Scott M. Tabakman

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.

Atomic layer deposition of metal oxides on pristine and functionalized graphene.

We investigate atomic layer deposition (ALD) of metal oxide on pristine and functionalized graphene. On pristine graphene, ALD coating can only actively grow on edges and defect sites, where dangling bonds or surface groups react with ALD precursors. This affords a simple method to decorate and probe single defect sites in graphene planes. We used perylene tetracarboxylic acid (PTCA) to functionalize the graphene surface and selectively introduced densely packed surface groups on graphene. Uniform ultrathin ALD coating on PTCA graphene was achieved over a large area. The functionalization method could be used to integrate ultrathin high-kappa dielectrics in future graphene electronics.

PEG branched polymer for functionalization of nanomaterials with ultralong blood circulation.

Nanomaterials have been actively pursued for biological and medical applications in recent years. Here, we report the synthesis of several new poly(ethylene glycol) grafted branched polymers for functionalization of various nanomaterials including carbon nanotubes, gold nanoparticles (NPs), and gold nanorods (NRs), affording high aqueous solubility and stability for these materials. We synthesize different surfactant polymers based upon poly(gamma-glutamic acid) (gammaPGA) and poly(maleic anhydride-alt-1-octadecene) (PMHC18). We use the abundant free carboxylic acid groups of gammaPGA for attaching lipophilic species such as pyrene or phospholipid, which bind to nanomaterials via robust physisorption. Additionally, the remaining carboxylic acids on gammaPGA or the amine-reactive anhydrides of PMHC18 are then PEGylated, providing extended hydrophilic groups, affording polymeric amphiphiles. We show that single-walled carbon nanotubes (SWNTs), Au NPs, and NRs functionalized by the polymers exhibit high stability in aqueous solutions at different pH values, at elevated temperatures, and in serum. Moreover, the polymer-coated SWNTs exhibit remarkably long blood circulation (t(1/2) = 22.1 h) upon intravenous injection into mice, far exceeding the previous record of 5.4 h. The ultralong blood circulation time suggests greatly delayed clearance of nanomaterials by the reticuloendothelial system (RES) of mice, a highly desired property for in vivo applications of nanomaterials, including imaging and drug delivery.

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/cm2) 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.

Carbon materials for drug delivery & cancer therapy

Carbon nanotubes and graphene are both low-dimensional sp2 carbon nanomaterials exhibiting many unique physical and chemical properties that are interesting in a wide range of areas including nanomedicine. Since 2004, carbon nanotubes have been extensively explored as drug delivery carriers for the intracellular transport of chemotherapy drugs, proteins, and genes. In vivo cancer treatment with carbon nanotubes has been demonstrated in animal experiments by several different groups. Recently, graphene, another allotrope of carbon, has also shown promise in various biomedical applications. In this article, we will highlight recent research on these two categories of closely related carbon nanomaterials for applications in drug delivery and cancer therapy, and discuss the opportunities and challenges in this rapidly growing field.

Preparation of carbon nanotube bioconjugates for biomedical applications

Biomedical applications of carbon nanotubes have attracted much attention in recent years. Here, we summarize our previously developed protocols for functionalization and bioconjugation of single-walled carbon nanotubes (SWNTs) for various biomedical applications including biological imaging; using nanotubes as Raman, photoluminescence and photoacoustic labels; sensing using nanotubes as Raman tags and drug delivery. Sonication of SWNTs in solutions of phospholipid-polyethylene glycol (PL-PEG) is our most commonly used protocol of SWNT functionalization. Compared with other frequently used covalent strategies, our non-covalent functionalization protocol largely retains the intrinsic optical properties of SWNTs, which are useful in various biological imaging and sensing applications. Functionalized SWNTs are conjugated with targeting ligands, including peptides and antibodies for specific cell labeling in vitro or tumor targeting in vivo. Radio labels are introduced for tracking and imaging of SWNTs in real time in vivo. Moreover, SWNTs can be conjugated with small interfering RNA (siRNA) or loaded with chemotherapy drugs for drug delivery. These procedures take various times ranging from 1 to 5 d.

Protein microarrays with carbon nanotubes as multicolor Raman labels

The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM—a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegeners granulomatosis, diluted up to 107-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure 12C and 13C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.

Photothermally enhanced drug delivery by ultrasmall multifunctional FeCo/graphitic shell nanocrystals.

FeCo/graphitic carbon shell (FeCo/GC) nanocrystals (∼4-5 nm in diameter) with ultrahigh magnetization are synthesized, functionalized, and developed into multifunctional biocompatible materials. We demonstrate the ability of this material to serve as an integrated system for combined drug delivery, near-infrared (NIR) photothermal therapy, and magnetic resonance imaging (MRI) in vitro. We show highly efficient loading of doxorubicin (DOX) by π-stacking on the graphitic shell to afford FeCo/GC-DOX complexes and pH sensitive DOX release from the particles. We observe enhanced intracellular drug delivery by FeCo/GC-DOX under 20 min of NIR laser (808 nm) induced hyperthermia to 43 °C, resulting in a significant increase of FeCo/GC-DOX toxicity toward breast cancer cells. The synergistic cancer cell killing by FeCo/GC-DOX drug delivery under photothermal heating is due to a ∼two-fold enhancement of cancer cell uptake of FeCo/GC-DOX complex and the increased DOX toxicity under the 43 °C hyperthermic condition. The combination of synergistic NIR photothermally enhanced drug delivery and MRI with the FeCo/GC nanocrystals could lead to a powerful multimodal system for biomedical detection and therapy.

Separation of nanoparticles in a density gradient: FeCo@C and gold nanocrystals.

Size and geometric control of nanomaterials are important to the discovery of intrinsic size/shape dependent properties and bottom up approaches for the fabrication of functional nanodevices. Two general strategies have been employed to create size-uniform nanocrystals. One method is direct particle size control during synthesis by adjusting growth parameters; 7–9] the other is post-synthesis separation. Much capacity exists to improve size separation efficacy in the latter case. Differential centrifugation can remove large and unstable particles from colloidal systems, but lacks precise control over particle size. Addition of adjustable amounts of “anti-solvent” (including CO2) [12] into colloidal systems may make precipitation processes more controllable. Other methods include filtration (including diafiltration), electrophoresis, and chromatographic methods that can produce particle fractions with narrow shape and size distributions. To maintain or improve the quality of nanoparticle (NP) separation, whilst addressing the issues of adhesion and clogging in liquid–solid phase separation processes, a completely liquid phase separation method is highly appealing. Isopycnic centrifugation, which is often used for biomacromolecule separation, relies upon a density gradient and ultracentrifugation to separate components according to subtle density differences, and has been applied for diameter and electronic-dependent separation of single-walled carbon nanotubes (SWNT). However, the isopycnic densitygradient centrifugation method reaches a limitation when it is extended to the separation of metal nanoparticles. Such a method requires that the components for separation have densities within a gradient range. Aqueous density gradient media usually have densities less than 1.4 gcm , which is much less than the density of metal nanoparticles. Size or shape separation of such heavy nanocrystals remains an issue, both in their preparation and utility for various applications. In contrast to isopycnic separation, ultracentrifugal rate separation can utilize density gradients to separate nanocrystals with higher densities than the gradient media itself. We have previously applied such a method to achieve length separation of suspended SWNTs and pegylated graphene oxide. In this report, the method was extended to metallic NP size separation. Nanoparticles of various size, suspension chemistry, and composition, including FeCo@C and gold nanoparticles (Au NPs), were separated using the method. FeCo nanocrystals coated in graphitic shells have superior magnetic properties, and have shown promise for applications in biolabeling and magnetic resonance imaging (MRI). However, the chemical deposition method used for their preparation produces nanocrystals with wide size distribution. They are thus ideal candidates for post-synthesis separation. FeCo@C NPs with average diameters of about 4 nm were separated first by our density gradient rate (DGR) separation method by using a 10+ 20+ 30+ 40% gradient and centrifugation for 3.5 h. TEM results of typical fractions (Figure 1A) indicate that fraction 8 (labeled as “f8” in Figure 1B) contained circa 1.5 nm NPs. The average particle diameter of subsequent fractions (f11, 15, 19, 24, and 27) gradually increased from 2.5 to 5.6 nm. By varying the step gradient densities and centrifuge exposure time, this method could be used for separation of nanoparticles of a larger size range, which was demonstrated by (on average) 7 nm FeCo@C NP separation (see TEM images of initial 7 nm FeCo@C NPs in the Supporting Information). A gradient of higher density steps (20+ 30+ 40+ 60%) was used. The use of higher density gradient media helps to control the sedimentation rate by reducing the density difference between the NPs and the environmental medium, and increasing the medium viscosity. After centrifugation for 2.5 h, several bands formed in the centrifuge vessel (Figure 2A), just as in the 4 nm NP case. Sampling fractions along the centrifuge vessel yielded nanoparticles of increasing size (with increasing density), as revealed by TEM (Figure 2B). From f5 to f16, the average particle size increased from 2 to 6.5 nm. It is noteworthy that the Fe/Co atomic ratios of f8 and f16 were measured by energy dispersive spectra (EDS) and found to be different (Figure 2B, bottom right). For f8, the ratio Fe/Co= 48:52; for f16, Fe/Co= 40:60. Previously such stoichiometry was analyzed by calcination/burning of the graphitic shells at 500 8C, dissolving the metal species in an HCl solution, and measuring the iron and cobalt concentrations on the basis of the ultraviolet[*] Prof. Dr. X. M. Sun, L. Bai State Key Laboratory of Chemical Resource Engineering Beijing University of Chemical Technology Beijing 100029 (China)

Multiplexed five-color molecular imaging of cancer cells and tumor tissues with carbon nanotube Raman tags in the near-infrared

AbstractSingle-walled carbon nanotubes (SWNTs) with five different C13/C12 isotope compositions and well-separated Raman peaks have been synthesized and conjugated to five targeting ligands in order to impart molecular specificity. Multiplexed Raman imaging of live cells has been carried out by highly specific staining of cells with a five-color mixture of SWNTs. Ex vivo multiplexed Raman imaging of tumor samples uncovers a surprising up-regulation of epidermal growth factor receptor (EGFR) on LS174T colon cancer cells from cell culture to in vivo tumor growth. This is the first time five-color multiplexed molecular imaging has been performed in the near-infrared (NIR) region under a single laser excitation. Near zero interfering background of imaging is achieved due to the sharp Raman peaks unique to nanotubes over the low, smooth autofluorescence background of biological species.