Graphene oxide nanoflakes exhibited very high affinity and ability of adhesion to cell membrane of cell bodies. Death rate of GM cells upon contact with graphene oxide flakes was proportional to its concentration, and with a concentration of 100p/ml, it was 42 % for GM U- 87 cells, and 52% for GM U- 118 cells. A significant disintegration of cell membrane was also found. As a result, it was found that graphene nanoflakes were the cause of apoptosis of glioblastoma cells in 67.5% in GM U- 87 cells and in 99 % in GM U- 118 cells. Moreover, genotoxic effect was also observed, as a consequence of penetration of cells by graphene platelets [24]. To sum up, graphene oxide nanoflakes exhibit antitumor activity, as determined based on in vivo study on U-87, U- 118 cells of glioblastoma multiforme. Tests on death rate, viability, membrane integrity, proliferation rate, DNA degradation exhibited toxic effects of graphene oxide nanoflakes on glioblastoma multiforme cells [25, 26].

Pt 0 platinum nanoparticles as compared to cisplatin affected, to a similar extent, cell survival rate, integrity of cell membranes, proliferation rate of cells and death rate; applied dose of platinum (as an element) for both administrations were on the same level. As a result of the use of hydrocolloid of platinum nanoparticles in the form of direct injection into the tumour tissue, a significant reduction in tumour weight and volume and activation of apoptosis signalling pathways were WO 2015/121150 PCT/EP₂₀₁₅/052429 observed (fig. 1). Step I ll-summarising study-determination of the effect of the suspension of graphene oxide and platinum nanoparticles (GO/Pt) on the morphology of glioblastoma multiforme tumour, and determination of mechanisms of antitumor activity on the level of protein, gene expression and visualization of microstructure and ultrastructure of the tumour tissue. Preparation of Graphene-Pt complexes as "drug delivery system" by the method of forced self-organisation verified by TEM, SEM visualisations and Zeta potential, UV absorbance). 24 experiments in 2 repetitions were conducted, and a high affinity of platinum nanoparticles to graphene oxide nanoflakes was found, independently of the concentration used, chaotic decoration of graphene oxide nanoflakes by platinum metal nanoparticles, sustainability of the suspensions prepared. 10 experiments were conducted, in which studied suspensions were administered to GM tumour derived from the cells of U- 87 line, implanted and grown on the chorioallantoic membrane of the chicken embryo. It was found that:

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) did not affect the induction of inflammation in GM tumour (fig. 5);

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) affected the reduction of the weight of GM tumour on the level of 8.64% to 82% of the weight of control tumour (figs. 6, 7 and 8); WO 2015/121150 PCT/EP₂₀₁₅/052429

Our own (in vitro and in vivo) study showed that, in comparison to the control group, positive control group (cisplatin) and group with platinum nanoparticles and with graphene oxide flakes, graphene oxide nanoflakes with platinum nanoparticles attached exhibit higher toxicity in relation to cells and tumours of U- 87 glioblastoma multiforme. At the same time, they exhibit lower toxicity for adjacent tissues due to its activity which is limited and concentrated in the tumour area. The solution according to the present invention has the following advantages: Platinum is present in the form of Pt(0) metal nanoparticles, which reduces its solubility and distribution in the organism, and is deposited on graphene oxide flakes, which allows for its safe transport and deposition within the tumour and successive releasing and binding to cellular DNA, which activates tumour cell apoptosis and tumour regression. o Pt nanoparticles, as opposed to platinum salts, have metal properties, do not dissolve in water, do not form salts. This limits the distribution of platinum via biological fluids and significantly reduces the toxicity of platinum for healthy tissues. o Platinum nanoparticles are transported across cell and nuclear membranes within 1-24h. o As metal nanoparticles, they are distributed in the organism through a route alternative to the blood (from cell to cell through cell membrane), which may considerably limit the toxicity of platinum to the place of introduction of nanoparticles and its small surrounding areas. o Platinum nanoparticles react with DNA, which results in its degradation and activation of the process of cell death by apoptosis. o Graphene oxide allows targeted administration of platinum nanoparticles, limited to the location of graphene oxide. o Graphene oxide exhibits an affinity to the cell membrane of tumour cells, when introduced into the tumour, it tends to position itself at its central point, with minimal tendency to migration within the body. WO 2015/121150 PCT/EP₂₀₁₅/052429 o Graphene oxide performs functions of not only the carrier of Pt nanoparticles but it also exhibits activation properties in tumour cells of the death process (apoptosis). o Combination of platinum nanoparticles with graphene oxide flakes conditions local activity of Pt(0) in place of its introduction. o Graphene oxide, being a material having very active surface and, in particular, edges, combines with platinum nanoparticles and it acts for them as a kind of raft and allows direct delivery of platinum nanoparticles to tumour cells by adhesion to their cell membranes. Innovation in the use of platinum nanoparticles bound to graphene oxide flakes in therapy directed against tumours is manifested by controlling the area of activity, i.e. limitation of activation site of the mechanism of programmed cell death to the area of the tumour and, to a negligible extent, healthy cells (i.e. not undergone neoplastic transformation), adjacent to the tumour tissue. Control of the activity site allows limitation of toxic effects to the site of administration and elimination of toxic side effects resulting from the penetration of platinum nanoparticles into adjacent tissues and the bloodstream. Further, the nanoparticles, as a fragmented metal, exclude the possibility of creating toxic compounds in the bloodstream and of interacting with other medicines used collaterally in chemotherapy. References [1] W. S. Hummers and R. E. Offeman, "Preparation of Graphitic Oxide", J. Am. Chem.(Soc.,vol.80 1958) 1339. [2] D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu and J. M. Tour, "Improved synthesis of graphene oxide", ACS Nano, vol. 4 (2010) 4806-4814. [3] 0. C. Compton and S. T. Nguyen, "Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials", Small, vol. 6 (2010) 711-723. WO 2015/121150 PCT/EP₂₀₁₅/052429 [4] A. Fakhri, "Adsorption characteristics of graphene oxide as a solid adsorbent for aniline removal from aqueous solutions: Kinetics, thermodynamics and mechanism studies", Journal of Saudi chemical Society, in press. [5] S.H. Hsieh, M.C. Hsu, W.L. Liu, W. J. Chen, "Study of catalyst on graphene and its application to fuel cell", Applied surface Science, vol. 277 (2013) 223 -230. [6] B.F. Machado, P. Serp, "Graphene-based materials for catalysis", Catalysis Science and Technology, vol. 2 (2012) 54 -75. [7] B. Seger, P.V. Kamat, "Electrocatalically active graphene-platinum nanocomposites. Role of 2-D carbon support in PEM fuel cells, The Journal of Chemical Chemistry C, vol. 113 (2009) 7990 -7995. [8] Y. Wang, J. Liu, D.D. Sun, "high-quality reduced graphene oxide-nanocrystalline platinum hybrid materials prepared by simultaneous co-reduction of grapheme oxide and chloroplatinic acid, Nanoscale Research Letters, vol.(6:241 2011). [9] Ch. Wang et al., "A unique platinum-graphene hybrid structure for high activity and durability in oxygen reduction reaction", Scientific Reports, vol.(3:2580 2013). [10] H. W. Chang et al., "Preparation of graphene-supported platinum nanoparticles in aqueous solutions by femtosecond laser pulses for methanol oxidation", Journal of Power Sources, vol. 239 (2013) 164 -168. [11] G. Yue et al., "Platinum/graphene hybrid film as a counter electrode for dye-sensitized solar cells", Electrochimica Acta, vol. 92 (2013) 64 -70. [12] Ch. Chen, L. MingCe, W. HaoDong, C. WeiMin, "One-step synthesis of Pt nanoparticles/reduced graphene oxide composite with enhanced electrochemical catalytic activity, Science China-Chemistry, vol. 56 (2013) 354 -361. [13] L. AiRong, H. ShiMing, "A glucose biosensor based on direct electrochemistry of glucose oxidase immobilized onto platinum nanoparticles modified graphene electrode, Science China-Physics, Mechanics and Astronomy, vol. 55 (2012) 1163 -1167. [14] F. Zhang et al., "Microwave-assisted synthesis of Pt/graphene nanocomposites for non enzymatic hydrogen peroxide sensor" Int. J. Electrochem. Sci., vol. 7 (2012) 1968 -1977. WO 2015/121150 PCT/EP₂₀₁₅/052429 [15] Brandes AA, Basso U, Reni M, Vastola F, Tosoni A, Cavallo G, Scopece L, Ferreri A J, Panucci MG, Monfardini S, Ermani M. First-line chemotherapy with cisplatin plus fractionated temozolomide in recurrent glioblastoma multiforme: a phase I I study of the Gruppo Italiano Cooperativo di Neuro-Oncologia. J Clin Oncol. 2004, 1;22(9):1598-604. [16] Boulikas T., Vougiouka M., Recent clinical trials Rusing cisplatin, carboplatin and their combination chemotherapy drugs. Oncol Rep 2004, 11: 559- 595. [17] Bencokova Z, Pauron L, Devic C, J oubert A, Gastaldo J, Massart C, Balosso J, Foray N. Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin. J Neurooncol. 2008, 86(1):13-21. [18] Rachlin K, Moore DH, Yount G. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis. I ntegr Cancer Ther. 2013, 12(6):517-27. [19] Jung Y, Lippard S J. Direct cellular responses to platinum-induced DNA damage. Chem Rev. 2007,107(5):1387-407. [20] Giavini, E., Lemonica, I. P., Lou, Y., Broccia, M. L. and Prati, M. Induction of micronuclei and toxic effects in embryos of pregnant rats treated before implantation with anticancer drugs: Cyclophosphamide, Cis-platinum, adriamycin. Teratog. Carcinog. Mutagen., 1990, 10: 417- 426 [21] Ognio E., Lapide M., Ottone M., Mandys V., Peterka M., Parodi B., Viale M. Embryo-lethal and teratogenic effect of the new platinum compound DPR in pregnant mice. Arch. of Tox., 2003, 77: 584- [22] Podratz JL, Knight AM, Ta LE, Staff NP, Gass J M, Genelin K, Schlattau A, Lathroum L, Windebank A J. Cisplatin induced mitochondrial DNA damage in dorsal root ganglion neurons. Neurobiol Dis. 2011, 41: 661- 8 [23] Prasek, M., Sawosz, E., J aworski, S., Grodzik, M., Ostaszewska, T., Komaszewski, M., Wierzbicki, M., Chwalibog, A. (2013) Influence of nanoparticles of platinum on chicken embryo development and brain morphology. Nanoscale Research Letters, 8, 251- 260 [24] Sawosz E., J aworski S., Kut w in M., Hotowy A., W ierzbicki M., Grodzik M., Kurantowicz N., Stro j ny B., Lipinska L., Chwalibog A. 2014 Toxicity of pristine graphene in experiments in a chicken embryo model. Internation al J ournal of Nano m edicine, 9, 3913-3922 WO 2015/121150 PCT/EP₂₀₁₅/052429 [25] J aworski, S., Sawosz, E., Grodzik, M., Winnicka, A., Prasek, M., Wierzbicki, M., Chwalibog, A. 2013. In vitro evaluation of the effects of graphene platelets on globlastoma multiforme cells. International Journal of Nanomedicine, 8, 413- 420 [26] H inzmann M., J aworski S., Kut w in M., Jagiello J., Kozinski R., Wierzbicki M., Grodzik M., Lipinska L., Sawosz E., Chwalibog A. 2014. Nanoparticles containing allotropes of carbon have genotoxic effects on glioblastoma multiforme cells. International J ournal of Nano m edicine, 9, 2409-2417 Claims What is claimed is:

Canceled

A method of treating human or animal tumors, the method comprising: depositing nanoparticles of aqueous colloidal metallic platinum in zero oxidation state on a surface of graphene oxide nanoflakes and forming a water suspension by using ultrasonic energy in an ultrasound bath thereof, wherein graphene oxide nanoflakes are present in the suspension in a ran g e of about 1 00q/ml to about 500p/ml and nanoparticles of metallic platinum are present in the suspension in a ran ge of about Suq/ml to about 5 0q/ml, wherein platinum nanoparticles and graphene oxide nanoflakes are bound together through electronic interactions and graphene oxide nanoflakes and platinum nanoparticles are non-reduced in the water suspension, administering the water suspension of non-reduced graphene oxide nanoflakes and nanoparticles of metallic platinum to human or animal tumors, wherein the water suspension functions as an antitumor agent. Currently amended

Page 2 of 9 IIPG-1-59512 Canceled

Graphene oxide nanoflakes exhibited very high affinity and ability of adhesion to cell membrane of cell bodies. Death rate of GM cells upon contact with graphene oxide flakes was proportional to its concentration, and with a concentration of 100p/ml, it was 42 % for GM U- 87 cells, and 52% for GM U- 118 cells. A significant disintegration of cell membrane was also found. As a result, it was found that graphene nanoflakes were the cause of apoptosis of glioblastoma cells in 67.5% in GM U- 87 cells and in 99 % in GM U- 118 cells. Moreover, genotoxic effect was also observed, as a consequence of penetration of cells by graphene platelets [24]. To sum up, graphene oxide nanoflakes exhibit antitumor activity, as determined based on in vivo study on U-87, U- 118 cells of glioblastoma multiforme. Tests on death rate, viability, membrane integrity, proliferation rate, DNA degradation exhibited toxic effects of graphene oxide nanoflakes on glioblastoma multiforme cells [25, 26].

Pt 0 platinum nanoparticles as compared to cisplatin affected, to a similar extent, cell survival rate, integrity of cell membranes, proliferation rate of cells and death rate; applied dose of platinum (as an element) for both administrations were on the same level. As a result of the use of hydrocolloid of platinum nanoparticles in the form of direct injection into the tumour tissue, a significant reduction in tumour weight and volume and activation of apoptosis signalling pathways were WO 2015/121150 PCT/EP₂₀₁₅/052429 observed (fig. 1). Step I ll-summarising study-determination of the effect of the suspension of graphene oxide and platinum nanoparticles (GO/Pt) on the morphology of glioblastoma multiforme tumour, and determination of mechanisms of antitumor activity on the level of protein, gene expression and visualization of microstructure and ultrastructure of the tumour tissue. Preparation of Graphene-Pt complexes as "drug delivery system" by the method of forced self-organisation verified by TEM, SEM visualisations and Zeta potential, UV absorbance). 24 experiments in 2 repetitions were conducted, and a high affinity of platinum nanoparticles to graphene oxide nanoflakes was found, independently of the concentration used, chaotic decoration of graphene oxide nanoflakes by platinum metal nanoparticles, sustainability of the suspensions prepared. 10 experiments were conducted, in which studied suspensions were administered to GM tumour derived from the cells of U- 87 line, implanted and grown on the chorioallantoic membrane of the chicken embryo. It was found that:

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) did not affect the induction of inflammation in GM tumour (fig. 5);

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) affected the reduction of the weight of GM tumour on the level of 8.64% to 82% of the weight of control tumour (figs. 6, 7 and 8); WO 2015/121150 PCT/EP₂₀₁₅/052429

Our own (in vitro and in vivo) study showed that, in comparison to the control group, positive control group (cisplatin) and group with platinum nanoparticles and with graphene oxide flakes, graphene oxide nanoflakes with platinum nanoparticles attached exhibit higher toxicity in relation to cells and tumours of U- 87 glioblastoma multiforme. At the same time, they exhibit lower toxicity for adjacent tissues due to its activity which is limited and concentrated in the tumour area. The solution according to the present invention has the following advantages: Platinum is present in the form of Pt(0) metal nanoparticles, which reduces its solubility and distribution in the organism, and is deposited on graphene oxide flakes, which allows for its safe transport and deposition within the tumour and successive releasing and binding to cellular DNA, which activates tumour cell apoptosis and tumour regression. o Pt nanoparticles, as opposed to platinum salts, have metal properties, do not dissolve in water, do not form salts. This limits the distribution of platinum via biological fluids and significantly reduces the toxicity of platinum for healthy tissues. o Platinum nanoparticles are transported across cell and nuclear membranes within 1-24h. o As metal nanoparticles, they are distributed in the organism through a route alternative to the blood (from cell to cell through cell membrane), which may considerably limit the toxicity of platinum to the place of introduction of nanoparticles and its small surrounding areas. o Platinum nanoparticles react with DNA, which results in its degradation and activation of the process of cell death by apoptosis. o Graphene oxide allows targeted administration of platinum nanoparticles, limited to the location of graphene oxide. o Graphene oxide exhibits an affinity to the cell membrane of tumour cells, when introduced into the tumour, it tends to position itself at its central point, with minimal tendency to migration within the body. WO 2015/121150 PCT/EP₂₀₁₅/052429 o Graphene oxide performs functions of not only the carrier of Pt nanoparticles but it also exhibits activation properties in tumour cells of the death process (apoptosis). o Combination of platinum nanoparticles with graphene oxide flakes conditions local activity of Pt(0) in place of its introduction. o Graphene oxide, being a material having very active surface and, in particular, edges, combines with platinum nanoparticles and it acts for them as a kind of raft and allows direct delivery of platinum nanoparticles to tumour cells by adhesion to their cell membranes. Innovation in the use of platinum nanoparticles bound to graphene oxide flakes in therapy directed against tumours is manifested by controlling the area of activity, i.e. limitation of activation site of the mechanism of programmed cell death to the area of the tumour and, to a negligible extent, healthy cells (i.e. not undergone neoplastic transformation), adjacent to the tumour tissue. Control of the activity site allows limitation of toxic effects to the site of administration and elimination of toxic side effects resulting from the penetration of platinum nanoparticles into adjacent tissues and the bloodstream. Further, the nanoparticles, as a fragmented metal, exclude the possibility of creating toxic compounds in the bloodstream and of interacting with other medicines used collaterally in chemotherapy. References [1] W. S. Hummers and R. E. Offeman, "Preparation of Graphitic Oxide", J. Am. Chem.(Soc.,vol.80 1958) 1339. [2] D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu and J. M. Tour, "Improved synthesis of graphene oxide", ACS Nano, vol. 4 (2010) 4806-4814. [3] 0. C. Compton and S. T. Nguyen, "Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials", Small, vol. 6 (2010) 711-723. WO 2015/121150 PCT/EP₂₀₁₅/052429 [4] A. Fakhri, "Adsorption characteristics of graphene oxide as a solid adsorbent for aniline removal from aqueous solutions: Kinetics, thermodynamics and mechanism studies", Journal of Saudi chemical Society, in press. [5] S.H. Hsieh, M.C. Hsu, W.L. Liu, W. J. Chen, "Study of catalyst on graphene and its application to fuel cell", Applied surface Science, vol. 277 (2013) 223 -230. [6] B.F. Machado, P. Serp, "Graphene-based materials for catalysis", Catalysis Science and Technology, vol. 2 (2012) 54 -75. [7] B. Seger, P.V. Kamat, "Electrocatalically active graphene-platinum nanocomposites. Role of 2-D carbon support in PEM fuel cells, The Journal of Chemical Chemistry C, vol. 113 (2009) 7990 -7995. [8] Y. Wang, J. Liu, D.D. Sun, "high-quality reduced graphene oxide-nanocrystalline platinum hybrid materials prepared by simultaneous co-reduction of grapheme oxide and chloroplatinic acid, Nanoscale Research Letters, vol.(6:241 2011). [9] Ch. Wang et al., "A unique platinum-graphene hybrid structure for high activity and durability in oxygen reduction reaction", Scientific Reports, vol.(3:2580 2013). [10] H. W. Chang et al., "Preparation of graphene-supported platinum nanoparticles in aqueous solutions by femtosecond laser pulses for methanol oxidation", Journal of Power Sources, vol. 239 (2013) 164 -168. [11] G. Yue et al., "Platinum/graphene hybrid film as a counter electrode for dye-sensitized solar cells", Electrochimica Acta, vol. 92 (2013) 64 -70. [12] Ch. Chen, L. MingCe, W. HaoDong, C. WeiMin, "One-step synthesis of Pt nanoparticles/reduced graphene oxide composite with enhanced electrochemical catalytic activity, Science China-Chemistry, vol. 56 (2013) 354 -361. [13] L. AiRong, H. ShiMing, "A glucose biosensor based on direct electrochemistry of glucose oxidase immobilized onto platinum nanoparticles modified graphene electrode, Science China-Physics, Mechanics and Astronomy, vol. 55 (2012) 1163 -1167. [14] F. Zhang et al., "Microwave-assisted synthesis of Pt/graphene nanocomposites for non enzymatic hydrogen peroxide sensor" Int. J. Electrochem. Sci., vol. 7 (2012) 1968 -1977. WO 2015/121150 PCT/EP₂₀₁₅/052429 [15] Brandes AA, Basso U, Reni M, Vastola F, Tosoni A, Cavallo G, Scopece L, Ferreri A J, Panucci MG, Monfardini S, Ermani M. First-line chemotherapy with cisplatin plus fractionated temozolomide in recurrent glioblastoma multiforme: a phase I I study of the Gruppo Italiano Cooperativo di Neuro-Oncologia. J Clin Oncol. 2004, 1;22(9):1598-604. [16] Boulikas T., Vougiouka M., Recent clinical trials Rusing cisplatin, carboplatin and their combination chemotherapy drugs. Oncol Rep 2004, 11: 559- 595. [17] Bencokova Z, Pauron L, Devic C, J oubert A, Gastaldo J, Massart C, Balosso J, Foray N. Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin. J Neurooncol. 2008, 86(1):13-21. [18] Rachlin K, Moore DH, Yount G. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis. I ntegr Cancer Ther. 2013, 12(6):517-27. [19] Jung Y, Lippard S J. Direct cellular responses to platinum-induced DNA damage. Chem Rev. 2007,107(5):1387-407. [20] Giavini, E., Lemonica, I. P., Lou, Y., Broccia, M. L. and Prati, M. Induction of micronuclei and toxic effects in embryos of pregnant rats treated before implantation with anticancer drugs: Cyclophosphamide, Cis-platinum, adriamycin. Teratog. Carcinog. Mutagen., 1990, 10: 417- 426 [21] Ognio E., Lapide M., Ottone M., Mandys V., Peterka M., Parodi B., Viale M. Embryo-lethal and teratogenic effect of the new platinum compound DPR in pregnant mice. Arch. of Tox., 2003, 77: 584- [22] Podratz JL, Knight AM, Ta LE, Staff NP, Gass J M, Genelin K, Schlattau A, Lathroum L, Windebank A J. Cisplatin induced mitochondrial DNA damage in dorsal root ganglion neurons. Neurobiol Dis. 2011, 41: 661- 8 [23] Prasek, M., Sawosz, E., J aworski, S., Grodzik, M., Ostaszewska, T., Komaszewski, M., Wierzbicki, M., Chwalibog, A. (2013) Influence of nanoparticles of platinum on chicken embryo development and brain morphology. Nanoscale Research Letters, 8, 251- 260 [24] Sawosz E., J aworski S., Kut w in M., Hotowy A., W ierzbicki M., Grodzik M., Kurantowicz N., Stro j ny B., Lipinska L., Chwalibog A. 2014 Toxicity of pristine graphene in experiments in a chicken embryo model. Internation al J ournal of Nano m edicine, 9, 3913-3922 WO 2015/121150 PCT/EP₂₀₁₅/052429 [25] J aworski, S., Sawosz, E., Grodzik, M., Winnicka, A., Prasek, M., Wierzbicki, M., Chwalibog, A. 2013. In vitro evaluation of the effects of graphene platelets on globlastoma multiforme cells. International Journal of Nanomedicine, 8, 413- 420 [26] H inzmann M., J aworski S., Kut w in M., Jagiello J., Kozinski R., Wierzbicki M., Grodzik M., Lipinska L., Sawosz E., Chwalibog A. 2014. Nanoparticles containing allotropes of carbon have genotoxic effects on glioblastoma multiforme cells. International J ournal of Nano m edicine, 9, 2409-2417 Claims What is claimed is:

Canceled

A method of treating human or animal tumors, the method comprising: depositing nanoparticles of aqueous colloidal metallic platinum in zero oxidation state on a surface of graphene oxide nanoflakes and forming a water suspension by using ultrasonic energy in an ultrasound bath thereof, wherein graphene oxide nanoflakes are present in the suspension in a ran g e of about 1 00q/ml to about 500p/ml and nanoparticles of metallic platinum are present in the suspension in a ran ge of about Suq/ml to about 5 0q/ml, wherein platinum nanoparticles and graphene oxide nanoflakes are bound together through electronic interactions and graphene oxide nanoflakes and platinum nanoparticles are non-reduced in the water suspension, administering the water suspension of non-reduced graphene oxide nanoflakes and nanoparticles of metallic platinum to human or animal tumors, wherein the water suspension functions as an antitumor agent. Currently amended

Page 2 of 9 IIPG-1-59512 Canceled

Graphene oxide nanoflakes exhibited very high affinity and ability of adhesion to cell membrane of cell bodies. Death rate of GM cells upon contact with graphene oxide flakes was proportional to its concentration, and with a concentration of 100p/ml, it was 42 % for GM U- 87 cells, and 52% for GM U- 118 cells. A significant disintegration of cell membrane was also found. As a result, it was found that graphene nanoflakes were the cause of apoptosis of glioblastoma cells in 67.5% in GM U- 87 cells and in 99 % in GM U- 118 cells. Moreover, genotoxic effect was also observed, as a consequence of penetration of cells by graphene platelets [24]. To sum up, graphene oxide nanoflakes exhibit antitumor activity, as determined based on in vivo study on U-87, U- 118 cells of glioblastoma multiforme. Tests on death rate, viability, membrane integrity, proliferation rate, DNA degradation exhibited toxic effects of graphene oxide nanoflakes on glioblastoma multiforme cells [25, 26].

Pt 0 platinum nanoparticles as compared to cisplatin affected, to a similar extent, cell survival rate, integrity of cell membranes, proliferation rate of cells and death rate; applied dose of platinum (as an element) for both administrations were on the same level. As a result of the use of hydrocolloid of platinum nanoparticles in the form of direct injection into the tumour tissue, a significant reduction in tumour weight and volume and activation of apoptosis signalling pathways were WO 2015/121150 PCT/EP₂₀₁₅/052429 observed (fig. 1). Step I ll-summarising study-determination of the effect of the suspension of graphene oxide and platinum nanoparticles (GO/Pt) on the morphology of glioblastoma multiforme tumour, and determination of mechanisms of antitumor activity on the level of protein, gene expression and visualization of microstructure and ultrastructure of the tumour tissue. Preparation of Graphene-Pt complexes as "drug delivery system" by the method of forced self-organisation verified by TEM, SEM visualisations and Zeta potential, UV absorbance). 24 experiments in 2 repetitions were conducted, and a high affinity of platinum nanoparticles to graphene oxide nanoflakes was found, independently of the concentration used, chaotic decoration of graphene oxide nanoflakes by platinum metal nanoparticles, sustainability of the suspensions prepared. 10 experiments were conducted, in which studied suspensions were administered to GM tumour derived from the cells of U- 87 line, implanted and grown on the chorioallantoic membrane of the chicken embryo. It was found that:

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) did not affect the induction of inflammation in GM tumour (fig. 5);

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) affected the reduction of the weight of GM tumour on the level of 8.64% to 82% of the weight of control tumour (figs. 6, 7 and 8); WO 2015/121150 PCT/EP₂₀₁₅/052429

Our own (in vitro and in vivo) study showed that, in comparison to the control group, positive control group (cisplatin) and group with platinum nanoparticles and with graphene oxide flakes, graphene oxide nanoflakes with platinum nanoparticles attached exhibit higher toxicity in relation to cells and tumours of U- 87 glioblastoma multiforme. At the same time, they exhibit lower toxicity for adjacent tissues due to its activity which is limited and concentrated in the tumour area. The solution according to the present invention has the following advantages: Platinum is present in the form of Pt(0) metal nanoparticles, which reduces its solubility and distribution in the organism, and is deposited on graphene oxide flakes, which allows for its safe transport and deposition within the tumour and successive releasing and binding to cellular DNA, which activates tumour cell apoptosis and tumour regression. o Pt nanoparticles, as opposed to platinum salts, have metal properties, do not dissolve in water, do not form salts. This limits the distribution of platinum via biological fluids and significantly reduces the toxicity of platinum for healthy tissues. o Platinum nanoparticles are transported across cell and nuclear membranes within 1-24h. o As metal nanoparticles, they are distributed in the organism through a route alternative to the blood (from cell to cell through cell membrane), which may considerably limit the toxicity of platinum to the place of introduction of nanoparticles and its small surrounding areas. o Platinum nanoparticles react with DNA, which results in its degradation and activation of the process of cell death by apoptosis. o Graphene oxide allows targeted administration of platinum nanoparticles, limited to the location of graphene oxide. o Graphene oxide exhibits an affinity to the cell membrane of tumour cells, when introduced into the tumour, it tends to position itself at its central point, with minimal tendency to migration within the body. WO 2015/121150 PCT/EP₂₀₁₅/052429 o Graphene oxide performs functions of not only the carrier of Pt nanoparticles but it also exhibits activation properties in tumour cells of the death process (apoptosis). o Combination of platinum nanoparticles with graphene oxide flakes conditions local activity of Pt(0) in place of its introduction. o Graphene oxide, being a material having very active surface and, in particular, edges, combines with platinum nanoparticles and it acts for them as a kind of raft and allows direct delivery of platinum nanoparticles to tumour cells by adhesion to their cell membranes. Innovation in the use of platinum nanoparticles bound to graphene oxide flakes in therapy directed against tumours is manifested by controlling the area of activity, i.e. limitation of activation site of the mechanism of programmed cell death to the area of the tumour and, to a negligible extent, healthy cells (i.e. not undergone neoplastic transformation), adjacent to the tumour tissue. Control of the activity site allows limitation of toxic effects to the site of administration and elimination of toxic side effects resulting from the penetration of platinum nanoparticles into adjacent tissues and the bloodstream. Further, the nanoparticles, as a fragmented metal, exclude the possibility of creating toxic compounds in the bloodstream and of interacting with other medicines used collaterally in chemotherapy. References [1] W. S. Hummers and R. E. Offeman, "Preparation of Graphitic Oxide", J. Am. Chem.(Soc.,vol.80 1958) 1339. [2] D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu and J. M. Tour, "Improved synthesis of graphene oxide", ACS Nano, vol. 4 (2010) 4806-4814. [3] 0. C. Compton and S. T. Nguyen, "Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials", Small, vol. 6 (2010) 711-723. WO 2015/121150 PCT/EP₂₀₁₅/052429 [4] A. Fakhri, "Adsorption characteristics of graphene oxide as a solid adsorbent for aniline removal from aqueous solutions: Kinetics, thermodynamics and mechanism studies", Journal of Saudi chemical Society, in press. [5] S.H. Hsieh, M.C. Hsu, W.L. Liu, W. J. Chen, "Study of catalyst on graphene and its application to fuel cell", Applied surface Science, vol. 277 (2013) 223 -230. [6] B.F. Machado, P. Serp, "Graphene-based materials for catalysis", Catalysis Science and Technology, vol. 2 (2012) 54 -75. [7] B. Seger, P.V. Kamat, "Electrocatalically active graphene-platinum nanocomposites. Role of 2-D carbon support in PEM fuel cells, The Journal of Chemical Chemistry C, vol. 113 (2009) 7990 -7995. [8] Y. Wang, J. Liu, D.D. Sun, "high-quality reduced graphene oxide-nanocrystalline platinum hybrid materials prepared by simultaneous co-reduction of grapheme oxide and chloroplatinic acid, Nanoscale Research Letters, vol.(6:241 2011). [9] Ch. Wang et al., "A unique platinum-graphene hybrid structure for high activity and durability in oxygen reduction reaction", Scientific Reports, vol.(3:2580 2013). [10] H. W. Chang et al., "Preparation of graphene-supported platinum nanoparticles in aqueous solutions by femtosecond laser pulses for methanol oxidation", Journal of Power Sources, vol. 239 (2013) 164 -168. [11] G. Yue et al., "Platinum/graphene hybrid film as a counter electrode for dye-sensitized solar cells", Electrochimica Acta, vol. 92 (2013) 64 -70. [12] Ch. Chen, L. MingCe, W. HaoDong, C. WeiMin, "One-step synthesis of Pt nanoparticles/reduced graphene oxide composite with enhanced electrochemical catalytic activity, Science China-Chemistry, vol. 56 (2013) 354 -361. [13] L. AiRong, H. ShiMing, "A glucose biosensor based on direct electrochemistry of glucose oxidase immobilized onto platinum nanoparticles modified graphene electrode, Science China-Physics, Mechanics and Astronomy, vol. 55 (2012) 1163 -1167. [14] F. Zhang et al., "Microwave-assisted synthesis of Pt/graphene nanocomposites for non enzymatic hydrogen peroxide sensor" Int. J. Electrochem. Sci., vol. 7 (2012) 1968 -1977. WO 2015/121150 PCT/EP₂₀₁₅/052429 [15] Brandes AA, Basso U, Reni M, Vastola F, Tosoni A, Cavallo G, Scopece L, Ferreri A J, Panucci MG, Monfardini S, Ermani M. First-line chemotherapy with cisplatin plus fractionated temozolomide in recurrent glioblastoma multiforme: a phase I I study of the Gruppo Italiano Cooperativo di Neuro-Oncologia. J Clin Oncol. 2004, 1;22(9):1598-604. [16] Boulikas T., Vougiouka M., Recent clinical trials Rusing cisplatin, carboplatin and their combination chemotherapy drugs. Oncol Rep 2004, 11: 559- 595. [17] Bencokova Z, Pauron L, Devic C, J oubert A, Gastaldo J, Massart C, Balosso J, Foray N. Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin. J Neurooncol. 2008, 86(1):13-21. [18] Rachlin K, Moore DH, Yount G. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis. I ntegr Cancer Ther. 2013, 12(6):517-27. [19] Jung Y, Lippard S J. Direct cellular responses to platinum-induced DNA damage. Chem Rev. 2007,107(5):1387-407. [20] Giavini, E., Lemonica, I. P., Lou, Y., Broccia, M. L. and Prati, M. Induction of micronuclei and toxic effects in embryos of pregnant rats treated before implantation with anticancer drugs: Cyclophosphamide, Cis-platinum, adriamycin. Teratog. Carcinog. Mutagen., 1990, 10: 417- 426 [21] Ognio E., Lapide M., Ottone M., Mandys V., Peterka M., Parodi B., Viale M. Embryo-lethal and teratogenic effect of the new platinum compound DPR in pregnant mice. Arch. of Tox., 2003, 77: 584- [22] Podratz JL, Knight AM, Ta LE, Staff NP, Gass J M, Genelin K, Schlattau A, Lathroum L, Windebank A J. Cisplatin induced mitochondrial DNA damage in dorsal root ganglion neurons. Neurobiol Dis. 2011, 41: 661- 8 [23] Prasek, M., Sawosz, E., J aworski, S., Grodzik, M., Ostaszewska, T., Komaszewski, M., Wierzbicki, M., Chwalibog, A. (2013) Influence of nanoparticles of platinum on chicken embryo development and brain morphology. Nanoscale Research Letters, 8, 251- 260 [24] Sawosz E., J aworski S., Kut w in M., Hotowy A., W ierzbicki M., Grodzik M., Kurantowicz N., Stro j ny B., Lipinska L., Chwalibog A. 2014 Toxicity of pristine graphene in experiments in a chicken embryo model. Internation al J ournal of Nano m edicine, 9, 3913-3922 WO 2015/121150 PCT/EP₂₀₁₅/052429 [25] J aworski, S., Sawosz, E., Grodzik, M., Winnicka, A., Prasek, M., Wierzbicki, M., Chwalibog, A. 2013. In vitro evaluation of the effects of graphene platelets on globlastoma multiforme cells. International Journal of Nanomedicine, 8, 413- 420 [26] H inzmann M., J aworski S., Kut w in M., Jagiello J., Kozinski R., Wierzbicki M., Grodzik M., Lipinska L., Sawosz E., Chwalibog A. 2014. Nanoparticles containing allotropes of carbon have genotoxic effects on glioblastoma multiforme cells. International J ournal of Nano m edicine, 9, 2409-2417 Claims What is claimed is:

Canceled

A method of treating human or animal tumors, the method comprising: depositing nanoparticles of aqueous colloidal metallic platinum in zero oxidation state on a surface of graphene oxide nanoflakes and forming a water suspension by using ultrasonic energy in an ultrasound bath thereof, wherein graphene oxide nanoflakes are present in the suspension in a ran g e of about 1 00q/ml to about 500p/ml and nanoparticles of metallic platinum are present in the suspension in a ran ge of about Suq/ml to about 5 0q/ml, wherein platinum nanoparticles and graphene oxide nanoflakes are bound together through electronic interactions and graphene oxide nanoflakes and platinum nanoparticles are non-reduced in the water suspension, administering the water suspension of non-reduced graphene oxide nanoflakes and nanoparticles of metallic platinum to human or animal tumors, wherein the water suspension functions as an antitumor agent. Currently amended

Page 2 of 9 IIPG-1-59512 Canceled

Graphene oxide nanoflakes exhibited very high affinity and ability of adhesion to cell membrane of cell bodies. Death rate of GM cells upon contact with graphene oxide flakes was proportional to its concentration, and with a concentration of 100p/ml, it was 42 % for GM U- 87 cells, and 52% for GM U- 118 cells. A significant disintegration of cell membrane was also found. As a result, it was found that graphene nanoflakes were the cause of apoptosis of glioblastoma cells in 67.5% in GM U- 87 cells and in 99 % in GM U- 118 cells. Moreover, genotoxic effect was also observed, as a consequence of penetration of cells by graphene platelets [24]. To sum up, graphene oxide nanoflakes exhibit antitumor activity, as determined based on in vivo study on U-87, U- 118 cells of glioblastoma multiforme. Tests on death rate, viability, membrane integrity, proliferation rate, DNA degradation exhibited toxic effects of graphene oxide nanoflakes on glioblastoma multiforme cells [25, 26].

Pt 0 platinum nanoparticles as compared to cisplatin affected, to a similar extent, cell survival rate, integrity of cell membranes, proliferation rate of cells and death rate; applied dose of platinum (as an element) for both administrations were on the same level. As a result of the use of hydrocolloid of platinum nanoparticles in the form of direct injection into the tumour tissue, a significant reduction in tumour weight and volume and activation of apoptosis signalling pathways were WO 2015/121150 PCT/EP₂₀₁₅/052429 observed (fig. 1). Step I ll-summarising study-determination of the effect of the suspension of graphene oxide and platinum nanoparticles (GO/Pt) on the morphology of glioblastoma multiforme tumour, and determination of mechanisms of antitumor activity on the level of protein, gene expression and visualization of microstructure and ultrastructure of the tumour tissue. Preparation of Graphene-Pt complexes as "drug delivery system" by the method of forced self-organisation verified by TEM, SEM visualisations and Zeta potential, UV absorbance). 24 experiments in 2 repetitions were conducted, and a high affinity of platinum nanoparticles to graphene oxide nanoflakes was found, independently of the concentration used, chaotic decoration of graphene oxide nanoflakes by platinum metal nanoparticles, sustainability of the suspensions prepared. 10 experiments were conducted, in which studied suspensions were administered to GM tumour derived from the cells of U- 87 line, implanted and grown on the chorioallantoic membrane of the chicken embryo. It was found that:

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) did not affect the induction of inflammation in GM tumour (fig. 5);

graphene oxide flakes decorated with Pt nanoparticles (at all of the studied concentrations of graphene and platinum) affected the reduction of the weight of GM tumour on the level of 8.64% to 82% of the weight of control tumour (figs. 6, 7 and 8); WO 2015/121150 PCT/EP₂₀₁₅/052429

Our own (in vitro and in vivo) study showed that, in comparison to the control group, positive control group (cisplatin) and group with platinum nanoparticles and with graphene oxide flakes, graphene oxide nanoflakes with platinum nanoparticles attached exhibit higher toxicity in relation to cells and tumours of U- 87 glioblastoma multiforme. At the same time, they exhibit lower toxicity for adjacent tissues due to its activity which is limited and concentrated in the tumour area. The solution according to the present invention has the following advantages: Platinum is present in the form of Pt(0) metal nanoparticles, which reduces its solubility and distribution in the organism, and is deposited on graphene oxide flakes, which allows for its safe transport and deposition within the tumour and successive releasing and binding to cellular DNA, which activates tumour cell apoptosis and tumour regression. o Pt nanoparticles, as opposed to platinum salts, have metal properties, do not dissolve in water, do not form salts. This limits the distribution of platinum via biological fluids and significantly reduces the toxicity of platinum for healthy tissues. o Platinum nanoparticles are transported across cell and nuclear membranes within 1-24h. o As metal nanoparticles, they are distributed in the organism through a route alternative to the blood (from cell to cell through cell membrane), which may considerably limit the toxicity of platinum to the place of introduction of nanoparticles and its small surrounding areas. o Platinum nanoparticles react with DNA, which results in its degradation and activation of the process of cell death by apoptosis. o Graphene oxide allows targeted administration of platinum nanoparticles, limited to the location of graphene oxide. o Graphene oxide exhibits an affinity to the cell membrane of tumour cells, when introduced into the tumour, it tends to position itself at its central point, with minimal tendency to migration within the body. WO 2015/121150 PCT/EP₂₀₁₅/052429 o Graphene oxide performs functions of not only the carrier of Pt nanoparticles but it also exhibits activation properties in tumour cells of the death process (apoptosis). o Combination of platinum nanoparticles with graphene oxide flakes conditions local activity of Pt(0) in place of its introduction. o Graphene oxide, being a material having very active surface and, in particular, edges, combines with platinum nanoparticles and it acts for them as a kind of raft and allows direct delivery of platinum nanoparticles to tumour cells by adhesion to their cell membranes. Innovation in the use of platinum nanoparticles bound to graphene oxide flakes in therapy directed against tumours is manifested by controlling the area of activity, i.e. limitation of activation site of the mechanism of programmed cell death to the area of the tumour and, to a negligible extent, healthy cells (i.e. not undergone neoplastic transformation), adjacent to the tumour tissue. Control of the activity site allows limitation of toxic effects to the site of administration and elimination of toxic side effects resulting from the penetration of platinum nanoparticles into adjacent tissues and the bloodstream. Further, the nanoparticles, as a fragmented metal, exclude the possibility of creating toxic compounds in the bloodstream and of interacting with other medicines used collaterally in chemotherapy. References [1] W. S. Hummers and R. E. Offeman, "Preparation of Graphitic Oxide", J. Am. Chem.(Soc.,vol.80 1958) 1339. [2] D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu and J. M. Tour, "Improved synthesis of graphene oxide", ACS Nano, vol. 4 (2010) 4806-4814. [3] 0. C. Compton and S. T. Nguyen, "Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials", Small, vol. 6 (2010) 711-723. WO 2015/121150 PCT/EP₂₀₁₅/052429 [4] A. Fakhri, "Adsorption characteristics of graphene oxide as a solid adsorbent for aniline removal from aqueous solutions: Kinetics, thermodynamics and mechanism studies", Journal of Saudi chemical Society, in press. [5] S.H. Hsieh, M.C. Hsu, W.L. Liu, W. J. Chen, "Study of catalyst on graphene and its application to fuel cell", Applied surface Science, vol. 277 (2013) 223 -230. [6] B.F. Machado, P. Serp, "Graphene-based materials for catalysis", Catalysis Science and Technology, vol. 2 (2012) 54 -75. [7] B. Seger, P.V. Kamat, "Electrocatalically active graphene-platinum nanocomposites. Role of 2-D carbon support in PEM fuel cells, The Journal of Chemical Chemistry C, vol. 113 (2009) 7990 -7995. [8] Y. Wang, J. Liu, D.D. Sun, "high-quality reduced graphene oxide-nanocrystalline platinum hybrid materials prepared by simultaneous co-reduction of grapheme oxide and chloroplatinic acid, Nanoscale Research Letters, vol.(6:241 2011). [9] Ch. Wang et al., "A unique platinum-graphene hybrid structure for high activity and durability in oxygen reduction reaction", Scientific Reports, vol.(3:2580 2013). [10] H. W. Chang et al., "Preparation of graphene-supported platinum nanoparticles in aqueous solutions by femtosecond laser pulses for methanol oxidation", Journal of Power Sources, vol. 239 (2013) 164 -168. [11] G. Yue et al., "Platinum/graphene hybrid film as a counter electrode for dye-sensitized solar cells", Electrochimica Acta, vol. 92 (2013) 64 -70. [12] Ch. Chen, L. MingCe, W. HaoDong, C. WeiMin, "One-step synthesis of Pt nanoparticles/reduced graphene oxide composite with enhanced electrochemical catalytic activity, Science China-Chemistry, vol. 56 (2013) 354 -361. [13] L. AiRong, H. ShiMing, "A glucose biosensor based on direct electrochemistry of glucose oxidase immobilized onto platinum nanoparticles modified graphene electrode, Science China-Physics, Mechanics and Astronomy, vol. 55 (2012) 1163 -1167. [14] F. Zhang et al., "Microwave-assisted synthesis of Pt/graphene nanocomposites for non enzymatic hydrogen peroxide sensor" Int. J. Electrochem. Sci., vol. 7 (2012) 1968 -1977. WO 2015/121150 PCT/EP₂₀₁₅/052429 [15] Brandes AA, Basso U, Reni M, Vastola F, Tosoni A, Cavallo G, Scopece L, Ferreri A J, Panucci MG, Monfardini S, Ermani M. First-line chemotherapy with cisplatin plus fractionated temozolomide in recurrent glioblastoma multiforme: a phase I I study of the Gruppo Italiano Cooperativo di Neuro-Oncologia. J Clin Oncol. 2004, 1;22(9):1598-604. [16] Boulikas T., Vougiouka M., Recent clinical trials Rusing cisplatin, carboplatin and their combination chemotherapy drugs. Oncol Rep 2004, 11: 559- 595. [17] Bencokova Z, Pauron L, Devic C, J oubert A, Gastaldo J, Massart C, Balosso J, Foray N. Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin. J Neurooncol. 2008, 86(1):13-21. [18] Rachlin K, Moore DH, Yount G. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis. I ntegr Cancer Ther. 2013, 12(6):517-27. [19] Jung Y, Lippard S J. Direct cellular responses to platinum-induced DNA damage. Chem Rev. 2007,107(5):1387-407. [20] Giavini, E., Lemonica, I. P., Lou, Y., Broccia, M. L. and Prati, M. Induction of micronuclei and toxic effects in embryos of pregnant rats treated before implantation with anticancer drugs: Cyclophosphamide, Cis-platinum, adriamycin. Teratog. Carcinog. Mutagen., 1990, 10: 417- 426 [21] Ognio E., Lapide M., Ottone M., Mandys V., Peterka M., Parodi B., Viale M. Embryo-lethal and teratogenic effect of the new platinum compound DPR in pregnant mice. Arch. of Tox., 2003, 77: 584- [22] Podratz JL, Knight AM, Ta LE, Staff NP, Gass J M, Genelin K, Schlattau A, Lathroum L, Windebank A J. Cisplatin induced mitochondrial DNA damage in dorsal root ganglion neurons. Neurobiol Dis. 2011, 41: 661- 8 [23] Prasek, M., Sawosz, E., J aworski, S., Grodzik, M., Ostaszewska, T., Komaszewski, M., Wierzbicki, M., Chwalibog, A. (2013) Influence of nanoparticles of platinum on chicken embryo development and brain morphology. Nanoscale Research Letters, 8, 251- 260 [24] Sawosz E., J aworski S., Kut w in M., Hotowy A., W ierzbicki M., Grodzik M., Kurantowicz N., Stro j ny B., Lipinska L., Chwalibog A. 2014 Toxicity of pristine graphene in experiments in a chicken embryo model. Internation al J ournal of Nano m edicine, 9, 3913-3922 WO 2015/121150 PCT/EP₂₀₁₅/052429 [25] J aworski, S., Sawosz, E., Grodzik, M., Winnicka, A., Prasek, M., Wierzbicki, M., Chwalibog, A. 2013. In vitro evaluation of the effects of graphene platelets on globlastoma multiforme cells. International Journal of Nanomedicine, 8, 413- 420 [26] H inzmann M., J aworski S., Kut w in M., Jagiello J., Kozinski R., Wierzbicki M., Grodzik M., Lipinska L., Sawosz E., Chwalibog A. 2014. Nanoparticles containing allotropes of carbon have genotoxic effects on glioblastoma multiforme cells. International J ournal of Nano m edicine, 9, 2409-2417 Claims What is claimed is:

Canceled

A method of treating human or animal tumors, the method comprising: depositing nanoparticles of aqueous colloidal metallic platinum in zero oxidation state on a surface of graphene oxide nanoflakes and forming a water suspension by using ultrasonic energy in an ultrasound bath thereof, wherein graphene oxide nanoflakes are present in the suspension in a ran g e of about 1 00q/ml to about 500p/ml and nanoparticles of metallic platinum are present in the suspension in a ran ge of about Suq/ml to about 5 0q/ml, wherein platinum nanoparticles and graphene oxide nanoflakes are bound together through electronic interactions and graphene oxide nanoflakes and platinum nanoparticles are non-reduced in the water suspension, administering the water suspension of non-reduced graphene oxide nanoflakes and nanoparticles of metallic platinum to human or animal tumors, wherein the water suspension functions as an antitumor agent. Currently amended

Page 2 of 9 IIPG-1-59512 Canceled