Products containing exosomes of plant origin

Abstract

The present invention relates to products containing exosomes of plant origin, which can be used for cancer treatment and wound healing. The invention makes it possible to provide a low-cost product which does not cause toxic effects in the human body, does not cause damage in healthy cells during the course of cancer treatment, does not cause any risk of infection, since it does not require any procedure such as radiation therapy or surgery, and in which there is no side effects present in chemotherapy treatment. In the invention, wheatgrass, garlic and ginger can be used alone or in combination as plant sources.

Description

[0001] This application is a divisional application of the international application PCT / TR2018 / 050034, filed on January 31, 2018, which entered into China with application number 201880020740.8 and entitled "Product containing plant-derived ectospheres". Technical Field

[0002] This invention relates to products containing plant-derived exosomes, which can be used for cancer treatment and wound healing. Background Technology

[0003] In multicellular organisms, communication is fundamental to intercellular communication through the binding of proteins released from one cell to receptors on the surface of another cell. Despite limited information about them, ectosomes are currently the essential communication molecules needed (1-3). Although they were initially named as unwanted cellular material and cellular waste when first discovered, subsequent studies have demonstrated their important role in the immune system (4). Ectosomes are vesicles ranging in size from 20 to 130 nm (nanometers) that are produced by all cells in the plasma and released from the cells (5). As recently discovered, these vesicles function in the endocrine system to deliver autocrine and paracrine signals to local or distant host cells. These ectosomes are also responsible for carrying proteins, lipids, and nucleic acid material (all RNA types) and transferring them to recipient cells. In this way, messages to be transmitted from the cell are delivered to other neighboring cells (3). The family of four-transmembrane proteins is the most frequently encountered protein group on the surface of ectosomes. This family includes CD9, BCD63, CD81, CD82, and CD83 proteins. The four-transmembrane protein family of proteins has the ability to interact with MHC and integrin (6). However generalized, ectosome protein complexes are quite cell-specific, and therefore dependent on the cell that releases them. Thus, various (heat shock) proteins are members of this complex (hsp70). Meanwhile, proteins found in the cytoskeleton, namely β-actin, tubulin, myosin, filoproteinases, basic histocompatibility components I-II, and glyceraldehyde-3-phosphate dehydrogenase, are the main common proteins in ectosome protein complexes. In order for an ectosome to be received into the cell to which it will be delivered, it must stimulate cell surface receptors on other cells. Therefore, Wnt-β-catenin signaling proteins, notchligang, delta-like 4 proteins, and interleukins must be present on the ectosome surface. Otherwise, the ectosome cannot stimulate the host cell to which it will transmit the information.

[0004] One study demonstrated the potential of ectosomes derived from mammalian cells for cancer treatment (Reghu et al., 2016). Furthermore, ectosomes showed anticancer activity against mammalian cancer cells (Ohno et al., 2013). In another similar study, ectosomes derived from brain epithelium were identified as exhibiting activity against brain cancer (Reriro et al., 2015).

[0005] Wheat (Triticum aestivum) plays a vital role in the human diet within the plant-based dietary classification. Epidemiological studies have demonstrated its protective effects in patients with chronic thalassemia and cancer. Numerous forms of wheat (wheatgrass or seeds (germs)) have been developed for cancer treatment. Similarly, ginger and garlic are antioxidants. Animal-derived ectosomes have been used in various applications in ongoing research. Furthermore, although plant-derived ectosomes have been identified as antioxidants, research on them is very limited. To the best of our knowledge, plant ectosomes exhibit properties similar to mammalian ectosomes.

[0006] Treatments targeting dividing cells, such as surgery, radiation therapy, and chemotherapy, have repeatedly led to cancer recurrence or numerous side effects (Kubota, 2012).

[0007] US Patent No. US2016346334 discloses effluxed somatic cells isolated from healthy cells for the treatment of breast cancer.

[0008] Because of this invention, alternative treatment methods have been developed to induce cancer cell death without harming healthy cells using products containing phytoexcitonins. Furthermore, the fact that effluxcitonins are cell-free products makes it possible to prevent problems that may arise from cell therapy. Effluxcitonins are ready-to-use therapeutic agents. They can be stored at low temperatures without the use of toxic cryoprotectants and ultra-freezers, and can be safely delivered to patients. Based on this information, the aim is to use wheatgrass, garlic, and ginger, which are used in alternative medicine, cancer treatment, and as wound healing agents. Summary of the Invention

[0009] The purpose of this invention is to provide a product containing plant-derived efflux compounds that can be used for cancer treatment.

[0010] Another object of the present invention is to provide a product that does not cause toxicity in the human body.

[0011] A further object of the present invention is to provide a product that is effective in wound healing and repair of damaged tissue.

[0012] Another object of the present invention is to provide a product that does not involve the side effects present during chemotherapy treatment during its use.

[0013] Another object of the present invention is to provide a product that does not cause damage to healthy cells during the course of cancer treatment.

[0014] A further object of the present invention is to provide a product that does not pose any risk of infection because it does not require any procedures such as radiation therapy or surgery.

[0015] Another objective of this invention is to provide a low-cost product.

[0016] A further object of the present invention is to provide natural products that do not involve chemical contamination in the body as is caused by synthetic drugs.

[0017] Another object of the present invention is to provide a practical and readily available product, since the plant to be used for treatment is easy to grow and produces a large number of effluxes in a short period of time. Attached Figure Description

[0018] The "product containing plant-derived effluxes" developed to achieve the objectives of this invention is illustrated in the accompanying drawings, wherein... Figure 1 -A is a view of the flow cytometry values ​​of the control group of A498 renal cell carcinoma cells.

[0019] Figure 1 -B is a view of the flow cytometry values ​​of the control group of A498 renal cell carcinoma cells stained with annexin V.

[0020] Figure 1 -C is a view of the flow cytometry values ​​of the control group of A498 renal cell carcinoma cells with PI staining.

[0021] Figure 1 -D is a flow cytometry view of the cell death values ​​of A498 renal cancer cells to which 15 mg / ml wheat efflux was applied.

[0022] Figure 1 -E is a flow cytometry view of the cell death values ​​of A498 renal cell carcinoma cells to which 15 mg / ml ginger efflux bodies were applied.

[0023] Figure 1 -F is a flow cytometry view of the cell death values ​​of A498 renal cell carcinoma cells to which 15 mg / ml garlic efflux was applied.

[0024] Figure 2 -A is a flow cytometry view of the control group of HEK 293 renal epithelial cells.

[0025] Figure 2 -B is a flow cytometry view of the control group of HEK 293 renal epithelial cells with PI staining.

[0026] Figure 2 -C is a flow cytometry view of the control group of HEK 293 renal epithelial cells stained with annexin V.

[0027] Figure 2 -D is a flow cytometry view of the death values ​​of HEK 293 renal epithelial cells to which 15 mg / ml wheat efflux was applied.

[0028] Figure 2 -E is a flow cytometry view of the death values ​​of HEK 293 renal epithelial cells after application of 15 mg / ml ginger efflux.

[0029] Figure 2 -F is a flow cytometry view of the death values ​​of HEK 293 renal epithelial cells to which 15 mg / ml garlic efflux was applied.

[0030] Figure 3 -A is a flow cytometry view of the control group of 22RV1 prostate cancer cells.

[0031] Figure 3 -B is a view of the flow cytometry values ​​of the control group of 22RV1 prostate cancer cells stained with annexin V.

[0032] Figure 3 -C is a view of the flow cytometry values ​​of the control group of 22RV1 prostate cancer cells with PI staining.

[0033] Figure 3 -D is a flow cytometry view of the cell death values ​​of 22RV1 prostate cancer cells to which 15 mg / ml wheat efflux was applied.

[0034] Figure 3 -E is a flow cytometry view of the cell death values ​​of 22RV1 prostate cancer cells to which 15 mg / ml ginger efflux bodies were applied.

[0035] Figure 3 -F is a flow cytometry view of the cell death values ​​of 22RV1 prostate cancer cells to which 15 mg / ml garlic efflux was applied.

[0036] Figure 4 -A is a flow cytometry view of the control group of PNT1A prostate epithelial cells.

[0037] Figure 4 -B is a flow cytometry view of the control group of PNT1A prostate epithelial cells stained with PI.

[0038] Figure 4-C is a flow cytometry view of the control group of PNT1A prostate epithelial cells stained with annexin V.

[0039] Figure 4 -D is a flow cytometry view of the death values ​​of prostatic epithelial cells to which 15 mg / ml wheat efflux PNT1A was applied.

[0040] Figure 4 -E is a flow cytometry view of the death values ​​of prostatic epithelial cells to which 15 mg / ml ginger efflux bodies were applied.

[0041] Figure 4 -F is a flow cytometry view of the death values ​​of prostate epithelial cells to which 15 mg / ml garlic efflux bodies PNT1A were applied.

[0042] Figure 5 -A is a view of the flow cytometry values ​​of the control group of MCF 7 breast cancer cells.

[0043] Figure 5 -B is a view of the flow cytometry values ​​of the control group of MCF 7 breast cancer cells stained with Annexin V.

[0044] Figure 5 -C is a flow cytometry view of the control group of MCF 7 breast cancer cells with PI staining.

[0045] Figure 5 -D is a flow cytometry view of the death values ​​of MCF 7 breast cancer cells to which 15 mg / ml wheat efflux was applied.

[0046] Figure 5 -E is a flow cytometry view of the death values ​​of MCF 7 breast cancer cells to which 15 mg / ml ginger efflux bodies were applied.

[0047] Figure 5 -F is a flow cytometry view of the death values ​​of MCF 7 breast cancer cells to which 15 mg / ml garlic efflux was applied.

[0048] Figure 6 -A is a flow cytometry view of the control group of MCF 10A mammary epithelial cells.

[0049] Figure 6 -B is a view of the flow cytometry values ​​of the control group of MCF 10A mammary epithelial cells with PI staining.

[0050] Figure 6 -C is a flow cytometry view of the control group of MCF 10A mammary epithelial cells stained with annexin V.

[0051] Figure 6 -D is a flow cytometry view of the cell death values ​​of MCF 10A mammary epithelial cells to which 15 mg / ml wheat efflux was applied.

[0052] Figure 6 -E is a flow cytometry view of the death values ​​of MCF 10A mammary epithelial cells to which 15 mg / ml ginger efflux bodies were applied.

[0053] Figure 6 -F is a flow cytometry view of the death values ​​of MCF 10A mammary epithelial cells to which 15 mg / ml garlic efflux was applied.

[0054] Figure 7 These are electron microscope images that confirm the presence of plant ectosomes obtained from wheat plants.

[0055] Figure 8 This is a flow cytometry view in which the presence of plant effluxes obtained from wheat plants is determined by HSP70 surface markers.

[0056] These results show that plant effluxes only induce apoptosis in cancer cells, causing the death of those cells, but do not cause any cell death in healthy cells. Detailed Implementation

[0057] Experimental research Preparation of plant-derived efflux bodies In the products of this invention containing plant-derived efflux compounds, wheatgrass, ginger, and garlic can be used alone or in combination as plant sources. Preferably, the wheatgrass selected in the experimental studies was obtained from seeds of Adana Ceyhan 69 in Turkey. During the work used for preliminary sample preparation, a seed growth period of 1.5 weeks was determined to be sufficient.

[0058] First, the collected wheatgrass was ground in 1% PBS (phosphate-buffered saline) and then filtered. The obtained filtrate wheatgrass was centrifuged at 1000xg for 10 minutes, 3200xg for 20 minutes, and 15000xg for 60 minutes, and then the cell cultures were separated using an ectosome isolation kit. The separated ectosomes were dissolved in 0.9% isotonic slurry.

[0059] The product efflux of the present invention, separated and in slurry form, was observed using a scanning electron microscope. Figure 7 They were then incubated with the HSP70 marker, and the presence of efflux bodies was observed in flow cytometry.

[0060] Development of treatment Assay for cytotoxicityThe toxicity of the prepared product of the present invention was determined by using the MTS method described in the literature (Yalvac et al., 2009).

[0061] Chemical molecules were prepared in culture medium at specified concentrations and applied to HEK (human kidney epithelial cells), MCF 10A (human mammalian epithelial cells), and PNT1A (human prostate epithelial cells) cell lines seeded on 96-well plates (5000 cells / well). Cellular response to chemical toxicity was determined by measuring cell viability for 4 days. Cell viability was analyzed according to the MTS method, a colorimetric method for measuring mitochondrial enzyme activity. Different doses were applied to pre-seeded cells in wells, and the effect of the applied substance on viability was examined over defined time periods. After an increase in viable cell count, the MTS reagent applied after mixing with culture medium yielded a dark color. The resulting color change was assessed based on absorbance measurements using an ELISA reader. Products developed within the scope of this invention were prepared to achieve results in healthy cells at concentrations of 5 ng / ml, 10 ng / ml, and 15 ng / ml at 24, 48, 72, and 96 hours.

[0062] Cancer cell death analysis To examine the effects of the product of this invention on cancer, the presence of annexin V protein (one of the most important proteins in the apoptosis pathway, a mechanism of suicide) was examined. PI (propidium iodide) staining was used to determine the presence of necrosis.

[0063] 22RV1 (prostate cancer cells), MCF7 (breast cancer cells), and A498 (renal cancer cells) cells were seeded at 300,000 cells / well in 6-well culture plates. Cells were allowed to grow by adding 10% FBS (fetal bovine serum) and 1% PSA (penicillin-streptomycin-amphotericidal) to either high-glucose medium or RPMI (for the 22RV1 cell line) and DMEM (for the MCF7 and A498 cell lines). Cell mortality was assessed after 24 hours at the specified concentrations. Results showed that cells binding to the annexin V antibody underwent or were about to undergo apoptosis, and cells binding to the PI antibody resulted in or were about to undergo necrosis. The application of these antibodies determined the treatments that killed the cells.

[0064] The same experimental protocol was also applied to the MCF10-A, PNT1-A and HEK cell lines, which were used as healthy cell lines.

[0065] Examination of wound healing In addition to its use in cancer treatment, the application of the product of this invention was also examined in wound healing. In experimental studies conducted for this purpose, healthy HEK cell lines were seeded in 6-well culture plates. Wound models were formed by creating scratches through the centers of the wells. The product containing efflux bodies was applied to the wound-modeled cells. The cells were incubated for 48 hours in DMEM medium containing 2% FBS at a concentration of 15 ng / ml of the product. The wound healing process was observed progressively by taking photographs daily under a microscope.

[0066] Experimental results The efflux vesicles were preferably obtained using wheatgrass via a developed separation method. Characterization experiments were conducted on the product, and the results demonstrated its cancer activity and wound healing properties. Data obtained from electron microscopy images revealed a similarity between the observed vesicles and electron microscopy images of efflux vesicles in the literature. Figure 7 Flow cytometry results have shown that ectosomes express the surface marker at 84.16%. This is one piece of evidence that the molecule we obtained is an ectosome. Figure 8 ).

[0067] MTS results demonstrated that exposure of healthy cells to plant efflux sap induced cell proliferation. The increased absorbance compared to the negative control indicated increased cell viability. Corresponding analyses demonstrated that wheat efflux increased the cell division rate of healthy cells. Figure 3 ).

[0068] 22RV1 cells were treated with plasma at concentrations of 5 μg / ml, 10 μg / ml, and 15 μg / ml for 24 hours; cell death was observed using annexin V assay by flow cytometry. Consistent with these results, when calculating the necrosis and apoptosis ratios, cell death rates of 26.02%, 40.08%, and 34.36% were observed at the above concentrations, respectively. Figure 3 ).

[0069] PNT1A cells were treated with plasma at concentrations of 5 μg / ml, 10 μg / ml, and 15 μg / ml for 24 hours; cell death was observed using annexin V assay by flow cytometry. Consistent with these results, cell death rates of 1.81%, 3.26%, and 3.22% were observed when calculating necrosis and apoptosis ratios, respectively. Figure 4 ).

[0070] MCF-7 cells were treated with plasma at concentrations of 5 μg / ml, 10 μg / ml, and 15 μg / ml for 24 hours; cell death was observed using annexin V assay via flow cytometry. Consistent with these results, cell death rates of 24.96%, 31.13%, and 23.49% were observed when calculating necrosis and apoptosis ratios, respectively. Figure 5 ).

[0071] MCF-10A cells were treated with plasma at concentrations of 5 μg / ml, 10 μg / ml, and 15 μg / ml for 24 hours; cell death was observed using annexin V assay via flow cytometry. Consistent with these results, cell death rates of 1.57%, 1.25%, and 2.74% were observed when examining necrosis and apoptosis ratios, respectively. Figure 6 A498 cells were treated with plasma at concentrations of 5 μg / ml, 10 μg / ml, and 15 μg / ml for 24 hours; cell death was observed using annexin V assay by flow cytometry. Consistent with these results, cell death rates of 51.05%, 38.01%, and 42.76% were observed when calculating necrosis and apoptosis ratios, respectively. Figure 1 ).

[0072] HEK 293 cells were treated with plasma at concentrations of 5 μg / ml, 10 μg / ml, and 15 μg / ml for 24 hours; cell death was observed using annexin V assay by flow cytometry. Consistent with these results, cell death rates of 1.79%, 1.63%, and 3.16% were observed when examining the necrosis and apoptosis ratios, respectively. Figure 2 ).

[0073] As a result of experimental research, it has been demonstrated that the product of this invention triggers a cell suicide mechanism in vivo, causing the death of cancer cells. Treatment using this product has been observed to eliminate the drawback often encountered in chemotherapy treatment, which involves killing cancer cells while simultaneously damaging healthy cells.

[0074] As a result of wound models based on MTS results, wound models in cells treated with the product of this invention showed significantly faster healing compared to untreated control cells. This demonstrates that the obtained product, particularly those derived from wheat ectosomes, positively influences and accelerates processes in wound healing and tissue repair models.

[0075] Application of the present invention This invention is mainly applied to prostate cancer, breast cancer, and kidney cancer; it is also active in pancreatic, liver, bone, skin, brain, lung, pleura, uterus, ovary, stomach, intestine, bladder, blood, lymph, and thyroid cancer types.

[0076] The products of this invention can be produced primarily in liquid slurry form, but they can also be produced in solid or hydrogel form. These can be in the form of slurry, syrup, tablet, pharmaceutical, gel, and cream. Additionally, another active substance (such as a drug or chemical) can be delivered into cells using nano-carrier molecules. This delivery method can be used in various forms and formulations as described above. References 1. Johnstone, RM, Adam, M, Hammond, JR, Orr L and Turbide, C (1987). Vesicleformation during reticulocyte maturation. Association of plasma mebraneactivities with released vesicles (exosomes). / BiolChem 262: 9412-9460 2. Valadi, H, K, Bossios, A M, Lee JJ and JO(2007).Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism ofgenetic Exchange between cells.Nat Cell Bio19: 654-659 3. Thery, C, Ostrowski, M and Segura, E (2009). Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9: 581-593 4. Gogolak P, Rethi B, Hajas G, Rajnavolgyi E. (2003) Targeting dendritic cells for priming cellular immune responses. J. Mol. Recognit.; 16: 299-317. 5. An, Q., Hücvkelhoven, R., Kogel, K. H., and van Bel, A. J. (2006). Multi vesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbia 18: 1009 - 1091 6. Escola, J. M., M. J. Kleijmeer, W. Stoorvogel, J. M. Griffth, O. Yoshie, and H. J. Geuze. (1998). Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273: 20121 - 20127. 7. Buschow, S. I., B. W. van Balkom, M. Aalberts, A. J. Heck, M. Wauben, and W. Stoorvogel. (2010). MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis. Immunol. Cell Biol. 88: 851 - 856 8. Simpson RJ, Lim JW, Moritz RL, Mathivanan S. (2009) Exosomes: proteomic insights and diagnostic potential. Expert. Rev. Proteomics.; 6: 267 - 283. 9. Gogolak P, Rethi B, Hajas G, Rajnavolgyi (2003) E. Targeting dendritic cells for priming cellular immune responses. J. Mol. Recognit.; 16:299 - 317 10. Vermeulen, P.B., Verhoeven, D., Hubens, G., Van Marck, E., Goovaerts, G., Huyghe, M., De Bruijn, E.A., Van Oosterom, A.T. and Dirix, L.Y. (1995). Microvessel density, endothelial cell proliferation and tumour cell proliferation in human colorectal adenocarcinomas. Annals of oncology, 6(1), 59 - 64. 11. Yalvac, M.E., Ramazanoglu, M., Gumru, O.Z., Sahin, F., Palotas, A. and Rizvanov, A.A. (2009). Comparison and optimisation of transfection of human dental follicle cells, a novel source of stem cells, with different chemical methods and electro - poration. Neuro chemical research, 34(7), 1272 - 1277.

Claims

1. Plant-derived ectosomes used to treat cancer by increasing cell death in cancer cells while increasing cell proliferation in healthy cells.

2. The plant-derived efflux molecule according to claim 1, characterized in that... The plant-derived efflux source is wheatgrass.

3. The plant-derived efflux molecule according to claim 1, characterized in that... The plant-derived efflux source is garlic.

4. The plant-derived efflux molecule according to claim 1, characterized in that... The plant-derived efflux source is ginger.

5. The plant-derived efflux molecule according to claim 1, characterized in that... The plant-derived efflux sources consist of a combination of wheatgrass, ginger, and garlic.

6. Use of plant-derived ectosomes in the preparation of drugs for treating cancer by increasing cell death of cancer cells while increasing cell proliferation of healthy cells.

7. The use according to claim 6, characterized in that... The drug is in the form of a liquid, syrup, tablet, gel, or cream.

8. The use according to any one of claims 6 to 7, characterized in that... The cancers mentioned are selected from prostate, breast, kidney, pancreas, liver, bone, skin, brain, lung, pleura, uterus, ovary, stomach, intestine, bladder, blood, lymph, and thyroid cancer types.

Images