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. 2007 Jan 16;104(3):932-6.
doi: 10.1073/pnas.0610298104. Epub 2007 Jan 10.

Biomimetic amplification of nanoparticle homing to tumors

Dmitri Simberg  1 , Tasmia Duza, Ji Ho Park, Markus Essler, Jan Pilch, Lianglin Zhang, Austin M Derfus, Meng Yang, Robert M Hoffman, Sangeeta Bhatia, Michael J Sailor, Erkki Ruoslahti
Affiliations

Biomimetic amplification of nanoparticle homing to tumors

Dmitri Simberg et al. Proc Natl Acad Sci U S A. .

Abstract

Nanoparticle-based diagnostics and therapeutics hold great promise because multiple functions can be built into the particles. One such function is an ability to home to specific sites in the body. We describe here biomimetic particles that not only home to tumors, but also amplify their own homing. The system is based on a peptide that recognizes clotted plasma proteins and selectively homes to tumors, where it binds to vessel walls and tumor stroma. Iron oxide nanoparticles and liposomes coated with this tumor-homing peptide accumulate in tumor vessels, where they induce additional local clotting, thereby producing new binding sites for more particles. The system mimics platelets, which also circulate freely but accumulate at a diseased site and amplify their own accumulation at that site. The self-amplifying homing is a novel function for nanoparticles. The clotting-based amplification greatly enhances tumor imaging, and the addition of a drug carrier function to the particles is envisioned.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Tumor homing of CREKA pentapeptide. (A and B) Fluorescein-conjugated CREKA peptide (200 μg per mouse) was injected into mice bearing syngeneic B16F1 melanoma tumors. Representative microscopic fields are shown to illustrate homing of fluorescein-CREKA to fibrin-like structures in tumors in wild-type mice (A, arrow) and lack of homing in fibrinogen null mice (B). (C) The CREKA phage binds to clotted plasma proteins in a test tube, whereas nonrecombinant control phage shows little binding. (D) Dextran-coated iron oxide nanoparticles conjugated with fluorescein-CREKA bind to clotted plasma proteins, and the binding is inhibited by free CREKA peptide. (D Inset) The microscopic appearance of the clot-bound CREKA-SPIO. [Magnification: ×200 (A and B) and ×600 (D Inset).]
Fig. 2.
Fig. 2.
Tumor homing of CREKA-conjugated iron oxide particles. CREKA-SPIO particles were intravenously injected (4 mg of Fe per kg) into BALB/c nude mice bearing MDA-MB-435 human breast cancer xenograft tumors measuring 1–1.5 cm in diameter. The mice were killed by perfusion 5–6 h later, and tissues were examined for CREKA-SPIO fluorescence (green). Nuclei were stained with DAPI (blue). (A) Distribution of CREKA-SPIO in tissues from MDA-MB-435 tumor mice that 2 h earlier had received an injection of PBS (Upper) or Ni/DSPC/CHOL liposomes (Ni-liposomes) containing 0.2 μmol Ni in 200 μl of PBS (Lower). (B) Plasma circulation half-life of CREKA-SPIO after different treatments. At least four time points were collected. Data were fitted to monoexponential decay by using Prizm software (GraphPad, San Diego, CA), and the half-life values were compared in unpaired t test (∗∗∗, P < 0.0001 relative to PBS control; n = 10). (C) Accumulation of CREKA-SPIO nanoparticles in tumor vessels. Mice were injected and tissues were collected as in A. Fluorescent intravascular CREKA-SPIO particles overlap with iron oxide viewed in transmitted light. (Magnification: ×600.) (D) Control organs of Ni-liposome/CREKA-SPIO-injected mice. Occasional spots of fluorescence are seen in the kidneys and lungs. The fluorescence seen in the heart did not differ from uninjected controls, indicating that it is autofluorescence. Representative results from at least three independent experiments are shown. [Magnification: ×200 (A and D) and ×600 (C).]
Fig. 3.
Fig. 3.
Accumulation of CREKA-SPIO nanoparticles in tumor vessels. Mice bearing MDA-MB-435 xenografts were injected with Ni-liposomes and CREKA-SPIO nanoparticles as described in the legend to Fig. 2. The mice were perfused 6 h after the nanoparticle injection, and tissues were collected. (A Top) Colocalization of nanoparticle fluorescence with CD31 staining in blood vessels. (A Middle) Colocalization of nanoparticle fluorescence and antifibrin(ogen) staining in tumor blood vessels. (Inset) An image showing CREKA-SPIO distributed along fibrils in a tumor blood vessel. (A Bottom) Lack of colocalization of nanoparticle fluorescence with anti-CD41 staining for platelets. (B) Intravital confocal microscopy of tumors using DiI-stained red blood cells as a marker of blood flow. The arrow points to a vessel in which stationary erythrocytes indicate obstruction of blood flow. Blood flow in the vessel above is not obstructed. Six successive frames from a 1-min movie (SI Movie 1) are shown. (C) CREKA-coated liposomes colocalize with fibrin in tumor vessels. The results are representative of three independent experiments. [Magnification: ×600 (A and C) and ×200 (B).]
Fig. 4.
Fig. 4.
Effect of blood clotting on nanoparticle accumulation in tumors. Mice bearing MDA-MB-435 human breast cancer xenografts were intravenously injected with PBS or a bolus of 800 units/kg of heparin, followed 120 min later by Ni-liposomes (or PBS) and CREKA-SPIO (or control nanoparticles). The mice received additional heparin by i.p. injections (a total of 1,000 units/kg) or PBS throughout the experiment. (A) Tumors were removed 6 h after the nanoparticle injection, and magnetic signal in the tumor after different treatments was determined with SQUID. Aminated dextran SPIO served as a particle control (control SPIO). SPIO nanoparticle concentration in tissues is represented by the saturation magnetization value (electromagnetic unit, emu) of the tissue at 1T magnetic field after the subtraction of the diamagnetic and the paramagnetic background of blank tissue. The magnetization values were normalized to dry weight of the tissue. Results from three experiments are shown. (B) Quantification of heparin effect on clotting in blood vessels. Mice were pretreated with PBS (open bars) or heparin (filled bars) as described above, followed by Ni liposomes/CREKA-SPIO nanoparticles. Three sections from two tumors representing each treatment were stained with anti-CD31 for blood vessels, and the percentage of vessels positive for fluorescence and fluorescent clots was determined. Note that heparin did not significantly change the percentage of blood vessels containing particles, but dramatically decreased the incidence of the lumens that are filled with fluorescence. (C) A representative example of the appearance of CREKA-SPIO particles in tumor vessels of mice treated with heparin. (D) Near-infrared imaging of mice that received Ni-liposomes, followed by Cy7-labeled CREKA-SPIO, with or without heparin pretreatment. The images were acquired 8 h after the injection of the CREKA-SPIO particles by using an Odyssey 2 NIR scanner (Li-COR Biosciences, Lincoln, NE). The images shown are composites of two colors, red (700-nm channel, body and chow autofluorescence) and green (800-nm channel, Cy7). Arrows point to the tumors, and arrowheads point to the liver. Note the strong decrease in signal from the tumor in the heparin-pretreated mouse. One representative experiment of three is shown.
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