Receptor- and magnetic field-targeted drug carrier system for liver cancer

Abstract

The invention relates to a drug carrier system, which is an acyaloglycoprotein receptor and magnetically targeted, arabinogalactan-functionalised and doxorubicin-loaded magnetic nanoparticle.

Description

[0001] RECEPTOR- AND MAGNETIC FIELD-TARGETED DRUG CARRIER SYSTEM FOR LIVER CANCER

[0002] Technical Field of the Invention

[0003] The invention relates to an acyaloglycoprotein receptor-selective and magnetic fieldsensitive dual-targeted drug carrier system for use in the health sector in general and for use in the treatment of liver cancer in particular.

[0004] State of the Art Regarding the Invention (Prior Art)

[0005] World statistics indicate that the number of cancer patients in 2020 will be more than 15 million. Liver cancer is the fifth most common cancer in men and the ninth most common cancer in women. According to the World Health Organization (WHO), liver cancer ranks 4thamong the most common types of cancer with 782,000 deaths in 2018. Currently, liver cancer treatment includes chemotherapy, transcatheter therapy, radiotherapy and, if appropriate, organ transplantation, depending on the location and stage of the cancer.

[0006] Due to factors such as tumor size, multifocality and vascular invasion, only 15% of patients with liver disease can undergo surgery. Although liver transplantation is useful in the treatment of early-stage liver cancer, it is only used in a small number of patients due to poor donor organ compatibility. Transcatheter treatments are one of the effective treatment methods for patients who cannot undergo surgery. Transcatheter treatment methods include transarterial chemotherapy (TAC), transarterial radioembolization (TARE), transarterial embolization (TAE) and transarterial chemoembolization (TACE). TACE is one of these commonly applied methods and commercial forms are available for implementation. Today, doxorubicin is frequently used as a chemotherapy drug in TACE systems. Doxorubicin, an anthracycline antibiotic, acts on the cell by entering between DNA base pairs, causing inhibition of DNA synthesis and inhibition of DNA-dependent RNA synthesis. Doxorubicin, used in the treatment of many types of cancer, including liver cancer, is an anthracycline-type drug that exerts its proliferation inhibitory activity on cancer cells through two different mechanisms, intercalation and enzyme inhibition. This widely used drug is known to have many side effects including cardiotoxicity, myelosuppression, neuropathy, handfoot syndrome, nausea, vomiting and hair loss. This limits the maximum dose of the drug that can be taken. In addition, rapid elimination and wide distribution require the administration of large amounts of the drug. However, this is uneconomical and can lead to undesirable effects.

[0007] For these reasons, there is a need for the development of drug carrier systems that will allow Doxorubicin to be directed to the target and used at a lower dose, thus eliminating side effects and reducing the clearance rate of the drug.

[0008] Summary and Objects of the Invention

[0009] The present invention relates to a dual drug carrier system loaded with an acyaloglycoprotein receptor-selective and magnetic field-sensitive doxorubicin cancer drug designed for use in the treatment of liver cancer, which meets the above- mentioned requirements, eliminates the disadvantages and brings some additional advantages.

[0010] The primary object of the invention is to eliminate the drawbacks observed in conventional chemotherapy in cancer treatment, to increase drug transport to cellular levels, to increase the concentration of the drug in circulation or tissues, to optimize release kinetics, to change the pharmacokinetic and pharmacodynamic properties of the drug, to provide effective and safe treatment at low doses, to increase the stability of the drug and to achieve the desired level of pharmacological response of the drug at the target site without damaging healthy tissues.

[0011] With the drug carrier system of the invention, both ligand-receptor and magnetic targeting of the anti-cancer agent doxorubicin has been achieved. The object of targeting is, in general, to eliminate or minimize the adverse effects of conventional therapy, to increase drug transport to cellular levels, to optimize the concentration and release kinetics of drugs in circulation or other biological fluids, to alter the pharmacokinetic and pharmacodynamic properties of drugs, to provide effective and safe treatment at low or high doses, to eliminate or minimize toxic and immunogenic properties, to increase the stability of drugs, and to achieve the desired level of pharmacological response at the target site without causing any unwanted interactions in other parts of the body.

[0012] The magnetic targeting approach involves intravenous injection of a therapeutic agent attached to a magnetic drug carrier or encapsulated in the carrier and directing to tumor tissue by external application of a localized magnetic field. Studies involving the delivery of cancer drugs to the target site by magnetic targeting are very important and have been ongoing for years. The mechanism of receptor-mediated endocytosis is an important approach for the delivery of drugs to specific cell types. The Asioglycoprotein receptor (ASGPR) was the first mammalian lectin to be discovered and is one of the most prominent targets in drug targeting studies to the liver. One of the limiting factors in the treatment of hepatocellular carcinoma is the retention of particles by Kupffer cells, preventing their accumulation in hepatocytes. Targeted delivery to hepatocytes constitutes an important strategy in the treatment of liver cancer and this approach can be made possible by targeted delivery to receptors overexpressed in hepatocytes. As a hepatic lectin, the acyaloglycoprotein receptor provides an ideal target for hepatocytespecific delivery.

[0013] Within the scope of the invention, the potential of liver cancer treatment by both i.v. injection and transcatheter administration using magnetic nanoparticles containing doxorubicin targeted to the acyaloglycoprotein receptor was investigated and the two routes of administration were compared. Thus, with this drug delivery system, both ligand-receptor and magnetic targeting of the active substance is provided, the bioavailability of the drug is increased, and an active targeted drug delivery system has been developed by using a lower dose of chemotherapy agent. Thus, a more effective treatment was realized.

[0014] The object of the invention is to reduce the amount of the cancer drug doxorubicin, which is effective in the treatment of liver cancer, and to reduce the side effects to which cancer patients are exposed during chemotherapy treatments. With the drug carrier system of the invention, the drug is delivered directly to the target, thus reducing the drug dose.

[0015] With the invention, with the development of an acyaloglycoprotein receptor-selective and magnetic field-sensitive dual target dual drug carrier system containing the anticancer agent doxorubicin, which is widely used in the treatment of liver cancer, it is aimed primarily to provide cancer patients, whose number is increasing day by day, with a better quality of life and treatment without being exposed to the side effects of chemotherapy treatments.

[0016] Another object of the invention is to ensure that patients withdrawing from social life during treatment can continue their normal lives in social life during treatment, and to reduce treatment costs due to the reduction in drug dose by delivering the drug to the target.

[0017] Another object of the invention is to develop arabinogalactan-coated magnetic nanoparticle formulations loaded with doxorubicin to be used parenterally in liver cancer, in order to ensure that it is selective to the asiaglycoprotein receptor, and to provide comparative administration intravenously (i.v.) and via hepatic artery. Thus, with this drug carrier system, both ligand-receptor and magnetic targeting of the active substance is provided, increasing the bioavailability of the drug and providing a more effective treatment.

[0018] Within the scope of the invention, magnetic functional nanoparticles targeted to the acyaloglycoprotein receptor found in liver cancer cells are synthesized and Doxorubicin is loaded into this structure. In order to obtain magnetic field sensitive nanoparticles, magnetite nanoparticles are synthesized based on iron salts. After the synthesized magnetic nanoparticles are modified with 2-aminoethyl phosphonic acid (APA) to carry an amino group, the magnetic nanoparticle is functionalized with arabinogalactan to bind to the acyaloglycoprotein receptor, the level of which increases in liver cancer, and the structure is loaded with doxorubicin in order for the system to carry drugs more selectively in liver cancer.

[0019] Definitions of Drawings Describing the Invention

[0020] Fig. 1 : FTIR spectra of magnetic nanoparticles.

[0021] Fig. 2 : SEM image of magnetic nanoparticles

[0022] Fig. 3 : TEM image of magnetic nanoparticles

[0023] Fig. 4: Thermogram curve of magnetic nanoparticles

[0024] Fig. 5: FTIR spectrum of APA-MNP

[0025] Fig. 6: Thermogram curve of APA-MNP Fig. 7: SEM image of APA-MNP

[0026] Fig. 8: TEM image of APA-MNP

[0027] Fig. 9: VSM graph of A) MNP and B) APA-MNP

[0028] Fig. 10: Hydrodynamic size graphs of A) MNP and B) APA-MNP

[0029] Fig. 11 : Zeta potential graphs of A) MNP and B) APA-MNP

[0030] Fig. 12: FTIR graph of acetate form of Dowex A) before and B) after the treatment with acetic acid

[0031] Fig. 13: FTIR graph of A) Arabinogalactan (AG) and B) Oxidized arabinogalactan.

[0032] Fig. 14: FTIR spectrum of ANP.

[0033] Fig. 15: Thermogram curve of ANP.

[0034] Fig. 16: SEM image of ANP.

[0035] Fig. 17: TEM image of ANP

[0036] Fig. 18: VSM graph for ANP.

[0037] Fig. 19: Binding efficiency (%) and binding amount (pg Dox / mg np) of doxorubicin to ANPs at different concentrations .

[0038] Fig. 20: FTIR spectrum of DANP formulation.

[0039] Fig. 21 : Thermogram curve of DANP formulation

[0040] Fig. 22: SEM image of DANP formulation

[0041] Fig. 23: TEM image of DANP formulation

[0042] Fig. 24: VSM graph for DANP.

[0043] Fig. 25: Release graph of doxorubicin in free form at 37°C at different pH values.

[0044] Fig. 26: pH-dependent drug release graph of doxorubicin from DANP formulation at 37 C.

[0045] Fig. 27: Viability (%) created by the increasing concentrations of A) free doxorubicin and B) DANP formulation on HepG2 cells after 48-hour incubation

[0046] Fig. 28: I VIS images of heart, kidney, liver and renal tissues of CD-1 mice treated i.v. with DANP formulation after A) 1 hour ve B) 3 hours of sacrification.

[0047] Fig. 29: I VIS images of heart, kidney, liver and renal tissues of CD-1 mice subjected to TACE administration of DANP formulation after A) 1 hour ve B) 3 hours of sacrification.

[0048] Fig. 301. Histology of liver tissues of mice in the pharmacokinetic study groups: Hematoxylin-Eosin Staining X4-40 magnification images of livers taken at A1. 15 min, A2. 30 min, A3. 1 hour, A4. 3 hours, A5. 9 hours, A6. 12 hours, A7. 24 hours after intravenous administration of DANP formulation. Hematoxylin-Eosin Staining X4-40 magnification images of livers taken at B1 . 15 min, B2. 30 min, B3. 1 hour, B4. 3 hours, B5. 9 hours, B6. 12 hours, B7. 24 hours after portal vein administration of DANP formulation.

[0049] Fig. 31. Histology of the liver tissues of cancer-modeled mouse treatment groups Hematoxylin-Eosin Staining X4-40 magnification of the liver after A) cancer control treatment group treated with PBS B) Intravenous administration of DANP formulation C) Portal vein administration of DANP formulation D) Treatment group of intravenous administration of free doxorubicin E) Portal vein administration of free doxorubicin.

[0050] Fig. 32. Histology of liver tissues of mice in the pharmacokinetic study groups: Anti Ki- 67 Staining X40 magnification images of livers taken at A1. 15 min, A2. 30 min, A3. 1 hour, A4. 3 hours, A5. 9 hours, A6. 12 hours, A7. 24 hours after intravenous administration of DANP formulation. Anti Ki-67 Staining X40 magnification images of livers taken at B1. 15 min, B2. 30 min, B3. 1 hour, B4. 3 hours, B5. 9 hours, B6. 12 hours, B7. 24 hours after portal vein administration of DANP formulation.

[0051] Fig. 33. Histology of liver tissues of treatment groups of cancer-modeled mice: Anti Ki- 67 Staining X40 magnification images of the liver after A) cancer control treatment group treated with PBS B) Intravenous administration of DANP formulation C) Portal vein administration of DANP formulation D) Treatment group of intravenous administration of free doxorubicin E) Portal vein administration of free doxorubicin.

[0052] Fig. 34. Histology of liver tissues of mice in the pharmacokinetic study groups: Anti myeloperoxidase Staining X40 magnification images of livers taken at A1. 15 min, A2. 30 min, A3. 1 hour, A4. 3 hours, A5. 9 hours, A6. 12 hours, A7. 24 hours after intravenous administration of DANP formulation. Anti myeloperoxidase Staining X40 magnification images of livers taken at B1 . 15 min, B2. 30 min, B3. 1 hour, B4. 3 hours, B5. 9 hours, B6. 12 hours, B7. 24 hours after portal vein administration of DANP formulation.

[0053] Fig. 35. Histology of the liver tissues of cancer-modeled mouse treatment groups Anti myeloperoxidase Staining X40 magnification of the liver after A) cancer control treatment group treated with PBS B) Intravenous administration of DANP formulation C) Portal vein administration of DANP formulation D) Treatment group of intravenous administration of free doxorubicin E) Portal vein administration of free doxorubicin.

[0054] Detailed Description of the Invention

[0055] The invention relates to a magnetic field-targeted drug carrier system comprising a nanoparticule which is conjugated with oxidized arabinogalactan to bind to the increased levels of the acyaloglycoprotein receptor in liver cancer, oxidized using arabinogalactan potassium periodate, surface-coated with 2-aminoethyl phosphonic acid with oxidized arabinogalactan, and containing doxorubicin, and to a preparation method thereof. Said arabinogalactan and potassium periodate are in the ratio of 0.5- 4:0.5-4 by weight (Arabinogalactan otassium periodate), respectively, and said oxidized arabinogalactan and 2-aminoethyl phosphonic acid-coated magnetic nanoparticles are in a ratio of 0.5-4:0.5-4 by weight (oxidized arabinogalactan :2- aminoethyl phosphonic acid-coated magnetic nanoparticle), respectively, and said Doxorubicin is in a dose range of 10-1500 pg / mL.

[0056] A method for preparing a magnetic field-targeted drug carrier system according to the invention comprises the process steps of i. incorporating oxidized arabinogalactan into the nanoparticle structure by conjugation with oxidized arabinogalactan for binding to the increased levels of the acyaloglycoprotein receptor in liver cancer, ii. oxidizing arabinogalactan by mixing with potassium periodate, iii. realizing bond formation between oxidized arabinogalactan and surface- coated magnetic nanoparticles with 2-aminoethyl phosphonic acid by mixing in aqueous medium, iv. loading doxorubicin into nanoparticles.

[0057] In an embodiment of the invention, a method for preparing a magnetic field-targeted drug carrier system according to the invention comprises the process steps of i. incorporating oxidized arabinogalactan into the nanoparticle structure by conjugation with oxidized arabinogalactan for binding to the increased levels of the acyaloglycoprotein receptor in liver cancer, ii. oxidizing arabinogalactan by mixing with potassium periodate in a ratio of 0.5-4:0.5-4 (arabinogalactan otassium periodate) by weight, iii. realizing bond formation between oxidized arabinogalactan and surface-coated magnetic nanoparticles with 2-aminoethyl phosphonic acid by mixing in aqueous medium in a ratio of 0.5- 4:0.5-4 (oxidized arabinogalactan: surface-coated magnetic nanoparticle with 2-aminoethyl phosphonic acid) by weight, iv. loading Doxorubicin into the nanoparticle comprises a dose range of 10-1500 pg / mL.

[0058] Preparation of doxorubicin-loaded arabinogalactan-coated magnetic nanoparticles

[0059] Synthesis of magnetic nanoparticles and their modification with APA

[0060] The synthesis of magnetic nanoparticles (MNPs) and their functionalization with 2- aminoethyl phosphonic acid (APA) was carried out according to the method known in the art. To synthesize the magnetic nanoparticle, 1 mol PeCl2 and 2 mol FeCh were dissolved in purified water passed through nitrogen gas and under nitrogen gas at a stirring speed of 3000 rpm under constant temperature of 60°C. Then the concentrated NH4OH solution was added to the solution and the pH of the environment was adjusted to 10. It is allowed to be incubated for 2 hours for nucleation. At the end of the time, the nanoparticles were separated by magnetic decantation method and washed with distilled water and ethanol. The washed nanoparticles were dried overnight in an oven at 60°C and magnetic nanoparticles were obtained. Then FTIR, TGA, ZetaSizer, VSM and SEM analysis were performed for characterization analysis. The nucleophilic character of APA was exploited in the binding reaction of the synthesized nanoparticles with APA.

[0061] 400 mg magnetic nanoparticles were dispersed in 300 mL distilled water. Freshly prepared APA solution (0.5 mmol) was added to this mixture and stirred at 2,000 rpm for 6 hours at 80°C. At the end of the time, nanoparticles were precipitated by centrifugation at 12000 rpm and washed with distilled water. Then, APA-MNPs were dried in an oven at 60°C. The obtained structures were characterized by FTIR, TGA, Zeta Sizer, VSM, SEM and their structures were confirmed. In addition, surface amino groups in the structure of APA functionalized magnetic nanoparticles were quantitatively determined using the trinitrobenzenesulfonic acid (TNBS) method. Oxidation of arabinogalactan and its conjugation to magnetic nanoparticles

[0062] Oxidation of arabinogalactan

[0063] In order for arabinogalactan to bind to nanoparticles with amino groups, the hydroxyl groups must first be oxidized to aldehyde form. This technique took place according to the known method. Arabinogalactan (AG) was dissolved in distilled water.

[0064] To this solution potassium periodate was added separately in the appropriate weight ratio (IO4 / AG units) and the mixture was stirred for 2 hours at room temperature in the dark. The resulting polyaldehyde was purified from iodate (IO3 ) and unreacted periodate (IO4 ) by Dowex-1 (acetate form) anion exchange chromatography. It was then transferred into a glass column. The column pH needs to be adjusted to 7 before the removal of unreacted period and formate ions from the oxidized AG. Therefore, the column pH was adjusted to 7 by passing 10 mM pH:7 Tris buffer in a continuous flow. The arabinogalactan solution reacted with potassium periodate was then passed through the column. After purification, oxidized arabinogalactan was dialyzed against distilled water (12 000 Da MWCO cellulose membrane) at +4°C for 3 days. The purified polyalkaldehyde was dried in an oven at 45°C. Aldehyde content was determined by hydroxylamine hydrochloride method. The amount of aldehyde was calculated. The presence of aldehyde groups was also examined by FTIR analysis.

[0065] Conjugation of oxidized arabinogalactan to magnetic nanoparticles

[0066] For the binding of oxidized arabinogalactan to nanoparticles containing amino groups, the method of Ehrenfreund-Kleinman et al. (2002) was used. For conjugation, 0.1 -0.8 g Ox-AG was dissolved in 0.1 M pH 11 20-90 mL borate buffer. ARA was combined with bound magnetic nanoparticles in a ratio of 0.5-4:0.5-4 and mixed in the dark for 1 -6 days. The pH of the mixture was kept at 11 during the reaction. At this time, Schiff base formation is expected to occur. The imine conjugate was then transferred to a dialysis membrane (12 000-14 000 MWCO cellulose membrane) and dialyzed against distilled water at 1 -10°C for 1 day. The final structure was centrifuged and washed with distilled water at 13 000 rpm to remove excess reagents from the environment. Sodium borohydride (NaBH4) was used to convert the imine conjugate to the more stable amine conjugate form and to reduce the remaining aldehydes to hydroxyl group. The imine conjugate and NaBH4were reacted in appropriate ratios and stirred overnight at room temperature. The amine conjugate was then transferred to a dialysis membrane (12 000-14 000 MWCO cellulose membrane) and dialyzed against distilled water at 1 - 10°C for 1-6 days. The dialysate was centrifuged and washed with distilled water at 13 000 rpm to remove excess reagents from the environment. The obtained arabinogalactan-coated nanoparticles (ANP) were dried in an oven at 40-90°C. Characterization analyses were performed by FTIR, TGA, zeta sizer, and SEM. In addition, the binding efficiency of arabinogalactan to nanoparticles was determined by calculation based on the reduction of the number of amino groups present in the nanoparticles using the TNBS method.

[0067] Loading of doxorubicin onto arabinogalactan-coated magnetic nanoparticles

[0068] Doxorubicin was loaded onto arabinogalactan-coated magnetic nanoparticles via adsorption. Based on the study by Yang et al. (2009) and a previous study by our group, adsorption of the drug on nanoparticles was realized. For this purpose, firstly, 1 - 8 mg nanoparticles were dispersed in 0.05-3 mL distilled water with the help of an ultrasonic probe (Bandelin, Sonoplus-HD 2070.2) purchased within the scope of the project. Then, 0.5-3 mL doxorubicin solution (10-1500 pg / mL) was added at varying concentrations. The resulting solution was stirred with an orbital mixer (Heidolph, Unimax 1010) at 100-400 rpm for 12-20 hours at 15-50°C. At the end of the time, the nanoparticles were centrifuged at 13 000 rpm for 20 minutes to separate the supernatant (drug not loaded on the nanoparticles). Nanoparticles were washed in distilled water. Doxorubicin was determined in the supernatant and wash water samples by HPLC analysis. In addition to HPLC analysis, doxorubicin determination was performed by absorbance measurements in UV-Vis spectrophotometer (480 nm). The amount of drug bound per mg of nanoparticles and the percentage of drug bound in the samples were calculated. In addition, the surface functional groups of doxorubicin-loaded arabinogalactan-coated nanoparticles (DANP) were determined by FTIR analysis, size and morphology by SEM analysis, hydrodynamic size and zeta potential analysis by ZetaSizer, thermal stability by TGA and the formulation containing the optimal concentration of drug was determined. After centrifugation and washing, the prepared DANP formulations were dried overnight in an oven at 10-50°C. Analysis and validation of doxorubicin by HPLC

[0069] SHIMADZU LC-20AT (High Performance Liquid Chromatography) system was used to determine the amount of doxorubicin in the samples. Inertsil ODS 3V (150*4.0) was selected as the analytical column. The mixture of Water containing perchloric acid at pH 2 for the mobile phase: Acetonitrile: Tetrahydrofuran (76:24:0.5, v / v / v) was prepared. The flow rate was set to 1.25 mL / min, the column oven temperature to 50°C and the injection volume to 20 pL. The wavelength used in the detector was determined as 480 nm (Excitation) and 560 nm (Emission). Selectivity, LOD-LOQ, linearity and reproducibility parameters were analyzed in HPLC validation studies.

[0070] In vitro release studies of prepared doxorubicin-loaded arabinogalactan coated magnetic nanoparticle (DANP) formulation

[0071] In vitro release studies with DANP formulation

[0072] After dissolution / dispersion of free doxorubicin and the developed DANP formulation containing 0.1-3 mg / mL doxorubicin in buffer solution, release studies were performed by dialysis bag method. Cellulose membrane with 12 000-14 000 MWCO was used in in vitro drug release studies.

[0073] In vitro release study using a dialysis bag: Free doxorubicin and the developed DANP formulation (containing 0.1 -3 mg / mL Doxorubicin) were transferred into a dialysis membrane (12 000- 14 000 MWCO) and dialysis was performed against 10 mL of pH 5.5±0.05 acetate buffer and pH 6.5±0.05 and pH 7.4±0.05 phosphate buffer. Dialysis was performed at a temperature of 37°C±0.5°C and a mixing speed of 300 rpm.

[0074] Samples were taken at specific time intervals (0.5-48 h) and ambient conditions were kept constant by adding buffer at the same temperature as the volume of sample taken. The sink condition was taken into account during these studies. The amount of Dox released in the samples was determined by HPLC analysis. However, the amount of doxorubicin released was confirmed by measuring the absorbance of the same samples at 480 nm in a UV-Vis Spectrophotometer. Sitotoxicity Test

[0075] In order to examine the cytotoxicity and effect of free doxorubicin, ANP and DANP formulation on the viability of HepG2 and THLE-2 cells with luciferase activity, 100 pL of each cell at a concentration of 1xio4cells / mL was seeded separately into 96-well cell culture plates. These cells were incubated for 24 hours at 37°C in 5% CO2 and humid environment. After 24 hours, the enviroments in 96-well cell culture plates were removed and cytotoxicity studies were started. First, cells were washed with 100 pL saline phosphate buffer. For cytotoxicity assays, all drug groups were firstly dispersed / dissolved in distilled water and then dilutions were prepared by diluting / dispersion in Prigrow III, THLE-2 BEGM Bronchial Epithelial Bullet Kit cell growth medium for HepG2 cells with luciferase activity. The dose range of free doxorubicin was prepared as 0.1-100 pg / mL. The dose range of ANP and DANP formulation was prepared as 1 -250 pg / mL. Cells were incubated for 48 hours after administration of drug groups at the determined dose range. Apart from these samples, control groups containing only broth were also created. At the end of this period, 100 pL MTT) was added to each well in the cell culture plates and incubated for 4 hours.

[0076] The media containing the samples tested for cytotoxicity (solutions of different concentrations of the active substance and colloidal system formed by dispersing DANP in distilled water) were removed and 100 pL DMSO was added to each well of 96-well cell culture plates. The first row of 96-well cell culture plates was used as the control group. To test cell viability, absorbance values were read and evaluated by UV spectrophotometer with ELISA (Thermo, Multiskan Fc) microplate reader at 540 nm. Cell viability was calculated with the following formula:

[0077] Cell Viability (%)=(T / R)x100

[0078] In the formula, T: Absorbance value read from the tested samples, and R: Absorbance value read from the control group.

[0079] The percentage of cell viability above 95% indicates that the applied sample has no cytotoxic effect on the cells. Using Calcusyn 2.0 program, the concentration values (IC50) that reduced cell growth by 50% under laboratory conditions were determined with standard deviations. Biocompatibility analysis of DANP formulation

[0080] Binding of DANP formulation to serum proteins

[0081] The binding of the prepared nanoparticles to serum proteins was analyzed by modifying the method described by Cole et al. (Cole et aL, 2011 ). Nanoparticles were added to fetal bovine serum with serummanoparticle volume ratios (v:v) of 10:90, 20:80, 40:60, 60:40 and total volume of 600 pL, respectively. Samples were incubated at 37°C for 2 hours at 140 rpm. After incubation, the samples were centrifuged at 13 000 rpm for 1 hour. After centrifugation, protein was determined in the supernatant according to the Bradford method (Bradford, 1976) and the amount of protein bound to mg of drug carrier and % binding values were calculated.

[0082] Hemolysis effect of DANP formulation

[0083] The damage of the prepared nanoparticles on erythrocytes was determined by in vitro hemolysis studies and according to the method applied by Mayer et al. (Mayer et aL, 2009). Accordingly, erythrocytes separated from whole blood were suspended in PBS at 2% concentration. Nanoparticles of varying concentrations (0.025-1 mg / mL) and erythrocyte suspension in a 1 :1 ratio were allowed to be incubated at 37°C for 2 hours. At the end of the time, the samples were centrifuged for 5 min and free hemoglobin in the supernatant was determined spectrophotometrically at 540 nm. The dispersing system of nanoparticles (PBS) was used as a negative control and 1% Triton X-100 ensuring 100% lysis was used as a positive control.

[0084] Stability tests of DANP formulation

[0085] Stability studies of the doxorubicin-loaded arabinogalactan-coated magnetic nanoparticle formulation were carried out with the lyophilized product in accordance with stability guidelines. They were carried out at 5 ± 3°C (refrigerated), 25 ± 2°C at 60± 5% relative humidity and 40 ± 2°C at 75± 5% relative humidity. In the stability study, the samples were checked at t=0, i.e. at the baseline and at Month 1 , 3, 6. DANP formulations were taken into stability studies in colored glass vials and the stability of these systems were evaluated. The parameters monitored in stability tests are appearance, particle size, moisture content and active substance quantification and dosage homogeneity. In addition, quality control parameters of lyophilized DANP formulations at 5 ± 3°C (refrigerated), 25 ± 2°C at 60± 5% relative humidity and 40 ± 2°C at 75± 5% relative humidity were also monitored after dispersion in 5% dextrose solution at Month 1 , 3, 6. The parameters monitored in these colloidal dispersions are pH, appearance, particle size, zeta potential, viscosity, active substance quantification, redispersibility time. Doxorubicin quantification was monitored by HPLC.

[0086] Investigation of the possibilities of using the prepared DANP formulation in liver cancer treatment under in vivo conditions

[0087] The studies were conducted at Ege University ARGEFAR Pre-Phase Research Unit in which the experimental animals were cared for and controlled conditions were provided. Ethics committee approval document (2019-030) for in vivo studies was obtained from Ege University Animal Experiments Local Ethics Committee. The mice used were obtained from the company KOBAY A.§ (Ankara). The environment was maintained at a temperatur of 24±1°C, 50±10% humidity, 12 / 12 hours light / dark period.

[0088] For in vivo studies, the DANP formulation was comparatively administered to mice via both intravenous (i.v) and hepatic arterial administration, and the formulation was dispersed in saline prior to administration.

[0089] The dose of doxorubicin administered intravenously and through the hepatic artery was determined by considering the IC5o value.

[0090] TACE administration

[0091] 6-8 weeks old male and female CD-1 mice were anesthetized with intraperitoneal injections of 40 mg / kg ketamine S and 10 mg / kg xylazine. The abdominal regions of the mice were shaved, sterilized with alcohol and betadine, and a midline cut of 3 mg was made. In the abdomen opened according to the technique, the visceral membrane was slightly shifted to the left side of the abdominal cavity to reveal the portal vein (PV), abdominal inferior vena cava, bile duct and celiac artery. Magnification of the portal vein was achieved using surgical goggles (magnification 6.5x45) and DANP formulation was administered via a sterile microinjection. After the injection, an antihemorrhagic (oxidized regenerated cellulose) was applied to the proximal and distal parts of the arteriotomy site to prevent bleeding and the operation site was closed with 4.0 vicril suture (Kim et al., 2017). After the administration, the vital functions of the mice were observed. Within the scope of our project, DANP formulation containing 0.5- 10 pg Dox was administered through the portal vein for TACE administration.

[0092] Biodistribution studies To determine the biodistribution of the formulation, nanoparticles were administered to mice intravenously (i.v.) and via catheter administration, taking into account the administration doses of doxorubicin. A total of 12 CD-1 male / female mice, 6-8 weeks old, were needed for biodistribution studies. In the biodistribution study, DANP formulation was administered to healthy CD-1 mice in two different administration groups. DANP formulation containing 10-100 pg Dox was administered in 10-300 pL for i.v. administration. Since this concentration caused death of mice in TACE administration, DANP formulation containing 0.5-10 pg Dox was administered in an amount of 1 -30 pL in TACE administration. After the administration of the magnetic DANP formulation, mice were administered with a neomidium magnet with a strength of 0.1 Tesla in the abdomen for 10-70 minutes. At Hour 1 and 3 following administration, mice were sedated under isofluorane and fluorescently imaged (Aex 465 nm / Aem 600 nm) in dorsal decubitus in an in vivo imaging system. In addition, the organs (kidney, lung, liver, heart) removed from the mice at the end of the period were imaged with IVIS-Spect. After homogenization of the collected organs (kidney, lung, liver, heart) and blood samples, active substance analyses were performed by HPLC to determine the biodistribution of the drug.

[0093] Tissue Homogenization Organs collected from sacrificed mice were first washed with PBS. Then the organs were weighed. Weighed organs were homogenized in Ultraturax 5000-10000 rpm for 0-5 min in PBS. Samples were then diluted with 1 / 10 methanol and passed through a 0.45 pm pore syringe filter (CH ROM -Fl L). HPLC analysis of the filtered samples was then performed.

[0094] Pharmacokinetic studies A total of 42 CD-1 male-female mice, 6-8 weeks old, were used for pharmacokinetic studies. For the analyses, mice were administered with DANP formulation containing 0-100 pg Dox for i.v. administration and DANP formulation containing 0-50 pg Dox for portal vein administration. Following nanoparticle administration, a magnet was again applied to the liver area and the mice were placed in metabolic cages after the administration. Blood, urine and feces samples taken from the mice at certain times (Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24; n=3) were collected and these samples were analyzed for drugs by HPLC. Mice were sacrificed at the end of the period and their livers were placed in formol solution. Tissue fixation was performed in the removed liver and ex- vivo analysis studies were initiated.

[0095] Establishment of a liver cancer model and investigation of its therapeutic potential 35 male CD-1 nude mice aged 6-8 weeks were used to monitor the treatment process. HepG2 cells with luciferase activity were used to induce liver tumors in CD-1 nude male mice. While the mice were under 40 mg / kg ketamine S and 10 mg / kg xylazine anesthesia, 1x10A3-10 luciferase active HepG2 cells in 0-200 pL PBS were slowly injected into the right upper lobe of the liver to create a tumor model. Mice were kept under observation from the first day of cell implantation to monitor their daily activities and general condition. Imaging was performed 15 days after HepG2 cells were administered to nude CD-1 mice. Tumor size was examined on an imaging device in the in vivo imaging system (IVIS, Caliper Perkin Elmer) using both luciferin substrate and luciferase enzyme activity of tumor cells and glucose probe (IVISense 2DG 750 Fluor RediJect). Measurements were taken on an IVIS device (Aex 745 nm / Aem 820 nm) 3 hours after injection of the glucose probe. Imaging studies were continued using a glucose probe because of better results. In order to determine the therapeutic efficacy of the nanoparticles, doses were repeated three times a week for 14 days for the i.v. route, while a single dose of drug was given for transcatheter administration. After magnetic nanoparticle administrations, a magnet was attached to the tumor liver area for 30 minutes. The experimental groups are detailed below:

[0096] • Saline for control purposes

[0097] • Doxorubicin i.v. group

[0098] • Doxorubicin portal vein group

[0099] • DANP i.v. group

[0100] • DANP portal vein group

[0101] Tumor size measurements and changes in size were monitored with the IVIS device using a glucose probe. Tumor size was calculated using Equation. Survival of mice with tumor treatments was monitored throughout the experiment. At the end of the experiment, mice were sacrificed and blood samples were collected in lithium heparinized tubes. Biochemistry analyses (ALB, ALT, AST, BUN, CRE, etc.) were performed on whole blood samples using Preventive Care Profile Plus rotors on the Vetscan VS2 device. Livers were removed from the sacrificed mice and placed in formol solution. Tissue fixation was performed in the removed liver and ex-vivo analysis studies were initiated.

[0102] Tumor size (mm3)= (((tumor width)2x (tumor length))) / 2

[0103] Ex-vivo studies and pathological evaluation

[0104] At the end of the administration period, the liver tissue obtained from the sacrificed subjects was fixed and blocked after routine histological tissue follow-up procedures. The collected sections were stained with histochemical and immunohistochemical stains and examined by light microscopy and data were obtained.

[0105] Fixation The kidney, placenta and brain tissues obtained after sacrification were placed in 4% paraformaldehyde fixative and routine histologic tissue follow-up protocol was applied. After fixation, the tissues were removed from the fixative by placing them in PBS for 24 hours and the follow-up protocol was started (Table 1 ).

[0106] Table 1. Process steps of routine histological tissue follow-up.

[0107] After removal of the tissues from paraffin 2, the tissues were suitably placed in tissue embedding molds and blocked with clean paraffin. The paraffin in the embedding molds containing the tissues was kept at room temperature for 1 day to freeze. Sectioning Process

[0108] Paraffin blocks were kept in the refrigerator at +4oC for 1 hour before sectioning process. For histochemical and immunohistochemical analysis by light microscopy, the sections of 5 pm were taken on a Leica RM 2145 microtome. The sections obtained were allowed to open a little in a 37oC water bath and were transferred to polylysine slides. The slides were allowed to dry and then the staining stages were started.

[0109] Deparaffinization Process

[0110] For light microscopic imaging, the preparations were kept in an oven at 57°C for 1 hour to deparaffinize the tissues before all staining. After cooling, the preparations removed from the oven were placed in xylol and kept in xylol overnight. Histochemical staining procedures were then performed.

[0111] Histological method: Hematoxylin-Eosin Staining Protocol

[0112] Deparaffinized preparations were subjected to Hematoxylin-Eosin staining process following the process steps in Table 2.

[0113] Table 2. Process steps for Hematoxylin-Eosin staining.

[0114] After the process steps in the table, the preparations were treated with xylol for 30 minutes, dripped with entellan, covered with lamella and prepared for imaging. Results obtained after the applied protocol: Nucleus: Blue, Cytoplasm: Pink. Immunohistochemical method After deparaffinization processes, which is classically performed before staining, the preparations, which had been kept in xylol overnight, were removed from xylol and dried. Indirect immunohistochemical method was performed by Avidin-Biotin-Peroxidase technique. After being transferred to polylysine slides, the preparations were allowed to dry and adhere to the slide and kept in an oven at 60°C for 1 hour before staining. After removal from the oven, the preparations were allowed to cool and kept in xylol overnight for deparaffinization process. After deparaffinization process, immunohistochemical staining was performed by following the process steps in Table 3. After the immunohistochemistry staining steps, they were treated with xylol for 30 minutes for transparency. After xylol, the preparations were covered with entellan and prepared for imaging. PCNA polyclonal antibody as primary antibody, myeloperoxidase, anti-her par 1 and ki-67 were used in immunohistochemistry method.

[0115] Table 3. Necessary process steps for immunohistochemical staining.

[0116] Microscopic evaluation: Immunohistochemical evaluation of the sections in each group was performed on a scale from 0 to 3+ according to an immunoreactive score of immunoexpression distribution and intensity on systematically randomly selected digitized images. Accordingly, it was evaluated as 0 in the absence of any staining, 1 + in weak immunoreactivity, 2+ in the presence of moderate immune reactivity and 3+ in the presence of strong (increased) staining. A binocular light microscope with a color digital camera (Olympus C-5050) integrated into a light microscope (Olympus BX-51 ) was used for image analysis. Histopathological evaluation was performed by two histologists at X10, X20, X40 and x100 magnifications without knowing the contents of the groups. H Skoru: Immunohistochemistry results could be assessed by a semi-quantitative approach used to assign an H-score (or “histo” score) to samples. First, membrane staining intensity (0, 1+, 2+ or 3+) was determined for each cell in a fixed area. With this method, the percentage of cells at each staining intensity level is calculated and finally, an H score was determined using the following formula:

[0117] [1 x (% cell 1+) + 2 x (% cell 2+) + 3 x (% cell 3+)]

[0118] The score ranges from 0 to 300.

[0119] Statistical analysis:

[0120] Size, zeta, PDI, viscosity and Ph measurements obtained at different temperature (4°C, 25°C and 40°C) and time points (baseline, Month 1, 3, 6 and 9) after stability tests were summarized using descriptive statistics (n, Mean, Standard Deviation, Change Range, Median, Minimum and Maximum values).

[0121] One-way analysis of variance (ANOVA) followed by Dunnet's test and Tukey's studentized range test and Student's t-test (only for the comparison of 4°C and 25°C averages at Month 9) were used to determine the mean changes in size, zeta, PDI, viscosity and pH measurements over time and to compare their means at different temperatures. Homogeneity of variances in ANOVA models was evaluated using Levene’s test. The fit of the models was examined and evaluations were made under the assumption of normal distribution. Type I error was accepted as 0.05 in statistical evaluations.

[0122] Evaluation of Change Over Time Compared to the Baseline: At each temperature level (4°C, 25°C and 40°C), one-way analysis of variance (ANOVA) followed by Dunnet's test was used to determine whether the mean change from baseline (0) was statistically significant.

[0123] Evaluation of Differences between Temperatures at the Same Time Point: One-way analysis of variance (ANOVA) followed by Tukey's studentized range test (HSD) was used to determine whether the differences between the mean temperature levels (4°C, 25°C and 40°C) for each time level (Month 1, 3 and 6) were statistically significant.

[0124] Student-t test was used to compare 4°C and 25°C means at Month 9.

[0125] The FTIR spectrum of the magnetic nanoparticle is given in Fig. 1. The characteristic Fe-0 bond of the magnetic nanoparticle was observed at 550-600 cm1. SEM image of the magnetic nanoparticle is given in Fig. 2. The size of the magnetic nanoparticle is between 15-23 nanometers and has a spherical shape. The sizes and morphology of the structures obtained by TEM analysis are consistent with the SEM results (Fig. 3).

[0126] TGA was used to investigate the thermal degradation of MNPs in the temperature range from 20 to 600°C. The thermal degradation graph of the MNP structure is given in Fig. 4. According to the thermogram curve, the weight loss of the MNP core is about 8%. This loss is thought to be due to the evaporation of water bound to the magnetic nanoparticle.

[0127] The FTIR spectrum of APA-functionalized MNP (APA-MNP) is shown in Fig. 5. In the FTIR spectrum, N-C band at 1131 cm-1and N-H band at 1637 cm-1were observed. These bands confirmed that APA interacts with the surface of the magnetic nanoparticle. In addition, the structure of the band belonging to the Fe-0 bond between 550-600 cm-1, which shows the magnetic character, is maintained. Thus, it was determined that the structure maintained its magnetic character. As can be seen from the thermogram curve of APA-MNP (Fig. 6), evaporation of water took place first (about 2%). Subsequently, a weight loss of APA-MNPs was also observed around 290°C. This loss is due to the APA reaching its melting temperature. The total weight loss of APA- MNPs is approximately 4%. The weight loss of the APA molecule alone is thought to be about 2%. The decrease in weight loss compared to MNP is thought to be due to the decrease in the interactions of water molecules on the magnetic nanoparticle surface with the addition of APA to the medium.

[0128] Surface amino groups in the structure of APA-modified magnetic nanoparticles were quantitatively determined using the trinitrobenzenesulfonic acid (TNBS) method. When the data obtained from TGA and the data obtained by TNBS method were compared, it was seen that the two methods were compatible with each other. SEM image of APA-surface functionalized magnetic nanoparticle is given in Fig. 7. APA-MNP sizes are between 17-23 nm and have a spherical structure. No difference in size and structure was observed between MNPs and APA-MNPs. The sizes and morphology of the structures obtained by TEM analysis are consistent with the SEM results (Fig. 8).

[0129] Magnetic characterization of MNPs and APA-MNPs was performed by VSM analysis. The magnetization curves measured at room temperature for magnetic nanoparticles and APA-modified magnetic nanoparticles are compared in Fig. 9. The magnetic saturation value for MNP and APA-MNP was 57 emu / g and did not differ. In this case, it was observed that there was no decrease in magnetic characteristics after functionalization with APA.

[0130] The change in zeta potential, hydrodynamic size and polydispersity (PD) due to surface modification of magnetic nanoparticle with APA molecule was observed and given in Table 4.

[0131] Table 4. Hydrodynamic dimension, zeta potential and PD! values of MNP and APA- MNP. (n=3)

[0132] The zeta potential value of MNP was found to be 7.03±4.52 and the zeta potential graph is given in Fig. 11 A. It is known from the literature that in neutral biological media, empty magnetite nanoparticles are almost uncharged (+5 mV) and tend to agglomerate. The higher the zeta potential, the more charge is present on the surface of the nanoparticles. The zeta potential of the magnetite surface was significantly increased through APA modification (22.83±1.72 mV). This is due to the fact that the amino groups give a continuous positive charge throughout the entire low pH range due to the presence of NH3+groups exposed to the surface.

[0133] FTIR graph of Dowex anion exchange resin (chloride form) is given in Fig. 12A. The band around 3400 cm1in the graph is due to stretching and bending vibrations of hydroxyl groups. The band formations at 3020 cm-1are due to the aromatic stretching between C-H. The band observed at 2900 cm-1is thought to be due to the C-H bond in the polymeric chain of the anion exchanger. The C=C stretching, which provides band formation at 1600 cm-1, is due to the aromatic ring in the structure of the resin. The characteristic peak at 1475 cm-1is thought to be due to the C-H bending and C-N stretching vibration of the quaternary ammonium functional group in the anion exchange resin. The FTIR graph of Dowex anion exchange resin in chloride form after treatment with acetic acid is given in Fig. 12B. Due to the characteristic feature of acetic acid, band formation was observed at 1213 cm1due to CH3symmetric deformation in the COH bond. The band observed at 1709 cm-1is due to the C=O stretching in acetic acid. In addition, the intensity of the band around 582 cm-1due to the chlorine element observed in Fig. 12A decreased. Considering all these results, it was determined that the conversion from Dowex chloride form resin to acetate form was successfully realized.

[0134] In the FTIR spectrum of the AG fraction, the broad band between 3600 and 3000 cm1corresponds to the vibration of hydroxyl groups (Fig. 13A). The band observed around 2878 cm’1corresponds to the methyl group vibrations in the structure. The band at 1598 cm’1is due to the C=O double bond in the uronic acid in the structure of arabinogalactan and the band at 1031 cm1is due to C-0 interactions in the glycoside bond in the structure of the polysaccharide. As arabinogalactan oxidizes, it loses H ions and turns into aldehyde form. The decrease in the intensity of the bands arising from hydroxyl groups in oxidized arabinogalactan was considered as an indication that oxidation had occurred (Fig. 13B).

[0135] The FTIR spectrum of the magnetic nanoparticle (ANP) conjugated with oxidized arabinogalactan is given in Fig. 14. The band observed between 550-600 cm1in the structure of ANP belongs to the Fe-0 bond and shows that the structure retains its magnetic character. However, there was a decrease in the intensity of the band. This decrease indicates that the conjugation of the obtained APA-MNPs with Ox-AG was successful. The band observed at 1334 cm1in the ANP spectrum is due to the C-0 bond in the structure of polysaccharides. The band originating from amine groups in APA-MNP and observed around 1630 cm-1disappeared in the structure of ANP. This indicates that Ox-AG successfully binds with the amine groups in the structure of APA- MNP. In addition, the band observed at 1724 cm’1in the structure of oxidized arabinogalactan disappeared in the ANP spectrum. This is thought to be due to the bond formation of amino groups on the surface of the magnetic nanoparticle with the dialdehyde structure formed as a result of oxidation. In addition, the band observed at 2878 cm-1belongs to the C-H bond in the aldehyde structure and a decrease was observed in this band. All these results confirm that conjugation was successfully achieved.

[0136] The thermogram curve of ANP is given in Fig. 15. As can be seen from the thermogram curve, evaporation of water took place first (about 5%). Subsequently, mass loss continued in a rapid and uninterrupted way. It is known in the literature that the melting temperature of arabinogalactan and APA group is in the same range. Considering this information, it is estimated that the mass loss continued uninterruptedly. The total weight loss of ANPs is about 16%. The weight loss of APA and arabinogalactan is thought to be about 11%. The higher total weight loss in ANP compared to APA-MNP is thought to be due to the incorporation of arabinogalactan into the structure.

[0137] In addition, the binding of arabinogalactan to nanoparticles was quantitatively controlled by the TNBS method. The decrease in the number of amino groups present after coating of magnetic nanoparticles not coated with arabinogalactan indicates that some of the free amino groups of the nanoparticles are bound by arabinogalactan.

[0138] SEM image of ANP is given in Fig. 16. The ANP size is between 25-35 nm and the structures have a spherical shape. The sizes and morphology of the structures obtained by TEM analysis are consistent with the SEM results.

[0139] The zeta potential, hydrodynamic size and PDI value of the ANP structure were analyzed with the ZetaSizer device. When the zeta potential values were examined, the zeta potential of APA-MNP was measured as +22.83±1.72 mV and the zeta potential value of ANP obtained after coating with arabinogalactan changed to - 27.03±5.87 mV and a significant decrease in the zeta potential value was determined. The presence of negatively charged oxidized arabinogalactans, which do not participate in the structure during ANP formation, gave the structure a significant negative charge. At the same time, the reduction of amine groups due to the binding of APA-MNP and Ox-AG caused a decrease in the positive charge. This also confirms that MNPs were successfully coated with arabinogalactan.

[0140] When the hydrodynamic size analysis of the obtained ANPs was examined, it was observed that the size of 184.7±6.36 nm measured in APA-MNPs decreased to 78±1 .47 nm as a result of arabinogalactan coating. This significant decrease is linked to the fact that arabinogalactan coats the surface of the magnetic nanoparticle, transforming it into a more stable structure. MNPs without any coating tend to cluster and agglomerate. The coating of arabinogalactan minimized the tendency for aggregation in the structure, thus leading to a significant reduction in nanoparticle size. As a result of coating the magnetic nanoparticle with a polymeric structure, its hydrodynamic sizes decreased. The sizes and morphology of the structures obtained by TEM analysis are consistent with the SEM results (Fig. 17).

[0141] VSM analysis was performed to characterize the magnetic properties of the ANP. Magnetization curves measured at room temperature for ANP are given in Fig. 18. The magnetic saturation value for APA-MNP was 57 emu / g, while the magnetic saturation value for ANP was 43 emu / g. It is seen that the magnetic saturation value decreases with the coating of the surface of the magnetic nanoparticle with oxidized arabinogalactan.

[0142] The graph of binding efficiency (%) and binding amount (pg Dox / mg np) of different concentrations of doxorubicin to ANPs obtained from HPLC analysis are shown in Fig. 19. Using a concentration of 500 pg / mL Dox, ANP contained 79.22 pg Dox per mg nanoparticle and the drug binding efficiency was 78.17%. It was planned to select a concentration of 500 pg / mL as the optimum drug concentration due to both the high binding efficiency and the high amount of drug bound compared to other lower concentrations. The optimum concentration should be decided by considering the size results.

[0143] The hydrodynamic size (nm) and PDI values of DANPs obtained by performing drug loading experiments at different concentrations (50, 100, 250, 500, 1000 pg / mL) are given in Table 5. The hydrodynamic size of ANPs is 78.82±1.88 nm. It was observed that the hydrodynamic sizes of DANPs gradually increased with increasing drug concentrations as a result of Dox loading. The hydrodynamic size of the nanoparticles obtained with 500 pg / mL initial doxorubicin concentration was 96.91 ±7.03 nm. Considering both size results and Dox binding efficiency, 500 pg / mL was considered the optimum concentration value and will be used in future in vitro and in vivo studies.

[0144] Table 5. Hydrodynamic dimension (nm) and PDI values of ANPs bound to doxorubicin at different concentrations of (n=3).

[0145] FTIR graph of DANP formulation is given in Fig. 20. In the spectrum, bands at 2930 cm'1(C-H), 1620 cm-1(N-H), 1400 cm-1(C-C) and 1055 cm-1(C-O) of doxorubicin were observed. These results confirm the presence of doxorubicin on arabinogalactan- coated ANPs.

[0146] TGA graph of DANP formulation is given in Fig. 21. The first mass loss of DANP formulation occurred between 200-300°C. In this range, there was a mass decrease due to APA, AG and Dox. At a temperature of 300 and above, there was a mass loss due to AG and Dox. SEM image of DANP formulation is given in Fig. 22. DANP size is between 30-35 nm and its morphology is spherical in shape.

[0147] When the TEM image of the DANP forrmulation was examined, it was seen that its structure had a spherical morphology and sizes ranging between 20-30 nm. The clustering and aggregations observed in SEM analysis are not present in TEM images and spherical structures are clearly seen. This fully confirms the morphology of the obtained DANP formulation.

[0148] The zeta potential of the DANP sample at the optimum concentration was studied. It is known that at pHs lower than neutral pH, the primary amine group in the drug structure induces a positive charge on the doxorubicin molecule (cationic Dox). Zeta potential measurement revealed that the surface charge of ANP was negative (-27.03±5.87 mV). After drug loading, an increase in the surface charge of DANP from -27.03±5.87 mV to -15±3.67 mV was observed. This result reveals that positively charged drug molecules successfully bind to negatively charged ANPs through electrostatic interactions. It has been reported that particles with zeta potential values between |5| and |15| mV are at the flocculation limit; particles with potential values between |5| and |3| mV can undergo maximum flocculation. It was concluded that the zeta value of DANPs, which is - 15±3.67 mV, is suitable for use in in vitro and in vivo studies.

[0149] The magnetic characterization of the obtained DANP formulation was determined by VSM analysis and is shown in Fig. 24. It was observes that the magnetic saturation value for DANP was 41 emu / g.

[0150] As a result, it was observed that the coating of the surface of the magnetic nanoparticle synthesized by precipitation method with arabinogalactan was successfully realized together with the characterization studies. Then, the binding of doxorubicin to the structure by adsorption method was confirmed by FTIR, TGA, hydrodynamic size and zeta potential analysis. As a result of drug binding optimization, 500 pg / mL Dox concentration with a binding efficiency of 78.17% was determined as the optimum concentration. The hydrodynamic size of the final formulation obtained was determined as 96.91±7.03 nm and the zeta potential as -15±3.67 mV. According to the results of the characterization studies of DANP, which was determined as the final formulation, the structure obtained was found to be suitable for use in in vitro and in vivo studies. Subsequent studies were continued at the determined optimum concentration.

[0151] SerurmWhen the nanoparticle ratio is taken into account, it is seen in Table 6 that the increase in serum ratio does not have a great effect on the protein binding ratio. When all the data are examined, it is seen that the binding percentages of APA-functionalized nanoparticles, drug-containing and drug-free nanoparticles with serum proteins vary in the range of 12-22% and the amount of adsorbed protein increases with increasing plasma amount. Table 6. Amounts and percentages of serum protein bound to APA-MNP, ANP and DANP at varying rates.

[0152] APA-MNP, ANP and DANP formulations administered at varying concentrations have no hemolysis effect.

[0153] Free doxorubicin was placed in pre-wetted dialysis bags and drug release study was performed at 300 rpm in 10 mL of environments at pH 5.5±0.05, pH 6.5±0.05 and pH 7.4±0.05 at 37°C±0.5°C. The results of pH-dependent drug release study of free doxorubicin are given in Fig. 19. When the data obtained were analyzed, it was observed that the drug release continued rapidly and increasingly at pH 5.5 and pH 6.5, while it was slower and less at pH 7.4. The percentages of free doxorubicin release at pH 5.5, pH 6.5, and pH 7.4 in the first five hours were 61.82%, 86.72%, and 87.75%, respectively. At the end of Hour 24, the release percentages were 64.80; 89.54 and 90.44. The accelerated release of doxorubicin at lower pH is probably due to the elemental nature of doxorubicin with high solubility at low pH. Accordingly, doxorubicin release is hardly affected by a neutral pH.

[0154] The results of the release study of doxorubicin-containing arabinogalactan-coated magnetic nanoparticles using dialysis bag and water bath are given in Fig. 26. According to the data obtained, at the end of Hour 5, the percentages of doxorubicin release from magnetic nanoparticles at pH 5.5, pH 6.5 and pH 7.4 were 4.6%, 5.9% and 12%, respectively. At the end of Hour 24, the release percentages were 9.8 for pH 7.4, 12.3 for pH 6.5 and 15.9 for pH 5.5. When the release data of Hour 50 is analyzed, it is seen that the release at pH 5.5 reached 22.5%, higher than the other pHs. Doxorubicin release from DANPs started rapidly in the first five hours and continued in a controlled manner. These results suggest that bound drug molecules will be released in significant quantities and in a controlled manner in the slightly acidic microenvironment (pH 5.5) of the tumors.

[0155] The graph of the viability (%) values of free doxorubicin and DANP formulation on HepG2 cell line after 48-hour incubation is given in Fig. 27, respectively. When the percent viability graphs of free doxorubicin and DANP formulation were analyzed, an increase in the cytotoxic effect was observed with increasing concentrations and it was observed that DANP formulation produced a more controlled effect than free doxorubicin. At the same time, the effect of the drug-free ANP formulation on cell lines was also examined and it was observed that the ANP formulation did not have any cytotoxic effect.

[0156] IC50 values of free doxorubicin and DANP formulation in HepG2 cells are given in Table 7.

[0157] Table 7. IC50 values of free doxorubicin and DANP formulation in HepG2 cells after 48- hour incubation.

[0158] In our study, we aimed to achieve controlled release of Dox from magnetic nanoparticles. As seen in the literature, free drugs have higher cytotoxicity in in vitro administrations. In current cancer treatments, high doses of drugs are required for effective treatment at the targeted site. These high doses damage healthy cells as well as cancer cells, causing many unwanted side effects. The main object of drug carrier systems is to deliver long-term treatment with a controlled and slow release directly to the cancerous area, while at the same time achieving maximum effect with minimum dose. In drug carrier systems, IC50 values are higher because drugs are not released directly into the cell environment.

[0159] It was also found that even at the highest doses of the DANP formulation applied in the THLE-2 cell line, the cells had more than 99% viability. Within the scope of the studies, liver cancer cell line and healthy cell line data were compared and it was determined that doxorubicin-loaded arabinogalactan-coated magnetic nanoparticles caused cytotoxicity in HepG2 cell line, while they did not cause any cytotoxic effect in THLE-2 cell line.

[0160] The results of the hydrodynamic size, zeta potential, PDI, viscosity and pH measurements of the product at Time 0, Month 1 , 3 and 6 after dispersing / dilution of DANP formulations with 5% dextrose solution with the help of ultraso nicator are given in Tables 8-13 respectively. The zeta potential value measured in the DANP formulation diluted with water was found to be -15±3.67 mV, and it was observed that as a result of dilution with 5% dextrose, these values decreased to -36.49 mV for the measurement at Time 0, and when the measurements at 1 , 3 and 6 months were examined, a decrease occurred again and ranged between -17.96 and -27.34. Slightly acidic dextrose produces a good isotonic environment for negatively charged nanoparticles (in terms of electrostatic stability) and it has been emphasized in the literature data that the decrease in the zeta potential of negatively charged nanoparticles to more negative values is due to dextrose (Kaasalainen et aL, 2012).

[0161] When the stability results are examined, it can be said that the stability of the magnetic nanoparticle is maintained with the zeta potential below -25 mV in Month 1 and 3. As of Month 6, the zeta potential of the magnetic nanoparticle shows a tendency to form aggregation by decreasing in absolute value. When size analysis results of Month 6 were analyzed, it was seen that the magnetic nanoparticle reached the maximum size (173-183 nm). The interpretation of increased aggregation due to the absolute decrease in zeta potential and the increase in hydrodynamic size in Month 6 support each other. The zeta potential value continued to decrease in absolute value in Month 9 and 12. Therefore, it can be said that aggregation continues. This is supported by the result that the heterogeneity of the magnetic nanoparticle increases with the increase in PDI values with time. When zeta potential values were examined as a function of temperature, no significant difference was observed between these values. It is thought that the absolute value of the zeta potentials of magnetic nanoparticles decreases with time due to the increase in the size with an increase in the tendency of the nanoparticles to aggregate. It has been reported in the literature that the zeta potential is affected not only by the properties of the nanoparticles but also by the nature of the solution such as pH and ionic strength (Gumustas et aL, 2017). Therefore, another reason for the difference in zeta potential is thought to be due to the differences in the pH of the nanoparticles. It is seen that the zeta potential values are also compatible with the pH results. The decrease in the absolute value of the zeta potential value, i.e. approaching the positive value, is in parallel with the pH values reaching slightly more acidic data.

[0162] When the hydrodynamic size results were compared in terms of temperatures, it was observed that the temperature value in the storage conditions did not have a great effect on the particle size in the data obtained. When size analysis results of the magnetic nanoparticle of Month 9 and 12 were examined, it was observed that the size, which reached the maximum value in Month 6, was fixed at 151 -158 nm values in both months.

[0163] In intravenous administration, the drug enters the bloodstream directly. Normal blood pH is between 7.35-7.45. The pH values of the fluids and drugs used may differ from the pH value of the blood. Therefore, acidic or basic drugs can damage the tunica intima of the vein by sensitizing this layer. When the pH values in the stability results are examined, it is seen that until the Month 6, the values are around 6, which is close to the physiological pH, and as of Month 9, it becomes acidic and decreases to around pH 4.

[0164] HPLC analyses were performed to investigate the chemical stability of doxorubicin in the DANP formulation, however, since no peak of doxorubicin was observed in the samples, it is thought that there is no degradation in the structure of the magnetic nanoparticle in this process and doxorubicin remains inside the nanoparticles. This was thought to maintain the chemical stability of the DANP formulation.

[0165] When the viscosity results of the DANP formulation were analyzed, no significant difference was observed in the viscosity value during the first 6 months. By Month 9, the viscosity value increased from the range of 6-7 to the range of 17-18. The difference in viscosity is thought to be due to the degradation in the structure of the DANP formulation.

[0166] When the appearance and redispersibility parameter results of the DANP formulation were examined, it was observed that there was no difference in the appearance and redispersibility parameters of the magnetic nanoparticles during Month 12.

[0167] When all stability results are evaluated in terms of zeta values, hydrodynamic size, PDI and pH, it is considered that the optimum storage time of DANP formulation is 6 months and storage temperature is 4°C. The reason why it was determined as the first 6 months is due to the fact that nanoparticles below 200 nm are determined as appropriate in the literature for the treatment of hepatocellular carcinoma, although the sizes increase to the range of 170-180 nm (D'Souza and Devarajan, 2015). After Month 9, it is seen that the storage stability is lost due to the increasing PDI values and the decrease in the absolute value of the zeta potential.

[0168] Table 8. Measurement results of DANP formulation for Time 0.

[0169] Table 9. Measurement results of the DANP formulation for month 1.

[0170] Table 10. Measurement results of the DANP formulation for month 3. Table 11. Measurement results of the DANP formulation for month 6.

[0171] Table 12. Measurement results of the DANP formulation for month 9. Table 13. Measurement results of the DANP formulation for month 12.

[0172] Ex vivo IVIS-Spect imaging of heart, lung, liver and kidneys from mice sacrificed at Hour 1 and 3 after intravenous administration is given in Fig. 28. In ex-vivo images, the organs are, from left to right, heart, lung, liver, kidney. When IVIS-Spect images obtained at Hour 1 and 3 were compared, it was observed that DANE formulation accumulated more in the liver depending on time. In addition, while no irradiation was observed in the kidney at Hour 1 of IVIS-Spect imaging, DANP-induced irradiation was observed when the images obtained at Hour 3 were analyzed. This indicates that excretion of the DANE formulation through the kidneys has begun to occur. In addition, no irradiation was observed in the lung and heart in the I VIS imaging performed both at the end of Hour 1 and Hour 3, while irradiation was observed in the liver region where the magnet was intensely kept.

[0173] After TACE administration, heart, lung, liver and kidney were removed from the mice sacrificed at the end of Hour 1 and 3, similar to i.v. administration, and ex vivo IVIS- Spect imaging was performed and the images are shown in Fig. 29. When Hour 1 and 3 of administration were compared, it was observed that DANP formulation accumulated more in the liver in a time-dependent manner. Unlike intravenous administration, renal irradiation was also observed after TACE administration. This is thought to be due to the fact that the formulation is administered through the portal vein in TACE administration. As with intravenous administration, no DANP formulation was observed in the lung and heart on I VIS imaging both at the end of Hour 1 and Hour 3. Furthermore, when I VIS images of intravenous administration and TACE administration were compared, it was observed that DANP intensity in the liver was higher in TACE administration.

[0174] Intravenous administration and administration by TACE showed that the DANP formulation successfully reached the liver. When the Dox intensity in I VIS images was analyzed, it was observed that the formulation accumulated in the liver was more intense in TACE administration. Therefore, when the two routes of administration are compared with each other, TACE is considered to be a more advantageous route of administration. After intravenous administration of DANP formulation, time-dependent plasma concentrations of the drugs were determined by determining doxorubicin by HPLC analysis in plasma samples collected at certain time intervals (Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24). Noncompartmental pharmacokinetics of the results obtained from blood samples were evaluated in Phoenix WINNONLIN 7.0 (Pharsight, USA). After intravenous administration of doxorubicin, the parameter results such as plasma versus concentration plots and volume of distribution (Vz f obs), clearance (CI_F_obs), half-life (t1 / 2) are given in Tables 14 and 15.

[0175] Table 14. Pharmacokinetic parameters of intravenous administration of doxorubicin.

[0176] Table 15.Continuation of pharmacokinetic parameters of intravenous administration of doxorubicin.

[0177] After the administration of DANP formulation via portal vein, time-dependent plasma concentrations of the drugs were determined by determining doxorubicin by HPLC analysis in plasma samples collected at certain time intervals (Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24). Noncompartmental pharmacokinetics of the results obtained from blood samples were evaluated in Phoenix WINNONLIN 7.0 (Pharsight, USA). After intravenous administration of doxorubicin, the parameter results such as plasma versus concentration plots and volume of distribution (Vz f obs), clearance (CI_F_obs), half-life (t1 / 2) are given in Tables 16 and 17. Table 16. Pharmacokinetic parameters of portal vein administration of doxorubicin.

[0178] Table 17.Continuation of pharmacokinetic parameters of portal vein administration of doxorubicin.

[0179] In our study, the C max value of DANP formulation was found to be 1.28 pg / mL after portal vein administration. Cmax is the highest concentration monitored in plasma and is a very important parameter. It is considered an indicator of the speed of absorption. It is also evaluated in bioavailability (Agabeyoglu, 2009). Comparing each route of administration, it was observed that the Cmax value decreased to 1.28 pg / mL in portal vein administration, which was 12.33 pg / mL in intravenous administration. Based on the result of this parameter, it can be said that the absorption, i.e. bioavailability, of DANP formulation increases via portal vein administration.

[0180] Tmax value in both routes of administration, i.e. the time to reach maximum doxorubicin in plasma, was found to be 1 hour. Bobde et al. prepared N- 2hydroxypropyl methacrylamide polymeric micelles loaded with doxorubicin and coated with PEG. When the pharmacokinetic parameters of the prepared drug carrier system were examined, it was observed that Tmax value was 1 hour (Bobde et aL, 2021). It is seen that Tmax value found in the study is consistent with the literature.

[0181] In the literature, the half-life of doxorubicin in intravenous administration has been reported as 20-48 hours (Pfizer). The half-life of DANP formulation after intravenous administration was found to be 22.63 hours, while this value was found to be 9.3 hours in portal vein administration. In intravenous administration, the half-life was found to be 22.63 as the DANP formulation is distributed throughout the body by entering the systemic circulation.

[0182] While mice were under ketamine (120 mg / kg) and xylazine (16mg / kg) anesthesia, 1 x106luciferase-active HepG2 cells in 50 pL PBS were slowly injected into the right upper lobe of the liver to create an orthotopic tumor model (Fang et aL, 2017). The tumor size of the mice was then determined on an imaging device ( I VIS, Caliper Perkin Elmer) with in vivo imaging system and calculated using Equation 7. Mice with the determined tumor size were divided into treatment groups as i.v and portal vein administration. To determine the therapeutic efficacy of DANP formulation in the treatment groups, Dox-containing DANP formulation was administered i.v. 3 times a week for 14 days via tail vein. In portal vein administration, a single dose of Dox- containing DANP formulation was administered. Following DANP administration, a magnet was applied to the tumor liver area for 30 minutes.

[0183] After HepG2 cells were injected into mice and tumors were successfully formed, the tumors were imaged in the I VIS device and the initial tumor sizes were calculated. For IV administration, mice were grouped as free Dox group and DANP group. Treatment was started after the mice were grouped. On Day 7 and 14 of treatment, images of the mice were taken with the IVIS device and tumor sizes were calculated from these images. Tumor sizes for mice of IV treatment group are given in Table 18.

[0184] When Table 18 is examined, after free Dox treatment via intravenous administration, metastasis formation as well as tumor size increase occurred in Mouse (1 ) due to the administration. In Mouse (2), it was observed that death occurred due to the administration. In Mouse (3), the tumor size decreased and the tumor disappeared completely at the end of Day 14, while in Mouse (4), the tumor size calculated as 1403 mm3decreased to 1244 mm3on Day 7 and 872 mm3on Day 14 due to the administration. In Mouse (5), metastasis formation was observed despite a decrease in tumor size from 1285 mm3to 795 mm3due to the administration.

[0185] When the data of DANP treatment via intravenous administration were analyzed, it was observed that Mouse (1 ) died on Day 7 due to tumor. The tumor size of Mouse (2), Mouse (4) and Mouse (5) decreased with time and the tumors disappeared completely at the end of Day 14. In Mouse (3), the tumor size decreased from 1199 mm3to 11 1 m3at the end of Day 14 due to the administration. Intravenously administered DANP formulation resulted in more successful treatment than free Dox. Within the scope of these data, it can be said that the use of doxorubicin-containing dual-targeted drug carrier system for the treatment of hepatocellular carcinoma has advantages over free drug and creates an anticancer effect on the tumor.

[0186] Table 18. Tumor sizes (mm3) of mice in the intravenous treatment group, which are measured at baseline, Day 7 and Day 14.

[0187] After HepG2 cells were injected into mice and tumors were successfully formed, the tumors were imaged in the I VIS device and the initial tumor sizes were calculated. For P.V. administration, mice were grouped as free Dox group and DANP group. Treatment was started after the mice were grouped. On Day 7 of treatment, images of the mice were taken with the I VIS device and tumor sizes were calculated from these images. Tumor sizes for mice of P.V treatment group are given in Table 19.

[0188] When Table 19 was examined, it was observed that after free Dox treatment via portal vein administration, Mouse (1 ) died due to administration. In Mouse (2), tumor size decreased from 1198 mm3 to 695 mm3, while tumor metastasis to other regions was observed despite the decrease in tumor size in Mouse (3). When the data of DANP treatment via portal vein administration were examined, it was observed that the tumor size of Mouse (1 ), which was calculated as 698 mm3, decreased to 99 mm3, and the tumor size of Mouse (3) decreased from 1149 mm3 to 45 mm3. The tumor of 667 mm3 in size, which was seen in mouse (2), disappeared. Considering all these results, the therapeutic potential of the DANP formulation appears to be high.

[0189] When the anticancer effects of free Dox and DANP formulation are analyzed as a result of treatment with portal vein administration, it can be said that free Dox is inadequate in treatment and causes deaths, while DANP formulation has a high therapeutic potential. The fact that the DANP formulation is targeted to the acyaglycoprotein receptor, the expression levels of which increase in hepatocellular carcinoma, and that magnetic targeting is performed in the tumor area by applying a magnetic field has enabled more effective treatment than free drug.

[0190] Table 19. Tumor sizes (mm3) of mice in the portal vein treatment group, which are measured at baseline and Day 7.

[0191] After cell administration to nude mice using an orthotopic model, tumor size and survival were monitored by imaging the control group with initial sizes ranging between 600-1000 mm3 at certain intervals. On Day 7 of Control 1 , the tumor size of the mouse in the control group was found to be 6153 mm3 and it was sacrificed. On Day 7 of Control 2, multiple metastases were observed and the mouse was removed from the experiment and sacrificed. The other 2 nude mice in the control group died 10 days after cell administration. When all the data were analyzed, it was seen that DANP formulation had a higher therapeutic potential on tumor than free doxorubicin in both routes of administration. The biggest advantage of Fe3O4 nanoparticles as a drug carrier system is that they can be delivered to the desired area by applying a magnetic field from the outside. The main reason for choosing the magnetic nanoparticle as a drug carrier system in our study is to quickly create nanoparticles in the desired area and to provide a more effective treatment in that area. In the light of the information obtained from the literature and our study, a more effective treatment of doxorubicin in hepatocellular carcinoma was realized using a magnetic and receptor-targeted drug carrier system.

[0192] When the two routes of administration were compared, it was observed that although portal vein administration was lower and single dose, it was more effective than intravenous administration of 6 doses. Since it is known that the expression of the acyaglycoprotein receptor is increased in hepatocytes in hepatocellular carcinoma, it was thought that the arabinogalactan-bound drug carrier has the potential to travel magnetically and through the acyaglycoprotein receptor to the target site. In addition, it is thought that the difficulties observed in portal vein administration due to the small size of the mouse can be overcome in human administration and may be more advantageous with the magnetic target. When the in vivo results are fully evaluated, it is observed that the developed dual-targeted DANP formulation can be used effectively in the treatment of hepatocellular carcinoma.

[0193] Table 20 summarizes the mean blood parameter values of mice after intravenous administration of free doxorubicin and DANP formulation. Table 21 summarizes the mean blood parameter values of mice after portal vein administration of free doxorubicin and DANP formulation.

[0194] Table 20. Mean biochemistry values after intravenous administration of free doxorubicin and DANP formulation.

[0195] Table 21. Mean biochemistry values after portal vein administration of free doxorubicin and DANP formulation.

[0196] Among the biochemical parameters, ALT, AST and ALP are the parameters related to liver damage. These parameters show an increase or decrease in levels depending on liver damage. ALT, AST and ALP levels of mice administered intravenously and with portal vein administration increased in a tumor-dependent manner.

[0197] When the results of intravenous administration were evaluated, ALP and AST levels of mice administered with free doxorubicin increased compared to mice administered with DANP formulation. No differences were observed for other biochemical parameters in free doxorubicin and DANP formulation given via intravenous administration. When the results of portal vein administration were evaluated, ALT and AST levels of mice administered with free doxorubicin increased compared to mice administered with DANP formulation. In addition, it was observed that the BUN parameter, a marker of kidney damage, increased more in mice administered with free doxorubicin. No differences were observed for other biochemical parameters in free doxorubicin and DANP formulation.

[0198] In the literature, it has been confirmed in terms of biochemical parameters (ALT and AST) that free doxorubicin administration causes more damage compared to drug carrier system administration. The data obtained in our study are consistent with the literature. In both routes of administration, DANP formulation was found to have lower liver damage compared to free doxorubicin. In our project, our object to minimize the damage caused by traditional chemotherapy treatment to healthy tissues and to create a more effective treatment profile by using drug carrier systems was supported in terms of biochemical parameters.

[0199] Ex-vivo studies and pathological evaluation

[0200] Hematoxylin-Eosin staining was performed as histochemical staining. Ki-67 and myeloperoxidase staining was performed as immunohistochemical staining.

[0201] Ex-vivo analysis of pharmacokinetic study

[0202] There were minimal signs of dilatation of the vena centralis and sinusoids in the subjects of the intravenous administration group of DANP formulation. Moderate intracytoplasmic edema in hepatocytes and minimal dilatation in portal triads were observed. After the administration, it was observed that the effects decreased due to the increase in time. Fig. 30 A1 -A7 shows the hematoxylin eosin staining images of the livers of mice sacrificed at Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24 after intravenous administration of DANP formulation, respectively. Fig. 30 B1 -B7 shows the hematoxylin eosin staining images of the livers of mice sacrificed at Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24 after portal vein administration of DANP formulation, respectively.

[0203] There were moderate signs of dilatation of the Vena centralis and sinusoids in the subjects of the portal vein administration group of DANP formulation. Moderate / high intracytoplasmic edema in hepatocytes and minimal dilatation in portal triads were observed. It was observed that the effects decreased as the hours after the administration increased. Minimal number of pyknotic hepatocytes were found in this group. It was found that the number of cells with pyknotic nuclei increased with time. In the samples in this group, the intense intracytoplasmic edema was observed and glycogen granules were found to be dispersed in the hepatocytes. In this group, especially at Hour 18 and Hour 24, the alignment of cell cords was disrupted, and the cytoplasmic vacuolization and microvesicular steatosis were detected.

[0204] Fig. 31 A-E shows the hematoxylin eosin staining images of the liver of the treatment groups and control group in the formation of liver cancer model after intravenous administration of DANP formulation, portal vein administration of DANP formulation, intravenous administration of free doxorubicin and portal vein administration of free doxorubicin, respectively.

[0205] The livers of cancer control mice had an appearance consistent with hemorrhagic necrosis at X4 magnification. In addition, an intense sinusoidal congestion was found in these subjects in this group. Hepatocytes with the granular eosinophilic cytoplasms were observed in significantly higher numbers than normal. The presence of thick cell cords was detected. Moderate / high dilatation of the portal triads was observed.

[0206] Focal ischemic areas were locally detected in the livers of mice in the intravenous administration group of DANP formulation. There are signs of moderate dilatation in the vena centralis and sinusoids. Moderate intracytoplasmic edema in hepatocytes and minimal dilatation in portal triads were observed. In the samples in this group, the moderate intracytoplasmic edema and glycogen granules were found to be dispersed in the hepatocytes. Minimal dilatation of the portal triads was observed.

[0207] There were moderate / low signs of dilatation of the Vena centralis and sinusoids in the cases of the portal vein administration group of DANP formulation. Hepatocytes showed moderate / low level of intracytoplasmic edema and nuclei were deleted locally. There are signs similar to centroacinar necrosis. Minimal dilatation of the portal triads was observed.

[0208] The cases of intravenous administration group of free doxorubicin resulted in moderate / high dilatation of the vena centralis and sinusoids. There were deletion in hepatocyte nuclei. Moderate / low dilatation of the portal triads was observed.

[0209] In the cases of the portal vein administration group of free doxorubicin, dilatation of the vena centralis and sinusoids was detected. In hepatocytes, more nuclear deletion was observed compared to the treatment groups of intracytoplasmic edema, intravenous administration of DANP formulation, portal vein administration of DANP formulation and intravenous administration of free doxorubicin. Moderate / low dilatation of the portal triads was observed.

[0210] Fig. 32 A1 -A7 shows ki-67 staining images of the livers of mice sacrificed at Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24 after intravenous administration of DANP formulation, respectively. Fig. 32 B1 -B7 shows the anti-ki-67 staining images of the livers of mice sacrificed at Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24 after portal vein administration of DANP formulation, respectively. Since liver tissue has a reserve regenerative capacity, Ki-67 expression was detected in the minimal number of hepatocyte nuclei in all sections. Interestingly, Ki-67 expression increased in hepatostellate cells (ITO cells) located in the Disse space, which is the structure between hepatocytes and sinusoid endothelium, in a timedependent manner, suggesting that it may trigger the proliferation of cells that may have a possible liver fibrosis effect.

[0211] Fig. 33 A-E shows the Anti Ki-67 staining images of the liver of the treatment groups and control group in the formation of liver cancer model after intravenous administration of DANP formulation, portal vein administration of DANP formulation, intravenous administration of free doxorubicin and portal vein administration of free doxorubicin, respectively. The cancer control group was the group with the highest Ki- 67 expression for all experimental groups. Ki-67 expression was seen in hepatocytes and hepatostellate cells forming thick cords with granular eosinophilic cytoplasm.

[0212] In the intravenous administration group of DANP formulation, expression was found in hepatostellate cells located in dilated Disse spaces rather than hepatocytes. It was observed that in the intravenous administration group of DANP formulation, the expression was observed in significantly fewer cells compared to the cancer control group.

[0213] The portal vein administration group of DANP formulation had a histologic appearance similar to the intravenous administration group of DANP formulation and Ki-67 expression was found in hepatostellate and Kupffer cells. Ki-67 expression in the portal vein administration group of DANP formulation was lower than that in the intravenous administration group of DANP formulation.

[0214] In parallel with moderate / high level dilatation in the vena centralis and sinusoids and deletion of hepatocyte nuclei in the subjects belonging to the intravenous administration group of free doxorubicin, it was found that the proliferative effect on hepatocytes was the highest in all experimental groups except the cancer control group.

[0215] The portal vein administration group of free doxorubicin was similar to the intravenous administration group of free doxorubicin, but with higher levels of intracytoplasmic edema and more intense Ki-67 expression in hepatostellate cell.

[0216] Fig. 34 A1 -A7 shows Anti myeloperoxidase staining images of the livers of mice sacrificed at Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24 after intravenous administration of DANP formulation, respectively. Fig. 34 B1 -B7 shows the Anti myeloperoxidase staining images of the livers of mice sacrificed at Minute 15, Minute 30, Hour 1 , Hour 3, Hour 9, Hour 18 and Hour 24 after portal vein administration of DANP formulation, respectively. A time-dependent increase in myeloperoxidase activation was detected around the vena centralis and in hepatocyte cell cords due to Dox administration to liver tissue. Investigation of liver cancer therapeutic potential

[0217] Fig. 35 A-E shows the Anti myeloperoxidase staining images of the liver of the treatment groups and control group in the formation of liver cancer model after intravenous administration of DANE formulation, portal vein administration of DANE formulation, intravenous administration of free doxorubicin and portal vein administration of free doxorubicin, respectively.

[0218] Among the experimental groups, the highest myeloperoxidase activity was detected in the EBS-treated cancer control group. This was followed by intravenous administration of free doxorubicin, then portal vein administration group of free doxorubicin. Among the treatment groups, the groups showing the lowest myeloprexidase activity were intravenous and portal vein administration groups of DANE formulation.

[0219] The statistical results of the stability studies are detailed below. When the size parameter results were examined, it was seen that the differences between the samples were statistically significant when Month 1 , 3 and 6 of the samples at 40°C were compared with t=0 using the Dunnet test / Wilcoxon exact tests. In the size analysis results in Month 1 , when different temperature values were internally analyzed using the Tuskey HD test, the differences between 4-25°C and 4-40°C were statistically significant. In the size analysis results in Month 3, when different temperature values were internally analyzed using the Tuskey HD test, the differences between 4-25°C and 4-40°C were statistically significant. In the size analysis in Month 6, 9 and 12, when the different temperature values within each month were compared using the Tuskey HD test, it was seen that the difference was not statistically significant.

[0220] Table 22. Descriptive statistics data for size parameter in stability studies.

[0221] When the Zeta potential parameter results were examined, it was seen that the differences between the samples were statistically significant when Month 3, 6, 9 and 12 of the samples at 4°C were compared with t=0 using the Dunnet test / Wilcoxon exact tests. It was seen that the differences between the samples were statistically significant when Month 6, 9 and 12 of the samples at 25°C were compared with t=0 using the Dunnet test / Wilcoxon exact tests. It was seen that the differences between the samples were statistically significant when Month 3 and 6 with the samples at 40°C were compared with t=0 using the Dunnet test / Wilcoxon exact tests. In the Zeta potential analysis in Month 1 , 3, 9 and 12, when the different temperature values within each month were compared using the Tuskey HD / Exact Wilcoxon test, it was seen that the difference was not statistically significant. In the Zeta potential analysis results in Month 6, when different temperature values were internally analyzed using the Tuskey HD test, the differences between 4-25°C were statistically significant.

[0222] Table 23. Descriptive statistical data for zeta potential parameter in stability studies.

[0223] When the PDI parameter results were examined, it was seen that the differences between the samples were statistically significant when Month 3 and Month 6 of the samples at 40°C were compared with t=0 using the Dunnet test. In the PDI analysis in Month 1 , 3, 6, 9 and 12, when the different temperature values within each month were compared using the Tuskey HD / Exact Wilcoxon test, it was seen that the difference was not statistically significant.

[0224] Table 24. Descriptive statistics data for PDI parameter in stability studies.

[0225] When the viscosity parameter results were examined, it was seen that the differences between the samples were statistically significant when Month 1 and 3 of the samples at 40°C were compared with t=0 using the Dunnet test / Wilcoxon exact tests. In the viscosity analysis results in Month 1 , when different temperature values were internally analyzed using the Tuskey HD test, the differences between 25-40°C and 4-40°C were statistically significant. In the viscosity analysis results in Month 3, when different temperature values were internally analyzed using the Tuskey HD test, the differences between 25-40°C were statistically significant. In the viscosity analysis results in Month 6, when different temperature values were internally analyzed using the Tuskey HD test, the differences between 4-25°C and 25-40°C were statistically significant. In the viscosity analysis in Month 9 and 12, when the different temperature values within each month were compared using the Tuskey HD test, it was seen that the difference was not statistically significant.

[0226] Table 25. Descriptive statistics data for viscosity parameter in stability studies.

[0227]

[0228] When the pH parameter results were examined, it was seen that the differences between the samples were statistically significant when Month 3 and 6 of the samples at 40°C were compared with t=0 using the Dunnet test / Wilcoxon exact tests. In the pH analysis results in Month 3, when different temperature values were internally analyzed using the Tuskey HD test, the differences between 4-25°C, 25-40°C and 4-40°C were statistically significant. In the pH analysis results in Month 6, when different temperature values were internally analyzed using the Tuskey HD test, the differences between 4-25°C and 25-40°C were statistically significant. In the pH analysis in Month 1 , 9 and 12, when the different temperature values within each month were compared using the Tuskey HD / Exact Wilcoxon test, it was seen that the difference was not statistically significant.

Claims

CLAIMS1 . A magnetic field-targeted drug carrier system, characterized in that it comprises a nanoparticule which is conjugated with oxidized arabinogalactan to bind to the increased levels of the acyaloglycoprotein receptor in liver cancer, oxidized using Arabinogalactan and potassium periodate, surface -coated with 2- aminoethyl phosphonic acid with oxidized arabinogalactan, and containing Doxorubicin.

2. A magnetic field-targeted drug carrier system according to claim 1 , characterized in that the ratio of said Arabinogalactan to potassium periodate is 0.5-4:0.5-4 (Arabinogalactan otassium periodate) by weight.

3. A magnetic field-targeted drug carrier system according to claim 1 , characterized in that the ratio of said oxidized arabinogalactan to a suitable magnetic nanoparticle, which is surface-coated with 2-aminoethyl phosphonic acid, is 0.5-4:0.5-4 (oxidized arabinogalactan :magnetic nanoparticle which is surface-coated with 2-aminoethyl phosphonic acid) by weight.

4. A magnetic field-targeted drug carrier system according to claim 1 , characterized in that said Doxorubicin is in the dose range of 10-1500 pg / mL.

5. A drug carrier system according to any one of claims 1 -4 for use in carrying a drug effective in the treatment of liver cancer.

6. A preparation method of the magnetic field-targeted drug carrier system, characterized in that it comprises the steps of: i. incorporating oxidized arabinogalactan into the nanoparticle structure by conjugation with oxidized arabinogalactan for binding to the increased levels of the acyaloglycoprotein receptor in liver cancer, ii. oxidizing Arabinogalactan by mixing with potassium periodate, iii. realizing bond formation between oxidized arabinogalactan and surface-coated magnetic nanoparticles with 2-aminoethyl phosphonic acid by mixing in aqueous medium,iv. loading Doxorubicin into nanoparticles.

7. A preparation method according to claim 6, characterized in that it comprises the process steps of: i. incorporating oxidized arabinogalactan into the nanoparticle structure by conjugation with oxidized arabinogalactan for binding to the increased levels of the acyaloglycoprotein receptor in liver cancer, ii. oxidizing Arabinogalactan by mixing with potassium periodate in a ratio of 0.5-4:0.5-4 (Arabinogalactampotassium periodate) by weight, iii. realizing bond formation between oxidized arabinogalactan and surface-coated magnetic nanoparticles with 2-aminoethyl phosphonic acid by mixing in aqueous medium in a ratio of 0.5- 4:0.5-4 (oxidized arabinogalactan :surface-coated magnetic nanoparticle with 2-aminoethyl phosphonic acid) by weight, iv. loading Doxorubicin into the nanoparticle at a dose range of IQ- 1500 pg / mL.

8. A magnetic field-targeted drug carrier system prepared by a method according to any one of claims 6-7.

9. A drug carrier system according to claim 8 for use in carrying a drug effective in the treatment of liver cancer.

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