In vivo fluorescence imaging in the second near-infrared window with long circulating carbon nanotubes capable of ultrahigh tumor uptake.

Cancer imaging requires selective high accumulation of contrast agents in the tumor region and correspondingly low uptake in healthy tissues. Here, by making use of a novel synthetic polymer to solubilize single-walled carbon nanotubes (SWNTs), we prepared a well-functionalized SWNT formulation with long blood circulation (half-life of ∼30 h) in vivo to achieve ultrahigh accumulation of ∼30% injected dose (ID)/g in 4T1 murine breast tumors in Balb/c mice. Functionalization dependent blood circulation and tumor uptake were investigated through comparisons with phospholipid-PEG solubilized SWNTs. For the first time, we performed video-rate imaging of tumors based on the intrinsic fluorescence of SWNTs in the second near-infrared (NIR-II, 1.1-1.4 µm) window. We carried out dynamic contrast imaging through principal component analysis (PCA) to immediately pinpoint the tumor within ∼20 s after injection. Imaging over time revealed increasing tumor contrast up to 72 h after injection, allowing for its unambiguous identification. The 3D reconstruction of the SWNTs distribution based on their stable photoluminescence inside the tumor revealed a high degree of colocalization of SWNTs and blood vessels, suggesting enhanced permeability and retention (EPR) effect as the main cause of high passive tumor uptake of the nanotubes.

Chirality enriched (12,1) and (11,3) single-walled carbon nanotubes for biological imaging.

The intrinsic band gap photoluminescence of semiconducting single-walled carbon nanotubes (SWNTs) makes them promising biological imaging probes in the second near-infrared (NIR-II, 1.0-1.4 µm) window. Thus far, SWNTs used for biological applications have been a complex mixture of metallic and semiconducting species with random chiralities, preventing simultaneous resonant excitation of all semiconducting nanotubes and emission at a single well-defined wavelength. Here, we developed a simple gel filtration method to enrich semiconducting (12,1) and (11,3) SWNTs with identical resonance absorption at ~808 nm and emission near ~1200 nm. The chirality sorted SWNTs showed ~5-fold higher photoluminescence intensity under resonant excitation of 808 nm than unsorted SWNTs on a per-mass basis. Real-time in vivo video imaging of whole mouse body and tumor vessels was achieved using a ~6-fold lower injected dose of (12,1) and (11,3) SWNTs (~3 µg per mouse or ~0.16 mg/kg of body weight vs 1.0 mg/kg for unsorted SWNTs) than a previous heterogeneous mixture, demonstrating the first resonantly excited and chirality separated SWNTs for biological imaging.

In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region.

Hits the dot: Ag(2)S quantum dots (QDs) with bright near-infrared-II fluorescence emission (around 1200 nm) and six-arm branched PEG surface coating were synthesized for in vivo small-animal imaging. The 6PEG-Ag(2)S QDs afforded a tumor uptake of approximately 10 % injected dose/gram, owing to a long circulation half-life of approximately 4 h. Clearance of the injected 6PEG-Ag(2)S QDs occurs mainly through the biliary pathway in mice.

Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy.

We developed nanosized, reduced graphene oxide (nano-rGO) sheets with high near-infrared (NIR) light absorbance and biocompatibility for potential photothermal therapy. The single-layered nano-rGO sheets were ∼20 nm in average lateral dimension, functionalized noncovalently by amphiphilic PEGylated polymer chains to render stability in biological solutions and exhibited 6-fold higher NIR absorption than nonreduced, covalently PEGylated nano-GO. Attaching a targeting peptide bearing the Arg-Gly-Asp (RGD) motif to nano-rGO afforded selective cellular uptake in U87MG cancer cells and highly effective photoablation of cells in vitro. In the absence of any NIR irradiation, nano-rGO exhibited little toxicity in vitro at concentrations well above the doses needed for photothermal heating. This work established nano-rGO as a novel photothermal agent due to its small size, high photothermal efficiency, and low cost as compared to other NIR photothermal agents including gold nanomaterials and carbon nanotubes.

Simultaneous nitrogen doping and reduction of graphene oxide.

We developed a simple chemical method to obtain bulk quantities of N-doped, reduced graphene oxide (GO) sheets through thermal annealing of GO in ammonia. X-ray photoelectron spectroscopy (XPS) study of GO sheets annealed at various reaction temperatures reveals that N-doping occurs at a temperature as low as 300 degrees C, while the highest doping level of approximately 5% N is achieved at 500 degrees C. N-doping is accompanied by the reduction of GO with decreases in oxygen levels from approximately 28% in as-made GO down to approximately 2% in 1100 degrees C NH(3) reacted GO. XPS analysis of the N binding configurations of doped GO finds pyridinic N in the doped samples, with increased quaternary N (N that replaced the carbon atoms in the graphene plane) in GO annealed at higher temperatures (> or = 900 degrees C). Oxygen groups in GO were found responsible for reactions with NH(3) and C-N bond formation. Prereduced GO with fewer oxygen groups by thermal annealing in H(2) exhibits greatly reduced reactivity with NH(3) and a lower N-doping level. Electrical measurements of individual GO sheet devices demonstrate that GO annealed in NH(3) exhibits higher conductivity than those annealed in H(2), suggesting more effective reduction of GO by annealing in NH(3) than in H(2), consistent with XPS data. The N-doped reduced GO shows clearly n-type electron doping behavior with the Dirac point (DP) at negative gate voltages in three terminal devices. Our method could lead to the synthesis of bulk amounts of N-doped, reduced GO sheets useful for various practical applications.

PEGylated nanographene oxide for delivery of water-insoluble cancer drugs.

It is known that many potent, often aromatic drugs are water insoluble, which has hampered their use for disease treatment. In this work, we functionalized nanographene oxide (NGO), a novel graphitic material, with branched polyethylene glycol (PEG) to obtain a biocompatible NGO-PEG conjugate stable in various biological solutions, and used them for attaching hydrophobic aromatic molecules including a camptothecin (CPT) analogue, SN38, noncovalently via pi-pi stacking. The resulting NGO-PEG-SN38 complex exhibited excellent water solubility while maintaining its high cancer cell killing potency similar to that of the free SN38 molecules in organic solvents. The efficacy of NGO-PEG-SN38 was far higher than that of irinotecan (CPT-11), a FDA-approved water soluble SN38 prodrug used for the treatment of colon cancer. Our results showed that graphene is a novel class of material promising for biological applications including future in vivo cancer treatment with various aromatic, low-solubility drugs.

Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement.

BACKGROUND: Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths. METHODS AND RESULTS: Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. CONCLUSIONS: The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease.

Ultra-low doses of chirality sorted (6,5) carbon nanotubes for simultaneous tumor imaging and photothermal therapy.

Single-walled carbon nanotubes (SWCNTs) exhibit intrinsic fluorescence and strong optical absorption in the near-infrared (NIR) biological window (0.7-1.4 µm), rendering them ideal for in vivo imaging and photothermal therapy. Advances in SWCNT sorting have led to improved nanoelectronics and are promising for nanomedicine. To date, SWCNTs used in vivo consist of heterogeneous mixtures of nanotubes and only a small subset of chirality nanotubes fluoresces or heats under a NIR laser. Here, we demonstrate that separated (6,5) SWCNTs exchanged into a biocompatible surfactant, C18-PMH-mPEG, are more than 6-fold brighter in photoluminescence on the per mass basis, afford clear tumor imaging, and reach requisite photothermal tumor ablation temperatures with a >10-fold lower injected dose than as-synthesized SWCNT mixtures while exhibiting relatively low (6,5) accumulation in the reticuloendothelial system. The intravenous injection of ∼4 µg of (6,5) SWCNTs per mouse (0.254 mg/kg) for dual imaging/photothermal therapy is, by far, the lowest reported dose for nanoparticle-based in vivo therapeutics.

Single-walled carbon nanotube surface control of complement recognition and activation.

Carbon nanotubes (CNTs) are receiving considerable attention in site-specific drug and nucleic acid delivery, photodynamic therapy, and photoacoustic molecular imaging. Despite these advances, nanotubes may activate the complement system (an integral part of innate immunity), which can induce clinically significant anaphylaxis. We demonstrate that single-walled CNTs coated with human serum albumin activate the complement system through C1q-mediated classical and the alternative pathways. Surface coating with methoxypoly(ethylene glycol)-based amphiphiles, which confers solubility and prolongs circulation profiles of CNTs, activates the complement system differently, depending on the amphiphile structure. CNTs with linear poly(ethylene glycol) amphiphiles trigger the lectin pathway of the complement through both L-ficolin and mannan-binding lectin recognition. The lectin pathway activation, however, did not trigger the amplification loop of the alternative pathway. An amphiphile with branched poly(ethylene glycol) architecture also activated the lectin pathway but only through L-ficolin recognition. Importantly, this mode of activation neither generated anaphylatoxins nor induced triggering of the effector arm of the complement system. These observations provide a major step toward nanomaterial surface modification with polymers that have the properties to significantly improve innate immunocompatibility by limiting the formation of complement C3 and C5 convertases.

Three-dimensional imaging of single nanotube molecule endocytosis on plasmonic substrates.

Investigating the cellular internalization pathways of single molecules or single nano objects is important to understanding cell-matter interactions, and to applications in drug delivery and discovery. Imaging and tracking the motion of single molecules on cell plasma membranes require high spatial resolution in three dimensions. Fluorescence imaging along the axial dimension with nanometre resolution has been highly challenging, but critical to revealing displacements in transmembrane events. Here, utilizing a plasmonic ruler based on the sensitive distance dependence of near-infrared fluorescence enhancement of carbon nanotubes on a gold plasmonic substrate, we probe ~10 nm scale transmembrane displacements through changes in nanotube fluorescence intensity, enabling observations of single nanotube endocytosis in three dimensions. Cellular uptake and transmembrane displacements show clear dependences to temperature and clathrin assembly on cell membrane, suggesting that the cellular entry mechanism for a nanotube molecule is via clathrin-dependent endocytosis through the formation of clathrin-coated pits on the cell membrane.

Near infrared imaging and photothermal ablation of vascular inflammation using single-walled carbon nanotubes.

BACKGROUND: Macrophages are critical contributors to atherosclerosis. Single-walled carbon nanotubes (SWNTs) show promising properties for cellular imaging and thermal therapy, which may have application to vascular macrophages. METHODS AND RESULTS: In vitro uptake and photothermal destruction of mouse macrophage cells (RAW264.7) were performed with SWNTs (14.7 nmol/L) exposed to an 808-nm light source. SWNTs were taken up by 94 ± 6% of macrophages, and light exposure induced 93 ± 3% cell death. In vivo vascular macrophage uptake and ablation were then investigated in carotid-ligated FVB mice (n=33) after induction of hyperlipidemia and diabetes. Two weeks postligation, near-infrared fluorescence (NIRF) carotid imaging (n=12) was performed with SWNT-Cy5.5 (8 nmol of Cy5.5) given via the tail vein. Photothermal heating and macrophage apoptosis were evaluated on freshly excised carotid arteries (n=21). NIRF of SWNTs showed higher signal intensity in ligated carotids compared with sham, confirmed by both in situ and ex vivo NIRF imaging (P<0.05, ligation versus sham). Immunofluorescence staining showed colocalization of SWNT-Cy5.5 and macrophages in atherosclerotic lesions. Light (808 nm) exposure of freshly excised carotids showed heating and induction of macrophage apoptosis in ligated left carotid arteries with SWNTs, but not in control groups without SWNTs or without light exposure. CONCLUSIONS: Carbon nanotubes accumulate in atherosclerotic macrophages in vivo and provide a multifunctional platform for imaging and photothermal therapy of vascular inflammation.

Multifunctional in vivo vascular imaging using near-infrared II fluorescence.

In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (∼30 µm) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1-3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.

Graphite-coated magnetic nanoparticle microarray for few-cells enrichment and detection.

Graphite-coated, highly magnetic FeCo core-shell nanoparticles were synthesized by a chemical vapor deposition method and solubilized in aqueous solution through a unique polymer mixture modification, which significantly improved the biocompatibility and stability of the magnetic nanoparticles (MNPs). Such functionalized MNPs were proven to be very stable in different conditions which would be significant for biological applications. Cell staining, manipulation, enrichment, and detection were developed with these MNPs. Under external magnetic manipulation, the MNP-stained cells exhibited directed motions. Moreover, MNPs were printed on substrates to modulate the magnetic field distribution on the surface. Capture and detection of sparse populations of cancer cells spiked into whole blood has been explored in a microarray fashion. Cancer cells from hundreds down to only two were able to be simply and efficiently detected from 1 mL of whole blood on the MNP microarray chips. Interestingly, the cells captured through the MNP microarray still showed viability and adhered to the MNP spots after incubation, which could be utilized for cancer cell detection, localized growth, and proliferation.

Plasmonic substrates for multiplexed protein microarrays with femtomolar sensitivity and broad dynamic range.

Protein chips are widely used for high-throughput proteomic analysis, but to date, the low sensitivity and narrow dynamic range have limited their capabilities in diagnostics and proteomics. Here we present protein microarrays on a novel nanostructured, plasmonic gold film with near-infrared fluorescence enhancement of up to 100-fold, extending the dynamic range of protein detection by three orders of magnitude towards the fM regime. We employ plasmonic protein microarrays for the early detection of a cancer biomarker, carcinoembryonic antigen, in the sera of mice bearing a xenograft tumour model. Further, we demonstrate a multiplexed autoantigen array for human autoantibodies implicated in a range of autoimmune diseases with superior signal-to-noise ratios and broader dynamic range compared with commercial nitrocellulose and glass substrates. The high sensitivity, broad dynamic range and easy adaptability of plasmonic protein chips presents new opportunities in proteomic research and diagnostics applications.

High Performance In Vivo Near-IR (>1 µm) Imaging and Photothermal Cancer Therapy with Carbon Nanotubes.

Short single-walled carbon nanotubes (SWNTs) functionalized by PEGylated phospholipids are biologically non-toxic and long-circulating nanomaterials with intrinsic near infrared photoluminescence (NIR PL), characteristic Raman spectra, and strong optical absorbance in the near infrared (NIR). This work demonstrates the first dual application of intravenously injected SWNTs as photoluminescent agents for in vivo tumor imaging in the 1.0-1.4 µm emission region and as NIR absorbers and heaters at 808 nm for photothermal tumor elimination at the lowest injected dose (70 µg of SWNT/mouse, equivalent to 3.6 mg/kg) and laser irradiation power (0.6 W/cm(2)) reported to date. Ex vivo resonance Raman imaging revealed the SWNT distribution within tumors at a high spatial resolution. Complete tumor elimination was achieved for large numbers of photothermally treated mice without any toxic side effects after more than six months post-treatment. Further, side-by-side experiments were carried out to compare the performance of SWNTs and gold nanorods (AuNRs) at an injected dose of 700 µg of AuNR/mouse (equivalent to 35 mg/kg) in NIR photothermal ablation of tumors in vivo. Highly effective tumor elimination with SWNTs was achieved at 10 times lower injected doses and lower irradiation powers than for AuNRs. These results suggest there are significant benefits of utilizing the intrinsic properties of biocompatible SWNTs for combined cancer imaging and therapy.