WO2025151105A1 - 5-fluorouracil and gentisic acid loaded nanoparticle coated with biotin - Google Patents

Abstract

The present application provides one or more nanoparticles comprising 5-fluorouracil and gentisic acid, furnished with one or more coatings comprising biotin. The present application also relates to one or more methods of manufacturing nanoparticles comprising the steps of providing 5-fluorouracil, gentisic acid and biotin, encapsulating the provided 5-fluorouracil and gentisic acid, thereby providing one or more nanoparticles comprising 5-fluorouracil and gentisic acid, providing a coating mixture comprising biotin, coating one or more nanoparticles encapsulated with 5-fluorouracil and gentisic acid using the provided coating mixture, thereby obtaining one or more nanoparticles furnished with one or more coatings containing biotin and containing 5-fluorouracil and gentisic acid.

Description

5-FLUOROURACIL AND GENTISIC ACID LOADED NANOPARTICLE COATED WITH BIOTIN

Technical Field

The present invention relates to nanoparticular systems, in particular to targeted nanoparticular systems and methods of their manufacture.

Background of the Invention

Although the main goal is to eliminate cancer cells during chemotherapy and radiotherapy treatment applications, healthy cells are also negatively affected along with cancer cells. For this reason, polymeric carrier systems are used in cancer treatment as an alternative to traditional treatments such as radiotherapy and chemotherapy, which have high side effects. The polymers supplied for polymeric carrier systems are selected among polymers that are biocompatible, biodegradable, do not cause antigenic response and can be excreted from the body. For example, PLGA (poly(lactic-co-glycolic acid)), approved by the EMA and FDA, may be a preferred polymer for polymeric carrier systems. PLGA is synthesized by ring opening co-polymerization of two different monomers, cyclic dimers of glycolic acid and lactic acid (l,4-dioxane-2, 5-diones). For this reason, PLGA should be preferred for use in various applications by considering the lactic acid/glycolic acid ratio.

During the synthesis of polymeric nanocarriers, surface-active components (e.g. polyvinyl alcohol (PVA)) can be used to become targeted drug active substance carriers with various surface modifications. For example, the surface properties of nanoparticles can be modified by coating with various agents. After the coating process, the nanoparticular system can ensure targeting and controlling the release of the drug active substance it carries.

The main objective for the synthesis of nanoparticles with various chemical and physical properties is to obtain a population of particles with a uniform distribution, i.e. a particle population with similar surface charge and zeta potential. The methods used in nanoparticle production (e.g. solvent volatilization, ionic gelation) may vary depending on the intended use of the particle and the active substance to be used.

Gentisic acid (GA), a metabolite of aspirin, is a phenolic compound with a molecular weight of 154.12 g/mol containing two (-OH) groups at para positions. Gentisic acid is a bioactive compound with a broad pharmacological spectrum, e.g. analgesic, antiarthritic, anti-inflammatory, antimutagenic, anticancer, antirheumatic, antispasmodic, antioxidant, antiparkinsonian, antifungal, siderophoricantigenotoxic activity. In addition, it may provide iron chelating and fibroblast growth factor (FGF) inhibition. Today, gentisic acid is preferred in cardiovascular applications due to its antioxidant activity and free radical scavenging properties. In addition, it is preferred for its pro- apoptotic and antiproliferative activity in various cancer cells after chemical carcinogenesis process. 5-fluorouracil is an antimetabolite that acts as a pyrimidine antagonist and is an anticancer agent. The mechanism of action of 5-fluorouracil is to interfere with DNA synthesis and mRNA translation. The active substance 5-fluorouracil has three mechanisms of action, as follows:

The 5-fluorouracil metabolite, fluorodeoxyuridine monophosphate (FdUMP), competes with uracil, one of the four organic bases in the structure of RNA, for binding to thymidylacetase (TS) and folate cofactor. This competition leads to a decrease in the production of thymidine, which is formed as a result of the combination of thymine, one of the four organic bases in the structure of DNA, and deoxyribose sugar, and thus cell proliferation.

By incorporating fluorodeoxyuridine triphosphate into the structure of DNA, the 5-fluorouracil metabolite interferes with DNA replication, the process by which the DNA molecule copies itself and doubles its amount.

5-The 5-fluorouracil metabolite interferes with the process of protein synthesis by incorporating fluorouridine-5-triphosphate (FUTP) into the RNA chain instead of uridine triphosphate (UTP).

Widely used in the treatment of various types of cancer (e.g. colorectal, stomach, lung, ovarian and breast cancers), 5-fluorouracil is also effective against precancerous skin lesions, actinic keratosis, viral warts and basal cell carcinoma. The 5-Fluorouracil molecule kills both cancerous and healthy cells in the body, even at the lowest doses (i.e. minimum dose) expected to ensure its effectiveness. Studies conducted by developing oral and topical dosage forms of the active substance 5-fluorouracil have concluded that the active substance has low bioavailability (i.e. the amount of the drug that can reach the target area from the application area) and low permeability (i.e. absorption to the applied area). (Ahmad, N., Albassam, A. A., FaiyazKhan, M., Ullah, Z., MohammedBuheazah, T., Salman AlHomoud, H., & AH Al-Nasif, H. (2022). A novel 5-Fluorocuracil multiple-nanoemulsionusedfortheenhancement of oral bioavailability in thetreatment of colorectalcancer. Saudijournal of biologicalsciences, 29(5), 3704-3716. https://doi.Org/10.1016/j.sjbs.2022.02.017). Due to these problems, 5-fluorouracil is used in therapeutic applications integrated with various carriers (e.g. nanoparticles).

Biotin, also known as vitamin H and vitamin B₇ in the literature, supports cellular growth. Its growthenhancing effect on cancer cells is higher compared to healthy cells. When cancer cells exhibit excessive growth as a result of biotin exposure, biotin-specific receptors are also overexpressed on the cancer cell surface. The specific interaction between biotin and receptors expressed on the cancer cell surface may enable targeted drug delivery. Short Description of the Invention

The primary object of the invention is to overcome deficiencies in the state of the art. Another object of the invention is to provide a targeted nanoparticle. These objects are achieved by means of the set of elements described in the independent claims.

The development subject to the present application comprises one or more nanoparticles comprising 5-fluorouracil and gentisic acid, provided with one or more coatings comprising biotin, said one or more nanoparticles may be considered as a nanoparticular system.

In the development subject of the application, the active ingredients are synthesized in the form encapsulated in nanoparticular carriers to increase the solubility and bioavailability of 5-fluorouracil and gentisic acid and to reduce their cytotoxicity.

In an exemplary application of the invention, the nanoparticle may comprise one or more coatings of biotin which equip the nanoparticle for encapsulation of 5-fluorouracil and gentisic acid. In a preferred application of the invention, said one or more coating materials may be directly selected as biotin. According to the data obtained as a result of the studies carried out within the scope of the present invention, the efficacy of 5-fluorouracil and gentisic acid encapsulated nanoparticles on cancer cells is lower compared to nanoparticles targeted by coating with biotin. The present development therefore comprises one or more nanoparticles furnished with one or more biotin-containing coatings.

In an exemplary application of the invention, said coating may be a biotin-containing coating product, or may be preferred as direct biotin. Within the scope of the invention, nanoparticles prepared by surface modification with biotin-containing coating material and/or directly with biotin may be developed specifically for biotin positive cancer types.

The method of the present application for producing one or more nanoparticles provided with one or more biotin-containing coatings and containing 5-fluorouracil and gentisic acid comprises the following steps: a. Procurement of 5-fluorouracil, gentisic acid and biotin, b. encapsulating 5-fluorouracil and gentisic acid provided in step a, thereby providing one or more nanoparticles comprising 5-fluorouracil and gentisic acid, c. obtaining a coating mixture containing biotin, d. coating one or more nanoparticles obtained in step b using the coating mixture provided in step c, thereby providing one or more nanoparticles furnished with one or more coatings comprising biotin and containing 5-fluorouracil and gentisic acid.

In an exemplary application of the present application, the binary emulsion solvent volatilization method may be preferred as a nanoparticle synthesis method. The method in question is based on the dissolution of the active ingredients to form an aqueous phase and then transferring the polymer to the other phase, which is the solution in which the polymer is present. In a preferred application of said method, step b may comprise the step of dissolving one or more polymers suitable for encapsulation in one or more solvents together with the 5-fluorouracil and gentisic acid provided in step a.

In a possible application of the method, poly(lactic-co-glycolic acid) may be preferred as a polymer. The PLGA polymer tailored for the encapsulation of 5-fluorouracil and gentisic acid can be selected by considering parameters such as dissolution rate, compatibility with active pharmaceutical ingredients and lactic acid/glycolic acid ratio.

In a possible application of the method, polyvinyl alcohol may be preferred as the surfactant.

In a possible application of the method, it may comprise using dichloromethane as solvent.

In a possible application of the method, the encapsulation of 5-fluorouracil and gentisic acid in step b may comprise sonication.

In a possible application of the method, step d may comprise mixing .

A possible application of the method could include step d followed by the following step: e. washing the resulting coated nanoparticle.

In a possible application of the method, the washing process in step e may comprise the following elements:

Subjecting one or more nanoparticles containing gentisic acid with 5-fluorouracil to one or more consecutive centrifugations in combination with a washing liquid and separation of a subphase formed after one or more centrifugations.

A possible application of the method involves the use of distilled/pure water as a washing liquid prior to one or more centrifugations.

A possible application of the method may comprise, following step e, combining the one or more coating mixtures comprising biotin obtained in step c with one or more nanoparticles comprising 5- fluorouracil and gentisic acid obtained in step b to provide one or more nanoparticles comprising 5- fluorouracil and gentisic acid, which are furnished with the one or more coating mixtures comprising biotin obtained in step d.

In the common context of the present application, nanoparticles loaded with 5-fluorouracil and gentisic acid are synthesized and coated with biotin as a targeting molecule, thus overcoming the shortcomings of the state of the art.

In connection with the above, the present application further provides a medicament for the treatment of biotin-positive cancer types. The medicinal product comprises one or more nanoparticles containing 5-fluorouracil and gentisic acid in encapsulated form and coated on their outer surface with one or more coating layers containing biotin. Brief Description of Drawings

The figures, brief descriptions of which are provided herein, are intended only for a better understanding of the invention and are not intended to define the scope of protection or the context of such scope, regardless of the specification.

Figure 1 shows the size analysis of the nanoparticles whose surface has not yet been coated, following step b of the method disclosed in the present application for producing one or more nanoparticles. The abscissa axis shows the particle size in diameter in nanometers and the ordinate axis shows the nanoparticle distribution in percent (Eng.: intensity percent).

Figure 2 shows a zeta analysis of nanoparticles whose surface has not yet been coated, following step b of the method disclosed in the present application for producing one or more nanoparticles. Abscissa axis, (Eng.: apparent Zeta potential) shows the zeta potential in mV of the nanoparticles produced in step b, the ordinate axis (in English: total counts) shows the total number of counts.

Figure 3 shows x30,000 magnified SEM analysis images of 5-fluorouracil and gentisic acid loaded nanoparticles after step b of the method described in the present application to produce one or more nanoparticles.

Figure 4 shows x40,000 magnified TEM analysis images of 5-fluorouracil and gentisic acid loaded nanoparticles after step b of the method disclosed in the present application to produce one or more nanoparticles.

Figure 5 shows the results of FT-IR analysis of 5-fluorouracil and gentisic acid loaded nanoparticles following step b of the method described in the present application to produce one or more nanoparticles.

Figure 6 shows the cytotoxicity analysis of the 5-fluorouracil molecule in the present application on A549 cells after 48 hours. The abscissa axis shows the gentisic acid concentration in milliMol/liter (mmol/L) and the ordinate axis shows the cell viability %.

Figure 7 shows the cytotoxicity analysis of the gentisic acid molecule in the present application on A549 cells after 48 hours. The abscissa axis shows the gentisic acid concentration in mmol/L and the ordinate axis shows the cell viability %. Figure 8 shows the cytotoxicity analysis of the biotin molecule in the present application on A549 cells after 48 hours. The abscissa axis shows the gentisic acid concentration in mmol/L and the ordinate axis shows the cell viability %.

Figure 9 shows the cytotoxicity assay of the combination of 5-fluorouracil and gentisic acid molecules on A549 cells after 48 hours in the present application. The abscissa axis shows the gentisic acid concentration in mmol/L and the ordinate axis shows the cell viability %.

Detailed Description of the Invention

The nanoparticulare system of the present application offers biotin coating and targeting of nanoparticles loaded with 5-fluorouracil and gentisic acid.

Nanoparticles are preferred for active substance delivery or cell targeting.

A polymeric carrier system produced as nanoparticles can be considered for use in cancer treatment. The polymers that form the skeleton of drug carrier and targeting nanoparticles can preferably be selected from biodegradable polymers that can be directly excreted from the body without undergoing metabolic transformations and whose degradation products are non-toxic. Examples of these polymers include chitosan, PLGA, poly(dimethylsiloxane) (PDMS for short).

In the present application, one or more nanoparticles comprising 5-fluorouracil and gentisic acid are produced. In an exemplary embodiment, the one or more nanoparticles produced may be polymer- based (i.e., polymeric, or substantially polymeric) and PLGA may be preferred as the polymer. PLGA is an FDA approved copolymer with high biodegradability and biocompatibility compared to other polymers and copolymers.

For one or more nanoparticles based on biodegradable polymers in the context of the present application, it is possible to state the following:

- may have a combined effect with various therapeutic components,

- allows targeting molecules (e.g. biotin) to be incorporated into the structure,

- can protect active substances classified as active ingredients against degrading forces,

- By forming a skeletal structure, it may provide the opportunity to control the diffusion and distribution of the drug active ingredients that it encapsulates at the site of administration.

5-fluorouracil and gentisic acid have been selected as the pharmaceutical active ingredients targeted for carriage under the current application. Although 5-fluorouracil, the preferred active pharmaceutical ingredient in the application, is used as a pyrimidine antagonist as a chemotherapeutic agent, it has a low solubility, which may reduce the bioavailability of 5-fluorouracil. In addition, the 5- fluorouracil molecule can kill cancerous cells and damage healthy cells in the body even at the lowest doses (i.e. a minimum dose) expected to ensure its efficacy. In order to overcome these technical problems, the present application provides the active ingredient 5-fluorouracil encapsulated in a polymeric nanoparticle.

As a result of the 5-fluorouracil encapsulation performed within the scope of the present application, it is possible to suggest the following assessments:

- Encapsulation into polymeric nanoparticles can increase the water solubility of 5- fluorouracil.

- Increased water solubility of 5-fluorouracil allows for increased bioavailability in topical and oral applications.

- While the controlled release of encapsulated 5-fluorouracil occurs in cancerous cells, the encapsulated active substance does not show toxic effects on healthy cells.

Gentisic acid, which is preferred to be used in combination with 5-fluorouracil within the scope of the current application, is an aspirin metabolite and can show biological activities such as analgesic, antiarthritic, anti-inflammatory, antimutagenic, anticancer, antirheumatic, antispasmodic, antioxidant, antiparkinsonian, antifungal, antigenotoxic effects. The aforementioned gentisic acid molecule is used for chemical carcinogenesis and shows pro-apoptotic and antiproliferative effects in various cancerous tissues. In the scope of the application, gentisic acid and 5-fluorouracil molecules were used in combination to administer conjugated drug therapy to various cancer cells. The use of active substances in combination or the use of active substances encapsulated in combination may increase their bioavailability, reduce their cytotoxic effects, increase their antioxidant activity, increase apoptosis on the cells to which they are administered compared to their administration alone.

In the context of the present application, the 5-fluorouracil and gentisic acid encapsulated nanoparticles obtained after encapsulation, furnished with one or more biotin-containing coatings, are adapted to target the cancer cells to which they are to be applied. Receptors on the surface of cancer cells are able to recognize and directly bind to various targeting molecules. With nanoparticles encapsulated with the active ingredient of the drug, cancer cells can be eliminated. Nanoparticles developed as carrier and targeting systems can be made specific to cancer types with the surface modification described below.

Surface modification allows biotin to better adhere to the nanoparticle surface, thus providing a highly effective targeting; thus reducing the possibility of biotin release and preventing it from contacting healthy cells around the tumor and damaging these healthy cells due to the effect of drug active substances.

In an exemplary embodiment, said coating may comprise or consist of biotin. In the context of the present application, the concept of surface modification may be considered either as a biotincontaining coating or biotin coating process on the surfaces of nanoparticles, or as a process for the appropriate use of a biotin-containing coating material or a surfactant that improves the adhesion of biotin to the nanoparticle surface. In the present context, PVA may be selected as a surfactant that improves the adhesion of the biotin-containing coating material or biotin to the nanoparticle surface, and the selected surfactant may be used as described in the present disclosure.

It can be said that the nanoparticles in the present application have been developed specifically for biotin positive cancer types; therefore, the nanoparticles subject to the application may offer a specific recognition mechanism. Examples of biotin positive cancer types include breast cancer, cervical cancer, lung cancer. In an exemplary embodiment, the specific recognition mechanism may be that the receptors on the surface of biotin positive cancer cells recognize the biotin covering the surface of the 5-fluorouracil and gentisic acid encapsulated nanoparticles produced within the scope of the present invention and specific binding is achieved.

In the context of the present application, with regard to nanoparticles coated with biotin in a targeted structure and 5-fluorouracil and gentisic acid encapsulated in nanoparticles, it is possible to say the following

It can be ensured that the active pharmaceutical ingredients 5-fluorouracil and gentisic acid provide relatively high efficacy at relatively low doses,

- Reduction of side effects that may occur due to the reduction of the dose of the active substance of the drug can be achieved,

- the active ingredients of these drugs can be directed to the target sites where cancer cells are located,

- by preserving molecules with weak chemical bonds (here, for example, 5-fluorouracil) in the blood and lymph circulation, their interaction with off-target receptors can be prevented.

Under the present application, the biotin molecule can be administered at an effective dose derived from cytotoxicity assays. The administration of the biotin molecule at an effective dose may prevent the occurrence of an unexpected toxic effect in the cancer cells to which the therapeutic application is administered.

The present application further provides a method for obtaining one or more nanoparticles provided with one or more coatings comprising biotin and containing 5-fluorouracil and gentisic acid. The method comprises the following steps: a. Providing 5-fluorouracil, gentisic acid and biotin, b. encapsulating the 5-fluorouracil and gentisic acid provided in step a, thereby providing one or more nanoparticles comprising 5-fluorouracil and gentisic acid, c. obtaining a coating mixture containing biotin, d. coating one or more nanoparticles obtained in step b by using the coating mixture provided in step c, thereby providing one or more nanoparticles provided with one or more coatings comprising biotin and containing 5-fluorouracil and gentisic acid. For the purpose of encapsulating 5-fluorouracil and gentisic acid, a person skilled in the relevant art, starting from the present specification, can achieve this by any of the means known in the relevant art. Examples of encapsulation methods known in the relevant art are solvent evaporation or, alternatively, ionic gelation methods. In an exemplary embodiment of the invention, as a solvent volatilization method, a binary emulsion solvent volatilization method may be preferred.

In an exemplary embodiment of the present application, one or more polymers and one or more polymeric surfactants suitable for encapsulation with 5-fluorouracil and gentisic acid provided may be dissolved in one or more solvents. Solvents with a relatively low boiling point may be preferred, as solvents with a low boiling point take longer to remove than solvents with a high boiling point.

In an exemplary embodiment of the invention, dichloromethane (in short: DCM) may be used as the solvent (i.e. solvent). DCM has a boiling point of 39°C, and this relatively low boiling point value makes DCM easy to remove compared to solvents with a higher boiling point.

In an exemplary embodiment of the invention, it may be preferred to use poly(lactic-co-glycolic acid) (in short: PLGA) as a polymer. PLGA is soluble in DCM. Polymers for use in nanoparticles are preferred based on parameters such as dissolution rate and compatibility with active pharmaceutical ingredients. The PLGA polymer proposed for use within the scope of the current application can be selected for use by considering the molecular weight, lactic acid/glycolic acid ratio.

5-fluorouracil and gentisic acid are soluble in water (e.g. distilled water, purified water) in combination. Therefore, in addition to DCM, water may be selected as another solvent suitable for use in the method of reference.

In an exemplary embodiment of the invention, it may be preferred to use one or more surfactants. In the context of the present application, it is possible to state the following for the properties acquired by the nanoparticles as a result of the use of surfactants (e.g. polyvinyl alcohol (PVA), polyoxyethylene):

- may enhance the catalytic activity of nanoparticles,

- drugs can reduce the toxic potential of active substances,

- may enable the nanoparticle to maintain its stability for a longer period of time,

- can prevent moisture loss that can occur when nanoparticles are stored in lyophilized or wet form.

Thanks to the properties of PVA such as having hydrophilic and hydrophobic parts at the same time, aqueous solutions can be prepared as pH neutral or acidic if desired, and it has high adhesion due to the binding fibers in its structure, it can be recommended to be used as a surface active component within the scope of the current application.

In an exemplary embodiment of the method, for the process of obtaining one or more nanoparticles comprising 5-fluorouracil and gentisic acid by encapsulation of 5-fluorouracil and gentisic acid, a sonication followed by mixing may be preferred. For mixing, a magnetic stirrer can be used, for example. Sonication enhances dispersion and can eliminate toxicity due to aggregation of nanoparticles. In an exemplary application, sonication followed by mixing can ensure that each nanoparticle produced has similar character and durability. In the present context, the term character can refer to one or more of properties such as zeta potential, polydispersity index (PDI), particle size and morphology. Particle size can be measured, for example, with a scanning electron microscope (SEM), transmission electron microscope (TEM), or zeta size meter (English: Zetasizer). Morphology can be assessed, for example, by SEM or TEM.

In an exemplary embodiment, one or more nanoparticles formed as a result of encapsulating 5- fluorouracil and gentisic acid can be washed to remove free circulating molecules not included in the nanoparticular system of the present application. Thus, the nanoparticles can be free of unencapsulated 5-fluorouracil and gentisic acid, PLGA polymer that does not form a nanoparticle skeleton, and free circulating surfactant.

In an example application, the flushing process in question may include the following steps:

Subjecting one or more nanoparticles containing gentisic acid with 5-fluorouracil to one or more consecutive centrifugations in combination with a washing liquid, separation of a subphase formed after one or more centrifugations.

In an exemplary application, one or more washing liquids selected from water, for example distilled water or ultrapure water, may be used as the washing liquid prior to one or more centrifugation steps. An exemplary application may comprise, following step e, contacting one or more coating mixtures comprising biotin obtained in step c with one or more nanoparticles comprising 5-fluorouracil and gentisic acid obtained in step b. Thereby providing one or more nanoparticles comprising 5- fluorouracil and gentisic acid, which are provided with the one or more biotin-containing coatings of step d.

In an exemplary embodiment, step d may be preceded by a washing operation as described in this specification. Following said washing, step d may comprise the following:

Coating one or more coating mixtures containing biotin provided in step c with one or more nanoparticles encapsulated with 5-fluorouracil and gentisic acid encapsulated in step b, thereby bringing the coating mixture into contact with the nanoparticles encapsulated with the active pharmaceutical ingredient,

Adding to the medium one or more crosslinkers (e.g., tripolyphosphate) adapted to help equip the nanoparticle surface with one or more biotin-containing coatings, thereby providing one or more nanoparticles containing 5-fluorouracil and gentisic acid, which are furnished with one or more biotin-containing coatings. e 1

In this example, a solvent volatilization method, e.g. binary emulsion solvent volatilization, can be used. Following the synthesis of nanoparticles, characterization and cell culture studies can be performed.

Nanoparticle synthesis by the binary emulsion solvent volatilization method can be carried out in the following steps:

1. providing 5-fluorouracil and gentisic acid molecules as active pharmaceutical ingredients (step a),

2. to providing PLGA polymers as one or more polymers suitable for encapsulation of active pharmaceutical ingredients,

3. providing the solution of the active ingredients of a medicinal product in water, thereby producing a solution of the active ingredient of the medicinal product,

4. Dissolution of the PLGA polymer in DCM, thus providing a polymer solution,

5. providing one or more surfactants, e.g. providing PVA as a surfactant,

6. Preparation of PVA in one or more solvents, for example in water and/or DCM, for example at a concentration of 5 milligrams / 100 milliliters, thereby obtaining a surfactant solution,

7. adding the drug active ingredient solution and the polymer solution to the surfactant solution to obtain a first mixture in which one or more nanoparticles encapsulated with the drug active ingredient are formed and contained,

8. sonicating the first mixture, thereby obtaining a sonicated version of the first mixture,

9. preparation of a second solution comprising, for example, one or more surfactants (here, PVA), for example at a concentration of 1 milligram/liter,

10. transferring the sonicated first mixture into the second solution, thereby obtaining a stabilization mixture; stirring said stabilization mixture for a stirring time and under a stirring temperature (here, a stirring time of 4 hours and a stirring temperature of 20°C), thereby providing stabilized nanoparticles.

In the exemplary embodiment of the present invention, the washing process is carried out in the following steps by precipitating the nanoparticles in a centrifuge by mixing them with water:

11. stabilized nanoparticles at a centrifugation temperature (here, +4 °C) and a centrifugation speed (it was found useful to keep the centrifugation speed as high as possible in order to incorporate relatively small particles into the pellet, e.g. 6000 or higher rpm, preferably 9000 or higher rpm, here 10000 rpm) for a centrifugation time (e.g. 30 minutes or longer, here 40 minutes), thereby obtaining a pellet containing nanoparticles as the lower phase and removing the upper phase from the pellet,

12. Subjecting the pellet to one or more washing processes, e.g., centrifuging the pellet by contacting it with a washing liquid before and/or after each washing process (e.g., +4 oC, 10000 rpm, 30 minutes, and here 3 times); separating the washing liquid and thereby obtaining washed pellets,

13. lyophilization of the washed pellet and thus obtaining nanoparticles in solid and/or powder form,

14. contacting the solid and/or powdered nanoparticle with a coating mixture which may comprise biotin as well as a crosslinker, thereby obtaining an exemplary configuration of the nanoparticles in the context of the present application.

As a result of the analysis performed with a ZetaSizer device, it was found that after step b of the method, the nanoparticles, whose surface had not yet been coated, had an average particle size of 192.27 nm (see Figure 1), and SEM analysis referring to Figure 3 and TEM analysis referring to Figure 4 were performed to support this result.

As a result of SEM analysis, the size measurements of 5-fluorouracil or gentisic acid encapsulated nanoparticles were made from three different regions (see Figure 3): Region 1 - 178 nm, Region 2 - 159 nm, Region 3 - 172 nm.

As a result of TEM analysis, size measurements of 5-fluorouracil or gentisic acid encapsulated nanoparticles were made from three different regions (see Figure 4). The measurement results obtained are as follows: Region 1 - 151 nm, Region 2 - 140 nm, Region 3 - 153 nm.

Referring to Figure 2, it is understood that they have a zeta potential of -24.3 mV and a PDI value of 0.110. These values were considered to indicate "optimized" nanoparticles in line with the choices made in Example 1.

According to (UV-) spectroscopic analysis of the supernatant separated at the end of the centrifugation step of the stabilized nanoparticles in Example 1, 5-fluorouracil and gentisic acid were found to have 90.6% (w/w) and 89.3% (w/w) encapsulation efficiencies, respectively.

As a result of FT-IR analysis of the nanoparticles, referring to Figure 5, it was observed that the nanoparticles were in the characteristic of PLGA, no peak from 5-fluorouracil or gentisic acid was observed on the nanoparticle surfaces, thus it was determined that the active ingredients were successfully encapsulated in the nanoparticle. Cellular uptake studies were performed to check whether the nanoparticles enter the cell. In this context, a ternary NP system containing 5-fluorouracil, gentisic acid and fluoresceinothiocyanate (FITC) fluorescent dye was synthesized and administered to cells (here, A549 cells); cellular uptake of nanoparticles was found to be present. Another fluorescent dye, DAPI (i.e., 4',6-diamidino-2- phenylindole), was stained in the nuclei of cells (here, A549 cells) and cytoplasmic staining was observed upon nanoparticle entry into the cell.

Example 3

Cell Culture Studies

Cytotoxic analyses were performed for 5-fluorouracil and gentisic acid individually and for both agents in combination on commercially available lung cancer cells, designated A549. The aforementioned cytotoxicity assay was performed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a colorimetric assay to assess the cell metabolic activity of living cells through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to insoluble formazan crystals by mitochondrial activities, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method and the half maximum inhibitory concentration (IC$o) values, which is the effective dose, were determined. The IC50 value is a measure of the potential of a substance to inhibit a specific biological or biochemical function. This IC50 value is a quantitative measurement value indicating the effective dose of a specific inhibitory substance, such as a pharmaceutical active ingredient, to inhibit a specific biological process or biological component by 50% in vitro.

While determining the dose ranges, a wide range of concentrations between pM (micromolar) and mM (millimolar) were tested and the IC₅₀ value was obtained after determining the ideal doses with 3 replicate experiments. Figure 6 shows the results of the cytotoxicity assay of 5-fluorouracil molecule on A549 cells after 48 hours. Figure 7 shows the results of cytotoxicity analysis of gentisic acid molecule on A549 cells after 48 hours. Figure 8 shows the results of the cytotoxicity analysis of the biotin molecule on A549 cells after 48 hours. Figure 9 shows the results of the cytotoxicity analysis of the combination of 5-fluorouracil and gentisic acid molecules on A549 cells after 48 hours.

The IC50 values obtained as a result of cytotoxicity analysis performed by MTT method are given in the table below:

Claims

1. One or more nanoparticles containing 5-fluorouracil andgentisic acid, wherein it is furnished with one or more coatings containing biotin.

2. Nanoparticle according to Claim 1, wherein one or more of the coatings in question contain biotin.

3. Nanoparticle according to any one of Claim 1 or Claim 2, one or more of the coatings in question is biotin.

4. A drug for the treatment of biotin-positive cancers containing one or more nanoparticles containing 5-fluorouracil and gentisic acid in encapsulated form and coated on their outer surface with one or more biotin-containing coating layers.

5. A method for obtaining one or more nanoparticles provided with one or more biotincontaining coatings and containing 5-fluorouracil and gentisic acid, wherein the method comprising the following steps: a. Providing 5-fluorouracil, gentisic acid and biotin, b. encapsulating the 5-fluorouracil and gentisic acid provided in step a, thereby providing one or more nanoparticles comprising 5-fluorouracil and gentisic acid, c. obtaining a coating mixture containing biotin, d. coating one or more nanoparticles obtained in step b by using the coating mixture provided in step c, thereby providing one or more nanoparticles provided with one or more coatings comprising biotin and containing 5-fluorouracil and gentisic acid.

6. The method according to claim 5, wherein step b comprises the following elements: dissolution of one or more polymers suitable for encapsulation in one or more solvents together with 5-fluorouracil and gentisic acid provided in step a.

7. The method according to claim 6, wherein it comprises the usage of poly(lactic-co-glycolic acid) as the polymer.

8. The method according to claim 7, wherein the surfactant comprises polyvinyl alcohol.

9. The method according to any one of claims 6 to 8, wherein it comprises the usage of dichloromethane as the solvent.

10. The method according to any one of claims 5 to 9, wherein the process of encapsulating 5- fluorouracil and gentisic acid in step b comprises the application of sonication.

11. The method according to any one of claims 5 to 10, wherein step d comprises the application of mixing.

12. The method according to any one of claims 5 to 11, wherein step d comprises the following step: e. washing of the resulting coated nanoparticle.

13. The method according to claim 12, wherein step e comprises the following elements:

Subjecting one or more nanoparticles containing gentisic acid with 5-fluorouracil to one or more consecutive centrifugations in combination with a washing liquid and separation of a subphase formed after one or more centrifugations.

14. The method according to claim 13, wherein the one or more centrifugations are preceded by at least one of water, distilled water or ultrapure water as the washing liquid.

15. A method according to claim 14, wherein step e comprises contacting one or more coating mixtures comprising biotin obtained in step c with one or more nanoparticles comprising 5- fluorouracil and gentisic acid obtained in step b.

16. A method according to any one of claims 1 to 15, wherein a washing process is carried out before step d, and wherein step d comprises the following:

- placing one or more coating mixtures comprising biotin provided in step c in the same medium as one or more nanoparticles encapsulated with gentisic acid with 5-fluorouracil in step b,

- the insertion of one or more cross-linkers into the medium in question.

17. A method according to claim 16, wherein the crosslinker comprises tripolyphosphate.