a calibration curve recorded underthe same conditions, allowing the drug loading efficiency to be estimated.

2.8. DOX release from the nanotubes

Suspensions of the DOX loaded SWCNTs (1 mg) were allowed to stand at 37 �C inpH 7.4 and pH 5.5 PBS buffered solutions (5 mL). After different time intervals, the

Fig. 3. TEM images of modified SWCNTs. (a) Cut SWCNTs, (b) ALG-SWCNTs, (c) CHI-SWCNTs, (d) CHI/ALG-SWCNTs, (e) DOX-SWCNTs, (f) DOX-ALG-SWCNTs, (g) DOX-CHI-SWCNTsand (h) DOX-CHI/ALG-SWCNTs.

X. Zhang et al. / Biomaterials 30 (2009) 6041–6047 6043

nanotubes were separated from the buffer by ultracentrifugation, and the concen-tration of released DOX in the supernatant was estimated by UV-Vis spectroscopy.

2.9. Incubation of HeLa cells with DOX loaded nanotubes

HeLa cells were cultured in DMEM supplemented with 10% FBS in a humidifiedincubator (MCO-15AC, Sanyo) at 37 �C in which the CO2 level was maintained at 5%.For cells incubated with the DOX loaded nanotubes or free DOX, the cells werecultured overnight to allow attachment, washed with FBS-free DMEM and thenincubated with 20 mg/mL DOX-FA-CHI/ALG-SWCNTs, 20 mg/mL DOX-CHI/ALG-SWCNTs or 50 mg/mL DOX at 37 �C for 1 h in FBS-free medium. After incubation, thecells were washed repeatedly with sterilized PBS before further analysis.

To further evaluate the role of FA in the cellular uptake of DOX-FA-CHI/ALG-SWCNTs, the cells were pretreated with free FA (0.5 mg/mL) for 2 h [35], DOX-FA-CHI/ALG-SWCNTs (20 mg/mL) was then added and the cells were cultured foranother 1 h, washed with sterilized PBS and analyzed by fluorescence microscopy.

For TEM analysis, HeLa cells were washed with PBS, and fixed with 2% glutar-aldehyde and 1% osmium tetroxide for 2 h at 4 �C. The cells were then dehydrated ina graded ethanol series (30%, 50%, 70% with 3% uranyl acetate, 80%, 95%, and 100%)for 10 min at each concentration and followed by two changes in 100% propyleneoxide. After infiltration and embedding in epoxy resins at 60 �C for 48 h, the sectionswere stained with lead citrate and analyzed by TEM [36].

Table 1Zeta potentials of the modified SWCNTs.

Zeta potential (mV)

Modified SWCNTs without DOXa DOX loadeda

Cut SWCNTs �63.23� 1.74 �17.00� 0.14ALG-SWCNTs �76.20� 2.07 �2.06� 0.48CHI-SWCNTs 2.56� 0.35 6.57� 0.18CHI/ALG-SWCNTs �5.87� 0.08 16.60� 1.41

a Values are averaged from three measurements.

2.10. Cell viability test

The WST-1 assay was used to measure cell viability [8,33]. In brief, HeLa cellswere seeded into a 24-well flat culture plate (Corning). After culturing overnight thecells were washed with FBS-free DMEM and incubated with a specific concentrationof DOX-FA-CHI/ALG-SWCNTs (10, 25, 50 mg/mL), FA-CHI/ALG-SWCNTs (50 mg/mL)and free DOX (100 mg/mL) in FBS-free culture medium at 37 �C for 1 h. The cells werethen washed three times with sterilized PBS and incubated with fresh mediumcontaining 10% FBS for the indicating times. The cells were then washed with PBSand FBS-free DMEM (500 mL) was used to substitute the culture medium beforeadding 1/10 (V/V) of WST-1 reagent. After incubation for 2 h at 37 �C, the absorbancewas measured at 450 nm using a microplate reader (Model 680, Bio-Rad). Thebackground absorbance was measured at 450 nm before adding the WST-1 reagentand the cells cultured in the absence of a drug were used as controls.

3. Results and discussion

3.1. Modification of the SWCNTs with polysaccharides and DOX

Functionalization of SWCNTs with polysaccharides is a relativelystraightforward process based on methods previously described inthe literature [22,29–32] (Fig. 2a). In brief, the SWCNTs wereinitially oxidized and cut and then sonicated in aqueous solutions ofthe appropriate polysaccharide (ALG or CHI), leading to theencapsulation of the SWCNTs by the polysaccharides [37]. However,in a two-step process involving initial treatment with ALG followedby CHI, the SWCNT core could be ‘doubly’ wrapped by both CHI andALG. Subsequent mixing of the polysaccharide modified SWCNTswith DOX in aqueous solution also allows the DOX to be attached tothe nanotubes.

All the SWCNT derivatives form stable suspensions in water withthe DOX loaded systems being lighter in colour (red-brown) thantheir precursors. Consequently, DOX loading was monitored by UV-Vis absorption spectroscopy (Fig. 2b). DOX inwater displays two mainabsorptions at 490 and 232 nm, and upon treatment of DOX with thepolysaccharide modified nanotubes, these two peaks are slightly red-shifted, indicative of interactions between DOX and SWCNTs.

Fig. 4. Drug loading and release of the modified SWCNTs. (a) Drug loading efficiency ofmodified SWCNTs. DOX release in PBS buffer at 37 �C at (b) pH 7.4 and (c) at pH 5.5.

X. Zhang et al. / Biomaterials 30 (2009) 6041–60476044

The structures of the modified SWCNTs were investigated byHR-TEM. Fig. 3a shows the cut nanotubes, after cutting and clean-ing, which appear to be smooth and without impurities, indicatingthat metal particles and amorphous carbon have been completelyremoved. The cut and purified SWCNTs are generally short(< 500 nm), well separated, and only form small bundles. Aftercoating with ALG or CHI, the polysaccharide chains can be observedon the sidewalls of SWCNTs (Fig. 3b, 3c). Moreover, the doublelayered polysaccharide present in CHI/ALG-SWCNTs (Fig. 3d) isthicker than the coatings in CHI-SWCNTs and ALG-SWCNTs. Thesidewalls of SWCNTs in Fig. 3d are poorly defined, presumably dueto the thick bilayer of bound CHI/ALG polysaccharides.

3.2. DOX loading onto the modified SWCNTs

As CHI contains cationic groups, whereas ALG is composed of ananionic structure, the two polysaccharides can adhere to the SWCNTsvia not only p-p interactions, but also via electrostatic interactions,thereby allowing the overall surface charge density to be tuned. Thezeta potentials of the modified SWCNTs were investigated, with thecut SWCNTs having a negative potential due to the presence of COO�

groups on the sidewalls, and the potentials changing significantlyafter deposition of the polysaccharides (Table 1).

Previously, it has been shown that DOX can be adsorbed onto thesidewalls of SWCNTs via p-p stacking interactions [18], by simplymixing the SWCNTs with DOX, albeit yielding nanotubes with veryheterogeneous surfaces (Fig. 3e). In contrast, when DOX is loadedonto polysaccharide modified SWCNTs, uniformly coated nanotubescould be obtained. Fig. 3f-h shows the TEM images of polysaccharidemodified SWCNTs loaded with DOX. The diameters of the nanotubesare increased and the clarity of the sidewalls of the nanotubes isreduced due to screening by both the DOX and the polysaccharides.

The zeta potentials of the nanotubes increase dramaticallyfollowing loading with DOX (Table 1), indicating that the cationic DOXions are adsorbed onto the sidewalls of the modified SWCNTs. TheDOX loading efficiencies (defined as the weight ratio of DOX to themodified SWCNTs) onto modified SWCNTs were found to be in excessof 120% (Fig. 4a) and follow the order: DOX-ALG-SWCNTs>DOX-SWCNTs>DOX-CHI/ALG-SWCNTs>DOX-CHI-SWCNTs, which cor-responds to the ascending order of zeta potentials of the modifiedSWCNTs (Table 1). The results indicate that the positively chargedDOX molecules are more readily adsorbed onto the surfaces withlower surface potentials, suggesting that electrostatic interactions aswell as p-p stacking interactions play an important role with respectto DOX loading.

3.3. DOX release from the modified SWCNTs

The release of the DOX loaded nanotubes is pH-triggered; drugrelease curves (Fig. 4b, 4c) show that the DOX on all the modifiedSWCNT supports is stable in PBS buffer at pH 7.4 at 37 �C. In slightlyacidic solutions, i.e. pH 5.5, corresponding to lysosomal pH, anappreciable release of DOX from all the materials was observed overa 72 h period. While loading efficiency is greatest with the ALG-SWCNTs the release of the DOX is slowest. In contrast, with CHI-SWCNTs the DOX loading is the lowest and its release is the fastest.Therefore, the CHI/ALG-SWCNTs are an ideal compromise in termsof loading and release of DOX. Clearly, if slow release is required thenthe ALG-SWCNTs would be the ideal choice and for fast release theCHI-SWCNT system would be preferred and the system could be finetuned to specific release rates by varying the ALG/CHI ratios.

The pH-triggered drug release from the modified nanotubes, inwhich the DOX is bound onto the SWCNT surface under normalphysiological conditions and released at reduced pH typical ofmicro-environments of intracellular lysosomes or endosomes or

cancerous tissue, more generally, provide an in-built mechanismfor selective drug release. To explore this hypothesis, and moreover,to endow the SWCNTs with tumor targeting properties, FA was alsoapplied to the nanotube surface. FA accumulates in certain cancers,in particular, cancers of the ovary, cervix, endometrium, kidney,breast, brain, lung, and colon, due to over-expression of FA recep-tors on their surfaces [38]. CHI reacts more efficiently with FA via anamidation reaction and therefore the CHI/ALG-SWCNTs offersa further advantage over the homo-polysaccharide coated nano-tubes in that one polymer has an affinity for the DOX drug and onefor the FA targeting molecule.

3.4. Cellular uptake of the DOX loaded SWCNTs

The DOX-FA-CHI/ALG-SWCNTs, DOX-CHI/ALG-SWCNTs and DOX(used as a control) were incubated with human cervical carcinomaHeLa cells and analyzed by fluorescence microscopy allowing the

Fig. 5. Fluorescence images of cells incubated with (a) DOX-FA-CHI/ALG-SWCNTs (20 mg/mL), (b) DOX-CHI/ALG-SWCNTs (20 mg/mL), (c) FA for 2 h followed by DOX-FA-CHI/ALG-SWCNTs (20 mg/mL), and (d) free DOX (50 mg/mL) at 37 �C for 1 h.

X. Zhang et al. / Biomaterials 30 (2009) 6041–6047 6045

DOX to be located as it emits red fluorescence under irradiation withgreen light. The DOX-FA-CHI/ALG-SWCNTs treated cells (Fig. 5a)show a brighter red fluorescence than the other two samples indi-cating that the FA conjugated nanotubes are taken up more effi-ciently into the HeLa cells. It is also apparent from Fig. 5a, 5b that thefluorescence is concentrated in the cytoplasm, in accordance withthe previous reports [21,22], and suggesting that the nanotubescannot cross intracellular membranes.

Fig. 6. TEM images of (a) untreated HeLa cells, (b) a DOX-FA-CHI/ALG-SWCNTs treated HeLa carrow points to a SWCNT-containing vesicle, and the white arrow points to aggregated SW

To further evaluate the role of FA in the cellular uptake ofnanotubes, the FA receptor was blocked on the surface of HeLa cellsby pre-treating HeLa cells with free FA for 2 h [22,33]. As shown inFig. 5c, following such treatment a much weaker red fluorescence isobserved, demonstrating that the uptake of DOX-FA-CHI/ALG-SWCNTs and FA is competitive, and suggesting that the DOX-FA-CHI/ALG-SWCNTs are internalized via the FA receptor-mediatedpathway.

ell, (c) magnified image of (b), (d) magnified image of the boxed region in (c). The blackCNTs inside a lysosome.

Fig. 7. Cytotoxicity of the modified SWCNTs. (a) Viability of HeLa cells treated with FA-CHI/ALG-SWCNTs, DOX-FA-CHI/ALG-SWCNTs and free DOX for 1 h, washed with sterilizedPBS, then continued incubating in fresh culture media (10% FBS) for another 24, 48 and 72 h. Fluorescence images of HeLa cells treated with DOX-FA-CHI/ALG-SWCNTs (20 mg/mL)for (b) 1 h and (c) the treated cells continued culturing in fresh media (10% FBS) after 72 h.

X. Zhang et al. / Biomaterials 30 (2009) 6041–60476046

To establish whether the DOX-FA-CHI/ALG-SWCNTs wereinternalized in HeLa cells, rather than being bound to the cellsurface, TEM was used to analyze the DOX-FA-CHI/ALG-SWCNTstreated cells. Unlike the untreated cells (Fig. 6), some aggregates ofthe nanotubes are observed as black patches inside the cell cyto-plasm (Fig. 6b). Based on the cell morphology (Fig. 6c, 6d), it isplausible that the majority of the internalized nanotubes accumu-lates inside the lysosomes [39].

3.5. Cell viability studies

Cell viability of the various nanotubes and DOX was establishedin HeLa cells using the WST-1 assay (Fig. 7a) with the DOX-FA-CHI/ALG-SWCNTs proving to be the most cytotoxic. Incubations with theunloaded nanotubes or the free drug did not result in appreciablecytotoxicity at the administered concentrations, indicating that theDOX-FA-CHI/ALG-SWCNTs are not only cytotoxic, but also selective.Fluorescence images of the DOX-FA-CHI/ALG-SWCNTs treated cellsafter 1 h (Fig. 7b) and after 72 h (Fig. 7c) show that the drug rapidlyaccumulates inside the cells and ultimately targets the cell nuclei. Asthe nanotubes themselves do not seem to cross the nuclearmembrane, these data suggest the DOX is been released in the lowpH environment of the lysosomes and then migrates into nucleus tobind DNA. This latter interaction has been shown to inhibit tran-scription and ultimately leads to cell death [40,41].

4. Conclusions

A highly effective drug delivery system based on functionalizedSWCNTs has been developed that overcomes the limitations of

other carbon nanotube-based systems. The system employs twodifferent polysaccharides (ALG and CHI) in a complementaryfashion to facilitate further functionalization with a targeting group(FA) and an anticancer drug (DOX). The complete system displaysexcellent stability under physiological conditions, but at reducedpH typical of the tumor environment and intracellular lysosomesand endosomes, the DOX is efficiently released and enters the cellnucleus and induces cell death. Based on a number of pertinentcontrol experiments it is possible to conclude that the overallnanoscale drug system is more selective and effective than the freedrug and it should result in reduced general toxicity, and hencereduced side-effects in patents, and also allow a lower amount ofthe drug to be applied.

Acknowledgements

We gratefully acknowledge financial supports from the NationalNatural Science Foundation of China (Grant number: 60577049),the Shanghai Municipal Science and Technology Commission(Grant number: 05JC14019 and 0652nm01), the Shanghai LeadingAcademic Discipline Project (No: B202), and Hi-Tech Research andDevelopment Program of China (Grant number: 2009AA03Z329).

Appendix

Figures with essential colour discrimination. Figs. 2, 4, 5 and 7 inthis article may be difficult to interpret in black and white. The fullcolour images can be found in the on-line version, at doi:10.1016/j.biomaterials.2009.07.025.

X. Zhang et al. / Biomaterials 30 (2009) 6041–6047 6047

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