Use of mir-342 enriched, tamoxifen loaded, mesenchymal stem cell derived exosomes for overcoming tamoxifen resistance in breast cancer

Abstract

The invention relates to a pharmaceutical composition comprising tamoxifen-loaded mesenchymal stem cell-derived exosomes enriched with miR-342 for use in the pharmaceutical and vaccine industry, in the treatment of cancer, in the treatment of breast cancer, in overcoming tamoxifen resistance in the treatment of estrogen receptor positive breast cancer using tamoxifen.

Description

[0001] DESCRIPTION

[0002] Use of miR-342 Enriched, Tamoxifen Loaded, Mesenchymal Stem Cell Derived Exosomes for Overcoming Tamoxifen Resistance in Breast Cancer

[0003] Field of the Invention

[0004] The present invention relates to a new pharmaceutical composition that overcomes tamoxifen resistance in the treatment of breast cancer.

[0005] The invention relates in particular to a pharmaceutical composition comprising tamoxifen- loaded mesenchymal stem cell-derived exosomes enriched with miR-342 for use in the pharmaceutical and vaccine industry, in the treatment of cancer, in the treatment of breast cancer, in overcoming tamoxifen resistance in the treatment of estrogen receptor positive breast cancer using tamoxifen.

[0006] State of the Art

[0007] Breast cancer is a heterogeneous disease in which genetic and environmental factors play a combined role and treatment decisions and prognosis are traditionally guided by tumour size, tumour grade and nodal status, as well as immunohistochemical (IHC) markers such as ER, progesterone receptor (PR), human epidermal growth receptor 2 (HER2) and Ki67 (proliferation index, threshold 14%). Molecular breast cancer subtypes are classified as luminal A, luminal B, human epidermal growth factor receptor-2 (HER2), HER2 enriched, basal-like, normal breast-like and low claudin. In the treatment of ER+ breast cancer, endocrine therapy methods that block the activity of estrogen receptors, especially tamoxifen, have an important place. Tamoxifen ( Chemical Name: (Z)-2-[4-(1 ,2-Diphenyl-1- butenyl)phenoxy]-N,N-dimethylethanamine, IUPAC Systematic name: (Z)-2-[para-(1 ,2- Diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine), although it was the first selective estrogen receptor modulator used, it was developed for use in the treatment of breast cancer in the early 1970s. Tamoxifen blocks the effects of estrogen on breast cancer cells by binding to estrogen receptors. However, the development of resistance to treatment is an important problem encountered in clinical practice, and new therapeutic strategies need to be developed to overcome this resistance. Mesenchymal stem cells (MSCs) have a wide range of cellular and molecular functions and have significant potential in regenerative medicine and cancer therapy. Exosomes derived from MSCs play critical roles in intercellular communication and have the potential to be used as drug carrier systems, thanks to their capacity to carry various molecules. Therefore, exosomes can modify the behaviour of cancer cells, especially by transporting microRNAs such as miR-342 and drugs used in cancer therapy to target cells, and thus target treatment-resistant cell populations.

[0008] In recent years, there has been a significant increase in the use of therapeutic nanocarriers for drug delivery. Despite major advances in drug delivery systems, the clinical applicability of these cells is still an evolving field due to the immunogenicity of the recipient cells, rapid blood clearance, cytotoxicity of the carrier construct and poor bio-distribution. Liposomes, which are widely used and represent one of the most successful therapeutic nanocarriers, are a biocompatible carrier system containing hydrophobic and hydrophilic regions, capable of encapsulating DNA or RNA and surrounded by a phospholipid monolayer, nano-sized, capable of targeting specific regions, low toxicity. Under specific conditions, the liposome- nucleic acid complex is integrated into the cell membrane, taken into the cell by endocytosis and releases the nucleic acid in the cytoplasm. The main advantages of this method are that it enables transfection of different cell types with high efficiency and low cost, but this method is often criticized due to short blood circulation and inefficient intracellular drug delivery. To overcome the complications associated with synthetic nanocarriers, new endogenous delivery systems need to be investigated. Endogenous particles show greater compatibility and lower cytotoxicity, paving the way for the effective use of endogenous tools in vivo. Exosomes are intercellular cargo and communication systems secreted from cancer cells as well as normal cells, and whose functions have been discovered in recent years. While early studies showed that exosomes were only involved in the removal of wastes from the cell, recent studies have shown that the cargo contents of exosomes do not only consist of waste molecules, but also carry nucleic acids, proteins, miRNA, IncRNA, mRNA, nucleoproteins and various enzymes to be used in important roles such as intercellular communication and signal transduction. Exosomes; can be isolated from various body fluids, including tissue, plasma, serum, sperm, urine, saliva, breast milk, amniotic fluid, cerebrospinal fluid and bile, and is stable when stored at -20°C. The main reason why exosomes, which function as endogenous nanocarriers, have recently begun to be seen as a new drug delivery system, is that exosomes have a biocompatible structure due to their formation. These structures have the potential to reduce the negative effects of targeted drug applications due to their noncytotoxicity, low immunogenicity and ability to target specific cells. However, there are shortcomings in the widespread therapeutic applicability of exosomes due to difficulties in the isolation steps and deficiencies in drug loading techniques.

[0009] Current studies on genomic analysis have shown that drug resistance poses a significant problem in the development of complete response to treatment, considering the polygenic nature and tumor heterogeneity. For this reason, multiple gene or protein profiles provide a much more realistic approach to determining drug resistance compared to single biomarker determination. However, there is insufficient information to support the use of this information in the clinic. Tamoxifen is one of the most commonly used treatments for breast cancer. In most ER+ patients using tamoxifen, the response to treatment is high initially and decreases over time. Additionally, long-term and high-dose use of tamoxifen leads to the development of endometrial cancer and thromboembolic diseases. In addition, tamoxifen has side effects in patients such as nausea, vaginal dryness, irritability, headache, fever, concentration impairment and fatigue. Considering that one of the most serious consequences of long-term tamoxifen use is the occurrence of life-threatening cardiomyopathy, the protective effect of exosomal formulations by eliminating these side effects is a clinically significant innovation. In addition, delivering long-term treatment doses within exosomal structures is advantageous in terms of seeing high effects at low doses. Although successful results have been achieved with endocrine therapy in ER+ breast cancer patients today; there are limitations to this treatment due to de novo and acquired drug resistance. Therefore, it is of great importance to understand the molecular mechanisms that lead to the development of resistance in endocrine therapy.

[0010] The national patent application numbered TR2017 / 18258 is about phosphodiesterase 4D (PDE4D) modulators that enable the induction of cyclic adenosine monophosphate (cAMP), which can be used in the treatment of breast cancer and especially in patients who develop tamoxifen resistance. In the relevant document, unlike the present invention, the use of aspirin + tamoxifen, dipirimadol + tamoxifen or Gebr-7b + tamoxifen is recommended.

[0011] US patent numbered US11357798B2, titled ‘Mesenchymal stem cell-derived exosome’ is related to microparticles derived from mesenchymal stem cells that have the activity that supports the growth of corneal epithelial stem cells and / or corneal epithelial cells, the activity that keeps corneal epithelial stem cells in an undifferentiated state or supports the formation of colonies in this way, and the function of protecting the corneal epithelium. However, the relevant document does not contain a recommendation for the use of exosomes as a carrier system in overcoming tamoxifen resistance.

[0012] As a result due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

[0013] Purpose of the Invention

[0014] The present invention aims to solve the abovementioned disadvantages by being inspired from the current conditions. The main purpose of the present invention is to provide a new pharmaceutical composition for the treatment of hormone receptor positive breast cancer by delivering miR-342 and 4- hydroxy tamoxifen, which increases therapeutic efficacy in endocrine resistance, to target cells via mesenchymal stem cell-derived exosomes.

[0015] Purpose of the invention is to use exosomes obtained by using mesenchymal stem cells of human bone marrow origin as a drug carrier system and to realise exosomal structures together with miR-342, which contributes to the opening of resistance in cells resistant to 4- Hydroxy Tamoxifen with known effect, in addition to its own potential therapeutic efficacy.

[0016] The drug carrier system in the composition of the invention is a naturally occurring carrier structure produced by mesenchymal stem cells. Compared to synthetic and chemical drug delivery systems, the use of exosomes, which are involved in intercellular communication, in the drug delivery system increases the effectiveness of targeted treatments, reduces side effects and contributes to the treatment potential. The miR-342 mimic microRNA structure added into it provides positive effects in the treatment of breast cancer, especially in the treatment response of tamoxifen-resistant patients. The combination of exosome+miR- 342+4-OH-Tamoxifen increases the treatment effect in drug resistance against tamoxifen.

[0017] In order to fulfill the purposes described above, the invention is a pharmaceutical composition for use in overcoming tamoxifen resistance in the treatment of estrogen-positive breast cancer and comprises mesenchymal stem cell-derived exosomes loaded with miRNA and tamoxifen.

[0018] One embodiment of the invention comprises 1 x 108particles / ml exosome, 10-50 pmol miRNA, 7-25 pM tamoxifen.

[0019] One embodiment of the invention comprises 1 x 108particles / ml exosome, 10-50 pmol miRNA, 7 M tamoxifen.

[0020] One embodiment of the invention comprises 1 x 108particles / ml exosome, 10-50 pmol miRNA, 25 pM tamoxifen.

[0021] One embodiment of the invention comprises at least one pharmaceutical excipient.

[0022] According to one embodiment of the present invention, said miRNA is miR-342.

[0023] According to one embodiment of the present invention, said tamoxifen is 4-OH-Tamoxifen. According to one embodiment of the invention, said exosomes are derived from human bone marrow mesenchymal stem cells and their size distribution is approximately 50-200 nm.

[0024] The invention also includes the production method of the above-mentioned pharmaceutical compositions, comprising; the isolation of exosomes from mesenchymal stem cells and the loading of miRNA and Tamoxifen into exosomes by electroporation.

[0025] The structural and characteristic features of the present invention will be understood clearly by the following figures and the detailed description made with reference to these figures and therefore the evaluation shall be made by taking these figures and the detailed description into consideration.

[0026] Figures to Help Understand the Invention

[0027] Figure 1. Workflow representation.

[0028] Figure 2. Average particle diameter and particle concentration of exosomes.

[0029] (a) Repeated analysis graph of control exosomes, (b) size and concentration graph of control exosomes. (c) Repetitive analysis graph of 4-OHT loaded exosomes, (d) Size and concentration graph of 4-OHT loaded exosomes. (e) Repeated analysis graph of 4- OHT+miR-342 loaded exosomes, (f) Size and concentration graph of 4-OHT+miR-342 loaded exosomes.

[0030] Figure 3. Density graph and particle analysis image of exosomes.

[0031] (a) Density graph of control exosomes, (b) particle analysis image of control exosomes.

[0032] (c) Density graph of 4-OHT loaded exosomes, (d) Particle analysis image of 4-OHT loaded exosomes. (e) Density graph of 4-OHT+miR-342 loaded exosomes, (f) Particle analysis image of 4-OHT+miR-342 loaded exosomes.

[0033] Figure 4. Protein image showing the presence of CD9, CD63, and CD81 , surface markers specific to exosomes.

[0034] Figure 5. Fluorescence microscope image of the uptake of BODIPY™ TR ceramide-labeled 4-OHT+miR-342-loaded MSC-derived exosomes into MCF7 and MCF7-Tam1 cells at 48thhour. Exosomes labeled with BODIPY™ TR ceramide, taken into living cells, are colored red. (a) MCF-7 control cells before exosome treatment (b), MCF-7 Tam1 control cells before exosome treatment (c) Unfiltered image of MCF-7 cells at 48thhour after 4-OHT+miR- 342+exo treatment (d) Unfiltered image of MCF-7-Tam1 cells at 48thhour after 4-OHT+miR- 342+exo treatment (e) Intracellular image of BODIPY™ TR ceramide labeled exosomes at 48thhour after 4-OHT+miR-342+exo treatment of MCF-7 cells (red regions), (f) Intracellular image of BODIPY™ TR ceramide labeled exosomes at 48thhour after 4-OHT+miR-342+exo treatment of MCF-7-Tam1 cells (red regions).

[0035] Figure 6. Electron microscope images of isolated exosomes showing a rounded morphology. Cryo-EM: (a) Control exosome, bar: 100 nm (X49000), (b) Control exosome, bar: 50 nm (X98000). Transmission electron microscopy: (c) 4-OHT loaded exosome, bar: 100 nm, (X40000) (d) Exosome loaded with 4-OHT+miR-342, bar: 200 nm (X30000).

[0036] Figure 7. Expression analysis of miR-342 loaded into MSC-derived exosomes (Compared with the control group; *p<0.05, **p<0.01 , ***p<0.001 , significant).

[0037] Figure 8. Expression analysis of miR-342 loaded into MCF7-derived exosomes (Compared with the control group; ***p<0.001 , significant).

[0038] Figure 9. Expression analysis of miR-342 loaded into MSC-derived exosomes (Compared with the control group; **p<0.01 , significant).

[0039] Figure 10. Effect of exosome, 4-OHT, 4-OHT+Exosome, miR-342+exosome, anti- miR342+exosome and 4-OHT+miR-342 loaded exosome groups on cell cytotoxicity in (a) MCF7 and (b) MCF-7 Tam1 cells. (Compared with the control group; *p<0.05, **p<0.01 , ***p<0.001 , significant).

[0040] Figure 11. Results obtained by transwell cell invasion analysis of exosome, 4-OHT, 4- OHT+Exosome, miR-342+exosome, anti-miR342+exosome and 4-OHT+miR-342 loaded exosome groups applied to MCF7 cells (20X images, 100 pm).

[0041] Figure 12. Results obtained by transwell cell invasion analysis of exosome, 4-OHT, 4- OHT+Exosome, miR-342+exosome, anti-miR342+exosome and 4-OHT+miR-342 loaded exosome groups applied to MCF7-Tam1 cells (20X images, 100 pm).

[0042] Figure 13. Results of the effects of exosome, 4-OHT, 4-OHT+Exosome, miR-342+exosome, anti-miR342+exosome and 4-OHT+miR-342 loaded exosome groups applied to MCF7 cells on wound healing at 24thand 48thhours (4X image). Figure 14. Results of the effects of exosome, 4-OHT, 4-OHT+Exosome, miR-342+exosome, anti-miR342+exosome and 4-OHT+miR-342 loaded exosome groups applied to MCF7-Tam1 cells on wound healing at 24thand 48thhours (4X image).

[0043] Figure 15. a) Annexin V-PI results in MCF7 cells (a) control gate, (b) control cells (c) exosome, (d) 4-OHT, (e) 4-OHT-Exo, (f) miR-342-Exo (g) anti-miR-342-Exo (h) 4-OHT+miR- 342+Exo.

[0044] Figure 16. a) Annexin V-PI results in MCF7 Tam1 cells (a) control gate, (b) control cells (c) exosome, (d) 4-OHT, (e) 4-OHT-Exo, (f) miR-342-Exo (g) anti-miR-342-Exo (h) 4-OHT+miR- 342+Exo.

[0045] Figure 17. (a) Effect of exosome, 4-OHT, 4-OHT+Exo, miR-342+Exo, anti-miR342+Exo and 4-OHT+miR-342+Exo groups on protein expression of ERa in MCF-7 and MCF7-Tam1 cells, (b) Western-blot analysis band signal graph.

[0046] Figure 18. Pathway enrichment analysis. Pathway enrichment of differentially expressed genes; the y-axis shows the pathway name and the x-axis shows the enrichment factor in each pathway. The sphere size indicates the number of genes. In the colour bar, green represents higher p-value and purple represents lower p-value, (a) Enrichment analysis of MCF7-Tam1 and MCF7-Tam1 cells treated with 4-OHT+miR342+Exo; pathways involving differentially expressed genes with statistically increased expression and (b) pathways involving statistically decreased expression are shown.

[0047] Figure 19. Pathway enrichment analysis. Pathway enrichment of differentially expressed genes; the y-axis shows the pathway name and the x-axis shows the enrichment factor in each pathway. The sphere size indicates the number of genes. In the colour bar, green represents higher p-value and purple represents lower p-value, showing (a) pathways containing genes from MCF7 and 4-OHT+miR342+Exo treated MCF7 cells and (b) pathways containing genes with statistically reduced expression.

[0048] Detailed Description of the Invention

[0049] In this detailed description, the preferred embodiments of the inventive pharmaceutical composition is described by means of examples only for clarifying the subject matter. The invention is a pharmaceutical composition containing tamoxifen-loaded mesenchymal stem cell-derived exosomes enriched with miR-342 for use in overcoming tamoxifen resistance in ER+ breast cancer.

[0050] According to an embodiment of the invention comprises 1 x 108particles / ml exosomes of mesenchymal stem cell-derived exosomes, 10-50 pmol miR-342-mimic and 7-25 M 4-OH- Tamoxifen.

[0051] According to an embodiment of the invention comprises the 1 x 108particles / ml mesenchymal stem cell-derived exosome for Tamoxifen sensitive 10-50 pmol miR-342-mimic and 7 M 4-OH-Tamoxifen for Tamoxifen sensitive.

[0052] According to an embodiment of the invention, the 1 x 108particles / ml mesenchymal stem cell-derived exosome for Tamoxifen resistant contains 10-50 pmol miR-342-mimic and 25 M 4-OH-Tamoxifen for Tamoxifen resistant.

[0053] The present invention is based on the synergistic effect of these three components in overcoming tamoxifen resistance. While mesenchymal stem cell-derived exosomes take part in the transport of tamoxifen, miR-342 estrogen-positive breast cancer cell selective feature provides effective treatment of 4-OH-Tamoxifen at the target cell level.

[0054] The method of obtaining miR-342-enriched tamoxifen-loaded mesenchymal stem cell- derived exosomes enriched with miR-342 in the most basic form involves isolation of exosomes from mesenchymal stem cells and loading miRNA and Tamoxifen into the exosome by electroporation. According to one embodiment, the invention also comprises adding at least one pharmaceutical excipient.

[0055] In the preparation of the pharmaceutical composition of the invention, ready-made kits were used and tested at the cellular level in the treatment of tamoxifen-resistant estrogen receptor positive breast cancer. Studies conducted within the scope of the invention have shown that miR-342 and tamoxifen together are effective in resistant estrogen receptor positive breast cancer cells within the exosome. The combination of exosome+miR-342+4-OH-Tamoxifen increases the treatment effect in drug resistance against tamoxifen.

[0056] Despite the vast opportunities for exosomes in clinical therapy, the heterogeneity of isolated exosomes remains a significant obstacle. Therefore, it is crucial to understand the advantages and limitations of existing technologies that can isolate pure and homogeneous exosome populations and then perform a precise characterization. In addition to being used as a carrier system, exosomal structures also have therapeutic activities. In alternative embodiments, they can also be used as stand-alone treatment. In addition, the drug and microRNA added into it can be changed in alternative applications suggested by the person skilled in the art. The uniqueness of the present invention is that in vitro studies have demonstrated that tamoxifen resistance is overcome by combining mesenchymal stem cell-derived exosomes with 4-OH-Tamoxifen and miR-342 in tamoxifen- resistant cells.

[0057] EXPERIMENTAL STUDIES

[0058] The miRNA mimic primers used in the experimental stages were selected as Anti-miR™ miRNA lnhibitor-miR-342 mimic (MC13066) and miR-342 mimic (AM13066).

[0059] In the present study, exosomes were isolated from mesenchymal stem cell (MSC) medium using the ‘Total Exosome Isolation Reagent (from cell luminal media)’ (Thermo Fisher Scientific, USA) kit in order to isolate exosomes used as a nanocarrier system with high purity and in large amounts (Patel et al., 2019). Human bone marrow mesenchymal stem cells grown up to the 5thpassage were used in the study. The medium for exosome isolation was prepared without FBS. The media were collected and isolated after incubating for 3 days in 25 cm2flasks and for 24 hours in 75 cm2flasks. The kit procedure was followed after the medium collected at the end of the incubation was filtered through a 0,22 pm filter.

[0060] Electroporation is a widely used method to study the functions and mechanisms of endogenous miRNAs. In the current study, chemically synthesized miRNA mimics and 4- OHT were used to transfer to exosomes.

[0061] Table 1. Feature of loaded miR-342:

[0062] It was determined that drug-loaded exosomes were in high concentration below 200 nm at 1000 V in terms of size and purity. After electroporation, the exosomes were incubated at 370 for 1 hour in order to restore the membrane st ructure to its previous stable state. “MW 3000 Spin Column” was used to eliminate the unloaded drug.

[0063] Table 2. Exosome sample Exosome was applied to cells at concentrations of 1 x 108particles / ml.

[0064] Exosome + 4-OHT + miR-342 1 x 108particles / ml exosomes, 7 pM tamoxifen, 10 pmol miRNA were applied to the cells for MCF7 cells. Exosome + 4-OHT + miR-342 1 x 108particles / ml exosomes, 25 pM tamoxifen, 50 pmol miRNA were applied to the cells for MCF7-Tam1 cells.

[0065] The molecular structure of 4-OH-Tamoxifen loaded into the exosome is shown below. A. Confirming Exosomes

[0066] These experiments are findings indicating the presence of exosomes.

[0067] 1-NTA analysis

[0068] Exosome size and concentration distribution analyses before and after drug loading were performed at 20-220 using NTA (Malvern Instruments Ltd., NanoSight NS300) with 10 replicates of each reading for each sample. Sample size distributions were obtained in a liquid suspension (1 :50 dilution in PBS- / -, 1 :1000 dilution) by analyzing Brownian motion via light scattering. The camera level was set to 16 and the viscosity to 1 with a filterless Blue488 nm laser unit in accordance with the manufacturer's instructions. Data were analyzed using NTA software version 3.4 (NanoSight, United Kingdom).

[0069] Results: As a result of the NTA analysis of the isolated exosomes in DPBS, their average size distributions and concentrations were determined (Table 3). As seen in the NTA data in Figure 2, the size distribution of exosomes was determined to be approximately 50-200 nm. Additionally, the particle concentration of exosomes was measured by NTA. The exosome concentration isolated from MSCs was 6,78e+09 + / - 1 ,15e+08 particles / ml. The concentration of 4-OHT loaded exosomes was determined as 2,35e+09 + / - 4,81 e+08. The concentration of exosomes containing drug and miRNA was determined as 3,40e+10 + / - 6,20e+08 particles / ml. Density graph and particle analysis images of exosomes are given in Figure 3.

[0070] Table 3. NTA analysis results of exosomes.

[0071] 2-Western-blot analysis

[0072] The presence of CD9, CD81 , CD63 tetraspanins on the surface of isolated exosomes is a characteristic feature of exosomes, and the presence of these tetraspanins at the protein level is shown by western blot analysis. In order to confirm the isolation of exosomes, anti- CD9 (D3H4P, Rabbit mAb), CD63 (ab68418, Rabbit mAb) and CD81 (D3N2D, Rabbit mAb) monoclonal antibodies were examined at the protein level by western-blot analysis. 100 and 150 pg / ml protein from the obtained sample was denatured with "SDS-sample buffer" by keeping it at 100°C for 5 minutes. Denatured protein samples were loaded onto a 4-20% polyacrylamide gel, 20 pL into each well, and carried out in vertical gel electrophoresis at 110 V for 2 hours. Then, the protein bands in the gel were transferred onto the PVDF membrane on ice for 2 hours at 250 mA. After the transfer process, the milk mixture prepared with 5% skim milk and 1 g BSA in 1X DPBS was blocked at room temperature for 1 hour. In the next step, the membrane was washed with 1X TBS-Tween 20 (TBST) solution. It incubated with monoclonal primary antibodies diluted to the appropriate dilution with 2% BSA overnight at +4°C on a shaker at 35 rpm.After incubation, the membrane was washed with TBST solution 3 times for 10 minutes and incubated with secondary antibodies (anti-rabbit) appropriate to the primary antibodies for 3 hours at room temperature and at 45 rpm in a shaker. Finally, the membrane was washed with 1X TBST in a shaker at 45 rpm for 3 times for 10 minutes, and chemiluminescence imaging was performed on the LI-COR C-DiGit device with the Pierce ICL kit.

[0073] Results: Western-blot analysis images performed for the presence of CD9, CD63 and CD81 , which are surface biomarkers of exosomes, are presented in Figure 4 ([3-actin was used as a control). Band images were obtained from all three of the exosomal membrane proteins analyzed. The presence of exosomes was thus proven at the protein level.

[0074] 3-Monitoring intracellular uptake of exosomes via fluorescently labelled membrane lipids

[0075] The intracellular uptake of exosomes via fluorescently labeled membrane lipids was examined in vitro (Figure 5). This protocol allows for labeling of exosomes separated from cell culture media or any body fluid including blood, urine, CSF or saliva, or isolated from any source (Liang et al., 2018).

[0076] 4-Cryo-EM and transmission electron microscopy (TEM) imaging of exosomes

[0077] Within the scope of the current study, imaging of exosomes with cryo-EM was carried out by procuring services from METU Central Laboratory (MERLAB) (Figure 6).

[0078] NTA, western-blot and TEM analysis findings of exosomes used as a nanocarrier system in the current study confirmed that exosomes were successfully isolated and collected.

[0079] As a result of the analysis carried out to determine the expression level of mimic-miR342, which was loaded into the exosome (1 x 108particles / ml) by electroporation with 20 ms, 2 pulse current at 1000 V, in exosomes, the expression level of miR-342 (50 pmol) in the loaded exosomes was significantly increased 18,21-fold compared to the control group (p<0,001), whereas miR-342 expression was significantly decreased -4,39-fold in the experimental group to which anti-miR-342 mimic miRNA was applied (p=0,045) (Figure 7). It was observed that the determined electroporation parameters enabled the efficient loading of mimic-miRNAs into exosomes.

[0080] As a result of the expression analysis performed with total RNA samples isolated at 48thhour of MCF7 and tamoxifen resistant MCF7-Tam1 cells treated with miR-342 loaded exosomes in order to confirm the uptake of miR-342 loaded exosomes into the cell; the expression level of miR-342 in MCF7 cells was significantly increased 14,93-fold compared to the control group (p<0,001), (Table 4 and Figure 8), while it was significantly increased 8-fold in MCF7- Tam1 cells (p<0,01 ), (Table 5 and Figure 9). Table 4. Fold change of miR-342 in MCF7 cells after miR-342 loaded exosome treatment.

[0081] Table 5. Fold change of miR-342 in MCF7-Tam1 cells after miR-342 loaded exosome treatment.

[0082] Cytotoxicity Analyses: When cell viability rates of MCF7-Tam1 cells for the same groups were examined, at the end of 24 hours; the viability rates of cells treated with 4-OHT, 4-OHT+Exo, and 4- OHT+miR-342+Exo are respectively; 64,77%, 34,78%, 48,12%, at the end of 48 hours; 44,73%, 32,08%, 24,96% and at the end of 72 hours 47,75%, 21 ,84%, 19,98% and all groups were determined to be statistically significant (p<0.05 and p<0.01) (Figure 10). No statistical significance was determined in the viability rates of exosome, miR-342+exo and anti-miR-342+exo applied cells. Transwell Invasion Analysis

[0083] Cell migration is an important feature of the living cell and the transwell migration / invasion and metastasis assay is used to analyse the ability of single cells to respond directionally to chemoattractants and therapies and to test the capacity to bind to the extracellular matrix. According to transwell invasion analysis, it was determined that miR-342+4-OHT loaded exosomes were more effective in preventing invasion in MCF-7 cells and tamoxifen resistant MCF-TAM1 cells compared to the other experimental groups, however, the group in which only exosomes originating from MCPs were applied was less invasive than the control group. It was determined that both MCF7 and MCF7-TAM1 invasive cells applied with anti-miR-342 loaded exosome formed denser cell colonies compared to the other groups (Figure 11 and Figure 12).

[0084] Determining the Effect of Exosomal Structures Enriched in Cargo Contents on Wound Healing

[0085] When the effect of exosomal constructs carrying loaded drugs and miRNA on the migration abilities of cells in MCF7 and MCF7-Tam1 cells was examined, it was found that although the wound area was closed in the control groups 48 hours after the wound healing area was obtained in each experimental group, there was a dose-dependent decrease in the migration abilities of the cells applied to exosome, 4-OHT, 4-OHT+Exo and 4-OHT+miR-342+Exo. When the effect of 4-OHT+miR-342+Exo was examined, especially on MCF7 and MCF7- tam1 cells, it was observed that the migration speed of the cells decreased significantly and the migration (wound healing) area increased compared to the control group (Figure 13 and Figure 14). In MCF-7 and MCF-7 Tam1 cells, it was observed that anti-miR342+exosome treatment caused an increase in migration rate and a decrease in migration distance. The dose-dependent changes in the migration ability of the cells are in parallel with the results obtained in other experiments.

[0086] Determining the Apoptotic Effect of Exosomal Structures Enriched with Cargo Contents

[0087] To determine the apoptotic effect of exosome, 4-OHT, 4-OHT+Exo, miR-342+exo, anti- miR342+exo and 4-OHT+miR-342+Exo groups on MCF-7 and MCF-7-Tam1 cells, he apoptotic effect at 48 hours was evaluated in accordance with the results obtained from WST-1 assay. Among the experimental groups; when the apoptotic effect of 4-OHT, 4- OHT+Exo and 4-OHT+miR-342+Exo on MCF7 and MCF-7-Tam1 cells was examined, it was observed that there was a significant increase in the percentages of early and late apoptosis in general (p< 0.01). However, when the apoptotic effect in miR-342+Exo and anti- miR342+Exo applied cells was evaluated; a lower rate of apoptotic death was detected in these cells compared to both MCF7 and MCF7-Tam1 parental cells. In particular, it was determined that anti-miR342+Exo, which binds to miR-342 and blocks the function of miR- 342 in the cell, positively contributed to the viability of MCF7 and MCF-7-Tam1 cells.

[0088] When the apoptotic effect of exosomal structures enriched in cargo content was examined in MCF7 and MCF-7-Tam1 cells at the 48thhour, in control group, early apoptosis rates in MCF7 cells treated with exosome, 4-OHT, 4-OHT+Exo, miR-342+Exo, anti-miR342+Exo and 4-OHT+miR-342+Exo is determined respectively as follows; 1 ,00%, 3,35%, 5,95%, 6,70%, 1 ,10%, 1 ,20% and 0,20%, late apoptosis rates was determined respectively as 4.80%, 7.35%, 11.55%, 12.30%, 5.00%, 3.90% and 12.65% (Figure 15).

[0089] In MCF-7-Tam1 cells, early apoptosis rates applied to control cells, exosome, 4-OHT, 4- OHT+Exo, miR-342+Exo, anti-miR342+Exo and 4-OHT+miR-342+Exo was determined as 3,27%, 1 ,91%, 2,87%, 2,43%, 2,77%, 2,1% and 18,55%. Late apoptosis rates was determined respectively as 5,79%, 7,49%, 17,76%, 19,84%, 5,9%, 4,6% and 3,35% (Figure 16).

[0090] While 12,65% late apoptosis and 22,60% dead cells were detected in MCF7 cells administered 4-OHT+miR-342+Exo among exosomal structures enriched in cargo contents, in tamoxifen-resistant MCF7-Tam1 cells, the early apoptosis rate was determined as 18,55% and the dead cell rate was 1 ,10%. When evaluated together with other death rates, it was determined that the applied exosomal content caused early apoptotic death in resistant cells.

[0091] Effect of Exosomal Structures Enriched in Cargo Contents on ERa at the Protein Level Protein expression level of ERa (ESR1), the main therapeutic target, in ER+ cells such as MCF7 and MCF7-Tam1 treated with exosome, 4-OHT, 4-OHT+Exo, miR-342+Exo, anti- miR342+Exo and 4-OHT+miR-342+Exo groups, was evaluated by western-blot analysis by normalizing with the reference protein beta-actin. The findings are presented in Figure 17. When the protein levels obtained were evaluated compared to the control, it was determined that ERa protein expression was decreased in the group in which 4-OHT loaded exosome was applied, compared to the groups in which only exosome or 4-OHT was applied to MCF7- Tam1 cells. In addition, it was determined that the protein level in the 4-OHT+miR-342+Exo applied group decreased compared to the 4-OHT only applied group. T ranscriptome Analysis

[0092] Transcriptome is the amount of all transcripts in a cell at a particular developmental stage or in a particular physiological state. Since genome data alone is not sufficient to understand the functions of genes, transcriptome data is needed. With RNA-Seq, expressed regions on the genome can be determined quantitatively with high reliability and sensitivity, and maps of these regions can be created. In order to understand the differences in the molecular mechanisms of MCF7 and tamoxifen-resistant MCF-7 Tam1 cells and to determine the mechanisms of action of the cargo-rich exosomes examined in our study in the resistance to tamoxifen, these two cell lines were analysed together with MCF7-Tam1 cells treated with exosomes, 4-OHT+Exo, 4-OHT+miR-342+Exo and MCF7 cells treated with 4-OHT+miR- 342+Exo (Groups used in the study: N1 : MCF7, N4: MCF7, 4-OHT+miR342+Exo applied, D1 : MCF7-Tam1 , D2: 4-OHT+Exo, D3: Exosome, D4: 4-OHT+miR342+Exo applied group).

[0093] In line with the pathway enrichment analysis in which MCF7-Tam1 and 4-OHT+miR342+Exo treated MCF7-Tam1 cells were compared; it was determined that the expression of the pathway including stimulus response genes pathway, cell communication pathway, genes regulating the binding of signal molecules (ligands) coming from outside the cell with the receptor on the cell surface or inside the cell, expressed as signal receptor binding, increased. However, cell cycle genes, neutrophil extracellular trap formation pathway, ESR- mediated signalling pathway, estrogen-dependent gene expression pathway genes and p53 signalling pathway were significantly downregulated (Figure 18).

[0094] Pathway enrichment analysis comparing MCF7 and 4-OHT+miR342+Exo treated MCF7 cells revealed that plasma membrane activation, cell communication pathway genes, genes related to calcium ion binding, and pathways including potassium activation genes were statistically significantly increased. Statistically significantly downregulated pathways were determined as extracellular exosome pathway, cell cycle, ROBO receptor and WNT mediated signalling pathways, ESR mediated signalling pathway, estrogen dependent gene expression pathway genes, mitochondrial gene expression related genes, pre-NOTCH expression and processing genes and neutrophil extracellular trap (NET) formation pathway as in MCF7-Tam1 (Figure 19).

[0095] Among the identified pathways, considering the limited studies related to breast cancer, the role of tumour-derived NETs in complications such as potential malignancy promoters, thrombosis and systemic inflammation, it was determined that the cargo-enriched exosomes of the M SC-derived exosomes offer a therapeutic approach to suppress NETosis in both MCF7 and MCF7-Tam1 cells.

Claims

CLAIMS1. A pharmaceutical composition intended for use in overcoming tamoxifen resistance in the treatment of estrogen-positive breast cancer, characterzed by comprising; miRNA and tamoxifen-loaded mesenchymal stem cell-derived exosomes.

2. The pharmaceutical composition according to claim 1 , characterzed by comprising; 1 x 108particles / ml exosome, 10-50 pmol miRNA, 7 -25 pM tamoxifen.

3. The pharmaceutical composition according to claim 1 , characterzed by comprising; 1 x 108particles / ml exosome, 10-50 pmol miRNA, 7 pM tamoxifen.

4. The pharmaceutical composition according to claim 1 , characterzed by comprising; 1 x 108particles / ml exosome, 10-50 pmol miRNA, 25 pM tamoxifen.

5. The pharmaceutical composition according to claim 1 , characterzed by comprising; at least one pharmaceutical excipient.

6. The pharmaceutical composition according to claim 1 , characterised in that; the said miRNA is miR-342.

7. Pharmaceutical composition according to claim 1 , characterised in that; the said tamoxifen is 4-OH-Tamoxifen.

8. The pharmaceutical composition according to claim 1 , characterised in that; the said exosomes are derived from human bone marrow mesenchymal stem cells.

9. The pharmaceutical composition according to claim 1 , characterised in that; the size distribution of exosomes is approximately 50-200 nm.

10. A method of producing a pharmaceutical composition according to any one of the preceding claims, characterised by comprising; isolating exosomes from mesenchymal stem cells, loading miRNA and Tamoxifen into the exosomes by electroporation.

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