IOP PUBLISHING NANOTECHNOLOGY

Nanotechnology 22 (2011) 465102 (8pp) doi:10.1088/0957-4484/22/46/465102

Single-walled carbon nanohorns as drugcarriers: adsorption of prednisolone andanti-inflammatory effects on arthritis

Maki Nakamura1,6, Yoshio Tahara1, Yuzuru Ikehara1,Tatsuya Murakami2, Kunihiro Tsuchida3, Sumio Iijima1,4,Iwao Waga5 and Masako Yudasaka1,6

1 National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi,Tsukuba 305-8565, Japan2 Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8501, Japan3 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake 470-1192,Japan4 Faculty of Science and Technology, Meijo University, Shiogamaguchi, Tempaku, Nagoya468-8502, Japan5 VALWAY Technology Center, NEC Soft, 1-18-7 Shinkiba, Koto-Ku, Tokyo 136-8627, Japan

E-mail: ma-ki-nakamura@aist.go.jp and m-yudasaka@aist.go.jp

Received 13 July 2011, in final form 17 August 2011Published 24 October 2011Online at stacks.iop.org/Nano/22/465102

AbstractPrednisolone (PSL), an anti-inflammatory glucocorticoid drug, was adsorbed on oxidizedsingle-walled carbon nanohorns (oxSWNHs) in ethanol–water solvent. The quantity ofadsorbed PSL on the oxSWNHs was 0.35–0.54 g/g depending on the sizes and numbers ofholes on the oxSWNHs. PSL was adsorbed on both the outside and the inside of the oxSWNHs,and released quickly in a couple of hours and slowly within about one day from the respectiveplaces. The released quantity in culture medium strongly depended on the concentration of thePSL–oxSWNH complexes, suggesting that PSL adsorbing on oxSWNHs and PSL in the culturemedium were in concentration equilibrium. The local injection of PSL–oxSWNHs into thetarsal joint of rats with collagen-induced arthritis (CIA) slightly retarded the progression of thearthritis compared with controls. By histological analysis of the ankle joint, theanti-inflammatory effect of PSL–oxSWNHs was also observed.

S Online supplementary data available from stacks.iop.org/Nano/22/465102/mmedia

(Some figures in this article are in colour only in the electronic version)

1. Introduction

Carbon nanotubes have been attracting much attention inmedicinal areas because of their potential utility in drugdelivery, imaging, and photodynamic therapy [1–5]. Single-walled carbon nanohorns (SWNHs) [6], a new type of single-graphene nanotubule, are potential carriers of drug deliverysystems (DDSs) [7–13]. SWNHs are stable graphitic tubules

6 Address for correspondence: Nanotube Research Center, National Instituteof Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1Higashi, Tsukuba 305-8565, Japan.

with diameters of 2–5 nm and lengths of 40–50 nm, whichhave closed ends with cone-shaped caps (horns). Usually, theSWNHs do not exist individually, and around 2000 SWNHsassemble to form a spherical aggregate with a diameterof 80–100 nm (figure 1(a)). From the viewpoint of drugcarriers, SWNHs have several advantages over other carbonnanotubes. SWNHs can be obtained in large quantities bya simple procedure at high purity [14]. Since no metalcatalysts are necessary during their synthetic process, they donot contain any contaminating metals that may cause serioustoxicity. Actually, SWNHs do not show any serious toxicity in

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Nanotechnology 22 (2011) 465102 M Nakamura et al

Figure 1. TEM images of asSWNHs (a) andPSL–oxSWNH(560) (b), and preparation process ofPSL–oxSWNH(Tox) (c). Abbreviations: asSWNH, as-grown SWNH;oxSWNH, oxidized SWNH; PSL, prednisolone.

animal tests [15], making them potentially applicable as drugcarriers. Moreover, the structural features of SWNHs are alsoadvantageous. The inner spaces of SWNHs become accessibleby opening the holes on the walls of the SWNHs by oxidativetreatment, and such SWNHs are called ‘oxidized SWNHs(oxSWNHs)’ [16–19]. The inner nanospaces of oxSWNHsare large enough to store large amounts of drug molecules.In addition, SWNHs are chemically stable enough for anyuse in the ordinary physiological environment, since they aregraphene-based materials.

We have been studying the use of SWNHs as drug carriers.Dexamethasone, an anti-inflammatory drug, has been reportedto be adsorbed on oxSWNHs and slowly released from themin mammalian cells [7]. The ability of oxSWNHs to bind andrelease vancomycin hydrochloride, an antibiotic drug, has beenalso investigated [13]. Local injection of cisplatin-incorporatedoxSWNHs into subcutaneously transplanted tumors showeda higher antitumor effect than that of intact cisplatin, ananticancer drug [11].

In this paper, we have investigated the adsorption ofprednisolone (PSL), an anti-inflammatory glucocorticoid drug,on oxSWNHs and the release of PSL from them. Furthermore,the anti-inflammatory effect of the obtained PSL–oxSWNHcomplexes has been examined in vivo by locally administeringthem to rats with collagen-induced arthritis (CIA), a rat modelof rheumatoid arthritis [20, 21].

2. Experimental section

2.1. Materials and structural analysis

SWNHs were prepared by CO2 laser ablation of graphitewithout any metal catalysts in Ar gas (760 Torr) at roomtemperature [6, 14]. The SWNHs were used withoutany purification (as-grown SWNHs (asSWNHs)). To openholes, the SWNHs were oxidized by the slow combustionmethod [19], namely, heated in dry air from room temperatureto the target oxidation temperatures (Tox = 500, 550, 560and 570 ◦C) at a heating rate of around 1 ◦C min−1 and cooleddown naturally. The obtained oxidized SWNHs (oxSWNHs)are referred to as oxSWNH(Tox) in this paper. Prednisolone(PSL, a biochemical agent), ethanol (HPLC grade) andmethanol (HPLC grade) were purchased from Wako. Culturemedium (RPMI 1640) was purchased from Invitrogen. PBSwas purchased from Sigma. Water was purified using a waterpurification system (Millipore).

The structures of the asSWNHs and PSL-loadedoxSWNHs (PSL–oxSWNHs) were observed by transmissionelectron microscopy (TEM, Topcon EM-002B) operated at120 kV. X-ray diffraction (XRD, Rigaku RINT2100) analyseswere also performed. The hole opening of the oxSWNHswas evaluated by measuring the m-xylene adsorption quantityaccording to our previously reported method [22]. Briefly,the sample was exposed to m-xylene atmosphere for30 min at room temperature in a closed container. Usingthemogravimetric analysis (TGA, TA Instruments TGA Q500)in helium, the quantity of adsorbed m-xylene was estimatedfrom the weight loss occurring below 300 ◦C.

2.2. Adsorption of PSL on SWNHs

Preparation of PSL–SWNHs was carried out in accordancewith the previously reported nano-extraction method [23],which is outlined in figure 1(c). PSL was dissolved inethanol/water (1:2) solvent with a concentration of 2 mg ml−1.Into this solution, oxSWNH(Tox) was added (ca. 10 mg,1 mg ml−1), sonicated for 5 min, and stirred at roomtemperature overnight. The resultant mixture was filtered, andthe obtained black powder on a filter paper was dried in flowingN2 gas at room temperature to give PSL–oxSWNH(Tox). Theamount of PSL adsorbed on the oxSWNH(Tox) was determinedby subtracting the amount of PSL in the filtrate, which wasquantified by using high performance liquid chromatography(HPLC), from the starting amount of PSL. The HPLCequipment consisted of a Shimadzu DGU-14A degasser, aShimadzu LC-10AD pump, a Shimadzu CTO-10AC columnoven, a Shimadzu SPD-10AV UV–vis detector, and a ShodexC18P 4D column. The mobile phase consisted of methanoland water, 48:52 (v/v). The flow rate of the mobile phase was1 ml min−1. The column temperature was maintained at 25 ◦C.PSL was detected by the UV absorption at 247 nm.

To find the effect of holes on the adsorption and releaseof PSL, the complex of PSL and asSWNHs (PSL–asSWNHs)was similarly prepared and analyzed.

For a large scale preparation of PSL–oxSWNH(560)for animal experiments, PSL adsorption on oxSWNH(560)

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(ca. 60 mg) was repeated five times and the obtained PSL–oxSWNH(560) was mixed in a vial. The quantity of PSLadsorbed on oxSWNH(560) was 0.43 g/g, that is, PSL–oxSWNH(560) contained PSL at about 30 wt%.

2.3. In vitro release of PSL from PSL–SWNHs

To ca. 0.75 mg of PSL–oxSWNH(Tox) or PSL–asSWNHs ina vial, culture medium or PBS was added (0.05 mg ml−1,ca. 15 ml). After shaking for 5 s, the vial was stood still ina 37 ◦C incubator. At certain time points (1, 4, 24 and 48 h),the vial was taken out from the incubator, shaken for 5 s, and0.5 ml of the mixture was sampled. The sampled mixture wasfiltered and the amount of PSL in the filtrate was quantified byusing HPLC.

2.4. Dependence of PSL–oxSWNH(560) concentration on invitro PSL release

PBS was added to ca. 5 mg of PSL–oxSWNH(560)(10 mg ml−1), and it was sonicated for 3 min. The mixture(10, 50 and 200 μl) was diluted with 10 ml of culturemedium in a vial (final concentration: ca. 0.01, 0.05 and0.2 mg ml−1, respectively), and 0.5 ml of the diluted solutionwas immediately sampled (0 h). The rest of the solution inthe vial was stood still in a 37 ◦C incubator. According tothe above-mentioned method, the mixture was sampled at timepoints of 1, 4, 24 and 48 h, and the amount of PSL in thesampled solution was quantified.

A similar experiment using PBS instead of culturemedium was also carried out at 0.05 mg ml−1 of PSL–oxSWNH(560).

2.5. Animal experiment

The animal experiments were carried out by KAC Co.,Ltd. Female DA/Slc rats aged eight weeks were purchasedfrom Japan SLC and housed individually under semi-barrierconditions (20–26 ◦C, 38–65% relative humidity, 12 h/12 hlight/dark cycle). They were allowed free access to standardpellets (CRF-1, Oriental Yeast Co., Ltd) and normal water.The animals were acclimated to this environment for eight daysprior to the administration on day 0. On day 0, they were nineweeks old and weighed 120–140 g. The animal experimentswere carried out in accordance with the regulations of thecommittees on the Use and Care of the National Instituteof Advanced Industrial Science and Technology and KACCo., Ltd.

On day 0, to induce arthritis, type II collagen(3 mg ml−1) emulsified 1:1 in incomplete Freund’s adjuvantwas intradermally injected (0.3 ml) at the base of the tail.The rats were allocated into four groups (five animals pergroup), receiving following administration by local injectioninto their tarsal joints (in both the right and left joints):(i) PBS (control group, 0.01 ml per joint); (ii) oxSWNH(560)(0.07 mg in 0.01 ml PBS per joint); (iii) PSL (0.03 mg in0.01 ml PBS per joint); (iv) PSL–oxSWNH(560) (0.1 mg in0.01 ml PBS (10 mg ml−1) per joint). These materials inPBS were sonicated for 3 min (premixing) before injection and

administered once a week (days 0, 7, 14 and 21). Arthritis wasquantified by a clinical scoring system ranging from 0 to 8. Thedegree of swelling and redness in each hind paw was scored asfollows: 0 = no; 1 = mild; 2 = moderate; 3 = severe;4 = very severe. The total score for each rat was the sum ofthe scores for the right and left hind paws.

On day 28, the rats were sacrificed by removingall their blood via the abdominal aorta under anesthesia.Leukocyte numbers in peripheral blood were examined usinga blood cell analyzer, and amounts of CRP in plasma werequantified with commercially available ELISA kits (Rat C-Reactive Protein ELISA (BioVendor)). The hind pawswere fixed in 10% phosphate-buffered formalin solution,decalcified, and embedded in paraffin blocks to performhistological examination. A 2 μm thickness section wasprepared and subjected to hematoxylin and eosin (HE) stainand immunohistochemical analysis with anti-CD68 antibody(Serotec). The antibody reaction was visualized with aHistofine Simple Stain Rat MAX-PO and diaminobenzidine(DAB) solution (Nichirei Bioscience). A counterstainingwas performed with hematoxylin. One-way ANOVA withDunnett’s test was used for significance test of the measuredvalues.

3. Results

We show in the following that, among the prepared PSL–oxSWNH(Tox), PSL–oxSWNH(560) was preferable for theanimal test in terms of the large released quantity of PSL inthe culture medium. Further, the treatment of CIA rats withPSL–oxSWNH(560) was shown to have certain effects for thealleviation of arthritis.

3.1. Adsorption and release of PSL on/from SWNHs

We previously reported that dexamethasone, having ahydrophobic steroid structure, was adsorbed on both theinside and outside surfaces of oxSWNHs, and the release ofdexamethasone from oxSWNHs was very slow in PBS, whileconsiderably faster in the culture medium [7]. Having a similarstructure to dexamethasone, PSL (for the chemical structure ofPSL, see figure 1(c)) showed more or less similar adsorptionand release properties on/from oxSWNHs, as shown below.

The amount of PSL adsorbed on the outside surfaces ofasSWNHs (closed tubules) was 0.14 g/g, while that on bothinside and outside surfaces of oxSWNH(560) was 0.45 g/g(table 1 and figure 2). TEM observation showed that PSL–oxSWNH(560) aggregate retained the spherical aggregatestructure with many horn tips sticking out at the aggregatesurfaces (figure 1(b)). Probably due to the adsorbed PSL,PSL–oxSWNH(560) appeared smoked in figure 1(b). Noexcess PSL crystals were found in the TEM observation,and PSL crystal peaks were absent in the XRD data(see figure S1 available at stacks.iop.org/Nano/22/465102/mmedia), suggesting that almost all of the PSL molecules inPSL–oxSWNH(560) adsorbed on oxSWNH(560).

To optimize the sizes of the holes in the oxSWNHsfor the controlled release of PSL in large quantity, we

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Figure 2. Quantities of PSL adsorbed on SWNHs (black) andreleased from SWNHs in culture medium in 24 h (red).

Table 1. Quantities of PSL and m-xylene adsorbed on SWNHs.

SWNHs PSL (g/g) m-xylene (g/g)

asSWNHs 0.14 0.09oxSWNH(500) 0.54 0.40oxSWNH(550) 0.50 0.34oxSWNH(560) 0.45 0.30oxSWNH(570) 0.35 0.26

tested oxSWNH(Tox) with various Tox, which was not donepreviously in the case of dexamethasone [7]. Incidentally,it was known that the pore volume of oxSWNHs changedwith Tox and maximized at Tox of 500 ◦C [19], while thehole size increased with Tox [24] up to 1.5 nm or largerat Tox of 570 ◦C [25]. In this context, we examined PSLadsorption and release on/from oxSWNH(Tox) specifically forTox higher than 500 ◦C (Tox = 500, 550, 560 and 570 ◦C),because the larger hole size would be preferable for PSLmolecules (size: ∼1.5 nm) to enter and leave the inner spacesof oxSWNHs. The results showed that the pore volume ofoxSWNH(Tox), evaluated by the m-xylene adsorption quantity,decreased with increasing Tox from 500 to 570 ◦C (table 1), andthe adsorption quantity of PSL decreased as well (table 1 andfigure 2). However, the amount of released PSL from PSL–oxSWNH(Tox) in culture medium increased with increasingTox from 500 to 570 ◦C, as shown in figure 2 (red line) andfigure 3(a). This tendency would be plausibly explained by thehole size of oxSWNH(Tox) becoming larger with increasingTox [24], and then the PSL molecules as well as the solventmolecules being able to pass through the holes more easily,leading to the larger amount of PSL coming out of the innerspaces of oxSWNH(Tox). For the animal test to study the effectof PSL–oxSWNH(Tox), we chose PSL–oxSWNH(560) for thesubstantial released amount of PSL (figure 2, 0.17 g/g in 24 h).

It should be noted that there was a fast release within thefirst few hours and a slow release reaching equilibrium within24 h in culture medium (figure 3(a)). Since the PSL releasefrom PSL–asSWNHs equilibrated within 4 h (figure 3(a), blackline), the quick release of PSL from PSL–oxSWNH(Tox) wasmainly from those attached to the outside of oxSWNH(Tox),and the slow release of PSL was from inside oxSWNH(Tox).

Figure 3. Time course of the release of PSL from PSL–SWNHs inculture medium (a) and PBS (b) at 37 ◦C (0.05 mg ml−1);PSL–asSWNHs (black), PSL–oxSWNH(500) (blue),PSL–oxSWNH(550) (green), PSL–oxSWNH(560) (red), andPSL–oxSWNH(570) (purple). The amounts of released PSL wereplotted as percentages of the total PSL bound to the PSL–SWNHs. Inthe repeatability test, the percentages of PSL released fromPSL–oxSWNH(560) in culture medium within 1, 4, and 24 h were13.9 ± 1.1, 24.8 ± 1.3, and 39.9 ± 1.3%, respectively (n = 6, mean± standard error).

The release of PSL from PSL–oxSWNH(Tox) wasdrastically suppressed in PBS (figure 3(b)). One possiblereason was that organic compounds in the culture medium(e.g. hydrophobic amino acids) were competitively adsorbedon the surfaces of the SWNHs, which had the effect ofdetaching PSL from the walls of the SWNHs, whereas PBSdid not contain such materials. Due to the small detachmentquantity of PSL, we chose PBS as an appropriate solvent forthe premixing of PSL–oxSWNH(560) before injection into therats to minimize the fast release of PSL.

3.2. Dependence of PSL–oxSWNH(560) concentration onin vitro PSL release

It was predicted that, in the tissue, the local concentrationof PSL and PSL–oxSWNH(560) influences the PSL releasefrom PSL–oxSWNH(560). Therefore, we carried out a

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Figure 4. Time course of the release of PSL fromPSL–oxSWNH(560) at 37 ◦C; 0.01 mg ml−1 in culture medium(green), 0.05 mg ml−1 in culture medium (red), 0.2 mg ml−1 inculture medium (blue), and 0.05 mg ml−1 in PBS (black). Thesample solutions were prepared by diluting 10 mg ml−1

PSL–oxSWNH(560) premixed with PBS (for details, see section 2).The amounts of released PSL were plotted as percentages of the totalPSL bound to PSL–oxSWNH(560).

further release experiment in the culture medium at differentconcentrations of PSL–oxSWNH(560). As shown in figure 4,the amount of released PSL strongly depended on theconcentration of PSL–oxSWNH(560). The percentage ofPSL released from PSL–oxSWNH(560) in 24 h increasedfrom 31% to 86% with the decrease of PSL–oxSWNH(560)concentration from 0.2 to 0.01 mg ml−1. It seemed that thePSL maintained the equilibrium between the PSL adsorptionon oxSWNH(560) and the PSL dissolution in the culturemedium. We believe that the considerable quantity of PSL inPSL–oxSWNH(560) would be gradually released in the bodyaccording to the change in the local concentration of PSL, andthis release process would increase the therapeutic effects ofthe PSL.

Here, the time course of the released PSL from PSL–oxSWNH(560) in the culture medium (0.05 mg ml−1) infigure 4 did not coincide with that in figure 3(a). The reason forthis difference was that the PSL–oxSWNH(560) was sonicatedin the case of figure 4, while it was not sonicated in the caseof figure 3(a). During the sonication, the PSL moleculeswere partly released from PSL–oxSWNH(560), resulting in anincrease of the first release of PSL (figure 4).

3.3. Animal experiment

PSL has been widely used in the treatment of chronicinflammatory diseases, such as rheumatoid arthritis, because ofits potent anti-inflammatory effect. To reduce the undesirableside effects, local release of PSL at the diseased area wouldbe effective. To confirm this, we locally injected PSL–oxSWNH(560) into the tarsal joints of CIA rats with theinflammation induced at the hind paws. The rats wereimmunized with type II collagen on day 0, and a series ofadministration of the materials (PSL–oxSWNH(560), PSL,oxSWNH(560) and PBS) was started at the same time (on days0, 7, 14 and 21).

Collagen-induced arthritis was manifested from betweenday 9 and day 13 in all rats, regardless of the injected materials.The arthritis score showed that the arthritis progression tendedto be slightly retarded in PSL–oxSWNH(560) treated ratscompared with PBS and PSL treated rats, and on day 17the arthritis score differed significantly between the PSL–oxSWNH(560) and control groups (figure 5(a)). In addition,leukocyte numbers and CRP level, which were used toestimate how active the inflammation was, were investigated.Leukocyte numbers tended to be lower in both the PSL andthe PSL–oxSWNH(560) groups than in the control group(figure 5(b)). Moreover, the CRP level was significantly lowerin the PSL–oxSWNH(560) group compared with the controlgroup (figure 5(c)). These results suggested that PSL releasedfrom PSL–oxSWNH(560) maintained its ability in vivo andthe administration of PSL–oxSWNH(560) could effectivelyreduce the disease activity.

Figure 5. Arthritis score (a), leukocyte number (b) and CRP level (c) of CIA rats. Leukocyte number and CRP level were determined on day28. The rats were treated with PBS (black), oxSWNH(560) (blue), PSL (green) and PSL–oxSWNH(560) (red). One-way ANOVA withDunnett’s test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

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Figure 6. Histological analysis of ankle joints after the development of arthritis (day 28) with HE staining ((a) and (b)) and CD68 staining((a-1), (a-2), (b-1), (b-2), (b-3) and (b-4)). The rats were treated with PBS (a) and PSL–oxSWNH(560) (b). S, synovial proliferation; B, bonedestruction; I , inflammatory cell infiltration.

It was noted that oxSWNH(560) treatment tended toaccelerate the arthritis manifestation (figure 5(a)), but tendedto lower the leukocyte numbers and CRP level (figures 5(b)and (c)) compared with PBS treatment (control). We thereforeconsidered that oxSWNH(560) could have some effect on themanifestation of arthritis.

To make clear the effect of using PSL–oxSWNH(560)and its exact distribution in ankle joints, we also performedhistopathological analysis. In the control group, synovialproliferation, bone destruction, and inflammatory cell infil-tration were histologically apparent and moderate to severearthritis was observed in the ankle joints (figure 6(a)). On theother hand, moderate synovitis, and minimal to moderate bonedestruction and inflammatory cell infiltration were observed inthe PSL–oxSWNH(560) group (figure 6(b)), suggesting thatPSL–oxSWNH(560) could effectively suppress the arthritisprogression compared with PBS (control).

Immunohistochemical analysis showed that CD68 posi-tive multinucleated giant cells accumulated in the bone areaof the ankle joint of the control group (figures 6(a-1) and (a-2)). They were considered to be osteoclastic cells, which wererelated to the bone destruction of arthritis. Treatment withPSL–oxSWNH(560) decreased the CD68 positive osteoclastic

cells (figures 6(b-1) and (b-2)). These tendencies againsuggested the favorable effect of PSL–oxSWNH(560) onarthritis.

Deposited black materials were found in thePSL–oxSWNH(560) group (figure 6(b)) and the oxSWNH(560)group (not shown), but not in the control group (figure 6(a)),indicating that they were the agglomerates of injectedPSL–oxSWNH(560) or oxSWNH(560). From the histologicalobservation, it was presumed that the depositedPSL–oxSWNH(560) gradually released PSL fromoxSWNH(560) at the ankle joints and suppressed theautoimmune response, resulting in the favorable effectagainst arthritis. Moreover, the black materials withsmall sizes (micrometer order) were often stored in CD68positive cells (figures 6(b-3) and (b-4)), suggesting thatmacrophage-like cells engulfed PSL–oxSWNH(560). If so, theimmunosuppressive effect of the released PSL inside the CD68positive cells could be increased.

4. Discussion

We previously reported that SWNHs intravenously admin-istered into mice accumulated in macrophages in liver,

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spleens, lungs and other reticuloendothelial systems withoutadverse histological changes [26, 27]. Macrophages havevarious immunological functions and are involved in thedevelopment of a lot of diseases, such as arteriosclerosis,inflammatory disorder, pulmonary disease, and so on. For thetreatment of diseases where macrophages are related to theirprogress, drug delivery systems for targeting macrophageshave been reported. For example, Chono et al investigatedthe drug delivery of liposomes containing dexamethasone tothe macrophages of atherosclerotic lesions, and their anti-atherosclerotic effects were confirmed following intravenousadministration in atherogenic mice [28]. Ryu et al showed thatamphiphilic poly(γ -glutamic acid) nanoparticles containingdexamethasone (DEX-NPs) accumulated in macrophages andmicroglia, and suppressed the inflammation both in vitroand in vivo. Moreover, intravitreal injections of DEX-NPsinto pathological rat eyes exhibited a neuroprotective effect,suggesting their potential for immunosuppressive treatmentof macrophages and microglia in damaged retinas [29].Chakravarthy et al delivered doxorubicin-conjugated quantumdots (QD-Dox) into the pulmonary airspaces of rats by oropha-ryngeal aspiration and successfully observed the uptake of QD-Dox into alveolar macrophages without any evidence of acutelung injury or increased cytokine levels [30]. In our study, weexpected that the PSL–oxSWNHs injected into the tarsal jointswould be engulfed by the macrophages related to articular in-flammation and selectively release PSL in the macrophages toenhance the medicinal effects. Actually, some PSL–oxSWNHswere observed in CD68 positive cells (figures 6(b-3)and (b-4)), indicating the possibility of oxSWNHs as drugcarriers of immunosuppressant drugs to the CD68 positivemacrophage-like cells.

Corticosteroids are used extensively and successfully inthe treatment of many inflammatory conditions, includingrheumatoid arthritis, but there are still problems with theirunfavorable and unavoidable side effects. Therefore, specifictargeting and sustained release of steroidal drugs is believed tobe beneficial to minimize the systemic side effects [31–33].Here, we have made a proof-of-idea study to show thatoxSWNHs are carriers that have possibilities of specificaccumulation in inflammatory macrophages (figures 6(b-3)and (b-4)) and controlled release of PSL from them accordingto the concentration dependence (figure 4). Progression ofthe study of corticosteroid delivery by using oxSWNHs wouldadvance the treatment of arthritis.

In summary, we have prepared PSL-attached oxSWNHsby immersing oxSWNHs in an ethanol–water solution of PSL.PSL–oxSWNH(560) released PSL in culture medium, andthe released quantity drastically increased with decrease inthe PSL concentration in the medium. In vivo, the anti-inflammatory effect of PSL–oxSWNH(560) was found inCIA rats with smaller values of arthritis scores. Milderprogression of arthritis and decreased numbers of osteoclasticcells in the bone area of the ankle joint were observed inthe histological examination. From these results, we proposethat oxSWNHs can act as potential PSL carriers for controlleddrug release inside CD68 positive cells which might have animmunoregulatory function.

Acknowledgments

MN thanks the Japan Society for the Promotion of Sciencefor a Grant-in-aid for Young Scientists (B) (No. 22710112).YI acknowledges grant support by the Program for Promotionof Basic Research Activities for Innovative Biosciences(PROBRAIN).

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