Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery · Translational Science Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery Yuan Wan1,2,

Translational Science

Aptamer-Conjugated Extracellular Nanovesiclesfor Targeted Drug DeliveryYuan Wan1,2, Lixue Wang3,4, Chuandong Zhu3, Qin Zheng3, Guoxiang Wang5,Jinlong Tong3, Yuan Fang3, Yiqiu Xia1,2, Gong Cheng1,2, Xia He4, andSi-Yang Zheng1,2,6,7

Abstract

Extracellular nanovesicles (ENV) released by many cells con-tain lipids, proteins, and nucleic acids that contribute to inter-cellular communication. ENVs have emerged as biomarkers andtherapeutic targets but they have also been explored as drugdelivery vehicles. However, for the latter application, clinicaltranslationhasbeen limitedby lowyield and inadequate targetingeffects. ENV vectors with desired targeting properties can beproduced from parental cells engineered to express membrane-bound targeting ligands, or they can be generated by fusionwith targeting liposomes; however, neither approach has met

clinical requirements. In this study, we demonstrate thatmechan-ical extrusion of approximately 107 cells grafted with lipidatedligands can generate cancer cell–targeting ENV and can be pre-pared in approximately 1 hour. This rapid and economicapproach could pave the way for clinical implementation inthe future.

Significance: A new and rapid method for production ofdrug-targeting nanovesicles has implications for cancer treat-ment by chimeric antigen receptor T cells and other therapies.Cancer Res; 78(3); 798–808. �2017 AACR.

IntroductionExtracellular nanovesicles (ENV) are cell-derived lipid bilayer–

enclosed entities with size ranging from30 to300nm(1). ENVs aresecretedbymany cell types andhavebeen identified indiversebodyfluids (2). They are specialized in intercellular communicationsfacilitating transferof cargoproteins andnucleicacids (3). In tumor,growing evidence indicates that ENVs can regulate tumor immuneresponse, initiate formation of premetastatic niche, determineorganotropic metastasis, contribute to chemotherapeutic resis-tance, and enable liquid biopsy for cancer diagnostics (4). More-over, ENVs have been exploited as drug vehicles for drug delivery(5). Compared with micelles, liposomes, and polymeric nanopar-ticles, ENVs as a natural delivery system can evade phagocytosis,

have extended blood half-life, and exhibit optimal biocompatibil-itywithoutpotential long-term safety issues (6). ENVs can fusewiththe cell membrane and deliver drugs directly into cytoplasm. Byevading the engulfment by lysosomes, ENVs remarkably enhancedelivery efficiencyof vulnerablemolecules (5, 7, 8). In addition, thesmall size of ENVs facilitates their extravasation, translocationthrough physical barriers, and passage through extracellular matrix(9). Although ENV-based drug delivery is promising, two majorchallenges exist. First, the yieldofENVs is low (10). Ingeneral, ENVscan be collected from either cultured cell supernatant or autop-lasma of patients. Given the secretion rate of ENVs ranges from 50to 150 per cell per hour, themass production of ENVs for potentialclinical translationwouldbe costly (11, 12).On theother hand, theprevalent ENV isolation techniques, such as ultracentrifugation, failto generate highly pure ENVs (13), let alone the low isolationefficiency. Second, the preparation procedure for current ENV-based targeted drug delivery systems is tedious and labor-intensive.The typical approach is to secrete targeting peptide- or protein-grafted ENVs by transfecting the donor cells with correspondingrecombinant virus or plasmid (14, 15). Of note, these transfectionapproaches have been criticized for inefficiency, potential toxicity,and insertional mutagenesis (16). The "click chemistry" has beenused to conjugate targeting moieties onto EV surface in one-step(17). However, the conjugation procedure is time consuming, andthe reaction conditions must be well controlled to avoid EVdisruption and aggregation (18, 19). Hence, direct surface modi-fication of EV is nontrivial either. In brief, for cancer treatment,nevertheless, simple approaches for large-scale production oftumor-targeting ENVs are desirable.

Here, we report a method for preparing aptamer-grafted ENVsfor paclitaxel loading (Fig. 1). A nucleolin-targeting aptamerAS1411 was covalently conjugated to cholesterol-poly(ethyleneglycol) (cholesterol-PEG) followed by anchoring the compoundsonto livingmouse dendritic cell (DC)membrane. Afterward, cellswere extruded by passing through microconstrictions to obtain

1Department of Biomedical Engineering, Micro and Nano Integrated Biosystem(MINIBio) Laboratory, The Pennsylvania State University, University Park, Penn-sylvania. 2Penn State Materials Research Institute, The Pennsylvania StateUniversity, University Park, Pennsylvania. 3The Second Affiliated Hospital ofSoutheast University, Nanjing, Jiangsu, China. 4Jiangsu Cancer Hospital &Jiangsu Institute of Cancer Research & Nanjing Medical University AffiliatedCancer Hospital, Nanjing, Jiangsu, China. 5Zetect Biomedical Company, Nanjing,Jiangsu, China. 6The Huck Institutes of the Life Sciences, The Pennsylvania StateUniversity, University Park, Pennsylvania. 7Department of Electrical Engineering,The Pennsylvania State University, University Park, Pennsylvania.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Y. Wan and L. Wang contributed equally to this article.

Corresponding Authors: Si-Yang Zheng, Penn State University, 205 HallowellBuilding, University Park, PA 16802-6804. Phone: 814-865-8090; Fax: 814-863-0490; E-mail: [email protected]; and Xia He, Jiangsu Cancer Hospital & InstituteAffiliated to Nanjing Medical University, No. 42 Baiziting Road, Nanjing, Jiangsu210009, China. Phone: 8625-8328-3565; Fax: 8625-8328-3565; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-2880

�2017 American Association for Cancer Research.

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exosome-mimetic ENVs, and paclitaxel was subsequently loadedinto ENVs with sonication. Using this aptamer-grafted ENVs, weinvestigated the ENVs biodistribution and whether paclitaxel canbe more effectively delivered both in vitro and in vivo.

Materials and MethodsCell culture

Cells were cultured in a humidified atmosphere of 5% CO2 at37�C. The MDA-MB-231 cells (ATCC, received in May 2016,passed fluorescence testing for Mycoplasma contamination onAugust 1, 2017, no more than 50 passages) were maintained inDMEM supplied with 10% FBS. Immature mouse DCs wereisolated from bone marrow (20). BALB/c mouse was euthanized.After removal of the femurs, both ends of femurs were trimmed.The contents of marrow were flushed with 2 mL of HBSS with aneedle, and bone marrow cells were washed with HBSS thrice.Approximately 2� 106 cells were cultured in RPMI1640 contain-ing 10% FBS and 0.2 mg rmGM-CSF. Surface expression of CD11c(Santa Cruz Biotechnology, sc-23951) was analyzed.

Selection of lipidFITC-tagged stearoyl-PEG (C18-PEG2000), distearoyl phos-

phethanolamine-PEG (DSPE-PEG2000), and cholesterol-PEG(chol-PEG2000) were dissolved in ethanol at 1 mmol/L. A totalof 3 nmol of each lipid probe was added to 107 DCs in 250 mL ofPBS, respectively. The samples were incubated at 4�C for 5minutes followed by washing thrice. Cells were fixed, stainedwith DAPI, rinsed with PBS, and finally resuspended in 500 mL ofPBS. Cell suspension (20 mL) was added onto slips for imaging.

Synthesis of AS1411-PEG2000-chol and DC labelingThe 100 nmol of disulphide-tagged AS1411 aptamers (San-

gon) were cleaved by TCEP solution on ice for 30 minutes (21),followed by incubation with 500 nmol of chol-PEG2000-mal-eimide (NanoCS) in HEPES buffer (Sigma-Aldrich) overnightat 4�C. The excess chol-PEG2000-maleimide was removedby filtration (MWCO 5000) at 10,000 � g for 15 minutes. Thepurified FITC-AS1411-PEG2000-chol was examined by matrix-assisted laser desorption/ionization—time-of-flight—massspectrometry (MALDI-TOF-MS). A total of 1–5 nmol ofFITC-AS1411-PEG2000-chol was added to 107 DCs in 250 mLof PBS, respectively. Cells were incubated at 4�C for 5 minutesfollowed by washing with PBS thrice. The fluorescence intensitywas measured using an Infinite M200.

Collection and characterization of nanovesiclesA total of 107 AS1411-PEG2000-chol labeled DCs in 1-mL PBS

were continuously filtered with 10-mm and 5-mm track-etchedmembrane (Whatman) at 2,000 rpm for 5 times, and eachfiltration took 5 minutes. The supernatant was centrifuged at300 � g for 5 minutes followed by 16,500 � g for 20 minutes.The supernatantwasfilteredusing0.22-mmfilter. The collectionofENVs spontaneously secreted from DCs follows a general proto-col. In brief, DCs were cultured in serum-free medium (SFM) for48 hours. The medium was centrifuged at 16,500 � g for 20minutes. Afterwards, the medium was filtered using a 0.22-mmfilter followed by ultracentrifugation at 100,000 � g and 4�C for2 hours. The ENV pellets were suspended in 500 mL of PBS.

Five microliters of AS1411-ENV samples were seeded ontosubstrate and fixed. The morphology of AS1411-ENVs was

Figure 1.

Schematics showing steps of experiments (not drawn to scale). The DCs were isolated frommouse bone marrow and multiplied in vitro. Cells were homogeneouslylabeled with aptamer-conjugated PEGylated cholesterol and then repeatedly filtrated through two filters with 10- and 5-mm pore size for generating ENVs.After loading paclitaxel, tumor-targeting ENVs were intravenously administered for tumor treatment in vivo.

Preparation of Cancer-Targeted Extracellular Nanovesicles

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confirmed under Zeiss FESEM. Five microliters of AS1411-ENVsamplewas placedon400mesh grids and incubated for 3minutesat roomtemperature. Excess sampleswere blottedwithfilter paperand stained with 1% uranyl acetate for 1 minute. Samples werethen examined in a FEI Tecnai TEM. For cryo-TEM, 5 mL ofAS1411-ENV sample was applied to a 200 mesh grids blottedfor 1 secondwith FEI Vitrobot before plunging into liquid ethane.Samples were visualized in FEI Tecnai F20 TEM. The number ofAS1411-ENVs was measured using Nanosight LM10 (Malvern).The characterization of ENVs follows the same protocol.

After lysis with RIPA buffer, protein amount in AS1411-ENVsand ENVs were determined using Micro BCA Protein Assay(Pierce). Protein samples were analyzed using acrylamide gels,and then transferred onto polyvinylidene difluoride membranes.The protein blot was blocked for 1 hour at room temperature with5%nonfat drymilk in PBS/0.05%Tween and incubated overnightat 4�Cwith Santa Cruz Biotechnology antibodies against AnnexinII (sc-28385), TSG101 (sc-7964), HSC70 (sc-7298), CD9 (sc-13118), CD59 (sc-133170), and CD55 (sc-51733). Afterward,secondary antibodies were incubated for 1 hour at room temper-ature. Samples were washed with PBS/0.05% Tween 20 for 10minutes thrice. Blots were developed with chemiluminescence.

Ten microliters of AS1411-ENV or ENV samples were seededonto slips and incubated at 4�C for 1 hour. The slip was blockedwith BSA (10 mg/mL) for 20 minutes followed by PBS rinsing. Acomplementary DNA probe of AS1411 aptamer (Cy5-50-CCACCA CAA CCA CC-30) and a control random DNA probe (Cy5-50-TGC TGT GAG TGA ACC TGC TGT GTT GA-30) in 50 mL ofsolution (pH 8.4, 50 mmol/L KCl and 1.5 mmol/L MgCl2) wasincubatedwithAS1411-ENVs or ENVs in a humidified chamber at37�C for 2 hours (22–24). After hybridization, the slip waswashed with PBS for fluorescence analysis.

Drug loading and releasePaclitaxel and nanovesicles weremixed in number ratio of 106.

The mixture was sonicated using a Model 505 Sonic Dismem-brator with 0.25-inch tip with the following settings: 20% ampli-tude, 6 cycles of 30 seconds on/off for 3 minutes with a 2-minutecooling period between each cycle. After sonication, the mixturewas incubated at 37�C for 1 hour. Alternatively, paclitaxel andnanovesicles were mixed in the same ratio, stirred, and incubatedfor 1 hour at room temperature. Excess free drug was removedfrom paclitaxel-loaded AS1411-ENVs or ENVs with a SephadexG25 column. The amount of loaded paclitaxel in respective groupwas measured by high-performance liquid chromatography(HPLC). To measure paclitaxel release, freshly prepared paclitax-el-loaded AS1411-ENVs or ENV were placed in a 300K MWCOfloat-A-lyzer G2 device (Spectrum Laboratories). The device wasthen placed in PBS at room temperature with stirring. Sampleswere taken at time points and analyzed by HPLC.

Therapeutic efficacy in vitro and in vivoThe cytotoxicity of paclitaxel, paclitaxel-loaded ENVs, and

paclitaxel-loaded AS1411-ENVs in MDA-MB-231 cells was eval-uated using MTT assay (Thermo Fisher Scientific). Approximately4,000 MDA-MB-231 cells were treated with paclitaxel in variousconcentrations, ranging from 0 to 3,300 nmol/L. After 24 hours,20 mL of MTT solution (5 mg/mL) was added to each well andincubated for 4 hours. Medium was discarded, and formazanprecipitate was dissolved in 10% SDS in DMSO containing 0.6%acetic acid. The microplates were shaken in the dark for 30

minutes, and absorbance at 570 nm was measured. The IC50

value was determined from the dose–response curves.MDA-MB-231 cells in 6 groups were, respectively, treated

with PBS, ENVs without paclitaxel, AS1411-ENVs withoutpaclitaxel, 200 nmol/L bare paclitaxel, paclitaxel-loaded ENVs,and paclitaxel-loaded AS1411-ENVs for 24 hours. A total of 5 �105 harvested MDA-MB-231 cells in 100 mL 1 � binding buffer(BD Biosciences) were incubated with 10 mL PI and 5 mL ofAnnexin V-FITC for 30 minutes at room temperature. Eachsample was analyzed by flow cytometry. With the same exper-imental setup, cells in 6 groups were stained with PI only andphotographed with high-content imaging system (MolecularDevices).

Approximately 2 � 106 MDA-MB-231 cells in 50-mL PBSmixed with 50 mL of Matrigel were inoculated subcutaneouslyto the flanks of BALB/c mice (�18–22 g, 6 weeks), and allowedto grow to a tumor size approximately 100 mm3. The mice werethen randomly divided into 6 groups. Drug was intravenouslyadministrated every 2 days (7.5 mg of paclitaxel-equivalent perkg of body weight per dose) for 3 weeks. Mice were euthanizedto harvest tumors. To study pharmacokinetics and biodistribu-tion of paclitaxel, 0.1–0.3 g tissue samples were collected at 6time points after treatment. The analysis of paclitaxel by HPLCfollows the published protocol (25). All animal experimentswere approved by and performed in accordance with guidelinesfrom the Institutional Animal Care and Use Committee(IACUC) of the Model Animal Research Center of the SecondAffiliated Hospital of Southeast University (Nanjing, Jiangsu,China).

Statistical analysesResults are presented as mean � SD. Statistical comparisons

were performed by paired Student t test or ANOVA.

ResultsPreparation and characterization of AS1411-ENVs

To efficiently immobilize lipid-conjugated aptamers ontocell membrane for subsequent generation of aptamer-graftedENVs, we first determined the lipid molecule type and optimaldose. PEGylated monoacyl lipid, diacyl lipid, and cholesterolare frequently used (26, 27). Hence, we chose FITC-tagged C18-PEG2000, DSPE-PEG2000, and cholesterol-PEG2000 for optimi-zation. The three lipids can spontaneously insert into cellularlipid bilayer, but show differential labeling effects (Fig. 2A).The average fluorescence intensity of cells labeled with chol-PEG2000 was higher than that of the others (P < 0.01, Fig. 2B).There was no difference in fluorescence intensity between C18-PEG2000 and DSPE-PEG2000. Because C18-PEG2000 and DSPE-PEG2000 can readily assemble forming micelles or liposomesdue to the long hydrophobic tail(s) and hydrophilic head, theself-assembly impairs cell labeling effect. In contrast, chol-PEG2000 can alleviate that to a certain extent as cholesterolitself is amphiphilic and relatively rigid. In addition, choles-terol also can increase the rigidity and stability of liposomes orEVs by enhancing the hydrophobic–hydrophobic interactionsin lipid bilayers (28).

Because the stable retention of lipid-conjugated aptamer oncell surface favors generation of AS1411-ENVs, the retention ofthree lipids over time was evaluated with the correspondingdecay of fluorescence at 4�C and 37�C, respectively. At 4�C, the

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Figure 2.

Optimization of lipid-conjugated aptamer and characterization of nanovesicles.A andB, Fluorescent images of DCs labeledwith three types of PEGylated lipids (C18,DSPE, and cholesterol) in PBS (scale bar, 10 mm) and quantified data of fluorescence intensity of each group (n¼ 150). C,Changes of fluorescence intensity of cells ineach group at 4�C and 37�C, respectively, reflecting retention of each type of lipid on cell membrane (n¼ 150).D, Fluorescence intensity of cells labeled with variousamount of cholesterol (n¼ 150). E,Confirmation of conjugation of AS1411 aptamerwith PEGylated cholesterol withMALDI-TOF-MS. F, Fluorescence image of AS1411-PEG-cholestrol–labeled DCs (scale bar, 10 mm). G, Fluorescence image of AS1411-conjugated ENVs (scale bar, 5 mm). H, Morphologic characterization by electronmicroscopy (scale bar, 100 nm). I, Annexin II, TSG101, HSC70, CD59, CD9, and CD55 were extracted from spontaneously released ENVs and extruded AS1411-ENVs,respectively, and identified by Western blotting. J, Changes in size of ENVs and AS1411-ENVs stored at �80�C in PBS for up to 6 months (n ¼ 5). K, Fluorescenceintensity of dots of ENVs and AS1411-ENVs hybridizedwith h-probes and c-probes, respectively (n¼ 20). L–M, Fluorescence images showing ENVs and AS1411-ENVstaken up by MDA-MB-231 cells in 30 minutes, respectively (scale bar, 10 mm), and quantified data of fluorescence intensity of each group (n ¼ 25).






fluorescence intensity of cells labeled with C18-PEG2000, DSPE-PEG2000, and chol-PEG2000, respectively, decreased to 93.2%,97.71%, and 95.74%, indicating excellent retention over 100minutes at low temperature (Fig. 2C). In contrast, at 37�C, thefluorescence intensity of cells labeled with C18-PEG2000, DSPE-PEG2000, and chol-PEG2000, respectively, gradually decreasedto 48.63%, 56.98%, and 65.04% over the same period (Fig.2C). We did not further measure the fluorescence intensitiesbeyond 100 minutes as the extrusion of labeled cells for gener-ating adequate ENVs only takes less than 30minutes. Consideringchol-PEG2000 demonstrated a superior labeling effect and anexcellent retention at low temperature, we chose chol-PEG2000

for the following studies.Next, we optimized the dose of chol-PEG2000 for cell mem-

brane labeling. The insufficient label with cholesterol-conjugatedaptamers causes unsaturated aptamer immobilization on cellsurface. On the contrary, overlabeling might trigger plasmamem-brane bubbling and destroy the integrity of the lipid bilayer. Thefluorescence intensity significantly increased with the dose ofchol-PEG2000 ranging from 1 to 4 nmol (P < 0.05; Fig. 2D). Toidentify the labeling efficiency of chol-PEG2000, we compared thefluorescence intensity of FITC-tagged chol-PEG2000 at variousdoses before and after labeling (Supplementary Fig. S1A). Thelabeling efficiency (fraction of fluorescence intensity differenceover initial FITC-tagged chol-PEG2000) of chol-PEG2000 rangingfrom 1 to 5 nmol (1–5 mL of 1 mmol/L chol-PEG2000 added into250 mL of cell suspension, Supplementary Fig. S1B) was 15.33%,20.03%, 20.05%, 22.09%, and18.73%, respectively (Supplemen-tary Fig. S1C). Therefore, we chose 4 nmol of chol-PEG2000 forlabeling of approximately 107 cells, and accordingly we deter-mined that approximately 5.3 � 107 chol-PEG2000 moleculeswere incorporated into an individual DC.

AS1411-PEG2000-chol was synthesized and confirmed byMALDI-TOF-MS (Fig. 2E). Excess free lipids were removed, andthen the amount of AS1411-PEG2000-chol was determined byUV-visible spectroscopy. Meanwhile, DCs were isolated and expand-ed in vitro in 5 days (Supplementary Fig. S2A). Following theoptimized protocol of cell labeling, we identified FITC-taggedAS1411-PEG2000-chol can be grafted onto DC membrane(Fig. 2F). After labeling, we loaded approximately 107 DC cells(purity: approximately 90%, characterized with CD11c, Supple-mentary Fig. S2b) into an extruder for generating AS1411-ENVs(29). The morphology of partial AS1411-ENV was observed witha super-resolution microscopy (Nikon) in SIM mode (Fig. 2G).The average diameter of AS1411-ENV measured by microscopyranged from 110 to 310 nm. Nanosight revealed the size distri-bution of AS1411-ENVs ranged from 50 to 270 nm with peakconcentration at 103 nm. In contrast, in the control group, thediameter of ENVs spontaneously secreted from unlabeled DCswas 30–240 nm with peak concentration at 98 nm. In addition,approximately 8.7 � 1010 AS1411-ENVs were prepared by extru-sion of approximately 107 cells in 25minutes. Under the assump-tion that the whole-cell membrane would participate in formingENVs during extrusion, the production efficiency of AS1411-ENVsusing our protocol was nearly 30%, and there is still room forimprovement. In comparison, only 3.4 � 1010 ENVs were har-vested fromapproximately 6.75� 107 cells cultured in SFM for 48hours. The generation efficiency of nanovesicles by extrusion wasapproximately 17-fold higher and 115-fold faster than that ofspontaneous secretion from cells. Under electron microscopy,there was no significant difference in morphology between

AS1411-ENVs and ENVs (Fig. 2H). Both displayed a typicalsaucer-shaped morphology. The cryo-TEM and cryo-SEM imagesalso demonstrate that both were spherical and enclosed by a lipidbilayer.

We also directly incubated bare ENVs with FITC-taggedAS1411-PEG2000-chol and determined the labeling efficiency.Given we used 4 nmol to label approximately 107 cells, followingthe same ratio of lipid amount to totalmembrane surface area, welabeled approximately 7 � 1010 bare ENVs with 2 nmol/L ofAS1411-PEG2000-chol in 250-mL suspension at 4�C for 5minutes.After removal of free aptamer–conjugated lipids with ultracen-trifugation, we recovered approximately 3 � 1010 AS1411-ENVs.The size at peak concentration was 105 nm (Supplementary Fig.S3A). Moreover, we found the fluorescence intensity of AS1411-ENVs prepared by extrusion (�5� 108 in 10 mL) was higher thanthat of equivalent AS1411-ENVs prepared by direct labeling (P <0.05, Supplementary Fig. S3B).We speculated that under the sameexperimental setting labeling ENVs with nanoscale lipids mightbe inefficient thanmicroscale cells' labeling. Theoperationof ENVlabeling is not comparable with cell labeling either. The addi-tional purification with ultracentrifugation not only requires atleast 2-hour processing time but also further decreases the finaloutput. Therefore, direct ENV labeling with targeting ligandsworks despite low efficiency.

Cargo proteins extracted from approximately 2� 108 AS1411-ENVs and ENVs were analyzed by Western blot analysis. Threecytosolic proteins, including Annexin II, TSG101, andHSC70 andthree surface proteins, including CD59, CD9, and CD55, wereidentified in both AS1411-ENVs and ENVs (Fig. 2I), indicatingthat these proteins can be enclosed into AS1411-ENVs duringextrusion of cells. The CD55 and CD59 protect extracellularvesicle (EV) from attack of the complement system, making themstable in the blood (30, 31). Tetraspanin CD9 facilitates directmembrane fusion with the target cell and contents-deliverydirectly into cytosol (32). In addition, we chose immature DCsas they are devoid of lymphocyte stimulatory molecules such asMHC II, CD80 or CD86 (33). The stability of AS1411-ENVs andENVs were examined by monitoring changes in size at respectivepeak concentration. The cryopreserved AS1411-ENVs and ENVswith approximately 100 nm in diameter did not show significantaggregation or degradation in PBS at �80�C over 6 months(Fig. 2J), indicating that AS1411-ENVs could be prepared inlarge-scale, cryopreserved in batches for at least 6 months, andthen thawed for use.

We further used Cy5-conjugated hybridization probe (h-probe) of AS1411 and random probe as a control (c-probe) toreconfirm the existence of AS1411 on conjugated ENVs. Thefluorescence intensities demonstrated only the dot blots ofAS1411-ENVs incubated with h-probes showed strong red fluo-rescence signals (Fig. 2K). In comparison, due to aminute amountof residual probes, weak fluorescence signals were detected in therest three groups. Next, approximately 8� 105 AS1411-ENVs andENVs were incubated with MDA-MB-231 cells expressing nucleo-lin on cell membrane, respectively (34). We identified thatAS1411-ENVs efficiently bound MDA-MB-231 cells in only 10minutes (Fig. 2L and M). In contrast, equal amount of PKH67-labeled ENVs barely fused with MDA-MB-231 cells at the sametime point. After 30minutes, a few ENVs attached and fused withcell membrane, while significant amount of AS1411-ENVs wereobserved in the experimental group (P < 0.01). The findingsindicate that AS1411-ENVs can rapidly recognize and bind onto

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cancer cell surface nucleolin, and thus would facilitate targeteddrug delivery.

Characterization of paclitaxel-loaded nanovesiclesPaclitaxelwas loaded intoAS1411-ENVs andENVs (in absolute

number ratio of paclitaxel/vesicle: 106), respectively, using eithersonication or simple incubation at room temperature. The aver-age paclitaxel-loading efficiency with sonication was 21.33% �1.53% in AS1411-ENVs and 26.4% � 2.67% in ENVs. In com-parison, paclitaxel loading efficiency with simple mixing, stirringand incubation was 5.06%� 1.12% in AS1411-ENVs and 7.73%� 2.38% in ENVs, respectively. During sonication, themembranemicroviscosity significantly decreases, allowing drugmolecules toenter vesicle, and the membrane integrity can be restored imme-diately after sonication. It does not significantly affect the mem-brane-bound proteins or the lipid contents of the ENVs (35). Ofnote, the negatively charged aptamers on the outer surface mightaffect encapsulation of negatively charged paclitaxel, causingslightly lower loading efficiency in comparison with that of ENVs.As the loading efficiency of sonication was much higher than thatof incubation, we used sonication for paclitaxel loading in thefollowing studies. After paclitaxel loading, the whole-size distri-bution showed a slight rightward shift, and the size of AS1411-ENVs and ENVs at peak concentration increased to 111 and109 nm (black curves), respectively (Fig. 3A). In addition, afterloading of paclitaxel, the mean zeta potential of AS1411-ENVsdecreased from �23.7 mv to �25.6 mv, and that of ENVsdecreased from �11.8 mv to �16.1 mv (Fig. 3B). The threecytosolic proteins including Annexin II, TSG101, and HSC70were barely detectable in approximately 2 � 108 AS1411-ENVsand ENVs after paclitaxel loading, respectively. These synteninsmight be released from vesicles during sonication (Fig. 3C).

Moreover, we investigated the release kinetics of paclitaxel fromAS1411-ENVs and ENVs at 37�C, respectively (Fig. 3D). Bothpaclitaxel-loaded nanovesicles showed burst release within thefirst 1 hour, and then displayed a sustained release profile there-after. At 24-hour time point, paclitaxel-loaded AS1411-ENVs andENVs released approximately 71.47% and 86.29% paclitaxel,respectively. The slow release of paclitaxel from nanovesicles isfavorable for drug administration. Especially in targeted drugdelivery, more drugs in vectors thus can be effectively deliveredto the lesion. The ability to deliver paclitaxel into target cells invitrowas studied by identifying the IC50 value of paclitaxel-loadedAS1411-ENVs, paclitaxel-loaded ENVs, and bare paclitaxel,respectively, after cell treatment for 24 hours, and we determinedthe respective IC50 value of paclitaxel in the above three groups as14.24, 35.08, and 212.69 nmol/L (Fig. 3E). The treatment efficacyof paclitaxel-loaded AS1411-ENVs and paclitaxel-loaded ENVsexhibited a 15-fold and 6-fold enhancement compared with barepaclitaxel in vitro. Of note, AS1411 as therapeutic effects, andAS1411 for acute myelogenous leukemia is being evaluated inphase II clinical trial (36). On the basis of generated AS1411-ENVamount and AS1411 grafting amount, we deduced that eachindividual AS1411-ENV bears approximately 2,000 AS1411molecules. The amount is adequate for tumor targeting in vivo(37), but has very limited therapeutic effects. The dose of AS1411in tumor therapy in vivo is 10mg/kg/d or even higher, whereas theamount of AS1411 for targeting is less than 4% of the therapeuticdose. We speculated the significant decrease of the IC50 value ingroup of AS1411-ENVs was mainly attributed to high deliveryefficiency of paclitaxel to cells through the targeted ENVs.

Cancer treatment efficacy in vitro and in vivoWe used 200 nmol/L paclitaxel-loaded AS1411-ENVs, pacli-

taxel-loaded ENVs, bare paclitaxel, and three negative controls totreat MDA-MB-231 cells, respectively. In groups of paclitaxel-loaded AS1411-ENVs and paclitaxel-loaded ENVs, approximately90% of cells were effectively blocked in G2–M phase, whereas inbare paclitaxel group, approximately 60% of cells had beenretained in G2–M phase (Fig. 3F and G). The paclitaxel-loadedAS1411-ENVs and paclitaxel-loaded ENVs demonstrated highertherapeutic efficacy in comparison with bare drugs as paclitaxel-loaded nanovesicles could efficiently deliver drugs to target cellsby membrane fusion and AS1411-mediated internalization. Incontrast, bare negatively charged paclitaxel molecules rely onpassive diffusion for crossing negatively charged cell membrane.The entry mode bypasses the endosomal–lysosomal pathway(38), and thus both AS1411-ENVs and ENVs as delivery vehiclescan circumvent the need for endosomal escape strategies. Thehigh-content screening also confirmed significantly more deadcells (stained in red) in groups of paclitaxel-loaded AS1411-ENVsand paclitaxel-loaded ENVs in comparison with that of barepaclitaxel group and three negative control groups (Fig. 3H). Ofnote, after paclitaxel treatment, dead cells detached from flasksurface during staining and rinsing, and thus fewer cells were leftin the three experimental groups.

Next, we investigate the potential of paclitaxel-loaded AS1411-ENVs andpaclitaxel-loaded ENVs in drug delivery using theMDA-MB-231 xenograft BALB/c mouse model. Tissue samples (heart,liver, spleen, lung, kidney, and tumor) were collected, and pac-litaxel concentration in eachorganwas analyzedwithHPLC. At 30minutes postadministration, paclitaxel underwent rapid distribu-tion to the main organs in each group (Fig. 4A). In bare paclitaxelgroup, paclitaxel easily diffused into other organs but less pacli-taxel accumulated in tumors. In the next 12 hours, bare paclitaxelwas readily cleared from main organs and tumors. In groups ofpaclitaxel-loaded AS1411-ENVs and paclitaxel-loaded ENVs, pac-litaxel concentration in tumors was significantly higher than thatin bare paclitaxel group at all time points, indicating encapsula-tion of paclitaxel into AS1411-ENVs and ENVs can achieve in vivoredistribution of paclitaxel. The clearance rate of paclitaxel-loadedAS1411-ENVs and paclitaxel-loaded ENVs were also slower thanthat of bare paclitaxel. We noted that both paclitaxel-loadednanovesicles show relatively high concentration of paclitaxel inliver and spleen at the 12-hour time point. A previous study alsoreported that EVs displayed a predominant localization of intra-venously administered EVs in the spleen and the liver (39). Ingeneral, the intravenous injection of nanoparticles with size over50 nm results in intensive recognition by microphages that takenanoparticles to the liver, meanwhile 150–250 nm nanoparticlesalso frequently accumulate in the spleen (40). In addition, pac-litaxel-loaded AS1411-ENVs continued to accumulate in tumorsand reached peak concentration at 12-hour time point, showingsignificantly different (P < 0.01) pharmacokinetics comparedwith the other two groups (Fig. 4A). The tumor volumes of micetreated with PBS (from 167.06 � 12.20 to 1,041.59 � 238.3mm3), ENVswithout paclitaxel (from167.55� 12.36 to 1,059.67� 339.68 mm3), and AS1411-ENVs without paclitaxel (from168.53 � 18.04 to 1,110.32 � 284.27 mm3) rapidly increased,and there was no significant difference among the three negativecontrols (Fig. 4B and C). The final tumor weight of mice treatedwith PBS, ENVs without paclitaxel, and AS1411-ENVs withoutpaclitaxel were 0.94 � 0.20 g, 0.96 � 0.32 g, and 1.00 � 0.26 g,






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respectively. Tumors that received bare paclitaxel showed a slightgrowth inhibition due to low bioavailability, and thus tumorvolume and weight increased from 168.96 � 16.56 mm3 to283.28 � 90.65 mm3 by the end of treatment. paclitaxel-loadedENVs inhibit the growthof tumor, and the volumedecreased from169.7 � 17.01 mm3 to 97.99 � 30.08 mm3. Paclitaxel-loadedAS1411-ENVs significantly restrained growth of grafted tumors,and the tumor volume decreased from 169.58 � 18.04 mm3 to35.34 � 3.38 mm3 (P < 0.05), which could be attributed to theactive targeting effect in tumor treatment (Fig. 4B and C). At theend of treatment, the relative tumor volume of each group was6.19 � 1.01 (without paclitaxel), 6.24 � 1.49 (ENVs withoutpaclitaxel), 6.58� 1.4 (AS1411-ENVs without paclitaxel), 1.66�0.43 (bare paclitaxel), 0.57� 0.15 (paclitaxel-loaded ENVs), and0.21� 0.04 (paclitaxel-loaded AS1411-ENVs), respectively. Aver-age tumor weight of paclitaxel-treated three groups was 0.18 �0.089 g (bare paclitaxel), 0.05 � 0.016 g (paclitaxel-loadedENVs), and 0.02 � 0.002 g (paclitaxel-loaded AS1411-ENVs),respectively (Fig. 4D). The in vivo therapeutic efficacy of paclitaxel-loaded AS1411-ENVs was improved approximately 9- and 3-foldcomparedwith that of bare paclitaxel andpaclitaxel-loaded ENVs,respectively (P < 0.05). There was no significant difference inmouse body weight during the 3-week administration (Fig. 4E).Furthermore, tumor proliferation and the histologic structures oftumors and organs from all groups were analyzed (Fig. 4F;Supplementary Fig. S4). Compared with other formulations,paclitaxel-loaded AS1411-ENVs caused remarkable tumor tissuedamage and reduced the percentage of proliferating Ki67-positivetumor cells, indicating the enhanced treatment efficacy. We alsomeasured the systemic toxicity. Bone marrow suppression is themajor dose-limiting toxicity of paclitaxel, and dose-/schedule-dependent neutropenia is the typical hematologic toxicity. Over10 days after intravenous administration, a routine blood test wasperformed to exam leukocytes counting and function enzymes ofliver and kidney. Themean counts of leukocytes, erythrocytes, andplatelets were relatively lower in bare paclitaxel-treated mice;meanwhile, the average level of five major indexes of kidney andliver function was also relatively higher (Fig. 4G). However, nosignificant difference was observed among 6 groups, and theirlevels werewell within the normal physiologic range. Thefindingsalso indicated paclitaxel-loaded AS1411-ENVs or paclitaxel-load-ed ENVs overcame the systemic toxicity. The tumor-bearing micecould tolerate higher dose of paclitaxel-encapsulated in targetingENVs without the occurrence of severe systemic toxicities. Theencapsulation would increase the chemotherapeutic effects whileminimizing exposure to normal and healthy cells.

DiscussionThe field of EV-based drug delivery has greatly expanded over

the last few years, and many studies have eloquently demon-strated that EV can function as therapeutic nanocarriers for

delivering a variety of cargos, including siRNAs, miRNAs,proteins, and chemotherapeutics to particular tissue (41–44).Compared with existing delivery systems, the outstandingadvantage of EVs as delivery vehicles is their favorable lipidand surface protein composition. Currently, cells such as epi-thelial cells and model cancer cell lines have been used asdonor cells for harvesting derived EVs. However, immunoge-nicity and biosafety concerns arise for in vivo applications ofthese donor cells derived EVs. Particularly, cancer cell line–derived EVs as drug delivery vehicles might possess tumorigenicpotential due to incomplete depletion of cargos and/or inherenttumor-associated proteins imbedded on EV membrane. In com-parison, DCs can be harvested from a patient's bone marrow orperipheral blood by standard protocols (45). They should thus beless immunogenic than any foreign delivery vehicle. The in vitromultiplication of DCs can constantly provide EVs, which isextremely important for multiple episodes of treatment. On theother hand, at present, only a small portion of published reportsutilize a targeting strategy for direct delivery of the therapeuticcargos. The mainstream routine for harvesting derived EVs relieson expression of targeting protein or peptide on cell membraneafter sophisticated gene transfection (8, 14, 33, 46). Alternatively,targeting ligands can be covalently immobilized onto membraneproteins via click chemistry.However, both technique routines arequite challenging.Moreover, theworkload required to upscale theproduction of cells and the resulting yield of EVs is currently notsuitable for clinical use.

The method we developed can easily create adequate targetingligand–grafted ENVs in 1hour for drug loading.Our approach hasmultiple advantages. First, both targeting ligands (lipid-conjugat-ed aptamers or palmitoylated antibodies) and donor cells(immune cells, stem cells, or others) can be arbitrarily geared tomeet actual need. Theoretically, lipidated ligands targeting estro-gen-related receptor beta 2 (ERRB2), prostate-specific antigen(PSA), carbonic anhydrase IX (CA IX), or other targets frequentlyused in chimeric antigen receptor (CAR) T-cell therapy could beproduced in large-scale andgraftedonto autologous immune cellsfor targeted therapy of different cancers. The whole procedurewould be very flexible. On the contrary, the bioengineered cellsthrough gene transfection only express designated protein orpeptide. To construct cells expressing another specific membraneprotein, the preparation of viral or plasmid vectors, transfection,and selection have to be performed once again. Second, donorcells potentially canbeobtained frompatient's autologous cells togenerate ENVs for drug loading. Genetic engineering is not nec-essary. Therefore, it is much safe than cell-based immunotherapy,such as CAR T-cell therapy. Although CAR T-cell therapy wasrecently approved, the efficacy, toxicity, potential mutagenesisand easy-to-operation are still big concerns (47, 48). Third, wedemonstrated generation efficiency and rate of ENVs via extrusionof cells is 17- and 115-fold higher than that of ENVs spontane-ously released by donor cells. Cells harvested from a T75 flask can

Figure 3.Characterization of paclitaxel (PTX)-loaded nanovesicles and cancer cell treatment in vitro. A, Size distribution of AS1411-ENVs and ENVs before paclitaxelloading (red curve) and after paclitaxel loading (black curve). B, Changes of zeta potential of ENVs and AS1411-ENVs before and after paclitaxel loading. C,Annexin II, TSG101, and HSC70 extracted from paclitaxel-loaded ENVs and AS1411-ENVs, respectively, and confirmed with Western blotting. D, In vitro release ofpaclitaxel from ENVs and AS1411-ENVs at 37�C, respectively. E, Respective IC50 values of bare paclitaxel, paclitaxel-loaded ENVs, and paclitaxel-loaded AS1411-ENVsafter 24-hour cell incubation. F and G, Cell cycle of MDA-MB-231 cells treated with 200 nmol/L bare paclitaxel or paclitaxel equivalent in nanovesicles for24 hours, respectively, and quantified data of cell cycles of each group. H, Fluorescence images of MDA-MB-231 cells treated with 200 nmol/L bare paclitaxel, orpaclitaxel equivalent–loaded ENVs, and paclitaxel-loaded AS1411-ENVs, stained with propidium iodide and scanned with high-content screening platform.






Figure 4.

Tumor treatment with paclitaxel-loaded nanovesicles in vivo. A, Biodistribution of paclitaxel in heart, liver, spleen, lung, kidney, and tumor in three groups,respectively, after an intravenous administration (n¼ 6).B,MDA-MB-231 tumor tissues obtained from each group of euthanizedmice 20 days after administration ofpaclitaxel� (i), ENV paclitaxel� (ii), AS1411-ENV paclitaxel� (iii), paclitaxelþ (iv), ENV paclitaxelþ (v), and AS1411-ENV paclitaxelþ (vi), respectively (n¼ 6). C and D,Tumor volume and weight of MDA-MB-231 tumor xenograft in mice from each group after drug or placebo administration (n ¼ 6). E, Average mass of micefrom each group without significant difference (n ¼ 6). F, Typical histologic sections of tumors stained with hematoxylin and eosin and Ki67, respectively. Thepaclitaxel-loaded AS1411-ENVs group demonstrated significant tumor tissue damage and the lowest Ki67 index, indicating high treatment efficacy (n ¼ 6). G,Systemic toxicity profile of bare paclitaxel, paclitaxel-loaded ENVs, and paclitaxel-loaded AS1411-ENVs. There was no significant difference in levels of leukocytes,erythrocytes, and liver and kidney function enzymes (n ¼ 6).

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generate approximately 8 � 1010 ENVs via extrusion, satisfying asingle dose, and the production can easily be scaled up. Therelatively low cost and the ease of operation would benefit theclinical implementation. Fourth, using our method, donor cellscan be safely, efficiently, and homogenously labeled with lipid-conjugated ligands in assigned ratio within 5 minutes, ensuringderived individual ENVs grafted with targeting ligands. Celllabelingwith aptamer-conjugated cholesterol only needs to deter-mine the amount of cells and lipidated aptamers. In contrast,neither immobilization of targeting ligands onto EV membranevia click chemistry nor in fusion of EVs with ligands graftedliposomes can be easily and precisely performed at nanoscale(49). Altogether, the extrusion of targeting ligands grafted cells forgenerating ligands functionalized ENVs is a rapid, easy, andeconomic approach to prepare sufficient drug delivery vehicles.The enhanced in vivo therapeutic efficacy and low systemic toxicitymake this approach conductive to potential clinical translation inthe future.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Wan, C. Zhu, Q. Zheng, G. Wang, J. Tong, Y. Fang,Y. Xia, G. Cheng, X. He, S.-Y. ZhengDevelopment of methodology: Y. Wan, L. Wang, C. Zhu, Q. Zheng, G. Wang,J. Tong, Y. Fang, Y. Xia, G. Cheng, X. He, S.-Y. Zheng

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Wan, L. Wang, C. Zhu, Q. Zheng, G. Wang, J. Tong,Y. Fang, Y. Xia, G. Cheng, X. He, S.-Y. ZhengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.Wan, L. Wang, C. Zhu, Q. Zheng, G.Wang, J. Tong,Y. Fang, Y. Xia, G. Cheng, X. He, S.-Y. ZhengWriting, review, and/or revision of the manuscript: Y. Wan, L. Wang, C. Zhu,Q. Zheng, G. Wang, J. Tong, Y. Fang, Y. Xia, G. Cheng, X. He, S.-Y. ZhengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Wan, C. Zhu, Q. Zheng, G. Wang, J. Tong,Y. Fang, Y. Xia, G. Cheng, X. He, S.-Y. ZhengStudy supervision:C.Zhu,Q. Zheng,G.Wang, J. Tong, Y. Fang, Y. Xia,G.Cheng,X. He, S.-Y. Zheng

AcknowledgmentsS. Zheng thanks the Penn State Materials Research Institute, the Huck

Institute of Life Sciences, the Penn State Microscopy and Cytometry Facility.This work was supported by Nanjing Science and Technology DevelopmentFoundation Number 201605031 (to Q. Zheng), Jiangsu Provincial MedicalYouth Talent AwardQNRC2016054 (to L.Wang), NanjingMedical Science andTechnology Development Foundation-Youth Program Number ZDX16008 (toX. He), Natural Science Foundation of Jiangsu Province BK20170134 (to C.Zhu), and National Cancer Institute of the NIH under award numberDP2CA174508 (to S. Zheng).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 20, 2017; revised October 26, 2017; accepted December1, 2017; published OnlineFirst December 7, 2017.

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2018;78:798-808. Published OnlineFirst December 7, 2017.Cancer Res Yuan Wan, Lixue Wang, Chuandong Zhu, et al. DeliveryAptamer-Conjugated Extracellular Nanovesicles for Targeted Drug

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Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery · Translational Science Aptamer-Conjugated Extracellular Nanovesicles for Targeted Drug Delivery Yuan Wan1,2,