WO2023119317A1

WO2023119317A1 - A target specific exosome based delivery vehicle and a bio-formulation to obtain the same

Info

Publication number: WO2023119317A1
Application number: PCT/IN2022/051088
Authority: WO
Inventors: Abhishek Dutta; Swastika Paul
Original assignee: Abhishek Dutta; Swastika Paul
Priority date: 2021-12-20
Filing date: 2022-12-17
Publication date: 2023-06-29

Abstract

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

Description

A TARGET SPECIFIC EXOSOME BASED DELIVERY VEHICLE AND A BIO¬

FORMULATION TO OBTAIN THE SAME

FIELD OF INVENTION

The present invention generally relates to a delivery vehicle, and more particularly it relates to an exosome based delivery vehicle and a bio-formulation composition to increase the specificity of the exosome based delivery vehicle.

BACKGROUND

Cancer is a disease characterized by uncontrolled growth of abnormal cells. Standard treatment for cancer includes but in not limited to surgery, radiation therapy and chemotherapy. However, for metastasized cancers surgery and radiation is of no help. Chemotherapy is the mainstay treatment for such metastasized cancers. However, chemotherapy is associated with a list of disadvantages including but not limited to poor bio-distribution to tumor and inability to differentiate normal cells and cancer cells thereby killing the both. Chemotherapy can reduce the tumor bulk by killing the non-stem cancer cells but fails to target the cancer stem cells. These chemo-resistant sub-population of cells which resides within the tumor survives the chemotherapy and can regenerate the heterogeneous tumor mass is the major cause behind tumor relapse. Chemotherapy is further associated with several serious side-effects and toxicity as there is no control over its bio-distribution. Effective targeting of the chemotherapeutic drug to the cancer cells as well as cancer stem cells can be a potential solution to the above listed problems. Exosome based delivery vehicle works on the similar lines and effectively targets the cancer cells as well as cancer stem cells. Exosome based delivery vehicle offers a safe passage of active substance to the target.

Exosomes are extracellular vesicles secreted by the cell. Exosomes acts as an excellent intercellular communicator owing to its attributes including but not limited to biocompatibility, high permeability and biodegradability. The composition of exosomes includes a list of conserved proteins that are responsible for its biological activities. The conservative protein includes but is not limited to tetraspanins (CD81, CD63, and CD9), TsglOl, and Alix. Furthermore, exosomes also have specific components depending on their parental cell types. The parental cells include but are not limited to mesenchymal stem cells (MSC), embryonic stem cells, and immune cells. Exosomes reflects the intrinsic properties of its parental cells and are now known to mediate the therapeutic effects of their parental cells. Over the years exosomes have been extensively used in the field of therapeutics. Exosomes are either used as naive exosomes isolated from parental cells without any interventions or genetically engineered exosomes. Exosomes isolated from their respective parental cells are directly used. However, these naive exosomes displays low specificity and yield. Genetically engineered esosomes are playing its part in many fields including but not limited to cancer, inflammation and neurological diseases. Emerging tumor therapy such as cell therapy utilizes the patient’s immune cells to target the cancer cells but these infused immune cells can be modulated by the tumor cells via secretion of cytokines such as TGF-P, IL-10, which can render these cells ineffective against the tumor. Also, cancer cells over express CD47 which makes the cancer cell appear to be healthy and thus avoid recognition by the immune cells. Using cell-free exosome-based therapies overcomes this drawback of cell therapy as the tumor cells cannot modulate these exosomes and thus they can be more effective in-vivo. Another emerging technique is immunotherapy which uses antibodies to block specific receptors on the tumor cells to prevent them from blocking the immune cells. But often these therapies fail and lead to more progressive disease. Cancer cells divide rapidly and due to this accumulate numerous mutations. Often these cancer cells accumulate specific genomic mutations that help them developed resistance to immunotherapy drugs. The use of exosomes can help us mitigate these unforeseen side-effects of immunotherapy, as exosomes utilize multiple cell surface receptors to enhance endocytosis and deliver their active substance inside the tumor cells thereby eliminating the use of a single cell surface receptor to identify the cancer cells.

With the advent of biomedicine, exosomes are rapidly developing as a new solution against tumors. In the recent years, exosomes loaded with bioactive substances are found to perform functions by delivering these bioactive substances to tumor cells. In addition, exosomes can also inhibit tumors by delivering chemical drugs. They show enhanced permeation and retention effect. Exosomes as natural carriers of chemical drugs can avoid phagocytosis by macrophages and prolong the half-life of chemical drugs compared with other artificial carriers. Consequently, in the process of delivery, exosomes significantly improve the delivery efficiency of certain biodegradable drugs. Since, most of the drugs are hydrophobic in nature; loading them inside an exosome based delivery vehicle also increases their stability, solubility and half-life.

In a review paper, entitled “Platform technologies and human cell lines for the production of therapeutic exosomes” Kim et. al. demonstrated how macrophage-derived exosomes loaded with anticancer therapeutic compound PTX to treat multi-drug resistant cancer. PTX-loaded exosomes demonstrated an increased anti-cancer effect in murine lung tumor models. However, these carrier vehicles showed poor yield and loading capacity and hence are not completely effective against the tumors.

To overcome the above mentioned obstacles, exosome mimetic are developed. One such mimetic is liposome-exosome hybrid as mentioned in Patent WO 2018/170398. The hybrid carrier vehicles manifest higher loading capacity and yield. They also exhibit easy encapsulation of larger molecules. These vehicles however have a list of drawbacks including but not limited to higher cytotoxicity and lower specificity.

Exosomes deliver biomolecules or chemical drugs into the pathological sites of recipient, which can effectively inhibit the progression of tumors. However, the treatments of tumors through the delivery of exosomes are not sufficiently accurate or efficient and various challenges need to be overcome. Scalability, consistency, and controllable manufacturing methods for culture will need to be established to produce clinical -grade exosomes. Additionally, the target specificity of exosome based delivery vehicle needs to be achieved for effective treatment of cancers. Specificity not only towards cancer cells but also towards cancer stem cells will provide a holistic solution to mitigate cancer completely. There is a long felt need to develop an efficient exosome based delivery vehicle for effective delivery of active substances to cancer cells and particularly to cancer stem cells for improved treatment of cancer through sustainable means. Also, none of the prior publication known in the art is directed to method of increasing the specificity of the exosomes based delivery vehicle via use of an effective formulation to elevate its specificity towards cancer cells and more particularly cancer stem cells. The present invention provides a vehicle, method and formulation to combat cancer holistically which ameliorates the aforesaid shortcomings of the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to the following description taken in combination with the drawing. Certain embodiments of the present invention are illustrated in the appended drawing. In the drawings, like numerals or alphabets indicate like elements throughout. It is to be noted, however, that the appended drawing illustrates only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments. In the drawing:

Fig.: 1 shows schematic representation of efficacy of the exosome based delivery vehicle loaded with a chemotherapeutic drug, according to an example of the present invention.

Fig.: 2 shows Flow cytometric analysis Annexin V cells following culture of PBMC with exosomes based delivery vehicle loaded with Doxorubicin (EXO-DOX) and free Doxorubicin (DOX), according to an embodiment of the present invention.

Fig.: 3 shows analysis of ICAM1 expression in targeted exosomes, according to an embodiment of the present invention.

Fig.: 4 shows internalization efficiency of the present delivery vehicle by cancer cells and cancer stem cells, according to an embodiment of the present invention.

Fig.: 5a, 5b and 5c shows specific targeting of cancer cells and cancer stem cells by the present delivery vehicle, according to an embodiment of the present invention.

Fig.: 6 shows enhanced targeting of cancer stem cells by the present delivery vehicle, as compared to the equal concentration of free drug, according to an embodiment of the present invention.

OBJECT OF THE INVENTION It is an object of the present invention to provide an exosome -based delivery vehicle for targeted delivery of active substance to cancer cells and cancer stem cells.

It is another object of the present invention to provide a method of preparing exosome based delivery vehicle for targeted delivery of active substance to cancer cells and cancer stem cells.

It is yet another object of the present invention to provide an effective bio-formulation to increase the specificity of the exosome-based delivery vehicle by over-expressing a transmembrane protein and/or a mRNA and/or a shRNA and/or a cDNA for targeted delivery of active substance to cancer cells and cancer stem cells.

It is yet another object of the present invention to provide an exosome based delivery vehicle that targets cancer stem cells that conventional chemotherapeutic drugs fails and also reduce the number of chemo-cycles required to achieve a similar reduction in tumor volume thus reducing the treatment cost as well as physical and the psychological effect on the patients.

It is still another object of the present invention to provide a bio-formulation which is being used to increase ICAM1 production in the immune cells, which is a much safer way to over-express a gene rather than using over expression plasmid by transfection, which can induce substantial stress on the immune cells.

It is still another object of the present invention to provide a bio-formulation which is being used to increase Galectin-3 production in the immune cells.

SUMMARY OF THE INVENTION

According to an aspect, the present invention provides an exosome based delivery vehicle for targeted delivery of active substances to cancer cells and cancer stem cells. The delivery vehicle is obtained by the method including the steps of incubating monocytes in a culture medium containing a bio-formulation to obtain a dendritic cells, adding an active substance into the culture medium containing the previously obtained dendritic cells to obtain an exosome based delivery vehicle loaded with active substances and isolating the exosome based delivery vehicle from the culture medium for targeted delivery of active substances specific to cancer cells and cancer stem cells.

Another aspect of the invention provides a bio-formulation to increase the specificity of exosome-based delivery vehicle to cancer cells and cancer stem cells. The bio -formulation includes a granulocyte macrophage colony-stimulating factor, an interleukin 4, an ionomycin, a tumor necrosis factor alpha, an IL10, a LPS and an angiotensin II.

BRIEF DESCRIPTION OF THE INVENTION

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 word “exemplary” or “illustrative” means “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 now will be described hereinafter with reference to the accompanying examples and/or drawings, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the scope of the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Various embodiment of the invention provides an exosome based delivery vehicle for targeted delivery of active substance to cancer cells and particularly to cancer stem cells and a method of obtaining the same. The present invention also provides a bio-formulation to increase the specificity of exosome -based delivery vehicle to cancer cells and cancer stem cells. The bio-formulation increases ICAM1 in the exosomes and also up-regulates galectin-3 which increases the specificity of the exosome based delivery vehicle.

The exosome based delivery vehicle in the present invention are bio -engineered to target cancer cells including but not limited to solid tumor cancers, hematologic cancers and metastatic cancers. A cancer may be a non-solid tumor type or a solid tumor. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, breast cancer, prostate cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, gastric cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, hematologic malignancies, acute myeloid leukemia, lymphoma and leukemia, metastases of the pancreas, breast, lung, colon, and melanoma, among others. The present exosome -based delivery vehicle is also capable of targeting cancer stem cells and hematological malignancies like CML and inhibit cancer cell growth. The delivery vehicle size ranges from 30nm to 150nm and carry active molecules in the size range of 30 kDa to 90 kDa. The shelf life of the vehicles is 1 to 3 months at -20°C and 6 months at -80°C. These delivery vehicles can successfully carry active substance within biological condition for about 30 mins to 3 hours time period and increases the half life of the active substance by 24hrs within biological condition.

The active substance is any substance or mixture of substances that directly inhibits abnormal cell growth. The substance alone or in combination with any other substance may enhance or inhibit a biological function that results in restriction of abnormal cell growth. The active substance alone or in combination with any other active substance may control the secretion of substances that regulate the function of the human body exhibiting abnormal conditions. The active substance in the present invention is selected from but not limited to a protein, a drug, an enzyme, a nucleic acid, a chemical and a mixture thereof. The chemotherapeutic drug includes but is not limited to Paclitaxel, Cisplatin, Doxorubicin. In one example of the present invention, the exosome -based delivery vehicle is used to carry biomolecules like mRNA, shRNA, siRNA, and DNA. In another example, the exosomes 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 a chemotherapeutic drug Doxorubicin. Fig.l shows schematic representation of efficacy of the present exosome based delivery vehicle loaded with chemotherapeutic drug, according to an example of the invention. The present vehicle also reduces the number of chemo-cycles required thus reducing the treatment cost as well as physical and the psychological effect on the patients.

The exosomes-based delivery vehicle successfully avoids phagocytosis by macrophages and prolongs the half-life of the active substance when loaded into vehicle owing to its clathrin-coating, so that the delivery vehicle can safely reach the target cell.

Another embodiment of the present invention provides a method of producing exosome- based delivery vehicle for targeted delivery of active substance to cancer cells and cancer stem cells. These exosomes are bio-engineered to increase its specificity towards the cancer cells and cancel stem cells. The method includes the steps of incubating monocytes in a culture medium containing a bio-formulation to obtain a dendritic cells, adding an active substance into the culture medium containing the previously obtained dendritic cells to obtain an exosome based delivery vehicle loaded with active substances and isolating the exosome based delivery vehicle from the culture medium for targeted delivery of active substances specific to cancer cells and cancer stem cells. The steps described herein are described in details below:

Initially, monocytes are incubated in a culture medium to obtain dendritic cells, herein after referred to as DCs. The culture medium is selected from the list including but not limited to RPMI, DMEM, L-15 or DMEM-high glucose. In one example, THP-1 cell line (a human leukemia monocytic cell line), procured from NCCS Pune, is maintained in DMEM supplemented with 10% FBS and IX antibiotic and antimycotic mix (Himedia) in a humidified chamber at 370C at 95% air and 5% CO2. Cells in the exponential growth phase are then used for further experiments. Next step is the generation of DCs from THP- 1 monocyte cell lines. In one example, mature DCs are induced from ThPl cells following incubation in RPMI media containing the present bio-formulation. The period of incubation is about 48 hours to 50 hours. The cells are incubated in a humidified chamber maintained at around 37° C at around 95% air and about 5% CO2. The DCs, hence obtained are then loaded with active substance to obtain exosome based delivery vehicle loaded with active substances. The active substance is packed with in the exosome via endogenous or exogenous packaging method. In one example, to package active substance within exosomes derived from DCs, monocyte-derived DCs are incubated with the active substance to be packaged within exosome in RPMI media containing the bio-formulation. The culture media is then harvested after about 24 hours for exosome isolation to obtain the exosome based delivery vehicle for targeted delivery of active substances to cancer cells and cancer stem cells. In one example, doxorubicin loading is performed by treatment of mature DCs with doxorubicin at a concentration of 1.8pg/ml or 2.0pg/ml for about 24 hours. Thereafter, the exosomes are isolated from the cell culture media using Total Exosome Isolation Reagent (Cat No. 4478359) using protocol given in the product manual. Exosomes, according to an example of the present invention is isolated from 2*10⁸ matured DC-CM cultured in exosome-depleted media for 48 hours by Total exosome isolation reagent. The harvested media is centrifuged at 2,000 rpm for 3 min at 4°C, and the supernatant is collected and centrifuged at around 4,000 rpm for about 30 minutes at around 4 °C. Exosomes are isolated following manufacturer’s instruction. The exosome pellet is then washed once with calcium and magnesium free PBS and re-suspended in 50- 100 pl PBS. Total exosomal protein is measured by the Bradford Protein Assay. In one example, the yield is 6-7 g/pl from 1ml of culture media.

Yet another embodiment provides a bio-formulation that increases the specificity of the exosome based delivery vehicle to cancer cells and cancer stem cells. The bio-formulation is a cocktail of ingredients that are mixed with the culture media in which the exosome based delivery vehicles are developed. The ingredients are selected from the list including but not limited to lipopolysaccharide, hereinafter referred to as LPS, Tumor Necrosis Factor-a, hereinafter referred to as TNF-a, Angiotensin II, Interleukin ip, hereinafter referred to as IL-ip and Interferon- Y, hereinafter referred to as IFN-Y. In particular, the composition of the present bio-formulation includes granulocyte macrophage colonystimulating factor in the concentration range of 100 ng/mL to 150ng/mL, interleukin 4 in the concentration range of 100 ng/mL to 150 ng/mL, ionomycin in the concentration range of 200 ng/mL to 250ng/mL, tumor necrosis factor alpha in the concentration range of 100 ng/mL to 150 ng/mL, IL1 P in the concentration range of 20 ng/mL to 40 ng/mL, LPS in the concentration range of 200 ng/mL to 250 ng/mL and Angiotensin II in the concentration range of 50 ng/mL to 100 ng/mL.

The process of preparation of bio-formulation includes the steps of adding the respective ingredients in their respective concentration in 100ml of culture media. The culture media in the present invention is any media wherein the monocytes of the present invention are incubated followed by generation of mature dendritic cells and eventually the exosome based delivery vehicle, including but not limited to RPMI, DMEM, L15 media. In one example, the culture media is high glucose DMEM media. The formulation hence obtained is then slowly stirred in a sterile environment at around 4°C for about 15 minutes to 20 minutes.

The source of the ingredients of the bio-formulation are listed below

According to an embodiment of the invention, incubation of the monocytes with bioformulation over-expresses a ICAM-1. These over-expressed proteins attributes to the target specificity of the obtained exosome based delivery vehicle. ICAM-1 over-expression counteracts immune-suppression by cancer cell-derived PGE2 to restore Cytotoxic T cell (CTL) function. Effectively the delivery vehicle of the present invention targets cancer stem cells and cancer cells and specifically getting internalized by cancer stem cells and cancer cells and at the same time the present invention also facilitates to increase the surface ICAM1 on these cells to increase their immunogenic profile, thereby enhances detection by immune cells such as CTLs. ICAM-1 also binds LFA-1 on CTLs and activate them in-vivo. The over expressed ICAM-1 also targets CD43 in the cancer stem cells to increase its specificity. The bio-formulation also up-regulates galectin-3 along with ICAM1 which helps targeting cancer cells and cancer stem cells. Galectin-3, hereinafter referred to as GAL3, is a structurally unique beta-galactoside-binding lectin, through the specific protein-protein and protein-carbohydrate interactions it participates in numerous biological processes, such as cell proliferation and apoptosis, adhesion and activation. Lipopolysaccharide (LPS) in the present bio-formulation provokes up-regulation of GAL3 expression on both gene and protein level in monocyte-like THP-1 cells and simultaneously increases the production of exosomes. MUC1 is a natural ligand of GAL3. The interaction between MUC1 and GAL3 promotes internalization of the present exosomes based delivery vehicle by cancer stem cells and cancer cells, thereby increasing the effectiveness.

Efficacy Studies:

Analyzing the toxicity of the present exosomes on patient derived and healthy individual derived PBMC.

The exosome based delivery vehicle of the present invention has a multi-pronged effect whereby it reduces the toxicity of chemotherapeutic drugs. To analyze the toxicity of free DOX and DOX-loaded exosomes (EXO-DOX) on PBMCs, PBMCs is isolated from healthy individual and tumor patients. Following isolation, they are cultured with DOX- loaded exosomes or free DOX for about 48 hours. The packaging of DOX within exosomes reduced its toxic side effects. In healthy patient blood the toxicity is reduced by -2 fold whereas in patient blood the toxicity was reduced by ~1.3 fold. Fig. 2 shows Flow cytometric analysis Annexin V cells following culture of PBMC with EXO -DOX and free DOX.

Analyzing the internalization efficiency of the present delivery vehicle by cancer cells and cancer stem cells:

To understand the internalization efficiency of the present delivery vehicle, the matured DC-derived exosomes and THP1 -monocyte derived exosomes 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 present result shows that matured DC-derived exosomes has a greater potency to get internalized in cancer cells (-12 fold) and CSCs (-2.8 fold) starting at around 6 hours as compared to THPl-monocyte derived exosomes. This establishes the enhanced targeting capabilities of the present delivery vehicle as compared to THPl-monocyte derived exosomes as shown in Fig. 3.

Analysis of ICAM1 expression in targeted exosomes

To validate the increase in expression of ICAM1 in targeted exosomes western blot analysis is conducted. To that end THP1 cells are treated with the present bio-formulation for about 48 hours thereafter exosomes are isolated. Following isolation of exosomes, total exosomal protein is isolated. ICAM1 expression on the targeted exosomes is analyzed by western blot analysis. The results depicts that following incubation of THP1 cells with the bio-formulation, the expression of ICAM1 increased -4 fold as compared to exosomes isolated from untreated THP1 cells as shown in Fig. 4.

Analysis of specific targeting of CSCs and cancer cells

To analyze the specificity of the present exosome -based targeted delivery vehicle, the present targeted exosomes is packaged with Doxorubicin (EXO-DOX). Thereafter, the breast cancer derived cancer stem cells, breast cancer cells and HEK293 cell are treated with EXO-DOX (4ng/ml) and free DOX (1800ng/ml). Following treatment, a comparable percentage of Annexin V cells in free DOX treated set as well as EXO-DOX treated set is found. This suggests that EXO-DOX is equally efficacious as free DOX at a -450 fold less concentration. Interestingly, when HEK293 cells are treated with EXO-DOX (4ng/ml) and free DOX (1800ng/ml), EXO-DOX failed to induce apoptosis in HEK293 cells where free DOX induced apoptosis in HEK293 cells. This confirms the specificity of the present invention to CSCs and cancer cells. Fig. 5a, 5b and 5c shows specific targeting of cancer cells and cancer stem cells by the present delivery vehicle.

Analysis of cytotoxicity in CSCs following incubation with equal concentration of free DOX and EXO-DOX

To analyze the enhanced effectivity of EXO-DOX as compared to free-DOX, cancer stem cells generated from MDA-MB-231 cells and MDA-MB-468 cells is cultured and treated them with 5ng/ml of free DOX and 5ng/ml of EXO-DOX. Annexin V analysis via flow cytometry reveals a ~2fold and ~6fold increase in apoptosis of MDA-MB-231 and MDA- MB-468 cell-derived CSCs respectively as shown in Fig. 6.

Because many modifications, variations, and changes in detail can be made to the described preferred embodiments of the invention, it is intended that all matters in the foregoing description and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense. Thus, the scope of the invention should be determined by the appended claims and their legal equivalence.

Claims

We Claim,

1) An exosome based delivery vehicle for targeted delivery of active substances to cancer cells and cancer stem cells, wherein the exosomes are produced by the method comprising the steps of:

Incubating monocytes in a culture medium containing a bio-formulation to obtain a dendritic cells, wherein the monocytes are incubated for 48 hours to 50 hours at a temperature range of 36°C to 38°C at 5% CO2;

Adding an active substance into the culture medium containing the dendritic cells to obtain an exosome based delivery vehicle loaded with active substances, wherein the dendritic cell is incubated with the active substance for 20 hours to 24 hours; and,

Isolating the exosome based delivery vehicle from the culture medium for targeted delivery of active substances specific to cancer cells and cancer stem cells.

2) The exosome based delivery vehicle as claimed in claim 1, wherein the size of the exosome based delivery vehicle ranges from 30 nm to 150 nm.

3) The exosome based delivery vehicle as claimed in claim 1, wherein the bioformulation decreases the incubation period for the monocytes to obtain the dendritic cells.

4) The exosome based delivery vehicle as claimed in claim 1 , wherein the active substance is an anti-cancer drug or anti cancer- RNA or DNA or proteins or combination thereof.

5) The exosome based delivery vehicle as claimed in claim 1, wherein the culture media is any culture media used for incubation of monocytes to obtain exosome based delivery vehicle.

6) The exosome based delivery vehicle as claimed in claim 1, wherein the molecular weight of the active substance is in the range of 300 g/mol to 12,000 g/mol. 7) The exosome based delivery vehicle as claimed in claim 1, wherein the bio -formulation increases ICAM1 in the exosomes for targeted delivery of active substance to cancer stem cells and cancer cells.

8) The exosome based delivery vehicle as claimed in claim 1, wherein the bio -formulation up-regulates galectin-3.

9) The exosome based delivery vehicle as claimed in claim 1, wherein the biocomposition comprises granulocyte macrophage colony-stimulating factor in the concentration range of 100 ng/mL to 150ng/mL, interleukin 4 in the concentration range of 100 ng/mL to 150 ng/mL, ionomycin in the concentration range of 200 ng/mL to 250ng/mL, tumor necrosis factor alpha in the concentration range of 100 ng/mL to 150 ng/mL, ILip in the concentration range of 20 ng/mL to 40 ng/mL, LPS in the concentration range of 200 ng/mL to 250 ng/mL and Angiotensin II in the concentration range of 50 ng/mL to 100 ng/mL, wherein the bioformulation is mixed in 100ml of culture media.

10) A bio-formulation to increase the specificity of exosome -based delivery vehicle to cancer cells and cancer stem cells, the bio-composition comprises granulocyte macrophage colony-stimulating factor in the concentration range of 100 ng/mL to 150ng/mL, interleukin 4 in the concentration range of 100 ng/mL to 150 ng/mL, ionomycin in the concentration range of 200 ng/mL to 250ng/mL, tumor necrosis factor alpha in the concentration range of 100 ng/mL to 150 ng/mL, ILip in the concentration range of 20 ng/mL to 40 ng/mL, LPS in the concentration range of 200 ng/mL to 250 ng/mL, Angiotensin II in the concentration range of 50 ng/mL to 100 ng/mL and/or combination thereof, wherein the bioformulation is mixed in 100ml of culture media. 11) The bio-formulation as claimed in claim 10, wherein the bio-formulation increases ICAM1 in the exosomes for targeted delivery of active substance to cancer stem cells and cancer cells.

12) The bio-formulation as claimed in claim 10, wherein the bio-formulation decreases the incubation period for the monocyts.

13) The bio-formulation as claimed in claim 10, wherein the bio-formulation up-regulates galectin-3.

14) The bio-formulation as claimed in claim 10, wherein the culture media any culture used for incubation of monocytes to obtain exosome based delivery vehicle.

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