There is growing evidence that ¯lamentous nanoparticles o®er advantages over spherical ones indrug delivery applications. The purpose of this study was to assess the potential of rod-like, plant-derived cellulose nanocrystals (CNCs) for nanomedical uses. Besides a nonspherical morphology,their facile bioconjugation, surface hydrophilicity and small size render CNCs promising drugcarriers. The cytotoxicity of CNCs against nine di®erent cell lines (HBMEC, bEnd.3, RAW 264.7,MCF-10A, MDA-MB-231, MDA-MB-468, KB, PC-3 and C6) was determined by MTT and LDHassay. CNCs showed no cytotoxic e®ects against any of these cell lines in the concentration rangeand exposure time studied (0�50 �g/mL and 48 h, respectively). Cellular uptake of °uorescein-5 0-isothiocyanate-labeled CNCs by these cell lines, quanti¯ed with a °uorescence microplate reader,was minimal. The lack of cytotoxicity and the low nonspeci¯c cellular uptake support ourhypothesis that CNCs are good candidates for nanomedical applications.
Keywords: Nanoparticles; drug carriers; FITC conjugation; nanomedicine; animal cell cultures.
1. Introduction
Nanoscale drug delivery systems hold great promisefor the pharmaceutical treatment of diseases be-cause they enable the targeting of therapeuticagents to speci¯c organs or tissues, thus reducing oreliminating undesirable drug side e®ects. To date,
about two dozen nanoscale drug delivery systems are
in clinical use, and many more are being evaluated
in preclinical studies.1 Nanoscale drug carriers can
be broadly divided into liposomes; polymer�drug
conjugates; micelles; dendrimers; nanoshells, such
nanoparticles. Among the key factors that govern thesuitability of a nanoscale carrier for drug deliveryapplications are its in vivo toxicity, and the rate andmechanism of clearance from the body. The phar-macological pro¯le of nanoscale drug delivery sys-tems is known to depend on their chemicalcomposition, surface hydrophilicity, surface charge,size and morphology.2 The majority of carrier sys-tems currently in use or under investigation arespherical or irregular in shape. However, there is anincreasing amount of evidence that elongated or ¯l-amentous morphologies o®er distinct pharmacologi-cal advantages.3 Nonspherical carriers in generalhave a better surface area-to-volume ratio thanspherical ones and therefore provide a larger surfacearea for conjugation of therapeutic and targetingagents. Filamentous carriers have been shown tomore successfully evade rapid clearance from theblood stream by the mononuclear phagocyte system(MPS) — typically a matter of minutes4 — byaligning with the blood °ow, thus avoiding vascularcollisions, ¯ltration and phagocytosis.5 Moreover,cylindrical carriers as large as 30 nm in diameter and2�m in length have been shown to translocatethrough the glomerular ¯ltration barrier of the kid-neys by orienting themselves with their long axesperpendicular to the endothelial fenestrations.6 As aresult, compared to spherical carriers, cylindrical or¯lamentous carriers are more readily cleared from thebody by the renal system. When it allows su±cienttime for carrier circulation, the renal clearance routeis preferable over the hepatic one because it mini-mizes carrier toxicity and retention.7
Cellulose nanocrystals (CNCs) are plant-derived,elongated nanoparticles that have gained attentionfor their comparatively low cost, high strength andsti®ness and tendency to form colloidal liquidcrystalline phases.8,9 Over the past decade, CNCshave been extensively studied as reinforcing ¯llers inpolymer nanocomposites.10,11 In a recent ecotoxi-cology study involving rainbow trout hepatocytesand nine aquatic species,12 CNCs have been foundto have low toxicity potential and present a lowenvironmental risk, rendering them attractive can-didates for nanoparticle applications. The presentstudy explores the potential of CNCs in therapeuticand diagnostic medicine. We have previously shownthat CNCs can be °uorescently labeled for bioima-ging applications13 and that they have promise foruse as drug carriers.14 Besides their elongatedmorphology, CNCs have several properties that
make them good candidates for drug deliveryapplications. CNCs are composed of cellulose, a�-1,4-glucan. Within the nanoparticle, the cellulosemolecules are oriented with their long axes parallelto the long axis of the particle. The surface chem-istry of CNCs is governed by the protruding hy-droxyl groups of the outermost cellulose molecules.The surface hydroxyl groups of CNCs can be readilyconverted into other functional groups, such asamino13 or carboxyl groups,15 or used directly forcovalent or noncovalent binding of targeting and/ordrug moieties to the CNC surface. Furthermore, inaqueous media, the surface hydroxyl groups bindwater molecules, thus creating a hydrophilic hy-dration shell around the particle.16,17 A high surfacehydrophilicity is known to impede adsorption ofopsonin proteins and therefore delay clearance ofthe drug carrier from the blood stream by theMPS.4,18�22 The size of CNCs depends strongly onthe cellulose source with cotton and wood pulpyielding the smallest particle dimensions.23 Wood-derived CNCs have average heights of 4.5�5.0 nmand lengths in the range of 60�300 nm with averagelengths between 100 nm and 150 nm.23 CNCsobtained from wood pulp are therefore small enoughto pass through the interendothelial slits in the wallsof venous sinuses — typically below 200�500nm inwidth24 — and thus avoid ¯ltration from the bloodstream by the spleen. In summary, their facile bio-conjugation, surface hydrophilicity, rod-like shape,and small size render CNCs promising nanocarriersfor bioimaging and drug delivery applications.
To test our hypothesis that CNCs are goodcandidates for nanomedical uses, we studied theircytotoxicity against nine mammalian cell lines.Additionally, we performed cellular uptake studieswith °uorescently labeled CNCs. The cell lines thatwere used are listed in Table 1. Brain microvascularendothelial cells, which line the inside of cerebralcapillaries, are a critical component of the blood�brain barrier and the main obstacle to the deliveryof diagnostic and therapeutic agents to the centralnervous system.25 The development of chemicalagents that can penetrate the blood�brain barrier isexpected to bring about signi¯cant advances in thediagnosis and therapy of acute brain trauma, braincancer and neurodegenerative disorders, includingAlzheimer's and Parkinson's disease, stroke andmultiple sclerosis. Mouse macrophages were studiedfor their role in the body's immune response andclearance of nanoparticles from the blood stream or
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tissues. The remaining cell lines, except MCF-10A,are malignant cell lines, representing di®erent can-cer types. MCF-10A was included in this study forcomparison with MDA-MB-231 and MDA-MB-468.
2. Experimental Section
2.1. Materials
Dissolving-grade softwood sul¯te pulp (Temalfa 93A-A) was kindly provided by Tembec, Inc. Ammoniumhydroxide (29.4%, Certi¯ed ACS Plus, FisherChemical), epichlorohydrin (99%, Acros Organics),ethylene glycol tetraacetic acid (99%, Acros Organ-ics), sodium borate (decahydrate, reagent grade),sodium chloride (Certi¯ed ACS), sodium hydroxide(50% (w/v), analytical reagent grade), sucrose(reagent grade), and sulfuric acid (95.9% (w/w),Certi¯ed ACS Plus) were purchased from FisherScienti¯c. Dimethylsulfoxide, °uorescein-5 0-isothio-cyanate (FITC, 90%, Aldrich), heparin, insulin, and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT, � 97.5% (TLC), powder) were pur-chased from Sigma-Aldrich. Dulbecco's modi¯cationof Eagle's medium (DMEM), DMEM/Ham's F-1250/50 mix, fetal bovine serum (FBS), L-glutamine,Hank's balanced salt solution (HBSS), donor horseserum, Leibovitz's L-15 medium, minimum essentialmedium (MEM), MEM vitamins, MEM nonessentialamino acids, penicillin�streptomycin, phosphate-bu®ered saline (PBS), RPMI 1640 medium, sodiumpyruvate, and trypsin/EDTA were purchased fromMediatech, Inc. Endothelial cell growth supplement,epidermal growth factors and BD Nu-Serum werepurchased from BD Biosciences. Lactate dehydroge-nase (LDH) cytotoxicity detection kits were pur-chased from Roche.
2.2. CNC preparation
CNCs were prepared according to the method ofRevol et al.26 with some minor modi¯cations. Lap-sheets of the wood pulp were cut into small pieces ofapproximately 1� 1 cm and milled in a Wiley mill(Thomas Wiley Mini-Mill) to pass a 60 mesh screen.The milled pulp was hydrolyzed under stirring with64wt.% sulfuric acid (10mL/g cellulose) at 45�Cfor 60min. The hydrolysis was stopped by dilution ofthe reaction mixture 10-fold with cold (� 4�C)deionized water (Millipore Direct-Q 5, 18.2M��cm).The nanocrystals were collected and washed oncewith deionized water by centrifugation for 10minat 4�C and 4550 � g (Thermo IEC Centra-GP8R)and then dialyzed (Spectra/Por 4 dialysis tubing)against deionized water until the pH of fresh dialysismedium stayed constant over time. The nanocrystalsuspension was sonicated (Sonics & Materials ModelVC-505) for 10min at 200W under ice-bath coolingand ¯ltered through a 0.45�m polyvinylidene °uo-ride (PVDF) syringe ¯lter (Whatman) for removalof potentially present aggregates. The ¯ltered sus-pension had a concentration of 0.8 wt.%.
2.3. Fluorescent labeling of CNCs
CNCs were labeled with FITC as previously de-scribed.13 First, the surface of the nanocrystals wasdecorated with epoxy functional groups via reactionwith epichlorohydrin (6mmol/g cellulose) in 1Msodium hydroxide (132mL/g cellulose) according tothe method of Porath and Fornstedt.27 After 2 h at60�C, the reaction mixture was dialyzed (Spectra/Por 4 dialysis tubing) against deionized water untilthe pH was below 12. Next, the epoxy ring wasopened with ammonium hydroxide to introduce
HBMEC Human Brain Endothelial Isolated from tissuebEnd.3 Mouse Brain, cerebral cortex Endothelial Endothelioma (benign) ATCC (CRL-2299)RAW 264.7 Mouse Ascites (abdominal °uid) Monocyte/macrophage Virus-induced tumor ATCC (TIB-71)MCF-10A Human Mammary gland, breast Epithelial Fibrocystic (benign) ATCC (CRL-10317)MDA-MB-231 Human Mammary gland, breast Epithelial Adenocarcinoma ATCC (HTB-26)MDA-MB-468 Human Mammary gland, breast Epithelial Adenocarcinoma ATCC (HTB-132)KB Human HeLa contaminant Epithelial ATCC (CCL-17)PC-3 Human Prostate Epithelial Adenocarcinoma ATCC (CRL-1435)C6 Rat Brain Fibroblast Glioma (malignant) ATCC (CCL-107)
Abbreviations: HBMEC, human brain microvascular endothelial cells; ATCC, American Type Culture Collection.
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primary amino groups. After adjustment of the pHto 12 with 50% (w/v) sodium hydroxide, ammoni-um hydroxide (29.4%, 5mL/g cellulose) was addedand the reaction mixture heated to 60�C for 2 h.The reaction mixture was dialyzed until the pHwas 7. Finally, the primary amino group was reac-ted with the isothiocyanate group of FITC to form athiourea. Following the method of Swoboda andHasselbach,28 FITC (0.32mmol/g cellulose) wasadded to the aminated nanocrystals in 50mM so-dium borate bu®er solution (50mL/g cellulose),containing ethylene glycol tetraacetic acid (5mM),sodium chloride (0.15M), and sucrose (0.3M). Thereaction mixture was stirred overnight in the darkand then dialyzed against deionized water untilFITC was no longer detected in the dialysate byUV/Vis spectroscopy. The suspension was sonicat-ed (10min, 200W, ice bath cooling), centrifuged(10min, 4550 � g, 25�C), and ¯ltered through aPVDF syringe ¯lter (0.45�m) for removal of po-tentially present aggregates. The ¯ltered suspensionhad a concentration of 0.05wt.% and a pH of 6.
2.4. Atomic force microscopy (AFM)
CNC characterization by AFM was performed withan Asylum Research MFP-3D mounted onto anOlympus IX 71 inverted °uorescence microscope.Sample substrates were prepared by fastening micadiscs (Ted Pella, ? ¼ 9mm) with two-componentepoxy onto regular 3� 1 in. microscopy glass slidesand peeling o® of several layers of the mica withconventional sticky tape. Once a smooth surfacewas obtained, one drop of a 0.001wt.% suspensionof CNCs in water was deposited onto the fresh micasurface and allowed to dry in air under ambientconditions. Samples were scanned in intermittentcontact mode in air with Olympus OMCL-AC160TStips (nominal tip radius: 9� 2 nm). Reported meanparticle heights and lengths were determined fromAFM height images according to a previously pub-lished procedure.29
2.5. Dynamic light scattering
Dilute aqueous suspensions (0.01wt.%) of CNCswere ¯ltered through a 0.45 �m PVDF syringe ¯lterinto a 12mm square glass cuvette (Malvern PCS1115). The sphere-equivalent hydrodynamic diam-eter of the particles was measured in triplicate witha Malvern Zetasizer Nano ZS. Reported values are
mean Peak 1 diameters obtained with the GeneralPurpose algorithm of the Zetasizer Nano software.Measurements were done at 25�C in the absence ofadded electrolyte.
2.6. ³-Potential measurements
Dilute aqueous suspensions (0.01wt.%) of CNCswere sonicated for 10min at 70W in a Cole-Parmer8890 ultrasonic cleaner and transferred to MalvernDTS1060 folded capillary cells. The �-potential ofthe particles was measured in triplicate with aMalvern Zetasizer Nano ZS using the Hückel model.Measurements were done at 25�C in the absence ofadded electrolyte.
2.7. Fluorescence measurementby microplate reader
Di®erent amounts of the stock suspension of FITC-labeled CNCs in water (0.5mg/mL) were addedto PBS in wells of a 96-well plate (Corning Costar)to produce the following concentrations: 0.5, 1, 5,10, 50, 100 and 500 �g/mL and a total volume of100 �L in each well. The °uorescence intensity fromeach well was determined with a Molecular DevicesSpectraMax M2e °uorescence microplate reader atexcitation and emission wavelengths of 485 nm and530 nm, respectively.
2.8. Cell cultures
All cells, except the human brain microvascularendothelial cells (HBMEC), were obtained from theAmerican Type Culture Collection. HBMEC wereoriginally isolated, cultivated and puri¯ed as previ-ously described.30,31 All cell lines used in this studywere cultured at 37�C in a humidi¯ed atmosphere of5% CO2 and 95% air. The respective growth mediaare listed in Table 2. For plating of the cells, theculture medium was removed and the cells wererinsed with HBSS. The cells were detached by addi-tion of 1.5mL of trypsin/EDTA. When the cells haddetached, medium was added to stop trypsinization,the cells were resuspended by repeated pipetting, andthen counted and plated.
2.9. MTT assay
Cells were plated at 25� 103 cells/well in a 48-wellplate (Corning Costar) and allowed to adhere for
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24 h at 37�C in a humidi¯ed atmosphere of 5% CO2
and 95% air. The stock suspension of CNCs (5mg/mL) was sterilized by ¯ltration through a 0.22�mPVDF syringe ¯lter. The cells were washed withPBS and the culture medium was replaced. CNCswere added at concentrations ranging from 0 to50�g/mL and the cells were incubated for 48 h.After 48 h, the culture medium was removed andreplaced with basal medium containing 0.5mg/mLof MTT. The cells were incubated for another 4 h,after which dimethylsulfoxide (500 �L) was added todissolve the formazan crystals for spectrophotomet-ric quanti¯cation. The absorbance at 570 nm wasdetermined for each well with the microplate reader.
2.10. LDH assay
Cells were plated at 25� 103 cells/well in a 48-wellplate and allowed to adhere for 24 h at 37�C in ahumidi¯ed atmosphere of 5% CO2 and 95% air.The stock suspension of CNCs was sterilized by¯ltration through a 0.22�m PVDF syringe ¯lter.The cells were washed with PBS and the culturemedium was replaced. CNCs were added at con-centrations ranging from 0 �g/mL to 50 �g/mL andthe cells were incubated for 48 h. After 48 h, a por-tion of the culture medium (100�L) was removedand transferred to a 96-well plate. Assay solution(100 �L) was added to each well and the plate wasincubated for 30min at room temperature. Theabsorbance at 490 nm was determined for each wellwith the microplate reader.
2.11. Cellular uptake assay
For cellular uptake studies, cells were plated at 25�103 cells/well in a 48-well plate and allowed to ad-here for 72 h at 37�C in a humidi¯ed atmosphere of5% CO2 and 95% air. The stock suspension ofFITC-labeled CNCs (0.5mg/mL) was sterilized by¯ltration through a 0.22�m PVDF syringe ¯lter.The cells were washed with PBS and the culturemedium was replaced. FITC-labeled CNCs wereadded at a concentration of 50�g/mL (26.3�g/cm2Þ and the cells were incubated for up to 48 h.After washing the cells three times with PBS, forremoval of CNCs not taken up, and covering thecells with fresh PBS, the °uorescence intensity fromeach well was determined with the °uorescencemicroplate reader at excitation and emission wave-lengths of 485 nm and 530 nm, respectively.
3. Results and Discussion
3.1. CNC characterization
Cell�nanoparticle interactions depend strongly onthe physicochemical properties of the nanoparticles.Careful characterization of the nanoparticles istherefore essential for the correct interpretation ofbiological test results. For the cellular uptakestudies, the CNCs had to be labeled with a °uorescentmarker, which might have a®ected their physico-chemical properties. To determine the physicochem-ical properties of the CNCs and whether theFITC-labeling had a®ected them, we characterized
Table 2. Growth media for the studied cell lines.
Cell line Growth medium
HBMEC RPMI 1640 medium, supplemented with 10% FBS, 10% NuSerum, 30�g/mL of endothelial cell growthsupplement, 15U/mL of heparin, 2mM L-glutamine, 2mM sodium pyruvate, nonessential amino acids,vitamins and 1% penicillin�streptomycin
bEnd.3 DMEM, supplemented with 10% FBS and 1% penicillin�streptomycinRAW 264.7 RPMI 1640 medium, supplemented with 10% FBS and 1% penicillin�streptomycinMCF-10A DMEM/Ham's F-12 50/50 mix, supplemented with 10% horse serum, epidermal growth factors (20 ng/mL),
insulin (0.01mg/mL), hydrocortisone (500 ng/mL), and 1% penicillin�streptomycinMDA-MB-231 Leibovitz's L-15 medium, supplemented with 10% FBS and 1% penicillin�streptomycinMDA-MB-468 RPMI 1640 medium, supplemented with 10% FBS and 1% penicillin�streptomycinKB MEM, supplemented with 10% FBS and 1% penicillin�streptomycinPC-3 RPMI 1640 medium, supplemented with 10% FBS and 1% penicillin�streptomycinC6 DMEM, supplemented with 10% FBS and 1% penicillin�streptomycin
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the CNCs before and after labeling in terms of mor-phology, mean dimensions, sphere-equivalent hydro-dynamic diameter — as a relative size measure, and�-potential — as a measure of surface charge.
Figure 1 shows AFM height images of unlabeledand FITC-labeled CNCs. The labeled CNCsexhibited the rod-like morphology of unlabeledCNCs but appeared slightly larger. The meanlengths and heights of labeled and unlabeled CNCs,determined from the AFM height data, are listed inTable 3. The mean length of the unlabeled CNCswas below the recommended maximum size of200 nm for engineered long-circulatory particles.24
Thus, unlabeled CNCs can be expected to avoidrapid clearance from the blood stream by splenic¯ltration. The mean length of the FITC-labeledCNCs was slightly larger than that of unlabeledCNCs but the di®erence was not statistically sig-ni¯cant. The mean height of the FITC-labeledCNCs was also slightly larger. FITC molecules areapproximately 1 nm in size,32 and consequently aheight increase of up to 2 nm might be expected
upon labeling. The observed height di®erence of0.6 nm could therefore be due to the FITC mole-cules on the surface of the labeled CNCs. To com-pare the sizes of the labeled and unlabeled CNCs insuspension, we measured their sphere-equivalenthydrodynamic diameters by dynamic light scatter-ing. The sphere-equivalent hydrodynamic diameterof the FITC-labeled CNCs was slightly larger thanthat of the unlabeled CNCs (Table 3) but the dif-ference was not statistically signi¯cant. On the basisof these results, we concluded that labeling had nosigni¯cant e®ect on the particle dimensions.
The �-potentials of the CNCs before and afterlabeling were negative and showed no statisticallysigni¯cant di®erence (Table 3). A negative�-potential signi¯es a negative surface charge.CNCs prepared by sulfuric acid hydrolysis areknown to have a negative surface charge due topartial esteri¯cation of the surface hydroxyl groupsby sulfuric acid molecules during the hydrolysis.33
In aqueous media, the resulting sulfate groups aredissociated and negatively charged. The negative
(a) (b)
Fig. 1. 3D AFM height images of (a) cellulose nanocrystals and (b) FITC-labeled cellulose nanocrystals.
Table 3. Properties of unlabeled and FITC-labeled cellulose nanocrystals.
Abbreviations: SEM, standard error of the mean; CNCs, cellulose nanocrystals.aNumbers in parentheses are standard deviations.
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�-potential of the labeled CNCs could indicate thatthe sulfate groups were still present on the particlesurface after the labeling procedure. Alternatively,the negative charge could stem from the FITCcarboxyl group. The aqueous suspension of labeledCNCs was slightly acidic with a pH of around 6,which is well above the pKa of the FITC carboxylgroup (� 4.3).34,35 Under these conditions, most ofthe FITC carboxyl groups should be dissociated andnegatively charged.
The density of FITC moieties on the labeledCNCs, determined previously by UV/Vis spectros-copy, was 0.03mmol per gram of cellulose or oneFITC moiety per 27 nm2 of particle surface area.13
To assess the °uorescence of the labeled CNCs inPBS, we measured the mean °uorescence intensity(MFI) at di®erent concentrations with a °uorescencemicroplate reader. As shown in Fig. 2, increasedconcentrations of FITC-labeled CNCs were corre-lated with increased MFI levels according to a nega-tive inverse exponential function. The MFI at 50 �g/mL (654� 12), which was the concentration used forcell incubation, was high enough for cellular uptakestudies.
3.2. Cytotoxicity assays
The cytotoxicity of unlabeled CNCs was measuredby MTT and LDH assay. CNCs were added tothe cell cultures at a concentration of 10�g/mL(5.3 �g/cm2Þ, 25�g/mL (13.2 �g/cm2Þ, or 50�g/mL (26.3�g/cm2Þ, and the cells were incubated for
48 h. After 48 h, the culture medium was removedand the respective test (MTT or LDH) performedaccording to standard procedures. Kroll et al. haverecently shown that at concentrations of 50�g/cm2
and above, nanoparticles may interfere withcommon cytotoxicity assays, including the MTTassay. The concentration range of 10�50�g/mL(5.3�26.3 �g/cm2Þ was chosen to avoid nanoparti-cle interference and aggregation during the assay.36
The results of the cytotoxicity assays are shownin Fig. 3. In both assays, CNCs showed no toxicityagainst any of the tested cell lines over the con-centration range and exposure time examined(0�50�g/mL and 48 h, respectively). The lack oftoxicity against brain microvascular endothelialcells (HBMEC and bEnd.3) is a necessary conditionfor the applicability of CNCs in the treatment ofneurodegenerative diseases. Similarly, the lack oftoxicity against ¯brocystic breast epithelial cells(MCF-10A) is a requirement for the safe use ofCNCs in the treatment of diseases of the breast,such as breast cancer.
The observed lack of toxicity of CNCs againstthe tested cell lines is in contrast with the reportedtoxicities of many other types of nanoparticles.According to Nan et al., for example, 200 nm longsilica nanotubes cause a decrease of about 30% inthe viability of MDA-MB-231 cells at a concentra-tion of 5 �g/mL and 72 h of exposure.37 Sohaebuddinet al. reported that silicon oxide nanoparticles at aconcentration of 10 �g/mL reduced the cell viabilityof RAW 264.7 macrophages in 24 h to 60%.38 Simi-larly, Soto et al. reported that multi-walled carbonnanotubes, iron oxide nanoparticles and silvernanoparticles lowered the cell viability of RAW264.7 macrophages in 48 h to 50% at concentrationsof ca. 6, 5, and 1 �g/mL, respectively.39 The identityof the cell line in that study is, however, somewhatquestionable. The authors refer to the cells as murinealveolar macrophages, whereas the cell line RAW264.7 has been established from the ascites of atumor induced by intraperitoneal injection of a leu-kemia virus. Furthermore, in two previous studies,the authors had used the cell line RAW 267.9, not264.7.40,41
In addition to the studies mentioned above, manyother nanoparticle cytotoxicity studies can be foundin the literature. However, because they typicallyinvolve di®erent cell lines, the results of these studiescannot be compared with the results obtained here.Our search for comparable cytotoxicity studies of
Fig. 2. Semilogarithmic plot of MFI versus concentrationof FITC-labeled cellulose nanocrystals in phosphate-bu®eredsaline. Data are the means � standard deviation of four mea-surements. The dotted line is a ¯t of a negative inverse expo-nential function to the data.
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recognized drug delivery systems, such as liposomesor polymeric carriers, for a more meaningful com-parison, has yielded no results.
3.3. Cellular uptake assay
In the cellular uptake assay, FITC-labeled CNCswere added at a concentration of 50�g/mL (26.3�g/cm2) to the cell cultures, and the cells were in-cubated for 1 h to 8 h. Given that the blood circu-lation half-life of covalently functionalized carbonnanotubes, another type of ¯lamentous nano-particles, has been reported to be in the range of1�3 h,42 exposure times beyond 8 h are likelymedically irrelevant.
After washing the cells thrice with PBS, for re-moval of CNCs not taken up, and covering of thecells with fresh PBS, the °uorescence intensity from
each well was determined with a °uorescencemicroplate reader at an excitation and emissionwavelength of 485 nm and 530 nm, respectively. Thecellular uptake in percent was calculated as
where MFICNC;uptake, MFIcontrol, and MFICNC;initialare the MFI of cells incubated with FITC-labeledCNCs, the MFI of cells incubated without FITC-labeled CNCs (cellular auto°uorescence), and theMFI of FITC-labeled CNCs initially added to thewells, respectively.
The results of the cellular uptake assay are shownin Fig. 4. Except for the cell lines HBMEC, MCF-10A and PC-3, all cell lines showed a more or lessprominent increase in uptake with exposure time.However, because the uptake after 8 h was still
(a)
(b)
Fig. 3. (a) MTT cell viability assay and (b) LDH cytotoxicity assay results, showing the e®ect of 48 h of exposure to cellulosenanocrystals at di®erent concentrations on various cell lines. Data are the means � standard deviation of four measurements.
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below 1% for most cell lines and below 3% for RAW264.7, the main conclusion from the data is thatcellular uptake of CNCs is physiologically insigni¯-cant at the concentration and exposure times ex-amined (50�g/mL and 1�8 h, respectively). Evenlonger exposure times (12�48 h) did not signi¯-cantly increase cellular uptake values. For KB cells(Fig. 5), for example, total uptake remained below2% after an exposure time of 48 h, which was theexposure time used in the cytotoxicity assays.
The low cellular uptake could be an indication ofparticle aggregation in the cell culture medium. Todetermine whether CNCs aggregate in cell culturemedium, we analyzed a suspension of FITC-labeledCNCs in RPMI 1640 medium (25 �g/mL) by dy-namic light scattering. The analysis revealed thepresence of larger scatterers, relative to CNCs in DIwater, which could be CNC aggregates. FITC-la-beled CNCs have a characteristic UV/Vis absorption
band at 495 nm. A comparison of the absorbances at495 nm of the FITC-labeled CNC suspension in me-dium and the aggregate-free supernatant after cen-trifugation showed that the absorbance of thesupernatant was 98% of that of the suspension beforecentrifugation. The fact that the absorbance of thesupernatant was nearly as high as that of the sus-pension before centrifugation suggested that the ex-tent of CNC aggregation in the cell culture mediumwas small. CNC aggregation was therefore unlikelythe reason for the observed low cellular uptake.
The uptake of nanoparticles by cells may occurby several mechanisms. Macrophages, such as RAW264.7, are able to engulf nanoparticles by a processcalled phagocytosis. This process starts with theadsorption of opsonin proteins on the particle sur-face. The opsonin-coated nanoparticles then adhereto the cell surface through speci¯c receptor�ligandinteractions, which trigger the spreading of cellmembrane extensions (pseudopodia) across theparticle surface, followed by internalization of theresulting vacuole (phagosome) by the cell. The factthat uptake of CNCs by RAW 264.7 was low after8 h of exposure suggests that clearance of CNCsfrom the blood stream by macrophages will be slow.Delayed clearance of nanoparticles by the MPS isdesired in drug delivery applications because itincreases the bioavailability of the drug.4,22
Nonphagocytic cells, such as endothelial cells,epithelial cells and ¯broblasts, take up nanoparticlesprimarily by receptor-mediated or nonspeci¯c ad-sorptive endocytosis.43 Receptor-mediated endocy-tosis may occur if the particles are decorated withhigh a±nity ligands that target speci¯c receptortypes, which was not the case in this study.
Fig. 4. Cellular uptake of FITC-labeled cellulose nanocrystals by various cell lines after 1 h, 2 h, 4 h and 8 h of exposure. Data arethe means � standard deviation of four measurements.
Fig. 5. Uptake of FITC-labeled cellulose nanocrystals by KBcells after 12, 24 and 48 h of exposure. Data are the means�standard deviation of 4 measurements.
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Nonspeci¯c endocytosis mechanisms are governedby the morphological and physicochemical proper-ties of the nanoparticles, including size, surface cur-vature and roughness, hydrophilicity and surfacecharge.44 While most of these factors are incom-pletely understood, conclusive evidence exists thatelectrostatic interactions at the negatively chargedcell surface play an important role in nonspeci¯ccellular uptake mechanims.43�45 Mahmoud et al.compared the uptake of FITC-labeled and rhoda-mine B isothiocyanate (RBITC)-labeled CNCs fromenzyme treated °ax ¯bers by human embryonickidney 293 (HEK 293) and Spodoptera frugiperda(Sf9) cells.46 In accordance with our results, theyobserved no uptake of FITC-labeled CNCs at phys-iological pH. They did, however, observe uptake ofRBITC-labeled CNCs, which were positivelycharged, and of FITC-labeled CNCs at pH 5, atwhich only a fraction of the FITC carboxyl groupsare ionized and therefore the negative charge ofFITC-labeled CNCs is reduced. On the basis of theirresults, the authors attributed the lack of cellularuptake of FITC-labeled CNCs at physiological pH tothe negative surface charge of the particles. The lowuptake of FITC-labeled CNCs by nonphagocyticcells may therefore be due to repulsive forces betweenthe negatively charged particles and cell membrane.Interestingly, Liebert et al. recently reported thecellular uptake of spherical FITC-labeled cellulosenanoparticles by human foreskin ¯broblasts.47 Thisseemingly contradictory result suggests that theparticle shape and the cell type are also importantfactors governing the cellular uptake of nano-particles. A low nonspeci¯c uptake, as observed forCNCs, is bene¯cial for drug delivery applicationsbecause it enables the use of speci¯c uptakemechanisms, such as receptor-mediated endocytosis,for drug targeting.
4. Conclusions
Our study has shown that CNCs are nontoxic to avariety of mammalian cells at concentrations ashigh as 50�g/mL and 48 h of exposure and thatcellular uptake of FITC-labeled CNCs under theseconditions is physiologically insigni¯cant. The dem-onstrated lack of cytotoxicity and the low nonspeci¯ccellular uptake of CNCs support our hypothesisthat CNCs are good candidates for nanomedicalapplications.
Acknowledgments
This material is based upon work supported by theNational Research Initiative of the USDA Cooper-ative State Research, Education and ExtensionService under Grant 2005-35504-16088 and theNational Science Foundation under Grants CHE-0724126 and DMR-0907567. Partial funding fromthe Institute for Critical Technology and AppliedScience, the Macromolecules and Interfaces Instituteand Omnova Solutions, Inc., are also acknowledged.The authors furthermore thank Tembec, Inc., for thepulp sample and F. Navarro for help with analyzingthe AFM data.
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