Layered hybrid nanofiber structured drug delivery system loaded chemotherapy drug and / or bioactive molecule for local use tumors in the surgical resection area to prevent recurrence in brain tumors

Abstract

The invention relates to a drug delivery system in a layered hybrid nanofiber structure containing chemotherapy drug and / or bioactive molecule, which can be applied locally in the pharmaceutical industry in the healthcare field, especially in the treatment of brain tumors, where long-term-regular drug release can be made, its production and use. The invention, in its most basic form, is a drug delivery system for local application in active ingredient loaded layered hybrid nanofiber structure which comprises PLA shell nanofibre layer with at least one active ingredient extract (10), PVA nanofiber layer loaded with at least one active ingredient (3), at least one active ingredient that electrosprayed (20) between said layers.

Description

[0001] LAYERED HYBRID NANOFIBER STRUCTURED DRUG DELIVERY SYSTEM LOADED CHEMOTHERAPY DRUG AND / OR BIOACTIVE MOLECULE FOR LOCAL USE TUMORS IN THE SURGICAL RESECTION AREA TO PREVENT RECURRENCE IN BRAIN TUMORS

[0002] Field of the Invention

[0003] The invention relates to a drug delivery system in a layered hybrid nanofiber structure containing chemotherapy drug and / or bioactive molecule, which can be applied locally in the pharmaceutical industry in the healthcare field, especially in the treatment of brain tumors, where long-term-regular drug release can be made, its production and use.

[0004] State of the Art

[0005] Glioblastoma (GB) is the most malignant brain tumor, causing 3-4% of all cancer related deaths (Louis et al, 2007). The World Health Organization defines GB as a stage IV tumor group characterized as malignant, mitotically active, and predisposed to necrosis. The 5 year survival rate of GB is 4-5% and has a very poor prognosis (McLendon and Halperin, 2003). Standard treatment of GB consists of surgical resection, radiotherapy, and chemotherapy. Despite this treatment protocol, patients only have a median survival of 12.6 months (Louis et al. 2007). The maximum surgical resection is an effective treatment method that prolongs the survival of patients with this disease. However, due to the invasive nature of GB and the location of the tumor in the brain, maximum resection cannot be performed in many cases. Alkylating chemotherapeutic agents such as carmustine and temozolomide (TMZ) are recommended in chemotherapy treatment guidelines (Alan and Yumuk, 2019). TMZ which is an alkylating agent, is the most common chemotherapy medicine used to treat GB, which can cross the blood-brain barrier (BBB) and is effective in oral use. The penetration rate of TMZ from the blood to the cerebrospinal fluid is only 20-30% and even lower into the cerebral intestine (Belter et al., 2020). This makes it difficult for therapeutic regimens to achieve effective drug concentrations with minimal toxicity and minor side effects. For this reason, despite TMZ treatment, there is only a 2 month increase in the survival time of the patients and the tumor develops recurrence. Another alkylating agent, carmustine {BCNU, [1 ,3-bis (2-chloroethyl) -1 -nitrosourea]} has also been used in tumor recurrence by intravenous administration or wafer implantation. Biodegradable wafers impregnated with carmustine (Gliadel® wafer) and implanted in the surgical bed on the walls of the resection cavity were originally developed to prevent toxicity associated with systemic administration of the carmustine system (Lillehei et al., 2018). It was thought that carmustine wafer implantation in GB patients who underwent surgical resection provided a therapeutic bridge between surgical resection and the beginning of radiotherapy. Previous studies evaluating the effectiveness of carmustine wafer have found an increase in overall survival (OS) of 2- 4 months in newly diagnosed GB patients, but the limited tissue penetration (1-5 mm) and short half life (20 minutes) of this medicine are the main limiting factors (Giese et al., 2004). Although the effectiveness of carmustine administration has been established in seminal studies, its safety remains controversial. Common side effects of carmustine based chemotherapy include nausea / vomiting and hematotoxicity with a rare delay after 4-6 weeks, and pulmonary fibrosis, the most terrible side effect (Roux et al., 2017). For carmustine wafer implantation, its impact on postoperative infections, quality of life, and feasibility of adjuvant oncological treatments is also controversial. A systematic understanding of whether carmustine wafer contributes to survival in glioma patients is still lacking.

[0006] Today, there are many reasons why newly developed drug candidates for GB treatment have negative results: (i) limited ability of drugs to pass BBB in glioma patients, (ii) poor accumulation of drugs on tumor tissue, (iii) inability to reach sufficient medicine concentration in the tumor due to early spread to cerebrospinal fluid and interstitial fluid, (iv) short half-life of the drug (Ramachandran et al., 2017).

[0007] Since the development of effective treatments is difficult due to the aggressiveness of the tumor, the observation of recurrence and metastasis, and the heterogeneity of the tumor, almost no significant improvements have been achieved in survival rate increase of GB patients (Stewart, 2002). Today, there is a need to find new therapeutic approaches that have less side effects and are more effective to be used in the local treatment of tumor cells remaining in the surgical resection area after GB, which are resistant to medicines and aggressive, and to develop alternative treatment strategies that have the potential to improve survival rates for GB patients with studies to be conducted in this context.

[0008] In the US patent application with the publication number US2020016277A1 encountered in the literature research, nanoparticles for the distribution of active ingredients to brain cancers are explained. The nanoparticle (NP) based invention, in which Temozolomide is used as the active ingredient, is directed to the targeted micellar active ingredient carriers. The micelle known in the present art is a component of the NP carrier system and cannot solve said disadvantages.

[0009] In the international patent application with publication number WO2021232072A1 , the three component solid lipid nanocomposition for killing cancer cells comprising curcumin, ginger oleoresin and rutin is disclosed. Here, a three component solid lipid nanocomposition loaded with phytochemical active ingredients, including the Rutin molecule, is proposed. In vitro cytotoxicity tests have shown that it exhibits cytotoxicity in Lewis Lung Carcinoma and Lung cancer, breast cancer, colorectal adenocarcinoma and human leukemia cells. However, the drug delivery system described in the relevant application is nanoparticle carrier known in the current art, and its effect on brain tumors has not been examined.

[0010] As a result due to the said disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

[0011] Objective of the Invention

[0012] The object of the invention is to solve said disadvantages by being inspired from the existing conditions.

[0013] The main object of the invention is to provide the drug delivery system, production and use, which can especially be applied brain tumor treatment locally, can do long termregular drug release, in layered hybrid nanofiber structure comprising chemotherapy drug and / or bioactive molecule.

[0014] An object of the invention is to provide control of the area of release on the body, while minimizing release speed and drug degradation or loss by applying layered hybrid nanofiber drug delivery system that is applied locally directly on the tumor bed following surgical resection unlike traditional chemotherapy agents used orally or intravenously in the present art.

[0015] In order to achieve said objects, the invention is drug delivery system for local use in a layered hybrid nanofiber structure, loaded at least one active ingredient and comprises the following:

[0016] PLA shell nanofibre layer with at least one active ingredient core PVA nanofiber layer loaded with at least one active ingredient At least one active ingredient that electrosprayed between said layers.

[0017] According to an embodiment of the invention, said active ingredient is an individual or combination selected from the group consisting of chemotherapy drugs, drug candidates and natural compounds which have a glycosidic chemical structure with a Rutin-like hydroxyl (-OH) polar group. Preferably said active ingredient is Rutin and / or Temozolomide, more preferably said ingredient is Rutin and Temozolomide.

[0018] In order to achieve said objects, the invention is the production method of a drug delivery system for local use in a layered hybrid nanofiber structure, loaded at least one active ingredient, and comprises the following: i. Formation of a PLA shell nanofiber layer with at least one active ingredient core by Core / Shell electrospinning method; ii. Electrospraying at least one active ingredient on said layer; iii. Formation of an PVA nanofiber layer loaded with at least one active ingredient on said active material by using blending electrospinning method.

[0019] In order to fulfill the objects described above, the invention also includes the use of a layered hybrid nanofiber loaded with Rutin and / or Temozolomide as a local drug delivery system for the field of surgical resection of brain tumors.

[0020] The structural and characteristic features and all the advantages of the invention will be understood more clearly by means of the figures and the detailed description with reference to these figures provided below and therefore, the evaluation should be made by taking these figures and the detailed description into consideration. Figures for a Better Understanding of the Invention

[0021] Figure 1, a schematic representation of the active ingredient loaded layered hybrid nanofiber drug delivery system of the invention.

[0022] Figure 2, In-vitro release profiles of LHNs loaded with Rutin (A) and TMZ (B) in PBS solution.

[0023] TMZ: Temozolomide.

[0024] Figure 3, SEM images of LHNrutinat different magnifications, A) surface (3 kX), B) section (300 X), C) section (500 X), D) lower layer section (5 kX), respectively.

[0025] LHN: Layered hybrid nanofiber.

[0026] Figure 4, SEM images of LHNTMZ at different magnifications, A) surface (1 kX), B) upper surface section (500 X), C) upper and middle layer section (1.5 kX), D) lower layer section (5 kX), respectively.

[0027] LHN: Layered hybrid nanofiber.

[0028] Figure 5, the effect of rutin and TMZ encapsulated LHNs on the cell reproduction speed of GB cells at IC50 doses. The values specified as cut nanofibers refer to those that have been cut to achieve an IC50 concentration and directly applied to the cells. Their p-values have been measured in comparison to UNT using One-way ANOVA and TUKEY test. The data were shown as average ± SD. ** p<0.0001

[0029] UNT: Untreated, TMZ: Temozolomide. LHN: Layered hybrid nanofiber.

[0030] Figure 6, the effect of LHNTMZ, LHNrutinand [_HNTMZ+rutinon the wound healing rate of T98G cells. The in vitro closure of the wound healing area was captured before treatments (Oh) and after 24-48 hours of treatments with LHN. The value of p was calculated at the 48 hour time point using the ANOVA test. The size of the wounded area was analyzed using Imaged software the data were shown as average ± SD. ** p<0.0001.

[0031] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide.

[0032] Figure 7, the effect of LHNTMZ, LHNrutin and LHNTMZ+rutin on the endothelial cell tube formation. The value of p was calculated at the 24 hour time point using the ANOVA test. The data were shown as average ± SD. ** p<0.0001.

[0033] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide. Figure 8, LHNTMZ, LHNrutin, and LHNTMZ+rutin increased the suppressive capacity on the colony forming ability of T98G cells. The data are shown graphically as a percentage of the number of colonies formed. **:p<0.0001.

[0034] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide. % clone number: % colony number.

[0035] Figure 9, the effect of LHNTMZ, LHNrutin, and LHNTMZ+rutin on the sphere formation of T98G cells. The T98G cells were seeded into the 96 well round-bottom ultra low junction plate and began forming spheres within 3 days. The spheres were treated with loaded LHN networks on day 3 and changes in sphere size were measured on day 8. Calcein-AM / PI staining detected the vital cell content of the spheres. The average integrated optical density (IOD) of the images were measured and analyzed using the Image-J software, **p<0.001.

[0036] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide.

[0037] Figure 10, changes in the RNA expression of EMT marker genes CDH2, TWIST, and ZEB1 in T98G cells due to LHNTMZ, LHNrutin, and LHNTMZ+rutin. The p-values for RT-qPCR were calculated using an independent sample t-test. **p<0.0001 compared to untreated cells; n=3.

[0038] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide.

[0039] Figure 11, LHNTMZ, LHNrutin and LHNTMZ+rutin related changes in RNA expression of CSC marker genes CD133, OCT4, SOX2 and NANOG in T98G cells. The p-values for RT-qPCR were calculated using an independent sample t-test. **p<0.0001 compared to untreated cells; n=3.

[0040] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide.

[0041] Figure 12, LHNTMZ, LHNrutin and LHNTMZ+rutin related changes of LncRNA expression levels in T98G cells. The p-values for RT-qPCR were calculated using an independent sample t-test. **p<0.0001 compared to untreated cells; n=3.

[0042] LHN: Layered hybrid nanofiber, UNT: Untreated, TMZ: Temozolomide.

[0043] Figure 13, MRI demonstration of GB tumor formation with C6 cancer cell in Wistar Albino rats.

[0044] Figure 14, application of a rutin and / or TMZ loaded layered hybrid nanofiber to the brain tumor resection area. Figure 15, macroscopic comparison of in-vivo GB model groups. a)Untreated control group with induced brain tumors b) Group treated with TMZ-loaded layered nanofibers following induction of brain tumors

[0045] Figure 16, demonstrating that the rutin and / or TMZ-loaded layered hybrid nanofiber is protected from degradation 15 days after placement at the resection area during resection.

[0046] Descriptions of Part References

[0047] 10 PLA shell nanofibre layer with at least one active ingredient core

[0048] 30 PVA nanofiber layer loaded with active ingredient

[0049] 20 active ingredient that electrosprayed

[0050] Detailed Description of the Invention

[0051] In this detailed description, the drug delivery system and its preferred embodiments explained only for a better understanding of the subject.

[0052] The invention, in its most basic form, is a drug delivery system for local use in a layered hybrid nanofiber structure loaded with at least one active ingredient and comprises the following:

[0053] PLA shell nanofibre layer with at least one active ingredient core (10)

[0054] PVA nanofiber layer loaded with at least one active ingredient (30)

[0055] - At least one active ingredient that electrosprayed between said layers (Figure 1).

[0056] A preferred embodiment of the invention is a biocompatible nanofiber based local drug delivery system to which the chemotherapy drug TMZ and / or Rutin which is a natural flavonoid can be loaded in desired concentration, which can be applied to the tumor cells following brain tumor surgical resection, which prevents recurrence by providing long term-controlled release of TMZ and / or rutin in the tumor location.

[0057] The preferred embodiment of the invention comprises a two layered structure. One layer is comprised of TMZ and / or rutin loaded PVA nanofiber. The other layer is comprised of TMZ and / or rutin core PLA shell core-shell layer. There is electrosprayed TMZ and / or rutin between said layers.

[0058] Preferred embodiments of the invention comprises 28-32% TMZ and / or rutin loaded PVA nanofiber layer by weight; 48-52% TMZ and / or rutin core PLA shell core-shell nanofiber layer by weight and 18-22% electrosprayed TMZ and / or rutin between said layers by weight.

[0059] A preferred embodiment of the invention comprises 30% TMZ and / or rutin loaded PVA nanofiber layer by weight; 50% TMZ and / or rutin core PLA shell core-shell nanofiber layer by weight and 20% electrosprayed TMZ and / or rutin between said layers by weight.

[0060] The active ingredient loaded PVA (Polyvinyl Alcohol) nanofiber in the first layer; is biocompatible, biodegradable and degraded upon release by itself on local application (Parni and Yildirim, 2021). It’s effective in the release of hydrophilic and hydrophobic drugs. The drug release kinetics can be optimized by adjusting the drug release kinetics, fiber diameter, morphology and / or porosity and drug / polymer ratio. The first layer shows effect by causing sudden release in the cerebrospinal fluid and rapidly deteriorating.

[0061] The PLA shell / core-shell nanofiber with active ingredient core located in the second layer, a synthetic polyester, PLA(Polylactic Acid) is an FDA approved nontoxic biocompatible and biodegradable polymer and it possesses many good mechanical properties for tissue engineering applications and many other biomedical applications (Ghorbani et al., 2018).

[0062] PLA core-shell is a modified system which also known as co-axial electrospinning and makes up the core structure (Rahmani et al. ,2017). This structure provides encapsulation of TMZ and / or rutin to the drug delivery system.

[0063] The outer part of the core-shell fibers produced for controlled drug release consists of polymer and the inner part consists of the active ingredient to be released. TMZ and / or rutin are encapsulated with polymer and controlled release happens depending on the permeability of the polymer structure. This process carries out long term release by protecting the effectiveness of TMZ and / or rutin against environmental factors (heat, light, moisture, pH).

[0064] The drug delivery system of the invention provides control of the area of release of the oral or intravenously used traditional TMZ chemotherapy on the body by being applied locally, while minimizing release speed and drug degradation or loss.

[0065] In recent years, flavonoid compounds have been frequently used in the determination of new treatment methods in many cancer types, and efforts have been made to make the existing treatments more effective and to reduce their cytotoxic effects. In addition, there are studies stating that the use of flavonoids with certain therapeutic properties with chemotherapeutic agents creates a synergistic effect and increases the effectiveness of the chemotherapeutic agent, thus reducing the dose of the existing agent in the treatment and reducing the side effects that this agent may cause. Rutin, a flavonoid, is a glycoside chemically containing flavonol aglycone quercetin with disaccharide rutinose. Rutin has a preventive effect against cancer cell proliferation, angiogenesis, drug resistance, and escape from apoptosis.

[0066] In the studies carried out by the inventors during the preparation process before the invention, it suppresses the aggressiveness of the in-vitro GB cancer cell by creating a doped effect in the use of the non-nanofiber-loaded rutin both alone and in combination with TMZ (Ercelik et al., 2023). With the Rutin loaded layered hybrid nanofiber developed by the inventors, it was determined that it prevented the proliferation of in- vitro GB cancer cells, killed cancer cells in an apoptotic manner, suppressed invasion, and reduced the size of 3D cancer spheres. With the rutin loaded drug delivery system developed by the inventors, systematic side effects caused by chemotherapy drugs can be prevented with natural flavonoid compound.

[0067] The main object of the invention is to provide gradual controlled drug release. For this reason, the use of layered hybrid structure was necessitated. In order to determine the release behavior of each layer, each layer was produced separately and the release behavior and the interaction of the active ingredient with the cell were measured over time. In this context, core nanofiber non-woven surfaces formed by encapsulating the active ingredient with the lowest release rate over time with the polymer were produced. In the production of this layer, PLA polymer which is insoluble in brain fluid was selected as the polymer and it was decided that the fiber section shape would be core-shell. In this way, the release of the active ingredient to the environment at low speed was ensured. After the application, the use of this layer as a lower layer was determined in order to release the continuous active ingredient at a low dose. The PVA polymer which dissolves in brain fluid was determined as the upper layer. Because PVA dissolves in brain fluid, a loaded nanofiber was produced by adding the active ingredient into the polymer. In this way, since the PVA fiber, which retains its solid state during the application process, will be dissolved by the brain fluid immediately after the application, the active ingredient loaded to the nanofiber will interact with the cell with a sudden release. Between the two layers, the active ingredient was sprayed with the electrospray method to adhere to the lower and upper layer surface by adhesion. In this way, after the upper layer, PVA, dissolves in the brain fluid and releases the active ingredient into the environment, the active ingredient between the two layers, which is free between the layers with the dissolution of the upper layer, will also suddenly interact with the cell. After this intense active ingredient loading, cell deaths will be rapid, but the PLA nanofibrous surface will remain in the environment, which will do long term release of the active ingredient at low speed to counter the risk of regeneration of cells. Since the amount of encapsulated active ingredient in PLA is high and the release rate is low, long term protection will be provided. In this way, a rapid release of the active ingredient after the dissolution of PVA in a short time after the application, which does not release any active ingredient during the application, the release of the free active ingredient that will enable the cell to interact with the active ingredient will be ensured, and then the release of the active ingredient to that region will be ensured with the slow release mechanism.

[0068] Rutin is a unique antioxidant flavonoid found mainly in fruit, vegetables, grains, and many other plants based human diets. Studies have highlighted the in vitro anticancer properties of rutin, including combination therapeutic strategies. Rutin has been shown to inhibit the proliferation of breast, colon, lung, and prostate cancers and other tumors. In addition, rutin, alone or in combination with other therapeutic agents, regulates carcinogenesis and apoptosis induction in a variety of mechanisms. The combination of rutin with other chemotherapy drugs is beneficial in preventing tumor cells by reducing drug resistance and chemotherapy side effects. At the same time, since the use of chemotherapeutic drugs have many various side effects and rutin has come to light as a safe anticancer agent since it has little side effects. With the rutin loaded layered hybrid nanofiber developed in the invention, it increases the anti-cancer effect of the chemotherapy drug by making a controlled release in the tumor area at the desired dose.

[0069] TMZ is the most commonly used chemotherapy drug in GB treatment which is effective in oral use. However, the drug half-life of TMZ is quite low, and the penetration rate from the blood into the cerebrospinal fluid is only 20-30%. This causes insufficient drug concentration in the tumor area and recurrence of the disease. With the TMZ-loaded layered hybrid nanofiber drug delivery system developed in the invention, control of the area of release in the body, release rate and minimization of drug degradation or loss is ensured by direct application to the tumor bed after surgical resection.

[0070] The technical specifications and production parameters of the drug delivery system of the invention, results of in vitro drug release and the proliferation effects parallel to the release:

[0071] The preparation of the Rutin and TMZ which are loaded into the Layered hybrid nanofiber:

[0072] TMZ (Cat. No.: T2577; 10 mg / mL), was dissolved in dimethyl sulphoxide (DMSO) in ultrasonic bath for 2 minutes.

[0073] Rutin (Cat. No.: PHL89270; 6.25 mg / 25 mL), was dissolved in distilled water in ultrasonic bath for 30 minutes.

[0074] The production of Rutin and / or TMZ loaded doped nanofibrous non-woven surfaces with Blending Electrospinning Method:

[0075] The production of doped PVA nanofibrous nonwoven surfaces was carried out with the INOVENSO Nanospinner24 electrospinning device. The production principle of the device is provided below schematically. In the study, nanofibrous non-woven surfaces were produced by separately mixing TMZ and Rutin in polymer. Cat. No.: T2577; The TMZ which was dissolved in 10 mg / mL dimethyl sulphoxide (DMSO) in ultrasonic bath for 2 minutes, Cat. No.: PHL89270; 6.25 mg / 25 mL, the Rutin which was dissolved in distilled water in ultrasonic bath for 30 minutes, the amounts of 2.0904 ml (rutin) and 0.6ml (TMZ) were added into the polymer and mixed for 24 hours. The PVA in which the active substance was mixed was 10% aqueous solution, 3.5 ml of which was taken and the active ingredient was mixed in above amount.

[0076] The resulting mixture was taken into the syringe to be worked in the electrospinning device and placed in the polymer feeding unit of the device and the doped nanofibrous non-woven surface was formed in the drum of the device.

[0077] Electrospinning working conditions;

[0078] Flow rate: 0.4 ml / hour for TMZ, 0.6 ml / hour for Rutin

[0079] Height: 110 mm

[0080] Voltage: 26 kV

[0081] Drum revolution speed: 250 rpm

[0082] The production of Rutin and / or TMZ loaded core PLA Core-shell nanofibrous non-woven surfaces by the Core-Shell Electrospinning method:

[0083] The production of cored PLA nanofibrous nonwoven surfaces was carried out with the INOVENSO Nanospinner24 electrospinning device. The production principle of the device is provided below schematically. In the study, nanofibrous non-woven surfaces consisting of core nanofibers were produced by encapsulating TMZ and Rutin with fibers separately. Cat. No.: T2577; 10 mg / mL, The TMZ which was dissolved in dimethyl sulphoxide (DMSO) in ultrasonic bath for 2 minutes Cat. No.: PHL89270; 6.25 mg / 25 mL, Rutin which had been dissolved in distilled water in an ultrasonic bath for 30 minutes was spun together with the polymer in the electrospinning device. The amount of Rutin used was 5.226 ml and the amount of TMZ used is 1.5 ml. In the study method, the flat system which was developed for making core-shell production was used. Dual feeding is used in this system: The first feeding forms the shell section while the second feeding forms the core section. PLA polymer was produced from the first feed and non-woven surfaces with encapsulated active ingredient were produced with PLA nanofiber by feeding active ingredient from the second feed. The polymer used is 5 grams of PLA at a concentration of 10%. PLA was dissolved in 40ml DMF (Dimethylformamide) and 10 ml chloroform.

[0084] Electrospinning working conditions; Flow rate: For TMZ; shell section 0.85 ml / hour, core section 0.2 ml / hour, for Rutin shell section 2 ml / hour, core section 0.3 ml / hour

[0085] Height: 143 mm

[0086] Voltage: 26 kV

[0087] Drum revolution speed: 250 rpm

[0088] Core-Shell Electrospinning, The production of Rutin and TMZ loaded Layered hybrid nanofibrous (LHN) non-woven surfaces using Blending and Electrospray:

[0089] The first layer of the layered structure is nanofibrous non-woven surface made up of the polymer PLA in core-shell structure. TMZ and Rutin has been sprayed separately on this surface with the electrospraying method.

[0090] Cat. No.: T2577; 10 mg / mL, The TMZ which was dissolved in dimethyl sulphoxide (DMSO) in ultrasonic bath for 2 minutes Cat. No.: PHL89270; 6.25 mg / 25 mL, 3.1356 ml (rutin) Rutin and 0.9ml (TMZ) which was dissolved in distilled water in an ultrasonic bath was sprayed.

[0091] Electrospinning working conditions;

[0092] Flow rate: 1.5 ml / hour

[0093] Height: 143 mm

[0094] Voltage: 26 kV

[0095] Drum revolution speed: 250 rpm

[0096] Release analysis of Loaded Layered Hybrid Nanofibers (LHN) in PBS:

[0097] The release analysis of loaded LHN structures produced was carried out (Figure 2). A partially controlled release has taken place during the release of Rutin. 6th Hour release is the highest, while the lowest release is the release at 1 hour as expected. Unexpectedly, the release at the 6. hour is the form of burst release, the reason for this is thought to originate from the release test mechanism being unable to simulate the layered structure’s application method. In the layered structure application, the nanofiber structure is designed so that the PVA coated surface comes into contact with the tissue. In this way, the brain fluid in the tissue will first dissolve the PVA and release the active ingredient mixed into the PVA onto the environment, and after the PVA layer is completely dissolved, the active ingredient which had formed an intermediate layer with electrospraying will have a high amount of release in the form of an explosion, which aims to do a low amount of long term release from the PLA / core shell layer to prevent the reviving of the cancer cells whose growth had been stopped completely. However, since there is no mechanism to simulate this type of application in the sample preparation mechanisms to be made for release tests and since a certain amount of cut nanofiber is thrown into the PBS liquid in a tube and the solvent interacts with the entire surface and does not gradually start to dissolve the entire surface at the same time, sudden release has been observed at an early time. The reason for the low amount of release in the 6th hour is thought to be that the active ingredient is not distributed homogeneously to the nanofiber structure during the production process. According to the test setup, specimens of each release period were prepared using different sections of the nanofiber. From the SEM images , it was seen that the LHNrutinstructure was formed properly (Figure 3). A nanofibrous structure was formed on the upper surface, and a sediment shaped surface was formed in the middle to form a layered structure of active substance. In the lower layer, a surface was formed to have a fibrous and layered structure.

[0098] When LHNTMZrelease was examined, controlled release was observed (Figure 2). All releases other than 18 hours were increased over time. The reason for the low amount of release in the 18th hour is thought to be that the active ingredient is not distributed homogeneously to the nanofiber structure during the production process. Although LHNTMZwas not as smooth as in the rutin, it was observed that a fibrous structure was formed. Especially in the upper part, the fibrous structure dominates the surface, while the fibrous structure in the lower part has been observed to turn into a layered structure during production. In the middle part, it was observed that the active ingredient formed a layer as in rutin. It was observed that the interaction between the middle layer and the upper layer was high, so the fibrous structure passed into the middle layer. Due to this interaction, the fibrous structures formed in the upper layer were more irregular, thicker and in some regions formed in a film-shifting form (Figure 4). However, this structural impairment had no effect on the controlled release of TMZ. The sample preparation process for the release test of the active ingredient TMZ was conducted in a similar manner Therefore, although it did not simulate the targeted application, it was deemed appropriate that it achieved the targeted controlled release. Average fiber diameters measured from 20 fibers were found to be =165±37 nm for LHNrutinImage J (version 1.520 software). Since the measurements take time, the creation of histogram curves continues. Likewise, the average fiber diameters measured from 20 fibers were found to be =315±82.8 nm for LHNTMZ. In addition, SEM images of LHNTMZshow that film and fibrous structures are formed together on the surface. Although the formation of the film structure is not desired, in-vitro release results from these nanofibers (LHNTMZ) indicate a controlled release for 72 hours.

[0099] Evaluating the effects of TMZ and Rutin loaded, Loaded Layered Hybrid nanofibers (LHN) on cell viability:

[0100] Rutin and TMZ’s IC50 concentrations were encapsulated per 0.5 cm2. To observe the effect of LHNTMZand LHNrutinnetworks on T98G cell profileration rate, T98G cells were treated with cut nanofibers containing IC50 doses and culture medium released from nanofibers, and cell viability was monitored for 120 hours. The viability of T98G cells treated with LHNTMZdecreased to 50.4% at 48 hours compared to cells grown in untreated cells (p<0.0001 ; Figure 5). The viability of T98G cells treated with LHNrutindecreased to 18.2% compared to cells grown in untreated cells (p<0.0001 ; Figure 5) and this nanofiber suppressed proliferation in the same way at 72-96th hours. In T98G cells treated with |_HNTMZ+rutincombination, it was observed that it reduced cell proliferation to 6.8% compared to the untreated cell group (p<0.0001 ; Figure 5).

[0101] The UV lamp of a laminar flow cabinet is used for the sterilization of the loaded layered hybrid nanofibers produced. Loaded nanoparticles are placed in clean petri dishes and these are placed by opening the laminar flow cabinet openings. While the airflow is running in the laminar flow cabinet, the UV lamp (254 nm) is turned on and the nanofibers are kept under UV light for 30 minutes - 1 hour.

[0102] Sterile loaded layered hybrid nanofibers are taken back into sterile petri dishes for use. For short term storage, it should be stored at room temperature in a dark and moisture- free environment, and for long term storage, it should be stored in a refrigerator at +4‘C. During the surgery in a sterile environment, it is to be locally placed in the area where the tumor was extracted from. Since Rutin and TMZ are encapsulated in layered hybrid nanofibers, they are used for short term storage at room temperature in a dark and moisture-free environment. For long term storage, it should be stored in a refrigerator at +4‘C. Loaded layered hybrid nanofibers, can be stored up to 6 months.

[0103] If needed, different chemotherapy drugs, drug candidates and natural compounds with a glycosidic chemical structure with Rutin like hydroxyl (-OH) polar group can be loaded individually and in combination into the same drug delivery system for local use in different types of cancer.

[0104] The inventors first demonstrated that rutin, when not loaded onto nanofibers, is effective on GB cells and can enhance the effectiveness of TMZ. With the said study, it was emphasized that rutin should be included as a suitable leading candidate in drug development studies since it suppresses the aggressiveness of GB tumors (Ercelik et al., 2023).

[0105] Afterwards, it was demonstrated with an in vitro study that rutin could be loaded into PLA core-shell nanofiber that were developed differently to the drug delivery system of the invention and rutin not loaded to nanofiber retains its effectiveness (Ercelik et al., 2023). In these in vitro studies, it has been shown that core-shell nano-nets, which are different from the drug delivery system of the invention, can be a new and effective therapeutic tool against recurrent GB cells. The nanofiber used in the said study is only comprised of the core-shell structure. The drug delivery system, which is the subject of the relevant invention application, is different from the core-shell nanofiber, which is a special nanofiber design comprising two layers and has a hybrid surface loaded with active substance in the middle. The present drug delivery system, which is the subject of the invention application, is a specific model produced for in vivo applications, allowing long term release (Figure 1)

[0106] In-vitro analysis results:

[0107] With the rutin and / or TMZ-loaded layered hybrid nanofiber we developed, it was found to inhibit the proliferation of in-vitro GB cancer cells, suppress invasion (Figure 6), inhibit vessel formation (Figure 7), inhibit colony formation (Figure 8), and shrink the size of 3D cancer spheres (Figure 7) (unpublished data). It was also observed that rutin and / or TMZ-loaded layered hybrid nanofiber had the ability to inhibit invasion by suppressing EMT markers (Figure 10), could prevent tumor aggressiveness by suppressing cancer stem cell markers (Figure 11), and rutin suppressed tumor aggressiveness through epigenetic mechanism by suppressing LncRNA markers (Figure 12).

[0108] In-vivo analysis results:

[0109] GB tumors were induced with C6 cancer cells in Wistar Albino rats, and tumor formation was visualized using MRI (Figure 13). After the tumor formation was successfully carried out, one group of tumors was not treated, while the other groups were treated with the rutin and / or TMZ loaded nanofiber we developed. It was left in the tumor resection area by stereotactic surgery method in a rutin and / or TMZ loaded nanofiber operating room environment (Figure 14). At the end of 15 days, the rat brain was removed and evaluated macroscopically and pathologically in terms of tumor size and tumor aggressiveness. While the size of the tumor formed in the untreated control group in the experimental animals transplanted with the same number of cancer cells was 1 ,4cm3, the tumor size formed in the experimental group treated with TMZ-loaded layered hybrid nanofiber was determined as 1.1cm3(Figure 15).

[0110] As a result, it was observed that the treatment applied with the local drug delivery system of the invention reduced the tumor size by 21.4% compared to the control group. The fact that nanofiber can still exist in the brain tissue removed after 15 days of application proved that the nanofiber we developed is suitable for long term controlled release (Figure 16).

[0111] REFERENCES

[0112] • Ercelik M, Tekin C, Tezcan G, Ak Aksoy S, Bekar A, Kocaeli H, Taskapilioglu MO, Eser P, Tunca B. Olea europaea Leaf Phenolics Oleuropein, Hydroxytyrosol, Tyrosol, and Rutin Induce Apoptosis and Additionally Affect Temozolomide against Glioblastoma: In Particular, Oleuropein Inhibits Spheroid Growth by Attenuating Stemlike Cell Phenotype. Life (Basel). 2023 Feb 8;13(2):470. doi: 10.3390 / life13020470.

[0113] • Ercelik M, Tekin C, Parin FN, Mutlu B, Dogan HY, Tezcan G, Aksoy SA, Gurbuz M, Yildirim K, Bekar A, Kocaeli H, Taskapilioglu MO, Eser P, Tunca B. Co-loading of Temozolomide with Oleuropein or rutin into polylactic acid core-shell nanofiber webs inhibit glioblastoma cell by controlled release. Int J Biol Macromol. 2023 Sep 8;253(Pt 2):126722. doi: 10.1016 / j.ijbiomac.2023.126722.

[0114] • Louis, D.N. Ohgaki, H. Wiestler, O.D. Cavenee, W.K. Burger, P.C. Jouvet, A. Scheithauer, B.W. Kleihues, P. 2007. “The 2007 WHO classification of tumours of the central nervous system”. Acta Neuropathol. 114:97-109.

[0115] • McLendon, R.E. Halperin, E.C. 2003. “Is the long-term survival of patients with intracranial glioblastoma multiforme overstated?”, Cancer, 98:1745-1748.

[0116] • Alan, O. Yumuk, P.F. 2019. “Glioblastomda Nuks Hastahkta Sistemik Tedavi Segenekleri”, Eri§kin Santral Sinir Sistemi Tumbrleri, Turkiye Klinikleri, p.61-5.

[0117] • Belter A, Barciszewski J, Barciszewska AM. Revealing the epigenetic effect of temozolomide on glioblastoma cell lines in therapeutic conditions. PLoS One. 2020 Feb 26;15(2):e0229534.

[0118] • Lillehei KO, Kalkanis SN, Liau LM, Mydland DE, Olson J, Paleologos NA, et al.. Rationale and design of the 500-patient, 3-year, and prospective Vigilant Observation of GlladeL WAfer ImplaNT registry. CNS Oncol. (2018) 7:CNS08.

[0119] • Giese, A. Kucinski, T. Knopp, U. Goldbrunner, R. Hamel, W. Mehdorn, H.M. Tonn, J.C. Hilt D. Westphal M. 2004. “Pattern of Recurrence following Local Chemotherapy with Biodegradable Carmustine (BCNU) Implants in Patients with Glioblastoma”, J. Neurooncol, 66, 351-360.

[0120] • Roux A, Peeters S, Zanello M, Bou Nassif R, Abi Lahoud G, Dezamis E, et al.. Extent of resection and Carmustine wafer implantation safely improve survival in patients with a newly diagnosed glioblastoma: a single center experience of the current practice. J Neurooncol. (2017) 135:83-92.

[0121] • Ramachandran, R. Junnuthula, V.R. Gowd, S. et al. 2017. ’’Theranostic 3- Dimensional nano brain-implant for prolonged and localized treatment of recurrent glioma”, Sci Rep, 7: 43271.

[0122] • Stewart, L.A. 2002. “Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual patient data from 12 randomised trials”, Lancet, 359:1011-1018.

[0123] • Parin, F.N. Aydemir, Q.l. Taner, G. Yildinm, K. 2021. “Co-electrospun- electrosprayed PVA / folic acid nanofibers for transdermal drug delivery: preparation, characterization and in vitro cytocompatibility”, Journal of Industrial Textiles.

[0124] • Ghorbani, S. Eyni, H. Tiraihi.T Asl, L.S. Soleimani, M. Atashi, A. Pour, S. Majid, B. Warkiani, E. 2018. “Combined effects of 3D bone marrow stem cell-seeded wet-electrospun poly lactic acid scaffolds on full-thickness skin wound healing”, International Journal of Polymeric Materials and Polymeric Biomatenals, 67:15, 905- 912.

[0125] • Rahmani, M. Bidgoli, S.A. Rezayat, S.M. 2017. “Electrospun Polymeric Nanofibers for Transdermal Drug Delivery”, Nanomed. J., 4 (2), 61-70.

Claims

CLAIMS1. Drug delivery system for local application in a layered hybrid nanofiber structure loaded with at least one active ingredient characterized by comprising the following layers:PLA shell nanofiber layer with at least one active ingredient core PVA nanofiber layer loaded with at least one active ingredient At least one active ingredient that electrosprayed between said layers.

2. The drug delivery system according to claim 1 , characterized in that; said active ingredient is an individuals or combinations selected from the group comprising chemotherapy drugs, drug candidates and natural compounds which have a glycosidic chemical structure with a Rutin-like hydroxyl (-OH) polar group.

3. The drug delivery system according to claim 1 or 2, characterized in that; said active ingredient is Rutin and / or Temozolomide.

4. The drug delivery system according to any claims 1 to 3, characterized in that; said active ingredient is Rutin and Temozolomide.

5. A method for producing drug delivery system for local application in a layered hybrid nanofiber structure loaded with at least one active ingredient, characterized by comprising the following process steps: i. Formation of an PLA shell nanofibre layer with at least one active ingredient core by Core / Shell electrospinning method; ii. Electrospraying at least one active ingredient on said layer; iii. Forming PVA nanofiber layer loaded with at least one active ingredient on said active ingredient by using blending electrospinning method.

6. The method according to claim 5, characterized in that; said active ingredient is an individuals or combinations selected from the group comprising chemotherapy drugs, drug candidates and natural compounds which have a glycosidic chemical structure with a Rutin-like hydroxyl (-OH) polar group.

7. The drug delivery system according to claim 5 or 6, characterized in that; said active ingredient is Rutin and / or Temozolomide.

8. The drug delivery system according to any claims 5 to 7, characterized in that; said active ingredient is Rutin and Temozolomide.

9. The method according to Claim 8, characterized in that; process step (i) comprises the following processes:The preparation of the solution of TMZ in DMSO at 10 mg / ml concentration, -The preparation of the solution of Rutin in distilled water at 6.25 / 25 mL concentration,The preparation of the solution of PLA in DMF and chloroform at 5g / 40ml / 10ml concentration,Formation of Rutin and TMZ loaded nanofiber layers separately by using Core / Shell electrospinning method, whereinElectrospinning working conditions;Flow rate: For TMZ; shell section 0.85 ml / hour, core section 0.2 ml / hour, for Rutin shell section 2 ml / hour, core section 0.3 ml / hourHeight: 143 mmVoltage: 26 kVDrum revolution speed: is 250 rpm.

10. The method according to claim 9, characterized in that; The amount of Rutin solution used is 5.226 ml and the amount of TMZ solution used is 1 .5 ml.

11. The method according to claim 8, characterized in that; process step (ii) comprises the following processes:- The preparation of the solution of TMZ in DMSO at 10mg / mL concentration,- The preparation of the solution of Rutin in distilled water at 6.25 / 25 mL concentration.- Rutin and TMZ solutions being sprayed with the electrospraying method,whereinElectrospinning working conditions;Flow rate: 1.5 ml / hourHeight: 143 mmVoltage: 26 kVDrum revolution speed: is 250 rpm.

12. The method according to claim 11 , characterized in that; 3.1356 ml Rutin and 0.9 ml TMZ solution being sprayed.

13. The method according to claim 8, characterized in that; process step (iii) comprises the following processes:- The preparation of the solution of TMZ in DMSO at 10 mg / ml concentration,- The preparation of the solution of Rutin in distilled water at 6.25 / 25 mL concentration,- Mixing of 2.0904 ml / 0.6ml / 3.5 ml concentrations of the Rutin solution, TMZ solution and 10% aqueous solution of PVA.- Formation of Rutin and TMZ loaded nanofiber layers separately by using Core / Shell electrospinning method, whereinElectrospinning working conditions;Flow rate: 0.4 ml / hour for TMZ, 0.6 ml / hour for RutinHeight: 110 mmVoltage: 26 kVDrum revolution speed: is 250 rpm.

14. The use of Rutin and / or Temozolomide loaded layered hybrid nanofiber as the local drug delivery system in the field of brain tumor surgical resection.

Images