A mucoadhesive nanoparticulate drug delivery system and its synthesis method.

Abstract

The invention relates to a nanoparticle for use as a drug carrier and the method of synthesizing this nanoparticle. In the invention, magnesium / aluminum (Mg / Al) mucoadhesive thiolated bilayer hydroxide (nLDHT) nanoparticles are thiolated with L-cysteine ​​and / or thioglycolic acid and / or 2-iminotiolane hydrochloride and / or glutathione and / or N-acetylcysteine ​​and / or 3-mercaptopropionic acid and / or 6-mercaptohexanoic acid. Thus, nLDHT interacts with mucin on the surface of the target mucosal tissue for a longer period and acts with fewer side effects in the target tissues.

Description

1 TARIFF A nanopartic drug delivery system with mucoadhesive properties. METHOD OF SYNTHESIS Technical Area 5 The invention is thiolated for use as a drug delivery system. With magnesium / aluminum (Mg / Al) bilayer hydroxide nanoparticles (nLDHT) It is related to the invention. Thanks to the nanoparticles that are the subject of the invention, mucoadhesives are used in drug delivery. The properties are being enhanced, and drugs are being targeted in a more specific way with fewer side effects. It is being moved. 10 State of the Art Drug delivery systems deliver therapeutic active ingredients directly to targeted areas. These are systems designed to facilitate the transport of goods. These systems are designed to reach the target cell. and depending on the active substance carried, micelle, dendrimer, liposome, nanoparticle or 15 They may have a carbon nanotube structure. However, some current drug delivery systems It had fundamental limitations. Some of these limitations were the first-pass effect and high doses. the need, fluctuations in plasma drug levels and sustained drug release or effect These limitations can be listed as the inability to provide certain things. Many studies are being conducted to eliminate the disadvantages. These studies number 20 As a result, nano-sized carrier systems are able to overcome these limitations. It has been recognized that it has potential. Bilayer hydroxides (LDH) are an important inorganic group with a two-dimensional structure. It is a nanomaterial. It has a high surface-to-volume ratio and can accommodate large molecules. 25 Thanks to its capabilities, it is a good carrier for drug and gene delivery [1]. The LDH structure consists of two It contains valence and trivalent metal ions. These ions are positively charged. It forms layers. Exchangeable anions are located between these layers. It receives and thanks to these ions, the release of the drug is regulated [2]. This feature, It reduces the frequency of drug administration while increasing the therapeutic efficacy of the drugs. 30 Other advantages of LDH include ease of production, low cost, and biocompatibility. These include low toxicity and protection of the intercalated drug, etc. LDHs They also possess high drug / active ingredient loading capacity and stability. Furthermore, 2 LDH has good biodegradability and biocompatibility compared to other carrier systems. It also has many advantages, such as low cytotoxicity. LDHs are available at a specific pH. because they have the ability to continuously release the drug within this range It allows the body to release the substance in the desired area. This controlled release is different from other treatments. This reduces the likelihood of side effects commonly encountered in these methods. Appendix 5 In this way, drug-loaded LDHs are rapidly excreted in the body and do not accumulate in organs [3]. Therefore, LDH is a promising nanomaterial for various pharmaceutical applications. However, there are still no ways to make it usable across a wide range of applications. No studies have been conducted. Although drug delivery systems are used in the treatment of many diseases, when applied directly to the target tissue or closer to the target They are much more effective when applied at specific points. Today, therapeutic... oral administration of agents using conventional drug delivery systems Despite being widely accepted, these drugs may not reach the target tissues for 15 days. Their dissolution or enzymatic degradation prevents the drugs from acting as intended. This prevents it from doing so. Therefore, in order to shorten the route of drug administration... Studies have begun on different application methods (for example, (Direct application of medications to the areas they are intended to treat). Oral medications. rather than the urinary tract, the gastrointestinal system, the female reproductive system, the eyes, the mouth, the nose, and 20 Administering via the surface of mucosal tissues such as the lungs is preferable to oral administration. It is seen that it has many advantages [4]. This method increases the bioavailability of drugs. It increases, provides target-specific distribution, and reduces side effects. It can reduce [the volume of tissue]. In this application method, mucosal tissues are commonly preferred. is being done. 25 Mucosal tissues (gastrointestinal system, female reproductive system, eye, mouth, nose and The lungs, etc., have a rich network of blood vessels. This rich vascular network allows for the rapid and efficient absorption of medication. This ensures effective absorption. Mucosal administration of the drug allows the drug to be administered directly. It ensures that the drug reaches the target area and is exposed to 30% of the digestive tract. It protects against reactions. Studies using current techniques show that LDH protects the ocular The advantages of administering it via this route are being demonstrated. LDH is positively charged when applied to the cornea and... The conjunctiva is negatively charged. However, the interaction of drug-loaded LDHs with the cargo molecule... This can cause a decrease in the positive charges of LDH and the connection between it and the target region. 3 Non-covalent bonds, such as electrostatic interaction, are insufficient for the vehicle. This can limit the system's attachment to the target surface [5]. Overcoming this problem to be able to reach and increase particle-mucosal interaction from mucoadhesive strategy (mucoadhesion) is utilized [6]. Mucin is mucus that plays a role in mucoadhesion. It is the main component of the liquid. Based on the interaction of nanoparticles and mucin, 5 Nanoparticles aimed at extending the residence time of nanoparticles in mucosal tissues. Drug delivery systems can be developed. Physicochemical properties of mucin fibers. thanks to this, electrostatic interaction and hydrophobic interaction occur between the nanoparticles and them. non-covalent bonds such as hydrogen bonds, van der Waals bonds, or thiol groups covalent bonds form between nanoparticles containing mucin and cysteine ​​molecules found in mucin. Disulfide bonds are formed, and nanoparticles adhere to the mucosa via adhesive forces. They are able to adhere. Mucoadhesion of nanoparticles is generally achieved through ionic interactions and secondary interactions include hydrogen bonding, dipole interactions, and molecule-molecule interactions. While bonding is achieved through covalent bonds, thiolated nanoparticles are achieved through covalent bonds. It can be formed via thiol / disulfide exchange reactions or a simple 15 Through the oxidation process, biological surfaces are formed into disulfide and thiol substrates with disulfide. They are able to form bonds. By thiolation of drug-carrying nanoparticles. The significant enhancement of mucoadhesive properties is highlighted [7]. However The literature is insufficient regarding studies on the thiolation of the LDH system. remains. 20 Patent application number US2022250929A1 functionalized hybrid layered Synthesis of LDH structure containing [Sn2S6]4− anions in its layer and interlayers It is explained. However, the use of LDH for drug delivery purposes is not recommended. It does not contain. In addition, the aforementioned structure contains mucosal 25, primarily mucin. also what kind of interaction it can have with other structures located in the tissues It is insufficient. The limitations and inadequacies of the solutions in the current technology, in the known state of the technology... In commonly used methods, drugs must reach the target tissue within 30 days. degradation of drug delivery systems applied directly to mucosal tissues The fact that its ability to hold on is limited and therefore its impact rate is low is promising. the interaction of LDH structures with target tissues due to the cargo they carry 4 Due to reasons such as the decrease, it is necessary to make improvements in this area. It has been made. Brief Description and Objectives of the Invention The invention describes a magnesium / aluminum (Mg / Al) mucoadhesive thiolated bilayer hydroxide 5. (nLDHT) nanoparticles are used as drug delivery systems. The invention refers to... Thiolation of the L-cysteine ​​amino acid is performed using different structures. This occurs with more mucin located on the surface of the mucosal tissue. It interacts with the target mucosal tissue, allowing nLDHT to remain there for a longer period. By interacting for a limited time, it acts on target tissues with fewer side effects. Active ingredient 10 Thanks to the direct application of these substances to mucosal tissues, this active ingredient Interaction with other tissues and organs is minimized, and in this way, these tissues and This prevents potential side effects in the organs. The main aim of the invention is to create a 15-year-old that can adhere for a long time to the mucosal tissue to which it is directly applied. The goal is to provide a drug delivery system with minimal side effects. The drug carrier system that is the subject of this invention. In this system, the nLDHT structure consists of Mg / Al layers. Double-layered hydroxide. Structures added to the content of nanoparticles (nLDH), primarily L-cysteine. Thiolation occurs thanks to this. Thiol groups are present on the surface of nanoparticles. It forms covalent disulfide bonds with glycoproteins such as mucin, which is 20 stronger and longer-lasting adhesion of nanoparticles to mucosal surfaces This strong binding ensures that nanoparticles remain on mucosal surfaces. extending the duration, allowing drugs to reach target areas more effectively. It provides and increases absorption. At the same time, it allows nanoparticles to adhere to the cell membrane. It facilitates adhesion and uptake into the cell. Thiol groups are located on the cell membrane at 25°C. By forming disulfide bonds with proteins, nanoparticles can enter cells more easily. This process facilitates the passage of therapeutic agents by increasing their cellular uptake. It enhances the intracellular bioavailability of drugs. This interaction is explained in the invention. This is possible thanks to the -SH groups found in structures such as L-cysteine ​​used. Thus, nLDHT coats the mucosal tissue to which it is applied, and penetrates the tissue for 30 minutes. interacts with mucin, which is found in mucus and gives it a moist and slippery texture. This involves the thiolation process of thioglycolic acid, 2-iminotiolane hydrochloride, glutathione, N-acetyl cysteine, 3-mercaptopropionic acid, or 6-mercaptohexanoic acid This can also be achieved using any of these structures: nLDHT and mucin. The interaction that occurs between them is much more pronounced compared to non-thiolated nLDH structures. It can hold on for a long time. This hold allows it to carry its cargo (therapeutic) to nLDHT. sufficient time for the active ingredient to be effectively transferred to the mucosal tissue It recognizes this. In this way, mucosal tissues interact directly with the active substance. It can pass through, and thanks to this direct interaction, the probability of side effects occurring is 5%. This is being prevented. Explanation of the Figures Figure 1: FTIR spectrum (1: FTIR spectrum of LDH sample, 2: LDHCys) (FTIR spectroscopy of the sample) 10 Figure 2: Graph of mucin binding activity (1: mucin of nLDH formulation) binding activity, 2: mucin binding activity of nLDHCys formulation) Figure 3: Zeta Potential Graph (1: nLDH formulation – mucin solution) Zeta potential value measured after mixing, 2: nLDHCys formulation – (Zeta potential value measured after mixing the mucin solution) 15 Figure 4: HeLa Cell Viability-Concentration Graphs (1: nLDH formulation Results of its effect on cell viability in the HeLa cell line after 24 hours, 2: Effect of nLDHCys formulation on cell viability in HeLa cell line The results after 24 hours, 3: nLDH formulation in HeLa cell line cells Results of its effect on viability after 48 hours, 4: nLDHCys formulation 20 Results of the effect on cell viability in the HeLa cell line after 48 hours. Figure 5: HT29 Cell Viability-Concentration Graphs (1: nLDH formulation Results of the effect on cell viability in the HT29 cell line after 24 hours, 2: Effect of nLDHCys formulation on cell viability in HT29 cell line Results after 24 hours, 3: nLDH formulation in cell 25 of HT29 cell line Results of its effect on viability after 48 hours, 4: nLDHCys formulation Results of the effect on cell viability in the HT29 cell line after 48 hours. Figure 6: L929 Cell Viability-Concentration Graphs (1: nLDH formulation Results of the effect on cell viability in the L929 cell line after 24 hours, 2: The effect of nLDHCys formulation on cell viability in H929 cell line 24 30 The results at the end of the hour, 3: nLDH formulation in H929 cell line cells Results of its effect on viability after 48 hours, 4: nLDHCys formulation Results of the effect on cell viability in the H929 cell line after 48 hours) 6 Detailed Description of the Invention The invention uses thiolated material for drug delivery systems. magnesium / aluminum (Mg / Al) mucoadhesive bilayer hydroxide nanoparticles (nLDHT) and its synthesis method are related. The thiolation mentioned in the invention. Thanks to this, nLDHT interacts more with mucin located on the surface of the mucosal tissue it targets. It engages in long-term interaction and acts with fewer side effects in target tissues. is doing. The invention is for use in drug delivery, and can be applied directly to mucosal tissue. and composed of magnesium / aluminum (Mg / Al) structures, L-cysteine ​​and / or thioglycolic 10 acid and / or 2-iminotiolane hydrochloride and / or glutathione and / or N-acetylcysteine and / or thiolated with 3-mercaptopropionic acid and / or 6-mercaptohexanoic acid It is a double-layered hydroxide nanoparticle (nLDHT). In one application of the invention, L-15 is used for the thiolation of double-layered hydroxide nanoparticles. Cysteine ​​is used. The synthesis method of the drug delivery system described in this invention is as follows: i. magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and aluminum nitrate nonahydrate Al(NO3)3.9H2O is dissolved in deionized water (DI water) to form salt 20 preparation of the solution, ii. Dissolving sodium hydroxide (NaOH) in deionized water, iii. While mixing the salt solution prepared in step (i), nitrogen Added to NaOH solution under atmospheric conditions and stirred continuously, iv. After mixing, the mixture is centrifuged and the LDH precipitate is separated from the DI water by 25 washing, v. Repeating step (iv) three times, vi. The LDH precipitate obtained after step (v) is mixed with DI water. Nitrogen is distributed inside and loaded into a pressure-resistant autoclave reactor. by subjecting it to atmospheric conditions and hydrothermal treatment, the nLDH structure is 30 obtaining, vii. L-cysteine ​​hydrochloride monohydrate and / or thioglycolic acid and / or 2- iminotianoline hydrochloride and / or glutathione and / or N-acetyl cysteine ​​and / or 3-mercaptopropionic acid and / or 6-mercaptohexanoic acid structure, 7 DI with solid form 1-Ethyl-3-(3ʹ-dimethylaminopropyl)carbodiimide hydrochloride Activation in a nitrogen atmosphere in water, viii. The mixture activated in step (vii) of process (vi) The nLDH structure obtained after this step is subjected to a nitrogen atmosphere. 5 added while mixing Mixing the mixture in step ix. (viii), x. After mixing, the mixture is placed on the dialysis membrane and dialysis is performed against DI water. purification of nLDHT structure It includes the steps involved in the process. An application of the synthesis method for the drug delivery system described in the invention; i. magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and aluminum nitrate nonahydrate Al(NO3)3.9H2O is dissolved in deionized water (DI water) to form salt. Preparation of the solution, 15 ii. Dissolving sodium hydroxide (NaOH) in deionized water, iii. While mixing the salt solution prepared in step (i), nitrogen Added to NaOH solution under atmospheric conditions and stirred continuously, iv. After mixing, the mixture is centrifuged and the LDH precipitate is separated from the DI water. washing, 20 v. Repeating step (iv) three times, vi. The LDH precipitate obtained after step (v) is mixed with DI water. Nitrogen is distributed inside and loaded into a pressure-resistant autoclave reactor. in the atmosphere and by subjecting it to hydrothermal treatment, the nLDH structure obtaining, 25 vii. The structure of L-cysteine ​​hydrochloride monohydrate (Cys) in its solid form is 1-Ethyl-3-(3ʹ- (dimethylaminopropyl)carbodiimide hydrochloride with DI water nitrogen activation in the atmosphere, viii. The mixture activated in step (vii) of process (vi) After this step, the resulting nLDH structure was subjected to 30 hours of nitrogen hydroxide treatment. adding while mixing Mixing the mixture in step ix. (viii), 8 x. After mixing, the mixture is placed on the dialysis membrane and dialysis is performed against DI water. bilayer hydroxide nanoparticles (nLDHCys) made and thiolated with L-cysteine purification of its structure It includes the steps involved in the process. An application of the synthesis method for the drug delivery system described in the invention; i. 0.3-300 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 0.1-100 mmol aluminum nitrate nonahydrate Al(NO3)3.9H2O 1-1000 mL deionized water (DI water) to prepare a salt solution, ii. 0.6–600 mmol sodium hydroxide (NaOH) in 0.4–4000 mL deionized water (10°C) dissolution, iii. The salt solution prepared in step (i) is heated at 100-3000 rpm While stirring, add to NaOH solution under nitrogen atmosphere, 1-30 minutes mixing throughout, iv. After mixing, the mixture is left to ferment at a speed of 1000-15000 rpm for 1-60 minutes. centrifuging and washing away the LDH precipitate, v. Repeating step (iv) three times, vi. The LDH precipitate obtained after step (v) is 10-10000 mL of DI is dissolved in water and loaded into a pressure-resistant autoclave reactor. 20 subjected to hydrothermal treatment in a nitrogen atmosphere and at 70-120°C for 6-48 hours Obtaining the nLDH structure by retention, vii. The structure of 0.004-4 mmol L-cysteine ​​hydrochloride monohydrate (Cys) is 0.004-4 mmol of solid form 1-Ethyl-3-(3ʹ-dimethylaminopropyl)carbodiimide hydrochloride Activation by 0.2-200 ml DI in water for 0.25-2 hours in a nitrogen atmosphere, viii. The mixture activated in step (vii) is processed in step (vi) 25 After this step, the resulting nLDH structure is mixed with nitrogen in a volume of 1-1000 ml. added while being mixed under the atmosphere Mixing the mixture in step ix. (viii) for 0.5-12 hours, x. dialysis with molecular weight separation limit of 1-15 kDa at the end of mixing. The membrane is placed and dialysis is performed for one day against DI water, then L-cysteine ​​is added for 30 days. thiolated bilayer hydroxide nanoparticle (nLDHCys) structure purification It includes the steps involved in the process. 9 Another application of the invention is the synthesis method of the drug delivery system; i. 3 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 1 mmol aluminum nitrate nonahydrate Al(NO3)3.9H2O 10 mL deionized water (DI water) preparing a salt solution by dissolving it in it, ii. Dissolving 6 mmol of sodium hydroxide (NaOH) in 40 mL of deionized water, 5 iii. While the salt solution prepared in step (i) is being mixed at 750 rpm, Added to NaOH solution under nitrogen atmosphere and left for 10 minutes. mixing, iv. After mixing, the mixture is centrifuged at 4500 rpm for 5 minutes. Washing of LDH precipitate, 10 v. Repeating step (iv) three times, vi. The LDH precipitate obtained after step (v) is mixed with 50 mL of DI Nitrogen is dissolved in water and loaded into a pressure-resistant autoclave reactor. by being subjected to hydrothermal treatment in the atmosphere and at 80°C for 24 hours Obtaining the nLDH structure, 15 vii. The structure of 0.04 mmol L-cysteine ​​hydrochloride monohydrate (Cys) is 0.04 mmol 1- 1 ml of ethyl-3-(3ʹ-dimethylaminopropyl)carbodiimide hydrochloride in 2 ml of DI water activation in a nitrogen atmosphere for one hour, viii. The mixture activated in step (vii) of process (vi) After this step, the resulting nLDH structure is mixed into a 10 ml volume of nitrogen atmosphere for 20 minutes. added while stirring underneath Mixing the mixture in step ix. (viii) for 1 hour, x. dialysis with molecular weight separation limit of 12 kDa at the end of mixing. The membrane is placed and dialysis is performed for one day against DI water with L-cysteine. 25 thiolated bilayer hydroxide nanoparticle (nLDHCys) structure purification It includes the steps involved in the process. The synthesis of nanoparticles used in the delivery of pharmaceuticals, which is the subject of this invention, takes 30 years. In the application, firstly Mg / Al NO3 bilayer hydroxide (LDH) nanoparticles (nLDH) synthesis is carried out. 3 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 1 mmol aluminum nitrate nonahydrate Al(NO3)3.9H2O 10 mL A salt solution is prepared by dissolving it in deionized water (DI water). Another method... On the other hand, 6 mmol of sodium hydroxide (NaOH) is dissolved in 40 mL of deionized water. It dissolves. The prepared salt solution is stirred at 750 rpm in a nitrogen atmosphere. It is added to the NaOH solution below and stirred for 10 minutes. After mixing, the mixture is centrifuged at 4500 rpm for 5 minutes and LDH is applied. The precipitate is washed. This process is repeated 3 times, and finally, the resulting 5 LDH precipitate is dispersed in 50 mL of DI water and transferred to a pressure-resistant autoclave reactor. loaded and subjected to hydrothermal treatment in a nitrogen atmosphere and at 80°C for 24 hours. The size, particle size distribution (PDI) of the resulting LDH suspension is measured. Zeta potential measurements were performed using the device. The results of these measurements are given in Table 1. is given. 10 Table 1. Size, PDI, and zeta potential of nLDH and nLDHCys formulations. measurement results Size (nm) PDI Zeta Potential (mV) nLDH 68.9 ± 2.4 0.174 ± 0.029 37.1 ± 1.4 nLDHCys 83.8 ± 2.7 0.260 ± 0.018 30.9 ± 2.3 The invention concerns the synthesis of nanoparticles used in drug delivery. In the continuation of the application, the thiol group of the nLDH structure synthesized in the first stage Modification is carried out with a compound containing 0.04 mmol of thiol group. The compound L-cysteine ​​hydrochloride monohydrate (Cys) is 0.04 mmol 1-Ethyl-3-(3ʹ- in solid form. (dimethylaminopropyl)carbodiimide hydrochloride in 2 ml DI for 1 hour in a nitrogen atmosphere 20 After activation, add 10 mL of nLDH suspension to a nitrogen atmosphere and stir. It is added below. After 1 hour of mixing, the molecular weight separation limit is 12. The dialysis membrane, which has a volume of kDa, is placed and dialysis is performed against DI water for one day. The size, PDI, and zeta of the nLDHCys formulation obtained after purification Its potential has been explored. The results are given in Table 1. 25 In the aforementioned application of the invention, the synthesized nLDH and nLDHCys samples The analyses were performed using an FTIR instrument. The samples were analyzed using IR-pure potassium. diluted with bromide (KBr) at a ratio of 1 / 200 by weight (mg) (sample / KBr) After grinding, the pellets were prepared using a press and the spectra were 4000-750 30 FTIR data for nLDH and nLDHCys samples was recorded in the cm-1 wavelength range. 11 The spectrum is given in Figure 1. The FTIR spectrum of the LDH sample is between 3600-3100. Sharp and broad absorption with a maximum of approximately 3467 cm-1 in the cm-1 region. The band originates from the asymmetric and symmetric vibrations of the -OH groups. Maximum A small, sharp band, approximately 1631 cm-1, indicates the deformation of H2O molecules. This is caused by bending vibrations. 5 in the 1500-1250 cm-1 region. long and sharp absorption bands with a maximum of 1383 cm-1 between the layered structure It originates from intercalated carbonate and nitrate ions. 1000-500 cm-1 The small, sharp or broad shoulder-shaped bands in this region are skeletal signs of LDH. vibrations, especially deformation and translation of Al-OH and Mg-OH structures This is caused by vibrations. In the FTIR spectroscopy of the nLDHCys sample, 10 Modification of LDH with Cys has resulted in the formation of new absorption bands, and they exist. Changes occurred in the absorption bands. FTIR of the LDH sample its maximum in the spectrum is approximately 3467 cm-1, in the 3600-3100 cm-1 region. sharp and wide absorption band, asymmetric and symmetric of -OH groups It results from vibrations. The small, sharp band 15 has a maximum of approximately 1631 cm-1. It results from the deformation and bending vibrations of H2O molecules. Long and sharp, with a maximum of 1383 cm-1 in the 1500-1250 cm-1 range. absorption band layered structure with intercalated carbonate and nitrate It originates from ions. Small sharp or insignificant spots in the 1000-500 cm-1 region. The broad shoulder bands, on the other hand, are particularly affected by the skeletal vibrations of LDH, especially Al-OH and 20 This results from deformation and translational vibrations of Mg-OH structures. In the FTIR spectroscopy of the LDHCys sample, the modification of LDH with Cys is observed. New absorption bands have formed and changes have been made to existing absorption bands. It occurred. 25 With the addition of a primary amine to the structure, the primary amine in the 3500-3100 cm-1 region absorption in that region due to the effect of asymmetric and symmetrical stretching vibrations An increase in the intensity of the band (max. 3462 cm-1) has been observed in the 3000-2800 cm-1 region. The wide range of 30 resulting from asymmetric and symmetric stretching vibrations of -CH2- groups. A new absorption band has formed in the shape of a shoulder. The carboxylic acid group in L-cysteine ​​reacts with the hydroxyl group in LDH. C=O stress due to carboxylic acid entering the 1740-1700 cm-1 region. 12 No band corresponding to its vibrations is observed. Primary amine N- in the 1650-1580 cm-1 region. H deformation vibrations and carboxylate -COO- structure in the 1695-1540 cm-1 region. Small sharp vibrations with a maximum of 1631 cm-1 due to the effect of asymmetric stress vibrations. The intensity of the band has increased. Carboxylate -COO- formed again in the 1440-1335 cm-1 region. The structure exhibits symmetrical tensile vibrations and CH5 formation in the 1410-1350 cm-1 region. Due to the effect of deformation vibrations, the maximum in the LDH spectrum is approximately 1383. The intensity of the cm-1 absorption band has increased and shifted slightly to the right (maximum approximately 1365 cm-1). The small shoulder-shaped band -CH2- with a maximum of approximately 1149 cm-1. The vibrations of the NH2 group were generated. Small sharp vibrations in the 1000-500 cm-1 region. and the broad shoulder bands are due to the CN stretching of the primary amine group and NH 10. Their intensity has increased due to the effect of out-of-plane bending vibrations. FTIR spectra LDH The intended relationship between the -OH groups in its structure and the -COOH groups in the Cys molecule FTIR analysis shows that the reaction occurred in the Cys molecule. The existence of SH groups has not been proven. Therefore, -SH groups Its presence has been proven by thiol group determination. 15 Thiol group determination was performed using the iodometric method [8]. LDH and 10 mg of the thiolated LDHCys product was dispersed in 3 ml of DI water and the pH was determined. The value was adjusted to a range of 2-3 with 1 N HCl. 150 µl of 1% was used as an indicator. (weight / volume) aqueous starch solution was added and the sample was mixed with 1.0 mM iodine solution for 20 minutes. Titrate was made to a light blue color. Thiol group of samples subjected to thiol analysis The amount is calculated according to Formula 1. Formula 1. Thiol Group Quantitative Analysis Formula 25 𝑇 𝑁 𝐹 𝑆1000 T: Thiol group content (µmol / g LDH) N: Normality of the iodine solution (mmol / mL) F: Factor of iodine solution S: Consumption (mL) 30 13 Calculations based on Formula 1 show that the nLDH sample does not contain a thiol group, The amount of -SH groups in the nLDHCys sample was determined to be 102.95 ± 7.1 µmol / g. In one application of the invention, the synthesized nLDH and LDHCys formulations were used with mucin. Analyses were conducted to determine the binding effectiveness, and the results of these analyses are 5. The mucoadhesive properties of nLDH and LDHCys formulations were compared with each other. its effectiveness is due to the interaction between mucin and nanoparticles in aqueous solution It was evaluated by examining [9]. Different amounts of mucin DI dissolved in water. (0.1-0.5 mg / mL) equal volumes of nLDH and nLDHCys nanosuspensions at 37°C After incubation for 10 minutes, it is centrifuged and the free 10 particles found in the supernatant are removed. The amount of mucin was determined by UV-Vis spectrophotometer (260 nm wavelength) and Nanoformulations for varying mucin concentrations using Formula 2 Mucin binding activities were determined. The results are given in Figure 2. The results, Interaction of nLDHCys formulation with mucin at all specified concentrations According to nLDH, it was significantly higher, meaning its mucoadhesive property was better. 15 This shows that. Formula 2. Mucin Binding Efficiency Equation 𝑀ü𝑠𝑖𝑛 𝐵𝑎ğ𝑙𝑎𝑚𝑎 𝐸𝑡𝑘𝑖𝑛𝑙𝑖ğ𝑖 𝑇𝑜𝑝𝑙𝑎𝑚 𝑚ü𝑠𝑖𝑛 𝑚𝑖𝑘𝑎𝑡𝚤 𝐵𝑎ğ𝑙𝑎𝑛𝑚𝑎𝑦𝑎𝑛 𝑚ü𝑠𝑖𝑛 𝑚𝑖𝑘𝑡𝑎𝑟𝚤𝑇𝑜𝑝𝑙𝑎𝑚 𝑚ü𝑠𝑖𝑛 𝑚𝑖𝑘𝑡𝑎𝑟𝚤 For an application of the invention, the synthesized nLDH and nLDHCys formulations were used to synthesize mucin. Its interaction was also monitored through changes in zeta potential. Positive zeta potential Formulations of nLDH and nLDHCys with negatively charged mucin having the specified values Changes in the zeta potentials of nanoparticles as a result of interaction 25 This was expected. Positive zeta was observed due to the adsorption of mucin onto the nanoparticle surface. A decrease in its potential value is anticipated. This situation For the purpose of analysis, different amounts of mucin DI were dissolved in water (0.1-0.5 mg / mL) in equal proportions. mixed with mucin by nano-suspensions of nLDH and nLDHCys in volumes The changes in zeta potentials after interaction were examined and the results obtained were 30 This is shown in Figure 3. According to the results, mucin levels, especially those higher than 0.1 mg / mL, were particularly high. decrease in zeta potential value of nLDHCys formulation at concentrations It was determined that it was higher than the nLDH formulation. This situation is nLDHCys 14 The formulation interacts better with mucin, meaning it is more mucoadhesive. It has been shown that its feature is better. Formulations of nLDH and nLDHCys synthesized for an application of the invention. Tests were conducted to determine its retention in mucosal tissue. 5 Two 2x1 cm pieces of thawed sheep vagina. The pieces were cut, moistened with phosphate-buffered saline (PBS), and 100 µL of LDH was added to them. and LDHCys were added separately. After incubating at 37°C for 1 hour, 2 mL of PBS was added. Washed while held at a 45° angle, and rinsed after every 200 µL of washing consumption. The solution was collected

[10] . Washing with UV-Vis spectrophotometer (296 nm wavelength) 10 The amount of nLDH and nLDHCys nanoparticles that passed into the solution was determined. Blind A washing solution without added nanoparticles was used as the (blank / baseline) solution. According to the results obtained, approximately 68% of nLDH is lost at the end of the washing process. When it passes into the solution, approximately 41% of the LDHCys passes through, meaning that 59% remains in the tissue. It has been determined that the LDHCys formulation remains in mucosal tissue for 15 days. It has been proven that its retention is higher. For one application of the invention, formulations of nLDH and nLDHCys were synthesized and used in cells. The effects on viability were determined using the MTT method. Cervical cancer 20 cell lines: HeLa cell line, colon cancer cell line (HT29) and healthy cell line. nLDH and nLDHCys formulations were studied on the mouse fibroblast cell line (L929). Incubation for 24 and 48 hours at varying concentrations (14.06-450 µg / mL) The resulting cell viability rates were determined. Belonging to the HeLa cell line. The results are shown in Figure 4, the results for the HT29 cell line are shown in Figure 5, and the results for the L929 cell line are shown in Figure 5. The results are given in Figure 6. Studies in the literature show that nLDH 25 It is stated that the formulations are biocompatible and exhibit low cytotoxic properties. The invention confirms this, however, the thiolated material obtained after modification. The nLDHCys formulation was also found to exhibit a very similar profile. Thiolation modification performed to enhance mucoadhesive properties It was determined that the formulation had no negative effect on the cytotoxic effect. 30 REFERENCES [1] Taviot-Guého, C., Prévot, V., Forano, C., Renaudin, G., Mousty, C., Leroux, F. 2018. “Tailoring hybrid layered double hydroxides for the development of innovative applications", Advanced Functional Materials, 28(27), 1703868. [2] Şentürk, S., Gök, MK, Demir, K., Pabuccuoğlu, S., Özgümüş, S. 2024. 5 "Optimized Zn / Al–NO3 LDHs nanosuspensions as a new generation inorganic gene carrier systems for primary cell lines", Journal of Dispersion Science and Technology, 1-13. [3] Ma, K., Chen, K.-Z., Qiao, S.-L. 2024. "Advances of Layered Double Hydroxide- Based Materials for Tumor Imaging and Therapy", The Chemical Record, 24(4), 10 e202400010. [4] Liu, C., Jiang, X., Gan, Y., Yu, M. 2021. "Engineering nanoparticles to overcome the mucus barrier for drug delivery: Design, evaluation and state-of-the-art", Medicine in Drug Discovery, 12, 100110. [5] Ameena Shirin, V. K., Sankar, R., Johnson, A. P., Gangadharappa, H. V., 15 Pramod, K. 2021. "Advanced drug delivery applications of layered double hydroxide", Journal of Controlled Release, 330, 398-426. [6] Liu, C., Jiang, X., Gan, Y., Yu, M. 2021. "Engineering nanoparticles to overcome the mucus barrier for drug delivery: Design, evaluation and state-of-the-art", Medicine in Drug Discovery, 12, 100110. 20 [7] Hock, N., Racaniello, G. F., Aspinall, S., Denora, N., Khutoryanskiy, V. V., Bernkop-Schnürch, A. 2022. "Thiolated Nanoparticles for Biomedical Applications: Mimicking the Workhorses of Our Body", Advanced Science, 9(1), 2102451. [8] Bernkop-Schnürch, A., & Steininger, S. 2000. "Synthesis and characterisation of mucoadhesive thiolated polymers." International Journal of Pharmaceutics, 194(2), 25 239–247. [9] Dyawanapelly, S., Koli, U., Dharamdasani, V., Jain, R., & Dandekar, P. 2016. "Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins." Drug Delivery and Translational Research, 6(4), 365–379. 30

[10] Eliyahu, S, Aharon, A., Bianco-Peled, H. 2018. " Acrylated chitosan nanoparticles with enhanced mucoadhesion" Polymers, 10, 106.

Claims

16 REQUESTS 1. It is a drug delivery system that can be applied directly to mucosal tissue, and its characteristic feature is: magnesium / aluminum (Mg / Al) structures, consisting of L-cysteine ​​and / or 5 thioglycolic acid and / or 2-iminotiolane hydrochloride and / or glutathione and / or 3- thiolated doublet with mercaptopropionic acid and / or 6-mercaptohexanoic acid It is a layered hydroxide nanoparticle (nLDHT).

2. A drug delivery system according to claim 1, characterized by its magnesium / aluminum 10-cell compound. Bilayer hydroxides consisting of (Mg / Al) structures, thiolated with L-cysteine. It is a nanoparticle (nLDHT).

3. Synthesis of a drug delivery system that can be applied directly to mucosal tissue. The method and its characteristic is; 15 i. 0.3-300 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 0.1- 100 mmol aluminum nitrate nonahydrate Al(NO3)3.9H2O 1-1000 mL Preparation of salt solution by dissolving in deionized water (DI water), ii. 0.6–600 mmol sodium hydroxide (NaOH) in 4–4000 mL deionized water dissolution, 20 iii. The salt solution prepared in step (i) is heated at 100-3000 rpm While being mixed, it is added to the NaOH solution under a nitrogen atmosphere, 1-30 stirring for a few minutes, iv. After mixing, the mixture is left to cool at a speed of 1000-15000 rpm for 1-60 minutes. Centrifuging and washing off the LDH precipitate, 25 v. Repeating step (iv) three times, vi. The LDH precipitate obtained after step (v) is 10- Dissolved in 10,000 mL of DI water and placed in a pressure-resistant autoclave reactor. loaded with water and hydrothermal filtration in a nitrogen atmosphere at 70-120°C for 6-48 hours. Obtaining the nLDH structure by subjecting it to treatment, 30 vii. The structure of 0.004-4 mmol L-cysteine ​​hydrochloride monohydrate (Cys), 0.004- 4 mmol 1-Ethyl-3-(3ʹ-dimethylaminopropyl)carbodiimide hydrochloride with 0.2-200 Activation of mL DI in water for 0.25-2 hours in a nitrogen atmosphere, 17 viii. The mixture activated in step (vii) of process (vi) After this step, the resulting nLDH structure is mixed with nitrogen in a volume of 1-1000 ml. added while being mixed under the atmosphere, Mixing the mixture in step ix. (viii) for 0.5-12 hours, x. dialysis 5 with molecular weight separation limit of 1-15 kDa at the end of mixing. The membrane is placed and dialysis is performed for one day against DI water, and L-cysteine ​​is removed. with thiolated bilayer hydroxide nanoparticle (nLDHCys) structure purification It includes the steps of the process.

4. It is a synthesis method according to claim 3, and its characteristic is; i. 3 mmol magnesium nitrate hexahydrate (Mg(NO3)2.6H2O) and 1 mmol aluminum nitrate nonahydrate Al(NO3)3.9H2O 10 mL deionized water (DI water) preparing a salt solution by dissolving it in it, ii. Dissolving 6 mmol of sodium hydroxide (NaOH) in 40 mL of deionized water, 15 iii. The salt solution prepared in step (i) is heated at a speed of 750 rpm. While stirring, it is added to the NaOH solution under a nitrogen atmosphere and left for 10 minutes. mixing throughout, iv. After mixing, the mixture is centrifuged at 4500 rpm for 5 minutes. Washing of LDH precipitate, 20 v. Repeating step (iv) three times, vi. 50 mL of LDH precipitate obtained after step (v) DI is dissolved in water and loaded into a pressure-resistant autoclave reactor. Subjected to hydrothermal treatment for 24 hours in a nitrogen atmosphere and at 80°C. Obtaining the nLDH structure by retention, 25 vii. The structure of 0.04 mmol L-cysteine ​​hydrochloride monohydrate (Cys), 0.04 mmol 1-Ethyl-3-(3ʹ-dimethylaminopropyl)carbodiimide hydrochloride with 2 mL DI water Activated in a nitrogen atmosphere for 1 hour, viii. The mixture activated in step (vii) of process (vi) After this step, 30% nitrogen is added to 10 ml of the resulting nLDH structure. added while being mixed under the atmosphere, Mixing the mixture in step ix. (viii) for 1 hour, x. dialysis with molecular weight separation limit of 12 kDa at the end of mixing. The membrane is placed and dialysis is performed for one day against DI water, and L-cysteine ​​is removed. 18 with thiolated bilayer hydroxide nanoparticle (nLDHCys) structure purification It includes the steps of the process.

5. A drug delivery system synthesized by a method according to claim 3 or 4. 5

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