Dual drug-containing PH-sensitive polymeric nanoconjugate and synthesis method thereof

Abstract

The invention relates to a dual drug loaded, targeted polymer drug nanoconjugate affecting multiple signaling pathways for use as a drug delivery system in the treatment of ovarian cancer. Said nanoconjugate comprises Cabozantinib (CBZ) as a VEGFR (Vascular endothelial growth factor receptor) inhibitor and Ki16425 as a LPAR (Lysophosphatidic acid receptor) 1,2,3 inhibitor. In the nanoconjugate of the invention, synergistic effect is obtained with dual drug use.

Description

[0001] DUAL DRUG-CONTAINING PH-SENSITIVE POLYMERIC NANOCONJUGATE AND SYNTHESIS METHOD THEREOF

[0002] Technical Field of the Invention

[0003] The invention relates to a dual drug loaded, targeted polymer drug nanoconjugate affecting multiple signaling pathways for use as a drug delivery system in the treatment of ovarian cancer. Said nanoconjugate comprises Cabozantinib as a VEGFR(Vascular endothelial growth factor receptor) inhibitor and Ki16425 as a LPAR (Lysophosphatidic acid receptor) 1 ,2,3 inhibitor, and synergistic effect is achieved with dual drug use.

[0004] State of the Art

[0005] The ovary is part of the female reproductive system and is the organ where eggs are produced and hormones related to the menstrual cycle and fertility are released. Ovarian cancer is caused by abnormal growth and proliferation of ovarian cells and is the most lethal gynecologic malignancy, accounting for 2.5% of all female cancers and 5% of female cancer-related deaths. Globally, ovarian cancer is the seventh most common cancer in women and the eighth most common cause of cancer-related deaths, with five- year survival rates of less than 45%. Around 140,000 women worldwide die from ovarian cancer each year. Ovarian cancer patients have high mortality rates due to late stage (III or IV) diagnosis.

[0006] Treatment for ovarian cancer is determined depending on the stage and type of cancer, the patient's general health, and other factors. Treatment of ovarian cancer usually involves surgery, chemotherapy, radiotherapy, or a combination of these. Surgery involves removal of the ovaries, fallopian tubes, and uterus (hysterectomy), along with removal of lymph nodes and other associated tissues, and is preferably the first line of treatment to prevent the spread of cancer and reduce the size of the tumor. However, surgical intervention carries certain risks. These include bleeding, infection, anesthesia- related complications, and other surgical complications. Furthermore, ovarian cancer surgery involves the removal of the ovaries and sometimes the uterus, which can permanently affect fertility, reducing or completely eliminating the chance of pregnancy. In addition, ovarian removal can cause the hormonal balance to change, and these hormonal changes can trigger menopausal symptoms and affect quality of life. Another treatment option for ovarian cancer is radiotherapy, which uses high-energy rays to destroy or control cancer cells. However, while radiotherapy kills cancer cells, it carries the risk of damaging surrounding healthy tissues. In the state of the art, it involves mass reduction surgery with platinum-taxane maintenance chemotherapy. Following first-line treatment, recurrence occurs in 60-70% of patients when the optimal mass reduction result is <1 cm residual and in 80-85% of patients when >1 cm residual.

[0001] . In addition to all these treatment options, polymer drug nanoconjugate systems in ovarian cancer treatment refer to nano-sized structures formed by binding polymers that function as drug carriers to cancer drugs. These systems can improve therapeutic efficacy by enabling targeted drug release in cancer treatment, while also reducing drug toxicity and minimizing side effects. Since polymers generally have a high drug loading capacity, more drugs can be transported into cancer cells and polymer nanoconjugates provide controlled and prolonged release of the drug, allowing the drug to remain in the bloodstream longer and increase its efficacy. The types of polymers used in said systems vary depending on the design of the nanoconjugate, the properties of the drugs to be transported, and the target to be applied, however generally poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), poly(N-vinyl pyrrolidone) (PVP), polyethyleneimina (PEI), hyaluronic acid (HA) or polyethyleneimine (PEI) are used [2],

[0007] Dysregulated signaling pathways in ovarian cancer refer to the disruption of molecular mechanisms that control the normal cell cycle of cancer cells and regulate intercellular communication. These dysregulated signaling pathways are highly influential in the development and progression of ovarian cancer, and VEGFR and LPA pathways are important signaling pathways that regulate cancer cell growth, metastasis, and tumor formation. VEGFR is a receptor protein that interacts with vascular endothelial growth factor (VEGF). VEGF promotes the formation of new blood vessels, providing the blood and nutrients needed for tumor growth. Solid tumors, such as ovarian cancer, can produce high levels of VEGF, which promotes tumor growth and metastasis. VEGF and its receptor VEGFR are important factors in both physiologic and pathologic angiogenesis that have been identified in ovarian cancer progression and metastasis development. Many studies have shown that VEGF signaling shows very high activity in ovarian cancer and is associated with tumor grade and poor prognosis. Therefore, drugs targeting VEGFR signaling pathways are being investigated in the present art to inhibit tumor growth and improve treatment. LPA is a lipid molecule that regulates intercellular communication and signal transduction and is an essential part of signaling pathways involved in cellular proliferation, migration, survival, vascular homeostasis, stromal remodeling, and immune regulation. LPA triggers cellular responses through LPA receptors and regulates various biological effects. LPA can promote the growth, invasive properties, and metastasis of tumor cells in ovarian cancer and other types of cancer. Therefore, therapeutic strategies targeting LPA pathways are also being studied in the present art to control the spread of cancer cells and tumor growth.

[0008] VEGFR inhibitors block VEGFR signaling pathways to inhibit the growth and spread of cancer cells and tumors. This reduces the ability of tumors to form and feed new blood vessels, reducing the cancer cells' need for oxygen and nutrients. VEGFR inhibitors also prevent tumors from metastasizing and spreading to surrounding tissues. Sunitinib, a VEGFR inhibitor, is a drug that targets VEGFR as well as other receptor tyrosine kinases and is used to treat kidney cancer, gastrointestinal stromal tumors (GIST), and other types of cancer. Sorafenib, another VEGFR inhibitor, is a tyrosine kinase inhibitor that targets VEGFR-2 and VEGFR-3 and is used to treat cancer types such as liver cancer, kidney cancer, and thyroid cancer. Bevacizumab targets VEGF and prevents cancer cells from forming new blood vessels. This reduces tumor growth and metastasis. It is used to treat many types of cancer, including colon cancer, breast cancer, lung cancer, and renal cell carcinoma. Cabozantinib is a targeted kinase inhibitor with anti-ovarian cancer activity and inhibits the growth and spread of cancer cells by targeting receptor tyrosine kinases such as VEGFR-2, MET, AXL, and RET [3]. This drug controls tumor growth and prevents metastasis by preventing cancer cells from forming new blood vessels. It also inhibits cancer cell survival and metastasis by blocking the over-activation of tyrosine kinases such as MET and AXL.

[0009] LPAR inhibitors aim to inhibit the growth, metastasis, and invasion of cancer cells by blocking over-activation of LPA receptors. Therefore, LPAR inhibitors play a potentially important role in cancer treatment. Today, clinical trials and studies on the use of LPAR inhibitors in cancer treatment are still ongoing. More research is needed on the efficacy and safety of such inhibitors in cancer treatment. However, given the importance of LPA and LPA receptors in cancer development, it is contemplated that LPAR inhibitors may play an important role in cancer treatment in the future. BMS-986020 is an LPA inhibitor that selectively inhibits the LPA1 receptor. AM095, another LPAR inhibitor that selectively inhibits the LPA1 receptor, plays a potential role in reducing cancer metastasis and invasion. Furthermore, Ki16425, which can also be used in the treatment of ovarian cancer, is shown in the present art to inhibit LPA1 , LPA2 and LPA3 receptors in a concentration-dependent manner, thereby blocking LPA-induced responses [4],

[0010] Reasons such as the limitations and inadequacies of the present art solutions, the low solubility of drugs used in the treatment of ovarian cancer, their short circulation time, their high toxicity in healthy tissues, the low efficacy of treatment options targeting a single pathway, and the need for high doses of drugs in relation to this, in addition, increased side effects due to high doses, damage to normal tissues alongside the treatment of cancerous tissue, resistance of the patient to existing therapies, and high recurrence rates even if complete remission is achieved have made it necessary to improve the treatment of ovarian cancer and to offer alternative treatments.

[0011] Summary and Objects of the Invention

[0012] The invention describes a dual drug loaded targeted polymer drug nanoconjugate affecting multiple signaling pathways for use as a drug delivery system in the treatment of ovarian cancer. Said nanoconjugate comprises Cabozantinib as a VEGFR(Vascular endothelial growth factor receptor) inhibitor and Ki16425 as a LPAR (Lysophosphatidic acid receptor) 1 ,2,3 inhibitor. In the nanoconjugate of the invention, synergistic effect is obtained with dual drug use.

[0013] The aim of the invention is to provide effective treatment of ovarian cancer. In the nanoconjugate of the invention, a synergistic effect is observed with the simultaneous use of Cabozantinib and Ki 16425 inhibitors. Furthermore, since said inhibitors target two different pathways (VEGF and LPA) in combination and have a simultaneous inhibition effect on these, the nanoconjugate of the invention provides high toxicity in cancer cells.

[0014] An object of the invention is to ensure greater drug release at a tumor acidic pH of the than at physiological pH and low toxicity in healthy tissues. In the invention, thanks to hydrazone and ester bonds that can be cleaved at tumor acidic pH, more drug is released at tumor acidic pH than at physiological pH, thus achieving a more targeted system and reducing the level of toxicity that may occur in healthy tissues. Another object of the invention is to affect ovarian cancer cells at a lower dose compared to free drugs and free combinations thereof. This is achieved by the synergistic effect of Cabozantinib and Ki16425 on each other in the ovarian cancer cell lines studied, and accordingly, it is more effective at a lower dose compared to single use. In A2780 and OVCAR3 cell lines, Ki-PEG-CBZ nanoconjugate was effective at a lower dose compared to single free and combination applications, while only in SKOV3 cell line, the IC50 value of the nanoconjugate was determined to be higher than other cells, compared to single and combined application. This situation was evaluated that the intracellular uptake profile of the nanoconjugate may differ and SKOV3 cell line is more metastatic and invasive than other cells.

[0015] Another object of the invention is to provide a high-resolution drug delivery system with a long residence time in the bloodstream for the treatment of ovarian cancer. PEGylation protects drugs and nanoparticles from aggregation, opsonization and phagocytosis, prolonging the duration of systemic circulation, increasing the stability of drugs, reducing proteolysis and renal excretion. PEGylation alters the physical and chemical properties of the biomedical molecule, such as its conformation, electrostatic binding, and hydrophobicity, leading to an improvement in the pharmacokinetic behavior of the drug. In general, PEGylation increases drug solubility and decreases immunogenicity.

[0016] Description of the Drawings

[0017] Fig. 1.1H-NMR spectrum of PEG (a) and HPEG (b)

[0018] Fig. 2. Comparative FTIR spectrum of PEG and HPEG

[0019] Fig. 3. Graph of CBZ binding efficiency at varying polymer:drug ratios (ns: p > 0.05, **** p<0.0001 )

[0020] Fig. 4.1H-NMR spectrum of CBZ-PEG conjugate

[0021] Fig. 5. Comparative FTIR spectrum of HPEG and CBZ-PEG conjugates

[0022] Fig. 6. Ki16425 binding efficiency at varying polymer: Ki16425 ratios (*: p<0.05, ns: p>0.05)

[0023] Fig. 7. 1 H-NMR spectrum of Ki-PEG-CBZ nanoconjugate

[0024] Fig. 8. Comparative FTIR spectrum of CBZ-PEG and Ki-PEG-CBZ nanoconjugates

[0025] Fig. 9. Hydrodynamic size (a) and zeta potential (b) graphs of the nanoconjugate (Ki- PEG-CBZ nanoconjugate) of the invention Fig. 10. TGA graphs. Temperature dependent mass losses of PEG, HPEG, CBZ-PEG and Ki-PEG-CBZ conjugates

[0026] Fig. 11. SEM images of PEG (a), HPEG (b), CBZ-PEG (c), and Ki-PEG-CBZ (d) conjugates

[0027] Fig. 12. pH and time dependent cumulative drug release graphs of free CBZ and Ki16425 and polymer-bound CBZ and Ki16425

[0028] Fig. 13. Amounts of TNF-a released from RAW 264.7 macrophage cells in response to Ki-PEG-CBZ nanoconjugate compared to free CBZ-Ki 16425 combination

[0029] Fig. 14. Comparison of the intracellular uptake of Ki-PEG-CBZ nanoconjugate with free

[0030] Fig. 18. % viability in HPEG-treated A2780, OVCAR3, and SKOV3 cell lines

[0031] Fig. 19. Dose-effect and conservative isobologram graphs of CBZ and Ki16425 combination in A2780 cell line

[0032] Fig. 20. Dose-effect and conservative isobologram graphs of CBZ and Ki16425 combination in OVCAR3 cell line

[0033] Fig. 21. Dose-effect and conservative isobologram graphs of CBZ and Ki16425 combination in SKOV3 cell line

[0034] Fig. 22. Dose-effect graphs of Ki-PEG-CBZ nanoconjugate in A2780, OVCAR3, and SKOV3 cell lines

[0035] Fig. 23. Apoptosis graphs for the A2780 cell line. (Control, CBZ, Ki 16425, CBZ-Ki16425, Ki-PEG-CBZ)

[0036] Fig. 24. Apoptosis graphs for the OVCAR3 cell line. (Control, CBZ, Ki16425, CBZ+ Ki 16425,+ PEG-CBZ)

[0037] Fig. 25. Apoptosis graphs for the SKOV3 cell line. (Control, CBZ, Ki16425, CBZ+Ki16425,+ PEG-CBZ)

[0038] Fig. 26. Effect graph of CBZ, Ki16425, CBZ+Ki16425, and Ki-PEG-CBZ nanoconjugate on invasion in A2780 cell line (****: p<0.0001)

[0039] Fig. 27. Effect graph of CBZ, Ki16425, CBZ+Ki16425, and Ki-PEG-CBZ nanoconjugate on invasion in OVCAR3 cell line (ns: p>0.05)

[0040] Fig. 28. Effect graph of CBZ, Ki16425, CBZ+Ki16425, and Ki-PEG-CBZ nanoconjugate on invasion in SKOV3 cell line (****: p<0.0001) Fig. 29. Microscope images of migration after CBZ, Ki16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment in A2780 cell line (A: 0 Hours, B: 24 Hours CBZ, C: 24 Hours Ki16425, D: 24 Hours CBZ-Ki16425, E: 24 Hours Ki-PEG-CBZ, F: 24 Hours Control)

[0041] Fig. 30. Microscope images of migration after CBZ, Ki16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment in OVCAR3 cell line for 24 hours (A: 0 Hour, B: 24 Hours CBZ, C: 24 Hours Ki 16425, D: 24 Hours CBZ-Ki16425, E: 24 Hours Ki-PEG- CBZ, F: 24 Hours Control)

[0042] Fig. 31. Microscope images of migration after CBZ, Ki 16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment in OVCAR3 cell line for 48 hours (A: 48 Hours Control, B: 48 Hours CBZ, C: 48 Hours Ki 16425, D: 48 Hours CBZ-Ki16425, E: 48 Hours Ki-PEG-CBZ)

[0043] Fig. 32. Microscope images of migration after CBZ, Ki16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment in OVCAR3 cell line for 72 hours (A: 72 Hours Control, B: 72 Hours CBZ, C: 72 Hours Ki 16425, D: 72 Hours CBZ-Ki16425, E: 72 Hours Ki-PEG-CBZ)

[0044] Fig. 33. Microscope images of migration after CBZ, Ki16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment in SKOV3 cell line for 24 hours (A: 0 hours, B: 24 Hours control, C: 24 Hours CBZ, D: 24 Hours Ki 16425, E: 24 Hours CBZ-Ki16425, F: 24 Hours Ki-PEG-CBZ)

[0045] Fig. 34. Microscope images of migration after CBZ, Ki 16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment in SKOV3 cell line for 48 hours (A: 48 Hours Control, B: 48 Hours CBZ, C: 48 Hours Ki 16425, D: 48 Hours CBZ-Ki16425, E: 48 Hours Ki-PEG-CBZ)

[0046] Detailed Description of the Invention

[0047] The invention relates to a dual drug loaded, targeted polymer drug nanoconjugate affecting multiple signaling pathways for use as a drug delivery system in the treatment of ovarian cancer. Said nanoconjugate comprises Cabozantinib (CBZ) as a VEGFR(Vascular endothelial growth factor receptor) inhibitor and Ki16425 as a LPAR (Lysophosphatidic acid receptor) 1 ,2,3 inhibitor. In the nanoconjugate of the invention, synergistic effect is obtained with dual drug use. The polymer drug nanoconjugate of the invention comprises polymer, Cabozantinib, and Ki16425 such that the ratio of hydrazinated polymer:CBZ is 1 :5 and the ratio of polymer:Ki is 1 :2. Here, the polymer in question is PEG (polyethylene glycol).

[0048] For single free drugs, IC50 values were determined as 4.58 pg / ml for CBZ and 492.15 pg / ml for Ki16425 at 72 hours in A2780 cell line. In 0VCAR3 cell line, IC50 values for CBZ and Ki16425 at 72 hours are calculated as 129.50 pg / ml and 109.42 pg / ml, respectively. In SKOV3 cell line, IC50 values were determined as 18.50 pg / ml for CBZ and 778.24 pg / ml for Ki16425 at 72 hours. In the free CBZ-Ki16425 combination, a combination study of CBZ and Ki16425 is performed for 72 hours. At the end of the analysis, dose reduction indices (DRI), combination indices (Cl), and effective doses (ED) are calculated for each cell line. As a result of CBZ-Ki16425 combination in A2780 cell line, Cl value for ED50 was calculated as 0.98 (additive), CBZ value was determined as 4.43 pg / ml with DRI 1.035 and Ki16425 value was determined as 8.4 pg / ml with DRI 74.135. In OVCAR3 cell line, as a result of CBZ-Ki16425 combination Cl value of 0.661 (synergistic) for ED50 was determined, while CBZ value was calculated as 96.43 pg / ml with DRI 1.343 and Ki16425 value was calculated as 4.76 pg / ml with DRI 22.98. In SKOV3 cell line, as a result of CBZ- Ki16425 combination Cl value as found as 0.512 (synergistic) for ED50, while CBZ value was determined as 7.94 pg / ml with DRI 2.33 and Ki16425 value was determined as 65.84 pg / ml with DRI 11.820. The IC50 value of the Ki- PEG-CBZ nanoconjugate of the invention is in the range of 2-378 pg / ml and in one embodiment of the invention, the IC50 value of said Ki-PEG-CBZ nanoconjugate for 72 hours is determined as 2.66 pg / ml for A2780 cell line, 90.16 pg / ml for OVCAR3 cell line, and 377.9 pg / ml for SKOV3 cell line (Table 1).

[0049] Table 1 : IC50 and ED50values in free Cabozantinib, free Ki16425, free CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treated groups

[0050] For A2780 and 0VCAR3 cell lines, Ki-PEG-CBZ nanoconjugate is found to be more effective than the free drug combination. It is determined that non-drug-bound HPEG does not have any cytotoxic effect on the cell lines studied. In the HPEG-treated group, 88.84% viability was detected in A2780 cell line, 98.9% in OVCAR3 cell line, and 83% in SKOV3 cell line at 72 hours.

[0051] Synthesis method of the polymer drug nanoconjugate of the invention comprises the process steps of: i. to convert the carbonyl group in the structure of PEG5000 to acyl hydrazine, dissolving PEG5000 in deionized water with EDC (N-(3-dimethyl amino propyl)-N’- ethyl carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) and activating and incubating at 280 rpm, ii. adding hydrazine hydrate (NH2NH2■ H2O) and re-incubating, iii. at the end of the period, performing dialysis against distilled water with a dialysis membrane having a MWCO (Molecular weight limit): 1000 Da to separate the reaction components, iv. drying the hydrazinated polymer in an oven, v. reacting HPEG: CBZ to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ (Cabozantinib), vi. reacting Cabozantinib with polymer at 280 rpm 25°C by dissolving in DMSO, vii. performing dialysis with a dialysis membrane of MWCO: 5000 Da for purification of nanoconjugate and obtaining polymer-drug conjugate (CBZ-PEG), viii. mixing CBZ-PEG conjugate and DMAP (4-Dimethylaminopyridine) in DMSO (Dimethyl sulfoxide), ix. after mixing Ki16425 with DCC in DMSO, adding the activated Ki16425-DCC mixture into the CBZ-PEG solution, x. reacting the mixture by stirring, xi. performing dialysis using a dialysis membrane of MWCO:1000 Da to separate the reaction components and obtaining Ki-PEG-CBZ nanoconjugate xii. drying Ki-PEG-CBZ nanoconjugate in an oven.

[0052] In another embodiment of the invention, the method of synthesis of the polymer drug nanoconjugate of the invention comprises the process steps of: i. to convert the carbonyl group in the structure of PEG5000 to acyl hydrazine, dissolving 1-10 mg of PEG5000 in 2-10 ml of deionized water with 6-65 mg of EDC (N-(3-dimethyl amino propyl)-N’-ethyl carbodiimide hydrochloride) and 0.7-7.5 mg of NHS (N-hydroxysuccinimide) and activating at 280 rpm at 25 °C for 1 hour and incubating for an hour, ii. adding 6-64 pl of hydrazine hydrate (NH2NH2 ■ H2O) and re-incubating at 280 rpm and 25°C for 24 hours, iii. at the end of the period, performing dialysis against distilled water with a dialysis membrane having a MWCO (Molecular weight limit): 1000 Da to separate the reaction components, iv. drying the hydrazinated polymer in an oven, v. reacting HPEG: CBZ at a ratio of 1 :1 , 1 :2, 1 :5, 1 :10 to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ (Cabozantinib), vi. reacting Cabozantinib with polymer at 280 rpm 25°C for 48 hours by dissolving in DMSO, vii. performing dialysis with a dialysis membrane of MWCO: 5000 Da for 48 hours for purification of nanoconjugate and obtaining polymer-drug conjugate (CBZ- PEG), viii. mixing CBZ-PEG conjugate and 1 pmol of DMAP (4-Dimethylaminopyridine) in 3 mL of DMSO (Dimethyl sulfoxide) for 30 minutes, ix. after mixing Ki16425 with DCC at a Ki16425 (Ki) :DCC (N,N'- Dicyclohexylcarbodiimide) molar ratio of 1 :1 in 2 mL of DMSO for 30 minutes, adding the activated Ki16425-DCC mixture to the CBZ-PEG solution, x. reacting the mixture by stirring for two days, xi. performing dialysis using a dialysis membrane of MWCO:1000 Da to separate the reaction components and obtaining Ki-PEG-CBZ nanoconjugate xii. drying Ki-PEG-CBZ nanoconjugate in an oven.

[0053] In a further embodiment of the invention, the method of synthesis of the polymer drug nanoconjugate of the invention comprises the process steps of: i. to convert the carbonyl group in the structure of PEG5000 to acyl hydrazine, dissolving 1 pmol of PEG5000 (5 mg) in 2 ml of deionized water with 166.5 pmol (32 mg) of EDC (N-(3-dimethyl amino propyl)-N’-ethyl carbodiimide hydrochloride) and 32.25 pmol (3.7 mg) of NHS (N-hydroxysuccinimide) and activating at 280 rpm at 25 °C for 1 hour and incubating for an hour, ii. adding 32 pl of hydrazine hydrate (NH2NH2 ■ H2O) and re-incubating at 280 rpm and 25°C for 24 hours, iii. at the end of the period, performing dialysis against distilled water with a dialysis membrane having a MWCO (Molecular weight limit): 1000 Da to separate the reaction components, iv. drying the hydrazinated polymer in an oven, v. reacting HPEG: CBZ at a ratio of 1 :1 , 1 :2, 1 :5, 1 :10 to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ (Cabozantinib), vi. reacting Cabozantinib with polymer at 280 rpm 25°C for 48 hours by dissolving in DMSO, vii. performing dialysis with a dialysis membrane of MWCO: 5000 Da for 48 hours for purification of nanoconjugate and obtaining polymer-drug conjugate (CBZ- PEG), viii. mixing CBZ-PEG conjugate and 1 pmol of DMAP (4-Dimethylaminopyridine) in 3 mL of DMSO (Dimethyl sulfoxide) for 30 minutes, ix. after mixing Ki16425 with DCC at a Ki16425 (Ki) :DCC (N,N'- Dicyclohexylcarbodiimide) molar ratio of 1 :1 in 2 mL of DMSO for 30 minutes, adding the activated Ki16425-DCC mixture to the CBZ-PEG solution, x. reacting the mixture by stirring for two days, xi. performing dialysis using a dialysis membrane of MWCO:1000 Da to separate the reaction components and obtaining Ki-PEG-CBZ nanoconjugate xii. drying Ki-PEG-CBZ nanoconjugate in an oven. In the invention, the carbonyl group in the structure of PEG5000 is converted into acyl hydrazine. For this, EDC / NHS chemistry is carried out with hydrazine hydrate. 1 pmol of PEG5000 (5 mg) is dissolved in 2 ml of deionized water with 166.5 pmol (32 mg) of EDC (N-(3-dimethyl amino propyl)-N’-ethyl carbodiimide hydrochloride) and 32.25 pmol (3.7 mg) of NHS (N-hydroxysuccinimide) and activated at 280 rpm at 25 °C for 1 hour. After an incubation period of one hour, 32 pl of hydrazine hydrate (NH2NH2 ■ H2O) is added and then re-incubated at 280 rpm and 25°C for 24. At the end of the period, dialysis is performed against distilled water with a dialysis membrane having a MWCO: 1000 Da to separate the reaction. The hydrazine polymer is dried in an oven and characterized by NMR and FTIR spectroscopy methods. HPEG: CBZ is reacted at a ratio of 1 :1 , 1 :2, 1 :5, 1 :10 to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ. The drug and polymer are reacted at 280 rpm 25°C for 48 hours by dissolving in DMSO. To purify the product, it is dialyzed with a dialysis membrane of MWCO: 5000 Da for 48 hours and at the end of the period, the polymer-drug conjugate (CBZ-PEG) is characterized by NMR and FTIR and the amount of CBZ bound to the polymer is determined by HPLC. To bind the LPA receptor inhibitor Ki16425 to the CBZ-bound conjugate, an esterification reaction is carried out with N,N'-dicyclohexyl carbodiimide (DCC) in the presence of N,N-dimethyl aminopyridine (DMAP). CBZ-PEG conjugate containing known amount of CBZ and 1 pmol of DMAP are mixed in 3 mL of DMSO for 30 minutes, and on a separate side, Ki16425 (polymer-CBZ: Ki16425) is mixed with DCC in DMSO in varying ratios of 1 :1 , 1 :2, 1 :5, 1 :10 to activate the carboxyl group of the inhibitor. After mixing Ki 16425 with DCC at a molar ratio of 1 :1 in 2 mL of DMSO for 30 minutes, the activated Ki16425-DCC mixture is added to the polymer-CBZ solution. It is reacted by stirring for two days. At the end of the period, dialysis is performed using a dialysis membrane of MWCO:1000 Da to separate the reaction components. The amount of Ki16425 bound to the polymer is determined by LC-MS / MS. The obtained Ki-PEG- CBZ nanoconjugate is dried in an oven and structural and morphological characterization is carried out by NMR, FTIR, SEM, TGA, zeta potential, hydrodynamic dimension analysis. The amounts of drug bound for both CBZ and Ki16425 are calculated according to Formula 1 .

[0054] Formula 1 : Drug release studies of CBZ and Ki16425 bound nanoconjugate are carried out with dialysis membrane at 37°C and 100 rpm. Samples are collected at specified intervals and fresh buffers are added under the same conditions and amount. Acetate buffer of 10 mM is used for pH 5.0, phosphate buffer of 10 mM is used for pH 6.0, pH 6.5 and pH 7.4, and at the end of the period, the amount of CBZ is determined by HPLC and the amount of Ki16425 is determined by LC-MS / MS. The cumulative amounts of drug released are calculated according to Formula 2. 100

[0055] The efficacy of the Ki-PEG-CBZ nanoconjugate of the invention is evaluated in A2780, OVCAR3, and SKOV3 ovarian cancer cell lines and primarily in vitro biocompatibility and intracellular uptake studies are carried out. For in vitro efficacy evaluation, cytotoxicity analysis is performed by MTT and apoptosis analysis by Annexin V FITC / PI staining. The effect of the synthesized polymer-drug nanoconjugate of the invention on invasion and migration in ovarian cancer cells and its effect on VEGF signaling pathway related genes are evaluated. Cells are incubated in 75 cm2cell culture flasks in media containing 10% of FBS, 1 % of 2 mM L-Glutamine and 1 % of Penicillin- Streptomycin in an incubator at 37°C, 95% humidity and containing 5% CO2 until they reach a certain number and become suitable for experimentation. RPMI-1640 medium is used for A2780 and OVCAR3 cells and Me Coy's 5A medium is used for SKOV3 cells. When the cells cover 95-98% of the flask, monolayer cells are removed and passaged with Trypsin-EDTA. Cell viability is checked under a light microscope with Trypan Blue stain. For the intracellular uptake study of Ki-PEG-CBZ nanoconjugate and free CBZ - Ki16425 combination in A2780, OVCAR3, and SKOV3 cells, 5 x 105cells are seeded in 25 cm2cell culture flasks. Fifty pg / ml Ki-PEG-CBZ nanoconjugate and 50 pg / ml free CBZ- Ki16425 combination are applied to the cells. After six hours of incubation, the media is removed and the cells are washed 3 times with cold 1 x PBS. After the cells are removed with trypsin, they are washed once more with 1 xPBS and centrifuged. After centrifugation, cells were placed in 250 pL of 1 % T riton-X 100 solution for 30 minutes for lysis followed by sonication at 40 W for 30 seconds. The lysed cells are centrifuged at 13000 rpm for 10 minutes and the amount of drug taken into the cell is determined. To determine the biocompatibility of the synthesized polymer drug nanoconjugate, inflammatory response and hemolysis rate determination experiments are performed. For this, TNF-a ELISA assay and hemolysis analysis are performed respectively. For the TNF-a ELISA assay, RAW 264.7 cells (Mus musculus macrophage cell) are used. RAW 264.7 cells are incubated at 37°C in a medium containing high glucose DMEM, 10% fetal bovine serum (FBS), 1 % L-glutamine, and 1 % antibiotics (penicillin and streptomycin). For the experiment, 12-well plates are seeded with 40,000 cells per well and after 24 hours of incubation required for the cells to attach, the cells are treated with a medium containing CBZ and Ki16425 bound polymeric nanoconjugate at a dose range of 500 pg / ml, 100 pg / ml, 50 pg / ml, 5 pg / ml, and 1 pg / ml and free CBZ and Ki16425 at the same ratio for 48 hours. To determine the amount of TNF- a released into the medium, the medium of the cells is collected and centrifuged at 2000 rpm for 10 minutes. The supernatant is used for ELISA analysis. In order to determine the amount of hemolysis that may be caused by the polymer drug nanoconjugate, firstly, the blood taken into the tube with EDTA is centrifuged at 2000 rpm for 10 minutes and after centrifugation, the plasma part is removed and the erythrocytes are washed 3 times with 1 x PBS. A 2% erythrocyte solution is prepared in PBS. On the other hand, Ki-PEG-CBZ nanoconjugate dissolved in PBS is prepared at concentrations of 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml. The conjugate and erythrocyte solution are mixed at a ratio of 1 :1 (v / v) and incubated at 150 rpm for 2 hours at 37 °C and at the end of the period, the samples are centrifuged at 13000 rpm for 10 minutes. The supernatant is measured spectrophotometrically (Agilent Technologies, Carry 60 UV-Vis) at 540 nm. 1 % Triton-X is used as positive control and PBS is used as negative control.

[0056] The cytotoxicity of free CBZ, free Ki 16425, combination of free CBZ and Ki 16425 with non-drug bound PEG with hydrazine and synthesized nanoconjugate is evaluated by MTT assay in A2780, OVCAR3, SKOV3 cell lines. Cells are seeded in 96-well plates at 1 x104cells / 100 pl per well. After a 24-hour incubation for cell adhesion, free drugs and nanoconjugate were applied in the dose range of 100 pg / ml to 0.78 pg / ml. Herein, nondosed cells are considered as control group. MTT assay is set up for 3 days for 24, 48, and 72 hours and following the incubation periods, MTT solution prepared as 5 mg / ml is applied and incubated for 4 hours. At the end of the incubation, 100 pl DMSO is added to dissolve the formazone crystals and measured in a microplate reader at a wavelength of 595 nm. IC50 values are calculated with the combined drug effect analyzer program with obtained absorbance values in a dose and time dependent manner. The IC50 values of CBZ and Ki-PEG-CBZ obtained from the cytotoxicity analysis, the ED50 values of CBZ-Ki16425 combination, and 10 pM Ki16425 are used in apoptosis experiments in ovarian cancer cell lines. The experiments are conducted using Annexin V FITC / PI staining and analyzed by flow cytometry. For this purpose, the cells are removed from the surface with Trypsin-EDTA and after the cells are removed from the surface, the cells are taken into an Eppendorf tube after adding medium, washed 2 times with PBS and centrifuged at 2000 rpm for 5 minutes. Cells are resuspended with 500 pl 1x binding buffer. Then 5 pl of Annexin V-FITC and 5 pl of propidium iodide (PI) are applied and one group receives no stain to be used as a control for gate acquisition, one group receives only FITC Annexin V stain, and the other group receives only PI stain. The mixtures are incubated at room temperature in the dark for 10 minutes and apoptosis analysis is performed with BD Accuri- C6 flow cytometry device.

[0057] Before starting the invasion assay, cells are incubated in serum-free medium for 24 hours. After 24 hours, cells acclimatized to serum-free medium are treated with 4.75 pg / ml (10 pM) Ki16425 for 24 hours with IC50 values of CBZ and Ki-PEG-CBZ and ED50 values of CBZ-Ki16425 combination. The invasion kit (Cellbiolabs, CBA-106-C) instructions are then followed.

[0058] The invasion plate is kept under sterile conditions at room temperature for 10 minutes. After moistening the basal membrane inside the inserts with 100 pl of warm and serum- free medium, cells are counted to 2x106cells / ml. Serum-free media containing specified doses of substances are suspended together with the cells. The serum-free medium in the inserts is removed and under sterile conditions, the feeder tray at the bottom of the plate is separated and 150 pl of medium containing 10% serum is added as chemoattractant. The chamber with the membranes is gently lowered back onto the feeder tray. The prepared cell / drug solutions are added to the membranes in 100 pl and left to incubate for 24 hours. Before finishing the incubation, 150 pl of the warmed cell separation solution is added to a clean cell collection tray. The cell / media solutions in the membrane compartment of the plate are removed and incubated for 30 minutes at 37°C in the cell collection tray containing cell separation solution. At the end of the period, the invaded cells remaining under the membrane are transferred into the cell separation solution by tilting several times. Prepare 4* Lysis buffer / cell proliferation test kit dye solution at a ratio of 1 :75 as needed and add 50 pl to the wells comprising cell separation solution. After incubation at room temperature for 20 minutes, 150 pl of the mixture was transferred to a 96-well plate suitable for fluorescence measurement and measured with a fluorescence plate reader at 480 nm / 520 nm.

[0059] A wound healing assay is performed to examine the effect of Ki-PEG-CBZ nanoconjugate on migration of ovarian cancer cells. For this purpose, cells from A2780, OVCAR3, and SKOV3 cell lines are seeded in 6-well plates at 2x105cells / well. After 24 hours of incubation for the cells to attach, a sterile 200 pl pipette tip is used to scratch the cells and a gap is created. The image at 0 hours is taken immediately before drug administration. Each cell line is then treated with media containing Ki-PEG-CBZ nanoconjugate and CBZ at their respective IC50 values and 4.75 pg / ml Ki 16425 and CBZ- Ki16425 combination at its ED50 value. The wound closure status of the cells was visualized at 24 hours, 48 hours and 72 hours under an inverted microscope with a DP72 model camera. For each cell line, the experiment is terminated when the control group is completely closed and the images are analyzed to quantitatively assess the relative openness compared to the control.

[0060] RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) is performed to evaluate the effect of the polymer drug nanoconjugate of the invention on the expression of genes associated with VEGF signaling pathway in A2780, OVCAR3, and SKOV3 ovarian cancer cell lines. Each cell line is treated with Ki-PEG-CBZ nanoconjugate at its IC50 value at 72 hours and CBZ-Ki16425 combination at their ED50values. After total RNA isolation, cDNA (Complementary DNA) is synthesized and RT-PCR is performed. Gene expression changes are quantitatively calculated and cells are seeded at 1 x 106cells / well in 6-well plates for total RNA isolation. Each cell line is treated with Ki-PEG- CBZ nanoconjugate at its IC50 value at 72 hours and CBZ-Ki16425 combination at their ED50values. For total RNA isolation, cells collected in a flask are centrifuged at 1200 rpm for 5 minutes. After centrifugation, the supernatant is removed and 350 pl RLT buffer is added to the pellet and vortexed. 70% ethanol in the same volume is added as the RLT buffer and the cell pellet is suspended. 700 pl of sample is transferred to an RNeasy spin column placed in a 2 ml collection tube and centrifuged at 8000 x g for 15 seconds. The liquid passing into the collection tube is removed and the DNase cutting part protocol of the RNA isolation kit protocol is applied. 350 pl of buffer RW1 is added to the RNeasy column, capped, and centrifuged at 8000 x g for 15 seconds and the liquid passing down is discarded. 10 pl of DNase I stock solution is added to 70 pl of Buffer RDD, the tube is gently mixed, and briefly centrifuged. 80 pl of the DNase I incubation mixture is added directly to the RNeasy column membrane and incubated for 15 minutes at room temperature. 350 pl of buffer RW1 is added to the RNeasy column, centrifuged at 8000 x g for 15 seconds and the liquid at the bottom is removed. 500 pl of RPE buffer is added into the spin column and centrifuged again at 8000 x g for 15 seconds. The liquid passing into the collection tube is removed. 500 pl of RPE buffer is added to the spin column and centrifuged at 8000 x g for 2 minutes. After centrifugation, a clean collection tube is placed under the spin column and centrifuged empty for 1 minute at 8000 x g to dry the column. After transferring the spin column to a clean 1 .5 ml Eppendorf tube, 30-50 pl of RNase-free water is added and centrifuged at 8000 x g for 1 minute. The amount and purity of the total RNAs obtained are measured. Samples with an absorbance value between 1 .8- 2 at 260 / 280 nm, and samples with an absorbance value of > 1 .8 at 260 / 230 nm are included in the study and the total RNAs obtained are stored at -80°C. After isolation of total RNAs, cDNA synthesis is performed. The vials in the kit are briefly centrifuged before use and then the genomic DNA elimination mix as indicated in the table below is prepared (Table 2).

[0061] Table 2: Genomic DNA elimination mix

[0062] After the genomic DNA elimination mix is prepared, it is incubated at 42°C for 5 minutes. Immediately afterwards, after holding for 1 minute on ice, the reverse-transcription mix is prepared according to Table 3.

[0063] Table 3: Reverse-transcription mix for cDNA synthesis

[0064] § Component Volume

[0065] 10 pl of reverse-transcription mix is added to tubes containing 10 pl of genomic DNA elimination mix. After pipetting slowly it is incubated at 42°C for 15 minutes and 95°C for 5 minutes and the reaction for cDNA synthesis is completed. 91 pl of RNase-free water is added to the samples and pipetted. The cDNA samples are stored at -20°C until the PCR protocol.

[0066] The mRNA levels of VEGF signaling pathway related genes are determined by qRT- PCR. Quantitative evaluation is performed using SYBR-Green qPCR Mastermixes with the customized plate containing 84 genes including VEGF-related genes specified in

[0067] Table 4 and 12 controls. The reaction is carried out by preparing the mix as indicated in Table 5.

[0068] Table 4. VEGF Associated RT2Profiler PCR Array

[0069] Table 5. Preparation of PCR components

[0070] Component Amount

[0071] RT2SYBR® Green Mastermix 1350 pl cDNA 102 pl

[0072] RNase-free water 1248 pl

[0073] Total volume 2700 pl 25 pl of the prepared mixture is added to the 96-well plate in which the genes were embedded. After the plate is covered with a sealer and centrifuged, the reaction is started by placing the 96-well plate in the PCR device. Gene expression normalization is performed according to the housekeeping genes (reference genes) in the H row of the plate such as ACTB, B2M, GAPDH, HPRT1 , RPLPO in the plate. The protocol used for the PCR step is described in Table 6. Results are calculated by threshold cycle (<CT) for each well using real time cycler software. The results are evaluated with the 2-AACtmethod.

[0074] Table 6. PCR run protocol

[0075] Statistical Analysis

[0076] Descriptive statistics (n, A. Mean, minimum and maximum value, standard deviation) are used to summarize continuous data. Differences between groups are evaluated by student t test and ANOVA test.

[0077] The first step of polymeric conjugate synthesis is the acylation stage of the carboxylic acid group of PEG5000, which is carried out by EDC / NHS chemistry. The addition of - NH2NH2 group to the structure as a result of the reaction is confirmed by the difference1H-NMR result between PEG5000 and HPEG (Fig. 1 ). According to the1H-NMR result, the broad peak at 3.58 ppm is determined to be the proton peaks belonging to the -CH2CH2 repeats from PEG5000, while the peak at 2 ppm confirms the incorporation of NH2NH2into the structure and its hydrazination. When the spectra of PEG and HPEG are examined, unlike in the PEG spectrum, the presence of the N-H stretching band at 3286 cm'1, the C=N stretching band at 1635 cm-1, and the N-H bending band at 1545 cm'1in the HPEG spectrum, confirms the hydrazination of PEG. Furthermore, the disappearance of the C=O stretching band at 1732 cm'1from the carboxylic acid structure of PEG in HPEG confirms the hydrazination of the structure (Fig. 2). Polymer:drug ratios of 1 :1 , 1 :2, 1 :5 and 1 :10 ratios (1 pmol CBZ: 0.5 mg, 2 pmol CBZ: 1 mg, 5 pmol CBZ: 2.5 mg, 10 pmol CBZ: 5.01 mg) are tested to determine the most efficient binding ratio.

[0078] It is determined that the binding efficiency increases with increasing amounts of drug, and the binding efficiency starts to decrease after 1 :5 ratio. For 1 :1 ratio, the binding efficiency is determined as 52.55 ± 1.47%, for 1 :2 ratio it is determined as 60.42 ± 2.79%, for 1 :5 ratio it is determined as 73.71 ± 0.53%, and for 1 :10 ratio it is determined as 40.06 ± 1.50% (Fig. 3). When the1H-NMR spectra are examined, the CBZ-PEG conjugate is confirmed by the presence of peaks corresponding to CH2CH2repetitions from PEG at 3.62 ppm (a), the required NH groups in the structure at 7.5-10 ppm (c,d,e), the methoxy (-OCH3) group at 4 ppm (b), the -OH group at 8.48 ppm (f), and the aromatic proton peaks between 6.4 ppm and 7.5 ppm (Fig. 4). In the literature, as a rare example of formulation studies conducted with CBZ, Yang et al.'s study on CBZ-loaded DSPE- PEG2OOO micelles reports1H-NMR results where the protons of methoxy groups appear at 4.1240 ppm and 4.1834 ppm, the protons of aromatic rings between 6.7053 - 8.4693 ppm, the protons of amino groups at 10.24442 ppm, and the protons of cyclopropane at 1.2754 ppm [5]. FT-IR results of CBZ-PEG conjugate when examined show N-H stretching band at 3292 cm1, C=O stretching band at 1641 cm1, C=N stretching band due to heterocyclic component at 1532 cm-1. The C-F stretching band, which reveals CBZ binding to the structure outside the annular structures, is detected at 1305 cm1(Fig. 5). The efficiency and amount of Ki 16425 binding to the CBZ-PEG conjugate is tested at ratios of 1 :1 , 1 :2, 1 :5, and 1 :10. According to LC-MS / MS results, Ki16425 binding efficiency is determined as 61.19 ± 0.14% for 1 :1 ratio, 77.72 ± 2.51% for 1 :2 ratio, 67.05 ± 6.09% for 1 :5 ratio, 60.39 ± 3.91% for 1 :10 ratio, respectively (Fig. 6). The highest drug binding efficiency is found to be at a ratio of 1 :2 and further studies are continued at a ratio of 1 :2 (polymer: Ki 16425). In a study conducted by Cai et al. on camptothecin (CPT)- loaded and Dox-loaded mPEG-PLA dimeric polymeric drug nanoparticles, the binding percentage was reported as 52% for CPT-SS-CPT conjugate and 53% for Dox-SS-Dox conjugate [6]. In another study on acetal-bound PEGylated paclitaxel loaded pH- sensitive prodrug (PAP + 40% PTX), PTX binding percentage is reported as 60.3% [7], Similarly, the study conducted by Lee et al. on PEGylated-peptide-Dox conjugate, Dox binding efficiency in the structures named P5D / D and P7D / was reported as 63.7% and 64.2%, respectively [8]. The incorporation of Ki 16425 into the CBZ-PEG structure is confirmed by1H-NMR. The NMR results confirm that Ki16425 binds to the conjugate in a very high yield for the 1 :2 ratio Ki-PEG-CBZ. According to the1H-NMR spectrum, CH2CH2 protons from PEG are detected at 3.62 ppm (a), protons in the -NH structure of the conjugate at 9.26 ppm and 9.03 ppm respectively (f+d), OH group proton at 8.47 ppm (b), and the -NH structure where the first binding occurred at 7.65 ppm (c). It is also observed that the -OCH3structure (e) shifts to a different ppm than CBZ-PEG due to the addition of Ki 16425 (Fig. 7). The final product, Ki-PEG-CBZ, is compared to the previous step, CBZ-PEG. According to the FT-IR spectrum, it is determined that the peak at 3279 cm’1corresponds to N-H stretching, the peak at 1732 cm1to C=O stretching, the peak at 1530cm1to N-0 stretching, the peak at 1344 cm1to C-N stretching from the aromatic ring, and the peak at 759 cm’1to C-CI stretching formed by the addition of Ki 16425 to the structure (Fig. 8). Hydrodynamic size and zeta potential analyses of Ki-PEG-CBZ nanoconjugate are performed. The hydrodynamic size of the nanoconjugate is determined as 169 ± 15.23 nm. The zeta potential is determined as -13.95 ± 1.21 mV and the PDI value as 0.556 (Fig. 9). The structure synthesized by Bobde et al. as a 2000 Da mPEG-PEGylated Dox-loaded nanoparticle (p(HPMA)-(HPMA-NH-N-DOX)-b-PEG (P6)) was found to have a size of approximately 160 nm and a zeta potential of -16.6 mV [9]. As an example of the dimension and surface charge of PEGylated conjugates containing dual drugs such as the structure synthesized in the invention, the LHRH- conjugated, PEGylated, poly-lactide-co-glycolide nanoparticle (PPL-DTX:QU) loaded with Docetaxel and Quercetin synthesized to study the treatment efficacy on prostate cancer is found to have a hydrodynamic dimension of 135.36 ± 6.9 nm and a zeta potential of -36.5 mV

[0010] . Thermogravimetric analysis (TGA) of the obtained Ki-PEG- CBZ nanoconjugate is evaluated. It is observed that PEG has a weight loss of 89.63% between 190 °C - 430 °C, HPEG has a weight loss of 96.93% between 290 °C - 442 °C, CBZ-PEG's first weight loss of 16% is between 246 °C-362 °C, and the 2nd loss is between 362 °C-445 °C, Ki-PEG-CBZ has a weight loss of 2.64% between 35 °C - 164 °C, followed by a second weight loss of 18.29% between 164 °C - 371 °C, and a final loss of 68.14% between 371 °C - 440 °C (Fig. 10). When SEM images of PEG, HPEG, CBZ-PEG and Ki-PEG-CBZ conjugates are examined, it is determined that the morphology of PEG and HPEG is spherical while the CBZ-PEG structure changes to rod-like structure upon CBZ binding. With the addition of Ki 16425 to the CBZ-PEG conjugate, it is observed that the structure takes a spherical shape again (Fig. 11 ). In a study with CBZ-loaded nanoparticles, SEM images revealed that CZ-PLGA-NP has a spherical appearance

[0011] . The cumulative drug release amounts of free CBZ and Ki16425 and Ki-PEG-CBZ nanoconjugate-bound CBZ and Ki16425 at pH 5.0, pH 6.0, pH 6.5, and pH 7.4 are evaluated. According to the in vitro drug release results, free CBZ was released at rates of 80.74 ± 0.5%, 73.43 ± 1 .45%, 76.43 ± 2.92%, and 70.02 ± 0.69% for pH 5.0, pH 6.0, pH 6.5 and pH 7.4, respectively, during the 24 hour period. The amount of CBZ released from the polymeric nanoconjugate was 15.83 ± 2.47%, 10.11 ± 0.001%, 8.70 ± 1.41% and 6.99 ± 0.4% for pH 5.0, pH 6.0, pH 6.5 and pH 7.4, respectively, for 48 hours. It is determined that free Ki16425 shows a release profile of 70.88 ± 0.345%, 70.93 ± 1.45%, 72.15 ± 0.03%, and 74.69 ± 0.115% for pH 5.0, pH 6.0, pH 6.5, and pH 7.4, respectively, for 24 hours. The amount of Ki 16425 released from the polymeric conjugate was 32.80 ± 0.29%, 15.64 ± 0.075%, 14.51 ± 0.58% and 12.77 ± 0.045% for pH 5.0, pH 6.0, pH 6.5 and pH 7.4, respectively, for 48 hours (Fig. 12).

[0079] The biocompatibility and hemolytic effect of the synthesized Ki-PEG-CBZ nanoconjugate are evaluated and accordingly it is determined that it is biocompatible and does not cause hemolysis compared to the free CBZ-Ki16425 combination. According to the results of TNF-a measurement by ELISA within the scope of biocompatibility tests, it is determined that the nanoconjugate applied at doses of 500 pg / ml, 100 pg / ml, 50 pg / ml, 5 pg / ml, and 1 pg / ml does not cause inflammatory response (Fig. 13). Within the scope of cell culture studies of the synthesized nanoconjugate, intracellular uptake, cytotoxicity, and apoptosis analyzes are performed and ovarian cancer cell lines A2780, OVCAR3, and SKOV3 cell lines are used for cell culture studies.

[0080] Intracellular uptake of Ki-PEG-CBZ nanoconjugate compared to CBZ-Ki 16425 combination treatment for ovarian cancer cell lines A2780, OVCAR3, and SKOV3 is evaluated. In A2780 and SKOV3 cell lines, the intracellular uptake of Ki-PEG-CBZ nanoconjugate was determined to be higher compared to the free drug combination, while for OVCAR3 cell line, the drugs in free combination were internalized into the cell more than the nanoconjugate. For A2780, Ki-PEG-CBZ is found to be approximately 5.75 times more internalized into the cell in quantity compared to the CBZ-Ki 16425 combination. For OVCAR3 cells, CBZ-Ki16425 free combination drugs were found to be taken 1 .69 times more into the cell than the Ki-PEG-CBZ nanoconjugate. For SKOV3 cells, it was found that the intracellular uptake of Ki-PEG-CBZ nanoconjugate was significantly higher than that of free CBZ-Ki16425. In terms of quantity, Ki-CBZ-PEG nanoconjugate was found to be taken 3.87 times more into the cell than the free form (Fig. 14). The cytotoxic effect of the synthesized nanoconugate in ovarian cancer cell lines is evaluated by MTT assay. The efficacy of the nanoconjugate is compared with free CBZ, free Ki 16425, free CBZ-Ki16425 combination. It is also examined whether the non-drugbound PEG structure with hydrazine has a cytotoxic effect on cells. According to MTT results, it is determined that the nanoconjugate shows a lower dose effect in A2780 and OVCAR3 cell lines compared to free drugs, and the PEG structure with hydrazine without any drug attached does not show a cytotoxic effect.

[0081] In A2780 cell line, IC50 values were determined as 4.58 pg / ml for CBZ and 492.15 pg / ml for Ki16425 at 72 hours (Fig.15). In OVCAR3 cell line, IC50 values for CBZ and Ki16425 at 72 hours are calculated as 129.50 pg / ml and 109.42 pg / ml, respectively (Fig. 16). In SKOV3 cell line, IC50 values were determined as 18.50 pg / ml for CBZ and 778.24 pg / ml for Ki16425 at 72 hours (Fig. 17). It is determined that non-drug-bound HPEG does not have any cytotoxic effect on the cell lines studied. In the HPEG-treated group, 88.84% viability was detected in A2780 cell line, 98.9% in OVCAR3 cell line, and 83% in SKOV3 cell line at 72 hours (Fig. 18). When the IC50 values of CBZ in the literature are examined, in the study conducted by Hoang et al. to evaluate the activity of CBZ with H1 , H3, and H10 melanoma cells, the IC50 values in the mentioned cell lines were determined as 71 .8 pM, 33.4 pM, and 70.4 pM, respectively

[0012] , In the study conducted with 14 HCC cell lines in which the single and combined effects of CBZ on hepatocellular carcinoma cells were evaluated, as a result of single application of CBZ, IC50 values in MHCC97-H, SNU- 182, HLE, HLF, Focus, SNU-449, SNU-423, SNU-475, PCL / PRF / 5, HepG2, SNU-387, Hep40, Huh7, Hep3B cell lines were determined as 6.3 pmol / L, 7.5 pmol / L, 17.8 pmol / L, 21.5 pmol / L, 29.3 pmol / L, 37.1 pmol / L, 43,7 pmol / L, 67.7 pmol / L, 109 pmol / L, 139.1 pmol / L, 272.8 pmol / L, 297.7 pmol / L, 380.2 pmol / L, and 446.7 pmol / L, respectively

[0013] . The IC50 values obtained in the invention are similar to the literature in terms of having different values in the same cancer type. The combination study of CBZ and Ki16425 is performed for 72 hours. At the end of the analysis, dose reduction indices (DRI), combination indices (Cl), and effective doses (ED) are calculated for each cell line. As a result of CBZ-Ki 16425 combination in A2780 cell line, Cl value for ED50was calculated as 0.98 (additive), CBZ value was determined as 4.43 pg / ml with DR1 1 .035 and Ki16425 value was determined as 8.4 pg / ml with DRI 74.135 (Fig. 19). In OVCAR3 cell line, as a result of CBZ-Ki16425 combination Cl value of 0.661 (synergistic) for ED50was determined, while CBZ value was calculated as 96.43 pg / ml with DR1 1.343 and Ki16425 value was calculated as 4.76 pg / ml with DRI 22.98 (Fig. 20). In SKOV3 cell line, as a result of CBZ- KH 6425 combination Cl value as found as 0.512 (synergistic) for ED5o, while CBZ value was determined as 7.94 pg / ml with DRI 2.33 and Ki16425 value was determined as 65.84 pg / ml with DRI 11.820 (Fig. 21). The IC50 value of Ki-PEG-CBZ nanoconjugate for 72 hours is determined as 2.66 pg / ml for A2780 cell line, 90.16 pg / ml for OVCAR3 cell line, and 377.9 pg / ml for SKOV3 cell line. For A2780 and OVCAR3 cell lines, Ki-PEG-CBZ nanoconjugate is found to be more effective than the free drug combination (Fig. 22). In a cytotoxicity study conducted by Yoo et al. with doxorubicinpolyethylene glycol-folate (DOX-PEG-FOL) conjugate in KB and A549 cell lines, it was determined that DOX / FOL nano-aggregates were more cytotoxic than free Dox

[0014] , It was found that the DOX-PEG-FOL conjugate showed a more effective cytotoxicity due to the rapid elimination of free DOX in the cytoplasm by the effect of P-glycoprotein pumps. Similar to the results obtained in the invention for A2780 and OVCAR3 cell lines, Ki-PEG-CBZ nanoconjugate shows a more effective cytotoxicity at a lower dose compared to free CBZ and Ki16425.

[0082] Apoptosis studies are performed with IC50, ED50 values obtained for each cell line and 10 pM Ki16425. In the ovarian adenocarcinoma cell line A2780 cell line, more apoptosis was detected in the Ki-PEG-CBZ nanoconjugate treated group compared to the control and other free drug treated groups. In the A2780 cell line, compared to control, CBZ induced apoptosis by 1 1 .9%, Ki16425 by 8.7%, CBZ-Ki16425 combination by 12%, while Ki-PEG-CBZ nanoconjugate induced apoptosis by 28.1 %. (Fig. 23). In the high-grade serous ovarian adenocarcinoma cell line OVCAR3 cell line, compared to control, CBZ induced apoptosis by 14.1%, Ki16425 by 9.5%, CBZ-Ki16425 combination by 16.6%, while Ki-PEG-CBZ nanoconjugate induced apoptosis by 21 .1 % (Fig. 24). In the SKOV3 cell line, compared to control, CBZ induced apoptosis by 15.4%, Ki16425 by 6.2%, CBZ- Ki 16425 combination by 18.4%, while Ki-PEG-CBZ nanoconjugate induced apoptosis by 34.4% (Fig. 25). Suo et aL, in their study with MCF-7 cells and Dox-loaded Folatedecorated PEGylated triblock copolymer, found that Dox-NMs containing 2 pg / ml free Dox and the same amount of Dox induced apoptosis in MCF-7 cells by 25.6% and 33.3%, respectively. It was shown that PEGylated nanoparticles loaded with Dox induced MCF- 7 cells to apoptosis more than free Dox

[0015] . Apoptosis and necrosis percentages after free CBZ, Ki16425, CBZ-Ki16425 and Ki-PEG-CBZ treatment are shown in Table 7. Table 7. Percentages of apoptosis and necrosis of CBZ, Ki16425, CBZ-Ki16245 combination and Ki-PEG-CBZ in A2780, OVCAR3, and SKOV3 cell lines

[0083] Ki-PEG-CBZ nanoconjugate applied to the A2780 cell line is found to reduce the number of invasive cells by 2.16-fold compared to the control. It is determined that while free CBZ decreased the number of invasive cells 1 .83-fold and Ki16425 1 .33-fold compared to the control, CBZ-Ki 16425 combination decreased the number of invasive cells 1.51 - fold. In the A2780 cell line, it is detected that Ki-PEG-CBZ nanoconjugate suppressed the number of invasive tumor cells more than single drugs in free form and the combination. (Fig. 26).

[0084] For the OVCAR3 cell line, it was determined that free drugs and Ki-PEG-CBZ nanoconjugate had no significant effect on the suppression of cell invasion compared to control (Fig.27). In SKOV3 cell line, it is observed that, compared to control, free Ki16425 suppressed invasion 1.3-fold, CBZ 1.82-fold, CBZ-Ki16425 combination 2.17-fold, and Ki-PEG-CBZ nanoconjugate reduced the number of invasive cells 2.65-fold (Fig. 28). The effect of CBZ, Ki16425, Ki-PEG-CBZ nanoconjugate, and CBZ-Ki16425 combination applied to human ovarian adenocarcinoma cell line A2780 on cell migration is examined and according to the microscope images and quantitatively evaluated results, at 24 hours, 24% open area was determined in CBZ-treated cells, 4% in Ki 16425- treated cells, 9% in CBZ-Ki16425 combination-treated cells, while 84% open area was determined in Ki-PEG-CBZ nanoconjugate-treated cells. The control group is observed to be completely closed at 24 hours. As a result of the wound healing assay, it was determined that Ki-PEG-CBZ nanoconjugate inhibited tumor cell migration more significantly than free drugs in the A2780 cell line (Fig. 29). The effects of CBZ, Ki16425, Ki-PEG-CBZ nanoconjugate and CBZ-Ki16425 combination on cell migration in OVCAR3 cell line are examined. In the OVCAR3 cell line, for 24 hours, 19.5% open area in control cells, 17% open area in CBZ-treated cells, 10.6% open area in Ki 16425-treated cells, 12.6% open area in CBZ-Ki 16425 combination-treated group were found, and 23% open area was determined in Ki-PEG-CBZ-treated group. Control cells had about 1% open area at 48 hours, while CBZ, Ki16425, CBZ-Ki16425 combination and Ki-PEG- CBZ-treated groups measured 1.3%, 0.4%, 12.6% and 16% open area, respectively.

[0085] While control cells and Ki16425-treated cells were completely closed at 72 hours, 0.7% open area was detected in the CBZ-treated group, 13% open area in the CBZ-Ki 16425 combination-treated group, and 17% open area in the Ki-PEG-CBZ nanoconjugate- treated cell group (Figs. 30, 31 , 32). As a result of the migration experiment conducted for the SKOV3 cell line, the control group is almost completely closed at 24 hours and approximately 5% open area is detected. The percentages of open area measured in CBZ, Ki16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treated groups were 65%, 35%, 79%, and 88%, respectively. At 48 hours, it was detected as 51 .5%, 2.4%, 55.3%, and 66% respectively, in CBZ, Ki 16425, CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treated groups, while the control group was completely closed at 48 hours (Figs. 33, 34).

[0086] In the study of the invention, when the migration and invasion results are evaluated, the preservation of wound gap in ovarian cancer cell lines and the decrease in the number of invasive cells indicate that the Ki-PEG-CBZ nanoparticle has the potential to show antimetastatic properties.

[0087] The gene expression changes of Ki-PEG-CBZ nanoconjugate applied at IC5o value and CBZ-Ki16425 combination applied at ED50value to A2780 cell line at 72 hours compared to the control group are shown in Table 8. It is determined that Ki-PEG-CBZ nanoconjugate causes a more pronounced change in the expression of many genes in cells to which it is applied. In gene expression analyses, differences with p values less than 0.05 and differences with more than 2-fold or less fold change in expression levels are considered statistically significant.

[0088] Table 8. Expression changes and p values of VEGF-related genes in A2780 cell line as a result of CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment compared to control

[0089] The effect of CBZ-Ki16425 combination applied at ED5oand Ki-PEG-CBZ nanoconjugate applied at IC50 in OVCAR3 cell line at 72 hours on the expression of VEGF pathway- associated genes compared to the control group is shown in Table 9.

[0090] Table 9. Expression changes and p values of VEGF-related genes in OVCAR3 cell line as a result of CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment compared to control

[0091] The gene expression fold changes and p values of Ki-PEG-CBZ nanoconjugate applied at IC50 value and CBZ-Ki16425 combination applied at ED50value to SKOV3 cell line at 72 hours compared to the control group are shown in Table 10. Table 10. Expression changes and p values of VEGF-related genes in SKOV3 cell line as a result of CBZ-Ki16425 combination and Ki-PEG-CBZ nanoconjugate treatment compared to control

[0092] Industrial Applicability of the Invention

[0093] The invention relates to a dual drug loaded, targeted polymer drug nanoconjugate affecting multiple signaling pathways for use as a drug delivery system in the treatment of ovarian cancer, and is industrially applicable.

[0094] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims.

[0095] REFERENCES

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Claims

CLAIMS1. A polymer drug nanoconjugate, characterized in that it comprises polymer, Cabozantinib (CBZ), and Ki16425 (Ki) such that the ratio of hydrazinated polymer:CBZ is 1 :5 and the ratio of polymer:Ki is 1 :2.

2. The polymer drug nanoconjugate according to claim 1 , characterized in that said polymer is PEG (polyethylene glycol).

3. The polymer drug nanoconjugate according to claim 1 or 2 for use in the treatment of ovarian cancer.

4. The polymer drug nanoconjugate according to claim 3, characterized in that the IC50 value of said nanoconjugate is in the range of 2-378 pg / ml.

5. A method of synthesizing the polymer drug nanoconjugate according to any one of claims 1-4, characterized in that it comprises the process steps of: i. to convert the carbonyl group in the structure of PEG5000 to acyl hydrazine, dissolving PEG5000 in deionized water with EDC (N-(3-dimethyl amino propyl)-N’-ethyl carbodiimide hydrochloride) and NHS (N- hydroxysuccinimide) and activating and incubating at 280 rpm, ii. adding hydrazine hydrate (NH2NH2■ H2O) and re-incubating, iii. at the end of the period, performing dialysis against distilled water with a dialysis membrane having a MWCO (Molecular weight limit): 1000 Da to separate the reaction components, iv. drying the hydrazinated polymer in an oven, v. reacting HPEG: CBZ to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ (Cabozantinib), vi. reacting Cabozantinib with polymer at 280 rpm 25°C by dissolving in DMSO, vii. performing dialysis with a dialysis membrane of MWCO: 5000 Da for purification of nanoconjugate and obtaining polymer-drug conjugate (CBZ-PEG), viii. mixing CBZ-PEG conjugate and DMAP (4-Dimethylaminopyridine) in DMSO (Dimethyl sulfoxide),ix. after mixing Ki16425 with DCC in DMSO, adding the activated Ki16425- DCC mixture into the CBZ-PEG solution, x. reacting the mixture by stirring, xi. performing dialysis using a dialysis membrane of MWCO:1000 Da to separate the reaction components and obtaining Ki-PEG-CBZ nanoconjugate xii. drying Ki-PEG-CBZ nanoconjugate in an oven.

6. The method according to claim 5, characterized in that it comprises the process steps of: i. to convert the carbonyl group in the structure of PEG5000 to acyl hydrazine, dissolving 1 -10 mg of PEG5000 in 2-10 ml of deionized water with 6-65 mg of EDC (N-(3-dimethyl amino propyl)-N’-ethyl carbodiimide hydrochloride) and 0.7-7.5 mg of NHS (N-hydroxysuccinimide) and activating at 280 rpm at 25 °C for 1 hour and incubating for an hour, ii. adding 6-64 pl of hydrazine hydrate (NH2NH2 ■ H2O) and re-incubating at 280 rpm and 25°C for 24 hours, iii. at the end of the period, performing dialysis against distilled water with a dialysis membrane having a MWCO (Molecular weight limit): 1000 Da to separate the reaction components, iv. drying the hydrazinated polymer in an oven, v. reacting HPEG: CBZ at a ratio of 1 :1 , 1 :2, 1 :5, 1 :10 to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ (Cabozantinib), vi. reacting Cabozantinib with polymer at 280 rpm 25°C for 48 hours by dissolving in DMSO, vii. performing dialysis with a dialysis membrane of MWCO: 5000 Da for 48 hours for purification of nanoconjugate and obtaining polymer-drug conjugate (CBZ-PEG), viii. mixing CBZ-PEG conjugate and 1 pmol of DMAP (4- Dimethylaminopyridine) in 3 mL of DMSO (Dimethyl sulfoxide) for 30 minutes, ix. after mixing Ki16425 with DCC at a Ki16425 (Ki) :DCC (N,N'- Dicyclohexylcarbodiimide) molar ratio of 1 :1 in 2 mL of DMSO for 30minutes, adding the activated Ki16425-DCC mixture to the CBZ-PEG solution, x. reacting the mixture by stirring for two days, xi. performing dialysis using a dialysis membrane of MWCO:1000 Da to separate the reaction components and obtaining Ki-PEG-CBZ nanoconjugate xii. drying Ki-PEG-CBZ nanoconjugate in an oven.

7. The method according to claim 6, characterized in that it comprises the process steps of: i. to convert the carbonyl group in the structure of PEG5000 to acyl hydrazine, dissolving 1 pmol of PEG5000 (5 mg) in 2 ml of deionized water with 166.5 pmol (32 mg) of EDC (N-(3-dimethyl amino propyl)-N’-ethyl carbodiimide hydrochloride) and 32.25 pmol (3.7 mg) of NHS (N-hydroxysuccinimide) and activating at 280 rpm at 25 °C for 1 hour and incubating for an hour, ii. adding 32 pl of hydrazine hydrate (NH2NH2 ■ H2O) and re-incubating at 280 rpm and 25°C for 24 hours, iii. at the end of the period, performing dialysis against distilled water with a dialysis membrane having a MWCO (Molecular weight limit): 1000 Da to separate the reaction components, iv. drying the hydrazinated polymer in an oven, v. reacting HPEG: CBZ at a ratio of 1 :1 , 1 :2, 1 :5, 1 :10 to form a hydrazone bond between the hydrazinated polymer (HPEG) and CBZ (Cabozantinib), vi. reacting Cabozantinib with polymer at 280 rpm 25°C for 48 hours by dissolving in DMSO, vii. performing dialysis with a dialysis membrane of MWCO: 5000 Da for 48 hours for purification of nanoconjugate and obtaining polymer-drug conjugate (CBZ-PEG), viii. mixing CBZ-PEG conjugate and 1 pmol of DMAP (4- Dimethylaminopyridine) in 3 mL of DMSO (Dimethyl sulfoxide) for 30 minutes, ix. after mixing Ki16425 with DCC at a Ki16425 (Ki) :DCC (N,N'- Dicyclohexylcarbodiimide) molar ratio of 1 :1 in 2 mL of DMSO for 30minutes, adding the activated Ki16425-DCC mixture to the CBZ-PEG solution, x. reacting the mixture by stirring for two days, xi. performing dialysis using a dialysis membrane of MWCO:1000 Da to separate the reaction components and drying Ki-PEG-CBZ nanoconjugate in an oven.

8. The polymer drug nanoconjugate synthesized by the method according to any one of claims 5-7 for use in the treatment of ovarian cancer.

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