Signaling molecules establish trigger coagulation cascade to attract therapeutic nanoparticles to cancer site.

Scientists have developed a two-stage communicating nanoparticle approach to tissue targeting and drug delivery that they claim can target 40 times higher doses of chemotherapeutics to tumors than traditional nanoparticles. Basing the new approach on communications systems found in nature such as insect swarming, immune-cell trafficking, and platelet self-assembly, the collaborative team led by Massachusetts Institute of Technology professor Sangeeta Bhatia, M.D., has devised a system that combines signaling nanoparticles or engineered proteins with receiving nanoparticles that carry the diagnostic or therapeutic agents.

Essentially, the signaling molecules home in on the tumor and activate the coagulation cascade, which results in recruitment of the clot-targeting receiving nanoparticles to the tumor site to deliver their cargo. Reporting in Nature Materials, the researchers claim their work provides the groundwork for a systems nanotechnology approach to targeting that could lead to more sensitive location, diagnosis, and treatment of tissue- or cell-specific diseases. Their paper is titled “Nanoparticles that communicate in vivo to amplify tumour targeting.”

Current approaches for targeting nanomaterials in vivo have focused on tuning the properties of individual nanoparticles (NPs) including their geometry, surface chemistry, ligand type, and ligand density, Dr. Bhatia and team notes. In contrast, the two-stage approach led by the MIT researchers and their collaborators uses two types of nanoparticles that work in concert.

They hypothesized that two signaling modules could selectively activate the coagulation cascade in tumors: NPs (gold nanorods, NRs) that target tumors and convert external electromagnetic energy into heat to locally disrupt tumor vessels, and engineered human proteins—more specifically tumor-targeted tissue factor, tTF—which autonomously survey host vessels for angiogenic tumor receptors and, in their presence, activate the extrinsic coagulation pathway. The receiving modules were constructed using a prototype imaging agent (magnetofluorescent iron oxide nanoworms, NWs) and a prototypical therapeutic agent comprising doxorubicin-loaded liposomes (LPs).

The first step was to test the capacity of the signaling modules to induce coagulation in tumors. Previous work had confirmed that PEG-coated gold NRs have a circulation half-life of over 17 hours in mice and can passively target tumors. The researchers intravenously administered PEG-NRs into tumor-bearing mice, and subsequently irradiated the treated tumors with near-infrared (NIR) light to increase focal tumor surface temperatures. After 24 hours the irradiated tumors in NR-treated mice demonstrated evidence that tumor blood vessel disruption had activated extravascular coagulation.

The researchers then moved on to investigate the potential for a biological signaling module to autonomously survey the host vasculature for angiogenic tumor receptors and, in their presence, engage the extrinsic coagulation cascade. This type of system would operate without the need for any external electromagnetic inputs (such as NIR energy) and could potentially amplify NP targeting to deep-seated and disseminated cancers, they note. For this the team used a truncated, tumor-targeted version of the human protein tissue factor (tTF-RGD), which harnesses an RGD peptide motif to induce coagulation on binding to angiogenic αvβ3 receptors. As occurred following PEG-NR administration, the tumors of mice injected with t-TF-RGD proteins also demonstrated vascular coagulation.

The next step was to construct receiving NPs that could efficiently target regions of coagulation and deliver therapeutics or imaging agents. To this end, a peptide substrate for the coagulation transglutaminase FXIII was tagged to magnetofluorescent iron oxide nanoworm imaging agents to act as an FXIII-NW imaging receiver.

Having verified the coagulation-targeting properties of the receiving FXIII-NW NPs in isolation, the researchers evaluated the two-stage signaling-receiving approach in combination. PEG-NRs were intravenously injected into mice bearing bilateral tumors. After NR clearance from circulation mixtures of active and inactive receiving NPs (FXIII-NWs and FXIIIControl-NWs) labelled with distinct NIR fluorochromes were co-injected intravenously, followed by NIR irradiation of just the right flank of the mouse.

Ninety-six hours later, imaging studies revealed pronounced homing of FXIII-NWs to the NR-heated tumors on the right flank, when compared both with the unirradiated tumor-bearing left flank, and with control, saline-injected mice. Histological examination showed that integrated NP generated intense regions of FXIII-NW fluorescence relative to controls, particularly in tumor boundaries where blood vessels were well perfused. Equivalent results were confirmed in different xengraft tumor models, which also demonstrated a several-fold amplification in the homing of targeted receiving NPs compared with untargeted controls.

In a separate set of experiments, the researchers evaluated the ability of autonomous communication between tTF-RGD signaling modules and FXIII-NW receiving modules to amplify tumor targeting. When co-injected alongside FXIII-NW receivers, the tTF-RGD signaling modules where shown to amplify the receiver targeting response several-fold compared with both noncommunicating controls and with NWs that are directly targeted by RGD-targeting ligands. Closer examination demonstrated that FXIII-NW receivers injected alongside tTF-RGD proteins produced a dendritic pattern of accumulation in tumors, corresponding to abundant intravascular localization immunohistochemically, the authors note. This amplified vascular targeting was found to be specific for tumors and was absent when the coagulation inhibitor heparin was administered alongside signaling and receiving modules.

To demonstrate proof of principal that the approach could improve tumor drug delivery and therapy, the researchers evaluated the efficacy of a therapeutic communicating nanosystem in which the receiver comprised doxorubicin-loaded LPs with tethered active FXIII. When these therapeutic receivers were used in place of the imaging receivers, the team was able to show that communication between NR signaling modules and FXIII-LP receivers amplified the accumulation of doxorubicin in NR-heated tumors by over 40-fold compared with LPs alone, and more than sixfold when compared with an optimized LP formulation that targeted endogenous vascular receptors (αvβ3 for high-affinity cyclic-RGD peptide-targeted LPs).

This amplification of drug delivery probably has at least two components, the authors suggest. Heat-dependent increases in passive accumulation due to improved extravasation in tumors, and specific biochemical recognition of the coagulation process by the peptide coating.

In a final set of studies the team evaluated the therapeutic efficacy of the communicating strategy using PEG-NRs as the signaling molecules and an intravenous dose of FXII-LPs in mice bearing a single human tumor. The PEG-NRs were injected into mice, and once cleared from the circulation, a single intravenous dose of FXIII-LP was given followed immediately by irradiation with NIR energy. The treatment resulted in prolonged inhibition of tumor growth that was significantly more effective than system components in isolation, without any detectable weight loss due to system toxicity.

“Given the diverse NP and synthetic biological ‘building blocks’ under development, coupled with the plethora of robust biological cascades that could be repurposed to enable communication between synthetic components, we believe that a wide array of nanosystems could be engineered to more sensitively locate, diagnose, and treat a diversity of focal human diseases,” the authors conclude. “We believe that this work motivates a paradigm of systems nanotechnology directed toward the construction of communicative diagnostic and therapeutic agents with sophisticated in vivo behaviors.”

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