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NATURE MATERIALS DOI: 10.1038/NMAT3049 ...
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Nanoparticles that communicate in vivo to amplify tumour targeting

Published on: Mar 3, 2016
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Transcripts - Nanoparticles that communicate in vivo to amplify tumour targeting

  • 1. ARTICLES PUBLISHED ONLINE: 19 JUNE 2011 | DOI: 10.1038/NMAT3049Nanoparticles that communicate in vivo toamplify tumour targetingGeoffrey von Maltzahn1,2 , Ji-Ho Park3 , Kevin Y. Lin4 , Neetu Singh1 , Christian Schwöppe5 ,Rolf Mesters5 , Wolfgang E. Berdel5 , Erkki Ruoslahti6,7 , Michael J. Sailor8,9and Sangeeta N. Bhatia1,10,11,12 *Nanomedicines have enormous potential to improve the precision of cancer therapy, yet our ability to efficiently home thesematerials to regions of disease in vivo remains very limited. Inspired by the ability of communication to improve targetingin biological systems, such as inflammatory-cell recruitment to sites of disease, we construct systems where syntheticbiological and nanotechnological components communicate to amplify disease targeting in vivo. These systems are composedof ‘signalling’ modules (nanoparticles or engineered proteins) that target tumours and then locally activate the coagulationcascade to broadcast tumour location to clot-targeted ‘receiving’ nanoparticles in circulation that carry a diagnostic ortherapeutic cargo, thereby amplifying their delivery. We show that communicating nanoparticle systems can be composedof multiple types of signalling and receiving modules, can transmit information through multiple molecular pathways incoagulation, can operate autonomously and can target over 40 times higher doses of chemotherapeutics to tumours thannon-communicating controls.A dvances in nanotechnology have produced a diverse toolkit amplification, positive feedback, ubiquitous presence in plasma and of individual nanodevices with unique electromagnetic potential to operate across multiple tumour types (Fig. 1c). We properties1–3 and the ability to encapsulate and pro- hypothesized that two signalling modules could selectively activategrammably release a diversity of therapeutics4–9 , yet the ultimate the coagulation cascade in tumours: NPs (gold nanorods, NRs)biomedical efficacy of such devices largely depends on their in that target tumours and convert external electromagnetic energyvivo fate. Over the past three decades, approaches to targeting into heat to locally disrupt tumour vessels, and engineered humannanomaterials in vivo have focused on tuning the properties of indi- proteins (tumour-targeted tissue factor, tTF) that autonomouslyvidual nanoparticles (NPs) including geometry, surface chemistry, survey host vessels for angiogenic tumour receptors and, in theirligand type and ligand density10–18 . These materials are typically presence, activate the extrinsic coagulation pathway (Fig. 1c).administered as populations of >1 trillion NPs to carry out iden- Receiving modules were constructed using two nanomaterialtical, competitive functions in vivo. Here, inspired by the power of platforms: a prototypical imaging agent (magnetofluorescent ironcommunication to improve targeting across multiple length scales oxide nanoworms, NWs) and a prototypical therapeutic agentin biological systems (for example, insect swarming, immune-cell (doxorubicin-loaded liposomes, LPs). We explored the potential totrafficking, platelet self-assembly), we considered the design of route communication to receivers through two molecular pathwaysNP systems that communicate to enhance in vivo diagnostics, in coagulation by developing peptide coatings that recognize fibrinregenerative medicines and therapeutics. directly and peptides that target coagulation enzyme activity by We set out to construct two-component nanosystems from well- acting as a substrate for the coagulation transglutaminase factorcharacterized NP and biological components, wherein signalling XIII (FXIII) (Fig. 1c).modules would first target tumours and then broadcast the We first set out to examine the capacity of signalling modulestumour’s location to receiving NPs in circulation (Fig. 1a). To to precisely induce coagulation in tumours (Fig. 2a). To test ourfacilitate rapid and robust signal transmission in regions of tumour hypothesis that photothermal heating of gold NRs could disruptgrowth, we sought to harness the machinery of an endogenous tumour blood vessels to initiate extravascular coagulation19–22 ,multistep biological cascade to transmit communications (Fig. 1b) we examined the transduction of tumour heating into localizedand selected the coagulation cascade due to its powerful signal coagulation by evaluating fibrin deposition in tumours as a function1 Harvard–MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts02139, USA, 2 Flagship VentureLabs, Flagship Ventures, 1 Memorial Dr. 7th Fl, Cambridge, Massachusetts 02142, USA, 3 Department of Bio and BrainEngineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea, 4 Department of ChemicalEngineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA, 5 Department ofMedicine/Hematology and Oncology, University Hospital Muenster, D-48129 Muenster, Germany, 6 Vascular Mapping Laboratory, Center forNanomedicine, Sanford-Burnham Medical Research Institute at UCSB, 3119 Biology II Bldg, University of California, Santa Barbara, California 93106-9610,USA, 7 Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA, 8 Materials Science and EngineeringProgram, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman, La Jolla, California 92093, USA, 9 Department ofChemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, USA, 10 Electrical Engineering and Computer Science,MIT, Massachusetts 02142, USA, 11 David H. Koch Institute for Integrative Cancer Research, MIT, Massachusetts 02142, USA, 12 Department of Medicine,Brigham and Women’s Hospital, Howard Hughes Medical Institute, Massachusetts 02115, USA. *e-mail: sbhatia@mit.edu.NATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials 545 © 2011 Macmillan Publishers Limited. All rights reserved
  • 2. ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT3049 a that heat specifically directed coagulation-cascade activation in tumours (Fig. 2b, Supplementary Fig. S1). Immunohistochemical staining for fibrin(ogen) in tumours from uninjected mice Blood vessel corroborated these findings, demonstrating that exogenous fibrinogen administration did not artificially drive accumulation in heated tumours (Supplementary Fig. S1). Having probed the thermal sensitivity of coagulation in tumours, we next investigated whether tumour-targeted gold NRs could specify coagulation to occur in tumour tissues. Rod-shaped gold NPs are precisely tunable plasmonic nanomaterials that may be Tumour synthesized in bulk, have narrow size distributions and have optical absorption coefficients 104 –106 times higher than those of con- ventional organic fluorochromes19–21 . Previously, we demonstrated b Input c Signalling that polyethylene glycol-coated gold NRs (PEG–NRs) have >17 h (energy, tumour receptor) circulation half-lives in mice and can passively target tumours in mice through their fenestrated angiogenic blood vessels to direct or precise tumour heating with otherwise benign NIR energy (Supple- Nanorods Targeted mentary Fig. S2; refs 23–25). Because NIR light can penetrate several tissue factor centimetres in human tissue, it provides an attractive external Signalling input to actuate vascular disruption in tumours26 . To examine XII XIIa NR-directed coagulation, PEG–NRs (10 mg Au kg−1 ) or saline were XI XIa TF intravenously administered to mice bearing bilateral MDA-MB-435 IX IXa VIIa VII VIIIa tumours (Fig. 2c,d). After PEG–NR clearance from circulation Biological (72 h post-injection), fluorescent fibrinogen was intravenously cascade X Xa X injected and the right flanks of mice were irradiated with NIR Va XIII light (∼1 W cm−2 ), generating focal tumour surface temperatures II IIa XIIIa of ∼49 ◦ C in PEG–NR-injected mice, while saline-injected tumour surface temperatures remained below ∼37 ◦ C (Fig. 2c). At 24 h Fibrinogen Fibrin post-injection, irradiated tumours on NR-injected mice displayed localized accumulation of fibrinogen (Fig. 2d), while tumours Receiving with PEG–NRs or NIR energy alone and peripheral tissues lacked or this feature. Histopathological analysis revealed that fibrin(ogen) Output Nanoworms Liposomes deposition formed a broad interstitial mesh in heated tumours, (diagnosis, therapy) Receiving indicating that NR heating could disrupt tumour blood vessels to activate extravascular coagulation (Supplementary Fig. S2).Figure 1 | Nanoparticles communication for amplified tumour targeting. We next investigated the potential for a biological signallinga, Schematic representation of communication between system module to autonomously survey the host vasculature for angiogeniccomponents. Tumour-targeted signalling NPs broadcast the tumour tumour receptors and, in their presence, engage the extrinsiclocation to receiving NPs in circulation. b, Harnessing a biological cascade coagulation cascade. Such a system would operate without theto transmit and amplify NP communications. c, Molecular signalling need for any external electromagnetic inputs (for example NIRpathway between the signalling and receiving components. Signalling and energy) and could potentially amplify NP targeting to deep-seatedreceiving components act as unnatural inputs and outputs to the and disseminated cancers. We used a truncated, tumour-targetedcoagulation cascade, respectively. Signalling components are either version of the human protein tissue factor (tTF–RGD), whichtumour-targeted plasmonic gold NRs, which initiate coagulation cascade harnesses an RGD peptide motif to induce coagulation on bindingactivation in tumours by photothermally disrupting tumour vessels and to angiogenic αv β3 receptors27–31 (Fig. 2e). When tTF is separatedactivating the extrinsic and intrinsic coagulation pathways, or from essential cell surface lipid co-factors, its activity towards FXtumour-targeted truncated tissue factor proteins, which are latent in activation diminishes by five orders of magnitude32 . This nearlycirculation and activate the extrinsic coagulation pathway on binding to digital dependence on cell-surface localization has enabled tumour-tumour receptors. Communication is exploited to recruit inorganic (iron targeted tTFs to specifically activate coagulation in mouse canceroxide nanoworms) or organic (drug-loaded LPs) receiving components models and, recently, in human cancer patients27,28 . As with PEG–through activity of the coagulation transglutaminase FXIII or through NR signalling modules, we first probed the relative accumulationtargeting of polymerized fibrin. of fluorescently labelled fibrinogen and albumin in MDA-MB-435 tumours of mice injected with varying doses of tTF–RGD proteins.of temperature (Fig. 2a). Purified fibrinogen (the precursor to At 24 h after injection, we observed a macroscopic appearancefibrin) and albumin (an abundant blood protein unrelated to of haemorrhage in tumours on mice injected with tTF–RGDcoagulation) were labelled with unique near-infrared (NIR) (>15 µg tTF–RGD/mouse), corresponding to the tumour-specificfluorochromes to allow simultaneous assessment of coagulation- accumulation of fibrinogen in dendritic, vascular patterns, whichdependent and independent protein tropism to heated tumours. were absent from control tumours (Fig. 2f,g, Supplementary Fig.Mixtures of fibrinogen and albumin were intravenously injected S3). Microscopically, this appearance of vascular coagulation wasinto athymic (nu/nu) mice bearing bilateral human MDA-MB- corroborated by the abundant localization of fibrin(ogen) within435 tumours, after which one tumour was heated using a tumour blood vessels (Fig. 2g).temperature-controlled water bath. At 24 h, mice were killed and Together, we found that both PEG–NR and tTF–RGD signallingthe relative levels of tumour fibrin(ogen) and albumin were assessed modules produced tumour-specific coagulation, highlighting thefluorescently. We observed a marked induction of fibrin(ogen) potential for localized coagulation to communicate the tumour’saccumulation in tumours between 45 ◦ C and 53 ◦ C, with little location to receivers in circulation. We next set out to developaccompanying increase in albumin accumulation, indicating receiving NPs that could efficiently target regions of coagulation546 NATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials © 2011 Macmillan Publishers Limited. All rights reserved
  • 3. NATURE MATERIALS DOI: 10.1038/NMAT3049 ARTICLES a Input e Input f Saline near-infrared light tumour receptor phosphatidylserine 50 nm tTF¬RGD Oscillations of Signal: electron cloud local Signal: ∆T coagulation Fibrin local coagulation Fibrin Signalling Signalling b ¬ 41 °C 45 °C 49 °C 53 °C g tTF¬RGD ¬ 15 µg 25 µg 50 µg Fibrinogen = green; albumin = red c Signalling NPs: PEG¬NRs Saline 50 Fibrinogen = green; albumin = red 40 T (°C) h ¬ tTF¬RGD 30 25 High d Fluorescence Low Fibrinogen = green; CD31 = red; nuclei = blueFigure 2 | ‘Signalling’ component characterization. a, Schematic representation of NR-directed coagulation and transmission electron microscopy ofNIR-absorbing NRs. Gold NRs are targeted to tumours to specify local coagulation-cascade activation through photothermal conversion of NIR energy.b, Probing the coagulation-dependent and independent protein tropism to heated tumours. Fibrinogen and albumin were labelled with unique NIRfluorochromes and injected into mice bearing bilateral MDA-MB-435 tumours. Immediately following injection, one tumour on each mouse was heatedusing a temperature-controlled water bath. At 24 h post-injection, mice were dissected and tumours imaged for the relative abundance of fibrinogen(green) and albumin (red). c, Thermographic imaging of PEG–NR- and saline-injected mice under NIR irradiation of the right flank. d, Fluorescencereflectance imaging of mice to visualize fibrinogen tropism to PEG–NR-heated tumours. e, Schematic representation of tumour-targeted tissue factorstimulation of the coagulation cascade in response to tumour receptors. Signalling components are ligand-targeted, truncated human tissue factor proteins(tTF–RGD) proteins that are latent in circulation and autonomously gain coagulation-inducing activity on binding to αv β3 receptors in tumour blood vesselsand associating with endothelial cell surface phosphatidylserine. f, Intraoperative images at 24 h post tissue factor injection revealing tTF–RGD-mediatedhaemorrhaging. g, Probing the coagulation-dependent and independent protein tropism to tumours on tTF–RGD-injected mice. tTF–RGD signallingcomponents were injected intravenously at varying doses alongside mixtures of fluorescent fibrinogen (green, VT750) and albumin (red, VT680) tomonitor tTF–RGD-mediated coagulation in tumours. h, Histopathologic analysis of tumour fibrinogen distribution without (left) and with (right) 25 µgtTF–RGD signalling component co-injection (red = CD31 blood-vessel stain; green = injected fibrinogen fluorescence; blue = nuclear stain; scalebars = 100 µm).to deliver therapeutics or act as imaging agents (Fig. 3a). Initially, tumours. The specificity of heat-induced targeting to coagulationmagnetofluorescent iron oxide nanoworm imaging agents (NWs; persisted up to 53 ◦ C, although the magnitude of accumulationFig. 3b top) were derivatized with a peptide substrate for the decreased (Fig. 3e), probably indicating that higher temperaturescoagulation transglutaminase FXIII (G–N– Q–E–Q–V–S–P–L–T– accelerated intravascular coagulation and occlusion, diminishingL–L–K–X–C–fluorescein)33–35 to enable receiver incorporation into the perfusion required for delivery of receiving NWs into tumours.regions of active coagulation (Fig. 3b bottom, Supplementary Histopathologically, FXIII–NWs showed marked extravasation andFig. S4). Having observed that external heating of tumours interstitial spreading in heated tumours compared with controlsproduced localized coagulation, we used this response in an assay (Fig. 3d, Supplementary Fig. S5), illustrating the capacity of thermalto assess the ability of receivers to target tumour coagulation before energy to dismantle tumour vascular barriers and direct abundantintegrating them with signalling modules. Mixtures of targeted interstitial receiver accumulation.and untargeted NWs, labelled with unique NIR fluorochromes, We also explored the feasibility of channelling communicationswere intravenously injected into mice bearing two MDA-MB- through an alternative molecular pathway in the coagulation cas-435 tumours. Immediately following injection, one tumour was cade. NWs were derivatized with a fibrin-binding peptide (Ac–d–d–submerged in a temperature-controlled water bath for 20 min and d–G–Y–e–C–hyP–cY–G–L–C–Y–I–Q–K–fluorescein; Fig. 3b) andmice were dissected at 24 h for fluorescent organ imaging. We tested in a similar assay. Fibrin-binding receiving NWs also exhib-found that the accumulation of FXIII–NW receivers was sharply ited nearly a tenfold amplification of targeting to heated tumoursamplified at 45 ◦ C compared with FXIIIControl–NW-bearing (Fig. 3c, Supplementary Fig. S5), with prominent extravascularpeptides without the essential glutamine for FXIII cross-linking accumulation histopathologically (Supplementary Fig. S5).(Fig. 3c,d, Supplementary Fig. S5), enabling nearly an order-of- We next constructed model therapeutic receiving modulesmagnitude increase in tumour targeting compared with unheated to provide amplified drug delivery to regions of tumourNATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials 547 © 2011 Macmillan Publishers Limited. All rights reserved
  • 4. ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT3049 a Output b Receiving modules: c remote imaging, drug delivery ¬ + Fibrin-binding NWs Untargeted NWs ∆T or Signal: Iron oxide NWs LPs local coagulation Fibrin-binding peptide ¬ + Ac–d–d–d–G–Y–e–C–hyP–cY–G–L–C–Y–I–Q–K–FI Factor XIII substrate FXIII¬NWs G–N–Q–E–Q–V–S–P–L–T–L–L–K–X–C–FI FXIIIControl¬NWs Receiving d e f FXIII¬NWs FXIII¬LPs 12 FXIIIControl¬NWs 80 FXIIIControl¬LPs over unheated tumour ∗ over unheated tumour ∗ Fold accumulation Fold accumulation 10 60 8 ∗ ∗ 6 40 ∗ 4 20 2 0 0 37 41 45 49 53 37 41 45 49 53 Tumour temperature (°C) Tumour temperature (°C) FXIII¬NWs = green CD31 = red Nuclei = blueFigure 3 | ‘Receiving’-component synthesis and testing. a, Schematic representation of receiving-NP homing to regions of coagulation. NW imagingagents and drug-loaded LPs (top and bottom, respectively) were derivatized with coagulation-targeting peptides to form receiving NPs. b, Nanostructureand targeting ligands of receiving NPs. Transmission electron microscopy images of the two classes of nanomaterials used in receiving-NP synthesis: ironoxide NWs (scale bar = 50 nm) and doxorubicin-loaded LPs (scale bar = 400 nm). Two peptides were used to generate receiving NPs: a fibrin-bindingpeptide and a glutamine-containing substrate for the coagulation transglutaminase FXIII to respectively direct particle binding and covalent attachment inregions of coagulation. c, Fluorescence reflectance imaging of receiving-NP homing to externally heated tumours. Mixtures of targeted (green) anduntargeted (red) NWs, labelled with the unique NIR fluorochromes VT750 and VT680, respectively, were intravenously injected into mice bearing bilateralMDA-MB-435 tumours. Immediately following injection, one tumour was submerged in a temperature-controlled water bath for 20 min and mice weredissected at 24 h for fluorescent organ imaging. Overlaid fluorescence images are shown for targeted (green) and untargeted (red) receiving-NPaccumulation in both heated (+, 45 ◦ C heating) and naive (−) tumours from the same mouse. d, Histopathological analysis of receiving-NP homing toheated tumours. Histological sections from naive (top) and heated (bottom, 45 ◦ C) tumours in FXIII–NW-injected mice were stained for CD31 (red) andnuclei (blue) and imaged to reveal receiving-NP distribution (green). (Scale bars = 100 µm.) e, Quantifying the amplification of FXIII–NW andFXIIIControl–NW receiver homing to heated over unheated tumours. The fold enhancement of NW targeting is plotted across the range of temperaturestested (P = 0.02, 0.03 and 0.04 for the difference between FXIII–NWs and FXIIIControl–NWs at 45 ◦ C, 49 ◦ C and 53 ◦ C, respectively; paired, two-sidedt-test, n = 4; error bars = s.d.). f, Quantifying the amplification of FXIII–LP and FXIIIControl–LP receiver homing to heated over unheated tumours. The foldenhancement of doxorubicin accumulation in tumours is plotted across the range of temperatures tested for FXIII–LPs and FXIIIControl–LPs (P = 0.025 andP = 0.049 for the difference between FXIII–NWs and FXIIIControl–NWs at 45 ◦ C and 49 ◦ C, respectively; unpaired, two-sided t-test, n = 3; errorbars = s.d.).coagulation. Therapeutic receivers were developed by synthesizing mouse and then the individual explanted organs were fluorescentlydoxorubicin-loaded LPs with tethered active (FXIII) or inactive imaged (Fig. 4b). Thermographic surveillance of photothermal(FXIIIControl) substrates (Supplementary Figs S4, S6). Here, heating showed focal tumour heating only in NR-injected micetumour heating to 45 ◦ C directed the accumulation of over 40 (Fig. 4c) and whole-animal imaging at 96 h revealed pronouncedtimes higher doses of doxorubicin in tumours compared with homing of FXIII–NWs to NR-heated tumours, with over anunheated controls and significantly enhanced targeting over order-of-magnitude increase in accumulation above unirradiatedinactive FXIIIControl substrate-modified LPs (Fig. 3f). contralateral tumours and tumours on saline-injected mice Having developed signalling and receiving modules and (Fig. 4d,e, Supplementary Fig. S7). Histologically, integrated NPcharacterized their functions in isolation, we proceeded to study systems produced intense regions of FXIII–NW fluorescencethe ability of integrated NP systems to communicate and amplify relative to controls, particularly in tumour boundaries where bloodtumour targeting in vivo (Fig. 4a). We began by testing the ability vessels were well perfused (Supplementary Fig. S8). NP systemsfor communication to amplify the targeting of magnetofluorescent were found to be effective in xenograft cervical tumour modelsFXIII–NW receiving modules to tumours. PEG–NRs (or saline) as well, directing several-fold amplification in homing of targetedwere intravenously injected into mice bearing bilateral MDA- receiving NPs over untargeted controls (Supplementary Fig. S9).MB-435 tumours. After NR clearance from circulation (72 h), We next probed the ability of autonomous communicationmixtures of active and inactive receiving NPs (FXIII–NWs and between tTF–RGD signalling modules and FXIII–NW receivingFXIIIControl–NWs) labelled with distinct NIR fluorochromes modules to amplify tumour targeting (Fig. 4f). When co-injectedwere co-injected intravenously, followed by NIR irradiation of the alongside FXIII–NW receivers (Fig. 4g), we find that tTF–RGDentire right flank of the mouse (∼0.75 W cm−2 , 810 nm, 20 min) signalling modules amplify receiver targeting by several-foldunder infrared thermographic observation. At 96 h, the entire over non-communicating controls and over NWs that are548 NATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials © 2011 Macmillan Publishers Limited. All rights reserved
  • 5. NATURE MATERIALS DOI: 10.1038/NMAT3049 ARTICLES a Input Output b Inject signalling NPs Inject receiving NPs c Signalling NP: imaging near-infrared light (PEG¬NRs or saline) (FXIII¬/control¬NWs) NRs Saline 46 42 0h 72 h 96 h 38 T (°C) Irradiate Image/dissect Oscillations of Signal: 34 electron cloud local 30 ∆T coagulation f Input Output Signalling Receiving tumour receptor imaging 26 phosphatidylserine e 20 ∗ Receiving NPs: Fold increase in targeting over unirradiated tumour 16 FXIII¬NWs d FXIIIControl¬NWs 12 Signal: local 8 coagulation 4 Signalling Receiving g Co-inject: tumour-targeted tissue factor 0 and FXIII¬NWs NRs Saline Signalling NPs h Signalling: tTF¬RGD tTF¬RGD ¬ ¬ 0h 24 h Receiving: FXIII¬NWs FXIIICont¬NWs FXIII¬NWs RGD¬NWs Image/dissect Receiving FXIII¬NWs = green NPs: FXIIIControl¬NWs = red High i Fluorescence Lung Heart Low High Fluorescence Fat Brain j Signalling: tTF¬RGD tTF¬RGD ¬ ¬ Receiving: FXIII¬NWs FXIIIControl¬NWs FXIII¬NWs RGD¬NWs Skin Muscle Low Tumour Tumour Receivers = green; CD31 = red; nuclei = blueFigure 4 | Amplified tumour targeting with two systems of communicating NPs. a, Schematic representation of communicating NPs. b, Experimentaltimeline for testing communicating NPs. c, Thermographic imaging of photothermal PEG–NR heating. At 72 h post NR or saline injection (10 mg Au kg−1 ),mice were co-injected with coagulation-targeted FXIII–NWs and untargeted FXIIIControl–NWs and their right flanks were broadly irradiated (810 nm,∼0.75 W cm−2 , 20 min) under infrared thermographic surveillance to reveal surface temperatures. d, Overlaid fluorescence reflectance image of targetedand untargeted receiving-NP homing. At 24 h post-irradiation, whole-animal fluorescence imaging revealed the distributions of coagulation-targeted(FXIII–NWs, green) and untargeted (FXIIIControl–NWs, red) receiving NPs. e, Quantification of receiving-NP homing in irradiated versus contralateralunirradiated tumours. After whole-animal imaging, mice were dissected and the fluorescence of each tumour was measured to quantify the homing ofreceiving NPs. (∗ indicates P = 0.02, paired, two-sided t-test; n = 4; error bars = s.d.) f, Schematic representation of a nanosystem that communicatesautonomously in the presence of tumour receptors. g, Experimental timeline for testing the autonomous nanosystem in vivo. h, Intraoperative imaging ofNW receivers. Nu/nu mice bearing a single MDA-MB-435 tumour were intravenously injected with communicating (tTF–RGD+ FXIII–NWs) or control(tTF–RGD + FXIIIControl–NWs) systems, FXIII–NWs alone or NWs targeted by the peptide used to direct signalling-component tumour homing (1 mg kg−1tTF–RGD). At 24 h post-injection, tumours were surgically exposed for fluorescent intraoperative imaging of NW homing. (FXIIICont–NWs =FXIIIControl–NWs.) i, Tumour specificity of the autonomous nanosystem. Excised organs from mice injected with autonomously communicatingnanosystems (tTF–RGD + FXIII–NWs) were imaged for NW fluorescence at 24 h post-injection (1 mg kg−1 tTF–RGD). j, Histopathological analysis of NWreceivers. Histopathological sections from experiments in h. At 24 h post-NW injection, mice were killed and tumours were analysed for NW receiverdistribution in histology. (Red = CD31 blood-vessel stain, blue = 4,6-diamidino-2-phenylindole nuclear stain, green = NW receiver distribution, RGD–NWscale bar = 100 µm; all others = 200 µm.)directly targeted by RGD-targeting ligands (Fig. 4h, Supplementary receiving modules (Fig. 4i, Supplementary Fig. S10). Further, weFig. S10). Similar to the fibrin(ogen) distribution observed found that tTF–NGR signalling modules, which target CD13during tTF–RGD signalling-module testing, FXIII–NW receivers angiogenic receptors, were also able to amplify receiver targetinginjected alongside tTF–RGD proteins produced a dendritic to tumours (Supplementary Fig. S10), highlighting the capacitypattern of accumulation in tumours, corresponding to abundant for autonomously communicating systems to be customized forintravascular localization immunohistochemically (Fig. 4h–j). specific molecular cancer signatures.This amplified vascular targeting was found to be specific for As a proof of principle that NP communication could improvetumours over normal organs and was absent when the coagulation tumour drug delivery and therapy, we studied the efficacy of ainhibitor heparin was administered alongside signalling and therapeutic communicating nanosystem (Fig. 5a). We found thatNATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials 549 © 2011 Macmillan Publishers Limited. All rights reserved
  • 6. ARTICLES NATURE MATERIALS DOI: 10.1038/NMAT3049 a Input Output b 12 c near-infrared light drug delivery ∗ Receiving NPs 10 FXIII¬LPs %ID doxo/g tumour FXIIIControlLPs 8 6 Signal: 4 Oscillations of electron cloud local 2 ∆T coagulation Signalling Receiving 0 Laser ¬ + ¬ + NRs Saline Signalling NPs FXIII¬LPs = green Doxorubicin = red Nuclei = blue d e 6 Signalling/receiving NRs + FXIII¬LPs 5 NRs + FXIIIcont¬LPs NRs Relative tumour volume Saline + FXIII¬LPs 4 Saline + FXIIIcont¬LPs Saline (–laser) Saline 3 2 1 ∗ ∗ ∗ ∗ ∗ ∗ 0 NRs + FXIII¬LPs 0 5 10 15 20 Time (days)Figure 5 | Amplified tumour therapy with communicating NPs. a, Schematic representation of a therapeutic system of communicating NPs.b, Quantification of doxorubicin-loaded LP receiver homing in irradiated versus contralateral unirradiated tumours. At 96 h after signalling NP injection,mice were dissected and the doxorubicin fluorescence of each tumour homogenate in acidic ethanol was measured to quantify the homing of receivingNPs. (∗ indicates P = 0.021, unpaired, two-sided t-test, n = 4; error bars = s.d.). c, Histopathological analysis of NR-directed FXIII–LP targeting anddoxorubicin delivery. Histopathological sections from the integrated NP signalling experiments in b. At 24 h post NW injection, mice were killed andtumours were analysed for FXIII–LP and doxorubicin distributions in histology. (Red = doxorubicin, blue = 4,6-diamidino-2-phenylindole nuclear stain,green = FXIII–LP distribution.) (Scale bars = 100 µm.) d, Tumour volumes following a single treatment with communicating NP systems and controls.Tumours in all treatment groups except ‘Saline (− laser)’ were exposed to NIR irradiation for 20 min (∼0.75 W cm−2 , ∼810 nm, arrow) 72 h afterintravenous NR or saline injection (P < 0.05 for NR + FXIII–LPs and all other treatment sets between days 8 and 24; analysis of variance, n = 7 mice in eachset; error bars = s.d.). e, Representative images of mice treated with communicating NPs (NRs+ FXIII–LPs, below) compared with untreated controls(Saline, above) (20 d post-treatment).communication between NR signalling modules and FXIII–LP irradiation with NIR energy (∼0.75 W cm−2 , 810 nm, 20 min). Wereceivers amplified the accumulation of doxorubicin in tumours found that a single treatment with communicating NPs directedby over 40-fold (∼8% ID g−1 ) as compared with the LPs alone a prolonged inhibition of tumour growth that was significantly(Fig. 5b) and more than sixfold when compared with an optimized more effective than system components in isolation (FXIII–LPs,LP formulation that targeted endogenous vascular receptors (αv β3 FXIIIControl–LPs, NRs) and non-communicating control systemsfor high-affinity cyclic-RGD peptide-targeted LPs), illustrating the (NRs + FXIIIControl–LPs; Fig. 5d,e; p < 0.05 for NR + FXIII–LPspotential for NP communication to amplify drug delivery over NPs compared with all other treatment groups at each day from 5 to 24directly targeted to tumour receptors (Supplementary Fig. S11). after treatment; one-sided t -test) without detectable weight loss dueThis amplification of drug delivery probably has at least two to systemic toxicity (Supplementary Fig. S11).components: heat-dependent increases in passive accumulation Inspired by communication in biological systems, we deviseddue to improved extravasation in tumours (as indicated by NP systems that communicate to amplify tumour targeting. WeFXIIIControl–LPs and consistent with previous observations22 ) demonstrated that systems of synthetic human proteins andand specific biochemical recognition of the coagulation process simple NPs can be engineered to transmit information throughby the peptide coating. Histologically, FXIII–LPs formed a broad endogenous biological pathways by acting as artificial inputs andinterstitial mesh in NR-heated tumours, with released doxorubicin outputs to the coagulation cascade. In contrast with ‘combination’fluorescence emanating from the nuclei of surrounded tumour therapies, where multiple disease pathways in the host are targetedcells, confirming the delivery and release of active drug within simultaneously, our strategy was composed of components thattumour tissues (Fig. 5c). communicate with one another to more efficiently target regions We finally evaluated the therapeutic efficacy of communicating of disease. We found that communication through the coagulationNPs in mice bearing single MDA-MB-435 human carcinoma cascade enhanced the accumulation of receiving modules intumours. PEG–NRs (10 mg kg−1 ) or saline were injected into tumours by up to 40-fold relative to receiving modules tested in themice and, once NRs had cleared from circulation (72 h), a absence of communication (Supplementary Fig. S12). Further, wesingle intravenous dose of FXIII–LPs, FXIIIControl–LPs or saline found that, after subtracting the baseline targeting of receivers with-(2 mg kg−1 doxorubicin) was given, followed immediately by out communication, each NR signalling module in a host tumour550 NATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials © 2011 Macmillan Publishers Limited. All rights reserved
  • 7. NATURE MATERIALS DOI: 10.1038/NMAT3049 ARTICLESwas able to recruit more than 150 FXIII–NWs or more than 35,000 in PBS (2 mg Fe kg−1 ) to tumour-bearing nu/nu mice to provide an internaldoxorubicin molecules encapsulated within FXIII–LPs (Supple- control reference for coagulation-specific NW homing. At 24 h post NW injection,mentary Fig. S12), demonstrating the capacity for signal amplifica- mice were killed and organs were analysed for both NIR fluorophores (LI-COR Odyssey Infrared Imaging System). For integrated NP-system characterization,tion in our approach. Similarly, each tTF–RGD signalling module mice were additionally imaged under isofluorane anaesthetic before killingthat accumulated in a host tumour was able to recruit more than using a whole-animal fluorescence reflectance imaging system (Xenogen,ten FXIII–NWs through induction of localized coagulation (Sup- IVIS Imaging System) to visualize the specificity of NW homing to tumours.plementary Fig. S12). The ability for each tumour-receptor-targeted Images from both organ scanning and whole-animal imaging are displayed throughout the manuscript as overlaid green–red images from both fluorescencetTF–RGD signalling module in our system to recruit many NP channels (VT750 = green and VT680 = red). For autonomously communicatingreceiving modules contrasts to conventional strategies for ligand- nanosystems, NIR-fluorophore-labelled peptide-bearing NWs (bearing VT750mediated NP targeting, where, depending on NP valency, one or fluorophores) were intravenously (2 mg Fe kg−1 ) in PBS to unanaesthetizedfewer NPs are delivered per ligand-bound receptor in tumours. MDA-MB-435 tumour-bearing nu/nu mice alone or alongside various tTF We believe that this work motivates a paradigm of ‘systems signalling modules (25 µg). At 24 h post NW injection, mice were killed and organs were analysed for NIR-receiver fluorescence (LI-COR Odyssey Infrared Imagingnanotechnology’ directed toward the construction of communica- System). For intraoperative fluorescent tumour imaging, mice were anaesthesizedtive diagnostic and therapeutic agents with sophisticated in vivo and tumours were surgically exposed to reveal detailed tumour fluorescencebehaviours. Given the diverse NP and synthetic biological ‘building (LI-COR). For whole-animal organ distribution, tTF–RGD signalling modulesblocks’ under development13–18,36–38 , coupled with the plethora of were administered intraperitoneally (25 µg) and FXIII–NWs were administeredrobust biological cascades that could be re-purposed to enable intravenously (2 mg Fe kg−1 ) to mice bearing a single MDA-MB-435 tumour. Quantification of receiver homing to tumours. Protocols for fluorescentcommunication between synthetic components, we believe that a quantification of NW and doxorubicin quantification are provided inwide array of nanosystems could be engineered to more sensitively Supplementary Information.locate, diagnose and treat a diversity of focal human diseases. Therapeutic assessment of communicating and control NP systems. Therapeutic studies were conducted by first intravenously administeringMethods PEG–NRs or saline into nu/nu mice bearing a single MDA-MB-435 tumour.Signalling-module synthesis. Long-circulating PEG–NRs were synthesized with At 72 h post-injection, mice were intravenously administered FXIII–LPs,5 kDa methoxy PEG–thiol coatings as described previously23 and tTF–RGD and FXIIIControl–LPs or saline (in a ∼150 µl bolus) and broadly irradiated in thetTF–NGR signalling modules were expressed in engineered Escherichia coli, purified vicinity of the tumour with NIR light (810 nm, ∼1 W cm−2 , 20 min). An additionaland tested in vitro to verify purity (>95%) and activity (factor-X coagulation test) cohort of mice was administered saline at 0 and 72 h and not exposed to NIR lightas described in Supplementary Information. to isolate any therapeutic efficacy of this input in isolation. At regular intervals after treatment, tumours were measured and mice were weighed. Mice were killed whenReceiving-module synthesis. Peptide synthesis. The three peptides used in tumours exceeded 500 mm3 .this work were synthesized through 9-fluorenyl-methoxycarbonyl solid-phasepeptide synthesis, purified by high-performance liquid chromatography to Received 15 November 2010; accepted 17 May 2011;>90% purity, and characterized through mass spectrometry as described in published online 19 June 2011Supplementary Information. Iron oxide NW synthesis. Superparamagnetic, dextran-caged iron oxide NWs Referenceswith a longitudinal size of ∼55 nm were synthesized, aminated using 20% v/v 1. Chan, W. C. & Nie, S. Quantum dot bioconjugates for ultrasensitiveammonium hydroxide, and derivatized with NIR fluorophores as described nonisotopic detection. Science 281, 2016–2018 (1998).previously11 . All peptide-functionalized NWs were characterized through dynamic 2. Park, J. H. et al. Magnetic iron oxide nanoworms for tumour targeting andlight scattering and intravenously injected in vivo to ensure all targeted NWs and imaging. Adv. Mater. 20, 1630–1630 (2008).control NWs exhibited similar circulation times. NIR-fluorophore and peptide 3. Xia, Y. N. & Halas, N. J. Shape-controlled synthesis and surface plasmonicattachment protocols, along with NW purification methods, are provided in properties of metallic nanostructures. MRS Bull. 30, 338–344 (2005).Supplementary Information. 4. Gref, R. et al. Biodegradable long-circulating polymeric nanospheres. Science Doxorubicin-loaded LP synthesis. Hydrogenated soy 263, 1600–1603 (1994).sn-glycero-3-phosphocholine (HSPC), cholesterol and 1,2-distearoyl-sn-glycero- 5. Sengupta, S. et al. Temporal targeting of tumour cells and neovasculature with3-phosphoethanolamine-N -polyethylene glycol 2000 (DSPE-PEG(2k)) and a nanoscale delivery system. Nature 436, 568–572 (2005).1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene 6. Park, J. H., von Maltzahn, G., Ruoslahti, E., Bhatia, S. N. & Sailor, M. J.glycol 2000)] (DSPE-PEG(2k)-MAL) were purchased from Avanti Polar Lipids. Micellar hybrid nanoparticles for simultaneous magnetofluorescent imagingDoxorubicin was purchased from Sigma Chemical. Briefly, for targeted LP and drug delivery. Angew. Chem. Int. Ed. 47, 7284–7288 (2008).synthesis, LPs with maleimide groups were prepared from HSPC, cholesterol, 7. Litzinger, D. C. & Huang, L. Phosphatidylethanolamine liposomes—drugDSPE-PEG(2k) and DSPE-PEG(2k)-MAL by lipid film hydration and membrane delivery, gene-transfer and immunodiagnostic applications.extrusion. Encapsulation of doxorubicin into the LPs was then carried out Biochim. Biophys. Acta 1113, 201–227 (1992).using a pH-gradient-driven loading protocol. Free doxorubicin was removed 8. Akinc, A. et al. A combinatorial library of lipid-like materials for delivery ofby gel filtration on Sephadex G-50 and the maleimide-terminated LPs were RNAi therapeutics. Nature Biotechnol. 26, 561–569 (2008).reacted with thiols on peptides (FXIII and FXIIIControl) for 2 h and then 9. Anderson, D. G., Lynn, D. M. & Langer, R. Semi-automated synthesis andpurified by gel filtration. screening of a large library of degradable cationic polymers for gene delivery. Angew. Chem. Int. Ed. 42, 3153–3158 (2003).In vivo studies. All studies in mice were approved by the Massachusetts Institute of 10. Leserman, L. 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USA 99, 12617–12621 (2002).sections of tumours were prepared. The sections were first fixed with acetone. Rat 13. Hood, J. D. et al. Tumour regression by targeted gene delivery to theanti-mouse CD-31 (1:50, BD PharMingen) and biotinylated mouse fibrin(ogen) neovasculature. Science 296, 2404–2407 (2002).antiserum (1:50, Nordic) were used for immunochemical staining of tumour tissue 14. Farokhzad, O. C. et al. Nanoparticle–aptamer bioconjugates: A new approachsections. The corresponding secondary antibodies were added and incubated for for targeting prostate cancer cells. Cancer Res. 64, 7668–7672 (2004).1 h at room temperature: AlexaFluor-594 goat anti-rat or rabbit IgG (1:1,000; 15. Weissleder, R., Kelly, K., Sun, E. Y., Shtatland, T. & Josephson, L. 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Work in the Muenster 316–317 (2001). laboratory is supported by Deutsche Forschungsgemeinschaft (SFB 656/C8 Mesters)27. Kessler, T. et al. Inhibition of tumour growth by RGD peptide-directed delivery and German Cancer Aid (109245 Berdel). G.v.M. acknowledges support from Whitaker of truncated tissue factor to the tumour vasculature. Clin. Cancer Res. 11, and NSF Graduate Fellowship. The authors thank P. Caravan for assistance with 6317–6324 (2005). the fibrin-binding peptide selection and testing, D. Kim, S. Mo, L. Ong and M. Xu28. Bieker, R. et al. Infarction of tumour vessels by NGR-peptide directed targeting for assistance with in vivo studies and R. Weissleder for assistance with preliminary of tissue factor. Experimental results and first-in-man experience. Blood 113, fluorescent imaging studies. 5019–5027 (2009).29. Huang, X. M. et al. Tumour infarction in mice by antibody-directed targeting Author contributions of tissue factor to tumour vasculature. Science 275, 547–550 (1997). G.v.M. and S.N.B. conceived the communication strategy, analysed results and wrote the30. El-Sheikh, A., Borgstrom, P., Bhattacharjee, G., Belting, M. & Edgington, T. S. manuscript; G.v.M., J-H.P., K.Y.L. and N.S. designed and carried out experiments; C.S., A selective tumour microvasculature thrombogen that targets a novel receptor R.M., W.E.B., E.R. and M.J.S. contributed reagents and technical expertise. complex in the tumour angiogenic microenvironment. Cancer Res. 65, 11109–11117 (2005).31. Persigehl, T. et al. Antiangiogenic tumour treatment: Early noninvasive Additional information monitoring with USPIO-enhanced MR imaging in mice. Radiology 244, The authors declare no competing financial interests. Supplementary information 449–456 (2007). accompanies this paper on www.nature.com/naturematerials. Reprints and permissions32. Paborsky, L. R., Caras, I. W., Fisher, K. L. & Gorman, C. M. Lipid association, information is available online at http://www.nature.com/reprints. Correspondence and but not the transmembrane domain, is required for tissue factor activity. requests for materials should be addressed to S.N.B.552 NATURE MATERIALS | VOL 10 | JULY 2011 | www.nature.com/naturematerials © 2011 Macmillan Publishers Limited. All rights reserved

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