Target-specific exosome-based delivery vehicles and bioformulations for obtaining same

Abstract

The present invention discloses an exosome-based delivery vehicle for targeted delivery of an active substance to cancer cells and cancer stem cells. The delivery vehicle is obtained by a method including the steps of incubating monocytes in a culture medium containing a biologic to obtain dendritic cells, adding an active substance to the culture medium containing the dendritic cells to obtain an exosome-based delivery vehicle containing the active substance, and isolating the exosome-based delivery vehicle from the culture medium for targeted delivery of the active substance specific to cancer cells and cancer stem cells. The present invention also discloses a biologic for enhancing the specificity of the exosome-based delivery vehicle to cancer cells and cancer stem cells. The biologic includes granulocyte macrophage colony stimulating factor, interleukin-4, ionomycin, tumor necrosis factor alpha, IL1β, LPS, and angiotensin II.

Description

[Technical field]

[0001] The present invention relates generally to delivery vehicles, and more specifically to exosome-based delivery vehicles and biopharmaceutical compositions for enhancing the specificity of exosome-based delivery vehicles. [Background technology]

[0002] Cancer is a disease characterized by the uncontrollable growth of abnormal cells. Standard treatments for cancer include, but are not limited to, surgery, radiation therapy, and chemotherapy. However, for metastatic cancer, surgery and radiation therapy are not useful. Chemotherapy is the main treatment for such metastatic cancer. However, chemotherapy has multiple drawbacks, such as poor biodistribution to tumors and the inability to distinguish between normal and cancer cells, resulting in the death of both. Chemotherapy can reduce tumor volume by killing non-stem cell cancer cells, but it cannot target cancer stem cells. These chemotherapy-resistant cell subpopulations present in tumors can survive chemotherapy and regenerate heterogeneous tumor mass, which is the main cause of tumor recurrence. Chemotherapy further involves several serious side effects and toxicities due to the inability to control its biodistribution. Efficient targeting of chemotherapeutic drugs to cancer cells and cancer stem cells could be a potential solution to the above problems. Exosome-based delivery vehicles also work in a similar way, efficiently targeting not only cancer cells but also cancer stem cells. Exosome-based delivery vehicles provide safe passage of active substances to their targets.

[0003] Exosomes are extracellular vesicles secreted by cells. Exosomes act as excellent intercellular communication devices due to their characteristics such as, but not limited to, biocompatibility, high permeability, and biodegradability. The composition of exosomes contains multiple conserved proteins that are responsible for their biological activities. The conserved proteins include, but are not limited to, tetraspanins (CD81, CD63, and CD9), Tsg101, and Alix. In addition, exosomes also have specific components depending on the type of parent cell. The parent cells include, but are not limited to, mesenchymal stem cells (MSCs), embryonic stem cells, and immune cells. Exosomes reflect the unique properties of their parent cells and are now known to mediate the therapeutic effects of the parent cells. Over the years, exosomes have been widely used in the field of therapeutics. Exosomes are used either as naive exosomes isolated from parent cells without any intervention, or as genetically modified exosomes. Exosomes isolated from the respective parent cells are used directly. However, these naive exosomes show low specificity and yield. Genetically engineered ethosomes have a role in many areas, including but not limited to cancer, inflammation, and neurological diseases. New tumor treatments such as cell therapy utilize the patient's immune cells to target cancer cells, but these injected immune cells can be regulated by the tumor cells through the secretion of cytokines such as TGF-β, IL-10, which can render these cells ineffective against the tumor. Cancer cells also overexpress CD47, making them appear healthy, thus avoiding recognition by immune cells. By using cell-free exosome-based therapies, this drawback of cell therapy can be overcome, as tumor cells cannot prepare these exosomes, making them more effective in-vivo. Another new technology is immunotherapy, which uses antibodies that block specific receptors on tumor cells, preventing immune cells from being blocked. However, in many cases, these therapies fail, resulting in a more aggressive disease. Cancer cells divide rapidly, which causes a large number of mutations to accumulate.In many cases, these cancer cells accumulate certain genomic mutations and develop resistance to immunotherapy drugs. The use of exosomes may be useful in mitigating these unexpected side effects of immunotherapy, since exosomes utilize multiple cell surface receptors to facilitate endocytosis and deliver their active substances into tumor cells, thus eliminating the need to identify cancer cells using a single cell surface receptor.

[0004] With the advent of biomedicine, exosomes are rapidly developing as a new solution for tumors. In recent years, it has been found that exosomes, including bioactive substances, function by delivering their bioactive substances to tumor cells. In addition, exosomes can also inhibit tumors by delivering chemical drugs. They show improved penetration and retention effects. Exosomes, as natural carriers of chemical drugs, can avoid phagocytosis by macrophages and extend the half-life of chemical drugs compared with other artificial carriers. As a result, in the delivery process, exosomes significantly improve the delivery efficiency of certain biodegradable drugs. Since most drugs are hydrophobic in nature, the addition of drugs into exosome-based delivery vehicles also improves the stability, solubility, and half-life of the drugs.

[0005] In a review article entitled "Platform technologies and human cell lines for the production of therapeutic exosomes," Kim et. al. showed how macrophage-derived exosomes containing the anticancer therapeutic compound PTX can treat multidrug-resistant cancers. PTX-containing exosomes showed increased anticancer efficacy in a mouse lung tumor model. However, these carrier vehicles have low yields and loading capacities, and therefore are not completely effective against tumors.

[0006] To overcome the above obstacles, exosome mimics have been developed. One such mimic is the liposome-exosome hybrid described in WO 2018 / 170398. Hybrid carrier vehicles exhibit higher content and yield. They also show easier encapsulation of larger molecules. However, these vehicles have multiple drawbacks, including but not limited to high cytotoxicity and low specificity.

[0007] Exosomes can deliver biomolecules or chemicals to the pathological site of the recipient and effectively inhibit tumor progression. However, the treatment of tumors by exosome delivery is not precise and efficient enough, and various challenges need to be overcome. To produce clinical grade exosomes, it is necessary to establish a scalable, consistent, and controllable manufacturing method for culture. In addition, effective treatment of cancer requires achieving target specificity of exosome-based delivery vehicles. Specificity to not only cancer cells but also cancer stem cells will bring about a comprehensive solution to completely alleviate cancer. In order to improve cancer treatment by sustainable means, the need to develop an efficient exosome-based delivery vehicle to effectively deliver active substances to cancer cells, especially cancer stem cells, has long been sought. In addition, none of the prior literature known in the art is related to a method of increasing the specificity of exosome-based delivery vehicles by using a formulation effective for increasing the specificity to cancer cells, more specifically cancer stem cells. The present invention improves the aforementioned shortcomings of the prior art and provides a vehicle, method, and formulation for fighting cancer overall.

[0008] The present invention will be better understood by reference to the following description in conjunction with the drawings, in which: Particular embodiments of the present invention are illustrated in the accompanying drawings, in which like numerals or letters refer to like elements throughout. It should be noted, however, that the accompanying drawings depict only typical embodiments of the present invention and are therefore not to be considered as limiting the scope of the invention, which may tolerate other embodiments that are equally effective. Summary of the Invention [Problem to be solved by the invention]

[0009] Objective of the invention An object of the present invention is to provide an exosome-based delivery vehicle for targeted delivery of active substances to cancer cells and cancer stem cells.

[0010] Another object of the present invention is to provide a method for preparing an exosome-based delivery vehicle for targeted delivery of active substances to cancer cells and cancer stem cells.

[0011] Yet another object of the present invention is to provide an effective biologic to enhance the specificity of exosome-based delivery vehicles by overexpressing transmembrane proteins and / or mRNA and / or shRNA and / or cDNA for targeted delivery of active substances to cancer cells and cancer stem cells.

[0012] Yet another object of the present invention is to provide an exosome-based delivery vehicle that targets cancer stem cells that are refractory to conventional chemotherapy drugs, reducing the number of chemotherapy cycles required to achieve a similar reduction in tumor volume, reducing the cost of treatment and the physical and psychological impact on the patient.

[0013] Yet another object of the present invention is to provide a biologic that can be used to increase the production of ICAM1 in immune cells, which is a much safer way of overexpressing the gene than using overexpression plasmids by transfection, which can induce significant stress in immune cells.

[0014] It is yet another object of the present invention to provide a biologic that can be used to increase the production of galectin-3 in immune cells. [Means for solving the problem]

[0015] According to one aspect, the present invention provides an exosome-based delivery vehicle for targeted delivery of an active substance to cancer cells and cancer stem cells, which is obtained by a method comprising the steps of: incubating monocytes in a culture medium containing a biologic to obtain dendritic cells, adding an active substance to the culture medium containing the previously obtained dendritic cells to obtain an exosome-based delivery vehicle containing the active substance, and isolating the exosome-based delivery vehicle from the culture medium for targeted delivery of the active substance specific to cancer cells and cancer stem cells.

[0016] Another aspect of the present invention provides biologics for enhancing the specificity of exosome-based delivery vehicles to cancer cells and cancer stem cells, including granulocyte-macrophage colony-stimulating factor, interleukin-4, ionomycin, tumor necrosis factor alpha, IL1β, LPS, and angiotensin II. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows a schematic diagram illustrating the efficacy of an exosome-based delivery vehicle containing a chemotherapeutic drug, according to an embodiment of the present invention. [Diagram 2]FIG. 1 shows flow cytometry analysis of Annexin V cells following PBMC culture using an exosome-based delivery vehicle containing doxorubicin (EXO-DOX) and free doxorubicin (DOX) according to one embodiment of the present invention. [Diagram 3] 1 shows an analysis of ICAM1 expression in targeted exosomes according to one embodiment of the present invention. [Figure 4] 1 shows the internalization efficiency of a delivery vehicle of the present invention by cancer cells and cancer stem cells, according to one embodiment of the present invention. [Figure 5a] 1 shows specific targeting of cancer cells and cancer stem cells by a delivery vehicle of the present invention, according to one embodiment of the present invention. [Figure 5b] 1 shows specific targeting of cancer cells and cancer stem cells by a delivery vehicle of the present invention, according to one embodiment of the present invention. [Figure 5c] 1 shows specific targeting of cancer cells and cancer stem cells by a delivery vehicle of the present invention, according to one embodiment of the present invention. [Figure 6] 1 shows enhanced targeting of cancer stem cells by a delivery vehicle of the present invention compared to an equivalent concentration of free drug, according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The following detailed description is merely exemplary in nature and is not intended to limit the described embodiments or the application and uses of the described embodiments. As used herein, the words "exemplary" or "illustrative" mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" or "illustrative" is not necessarily to be construed as preferred or advantageous over other implementations. The present invention will now be described with reference to the accompanying examples and / or drawings, in which embodiments of the invention are shown. The description is not intended to detail all the different ways in which the invention may be practiced or all the features that may be added to the invention. For example, features shown with respect to one embodiment may be incorporated in other embodiments, and features shown with respect to a particular embodiment may be deleted from that embodiment. Thus, the present invention contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted. Moreover, numerous modifications and additions to the various embodiments proposed herein will be apparent to those skilled in the art in light of this disclosure, and such modifications and additions do not depart from the scope of the invention. Thus, the following description is intended to describe some particular embodiments of the present invention, without exhaustively specifying all permutations, combinations, and variations thereof.

[0019] Various embodiments of the present invention provide an exosome-based delivery vehicle for targeting and delivering active substances to cancer cells, particularly cancer stem cells, and a method for obtaining the same. The present invention also provides a biologic for increasing the specificity of the exosome-based delivery vehicle to cancer cells and cancer stem cells. The biologic increases ICAM1 in exosomes and upregulates Galectin-3, which increases the specificity of the exosome-based delivery vehicle.

[0020] The exosome-based delivery vehicle of the present invention is biologically engineered to target cancer cells, including, but not limited to, solid tumor cancer, blood cancer, and metastatic cancer. Cancer can be a non-solid tumor type or a solid tumor. Examples of such cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, breast cancer, prostate cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, gastric cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, liver cancer, vulvar cancer, thyroid cancer, liver cancer, various head and neck cancers, hematological malignancies, acute myeloid leukemia, pancreatic cancer, breast cancer, lung cancer, colon cancer, and melanoma metastasis. The exosome-based delivery vehicles of the present invention are also capable of targeting hematological malignancies such as cancer stem cells and CML and inhibiting the growth of cancer cells. The size of the delivery vehicles ranges from 30 nm to 150 nm and carries active molecules in the size range of 30 kDa to 90 kDa. The shelf life of the vehicles is 1-3 months at -20°C and 6 months at -80°C. These delivery vehicles successfully carry the active substances for approximately 30 minutes to 3 hours under biological conditions, extending the half-life of the active substances under biological conditions by 24 hours.

[0021] An active substance is any substance or mixture of substances that directly inhibits abnormal cell growth. This substance, alone or in combination with any other substance, can enhance or inhibit the biological function that results in limiting abnormal cell growth. The active substance, alone or in combination with other active substances, can control the secretion of substances that regulate the function of the human body that exhibits abnormal conditions. The active substance in the present invention is selected from, but is not limited to, proteins, drugs, enzymes, nucleic acids, chemicals, and mixtures thereof.

[0022] Chemotherapy drugs include, but are not limited to, paclitaxel, cisplatin, and doxorubicin. In one embodiment of the present invention, exosome-based delivery vehicles are used to carry biomolecules such as mRNA, shRNA, siRNA, and DNA. In another example, the exosome-based delivery vehicle can transport drugs that inhibit the BCR-ABL fusion protein and specifically target IL3-R. In another example of the present invention, the exosome-based delivery vehicle is loaded with the chemotherapy drug doxorubicin. Figure 1 shows a schematic diagram illustrating the efficacy of the exosome-based delivery vehicle of the present invention with a chemotherapy drug according to an embodiment of the present invention. The vehicle of the present invention also reduces the number of chemotherapy cycles required, thereby reducing not only the cost of treatment, but also the physical and psychological effects on the patient.

[0023] When added to the vehicle, exosome-based delivery vehicles can avoid phagocytosis by macrophages due to their clathrin coating and extend the half-life of the active substance, so that the delivery vehicle can safely reach the target cells.

[0024] Another embodiment of the present invention provides a method for producing an exosome-based delivery vehicle for targeted delivery of an active substance to cancer cells and cancer stem cells. These exosomes are biologically engineered to enhance specificity for cancer cells and cancer stem cells. The method includes the steps of incubating monocytes in a culture medium containing a biologic to obtain dendritic cells, adding an active substance to the culture medium containing the previously obtained dendritic cells to obtain an exosome-based delivery vehicle containing the active substance, and isolating the exosome-based delivery vehicle from the culture medium for targeted delivery of the active substance specific to cancer cells and cancer stem cells. The steps described herein are described in detail below.

[0025] First, monocytes are incubated in culture medium to obtain dendritic cells (hereafter referred to as DCs). The culture medium is selected from a list including but not limited to RPMI, DMEM, L-15, or DMEM high glucose. As an example, THP-1 cell line (human leukemia monocytic cell line) obtained from NCCS Pune is maintained in DMEM supplemented with 10% FBS and 1X antibiotic and antimycotic mix (Himedia) in a humidified chamber at 370C, 95% air, and 5% CO2. The cells in the exponential growth phase are then used for further experiments. The next step is the generation of DCs from the THP-1 monocytic cell line. In one example, mature DCs are derived from ThP1 cells after incubation in RPMI medium containing the bioformulation of the present invention. The incubation period is about 48 hours to 50 hours. The cells are allowed to incubate in a humidified chamber maintained at about 37°C in about 95% air and about 5% CO2. An active substance is added to the DCs thus obtained to obtain an exosome-based delivery vehicle containing the active substance. The active substance is packaged in exosomes by endogenous or exogenous packaging methods. In one example, to package an active substance in DC-derived exosomes, monocyte-derived DCs are incubated with the active substance to be packaged in exosomes in RPMI medium containing a biologic. Then, the culture medium is collected after about 24 hours to isolate exosomes, to obtain an exosome-based delivery vehicle for targeted delivery of the active substance to cancer cells and cancer stem cells. In one example, adding doxorubicin is performed by treating mature DCs with doxorubicin at a concentration of 1.8 μg / ml or 2.0 μg / ml for about 24 hours. Then, exosomes are isolated from the cell culture medium using Total Exosome Isolation Reagent (Cat. No. 4478359) using the protocol described in the product manual. According to one embodiment of the present invention, exosomes are isolated from 2×10 6-cell cultured in exosome-depleted medium for 48 hours. 8From mature DC-CM, exosomes are isolated using Total Exosome Isolation Reagent. The collected medium is centrifuged at 2,000 rpm for 3 min at 4°C, and the supernatant is collected and centrifuged at approximately 4,000 rpm for approximately 30 min at 4°C. Exosomes are isolated according to the manufacturer's instructions. The exosome pellet is then washed once with calcium- and magnesium-free PBS and resuspended in 50-100 μl of PBS. Total exosomal protein is measured by Bradford protein assay. In one example, a yield of 6-7 μg / μl is obtained from 1 ml of culture medium.

[0026] Yet another embodiment provides a biologic that enhances the specificity of exosome-based delivery vehicles to cancer cells and cancer stem cells. The biologic is a cocktail of components that is mixed with the culture medium in which the exosome-based delivery vehicle is developed. These components are selected from a list including, but not limited to, lipopolysaccharide (hereinafter referred to as LPS), tumor necrosis factor-α (hereinafter referred to as TNF-α), angiotensin II, interleukin-1β (hereinafter referred to as IL-1β), and interferon-Y (hereinafter referred to as IFN-Y). In particular, the composition of the biopharmaceutical preparation of the present invention includes granulocyte-macrophage colony-stimulating factor in a concentration range of 100ng / mL to 150ng / mL, interleukin-4 in a concentration range of 100ng / mL to 150ng / mL, ionomycin in a concentration range of 200ng / mL to 250ng / mL, tumor necrosis factor alpha in a concentration range of 100ng / mL to 150ng / mL, IL1β in a concentration range of 20ng / mL to 40ng / mL, LPS in a concentration range of 200ng / mL to 250ng / mL, and angiotensin II in a concentration range of 50ng / mL to 100ng / mL.

[0027] The process of preparing the biopreparation includes adding each component at its respective concentration to 100 ml of culture medium. The culture medium in the present invention is any medium, including but not limited to RPMI, DMEM, and L15 medium, in which the monocytes of the present invention are incubated, then mature dendritic cells are generated, and finally exosome-based delivery vehicles are generated. In one example, the culture medium is high glucose DMEM medium. The preparation thus obtained is slowly stirred in a sterile environment at about 4° C. for about 15 to 20 minutes. The sources of the components of the biologics are listed below.

[0028] [Table 1]

[0029] According to one embodiment of the present invention, incubation of monocytes with the biologic results in overexpression of ICAM-1. These overexpressed proteins are due to the targeting specificity of the resulting exosome-based delivery vehicle. Overexpression of ICAM-1 prevents immune suppression by cancer cell-derived PGE2 and restores cytotoxic T cell (CTL) function. The delivery vehicle of the present invention effectively targets and is specifically internalized by cancer stem cells and cancer cells, while the present invention increases surface ICAM1 on these cells to increase their immunogenic profile, thereby improving detection by immune cells such as CTL. ICAM-1 also binds to and activates LFA-1 on CTL in vivo. Overexpressed ICAM-1 also targets CD43 in cancer stem cells, enhancing its specificity. The biologic also upregulates ICAM1 as well as galectin-3, aiding in targeting of cancer cells and cancer stem cells. Galectin-3 (hereinafter abbreviated as GAL3) is a structurally unique beta-galactoside-binding lectin that is involved in numerous biological processes, such as cell proliferation and apoptosis, adhesion and activation, through specific protein-protein and protein-carbohydrate interactions. Lipopolysaccharide (LPS) in the biopreparation of the present invention causes upregulation of GAL3 expression at both gene and protein levels in monocytic THP-1 cells, concomitantly increasing the production of exosomes. MUC1 is a natural ligand for GAL3. The interaction between MUC1 and GAL3 promotes the internalization of the exosome-based delivery vehicle of the present invention by cancer stem cells and cancer cells, improving its efficacy.

[0030] Efficacy Testing: The toxicity of the exosomes of the present invention to PBMCs derived from patients and healthy individuals will be analyzed. The exosome-based delivery vehicle of the present invention has pleiotropic effects, thereby reducing the toxicity of chemotherapy drugs. To analyze the toxicity of free DOX and exosomes containing DOX (EXO-DOX) on PBMCs, PBMCs are isolated from healthy individuals and tumor patients. After isolation, they are cultured with DOX-containing exosomes or free DOX for about 48 hours. By packaging DOX within exosomes, the toxic side effects were reduced. In healthy patient blood, the toxicity was reduced by 2-fold, while in patient blood, the toxicity was reduced by about 1.3-fold. Figure 2 shows the flow cytometry analysis of Annexin V cells after culture of PBMCs with EXO-DOX and free DOX.

[0031] Analysis of internalization efficiency of the delivery vehicle of the present invention by cancer cells and cancer stem cells: To understand the internalization efficiency of the delivery vehicle of the present invention, exosomes derived from mature DCs and exosomes derived from THP1 monocytes are tagged with CFSE. After isolating the CFSE-tagged exosomes, CSCs (cancer stem cells) and cancer cells are cultured with these tagged exosomes in a time-dependent manner. The results show that exosomes derived from mature DCs are more likely to be internalized by cancer cells (about 12-fold) and CSCs (about 2.8-fold) in about 6 hours compared to exosomes derived from THP1 monocytes. This reveals the improved targeting ability of the delivery vehicle of the present invention compared to exosomes derived from THP1 monocytes, as shown in Figure 3.

[0032] Analysis of ICAM1 expression on targeted exosomes Western blot analysis is performed to verify the increased expression of ICAM1 on the target exosomes. To do so, THP1 cells are treated with the bioformulation of the present invention for about 48 hours, and then exosomes are isolated. After exosome isolation, total exosomal proteins are isolated. The expression of ICAM1 on the target exosomes is analyzed by Western blot analysis. The results show that after incubating THP1 cells with the bioformulation, the expression of ICAM1 increased about 4-fold compared to exosomes isolated from untreated THP1 cells, as shown in Figure 4.

[0033] Analysis of specific targeting of CSCs and cancer cells To analyze the specificity of the exosome-based targeted delivery vehicle of the present invention, the targeted exosome of the present invention is packaged with doxorubicin (EXO-DOX). Then, breast cancer-derived cancer stem cells, breast cancer cells, and HEK293 cells are treated with EXO-DOX (4ng / ml) and free DOX (1800ng / ml). After treatment, the proportion of Annexin V cells is found to be similar in the free DOX treatment set and the EXO-DOX treatment set. This suggests that EXO-DOX is as effective as free DOX even at a 450-fold lower concentration. Interestingly, when HEK293 cells are treated with EXO-DOX (4ng / ml) and free DOX (1800ng / ml), free DOX induced apoptosis in HEK293 cells, but EXO-DOX failed to induce apoptosis in HEK293 cells. This confirms the specificity of the present invention to CSCs and cancer cells. Figures 5a, 5b and 5c show the specific targeting of cancer cells and cancer stem cells by the delivery vehicle of the present invention.

[0034] Analysis of cytotoxicity in CSCs after incubation with equal concentrations of free DOX and EXO-DOX To analyze the enhanced efficacy of EXO-DOX compared to free DOX, cancer stem cells generated from MDA-MB-231 and MDA-MB-468 cells were cultured and treated with 5ng / ml free DOX and 5ng / ml EXO-DOX. Annexin V analysis by flow cytometry revealed approximately 2-fold and 6-fold increased apoptosis of CSCs derived from MDA-MB-231 and MDA-MB-468 cells, respectively, as shown in Figure 6.

[0035] Because the described preferred embodiment of the invention is susceptible to many modifications, variations and changes in detail, it is intended that all matters set forth in the foregoing description and accompanying drawings be interpreted in an illustrative and not a limiting sense, and the scope of the invention is therefore to be determined by the appended claims and their legal equivalents.

Claims

1. An exosome-based delivery vehicle for targeted delivery of an active substance to cancer cells and cancer stem cells, comprising: Incubating monocytes in a culture medium containing the biologic to obtain dendritic cells, said monocytes being incubated at 5% CO ２ and incubating at a temperature range of 36°C to 38°C for 48 to 50 hours; adding an active substance to the culture medium containing the dendritic cells to obtain an exosome-based delivery vehicle containing the active substance, wherein the dendritic cells are incubated with the active substance for 20 to 24 hours; isolating the exosome-based delivery vehicle from the culture medium for targeted delivery of an active substance specific to cancer cells and cancer stem cells; An exosome-based delivery vehicle produced by a method comprising the steps of:

2. 2. The exosome-based delivery vehicle of claim 1, wherein the size of the exosome-based delivery vehicle is in the range of 30 nm to 150 nm.

3. 2. The exosome-based delivery vehicle of claim 1, wherein the biologic shortens the incubation period for the monocytes to obtain the dendritic cells.

4. 2. The exosome-based delivery vehicle of claim 1, wherein the active agent is an anti-cancer drug or an anti-cancer RNA or DNA or protein or a combination thereof.

5. 2. The exosome-based delivery vehicle of claim 1, wherein the culture medium is any culture medium used for incubating monocytes to obtain the exosome-based delivery vehicle.

6. 2. The exosome-based delivery vehicle of claim 1, wherein the molecular weight of the active agent is in the range of 300 g / mol to 12,000 g / mol.

7. 2. The exosome-based delivery vehicle of claim 1, wherein the biologic increases ICAM1 in the exosomes for targeted delivery of active agents to cancer stem cells and cancer cells.

8. 2. The exosome-based delivery vehicle of claim 1, wherein the biologic upregulates galectin-3.

9. 2. The exosome-based delivery vehicle of claim 1, wherein the biologic comprises granulocyte-macrophage colony-stimulating factor in a concentration range of 100 ng / mL to 150 ng / mL, interleukin-4 in a concentration range of 100 ng / mL to 150 ng / mL, ionomycin in a concentration range of 200 ng / mL to 250 ng / mL, tumor necrosis factor alpha in a concentration range of 100 ng / mL to 150 ng / mL, IL1β in a concentration range of 20 ng / mL to 40 ng / mL, LPS in a concentration range of 200 ng / mL to 250 ng / mL, and angiotensin II in a concentration range of 50 ng / mL to 100 ng / mL and / or combinations thereof, wherein the biologic is mixed into 100 ml of culture medium.

10. A biologic for increasing the specificity of an exosome-based delivery vehicle to cancer cells and cancer stem cells, comprising: the biologic comprises granulocyte macrophage colony stimulating factor in a concentration range of 100 ng / mL to 150 ng / mL, interleukin-4 in a concentration range of 100 ng / mL to 150 ng / mL, ionomycin in a concentration range of 200 ng / mL to 250 ng / mL, tumor necrosis factor alpha in a concentration range of 100 ng / mL to 150 ng / mL, IL1β in a concentration range of 20 ng / mL to 40 ng / mL, LPS in a concentration range of 200 ng / mL to 250 ng / mL, angiotensin II in a concentration range of 50 ng / mL to 100 ng / mL, and / or combinations thereof; Wherein the biologic is mixed into 100 ml of culture medium.

11. 11. The biologic of claim 10, wherein the biologic increases ICAM1 in the exosomes for targeted delivery of active agents to cancer stem cells and cancer cells.

12. 11. The biologic of claim 10, wherein the biologic reduces the incubation period of the monocytes.

13. The biologic of claim 10, which upregulates galectin-3.

14. 11. The bioformulation of claim 10, wherein the culture medium is any culture medium used to incubate monocytes to obtain the exosome-based delivery vehicle.

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