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[ 33 ] [ 28,29 ] accu- [ 20–23 ] including A heavy build- [ 7–19 ] wileyonlinelibrary.com www.afm-journal.de [ 23,24 ] nanoparticles inhibit CYP450 [ 32 ] ammation and may adversely affect a mulate primarily in the liver regardless of nano- administration route. Furthermore, particles have been shown to remain in the liver for an extended period of time, often months or years. up of nanoparticles in the liver causes infl number of liver functions, including meta- multiwalled CNTs (MWCNTs), (MWCNTs), multiwalled CNTs body through respiration, ingestion, orbody through respiration, can be absorbedskin contact, nanoparticles system andinto the human circulatory deposited into theare then predominantly system,organs of the reticuloendothelial such as the liver and spleen. Almost all tested nanoparticles, Ex vivo studies have indicated that gold, Ex vivo studies have indicated that gold, [ 25–27 ] ed and polystyrene ed by nanocombinatorial chemistry tuned cellular functions [ 30,31 ] The high surface area of nanoparticles, such as MWCNTs, sug- nanoparticles, such as MWCNTs, The high surface area of Dr. Y. Zhang Zhang Y. Dr. University of Chinese Academy of Sciences Beijing 100049 , China Prof. H. Zhu Wang, W. for Computational The Rutgers Center and Integrative Biology , Camden NJ 08102 , USA Prof. H. Zhu Department of Chemistry Rutgers University , Camden NJ 08102 , USA Therefore, it is an effective way to derive novel functions fromTherefore, it is an effective way to derive have shown that functionalized MWCNTs nanomaterials. We modifi silver, silver, bolic functions. enzyme activities. Despite multiple indications that nanoparticlesenzyme activities. Despite multiple indications bind MWCNTs perturb CYP450 activities, it is unknown whether An even moreto CYP450 enzymes and perturb their activities. functionalization can mod- pressing issue is whether MWCNTs’ function.uences on CYP450 ulate their infl alter their interac- cation may gests that surface chemical modifi acation is modifi tions with biological systems. Combinatorial surface chemistries. powerful approach to diversify MWCNT At the At [ 5 ]

[ 2 ] creating multiple 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim & Co. GmbH 2015 WILEY-VCH Verlag [ 6 ] © and tissue engineering. and tissue engineering. [ 3 ] The perturbation of CYP450 functions The perturbation [ 1 ] imaging, 26, 841–850 [ 3,4 ] 2016,

In the past decade, nanotechnology has been explored for appli- In the past decade, nanotechnology has been explored for DOI: 10.1002/adfm.201504182 routes for nanoparticle exposure. After entering the human seriously compromises human health and safety. safety. seriously compromises human health and Mater. Funct. Adv. Cytochrome P450 (CYP450) is a large family of membrane- Cytochrome They play a key liver. bound enzymes primarily residing in the including 75% role in the metabolism of xenobiotic substances, of all marketed drugs. 1. Introduction The liver plays an important role in metabolizing foreign materials, such as role in metabolizing foreign materials, The liver plays an important hydrophobic of carbon nanotubes and other drugs. The high accumulation may liver has raised concerns that nanoparticles nanoparticles in the nanotubes report here that carbon function. We interfere with liver metabolic and interact with CYP enter hepatic cells after intravenous introduction cations alter the carbon modifi enzymes, including CYP3A4. Surface chemical in human liver microsomes. nanotubes’ interactions with CYP450 enzymes on the enzymatic function of CYP3A4. They enhance, inhibit, or have no effect chemical structures are identifi Using a cheminformatics analysis, certain

Metabolic Activity of CYP3A4 in the Liver in the of CYP3A4 Activity Metabolic Yi Zhang , Yabin , Wang Aijuan Liu , Wenyi , Sherry Wang Hao Li , Zhu Xu , and Bin Bing Zhao , * Yan Yi Zhang , Hanfa , Zou Modulation of Carbon Nanotubes’ Perturbation to the Perturbation Nanotubes’ Carbon of Modulation that induce an enzyme inhibitory on the surface of the carbon nanotubes enzymes. effect or prevent disruption of CYP3A4 same time, more than 1826 consumer products have been mar- same time, more than 1826 consumer products keted and are now used in our daily lives, cation in a variety of areas, including biomedicine. For example, For cation in a variety of areas, including biomedicine. been investigated for applications have carbon nanotubes (CNTs) in drug delivery, www.MaterialsViews.com Dr. Y. Zhang, Y. Wang, A. Liu, Prof. B. Yan A. Liu, Prof. B. Yan Wang, Y. Zhang, Y. Dr. School of Chemistry and Chemical Engineering Shandong University Jinan 250100 , China E-mail: [email protected] S. L. Xu, Prof. B. Zhao Dr. State Key Laboratory of Environmental Chemistry and Ecotoxicology CAS for Eco-Environmental Sciences Research Center Beijing 100085 , China Prof. H. Zou Zhang, Y. Dr. CAS Key Lab of Separation Sciences for Analytical Chemistry National Chromatographic R&A Center Dalian Institute of Chemical Physics Chinese Academy of Sciences Dalian 116011 , China FULL PAPER 842 H, hepatocytes;S,sinusoid;N,nuclear; M,mitochondria;E,endosome;Cyto, cytoplasm;ER,endoplasmicreticulum;G,Golgi apparatus; L,lysosome. (D–F)) areidentified byredarrows.MWCNTs arefound inD)endosomes,E)thecytoplasm,andassociatedwithERmembrane. K,Kupffercells; granules. TheMWCNTs intheKupffercell(panels(C))arehighlighted byasquareandenlargedintheinset.TheMWCNTs inthe hepatocyte (panels C–E) MWCNT Figure1. wileyonlinelibrary.com way tomodulatetheirenzymeperturbationsintheliver. strates thatthesurfacechemistryofMWCNTs providesafeasible avoided soastonotdisruptCYP3A4activity. Ourresearchdemon- to identifythechemicalfeaturesofsurfaceligandsthatshouldbe titative structure-activityrelationship(QSAR)studywasconducted late CYP3A4activityinquitedifferentmanners.Moreover, aquan- modifi the abilityofCYP3A4tometabolizedrugs,and(3)variouschemical CYP3A4, changingtheenzyme’s conformationandcompromising (2) MWCNTs bindtoavarietyofCYP450enzymes,including MWCNTs enterhepatocytesafterintravenousinjectioninmice; ized MWCNTs andhumanlivermicrosomes.We found that(1) work, weinvestigatedtheinteractionsbetweensurfacefunctional- liver. MWCNTs absorbedbyanimalslargelyaccumulateinthe Hepatocytes Enter MWCNTs 2.1. Discussion and Results 2. by reprogrammingthecellularsignalingmachinery. www.afm-journal.de [ 24,35] cations oftheMWCNTsurfaceallownanotubestomodu- Internalization ofMWCNTs inKupffercellsandhepatocytestheliver. Representativeimages ofA,B)liverslicesfromcontroland BecausetheCYP450enzymesresponsiblefordrug 1 treatedmice areshown.Kupffercellintheliverisoutlinedbydottedlines(panels (A,C)).Thedarkmassinpanel(C)(arrow)isglycogen © 2015WILEY-VCHVerlag GmbH &Co. KGaA,Weinheim [ 34]

Inthis microscopy the liverhasbeenobservedusingmultiphotonfl particles andpolystyrenenanoparticles)byhepatocytesin 1F). (Figure membrane reticulum endoplasmic the cytoplasmofhepatocytes(Figure 1 E) andassociatewiththe (Figure 1 D). Moreover, individual MWCNTs wereabletoenter clusters werefoundinendocytosisvesicleshepatocytes an endocytosisvesicle(Figure 1 C). Meanwhile, fewerMWCNT cells, alargequantityofMWCNTs wasuptakenasaclusterin in bothKupffer cellsandhepatocytes( TEM imagesshowedthatbothtypesofMWCNTs accumulated prepared fortransmissionelectronmicroscopy(TEM)analysis. livers wereharvested,andthinsectionsofthelivertissues either carboxylatedMWCNTor entered hepatocytes.Two hoursafteri.v. administrationof fi metabolism primarilyresideinhepatocytestheliver, foreign particlesviaanendocytosismechanism. after hepatocyteuptake. that well-dispersedCNTs areexcretedviaabiliarypathway rst investigatedwhetherMWCNTs accumulatedintheliver The uptakeofothernanoparticles(mesoporoussilicanano- [ 37] andTEM. [ 39,40] [ 38] Previousstudieshavealsoshown Hepatocytes inthelivercanclear Adv. Funct. . n Kupffer In 1). Figure www.MaterialsViews.com 1 inICRmice,the Mater. [ 41,42] 2016, uorescence OurTEM 26, 841–850 [ 36] we FULL PAPER 843 wileyonlinelibrary.com www.afm-journal.de CYP3A4 , CYP2E1, P450 enzymes bound by the f-MWCNT P450 enzymes CYP2E1, CYP2A6, CYP1A2, CYP2C8, CYP2C9, , CYP2D6, CYP4F2, CYP8B1, CYP3A4 CYP4A11, CYP2B6, CYP51A1, CYP27A1, CYP4F3, CYP4V2, CYP4F11 CYP3A4 , CYP2E1, CYP2A6, CYP2C9, CYP1A2, CYP3A4 CYP2D6, CYP4A11, CYP4F2, CYP2C8, CYP8B1, CYP2B6, CYP27A1, CYP4F11, CYP2C19 CYP2E1, CYP2A6, CYP2C9, CYP1A2, CYP2C8, , CYP4F11, CYP2B6, CYP3A4 CYP4A11, CYP2D6, CYP3A5, CYP2C19, CYP3A7, CYP4A22 CYP2E1, CYP2A6, CYP1A2, CYP4A11, CYP2C8, , CYP2B6, CYP4F11, CYP3A4 CYP2D6, CYP2C9, CYP3A5, CYP2C19, CYP3A7, CYP4V2, CYP20A1 , CYP2E1, CYP2A6, CYP1A2, CYP2D6, CYP3A4 CYP2C8, CYP4A11, CYP2C9, CYP2B6, CYP4F2, CYP27A1, CYP8B1, CYP4F3, CYP51A1, CYP2C19, CYP4F11, CYP4V2, CYP20A1 , CYP2A6, CYP4A11, CYP2E1, CYP2C9, CYP3A4 CYP2C8, CYP1A2, CYP2D6, CYP4F2, CYP4F3, CYP2B6, CYP8B1, CYP7B1, CYP27A1 CYP2E1, CYP2A6, CYP2C9, CYP1A2, CYP2C8, CYP2C9, CYP1A2, CYP2C8, CYP2E1, CYP2A6, CYP2B6, , CYP4A11, CYP4F11, CYP3A4 CYP2D6, CYP4V2, CYP20A1 CYP3A5, CYP2C19, CYP3A7, CYP2C8, CYP2A6, CYP2C9, CYP4F3, CYP2D6, CYP1A2, CYP4A11, CYP2B6, CYP8B1, CYP2J2 CYP27A1, CYP3A5, CYP4F2, , CYP2A6, CYP2E1, CYP3A4 CYP2C8, CYP2C9, CYP4F3, CYP4A11, CYP1A2, CYP2D6, CYP27A1, CYP2B6, CYP4F2, CYP3A5, CYP8B1, CYP2J2, CYP4F11, CYP51A1 CYP2E1, CYP2A6, CYP2C9, CYP2C8, CYP1A2, , CYP2B6, CYP3A5, CYP3A4 CYP4A11, CYP2D6, CYP4F11, CYP3A7, CYP2C19, CYP20A1 Proteomic identifi cation of f-MWCNT-bound P450 enzymes. P450 enzymes. of f-MWCNT-bound cation Proteomic identifi Table 1. 1. Table f-MWCNT

2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim & Co. GmbH 2015 WILEY-VCH Verlag 9 [ 46 ] © . Because all . Because all 3 Ten function- Ten cations should 1–10 ) (Figure 5 ) [ 45 ] , all f-MWCNTs and 2 Table 1 26, 841–850 To investigate such inter- To and functionalized MWCNTs MWCNTs and functionalized 2016, [ 36 ] [ 43,44 ] les. Some abundant and functionally les. Some abundant and functionally As shown in As shown in The enzymes in each row are listed CYP3A4 is the most abundant CYP450 and is particularly important, being respon- sible for the metabolism of almost half of Mater. Funct. Adv. Because CYP450 enzymes are located in in CYP450 enzymes are located Because hepatocytes 2.2. Functionalized MWCNTs Bind CYP450 Microsomes Enzymes in Human Liver analysis provided the fi rst direct observation rst direct the fi analysis provided in hepatocytes. of MWCNTs offer MWCNTs different physiochemical different physiochemical offer MWCNTs properties. liver microsomes actions, we used human (HLMs) as an ex vivo system to test whether enzymes and bind CYP450 MWCNTs whether the surface chemistry of MWCNTs plays a role in such binding. In an HLM to system, CYP450 enzymes are tethered or the inner membranes of mitochondria to in the endoplasmic reticulum, similar their locations in the liver. (f-MWCNTs) are also internalized by hepat- are (f-MWCNTs) of this study investi- ocytes, the next part interact with CYP450 gated how MWCNTs modifi enzymes. Surface alized MWCNTs (f-MWCNTs) with different with different (f-MWCNTs) alized MWCNTs surface chemistries (Nos. and bound CYP450 enzymes extensively, had identical binding no two f-MWCNTs profi and important enzymes, such as CYP3A4 members of the CYP2 family (CYP2C8, CYP2C9, CYP2D6 and CYP2E1) were asso- On the ciated with all the tested f-MWCNTs. other hand, other enzymes, such as CYP7B1 For and CYP2J2, exhibited higher selectivity. example, CYP7B1 bound only to MWCNT while CYP2J2 was but not other MWCNTs, only bound by MWCNT were incubated in HLM for 30 min. The The were incubated in HLM for 30 min. bound enzymes were isolated and analyzed by liquid chromatography–tandem mass S1, Sup- spectrometry (LC–MS–MS) (Table porting Information). have comparable physical tested f-MWCNTs S2, and morphological properties (Table Information), the above results Supporting indicate that the binding of CYP450 was selective and enzymes by f-MWCNTs surface depended only on the f-MWCNT chemistry. order of their number of unique peptides. The chemical structures of each f-MWCNT are also shown. enzyme (30%–60% of all CYP450 enzymes) www.MaterialsViews.com FULL PAPER 844 wileyonlinelibrary.com structures inCYP3A4afterf-MWCNT binding,ascalculatedbyCDproonthebasisofeachCDspectrum. and apositivebandat193nm.MWCNT Figure3. centrations (from0.1to0.4mgmL and quantifi ed bydensitometryofdots.f-MWCNTs wereincubatedwithHLMatvariouscon- between f-MWCNTs andCYP3A4wereanalyzedbydotblotusingaspecifi c antibody Figure2. www.afm-journal.de f-MWCNTs andCYP3A4. absolute intensityofthedotswasdeterminedbyImageJ.B)Dose-dependentbindingsbetween f-MWCNT bindingpartiallyunfoldsCYP3A4protein. A)CYP3A4exhibitsacharacteristicCDspectrumwithnegative bandsat222 and208nm Bindings off-MWCNTs toCYP3A4withdifferentaffi nities. A)Thebindingaffi nities −1 ) beforeblottingwiththeCYP3A4-specifi c antibody. The 1 treatment decreasestheellipticityofCDspectrumat222and208nm. B) Content ofproteinsecondary © 2015WILEY-VCHVerlag GmbH &Co. KGaA,Weinheim drugs, proteintherapeuticsandgenes. as carriersforthedeliveryofsmall-molecule cially importantwhenf-MWCNTs areused modulate suchperturbations.Thisisespe- perturb thefunctionofCYP3A4andhowto we aimedtounderstandhowf-MWCNTs maintaining normalphysiologicalconditions, Because oftheessentialroleCYP3A4in Conformation CYP3A4 Changes Binding MWCNT 2.3. f-MWCNTs. mined thebindingaffi petitive conditions,surfacechemistrydeter- all MWCNTs, suggestingthatundercom- CYP3A4 bindingselectivitywasapparentfor MWCNT and different affi dose-dependent manner( show thatf-MWCNTs boundtoCYP3A4ina the densitometryofdots.Ourresults The bindingaffi using aCYP3A4-specifi c antibody(dotblot). binding amountsofCYP3A4tof-MWCNTs alized theenzymeconcentration-dependent quantitatively analyzethisbinding,wevisu- diverse surfacechemistries(Table 1 CYP3A4 boundtof-MWCNTs modifi ). To therapeutic drugs. of 0.4mgmL almost saturatedatanHLMconcentration CYP3A4 proteinsboundbyf-MWCNTs was When theCYP3A4proteinwasincubated (CD) techniques, suchascirculardichroism tein canbeobtainedusingspectroscopic Evidence ofnanoparticlesbindingtoapro- concentrations (suchas0.2mgmL As showninFigure 2 , theamountof 7 showedhigherbindingaffi [ 43] andfl 5 hadaloweraffi nities. For example,MWCNTs −1 ursec spectroscopy. uorescence nity wasalsoestimatedby . However, forunsaturated Adv. Funct. [ 47] Ourresultsshowthat www.MaterialsViews.com nity ofCYP3A4to ) n with and 2 A) Figure Mater. iy Fgr 2B). (Figure nity 2016, nities, while 26, 841–850 −1 e by ed ) the ), [ 21,48] [ 49] 1

FULL PAPER 845 1 is ) or keto- −1 ; nimodipine, −1 wileyonlinelibrary.com 1 mg mL www.afm-journal.de ; nifedipine, 6.9 µg mL ; nifedipine, −1 . Mass spectra of D) the parent drug nifedipine 1 Based on these studies, we cannot exclude Based on these studies, we cannot exclude [ 50,51 ] ; HLM, 0.5 mg mL ; HLM, −1 the possibility of non-specifi c interactions between CNTs between CNTs c interactions the possibility of non-specifi and lipid molecules and such interactions might perturb the our data showed that However, activity of CYP3A4 indirectly. on CYP3A4 were surface chem- perturbations of f-MWCNTs bindings istry-dependent, indicating that direct CNT-CYP450 to CYP450 played a major role. The binding of f-MWCNTs affected enzymes raised the question of whether f-MWCNTs therefore the metabolic functions of these enzymes. We binding changed the enzymatic tested whether f-MWCNT activity of CYP3A4. MWCNT surface. CNTs were reported to also bind cell mem- MWCNT surface. CNTs brane lipids. -helix α ; ketoconazole (KCZ), 1.3 µg mL ; ketoconazole −1 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim & Co. GmbH 2015 WILEY-VCH Verlag © , 0.2 mg mL , 0.2 mg 1 26, 841–850 , their interaction induced a large reduction in 1 2016, to CYP3A4 altered the secondary structure of 1 ). This result indicates that the binding Figure 3 . −1 f-MWCNT binding changes the metabolic activity of CYP3A4 as measured by HPLC. The HPLC chromatogram of MWCNT metabolic activity of CYP3A4 as measured by HPLC. The HPLC chromatogram of MWCNT f-MWCNT binding changes the The above results provide compelling evidence that results provide compelling evidence that The above C after which nifedipine was added. After 15 min, the remaining nifedipine concentration min at 37 °C after which nifedipine was added. After 15 min, conazole solution was incubated in HLM for 20 as in panel and the drug nifedipine. B) Same mixture was determined by HPLC with nimodipine as an internal standard (IS). A) Incubation of HLM (A) with the addition of ketoconazole. C) Same mixture as in panel (A) with the addition of MWCNT all reaction systems were the HPLC assay, binding. For (D) and E) its metabolite. Summary of the relative CYP3A4 activity induced by F) f-MWCNT 125 µL. Final concentrations: MWCNT 0.5 µg mL Mater. Funct. Adv. with MWCNT CD intensity at 208 and 222 nm, which is typical for Figure 4. presented as an example of the effect of f-MWCNTs on the metabolic activity of CYP3A4. A fraction (25 µL) of f-MWCNT solution ( on the metabolic activity of CYP3A4. A fraction (25 µL) of f-MWCNT solution presented as an example of the effect of f-MWCNTs structures ( of MWCNT the enzyme. The altered protein structural domain might be responsible for the enzymatic function; thus, this alteration may explain the reduction in CYP3A4 enzymatic activity. bound to CYP enzymes and thereby altered f-MWCNTs to nity of f-MWCNTs conformation. The affi the enzyme’s CYP3A4 was governed by the physicochemical nature of the chemical properties of the cally, specifi the f-MWCNTs: www.MaterialsViews.com FULL PAPER 846 wileyonlinelibrary.com Detailed knowledgeonsuch reactions iscurrentlylacking. on MWCNTsurfacemightbe metabolizedbyliverenzymes. HLM. For suchstudies,there isalwaysdoubtwhetherligands dominant roleinregulatingthe enzymaticactivityofCYP3A4in onstrated thatthesurfacechemistry off-MWCNTs didplaya on thesurfacechemistryof MWCNTs. Theseresultsdem- pressed, orcausednoeffectontheCYP3A4activity, depending ment as0%.Asshownin as 100%andthatwithCYP3A4inhibitorketoconazoletreat- was determinedbydefi ning enzymeactivitywithoutMWCNT or thepresenceoff-MWCNTs. InhibitionofCYP3A4activity relative toaninternalstandardsnimodipineintheabsence analyzing theamountofnifedipinemetabolizedbyCYP3A4 the effectsofsevenf-MWCNTs ontheactivityofCYP3A4by can beassessed(seeExperimentalSection).We thenevaluated nifedipine usingLC–MS,theenzymaticactivityofCYP3A4 the concentrationsofremainingparentdrugoroxidized by CYP3A4canbeinhibitedketoconazole.By determining nifedipine byoxidationreactions.Theof One ofCYP3A4’skeyfunctionsistometabolizeadrugcalled CYP3A4 2.4. MWCNT BindingChangestheEnzymaticActivityof www.afm-journal.de area ofNFPinketoconazolegroup−peakHLM-onlygroup). following equation:f-MWCNT’seffectonCYP3A4activity=(peakareaofNFPinketoconazolegroup−peakf-MWCNTgroup)/(peak as 100%,andthatintheketoconazolegroupwasdefi ned as0%.TheactivityofCYP3A4in f-MWCNT-treated groupswascalculated according tothe Figure5. CYP3A4 activityismodulatedbyf-MWCNTs fromacombinatorialMWCNTlibrary. TheCYP3A4activityintheHLM-onlygroupwasdefi ned 4 Figure , f-MWCNTs enhanced, sup- © 2015WILEY-VCHVerlag GmbH &Co. KGaA,Weinheim were metabolizedunderourexperimentalconditions. activities, itwasnotlikelythatsurfaceligandsonMWCNTs However, sincef-MWCNTs causeddifferenteffectsonCYP3A4 MWCNT library. was accomplishedbythesynthesisofananocombinatorial modifi Systematic Functional the Regulation ofCYP3A4 Affects Chemistry Surface MWCNT 2.5. medical applicationsorviaaccidental exposurefrompolluted activity ofCYP3A4(activitybetween 90%and110%). (activity <90%), and14outof80MWCNTs enhancedCYP3A4 activity are 32outof80MWCNTs inhibitedCYP3A4activity(activity activity ofCYP3A4overabroadrange(60%–131%).There f-MWCNTs withdiversechemistriesmodulatedtheenzymatic members withNFPinHLM.Asshown remaining parentdrugconcentrationafterincubatinglibrary activity inahigh-throughputformat,wedeterminedthe f-MWCNT library, whichcontains 80members,onCYP3A4 Whether throughtheirpurposeful introductionforbio- >110%). Noticeably, 34f-MWCNTs didnotperturbthe [ 33] cation ofthesurfacechemistryMWCNTs To testtheeffectsofthiscombinatorial Adv. Funct. www.MaterialsViews.com Mater. multiple 5 , Figure 2016,

26, 841–850 FULL PAPER 847 k NN models, 75 95.3 98.2 94.5 68.8 k NN modeling, wileyonlinelibrary.com www.afm-journal.de Substructures favored for binding Substructures 110%, 14 MWCNTs), the neu- 110%, 14 MWCNTs), 90%, 32 MWCNTs). Because the Because the 90%, 32 MWCNTs). NN) is a QSAR modeling approach k 89.6 81.33 83.14 81.75 85.82 Avg. activityAvg. cance [%] Statistical signifi nearest neighbor compounds in chem- nearest neighbor compounds k First, according to the activity readouts, activity readouts, according to the First, Based on the Dragon chemical descriptors, Based on the Dragon chemical descriptors, [ 55,56 ] 8 6 3 3 [ 57,58 ] 12 cient, only QSAR analyses based on a combination analyses based on a combination cient, only QSAR NN models were built with a fi vefold cross validation vefold a fi NN models were built with k Description ned as 0) groups was conducted using molecular descrip- ned as 0) groups nearest neighbor ( The k 100 tral group (activity from 90 to 110%, 34 MWCNTs) and the and the from 90 to 110%, 34 MWCNTs) tral group (activity < inhibition group (activity for the MWCNT enhancement group amount of data available was insuffi ned as 1) and neutral (activity defi of the inhibition (activity defi 6.0. In total, 503 chemical tors calculated by Dragon ver. molecules attached descriptors were generated for the surface to the MWCNTs. based on the class used to classify unknown compounds membership of its ical space. we classifi ed MWCNTs in the library into three groups: the groups: the in the library into three ed MWCNTs we classifi (activity > enhancement group the above descriptors were prioritized in terms of the fre- the above descriptors were prioritized all 100 quency with which they appeared among and a combined accuracy of 78.77%. After the and a combined accuracy of 78.77%. After nanoparticles. c 4 9 7 5 17 ndings Thus, the It has been [ 54 ] [ 53 ] F10 O–F of O–F at topological distance 10 Frequency O & F pairs at distance 10 Fewer F02 C–N of C–N at topological distance 2 Frequency & N pairs at distance 2 C Fewer F05 N–O 5 of N–O at topological distance Frequency N & O pairs at distance 5 Fewer Alter-containing molecule No. Actives 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim & Co. GmbH 2015 WILEY-VCH Verlag © or drug intoxication, as has 1.30 0.99 2.02 To our knowledge, the fi To [ 52 ] [ 28 ] cacy 1.31 0.64 0.86 26, 841–850 Active Inactive Descriptor 2016, others can also activate this enzyme. others can also activate [ 52 ] Analysis of important descriptors. of important descriptors. Analysis Structure alerts for CYP3A4 inhibition. Structure alerts for CYP3A4 inhibition. Although some small molecules deactivate CYP3A4 small molecules deactivate CYP3A4 Although some The systematic modifi cation of f-MWCNTs modulated their their modulated cation of f-MWCNTs The systematic modifi Mater. Funct. Adv. suggested that, like small molecules, nanoparticle binding may molecules, nanoparticle binding may suggested that, like small activity. also enhance CYP450 environment, it is desirable that these materials do not perturb that these materials do not perturb environment, it is desirable discovered in CYP3A4 inert f-MWCNTs CYP3A4 function. The great potential for biomedicine and other this work may have a applications. enzyme, enhancement of CYP3A4 activity by f-MWCNTs is as toxic as enhancement of CYP3A4 activity by f-MWCNTs ed and avoided. enzyme inhibition and should be identifi nding raised the question interactions with CYP3A4. This fi was correlated to the surface chemistry of how the f-MWCNT question, we answer this To perturbations in CYP3A4 activity. which has recently been used to pre- conducted a QSAR study, cytotoxicity of specifi dict the physiochemical properties and presented herein provide the fi rst experimental evidence to sup- presented herein provide the fi enzyme activity may port this hypothesis. Enhanced CYP3A4 lead to low therapeutic effi been observed in the case of enzymatic inhibition. been observed in the case of enzymatic inhibition. www.MaterialsViews.com Alter Table 3. Table 28.85% 35.51% 14.86% Table 2. Table P -value FULL PAPER 848 both endsofthesidechain,andredarrowsindicatetwo show similarinhibitoryeffectstowardCYP3A4activityusedforpharmacophoresearch.Bluearrowsindicatethebenzene/aromatic ringson Figure6. hydrophobic centroid (green dots); Acc: hydrogen bond acceptor (blue dots); Don: hydrogen bond donor (purple dots); Aro: aromatic ring (orange dots). hydrophobic centroid(greendots);Acc:hydrogenbondacceptor(blueDon:donor(purpleAro:aromatic ring(orangedots). on MWCNTs thatdonotchangeCYP3A4activity. Greenarrowsindicatereducedaromaticityandfewertertiarynitrogensinspecifi wileyonlinelibrary.com rings shouldbeavoided( CYP3A4 activity, longandcomplexsidechainsaromatic Modeling. Ourstudyshowsthattominimizetheinhibition of tors thatoverlappedwiththoseidentifi ed intheCase Ultra using f-MWCNT surfacemoleculeswith keyDragondescrip- activity ( functional groupalertsthatsignifi cantly inhibitedCYP3A4 corresponding CYP3A4activities.Themodelingidentifi to analyzetherelationshipsbetweenf-MWCNTs andthe tive MWCNTs (Table 2 ). are consideredtodiffersignifi are shownin the threetopologicallybaseddescriptorswith The 20-descriptor-reduced poolwassubmittedtoa and the20highestdescriptorswerechosenforthisanalysis. www.afm-journal.de WNs n Y34 Fgr 6D). (Figure CYP3A4 on MWCNTs fewer tertiarynitrogensinspecifi macophore analysisindicate that feweraromaticringsand for theresultsofaboveanalysis. Theresultsofthephar- MWCNTs notaffectingCYP3A4,providesadditionalsupport which wasbasedonamapping ofthesurfacemolecules be avoided(Table 3 ). Meanwhile, thepharmacophoreanalysis, and more Next, theCase Ultraprogram(version1.5)wasemployed Identifi 3 Table ). We thenconductedapharmacophoresearch CONR 2 Table . Basedontheir cation ofpharmacophoresonMWCNTsurfacemolecules.A–C)Thestructuresthreegroupsmolecules onMWCNTs that 2 alsoleadtoCYP3A4bindingand should –) Mroe, fewer Moreover, 6 A–C). Figure cantly betweenactiveandinac- c positionsreducetheeffectof P values,thesedescriptors © 2015WILEY-VCHVerlag GmbH &Co. KGaA,Weinheim value P -et and t -test, CONR2 groups.D)Theidentifi ed fi ed ve < 40% NH 2

in biomedicineandconsumer products. in guidingtherationaldesignof safeMWCNTs forapplications CYP3A4 functionintheliver. Thisinformationwillbehelpful physicochemical featuresoff-MWCNTs andregulationofthe insights intotheunderstanding oftherelationshipsbetween likely togeneratesafeMWCNTs. Ourfi ndings providekey lower aromaticityandfewertertiarynitrogenatomsaremore f-MWCNTs. We alsodiscoveredthatthepharmacophoreswith surfaces areresponsibleforinducingtheinhibitoryeffects of complex hydrophobicoraromaticsidechainsonthenanotube the liver. BasedonaQSARanalysis,wefoundthatlong and can modulatef-MWCNT perturbations ofCYP3A4activityin shown thatthechemicalmodifi cation ofMWCNTsurfaces binding totheproteinandalteringitsconformation.We have that f-MWCNTs perturbtheenzymaticactivityofCYP3A4by matic functions.Inthisinvestigation,wehavedemonstrated able perturbationstometabolicfunctions,includingCYPenzy- The accumulationofnanoparticlesinthelivercausesundesir- or accidentalexposurestonanoparticlesintheenvironment. quence oftheusenanomaterialsforbiomedicalapplications nanomaterials, regardlessofwhetherexposureisadirectconse- Extensive nanoparticledepositionintheliveriscommonforall Conclusion 3. cation of pharmacophores in parts of the surface molecules cationofpharmacophoresinpartsthesurfacemolecules Adv. Funct. www.MaterialsViews.com Mater. c locations.Hyd: 2016, 26, 841–850 FULL PAPER 849 owing mobile 2015 , 12 , 563 . J. Am. Chem. Soc. wileyonlinelibrary.com www.afm-journal.de , and the absorbance , and the absorbance 1 − Revised: November 13, 2015 Revised: 2003 , 192 , 23 . Received: September 30, 2015 Received: 2007 , 21 , 70 . 2007 , 28 , 344 . Published online: December 21, 2015 Published online: December 21, 2015 2007 , 168 , 176 . 2011 , 32 , 206 . 2008 , 68 , 6652 . 2009 , 236 , 16 . 1990 , 42 , 821 . 2011 , 7 , 2232 . Cancer Res. Cancer G. Jia , Y. X. Gao , Lett. Toxicol. B. Li , J. , Sun Y. F. Li , F. Jiao , Y. L. Zhao , Z. F. Chai , 2008 , 130 , 11467 . (accessed: September 2015). cpi/products/ A. Barazza , E. A. , Fisher Biomaterials R. E. Gordon , Z. A. , Fayad W. J. M. A. , Mulder S. , Thakor C. Small H. Nielsen , R. , Sinclair Z. Cheng , S. S. H. S. Kim , H. , Gambhir O. Pharmacol. Kim , Y. T. Lim , B. H. Chung , J. Jeong , Appl. Toxicol. macol. : Based on the : Relationship Analysis Quantitative Structure-Activity [1] F. P. Guengerich , Chem. Res. Toxicol. [2] M. Pirmohamed , B. [3] K. Park , M. Toxicology , Martincic G. , [4] Tobias Expert Opin. Drug Delivery S. , Dhar http://www.nanotechproject.org/ Z. Liu , J. Inventory, Products Consumer Thomale , H. [5] Dai , S. B. J. S. Lippard , Harrison , [6] A. Atala , Biomaterials [7] T. Skajaa , D. P. , Cormode P. A. Jarzyna , A. [8] Delshad , C. C. Blachford , L. , Zavaleta K. B. Hartman , Z. Miao , M. [9] L. W.-S. James , Cho , P. Kempen , M. Cho , J. , Jeong M. Choi , H.-Y. Cho , B. S. Han , [11] P. Jani , G. W. Halbert , J. Langridge , A. T. Florence , J. Pharm. Phar- [10] Z. Liu , K. Chen , C. Davis , S. Sherlock , Q. , Cao X. [12] Chen , J. H. X. , Wang Dai , G. Q. Zhou , C. Y. Chen , H. W. , Yu T. C. , Wang Y. M. Ma , Acknowledgements contributed equally to this work. The authors thank and Y.W. Y.Z. early phase of this project. for technical assistance in the Lei Yang Dr. of China Foundation This work was supported by the Natural Science Priority Research (21137002, 91543204 and 21525730), the Strategic (XDB14030400 and Program of the Chinese Academy of Sciences and the Colgate- XDB14030401), the NIEHS of the NIH (R15ES023148), Palmolive Grant for Alternative Research. supernatant was measured by high performance liquid chromatography performance liquid chromatography was measured by high supernatant column was packed Japan). The HPLC Shimadzu, 2010EV, (HPLC-MS bonded silica. The fl silane chemically with octadecyl gradient and water with the following of methanol phase was composed 9–12, and min 5–8, 75:25 for min for min 0–4, 65:35 for elution: 50:50 mL min ow rate was 0.3 fl 50:50 for min 13). The Supporting Information from the Wiley Online Library or Supporting Information is available from the author. of the target was detected at 238 nm. of the target was detected ed into inhibition were classifi f-MWCNTs enzymatic activity of CYP3A4, Ultra modeling were then modeling and Case and neutral groups. kNN selected functional group features of MWCNTs applied to analyze the were kNN modeling, 503 Dragon descriptors from each group. For ed based 20 important descriptors were identifi generated, and the top among all qualifying kNN models. Case on the occurrence frequency to analyze the relationships between f-MWCNTs Ultra models were built CYP3A4 activities. The conclusions of the QSAR and their corresponding overlap between important Dragon descriptors study were based on the the models. Finally, Ultra the Case ed in and the structures identifi ed to emphasize important features. pharmacophores were identifi

, 1 ) ) 1 − O M − −1 2 body ) and M −1 3 − ) of HLM 1 Tris-HCl. − M 10 , 3.13 µL) was 1 − c antibody for 1 h. 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim & Co. GmbH 2015 WILEY-VCH Verlag ve times with PBS and © ) and alkylated by adding M lm processor. The gray level lm processor. urea and 50 m M C. The reaction was quenched nal concentration, 0.18 mg mL 5:1) were added; the reaction was ) in tubes and blended at 800 rpm 1 − ) was added dropwise while the above 1 − ). After that, the solution was ultrafi ltered ltered ). After that, the solution was ultrafi ed by ImageJ software. M C. Next, nifedipine (2.5 µL, 10 × For animal experiments, the suspensions of animal experiments, For [ 33 ] for 10 min. The quantity of nifedipine in the 1 26, 841–850 − C. The mixture was then centrifuged at 15 000 rpm min 2016, c. solution. Next, tissues were washed three times in ddH ) was incubated with an f-MWCNT suspension (70 µg mL ) was incubated with an f-MWCNT 4 −1 for 20 min at 37 ° 1 − : For electron microscopy, tissue electron microscopy, : For Electron Microscopy Transmission : First, HLM (20 mg mL Nifedipine Metabolism in HLM : The Protein was dissolved in : The cation LC–MS–MS for Protein Identifi : Different concentrations (0.4, 0.2, 0.1, and 0 mg mL Dot Blot : Human cytochrome P450 3A4 : Human Circular Dichroism Analysis : Nifedipine (N7634), nimodipine (N149), (N7634), nimodipine : Nifedipine Antibodies Reagents and for : The synthesis methods MWCNT Preparation Functionalized : The animals used in this study were 5 week old : The animals used in Animal Experiments weight. Two hours after injection, animals were sacrifi ced, and the liver injection, animals were sacrifi hours after weight. Two xed for super-thin section preparation. was fi overnight in glutaraldehyde (2.5%) in PBS (0.1 xed specimens were fi and dehydrated stepwise in EtOH. Tissues were polymerized with Spurr and dehydrated stepwise in EtOH. Tissues were resin at 68 °C for 15 h. The embedded specimens were subsequently a Hitachi H-7650 TEM thin-sectioned into 70 nm slices and viewed on operating at 80 kV. denaturing buffer (pH 7.4) containing 8 (J-810, JASCO, Japan) in PBS for 30 min before circular dichroism and without f-MWCNTs analysis. The conformations of CYP3A4 with structure content of were recorded at room temperature. The secondary CYP3A4 was calculated using CDPro software. for 30 min at 37 ° for 10 min. The f-MWCNT sediment was washed fi resuspended in PBS (100 µL), of which 1 µL was blotted to form the dot on the nitrocellulose (NC) membrane. The blots were blocked with 5% milk for 1 h and then incubated with the CYP3A4-specifi 20, the blots After washing three times with PBS containing 0.5% Tween After were incubated in the secondary antibody for an additional hour. washing again, the blots were immersed in the chemiluminescent solution for visualization on a 2600C automatic X-ray fi of the dots was semiquantifi iodoacetamide (IAA) (4 µ (fi in PBS were incubated with f-MWCNTs pH 7.4). After washing three times in PBS, the tissues were fi xed for 1 h tissues were fi pH 7.4). After washing three times in PBS, the in 2% OsO The protein was reduced by adding DTT (2 µ and then digested by trypsin and desalted with a C18 SPE column. The and then digested by trypsin and desalted with (Thermo Velos resulting peptides were analyzed using the LTQ-OrbiTrap was completed by Fisher Scientific Inc., San Jose, CA). The data analysis searching with MaxQuant (version 1.1.1.36). (26 µg mL NADPH preparation (7.5 µL, A:B = allowed to continue for 15 min at 37 ° by immersing the tubes in ice. Next, the internal standard nimodipine solution (125 µL, 5 µg mL Mater. Funct. Adv. 4. Experimental Section (Stpurchased from Sigma-Aldrich (K1003) were and ketoconazole from Cell antibody was purchased USA). The CYP3A4 Louis, MO, USA). Horseradish peroxidase conjugatedSignaling (Boston, MA, purchased from BioRad (Richmond, CA, USA).secondary antibody was and luminescence reagents were purchasedNitrocellulose membranes Regenerating HLMs and NADPH from EMD Millipore Corporation. Solution B) were commercially available fromSystem (Solution A and cytochrome P450 3A4 (H5042) was purchasedBD Biosciences. Human reagents for HPLC were purchased from Thermofrom Sigma-Aldrich. All Fisher Scientifi in detail in the combinatorial library have been reported f-MWCNTs in our previous study. min diluted in PBS (89 µL). 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