WO2021096464A1 - A novel nanotechnologic approach to glioblastoma treatment with solid lipid carriers - Google Patents

Abstract

The invention is to a solid lipid nanoparticle, which is a targeted drug delivery system that can cross the blood-brain barrier for use in the treatment of Glioblastoma. With the invention, solid lipid nanoparticles (SLN) containing carmustine (BCNU) and temozolomide (TMZ) are prepared and the surface of these nanoparticles is coated with polysorbate 80 to cross the blood brain barrier (BBB). The drug system is formulated for intranasal administration in order to reach the brain with a higher concentration of solid lipid nanoparticles loaded with the prepared dual drug system.

Description

A NOVEL NANOTECHNOLOGIC APPROACH TO GLIOBLASTOMA TREATMENT WITH SOLID LIPID CARRIERS

Technical Field of the Invention

The invention is related to a MCT-1 (Monocarboxyl ate transporter 1) receptor targeted solid lipid nano drug delivery system that can cross the blood-brain barrier for use in the treatment of Glioblastoma (GBM).

Known State of the Invention (Prior Art)

According to the statement made by the American Brain Tumor Association, brain tumor is the most common type of cancer after leukemia in individuals aged 0 to 19. In addition, brain tumors are the second most common cause of death after leukemia in individuals aged 1 to 19 years. Meningiomas take the first place among the brain tumors with a rate of 36.4%. This is followed by glioma with a rate of 27% and it constitutes 80% of malignant brain tumors.

Grade IV astrocytoma glioblastoma (GBM), which is one of the astrocytic tumors within neuroepithelial tissue tumors, it is perceived approximately four times more than grade III anaplastic astrocytoma. GBM has two to three incidences per 100.000 adults in a year and it accounts for 52 percent of all primary brain tumors. Overall, GBM accounts for about 17 percent of all brain tumors (primary and metastatic). Although the treatment approach is a standard multimodal treatment for newly diagnosed GBM, the prognosis is poor and the average survival is 14,6 months. Patients receive radiotherapy and chemotherapy treatment. They can receive temozolomide (TMZ) treatment as primary or adjuvant chemotherapy. This treatment results in the current prognosis for an average survival of 14,6 months following diagnosis.

The prior art is to apply a radiotherapy treatment regimen with the FDA's recommendation carmustine (BCNU) in the treatment of recurrent glioblastoma. This application can increase the survival by only 2,5 months on average in glioblastoma treatment. Therefore, there is a need to develop new therapeutic approaches for glioblastoma, which covers a very high proportion of malignant brain tumors. The American Cancer Society defines cancer treatment methods as chemotherapy, radiotherapy, immunotherapy, hormone therapy, targeted therapy, stem cell or bone marrow transplantation, and surgery.

Carmustine (BCNU) and temozolomide (TMZ) are drugs belonging to the group of anti cancer agents that have alkylating agent properties that slow the growth of cancer cells. It is an accepted view by scientists that inhibition of a single pathway in cancer treatment cannot give very successful results. Therefore, there is a need for treatment methods using innovative and combined drugs.

In a study conducted by Alex et al. in 2016 in which carboplatin chemotherapeutic agent was used for the treatment of glioma, a nanoparticle that was created using biodegradable polymer polycaprolactone was loaded with carboplatin and it was administered intranasally to Wistar rats. These nanoparticles have been shown to increase the nasal absorption of carboplatin.

In the patent document numbered WO16026942A1 titled "Methods for enhancing permeability to blood-brain barrier and uses thereof', it is aimed to cross the blood brain barrier by using TMZ and VEGF combined drugs for the treatment of brain tumors. In this study, drugs were given intravenously and intranasal administration was not used. Since the plasma half-life of the TMZ drug is 1,8 hours, the effectiveness of the drug decreases when the drug enters the systemic circulation.

Among the current treatment methods, targeted cancer treatment has not yet become widespread. The targeting strategies that are being used are also very limited. It is known in the prior art that there are different drug delivery systems containing TMZ or BCNU for the treatment of GB and some of them have been developed as being receptor-targeted. In a study by Chen Jiang et al., transferrin receptors instead of MCT-1 were targeted for the treatment of glioblastoma. Carmustine is loaded into PEG-PLGA micelles, and it binds the T7 peptide which can interact with the transferrin receptor to the structure. In the study, the drug delivery system was administered intravenously on BALB/c nude mice. The technical problem that this system cannot solve is that it takes time for the drug to reach the brain as the drug is administered intravenously, and the drug's effectiveness is reduced when it reaches the brain. At the same time, phase I and II studies in which erlotinib, gliadel, XRT, avastine, edotecarin, celecoxib, capecitabine, lomustine were used in combination with TMZ or BCNU were found in the prior art, and TMZ and BCNU were used together in only one study. However, in said studies, no drug delivery system was used and no targeting was made on drugs. For this reason, said studies have disadvantages such as the unwanted effects of the drugs used in non- target tissues due to the introduction of the drugs into the systematic circulation of the body by intravenous injection, as well as the decrease in the effectiveness of the drugs reaching the brain.

In another study, repamycin-bound albumin nanoparticles were prepared and stated to be administered to patients after treatment with TMZ and irinotecan.The technical problem that this system cannot solve is that since the drug is administered intravenously and the delivery system is not targeted, some of the drug is metabolized in the liver and eliminated from the body after it enters the systemic circulation. It takes time for the administered drug to reach the brain as it is distributed throughout the body, and the effectiveness of the drug decreases by the time the drug reaches the brain.

In another study, paclitaxel loaded albumin nanoparticles were prepared and a phase II study was carried out using carboplatin and TMZ. This study was used for advanced malignant melanoma. Its effectiveness in the treatment of glioblastoma has not been investigated. In the phase II study that started in 2014 and completed this year, TMZ was used in combination with SGT-53 gene therapy. The drug systems used in these studies were given by intravenous administration. Therefore, with the addition of the drug to the systemic circulation, loss of the drug amount and side effects on the tissues in the systemic circulation can be seen.

For these reasons, there is a need to develop treatment methods using innovative and combined drugs with high efficiency and targeting.

Summary and Aims of the Invention

With the invention, solid lipid nanoparticles (SLN) containing carmustine (BCNU) and temozolomide (TMZ) are prepared and their surface is coated with polysorbate 80 to cross the blood brain barrier (BBB). The drug system is formulated for intranasal administration in order to reach the brain with a higher concentration of solid lipid nanoparticles loaded with the prepared dual drug system and in vitro and in vivo studies have been conducted. The invention can also be administered intravenously. Since the permeability of the structure of the solid lipid nanoparticles used through cell membranes is higher compared to other carrier systems, intranasal application can be performed. In addition, by adding polysorbate 80 to the solid lipid nanoparticle structure, membrane permeability was further increased.

The target group of the invention is patients with glioblastoma, and by means of the invention, drug release and MCT-1 targeting are performed in a controlled manner, providing more benefits to the patient with low-dose drugs with increased therapeutic index of drugs in the treatment, and this allows both an increase in patient welfare and a decrease in cost.

The purpose of targeting the MCT-1 protein in the invention is to increase the receptor of this protein in tumor tissue and the role of this protein in the transport of monocarboxylic acids in energy metabolism. MCT-1 protein plays a role as a carrier for monocarboxylic acids.

Easy penetration of the solid lipid nanostructure into the tumor tissue is provided with dual drug content of the system used with the invention and through physical targeting by utilizing the increase of MCT-1 receptor in the tumor tissue. With the structure of the invention that can cross the blood brain barrier, the possibility of delivering controlled drugs, the intranasal applicability of the system to the patient and the combination of all these advantages on a single system are the differences and superior features of the invention when compared with other known methods. The MCT-1 receptor targeting of the invention can also be used with receptors that accept the HBA-SA conjugate as a substrate and increase in tumor tissue.

The created system can be defined as targeted chemotherapy. Unlike the existing methods, more than one targeting strategy is used in the developed system. Clinical database searches have shown that there is no study involving a system in which TMZ and BCNU are used together and loaded into nanoparticles. Also, inhaler therapy applications of this nanoparticle system are not encountered. Many chemotherapeutic agents are administered as intravenous treatment, where the liquid substance is given directly through the vein, but inhaler therapy, where the drug is administered to the body by inhalation, helps the drug to reach the brain by passing the BBB, in other words, it allows the chemotherapeutic agent to reach the malignant tissue directly. This situation provides an advantage in treatment. Considering the success of solid lipid nanoparticles passing through BBB, toxicity and stability characteristics, it is seen as one of the most suitable systems for drug delivery to the brain. In addition, in the invention, by targeting the solid lipid nanoparticle to the MCT-1 receptor, it is aimed to pass drugs that normally cannot pass through the blood-brain barrier easily and to provide an effective treatment in the cancerous area without damaging healthy cells as much as possible.

With the invention, a MCT-1 receptor targeted solid lipid nano drug carrier system that can cross the blood brain barrier is created for use in the treatment of glioblastoma. Solid lipid nanoparticles are synthesized with polysorbate-80 surface coating to cross the blood brain barrier, and it is also present in healthy tissue in order for the system to deliver drugs more selectively for glioblastoma treatment, but the number of MCT-1 receptors in solid brain tumor tissue increases compared to healthy tissue. Stearylamine (SA) and b-Hydroxybutyrate (HBA) conjugate (SA-HBA) are designed and attached to the solid lipid nanoparticle structure for targeting the increased MCT-1 receptor. The system created with SA-HBA acts as a substrate for the MCT-1 receptor and thus interacts with the MCT-1 receptor increased in glioblastoma and the HBA-SA conjugate, enabling targeted therapy to be formed.

Since it is an accepted view by scientists that inhibition of a single pathway in cancer treatment will not produce sufficiently successful results, with the invention, temozolomide and carmustine chemotherapy agents used in the prior art are combined and loaded into solid lipid nanoparticles to provide dual therapy.

For the formation of solid lipid nanoparticles, cetyl palmitate, polysorbate 80, SA-HBA conjugate, temozolomide and carmustine are added to the medium, and then the structure is formed with the ultraturax device and sonic bath. With in vivo biodistribution studies, it is seen that the MCT-1 targeted nano-carrier system, which can cross the blood-brain barrier, accumulates at a high rate in the brain tissue, and its accumulation in other organs is below the detectable level. These results support the therapeutic efficacy of the invention.

The biocompatibility, binding to serum proteins and the amount of hemolysis of the invention are tested, and the effect of cytotoxicity and apoptosis is determined by cell culture studies. It is determined that the system is directed to the targeted tissue for its purpose by conducting single dose and repeated dose toxicity studies, biodistribution and pharmacokinetic trials and it provides therapeutic effect by controlled release in that region.

The invention differs from the prior art in that it allows the synthesized nanoparticle to cross the blood-brain barrier, its ability to accumulate only in the area where the system will act with MCT-1 targeting, allowing controlled release of drugs, and being administered intranasally. Unlike the prior art document numbered WO16026942A1, in the invention, targeted therapy is used with a drug delivery system and this treatment is administered intranasally. As the drugs are loaded into the solid lipid nanoparticle and these nanoparticles are increasingly targeted to the MCT-1 receptor, the passage through the blood-brain barriers is facilitated, and after the intranasal administration of the drug, approximately 49% of the drug has been observed to accumulate in the brain. In addition, the drug is provided to reach the brain faster with intranasal administration.

The conducted clinical database searches show that there is no study involving a nanoparticular system in which TMZ and BCNU within the scope of the invention are used together and literature searches, clinical studies and patent studies show that the nano preparation designed within the scope of the invention is unique.

Definitions of Drawings Illustrating the Invention

Figure 1: FTIR spectrum of stearylamine and HBA-SA conjugate.

Figure 2: FTIR spectrum of b-hydroxy butyrate (HBA) and HBA-SA conjugate.

Figure 3: H-NMR spectra: A) stearylamine B) HBA C) HBA-SA samples.

Figure 4: SEM image of solid lipid nanoparticles.

Figure 5: A) FTIR spectrum of Polysorbate 80 B) Overlapped FTIR spectra of SLN, Cetyl Palmitate and HBA-SA conjugate.

Figure 6: TGA analysis result of drug-free solid lipid nanoparticles.

Figure 7: A) Hydrodynamic diameter B) ZetaPotential result of solid lipid nanoparticles.

Figure 8: HPLC chromatogram of the supernatant of nanoparticles prepared at 0,1% Temozolomide concentrations.

Figure 9: HPLC chromatogram of the supernatant of nanoparticles prepared at 0,1% Carmustine concentration.

Figure 10: A) hydrodynamic diameter and B) zeta potential result of a SLN sample containing 0,1% drug

Figure 11: SEM image of a SLN sample containing at 0,1% drug concentration.

Figure 12: FTIR spectrum of nanoparticles containing a dual drug system.

Figure 13: Thermogram curve of nanoparticles containing a dual drug system.

Figure 14: Time dependent release curve of temozolomide and carmustine from nanoparticles.

Figure 15: Drug Release Graph in Solid Lipid Nanoparticles containing TMZ and BCNU made using Franz cell

Figure 16: Percentages of viability versus doses of solid lipid nanoparticles containing BCNU, solid lipid nanoparticles containing TMZ, and solid lipid nanoparticles containing BCNU-TMZ, applied to U87MG cells for 48 hours. Figure 17: Percentages of viability versus doses of solid lipid nanoparticles containing BCNU, solid lipid nanoparticles containing TMZ, and solid lipid nanoparticles containing BCNU-TMZ, applied to U87MG cells for 72 hours.

Figure 18: A) Conjugate-bearing nanoparticle B) non-conjugate-bearing nanoparticle C) chromatograms for TMZ analysis showing uptake amounts of free drug samples into bEnd.3 cells.

Figure 19: IVIS image detected at the end of 3 hours in a solid lipid nanoparticle containing FITC loaded conjugate and nude mice given FITC only.

Figure 20: IVIS images of brain tissue extracted after sacrificing of mice applied with solid lipid nanoparticles containing FITC-labeled temozolomide and carmustine at the end of 3 hours

Figure 21: Time dependent plasma concentration graph for TMZ Figure 22: Time dependent plasma concentration graph for BCNU Figure 23: 28-day Subacute Toxicity Weight Change Graph Figure 24: Cancer control group IVIS images.

Figure 25: IVIS images of the low-dose solid lipid nanoparticle group.

Figure 26: IVIS images of the high-dose solid lipid nanoparticle group.

Figure 27: IVIS images of free drug group.

Figure 28: Ex vivo staining images of the control group.

Figure 29:Ex vivo staining images of the free drug group.

Figure 30: Ex vivo staining images of the low dose nano group.

Figure 31: Ex vivo staining images of the high dose nano group.

Figure 32: A) Control group B) Free drug group C) Low dose nano group and D) Ki67 staining results of high dose nanoparticles.

Detailed Description of the Invention

The detailed description of the invention, which is an MCT-1 receptor targeted solid lipid nano drug delivery system that can cross the blood-brain barrier for use in the treatment of glioblastoma, is given below.

Conjugation of Stearylamine (SA) and b-Hydroxybutyrate (HBA)

Stearylamine and b-hydroxybutyrate act as substrates for the MCT-1 receptor. In many mammalian cells, the monocarboxylate carrier MCT-1 mediates the transport of lactate, acetoacetate and pyruvate in addition to b-hydroxybutyrate. In the invention, as an alternative to b-hydroxybutyrate, preferably lactate, acetoacetate and pyruvate can be used b- hydroxybutyrate, lactate, acetoacetate, and pyruvate can be referred to as ketone bodies transported with the MCT-1 receptor. By using stearylamine and ketone bodies transported with said MCT-1 receptor, the substrate system for the MCT-1 receptor can be formed.

The main purpose of using stearylamine is to act as a spacer arm to create a better interaction of b-hydroxybutyrate substrate (or ketone bodies transported with the alternative MCT-1 receptor) with the increased MCT-1 receptors in cancer cells.

In an embodiment of the invention containing HBA, 150 mg of b-hydroxybutyrate is dissolved in 5 mL of N, N-dimethyl formamide (DMF) in order to provide the conjugation of SA and HBA, which is available in the state of the art. Then, 40 mg of N-hydroxy succinimide (NHS) and 38 mg of 1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) are dissolved in DMF, respectively, and added to the b-hydroxy butyrate solution to reach the final volume of 10 mL, and stirred at room temperature for 2 hours. At the end of the time, 100 mg of stearylamine and 500 pL of pyridine are added and left to react under stirring overnight in the dark. After the reaction is completed, the SA-HBA conjugate is precipitated by adding 50 mL of purified water to the reaction medium. Then, the conjugate is dialyzed against distilled water for 3 days in order to remove the contaminants in the medium. After dialysis, the dialysate is filtered by means of the por 3 nuche funnel. The conjugate remaining on the filter is dried in a 30°C oven for 3-5 days and stored in dry state at +4°C for the next reactions.

Synthesis of Solid Lipid Nanoparticles

Cetyl palmitate, one of the elements that constitutes the solid lipid nanoparticle, is used to form the lipid core. As an alternative to cetyl palmitate, mono-, di- and tri-glycerides such as glyceryl monostearate, soy lecithin, tripalmitine, docosanoic acid, stearic acid can be used to form the lipid core.100 mg of cetyl palmitate is melted in a water bath at 65°C. Then, to this organic phase is added 5 mg of HBA-SA conjugate dissolved in 250 pL of chloroform. HBA- SA is added to solid lipid nanoparticle structure for MCT-1 targeting.

In a separate place, an aqueous solution of polysorbate 80 is prepared so that the final concentration is 2% (w/w) and the solution is heated in a water bath to a temperature of 65°C. As an alternative to polysorbate 80, polysorbate 20 can also be used, but better encapsulation efficiency is obtained with polysorbate 80. The aqueous phase is added to the lipid phase at the same temperature and homogenized for 2 minutes at 22000 rpm by means of a high speed blender (Ultraturrax, ISOLAB) to obtain a pre-emulsion. This pre-emulsion is continued to be homogenized for 1 minute in the sonic bath that reaches 65 °C. The emulsion obtained at the end of the period is rapidly cooled to 4°C and then the temperature is increased to 25°C. The mixture is centrifuged at 12000 rpm and the pellet is collected by separating the supernatant.

Synthesis of Solid Lipid Nanoparticles Containing Temozolomide and Carmustine

100 mg of cetyl palmitate is dissolved with temozolomide and carmustine at concentrations of 0,1%. After the addition of conjugate dissolved in chloroform, the polysorbate 80 solution is also added to the system. The mixture homogenized by means of Ultraturrax and sonic bath is rapidly cooled to 4°C and the temperature is increased to 25°C. The mixture is centrifuged at 12000 rpm and the pellet is collected by separating the supernatant. Drug amounts in supernatants are analyzed using HPLC and characterization studies are performed in the pellet. The solid lipid nanoparticle containing BCNU and TMZ obtained in the invention is synthesized by mixing all the chemicals mentioned above.

In vitro Drug Release

In vitro release studies are performed with MWCO 14.000 dialysis membrane (Sigma Aldrich) at a temperature of 37°C by using modified Franz diffusion cell (Logan Instruments Corp., FDC-6T). Solid lipid nanoparticles containing BCNU and TMZ at a concentration of 3,73 mg/mL in a total volume of 300 pL are placed in the donor compartment and receptor compartment is individually filled with 10 mM phosphate buffer at pH 7,4. At different time intervals for 72 hours, 5 mL of buffer from the receptor compartment was withdrawn and fresh 5 mL of buffer is added each time to maintain constant volume. The amount of drug in samples taken from the receptor cell is determined by BCNU and TMZ HPLC method. Determination of Biocompatibility of Solid Lipid Nanoparticles Containing a Dual Drug System

The amount of binding of solid lipid nanoparticles containing a dual drug system to serum proteins is analyzed using the methods explained by Cole et al. 2010 and Semete et al. 2011. Solid lipid nanoparticles containing a dual drug system attached with b-hydroxybutyrate ligand and drug-free solid lipid nanoparticles (0,1%) are dispersed in PBS. Since PBS is a physiological phosphate buffer, it is used to create minimum irritation during drug administration to live animals. Nanoparticles are added to fetal bovine serum at a serum: nanoparticle volume ratio (v: v) of 10:90, 20:80, 40:60, 60:40, respectively, and a total volume of 600 pL. The samples are left to incubate at 37°C for 2 hours at 160 rpm and after incubation the samples are centrifuged at 13000 rpm for 40 minutes. After centrifugation, protein was determined in the supernatant according to the Bradford method (Bradford, 1976) and the amount of protein bound to mg drug carrier and % binding values were calculated.

The damage that can be caused by solid lipid nanoparticles containing dual drugs on erythrocytes is determined by in vitro hemolysis studies and the methods applied by Mayer et al., 2009 and Yallapu et al. 2015 are used for the method. Accordingly, erythrocytes are separated from whole blood, washed with PBS and suspended in PBS to a final concentration of 2%. dual drug-containing and drug-free nanoparticles are dispersed in PBS at concentrations of 0,5; 0,1; 0,05; 0,01 mg nanoparticles/mL. The nanoparticle and erythrocyte suspension are mixed in a ratio of 1 : 1 (v.v) and incubated at 160 rpm for 2 hours at 37°C. PBS is used as negative control and 1% Triton X-100 solution is used as positive control. At the end of the period, the samples are centrifuged at 1000 rpm and the cells are separated from the medium, and then the nanoparticles are removed by centrifugation at 13000 rpm for 40 minutes. Hemoglobin is determined at 540 nm in supernatants.

Stability Studies

In order to investigate the stability of solid lipid nanoparticle formulations, properties such as physical appearance, average particle size, zeta potential, polydispersity index of the formulations are examined. In addition, the chemical stability of the active substance in the nanoparticle is also investigated.

Stability studies of the formulations are carried out in accordance with the stability guide at 5±3°C, 25±2°C 60±5% relative humidity and at 40±2°C at 75±5% relative humidity. In the stability study, samples are controlled for 12 months at t=0, i.e. at the initial moment, at months 1, 2, 3, 6, 9 and 12. The formulations are taken for stability study in colored glass vials and the stability of the nanoparticles is evaluated. The quantity of active substance was assayed by HPLC.

Cell Culture Studies of Solid Lipid Nanoparticles Containing Dual Drug

Cells are set to an initial density of 5xl0³ cells/100 pL in 96-well plates and are incubated to adhere for 24 hours before sample groups (free TMZ, free BCNU, mixture of free TMZ and BCNU, SLN containing BCNU and TMZ, SLN containing BCNU and SLN containing TMZ) are added. Dosage ranges of free temozolomide, free carmustine and carmustine- temozolomide combination are prepared as 1,563-100 pg/mL. The dose range of SLN containing carmustine is 119-1900 pg/mL, the dose range of SLN containing Temozolomide is 124-3975 pg/mL, and the dose range of SLN containing carmustine and temozolomide is 87,5-2800pg/mL. In cell culture studies, it was observed that the dose range of 400-1400 pg/mL of solid lipid nanoparticles containing BCNU and TMZ was more effective.

After the 24, 48 and 72 hours of incubations after the application of the drug groups in the appropriate dose range to the cells, the media containing the drug is removed from the wells and 90 pL of FBS-free fresh media is added instead. 10 pL of MTT (5 mg/mL in sterile PBS- 7.4) solution is added and incubated for 4 hours at 37°C. After the incubation period, formazan crystals formed by discarding the supernatant containing MTT are dissolved in 100 pL of DMSO. Quantitative measurement is evaluated colorimetrically by measuring at 540 nm wavelength in a microplate reader (Polarstar Omega) at the end of 15 minutes. The results are compared by determining IC50 values of the drug groups on the cells.

Cell acquisition studies

Brain endothelial cells, bEnd.3 cells, expressing the MCT-1 receptor are plated into a 6-well plate at 76000 cells per well and incubated for 24 hours to adhere and grow. The conditions of 5% C0₂ and temperature of 37°C are provided for the growth of cells. At the end of the period, diluted free drugs and solid lipid nanoparticles carrying dual drug and solid lipid nanoparticles carrying dual drug without target molecule (conjugate) are added to the wells whose supernatant has been removed with 2 mL. Drug groups containing 154 pg of TMZ and 126 pg of BCNU are applied to the cells. Drug-free cells are considered a control. Cells are incubated at 5% CO2 and 37°C conditions. The supernatant of the cells is removed after 2, 4, 6 and 16 hours for all three substances following drug administration. After washing with cold PBS four times, the cells removed with the cell scraper are centrifuged at 1200 rpm and the supernatant is removed. For cell lysis, 200 pL 0.5% (v/v) Triton X-100 and 200 pL water mixture is added to the cells. After 10 minutes of incubation at room temperature, it is centrifuged at 14000 rpm to extract the cell contents into the aqueous phase. Carmustine and temozolomide in the collected supernatants are determined by HPLC.

Apoptosis tests in cancer cell lines

In apoptosis tests in cancer cell lines, the apoptotic effects of solid lipid nanoparticles containing TMZ and BCNU, a mixture of free TMZ and BCNU and control groups on the U87MG cell line are determined by two separate methods (Annexin V and ApoDirect). Nanoparticles and free drug mixture are applied taking into account the IC₅₀ dose found in the cytotoxicity analysis. Accordingly, the nanoparticles containing BCNU and TMZ are used by being diluted at a concentration of 100, 300, 500 pg/mL, and the mixture of free BCNU and TMZ at a concentration of 2,5, 7,5, 12,5 pg/mL in EMEM medium, which is a cell growth medium. Drug-free cells are considered a control. Apoptosis analysis is performed with 2 different methods.

1) Annexin V Method: The protocol of Annexin V-FITC method is as follows:

1. U87MG cells are expected to induce apoptosis for 48 hours.

2. Cells (5x105) are collected by centrifugation.

3. In the next step, the cells are resuspended with 400 pi of IX Binding Buffer.

4. Then 5 mΐ of Annexin V-FITC and 5 mΐ Propidium Iodide is added.

5. In the last step, samples are kept for 15 minutes at room temperature in the dark.

6. Analysis is performed by flow cytometry.

2) APO-DIRECT™ Kit:

The protocol of APO-DIRECT™ Method is stated below:

1. U87MG cells are expected to induce apoptosis for 48 hours. 2. Cells are collected by centrifugation at 2000 rpm for 5 minutes.

3. 1 ml of PBS is added onto the cell pellet and resuspended.

4. Cells are centrifuged at 2000 rpm for 5 minutes and the supernatant is removed.

5. 990 pL of PBS and 10 pL of formaldehyde are added to the cell pellet at room temperature and incubated for 30 minutes.

6. Cells are centrifuged at 2000 rpm for 5 minutes and the supernatant will be removed. It is resuspended with 1 ml of PBS.

7. 300 pL of PBS and 700 pL of ethanol (-20°C) are added to the cell pellets.

8. Cells are centrifuged at 2000 rpm for 5 minutes and the supernatant is removed.

9. 1000 pL of wash buffer is added to the cell pellets. The supernatant is removed by centrifugation at 2000 rpm for 5 minutes. This process is repeated 2 times.

10. DNA labeling solution is added on the cell pellets and incubated for 60 minutes at room temperature.

11. After adding 1000 pL of rinse buffer, it is centrifuged at 2000 rpm for 5 minutes and the supernatant is removed. This process is repeated 2 times.

12. 500 pL of PI/RNAase staining buffer is added and incubated for 30 minutes at room temperature.

13. Analysis is performed by flow cytometry.

Biodistribution and Pharmacokinetic Studies

Biodistribution studies are carried out in 2 separate stages. While healthy CD-I mice are used in the first group study, in the second stage, studies are carried out with nude CD-I mice that have been created with glioblastoma model. First, the prepared drug loaded SLNs are administered intranasally to CD-I mice. At 1 and 3 hours (n=3) after the administration, mice are sacrificed and heart, liver, spleen, kidney, brain and blood samples are collected. The received organs are homogenized. After homogenization processes, the biodistribution of the drug is determined by performing TMZ and BCNU analysis with HPLC method on all organs and blood samples. Second, FITC marked solid lipid nanoparticles are intranasally applied to nude mice carrying glioblastoma. For comparison purposes, 3 nude mice are given FITC by only intranasal application. After the application, images are taken with the IVIS imaging device in 1 and 3 hours (n=3). Then, the brains of sacrificed mice are removed and the organs are imaged with the IVIS device.

For pharmacokinetic studies, samples of heart, liver, spleen, kidney, brain tissues and feces taken from mice placed in metabolism cages and sacrificed at the end of certain periods are homogenized. The determination of drugs in homogenates is carried out by HPLC method. In addition, drug analyzes are carried out by HPLC method on plasma samples obtained from blood taken into tubes with EDTA. In addition, drug analysis is performed on urine samples collected in metabolism cages.

Toxicology

For single dose acute toxicity, the highest dose of the drug delivery system, 500 pg/mL, is administered intranasally to 1 male and 1 female CD-I mouse and the mice are observed for 48 hours. As it was observed that the mice did not die as a result of the experiment, the substance was accepted as non-toxic and repeated dose toxicity application was started. In the repeated dose (Subacute) toxicity study, 6-8 weeks old (20-25 g) female and male CD-I mice are used. Solid lipid nanoparticles carrying BCNU and TMZ are distributed in 3 separate doses; 100 pg/mL, 300 pg/mL and 500 pg/mL in 10 mM pH=7,4 phosphate buffer. Dose groups (10 mice in each group; 5 males and 5 females) are administered intranasally for 4 weeks, in 3 replicates per week. The mice are weighed every week during the application and their situations are monitored. When the study is completed (at the end of 4 weeks), the mice are sacrificed and their blood is taken into lithium heparin tubes and blood parameters (such as ALB, ALT, GLU, TP, BUN) are examined on the Vetscan VS2 device using the Comphrehensive Diagnostic Profile rotor. Toxicological evaluation is made using all the data obtained.

Implantation of U87MG cells and formation of a glioblastoma model

In order to create an orthotopic model, nude mice placed in a stereotactic frame were given 5 pL and 2x105 U87MG cells suspended in nutrient medium (Geletneky et ah, 2010). First, mice are anesthetized by applying 3% isofluorane anesthesia (60-80 seconds). For this purpose, the mice are firstly placed in the chamber, and then in 1,5% isofluorane anesthesia by masking and fixed to the stereotactic frame. A small amount of ophthalmic ointment is then applied to the mice's cornea to keep their eyes moist, and the effective antibiotic is administered subcutaneously to the mice for 2 weeks. Then, the operation area is first cleaned with alcohol and then povidone. The operation area is exposed by incision with scissors and blunt-tipped tissue forceps. The area to be operated is adjusted to be 1 mm anterior and 2 mm lateral of the bregma.In the skull, a hole is drilled by means of a 0.6 mm tip and a drill to create a hole with a lateral diameter of 2 mm. 5 pL of cell suspension is injected into 1-3 mm depth within 1 minute using a 10 pL Hamilton glass injector. The needle is moved very slowly as the tip is withdrawn. Haemostat (oxidized regenerated cellulose) is applied to the area where the cells are injected, and after the operation area is closed with 4.0 vicryl suture, the area is dressed with antibiotic cream. Body temperatures of the treated mice are measured with a digital non-contact thermometer. The mice taken for postoperative care are observed until they come out of anesthesia and liquid analgesic is added to their drinking water. From the first day of cell implantation, it is monitored for the purpose of following daily activities and general conditions. Regular dressings are made, body weight and body temperature are measured.

Monitoring the treatment process

Tumor formation is analyzed at 7-10 days following cell implantation of tumor-induced mice with the orthotopic model. Tumor size is examined using the luciferin substrate in an IVIS (Caliper Perkin Elmer) imaging device, utilizing the luciferase activity of U87MG tumor cells. Since the best image in IVIS imaging is obtained with the BIN 8, FI device setting, this setting is used throughout the studies. Luciferin solution prepared in 200 pL 12 mg/mL water is given subcutaneously from the neck of the mice, and 5 minutes after the application, IVIS imaging is performed under isofluorane anesthesia. Tumor size is calculated with the formula 0.5x (Width)2xLength. Mice with tumor size over 200 mm3 are randomly grouped as 5-7 mice in each group. The details of the groups and the dose of intranasal therapy are as follows:

1. Control group: 20 pL of PBS is applied 3 times a week.

2. Low-dose nanoparticles group: Solid lipid nanoparticles prepared in PBS and at concentration of 300 pg/mL are applied 3 times a week at 20 pL.

3. High-dose nanoparticles group: Solid lipid nanoparticles prepared in PBS and at concentration of 1000 pg/mL are applied 3 times a week at 20 pL. 4. Free drug group: Free drug mixture containing 35,33 pg/mL BCNU and 30,53 pg/mL TMZ prepared in PBS is applied 3 times a week at 20 pL.

At the end of the treatment period, the cardiac blood of the mice sacrificed under anesthesia is first collected and then the brain tissues are collected for ex vivo studies. In order to determine blood parameters such as ALB, ALT, GLU, TP, BUN in blood samples taken into tubes with lithium heparin, analyzes are performed using the Comphrehensive Diagnostic Profile rotor in the Vetscan VS2 device.

Ex vivo studies and Pathological evaluation

Brain tissues are examined in gross after fixing in 10% formalin between 24-48. Serial sections are applied and the entire tissue is processed in an automatic tissue tracking device to create formalin-fixed paraffin-embedded tissue. 4 micron sections are taken on positively charged slides and stained with Hematoxylin eosin and Ki67 (Dako- MIB1) using Ventana automatic Stainer. All tissues are evaluated by a single neuropathologist under a light microscope, without the sample source known (blind). Slides were scanned with 3DHISTECH Pannoramic P250- Flash III Slide Scanner. Ki67 ratio is determined by "Cell- Quant" method in "Digital Quant Center" module in 3DHISTECH-CaseViewer. In addition, the longest diameter of the tumor, the longest cross-sectional length of the specimen, the widest area of necrosis are measured in 3DHISTECH-CaseViewer. Ki67 is an antibody that displays proliferating cells within the tumor. Ki67 ratio is known as a poor prognostic factor. Therefore, it is very important to determine this ratio in treatment groups.

As shown in Figure 1, the N-H stretching peaks belonging to the primary amine group in the structure of stearyl amine are seen at 3331 cm ¹ and 3251 cm ¹. After the conjugate formation, the primary amine group is expected to be replaced by the secondary amine group. The broad and single peak observed between 3325 cm ¹ and 3215 cm ¹ is the N-H stress peak belonging to the secondary amine group. In addition, it has been stated that the peak seen at —1550 cm ¹ in the literature data is the N-H bending peak of the secondary amine group, which is expected to be in the HBA-SA conjugate (Venishetty et ak, 2013). In addition, N-H bending peak of the primary amine group is observed at 1606 cm-1 in the spectrum of stearyl amine. In both spectra of conjugate and stearyl amine, C-C (sp3) asymmetric bending peak at 1462 cm ¹ and C-H stretch peaks at 2916 and 2848 cm ¹ belonging to the SA structure are observed. The findings are compatible with the literature data (Varshosaz et ak, 2012). As shown in Figure 2, FTIR spectrum of HBA is parallel to FTIR spectrum of poly beta- hydroxy butyrate in the study conducted by Ramezani (2015). It is the O-H stress peak of the flat peak carboxylic acid observed at 3400 cm ¹ and 3100 cm ¹. This peak overlapped with the N-H stress peak in the spectrum of the conjugate. Peaks seen at 1404 cm ¹ and 1379 cm ¹ indicate C-H bending. In addition, the peak seen at 1288 cm ¹ is the C-0 stress peak and is consistent with the literature data (Ramezani et ak, 2015). In addition, the C=0 stress peak in the spectrum of HBA is observed at 1700 cm ¹ and belongs to the carboxyl group. The C=0 stress in the conjugate is expected to result from the amide group as a result of amide bond formation, and this peak amide shifted to 1633 cm ¹ as seen in the spectrum. This confirms that amide bond formation has taken place. Results were found compatible with the literature data (Venishetty et ak, 2013).

According to Figure 3, according to the H-NMR spectrum of the conjugate, it is seen that the NH signal of the amide bond formed in accordance with the literature data (Venishetty et ak, 2013) is at 5H 7,27 and the CH group containing the hydroxyl group is at 5H 4,20. FTIR and NMR results confirm that the conjugate has been achieved successfully.

In Figure 4, SEM images of the particles are given and the size of the nanoparticles was observed around 120-150 nm and with a shape close to the sphere.

According to Figure 5, the C-H stretching peak seen at 2916 and 2848 cm ¹ in Cetyl palmitate, conjugate and polysorbate 80 structures was also observed in SLN structure. C=0 stretch band at 1734 cm ¹ determined in the spectra of cetyl palmitate and polysorbate 80 containing ester groups are also found in the SLN spectrum. The peak of the conjugate containing amide bond carbonyl at 1633 cm ¹ was also detected in the SLN spectrum. The C- O stretch signal in the range of 1090-1300 cm ¹ was seen in SLN structure as well as cetyl palmitate, conjugate and polysorbate 80. In addition, the C-H bending peak at 1462 cm ¹ is found in spectra of cetyl palmitate, conjugate, polysorbate 80 components and SLN. According to the obtained results, it can be said that the starting components are included in the nanoparticle structure.

According to the thermogram curve seen in Figure 6, no weight loss was observed in the structure up to approximately 200°C. It was found that the structure was completely degraded at approximately 360°C and the maximum degradation temperature was 362°C according to DTGA data. As shown in Figure 7, it is known that the size of the aqueous form of nanoparticles can vary according to the dry form. The hydrodynamic diameter of SLNs prepared under optimum conditions was found to be 220.9±35,73 nm and the zeta potential as -7,55 mV (Figure 10.) In the findings obtained by Lockman et al. (2004), it has been observed that negatively charged nanoparticles cross the blood-brain barrier faster at high concentrations and show more effect in this region. It is stated in the literature that it is possible to cross the blood brain barrier (BBB) with nanoparticles with an average diameter of 100-400 nm. In the study, it was observed that liposomes with a particle size of 40-80 nm could not pass the BBB, but nanoparticles with a larger particle size and covered with materials such as polysorbate 80 could pass the BBB ( etin and apan, 2004).

According to Figure 8, the retention time (rt) of the sample is 3.147.

According to Figure 9, the retention time (rt) of the sample is 4.434.

Table 1. Drug amounts in SLN content (pg drug/mg SLN) and drug loading efficiency (%).

As shown in Table 1 in Figure 10, there are hydrodynamic diameter and zeta potential data of SLNs loaded with drugs at a concentration of 0,1% by weight (pg drug/mg SLN). Blank SLNs and SLNs containing 0,1% drug were observed to be very close in size and anionic in potential. In another study conducted by Pandya et al. (2018), it has been shown that the zeta potentials of produced SLNs are negative and this negative charge will increase the stability of the drug delivery system. In another study conducted by Kumar et al. (2018), the hydrodynamic size of the particles was determined as 323.35±24.56 nm.This situation showed that the size of the obtained nanoparticles was in accordance with the literature data.

According to Figure 11, it has been determined that SLNs have a spherical/ellipsoidal shape and a smooth surface. The hydrodynamic diameter of SLNs containing drugs at a concentration of 0,1% by weight (pg drug/mg SLN) was determined as 227±46,80 nm, and the dimensions of the dry form measured by SEM were approximately 170-180 nm. Similar to the obtained results, in the study conducted by Padya et al. (2018), it has been shown that SLNs differ from their dry dimensions measured by SEM and hydrodynamic diameter measured with zeta sizer in aqueous form. In the study, the size of SLNs loaded with olmesartan medoxomil was approximately 131 nm, while the hydrodynamic dimension was observed as 152.40±2.92 nm.

According to Figure 12, the FTIR spectrum of the nanoparticles loaded with temozolomide and carmustine is similar to the spectrum of non-drug loaded, empty nanoparticles and the peaks coincide with each other. It is thought that carbonyl stretch, C-H bending, C-0 stretching signals increase due to the structure of drugs.

According to Figure 13, weight loss was not observed up to approximately 200°C in the nanoparticle structure carrying dual drugs as in the empty nanoparticles. It was found that the structure was completely degraded at 360°C and the maximum degradation temperature was 367°C according to DTGA data. It is estimated that this value has shifted by 5°C compared to the empty nanoparticles and this is due to the drugs added to the structure. As with empty nanoparticles, it is believed that solid lipid nanoparticles containing drugs will retain their properties under operating conditions.

According to the data obtained according to Figure 14, temozolomide was released at the rate of 9,78% and 18,4%, and carmustine at 9,23% and 28,1% in 1 and 5 hours, respectively. At the end of 25 hours, the release percentages are 19,82 for temozolomide and 30,22 for carmustine. It is observed that the release of drugs from solid lipid nanoparticles begins rapidly in the first 5 hours and continues in a controlled manner. As is known, nanoparticles can provide slow and controlled release of active ingredients.

As shown in Figure 15, it is observed that the release of drugs from solid lipid nanoparticles begins rapidly in the first 6 hours and continues in a controlled manner. As is known, nanoparticles can provide slow and controlled release of active ingredients. At the end of the 72 hour release experiment, a release from solid lipid nanoparticles was 26,71% for BCNU and 10,29% for TMZ.

Protein binding amounts and binding percentages are given in Table 2. As can be seen from the table, the amount of protein bound to the structures of the empty nanoparticles and the nanoparticles containing the drug mixed in the ratios of 10:90 and 20:80 was not found. In systems prepared to contain more serum, the percentage of bound protein is approximately the same for empty and drug-loaded nanoparticles and ranges from 21-28%. Table 2. The amount and percentage of nanoparticles binding to serum proteins.

Hemolysis testing was performed with positive control, negative control, SLN samples containing drug at a concentration of 0,5; 0,1; 0,05; 0,01 mg/mL, drug-free SLN samples at a concentration of 0,5; 0,1; 0,05; 0,01 mg/mL and it was found that nanoparticles at varying concentrations did not cause any hemolysis. It is an expected result that the used empty and drug-loaded nanoparticles, considering they are loaded, do not create hemolysis. When looking at all the protein binding and hemolysis results, it can be said that nanoparticles are biocompatible.

Cytotoxicities of BCNU-SLN, TMZ-SLN and BCNU-TMZ-SLN groups on U87MG cell are shown in Figures 16 and 17 for 48 and 72 hours, respectively. It was determined that more cytotoxicity occurred with increasing drug concentration of all drug groups. The highest lethal effect in the drug-containing solid lipid particle group was achieved in the solid lipid particle containing BCNU-TMZ.

Table 3. IC50 values of free drug groups and drug containing nanoparticles after 48 and 72 hours incubation on U87MG cells.

When looking at the effect of drug containing nanoparticles on viability, the toxic effect of nanoparticles was observed at higher doses than free drugs due to the slow release of drugs from nanoparticles. When the effects of solid lipid nanoparticles containing drugs on vitality are listed, it can be expressed as BCNU-TMZ-SLN> BCNU-SLN> TMZ-SLN. As in free drug trials, nanoparticles containing only carmustine at the same dose create more toxicity than nanoparticles containing only temozolomide. Nanoparticles containing both carmustine and temozolomide have a much higher toxicity. IC₅₀ value of the solid lipid nanoparticle containing dual drug was calculated separately for both drug types. IC50 values of the solid lipid nanoparticle containing dual drug in terms of carmustine in 48 and 72 hours were 36,23 mM±4,65 and 40,16 mM±3,23, respectively; IC50 values for temozolomide in 48 and 72 hours were calculated as 57,64 mM±7,40 and 63,90 mM±5,13, respectively. bEnd.3 healthy brain cells and dual drug loaded nanoparticles have been found to have more than 90% viability in cells even at the highest drug doses. In the free drug combination, it was determined that the viability decreases to 74-82% at concentrations of 50 and 100 pg/mL, respectively. When the cancer cell line and healthy cell line data were compared, it was determined that dual drug loaded nanoparticles showed cytotoxicity in U87MG cells, but did not cause toxicity in bEnd.3 cells. These results suggest that although nanoparticles may cause minimal damage to healthy endothelial cells, they may inhibit cell growth and show anticancer activity in glioblastoma cells and support the main goal of our project.

According to the results of cell uptake studies, drug amounts could not be determined in cell samples 16 hours ago, and the chromatograms obtained after 16 hours of incubation are given in Figure 18. As can be seen from the figure, no temozolomide peak was found in the control group cell that was not treated with drug. According to the obtained results, the intake percentage of bEnd.3 cells is 0,664%, 0,394% and 0,186% for conjugate loaded solid lipid nanoparticles containing dual drug, non-conjugate loaded solid lipid nanoparticles containing dual drug and free drug mixture, respectively. The penetration of nanoparticles containing conjugates specifically to MCT-1 carrier is 1.68 times higher than non-conjugate-carrying nanoparticles and 3,57 times higher than free drug. These results obtained on the bEnd.3 model cell show us that the nanoparticles can enter the MCT-1 expressing cells specifically to the carrier.

According to our apoptosis results; apoptosis rates of cells treated with free drug (TMZ+BCNU Combination) (2.5-7.5-12.5 pg/ml) and SLN (100-300-500 pg/ml) are 1%, 1.4% and 1.9%, respectively, in free drug, while they were determined as 1%, 1.7% and 2.3% as a result of SLN application. As a result, when the apoptotic rates of cells treated with free drug and nanoparticle bound drug were compared, it was observed that they increased in a concentration-dependent manner. As a result of treatment with nanoparticle-bound drug, it was determined that the apoptosis rates of cells increased 21.4 and 26.3 times with the increase in concentration compared to free drug. Accordingly, it is thought that the use of drug delivery systems in drug treatment methods will reduce the stress on the cell by administering drugs to the medium more slowly, leading to apoptosis, which is a more controlled form of cell death, and provides an effective treatment.

According to the Apodirect In Situ DNA fragmentation method, the apoptosis rates of cells treated with free drug (TMZ+BCNU Combination) and SLN were determined as 2.1%, 3.2%, 4.7% and 1.1%, 2.2% and 3.5%, respectively. According to this method, it was found that apoptosis rates are increased in increasing concentrations, but the apoptosis rates of cells treated with the free drug combination were higher than the nanoparticle-bound drug. As a result of the treatment with nanoparticle-bound drug, it was determined that the apoptosis rates of the cells increased with the increase in concentration compared to the free drug. Since both early and late apoptosis rates were shown in the Apodirect In Situ DNA fragmentation method, it was observed that the apoptosis rates of the cells treated with the free drug combination were higher than the nanoparticle-bound drug.

Table 4. Zero time-measurement results of nanoparticles.

Table 5. 3rd Month measurement results of nanoparticles.

Table 6. 6'th Month measurement results of nanoparticles.

Table 7. 9th Month measurement results of nanoparticles.

Table 8. 12th Month measurement results of nanoparticles.

Table 9. Biodistribution results

The biodistribution results are shown in table 9. While creating the table values, the average of the data of 3 mice belonging to each period and organ was calculated. When the table data was examined, the drug could not be determined by the method applied in spleen, heart and lung samples. It can be said that there is no accumulation of solid lipid nanoparticles containing drugs in these organs. However, accumulation is seen when drug analysis is performed in the brain and kidney.

Table 9. % Distributions for time dependent TMZ in the samples.

Based on the TMZ determination, when the average % distribution data of solid lipid nanoparticles administered intranasally to mice were examined (Table 9), it was seen that there was approximately 49% accumulation in the brain, and the kidney was the organ where the most drug was deposited. When the literature data are examined, it has been stated that the TMZ plasma half-life is approximately 1.8 hours and its elimination occurs mainly in the kidneys (Portnow I, et ah, 2009).

Table 10. % Distributions for time dependent TMZ in the samples.

Based on the BCNU determination, when the average % distribution data of solid lipid nanoparticles administered intranasally to mice were examined (Table 10), it was seen that there was approximately 60% accumulation in the brain, and the brain and then the kidney were found to be the organ where the most drug was deposited. Both drugs could not be detected in HPLC determination in spleen, heart and lung samples, and it can be said that the drugs and therefore the solid lipid nanoparticle did not distribute to these organs.

In order to fully evaluate the biodistribution study, a trial was also conducted in nude mice for which a glioblastoma model was created. 20 pL of FITC loaded solid lipid nanoparticles were administered to 3 nude mice with cancer, and FITC was given intranasally to 3 nude mice. The image detected at the end of the 3 hours in nude mice is shown in Figure 19. In addition, in order to determine whether conjugated solid lipid nanoparticles allow for targeted therapy, only FITC was given to 3 nude mice with cancer by nasal application and FITC-induced radiation was determined by IVIS imaging. The Nude mice treated intranasally were sacrificed at the end of 3 hours, their brain tissues were removed and visualized by IVIS. The accumulation of nanoparticles in nude mouse brains after 3 hours is seen in Figure 20. As seen in Figure 20, when only FITC was given to nude mice, the spread was seen in the whole body, while the spread of the FITC loaded solid lipid nanoparticle was detected in the brain and lung.

Table 11. Pharmacokinetic parameters

The pharmacokinetic parameters of TMZ and BCNU active substances were determined by drawing, time-dependent plasma concentration graphs with the specified program (Table 11). Plasma concentration graph for TMZ is given in Figure 21 and for BCNU in Figure 22.

In single-dose (acute dose) toxicity studies, it was observed that there was no acute toxic effect at a concentration of 500 pg/mL. During the 28-day repeated dose toxicology studies, all mice were regularly weighed every week. In Figure 23, the 28-day subacute toxicity weight change graph is given. In addition, in Table 12, the difference in average mouse weights for each dose group at the beginning of the trial and at the end of the trial is given. It is obvious that there was no weight loss in the three drug group and control group mice, and there was no weight gain. Table 12: Toxicology studies control and dose groups mean weight difference

Table 13: Average Biochemistry Values in Toxicology Studies.

According to Table 13, when biochemistry parameters were examined, no significant difference was observed in terms of intranasal application compared to the healthy control group. There was no significant difference in ALT enzyme activity in healthy mice when the dose groups were compared. While ALB level decreases in liver diseases, GLOB protein level is known to increase in liver diseases. In a study by Serfilippi L.M. et al. (2003), ALT enzyme activity was determined as 25-76 U/L, ALB 3,0-4, lg/dL and GLOB 1,8-2, 3 g/dL. Considering these reference values, it can be said that liver damage was not encountered in mice in the dose group 3.

In the invention, Na+, K+, BUN and CRE values were examined and whether the mice had impaired renal function were compared by comparing the dose groups with healthy mice. Since no significant difference was observed in the trial, it can be said that nephrotoxicity, which is a side effect of carmustine, did not occur. BCNU-TMZ-SLN structure synthesized as a result of repeated dose toxicity studies was found to have no toxic effect.

Table 14. Cancer control group tumor sizes

Nude mice whose tumor size exceeds 2000 mm³ within the framework of ethical rules are excluded from the experiment. The * sign in the table indicates that sacrification was done to be excluded from the trial. As seen in Table 14 and Figure 24, tumor size of control nude mice with cancer in the experimental group given intranasal 20 pL of PBS 3 times a week increased very rapidly and some nude mice were found dead in their cages during the experiment.

A two-stage treatment protocol was created to determine the cancer treatment potential of solid lipid nanoparticles containing dual drugs. Initially, solid lipid nanoparticles prepared in PBS and at a concentration of 300 pg/mL were administered intranasally to 5 nude mice with cancer, 3 times a week, and survival was monitored and tumor sizes were calculated by IVIS imaging at certain intervals. In Table 15, nude mouse tumor sizes given low dose solid lipid nanoparticles are shown. In Figure 25, IVIS images are given. Table 15. Low-dose solid lipid nanoparticle group.

As seen in Table 15 and Figure 25, it was determined that the application of solid lipid nanoparticles at a concentration of 300 pg/mL was not effective in the treatment of glioblastoma. For this reason, a new set of experiments has been established using higher doses of nanoparticles in intranasal application.

Table 16. High-dose solid lipid nanoparticle group.

When Table 16 values are examined, it can be said that solid lipid nanoparticles are effective in cancer treatment, except for 1 nude mouse. Applying a solid lipid nanoparticle at a concentration of 1000 pg/mL appears to reduce the size of the tumor and even completely eliminate the tumor in the nude mouse 7 (Figure 26). Considering that glioblastoma is an aggressive tumor and spreads in a very short time, the conjugated solid lipid nanoparticle containing dual drug prepared in the treatment of glioblastoma is promising.

As a result of the high dose solid lipid nanoparticle found to be effective in the treatment of glioblastoma, the free drug containing the mixture of drugs trapped in the solid lipid nanoparticle at the specified concentration (35,33 pg/mL BCNU and 30,53 pg/mL TMZ) was administered to 7 nude mice with cancer by intranasal administration and tumor sizes and survival were monitored.

Table 17. Free drug group.

As seen in Table 17 and Figure 27, when the free drug mixture was given nasally to nude mice with cancer, other nude mice included in the group found dead in 4 mouse cages were excluded from the trial because of metastasis and tumor size over 2000 mm³. The free drug mixture was not effective in the treatment of glioblastoma and the subjects died in a short time.

The results of tumor size analysis of cancer control, free drug group, low-dose solid lipid nanoparticle group, and low-dose solid lipid nanoparticle groups were evaluated statistically using an independent sample t test.

It was investigated whether there is a difference between the low dose solid lipid nanoparticle group and the high dose solid lipid nanoparticle group, free drug group and cancer control group. According to the results, it was observed that the differences between the low dose solid lipid nanoparticle group, free drug (p=0.072>0.05) and cancer control group (p=0.147>0.05) were not statistically significant. In addition, it was observed that the difference between high-dose solid lipid nanoparticle group and low-dose solid lipid nanoparticle group was statistically significant.

It was investigated whether there is a difference between the high-dose solid lipid nanoparticle group and the free drug group and the cancer control group. According to the results, it was observed that the differences between the low dose solid lipid nanoparticle group, free drug (p=0.011<0.05) and cancer control group (p=0.008<0.05) were statistically significant. It was investigated whether there is a difference between the free drug group and the cancer control group. According to the results, it was seen that the difference between the free drug group and the cancer control group was not statistically significant. (p=0.58>0.05). Descriptive statistical parameters of free drug, cancer control, low dose solid lipid nanoparticle and high dose solid lipid nanoparticle groups were also calculated for the differences in tumor size depending on time.

Cardiac blood was drawn from mice that completed or failed to complete the treatment process, and whole blood analyzes were performed on a biochemistry analyzer (VetScan VS2) using a rotor (Comphrehensive Diagnostic Profile), and the results are given in Table 18.

Table 18. Data of biochemical analysis results on blood.

Biochemistry results of healthy control, cancer control groups, free drug group and solid lipid nanoparticle group were evaluated statistically using an independent sample t test.

While no statistically significant difference was observed between the biochemical parameters of TBIL (p= 0.423>0.05) and Cl (p= 0.16>0.05) in the free drug group and in the solid lipid nanoparticle group, the statistical differences between other biochemical parameters (ALB, ALP, CA, TP, GLOB, BUN, ALT, GLU, NA, K CRE) are significant. This indicates that free drug in particular can cause tissue damage. For all biochemical parameters (ALB, ALP, CA, TP, GLOB, BUN, ALT, GLU, NA, K CRE, TBIL, Cl) between free drug group and cancer control groups, p<0.05 indicates that the difference between groups is statistically significant. The difference between the healthy control group and the group treated with free drug was statistically significant in all groups, except for the ALB (p=0.123) parameter (p<0.05).

While no statistically significant difference was observed between the biochemical parameters of TBIL (p= 0.423>0.05) and Cl (p= 0.16>0.05) in the free drug group and in the solid lipid nanoparticle group, the statistical differences between other biochemical parameters (ALB, ALP, CA, TP, GLOB, BUN, ALT, GLU, NA, K CRE) are significant. For all biochemical parameters (ALB, ALP, CA, TP, GLOB, BUN, ALT, GLU, NA, K CRE, TBIL, Cl) between cancer control group and the group treated with solid lipid nanoparticles, p<0.05 indicates that the difference between groups is statistically significant. The difference between the healthy control group and the group treated with solid lipid nanoparticle was statistically significant in all parameters except for the K parameter (p=0.904) (p<0.05).

For all biochemical parameters (ALB, ALP, CA, TP, GLOB, BUN, ALT, GLU, NA, K CRE, TBIL, Cl) between cancer control group and healthy control group, p<0.05 indicates that the difference between groups is statistically significant.

The ratio of tumor to the whole tissue calculated after the histopathological method is given in Table 19.

Table 19. Tumor/tissue ratio in the brain in nude mice

The values found for tumor/tissue ratio; Independent sample t test analysis results of the difference between free and control, low dose nano and control, high dose nano and control, free and low dose nano, free and high dose nano and low dose and high dose nano are shown in Table 20. The difference between control - high dose nano for tumor/tissue ratio is statistically significant (p=0.042<0.05). The difference between other groups is not significant (p>0.05).

Table 20. Independent sample t test results

Ki67 ratio in samples stained with Ki67 (Dako-MIBl) using hemotoxylin eosin and Ventana automatic staining device was determined by "Cell-Quant" method in "Digital Quant Center" module in 3DHISTECH-CaseViewer and the findings are given in Table 21. Table 21. Ki67 ratio

The values found for Ki67 ratio; Mann-Whitney U test of the difference between free and control, low dose nano and control, high dose nano and control, free and low dose nano, free and high dose nano and low dose and high dose nano was examined. According to the results, the difference between control and high dose nano groups was found to be statistically significant (p=0.017<0.05). The difference between other groups is not statistically significant (p>0.05). Mann-Whitney U test data are shown in Table 22.

Table 22. Mann-Whitney U test

Images selected from staining samples in ex-vivo studies are shown in Figures 28, 29, 30 and 31. Selected images of Ki67 staining index are shown in Figure 32. Glioblastoma are tumors that consist of astrocytic cells, have high mitotic activity and a parallel high Ki67 staining index, and often show signs of necrosis. They are characterized by the formation of a large mass in a short time because they have a very fast growth potential. The most important factors in evaluating the treatment response in glioblastoma are reduction in tumor size and decrease in Ki67 staining index. Since the presence of necrosis may reflect both the characteristics of the tumor and the response to treatment, it is not appropriate to use it as a treatment response criterion. The tumors created in our study are phenotypically identical to glioblastoma. When Ki67 staining index, which shows the growth rates of tumors, was evaluated, statistically significant lower results were found in the group treated with high- dose nanoparticles compared to the other groups. These results show that the tumor responds with conjugated solid lipid nanoparticle treatment.

In addition, when the ratio of the tumor size in the whole brain tissue is examined, it is seen that the size of the tumor is smaller than the whole brain size in animals treated with high- dose nanoparticles. Again, this result shows that a response is obtained and tumor growth rate is slowed in tumors treated with conjugated solid lipid nanoparticles.

Claims

1. A solid lipid nanoparticle (SLN) targeted to the monocarboxylate transporter 1 (MCT- 1) receptor, characterized by comprising the conjugate of Stearylamine (SA) and ketone bodies transported with the MCT-1 receptor and also carmustine (BCNU) and temozolomide (TMZ).

2. A solid lipid nanoparticle according to claim 1, characterized in that the ketone bodies transported with said MCT-1 receptor are b-hydroxybutyrate (HBA), lactate, acetoacetate or pyruvate.

3. A solid lipid nanoparticle according to claim 2, characterized in that the ketone bodies transported with the said MCT-1 receptor are b-hydroxybutyrate.

4. A solid lipid nanoparticle according to claim 1, characterized in that the lipid core of said solid lipid nanoparticle comprises cetyl palmitate, monoglyceride, diglyceride, or triglyceride.

5. A solid lipid nanoparticle according to claim 4, characterized in that the lipid core of said solid lipid nanoparticle comprises cetyl palmitate.

6. A solid lipid nanoparticle according to claim 1, characterized in that the surface of said solid lipid nanoparticle is coated with polysorbate.

7. A solid lipid nanoparticle according to claim 6, wherein said polysorbate is polysorbate 20 or polysorbate 80.

8. A solid lipid nanoparticle according to any of the preceding claims, characterized in that the sizes of dry form are 170-180 nm.

9. A solid lipid nanoparticle according to any of the preceding claims, comprising the carmustine (BCNU) and temozolomide (TMZ) drug at a concentration of 0.04-0.13% by weight (pg drug/mg SLN).

10. A solid lipid nanoparticle according to any of the preceding claims, characterized in that the solid lipid nanoparticle is used in the preparation of the drug to be used in, in vitro studies.

11. A solid lipid nanoparticle according to claim 10, characterized in that the dose range of said drug is 400-1400 pg/mL.

12. A solid lipid nanoparticle according to any of the preceding claims 1-9, characterized in that the solid lipid nanoparticle is used in the preparation of the drug to be used in the treatment of glioblastoma.

13. A solid lipid nanoparticle according to claim 12, characterized in that the administration route of said drug is intranasal.

14. A solid lipid nanoparticle according to claim 12, characterized in that the administration route of said drug is intravenous.

15. A production method of a solid lipid nanoparticle targeted to the monocarboxylate transporter 1 (MCT-1) receptor, comprising the process steps of: i. Selecting at least one of from group containing cetyl palmitate, monoglyceride, diglyceride and triglyceride, and adding the conjugate containing Stearylamine (SA) and ketone bodies transported with the MCT-1 receptor, ii. Synthesis of solid lipid nanoparticles by adding temozolomide, carmustine and polysorbate to the resulting mixture.

16. A production method of a solid lipid nanoparticle according to claim 15, comprising the process steps of: i. Adding stearylamine (SA) and conjugate containing ketone bodies transported by MCT-1 receptor on cetyl palmitate, ii. Synthesis of solid lipid nanoparticles by adding temozolomide, carmustine and polysorbate to the resulting mixture.

17. A production method of a solid lipid nanoparticle according to claim 15 or 16, characterized in that the ketone bodies transported with said MCT-1 receptor are b- hydroxybutyrate, lactate, acetoacetate or pyruvate.

18. A production method of a solid lipid nanoparticle according to claim 17, characterized in that the ketone bodies transported with said MCT-1 receptor are b-hydroxybutyrate.

5 19. A production method of a solid lipid nanoparticle according to claim 15 or 16, wherein said polysorbate is polysorbate 20 or polysorbate 80.