[go: up one dir, main page]

WO2007094870A2 - Toxicologie et effet cellulaire de nanomatériaux fabriqués - Google Patents

Toxicologie et effet cellulaire de nanomatériaux fabriqués Download PDF

Info

Publication number
WO2007094870A2
WO2007094870A2 PCT/US2006/060369 US2006060369W WO2007094870A2 WO 2007094870 A2 WO2007094870 A2 WO 2007094870A2 US 2006060369 W US2006060369 W US 2006060369W WO 2007094870 A2 WO2007094870 A2 WO 2007094870A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
genes
nanomaterials
nanomaterial
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2006/060369
Other languages
English (en)
Other versions
WO2007094870A3 (fr
Inventor
Fanqing Chen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Publication of WO2007094870A2 publication Critical patent/WO2007094870A2/fr
Priority to US12/111,026 priority Critical patent/US8785505B2/en
Anticipated expiration legal-status Critical
Publication of WO2007094870A3 publication Critical patent/WO2007094870A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns

Definitions

  • This work was performed under the auspices of the U.S. Department of Energy, at the University of California/Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, and University of California/Lawrence Berkeley National Laboratory, under Contract No. DE-AC03-76SF00098, now Contract No. DE- AC05-CH11231. The government has certain rights in this invention.
  • the present invention relates to carbon nanomaterials and their therapeutic and cytotoxic uses thereof. More specifically, the present invention relates to using multiwall carbon nanomaterials for the treatment of cancer and other diseases. The present invention also relates to carbon nanomaterials and methods for measuring their toxicity thereof.
  • Material that does not manifest toxic or carcinogenic characteristics in regular forms may have altered physical/chemical properties due to the quantum effect when their feature sizes fall in the 1-lOOnm range that define them as nanomaterials.
  • the transport and persistence of nanomaterials in the environment might be drastically different from the bulk material that we are familiar with, and new biological mechanisms for interaction, uptake and metabolism of nanomaterials have begun to emerge in the last few years.
  • the unique properties of the nanomaterials include the increased surface/mass ratio, different shapes with size scale at the same range as biomolecules, altered mechanical and electromagnetic properties. It is critical to identify potential toxic/carcinogenic features of manufactured nanomaterial early in the process so that proper precautions can be taken before long term damages are done.
  • Carbon nano-materials including carbon nanoparticles and nanotubes, have been one of the most extensively used nanoparticles, because of their unique and superior properties, including large surface areas, high electrical conductivity, and excellent strength.
  • Multiwall carbon nanotubes MWCNTs
  • MWCNOs multiwall carbon nano-onions
  • Single-walled, double-walled and multi-walled MWCNTs have led them to be used in applications ranging from nanowires, electronic components , catalyst supports, electronic displays to drug delivery, and may even be used for hydrogen storage.
  • Giant nested fullerenes, generally called nano-onions (MWCNOs)
  • MWCNOs nested fullerenes
  • MWCNOs are usually produced by an underwater carbon-arc discharge.
  • the applications of MWCNOs have lagged behind those of MWCNTs, they have been used as components of nanocomposites for applications including solar cells, light-emitting devices, and in fuel-cell electrodes.
  • SWCNT single-walled carbon nanotubes
  • C 2 Bi 0 carborane cage-coated SWCNT has been constructed as the delivery vehicle for boron neutron capture therapy for cancer (Yinghuai, Z. et al.
  • the present invention is directed tobiomarkers whose gene expression profiles are changes upon exposure to nanomaterials.
  • the biomarkers are found in the biological pathways of inflammation, apoptosis, immune response, ubiquitination, cell proliferation, cell cycle regulation, cell differentiation, golgi vesicle transport, membrane fusion, secretory pathway, intracellular transport, nucleocytoplasmic transport, apoptosis, response to DNA damage, response to stress and stimuli.
  • biomarkers described herein and in the Examples can be used to select and/or develop the suitable instruments and methods for measuring exposure to particles according to the health affects.
  • An ideal instrument would be a biosensor or lab on a chip device that industry could use to batch test materials.
  • biomarkers identified that are associated with particular nanoparticles it is possible to evaluate the cytotoxicity of various nanomaterials that are carbon- based (such as carbon nanotubes and spherical carbon nanoparticles) or semiconductor-based (such as semiconductor nanocrystals), or metal-based, using the biomarkers and biomarker temporal change patterns as predictors for other nanoparticles. It was found that particular biological pathways are activated or perturbed by nanoparticle, these pathways and the nanoparticle specific biomarkers are listed in tables in the publications, including apoptosis, inflammation, cellular transporter, ubiquitination, etc. The changes in these biomarkers can be used as indicators or predictors for nanotoxicity.
  • Another aspect of the invention is the sensitivity of the assay system on nanotoxicity, when microarray technology is used here.
  • the invention here provides guidelines for the threshold of cytotoxicity and the correlation with gene expression profile changes.
  • the threshold is defined as 1% of total genes are changed more than two-fold.
  • Nanomaterials such as quantum dots are well attenuated by the protective, polyethylene glycol, and the genes changed are less than 0.5% of total genes. This number can be used as a quantitative measurement on whether the protective coating is effective for any other nanoparticles, or nanomaterials.
  • microarray gene expression analysis provides are quantitative and comprehensive measurement matrix for determine the effectiveness of the protective coating. This quantitative measurement can be used for any other nanoparticles that might have toxicity against the cells, tissues, or organs.
  • the gene ontology classification methods used also are very powerful indicators for the primary effect of the nanoparticles/nanomaterials. For instance (Table 1), 2% percentage of genes in the Golgi body tranport pathways are changed after treatment, with P value ⁇ 0.0001. These markers identified can be used as indicators of the nanotoxico logical effects of nanomaterials, and more specifically nanocrystals, multiwall carbon nano-onions (MWCNOs) and multiwall carbon nanotubes (MWCNTs).
  • MWCNOs multiwall carbon nano-onions
  • MWCNTs multiwall carbon nanotubes
  • the genes identified and similar gene profiles identified using similar assay systems can be used as guidelines for attenuating the toxic effects.
  • the biomarker changes should be eliminated or weakened, with the elimination of the nanotoxicity. This can be used as a measurement on the efficiency of toxicity control.
  • Another related aspect of the present invention is directed to using the cytotoxicity of nanoonions, by attaching proper targeting mechanisms (such as antibodies, small molecules, or peptides) to the nanoonions, for delivery to cancer cells in a tumor to kill the cancer cells.
  • the nanoonions can be coated by liposomes, which can be attached to other targeting mechanisms and allow specific delivery.
  • the nano-onions can also be directly inject into solid tumor by intratumoral injection or catheter-directed injection, accumulate in the tumor and kill the tumor over a 1-2 week period by staying in the interstitial fluid within the tumor and taken up by the tumor.
  • IR laser can be used to further enhance the killing of the cancer cells with nano-onion accumulated, by hyperthermal effects through absorption.
  • Carbon nanoonion can also be doped with Gadolinium and serve as a MRI contrasting reagent. It can also be doped with radionuclides for use in PET imaging. In the imaging-enhance version of the nano-onions, it can be used as a tool for image-guided intervention of tumor.
  • the nanoonions can be used for intratumoral injection to kill cancer when conjugated to tumor-targeting molecules, such as monoclonal antibodies, peptides, folate, etc.
  • tumor-targeting molecules such as monoclonal antibodies, peptides, folate, etc.
  • the targeted nanoonion can be carried to tumor and kill the tumor cells.
  • the nanoonions can be used bound to or encapsulated in immunoliposomes.
  • the present invention provides for a composition for the treatment of cancer, comprising: a multiwall carbon nanomaterial conjugated to a tumor-targeting molecule and an imaging reagent.
  • the multiwall carbon nanomaterial is a nanoonion having a diameter of 10 to 50 nm.
  • the tumor-targeting molecule can be selected from the group consisting of monoclonal antibodies, oligonucleotides, peptides, and small molecules.
  • the tumor-targeting molecule is a monoclonal antibody that is specific for Erb ⁇ 2.
  • the tumor-targeting molecule is a small molecule selected from the group consisting of folate, a vitamin, and a drug.
  • the imaging reagent can be selected from the group consisting of a radiolabel, radionuclide, fluorescent probe and chemiluminescent probe.
  • composition further comprises an immunoliposome bound to or encapsulating the multiwall carbon nanomaterial.
  • the present invention also provides a method of treating cancer comprising delivering the multiwall carbon nanomaterial conjugated to a tumor-targeting molecule and an imaging reagent to a subject in an effective therapeutic amount.
  • the effective therapeutic amount does not induce greater than 25% overexpression or underexpression of a gene following treatment.
  • FIG. 1 Scanning electron microscopy (SEM) images and high resolution transmission electron microscopy (HRTEM) images of carbon nanomaterials used in this study.
  • A. SEM image of multi-walled carbon nanotubes (scale bar 200nm).
  • B. SEM image of carbon nano-onions (scale bar 200 nm).
  • C. HRTEM images of multi-walled carbon nanotubes (MWCNTs) (scale bar 5 nm).
  • Figure 2. Cell viability measurements after treatment with carbon nanomaterials at cytotoxic doses.
  • the numbers of low doses (0.6 ⁇ g/mL for MWCNO and 0.06 ⁇ g/mL for MWCNT), and high doses (6 ⁇ g/mL for MWCNO and 0.6 ⁇ g/mL for MWCNT) represent the nanomaterials concentration used for treatment. Bars represent the mean of cell numbers from 10 imaged viewfields in 10 treated wells and error bars represent a 95% confidence interval. Each nuclei imaged by the KSR was identified with the Cell Health Profiling software in the blue channel by Hoechst staining. B and C.
  • YO-PRO 1 is visualized in the green channel and PI is visualized in the red channel, where measurement such as dye intensity and area can be made using the Cell Health Profiling algorithm.
  • D Average intensity of YO-PRO 1 intensity and PI intensity of mock treated and treated skin fibroblasts at 48 hours. The YO-PRO 1 intensity is proportional to apoptosis and the PI intensity correlates to necrosis. Bars represent the mean of cell numbers from eight treated wells and the error bars represent a 95% confidence interval. Data for lung fibroblast treated under the same condition is presented in Figure 7. [031] Figure 3. Measurement of cell proliferation after treatment with carbon nanomaterials at cytotoxic doses.
  • FIG. 1 An average of 20,000 cells was measured for each treatment condition.
  • Figure 4. A Numbers of genes whose expression levels changed after treatment with carbon nanomaterials at cytotoxic doses.
  • B-E Venn diagrams comparing numbers of genes that showed expression changes. Each Venn diagram is divided into 3 areas and labeled as I, II and III. Area II is the overlapping area of two circles, represent commonly changed genes in both conditions. Area I and III represent genes that changed only in the condition specified in the circle. Bioconductor software was used to perform significance analysis to determine the difference between expression levels in treated sample and control sample possesses statistical significance. The empirical Bayesian model was used with Bonferroni's multi-test correction.
  • the cutoff of p-values produced through the analysis was determined by at least 10 times less than the p-values of the smallest p-value of control probe sets on the chip.
  • B comparing different doses for the nano-onions.
  • C comparing different doses for the nanotubes.
  • D comparing different particles at low doses (0.6 ⁇ g/mL for MWCNO and 0.06 ⁇ g/mL for MWCNT).
  • E comparing different particles at high doses (6 ⁇ g/mL for MWCNO and 0.6 /zg/mL for MWCNT).
  • FIG. 5 Promoter analysis.
  • A. The interaction matrix for the differentially expressed genes (horizontal) and transcription regulatory elements (vertical) in the up- and down-regulated gene sets at different dosage using different carbon nano-particles. Individual elements of the matrix are colored by the significance/?- values: over-representation in the matrix is colored in red. The brightest red represents low/?-value (most significantly over-represented).
  • B. Enriched transcription regulatory elements for the nano-particle dataset.
  • Figure 6 A comparison of activated signal transduction networks for higher dose responses to carbon tubes and carbon onions.
  • PathwayBuilder software (Arkin Group, LBNL) is used to analyze and create pathways differentially activated with the treatment matrix based on published literature.
  • Figure 7. A. Same as in Figure 2, except lung fibroblasts were treated. B. Average intensity of YO-PRO 1 intensity and PI intensity of mock treated and treated lung fibroblasts at 48 hours. Bars represent the mean of cell numbers from eight treated wells and the error bars represent a 95% confidence interval. [036] Figure 8. Scatter plot of normalized GeneChip data. The average signal intensities of
  • X-axis represents the signal intensity of ethanol control sample
  • Y-axis represents samples treated with Carbon nanoparticles at different doses.
  • the line through the center of gene populations indicates exactly same intensities between Carbon and control samples, two lines flanking the center line indicates 2 folds differences between the two conditions. Genes that fall in the area outside the 2-fold difference lines have more than 2- fold gene expression changes.
  • FIG. 9 A. Schematics of the PEG-silica embedded semiconductor nanocrystals.
  • the silica shell is functionalized with -SH groups and with PEG group. The latter provide additional stability and reduced non-specific bindings.
  • the scale bar ( ⁇ 3 nm) provides a qualitative comparison between the overall size of the silanized dot ( ⁇ 8nm) and the size of the semiconductor core ( ⁇ 3nm).
  • FIG. 10 Cell counts for IMR-90 and HSF-42 cells after treatment with silanized semiconductor nanocrystals in various doses. When treated with PEG-silane-semiconductor nanocrystals, the survival rate of both cell lines is mostly unaffected. The statistically insignificant reduction in the cell number may be explained by a mild block of the G2/M phase (see Fig.2.III). This contrasts with the marked effect that organic nanostructures (carbon nanotube and nano-onions) have on IMR-90 and HSF42 cells. II. a.
  • Figure 11 A. Scatter plots for the two doses of PEG-silane-Qdot treatment, in a loglO scale.
  • the Y-axis represents treated cells, the X-axis represent the control.
  • the dashed lines correspond to changes of level of expression by a factor of 2. The tightness of the plot, indicates that most of the genes do not change significantly after PEG-silane-semiconductor nanocrystals treatment. .
  • Figure 12 Analysis of Transcription Regulatory Elements (TREs) in the promoters of the altered genes.
  • the TREs for different transcription factors on the promoter regions of the altered genes are analyzed for over/under-representation relative to all promoters in PAINT database.
  • the relationship of TREs and input genes are represented as an image of the interaction matrix: the columns of the interaction matrix correspond to the enriched TREs and each row corresponds to a gene from the input list. Individual elements of the matrix are colored by the significance p-values: over-representation in the matrix is indicated in red.
  • FOX transcription binding elements There is an enrichment of FOX transcription binding elements on the high dose responsive genes. In low dose responsive genes that are down regulated, there's enrichment of DEC/BHLHB2, and COMPl (cooperates with myogenic protein 1).
  • Figure 14 Human epithelial cells with foci of activated DNA damage response proteins.
  • Figure 15 Automated cell culture and manipulation system that can be used to assess responses of fibroblasts, keratinocytes and epithelial cells to nanomaterials.
  • Figure 16 Two-dimensional gel electrophoresis analysis of multi-wall carbon nano- onion. A. untreated cell; B. treated cell; C andD. Mass spectrometry for two of the genes identified
  • Figure 19 Reverse phase protein array, probed with anti-pAkt.
  • Table 1 Most significantly changed gene categories after treating HSF42 cells with carbon nanomaterials at cytotoxic doses.
  • the categories are generated by GoMiner program (Materials and Methods, Supporting Information), using p-value as the evaluation criteria of statistically significant changes.
  • p-value was calculated by conducting two-sided Fisher's exact test, which reflects the statistical significance for that category being enriched in changed genes. The p-values were used to sort categories to identify those gene functional groups that have responded the most after treatments.
  • transport Golgi vesicle transport, membrane fusion, secretory pathway, intracellular transport.
  • Fold change of gene expression is given for the low dose (0.6 ⁇ g/mL for MWCNO and 0.06 ⁇ g/mL for MWCNT). Fold changes represent the ratio of mRNA amount of treated samples divided by those of control samples.
  • Table 4 Genes changed by nano-onion and carbon nanotubes but fall in the category of cell cycle regulatory genes (Gl /S transition of mitotic cell cycle, mitotic cell cycle, and cell growth of maintenance). Fold change of gene expression is given for the low dose (0.6 ⁇ g/mL for MWCNO and 0.06 ⁇ g/mL for MWCNT). Fold changes represent the ratio of mRNA amount of treated samples divided by those of control samples.
  • Fold changes represent the ratio of mRNA amount of treated samples divided by those of control samples.
  • Fold changes represent the ratio of mRNA amount of treated samples divided by those of control samples.
  • Table 7 The functional categories of the genes affected by low and high doses of PEG- silane-Semiconductor nanocrystals. All functional categories affected by high doses are also affected by a low dose treatment. A significant portion of the down-regulated genes are related to M-phase of mitotic cell cycle, especially the spindle assembly and cytokinesis.
  • the up-regulated genes include those for carbohydrate binding proteins (possibly in recognition of the PEG coating of
  • Semiconductor nanocrystals include intracellular organelle (especially vacuole and intracelluar vesicle) related proteins (possibly involved in intracellular transport of semiconductor nanocrystals), and stress-response genes (possibly due to the slight stress induced by treatment).
  • Table 8 Significantly changed genes after treatment with PEG-silane-semiconductor nanocrystals.
  • the genes presented in the table are the ones with fold change more than 2, and P value less than 0.05.
  • Table 10 Genes in Area II of Figure 4B. Common genes changed in both the high and low dose treatment with carbon nano-onions.
  • Carbon nano-materials including carbon nanoparticles and nanotubes, have been one of the most extensively used nanoparticles, because of their unique and superior properties, including large surface areas, high electrical conductivity, and excellent strength. It is postulated that there are size-specific, shape-specific, and surface-specific effects and effectors for particles at the quantum range (1-lOOnm). These effects are different from the effects observed for micro-sized particulates, and these quantum properties unique to nanomaterial play important role in determine toxicity, with altered genomic, proteomic and cellomic profiles, altered mutagenesis and carcinogenesis potentials, and different cellular level transport mechanisms.
  • Toxicity of nanomaterials is a major healthcare concern that may impact the nanotechnology industry.
  • Concern has been rising following studies on the toxicity of carbon nanophase materials, some of which are found in flames, welding fumes, diesel exhausts and other petrol byproducts See Maynard, A. D., et al.,. J Toxicol Environ Health A 2004, 67, 87-107; Silva, V. M, et al., Toxicol Sci 2005; Frampton, M. W., et al., Res Rep Health Efflnst 2004, 1-47; discussion 49-63; Block, M. L., et al., Faseb J 2004, 18, 1618-1620).
  • Ceo is an excellent electron acceptor that can readily react with available oxygen and water to generate free radicals leading to oxidative damage of the cellular membrane. Derivatized fullerenes are less efficient in producing oxygen radicals, therefore Ceo derivatized with hydroxyl groups is much less toxic. Less is known about the toxicity of fluorescent semiconductor nanocrystals (commercially sold as QUANTUM DOTS by Invitrogen). Semiconductor nanocrystals are CdSe/ZnS core/shell nanocrystals and the heavy elements that make up the core may induce a more pronounced and acute cytotoxic response than carbon nanostructures.
  • assays detecting toxicity, stress response and DNA damage as a result of nanomaterial exposure are examined in any cell type, preferably in human epithelial cells, normal human keratinocytes (NHK) and human fibroblasts (HSF).
  • each assay is first calibrated against nanomaterial known to elicit toxic, stress and/or DNA damage responses. For instance, the examples show that MWCNT induces inflammatory response, and titanium dioxide induces DNA damage.
  • the dose range for each of the nanomaterials tested by the assay is decided using cell proliferation, apoptosis/necrosis, cell cycle assays using the cytometry and Cellomics.
  • Nanomaterial assessment In the present method, nanomaterials are assessed for toxicity and ability to elicit stress and/or DNA damage using calibrated materials as described herein. All analyses should be performed in triplicate for the three test cell types. Depending on composition, nanomaterials will be resuspended in water, ethanol or DMSO or any other appropriate solvent and sonicated for one hour prior to biological assessment. The exact assessment strategies will depend on the results of the calibration studies. However, the assays will be optimized to achieve maximum sensitivity to induced toxicity, stress and DNA damage. Surface chemistry will be an important parameter to be explored since published reports suggest this is one of the critical determinants in physiological impact (See Sayes, C. et al.
  • the reverse phase protein array can be used (Fig. 15) as described by Shingyoji, M., Gerion, D., Pinkel, D., Gray, J.W. & Chen, F. Quantum Dots-based Reverse Phase Protein Microarray. TALANTA 67, 472-478 (2005), hereby incorporated by reference, to quantitate proteins in treated cells to determine toxicity. It may be preferred to use antibodies already tested for epithelial cancer cells in the NCI ICBP P50 program, to minimize efforts validating the assay.
  • one of the following approaches can be used to evaluate toxicity in nanomaterial exposed cells: (i) the measurement of phenotypic changes in large populations of cells by high content analysis and (ii) gene expression array analysis in exposed cells. For instance, it was found that carbon nanomaterials generated mRNA level changes in exposed skin fibroblasts, including changes in mRNA levels from genes involved in metabolism, apoptosis, cell cycle, stress response, cellular transport, and inflammatory response. Thus, in a preferred embodiment, toxicity is measured by profiling the transcription levels of genes, specifically those found in the Tables as potentially being most affected by exposure to nanomaterials. Genes that demonstrate expression level changes after nanomaterial treatment are placed into Gene Ontology categories using GOMiner, evaluated for statistical significance, and then sorted by significance (See Table 1 in Appendix for example).
  • transcription profiling is carried out using methods and systems known in the art.
  • transcription profiling is carried out using Affymetrix Ul 33 A arrays in the High Throughput Array (HTA) system.
  • HTA High Throughput Array
  • the HTA that processes arrays in a standard 96 well microplate format.
  • a Sciclone microfluidics platform (Caliper Life Sciences, Hopkinton, MA) integrated into this system performs standardized protocols for cRNA probe preparation, quantification and normalization; hybridization setup, and array washing and staining.
  • a Zymark Twister (Caliper Life Sciences, Hopkinton, MA)arm moves plates (and tips) onto and off of the deck and into the thermal cycler for all temperature-dependent steps.
  • An Axon scanner and ImageXpress 5000 (Molecular Devices, Sunnyvale, CA) console application, tightly integrated with this system, generates 25 GB of data from one plate of 96 human U133AofAv.2 arrays (Affymetrix, Santa Clara, CA) in approximately 8 hours.
  • This new format automates the most labor-intensive steps resulting in much higher throughput (five fold increase) at a much reduced cost.
  • nanomaterials that are carbon-based (such as carbon nanotubes and spherical carbon nanoparticles) or semiconductor-based (such as semiconductor nanocrystals), or metal oxide -based, or any nanomaterials made from any combination of these or derivative thereof, having any surface or other modification thereof.
  • the nanomaterials to be evaluated can be in the size range of 1-lOOnm.
  • biomarkers and biomarker temporal change patterns herein described and further obtained can be used as predictors for other nanoparticles. It was found that particular biological pathways are activated or perturbed by nanoparticles, including apoptosis, inflammation, cellular transporter, ubiquitination, etc. These pathways and the nanoparticle specific biomarkers are listed in Tables 2-7 and 8-21 attached and incorporated by reference. Thus, the changes in these biomarkers can be used as indicators or predictors for nanotoxicity.
  • the Tables and results herein described are used as guidelines for the threshold of cytotoxicity and the correlation with gene expression profile changes. For example, comparison between gene expression profiles of cell exposed to the nanomaterial being tested and the gene expression profile of semiconductor nanocrystals (Table 7-8) and multi-wall carbon nanotubes and multi-wall carbon nanoonions (Tables 1-6) can be used aid in the prediction of toxicity of a nanomaterial.
  • the threshold is defined as 1% of total genes are changed more than two-fold. In other embodiments, the threshold may be defined by observing a percent change (e.g., 20% to 50% or more change) in gene expression in a predetermined set of genes.
  • Nanomaterials such as semiconductor nanocrystals are well attenuated by the protective, polyethylene glycol, and the genes changed are less than 0.5% of total genes. In one embodiment, this number can be used as a quantitative measurement on whether a protective coating is effective for any other nanoparticles, or nanomaterials.
  • chemically induced toxicity is measured in cell lines for the nanomaterials being tested plus other compounds selected to induce stress or DNA damage, such as MNNG etc., which was used in previous studies (Yu, Y. et al. A comparative study of using comet assay and ⁇ H2AX foci formation in the detection of N-methyl-N'-nitro-N-nitrosoguanidine- induced DNA damage. Toxicology In Vitro In press (2006); Zhou, C. et al. DNA damage evaluated by ⁇ H2AS foci formation by a selective group of chemical/physical stressors. Mutation Res. In press (2006)).
  • epithelial cells, keratinocytes and fibroblasts will be grown in 24 well format and IC50 values will be determined by measuring changes in cell number induced by each nanomaterial and reference compound.
  • cells will be analyzed in 24 well cultures established and maintained using the automated cell culture and manipulation instrumentation (Fig 15).
  • Cell number will be determined by staining cultures with 4',6-diamidino-2-phenylindole (DAPI), a fjuorgscgnt stain that binds strongly to DNA, automatically acquiring images of cells in each well and counting the number of DAPI stained cells in each well using the Cellomics Arrayscan V TI (Cellomics, Inc., Pittsburgh, PA) and associated software.
  • DAPI 4',6-diamidino-2-phenylindole
  • An evaluation matrix can be used that focuses on one variable at a time for each set of nanomaterials tested, and using PEG-passivated semiconductor nanocrystals as a control because the PEG passivated semiconductor nanocrystal induces minimal changes in gene expression, and can be a very good negative control (Data shown in Examples).
  • the stress response of cells in response to nanoparticle treatment is evaluated.
  • Responses to toxic chemicals typically include repression of protein synthesis and cell-cycle-regulated genes and induction of DNA damage and oxidative stress- responsive genes. These responses manifest at several levels. Others have shown, for example, that these responses can be revealed using microarray based analysis of gene expression and suggest the utility of assessing changes in gene expression as a sensitive way of identifying nanomaterial- induced stress.
  • Transcript profiling technology or high-throughput 2D gel - mass spectrometry enable quantitative measurement of the transcriptional activity of thousands of genes and many proteins in biological samples.
  • inflammation plays a central role in development of cancer. Inflammatory cells in "inflamed" tissues produce a variety of free radicals and Reactive Oxygen Species (ROS); free radicals and ROS exert effects on cells.
  • ROS Reactive Oxygen Species
  • the Examples demonstrate that stress response genes are perturbed by treatment with carbon nanomaterials. See Table 6, for example. For instance, of interest is the observation that MWCNTs appear to induce a greater amount of stress upon the cells than MWCNOs, even though the dosage is 1/10 th by weight/volume concentration.
  • the present toxigenomic approach also find genes involved in inflammatory and innate immune response affected by nanoparticles.
  • Mammals respond to wounding, pathogens, foreign particles and non-self proteins by activation of innate and adaptive immune systems. Chronic presence of a proinflammatory pathogen/particle leading to chronic activation of granulocytes is accompanied by production Of H 2 O 2 that can result in suppression of adaptive immune functions, specifically release of ROS. It will be important to investigate whether nanomaterials initiate "inflammatory-type" responses. Most importantly, with bioinformatics tools, the spatial-temporal activation of the immune response can be categorized by data clustering software, and expression patterns can be associated with specific size, shape and surface chemistry, and used as biomarkers.
  • the stress responses of any cell type that can be cultured are measured.
  • epithelial cells, keratinocytes and fibroblasts to agents known to induce stress responses e.g., doxorubicin, 5-fluorouracil, mitomycin C and radiation
  • agents known to induce stress responses e.g., doxorubicin, 5-fluorouracil, mitomycin C and radiation
  • Changes in global gene expression patterns, p38 phosphorylation and COX-2 expression are assessed using microarray technologies and high content imaging, respectively.
  • cells are exposed to the stress-inducing agents, such as doxorubicin, 5-fluorouracil, mitomycin C and radiation, at six different concentrations in 24 well cultures, preferably using automated cell culture and manipulation instrumentation as shown in Fig.
  • RNA can be harvested automatically.
  • Global changes in gene transcription can be analyzed using the Ul 33 A expression microarray platform via the Affymetrix HTA system. These data are analyzed to identify stress response transcriptional signatures that are common to all agents.
  • changes in COX-2 and phospho-p38 levels can be assessed using a high content imaging system after immunocytochemical staining for COX-2 and phospho-p38.
  • a high-content fluorescence image analysis system such as Cellomics ArrayScan (Cellomics, Inc., Pittsburgh, PA) is used to measure cellular responses to chemical and nanomaterials.
  • the ArrayScan is an automated imaging instrument that scans through the bottom of clear-bottom 24-well plates, focuses on a field of cells, and acquires images at each selected color channel.
  • the Cellomics software identifies and measures individual features and structures within each cell in a field of cells, so that up to hundreds of cell samples can be analyzed in parallel.
  • the software then tabulates and presents the results in user-defined formats. In a preferred embodiment, these data are analyzed to identify COX-2 and phospho-p38 staining characteristics that are common to all chemicals. These signatures are then compared to nanomaterials-induced changes in order to identify nanomaterials that induce stress responses. For microarray experiments, it is preferred that 2 time points, once each at 2 hours and 8 hours is used to capture acute response.
  • the nanomaterials are assessed using 2D-gels and/or mass spectrometry.
  • treated HSF cells are lysed and electrophoresed on the 2D gel as described below for Figure 16.
  • the first-dimension IEF was performed by using an Ettan IPGphor unit (Amersham Biosciences) with a power supply EPS 350 IXL.
  • the second-dimension SDS-PAGE is carried out in an Ettan DALTsix system (Amersham Biosciences). IPG strips were equilibrated and sealed on the top of 10% SDS gels with 0.5% SeaKem LE-agarose (Cambrex Corp.). SDS-PAGE was performed and the 2D gel silver stained (Fig. 12A).
  • Apoptosis Apoptotic cells can be detected based on nuclear morphology, mitochondrial mass and/or membrane potential and f-actin content after staining with the Cellomics Multiparameter Apoptosis 1 HitKitTM (Cellomics, Inc., Pittsburgh, PA). Nuclear morphology (condensation or fragmentation) is measured after staining with a stain such as Hoechst 33342.
  • Mitochondrial membrane potential and mitochondrial mass is measured after staining with, for example, Mito Tracker® Red (Molecular Probes, Invitrogen, Carlsbad, CA). F-actin can be measured after staining with an Alexa Fluor® 488 conjugate of phalloidin (Ax488-ph) (Molecular Probes, Invitrogen, Carlsbad, CA). An additional measure of apoptosis will include staining with Alexa Fluor® 488 conjugate of annexin V (Molecular Probes) and staining with propidium iodide (PI). PI cannot permeate apoptotic cells and live cells but can enter and bind nucleic acids in necrotic cells. When Hoechst 33342 is used as a counterstain, apoptotic cells fluoresce green, necrotic cells both green and red, and live cells only blue from the Hoechst stain.
  • DNA damage In a preferred embodiment, mutagenic potential will be indexed by measuring induction of DNA damage, mutagenesis, and performing gammaH2AX foci formation and comet assays. DNA damage elicits several responses that can be quantified as an indication of the extent of damage. These include recruitment of p-ATM, p-Chk2 and gammaH2AX (Fig .14) to sites of DNA damage, apoptosis and cell cycle inhibition.
  • DNA damage induced by radiation known mutagens, carcinogens or other materials is measured in order to calibrate DNA damage assays that will be used to assess DNA damage induced by nanomaterials. For example, one can stain p-ATM, p-Chk2 and gammaH2AX in interphase cells immunocytochemistry and determine number of fluorescently stained foci/nucleus using the Cellomics Arrayscan., whereby the number of foci will be used as an indication of extent of DNA damage (see Yu, Y.
  • DNA damage and chromosomal aberration is measured by using comparative genomic hybridization (CGH).
  • CGH comparative genomic hybridization
  • ArrayCGH is performed to allow global profiling of gene amplification/deletion to generate genome instability data after chronic treatment by the nanomaterial.
  • Another aspect associated with oxidative damage is mutagenesis and carcinogenesis. It is still unknown whether chronic exposure to nanomaterials will induce meaningful genome instability to be mutagenic and carcinogenic.
  • Mutagen sensitivity measured as mutagen-induced chromatid breaks per cell has been used to study susceptibility to various epithelial cancers.
  • CGH comparative genomic hybridization
  • CGH is a powerful genome- wide method for molecular cytogenetic analysis of cancer as described in Kallioniemi, A. et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818-821 (1992), and incorporated by reference. It has been used successfully in molecular classification and diagnosis of carcinomas, such as melanoma, breast cancer, ovarian cancer, lung cancer, etc.
  • CGH allows detection and mapping of allelic imbalance by simultaneous in situ hybridization of differentially labeled tumor genomic DNA (green fluorescing) and normal reference DNA (red fluorescing) to a normal human metaphase spread.
  • Regions of increased or decreased copy number in the tumor are mapped onto the normal metaphase chromosomes as increases or decreases in the green to red fluorescence ratio for each locus. Fluorescence ratios along the length of the chromosomes provide a cytogenetic representation of DNA copy-number variation. [099] Specific methods for earring out each of these assays are described below and in Example
  • the treatment experiments are carried out using an automated cell culture system as shown in Figure 15. Genome instability index will serve as an additional indicator for carcinogenesis/mutagenesis potential.
  • the cytotoxicity of nanomaterials can be determined using three- dimensional tissue culture models.
  • the stromal microenvironment contributes significantly to establishment of cancer and can modulate metastatic dissemination; thus, it is speculated that nanomaterial disruption of this microenvironment may be carcinogenic.
  • the extent to which nanomaterials modulate signaling from the microenvironment to epithelial cells can be explored by investigating the extent to which nanomaterials influence proteins and phosphoproteins involved in signaling from the microenvironment in breast epithelial cells.
  • 3DBM cultures can be used to assess impact of nanomaterials with emphasis on those that are found to be toxic, induce stress or DNA damage OR that are intended for interrogation of living systems.
  • Endpoints that can be assessed include (a) transcript profiles as measured on the Affymetrix HTA system using U 133 A arrays, (b) proteins involved in signaling from the ECM including ⁇ l integrin, EGFR, ⁇ v ⁇ 6 integrin, MAPK, PI3K, ErbB2,CAR, PDGF, Src, Fnl4 and LT ⁇ (c) Cell morphology will be imaged daily for 6 days. Cellular response to treatment will be assessed by morphological criteria, e.g.
  • Day 6 controls and wells which are deemed of interest will be treated with Matrisperse so that cell structures can be dispersed onto glass slides, fixed and assessed for cell proliferation, polarity and apoptosis.
  • Matrisperse For microarray expression analysis, four 35mm plates of cells will be cultured in 3D as described above. Again, representative cultures will be viewed daily and on day 6 plates treated with PBS w/o Ca & Mg+EDTA to release cellular structures.
  • Disaggregation to single cells will be accomplished enzymatically so that epithelial and stromal cells can be purified by magnetic beads using cell surface antibodies as described above. Purified cells will be used to isolate RNA using Qiagen RNAeasy kit or to make protein Iy sates.
  • a study is performed on whole organisms.
  • a living system may have several lines of defense to prevent or minimize some of the toxic effects of exposure to small particles, thus in a preferred embodiment, animal and human studies should be carried out.
  • nanomaterials fail to be efficiently cleared, their risk of cellular contact will be enhanced. Inappropriate cellular contacts may stimulate inflammatory and/or oxidative stress responses that could then be potentiated by large surface areas (relatively) of nanomaterials and result in specific or systemic dysfunction.
  • the present approach will be to use well-characterized models of cell physiology of increasing relevance and complexity to investigate nanoparticle-induced changes that may indicate toxic or carcinogenic effects.
  • high throughput genomic and proteomic analysis strategies to identify physiologic effects of nanoparticles in epithelial cells, keratinocytes and fibroblasts in two- dimensional (2D) cell cultures are carried out.
  • Nanomaterials that regulate gene expression associated with toxicity or carcinogenesis will be tested as well.
  • the nanomaterials should be tested using three dimensional (3D) cultures that mimic microenvironments in vivo (i.e. cell-ECM, cell-cell- myoepithelial and stromal- interactions.
  • 3D three dimensional
  • the present invention further provides kits for diagnosing the cytotoxicity of a nanomaterial.
  • the biomarkers described herein and in the Examples can be used to select and/or develop the suitable instruments and methods for measuring exposure to particles according to the health affects.
  • An ideal instrument would be a biosensor or lab on an array chip device that industry could use to batch test materials.
  • MWCNO and MWCNT exposure activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response.
  • MWCNTs induce genes indicative of a strong immune and inflammatory response within skin fibroblasts, while MWCNO changes are concentrated in genes induced in response to external stimuli.
  • Promoter analysis of the microarray results demonstrate that interferon and p38/ERK-MAPK cascades are critical pathway components in the induced signal transduction contributing to the more adverse effects observed upon exposure to MWCNTs as compared to MWCNOs.
  • the unique genes flanking the overlapping area in Figure 4D and 4E may indicate cellular responses unique to exposure with MWCNOs or MWCNTs (Supplement Tables 15, 17, 18, and 20).
  • MWCNOs or MWCNTs Selection Tables 15, 17, 18, and 20.
  • the results presented here show for the first time both a phenotypic response of cells to carbon nanomaterials (apoptosis, necrosis, cell cycle perturbation, and anti-proliferation) and a global gene expression response at a cellular level. Phenotypic effects were confirmed two different fibroblast cell types, human skin fibroblast (HSF, see Figures and Tables in text) and IMR-90 ( Figure 7). This information will be important for elucidating possible mechanisms responsible for the toxicity observed after exposure to these particles.
  • HCA high content analysis
  • HCA of cells treated with MWCNOs, MWCNTs and semiconductor nanocrystals showed significant changes in cell number that, upon further investigation, was shown to be due to apoptosis, cell death and proliferation changes. Therefore it can be concluded that nanomaterials in general do demonstrate toxicity, especially at higher concentrations. Size and shape of the nanomaterials also appears to affect toxicity levels..
  • the present invention establishes sets of biomarkers whose gene expression levels are changed in response to exposure to carbon nanomaterials. It was found that particular biological pathways are activated or perturbed by nanoparticle.
  • the biological pathways activated or perturbed include the pathways of inflammation, apoptosis, immune response, ubiquitination, cell proliferation, cell cycle regulation, cell differentiation, golgi vesicle transport, membrane fusion, secretory pathway, intracellular transport, nucleocytoplasmic transport, apoptosis, response to DNA damage, response to stress and stimuli. These pathways and the nanoparticle specific biomarkers are listed in Tables 2-7 and 8-21.
  • biomarkers identified that are associated with particular nanoparticles it is possible to evaluate the cytotoxicity of various nanomaterials using the biomarkers and biomarker temporal change patterns as predictors for other nanoparticles.
  • Any nanomaterial can be evaluated including, but not limited to, nanomaterals that are carbon-based (such as carbon nanotubes and spherical carbon nanoparticles) or semiconductor-based (such as semiconductor nanocrystals), or metal-oxide based,any nanomaterial comprised of combinations, and derivates thereof, having any contemplated modification thereof.
  • the biomarkers identified in the Tables will prove useful as a baseline for future studies or assessment of nanomaterials.
  • gene expression changes in human skin fibroblasts serve as a readout for cellular responses to the stimulus of carbon nanomaterials.
  • gene expression is used in a broad sense. It comprises an increase or decrease of gene copy number; it can also comprise assessment of amplification or decrease in levels of the gene, and/or gene products. Thus levels of gene expression, as well as corresponding protein expression can be evaluated. In the embodiments that follow, it is understood that assessment of gene expression can be used to assess level of gene product such as RNA or protein.
  • Another aspect of the invention is the sensitivity of the assay system on nanotoxicity, when microarray technology is used here.
  • the invention here provides guidelines for the threshold of cytotoxicity and the correlation with gene expression profile changes.
  • the threshold is defined as 1% of total genes are changed more than two-fold.
  • measuring a two-fold or more change in the gene expression of a specific gene or set of genes listed in Tables 2-7 or Table 8-21 in response to exposure to a nanomaterial is an indicator of nanotoxicity of the nanomaterial.
  • protective outer coatings such as polyethylene glycol
  • the data uncovers a surprising observation, that low or high dosages of semiconductor nanocrystals ("Qdots") during the incubation step does not induce a marked difference in the phenotypic response of cells.
  • the higher dosage of semiconductor nanocrystals during incubation does however result in a higher degree of particle uptake as measured by a stronger fluorescent signal. It is unclear, however, if the 10-fold increase of PEG-silane- semiconductor nanocrystals used for the incubation period results in a 10-fold increase of particle uptake.
  • the high concentration of Semiconductor nanocrystals used in this study corresponds to an approximately 5 -fold greater concentration than reported previously in toxicity studies using non-PEGalated semiconductor nanocrystals.
  • skin HSF-42 and lung IMR-90 cells only show a mild phenotypic response to PEG-silane- semiconductor nanocrystals, as measured by changes in cell proliferation, cell cycle regulation and cell death and shown in Tables 7-8.
  • the gene expression change level of less than 0.5% of total genes can be used as a quantitative measurement on whether the protective coating is effective for any other nanoparticles, or nanomaterials.
  • the microarray gene expression analysis provides quantitative and comprehensive measurement matrix for determine the effectiveness of the protective coating. This quantitative measurement can be used for any other nanoparticles that might have toxicity against the cells, tissues, or organs.
  • Surface modifications can include charge density alteration by introducing positively or negatively charge groups, encapsulation by polymers, lipids, inorganic thin films, biocompatible materials, and biomolecules including biopids, biominerals, polysaccharides, nucleic acids, dendrimers, aptamers, polypeptides, proteins. And nanocomposites which are a combination of more than two of the above variations.
  • the enrichment of certain gene ontology classes above the background percentage levels of the total genome indicates a likelihood that there is an increase in expression of certain impacted groups.
  • the fold change of enrichment will be a useful quantitiative index for determining the relative toxico logical impact of a particular nanomaterial on an affected gene class relative to the impact on the overall genome.
  • One way of looking at this fold change is by determining the ratio of affected genes to the number of genes in the pathway compared to the ratio of genes in the pathway to the overall genome.
  • Example 1 gene ontology analysis gave further evidence supporting the qualitative differences of cell responses to low and high doses of carbon nanomaterials.
  • the genes identified and similar gene profiles identified using similar assay systems can be used as guidelines for attenuating the toxic effects.
  • the biomarker changes should be eliminated or weakened, with the elimination of the nanotoxicity. This can be used as a measurement on the efficiency of toxicity control.
  • embodiments of the invention include: A method for prognosing the cytotoxic effect of a nanomaterial upon a cell, said method comprising: providing a cell; exposing said cell to a nanomaterial; detecting from the provided cell, the level of gene amplification or gene expression for at least one gene set forth in Tables 2-21 in response to said exposure; identifying at least twofold change in gene expression of said gene; whereby, when the two-fold change in gene expression is identified, this is an indication that the nanomaterial is cytotoxic to said cell.
  • This method can comprise that the gene or gene product is involved in ERK and p38 MAPK activities and the induction of interferon signaling.
  • the detecting step can comprise use of a methodology selected from the group consisting of transcription profiling, the measurement of phenotypic changes in large populations of cells by high content analysis, gene expression array analysis in exposed cells, measuring mRNA level changes, promoter analysis, chemically induced toxicity, 2D gel electrophoresis, mass spectrometry, reverse phase protein lysate arrays for protein, [0122]
  • specific cellular response to nanomaterial exposure is measured by determining, (a) toxicity of the nanomaterials by (i) the measurement of phenotypic changes in large populations of cells by high content analysis and (ii) gene expression array analysis in exposed cells; (b) DNA damage and chromosomal aberration caused by the nanomaterials and measured by using comparative genomic hybridization (CGH), and performing gammaH2AX foci formation and comet assays, (c) stress response due to nanomaterial exposure by measuring changes in global gene expression patterns, p38 phosphorylation and COX-2 expression using microarray technologies and high content imaging, and
  • the present invention further provides multi-walled carbon nanomaterials and therapeutic uses thereof.
  • the multi-walled carbon nanomaterials used herein are carbon nanotubes or nanoonions, more preferably nanoonions.
  • the regulation of p38/ERK and the EGFR also provides for the use of carbon nano-onions and potentially other carbon nanomaterials to be exploited as a nanomedicine platform for cancer therapy, especially epithelially derived cancers.
  • Mutiwall Carbon Nanomaterials are synthesized by using a chemical vapor deposition (CVD) method as described in Service, R.F. American Chemical Society meeting. Nanomaterials show signs of toxicity.
  • CVD chemical vapor deposition
  • Example 1 also describes a preferred method for synthesizing multiwall carbon nanotubes.
  • multi-walled carbon nanoonions are synthesized by using the direct-current electric-arc discharge method described in Sano, N., Wang, H., Chhowalla, M., Alexandrou, I. & Amaratunga, G.A.J. Nanotechnology: Synthesis of carbon Onions' in water. Nature (London) 414, 506 - 507 (2001) and hereby incorporated by reference.
  • a preferred method described in Examples is used for synthesizing the carbon MWCNOs.
  • Figure IB and ID show the carbon nanoonions produced by the preferred method.
  • the multiwall carbon nanonions are approximately 10-50 nm in diameter, more preferably about 30 nm. By the term “about” it is meant, that it is contemplated that the size can be within ⁇ 5, 10, 15, 20, or 25 units or 5, 10, 15, 20, or 25% of the stated values.
  • the multiwall carbon nanomaterials may be "conjugated” (i.e., linked) to a biological molecule or composition, directly or via one or more linking agents.
  • Linking agent refers to any compound that forms a bond between the nanomaterial and the biological molecule and includes e.g., a functional group, an affinity agent, or a stabilizing group.
  • Suitable bonds include ionic interactions, covalent chemical bonds, physical forces such van der Waals or hydrophobic interactions, encapsulation, embedding, binding affinity, attraction or recognition, and various types of primary, secondary, tertiary linkages including but not limited to, peptide, ether, ester, acryl, aldehyde, ketone, acryloyl, thiol, carboxyl, hydroxyl, sulfhydryl and amine linkages or the like.
  • the biological molecule or composition can be a radioactive label, such as Gd-DPTA, 19 F, 1 H, or 125 I, and serve as a MRI contrasting reagent; radionuclides, such a 64 Cu, F, I, Cl, Br, for use in PET imaging; or imaging reagents such as fluorescent or chemiluminscent probes for use in infrared imaging.
  • a radioactive label such as Gd-DPTA, 19 F, 1 H, or 125 I
  • radionuclides such as 64 Cu, F, I, Cl, Br
  • imaging reagents such as fluorescent or chemiluminscent probes for use in infrared imaging.
  • imaging-enhanced versions of the nano-onions it is contemplated for uses such as, as a tool for image-guided intervention of tumors.
  • the multiwall carbon nanomaterials can also be conjugated to tumor-targeting molecules, such as monoclonal antibodies, nucleic acids, peptides, small molecules, etc., whereby the targeted nano-onion can be carried in vivo to a tumor to kill the tumor cells.
  • tumor-targeting molecules such as monoclonal antibodies, nucleic acids, peptides, small molecules, etc.
  • the nanomaterial is conjugated one or more antibody, composition, small molecule, nucleic acid or peptide that binds to any one of the genes known to be upregulated in cancer cells, such as Erb ⁇ 2 and EGFR.
  • the multiwall carbon nanomaterial is conjugated to a small molecule such as folate, any vitamin specific for a disease, or a drug such as quinazoline derivatives which act as tyrosine kinase inhibitors (e.g., Erlotinib and Gefitinib).
  • the nucleic acid or peptide is an antisense oligonucleotide, aptamer or siRNA specific for a cancer marker.
  • the multiwall carbon nanomaterial is conjugated to a monoclonal antibody anti-Erb ⁇ 2, which targets tumor cells or inserted into a delivery vehicle having an anti-Erb ⁇ 2 monoclonal antibody.
  • ErbB-targeted therapy has been validated with FDA approval of the ErbB2 binding Mab, trastuzumab (HERCEPTIN) for treatment of advanced breast cancer.
  • trastuzumab HERCEPTIN
  • Trastuzumab binds to the receptor extracellular domain resulting in tumor growth inhibition via poorly understood mechanisms, although both antibody dependent cellular cytotoxicity and interference with receptor signaling probably play a role in therapeutic efficacy (Albanell, J., Codony, J., Rovira, A., Mellado, B., and Gascon, P. (2003).
  • Mechanism of action of anti- HER2 monoclonal antibodies scientific update on trastuzumab and 2C4.
  • IRESSA small molecule kinase inhibitor gefitinib
  • the carbon nanomaterials are multi-modality and are conjugated to both an imaging reagent and a tumor-targeting molecule.
  • these multi-modality carbon nanomaterials are also crosslinked, bound or encapsulated with an immunoliposome as described in Example 6, whereby the immunoliposome is conjugated to tumor- targeting molecules, such as monoclonal antibodies, peptides, small molecules, etc., whereby the targeted nanomaterial can be carried in vivo to a tumor to kill the tumor cells.
  • tumor- targeting molecules such as monoclonal antibodies, peptides, small molecules, etc.
  • the nanomaterial immunoliposome is conjugated to one or more antibody, composition, small molecule, nucleic acid or peptide that binds to any one of the genes known to be upregulated in cancer cells, such as Erb ⁇ 2 and EGFR.
  • the nanomaterial immunoliposome is conjugated to a small molecule such as folate, any vitamin specific for a disease, or a drug such as quinazoline derivatives which act as tyrosine kinase inhibitors (e.g., Erlotinib and Gefitinib).
  • a small molecule such as folate, any vitamin specific for a disease, or a drug such as quinazoline derivatives which act as tyrosine kinase inhibitors (e.g., Erlotinib and Gefitinib).
  • the nucleic acid or peptide is an antisense oligonucleotide, aptamer or siRNA specific for a cancer marker.
  • compositions of the present invention may be formulated for delivery either encapsulated in or bound to a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • liposomes are generally known to those of skill in the art (see for example, Couvreur et al, 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases).
  • liposomes were developed with improved serum stability and circulation half-times (Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741,516).
  • various methods of liposome and liposome like preparations as potential drug carriers may be used (Takakura, 1998; Chandran et al, 1997; Margalit, 1995; U. S. Patent 5,567,434; U. S.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the nanomaterial compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
  • Targeting is generally not a limitation in terms of the present invention. However, should specific targeting be desired, methods are available for this to be accomplished. For example, antibodies may be used to bind to the liposome surface and to direct the liposomes and its contents to particular cell types. Carbohydrate determinants (glycoprotein or glyco lipid cell-surface components that play a role in cell-cell recognition, interaction and adhesion) may also be used as recognition sites as they have potential in directing liposomes to particular cell types. For example, in one embodiment, the multiwall carbon nanomaterials are crosslinked to an immuno liposome. In a preferred embodiment, the immuno liposome and methods of use as described in U. S. Patent Nos.
  • the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987; Quintanar-Guerrero et al, 1998; Douglas et al, 1987).
  • ultrafine particles sized around 0.1 m
  • Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
  • Such particles may be easily made, as described (Couvreur et al, 1980; 1988; zur Muhlen et al, 1998; Zambaux et al 1998; Pinto-Alphandry et al, 1995 and U. S. Patent Nos. 5,145,684). Others have described nanoparticles in U.S. Patent Nos. 6,602,932; 6,071,533.
  • the multi-wall carbon nanomaterials of the present invention is delivered to cancerous cells in a subject using other microparticles, nanostructures and nanodevices.
  • microspheres may be used such as those available from PolyMicrospheres, Inc. (Indianapolis, IN).
  • FDA PolyMicrospheres, Inc.
  • the nanomaterials of the present invention can be used to treat or prevent a variety of disorders associated with cancer.
  • the nanoonions are administered to a patient in an amount sufficient to elicit a therapeutic response in the patient ⁇ e.g. , inhibiting the development, growth or metastasis of cancerous cells; reduction of tumor size and growth rate, prolonged survival rate, reduction in concurrent cancer therapeutics administered to patient).
  • the nanomaterials of the invention can be administered directly to a mammalian subject using any route known in the art, including e.g., by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intratumoral or intradermal), inhalation, transdermal application, rectal administration, or oral administration.
  • injection e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intratumoral or intradermal
  • inhalation e.g., transdermal application, rectal administration, or oral administration.
  • compositions of the invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the nanoonions are formulated with a pharmaceutically acceptable carrier prior to administration.
  • compositions of the present invention are determined in part by the particular composition being administered (e.g., nucleic acid or polypeptide), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular multi-wall carbon nanomaterial (e.g. nanotube or nanoonion) employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular peptide or nucleic acid in a particular patient, such as the increase in gene expression of proteins involved in inflammatory responses as shown in the Tables.
  • the physician evaluates circulating plasma levels of the multi-wall carbon nanomaterial, multi-wall carbon nanomaterial toxicities, progression of the disease (e.g., ovarian cancer), and the production of antibodies that specifically bind to the multi-wall carbon nanomaterial.
  • the dose equivalent of a polypeptide is from about 0.1 to about 50 mg per kg, preferably from about 1 to about 25 mg per kg, most preferably from about 1 to about 20 mg per kg body weight.
  • the dose equivalent of a naked c acid is from about 1 ⁇ g to about 100 ⁇ g for a typical 70 kilogram patient, and doses of vectors which include a viral particle are calculated to yield an equivalent amount of therapeutic nucleic acid. Dosages of multi-wall carbon nanomaterials administered to a patient can be based upon these dose equivalents for other therapeutics.
  • the 2 fold increase of apoptosis/necrosis from the baseline was an artificially defined point.
  • a different scheme of dosing may be required.
  • multi-wall nanomaterials that are coated or contain surface modifications as contemplated, should have attenuated cellular responses to toxicity, stress and damage. Using the Tables showing Determination of a therapeutically effective amount is also affected by the number and percentage of genes showing statistically significant changes in overexpression and underexpression after cytotoxic dose and exposure to the multiwall carbon nanomaterials.
  • the dosage of the multiwall carbon nanomaterials delivered should not effect more than a 2%, 5%, 7%, 10%, 15%, 20%, 25%, 40%, or 50% change in overexpression or underexpression of a specific gene, or overall in a gene functional family after cytotoxic dose and exposure. See Tables 1-7 and Tables 8-21.
  • multi-wall carbon nanomaterial of the present invention can be administered at a rate determined by the LD-50 of the multi-wall carbon nanomaterial, and the side- effects of the multi-wall carbon nanomaterial at various concentrations, as applied to the mass and overall health of the patient.
  • Administration can be accomplished via single or divided doses, e.g., doses administered on a regular basis (e.g., daily or weekly) for a period of time (e.g., 2, 3, 4, 5, 6, days or 1-3 weeks or more).
  • the multi-wall carbon nanomaterials of the invention are cytotoxic and slowly release each layer of the carbon nanomaterial, therefore, doses may be spaced out according to the release time determined for each dosage delivered.
  • compositions comprising the multi-wall carbon nanomaterial of the present invention parenterally, intravenously, intramuscularly, or even intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington 's Pharmaceutical Sciences, 15th Edition, pp.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • compositions disclosed herein may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides
  • organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • the multi-wall carbon nanomaterials are administered in combination with a second therapeutic agent for treating or preventing cancer.
  • multi-wall carbon nanomaterials may be administered in conjunction with a second therapeutic agent, such as radiation or chemotherapy, for treating or preventing any cancer.
  • a second therapeutic agent such as radiation or chemotherapy
  • multi-wall carbon nanomaterials may be administered in conjunction with any of the standard treatments for ovarian cancer including, but not limited to, paclitaxel, cisplatin, carboplatin, chemotherapy, and radiation treatment.
  • the multi-wall carbon nanomaterials and the second therapeutic agent may be administered simultaneously or sequentially.
  • the multi-wall carbon nanomaterials may be administered first, followed by the second therapeutic agent.
  • the second therapeutic agent may be administered first, followed by the multi-wall carbon nanomaterials.
  • the multi-wall carbon nanomaterials and the second therapeutic agent are administered in the same formulation.
  • the multi-wall carbon nanomaterials and the second therapeutic agent are administered in different formulations.
  • their administration may be simultaneous or sequential.
  • the multi-wall carbon nanomaterials can be used to target therapeutic agents to cells and tissues expressing any candidate genes that are related to reduced survival rate.
  • Therapeutic Kits The present invention further provides kits for therapeutic uses.
  • the subject composition of the present invention may be provided, usually in a lyophilized form, in a container.
  • the multi-wall carbon nanomaterials described herein are included in the kits with instructions for use, and optionally with buffers, stabilizers, biocides, and inert proteins.
  • kits may further comprise a second therapeutic agent, e.g., paclitaxel, carboplatin, a chemotherapeutic agent.
  • Example 1 Materials and Methods of measuring toxicology of MWCNOs and MWCNTs
  • the carbon MWCNOs used in this study were produced by using a modified direct- current electric-arc discharge method based on the methods described by Sano, N., Wang, H., Chhowalla, M., Alexandrou, I. & Amaratunga, G.A.J. Nanotechnology: Synthesis of carbon Onions' in water. Nature (London) 414, 506 - 507 (2001), hereby incorporated by reference. (See Figure 1 and infra).
  • the multi-walled carbon nanotubes (MWCNTs) were synthesized by using a chemical vapor deposition (CVD) method as described in Andrews, R., Jacques, D., Qian, D. & Rantell, T. Multiwall carbon nanotubes: synthesis and application.
  • HCA High Content Image Analysis
  • the DNA stain, YO-PRO-I can transverse the slightly permeable membranes of apoptotic cells while propidium iodide requires the greater membrane permeability of necrotic cells.
  • An Affymetrix High- Throughput Analysis (HTA) automated GeneChip system was used for acquisition of the microarray data for the gene expression profiling. Target preparation, washing, and staining have been carried on an Affymetrix/Caliper robotic system, and scanning was performed on a CCD-based Affymetrix High Throughput (HT) scanner, which is a fully automated epiflourescent imaging system. More details can for the HTA protocols can be found in the Supplement.
  • HT Affymetrix High Throughput
  • MWCNOs The carbon MWCNOs used in this study were produced by using a modified direct-current electric-arc discharge method. Three liters of deionized Milli-Q (Millipore) water were degassed by vigorous magnetic stirring under vacuum for at least 1 hour. Then the water was cooled in an ice bath. Two graphite rods with a purity of 99.99% were used as electrodes.
  • a 5 -mm diameter rod was connected to the positive output of a power supply (DUAL MIG 151 T/2, Chicago, USA), and a section of a 12-mm rod was connected to the negative output and placed in a fixed position at the bottom of the water container.
  • the two electrodes were submerged in the deionized water, and placed in fixed positions near the bottom of the water container.
  • Helium gas was bubbled through the water at a rate of about 0.3LmUi '1 to obtain an inert atmosphere.
  • a plastic film was used to seal the mouth of the water container. The anode was gradually moved towards the cathode until the arc initiated, and the arc was maintained by continuously adjusting the anode-cathode distance.
  • MWCNTs multi- walled carbon nanotubes
  • the multi-walled carbon nanotubes (MWCNTs) were synthesized by using a chemical vapor deposition (CVD) method. Ferrocene (sublimation temperature, ⁇ 140°C) was chosen to produce Fe catalyst particles to seed nanotube growth.
  • Xylene was selected as a hydrocarbon source because it has a boiling point of 140 0 C, well below the decomposition temperature of ferrocene ( ⁇ 190°C). Approximately 6.5 mol% of ferrocene was dissolved in xylene to obtain a feed solution with -0.75% Fe/C ratio, and the liquid was fed continuously into a two-stage tubular quartz reactor (diameter, ⁇ 34 mm) using a syringe pump. The liquid feed is passed through a capillary tube and preheated to ⁇ 175°C prior to entering into the furnace. At this temperature, the liquid exiting the capillary is immediately volatilized and swept into the reaction zone of the furnace by a flow of argon with 10% hydrogen.
  • the preheater and the furnace were allowed to cool to room temperature in flowing argon.
  • MWCNOs formed on the walls of the quartz furnace tube and on plain quartz substrates were collected.
  • the reactor was operated at 1" OfH 2 O pressure above atmospheric pressure to prevent any influx of oxygen.
  • BrdU was added to the cells in media at a final concentration of lO ⁇ M for 1 hour, cells were then fixed with 70% ethanol and put at 4°C overnight. Staining was performed using anti-BrdU (cat. 555627 BD Bioscience) at a 1 :100 dilution and a secondary rabbit anti-mouse AlexaFluor 488 (cat. A-11059 Molecular Probes) diluted 1 :500, both in PBS/0.5% tween-20. Propidium iodide, 0.5 ⁇ g/ml, was used as a second stain to obtain DNA content information. Stained culture plates were scanned/analyzed on a Cellomics High Content Imaging system (Cellomics, KineticScan).
  • the KineticScan is an automated imaging instrument that scans through the bottom of clear-bottom 96-well plates, focuses on a field of cells, and acquires images at each selected fluorescence channel.
  • the Cellomics software Cell Health Profiling
  • Intensity measurements for BrdU antibody staining and DNA staining with propidium iodide were obtained for each identified cell and these measurements were plotted by scatter plot, to obtain percentage of cells in G0/G1, S, and G2/M phases. Approximately 20,000 cells were plotted per treatment. A student t-test was performed to assess the significance of differences between treated and control cells.
  • the number of apoptotic and necrotic cells were also measured 48 hours after treatment. Apoptotic cells and necrotic cells were detected using DNA dyes that only traverse membranes of necrotic or apoptotic cells.
  • the DNA stain, YO-PRO-I (Molecular Probes, Y3603) is a dye that can transverse the slightly permeable membranes of apoptotic cells while propidium iodide requires the greater membrane permeability of necrotic cells. Live cells were exposed to these dyes for 30 minutes and then immediately analyzed on the KineticScan where the intensities of these dyes were measured for each cell. Greater intensities are measured with increasing membrane permeability.
  • the Cellomics software (Cell Health Profiling) was used to quantify these intensities and then these were averaged for all the measured cells. Eight wells were done per condition and the results from these analyses were used in a t-test to assess if the treated group showed significantly different staining from the control group.
  • RNA isolation Cell cultures of HSF42 cells were incubated at 37°C in humidified 5% CO 2 . Plates were harvested 24 hrs after treatment. One T75 flask was used for each treatment, and each treatment was performed in triplicate. Cells were homogenized in TRIZOL reagent (Gibco BRL) for the isolation of total RNA following the manufacturer's instructions. The TRIZOL-isolated RNA were further purified with RNeasy kit (Qiagen) and resuspended in DEPC- treated water (SIGMA- Aldrich). [0163] Microarray hybridization and data acquisition; Target preparation.
  • the target preparation protocol of the GeneChip ® assay (Affymetrix, Santa Clara, CA) were broken down into sections of methods and adapted to the robotic station as follows: For each sample, the RNA target is prepared by putting 2.5 ⁇ g of total RNA in 5 ⁇ l water and 5 ⁇ l of lO ⁇ M T7 (dt)24 primer into a 96- well reaction plate (MJ Research, Waltham, MA). The total RNA undergoes an annealing step at 70 C for 10 minutes followed by a 4 C cooling step for 5 minutes. The plate is transferred back to the deck position and undergoes first strand cDNA synthesis.
  • lO ⁇ l of First Strand cDNA Synthesis cocktail (4 ⁇ l of Affymetrix 5X 1 st strand buffer (250 mM Tris-HCl, pH 8.3 at room temperature; 375 mM KCl; 15 mM MgCl 2 ), is mixed with 2 ⁇ l 0.1M DTT, l ⁇ l 1OmM dNTP mix, l ⁇ l Superscript II (200U/ul), and 2 ⁇ l nuclease free water per reaction) is added, and the plate is then transferred to the thermal cycler and incubated at 42 C for 60 minutes and 4 C for 5 min.
  • Affymetrix 5X 1 st strand buffer 250 mM Tris-HCl, pH 8.3 at room temperature; 375 mM KCl; 15 mM MgCl 2
  • 2 ⁇ l 0.1M DTT 2 ⁇ l ⁇ l 1OmM dNTP mix
  • l ⁇ l Superscript II 200U/ul
  • 2 ⁇ l nuclease free water per reaction
  • T4 Polymerase cocktail comprised of 2 ⁇ l T4 DNA Polymerase plus 2 ⁇ l IX T4 DNA Polymerase Buffer (165 mM Tris-acetate (pH 7.9), 33OmM Sodium-acetate, 5OmM Magnesium-acetate, 5mM DTT) is added and the plate is taken back to the thermal cycler where it is cycled at 16 0 C for 10 minutes, 72 0 C for 10 minutes, and cooled to 4 0 C for 5 minutes.
  • IX T4 DNA Polymerase Buffer 165 mM Tris-acetate (pH 7.9), 33OmM Sodium-acetate, 5OmM Magnesium-acetate, 5mM DTT
  • the plate is transferred back to the deck and Agencourt Magnetic Beads (Beverly, MA) are used for the cDNA clean-up.
  • 162 ⁇ l of magnetic beads are mixed with 90 ⁇ l of in the cDNA Clean-Up Plate and incubated for 5 minute.
  • Post incubation the cDNA bound to the beads in the cDNA Clean-Up Plate is moved to the Agencourt magnetic plate.
  • Another 115 ⁇ l of magnetic beads is mixed with 64 ⁇ l cDNA incubated for 5 minutes, and then moved to the Agencourt magnetic plate.
  • Post incubation the supernatant is removed and two washes with 75% EtOH are performed using 200 ⁇ l solution. The EtOH is then removed and the beads sit for 5 minutes.
  • the purified cRNA is taken to the spectrophotometer and read concentration in each of well of a 96 well plate is adjusted to a nominal value of 0.625 ⁇ g/ ⁇ l. A second reading is taken to verify the normalization process. 30 ⁇ l of cRNA was transferred from the cRNA Normalization Plate and dispensed in the Fragmented cRNA Plate. 7.5 ⁇ l of 5x fragmentation buffer per sample is added. The plate is then transferred to the thermal cycler where it is cycled at 94 0 C for 35 minutes followed by a cooling step at 40 0 C for 5 minutes.
  • the sample is then mixed with 90 ⁇ l of hybridization cocktail (3 ⁇ l of 2OX bioB, bioC, bioD, and creX hybridization controls mixed with 1.6 ⁇ l 3nM oligo- B2, l ⁇ l lOmg/ml Herring sperm DNA, l ⁇ l 50mg/ml acetylated BSA, and 83.4 ⁇ l 1.2X Hybridization Buffer).
  • hybridization cocktail 3 ⁇ l of 2OX bioB, bioC, bioD, and creX hybridization controls mixed with 1.6 ⁇ l 3nM oligo- B2, l ⁇ l lOmg/ml Herring sperm DNA, l ⁇ l 50mg/ml acetylated BSA, and 83.4 ⁇ l 1.2X Hybridization Buffer).
  • Hybridization The sample is then ready to be hybridized.
  • the peg array plate is incubated in 60 ⁇ l pre-hybridization cocktail (l ⁇ l 10mg/ml Herring sperm DNA, l ⁇ l 50mg/ml Acetylated BSA, 84 ⁇ l Hybridization buffer, 15 ⁇ l nuclease free H 2 O per reaction).
  • the hybridization- ready sample is taken to the thermal cycler and denatured for 95 0 C for 5 minutes.
  • the plate Upon completion of this step, the plate is returned to the deck where 70 ⁇ l of sample is transferred to a hybridization tray.
  • the peg plate is then lifted off of the pre-hybridization tray and taken to the hybridization plate where it is placed. This "hybridization sandwich" is then manually transferred to a hybridization oven where it incubates at 48 0 C for 16-18 hours.
  • the plate is then transferred to the first stain (31.5 ⁇ l nuclease free H 2 O, 35 ⁇ l 2X MES stain buffer, 2.8 ⁇ l 50mg/ml Acetylated BSA, 0.7 ⁇ l R-Phycoerythrin Streptavidin), where it will incubate at room temperature for 10 minutes. At the end of the 10 minute incubation, the peg plate undergoes another 4 cycles of dip washing method. The peg tray is then transferred to stain 2 (2.8 ⁇ l 50mg/ml Acetylated BSA, 0.7 ⁇ l reagent grade goat IgG, 0.4 ⁇ l biotinylated goat Anti-streptavidin antibody per reaction).
  • stain 2 2.8 ⁇ l 50mg/ml Acetylated BSA, 0.7 ⁇ l reagent grade goat IgG, 0.4 ⁇ l biotinylated goat Anti-streptavidin antibody per reaction.
  • the 96 well peg plate is scanned by the Affymetrix High Throughput (HT), which is a fully automated epiflourescent imaging system with an excitation wavelength range of 340nm to 675 nm and a cooled 1280X1024 CCD camera with 12 bit readout and resolutions of l.O ⁇ m/pixel with the 1OX objective.
  • the images are captured at two different exposure times. Each well will have 49 sub-images/exposure time.
  • the software program then convert these .dat files into mini .eel files and then into composite eel files where the information can be analyzed in the Affymetrix GCOS 1.2 software.
  • n is the total number of genes on the chip that belong to the category
  • N f is the number of changed genes on chip
  • N is the total number of genes on chip.
  • p-value was calculated by conducting two-sided Fisher's exact test, which reflects the statistical significance for that category being enriched in changed genes. The p- values were used to sort categories to identify those gene functional groups that have responded the most after treatments.
  • TRE Core Similarity threshold 1.00; including TREs found on complementary strand.
  • the software then computes /rvalues to look for the overrepresented TREs in the set of promoters analyzed in reference to all the genes in the PAINT database to generate filtered (p-value value ⁇ 0.1) interaction matrices.
  • the hierarchical clustering was conducted using Cluster 3.0, a modified version developed at Tokyo University, based on Michael Eisen's original software (Eisen, M. B., Spellman, P. T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95, 14863-14868 (1998)). The analysis was performed with complete linkage using similarity matrix of centered correlation.
  • Example 2 Evaluating toxicity in nanomaterial exposed cells
  • cells were added to 96-well plates (BD Biosciences), grown to approximately 70% confluency in a CO 2 incubator and then exposed to several concentrations of MWCNOs and MWCNTs (Figure 1).
  • Figure 1 To determine the cytotoxic dose to be used for this study, cells were treated with serial dilutions of MWCNO and MWCNT (data not shown), and we chose doses of 0.6 mg/L and 6 mg/L for MWCNO, and doses of 0.06 mg/ml and 0.6 mg/L for MWCNT, so that the cells show approximately 2 fold increase in apoptosis/necrosis from the untreated baseline cells, and a -50% reduction in proliferation (measured by end point cell numbers) after a treatment of 48 hours at the low dose.
  • the 2 fold increase of apoptosis/necrosis from the baseline is an artificially defined point, an approach previously used in Ding, L.H. et al.
  • Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation a comparative study of low and high doses. Radiat Res 164, 17-26 (2005).
  • the high doses are chosen as 10 times of the low dose, so that pronounced gene expression changes can be observed to mimic the acute exposure to carbon nanomaterials.
  • Cells were exposed for 24 or 48 hours, counted and various measurements were made to evaluate cytotoxicity and proliferation.
  • the MWCNT seem to be ten times more toxic than the MWCNO, which is the reason that the amount of MWCNT used in our studies is only one tenth of the amount of MWCNO used, at both the low dose and high dose levels .
  • Results Whole genome expression array analysis and high content image analysis-based phenotypic measurements were performed on human skin fibroblast cell populations exposed to multiwall carbon nano-onions (MWCNOs) and multiwall carbon nanotubes (MWCNTs).
  • MWCNOs multiwall carbon nano-onions
  • MWCNTs multiwall carbon nanotubes
  • Some of the genes that are typically induced by an interferon type I response include Irf7, IsgOg, Statl Adar, CxcllO, Irfl, IsgOg, IFITl, MX2, all found in Table 2.
  • the dimension of carbon tubes is similar to virus and the cellular response may mimic the response observed with viral infection.
  • kerotinocytes HEKs exposed to chemically unmodified MWCNT released interleukin-8, a proinflammatory cytokine, which was postulated to result in the skin irritation associated with exposure 39 .
  • AHR intracellular aryl hydrocarbon
  • TNFRSFlOB TRAILR2
  • TRAILR2 TNF family member
  • BCL2L2 and MCLl additional apoptosis genes involved include BCL2L2 and MCLl.
  • RIPK2 and TNFAIP3 genes that contribute to the induction of apoptosis, were also observed to be up-regulated in these treated cells.
  • Virus also impinges upon the signal transduction pathway in the sense that their binding to the receptor perturbs the normal receptor- coupled signal transduction pathways.
  • Many of receptors e.g. EGFR, are potent stimulators of the mitogen-activated protein kinase (MAPK) signaling pathway.
  • MAPK mitogen-activated protein kinase
  • Chronic stimulation of EGFR and of multiple steps in the MAPK signaling pathway is involved in multiple cellular processes, especially in the interaction between viruses and tyrosine kinase pathways 91 .
  • One interesting observation is the downregulation of EGFR by >4 fold, which indicate that the nano-onion and nanotubes might serve as therapeutics for EGFR-overexpressing epithelial cancers, such as >20% of the breast cancer.
  • the upstream events leading to the different expression pattern seem to be related to ERK and p38 MAPK activities and the induction of interferon signaling.
  • the pathway responses shown here are similar to the response of human bronchial epithelial cells to combustion-derived metals.
  • Example 3 Evaluating Apoptosis and Necrosis in Nanomaterial Exposed Cells.
  • Cytotoxicity was evaluated by staining live cells for 30 minutes with YO-PRO 1 (Invitrogen, Molecular Probes), propidium iodide (PI, Sigma) and Hoechst. Live cells are impermeable to YO-PRO 1 and PI, both of which are intercalating DNA dyes. Apoptotic cells are permeable to YO-PRO 1, whilst PI only stains necrotic cells. Stained culture plates were analyzed using the KSR and images were acquired at each appropriate fluorescence channel for Hoechst, YO- PRO 1, and PI. The image analysis software establishes average and total intensity for each nucleus in all channels.
  • FIG. 3A shows images from one field, generated by the KSR for image analysis, with PI staining pictured in channel 1, BrdU antibody staining in channel 2, and the composite is pictured in the middle. After images from stained culture plates were obtained using the KSR intensity measurements for both BrdU and DNA staining were made for each identified cell to generate a scatter plot with the intensity of BrdU antibody staining on the Y-axis and PI intensity on the X-axis.
  • MWCNO and MWCNT treatment caused expression changes in similar groups of genes, including Golgi vesicle transport, secretory pathway, fatty acid biosynthesis, protein metabolism and Gl /S transition of mitotic cell cycle (Table 1), with down-regulated genes dominating in all of these categories.
  • IFN7 was recently demonstrated to regulate all elements of IFN responses, including the systemic production of IFN in innate immunity.
  • IRFl also up-regulated, has been demonstrated to play an important role in transcription activation of type I IFN genes.
  • most of the genes in Table 2 are IFN inducible including ADAR, CXCLlO, G1P2, G1P3, IFI44, IFITl, IFIT2, IFIT3 6 , and IFIT5 among others (Table 2).
  • Several induced genes are also specifically associated with an antiviral response including MXl, MX2, OASl, OAS2 and OAS3. .
  • the MX proteins are related to an interferon-regulated mouse protein induced by influenza virus and the OAS proteins have been observed to be induced as a response to the yellow fever vaccine. These data indicate that MWCNTs may interact with cells differently than MWCNOs and this type of interaction influences the cellular response. Based on the large number of genes associated with cellular response to viral infection and an IFN type I response MWCNT treatment may mimic viral infection in some respects. [0195] Many of the genes altered in expression after treatment with the lower concentration of nanomaterials are those involved in transport, membrane fusion, and secretion (Table 3). These genes did not show discernable changes in expression with higher concentrations of MWCNOs and MWCNTs.
  • genes in this category are involved in the process of docking and fusion of vesicles to their target membranes. Most of the genes in this category are under expressed indicating that the cells may be slowing secretion of proteins. Treatment of cells with the lower concentrations of nanomaterials also has an impact on the expression of cell cycle genes (Table 4) and genes involved in ubiquitination (Table 21). Again, many of these genes are down-regulated, indicating a slowing of cell proliferation and protein degradation.
  • Table 5 lists genes involved in apoptosis that were induced or repressed with nanomaterial treatment. A greater number of genes involved in apoptosis were observed to be up- regulated with MWCNT treatment at the higher dose, possibly explaining the greater number of apoptotic and dead cells observed with high content screening (Figure 2). Of interest was the up- regulation of the cytokine and TNF family member, TNFRSFlOB (TRAILR2) in cells treated with the highest concentration of MWCNTs, which is known to induce apoptosis. Also, the RIPK2 73 gene contributes to the induction of apoptosis and was observed to be up-regulated in these treated cells.
  • TNFRSFlOB TNFRSFlOB
  • Promoter Analysis According to our analysis of regulatory elements (cis elements) within the promoters of genes altered in expression upon carbon nanomaterial treatment, different pathways appear to be activated depending upon the nanomaterial dosage. As gene expression patterns observed in microarray experiments reflect the activity of transcription factors (TFs) in trans, we can trace back the regulatory cascades upstream of the physiological effect. This is performed by identifying the enriched transcription regulatory elements on the promoters of genes demonstrating altered expression profiles. These analyses were performed using the microarray data from MWCNT and MWCNO treated HSF cells at low and high dosages.
  • TFs transcription factors
  • the interaction matrix is shown for the differentially expressed genes (horizontal) and transcription regulatory elements (vertical) in the up- and down-regulated gene sets at different dosage using different carbon nano-particles.
  • the PAINT software then computes /rvalues to look for the overrepresented TREs in the set of promoters analyzed in reference to all the genes in the PAINT database to generate filtered (p-value value ⁇ 0.1) interaction matrices.
  • Individual elements of the matrix are colored by the significance /rvalues: over-representation in the matrix is colored in red. The brightest red represents low/?-value (most significantly over-represented).
  • the enriched transcription regulatory elements for the nano-particle dataset are specifically called out in the figure.
  • Promoter analysis of the predominantly down-regulated genes at the lower dosages points to the enrichment of EGRl(KROXl), GATA4, ELKl and USF regulatory elements in cells treated with MWCNO versus GATA4, ELKl and USF regulatory elements in cells treated with MWCNTs (Figure 5). Promoters in genes of up-regulated transcripts demonstrate the enrichment of EGRl binding elements. However, the transcription of EGRl is down-regulated after MWCNO treatment indicating that up-regulation of some transcripts may be a consequence of relieved repression as opposed to activation. GAT A4, EGRl, USF and ELKl TFs have all been shown to be phosphorylated and activated by ERK and p38 MAPK cascades.
  • CCAAT enhancer binding protein delta (C/EBPdelta), enriched in MWCNO treated cells, is a target of p38 MAPK and is associated with growth arrest in epithelial cells.
  • C/EBPdelta CCAAT enhancer binding protein delta
  • the expression pattern of higher dose MWCNT treatment differs significantly from that of MWCNO treatment: For example, a robust IFN response is observed in MWCNT treated cells, but not in MWCNO treated cells.
  • the presence of IRF elements contained within the promoters of many of the up-regulated genes may explain this response.
  • IRF7 is one of the up-regulated genes observed (Table 2) and is believed to be central to an IFN response along with STATl (Table 5), another up-regulated gene discussed above, and one of the central signal transduction factors needed for an IFN response.
  • Transcriptional regulatory elements present in the down-regulated genes of cells treated with MWCNOs may also contribute to the differences in gene transcription observed.
  • FOS gene expression is also reduced, leading to a lowered activity of API (FOS/JUN) transcription factors. These differences may be responsible for the difference in the magnitude of response between these particles, observed phenotypically by high content analysis. Additional experiments monitoring the kinase activities should give us better understanding the underlying mechanism.
  • Example 6 Cytotoxic and Kinetic Studies for Nanoonions for Therapeutic Use In vivo.
  • 8- isoprostane levels will be examined as a marker of oxidative stress followed by assessment of the nature of inflammation, e.g., tissue retention of nanomaterial, cell death due to localized cytotoxicity, altered vascular homeostasis, ischemia (Miles, A.A., and Miles, E. M. (1952). Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea pigs. Journal of Physiology 118, 228-257) or elevated IFP (Eichten, A.E., Hyun, W.C., and Coussens, L.M. (2005).
  • Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 64, 3731-3736). These analyses will reveal if inflammation is a primary response in specific tissues where nanomaterials are retained and immunogenic, or secondary to altered vascular homeostasis, and subsequent changes in capillary permeability, impaired clearance by lymphatics and elevated IFP. If capillary permeability is found to be altered, we would assess to what degree lymphatic dysfunction follows as demonstrated by IFP, edema or enlarged lymphatics by lymphatic image analysis and/or MRI (Eivier, A.E., Shen, H. -C. J., and Coussens, L.M.
  • Toxicity Nanomaterials will be used in toxicity studies in healthy mice at multiple dosing levels, delivered by various routes, to determine MTD and evidence of induced organ/tissue toxicities. MTD of nanomaterial formulations will be determined in groups of 3 mice per concentration and route of compound to be tested. On the day of the experiment, mice will be randomly grouped and individually marked in appropriately labeled cages. After single exposures, survival, morbidity and body weights will be monitored. Individual body weights will be recorded 3- times/week for 14-days. All animals will be observed for signs of ill health based on body weight, appetite, rough coat, grooming, behavioral changes such as altered gait, lethargy and gross manifestations of stress.
  • mice will receive a single exposure to saline or nanomaterial immunoliposome reconstituted in saline at the MTD. Blood, urine and organs will be collected from saline and nanomaterialtreated mice at 5-min, 15-min, 1-, 3-, 6-, 12-, 24- and 48-hrs post injections. Where possible, urine will be collected from mice prior to termination and at other time points.
  • All blood, urine and organs (liver, spleen, lung, heart, kidney and tumor if present) will be flash frozen and stored at -80 0 C prior to evaluation where tissue, cells and/or plasma will be tested for total nanomaterial content by HPLC, using a method based on that of Seymour et al, in The pharmacokinetics of polymer-bound adriamycin, Biochem Pharmacol 39, 1125-1131(1990), hereby incorporated by reference. Time points will contain a minimum of 5 age-matched animals.
  • mice In vivo MRI of tissue: Animals subjected to MRI scanning prior to nanomaterial injection and at several times after injection will be performed on a Varian system equipped with a 7.0-Tesla, 18.3-cm horizontal bore magnet (300-MHz proton frequency) inside the MT Zion the barrier at UCSF.
  • mice will be anesthetized with sodium pentobarbital (70 mg/kg i.p) and maintained at 37°C inside the magnet using a heated circulation water blanket, with pelvis motion minimized by a plastic support placed before insertion into a 3-cm diameter quadrature birdcage coil.
  • Each set will contain 9-25 slices and enough sets obtained to provide contiguous image data of the tissue.
  • Tissue volumes obtained from final MRIs will be compared with findings from direct anatomical inspection at tissue dissection/necropsy.
  • Cd 2+ release is noticeably slowed if nanocrystals are embedded in a cross-linked shell reducing toxicity.
  • cells treated with CdSe/ZnS nanoparticles embedded in a silica shell do not show signs of toxicity, even when treated with dosages 6-12 times higher that the toxicity-inducing dosage of mercaptoacid coated CdSe/ZnS semiconductor nanocrystals.
  • Kirchner, C Liedl, T.; Kudera, S.; Pellegrino, T.; Munoz Javier, A.; Gaub, H. E.; St ⁇ lzle, S.; Fertig, N.; Parak, W. J. Nano Letters 2005, 5, 331-338.
  • silane shell adds -2-3 nm in thickness and thus silanized semiconductor nanocrystals are -8-10 nm in diameter.
  • Such semiconductor nanocrystals chemistry was observed in Kirchner, C; Liedl, T.; Kudera, S.; Pellegrino, T.; Muiioz Javier, A.; Gaub, H. E.; St ⁇ lzle, S.; Fertig, N.; Parak, W. J. Nano Letters 2005, 5, 331-338, to pose minimal toxicity to breast cancer cells when the cells were exposed to a solution containing 2-10 nM of PEG-silane- semiconductor nanocrystals.
  • HCA High Content Image Analyzer
  • YO-PRO-I a green dye
  • Propidium Iodide (PI) a red dye
  • Cell cycle distribution was performed by adding bromo-deoxyuridine (BrdUrd) to the cell medium and subsequently staining the cells using anti-BrdUrd antibody labeled with AlexaFluor 488 and PI to obtain DNA content information.
  • intensity measurements for both BrdUrd and DNA staining were made for each identified cell to generate a scatter plot with BrdUrd intensity on the Y-axis and PI intensity on the X-axis. Analysis of these scatter plots allow estimation of the percentages of these cells in G0/G1, S, and G2/M phases.
  • Gene expression profiling was obtained with an Affymetrix High Throughput Analysis automated Genechip system. Target preparation, washing and staining were carried out on a Affymetrix/Caliper robotic system, and scanning was performed on a CCD-based High Throughput scanner. The chip contains -22,000 probe set, among which 18,400 are known genes or probe sets. Data analysis has been performed using Genesping, Bioconductor, Gene Traffic, Cluster 3.0, PAINT, GoMiner and Pathway Assist.
  • MSCs human bone marrow mesenchymal stem cells
  • TGF- ⁇ l transforming growth factor ⁇ l
  • MSCs can be differentiated into a variety of cell types in response to TGF- ⁇ l, with increased smooth muscle (SM) ⁇ -actin expression in MSCs (Kinner, B., Zaleskas, J.M. & Spector, M. Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 278, 72-83 (2002); Wang, D. et al.
  • MSCs were obtained from Cambrex Corp (Walkersville, MD). The surface markers and differentiation potential of these MSCs have been well characterized, i.e. positive for CD105, CD166, CD29, and CD44, but negative for CD34, CD14, and CD45 (Fig. S3).
  • MSCs Mesenchymal Stem Cell Growth Medium
  • MSCGM Mesenchymal Stem Cell Growth Medium
  • 1OmM L-glutamine 10% pre-screened fetal bovine serum (Cambrex Corp.) and 1% Penicillin-Streptomycin (Invitrogen) to allow for cell proliferation without differentiation.
  • Cell culture products and other consumable laboratory supplies were purchased from Fisher Scientific Corp. (Fairlawn, NJ) and VWR International (Brisbane, CA).
  • MSCs up to passage 14 were used in our experiments for gene expression analysis.
  • TGF- ⁇ l Sigma- Aldrich Corp.
  • 5ng/ml was used to treat MSCs for 24 hours.
  • One 100mm dish was used for each treatment, which was performed in triplicate.
  • RNA Stat 60 Tel-Test Inc, Friendswood, TX. RNA was extracted using chloroform and phenol extraction steps. RNA was resuspended in DEPC- treated water and quantified using a RiboGreen® RNA quantification assay (Molecular Probes Inc, Eugene, OR).
  • RNA generation and biotin labeling As determined by the RiboGreen ® dye assay, lOOng of total RNA was used in a MessageAmpTM II aRNA (Ambion, Austin, TX) reaction, (antisense RNA is referred to as cRNA). Total RNA was reversed transcribed with an oligo(dT) primer bearing a T7 promoter into first strand cDNA and used as a template for second strand cDNA synthesis. The resulting cDNA was then column purified and used in an in vitro transcription reaction with T7 RNA Polymerase to generate cRNA copies of each mRNA in the sample.
  • cRNA MessageAmpTM II aRNA (Ambion, Austin, TX) reaction
  • RNA labeling 25% of the UTPs in the in vitro transcription reaction was replaced by Biotin- 16-UTPs (Roche Molecular Biochemicals, Mannheim, Germany) to generate biotinylated cRNAs.
  • the cRNA sample was then column purified, quantified with RiboGreen ® dye, and qualified with the RNA 6000 Pico LabChip ® assay.
  • Successful cRNA samples showed a broad hump with no presence of ribosomal RNA.
  • the final gene panel used for hybridizations contains a hundred different encoded beads. 100 different gene specific probes were conjugated to 100 different bead codes while 20 control and calibrator sequences were conjugated to another 20 different beads. Hybridization to complement gene-specific oligonucleotide targets was used to verify that the correct probe was conjugated to the expected encoded bead. [0215] QBead System Hybridization. Hybridizations were performed in 96-well PCR plates (Axygen Scientific Inc., Union City, CA), where one well contained a different sample hybridized to the 100-plex gene panel.
  • One microgram of the biotin-labeled cRNA sample was hybridized in a 50 ⁇ L 3X SSC / 0.2% SDS hybridization solution at 65 0 C for 2 hours. Post-hybridization washes were performed using a 96 channel Biomek® FX Laboratory Automation Workstation (Beckman Coulter, Inc., Fullerton, CA). Hybridized beads were washed 5 times in a 0.5X SSC / 0.05% SDS solution at room temperature, followed by 3 washes with IX TBS / 0.1% BSA / 0.1% sodium azide staining buffer solution at room temperature. The quantification reporter used is a Qdot 655 Streptavidin Conjugate.
  • qPCR Quantitative Polymerase-Chain-Reaction
  • RNA for each gene was normalized with the amount of 18S RNA in the same sample.
  • Affymetrix GeneChip ® microarray hybridization and data acquisition An Affymetrix High- Throughput Automation (HTA) GeneChip ® system was used for acquisition of the microarray data for the gene expression profiling 5 .
  • Target preparation, washing, and staining have been performed on an Affymetrix GeneChip ® Array Station (GCAS), and scanning was performed on a CCD-based Affymetrix High- Throughput (HT) scanner, which is a fully automated epifluorescent imaging system. More details for the HTA protocols can be found in Examples 1-5 .
  • Qdots PEG-coated silanized Qdots
  • Fig.9A PEG-coated silanized Qdots
  • Human Skin Fibroblasts and Lung Fibroblasts were selected as model systems because entry of nanomaterials through the skin and respiratory track is the most likely route of human exposure to nanomaterials.
  • genotoxicity data of carbon nanotubes and nano-onions are available for these cells and can be used for comparison purposes. See Ding, L.; Stilwell, J.; Zhang, T.; Elboudwarej, O.; Jiang, H.; Selegue, J. P.; Cooke, P. A.; Gray, J. W.; Chen, F. F. Nano Letters 2005, In press.
  • Fig. 9B Human Skin Fibroblast and Lung Fibroblast cells exposed to 8 nM or 80 nM of PEG-silane-Qdots for 48 hours internalize them. As shown in Fig IB, all cells are labeled by Qdots. The entry mechanism is likely endocytosis, as observed previously by Jaiswal et al. for HeIa and D. discoideum cells. 30 ' 31 The nanoparticles are stored in the perinuclear region, as most studies report, 23 ' 26 but we also observed PEG-silane-Qdots dispersed in the cytoplasm (Fig. 9B). A careful look at Fig. 9 and comparable images indicate a slightly elevated number of labeled cells are in the cytokinesis stage of mitotic cell cycle. This warrants further quantitative analysis of the cell cycle profile.
  • Apoptosis/necrosis Quantifying apoptotic or necrotic cells generated further information on cell cytotoxicity. Live cells are impermeable to YO-PRO 1 and PI, two DNA staining dyes, but apoptotic cells are permeable to YO-PRO 1 (a green dye), and necrotic cells are permeable to PI (a red dye). Thus, we could count and differentiate cells undergoing apoptosis or necrosis. The results of large scale analysis over more than 20,000 cells, replicated 10 times, are reported in Fig.1 Oil as the percentage of all cells exhibiting apoptosis or necrosis.
  • Ill shows the relative percentage of treated cells compared to control cells in each of the three phases of the cell cycle.
  • the ratio of PEG-silane-Qdot treated cells to control cells in G0/G1 is close to one, indicating that PEG-silane-Qdot treatment does not induce a block in Gl.
  • the ratio of cells in S-phase of treated to control is -0.94, with a student t-test demonstrating only borderline statistical significance.
  • the largest difference in ratio occurs at the G2/M phase, where the ratio of cells treated with PEG-silane-Qdot vs control is -1.1, possibly indicating a block in G2/M.
  • the Affymetrix High Throughput Array (HTA) GeneChip ® system was used to profile gene expression changes in Human Skin Fibroblasts labeled with PEG-silane- semiconductor nanocrystals. The results are plotted in a 2D diagram in Fig.3 where each gene is represented by an (X,Y)-value in a log scale.
  • the Affymetrix HG-Ul 33 Av2.0AofA GeneChip ® contains 25mer oligoprobes, in sets for identification of transcripts from -22,000 genes and ESTs in the human genome.
  • Each dot on the graph represents a gene where the X-value corresponds to the level of expression in control cells, while the Y-value corresponds to the level of expression of that same gene in the PEG-silane-semiconductor nanocrystals labeled cells.
  • a dot that lands on the graph where the slope is 1 (red line) indicates no difference between the gene expression level of the treated and control samples.
  • the two dotted lines flanking the central line indicate the cutoff for two- fold up-regulation (top line) or down-regulation (bottom line) of the sample vs. the control.
  • Dots above or below the 2-fold box lines represent genes with a greater than two-fold change in gene expression and are discussed below.
  • the MWCNT at a concentration of 0.6mg/L induced significant changes in 216 genes, while the PEG-silane-Qdot induced changes in twenty times less genes at a much higher concentration of 40mg/L (8OnM, with molecular weight approximately 500KDa).
  • the functional categories of the changed genes are consistent across the two different dosages. Genes overexpressed are mostly related to carbohydrate binding (CHI3L1, GPNMB, PRELP, TNXB), intracellular vesicle localization (CTSF, CTSH, GPNMB, PTGIS/CYP8A1) and cell membrane-associated and intracellular vesicular proteins involved in cellular response to stress (CLU, MAP2K6/MKK6, FST). Interestingly, both MAP2K6 and CLU are both implicated in the inhibition of apoptosis 33 ' 34 and induction of senescence 35 ⁇ 38 , while CLU is a sulfated glycoprotein on the cell surface 39 .
  • the majority of the down-regulated genes fall into the functional categories controlling the M-phase progression in mitosis, spindle formation and cytokinesis (BUB 1 , CyclinA2/CCNAl, CDC20, KIF2A, KIF2C, NEK2, PLKl, PTTG, TACC3 for low dose and BUBl, MPHOSPHl for high dose), 40"52 indicating that these proteins might account for the limited perturbation of M-phase progression by PEG-silane-semiconductor nanocrystals.
  • the expression of the transcription factors FOXMl and BHLHB2/Decl are also down-regulated in low dose treated cells.
  • PEG-silane-semiconductor nanocrystals treatment does not seem to illicit any genes involved in wound healing or the immune response, contrary to both the responses we observed in human skin fibroblasts treated with carbon nano-tubes 27 and the response of dendritic cells to nanosphere treatment by others. 53
  • the lack of induction of these genes may underscore the negligible toxic effects of PEG-silane-Qdot treatment in this cell line. This observation also counters a widely held preconception that semiconductor nanocrystals are toxic to cells because of the presence of Cd in the nanocrystal.
  • the promoter analysis of the down-regulated genes at the lower dosage points to the enrichment of two transcriptional regulatory elements: DEC and COMPl.
  • Genes under-expressed in response to low-dose PEG-silane-Qdot treatment include BHLHB2/DEC1/STRA13. This gene is involved in transcriptional repression, differentiation, hypoxia-induced stress response, and circadian clock regulation. It was recently proposed to have a role in differentiation by promoting cell cycle exit. 55"58 There is not enough information about COMPl to deduce its putative role in PEG-silane- Qdot response.
  • ⁇ 5xlO 10 particles / mm 3 in lungs or skin fibroblast cells represent a dosage that would be extreme and unlikely in cases of an accidental inhalation or exposure to semiconductor nanocrystals.
  • semiconductor nanocrystals solutions are typically stored in micro-molar concentrations and if inhaled will be spontaneously diluted below toxic concentrations.
  • the high concentration of semiconductor nanocrystals used in this study corresponds to an approximately 5 -fold greater concentration than reported previously in toxicity studies using non-PEGalated semiconductor nanocrystals in Kirchner, C; Liedl, T.; Kudera, S.; Pellegrino, T.; Munoz Javier, A.; Gaub, H. E.; St ⁇ lzle, S.; Fertig, N.; Parak, W. J. Nano Letters 2005, 5, 331-338.
  • skin HSF-42 and lung IMR-90 cells only show a mild phenotypic response to PEG-silane-semiconductor nanocrystals, as measured by changes in cell proliferation, cell cycle regulation and cell death.
  • Example 7 Using Nanoonions for Therapeutic Treatment of Cancer in Mammals
  • Immuno liposomes have been constructed using a modular strategy in which components (mAb fragments, conjugation method, liposome, drugs) were optimized for internalization and intracellular drug delivery (Harding, J.A., Engbers, CM., Newman, M.S., Goldstein, N.I., and Zalipsky, S. (1997). Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes.
  • Immunoliposome conjugation ILs were prepared using small unilamellar liposomes (SUV;70-100 nm) consisting of disteroyl phosphatidylcho line/cholesterol (DSPC/Chol, 3:2 molar ratio) and polyethylene glycol (PEG2000)- derivatized disteroyl phosphatidylethanolamine (PEG- PE).
  • SUV small unilamellar liposomes
  • PEG2000 polyethylene glycol
  • PEG- PE polyethylene glycol
  • Anti-HER2 MAb fragments consisted of trastuzumab-Fab', scFv C6.5, scFv F5, or variants; and contained a C-terminal cysteine for covalent conjugation (a-c) or hexahistidine for chelation(d).
  • Ls-MAb linkage MAb fragments were conjugated to maleimide moieties (M-PE) at the liposome surface
  • PEG-MAb linkage MAb fragments were conjugated to maleimide-terminated PEG-PE (M-PEG-PE), resulting in MAb fragments at the distal ends of PEG chains
  • M-PEG-PE maleimide-terminated PEG-PE
  • Micellar Insertion Preformed liposomes lacking functional sites for conjugation were converted into ILs by insertion of modified MAb fragments.
  • MAb fragments were first coupled to M-PEG-PE, forming micelles for subsequent insertion into liposomes at high efficiency under controlled heating,
  • Ni-NTA Chelation Phage scFv were shuttled to liposomes by recombinant addition of a C-terminal hexahistidine sequence, then chelation between this sequence and nitrilotriacetic acid-nickel (Ni- NTA) complex anchored to the liposome surface. This enables "instant" ILs by mixing of scFv- containing supernatants with Ni-NT A-containing liposomal probes or drugs to expedite in vitro screens.
  • Multiwall Carbon Nanoonion Immuniliposomes Preparation: Multiwall carbon nanoonions made as in Example 1 are inserted into the immunoliposomes using any means known in the art which generates a high yield of multiwall carbon nanoonion immuniliposomes. Known methods include passive insertion, sonication and microemulsion or inverse microemulsion, and precipitation. If the subjects will be subject to MRI or other imaging, the multiwall carbon nanoonions are modified and conjugated to the appropriate imaging radionuclide or radiolabel.
  • mice receiving nanomaterials at multiple doses via distinct routes of administration e.g., intravenous, intraperitoneal, intramuscular, topical, oral and inhaled (where possible).
  • routes of administration e.g., intravenous, intraperitoneal, intramuscular, topical, oral and inhaled (where possible).
  • CSF cerebral spinal fluid
  • Nanomaterials emerging from Example 1 and intended for large scale use or interofation of living systems will be further assessed for their in vivo effects by toxicogenomics as assessed by mRNA expression of drug metabolism genes (genes within the cytochrome P-450 subfamily), genes that regulate toxicologic events (HSP70 and SODxc) and genes that regulate sugar and lipid metabolism as previously reported (Gerhold, D., Lu, M., Xu, J., Austin, C, Caskey, C. T., and Rushmore, T. (2001). Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays. Physiol Genomics 5, 161-170). The rationale for this analysis is that transcriptional changes in gene expression in the liver may provide clues to mechanisms of toxic insult.
  • Such insults may be oxidative, tumor initiating or promoting, or inflammatory for example.
  • Analyses of livers from nanomaterial-exposed animals will be compared to livers of mice exposed to a known xenobiotic, e.g., 3-methylcholanthrene, phenobarbitol, dexamethasone or clofibrate.
  • a known xenobiotic e.g., 3-methylcholanthrene, phenobarbitol, dexamethasone or clofibrate.
  • We will isolate total RNA by standard methodology from the livers of control versus treated mice, and subject that RNA to microarray analysis. These analyses will allow us to evaluate complex transcriptional responses to nanomaterials as compared to xenobiotics and subsequently make predictions of their physiological effects in acute versus chronic disease states that will help guide additional analysis of nanomaterials in vivo in concert with results obtained by HR-MAS in collaboration.
  • 8- isoprostane levels will be examined as a marker of oxidative stress followed by assessment of the nature of inflammation, e.g., tissue retention of nanomaterial, cell death due to localized cytotoxicity, altered vascular homeostasis, ischemia (Miles, A.A., and Miles, E. M. (1952). Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea pigs. Journal of Physiology 118, 228-257) or elevated IFP (Eichten, A.E., Hyun, W.C., and Coussens, L.M. (2005).
  • Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 64, 3731-3736). These analyses will reveal if inflammation is a primary response in specific tissues where nanomaterials are retained and immunogenic, or secondary to altered vascular homeostasis, and subsequent changes in capillary permeability, impaired clearance by lymphatics and elevated IFP. If capillary permeability is found to be altered, we would assess to what degree lymphatic dysfunction follows as demonstrated by IFP, edema or enlarged lymphatics by lymphatic image analysis and/or MRI (Eivier, A.E., Shen, H. -C. J., and Coussens, L.M.
  • Nanomaterials will be used in toxicity studies in healthy mice at multiple dosing levels, delivered by various routes, to determine MTD and evidence of induced organ/tissue toxicities. MTD of nanomaterial formulations will be determined in groups of 3 mice per concentration and route of compound to be tested. On the day of the experiment, mice will be randomly grouped and individually marked in appropriately labeled cages. After single exposures, survival, morbidity and body weights will be monitored. Individual body weights will be recorded 3- times/week for 14-days. All animals will be observed for signs of ill health based on body weight, appetite, rough coat, grooming, behavioral changes such as altered gait, lethargy and gross manifestations of stress.
  • mice will receive a single exposure to saline or nanomaterial immunoliposome reconstituted in saline at the MTD. Blood, urine and organs will be collected from saline and nanomaterialtreated mice at 5-min, 15-min, 1-, 3-, 6-, 12-, 24- and 48-hrs post injections. Where possible, urine will be collected from mice prior to termination and at other time points.
  • All blood, urine and organs (liver, spleen, lung, heart, kidney and tumor if present) will be flash frozen and stored at -80 0 C prior to evaluation where tissue, cells and/or plasma will be tested for total nanomaterial content by HPLC, using a method based on that of Seymour et al, in The pharmacokinetics of polymer-bound adriamycin, Biochem Pharmacol 39, 1125-1131(1990), hereby incorporated by reference. Time points will contain a minimum of 5 age-matched animals.
  • mice In vivo MRI of tissue: Animals subjected to MRI scanning prior to nanomaterial injection and at several times after injection will be performed on a Varian system equipped with a 7.0-Tesla, 18.3-cm horizontal bore magnet (300-MHz proton frequency) inside the MT Zion the barrier at UCSF.
  • mice will be anesthetized with sodium pentobarbital (70 mg/kg i.p) and maintained at 37°C inside the magnet using a heated circulation water blanket, with pelvis motion minimized by a plastic support placed before insertion into a 3-cm diameter quadrature birdcage coil.
  • Each set will contain 9-25 slices and enough sets obtained to provide contiguous image data of the tissue.
  • Tissue volumes obtained from final MRIs will be compared with findings from direct anatomical inspection at tissue dissection/necropsy.
  • Suspensions of the multi-wall carbon nanoonion immunoliposomes can be prepared by combining the nanoonion immunoliposomes and a buffer or detergent to prepare suspensions in a therapeutic concentration range.
  • the nanoonion immunoliposomes are synthesized as described above, weighed and can be dissolved in low salt buffer through mixing and sonication. Solubilizing and delivery agents can be added to the solution. Dilutions can be made from a stock solution and the final excipient, such as 0.9% NaCl at 37° C, is added to each dose formulation just prior to dosing.
  • the final ratio of liquid components can be, for example, 5:5:90, respectively.
  • a sample dosage may be, for example, 0.1 to 0.5 ml, one to five times/week, using a syringe and a needle.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'utilisation croissante des nanotechnologies dans les produits grand public et les applications médicales souligne l'importance d'en comprendre les effets toxiques potentiels pour les personnes et pour l'environnement. L'invention concerne des procédés et des essais permettant de prédire et d'évaluer les effets cellulaires de l'exposition à des nanomatériaux. Nous avons effectué une analyse sur matrice d'expression du génome entier et des mesures phénotypiques basées sur l'analyse d'images à contenu élevé sur des populations de cellules fibroblastiques de peau humaine exposées à des nano-oignons de carbone à parois multiples (MWCNO), à des nanotubes de carbone à parois multiples (MWCNT) et à des nanocristaux semi-conducteurs. Nous démontrons ici que l'exposition de cellules à des nanomatériaux à des doses cytotoxiques induit un arrêt du cycle cellulaire et augmente l'apoptose/la nécrose, active des gènes impliqués dans le transport cellulaire, le métabolisme, la régulation du cycle cellulaire et la réponse au stress. Certains nanomatériaux induisent des gènes qui sont le signe d'une forte réponse immunitaire et inflammatoire dans les fibroblastes cutanés. En outre, du fait de leur cytotoxicité, les MWCNO selon l'invention peuvent être utilisés comme agents thérapeutiques dans le traitement du cancer.
PCT/US2006/060369 2005-10-28 2006-10-30 Toxicologie et effet cellulaire de nanomatériaux fabriqués Ceased WO2007094870A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/111,026 US8785505B2 (en) 2005-10-28 2008-04-28 Toxicology and cellular effect of manufactured nanomaterials

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US73155705P 2005-10-28 2005-10-28
US60/731,557 2005-11-04
US79217106P 2006-04-12 2006-04-12
US60/792,171 2006-04-12

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/111,026 Continuation US8785505B2 (en) 2005-10-28 2008-04-28 Toxicology and cellular effect of manufactured nanomaterials

Publications (2)

Publication Number Publication Date
WO2007094870A2 true WO2007094870A2 (fr) 2007-08-23
WO2007094870A3 WO2007094870A3 (fr) 2008-12-04

Family

ID=38371956

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/060369 Ceased WO2007094870A2 (fr) 2005-10-28 2006-10-30 Toxicologie et effet cellulaire de nanomatériaux fabriqués

Country Status (1)

Country Link
WO (1) WO2007094870A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008010843A3 (fr) * 2005-12-09 2008-04-03 Univ California Évaluation du potentiel toxique de nanomatériaux
WO2008118960A3 (fr) * 2007-03-26 2009-05-07 Univ Rice William M Protection contre les rayonnements à l'aide de dérivés de nanotubes de carbone
US7680553B2 (en) 2007-03-08 2010-03-16 Smp Logic Systems Llc Methods of interfacing nanomaterials for the monitoring and execution of pharmaceutical manufacturing processes
US8784866B2 (en) 2007-03-26 2014-07-22 William Marsh Rice University Water-soluble carbon nanotube compositions for drug delivery and medicinal applications
KR101469703B1 (ko) * 2011-01-20 2014-12-08 한양대학교 산학협력단 유세포 분석을 이용한 나노 물질 위해성 평가 방법
CN106018688A (zh) * 2016-05-17 2016-10-12 中国水产科学研究院黄海水产研究所 一种金属纳米颗粒离子和纳米效应毒性贡献率的估算方法
CN119685438A (zh) * 2025-02-24 2025-03-25 北京大学口腔医学院 用于评价纳米颗粒对细胞毒性的方法及应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DING ET AL.: 'Molecular Characterization of the Cytotoxic Mechanism of Multiwall Carbon Nanotubes and Nano-Onions on Human Skin Fibroblast' NANO LETTERS vol. 5, no. 12, 2005, pages 2448 - 2646 *
JIN ET AL.: 'Toxicity of Luminescent Nanoparticles to Living Cells' TOXICITY vol. 20, no. 8, 2007, pages 1126 - 1133 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008010843A3 (fr) * 2005-12-09 2008-04-03 Univ California Évaluation du potentiel toxique de nanomatériaux
US7680553B2 (en) 2007-03-08 2010-03-16 Smp Logic Systems Llc Methods of interfacing nanomaterials for the monitoring and execution of pharmaceutical manufacturing processes
WO2008118960A3 (fr) * 2007-03-26 2009-05-07 Univ Rice William M Protection contre les rayonnements à l'aide de dérivés de nanotubes de carbone
US8784866B2 (en) 2007-03-26 2014-07-22 William Marsh Rice University Water-soluble carbon nanotube compositions for drug delivery and medicinal applications
KR101469703B1 (ko) * 2011-01-20 2014-12-08 한양대학교 산학협력단 유세포 분석을 이용한 나노 물질 위해성 평가 방법
CN106018688A (zh) * 2016-05-17 2016-10-12 中国水产科学研究院黄海水产研究所 一种金属纳米颗粒离子和纳米效应毒性贡献率的估算方法
CN106018688B (zh) * 2016-05-17 2018-02-06 中国水产科学研究院黄海水产研究所 一种金属纳米颗粒离子和纳米效应毒性贡献率的估算方法
CN119685438A (zh) * 2025-02-24 2025-03-25 北京大学口腔医学院 用于评价纳米颗粒对细胞毒性的方法及应用

Also Published As

Publication number Publication date
WO2007094870A3 (fr) 2008-12-04

Similar Documents

Publication Publication Date Title
US8785505B2 (en) Toxicology and cellular effect of manufactured nanomaterials
He et al. Total internal reflection-based single-vesicle in situ quantitative and stoichiometric analysis of tumor-derived exosomal microRNAs for diagnosis and treatment monitoring
Kedziorek et al. Gene expression profiling reveals early cellular responses to intracellular magnetic labeling with superparamagnetic iron oxide nanoparticles
Hillegass et al. Assessing nanotoxicity in cells in vitro
Xue et al. Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon
Yang et al. Aptamer-based FRET nanoflares for imaging potassium ions in living cells
Zhu et al. Dihydroartemisinin suppresses glycolysis of LNCaP cells by inhibiting PI3K/AKT pathway and downregulating HIF-1α expression
Rabiee et al. Aptamer hybrid nanocomplexes as targeting components for antibiotic/gene delivery systems and diagnostics: a review
Zhai et al. Turning on/off the anti-tumor effect of the Au cluster via atomically controlling its molecular size
EP3308164B1 (fr) Optimisation à haut rendement de nanoparticules chargées en contenu
Wang et al. Emerging extracellular vesicle-based carriers for glioblastoma diagnosis and therapy
Geinguenaud et al. Electrostatic assembly of a DNA superparamagnetic nano-tool for simultaneous intracellular delivery and in situ monitoring
CN105555956B (zh) 针对神经胶质瘤细胞的适体
Yu et al. A common anchor facilitated GO-DNA nano-system for multiplex microRNA analysis in live cells
Malile et al. DNA-conjugated gold nanoparticles as high-mass probes in imaging mass cytometry
Ding et al. The influence of physiological environment on the targeting effect of aptamer-guided gold nanoparticles
WO2007094870A2 (fr) Toxicologie et effet cellulaire de nanomatériaux fabriqués
CN116287272B (zh) Prdx5在诊断及治疗胶质瘤中的应用
Jabir et al. Combined oncolytic virotherapy gold nanoparticles as synergistic immunotherapy agent in breast cancer control
CN103278634A (zh) Cd73作为肾透明细胞癌干细胞表面标志物的应用
EP2585612B1 (fr) Analyses de l'expression de gènes pour caractériser et identifier des composés génotoxiques
Eustaquio et al. Nanobarcoding: detecting nanoparticles in biological samples using in situ polymerase chain reaction
CN114813682A (zh) 一种复合荧光纳米探针及其制备方法与应用
Wang et al. Techniques for investigating molecular toxicology of nanomaterials
US20160361266A1 (en) Modified silica shell particles, and methods of making and using the same

Legal Events

Date Code Title Description
NENP Non-entry into the national phase in:

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06850083

Country of ref document: EP

Kind code of ref document: A2