CARBONNANOTUBE NANOBOMB

Reference to Related Applications

[0001] The present application claims the benefit of U.S. Provisional Application No.

60/696,473 filed July 1, 2005.

Reference to Government Support

[0002] The present invention was supported in part by DOD/CDMRP grant BC024244 and DOD/BER grant ER63055. The United States government has certain rights in the invention.

Field of the Invention

[0003] The invention is related to applications of carbon nanotubes, preferably, single wall carbon nanotubes (SWCNT), as potent therapeutic nano-bomb agents for killing or damaging cells, such as cancer cells.

Background of the Invention

[0004] Conventional surgical treatment of solid tumors is an effective therapy for the removal of well-defined, accessible, primary tumors located within non vital tissue regions.

However, the high morbidity and invasive nature associated with surgical resection renders this therapy unsuitable for treatment of small, poorly defined metastases or other tumors embedded within vital regions. Thermal ablative therapies using nanoscale materials can provide a minimally invasive alternative to conventional surgical treatment of solid tumors.

In addition to their minimally invasive nature, thermal therapeutic procedures are relatively simple to perform, and therefore have the potential of improving recovery times, reducing complication rates and hospital stays.

[0005] Loosely packed single wall carbon nanotubes have been known to burn on exposure of light with a sound in the presence of oxygen, P. M. Ajayan et al., "Nanotubes in a Flash: Ignition and Reconstruction", Science 296: 705 (26 April 2002).

[0006] Carbon exists in various molecular configurations, including diamond, graphite, fullerene, and carbon nanotube (CNT). There are several U.S. patents that discuss carbon nanotubes, including U.S. 6,740,224; 5,753,088; 5,783,263; 6,063,243; 6,149,775; 6,451,175;

4,855,091 ; 5,591,382; 5,643,502 and 5,171,560.

[0007] The nanotube form of carbon was discovered relatively recently. Each carbon nanotube is a long, thin cylinder of carbon, with a diameter that can be as small as 1 nm and a length that can range from a few nanometers to one or more microns. A carbon nanotube may be thought of as a sheet of graphite, i.e., a hexagonal lattice of carbon, rolled into a cylinder. A carbon nanotube may have a single cylindrical wall, or it may have multiple walls, giving it the appearance of cylinders inside other cylinders. A single wall carbon nanotube has only a single atomic layer, whereas a multiple wall carbon nanotube may contain, for example, from 100 to 1,000 atomic layers.

[0008] L. R. Hirsch et ah, "Nanoshell-mediated near infra-red thermal therapy of tumors under magnetic resonance guidance", Proceedings of National Academy of Sciences, 100 (23): 13549-13554, Nov.1 1, 2003, used silica-gold nanoshells to deliver a therapeutic dose of heat to breast carcinoma cells. Summary of the Invention

[0009] The present invention provides methods and compositions for delivering therapeutic treatments to cells and tissues. In the methods of the invention, carbon nanotubes are delivered to targeted cells or tissues wherein treatment is needed or desired. The nanotubes are hydrated with water or an aqueous solution prior to or after delivery to the site where treatment is needed or desired. The nanotubes are then exposed to light of sufficient intensity and for sufficient duration to cause explosion of the nanotubes. Explosion of the nanotubes exposes the targeted cells to a lethal or damaging dose of heat. While not wishing to be bound to any particular mode or theory of action, at the present time it is believed that exposure of the hydrated nanotubes to laser light heats the water adhering to the nanotubes to temperatures greater than 100 ⁰ C and the resulting pressure from the heated water causes the nanotubes to explode. The water released from the nanotubes heats exposed cells to temperatures that are lethal or damaging to the cells and cell membrane. [00010] The methods of the invention are a minimally invasive technique for thermal ablative surgery. The methods of the invention direct a lethal or damaging dose of heat to specific cells or tissues, such as a tumor, with little damage to surrounding tissues. [00011] The methods of the invention can be used to kill or damage any type of cells in vitro or in vivo. In a preferred embodiment of the invention the methods of the invention are

used to kill or damage mammalian cells in vivo, on the surface or skin of the mammal or internally.

[00012] A further aspect of the invention provides carbon nanotubes containing water or other aqueous solution and a targeting molecule. The carbon nanotubes can also optionally contain one or more therapeutic compounds in the water or other aqueous solution or adherent to the carbon nanotube.

[00013] Another aspect of the invention provides a composition comprising bundles of carbon nanotubes and a targeting molecule bound to the nanotubes. Preferably, the carbon nanotubes are single wall carbon nanotubes. A preferred type of targeting molecule is an antibody or cell surface receptor. The composition can optionally further contain a therapeutic compound, such as a chemotherapy drug, and/or water or other aqueous solution.

[00014] The methods of the invention have a number of applications. The methods of the invention can be used for ablative surgery to kill or damage cancer cells or for cauterization of tissue. Other uses include delivery of therapeutic compounds, such as chemotherapy drugs, to cells and tissues. The present invention thus also provides methods of treating cancer or other diseases or conditions.

Brief Description of the Drawings

[00015] Figure 1 shows the photomicrographs of cells before and after light exposure.

[00016] Figure 2 illustrates that only the cells that received light were destroyed showing the localized heating that is achievable using this approach.

[00017] Figure 3A shows cancer cell tissues treated with nanotubes before light exposure.

[00018] Figure 3B shows the image in Figure 3A after light exposure.

[00019] Figure 4 shows cancer cell tissues after light exposure.

[00020] Figure 5A shows BT474 cells killed after treatment with Her2 antibody-labeled carbon nanotubes and NIR radiation for 3 minutes. The cells appear blue indicating that the cells are dead.

[00021] Figure 5B shows BT474 cells treated with non-specific antibody labeled carbon nanotubes and NIR treatment for 3 minutes. The cells do not appear blue, indicating that the cells were not killed by the treatment.

Detailed Description of the Invention

[00022] The present invention provides nanobombs by adsorbing water or saline solution to

carbon nanotubes and exposing the hydrated nanotubes to near infra-red light, or other intensity of light sufficient to cause explosion of the nanotubes. The nanobombs are created due to extreme pressures that are generated internally when the adherent water molecules are heated, to create explosions. The methods of the invention solve the problem of killing cancer cells while at the same time causing minimal damage to neighboring non-cancerous cells. Further, the nanobombs are benign as the bomb is created with water molecules or saline solutions that can potentially have no side effects when applied in clinical stage. [00023] Applicant has found that by adsorbing water molecules in nanotube sheets, potent nanobombs can be created by heating the water molecules to more than 100 C° that causes the nanotubes to explode due to the extreme pressures that are created inside the nanotubes on exposure to light. Explosion of carbon nanotubes has been utilized to kill BT474 cells, a breast carcinoma cell line, in a saline buffer solution by co-localization of nanotubes to the cancer cells. Nanobombs were created by the application of laser light of 800 nm at light intensities of ~ 200 mW/cm ² .

[00024] Applicant has also found that carbon nanotubes labeled with an antibody specific for a cancer cell receptor can target the cancer cells, and, when the nanotubes are exploded, kill cells to which they are bound.

[00025] The nanotubes can be directed to specific cells using targeting molecules attached to the nanotube, such an antibodies specific for cell-surface antigens. Nanotubes coated with antibodies will attach specifically to the cells and once attached the nanotubes will be exploded on exposure to light.

[00026] The term carbon nanotubes refers to various tubes or fibers, particularly carbon fibers, having very small diameters including fibrils, whiskers, buckytubes, etc. Preferably, the nanotubes used in the present invention have a diameter less than 1 micron, preferably less than about 0.5 micron, and even more preferably less than 0.1 micron and most preferably less than 0.05 micron. Preferably, single wall carbon nanotubes are used in the methods of the invention; however, multiple wall carbon nanotubes can also be used. Carbon nanotubes can be prepared by any suitable process. Carbon nanotubes are currently manufactured in laboratories via laser ablation, electric-arc, or chemical vapor deposition processes. [00027] Bundles of nanotubes refers to groups of 2 to about 1,000 individual nanotubes

held together by van der Waal's forces or otherwise bound together. Useful diameters for bundles range from about 2 nm for bundles of a few nanotubes up to about 100 nm.

Preferred are bundles having diameters from about 2 nm to about 50 nm; more preferably, diameters of about 5 nm to about 20 nm. Clumps of individual nanotubes or bundles are also useful in the methods and compositions described herein.

[00028] For some embodiments of the present invention, the carbon nanotubes are preferably targeted to the cells to be killed or damaged. For targeting the nanotubes to cells or tissues, the nanotubes can be labeled with a targeting molecule such as an antibody or other molecule that specifically binds to the cells to be killed or damaged. For example, for targeting the nanotubes to cancer cells, the nanotubes can be labeled with antibodies specific for antigens on the surface of the cancer cells.

[00029] The targeting molecule can be any type of molecule that can bind to a normal or cancer cell, such as a dye, an antibody or a radiolabel. Additional types of targeting molecules include receptors, DNA, protein, peptides and RNA. The targeting molecule can be attached to the carbon nanotubes by noncovalent adsorption, covalent linkage or other means appropriate for the target molecule by methods known in the art.

[00030] In other embodiments of the invention, the nanobomb further comprises a therapeutic compound. The therapeutic compound can be mixed with water or aqueous solution or attached to the nanotube. For example, the nanobomb can include a chemotherapy drug for treatment of cancer.

[00031] Examples of chemotherapy drugs include paclitaxel, doxorubicin, vincristine, actinomycin D, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, daunorubicin, etoposide, fluorouracil, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, vinblastine and vindesine.

[00032] The compositions of the invention comprise bundles of nanotubes and a targeting molecule bound on the nanotubes. Targeting molecules can be bound to all or a portion of the nanotubes in the bundles. The compositions can further comprise a pharmaceutically acceptable carrier or diluent such as water or an aqueous buffer, such as phosphate buffered saline (PBS).

[00033] Nanotubes will explode when exposed to laser light in the presence of oxygen. (P.

M. Ajayan et al, "Nanotubes in a Flash: Ignition and Reconstruction", Science 296: 705, (26

131-435

April 2002)). It is not necessary in the present invention to supply oxygen to the nanotubes since oxygen is present in the air surrounding the cells or tissues. However, for some embodiments of the invention, it may be advantageous to supply oxygen to the nanotubes to supplement the oxygen present in the air.

[00034] The nanotubes can be hydrated prior to or after delivery to the cells or tissue. The nanotubes are hydrated by application of water or aqueous solution to the nanotubes where it is adsorbed. The nanotubes are hydrated by mixing with water or aqueous solution, or by applying water or aqueous solution to the nanotubes. A preferred aqueous solution is a buffer such as phosphate buffered saline (PBS).

[00035] The methods of the invention can be used to treat any type of condition where removal of or killing of tissue or cells is desired or necessary, such as, for example, removal of tumors or tumor cells. Another application is to use the nanotubes as cauterization agents to kill tissues and also use nanotubes as surgical tools to remove blocks from the arterial veins and also do surgery in parts of the body considered inaccessible in the past. [00036] The nanotubes can be delivered to the site where treatment is needed or desired by topical application to the site, by injection or other means. For example, nanobombs can be delivered to cancer sites by either directly injecting them to the cancer site or using magnetic resonance imaging (MRI) to guide nanotubes to the cancer cell site through the blood vessels. Hence targeted guidance of the nanobombs using conventional MRI techniques already used in hospitals can be used to deliver the nanobombs selectively to cancer sites. Further, by application of antibody specific to cell surface receptors in cancer cells on top of the nanotubes and using MRI techniques for guidance, the nanobombs can be delivered specifically to the cancer sites owing to the attachment of antibody to the surface receptors in cancer cells thereby attaching the nanotube intimately to cancer cells. [00037] A sufficient amount of carbon nanotubes is delivered to the treatment site to result in cell damage or death upon explosion of the nanotubes. The amount of carbon nanotubes delivered to the treatment site will depend on factors such as the location of the treatment site in or on the body, and the size of the area to be treated. Suitable amounts of the nanotubes range from about 1 microgram to about 1 gram, although smaller or larger amounts can also be used. [00038] The nanotubes are exposed to light of sufficient intensity and for a sufficient length

131-435 of time to cause the hydrated nanotubes to explode. At the present time, near infra-red light

(700-1000 nm) is preferred for the methods of the invention. Most mammalian tissues have a naturally occurring deficit of near infra-red absorbing chromophores permitting transmission of near infra-red light through tissue with scattering-limited attenuation and minimal heating.

Light within this spectral region has been shown to penetrate tissue at depths beyond 1 cm with no observable damage to the intervening tissue. Other wavelengths of light that do not produce lethal damage to cells can also be used in the methods of the invention.

[00039] The type of light sources used is coherent radiation from laser sources. Lasers are highly effective as they have smaller spot size, and are highly wavelength specific. It should be mentioned that the laser energy in the near infra-red region that is used is not harmful to normal and healthy cells. Only the presence of the nanotubes and the absorption of photons by the nanotube upon exposure to light causes the explosion. The equipment that will be used in a clinical setting will use a laser source using a semiconducting laser that is commercially available. The lasers will be tuned to different frequencies (10 Hz-100 KHz) as high frequencies can be faster in causing explosion of the nanobombs. Further, this will also ensure that no heat dissipation occurs to the surrounding tissues and cells that will ensure that the bombing is highly selective to even single cells.

[00040] The nanobombs are exposed to the light for a length of time sufficient to allow explosion; i.e., until explosion of the nanobombs occurs. In the example herein, cells were exposed to the light for sixty seconds. However, the length of time for exposure to light can be longer or shorter.

[00041] All the references described above are incorporated by reference in its entirety for all useful purposes.

[00042] While there is shown and described certain specific structures embodying the invention, it will be manifest to those skilled in the art that various modifications and rearrangements of the parts may be made without departing from the spirit and scope of the underlying inventive concept and that the same is not limited to the particular forms herein shown and described.

EXAMPLE 1

[00043] Single wall carbon nanotubes were fabricated using methane-based chemical vapor

deposition process at 1000 ⁰ C using iron nanoparticles as catalysts according to the process of K. Sivakumar, and B. Panchapakesan, "Electric Field Assisted Growth of Nanowires using Carbon Nanotubes for Nanoelectronics and Sensor Applications", Journal of Nanoscience and Nanotechnology, Volume 5, Number 2, 313-318 (2005). The single wall carbon nanotube sheets were made by vacuum filtration of 20 mL of a 0.6 mg/mL single wall carbon nanotube suspension through a poly(tetrafluoroethylene) filter (Millipore LS, 47 mm in diameter, 5-Dm pores). The single wall carbon nanotube sheet was washed with 200 mL of deionized water and then 100 mL of methanol to remove residual NaOH and surfactant, respectively. The sheets were allowed to dry under continued vacuum purge for one hour before being peeled from the filter. The typical single wall carbon nanotube sheet was fabricated into circular discs about 100 mm in diameter and about 50 μm thick. [00044] Two wells were created on the single wall carbon nanotube sheet by pulsing 800 nm photons at 300 mW/cm ² for 10 seconds that burned only the surface of the single wall carbon nanotube sheet to form wells.

[00045] BT474 cells (a breast cancer cell line) were maintained in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin, 5 μg/mL streptomycin, and 2 mM glutamine under 5% CO ₂ in a humidified incubator at 37 ⁰ C. For deposition into wells, the BT474 cells were removed from the titer plates using 0.5 mM EDTA, pH 7.4, and counted using a hemacytometer under an optical microscope after staining with 0.4% Trypan blue in PBS. About 95 % (percent) of cells were viable. [00046] 1.0 mL suspensions of approximately 1x10 ⁵ BT474 cells in phosphate buffered saline (PBS) were deposited in the well.

[00047] Clusters of BT474 cells on the bottoms of the wells were stained with Trypan blue dye to investigate membrane permeability and cell damage before and after light exposure. [00048] The BT474 cells in one well were illuminated with 800 nm laser light at 200 mW/cm ² for 60 seconds.

[00049] Figure 1 shows photomicrographs of BT474 cell clusters before and after light exposure. Only the cells that were co-localized with the single wall carbon nanotubes and received light exposure were destroyed. The cells that were co-localized with single wall carbon nanotubes and received no light were viable. Nanotube-free control samples showed

no cell damage upon light exposure.

[00050] Figure 2 illustrates that, although the cell clusters were right next to each other, only those cells that received light were destroyed, demonstrating the localized heating that is achievable using this approach. The dramatic explosions of the cells that received light exposure show the temperatures within this area must have been in excess of 100 ⁰ C. Bubbles can be seen around the dead cells showing the boiling effect of PBS with in this co- localized area. Photo-thermal excitation of nanotubes can potentially be used not only for killing cells but also as a nano-surgical tool for repairing cells, organs and tissues in parts of the body that were considered inaccessible in the past. The high efficiency of opto-thermal transitions and the higher achievable temperatures in a localized area at low power makes carbon nanotubes a potent therapeutic agent compared to any other nanotechnological approaches for killing cells.

[00051] Figure 3 A shows another view of cancer cell tissues treated with nanotubes before light exposure. Figure 3B shows the tissues after light exposure.

[00052] Figure 4 shows a close up view of cells exposed to light. The blue area indicates cell death. EXAMPLE 2

[00053] Immunotargeted Nanobombs for Selective Killing of Cancer Cells [00054] Bundles of single wall carbon nanotubes (~ 20 nm) were prepared according to the following method. Single-wall carbon nanotubes (SWCNT) are first grown in-house using methane-based thermochemical vapor deposition technique at 900 ⁰ C and atmospheric pressures using iron and nickel as catalyst metals. The growth is set in the reaction rate- limited regime with high temperatures facilitating high kinetic energy of the gas molecules and with low supply of carbon, allowing the formation of SWCNT. The grown SWCNTs are purified by first heating in dry air at 400 ⁰ C to remove the soot and an acid reflux (3 MHCl for 10 h) to remove the catalyst particles.

[00055] Nonspecific mouse myeloma IgGl (EMD Biosciences) and specific anti-IGFl receptor mouse mAb Ab-3 (33255.111,EMD Biosciences, MA) were prepared in 0.138 M NaCl, 0.0027 MKCl, pH 7.0 (phosphate-buffered saline, PBS) by diluting a 1 mg/mL mAb solution with PBS to a ratio of 1 : 10 (mAb:PBS). Nanotubes were labeled with antibodies by noncovalent adsorption by mixing nanotubes with the antibody solution. The antibody

labeled nanotubes were then incubated with BT474 cells for one hour. BT474 is a human breast cancer cell line that overexpresses HER2 (Her2+) and cMYC oncogenes. [00056] Nanotube bundles were coated with Her2 specific antibodies by mixing the nanotubes with an aqueous buffered solution of antibodies and incubating for up to two hours. The Her2 specific antibody coated nanotube bundles were mixed with BT474 cells in buffer (the buffer solution used was phosphate buffered saline 0.138 MNaCl, 0.0027 λ/KC1, pH 7.0) and allowed to incubate for one hour.

[00057] As a control, nanotube bundles were also coated with a non-specific antibody (i.e., not specific for a receptor or other structure on BT474 cells), as described above, and incubated with BT474 cells for one hour.

[00058] The mixtures of cells and antibody coated nanotube bundles were then subjected to near infrared (NIR) laser radiation at 808 nm and ~800 mW/cm ² intensity for three minutes. Following laser treatment, the cells were treated with Trypan blue dye to capture the uptake of the blue dye due to membrane damage caused by nanotube explosions. Some of the cells around the main cell cluster exploded causing cellular debris stained blue. Her2 specific antibody targets the nanotube bundles to cancer cells by docking with its cell surface receptor on the BT474 cells. Radiation treatment of the Her2 coated nanotube bundles and BT474 cells results in explosion of the nanotubes and destruction of cells. By contrast, radiation treatment of the mixture of non-specific antibody coated bundles of nanotubes and BT474 cells did not kill the cells.

[00059] Figure 5A shows nanotubes treated with Her2 antibodies and incubated with BT474 cells in PBS for 1 hour followed by NIR radiation for 3 minutes. The cells appear blue indicating that they were killed. Figure 5B shows nanotubes treated with non-specific antibody, incubated with BT474 cells for one hour and followed by NIR treatment for 3 minutes. The cells do not appear blue indicating that they were not killed.