WO2022125031A1 - Trimethyl chitosan nanoparticles containing magnetic- targeted gemcitabine - Google Patents
Trimethyl chitosan nanoparticles containing magnetic- targeted gemcitabine Download PDFInfo
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- WO2022125031A1 WO2022125031A1 PCT/TR2021/051205 TR2021051205W WO2022125031A1 WO 2022125031 A1 WO2022125031 A1 WO 2022125031A1 TR 2021051205 W TR2021051205 W TR 2021051205W WO 2022125031 A1 WO2022125031 A1 WO 2022125031A1
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- gemcitabine
- chitosan
- tmc
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 63
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 52
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 title claims abstract description 47
- 229960005277 gemcitabine Drugs 0.000 title claims abstract description 47
- 238000011282 treatment Methods 0.000 claims abstract description 21
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 9
- 201000005202 lung cancer Diseases 0.000 claims abstract description 9
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
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- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 claims description 19
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 9
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- CKTSBUTUHBMZGZ-ULQXZJNLSA-N 4-amino-1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-tritiopyrimidin-2-one Chemical compound O=C1N=C(N)C([3H])=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-ULQXZJNLSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
- A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
- A61K9/1676—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface having a drug-free core with discrete complete coating layer containing drug
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention relates to trimethyl chitosan nanoparticles containing magnetic -targeted gemcitabine developed for use in the treatment of lung cancer.
- Lung cancer accounts for 15% of new cancer cases every year and also covers 18% of cancer- related deaths. It is the cancer type having the highest incidence in the world according to World Health Organization (WHO) data. Therefore, it is an issue that needs to be taken precautions.
- WWO World Health Organization
- One of the most important challenges in the pharmacological treatment of the disease is the mobilization of the active agents used for the treatment to reach the targeted area in particular and to ensure continuous drug release.
- Gemcitabine is an analog of deoxycytidine, which has high activity against lung cancer tumors as well as many different types of solid tumors.
- Gemcitabine has a very short plasma half-life and exhibits certain limitations, such as rapid metabolism to its inactive form by cytidine deaminase.
- common side effects such as systemic toxicity caused by high dosage and lack of specificity regarding healthy cells also limit their antineoplastic properties.
- Many drug delivery system treatments have been developed for controlled and targeted administration.
- a magnetic drug transport system that is soluble in body pH has been developed by modifying the biocompatible and biodegradable chitosan as a result of our study.
- Chemotherapy, radiotherapy, and surgical intervention are generally used in the treatment of lung cancer.
- Chemotherapy has long been an effective treatment for lung cancer and remains one of the most important elements of treatment for many patients. Tumors or sections of the lung affected by cancer can be removed with small incisions by surgical methods.
- the treatment of spreading tumors and tumor metastases requires extensive chemotherapy even though localized primary solid tumors can be successfully removed by surgical procedure.
- High toxicity, low bioavailability, low therapeutic effect, non-specific biodistribution, and the effect of an anticancer agent on both normal and cancerous cells are the main deficiencies of traditional methods in cancer treatment.
- Patent document US8236752B 1 relates generally to the pharmaceutical compositions of nanoparticles.
- Example 22 describes the encapsulation of gemcitabine drug substance in nanoparticles.
- the chitosan used in this patent has a low molecular weight. Differently, high molecular weight chitosan was used in our study.
- Gemcitabine is the preferred chemotherapeutic agent for the treatment of cancer. Therefore, it is frequently encountered in studies. It was aimed to transport the gemcitabine more effectively and to reach the target area more easily in our study. In addition, it is thought that the system developed will show higher activity at a lower concentration in the target area due to the slow release of the gemcitabine. Current drug release studies support this view. Finally, the system developed with magnetic targeting is planned to reach the target region (cancerous tissue) as soon as possible.
- Patent document WO2015136477A1 relates to a targeted drug delivery system consisting of nanoparticles having a core comprising one or more polymers and one or more lipids.
- the receptor-ligand method was chosen as the targeting strategy, but no study was conducted on magnetic targeting in this patent. Differently, magnetic targeting was preferred in our study, so magnetic nanoparticles were used.
- This drug delivery system will minimize the side effects of the chemotherapeutic agent used and provide patients with better living conditions and treatment. It will also contribute to the reduction of treatment costs thanks to the decrease in the dose amount by delivering the drug to the desired area.
- the magnetic properties of the system used, the solubility of the chitosan in the body pH by modification of the chitosan, obtaining the desired effect at lower and lower doses directly to the targeted tissue and the controlled release of the drug is different and superior features of this system compared to other existing systems.
- a magnetic trimethyl chitosan drug transport system with gemcitabine-loaded, magnetic properties, and easily soluble in body pH was developed for use in the treatment of lung cancer.
- Chitosan a biodegradable and non-toxic polymer
- the water solubility of chitosan is poor and its use in physiological conditions is a major problem due to the loss of penetration-enhancing activity at values above pH 6.
- Chitosan was trimethylated using methyl iodide to solve this problem.
- Trimethyl chitosan (TMC) which is formed as a result of the trimethylation process, has high solubility, low toxicity, and biodegradability, but is highly bioadhesive compared to chitosan. Therefore, TMC can be used as a very useful nano-transporter system for pharmaceutical applications. Magnetic nanoparticles were synthesized using the co- sedimentation method.
- TMC and chitosan nanoparticles were prepared by cross-linking with tripolyphosphate. Gemcitabine was loaded into these nanoparticles synthesized using the adsorption technique. Then, characterization studies and in vitro drug release tests were performed. MTT tests were performed to determine their cytotoxicity against A549-Luc-C8 and CRL5809 (non-small cell lung cancer cells) cell lines.
- Figure 1 a - d. FTIR spectra; TMC and chitosan (a), TMCN (trimethyl chitosan nanoparticles) and CN (chitosan nanoparticles) (b). Hydrodynamic size distributions; TMCN (c), CN (d).
- Figure 2. a - d. FTIR spectra; magnetite (a) MTMC (magnetic trimethyl chitosan nanoparticles) and MC (magnetic chitosan nanoparticles) (b). Hydrodynamic size distributions; MTMC (c) and MC (d).
- FIG. 3 a - d. Specific drug loading efficiency of both nano systems: GMTMC (Gemcitabine-Loaded Magnetic TMC Nanoparticles) and GMC (Gemcitabine-Loaded Magnetic Chitosan Nanoparticles) (a). FTIR spectra of gemcitabine, GMTMC, and GMC (b). Hydrodynamic size distributions; GMTMC (c), GMC (d).
- FIG. 1 a, b. TEM images; GMTMC (a), GMC (b).
- Figure 5. a, b. Gemcitabine release profile (%); in buffers pH 6 (a) and pH 7.4 (b) from GMTMC and GMC
- the synthesized nanoparticle can only be concentrated in the cancerous tissue desired to act due to its magnetic properties, allows it to release in a controlled manner, and that TMC is preferred instead of chitosan due to its limited solubility.
- Chemotherapeutic agents used in the field of cancer therapy have toxic side effects on normal cells as well as tumor cells, as is known.
- the main objective of drug targeting and controlled drug release is to eliminate or minimize the negative effects occurring in traditional treatment methods and to increase reaching cellular levels.
- Trimethyl chitosan (TMC) was synthesized by chitosan methylation in an alkaline environment. Chitosan was treated with sodium iodide by stirring in the presence of sodium hydroxide (NaOH) and N-methyl-2-pyrrolidone (NMP) for 20 minutes at 60°C. Then, iodomethane (CH3I) was added to the solution and stirred at a constant rate under the condenser. Then, CH3I and NaOH were added to the mixture again. Diethyl ether was added and dissolved with sodium chloride solution to precipitate the polymer. The TMC obtained was dialyzed against distilled water for 2 days and dried. TMC was characterized by an infrared spectrophotometer of Fourier transformation.
- TMC nanoparticles (TMCN) and chitosan nanoparticles (CN) were prepared by the ionic gelation method.
- TMC (2 mg/mL) was dissolved in distilled water and 10 N NaOH was added until the pH reached around 4-5.
- Chitosan (2mg/mL) was dissolved in acetic acid and 10 N NaOH was added similarly until the pH reached around 4-5.
- Different volumes of 1 mg/mL tripolyphosphate (TPP) solution (2-8 mL) were added dropwise to the reaction mixtures and stirred at 480 rpm for 20 minutes at a constant rate. These solutions were centrifuged at 13000 rpm and the pellets were washed with distilled water.
- the optimum TPP amount was determined by hydrodynamic size analysis using a zetasizer.
- Magnetites were synthesized by the co- sedimentation method and their structures were confirmed by FTIR analysis. Magnetite dispersions (2-6 mL) in various concentrations were added into TMC and chitosan solutions. Then, 4 mL of TPP solution was added dropwise to the reaction mixture and centrifuged. Thus, magnetic TMC nanoparticles (MTMC) and magnetic chitosan nanoparticles (MC) were obtained. The appropriate magnetic concentration was determined by hydrodynamic size analysis.
- Gemcitabine was loaded to MTMC and MCs by adsorption technique.
- Various concentrations of gemcitabine solution (1-3,5 mg/mL) were added to the synthesized nanoparticles under optimum conditions.
- the obtained mixture was incubated at 37°C for 15 hours.
- the nanoparticles were centrifuged at 13000 rpm and the unbound drug was removed from the nanoparticles. Meanwhile, the nanoparticles were washed with distilled water.
- Gemcitabine- loaded magnetic TMC nanoparticles (GMTMC) and gemcitabine-loaded chitosan nanoparticles (GMC) were obtained as a result.
- Drug binding efficiencies were calculated as a result of spectrophotometric measurements of the supernatants at 268 nm. Optimization studies were carried out with FTIR and TEM analyses. Drug release characteristics for GMTMC and GMC in vitro drug release studies were calculated in 10 mM pH 7.4 and pH 6 phosphate buffer. The nanoparticles were taken into the dialysis membrane (MW: 14000) and dialysis was initiated in a water bath at 37°C. The dialysis medium was replaced with a new buffer at regular intervals. Meanwhile, the release profile of the free gemcitabine formulation was examined. The amount of drug released was calculated.
- GMTMC g gemcitabine/mE
- free drug 0.16-5 pg gemcitabine/Ml concentration
- TMC Trimethyl Chitosan Synthesis
- TMC has a higher water solubility than chitosan in the wider pH and concentration range and higher stability under various ionic conditions.
- TMC is more protective against hydroxyl radicals than other chitosan derivatives and is more prone to adsorption. Therefore, the use of TMC was preferred.
- Chitosan was trimethylated by reacting with excess iodomethane in N-methyl 2-pyrrolidone and using sodium iodide in this study. All these reactions produced permanently positively charged regions in the TMC structure.
- Chitosan has an N-H stretching peak at 2850 cm' 1 as can be seen from Figure la. However, this peak was not observed in the TMC structure.
- TMC also has a C-H peak belonging to the methyl group at 1450 cm' 1 in its structure; however, this peak cannot be seen in the chitosan structure. This indicates that the chitosan has been successfully methylated. Furthermore, the angular deformation of the N-H bonds of the amino groups occurs in both chitosan and TMC at 1577 cm' 1 for chitosan and 1559 cm' 1 for TMC, but is weaker or disappears due to the occurrence of N-methylation. In addition, a new peak appears at a high wave of 1630-1660 cm' 1 of a quaternary ammonium salt.
- Chitosan nanoparticles consist of intramolecular and intermolecular cross-links by anionic molecules. This method is called ionic gelling. Spherical- shaped nanoparticles can be selfformed by electrostatic interactions with TPP, a polyanion that acts as a chitosan crosslinker. This method is one of the most important advantages of the ionic gelling method in forming nanoparticles at room temperature and under mild conditions.
- TMCN can be prepared like chitosan nanoparticles.
- Magnetite is a biocompatible and FDA30 approved magnetic structure and is clinically used as a Magnetic Resonance Imaging (MRI) contrast agent with commercial forms such as EndoremTM, Feridex ®, and Resovist ®, so its use has been preferred.
- MRI Magnetic Resonance Imaging
- Magnetic nanoparticles provide drug deposition with a magnetic gradient at the tumor site, so that the anticancer effect of the chemotherapeutic agent may increase and the systemic toxicity of the agent may decrease to acceptable levels.
- the chemical structure of magnetite nanoparticles was characterized by FTIR in this study. The sharp peak at 550-600 cm' 1 is the characteristic peak representing the Fe-0 bond according to the FTIR spectrum ( Figure 2a). These data confirmed the structure of magnetite and were also consistent with previous studies. A magnetite concentration of 4 mg/mL and 3 mg/mL was optimized, respectively, depending on the minimum PDI value and size for TMC and chitosan nanoparticles (Table 2).
- Gemcitabine was loaded into both magnetic nanoparticle systems with weak interactions. Maximum drug loading occurred for MTMC and MC with initial gemcitabine concentrations of 1,5 and 2,5 mg/mL (with 54,7% and 30,3% adsorption efficiencies) as seen in Figure 3a. The gemcitabine adsorption efficiency for MTMC was found to be higher than that of MC. 82.05 pg of drug per MTMC mg was loaded and this value for MC was 75.75 pg. MTMC was found to have a higher drug loading capacity due to the trimethylated structure.
- Figure 3b shows the FTIR spectra of gemcitabine, GMTMC, and GMC. The signal at 1020 cm' 1 belongs to the fluoride group of the drug found in the structure of GMTMC.
- CRL5807 and A549-Luc-C8 (non-small cell lung cancer cells) cell lines were selected for cytotoxicity studies. Blank chitosan and TMC nanoparticles were tested as blank (data not shown) and did not lead to cell death at these concentrations.
- Figure 6 shows the viability (%) of the cells treated with the drug groups for 72 hours. All data showed that GMTMC and GMC-treated cell survival decreased with increased doses of gemcitabine in nanoparticles; suggesting cytotoxicity caused only by the active substance.
- the IC50 values of gemcitabine, GMTMC, and GMC for cell lines at 72 hours can be seen in Table 3. There were statistically significant differences in IC50 values of all drug groups for both cell lines (p ⁇ 0.05).
- IC50 values of gemcitabine against the A549-Luc-C8 cell line were consistent with a previous study. Polymers can increase the internalization of gemcitabine by affecting passive diffusion through bio-membranes. IC50 values of nanoparticles are higher than free drug due to their slower release than nanostructures, especially GMC shown in the previous section (p ⁇ 0.05).
- the IC50 value of GMTMC for the A549-Luc-C8 cell line is 1.75 times lower than GMC and 3.48 times lower for the CRL5807 cell line.
- the 25-cell survival difference between cells can be explained by the drug susceptibility of the cells as well as the uptake of the drug carrier into the cell. GMTMC was probably more effective than GMC for drug release testing for both cell lines.
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Abstract
The invention relates to trimethyl chitosan nanoparticles containing magnetic -targeted gemcitabine developed for use in the treatment of lung cancer.
Description
TRIMETHYL CHITOSAN NANOPARTICLES CONTAINING MAGNETIC- TARGETED GEMCITABINE
Technical Field of the Invention
The invention relates to trimethyl chitosan nanoparticles containing magnetic -targeted gemcitabine developed for use in the treatment of lung cancer.
State of the Art of the Invention (Prior Art)
Lung cancer accounts for 15% of new cancer cases every year and also covers 18% of cancer- related deaths. It is the cancer type having the highest incidence in the world according to World Health Organization (WHO) data. Therefore, it is an issue that needs to be taken precautions. There are three types of treatment: surgery, radiotherapy, and/or chemotherapy even though the treatment approaches vary according to the stages of cancer and histopathological types. One of the most important challenges in the pharmacological treatment of the disease is the mobilization of the active agents used for the treatment to reach the targeted area in particular and to ensure continuous drug release. Gemcitabine is an analog of deoxycytidine, which has high activity against lung cancer tumors as well as many different types of solid tumors. However, Gemcitabine has a very short plasma half-life and exhibits certain limitations, such as rapid metabolism to its inactive form by cytidine deaminase. In addition, common side effects such as systemic toxicity caused by high dosage and lack of specificity regarding healthy cells also limit their antineoplastic properties. Many drug delivery system treatments have been developed for controlled and targeted administration. A magnetic drug transport system that is soluble in body pH has been developed by modifying the biocompatible and biodegradable chitosan as a result of our study.
Chemotherapy, radiotherapy, and surgical intervention are generally used in the treatment of lung cancer. Chemotherapy has long been an effective treatment for lung cancer and remains one of the most important elements of treatment for many patients. Tumors or sections of the
lung affected by cancer can be removed with small incisions by surgical methods. However, the treatment of spreading tumors and tumor metastases requires extensive chemotherapy even though localized primary solid tumors can be successfully removed by surgical procedure. High toxicity, low bioavailability, low therapeutic effect, non-specific biodistribution, and the effect of an anticancer agent on both normal and cancerous cells are the main deficiencies of traditional methods in cancer treatment.
The following documents have been found as a result of the studies made in the present art;
Patent document US8236752B 1 relates generally to the pharmaceutical compositions of nanoparticles. Example 22 describes the encapsulation of gemcitabine drug substance in nanoparticles.
The chitosan used in this patent has a low molecular weight. Differently, high molecular weight chitosan was used in our study. Gemcitabine is the preferred chemotherapeutic agent for the treatment of cancer. Therefore, it is frequently encountered in studies. It was aimed to transport the gemcitabine more effectively and to reach the target area more easily in our study. In addition, it is thought that the system developed will show higher activity at a lower concentration in the target area due to the slow release of the gemcitabine. Current drug release studies support this view. Finally, the system developed with magnetic targeting is planned to reach the target region (cancerous tissue) as soon as possible.
Patent document WO2015136477A1 relates to a targeted drug delivery system consisting of nanoparticles having a core comprising one or more polymers and one or more lipids.
The receptor-ligand method was chosen as the targeting strategy, but no study was conducted on magnetic targeting in this patent. Differently, magnetic targeting was preferred in our study, so magnetic nanoparticles were used.
There is no study on magnetic trimethyl chitosan nanoparticles containing gemcitabine even though gemcitabine and trimethyl chitosan are included in the list in the current documents. Gemcitabine was loaded into magnetic trimethyl chitosan nanoparticles in our invention, and
the unexpected technical effect of the invention on the drug delivery system was proven by the test results.
Brief Description and Objectives of the Invention
This drug delivery system will minimize the side effects of the chemotherapeutic agent used and provide patients with better living conditions and treatment. It will also contribute to the reduction of treatment costs thanks to the decrease in the dose amount by delivering the drug to the desired area. The magnetic properties of the system used, the solubility of the chitosan in the body pH by modification of the chitosan, obtaining the desired effect at lower and lower doses directly to the targeted tissue and the controlled release of the drug is different and superior features of this system compared to other existing systems. As a result of our study, a magnetic trimethyl chitosan drug transport system with gemcitabine-loaded, magnetic properties, and easily soluble in body pH was developed for use in the treatment of lung cancer. Chitosan, a biodegradable and non-toxic polymer, is a polymer widely used in drug transport systems. However, the water solubility of chitosan is poor and its use in physiological conditions is a major problem due to the loss of penetration-enhancing activity at values above pH 6. Chitosan was trimethylated using methyl iodide to solve this problem. Trimethyl chitosan (TMC), which is formed as a result of the trimethylation process, has high solubility, low toxicity, and biodegradability, but is highly bioadhesive compared to chitosan. Therefore, TMC can be used as a very useful nano-transporter system for pharmaceutical applications. Magnetic nanoparticles were synthesized using the co- sedimentation method. TMC and chitosan nanoparticles were prepared by cross-linking with tripolyphosphate. Gemcitabine was loaded into these nanoparticles synthesized using the adsorption technique. Then, characterization studies and in vitro drug release tests were performed. MTT tests were performed to determine their cytotoxicity against A549-Luc-C8 and CRL5809 (non-small cell lung cancer cells) cell lines.
Definitions of Figures Describing the Invention
Figure 1. a - d. FTIR spectra; TMC and chitosan (a), TMCN (trimethyl chitosan nanoparticles) and CN (chitosan nanoparticles) (b). Hydrodynamic size distributions; TMCN (c), CN (d).
Figure 2. a - d. FTIR spectra; magnetite (a) MTMC (magnetic trimethyl chitosan nanoparticles) and MC (magnetic chitosan nanoparticles) (b). Hydrodynamic size distributions; MTMC (c) and MC (d).
Figure 3. a - d. Specific drug loading efficiency of both nano systems: GMTMC (Gemcitabine-Loaded Magnetic TMC Nanoparticles) and GMC (Gemcitabine-Loaded Magnetic Chitosan Nanoparticles) (a). FTIR spectra of gemcitabine, GMTMC, and GMC (b). Hydrodynamic size distributions; GMTMC (c), GMC (d).
Figure 4. a, b. TEM images; GMTMC (a), GMC (b).
Figure 5. a, b. Gemcitabine release profile (%); in buffers pH 6 (a) and pH 7.4 (b) from GMTMC and GMC
Figure 6. a - d. Dose-dependent cytotoxic effects of drug groups at 72 hours: Gemcitabine against A549-Luc-C8 cells (a), gemcitabine against CRL5807 cells (b), GMTMC and GMC against A549-Luc-C8 cells (c), and GMTMC and GMC against CRL5807 cells (d).
Detailed Description of the Invention
The original features of the invention are that the synthesized nanoparticle can only be concentrated in the cancerous tissue desired to act due to its magnetic properties, allows it to release in a controlled manner, and that TMC is preferred instead of chitosan due to its limited solubility. Chemotherapeutic agents used in the field of cancer therapy have toxic side effects on normal cells as well as tumor cells, as is known. The main objective of drug targeting and controlled drug release is to eliminate or minimize the negative effects occurring in traditional treatment methods and to increase reaching cellular levels. In addition, it is to provide optimization of the kinetic properties of the concentration and release of drugs in the circulatory system or different biological fluids, to change the pharmacokinetic- pharmacodynamic properties of drugs, to provide effective and reliable treatment at low or high dosages, to eliminate or minimize toxic effects and immunogenic factors, to increase the stability of drugs, and to provide a pharmacological response with the intended level within the targeted area without causing any harmful interaction in different parts of the body. The increasing number of cancer patients will achieve a better quality of life and treatment without being exposed to the side effects of the chemotherapy treatments they are using as a result of the study.
Trimethyl Chitosan Synthesis
Trimethyl chitosan (TMC) was synthesized by chitosan methylation in an alkaline environment. Chitosan was treated with sodium iodide by stirring in the presence of sodium hydroxide (NaOH) and N-methyl-2-pyrrolidone (NMP) for 20 minutes at 60°C. Then, iodomethane (CH3I) was added to the solution and stirred at a constant rate under the condenser. Then, CH3I and NaOH were added to the mixture again. Diethyl ether was added and dissolved with sodium chloride solution to precipitate the polymer. The TMC obtained was dialyzed against distilled water for 2 days and dried. TMC was characterized by an infrared spectrophotometer of Fourier transformation.
Preparation of Gemcitabine-Loaded Magnetic TMC and Chitosan Nanoparticles
Optimization of TPP amount TMC nanoparticles (TMCN) and chitosan nanoparticles (CN) were prepared by the ionic gelation method. TMC (2 mg/mL) was dissolved in distilled water and 10 N NaOH was added until the pH reached around 4-5. Chitosan (2mg/mL) was dissolved in acetic acid and 10 N NaOH was added similarly until the pH reached around 4-5. Different volumes of 1 mg/mL tripolyphosphate (TPP) solution (2-8 mL) were added dropwise to the reaction mixtures and stirred at 480 rpm for 20 minutes at a constant rate. These solutions were centrifuged at 13000 rpm and the pellets were washed with distilled water. The optimum TPP amount was determined by hydrodynamic size analysis using a zetasizer.
Optimization of Magnetite Concentration
Magnetites were synthesized by the co- sedimentation method and their structures were confirmed by FTIR analysis. Magnetite dispersions (2-6 mL) in various concentrations were added into TMC and chitosan solutions. Then, 4 mL of TPP solution was added dropwise to the reaction mixture and centrifuged. Thus, magnetic TMC nanoparticles (MTMC) and magnetic chitosan nanoparticles (MC) were obtained. The appropriate magnetic concentration was determined by hydrodynamic size analysis.
Optimization of Gemcitabine Concentration
Gemcitabine was loaded to MTMC and MCs by adsorption technique. Various concentrations of gemcitabine solution (1-3,5 mg/mL) were added to the synthesized nanoparticles under optimum conditions. The obtained mixture was incubated at 37°C for 15 hours. The nanoparticles were centrifuged at 13000 rpm and the unbound drug was removed from the nanoparticles. Meanwhile, the nanoparticles were washed with distilled water. Gemcitabine- loaded magnetic TMC nanoparticles (GMTMC) and gemcitabine-loaded chitosan nanoparticles (GMC) were obtained as a result. Drug binding efficiencies were calculated as a result of spectrophotometric measurements of the supernatants at 268 nm. Optimization studies were carried out with FTIR and TEM analyses. Drug release characteristics for GMTMC and GMC in vitro drug release studies were calculated in 10 mM pH 7.4 and pH 6 phosphate buffer. The nanoparticles were taken into the dialysis membrane (MW: 14000) and dialysis was initiated in a water bath at 37°C. The dialysis medium was replaced with a new buffer at regular intervals. Meanwhile, the release profile of the free gemcitabine formulation was examined. The amount of drug released was calculated.
Cytotoxicity Tests
Cell lines A549-Luc-C8 and CRL5807 were cultured in RPMI 1640 medium containing 10% FBS, Penicillin Streptomycin, L-glutamine and incubated at 37 °C in 5% CO2 medium. Cytotoxicity analyses were performed by MTT test (n=3). For all cell lines, 5 x 103 cells per well were plated in a 96-well plate and incubated for 24 hours. 100 pF drug group (GMTMC, GMC at 0.625-40 pg gemcitabine/mE and free drug at 0.16-5 pg gemcitabine/Ml concentration) was added to the cells and incubated at 37°C for 72 hours in 5% CO2 medium after the cells were retained. The medium was removed and added to each well with MTT solution (10:1) (V/V) after incubation. The cells were incubated for 4 hours and the formed formazan crystals were dissolved in DMSO solution. Absorbance measurement was taken at 450 nm in the microplate reader. IC50 values were calculated using GraphPad Prism 8 software and the data were expressed as mean ± standard deviation. In addition, the data were analyzed and compared by one-way ANOVA test using the SPSS Statistics v25 software.
Trimethyl Chitosan Synthesis
TMC has a higher water solubility than chitosan in the wider pH and concentration range and higher stability under various ionic conditions. In addition, TMC is more protective against hydroxyl radicals than other chitosan derivatives and is more prone to adsorption. Therefore, the use of TMC was preferred. Chitosan was trimethylated by reacting with excess iodomethane in N-methyl 2-pyrrolidone and using sodium iodide in this study. All these reactions produced permanently positively charged regions in the TMC structure. Chitosan has an N-H stretching peak at 2850 cm'1 as can be seen from Figure la. However, this peak was not observed in the TMC structure. TMC also has a C-H peak belonging to the methyl group at 1450 cm'1 in its structure; however, this peak cannot be seen in the chitosan structure. This indicates that the chitosan has been successfully methylated. Furthermore, the angular deformation of the N-H bonds of the amino groups occurs in both chitosan and TMC at 1577 cm'1 for chitosan and 1559 cm'1 for TMC, but is weaker or disappears due to the occurrence of N-methylation. In addition, a new peak appears at a high wave of 1630-1660 cm'1 of a quaternary ammonium salt.
Preparation of Gemcitabine-Loaded Magnetic TMC and Chitosan Nanoparticles
Chitosan nanoparticles consist of intramolecular and intermolecular cross-links by anionic molecules. This method is called ionic gelling. Spherical- shaped nanoparticles can be selfformed by electrostatic interactions with TPP, a polyanion that acts as a chitosan crosslinker. This method is one of the most important advantages of the ionic gelling method in forming nanoparticles at room temperature and under mild conditions. In addition, TMCN can be prepared like chitosan nanoparticles. Magnetite is a biocompatible and FDA30 approved magnetic structure and is clinically used as a Magnetic Resonance Imaging (MRI) contrast agent with commercial forms such as Endorem™, Feridex ®, and Resovist ®, so its use has been preferred.
Optimization of TPP Amount
Both nano-carrier systems were not formed in the reaction medium containing 2 and 3 mL of TPP, and unwanted pellet formations were observed in those containing 8 mL of TPP. According to the zeta size analysis for both systems, the optimum TPP amount was found to be 4 mL TPP (Table 1). The poly dispersity index (PDI) and particle size results for TMCN
were 0.246 and 294.1 ± 18.85 nm, respectively (Figure 1c). PDI and particle size for CN were 0.341 and 226.2 ± 53.56 nm, respectively (Figure Id). The results were compared with the literature data and found to be compatible. Therefore, the optimum TPP amount in both systems was determined to be 4 mL and this volume was used in future studies.
Table 1. Hydrodynamic size results of TMC and chitosan and nanoparticles prepared with different TPP concentrations
FTIR analyses confirmed that the N-H bending peak at 1640 cm'1 indicates the chitosan structure. This peak is visible in the chitosan. For chitosan nanoparticles, this peak shifted to 1520 cm'1 (Figure lb). This indicates that the NH2 groups are cross-linked with TPP. Furthermore, the P-0 stretching peak at 786 cm'1 and the P=O peaks between 1200 and 1270 cm'1 confirmed that chitosan nanoparticles were composed of TPP. In addition, the P=O peak between 1116 and 1216 cm'1 confirmed the TMC nanoparticle structure with TPP. These data obtained were found to be consistent with a previous study.
Optimization of Magnetite Concentration
Magnetic nanoparticles provide drug deposition with a magnetic gradient at the tumor site, so that the anticancer effect of the chemotherapeutic agent may increase and the systemic toxicity of the agent may decrease to acceptable levels. The chemical structure of magnetite nanoparticles was characterized by FTIR in this study. The sharp peak at 550-600 cm'1 is the characteristic peak representing the Fe-0 bond according to the FTIR spectrum (Figure 2a). These data confirmed the structure of magnetite and were also consistent with previous studies. A magnetite concentration of 4 mg/mL and 3 mg/mL was optimized, respectively,
depending on the minimum PDI value and size for TMC and chitosan nanoparticles (Table 2). Particle size distributions of MTMC and MC prepared with optimum magnetite concentration are given in Figure 2c and Figure 2d. In addition, the peak observed at 558 cm'1 in MTMC and 550 cm'1 in MC represents the characteristic bands for Fe-0 according to FTIR spectra (Figure 2b), and this showed that these nano-mixing systems were encapsulated with magnetic nanoparticles.
Table 2. Hydrodynamic size results of magnetic TMC and chitosan nanoparticles prepared with different concentrations of magnetite dispersion
Optimization of Gemcitabine Concentration
Gemcitabine was loaded into both magnetic nanoparticle systems with weak interactions. Maximum drug loading occurred for MTMC and MC with initial gemcitabine concentrations of 1,5 and 2,5 mg/mL (with 54,7% and 30,3% adsorption efficiencies) as seen in Figure 3a. The gemcitabine adsorption efficiency for MTMC was found to be higher than that of MC. 82.05 pg of drug per MTMC mg was loaded and this value for MC was 75.75 pg. MTMC was found to have a higher drug loading capacity due to the trimethylated structure. Figure 3b shows the FTIR spectra of gemcitabine, GMTMC, and GMC. The signal at 1020 cm'1 belongs to the fluoride group of the drug found in the structure of GMTMC. In addition, an N-0 stretching peak was observed at 1535 cm'1 in the GMC spectrum. This peak is specific to gemcitabine. It was noted based on these data that gemcitabine successfully binds to MTMC and MC, with even higher amount of drug analyzed in GMTMC. The hydrodynamic sizes of GMTMC and GMC were found to be 462,1 ± 201,5 nm and 345,0 ± 96,2 nm (Figure 3c and d) with PDI values of 0,247 and 0,302, respectively. In addition, TEM images (Figure 4)
showed global shapes of both GMTMC and GMC. These data were consistent with previous studies.
In vitro Drug Release
In vitro drug release studies were performed by the dialysis method. The maximum percent release from GMTMC and GMC at pH 6 at 28 hours was 88% and 9%, respectively, while these values were calculated as 53% and 5% at pH 7.4 as can be seen in Figures 5a and b. Drug release from free formulation reached 95% after three and a half hours in both buffer media. Drug release from nanoparticles was performed in a controlled manner during the study period and also more slowly than free gemcitabine. Drug release profiles from nanoparticles depend on the nature of the carrier system and the structure of the active substance. The initial rapid release of gemcitabine may be due to gemcitabine adsorbed on the surface of the nanoparticles. Drug release from GMC was found to be slower toxic to cancer cells compared to GMTMC in both pH 6 and 7.4 buffers in our study. On the other hand, higher drug release was detected in the acidic environment compared to the physiological environment, meaning that there may be higher drug release in the cancerous tissue compared to the circulation and healthy tissues.
Cytotoxicity Tests
CRL5807 and A549-Luc-C8 (non-small cell lung cancer cells) cell lines were selected for cytotoxicity studies. Blank chitosan and TMC nanoparticles were tested as blank (data not shown) and did not lead to cell death at these concentrations. Figure 6 shows the viability (%) of the cells treated with the drug groups for 72 hours. All data showed that GMTMC and GMC-treated cell survival decreased with increased doses of gemcitabine in nanoparticles; suggesting cytotoxicity caused only by the active substance. The IC50 values of gemcitabine, GMTMC, and GMC for cell lines at 72 hours can be seen in Table 3. There were statistically significant differences in IC50 values of all drug groups for both cell lines (p<0.05).
Table 3. IC50 values of gemcitabine and gemcitabine-loaded nanoparticle systems against A549-Luc-C8 and CRL5807 cell lines at 72 hours (ug/mL)
IC50 values of gemcitabine against the A549-Luc-C8 cell line were consistent with a previous study. Polymers can increase the internalization of gemcitabine by affecting passive diffusion through bio-membranes. IC50 values of nanoparticles are higher than free drug due to their slower release than nanostructures, especially GMC shown in the previous section (p<0.05). The IC50 value of GMTMC for the A549-Luc-C8 cell line is 1.75 times lower than GMC and 3.48 times lower for the CRL5807 cell line. The 25-cell survival difference between cells can be explained by the drug susceptibility of the cells as well as the uptake of the drug carrier into the cell. GMTMC was probably more effective than GMC for drug release testing for both cell lines.
Claims
CLAIMS Trimethyl chitosan nanoparticles, containing magnetic-targeted gemcitabine developed for use in the treatment of lung cancer. Trimethylchitosan nanoparticles containing magnetic-targeted gemcitabine according to claim 1, characterized in that the polydispersity index (PDI) is 0.246. A trimethyl chitosan nanoparticle containing magnetic -targeted gemcitabine according to claim 1, is characterized in that the particle size is 294.1 ± 18.85 nm. A method of manufacturing trimethyl chitosan nanoparticles containing magnetic- targeted gemcitabine developed for use in the treatment of lung cancer, characterized in that it comprises the following steps:
- Synthesizing trimethyl chitosan (TMC) by chitosan methylation in an alkaline environment
- Treating chitosan with sodium iodide by stirring in the presence of sodium hydroxide (NaOH) and N-methyl-2-pyrrolidone (NMP) for 20 minutes at 60°C
- Then, adding iodomethane (CH3I) to the solution and stirring at a constant rate under the condenser
- Then, adding CH3I and NaOH to the mixture again
- Adding diethyl ether and dissolving with sodium chloride solution to precipitate the polymer
- Dialyzing the TMC obtained against distilled water for 2 days and drying
- Dissolving TMC (2 mg/mL) in distilled water and adding 10 N NaOH until the pH reaches around 4-5
- Adding the appropriate concentration of gemcitabine solution to the TMC solution containing the appropriate concentration of magnetic nanoparticle
- Adding 1 mg/mL tripolyphosphate (TPP) solution dropwise to reaction mixtures at appropriate volumes and stirring at 480 rpm for 20 minutes at a constant rate
- Reacting the solution at 37°C for 15 hours
- At the end of the period, centrifuging the solution at 13000 rpm and washing the pellets with distilled water.
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