Research ArticleThe Effect of Triptolide-Loaded Exosomes on the Proliferationand Apoptosis of Human Ovarian Cancer SKOV3 Cells

Huan Liu 1 Ming Shen2 De Zhao2 Dan Ru1 Yourong Duan2

Chenhuan Ding 1 and He Li 1

1Traditional Chinese Medicine Department Renji Hospital School of Medicine Shanghai Jiao Tong UniversityShanghai 200127 China2State Key Laboratory of Oncogenes and Related Genes Shanghai Cancer Institute Renji Hospital School of MedicineShanghai Jiao Tong University Shanghai 200032 China

Correspondence should be addressed to Chenhuan Ding huanhuan0619msncom and He Li lihe1972hotmailcom

Received 1 January 2019 Revised 12 March 2019 Accepted 23 April 2019 Published 21 May 2019

Academic Editor Susan Sandeman

Copyright copy 2019 Huan Liu et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Triptolide has been proven to possess anticancer efficacy however its application in the clinical practice was limited by poor watersolubility hepatotoxicity and nephrotoxicity In this study a triptolide-loaded exosomes delivery system (TP-Exos) was constructedand its effects on the proliferation and apoptosis of SKOV3 cells in vitro and in vivo were observed SKOV3-exosomes (SK-Exos)were collected by ultracentrifugation and ultrafiltration centrifugation TP-Exos was constructed by sonication and ultrafiltrationcentrifugation SK-Exos and TP-Exos were characterized by transmission electron microscopy western blotting nanoparticle-tracking analysis and high-performance liquid chromatography Cellular uptake of exosomes 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay bromodeoxyuridine (BrdU) cell proliferation assay and cell apoptosis experiment wereused to study the effect of TP-Exos on ovarian cancer in vitro Tumor-targeting study of exosomes monitoring the tumor volumeof mice and TdT-mediated dUTP Nick-End labeling (TUNEL) assay were used to evaluate the effect of TP-Exos on ovarian cancerin vivo The toxicity of TP-Exos in vivo was evaluated by liver and kidney function and histopathology of major organs (heartliver spleen lung kidney and ovary) The results revealed that TP-Exos not only have the general characteristics of exosomes butalso have high drug encapsulation efficiency Besides PKH26 labeled exosomes (PKH26-Exos) could be uptaken by SKOV3 cellsand Dir labeled exosomes (Dir-Exos) could be enriched to the tumor site of tumor bearing mice Furthermore the cytotoxic andapoptotic effects on SKOV3 cells of TP-Exos were weaker than those of free TP and tumor cell proliferation inhibition and tumorgrowth inhibition were stronger than that of free TP Moreover TP-Exos have toxic effect on liver and spleen In conclusion theTP-Exos could be a promising strategy for ovarian cancer but they need to be further optimized to attenuate the damage to liverand spleen

1 Introduction

Triptolide (TP) is a major active ingredient of the Chineseherbal medicine Tripterygium wilfordii Hookf It has beenreported that TP has not only immunosuppressive and anti-inflammatory effects but also an antitumor activity whichcan inhibit the proliferation of various types of cancer cellsin vitro and reduce the growth and metastasis of somesolid tumors in vivo [1ndash6] It has been found that TP has agreat potential against ovarian cancer and could inhibit theproliferation of such cells and induce their apoptosis [7ndash10]

Unfortunately some studies have revealed that TP not onlyhas poor solubility but also could cause severe reproductive-system toxicity [11 12] hepatotoxicity [13ndash15] andnephrotox-icity [16 17] Generally the problemsmentioned above hinderthe potential clinical applications of TP to ovarian cancertreatment

In recent years controlled-release delivery systems andtargeted drug delivery systems have become some of themost promising innovations in the field of drug delivery forexample liposomes and polymersome micelles which areused to increase drug solubility improve drug bioavailability

HindawiBioMed Research InternationalVolume 2019 Article ID 2595801 14 pageshttpsdoiorg10115520192595801

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and to reduce toxicity (adverse effects) of drugs [18] Inour previous studies two types of TP delivery systemsTP-loaded micelles [19] and triptolide-loaded liposomes-CaP nanoparticles (TPLips-CaP) [20] have been con-structed The results revealed that they can enhance theantitumor effect and reduce the toxicity of TP Nonethe-less the TP-loaded micelles are material of xenobiotic ori-gin and are easily cleared by the mononuclear phagocytesystem (MPS which is mainly composed of monocytesand macrophages) Regarding TPLips-Cap although theuptake by MPS was reduced by polyethylene glycol (PEG)the interaction between target cells and barrier cells wasalso reduced Because of the deficiencies in the drug deliv-ery system of two types of nanoparticles we continuouslyseek innovative nanosized materials of biological originto further promote the use of TP in antindashovarian cancertherapy

Exosomes are natural nanosized vesicles (30 sim 150 nm)secreted by a variety of cells and also a promising drugdelivery carrier Exosomes as drug delivery carrier havemanyadvantages for example a small particle size biocompati-bility biological barrier penetration a phospholipid bilayersimilar to that of liposomes low toxicity and low immuno-genicity [21ndash23] At present exosomes derived from B cellsdendritic cells T cellsmacrophagesmesenchymal stem cellsand tumor cells are often employed as drug delivery carriers[24ndash28] Exosomes have already been tested for the deliveryof different antitumor agents such as curcumin [29 30]paclitaxel [31 32] and doxorubicin [25 33] These exosomescan deliver drugs to recipient cells and alter their functionAt present there are few studies on the treatment of ovariancancerwith drug-loaded exosomes In this study we preparedTP-Exos and investigated the effects of TP-Exos on theovarian cancer cell line SKOV3 in vitro and in vivo

2 Materials and Methods

21 Reagents Dulbeccorsquosmodified Eaglersquosmedium (DMEM)fetal bovine serum (FBS) and a penicillinstreptomycinsolutionwere purchased fromGibco (Invitrogen USA)TheBCA Protein Assay Kit Hematoxylin and Eosin Staining Kitand SDS-PAGE Preparation kit were purchased from Bey-otime (Shanghai China) TP was bought from the ChengduMust Bio-technology and FITC Annexin V Apoptosis Detec-tion Kit I In Situ Cell Death Detection Kit and BD Pharmin-gen BrdU Flow Kit from BD Bioscience (Shanghai China)Acetonitrile and 10 formalin neutral buffer solution werepurchased from Sangon Biotech (Shanghai) Co Ltd 111015840-dioctadecyl-333101584031015840-tetramethylindotricarbocyanine iodide(Dir) PKH26 Red Fluorescent Cell Linker Kit dimethylsulfoxide (DMSO) 46-diamidino-2-phenylindole (DAPI)trypsin phosphate buffered saline (PBS) and MTT fromSigma-Aldrich (Shanghai China) whereas a horseradishperoxidase- (HRP-) conjugated anti-rabbit IgG antibodyand anti-CD9 and anti-CD81 monoclonal antibodies wereacquired from Abcam (Cambridge UK) The female Balbcnude mice (4 sim 6 weeks old) were purchased from theShanghai Renji Hospital Animal Facility

22 Cell Culture Thehuman ovarian cancer cell lines SKOV3and A2780 were acquired from the National Laboratoryfor Ontogenesis and Related Genes Cancer Institute ofShanghai Jiao Tong University and were cultured in DMEMwhich was supplemented with 10 of FBS and 1 of thepenicillinstreptomycin solution All cells were maintained at5 CO

2and 37∘C

23 Isolation of Exosomes Isolation of exosomes was per-formed according to the literature [34] with some modi-fications To avoid contamination by the FBS-derived exo-somes FBS was centrifuged at 110000 times g for 6 h toremove exosomes before the experimentThe human ovariancancer SKOV3 cells were cultured in DMEM which wassupplemented with 10 of exosome-free FBS and 1 ofthe penicillinstreptomycin solutionThe culture supernatantfrom SKOV3 cells grown in a T75 flask was harvested at48 h and exosomes were isolated by ultracentrifugation andultrafiltration centrifugation Briefly the culture supernatantswere cleared of cell debris and large vesicles by sequentialcentrifugation at 300 times g for 10 min 1000 times g for 20 min and10000 times g for 30 min followed by filtration using 022 120583msyringe filters Then the sample was centrifuged at 110000 timesg for 6 h to obtain an exosomal pelletThe collected exosomeswere washed twice with PBS by means of an Aicon Ultra-15Centrifugal Filter (10 kDa Millipore) then passed through a022 120583m syringe filter for sterility and stored at ndash80∘C untiluse for studies All procedures were carried out at 4∘C

24 Loading of Exosomes Preparation of TP-Exos was con-ducted according to the literature [26] with some modifi-cations Namely 3 mg (protein concentration) of purifiedexosomes dispersed in 900 120583l PBS (pH 72 - 74) was mixedwith 100 120583g TP dissolved in 100 120583l DMSO and then themixture was sonicated by an ultrasonic cell crusher (JY92-II China) (20 power 15 cycles of a 2 s pulse2 s pause)After sonication the mixture was incubated at 37∘C for 60min to allow for recovery of the exosomal membrane Excessfree TP and DMSO were separated from the mixture bycentrifugation at 12000 times g for 30 min using the AmiconUltra-05 Centrifugal Filter (10 kDa Millipore) and thenwashed twice with precooled PBS Finally the pellet wassuspended in PBS passed through a 022 120583m syringe filter forsterility and stored at ndash80∘C until use for studies

25 Characterization of Exosomes The morphology of SK-Exos and TP-Exos was examined by transmission electronmicroscopy (TEM) Firstly the 200 mesh formvar coppergrids were exposed to theGlowDischarge technique for 1minand purified exosomes were fixed with 2 Paraformaldehydefor 5 min And then 5 120583l of exosomes suspension solutionwas added onto the grids and incubated for 1 min at 4∘CSubsequently the grids were negatively stained with 1uranyl acetate for 2 min washed with distilled water anddried out in darkness for 10 min under room temperatureFinally the samples were analyzed by TEM (Tecnai G2 SpiritTWIN) at 80 Kv

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Nanoparticle-tracking analysis (NTA) was performedto analyze the size distribution of SK-Exos and TP-Exosaccording to the literature [35 36] Firstly the exosomessamples were diluted in PBS (110 dilution) And then three30 s videos were recorded Finally particle size was analyzedby NTA software (version 32 NanoSight)

Western blotting (WB) was carried out to detect theexpression of tetraspanins such as CD9 and CD81 on thesurface of exosomes Proteinwas extracted fromSKOV3 cellsSK-Exos and TP-Exos using a cell lysis buffer and the totalprotein concentrations of three samples were determinedusing the BCA Protein Assay Kit according to manufacturerrsquosrecommendations Then 5 times loading Buffer was added tothe protein samples After boiling 10 min 20 120583g of proteinswas electrophoresed and transferred to a polyvinylidenedifluoride membrane Furthermore the membranes wereblocked with 5 (wv) skim milk powder and then treatedwith a primary CD9 and CD81 antibody (11000 dilution)Finally the membranes were incubated with a secondaryantibody (11000 dilution) and the protein bands were visu-alized by Western Lightening Chemiluminescence reagents(Millipore)

The concentration of TP in the TP-Exos was determinedby high-performance liquid chromatography (HPLC) (Agi-lent Technologies 1200 series)TheHPLC profiles of TP stan-dard solution SK-Exos solution and TP-Exos solution weredetected by HPLC to investigate the interference of SK-Exossolution on the determination of TP A series of TP solutionswere prepared and 20 120583l samples solution was injected forchromatographic analysis The standard curve was drawnwith the concentration of TP as the transverse coordinateand the average peak area as the longitudinal coordinate50 120583l of TP-Exos solution was added to 1950 120583l acetonitrileto extract the drug and precipitate the exosomal proteinsThe precipitated proteins were separated by centrifugation(12000 timesg for 10 min) supernatant was separated and 20 120583lsupernatant samples were analyzed on HPLC system using areversed-phase column (Zorbax SB-C18 150times46 mm 5120583m)Themobile phase consisted of a mixture of acetonitrile-water(4060 vv) and the flow rate was 10 mlmin The columneffluent was detected at 218 nm and the column temperaturewas 30∘C Entrapment efficiency () was calculated via theformula (Drug entrappedTotal amount of drug) times 100

26 Exosome Labeling Purified exosomes were incubatedwith 4 120583l PKH26 for 15 min at 37∘C Unbound dye wasremoved by centrifugation at 12000 times g for 30 min bymeans of the Amicon Ultra-05 Centrifugal Filter (10 kDaMillipore) and then the pellet was washed twice with pre-cooled PBS The obtained PKH26-Exos were resuspended inprecooled PBS and stored at minus80∘C protected from light priorto use

27 Cellular Uptake of Exosomes SKOV3 and A2780 cellswere incubated with PKH26-Exos (20 120583gml protein con-centration) at 37∘C and 5 CO

2for various periods After

each time point the medium was removed and the cellswere washed 3 times with PBS and then stained with 1 mlDAPI (25 120583gml) for 5min Subsequently the cells were fixed

with 1 ml 4 paraformaldehyde for 10 min Internalizationof PKH26-Exos by SKOV3 and A2780 cells was observedby fluorescence microscopy (Olympus IX51 Japan) Thefluorescence intensity of PKH26 was measured with ImageJ Software

28 A Cytotoxicity Assay Cytotoxicity of free TP and TP-Exos was measured by an MTT assay SKOV3 cells wereseeded onto 96-well plates at a density of 5 times103 cells perwell 24 h prior to drug treatment Various concentrationsof free TP or TP-Exos were added to cells for 24 or 48 hincubation Subsequently free TP- and TP-Exo-containingmedia were removed and the cells were incubated at 37∘C for4 h with anMTT solution (05mgml) FinallyMTT solutionwere removed and 150 120583l DMSO solution per well was addedto dissolve formazan crystals Formazan concentration wasdetermined bymeasuring absorbance at 490 nmon an ELISAmicroplate reader (Bio TEK ELX800 USA) and the numberof living cells was calculated from formazan optical density at490 nm (OD

490) values

29 A Proliferation Assay The influence of SK-Exos freeTP and TP-Exos on the proliferation of SKOV3 cells wasevaluated by BrdUDNA double-parameter flow cytometryAccording to the results of MTT assay the concentrationsof TP in free TP and TP-Exos groups was 10 ngml Theprotein concentration in SK-Exos group was the same asthe SK-Exos protein concentration in 10 ngml TP-Exosgroup The protein concentrations of SK-Exos in 10 ngmlTP-Exos was determined using the BCA Protein Assay Kitaccording to manufacturerrsquos recommendations Free TP-TP-Exondash and SK-Exondash containing media were added toSKOV3 cells for 24 or 48 h incubation After treatment cellswere collected and processed according to the respectivemanufacturersrsquo instructions for the BD Pharmingen BrdUFlow Kit The samples were analyzed on a BD flow cytometer(BD FACSCelesta USA) The data were analysed withFlowJo 76 SoftwareThe cell proliferation index (PI) was usedto indicate the proliferation state of the cell population PI =(S + G2M)(G0G1 + S + G2M) times 100 where the lettersdenote a percentage of cells in a respective phase of the cellcycle

210 An Apoptosis Assay The effect of SK-Exos free TPand TP-Exos on the apoptosis of SKOV3 cells was assessedby double-staining flow cytometry with propidium iodide(PI) and an annexin Vndashfluorescein isothiocyanate (FITC)conjugate The concentration of SK-Exos free TP and TP-Exos was the same as Proliferation Assay Free TP- TP-Exondashand SK-Exondashcontainingmediawere added to SKOV3 cells for24 or 48 h incubation After that the cells were processedaccording to the respective manufacturersrsquo instructionsfor the FITC Annexin V Apoptosis Detection Kit IThesamples were analyzed on the BD Flow cytometer (BDFACSCelesta USA)The data were analysed with FlowJo 76Software

211 Tumor Targeting of Exosomes In Vivo Preparation ofDir-Exos was conducted according to the literature [24] with

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some modifications Purified exosomes were incubated with25 120583gml fluorescent lipophilic tracer Dir for 15 min at 37∘CUnbound dye was removed by centrifugation at 12000times g for30 min by means of the Amicon Ultra-05 Centrifugal Filter(10 kDaMillipore) and then the pellet waswashed twicewithprecooled PBS The obtained Dir-Exos were resuspendedin precooled PBS and the particle concentration of it wasmeasured by NTA Dir-Exos were diluted in PBS to achieve aparticle concentration of 75 times 1010 per ml

To ensure the number of tumor-bearing mice 10 femaleBalbc nude mice were subcutaneously injected with SKOV3cells in 100120583l of PBS (2times106 cells) in the right flankThe tumorsize was measured by a Vernier caliper The tumor volumewas calculated as V = (d2times D)2 where d and D representthe shortest diameter of the tumor and the longest diameterperpendicular to the short diameter in mm respectivelyWhen the tumor volume reached approximately 500 mm3 6tumor-bearing mice were randomly divided into two groups(n = 3) Free-Dir group and Dir-Exos group And then200 120583l Free-Dir (25120583gml) and 200 120583l Dir-Exos (75 times 1010per ml) were administered via peritoneal injection 4 824 h and 48 h after injection tumor-bearing mice wereanesthetizedwith isoflurance and then the distribution ofDirwas detected by an in vivo imaging system (Night OWL IILB983) After 48 h of treatment tumor-bearing mice weresacrificed and the main organs such as heart liver spleenlung kidney and tumors were excised for fluorescenceimagingThe data were analysed with the Night OWL IndigoSoftware

212 Antitumor Efficacy of TP-Exos in Tumor-XenografedNudeMice To ensure the number of tumor-bearingmice 40female Balbc nude mice were subcutaneously injected withSKOV3 cells in 100 120583l of PBS (2times106 cells) in the right flankWhen the tumor volume reached approximately 100mm3 24tumor-bearing mice were randomly divided into four groups(n = 6) control group SK-Exos group free TP group and TP-Exos group The control group was treated with 200 120583l PBSThe concentrations of TP in free TP andTP-Exos groupswere02 mgkg The protein concentration in SK-Exos group wasthe same as the SK-Exos protein concentration in TP-Exosgroup

PBS solution SK-Exos solution free TP solution andTP-Exos solution were injected into the tumor-bearing micevia peritoneal injection The mice were treated twice a weekfor 4 weeks and the tumor size was measured once a weekfor 4 weeks by a Vernier Caliper The tumor volume wascalculated as V = (d2times D)2 where d and D represent theshortest diameter of the tumor and the longest diameterperpendicular to the short diameter in mm respectivelyAfter treatment blood was collected by submandibular veinplexus and then mice were sacrificed Subsequently thetumors and the vital organs (such as heart liver spleenlung kidney and ovary) of mice were resected and adigital camera was used for the imaging of tumor Thetumors and the vital organs were fixed in 10 formalinneutral buffer solution for TUNEL assay and histologicalanalysis

213 TUNEL Assay of Tumor Tissue The tumor tissue wasembedded in paraffin and sliced into 5 microns Apoptoticcells in tumor tissue were determined by the TUNELmethodusing In Situ Cell Death Detection Kit according to themanufacturerrsquos instructions Apoptotic cells were observedand photographed under fluorescence microscope Greenfluorescence was positive cell and blue fluorescence wasnucleus TUNEL-positive cells and total cells in paraffin sec-tions of tumor tissue were measured with Image J Software

214 Systemic Toxicity Evaluation of TP-Exos Blood wascentrifuged at 1000 times g for 10 min at 4∘C and then serumwas collected and stored at - 20∘C until analysis The levels ofblood urea nitrogen (BUN) creatinine (Cr) aspirate amino-transferase (AST) and alanine aminotransferase (ALT) weremeasured in serum samples

The vital organs (such as heart liver spleen lungkidney and ovary) were embedded in paraffin and slicedinto 5 microns The paraffin sections were stained withHematoxylin and Eosin Staining Kit according to the man-ufacturerrsquos instructions and examined with a fluorescentmicroscope

215 Statistical Analysis For all the experiments data arepresented as the mean plusmn SD (standard deviation) Testsfor significance of differences between the groups wereperformed by the t test (two groups) or one-way analysisof variance (one-way ANOVA)(multiple comparisons) inGraphPad Prism 70 (GraphPad Software USA) Data withP lt 005 were considered significantly different

3 Results and Discussion

31 Characterization of SK-Exos and TP-Exos TEM showedthat SK-Exos had a typical cup-shaped structure and TP-Exoshad an ellipse shape (Figures 1(a) and 1(b)) NTA indicatedthat the sizes of SK-Exos and TP-Exos were 1296 plusmn 19 nmand 1599 plusmn 27 nm respectively (Figure 1(c)) WB suggestedthat SK-Exos and TP-Exos were rich in tetraspanins such asCD9 and CD81 (Figure 1(d)) The encapsulation efficiencyof TP in TP-Exos was analyzed by HPLC The resultsdisplayed that the HPLC profiles for TP extracted from TP-Exos and TP reference showed no distinction indicatingSK-Exos solution did not interfere with the determinationof TP (Supplementary Figure 21) The linear regressionequation of TP standard curve was Y = 46225 X + 32738r = 09964 (Supplementary Figure 22) indicating that theconcentration of TP in the range of 025 sim 50 120583gml hada good linear relationship with its peak area integral valueThe encapsulation efficiency of TP in TP-Exos was 765 plusmn18 (Supplementary Table 1) The results of TEM WB andNTA showed that SK-Exos had the typical characteristicsof exosomes which indicated that the extraction method ofexosomes was feasible Characterization results of TP-Exosshowed that TP-Exos not only had the general characteristicsof exosomes but also had high entrapment efficiency of TPindicating that TP could be loaded into SK-Exos effectivelyby sonication

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SK-Exos(a)

TP-Exos(b)

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ml)

(c)

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CD9

CD81

TP-Exos

(d)

Figure 1 Characterization of SK-Exos and TP-Exos (a b) TEM images of SK-Exos and TP-Exos Scale bar = 100 nm (c) Size distribution ofSK-Exos and TP-Exos measured by NTA (d) Western blot analysis of SK-Exos and TP-Exos protein expression

Several methods such as incubation at room temper-ature electroporation surfactant permeabilization sonica-tion hypotonic dialysis freezendashthaw cycles extrusion andcell-mediated packaging can be utilized to encapsulate adrug in exosomes [23] Studies have shown that exosomes canincrease their drug-loading capacity after sonication MyungSoo Kim et al [26] tried different methods to prepare drug-loaded exosomes and the amount of paclitaxel loaded intoexosomes increases in the following order incubation atroom temperature lt electroporation lt sonication MatthewJ Haney et al [37] tested a variety of methods for loadingcatalase into exosomes and the amount of catalase loadedinto exosomes increased in the following order incubation atRT lt freezethaw cycles lt sonication asymp extrusion Accordingto the above observations TP was loaded into exosomes bysonication in this study

After TPwas loaded into SK-Exos by sonication TP-Exoswere collected by ultracentrifugation and ultrafiltration cen-trifugation respectivelyThen we compared the difference ofTP-Exos obtained by two methods by NTA (SupplementaryFigure 1(a)) The results showed that the D 10 D 50 and D90 of TP-Exos collected by two methods had no significantdifference indicating that the two methods have no signif-icant effect on the particle size of TP-Exos (SupplementaryFigure 1(b)) D 90 is the corresponding particle size whenthe cumulative particle size distribution of a sample is upto 90 Its physical meaning was that the size of particlessmaller than 220 nmaccounted for 90of the total number ofparticles in this study The particle concentration of TP-Exosin ultracentrifugation group andultrafiltration centrifugationgroupwas significantly lower than that in SK-Exos group (Plt0001) and that in ultracentrifugation group was significantly

6 BioMed Research International

lower than that in ultrafiltration centrifugation group (P lt0001) (Supplementary Figure 1(c))The results indicated thatthere was a loss of exosomes in the process of collectingTP-Exos by the two methods but the amount of exosomeslost by ultrafiltration centrifugation was lower Thereforeultrafiltration centrifugation was used to collect TP-Exos inthis study

In Figure 1 the size of the sample measured by TEMwas not consistent with the size of the sample measured byNTA The reasons for this phenomenon are as follows TEMcan describe the diameter of exosomes and clearly displaythe structure of exosomes by direct measurement Howeverthe size distribution data obtained by TEM are often notrepresentative because of the limited scope observed at onetime In addition the samples of TEM often need to bepretreated by drying fixation and freezing which mayaffect the size of exosomes In contrast NTA can detectthe size of exosomes in solution which provides struc-tural and functional protection for exosomes NTA allowsthe exosomes to be measured closer to its original statewhich ensures the authenticity and validity of the measureddata

The encapsulation efficiency of TP in TP-Exos was com-pared with that of two TP delivery systems in our previousstudy TPmicelles and TPLips-CaP nanoparticles [19 20]The encapsulation efficiency of TP in TP-micelle TPLips-CaP nanoparticles and TP-Exos was 621 plusmn 33 7231plusmn 311 and 765 plusmn 18 respectively The encapsulationefficiency of TP in TP-Exos was similar to that of in TPLips-CaP nanoparticlesThis phenomenonmay be related to lipidbilayer structure in exosomes Exosomes have similar lipidbilayer structure with liposomes [38] The hydrophobic layercan load the hydrophobic substances while the inner aqueouscavity can load water-soluble substances Triptolide is solublein organic solvents such as ethanol methanol dimethylsul-foxide (DMSO) and acetonitrile but not in water Thereforethrough the hydrophobic affinity between the hydrophobicstructure of exosomes and the hydrophobic drug triptolidemay be self-assembled into the lipid bilayers In additionit has been shown that hydrophobic drug encapsulationefficiency of exosomes was at least two fold higher than forstandard liposomes [39]This may be the reason why the TP-Exos collected by ultrafiltration centrifugation still has a highincorporation efficiency of TP

32 Accumulation of SK-Exos in Cells The ability of exo-somes to deliver a drug into target cells is crucial forthe therapeutic efficiency of exosomal formulations Wetested whether tumor cells had ability to take up their ownexosomes in vitro The results displayed that both SKOV3and A2780 cells could uptake PKH26-Exos (Figures 2(a)and 2(c)) but accumulation of PKH26-Exos in SKOV3cells was approximately twice that in A2780 cells (Figures2(b) and 2(d)) The results suggested that SK-Exos couldbe taken up by SKOV3 cells which was similar to theresults of Heikki Saari et al [40] showing that SK-Exosmay be able to efficiently deliver a drug to their parentalcells

33 Cytotoxicity of TP-Exos The MTT assay was used toassess the cytotoxicity of different concentration of free TPand TP-Exos on SKOV3 cells after 24 h and 48 h Statisticalanalysis of the survival rate of cells was given in Figure 3 After24 h of treatment the survival rate of cells in TP-Exos groupwas significantly higher than that in TP group and the half-maximal inhibitory concentration (IC50) values of free TPandTP-Exos groupswere 2568 and 2703 ngml respectivelyHowever after 48 h the cell survival rate in TP-Exos groupwas slightly higher than that in TP group and the IC50values of free TP and TP-Exos groups were 106 and 1893ngmL respectively We can conclude that encapsulationinto exosomes may attenuate or delay the cytotoxicity of TPtoward SKOV3 cells in vitro which was similar to the resultsof Tang K et al [41]

34 13e Effect of TP-Exos on Proliferation of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the proliferation of parental cells [42] ThereforeBrdUDNAdouble-parameter flow cytometrywas conductedto detect antiproliferative action of TP-Exos toward SKOV3cellsThe results of flow cytometry were shown in Figure 4(a)We first analyzed the effect of SK-Exos free TP and TP-Exoson the cell cycle of SKOV3 cells based on Figure 4(a) and theresults were shown in Figures 4(b) 4(c) and 4(d) Then weanalyzed the effect of SK-Exos free TP and TP-Exos on thePI of SKOV3 cells according to Figures 4(b) 4(c) and 4(d)and the results were shown in Figure 4(e) At 24 h SK-Exoshad no effect on cell cycle but at 48 h SK-Exos blocked cellsin G0G1 phase At 24 h TP blocked cells in G0G1 phasebut at 48 h the number of cells in G0G1 phase S phase andG2M phase was lower than that in control group indicatingthat TP induced cell apoptosis At 24 h TP-Exos blocked cellsin S phase but at 48 h TP-Exos blocked cells in G0G1 phaseAfter 24 h the PI of SKOV3 cells treated with SK-Exos wasnot significantly different from that of the control group (pgt 005) The PI of the free TP group was lower than that ofthe control group (P lt 0001) however the TP-Exos groupyielded a greater result than that of the control group (P lt0001) After 48 h the PI of SK-Exos group free TP groupand TP-Exos group was lower than that of the control group(P lt 005) and the TP-Exos group had a lower PI than did thefree TP group (P lt 001)

In Figure 4(e) PI increased significantly in TP-Exosgroup at 24 h which was due to the following reasons Inthis study 10 ngml TP-Exos blocked cells in S phase at 24h and G0G1 phase at 48 h S phase arrest may be the reasonfor the increase of proliferation index in TP-Exos group at 24h The arrest of cell cycle by TP-Exos may be related to theconcentration of free TP Wang Y et al [43] found 125 nML(4505 ngml) TP could block the cells in S phase at 24 h Liu Let al [44] found 10 ngml TP could block cells in G0G1 phaseat 24 h The cell cycle results of the 10ngml TP-Exos treatedgroup were consistent with those of Wang et al at 24 h andwere consistent with those of Liu et al at 48 h These resultssuggested that TP may be released less from TP-Exos at 24h and almost the entire after 48 h Studies have shown thatdrug-loaded exosomes exhibited a time-dependent releaseand the cumulative release of drugs were over 50 after 24 h

BioMed Research International 7

DAPI PKH26-Exos Merge

24h

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(c)A2780

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Figure 2 (a c) Fluorescence microscopy images of exosomes labeled with PKH-26 after incubation with SKOV3 and A2780 cells for 4 8 12or 24 h Original magnification 200times (b d) Fluorescence intensity statistical analytical diagram of panels (a) and (c) Data are presented asmean plusmn SD (n = 3)

[45] and over 90 at 48 h [46] Furthermore exosomes maycarry more drugs into the cells so that the inhibitory effect ofTP-Exos on cell proliferation is superior to that of free TP at48 h

35 13e Influence of TP-Exos on Apoptosis of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the apoptosis of parental cells [47] ThereforePIAnnexin VndashFITC double staining flow cytometry wasconducted to detect the effect of TP-Exos on the apoptosisof SKOV3 cells The results were illustrated in Figure 5 After24 h of treatment the apoptosis rate of the SK-Exos groupwas not significantly different from that of the control group(P gt 005) The percentages of apoptotic cells in the free TPgroup and TP-Exos group were higher than this parameter

of the control group (P lt 005) After 48 h of treatment theapoptotic rates of the SK-Exos group free TP group and TP-Exos group were significantly higher relative to the controlgroup (Plt 005) At the sameTP concentration the apoptosisrate of the free TP group was higher than that of TP-Exosgroup after 24 and 48 h (P lt 005) The above experimentalresults suggest that TP-Exos can induce apoptosis of SKOV3cells but the effect is weaker in comparison with free TPwhich was consistent with the results of MTT assay

Both SK-Exos and free TP could induce apoptosis butTP-Exosndashinduced apoptosis is less extensive than that causedby free TP Encapsulation into exosomes may delay thecytotoxicity of TP and then weaken its ability to induceapoptosis in vitro However the delayed cytotoxicity of TP inTP-Exos may be related to the time required for TP-Exos to

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Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

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)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

BioMed Research International 3

Nanoparticle-tracking analysis (NTA) was performedto analyze the size distribution of SK-Exos and TP-Exosaccording to the literature [35 36] Firstly the exosomessamples were diluted in PBS (110 dilution) And then three30 s videos were recorded Finally particle size was analyzedby NTA software (version 32 NanoSight)

Western blotting (WB) was carried out to detect theexpression of tetraspanins such as CD9 and CD81 on thesurface of exosomes Proteinwas extracted fromSKOV3 cellsSK-Exos and TP-Exos using a cell lysis buffer and the totalprotein concentrations of three samples were determinedusing the BCA Protein Assay Kit according to manufacturerrsquosrecommendations Then 5 times loading Buffer was added tothe protein samples After boiling 10 min 20 120583g of proteinswas electrophoresed and transferred to a polyvinylidenedifluoride membrane Furthermore the membranes wereblocked with 5 (wv) skim milk powder and then treatedwith a primary CD9 and CD81 antibody (11000 dilution)Finally the membranes were incubated with a secondaryantibody (11000 dilution) and the protein bands were visu-alized by Western Lightening Chemiluminescence reagents(Millipore)

The concentration of TP in the TP-Exos was determinedby high-performance liquid chromatography (HPLC) (Agi-lent Technologies 1200 series)TheHPLC profiles of TP stan-dard solution SK-Exos solution and TP-Exos solution weredetected by HPLC to investigate the interference of SK-Exossolution on the determination of TP A series of TP solutionswere prepared and 20 120583l samples solution was injected forchromatographic analysis The standard curve was drawnwith the concentration of TP as the transverse coordinateand the average peak area as the longitudinal coordinate50 120583l of TP-Exos solution was added to 1950 120583l acetonitrileto extract the drug and precipitate the exosomal proteinsThe precipitated proteins were separated by centrifugation(12000 timesg for 10 min) supernatant was separated and 20 120583lsupernatant samples were analyzed on HPLC system using areversed-phase column (Zorbax SB-C18 150times46 mm 5120583m)Themobile phase consisted of a mixture of acetonitrile-water(4060 vv) and the flow rate was 10 mlmin The columneffluent was detected at 218 nm and the column temperaturewas 30∘C Entrapment efficiency () was calculated via theformula (Drug entrappedTotal amount of drug) times 100

26 Exosome Labeling Purified exosomes were incubatedwith 4 120583l PKH26 for 15 min at 37∘C Unbound dye wasremoved by centrifugation at 12000 times g for 30 min bymeans of the Amicon Ultra-05 Centrifugal Filter (10 kDaMillipore) and then the pellet was washed twice with pre-cooled PBS The obtained PKH26-Exos were resuspended inprecooled PBS and stored at minus80∘C protected from light priorto use

27 Cellular Uptake of Exosomes SKOV3 and A2780 cellswere incubated with PKH26-Exos (20 120583gml protein con-centration) at 37∘C and 5 CO

2for various periods After

each time point the medium was removed and the cellswere washed 3 times with PBS and then stained with 1 mlDAPI (25 120583gml) for 5min Subsequently the cells were fixed

with 1 ml 4 paraformaldehyde for 10 min Internalizationof PKH26-Exos by SKOV3 and A2780 cells was observedby fluorescence microscopy (Olympus IX51 Japan) Thefluorescence intensity of PKH26 was measured with ImageJ Software

28 A Cytotoxicity Assay Cytotoxicity of free TP and TP-Exos was measured by an MTT assay SKOV3 cells wereseeded onto 96-well plates at a density of 5 times103 cells perwell 24 h prior to drug treatment Various concentrationsof free TP or TP-Exos were added to cells for 24 or 48 hincubation Subsequently free TP- and TP-Exo-containingmedia were removed and the cells were incubated at 37∘C for4 h with anMTT solution (05mgml) FinallyMTT solutionwere removed and 150 120583l DMSO solution per well was addedto dissolve formazan crystals Formazan concentration wasdetermined bymeasuring absorbance at 490 nmon an ELISAmicroplate reader (Bio TEK ELX800 USA) and the numberof living cells was calculated from formazan optical density at490 nm (OD

490) values

29 A Proliferation Assay The influence of SK-Exos freeTP and TP-Exos on the proliferation of SKOV3 cells wasevaluated by BrdUDNA double-parameter flow cytometryAccording to the results of MTT assay the concentrationsof TP in free TP and TP-Exos groups was 10 ngml Theprotein concentration in SK-Exos group was the same asthe SK-Exos protein concentration in 10 ngml TP-Exosgroup The protein concentrations of SK-Exos in 10 ngmlTP-Exos was determined using the BCA Protein Assay Kitaccording to manufacturerrsquos recommendations Free TP-TP-Exondash and SK-Exondash containing media were added toSKOV3 cells for 24 or 48 h incubation After treatment cellswere collected and processed according to the respectivemanufacturersrsquo instructions for the BD Pharmingen BrdUFlow Kit The samples were analyzed on a BD flow cytometer(BD FACSCelesta USA) The data were analysed withFlowJo 76 SoftwareThe cell proliferation index (PI) was usedto indicate the proliferation state of the cell population PI =(S + G2M)(G0G1 + S + G2M) times 100 where the lettersdenote a percentage of cells in a respective phase of the cellcycle

210 An Apoptosis Assay The effect of SK-Exos free TPand TP-Exos on the apoptosis of SKOV3 cells was assessedby double-staining flow cytometry with propidium iodide(PI) and an annexin Vndashfluorescein isothiocyanate (FITC)conjugate The concentration of SK-Exos free TP and TP-Exos was the same as Proliferation Assay Free TP- TP-Exondashand SK-Exondashcontainingmediawere added to SKOV3 cells for24 or 48 h incubation After that the cells were processedaccording to the respective manufacturersrsquo instructionsfor the FITC Annexin V Apoptosis Detection Kit IThesamples were analyzed on the BD Flow cytometer (BDFACSCelesta USA)The data were analysed with FlowJo 76Software

211 Tumor Targeting of Exosomes In Vivo Preparation ofDir-Exos was conducted according to the literature [24] with

4 BioMed Research International

some modifications Purified exosomes were incubated with25 120583gml fluorescent lipophilic tracer Dir for 15 min at 37∘CUnbound dye was removed by centrifugation at 12000times g for30 min by means of the Amicon Ultra-05 Centrifugal Filter(10 kDaMillipore) and then the pellet waswashed twicewithprecooled PBS The obtained Dir-Exos were resuspendedin precooled PBS and the particle concentration of it wasmeasured by NTA Dir-Exos were diluted in PBS to achieve aparticle concentration of 75 times 1010 per ml

To ensure the number of tumor-bearing mice 10 femaleBalbc nude mice were subcutaneously injected with SKOV3cells in 100120583l of PBS (2times106 cells) in the right flankThe tumorsize was measured by a Vernier caliper The tumor volumewas calculated as V = (d2times D)2 where d and D representthe shortest diameter of the tumor and the longest diameterperpendicular to the short diameter in mm respectivelyWhen the tumor volume reached approximately 500 mm3 6tumor-bearing mice were randomly divided into two groups(n = 3) Free-Dir group and Dir-Exos group And then200 120583l Free-Dir (25120583gml) and 200 120583l Dir-Exos (75 times 1010per ml) were administered via peritoneal injection 4 824 h and 48 h after injection tumor-bearing mice wereanesthetizedwith isoflurance and then the distribution ofDirwas detected by an in vivo imaging system (Night OWL IILB983) After 48 h of treatment tumor-bearing mice weresacrificed and the main organs such as heart liver spleenlung kidney and tumors were excised for fluorescenceimagingThe data were analysed with the Night OWL IndigoSoftware

212 Antitumor Efficacy of TP-Exos in Tumor-XenografedNudeMice To ensure the number of tumor-bearingmice 40female Balbc nude mice were subcutaneously injected withSKOV3 cells in 100 120583l of PBS (2times106 cells) in the right flankWhen the tumor volume reached approximately 100mm3 24tumor-bearing mice were randomly divided into four groups(n = 6) control group SK-Exos group free TP group and TP-Exos group The control group was treated with 200 120583l PBSThe concentrations of TP in free TP andTP-Exos groupswere02 mgkg The protein concentration in SK-Exos group wasthe same as the SK-Exos protein concentration in TP-Exosgroup

PBS solution SK-Exos solution free TP solution andTP-Exos solution were injected into the tumor-bearing micevia peritoneal injection The mice were treated twice a weekfor 4 weeks and the tumor size was measured once a weekfor 4 weeks by a Vernier Caliper The tumor volume wascalculated as V = (d2times D)2 where d and D represent theshortest diameter of the tumor and the longest diameterperpendicular to the short diameter in mm respectivelyAfter treatment blood was collected by submandibular veinplexus and then mice were sacrificed Subsequently thetumors and the vital organs (such as heart liver spleenlung kidney and ovary) of mice were resected and adigital camera was used for the imaging of tumor Thetumors and the vital organs were fixed in 10 formalinneutral buffer solution for TUNEL assay and histologicalanalysis

213 TUNEL Assay of Tumor Tissue The tumor tissue wasembedded in paraffin and sliced into 5 microns Apoptoticcells in tumor tissue were determined by the TUNELmethodusing In Situ Cell Death Detection Kit according to themanufacturerrsquos instructions Apoptotic cells were observedand photographed under fluorescence microscope Greenfluorescence was positive cell and blue fluorescence wasnucleus TUNEL-positive cells and total cells in paraffin sec-tions of tumor tissue were measured with Image J Software

214 Systemic Toxicity Evaluation of TP-Exos Blood wascentrifuged at 1000 times g for 10 min at 4∘C and then serumwas collected and stored at - 20∘C until analysis The levels ofblood urea nitrogen (BUN) creatinine (Cr) aspirate amino-transferase (AST) and alanine aminotransferase (ALT) weremeasured in serum samples

The vital organs (such as heart liver spleen lungkidney and ovary) were embedded in paraffin and slicedinto 5 microns The paraffin sections were stained withHematoxylin and Eosin Staining Kit according to the man-ufacturerrsquos instructions and examined with a fluorescentmicroscope

215 Statistical Analysis For all the experiments data arepresented as the mean plusmn SD (standard deviation) Testsfor significance of differences between the groups wereperformed by the t test (two groups) or one-way analysisof variance (one-way ANOVA)(multiple comparisons) inGraphPad Prism 70 (GraphPad Software USA) Data withP lt 005 were considered significantly different

3 Results and Discussion

31 Characterization of SK-Exos and TP-Exos TEM showedthat SK-Exos had a typical cup-shaped structure and TP-Exoshad an ellipse shape (Figures 1(a) and 1(b)) NTA indicatedthat the sizes of SK-Exos and TP-Exos were 1296 plusmn 19 nmand 1599 plusmn 27 nm respectively (Figure 1(c)) WB suggestedthat SK-Exos and TP-Exos were rich in tetraspanins such asCD9 and CD81 (Figure 1(d)) The encapsulation efficiencyof TP in TP-Exos was analyzed by HPLC The resultsdisplayed that the HPLC profiles for TP extracted from TP-Exos and TP reference showed no distinction indicatingSK-Exos solution did not interfere with the determinationof TP (Supplementary Figure 21) The linear regressionequation of TP standard curve was Y = 46225 X + 32738r = 09964 (Supplementary Figure 22) indicating that theconcentration of TP in the range of 025 sim 50 120583gml hada good linear relationship with its peak area integral valueThe encapsulation efficiency of TP in TP-Exos was 765 plusmn18 (Supplementary Table 1) The results of TEM WB andNTA showed that SK-Exos had the typical characteristicsof exosomes which indicated that the extraction method ofexosomes was feasible Characterization results of TP-Exosshowed that TP-Exos not only had the general characteristicsof exosomes but also had high entrapment efficiency of TPindicating that TP could be loaded into SK-Exos effectivelyby sonication

BioMed Research International 5

SK-Exos(a)

TP-Exos(b)

125

145

TP-ExosSK-Exos

200 800600400 10000Size (nm)

0

2

4

6

8

10

Con

cent

ratio

n (times

109 pa

rtic

les

ml)

(c)

SKOV3 SK-Exos

25KDa

26KDa

CD9

CD81

TP-Exos

(d)

Figure 1 Characterization of SK-Exos and TP-Exos (a b) TEM images of SK-Exos and TP-Exos Scale bar = 100 nm (c) Size distribution ofSK-Exos and TP-Exos measured by NTA (d) Western blot analysis of SK-Exos and TP-Exos protein expression

Several methods such as incubation at room temper-ature electroporation surfactant permeabilization sonica-tion hypotonic dialysis freezendashthaw cycles extrusion andcell-mediated packaging can be utilized to encapsulate adrug in exosomes [23] Studies have shown that exosomes canincrease their drug-loading capacity after sonication MyungSoo Kim et al [26] tried different methods to prepare drug-loaded exosomes and the amount of paclitaxel loaded intoexosomes increases in the following order incubation atroom temperature lt electroporation lt sonication MatthewJ Haney et al [37] tested a variety of methods for loadingcatalase into exosomes and the amount of catalase loadedinto exosomes increased in the following order incubation atRT lt freezethaw cycles lt sonication asymp extrusion Accordingto the above observations TP was loaded into exosomes bysonication in this study

After TPwas loaded into SK-Exos by sonication TP-Exoswere collected by ultracentrifugation and ultrafiltration cen-trifugation respectivelyThen we compared the difference ofTP-Exos obtained by two methods by NTA (SupplementaryFigure 1(a)) The results showed that the D 10 D 50 and D90 of TP-Exos collected by two methods had no significantdifference indicating that the two methods have no signif-icant effect on the particle size of TP-Exos (SupplementaryFigure 1(b)) D 90 is the corresponding particle size whenthe cumulative particle size distribution of a sample is upto 90 Its physical meaning was that the size of particlessmaller than 220 nmaccounted for 90of the total number ofparticles in this study The particle concentration of TP-Exosin ultracentrifugation group andultrafiltration centrifugationgroupwas significantly lower than that in SK-Exos group (Plt0001) and that in ultracentrifugation group was significantly

6 BioMed Research International

lower than that in ultrafiltration centrifugation group (P lt0001) (Supplementary Figure 1(c))The results indicated thatthere was a loss of exosomes in the process of collectingTP-Exos by the two methods but the amount of exosomeslost by ultrafiltration centrifugation was lower Thereforeultrafiltration centrifugation was used to collect TP-Exos inthis study

In Figure 1 the size of the sample measured by TEMwas not consistent with the size of the sample measured byNTA The reasons for this phenomenon are as follows TEMcan describe the diameter of exosomes and clearly displaythe structure of exosomes by direct measurement Howeverthe size distribution data obtained by TEM are often notrepresentative because of the limited scope observed at onetime In addition the samples of TEM often need to bepretreated by drying fixation and freezing which mayaffect the size of exosomes In contrast NTA can detectthe size of exosomes in solution which provides struc-tural and functional protection for exosomes NTA allowsthe exosomes to be measured closer to its original statewhich ensures the authenticity and validity of the measureddata

The encapsulation efficiency of TP in TP-Exos was com-pared with that of two TP delivery systems in our previousstudy TPmicelles and TPLips-CaP nanoparticles [19 20]The encapsulation efficiency of TP in TP-micelle TPLips-CaP nanoparticles and TP-Exos was 621 plusmn 33 7231plusmn 311 and 765 plusmn 18 respectively The encapsulationefficiency of TP in TP-Exos was similar to that of in TPLips-CaP nanoparticlesThis phenomenonmay be related to lipidbilayer structure in exosomes Exosomes have similar lipidbilayer structure with liposomes [38] The hydrophobic layercan load the hydrophobic substances while the inner aqueouscavity can load water-soluble substances Triptolide is solublein organic solvents such as ethanol methanol dimethylsul-foxide (DMSO) and acetonitrile but not in water Thereforethrough the hydrophobic affinity between the hydrophobicstructure of exosomes and the hydrophobic drug triptolidemay be self-assembled into the lipid bilayers In additionit has been shown that hydrophobic drug encapsulationefficiency of exosomes was at least two fold higher than forstandard liposomes [39]This may be the reason why the TP-Exos collected by ultrafiltration centrifugation still has a highincorporation efficiency of TP

32 Accumulation of SK-Exos in Cells The ability of exo-somes to deliver a drug into target cells is crucial forthe therapeutic efficiency of exosomal formulations Wetested whether tumor cells had ability to take up their ownexosomes in vitro The results displayed that both SKOV3and A2780 cells could uptake PKH26-Exos (Figures 2(a)and 2(c)) but accumulation of PKH26-Exos in SKOV3cells was approximately twice that in A2780 cells (Figures2(b) and 2(d)) The results suggested that SK-Exos couldbe taken up by SKOV3 cells which was similar to theresults of Heikki Saari et al [40] showing that SK-Exosmay be able to efficiently deliver a drug to their parentalcells

33 Cytotoxicity of TP-Exos The MTT assay was used toassess the cytotoxicity of different concentration of free TPand TP-Exos on SKOV3 cells after 24 h and 48 h Statisticalanalysis of the survival rate of cells was given in Figure 3 After24 h of treatment the survival rate of cells in TP-Exos groupwas significantly higher than that in TP group and the half-maximal inhibitory concentration (IC50) values of free TPandTP-Exos groupswere 2568 and 2703 ngml respectivelyHowever after 48 h the cell survival rate in TP-Exos groupwas slightly higher than that in TP group and the IC50values of free TP and TP-Exos groups were 106 and 1893ngmL respectively We can conclude that encapsulationinto exosomes may attenuate or delay the cytotoxicity of TPtoward SKOV3 cells in vitro which was similar to the resultsof Tang K et al [41]

34 13e Effect of TP-Exos on Proliferation of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the proliferation of parental cells [42] ThereforeBrdUDNAdouble-parameter flow cytometrywas conductedto detect antiproliferative action of TP-Exos toward SKOV3cellsThe results of flow cytometry were shown in Figure 4(a)We first analyzed the effect of SK-Exos free TP and TP-Exoson the cell cycle of SKOV3 cells based on Figure 4(a) and theresults were shown in Figures 4(b) 4(c) and 4(d) Then weanalyzed the effect of SK-Exos free TP and TP-Exos on thePI of SKOV3 cells according to Figures 4(b) 4(c) and 4(d)and the results were shown in Figure 4(e) At 24 h SK-Exoshad no effect on cell cycle but at 48 h SK-Exos blocked cellsin G0G1 phase At 24 h TP blocked cells in G0G1 phasebut at 48 h the number of cells in G0G1 phase S phase andG2M phase was lower than that in control group indicatingthat TP induced cell apoptosis At 24 h TP-Exos blocked cellsin S phase but at 48 h TP-Exos blocked cells in G0G1 phaseAfter 24 h the PI of SKOV3 cells treated with SK-Exos wasnot significantly different from that of the control group (pgt 005) The PI of the free TP group was lower than that ofthe control group (P lt 0001) however the TP-Exos groupyielded a greater result than that of the control group (P lt0001) After 48 h the PI of SK-Exos group free TP groupand TP-Exos group was lower than that of the control group(P lt 005) and the TP-Exos group had a lower PI than did thefree TP group (P lt 001)

In Figure 4(e) PI increased significantly in TP-Exosgroup at 24 h which was due to the following reasons Inthis study 10 ngml TP-Exos blocked cells in S phase at 24h and G0G1 phase at 48 h S phase arrest may be the reasonfor the increase of proliferation index in TP-Exos group at 24h The arrest of cell cycle by TP-Exos may be related to theconcentration of free TP Wang Y et al [43] found 125 nML(4505 ngml) TP could block the cells in S phase at 24 h Liu Let al [44] found 10 ngml TP could block cells in G0G1 phaseat 24 h The cell cycle results of the 10ngml TP-Exos treatedgroup were consistent with those of Wang et al at 24 h andwere consistent with those of Liu et al at 48 h These resultssuggested that TP may be released less from TP-Exos at 24h and almost the entire after 48 h Studies have shown thatdrug-loaded exosomes exhibited a time-dependent releaseand the cumulative release of drugs were over 50 after 24 h

BioMed Research International 7

DAPI PKH26-Exos Merge

24h

12h

8h

4h

(a)SKOV3

0

20

40

60

80

100

MFI

(AU

)

241284Time (h)

(b)

DAPI

24h

12h

8h

4h

PKH26-Exos Merge

(c)A2780

0

20

40

60

80

100M

FI (A

U)

241284Time (h)

(d)

Figure 2 (a c) Fluorescence microscopy images of exosomes labeled with PKH-26 after incubation with SKOV3 and A2780 cells for 4 8 12or 24 h Original magnification 200times (b d) Fluorescence intensity statistical analytical diagram of panels (a) and (c) Data are presented asmean plusmn SD (n = 3)

[45] and over 90 at 48 h [46] Furthermore exosomes maycarry more drugs into the cells so that the inhibitory effect ofTP-Exos on cell proliferation is superior to that of free TP at48 h

35 13e Influence of TP-Exos on Apoptosis of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the apoptosis of parental cells [47] ThereforePIAnnexin VndashFITC double staining flow cytometry wasconducted to detect the effect of TP-Exos on the apoptosisof SKOV3 cells The results were illustrated in Figure 5 After24 h of treatment the apoptosis rate of the SK-Exos groupwas not significantly different from that of the control group(P gt 005) The percentages of apoptotic cells in the free TPgroup and TP-Exos group were higher than this parameter

of the control group (P lt 005) After 48 h of treatment theapoptotic rates of the SK-Exos group free TP group and TP-Exos group were significantly higher relative to the controlgroup (Plt 005) At the sameTP concentration the apoptosisrate of the free TP group was higher than that of TP-Exosgroup after 24 and 48 h (P lt 005) The above experimentalresults suggest that TP-Exos can induce apoptosis of SKOV3cells but the effect is weaker in comparison with free TPwhich was consistent with the results of MTT assay

Both SK-Exos and free TP could induce apoptosis butTP-Exosndashinduced apoptosis is less extensive than that causedby free TP Encapsulation into exosomes may delay thecytotoxicity of TP and then weaken its ability to induceapoptosis in vitro However the delayed cytotoxicity of TP inTP-Exos may be related to the time required for TP-Exos to

8 BioMed Research International

Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

20

40

60

80

100

Cel

l via

bilit

y (

)

TP-ExosTP

TP-ExosTP

24h

25 5 10 1005025 5001 250Concentration (ngml)

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

4 BioMed Research International

some modifications Purified exosomes were incubated with25 120583gml fluorescent lipophilic tracer Dir for 15 min at 37∘CUnbound dye was removed by centrifugation at 12000times g for30 min by means of the Amicon Ultra-05 Centrifugal Filter(10 kDaMillipore) and then the pellet waswashed twicewithprecooled PBS The obtained Dir-Exos were resuspendedin precooled PBS and the particle concentration of it wasmeasured by NTA Dir-Exos were diluted in PBS to achieve aparticle concentration of 75 times 1010 per ml

To ensure the number of tumor-bearing mice 10 femaleBalbc nude mice were subcutaneously injected with SKOV3cells in 100120583l of PBS (2times106 cells) in the right flankThe tumorsize was measured by a Vernier caliper The tumor volumewas calculated as V = (d2times D)2 where d and D representthe shortest diameter of the tumor and the longest diameterperpendicular to the short diameter in mm respectivelyWhen the tumor volume reached approximately 500 mm3 6tumor-bearing mice were randomly divided into two groups(n = 3) Free-Dir group and Dir-Exos group And then200 120583l Free-Dir (25120583gml) and 200 120583l Dir-Exos (75 times 1010per ml) were administered via peritoneal injection 4 824 h and 48 h after injection tumor-bearing mice wereanesthetizedwith isoflurance and then the distribution ofDirwas detected by an in vivo imaging system (Night OWL IILB983) After 48 h of treatment tumor-bearing mice weresacrificed and the main organs such as heart liver spleenlung kidney and tumors were excised for fluorescenceimagingThe data were analysed with the Night OWL IndigoSoftware

212 Antitumor Efficacy of TP-Exos in Tumor-XenografedNudeMice To ensure the number of tumor-bearingmice 40female Balbc nude mice were subcutaneously injected withSKOV3 cells in 100 120583l of PBS (2times106 cells) in the right flankWhen the tumor volume reached approximately 100mm3 24tumor-bearing mice were randomly divided into four groups(n = 6) control group SK-Exos group free TP group and TP-Exos group The control group was treated with 200 120583l PBSThe concentrations of TP in free TP andTP-Exos groupswere02 mgkg The protein concentration in SK-Exos group wasthe same as the SK-Exos protein concentration in TP-Exosgroup

PBS solution SK-Exos solution free TP solution andTP-Exos solution were injected into the tumor-bearing micevia peritoneal injection The mice were treated twice a weekfor 4 weeks and the tumor size was measured once a weekfor 4 weeks by a Vernier Caliper The tumor volume wascalculated as V = (d2times D)2 where d and D represent theshortest diameter of the tumor and the longest diameterperpendicular to the short diameter in mm respectivelyAfter treatment blood was collected by submandibular veinplexus and then mice were sacrificed Subsequently thetumors and the vital organs (such as heart liver spleenlung kidney and ovary) of mice were resected and adigital camera was used for the imaging of tumor Thetumors and the vital organs were fixed in 10 formalinneutral buffer solution for TUNEL assay and histologicalanalysis

213 TUNEL Assay of Tumor Tissue The tumor tissue wasembedded in paraffin and sliced into 5 microns Apoptoticcells in tumor tissue were determined by the TUNELmethodusing In Situ Cell Death Detection Kit according to themanufacturerrsquos instructions Apoptotic cells were observedand photographed under fluorescence microscope Greenfluorescence was positive cell and blue fluorescence wasnucleus TUNEL-positive cells and total cells in paraffin sec-tions of tumor tissue were measured with Image J Software

214 Systemic Toxicity Evaluation of TP-Exos Blood wascentrifuged at 1000 times g for 10 min at 4∘C and then serumwas collected and stored at - 20∘C until analysis The levels ofblood urea nitrogen (BUN) creatinine (Cr) aspirate amino-transferase (AST) and alanine aminotransferase (ALT) weremeasured in serum samples

The vital organs (such as heart liver spleen lungkidney and ovary) were embedded in paraffin and slicedinto 5 microns The paraffin sections were stained withHematoxylin and Eosin Staining Kit according to the man-ufacturerrsquos instructions and examined with a fluorescentmicroscope

215 Statistical Analysis For all the experiments data arepresented as the mean plusmn SD (standard deviation) Testsfor significance of differences between the groups wereperformed by the t test (two groups) or one-way analysisof variance (one-way ANOVA)(multiple comparisons) inGraphPad Prism 70 (GraphPad Software USA) Data withP lt 005 were considered significantly different

3 Results and Discussion

31 Characterization of SK-Exos and TP-Exos TEM showedthat SK-Exos had a typical cup-shaped structure and TP-Exoshad an ellipse shape (Figures 1(a) and 1(b)) NTA indicatedthat the sizes of SK-Exos and TP-Exos were 1296 plusmn 19 nmand 1599 plusmn 27 nm respectively (Figure 1(c)) WB suggestedthat SK-Exos and TP-Exos were rich in tetraspanins such asCD9 and CD81 (Figure 1(d)) The encapsulation efficiencyof TP in TP-Exos was analyzed by HPLC The resultsdisplayed that the HPLC profiles for TP extracted from TP-Exos and TP reference showed no distinction indicatingSK-Exos solution did not interfere with the determinationof TP (Supplementary Figure 21) The linear regressionequation of TP standard curve was Y = 46225 X + 32738r = 09964 (Supplementary Figure 22) indicating that theconcentration of TP in the range of 025 sim 50 120583gml hada good linear relationship with its peak area integral valueThe encapsulation efficiency of TP in TP-Exos was 765 plusmn18 (Supplementary Table 1) The results of TEM WB andNTA showed that SK-Exos had the typical characteristicsof exosomes which indicated that the extraction method ofexosomes was feasible Characterization results of TP-Exosshowed that TP-Exos not only had the general characteristicsof exosomes but also had high entrapment efficiency of TPindicating that TP could be loaded into SK-Exos effectivelyby sonication

BioMed Research International 5

SK-Exos(a)

TP-Exos(b)

125

145

TP-ExosSK-Exos

200 800600400 10000Size (nm)

0

2

4

6

8

10

Con

cent

ratio

n (times

109 pa

rtic

les

ml)

(c)

SKOV3 SK-Exos

25KDa

26KDa

CD9

CD81

TP-Exos

(d)

Figure 1 Characterization of SK-Exos and TP-Exos (a b) TEM images of SK-Exos and TP-Exos Scale bar = 100 nm (c) Size distribution ofSK-Exos and TP-Exos measured by NTA (d) Western blot analysis of SK-Exos and TP-Exos protein expression

Several methods such as incubation at room temper-ature electroporation surfactant permeabilization sonica-tion hypotonic dialysis freezendashthaw cycles extrusion andcell-mediated packaging can be utilized to encapsulate adrug in exosomes [23] Studies have shown that exosomes canincrease their drug-loading capacity after sonication MyungSoo Kim et al [26] tried different methods to prepare drug-loaded exosomes and the amount of paclitaxel loaded intoexosomes increases in the following order incubation atroom temperature lt electroporation lt sonication MatthewJ Haney et al [37] tested a variety of methods for loadingcatalase into exosomes and the amount of catalase loadedinto exosomes increased in the following order incubation atRT lt freezethaw cycles lt sonication asymp extrusion Accordingto the above observations TP was loaded into exosomes bysonication in this study

After TPwas loaded into SK-Exos by sonication TP-Exoswere collected by ultracentrifugation and ultrafiltration cen-trifugation respectivelyThen we compared the difference ofTP-Exos obtained by two methods by NTA (SupplementaryFigure 1(a)) The results showed that the D 10 D 50 and D90 of TP-Exos collected by two methods had no significantdifference indicating that the two methods have no signif-icant effect on the particle size of TP-Exos (SupplementaryFigure 1(b)) D 90 is the corresponding particle size whenthe cumulative particle size distribution of a sample is upto 90 Its physical meaning was that the size of particlessmaller than 220 nmaccounted for 90of the total number ofparticles in this study The particle concentration of TP-Exosin ultracentrifugation group andultrafiltration centrifugationgroupwas significantly lower than that in SK-Exos group (Plt0001) and that in ultracentrifugation group was significantly

6 BioMed Research International

lower than that in ultrafiltration centrifugation group (P lt0001) (Supplementary Figure 1(c))The results indicated thatthere was a loss of exosomes in the process of collectingTP-Exos by the two methods but the amount of exosomeslost by ultrafiltration centrifugation was lower Thereforeultrafiltration centrifugation was used to collect TP-Exos inthis study

In Figure 1 the size of the sample measured by TEMwas not consistent with the size of the sample measured byNTA The reasons for this phenomenon are as follows TEMcan describe the diameter of exosomes and clearly displaythe structure of exosomes by direct measurement Howeverthe size distribution data obtained by TEM are often notrepresentative because of the limited scope observed at onetime In addition the samples of TEM often need to bepretreated by drying fixation and freezing which mayaffect the size of exosomes In contrast NTA can detectthe size of exosomes in solution which provides struc-tural and functional protection for exosomes NTA allowsthe exosomes to be measured closer to its original statewhich ensures the authenticity and validity of the measureddata

The encapsulation efficiency of TP in TP-Exos was com-pared with that of two TP delivery systems in our previousstudy TPmicelles and TPLips-CaP nanoparticles [19 20]The encapsulation efficiency of TP in TP-micelle TPLips-CaP nanoparticles and TP-Exos was 621 plusmn 33 7231plusmn 311 and 765 plusmn 18 respectively The encapsulationefficiency of TP in TP-Exos was similar to that of in TPLips-CaP nanoparticlesThis phenomenonmay be related to lipidbilayer structure in exosomes Exosomes have similar lipidbilayer structure with liposomes [38] The hydrophobic layercan load the hydrophobic substances while the inner aqueouscavity can load water-soluble substances Triptolide is solublein organic solvents such as ethanol methanol dimethylsul-foxide (DMSO) and acetonitrile but not in water Thereforethrough the hydrophobic affinity between the hydrophobicstructure of exosomes and the hydrophobic drug triptolidemay be self-assembled into the lipid bilayers In additionit has been shown that hydrophobic drug encapsulationefficiency of exosomes was at least two fold higher than forstandard liposomes [39]This may be the reason why the TP-Exos collected by ultrafiltration centrifugation still has a highincorporation efficiency of TP

32 Accumulation of SK-Exos in Cells The ability of exo-somes to deliver a drug into target cells is crucial forthe therapeutic efficiency of exosomal formulations Wetested whether tumor cells had ability to take up their ownexosomes in vitro The results displayed that both SKOV3and A2780 cells could uptake PKH26-Exos (Figures 2(a)and 2(c)) but accumulation of PKH26-Exos in SKOV3cells was approximately twice that in A2780 cells (Figures2(b) and 2(d)) The results suggested that SK-Exos couldbe taken up by SKOV3 cells which was similar to theresults of Heikki Saari et al [40] showing that SK-Exosmay be able to efficiently deliver a drug to their parentalcells

33 Cytotoxicity of TP-Exos The MTT assay was used toassess the cytotoxicity of different concentration of free TPand TP-Exos on SKOV3 cells after 24 h and 48 h Statisticalanalysis of the survival rate of cells was given in Figure 3 After24 h of treatment the survival rate of cells in TP-Exos groupwas significantly higher than that in TP group and the half-maximal inhibitory concentration (IC50) values of free TPandTP-Exos groupswere 2568 and 2703 ngml respectivelyHowever after 48 h the cell survival rate in TP-Exos groupwas slightly higher than that in TP group and the IC50values of free TP and TP-Exos groups were 106 and 1893ngmL respectively We can conclude that encapsulationinto exosomes may attenuate or delay the cytotoxicity of TPtoward SKOV3 cells in vitro which was similar to the resultsof Tang K et al [41]

34 13e Effect of TP-Exos on Proliferation of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the proliferation of parental cells [42] ThereforeBrdUDNAdouble-parameter flow cytometrywas conductedto detect antiproliferative action of TP-Exos toward SKOV3cellsThe results of flow cytometry were shown in Figure 4(a)We first analyzed the effect of SK-Exos free TP and TP-Exoson the cell cycle of SKOV3 cells based on Figure 4(a) and theresults were shown in Figures 4(b) 4(c) and 4(d) Then weanalyzed the effect of SK-Exos free TP and TP-Exos on thePI of SKOV3 cells according to Figures 4(b) 4(c) and 4(d)and the results were shown in Figure 4(e) At 24 h SK-Exoshad no effect on cell cycle but at 48 h SK-Exos blocked cellsin G0G1 phase At 24 h TP blocked cells in G0G1 phasebut at 48 h the number of cells in G0G1 phase S phase andG2M phase was lower than that in control group indicatingthat TP induced cell apoptosis At 24 h TP-Exos blocked cellsin S phase but at 48 h TP-Exos blocked cells in G0G1 phaseAfter 24 h the PI of SKOV3 cells treated with SK-Exos wasnot significantly different from that of the control group (pgt 005) The PI of the free TP group was lower than that ofthe control group (P lt 0001) however the TP-Exos groupyielded a greater result than that of the control group (P lt0001) After 48 h the PI of SK-Exos group free TP groupand TP-Exos group was lower than that of the control group(P lt 005) and the TP-Exos group had a lower PI than did thefree TP group (P lt 001)

In Figure 4(e) PI increased significantly in TP-Exosgroup at 24 h which was due to the following reasons Inthis study 10 ngml TP-Exos blocked cells in S phase at 24h and G0G1 phase at 48 h S phase arrest may be the reasonfor the increase of proliferation index in TP-Exos group at 24h The arrest of cell cycle by TP-Exos may be related to theconcentration of free TP Wang Y et al [43] found 125 nML(4505 ngml) TP could block the cells in S phase at 24 h Liu Let al [44] found 10 ngml TP could block cells in G0G1 phaseat 24 h The cell cycle results of the 10ngml TP-Exos treatedgroup were consistent with those of Wang et al at 24 h andwere consistent with those of Liu et al at 48 h These resultssuggested that TP may be released less from TP-Exos at 24h and almost the entire after 48 h Studies have shown thatdrug-loaded exosomes exhibited a time-dependent releaseand the cumulative release of drugs were over 50 after 24 h

BioMed Research International 7

DAPI PKH26-Exos Merge

24h

12h

8h

4h

(a)SKOV3

0

20

40

60

80

100

MFI

(AU

)

241284Time (h)

(b)

DAPI

24h

12h

8h

4h

PKH26-Exos Merge

(c)A2780

0

20

40

60

80

100M

FI (A

U)

241284Time (h)

(d)

Figure 2 (a c) Fluorescence microscopy images of exosomes labeled with PKH-26 after incubation with SKOV3 and A2780 cells for 4 8 12or 24 h Original magnification 200times (b d) Fluorescence intensity statistical analytical diagram of panels (a) and (c) Data are presented asmean plusmn SD (n = 3)

[45] and over 90 at 48 h [46] Furthermore exosomes maycarry more drugs into the cells so that the inhibitory effect ofTP-Exos on cell proliferation is superior to that of free TP at48 h

35 13e Influence of TP-Exos on Apoptosis of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the apoptosis of parental cells [47] ThereforePIAnnexin VndashFITC double staining flow cytometry wasconducted to detect the effect of TP-Exos on the apoptosisof SKOV3 cells The results were illustrated in Figure 5 After24 h of treatment the apoptosis rate of the SK-Exos groupwas not significantly different from that of the control group(P gt 005) The percentages of apoptotic cells in the free TPgroup and TP-Exos group were higher than this parameter

of the control group (P lt 005) After 48 h of treatment theapoptotic rates of the SK-Exos group free TP group and TP-Exos group were significantly higher relative to the controlgroup (Plt 005) At the sameTP concentration the apoptosisrate of the free TP group was higher than that of TP-Exosgroup after 24 and 48 h (P lt 005) The above experimentalresults suggest that TP-Exos can induce apoptosis of SKOV3cells but the effect is weaker in comparison with free TPwhich was consistent with the results of MTT assay

Both SK-Exos and free TP could induce apoptosis butTP-Exosndashinduced apoptosis is less extensive than that causedby free TP Encapsulation into exosomes may delay thecytotoxicity of TP and then weaken its ability to induceapoptosis in vitro However the delayed cytotoxicity of TP inTP-Exos may be related to the time required for TP-Exos to

8 BioMed Research International

Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

20

40

60

80

100

Cel

l via

bilit

y (

)

TP-ExosTP

TP-ExosTP

24h

25 5 10 1005025 5001 250Concentration (ngml)

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

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Com

p-PI

-A

0

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102

Com

p-PI

-A

0

104

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p-PI

-A

0

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Com

p-PI

-A

0

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p-PI

-A

0

104

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105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

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1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

BioMed Research International 5

SK-Exos(a)

TP-Exos(b)

125

145

TP-ExosSK-Exos

200 800600400 10000Size (nm)

0

2

4

6

8

10

Con

cent

ratio

n (times

109 pa

rtic

les

ml)

(c)

SKOV3 SK-Exos

25KDa

26KDa

CD9

CD81

TP-Exos

(d)

Figure 1 Characterization of SK-Exos and TP-Exos (a b) TEM images of SK-Exos and TP-Exos Scale bar = 100 nm (c) Size distribution ofSK-Exos and TP-Exos measured by NTA (d) Western blot analysis of SK-Exos and TP-Exos protein expression

Several methods such as incubation at room temper-ature electroporation surfactant permeabilization sonica-tion hypotonic dialysis freezendashthaw cycles extrusion andcell-mediated packaging can be utilized to encapsulate adrug in exosomes [23] Studies have shown that exosomes canincrease their drug-loading capacity after sonication MyungSoo Kim et al [26] tried different methods to prepare drug-loaded exosomes and the amount of paclitaxel loaded intoexosomes increases in the following order incubation atroom temperature lt electroporation lt sonication MatthewJ Haney et al [37] tested a variety of methods for loadingcatalase into exosomes and the amount of catalase loadedinto exosomes increased in the following order incubation atRT lt freezethaw cycles lt sonication asymp extrusion Accordingto the above observations TP was loaded into exosomes bysonication in this study

After TPwas loaded into SK-Exos by sonication TP-Exoswere collected by ultracentrifugation and ultrafiltration cen-trifugation respectivelyThen we compared the difference ofTP-Exos obtained by two methods by NTA (SupplementaryFigure 1(a)) The results showed that the D 10 D 50 and D90 of TP-Exos collected by two methods had no significantdifference indicating that the two methods have no signif-icant effect on the particle size of TP-Exos (SupplementaryFigure 1(b)) D 90 is the corresponding particle size whenthe cumulative particle size distribution of a sample is upto 90 Its physical meaning was that the size of particlessmaller than 220 nmaccounted for 90of the total number ofparticles in this study The particle concentration of TP-Exosin ultracentrifugation group andultrafiltration centrifugationgroupwas significantly lower than that in SK-Exos group (Plt0001) and that in ultracentrifugation group was significantly

6 BioMed Research International

lower than that in ultrafiltration centrifugation group (P lt0001) (Supplementary Figure 1(c))The results indicated thatthere was a loss of exosomes in the process of collectingTP-Exos by the two methods but the amount of exosomeslost by ultrafiltration centrifugation was lower Thereforeultrafiltration centrifugation was used to collect TP-Exos inthis study

In Figure 1 the size of the sample measured by TEMwas not consistent with the size of the sample measured byNTA The reasons for this phenomenon are as follows TEMcan describe the diameter of exosomes and clearly displaythe structure of exosomes by direct measurement Howeverthe size distribution data obtained by TEM are often notrepresentative because of the limited scope observed at onetime In addition the samples of TEM often need to bepretreated by drying fixation and freezing which mayaffect the size of exosomes In contrast NTA can detectthe size of exosomes in solution which provides struc-tural and functional protection for exosomes NTA allowsthe exosomes to be measured closer to its original statewhich ensures the authenticity and validity of the measureddata

The encapsulation efficiency of TP in TP-Exos was com-pared with that of two TP delivery systems in our previousstudy TPmicelles and TPLips-CaP nanoparticles [19 20]The encapsulation efficiency of TP in TP-micelle TPLips-CaP nanoparticles and TP-Exos was 621 plusmn 33 7231plusmn 311 and 765 plusmn 18 respectively The encapsulationefficiency of TP in TP-Exos was similar to that of in TPLips-CaP nanoparticlesThis phenomenonmay be related to lipidbilayer structure in exosomes Exosomes have similar lipidbilayer structure with liposomes [38] The hydrophobic layercan load the hydrophobic substances while the inner aqueouscavity can load water-soluble substances Triptolide is solublein organic solvents such as ethanol methanol dimethylsul-foxide (DMSO) and acetonitrile but not in water Thereforethrough the hydrophobic affinity between the hydrophobicstructure of exosomes and the hydrophobic drug triptolidemay be self-assembled into the lipid bilayers In additionit has been shown that hydrophobic drug encapsulationefficiency of exosomes was at least two fold higher than forstandard liposomes [39]This may be the reason why the TP-Exos collected by ultrafiltration centrifugation still has a highincorporation efficiency of TP

32 Accumulation of SK-Exos in Cells The ability of exo-somes to deliver a drug into target cells is crucial forthe therapeutic efficiency of exosomal formulations Wetested whether tumor cells had ability to take up their ownexosomes in vitro The results displayed that both SKOV3and A2780 cells could uptake PKH26-Exos (Figures 2(a)and 2(c)) but accumulation of PKH26-Exos in SKOV3cells was approximately twice that in A2780 cells (Figures2(b) and 2(d)) The results suggested that SK-Exos couldbe taken up by SKOV3 cells which was similar to theresults of Heikki Saari et al [40] showing that SK-Exosmay be able to efficiently deliver a drug to their parentalcells

33 Cytotoxicity of TP-Exos The MTT assay was used toassess the cytotoxicity of different concentration of free TPand TP-Exos on SKOV3 cells after 24 h and 48 h Statisticalanalysis of the survival rate of cells was given in Figure 3 After24 h of treatment the survival rate of cells in TP-Exos groupwas significantly higher than that in TP group and the half-maximal inhibitory concentration (IC50) values of free TPandTP-Exos groupswere 2568 and 2703 ngml respectivelyHowever after 48 h the cell survival rate in TP-Exos groupwas slightly higher than that in TP group and the IC50values of free TP and TP-Exos groups were 106 and 1893ngmL respectively We can conclude that encapsulationinto exosomes may attenuate or delay the cytotoxicity of TPtoward SKOV3 cells in vitro which was similar to the resultsof Tang K et al [41]

34 13e Effect of TP-Exos on Proliferation of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the proliferation of parental cells [42] ThereforeBrdUDNAdouble-parameter flow cytometrywas conductedto detect antiproliferative action of TP-Exos toward SKOV3cellsThe results of flow cytometry were shown in Figure 4(a)We first analyzed the effect of SK-Exos free TP and TP-Exoson the cell cycle of SKOV3 cells based on Figure 4(a) and theresults were shown in Figures 4(b) 4(c) and 4(d) Then weanalyzed the effect of SK-Exos free TP and TP-Exos on thePI of SKOV3 cells according to Figures 4(b) 4(c) and 4(d)and the results were shown in Figure 4(e) At 24 h SK-Exoshad no effect on cell cycle but at 48 h SK-Exos blocked cellsin G0G1 phase At 24 h TP blocked cells in G0G1 phasebut at 48 h the number of cells in G0G1 phase S phase andG2M phase was lower than that in control group indicatingthat TP induced cell apoptosis At 24 h TP-Exos blocked cellsin S phase but at 48 h TP-Exos blocked cells in G0G1 phaseAfter 24 h the PI of SKOV3 cells treated with SK-Exos wasnot significantly different from that of the control group (pgt 005) The PI of the free TP group was lower than that ofthe control group (P lt 0001) however the TP-Exos groupyielded a greater result than that of the control group (P lt0001) After 48 h the PI of SK-Exos group free TP groupand TP-Exos group was lower than that of the control group(P lt 005) and the TP-Exos group had a lower PI than did thefree TP group (P lt 001)

In Figure 4(e) PI increased significantly in TP-Exosgroup at 24 h which was due to the following reasons Inthis study 10 ngml TP-Exos blocked cells in S phase at 24h and G0G1 phase at 48 h S phase arrest may be the reasonfor the increase of proliferation index in TP-Exos group at 24h The arrest of cell cycle by TP-Exos may be related to theconcentration of free TP Wang Y et al [43] found 125 nML(4505 ngml) TP could block the cells in S phase at 24 h Liu Let al [44] found 10 ngml TP could block cells in G0G1 phaseat 24 h The cell cycle results of the 10ngml TP-Exos treatedgroup were consistent with those of Wang et al at 24 h andwere consistent with those of Liu et al at 48 h These resultssuggested that TP may be released less from TP-Exos at 24h and almost the entire after 48 h Studies have shown thatdrug-loaded exosomes exhibited a time-dependent releaseand the cumulative release of drugs were over 50 after 24 h

BioMed Research International 7

DAPI PKH26-Exos Merge

24h

12h

8h

4h

(a)SKOV3

0

20

40

60

80

100

MFI

(AU

)

241284Time (h)

(b)

DAPI

24h

12h

8h

4h

PKH26-Exos Merge

(c)A2780

0

20

40

60

80

100M

FI (A

U)

241284Time (h)

(d)

Figure 2 (a c) Fluorescence microscopy images of exosomes labeled with PKH-26 after incubation with SKOV3 and A2780 cells for 4 8 12or 24 h Original magnification 200times (b d) Fluorescence intensity statistical analytical diagram of panels (a) and (c) Data are presented asmean plusmn SD (n = 3)

[45] and over 90 at 48 h [46] Furthermore exosomes maycarry more drugs into the cells so that the inhibitory effect ofTP-Exos on cell proliferation is superior to that of free TP at48 h

35 13e Influence of TP-Exos on Apoptosis of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the apoptosis of parental cells [47] ThereforePIAnnexin VndashFITC double staining flow cytometry wasconducted to detect the effect of TP-Exos on the apoptosisof SKOV3 cells The results were illustrated in Figure 5 After24 h of treatment the apoptosis rate of the SK-Exos groupwas not significantly different from that of the control group(P gt 005) The percentages of apoptotic cells in the free TPgroup and TP-Exos group were higher than this parameter

of the control group (P lt 005) After 48 h of treatment theapoptotic rates of the SK-Exos group free TP group and TP-Exos group were significantly higher relative to the controlgroup (Plt 005) At the sameTP concentration the apoptosisrate of the free TP group was higher than that of TP-Exosgroup after 24 and 48 h (P lt 005) The above experimentalresults suggest that TP-Exos can induce apoptosis of SKOV3cells but the effect is weaker in comparison with free TPwhich was consistent with the results of MTT assay

Both SK-Exos and free TP could induce apoptosis butTP-Exosndashinduced apoptosis is less extensive than that causedby free TP Encapsulation into exosomes may delay thecytotoxicity of TP and then weaken its ability to induceapoptosis in vitro However the delayed cytotoxicity of TP inTP-Exos may be related to the time required for TP-Exos to

8 BioMed Research International

Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

20

40

60

80

100

Cel

l via

bilit

y (

)

TP-ExosTP

TP-ExosTP

24h

25 5 10 1005025 5001 250Concentration (ngml)

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

6 BioMed Research International

lower than that in ultrafiltration centrifugation group (P lt0001) (Supplementary Figure 1(c))The results indicated thatthere was a loss of exosomes in the process of collectingTP-Exos by the two methods but the amount of exosomeslost by ultrafiltration centrifugation was lower Thereforeultrafiltration centrifugation was used to collect TP-Exos inthis study

In Figure 1 the size of the sample measured by TEMwas not consistent with the size of the sample measured byNTA The reasons for this phenomenon are as follows TEMcan describe the diameter of exosomes and clearly displaythe structure of exosomes by direct measurement Howeverthe size distribution data obtained by TEM are often notrepresentative because of the limited scope observed at onetime In addition the samples of TEM often need to bepretreated by drying fixation and freezing which mayaffect the size of exosomes In contrast NTA can detectthe size of exosomes in solution which provides struc-tural and functional protection for exosomes NTA allowsthe exosomes to be measured closer to its original statewhich ensures the authenticity and validity of the measureddata

The encapsulation efficiency of TP in TP-Exos was com-pared with that of two TP delivery systems in our previousstudy TPmicelles and TPLips-CaP nanoparticles [19 20]The encapsulation efficiency of TP in TP-micelle TPLips-CaP nanoparticles and TP-Exos was 621 plusmn 33 7231plusmn 311 and 765 plusmn 18 respectively The encapsulationefficiency of TP in TP-Exos was similar to that of in TPLips-CaP nanoparticlesThis phenomenonmay be related to lipidbilayer structure in exosomes Exosomes have similar lipidbilayer structure with liposomes [38] The hydrophobic layercan load the hydrophobic substances while the inner aqueouscavity can load water-soluble substances Triptolide is solublein organic solvents such as ethanol methanol dimethylsul-foxide (DMSO) and acetonitrile but not in water Thereforethrough the hydrophobic affinity between the hydrophobicstructure of exosomes and the hydrophobic drug triptolidemay be self-assembled into the lipid bilayers In additionit has been shown that hydrophobic drug encapsulationefficiency of exosomes was at least two fold higher than forstandard liposomes [39]This may be the reason why the TP-Exos collected by ultrafiltration centrifugation still has a highincorporation efficiency of TP

32 Accumulation of SK-Exos in Cells The ability of exo-somes to deliver a drug into target cells is crucial forthe therapeutic efficiency of exosomal formulations Wetested whether tumor cells had ability to take up their ownexosomes in vitro The results displayed that both SKOV3and A2780 cells could uptake PKH26-Exos (Figures 2(a)and 2(c)) but accumulation of PKH26-Exos in SKOV3cells was approximately twice that in A2780 cells (Figures2(b) and 2(d)) The results suggested that SK-Exos couldbe taken up by SKOV3 cells which was similar to theresults of Heikki Saari et al [40] showing that SK-Exosmay be able to efficiently deliver a drug to their parentalcells

33 Cytotoxicity of TP-Exos The MTT assay was used toassess the cytotoxicity of different concentration of free TPand TP-Exos on SKOV3 cells after 24 h and 48 h Statisticalanalysis of the survival rate of cells was given in Figure 3 After24 h of treatment the survival rate of cells in TP-Exos groupwas significantly higher than that in TP group and the half-maximal inhibitory concentration (IC50) values of free TPandTP-Exos groupswere 2568 and 2703 ngml respectivelyHowever after 48 h the cell survival rate in TP-Exos groupwas slightly higher than that in TP group and the IC50values of free TP and TP-Exos groups were 106 and 1893ngmL respectively We can conclude that encapsulationinto exosomes may attenuate or delay the cytotoxicity of TPtoward SKOV3 cells in vitro which was similar to the resultsof Tang K et al [41]

34 13e Effect of TP-Exos on Proliferation of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the proliferation of parental cells [42] ThereforeBrdUDNAdouble-parameter flow cytometrywas conductedto detect antiproliferative action of TP-Exos toward SKOV3cellsThe results of flow cytometry were shown in Figure 4(a)We first analyzed the effect of SK-Exos free TP and TP-Exoson the cell cycle of SKOV3 cells based on Figure 4(a) and theresults were shown in Figures 4(b) 4(c) and 4(d) Then weanalyzed the effect of SK-Exos free TP and TP-Exos on thePI of SKOV3 cells according to Figures 4(b) 4(c) and 4(d)and the results were shown in Figure 4(e) At 24 h SK-Exoshad no effect on cell cycle but at 48 h SK-Exos blocked cellsin G0G1 phase At 24 h TP blocked cells in G0G1 phasebut at 48 h the number of cells in G0G1 phase S phase andG2M phase was lower than that in control group indicatingthat TP induced cell apoptosis At 24 h TP-Exos blocked cellsin S phase but at 48 h TP-Exos blocked cells in G0G1 phaseAfter 24 h the PI of SKOV3 cells treated with SK-Exos wasnot significantly different from that of the control group (pgt 005) The PI of the free TP group was lower than that ofthe control group (P lt 0001) however the TP-Exos groupyielded a greater result than that of the control group (P lt0001) After 48 h the PI of SK-Exos group free TP groupand TP-Exos group was lower than that of the control group(P lt 005) and the TP-Exos group had a lower PI than did thefree TP group (P lt 001)

In Figure 4(e) PI increased significantly in TP-Exosgroup at 24 h which was due to the following reasons Inthis study 10 ngml TP-Exos blocked cells in S phase at 24h and G0G1 phase at 48 h S phase arrest may be the reasonfor the increase of proliferation index in TP-Exos group at 24h The arrest of cell cycle by TP-Exos may be related to theconcentration of free TP Wang Y et al [43] found 125 nML(4505 ngml) TP could block the cells in S phase at 24 h Liu Let al [44] found 10 ngml TP could block cells in G0G1 phaseat 24 h The cell cycle results of the 10ngml TP-Exos treatedgroup were consistent with those of Wang et al at 24 h andwere consistent with those of Liu et al at 48 h These resultssuggested that TP may be released less from TP-Exos at 24h and almost the entire after 48 h Studies have shown thatdrug-loaded exosomes exhibited a time-dependent releaseand the cumulative release of drugs were over 50 after 24 h

BioMed Research International 7

DAPI PKH26-Exos Merge

24h

12h

8h

4h

(a)SKOV3

0

20

40

60

80

100

MFI

(AU

)

241284Time (h)

(b)

DAPI

24h

12h

8h

4h

PKH26-Exos Merge

(c)A2780

0

20

40

60

80

100M

FI (A

U)

241284Time (h)

(d)

Figure 2 (a c) Fluorescence microscopy images of exosomes labeled with PKH-26 after incubation with SKOV3 and A2780 cells for 4 8 12or 24 h Original magnification 200times (b d) Fluorescence intensity statistical analytical diagram of panels (a) and (c) Data are presented asmean plusmn SD (n = 3)

[45] and over 90 at 48 h [46] Furthermore exosomes maycarry more drugs into the cells so that the inhibitory effect ofTP-Exos on cell proliferation is superior to that of free TP at48 h

35 13e Influence of TP-Exos on Apoptosis of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the apoptosis of parental cells [47] ThereforePIAnnexin VndashFITC double staining flow cytometry wasconducted to detect the effect of TP-Exos on the apoptosisof SKOV3 cells The results were illustrated in Figure 5 After24 h of treatment the apoptosis rate of the SK-Exos groupwas not significantly different from that of the control group(P gt 005) The percentages of apoptotic cells in the free TPgroup and TP-Exos group were higher than this parameter

of the control group (P lt 005) After 48 h of treatment theapoptotic rates of the SK-Exos group free TP group and TP-Exos group were significantly higher relative to the controlgroup (Plt 005) At the sameTP concentration the apoptosisrate of the free TP group was higher than that of TP-Exosgroup after 24 and 48 h (P lt 005) The above experimentalresults suggest that TP-Exos can induce apoptosis of SKOV3cells but the effect is weaker in comparison with free TPwhich was consistent with the results of MTT assay

Both SK-Exos and free TP could induce apoptosis butTP-Exosndashinduced apoptosis is less extensive than that causedby free TP Encapsulation into exosomes may delay thecytotoxicity of TP and then weaken its ability to induceapoptosis in vitro However the delayed cytotoxicity of TP inTP-Exos may be related to the time required for TP-Exos to

8 BioMed Research International

Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

20

40

60

80

100

Cel

l via

bilit

y (

)

TP-ExosTP

TP-ExosTP

24h

25 5 10 1005025 5001 250Concentration (ngml)

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

BioMed Research International 7

DAPI PKH26-Exos Merge

24h

12h

8h

4h

(a)SKOV3

0

20

40

60

80

100

MFI

(AU

)

241284Time (h)

(b)

DAPI

24h

12h

8h

4h

PKH26-Exos Merge

(c)A2780

0

20

40

60

80

100M

FI (A

U)

241284Time (h)

(d)

Figure 2 (a c) Fluorescence microscopy images of exosomes labeled with PKH-26 after incubation with SKOV3 and A2780 cells for 4 8 12or 24 h Original magnification 200times (b d) Fluorescence intensity statistical analytical diagram of panels (a) and (c) Data are presented asmean plusmn SD (n = 3)

[45] and over 90 at 48 h [46] Furthermore exosomes maycarry more drugs into the cells so that the inhibitory effect ofTP-Exos on cell proliferation is superior to that of free TP at48 h

35 13e Influence of TP-Exos on Apoptosis of SKOV3 CellsExosomes derived from tumor cells may have effects topromote the apoptosis of parental cells [47] ThereforePIAnnexin VndashFITC double staining flow cytometry wasconducted to detect the effect of TP-Exos on the apoptosisof SKOV3 cells The results were illustrated in Figure 5 After24 h of treatment the apoptosis rate of the SK-Exos groupwas not significantly different from that of the control group(P gt 005) The percentages of apoptotic cells in the free TPgroup and TP-Exos group were higher than this parameter

of the control group (P lt 005) After 48 h of treatment theapoptotic rates of the SK-Exos group free TP group and TP-Exos group were significantly higher relative to the controlgroup (Plt 005) At the sameTP concentration the apoptosisrate of the free TP group was higher than that of TP-Exosgroup after 24 and 48 h (P lt 005) The above experimentalresults suggest that TP-Exos can induce apoptosis of SKOV3cells but the effect is weaker in comparison with free TPwhich was consistent with the results of MTT assay

Both SK-Exos and free TP could induce apoptosis butTP-Exosndashinduced apoptosis is less extensive than that causedby free TP Encapsulation into exosomes may delay thecytotoxicity of TP and then weaken its ability to induceapoptosis in vitro However the delayed cytotoxicity of TP inTP-Exos may be related to the time required for TP-Exos to

8 BioMed Research International

Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

20

40

60

80

100

Cel

l via

bilit

y (

)

TP-ExosTP

TP-ExosTP

24h

25 5 10 1005025 5001 250Concentration (ngml)

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

8 BioMed Research International

Concentration (ngml)48h

1 25 5 10 25 50 100 250 5000

20

40

60

80

100

Cel

l via

bilit

y (

)

TP-ExosTP

TP-ExosTP

24h

25 5 10 1005025 5001 250Concentration (ngml)

0

20

40

60

80

100

Cel

l via

bilit

y (

)

Figure 3 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h The statistical analysis diagram of the results of the MTT assayData are presented as mean plusmn SD (n = 3)

release TP In this study 10 ngml TP blocked cells in G0G1phase after 24 h but 10 ngml TP-Exos blocked cells in G0G1phase after 48 h Therefore the time of apoptosis induced byTP-Exos may be 24 hours later than that of free TP

36 TumorTargeting of SK-Exos InVivo To establishwhetherexosomes can be used effectively for drug delivery under-standing of their tumor targeting and distribution in vivoare important It has been proved that intraperitoneal injec-tion increased accumulation of drugs in tumor comparedwith intravenous injection [24] Therefore intraperitonealinjection has been used in this study As shown in Figure 6Dir was mainly distributed in the liver and spleen andthe fluorescent intensity in the tumor for Dir-Exos groupincreased with time while that for Free-Dir group did notsignificant increase The results demonstrate that exosomescan be rapidly uptaken by the liver and spleen which isconsistent with previous studies on the biodistribution ofexosomes [25 48ndash50] We found that Dir-Exos could berecruited to the tumour site which was consistent with theresults of Ke Tang et al [41] suggesting that SK-Exos maytransport drugs to tumors via the circulatory system

37 13e Effect of TP-Exos on Ovarian Cancer In VivoThe antitumor efficacy of SK-Exos free TP and TP-Exoswas evaluated based on the tumor volumes of the SKOV3tumor-bearing mice As shown in Figure 7 after 4 weeksof treatment the tumor volume of TP-Exos group controlgroup SK-Exos group and free TP group was 22251 plusmn 2341mm3 97735 plusmn 8162 mm3 87759 plusmn 3025 mm3 and 50879plusmn 1796 mm3 respectively These results suggested that tumorgrowth was inhibited in SK-Exos free TP and TP-Exos groupcompared with control group and tumor suppression of TP-Exos group was significantly better than that of free TPgroups

The cytotoxicity of TP-Exos group is weaker than thatof free TP group in vitro but its antitumor effect in vivo issuperior to free TP group Exosomes derived from tumorcells carried tumor specific antigen proteins and miRNAwhich could activate immune cells in vivo to play a synergisticantitumor effect In addition triptolide could significantincrease the cell populations of T cells B cells monocytesand macrophage Furthermore triptolide also could increasethe phagocytosis of macrophage from peripheral bloodmononuclear cells [51]

The effect of TP-Exos on apoptosis of cells in transplantedtumors was examined by the TUNEL staining assay As seenin Figure 8 the apoptotic cells displaying green fluorescencewere found Except for SK-Exos group TUNEL-positive cellsratio in free TP group and TP-Exos group were higher thanthat in control group (p lt 0001) In addition TUNEL-positive cells ratio in free TP group was higher than thatin TP-Exos group (p lt 005) which suggested that TP-Exoscan induce apoptosis of cells in transplanted tumors but theeffect is weaker in comparison with free TP The effect of TP-Exos on the proliferation and apoptosis of cells in vivo wasconsistent with that in vitro

38 Toxicity of TP-Exos InVivo In this study the effect of TP-Exos on liver and kidney function was evaluated by detectingthe level of AST ALT BUN and Cr in serum of mice Asshown in Figure 9 compared with the control group SK-Exos free TP and TP-Exos groups had no effect on the levelof BUN and Cr (p gt005) Except for free TP group the levelsof AST and ALT in SK-Exos and TP-Exos groups were higherthan that in control group (p lt 0001) In addition the level ofAST and ALT in TP-Exos group was not different from thatin SK-Exos group (p gt005) but significantly higher than thatin TP group (p lt 0001)

The toxicity of TP-Exos to major organs was detectedby histopathology in this study As shown in Figure 10 no

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

BioMed Research International 9

TP-ExosTP

7-ADD

SK-ExosControl

48h

24hBr

dU-A

PC

102

101

103

104

100

0 200 400 600 800 1K

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

102

101

103

104

100

APC

-A

7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

0 200 400 600 800 1K7-AAD-A

P1413P2301P3554P4580

P1220P2860P3237P4151

P1157P2635P3203P4868

P1288P2758P3338P4125

P1122P2746P3148P4486

P10572P2642P3221P4820

P1155P2660P3178P4772

P10440P2648P3258P4417

(a)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

24h48h

SK-Exos TP TP-ExosControlG0G1 phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(b)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

24h48h

0

20

40

60

80

100

Cel

l pro

port

ion

()

SK-Exos TP TP-ExosControlS phase

(c)

lowastlowast

lowast

lowast

24h48h

SK-Exos TP TP-ExosControlG2M phase

0

20

40

60

80

100

Cel

l pro

port

ion

()

(d)

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowast

NS

24 48Time (h)

ControlSK-Exos TP-Exos

TP

0

20

40

60

80

100

Cel

l pro

lifer

atio

n in

dex

()

(e)

Figure 4 (a) Results of BrdUDNA double-parameter flow cytometry P1 cells in sub-G0G1 phases P2 cells in G0G1 phases P3 cells in Sphase and P4 cells in G2M phase (b c d) Quantitative statistical analysis of the cell cycle based on panel (a) (e) Statistical analysis of thePI based on panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 lowastlowastp lt 001 and lowast lowast lowastp lt 0001

significant pathological changes were found in the heartlung kidney and ovary in SK-Exos free TP and TP-Exosgroups There were obvious areas of necrosis in liver tissuein SK-Exos and TP-Exos group and the area of the germinalcenter of spleen tissuewas not obvious in freeTP andTP-Exosgroup

All the above results showed that TP-Exos had no effecton renal function but could lead to liver dysfunction TP-Exos have no obvious pathological damage to heart lungkidney and ovary but has pathological damage to liver and

spleen In addition we found that SK-Exos in TP-Exos maybe the cause of liver injury and TP in TP-Exos may be thecause of spleen injury indicating that TP-Exos may need tobe further optimized to solve the problem of liver and spleeninjury

4 Conclusions

In this study based on exosomes derived from SKOV3 cellswe have developed and characterized a novel anticancer

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

10 BioMed Research International

TP-ExosComp-FITC-A

TPSK-ExosControl

Annexin V-FITC

PI

48h

24h

102

103

104

105

0

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Comp-FITC-A10

210

310

410

50

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

Com

p-PI

-A

0

104

103

105

102

(a)

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

48h

0

20Apop

tosis

rate

()

40

60

80

100

TP-ExosTPSK-ExosControl

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

NS

24h

0

20Apop

tosis

rate

()

40

60

80

100

(b)

Figure 5 SKOV3 cells were incubated with TP or TP-Exos for 24 and 48 h (a) Results of PIannexin V double-staining flow cytometry (b)Statistical analysis of the apoptosis rate in panel (a) Data are presented as mean plusmn SD (n = 3) NS means p gt 005 lowastp lt 005 and lowast lowast lowastp lt0001

Free-Dir

4h 8h 24h 48h

Dir-Exos

times109

10Tu

Tu

K

K

Lu

Lu

Sp

Sp

Li

Li

H

H

20

30

40

50

60

(a)

0

200

400

600

800

1000

Free-DirDir-Exos

MFI

(AU

)

4 8 24Time (h)

48

(b)Figure 6 (a) Fluorescent images of SKOV3-tumor-bearing mice at 4 8 24 and 48 h postinjection of Free-Dir and Dir-Exos and fluorescentimages of major organs (H heart Li liver Sp spleen Lu lung K kidney Tu tumor) The blue circle is where the tumor is (b) Statisticaldiagram of fluorescence intensity in tumor Data are presented as mean plusmn SD (n =3)

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

BioMed Research International 11

TP-Exos

TP

SK-Exos

Control

(a)TP-Exos

TPSK-Exos

Control

0

200

400

600

800

1000

1200

Tum

or V

olum

e (m

m3)

1 2 3 4 50Time (weeks)

(b)

Figure 7 In vivo antitumor effect of the different drugs in SKOV3-tumor-bearing mice (a) Photograph of tumor dissected from the miceafter the last treatment (b) The change of relative tumor volume (VV initial) with treatment time Data are presented as mean plusmn SD (n =6)

TP-ExosTP

SK-ExosControl

(a)

0

5

10

TUN

EL p

ositi

ve ce

ll ra

tio (

)

15

20

TP-ExosTPSK-ExosControl

NS

lowastlowastlowast

lowastlowastlowast lowast

(b)

Figure 8 (a) Fluorescence image of TUNEL assay of the tumor tissue after treatment of different drugs (b) Statistical analysis of TUNEL-positive cell ratio based on panel (a) Original magnification 200times Data are presented as mean plusmn SD (n = 6) NS means p gt 005 lowastp lt 005and lowast lowast lowastp lt 0001

drug formulation TP-Exos Then we examined the effectsof TP-Exos on proliferation and apoptosis of SKOV3 cellsin vitro and in vivo The results showed that TP-Exos mayattenuate the cytotoxicity and apoptosis-inducing capabilityof TP toward SKOV3 cells but enhance the inhibitory effectof TP on cell proliferation However we further studied thetoxicity of TP-Exos in vivo and the results displayed that TP-Exos has toxic effect on liver and spleen It proposed that the

TP-Exos may be a promising strategy for ovarian cancer butit needs to be further optimized to attenuate the damage toliver and spleen

Data Availability

The data used to support the findings of this study areincluded within the article

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

12 BioMed Research International

TP-ExosTP

NS

NS

NS

NS

AST

SK-ExosControl

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

0

200

400

600

800

1000

UL

SK-Exos TP TP-ExosControlALT

0

200

400

600

800

1000

UL

(a)

0

50

100

150

1200

umol

L

SK-Exos TP TP-ExosControlCr

SK-Exos TP TP-ExosControlBUN

0

10

20

30

40

mg

dL

(b)

Figure 9 (a) Serum levels of ALT AST in liver function (b) Serum levels of BUN and Cr in renal function Data is presented as mean plusmn SD(n = 3) NS means P gt 005 lowast lowast lowastP lt 0001

TP-ExosTPSK-ExosControl

Heart

Liver

Spleen

Lung

Kindey

Ovary

Figure 10 Histopathology of heart liver spleen lung kidney and ovary from mice after treatment Original magnification 200times

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

BioMed Research International 13

Conflicts of Interest

The authors claim that there are no conflicts of interestassociated with this work

Acknowledgments

This research was supported by the National Natural ScienceFoundation of China (grant Nos 81874479 81502692)

Supplementary Materials

Supplementary 1 Supplemental Figure 1 Comparison ofTP-Exos collected by ultracentrifugation (TP-Exos-UC) andultrafiltration centrifugation (TP-Exos-UF) (a) AveragedFTLA (Fibre to the Last Active) ConcentrationSize for NTAexperiment (b) Size distribution of SK-Exos and TP-Exosmeasured by NTA (c) Particle concentration of SK-Exos andTP-Exosmeasured by NTA Data are presented asmean plusmn SD(n = 3) lowast lowast lowastp lt 0001Supplementary 2 Supplemental Figure 21 TheHPLCprofilesof TP standard solution SK-Exos solution and TP-Exossolution Supplemental Figure 22 Calibration curve of TP byHPLCSupplementary 3 Supplemental Table 1 Results of encapsula-tion efficiency of TP-Exos

References

[1] E Wai-Ching Chan S Chak-Sum Cheng F Wan-Yee Sin andY Xie ldquoTriptolide induced cytotoxic effects on human promye-locytic leukemia T cell lymphoma and human hepatocellularcarcinoma cell linesrdquo Toxicology Letters vol 122 no 1 pp 81ndash87 2001

[2] B Wang J Cao J Chen et al ldquoTriptolide induces apoptosis ofgastric cancer cells via inhibiting the overexpression ofMDM2rdquoMedical Oncology vol 31 no 11 article no 270 2014

[3] H Gao Y Zhang L Dong et al ldquoTriptolide induces autophagyand apoptosis through ERK activation in human breast cancerMCF-cellsrdquo Experimental and13erapeutic Medicine vol 15 no4 pp 3413ndash3419 2018

[4] Y-Y Sun L Xiao D Wang et al ldquoTriptolide inhibits viabilityand induces apoptosis in liver cancer cells through activationof the tumor suppressor gene p53rdquo International Journal ofOncology vol 50 no 3 pp 847ndash852 2017

[5] Y Tao M-L Zhang P-C Ma et al ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific Journal of Cancer Prevention vol 13 no 4pp 1611ndash1615 2012

[6] P A Phillips V Dudeja J A McCarroll et al ldquoTriptolideinduces pancreatic cancer cell death via inhibition of heat shockprotein 70rdquoCancer Research vol 67 no 19 pp 9407ndash9416 2007

[7] HHu L Luo F Liu et al ldquoAnti-cancer and sensibilisation effectof Triptolide on human epithelial ovarian cancerrdquo Journal ofCancer vol 7 no 14 pp 2093ndash2099 2016

[8] Y Zhong H Chen B Tan et al ldquoTriptolide avoids cis-platin resistance and induces apoptosis via the reactive oxygen

speciesnuclear factor-120581B pathway in SKOV3PT platinum-resistant human ovarian cancer cellsrdquo Oncology Letters vol 6no 4 pp 1084ndash1092 2013

[9] J Wu Q Q Li H Zhou et al ldquoSelective tumor cell killing bytriptolide in p53wild-type and p53mutant ovarian carcinomasrdquoMedical Oncology vol 31 no 7 article no 14 2014

[10] H Hu G Huang H Wang et al ldquoInhibition effect of triptolideon human epithelial ovarian cancer via adjusting cellularimmunity and angiogenesisrdquo Oncology Reports vol 39 no 3pp 1191ndash1196 2018

[11] J Liu Z Jiang Y Zhang et al ldquoTriptolide induces adverse effecton reproductive parameters of female Sprague-Dawley ratsrdquoDrug and Chemical Toxicology vol 34 no 1 pp 1ndash7 2011

[12] X Chen and S-L Chen ldquoA woman with premature ovarianfailure induced by Tripterygium wilfordii Hookf gives birth toa healthy childrdquo Fertility and Sterility vol 96 no 1 pp e19ndashe212011

[13] Y Chen X-M Zhang F-M Han and Q-S Xia ldquoGene expres-sion profile analyses of mice livers injured by LeigongtengrdquoWorld Journal of Gastroenterology vol 13 no 26 pp 3619ndash36242007

[14] J Yao Z Jiang W Duan et al ldquoInvolvement of mitochondrialpathway in triptolide-induced cytotoxicity in human normalliver L-02 cellsrdquoBiological amp Pharmaceutical Bulletin vol 31 no4 pp 592ndash597 2008

[15] Q Fu X Huang B Shu et al ldquoInhibition of mitochondrialrespiratory chain is involved in triptolide-induced liver injuryrdquoFitoterapia vol 82 no 8 pp 1241ndash1248 2011

[16] F Yang L Zhuo S Ananda T Sun S Li and L Liu ldquoRoleof reactive oxygen species in triptolide-induced apoptosis ofrenal tubular cells and renal injury in ratsrdquo Journal of HuazhongUniversity of Science and Technology (Medical Sciences) vol 31no 3 pp 335ndash341 2011

[17] F Yang L Ren L Zhuo S Ananda and L Liu ldquoInvolvementof oxidative stress in themechanism of triptolide-induced acutenephrotoxicity in ratsrdquo Experimental and Toxicologic Pathologyvol 64 no 7-8 pp 905ndash911 2012

[18] M Kotmakcı and V B Cetintas ldquoExtracellular vesicles asnatural nanosized delivery systems for small-molecule drugsand genetic material steps towards the future nanomedicinesrdquoJournal of Pharmacy amp Pharmaceutical Sciences vol 18 no 3pp 396ndash413 2015

[19] H Li X-S Wen and W Di ldquoIn vitro and in vivo evaluationof Triptolide-loaded Pluronic P105 polymeric micellesrdquo DrugResearch vol 62 no 7 pp 340ndash344 2012

[20] D Ru C Ding L Liu et al ldquopH sensitive triptolide-loadedliposome calcium phosphate nanoparticles exhibit enhancedanti-tumor activities against ovarian cancer without damagingthe reproductive systemrdquo Journal of Biomedical Nanotechnologyvol 13 no 11 pp 1413ndash1424 2017

[21] A Srivastava A Babu J Filant et al ldquoExploitation of exosomesas nanocarriers for gene- chemo- and immune-therapy ofcancerrdquo Journal of Biomedical Nanotechnology vol 12 no 6 pp1159ndash1173 2016

[22] L Barile and G Vassalli ldquoExosomesTherapy delivery tools andbiomarkers of diseasesrdquo Pharmacology amp13erapeutics vol 174pp 63ndash78 2017

[23] J P K Armstrong and M M Stevens ldquoStrategic design ofextracellular vesicle drug delivery systemsrdquo Advanced DrugDelivery Reviews vol 130 pp 12ndash16 2018

14 BioMed Research International

[24] O PWiklander J ZNordin AOrsquoLoughlin et al ldquoExtracellularvesicle in vivo biodistribution is determined by cell sourceroute of administration and targetingrdquo Journal of ExtracellularVesicles (JEV) vol 4 Article ID 26316 2015

[25] Y Tian S Li J Song et al ldquoA doxorubicin delivery platformusing engineered natural membrane vesicle exosomes for tar-geted tumor therapyrdquo Biomaterials vol 35 no 7 pp 2383ndash23902014

[26] M S Kim M J Haney Y Zhao et al ldquoDevelopment ofexosome-encapsulated paclitaxel to overcome MDR in cancercellsrdquo Nanomedicine Nanotechnology Biology and Medicinevol 12 no 3 pp 655ndash664 2016

[27] M S Kim M J Haney Y Zhao et al ldquoEngineering ma-crophage-derived exosomes for targeted paclitaxel deliveryto pulmonary metastases in vitro and in vivo evaluationsrdquoNanomedicine Nanotechnology Biology and Medicine vol 14no 1 pp 195ndash204 2018

[28] R C Lai R W Y Yeo K H Tan and S K Lim ldquoExosomes fordrug deliverymdasha novel application for the mesenchymal stemcellrdquo Biotechnology Advances vol 31 no 5 pp 543ndash551 2013

[29] DM Sun X Y Zhuang X Y Xiang et al ldquoA novel nanoparticledrug delivery system the anti-inflammatory activity of cur-cumin is enhanced when encapsulated in exosomesrdquoMolecular13erapy vol 18 no 9 pp 1606ndash1614 2010

[30] X Zhuang X Xiang W Grizzle et al ldquoTreatment of braininflammatory diseases by delivering exosome encapsulatedanti-inflammatory drugs from the nasal region to the brainrdquoMolecular 13erapy vol 19 no 10 pp 1769ndash1779 2011

[31] T Yang P Martin B Fogarty et al ldquoExosome deliveredanticancer drugs across the blood-brain barrier for brain cancertherapy in Danio Reriordquo Pharmaceutical Research vol 32 no 6pp 2003ndash2014 2015

[32] L Pascucci V Cocce A Bonomi et al ldquoPaclitaxel is incorpo-rated by mesenchymal stromal cells and released in exosomesthat inhibit in vitro tumor growth a new approach for drugdeliveryrdquo Journal of Controlled Release vol 192 pp 262ndash2702014

[33] S C Jang O Y Kim C M Yoon et al ldquoBioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeu-tics tomalignant tumorsrdquoACSNano vol 7 no 9 pp 7698ndash77102013

[34] CThery S Amigorena G Raposo and A Clayton ldquoUNIT 322isolation and characterization of exosomes from cell culturesupernatants and biological fluidsrdquo Current Protocols in CellBiology vol 3 pp 3ndash22 2006

[35] R A Dragovic C Gardiner A S Brooks et al ldquoSizing andphenotyping of cellular vesicles using nanoparticle trackinganalysisrdquoNanomedicine Nanotechnology Biology andMedicinevol 7 no 6 pp 780ndash788 2011

[36] P Carnell-Morris D Tannetta A Siupa P Hole and RDragovic ldquoAnalysis of extracellular vesicles using fluorescencenanoparticle tracking analysisrdquo Methods in Molecular Biologyvol 1660 pp 153ndash173 2017

[37] M J Haney N L Klyachko Y Zhao et al ldquoExosomes as drugdelivery vehicles for Parkinsonrsquos disease therapyrdquo Journal ofControlled Release vol 207 pp 18ndash30 2015

[38] R van der Meel M H Fens P Vader W W van Solinge OEniola-Adefeso and R M Schiffelers ldquoExtracellular vesicles asdrug delivery systems lessons from the liposome fieldrdquo Journalof Controlled Release vol 195 pp 72ndash85 2014

[39] G Fuhrmann A Serio M Mazo R Nair and M M StevensldquoActive loading into extracellular vesicles significantly improves

the cellular uptake and photodynamic effect of porphyrinsrdquoJournal of Controlled Release vol 205 pp 35ndash44 2015

[40] H Saari E Lazaro-Ibanez T Viitala E Vuorimaa-LaukkanenP Siljander and M Yliperttula ldquoMicrovesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxelin autologous prostate cancer cellsrdquo Journal of ControlledRelease vol 220 pp 727ndash737 2015

[41] K Tang Y Zhang H Zhang et al ldquoDelivery of chemother-apeutic drugs in tumour cell-derived microparticlesrdquo NatureCommunications vol 3 article no 1282 2012

[42] J-L Qu X-J Qu M-F Zhao et al ldquoGastric cancer exo-somes promote tumour cell proliferation throughPI3KAkt andMAPKERK activationrdquoDigestive and Liver Disease vol 41 no12 pp 875ndash880 2009

[43] Y Wang T Liu and H Li ldquoEnhancement of triptolide-loadedmicelles on tumorigenicity inhibition of humanovarian cancerrdquoJournal of Biomaterials Science Polymer Edition vol 27 no 7pp 545ndash556 2016

[44] L Liu X Xiong M Shen et al ldquoCo-delivery of triptolideand curcumin for ovarian cancer targeting therapy via mPEG-DPPECaP nanoparticlerdquo Journal of Biomedical Nanotechnol-ogy vol 14 no 10 pp 1761ndash1772 2018

[45] R Munagala F Aqil J Jeyabalan and R C Gupta ldquoBovinemilk-derived exosomes for drug deliveryrdquo Cancer Letters vol371 no 1 pp 48ndash61 2016

[46] A K Agrawal F Aqil J Jeyabalan et al ldquoMilk-derivedexosomes for oral delivery of paclitaxelrdquo Nanomedicine Nan-otechnology Biology and Medicine vol 13 no 5 pp 1627ndash16362017

[47] E Ristorcelli E Beraud P Verrando et al ldquoHuman tumornanoparticles induce apoptosis of pancreatic cancer cellsrdquo 13eFASEB Journal vol 22 no 9 pp 3358ndash3369 2008

[48] Y Takahashi M Nishikawa H Shinotsuka et al ldquoVisualizationand in vivo tracking of the exosomes of murine melanomaB16-BL6 cells in mice after intravenous injectionrdquo Journal ofBiotechnology vol 165 no 2 pp 77ndash84 2013

[49] C P Lai O Mardini M Ericsson et al ldquoDynamic biodistribu-tion of extracellular vesicles in vivo using amultimodal imagingreporterrdquo ACS Nano vol 8 no 1 pp 483ndash494 2014

[50] T Smyth M Kullberg N Malik P Smith-Jones M W Granerand T J Anchordoquy ldquoBiodistribution and delivery efficiencyof unmodified tumor-derived exosomesrdquo Journal of ControlledRelease vol 199 pp 145ndash155 2015

[51] S-F Chan Y-Y Chen J-J Lin et al ldquoTriptolide induced celldeath through apoptosis and autophagy in murine leukemiaWEHI-3 cells in vitro and promoting immune responses inWEHI-3 generated leukemia mice in vivordquo EnvironmentalToxicology vol 32 no 2 pp 550ndash568 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom