A nanoparticular drug delivery system with mucoadhesion properties and the synthesis method thereof

Abstract

The invention relates to a nanoparticle for use as a drug carrier and the synthesis method of this nanoparticle. In the invention, magnesium / aluminium (Mg / AI) mucoadhesive thiolated layered double hydroxide (nLDHT) nanoparticles are thiolated with L-cysteine and / or thioglycolic acid and / or 2-iminothiolane hydrochloride and / or glutathione and / or N-acetyl cysteine and / or 3-mercaptopropionic acid and / or 6- mercaptohexanoic acid. Thus, nLDHT interacts with the mucin on the surface of the targeted mucosal tissue for a longer period of time and acts with fewer side effects in the target tissues.

Description

[0001] A NANOPARTICULAR DRUG DELIVERY SYSTEM WITH MUCOADHESION PROPERTIES AND THE SYNTHESIS METHOD THEREOF

[0002] Technical Field

[0003] The invention relates to thiolated Magnesium / Aluminium (Mg / AI) layered double hydroxide nanoparticles (OLDHT) for use as a drug delivery system. By means of said nanoparticles, mucoadhesive properties are increased in drug delivery and drugs are transported to targets in a more specific manner with fewer side effects.

[0004] State of the Art

[0005] Drug delivery systems are systems designed to provide direct delivery of therapeutic active ingredients to targeted areas. These systems can be in the form of micelles, dendrimers, liposomes, nanoparticles or carbon nanotubes, depending on the targeted cell and the active ingredient being delivered. However, existing drug delivery systems had some fundamental limitations. Some of these limitations can be listed as first-pass effect, high dose requirement, fluctuations in plasma drug levels and inability to provide continuous drug release or effect. Many studies are being conducted to eliminate the disadvantages of these limitations. As a result of these studies, it has been realised that nano-sized delivery systems have the potential to eliminate these limitations.

[0006] Layered double hydroxides (LDH) are important inorganic nanomaterials with a two- dimensional structure. They are good carriers for drug and gene delivery with their high surface-to-volume ratio and ability to accommodate large molecules [1]. The LDH structure contains divalent and trivalent metal ions. These ions form positively charged layers. There are exchangeable anions between these layers and the release of the drug is regulated by these ions [2], This feature reduces the frequency of drug application and increases the therapeutic efficacy of drugs. Other advantages of LDH include ease of production, low cost, biocompatibility, low toxicity and protection of the intercalated drug, etc. LDHs also have high drug / active substance loading capacity and stability. In addition, LDH has many advantages over other carrier systems such as good biodegradability, biocompatibility and low cytotoxicity. LDHs have the ability to continuously release the drug within a certain pH range, allowing the drug to be released in the desired area of the body. This controlled release reduces the likelihood of side effects that are frequently encountered in other treatment methods. In addition, drug-loaded LDHs are not rapidly excreted in the body and do not accumulate in organs [3]. For this reason, although LDH is a promising nanomaterial for various pharmaceutical applications, studies have not yet been conducted to enable its use in a wide range.

[0007] Although drug delivery systems are used in the treatment of many diseases, they are much more effective when applied directly to the target tissue or when applied from points closer to the target. Although the oral administration of therapeutic agents using traditional drug delivery systems is widely accepted today, the dissolution or enzymatic degradation of these drugs before they reach the target tissues prevents the drugs from acting as desired. For this reason, studies have begun to be conducted on different application methods to shorten the route of drug administration (for example, direct application of drugs to the areas they are intended to treat). It is seen that administering drugs from the surface of mucosal tissues such as the gastrointestinal system, female reproductive system, eye, mouth, nose and lung rather than orally has many advantages over oral administration [4], This method increases the bioavailability of drugs, provides target area-specific distribution and reduces side effects. Mucosal tissues are widely preferred in this application method.

[0008] Mucosal tissues (gastrointestinal system, female reproductive system, eye, mouth, nose and lung etc.) have a rich blood vessel network. This rich vascular network ensures rapid and effective absorption of the drug. Mucosal drug delivery ensures that the drug reaches the target area directly and protects the drug from the reactions it is exposed to in the digestive tract. Studies in the state of the art reveal the advantages of administering LDH via the ocular route. LDH is positively charged, while the cornea and conjunctiva are negatively charged. However, the interaction of drug-loaded LDHs with the cargo molecule can cause the positive charges of LDH to decrease and non- covalent bonds such as electrostatic interaction between the target area and the carrier system may be insufficient, limiting the retention of the vehicle system on the target surface [5]. In order to overcome this problem and increase particle-mucosa interaction, mucoadhesive strategy (mucoadhesion) is used [6]. Mucin is the main component of mucus fluid that plays a role in mucoadhesion. Based on the interaction between nanoparticles and mucin, nanoparticle drug delivery systems can be developed to extend the residence time of nanoparticles in mucosal tissues. Due to the physicochemical properties of mucin fibres, nanoparticles can be held to the mucosa by adhesion forces, such as electrostatic interaction, hydrophobic interaction, hydrogen bonding, van der Waals bonding, and non-covalent bonds , or covalent disulphide bonds between nanoparticles containing thiol groups and cysteine molecules in the mucin. While the mucoadhesion of nanoparticles is generally provided through ionic interactions and secondary bonds including hydrogen bonding, dipole interactions, and molecule-molecule interactions, thiolated nanoparticles can form covalent bonds. They can form disulphide bonds with the disulphide and thiol substructures of biological surfaces through thiol / disulphide exchange reactions or through a simple oxidation process. Thiolation of drug carrier nanoparticles significantly increases their mucoadhesive properties [7], However, the literature regarding studies on thiolation of the LDH system is insufficient.

[0009] In the patent application numbered US2022250929A1 , the synthesis of the LDH structure containing a functionalised hybrid layered layer and [Sn2Se]4" anions in its intermediate layers is described. However, said LDH does not include its use for drug delivery purposes. In addition, it is insufficient regarding the type of interaction that said structure can provide with other structures in mucosal tissues, especially mucin.

[0010] Due to the limitations and inadequacies of the solutions in the state of the art, the degradation of drugs before they reach the target tissue in the methods commonly used in the state of the art, the limited adhesion of drug carrier systems directly applied to mucosal tissues to the tissue and therefore the decrease in the effectiveness rate, the decrease in the interaction of promising LDH structures with the target tissues due to the cargo they carry, etc., it has become necessary to make developments in this field.

[0011] Brief Description and Aims of the Invention In the invention, magnesium / aluminium (Mg / AI) mucoadhesive thiolated layered double hydroxide (nLDHT) nanoparticles are used as drug carrier systems. The thiolation mentioned in the invention is realised by using different structures including L-cysteine amino acid, interacts more with mucin on the surface of mucosal tissue and thus nLDHT interacts with the targeted mucosal tissue for a longer period of time and affects the target tissues with fewer side effects. By means of the direct application of active substances to mucosal tissues, the interaction of this active substance with other tissues and organs is minimised and thus the side effects that may occur in these tissues and organs are prevented.

[0012] The main aim of the invention is to provide a drug carrier system that can hold onto the mucosal tissue to which they are directly applied for a long time and has few side effects. In the drug carrier system of the invention, the nLDHT structure consists of Mg / AI layers. Thiolation occurs by means of the structures added to the content of layered double hydroxide nanoparticles (nLDH), especially L-cysteine. Thiol groups form covalent disulphide bonds with glycoproteins such as mucin on the surface of nanoparticles, which allows nanoparticles to adhere more strongly and for a longer period of time to mucosal surfaces. This strong bonding prolongs the time that nanoparticles remain on mucosal surfaces, allows drugs to reach target areas more effectively and increases their absorption. At the same time, it facilitates the adhesion of nanoparticles to the cell membrane and their uptake into the cell. Thiol groups also establish disulphide bonds with proteins in the cell membrane, allowing nanoparticles to pass more easily into the cell. This process increases the cellular uptake of therapeutic agents and increases the bioavailability of drugs within the cell. This interaction is possible because of the -SH groups in structures such as L-cysteine used in the invention. Thus, nLDHT interacts with the mucin in the mucus that covers the mucosal tissue to which it is applied and provides a moist and slippery structure to the tissue. Said thiolation process can also be carried out using any of the structures such as thioglycolic acid, 2-iminothiolane hydrochloride, glutathione, N-acetyl cysteine, 3- mercaptopropionic acid or 6-mercaptohexanoic acid. The interaction between nLDHT and mucin can hold on for much longer than nLDH structures without thiolation. This retention gives nLDHT enough time to effectively transfer its cargo (therapeutic active ingredient) to the mucosal tissue. In this way, mucosal tissues can interact directly with the active substance and this direct interaction prevents the possibility of side effects. Description of Drawings

[0013] Figure 1 : FTIR spectra (1 : FTIR spectra of LDH sample, 2: FTIR spectra of LDHcys sample)

[0014] Figure 2: Mucin binding activity graph (1 : mucin binding activity of nLDH formulation, 2: mucin binding activity of nLDHcys formulation)

[0015] Figure 3: Zeta Potential Graph (1 : zeta potential value measured after mixing nLDH formulation - mucin solution, 2: zeta potential value measured after mixing nLDHcys formulation - mucin solution)

[0016] Figure 4: HeLa Cell Viability-Concentration Graphs (1 : The results of the effect of nLDH formulation on cell viability in HeLa cell line after 24 hours, 2: The results of the effect of nLDHcys formulation on cell viability in HeLa cell line after 24 hours, 3: The results of the effect of nLDH formulation on cell viability in HeLa cell line after 48 hours, 4: The results of the effect of nLDHcys formulation on cell viability in HeLa cell line after 48 hours)

[0017] Figure 5: HT29 Cell Viability-Concentration Graphs (1 : The results of the effect of nLDH formulation on cell viability in HT29 cell line after 24 hours, 2: The results of the effect of nLDHcys formulation on cell viability in HT29 cell line after 24 hours, 3: The results of the effect of nLDH formulation on cell viability in HT29 cell line after 48 hours, 4: The results of the effect of nLDHcys formulation on cell viability in HT29 cell line after 48 hours)

[0018] Figure 6: L929 Cell Viability-Concentration Graphs (1 : The results of the effect of nLDH formulation on cell viability in L929 cell line after 24 hours, 2: The results of the effect of nLDHcys formulation on cell viability in L929 cell line after 24 hours, 3: The results of the effect of nLDH formulation on cell viability in L929 cell line after 48 hours, 4: The results of the effect of nLDHcys formulation on cell viability in L929 cell line after 48 hours)

[0019] Detailed Description of the Invention

[0020] The invention relates to thiolated magnesium / aluminium (Mg / AI) mucoadhesive layered double hydroxide nanoparticles (nLDH?) for use as a drug delivery system and the synthesis method thereof. By means of the thiolation mentioned in the invention, JILDHT interacts with the mucin on the surface of the targeted mucosal tissue for a longer period of time and acts with fewer side effects in the target tissues.

[0021] The invention is a layered double hydroxide nanoparticle (nLDH?) thiolated with L- cysteine and / or thioglycolic acid and / or 2-iminothiolane hydrochloride and / or glutathione and / or N-acetyl cysteine and / or 3-mercaptopropionic acid and / or 6- mercaptohexanoic acid, which can be directly applied to the mucosal tissue and consists of magnesium / aluminium (Mg / AI) structures for use in drug delivery.

[0022] In one embodiment of the invention, L-cysteine is used for thiolation of the layered double hydroxide nanoparticle.

[0023] The synthesis method of the drug delivery system that is the subject of the invention comprises the process steps of: i. preparing salt solution by dissolving magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and aluminium nitrate nonahydrate AI(NO3)3.9H2O in deionised water (DI water), ii. dissolving sodium hydroxide (NaOH) in deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring and mixing, iv. centrifuging the mixture at the end of mixing, and washing the LDH precipitate with DI water, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere, vii. activating L-cysteine hydrochloride monohydrate and / or thioglycolic acid and / or 2-iminothiolane hydrochloride and / or glutathione and / or N-acetyl cysteine and / or 3-mercaptopropionic acid and / or 6-mercaptohexanoic acid structure with solid form 1 -Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in DI water under nitrogen atmosphere, viii. adding the mixture activated in the process step (vii) to the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii), and X. placing the mixture in a dialysis membrane after mixing and purifying the nLDHT structure by dialysis against DI water.

[0024] An embodiment of the synthesis method of the drug delivery system that is the subject of the invention comprises the process steps of: i. preparing salt solution by dissolving magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and aluminium nitrate nonahydrate AI(NO3)3.9H2O in deionised water (DI water), ii. dissolving sodium hydroxide (NaOH) in deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring and mixing, iv. centrifuging the mixture at the end of mixing, and washing the LDH precipitate with DI water, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere, vii. activating L-cysteine hydrochloride monohydrate (Cys) structure with 1 - Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in solid form in DI water under nitrogen atmosphere, viii. adding the mixture activated in the process step (vii) to the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii), and x. placing the mixture in a dialysis membrane after mixing, and dialysing it against DI water to purify the L-cysteine and thiolated layered double hydroxide nanoparticle (nLDHcys) structure.

[0025] An embodiment of the synthesis method of the drug delivery system that is the subject of the invention comprises the process steps of: i. preparing salt solution by dissolving 0.3-300 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 0.1 -100 mmol aluminium nitrate nonahydrate AI(NO3)3.9H2O in 1 -1000 mL deionised water (DI water), ii. dissolving 0.6-600 mmol sodium hydroxide (NaOH) in 0.4-4000 mL deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring at 100-3000 rpm and mixing for 1 -30 minutes, iv. centrifuging the mixture at 1000-15000 rpm for 1 -60 minutes at the end of mixing and washing the LDH precipitate, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in 10-10000 mL DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere for 6-48 hours at 70-120°C, vii. activating 0.004-4mmol L-cysteine hydrochloride monohydrate (Cys) structure with 0.004-4 mmol 1 -Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in solid form in 0.2-200 ml DI water under nitrogen atmosphere for 0.25-2 hours, viii. adding the mixture activated in the process step (vii) to 1 -1000 ml volume of the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii) for 0.5-12 hours, and x. placing the mixture in a dialysis membrane with a molecular weight separation limit of 1 -15 kDa after mixing, and dialysing it against DI water for 1 day to purify the L-cysteine and thiolated layered double hydroxide nanoparticle (nLDHcys) structure.

[0026] The synthesis method of the drug delivery system in another embodiment of the invention comprises the process steps of: i. preparing salt solution by dissolving 3 mmol magnesium nitrate hexahydrate (Mg(NOa)2.6H2O) and 1 mmol aluminium nitrate nonahydrate AI(NO3)3.9H2O in 10 mL deionised water (DI water), ii. dissolving 6 mmol sodium hydroxide (NaOH) in 40 mL deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring at 750 rpm and mixing for 10 minutes, iv. centrifuging the mixture at 4500 rpm for 5 minutes at the end of mixing and washing the LDH precipitate, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in 50 mL DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere for 24 hours at 80°C, vii. activating 0.04mmol L-cysteine hydrochloride monohydrate (Cys) structure with 0.04 mmol 1 -Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in solid form in 2 ml DI water under nitrogen atmosphere for 1 hour, viii. adding the mixture activated in the process step (vii) to 10 ml volume of the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii) for 1 hour, and x. placing the mixture in a dialysis membrane with a molecular weight separation limit of 12 kDa after mixing, and dialysing it against DI water for 1 day to purify the L-cysteine and thiolated layered double hydroxide nanoparticle (nLDHcys) structure.

[0027] In one application of the synthesis of nanoparticles used in drug delivery, which is the subject of the invention, first the synthesis of Mg / AI NO3 layered double hydroxide (LDH) nanoparticles (nLDH) is carried out. 3 mmol of magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 1 mmol of aluminum nitrate nonahydrate AI(NO3)3.9H2O are dissolved in 10 mL of deionised water (DI water) to prepare the salt solution. In another place, 6 mmol of sodium hydroxide (NaOH) is dissolved in 40 mL of deionised water. The prepared salt solution is added to the NaOH solution under nitrogen atmosphere while stirring at 750 rpm and stirred for 10 minutes. At the end of mixing, the mixture is centrifuged at 4500 rpm for 5 minutes and the LDH precipitate is washed. This process is repeated 3 times and finally the LDH precipitate obtained is dispersed in 50 mL DI water and loaded into a pressure-resistant autoclave reactor and subjected to hydrothermal treatment in a nitrogen atmosphere at 80°C for 24 hours. The size, particle size distribution (PDI) and zeta potential measurements of the obtained LDH suspension were made in the device. The results of these measurement are given in Table 1 . Table 1. Size, PDI and zeta potential measurement results of nLDH and nLDHcys formulations

[0028] In the continuation of an embodiment of the synthesis of nanoparticles used in drug delivery, the modification of the nLDH structure synthesised in the first stage with a thiol group-containing compound is carried out. 0.04 mmol of the thiol group-containing compound L-cysteine hydrochloride monohydrate (Cys) is activated with 0.04 mmol of 1 -Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in solid form in 2 ml of DI for 1 hour under a nitrogen atmosphere and then added to 10 mL of nLDH suspension under nitrogen atmosphere and stirring. After 1 hour of stirring, the size, PDI and zeta potential of the obtained nLDHcys formulation were performed after being placed in a dialysis membrane with a molecular weight separation limit of 12 kDa and purified by dialysis against DI water for one day. The results are given in Table 1 .

[0029] In said embodiment of the invention, the synthesised nLDH and nLDHcys samples were analysed on the FTIR device. After the samples were ground by diluting them with IR purity potassium bromide (KBr) at a weight ratio of 1 / 200 (sample / KBr), their pellets were prepared with the help of a press and the spectra were recorded in the wavenumber range of 4000-750 cm’1. The FTIR spectra of the nLDH and nLDHcys samples are given in Figure 1 . In the FTIR spectra of the LDH sample, the sharp and broad absorption band in the 3600-3100 cm’1region with a maximum of approximately 3467 cm’1comes from the asymmetric and symmetric vibrations of the -OH groups. The small sharp band with a maximum of approximately 1631 cm’1is due to the deformation and bending vibrations of the H2O molecules. The long and sharp absorption band with a maximum of 1383 cm’1in the 1500-1250 cm’1region is due to carbonate and nitrate ions intercalated between the layered structure. The small sharp or broad shoulder bands in the 1000-500 cm’1region are formed due to the skeletal vibrations of LDH, especially the deformation and translational vibrations of the AI-OH and Mg-OH structures. In the FTIR spectra of the LDHcys sample, new absorption bands were formed with the modification of LDH with Cys and changes occurred in the existing absorption bands.

[0030] With the addition of primary amine to the structure, an increase in the intensity of the absorption band (max. 3462 cm’1) was observed in the 3500-3100 cm’1region due to the asymmetric and symmetric stretching vibrations of the primary amine in that region. A new absorption band in the form of a wide shoulder was formed in the 3000-2800 cm’1region due to the asymmetric and symmetric stretching vibrations of the -CH2- groups.

[0031] Since the carboxylic acid group in L-cysteine reacts with the hydroxyl group in LDH, the band belonging to the C=O stretching vibrations originating from the carboxylic acid is not observed in the 1740-1700 cm’1region. The intensity of the small sharp band with a maximum at 1631 cm’1increased due to the effect of the primary amine N-H deformation vibrations in the 1650-1580 cm’1region and the carboxylate -COO- structure asymmetric stretching vibrations in the 1695-1540 cm’1region. Again, the intensity of the absorption band with a maximum at approximately 1383 cm’1in the LDH spectra increased and shifted slightly to the right (maximum at approximately 1365 cm’1) due to the effect of the carboxylate -COO- structure symmetric stretching vibrations formed in the 1440-1335 cm’1region and the C-H deformation vibrations formed in the 1410-1350 cm’1region. The small shoulder-shaped band with a maximum at approximately 1 149 cm’1was formed by the vibrations of the -CH2-NH2 group. The small sharp or broad shoulder bands in the 1000-500 cm’1region increased in intensity due to the C-N stretching and N-H out-of-plane bending vibrations of the primary amine group. FTIR spectra show that the intended reaction between the -OH groups in the LDH structure and the -COOH groups in the Cys molecule occurred. FTIR analysis was insufficient to prove the presence of -SH groups in the Cys molecule. Therefore, the presence of -SH groups was proven by the determination of the thiol group.

[0032] Thiol group determination was carried out using the iodometric method [8]. 10 mg of LDH and thiolated LDHcys product were taken and dispersed in 3 ml of DI water and the pH value was adjusted to the range of 2-3 with 1 N HCI. 150 pl of 1 % (w / v) aqueous starch solution was added as an indicator and the sample was titrated with 1.0 mM iodine solution until a light blue colour. The amount of thiol groups in the samples subjected to thiol analysis is calculated according to Formula 1 .

[0033] Formula 1 . Thiol Group Quantity Analysis Formula

[0034] N x F x S T ~ 1000

[0035] T : Amount of thiol group content (pmol / g LDH)

[0036] N : Normality of iodine solution (mmol / mL)

[0037] F : Factor of iodine solution

[0038] S : Consumption [ml

[0039] In the calculations made according to Formula 1 , there was no thiol group in the nLDH sample, while the amount of -SH group in the nLDHcys sample was determined as 102.95 ± 7.1 pmol / g.

[0040] In an application of the invention, analyses were performed to determine the mucin binding activity of the synthesised nLDH and LDHcys formulations and the results of these analyses were compared with each other. The mucoadhesive activities of the nLDH and LDHcys formulations were evaluated by examining the interaction between mucin and nanoparticles in aqueous solution [9]. Different amounts of mucin were dissolved in DI water (0.1 -0.5 mg / mL) and incubated with equal volumes of nLDH and nLDHcys nanosuspensions at 37°C for 10 minutes, then centrifuged and the amount of free mucin in the supernatant was determined by UV-Vis spectrophotometer (260 nm wavelength) and the mucin binding activities of the nanoformulations were determined for varying mucin concentrations using Formula 2. The results are given in Table 2. The results show that the nLDHcys formulation interacts significantly more with mucin than nLDH at all concentrations, meaning that it has better mucoadhesive properties.

[0041] Formula 2. Mucin Binding Activity Formula

[0042] Total mucin amount — Amount of unbound mucin Mucin Binding Activity = - - -

[0043] Total mucin amount

[0044] For an embodiment of the invention, the interaction of the synthesised nLDH and nLDHcys formulations with mucin was also monitored by the change in zeta potential. It was expected that the zeta potentials of the nanoparticles would change as a result of the interaction of the nLDH and nLDHcys formulations with positive zeta potential values with the negatively charged mucin. It was predicted that the positive zeta potential would decrease due to the adsorption of mucin onto the nanoparticle surface. In order to examine this situation, different amounts of mucin were dissolved in DI water (0.1 -0.5 mg / mL) and mixed with equal volumes of nLDH and nLDHcys nano suspensions, and the change in zeta potentials after interaction with mucin was examined and the results obtained are given in Figure 3. According to the results, it was determined that the decrease in the zeta potential value of the nLDHcys formulation was greater than the nLDH formulation, especially at mucin concentrations higher than 0.1 mg / mL. This situation showed that the nLDHcys formulation had better interaction with mucin, that is, better mucoadhesive properties.

[0045] For an application of the invention, tests were conducted to determine the adhesion of the synthesised nLDH and nLDHcys formulations in mucosal tissue. Two 2x1 cm pieces were cut from frozen sheep vagina after thawing, and soaked with Phosphate buffered saline (PBS) and 100 pL of LDH and LDHcys were added separately. After incubation at 37°C for 1 hour, it was washed with 2 mL of PBS by holding it at a 45° angle and the washing solution was collected after every 200 pL of washing consumption

[0010] , The amount of nLDH and nLDHcys nanoparticles passing into the washing solution was determined with UV-Vis spectrophotometer (296 nm wavelength). The wash solution without nanoparticle addition was used as a blank (baseline). According to the results obtained, it was determined that approximately 68% of nLDH passed into the wash solution at the end of the wash process, while approximately 41 % of LDHcys passed, i.e. 59% remained in the tissue. This study also proved that the LDHcys formulation has higher adherence in the mucosal tissue.

[0046] The effects of the nLDH and nLDHcys formulations synthesised for an embodiment of the invention on cell viability were performed using the MTT method. The cell viability rates obtained as a result of 24 and 48 hours of incubation of nLDH and nLDHcys formulations at varying concentrations (14.06-450 pg / mL) on the cervical cancer cell line (HeLa), colon cancer cell line (HT29) and mouse fibroblast cell line (L929) as the healthy cell line were determined. The results for the HeLa cell line are given in Figure 4, the results for the HT29 cell line are given in Figure 5, and the results for the L929 cell line are given in Figure 6. Studies in the literature indicate that nLDH formulations are biocompatible and have low cytotoxic properties. The invention confirms this, and it has been observed that the thiolated nLDHcys formulation obtained after modification also exhibits a very similar profile. It has been determined that the thiolation modification performed to increase the mucoadhesive property does not have a negative effect on the cytotoxic effect of the formulation.

[0047] REFERENCES

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[0049] [2] §enturk, S., Gbk, M. K., Demir, K., Pabuccuoglu, S., Ozgumu§, S. 2024. "Optimized Zn / AI-NO3 LDHs nanosuspensions as a new generation inorganic gene carrier systems for primary cell lines", Journal of Dispersion Science and Technology, 1 -13.

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Claims

CLAIMS1. A drug delivery system that can be directly applied to the mucosal tissue wherein it is a layered double hydroxide nanoparticle (nLDHT) consisting of magnesium / aluminium (Mg / AI) structures and thiolated with L-cysteine and / or thioglycolic acid and / or 2-iminothiolane hydrochloride and / or glutathione and / or N-acetyl cysteine and / or 3-mercaptopropionic acid and / or 6-mercaptohexanoic acid.

2. A drug delivery system according to Claim 1 , wherein it is an layered double hydroxide nanoparticle (nLDHT) thiolated with L-cysteine, consisting of magnesium / aluminium (Mg / AI) structures.

3. Synthesis method of a drug delivery system that can be directly applied to mucosal tissue, comprising the process steps of: i. preparing salt solution by dissolving magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and aluminium nitrate nonahydrate AI(NO3)3.9H2O in deionised water (DI water), ii. dissolving sodium hydroxide (NaOH) in deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring and mixing, iv. centrifuging the mixture at the end of mixing, and washing the LDH precipitate with DI water, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere, vii. activating L-cysteine hydrochloride monohydrate and / or thioglycolic acid and / or 2-iminothiolane hydrochloride and / or glutathione and / or N-acetyl cysteine and / or 3-mercaptopropionic acid and / or 6-mercaptohexanoic acid structure with 1 -Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in DI water under nitrogen atmosphere,viii. adding the mixture activated in the process step (vii) to the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii), and x. placing the mixture in a dialysis membrane after mixing and purifying the nLDH? structure by dialysis against DI water.

4. A synthesis method according to claim 3, comprising the steps of: i. preparing salt solution by dissolving magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and aluminium nitrate nonahydrate AI(NO3)3.9H2O in deionised water (DI water), ii. dissolving sodium hydroxide (NaOH) in deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring and mixing, iv. centrifuging the mixture at the end of mixing, and washing the LDH precipitate with DI water, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere, vii. activating L-cysteine hydrochloride monohydrate (Cys) structure with 1 - Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in solid form in DI water under nitrogen atmosphere, viii. adding the mixture activated in the process step (vii) to the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii), and x. placing the mixture in a dialysis membrane after mixing, and dialysing it against DI water to purify the L-cysteine and thiolated layered double hydroxide nanoparticle (nLDHcys) structure.

5. A synthesis method according to claim 4, comprising the steps of: i. preparing salt solution by dissolving 0.3-300 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 0.1 -100 mmol aluminium nitrate nonahydrate AI(NO3)3.9H2O in 1 -1000 mL deionised water (DI water), ii. dissolving 0.6-600 mmol sodium hydroxide (NaOH) in 4-4000 mL deionised water, iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring at 100-3000 rpm and mixing for 1 -30 minutes, iv. centrifuging the mixture at 1000-15000 rpm for 1 -60 minutes at the end of mixing and washing the LDH precipitate, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in 10-10000 mL DI water, loading it into a pressureresistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere for 6-48 hours at 70-120°C, vii. activating 0.004-4mmol L-cysteine hydrochloride monohydrate (Cys) structure with 0.004-4 mmol 1 -Ethyl-3-(3'- dimethylaminopropyl)carbodiimide hydrochloride in 0.2-200 ml DI water under nitrogen atmosphere for 0.25-2 hours, viii. adding the mixture activated in the process step (vii) to 1 -1000 ml volume of the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii) for 0.5-12 hours, and x. placing the mixture in a dialysis membrane with a molecular weight separation limit of 1 -15 kDa after mixing, and dialysing it against DI water for 1 day to purify the L-cysteine and thiolated layered double hydroxide nanoparticle (nLDHcys) structure.

6. A synthesis method according to claim 5, comprising the steps of: i. preparing salt solution by dissolving 3 mmol magnesium nitrate hexahydrate (Mg(NOs)2.6H2O) and 1 mmol aluminium nitrate nonahydrate AI(NO3)3.9H2O in 10 mL deionised water (DI water), ii. dissolving 6 mmol sodium hydroxide (NaOH) in 40 mL deionised water,iii. adding the salt solution prepared in step (i) to the NaOH solution under nitrogen atmosphere while stirring at 750 rpm and mixing for 10 minutes, iv. centrifuging the mixture at 4500 rpm for 5 minutes at the end of mixing and washing the LDH precipitate, v. repeating step (iv) 3 times, vi. obtaining the nLDH structure by dispersing LDH precipitate obtained after process step (v) in 50 mL DI water, loading it into a pressure-resistant autoclave reactor and subjecting it to hydrothermal treatment in a nitrogen atmosphere for 24 hours at 80°C, vii. activating 0.04mmol L-cysteine hydrochloride monohydrate (Cys) structure with 0.04 mmol 1 -Ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride in solid form in 2 ml DI water under nitrogen atmosphere for 1 hour, viii. adding the mixture activated in the process step (vii) to 10 ml volume of the nLDH structure obtained after step (vi) while stirring under nitrogen atmosphere, ix. mixing the mixture of the process step (viii) for 1 hour, and x. placing the mixture in a dialysis membrane with a molecular weight separation limit of 12 kDa after mixing, and dialysing it against DI water for 1 day to purify the L-cysteine and thiolated layered double hydroxide nanoparticle (nLDHcys) structure.

7. Drug carrier system synthesised by a method according to any one of claims 3 to 6.

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