POTENTIAL CLINICAL RELEVANCE

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Original A

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 427–441

Pharmacological and toxicological target organelles and safe use ofsingle-walled carbon nanotubes as drug carriers in

treating Alzheimer diseaseZhong Yang, PhDa, Yingge Zhang, PhD, MDa,⁎, Yanlian Yang, PhDb, Lan Sun, PhDa,

Dong Han, PhDb, Hong Li, PhDa, Chen Wang, PhD, MDb

aKey Laboratory of Nanopharmacology and Nanotoxicology, Beijing Institute of Pharmacology and Toxicology, Beijing, ChinabNational Center for Nanoscience and Nanotechnology, Beijing, China

Received 20 August 2009; accepted 18 November 2009

www.nanomedjournal.com

Abstract

Identification of pharmacological and toxicological profiles is of critical importance for the use of nanoparticles as drug carriers innanomedicine and for the biosafety evaluation of environmental nanoparticles in nanotoxicology. Here we show that lysosomes are thepharmacological target organelles for single-walled carbon nanotubes (SWCNTs) and that mitochondria are the target organelles for theircytotoxicity. The gastrointestinally absorbed SWCNTs were lysosomotropic but also entered mitochondria at large doses. Genes encodingphosphoinositide-3-kinase and lysosomal-associated membrane protein 2 were involved in such an organelle preference. SWCNTadministration resulted in collapse of mitochondrial membrane potentials, giving rise to overproduction of reactive oxygen species, leading todamage of mitochondria, which was followed by lysosomal and cellular injury. Based on the dosage differences in target organelles,SWCNTs were successfully used to deliver acetylcholine into brain for treatment of experimentally induced Alzheimer disease with amoderate safety range by precisely controlling the doses, ensuring that SWCNTs preferentially enter lysosomes, the target organelles, and notmitochondria, the target organelles for SWCNT cytotoxicity.

From the Clinical Editor: Single wall carbon nanotubes (SWCNT) could make excellent targeted delivery systems for pharmaceuticals.Inside the cells, lysosomes are the pharmacological target organelles of SWCNT, but in large doses mitochondria also take up SWCNTand mitochondrial toxicity becomes the reason for overall toxicity of this approach. In this paper, SWCNT were successfully usedto deliver acetylcholine in Alzheimer’s disease brains with high safety range by controlling the doses to ensure lysosomal but notmitochondrial targeting.© 2010 Elsevier Inc. All rights reserved.

Key words: Single-walled carbon nanotubes; Lysosomes; Mitochondria; Organelles; Target treatment; Alzheimer disease

To successfully apply the new generation of nanomaterials asdrug carriers in the treatment of diseases, it is essential todetermine their pharmacological and toxicological profiles.Carbon nanotubes (CNTs) are nanoparticles with great promisein biomedicine as drug carriers, although their biosafety is of greatconcern. CNTs can interact with mammalian cells and enter cellsvia cytoplasmic translocation1-4; they therefore can deliver a

This work was supported by National Basic Research Program of China(2006CB933304, 2006CB705602-7, and 2010CB933904).

⁎Corresponding authors: 27 Taiping Road, Beijing 100850, China.E-mail address: zhangygm@126.com (Y. Zhang).

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2009.11.007

Please cite this article as: Z. Yang, et al, Pharmacological and toxicological targein treating Al.... Nanomedicine: NBM 2010;6:427-441, doi:10.1016/j.nano.200

range of therapeutic reagents into the cell. For example, plasmidDNA has been internalized by the cell, and the expression of theplasmid-carried marker genes has been enhanced.4-7 Othermacromolecules, including proteins,8 polymers,9 and single-stranded DNA10 have also been internalized by coating ontoCNTs and through the interaction of CNTswithmammalian cells.Despite all of their proven potentials as drug carriers, CNTs haveshown toxicity on cultured cells such as human keratinocytes,11,12

T lymphocytes,13 kidney cells,14 and alveolar macrophages,15-18

as well as in animals.19,20 If inhaled into lungs, CNTs can bemoretoxic than carbon black or as toxic as quartz.15,18 The cytotoxicityof SWCNTs has been attributed to the cellular oxidative stressesinduced by this material.21-24 However, other studies have foundSWCNTs to have limited toxicity for endothelial cells in vitro25

t organelles and safe use of single-walled carbon nanotubes as drug carriers9.11.007

Figure 1. Transmission electron microscopy (TEM) images and Raman spectra of single-walled carbon nanotubes (SWCNTs). (A) TEM images of SWCNTsand (B) of the shearing-shortened SWCNTs. (C) Raman spectra of SWCNTs before gastrogavage, (D) of the shearing-shortened SWCNTs, and (E) of theSWCNTs retrieved from the lysosomes and mitochondria of the intestinal cells.

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and to lead to no serious histopathological changes whenimplanted in the bodies of rats for 90 days.26 Obviously there iscontroversy about the toxic effects of CNTs, which must beresolved before CNTs can be acceptable for practical use as drugcarriers. Unfortunately, the present data are far from sufficient tosolve this controversy, although there have been some studies onthe excretion of SWCNTs from the bodies of animals.27,28 Thepresent study is designed to investigate the in vivo pharmacolog-ical and toxicological profiles of SWCNTs from basic mechan-

isms to experimental therapeutics, and to discover a method to usethem as drug carriers.

Methods

Single-walled carbon nanotubes

SWCNTs were provided by Nachen Science and TechnologyCompany (Beijing, China). A single tube has a diameter of 0.8–

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1.2 nm and a length of several microns (Figure 1, A). TheirRaman spectrum showed the chemical composition of 99.455%carbon, 0.439% oxygen, 0.106% silicon atoms, and no iron(Figure 1, C). To obtain a suspension for gastrogavage,SWCNTs were sheared in dimethyl formamide by a XHF-Dhi-speed dispersator (Ningbo Scientific Biotechnology, Ningbo,China) at 20,000 rpm and were then centrifuged (1000 g, 10minutes). The precipitate was suspended in normal saline bysonication29 and allowed to stand for 10 minutes. Thesupernatant was then collected and recentrifuged (1000 g, 10minutes), and shortened SWCNTs were obtained; these weredried, resuspended in normal saline by sonication, and incubatedfor 10–20 minutes before the gastrogavage. The shortenedSWCNTs had a length of about 50–300 nm (Figure 1, B).Shearing and brief sonication did not change the chemicalcomposition of SWCNTs, as shown by the similar Ramanspectra before and after the shearing (Figure 1, D).20 Sampleswere freshly prepared on the day of dosing. The composition ofSWCNTs was determined by a RFS-100/S Raman spectrometer(Bruker, Fallenden, Switzerland) with excitation of 514 nm andpower of 400 mW.

Handling of animals

Kunming mice obtained from the Animal Center of BeijingAcademy of Medical Sciences were constantly monitored in theRodent Health Monitoring Department of the center and fed witha fluid nutritional diet free of any pathogen, chemicals withpotentials to cause organelle diseases,30 or particulate materials,so as to avoid any potential interference with observation results.All animals were allowed to acclimate with a 12-hour light-darkcycle for at least 1 week before being used in the study and werecared for and used humanely according to National Aeronauticsand Space Administration (NASA) Animal Care and UseProgram Guidelines. The use of the animals was approved bythe Animal Subject Review Committee of the Beijing Academyof Medical Science.

SWCNT gastrogavage

A total of 120 Kunming strain male mice (20–22 g) wereevenly and randomly divided into six groups, of which fivewere to receive gastrogavage of 5, 50, 100, 300, 400, and 500mg/kg SWCNTs and one was to receive normal salinegastrogavage as control. To ensure that sufficient SWCNTswould enter into the body for ease of detection, these doseswere given as a bolus once a day for 10 days. A 0.3-mLvolume of freshly ultrasonicated SWCNTs suspension per 10g body weight was gastrogavaged via a 24-gauge gastro-gavage injector, and care was taken to avoid injury to themouth, throat, and esophagus, or introduction of thesuspension into the trachea. The entire operation was carriedout under sterile conditions.

Transmission electron microscopy (TEM)

The intestinal tissues were sampled from two mice in eachgroup at 30 minutes after gastrogavage every day for TEMobservation. A 1-mm3 sample of internal organs was removedand immediately fixed in 3% glutaraldehyde, dehydrated in

acetone, filtered, and embedded in epoxy resin. Ultrathinsections of 40 nm were made by a TY9334 ultramicrotome(Leica Microsystems, Weltzer, Germany) and stained in uranylacetate according to the standard methods for the preparation ofTEM samples. TEM was performed on a CM-120 microscope(Philips, Eindhoven, The Netherlands) at different acceleratingvoltages andmagnifications, and images were obtained by a high-resolution charge-coupled device camera (AMT; NatureGene,Medford, New Jersey).

Collection and examination of SWCNTs in nucleus, lysosomes,and mitochondria of intestinal cells

Within 24 hours after the last gastrogavage, parts of theintestines were removed from two mice in each group, choppedinto 2-mm3 pieces, and then homogenized with a XHF-D hi-speed dispersator (Ningbo Scientific Biotechnology). Nucleus,lysosomes, and mitochondria were isolated with correspondingSigma isolation kits respectively, according to the methodprovided by Sigma (St Louis, Missouri) in the technicalbulletin. The lysosomal and mitochondrial pellets were eachweighed in a 1/100,000 balance and placed in a glass tubecontaining 10 mL 10 M nitric acid and digested at 90°C for 2hours to remove the tissues and organic materials. The digestedfluid was centrifuged, and the indigestible pellet was examinedby Raman spectrometry (System RM2000; Renishaw, HoffmanEstates, Illinois) with 532-nm laser excitation. The SWCNTscollected from every mouse in a group were combined andscaled in a 1/100,000 balance. The SWCNT contents wereexpressed as the ratio between the weights of SWCNTs andthose of corresponding organelles.

Enzymatic detection of the influences of mitochondria onlysosomal damage

The integrity of lysosomes was assessed by assaying releasedβ-galactosidase.49 One hundred milligrams of lysosomes(expressed as proteins) were incubated for 3 hours at 37°C in asolution of phospholipase A2 (0.2 U mL–1), 30 μM lysosomo-tropic detergent O-methyl-serine dodecylamide hydrochloride(MSDH), with 2.5 μg mL–1 of either cathepsin B or cathepsin D(stock solutions of the cathepsins were in NaCl–inorganicphosphate pH 6.0, whereas MSDH and phospholipase A2 werein NaCl–inorganic phosphate pH 7.4), and then centrifuged at14,000 g for 10 minutes. The supernatants were removed, and1 mL 0.1% Triton X-100 in distilled water (vol/vol) was added tolyse the remaining intact lysosomes. Activities of β-galactosidasein the ruptured lysosomal pellet and in the supernatant wererespectively measured using Lysosomal β-Galactosidase AssayKit (Beyotime, Haimen, China). The reaction was carried out in a96-well plate. The reaction solution in each well contained 200 μLassay buffer, 10 μL lysosomal lysates, and 1.6 μg O-nitrophenyl-β-D-galactopyranoside, which were added into the well andincubated at 37°C for 20–60 minutes until a yellow color wasobserved. The optical density (OD) was measured at 420 nm in aUV-2800 ultraviolet-visible (UV-Vis) spectrometer (Unico,Shanghai, China) after 100 μL 1 M Na2CO3 had been added tostop the reaction. The β-galactosidase activity was calculatedusing the formula: β-gal (unit) = (OD 420 nm × 380 × 10)/

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(incubation time in minutes × volume in milliliters of cell extract).The degree of lysosome integrity was expressed as the percentageof the supernatant β-galactosidase activities over total activities.The influences of mitochondria were determined by addingmitochondria homogenate (the quantity was expressed in proteincontents) into the lysosome samples and assaying the changes inlysosome integrity.

Measurement of reactive oxygen species (ROS) in isolatedlysosomes and mitochondria

Oxidation-sensitive fluorescent dye 2′, 7′-dichlorodihydro-fluorescein diacetate (DCFD; Molecular Probes, Eugene, Oregon)was used to measure the production of ROS, mainly hydrogenperoxide and hydroxyl radicals, in isolated lysosomes andmitochondria.31 Samples of lysosomes or mitochondria (200 mgproteins) were added into wells of a 96-well culture plate and thentreated with SWCNTs (5, 10, 20, and 40 μg/mL, respectively)alone or in combination with the antioxidants vitamin E (100mM)or N-acetylcysteine (10 mM). After 30 minutes organelles werewashed and incubated at 37°C with 10 μg mL–1 DCFD for 5–40min. After washing twice, fluorescence intensity was measured at530 nmwith excitation at 485 nm in a FL500 fluorimeter (BioTek,Winooski, Vermont). Four wells were used for each measurement.

Measurement of mitochondrial membrane potential

Mitochondria (50 μg) were incubated with SWCNTs for 20minutes in a respiratory buffer (0.07 M sucrose, 0.23 Mmannitol, 30 mM Tris HCl, 4 mM MgCl2, 5 mM KH2PO4,1mM ethylenediamine tetraacetic acid, 0.5% bovine serumalbumin, pH 7.4) in a spectrofluorophotometer cuvette at 37°C.The fluorescence was observed under a TCS-SP2 laser-scanning confocal microscope (Leica Microsystems) afteraddition of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimida-zolyl-carbocyanide iodine (JC-1; Molecular Probes) in a finalconcentration of 3 μM. JC-1 is a unique cationic dye to signalthe loss of mitochondrial membrane potential (MMP).32 Thenegative charge established by the intact MMP allows JC-1 toaggregate in the mitochondrial matrix and become fluorescentred. When MMP collapses the JC-1 cannot aggregate within themitochondria, remaining in a green fluorescent monomericform. To quantitatively measure the influences of SWCNTs onMMP, 50 μg mitochondria were added into 0.9 mL five times–diluted JC-1 staining working solution (50 μL JC-1 + 8 mLultrapure water + 2 mL staining buffer) for each well of the six-well plate, mixed evenly, and incubated at 37°C for 20 minutes.The red and green fluorescence intensity were determinedseparately at 600 nm with an exciting wavelength of 550 nmand at 535 nm with an excitation of 485 nm in a FL500fluorimeter (BioTek). MMPs were expressed as the ratio of redto green fluorescence intensity.

Blood biochemical measurements

Sera were collected by centrifuging the blood samplescollected from the carotid artery for a series of biochemicaltests. The activities of serum enzymes, total proteins, albumin,blood urea nitrogen, creatine, and creatine kinase in the serumwere determined in a 7020 automatic biochemistry analytical

instrument (Hitachi, Tokyo, Japan). The data from six sampleswere statistically analyzed.

Mouse models of Alzheimer disease (AD)

Thirty Male Kunming strain mice (25–30 g, 9 weeks old) weretrained for the learning andmemory capability tests. Six were usedas normal controls, which received normal saline injection.Twenty-four were used for drug treatment. The learning andmemory capabilitieswere represented by the behaviormerits of thetrained mice in one trial avoidance test,33 shuttle test,34 and watermaze test35 24 hours before the experiment. ADmodelsweremadeby an intraperitoneal single-dose injection of 20 mg/kg kainic acid(KA).36 KA is a neurotoxic amino acid that can damage neurons tocause dementia and has been used as an agent in the preparation ofanimal models of AD.36 The learning and memory capabilitieswere retested 24 hours after KA injection. AD mice would havelower merits in comparison with themselves before KA injectionas well as in comparison with the normal mice.

Therapeutic effects of SWCNT-acetylcholine on AD mice

SWCNTs were loaded with acetylcholine (ACh) by adding100 mg SWCNTs into 10 mL of 1 M ACh solution. Then themixture was placed in an ultrasonic field at 4°C for 30 minutes toallow the adsorption of ACh onto SWCNTs, followed bycentrifuging at 3000 rpm, after which the pellet of ACh-loadedSWCNTs (SWCNT-ACh) was collected. The ACh loading ofSWCNTs was confirmed by Raman spectrum in a RM2000Raman spectrometer (Renishaw). The quantity of ACh adsorp-tion was determined by measuring the ACh OD at 492 nm in thesupernatant after adsorption with a UV-2800 UV-Vis spectrom-eter (Unico) and calculated by the formula: Q = (TACh – RCACh ×V), where Q (mg/g) was the quantity of the adsorbed ACh onSWCNTs, TACh was the total quantity of ACh in 100 mLsolution, RCACh was the residual ACh at the end of theadsorption, and Vwas the total volume of the adsorption mixture.A Q of 200 mg ACh per gram SWCNTs could be obtained afterthe nonspecifically adsorbed ACh was washed off by centrifug-ing SWCNT-ACh three times in pH 7.4 phosphate buffersolution. The dependence of the release of ACh from SWCNT-ACh complex on pH was confirmed by a Q value of 5 mg AChper gram SWCNTs when the washing was carried out in pH 4.5phosphate buffer solution.

Twenty-four AD mice trained as mentioned above weredivided into five groups including one control group receivingnormal saline, one free-ACh 5 mg/kg, one SWCNTs alone, andone SWCNT-ACh 25 mg/kg. All of the drugs, respectively, wereadministered by gastrogavage 24 hours after KA injection.Learning and memory capabilities were tested 8 hours afterdrug administration.

Coefficient of the safety range of SWCNTs in treating AD mice

One hundred-twenty AD male mice (25–30 g) were dividedinto two groups of 60 mice each for the calculation of the 99%effective dose (ED99) for SWCNTs in the treatment of AD, andfor the toxic dose of SWCNTs expressed as the damage of 1% ofthe mitochondria (TD1).

37 The safety range was expressed as theratio of ED99 to TD1.

38

Figure 2. Single-walled carbon nanotubes (SWCNTs) in live cells and the content differences in different intestinal segments examined by transmission electronmicroscopy. (A) Intestinal wall tissues. An absorption cell with SWCNTs in the intestinal mucous layer (arrow a); two macrophages with SWCNTs in thesubmucous layer (arrows b and c). (B)Magnification of the absorption cell indicated by arrow a in A. Ve, absorption vesicle; Vi, microvilli. (C) The double-layer membrane (arrow) enclosing the Ve. (D) Magnification of macrophage indicated by arrow b in A. Many SWCNTs can be seen (arrows). (E)Magnification of macrophage indicated by arrow c in A. Many SWCNTs can be seen (arrows). (F) SWCNT-containing lysosomes in one cell (SCLCs) fromjejunum; (G) SCLCs from ileum. (H) The relationship between the ratio taken by the lysosomes containing SWCNTs (as FLSs) in the total number of theobserved lysosomes and the times of gastrogavage. (I) Comparison of the average number of SWCNTs in one cell (ANSOC) from jejunum with that from ileum.***P b 0.001 compared with jejunum. (J) Comparison of numbers of SWCNT-containing cells (SCC) in jejunum with those in ileum. ***P b 0.001 comparedwith jejunum.

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Statistical analysis

The results are reported as means ± standard deviation.Groups were analyzed by analysis of variance, and pairwisecomparison was performed using the Tukey test with signifi-cance defined by a P value of less than 0.05.

Results

Confirmation of SWCNTs in tissues

On different days of SWCNT gastrogavage treatment with 50–300 nm SWCNTs, fiberlike structures (FLSs) were found in theabsorption cells in the intestinal tissues (Figure 2, A) of the miceunder TEM. No FLSs can be found in normal saline-gastro-gavaged mice, excluding the possibility of non-SWCNTmaterialsforming FLSs in the intestinal tissues and suggesting that the FLSsare SWCNTs from the intestinal cavity of SWCNT-gastrogavagedmice. Most of the FLSs had a diameter of 2–10 nm (Figure 2, D–G), with some distinguishable 1-nm individuals, and a length of50–300 nm (Figure 2, C), which was identical with the diametersand the lengths of the SWCNTs in the gastrogavage suspension.There were good positive correlations between FLS incidence

(FLS-I) and SWCNT doses (SWCNT-D), and between FLS-I andSWCNT gastrogavage times (SWCNT-GT) (Figure 2,H). FLS-I–SWCNT-GT curves shifted toward the upper left with an increasein SWCNT-D (Figure 2, H), further showing that FLSs areSWCNTs. The SWCNTs recollected from in vivo cells were alsofiberlike, with diameters of 0.8–1.6 nm and lengths of 5–300 nm.The chemical examination demonstrated a Raman spectrum quitesimilar to the original ones (Figure 1, E), further indicating that theFLSs in in vivo cells are SWCNTs.

Tissue and cell distribution of SWCNTs absorbed via thegastrointestinal route

SWCNTs were detected only in the absorption cells of themucous membrane (Figure 2, A, arrow a) and the macrophagecells (Figure 2, A, arrows b, c) in the submucous layer ofintestinal wall, but not in any other cells. Absorption cells wereconfirmed by the microvilli in their cell margin (Figure 2, B) andtheir position in the absorptive mucous membranes (Figure 2, A,arrow a). The macrophages were identified by their multiplenuclei, rich in lysosomes and dense-stained chromatins along theinner margin of the nuclear membrane (Figure 2, D–G), andtheir position in the submucous-layer tissue space (Figure 2, A).SWCNTs in the absorption cells formed vesicles (Figure 2, B),

Figure 2 (continued).

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the transportation organelles in the absorption cells. SWCNTsinside the vesicles aggregated into bundles, whereas some singleSWCNT fibers were recognizable (Figure 2, C) and those in themacrophages aggregated into one or two bundles (Figure 2, F,G). The numbers of SWCNTs in the intestinal cells increaseddose- and time-dependently (Figure 2, H). SWCNT contents indifferent segments of intestines were different. None were foundin duodenum, some in jejunum (Figure 2, F, G), and most inileum (Figure 2, D, E). Both the average number of SWCNTs in

one cell (Figure 2, I) and the percentages of cells containingSWCNTs (Figure 2, J) showed that the distribution of SWCNTsin ileum was richest and that in jejunum secondary.

SWCNTswere also detected in other internal organs includingliver, brain, and heart. Interestingly, SWCNTs were seen only inneurons (Figure 3, A, B) and neurites (Figure 3, E, F), but not inglial cells (Figure 3, C). In neurons, SWCNTs localized inlysosomes (Figure 3, A, B). In neurites SWCNTs manifested asrodlike in the sections parallel to neurites (Figure 3, D) and asdots in the cross sections of the neurites (Figure 3, E).

Subcellular distribution of SWCNTs

At doses of 5–300 mg/kg, SWCNTs were detectedexclusively in lysosomes (Figures 2, D–G, 3, B, and 4, A),but at doses above 400 mg/kg SWCNTs began to appear inmitochondria (Figure 4, C). SWCNTs in lysosomes wereparallel to the longitudinal axis of the organelles (Figure 4, A),but those in mitochondria had no determined orientation(Figure 4, C). There may have been one or two bundles ofSWCNTs in one lysosome or mitochondrion. Figure 4, Dquantitatively illustrates the relationships between doses andpercentages of organelles containing SWCNTs in lysosomesand mitochondria, which revealed the lysosomotropic propertyof SWCNTs by two curve features: the first is the dose-dependent increase SWCNT-containing rate only in lysosomesat doses less than 300 mg/kg; the second is that the curverelating dose to the SWCNT-containing rate of lysosomes wasconsiderably higher than that of mitochondria when dosesexceeded 400 mg/kg.

The influences of autophagy inhibitor on subcellulardistribution of SWCNTs

3-MA, an inhibitor of autophagosome formation,39 in anintraperitoneal injection of 20 mg/kg, decreased the number ofSWCNTs in lysosomes significantly and increased that inmitochondria significantly (Figure 4, E). Similar results wereobtained by intraperitoneal injection of 20 mg/kg smallinterfering RNA (siRNA) (the sequences of the regions targetedby the siRNA in the exon 8a of the gene encoding lysosomal-associated membrane protein 2 were 5′-GACTGCAGTGCA-GATGAAG-3′, 5′-CTGCAATCTGATTGATTA-3′, and 5′-TAAACACTGCTTGACCACC-3′, corresponding to bases1198–1216, 1331–1359, and 1678–1700) that was specific toinhibit the function of chaperone-mediated autophagy (CMA)(Figure 4, E).40 Under energy deprivation by subtracting energy-producing components from the fluid diet, the distribution ofSWCNTs increased in lysosomes but decreased in mitochondria(Figure 4, E). These results revealed a dynamic equilibriumbetween the distribution in lysosomes and that in mitochondria.

The different damage effects of SWCNTs on lysosomesand mitochondria

At doses of 5–300 mg/kg, SWCNTs did not induce anysignificant damage, even to the SWCNT-containing lysosomes.At doses of 400–500 mg/kg they caused different degrees ofdamage to the mouse organelles. The damage was mainlylocalized in the lysosomes (Figure 5, A–E) and mitochondria

Figure 3. The cell distribution of single-walled carbon nanotubes (SWCNTs) in brain examined by transmission electron microscopy. (A) A neuron containingSWCNTs (arrowheads); (B) SWCNTs in two lysosomes indicated by arrows in A. (C) A glial cell containing no SWCNTs. (D) Section parallel to thelongitudinal axis of one neurite, which was confirmed by the sheath (arrowhead). SWCNT is fiberlike. (E) Cross section of neurite, which was confirmed by thesheath (arrowhead). SWCNTs are dotlike.

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(Figure 5, F–H). Damaged lysosomes dilated with a decreasein their contents (Figure 5, B–D), destruction of membranes(Figure 5, D), excretion or dissolution of contents (Figure 5,D), and cavity formation (Figure 5, E). The damage rate canreach more than 30% of the total numbers of SWCNT-containing lysosomes (Figure 5, I). Almost all of the SWCNT-containing mitochondria were injured (Figures 4, C, 5, F–H),so that the damage rate was nearly equal to the percentagecontaining SWCNTs (Figure 5, J). The injured mitochondriamanifested swelling, decrease or disappearance of their cristae,and complete destruction (Figures 4, C and 5, F–H). At 400–500 mg/kg the proportion of mitochondria containingSWCNTs and the proportion showing damage may becomehigher than 30% (Figure 5, J). No damage effects wereobserved in other organelles.

The target organelles for SWCNT damage

SWCNTs did not induce ROS production in lysosomes(Figure 6, A) but did so in mitochondria (Figure 6, B),demonstrating that mitochondria are the direct-target organellesof SWCNT damage. In another experiment measuring the releaseof β-galactosidase, without the presence of mitochondriaSWCNTs did not induce significant release of β-galactosidasefrom lysosomes, but the addition of mitochondria homogenategreatly increased the β-galactosidase activities by three to eighttimes dose-dependently (Figure 6, C), demonstrating that

lysosomal damage was secondary to mitochondria damage.Confocal laser-scanning microscopy with JC-1 fluorescent dyedemonstrated that SWCNTs significantly decreased MMP(Figure 6, D, E), which was followed by the increase of ROSproduction (Figure 6, F), demonstrating that ROS overproduc-tion was caused by collapse of MMP. These results incombination revealed that mitochondria are the target organellesof SWCNT toxicity.

The target organelles of SWCNTs as drug carriers

Because SWCNTs can enter the brain (Figure 3, A–C),preferentially enter lysosomes (Figure 3, A,B), and aredistributed specifically in neurons and neurites (Figure 3, D,E), they present great potential for use as drug carriers to deliverdrugs inside the brain for the treatment of neurodegenerativediseases. The therapeutic effects of SWCNT-ACh on experi-ments with AD mice revealed that lysosomes are the targetorganelles of SWCNTs as drug carriers.

ACh was successfully loaded onto SWCNTs, as verified byRaman spectrum (Figure 7, A). Using SWCNT-ACh, AD micerecovered their learning ability to the normal levels asindicated by the results of the step test (Figure 7, B), shuttletest (Figure 7, C), and Morris water maze test (Figure 7, D).In these experiments neither the free-ACh group nor the free-SWCNT alone group (Figure 7, B–D) achieved such effects.The effects of SWCNT-ACh on recovery of learning and

Figure 4. Subcellular distribution of single-walled carbon nanotubes (SWCNTs) examined by transmission electron microscopy. (A) SWCNTs in lysosomes; (B)magnification of one SWCNT-containing lysosome. The single-layer membrane (arrow) and even distribution of its contents indicate a lysosome. (C) SWCNTs inmitochondria. The double-layer membrane and the cristae in them indicate mitochondria. (D) Relationship between the percentages of SWCNT-containing organellesand the doses of SWCNTs, which can rise above 80% for lysosomes and 30% for mitochondria. SWCNTs were visible in mitochondria only when doses were higherthan 400 mg/kg. ***P b 0.001 compared with control. (E) Influences of autophagy blockage (see Results section) and energy deprivation (ED) on SWCNT subcellulardistribution. ***P b 0.001. SWCNTs were gastrogavaged in a dose of 500 mg/kg, and drugs were intraperitoneally administered.

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memory ability of AD mice showed good dose-effect relationsin all three of the tests.

Effects of SWCNTs on the overall physiology and health statusof mice

SWCNTs administered at doses under 300–500 mg/kgcaused no significant changes in the activities of serum enzymesincluding glutamic-pyruvic transaminases, glutamic oxaloacetic

Figure 5. The damage effects of single-walled carbon nanotubes (SWCNTs) on lyswith SWCNT-damaged lysosomes (arrows). (B)Magnification of the SWCNT-damdamaged lysosome indicated by arrow B in A. (D)Magnification of the SWCNT-d(arrow). (E)magnification of the lysosome indicated by arrow d inA. The whole lysbegin to appear in cells. (G)Mild mitochondrial damage: the cristae are smaller. (Hhave disappeared. The residual cristae reveal that the structures are mitochondria. Spercentages of lysosomes containing SWCNTs and of lysosomes showing damage.percentages of mitochondria containing SWCNTs and of mitochondria showing da

transaminases, creatine kinase, and lactate dehydrogenase(Figure 8, A); neither did they have significant influences onthe blood cell counts (Figure 8, B), total protein, or blood ureanitrogen (Figure 8, A). At the tested range of doses, SWCNTsalso did not show any adverse effects on the overall growth statusof mice, in that mice in all treatment groups had body weightincrements similar to those of the control group during the 10-day experiment (Figure 8, C). These results reflected theinsidiousness of SWCNT damage, because there were still no

osomes and mitochondria under transmission electron microscopy. (A) A cellaged lysosome indicated by arrow A inA. (C)Magnification of the SWCNT-amaged lysosome indicated by arrow c in A. Some contents are being excretedosome became a large cavity. (F) The SWCNT-damaged mitochondria (arrow)) Severe mitochondrial damage. The cristae are nearly completely destroyed orWCNTs were found in some of them. (I) The relationship of SWCNT dose to***P b 0.001 compared with control. (J) The relationship of SWCNT dose tomage. ***P b 0.001 compared with control.

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igure 6. Relationships between lysosomal damage and mitochondrialamage studied in vitro. (A) Effects of SWCNTs on the production ofactive oxygen species (ROS) in lysosomes. (B) Effects of SWCNTs one production of ROS in mitochondria. NAC, N-acetylcysteine (10 μmol–1); Vit E, vitamin E (10 μmol L–1). (C) The influence of mitochondrian the lysosomal damage by SWCNTs. Mitochondria were added at thedicated concentrations, and the activities of β-galactosidase wereeasured. Lysosomal damage was expressed as the activity of the-galactosidase leaked from lysosomes. (D) Fluorescence images showinge influences of SWCNTs on the mitochondrial membrane potentialMP). Green fluorescence increased and red fluorescence decreased withcubation time. (E) Changes in the red-to-green fluorescence ratios ine mitochondrial membranes over 25 minutes of incubation withWCNTs. SWCNTs significantly decreased the ratio time-dependently.F) The time courses for the changes of both MMP and mitochondrialOS production. ***P b 0.001 compared with control. &&&P b 0.001ompared with SWCNT.

Figure 6 (continued).

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FdrethLoinmβth(MinthS(Rc

manifestations of disease although considerable pathologicalchanges had taken place in the ultrastructure of cells at doses of400–500 mg/kg.

The safe range for SWCNT dosing in treatment of AD mice

Because of the lack of symptoms or systematic signs ofdisease, it is difficult to determine a safe range for SWCNTdosage in the treatment of diseases in biomedicine and for theexposure limit in nanotoxicology. Here we propose to evaluatethe safe range or the exposure limit by virtue of the pathologicalchanges we observed in organelle ultrastructures.

Figure 7. Effects of SWCNT-delivered acetylcholine (ACh) on learning andmemory in AD mice. (A) Raman spectra of the adsorption of ACh ontoSWCNTs. Spectrum A, SWCNTs; spectrum B, SWCNTs loaded with ACh.(B) Effects of SWCNT-ACh on learning and memory of AD mice measuredby the step-down test. Latency is the time that the AD mice remain on the platethat protects them from electrical stimulation. ACTS, the accumulated time ofthe mice being stimulated; AE, the number of errors made by mice. (C) Effectsof SWCNT-ACh on the learning and memory of AD mice measured by theshuttle-box test. TI, the accumulative interval times that the animal stayed in thesafe area; NI, number of active escapes; NS, number of instances of passiveresponse. (D) Effects of SWCNT-ACh on the learning and memory of ADmice measured by the Morris water maze test on days 1, 2, and 3 after theinjection of kainic acid (KA). ***P b 0.001 compared with control.

Figure 7 (continued).

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Based on the experiments on the treatment of AD mice, theeffective doses of SWCNT-ACh were determined to be in therange of 20–50 mg/kg, corresponding to the doses of ACh at 4–10 mg/kg. The ED99 value of SWCNTs was determined to be25 mg/kg, corresponding to that of ACh at 5 mg/kg. Based onED99 and the maximal safe dose of 300 mg/kg for mitochondria

and lysosomes, the safe dose range for SWCNTs was proposedas 12 mg/kg in the treatment of AD in mice.

Discussion

The significance of gastrointestinal absorption of SWCNTs

The gastrointestinal tract is the most convenient avenue fordrug administration. The distribution of SWCNTs in internal

Figure 8. Influences of SWCNTs on the blood biochemistry, blood cellcounts, and body weight of mice. (A) Changes in enzyme activities andconcentrations of metabolites in the serum. ALB, albumin; BUN, blood ureanitrogen; CK, creatine kinase; CRE, creatine; GOT, glutamic oxaloacetictransaminases; GPT, glutamic-pyruvic transaminases; LDH, lactate dehy-drogenase; TP, total protein. (B) Influence of SWCNTs on the numbers ofred blood cells, white blood cells, and platelets. (C) Influence of SWCNTson the body weight of mice during the experiment.

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organs shows that they are gastrointestinally absorbable,which is the prerequisite for their application as oral drugcarriers. The gastrointestinal tract is also the first exposure ofthe body to nanoparticles. The gastrointestinal absorption of

SWCNTs demonstrated the importance of gastrointestinalexposure, which should be avoided if at all possible.Although some studies have dealt with the effect of exposureto modified CNTs, there have been no studies on gastroin-testinal exposure to pristine SWCNTs. Because mostenvironmental SWCNTs are pristine ones, our results are ofmore practical importance in comparison with results with themodified CNTs.

Mechanisms for gastrointestinal SWCNT absorption

There have been many studies reporting the lymphaticabsorption of nanoparticles,41-43 but our studies have shown, toour knowledge for the first time, that SWCNTs can beabsorbed through the columnar epithelial cells of intestines, forwhich the selective distribution of SWCNTs in transportvesicles of the intestinal columnar epithelial cells providesstrong evidence. The lack of SWCNT distribution in otherintestinal cells besides absorption cells and macrophagesexcluded the possibilities of other absorption mechanismssuch as lymphatic absorption and the passive absorptionresulting from the syringe effects of SWCNTs.

The lysosomotropic property of SWCNTs, its mechanismand meaning

Several studies have reported the distribution of SWCNTs inlysosomes, but few have addressed their distribution inmitochondria and the relations between organelles in SWCNTdistribution. The present study demonstrated that lysosomes arethe preferred organelles for SWCNT distribution. The mito-chondria are organelles for SWCNT distribution only at highdoses that are beyond the capacity of lysosomes. Thelysosomotropic property of SWCNTs may arise becauseSWCNT-containing vesicles can enter lysosomes by simplemembrane fusion.44 However, we found that the genes encodingphosphoinositide-3-kinase and lysosomal-associated membraneprotein 2 were involved in the process of SWCNT entry, asdemonstrated by the experiments with 3-MA and siRNA. Moreimportantly, there is equilibrium between lysosomes andmitochondria in SWCNT distribution. Blockage of the auto-phagy of lysosomes can decrease the quantity of SWCNTs inlysosomes and increase that in mitochondria, and vice versa.These results mean that the quantity of SWCNTs in lysosomesand mitochondria can be controlled or regulated with the aid ofautophagy regulators used deliberately according to the thera-peutic need. This offers hope for the design of clinical protocolsin the treatment of diseases and SWCNT overexposure.

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The target organelles for SWCNTs as drug carriers and astoxic agents

We found that SWCNTs did not damage lysosomes, althoughlysosomes are their preferred organelles in distribution, meaningthat lysosomes may be employed as the pharmacological targetorganelles of SWCNTs. Lysosomes, with an interior pH lowerthan that in cytoplasm, are favorable targets for drug delivery,especially for pH-sensitive target drug delivery systems, inwhich the drug release can be triggered by a mild acidicenvironment, such as that occurring in solid tumors andinflammatory tissues.45-47 SWCNT-ACh was such a pH-sensitive drug delivery system that successfully delivered AChinto brain with good curative effects on experimental AD,providing good evidence for lysosomes as the pharmacologicaltarget organelles of SWCNTs. The selective distribution ofSWCNTs in the lysosomes in neurons and neurites is favorablefor the release of ACh to develop transmitter function.

Many studies have reported the cytotoxicity ofSWCNTs,13-20,23-27 but none of them investigated the relationsbetween different organelles, and we still do not know whichorganelles are responsible for the initiation of SWCNT damage.On TEM examination it was noted that lysosomes were neverdamaged unless mitochondria were damaged also, implying thatlysosomal damage was secondary to that of mitochondria.Experiments with isolated lysosomes and mitochondria foundthat SWCNTs failed to induce the production of ROS in isolatedlysosomes but significantly induced overproduction of ROS inisolated mitochondria, indicating that SWCNTs can directlydamage mitochondria but not lysosomes. Further experimentsdemonstrated that SWCNTs significantly induced the productionof ROS in lysosomes when mitochondria homogenate was addedinto the isolated lysosomal experimental system. These resultsindicate that mitochondria are the original organelles forultrastructural damage of the cells. These isolated organelleexperiments in combination with the in vivo TEM examinationrevealed that mitochondria are the target organelles of SWCNTtoxicity. The significance of the elucidation of the targetorganelles for toxicity and for pharmaceutical delivery is thatwe can direct SWCNTs (based on the target mechanisms) toselectively enter lysosomes but not mitochondria, so as to obtainthe ideal therapeutic effects while avoiding the toxic effects.

Mechanism for the damaging effects of SWCNTs onmitochondria and lysosomes

There have been no available data on the mechanism bywhich SWCNTs injure mitochondria. Our experiments haveanswered this question primarily, although further study isrequired. SWCNTs induced the collapse of MMP, which wasfollowed by the overproduction of ROS, indicating that theinfluence of SWCNTs on MMP is the cause for mitochondrialdamage. Mitochondria are the power station of cells, wherebiological oxidation develops. ROS can be produced byelectrons leaking from the electron transport chains and maybe eliminated by the enzyme system in cells under normalconditions.48,49 SWCNTs have many π electrons in theirmacromolecules that interact with one or some of theoxidation-reduction pairs such as NAD/NADH and NADP/

NADPH in the electron transport chain, blocking the electrontransportation and increasing the leaking of electrons, leading tothe collapse of MMP and further to the increment of ROS.50

ROS would damage mitochondria by peroxidation of lipids,proteins, and DNA. The damaged mitochondrial membranesallow ROS to diffuse out to attack lysosomes as well as the othercell structures. The damaged lysosomes release various digestiveenzymes, finally leading to the cell's destruction.

The insidiousness of SWCNT damage on animals

Interestingly, one of the features of SWCNT toxicity is theirinsidiousness, which may be the reason some experiments foundtoxic effects of SWCNTs whereas others did not. At certain doselevels they caused significant subcellular damage revealed asultrastructural pathological changes but did not affect overallphysiology and well-being of the animals, in that the animalsshowed no signs of clinical abnormality by symptom observationand growth assessment. Traditionally, drug toxicity in the clinic islargely evaluated by symptoms, histopathological observations,and blood panels including hematology. In animal experimentstoxicity is often evaluated as the dosage for the death of half of theanimals (LD50), the symptoms, and the organ injuries. Obviouslythese indexes cannot accurately evaluate the toxicity of SWCNTsbecause of the insidiousness of their toxicity. Based on the uniquepathological feature of SWCNTs, the safety range of somenanomaterials should be re-evaluated by the ultrastructuralpathological changes at the subcellular level in preference tousing traditional histopathological and symptomatic indexes.

The mechanism for the insidiousness of SWCNT damagemay be related to SWCNTs' giant molecules. Individualmolecules of SWCNTs may be several hundreds or thousandstimes larger than the molecules of substances in general, so thatthey cannot distribute as evenly as do usual chemicals in tissuesand cells. This uneven distribution leads to their presence inonly a few organelles in cells or only a few cells in tissues,which results in high concentrations in single organelles orsingle cells, although the SWCNT-containing organelles andcells may account for only a very small proportion of the totalorganelles and cells. The high concentration in singleorganelles or cells causes considerable ultrastructural patholog-ical changes to develop in single organelles and single cells,but is not enough to cause systematic symptoms. Althoughsystematic symptoms are lacking, the effects of damage bySWCNTs on individual organelles and cells should be avoided,because such damage may be the cause of those diseases thatdevelop from a single or a few cells, such as tumors. Therelations between the damage of mitochondria and the tumordevelopment are being established.51

The use of SWCNTs as drug carriers in the treatment of AD

All nanomaterials may be toxic in a strict sense, but theirtoxicity does not necessarily militate against their application innanomedicine. The key is to control the dosage. Our studyshowed that only at high dosages did SWCNTs cause thepathological changes in the ultrastructures of lysosomes andmitochondria; they are highly safe at low doses, providing thebasis for their use as drug carriers. In the present study SWCNTs

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were successfully used to deliver drugs for the treatment ofexperimental AD and achieved satisfactory results with the dosesprecisely controlled. These results exemplified the importance ofunderstanding the therapeutic and toxicological profiles ofnanomaterials for their application in modern biomedicine.

AD is a neurodegenerative disease caused by a decrease inneurotransmitter ACh of the cholinergic nervous system due tothe inability of neurons to synthesize it.52 Administration ofACh into the brain can improve the dementia of patients, butfree ACh cannot enter the brain because of its strong polaritiesand ease of decomposition in blood. Therefore, currently brainACh can only be increased by clinical administration of certainmild inhibitors of acetylcholinesterases that hydrolyze ACh.53

Because SWCNTs have the ability to absorb inorganic54,55 andorganic chemicals,56-60 in addition to the ability to enterthe brain via nerve axons, so they are ideal for the delivery ofACh molecules. At the same time, ACh is composed of anacetyl group and a quaternary ammonium group, which allowsfor its easy absorption by SWCNTs. The good curativeeffects on experimental AD indicated that SWCNTs success-fully delivered ACh into the brain. Once inside the brain, theyenter the lysosomes of the neurons and then release thedrug ACh as a result of the increased polarity and hydro-philicity of ACh at the low lysosomal pH, bringing ACh intoplay as a neurotransmitter.

We investigated the target organelles of SWCNTs and how tosafely use SWCNTs as drug carriers in the treatment ofexperimental AD. Both in vivo and in vitro experimentsdemonstrated that SWCNT are excellent drug carriers but haveinsidious damage effects on cells by causing the collapse ofMMP. Different target organelles are responsible for thepharmacological and toxicological effects of SWCNTs. Thelysosomes are the pharmacological target organelles, whereas themitochondria are the target organelles of SWCNT toxicity. Thereare differences in doses for SWCNTs to target these two kinds oforganelles, which is the key for the safe use of SWCNT as drugcarriers. As a toxicological feature, the damage effects ofSWCNTs are insidious, so we propose that the safe range ofSWCNT should be evaluated by the structural changes inorganelles instead of by traditional parameters. To evaluate theability of nanomaterials to deliver drugs, it is important tocompare their pharmacological and toxicological effects, todetermine whether their negative effects can be avoided by anymethod. SWCNTs can carry ACh into the lysosomes of theneurons and achieve excellent therapeutic effects, whereas theirtoxicological effects can be avoided by precisely maintaining thedoses under 300 mg/kg, thus ensuring that SWCNTs enter onlylysosomes, the pharmacological target organelles, whereas fewor none enter mitochondria, the target organelles for toxicity. Ourresults showed, to our knowledge for the first time, thatSWCNTs are good drug carriers in the treatment of diseases ofthe central nervous system.

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