THERAPY

Technical Field

[1] The subject matter of the invention is a method of making a system comprising gold nanoparticles functionalized with temozolomide, being a complex chemotherapeutic drug and intended in particular for the treatment of a brain tumor with a high degree of malignancy, which is glioblastoma multiforme.

[2] The subject matter of the invention is also the use of this system in anticancer therapy, in particular for the treatment of glioblastoma multiforme, in combination with radiation therapy.

Background Art

[3] Glioblastoma multiforme (GBM) is a particularly malignant form of a primary brain tumor, which is characterized by a differentiated histological and cellular system. A characteristic feature of GBM is the pleomorphism of neoplastic cells, which can build various low-differentiated forms, such as e.g. multinucleated giant cells, as well as more mature gemistocytes or fibrillary astrocytes. As a consequence, in glioblastoma multiforme there are cells of different size, shape and degree of differentiation. GBM rapidly spreads to the surrounding parts of the brain because of its increased proliferative potential, which causes its aggressive growth and a very unfavorable prognosis. As described in A. Rojek et al. , “Wieloletnie przezycie chorych z glejakiem wielopostaciowym — opisy przypadkow”, Polski Przeglqd Neurologiczny 2015, 12 (2): 107-115, the median survival time of patients with this type of tumor is only 5 months, over 80% of patients die within a year of diagnosis and only about 3-8% of patients survive 2 years.

[4] Currently, the treatment of patients with GBM involves surgical excision of the tumor and then undergoing chemotherapy or radiation therapy or radiation therapy in combination with chemotherapy.

[5] Chemotherapy with the use of drug temozolomide (TMZ) has been used for years. As described in, inter alia, Sang Y. Lee “Temozolomide resistance in glioblastoma multiforme”, Genes & Diseases (2016) 3, 198-210, TMZ is a well-tolerated oral agent which passes in an intact form through the blood-brain barrier. However, the resistance to TMZ is a serious problem in the treatment of malignant tumor glioma cells, since at least 50% of patients treated with TMZ do not respond to this treatment. The publication also discloses that combination therapy with the use of both temozolomide and radiation improves the overall survival of patients compared to the therapy of patients treated with radiation alone. To treat patients with GBM, the usual dose of temozolomide is 75 mg/m2 daily for 6 weeks, combined with 60 Gy focal radiation therapy, followed by 6 cycles, in which 150 mg/m2 is administered once daily for 5 days in a row, with a 23-day rest period before the next cycle.

[6] As the traditional therapeutic strategy is often associated with significant toxicity and limited efficacy, new treatment methods are still being investigated. In recent years, particularly attractive opportunities have been offered by combining traditional methods with nanotechnology-based methods, through the use of metal nanoparticles in drug delivery systems.

[7] For example, patent application PL408781 A1 discloses the use of platinum metal nanoparticles for the manufacture of a drug intended for the treatment of central nervous system tumors, particularly glioma. The drug, being in the form of a colloid, is intended to be administered by direct injection into the tumor and/or into the immediate vicinity of the tumor and/or into the blood.

[8] The publication of the international application WO2012089768 A1 discloses a system for selective and controlled release of a therapeutic agent, which is intended, inter alia, for the treatment of tumors. The system comprises a carrier in the form of metal nanoparticles with a hydrophilic coating, the carrier being bonded with at least one therapeutic agent. The nanoparticles are selected from the group comprising Au, Ag, Pt, Co, Fe, oxides thereof, Ti02 and mixtures thereof and preferably the nanoparticles are gold nanoparticles with a size between 5 and 30 nm. The hydrophilic coating consists of hyaluronic acid oligomers and the therapeutic agent is selected from the group comprising anti-inflammatory agents, antibodies, antibiotics, analgesic agents, antineoplastic agents, immunotherapeutic agents and combinations thereof. Numerous compounds such as cisplatin, streptozotocin, dacarbazine, temozolomide, bleomycin, mitomycin, paclitaxel and many others are used as antineoplastic agents. As demonstrated by the description of the invention, radiation therapy can enhance the therapeutic effect of the system in the treatment of tumors.

[9] K. Kalimuthu et al. , “Gold nanoparticles stabilize peptide-drug-conjugates for sustained targeted drug delivery to cancer cell”, J Nanobiotechnol (2018) 16:34, disclose systems of peptide-drug conjugates intended to deliver drugs to carcinoma cells based on the example of lymphoma. Because of the low stability of peptides in the blood, they were linked to gold nanoparticles as a stabilizer. The nanoparticles were coated first with citrate, then polyethylene glycol (PEG) and then with polypeptide-drug conjugate, the drug being, inter alia, chlorambucil, melphalan and bendamustine. The gold nanoparticles were prepared from a solution of gold chloride which was added to deionized water and heated to boiling, then sodium citrate was added and after the solution turned dark red it was allowed to cool down to room temperature and the gold nanoparticles were separated from water and the rest of the substances dissolved in water by centrifugation. They were then combined with PEG, which replaced the citrate on the surface of the gold nanoparticles. In the final step, the polypeptide-drug conjugate was added and whole mixture was stirred for 30 minutes at room temperature.

[10] From M. U. Farooq et al., “Gold Nanoparticles-enabled Efficient Dual Delivery of Anticancer Therapeutics to FleLa Cells, Scientific Reports” (2018) 8:2907, a system is known intended for the treatment of cervical neoplasm, containing gold nanoparticles with a spherical shape and a diameter of about 13 nm, with PEG as a linking layer, the nanoparticles being functionalized with bleomycin and doxorubicin as chemical therapeutic agents.

[11] A. Orza et al., “Reversing chemoresistance of malignant glioma stem cells using gold nanoparticles”, Int J Nanomedicine. 2013; 8: 689-702, describe studies where aspartate-stabilized gold nanoparticles were used to bind temozolomide to form a desired final drug delivery system intended for the treatment of glioma. The conducted studies showed that the system of gold nanoparticles - L-aspartic acid - TMZ gave better results than TMZ alone. The gold nanostructures were in the shape of triangles of about 55 nm and were synthesized by reducing the gold salt of FIAuCI4 in a strong acid medium (pH=2) in the presence of L-aspartic acid molecules, under continuous stirring at room temperature for 24 hours. The resulting product was then centrifuged and purified with water. TMZ was added to the product and stirred for 1 hour.

[12] Further, J. Verma “Delivery and cytotoxicity of doxorubicin and temozolomide to primary glioblastoma cells using gold nanospheres and gold nanorods”, Eur. J. Nanomed. 2016; 8(1): 49-60, describes silica coated gold nanoparticles of an elongated shape, whose length was 49-65 nm and diameter was 8.5-14 nm, and gold nanoparticles in the shape of nanospheres of a diameter of 9.5-37 nm, which were functionalized with doxorubicin or temozolomide. Their antitumor effects, especially towards glioma cells, was also disclosed.

[13] On the other hand, as described in J. Rzeszutek et al. , “Zastosowanie nanocz¾stek i nanomaterialow w medycynie”, Hygeia Public Health 2014, 49(3): 449-457, despite their many advantages, gold nanoparticles may exhibit toxic effects towards normal, healthy tissues, which is influenced by their size, shape and surface modifications.

[14] For example, in Schleh C. et al. , “Size and surface charge of gold nanoparticles determine absorption across intestinal barriers and accumulation in secondary target organs after oral administration”, Nanotoxicology 2012;6(1):36-46, the authors described their studies with rats which were administered gold nanoparticles of different size and charge by the esophagus. Particles with a diameter of 1.4 nm, 5 nm, 18 nm, 80 nm or 200 nm and a diameter of 2.8 nm and a positive or negative charge were used. The animals were tested after 24 hours. Generally, the largest accumulation in the secondary organs was observed with the smallest 1.4 nm particles. However, most particles with a diameter of 18 nm were detected in the brain and heart.

[15] Sun Y.N. et al., “Shape dependence of gold nanoparticles on in vivo acute toxicological effects and biodistribution”, J. Nanosci. Nanotechnol. 2011, 11 (2): 1210—1216, investigated the influence of the shape of gold nanoobjects on toxic effects in mice injected with a single dose of gold nanoparticles in the shape of spheres, cubes or rods and then their distribution in the liver, spleen, lungs and kidneys was observed. The rod-shaped nanogold produced the most toxic effects, while the spherical gold nanoparticles had the mildest effect. [16] Based on the prior art analysis, despite clinical and technological advances, there is still no effective method of treating glioblastoma multiforme because of its being diffuse and its infiltrating growth pattern hindering complete surgical resection. Standard care for patients diagnosed with a highly malignant glioma includes post-operative treatment with temozolomide and the use of radiation therapy. Unfortunately, this therapeutic strategy is associated with significant toxicity and limited efficacy. This is because malignant glioma stem cells are resistant to chemotherapeutics and ionizing radiation due to, inter alia, their high ability to repair DNA damage. Therefore, more complex, advanced and targeted treatment methods for glioblastoma multiforme are sought, with ongoing research in this area.

[17] Due to their beneficial properties, gold nanoparticles show great application possibilities in drug delivery systems. The functionalization of their surfaces enables them to be used in the area of tumor cell control. However, in the prior art methods of linking gold nanoparticles with antitumor drugs, an intermediate linking layer has usually been used, such as e.g. silica, oligomers of hyaluronic acid or polyethylene glycol, which hermetically covers the entire surface of gold nanoparticles. Thus, despite their therapeutic properties, gold nanoparticles are usually regarded primarily as a carrier of the drug they are coated with.

[18] In addition, the shapes and sizes of gold nanoparticles and surface modifications clearly influence their biological effect, absorption, distribution or interaction with tumor cells and what is more, particles of some shapes can also be toxic to normal tissues.

Summary of Invention

[19] The object of the invention is to develop a method of making a system comprising gold nanoparticles functionalized with an antitumor drug, with the effect of the drug in the said system being further enhanced by the behavior of the nanoparticles themselves. The use of the system together with radiation therapy will enable the treatment of glioblastoma multiforme to be more effective than before, with lower drug doses used in combination therapy and a reduction in side effects of therapy.

Technical Problem [20] The aim of the invention is to provide an alternative drug delivery system which enable the treatment of neoplasm, in particular glioblastoma multiforme.

Solution to Problem

[21 ] The essence of the method of making a system comprising gold nanoparticles, consisting in making gold nanoparticles by chemical reduction of gold ions dissolved in water and functionalizing their surface with antitumor drug temozolomide, through a linking substance, is that gold nanoparticles are prepared in a two-step process, wherein a solution containing gold nuclei is obtained in the first step in such a way that to an aqueous solution of hexadecyltrimethylammonium bromide CTAB at a concentration of 4.5x10 ^-3 - 5.5x1 O ³ mM, heated to a temperature of 25-35°C, is added an aqueous solution of chloroauric acid HAuCU at a concentration between 0.46 mM and 0.50 mM, in a 1:1 volume ratio. Subsequently, after a clear mixture is obtained, an aqueous solution of sodium borohydride NaBhU, cooled to a temperature of 4-8°C and at a concentration between 0.10 and 0.15 mM, is added to the mixture in such a volume ratio that for 0.9-1.0 of the mixture of CTAB and HAuCU there is 0.06-0.07 NaBH4 and the mixture is stirred for another 30-60 minutes from the moment the solution changes its color to pink/ violet. The stirring is carried out at a temperature of 25- 35°C at a speed of 500-700 rpm. In the second step, gold nuclei obtained in the first step grow in such a way that to an aqueous solution of CTAB at a concentration of 4.5x10 ³ -5.5x1 O ³ mM, heated to a temperature of 25-35°C, is added a solution of silver nitrate AgNCb at a concentration of 3.97-4.00 mM, in such a volume ratio that for 0.9-1.0 CTAB there is 0.04-0.05 AgNCb. After thorough stirring, an aqueous solution of HAuCU at a concentration of 0.46-0.50 mM is added to this mixture in such a volume ratio that for 0.9-1.0 of the mixture of CTAB and AgNCb there is 0.96-1.00 HAuCU and the stirring is carried out further and then an aqueous solution of ascorbic acid CeHsCte at a concentration of 78.70-79.00 mM is added in such a volume ratio that for 0.9-1.0 of the mixture of all previous components there is 0.014-0.017 of CeHeOe. Subsequently, a solution containing gold nuclei obtained in the first step is introduced to the whole mixture, in such a volume ratio that for 0.9-1.0 of the whole mixture there is 0.0030-0.0035 of the solution containing gold nuclei and the mixture is stirred for at least 3 hours from the moment the solution changes its colour to pink/ violet, at a temperature of 25-35°C, at a speed of 500- 700 rpm, wherein at least/up to 1 % of the volume of the solution is drawn off within 3 hour, to afford a suspension of gold nanoparticles, which in at least 65% by weight exhibit essentially the shape of Cassini oval, of a size of 55-65 nm along x- axis and 24-34 nm along y-axis and the width of the narrowing in the middle part of the oval is 22-28 nm. The obtained shape of the nanoparticles results from the conducted method of synthesis. The addition of CTAB causes it to bind to the surface of the gold nanoparticles and reduces their energy. This makes it possible to control the evolution of the shape and growth of the nanoparticles. However, the obtained shape is most heavily influenced by the final step in which part of the solution is drawn off according to a non-obvious procedure. The solution contains Ag ⁺ silver ions which are responsible for depositing gold atoms in the most energy- efficient place, i.e. along the crystallographic plane {111}. The gradual removal of the solution and the Ag ⁺ ions contained therein causes the gold atoms to begin to deposit on the plane {111} and so the nanoparticle begins to expand in the lateral direction. The resulting suspension of the gold nanoparticles, after prior 10- to 10,000-fold dilution with water, is mixed with the linking substance, being 16- mercaptohexadecanoic acid MHDA at a concentration of 5 mM, in such a volume ratio that for 0.9-1 .0 of the suspension of the gold nanoparticles there is 0.05-0.06 MHDA and left for 20-24 hours at a temperature of 4-8°C, then the whole mixture is centrifuged to remove the excess MHDA and washed with dimethylformamide at a concentration of 20-21 mM. Subsequently, the reaction mixture is added in such a volume ratio that for 0.5-1.0 of the suspension of the nanoparticles and MHDA there is 0.5-1 .0 of the reaction mixture. The reaction mixture activates the carboxyl group in MHDA. The reaction mixture consists of solutions of: carboxymethylcellulose at a concentration of 20-21 mM, phosphoenolpyruvate at a concentration of 20-21 mM, ethyldiisopropylamine at a concentration of 20-21 mM and dimethylformamide at a concentration of 20-21 mM all used in a 1 : 1 : 1 :2 molar ratio. The whole mixture is left at room temperature for a period of at least 30 minutes. After this time, the mixture is centrifuged and washed with dimethylformamide and antitumor drug temozolomide TMZ in a concentration of 1 - 20 mM is added in a 1 :1 volume ratio in relation to the previously obtained diluted suspension containing the gold nanoparticles. The mixture is left for a period of 25- 40 minutes at room temperature, then centrifuged at a speed of 10,000-14,000 rpm and washed with distilled water at least 2 times. In the final step, a solution of N- benzoyl-L-arginine ethyl ester at a concentration of 100 mM, dissolved in a 1:1 molar ratio in a carboxyl buffer of pH=7-9 is added and the mixture is centrifuged at a speed of 10,000-14,000 rpm. The addition of the latter components blocks TMZ binding sites, thereby preventing the adjacent TMZ molecules from linking with each other, both within the same gold nanoparticle and adjacent nanoparticles, which could result in the loss of their therapeutic properties. The obtained system is suspended in water, sealed and stored under sterile conditions.

[22] The essence of the invention is also the use of the system containing gold nanoparticles and made by the method of the present invention in antitumor therapy, in particular for the treatment of glioblastoma multiforme, in combination with radiation therapy, wherein during the entire therapy, the system containing gold nanoparticles according to the invention is used in the amount of 110 m I for every 100-9,000 cells and the irradiation is carried out to the therapeutic volume after 30 minutes to 24 hours from administering the system, for a period of 15 seconds to 3 hours.

[23] Preferably, the radiation therapy is teleradiotherapy with MV X-rays of energy of 3-25 MeV or with electron beams of energy of up to 50 MeV or with gamma radiation or X-rays of maximum energy of 300 kVp or with proton beam of energy of up to 250 MeV.

[24] Also, the antitumor therapy according to the invention may be used in combination with brachytherapy with gamma radiation or X-rays of maximum energy of 300 kVp, within the fractional dose range of 0.15-15.00 Gy, with a total dose of up to 80 Gy, with different fractionation regimens considered.

[25] Preferably, the single fraction irradiation is carried out, using X-rays at a dose of 2 Gy or proton radiation at a dose of 2 Gy.

[26] Because of the characteristic heterogeneity of glioma cells, the gold nanoparticles prepared by the method of the invention due to their unique irregular shape with the outline of Cassini oval, i.e. rod-shaped with a recess in the middle part and spherical at the ends, which is exhibited by at least 65% by weight of all nanoparticles obtained, can more effectively affect this type of tumor cells and lead to their death effectively as compared to the previously used only spherical or rod shaped nanoparticles, with the least toxicity of the nanoparticles in the body being maintained at the same time. Their shape and size also enable them to easy penetrate the blood-brain barrier, which is extremely important for the treatment of brain glioma. A particularly important role in the treatment is played by the recess formed in the central part of the oval of the gold nanoparticles during the synthesis and the size of the recess may correspond to the major DNA groove of tumor cells, which increases the likelihood that DNA-nanoparticle-drug adducts will form and thus increases the genotoxic effect of this nanosystem leading in consequence to the death of tumor cells.

[27] Additionally, the use of the CTAB solution as a stabilizer, which coats the gold nanoparticles, prevents their agglomeration during storage and application. This mechanism is based on a complex between Br - and Ag ⁺ ions being formed, which changes the reactivity of the surface of the nanoparticles and affects their growth process.

Advantageous Effects of Invention

[28] An important advantage of the whole system made by the method according to the invention is the fact that the use of 16-mercaptohexadecanoic acid as the linking substance between the gold nanoparticles and antitumor drug TMZ does not block the surface of the nanoparticles and does not separate the said surface from the biological environment, as is the case in the previously used systems. This is because MHDA does not completely cover the entire surface of the nanoparticles, but links pointwise with their surface with its one end to the thiol group -SH and with its other end, being a carboxyl group, with TMZ. In this way the gold nanoparticles in the uncovered places can come into direct contact with biological membranes and enhance the treatment process and additionally interact with ionizing radiation. Due to the high value of the atomic number Z=79, the gold nanoparticles are an excellent factor enhancing radiation therapy, including in particular proton radiation therapy. The main advantage of the proton radiation therapy is the very high precision with which the beam of radiation reaches the tumor. This is due to the physical properties of the protons themselves (Bragg peak) and because of the favorable dose distribution in the tissues.

[29] Thus, there is a triple effect of the system, namely the effect of antitumor drug TMZ, the effect of the gold nanoparticles themselves in the unmasked areas where the drug is not attached and the effect of the proton radiation therapy, which is additionally enhanced by the effect of the gold nanoparticles in the uncovered places.

[30] The use of the system improves the efficacy of antitumor therapies and reduces side effects on normal cells, compared to the previously used antitumor therapies because of it being possible, inter alia, to adjust the target dose of proton radiation to the volume of the tumor, which is known e.g. from M. Hu et al. , “Proton beam therapy for cancer in the era of precision medicine”, J Hematol Oncol. 2018; 11: 136, as well as to reduce the doses of the drug administered to patients in relation to those used currently, i.e. in the amount of 20-140 mM, as indicated, inter alia, in J. Gaspar et al., “Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts”, Neuro Oncol. 2009 Jun; 11(3): 281-291. As a result, the duration and cost of such a therapy may also prove to be lower.

[31 ] It can also be assumed that the gold nanoparticle systems made by the method according to the present invention in combination with other drugs will be active against other types of tumors by the use of combination therapy and in particular when combined with the proton radiation therapy.

Brief Description of Drawings

[32] The method of making a system comprising gold nanoparticles functionalized with temozolomide and the use of this system for the treatment of glioblastoma multiforme is explained below in practical embodiments of the invention and in the drawing.

[33] Fig. 1 shows an image of the gold nanoparticles obtained by a scanning transmission electron microscope.

[34] Fig. 2 shows a diagram of the process of functionalizing the surface of the gold nanoparticles with antitumor drug TMZ, through the linking substance MHDA.

[35] Fig. 3 shows FT-Raman spectra for TMZ (a), for MHDA (b), for the gold nanoparticles linked to MHDA (c), for the system containing the gold nanoparticles linked to MHDA and TMZ (d).

[36] Fig. 4 shows graphs of temperature stability of the system as a function of time: TGA (a) and DSC (b). [37] Fig. 5 shows electrophoretic separation and assessment of the integrity of RNA isolated from U 118 and U251 glioma cell lines irradiated with protons.

[38] Fig. 6 shows electrophoretic separation and assessment of the integrity of RNA isolated from U118 and U251 glioma cell lines treated successively with TMZ, gold nanoparticles and the system containing gold nanoparticles and TMZ and then irradiated with protons.

[39] Fig. 7 shows demonstrative photographs of U251 line cells (400x magnification) treated with TMZ (b), gold nanoparticles (c), the system containing gold nanoparticles and TMZ (d), as well as additionally irradiated with protons (f-h) and an image of the control sample of normal U251 line cells (a) and of the latter irradiated with protons (e).

Description of Embodiments

[40] The examples shows the method of preparation of gold nanoparticles and use thereof.

Examples

[41] Example 1

[42] The gold nanoparticles were prepared in a two-step process. In the first step, gold nuclei of a spherical shape were obtained and in the second step, these nuclei grew to form nanopeanuts.

[43] Step 1 - obtaining gold nuclei

The following solutions were prepared:

[44] A - a solution of hexadecyltrimethylammonium bromide CTAB at a concentration of 5x10 ³ mM, obtained in such a way that 0.364 g of CTAB surfactant was weighed and then dissolved in 5 ml of water under slight heating to a temperature of 30°C until a transparent solution was obtained.

[45] B - a solution of chloroauric acid FIAuCU at a concentration of 0,46 mM, obtained in such a way that 0.0085 g FIAuCU was weighed and dissolved in 5 ml of distilled water.

[46] C - a solution of sodium borohydride NaBFU at a concentration of 0.1 mM, obtained in such a way that 0,0038 g NaBFU was weighed and then dissolved in 10 ml of water and immediately placed in a refrigerator to reduce its temperature to 4°C.

[47] 5 ml of solution B was added to 5 ml of solution A and stirred thoroughly and after 3 minutes with a clear mixture obtained, 0.6 ml of solution C was added and stirred further for 15 minutes until the solution changed its color to pink/ violet and then for another 30 minutes. Stirring was carried out on a heating plate with a magnetic stirrer, at a temperature of 30°C with a stirring speed of 700 rpm.

[48] Step 2 - growth of gold nanopeanuts

Fresh solutions A and B were prepared again and in addition:

[49] D - a solution of AgNCb at a concentration of 3.97 mM, obtained in such a way that 0.0135 g AgNCb was weighed and then dissolved in 20 ml of distilled water.

[50] E - a solution of ascorbic acid CeFIsCteat a concentration of 78.7 mM, obtained in such a way that 0.1386 g of ascorbic acid was weighed and then dissolved in 10 ml of distilled water.

[51 ] 0.2 ml of solution D was added to 5 ml of solution A and stirred thoroughly and then after 3 minutes, 5 ml of solution B was added under continuous stirring and then after another 3 minutes, 70 pi of solution E was added. After a clear mixture was obtained, 30 mI of the solution containing gold nuclei obtained in step 1 was introduced into the mixture and further stirred for 5 minutes until the solution changed its color to pink and violet and then stirred for another 3 hours. Stirring was carried out on a heating plate with a magnetic stirrer, at a temperature of 30°C with a stirring speed of 700 rpm. After successively 1 minute, 15 minutes, 30 minutes and 1 hour 0.25% of the solution volume was drawn off each time.

[52] A suspension of gold nanoparticles was obtained, which in at least 65% by weight exhibited the shape of Cassini oval, of a size of 55 - 65 nm along x-axis and 24 - 34 nm along y-axis and the width of the narrowing in the middle part of the oval was 22 - 28 nm.

[53] The morphology of the obtained gold nanoparticles was tested by means of scanning transmission electron microscopy (STEM) using a high angle annular dark field detector, in conventional mode and at high resolution. FEI Titan electron microscope, operating at a voltage of 300 kV and equipped with a FEG cathode, was used for the test. The obtained STEM image of the nanoparticles is shown in Fig. 1.

[54] Subsequently, 1 ml of the resulting suspension of the gold nanoparticles, after prior 1,000-fold dilution with water, was mixed with 50 pi of 16- mercaptohexadecanoic acid MHDA at a concentration of 5 mM and left for a period of 24 hours at a temperature of 4°C.

[55] After this time, the excess MHDA was centrifuged, the nanoparticles were washed with a solution of dimethylformamide DMF at a concentration of 20 mM and centrifuged again and then 100 mI of a reaction mixture was added consisting of solutions of: carboxymethylcellulose at a concentration of 20 mM, phosphoenolpyruvate at a concentration of 20 mM, ethyldiisopropylamine at a concentration of 20 mM and DMF at a concentration of 20 mM, all mixed in a 1 : 1 : 1 :2 molar ratio. The whole mixture was left at room temperature for a period of 30 minutes, then centrifuged, washed with DMF solution and centrifuged again until DMF was completely removed.

[56] In the final step, 1 ml of TMZ at a concentration of 5 mM was added to the mixture and this was left at room temperature for 30 minutes, then centrifuged for 10 minutes at a speed of 14,000 rpm and washed twice with distilled water and then a solution of N-benzoyl-L-arginine ethyl ester at a concentration of 100 mM, dissolved in a 1:1 molar ratio in a carboxyl buffer of pH=8 was added and the mixture was centrifuged under the same conditions as before and the filtrate was removed. The obtained system was suspended in water, sealed and stored under sterile conditions. The process of functionalizing the surface of gold nanoparticles is shown schematically in Fig. 2.

[57] To verify the functionalization process, the obtained system was tested using FT-Raman Nicolet NXR 9650 spectrometer equipped with a Nd: YAG (1064 nm) laser and a germanium detector. The measurements were taken in the range from 150 to 3700 cm ¹ at a laser power of 1 W. A unfocussed laser beam with a diameter of about 100 pm and a spectral resolution of 8 cm ^-1 was used. Each individual spectrum was collected using 128 scans. Raman spectra were analyzed using OPUS 7.0.129 software. The obtained results are shown in Fig. 3, in which the FT- Raman spectrum of pure TMZ (a), pure MHDA (b), nanoparticles linked to MHDA (c) and the gold nanoparticles linked with MHDA and TMZ (d) was marked. MHDA is a chemical compound which has a thiol group -SH at one end of the chain and a carboxyl group -COOH at the other end. Fig. 3 indicates Raman shifts for these two groups. The oscillation of the thiol group is at the Raman shift of 2900 cm ¹ . On the other hand, the oscillation at the Raman shift of 1680 cm ^-1 is characteristic of group C=0. Signals from the aforementioned functional groups can be seen in the FT-Raman spectrum characteristic of pure MFIDA, while in the spectrum for gold nanoparticles linked to MFIDA, no signal coming from the thiol group was observed, which confirms that MFIDA linked through the thiol group to the surface of gold nanoparticles. By contrast, in the spectrum for gold nanoparticles linked with MFIDA and TMZ, no oscillation was observed either coming from the — SFH group or from the C=0 group. This confirms that MFIDA linked with its other end with the antineoplastic drug TMZ.

[58] The temperature stability of the obtained system was also tested and the results are shown in Fig. 4. The measurements were taken using DSC 2500 differential scanning calorimeter from TA Instruments equipped with LN2P liquid nitrogen pump. Samples weighing about 1 -3 mg were placed in aluminum dish and clamped with airtight lids. The thermal behavior of the samples was tested under dry purging with pure nitrogen N5.0 (25 ml/min) at a temperature range of 10°C to 200°C, at a heating rate of 5°C and 10°C/min and at a cooling rate of 5°C/min. TRIOS software was used to calculate peak temperatures and enthalpy values of the recorded thermal events. The results of the TGA (a) and DSC (b) experiments indicate that the system remains thermally stable up to a temperature of 100°C at a heating rate of 5°C/min. The shape of the TGA signal indicates strong solvent evaporation from the liquid sample, visible between about 68°C (5% mass loss) and 119°C (59.7% mass loss) followed by its chemical decomposition at a temperature range of 120°C to 138°C (mass loss 40.03%). The DSC thermogram shows no significant thermal anomalies up to 110°C. Anomalies recorded above this temperature, as well as the shift of the baseline, are the result of rapid boiling and decomposition of the sample or destabilization of MFIDA.

[59] Example 2 The system made by the method described in Example 1 was applied to two glioblastoma multiforme cell lines classified as grade IV of malignancy by World Health Organization (WHO):

[60] 1 ). U 118 MG (ATCC® HTB-15TM) purchased from The American Type Culture

Collection (ATCC);

[61 ] 2). U 251 MG, purchased from Public Health England (PHE) Culture Collections, cat. no. 09063001.

[62] U 118 MG cells were cultured according to ATCC procedure. Cells from the passage in the range 9-11 were used for seeding.

[63] U 251 MG cells were cultured according to the procedure attached by PHE. Cells from the passage in the range 7-8 were used for seeding.

[64] Cells were seeded in the amount of 2-3 thousand cells for each 200 pi of complete culture medium consisting of Dulbecco’s Modified Eagle’s Medium (ATCC, cat. no. 30-2002) in a 9:1 ratio to foetal serum (Sigma-Aldrich, cat. no. F9665). Cells were cultured at a temperature of 37°C, under a 5% CO2 atmosphere.

[65] 72 hours after the culture was established, the culture medium was exchanged with fresh medium and then 110 mI of the system, containing TMZ at a concentration of 5 mM and the gold nanoparticles in the form of a suspension at a concentration of 0.01487 mM, was added to each individual culture having 6-9 thousand cells.

[66] 2 hours after the system was added, the cells were irradiated. A single 1 -minute irradiation session was given to: one part with a beam of X-rays with maximum energy of 250 kVp at a dose of 2 Gy and the other part with a proton beam with energy of 70 MeV, also at a dose of 2 Gy.

[67] X-ray irradiations were carried out using an X-ray instrument with MCN 323 lamp from Philips, the apparatus operating at a voltage of 250 kVp, at an intensity of 10 mA, with a 2.5 mm thick filter. The distance from the lamp to the irradiated samples was 21 cm. Doses were determined using PTW Freiburg UNIDOS dosimeter with an ionization chamber of 0.125 cm ³ at 50cGy/min.

[68] Proton beam irradiation was carried out at the Bronowice Cyclotron Centre, at the Institute of Nuclear Physics in Krakow, at a gantry station enabling proton beam irradiation with the use of the Proteus C-235 cyclotron (IBA PT, Louvain-la-Neuve, Belgium), using scanning technique. A monoenergetic field with energy of 70 MeV and dimensions of 15 cm x 15 cm was used. The irradiation was carried out with water equivalent thickness of 1.1 cm.

[69] Fig. 5 shows electrophoretic separation and assessment of the integrity of RNA (RIN - RNA Integrity Number) isolated from U118 and U251 glioblastoma multiforme cell lines irradiated with 2 Gy proton radiation and non-irradiated cells (control), 3 hours after the end of irradiation. RNA isolation was made in accordance with QIAGEN protocol, RNeasy Mini Kit, cat. no. 74104. RNA integrity was measured from 1.5 pi sample at wavelengths of 260 nm and 280 nm using Tecan SPARK 10M spectrophotometer.

[70] The method used makes it possible to determine total RNA on a scale from 1 - 10, where 1 is the most degraded RNA and 10 is the best quality RNA. Cell lines are numbered from 1, starting from the left and “nt” symbol in the graph is the number of nucleotides.

[71 ] The following results were obtained Line 1 - fragment length standard

Line 2 - U 118 line, no irradiated (control) RIN 9.8

Line 3 - U118 line + proton radiation RIN 9.2

Line 4 - U251 line, no irradiated (control) RIN 9.3

Line 5 - U251 line + proton radiation RIN 9.8

[72] The obtained RIN values indicate no clear degradation of the genetic material, both for the control and glioma cells treated with proton radiation alone. This demonstrates that applying radiation therapy alone to this type of tumor cells damages the genetic material with little effectiveness.

[73] Fig. 6 shows electrophoretic separation and assessment of the integrity of RNA (RIN) isolated from U118 and U251 glioblastoma multiforme cell lines treated successively with TMZ, gold nanoparticles and the system and then irradiated with 2 Gy proton radiation. [74] The assessment was made 3 hours after the end of irradiation. Cell lines are numbered from 1 , starting from the left and “nt” symbol in the graph is the number of nucleotides.

[75] The following results were obtained:

Line 1 - fragment length standard

Line 2 - U118 line, TMZ + proton radiation RIN 8.4

Line 3 - U 118 line, gold nanoparticles + proton radiation RIN 5.3 Line 4 - U118 line, the system + proton radiation RIN 3.4

Line 5 - U251 line, TMZ + proton radiation RIN 9.1

Line 6 - U251 line, gold nanoparticles + proton radiation RIN 10.0 Line 7 - U251 line, the system + proton radiation RIN 5.0

[76] The obtained RIN values indicate the effect of the system together with proton radiation, leading to RNA degradation for both glioma cell lines tested, thus preventing further research in the field of gene expression. This is of great importance to therapy, as the system in combination with radiation is effective towards different types of glioma. As opposed to the use of the system, the use of TMZ alone at a concentration of 5 mM together with proton radiation did not lead to such drastic changes in RNA integrity. Also, the gold nanoparticles alone together with proton radiation led to disintegration only for the U 118 line and were not effective with U 251 line.

[77] Table 1 shows the percentage of viable cells with standard deviation obtained for U251 and U 118 glioma lines irradiated with 2Gy dose of X-ray and the same dose of proton radiation. Cell viability measurement was taken 48 and 72 hours after the end of irradiation. After 24 hours, there were no significant changes in cell viability with respect to control. Assays for cytotoxicity were performed based on the quantitative method of determining cell metabolic activity - MTT test (In Vitro Toxicology Assay Kit, MTT based, Sigma Aldrich, cat. no. TOX1-1KT). Tecan SPARK 10M plate reader was used for reading. [78] Table 1

[79] The obtained results show that radiation alone does not cause drastic changes in the viability of glioma cells even after 48 and 72 hours. Proton radiation was most effective with U 118 line but caused the highest mortality only for about 20% of cells after 72h.

[80] Table 2 shows the percentage of viable cells with standard deviation obtained for U251 and U 118 glioblastoma multiforme lines. The cells were treated successively with TMZ, gold nanoparticles and the system and then irradiated with 2 Gy dose of X-ray or 2 Gy dose of proton radiation. Cell viability measurement was taken 24 hours after irradiation and 26 hours after the administration of TMZ or the gold nanoparticles or the system, without irradiation. Assays for cytotoxicity were performed based on the quantitative method of determining cell metabolic activity - MTT test (In Vitro Toxicology Assay Kit, MTT based, Sigma Aldrich, cat. no. TOX1-1KT). Tecan SPARK 10M plate reader was used for reading.

[81] Table 2

[82] The obtained values clearly confirm that the system alone causes a decrease in cell viability for both tested GBM cell types. Ionizing radiation improves the effects of the system and the largest number of tumor cells killed is observed when the system is used in combination with proton radiation. The weakest effect was observed for the currently used therapy, i.e. the combination of TMZ with radiation therapy or TMZ alone.

[83] Fig. 7 shows demonstrative photographs of U251 line cells taken with Leica DMi8 microscope at 400x magnification 24 hours after the end of cell irradiation. The image of normal cells is shown in Fig. 7a. The cells were treated successively with TMZ (Fig. 7b), gold nanoparticles (Fig. 7c) and the system (Fig. 7d). Photographs were also taken after irradiation of the cells with 2 Gy dose of proton radiation, 2 hours after the administration of the above-mentioned substance. Fig. 7f shows an image of the cells treated with TMZ and radiation, Fig. 7g shows the cells treated with the gold nanoparticles and radiation, Fig. 7h shows the cells treated with the system and radiation and Fig. 7e shows the cells treated with radiation alone (control).

[84] With the system used (Fig. 7d and Fig. 7h), a characteristic change in the morphology (shape) of the cells can be seen, which are not flattened and do not stick to the basis as normal cells (Fig. 7a) but are detached from the substrate because of the cytotoxic and genotoxic action. They formed a suspension and changed their shape to a round one. This confirms the synergistic effect of the system which led directly to the killing of GBM tumor cells.