Patent Description:
Lung cancer accounts for <NUM> % of all types of cancer. <NUM> % of cancer-related deaths is resulted from lung cancer. Such high rate leads to need for making novel strategies in lung cancer treatment. The American Cancer Society defines the methods for cancer treatment as chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted therapy, stem cell or bone marrow transplantation and surgery. Among currently known treatment methods, targeted cancer therapy has not been commonly used yet. Additionally, targeting strategies that have already been used are quite limited.

Many chemotherapeutic agents are intravenously applied. Inhaled therapy is advantageous for lung cancer treatment as such therapy enables the chemotherapeutic agent to directly reach to malign tissues. However, many drugs are not applicable to such inhaled therapy applications.

In the present invention; a magnetic directed, ultrasound susceptible nano drug carrier system that comprises dual drug to be used in lung cancer therapies is provided. Unlike current methods, more than one targeting strategies are used in this newly developed system.

<NPL>, describes ultrasound/magnetic targeting with SPIODOX-microbubble complex for image-guided drug delivery in brain tumours.

<NPL>, describes SPIO-conjugated, doxorubicin-loaded microbubbles for concurrent MRI and focused-ultrasound enhanced brain-tumour drug delivery.

<NPL>, describes preparation and characterization of cancer cell targeted bionnimetic drug carrier systems and research their biopotential.

<NPL>, describes Bio-functionalization of magnetite nanoparticles using an aminophosphonic acid coupling agent: new, ultradispersed, ironoxide folate nanoconjugates for cancer-specific targeting.

<NPL>, describes new pemetrexed-peptide conjugates: synthesis, characterization and in vitro cytostatic effect on non-small cell lung carcinoma (NCI-H358) and human leukemia (HL-<NUM>) cells.

The primary aim of the invention is to provide an alternative and efficient solution to lung cancer treatment and thus a more effective therapy. With the drug system developed in the invention, a novel solution is oferred to lung cancer treatment, and this targeted chemotherapeutic therapy has numerous advantages as follows:.

The invention relates to the ultrasound susceptible magnetic directed nano drug carrier system designed to be used in lung cancer treatment. Magnetic nanoparticles are synthesized based on iron salts for magnetic targeting. A hexapeptide capable of disintegrating enzyme matrix metalloproteinase-<NUM> which is not available in healthy tissue but increases in solid lung tumors has been designed in an attempt to enable the system to release drug more selectively in lung cancers, and also is covalently bound to a surface of the magnetic nanoparticle. The drugs are pemetrexed and pazopanib.

Many scientists commonly agree on the idea that inhibition of only one pathway in cancer treatment would not yield fruitful outcomes that much. Therefore, in the drug carrier system subject to the present invention, the drugs that are routinely used in clinics are administered in combined manner. For this reason, two different chemotherapy agents, pemetrexed and pazopanib, are loaded to magnetic peptide nanoparticles for ensuring a dual therapy. Such dual use; when administered by combining antifolate effect of the pemetrexed and tyrosine kinase inhibition effect of the pazopanib, allows obtaining more amount of cytotoxic in cancer cells. Bondingbetween peptide and drugs is provided through thiol group of cysteine.

Magnetic conjugate obtained is retained within a lipid-structured liposome in order to enable the same to circumvent the circulation. The liposome; is formed using <NUM>,<NUM>-dipalmitoil-sn- glisero-<NUM>-fosfokolin(DPPC),<NUM>,<NUM>-dioleil-sn-glisero-<NUM>-fosfokolin(DOPC)and cholesterol, and is subsequently passed through membranes via an extruder and thereby made smaller in size. Following the size reduction process, chemically inert and biocompatible argon (Ar) gas is loaded into the liposome, which makes the later ultrasound susceptible, and thus bubble structure is formed. For increasing stability, the system is made ready by conducting a freezing and thawing process on dry ice. The ultrasound susceptible magnetic directed nano drug carrier system is directed to the tumor tissue with a magnetic area that is locally applied from the outside, and after targeting process, a nanoconjugate structure synthesized with the ultrasound that is locally applied is extruded from the nanobubble and thus enabled to accumulate on said region.

Biocompatibility of the system is tested based on bounding to serum proteins, haemolysis amount, and phagocytosis detection by macrophage cells, and also parameters such as determination of cytotoxicity and apoptosis effect are determined with cell culture experiments. In vivo tests are conducted by forming a tumor model with injection of the except small cell lung cancer line. Single dose and repeated dose toxicityexperiments, biodistribution and pharmacokinetics tests are made, in vivo tests are carried out and thus it is determined that the system is directed to the target tissue according to its aim and is capable of providing a therapeutic effect by being released in controlled manner over this region.

In the drug carrier system subject to the present invention; easier targeting with magnetic targeting, controlled drug release with ultrasound application, and specific targeting with matrix metalloproteinase-<NUM> is provided.

A method for preparing the ultrasound susceptible magnetic directed nano drug carrier system to be used in cancer treatment, particularly except small cell lung cancer treatment comprises the following process steps:.

Magnetic nanoparticles (MNP) are initiallysynthesized using the method of coprecipitation in order to obtain the magnetic nanoparticles carrying an active amine group. In the present invention, preferably <NUM>-<NUM> moles of iron (II) chloride (FeCl2) and <NUM>-<NUM> moles of iron (III) chloride (FeCl3) are dispersed in deoxygenated distilled water within a jacketed reactor at a constant temperature of <NUM>-<NUM>, preferably <NUM>°Cand at <NUM>-<NUM> rpm mixing speed. During this process, nitrogen (N<NUM>) gas is continuously passed through the medium. Subsequently, with addition of concentrated base solution, preferably concentrated ammonium hydroxide (NH<NUM>(OH)<NUM>) solution as precipitation agent to the solution, pH of the medium is adjusted to alkali (pH <NUM>-<NUM>). The solution is left to <NUM>-<NUM>-hour incubation for nucleation under the constant temperature (<NUM>-<NUM>) and powerful mixing. The nanoparticles formed at the end of the duration are separated with magnetic decantationand subsequently washed with distilled water and ethanol (EtOH). Washed nanoparticles are dried nightlong in an incubator at the temperature of <NUM>-<NUM>, preferably <NUM>, and thus MNP crystals are obtained. Scanning electron microscope (SEM) image as to MNP samples is given in <FIG>.

Numerous methods are provided for magnetite synthesis. The method used has a direct effect on character of the magnetite obtained. Coprecipitation method is preferred in the invention with regard to proper shape, size, magnetic power and simple applicability of the method.

Ammonium hydroxide is used as a precipitation agent in synthesis of magnetic nanoparticles by use of copresipitation method. Basic character of the ammonium hydroxide enables the medium to be alkali.

Oxygen is removed from the medium by passing nitrogen gas through the same in the invention. Removing oxygen from the medium is important for preventing oxidation to different derivatives of the magnetite. In addition, size of the magnetic nanoparticles generated in an oxygen-free medium is much smaller.

Magnetic nanoparticles are synthesized with various bases under alkali conditions. The reaction occurring when magnetic nanoparticle is formed is as the following;.

Fe+<NUM> + 2Fe+<NUM> + <NUM> OH- Fe<NUM>O<NUM> + <NUM><NUM>O.

As can be seen in the reaction, the mole ratio of <NUM>:<NUM> is significant in terms of magnetic nanoparticle formation.

In the invention, experiments are conducted preferably at the temperature of <NUM> in order to allow the reaction to occur.

A high speed is required in order to ensure crystallization and solid catalyzed reaction. As a higher speed allows smaller and homogeneous nanoparticle formation, higher speeds are preferred for the experiments conducted in the invention.

<NUM>-Aminoethylphosphonic acid (APA) solution is added to the medium comprising magnetic particle and having a pH of <NUM>-<NUM>. Subsequently, it is left to incubation for a reaction at <NUM>-<NUM> and <NUM>-<NUM> rpm. At the end of the duration, the MNPs carrying the active amine group are washed with distilled water with the help of magnetic decantation and made ready to disperse in phosphate-salt-buffer (PBS).

Prior to conjugation of the Pemetrexed drug with peptide, a modification experiment with the Pemetrexed is conducted and the drug is enabled to be chloro-acetylated. Therefore, two-step modification is applied. In the first step, methoxylation and in the second stepchloracetylenationis performed. In the first modification; <NUM>-<NUM> mmol of thionyl chloride (SOCl<NUM>) is slowly added to cold <NUM>-<NUM> of methanol (MeOH) at -<NUM>-(-<NUM>)°C and subsequently left to reaction at -<NUM> for <NUM> minutes. At the end of the duration, the temperature of the reaction medium is increased to <NUM>-<NUM> by adding <NUM>-<NUM> of pemetrexedin sodium salt, and incubated for <NUM>-<NUM> hours by being mixed under a condenser. Sodium chloride (NaCl) formed following the reaction is separated with filtration and evaporated out with methanol vacuum.

The excess of the compound SOCl<NUM> is removed by being successivelydissolved in hot methanol. The product obtained is crystallized in ethanol and the crystals formed are separated from the medium by filtration, and dried by being treated with diethylether ((C<NUM>H<NUM>)<NUM>O). Pem (OMe)<NUM> crystals in light green colorare obtainedas a result of the first-step modification of the Pemetrexed.

After completing this first step, chloroacetic acid (ClCH<NUM>CO<NUM>H) group is formed in the methoxylatedPemetrexed. In the second-step modification, Pem(OMe)<NUM>crystals are dissolved within <NUM>,<NUM>-<NUM>,<NUM> of dimethyl formamide (DMF). Subsequently, <NUM>-15µl of diisopropylethylamine and <NUM>-<NUM> of chloroacetic anhydride are added. This medium is left to reaction for <NUM>-<NUM> hours at the temperature of <NUM>-<NUM>. At the end of the duration, acetonitrile (ACN): distilled water (containing <NUM>-<NUM>% of trifluoroacetic acid (TFA)) and <NUM>-<NUM> of distilled water at the ratio of <NUM>%:<NUM>-<NUM>%:<NUM> (v/v) are added to the mixture and it is left to incubation for <NUM>-<NUM> hours at room temperature. Following the incubation, the yield is precipitated by adding distilled water to the medium and removed from the medium by means of centrifuge method. The yield is crystallized in ethanol and left to dry after being treated with dietylether. Consequently, chloroacetylated permetrexed (ClAc-PEM (OMe<NUM>)) yield is obtained. HPLC chromatograms as to Pemetrexed and modified species are given in <FIG>, <FIG>.

Pemetrexed shows activity over the carboxyl (-COOH) group available in its structure. The pemetrexed needs to be chloroacetylated to be able to be conjugated with peptide over thiol group. Not to conduct chloroacetylation over the -COOH groups, these groups are initially preserved by being methoxylated. This is the first-step modification. Subsequently, with the second-step modification, the chloroacetyl group through which it would be bonded to the peptide is formed.

In modification experiment of the pazopanib, the drug is converted to reactive form by bonding the chloroacetate with the method proposed in chloroacetylation reaction carried out in the second step of the pemetrexed modification to the amine (NH<NUM>) group bonded to sulfur dioxide (SO<NUM>) available on the structure. The first step of the the modification method proposed for the pemetrexed in the pazopanib modification is skipped and only the second step is applied. HPLC chromatograms as to the pazopanib and its modified state are given in <FIG>.

The pazopanib is bonded to peptide over thiol group. As it has no functional group that needs to be preserved, the only modification in the 2nd step of pemetrexed modificationis conducted in order to form the chloroacetyl group through which it would be bonded to the peptide.

With reference to areas of peaks of the Pemetrexed and Pem(OM<NUM>); it is discovered that the sample analyzed in calculations conducted in the equation of standard graphic has <NUM>,<NUM> ppm of free pemetrexed and <NUM>,<NUM> ppm of Pem (OMe<NUM>). According to this result, it is seen that the first-step modification yield of the drug is <NUM>%.

In the sample introduced to the HPLC analysis following the second-step modification of the drug Pemetrexed, peak of the Pem (OMe<NUM>) is observed, which is a free drug and a yield of the first modification. It is seen that the retention time in the yield analyzed is <NUM>,<NUM> and as a result of the calculation carried out from the area of this peak, ClAc-Pem(OMe2) is found, which is a second modification yield of <NUM> ppm. As seen from <FIG>, as the peak of the free and first modified drug is not observed, it is determined that the second modification of the drug occurred <NUM>%.

According to the calculations conducted from peak areas of the Pazopanib, it is found that the samplehas <NUM>,<NUM> ppm of free pazopanib and <NUM>,<NUM> ppm of modified pazopanib. When having yield calculation made, it is seen that the modification of the drug Pazopanib occurred at the rate of <NUM>%.

The peptide structure designed for the aim is like Ala-Lys-Ala-Leu-Arg-Cys. The modified pemetrexed and pazopanib are bonded over the thiol group of cysteine amino acid of peptide with application of the same method. In the peptide-drug conjugation, the reaction between the drug and the peptide involving cysteine, tris buffer: DMF (<NUM>:<NUM> - <NUM>:<NUM>, %v/v) solvent mixture is used. Initially, <NUM>,<NUM>-<NUM>,<NUM> mmol of modified drug is dissolved in <NUM>-<NUM> of DMF and subsequently, <NUM>-<NUM> <NUM>-<NUM> tris buffer (pH <NUM>-<NUM>) is added to the medium. Then, <NUM>-<NUM> mmol of peptide is slowly added to the reaction medium. The samples are left to incubation for a duration <NUM>-<NUM> hours in a shaking incubator at room temperature. At the end of the period, the solvent is removed and peptide-drug conjugations are obtained.

For this process, <NUM>-<NUM> of MNP-NH2; is mixed with sonication within PBS (pH: <NUM>-<NUM>)containing <NUM>-<NUM> of polysorbate <NUM> (tween <NUM>). Peptide-pemetrexed and peptide-pazopanib conjugatesare added to the medium such that the reaction medium has <NUM>-<NUM> of peptide-pemetrexed and <NUM>-<NUM> of peptide-pazopanib conjugates. Afterwards, by adding <NUM>-<NUM> mmol of N-ethyl-N'-(<NUM>-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and <NUM> - <NUM> mmol of N-hydroxysuccinimide (NHS), it is left to incubation under nitrogen gas at <NUM>-<NUM> for <NUM>-<NUM> hours for completing the reaction. At the end of the duration, it is centrifuged at <NUM>-<NUM> rpm, magnetite (Fe<NUM>O<NUM>) -peptide-drug conjugates are precipitated, and supernatant is removed. The unreacted excess peptide-drug conjugations are washed with nanoparticulates PBS for removing EDAC and NHS from the medium and are removed from the medium by being centrifuged at <NUM>-<NUM> rpm. FTIR spectrum of the magnetic nanoparticulate conjugate (MNP-PPP) bonded to peptide-pemetrexed-pazopanib is given in <FIG>.

As the magnetic conjugate can only be separated from supernatant by precipitating and purged from impurities at high rpms, experiments in the invention are conducted at high speeds.

When chemical structure of the magnetic-peptide-drugs conjugate is examined, with large peaks in the <NUM>-<NUM>-<NUM>of wavelength area of N-H stretching peaks of the amine (-NH<NUM>) groups available in structure of the drugs and peptide and the large peak in the <NUM>-<NUM>-<NUM>ofarea of the C-N groups, the presence of these groups is verified. Aromatic ring C-H stretching vibration at <NUM>-<NUM>, in-ring C-C and C-H stretching vibrations at <NUM>-<NUM> and <NUM>-<NUM>, available in the structure of drugs and peptide,were observed. CH stretching at <NUM>-<NUM>, O-H bending peak between <NUM>-<NUM>-<NUM>, and also C = N peak expected to be between <NUM>-<NUM>-<NUM> at <NUM>-<NUM> and <NUM>-<NUM>were seen at very weak intensity. That the <NUM>-<NUM>of vibration band showing the structure as to Fe-O link available in the Fe<NUM>-O<NUM>molecule free from FTIR spectrum is observed at <NUM>-<NUM>when it is bonded to the molecule structure was revealed in the magnetic nanoparticle structure synthesized by Lubambo et al. in <NUM> (Lubambo et al. The drugs for the magnetic structure prove that the magnetic structure is bonded with the drugs and peptide in accord with the peak literature in <NUM>-<NUM>of finger print region observed in FTIR spectrum of the nanoparticle synthesized with peptide and Fe<NUM>-O<NUM>.

<NUM>,<NUM>-dipalmitoyl-sn-glycero-<NUM>-phosphocholine (DPPC), <NUM>,<NUM>-dioleyl-sn-glycero-<NUM>-phosphocholine (DOPC), and cholesterol (CH) mixtures (<NUM>-<NUM> DPPC, <NUM>-<NUM> DPOC, <NUM>-<NUM> CH) are dissolved in <NUM>-<NUM> of chloroform. Then, organic solvent is evaporated out by passing nitrogen gas in a water bath at the temperature of <NUM>-<NUM> and this process is proceeded until a lipid film is formed around a vial wall. Afterwards, the entire solvent is left to rest for <NUM>-<NUM> hours under high pressure vacuum. At the end of the duration, the lipid film is left to rest for <NUM>-<NUM> minutes at room temperature with addition of nanoconjugate mixed in <NUM>-<NUM> <NUM>-<NUM> of mannitol solution. Then, this mixture is left to incubation in sonic bath for <NUM>-<NUM> minutes and thus liposome formation is acquired. In an aim to obtain nano-size liposomes, polycarbonate membranes and extruder system having a pore diameter of <NUM> and <NUM> nanometers are used. For synthesis of nanobubbles, compressed nitrogen gas at <NUM>-<NUM> atm is passed through the liposomes for <NUM>-<NUM> minutes. Then, they are freezed on dry ice for <NUM>-<NUM> minutes. Freezed nanobubbles are taken from dry ice and left to dissolve to be at room temperature. The step of freezing and thawing is repeated for <NUM>-<NUM> times. The image of high contrast permeable electron microscope (C-TEM) of NB-<NUM> (A) and NB-<NUM> (B) is given in <FIG>.

It was discovered that the hydrodynamic size of the bubble (NB-<NUM>) form that was passed through the membranes having a pore diameter of <NUM> was <NUM>; the hydrodynamic size of the bubble (NB-<NUM>) form that was passed through the membranes having a pore diameter of <NUM> was <NUM>. When considering the results, it can be concluded that NB-<NUM> is suitable to be used in inhalation applications, on the other hand, NB-<NUM> is suitable to be used in intravenous applications. That the nanobubbles obtained have a global shape is determined with reference to the TEM images of <FIG>.

The nanoconjugate comprising magnetite can be encapsulated to the microbubble structure at a ratio of <NUM>%. When nano is being formed liposome structures are opened and they are reformed in nano size. During this formation some part of the content is not encapsulated and the other part remains in the membrane. Due to this reason the encapsulation yield of MNP-PPP is reduced as the size is reduced.

It has been noted that the samples of MNP-PPP, liposome (sieved through a <NUM>'lik pore diameter membrane) and bubble (sieved through a <NUM>'lik pore diameter membrane) created high rates of cytotoxicity with increasing concentration for all cells. It has been determined that a toxic effect has been formed in the CCD-34Lu cell which is a healthy cell similar to the effect formed in cancerous cells. It is believed that the damage that may be caused by the drug carrier in healthy cells can be changed in vivo. It has been noted that MNP-PPP samples (<FIG>) created <NUM>-<NUM>%, liposome samples (<FIG>) created <NUM>-<NUM>% and nanobubble samples (<FIG>) created <NUM>-<NUM>% cytotoxic effect at aconcentration of <NUM>µg/mL for five cell lines after <NUM> hours.

The IC<NUM> values for <NUM> hours of the MNP-PPP sample has been recorded as <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the A549-luc cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the HTB- <NUM> cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the CRL5807 cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the CRL5826 cell and <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the CCD-34Lu cell.

The IC<NUM> values for <NUM> hours of the Liposome samples has been recorded as <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the A549-luc cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the HTB- <NUM> cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the CRL5807 cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the CRL5826 cell and <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the CCD-34Lu cell.

The IC<NUM> values for <NUM> hours of the NB-<NUM> samples has been recorded as <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the A549-luc cell; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for the HTB-<NUM><NUM> cell; CRL5807 hücresi için <NUM>,<NUM>±<NUM>,<NUM>µg/mL; CRL5826 hücresi için <NUM>,<NUM>±<NUM>,<NUM>µg/mL ve CCD-34Lu hücresi için <NUM>,<NUM>±<NUM>,<NUM>µg/mL'dir.

A brief period of ultrasound has been applied to cells following drug administration in order to see if the cytotoxicity of the NB-<NUM> sample was altered or not. In the analyses carried out on the A549-luc cell as it shall be working in in vivo assays the IC<NUM> value on the <NUM>th hour of the NB-<NUM> sample has been calculated as <NUM>,<NUM>±<NUM>,<NUM>µg/mL. When we look at the value if ultrasound was not applied, it has been noted that the IC50 value was decreased a little following ultrasound application (<NUM>,<NUM>±<NUM>,<NUM>µg/mL for <NUM> hours; <NUM>,<NUM>±<NUM>,<NUM>µg/mL for <NUM> hours). Therefore it has been decided that nanobubble structures are more effective in releasing the drug inside their structure as being susceptible to ultrasound.

The amounts of the cells at the live/necrotic/proapoptotic/apoptotic stages have been determined during the flow cytometry analysis carried out in order to understand the apoptosis effect of NB-<NUM> on the A549-luc cells. It has been noted that the NB-<NUM> sample at a concentration of <NUM>,<NUM>µg/ml had apoptotic effect at a ratio of <NUM>,<NUM>% necrotic, <NUM>,<NUM>% proapopotic and <NUM>,<NUM>% apoptotic in comparion to the control group cells.

As the plasma protein amount can vary from person to person, trials have been carried out in order to obtain nanobubble and plasma concentrations at ratios of <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM> (plazma:nanobubble). It has been noted that the serum protein bining amount increased depending on the increased amount of nanobubbles following the trial. The <NUM>:<NUM> ratio amount of binding has been determined as <NUM>%.

Haemolysis trials have been carried out by incubating the NB-<NUM> sample with erythrocytes at different concentrations. Haemolysis has not been observed in comparison to the positive controls at other concentrations up to <NUM>µg/ml concentration of nanobubbles that have been added into the blood samples at varying ratios that have been adjusted to the <NUM>% hematocrit rate. At a concentration of <NUM>µg/ml however a very low amount of haemolysis has been determined.

The cells have been incubated with nanobubbles for an hour in order to determined phagocytosis in macrophage cells of the NB-<NUM> sample and at the end of the one hour period the morphological structures of the cells have been examined and their numbers have been determined. Accordingly, it has been noted that following the incubation of the cells with nanobubbles, the morphological structures of the cells was not altered however their numbers had been somewhat decreased. In order to determine the amount of NB-<NUM> that was ingested into the cells, ICP-MS analysis has been used. The macrophage cells that were not administered drugs during analisys have been accepted as the control group and calculation has been made using the iron amount in cells that have been administered with drugs. It has been noted that <NUM>,<NUM>% of the nanobubbles were received by the macrophage cells and therefore have been phagocytized. When all the data obtained is taken into consideration, it is believed that the nanobubbles are biocompatible.

NB-<NUM> which was formed by encapsulating FITC which has fluorescence effect has been injected to nude mice through their tail veins and after <NUM> minutes the distribution of the NB-<NUM> sample inside the body was viewed using the in vivo imaging system (IVIS). Besides this NB-<NUM> that was encapsulated with FITC was injected to another nude mouse and following this <NUM> minutes of magnet application was carried out at the tumor region and its image was captured. Following the magnet application ultrasound was applied to the tumor region of the mouse and images were taken by IVIS. It has been noted that when the magnet was not applied to the mouse the FITC sourced radiation was distributed to the entire body of the nude mouse. However following the magnet application the nanobubble structures accumulated at the area where the magnet was applied to as said structures contained magnetite. Besides this, following the magnet application, and following the ultrasound application, the scattering of radiation observed showed us that the nanobubbles had bursted by means of ultrasound and their contents had been released. After the images were taken, the nude mice were sacrificed under ethical conditions and their organs were collected in order to carry out an ICP-MS analysis in order to determine the nanoubble amount for Fe analysis. It has been determined that approximately <NUM>% of the nanobubble strcutrure were accumulated inside the tumor found in the nude mice that were subjected to magnet application.

NB-<NUM> was given from the tail vein and following <NUM>. min, <NUM>. min, <NUM>. hrs, <NUM>. hrs and <NUM>. hrs the animals were sacrificed and Fe analysis was carried out in their organ, blood, urine and feces samples using ICP-MS and the % distribution in organs depending on time was calculated by means of the magnetic conjugate amounts found inside the prepared nanobubbles.

For intravenous treatment NB-<NUM> has been injected through the tail vein and magnet has been applied to the tumor region for <NUM> minutes and after the magnet has been removed ultrasound has been applied.

It can be seen from the table values that the nanobubbles that have been prepared according to Table <NUM> have IV application treatment potential. When the tumor size is small treatment potential is higher.

For inhaler treatment the NB-<NUM> sample has been placed into the inhalation device and inhaler treatment has been applied to mice with lung cancer <NUM> times a week. Following this magnet has been applied to the tumor region for <NUM> minutes and when the magnet was removed ultrasound application was carried out.

As it can be seen from Table <NUM>, it can be seen that there were alterations in the tumor sizes of the animals and different responses were given to treatment. It can be seen that treatment potential was high with the bubbles given by means of inhalation treatment if the tumor was small sized however when the tumor mass was higher it was perceived that treatment took longer even though it was successful. It can be seen that all of the animals within the group responded positively to treatment.

According to the "up and down" test carried out in relation to the "OECD Guideline Number <NUM>", the test agent did not show acute toxic effects at <NUM>/ml test concentrations.

It has been determined that the test agent did not have any kind of effect that may lead to mutation on the Salmonella typhimurium TA <NUM> andTA <NUM> strains at the tested concentrations.

<NUM>,<NUM>-<NUM>,<NUM> blood samples were obtained from nearly all mice under ketamine+xylazine anesthesia after the completion of the <NUM> day repeat dose toxicity trial by inhaler and IV applications and haemogram analysis was carried out by outsourced support. During the IV and inhaler application toxicology study, it has been observed that the blood parameter values were between reference ranges and deviation from the reference values were not observed during the haemogram analysis at <NUM> different doses of the preparation.

Claim 1:
An ultrasound susceptible magnetic directed nano drug carrier system preparation method, characterized by comprising the following process steps;
i) Synthesizing the magnetic nanoparticles (MNP), preferably by means of the coprecipitation method
ii) Amination of the synthesized magnetic nanoparticles,
iii) Modifying drugs, where drugs being pemetrexed and pazopanib
iv) Conjugating each of the modified drugs with different peptides,
v) Obtaining nanoconjugates by binding the peptide-drug conjugates to the aminated magnetic nanoparticles,
vi) Synthesizing the nanoconjugate loaded nanobubbles.